Use of Biomarkers in

Assessing Health and

Environmental Impacts of
Chemical Pollutants
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Use of Biomarkers in
Assessing Health and
Environmental Impacts of
Chemical Pollutants
Edited by

Curtis C. Travis
Oak Ridge National Laboratory
Oak Ridge, Tennessee

Springer Science+Business Media, LLC

Proceedings of a NATO Advanced Research Workshop on
Use of Biomarkers in Assessing Health and Environmental Impacts of Chemical POllutants,
held June 1-5, 1992,
in Luso, Portugal

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 DIRECTEDBY

Dr. Curtis Travis and Dr. Jose Amaral-Mendes

ADVISORY COMMITTEE

DR.LARSEHRENBERG DR. JOSE AMARAL-MENDES

Department of Radiobiology
University of Stockholm
Stockholm, Sweden S 106 91
(46) 8 162000
(46) 8 166488 (FAX)
DR. ROBERT LAUWERYS
Universite Catholique De Louvain
Faculte De Medecine
Clos Chapelle-Aux-Champs 30
Bte 30-4
1200 Bruxelles, Belgium
(32) 2 771 4772
(32) 2 764 5322 (FAX)
Environmental Health and Toxicological
Studies Center
National Institute of Health
Av. Padre Cruz
1699 Lisbon Codex, Portugal
(351) 1 6790217
(351) 1 607481 (FAX)
DR. CURTIS TRAVIS
Center for Risk Management
Oak Ridge National Laboratory
P.O. Box 2008, MS-6109, 4500S
Oak Ridge, Tennessee 37831-6109
(615) 576-2107
(615) 574-9887 (FAX)

v
 PREFACE

Biological markers (biomarkers) are useful tools for understanding the nature and
extent of human exposure and risk from environmental toxicants. Biomarkers are classified
into three basic categories: exposure, effect, or susceptibility. A marker of exposure is the
product of the interaction between a target cell or molecule and a foreign substance (NAS,
1989). These markers can be used to determine the biologically effective dose necessary
to elicit a particular physiological change in an organism. A marker of effect is a
biochemical or physiological change in an organism that can predict the onset of adverse
health effects resulting from a given exposure. Lastly, markers of susceptibility act as
indicators of an inherent or acquired tendency of an organism to experience an adverse
health effect (NAS, 1989).These markers are already used to detect a variety of diseases
and show great promise for developing a better understanding of the mechanicisms of
disease. Additionally, biomarkers can be used to establish a more rational basis for
quantitative risk extrapolation between species, as weIl as to obtain more precise estimates
of the time of critical exposure. These markers can also prove helpful in identifying
potentially damaging exposures before the onset of adverse health effects. Biomarkers serve
as a valuable exposure assessment tool because they take into account exposure from all
routes and integrate exposure from all sources. They have the potential to yield better risk
estimates than current monitoring and modeling protocols.
In lune 1992, Dr. Travis and Dr. Amoral-Mendes co-directed a NATO International
Scientific Exchange Programme Advanced Research Workshop in Luso, Portugal, on the
use of biomarkers in assessing health and environmental impacts of chemical pollutants. The
Advanced Research Workshop brought together international experts on biomarkers and
biomonitoring in an effort to devise a unified strategy for development and validation of
biomarkers as a means of assessing the status of environmental health. The workshop united
scientists from around the world to collaborate on the use of biomarkers in evaluating
human health and the environment, and to formulate the research needs for implementation
and interpretation of biological monitoring programs to define environmental health
problems.

*National Academy of Sciences (NAS), 1989, Biologie Markers in Reproduetive Toxieology. Subcommittee
on Reproductive and Neurodevelopmental Toxicology, Committee on Biologie Markers, National Academy
Press, Washington, D.C.

vii
 This volume contains manuscripts from each of the workshop speakers on the diverse uses
of biomarkers in assessing human health. The articles include such topics as the use of biomarkers
to prioritize community health problems, the use of ecological biomarkers (such as fish) as
indicators of human exposure, and the use of DNA adducts in quantitatively predicting cancer risk.

Curtis C. Travis

Center for Risk Management

Oak Ridge National Laboratory
Oak Ridge, Tennessee 37831-6109

vi
 CONTENTS

MOLECULAR DOSIMETRY

Introduction to Molecular Dosimetry

L. Ehrenberg

The 4-Aminobiphenyl Hemoglobin Adduct as a Biomarker of Effect . . . . . . . . . . . . . . . . .. 9

P. L. Skipper

Current Research on Hemoglobin Adducts and Cancer Risks: An Overview ............. 17

M. Trnqvist

Use of Biomarkers in Quantitative Risk Assessment ............................. 31

L. Rhomberg

DOSE-RESPONSE RELATIONSHIPS FOR MUTAGENIC

AND CARCINOGENIC ACTION OF GENOTOXIC AGENTS

Measurement of Mutation Spectra as a Molecular Dosimeter ....................... 47

W. G. Thilly

Biomarkers as Molecular Dosimeters of Genotoxic Substances ...................... 53

P. B. Farmer

BIOMARKERS OF ENVIRONMENTAL EXPOSURE

Public Health Assessment as a Tool in Identifying Human Exposure

to Environmental Pollutants .......................................... 63
M. M. Bashor

The Suitability of the Mosses Sphagna as Quantitative Indicators of Heavy Metal Levels
in Urban Atmospheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
M. T. Vasconcelos and H. M. Tavares

Epidemiologie Approach for the Assessment of Acceptable Exposure Levels to Cadmium

and Manganese ................................................... 73
R. R. Lauwerys, A. Bernard, H. Roels, and J. P. Buchet

Biological Monitoring of Exposure to Organie Compounds ........................ 83

M. Jakubowski

ix
 BIOMARKERS OF TOXICITY

Stress Proteins as Biomarkers of Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

P. L. Goering, B. R. Fisher, C. A Kimmei, G. L. Kimmel

Significance of Serum Ferritin Concentrations in Lung Cancer and Its Relation with
Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
O. S. Sardas, S. Sardas, and O. Sancaktar

BIOLOGICAL MARKERS IN REPRODUCTlVE TOXICOLOGY

Outcome Based Biomarkers of Female Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . .. 105

J. F. larrell

Factors Determining the Exposure of the Embryo and Fetus:Species Variation of

Teratogenesis and Placental Transfer of Xenobiotics . . . . . . . . . . . . . . . . . . . . . . . . . 121
H. Nau

Assessing Reproductive Risks with Biological Markers . . . . . . . . . . . . . . . . . . . . . . . . . . 137

T. Silvaggio and D. R. Mattison

BIOMARKERS OF NEUROTOXICITY

Behavioral Biomarkers to Identify Neurotoxic Effects 159

W. K. Anger

Mechanisms of and Biomarkers for Acute and Delayed Neuropathic Effects

of Organophosphorus Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
M. K. Johnson

Mechanisms and Biomarkers of Solvent-Induced Behavioral and Neuroendocrine Effects ... 183
AMutti

Biomarkers of Immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

L. D. Koller

Serum Biomarkers in the On-Site Evaluation of Suspected Cancer Risk in Humans

Residing Near Hazardous Waste Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
E. Pluygers, P. Gourdin, G. Dardenne, B. Scoubeau, and A Parfonry

The Use of Biomarkers in the Evaluation of Exposure and Health at a

Hazardous Waste Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
J. S. Reif, H. S. Ramsdell, N. M. DuTeau, W. K. Anger, and T. A Tsongas

ECOLOGICAL BIOMARKERS

State of the Art--Ecological Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

L. R. Shugart

Detection of Genotoxicity of Water and Air Pollutants Using

Tradescantia (Spiderwort) Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Te-Hsiu Ma

x
Animals and Plants as Bioindicators of Radionuclide
Contamination in Forest Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
R.T.Palo

TUMOR MARKERS

Tumor Markers in Effusions:A Comparative Study of Tumor Marker Levels

in Sera and Effusions ............................................. 265
I. Gullu, S. Yalcin, G. Tekuzman, A. Kars, E. Baltali, N. Guler,
I. Barista, D. Firat, C. Bekdik, and Z. Koray

Evaluation of Ceruloplasrnin Level in Women with Breast Disease:Prelirninary Results .... 273
O. Ozyilkan, E. Baltali, A. Kars, G. Tekuzman, and D. Firat

Contributors ........................................................ 279

Index ............................................................. 283

xi
INTRODUCTION TO "MOLECULAR DOSIMETRY"

L. Ehrenberg

Department ofRadiobiology, Stockholm University

S-106 91 Stockholm
Sweden

The main message of this introduction concerns the necessity of keeping the
quantitative aspects in mind in applications and development of methods for molecular
dosimetry and related biochemical methods.
In vivo dose monitoring by means of macromolecule adducts was originally suggested
as a possible way of solving aseries of problems posed by experience from the use of
current methods for risk assessment, particularly of mutagens and carcinogens. Of prevalent
techniques for this purpose, disease epidemiology and long-term animal tests suffer from low
sensitivity (low power) and epidemiological methods also from long latency times and
difficulties of identitying causative factors, the latter also a problem when cytogenetic
endpoints are determined as a measure of exposure. Short-term in vitro tests are able, in
many cases with a high power, to detect genotoxic properties, but cannot in their present
design generate data that permit risk quantification (risk estimation).
As far as can be judged today the hopes originally set on adduct measurement seem to
be fulfilled: ."'rom an observed adduct level the dose, properly defined (cf below), can be
calculated and the associated risk estimated. Also apriori unknown mutagens/cancer
initiators can be detected, identified and subjected to risk estimation. The method, which is
applicable to both animals and humans, permits a risk assessment ~tlready a few days after
the onset of an exposure. The power of the method is at present higher than those of disease
epidemiology and Iong-term animal tests by 3-4 orders of magnitude, an increase in
sensitivity that is in many cases sufficient to cover the whole range of risks that, at least for
individual compounds, might be considered unacceptable (Trnqvist et al., 1986). By
development of analytical methods a considerable further increase in power is within reach.
At present the number of laboratories adopting methods for measuremtlnt of adducts,
particulary to DNA but also to hemoglobin (Rb) and other macromolecules, is increasing
rapidly, however, with very few exceptions withouth those quantitative aspects that are
required for a realization of the meaning of observed adduct levels. Although those studies
have demonstrated differences between exposed and non-exposed populations - an
indication ofthe usefulness, in principle, ofthe procedures as epidemiological tools - there is
a certain danger in this development.
A major, if not predominant, effect of applications of nuc1ear energy are the psycho-
social disturbanees, with anxiety in exposed populations (Ehrenberg, 1978; International
Chernobyl Project, 1991ab) and skewed priority setting in consequence. This effect is to a

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993
large extent due to the smallness ofthe Bq unit and the great power ofmethods for radiation
dosimetry, coupled with the difficulties of picturing small risks (in the sense of probabilities
of contracting cancer). The development of methods for chemical dosimetry is now, in terms
of risk, approaching the sensitivity of those for radiation dosimetry. This renders it
increasingly important to translate measured adduct levels to the associated risks, not only in
order to optimize priorities for risk-reducing measures, but also to avoid alarms that could
lead to a banning ofvaluable products the use ofwhich is associated with negligible risks.

PRINCIPLES OF RISK ESTIMATION OF CHEMICALS

At this place seemingly important corner-stones of the complex process of risk

assessment (coHective term, for identification and estimation of risks; cf O'Riordan, 1979)
will be briefly summarized. Margareta Trnqvist will at this Workshop give a few practical
examples.
It has fust to be realized that there is at present no generally valid method for risk
estimation. In the present period of development of more sensitive methods it is therefore
needed to weigh together the molecular measurements with data from all sourees:
epidemiological studies (it is often overlooked that also negative results are of value; giving
an upper limit of a possible risk they are a useful check of the reasonableness of estimated
risks), animal tests (including data for metabolism) and short-term tests. This means that one
has to overcome what Barr (1985) called the "overcompartmentalization of the risk
assessment arena", meaning experts' tendency to overemphasize the ability of their own
approach, and neglecting those of others, to solve the problem.
A central question concerns dose-response relationships. At low doses frequencies of
induced mutation are compatible with a linear dependence on dose without no-effect
threshold, and data are accumulating to indicate that no such threshold exists, even if the
slope of the curve could be somewhat higher or lower at those very low doses where, for
statistical reasons, informative observations cannot be obtained (equation 1; cf Ehrenberg et
al., 1983).

P(D)=p o +bD (1)

(P probability; po background risk; b slope; D dose). Expression (1) is expected to hold -

n.b. at low doses - also for cancer incidence due to mutagens, acting as initiators in
interaction with promotive and modifying factors and conditions that are present for reasons
other than the exposure.
This linearity of the dose-risk relationships implies that, in contrast to acute toxic
effects, anyexposure, however small, leads to a risk increment, with the consequence that
monitoring methods have to be sensitive enough for the detectioll of risk factors down to
those low doses where the risks are becoming acceptable.
Due to the mentioned interaction, a high background incidence in a particular organ
would presuppose that, generally, there is a high level of promoter and other cancer-prone
conditions that may interact with "spontaneous" as weH as with induced mutations. In
concordance herewith a strong tendency towards proportionality between po and b in
equation (1) has been observed, primarily in comparative studies of radiogenic cancer in
different animal strains (Storer et al., 1988), but also in radiation-exposed human
populations (cf NRC, 1990). In risk estimation ofIow-LET radiation, the additive model (1)
has been shifted towards a multiplicative model which, in principle, could be written (cf
NRC,1990):

Pj(D) = (1 + ) Pj~ (2)

2
where j denotes organ (site), age, sex. This model seems also to be applicable to the
carcinogenie action of ethylene oxide in rodents and is probably valid also for other
chemieals (Ehrenberg et al., 1992).
Due to the mostly high incidences at higher ages, application of the multiplicative
model (2) leads to considerably higher lifetime projected radiation risks than the previously
used additive model (1) (UNSCEAR, 1988; NRC, 1990). If this applies also to mutagenic
chemieals - which is expected in view of the irreversibility of mutations - epidemiologie
studies of chemically exposed populations may lead to large underestimations of the cancer
risks, ifthe follow-up periods are too short.
For related reasons it seems more correct to calculate with the lifetime dose rather
than the dose per day, as is done, e.g., by U.S. EPA, in risk estimation from animal test data
(cf Trnqvist and Ehrenberg, 1992).

RELA TIVE-POTENCY METHODS

The number of potentially mutagenic/carcinogenic chernicals to which humans are

exposed amounts to thousands or tens of thousands, sometimes entering as components in
complex mixed exposures. Obviously, each of these compounds cannot be subjected to an
expensive and resource-consuming risk assessment. Certain simplified techniques will be
needed, involving identification of those compounds which are important contributors to risk
or which can be used as representative indicators of mixed exposures.
In this situation so-called relative-potency methods may present a simplified solution
of the problem. These methods are based on the determination of the relative genotoxic
effectiveness of studied compounds or mixtures using an agent with well-defined dose-risk
relationship as a reference standard. Two approaches of this kind are studied at present: J.
Lewtas (1992) bases risk estimation of air pollutants, particularly diesel exhausts, on their
potency compared to that of the occupational exposure in coke-oven and similar work, the
associated lung cancer risk of which has been estimated epidemiologically. So far these
studies have been carried out without in vivo dose monitoring. The Stockholm group tries to
express in vivo doses, measured by means of adduct monitoring, in radiation-dose
equivalents (rad-equ.), low-LET radiation being the environmental mutagenic factor the
cancer risks of which have been most intensely studied. Besides that the risk philosophy
developed, particularly by ICRP (1977, 1991), for radiological protection has a great value
as a model in the corresponding protection against chemical mutagens and carcinogens
(important aspects summarized in Table 1). The approach is based on the reasonable
assumption that the probability of an initiated cell giving rise to a tumour, in interaction with
prevailing pro motive and modifYing factors, which may be inherited or acquired, is the same,
irrespectively ofwhether the initiation was caused by radiation or a chemical mutagen.
The rad-equivalence approach is based on early studies of reaction-kinetic parameters
that are deterrninants of genotoxic potency (Turtoczky and Ehrenberg, 1969; Osterrnan-
Golkar et al., 1970; review: Ehrenberg, 1979). Results of studies of nucleophilic substitution
reactions

RX + Y ~ RY + X (3)
electrophile nucleophile product leaving
("adduct") group

indicated that at low dose the degree of alkylation, [RY]/[Y], inducing the same forward
mutation frequency as 1 rad of low-LET radiation was the same for different alkylating
agents. Since it was also the same in different biological systems including mammalian cells,
it was assumed that it might also be valid in man. Provided target doses could be measured
in humans, the risk-coefficients for radiogenic cancer could then be applied for risk

3
Table 1. Purposes of comparisons of chemicals and ionizing radiation with respect to health
effects

elllustration, in terms of a known factor, of the detection level (power) of epidemiological

studies or laboratory tests.

e Definition of "acceptable" risk, for instace as the required sensitivity of test procedures
(Tmqvist et al., 1986).

e Application of mIes for radiological protection (dose optimization, ALARA principle) to

chemical exposures (ICRP, 1977, 1991).

e Using experience from epidemiological studies of irradiated populations to (testable)

hypotheses of importance to risk estimation of genotoxic chemicals regarding expected
effects (e.g. effects on IQ?); influences of dose-rate, age, background incidence, etc. (cf
ICRP, 1991).

eExpression of chemical doses in radiation-dose equivalents (rad-equivalents) offers a

promising principle for risk estimation (the rad-equivalence approach).

e By giving different exposures in a common unit, possibilities of comparison are gained, e.g.
for solutions of technical problems (food preservation, energy sources). Further, this
permits common management of radiogenic and chemical cancer risks and may decrease
the gap between estimated risk and perceived risk.

estimation. In on-going work various etforts to validate the approach, including the difficult
problem of influences of dose rate (for chemicals, dose rate = concentration; cf Ehrenberg
et al., 1983), have so far been confirmatory.
According to this relative potency approach, the risk increment from an exposure is
calculated from expressions

P(D) = kstd . Q . Dtarg (4)

where kstd is the risk coefficient for the reference standard, Q the relative potency (such as
the "rad equivalence") and Dtarg the target dose, defined as the time-integral of
concentration ofthe proximal mutagen in (nuclei of) target cells (Ehrenberg et al., 1983)

D = J[RX](t)dt (5)
t
During human exposures, which are mostly chronic or intermittent, steady-state levels
of adducts are buHt up. The relationship of dose to the steady-state adduct level is
determined by the ratio, kJl4, of the rates of disappearance and formation, respectively, of
the adducts (cf Granath et al., 1993). Due to the variation in rate of repair between
chemicals, tissues, ceIls and chromosome regions, k.. is not known and doses cannot at
present be calculated from steady-state DNA adduct levels.
In contrast, protein adducts are, at least at low levels, not subjected to "repair" and the
value of k.. is thus weIl defined. Therefore, in the tissue which is generally most easily

4
accessible, the blood, the hemoglobin (Rb) and to some extent serum albumin appear more
useful than leukocyte DNA for in vivo dose monitoring. In the Stockholm group methods
for measurement ofHb adducts have been developed, with an analytical power sufficient to
cover the whole range where associated cancer risks may be considered unacceptable
(Trnqvist et al., 1986).
In view of certain indications of the existence of a fraction of long-lived lymphocytes
with a very slow or zero DNA repair (Trnqvist, this Workshop), it becomes an important
issue to characterize the kinetics ofDNA repair in white blood cells to an extent that renders
DNA directly useful for a dosimetry and risk estimation valid for much longer periods of
time than the four months covered by the life span ofHb.
The discussed monitoring systems give measures of the dose in the blood, Dbl. In
order to allow for dose gradients in the body, Dtarg in equation (4) has therefore to be
corrected by values for the ratios DtargiDbl as determined in animal models. The issue of
expressing risk (P(D in terms of exposure dose will be discussed below by M. Trnqvist.
It may be pointed out that the risk/dose function (equation 4) is, in principle, the same
as the one which has been used for radiations of qualities deviating from that ofthe reference
standard, gamma-radiation (ICRP, 1977).

ALOOKAHEAD

One lesson leamt from work with risk assessment is that for a long time there will be
no single routine method for risk estimation of environmental chemieals. At present, data for
exposures (which are often disturbingly shaky) have to be combined with knowledge ftom
epidemiological and experimental studies, including data on metabolism, to arrive at an
estimate that is most likely at the present state of the art. This means that no risk estimate
arrived at today should be considered final, but that it may be improved as our knowledge of
the mechanisms of carcinogenesis progresses. Although much remains to be known about
these mechanisms, present efforts to estimate risks are based on mathematical descriptions of
the kinetics ofthose steps in carcinogenesis which we think are rate-limiting (Ehrenberg and
Scalia-Tomba, 1991). In particular, deviations ftom linearity of dose-response curves at very
low doses or dose rates (Ehrenberg et al., 1983) including the question whether no-effect
thresholds exist (Granath, 1991) are important issues.
Another problem concerns the origin of the background cancer incidence in knowingiy
unexposed persons. From variations in cancer incidence and mortality between population
strata and geographie areas a large ftaction ofthe background incidence is "explainable" in a
statistical sense, and has partly been referred to life-style factors (Higginson and Muir, 1979;
Ehrenberg et al., 1990). It is a further, important question to what extent initiations in this
background carcinogenesis are caused by unknown reactive chemicals, partly of endogenous
origin, of the kinds observed in studies of background adduct levels (Trnqvist and
Kautiainen, 1992, and Trnqvist, this Workshop), in which case prcventive measures might
be invented, or to what extent they are due to unavoidable coding errors etc. in normal -
scheduled or unscheduled - DNA synthesis (Smith, 1992).
An important line of research that could shed light on these problems is the
"mutational spectrometry" by a combination of PCR and denaturing gradient gel
electrophoresis (Thilly, 1991). Due to the uniqueness ofthe spectra produced by different
mutagens it seems possible to clarity the relative role of electrophiles identified through their
adducts, and the method seems to be sensitive enough to give important data on dose-
response relationships at low doses.
Another challenging line of research - which, in fact, indicates a bridge on the level of
mechanisms between radiations and chemicals - concerns the immediate response to both
radiation and chemieals, leading to a reprogramming of cells, with, i.a. increased mutation-
proneness and non-targeted mutation in consequence (Herrlich, 1992). Within work aiming

5
at risk assessment it is important to clarify the involvement in the risks of these effects and
their dose-response relationships.

ACKNOWLEDGEMENT

The present review is mainly based on studies supported financially by U.S. Department of
Energy (Grant No. DE-FG02-89ER60784).

REFERENCES

Barr, J.T., 1985, The calculation and use of carcinogenic potency: A review, Regulat. Toxicol. Pharmacol.
5:432.
Ehrenberg, L., 1978, Dose-response relationships for biological effects of ionizing radiation; application in
risk estimation, Report to the Swedish Energy Commission, Swedish Govt., Ds I, 24: 1 (in Swedish
with English summary and table texts).
Ehrenberg, L., 1979, Risk assessment of ethylene oxide and other compounds, in: "Assessing Chemical
Mutagens: The Risk to Humans," V.K. McElheny, and S. Abrahamson, eds, Banbury Report 1,
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Ehrenberg, L., Ekman, G., and Svensson, A., 1990, Epiderniological studies of geographie variations of
cancer incidence in Sweden, Acta Oncol. 29:961.
Ehrenberg, L., Monstacchi, E., Osterman-Golkar, S., and Ekman, G.,1983, Dosimetry of genotoxic agents
and dose-response relationships oftheir effects, Mutat. Res. 123: 121.
Ehrenberg, L., and Scalia-Tomba, G.P, 1991, Mathematical models for the initiating and promotive action
of carcinogens, in: "Statistical Methods in Toxicology, Lecture Notes in Medical Informatics," L.
Hothorn, ed., Springer, Berlin.
Ehrenberg, L., Trnqvist, M., and Vaca, C., 1992, Cancer risks from low doses ofionizing radiation and
electrophilic ehernicals: sirnilarities and differences. Ms.
Granath, F., 1991, Statistical problems in estimating a threshold in a dose-response model., in: "Statistical
Methods in Toxicology," L. Hothorn, ed., Lecture Notes in Medical Informatics, Springer, Berlin.
Granath, F., Ehrenberg, L., and Trnqvist, M., 1993, Degree ofalkylation ofmacromolecules in vivo from
variable exposure. Mutat. Res. (in press).
Herrlich, P., 1992, Induction of gene expression by radiation, in: "Radiation Research: Vol. 2: Proceedings,"
W.C. Dewey et al., eds. (in press).
Higginson, J., and Muir, C.S., 1979, Environmental carcinogenesis: rnisconceptions and lirnitations to
cancer control, J. Natl. Cancer Inst. 63:1291.
ICRP, 1977, "ICRP Publication No. 26, Recommendations ofthe International Comrnission on Radiological
Protection," Pergamon, Oxford.
ICRP, 1991, "ICRP Publication No. 60, 1990 Recommendations ofthe International Comrnission on
Radiological Protection," Pergamon, Oxford.
International Chernobyl Project, 1991, "Assessment ofRadiological Consequences and Evaluation of
Protective Measures. Report by an International Advisory Comrnittee. a: An Overview; b: Technical
Report," IAEA, Vienna.
Lewtas, J., 1992, Carcinogenie risks ofpolycyclic organie matter (pOM): Development of a comparative
potency method, Appl. Occup. Environ. Hyg. (in press).
NRC, National Research Council, 1990, Comrnittee on the Biological Effects oflonizing Radiations. "Health
Effects ofExposure to Low Levels oflonizing Radiation. BEIR V RepOlt," National Academy Press,
Washington DC.
O'Riordan, T., 1979, Environmental impact analysis and risk assessment in a management perspective, in:
"Energy Risk Management," G.T. Goodman and W.D. Rowe, eds.,Acadernic Press, New York and
London.
Osterman-Golkar, S., Ehrenberg, L., and Wachtmeister, C.A, 1970, Reactionkinetics and biological action
in barley ofmono-functional methanesulfonic esters, Radiat. Bot. 10:303.
Srnith, K.C., 1992, Spontaneous mutagenesis: experimental, genetic and other factors, Mutat. Res. 277:139.
Storer, J.B., Mitchell, T.J., and Fry, R.J.M., 1988, Extrapolation ofthe relative risk ofradiogenic neoplasms
across mouse strains and to man, Radiat. Res. 114:331.

6
Thilly, W., 1991, Mutational spectrometry: opportunity and limitations in human risk assessment, in:
"Human Carcinogen Exposure: Biomonitoring and Risk Assessment," R.C. Garner, P.B. Farmer,
G.T. Steel and A.S. Wright, eds., Oxford University Press, Oxford.
Trnqvist, M., and Ehrenberg, L., 1992, On the cancer risk ofUIban air pollution, Environ. Health Perspect.
(submitted) .
Trnqvist, M., and Kautiainen, A., 1992, Adducted proteins for identification of endogenous electrophiles,
Environ. Health Perspect. (in press).
Trnqvist, M., Mowrer. J., Jensen, S., and Ehrenberg, L., 1986, Monitoring of environmental cancer
initiators through hemoglobin adducts by a modified Edman degradation method, Anal. Biochern.
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Effects and Risks ofIonizing Radiation," Report to the General Assembly, United Nations, New
York.

7
TUE 4-AMINOBIPHENYL HEMOGLOBIN ADDUCT

AS A BIOMARKER OF EFFECT

Paul L. Skipper

Division of Toxicology
Massachusetts Institute of Technology
Cambridge, MA 02138

INTRODUCTION

One of the questions raised repeatedly at this meeting was whether biochemical
markers such as DNA and protein adducts of chemical carcinogens were or could be
biomarkers of effect, which is to say that the concentration of the marker is
proportional in some way to the prob ability of developing cancer. The question was
left unresolved, principally for lack of data which could be used to attempt an
answer. In several studies of 4-aminobiphenyl-hemoglobin adducts and bladder
cancer in Prof. S. R. Tannenbaum's laboratory at MIT, we have now made
measurements of this biomarker in more than 500 subjects collectively. It is the
purpose of this paper to compile these data and analyze them with a view to
determining the predictive power of this biomarker.

ASSOCIATION OF RISK FACTORS AND ADDUCTS

The primary risk factor for bladder cancer, outside of occupational exposure to
aromatic amines and some of the products made from aromatic amines, such as dyes,
is cigarette smoking (Ross et al., 1988). The relative risk for smokers ranges from
1.5 to 3.0 compared to nonsmokers, with higher relative risks reported for certain
populations. It has been hypothesized that the aromatic amines in cigarette smoke
may be the principal causative agents, and among these are the human bladder
carcinogens 4-aminobiphenyl and 2-naphthylamine.
Two other risk factors have been identified. One is tobacco type. Smokers of
air-cured ("black") tobacco cigarettes are at elevated risk compared to smokers of
flue-cured ("blond") tobacco cigarettes and experience a relative risk compared to
nonsmokers of 5-8 (Isocovich et al., 1987; Vineis et al., 1988). Again, this may be

Use 0/ Biomar1cers in Assessing Health and Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Tmvis, Plenum Press, New York, 1993 9
related to the higher aromatic amine content of the black tobacco smoke. The
second risk factor is having the slow acetylator phenotype (Evans, 1986). Acetylation
of aromatic amines is considered detoxifying with regard to urinary bladder
carcinogenesis, so the slow acetylatar converts less of a dose of aromatic amine to
its "nontoxic" form. The slow acetylator phenotype is associated with a relative risk
of 1.3 (95% confidence interval, 1.02-1.71).
In cross-sectional studies, it has been found that the me an values of 4-ABP-Hb
adducts in population groups varies accarding to the relative risks observed for those
populations. Thus, all smokers have a mean adduct value of 136 pg/g Rb, which is
4.25 times the mean value of 32 for all nonsmokers. Mean adduct values in slow
acetylators are 1.6, 1.3 and 1.5 times high er than in fast acetylators in, respectively,
nonsmokers, blond tobacco smokers and black tobacco smokers. Among all black
tobacco smokers, adduct values are 5-6 fold higher than among all nonsmokers.
Thus, the principal question of this discussion: If these adducts are biomarkers
of effect, if they yield information concerning which of the individuals eventually
develop bladder cancer as a result 0/ their exposure to 4-aminobiphenyl, how can their
predictive power, if any, be described? Is it, as suggested by the cross-sectional
studies, that an individual's risk is proportional to his adduct level? Or, is the
proportionality of me an adduct values to relative risk simply fartuitous, failing to
reveal an actual situation in which only those individuals with the very highest
adducts are the specific persons who are at risk? A positive answer to the first
question would be consistent with a stochastic mechanism of 4-aminobiphenyl-
induced carcinogenesis, while a positive answer to the second would suggest a more
complex mechanism characterized by nonlinearity of the relationship between target
tissue dose and outcome.
Figure 1 illustrates separately the distribution of all adduct values measured thus
far in nonsmokers and in smokers. In both cases the data are best described as
having a log normal distribution and the fitted prob ability distribution functions are
also illustrated. These data will be examined in terms of the two alternative mod~ls
of carcinogenesis just described.

THE THRESHOLD MODEL

Bladder cancer is relatively infrequent, accounting far about 5% of all cancers

other than non-melanoma skin cancer and carcinoma in situ in the United States.
Since cancer strikes about 30% of the population, a random selection of individuals
as we have done in our cross-sectional studies would have about one person in sixty
who would eventually be co me abladder cancer case. Three out of ten people in the
U.S. are smokers and it will be assumed far the purpose of this discussion that their
relative risk for bladder cancer is 3 compared to nonsmokers. With these inputs, it
can be estimated that one smoker in 32 and one nonsmoker in 96 will become a
case.
Let us assurne that 4-ABP adducts are relatively constant throughout life, so that
a determination made at any stage in life would also characterize the individual when
he became a cancer case. This is probably an accurate assumption. Adduct values
and age have not been carrelated in any of our studies, and recent unpublished data
indicate a very high concardance among blood sampies taken from individuals before
and after a 3-month period. Then, in the set of individuals whose adduct values are
illustrated in Figure 1, there is a high probability that one or more are eventual
cases.

10
 Consider first the smokers, which comprise a set of 301 individuals. Since it was
estimated that 1 in 32 is the prob ability of becoming a case, we would expect this
group to include ab out 9 future cases. The 9 highest values range from about 330
to 600 pg/g Hb, with an average value of about 430. Likewise, the upper tail of the
prob ability density function which has 1/32 of the area begins at 368. Can we
therefore conc1ude that a 4-ABP-hemoglobin adduct of greater than about 350 pg/g
Hb signifies a very high risk of bladder cancer, while lower values such as those near
the mean are associated with a very low risk?

1.4

1.3 ~ ...

Nonsmokera

8.2 _ ...

111.1 '- .. i

>-
U
C
tD
:::I
111 l Ln i
l
u.. 8.1118

8.1118

Smokers

8.84

111.02 r-...'.... _....

['\

o ... iI ~ ';ft itfu - ~ .., m

8 50 llilB 150 200 250 31118 358 _ 458 500 550 _ 650

Adduct level (pg/g Hb)

Figure 1. Distribution of 4-aminobiphenyl hemoglobin adducts in nonsmokers (upper) and smokers

(lower). Log normal distribution functions fitted to the data are also illustrated.

11
 If this interpretation were correct, we might expect smoking cases to exhibit
adducts some 3 to 4-fold higher than smoking controls (ca. 350-600 compared to the
average of ca. 135 pg/g Hb). Quite different results were obtained, though, in a
recently completed case/control study of bladder cancer in smokers and 4-
aminobiphenyl-hemoglobin adducts (DelSanto et al., 1991). The average adduct
value in the cases in this study was not significantly higher than our historical average
value for smokers, and was only increased relative to controls because the controls
were matched according to the degree of tobacco exposure. Even then, the increase
was modest, the ratio of adducts in cases to adducts in controls being only 1.58.
Consideration of the data from nonsmokers also argues against the
interpretation that cases arise from a subpopulation having the highest adducts.
Above 350 pg/g Hb, the area in the tail of the distribution curve which describes the
nonsmoker adduct values is essentially zero, indicating that the relative risk of
nonsmokers compared to smokers would be infinitesimal. In reality, the relative risk
is about 0.3. Thus if the adduct distributions are truly log normal, and the fit is very
good, there would be no nonsmokers with sufficiently high adducts to be at risk,
insofar as such risk can be defined by the right hand tail of the distribution of
adducts in smokers.
It should be recognized, though, that this argument is only valid if the
distribution of adduct values is correctly described by a well-behaved probability
density function such as the log normal. It is also possible that more high adduct
values will be observed than would be predicted by the prob ability density function
genera ted by the set of lower adduct values, for reasons unknown. Do the present
data suggest this? In fact, they do not contradict it: The expectation for the number
of lifetime cases in a set of 225 nonsmokers is only about 2, and we have observed
one adduct value in a nonsmoker in the range suggested as critical. The possibility
that nonsmoker cases will also exhibit adducts as high as the (presumed) smoker
cases cannot be ruled out.

TUE STOCHASTIC MODEL

What could be expected if the adduct is simply a very accurate dosimeter,

reflecting the influence of all the interindividual differences in exposure, uptake and
pharmacokinetics so that it reports very nearly the target dose of the ultimate
carcinogenic metabolite? Taking this point of view and assuming that the
relationship between this accurately determined dose and eventual outcome is purely
probabilistic, some calculations can be made that agree quite weIl with observation.
Thus, assurne that the general population has the bio marker, A, which is the
hemoglobin adduct of 4-aminobiphenyl and whose probability density function has
been characterized. Consider the frequency distributions illustrated in Figure 1 as
being good estimates of the actual continuous probability density functions. This
biomarker is also assumed to be effectively constant throughout life and unaffected
by disease state.
Also assurne that the probability of being an eventual bladder cancer case as the
result 0/ exposure to 4-aminobiphenyl is determined by, and is linearly proportional to,
the value of the biomarker. Thus, if A were 0 the probability would be 0, and at
some as yet unknown value of A the prob ability would be co me 1 and remain 1 for
all greater values of A.
Further assurne that the probability of becoming abladder cancer case from
other causes is independent of the value of A.

12
 Then, the distribution of the biomarker in the subpopulation of individuals who
will be cases in their lifetimes can then be described by the function:

(1)

where P(Ac } is the probability that the marker will have the value A in cases

P(J\} is the prob ability that the marker has the value A in the entire
population; and

k is a proportionality constant; and

kx ~ is < 1 for ~ less than some limit value of A.

-...
"b
7

..
~
~ 5

C
4
'0

~ 3
:i5

..
111
.a
0
a.
i!

58 188 158 i!8II i!S8 388 3S8 .... 458 588 558 _ _

Adduct level (pg/g Hb)

Figure 2. Probability density functions for observed adduct values in all smokers (-) and the
distribution cxpectcd in cases (- - - -) by application of equation 1. The distribution for all smokers is
alm ost identical to that expected in non cases since bladder cancer is relatively rare.

Figure 2 illustrates the distribution of adduct values that would be expected in

cases who are smokers determined according to this model from the distribution of
adducts that we have observed in all smokers. The observed distribution of adducts
in all smokers is also illustrated for comparison. The most important feature of this
new distribution is that its mean value is increased by only about 40% from the mean
value of 136 pg/g Hb for all smokers to 190 pg/g Hb, which is in good agreement
with the results reported by DelSanto et al. in their case/control study.

13
 0_7

0 .1

0_5

0 .4

0.3

>-
U 0-2
r;;;
111
::::I
Z-
u: 0.1

0.3

0-2

0 .1

Adduct level (pg /g Hb)

Figure 3. Upper: distribution or 4-aminobiphenyl hemoglobin adducts expected

in a population composed of nonsmokers and smokers in the ratio of 7:3. Lower:
distribution of adducts expected in cases, based on transformation or the upper
distribution by equation 1. Darker shading represents smokers, while lighter
shading indicates nonsmokers. The fraction of smokers increases from 0.3 in the
general population to 0.68 in cascs.

A second exercise which is of interest is to calculate the proportion of smokers

expected in a sampie of bladder cancer cases, and thereby predict the odds ratio
expected in a case control study of bladder cancer. To do this it is necessary to
combine the adduct distributions for nonsmokers and smokers which are illustrated
in Figure 1, and to combine the two distributions requires adjustment of the values
in one or the other by a constant factor since the two population sizes of 225 and 301
are not representative of the actual ratio of nonsmokers to smokers. If it is assumed
that nonsmokers comprise 70% of the population, then each cell of nonsmokers
adducts can be multiplied by 702/225 for it to represent the correct frequency of
nonsmokers. This adjustment is based on the smoker sampie size of 301, which is

14
assumed to represent 30% of a total sampIe size of 1003. The combined sampIes
histogram is illustrated in the upper half of Figure 3.
Transformation of this data set by application of equation 1 generates the new
frequency distribution illustrated in the lower half of Figure 3, which represents the
hypothetical distribution of adduct values in cases. In this new distribution, smokers
comprise 62% of the total and nonsmokers 38%. Thus, the expected ratio of
smokers to nonsmokers in a sampIe of cases is predicted to be 1.63 and the
corresponding odds ratio if 30% of controls are smokers is 3.8. This value is at the
high end of most estimates of the relative risk for bladder cancer from smoking, but
is not unreasonable. Note that this estimate of the relative risk is not the same as
that obtained by simply taking the ratio of the mean adduct values in smokers and
nonsmokers (4.2) or the ratio of the geometric means (4.4).

CONCLUSION

It has been shown that a simple model of carcinogenesis correctly predicts the
relative risk of smoking for bladder cancer, as weIl as level of adducts in cases, from
the observed distribution of adduct values in the general population. If the
assumptions of the model are accepted, then it may be said that the adducts are
biomarkers of effect. In the model, neither is there a low-risk subgroup consisting
of individuals with adducts below a certain value, nor does having an adduct value
in the upper few percent of the range of values signify an exceptionally high risk; the
risk is simply proportional to the level of the adduct. f course, it is quite possible
that within any set of individuals with the same adduct value there will be a subset
at higher or lower risk than the rest. If the 4-aminobiphenyl adduct values are
independent of these other risk determinants, then the analysis presented in this
paper will be insensitive to their effects and will not reveal their existence. It
remains for future cohort or nested case/control studies to deterrnine the nature of
other such risk factors. Absent other independent risk factors, the 4-arninobiphenyl
hemoglobin adduct appears to be as good a biomarker of effect as possible. Its
ability to predict outcome in the presence of other independent risk factors will
depend on the weight of those factors.

REFERENCES
De!Santo, P., Moneti, G., Salvadori, M., SaItutti, C., DelleRose, A., and Dolara, P., 1991, Levels of
the adducts of 4-aminobiphenyl to hemogIobin in control subjects and bladlJer carcinoma
patients, Cancer Letters 60:245.
Evans, DA.P., 1986, Acetylation, ;11: "Ethnic Differences in Reactions to Drugs and Xenobiotics,"
H.W. Goedde & D.P. Agarwal, eds., Alan R. Liss, lnc., New York.
lsocovich; J., Castelleto, R., Esteve, J., Munoz, N., Colanzi, R., Coronel, A., Demeasola, 1., Tassi, V.,
and Arslan, A., 1987, Tobacco smoking, occupational exposure, and bladder cancer in
Argentina, [nt. J. Callcer 40:734.
Ross, R.K., Paganini-HilI, A., and Henderson, B.E., 1988, Epidemiology of bladder cancer, in:
"Diagnosis and Management of Genitourinary Cancer," D. Skinner & G. Lieskovsky, eds., W.B.
Saunders.
Vineis, P., Esteve, J., Hartge, P., Hoover, R., Silverman, D.T., and Terracini, B., 1988, Effects of the
timing and type of tobacco in cigarette-induced bladder cancer, Callcer Res. 48:3849.

15
CURRENT RESEARCH ON HEMOGLOBIN ADDUCTS AND CANCER RISKS:
ANOVERVIEW

Margareta Tmqvist

Department ofRadiobiology, Stockholm University

S-106 91 Stockholm
Sweden

INTRODUefION

Most known chemical carcinogens are electrophilically reactive compounds (RX) or

are formed in vivo from non-reactive precursors (A) (Miller and Miller, 1966). The
electrophiles react with nucleophilic atoms (Y) of biomacromoleculcs giving rise to adducts
(RY):

A ~ RX +Y; RY + X- (1)
(ky )
The measurement of adducts to sufficiently stable macromolecules may be used for
identification of electrophiles in vivo and for dose monitoring as a basis for risk estimation.
For reasons to be discussed below, the Stockholm group has used hemoglobin (Rb) for in
vivo dose monitoring of chemical carcinogens in animals and humans.
In early efforts to characterize the determinants of mutagenic effectiveness of
alkylating agents the Swain-Scott linear free energy relationship was employed. These
studies showed for monofunctional alkylating agents a proportionality between mutation
frequency and degree of alkylation at the nucleophilicity of DNA oxygens (Turt6czky and
Ehrenberg, 1969; Osterman-Golkar et al., 1970). This suggested, as indicated by Ehrenberg
(this Workshop), that ifthe in vivo dose ofthe carcinogen could be measured, cancer risks
could be estimated by a relative potency method using as reference standard an agent with
well-known cancer risk per unit of in vivo dose. In the work of the Stockholm group y-
radiation has been used as a standard since it is the environment al factor for which the
relationship between risk and dose in humans is best known. This comparison of chemical
carcinogens and radiation presumes that a cell initiated by mutation has the same chance of
leading to a tumour irrespectively ofthe nature of the initiator.
The present paper will deal with methods for dose monitoring and the usefulness of
data for risk estimation. The background of models for risk estimation are discussed by
Ehrenberg (this Workshop).

Use o[ Biomarkers in Assessing Health and EnvironmentalImpacts o[ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 17
DOSE MONITORING

In the development of methods for dose monitoring certain reaction-kinetic

considerations are essential. If the rate constant ky is known for the formation of the adduct
RY in reaction (1), the dose D from an acute exposure is calculated from eqn. (2):

D=J....~ (2)
ky [Y]

The dose is then defined as the time-integral ofthe concentration ofRX (3):
D= J [RX] (t) dt (3)
t
Most known human exposures are however not acute but chronie or intermittent, leading to
the establishment of steady-state adduct levels, [RY]s.s. / [Y]. These are determined both by
the rate offormation and by the rate of disappearance ofthe adduct (k_):
[RY]s.s. = ~ (4)
[Y] k_
where a is the incremental adduct level per unit of time.
In the case of monitoring of dose by DNA adducts, k., which depends on variable
rates of repair and ceH division, is not weH defined. In order to use steady-state adduct levels
for calculation of dose a monitor with known k_ is preferred. For the time being this fact that
adducted protein is not subjected to repair is a main reason for maintaining Rb (or serum
albumin) as a dose monitor.
From (4) the dose in the period oftime considered is calculated as
D=E a (5)
t
(Granath et al., 1992). For Rb, k. = 2/ter, where t er is the life span ofthe erythrocytes (ca. 4
months in man, 40 days in mice).
Monitoring of dose by Rb adducts gives the dose in the blood (as would also be the
case with monitoring by DNA adducts in leukocytes). In order to allow for dose gradients in
the body, ratios between target dose and blood dose have to be determined by the
measurement ofthe DNA adduct levels in the respective tissues after acute exposures.
Since DNA is the target molecule for genotoxic action the demonstration of DNA
adducts is, however, an important demonstration of a genotoxic exposure, even if the data
cannot at present be used for a precise calculation of the dose.

METHODS FOR ADDUCT DETERMINATION

Protein Adducts

Proteins contain several nucleophilic groups such as nitrogens ofN-termini (valines in

Rb), lysine residues and the imidazole ring of histidines; sulfurs of cysteine and methionine
residues; and oxygens in the carboxyl groups of aspartate and glutamate residues and also in
the hydroxyl groups of serine and threonine. The procedure for the identification and
quantitative determination of substituents of these atoms depends on the nature of the
adduct and its position in the protein (Table 1). Protein adducts can, foHowing suitable

18
 Table 1. Methods for protein adduct determination.

-Total hydrolysis to alkylated (etc.) amino acids (cysteines, histidines)

-Mild hydrolysis to release adducts (bound as esters to carboxyl groups or as

sulfinamides to cysteines)

-Modified Edman degradation for splitting off of alkylated N-termini (valines in Rb)

-Immunological methods

derivatization, be identified and quantified by mass-spectrometrical methods. Various

procedures for mass-spectrometrical analysis are reviewed by Tannenbaum et al. (1992). To
a limited extent also immunological and fluorimetric methods have been applied for protein
adduct determination.
Adducts to residues in the interior ofthe peptide chain have been determined following
total hydrolysis by acid or enzymes. This procedure is time-consuming (Calleman et al.,
1978) and apt to artifact formation (Calleman et al., 1979). Furthermore, there may be a
contribution of alkylated amino acids in the interior of the chain, at least of low-molecular
weight adducts, through misincorporation of substituted amino acids, e.g. rom the food, in
the protein synthesis (Kautiainen et al., 1986).
Adducts to carboxyl groups may be released by mild hydrolysis (Hecht et al., 1992;
Taghizadeh and Skipper, 1992; and refs. in Table 2). A similar procedure will also release
adducts rom aromatic amines bound as sulfinamides to cysteine residues (Skipper and
Stillwel1, 1992; and refs. in Table 2).
For a specific c1eavage of adducts to the N-terminal valines in Rb a modified Edman
degradation method has been developed (Trnqvist et al., 1986a; Trnqvist, 1992). In this
procedure the isolated globin is derivatized by a fluorinated Edman reagent,
pentafluorophenyl isothiocyanate, and in difference rom the unsubstituted N-termini the
adducted valine is split off as a phenylthiohydantoin already in the neutral derivatization
medium, rom which it is then extracted. As exemplified in Table 2, this method has been
used for the determination of several low-molecular weight adducts rom simple epoxides
and other alkylating agents. Also a number of aldehydes has been determined after reduction
by NaBlf4 ofthe unstable Schiffbases or enarnines to secondary amines.
Immunological methods have been developed for fast routine analysis of adducts, for
instance for the determination of adducts from ethylene oxide (Wraith et al., 1988).
The N-alkyl Edman procedure for gas chromatopraphic/mass-spectrometric (GC/MS)
analysis of adducts to N-terminal valines has a detection level in the range of 1-10 pmol/g
globin for several adducts and this level may be lowered by enrichment before analysis
(Trnqvist, 1992). The method has a high reproducibility (Trnqvist et al., 1992a).
Determination of adduct-carrying interior amino acids are less sensitive by one to two orders
of magnitude (Farmer et al., 1986). In the determination of aromatic amines and lipophilic
adducts to carboxyl groups a high sensitivity in the finol/g range is attainable because of the
ease of isolation of the adducts (Tannenbaum et al., 1992). Independently of the detection
level of the method, monitoring of adduct levels due to specific exposures are limited if there
occur background levels ofthe same adducts.

19
 Table 2. Protein adducts determined in humans. (List ofreferences not exhaustive.)

Simple alkylating agents and metabolites

- CH3 Occupational: Methyl bromide

(methyl) Tobacco smoke: NNK, DMN,
methyl chloride?
Background: Endogenous S-adenosyl-
methionine Val2
Misincorporation3 ? Cys4, HisS

-CH2CH20H Occupational: Ethylene oxide, Hi s6-8 VaI8- 11

(2-hydroxyethyl) ethene Val l2 '
Tobacco smoke: Ethene Val13,14
Cytostatics: 1-(2-Chloroethyl)-
-l-nitrosoureas Val IS
Background: Endogenous ethene
(role ofintestinal flora, fat diet) Val16,17

-CH2~~CH3 Occupational: Propylene oxide His Val I8

Tobacco smoke: Propene Vai14,19
(2-hydroxypropyl) Background: Endogenous, probably
propene Va1 14,19

-CH2yHCH=CH2 Occupational: Butadiene Val20

OH
(2-hydroxy-3-butenyl)

-CH2-CH-C()IIs Occupational: Styrene Val 2I

6H
(-hydroxyphenethyl)

Occupational: Benzene SA, Cys22

y
-CH2 H-<;'-NH2 Occupational: Glycidamide, metabolite
OHO ofacrylamide (see below) Val23

-CH2CHO Occupational: Vinyl chloride (due to

(formylmethyl) high background level no
significant effect) His, Val24
Background: Urethane, glycolaldehyde, etc.?

20
Unsaturated compounds

-CH2-CH2-C=N Occupational: Acrylonitrile

(2-cyanoethyl) Tobacco smoke: Acrylonitrile

-CH2-CH2-C-NH2 Occupational: Acrylamide

(carbamoylethyl)

-CH2-CH2-g0- Background: Acrylamide? Cys25

(carboxyethyl)

Aldehydes (adducts determined after reduction to stable alk;ylamines)

= CH2R Background: Endogenous formalde- Val26- 27

Alkylidene hyde, acetaldehyde, glycolaldehyde,
and methylglyoxal,malonaldehyde,
-CH=CHR hexanal. Role offat diet and intestinal
Alkenyl flora in the endogenous production
of malonaldehyde
Aromatic amines

Sulfinamides Occupational: Aniline Cys28

Tobacco smoke: Aminobiphenyls,
toluidines, 2-ethylaniline,
2-naphthylamine Cys29
Background: Unknown sources and
possibly contribution from
environmental tobacco smoke Cys29
Nitrosamines

4-(3-pyridyl)- Tobacco smoke: NNK, NNN COO-30

-4-oxobutyl Background: Unknown and possibly
contribution from environmental
tobacco smoke COO-30
Polycyclic compounds

from PAH-diol epoxides Background: benzo(a)pyrene, chrysene

in diet? COO-31,32
from Trp-P-l Background: Diet, also tobacco smoke? ?33
from aflatoxin Background: Diet SA, Lys34
IIwasaki et al., 1989; 2Trnqvist et al., 1992b; 3Kautiainen et al., 1986; 4Bailey et al., 1981;
5Trnqvist et al., 1988~Calleman et al., 1978; 7van Sittert et al2 1985; 8Farmer et al., 1986;
9Wraith et al., 1988; 1vuuus et al., 1989; llTates et al., 1991; 1 Trnqvist et al., 1989a;
13Trnqvist et al., 1986b; 14Trnqvist, 1989; 15Bailey et al., 1991; 16Trnqvist et al., 1989b;
17Filser et al., 1992; 18Hgstedt et al., 1991; 19Trnqvist and Ehrenberg, 1990; 20S. Osterman-
Golkar et al., current work; 21Christakopoulos et al. 1992; 22Bechtold et al., 1992; 23Bergmark et
al., 1992; 24Svensson and Osterman-Golkar, 1987; 25Bailey et al., 1986; 26Kautiainen et al., 1989,
1991; 27Trngvist and Kautiainen, 1992; 28Lewalter and Korallns, 1985; 29Bryant et al., 1987,
1988, 1989; 3 Cannella et al., 1990; 31Weston et al., 1989; 32Day et al., 1990; 33Umemoto et al.,
1992; 34Gan et al., 1988;

21
DNAAdducts

DNA adducts in humans have been determined by immunochemical, postlabeling and

physico-chemical methods (reviewed by Schut and Shiverick, 1992). Immunochemical
methods are applied to the whole or digested DNA, the latter methods involve analysis of
nucleotides, nucleosides or bases, and in some cases, the adduct released from the DNA.
The sensitivity of these methods are 1.10-9 - 1.10-7 per nucleotide, corresponding 3 - 300
pmoVg DNA. Limitations of these methods are, in the case of immunochemical method the
cross-reactivity, in the case of labeling methods that identification after chromatographie
separation is obtained by retention times only.

ADDUCTS MEASURED IN HUMANS

Protein Adducts

Protein adducts so far studied in humans are summarized in Table 2 with reference to
the methods used. Most adducts from low-molecular weight alkylating agents and aldehydes
have been most reliably determined as valine adducts, the aldehydes following reduction of
the primary reaction products to stahle alkylamines. Besides contribution from occupational
exposures and tobacco smoke background levels have been encountered in most cases.
Particularly high background levels have been seen in the measurement of cysteine and
histidine adducts, possibly due to misincorporation of substituted amino acids in the protein
synthesis. This background is a contributing factor that renders monitoring of low doses by
adducts to histidines and cysteines less suitable. The sources of the background of adducts
to N-terminal valines have been studied in some cases and electrophilic reagents of
endogenous origin are indicated to playamajor role (Trnqvist and Kautiainen, 1992).
In the dose-monitoring of benzene, S-phenylcysteine in serum albumin (SA) has been
observed. This adduct is probably formed by aromatization of the product of reaction of
benzene oxide.
As for the aromatic amines the sulfinamides formed with cysteine residues are due to a
specific reaction different from the one leading to mutagenesis. In most of the studies of
aromatic amines low background levels appear, so far ofunknown origin.
Nitrosamines give rise to alkylating species with low selectivity, i.e. relatively high
reactivity towards oxygens (Segerbck, 1990). The tobacco-specific nitrosamines N'-
nitrosonornicotine (NNN) and 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone (NNK) give
rise to methyl and 4-(3-pyridyl)-4-oxobutyl-ester groups with carboxyls in protein. After
hydrolysis, the latter are isolated and determined with high sensitivity by GC/MS.
Certain adducts from PAR-diol epoxides have also been demonstrated to be present in
non-smokers by measurement of the tetrahydrotetrols formed in hydrolysis. A contribution
from dietary PAR is likely (Vaessen et al., 1988; Waldman et al., 1991).

DNAAdducts

Raised levels ofDNA adducts have been observed in a number of exposure situations
(for refs., see review by Schut and Shiverick, 1992). In e.g. coke oven, iron foundry and
aluminum!tIant workers raised PAR adduct levels in white blood cells have been observed
by both 3 P-postlabeling and immunochemical methods. PAR adducts have also been seen
e.g. following tobacco smoking and consumption of charcoal broiled beef. Aflatoxin-
contaminated food have been shown to give rise to significant increase of adduct levels.

22
Comparison of Protein and DNA Adduct Levels

For several compounds the rates of formation of Rb and DNA adducts have been
shown to be proportional over a wide range of doses adminstered (Segerbck, 1985). This
primary requisite for using Rb as a substitute monitor is thus fulfilled.
For a few adducts the steady-state levels have been determined in both Rb and DNA
(Table 3). This concems background level of and effeet of smoking on methyl and 4-(3-
pyridyl}-4-oxobutyl (adduet from NNN and NNK, cf above). In addition, a background
value for 7-(hydroxyethyl)guanine in lymphocyte DNA has appeared in an investigated small
group of smokers and nonsmokers; for this adduet the levels in Rb are weIl known.
A look at this comparison gives the surprising impression that, throughout, the DNA-
adduct levels are much higher than the corresponding Rb-adduet levels. It would carry too
far to discuss in detail the reaetivities ofthe electrophilic species involved (which may not be
the same in reactions with Rb and DNA, particularly in the case of methyl and 4-
aminobiphenyl, 4-ABP) and of the respective nucleophilic groups. Suffice it too say that in
the case of hydroxyethylation, the causative faetor has been shown with high probability to
be ethylene oxide from mainly endogenous ethene (Filser et al., 1992), for which the ratio of
the rates offormation ofthe measured DNA and Rb adduets is about 2:1 (Segerbck, 1990).
The DNNHb ratio of steady-state adduet levels found in humans is on the order 100: 1.
Since sources of background 7-(hydroxyethyl)guanine other than ethene/ethylene oxide are
not known (and provided artifaet formation - cf Tmqvist, 1990 - can be exc1uded) this and
the other tabulated differences between DNA and Rb adduet levels should be taken to
indicate the existence in lymphocytes and in other tissues of a velY long-lived DNA with
stable adducts (cf Randerath et al., 1989). Due to the potential usefulness ofthis DNA for
long-term dose monitoring useful for risk estimation, the c1arification of the kinetics of
formation and decay ofDNA adduets is an urgent issue (cf Ehrenberg, this Workshop).

RISK ESTIMATION, EXAMPLE: ETHENE

Ethene occurs in certain occupational environments in chemical industry. Besides that

it occurs as a general pollutant, being formed in incomplete combustion of organic matter. It
is also formed by microorganisms and plants (Sawada and Totsuka, 1986). In humans the
background dose of ethene is mainly due to endogenous produetion (Filser et al., 1992). The
risk estimation of ethene is concemed with its reactive metabolite, ethylene oxide
(Segerbck, 1983). Since it is wanted to express the risk in terms of exposure dose, D exp'
the following relationships have to be determined: :

Exposure fl Blood f2 Target

dose ---+ dose ---+ dose
Dexp 14>100d Dtarg

According to the rad-equivalence approach the risk is then calculated from eqn. (6):

Risk = ky . Q . Dtarg (6)

Dtarg = f} . f2 . D exp

where ky is the risk coefficient for radiogenie cancer and Q the quality factor for relative
genotoxic effectiveness ofthe genotoxic chemieal.

23
 Table 3. Hb- and DNA-adducts: background levels and effects of smoking.

Adduct Background Increment in

(pmoVg) smokers (pmoVg)

N-Methylvaline in Hb 1 300 40 (10 cig./day)

7-Methylguanine in DNA
(total WBC)2 ~ 1000 1000
~

(lymphocytes)2 4000 3000

(lung)3 3000 (smokers)

N-(2-hydroxyethyl)valine in Hb4 20 85 (10 cig./day)

7-(2-hydroxyethyl)gt.1anine in DNA
(lymphocytes)5 8500 (smokers + nonsmokers)

NNN,NNK
Carboxyl ester in Hb6 0.03 0.05 (22 cig./day)
DNA addu~ hydrolysable
(lung) 0.9 10

4-Aminobiphenyl
Hb-Cys adduct8 0.2 0.7
DNAadduct9 300 (smokers + nonsmokers)

ITrnqvist et al,. 1992b; 2Mustonen and Hernminki, 1992; 3Shields et 31, 1990;
4Trnqvist and Ehrenberg, 1990; 5pst et al., 1989; 6Carrnella et al., 1990;
7Poiles et 31., 1991; 8Bryant et al., 1987; 9Wilson et al., 1989;

In the exposures so far studied (urban air, chemical industry, fruit-store workers) it has
turned out to be very difficult to obtain relevant long-term measures of exposure that could
be used for calculation of annual exposure dose etc. Mean values obtained are uncertain by
at least a factor oftwo. For these reasons the determination off} in (6) above has to a large
extent been based on the ethene uptake by cigarette smokers. This choice leaves, however,
the possibility open that smokers, due to enzyme induction, might metabolize ethene more
effectively than non-smokers.
For details ofthe determination ofthe parameters in equation 6, see Trnqvist (1989)
and Trnqvist and Osterman-Golkar (1991). The following values have been adopted for
ethene:
It may be estimated that a smoker inhales 250 Jlg ethene per cigarette smoked.
fl = 5.10-9 (mM h)(ppb ht 1
With dose defined by equation (3) the blood dose (as also the target dose) is expressed
in millimolar-hours (mMh) and the exposure dose in ppb-hours (ppbh)
f2 ~ 1 (Segerbck, 1985).
This means that the dose of ethylene oxide, according to animal experiments, is evenly
distributed within the body.

24
 For the radiation-dose equivalent of the ethylene oxide dose, a mean value rom
measurements in various biological systems,
Q = 80 rad-equ. (mM . h)-I,
is adopted (Ehrenberg et al., 1983; Kolman et al., 1988).
For cancer mortality we have applied a risk coefficient
k.y = 2 . 10-4 rad- l or rad-equ- l .
For cancer morbidity the risk would be approximately two times larger. These values allow
for the risks being ca. 5 times lower at low dose rates compared to the value, k.y ,.. 10 . 10-4
rad-I, valid at high dose and high dose rates (NRC, 1990). In Table 4 are given the steady-
state Rb-adduct levels and the associated risks of cancer death rom exposure to ethene in
different environments, including endogenous production.

Table 4. Steady-state levels of adducts tn N-terminal valines in Rb and estimated

cancer risks rom situations with ethene or ethylene oxide exposure. (Source:
Ehrenberg and Trnqvist, 1992).

Steady-state Rad-equiv. Associated lifetime risk

Exposure adduct level annual of cancer death rom one
(pmoVg) dose year's exposure, x 105

Ethylene oxide, 1 ppm

40 hlweek 2400 22 400
Ethene, 1 ppm, 40 hlweek 120 1 20
Ethene rom smoking
10 cig/day 85 0.8 16
Background 20 (8-25) 0.18 3.6

Comparison with Other Risk Estimates

The risk estimates in Table 4 are higher by ab out one order of magnitude than the ones
obtained by applying the unit risk factor for ethylene oxide of U.S. EPA (1990); cf
Trnqvist and Ehrenberg (1992). This discrepancy is partly due to the unit risk factor being
based on the dose per day whereas the lifetime dose seems to be more appropriate for an
irreversible effect such as cancer initiation. This leads to an underestimate by a factor of 35
(70 years in humans compared to 2 years for laboratory animals). This factor is partly
counteracted by EPA's calculation of dose per m2 body surface area, which renders man ca.
6 and ca. 14 times more sensitive than the rat and the mouse, respectively.

RISK IDENTIFICATION

It appears rom Table 2 that background levels of several adducts have been
encountered in human blood sampies. Since practically all electrophlles reactive towards
proteins are also able to react with DNA the observation of background adducts may be
taken as an indication of exposure to cancer initiators (Ehrenberg and Osterman-Golkar,
1980).
In the case of background hydroxyethyl adducts to N-terminal valine in Rb a level of
20 pmoVg Rb has been assumed to reflect a true level, artefacts of various kinds being
excluded (Kautiainen et al., 1986; Trnqvist, 1990). The level is compatible with the rate of

25
endogenous ethene production being a major source (Filser et a1., 1992). This production was
shown in animal experiments to be associated with intestinal bacteria and dietary factors
(Tornqvist et a1., 1989b).
The rad-equivalent annual dose of background ethene/ethylene oxide (Table 4) is
comparable to that of background radiation, 0.1 rad/year. The lifetime dose from this factor
0.18 rad-equ./year x 70 years = 12.6 rad-equ., would correspond to a risk of 250 10_5, i.e. ca.
1% of the total cancer mortality risk. The same value is obtained by applying the multiplicative
model (cf. Ehrenberg, this Workshop) with the risk coefficient 0.1% per rad or per rad-equ.
(Ehrenberg et a1., 1992).

USEFULNESS OF ADDUcr MONITORING

For the ultimate goal of cancer prevention, measurement of Rb adducts is applicable

in different ways.
Since there is a quantitative relationship between exposure dose, adduct level, target
dose and cancer risk, adduct determination becomes a relevant end point in epidemiological
investigations, directly responding to recent exposures.
Because of the proportionality between exposure dose (i.e. time-weighted average
exposure concentration) and adduct level, and since adduct levels can be measured much more
exactiy than exposure concentrations, the measurement of adduct levels is useful for
surveillance of work environments with exposure to mutagens/cancer initiators. This could be
accomplished by using a few ml blood, drawn at routine medical examinations, for adduct
determination. Adduct levels measured, may be used for an estimation of the magnitude of
cancer risks from specific compounds.
For related reasons, the measurement of adducts from mixed exposures may be used
to identify etiological factors and to estimate their contributions to cancer risk.
The identification of adducts in knowingly unexposed individuals allows the detection
of possible initiators. Quantification of these adduct levels and risk estimation of the causative
chemical species may thus be used to identify etiological factors in the background cancer
incidence, which still is the largest part of the total incidence.
Being measurements of dose at the individualleveI, adduct levels reflect the net effect
of bioactivation of precarcinogens and detoxification of reactive metabolie intermediates.
Adduct measurement may thus be used to identify individuals that are sensitive due to high
bioactivation rate or deficient detoxification, properties that may be heritable or acquired. (For
the identification of individuals with raised sensitivity because of defective DNA repair or
ability to cope with initiated celIs, complementary biochemicalor molecular-biological tests may
be carried out.)
In animal tests the measurement of adducts is a useful tool to cIarify metabolie
pathways leading to reactive intermediates, of value in extrapolation to humans. Adduct
measurement permits further, due to its high statistical power, the discIosure of false negatives
and also false positives (cf Ehrenberg, 1984) in animal tests and in vitro genotoxicity tests.
The demonstration of raised DNA adduct levels in various exposure situations shows
their potential usefulness in the same respects. As indicated above, DNA adducts may reflect
exposure during longer time, then the Rb adducts. For application to risk estimation, the
kinetics of the disappearance of DNA adducts due to repair and cell division has to be cIarified.
For studies of mixtures and "unknowns" the increased application of mass-spectrometric
techniques for identification-quantification, as is now done with protein adducts, is a wanted
development (cf. Fedtke and Swenberg, 1991).

ACKNOWLEDGEMENT

The present review is main1y based on studies supported financially by the Swedish
Environmental Protection Agency, the Shell Internationale Research Maatschappij B. V. and
U.S. Department ofEnergy (Grant No. DE-FG02-89ER60784).

26
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exposed fruit store workers, Scand. J. Work Environ. Health 15:436.
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30
USE OF BIOMARKERS IN QUANTITATIVE RISK ASSESSMENT

Lorenz Rhomberg 1

Office of Health and Environmental Assessment

D.S. Environmental Protection Agency
RD-689
401 M Street, SW
Washington, DC 20460

INTRODUCTION

This paper is about the use of biological markers in the quantitative analysis of the health
risks that may follow from exposure to environmental contaminants. That is, it emphasizes the
application of data on biomarkers to address questions that arise in the context of quantitatively
relating exposures to their toxic effects. To provide the context for these comments, abrief
review of the general framework of risk assessment is in order.

FRAMEWORK FOR RISK ASSESSMENT

Most risk assessment activity in the U.S. Federal government is structured around a
framework proposed in a seminal publication of the American National Academy of Sciences
(NAS, 1983). The U.S. Environmental Protection Agency (EPA) has implemented this structure
in its guidelines for conducting risk assessment (EPA, 1987). First of all, the NAS called on
practitioners to distinguish risk assessment-the objective characterization and evaluation of
knowledge about an agent's ability to cause toxie effects-from risk management-the process of
deciding what can and should be done to mitigate or control the risks that may be engendered.
Within the rea1m of risk assessment, the NAS identified four components. The first, hazard
identification, asks whether the agent has the ability to cause toxic effects of concern under same
circumstances of exposure, and if so, identifies the nature of those effects. That is, hazard
identification is the qualitative component of risk assessment, establishing the existence, but not
the degree, of adverse consequences from exposure to the agent. The second component, dose-
response assessment, addresses the quantitative issues, aiming at establishing the probability or

1 The views expressed in this document are those cf thc author and da not necessarily reneet the views and policies
of the U.S. Environmental Protection Agency. Mention of trade narnes, commercial products, or organizations does not imply
endorsement by the V.S. Govemment.

Use 0/ Biomarkers in Assessing Healrh anti Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Travis, Pl"num Press, New York, 1993 31
degree of response that may be expected from various levels of exposure. The third component,
exposure assessment, is the process of characterizing the intensity, frequency, and duration of
actual human exposures (or exposures to other targets of interest), as weH as projecting such
information for exposures that might occur under alternative courses of regulatory action. The
final component, ri"sk characterization, brings together the previous three elements; the quantitative
information on human exposure levels is combined with the results of dose-response analysis to
characterize the risks of adverse health effects that might be associated with those exposures,
providing a picture of the impact of the agent on the population of concern. The risk
characterization should include a discussion of the assumptions and uncertainties involved in
making such a projection, including an evaluation of the effect of adopting reasonable alternative
methods of analysis.
This structure provides a framework within which the toxicologic, epidemiologie, and
exposure data at hand can be brought to bear on the question of interest: the likelihood that
exposures to the agent as they exist (or as they might exist under specified courses of action) result
in harm to the health of people and the environment. The inference of the needed results
encounters many difficulties. Owing to limited data and incomplete scientific understanding, the
projection from the circumstances studied to those of interest engenders a great deal of uncertainty.
Biomarkers can provide helpful tools in each of the stages of risk assessment. Although a
comprehensive examination of these uses is beyond the scope of the present paper, it may be weIl
briefly to review the principle applications.

APPLICATION OF BIOMARKERS TO HAZARD IDENTIFICATION

In the qualitative phase of risk assessment-hazard identification-biomarkers may be of use

in several ways:

(a) establishing the existence, nature, or degree 0/ toxic injury engendered by an agent. Such
use is possible both in toxicological experiments and in epidemiological studies, where it may
be advantageous to be able to distinguish individuals with and without adverse respons-
es--even cryptic or early-stage responses-in an inexpensive, nondestructive, or noninvasive
way. An example is the use of serum glutamate-oxaloacetate transaminase (SGOT) as an
indicator of liver injury;

(b) providing insight into the mechanism oftoxic action. Biomarkers may aid in establishing that
a compound is capable of certain biological activities of concern or that its effects show
similarities to better known toxicants. For example, the production of DNA adducts raises
concern for a genotoxic mechanism of carcinogenesis (a concern that may be modulated by
the type of adduct produced). The ability of the so-called coplanar polychlorinated biphenyls
(PCBs) to bind to the Ah receptor, responsible for dioxin's toxic effects, raises concern about
the potential for similar effects from those PCBs;

(c) establishing (or refuting) the commonality 0/ toxicological processes in test animals and
humans (or among dose levels or exposure regimes). The process of inferring potential
hazard to the general human population from animal studies or from occupational
epidemiological studies can be aided by demonstrating parallel biomarker patterns, indicating
that similar underlying processes are operative in both cases. Conversely, the lack of
concordant biomarker patterns can sometimes constitute evidence that animal responses have
questionable relevance to humans. For example, the lack of induction of peroxisome
proliferation in human liver ceHs by agents that have this action in rodents raises questions

32
 about whether certain chemicals, which seem to have their hepatocarcinogenesis mediated by
this process, constitute a human cancer hazard.

In brief, in the hazard assessment phase of risk assessment, biomarkers can serve to help identify
toxic reactions and to modulate the supposition that humans in the general population may be at
some risk.

APPLICATION OF BIOMARKERS TO EXPOSURE ASSESSMENT

Perhaps the most extensive use of biomarkers has been in the arena of exposure assessment.
For this reason, the applications are more familiar, and I will treat them only briefly. Again, the
potential applications are several, including:

(a) establishing the fact of exposure. Monitoring, and especially modeling, infer the fact of
exposure by estimating environmental levels in the immediate vicinity of the presumably
exposed individuais, but biomarkers (such as the presence of a compound or its unique
metabolites in expired air, urine, or blood) can unequivocally demonstrate that those
individuais have suffered some uptake (the source of which, however, can still be
questioned);

(b) reducing exposure misclassijication in epidemiological studies. Exposure measures in

epidemiological studies are often very indirect and uncertain surrogates, such as job
classification or self-reported exposure. Placement of individuals in inappropriate exposure
categories can significantly reduce the power of a study to detect an adverse effect.
Biomarkers may provide a more dependable and direct exposure measure (at least of recent
exposure), and can serve as a check on self-reported behavior (e.g., by checking for cotinine,
a metabolite of nicotine, in the urine of self-reported non-smokers). The D.S. EPA (1992a)
used cotinine levels to estimate the relative levels of exposure to environmental tobacco
smoke among nonsmoking spouses of smokers and nonsmokers, which allowed the observed
differential in cancer risk among these groups to be related to exposure;

(c) population screening. This application is especially useful for long-lived compounds where
the concern is for the accumulation of a significant body burden. Biomarkers may provide
an efficient means for screening large numbers of the general population to identify highly
exposed individuals for treatment or intervention. A good marker for such a purpose should
be quick to determine, inexpensive, minimally invasive or disturbing to the subject, and
robust to use by a variety of practitioners (perhaps with little training) under conditions found
in the field. A familiar example is the determination of blood lead levels as an indicator of
high body burdens in children;

(d) reconstructive exposure assessment. For compounds with relatively simple pharmacokinetics
and temporal exposure patterns, it may be possible to reconstruct estimates of the
environmental levels that an individual was probably exposed to in order to result in an
observed body burden. Such an exercise can be useful in its own right, as a means to obtain
quantitative measures of exposure in an actual population, or it can help in validating models
that project such exposure levels from characteristics of the source of environmental
contamination. Since data are taken after the fact, reconstructive methods are useful for
assessing unexpected episodes of exposure, such as accidental releases. They may also be
useful in determining typical background levels of exposure in the general population. For
example, in a document on exposure to dioxins the D.S. EPA (1992b) has used such methods
to check the consistency of estimates of uptake by the general population with observations
of typical levels in body fat.

33
 (e) os an inregrative meosure 0/ exposure. Exposure assessment typically focuses on the analysis
of sources of environmental contamination by following the agent's distribution through the
environment. Such analyses tend to separate sources and routes of uptake, dealing only
awkwardly with exposure that varies temporally and spatially. In contrast, biomarkers focus
on the body burden-tbat is, on the integrated result of an individual's total exposure, being
the product of a variety of contamination sources, routes of uptake, and the varying patterns
in space and time-leading to a measure of the overall impact of the complex patterns on
individual people according to their various perSonal histories, habits, and behaviors.

In sum, biomarkers can be used in exposure assessment as both qualitative and quantitative
surrogates for exposures to environmental contaminants as they are actua1ly experienced by the
subject population.

APPLICATION OF BIOMARKERS TO DOSE-RFSPONSE ASSESSMENT

Tbis is the main subject of the present paper. Dose-response assessment concerns itself with
the qUll1Uitative aspects of dosimetry and toxic effects. Since these take place within the body
(where they are hard to observe and measure, especially if this is to be done without invasion or
destruction of the subject organisms) methods that are based on quantitative measures in biological
media (which biomarkers are, by definition) are particularly appropriate and indeed necessary.
Tbe balance of this paper will focus on how such indirect measures of the processes inside the
body can be used quantitatively in the projection of observed magnitudes of risk following
measured doses in animals (or in occupationally exposed humans) to expected magnitudes of risk
in the general human population (or in other populations of interest).
In particular, the dose-response assessment typically entails a number of extrapolations:

(1) from the high dose levels of toxicological experiments to the much lower exposure levels to
which people may be exposed in the environment;

(2) from genetically homogeneous, especially bred experimental animals maintained in controlled
environments to genetically diverse humans living under a variety of circumstances in the real
world;

(3) from controlled regimens of exposure to varying, intermittent, or occasional exposure; and

(4) from experimental routes of exposure to those occurring in the population of interest.

Biomarkers can aid in all of these extrapolations by providing quantitative insight into the
operation of underlying causative processes. Before examining such applications in detail, it is
well to address some general properties of biomarkers as they are used in quantitative analysis.

QUANTITATIVE BIOMARKERS: SOME GENERAL CONSIDERATIONS

Tbe issues surrounding the quantitative use of biomarkers are best understood by bearing in
mind that biomarkers are surrogate or proxy measures. That is, we seek information about
something that is difficult to measure (because the object may be inaccessible, or the measurement
may be technically difficult, unacceptably disruptive of the system, or unduly expensive) by
measuring something else that is cheaper, easier, more accessible, or more readily quantified.
What distinguishes biomarkers from other surrogate measures is that they constitute measurements
in biological media, taken with the aim of providing insight into the internal biological operations

34
of the system or into that system's interaction with the external environment. Clearly, the validity
of the information provided by such surrogate measures depends on the correlation of the unknown
"target" value and the measured "marker" value.
The considerations for using a biomarker in quantitative risk assessment, then, are similar to
those one would examine for any surrogate measure. One should examine the specijicity of the
marker-the degree to which the appearance of the marker reflects only the thing of interest, and
not other, extraneous or irrelevant factors. For example, if a compound causes DNA adducts of
a common type for which there are many possible sources, the usefulness of such adducts as a
marker of exposure to that particular compound may be compromised. One should also examine
the sensitivity of the marker-the degree to which changes in the thing of interest are reliably
reflected as changes in the marker. For example, blood levels of a compound with a short
biological half-lite may adequately reflect recent exposure, but detection may be insufficiently
sensitive to reflect an episodic exposure distant in time.
For quantitative applications, a third property is important to examine: the quantitative
correspondence of the marker and the endpoint of interest-the degree to which quantitative
variation in the one mirrors variation in the other. The correspondence need not be linear (Le.,
the marker maintaining direct proportionality to the object), but any non-proportionality should
be characterized sufficiently so that the implications of variation in the marker can be understood.

NATIONAL ACADEMY OF SCIENCES CLASSIFICATION

The American National Academy of Sciences (NAS, 1989) has developed a classification of
biomarkers recognizing three categories:

(a) a biomarker 0/ exposure is "an exogenous substance or its metabolite or the product of an
interaction between a xenobiotic agent and some target molecule or cell that is measured in
a compartment within an organism; "

(b) a biomarker 0/ effect is "a measurable biochemica1, physiological, or other alteration within
an organism that, depending on magnitude, can be recognized as an established or potential
hea1th impairment or disease; "

(c) a biomarker 0/ susceptibility is "an indicator of an inherent or acquired limitation of an

organism's ability to respond to the challenge of exposure to a specific xenobiotic substance. "

The first two categories are defined in terms of the types of measurements used as markers, but
the titles of the categories make clear that this is intended to be a functional classification, Le.,
one in which the categories correspond 10 the uses to which the marker is to be put. The U.S.
EPA has followed the NAS taxonomy in designing its biomarker research program. The Agency's
Office of Research and Development has laid out a research strategy (EPA, 1991a) in which
different laboratories have lead responsibility for development of the different biornarker
categories.
The ambiguity between defining categories in terms of what is measured versus why it is
measured can lead to some confusion when the taxonomy is applied to actual cases. This is
perhaps better illustrated by consideration of Figure 1, which is taken from the EPA research
strategy document. The diagram (a variant of which appears in most discussions of biomarkers
and risk assessment) depicts the "cascade of events" between exposure to a toxicant and the
consequent appearance of disease. The categories of biomarkers as defined by the NAS report are
shown as impinging on the cascade according to where they are measured. However, the
additional considerations relating to the practica1 application of these measurements has led the

35
diagram drawers to represent the exposure- and effect-biomarker categories as blended into a
continuum.
As an exarnple of the ambiguities encountered, consider a hypothetical measurement of
protein adducts in the target organ of toxicity following exposure to a toxicant. This is clearly a
biomarker of exposure under the NAS definitions. Under the apparent intentions of those
definitions, and under the "cascade of events" interpretation of Figure I, this measure is a

I Susceptlblllty
I
Exposure

I
~ Absorbed
Dose

I
f-~
Dellvered
or
Target Dose

I
Blologlcal
Effect

I
f-t-..
Health
Effect

I
Exposure Effect

Blomarkers
I 1
Figure 1. The "cascade of events" between exposure to a toxicant and the subsequent appearance of disease, as
depicted in EPA (1991a).

surrogate for "target dose," that is, one is interested in the toxieologieal consequences of the
measured entity. However, the same data could also be used as a marker of the amount of sub-
stance inhaled by the individual whose adducts are measured (in the manner of reconstructive
exposure assessment, discussed above). That is, instead of inference leading from the marker to
its consequences (moving to the right on the diagrarn) one is pursuing inference from the marker
to its antecedents (moving to the left). The use of bio markers in exposure assessment, as outlined
above, is primarily of the latter type, while applications of the former type are usually thought of
under the rubric of dosimetry.
A similar ambiguity arises in the case of biomarkers of effect. The NAS definition implies
use of the marker to predict subsequent health consequences (e.g., enzyme-altered foci preceding
tumors, receptor binding preceding the induction of effect), but most of the examples usually
adduced are cases where the marker is used as a means of diagnosing the existence of cryptic toxie
effects that have already occurred (e.g., forced expiratory volume as an index of pulmonary
toxicity, serum GOT as an index of liver injury).

BIOMARKER "ICONOGRAPHY"

My aim in raising these issues is not to criticize the NAS definitions or the "cascade"
diagram. Rather, it is to point out that these aids to structuring discussion have limitations that,
if not recognized, can inhibit exploration of other, equally important aspects of the problem.
When a diagram becomes as ubiquitous as has the structure depieted in Figure 1, there is a danger

36
that it will begin to channel our thinking. A diagram is agraphie analogy to the subject being
discussed, and, like any analogy, it is useful only insofar as the analogy holds. When extraneous
features of the diagram color thinking about the biological reality, one has moved past analogy to
iconography. It may be worthwhile, then, to undertake abrief examination of Figure I to spot
potentially misleading interpretations that could be made.
The purpose of the cascade diagram is to point out that the grossly observed connection of
exposure to effect is composed of achain of causa! relationships at the underlying mechanistic
level. Biomarkers, as indicators of the difficult-to-observe events that are transpiring, can help
to identify differences in these processes among species, dose levels, or exposure regimes that
would otherwise be difficult to characterize and account for in the dose-response assessment
extrapolations outlined earlier. Although at the overt level the proportionality between exposure
and response may not be maintained, the hope is that at a deeper, mechanistic level, phenomena
that are "elose" (in the sense of being removed by few intervening steps from one another) may
maintain such proportionality. For instance, cytotoxicity in the liver may more easily and reliably
be related to a toxicant's concentration in liver cens than to the concentration of the agent in
ingested water.
The series of linked boxes in Figure 1 represents the chain of causa! relationships. Among
the potential pitfalls in interpreting such a diagram are what could be called the faulty suppositions
of sequence, series, and proportionality. I shall discuss each in turn:

(a) sequence - the phenomena to the left in Figure I c1early beg in earlier than those on the right,
in the order of boxes in the diagram. But, especially in a situation of chronic exposure and
chronic toxie response, the entire suite of phenomena are, in the end, happening simulta-
neously. The diagram does not represent a sequence of events but rather a progression down
achain of cause and effect, with each element representing an ongoing process that bears on
those "downstream" and is influenced by those "upstream." The entire process can have
temporal variation, with the effects of changes at one level "sweeping down" the chain of
cause and effect (e.g., a momentary peak in the concentration of a toxicant in inhaled air will
lead to a peak in target tissue concentration, and perhaps a peak in cytotoxicity). There is
a tendency to simplify this process by thinking of the steps as happening one after the other,
with the output of each completed step serving as the input to the next. This can lead one
to ignore important temporal information-the moment-by-moment connections of the
elements in the causa! chain. Since many biomarkers are "integrative" measures, they tend
to encourage this squeezing-out of the time component. For example, protein adducts as a
surrogate measure of the presence of reactive compound at a partieular anatomie site will
characterize the average concentration of the agent over time, but not its temporal fluctuation,
whieh may be the key to interpreting the toxie effects engendered.

(b) series - In Figure 1 the elements are shown as comprising a single chain. In fact, the causal
patterns can be more complex, involving feedback loops (e.g., toxic reaction at the site of
uptake affecting absorption, substances that induce their own metabolism), multiple influences
impinging on a process (e.g., co-exposure to two agents inhibiting one another's metabolism),
effects on multiple endpoints (e.g., a carcinogen with a genotoxie metabolite and another
metabolite acting as a promoter), and so on. If diagrams such as Figure 1 are interpreted as
"influence diagrams" rather than flow diagrams (as I think they should be-that is, they
diagram the cause-and-effect relations among the entities rather than transfers), then it is elear
that a single series of boxes in tandem cannot represent all the important factors that impinge
on the process.

(c) proponionality - The goal of an analysis such as shown in Figure 1 is to examine dose-
response patterns in terms of "internal" phenomena that are mechanistically elosely connect-
ed, in the hope that extraneous sources of nonproportionality (appearlng elsewhere in the

37
 chain and tending to obscure the relationship of exposure to disease) will be eliminated.
Thus, there is a temptation to suppose that phenomena in adjacent boxes in the diagram are
proportional to one another-by getting "close" to a box (Le., by measuring an adjacent box)
one is necessarily approaching a simple direct relationship. Clearly, if each box were always
truly proportional to its adjacent ones, then all boxes would be proportional to one another,
and measuring any one would give complete information about the magnitude of all, from
exposure to disease. The point is that a good deal of nonproportionality can be squeezed into
the relationship between adjacent boxes, and the simple representation by an arrow should
not be taken to imply a necessarily simple relation between their behaviors. The proposition
that a tight causal link between particular phenomena leads to proportionality of their
magnitudes is an assertion that needs to be defended case-by-case. (As noted, the goal of the
analysis is to discovers such simple relationships as an aid to understanding the complex
whole.) A diagram such as Figure 1, however, is a means for framing such an assertion of
proportionality, and does not constitute evidence that the assertion is true.

Again, the points discussed above are not faults of the cascade diagram; rather, they constitute
potential misinterpretations of the meaning and intent of such diagrams. In the context of the
present discussion, the importance of recognizing them is that imprecise understanding of the
diagrammed relationships constitutes imprecise understanding of the impact and meaning of
biomarkers as they might be used in dissecting the overall dose-response relationship.

AN ALTERNATIVE CLASSIFICATION BASED ON CAUSAL CONNECTION

If we redraw the cascade diagram as showing a more complete pattern of relationships by

which the various factors influence the values of others, the simple, single chain of boxes in
Figure 1 becomes a complex nexus of cause-and-effect relationships as shown in Figure 2. The
specific identity of the various boxes is unimportant in this hypothetical example. Figure 2 is
intended to recognize explicitly that the values of elements in this nexus can be the product of
several influences, that elements can in turn influence several others, and that, as a result, the
cascade of causality proceeds not along a single, simple chain but (at least potentially) in feedback
loops, parallel tracks, and anastomosing networks.

Figure 2. Dose-response as a complex nexus of cause-and-effect relationships.

38
 It is fair to ask at this point what is to be gained by wallowing in all this complexity. After
all , the point of diagrams such as Figure I is to simplify a problem until it is tractable. The
answer , I think, is to remember the nature of biomarkers as surrogate measures. We measure
accessible elements in the complex nexus of Figure 2 in order to make inferences about less
accessible elements, and we do so in order to illuminate cause-and-effect relationships at a more
fundamental biological level. The value of biomarkers in such analysis, therefore, depends on
understanding the causallinks between the measured "marker" element and the "target" element
of interest. The impact of the marker measurement on the risk analysis depends on the
understanding of the target element's interaction with others in the production of the end effects
of concern. In practice, we may know very little about the network of biological influences and
consequences that underlies our dose-response observations, and drawing out a sufficiently full
diagram of cause-and-effect may be problematic. But it is important to layout what is known to
be important, to make explicit our assumptions about the connection of elements that are more
poorly understood, and to identify missing pieces of information of potentially major consequence.
Actually, despite the complexity evinced by Figure 2, there are only three possible funda-
mental types of relation between a biomarker and the element for which it is intended as a
surrogate. These three types could serve as an alternative classification of biomarkers-a
classification based not on the type of measurement being made nor on use in the risk assessment
process, but on the pathway of inference between marker and target, depending on the direction
of cause-and-effect. The three types are diagrammed in Figure 3.
In the first type (Figure 3a), the biomarker is a downstream biomarker-that is, one that is
downstream in the flow of cause and effect from the target of inference. In other words, one is
inferring astate or value of interest by measuring one of its consequences. It may be possible to
recognize and delimit a number of intermediate steps in the link between target and marker, but
the fundamental direction of causality from target to marker (and of inference from marker to
target) is unchanged. Figure 3a also recognizes that other (perhaps unrecognized) influences on
the value of the marker may exist. Thus, in using the marker, one must convince oneself that the
inference about the target is sufficiently robust that these extraneous factors can be overlooked.
Reconstructive exposure assessment, as described earlier, is an example of the application of
a downstream biomarker as envisaged here. One is using observations of current body burden (or
of adduct levels or a similar measure)-a consequence of previous exposure--to make inferences
about the magnitude of that exposure. One must be concerned about the possible impact of other
influences on the marker, e.g., background exposure, endogenous production of the compound,
the influence of pattern of exposure and duration since exposure on the current burden,
interindividual variation in uptake and clearance rates, and so on. Diagnostic biomarkers that
revea1 the existence of cryptic toxicity or disease are also examples of downstream markers. One
is inferring the presence of disease from its consequences (e.g., liver toxicity from elevated
SGOT). As a third example, measurement of amounts of a compound's end metabolite may be
a downstream marker of the rate of formation of a tran si tory reactive form that is present only
momentarily during the biotransformation process.
The second type of biomarker under this scheme, an upstream biomarker, is illustrated in
Figure 3b. Here, the marker is upstream from the target; one is inferring the state or value of
interest by measuring one of its causes. As before, several steps may intervene, and the possible
existence of other causes impinging on the target must be considered. One is using upstream
biomarkers when one measures adduct levels as a cause of subsequent mutation or cytotoxicity,
when one measures enzyme-altered foci in rat livers as an indicator of risk of subsequent tumor
formation, and when one empirically measures a patient's amount of body fat to predict a drug's
volume of distribution.
The third and final type of bio marker results from effects with a common cause and is
illustrated in Figure 3c. Both the marker element and the target element are consequences of some
common third element (although, as always, other influences can impinge), and the path of
inference goes first upstream in the flow of cause-and-effect, from the marker to the common

39
 A. "Downstream"

-+-~
B. "Upstream"

C. "Effects with Common Cause"

Figure 3. Types of biomarkers, classified by the pathway of inference from the marker to the objective.

40
element, and then downstream to the target. The correlation between marker and target derives
from their being influenced in common by the shared causa! factor, and interpretation of the
inference from one to the other must be tempered by recognition of the possible predominance of
influences unique to each branch of the forking pathway. This third type of biomarker is
important to recognize for two reasons. First, it is quite a common pattern (albeit one that is not
always recognized) among biomarkers in current use, and second, its existence is not obvious (or
easily accommodated) by the usual cascade diagram as in Figure 1.
The most clear cut example of this type of biornarker is the use of hemoglobin adducts to
infer a DNA-reactive dose of an agent. The adducts and the DNA reaction both depend on the
presence in the body of the reactive compound (which may have to be metabolica1ly activated to
show such reactivity), and so the readily measured amount of reaction with hemoglobin can serve
as a surrogate for the less observable amount available to react with DNA. Nonetheless, the
reactions are taking place in different anatomica1 locations and with different constituents of
different ceHs; one must assurne (or experimenta1ly establish) that the common dependence on the
overall body's burden of reactive compound is such that the marker and the target variable
maintain proportionality.
In general, when adducts are used as dosimeters, they constitute biomarkers of this third type.
That is, unless the adducts themselves are causatively involved in to toxic processes (as they may
be, for instance, in some genotoxic compounds binding to DNA), their use is to act as a surrogate
for the amount of reactive material available for some other interaction, based on the common
dependence of adduction rate and the rate of this other toxicological process on the presence in
the body of reactive agent. This is true even if adducts are measured in the target tissues. Of
course, the shorter the branch in the network of influences leading to the marker, the eloser
markers of the third type become to being markers of the first or second types.

VALIDATION AND USE IN EXTRAPOLATION

The purpose of the alternative classification of biomarkers developed above is to emphasize

the nature of biomarkers as surrogate or proxy measures; it is therefore important to establish the
quantitative connection between the measured marker variable and the unmeasured but
toxicologically interesting target quantity. Clearly, the best way to establish this linkage is to
develop a thorough mechanistic understanding of the underlying biologica1 processes, but in most
cases an empirica1 demonstration of correlation may suffice. Even if few data can be brought to
bear on the subject, reasonable inferences can often be drawn based on general principles and
background knowledge. In any case, it is important explicitly to articulate the basis for believing
the marker to be correlated with the target quantity so that its grounding in assumption and fact
can be clearly understood. This ineludes consideration of the possible role of factors and
circumstances that would tend to disrupt the correlation of marker and target.
The importance of explicitly examining the rationale for the surrogate nature of a biomarker
is underscored during the use of that biornarker in extrapolation. After all, the nature of
extrapolation is to make inferences beyond the range of observed data, based on the presumption
that interrelations among variables observed in the data continue to apply. The user of a
biomarker in extrapolation must ask whether the relationship of marker to target quantity may
reasonably be supposed to apply through the whole range of situations of concern.
In extrapolating dose-response relationships to low doses, it may be possible to establish a
linkage between the toxic effect of concern (say, tumor incidence) and a biomarker (say, DNA-
adducts). If the relationship established between these can be established at high doses can
plausibly be expected to apply at lower doses, and if the biomarker can be observed at dose levels
lower than tumor incidence can reliably be measured, then the marker can be used to extend the
empirica1 basis of the dose-response curve to dose levels lower than would otherwise be possible.
Such an approach has been suggested, for example, by Belinsky and coworkers (1990) for DNA-

41
adducts from the tobacco-specific nitrosamine NNK and pulmonary tumors in rats. Clearly, the
confidence placed in such an extrapolation depends on the rigor with which one can establish that
the marker and the toxic endpoint maintain their relationship at dose levels below those for which
the toxic endpoint can reliably be observed.
The principle use of biomarkers in extrapolation of the quantitative dose-response relationship
is as a dosimeter. That is, the "target" variable is some measure of delivered dose or exposure
of the target tissues at the site of toxic action. Rather than presuming that such adelivered dose
is proportional to the amount administered, one can use a biomarker of delivered dose to
demonstrate and characterize nonproportionality that may arise because of saturation of
metabolism, differing degrees of absorption, species differences in the uptake, metabolie process-
ing, and elimination of the agent, and so on. These sources of nonproportionality tend to
complicate and confound the overall relationship of exposure to its health consequences; if such
factors can be characterized and eliminated, a presumably simpler pattern is revealed in which the
physiological responses of the target tissues depend on their immediate and direct encounter with
the toxic agent.
As noted earlier, however, one should not presume that "delivered dose," as characterized
by a biomarker, is necessarily proportional to the target tissue's response. First, the response
depends on what may still be very complex biological interactions at the target, which may include
the balance of damage and repair, binding to specific receptors followed by complex induction of
programmed responses, perturbations of the normal balance of cellular constituents counteracted
by homeostatic mechanisms, and so on. The dose-response relationship at the level of the target
organ will still have to be characterized. Second, there are other features aside from the dosimet-
rie ones that vary during the extrapolation process. This is most important for cross-species
extrapolation; not only do species differ in their uptake and metabolism of a toxic substance, they
also differ in the exact nature of their target tissues. This includes, but goes beyond, differences
in "sensitivity" to the agent. One must also account for the different numbers of cells in the target
tissue, their different inherent turnover rates, repair rates, the time lags in feedback controls, and
the many other ways in which homologous tissues vary among species. Third (and most important
to the present discussion), since biomarkers are often integrating measures of the presence of the
agent, they tend to lose information about temporal patterns, information that may be crucial to
the resulting toxic effect. Monro (1992), for example, demonstrates that, even if the same amount
of an agent is absorbed from agavage dose and from exposure in the diet, either exposure could
prove the more potent, depending on whether the toxic response depends on achieving a high peak
concentration or on the duration of exceeding some lower concentration. A biomarker (such as
adduct formation) that reflects only total amount absorbed, and not the time course of that
absorption, would fail to differentiate between these exposures by different routes, even if it
correctly accounted for the amount of agent that "reached the target." These issues are discussed
further elsewhere (Rhomberg, in press).

AN EXAMPLE: CARCINOGENIC POTENCY OF INHALED FORMALDEHYDE

An example that illustrates some of the foregoing points may be found in the EPA's dose-
response analysis of rat nasal carcinomas produced by inhaled formaldehyde and the resulting
estimate of an upper bound cancer potency in humans (EPA, 1991b). Similar analyses have been
presented by Starr and Buck (1984). (The EPA analysis is still underway as the present paper
goes to press; the following discussion should be considered illustrative, and not the official policy
of the U.S. Environmental Protection Agency.)
The central issue of the formaldehyde risk assessment is the extraordinary steepness of the
curve of nasal carcinoma incidence with increasing air concentrations of formaldehyde among
chronically exposed rats in a bioassay by Kerns et al. (1983). The issue arises whether a dose-
related change in the availability of reactive formaldehyde to the nasal epithelium contributes to

42
this nonlinearity of response. In addition, the relative availability of reactive formaldehyde to
nasal tissues in rats and humans may help to illuminate formaldehyde's relative carcinogenic
potency in the two species.
A potential biomarker is available in the measurable concentration of DNA-protein cross-links
in the nasal epithelium. Formaldehyde exhibits bifunctional reactivity with nuc1eophiles, inc1uding
nuc1eophilic centers in DNA and cellular proteins, and a certain fraction of the formaldehyde that
reaches the target tissues reacts with one molecule of each, producing the DNA-protein cross-links
(DPX). The extent of such cross-linking following single exposures to various air concentrations
of formaldehyde (inc1uding those used in the bioassay) has been measured in the nasal epithelia
ofrats (Casanova, et al., 1989) and monkeys (Heck, et al., 1989). (No measurements have been
made in humans, nor have repeated exposures been examined.) The levels of nasal DPX are
indeed nonlinear with formaldehyde air concentration, although the curve is not nearly as steep
as that for nasal carcinoma incidence and becomes linear at lower doses. Moreover, monkeys
have markedly lower DPX levels at a given air concentration than do rats, although they show
DPX deeper in the respiratory tract, which the rats lack.
What sort of a biomarker is provided by the observed DNA-protein cross-links? According
to the National Academy of Sciences definitions (NAS, 1989), cross-links are c1early a marker of
exposure, being "the product of an interaction between a xenobiotic agent and some target
molecule... within an organism. " One hopes, of course, to use the cross-links to illuminate some
aspects of formaldehyde's carcinogenic effect as weIl. It is helpful to enumerate the forces
influencing formaldehyde's fate upon being inhaled, as weIl as the potential effects of the agent
on the tissues it encounters.
Some inhaled formaldehyde will remain in the lumen of the airways; being unavailable to
react with cells lining those airways, this fraction will be re-exhaled. Species differences in
breathing patterns and in the geometry of the nasal passages-in particular, the high degree of
convolution of the nasal turbinates in rats that is not seen in monkeys or humans-will influence
the turbulence of the airflow, and hence the opportunity for airborne formaldehyde to interact with
nasal tissues. Of the formaldehyde that does interact with the walls of the nasal passages, much
will react with components of the mucus layer overlying the epithelial cells themselves. Of the
remainder, some will be detoxified by the metabolic machinery of the epithelial cells (at rates that
may differ acrosS' species), and some will react spontaneously with a variety of intracellular
nuc1eophilic molecules. Only a fraction of the reacting formaldehyde will produce DNA-protein
cross-links; in addition, a variety of protein- and DNA-adducts may be expected to be formed.
Which among the many reaction products of formaldehyde are of toxicological relevance is
problematic. Mechanisms that can plausibly be envisaged inc1ude both very general ones (e.g.,
cytotoxicity attributable to general breakdown of ceIlular homeostatic processes as they lose
functionality under the load of formaldehyde adducts) and very specific ones (e.g., DNA-adducts
at a particular spot in a key genetic locus inducing a particular oncogene-activating mutation). It
should also be borne in mind that many of the various forms of damage induced by reactive
formaldehyde in the nasal epithelium are largely repairable, and that the amount of damage
existing at any particular moment is the product of the balance between rates of formation and
rates of repair. These rates (and hence the balance they strike) may vary among species and
among circumstances of exposure.
How do the above considerations affect the interpretation of DNA-protein cross-links as a
quantitative biomarker? Clearly, many assumptions about proportionality are involved; the cross-
links are a product of reactive formaldehyde in the target ceHs, and their rate of formation is
plausibly proportional to the intraceHular free-formaldehyde concentration. If the rate of cross-
link formation, dDPX/dt, varies directly with tissue concentration of formaldehyde, [HeHO],

d DPX k i [HCHO] (1)

(Jt

43
(where k; is a rate constant), then the total amount of cross-links formed over a span of time T
varies directly with the area under the curve of intracellular formaldehyde concentration (AUe)
over that interval.

JdDPX
T

DPXT = (2)
o

J
T

= k i [HCHO] dt (3)
o

= k i AUC (4)

Thus, the amount of DPX observed in a target tissue after a given period of formaldehyde
exposure could be used as an index of the area under the curve of the concentration of reactive
formaldehyde in that tissue during the timespan. This is true only if there is no significant repair
of cross-links; if such repair is significant, Equation 1 must contain arepair term, and the
proportionality of AUe to DPXT (which now represents the net retained, not the total formed) is
broken. (It is an interesting line of thought to consider, however, that the net DPX levels might
be the more appropriate measure to relate to toxic effects under some models of toxic action; this
interpretation depends on the cross-links being directly mechanistically involved in the toxic
process in a way that depends on their standing concentrations, not their rates of formation. Such
an interpretation contradicts the use of DPX as a dosimeter, however, for the reasons outlined
already.)
The foregoing suggests that, given some conditions, nasal tissue DPX should be a useful
dosimeter, providing insight into the area under the curve of reactive formaldehyde in the target
tissue. This is so whether or not the cross-links are actually involved in the toxic processes of
concern. As long as the toxicologically relevant reactions (whatever they may be) proceed at a
rate proportional to the tissue formaldehyde concentration (as in Equation 1), then the total amount
of such reaction accomplished over a timespan should be proportional to the DPX formed over
that same span. In the terminology developed above, DPX serve as a biomarker of the third type,
consequences of a common cause, since the "marker" cross-linking reaction and the "objective"
toxicologically relevant reaction are common consequences of the pattern of free, reactive
formaldehyde availability in the cells of the target tissue.
Conditions that would interfere with the proportionality of marker to objective are the
existence of significant cross-link repair rates and departure from first order reaction rates. The
latter would include saturable metabolic reactions, changes in the concentration of other reactants
(e.g., depletion of target sites for adduction, which seems unlikely), or compensatory reactions
by the cells involved affecting the reaction rates. To use DPX as a dosimeter across species, one
must assurne that the cross-linking rate is similar across species for a given intracellular
formaldehyde concentration, that the concentration of targets for cross-linking is similar, and that
the proportionality between the cross-linking reaction and the toxicologically relevant reactions is
also maintained.
In sum, when using DNA-protein cross-links as a bio marker in the risk assessment of
formaldehyde's potential carcinogenic effects, one hopes that the biomarker will be sensitive to
variation in those factors that one hopes to take into account through the biomarker's use--species-
and exposure-related differences in the delivery of reactive formaldehyde to the cells of target
tissues of toxicity-and that the biomarker will be relatively unaffected by factors that tend to
obscure the desired proportionality of marker to objective. To establish this, one should examine
the causal links underlying the various influences on the marker's quantitative behavior.

44
Frequently, much potentially important information will be missing, but one should explicitly
consider the assumptions (and their plausibiJity) needed to bridge these gaps. In particular, one
should note situations that may arise under which the value of the biomarker variable may give
misleading information about the state of the unknown objective variable.

CONCLUSIONS

I have argued that biomarkers are best understood as surrogate or proxy measures,
substituting measurements of something readily observable for the unknown value of key variables
that are difficult, disruptive, or expensive to observe or measure. In dose-response analysis, such
surrogate measures can provide valuable insight into the operation of the biologica1 processes that
underlie the grossly observed relationship of exposure to disease, and they can help extrapolations
of this relationship by extending observations of the marker to situations where the toxic endpoints
themselves cannot practica1ly be observed. For biomarkers to be properly used, however, the
complexity of the cause-and-effect relationships underlying the grossly observed dose-response
relationship must be appreciated, and thc:: rationale for presuming the biomarker-surrogate to give
useful information about the cryptic objective variables must be explicitly examined.

REFERENCES
Belinsky, S.A., Foley, J.F., Wbite, C.M., Anderson, M.W., and Maronpot, R.R., 1990, Dose-response relationship
between O"-methylguanine formation in Cl!lCa cells and induction of pulmonary neoplasia in the rat by 4-
(methylnitrosamino)-I-(3-pyridyl)-I-butanone, Cancer Res. 50:3772.

Casanova, M., Deyo, D.F., and Heck, H. d' A., 1989, Covalent binding of inbaled formaldehyde to DNA in the nasal
mucosa of Fischer-344 rats: Analysis of formaldehyde and DNA by high-performance liquid chromatography
and provisional pharmacokinetic interpretation,Fundam. Appl. Toxicol. 12:397.

EPA, 1987, "The Risk Assessment Guidelines of 1986," [EPAl600/8-87/0451, Office of Health and Environmental
Assessment, United States Environmental Protection Agency, Washington, DC. (also available in Federal
Register 51:33992-34054.)

EPA, 1991a, "ORD Health Biomarkers Program: Research Strategy Document," [EPA/600/9-9110091, Office of
Research and Development, United States Environmental Protection Agency, Washington, DC.

EPA, 1991b, "Formaldehyde Risk Assessment Update (Draft dated June 11, 1991)," Office of Toxie Substances,
United States Environmental Protection Agency, Washington, DC.

EPA, 1992a, "Respiratory Health Effects of Passive Smoking: Lung Cancers and Other Disorders," [EPA/600/6-
90/006Fl, Office of Research and Development, United States Environmental Protection Agency, Washington,
DC.

EPA, 1992b, "Estimating Exposure to Dioxin-Like Compounds (Review Draft) , " [EPA/600/6-88/005Bl, Office of
Research and Development, United States Environmental Protection Ageney, Washington, DC.

Heck, H.d'A., Casanova, M., Steinhagen, W.H., Everitt, J.I., Morgan, K.T., and Popp, J.A., Formaldehyde
toxieity-DNA-protein cross-linking studies in rats and nonhuman primates, in: "Nasal Carcinogenesis in
Rodents: Relevance to Human Health Risk," Feron, V.I. and Bosland; M.C., eds., Pudoc Wageningen, The
Netberlands.

Kerns, W.D., Pavkov, K.L., Donofrio, D.J., ""d Swenberg, I., 1983, Carcinogenicity of formaldehyde in rats and
mice after long-term inbalation exposure, Cancer Res. 43:4382.

Monro, A" 1992, What is an appropriate measure of exposure when testing drugs for carcinogenicity in rodents?,
Toxicol. Appl. Pharmacol. 112: 171.

45
NAS, 1983, "Risk Assessment in the Federal Govemment: Managing the Process," National Academy Press,
Washington, DC.

NAS, 1989, "Biologie Marlcers in Pulmonary Toxicology," National Academy Press, Washington, Oe.

Rhomberg, L.R., What Constitutes 'Dose"? (Definitions), in: "Dose-Response ReJatiQnships in Carcinogen Risk
Assessment,' In Press, ILSI Press, Washington, DC.

Starr, T.B., and Buck, R.D., 1984, The importance of delivered dose in estimating Jow-dose cancer risk from
inhalation exposure to formaldehyde, Fundmn. Appl. Toxicol. 4:740.

46
MEASUREMENT OF MUTATION SPECTRA AS A MOLECULAR DOSIMETER

William G. Thilly

Center for Environmental Health Sciences

E18-666 Massachusetts Institute of Technology
Cambridge, Massachusetts 02139

INTRODUCTION

Most presentations in this meeting take as an article of faith that knowledge

of the amount and kinds of human chemical exposure are of value in determining the
causes of human cancer. It can be argued however that while such knowledge may
be valuable, the presence of a chemical does not prove that it has significantly
contributed to genetic change in general or to cancer mutations specifically. To
make this link, analytical chemistry must be joined to analytical genetics. As a
potential means to create this joining, the concepts and present practices of
mutational spectrometry will be reviewed. Emphasis will be restricted to methods
which allow genetic measurements in humans such as denaturing gradient
separations or the newly developed mismatch amplification mutation assay (MAMA)
technology. Experiments aimed at determining when in a person's lifetime
oncomutations occur will also be discussed.

In presenting the argument of this paper, I hold the following facts to be true:

(1) - Human beings suffer a wide range of diseases as a result of genetic

changes in germinal or somatic cells

(2) - The human environment contains a large number of agents capable of

mutating human cells.

The problem of relating environmental agents to genetic change in humans

is not trivial. Genetic changes occur via the enzymatic machinery for replicating DNA.
Cells biochemistry creates a wide variety of chemically reactive substances which can
act on the genetics of the cells themselves or on the genetics of the other cells in the
body.
The sum of all causes of genetic changes must include spontaneous
replication error, reactions with endogenous chemicals and reaction with the
exogenous agents and their metabolites.

Use o[ Biomarkers in Assessing Health and Environmental Impacts o[ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 47
 The complexity of the environmental mixture to which humans are exposed is
intensified by the action of enzymatic metabolism - primary oxygenation or reduction,
conjugation reactions, etc. - applied in series and in parallel to any single chemical
to produce many different chemicals from a single environmentally derived parent
molecule. Each cell type in the body mayas a first approximation have a unique set
of these "drug metabolism enzymes" and these handle each environmental chemically
in a qualitative or quantitatively unique way. The same may be said for the set of
DNA repair enzymes present in each body cell type.
Furthermore, because humans are an outbred population of animals there will
be reasonably large differences expected among humans with regard to both the
biochemistry of xenobiotic metabolism and DNA repair. Needless to add, individual
habits of diet, drug usage, exposure to sunlight, occupation and regional air, food
and water contamination, would be expected to give rise to different health outcomes
if any critical agent or biochemical pathway is involved.
To this morass, the mutational spectrometrist arrives with one single fact to
bridge between exposure and genetic disease. For any homogenous cell population,
mutagen and condition of exposure, there exists a specific reproducible pattern of
genetic changes: the mutational spectrum.
This fact was discovered by Seymour Benzer (Benzer and Freese, 1958) who
showed that the position and frequency of mutations in a genome of the T4 bacterial
virus was reproducible among independent experiments and unique for untreated
and chemically treated virus populations.
In the following 34 years Benzer's observations have been confirmed and
extended to bacteria, fungi, rodent, and human cells. In the simplest form an
experiment involves treating a large number of cells with a mutagen and, one at a
time, determining the DNA sequence of surviving treated cells. In practice, a
sequence - a gene or part of a gene - is first defined, limiting a study to 100 to 1000
base pairs.
In practice up to this point, in time all experiments have used selective
conditions to kill off non-mutant cells and increase the efficiency by wh ich mutant
sequences may be isolated and mapped. Now (as is laboriously described in the
oral presentation) it is possible to separate mutant DNA sequences from non-mutant
sequences by physical-chemical means.
Thus one may contemplate obtaining mutational spectra in cells isolated from
animal tissues and organs (Thilly W.G., 1990).
Given the fact that mutational spectra exist and may be reproducibly obtained,
we should mention a few caveats for the new practitioner to consider:
1. Cells of a tissue are not homogenous with regard to kind. It will be
necessary to separate stroma, parenchyma and resident immune cells in order
to observe the mutational spectrum of a particular cell type.

2. Cells of a tissue are not homogenous as to position in the cell cycle

whether in tissue growth or turnover cell generations. A single treatment may
effect a subpopulation in a manner different from its effect on the bulk
population. Treatments continuing over one or several growth or turnover
generations would seem to overcome this difficulty.

3. The way cells respond to a particular chemical can depend both on dose
and dose rate. The mutational spectrum may likewise be affected by these
parameters. In order to obtain results in cell or animal experiments one will
have to determine the minimum dose rate wh ich gives rise to a recognizable
spectrum different from untreated cells or animals.

48
 These points, though not an exhaustive set, must be experimentally addressed
for any single mutagenic agent in any cell type in vive or in vitro.
With regard to real environmental mixtures of chemicals another concept is
useful. The concentrations of all chemicals are distributed such that some chemical
is present at the highest concentration, far above the mean. A simple variant is to
assume the log of chemical concentration is normally distributed.
The log normal concept is plastic and applies just as weil to the specific
mutagenic activity to wh ich the same certain cell type is exposed. Thus one of the
myriad mutagens present would be expected to have a mutational potency far above
that of the average mutagen present.
As a first approximation, the mutagenicity of a mixture specifically an
environmental mixture, is the sum of the products of the specific activity, Ai and
concentration, Ci of its constituent chemicals.

Mixture Mutagenicity = A 1C 1 + ~C2 + ... + AnCn + E'\ AiCi

But applying the log normal concept to both Ai and Ci, we note that the
product AiCi will also be log normally distributed with a much great dispersion.

Variance (AiCi) = Variance (Ai) + Variance (Ci)

This dispersion is so great that one expects one mutagen to produce the
plurality, majority or even nearly all the mutations induced by the mixture

EAiCi:::: Ax Cx

where "X" is the mutagen most responsible for the mutagenicity of the mixture. In
real experiments, this concept had found fair precedent. The mutagenicity for human
cells of moldy peanut meal is caused by aflatoxin B1, of turbulent combustion
exhausts by cyclo penta [c,d] pyrene and recently we found formal evidence for a
single primary human cell mutagen in extracts of smokeless tobacco.
To the mutational spectrometrist the "log normal universe" is a necessary
deconvoluting concept. It lays the theoretical basis for expecting that a particular
human's tissue will yield a single clear pattern of mutations induced primarily by a
single cause. The log normal concept stands in clear contrast to the "every mutagen
does its bit" nation which, however foggy, seems to represent majority opinion in the
field of environmental genetic toxicology.
These arguments - the existence of unique mutational spectra and the log
normal distribution of chemical concentration and mutagenic activities - serve as the
theoretical basis for the application of mutational spectrometry to human populations.
The technology is fairly straightforward. Actually two separate analytical
approaches are now available at the research bench.

DGGE - HiFi PCR

In this approach, DNA is isolated from a homogeneaus cell population and cut
with restriction enzymes to liberate the desired DNA segment. Non-mutant fragments
are separated from mutants in a 100bp sequence by the physical-chemical
processes active in denaturing gradient gel electrophoresis (Fischer and Lerman,
1983).

49
 Once isolated, these mutant fragments are increased in number using HiFi
polymerase chain reaction DNA amplification (Kleppe et al, 1971, Keohavong and
Thilly, 1989).
The amplified DNA is 32p labelIed and individual mutant bands may be
observed, measured, excised and sequenced using another gradient denaturing gel
electrophoresis step. Given the frequency (intensity of bands) position and kind
(sequences) of the set of predominant mutants one has obtained the point mutational
spectrum of the particular DNA sequence in the cell population of choice.

MISMATCH AMPUFICATION MUTATION ASSAY (MAMA)

This assay uses a variation on low fidelity PCR approaches. First "allele
specific" primers are defined by experiments for each specific mutation - position and
kind - that it is desired to measure. Theoretically, a set of primers could be defined
for all 300 base pair substitutions possible in a 100 base pair sequence. Since only
the mutant and not the wild type allele are amplified, one merely assures that
amplification efficiency for each allele is (a) known and (b) constant during
amplification in order to convert the number of mutant allele copies after amplification
into the number of mutant copies in the original mutant sampie.

(Number of Found Copies) - (Number of original copies) (1 + Vi)"

where Yi is the efficiency (constant) of the amplification for the mutant sequence, i,
and n is the number of amplification cycles.
These procedures are fairly straightforward and can be mastered in less than
a year of practice.
Simplification of the technology occurs at regular intervals and full automation
is easy to contemplate.

MUTATIONALSPECTROMETRY

The First Human Trials

Using clone forming mutation assays with T cells, Morley's and Albertini's
(McGinniss et al. 1990 and Grist et al. 1992) laboratories have shown that the rates
of accumulation of mutants of two separate nuclear genes (hprt, HLA) are constant
from birth to old age. If this is true, its also consistent with the idea of a principal
mutational pathway in a relatively homogeneous cell population in contact with an
inhomogeneous but essentially constant environment.
The first job for the human mutational spectrometrist is to see what is actually
happening. A variety of DNA sequences should be chosen to maximize the kind of
information to be gained. Certainly transcribed and untranscribed nuclear sequence
and most certainly mitochondrial sequences should be chosen. Because 109 copies
of genes are necessary for precise spectra given the known mutant fraction in human
T cells, one would have to work with autopsy or surgical specimens 109 copies
means isolating DNA from ab out 3-4 grams of tissue after separating the various cell
types to assure homogencity. For multicopy genes such as the ribosomal RNA
genes or mitochondrial genomes, the problem is eliminated as they occur at about
500 or 5000 copies per cell respectively.

50
 Actually, the first few human studies should tell us if clear mutational spectra
exist or not. If useful spectra exist, the pathway to future studies is clear. Lifetime
studies in multiple organs especially the primary organs of major cancers: lung,
stomaeh, and colon.
The problem of finding out what is responsible for mutation of ce1ls is an
important one. Society cannot afford to imagine a bogey-man in every breath of air
or bite of apple. Mutational spectrometry and concepts related to its use seem to
lay a reasonable theoretical basis for its application in human studies. It is possible
it will provide a means to firmly establish cause and effect relationships necessary
both in science and in law. Then again it may not. We will know soon.

REFERENCES

Benzer, S. and Freese, E., 1958, Induction of specific mutations with 5 bromouracil,
Proc. Nat. Acad. Sci. USA. 44: 112-119.
Fischer, S. G. and Lerman, L. S., 1983, DNA fragments differing by single base-pair
substitutions separated in denaturing gradient gels: correspondence with
melting theory, Proc. Nat. Acad. Sci. USA. 80:1579-1583.
Grist, S. A., McCarron, M., Kutlaca, A., Turner, D. R., and Morley, A. A., 1992, In vive
human somatic mutation: frequency and spectrum with age, Mutation Res.
189-196.
Keohavong, P. and Thilly, W. G., 1989, Fidelity of DNA amplification in vitro, in
"Polymerase Chain Reaction," H. A. Erlich, R. Gibbs and H. H. Kazaziam Jr.,
eds., Cold Spring Harbor Laboratory Press, New York, 19-24.
Kleppe, K. Ohtsuka, E., Kleppe, R., MOlineux, E. and Khorona, H. G., 1971, Studies
on polynucleotides. XCVI. Repair replications of short synthetic DNA's as
catalyzed by DNA polymerases, J. Mol. Biol. 56:341-361.
McGinniss, M. J., Falta, M. T., Sullivan, L. M., and Albertini, R. J., 1990, In Vivo hprt
mutant frequency in T cells of normal human newborns, Mutation Res.,
240:117-126.
Thilly, W. G., 1990, Mutational spectrometry in animal toxicity testing, Ann. Rev.
Pharmacol. Toxicol., 30:369-85.

51
BIOMARKERS AS MOLECULAR DOSIMETERS OF GENOTOXIC
SUBSTANCES

Peter B. Farmer
MRC Toxicology Unit
MRC Laboratories
Woodmansterne Road
Carshalton, Surrey
SM5 4EF, u.K.

INTRODUCTION
Exposure to genotoxic carcinogens results in the formation of covalently bound
adducts between the genotoxin and DNA, which may cause mutation and cytogenetic
alterations. The dose of the genotoxic carcinogenic compounds involved in the exposure
may be monitored by quantitative analysis of DNA adducts, and these data may also be
used as an indicator of genotoxic risk. The analytical techniques required for adduct
measurernents need to be of exceptional sensitivity and include 32P-postlabelling, mass
spectrornetry and immunoassay. Exposure to alkylating carcinogens mayaiso be
monitored by measurement of adducts formed with amino acids in haemoglobin (Rb).
Since, for a variety of compounds, the extent of protein-adduct formation relates
quantitatively with that of DNA adducts, measurement of the former may indicate the
biologicaIly-effective dose of the compound received.
Dose-response relationships for a wide variety of DNA and Rb adducts have been
determined, and these are reviewed in this article. The relationship between adduct
levels and carcinogenic risk is not as clearly established. For such a correlation to exist
it is necessary both that the adduct measured gives a representative estimate of the adduct
at the carcinogenic target site (wh ich is not normally measured) and also that the amount
of the target site adduct itself is proportional to tumorigenicity. Experimental data to
support either of these parameters is not yet widely abundant and clearly will be more
generally required if adduct ll1easurell1ents are to achieve their potential for use as risk
estimates.

1) Haemoglobin addllcts - dose response

There are many nucleophilic sites in the Rb which are capable of forming adducts
with electrophilic genotoxic cOll1pounds. Adducts at 4 of these sites (cysteine, histidine,
carboxylic acids and the N-terll1inal valine) have been used for human monitoring. Gas
chromatography-mass spectrometry (GC-MS) has been the method of choice for these
determinations.

A) Cysteine addllcts The main classes of cysteine adducts are formed by direct
alkylation of the sulphydryl group or by the interaction of this group with aryl nitroso
compounds, derived metabolically from arylamines. AdditionaIly, addition products of
cysteine with O',-unsaturated compounds have been ll10nitored.
Use 0/ Biomarkers in Assessing Health and Environmentallmpacts 0/ Chemical
Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 53
 The in vivo interaction of cysteine in Rb with simple methylating agents was one
of the first reactions monitored by GC-MS (Farmer et al., 1980). The product
S-methylcysteine was analysed as the n-butyl ester of its N-heptafluorobutyryl derivative
following its purification by ion-exchange chromatography from a hydrolysate of Rb. A
deuterated (d3 -) analogue was used as internal standard. The dose-response relationship
for formation of S-methylcysteine from methyl methanesulphonate in rat Rb was linear
(Bailey et al., 1981). For N-nitrosodimethylamine the dose-response curve showed an
upward curve, i.e. greater doses showed greater than linear increases in alkylation. This
phenomenon proved to be rather unusual for Rb alkylations and may be associated with
the fact that high doses (up to 30 mg/kg) of the nitrosamine were used. Other alkylations
of cysteine that have been investigated following in vivo exposures include ethylation
(Murthy et al., 1984), oxoethylation (Svensson, 1988), 2-hydroxyethylation (Ehrenberg
et al., 1977; Segerback, 1983) and phenylhydroxyethylation (Ting et al., 1990). More
recently the S-phenylation of cysteine has been used as an exposure monitor for benzene
(Bechtold et a1., 1992; Melikian er al., 1992). The dose-response relationships for these
exposures are summarised on Table 1.
The arylamine conjugates with cysteine are generated following the metabolic
activation of the amine to a hydroxylamine. The latter is oxidized in the erythrocyte to a
nitroso compound wh ich reacts spontaneously with the SH group of cysteine (Neumann,
1984). A relatively stable sulphinamide is formed following rearrangement of the
product. Neumann (1980) carried out an extensive dose-response study using eH]-trans-
4-dimethylaminostilbene at oral doses from 5 x 10. 10 to 1.8 X 10.4 moles/kg in the female
Wistar rat. Binding to Rb increased linearly with dose, the corre1ation only deviating at
the highest dose. Mild hydrolysis of aromatic amine sulphinamides regenerates the
parent amine, whose determination allows an assessment to be made of the exposure of
Hb to the arylnitroso compollnd.
GC-MS techniques have now been developed for this purpose for a variety of
amines including 4,4'-methylenebis (2-chloroaniline) (MBOCA) (Bailey et al., 1992;
Sabbioni and Nellmann, 1990; Chen er al., 1991), 4,4'-methylene dianiline (MDA)
(Bailey et a1., 1990), aniline (Lewalter and KoraIllls, 1985; Albrecht and Neumann,
1985), 4-aminobiphenyl (Bryant et al., 1987) and 0-, m- and p-toluidine (StillweIl et al.,
1987). Where stlldied (e.g. for MBOCA and MDA) the dose-response relationships
were essentially linear.
As indicated above, cysteine in Rb also forms addllcts with a,-unsaturated
compounds. Thus acrylamide binds extensively to Hb in vivo yielding an
S-(2-carboxamidoethyl) adduct with cysteine (Bailey er al., 1986). In rats, over an i. v.
dose range of 0-50 mg/kg, the amollnt of adduct increased at a greater than linear rate
with dose. It was speclllated that a competing process was possibly responsible for the
lack of linearity. Interestingly a similar dose-response was seen for the total binding to
Hb of 14C-acrylonitrile, which forms as a major prodllct S-(2-cyanoethyl)cysteine
(Fennell er aI., 1991). In contrast however Bergmark et al. (1991) found a linear dose-
response for adduct formation from i.p. acrylamide in the rat and a downward sloping
dose-response curve for its metabolite glycidamide.
B) Histidine adducts The analysis of histidine adducts in Hb follows the same
general procedure as that for S-alkylcysteines (see above), involving hydrolysis of Hb
followed by ion exchange chromatography, derivatisation and GC-MS. This aproach
(which is no longer extensively applied) was used in particlliar for the monitoring of
exposures to ethylene/ethylene oxide which yields N'-(2-hydroxyethyl)histidine and to
propylene/propylene oxide wh ich yields NT -(2-hydroxypropyl)histidine. Exposure of
animals to the epoxides by inhalation resulted in adduct formation linearly related to dose
(Osterman-Golkar er al., 1983; Farmer .et al., 1982; Potter et a1., 1989).
C) Carboxylic acid adducts Same electrophilic genotaxic carcinogens interact
with the carboxylate functions in Hb (aspartic acid, glutamic acid and carboxylic acid

54
 Tahle 1. Haemoglobin adducts.
Carcinogen Adduct
4-Methylnitrosamino- Pyridyloxobutyl esters
1-(3-pyridyl)-I-butanone
Methyl methane sulphonate S-methylcysteine
N-Nitrosodimethylamine S-methylcysteine
Ethyl methane sulphonate S-ethylcysteine
Urethane S-(2-oxoethyl)cysteine
N T-(2-oxoethyl)histidine
N -(2-oxoethyl)valine
Ethylene/ethylene oxide NT -(2-hydroxyethyl)histidine
N -(2-h ydrox yethy l)valine
Propylene/propylene oxide NT-(2-hydroxypropyl)histidine
N-(2-hydroxypropyl)valine
Styrene oxide S-(phenylhydroxyethyl)cysteine
N-(phenylhydroxyethyl)valine
Phenylhydroxyethyl esters
Benzene S-phenylcysteine
trans-4-dimethylaminostilbene Sulphinmnide with cysteine
4,4'-methylene bis (2-chloroaniline) Sulphinamide with cysteine
4,4'-methylene dianiline Sulphinamide with cysteine
Acrylamide S-(2-carboxamidoethyl)cysteine
Acrylonitrile S-(2-cyanoethyl)cysteine
Benzo(a)pyrene Benzo(a)pyrene tetrol esters
L linear
U upward deviation from linear response to dose
D downward deviation from linear response to dose
Dose-response Reference
relationship
L Murphy et al., 1990
L Bailey et al., 1981
U Bailey et al., 1981
LlU Murthy et al., 1984
L Svensson, 1988
L Svensson, 1988
L Svensson, 1988
L Osterman-Golkar
et al., 1983;
Polter er al., 1989
L Tornqvist et al., 1988,
Bailey et al, 1988
LlU Walker et al., 1992a
L Farmer et al., 1982
L Tornqvist er al., 1988
L Ting er al., 1990
L Tang et al.,
unpublished
L Sepai et al., 1992
LID Bechtold et al., 1992
L Neumann, 1980
L Bailey et al., 1992
LlU ehen et al., 1991
L Bailey et al., 1990
U Bailey et al., 1986
U Fennell er al., 1991
LID Bechtold et al., 1991
55
terminus) to yield esters. These may be hydrolysed in dilute base to the alcohol which
can be derivatised and determined by GC-MS as a biomonitor of exposure to the
alkylating agent. Exposure to methylating agents is difficult to detect owing to the
ubiquitous presence of traces of methanol, but has been achieved using stable isotope
labelled N-nitrosodimethylamine (Gan et al., 1989). The tobacco-specific nitrosamine
4-(methylnitrosamino)-I-(3-pyridyl)-I-butanone (NNK) forms an adduct with Hb which
releases 4-hydroxy-l-(3-pyridyl)-I-butanone on basic hydrolysis (Carmella et al., 1990).
Stable isotope labelling studies have indicated that this adduct is a carboxylic acid ester
(Carmella et al., 1992). Murphy et aI. (1990) have shown that the pyridyloxobutylation
of globin increases linearly with dose of NNK from 3 to 10000 j.tg/kg/day (rat, i.p.).
We have recently demonstrated that treatment of Hb with styrene oxide results in
the formation of carboxylic acid ester adducts, wh ich account for 15 % of the total
binding of the epoxide (Sepai er al., 1992). These may be hydrolysed yielding styrene
glycol which we have determined by GC-MS as its trimethylsilyl derivative using a d g-
labelIed internal standard. Rats treated i.p. with styrene oxide (0-833 j.tmole/kg) showed
adduct levels which increased with dose at a greater than linear rate. Higher molecular
weight hydrocarbons similarly produce ester adducts, and GC-MS methods have been
established for example to detect benzo(a)pyrene tetrol derived from the hydrolysis of the
ester formed by the active metabolite of benzo(a)pyrene with Hb (Day et al., 1990,
1991). Bechtold er al. (1991) has studied the dose-response of this adduct formation
using HPLC with fluorescence detection to detect the tetroI. The extent of adduct
formation was not linearly related to dose.
D) N-Terminal valine adducts The discovery by Tornqvist et al (1986) that a
modified Edman degradation procedure may be used to isolate carcinogen adducts with
N-terminal valine in Hb has stimulated extensive work on biomonitoring adducts at this
site, primarily those derived from low molecular weight epoxides. For example the
procedure has been used to monitor human exposure to hydroxyethylating agents from
occupational sources, cigarette smoke and anti-cancer agents. The dose-response
relationship for numbers of cigarettes smoked and production of N-terminaI
(2-hydroxyethyl)valine in Hb is linear (Bailey et al. 1988). The same dose-related
modification of Hb has been shown to be produced by the exposure of animals to exhaust
fumes (Tornqvist er al., 1988) although an upwards deviation from linearity was seen in
the dose-response curves of rats and mice treated repetitively with inhaled ethylene oxide
(Walker er al., 1992a). Dose-response studies for the interaction of higher molecular
weight alkylating agents with Hb N-terminal valine have been less weil studied. In
unpublished work from our laboratories we have demonstrated the production of the
styrene oxide-valine adduct in rats treated i.p. with this epoxide. Quantitation of this
adduct using GC-MS with a ds-Iabelled internal standard has shown that the amount of
adduct increases linearly with dose (0-833 j.tmole/kg).
E) Conclusion: dose-response of haemoglobin adduct formation The available
information on the dose-response relationship of the selection of Hb adducts considered
above is summarised in Table 1. The shape of the dose-response curve is most
commonly linear although there are several examples where the extent of Hb
modification increased at a greater than linear rate with respect to dose. The adduct
measurements are believed to reflect the dose of active electrophilic material reaching
Hb, and it may therefore be postulated that for some compounds there may be competing
reactions involving other nucleophiles which decrease the possibility of binding to Hb
occurring, and that these competing reactions may be saturable, leading to relatively
higher binding to Hb at higher doses of electrophile.

2) DNA adducts - dose response and lelationship to Hb adducts

Adducts are formed between electrophilic genotoxic carcinogens and the nitrogen
and oxygen atoms in nucleic acid bases. Highly sensitive detection of essentially all of
these adducts may be achieved by the technique of 32P-postlabelling, which should in
theory be capable of quantitation down to ca. 1 adduct per cello Alternative approaches
for DNA adduct determination in humans include immunoassay, GC-MS, HPLC and, in

56
 Table 2. DNA adducts.

Carcinogen Adduct Dose-response Reference

relationship'

4-Methylnitrosamino- N -7 -methylguanine LID Murphy et al., 1990

1-(3-pyridyl)-1-butanone

Ethyl methanesulphonate N-7-ethylguanine LlU Murthy er al., 1984

N-Nitrosodiethylamine 0 4 -ethylthymidine LID Boucheron et al., 1987

Ethylene oxide N-7-(2-hydroxyethyl)guanine L Osterman-Golkar

er al., 1983
Segerback, 1983
Potter er al., 1989
LlU Walker er al., 1992b

Urethane N-7 -(2-oxoethyl)guanine L Svensson, 1988

Trans-4-dimethy laminosti Ibene Total adducts L Neurnann, 1980

2-Acetylaminofluorene C-8 guanine adduct with L Beland er al., 1990

2-aminotluorene

L linear
U upward deviation from linear response to dose
D downward deviation from linear response to dose

experimental systems scintillation counting. Extensive work has been reported on dose-
response relationships: some representative examples are summarised below.
A) Methylating agents Exposure of rats to the tobacco-specifie nitrosamine
NNK results in both methylation and pyridyloxobutylation of DNA. One of the
methylation products, N-7-methylgllanine, was determined by Murphy et al. (1990) in
animals treated conseelltively for 4 days with NNK (i.p. 3-10000 I-tg/kg/day). In liver
and lung the 7-methylguanine levels increased linearly with dose between 3 and 600
I-tg/kg/day NNK. Above 600 I-tg/kg/day the lung DNA was relatively less weIl
methylated than liver DNA. Measllrements of pyridyloxobutylation of DNA also
revealed a non-linear dose-response relationship, especially for liver DNA alkylation.
As globin a1kylation by NNK is linearly related to dose (cf I (e) above) the eonsequenee
is that at high doses NNK adduct formation in globin is not linearly related to lung or
liver DNA adducts.

B) Ethylating agents Ethyl methanesulphonate is a directly acting alkylating

agent whieh ethylates both DNA and Rb. Over a dose range to mice of 0.32-100
I-tmole/kg bodyweight the extent of ethylation of guanine N-7 was linearly related to dose
(Murthy et 01., 1984). At higher doses a relatively higher degree of alkylation was
observed. Parallel experiments to measure total Rb binding and also S-ethylcysteine
formation in Rb showed similarly shaped dose-response relationships, indicating that Rb
a1kylation is a valid indicator of DNA alkylation for ethyl methanesulphonate.
A further well-studied ethylating carcinogen is N-nitrosodiethylamine wh ich
requires metabolism to generate the active alkylating speeies. Boucheron et 01. (1987)
administered N-nitrosodiethylamine (0.4-100 ppm in drinking water) continuously to rats

57
over periods up to 70 days. 04-Ethylthymidine, a major promutagenic DNA adduct, was
measured in liver DNA by radioimmunoassay, and was shown to reach plateau levels
after 7 days exposure. The dose-response relationship was essentially linear although the
highest dose showed a lower than linear extent of alkylation indicating either greater
repair of the adduct or less effective production of the active metabolite from the
nitrosamine.
C) Ethylene oxide Ethylene oxide has been extensively used as a model
aIkyIating genotoxic agent. Initially Ehrenberg et al. (1974), Segerbck (1983), and
Osterman-Golkar et al. (1983) provided evidence supporting the linearity of DNA
adduct/dose-response relationships and the fact that Hb adducts give a reasonable
estimate of DNA dose of ethylene oxide. These data were supported and confirmed by
Potter et al. (1989) who measured 2-hydroxylation of N-7 of guanine in a variety of rat
tissues following exposure to 1, 10 or 33 ppm ethylene oxide (6 hours). The extent of
alkylation in DNA from brain, lung, liver, spleen and kidney was similar and linearly
related to dose. As indicated above (1.B) alkylation of Hb histidine also was linearly
related to dose and is therefore a valid indicator of DNA dose of the epoxide. A major
recent study by Walker er ai. (l992b) extends the data over larger dose ranges and
exposure conditions in rats and mice. Upward deviations were seen from linearity in the
dose response relationship for production of N-7-(2-hydroxyethyl)guanine in tissue DNA.
The correlation between Hb adducts (N-terminal valine) and DNA adducts changes over
time during multiple exposures to ethylene oxide (Swenberg et ai., 1992)
D) Urethane Urethane (ethyl carbamate) is believed to be metabolised to
epoxyethyl carbamate which introduces 2-oxoethyl groups at guanine N-7 positions in
DNA. In mice administered [14C]-urethane there was an approximately
linear relationship between the degree of oxoethylation of guanine and dose of urethane
(i.p. 1-260 mg/kg) (Svensson, 1988). Alkylation of amino acids in Hb was also linearly
related to dose and thus correlated with DNA alkylation.
E) Trans-4-dimethylaminostilbene As indicated in l.A above, one of the
widest range dose-response studies carried out has been with trans-4-
dimethylaminostilbene (Neumann, 1980). Binding to t-RNA and to DNA in liver and
kidney of eH1-labelled trans-4-dimethylami~ostilbene increased linearly with dos~s to
rats of 5 x 10- 0 to 3.5 X 10-5 moles/kg. A hlgher dose showed a less than proportIOnal
increase in binding. Correlation with binding to blood proteins was good, providing
convincing evidence for the value of protein adducts as DNA dose monitors.
F) 2-Acetylaminofluorene A large DNA adduct dose-response study recently
conducted has been that with 2-acetylaminofluorene (Beland er al., 1990). Mice were
fed 5-150 mg 2-acetylaminofluorene/kg diet for 28 days and the concentration of the
adduct with C-8 of guanine in liver and bladder DNA measured by radioimmunoassay.
Both tissues showed a linear correlation between adduct and dose. Data for Hb adducts
is not available for this experiment. However relationships with tumour incidence in
bladder and liver could be drawn (see 3 be1ow).

G) Conclusion: dose-l'esponse of DNA adduct formation and relationship to

Hb adducts The examples discussed (2.A-2.F) were chosen to represent the
relationships of DNA adducts with Rb adducts and tumorigenesis (see below), and were
not intended to be a comprehensive survey (c. f. IARC, 1988 for a more compiete
review). As summarised in Table 2 the data considered here suggests that dose-response
reiationships for DNA adducts are generally linear although high doses may show
upward or downward derivations from linearity in adduct production. This indicates that
(notably at the lower doses studied) Rb adducts are a valid monitor of DNA adducts.
3) Relationship between addllcts and tllmOloigenesis
It seems c1ear that Rb and DNA adducts are generally good measures of dose of
active genotoxic compound received at these sites. Anomalies do exist, such as the fact
that white blood cell DNA adducts with polycyclic aromatic hydrocarbons do not

58
correlate with extent of cigarette smoking; however such a correlation does exist for lung
DNA (Phillips et al., 1988, 1990). Such situations could be explained by as yet
unappreciated tissue variations in metabolite activation or detoxification.
Many DNA adducts have been measured at non-target sites for the carcinogen
(commonly lymphocytes or placenta). The relationship between non-target site adducts,
target site adducts, and tumour incidence has not been extensively studied. The
importance of selecting the correct site when one wishes to use adducts as carcinogen
risk monitors was weIl exemplified by the work of Belinsky et al. (1990) on the tobacco-
specific nitrosamine, NNK. Steady state concentrations of the promutagenic adduct 0 6 _
methylguanine were measured by radioimmunoassay in lung ceUs of rats treated
repetitively with doses of 0.03-50 mg/kg NNK. A linear relationship existed between
tumour incidence and the degree of guanine methylation in Clara cell DNA, whereas the
relationship was not linear in type 11 cells or wh oie lung.
Even in cases when one has available the data on DNA adducts at the target
tissue, the relationship of this with tun10ur incidence is not always simple. Thus in the
case of 2-acetylaminotluorene (2.F) (Beland er al., 1990), the amount of the guanine C-8
adduct in liver is linearly correlated to Iiver tumour incidence. However in the bladder
there was a 'threshold' region where tumour incidence was not related to adduct levels.
It appears therefore that considerably more experimental studies are required to
clarify the quantifications of adduct : risk correlations for genotoxic carcinogens.
However this requirement should not limit our interest in acquiring human adduct data as
the latter are c1early al ready good exposure monitors for these carcinogens. Furthermore
current molecular epidemiology approaches are now showing indications of the
association between addllct levels and human cancer incidence (e.g. aflatoxin-guanine
adducts and liver cancer, Ross er al., 1992).

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Bailey, E., Brooks, A.G., Bird, 1., Farmer, P.B. and Street, B., 1990, Monitoring
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Farmer, P.., Gorf, S.M. and Bailey, E., 1982, Determination of hydroxypropyl-
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adduct formation by acrylonitrile in rats and mice, in "Human Carcinogen
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G.T. Steel and A.W. Wright, ed., Oxford University Press, Oxford, pp 241-246.

Gan, L.S., Wishnok, 1.S., Fox, IG. and Tannenbaum, S.R., 1989, Quantitation of
methylated hemoglobin via hydralysis of methyl esters to yie1d methanol. Anal.
Biochem. 179:326.
IARC Scientific Publication No. 89. 1988. Methods for detecting DNA damaging agents
in humans: applications in cancer epidemiology and prevention. H. Bartsch, K.
Hemminki and LK. O'Neill, eds., LA.R.C., Lyon.

Lewalter, land Korallus, u., 1985, Blood protein conjugates and acetylation of
aromatic amines. New findings on biological monitoring. Int. Arch. Occup.
Environ. Health, 56:179.
Me1ikian, A.A., Prahalad, A.K. and Coleman, S., 1992, Isolation and characterization
of two benzene-derived hemoglobin adducts in vivo in rats. Cancer Epidemiology
Biomarkers and Prevenrion. 1: 307.
Murphy, S.E., Palomino, A., Hecht, S.S. and Hoffmann, D., 1990, Dose-response
study of DNA and hemoglobin adduct formation by 4-(methy1nitrosamino)-I-(3-
pyridyl)-l-butanone in F344 rats. Cancer Res. 50:5446.

Murthy, M.S.S., Calleman, C.l, Osterman-Golkar, S., Sedgerbck, D. and Svensson,

K., 1984, Relationships between ethylation of hemoglobin, ethylation of DNA
and administered amount of ethyl methanesulfonate in the mouse. Mut.
Research. 127: 1.
Neumann, H.G., 1980, Dose-response relationship in the primary lesion of strang
electrophilic carcinogens. Areh. Toxicol. 3:69.

Neumann, H.G., 1984, Analysis of hemoglobin as a dose monitor for alkylating and
arylating agents. Areh. Toxieo!. 56: I.
Osterman-Golkar, S., Fanner, P.B., Segerbck, D., Bailey, E., Calleman, C.l.,
Svensson, K. and Ehrenberg, L., 1983, Dosimetry of ethylene oxide in the rat by
quantitation of alkylated histidine in hemoglobin. Teratog. Carcinog. Mutagen.
3:395.

Phillips, D.H., Hewer, A., Martin, C.N., Garner, R.C. and King, M.M., 1988,
Correlation of DNA adduct levels in human lung with cigarette smoking. Nature.
336:790.

Phillips, D.H., Schoket, B., Hewer, A., Bailey, E., Kostic, S. and Vincze, 1., 1990,
Influence of cigarette smoking on the levels of DNA adducts in human bronchial
epithelium and white blood cells. In!. J. Cancer. 46:569.

Potter, D., Blair, D., Davies, R., Watson, w.P. and Wright, A.S., 1989, The
relationships between alkylation of haemoglobin and DNA in Fischer 344 rats
e
exposed to 4C]ethylene oxide. Arch. Toxicol., Supp!. 13:254.

Ross, R.K., Yuan, Y-M., Yu, M.C., Wogan, G.N., Qian, O-S., Tu, J-T.,Oroopman,
J.D., Gao, Y-T. and Henderson, B.E., 1992, Urinary aflatoxin biomarkers and
risk of hepatocellular carcinoma. Lancet. 339:943.

61
Sabbioni, G. and Neumann, H.G., 1990, Quantification ofhaemoglobin binding of4,4'-
methylenebis (2-chloroaniline) (MOCA) in rats. Arch. Toxicol .. 64:451.
Segerbck, D., 1983, Alkylation of DNA and hemoglobin in the mouse following
exposure to ethene and ethene oxide. Chem. -Bio!. Interactions . 45: 139.

Sepai, 0., Anderson, D., Street, B., Bird, 1., Farmer, P.B. and Bailey, E., 1992, The
monitoring of exposure to styrene oxide by GC-MS analysis of
pheny1hydroxyethy1 esters in hemog1obin. Arch. Toxicol. in press.

Stillwell, w.G., Bryant, M.S. and J.S. Wishnok., 1987, GC/MS analysis ofbio1ogical1y
important aromatic amines. Application to human dosimetry. Biomed. Environ.
Mass Spectrom. 14:221.
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Carcinogenesis.9:2197. .
Swenberg, J.A., Walker, V.E., McNeela, J.P. and Fennell, T.R., 1992, Mo1ecular
dosimetry of DNA and hemoglobin adducts in mice and rats exposed to ethylene
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Ting, D., Smith, M. T., Doane-Setzer, P. and Rappaport, S,M., 1990, Analysis of
styrene oxide-globin adducts based upon reaction with Raney nickel.
Carcinogenesis. 11 :755.
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environmental cancer initiators through hemoglobin adducts by a modified Edman
Degradation method. Anal. Biochem. 154:255.

Tornqvist, M., Kautiainen, A., Gatz, R.N. and Ehrenberg, L., 1988, Hemoglobin
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Walker, V.E., MacNeela, 1.P., Swenberg, 1.A., Turner, M.J. and Fennell, T.R.,
1992a, Molecular dosimetry of ethylene oxide: formation and persistence of
N-(2-hydroxyethyl)valine in hemoglobin following repeated exposures of rats and
mice. Cancer Res. 52:4320.

Walker, V.E., FennelI, T.R., Upton, P.B., Skopek, T.R., Prevost, V., Shuker, D.E.G.
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exposures of rats and mice. Cancer Res. 52:4328.

62
PUBLIC HEALTH ASSESSMENTS AS A TOOL IN IDENTIFYING HUMAN
EXPOSURE TO ENVlRONMENTAL POLLUTANTS

Mark M. Bashor

Assoeiate Administrator for Federal Programs

Ageney for Toxie Substances and Disease Registry
1600 Clifton Road, N.E., Mai! Stop E-28
Atlanta, Georgia 30333

INTRODUCTION

This paper summarizes the presentation made at the NATO Advanced Research
Workshop on the "Use of Biomarkers in Assessing Hea1th and Environmental Impaets of
Chemica1 Pollutants" held in Luso, Portugal on June 1-5, 1992. This paper provides a
brief overview of the Ageney for Toxie Substances and Disease Registry, focusing
primarilyon the publie hea1th assessment program.

OVERVIEW

The Ageney for Toxie Substances and Disease Registry (ATSDR) is an ageney of the
Publie Hea1th Service, U.S. Department of Hea1th and Human Services. ATSDR was
ereated by the Comprehensive Environmental Response, Compensation, and Liability Aet
of 1980. Additional responsibilities for ATSDR were detailed under two subsequent pieces
of legislation; the Resource Conservation and Recovery Act of 1984 and the Superfund
Amendments and Reauthorization Aet of 1986. 1-5
The mission of ATSDR is to prevent or mitigate adverse human hea1th effects and
diminished quality of life resulting from exposure to hazardous substances in the
environment. In order to carry out its mission, ATSDR has developed programs consistent
with the legislative mandates. The eurrent major ATSDR program areas are: publie hea1th
assessments, hea1th studies, exposure and disease registries, emergeney response,
toxieologica1 profIles, applied research, and hea1th education.
The pUIpose of the hea1th studies program is to inerease understanding of the
relationship between exposure to hazardous substances and adverse human hea1th effects.
This is aeeomplished primarily through epidemiologie surveillance.
The exposure and disease registry program was established to eollect information in

Use 0/ Biomarken in Assessing Health and Environmental Impacts 0/ Chemical

Pollulants, Edited by C.C. Thlvis, Plenum Press, New York, 1993 63
 a systematic way on persons exposed to hazardous substances. The individuals are tracked
to ascertain the development of any serious diseases or illnesses. These registries will
facilitate future studies on the exposed persons and additionally provide a network for
communicating information to the exposed individuals.
The Agency's emergency response program provides support to states, local agencies,
and hea1th care providers in the event of a public hea1th emergency involving exposures
to hazardous substances. Furthermore, training is provided to individuals who are typically
the "fIrst responders" to an emergency event involving hazardous substances.
The toxicological profIles prepared by ATSDR summarize the available information
on speciftc hazardous substances. They are prepared to serve as a source of information
to the general public as well as convey technical information to the scientiftc community.
Additionally, they identify gaps in knowledge which provide the basis for developing the
applied research program. The applied research program serves to conduct or sponsor
research designed to increase scientiftc knowledge about the effects on human hea1th of
hazardous substances released into the environment.
ATSDR's hea1th education program was created to develop and disseminate
information on the hea1th effects of hazardous substances to physicians and other hea1th
care providers. Numerous environmental health education activities, training courses and
materials are generated to fulitll tbis pUlpose.
Finally, ATSDR's public hea1th assessment program was developed to evaluate data
and information on the release of hazardous substances into the environment in order to
assess any current or future impact on public hea1th, develop hea1th advisories or other
health recommendations, and identify studies or actions needed to mitigate or prevent
adverse human hea1th effects. Additionally, studies or actions needed to fully evaluate the
impact of arelease may be identifted. ATSDR conducts these public hea1th assessments
for all waste sites listed on the Environmental Protection Agency's National Priorities List.
Furthermore, ATSDR may conduct public hea1th assessments or health consultations in
response to petitions from concemed individuals and organizations.
As described in the Superfund Amendments and Reauthorization Act, the purpose of
the public health assessment is to assist in determining whether actions should be taken to
reduce human exposure to hazardous substances from a facility and to identify the need for
collecting additional information on human exposure and associated hea1th risks by
conducting epidemiological studies, establisbing a registry, establishing a hea1th
surveillance program or by other means.
The ATSDR public health assessment focuses on the release of hazardous substances
into the environment and identiftes ways in wbich these releases can reach and expose
human populations. The three primary sources of information are environmental data
describing the release of hazardous substances into the environment, hea1th outcome data
for populations exposed or potentially exposed to the release, and community hea1th
concems as expressed by persons living near or otherwise associated with arelease of
hazardous substances.
Environmental data are collected from site visit surveys and site inspection reports
conducted by ATSDR or other federal or state agencies, from geologieal surveys, and from
preliminary assessments and remedial investigation reports prepared by the U.S.
Environmental Protection Agency. Hea1th outcome data are obtained from records on birth
statistics, medical records, hea1th surveillance data, morbidity and mortality tables, tumor
and disease databases, and other sourees. Community hea1th concems are received by
ATSDR from news media (e.g. radio, television or newspaper reports) as well as
interviews, letters or telephone calls with individuals, community groups and loeal
officials.
The process by wbich ATSDR produces its public hea1th assessments is detailed in the
ATSDR Public Health Assessment Guidance Manual6 Briefly, the public hea1th

64
assessment process involves six steps: (1) evaluating information on the site's physical,
geographical, historical and operational setting, and identifying hea1th concerns of the
affected community or communities; (2) determining what contaminants are of particular
concern at the site; (3) identifying environmental pathways such as air, soll, food chain,
groundwater or surface water whereby the contaminants could reach a human population;
(4) identifying human exposure pathways such as ingestion, inhalation, or direct contact
and dermal absOIption; (5) determining the public hea1th implications based on available
medical and toxicological information; and (6) determining appropriate conclusions and
recommendations concerning the public hea1th impacts of the site. Information on the
demographics associated with the site, land use, groundwater, surface water, sediments,
soll, air, food chain, and the chemical and physical properties of the contaminants are
gathered. After the information has been analyzed, the Imdings are presented to the Hea1th
Activities Review Panel, a panel of scientists representing different areas of technical
expertise from each program within ATSDR. This panel helps to formulate the
conclusions and recommendations of the public hea1th assessment.
The public hea1th assessment will identify a site as belonging in one of five categories.
The category chosen is dependent on the nature of the public hea1th hazard posed by the
site, the sufficiency of the information that was available at the time the public hea1th
assessment was prepared, and the community health concems posed by the site. The Irrst
category is an "urgent public hea1th hazard." For these high priority sites, ATSDR
generally issues a health advisory to state, federal and Iocal agencies, apprising them of
the "urgent public hea1th hazard." The second category for sites is a "public hea1th
hazard." ATSDR generally recommends some type follow-up hea1th activity or study at
these sites. The third category, "indeterminate public hea1th hazard," identifies types of
missing information that are needed to make adetermination and leads to recommendations
to collect such information. The fourth category is "no apparent public hea1th hazard."
These sites hold a fairly low priority at ATSDR, although ATSDR continues to monitor
them periodically to insure that no changes have occurred or no new information has
become available which would prompt the need for re-evaluation of the site. The final
category are sites designated as "no public hea1th hazard." ATSDR generally has no
further involvement with these sites.
The types of recommendations which the public health assessment identifies fall into
three broad types: (1) recommendations to conduct actions to protect public health, (2)
recommendations to obtain additional information about the environmental release, and (3)
recommendations to conduct follow-up activities. These recommendations may include
issuing a public hea1th advisory or some other emergency response action, may call for
additional environmental characterization, may call far additional public health assessment
or hea1th consultation work or may call for a follow up hea1th investigation. The
investigation may take the form of a community hea1th investigation, a case study, a
disease prevalence study, a symptoms prevalence study, an exposure study, a cluster
investigation, an exposure and disease registry, a hea1th surveillance or other epidemiologie
study. The pUIpose of the activity would be to determine the association between arelease
of hazardous substances and the occurrence of adverse hea1th affects in the exposed human
populations.
While the public hea1th assessment of a hazardous waste site or facility is one method
for identifying the need for health investigations, there are other programs within ATSDR
which also plan and conduct health investigations. One such program involves the
investigation of priority hea1th conditions and their relationship to exposure to hazardous
substances in the environment. The priority hea1th conditions currently under investigation
at ATSDR are (in alphabeticalorder): birth defects and reproductive disorders, immune
function disorders, kidney dysfunction, liver dysfunction, lung and respiratory disease,
neurotoxic disorders and se1ected cancers.

65
REFERENCES

1. Comprehensive Environmental Remonse. Compensation and Liability Act of 1980.

42 U.S. Code 9401-9657. (1980).

2. Solid Waste Disposal Act. 42 U.S.Code 6901-6991i.

3. Superfund Amendments and Reauthorization Act of 1986. Pub. L. No 99-499. 100

Stat. 1613 (1986).

4. B.L. Johnson, Implementation of Superfund's hea1th-related provisions by the

Agency for Toxic Substances and Disease Registry, Envir L Rep.
20: 10277-10282 (1990).

5. A.S. Susten, Findings from ATSDR's hea1th assessments, J Environ Hea1th.

55(3): 17-31 (1992).

6. Agency for Toxic Substances and Disease Registry. ATSDR Public Hea1th
11

Assessment Guidance Manual," Lewis Publishers, Chelsea (1992).

66
TUE SUITABILITY OF TUE MOSSES SPHAGNA AS QUANTITATIVE
INDICATORS OF UEAVY METAL LEVELS IN URBAN ATMOSPUERES

M. Teresa Vasconcelos and Helena M. Tavares

Chemistry Department
Faculty of Science of porto
P4000 Porto, Portugal

INTRODUCTION
The aims of this paper are twofold. Firstly, to review briefly the properties of the
mosses that make them the most sensitive biological instruments for measuring the deposition
of heavy metals in terrestrial ecosystems and the ways in which mosses have been employed
to monitor patterns of heavy metal pollution. Secondly, on the basis of investigation in
progress, to discuss the suitability of Sphagnum auriculatum for lead monitoring in the urban
atmosphere of porto city.

REVIEW
Interest of bioindicators for the determination of heavy metals in atmosphere
Studies for assessing health and environmental impacts of heavy metals should include
the determination of their levels (and if possible their speciation) in diet, water and air.
Measurements of the heavy metal concentrations in air, especially over large areas, have
been made in limited number, mainly due to the lack of suitable, sufficiently sensitive and
inexpensive techniques permitting simultaneous measurements at a large number of stations
during long periods of time.
For monitoring heavy metal levels in the atmosphere of urban or industrial areas,
mechanical sampiers (low or high volume sampiers) are usually used. The replacement of
mecanical sampiers by biomonitors would be advantageous because these are cheaper and
required less attendence.
For the detection, recognition or accumulation of air pollution, biological organisms
which respond sensitivity and specifically to the pollutants under consideration have be used
for over a century. The major criteria for suitable bioindicators are as follows 1:
- The organism must be capable of accumulating metals in measurable amounts.
- The organism, or its relevant parts, must be readily available for the whole period of
study with relative ease of collection.
- For assessing airborne contamination, the organism must not be subjected to
substantial uptake or ingestion of metals from other sources.
- Repeatability is essential.
" Cost of collection and analysis should be acceptable.
- The organism should show a differential uptake/accumulation which is related to
exposure thus allowing either:
(1) The determination of relative pollution levels or

Use 0/ Biomarkers in Assessing Hea/th and Environmental Impacts 0/ Chemical

Pollu_Is, Edited by C.C. Travis, Plenum Press, New York, 1993 67
(2) The establishment of a more quantitative relationship with deposition rates or air
concentrations.
Low-level plants, especially mosses, accumulate high amounts of heavy metals. The
main reasons for this accumulation have been extensively discussed in reviews published in
recent years, e.g.I-4. Therefore, only some pertinent aspects are resumed here.

Mechanisms of metal uptake and retention

Most of the meta! content of mosses is accumulated extracellularly via ion exchange and
particulate trapping25. The cell walls of mosses possess ion exchange sites onto which meta!
ions bind in a way comparable to the binding of ions onto synthetic ion exchange resins. If a
moss is placed in a solution of a metal salt, ion exchange takes place quickly without
requirement of metabolic energy. The following equilibrium describes ion exchange in which
meta! ions (M2+) are being taken up and other meta! or hydrogen ions (Cx+) released:
M2+ + CA(n-x)- _ MA<n-2)- + cx+ (1)

where An- denotes a fIXed extracellular anionic functional group2.5.

Sphagna have large numbers of protonated anionic funcional groups (ion exchange
sites) in the form of uronic acids. In some species thesegroups constitute 25-30% of the
moss dry weight5.6 . Because of this characteristic, Sphagna are the most suitable mosses for
heavy meta! monitoring 5.
The retention efficiency order is Cu 2+, Pb 2+ > Ni 2+ > C 0 2+ > Zn 2+, Mn 2+, as shown
by Ruhling and Tyler7. Even in the presence of high concentrations of competing ions such
as Mg2+, Ca2+, K+ and Na+. Cu 2 + and Pb 2+ were completely absorbed 3 . The heavy metal
concentrations in Sphagnum species exposed to a contaminated area may be as high as
several hunders of mg/kg of dry moss.
Other important advantage of mosses as heavy meta! deposition monitors is that they
usually lack a protective cuticle and thickened epidermal cell walls, making their tissues easily
permeable to water and minerals, including metal ions. The large exchange capacity, absence
of cuticle and simple organization of the tissues render mosses incapable of avoiding heavy
metal uptake from deposition4 . A high surface to volume or weight ratio "lliso favours the
trapping of large particles though wind speed and other site characteristics may influence the
efficiency of the process4 . Mosses are ectohydric. which means that they absorb water over
their entire surface4. Water and, hence, meta! uptake in mosses is thus almost independent of
substrate composition. In addition, the transport of heavy metals to the plant surface in the
form of dust originating from the surrounding soil is negligible compared to the contribution
by atmospheric deposition4 . Therefore, during the last two decades, Sphagnum species have
been utilized in numerous studies of heavy metals atmospheric pollution, e. g)-5. 8-16.

Biomonitoring methods

The best-suited species for biomonitoring, i.e. for quantitative pollutant determination 3,
are usually absent from urban areas. In areas where the use of indigenous moss far
monitoring purpose is not applicable. exposure transplants, usually in moss bags8, are used.
Moss bags are simple and inexpensive to produce, do not require energy supplies or
maintenance during exposure periods and are of little interest to vandals2. Moss bags usually
consist 8-10 of a flat 10 cm by 10 cm package of 2 mm mesh nylon, in which 1-3 g dry
weight of weIl washed moss are placed. The transplant generally dies after so me weeks of
exposure to urban areas, partly due to an adverse climate or severe pollution conditions.
Despite this, it may continue to accumulate metals after death.
A number of intercalibration studies of the relationship between direct measurement of
heavy metal in rainfall and accumulated on mosses have been published in recent years, most
of them summarized in recent reviews, e.g. 1,4. Good correlation between annual rainfall and
the concentration of lead and copper in mosses have also been observed 4. Essentially, the
largest part of the previous studies have provided qualitative information about air quality,
namely, the determination of the intensity and distribution of heavy metal pollutants and the
reconaissance of the major fonts. Quantitative comparisons of the amounts of metals which

68
have accumulated in biomonitoring with air pollution measurements at the same site are very
scace in the literature. Such a comparison would enable the calibration of the biomonitoring
system to be made 3.

BIOMONITORING OF LEAD IN THE OPORTO URBAN A TMOSPHERE

In the Chemistry Department of the FacuIty of Science of Oporto a few projects, whose
main purpose is the determination of heavy metallevels in urban or occupacional atmospheres
have been developed 17 ,18. To collect the pollutants, low volume sampiers were used.
Recently, a project was launched to investigate the capability of the moss Sphagnum
auriculatum for biomonitoring of lead in urban atmosphere 19. For this purpose the moss bag
technique has been used. Biological and mechanical "sampiers" have been used in parallel for
comparison purpose. As analytical technique, atomic absorption spectroscopy has been
used 20,21.
In a first stage of this study, the influence of the moss pretreatment on the lead
bioaccumulation was investigated. Three types of treatment were carried out: (a) washing
with de-ionized water and drying at low tempearture (ca. 4QOC) in order to maintain the moss
alive; (b) washing and hot-drying; and (c) acid treatment. Moss treated by (a), (b) and (c)
procedures were exposed in parallel. Both hot-drying and acid treatments kill the moss, and
therefore uptake is purely passive 2. Acid washing also displaces preexisting metal ions.
However, for long exposure times (>30 days) no significant difference in the uptake capacity
of dead and alive mosses was observed (see fig.l), which confirms literature data 2,4.
Nevertheless, the accumulation was more regular for acid-pretreated plants in the first days of
exposure and, therefore, this type of pretreament was used for further experiments.

7.----------------------------------------.

6
,-.,.
>-
~5
x
::4
o
Hot-dry
E

*
Uve
0>3
~
..0
0.. 2
01
::J.

O+------.------~------~----~------.-----~
o 10 20 30 40 50 60
Exposure time (day)
Figure I. Influence of the pretreannent on lead accumulation in Sphagnum auriculatum

Results obtained by biomonitoring in dry weather conditions, of which typical results are
presented in fig. 2, show the following features. The lead uptake was linear for about a
month, as was observed before2. After this period of exposure, saturation of exchange sites
oecur. For more prolonged exposure dead material fragments start to decompose 2 and may
partly account for the decreasing in the metal content.

69
 140
n=8
0
120 hterc:apt = 1.3 2.2 llIJ/ g
Sklpe = 2.0 0.1 llIJ / (gxdJy)
R = 0.996
100 0
III
III 0
0
E 80
0)

"-
..c
Cl... 60
0)
::J.
40

20

10 20 30 40 50 60 70 80
91/04/17 Exposur e time (day)
Figure 2. Variation witb time of lead level in Sphagnum auriculatum

The amount of lead accumulated per gram of moss and per day obtained in two different
sampling periods, under similar weather conditions, are presented in fig.3, as weIl as the
dayly average vaIues of lead concentration in air over the same periods. The figure shows that

6.0~-----------------~1.0

,.....
>-
o
"0 4 .0
~---------~ 0.5
x

'"'"o
E

0.0

91/04/17 91/07/12
I
0.0..j...0--2~0--4O~-~60-~8'-/O ' 0 20
40 60 80
Exposur e time (day)
Figure 3. Dayly average values of lead concentration in air and tbe amounts of tbe meta! accumulated per
gram and per day on tbe Sphagnum auriculatum in two sampling periods, under similar weatber conditions

70
high signifieant eorreiations were obtained between the lead aeeumulated in moss and its
eoneentration assessed by the air filtration sam pIer. Furtherrnore, the reproducibility of the
biological monitoring was aeceptabie.
FinalIy, it was observed (fig.4) that rainfall influences markedIy the metal up take, as
was expected beeause the humidity favours ion-exchange on the cell walls. However, good
eorrelation between the lead accumulation and total rain fall over the exposure period was
found, as observed by Pilegaard22 far similar plants exposured to an industri'lli atmosphere.

7 ~---------------------------------.300

6
........
>-
0
"05 200 ........
x
E
(/)
(/) E
'--'
~4
......,
CI pg Pb/(g moss x day)
...."0c
"- Ci
0::
if3 100
CI
:J..

1 ~--.------.-------,------.-------.-~O
25/2 01/3 17/4 12/7 27/1
Beginnlng of exposure
1991 1992
Figure 4. Comparison of the lead accumulation rates in Sphagnum auriculatum and the total rainfall over
the exposure period. Results for five experiments are presented

CONCLUSIONS

From the present work it can be eoncluded that mossSphagnum auriculatum is suitable
for heavy metals monitoring (quantitative determination) in urban atmospheres. For lead the
retention efficiency is very high, whieh makes the method particularly sensitive for this metal.
However, the exposure time should not exceed one-two months in areas with high deposition
rates of metals or if the weather is dry. Detailed information about the humidity of the air and
rainfall during the sampling period is required. In addition, the method needs previous
calibration by parallel determination with a mechanical sam pier, for each sampling site. This
aspect is under investigation.
As far as we know, the suitability of biomonitors for speciation studies has never been
investigated, in spite of the determination of the bioavailability of heavy metals from the
different environmental sources being of great importance for health assessments. Therefore,
future research about this topic deserves mueh interest.
Acknowledgments

H.M. Tavares aeknowledges JNICT/PROGRAMA CII3.NCIA seholarships for

Doetorate Degree. Weather data were provided by the Geophysies Institute of the University
ofOporto

71
REFERENCES
1. K.J. Puckett, Bryophytes and lichens as monitors ofmetal deposition, in: "Lichens, Bryophytes and Air
Quality", T.H. Nash III and V. Wirth, eds., BibliothecaLichenologica, Berlin (1988)
2. D.H. Brown, Uptake of mineral elements and their use in pollution monitoring, in: "The Experimental
Biology ofBryophytes", A.F. Dyer and J.G. Duckett, eds., Academic Press, London (1984).
3. J. Hertz, Bioindicators for monitoring heavy metals in the environment, in: "Metals and Their Compounds
in the Environment", E. Merian, ed., VCH, New York (1991).
4. G. Tyler, Bryophytes and heavy metals: a literature review, Bot. J. Linnean Soc., 104:231 (1990).
5. D.H.S. Richardson, Air pollution, in: "The Biology of Mosses", Blackwell Scientific Publications,
London (1981).
6. R.S. Clymo, Ion exchange in Sphagnum and its relation to bog ecology, Ann. Bot. N.S., 27:309 (1963).
7. A. Ruhling, and G. Tyler, Sorption and retention of heavy metals in the woodland moss Hylocomium
splendens (Hedw.) Br. et Sch., Oikos, 21:92 (1970).
8. P. Little and H. Martin, Biological monitoring of heavy metal pollution, Environ. Pol/Jlt., 6:1 (1974).
9. A. Makinen, Sphagnum moss-bags in air pollution monitoring in the city of Helsinki, Symp. Biol.
Hungarica, 35:755 (1987).
10. V. Hynninen, Monitoring of airborne metal pollution with moss bags near an industrial source at
Harjavalta, southwest Finland, Ann. Bot. Fenn., 23:83 (1986).
11. P. Pakarinen, and K. Tolonen, Distribution of lead in Sphagnum fuscum profiles in Finland, Oikos,
28:69 (1977).
12. P. Pakarinen, Distribution of heavy metals in the Sphagnum layer of bog hummocks and hollows, Ann.
Bot. Fennici, 15:287 (1978).
13.N. Malmer, Patterns in the growth and the accumulation of inorganie constituents in the Sphagnum cover
on ombrotrophie bogs in Scandinavia, Oikos, 53:105 (1988).
14. M. Kirchhoff,and H. Rudolph, A sandwieh technique for the continuous monitoring of air pollutants with
the bryophyte Sphagnum, J. Hattori Bot. Lab., 67:423 (1989).
15. P. Ferguson, R.N. Robinson, M.C.Press, and J.A. Lee, Element concentrations in five Sphagnum
species in relation to atrnospheric pollution, J. Bryol., 13:107 (1984).
16. K.E. Percy, Heavy metal and sulphur concentrations in Sphagnum magellanicum brid. in the maritime
provinces, Canada, Water, Air, Soil Pollut.,19:341 (1983).
17. MJ. Moura, M.T.Vasconcelos, S. Sousa and A. Machado, Lead and other heavy metals in atrnospheric
aerosols ofOporto, Chemosphere, 17:2093 (1988).
18. M.T. Vasconcelos, M.S. Barbosa, P.A. Silva, and A. Machado, Influence of the welding parameters on
the pollutant levels at the breathing zone of welders working in a metallurgie welding plant, First
Internaional Scientific Conference of International Occupational Hygiene Association, Belgium, 1992
(communication accepted).
19. H. Ferreira, A. Seneca, C. Sergio and M.T. Vasconcelos, Integra~ao de indicadores biol6gicos na
monitoriza~o de churnbo na atrnosfera urbana do Porto-primeiros resultados, in Proccedings of "3'
Conferencia Nacional sobre a Qualidade do Ar", A.R.Pires, C. Pio, C. Boia and 1". Nogueira, eds.,
Universidade de Aveiro, Aveiro (1992).
20. M.J. Moura, M.T.Vasconcelos and A. Machado, Determination oflead in atrnospheric aerosols by
electrothermal atomisation atomic absorption spectrometry with direct introdution of filter in the
graphite furnace, J. Anal. At. Spectrom, 2:451 (1987).
21. H.M. Tavares, M. T. Vasconcelos and A. Machado, Aplication of hydride generation - atomic absorption
spectrometry to mechanical and biological monitoring of lead in urban atrnosphere, in preparation
(1992).
22. K. Pilegaard, Heavy metals in bulk precipitation and transplanted Hypogymnia physodes and
Dicranoweisia cirrata in the vicinity of a danish steelworks, Water, Air, Soil Pollut., 11:77 (1979).

72
 EPIDEMIOLOGIC APPROACH FOR THE ASSESSMENT OF ACCEPTABLE
EXPOSURE LEVELS TO CADMIUM AND MANGANESE

Robert R. Lauwerys, A. Bernard, H. Roels, and J.P. Buchet

Industrial Toxicology and Occupational Medicine Unit

Catholic University of Louvain
30.54. Clos Chapelle-aux-Champs
1200 Brussels, Belgium

INTRODUCTION

This paper deals with the problem of health risk assessment of long term
exposure to chemieals with special emphasis on two inorganie pollutants :
cadmium and manganese and illustrates the use of biologieal markers for assessing
the no-effect levels of these pollutants.

Although it is a diehe to state that experimental and epidemiologie studies

constitute two complementary approaches to assess the risk resulting from long
term exposure to chemicals, it may be relevant, as an introduction, to briefly
recall the main contribution of both disciplines for health risk assessment. For
newly developed chemicals, an experimental approach is the only one feasible.
The main outcome of experimental studies is the characterization of the pattern
of toxic effects induced by chemieals, Le. the identification of the main target
organs or functions, the understanding of the relationship between their metabolic
handling and interactions with target molecules and the identification of their
critical effects and the most relevant markers to assess their biologically active
internal doses. Unfortunately, extrapolation of dose-effect(s) relationships from
animals to humans is highly questionable, mainly with regard to systemic effects,
hence the practiee to apply arbitrary uncertainty factors to the dos es without
observed adverse effects in animals.
For chemieals or groups of chemieals already present in the environment, an
epidemiologie approach can be used which offers the advantage to establish the
dose-effect/dose-response relationships directly in humans. This approach,
however, is fraught with methodological difficulties, such as an approximate
assessment of the intensity of human exposure to environmental agents and the
limited sensitivity of the tools used to monitor health effects.

Use 0/ Biomarkers in Assessing Health anti Enllironmenlal Impacts 0/ Chemical

Pollutants, Edited by C.C. 'Ihlvis, Plenum Press, New York, 1993 73
 Indeed, as illustrated in Figure 1 the study in humans of dose-effeet(s) or dose-
response relationships with the aim to identify acceptable exposure levels
(threshold limit values - TLV - or biologieallimit values - BLV -) can be carried
out with the aid of different indieators of exposure or health impairment l
Numerous epidemiologie studies on the health effeets of environmental and
industrial pollutants have focused on the relationships between various external
indieators of pollution (e.g. presence of emission sources, ambient monitoring . )
and morbidity (clinieal signs) and/or mortality data. This type of studies suffers
from many limitations. Current and/or past exposure to pollutants is usually
estimated on a group basis. Furthermore diagnosed clinieal entities are often late
manifestations of pathophysiologieal processes, the evolution of whieh may have
been influenced by numerous exogenous and endogenous faetors. With such an
approach, environmental and/or industrial pollutants can only be identified as
etiologic agents of pathological eonditions when their role is predominant in
eomparison with that of other factors (e.g. lung cancer in asbestos exposed
workers) or when the time interval between exposure and diseases (Iatency period)
Is not too long (e.g. lung fibrosis in hard metal workers). But in general, exposure
to environmental or industrial chemicals is rarely of sueh intensity that their
clinieal effects become rapidly apparent. Furthermore, since many diseases are
multifactorial, the contributory role of environmental or occupational factors may
be easily overlooked with an epidemiologie approach based on a crude assessment
of exposure and a search for late effects (diseases, eauses of death).

Exposure Effects

Qualitative characterization Death

[ job classification

Metabolism
Ouestionnaire
Ambient +concentration
1
Diseases

.
Mechanism
of
Xenobiotics
+
Personal monitoring t of action of
Xenobiotics
Funetional
(. ehanges
Absorbed amount Internal dose)
(Active +metabolite)
1
Target +
dose Critical
Biologie effects

J
Relationship
Exposure Health
Intensity Risk
(Oose) (Effects response)

Acceptable exposure levels ITLV. BLV)

Figure 1. Exposure and health parameters used for dose-effect/dose-response
relationshi ps

74
 These difficulties can partly be overcome if the assessment of exposure is
carried out on an individual basis (e.g. by personal environmental monitoring
techniques or better by biological monitoring methods) and if early biological
effects are selected as health indicators. In summary, health risk assessment is
best performed by epidemiologie studies in which the individual dose (and ideally
the target dose) and the critical biological changes are monitored with sensitive
and specific markers. But the use of such markers of exposure and effects
requires the knowledge of the fate of the chemicaI(s) in the organism and its
mechanism of action (or its critical target organ(s)). These are the fields in which
experimental studies are most relevant.
So far, an epidemiologie approach relying on the measurement of the target dose
and/or the critieal biologieal changes has only been used for a limited number of
pollutants.
The assessment of the critical exposure level of the nephrotoxic chemical
cadmium illustrates this approach. Manganese is another example but for wh ich
the background information on metabolism and early biologieal effects is less
extensive than for Cd.

CADMIUM

Cadmium (Cd) is a very cumulative toxic chemieal. A biologieal indieator,

i.e. Cd in urine, is available to estimate its concentration in the main critical
organ, the kidney, and biologieal markers can also be used to detect the early
toxie effects in the kidney2. Although the proximal renal tubule is the main site
of Cd action, it is now reeognized that other sites of the nephron mayaiso be
affeeted such as the glomerulus, the loop of Henle, the distal tubule and possibly
the interstitium.

More than 15 years ago, we performed several cross sectional

epidemiologie studies on Cd workers to study the relationship between the
prevalence of renal tubular dysfunetion and Cd coneentration in urine, an indirect
indieator of Cd body burden.
As illustrated on figure 2 for 2-microglobulin ( 2-m) in urine, it was found that
tubular dysfunction (Le. an increased urinary excretion of 2-m) occurs when Cd
in urine exceeds 10 Ilg/g creatinine, corresponding to a Cd concentration in renal
cortex of 200 ppm 3.

In the framework of a collaborative EEC projeet, we have reeently

reexamined a eohort of workers exposed to Cd and a matched eontrol group for
the presenee of early renal changes 4. As listed in Table 1, a large battery of
biologieal markers was used in order to deteet changes at different renal sites
(glomerulus, proximal tubule, loop of Henle, distal tubule, and interstitium).
A multivariate eorrelation analysis revealed statistically significant association
between the Cd eoncentration in urine and the urinary output of albumin,
transferrin, 2-m, NAG, tubular antigens (BB50, BBA, HF, IAP), THG, 6-keto-
PGF l' PGE 2 and GAG and the eoneentration of 2-m in serum. The application
of a logistic regression model revealed that the urinary exeretion of albumin,
NAG, GAG, BBA and THG and the serum level of 2-m already tended to inerease

75
 from thresholds of urinary Cd less than 10 Ilg/g creatinine (Figure 3). In fact,
three main groups of thresholds for urinary Cd could be identified. A threshbld
around 2 Ilg Cd/g creatinine mainly associated with biochemical alterations
(prostagiandin 6-keto-PGF lll and sialic acid), one around 4 Ilg Cd/g creatinine for
the increased excretion of the renal tubular antigen BBA, the enzymes NAG and
IAP, and the high molecular weight proteins, albumin and transferrin, and another
threshold around 10 Ilg Cd/g creatinine for the increased excretion of TNAP, the
brush bord er antigen HF5, the low molecular weight proteins RBP and 2-m and
GAG. Serum 2-m and urinary THG had intermediate thresholds of 6 and 7 Ilg
Cd/g creatinine, respectively.

1 68
1
62
1
13S
1
6S
1
61S
1

-=C
Q.I
L-
u 0.4
=
~ 1
E 11 1- 1 - - ..1 -1- - -.l - 1 - ' - ------
::::> 1 1 2 1 11 1 1 1
c:
I 0.1 1
1 1
1 1 1
1 21
11 1
:; 1 ~1 11 1 1 1 1 11 11
1 111 1 1
1
1 11
1 1
L
.0
0 2 1
C,
0
<-
1 f1 1 1
1 1 1
21 1 1" 1 1 1
.!: 0.025 1 1 1 111 1
:E 1
I 1
N 11
<=
1 1
2.5 5 10 20 40 80
Cd - U (~g/g (reat.)
Figure 2. Relationship between urinary excretion of Cd and B2 -m

Which of these thresholds must be adopted as maximum permissible value

for occupational exposure to Cd depends on the health significance of the
observed renal effects, Le. whether they are predictive or not of a progressive
decline of the renal function. For low molecular weight proteinuria, for wh ich the
threshold seems to be around 10 Ilg Cd/g creatinine, it is now weil established
that their increased urinary excretion is irreversibleS. We have also found that the
age related dec1ine in the glomerular filtrate rate (GFR) is 5 times greater in
subjects with microproteinuria than in age-matched control subjects 6. However
the peak GFR after an oral protein-Ioad which reflects the filtration reserve
capacity of the kidney was not different between Cd exposed workers and control

76
 Table 1. Biological markers of renal effeets

Glomerulus

High moleeular weight plasma proteins (Mr > 40000) in urine

Albumin
Transferrin
IgG
Components of glomerular strueture In urine/plasma
Fibroneetin
Laminin
Si alle acids
Creatinine and 2-mieroglobulin ( 2-m) in serum
Anti-GBM antibodies in plasma
Red blood cell membrane negativecharges

Proximal tubule

Low molecular weight plasma pro teins in urine

2-:m
Retinol binding protein (RBP)
Alphal-microglobulin
Protein 1
Enzymes and antigens
N-acetyl--D-glucosaminidase (NAG)
Tissue nonspecific alkaline phosphat ase (TNAP)
Intestinal alkaline phosphatase (IAP)
Brush border antigens : BB 50 BBA, HF 5
Aminoacids (AA)
Glucose
Calcium (Ca)

Loop of Henle and distal tubule

Thamm-Horsfall glycoprotein (THG) in urine

Distal tubule

Kallikrein in urine

Various sites

Eicosanoids (6-keto-PGF lalpha. PGE 2 PGF 2alpha. TXB 2) in urine

Glyeosaminoglyeans (GAG) in urine

77
 0.6

l-m (serum)
0.5
Transfenin

0.4 Albumin
l-m

0.3 RBP

0.2

0.1

0.0
1 2 5 10 20
O.B

UI 0.7
CI>
:::I BBA
IV 0.6 IAP
> NAG

~0 0.5

-
c:: 0.4 TNAP
.J:2
ra HF5
0 0.3
>.
~
:ci 0.2
ra
e
.J:2
0.1
Il.
0.0
1 2 5 10 20
O.B

0.7 6-keto-PGF 1 ce

0.6
Sialic acid
0.5
THG
0.4 GAG

0.3

0.2

0.1

2 5 10 20
Cadmium in urine ( l19/g creatinine )

Figure 3. Probability of renal effects as a function of urinary Cd concentration (see Table

1 for the definition of the abbreviations)

78
as long as Cd indueed mieroproteinurla had not yet oeeurred7 . This finding ean
be interpreted as evidenee that the threshold of 10 ~g Cd/g ereatinine affords an
adequate proteetion. Renal effeets whieh were found to oeeur from a threshold
of urinary Cd lower than 10 ~g Cd/g ereatinine eonsist mostly in an inereased
leakage in urine of tubular antigens or enzymes and for most of these effeets, a
link with the subsequent development of renal insufficieney is not established.

A more stringent guideline, however, may be justified for the general

population. Until the renal ehanges indueed by cadmium have not been c1early
proven to be without long-term health consequences, it seems preferable to
prevent their occurrence in the general population. Furthermore, we have
reeently performed a large seale cross seetional epidemiologie study among more
than 2000 subjeets non-occupationally exposed to Cd and living in different urban
or rural areas of Belgium with different degrees of environmental pollution by Cd.
This study was ealled Cadmibel and was carried out in collaboration with 3 other
Belgian research teams 8. After exc1usion of subjects who had been oecupationally
exposed to heavy metals (Cd, zine, lead, mereury), those aged under 20 or 80
years, those who provided nonreliable information on smoking habits or
oeeupational exposure to heavy metals, and those whose 24-h urine eolleetion
were not eonsidered reliable, 1699 subjeets remained for the final statistical
analysis. A multivariate eorrelation analysis revealed that several urinary markers
(i.e. 2-m, RBP, NAG, AA, Ca) were signifieantly associated with cadmium level
in urine 9. A statistically signifieant assoeiation was found between cadmium level
in urine and the prevalence of elevated values of Z-m, RBP, NAG, AA and Ca
after standardization for the other predictors. A logistic regression model was
used to assess further the dose-response relationships between the cadmium
excretion and the renal effects. The probability that individual subjects, after
adjustment for the significant covariates, would have abnormal values of the renal
variables was related to the urinary cadmium excretion (Figure 4).
For 2-m excretion it was estimated that the probability of abnormal values was
about 10 % when cadmium in urine reached 3.1 J.1g/24h. The corresponding values
of urinary Cd (~g/24h) for the other renal markers were 2.9 for RBP, 2.7 for
NAG, 4.3 for AA and 1.9 for Ca. The results of this epidemiologie study led us to
conclude that environmental exposure to Cd in certain areas of Belgium may
induce slight renal tubular dysfunction and may probably also affect calcium
homeostasis. The probability of occurrenee of tubular dysfunction significantly
exceeds the background level when Cd in urine reaches 2 ~g/g creat. This value
should be regarded as the maximum tolerable internal dose of Cd for the general
population. This level corresponds to a renal cortex concentration of about 50
ppm. In view of our knowledge, on the toxicokinetics of Cd, it can be estimated
that in non-smokers, this level is attained after 50 years of an oral daily intake
of about 1 ~g Cd/kg body weight.

MANGANESE

In industry, workers are mainly exposed to manganese (Mn) containing dust

wh ich may be toxic for the respiratory tract and the central nervous system
(CNS) wh ich is considered as the critical organ. Indeed, following long-term
exposure, Mn may induee neurobehavioural symptoms and neurologie signs
charaeteristic of an extrapyramidal syndrome which has several similarities to
Parkinson's disease lO .

79
 For this metal, the situation is less favourable than for Cd since no
biochemical markers have yet been identified to assess the target dose and the
toxic effects after long term exposure. Since very efficient horneostatic
mechanisms prevent large f1uctuations of Mn concentration in whole blood and
since Mn is rnainly excreted by the biliary route, it has not yet been possible to
identify a biological marker to assess the intensity of exposure or the
concentration in the target organ. In industry, evaluation of individual exposure
to Mn is thus best carried out by monitoring its concentration in total (inspirable)
and respirable dust in the breathing zone of the workers. Furthermore, no
biochemical indicator is available for the detection of the early neurotoxic
effects of Mn. Presently, neurofunctional examination (e.g. the measurement of
visual reaction time, eye-hand coordination, hand steadiness) represents the most
sensitive approach for this purpose.

0.5
RETINOL BINDING PROTEIN 5
l::J 2 N-ACETYL- - GLUCOSAMINIDASE
:z
es
LU-J
0.4 3 2 MICROGLDBULIN
LU LU 4 AMINOACIDS
xw>
LU
5 CALCIURIA
LU-J
Vl O
0.3
~-,
-J 0
~Bj
u.. LU
00:
:x:: 0.2
>-I-
!=LU
::!:x::
co I-
~
0
0.1
0:
0..

0.0
005 0.1 0.2 05 1 2 5
Cd IN URINE ().Jg/24hl
Figure 4. Probability of renal dysfunction assessed by five urinary variables as a function
of urinary Cd excretion

These indicators of exposure and toxic effects were monitored in two

groups of workers exposed to Mn containing dust in a Mn salt producin~ plant and
a dry alkaline battery factory and in their matched control groups 11, 1 . Study of
the dose-response relationships indicated that the prevalence of same
neurofunctional disturbances was related to the integrated exposure to airborne
Mn (total and respirable dust).
A logistic regression analysis of the da ta also showed that tremor (hand
steadiness) was the most appropriate parameter to define a threshold effect level
(Figure 5).

This analysis sug~ested that a lifetime integrated exposure to Mn dust (as

Mn02) above 3575 j.1g/m .year (total dust) or 730 j.1g Mn/rn 3. year (respirable dust)

80
 040

0.35
~
030
E
L..
2
D
d
Q25 Respirable
VI

'"
.~ Q20
u
~
~ 015
u
c
d
.J::.

'15 010
~
: 0.05
d
D
0
C-
a.. O
25 50 100 200 400 800 1600 3200 6400 12800 25600
Lifetime integrated exposure to airborne Mn dust 1}J9 Mn/m 3 xyear I
Figure 5. Probability of abnormal hand steadiness as a function of lifetime integrated
exposure to respirable or total airborne Mn dust

may lead to an increased risk of tremor. It can therefore be concluded that for
a professional life of 40 years, the current occupational exposure limit for Mn
(total dust: 5 mg/m 3) is too high and should be reduced by about 60 fold in order
to protect the majority of workers from the neurotoxicity of Mn. Unfortunately,
for the reasons just explained, the risk of overexposure to Mn cannot yet be
defined at the individual level through biological monitoring methods and the
health surveillance of the workers must still rely on the search for functional
cbanges wbich, when present, probably reflect an important accumulation of the
metal in the central nervous system.

CONCLUSION

These studies highlight the fact that health risk assessment is best
performed by epidemiologie studies in which the individual dose (and ideally the
target dose) and the critical biological changes are monitored with sensitive and
specific markers. But usually, this is only feasible if fundamental work on the
metabolism and the mechanism of action of chemicals has first be carried out.

REFERENCES

1. R. Lauwerys, Occupational toxicology. Chapter 29 in "Casarett and Doull's

Toxicology : The Basic Science of Poisons" (C.D. Klaassen, M.O. Amdur,
J. Doull, ed, Fourth Edition, Macmillan, New York (1990).
2. A. Bernard, and R. Lauwerys, Effects of cadmium exposure in humans,
Chapter V in "Handbook of Experimental Pharmacology", E.C. Foulkes, ed.,
Springer Verlag, (1986).

81
 3. ) .P. Buchet, H. Roels, A. Bernard, and R. Lauwerys, Assessment of renal
function of workers exposed to inorganic lead, cadmium or mercury
vapour. ). Occup. Med. 22:741 (1980).
4. H. Roels, A.M. Bernard, A. Cardenas, ).P. Buchet, R.R. Lauwerys, G. Hotter,
I. Ramis, A. Mutti, 1. Franchini, 1. Bundschuh, H. Stolte, M.E. De Broe,
G.D. Nuyts, S.A. Taylor, and R.G. Price, Markers of early renal changes
induced by industrial pollutants. III. Application to workers exposed to
cadmium, Br. ). Ind. Med. in press.
5. H. Roels, ). Djubgang, ) .P. Buchet, A. Bernard, and R. Lauwerys,
Evolution of cadmium-induced renal dysfunction in workers removed from
exposure. Scand. ). Work Environ. Health 8:191 (1982).
6. H.A. Roels, R.R. Lauwerys, ).P. Buchet, A.M. Bernard, A. Vos, and
M. Oversteyns, Health significance of cadmium-induced renal dysfunction :
a five-year follow-up. Brit.). Ind. Med. 46:755 (1989).
7. H.A. Roels, R.R. Lauwerys, A.M. Bernard, ).P. Buchet, A. Vos, and
M. Oversteyns, Assessment of the filtration reserve capacity of the kidney
in workers exposed to cadmium. Brit.). Ind. Med. 48:365 (1991).
8. R. Lauwerys, A. Amery, A. Bernard, P. Bruaux, ).P. Buchet, F. CIaeys,
P. De Plaen, G. Ducoffre, R. Fagard, P. Lijnen, L. Nick, H. Roels,
D. Rondia, A. Saint-Remy, F. Sartor, ). Staessen, Health effects of
environmental exposure to cadmium. Objectives, design and organization
of the Cadmibel study : a cross-sectional morbidity study carried out in
Belgium from 1985 to 1989. Environmental Health Perspectives 87:283
(1990).
9. J.P. Buchet, R. Lauwerys, H. Roels, A. Bernard, P. Bruaux, F. Claeys,
G. Ducoffre, P. De Plaen, J. Staessen, A. Amery, P. Lijnen, L. Thijs,
D. Rondia, F. Sartor, A. Saint-Remy, L. Nick, Renal effects of cadmium
body burden of the general population. The Lancet 336:699 (1990).
10. R. Lauwerys, Manganese - Editions techniques - Encycl. Med. Chir. (Paris,
France), Toxicologie - Pathologie Professionnelle, 16003 A 30 , 5 p. (1992).
11. H. Roels, R. Lauwerys, J.P. Buchet, P. Genet, M.). Sarhan, I. Hanotiau,
M. de Fays, A. Bernard, D. Stanescu, Epidemiological survey among
workers exposed to manganese : effects on lung, central nervous system
and some biological indices. Am. J. Ind. Med. 11 :307 (I987).
12. H.A. Roels, P. Ghyselen, J.P. Buchet, E. Ceulemans, and R. Lauwerys,
Assessment of the permissible exposure level to manganese in workers
exposed to manganese dioxide dust. Br. J. Ind. Med. 49:25 (1992).

82
BIOLOGICAL MONITORING OF EXPOSURE TO ORGANIC
COMPOUNDS
Marek Jakubowski
Institute of Occupational Medicine
Sw. Teresy 8, Lodz, Poland

INTRODUCTION

In 1980 the participants of a seminar organized by CEC, NIOSH and OSHA (Berlin
et al.,1984) agreed upon the following definitions:
1. Monitoring (in preventive health care) is "a systematic continuous or repetitive health
related activity, designed to lead if neeessary to correetive action" .
2. Biological monitoring (BM) is "the measurement and assessment of workplace agents
or their metabolites either in tissues, seereta excreta or any combination of these to
evaluate exposure and health risk compared to an appropriate reference" .
In reeent years there has been a rapid development of methods to assess early,
possibly reversible biological effeets. In the past the determination of biological effects
was inc1uded in the biological monitoring. In 1986 Zielhuis and Henderson proposed
additional definition:
3. Biological effeet monitoring (BEM) "the measurement and assessment of early
biological effeets, of which the relationship to health impairment has not yet been
established in exposed workers to evaluate exposure and/or health risk compared to an
appropriate reference".
This paper concems the area of biological monitoring (BM). BM may be speeific for
speeified agent or is some cases groups of related agents. The example can be the
determination of I-hydroxypyrene in exposure to polycyc1ic aromatic hydrocarbons
(Jongeneelen et al. , 1988). BM primarily serves for assessing whether actual or previous
overexposure took place and, consequently, for assessing an increased health risk. In both
the occupational and environmental toxicology acceptable limits for the level of chemicals
have been set on assumption that there is an unacceptable risk at levels below the limit
values and at levels significantlyexceeding the limit values, the risk may be high enough
to justify action. Studies in industrial toxicology demonstrated that neither of these
assumptions was entirely correet. Examples were found where sufficiently low
concentrations of chemicals in the air of working premises did not seeure the health of
workers if chemicals could be absorbed through the skin (for example pesticides, aniline,
benzidine) or gastrointestinal tract (lead, cadmium). On the other hand, high concentration
in the air of working premises did not neeessarily increase the risk, especially if personal
proteetion had been instituted satisfactorily.
It has beeome c1ear that the relevant factor to be monitored is the actual exposure,
quantified in terms of doses absorbed daily through various routes, and possibly from
various sources.Biological monitoring of chemicals was at first developed in the field of

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemical

Pollutanls. Edited by C.C. Travis. Plenum Press. New York. 1993 83
industrial toxicology as a method for estimating exposure of individuals. BM encountered
the problem of estimating integrated exposure when environmental monitoring is not
sufficient because of the influence in the case of organic solvents of such parameters as:
alternative absorption through the skin, use of protective devices and their efficiency,
work load and personal working habits.
The need for estimating integrated exposure to chemicals has resulted in a high
demand for biological monitoring methods. In response to this demand the number of
chemicals subject to biologica1 monitoring has increased and so have the difficulties in
interpreting the results.
Tbe aim of this paper is to present some factors which can influence both the
development of the methods for biologica1 monitoring of exposure to organic solvents as
weIl as the interpretation of obtained results.

BIOKINETIC PROPERTIES OFCHEMICALS AND POSSmILITY OFEXPOSURE

ESTIMA TION.

The application and interpretation of biological moni- toring depend on the

toxicokinetic properties of the substance. Generally the folllowing possibilities can be
distinguished .

Evaluation of the Rate of Absorption. The half-life of the chemica1 in the given
compartment of the body is so short that the concentration in biological media reflects the
actual exposure. An example can be the concentration of volatile solvents in blood and
exhaled air in sampIes collected during exposure.

Evaluation of the Daily Exposure. The half-life of the chemical in the body is
sufficiently short and biologica1 level reflects the dose absorbed on a given day. In this
case, biological monitoring is useful in industrial toxicology even if the measured levels
fluctuate largely from day to day. Time of sampie collection is essential. The use of
biological monitoring in environments other than industry, where the source of exposure
has not been identified could give misleading results. Toluene, xylenes, phenol belong to
this group.

Evaluation ofthe Cumulated Exposure. Tbe half life of chemical is very long, allowing
substantial accumulation of the substances. Here, the fluctuations of levels from day to
day and within Olle day are small and, therefore, the exact location of the source of
exposure is not necessary. For this reason the biological monitoring can also be used for
exposure estimation in the general environment mainly for persistant chemicals like PCBs
with half-life of about 2-6 years (phillips et al., 1989).
Most chemica1s do not belong to either of these classes, having neither very short nor
very,long half-lives. For the substances such as nitrobenzene, trichloroethylene or tetra-
chloroethylene, biologicallevels depend both on the actual exposure during the day and
on the past exposure over the last week or so.
This situation is common in the case of elimination of organic solvents from the fat
tissue. None of the situations is usually clear enough to allow proper interpretation of the
data without adequate knowledge on the biokinetic properties of the substances.

EXISTING REFERENCE VALUES

There is a number of comprehensive reports in which interpretative data on the

application of biologica1 monitoring for the evaluation of organic compounds absorption

84
in the organism are presented. These reports comprise both a wide range of information
and discussion on toxicokinetics and possibilities arising in the field of BM, such as e.g.
the works published by the Commission of the European Communities (CEC, 1983),
NIOSH (Piotrowski, 1977), WHO (1981, 1982) as weIl as tabular data conceming the
recommended Biological Exposure Indices in different countries e.g. ACGIH (1991-92),
DFG (1990).
There are data for above 100 compounds and only for a part of them metabolie studies
in humans are availabie. The older tests were mainly based on the analysis of metabolites
in urine, whereas recent procedures often recommend the analysis of blood and expired
air and the unchanged substances are usually determined.
The reference values for occupationally exposed workers represent values regarded as
acceptable under working conditions. These reference values may be derived by two main
approaches:

Health-Based Reference Values. These are defined as levels in biological mlf,terial which
do not rise to any detectable adverse toxic effects. They are based on exposure-effect and
exposure-response relationships and do not consider technological or economic feasibility.
Such values are difficult to obtain and only a few have been proposed for organic
compounds. The WHO (1981) has recommended such values for trichloroethylene,
xylene and some pesticides. As for toluene, the opinions were devided.

Administrative Standards. Most administrative biologie reference values are indirectly

derived from workroom hygienic standards, such as a threshold limit values. This
approach is much easier than the previous one and according to Schulte et al. (1987)
during the period of 1981-1985 74% of the biological monitoring studies evaluated the
relationship between the level of environmental exposure and biological level of the
intoxicant or metabolite.
In majority of the studies the dependence between time-weighted average
concentration in the air and the concentration of the unchanged compound or its metabolite
in biological material were adopted. Other groups of authors considered adopting the
absorbed dose of the compound as the independent variable, to be more appropriate. In
this case the admissible dose is calculated from the formula:

(1)

in which: D = the absorbed dose, C = TLV value, T = duration of daily exposure, R

= fraction of substance retained in the respiratory tract, V = lung ventilation (may be
assumed as 1 m3/h at the 25 W workload).
In the case of the chemieals which can be absorbed through the skin in the form of
vapours, the admissible dose is calculated from the formula:

D =CT (R V + oe) (2)

where: oe = skin absorption coefficient expressed in volume of air cleared of given

compound per unit of time (e.g. in the case of phenol oe = 0.35 m3 /h) (Piotrowski,
1977).

PRECISION OF EVALUATION OF EXPOSURE TO ORGANIC SOLVENTS

AND PROBLEMS IN INTERPRETATION OF RESULTS

The precision with which the absorption of organie compounds can be assessed by
means of biological monitoring is debatable and depends on the conditions under which

85
 the studies are performed. Determinations of the dependence between exposure magnitude
and the concentration of the compound or its metabolite in biological material which are
performed under industrial conditions include apriori errors of exposure assessment.
Important data on the precision of exposure estimation come from the experimental
studies in human volunteers. Disperision of results for various individuals may be more
apparent when individual variations in retention and ventilation rate are not considered.
An example of the influence of different parameters on the precision of ~valuation of
exposure can be for xylenes the data published by Sedivec and Flek (1976) (Fig. 1).

(!) C C <D Ci) @ (!)

2:
'"E
";'" 2:~

~
.
~
0
u ;g
~ r
16
2'
2!
c;.
E
1c;.
E
16
120

12
12

80

1000

400 1000 40

CONCENTRATION OF XYLENE IN AIR

Figure 1. Comparison of dispersion of methods expressing excretion of metabolites. Regression line marked
by a thick, fullline, 90 % confidence limits by thin lines. All-shift urine: mg/I = amount of metabolite (in
mg) in 11 urine; mg/I corr. = amount ofmetabolite in 1 I ofurine of standard density 1.024 g/ml; mg/creat
= amount of metabolite recalculated to 1 g of excreted creatinine; mg = amount of metabolite excreted
within a given time; mg/weight = amount of metabolite excreted within a given time and recalculated to
1 kg body weight of examined subject; mg/height - 100 = amount of metabolite excreted within a given time
and recalculated to 1 kg "ideal" weight of examined subject; mg/ventilation = amount of metabolite excreted
within a given time and recalculated to unit lung ventilation (i.e., divided by minute lung ventilation in I).

If the absorbed doses are taken as the independent variable instead of concentrations,
a weIl controlled test can, as a rule,be characterized by a precision of about 20%.
Extreme cases as the exposure test for phenol (precision 10%) or nitrophenol (precision
30%) resulted from very simple metabolism and high excretion rate or complex
metabolism and acummulation in the adipose tissue. A usually attainable precision
of 20 % seems satisfactory as no other method can evaluate the uptake of toxic substances
with comparable results.
When "spot" sampIes are collected during the last hours of the workshift or the
strictly determined fraction of urine from the last two hours, the assessment of absorbed
dose in the case of non-uniform exposure (for instance mainly in the first or the last hour
of exposure) may be considerably different from observed in the controlled experiments.
This refers mainly to the compounds with short half-time of excretion. This problem was
discussed by Sedivec and Flek (1976). In order to improve the reliability of evaluation of
exposure these authors recommended basing exposure tests on the determinations made
in urine sampIes collected during the whole shift.Dispersion of results obtained by various
authors and their generalization without taking into consideration all contributions are very
important issues in the determination of biological exposure indices. Assessment of

86
exposure to benzene is a characteristic example from this field.
A number of studies aiming at the evaluation of a relationship between phenol
excretion in urine and the magnitude of occupational or experimental exposure to benzene
were carried out. Their results have been generalized in two studies by Lauwerys
(C.E.C., 1983) and Piotrowski (1977). It should be noted that recommendations in both
these papers differ significantly.
Piotrowski adopted as a basis the obtained by Dutkiewicz (1963) dependence between
phenol concentration in urine collected between the sixth and eighth hour of exposure and

300

100

300 400 500 Dose (mg)

25 Benzene in air( ppm)
Figure 2. The concentration of phenol in urine samples collected at the end of exposure as dependent on
the concentration of benzene in the air or absorbed dose of benzene. Convertions between the dose and
concentration of benzene in the air were made assuming mean lung ventilation of 1 ni'/h and retention 0.7.
Regression curves: 1 - Dutkiewicz (1963), 2 - Lauwerys (1983), 3 - WalkIey et al. (1961), 4 - Rush and
Ott (1977).

benzene dose determined under strictly controlled experimental conditions. The

dependence is determined by the equation:

x == 1.96 Y (3)

where: x == absorbed dose of benzene (mg), y == phenol concentration in urine (mg/I)

The data presented in Fig. 2 indicate the consistency of results obtained by Dutkiewicz
(1963) and Walkley et al.(1961). The results obtained by Roush and Ott (1977) under
industrial conditions in workers exposed to low benzene concentrations of 2 - 13 mg/m3
also approximate those obtained by Dutkiewicz.
Lauwerys (1983), on the other hand, based his conclusions on the Parkinson's (1975),
Sherwood's (1972) and Rainsford and Davies' (1965) reports disregarding the results
mentioned above. He proposed the following relationship as a result of generalization of
these data:

y == 20 + 0.33 x (4)

87
 where: y = phenol concentration in urine collected before cessation of exposure
x = the index of integrated exposure (benzene concentration in ppm multiplied
by duration of exposure in hours)

After recalculating ppm into mg/m3 and assesing that lung ventilation during light
work amounts to 1 m3/h, and benzene retention in lungs is 70%, the formula (4) has the
following form:

y = 20 + 0.143 x (5)

where: y = phenol concentration in urine (mg/I)

x = absorbed dose of benzene (mg)

As results from Fig. 2 these two sets of data differ considerably and the concentration
of phenol in urine, corresponding to concentration of benzene in the air of about 10 ppm,
amounts, according to Lauwerys, to about 50 mg/l and according to the results adopted
by Piotrowski, to about 100 mg/I.
The data included in WHO materials (WHO 1981) are an example of the proposal of
admissible concentration in biological material when the impact of physical effort is
disregarded. The Study Group recommended as the health-based occupational exposure
limit the concentration of xylene in the air of 215 mg/m3 The Group concluded from the
data reported by Ogata et al. (1970) and Sedivec and Flek (1976) that 8-h exposure to 200
mg of xylene/m3 corresponds to a methylhippuric acid concentration in urine of about 1,4
g/1 on the basis of the sampies collected from groups of workers at the end of a workshift,
corrected to a specific density of l.024. When adopting the data obtained by Sedivec and
Flek (1976) and Ogata et al. (1970) as a basis for a health-based limit for methylhippuric
acid in urine the fact that they refer to persons who were exposed at rest (lung ventilation
of about 9 11min.) was disregarded. During a light work (workload of about 25 W), which
may be taken as anormal state to which TLV corresponds, lung ventilation and thereby
the absorption increases about two times. This indicates that the value of the recommended
health-based limit of methylhippuric acid concentration in urine should amount rather to
about 2.8 g/l than to the proposed l.4 g/l. The value of l.4 gIg creat. has been also
adopted in ACGIH (1992).
These two examples show that the difficulties in determining biological exposure
indices for organic compounds metabolites in urine may occur even for the apparently
weIl tested compounds. These difficulties result chiefly from the lack of a uniform
protocol for carrying out investigations of that kind covering both the study model and the
methods used for determining the tested compounds both in the air and in biological
material. It seems that the best solution in the experimental studies in volunteers could be
adopting the absorbed dose as the independent variable. It would allow to avoiding many
misunderstandings as e.g. in the case of xylene. It would be also advisable to adopt a
uniform means of expressing the excretion of the compounds in urine. The existing
diversity (rate of excretion, concentrations corrected to specific density or creatinine
concentration) makes the comparison of results obtained by various authors difficult or
even impossible. As an example, the available data on methylhippuric acid excretion in
urine resulting from experimental and industrial exposure to xylene are presented in
Tablel.
It seems that there are numerous misunderstandings with regard to the application of
determinations of unchanged forms of organic solvents in blood for the assessment of
exposure. For instance when comparing the concentrations of toluene in blood sampies
collected at the end of the shift, with the mean concentrations of solvent vapours during
the whole 7-h shift Brugnone et al., (1985) found high correlation (r=0.84) between the
two values. This may only be due to uniform concentrations in the air during the whole

88
 Table 1. Excretion of methylhippuric acid after 8 hinhalation exposure to xylene
in concentration of 200mg/m3

Excretion of methylhippuric
acid in urine

Exposure Urine g/l gIg of Rate of Reference

mg/m3 fraction creat. excretion
mglh

E 4-8 h 1.263) 0.98 1) 64 Ogata et

435 of exposure 0.844) al.(1970)

E 4-8 h 105 + 7.5% Senczuk

and
100-600 of exposure 1.6 1) Orlowski
(1978)

E 0-8 h 1.573) 1.03 Sedivec

200-400 of exposure 1.044) and PIek
(1976)
6-8 h 2.13) 1.34
of exposure 1.44)

I Last 4-5 h Engstrom

3-300 of exposure 0.64 et al.
(1977)

I Second half Lundberg

of and
10-850 the shift 0.91) 602) Sollenberg
(1968)
100-200 1.51) 952)

E Experimental exposure
I Industrial exposure
I) Recalculated from the rate of excretion of methylhippuric acid

assuming mean excretion rate of creatinine of 0.065 mglh

2) Reca1culated from the rate of excretion in mg/h/kg body weight

assuming 70 kg as the average hody weight

3) sg corrected to 1.024

4) sg corrected to 1.016

shift. For a number of organic solvents the first half-time of elimination from blood
corresponds to the compartment of circulatory blood and is very short e.g. for toluene
approximating to about four minutes (Fig. 3).
Thus, the concentrations of toluene in blood sampies collected during or just before the
end of a workshift reflect the actual rate of absorption whereas concentrations in sampies
collected 15-20 minutes after the end of exposure reflect the exposure during a few
preceding hours. It seems, however, that for the compounds which are accumulated in
adipose tissue, e.g. tetrachloroethylene (t1/2 of the I, 11 and III phase of elimination from the
blood amounting to 15 min, 5 h and 54 h respectively the concentration of the unchanged
compound in blood sampies collected 15-20 min. after the end of exposure reflects the
whole shift exposure or even to a greater extent the exposure of the last 2-3 days
(Kostrzewski, 1985).
The same toxicokinetic consideration may concern determinations of volatile compounds
in the exhaled air. The possibility of applying determinations of volatiles in the expired air

89
 19C

3,0

ISA lt =0,6ge- 10,4t + O,2Se -O,38St +O,03e -0,028t

ISA 10

,,0't:-_ _ _ _~----_+_----___+_----_---
10 20 30 40
t (h J
Figure 3. Kinetic of elimination of toluene from capillary blood after termination of exposure. The half-life
for capillary blood amounted to t 1/2 = 4 min.; t 1/2 = 1,8 h; t 1/2 = 24,5 h for phases I, 11 and III, respectively.
(Kostrzewski and Piotrowski, 1991).

for the evaluation of exposure is even lower than in the case of determinations in blood
sampIes. A possible additional influence of the content of fat tissue and a very low
possibility of monitoring differences of exposure due to the workload should be taken into
account (Fig. 4). On the other hand, determinations of unchanged solvents in blood or in
exhaled air sampIes collected before the shift, after the state of equilibrium between the
adsorption, cumulation and excretion is reached, may provide valuable information on
exposure in the case of determining the health-based limits in biological material for
chemicals having effect on the CNS functions.

POSSIBLE INFLUENCE OF INDUCTION OF METABOLISM AND TOXICO-

KINETIC INTERACTIONS ON TUE RESUL TS OF BIOLOGICAL MONITORING

There is a number of reports with the experimental data pointing to the possible
influence of enymatic induction of metabolism on the toxicity and metabolism of organic
compounds (Robertson et al., 1989; Ohtskui and Ikeda, 1971; Comish et al., 1974).
I was suggested that apart from influence on the toxicity of industrial chemicals, the
induction of microsomal enzymes may also affect the results of biological monitoring of
exposure. However, taking into account the biotransformation capacity of the human liver,
this influence seems not to be so important in the case of industrial exposure below the
admissible concentrations. In two experiments performed in human volunteers exposed for
8 h to m-xylene of the concentration of 400 mglm3 with and without the pretreatment with
phenobarbital (2 mglkglday) for 11 days the excretion of m-methylbenzoic acid in urine
was the same (Fig. 5). Another example confirming this assumption is the simulation of the
influence of enzyme induction on TRI metabolism presented in Fig. 6. The effect of
enzyme induction on TRI metabolism in vivo depends on the exposure. At the
concentrations occuring in industrial conditions when the hepatic blood flow rate limits the
metabolism, a five-fold increase in Vmax caused only a marginal influence on the TRI
metabolism. In view of biological monitoring of exposure it is noteworthy that the enzyme
induction due to drug consumption or drinking ethanol may not affect the pharmacokinetic
behaviour of organic solvents as much as the animal studies conducted in vivo and in vitro
would suggest.

90
 Average alveolar
toluene concentration
25
.' Females 100 W (6)

7"0'"
20
Males 100 W (13)
ppm _ ...... :-:i.'Females 75 W (14)

15 75 W (4)

10~--~~~--------------
Rest Work
period period
Figure 4. The average concentration of toluene during the rest period and the period including work of males
and females separates according to excercise level. The numbers of subjects in each group is shown in
brackets. The average within group standard deviation of the values was 9,1 ppm (Baelum, 1990).

The majority of methods for the biologie al monitoring of exposure to organie solvents
have been developed for particular chemie als under experimental conditions with only one
substance present in the atmosphere. Their application in industrial conditions, where
workers are exposed to mixtures rather than single chemie als, has been based on the
assumption that the probability of toxicokinetic interactions at concentrations of solvents
in the air approximating TLV values is rather low. However, littIe is known about both the
pharmacokinetics of inhaled solvents in relation to atmospheric exposure concentrations, and

mg/h
o Control
Penobarbital
150 pretreatment

\
"C
'i 1
.S:!
o
N
c:
..2l 100
~
Q)
E
E

~
50

4 8 12 16 20 24 h

Exposure
Figure 5. Mean values of the excreted amount of conjugated m-methylbenzoic acid in five male volunteers
without (empty circles) and with (black dots) pretreatrnent by phenobarbital 2 mglkg/day for 11 days. Urine
was collected in seven 2-h intervals and in the last 10-h interval, total 24 h (David et al., 1979).

91
 300
o TTC in urine
32 mg/min ~_ _--~
250
<:.
CT>

.5
c:
.9 200
'"t;
)(

u'" 150
f-
f-
>-
-------------------0
~ 100
'
'
f;; 50 32 mg/min (normal)
a:
o----o---------o~--------~o~----------------~o
0
i i i I
0 500 1000 1500 2000 2500
Exposure concentration of TRI (ppm)

Figure 6. Dose dependent relation between enzyme induction and TRI metabolism. Enzyme induction by
ethanol was assumed to increase the Vrnax of TRI metabolism five (16 mg/min) or lO-fold (32 mglmin)
without changing the Km. Simulations were performed for a standard male worker who inhaled TRI at various
concentrations (0-2000 ppm) for eight hours (08.00 - 12.00 and 13.00 - 17.00) under the influence of enzyme
induction. The rate of urinary total ttichlorocompounds (TTC) excretion at the end of inhalation (17.00) are
plotted against the concentration in inhaled air (Sato et a1., 1991).

the possible interactions of industrial solvents. According to David et al., (1979) the human
capacity of biotransform m-xylene arnounts to the dose absorbed at about 800 mgln3 , and
according to Riihimaki et al., (1992) to 1300 mglm 3 Excretion of mandelic acid in urine
increases linearly with the increase of styrene concentrations in the air up to 600 mglm 3
(Engstrom; et al., 1976). Combined exposure of m-xylene and ethylbenzene in
concentrations of 655 mglm3 both 10wered the arnounts of metabolites (Engstrom et al.,
1984). Co-exposure to m-xylene and methyl ethkyl ketone (MEK) in concentrations of 100
ppm (435 mglm3 ) and 200 ppm (590 mglm3 ) resulted in inhibited xylene metabolism (Liira
et al., 1988). No significant differences in the excretion of methylbenzoie acid in urine were
observed after exposure to m-xylene in concentrations of about 45 and 70 ppm and m-
xylene together with other four solvents in combined concentrations of about 90 and 140
ppm. For both the single and combined exposure the kinetic of excretion of methylbenzoic
acid was similar (Jakubowski and Kostrzewski, 1989).
All these data suggest that toxicokinetic interactions between inhaled solvents may occur
at rather high levels of exposure approaching the metabolie capacity of the human liver.

CONCLUSIONS

1. Present research refers predominantly to the relationship between the concentrations of

chemical substances and metabolites in biological media and environmental exposure even
if it is disputable from the point of view of toxicokinetics and result interpretation.
2. It seems necessary to set up, on the international scale, the principles according to which
the biological indices of exposure magnitude should be developed. Tbe knowledge of
human toxicokinetics and the relations hip between the absorded dose and the rate of urinary
excretion should be considered aprerequisite for this kind of activity. Tbis can be achieved
under fully controlled experimental conditions.

92
3. The studies aiming at the determination of dose-effect and dose-response relationship and
consequently of the relationship between exposure and adverse effect are of primary
importance. In this case determinations of organic chemicals in blood and expired air, after
the equilibrium has been reached may be used as an index of accumulated dose.
4. In view of the high cost of carrying out the investigations as weH as the ethical problems
related to the involvment of human subjects either in experimental or field conditions it
seems indispensable to establish a priority list of chemical substances to be considered.

REFERENCES

ACGIH,1991-1992, Threshold Limit Values for Chemical Substances and Physical Agents and Biological
Exposure Indices.
Berlin A., Yodaiken R.E., Henman B.A., 1984, "Assessment oftoxic agents at the workplace. Roles of
ambient and biological monitoring" , Nijhoff, Boston, The Hague, Wordrecht, Lancaster.
Brugnone F., De Rosa E., Perbellini L., Bartolucci G.B.,1986, Toluene concentrations in the blood
and alveolar air of workers during the workshift and the moming after, Brit. J Ind Med. 43:56.
CEC, 1983, "Human Biological Monitoring of Industrial ChemicaIs Series", Alessio L., Berlin A., RoiR.,
Boni M.ed. Office for Official Publications of the European Communities, Luxemburg.
Cornish H.H., Ling B.P.,Barth M.L., 1974,Phenobarbital and organic solvent toxicity, Am Ind Hyg Assoc
J.34:487.
David A., Flek J.,Frantik E., Gut I.,Sedivec V.,1979,Influence of phenobarbitaI on xylene metabolism,
Int Arch Occup Environ Healrh. 44: 117.
DFG, 1990, Maximale Arbeitsplatzkonzentrationen und Biologische Arbeitsstofftoleranzwerte.
Dutkiewicz T., 1963, Quantitative exposure test for benzene,in:Intemational Congress Series No 62,
Int.Congr.Occup. Health, Excerpta Medica.
Engstrom K., Husman K., Rantanen 1., 1976, Urinary mandelic acid concentration after occupational
exposure to styreneand its use as a biological exposure test, Scand J Work Environ Health. 2:21.
Engstrm K., Husman K.,Riihimaki V.,1977,Percutaneous absorption of m-xylene in man, Inr Arch
Occup Environ Health. 39:181.
Engstrm K., Riihimaki V., Laine A., 1984, Urinary disposition of ethylbenzene and m-xylene in man
following separate and combined exposure, Int Arch Occup Environ Healrh. 54:355.
Jakubowski M., Kostrzewski P., 1989, Excretion of methyl- benzoic acid in urine as a result of single
and combined exposure to m-xylene, Polish J Occup Med. 2:238.
Jongeneelen F.J., Auzion R.B.M., Scheepers P.T.I., Bos R.P.,Henderson P.Th., Nijenhuijs E.H., Veenstra
S.I., Brouns R.M.E., Winkers A., 1988, I-Hydroxypyrene in urine as a biological indicator of
exposure to polycyc1ic aromatic hydrocarbons in several work environments, Ann Occup Hyg. 32:35.
Kostrzewski P., Jakubowski M., 1985, Application of determinations of volatiles in capillary blood sampies
fOT evaluation of industrial exposure: tetrachloroethylene, Ann Am Conf Ind Hyg. 12:269.
Kostrzewski P., Piotrowski l.K., 1991, Toluene determination in capillary blood as a biological indicator
of exposure to low levels of toluene, Pol J Occup Med Environ Health, 3:249.
Liira J., Riihimaki V.,Engstrom K.,Pfaffli P.,1988,Coexposure of man to m-xylene and methyl ethyl
ketone,Scand J Work Environ Health. 14:322.
Lundberg I.,Sollenberg J.,1986, COTTelation of xylene exposure and methylhippuric acid excretion in urine
among paint industrial workers, Scand J Work Environ Health. 12: 149.
Ogata M.,Tomokuni K.,Takatsuka Y.,1970, Urinary excretion of hippuric acid and m- or p-xylene as atest
of exposure, Brit J Ind Med, 27:43.
Ohtsui H., Ikeda M.,1971, The metabolism of styrene in the rat and the stimulatory effect of
phenobarbital,Toxicol Appl Phannacol. 18:321.
Piotrowski J.K., 1977, "Exposure Test for Organic Compounds in Industrial Toxicology", U.S. Department
of Health,Education and Welfare, NIOSH, Cincinnati.
Phillips D.L., Smith A.B., Burse V.W., Steele G.K., Needhan L.L.,Hannon W.H., 1989, Half-life of
polychlorinated biophenyls in occupationally exposed workers, Arch Environ Health. 44:351.
Riihimaki V.,Savolainen K.,Pfaffli P.,Pekari K.,Sippel H.W.,Laine A.,1982, Metabolic interaction between
m-xylene and ethanol, Arch Toxicol. 49:253.
Robertson P.Jr.,White E.L.,Bus J.S.,1989,Effects of methyl ethyl ketone pretreatment on hepatic mixed -
function oxidase activity and on in vivo metabolism of n-hexane, Xenobiotica. 19:721.
Roush G.J.,OIt G.,1977, A study of benzene exposure versus urinary phenol levels, Am Ind Hyg Assoc J.
38:67.
Schulte P., Halperin W.,Herrick M.,Connally B.,1987,The CUTTent focus on biological monitoring in:

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 "Occupational and Environmental Chemical Hazards: Cellular and Biochemical Indices for
Monitoring Toxicity", V.Foa, E.A. Emmett,M.Maroni ed., Ellis Horwoad LId. Chichester, England.
Sedivec V., Flek J., 1976, Exposure test for xylenes, Int Arch Occup Environ Health. 37:219.
Senczuk W., Orlowski J., 1978, Absorption rate of m-xylene vapours through the respiratory tract and
excretion of m-methylhippuric acid in urine, Brit J Ind Med. 35:50.
Sherwood R.J.,1972, Evaluation of exposure to benzene vapour during the loading of petrol, Brit J Ind Med.
29:65.
Walkley J.E., Pagnotto L.D., Elkins H.B., 1961, The measurement ofphenol in urine as an index of
benzene exposure,Amer Ind Hyg Assoc J. 22:362.
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94
STRESS PROTEINS AS BIOMARKERS OF TOXICITY

Peter L. Goering, Benjamin R. Fisher,

Carole A. Kimmel *, and Gary L. Kimmel *

Division of Life Sciences

Office of Science and Technology
Center for Devices and Radiological Health
Food and Drug Administration
Rockville, MD 20857

*Reproductive and Developmental Toxicology Branch

Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460

INTRODUCTION

The development of more sensitive and predictive test methods to characterize the
safety of drugs and chemicals is an important part of the missions of the U.S. Food and
Drug Administration and the U.S. Environmental Protection Agency. One approach is to
develop methodologies which would define biomarkers of exposure and toxicity.
Biomarkers have been proposed to be used to: 1) identify potential hazards, 2) establish
dose-response relationships, 3) estimate risk at low-dose exposures, 4) serve as short-term
in vitra toxicity tests, and 5) improve risk assessment and risk management capabilities
(Committee on Biological Markers, 1987).
We are investigating the possibility of using altered protein synthesis patterns as
biomarkers of exposure and toxicity. Our approach is to use well-known toxicants in order
to identify altered patterns of protein synthesis wh ich could serve as "biochemical
fingerprints" of exposure and/or toxicity. As relationships between the altered protein
synthesis patterns and exposures and/or toxicities become more defined, our goal will be
to study the "fingerprints" of test compounds, which may provide dues as to the potential
toxicity of these new or untested compounds if similar patterns are revealed.
Cells respond to various environmental stressors by enhancing the expression of
specific genes, the products of which comprise a family of proteins known as heat-shock,
or stress, proteins (for reviews, see Morimoto et al., 1990; Welch, 1990; Nover, 1991).

Use 0/ Biomarkers in Assessing Hea/th anti Environmenkll Impacts 0/ Chemica/

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 95
Although heat was the first insult which demonstrated this response, the chemical and
physical insults which produce this response are diverse (Nover, 1991); thus, the broader
and more inclusive term "stress proteins" is used. One feature of the stress protein
response is that the changes in protein synthesis are rapid. The up-regulation of stress
proteins and the down-regulation of constitutive proteins occurs within 1 to 3 hr after
exposure to many insults (Blake et al., 1990). Stress proteins have been induced in a wide
variety of mammalian cells in culture and tissues in vivo, including liver, heart, adrenal,
brain, spleen, and embryos.
These intriguing properties have led us and others to hypothesize that the stress protein
response: 1) could be exploited as a biomarker of exposure and toxicity, and 2) may
represent an early cellular response which may underlie or be related to mechanisms of cell
injury. To evaluate these hypotheses, we have pursued aseries of studies examining the
effects of well-known xenobiotics on stress protein synthesis, determining whether the
effects are target organ specific, and assessing the temporal relationships between altered
protein synthesis patterns and toxicity. Our experimental approaches for target organ
toxieology studies and developmental toxicology studies initially have involved in vivo
experiments whieh are conducted using agents whose target tissues are well-known. These
stressors are used as tools to assess protein synthesis patterns in target and non-target
tissues and toxicity. Studies in cultured cell or embryo systems are then carried out using
the same insults to compare altered protein synthesis patterns in vitro with the in vivo data.
Ultimately, new chemicals whose toxicity is unknown can be tested using this system in
order to predict the toxic potential of these compounds.

RESULTS

Target Organ Toxicology Studies

Using cadmium as a well-known hepatotoxicant and mercury as a well-known

nephrotoxicant, our initial studies attempted to characterize changes in stress protein
synthesis induced in target and non-target tissues in adult rats (Goering et al., 1991;
Goering et al., 1992). These studies utilized in vivo exposure of male rats, labeling
proteins in OA-mm tissue slices in vitro with 35S-methionine, and analysis of proteins
using SDS polyacrylamide gel electrophoresis and autoradiography. Dose- and time-
dependent alterations in gene expression in kidney and liver were demonstrated after in
vivo exposure of rats to mercuric chloride and cadmium chloride, respectively, as evidenced
by enhanced de novo synthesis of the 70-, 90-, and 1l0-kilodalton (kDa) stress proteins
2-4 hr after exposure. Synthesis of constitutive proteins of 68- and 38-kDa was inhibited
at the same time. Standard clinical, functional, and histopathologie indices were used to
assess renal and hepatic cell injury. Changes in kidney de novo protein synthesis occurred
prior to detectable elevations in blood urea nitrogen and decreases in the uptake of 3H_p_
aminohippurate in renal slices, and prior to any observable renal tubule necrosis. Altered
liver de novo protein synthesis occurred before any increases in plasma sorbitol
dehydrogenase and decreases in microsomal N-demethylase activities were observed, and
prior to any evidence of hepatocellular necrosis. In both studies using mercury and
cadmium, the early changes in protein synthesis appeared to be target organ specific. For
example, the nephrotoxicant mercury affected protein synthesis in kidney but not liver, and
did not produce hepatotoxicity (Goering et al. , 1992). Conversely, cadmium treatment
induccd changes in liver protein synthesis, but not in kidney, and ultimately produced
cellular injury in liver but not kidney (Goering et al., 1991).

96
Developmental Toxicology Studies

Developmental toxicology studies conducted in our laboratories have focused on

identification of changes in specific proteins that correlate with the appearance of
developmental abnormalities following heat shock. The data have demonstrated an
association between heat-induced alterations in embryonic proteins and developmental
defects in rat embryos. In initial studies, it was observed that exposure of pregnant rats to
heat on gestation day (GD) 10 resulted in disrupted development of somites, the
presumptive skeletal embryonic tissue, after 24 hr, and produced thoracic skeletal
malformations in neonatal rats (Cuff et a1., 1990; Kimmel et al., 1992). In these studies,
the altered somite development observed 24 hr after exposure to heat in utero and in vitro
was correlated with the localization of the skeletal defects observed three days postpartum.
Further studies (Fisher et a1., 1991; Kimmel et a1., 1991; Fisher et a1., 1992), demonstrated
enhanced de novo synthesis of the 70- and 90-kDa stress proteins in embryos 1-8 hrs after
in utero and in vitro heat exposure. Western blot analysis using specific antibodies was
used to detect changes in embryonic concentrations of heat shock proteins, actin
(microfilaments), tubulin (microtubules), and vimentin (intermediate filaments).
Immediately following heat shock, total vimentin was reduced to minimal detectable levels
and remained depressed for more than 2 hr, retuming to control levels 4-8 hr after
exposure. No changes in total tubulin or actin concentrations were observed. The data
demonstrated that heat-induced reduction in the concentrations of proteins comprising
intermediate filaments occurred concomitantly with the induction of stress proteins, both
of which preceded aberrant morphologic changes in rat embryos.

DISCUSSION

A biomarker is defined as a change which occurs in a biological system that is

qualitatively or quantitatively predictive of health impairment or potential impairment
resulting from toxicant exposure (Committee on Biological Markers, 1987). Several
important criteria for biomarkers are: 1) that the response should occur prior to the onset
of overt clinical symptoms of disease, and 2) for biomarkers of exposure, the response
should be specific so as to detect exposure to a single agent or class of chemicals, and for
biomarkers of effect or toxicity, the response should be related, although not necessarily
causally, to dynamic intracellular changes leading to overt toxicities (Fowler et a1., 1984).
Based on these criteria, the results of our studies in adult target tissues and embryos
suggest that the altered protein synthesis patterns observed may serve as biomarkers of
toxicity. These data extend a growing body of evidence which suggests that altered protein
synthesis patterns, including the well-characterized stress proteins, may be useful in
toxicology as biomarkers of exposure and toxicity. For example, Aoki et a1. (1990) found
that non-cytotoxic concentrations of the semiconductor component metal, gallium,
stimulated the synthesis of several proteins and inhibited the synthesis of others in cultured
kidney tubules. Anderson et a1. (1987) showed that each of several xenobiotics produced
unique chemical-specific protein synthesis patterns in mouse liver. Deaton et a1. (1990)
demonstrated that non-cytotoxic concentrations of sodium arsenite induced the synthesis
of hsp70 and hsp90, and phenyldichloroarsine, a skin vesicant, stimulated synthesis of
hsp70, but not hsp90, in cultured human epidermal keratinocytes. Gonzalez et a1. (1989)
found that kainic acid, a neurotoxin, induced hsp72 in regions of rat brain which are
injured. Expression of hsp70 mRNA and depression of protein synthesis after a transient
ischemic insult occurred in brain regions susceptible to injury (Nowak, 1990). Anson et
a1. (1991) found a correlation between terata detected in mice on GD 17 and the production
of stress proteins in embryonic target tissues 2.5 hr after treatment with retinoic acid on GD

97
11. No induction of stress proteins was evident in non-target tissues. Others have
demonstrated that stress proteins and altered protein synthesis patterns may be markers of
hepatic injury (Anderson et al., 1987; Van Dyke et al., 1992), hepatocarcinogenesis
(Anderson et al., 1992), developmental toxicities (Hansen et al., 1988; Mirkes and Doggett,
1992), and autoinunune diseases (Heufelder et al., 1992).
A concomitant inhibition of activity of other genes has been associated with the
activation of heat shock, or stress, protein genes (Morimoto et al., 1990). Since proteins
are responsible for critical intracellular functions, e.g., enzyme catalysis, cell structure, and
gene regulation, xenobiotie-indueed ehanges in protein synthesis may play a meehanistie
role in cell injury and represent early subcellular perturbations whieh eventually lead to
overt toxicity. Data from our studies demonstrated that the induction of stress proteins was
marked; however, a concomitant inhibition of synthesis of proteins whieh are constitutively
expressed in rat kidney and liver, i.e., 68- and 38-kDa proteins, and GD 10 embryos, i.e.,
vimentin, was observed. Although specifie gene produets were not identified in all the
studies, the demonstration that vimentin concentrations are transiently reduced in embryos
after heat insult suggests that stress protein synthesis may compromise important
intracellular funetions related to cell structure, growth, and homeostasis. While there is no
direct evidence that any of these affected proteins are meehanistically involved in the
adverse effeets resulting from exposure to metals or heat, the down-regulation of the
synthesis of eritical proteins due to a diversion of cellular metabolie energy towards stress
protein synthesis could contribute to cellular toxieity. Further eharaeterization and
identification of these affeeted proteins, especially incorporating the more comprehensive
two-dimensional gel eleetrophoresis techniques (Anderson et al., 1987; Hansen et al., 1988;
Aoki et al., 1990), may advance our understanding of the moleeular meehanisms involved
in cell injury indueed by specific chemicals.

CONCLUSION

Evidence is accumulating that enhanced synthesis of stress proteins and altered

synthesis of other constitutive proteins in response to chemical exposure may be useful to
exploit as biomarkers of exposure andlor toxicity for assessing exposure to and predieting
the toxie potential of xenobiotics. Further extensive analysis of these ehanges in protein
synthesis may be important in elucidating underlying meehanisms of eell injury and
toxicity.

REFERENCES

Anderson, N.L., Giere, F.A, Nance, S.L., GemmeU, M.A, Tollaksen, S.L., and Anderson, N.G., 1987,
Effects of taxie agents at the protein level: Quantitative measurement of 213 mouse liver proteins
foUowing xenobiotie treatment, Fund. Appl. Toxicol. 8:39.
Anderson, N.L., Coppie, D.C., Bendele, R.A, Probst, G.S., and Riehardson, F.C., 1992, Covalent protein
modifications and gene expression ehanges in rodent liver following administration of
methapyrilene: A study using two-dimensional electrophoresis, Fund. Appl. Toxicol. 18:570.
Anson, J.F., Laborde, J.B., Pipkin, J.L., Hinson, W.G., Hansen, D.K., Sheehan, D.M., and Young, J.F.,
1991, Target tissue specifieity of retinoie acid-induced stress proteins and malfonnations in mice,
.Teratol. 44:19.
Aoki, Y., Lipsky, M.M., and Fowler, B.A., 1990, Alteration in protein synthesis in primary cultures of rat
kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite, Toxicol.
Appl. Pharmaco1. 106:462.
Blake, M.J., Gershon, D., Fargnoli, J., and Holbrook, NJ., 1990, Discordant expression of heat shoek
protein mRNAs in tissues of heat-stressed rats, J. Biol. ehern. 265:15275.

98
Comminee on Biological Markers, 1987, Biological markers in environmental health research, Environ.
Hlth. Perspect. 74:3.
Cuff, J.M., Kimmei, C.A, Kimmei, G.L., Heredia, DJ., and Brown, N.T., 1990, Correlation of altered
somite morphology with effects of heat stress on the rat axial skeleton, Teratol. 41:546 (abstr.).
Deaton, M.A., Bowman, P.D., Jones, G.P., and Powanda, M.C., 1990, Stress protein synthesis in human
keratinocytes treated with sodium arsenite, phenyldichloroarsine, and nitrogen mustard, Fund.
Appl. Toxicol. 14:471.
Fisher, B.R., Brown, K.M., and Heredia, D.J., 1992, In vitro heat shock produces alterations in
cytoskeletal proteins in cultured rat embryos, Toxicologist 12:332 (abstr.).
Fisher, B.R., Kimme!, G.L., Kimmei, C.A, and Heredia, DJ., 1991, Tbe association of heat-induced
alterations in protein synthesis with somite defects in rat embryos, Teratol. 43:465 (abstr.).
Fowler, B.A, Abel, J., Elinder c.-G., Hapke, H.-J., Kagi, J.H.R., Kleiminger, J., Kojima, Y., Schoot-
Uiterkamp, AlM., Silbergeld, E.K., Silver, S., Summer, K.H., and Williams, RJ.P., 1984,
Structure, mechanism, and toxicity, in: "Changing Metal Cyc1es and Human Health," J.O. Nriagu,
ed., pp. 391-404, Springer-Verlag, New York.
Goering, P.L., Fisher, B.R., Chaudhary, P., and Diek, C.A, 1991, Stress protein synthesis induced in rat
liver by cadmium precedes hepatotoxicity, Toxicologist 11:42 (abstr.).
Goering, P.L., Fisher, B.R, Chaudhary, P.P., and Dick, C.A, 1992, Relationship between stress protein
induetion in rat kidney by mereuric chloride and nephrotoxicity, Toxicol. Appl. Pharmacol.
113:184.
Gonzalez, M.F., Shiraishi, K., Hisanaga, K., Sagar, S.M., Mandabaeh, M., and Sharp, F.R., 1989, Heat
shock proteins as markers of neural injury, Molec. Brain Res. 6:93.
Hansen, D.K., Anson, J.F., Hinson, W.G., and Pipkin, Jr., J.L., 1988, Phenytoin-induced stress protein
synthesis in mouse embryonic tissue, Proc. Soc. Exper. Biol. Med. 189:136.
Heufelder, AE., Goellner, J.R., Wenzel, B.E., and Bahn, R.S., 1992, Immunohistoehemical deteetion and
localization of a 72-kilodalton heat shoek protein in autoimmune thyroid disease, J. Clin.
Endocrinol. Metab. 74:724.
Kimmei, C.A., Cuff, J.M., Kimmei, G.L., Heredia, D.J., Tudor, N., Silverman, P.M., and Chen, J., 1992,
Skeletal development following heat exposure in the rat, Teratol. (in press).
Kimmei, C.A., Kimmei, G.L., Lu, c., Heredia, D.J., Fisher, B.R, and Brown, N.T., 1991, Stress protein
synthesis as a potential biomarker for heat-indueed developmental toxieity, Teratol. 43:465
(abstr.).
Mirkes, P.E., and Doggen, B., 1992, Aeeumulation of heat shoek protein 72 in postimplantation rat
embryos after exposure to various periods of hyperthermia in vitro: Evidenee that heat shoek
protein 72 is a biomarker of heat-indueed embryotoxieity, Teratol. 46:301.
Morimoto, R.I., Tissieres, A, and Georgopoulos, c., 1990, Tbe stress response, funetion of the proteins,
and perspeetives, in: "Stress Proteins in Biology and Medicine," RI. Morimoto, A Tissieres, and
C. Georgopoulos, eds., pp. 1-36, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York.
Nover, L., 1991, "Heat Shoek Response," CRC Press, (ne., Boea Raton, Florida.
Nowak, Jr., T.S., 1990, Protein synthesis and the heat shoek/stress response after isehemia, Cerebrovasc.
Brain Metab. Rev. 2:345.
Van Dyke, RA, Mostafapour, S. Marsh, H.M., Li, Y., and Chopp, M., 1992, Immunocytoehemical
detection of the 72-kDa heat shoek protein in halothane-indueed hepatotoxicity in rats, Life Sei.
50:PlA1.
Welch, W.J., 1990, Tbe mammalian stress response: Cell physiology and bioehemistry of stress proteins,
in: "Stress Proteins in Biology and Medicine," RI. Morimoto, A. Tissieres, and C. Georgoponlos,
eds., pp. 223-278, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

99
 SIGNIFICANCE OF SERUM FERRITIN CONCENTRA TIONS IN LUNG CANCER
AND ITS RELATION WITH CELLULAR IMMUNITY

Orhan S. Sardlll, Semra Sardll2, and Oktay Sancaktar1

1Ankara University Faculty of Medicine

Department of Hematology and Oncology

20azi University Faculty of Pharmacy

Department of Toxicology, Ankara, Turkey

INTRODUCTION

In the last decade we have seen an explosion of reports dealing with tumor markers
that are able to detect precocious signals of neoplasia. The possibility of detecting a lung
tumor at an early clinical stage and of assessing the completeness of surgical resection or
the effectiveness of radiation or chemotherapy would be of infinite value. For these reasons
the utility of lung cancer markers is widely examined, and studies currently in progress
address the major problem of laboratory diagnosis, the difficulty of detecting the tumor at
an early c1inical stage. The substances that have been described to date can be categorized
as either tumor-associated antigens or biochemical tumor markers; these are substances
normally present that occur at elevated levels in the serum of tumor-bearing hosts.
Similarly, many different proteins, hormones, and enzymes have been described as being
potentially valuable tumor markers (Burt et al. , 1978). Recently serum ferritin was
considered to be a reliable index in tumors and hematological malignancies (Iones et al.,
1973). Also, in vitra studies exhibited a disturbance in T-lymphocyte function caused by
ferritin (Papenhausen et al. , 1984). Many of the proliferative disorders in which high serum
ferritin levels are found are also associated with impaired cell-mediated immunity.

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemica/

Pollutants. Edited by C.C. Travis. Plenum Press. New York. 1993 101
METHODS
The high incidence of elevated ferritin levels observed in patients with cancer
prompted us to examine serum ferritin as a potential tumor marker in 27 lung cancer
patients. These levels were compared with those in a control group consisting of 12 healthy
subjects. Serum ferritin levels were determined by the radioimmunoassay technique (Walters
et al., 1973), and ferritin kits were supplied from Uppsala-Sweden-Phadebas. Peripheral
venous blood was layered with Ficoll-Hypaque for E-rosette tests. The lymphocyte layer,
which was separated by centrifuging at 1000 rpm for 40 min, was washed with Hanks
balanced salt solution, and a lymphocytc suspension was prepared. This was mixed with
washed and diluted sheep erythrocytes and again centrifuged for 5 min at 1000 rpm. The
tubes were kept at +4C for 24 hours and cells were counted. Lymphocytes which bound
at least three sheep erythrocytes were accepted to be E-rosette formed cells (Jondal et al.,
1972).

RESUL TS AND DISCUSSION

As shown in Table 1, the serum ferritin levels of pretreatment patients were
significantly different from healthy contro1 subjects (p<O.OOl). Also, the serum ferritin
levels of the same patients before undergoing any medical treatment differed significantly
(p<O.OOl) after therapy. These significant statistical differences also existed for E-rosette
formation when the pretreatment patient group was compared to the healthy control group
(p<O.OOl) and to the post-treatment patient group (p<O.OOl)
Statistical analysis was performed by the student t-test.
There have been different reports concerning the use of serum ferritin as a marker
in monitoring the therapy of pulmonary malignancies. Valpino et al. (1984) have measured
serum ferritin levels in lung cancer patients according to histological type, and the highest
levels were found in 1arge-cell tumors. The average percentage increase of the marker in
this type of tumor as compared with normal was 172.9. The same group also evaluated the
level of ferritin in relation to the stage of the disease in the same patients, and in third-stage
cancers the mean increase of the marker proved to be significantly higher than that found
in first-stage tumors. More recently, Pluygers et al. (1991) se1ected general markers to value
early detection of malignancy in the ear1y stages of mesothelioma and bronchogenic
carcinoma in occupationally asbestos-exposed workers in various industries, and increases
in ferritin levels were detected according to the grades of the cases. Groop et al. (1977)
demonstrated that 11 out of 28 lung cancer patients without clinically proven metastases

102
had significantly lower levels of ferritin than the group with clinically demonstrated
metastases. However, Cox et al. (1985) could not demonstrate any significant difference
between serum ferritin levels in patients with detectable metastases and those without,
although the ferritin levels were considerably elevated in patients with small-cell lung
cancer compared with controls. Dur findings confirm and extend the results of these studies.
Further clinical significance can also be seen from recent reports of Papenhausen et
al. (1984), Matzner et al. (1979), and Hancock et al. (1979), namely, that in malignancies,
as a result of the elevated serum ferritin level, the peptide covers the surface of T-
lymphocytes and inhibits its rosette formation with sheep erythrocytes, thereby causing a
defect in cellular immunity. The same lymphocytes when incubated with levamisol and
papain in vitro release the ferritin molecules on their surfaces and their functions return to
normal. Also, a clear negative correlation was demonstrated between serum ferritin level
and E-rosette formation in the present study (r:-O.676 p<O.OOl). Due to the rise in serum
ferritin level, E-rosette formation diminishes, but after therapy following the fall in ferritin
level, E-rosette formation returns to normal. If the presence of these proteins on the cell
membrane interferes with the functional aspects of cellular immunity, the quantities of these
proteins in the lung contribute to significant suppression. Further studies are needed to
assess the full potential of these combined assays.

Table 1. Pretreatment and Post-Treatment Serum Ferritin and E-Rosette Levels in Patient
and Control Groups

Groups Serum ferritin (mg/L) E-rosette formation (%)

meanSD meanSD

A Patient group Al: 385162 ~: 34.97.0

before therapy

B Patient group BI: 17565 B2 : 44.26.9

after therapy

C Healthy group Cl: 5633 C2 : 57.93.2

Comparisons: Al and Cl: p<O.OOl A2 and C 2 : p<O.OOl

BI and Cl: p<O.OOl B2 and C 2: p<O.OOl
Al and BI: p<O.OOl A2 and B2 : p<O.OOl

103
REFERENCES

Burt, R.W., Ratcliffe, J.G., Stack, B.H.R, Cuthbeet, J., Kennedy, R.S., Corker, C.S.,
Franchimont, P., Spilg, W.G.S., and Stimson, W.H., 1978, Serum biochemical
markers in lung cancer, Br.J. Cancer, 37:714.

Cox, R, Gyde, O.H., and Leyland, J., 1986, Serum ferritin levels in small celllung cancer,
Eur.J. Cancer, Clin. Oncology, 22:831.

Gropp, C., Lehmann, EG., Baver, H.W., and Havemann, K., 1977, Carcino embryonic
antigen, (Xj-fetoprotein, ferritin and ~-pregnancy associated glycoprotein in the
serum of lung cancer patients and its demonstration in lung tumor tissues,
Oncology, 34:267.

Hancock, B.W., Bruce, L., May, K., and Richmond, J., 1979, Ferritin, a senslUzmg
substance in the leucocyte migration inhibition test in patients with malignant
lymphoma, Br.I. Hematol., 43:223.

Jondal, M., Holm, G., and Wigzell, H., 1972, Surface markers on human and B
lymphocytes. A large proportion of lymphocytes forming nonimmune rosettes with
sheep red blood cells, Journal of Experimental Medicine, 136:207.

Jones, P.A.E., Miller, EM., Worwood, M., and Jacobs, A., 1973, Ferritinaemia in leukemia
and Hodgkin's disease, Br.J. Cancer, 27:212.

Matzner, Y., Hershko, c., Polliack, A., Konijn, A.M., and Izak, G., 1979, Suppressive effect
of ferritin on in vitra lymphocyte function, Br.J. Hematol., 42:345.

Papenhausen, P.R, Emeson, E.E., Craft, C.B., and Borowiecki, B., 1984, Ferritin-bearing
lymphocytes in patients with cancer, Cancer, 53:267.

Pluygers, E., Baldewyns, P., Minette, P., Beauduin, M., Gourdin, P., and Robinet, P., 1991,
Biomarker assessments in asbestos-exposed workers as indicators for selective
prevention of mesothelioma or bronchogenic carcinoma: Rationale and practical
implementations, European Journal of Cancer Prevention, 1:57.

Volpino, P., Cangemi, V., Caputo, V., and Galati, G., 1984, Clinical usefulness of serum
ferritin measurements in lung cancer patients, J.NucI.Med.A1I.Sci., 28:27.

Walters, G.O., Miller, EM., and Worwood, M., 1973, Serum ferritin concentration and iron
stores in normal subjects, J.Clin.Pathol., 26:770.

104
OUTCOME BASED BIOMARKERS OF FEMALE REPRODUCTION

John F. Jarrell
Clara Christie Professor and Head
Department of Obstetrics & Gynaecology
The University of Calgary
Calgary, Alberta, Canada, T2N 2T9

INTRODUCTION

In undertaking a review of female reproductive bio-

markers it is possible to identify at least two distinct
approaches, both of which are relevant to the sUbject and
may have generalized utility. The first is represented by
an approach of linking comprehensive lists of tests of the
physiology of reproduction within the various stages of the
reproductive process. This has been the major thrust of the
National Research council 1 . This technique is extremely
helpful to those not necessarily familiar with reproduction
or the most recent developments in reproductive technology.
We wish to introduce another approach which identifies
specific priority outcomes first, using epidemiologic
principles 2 and then develops specific programs which will
utilize, develop and characterize biomarkers appropriate to
the specific problem at hand. As toxicology in general and
reproductive toxicology in particular is developing into a
substantial interdisciplinary science, the use of outcomes to
drive the investigations and risk assessment is consistent
with modern clinical practise.
It is the intention of this review to present an overview
of the sUbject of female reproductive biomarkers in relation
to both normal and abnormal function; to describe female
reproductive biomarkers from the perspective of dose and
response and finally, provide several examples of the

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 105
attributes of the problem oriented approach directed to
premature menopause.

DEFINITIONS

A reproductive biomarker can be defined in many ways. It

defines a relationship between the exposure of a subject and
the resultant transient or permanent disability to
reproductive or developmental health. The term refers to the
use made of information gained from a diagnostic test, rather
than the specific type of information 3 A biomarker is a
change in a biological system that can be related to exposure
to, or effects from a specific xenobiotic.
The ideal biomarker is specific for a chemical, detec-
table in small quantities, measured by non-invasive tech-
niques, inexpensive, associated with prior exposure and which
has an excellent positive predictive value to a specific
disease state 3 . There will be few biomarkers with such
strengths, particularly in the reproductive field.
In addition to general definitions, exposure biomarkers
provide information regarding contamination of tissues com-
partments by xenobiotic (s) An example is the presence of
pesticides in seminal fluid. Exposures include external
sites (skin), interna I sites (serum) or actual critical sites
(ovarian follicular fluid, site of meiosis; fallopian tubal
fluid, site of fertilization)4. Appropriate use of exposure
biomarkers provides information on early (potentially rever-
sible) and late (potentially irreversible) effects.
Biomarkers in general have been grouped into those which
are used for environmental monitoring, biological monitoring
and health monitoring 4 . Environmental monitoring assesses
the potential for human exposure as weIl as the impact of
time, geography, social demographic variables and effec-
tiveness if regulation. Biological monitoring measures human
exposure in a variety of sites and is an integral part of the
process of risk assessment. Health monitoring attempts to
focus on the surveillance of disease states and sentineis of
health such as public health re cords relating to births and
deaths within a region.
It is relevant to indicate what may represent good
biomarker function. Particularly in reproduction, the closer

106
the marker is to the endpoint of interest the more economical
and relevant it will be. The biomarker will only be as good
as our ability to separate those who exhibit the outcome
under evaluation and those that do not.

SPECIAL CONSIDERATIONS INREGARD TO REPRODUCTION

Successful reproduction is a socially, economically and

physiologically defined health state related to the regen-
eration of the species. The social context is entirely rele-
vant for, in human, unlike any other species one must keep
track of desired outcome and for many, anything which
interferes with contraception can be considered a repro-
ductive toxicant.
Reproduction is also simul taneously a continuous and an
intermittent process. For example, spermatogenesis is con-
tinuous beginning very early in young boys and must be
sustained over many years to permit the limited number of
actual fertilizations which result in a clinical conception
and possibly a live birth. Thus significant toxicity could
theoretically be present and not appreciated until events are
desired. For practical purposes we wish to know if important
reproductive outcomes within a community are altered in some
measurable way due to the presence of one or more xenobiotic
exposures by the using certain markers. The driving force is
the identification of relevant reproductive outcomes.
In an excellent review of the selection of reproductive
outcomes, Savitz and Harlow identified scientific issues
which should be carefully consideredi severity of the out-
comes, relative sensitivity of outcomes to environmental
exposure, interrelationship of adverse outcomes, frequency of
outcomes, toxicological information and specificity of repro-
ductive effects 2 To this list one might add the strength of
the positive predictive value of the diagnostic test and
because positive predictive values are prevalence dependent,
the actual prevalence of the reproductive outcome(s) in the
community to be evaluated. certain practical considerations
include measurement considerations, cost vs benefit, power
analysis, relevance to the community and feasibility given
the intrusiveness of reproductive testing.

107
BIOMARKERS OF FEMALE REPRODUCTION

Biomarkers of female reproduction are presented in Table

one.

Table 1. Current and Potential Female Reproductive

Biomarkers (Modified from Reference 1)

Marker Limited Human Large Scale

Studies Studies

1. Exposure Markers
Blood urine saliva xx xx
Tissues xx xx
Follicular fluid xx
Peritoneal fluid xx
Placentae xx xx
Cord blood xx xx
CSF xx

2. Genotoxic Markers
Ovaries xx
Oocytes xx
Placentae xx xx
Maternal derived xx xx
Fetal blood
Fetal blood xx
Unscheduled DNA
synthesis
maternal
lymphocytes xx xx
fetal
lymphocytes xx
Sister chromatid
exchange
maternal
lymphocytes xx
fetal
lymphocytes xx
Chromosomes
abortus xx xx
chorionic biopsy xx xx
amniotic cells xx xx
maternally
derived fetal
cells xx xx
Micronuclei
maternal blood xx
Specific locus tests xx

108
 Table 1. (Continued)

Marker Limited Human Large Scale

Studies Studies

3. Development and Aging

Puberty xx
Thelarche xx
Adrenarche xx
Menarche xx
Menstrual cyclicity xx
Ultrasound xx
MRI of ovaries xx
Inhibin xx xx
Reproductive hormones xx xx
Ovarian perfusion

4. Menstrual Function
Cycle characteristics xx
Ovulation detection
ultrasound xx
BBT xx xx
progesterone xx xx
LH kits xx xx
cervical mucous xx
vaginal biophysics xx
endometrium
biopsy xx
receptors xx
enzymes xx
endocrine assays xx
in vitro assays xx
ovarian perfusion xx

5. Fertilization,
Implantation and Loss
Clinical Abortion xx
HCG xx xx
EPF xx
PEP xx
suppressor activity xx

6. Use of New Reproductive

Technologies in Biomarker
Formulation
Response to
induction xx
Oocyte recovery xx
Oocyte cleavage rate xx
Oocyte chromosomes xx
In vitro oocyte assays xx
Early embryonic
Development xx
Polyploid human embryos xx
Computerized sperm analysis xx

109
 Savitz and Harlow recommended reproductive risk assess-
ment include measures of fecundability (menstrual function
and time to pregnancy), fetal loss (clinical abortion rate,
infant health (birth weight and gestational age). Because of
the relevance to society in terms of cost and potential
disability we have engaged premature loss of oocytes or
premature ovarian failure as an additional priority.
This represents a modified summary of the female
reproductive biomarkers compiled by the National Research
council. This is an excellent source for identifying
strategies to pursue reproductive outcomes using established
methodology.

OUTCOHE BASED BIOMARKERS IN FEHALE REPRODUCTION

The term reproduction explicity expresses interest in a

product or an outcome. Emphasizing outcomes as the basis of
biomarker characterization and development is consistent with
an organized approach to the study of agents which interfere
with regeneration of the species.
There are several considerations to be made using this
approach;
a) priority of the outcome under review
b) relationships of the markers and outcome defined
c) aspects of diagnostic test assessment considered
sensitivity
specificity
positive predictive value
negative predictive value
receiver operating characteristic curves
d) presence of clinical confounders which also produce the
same outcome
e) prevalence of the outcome in different groups of
populations.
In proposing outcome based strategies there are two
potentially competing factors which have to be addressed.
For a biomarker to have utility and cost effectiveness, it
should have some relationship to the prediction of an
untimely event. Secondly, there are only so many ways that a

110
reproductive toxicant can be expressed clinically. These
patterns also overlap with important disease studies which
must be considered in terms of the ultimate interpretation of
the biomarker results. Rather than emphasizing the disease
status it is proposed these be considered focus group to
indicate the multiple causes (and diseases) expressed in
limited clinical situations.

Readily identifiable focus groups of female reproduction

include menstrual abnormalities, premature ovarian failure,
infertility and recurrent pregnancy loss. These are closely
linked and there is considerable overlap among several.
Nevertheless, reasons for isolating these are the relative
frequency of occurrence, ease of clinical recognition and
diagnosis, recognition of other confounding clinical
conditions and the ability to develop different strategies
for analysis of risk (Figure 1 - 3).

FEMALE MALE

PREGNANCY

Intrauterine
Growth
Restriction
Figure 1. Focus groups of reproductive toxicology

111
 INTEGRATION OF BIOMARKERS INTO FOCUS GROUPS
INFERTILITY REPRESENTATlVE BIOHARKERS

FEHALE - ANOVULATION

1. Biochemica1
serum P4, LH:FSH, PRL
sa1ivary P4

2. Physica1
Ultrasound
follicle tracking
corpus luteum
doppler
CONFOUNDING CLINICAL DIAGNOSIS endometrial
thickness
Unexplained inferti1ity biopsy

Female related 3. NRTs

Endometriosis estradiol response
Tubal disease oocyte recovery rate
oocyte cleavage rate
Male related embryo cleavage rate
Semen abnormally comparison to HPT
uncleaved oocytes
polypoid embryos

4. Epidemiological
time to pregnancy

5. Ovarian Perfusion
System
oxygen consumption
ATP
marker enzymes etc.
Fi.gure 2. Infertility focus group

It is important to recognize that this use of focus grouping

does not indicate those individuals to whom the biomarkers
should be administered. Restricted use to those exhibiting a
disease may in fact make it more difficult to identify an
effect in the general communi ty2 . For example, i t may be
much more difficult to establish that a certain chemical is
influencing the rate of spontaneous abortions by studying
only those who have recurrent pregnancy loss who have a
recurrence rate of clinical abortion of approximately 40%
compared to the general rate of 10 15%. This is also
relevant to measures of fecundity such as time to pregnancy
among the general population vs the infertile.

112
 INTEGRATION OF BIOMARKERS INTO FOCUS GROUPS
1. Biochemical
Serum FSH, LH, E2
Urine FSH, LH, E2
Salivary E2, P4
BBT
ovarian Perfusion
Inhibin
Follicle fluid
samples

2. Physical
Ovarian Ultrasound
Doppler
Volume
Follicle Measure
CONFOUNDING CLINICAL DIAGNOSIS Corpus luteum

Idiopathic premature ovarian 3. Epidemiological

failure Last menstrual per iod
Multiple endocrinopathics correlates of
Previous radiation follicle fluid
Previous chemotherapy
Galactosemia
Resistant gonad syndrome
Chromosomal abnormalities
ie. gonadal
dysgenesis
Figure 3. Premature ovarian failure focus group

USE OF OUTCOME BASED BIOMARKERS - PREMATURE OVARIAN FAlLURE

Our group has been directing its efforts at establishing

biomarkers of premature ovarian failure by concentrating on
1) the evaluation of serum FSH as a biomarker for ovarian
toxicity and 2) characterizing biological markers for
hexachlorobenzene, a potent toxicant of primordial germ cells
3) studying composite ovarian function by means of ovarian
perfusion to study mechanism of oocyte destruction.

SERUH FSH AS A BIOMARKER

The serum FSH has been shown to be elevated as a
consequence of ovarian failure although the actual specifics
of regulation with respect to the components of the ovary
have not been validated. Rats were sUbjected to either
direct ovarian radiation alone or in combination with

113
cyclophosphamide. The irradiated ovary of the rat is capable
of estrogen production despite widespread loss of follicles.
When the follicle counts were correlated to the resultant
serum FSH levels, it was apparent that the serum level of FSH
was highly correlated with the calculated volume of the
healthy antral follicles, determined by a bioquant system 5 .
There was no correlation with primordial germ cells. This
relationship was present only in those rats which had been

(I SD AaDYE MUN)
.8

>
t- .6
:;
~ MEAN FSH .1-
MlA.TIPLES OF 8D
Ci; .4
Z
W
U) .2

-.1 0 .2 .4 .6 .8 1
FALSE POSITIVES
Figure 4. Receiver operating characteristics curve (ROC
curve) of the best diagnostic cut of the relationship of log
10 volume of antral follicles and multiple of the standard
derivation of serum FSH.

treated with the combination of cyclophosphamide and radia-

tion. Under these circumstances, there was a dose-related
reduction in serum estradiol levels. The findings are
represented by receiver operating characteristic curves
(Figure 4).
The finding of the ROC curve is that under hypergonadotropic
hypergonadal influence, elevation of the serum FSH by 2
standard definitions indicates a significant reduction in
antral follicle volume.

114
NEW REPRODUCTIVE TECHOLOGIES AS BIOMARKERS OF REPRODUCTIVE
TOXICITY
Hexachlorobenzene, an ubiquitous organochlorine has been
identified as a priority chemical by the International Joint
Commission studying the Great Lakes of North America 6 .
Reason for concern relate to the persistence of HCB in the
ecosphere of the Great Lakes, landfill sites and certain
biological species. We have identified that HCB can be
isolated from ovarian follicular fluid of women receiving in
vitro fertilization in three cities in Canada 7 (Figure 5).
Although we cannot identify the source of contamination, the
concentrations do differ geographically7. These
concentrations were not identified to have a deleterious
effect on reproductive outcome in terms of oocyte recovery or
cleavage rate. We are now beginning to correlate findings
with pre-existing clinical conditions.

.....
(A) pc. (.) HC. (C) DDE

.-

. .-
....

,,, ..
....
. ~B$
,,,
~~$
--- ---.
--
(D) ALCH (E) OXCH (F) TOTAL

...
... ...

J~ j~.

... ...
...J J~
--- ~~cb
,,,---. ... J~ ~~~
--- ---. ,,,
IML ..... .,.,. ..... MMI .....

Figure 5. Boxplots of serum(s) and follicular fluid (FF)

samples in three Canadian cities; Halifax (HAL) , Hamilton
(HAM) and Vancouver (VAN).

115
 HeB has been shown to cause the destruction of primordial
germ cells of the ovary of the Rhesus monkey8. These
investigations were carried out using small numbers of
monkeys (n=5) and very high concentrations of HeB (up to 128
mg/kg BWt/day). In addition to the loss of germ cells, there
was a thickening of the surface of the ovary and a reduction
in the number of corpora lutea suggesting the induction of
anovulation. These findings were consistent with other
species which showed that HeB was taken up by the ovary in
general and the follicle in particular 9 . We initially
studied the effects of HeB in the cynomolgus monkey at
concentrations which were comparable to those of Iatropoulos
et al but the animals demonstrated generalized toxicity and
the doses had to be reduced. Nevertheless, there was
evidence from wet weight of the ovaries and pituitaries in
conjunction with follicle counts that the lesion was likely a
direct effect on the ovary8.
Subsequent investigations reduced the dose of admin-
istered HeB to 0.1, 1.0, and 10.0 mg/kg/ day10. Even at these
low doses, there was evidence of ul trastructural damage to
the primordial germ cell without any evidence of systemic
toxicity. It is of interest that the high dos es did effect
significant reductions in the numbers of primordial germ
ce11s. There were no effects on the menstrual function,
fertilization of oocytes collected or early embryo
development at in vitro fertilization (Table 2).
Thus the findings would indicate a relevant separation of
focus groups which separate the out comes of menstrual
function and premature ovarian failure or premature meno-
pause.
This indicates that oocyte destruction, is similar to
atresia i ts physiological counterpart, and remains a quie-
scent process which does not necessarily result in abnormal
menstrual function. This is consistent with the observations
of Mattison regarding the effects of smoking and beno (a)-
pyrene on oocyte destruction, infertility and onset of
menopause ll - 13 .

OVARIAN PERFUSION TO DEVELOP REPRODUCTIVE BIOMARKERS

In addition to these techniques we have been evaluating
the ovarian biomarkers of toxicity via the in vitro isolated

116
Table 2 The Effect of Hexachlorobenzene on Ovarian Histology
and In vitro Fertilization Outcome
Anima1s were treated with HeB in the doses indicated for 90 days and
subjected to in vitro ferti1ization.
* mean 1 SEM
REPROOUCTlVE PARAITER OOSE Cf HeB mo/kg

o 0.1 1.0 10.0

Ovarlan wt(g) 1.84. 0.31 2.66. 0.51 1.59. 0.22

Antra1 Fo111e1es 58. 11 115. 26 110 9 59. 26

Pnoantra1 Fo111e1es 1709. 206 1823. 326 1644. 309 1067:1: 491

Prlmordla1 Fo111e1es 26348. !11!60& 19473. 4504 24027. 3717 8737. 3047b

Corpora Lutea 1.75. 2.48 1.50:1: 0.29 1.50:1: 0.65 1.75:1: 0.65

Fo111eles Asplrat.ed 28.0 1.0 31.5:1: 5.2 31.75. 4.5 30.0 :I: 1.1

Ooe,ytes Reoovel"Old 15.5 :I: 2.7 18.5. 1.2 23.2:1: 0.~5 15.5 :I: 3.8

I Fertl11zatlon 1 14.88:1: 8.70 14.25. 4.29 21.52. 8.19 9.67:1: 6.62

I Fertl11zatlon 2 23.38. 7.04 18.13:1: 4.41 25.13:1: 11.67 11.25:1: 7.84

I Fertl11zatlon 3 30.75 :I: 16.42 25.25 :I: 13.43 34.0. 7.23 11.25. 7.89

1,2,3 refers to the assessment times of 22-24 h; 41-46 h; 65-70h

fo11owing insemination for the detection of either pronuc1ear formation
or ce11u1ar c1eavage as evidence of ferti1ization.
Note two embryos inc1uded in % ferti1ization 3 treated with 10 mgjkgjday
were disintegrating.

ovary perfusion system. This technique has been established

by Dr. P.O. Janson for physiogical studies of the ovary and
only recently have we identified its use in toxicity testing.
Of relevance to ovarian toxicity, we have determined that the
ovary is capable of the release of glutathione and the
release is dependent on the stage of the estrous cycle
(Figure 6), and the release can be inhibited by the perfusion
of buthionine sulfoximine, an inhibitor of glutathione
synthesis (Figure 7)14. These findings would indicate that
the release of glutathione from the ovarian perfusion system

117
 CONCENTRATION OF GLUTATHIONE
IN PERFUSATE
0.8
,...,.
E
"-

0.6
E
c
'-'
I.LJ
z 0.4
0
:i:
~
"'...J"
:::l 0.2
~

0.0
1 2 3 456 7 8
TIME(hours)

Figure 6. Adult cycling rats were sacrificed and the right ovaries
subjected to isolated in vitro organ perfusion. There is a significant
overall difference in rates of release of glutathione with the highest
levels present in estrus.

CONCENTRATION OF GLUTATHIONE
IN PERFUSATE

=
E
-::::::.
~ 0.8
1.0
O-Ocontrol
.-.aso 1.25nmol/.ml
/l.-/l. aso 12.5nmoll.ml
- aso 12.5nmoli'ml
added at T=2hrs.
J
c
""'
~ 0.6
o
I
!;( 0.4 ~:;/e_o
I/ft~~
3
(!)
I~O . ~
0.2 T~~ I_l_~"""""'"
~~_"' _ _

O.O~~~~=-~~--~~-~~~--=~~==~~==~r--
1 2 345 6 7 8
TIME(hours)
Figure 7. Adult cycling rats were sacrificed on estrous. Isolated
ovaries were perfused with buthionine sulfoximine. There was a time and
dose related reduction in ovarian gluatathione release.

118
may in fact be a biomarker for relevant toxification and
detoxification within the integrated follicle of the ovary.

SUMMARY

The outcome based biomarker principle permits a strategie

approach to defining a relationship between exposure and
disability. As a general principle new biomarkers are
selected to sharpen the approach. In the case of ovarian
senescence, this may invol ve computer enhanced ul trasound
imaging, and magnetic resonance imaging.

ACKNOWLEDGEMENTS

The author wishes to acknowledge the support of Health and

Welfare Canada, the National Cancer Institute of Canada and
the Nat Christie Foundation. The assistance of Glennis
Brittain is appreciated.

REFERENCES
1. National Research Council. Biologie Markers in Reproductive
Toxicology. Washington, D.C.: National Academy Press, (1989).
2. D.A. Savitz and S.D. Harlow, Selection of reproductive health end
points for environmental risk assessment. Environ Health Perspect
90:159 (1991).
3. R.F. Henderson, W.E. Bechtold, J.A. Bond and J.D. Sun, The use of
bio1ogica1 markers in toxico1ogy. Crit Reviews in Tox. 20:65 (1992).
4. D.R. Mattison, An overview on biological markers in reproductive and
deve10pmental toxicology: concepts, definitions and use in risk
assessment. Biomed and Environ Sei. 4:8-34 (1991).
5. J.F. Jarrel1, D.R. Mattison, E.V. YoungLai and A. McMahon, Serum FSH
is a biomarker for the volume of healthy antral follicles in rats
exposed to ovarian irradiation and cyclophosphamide. Soc for Gyne
Invest. 266P:166 (1987).
6. Government of Canada Toxic Chemicals in the Great Lakes and
Associated effects. ottawa: Minister of Supply and Services Canada
(1991).
7. J.F. Jarre11, D.C. Vi1leneuve, C.L. Franklin, et al. Contamination

119
 of Human Serum and Ovarian Follicular Fluid In Three Canadian
Cities. Can Med Assoc J. accepted (1992).
8. M.J. Iatropou10s, W. Hobson, V. Knauf and H.P. Adams. Morpho10gical
effects of hexachlorobenzene toxicity in female rhesus monkeys. Tox
& App Pharm. 37:433 (1976).
9. Ingebrigtsen K. Hexachlorobenzene: Proceedings of an International
symposium. In: C.R.Morris, J.R.P.Cabra1, eds. Comparative studies on
the distribution and excretion of 14C-hexachlorobenzene by wholebody
autoradiography. Lyon: IARC Scientific Publications. 277, (1986).
10. J.F. Jarrell, A. McMahon, D.C. Villeneuve et al. Ovarian germ cell
destruction in the monkey with hexachlorobenzene in the absence of
induced porphyria. Rep. Tox. accepted, (1992).
11. D.R. Mattison and S.S. Thorgeirsson. smoking and industrial
pollution, and their effects on menopause and ovarian cancer. The
Lancet. 187, (1978).
12. P.J. Thomford and D.R. Mattison. The effect of cigarette smoking on
female reproduction. J Arkansas Med Soc. 82:12;597, (1986).
13. K. Shiromizu and D.R. Mattison. Murine oocyte destruction following
intraovarian treatment with 3-methylcholanthrene or 7, 12-
dimethylbenz(a)anthracene: protection by alphanaphthoflavone.
Teratogenesis Carcinog Mutagen. 5:6;463, (1985).
14. N. Clague, M. Sevcik, G. Stuart, M. Brannstrom, P.O. Janson, J.F.
Jarrell. An evaluation of the effect of the estrous cycle and
buthionine sulfoximine on glutathiane release fram the in vitra
perfused rat ovary. Rep Tox, accepted, (1992)

lW
FACfORS DETERMINING TUE EXPOSURE OF TUE EMBRYO
AND FETUS: SPECIES VARIATION OF TERATOGENESIS
AND PLACENTAL TRANSFER OF XENOBIOTICS

HeinzNau

Institute of Toxicology and Embryopharmacology

Free University Berlin
Garystrae 5
D-lOoo Berlin 33
Germany

'~he fundamental and analytical aspects of a problem

are intimately interrelated;
they are almost like the two sides of the same coin"
Pete Kolthoff

INTRODUCTION

It becomes increasingly c1ear that the dose is often an unsatisfactory

correlate to the toxic effect produced by a drug or environmental agent. The effect
in a particular target organ is strictly dependent on the concentration-time
relationships of the drug and/or its active metabolite(s) in the target tissue, or, as
in the case of "irreversible" pharmacokinetics, on reactive metabolites formed or
specific binding phenomena (Momo, 1992). Thus, an effect is dependent on the
chemical structure of the xenobiotic (intrinsic activity), the exposure of the target
tissue, and the susceptibility or sensitivity toward a particular insult
(pharmacogenetics) (Scheme 1). This general concept will be discussed here with

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. 1iavis, Plenum Press, New York, 1993 121
regard to drug teratogenesis, where it is particularly helpful to explain and
overcome species differences, which are so extreme in this area of toxicology.
Emphasis will be placed on factors which determine the exposure of the embryo to
foreign substances.

Scheme I
DOSE

I CHEMICAL STRUCTURE

EFFECT I
intrinsie activity

embryonie exposure

susceptibility

Several aspects are of particular importance in developmental toxicology

and teratology: 1. the rapid change in the developing organism; 2. the relatively
"narrow window of sensitivity" of specific effects; and 3. the early stages of gestation
during which many major malformations are induced. In the human and monkey,
thalidomide was shown to induce the dramatic limb defects during the fourth week
of development when limb buds just become visible (Neubert et al., 1988, Merker
et al., 1988) (Fig. 1). The antiepileptic drug valproic acid can induce Spina bifida
aperta, a severe defect which is also induced in the human during early gestation
(4th week of gestation), in 1-2% of exposed cases (Robert, 1988; Lindhout and
Schmidt, 1986). In the mouse, another form of neural tube defect (exencephaly) is
produced by valproic acid around day 8 of gestation (Nau, 1985b; 1986b), while
Spina bifida is induced about one day later during a very short period of sensitivity
(Fig. 1) (Nau et al., 1991; Ehlers et al., 1992).
These short and early periods of sensitivity make it necessary to work with
very small amounts of tissue and to insure that appropriate exposure has occurred
during that time. Modern analytical techniques have made it possible to study the
concentrations of drugs and metabolites in sub-mg quantities of tissues, thus
facilitating the analysis of the distribution of substances within the early embryo,
although autoradiography is still the method of choice for distribution studies.

122
 6 4 5 7 9
month of pregnancy (human)

.
--
0

}. exencephaly
...
0

I I I I

0 2 4 6 8 10 12 14 16 18 20
day orgestalion (mouse)

Figure 1. Top: Human gestation; The maximum sensitivity towards the cbaracreristic limb
defects induced by thalidomide occurs during tbe fourtb week of development; during tbat period,
tbe antiepileptic drug valproic acid can produce Spina bifida aperta in 1-2% of exposed cases.
Bottom: Mouse gestation; valproic acid induced neural tobe defects; exencephaly is
produced by administtatiOll around day 8 of gestation, Spina bifida (aperta and occulta) is
produced on day 9 of gestation. (For references see Text).

123
SPECIES DIFFERENCES

Species differences is still one of the most important problems in toxicology,

and in particu1ar teratology. Thalidomide was shown to produce characteristic limb
defects in human and subhuman primates, but not in the rat and the mouse, even
with doses several orders of magnitude above human therapeutic doses (Table 1);
the rabbit appears to be responsive to thalidomide, at least at high dosing (Neubert
et al., 1988; Merker et al., 1988; Sterz et al., 1987; Schmahl et al., 1989).

Table 1. Teratogenic dosing regimen resulting in distinct malformations

Substance Species mg/kg/day

Thalidomide rat, mouse > > 100

(limb defects) monkey 0.11

Valproic acid mouse 200

(neural tube defects) rat ?
monkey ?
human 20-30

Isotretinoin mouse 100

rat 150
rabbit 10
monkey 2.5 - 5
human 0.4 -1

Isotretinoin, a retinoid used for treatment of severe acne and other skin conditions,
showed similar, if not as extreme species differences: Also here, the human was
very sensitive toward this drug, followed by the monkey and the rabbit; the mouse
and rat were again relatively insensitive (cf. Nau, 1990). Valproic acid was shown to
induce Spina bifida with high therapeutic doses; in the mouse, lO-fold higher doses
were needed (Nau et al., 1991).

124
 The reason behind the species difference and mechanism of thalidomide
teratogenesis is still unknown and pharmacokinetic studies have not yet added to
our knowledge here. Pharmacokinetic studies are well on the way to explain most,
if not all of the major species differences in regard to isotretinoin teratogenicity.
Valproic acid-induced neural tube defects can at least in part be explained by
pharmacokinetics, although differences in sensitivity also playa role here.

MATERNAL PHARMACOKlNETlC FACTORS

Exposure of the embryo will be determined by all pharmacokinetic factors in

the maternal organism, Le. absorption, distribution, binding to plasma and tissue
proteins, metabolism and excretion (Monro 1992; Ings, 1990). Difficulties with
absorption can usually be corrected by a choice of the site of administration and
the formulation used. Differences in the volume of drug distribution are also not
usually a major reason for species variation (Nau, 1986a).

The most drastic differences between species are found with regard to drug
elimination, and both excretion and metabolism can exhibit species differences
(Smith et al., 1990). Drugs and environmental agents are usually cleared much
more rapidly in experimental animals than in man: the half-lives and clearance
values can easily differ by at least one order of magnitude or more (Table 2). The
reasons behind these differences are not always clear, but probably lay in the
differences in body surfacejbody weight ratios which are much higher in laboratory
animals than in man (Table 2). Thus, small animals must have high metabolie rates
in order to maintain physiological body temperature. The concept of allometric
scaling has been developed for species comparison to scale the variable of interest
Y according to body weight W (a is proportionality factor) (Lutz et al., 1980;
Mordenti, 1986):

The exponent b is approximately 0.25 for biological times such as half-lives

and 0.7 for clearance and metabolie rates. This concept proved useful as an
empirical approach, but bears little physiological meaning. Thus, physiological
pharmacokinetic models have recently gained popularity. They are anatomically,
physiologically, thermodynamically and biochemically realistic, but are difficuIt and
expensive to perform. Here, a flow scheme is created where all important organ
and tissue compartments are connected via the circulatory system, and binding

125
parameters, lipid solubility, ionizational and partition coefficients of the drugs in
the various tissues come into play.

AUC and Cmax Values as Correlates to the Teratogenic Response

Due to the short half-lives of many drugs in experimental animals, the

pharmacokinetics differ greatly from that of the human. The human is often
exposed throughout the sensitive periods of gestation, although drug fluctuations
mayaiso occur here. However, these drug level fluctuations are much more drastic
in animals, and high drug concentration peaks alternate with periods of "drug
holidays" (Nau et al., 1981; Nau, 1985b; Scialli, 1992) (Fig. 2).

Table 2. Body weights, surface areas and conversion factors

of dosing from mg/kg into mgjm2.

Dose
Body Surface area Conversbon equivalent/
Species weight, kg m2 factor a, kg C

Mouse 0.02 0.0066 3.0 U.O

Rat 0.15 0.025 5.9 6.0
Dog 8 0.40 20 1.7
Monkey 3 0.24 12 3.0
Humans
Child 20 0.80 25 1.5
Adult 60 1.60 37 1.0

aLiterature data from Chodera and Feiler, 1978; Freireich et al., 1966
b.ro convert a mg/kg dose in a given species into an equivalent mg/m 2 dose, multiply the dose by the
conversion factor.
cDose equivalent for the adult human is set as 1.0.

It is therefore of importance to establish if peak levels or AUe values

correlate with the toxic effect observed (Table 3). For valproic acid it is dear that
peak levels are the decisive correlates as has been demonstrated by studies of the
concentration-response relationships of various dosing regimens induding infusion
via osmotic minipumps (Nau et a1., 1981; Nau, 1991a; Nau, 1985b). Thus, high
single daily doses should be avoided in therapy, and the daily dose should be
divided into several portions to minimize the risk.

126
 log Concentration

- ,,-
VPA (jJ9j ml)
Mouse

Human

30 ,
. . . . .. . ... . .
w o. ... ' . ' . ' , .. , ' , ' , ' , ' , ' . " , '

"
~

T , . ..
'

Human

4 5 7 ~ 9 h 1'1
Day of ges1ation
~N:i:nEX~:e~~aiY'f:tf~ !:~~r~~l ~
~

Figure 2. Comparison between plasma pharmacokinetics of antiepileptic therapy with valproic acid in
the human with the plasma kinetics following a teratogenic dosing regimen in the mouse. Note the
persisting exposure in the human vs. the considerable drug holidays intermittedly broken up by large
concentration peaks in the mouse.

127
 Table 3. Pharmacokinetic corre1ates of teratogenesis

Teratogenic effects correlate with:

Aue

valproic acid retinoids

caffein cyclophosphamide
ethanol salicylate
tetracycline

for references see reviews: Nau, 1986.

The AUC's are the decisive correlates with retinoid teratogenesis. Here,
relatively low concentrations in the ng/ml plasma range are teratogenic, if they are
maintained throughout the sensitive periods (Lfberg et al., 1990; Nau, 1990). It
has been estimated that the sensitive periods of the human are of longer duration
than of experimental animals [factor 3 to 4 (Neubert and Chahoud, 1985)]. This
could mean that the drug has a longer time to act on the developing human
embryo; the AUC accumulates, which may be one important reason for the
sensitivity of the human embryo as compared to the embryo of experimental
animals.

Because of the short half-lives of drugs in experimental animals and the

short developmental periods, the drugs may have to be administered several times
within a relatively short interval for a thorough study of particular defects ("Cmax
drugs"). For "AUC drugs", infusion throughout the sensitive periods of gestation
would be a good approach, although this technique is often technically difficult to
perform, and sometimes results in overwhelming unspecific effects (such as
embryolethality) which disguise specific malformations.

PLACENTAL TRANSFER

The transfer of most drugs and environmental agents occurs by simple

diffusion, and the concept of the placenta as lipoid membrane(s) is useful in
describing the influence of the physicochernical characteristics of the drugs on their
placental transfer. Rapid transfer will be related to high lipid solubility, low
ionization and low plasma protein binding of drugs with a molecular weight below
1000. Low placental transfer is related to extensive plasma protein binding,
extensive ionization (e.g. quarternary ammonium salts such as in tubocurarine;

128
elementary ions such as Cd, Hg) and large molecular weights (> 1(00) (cf. Mirkin
and Singh, 1976).

There are, however, many exceptions to tbis general concept. Many drugs
are weak acids or bases (see below) and are ionized extensively at physiologica1
pH; still, they generally transfer weIl to the embryo. Chlorinated aromatic
hydrocarbons such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) may have such a
high partition coefficient that they are tightly bound to membranes, and stored in
matemal liver and adipose tissue, thus limiting the placental transfer. The
concentrations of TCDD in the embryo following teratogenic dosing are in the nM
range, several orders of magnitude below corresponding matemaIliver levels (Nau
and Ba, 1981; Nau et al., 1986). These embryonic concentrations are sufficient to
interact with a receptor in the embryo which is possibly related to the toxic effect
produced.

The exposure of the embryo will depend on the rate as weIl the extent of
transfer. Factors determining the rate and extent of transfer are summarized in
Table 4.

Table 4. Factors determining the rate and extent of drug transfer to the
embryo/fetus.

Characteristics of the
Transfer
drug matemal/
placental/
fetal unit

Rate lipid solubility placental structure

molecular weight and function
structural char~teristics placental blood flow
protein binding thickness of
type of transfera placental membranes

Extent degree of ionization matemal/fetal

(PKa) pHgradient
protein binding
type of transfera

~ passive diffusion, facilitated or active transport, pinocytosis.

if transfer is relatively slow.
from: Mirkin and Singh, 1976; Nau, 1986a and 1991.

129
lonization oe Drugs

As mentioned previously, fully ionized substances are only to a very limited

degree transferred to the embryo and fetus. The ionization of weak acids or bases
does not appear to limit the transfer of such drugs. Salicylate or valproate,
essentially fully ionized at physiological pH, are transferred rapidly to the embryo
and fetus. The small portion of nonionized drug is apparently rapidly transferred,
and the equilibrium between the ionized and nonionized forms is rapidly re-
established for further transfer of the nonionized form (Nau, 1991b).

Thus, the extent of transfer at postdistributive equilibrium will depend on

the pH difference between the maternal blood and the embryo/fetus. Surprisingly,
the tissue pH of the embryo during early organogenesis has been found to be
higher than maternal plasma pH in experimental animals such as the mouse, rat
and monkey. Therefore, weak acids accumulate in the early embryo relative to
maternal plasma (Nau and Scott, 1986). It should be mentioned in this regard that
most human teratogens are weak acids. It is not known if the mechanism of these
acids is related to the relatively high embryonic pH during the sensitive periods.
Certainly, the intracellular pH is involved in regulation of a number of biological
functions such as cell proliferation, DNA, RNA and protein synthesis, intercellular
communication and possibly the switch from glycolytic pathways to oxidative
phosphorylation. Any alteration of this intracellular embryonic pH could therefore
carry grave consequences for development. It is unknown how acidic drugs alter
embryonic pH, but the very strict structural requirements for an acid to express
teratogenic activity imply that very specific mechanisms operate here. We have
recently observed, that the teratogenicity of branched, short-chain acids related to
the antiepileptic drug valproic acid is stereoselective; one enantiomer is highly
teratogenic, the other exhibits much lower activity, while both enantiomers reach
the embryo in comparable amounts (Hauck and Nau, 1989; Hauck et a1., 1990;
Hauck et al., 1992). These results indicate that the interaction of these
stereoisomers with a chiral structure in the embryo (enzyme? transport protein?
receptor?) is crucially involved in the teratogenic mechanism.

During the fetal period, the pH of tissues and fluids are generally lower than
maternal plasma pH. The basic compound nicotine therefore accumulates in am-
niotic fluid, placenta and fetal serum in comparison to maternal plasma (Luck et
al., 1985) (Table 5). The mother's milk is also more acidic than plasma, and nico-
tine accumulates in that compartment as weIl. The metabolite cotinine, often used
as exposure parameter for smoking, does not exhibit such effects. Small amounts of

130
nicotine and cotinine are also found in infant's serum and urine from passive
smoking as weH as transfer via mother's milk.

Protein Binding of Drngs

The plasma and tissue protein binding will effect both the rate, but
particularly the extent of transfer of drugs across the placenta. Since only the free
(unbound) portion of the drug can cross membranes, maternal plasma protein
binding can have a limiting effect on placental transfer. For drugs with high transfer
rates, maternal plasma pro tein binding can have a transport role, thus increasing
placental transfer rates by presenting more drug to the placenta (cf. Nau, 1991b).

Table 5. Maternal smoking: nicotine and cotinine levels.

Ratio to maternal
serum concentration
Specimen Period
Nicotine Cotinine

Amniotlc fluid week 16-24 1.5 0.72

Placenta week 10 1.5 0.6
birth 2.6 0.74
Fetal serum birth 1.12 1.05
Mother's milk postnatal 2.9 0.8
Infant serum postnatal 0.06 0.15

For references cf. text.

The extent of placental transfer will depend on the relative binding

capabilities on both sides of the placenta. There is convincing evidence that the
binding affinity and number of binding sites of fetal proteins are similar to
maternal pro teins. Therefore, the extent of binding is governed by the relative
concentrations of binding proteins and binding modulators such as free fatty acids
on both sides of the placenta. Both of the two major binding proteins, albumin and
al-acid glycoprotein, are relatively low during the early and middle fetal period.
Many drugs bound to these proteins therefore reach lower concentrations in fetal
as compared to maternal blood (Krauer et a1., 1986; Nau and Krauer, 1986). This
situation continues until birth for drugs bound to the al-acid glycoprotein (such as
local anaesthetic agents of lidocaine and bipivacaine type) (Nau, 1985a). Albumin
levels in fetal blood during late gestation and at birth, however, exceed
corresponding maternailevels (which drop during gestation). Therefore, drugs such

131
as benzodiazepines, salicylate and valproate accumulate in fetal blood relative
tomaternal blood during late gestation and at birth (Hamar and Levy, 1980; Kuhnz
and Nau, 1983; Nau et al., 1984).

The free concentrations, however, are governed by the maternal/fetal pH

gradient and do not necessarily follow the same pattern as the total levels (see
above).

Selective Transfer Mechanisms

There is Httle evidence that therapeutic or environmental agents are

transferred to the embryo and fetus via active ar facilitated transport mechanisms.
These transport mechanisms are important for endogeneous substances such as
amino acids, some proteins, vitamins, trace elements, fatty acids, steroids, some
sugars and hormones, (except peptide hormones, which are not generally
transferred.) It may, however, be that some drugs, which structurally resemble
endogeneous compounds, can indeed be transported by similar specific
mechanisIDS. Retinoids, which are structurally and functionally related to vitamin
A, could serve as an example here.

CONCLUSION

Tbe extrapolation of teratogenicity studies from experimental animals to the

human is still problematic. It is demonstrated that the exposure of the embryo to
xenobiotics, in addition to the sensitivity of the target tissue, is crucial for
expression of developmental toxicity. Tbe teratogenicity of some drugs correlates
with the AUC values, of other drugs with the Cmax values reached in the embryo.
Tbe placental transfer of xenobiotics is governed by the physicochemical
characteristics of the drugs (PKa, Hpophilicity, molecular weight) as weH as the pH-
and protein binding gradient across the placenta.

Acknowledgements

Tbe original work described here from our laboratory was supported by the
Deutsche Forschungsgemeinschaft (Sfb 174, C6) and the Bundesgesundheitsamt
Berlin (ZEBET).

132
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136
ASSESSING REPRODUCTIVE RISKS
WITH BIOLOGICAL MARKERS1

Teresa Silvaggio and Donald R. Mattison

Graduate School of Public Health

University of Pittsburgh
111 Parran Hall
130 DeSoto Street
Pittsburgh, PA 15261

INTRODUCTION

This discussion suggests a general framework for assessing reproductive risks using
biological markers (Figure 1). Reproductive terminology and summary statistics on
reproductive outcomes will be presented. The process of risk assessment will be defined,
with attention to the assessment of reproductive risks. In addition, the influence of
physiological differences between men, nonpregnant and pregnant women on
toxicokinetics, biomarkers and risk assessments is discussed. An example reproductive
risk assessment using sperm concentration as a biological marker of reproductive function
will be presented.
U.S. statistics suggest that 10-15% of couples are infertile, 15-20% of pregnancies
end in recognized spontaneous abortions, 10-15 % end in unrecognized spontaneous
abortions (Kline et al., 1989), and another 2% end in stillbirths (Bloom, 1981). Some
studies suggest that total pregnancy loss may be as high as 75 % of all conceptions (Kline
and Stein, 1985). These data suggest that human reproductive success is not assured.
To complicate the problem further are environmental and occupational exposures which
may impact adversely on reproduction.

IThis review prepared for the NATO Advanced Research Workshop, Luso, Portugal, June 1-5, 1992,
includes material from three publications: (1) An Overview of Biologica1 Markers in Reproductive and
Developmental Toxicology: Concepts, Definitions and Use in Risk Assessment, by Donald R. Mattison in
Biomedica1 and Environmental Sciences 4: 8-34, 1991; (2) A Comparative Approach to Toxicokinetics,
by Teresa Silvaggio and Donald R. Mattison in Occupational and Environmental Hazards: A Guide for
Clinicians, edited by Maureen Paul, to be published by Williams and Wilkins, 1992; and (3) Methods for
Quantitative Assessment of Reproductive Risks, by Marvin Meistrich and Donald R. Mattison, to be
published in Assessing the Risks of Adverse Reproductive Outcomes, Monograph 4, Conte lnstitute for
Environmental Hea1th, Diane Brenner and Arthur Bloom, eds., March of Dimes.

Use 0/ Biomarkers in Assessing Health anti EnvironmentDllmpacts 0/ Chemical

Pollulallts, Edited by C.C. navis, Plenum Press, New York, 1993 137
 Reproduction and development are intricate processes and because of their
complexity are vulnerable to a wide range of biologica1, chemica1 and physica1 insults.
The processes involved in reproduction and development encompass many different
integrated biologica1 phenomena in the male, female and conceptus. Disruptions may
therefore occur at multiple sites along the pathway of normal reproduction as a result of
toxic exposures. Reproduction is unique in comparison to other systems in that it is
intermittently expressed; it is therefore necessary to evaluate the relationship between
exposure and function in a couple (male and female) in order to fuHy assess reproductive
toxicity. In addition, many indicators of reproductive function can only be evaluated in
a couple attempting pregnancy.

male fecundity pre-implantation pregnancy 1088

I
couple based _
I
oonception ____ implantation _ unreoognized
factoj I pregnancy 1088

female fecundity c l i n i c a l . reoognized

pregnancy pregnancy loss

premature
delivery
~ delivery
full-term

I
I I
developmental normal
defect infant

Figure 1. Fertility model used for biomarker-based risk assessments.

Modified from Mattison et al., 1990a.

In the sexually mature couple, successful reproduction assurnes conception,

successful progression of the pregnancy to term (38-42 weeks), effective delivery and
normal growth and development of the infant. Successful reproduction is dependent on
both individual and couple factors (Figure 1). Fecundity is the capacity of a male,
female, or a couple to produce offspring; fertility is the actual production of offspring.
A reproductive toxicant is a chemical, physica1 or biologica1 agent which alters male
fecundity, female fecundity or couple factors which may result in altered fertility.
Reproductive toxicity alters the ability of a couple to successfuHy achieve a clinica1ly
recognized pregnancy or increases pregnancy loss. A developmental toxicant is a
chemical, physica1 or biological agent which acts before or during pregnancy to alter the
survival, structure or function of the embryo or fetus.

COMPARATIVE TOXICOKINETICS - INTERACTION WITH BIOMARKERS

Physiologic differences between men, nonpregnant and pregnant women may have

138
a substantial impact on toxicokinetics (Silvaggio and Mattison, 1992). Physiological
parameters which may be important in toxicokinetics include: body weight, body
composition, surface area, blood volumes, organ and tissue volumes, basal metabolism,
cardiac output, minute ventilation, alveolar ventilation, respiratory frequency, totallung
capacity , vital capacity , functional residual capacity , gastrointestinal function, renal
function, hematologic parameters, and vascular bed volumes.
Physiologically based toxicokinetics incorporates pharmacokinetic factors into risk
assessments. The basic assumption stimulating this more detailed approach is that
physiological differences need to be delineated for accurate assessment of risk. Pertinent
physiological and toxicokinetic functions in pregnant women are summarized and
compared to those of men and nonpregnant women, where data allow. This approach
provides a means of exploring how differences in these factors may contribute to
variations in exposures, target tissue doses and responses to chemicals.
Changes in pulmonary, cardiovascular, renal, gastrointestinal, and hepatic function
occur during pregnancy and can alter toxicokinetic factors and affect the response of the
pregnant woman and fetus to certain chemicals (Hytten and Chamberlain, 1980;
Mattison, 1986, 1990; Mattison et al., 1991, 1992). Toxicokinetics quantitates
absorption, distribution, metabolism, and excretion of chemicals. Different models have
been employed to describe the effects of physiological adaptations during pregnancy on
the kinetics of xenobiotics (Mattison, 1986; Mattison et al., 1991, 1992). By correlating
the external dose of a chemical with its concentration at the target tissue (internal dose
biomarker) and observing the effect (effect biomarker), better estimations of allowable
exposure limits can be determined.

ABSORYfION

Absorption is the process by which xenobiotics cross body surfaces and enter the
bloodstream. Major sites of absorption are the gastrointestinal tract, skin and the
respiratory tract. Physiological differences between men, women and pregnant women
which may alter absorption are summarized on Table 1.
The extent of transdermal absorption of toxicants is an important determinant of
systemic effects (Mattison, 1990). According to Fick's Law of Diffusion, as the surface
area of the epidermis exposed to the chemical increases, the amount transferred per unit
time increases. Since the total body surface area is greater for males, the rate of
transdermal absorption from a given exposure may also be greater, all other factors being
equal. As described by Fick's Law, as the thickness of the barrier decreases, the rate
of transport increases. Differences in the thickness of the epidermis, in particular the
stratum corneum, may therefore impact on transdermal absorption. Depending on the
location, there are variations in epidermal thickness between males and females. In the
arms and fingers, the epidermis is slightly thicker in males (Southwood, 1955) which
may decrease transdermal absorption in the workplace.
Increasing the hydration state of the stratum corneum increases transdermal
absorption of many polar molecules. While sex-specific differences in the hydration state
of the epidermis have not been investigated, inferences may be made from data on the
extracellular and intracellular body water content in males and females. Total body.
water, intracellular body water, and extracellular body water are alilarger in males than
females and greater in pregnant than nonpregnant women, but still less than males
(Silvaggio and Mattison, 1992). Pregnant females and males may thus have increased
transdermal absorption of certain toxicants based on the hydration state of the stratum
corneum.

139
 Table 1. Physiological differences between men and women which may alter
absorption .

PARAMETER PHYSIOLOGIC TOXICOKINETIC

CHANGE CHANGE

Gastric Juice pH acidity M>F>pregnant F altered absorption of acidlbases

Gastric Juice Flow M>F change in absorption

Intestinal Motility decreased in pregnant F absorption increased

Gastric Emptying prolonged in pregnant F absorption, gastric metabolism

increased

Dermal Hydration increased in pregnant F altered absorption in pregnant F

Dermal Thickness increased in M absorption decreased

Dermal Area increased in BSA* in M absorption increased

Skin Blood Flow increased in pregnant F absorption increased

Pulmonary Function increased in pregnant F pulmonary exposure increased

Cardiac Output increased in pregnant F absorption increased

*BSA = body surface area

+Data from Silvaggio and Mattison, 1992.

Blood flow to the skin may also affect transdermal absorption of toxicants.
According to Williams and Leggett (1989), the percentage of cardiac output to the skin
(5%) and blood perfusion rate (120 mllkg/min) do not differ between males and females.
However, there are changes in blood flow to different regions of the skin during
pregnancy. Blood flow to the hand increases approximately six-fold during pregnancy,
and blood flow to the foot doubles (deSwiet, 1980a). At the same time, there are only
small increases in blood flow to the forearm and leg. These alterations in dermal blood
flow during pregnancy may have a significant impact on transdermal absorption of
xenobiotics.
Pulmonary volumes and respiratory rates are important toxicokinetic parameters,
especially when the route of exposure is via inhalation, important in occupational
settings. Absorption, metabolism, and excretion may all be related to pulmonary
function. Gender differences in pulmonary function can alter the toxicokinetics of certain
chemicals. Lung volumes correlate with total body weight and surface area; therefore,
one would expect higher volumes in men. Total lung capacity , functional residual
capacity, vital capacity, and dead space are all higher in men than wmen.
Pulmonary function changes during pregnancy. Although the respiratory rate is
unaltered, the tidal volume is increased by 39%. Increases in minute ventilation parallel
increases in tidal volume. As a result, the amount of inhaled toxicants may be
significantly greater during pregnancy. During an 8-hour exposure, the largest
pulmonary dose would be delivered to the pregnant woman (Table 2).
The increases in tidal and minute volumes also suggest an increase in pulmonary
distribution and alveolar mixing of gases, lessening the time to reach alveolar steady
state. Gas transfer, however, appears to be decreased due to interstitial changes in

140
 Table 2. Effect of variation in pulmonary function on pulmonary dose.

COMPOUND RECOMMENDED VOLUME OF AIR PULMONARY

EXPOSURE EXCHANGED IN DOSE
LIMIT 8 HRS (m3) (mg)
(mg1m3) (RESTING)

M F Pregnant F M F PregnantF

ARSENIC 0.2 3.6 2.9 5.0 0.72 0.58 1.0

BENZENE 32 3.6 2.9 5.0 115 93 160

the lungs during pregnancy. For example, the pulmonary diffusion capacity of carbon
monoxide is reduced from 26.5 to 22.5/min/mm Hg (deSwiet, 1980b).
Cardiac output and regional distribution of blood flow are two important parameters
that will impact on toxicokinetics, especially absorption. Cardiac output increases
approximately 50% during pregnancy (deSwiet, 1980a). This increase occurs by the end
of the first trimester and remains elevated over the remainder of pregnancy. The
increase in cardiac output is accomplished by an increase in both stroke volume and heart
rate.
Distribution of cardiac output, or regional blood flow, can impact on toxicokinetics.
Williams and Leggett (1989) have proposed reference values for resting blood flow to
organs and tissues for typical 35 year old males and females. Significant differences
occur for resting blood flow as apercentage of cardiac output to skeletal muscle (greater
for men) and adipose tissue (greater for women). These differences may reflect
gender-based differences in the percentage of total body mass represented by each tissue
(Williams and Leggett, 1989).
During pregnancy, increased cardiac output leads to an increase in uterine blood flow
which approximates 150 ml/kg/min at term (Moawad and Lindheimer, 1982; Metcalf et
al., 1988). This value is comparable to uterine blood flows determined in experimental
animals. Blood flow to the skin and kidney is also increased during gestation. Renal
blood flow is increased by approximately 50% very early in pregnancy. No significant
changes are reported in cerebral or hepatic blood flows, however there is a paucity of
information regarding regional blood flow changes during pregnancy (Brinkman, 1989).

DISTRIBUTION

Distribution is the process by which a xenobiotic is translocated from sites of

absorption or metabolism to tissues and organs throughout the body. The total amount
of chemical in the body is the body burden and the apparent volume of distribution is
integral in determining the body burden ofaxenobiotic. Body composition parameters
are important toxicokinetically, especially in regard to volume of distribution (Table 3).
Sex- and gestation-specific differences in these parameters may account for differences
in the concentration ofaxenobiotic at the target site and result in varying responses.
Total body water, extracellular water, intracellular water, total blood volume, plasma
volume, and red blood cell volume are greater for men than nonpregnant women.
Therefore, if an average male and an average female are exposed to the same dose of a
water soluble xenobiotic, the greater total body water, plasma volume, extracellular
water, and intracellular water will increase the volume of distribution and thus decrease
the concentration of the xenobiotic in the male.

141
 Table 3. Physiological differences between men and women which may alter
distribution .

PARAMETER PHYSIOLOGIC TOXICOKINETIC

CHANGE CHANGE
Plasma Volume pregnant F > M > F decreased concentration

Total Body Water M > pregnant F > F decreased concentration

Plasma Proteins M,F>pregnant F concentration

Body Fat pregnant F > F > M increase body burden of lipid-

soluble xenobiotics

Cardiac Output M > pregnant F > F increase rate of distribution

Data from Silvaggio and Mattison, 1992.

During pregnancy, changes in body weight, plasma proteins, plasma volume,

extracellular fluid volume, total body water, and body fat may alter xenobiotic
distribution (Hytten and Chamberlain, 1980; Mattison, 1986, 1990; Mattison et al., 1991,
1992). Matemal weight increases from 50 kg at the start of pregnancy to approximately
63 kg at 40 weeks. Total body water increases from 25 to 33 liters at term, but remains
less than the adult reference male (42 liters). Matemal extracellular fluid volume
increases from 11 to 15 liters over the course of pregnancy, compared to 18.2 liters in
the adult reference male. Plasma volume increases from 2.5 liters to 3.8 liters in the
pregnant female at term, greater than the 3 liters in the adult reference male. These
volume measurements in the pregnant female exceed all nonpregnant female values and
also exceed total blood volume, plasma volume, and red blood cell volume for the adult
male. These differences may be important, especially for xenobiotics distributed into
plasma.
In many toxicokinetic models, matemal volume of distribution increases continuously
during gestation; however, it does not increase for all xenobiotics. At times, it is
inferred that an increase in volume of distribution has occurred because of decrease in
plasma concentration. However, other factors such as increase in the rate of elimination,
decrease in a binding protein, or a decrease in the rate of absorption may also reduce
serum concentrations. Decreases in plasma binding proteins, such as albumin, may
increase the amount of free xenobiotic available for extravascular distribution, for
delivery to target tissues, and for elimination. Total protein and serum albumin
concentrations do not significantly differ between males and nonpregnant females
(Keating et al., 1969), however total protein content of serum decreases in pregnancy,
mainly due to a decrease in albumin (Hytten and Chamberlain, 1980).
Body fat composition also impacts on toxicokinetics. The body can be divided into
fat containing and fat-free tissues. Body fat as apercentage of total body weight is
higher in women than men and increases by age in both sexes (Young et al., 1963). The
total body fat for an adult reference male is 13.5 kg. Matemal body fat increases by
about 25%, from 16.5 kg in the non-pregnant female to 19.8 kg at 40 weeks gestation
(Hytten, 1980a, 1980b; Mattison, 1986). The larger proportion of body fat in women
and increase in body fat in pregnant women may increase the body burden of
lipid-soluble, slowly metabolized toxicants, especially during gestation.

142
METABOLISM

Metabolism is a major factor in determining response to xenobiotics.

Biotransformation occurs predominantly in the liver, but there are also extrahepatic sites
of metabolism such as the lung, kidney, intestinal tract, and skin. Biotransformation can
also occur in the placenta and in fetal tissues.

Table 4. Physiological differences between men and women which may alter
metabolism .

TOXICOKINETIC
PARAMETER PHYSIOLOGIC
CHANGE

Hepatie higher BMR* in M inereased metabolism

hepatie metabolism in pregnant F
Extra-Hepatie metabolism by fetus/plaeenta metabolism
Plasma Proteins decreased in pregnant F metabolism

*BMR = Basal Metabolie Rate

tData from Silvaggio and Mattison, 1992.

Many factors impact on the rate of biotransformation of xenobiotics. Factors that

affect uptake of xenobiotics by target tissues are also important in metabolism, since the
toxicant must reach cellular sites of biotransformation. Thus, lipid solubility, protein
binding, dose, and route of exposure all affect the rate of biotransformation. In addition,
individuals show large variation in metabolism of xenobiotics. However, data regarding
the rates of metabolism of toxicants for males, nonpregnant and pregnant females are
scarce. In humans, sex-dependent differences in biotransformation have been observed
for a few specifie xenobioties sueh as nicotine, acetylsalicylie acid, and heparin (Sipes
and Gandolfi, 1986).
Metabolism of chemicals may be estimated by basal metabolie rates. For all ages,
men have a higher basal metabolie rate than women. Basal metabolie rate incorporates
a number of cellular funetions from different eell types. Since the metabolism of adipose
tissue differs from that of muscle tissue, some of the differenees between men and
women are attributed to body composition (Ljunggren, 1963). Cunningham (1982)
reviewed multiple studies and concluded that the lower basal metabolie rate per unit body
surface area reflects the reduetion of lean body mass in women due to a smaller skeletal
muscle eomponent. The altered hormonal milieu of pregnaney is associated with ehanges
in hepatic and extrahepatie xenobiotie metabolism (Mattison, 1986; Lewis, 1980, 1983).

ELIMINATION

Xenobiotics are generally eliminated from the body by renal, hepatic, or pulmonary
routes. Toxicants may also be exereted via bodily secretions sueh as sweat, tears, and
milk (Klaassen, 1986).

143
 Table S. Physiological differences between men and women which may alter
metabolism.
PARAMETER PHYSIOLOGIC TOXICOKINETIC CHANGE
CHANGE
Renal Blood Flow/GFR* pregnant F>M>F increase renal elimination
Pulmonary Function M>pregnant F>F increase pulmonary elimination
Plasma Proteins decrease in pregnant F elimination

*GFR = Glomerular filtration rate

+Data from Silvaggio and Mattison, 1992.

Renal function is important for elimination. Chemicals can be excreted into the urine
through glomerular filtration, passive diffusion, and active secretion. Increases in renal
blood flow and glomerular filtration will increase the elimination rate of xenobiotics
cleared by the kidneys. When standardized for body surfaee area, renal blood flow,
glomerular filtration, tubular secretion, and tubular reabsorption are all larger in men
than nonpregnant women (Hytten and Chamberlain, 1980; Silvaggio and Mattison, 1992).
During gestation, changes in renal blood flow, glomerular filtration rates, hepatic blood
flow, bile flow, and pulmonary function may alter matemal elimination ofaxenobiotic.
Matemal renal plasma flow increases from 500 to 700 ml/min/l.73 m2, a l.44-fold
increase over the nonpregnant female value and a l.1-fold inerease over the male value.
Glomerular filtration also increases during pregnancy. At the beginning of gestation,
glomerular filtration is approximately 100 ml/min/l.73 m2 By 20 weeks gestation, the
glomerular filtration rate has increased to approximately 150 mllmin/l.73 m2 , a l.5-fold
increase over the nonpregnant female value and a l.2-fold increase over the male value
(Davison, 1980).
Volume of distribution and elimination rates interact to modify the concentration of
a toxicant in the matemal organism during gestation. There is a paucity of data
regarding the impact of changes in pulmonary and hepatic function on elimination. As
a result of the increase in minute volume, the amount of inhaled toxicants significantly
increases. These same increases in pulmonary function during pregnaney may also
inerease pulmonary elimination. However, it is unknown whether these postulated
increases in pulmonary elimination are sufficient to override the inerease in pulmonary
absorption.
Physiological and toxicokinetic faetors must be considered in determining allowable
exposure limits for men, nonpregnant and pregnant women and in establishing guidelines
to protect reproductive and developmental health. Any method of quantitative risk
assessment must consider these physiological differences between and among the sexes.
Biomarkers are an integral element of a quantitative risk assessment model. The
variations in toxicokinetie parameters will impact on biomarkers. Data deseribing
physiological, toxicokinetie, and metabolie parameters for men, nonpregnant and
pregnant women needs to be developed. Onee the data are available, it will be possible
to institute quantitative risk assessment based on physiological and toxicokinetie
differenees. This data-based quantitative risk assessments approach will act to protect
all individuals.

RISK ASSESSMENT

The Institute of Medieine (1988) defined a framework for publie health objectives
whieh can be used to consider the protection of reproductive and developmental health.
That 10M framework includes three steps: (1) assessment; (2) poliey development; and

144
(3) assurance. This discussion will focus on assessment as it relates to reproductive
health. Assessment characterizes the reproductive health status of the community and
determines the potential or observed effect of exposure. Epidemiological data of
chemically-induced reproductive diseases are necessary to characterize the potential
hazards associated with exposure and the effect on reproductive function in the male,
female and subsequent impact on fertility. The background reproductive health status of
the community is essential in the assessment step in order to be able to delineate the
impact of exposure to a putative reproductive toxicant.
There are limitations in the assessment process which are inherent due to the privacy
surrounding reproduction. In addition, reproductive status can only be determined in
those couples attempting pregnancy. Gaps also exist in data characterizing the
reproductive health status of a community. Record-keeping and accurate recording of
many adverse reproductive events are inconsistent.

Table 6. Examples of factors and toxicants which may impair male and female
fecundity.

Male Fecundity Female Fecundity

Factors Toxicants Factors Toxicants
Vasectomy DBCP Contraception DDT & metabolites
Mumps EDB Tubal ligation Carbaryl
Fever Lead Infection Kepone
Varicocele Carbon disulfide Prescription drugs Lead
Diabetes Kepone Smoking PAH
Hypertension Boron Alcohol Ionizing radiation
Prescription drugs Carbaryl Age DES
Smoking Cadmium Abused substances 2-Ethoxy acetic
Alcohol Methylmercury Anesthetic gases
Abused substances Ionizing radiation Aniline
DES Benzene
Chloroprene Carbon disulfide
Ethylene oxide Chloroprene
Glycol ethers Glycol ethers
Hexane Formaldehyde
Vinyl chloride Methylmercury
Pesticides
Phthalic acid ester
PCBs
Styrene
Toluene
Vinyl chloride

Modified from Mattison et aI., 1990a, 1990b.

The epidemiology of reproductive disease is incomplete. The lack of formalized

reproductive health surveillance programs adds to the lack of epidemiological data in the
workplace. Referring back to Figure I we are reminded of the intricacies of the
reproductive process, the problems in identifying adverse outcomes for all endpoints and
the etiologies of these adverse outcomes.
The risk sciences are important in the field of environmental and occupational health.

145
They inc1ude: (1) risk assessment, (2) risk management, (3) risk perception, and (4) risk
communication. Risk assessment is used by many agencies in evaluating health risk and
setting allowable standards for workplace exposures. Risk assessment methods have been
used to set limits for exposure standards for airbome contaminants. The National
Academy of Science among others (1983) has developed models for risk assessment.
The process inc1udes hazard identification, hazard characterization, exposure assessment
and risk characterization.
One must identify the causal relationship of an observed adverse effect with
exposures to particular agents. Once it is determined that the agent does in fact cause
the effect, then the relationship between the response (adverse effect) and the dose
(exposure) must be ascertained. This is the step of hazard characterization and this
process may be complicated by multiple responses to a single exposure (dose).
Hazard identification and characterization are important steps in the risk assessment
process. There are also gaps in the experimental animal data identifying agents that are
reproductive and developmental toxicants. OECD has recently completed a survey of
toxicity testing data on high production volume chemicals (greater than 10,000 tons/yr)
among member countries (Brydon et al., 1990). Only 38% of all high production
volume chemicals underwent reproduction and/or developmental testing. Some factors
associated with impaired reproduction and some reproductive toxicants have been
identified (Table 6), others are under investigation and need to be characterized, and
additional chemicals remain that have not even been considered as putative reproductive
toxicants.
After the hazards have been identified and characterized, exposure assessment must
be carried out. Exposure assessment determines the actual or anticipated exposure and

Table 7. Endpoints for assessing human reproductive function.

Couple
Infertility or the absence of fertility throughout the entire reproductive
lifespan or loss of an essential component of the reproductive process.

Subfertility reflected by increased time to pregnancy, decreased

cumulative fertility and decreased standardized fertility ratio.
Reproductive loss or a decreased rate of continuing pregnancy.

Male
Number of motile sperm
Sperm morphology
Semen volume
Semen composition
Reproductive tract anatomy
Testicular volume
Hormone levels

Female
Alteration in reproductive lifespan
pubertal delay
premature menopause
Disturbance in ovarian function
ovulation!oligo-ovulation
dysfunctional cycles
diminished functionaI capacity of oocyte
Compromised genital tract
structural abnormalities
From Mattison et aI. , 1990b.

146
how much of the dose reached the target tissue. Risk characterization identifies the
actual or estimated risk of an adverse outcome in a given population in relation to the
given exposure (National Academy of Sciences, 1983). These steps must be carried out
within the framework of biologically based models developed specifically for
quantitative assessment of reproductive risk and should include the likelihood that a given
exposure will result in a specific adverse reproductive outcome and quantitative the
degree of alteration of successful reproductive.
There are many endpoints for assessment of reproductive function (Table 7). These
endpoints are important measures in the evaluation of a possible toxicant. Evaluation of
the effect of a potential hazard also requires evaluation of specific human endpoints of
reproductive and developmental toxicity (Table 8). Some progress has been made in
identifying endpoints; however, views vary on which endpoints need to be considered to
evaluate a population' s reproductive and developmental health. Proper selection of these
endpoints is essential for valid assessment of the impact of potential toxicants on humans.
Considerations involved in selection ofthese endpoints are provided by Savitz (1991) and
are listed on Table 9.
Severity refers to the nature of the outcome, such as stillbirths and major birth
defects. Delayed conception and subclinical pregnancy loss are not considered severe.
In the past, severe outcomes have been the focus of epidemiological study; currently,
some are suggesting that less severe outcomes may yield more information, and
may lead to earlier detection and prevention. For example, studying an endpoint

Table 8. Selected human endpoints of reproductive and developmental toxicity.

Male infertility
Female infertility
Couple infertility
Spontaneous abortion
Fetal death, early and late stillbirth, perinatal death
Placental, cord and fetal membrane abnormalities
Growth retardation (intrauterine, postnatal)
Altered sex ratio
Birth defects
Infancy and childhood morbidity and mortality
Abnormal maturation
Abnormal sexual development or function
Mental retardation/learning disability
Specific organ system dysfunction
Developmental disabilities
Transplacental carcinogenesis and mutagenesis
Modified from Mattison et al., 1989.

Table 9. Issues to consider in selection of reproductive and developmental

endpoints.
Severity of the outcomes
Relative sensitivity of different outcomes to environmental agents
Interrelationship among adverse outcomes
Baseline frequency of an event
Evidence from the literature
Frequency of occurrence of an event
Frequency of occurrence of aprerequisite event

Modified from Savitz and Harlow, 1991.

147
like "time to pregnancy" may allow detection of subtle changes of reproductive function
which are occurring within a population (see the example reproductive risk calculation).
Relative sensitivity of different outcomes to exposure to agents refers to the fact that
substances such as DBCP produce testicular toxicity in males, but the female
reproductive tract does not appear to be sensitive. The interrelationship among adverse
outcomes refers to the idea that varied pathways of reproduction and development can
be disturbed by a single agent.
Baseline frequency of an event involves consideration of the incidence of events.
Observing more frequent events may yield more information in smaller populations than
rare events. It is also necessary to know baseline incidence levels in a community to
detect any increases in relation to specific exposures. Some reproductive toxicologists
suggest that evidence from the literature such as animal studies may offer some
guidelines in selection of endpoints. Frequency of occurrence of an event refers to
outcomes that have naturally higher incidence levels and may be easier to study in a
smaller population. If one assurnes that because they occur more frequently, they may
be more sensitive at a baseline level and thus may be more sensitive to effects of
toxicants. The frequency of occurrence of aprerequisite event may be restrictive in a
smaller population if one chooses aprerequisite event such as pregnancy. It has been
suggested that selecting prerequisite events such as menstrual function or seminal fluid
may be less restrictive. One must be able to differentiate subtle changes, which are
statistically significant in these endpoints. Once these are detected, the causal
associations between the adverse outcomes and toxicants must then be established
(summarized from Savitz and Harlow, 1991).

BIOLOGICAL MARKERS IN RlSK ASSESSMENT

Successful reproduction is a complex sequence of many independent processes. The

biological and physiological principles of reproduction indicate that both sexes are at risk
for potential hazards in the workplace. Reproductive and developmental endpoints
represent the integration of these processes and incorporate male, female and conceptus
exposures. Inherent difficulties in the monitoring of these endpoints complicate medical
surveillance of reproductive and developmental health. Risk assessment for reproductive
and developmental outcomes requires the use of biomarkers to link exposures to disease.

The role of biomarkers in reproductive and developmental toxicology are currently

of much interest. It is generally accepted that valid biomarkers can improve toxicological
and epidemiological research and risk assessment (Schulte and Mazzuckelli, 1991; Hogue
and Brewster, 1991; Perera et al., 1991; Hattis, 1991). The term "biological marker"
refers to "cellular, biochemical or molecular alterations which are measurable in
biological media such as human tissues, cells or fluid and are indicative 0/ exposure to
environmental chemicals" (Hulka, 1988). Biological markers are indicators measured in
a biological specimen and refer to three types of events: exposure, effect, and
susceptibility (Schulte and Mazzuckelli, 1991). Biomarkers which estimate exposure can
differ from those used to estimate effect. Biomarkers of effect can be further divided
into those that characterize biologically effective doses (DNA adducts) and biological
responses (Hogue and Brewster, 1991).
Examples of potential biological markers are the formation of hemoglobin adducts
from alkylation of hemoglobin by ethylene oxide and ethylene-dibromide-DNA adducts.
Exposure bio markers that have been developed in reproductive epidemiology include
glucaric acid, thioethers and porphyrin patterns. Effect biomarkers of biological

148
responses include semen analysis and hormonal measures of early pregnaney loss. Hair
sampies for methylmereury (Clarkson, 1987) serve as an excellent biomarker for fetal
and adult absorbed doses of methylmereury. Examining maternal hair sampies for
methylmereury eoncentration have been used to estimate blood levels during pregnaney.
The development of appropriate biomarkers is important for the future of reproduetive
epidemiology. Using these biomarkers it is possible to define a biologically based risk
assessment methodology for reproduetive toxicity.
Identifieation of xenobioties which produce human reproductive disease is a
multistage process which involves three types of monitoring: environmental, biological,
and health. Environmental monitoring is the assessment of the amount of xenobiotic in
different media (e.g. air, soil, water). Biological monitoring is the assessment of the
absorbed dose, body burden, or target tissue levels of the xenobiotic or its metabolites.
Health monitoring includes the surveillance of populations. The goal of health
monitoring is to detect adverse health effects that result from interaction with a
xenobiotic in a preclinical state, so that one could intervene and prevent disease.
Biological markers are the tools of biological and health monitoring. They can be
utilized to define the relationship between xenobiotic exposure and effect. The effect
noted may be permanent or transient impairment of reproductive and developmental
health. Xenobiotic exposure, toxicokinetie factors and biologic markers all interrelate.
Toxicokinetic factors determine absorption, distribution, metabolism and elimination of
a xenobiotie after exposure in an individual. Biologieal markers attempt to bridge the
gap between exposure, effect, and disease.
Major routes of entry of xenobiotics after exposure are through the skin, respiratory
and digestive tract. Early after the exposure, biomarkers of internal dose, external dose
and biologically effective dose may be ehosen for monitoring. Later, after the exposure,
strueture and organ function can be monitored through biomarkers of effect which are
specifically related to the diseased organ or system. It is imperative to define and
validate biomarkers, in order to establish the relationship between exposure levels
(external dose), internal dose, and development of disease. Biomarkers can be affected
by xenobiotic-specific characteristics, individual charaeteristics and organ or target tissue
characteristics. Biomarkers are utilized to detect the biological effects which result from
interactions between the xenobiotic or its metabolites and the biological materials.

REPRODUCTIVE RISK ASSESSMENT USING BIOMARKERS

The use of biological markers in assessing reproductive risk is a developing area.

Biologie markers should be utilized to clarify relationships between exposure and disease.
The availability ofvalidated biological markers for reproductive processes (Figure 1) will
allow the development of biomarker-based risk assessment for reproduction (male
fecundity, female fecundity, couple specific faetors) and development (pregnancy loss,
stillbirth, premature birth, and developmental defect). The biomarker-based model of
risk assessment incorporates factors which may influence reproductive success; the
clinical pregnancy rate, male and female fecundity, couple-specific factors (frequency of
intercourse), and early pregnancy loss. Other factors important for developmental
risk assessment include the rate of stillbirth, rate of premature birth, and rate of
structural and functional developmental disease. In this approach to biomarker-based risk
assessment, the risk of reproductive disease will be a function of male fecundity, female
fecundity, eouple-specific factors, and developmental processes (Mattison, 1991).

149
 Male fecundity (MF) is a measure of the ability of the male to fertilize the female,
that is the reproduetive effectiveness of a given male. The contribution of male fecundity
to the fertility of a eouple may be affected by exposure to a toxicant that has an effect
on the male reproduetive system. An assessment of the effect of the toxicant on male
fecundity may be ascertained through biomarkers for male reproduetive toxicity. The
biomarkers whieh may be applicable are anatomie faetors (Afm), ejaeulate volume (Ev),
ejaeulate eomposition (Be), sperm number per ejaeulate (Sn), sperm motility (Sm), and
sperm morphology (Ss). These biomarkers all eontribute to a funetion:

male fecundity = MF(Afm, Ev, Ec, Sn, Sm, Ss) (Equation 1)

All of these biomarkers may not be inc1uded in an assessment depending on the

availability of data and the eontribution of a given biomarker. Male fecundity will vary
with the different biomarkers. The most well-defmed relationship is between male
fecundity and sperm eount (Meistrieh and Brown, 1983; Mattison, 1991).
Female fecundity (FF) is a measure of the IikeIihood of fertilization following
delivery of sperm to the female reproduetive !Taet. The biomarkers that appear most
appropriate are anatomie faetors (Aft) , ovulatory frequeney (Ot), follieular phase
eharaeteristies (Ft), Iuteal phase eharaeteristies (Lt), endometrial funetion (Et), and tubal
funetion (Tt). These biomarkers all eontribute to a funetion:

female fecundity = FF(Aff, Of, Ff, Lf, Ef, Tt) (Equation 2)

Couple-dependent faetors (CF) are those impaeting on fertility whieh reflect a

specifie eouple. CoupIe-dependent faetors inc1ude frequeney of intereourse (Fi), male-
female interaetions (Mt), and female-male interaetions (Fm). These biomarkers all
eontribute to the funetion:

eouple-dependent faetors = CF(Fi, Mf, Fm) (Equation 3)

At this time, frequency of intercourse is the onlycouple-dependent factor which is

evaluated in this risk assessment model.
In this approach, the unit of time considered is the cyc1e, therefore female fecundity
can be expressed as a single value. Reproduetive risk (RR) is a funetion of the
individual and eoupIe-specifie faetors and is represented by the equation:

reproduetive risk = RR(MF, FF, CF, EPL) (Equation 4)

where: MF = male fecundity, FF = female fecundity, CF = eouple faetors, and EPL

= earIy pregnaney loss.
Different measures of fertility may be used to represent reproduetive risk. Some
examples are eyc1e specifie fertility rate (CSFR), pereentage of eouples failing to
conceive after one year, and mean number of eyc1es required to conceive. Changes in
any of these measures may be the result of exposure to reproduetive toxieants and may
potentially be used to ealeulate reproduetive risk in human populations.
Animal and selected human data is needed to investigate the relationships between
the biomarkers and reproduetive risk. Methods to properly extrapolate this data is also
required. Interspecies extrapolation factors (IEF) can be utilized to convert animal dose-
response curves to the expected human dose-response curves (Meistrich and Brown,
1983; Meistrich, 1989a, 1989b). IEF ean be defined as the dose necessary to produce

150
a given change in reproductive biomarker in test animal divided by the dose necessary
to produce equivalent change in reproductive biomarker in humans (Meistrich and
Mattison, in press). Utilization of these extrapolation methods must be based on
information derived from valid data on the effects of the toxicants from animals and
humans.

EXAMPLE APPLICATION OF REPRODUCTIVE RISK ASSESSMENT USING

BIOMARKERS

Reproductive risk assessments utilizing biomarkers must be quantitative and

biologically-based. A couple-specific approach is necessary along with appropriate
selection of biomarkers and specific endpoints. As mentioned previously, outcome
measures such as time-to-pregnancy (TIP), cyc1e specific fertility rate (CSFR), or mean
number of cyc1es to pregnancy can be utilized. Quantitative reproductive risk assessment
involves determining reproductive efficiency and can be represented by Equation 4.
Welch et al. (1988) examined the effects of ethylene glycol ethers on male
reproduction. They determined that males with exposure to ethylene glycol ethers had
lower sperm counts per ejaculate than controls. An example reproductive risk assessment
utilizing glycol ethers and biomarkers can be constructed using this data. Sperm count
may be used as the biomarker of reproductive function. This relationship was defined
by Meistrich and Brown (1983) when they developed a two-distribution mathematical
model to provide estimates of increased risk of human infertility from variations in sperm
count.
Reproductive hazard identification for ethylene glycol ethers can be obtained from
animal and human data. Testicular atrophy, degeneration of the germinal epithelium,
abnormal sperm morphology, and infertility has been identified in experimental animals
(Creasy and Foster, 1984; Oudiz et al., 1984; Miller et al., 1983; Welch et al., 1988).
Reproductive effects noted from human data are decreased testicular size (Cook et al.,
1982), decreased sperm concentrations (NIOSH, 1986), and lower sperm count per
ejaculate (Welch et al., 1988).
Hazard characterization involves defining dose-response characteristics, site and
mechanism of action. Dose response data for ethylene glycol monomethyl ether (EGME)
is available from studies conducted by the National Toxicology Program (Table 10).

Table 10. Summary of data from sperm studies of the effects of EGME in C3H
mice.

TESTICULAR SPERM DENSITY FERTILITY

DOSE
WEIGHT (gm) (x Hf) INDEX'

CONTROL 0.084 .002 1298 54 93

0.03% 0.085 .002 1323 63 89

0.1% 0.085 .002 1216 86 84

0.3% 0.041 .003 e 518 156' Ob

Data from National Toxicology Program, National Institute of Environmental Health Sciences
(NTP-89-020), "Ethylene Glycol Monomethyl Ether Reproductive and Fertility Assessment in
C3H Mice When Administered in Drinking Water," January, 1989 (Tables 2, 8, 9).
'fertility index( %) = number fertile pairsInumber cohabiting pairs
hp< 0.01 "1>< 0.05 'p< 0.05

151
 Biomarkers of male fecundity such as sperm density are evaluated for their impact
on reproductive endpoints such as fertility index. Dose-response relationships are not
readily available for EGME in humans. Extrapolations can be made using an IEF and
animal dose-response data. As previously mentioned, the IEF is defined by the dose
necessary to produce a given change in a reproductive biomarker in a test animal divided
by the dose necessary to produce an equivalent change in a reproductive biomarker in
humans. Meistrich (19891, 1989b; Meistrich and Mattison, in press) suggests the IEF
for 2-ethoxyethanol to be < 12 and for 2-methoxyethanol to be < 32-35. An example
of a dose-response curve for humans derived from animal data using an IEF of 10 is
illustrated on Figure 2.

100

'0

80

70

...0
.H 60

8
. 50
C
&.
..
0
H

'0

2.

,.
0 2

Log Dc

o An1.J:nal + HWII&n

Figure 2. Dose response curves for a hypothetical reproductive toxicant in

animal and human subjects. Note that the human is more sensitive than the
animal model.

In this example the graph is defined by the equation:

Y = a-d)/(l + dose/c)"'b + d (Equation 5)

where the slope (b) = 3, the response at zero dose (a) = 100, the ED50 (c) = 400
(animals) and 40 (humans), and the response at infinite dose (d) = O.
Exposure assessment involves determination of the exposure level, duration of
exposure, and defining the population exposed. In the example for EGME (Welch et al.,
1988), the population exposed was male painters, estimated levels of exposure are listed
on Table 11.
Welch et al. (1988) used analysis of variance and compared sperm density and count
per ejaculate between exposed and unexposed men and determined that the measures of
sperm count were lower in the exposed group with p = 0.10 for sperm density and p =
0.11 for total sperm count (Table 12).

152
 Table 11. Exposure levels of 2-EE and 2-ME in exposed and unexposed
workers.

2-EE(8hr TWA) 2-ME(8hr TWA)

0-80.5 mg/ar 0-17.7 mg/ar

EXPOSED SUBJECfS
(0-20.5 ppm) (0-5.6 ppm)
MEAN = 9.9 mg/m) MEAN = 2.6 mg/m)
(2.6 ppm) (0.8 ppm)
CONTROLS o o
Data from Welch et al., 1988, Effects of exposure to ethylene glycol ethers on shipyard painters:
ll. Male reproduction, American Journal of Industrial Medicine, 14:509-526.

Table 12. Semen characteristics of exposed and unexposed men.

CHARACTERISTIC EXPOSED UNEXPOSED % CHANGE

SPERM
CONCENTRATION 66.5 40.3 78.6 53.9 16
(millions/ ce)

TOTALSPERM
COUNT 158 108 211 140 25
(millions/ejac.)

Data from Welch et al., 1988, Effects of exposure to ethylene glycol ethers on shipyard painters:
1I. Male reproduction, American Journal of Industrial Medicine, 14:509-526.

Risk characterization is the next step in the risk assessment process. This is often
a difficult step because of the lack of human data. Risk characterization may be
expressed by CSFR, CTP, percent pregnant at one year and percent infertile at one year.
If CSFR is chosen as the parameter to represent reproductive efficiency, the following
equation can be used to determine CSFR:

CSFR = MF x FF x CF x (l-EPL) (Equation 6)

Results of these example risk calculations are listed on Table 13.

Table 13. Reproductive risk calculation for glycol ethers.

EXPOSED UNEXPOSED

0.513 0.574
MALE FECUNDITY
(0.60 x 0.855) (0.60 x 0.957)
CSFR 0.131 0.146
% PREGNANT AT 1 YR 85 81
MEAN CYCLES TO
4.9 4.7
PREGNANCY

153
 Examples of applications of these calculations assuming FF = 0.6, CF = 0.5 and
EPL = 0.15 and MF represented as a function of varying sperm counts(million/ml) is
illustrated on Figure 3, which depicts the change in cycle specific fertility rate (CSFR)
as a function of sperm count (millions/ml).

0.2.

0.36

O.2.ft.

.0.22

0.2

0.1.

0.1.

.f
U
0.1.&

0.12

0.1

o.oe
0.0.

0.04.

0.0:2

0
0 20 .. 0 so 80 100

Figure 3. Relationship between cycle specific fertility rate and sperm count
(millions/ml).
(pF = 0.6, CF = 0.5, EPL = 0.15)

1.1 --~------~----------------------------------------~

0.9

.
i
0.7

...
H
c.s

C C.5

..
U
H
c ...

0.3

0.:0

0.1

C
20 60 80 100

Figure 4. Relationship between sperm count and cumulative percent pregnant at

one year.
154
 The relationship between cumulative percent pregnant and sperm count (millions/ml)
is depicted on Figure 4.
Quantitative risk assessment is necessary to accurately define reproductive risk.
Qualitatively, reproductive efficiency is a function of MF, FF, CF and EPL. We can
infer that if sperm count decreases, MF decreases and thus CSFR decreases, TTP
increases, and cumulative percent of couples pregnant at 1 year decreases. These
relationships are more difficult to define quantitatively, however one can estimate these
parameters using appropriate risk calculations based on eertain equations and using
proportionate changes in the observed biomarkers of reproductive function.

CONCLUSIONS

A general model has been presented integrating elements necessary for reproductive
risk assessment. Onee the relationship between exposure and fecundity, couple factors,
and early pregnancy loss has been defined, it is possible to explore the relationship to
fertility. The calculations presented are a first step in the development of quantitative
approaches for estimation of reproductive risks. Eventually it is hoped that factors of
male fecundity, female fecundity, couple interactions and early pregnancy loss could all
be combined into one model for reproductive risk assessment.

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Mattison, D.R., Blann, E., and Malek, A., 1991, Physiological alterations during pregnancy: impact
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Mattison, D.R., Working, P.K., Blazak, W.F., Hughes, Jr., C.L., Killinger, J.M., Olive, D.L., and
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Meistrich, M.L., 1989a, Interspecies comparison and quantitative extrapolation of toxicity to the human
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Perera, F., Mayer, J., Santella, R.M., Brenner, D., Jeffrey, A., Latriano, L., Smith, S., Warburton,
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Schulte, P., and Mazzuckelli, L.F., 1991, Validation of biological markers for quantitative risk
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158
BEHAVIORAL BIOMARKERS TO IDENTIFY NEUROTOXIC EFFECTS

W. Kent Anger
Center for Research on Occupational and Environmental
Toxicology, L606
Oregon Health Sciences University
3181 S.W. Sam JacksonPark Road
Portland, OR 97201

INTRODUCTION
Neurotoxic chemicals pose a particularly serious health threat due to the unique and
critical role of the nervous system in body function. Exacerbating this potential impact is
the vulnerability of tbe nervous system due to: (a) it's unparalleled complexity; (b) the
limited potential for repair of neurons; (e) the extensive energy dependence and metabolie
requirements unique to this system; and, (d) the tendeney of lipids to accumulate toxicants.
To further target this vulnerable system, some ebemicals used in the workplace and the
environment (e.g., pesticides) are specifically designed to destroy nervous system elements
(National Research Council, 1992). These and other factors led several House and Senate
Committees of the US Congress to commission the New Developments in Neuroscience
Advisory Panel to study issues surrounding neurotoxicity and suggest options for
Congressional action. The study "Neurotoxicity," published by the US Congress Office of
Technology Assessment (1990), notes that the entire US population "is at risk of being
adversely affected by neurotoxic substanees" (US Congress/OTA, 1990, p. 8) and
identified the unique value of human biological monitoring programs "to detect exposure to
toxic substanees and to aid in making deeisions about health risks" (US Congress/OTA,
1990, p. 12-13). This potential exposure and recognized need for biological monitoring
programs extends to every corner of the world.
Many environmental and occupational toxicants are known to affeet the nervous
system. Anger and Johnson (1985) identified 750 chemicals for wh ich, at some
concentration, there is evidence of adverse changes measured by established tests of
nervous system chemistry, structure, or function. Not surprisingly, neurotoxic chemicals
have been among the most significant in tbe development of occupational and
environmental standards. Approximately 40% of the National Institute for Oeeupational
Safety and Health (NIOSH) Recommended Exposure Limits (RELs) are based, in part, on
effects manifest in the nervous system. Of just under 200 chemicals to which 1 million or
more workers are exposed in the US (as of 1977 per NIOSH sampling), one-third affect tbe
nervous system at some exposure concentration (Anger, 1986). Of the 588 chemicals
identified by the American Conference of Govemmentallndustrial Hygienists (ACGIH) as
having toxicologic significance and found in the US workplace, ACGIH documented that
almost 30% affected the nervous system (Anger, 1984). At very high concentrations,

Use 0/ Biomarkers in Assessing Health and EnvironmentalImpacts 0/ Chemica/

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 159
these neurotoxicants have led to a wide variety of fatal occupational, food adulteration and
environmental incidents associated with effects on the nervous system. At lower
concentrations, these neurotoxicants have produced an extremely wide range of serious
nervous system deficits (Anger, 1990; Anger and Johnson, 1985). Neurotoxicity is thus
among the most significant and adverse health effects produced by environmental
chemicals that we face today.
While some target organs (e.g., the liver) have a relatively narrow range of chemically-
induced effects, the large number of neurotoxic chemicals found in industry and,
subsequently, the environment, have produced a very wide variety of effects in the nervous
system. For example, Anger and Johnson (1985) and Anger (1986) reviewed and described
the adverse effects, most heavily focused on animal research, that were associated with
each of 750 chemicals with evidence of neurotoxic effect. Some neurotoxic effects (e.g.,
weakness, tremor, equilibrium changes, sleep disturbances) are produced by a wide variety
of chemicals, while others (e.g., memory deficits, hearing loss, hallucinations) are
associated with a sm aller number of chemicals, and still other effects (e.g., personality
disturbances) are relatively rare. Anger and Johnson (1985) identified over 120 unique
behavioral or neurological effects associated with neurotoxic chemicals, as documented by
animal and human research. In exclusively human worksite cross-seetional research,
Anger (in a 1990 review) identified over 155 unique behavioral tests sensitive to group
differences produced by chemicals in this extensive research literature. Here the increasing
research focus on solvent exposures reveals a far greater involvement of cognitive effects,
including leaming, memory, concept shifting, attention, and complex tasks such as coding
(Anger, 1990).
It is clear that neurotoxic chemicals produce a very broad array of changes associated
with nervous system deficits. Given the nervous system's great complexity and its wide
variety of neurotransmitter systems and specialized structures, this diversity in effect is
predictable. Research on neurotoxic agents, however, has provided relatively limited
information about the meehanisms underlying the vast array of neurotoxic effeets. While
evidenee demonstrating the role of gamma diketones in hexacarbon neurotoxicity (e.g., St.
Clair er al., 1988) and the effeets of pyrethroids on sodium channels (e.g., Lund and
Narahashi, 1982) are examples whieh provide insight into some mechanisms at the cellular
and subeellular level, the meehanisms by whieh the 750 ehernieals with neurotoxic
potential (Anger and Johnson, 1985) produce their effects are largely unknown (National
Research Council, 1992). This combination of wide-ranging effects and a lack of
identifieation of mechanisms has seriously constrained the development of biomarkers for
neurotoxic chemicals.

MEASURES OF NERVOUS SYSTEM EFFECTS

Several specific measures of neurotoxic effeets have been established by painstaking
meehanistic and elinical research. For example, measures of nerve eonduetion rates and
more sophistieated neurophysiologie tests are well-known indicators of peripheral nerve
deficits (e.g., Albers, 1990). Two measures that have shown promise in reeent years to
identify broad-speetrum health effects are neurotoxic esterase (NTE) and glial fibrillary
acidic protein (GFAP). Lymphocyte NTE has been used to evaluate the eholinergic effects
of organophosphorous eompounds (Abou-Donia, 1981; Johnson, 1990) and GFAP refleets
central neuronal damage, although nervous system tissue is required for this measurement
(O'Callaghan, 1988).
Nondestructive cellular markers are available for only a few of the many chemicals
known to be neurotoxic. Given the diversity of the nervous system and its wealth of
potential endpoints, it is not surprising that the most widely-used broad-spectrum measures
of neurotoxic (health) effects in human populations are behavioral tests. Anger (1990), in a
comprehensive review of the internationalliterature, identified over 185 published studies
of chronic or long-term chemical exposures that had employed behavioral measures in
cross-sectional epidemiological research. Over 250 unique behavioral tests were identified
in Anger's 1990 review, and over 155 of these tests identified significant changes in
workers exposed to chemicals, when compared to unexposed subjects. Table 1 lists general
functional areas or modalities evaluated in behavioral worksite research in the 185 studies
reviewed in Anger (1990).

160
 Table 1. Nervous System Functions Assessed Most Frequently in Worksite Research

COGNlTlYE MOTOR SENSORY

Memory Vocabulary Coordination Vision-flicker
Intelligence Calculation Coordination/Speed Vision-color
Spatial Relations Concept Shifting Response Speed Vision-pattern
Coding Categorization Speed/Decision Equilibrium
Vigilance or Attention Distractibility Steadiness
Acquisition Grip Strength llIlI.ER
Personality
Affect
Source: Anger, 1990, 1992

Tbe somewhat arbitrary categorization of functions in Table 1 reveals the wide range
of cognitive, motor, sensory, affective and personality deficits now known to be associated
with exposure to chemicals found in industry and the environment. While the number of
functions assessed is smaHer than the number of tests, there is no widely accepted
taxonomy of human functions, nor have there been fuHy comprehensive attempts to
analyze the factors assessed by the tests used in this field.

Objectivity and Reliability of Behavioral Tests

Behavioral tests are often thought of as subjective or unreliable. Tbey are neither.
Most behavioral tests measure the performance (i.e., an objective measure) of the person
being tested. Three examples will demonstrate the objectivity of the measures.
In human worksite research the most widely used test of attention and memory is the
Digit Span. In this test, the examiner reads a sequence of numbers to the test subject who
is asked to repeat the numbers in sequence. Tbis test is often administered on a computer
in which the numbers are presented successively on the screen (visuaHy), and the subject
must then type the numbers on the computer keyboard.
In human worksite research the most frequently used test of coordination is the Santa
Ana. Tbe subject is presented with a board with 48 "pegs" resting on it. Tbe pegs are
round on top but their hidden base is square and it is resting in a square hole in the board.
The subject is required to grasp the peg, raise it above the board, rotate it 180 degrees and
place it back in the hole. The test is timed, so that the measure is the number of pegs
correcdy rotated 180 degrees in abrief period of time.
A test of response speed, often implemented on a computer, requires the subject to
press a button as rapidly as possible after a light appears.
In each of these examples, the measures are quite objective--they do not require the
tester's judgment. What leads some to consider such tests "subjective" is that responding is
entirely under the control of the person being tested--they can try very hard to do weH on
the tests or they can do very poorly (i.e., work slowly, remember very litde on a memory
test). Thus, motivation of the test subject is an important factor that can affect or modify
performance. Extensive baseline data have been collected on many tests to help identify
intentionally poor performance. Of course, purposefully poor performance by a subset of
subjects would increase data variability and decrease the chances of discriminating between
exposed and control populations in group comparison studies. It would also reduce the
likelihood of finding a pattern of deficits reflecting a unique neurotoxic effect. In
epidemiologic testing such as this, replication is what establishes a finding and can
substantiate a causal relationship between a given pattern of behavioral deficits and a given
chemical. These are the factors which limit or control the influence of the test subject in
behavioral neurotoxicology research.
The behavioral tests used in this field, as noted above, are reliable. In a selection of 10
tests employed in field research, Letz (1990) summarized test-retest reliability results from
six studies. These studies, conducted in the field and the laboratory, had reliability
coefficients of 0.55-0.91. Walsh (1989) considers 0.85 to be acceptable for a laboratory
test, and 6 of the 10 tests equalled or exceeded a correlation of 0.85. Thus, tbe tests are
reliable in repeat testing of the same individual.

161
 The behavioral tests used in field reseach reflect dose-related changes. That is, higher
doses of neurotoxic chemicals produce quantitatively larger deficits in test performance.
For example, low-Ievel mercury exposure in thermometer and chloralkalai plants, where
workers had elevated urine mercury levels of 0.24-1.4 ug/l were significantly associated
with incoordination and slight memory or attention deficits (Langolf et al., 1978; Roels et
al., 1985; Williamson et al., 1982), while a large dose results in clinically observable
tremor, c1umsiness and extended memory lapses at 10-20 ug mercury/l of urine (Wood et
al., 1973).

BIOMARKERS OF NERVOUS SYSTEM EFFECTS

Biologic markers (biomarkers) can be used to measure the impact of neurotoxic
cbemicals. Such biomarkers may provide measures of chemical exposure, measures of
[neurologic] effects of exposure, or measures indicating susceptibility to a chemical
(National Research Council, 1989). Markers of cbemical exposure are useful only when
the dose response curve is established for a given chemical, information which is very
limited for neurotoxic effects of chemical contaminants of the environment. Measures of
susceptibility require an understanding of cellular or molecular mechanisms, which exist
for only a handful of neurotoxicants (National Research Council, 1992). Therefore, the
best markers of most neurotoxic chemicals are measures of health effects.
Although many functions are affected by neurotoxic chemicals, behavioral tests have
not traditionally been seen as biomarkers. While no body fluid is directly employed in
behavioral tests, such tests nonetheless measure functional abilities which provide a
noninvasive measure of the integrity of nervous system structure and chemistry.
Behavioral tests measure the integrated activity of the nervous system. This involves
detection and recognition of a stimulus, relating to or generalizing from previously
recognized stimuli, and performing a response to the stimulus. Behavioral tests can
identify deficits in any of these systems or in the integrated response, and they thus reflect
or are predictive of the neuropathological damage or neurochemical change responsible for
the changing performance. This lack of specificity in defining the deficit is a benefit in a
complex system such as the nervous system which would require a huge number of specific
tests for a full evaluation and because the number of chemieals that may produce nervous
system changes is large. It is a detriment in the sense that the tests may not identify the
specific system element affected by the chemical of concem. While behavioral tests have
not in the past been considered biomarkers in tbe classic usage of tbat tenn, tbay do indeed
fit the definition because they identify or mark neurotoxic effects that cannot otherwise be
identified with noninvasive, nondestructive tests of the human nervous system.

BEHAVIORAL TEST BATTERIES

During the 1980's there was a huge growth in the number and diversity of behavioral
tests used in field research (Table 3 in Anger, 1990) and behavioral test batteries (i.e.,
collections of these tests) which have begun to serve as widely accepted (i.e., standardized)
biomarkers of neurotoxic effect (Anger, 1992). Such batteries are necessary to assess
health effects in any system as complex as the nervous system. The most difficult problem
for assessing a complex system efficiently is selecting the minimum number of tests to
identify the widest possible number of important health effects that could occur in the
system.
Several approaches have been used for selecting tests for such batteries. These can be
categorized as selecting tests with: (a) established sensitivity to the effects of known
neurotoxic chemicals; (b) relevance to psychological theory; and, (c) c1inical relevance.
The tests relevant to psychological theory have long term potential for explaining patterns
of results with various chemicals, just as do neuropsychological tests used for clinical
assessments. However, neither of these approaches have been used extensively in research
with neurotoxic chernicals, so their ability to answer these questions is unproven. Most
batteries have adopted tests of demonstrated sensitivity to neurotoxic chemicals--that is,
they discriminated between exposed and unexposed groups in human worksite research
with known neurotoxicants such as lead and mercury (Anger, 1990, 1992). Since

162
biomarkers should be universally applicable, only those available for administration in
several major languages is suitable.
Of the behavioral test batteries which have been translated into several languages
(see Anger, 1992; Anger and Johnson, 1992, for other batteries), two can be considered
suitable candidates as standardized biomarkers of neurotoxic effect. They are the WHO-
recommended Neurobehavioral Core Test Battery (NCTB) and the Neurobehavioral
Evaluation System (NES). The tests in each of these batteries are listed in Table 2.
Table 2. Behavioral Test Batteries (Multi-Language)

World Healtb 0manjzatjon fWHOl Neurobehayjoral Core Test Battery (NCTBl

SantaAna Digit Symbol

Aiming Benton Visual Retention Test
Simple Reaction Time Digit Span
Profile of Mood States (POMS)
Source' Johnson ef al 1987

Neurobebayjoral Eyaluatjon System (NES)

COGNITWE MEMORY AND LEARNING PSYCHOMOTOR
Vocabulacy Digit Span" Symbol-Digit ..
Horizontal Addition Paired-Associate Learning Hand-Eye Coordination
Switching Attention Paired-Associate Recall Simple Reaction Time ..
Grammatical Reasoning Visual Retention Continuous Performance Test
ColorWord Pattern Memory Finger Tapping
~ Memory Scanning PERCEPTUAL ABILITY
MoodTest Serial Digit Learning Pattern Comparison

.. Variant of WHO NCfB core test (top)

Source: Letz and Baker, 1986; Letz, 1990

Both the NCTB and NES batteries are used currently, have been widely applied in
occupational settings, and are suitable for screening. The NCTB, developed in English, has
been translated into Chinese, Dutch, Finnish, French, German, Italian, Polish, and Spanish.
The NES, also developed in English, has been translated into Chinese, Danish, Dutch,
Finnish, French, German, Italian, Japanese, and Spanish. Inspection of the tests used in
these (and other) batteries (see Anger, 1990, 1992) reveals considerable overlap, suggesting
a degree of consensus on behavioral biomarkers appropriate for screening purposes.

Neurobebavioral Core Test Battery (NCTB)

The Neurobehavioral Core Test Battery (NCTB) was developed by a group of expert
scientists at a 1983 meeting in Cincinnati sponsored by the World Health Organization
(WHO) and the U.S. National Institute for Occupational Safety and Health (NIOSH).
These experts included the world's most experienced investigators in the nascent research
field conducting worksite research in the 1970's and early 1980's. They were convened by
WHO and NIOSH to consider the possibility that a single test battery could be
recommended to screen for neurotoxic effects in working populations. They recommended
a core set of tests (Table 2, top) to be regularly used to evaluate people exposed to
neurotoxicants and also supplementary tests depending on the chemical exposure and the
setting where testing was to be carried out (Johnson et al., 1987). The NCTB is
administered in a one-on-one administrator-with-subject session, and its component tests
have consistently discriminated between chemical-exposed groups and controls not
exposed to chemicals (Anger, 1990). It was on the basis of their demonstrated ability to
discriminate exposed from unexposed subjects--sensitivity to chemicals or criterion
validity--that these tests were selected by the WHO/NIOSH experts (Johnson et al., 1987).
To determine the feasibility of using the NCTB in diverse cultures, a Cross-Cultural
Assessment (CCA) was developed under WHO auspices (Anger and Cassitto, in press). By

163
mid-1991, the NCTB had been administered to 2300 adults who were Il2l. exposed to
neurotoxic chemicals in their work. These subjects were between the ages of 16 and 65
years and lived in ten countries around the world. Representative results from the Digit
Span test are shown in Figure 1. The results reveal that mean perform:mce un the Simple
Reaction Time and Benton test of the NCTB were very similar in all countries, while
performance on the Digit Span, Digit Symbol, Santa Ana, and Aiming tests was more
variable between countries. In the male subject, the data from Nicaragua (rightmost bar)
were much lower than that in all other countries on all cognitive and psychomotor tests
except the Santa Ana test of coordination (Anger er al., in press).
The performance difference in Nicaragua could reflect ethnic or cultural differences,
but the more likely reason is the lack of education in the Nicaraguan subjects tested. About
74% of the Nicaraguan subjects had 0-3 years of education, while most subjects in the
other 9 countries where the assessment was conducted had 8 or more years of education.
Years of education correlated with test performance at 0.31 to 0.64 (Kendall's Tau) on the
various NCTB tests in Nicaraguan subjects (Anger er al., in press).
The variables of age and sex had a relatively small effect on NCTB performance in the
CCA, although small differences were seen on several tests. Figure 2 demonstrates the
relatively limited impact these variables had on one test, the Digit Span, in CCA data
collected in the USo
The NCTB clearly produced consistent results in many countries. However, it is also
obvious that baseline (unexposed subjects) data from one country cannot be used as a
control group (or as normative data) for subjects exposed to chemicals from another

25 Male 25 Digit Span

Digit Span
Subjects
..
'C 'C
~ oS! Female

20 0 PR China ~20 Subjects
u
GI GI
a: Poland a:

15 fZl Austrla
'"
Cl Netherlands
'"
Cl
15
D PR China
E 10 0 France .E
e; 10 ~ Austria

m
In j Italy 111 Netherlands

Cl
Hungary :> ~ Italy
5 EI USA c 5 Po land

Canada E;:J USA
Nicaragua
0 0
2635 26 - 35
Age Range Age Range

Figure 1. Mean (+ Isd) Digit Span perfonnance in NCfB Cross-Cultural Assessment (CCA).

25 USA 25 People's Republic

11 Males
G Females of China I!II Males
'C (;;] Females
'C
GI
~
;;; 20 :;; 20
u u
GI GI
a:
a: 15 15
'"c:
Cl '"Clc:
'5 10 '5
CI)
10
In

:> 5 'c, 5
c

' 0 o 1625 26-35 36 - 45 46 -55 56-65

1625 2635 36-45 46-55 5665
Age Range Age Range

Figure 2. Perfonnance (mean +lsd) on the Digit Span test by males and females from 16-65 years of age in
the US and the People's Republic of China.

164
country (Cassitto et al., 1990; Anger et al., in press). This is very likely also true of
research within a country. In this first large-scale attempt to evaluate the impact of human
subject variables on behavioral biomarkers, it appears, though replication is clearly needed,
that education is at least as important as any other subject variable studied thus far (Anger
et al., in press). Subject variables may thus have a larger effect on behavioral tests than is
the case with many other biomarkers.

Neurobehavioral Evaluation System (NES)

The Neurobehavioral Evaluation System (NES) is the most widely-used battery in
human behavioral neurotoxicology (Baker et al., 1985; Letz and Baker, 1986; Letz, 1990).
The NES (Table 2, bottom) battery of tests (only a sampie of the tests are used in any one
study) is implemented on IBM PC-type computers. Computer implementation provides
an efficient means of test administration to literate subjects (who can read the on-screen
instructions), although the administrator can also read the instructions to the non-literate
subject.
Computer-implemented behavioral tests have not been used in worksite research until
the past five years, and relatively few reports have been published (see Letz, 1990, for a
listing). Most such tests are derived from tests administered by a human (who says the
instructions and essentially re-explains them until they are understood). Since most such
computer-implemented variants of these tests have not yet discriminated, in cross-sectional
research, between exposed and unexposed populations, they do not yet have proven
sensitivity to chemicals (criterion validity). Further, one can question whether an
impersonal computer can test the functional capacity of a human subject as effectively as a
personable and insightful test administrator?
To shed light on this issue, approximately 900 baseline (unexposed) subjects were
administered comparable tests from both the computer-implemented NES and the human-
administered NCTB (counterbalanced order of administration). Figure 3 contains mean
performance data from that research. Performance in males and females reveals identical
trends on the Benton Visual Retention test and the comparable but longer NES Visual
Retention test across the age ranges of 16-65. NCTB data (left panel) shows that males and
females recall about the same number of test figures and that performance declines slightly
in the 55-65 age range. NES data (right panel) reflect the same trend, although the NES
had more trials and thus subjects had an opportunity to recall more figures. These
descriptive data require careful analysis, but they suggest that performance trends are
similar in related tests given by a human administrator (NCTB) or administered on a
computer with written instructions (NES). They are encouraging data which suggest that
computer-implemented tests are equivalent to human-administered tests in assessing certain
functional capabilities.

NCTB NES

18 Benion Visual Relenlion 18 NES Visual Retenlion

16 16 Males
"C Males "C
GI 14 EJ Females :!l 14 [21 Females
'e:
N
'e:0>
0
12 g' 12
u u
GI 10 ~ 10
:< .0.
,0,
:,0,
,~:
a:

~
" 0 ~.'.
0'0 ~:: ~:~
:0l ':. ;~:~ :;~;
8 .',',
:< ::~
~"' W "'
~
8 :~:
~:: { :::=
.:'

~~
:::> 6 I:;: ::> 6
...
.~
4
0>
: 4
;:
:: m
:;~
;:;~
',',
:::.
':':
0'0
',',
,>
,','
'0'

I::: ::: ,0, 00'

.;< ~::
2
:;;
:8:
2 ,>
:..'
':
,;,
>.
'0' ','.
>;. .<,'~':'... ,0,

:
0 ~
o ,','
1625 2635 3645 4655 5665 1 625 2635 3645 46-55 56-65
Age Range Age Range

Figure 3. Mean (+ I sd) figures recognized in the Visual Retention test {rom the NCTB and NES batteries
given to the same subjects.

165
 There are several computer-implemented behavioral biomarker batteries (Anger,
1990b), but each has significant limitations in implementation or availability. Laursen's
Cognitive Function Scanner (Laursen, 1990), Kennedy's (1987) Automated Performance
Test System (APTS) and Gamberale et al.'s (1990) Swedish Performance Tests System
(SPES) are also available and have a growing usage described in the literature. The NES is
the major computer-implemented behavioral biomarker battery in the field today (Letz,
1990), although few reports are yet published.

Other Batteries
The CNS/B (Bowler et al., 1986) and Pittsburgh Occupational Exposures Test (POET)
battery (Ryan et al., 1987) are neuropsychological batteries developed for individual
subject analysis of traditional neuropsychological problems. However, individual
neuropsychological assessment has been reported in only a small numbers of cases of
chemically-exposed individuals (e.g., White, 1987; White et al., 1990). As a result it is not
yet clear that the lengthy neuropsychological test batteries have greater population
screening potential than other established batteries such as the I-hour NCTB or that the
goal of individual assessment will be achieved. Also, the requirement for highly trained
individuals to administer the tests and lengthy per-subject administration times makes these
batteries very expensive to employ in large scale studies.
The (Information Theory) battery of Williamson (1990) was developed in Australia,
derived from Information Theory research in experimental psychology. This battery's
strength is that its basis for the selection of tests is a theoretical framework. Test results
can be related to theoretical psychological functions or constructs and, once a large
database is compiled, structure/function relations with chemical classes can be buHt.
However, Williamson and colleagues have studied only workers exposed to lead (1986)
and mercury (1982), and abalone divers with presumed oxygen deficits (1987). This
battery has the potential for relating nervous system effects to Information Theory.
However, a larger database than three studies is needed for Williamson's (Information
Theory) battery before it can be recommended in place of batteries with tests of
demonstrated sensitivity for detecting a range of neurotoxic chemicals.

New Developments
There has been little development or modification of behavioral test batteries in the
past five years. In September, 1991, the Agency for Toxic Substances and Disease
Registry (ATSDR) convened an expert group to consider the existing test batteries and to
counsel ATSDR on the most appropriate battery to screen for neurotoxic effects from 10w-
concentration exposures anticipated near hazardous waste sites. The expert group (chaired
by the author of this chapter and including the developer of the NES, and scientists from
the Environmental Protection Agency [EPA], NIOSH, ATSDR, and academia) proposed a
1.5-hour battery of tests which included elements of the NES and NCTB, as well as select
tests not included in existing batteries. Following these recommendations and other
information gathered from various sources, ATSDR (Hutchinson et al., in press) published
their test choices for the Adult Environmental Neurobehavioral Test Battery (AENTB). It
contains several computer-administered tests (drawn chiefly from the NES), some NCTB
tests, and other tests not presently incorporated in any test battery (ATSDR, 1992). This
newly-proposed battery does not break new theoretical ground. Rather it supports the
approach of using tests demonstrated sensitive to chemicals. This continues to be the
approach of choice in selecting tests or test batteries for unique problems (in this case,
hazardous waste sites), although some neuropsychologists gathered at the ATSDR meeting
favored using a traditional neuropsychological approach to selecting tests for neurotoxicity
assessments.

BEHAVIORAL TESTS AS BIOMARKERS

Behavioral tests are the most widely applied measures used in human eross-seetional
studies to reflect physiologie change or struetural alteration in the nervous system produced

166
by chemicals such as solvents and metals found in the workplace and environment.
Behavioral tests are objective, reliable, valid measures of nervous system function. They
are best seen as population biomarkers suitable for cross-sectional studies. They are less
suited for identifying a specific health effect or chemical exposure measurable on an
absolute scale across populations, as is the case for a measure like blood lead. Many
standardized behavioral tests and test batteries are widely used to identify neurotoxic
effects.

ACKNOWLEDGEMENTS
This manuscript is supported in part by EPA R-820111-01-0. Appreciation is
extended to Jo Brown for word processing the manuscript. David Chrislip of the National
Institute for Occupational Safety and Health collected the NES data in Figure 3.

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168
MECHANISMS OF AND BIOMARKERS FOR ACUTE AND DELAYED
NEUROPATHIC EFFECTS OF ORGANOPHOSPHORUS ESTERS

Martin K. Johnson

Toxicology Unit
MRC Laboratories
U.K. Medical Research Council
Carshalton, Surrey SM5 4EF
England

Toxicity of a chemical is dependent on several different but interconnected phases:

a) factors which influence the delivery of the ultimate toxic agent to its site of action
(absorption, distribution, storage, activation, detoxification); b) the reaction with the
primary target (reversible or irreversible); c) the biochemical and physiological
consequences and d) the clinical expression of toxicity. For the purposes of
biomonitoring we are fortunate that in the case of organophosphorus (OP) esters the
mechanism of their interaction with the primary targets, acetylcholinesterase (AChE) and
Neuropathy Target Esterase (NTE, formerly Neurotoxic Esterase) has been identified.
While OP-interaction with AChE causes acute effects (cholinergic syndrome), interaction
with NTE leads to the development of a completely different syndrome known as OP-
induced delayed polyneuropathy (OPIDP). Details of the mechanisms are given below.
Many other effects, caused by a single OP or by a group of related OPs, have been
reported, but not all are well substantiated. These include behavioural and chronic
effects on the central nervous system, mutagenic, carcinogenic, teratogenic, and
porphyric effects, effects on the immune system, on hormones, on the reproductive
system, on the retina, on lipid metabolism, etc. These effects have been reviewed
elsewhere (WHO, 1986) and will not be addressed here.

CHEMICAL PROPERTIES OF ORGANOPHOSPHORUS ESTERS

Table 1 illustrates various structures of OP insecticides. These chemicals are

normally esters, amides, or thiol derivatives of phosphoric, phosphonic,
phosphorothioic, or phosphonothioic acids. The general structural formula is:

Use 0/ Biomarkers in Assessing Health anti Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 169
 Table 1. Class and chemical structure of some OP insecticides (adapted from WHO,
1986)

PHOSPHATE
chlorfenvinphos, crotoxyphos, dichlorvos, dicrotophos
heptenphos mevinphos, monocrotophos, naled,
phospliamidon, TEPP, tetrachlorvinphos, triazophos

S-ALKYL PHOSPHOROfHIOATE o
R-S ....... II
profenfos, trifenfos /p-O-X
R-O

S.-ALKYL PHOSPHORODITHIOATE S
R-S ........... II
prothiofos, sulprofos ..,.......P-O-X
R-O

Q-ALKYL PHOSPHOROfHIOATE o11

amiton, demeton-S-methyl, omethoate (R-O)2-P-S-X
oxydemeton-methyl, phoxim, vamidothion
azothoate bromophos, bromophos-ethyl, chlorpyrifos S
chlorpyrifos-metliyl, coumaphos, diazmon, dichlofentl1ion, 11
fench1orphos, femtrothion, fenthion, iodofenphos, parathion, (R-O)2-P-O-X
parathion-methyl, f.yrazophos, pyrimiphos-ethyl,
pyrimiphos-methy , sulfotep, temephos, thionazin

PHOSPHORODITHIOATE S
amidithion, azinphos-ethyl, azinphos-methyl, dimethoate, (R-O)2-P-S-X
dioxathion, disulfoton, ethlOn, frmothion, malathion, mecarbam,
menazon, methidathion, morphothion, phenthoate, phorate,
phosalone, phosmet, prothoate, thiometon

PHOSPHOROAMIDATE
cruformate, fenamiphos, fosthietan

PHOSPHOROfHIOAMIDATE o11
methamidophos R-O)-~
S-alkyl

isofenphos S
11
(R-O)2-P-N~

PHOSPHONATE o
R-O ....... n
butonate, trichlorfon ,........P-O-X
R

PHOSPHONOfHIOATE S
R-O ........ n
EPN, trichlornat, leptophos, cyanofenphos ,........P-O-X
R

170
R1 and R2 are usually simple alkyl or aryl groups both of which may be bonded directly
to the phosphorus atom (in phosphinates), or linked via -0- or -S- (in phosphates), or R1
may be bonded directly and R 2 via one of the above groups (phosphonates). In
phosphoroamidates, carbon is linked to phosphorus through an -NH group. The group X
can be any one of a wide variety of substituted and branched aliphatic, aromatic, or
heteroeyclie groups, linked to phosphorus via a bond of some lability (usually -0- or -S-)
and it is referred to as the leaving group. The double bonded atom may be oxygen or
sulphur, and related compounds would be called phosphates or phosphorothioates (the
nomenclature 'thiophosphate' or 'thionophosphate' is now outdated). The p=o form of
a thioate ester, referred to as the oxon, is often incorporated in the trivial name (e.g.
parathion is the parent compound of paraoxon). The P=S compounds (when pure) are
not directly toxic: they require metabolic conversion to the equivalent oxons in order to
exert their effect as inhibitors of enzymes (see below).
Besides the insecticidal OP compounds a different type of OP ester is produced
commercially in massive quantities. Rather stable triesters of phosphoric acid with a
variety of phenols, xylenols etc. or with higher alcohols such as 2-ethyl-hexanol, butoxy-
butanol etc. are produced as plasticisers, flame retardants or hydraulic fluids. Few of
these compounds cause acute toxic effeets but some can cause OPIDP.

ACUTE AND DELAYED NEUROPATHIC EFFECTS OF SOME OP ESTERS

The acute effects of OP esters arise from inhibition of AChE in the nervous system
and subsequent accumulation of toxie levels of endogenous acetylcholine (ACh) in
nervous tissue and effector organs in both inseets and mammals. In mammals, ACh is
the chemical transmitter of nerve impulses at endings of post-ganglionic parasympathetic
nerve fibres, somatic motor nerves to skeletal muscle, pre-ganglionie fibres of both
parasympathetic and sympathetic nerves, and certain synapses in the central nervous
system. Thus, inhibition of AChE causes signs and symptoms that mimic nicotinic,
muscarinic and central nervous system actions of ACh. The clinical picture is deseribed
in detail elsewhere (Taylor, 1985; WHO 1986).
Regardless of the severity of anticholinesterase effects, some OPs can also induce a
quite different syndrome known as OPIDP. Features of this syndrome are: (1) a de1ay
of 1-3 weeks between ingestion of the agent and development of clinieal signs: (2) the
fact that a single dose may be sufficient to cause the effect even when the agent is
unstable and cleared from the body within hours; (3) the preferential invo1vement of
longer axons of spinal cord and peripheral nerves with degeneration in the most distal
regions; (4) irreversible clinieal effects in all but mildly-affected cases. Details of
history, clinical and morphological features are reviewed elsewhere (Cavanagh, 1973;
Johnson, 1975; Davis and Richardson, 1980) and the bioehemical mechanism is
discussed below.

MECHANISM OF INTERACTION OF OP ESTERS WITH ESTERASES

The chemistry of OP interactions with AChE and other esterases (e.g. NTE, liver
carboxylesterase, serum butyrlycholinesterase, trypsin and chymotrypsin etc) is common
to all the esterases and is illustrated in Figure 1. Following the formation of a Michaelis
complex (reaction 1), a specific serine residue in the protein is phosphorylated with loss
of the leaving group X (reaction 2). Two further reactions are possible. Reaction 3
(reactivation) may occur spontaneously at a rate that is dependent on the nature of the
attached group and on the protein: it is also influenced by pH and added nucleophilic
reagents, such as oximes, which may catalyse reactivation. Reaction 4 (aging) involves
cleavage of an R-O-P bond with the loss of Rand the formation of a charged

171
monosubstituted phosphoric acid residue still attached to protein. The reaction is called
aging because it is time-dependent, and the product is no longer responsive to
nucleophilic reactivating agents. Each step is discussed below and the textbook by
Aldridge and Reiner (1972) should be consulted for greater detail.

1
k+ 1 0
11 (1) ...... II
ENZYME-OR + X-P-(OR)2<;<======~ [ENZYME-OR--X-P-(OR)2]

k_ 1 (2) k+
20

11

EN;.;r:-~-(OR)'

~----<IIOi:~------- ENZYME-O-~-OR
b-
Fig. 1 Inhibition of an esterase by an OP compound. (1) Formation of Michaelis complex. (2)
Phosphorylation of the enzyme. (3) Reactivation reaction. (4) "Aging".

Inhibition

When cholinesterase is incubated with an OP ester, the activity of the enzyme will
decrease in a time-dependent manner and the rate of inhibition will be dependent on the
inhibitor concentration. Formation of inhibitor-enzyme complex with
[inhibitor] > > [enzyme] is a pseudo-first-order bimolecular reaction and the bimolecular
rate constant (k ) for the reaction is a characteristic of the enzyme and the particular OP
under defined c~:mditions of temperature, pR and composition of the medium and can be
determined experimentally in vitro.
Many OP compounds with biologically significant effects have an inhibition t'h not
higher than 20 min at 37C for concentrations lower than 10-5 M (i.e. k > 3,500 M -I
min- I ). Some compounds can be much more active, with a t,j, =20 m'in at 10 -9 M
concentration. Rowever, some effective insecticides such as dimethoate or
methamidophos appear to be rather poor inhibitors of AChE in vitra with their toxicity
depending on the fact that their disposal rate in viva is comparatively slow (i.e. half-life
of the active agent being up to 1-2 days rather than a few minutes as in the case of
dichlorvos, for instance).

Reactivation

The classification of esters into substrates and inhibitors is somewhat arbitrary; the
difference between them is in the velocity of reaction 3. Values of k+ 3 differ greatly
between substrate and inhibitors. For the hydrolysis of ACh by AChE, k+ 3 is
approximately 3x105 min- I so that the acyl-enzyme is rapidly deacetylated and catalytic
activity is regenerated; for OP compounds and AChE, k+ 3 is 10- 1 _10- 6 min- I and the
regeneration of active enzyme is very slow. The rate of spontaneous reactivation of
phosphorylated AChE depends on parameters such as pR and temperature, and also on
the chemical structure of the side-chain bound to the phosphorus atom. For

172
phosphorylated AChE k+ 3 values vary according to R as follows:
(2-ClEtO)2> (MeO)2 > (IsoProO)2 > (EtO)2 (Reiner, 1971) and the half-life (t'h) of the
inhibited enzyme ranges from less than 30 mins to more than 30 days. The presence of
a sulphur instead of oxygen greatly increases the rate of reactivation of the analogous
compound (Clothier et al, 1981). It is believed that no spontaneous reactivation of
AChE occurs after inhibition by phosphoroamidates. Reactivation of phosphorylated
AChE can be accelerated in vitro by nucleophilic agents such as NaOH, oximes
(RrC=NOH), hydroxamic acids (R.CO.NHOH) or fluoride ions. Reactivation
proceeds by the reaction of the nucleophile (water in the simplest case) with the
electrophilic phosphorus attached to the enzyme. For the therapeutic purpose of
increasing the rate of restoration of active AChE in vivo the only agents which combine
efficacy with few toxic side-effects are certain oximes.

"Aging"

The aging phenomenon, is the time-dependent loss of ability of the phosphorylated

enzyme to be reactivated by nucleophilic agents. The mechanism, described by the
reaction 4 on Fig. 1, consists of cleavage of one R group and formation of a charged
monosubstituted phosphoric acid residue on the protein. The rate of aging is, as for
reactivation, dependent on pH, temperature etc, and also on the structure of the R group.
For AChE the rate of aging is as follows: (highly branched alkyl group-O) > (Met-O)
> (isoPro-O) > (Et-O) (O'Brien, 1967).

In Vitro/In Vivo Extrapolations

Although in vitro data, described above, cannot be transposed direct1y to in vivo

situations, they are consistent with the weIl known fact that, after poisoning by a sub-
lethai dose of some dimethyl phosphates, recovery and disappearance of symptoms is
complete within a few hours. The value of k+ 3 for erythrocyte-AChE, taken from rats
dosed in vivo with dimethyl phosphates, was reported to be 57 x 10-4 (a half-life of
inhibited enzyme of 2 hours) (Vandekar and Heath, 1957). One day after a dose of
80% of LD 50 , most AChE was in the uninhibited form with a small fraction in the aged
inhibited form. By contrast, these workers found that less than 20% of the enzyme
activity was restored one day after poisoning with a diethyl phosphate: this was due to a
slow rate of spontaneous reactivation rat her than to significant aging of inhibited
enzyme. The same contrast in rates of reactivation and aging are found for human
AChE after inhibition by dimethyl or diethyl phosphates (Table 2) and the same contrast

Table 2. Half-lives (hours) of spontaneous reactivation and of aging of dimethyl

phosphoryl and diethyl phosphoryl human AChE and plasma ChE under approximately
normal physiological conditions (37" and pH 7): Data from Aldridge and Reiner, 1972;
Hobbiger, 1956; Skrinjavic-Spoljar et al, 1973; WHO, 1986.

AChE (Erythrocyte
Alkyl Group or neural) ehE (Serum)

Reactivation Reacti vati on

Methyl 0.85 4 ? ?

Ethyl 60 20-40 30 days ?

173
in speed of recovery from intoxication can be expected unless, of course, the persistence
of toxic agent in the body over-rides the reactivation effect.

OP Esters and Neuropathy Target Esterase

The first essential step in the initiation of the delayed neuropathie effeet of an OP is
phosphorylation of a target protein in the nervous system. The protein, whieh was first
identified by radiolabelling, has esteratie aetivity and phosphorylation can be
eonveniently monitored as a progressive inhibition of the aetivity of this enzyme, now
known as NTE (Johnson, 1982). It has beeome clear that the eonsequenees of OP-NTE
interaction are quite different from those of AChE inhibition. Acute toxicity arises
directly from the loss of catalytie aetivity of AChE, leading to exeessive aceumulation of
anormal physiologieal substrate (ACh). On the other hand, initiation of OPIDP requires
the generation of a eertain quantity of 'modified' NTE in the nervous system at some
point in time rather than mere loss of NTE eatalytie aetivity; there is no evidenee of
deleterious aeeumulation of physiological substrate or laek of hydrolysis produets in the
aetiology of OPIDP. The 'modification' of the NTE moleeule depends on the nature of
the ehemieal group derived from inhibitor whieh beeomes eovalently bound to the
eatalytie site. The most easily deseribed modification is brought about by aging of
organophosphorylated NTE (Reaetion 4 of Fig. 1). Compounds sueh as phosphinates
which inhibit NTE but cannot then engage in the aging reaetion bloek the proeess so that
they are not neuropathie themselves but aet as prophylactic agents when administered
before a neuropathie OP is given to test animals (Johnson, 1974). It is now elear that
inhibitors of NTE can be sub-divided as if they had a range of partial agonist effeets
when bound eovalently, and that the above description of aging and non-aging of
inhibited NTE may represent the two extremes with various degrees of charge
distribution around the phosphorus atom aeeounting for the range of partial effeets
(Lotti, 1991; Johnson, 1993). For the purposes ofbiomonitoring it is adequate to
measure ehanges in NTE eatalytie aetivity (see below).
In experiments with adult hens, deteetable neuropathie events are never seen after a
single dose/exposure of an OP unless at least 70% of the normally available NTE is
eonverted to the modified form. In single-dose experiments, the peak amount (measured
as loss of NTE catalytic activity) is usually reached within 1-72 hours of dosing: the time
depends on the speed of metabolie activation (if any) and disposal reactions for the
partieular eompound. Owing to the synthesis of fresh protein, this inhibition declines
markedly during the 8 to 14 day delay period, and there is no correlation between
neuropathy and NTE inhibition measured by the time clinical signs reach their peak.
The sequence of events leading from formation of the necessary quantum of modified
NTE to axonal degeneration and clinieal neuropathy is largely unknown. The threshold
above whieh inhibition of human NTE preeipitates clinieal neuropathy is not eertain but
it may not be too far removed from the threshold seen in hens and some mammals
(Johnson, 1982; Lotti el al, 1986).

BIOMONITORING

Bioehemical assays are available to monitor effeets of exposure to OPs on aetivity of

various esterases in accessible tissue from man or in post-mortem samples from man or
animals: in some eireumstanees these effeets may eorrelate with effects on health. At
the ehemicallevel analytieal proeedures are now available to detect the presenee of
residues of OP esters in food, tissue or environmental sampies at levels lower than those
known to relate to deleterious effeets on health.

174
TISSUE ESTERASES AS INDICATORS OF EFFECTS

Besides neural AChE and NTE other ~p-sensitive esterases occur in tissues. For
monitoring purposes the most interesting are those present in blood as an accessible
tissue. AChE is found in erythrocytes and, in general, the inhibitor-sensitivity of the
accessible AChE is similar to that of inaccessible target AChE found at nerve-endings :
it is therefore a useful 'effect' biomonitor. Significant depression of erythrocyte AChE
is a strong indication of a health hazard as weIl as of exposure to OP compounds.
Plasma contains an enzyme which also can hydrolyse AChE and is known variously as
Plasma cholinesterase, ChE, Pseudocholinesterase or butyrylcholinesterase: it has a
wider specificity for substrates than has AChE but has no known physiological function.
It is able to hydrolyse some substrates of AChE and is sensitive to inhibition by OP
compounds and was a popular alternative to erythrocyte AChE as a biomonitor in the
days when the red coloration of haemoglobin hindered colorimetric assays. However
BuChE can only be used as a monitor of exposure (not of effect) since there is no direct
relationship between inhibition of this enzyme and toxicity. Since structure/activity
relationships for inhibitors of AChE and BuChE are different, one or other of the two
may be the more sensitive indicator of exposure in any particular case (WHO, 1986).
Physiological variations in blood-ChE levels occur in a healthy person and are seen
among the population. It has been estimated that the coefficient of variation for AChE
activity in sampies from an individual is 8-11 %, and that a decrease of 23 % below pre-
exposure level may, therefore, be considered significant. If the average of several pre-
exposure values were available, then a decrease of 17% would be significant. It has
been recommended that, if measured activity is reduced by 30% or more of the pre-
exposure value, AChE measurements should be repeated at appropriate intervals to
confirm the results. Depressions of AChE or ChE in excess of 20-25% are considered
diagnostic of exposure but not, necessarily, indicative of hazard. Depressions of 30-
50% or more are considered indicators for removal of an exposed individual from
further contact with pesticides untillevels return to normal (WHO 1986). A major
problem with 'spot-checks' using BuChE as indicator is that activity of this enzyme
fluctuates much more widely than AChE activities and is affected by many disorders of
health. Therefore assays of BuChE are seldom of value diagnostically but may be useful
for regular screening of occupationally exposed workers.
The reports of the annual Joint FAO/WHO Working Parties on Pesticide Residues in
Food contain summaries of numerous controlled exposure studies. No cases appear to
be known of significant clinical effects in man in the absence of depression of plasma- or
erythrocyte-ChE levels. No-observed-adverse-effect levels have been calculated on this
basis, where the data are available, or have been estimated for man by extrapolation of
the available data for exposed animals.

Assay Procedures

By far the most satisfactory procedure for assay of AChE is that developed by
Ellman et al (1961) based on colorimetric measurement of thiocholine liberated from
acetylthiocholine : the widely used method of Michel (1949) has greater variability, and
radiometric assays are not suited to field work. A cheap, robust and apparently reliable
battery-operated spectrophotometer has undergone considerable field-testing (Magnotti
et al, 1987, 1988; Verschoyle and and Johnson, 1988; Verschoyle, 1989).
Monitoring of NTE activity in accessible tissue is possible since the enzyme is found
(at rather low levels) in human lymphocytes and platelets (Dudek and Richardson, 1980;
Bertoncin et al, 1985; Lotti 1987; Maroni and Bleeker 1986). So far as has been tested
the inhibitor-sensitivities of these accessible NTEs are similar to those of the neural
enzyme. Assay of NTE is not widely used for monitoring workers since in most cases
the AChE or BuChE will be more sensitive but one report exists where workers were

175
successfully monitored who had handled cotton from plants treated with an OP defoliant
wh ich has low anticholinesterase activity (Lotti et al, 1983). NTE assays have also
served to evaluate likelihood of neuropathie effects in suicidal persons who have ingested
OPs (Lotti et al, 1986).
It should be emphasized that the structure/activity relationships for inhibition by OP
esters of AChE and NTE are markedly different. Thus dichlorvos is at least l00x more
inhibitory to the former than the latter (k~ChE/~TE > 100) but for the di-n-pentyl
analogue of dichlorvos that ratio is < 10-3 (Lotti and Johnson, 1987).

Storage of Blood and Tissue Sampies Prior to Assay

The stability of erythrocyte AChE and plasma BuChE under various conditions
(with/without anticoagulant, buffer or saline) is discussed in detail by St. Omer and
Rottinghaus (1992) and by Duncan and Griffith (1992) but the special case of sampies
drawn from workers actually exposed to OPs requires extra precautions against (a)
ongoing inhibition during storage of the sampie due to the presence of residual OP in the
blood or to contamination from the client's skin, and (b) spontaneous reactivation of
inhibited enzyme: storage at 2-4 is advisable and assay should be done as soon as
0

possible and by a method (such as Ellman' s) which involves only brief incubations.
Some evidence exists from studies of human blood sam pies inhibited in vitro that,
provided sampies are assayed after a delay of not more than one day (i.e. allowing
transmission to an analytical laboratory by post) then assay results are not greatly
distorted (private communication from U.K. Health and Safety Executive). This
experience seems at odds with the reported half-lives of only one hour for spontaneous
reactivation of human dimethylphosphoryl AChE at 3r (Table 2). Further investigation
with serial analyses of sampies from exposed workers is clearly necessary (IPCS, 1993).
The experience of Wilhelm and Reiner (1973) should be noted : these workers found that
even methylcarbamylated AChE wh ich reactivates spontaneously at a high rate if the
blood sampie is stored undiluted could be stabilised when diluted in pH5 buffer and that
such dilution also prevented ongoing inhibition due to residual compound.

Duration of Inhibition of Esterases following OP Exposure

Three factors must be considered : persistence of the toxic compound, spontaneous

reactivation of inhibited enzyme(s) and synthesis of fresh enzyme.

a) Persistence of compound. OP insecticides are, in general, much less persistent than

typical organochlorine pesticides but the range of lifetimes in vivo is large. Thus
dichlorvos is probably virtually all degraded within a day in vivo but residues of
fenitrothion seemed to remain in fat stores of an intoxicated woman long enough to cause
recurrence of symptoms when she reduced her diet and lost weight about 2 months after
clinical recovery from her first experience (Ecobichon et al, 1977).

b) Spontaneous reactivation of inhibited esterases. Table 2 indicates that after a

single exposure to a dimethyl phosphate activity of blood AChE should return to normal
level rapidly. However, if the exposure is repeated through daily occupation or by
ingestion of a massive dose which is cleared only slowly then aging of the inhibited
AChE will supravene and the scope for reactivation will be reduced markedly.
Interestingly aIthough spontaneous return of activity after inhibition by diethylphosphates
is negligible (see Table 2) yet, since aging is almost nil, there remains good hope for
therapeutic use of oximes over a long period to force reactivation. Blood AChE and
neural enzyme seem to behave similarly to each other in these processes of reactivation
and aging. Although the data for human plasma ChE is not formally tabulated there is
anecdotal evidence that half-lives of reaetivation and aging of the inhibited enzyme are
not similar to those for AChE (see also (e) below).
176
(c) Synthesis of fresh enzyme(s). The li fe-time of human erythrocytes is about 120d
whereas AChE of brain (and, probably, of most neural tissue) turns over much faster (t'h
around 4-8d in various mammals). Thus, in the absence of spontaneous reactivation,
inhibition of blood AChE can be detected long after neural effects have regressed and it
is only in rather acute cases that the actual measured activities are of value in guiding
therapeutic procedures. For the purpose of indicating previous exposure the slow
disposal of red cells is an advantage. Plasma BuChE is turned over very rapidly and
measured activities are not likely to be depressed for long after metabolie disposal of a
contaminating compound is complete.

TISSUE ESTERASES AS ENVIRONMENTAL BIOMONITORS

This topic has been reviewed elsewhere (Thompson and Walker, 1992) and will not
be discussed here at length. Plasma of most mammals and birds contain various levels
of cholinesterase or carboxyesterases and, in principle, populations or individuals can be
monitored for evidence of exposure which may act both as a health guide and as an
indicator for post-spraying persistence of pesticide in the environment. While
procedures are essentially the same as for human enzymes, certain differences in stability
of both uninhibited and inhibited enzymes have been noted in sampies from birds.
Diurnal variation in plasma enzymes can be marked in some birds. Some discussion of
the value of assaying fish brain AChE levels as an aquatic environmental biomonitor is
made by Murty and Ramani (1992).

DETECTION OF METABOLITES AS INDICATORS OF EXPOSURE

A major pathway of metabolic degradation of OP esters is hydrolytic. Significant

amounts of di-alkyl phosphoric acid or di-alkyl phosphorothioc acid from typical OP
insecticides are excreted in urine of exposed humans or animals beginning within a day
of exposure and the amounts usually decline quickly over a few days. A limited number
of phosphorie acidic products are produced from a variety of insecticides so that one
analytical procedure may serve as the basis for monitoring exposure to many OP
insecticides. Some good procedures are available (see below) but require sophisticated
GLC or HPLC equipment. Another product of hydrolysis is derived from the 'X'
leaving group of OP insecticides : such products may be identifiable and quantifiable by
simpler colorimetric assays. Thus 4-nitrophenol derived from parathion can be
measured directly and many other phenolic compounds are detectable by the amino-
antipyrine reagent of Gottleib and Marsh (1946). Although essentially simple these
assays are not generic: they are limited to one or two pesticides only and may suffer
from interference from endogenous urinary metabolites uniess preliminary clean-up
procedures are applied.

Methods for Determining Dialkyl Phosphates and Related Compounds

This topic is weIl covered in arecent critical review (Murray and Franklin, 1992)
and therefore only a few crucial points will be reiterated here in note form and without
detailed reference to original methods. The majority of published monitoring studies
utilised procedures with varying amounts of clean-up followed by alkylation using a
diazoalkane (methane up to pentane) with GLC separation of products: the recent report
by Vasilic et al (1992) is a good example. Detection with phosphorus-sensitive detectors
enables some clean-up procedures to be omitted but the response-sensitivity of various
metabolites with such detectors is unpredicatable so that internal reference standards are
less meaningful. Apart from any problems of tedium, yield, sensitivity etc., a major
objection to all these methods is that diazomethane and its homologues are explosive

177
agents wh ich must be freshly prepared each time and handled in solution with extreme
caution. Furthermore they are volatile and among the most carcinogenic agents known.
The combined hazards of this group of agents makes them unacceptable in any but the
most specialised laboratory.
Alternatives to diazoalkane-based procedures have involved on-column
derivatisation with trimethylanilinium or trimethylammonium hydroxides or have used
lipophilic ion-pair extraction into methylene chloride containing pentafluorobenzyl
bromide or ethyl iodide as derivatising agent. While Murray and Franklin (1992)
commend several aspects of this approach, they state that the procedure does not cope
with the non-sulphur containing metabolites which are produced alongside the
phosphorothioate metabolites. They also comment that good qualitative identification but
not quantification of metabolites could be obtained by removing water from sampies by
azetropic distillation instead of trying to extract these polar metabolites out of the
aqueous phase.
HPLC separation systems are also discussed by Murray and Franklin (1992) but
they conclude that sensitivity of detection is generally a problem unless a special post-
column derivatisation system is added.

COMPARISONS OF URINARY METABOLITE AND BLOOD ENZYME ASSAYS

AND THEIR VALUE IN HUMAN HEALTH MONITORING

It is generally accepted that urinary metabolites can be detected at low levels in

sampies from occupationally exposed workers without the activity of their erythrocyte or
serum cholinesterases being markedly depressed. Furthermore the detection of
metabolites is a positive procedure whereas detection of inhibition of enzymes requires
either a pre-exposure reference value or aceess to mean control population values with
their inherent variability. However it cannot be emphasised too mueh that there is not
the slightest general correlation between urinary levels of a particular metabolite and
clinical response to all OPs which can generate that metabolite: precise callibration is
needed for each and every pesticide. Thus it is obviously likely that much more
dirn ethyl phosphorothioic acid will be excreted after ingestion of, say, one quarter of
LD so of malathion than after one quarter of LD so of methyl parathion [LDsos are about
1800 and 40 mg/kg respectively]. Furthermore, individuals vary greatly in the rates and
patterns of metabolic disposal of OP insecticides and no general guidelines based on
urinary metabolites have developed for guiding hygienie proeedures or for making
decisions (say to withdraw individuals from spraying activities). Vasilec et al (1992) set
metabolite and enzyme data side by side for a number of persons acutely poisoned by
ingestion of two OP pesticides and found no consistent relationship.

ELECTROPHYSIOLOGICAL EFFECTS AS BIOMONITORS

Electromyographic (EMG) studies using non-invasive surface electrodes have been

claimed to give sensitive indieations of exposure to organophosphorus pestieides, even in
situations where blood-ChE activity has returned to normal levels. The method requires
sophisticated equipment and a very skilIed praetitioner. There is still considerable doubt
about the validity of some published studies. Reprodueibility is known to be very
sensitive to loeal faetors such as temperature of the skin, and eonflieting results have
been published: so me report smaU increases and others claim small deereases in the
amplitude of evoked muscle action potential, in response to nerve stimulation (Jager
et al, 1970; Drenth et al, 1972; Roberts, 1977). These findings have been reviewed by

178
LeQuesne and Maxwell (1981), who noted that changes that have been reported tended
not to be dose-related. In addition, they evaluated the technique under controlled
circumstances. In a treatment to eradicate parasitic schistosomes, 55 children were
dosed orally with trichlorfon, 3 times, at 2-weekly intervals, at doses that measurably
depressed blood-ChE (mean 50%), but were not enough to cause overt toxic effects,
apart from mild cramps and diarrhoea in a few cases. Only 3 children showed a
significant alteration in electromyographic response. Shortly after the last (and highest)
dose of 10 mg/kg body weight, 3 children developed repetitive activity recorded over the
thenar muscles following supramaximal stimulation of the median nerve at the wrist.
The activity consisted of a small potential at the end of the main muscle response and
was characterized by being abolished by a second stimulus 30 or 80 milliseconds after
the first, or by maximum voluntary contraction for 10 seconds; the amplitude of the
response to the second stimulus was not reduced. These characteristics are necessary
criteria that distinguish (these) dose-related responses from pre-existing natural (and
idiosyncratic) responses, wh ich can otherwise confuse EMG studies in a population.
Changes in amplitude measured on 52 control subjects (mean 13.82.5 SD), on 2
occasions (2 weeks apart), ranged from +5 to -3 mV. Thus, EMG does not appear to
give a highly sensitive measure of exposure to an ingested organophosphorus compound.
Other neurophysiological function tests have been reviewed recently (Misra, 1982).
Several changes were claimed for workers who had been occupationally exposed on a
daily basis to fenthion [O.O-dimethyl 0-(4-methylmercapto-3-methylphenyl)
phosphorothioate] until 3 weeks before testing. (Misra ct al, 1988) : the changes were
associated with depressions of plasma cholinesterase. No clinical abnormalities were
noted, measurements of erythrocyte AChE were not made and late follow-up studies
have not been reported so the sensitivity and significance of these effects cannot be easily
assessed. Ring ct al (1985) claimed that EMG studies enabled them to pick out workers
likely to be particularly susceptible to exhibit clinical symptoms in the first 2-5 weeks of
a spraying season. The most sensitive parameter was the motor conduction velocity
(MCV) measured in the ulnar nerve above the elbow: they stated that effects on
amplitude of action potential were not significant in reflecting acute effects of OP
absorption in contrast to reports by Roberts (1976, 1977). The findings of effects on
MCV are in marked contrast with the experience of Maxwell Cf al (1981) who found no
changes in MCV in median nerves of the schoolchildren treated with the OP anti-
schistosome drug as described above.
From the above brief survey it seems that the procedures of electrophysiological
monitoring are not weIl adapted to field testing, although they may be applicable to
factory workers. No consistent effects are certain at doses which do not also affect
blood cholinesterases and no irreversible effects are proven which are not associated
with clear OPIDP.

BEHAVIOUR EFFECTS AS BIOMONITORS

The methodology and validation of these procedures are beyond the scope of this
Review. Behavioural tests involving many types of populations are reviewed in an
accompanying article (Anger, 1993). Neurological and behavioural differences between
groups of pesticide-exposed and non-exposed control workers are both reported (Savage
ct al, 1988) and denied (Maizlish ct ai, 1987). In the former case significant exposure
over a mean exposure of 39d was demonstrated by the detection of low levels of urinary
metabolites of diazinon but no measures of blood esteraseS were made and no clinical
intoxication had been experienced. In the latter report only frankly intoxicated
(hospitalised persons were examined at long but unspecified periods (years in many
cases) after the acute poisoning: few neurological differences were detected but

179
significant differences were seen in several neuropsychological tests. It is not clear
whether the terrifying experience of being poisoned might influence the outcome of such
tests regardless of the nature of the intoxicant. It seems that the value of screening for
behavioural effects in OP-exposed populations may be of some value but their relevance
or sensitivity for monitoring individuals is doubtful given the wide range of individual
responses.

CONCLUSION

Mechanisms of intoxication by OP insecticides are now weH understood and have

enabled biomonitoring methods to be developed. Health effects are best monitored by
serial assays of erythrocyte AChE and good assay procedures are available including one
utilising a robust field kit. Serial assays of plasma/serum cholinesterase can provide a
useful exposure indicator and a guide to re-entry decisions for agricultural workers.
Sensitive measurement of urinary metabolites at low levels is possible and may provide a
more sensitive indicator of exposure than enzyme assays. However aIl the methods for
metabolite determination require well-equipped laboratories and most have so me
technical drawbacks: the ideal simple and soundly quantitative method has yet to be
developed. Electrophysiological and behavioural tests are less convenient and have not
been shown to be more sensitive than the above tests in routine monitoring. The case
for long-term irreversible behavioural effects of exposure to OPs is difficult to establish
or disprove at present. Better selection of exposed groups is essential if such effects are
to be probed furthe:' - defined exposure to single compounds would help greatly
especially if the study were accompanied by enzyme assays and urinary metabolite
determination.

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182
MECHANISMS AND BIOMARKERS OF SOLVENT-INDUCED
BEHAVIORAL AND NEUROENDOCRINE EFFECTS

A. Mutti

Laboratory of Industrial Toxicology

Institute of Clinical Medicine and Nephrology
University of Parma Medical School
Via Gramsci 14
43100 Parma, Italy

INTRODUCTION

Organic solvents are a highly heterogeneous dass of chemical substances sharing

just a few common properties: (i) they are chemically inert compounds, Le. they do
not react with solutes; (ii) they are usually volatile, since they should rapidly disappear
from the surface once the desired effects have been obtained; (iii) they are usually
poorly soluble in water. Perhaps because of these properties, organic solvents have
long been considered as relatively safe chemicals and as such have been widely used
not only in industry, but even at horne, often in the absence of any hygienic measure
to limit exposure.
The reasons why the interpretation of toxicity data has long lead to a substantial
underestimation of the actual risk associated with solvent exposure belong to each
one of the four major components of comprehensive risk assessment: (i) hazard
evaluation has been influenced by the low toxicity of most solvents; (ii) dose-response
evaluation has been difficult because of the aspecific pattern of adverse effects caused
by most solvents; (iii) human exposure has long been underestimated because of the
poor knowledge of toxicokinetics and especially toxicodynamics of these compounds
and their metabolites: both absorption and bioactivation have long been considered to
be negligible; (iv) risk characterization has thus been rather poor, until some
outbreaks of specific endpoints (e.g. 'Y-diketone-induced neuropathy, leukemia

Use 0/ Biomarkers in Assessing Health and Environmental Impacts 0/ Chemical

Pollulants, Edited by C.C. Travis, Plenum Press, New York, 1993 183
associated with benzene exposure) drew attention of researchers and regulatory
agencies on the possible underestimation of the actual risk.
Although largely aspecific symptoms and dysfunctions have been reported, human
studies indieate that occupational exposure to organic solvents might cause brain
damage. Because of the widespread use of many solvents, often in a confined
environment, the actual risk might be much higher than that predictable on the basis
of toxicity data, thus giving rise to public health concern.
Recent findings on brain and neuroendocrine dysfunction associated with solvent
exposure are reviewed here. Mechanistie aspects of solvent neurotoxicity and possible
markers potentially applieable to human biomonitoring are also discussed. This paper
also attempts to provide an overview of current research findings, including some
unpublished results (details on methods available from the author). FinaIly, problems
in the application of biomarkers and research needs will be emphasized.

Behavioral etTects of organic solvents

Over the last ten years, it has been repeatedly shown that chronic exposure to
organic solvents may be associated with brain dysfunction. Whereas there is little
doubt about effects occurring during andjor shortly after exposure, the long-term
prognosis of early changes is still controversial. Solvent-induced behavioral changes
are mainly represented by reduced vigilance and slowered reaction times, consistently
found by many independent investigators (for review, see Anger, this volume).
Impairments in memory tests and other changes such as reduced visuo-constmctive
ability, which have also been found to be associated with solvent exposure, could
represent additional effects or, more likely, secondary effects due to a reduced
arousallevel, impairing vigilance as weIl as attention and ability to cope with many of
the assigned neurobehavioral tasks. Color vision loss (Mergler et a1., 1987b and 1988)
has also been reported as an early sign of neurotoxic effects.
Most such effects have been shown to be delayed (hoursjdays) with regard to
recent exposure (Cherry et a1., 1980 and 1981; Mutti et a1., 1984a) and to be reversible
after weeksjmonths from exposure (Mutti et a1., 1985a ). Although mainly exposure to
commercial mixtures based on aromatic hydrocarbons has been shown to be
associated with brain dysfunction, other compounds, e.g. chlorinated hydrocarbons
have been found to cause similar impairments (Ferroni et a1., 1992).

Neuroendocrine etTects

Neuroendocrine changes observed in laminators occupationaIly-exposed to styrene

(Mutti et a1., 1984b; Arfini et a1., 1987; Mutti, 1988a) and in dry cleaners exposed to
perchloroethylene (Ferroni et a1., 1992) mainly consisted of increased baseline and
stimulated prolactin (PRL) levels. At variance with neurobehavioral effects, any
possible influence of such extraneous variables as the educational level and the
cultural background may be mIed out as weIl as excessive alcohol intake. Thus, the
neuroendocrine changes associated (not necessarily correlated) with behavioral
effects are not accounted for by confounders or modifiers making the interpretation
of psychometrie tests very diffieult and sometimes even ambiguous. Furthermore, such

184
neuroendocrine changes may account, at least in part, for other solvent-related
effects, for example for their reproductive toxicity.
A cross-sectional study was carried out on thirty female laminators exposed to high
styrene concentrations (8h-TWA was about 130 ppm), who were compared to a
control group of female workers matched with respect to age, and the phase
(proliferative) of the menstrual cyele. The exposed subjects' basal serum PRL levels
were more than double the control values and were correlated to the urinary
excretion of styrene metabolites (mandelic and phenylglyoxylic acids) in the "next
morning" urine spot sampie. The serum levels of hGH (human growth hormone) were
also higher than in the reference group. Though within the reference values, the TSH
levels of the exposed subjects were significantly related to the urinary excretion of
styrene metabolites. No changes were seen in gonadotropins. It was coneluded that
"the styrene-induced neuroendocrine effects are mostly due to acute exposure", where
the term "acute" refers to "recent" exposure (Mutti et al., 1984b).
In workers exposed to lower styrene concentrations, exhibiting minor increases in
baseline serum PRL, the dynamic response to TRH stimulation was evaluated. Only
one out of 16 exposed subjects, as compared to 15 out of 16 control subjects, showed a
normal response. In the exposed workers, the median values of PRL at different times
after TRH administration were higher than the mean + three SO of a reference
population (Arfini et al., 1987). It was concluded that the dopaminergic modulation of
pituitary secretion is impaired among styrene-exposed workers, owing to the inability
of feedback mechanisms to inhibit the PRL secretion and/or release following TRH
stimulation. The impressive PRL response to TRH stimulation recorded among
styrene-exposed workers is consistent with abnormally high PRL stores within
lactotrope cells, thus suggesting that PRL production is not adequately inhibited by
the hypothalamic dopamine released into the portal system. Oata gathered from this
and subsequent surveys during which a control group was always simultaneously
examined indicate a prevalence of 4/80 (or 5%) subjects with PRL-secreting
microadenomas among styrene-exposed workers against none in the controls. This
finding suggests that not only may styrene exposure interfere with PRL production
(owing to a failure of dopamine to inhibit PRL synthesis), but also PRL release may
be affected in chronically exposed people. It is worth mentioning that, contrary to
most PRL secreting adenomas, in these cases the dopamine agonist bromocriptine
was unable to inhibit hyperprolactinemia (unpublished results).

MECHANISMS OF SOLVENT NEUROTOXICITY

One challenging question has long been why a diverse group of chemically inert
substances may cause similar effects on the central nervous system. One attracting
hypothesis has been the selective accumulation of the parent compounds in the brain,
due to the fact that most solvents are strongly lipophilie. Shifts in membrane fluidity
would then result in masking or unmasking reeeptor sites (Oave and Witorseh, 1984;
Wesemann et al., 1986). However, it should be reeognized that the brain has a
tissue/blood partition eoefficient similar to the remainder of the vessel rieh group.
Owing to the high blood flow, the half-life of solvents in the brain is not expeeted to

185
be higher than in other organs (Mutti and Franchini, 1987). As a result, the selective
vulnerability of the central nervous system is not accounted for by kinetic factors of
the parent compounds, owing to their short half-times, although a selective
accumulation within subcellular structures such as plasma membranes cannot be
excluded. Whereas the acute effects of heavy solvent exposure may weIl be due to
aspecific changes in membrane fluidity and hence in conformation of membrane
proteins, including ion channels and receptors, such solvent-induced changes in the
physieal properties of membranes seem not to account for more specifie effects
occuITing at low exposure levels and showing a tendency to accumulate over the
exposure period.
Toxicodynamic factors related to reactive metabolites rather than to the parent
compounds might play an important pathophysiologie role. If biotransformation is
however considered as a necessary step, it is very diffieult to identify metabolites,
mechanisms and targets common to such a varied class of chemicals, including
aliphatic, aromatie and substituted (halogenated) hydrocarbons, a1cohols, and
ketones, very often combined in the very complex mixtures commercially available.
The consideration that dopaminergie systems are very important in the regulation
of arousal and that other reported effects, such as changes in mood (depression) could
also be due to dopaminergie dysfunction lead us to investigate catecholamine
tumover during experimental exposure to styrene in rabbits (see below and Mutti et
al., 1984c and 1985b). That dopaminergic dysfunction is induced in man by exposure
to certain organie solvents is also suggested by increased PRL secretion. Whereas the
other dopaminergic pathways are protected by the bloodjbrain baITier and may be
affected only by those hydrophilic compounds whieh are formed in situ, the tubero-
infundibular dopaminergie system is also a target for metabolites dissolved into the
blood stream. This might imply a selective vulnerability of pituitary functions,
especially PRL secretion, whieh is under the direct dopaminergie control, tonically
inhibiting prolactin secretion within a feed-back loop.
More recently, other behavioral alterations have been described in workers
exposed to styrene and to other aromatic and chlorinated solvents which could also
recognize dopaminergic dysfunction as the underlying mechanism. For instance,
impairment in color vision could be related to peripheral rather than to central
impairment: amacryne ceIls, known to have dopaminergie terminals are important in
the regulation of retinic reaction to light stimulation and hence could represent an
additional target selectively vulnerable to certain solvents. Neuroendocrine effects are
also consistent with the hypothesis of a selective vulnerability of the tubero-
infundibular dopaminergie system to the effects of certain solvent metabolites.

Neurochemical etTects of solvent exposure

Neurochemieal studies carried out in our and in other laboratories showed that
dopaminergie systems are selectively affected by exposure to various aromatic
solvents. It has been shown that repeated exposure to styrene causes a dose-
dependent striatal and tuberoinfundibular dopamine depletion in rabbits (Mutti et al.,
1984c). An increase in dopamine receptor binding, possibly as areaction to dopamine
depletion, has also been reported (Zaida et al., 1985). Although in styrene-exposed

186
rabbits, the time course of striatal dopamine following administration of Q-methyl-
para-tyrosine, a selective blocking agent for tyrosine hydroxylase, showed the same
slope observed in the control group, the turnover rate turned out to be significantly
lower in the exposed animals because of the lower initial levels. It was however
apparent that the increased dopamine catabolism eoexisted with a substantially
normal turnover time, eonfirmed by the parallel inerease in dopamine levels after the
blockade of mono-amine oxidases by pargyline (Mutti et a1., 1984c). Reeent studies
(see below) indicate that both tyrosine hydroxylase and monoamine oxidases Bare
inhibited by tetra-hydro-isoquinolines. This could explain why no apparent changes
were seen in turnover time after their blockade by selective agents.
In a subsequent study, the time course of striatal dopamine and brain styrene
concentrations were assessed weekly following a 3-day exposure period to 1500 ppm
of styrene in the air. A further deerease in striatal dopamine was observed two days
after discontinuing exposure. The dopamine levels were still significantly reduced
three weeks later (Mutti et a1., 1985b). These effects were shown to be common to
other monocyc1ic hydrocarbons and to be predictable on the basis of the chemical
structure of metabolites rather than of the parent compounds. For instance,
vinyltoluene and 7-methyl-styrene are very similar, but the different loeation of the
methylic group is associated with a substantially different effeet on brain dopamine.
Whereas no effects were found following exposure to 7-methyl-styrene, dopamine
depletion was found after exposure to vinyltoluene. Owing to the methylie group on
the side chain, 7-methyl-styrene cannot be biotransformed into an Q-ketoacid. The
latter seems to be the reaetive group common to those solvent metabolites which are
effeetive in producing dopamine depletion (Mutti et a1., 1988b).

Pictet-Spengler reaction. Glyoxylic acid is used in histochemical studies to detect and

quantify dopamine stores. In fact, a fluorescent 6,7-hydroxy-tetrahydroisoquinoline
(TIQ) is formed non-enzymatically by condensation of glyoxylic acid with dopamine in
the Pictet-Spengler reaction of the carbonylic group of the Q-ketoacid with the aminie
group of dopamine (Sjoquist et a1., 1985).
Experiments in vitro showed that both glyoxylic and phenylglyoxylic acid condense
with dopamine, the concentration of which is markedly reduced by the presence of the
two Q-ketoacids (Mutti et a1., 1988b). Reactive carbonylic groups are common to a
number of solvents and c1asses of solvents biotransformed into either Q-ketoacids or
aldehydes. Interestingly, reactive intermediate metabolites of those solvents that have
been shown to cause narcotic or pre-narcotic effects are common or similar to those
of anesthetic gases (Mutti and Franchini, 1987). Both enflurane and methoxy-flurane
in vivo give rise to glyoxylic acid, an intermediate metabolite common to methyl-
cellosolve and to other solvents, e.g. styrene and ethylbenzene, biotransformed into
phenylglyoxylic acid. Halothane and fluroxene are both catabolised to trifluoroacetic
acid. It is reasonable to assurne that like trichloroacetic acid, this acidic compound is
also produced through an aldehyde ehemically and probably pharmacologically
similar to trichloroaeetaldehyde (or chloral), that is a major intermediate metabolite
of chlorinated hydroearbons sharing narcotic properties (fig. 1). Thus, from a
metabolie point of view, there are striking similarities between anesthetic gases and
solvents known to impair arousal and vigilance.

187
 The fact that reactive metabolites of all these solvents recognize doparnine as a
selectively vulnerable target suggests that doparnine depletion may play a role in
solvent toxicity to the central nervous system. However, it should be recognized that
most such solvent metabolites are more or less polar and hence should not cross the
blood-brain barrier. On the contrary, TIOs formed outside the nervous system easily
cross the blood-brain barrier (Niwa et al., 1988). Owing to their potent activity on
catecholarnine synthesis (see below), they are good candidates to explain the selective
vulnerability of doparninergic systems.

I
0 ()
R

CH R=
0-:/
//,H 2

F
OH F F
H
O=C
/ CIWCI
~

O=<)=_0
OH Various solvents give
rise to metabolites
enflurane --------------- > glyoxylic containing glyoxylic
acid: methyl-cellosolve.
methoxyflurane ------- > acid styrene. ethylbenzene
HO

Fluroxene Trich loroethylene

Perchloroethylene

1
CI
trifluoro- CI~CI
acetaldehyde HC trichloro-
HC "- ~o
'0 acetaldehyde

trifluoroacetic
1 1trichloro-
acid acetic acid

Figure 1. Chemical radicals combined with reactive carbonylic groups in metabolites derived from
biotransformation of various organie solvents. The lower part of the figure illustrates chemical similarities
between intermediate metabolites of certain organie solvents and those of anesthetic gases, which also
contain reactive carbonylic groups.

188
Biological actions of tetra-hydro-isoquinolines. Since a selective, irreversible
neurotoxin (methyl-phenyl-tetra-hydro-pyridine - MPTP) has been shown to cause
parkinsonism (Langston et al., 1987), other structurally similar compounds were
investigated. TIQs have been suggested as endogenous toxic substances possibly
involved in the pathogenesis of Parkinson's disease (Yoshida et al., 1990). Tbeir
occurrence in food, their formation from acetaldehyde and possibly from the
biotransformation of other substances either ingested or inhaled, their ability to cross
the bloodjbrain barrier, their occurrence in human brain support the hypothesis of
their contribution to the etiology of idiopatic Parkinson's disease (Booth et al., 1989).
Although their neurotoxic properties have been questioned, these o:-ketoacid and
aldehyde adducts can exert specific, though varied and diverse, actions on populations
of neurons in different regions of the brain (for review, see Myers, 1989). To illustrate,
they have been shown to: affect pre-synaptic dopamine receptors, accumulate in
synaptic vesicles, bind to 0!2-adrenergic and opiate receptors, evoke spontaneous
preference for ethanol. Interestingly, though similar effects have been reported for
different TIQs, small changes in the molecule result in differential specificity andjor
potency. For instance, 6,7-dihydroxy-1-benzyl-TIQ is known to possess -adrenergic
properties whereas other analogs act as antagonists.
Biochemical studies, including recent work from our laboratory (manuscript in
preparation), suggest that TIQs may interfere at different levels with catecholamine
synthesis (fig. 2). TIQs seem to induce dopamine depletion by inhibiting tyrosine
hydroxylase (Yoshida et al., 1990). Tbe specificity of the effect on dopamine would be
enhanced by the parallel increase in dopamine--hydroxylase, limiting possible effects
on norepinephrine levels. At higher concentrations, TIQs also inhibit monoamine
oxidases, which would explain why the effects on catecholamine turnover are self-
limiting. It ought to be noted that the relative potency is not the same for various
solvent-related TIQs, which would account for the different activity of some solvents
as compared to more polar molecules and in particular to ethanol.

Recent studies on pel2 ceUs. PC12 is adonai cell line derived from rat
pheochromocytoma exhibiting many of the properties of mature, terminally
differentiated sympathetic neurons and have become increasingly popular in
neurobiological research as a model for catecholamine biosynthesis, storage,
secretion, and re-uptake (Shafer and Atchinson, 1991; Veronesi, 1992).
6,7-Hydroxy-benzyl- and 6,7-hydroxy-phenyl-TIQ synthetized in our laboratory
were used in experiments with PC12 cells aimed at characterizing their possible
effects on dopamine synthesis (fig. 3). In the first experiment, PC12 were cultured in
plates coated with purified bovine dermal collagen, in RPMI - 1640 without L-Glu,
supplemented with inactivated horse (10%) and fetal calf (5%) serum, L-Glu 200
mmol and Pen-Strep 50 U. After stabilization, the cells were incubated with TIQ-
containing medium. At 4 and 24 h, catecholamine concentration was measured both
in the supernatant and in cell homogenates.
Dopamine concentration was markedly reduced by exposure to TIQ. At 24 h, the
calculated 1Cso for 6,7-hydroxy-benzyl- and 6,7-hydroxy-phenyl-TIQ ranging from 10
and 20 /Lmol. Equimolar amounts of salsolinol, aTIQ formed from ethanol, caused
much less pronounced effects, its ICSO being about 100 /Lmol. These in vitro

189
experiments are consistent with in vivo observations (Mutti et al., 1984c and 1985b)
and suggest that dopamine synthesis is inhibited by TIO.
The inhibitory activity of TIOs on tyrosine hydroxylase measured in pe12 cel1
homogenates accounts for their differential ability to induce the dopamine depletion
observed in pe12 cells. The affinity of TIOs formed by phenyl- and benzyl-aldehyde is
much greater (Ki = 2.0 and 5.5 Iilllol, respectively) than that of salsolinol (20 Iilllol).
The greater affinity for the enzyme - probably related to the aromatic ring in position
1 - results in a higher potency as competitive inhibitors of tyrosine hydroxylase.
At higher concentrations, TIOs are known to inhibit monoamine oxidases B, which
would explain why the effects found in vivo following solvent exposure by inhalation
appear to be self-limiting, dopamine depletion never exceeding 50% (Mutti et al.,
1984c).
Pictet-Spengler reaction

o HO

Rl R2 ~H,~
+ HO~
carbonytic Rl R2
dopamine
group 6.7-<lihydroxy-tetra-hydro-
isoquinoline (rIO)

o
HO~
" OH
HO 1..-:; NH 2

TYROSINE DOPA

~DDC

H
mH
O
HO

DOPAMINE

TIO
+-- +
OH

HO~ OH

HO
I"
..-:;
NH
I
.-- HO~
CH 3 NMT HoD NH 2

EPINEPHRINE NOREPINEPHRINE

Figure 2. Tetra-hydro-isoquinolines (TIQs) act as competitive inhibitors of tyrosine hydroxylase (TH)

and monoamine oxidases, whereas they enhance the activity of dopamine-p-hydroxylase (DBH). An
additional effect, not shown here, is represented by the inhibition of monoamine oxidases B.

190
a
HO
HO

+ HO ~H
(Y'rH

2
__ HO +H 2 0

-....::::

lY
H
HO
HO~
o + I NH
HO h 2 -- HO

Figure 3. Tetra-hydro-isoquinolines used in experiments in vitra with pel2 cells. 6,7-Hydroxy-benzyl-

tetrahydro-isoquinoline (a) and 6,7-hydroxy-phenyl-tetrahydro-isoquinoline (b) are non-enzymatically
formed by condensation of dopamine with benzylacetaldehyde and phenylacetaldehyde, which are
putative metabolites ofvery common aromatic solvents (toluene, ethylbenzene, styrene).

Another remarkable neurochemical effect caused not only by TIQs, but also by
certain solvent metabolites (e.g. mandelic and phenylglyoxylic acid) is the enhanced
activity of dopamine--hydroxylase, which would account for unchanged levels of
norepinephrine following solvent exposure (Mutti et al., 1984c).

Organic solvents and oxydative stress

The ability of various organic solvents, mainly chlorinated hydrocarbons to give

rise to reactive oxygen species (ROS) has long been known, but the brain has received
little attention in this context (Bondy, 1992). An aromatic organie solvent, toluene, has
recently been shown capable of induction of excess ROS in the brain both in vitro and
in vivo (Mattia et al., 1991). Other aromatic solvents of environmental significance,
namely styrene and xylenes, are able to induce free-radical formation. Although both
benzyl alcohol and benzoic acid had free-radical quenching properties, benzaldehyde
was a potent agent in enhancing oxygen radical formation.
This free-radical inducing property of solvents seems not to be associated with
neuronal hyperactivity, Le. increased intracellular calcium, owing to their anesthetic
rather than stimulating properties, and can be blocked by pre-treatment of rats with
an inhibitor of mixed function oxidases, namely methyrapone (Mattia et al., 1991).
Solvent-induced ROS may not enhance their acute toxicity, but rather may give rise to
subtle effects, such as an acceleration of normal aging processes (Bondy, 1992).
At variance with the formation of TIQs, known to cross the bloodjbrain barrier,
the hypothesis of the generation of ROS implies that solvents are taken-up by neurons
and that bioactivation occurs within the brain, since it seems unlikely that very
reactive or polar metabolites can easily get their targets in the central nervous system.

191
Secondary efTects

As discussed above, various mechanisms may underly the behavioral effects of

organic solvents. All such mechanisms are based on the oxidation of non-polar
substances to reactive metabolites, which on the one hand may give rise to the
formation of TIQs, accounting for a number of pharmacological (reversible) effects,
on the other hand might be implicated in more subtle mechanisms, e.g. the formation
of free radicals and subsequent oxidative stress, possibly involved in such subtle
processes as accelerated brain aging.
Owing to key role of the central nervous system in the regulation of a number of
vital functions, solvent-induced neurotoxic changes may lead to a variety of apparently
independent effects on peripheral organs and systems (fig. 4). Certain organic solvents
are biotransformed into reactive metabolites able to cause dopamine depletion, thus
impairing the dopamine contral on pituitary secretion and causing neuroendocrine
changes. Such effects are usually related to recent exposure, but seem to require a
latency period (several hours to a few days) before their appearance.
Secondary amenorrhea or irregular menstrual cycles have been reported in
styrene-exposed female workers (Arfini et al., 1987). This is consistent with the
reduced fertility recorded in a subsequent study (Mutti et al., 1989). More recently,
the distribution of lymphocyte subsets in styrene-exposed workers was also
investigated. Styrene-exposed workers showed changes consistent with the hypothesis
that hypothalamic-pituitary dysfunction may be associated with alterations of the
immune system (Mutti et al., 1992). Although the latter effects might weIl be due to
direct effects on the immune system, a growing body of evidence demonstrates that
multiple interactions and bi-directional communications exist between the
neuraendocrine and immune systems (for review, see Blalock, 1992), thus suggesting
that neuroendocrine changes mayaiso lead to secondary aIterations in the regulation
of immune function.

BIOLOGICAL MARKERS

Over the last ten years a great deal of effort has been devoted to the development
and validation of early markers of effect resulting from exposure to environmental
pollutants.
A biological effect is defined as a biochemical, functional or structural change
resulting from exposure to a given agent or to a particular condition. It may range
from a simple adaptive response to death, from subtle variations of a continuous
variable to sudden appearance of a dichotomous (quantal) condition. It may be acute,
i.e. following shortly after exposure to a substantial amount of a substance, or chronic,
i.e. showing an irreversible or progressive course. However, one should be careful
when using such adjectives as acute or chronic, since it is seldom possible to predict
the time course of a given condition on the basis of a time point estimate or of a few
measurements over time.

192
 ORGANIC SOLVENTS

blotranslormation

1
Reactlve Intermediate metabolites

o
Plctet-Spengler reactlon 1 TIQs (tetra-hydro-lsoqulnolines)

+
R1 R 2

Effects on catecholamlne synthesis:

DOPAMINE DEPLETION ...
4f----1 Inhibition 01: TYROSINE HYDROXYLASE
MONOAMINE OXIDASES
enhancement 01 dopamlne-b-hydroxylase

REPRODUCTIVE DYSFUNCTION
BEHAVIORAL
CHANGES
ALTERED IMMUNE FUNCTIONS

Figure 4. Schematic presentation of the mechanism of action of organic solvents on the central nervous
system and proposed multiple outcome subsequent to secondary effects on peripheral targets.

Biomarkers thus far identified belong to one of three general cathegories: (i)
markers of susceptibility (usually on a genetic basis); (ii) markers of effective dose
delivered at the target organ; (iii) markers of early effects on the critical organ.
Whereas rather precise knowledge of molecular mechanisms of toxicity is necessary to
identify markers of susceptibility and of effective dose, most behavioral or
biochemical markers of effect have been identified on the basis of logical or
pathophysiological reasoning, usually starting from clinical conditions and

193
extrapolating backward changes expected to precede illness. We should recognize that
clinical and epiderniological approaches are very often confused and the
methodological context is seldom considered when the health significance of
measured changes is evaluated. A critical discussion of conceptual aspects to be
considered when using biological markers could be useful to avoid the abuse or
rnisuse of available tests.

Diagnostic validity

The predictive value or diagnostic validity of a test is not a property of the test
alone. It results from the combination of the sensitivity and specificity of the test and
the prevalence of the disease in the population being exarnined (fig. 5). Positive
results even for a very specific test, when applied to subjects with a low likelihood of
being ill, will be largely false positive. In the c1inical setting, where people are referred
to because of symptomsjsigns suggesting the occurrence of a disease, the positive
predictive value of a diagnostic test (i.e. the probability that a subject with positive
test results is actually ill will be very high), whereas irrespective of the sensitivity and
specificity, its negative diagnostic value will be low. Conversely, in field investigations
the negative diagnostic value will be high and the positive diagnostic value will be
low, no matter how sensitive and specific a test might be. As a result, most tests
aimed at detecting early effects are useful to monitor health rather than to screen for
illness. From the above paragraph, one might argue that behavioral and
neuroendocrine tests are not useful because of their low positive diagnostic value.
This is wrong for a number of reasons. It is worth mentioning that the low prevalence
expected in field surveys refers to illness and not to behavioral or neuroendocrine
changes, which often occur at a relatively high rate. On the contrary, these changes
are likely to occur, but in interpreting their significance we should limit our tendency
to overdiagnose, i.e. we should control our clinical cultural background and carefully
consider the context in which the test result has been obtained.

Prognostic value

When people show any deviations from reference values for biochernical or
physiological parameters, then there will be a number of questions to be dealt with.
The main question is a prediction regarding the outcome: prediction of the course
of disease following its onset, prediction of the probability of illness following early
changes, prediction that no adverse effects will be observed in future. Unfortunately,
these predictions should be based on data which is usually unavailable to health
professionals, be they doctors, psychologists, researchers in general. In fact, when
exarnining people at work for possible behavioral changes, we should be prepared to
answer some relevant questions that may be anticipated. The first one is the risk
associated with behavioral changes, i.e. the probability that such changes will be
followed by a disease affecting the nervous system. The second one is the prognosis of
observed changes, i.e. their consequences in terms of disability, complications or even
death that mayor may not follow our investigation.

194
~ 1100
v
::>
0 80
> Sensitivity 01 the test: 95%
u Spec ilic ity 01 the test: 95%
:.;=
If)
0 60
c
CJ"l
0
-0 40
Q)
>
:.;=
If) 20
0
0.-

20 40 60 80 100
Prevalence of disease. %

Figure 5. Relationship between the positive diagnostic value (probability of ilness in subject positive at
the test) and the prevalence of disease in the examined population. The test characteristics (sensitivity
and specificity) are arbitrarily set at 95%. The positive diagnostic values reHes much more on the
prevalence of disease than on the test characteristics.

We must admit that most often we are not able to predict anything. Since we
actually don't know both the risk and the prognosis, we should avoid any indulgence
in unwarranted conclusions. Owing to their low incidence in the general population,
most degenerative diseases cannot be adequately studied using a prospective
approach. In fact, their occurrence even in specific groups at risk will be extremely
low. As a result, the use of markers sensitive enough to pick up early changes due to
re cent exposure seems now to be the only feasible approach to risk assessment.
Obviously, the prognostic value of such early changes is often unknown. Nevertheless,
it would be ethically unacceptable to "wait and see", when there are indications to
improve a potentially harmful working environment.
Neurotoxicology offers a wide array of examples suggesting that potentially serious
effects of chemical exposure might be understimated. A broad range of substances
have been implicated in neurodegenerative disorders afflicting patients in later life,
when the progressive loss of previously effective compensatory mechanisms unmasks
otherwise "silent neurotoxicity" (Reuhl, 1991). This also suggests a possible interaction
between neurotoxicity and aging, in terms of abiotrophic processes displacing age-
specific function and leading to an earlier onset of neurodegenerative diseases, such
as Parkinson's disease, Alzheimer's disease amyotrophic lateral sclerosis and others
(Weiss, 1991).
Although based on speculations rather than on data, the possible role of chemical
exposure in neurodegenerative disorders has been suggested by several authors and a

195
recent issue of Neurotoxicology has been devoted to these problems (Cranmer et al.,
1991).

BIOMARKERS OF SOLVENT NEUROTOXICITY

Computer-based behavioral test batteries have been successfully applied to detect

CNS dysfunction caused by occupational and/or environmental exposure to a variety
of neurotoxic substances (see Anger, this volume).
Baseline PRL levels are only marginally affected by exposure levels (doses) which
can be measured in current field situations, at least in most developed countries.
However, in particular situations such as in certain jobs exposing to styrene vapors,
baseline PRL levels were useful both as indirect markers of tubero-infundibular
dopaminergic dysfunction and as a screening tool to identify unusually bigh
prevalences of PRL-secreting adenomas. Dynamic tests (e.g., PRL response to
stimulation with TRH) are much more sensitive and may thus give unambiguous
evidence of neuroendocrine disturbances related to very low exposure levels (Arfini et
al., 1987). Unfortunately, tbey are botb impractical and ethically unacceptable for
health monitoring at the workplace. However, they are recommended for diagnostic
purposes in subjects exhibiting disturbances and/or abnormally high basal levels of
serum PRL, owing to the increased frequency of PRL-secreting adenomas in workers
exposed to certain solvents, and particularly to styrene vapors.
The measurement of serum PRL in styrene exposed workers could be used both as
a marker of dysfunction of the tubero-infundibular dopaminergi~ system and to screen
for the possible occurrence of PRL-secreting pituitary adeno~: .
Peripheral markers of neurochemical function include I?iochemic~ and hormonal
tests, as weIl as receptor binding studies. Although proposed as a potentially useful
test to assess dopamine receptors, 3H-spiperone binding to lymphocytes gave
unremarkable results in styrene-exposed workers (Checow~y et al., 1990). This is
probably due to the fact that sigma, rather than dopamine receptors (absent in
lymphocyt~s) are measured by this test.
Serotonin uptake by platelets has been proposed both as an indicator of oxidative
stress (Bosin, 1989) and as a biomarker of solvent-induced neurotoxic effects (Beving
et al., 1984). The results of this test were not always encouraging (Checoway et al.,
1992). Nor were the mechanisms leading to increased uptake of serotonin in platelets
clearly understood.
The activity of monoamine oxidases B (MAO-B) in platelets has recently been
proposed as a useful test in styrene-exposed workers (Checoway et al., 1992). A direct
inhibitory effect on MAO-B by TIOs may account for the reduced activity of MAO-B,
which has been experimentally found in styrene-exposed rats (Husain et al., 1980) and
empirically confirmed in styrene-exposed workers. Interestingly, in the study by
Checoway et al (1992) MAO-B activity was faund to be negatively related both to
exposure and subjective disturbances.
Be they biochemical ar functional in nature, biomarkers thus far available seem to
be suitable to identify groups at risk or harmful working environments, whereas
caution must be exercised in their application at the individual level.

196
RESEARCH NEEDS

Although the identification of accessible biological markers would be particularly

relevant to the study of neurotoxicity, when ethical and feasibility constraints preclude
direct measurements of exposure-related changes in the central nervous system, this
field of investigation is still an exciting research area with just a few encouraging
opportunities available for biochemical monitoring of humans for neurotoxicity.
Questions remaining to be answered include the relevance of known mechanisms
accounting for solvent induced brain dysfunctions. An even more important question
is the significance of such effects with regard to the long-term prognosis of solvent-
exposed workers. Although the neuroendocrine changes require several weeks for a
complete recovery and at least a few days are necessary to improve visuo-motor
performance, the long-term significance of early effects cannot be easily evaluated
and still remains achallenging area of research waiting for our contribution over the
years to come.
Experimental and epidemiological work should confirm some prornising results
obtained with the measurement of MAO-B in platelets from styrene-exposed workers
and extend observation to other potentially neurotoxic solvents. Other enzyme
activities measurable in accessible media, e.g. plasma dopamine--hydroxylase, could
be useful to obtain complementary information about solvent-induced interference
with catecholarnine metabolism.
The role of various tetra-hydro-isoquinolines (TIQs) in solvent neurotoxicity
should be further elucidated and attempts should be made to quantify their
concentration in accessible biological media. The validity of such indicators as
markers of effective dose delivered at the central nervous system also needs to be
assessed.

Acknowledgements: Part of the work described here bas been supported by the Commission of the
European Communities in the STEP (Science and Technology for Environment Protection) program.
RosseUa Alinovi, Alfredo Bacchini, Claudia Biagini and Stefania Cavazzini are gratefully acknowledged
for pieces of unpublished work reported here.

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199
BIOMARKERS OF IMMUNOTOXICOWGY

Loren D. Koller

College of Veterinary Medicine

Oregon State University
Corvallis, Oregon 97331

IMMUNE SYSTEM

The immune system is the body's main defense against invasion by foreign
materials and biological agents. The immune system is composed of cells, their
receptors and soluble products, tissues, and organs. Immunoreactive cells are
neutrophils, lymphocytes, macrophages, monocytes, eosinophils, basophils, and mast
cells, inc1usive of numerous subpopulations, each characterized by expression and/or
function. These cells are distributed throughout the body and concentrate in blood,
thymus, spleen, and associated lymphatics and lymph nodes. The immune system is
controlled by an array of intra- and interregulated circuits that involve other biological
systems such as the endocrine and central nervous systems. Thus, it is impossible, at
this time, to duplicate in vitra the multiplicity of the intact immune system.
Numerous reviews and texts have been written on the basic principles, cell
functions, and intricate interactions that orchestrate immune reactions. That
information will not be duplicated herein. However, one must be reminded that the
immune systems of animals and humans are similar, and thus, most information
collected in animals has relevance to humans.
The immune system reacts bimodally, Le., suppression can predispose the host
to become increasingly susceptible to infections and neoplastic agents while enhance-
ment can be expressed as hypersensitivity and autoimmunity. In a suppressed system,
adverse health consequences are those of severe disseminated disease that can be
exacerbated by numerous factors such as age, poor nutrition, and stress. When the
immune system becomes hyperactive, the result can be expressed as asthma, rhinitis,
pneumonitis, granulomatous pulmonary disorders, or autoimmune-like disorders such
as arthritis, glomerulonephritis, thyroiditis, hemolytic anemia, hepatitis, erythematosus,
and sc1erosis. Xenobiotics can either exacerbate existing disease or induce immune
dysfunction.

BIOLOGIC MARKERS

Biologie markers (biomarkers) can be broadly defined as indicators of events in

Use 0/ Biomarkers in Assessing Health anti Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 201
biological systems; i.e., variations in the number, structure or function of cellular or
biochemical components. Biologie markers can serve as surrogates to identify early
stages of disease and to cultivate an understanding of the basie mechanisms of bio-
logieal response resulting from exposure to environmental substances.
Immunologists have considered "markers" in respect to cell identifieation, receptor
activity, cytokine production, immunoglobulin type, and a sundry of other qualitative
and quantitative features that distinguish the cellular and soluble compartments of the
immune system. The definition of biologic markers is more encompassing and extends
to include indieators of events or conditions in biologie systems or sampies. Thus,
biomarkers can be classified as markers of exposure, effect, or susceptibility. These
markers progressively represent a continuation of events from exposure to clinical
disease.
The measurement of a chemical or its metabolite in a biologie specimen is a
marker of exposure. Indieators of exposure are measurable concentrations ofaxeno-
biotic in urine, blood, or body tissues and organs, including hair or nails. Markers of
exposure for the immune system would be antigen-specific antibodies or cellular
responses to a particular xenobiotic. However, since many environmental substances
are small molecules that do not elicit an immune response, they do not trigger antigen-
recognition pathways unless they are bound to larger carrier proteins. Also, small
doses of a chemical may not be recognized by the immune system while extremely
large dos es may paralyze it. Further, immune markers can decay after discontinuance
of exposure. Nevertheless, although little is known about chemical-induced immune
markers of exposure, the ability of the immune system to produce a specifie response
directly identifiable to exposure of a given chemical has promise as a method of
detecting exposure, especially when overt toxicity and/or analytieal methods fail to
confirm exposure.
A biomarker of effect is a measurable cellular or biochemical alteration within an
organism that can be associated with impaired health. Immunological markers of
effect can be any detectable change within the immune system and changes in other
tissues resulting from immune-mediated dysfunction. Immune system markers of effect
must be defined in terms of specifie health effects to have relevance as indieators of
effects. Immune dysfunction manifest in clinical disease is obvious but biomarkers of
effect become more obscure during the "potential" or inactive phases of immune-
mediated disease. Nevertheless, the greater the correlation between an immunological
marker and associated disease(s)--host resistance, hypersensitivity, or autoimmunity--
the greater the value of the marker in relation to etiology, diagnosis, and progression
of the disease.
The biologic marker of susceptibility is an indicator of an inherent or acquired
ability of an organism to respond to the challenge of exposure to a specific xenobiotic.
Susceptibility can be influenced by geneties, the environment, or biorhythms. These
influences can cause differences within individuals over time, as weIl as between
individuals and populations. Three common sources of variability are genetie, age-
related, and neuropsychologic factors. Environmental factors such as smoking, routine
use of pharmaceutieals for therapy, malnourishment, photosensitivity, and prolonged
diseases can signifieantly affect the immune system. Thus, "normal" reactivity of the
immune system can vary substantially from individual to individuaf.
Sensitivity and specificity are critieal components when validating immune
responses. The qualitative ability of markers of exposure or effect to be indieative of
actual exposure or development of disease, respectively, reflects sensitivity. Specificity
is quantitating a specific immune event that can be associated with exposure to a given
chemieal. Validation is bidirectional: from exposure to effect and vice versa. Once
a positive correlation is established, health risk can be predieted and pending disease
prevented.

202
IMMUNOTOXICOWGY

Immunotoxicology can be defined as the study of injury to the immune system that
can result from occupational, inadvertent, or therapeutic exposure to a variety of
environmental chemicals or biologic materials. The immune system can be a passive
target for suppressive xenobiotics and exposure can be manifest by increased incidence
of infectious disease or neoplasia. Other xenobioties stimulate the immune system,
expressed as hypersensitivity (asthma, rhinitis, and eontaet dermatitis) or autoimmune
disease.
Immunotoxieology eneompasses identification of immunotoxie ehemieals, develop-
ment of sensitive, quantitative assays, and determination of the mechanisms by whieh
xenobiotics ean alter immune funetion. Although it is widely aeeepted that numerous
ehemieals ean alter immune responses in animals, the immune system is the primary
target organ of only a small number of xenobioties. The dimension of immunotoxieol-
ogy in humans is less clear, and although the immune systems of animals and humans
are similar, the metabolie process may differ for some ehemieals. Thus, well-designed
epidemiologie investigations would provide valuable information for humans to
eonfirmjdispute the immunotoxie data that has been established in anima! models.

IMMUNE-MEDIATED DISEASE

Hypersensitivity reactions are the most eommon type of immunotoxicity associated

with xenobioties in the environment. Hypersensitivity and suseeptibility to autoimmune
disease is strongly influenced by geneties. Disorders of hypersensitivity are usually
associated with a specific antibody, receptor, eell population, or target tissue. Although
elinieal signs of hypersensitivity have generally provided adequate markers of effect,
their linkage to a specific chemical has been achalienge. The common routes of
exposure to sensitizing agents is through eontaet with the skin, the respiratory tree, and
the gastrointestinal traet.
Low molecular weight ehemieals are not detected by the immune system unless
eovalently coupled to body proteins. These larger molecu1es can sensitize an indivi-
dual, but sensitization does not always lead to disease. Inhalation of sensitizing
xenobodies ean result in asthma, rhinitis, pneumonitis or granulomatous disorders.
Ingestion of certain foods ean lead to food hypersensitivity or intoleranee. Allergie
contact dermatitis is an expression of ehemieal sensitivity following dermal exposure.
Biomarkers available to diagnose hypersensitivities include skin tests, assays for specifie
antibodies (1gB, IgG), cellular immune reactions, provocation challenges, and the
cytologic evaluation of bronehoalveolar lavage fluid.

AUTOIMMUNE DISEASE

Autoimmune disease occurs when an individual's own immune system attacks one
or more tissues or organs, resulting in funetional impairment, inflammation, and
sometimes permanent tissue damage. Autoimmunity basically originates from the loss
of immune toleranee to self, and the immune system is stimulated to reaet to self-
antigens that normallyare not reeognized by one's own immune system. Autoimmunity
ean be expressed direetly through the formation of circu1ating antibody to self,
indirectly through the formation of immune eomplexes, or as a eonsequence of eell-
mediated immunity.
Numerous xenobioties ean induee autoimmune disorders, some of which require
a genetie predisposition. Considerable knowledge has been aecumulated on drug-
induced autoimmunity but Httle is known about autoimmune diseases developing from
exposure to environmental substances or the influence of environmental factors on

203
spontaneous autoimmunity. Although the marker of exposure is more obscure, the
biomarker of effect from exposure to environmental xenobiotics emulates those that
arise spontaneously or from known pharmaceuticals. On the other hand, the human
leukocyte antigen (HLA) system, whieh is a classification of cell-surface glycoprotein
on lymphocytes and macrophages, has a potential to serve as a marker of susceptibility
to certain immune-mediated diseases ranging from ankylosing spondylitis to pemphigus
vulgaris. At this time, the HLA class 11 haplotype appears to be the best predietor of
susceptibility to autoimmune disease.

BIOLOGIe MARKERS OF IMMUNOSUPPRESSION

Aseries of human immunodeficiency disease syndromes have been characterized

and have established adefinite association between immunosuppression and an in-
crease in incidence of infectious and neoplastie disease. These infections can recur
throughout life. Children that are born without white blood cells generally die from
infectious disease in the first year of life. Animal models have been useful in charac-
terizing specific immunologie defects in many of these syndromes and have confirmed
the increased susceptibility to infectious, neoplastie and autoimmune disease. There
is also a well-established association between immune suppressive chemotherapeutie
agents and an increase in incidence of infectious and neoplastie disease in humans.
Rodent studies have a high degree of predietability, with few exceptions, for both
type of immune effects and the dose required to achieve the effects of immunosuppres-
sive therapeutic drugs developed for human medicine. An example is that data that
has been accumulated in animals for cyclosporine A is similar to the immunosuppres-
sive effects produced in humans by this drug. Rodent studies have cataloged a large
data base on potentially immunotoxic chemicals with some pursuing the mechanism of
action to a specific chemical. The utility of both prospective and retrospective
immunotoxieologieal studies in humans could give considerable credence to rodent
investigation if "conditions" were carefully controlled and/or documented.
Some classes of chemicals that contain known immunomodulating "chemicals" are
polyhalogenated aromatie hydrocarbons, aromatie hydrocarbons, polycyclic aromatic
hydrocarbons, aromatic amines, pesticides, metals, organotins, oxidant gases, particles,
natural products, drugs of abuse, and therapeutie drugs. Although eonsiderable know-
ledge has been acquired from specifie chemieal exposures, a paucity of information is
available for the immunotoxic interactions that could occur from complex mixtures.

ANIMAL MODELS

Animal models serve as surrogates for the detection of harmful effects of toxic
substances in humans. The immune and metabolie systems of animals resemble those
of humans, with some exeeptions, and are similar enough that data from animal models
provide a conduit of information to prediet immunotoxic xenobiotics for humans. Most
drugs and other chemicals that are known to suppress the immune response in humans
produce similar results in animals and the mechanism of action is similar for both
species. Thus, data acquired from animal immunotoxicity investigations are applicable
for predicting human health risks associated with environmental exposure to
xenobioties.
Bioassays must be validated before they can be used successfully to detect
immunotoxie agents. To be validated, an assay must be sensitive, specifie, reproduc-
ible, and measure alterations in immune function. Immune function can be defined
as the normal, special, or proper action of immunocytes and their secretory products.
Many immune procedures do not assess function, and thus, do not appropriately
represent immune reactivity unless a positive correlation is definitely established.

204
 Both the mouse and rat models are acceptable to assess the immunotoxic proper-
ties of xenobiotics. Each species has its pros and cons, but advances in technology
have reduced or eliminated many of the barriers that have existed for years and sensi-
tive methodology has improved the application of these animal models for human
disease. Nevertheless, animal models are essential to assess the health risks of xeno-
biotics and offer a high degree of predictability for humans when such information is
missing.

METHODS OF DETECTING IMMUNOTOXICANTS

The immunotoxic potential of xenobiotics can best be assessed in animals that

have been exposed via the route that simulates environmental exposure to humans.
Although some immune assays are performed in vivo, the majority are conducted in
vitro using cells and secretory products that have been exposed to the chemical in vivo.
Aseries of procedures have been characterized for their applicability of assessing
immunotoxicity in animals.
Pathological evaluation should be included in the initial screen for immunotoxic
chemieals. Pathology is neither sensitive nor predictive, unless lesions or associated
changes in hematopoietic and lymphoid tissues are evident. When changes are detec-
ted, they can indicate where to foeus the search for the target of the immunotoxicant.
However, for many xenobiotics, there is not a direct correlation between pathology and
the more sensitive immunotoxicological procedures.
One functional assay that has been shown to detect the majority of an immuno-
suppressive toxicants is the measurement of humoral antibody to a T-dependent
antigen. In such an assay, the B-Iymphocyte, T-Iymphocyte, and macrophage each
have a role in producing the optimum immune response. Chemical interference in any
one of these compartments could result in an inadequate immune response. Proce-
dures that are commonly used to test for the humoral immune response are the
antibody plaque-forming cell assay and the enzyme-linked immunosorbent assay.
Cell-mediated immunity originates in the thymus. Several chemicals are known
to cause hypoplasia of thymic cells. Reduced cellularity (histopathology) and thymic
weight are indicators of thymus atrophy. Three assays used to evaluate cell-mediated
immunity are the delayed-hypersensitivity, mixed lymphocyte reaction, and cytotoxic T-
cell procedures. These procedures evaluate the functional activity of this arm of the
immune system.
Non-specific immune procedures that have application to immunotoxicology
include natural-killer cell cytotoxicity, macrophage phagocytosis, antibacterial and anti-
tumor activity, bone marrow cellularity, the production/activity of numerous cytokines,
and host resistance to challenge. These methods have been validated and several have
been used for in-depth mechanistic investigations. Some methods, such as cell surface
antigens and receptors, tumor necrosis factor, lymphokine-activated killer cells, mRNA
levels, etc., required additional testing and confirmation before they can be accepted
as validated. Finally, the value of immunotoxicology becomes a concern if the immune
system is the primary target organ of a chemical. If not, other biological systems will
be damaged prior to development of immune dysfunction, and thus, immune dysregula-
tion is essentially a moot point.

HUMAN IMMUNE SYSTEM

Assessment of the human immune system for chemical-induced immune dysfunc-

tion is much more difficult than in animals. In most cases, intrusive techniques cannot
be used in humans, and thus, many of the procedures used in animals such as the
plaque-forming cell assay cannot be used for humans. However, several procedures
are available to evaluate immune activity in humans.

205
 Aseries of procedures have been developed to assess humoral and cellular
immunity as weIl as nonspecific immunity in humans. A tiered approach could be
used. The first tier could include assays to test for antibody levels to ubiquitous
antigens, secondary antibody responses to proteins, enumeration of B- and T-cells in
peripheral blood, evaluation of secondary delayed-type hypersensitivity reactions, and
antibody titers to "natural antigens." The second tier could include assessment of the
primary antibody response to protein and polysaccharide antigens, primary delay-type
hypersensitivity reaction, natural killer cell and monocyte activity, T- and B-cell
antigenic markers and receptors, cytokine activity, and detection of shed or secreted
cellular activation markers and receptors. A variety of tests could be used in tier three
that would better characterize abnormalities observed in tier two.
Although many of the above procedures have been used by clinical immunologists,
many of them have not been validated for immunotoxicology purposes. A dose-
responsive relationship must be established to confirm the applicability of these
proeedures.

EPIDEMIOLOGY

The effeets of environmental substances on the human immune system are rela-
tively unknown at this time. There is evidenee through epidemiological data that toxic
substances can alter the immune response of humans. However, well-delineated epide-
miological studies, either cross sectional, retrospective, or prospective, would provide
valuable information on this subject. Epidemiology, the study of disease patterns in
human populations, eould confirmjdispute animal data that is presently available and
demonstrates specific chemicals ean induce immune dysfunction. Groups of individuals
who are exposed to higher coneentrations of xenobioties than the normal population,
either accidentally or in the work place, could be compared for immune system irregu-
larities. The common problems that complicate epidemiology studies are quantifying
exposures to a known substance and minimizing biologieal variables and sundry of
other environmental factors that ean typieally alter immune response.
Biologie markers of the immune system can be utilized as indieators of exposure,
effect, and susceptibility in epidemiology. Considerable research needs to be
eondueted with each of these markers to better eharacterize the relevance of chemical-
induced immune-mediated disease in humans.

SUMMARY

The information presented herein is basieally an overview of the "Biologie Markers

in Immunotoxieology" book that was published by the National Academy Press,
Washington, D.C., 1992. Immunotoxicology has received global recognition in its
nearly twenty years of existence. The discipline has rapidly expanded from
fundamental to applied research with considerable interest in its applieation in
developing health standards and permissible levels for human exposure to xenobiotics.
Immunotoxieology explores the effects of physical, biologie, and chemieal agents on the
immune system. The immune system is extremely sophistieated; is intra- and inter-
regulated by other systems; and immunocytes, their reeeptors and secretory products
interaet in a network of circuits. The interaction between the nervous, endoerine and
immune systems makes it virtually impossible to duplicate these systems in vitra, and
thus, intaet animal systems are essential to assess the immunotoxie potential of
xenobioties for human health risks.
Biologie markers are indicators of events in biologieal systems whieh can serve as
surrogates to identify exposure, suseeptibility, and biologieal effects when an organism
is exposed to environmental substanees. Thus, biologie markers of immunity must be

206
able to recognize exposure, predict species susceptibility to exposure, and/or identify
biological effects or disease as a result of exposure to a xenobiotic. Methods to
measure biological markers must be sensitive and specific to have value in predicting
health risks and pending disease.
Several assays have been validated in animals to detect immunomodulation by
xenobioties. A large body of immunotoxicologieal information has been cataloged for
many chemicals using animal models. However, a paucity of immunotoxicological data
is available in humans that have been exposed to environmental substances. A battery
of tests does exist to assess, to a degree, immune competence in humans who have
been exposed to known or suspected immunotoxicants. Well-designed and controlled
epidemiologie investigations need to be conducted to gather information on the
immunotoxic properties of xenobioties in humans whieh will ascertain the potential
health effects of these compounds. Although immune markers offer promise in
assessing the effect of environmental toxicants on human health, there is presently
insufficient data to confirm that the immune system of individuals who are putatively
exposed to environmental toxicants is compromised to the extent that they have an
increased risk of disease. Development of biologieal markers as surrogates for human
disease will better characterize the immunotoxic potential of xenobiotics in humans.

REFERENCE
National Research Council, 1992, "Biologic Markers in Immunotoxicology," National
Academy Press, Washington, D.C.

207
SERUM BIOMARKERS IN THE ON-SITE EVALUATION OF
SUSPECTED CANCER RISK IN HUMANS RESIDING NEAR
HAZARDOUS WASTE SITES

Eric Pluygers,1 Paul Gourdin,1 Guy Dardenne,2

Brigitte Scoubeau, 2 and Alex parfonryl

1Scientific Board
2Field Team
UNEP-RISCAPE (Unit for Evaluation and Prevention
of Carcinogenesis Risks of occupational and
Environmental Origin)
Rue des Carrieres 52
7181 Arquennes, Belgium

INTRODUCTION

The purpose of this study is to report the results of cancer risk evaluations carried out
in populations residing in the vicinity of two hazardous waste sites, based on the biochemical
assessment of two tumor markers in serum. The use of tumor markers in the evaluation of
health impairments related to environmental pollution has rarely been reported previously
(Schlipkter et al 1978) ; our own research in this field proceeds from aseries of
observations in occupational settings - In order to define the rationale of this rather novel
approach, this article will be subvided in two distinct parts : the first will review the methods
presently available for cancer risk evaluation, emphasis being put on the techniques involving
the biochemical assessment of tumor markers ; the second will present the methodology and
results of two field studies.

CANCER RISK EVALUATION: RATIONALE FOR DIFFERENT APPROACHES

Huge quantities of domestic and industrial wastes are generated nowadays and their
disposal, often in poorly or totally uncontrolled conditions, is arousing major concem with
regard to environmental pollution and potential impact on human health. Hazardous or toxic
substances have often been identified in waste dumps; air pollution and leakage into ground
waters have been observed at considerable distances. Real or alleged health impairments are
regularly reported by residents in the vicinity of the dumps, and heavy mediatization has led
to "epidemics" of collective panic.
Whereas the official reactions have generally striven to rninimize the potential health etfects,

Use o[ Biomarkers in Assessing Health and Environmental Impacts o[ Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 209
the physicians, faced with the necessity of objectively informing and counseling their
patients, most often were unable to collect the scientifically unquestionable data that would
have coined their answers.Of course the toxic effects of some identified hazardous
substances such as heavy metals, benzene, vinyl chloride are known, but the evaluation and
prediction of toxicity (and lethality in experimental animals) become ineffective when
complex waste mixtures are considered (Simmons et al, 1989).
What is true for toxicity assessments is even more obvious when the long-term
carcinogenicity risk of exposure to complex waste mixtures has to be predicted.
The research unit UNEP-RISCAPE (Unit for Evaluation and Prevention of
Carcinogenesis Risks of occupational and Environmental Origin) has feit this as achalienge
and has had recourse to a biochemical approach of which the background rationale will first
be discussed together with an evaluation of the other available techniques, to finally present
some field data. The latter are related to two on-site evaluations near two waste-dumps
located respectively at Mellery (Brabant Province, Belgium) and Hensies (Hainaut Province,
Belgium) and will be completed by some recent data ab out the effects of the occupational
environment of agriculturists.
Among the available methods for cancer risk evaluation, we shall successively consider :
epidemiological approaches
multistage carcinogenesis mechanisms and their relevance to risk evaluation
markers of exposure to carcinogens
molecular identification of oncogene mutations and mutational spectra
markers oftumor promotion
assessment of oncogene products
assessment of biochemical changes induced by mechanisms of tumor promotion,
e.g. responsible for the clonal expansion of initiated cells.

EPIDEMIOLOGICAL DATA AND RISK EVALUATION

The medical literature contains a few reports on the relation between the presence of
chemical industries or of hazardous waste dumps and cancer mortality (Hoover and
Fraurneni, 1975; Harris et al., 1984; Butller et al, 1985; Griffith et al, 1989).
In the last mentioned study, the authors have compared the cancer mortality in 339
U.S. counties (representing 593 sites) in which hazardous waste sites had resulted in ground
water pollution and this ground water being the only available source of drinking water, with
that of the 3065 "clean" counties of the contiguous United States; they found a significant
excess of deaths in the HWS counties for cancers ofthe lung, bladder, esophagus,stomach,
large intestine and rectum in white males, and for cancers of the lung, breast, bladder,
stornach, large intestine and rectum in white females ; this is in fact for nearly all major
cancer sites. Doubtless, this excess of cancer deaths should not be assigned solely to the
presence of HWSs and aseries of confounding factors may exert their influence, such as
extent of industrialization in these counties and type of the industries, personal habits related
to social class, ethnic peculiarities, etc; it is nevertheless a striking finding, justi(ying
sustained research. The evaluation of these situations is not without reminding the search for
occupation-related diseases as made explicit by the Occupational Sentinel Health Event
(SHE (0 concept defined by Rutstein et al. (1983).
According to these authors, a SHE (0) is :
"A disease, disability, or untimely death which is occupationally related and whose
occurrence may :
1) provide the impetus for epidemiologie or industrial hygiene studies (Rutstein et al.,
1983).
2) serve as a warning signal that materials substitution, engineering control, personal
protection, or medical care may be required (Rutstein et al., 1983). We believe that,after

210
slight adaptation, this concept might give rise to the identification of Environmental Sentinel
Health Events that should initiate the same reactions. An updated list of 64 SHE (0)
diseases or conditions has been recently published (Mullan and Murthy, 1991), and it may be
taken for granted that much of the information aimed at making the house-physicians aware
of occupation-related conditions might similarly make them environment-conscious and
facilitate their evaluation of the environmental impact on human health. However, with
regard to the malignant conditions mentioned in the SHE (0) list, it has to be recalled-as
with other epidemiological surveys that, when the malignancy itself is considered as the end-
point for evaluation, this will usually leave only limited possibilities for curative treatment,
whereas any type of secondary prevention is definitive1y mied out.
This but stresses the severe limitations of the epidemiological approach in situations
characterized by long-term exposures to a multitude of potential (partly unidentified)
carcinogens ; the lack of power of classical epidemiology in these situations is well-known
and the results ofthis approach are further obscured by the absolute preclusion ofpreventive
outcomes.
In fact, in order to be really helpful, the risk evaluation should not evaluate cancer, but
carcinogenesis, e.g. the long sequence of complex events that, over many years, will convey
anormal cell into overt cancer.

MULTISTAGE CARCINOGENESIS

The detailed description of the different steps of carcinogenesis, finally resulting in the
appearance of a malignant cell, would reach far beyond the scope ofthis study ; nevertheless
a few fundamental data will be recalled, in order that the different approaches to cancer risk
evaluation could be correcdy positioned during the course of carcinogenesis. The major
phases of carcinogenesis have been duly characterized and include initiation, promotion,
conversion and progression. It is obvious that the earlier the detection, the better will be the
prospects for controlling and aborting the process.
As a first step normal cells become initiated after being exposed to genotoxic
carcinogens (mostly chemical) ; the genotoxic damage in1icted to the cells may be assessed
and a number of risk evaluation techniques are based on this approach. The main feature of
tumor promotion is the clonal expansion of the initiated cells, confering a selective growth
advantage to these cells, under the in1uence of substances called promoters ; contrarily to
initiators, promoters need not be cytotoxic (Travis and Belefant, 1992).
Promotion involves a complicated sequence of events, extending over many years,
characterized primarily by activation of protooncogenes to oncogenes, and inactivation of
tumor suppressor genes. Mutations are at the origin of these gene activations/inactivations
and will initiate a cascade of events including the production of altered oncoproteins, in turn
these will dysregulate intracellular signal transduction, growth factor and receptor
expression, and facilitate aseries of mechanisms responsible for the clonal expansion of the
initiated cells, such as cell-cell interactions (Trosko et al., 1983 ; Yamasaki, 1990) ; cell-
matrix interactions (Liotta, 1986) ; interactions between cells and growth factors (Goustin et
al., 1986) and finally dysregulations ofthe cell cycle (Strzbecher et al., 1990), ofterminal
differentiation and of programmed cell death (Evan et al., 1992).specific biomarkers
correspond to each one of these steps and their assessment, alone or in combination, might
provide reliable information about cancer risk. The biomarkers of the promotional phase of
carcinogenesis may be considered as markers 01 effect (in contrast to the markers of
exposure), and represent an integrated expression of the effects induced not only by all the
xenobiotics to which an organism is exposed, but also of their modulation (synergistic or
inhibitive) by endogenous or environmental factors.
This integrated expression of effects becomes especially valuable when an organism -a
human being- is exposed to complex mixtures of ill or unidentified compounds, a situation

211
obviously prevailing near HWSs ; in such conditions, the precise and exhaustive
identification ofall the compounds present in a mixture is not compulsory.
The markers of the later phases of carcinogenesis- conversion and progression- mainly
consist of major chromosomal alterations and will not be considered because they are less
amenable to preventive interventions.

MARKERS OF EXPOSURE TO CARCINOGENS

Although it may be of very short duration, the exposure to genotoxic carcinogens is

readily identified by modern technology, for instance through the quantitative assessments of
carcinogen-DNA adducts, or alternatively (and more easily) of protein-carcinogen adducts,
e.g. Haemoglobin-carcinogen adducts (Lee and Santella, 1988 ; Weston et al., 1989 ;
Wogan, 1989). These techniques have reached high levels of sensitivity, in spite of what
there is a consensus that the value of the isolated assessment of DNA-or protein-adducts in
predicting cancer risk still has to be validated (Vineis and Caporaso, 1988 ; Wogan, 1989)-
C. Harris (1991) writes "Although methods to measure carcinogen-macromolecular adducts
are an important contribution to improved carcinogen exposure assessment, carcinogen-
macromolecular adducts will not be a precise predictor 01 cancer risk because of the
multistage and complex nature of human carcinogenesis" (underlined by Pluygers).
Moreover, the assessments tend to evaluate total adducts and not only the critical ones
(perera, 1987). Similar comments apply to cytogenetic techniques commonly used to
evaluate the very early efIects of an exposure to carcinogens (they may be considered
initiation-early efIect markers), such as Sister Chromatid Exchanges and Micro-Nuc1eated
cells. As mentioned by Autrup (1991), not only are the observed cells (peripheral blood
cells) surrogates for the real target of the carcinogenic efIect, but overall these techniques
lack specifity and sufIer rom their uncertain biological relevance in relation to cancer
development. The picture is still further complicated by wide interindividual variations in
human carcinogenesis, occurring at the levels of carcinogen metabolism, efIectiveness of
detoxifYing enzymes, DNA repair fidelity after adduct formation and excision, genomic
instabiIity, etc (Harris et al., 1987). So, with Harris (1991) we feel prone to consider
"carcinogen-DNA adducts as only one piece ofthe puzzle-other pieces include determinants
of tumor promotion".

MARKERS OF TUMOR PROMOTION

Mutation Spectra

As already mentioned, tumor promotion is characterized by a succession of events in

which the intervention of genotoxic compounds is not aprerequisite, and in which activation
of proto-oncogenes to oncogenes, and inactivation of tumor suppressor genes play a
dominant role. The sequence of events is weil known in several tumor systems, and its early
identification would yield considerable benefits, in terms of cancer prevention, as promotion
may be inhibited by impairment of the c10nal expansion of the initiated cells, and so be
amenable to (chemo)prevention. Major features of promotion are the activation of an
oncogene ofthe ras family (generally an early event) and the inactivation ofthe p53 tumor
suppressor gene (often a later event). These activations/inactivations are the consequence of
mutations occurring at weIl defined co dons ofboth ras and p53 genes; the involved codons
are highly correlated with each chemical carcinogen and may vary according to tissue site
and cancer types. The codons targeted by specific carcinogens may be identified by the
DNA polymerase "fingerprint" assay and "mutational spectra" can be identified linking
carcinogenic agents, specific types ofDNA adducts and specific mutations (Harris, 1991).

212
These techniques presently enable to discriminate between the effects of exogenous or
endogenous carcinogens (Jones et al., 1991);as noted by Puisieux et al.(1991), ''p53
mutational hotspots identified in different tumors are selected targets specifically for the
etiologically defined environmental "carcinogens"; in the case of multiple tumors, p53
mutation patterns also allow to differentiate the multifocal or metastatic origin (Oda et al,
1992).
The p53 gene is particularly weIl suited for mutational spectrum analysis due to the
occurrence of a broad spectrum of mutations dispersed over a large portion of the gene.!t is
obvious, from these few examples, that mutation spectrum analysis will open fascinating
insights on the origins of human cancer, warranting the considerable amount of research
devoted to this field in recent years.
However, the techniques involved in this research are sophisticated, and not really
applicable to large-scale population evaluations.
So, it seems meaningful to develop markers of the promotional phase that might be
more readily applied to sized populations. Surprisingly, and in spite ofthe known limitations
of the exposure-inititation biomarkers that have been previously considered, markers of
promotion have received but little attention at the biochemical level. In agreement with
several authors, ce Harris (1991) recently wrote "Methods to identify human tumor
promoters and to predict responses to tumor promoters among different humans need to be
developed" .

Oncoproteins

As mentioned above, one of the features of promotion consists in the activation of

oncogenes ; the assessment of their products in body fluids might be a reflection of this
activation. This approach has been advocated by PW Brandt-Rauf (1988).
After observing that elevated expression of the p2I oncoprotein encoded by the ras
oncogene, or expression of abnormal p2I, had been detected in aseries of malignant as weIl
as premalignant conditions (Spandidos et al., 1984;Pelling et al., 1988; Bos 1988; Ohuchi et
al., 1986; Michelassi et al., 1987), and also considering the finding that ras activation is
often detected in benign conditions-its tissue levels paralle1ing malignant potential
(Michelassi et al., 1987)-thus suggesting that it is an early event in carcinogenesis (Vogelstein
et al., 1988 ; Weinberg 1989), Brandt-Rauf (1988) assessed the p2I oncoprotein-together
with others-in the serum ofworkers exposed to occupational carcinogens, and suggested the
higher levels of the oncoprotein to be predictive of higher cancer risk.
Sirnilar observations have been made for aseries of other oncoproteins (Brandt-Rauf
and Niman, 1988), of which elevated levels have been found in the serum of many cancer
patients ; however in a small series of treated cancer patients in whom 9 oncoproteins were
assessed, only the p2I oncoprotein was significantly elevated (perera et al, 1990) ; this
appears to be the target of choice for evaluations. Since then, this approach has been
extended to aseries of occupation-related conditions (Brandt-Rauf et al., 1992, and
references there in). Whereas the p53 gene seems to be a nearly-ideal target for mutational
spectrum analysis, this tumor suppressor gene is only poorly suited for the assessment of its
product(s).

Biochemical markers of other mechanisms involved in tumor promotion

Among the complex and intricated mechanisms that will progressively confer a more
decisive growth advantage to the initiated cells, we shall now briefly consider those most
prone to be identified by biomarkers and assessed in our field evaluations.
The inhibition of intercellular gap junctional comrnunication appears to be a major
factor in carcinogenesis (Trosko et al. 1989 ; Yamasaki 1990) known to be induced
experimentally by tumor promoters of the phorbol ester type. As aprerequisite for the

213
formation of these gap junctions is the occurrence of cell-to-cell adhesion, itself modulated
by adhesion molecules (Kanno 1985).
In such a context, it seems exceedingly important to notice that Carcinoembryonic
Antigen (CEA), a much used and studied human tumor marker, functions as an intercellular
adhesion moleeule (Benchimol et al. 1989) or as an accessory adhesion moleeule (Pignatelli
et al. 1990). Stucturally CEA belongs to the immunoglobulin superfarnily (paxton et al.
1987), that also comprises weIl characterized adhesion moleeules such as the Neural Cell
Adhesion Molecule (NCAM) (Rutishauser et al, 1988). Polysialylation of NCAM is fre-
quently observed in tumor cells and profoundly reduces the homophilic binding properties of
cells (Rutishauser et al., 1988 ; Rygaard et al., 1992) ; polysialylation is also observed in
those of the Lewis Blood Group Antigens more strongly correlated with malignancy (Kim et
al. , 1986) ; thus polysialylation might weIl be a good target for risk evaluation.
Ever since its discovery by Gold and Freedman (1965), CEA has been subjected to
innumerable clinical and experimental evaluations ; summarizing very abundant data, it may
be infered that higher CEA levels in serum are associated with aseries of conditions known
to carry an increased cancer risk, such as age, smoking, alcohol consumption, poor work
environment (Herbeth et al. 1980; Pluygers et al. 1986).
As early as 1978, Schlipkter et al (1978) had observed higher serum CEA levels in
non-smoking men residing in industrial districts as compared to residential areas, and they
also correlated CEA levels with air pollution. Elevated levels were found in the serum of
workers in the chemical industry (pluygers et al, 1988) as weIl as in other industrial settings
(pluygers et al, in press).
Moreover, the five-year follow-up of an asymptomatic population (n = 2 000) revealed
a ten-fold increase in cancer incidence in the screenees having presented CEA levels in the
highest percentile compared with those in the lowest (pluygers et al. 1986), whereas a
reduction in the availability of dietary promoters resulted in a decrease of the CEA levels
(Sadowska et al, unpublished data).
For all these reasons, and others related to the co-mapping - on chromosome 19 - of
the genes coding for CEA and those coding for aseries of factors involved in carcino-
genesis (Transforrning Growth Factor beta, Protein Kinase C, ... , the CEA levels in serum
may be considered as a marker of carcinogenesis (pluygers et al. 1990), and their assessment
may represent an easily accessible method for cancer risk evaluation. An indispensable
precaution is to adequately select the antibodies recognising several well-defined epitopes of
the antigen (pluygers et al, 1987), sothat an optimal sensitivity will be reached at the (very)
low concentrations of antigen observed in risk evaluation, be it at the expense of specifity.
A serious limitation, in risk evaluation, results from the fact that not alJ tumors-
probably not all carcinogenesis mechanisms-implicate the participation of CEA. An example
of this situation is provided by the analysis of sera from asbestos-exposed workers,
demonstrating the lack of CEA production in mesothelioma cases, whereas CEA is
expressed in a majority ofthe workers developing bronchogenic carcinoma ofnon-small-cell
type (pluygers et al 1991, 1992). (lnteresting to notice, neither p53 alteration nor K-ras
activation constitutes a critical step in the development of human mesothelioma (Metcalf et
al. 1992. A common feature after exposure to asbestos Ca non-initiating carcinogen, acting
through free-radical generation) is an increase in the serum levels of Tissue Polypeptide
Antigen (TPA), a cytoskeletal antigen recognised by antibodies against cytokeratins 8, 18
and 19 (Quinlan et al. 1985).
Cytokeratins are cytoskeletal proteins forrning intermediate-sized filaments and specific
junction proteins, functionally related to the cadherin farnily of adhesion molecules involved
in homotypic cell-cell interactions (Franke 1992).
In clinical oncology, CEA and TP A often are expressed in a complementary way,
making the CEA-TP A combination assay particularly efficient in the monitoring of a broad
spectrum ofmalignancies (Lthgens and Schlegel, 1989).
We suggest that the differential expression ofboth markers might result from different

214
specifities and potentialities in cellular expression, but it should also be considered that the
expression of different markers might be the consequence of different carcinogenesis
mechanisms. Considering the results already mentioned in asbestos-exposed workers
(pluygers et al, 1991-1992), we would suggest that the rise in serum TPA observed in
practically all the exposed workers is a free-radical effect-inflammatory and/or carcinogenic,
not accompanied by ras nor pS3 mutations in mesothelioma (Metcalf et al, 1992),whereas
the rise in serum CEA, observed only in smokers, is an expression of chemical
carcinogenesis induced by tobacco carcinogens; it would of course be of interest to assess
the mutational spectra in these latter cases.
Obviously, CEA and TPA assessments in serum only represent the very basic initial
stage of a biochemical approach that could become considerably more accurate if it were to
inc1ude a broader series ofmarkers comprising more specific ones such as enolase (smali cell
lung cancer), SCC-antigen (squamous cell carcinoma antigen), PSA (prostate specific
antigen), CA 15.3 (specific for mammary tissue), CA 12.5 (specific for seropapillar ovarian
tumors), and many others depending on identified or suspected target organs. As already
suggested by Franke as early as 1981, cytokeratin patterns also might bring their
contribution to the evaluation (Franke et al. 1981).
Finally, the biochemical approach should be extended so as to inc1ude markers
identifYing other mechanisms and pathways of promotion, among which we will mention
growth factors, protein kinases, cell-cyc1e regulators, matrix proteins, individual
susceptibility factors, modulations of the immune system, etc, of which the complex
interrelationships are gradually being brought to light, as exemplified by a few citations
(Sporn and Roberts 1985 ; Goustin et al. 1986 ; Liotta 1986 ; Chakrabarty et al. 1988 ;
Regg et al. 1989; Chakrabaty et al. 1989; Nishizuka 1989; Saiki et al. 1991 ; Hynes 1992,
Brandt-Rauf et al., 1992). A majority of these parameters are amenable to biochemical
evaluation in body fluids or in cellular compartments ; it does not seem unrealistic to believe
that "marker spectra" might be defined in the same way as the already mentioned "mutation
spectra".
The results ofthe "basic" biochemical approach, e.g. the assessment of CEA and TPA
in serum, in populations residing near two HWSs will now be reported, and discussed in
relation to data collected in a population of agriculturists exposed to phyto-pharmaceuticals.
General conc1usions will be drawn.

FlELD OBSERVATIONS

Description of Sites

Site 1 - Mellery - This is a rural site located in the southern part of Brabant province,
Belgium. The countryside is hilly and dotted with formerly exploited sandpits.One of the
huge excavations (ab out 800 x 500 metres,depth 20 metres) corresponding to a sandpit is
being filled up with wastes since the beginning of the eighties, and has elicited an ever
increasing choir of complaints from the inhabitants of Mellery village, stretched along a
valley west and northwest ofthe waste site (WS) but downwards ofit; the houses c10sest to
the WS are about ISO metres distant. The winds in the area are variable, with slightly
dominant south-west winds. The wastes were categorized as non-hazardous ("semi-
industrial") and included, among many unidentified,residues of the paint industry, aluminum
slag and sugarbeet pulp. After filling up the pit, the wastes were covered with a thick layer
of air-and watertight clay;unfortunately under the dry weather conditions prevailing last
years, this became fissured, allowing loathsome nauseous gases to escape; these were shown
to contain benzene,benzopyrene,toluene and several polychlorinated biphenyls. The weil
water collected closest to the site revealed the presence of high levels of lead and cadmium.
Considerable (and costly) works have been undertaken to rehabilitate the site.

215
Site 2 - Hensies - The second disposal site is located in Hainaut province (Belgium) on
industrial grounds formerly belonging to a coal mine (presently elosed down). The land-
scape is absolutaly flat ; the surroundings are mixed agricultural-industrial;heavy industrial
concentrations exist about 15 lans west (Valenciennes, France) or east (Tertre,
Belgium). The WS is situated elose to the France-Belgium boarder and is lined on one side
by the Paris-Brussels expressway, on the other by a broad canal. In this area the dominant
winds are from south-west;at the time of collecting blood, the waste dump had caught lire
spontaneously and all the area was covered with thick smoke for several weeks; it is
normally a foggy area. Since these events, the dump has been covered with earth. The
exact composition ofthe wastes is unknown.
The area is rather densely populated,although still being rural. For comparision purposes
between several population groups supposed to be at different risk levels related to their
orientation and remoteness from the WS, several sectors have been defined (see map, figure
1). Sector I,consisting in blocks of shabby-Iooking worker's flats ; distance 100 to 500
metres downwind from WS ; Sector n a and b : two hamlets belonging to Hensies
township, adjoining the WS but upwind ; Sector n c : the village of Pommeroeul 2-3
kilometres downwind from the WS ; Sector m North : the area extending north of the
expressway, with population mainly residing in the village of ViIIe-Pommeroeul ; Sector m
South : the village ofHensies (1 kilometre south ofthe WS) and the area to the east ofit,
south ofthe expressway.

Figure 1 - Location ofHensies waste site

For explanation of sectors : see text
Figures in boxes: number of participants per sector and mean age.
For sector I : mean age adults ouly and total population (adults + children)

216
Description of the Population

Mellery study - The selection of the volunteers participating in the study was carried out by
the Institute ofHygiene an Epidemiology (Brussels) and included 58 persons (24 females, 22
males and 12 children) scattered throughout the village and residing at distances ranging
from 200-800 metres from the WS. The blood was drawn by the consultants whereas the
UNEP-RISCAPE team personally interrogated the participants and filled in a questionary
related to personal life-style (smoking, alcohol consumption, diet), occupation, chronic
illnesses, medicine use, etc. A first evaluation was carried out in April-May 1990 and its
results will be presented in this paper ; a control evaluation is scheduled during the fall of
1992 and will be extended to population groups residing in nearby villages.

Hensies study - The Mellery experience prompted us to select larger population

groups,residing in well defined sectors,in order to achieve statistical significance. The
recruitment of the volunteers has been entrusted to the general practitioners who were
instructed to include a proportion of children, shown by the Mellery study to be especially
sensitive to the pollution in the vicinity of WSs;these general consultants also filled in a
questionary of the Mellery type. The distribution of the population between the different
sectors is mentioned on the map (figure 1), as well as the mean age in each sector ; the
proportion of children is particularly high in sector I, explaining that the mean age in that
sector is 25,1 years (39 years for the adults only).Another peculiarity of sector I is the high
proportion of Turkish immigrants (26/39);this population is rather homogenous, originating
from Karadeni province, in the Eastem Black Sea area ; the importance of these precisions
will be emphasized during the discussion. To be eligible for the study, a minimum 3 years of
residence in the sector was requested. A first evaluation of the entire Hensies area has been
carried out during the summer of 1991, and a control in sector I in July 1992.

Control population - The control population for all subgroups has been derived from the
asymptomatic population (n = 3000) attending a cancer screening unit, in whom the same
marker assessments had been carried out in about 300 screenees;106 of them have been
matched for gender, age and smoking habits to the population groups ; no control group was
available for the children.

The Selected Tumor Markers

For the reasons presented in the introduction, Carcino-Embryonic Antigen (CEA) and
Tissue Polypeptide Antigen (TP A) have been selected as the markers most suitable for a
first-step risk evaluation.The assessments have been carried out in the Radio-Immunology
Laboratory,Oncology Department, Jolimont Hospital, Haine Saint Paul, Belgium,using
commercially available kits. The specifications of the antibodies used in the radio-immuno-
metric assays are extremely important, as high sensitivity and good reproducibility have to be
ensured at the low marker values encountered in risk evaluation (for discussion see Pluygers
et al., 1987 ;Pluygers et al.,1992 a, in press) ; the following kits have been retained : for
CEA, the kit using a mixture of 3 antibodies (oligoclonal technique) manufactured by
MEDGENIX, SA, Brussels ; for TP A, the Prolifigen-Kit using a polyclonal antibody,
manufactured by SANGTEC, AB, Bromma (Sweden) and distributed in Belgium by Byk-
Sangtec SA, Brussels.
The assessments have been performed on frozen sera, no more than four weeks after
their collection;the same batch of Kits has been used for all determination;the same experi-
enced technician was in charge.
In the Mellery population, Sister Chromatid Exchanges have been evaluated in
circulating lymphocytes, and cadmium and lead have been determined in blood; these
analyses were the responsibility of the Institute of Hygiene and Epidemiology, and will

217
therefore not be presented; they will however be evoked during the discussion.

RESULTS

The results of the marker assessments may be evaluated as the mean of the absolute
marker values in the cons1idered populations, or as the prevalence (percent) ofindividuals in
whom the marker value exceeds a threshold considered as "normal", the "normal" level
being of course conventional. For CEA, this threshold is 3 ng/ml in serum (for children
under 15 years, 1 ng/ml); for TPA it is 55 Units/litre.
As the marker levels are influenced by some pathological conditions and some
occupational exposures,the participants in whom such a situation was identified have been
excluded from the evaluation;this occurred in 9 participants ofthe Hensies study (5 chronic
inflammatory diseases,1 Kidney failure,1 colon cancer, 1 stornach cancer, 1 exposure to
asbestos) ; occupation in the chernical industry was not considered as a cause of exelusion.

The Mellery evaluation - The lump data of this evaluation are presented in table 1.

Table 1. Absolute mean values of CEA (in ng/ml) andd TP A (in Ullitre) in residents living
elose to the Mellery site.

Population CEA TPA

group (number) Nonsmokers Smokers Total
------------------------------------------------------------------------------------------------------------
Controls(1 00) 1,02( 4)1 1,64(11,5)1 1,34(8)1 34,1(4)1
Mellery :
Men ( 22) 1,00(10) 2,17(16,6) 1,64(13,6) 39,9(27)
Women ( 24) 0,83(0) 2,17(25) 1,28( 8,3) 28,4(40)
Total
adults (46) 0,89(3,8) 2,17(20) 1,45(10,8) 33,9(10)
Children( 12) 0,46(16,6)2 0,46(16,6) 41 (25)

10 ratio in % of population having marker values in excess of the threshold (3 nglrnl for CEA, 55 U/litre for
TPA)
20 children under 15 years, threshold for CEA : 1 nglrnl.

At no level did these data reach statistical significance when compared with the controls ;
however there was a definite trend towards an increased prevalence ofhigher values for both
markers. Moreover when considered at the individual level, 7 participants (5 adults, 2
children) showed an above-threshold value for one or both markers ; this finding remained
totally unexplained by their personal or occupational history in three participants, ineluding
the two children (two boys aged respectively 11 and 14 years with CEA levels of2,26 ng/ml
and 4,77 ng/ml respectively). The children with the higher TPA levels (above 41 Ullitre
mean value in the group) tended to reside eloser to the WS : mean distance 260 metres
versus 375 metres for the other children.

The Hensies evaluation - Having been carefully planned on the site, this evaluation made it
possible to compare the results in different population groups corresponding to sectors
delineated on the map (figure 1) and believed to have undergone different exposure levels
because of relative orientation (upwind or downwind from the WS), or distance -
Quantitative measurements of air pollution were not carried out.

218
 Table 2. Absolute mean values ofCEA (in nglml) and TPA (in ngllitre) in residents living
elose to the Hensies site.

Population CEA TPA

subgroups
(number) Non smokers Smokers Total
------------------------------------------------------------------------------------------------------------
Controls( 106) 1,02 (4)2 1,64(11,5)2 1,34(8)2 34,1 (4)2

Hensies, seetor I I,n = 39

Men (ll) (1 ease) 1,55(0) 1,44(0) 44,6(36,4)
Women (11) 0,66 (0) 1,15(0) 0,78(0) 54,2(36,4)
Total
adults (22) 0,63 (0) 1,22(0) 0,87(0) 49,4(36,4)
Children(17) 0,60(23,5)3 0,60(23,5)3 54,2(52,9)

Hensies, seetors 11 a + b, n = 22
Men ( 8) (1 ease) 1,42(0) 1,37(14,3) 49,0(50)
Women (11) 0,89 1,23 0,99(9,1) 41,7(18,2)
Total
adults (19) 1,14(11,1) 1,36(0) 1,25(10,5) 44,8(27,3)
Children (3) 0,28(0) 36,0(0)

Hensies, seetor He, n = 28

Men (10) 2,46(20) 2,46(20) 53,9(40)
Women (18) 0,98(0) 1,02(0) 0,99(0) 42,1(29,4)
Total
population(28) 0,98(0) 1,97(13,3) 1,52(3,6) 47,2(33,3)

Hensies, sector III North, n = 29

Men (12) 1,28 1,08 1,15(8,3) 44,8(33)
Women(17) 1,02 0,40 0,86(5,9) 38,4(17,6)
Total
population(29) 1,08(6,25) 0,98(0) 1,04(6,9) 41,1(24,1)

Hensies, seetor III South, n = 17

Men (8) ND4 1,09(0) 46,9(25)
Women (9) ND 1,11(0) 34,I(ll)
Total
population( 17) ND ND 1,10(0) 40,1(17,6)

1For location of sectors, consult map - 0 2 ration in % of population having marker values in excess of the
thresho1d (3 ng/rul for CEA, 55 Ullitre for TPA - 0 3 for chi1dren under 15 years, thresho1d for CEA : 1
ng/rul - 4ND = not determined

The eomprehensive results of all seetors are displayed together in table 2 and in a more
eoneise form, in table 3.
The population of seetor I,the closest-downwind-to the WS, presents some interesting
peeularities : the Turkish origin of a majority of these subjeets has already been mentioned ;
the large proportion (17/39) of ehildren (3-14 years) is another characteristic of this
population, overwhelmingly eomposed ofyounger persons (mean age of adults : 39 years ;
of total population: 25 years). This very young mean age has somewwhat hampered the

219
comparison of CEA levels in sector I with those of the other sectors composed of older
populations (see map), as this marker is strongly influenced by ageing. Interestingly, for 4 of
the Turkish immigrant families, assessments were obtained for all the members of each
family ; the correlation between TPA levels and duration of residence (DR) are as follows :
family 0: 6 persons, DR 3-4 years; TPA 32 Ullitre; family A, 7 persons, DR 6 years; TPA
53.6 Ullitre ; family S, 5 persons, DR 9 years ; TPA 74 U/litre ; family E, 4 persons, DR
more than 10 years ; TP A 64 Ullitre. These results will be later discussed.
Because of the elevated TPA values observed in sector I and the concern they induced,
control assessments were carried out in July 1992 in all persons who had levels exceeding
the threshold on the first determination; a slight improvement has been noted, moderately
decreased levels being observed in 11/19 cases ; 3 cases showed no variation and in 5 cases
the TPA levels increased; this was the case for instance for family E , in whom the mean
TPA level rose from 64 to 69 U/litre.
During this one-year observation period, the mean TP A value among these 19 cases
dropped from 65,3 to 59,8 Ullitre, and the above-threshold values still numbered 11.
The figures for the other sectors are self-explanatory; sectors 11 a and 11 b, located
immediately south and south-west of the WS, have been merged because of small numbers
and comparable exposures.

Statistical analysis - The statistical power of the observed results has been calculated
for practically every possible relationship, using the Student t-test.
These detailed results are available upon request ; the statistical significance of the
differences between the results in the major population groups have been entered on table 3.

DISCUSSION

The concern raised by the nuisances of the Mellery WS and the fear of potential long-
term hea1th impairments shared by the general practitioners (GPs) of the area, incited the
Health Authorities to command a study aimed at searching for eventual health effects. It
was decided to carry out a cytogenetic study consisting in the evaluation of Sister Chroma-
tid Exchanges and at the request of the GPs, this was completed by the assessment of the
tumor markers CEA and TPA in the serum. The SCE evaluation, using a newly developed
technique (analysis of cells with high frequency of SCE's) gave appalling results, all the
children and a majority of non-smokers ending up with very worrying values. On releasing
these results, a nearly-hysterical panic developed, that the UNEP-RlSCAPE team (EP,PG)
feIt excessive,considering the marker values and emphasizing the differences in response
rates ofmarkers ofexposure-early effect,and markers oflater effect (see introduction).
A glance at the detailed results presented in table 2 and their synthesis in table 3 is
sufficient to persuade the observer that the situation is quite different at the Hensies site,
where large population groups show statistically significant differences from the control
group, at distances of several kilometres from the ws. The data are particularly
demonstrative for TPA. Downwind from the WS, a true gradiant is observed from the very
elose sector I through sector 11 c until sector III N, at a distance of at least 4 kilometres. The
residents living alongside the WS,but upwind, show intermediate values. In sector I,there
seems to exist a correlation with duration of residence; it should be noted that some of the
younger children were born in sector I and always lived there.
Even in the most distant sectors, the TP A levels still are significantly raised in regard to the
controls ; this corresponds to the finding of Schlipkter et al (1978), who detected higher
CEA levels in industrialized areas.

220
 Table 3. Summary of results and statistical significance

Populations CEA TPA

subgroups Mean Prevalence 1 Mean Prevalence1

Controls 1,34 8 34,11 4

-------------------------------------------------------------
Sector I(cbidren) 0,60 23,5 5422 5292
Sector I (adults) 0,87 0 51'5 2 36'3 2
Sector 11, a + b 1,15 10,5 44'8 3 27'33
Sector 11, c 1,5 23,6 47'2 2 33'3 2
Sector III, North 0,9 86,9 41' 13 24', 13
Sector III, South 1,10 0 40',13 17,6
Site I (Mellery) 1,45 10,8 35,4 12,5

1 Ratios ofmarker values in excess ofthreshold (see captions tables 1 and 2).
2 Statistically different from controls, p > 0,001
3 Statistically different from controIs, p > 0,01

Taken all together, these findings are very worrying, especially that TPA levels are only
weakly intluenced by smoking or ageing, and that the cases presenting conditions known to
intluence TPA levels were exeluded trom the study. Being a proliferation marker, it might
be anticipated that TPA levels would be raised in younger cbildren during their growth ;
however this has not been confirmed by sequential studies ; it is more likely that the bigher
levels in children proceed trom the fact that they are playing alongside the WS, or even on it
as it is only loosely fenced. A potential confounding factor might have been the Turkish
origin of several families, as environmental exposure to asbestos (tremolite, elosely related
to the ampbiboles) is known to occur in several regions in Central Anatolia (Cappadocia)
and induces "spontaneous" mesotheliomas (Yazicioglu, 1976; Baris et al., 1988). Now we
have shown TPA to be a serum marker of asbestos exposure (pluygers et al., 1991-1992 ;
Pluygers et al., 1992 b), so tbis might have intluenced the TPA levels. However the
Karadeni region in Turkey,trom wbich the families are originating,is not reported to possess
asbestos outcrops; moreover tbis would not explain the raised levels observed in cbildren
born in Belgium.
Another puzzling observation is the occurrence of significantly bigher TPA values in
women than in men, in sector I, whereas the opposite is true in all other sectors (and in po-
pulations analysed in other studies). We hypothesize that in sector I, the Turkish women
generally stay at horne and so are more permanently in elose contact with emanations of the
WS;in the other sectors the women generally work outside their hornes. The only other
circumstance in wbich a similar observation was made is in female agriculturists residing in
the billy Ardennes region (Belgium);it is believed (and presently monitored) that their raised
TP A levels might be a consequence of natural radon exposure (pluygers et al. ,in press). The
presence of radon emanations near the Hensies WS has not been investigated so far, but
radon emanations are known to occur in several small areas in Hainaut province.
At first glance, the results for CEA are all but convincing ; this is -to a large extent-the
consequence of the population characteristics in the different sectors. CEA levels are strongly
intluenced by age and unfortunately,the most exposed sector (I) also had by far the youngest
population, thus blurring the effect.When populations of similar mean age are compared,as
between sectors IIc and III North, again a significant trend appears.Moreover,when

221
considering the prevalence ofhigher values among the children in sector I (23,5 %),this has
to be compared with the 16,6 % observed at Mellery, and the 22,5 % detected in a cohort
exposed to chromium and nickel oxides (pluygers et al.,in press); at Mellery as well as in the
last cohort,these data correlated in a statistically significant way with the rate ofSCEs.
It appears from all the presented data that there exists a statistically significant increase
in the risk of developing health impairments in populations residing in the vicinity of WSs ;
the risk level follows a decreasing gradiant downwind, identifiable biological effects still
being observed at a distance of 5 kilometres. In the environmental conditions prevailing at
Hensies, TPA was more sensitive than CEA,partly for already mentioned reasons. It should
however also be noted that a rise in TP A levels might be indicative not only of an increased
cancer risk, but also of an increased risk for developing chronic inflammatory conditions
later facilitating the development of a malignancy, in a sequence comparable to the asbestos
exposure-asbestosis-mesothelioma sequence. It does not seem pertinent presently to try and
evaluate the relative risk suffered by the most heavily exposed population in sector
I;however it seems important to have objectively demonstrated that such a risk exists,to have
drawn the outline of the evaluation methods that may be applied, and to draw some practical
conc1usions.

CONCLUSIONS

The development of increasing numbers of WSs is a matter of growing concern not

only to the involved populations, but also to the medical authorities-and more precisely the
GPs- who are in charge of these populations. The GPs, in order to be able to answer the
numerous queries with which they are overwhelmed, need to be informed ab out the available
methods for risk evaluation, especially long-term risks such as cancer. To be fully
practicable in general medical practice, these methods should be easily performed during
daily practice, non-aggressive and inexpensive so as to allow for evaluations in large-sized
populations ; in spite of these preliminary requirements, the methods should be accurate and
reproducible, enabling follow-up studies.
This study has demonstrated the reality of an increase in risk caused by the proximity of
a WS ; it has shown this risk to persist at considerable distances from the ws. Among the
different evaluation methods that have been described in the introduction, this study brings
evidence that the biochemical assessment of selected parameters in body fluids (blood but
possibly also urine) represents an easy and reliable method far risk evaluation, that might be
recommended as a first approach in rather large-sized populations ; more sophisticated and
costly techniques such as oncoprotein determinations or mutational spectra assessments
might then be reserved for the cases necessitating more thorough investigations. It can be
imagined that the Sentinel Health Effect refered to in the introduction might well not only be
a physical sign(s) drawing the GP's attention to possible consequences, but that it might
quite as well be a biochemical signal leading to increased surveillance and, perhaps, to some
form of secondary prevention.
Is the biochemical tandem CEA-TPA ideal for risk evaluation? This and other studies
have at least demonstrated its usefulness as a primary approach ; it is however obvious that
its accuracy could be extended by including other parameters in the assay, such as a growth
factor, or an extracellular matrix protein, or an individual susceptibility factor such as
glutathione-transferase-mu, and others. This will inevitably increase the costs and eventually
restrain the use of the assay ; possibly the tandem evaluation might then be more efficiently
completed by one of the already mentioned more sophisticated techniques, even if then in
smaller populations.
Another major problem is that ofthe psychological impact ofthis kind of studies on the
implicated populations; this can induce a major feeling of insecurity and anxiety. To avoid
this as far as possible we plan, in the future, to carry out a preliminary exposure evaluation
using higher plants; we have selected the Tradescantia Micronuc1eus Test developed by T-H

222
Ma et al., 1984 (see also Sandhu et al.,1989 and T-H Ma in this volume), whieh shows a
high sensitivity for genotoxie exposure assessments; we believe that this might reduee the
number of populations to be assessed.
As a final conclusion, we want to stress the reality of the health impairments in humans,
related to the proximity of WSs ; the potential longterm effects with regard to carcino-
genesis are still speculative, but cannot be excluded.In selecting future WSs,it seems decisive
to locate them as remotely as possible from inhabited areas.When nevertheless populations
unavoidably reside in the vieinity of WSs, they should be provided with adequate and
compulsory medical surveillance at regular intervals, at distances up to severa1 kilometers.
We believe the CEA-TPA tandem to be well-suited to earry out the first-stage surveillance.

ACKNOWLEDGEMENTS

The authors want to express their sineere appreeiation to a1l the volunteers who
participated in this study, ineluding 32 ehlldren ofwhom the youngest was 3 years old. This
study would not have been possible without the active eooperation of the general
practitioners on the field ; our gratitude goes to doctors Jean-Gerard Pauluis; Olivier Berny;
Leon Deleuse, Roland Moens and Mare Willem for the Mel1ery evaluation; Pierre Riehez;
Pierre Vigin and others for the Hensies evaluation. It is our pleasure to aeknowledge the
support of BYK-SANGTEC, SA, Brussels; we appreeiate the teehnieal skills of Paul
Wadeleux and Veronique Cauehie (radio-immunology Department, Oncology Department
(Head : Dr Mare Beauduin), Jolimont Hospital).
We are grateful to Jaeques Groulard for drawing the map, and to Ms Sandra Cordier for
excellent seeretarial assistance in typing the manuseript.

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THE USE OF BIOMARKERS IN THE EVALUATION OF EXPOSURE
AND HEALTH AT A HAZARDOUS WASTE SITE

John S. Reif', Howard S. Ramsdell', Nancy M. DuTeau '

W. Kent Anger2 and Theodora A. Tsongas 3

'Department of Environmental Health

Colorado State University
Fort Collins, CO 80523 U.S.A.

2Center for Research on Occupational and Environmental Toxicology

Oregon Health Sciences University. Portland OR 97201

3Tsongas Consulting. 6764 E. Exposition

Denver, CO 80224

INTRODUCTION

Epidemiologie studies eondueted at hazardous waste sites present a number of

methodologie diffieulties in addition to those generally assoeiated with observation al
studies in human populations (Marsh and Caplan, 1987). First, the populations studied
are relatively smalI, making the identification of rare outcomes, such as cancer,
difficult. Second, the latent period required for many c1inical outcomes to be expressed
may be long and the duration of residenee for many members of the population
inadequate for disease expression. Migration into and out of the study area may make
preeise aseertainment of duration of exposure diffieult. Third, there may be a laek of
specificity for many potentialoutcomes. Fourth, confounding bias exerted by other
exposures (e.g., smoking, alcohol eonsumption, oecupational exposures to ehemieals)
may make it difficult to determine whether assoeiations deteeted are related to
exposure to the waste site.

Of paramount importance as a eonstraint in conducting epidemiologie studies

at hazardous was te sites is difficulty in obtaining aceurate information on exposure to
eontaminants. Information on dose level and duration of exposure is often missing
(Hulka and Wileosky, 1988). Exposure is most frequently defined by so me surrogate
sueh as proximity to the site. Misclassification of exposure leads to erroneous results,
with the effeet estimate biased towards the null when the misclassifieation is
nondifferential. Lack of adequate environmental exposure information and complex

Use 01 Biomarkers in Assessing Health and Environmental Impacts 01 Chemical

Pollutants, Edited by C.C. Travis, Plenum Press, New York, 1993 227
 exposure pathways to mixtures of chemicals contribute further to the difficulties in
classifying exposure for epidemiologie studies.

The purpose of this paper is to present three complementary approaches being

used in the evaluation of exposure and health effects in a population residing near a
hazardous waste site. A population-based epidemiologic study which ineorporates
biomonitoring, neurobehavioral testing and the use of biomarkers of exposure and
effect is described.

DESCRIPfION OF THE STUDY SITE

The Rocky Mountain Arsenal (RMA) is a Superfund hazardous waste site near
Denver, Colorado. It is unique in terms of its large size (27 square miles), levels of
contaminants and the complex mixture of chemicals documented in various media
onsite. The RMA was used by the U.S. Army to manufacture, assemble, test and
dispose of chemical and incendiary munitions including nerve gases, mustards and
rocket fuels. Other occupants manufactured pesticides including paradichloro-
diphenyltrichloroethane (DDT), dibromochloropropane (DBCP), dieldrin, aldrin,
endrin, and isodrin and chlorinated benzenes (CDH, 1989).

Approximately 80 contaminants have been identified in off-site sampling of

environmental media, particularly alluvial ground water (ATSDR, 1992). Human
exposure to volatile and semi-volatile organic compounds including benzene, carbon
tetrachloride, chloroform, trichloroethylene, tetrachloro-ethylene, 1,2-dichloroethane"
organochlorine pesticides, heavy metals and products associated with the manufacture
of chemical warfare agents is believed to have occurred via air, water or soi! exposure
pathways (CDH, 1989).

BIOMONITORING

Environmental measurements of xenobiotics provide important information

about the sources and relative concentrations of ambient exposures. However, these
measurements may not reflect concentrati.ons that are actually ingested or absorbed
from a11 routes of exposure. Biological monitoring can be used to determine the
internal exposure level or "absorbed dose" (Wogan and Tannenbaum, 1987). Biological
monitoring takes into account absorption by all routes from multiple exposure sources.
Total exposure ean be estimated for multiple ehemieals (Wog an and Tannenbaum,
1987). Appropriate biomoni tori ng of exposed populations at hazardous waste sites
should provide a more useful measure of the impact ofaxenobiotic than its external
measurement in the environment (Hulka and Margolin, 1992).

In response to evidence of elevated chemical concentrations at locations around

the RMA, populations presumably exposed by defined pathways, and anecdotal
information indicating that acute adverse health outcomes had taken pI ace, an
exposure study using biomonitoring for selected contaminants was initiated in 1989
(ATSDR, 1992). The analytes chosen for screening included arsenic and mercury, the
organochlorine pesticides dieldrin, endrin, aldrin, isodrin, DDE and DDT;
clorophenylmethylsulfone (CPMS0 2), an intermediate in the production of a herbicide
(planavin) manufactured at RMA, and diisopropylmethylphosphonate (DIMP) a by-

228
product of nerve agent manufacture produced at RMA. Tbe latter two chemieals were
intended to serve as site-specific "fingerprints" for exposure.

Tbe investigation was designed as a cross-sectional study of chemieal exposures

among residents of communities adjacent to RMA (Reif et a1., 1992). From an
epidemiological perspective, several features of the study design relevant to the
application of biomarkers in human populations, merit ~~,phasis. First, the study was
population-based to minimize the introduction of selection bias. Tbis was accomplished
by conducting a census in the areas considered exposed and in a comparison area. In
densely settled neighborhoods, blocks were chosen randomly for census, while in
sparsely settled areas the census was complete. An age and gender stratified sampie
was chosen randomly from strata which had been group matched across exposure
areas. Second, an eligibility criterion of two years was used to ensure that participants
had had ample opportunity for exposure. The mean duration of residence for
participants was approximately 13 years and was similar for the exposure and
comparison areas. Third, the comparison area was chosen carefully to ensure that the
residents had similar demographic and socioeconomic characteristics and that the area
was ecologically similar. Finally, an extensive interview was conducted to collect
information about potential confollnding factors and to deve10p hypotheses about the
factors which resllited in exposure to chemieals. The interview included demographie
information, residential history, occupational history, and information about water
supply and llse patterns, consllmption of horne grown fruits and vegetables, horne and
workplace exposures to pesticides and other organic and inorganic chemicals, and other
potential exposure pathways. Questions concerning education, housing characteristics,
smoking, a\cohol consumption, diet, play habits, hobbies, and activities were included
to control for potential confounding and search for effect modification. Limited data
were gathered on each participant's past medical history in order to be able to assess
pre-existing chronic disorders that could affect metabolic status or detoxification
capahility through hepatic or renal mechanisms.

NEUROEHA VTORAL MARKERS

\Ve introduced neurobehavioral testing as markers of effect at RMA since

neurotoxicologic and neurobehavioral effects have been reported with human exposure
to c1asses of chemicals which are major contaminants at RMA. These contaminants
include mercury, agricultural pesticides, especially the organophosphate insecticides,
and organic solvents. Neurobehavioral effects have been shown to be sensitive markers
of health effects for mercury (ATSDR, 1989), pesticides (Savage et al., 1988) and
organic solvents (Gamherale, 1985). The methods employed in neurobehavioral testing
are non-invasive, relatively simple to perform, cost-effective and reprodueible. Tbere
are consistent findings from over 185 epidemiologie behavioral neurotoxieology studies
that show a broad speetrum of cognitive, motor and affeetive or personality ehanges.
Thc most frequently reported functional deficits are in tests of intelligence, memory,
spatial relations, eoordination, and speed plus eoordination (Anger, 1990).

Neu rotoxicologic evaluation of persons exposed to mixtures of hazardous wastes

at dump sites has heen reported (Sehallmburg et a1., 1983). Nerve conduction velocity
was used as a marker of potential neurotoxicity. A significant reduction in nerve
conduction velocity was linked to exposure. Neurotoxicologic assays including c1inical
tests, nerve eonduetion studies, blink reflex measurements and neuropsyehologieal
testing were also eonducted in persons exposed to weil water eontaining

229
trichloroethylene and other solvents at the Woburn, Massachusetts site. A significant
difference between exposed and unexposed persons was found on several
neuropsychologieal tests and in blink reflex latency (Feld man et al., 1988)

We are eurrently studying approximately 300 adults with the Neuro-behavioral

Co re Test Battery (NCTB) recommended by the World Health Organization (Johnson
et al, 1987). They were screened by biomonitoring during the exposure study described
above. The prevalence of symptoms of neurologieal disorders will also be assessed in
this sampIe and compared among study areas. Tbe NCTB was seleeted to give a
relatively eomprehensive sampIe of the major areas of established neurotoxic effects
found in humans. In addition, the NCTB eonsists of tests proven sensitive in cross-
seetional field research to the chemicals found at RMA (Anger, 1990).
Neurobehavioral testing will be utilized to detect various individual functional deficits
and nervous system disorders due to neurotoxic chemieal exposures from RMA. Tbe
NCTB was seleeted with two major considerations. First, five of the NCTB tests, when
administered individually in various studies, have demonstrated sensitivity in field
evaluations of ehronie exposures to neurotoxie ehemieals known to be present at the
RMA. Second, the NCTB has been used worldwide in populations from varying
soeioeconomie strata (Anger et al., 1992); many of the subjects in the study population
have low levels of edueation.

The NCT includes seven behavioral tests: 1) Santa Ana; 2) Aiming; 3) Simple
Reaetion Time; 4) Digit Symbol; 5) Benton Visual Retention Test; 6) Digit Span, and
7) Profile of Mood States (POMS). The NCTB assesses various neurobehavioral
funetions; psyehomotor skills, visual perception, memory, learning and affect (Johnson
et a1., 1987; Cassitto et al., 1990). Tests of visual acuity and color discrimination are
also being assessed.

lOMARKERS

Biologic markers can be used to strengthen epidemiologie research designs and

to reduee misclassifieation of exposure and effeet (Hulka and Margolin, 1992). In the
broad sense, biologie markers are measurements on biologie speeimens that will
elueidate the relationship between environmental exposures and human disease, so that
such exposures and diseases can be prevented (NRC, 1992). Early deteetion leading
to prevention of disease and disability is the ultimate goal in the application of
biomarkers in human populations (NRC, 1992).

Classieal approaehes to assessing exposure to xenobioties involve measurement

of the agents in biologie fluids or tissues. However, numerous organic ehemieals are
rapklly mctabolized to other eompounds for which suitable assays do not exist; further,
the exposure may involve complex mixtures of chemieals. These circumstances suggest
that indirect assessment of exposure status through the use of biomarkers may improve
validity and reduee misclassification in epidemiologic studies. (Brewster 1988, Hulka
et al, 1990). The reduetion of misclassification is particularly relevant to the study of
populations living around hazardous waste sites where low levels of exposure, small
sampie sizes and weak relative risks are likely to be found.

Hulka and Wilcosky (1988) described several issues to be resolved in selecting

markers for environmental exposure studies including exposure complexity, marker

230
specificity, marker persistence, and the use of surrogate biologieal media. We
considered several of these in the design of this study. Biomarkers that can be
measured in urine offer distinct advantages for detection of exposure to toxic chemicals
in population studies since sampies are readily obtained. Biomarkers of exposure may
be non-specific with respect to particular chemicals, but may constitute appropriate
endpoints that reflect exposure to complex mixtures of contamina.nts. Biomarkers of
effect may be useful tools at hazardous waste sites to permit detection of early
biological responses in the absence of clinical evidence of disease.

Biochemical Markers

Three biochemieal markers of exposure detectable in urine have been

incorporated into the design of the current study at RMA: D-glucaric acid excretion,
methylxanthine metabolite ratios and retinol-binding protein. Each parameter is
potentially sensitive to contaminants found at RMA.

Urinary excretion of D-glucaric acid reflects the level of glucuronide formation,

which may be influenced by agents that induce hepatic microsomal enzyme activities.
Many xenobiotics are biotransformed by conjugation with glucuronic acid. Increased
levels of D-glucaric acid have been observed in the urine of human..'i exposed to dioxin
(Ideo et al., 1982) or to chlorinated cyclodiene insecticides such as dieldrin (Hunter
et al., 1972), manufactured at the RMA. Notten and Henderson (1977) studied the
effects of nllmerolls compounds including organic solvents, insecticides and herbicides
and conclllded that enhanced urinary excretion of D-glucaric acid is a useful non-
specific marker for exposure to xenobiotic compounds.

The chlorinated pesticides found at the RMA are known to induce cytochrome
P-450 isoenzymes. The pattern of methylxanthine derivatives found in urine as a
consequence of consumption of caffeine-containing beverages may be a useful indicator
of alterations of cytochrome P-450 isoenzyme content (CampbeH et a1., 1987).
Demethylation reactions appear to be mediated by PAH-inducible forms whereas
methylxanthine 8-hydroxylation is catalyzed by isoenzymes that are not induced by
PAH's (Camphell et al., 1987). ThllS, determination of the ratio of methylxanthine
metaholite concentrations in the urine should provide an indication of exposure to
cytochrome P-450 indllcers. A significantly altered ratio was observed in smokers
compared to non-smokers (Campbell et al., 1987). This could be a very useful
approach, because caffeine consllmption is very common, caffeine consumption can be
evaillated by interview, and measurement of the relative amounts of different
metabolites eliminates the need for administration of defined doses of caffeine or the
use of labeled compound. HPLC analysis of urine extracts provides ample sensitivity
for detection of methylxanthine metabolites in the urine of moderate coffee or cola
drinkers.

Several of the contaminants at RMA are known to cause toxie effects in the
proximal tubular cells of the kidney. They include metals (eg., cadmium, mercury,
chromium) and chlorinated compounds (eg., chloroform, trichloroethylene,
chlorobenzene, tetrachloroethylene). Active reabsorption of low molecular weight
proteins is decreased by exposure to agents that affect the renal tubular ceHs. Thus,
measurement of such a protein in the urine may provide a sensitive indicator of renal
toxicity. Retinol-binding protein (RBP) which has been found to be a useful early
indicator of renal damage (Bernard et a1., 1987). Urinary concentrations of RBP may
be readily measured with immulloassays using commercially available antibodies.

231
Genetic Markers

The current study of health effects at RMA incorporates three urinary

biomarkers which may provide evidence of genotoxicity or of exposure to mutagens
among persons residing near the site.

Urine Mutagenicity. The presence of mutagens in human body fluid has been
used as an indicator of systemic exposure to a wide range of mutagenic chemicals. A
variety of test systems are available to measure DNA mutations and chromosomal
changes or employ DNA repair assays. Tbe most common test systems utilize specially
constructed strains of Salmonella typhimurium (Ames test) and Esehenehia eoli
(tluctuation test). The biological basis of these tests is their ability to induce specific
mutations in selected bacteria. These mutations may be either base-pair or frameshift
mutations (McCann, 1983).

Mutagenicity testing is especially useful for analysis of sampies with multiple or

unknown substances. The urine mutagenesis assay using a bacterial test system is one
of the most widely used techniques to monitor populations for mutagenic exposures.
Urine represents a useful substrate for the evaluation of biomarkers since it is a major
route of elimination of xenobiotics, is readily available and stored, reflects previous
concentration by the kidney, and is suitable as an indicator of recent exposure (Rynard,
1990). Urine mutagenesis assays have been conducted to detect exposure to several
environmental risk factors in humans. Life-style exposures such as active and passive
cigarette smoking, coffee consumption and various dietary components have been
evaluated. A variety of occupational groups have been studied including health care
workers exposed tn cytostatic drugs, factory workers exposed to petroleum coke and
pitch, chemical and coke oven workers, rubber workers and sewage treatment workers
(Rynard, 1990). The urine mutagenesis assay is relatively simple and inexpensive to
perform, but may produce inconsistent findings and has not been linked directly to
subsequent risk of disease. Nonetheless, urine mutagenicity assays appear to be a
suitable screening biomarker for persons at hazardous wasle sites.

Chromosomal Aneuploidy. Screening programs for persons living near a

hazardous waste site where a potent bladder carcinogen, beta-naphthylamine, was
manufactured have been described recently (Marsh eL al. , 1990). The urinary
biomarkers used to screen persons at high risk of developing bladder cancer included
Papanicolaou cytology of exfoliated bladder cells as weil as a test for abnormal
cytology based on a quantitative fluorescence image analysis (QFIA) (Marsh et al.,
1990). Similar efforts are underway at the RMA. The DNA content of bladder ceHs
from urine is being estimated by measuring the tluorescence of each cell by flow
cytometry after staining with propidium iodide. Cells are considered abnormal with
fluorescence greater than two standard deviations above that of normal diploid,
dividing cells (aneuploidy). This technique measures very early, precancerous ceH
changes and may detect low-grade tumors missed by conventional Papanicolaou
cytology (Bass et al., 1987).

Cellular Micronuclei. The assessment of micronuclei in exfoliated ceHs is a

promising tool for the study of epithelial carcinogens (Tolbert et al., 1992). Tbe
exfoliated cellmicronucleus assay involves the examination of epithelial smears for the
prevalence of micronuclei in cells. Micronuclei are extranuclear bodies composed of
chromosomal fragments or entire chromosomes which are found in the cytoplasm when
chromatidj chromosomal fragments or whole chromosomes are not incorporated into

232
daughter nuc1ei during mitosis. The micronuc1eus assay may be used to detect
chromosome breakage (c1astogenesis) or interference with mitosis (Stich and Rosin,
1983), events believed to be associated with carcinogenesis (Jenssen and Ramel, 1980).
Micronuclei may result from exposure to agents that cause damage to the spindie
apparatus during cell division (Vine, 1990; Jenssen, 1982). The micronuc1eus assay may
be performed on exfoliated cell populations oi several origins, inc1uding the bladder
(Reali et. al., 1987). Micronuc1ei in exfoliated cells result from genotoxic events that
occurred in the dividing basal celllayer 1-3 weeks earlier. Tolbert et al. (1991) have
developed and used a set of criteria for c1assifying micronuc1ei and other nuc1ear
anomalies based on underlying causes of the observed abnormalities. This technique
is being applied to assess exposure of populations at RMA to genotoxic substances,
especially epithelial carcinogens.

SUMMARY

In this paper we have described an approach towards improving the usefulness

of epidemiologie studies at hazardous waste sites. The study underway at RMA makes
use of a population-based approach to identifying participants in the exposure and
comparison areas, control of potential confounders, and a two stage approach to
evaluating exposllre-disease relationships. In the first stage, biomonitoring was
condllcted to evaltlate the presence of arsenic, mercury, chlorinated pesticides, a
phosphonate prodllced dllring the manllfacture of nerve agent, and an intermediate in
the manllfacture of cyclodiene pesticides. In the second stage, health effects including
reprodllctive and neurobehavioral endpoints are being evaillated. Biochemical and
genetic bio markers have been incorporated irrto the study protocol to improve the
classification of ex pos ure amI to search for early measures of effect at the site.

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Feldman, RG., Chirico-Post, J., Proctor, S.P., 1988, Blink reflex latency after exposure
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Gamberale, F., 1985, Use of behavioral performance tests in the assessment of solvent
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Hulka, B.S. and Margolin, B.H., 1992, Methodological issues in epidemiologic studies
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Ideo, G., Bellati, G., Bellobuono, A, et al., 1982, Increased urinary D-glucaric acid
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University Press, New Yark pp. 57-77.

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Schaumburg, H.H., Spencer, P.S., Arezzo, J.c., 1983, Monitoring potential neurotoxic
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Tolbert, P.E., Shy, C.M., Allen, J.W., 1991, Micronuclei and other nuclear anomalies
in buccal smears: A field test in snuff users. Amer. J. Epidemiol. 134:840-850.

Vine, M.F., 1990, Micronuclei, in: "Biological Markers in Epidemiology", B.S. Hulka,
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235
 STATE OF mE ART - ECOWGICAL BIOMARKERS

Lee R. Shugart

PO Box 2008
Environmental Sciences division
Oak Ridge National Laboratory
Oak Ridge, TN 37831-6036, USA

BIOWGICAL MARKERS

It is especially difficult to demonstrate the effect of environmental pollution at

the Ecosystem level, where populations and communities are studied. Although
pollution can produces stress at the Ecosystem level, the response observed is latent and
so far removed from the initial event of exposure that causality is almost impossible to
establish. Risk assessment to date has relied on models that use toxicity data and
physical properties of chemicals. This approach has been ineffective at the Ecosystem
level because of the complexity of pollutants in the environment and the variability in
susceptibility of the numerous species present. The use of biological responses
(biomarkers) in organisms present in apolluted environment is an approach that may
resolve some of the problems outlined. Chemicals are known to eUcit measurable and
characteristic biological responses in exposed organisms. Such evidence can provide
a link between exposure and effect. Unfortunately, this area of endeavor has received
little attention until recently. It has only been within the past several years that
authoritative texts have become available to document the application of this promising
approach.

RELEVANT RESOURCE MATERIALS

The state-of-science in a particular discipline or field is reflected in the scientific

literature available. In Table 1 are listed several reference books, either published
recently or currently in press, whose subject matter bears directly on the biological
marker approach to evaluate the ecological and hea1th effects of environmental
contamination.

Use 0/ Biomarkers in Assessing Health anti Environmental Impacts 0/ Chemical

Pollutants, Edited by C.C. 'fravis, Plenum Press, New York, 1993 237
 Table 1. Relevant Resource Materials.

Biomarkers of Environmental Contamination

lohn MeCarthy and Lee Shugart (Eds.)
Lewis Publisher, Ine., (1990)
Biomarkers: Biochemical, Physiological, and Histological
Markers of Anthropogenie Stress
Robert Huggett, et al (Eds.)
Lewis Publisher, Ine., (1992)
Biomarkers: Research and Application in the
Assessment of Environmental Health
NATO ASI Series H: Cell Biology, Vol., 68
David Peakall and Lee Shugart (Eds.)
Springer-Verlag, (1993)
Animal Biomarkers as Pollution Indicators
David Peakall
Chapman and Hall, (1992)
Nondestruetive Biomarkers in Vertebrates
Christina Fossi and Claudio Leonzio (Eds.)
Lewis Publishers Ine., (1993, in press)
Environmental Epidemiology: Publie Health & Hazardous Waste
National Research CouncillNational Academy of Sciences
National Academy Press (1991)

In the Fall of 1988 the Environmental Chemistry Division of the Ameriean

Chemical Society sponsored a symposium on Biomarkers of Environmental
Contamination. The objective of that symposium was to review the state-of-science as
it pertained to the scientifie basis, eurrent state of development, validation, and use of
biological markers in environmental research. The papers presented at that symposium
were published in 1990 (McCarthy and Shugart, 1990). This work represents one of
the first attempts to bring together, in a cohesive manner, studies performed by a group
of scientists with varying technical specialties that focused on the health of species in
contaminated environments.
The Society of Environmental Toxieology and Chemistry (SETAC) sponsored
a Biomarker Workshop in 1989 to provide an in-depth review of biomarkers and their
present and future utility to either determine exposure to or effects of anthropogenie
stress. The proceedings of that workshop were published in 1992 as a volume
(Huggett, et al., 1992) in SETAC's Special Publication Series. The volume is divided
into several subject areas that present eritieal analyses of using biochemical,
physiological, and histological changes to estimate exposure of organisms to toxicants
or resultant effects of exposure. As such this book is a valuable resouree that offers
a eatalogue on biomarkers and their utility.
Even though the previous works raised the level of awareness about the
applieation of biomarkers to the investigation of environmental health, dialogue among
interested scientists revealed that the field ofbiomarker-based biomonitoring stilliaeked
direction, especially with regard to a unified strategy. To correct this situation, it was

238
decided that a panel of international experts should be convened to detail the conceptual
framework for the use of biomarkers in the assessment of environmental health. In
May of 1991 a NATO Advanced Research Workshop was held at The Nethedands
Institute of Sea Research in the Netherlands and was attended by 37 scientists from
Europe and North America. The participants were provided with a "straw" document
that outlined the major issues dealing with biomarkers that required a consensus within
the scientific community. Although the straw document was valuable in focusing
discussion at the workshop, it was largely rewritten and is currently being published in
book form in the NATO ASI Series (peakall and Shugart, 1993).
Dr. David Peakall has recently written a monograph (Peakall, 1992) on Animal
Biomarkers as Pollution Indicators. Dr. Peakall is a distinguished research scientist
who is internationally recognized for his work on the effects of pollutants on birds. His
book presents an in-depth and up-to-date account of the use of biomarkers in
ecotoxicology. The core of the book is a consideration of the major bio markers that
are available and documents their past and present use.
The concept of nondestructive testing has great merit and potential but progress
in its development and application to environmental health assessment has lagged
because no one has brought an the different methodologies and concepts of
nondestructive testing together in an organized way. In May of 1992 a Workshop was
convened at the University of Siena in ltaly to address an alternative strategy for hazard
assessment in vertebrates based on nondestructive testing. Many biomarkers commonly
used in environmental research require the analysis of tissues and organs such as liver,
kidney or brain and involve the destruction of living organisms. Apart from ethical
considerations, destructive testing may be undesirable in many situations; for example
the number of animals available at a site may be limited, it may be necessary to study
a protected species or sequential sampling from the same individual may be required
for time course studies. Furthermore, nondestructive testing can enable hazard
assessment in protected or threatened species whose existence cannot be further
jeopardized by the use of destructive methods. The biological materials required for
biomarker analyses can often be obtained without stress or damage to the animal, for
example collecting samples of blood, epithelial tissue, eggs, feathers, feces, etc. The
proceedings of the Siena Workshop are currently in press (Fossi and Leonzio , 1993).
The last resource material found in Table 1 is a recent book (CEElNRC, 1991)
published by the National Academy of Sciences in the United States. The document
was prepared by the Committee on Environmental Epidemiology of the National
Research Council for the Agency for Toxic Substances and Disease Registry. Although
the thrust of the book is effects on human health that could be linked with exposure to
hazardous-waste disposal sites, the concepts and approaches are applicable toward the
assessment of the health of all organisms in the environment. An entire chapter is
devoted to the use of biologieal markers in epidemiologie studies.

CONCERN FOR TIIE ENVIRONMENT

A recent editorial in the prestigious scientific journal Nature (Editorial, 1991)

entitled "Common Sense in the Environment" addressed remarks made in the National
Academy of Sciences' book (CEE/NRC, 1991) on Environmental Epidemiology
claiming that "the United States government may be wasting billions of dollars in its
zeal to clean up the environment simply because it had failed to conduct studies that
would provide asound scientific underpinning to judge whieh of the many potential

239
 toxins constituted a genuine and serious threat to human health". Furthermore, it was
noted that "biomarkers have not been adequately adopted for assessment of broad
environmental exposure to hazardous waste" .
Clean-up of the environment is one of the most difficult decisions facing society
today. The pressures to do something is immense, and a decision to do too little may
cause irreversible harm to the environment, while a decision to do more than is
necessary may waste large sums of money which could have better use elsewhere
(peakall, 1992). The correct decision will require that society and science address two
basic questions. First, how much damage to our health and that of the environment are
we prepared to tolerate; and second, how much proof is necessary before action is
taken? Pragmatica1ly, the biomarker approach would help to assess the health of the
environment, and, in addition, would enable scientists to defend the advice they provide
concerning environmental issues.

ENVIRONMENTAL MONITORING

Introduction

Biologica1 monitoring is an informative, cost-effective and 10gica1 complement

to chemica1 monitoring of toxicants in the environment (Shugart, et al., 1992). The
rationale for chemica1 monitoring is our concern for the potential threat that chemica1s
can pose to biologica1 resources. The study of biological responses in living organisms
to contamination in their environment is a complementary approach that is becoming
a reasonable and necessary component of many environmental monitoring programs.
The concept of "biomarkers" has recently received considerable attention among
environmental toxicologists as a new and potentially very powerful and informative tool
for detecting and documenting exposure to, and effects of, environmental contamination
(McCarthy and Shugart, 1990; Huggett et al. 1992; Peakall1992; Peakall and Shugart,
1993, Fossi and Leonzio, 1993).

Strategy

General Elements of a Biomarker Study. Regardless of the specific objectives

or motivation for a biomarker study, many elements of the study design remain
constant. The 10gica1 pathway for designing an environmental monitoring study is
indicated in Fig. 1. This chart is taken from Peakall and Shugart (1993) wherein each,
element in the pathway is dissected into aseries of relevant issues that are defined in
considerable detail. These issues form a "check-list" of major concems that must be
addressed in the design and implementation of a study.

Prioritization of Biomarkers. The prioritization process involves the selection

of biomarkers that are appropriate to the objectives of a given biologica1 monitoring
prograrn. The suitability of a particular biomarker should be judged on the anticipated
probability of obtaining information that will document either exposure, status of
cellular compensatory mechanisms, or potential for harm. An in-depth survey of
biomarkers was recently conducted. (Huggett, et al., 1992) From such a survey it may
be possible to identify a small sub set of biomarkers with which to examine and evaluate
both specific or general types of contamination problem. The identification of this
sub set and its supplementation represents the first step in the prioritization process. The

240
 Delining Study Statistical Analysis
Area and Selection and Interpretation
01 Relerence Sites 01 Results

Selection 01 Biological and

Sampling sites Chemical Analyses

Characterization 01
Studyand
Relerence Sites

Fig. 1. General elements of a biomarker-based monitoring study (laken from PeakaU and Shugart,
1993).

list of biomarkers described in Table 2 would constitute a reasonable generic core from
which to expand. The suite of biomarkers chosen span several levels of biological
organization, various types and temporal occurrence of biological responses. As with
any suite of biomarkers, the interpretation of the observed response must be tempered
by the current state of our scientific knowledge (Le., specific limitations and
restrictions). Some responses will be definitive indicators of exposure and even
predictive of long-term adverse effects; whereas other responses will only be a signal
of a potential problem (Shugart, et al., 1989).
The importance of integrating results from biomarker studies in the laboratory
with those from the field (and vise versa) can not be overemphasized (peakall and
Shugart, 1993; Shugart, et al., 1992). The elucidation of relationships between known
contaminants and biological response under controlled laboratory conditions will help
in the selection of biomarkers for field studies. Conversely, biomarker data from field
studies can direct the choice of biomarkers for more detailed laboratory studies.

Factors Affecting Biomarker Response. It is obvious, from both logistical and

economical points of consideration, that a biomarker-based biomonitoring project can
not inc1ude a large number of biomarkers. Established guidelines will dictate to a great
extent the type and number considered suitable, but even at this point in the selection
process, the list of potential biomarkers might be too large. If several general
attributes and characteristics of biomarkers are recognized and considered, then
selection can be fine-tuned without diminishing effectiveness. Factors to be considered
are:

(a) Response - It should not be anticipated a priori that exposure of an

organism to contaminants in its environment will elicit a biological
response, or that responses will be observed through different levels of

241
 Table 2. Biomarkers for Environmental Monitoring.

Biomarkers Biological Levelof Temporal Restriction2

Response Biological Occurrence'
Organization

Detection Limitations
Level
DNADamage Adducts Molecular Early Low Repairl
Analysis
DNADamage Strand Breaks Molecular Early Moderate Repair
Mixed Function Enzyme Induction Molecular Early Moderate Species
Oxidase Variability
Metabolites Xenobiotic BiochemicaI Early Low Chemical
Chemicals Speciation
Cholinesterase Enzyme Inhibition Molecular Early Moderate Species
Variability
Heme Abnormal Biochemical Middle Moderate Species
Biosynthesis Porphyrins Variability
Blood Chemistry Various Entities Biochemical Middle High
and Cellular
Condition Various Entities BiochemicaI MiddlelLate High Species
Indices CeIl\Tissues Availability
Immune Phagocytosis Cellular MiddlelLate Moderate Analysis
Competence
Chromosomal Abnormal DNA Sub-cellular Middle/Late High
Aberrations
Enzyme-Altered Neoplastic Lesions Sub-cellular Middle/Late Moderate Analysis
Foci
Cellular Necrosis/Neoplasia CelllTissue Late High
Transformation

ITemporal occurrence subsequent to exposure: early-hours to days; middle-days to weeks/months; late-

weeks/months to years.
2Constraints on biomarker application: (1) detection level - anticipated probability of detecting biomarker;
and (2) limitation - factor(s) contributing to detection or affecting use of biomarker.

biological organization. Where possible, biomarker selection should be

based on the known toxicological mechanism of action of the
contaminants present, thus maximizing the probability of observing a
biological response(s).
(b) Temporal Occurrence - Some biomarkers are measurable early (hours)
after exposure and are observable at the biochemicallmolecular level of
biological organization. These early responses may exhibit atemporal

242
 existence. Others appear much later (years) and are seen at higher levels
of organization. Implicit in the concept of biomarker use is the potential
for correlating responses among various levels ofbiological organization.
(c) Variability - Sources of variability that may influence the measurement
of a biological response generally fall into two categories: those that are
inherent to the laboratory method, procedure, or assay selected, and
those intrinsic to the species being sampled. Sampie collection,
preparation, and storage as well as reagent purity and instrument
selection and calibration are examples of the first category. They are
more easily controlled through adherence to quality assurance and quality
control policies. Age, sex, and disease state of the organism being
sampled, or environmental stresses such as climate or food availability
are examples of factors that contribute variability in the second category.
The effects of these latter factors on the biomarker assay are difficult to
predict; however, they can be documented and accounted for by
establishing baseline data from appropriate noncontaminated or reference
sites.
(d) Limitations - Specific limitations and restrictions apply to the use and
interpretation of many ofthe biomarkers, and these factors should be
verified by consulting the current scientific literature.

Impediments. There can be little doubt that measurement of biomarker

responses in organisms from contaminated sites offer great promise for providing
information that can contribute to monitoring programs designed for hazard
identification, hazard assessment, or risk prediction. The challenge is to develop and
implement a program that will permit this promise to be fully realized. Current
understanding and application of biomarkers justifies their immediate implementation
in an environmental monitoring program, however, the full potential of this
methodology will be realized only after a larger data base of field and laboratory studies
can be accumulated and analyzed. Furthermore it is important to recognize that our
current understanding of biomarker responses in environmental species is limited and
to achieve their full potential as a tool for environmental protection, a great deal of
research will be needed to develop, validate, and interpret biomarker-based monitoring.
To accomplish this task a research initiative is needed that provides not only the
required data n~ssary to validate, biomarkers but also the scientific understanding
necessary to interpret biomarker responses in environmental species. This initiative
should include an evolving monitoring strategy that focuses broadly on evaluation of
contamination in an array of Ecosystem types. The challenges and obstacles to be
addressed should include:
(a) Establish the quantitative and qualitative relationships between chemical
exposure, biomarker responses and adverse effects.
(b) Responses due to chemical exposure must be able to be distinguished
from natural sources of variability (ecological and physiological
variables, species-specific differences, and individual variability).
(c) Establish the validity of extrapolating between biomarker responses
measured in individual organisms and some higher-level effect at a
population of community level.
(d) Explore the use of exposure biomarkers in animal surrogates to evaluate
the potential for human exposure.

243
 FUTURE DIRECTIONS

As mentioned previously, it is difficult to demonstrate the direct effect of

environmental pollution at the Ecosystem level. To circumvent this deficiency
biomarker strategy has been to measure responses at various levels of biological
organization within the individual and to demonstrate relatedness, that is, exposure to
deleterious substances may induce a cascade of biological events that eventually lead
to some dysfunction. Once impairment at the individual level is observed, it becomes
less difficult to extrapolate the potential for harm to the population and community
levels.
Another way to approach this problem is to ask mechanistic questions about how
an organism relates to other organisms and in turn to its physica1 environment.
Ecosystems results from the dynamic interactions of living and inert matter where the
living material acclimates and adapts to environmental change. These processes are
physiologica1 and have a genetic basis therefore understanding change at the genetic
level should help define the more complex changes at the Ecosystem level. Recent
advances in the discipline of molecular biology provide the experimental tools with
which to precisely investigate key biologica1 mechanisms that regulate and limit the
response of an individual organisms to physica1 factors in their environment (e.g.,
chemica1s). This is a fruitful area for new biomarker research, as it offers an
opportunity to rapidly advance our knowledge and understanding of the effect of
pollution on Ecosystems.

SUMMARY

At the present time there is a strong drive to clean up the environment;

however, since costs increase rapidly with the degree of clean-up, there is a pressing
need to have a practical, defensible strategy that provides information for establishing
both priorities for environmental restoration and endpoints for regulatory compliance.
An informative approach to quantifying exposure and its potential impact at the
individual and Ecosystem levels is to monitor biological endpoints (biomarkers) as
indicators of exposure and effects to environmental contaminants (McCarthy and
Shugart, 1990). The biomarkers are any of aseries of biochemica1 or molecular
responses to compounds that have entered an organism, reached sites of toxic action,
and are exerting an effect on the organism. In this context, the organism function as
integrator of exposure, accounting for abiotic and physiologica1 factors that modulate
the dose of toxicant taken up from the environment. These biologica1 markers can be
used to quantify exposure to environmental insults (Shugart, et al., 1989).
The concept of biomarkers has been described, and a variety of biological
responses at the tissue, cellular and subcellular levels have been developed as markers
to evaluate the physiological condition of an organism to detect exposure to
contaminants in their environment (McCarthy and Shugart, 1990; Huggett, et al. 1992).
However, in order to predict hea1th risks at the Ecosystem level associated with
environmental exposure an understanding of the critical cellular events that may occur
between exposure and effects must be known. Therefore it is often necessary to regress
down the conceptual sequence of organismal responses to toxicant exposure and select
appropriate biologica1 responses that are causally related to, or predictive of, longer
term effects.

244
 The biomarkers approach might, for example, be employed to avoid
unacceptable and often irreversible effects at high levels of biologieal organization such
as disease in humans, mass mortality and loss of commercially or ecologically
important species. Because of the commonality of biochemieal and cellular structure
and function among organisms, biomarkers are potentially applicable over a broad range
of species and across most Ecosystem types. Detailed information on biomarkers and
their potential for application in evaluating environmental contamination is available in
several authoritative texts (Table 1).

Acknowledgement

The Oak Ridge National Laboratory is managed by Martin Marietta Energy

Systems under Contract DE-AC05-840R21400 for the D.S. Department of Energy.
This is Environmental Sciences Division publication no. 4018.

REFERENCES

1. CEE/NRC: Committee on Environmental Epidemiology/National Research

Council of the National Academy of Sciences, 1991, "Environmental
Epidemiology: Public Health and Hazardous Wastes, " National Academy Press,
Washington DC.
2. Editorial, 1991, Common sense in the environment, Nature 353:779.
3. Fossi, M. C., and Leonzio, C., 1993, "Nondestructive Biomarkers in
Vertebrates, " Lewis Publishers, Inc., Boca Raton, FL.
4. Huggett, R. J., Kimerle, R. A., Mehrle, P. M., and Bergman H. L., 1992,
"Biomarkers: Biochemieal, Physiological, and Histological Markers of
Anthropogenie, Stress," Lewis Publisher, Inc., Boca Raton, FL.
5. McCarthy, J. F., and Shugart, L. R., 1990, "Biomarkers of Environmental
Contamination, " Lewis Publishers, Inc., Boca Raton, FL.
6. Peakall, D. B., 1992. "Animal Biomarkers as Pollution Indicators," Chapman
and Hall, London.
7. Peakall, D. B. and Shugart L. R., 1993, "Biomarkers: Research and
Application in the Assessment of Environmental Health," NATO ASI Series H:
Cell Biology, Vol. 68, Springer-Verlag, Heidelberg.
8. Shugart, L. R., Adams, S. M., Jimenez, D. B., Talmage, S. S. and McCarthy,
J. F., 1989, Biological markers to study exposure in animals and bioavailability
of environmental contaminants, in: "Biological Monitoring for Pesticide
Exposure: Measurement, Estimation, and Risk Reduction," R. G. M. Wang,
C. A. Franklin, R. C. Honeycutt, and J. C. Reinert, eds. ACS Symposium
Series 382, American Chemical Society, Washington DC.
9. Shugart, L. R., McCarthy, J. F., and Halbrook, R. S., 1992, Biological markers
of environmental and eological contamination: an overview, J Risk Analysis,
12:353.

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245
DETBC~IOH OF GER~XICITY OF WA~ER AHD AIR POLLur~S

USIHG TradeBcan~ia (SPIDERWOR~) PLANTS

Te-Hsiu Ma
Department of Biological Sciences
Western Illinois University
Macomb, IL 61455 USA

IftRODUC~IOH

Gene mutation and chromosome breakage are the biomarkers of the damage
inflicted by environment al pollutants in Tradescan~ia (spiderwort) plants.
Gaseous or liquid agents in the environment can be easily absorbed by this
plant through gaseous diffusion or liquid translocation via its vascular
system to reach the target cells in the young flower buds. The special
spiderwort clone 4430 which is a hybrid and heterozygous (Pr/pr) for
purplish blue (Pr) and pink (pr) in its stamen hair cells. Mutation from
Pr to pr induced by mutagens will yield pink colored cells among the
dominant purplish blue colored cells in the stamen hair. Based upon this
principle, the Tradescantia-stamen-Hair-Mutation (Trad-SHM) bioassay was
developed4.,41,S7. In this clone of plants, broken pieces of chromosomes in
the meiotic microspore mother cells induced by clastogens will form
micronuclei (MCN) in the tetrads, the end products of meiosis. Formation
of MCNs which are visible under a microscope (400X magnification) serves as
the marker of the genetic damage. This was the foundation for the
development of the Tradescantia-Micronucleus (Trad-MCN) bioassay~. Since
the late 70s, the Tradescantia clone 4430 has been used as a dual test
system for in situ monitoring or in laboratory screening of environment al
mutagens and clastogens.
Extensive database was established in both of these simple and highly
sensitive short-term bioassays28,29,30,34,48,so. Generally speaking, both point
mutation or chromosome damage can lead to carcinogenesis, thus this dual
system can be applied to the preliminary screening of carcinogens. New
hazardous agents such as pesticides, drugs, and food additives which have
been created endlesly will eventually become environmental pollutants.
These pollutants have been widely dissipated and accumulated in the air,
water and soil. Recent studies on the efficiency and reliability of this and
other plant bioassays under the auspices of the International Program on
Chemical Safety, WHO, UN, suggested that this dual system is weIl suited for
on site monitoring pollutants in water and air, and the screening mutagens
and carcinogens 39,40. A world wide biomonitoring network could be established
to give the first alert of the presence of hazardous agents in these vital
elements of life and to safegaurd the ecosystem including the weIl being of
humans.

MATERIALS AHD METHODS

Tradescantia clone 4430 which has served as a cytogenetic materials

Use 0/ Biomarleers in Assessing Health anti EnvinmmentDl Impacts 0/ Chemical

Polluronts, Edited by C,C, Travis, Plenum Press, New York, 1993 247
for detection of chromosome aberration and gene mutation for more than 3
decades is exclusively used in this dual bioassay system. Two major ways
of exposure to environmental mutagen and clastogens are either on site
monitoring or in laboratory chemical screening.
For detection of mutagens, on site monitoring of gaseous agent can be
done simply by exposing the plant cuttings which contain young flower buds
in the pOlluted atmosphere for 6 - 24 h. On site monitoring of polluted
surface water can be done by exposing the plant cuttings on a floating
device called "Aquatoon". In laboratory treatment can be accomplished by
fumigation of the [lant cuttings with a known concentration of gaseous
agents in a daynamic flow chamber, or with a known concentration of aqueous
solution or a solution of some selected harmless solvents such as low levels
of dimethyl sulfoxide (DMSO) or ethanol. The exposed mitotic cells of the
stamen hair require a recovery per iod of 7 - 12 days to allow the stamen
hairs to reach maturity possessing around 24 cells. Pink mutation events
in the predominant purplish-blue cells can be scored under a 40X dissectin~
microscope. A detailed account of this bioassay was decribed in an earlier
and a recent~ publication.
For detection of chromosome damge, the same plant cuttings exposed to
clastogens in the atmosphere or in the surface water, or those in the
fumigation chamber or the solution can be used to analize the extent of
chromosome damage in the forms of micronuclei. A 24 h recovery per iod is
required in Trad-MCN assay to allow the treated early prophase I meiotic
cel1s to arrived at the tetrad stage (the four-cell stage at the end of
meiosis). At the end of the recovery period, flower buds are fixed in
Aceto-alcohol (1:3 ratio), and stored in 70% ethanol after 24 h fixation.
The fixed material can be used to make slides of the early tetrad stage
using aceto-carmina squash method for scoring of micronuclei frequencies.
The detailed procedure was described in a separate publication~
About 15 cuttings are needed to constitute an experimental group in
Trad-MCN bioassay, and about 20 cuttings are necessary per experimental in
Trad-SHM bioassay to provide the adequate population size for analysis of
statistical significance. Analysis of variance (ANOVA) F test and Dunnett's
t-statistic are commonly used to determine the significant diffeences among
a control groups versus aseries of treated groups in a given experiment.
Recent development of the Tradescantia-Micronucleus Image Analysis
system'" for scoring MCN frequencies and data analysis provides and automated
tool to increase the efficiency and reduce the human error or bias in data
collection. The proficiency tests 3 on the prototype of this machine proved
that the system is a time and labor saving and reliable device capable of
handling a large scaled experimentation with the least manual labor
involved.

DATABASE AND DISCUSSION

The current database covers the studies of in situ monitoring of the

gaseous mixtures at the outdoor and indoor sites, the in situ monitoring of
liquid mixtures. The studies on the pure gases in the controlled chambers,
and the wastewater or drinking water and the solution extracted from the
soil samples collected from the polluted sites are also reviewed. Recent
bioassay on seven chemicals selected from the Super fund Priotority 1 list
and earlier studies on the effects of intern al and extern al radiation,
especially the in situ monitoring at the nuclear power plants are included
In order to corroborate the efficiency and suitability of this dual system.
The location of the sites monitored, the sources of the samples collected
for laboratory tests and the references for each of these studies are listed
in Table 1.
Based upon the test results of the common environmental pollutants,
both Trad-MCN and Trad-SHM bioassays can detect very 10w concentration of
chemicals at uM level, and low dose of radiation at the pCi and mR levels.
This dual bio-monitoring system could be claimed to be the most sensitive
and easy to operate as well as cost effective~ one among the well known in
situ bio-monitors". The plant cuttings can survive in the in vitro
conditions for indefinite length of time and function as the intact live
plants. Thus the plant cuttings used for testing are the portable in vitro
test materials but at the same time they can provide effectively the results
comparable to the data obtained from the in vivo tests.

248
Tab1e 1. Literature pertaining to the studies on the ~ monitoring of
po11utants and 1aboratory testing of samp1es co11ected at the site of
pOllution, as we11 as the chemica1s common1y found at the hazardous
waste site.
Major groups Type of Site location Reference
of pOllution assays type of po11utants cited
Gas mixtures
Outdoor in situ Bus depot, PRC, US 21,23,28
monitor Parking garage, US 21,23,28
Trad-MCN Petro company, US 23,28,36
Trad-SHM Truck stops 21,23,28
Industrial district,
US 17,28,48,
49,50,51
PRC 9,23,28
Mexico 33
Japan 56
Indoor in situ College Dormitory 33
Trad-SHM Smoking room 23,28,31,
33
Trad-MCN Radon polluted house 9
Trailers 31
Chamber in situ Ethylene dibromide, EMS 18,44,53
Trad-MCN Pesticides Ma1athion 6,19,25,
28,43
Trad-SHM Formaldehyde fumes 43
Air fresheners 9,31
S02, 03, N02 23,53
Diesel exhaust fumes 22,23,26
Liquid mix
Wastewater in situ Lake Superior, Canada 3
Trad-MCN Sea water, PRC 3,4,5,7,
11
Trad-SHM Sludge, Chicago, US 12
Mexico 46
Wastewater Lab test Arena Canal, Mexico 37,45,46
Fujian, PRC 15,59
Tapwater Lab test US, PRC, Austria 16,27
Sha110w well, Lewistown 10,32
Super fund
chemicals Lab test Lead tetra acetate 35,39,47
Trad-MCN Tetrachlorethylene 35,47
Aldrin, Dieldrin 35,47
Benz(a) anthracene 28,35,47
Arsenic, Heptachlor 33,35,47
IPCS chemicals 39
Pesticides 43
Trad-SHM other mutagens, Japan 56
IPCS chemicals & others 28,33,40
Radiation
In situ
Trad-SHM Nuclear plant, Japan 13
Internal Trad-MCN P-32, H-3, 1-131 1,28,54,
55
External Trad-SHM X-rays 19,20,21,
44,52
Trad-MCN Gamma rays, Neutron 2,44,52
Trad-SHM Soil, Bikini Island 14

249
 Results of this dual system can elucidate the relationship between the
chromosome damage and gene mutation. The damage on the meiotic chromosomes
in the gametes of the Trad-MCN system can also serve as the indicator of
the genetic effects which may be passed on to the future generations. This
dual bio-monitoring system is specially suitable for monitoring hazardous
industrial waste sites where fumes, contaminated water and soil can be
monitored in the form of mixtures This bio-monitoring process accompanied
with chemical analysis would also be able to isolate the prime hazardous
agents from the mixtures. Application of this kind of bio-monitors to the
industrial waste site prior to the costly chemical analysis would be able
to rank the relative degree of hazardous conditions of many waste sites,
and set the priority for the abatement operations. This would make the waste
site removal operation more effective and economical.
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250
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251
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applicable to chemical mutagenesis, in: Chemical Mutagenesis:
Principles and Methods for their detection, Vol. 3, A. Hollaender
(Ed.), Plenum Press, NY pp.171-207(1973).
58. J. Xu, T. H. Ma, W. Xia, X. Jong, W. Sun, 1989, Image analysis system
for rapid data processing in Tradescantia-Micronuc1eus bioassay,
In: Plants for Toxicity Assessment, Amer. Soc. Test. Materials
(ASTM) Publ. Wang, Gorsuch and Lower (Eds.) STP 1091, ASTM
Philadelphia, PA pp. 346-356, 1990).
59. D. Zheng, Tradescantia-Micronucleus tests on the industrial
wastewater from a printing and dying factory in Fuzhou city. J.
Fujian Normal University, 21:5-7(1985).

253
ANIMALS AND PLANTS AS BIOINDICATORS OF RADIONUCLIDE
CONTAMINATION IN FOREST ECOSYSTEMS

R. Thomas Palo
Departrnent of Animal Ecology
Swedish University of Agricultural Sciences
S-901 83 Ume, Sweden

ABSTRACT
Risk assessment of radiological situations, such as nuclear accidents, would be
significantly enhanced by the analysis of organisms having the ability to accurately
reflect a deposition pattern. The ideal bioindicators would posses such properties as
high accumulation potential and reliable reflection of adeposition from which long and
short-term effects could be predicted. This is especially important when studying
natural ecosystems where transfer of contaminants are subtle. Unfortunately, few plants
and animals in the forest ecosystem fulfill these criteria. Organisms in natural
ecosystems are engaged in complex interactions that ren der data inconclusive. In order
to resolve ambiguities, we need to understand the ecology of each specific organism in
great detail. Without this knowledge, predictions of distribution pattern and changes
with time will remain inaccurate.
This paper investigate plant and animal species as potential indicators of
radionuclide contamination in boreal forest.

INTRODUCTION

The Chernobyl nuelear aecident aeeentuated the need for information about the
distribution of radionuelides in natural eeosystems. Radionuelide behaviour in forest
eeosystems is fundarnentally different from agro-eeosystems. Likewise, transfer data from
agro-eeosystems are not eompatible with those from forest eeosystems. Envirorunental
transfer of radionuelides has principally been studied in agrieultural eonditions, and models
have been constructed primarily using results from these studies (Desmet et al., 1990). Thus,
at the time of the Chernobyl accident, there was an apparent lack of data that could have been
usefull in models of forest eeosystems and for prediction of human exposure.
Forest ecosystems are considerably valuable to humans. They provide important
commodities such as wood, paper, recreation, berries, mushrooms, and garne animals. This
significant supplier of natural resourees is not without its perils. In Sweden, for exarnple,

UStl of Biomarkers in Assessing Health and EnvironmenUll Impacts of Chemical

PollulDllts, Edited by C.C. 'fiavis, Plenum Press, New York, 1993 255
consumption of game and wild berries are the major pathways of radionuclides from the
forest ecosystem to humans. Moreover, contaminants such as Cs137 have been shown to be
prevalent in the forest ecosystem for long periods of time.
A good bioindicator of radionuclide contamination should preferably have some of the
following properties. It should be easy to collect and evaluate, an accurate indicator of
contamination levels, and usefull as a predictors of short and long term effects. In this paper I
discuss animal and plant species as potential bioindicators of radionuclide contamination in a
forest ecosystem.

Table 1. List of typical mammalian species in the boreal forest of Sweden and their way of
living. Type:l=insectivorous, C=carnivorus, H=herbivorous, Occurrence: Ab=abundant,
Co=common,Un=uncommon

Species Type Occurence

Sorex araneus 1 Co
S. minutus I/C Un
S. minutissimus 1 Un
S. sinalis <fodiens) 1 Un
Neomys fodiens I/C Un
Microtus agrestis H Co
Clethrionomys glareolus H Ab
C. rufocanus H Co
Arvicola terrestris H Co
Myopus schisticolor H Un
Apodemus flavicollis H/I Co
Castor fiber H Co
Sciurus vulgaris H Co
Capreolus capreolus H Co
Lepus timidus H Ab
Rangifer tarandus H Ab
Alces alces H Ab
Vulpes vulpes C Co
Ursus arctos H/C Un

Animals as bioindicators

There are about 25 mammalian species commonly found in the Fennoscandian, but the
total number of vertebrates is much higher. Table 1 shows mammalian herbivorous and
carnivorous species in the Swedish boreal forest that potentially could serve as bioindicators.
The moose (Alces alces) constitute the most important hunting bag in Sweden. About
130, 000 animals were shot there in 1991. This corresponds to 20xl06 kg meat for

256
consumption. This large herbivore forage over relatively large areas and feed mainly on
woody shrubs and trees. About 80 percent of its diet in autumn is composed of the genus
Betula spp. Salix spp. and the species Vaccinium myrtillus (Cederlund et al.; 1980, Zach et
al., 1982; Palo and Wallin 1993). Few animals consume a large proportion of any one plant
species, as most have a rather mixed diet. Although, diet is the only source of uptake of
radionuclides, no relationship between the proportion of specific plant species in the rumen of
moose and activity concentration in flesh have been found (Palo et al., 1991). This is
probably due to a time lag between actual diet and manifestation in the flesh.

8
b
7

ii)
(1) r 2 = 0.44***
."
111
6 (2) r 2 = 0.51***
E
J:.
11)
5
!
CI

~ 4
'!!.
.E
3

I
1.5 2 2.5 3 3.5 4 4.5
In(KBq/m2 )
Figure 1. Regression of es 137 concentration in moose meat against ground deposition at site of collection.
Data for 1987. Calves (-) equation In Y=1.01In x +In 2.68, r=O.66, p<O.OOI, adult (---), In Y= 0.75 In x + In
2.35, r=O.75, p<O.OOI (Modified from Palo et al., 1991).

Tbe activity concentration in moose meat varied unsystematically between individual

animals and years. During the hunting seasons in September 1986, 1987, 1989, 1991 and
1992 activity concentrations were comparable, with a mean of 300 Bq/kg (Median=253,
SD=250). Tbe years 1988 and 1990 showed dramatically higher levels than other years (Palo
et al. 1991). Tbe activity concentration in 1988 was.640 Bq/kg in adults and 1300 Bq/kg in
calves. Corresponding figures in 1990 were 514 Bq/kg in adults and 1120 Bq/kg in calyes.
Generally, calves showed about 40% higher activity concentrations than adults.

257
 A multivariate linear estimation with Cs-137 concentration in moose meat as the
dependent variable and age, sex, year and ground deposition at the site of collection as
covariates showed that the total variability in Cs-137 concentration is largely explained by
deposition at the collection site (Figure 1). The best relationship was found for calves in 1987
which explained 57 percent of the variation in meat concentration. However, the relationship
was less apparent in other years. Age showed a negative relationship supporting the higher
levels found in calves.

6400

~ 3200
er
e


'0 1600
c:
.2
~
C

1lc:
o
u 800

400
9 11 13 17 20

Ground deposition (kBq 1m 2 )

Figure 2. Relationship between activity concentration of es 137 in bank vole and ground deposition at site
of collection. Tbe figure show mean values for sites and logaritmic scales on both axes. Tbe regression
equation Y=O.858+2.396lnx, r=O.443, p<O.OOl

Bank: vole (Clethrionomys glareolus) and the grey-sided vole (C. rujocanus) are small
rodents common in the boreal forest. These marnmals show pronounced periodic fluctuations
in numbers with a peak population density about every fourth year (Hrnfelt 1978; Hansson
and Henttonen 1988). They cover a horne range about 0.2 ha in size and would reflect
deposition in a small area (Larsson and Hansson 1977).
At the time of the Chemobyl accident the bank: vole population was about to increase and
peaked in 1987. The grey-sided vole peaked in 1988. However, the number of trapped grey-
sided vole was much lower than for bank: vole, irrespective year of the collection.

258
 The activity concentration in bank vole varied between years and between sites. The
highest concentration was found in 1988 with a mean of 1485 Bq/kg (SD=881). Vo1es in
clear cut sites showed about one third of the activity concentration found in voles in mature
forest. No relationship between body mass and activity concentration was apparent. A
stepwise linear regression eliminated age, sex, and body mass from an explanatory model for
Cs-137 concentration in voles. The year of collection and the amount of deposition explained
most ofthe variation in Cs-137 concentration in these small mammals (Figure 2).
The mean activity concentration of Cs-137 in the grey-sided vole was 877 Bq/kg
(SD=847) and for Cs-134 it was 259 Bq/kg (SD=257) during the period. No significant
differences in Cs-137 activity was found between the two species.

5000 0
0
0
4000

~
~

c 0
E 0 0
>-
.t; 3000 0
...
Cl
-"
<T
8
0
~

"'" 2000
~

1000 1989
o 1986

0
0
0 30 60 90

Ground deposition Cs-137 (kBq 1m 2 )

Figure 3. Regression of Cs 137 activity concentration in bilberry against ground deposition. Equations (1986)
Y=O.048x+1200, r=O.56, p<O.Ol, (1989) Y=0.0093x+350, r=0.42, p<0.05 (Data from NeUn and Ny1en 1993).

Plants as bioindicators

Characteristic of the boreal forest is its dominance of coniferous tree species, short
vegetation period, and relatively high range of temperature fluctuations. The Scandinavian
boreal forest is dominated by Picea abies and Pinus silvestris in which is mixed such
deciduous species as Sorbus aucuparia, Populus tremula, Betula spp. and Salix spp. Forest
ground vegetation in poor habitats is dominated by lichens (Cladonia spp.)and shrubs such as
Calluna vulgaris and Empetrum hermaphroditum while more rich habitats contain mainly

259
mosses (Dicranum spp., Pleurocumbens schreberi, Hylocomnum splendens) and Vaccinium
spp.
Many of these plant species are important food sources for herbivores. Early successional
species such as birch (Betula spp.) and aspen (Populus tremula) benefit from forestry
practices and are the species prefered by moose.
Bilberry (Vaccinium myrtillus) is an important food supplement during autumn while
young pines growing over large areas provide moose with high quality sustenance during
winter. Moreover, bilberry is extensively picked by humans for consumption. The
radionuclide concentration and turnover rate in these plant species are very important
considering their wide application as food sources.
In Vaccinium myrtillus there is a rapid dec1ine of Cs-137 concentration with time (Palo
et al. 1991; Ne1in and Nylen 1993), In contrast, concentrations in birch (Betula pubescens)
did not dec1ine significantly after 1986. Qnly a weak relationship between activity
concentration in these species and ground deposition was found (Figure 3).
Forest floor vegetation such as lichens and bryophytes receives radionuclides from
several diffrent sources. Apart from direct fallout via dry and wet deposition they also are
contaminated by litter fall, leaching from trees and the excrement left by herbivores.
Bryophytes and lichens could be considered cesspools of radionuclides in the forest that
significantly reduce nuclide turnover rate in the system.
The position of these plant taxa in the forest make them potential bioindicators capable of
refelecting total deposition of radionuclides. Nimis et al. (1986) showed that the moss
Clendium molluscum growing on calcarious rocks in northem Italy was an effective predictor
of ground deposition in that area. The mosses in the boreal forest showed a significant
relationship to activity in soil sampies, but the variation is poorly explained by the
relationship (Figure 4a). Another important relationship exist between Cs-137 per square
meter dry moss and water content in mosses (Figure 4b ).

DISCUSSION

The suitability of an organism as a bioindicator of radiocontamination is dependent on

several factors such as their availability, distribution, and general ecology of the species. The
pathways, spatial distribution, and time perspective of contaminants in the forest are in turn
dependent on the successional status, species composition, and productivity of the system. For
example, the type of vegetation is probably the most important variable controlling the input
of airborn pollutants into the forest ecosystem. At the individual plant level factors, such as
plant height, foliage density, leaf area index and surface characteristics are important
variables (Droppo 1976). Further, the proportion of ground cover and canopy height are other
factors to consider. In the boreal forest most plants are poor predictors of contamination
levels as concentration changes rapidly with time and geographical variation is large. The
complexity of the problem is indicated by the relationship between concentration of
es 137/m2 in dry moss and water content/m2 . This relationship probably reflects differences
in total surface area of the moss and associated water absorption capacity. However, more
than 90 percent of the activity in plants in the field layer are found in mosses.
Uptake in animals is equally complex and estimated transfer coefficients are often
missleading. Ultimately food composition and the position in the food chain determine
transfer rate. Types of food consumed vary greatly with animal species and respective habitat.
The proportion of plants in the diet change continually according to daily foraging decisions
and habitat use. Shlfts in eating patterns and crossing of habitat boundaries may be important
events affecting the uptake of radionuclides. For example, consider two habitat types with
different availabillities of forage. At the inferiour habitat with a low reward rate the animal

260
 15000
a
i
~

"0-
.:S
"- lOOOO 0
~ 0 0
0
'0
"
.~
"
C 5000
~

u
"
0

0 0
0
0
0
0
10000 15000 20000 25000 30000

Ground deposition (Bq/m 2 )

40000
b

0
30000
~

E
>-
~

'".....E
<T
co
.....
~
:.. lOOOO
u

10

Water content i moss 1m 2

Figure 4. a) Relationship between activity concentration in dry moss and soH activit at the site of collection.
Equation Y=0.247x +1172, r=0.44, p<0.035. b) Regression of deposition per square meter in dry moss with
water content in moss per square meter. Y=2449x + 170, r=O.76, p<O.OOOI, N=34.

261
might feed for longer time in that habitat. Alternatively, in a rich habitat the feeding time may
be shorter, but the harvesting rate will be higher, thus faciliating a higher uptake. All these
events render transfer coefficients into crude estimates that would be more realistically
expressed as transfer functions. It is difficult to obtain corroborating statistics from data
collected on animals due to the many variations among and within species. Incongruities will
not be easily resolved without detailed understanding of the ecology and the species involved.
The selection of animals and plants as pollution indicators must be done with great care
in order achieve information with high reliability. In forest ecosystems only a few animals
and plants are suitable for this purpose. Suitable animals are moose and voles which occur in
suffieient numbers to be easily collected. Among plants bryophytes an lichens are potential
candidates. However, in order to understand uptake and translocation of radionuclides and
other pollutants, we need to develop speeies specific models in radioecology. These models
should enable us to explain distribution of contamitants in particular animal populations
without losing site of the ecosystem in general.

Acknowledgement

1 thank K. Danell, and C. Travis for valuable comments on the manuscript. M. Simmons
improved the english. Thanks also to P. Nelin and T. Nylen for providing unpublished data.
This work was partly supported by Center for Environmental Research, CEC Radiation
Protection Programme and the Swedish Radiation Protection Institute.

REFERENCES

Cederlund, G., Ljungqvist, H., Markgren, G. and Stlfelt, F. 1980. Foods ofmoose and roe
deer at Grims in central Sweden. Swed., Wild1. Res. 11:171-247.

Desmet, G., Nassimbeni and Belli, M.,1990, Transfer of Radionuc1ides in Natural and Semi-
natural environments. Elsevier Science Publishing. New York(1990).

Droppo, J.G. 1976. Dry deposition processes on vegetation canopies. In: Atmospheric-surface
exchange ofparticulate and gaseous pollutants. (Eds. R.J. Engelman and G.A. Schmel)
US/ERDA Sero Conf. 740921,1974, Springfield. Va. Natl. Tech. Info. Servo 104-111.

Hansson, L. and Henttonen, H. 1988. Rodent dynarnic as a community process. Trends in

Ecology and Evolution 3: 195-200.

Hrnfelt, B, 1978, Synchronous population fluctuations in voles, small game, owls and
tularemia in northem Sweden, Oecologia 32: 141-152.

Larsson, T. and Hansson, L. 1977. Vole diet on experimentally managed afforestation areas
in northern Sweden. Oikos 28: 242-249.

Nelin, P. and Nylen, T. 1993. Factors influencing the change over time in Cs 137 1evels in
boreal forest plants in Sweden. J. Sei. Tot. Env. (accepted for publication).

Nimis, P-L., Giovani, N. and Padovani, R. 1986. La contarninazione de Cesio-134 e Cesio-

137 nei macromieceti deI Friuli-Venzia Giulia ne11986. Studia Geobotanica 6: 3-63.

Palo, R. T., Nelin, P., Nylen, T. and Wickman, G. 1991. Radiocesium levels in Swedish
moose in relation to deposition, diet and age. J. Env. Qual.20:690-695.

262
Palo, R. T. 1992. Radioactive caesium in aboreal forest ecosystem. Ecological concept in
radioecology. In: (Ed. L. Moberg) The Chemobyl fallout in Sweden. SSI, Stockholm.
p.457-466.

Palo, R. T. and Wallin, K. 1993. Diet breadth and radionuclide dynamic in moose. Submitted
manuscript.

263
TUMOR MARKERS IN EFFUSIONS : A COMPARATIVE STUDY OF TUMOR
MARKER LEVELS IN SERA AND EFFUSIONS

Ibrahim Gullu l , Suayip Yalcin l , Gulten Tekuzman l , Ayse Kars l ,

Esmen Baltaljl, Nilufer Gulerl,lbrahim Baristal, Dincer Firat l ,
Coskun Bekdik2 , Zehra Koray 2

Hacettepe University, School Of Medicine, Departments Of Oncology l

And Nuclear Medicine 2
Ankara, Turkey

SUMMARY

The tumor markers; CEA, CA 19-9, CA 125, CA 15-3 levels in effusions caused by
some common malignant tumors, such as breast, gastrointestinal tract and ovarian cancers
were compared to the serum levels of the same group of patients. Thirty three patients were
included in the study group. Comparison with benign effusions (12 patients) was also
carried out. Corresponding tumor markers designated for each tumor type were analyzed
besides CEA which was measured in all patients. Although we got more positive results in
malign effusions than the sera, this difference was not statistically significant (p>0.05). But
mean values of tumor markers were found to be statistically higher in malign effusions when
they are compared to the sera of the same patients and to the benign effusions of control
group (p < 0.05). As a result, determination of tumor markers in the effusions is more
sensitive than in the sera and this method is also useful for differential diagnosis of benign
and malign effusions.

Application of tumor marker levels in the diagnosis, treatment and follow-up of

malignant tumors is not a new concept. Serum acid phosphatase was utilized in evaluation
of men with cancer of prostate as early as 1938(1). Later, Bence-Jones protein was described
as a marker of Multiple Myeloma in 1948(2). Today many different tumor markers (such as
CEA, AFP, CA 125, CA 19-9, CA 15-3) have been proposed as clinically useful in
screening, diagnosis ;md patient management. However some of these markers not only
increase in malignant but also in nonmalignant diseases. Generally these markers are
measured in sera of patients, but are rarely investigated in effusions(1-8).

Key words: Tumor Markers, CEA, CA 19-9, CA 125, CA 15-3, Malign effusion, Benign
effusion

Use 0/ Biomarkers in Assessing Health artd Environmentallmpacls 0/ Chemical

PollutanIs, Edited by C.C. Travis, Plenum Press, New York. 1993 265
 In the present study we detennined some tumor marker levels; CEA, CA 19-9, CA 125,
and CA 15-3, in effusions caused by some common malignant tumors, such as breast,
gastrointestinal (GI) tract and ovarian cancers, and compared them with serum levels of the
same group of patients. Comparison with benign effusions was also carried out.

MATERIALS AND METHODS

Thirty-three patients with histopathologically diagnosed breast, over and GI tract

(stomach or colon) cancer and a control grOUp consisting of 12 patients with benign
diseases were included in the study (Tables 1. 2). In the study group; 13 patients had
ovarian cancer, 11 had stomach or colon cancer and 9 had breast cancer. In the control
group tumor marker levels were measured in either pleural or peritoneal effusions of 6
patients with chronic Iiver disease, 3 patients with nephrotic syndrome. 2 patients with
congestive heart failure and one patient with tuberculosis. In the study group an
accompanying benign pathology was ruled out on the basis of history, physical examination
and laboratory studies as an inappearent cause of the effusions in the patients with
malignant tumors. Blood and the effusion sampies were obtained simultaneously before the
treatment. Corresponding tumor markers designated for each tumor type were analyzed.
besides CEA which measured in all patients. Tumor marker levels were assessed by means
of immunoradiometrie commercial assays (Table 3)
Mann Whitney U and Chi-square tests were carried out for statistical analyses.

Table 1. Tumor localizations of the patients

Patients No
Ovarian cancer 13
GI ( stomach or colon) cancer 11
Breast cancer 9
Total 33

Table 2. Patients included in the benign control group

Control group No
Chronic Iiver disease {,
Nephrotic syndrome 3
Congestive heart failure 2
Tuberculosis 1
Total 12

266
 Table 3. The commercial immunoradiometric assay kits used and their accepted upper
limits
Tumor mark.er Kit used Accepted upper limit
CA 125 Immunoradiometric Assay of CA-125 33 U Iml
(CIS) France, ELSA CA 125

CA 19 - 9 Immunoradiome1ric Assay of CA-19-9 33 U Iml

( CIS) France, ELSA 19-9

CA15-3 Immunoradiometric Assay of CA 15-3 21.2 U I ml

(CIS) France, ELSA 15-3

CEA Amerwell CEA Assay. 5 ng/ml

Amersham, Amerlite, England

RESULTS

CEA and CA-125 levels were determined in the sera and effusions of 13 patients with
ovarian cancer (Tables 4, 5). Serum CEA levels were found to be elevated in five serum
sampies (38.46%) and in only two effusions (27.07%). The mean CEA concentration was
however found higher in the effusions compared to the sera (8.983.58 ng I ml in the
effusions .and 3.99O.99 in the sera ) but this difference was not found statistically
significant ep
> 0.05). When CA 125 levels were studied in the same group of patients, it
was found to be increased in the sera of six (46.15%) and the effusions of nine patients
(62.33%). The mean CA 125 levels were 78.8524.24 U/ml and 218.5354.76 U/ml
respectively. The difference was statistically significant (p < 0.05).

Table 4. CEA and CA 125 concentrations in the sera and effusions of patients with
ovarian cancer

CEA (ng/ml) CA 125 ( U I ml )

Patients Serum Effusion Serum Effusion
8.8 48.8 112 472
2 0.22 2.65 230.5 466
3 0.22 4.65 148.6 496.8
4 6.12 4.05 84.2 496.8
5 0.22 3.7 16.2 10.4
6 10.4 20.6 26.5 164.2
7 6.16 10.Q2 66.7 70.2
8 1.09 4.02 266.7 216.4
9 1.3 4.02 10.6 228.9
10 1.6 4.06 22.2 31
11 6.1 3.06 15.3 18.2
12 1.04 4.05 10.4 148.6
13 0.92 3.14 15.2 21.4
Mean 3.39 0.99 8.98 3.58 78.85 24.24 218.53 54.76

267
 Table 5. The results obtained from the patients with ovarian cancer
No of positive values Percentage Mean values p value
CEA Effusion 3 23.07 8.98 P > 0.05
Serum 5 38.46 3.39
CA 125 Effusion 9 69.23 218.53 p< 0.05
Serum 6 46.15 78.85

CEA and CA 19-9 levels were measured in the sera and the effusions of 11 patients
with GI tract cancer (Tables 6, 7). lncreased CEA levels were detected in seven sera
(63.63%) and nine effusions (81.81%). Mean CEA concentrations were 14.55.64 ng/ml in
the sera and 72.3123.68 ng/ml in the effusions (p<0.05). In the same group CA 19-9 levels
was high insix sera (54.54%) and ten effusions (90.9%) The mean CA 19-9 concentrations
were 37.507.33 U/ml in the sera and 81.408.60 U/ml in the effusions so that the mean
CA 19-9 levels was statistically high er in the effusions (p<O.OOl).

Table 6. CEA and CA 19-9 concentrations in the sera and effusions of patients with GI
tract (stornach or colon) cancer

CEA (ng/ml) CA 19-9 ( U I ml )

Patients Serum Effusion Serum Effusion
1.14 2.51 63.2 107.9
2 16.2 60 70.4 120
3 7.3 14.4 16.4 82.3
4 0.5 0.5 16.2 17.5
5 4.94 200 32.8 107.9
6 6.06 19.5 10.6 60.6
7 66 184.6 82.3 80.6
8 1.04 21.9 17.5 92.7
9 14.4 184.6 41.2 63
10 19.5 66 38.2 93.2
11 21.9 42 SI 70.4
Mean 14.5:t 5.64 72.31 :t 23.58 37.5:t 7.33 81.40:t 8.60

Table 7. The results obtained from the patients with gastrointestinal (stornach or colon)
cancer

No of positive values Percentage Mean values p value

CEA Effusion 9 81.81 72.31 p< 0.05
Serum 7 63.63 14.5
CA 19-9 Effusion 10 90.90 81.4 P < 0.001
Serum 6 54.54 37.5

268
 CEA and CA 15-3 levels were determined in the sera and the effusions of nine patients
suffering breast cancer (Tables 8, 9). CEA levels exceeded the upper limit accepted for the
normal range in six sera (66.6%) and seven effusions (77.7%). The mean CEA
concentrations were 8.022.49 ng/ml in the sera and 24.106.44 ng/ml in the effusions
(p<0.05). CA 15-3 levels were beyond the upper limit in the sera of five patients (55.55%)
and effusions of seven patients (77.7%). The mean CA 15-3 concentrations were
24.015.71 U/ml in the sera and 97.8619.88 U/ml in the effusions and the difference was
statistically significant (p<0.01). CEA. CA 15-3, CA 19-9 and CA 125 levels were
determined in the effusions of 12 patients with benign diseases ( Table 10 ), CEA levels
were higher than normal in four patients (33.3%), while CA 19-9 levels were beyond the
upper limit in two effusions (16.6%) and so was CA 15-3 (16.6%). A higher than normal
CA 125 level was detected only one effusion. The mean concentrations were 4.76 ng/ml,
15.45 U/ml, 11.62 U/ml and 20.08 U/rnl respectively. These values were statistically lower
than the respective tumor marker levels in the rnalign effusions (p<0.01). Although the
mean concentrations of tumor markers in the rnalign effusions were found to be statistically
higher than the sera, the occurrence of positive results in all of the groups did not corne up
as statistically significant (p<O.05).

Table 8. CEA and CA 15-3 concentrations in the sera and effusions of patients with
breast cancer

CEA (ng/ ml) CA 15-3 (U I mI )

Patients Serum Effusion Serum Effusion
9.00 36.00 60.00 168.00
2 1.04 3.73 24.7 85.00
3 1.02 10.42 34.00 61.2
4 6.22 21.10 9.2 172.00
5 5.Q2 9.12 7.4 21.2
6 20.4 56.00 10.2 85.00
7 3.04 4.22 11.2 19.8
8 6.07 48.04 34.2 164.00
9 20.44 29.19 25.2 104.6
Mean 8.Q2 1: 2..49 24.101:6.44 24.01 1: 5.71 97.86 1: 19.88

Table 9. The results obtained from the patients with breast cancer

No of positive values Percentage Mean values p value

CEA Effusion 7 77.7 24.10 P < 0.05
Serum 6 66.6 8.02
CA 15-3 Effusion 7 77.7 97.86 p< 0.01
Serum 5 55.5 24.01

269
 Table 10. Tumor marker levels in effusions of patients with chronic liver disease (CLD),
congestive heart failure (CHF), nephrotic syndrome (NS) or tuberculosis (Tbc)

Patients CEA CA 15-3 CA 19-9 CA 125

CLD 4.2 16.2 2.2
CLD 7.2 11 31.2
CLD 0.5 22.5 4.4 30.8
CLD 1.07 16.5 34.1 28
CLD 5.5 20 10.2 11.8
CLD 4.9 10.8 7.7 38.2
CHF 4.06 30.2 46.6
CHF 0.5 10.8 16.7 29.5
NS 3.82 21.3 19.7
NS 1.94 21 34.2 8.5
NS 6.02 11.5 12.1 25.5
Thc 8.4 16.9 17.7
Mean 4.76:t1.09 15.45:t3.18 11.62:t4.06 2O.O86.60

DISCUSSION

Tumor markers have already proved to be clinically useful by many studies in

screening, diagnosis, monitorization of the treatment and follow up of the patients with
some malignancies'<I.2, 9-12) Studies on tumor markers are usually carried out in the sera and
tumor tissue of the patients, but occasionally in effusions.
In this study; CEA and carbohydrate antigens in sera and effusions of the patients
with ovary. gastrointestinal tract (stomach and colon) and breast cancer were examined.
These markers increase not only in malignancies but also in benign diseases. High levels of
CEA is seen in gastrointestinal tract carcinomas, inflammatory lesions, collagen diseases,
benign liver diseases, renal impairment and smokingincreased levels of CA 125 is detected
in carcinoma of the ovary, lung, breast, endometrium, cervix and chronic liver diseases. CA
19-9 increases in gastrointestinal carcinomas, inflammatory bowel diseases. pancreatitis,
liver and biliary diseases and CA 15-3 increase in carcinoma and benign diseases of breast
and hepatic diseases,<9,lO)
Second primary cancers or any other benign diseases were not present in any of our
patients.
In the group with ovarian cancer, the CEA in sera was found to be high in 5 patients,
however only 3 patients had high CEA level in effusion. Although CEA level in effusions
were two to six times that of in sera, there was no statistical significance between them. CA
125 level was found to be increased in the sera of six patients and in the effusions of nine,
the level in effusions being considerably higher than that in the sera (p<O.05). Of the 9
patients with low level of CA-125 in the sera, 3 had high levels of CA-125 in effusions.
There weren't any patients with high CA-125 in serum and normal in effusion.
Increased CEA levels were observed in seven sera and nine effusions in the patients
with gastrointestinal cancer and the levels were higher in effusions than in sera. There was
correlation between CEA levels in sera and effusions. In six patients there were increased
CA 19-9 levels both in sera and effusions, but twice much in effusions. Although CA 19-9
was normal in sera of four patients, it was found to be increased in effusions. Most of the
patients with high CEA had high CA 19-9 as weIl (Table 6).
Six patients with breast cancer had high CEA levels both in sera and effusions. In
effusions this increase was observed to be two to eight times that in sera. CA 15-3 level of

270
three patients were high both in sera and effusions. On the other hand four patients had
high CA 15-3 levels in effusions. but normal levels in sera. The difference of mean CA 15-3
level in sera and effusions was statistically significant (p<O.01).
All of these observations show that CEA and carbohydrate antigens are found with
higher incidences and higher quantities in effusions. Tumor marker concentrations of
effusions were statistically higher than in sera and effusions due to benign causes. This
finding can be explained by the antigenic stimulation of the tumoral involvement of the
surrounding cavity. If a tumor causes an effusion. it is probable that it will give its antigenic
determinants into the fluid. Studies have shown that peritoneum and basement membrane
of a tumor form a partial barrier to macromolecules{14. 15), This may partially explain the high
levels of tumor markers in malign effusions. We suggest that determination of the tumor
markers in the effusions can be useful in diagnosis of malign diseases. We believe that the
studies including larger groups of patients are necessary to achieve more significant results.

REFERENCES

1. Schwartz MK: Tumor markers; Whafs their role. c.wc<'r uJvt:Sl{i(atioIJ, 8: 439-440 (1990).
1. Schwartz MK: Tumor markers in Diagnosis and Screening. !n Human Tumor Markers. Ting SW. Ches JS.
Schwartz MK (eds) Amsterdam. Elsevier Science Publishers. pp: 16-58. (1989).
3. New tumor associated antigens. 2nd Symposium on tumor markers. Hamburg. Greten H. et al (eds) Verlag
Stuttgard-New York.1986. pp: 16-86 (1984).
4. Van Nappel JR. Donaldson ES. Hanson MB. et al: Biochemical markers in the plasma and tumors of patients
with gynocological malignancies. c.wc('J', 48:485-503, (1981 ).
5. Quentmeier A. Mller P. Schwartz V. et al: CEA. CA 19-9 and CA 125 in nonnal and carcinomatous human
colorectal tissue. Cancer, 60: 2261-2266, (! 987).
6. Chedid A. Chejfec G. Eichorst M. et al: Antigenic markers of hepatocellular carcinoma. c.wcer, 65: 84-87.
(1990).
7. Garret PE. Kurtz JR: Clinical utility of oncofotal proteins and honnones as tumor markers. Mcd Gin NortIJ
Am. 70: 1295-1306. (1986).
8. Torosian MH: The clinical usefulness and limitations of tumor markers. SUIl? GynecolObstet. 166: 567-579.
(1988).
9. Oaar AS. Lennox ES: Tumor Markers and Antigens. In Tumor Markers in Clinical Practice. Oaar AS.
Woodruf M (eds). Blackwell Scientific Publications. Oxford. pp: 1-26, (1987).
10. Rustin GJS: Circulating Tumor Markers in the Management of Human Cancer. In Tumor Markers in Clinical
Practice. Daar AS, Woodruf M (eds). Blackwell Scientific Publications. Oxford, pp: 56-96. (1987).
11. Lahousen M. Stettner H, Pickel H. et al: Tbe predictive value of a combination of tumor markers in monitoring
patients with ovarian cancer. Cancer, 60: 2228-2232. (1987).
12. Bac OJ. Kole TC. Goast VA. Spider TA: Evaluation of CA 19-9 senlm levels for monitoring disease activity
during chemotherapy of pancreatic adenocarcinoma. J C.vICer Res Gin Oncol, 117: 223-265. (1991 ).
13. Sakamato K. Haga Y. Yoshimura R: Comparative effectiveness of the tumor diagnostics. CA 19-9. CA 125
and CEA in patients with diseases of the digestive system. GII!, 28: 323-32. (1987).
14. Daar AS, Lennox ES: Circulating Tumor Markers. In Tumor Markers in Clinical Practice. Daar AS. Woodruf
M (eds). Blackwell Scientific Publications, Oxford, pp: 17-19, (1987).
15. Hili R, Daunter B, Silburn PA, et al: Affinity chromatography separation of tumor associated antigen from
ascidic fluid of ovarian cancer patients. Gynr.colOncol, 67: 414-416, (1986).
16. Fleuren GJ. Nap M. Aalders JG. et al: Explanation ofthe limited correlation between tumor marker CA 125
content and serum CA 125 antigen levels in patients with ovarian tumors. Cancer, 60: 2427-2442. (1987).
17. Shibate S: Basement Membranes. In Proceedings of the International Symposium on Basement Membranes.
Shibate S (ed) Amsterdanl. Elsevier Scientific Publishers. pp: 6-36, (1988).

271
EVALUATION OF CERULOPLASMIN LEVEL IN
WOMEN WITH BREAST DISEASE: PRELIMINARY RESULTS

Ozgur Ozyilkan, Esmen Baltali, Ayse Kars, Gulten Tekuzman, Dincer Firat

Institute Of Oncology
Hacettepe University,
School Of Medicine
06100 Ankara. Turkey

ABSTRACT

In this study, the correlation between the ceruloplasmin and CA 15-31evels in breast
diseases was investigated. The average ceruloplasmin levels of 29 patients with active breast
cancer and 22 patients in remission were 82461 mg/L and 63018 mg/L, respectively.
The average ceruloplasmin level of 17 patients with benign breast diseases (BBD) was
55529 mg/L and of 18 healthy women in the control group it was 58417 mg/L. The
patients with active disease were found to have ceruloplasmin levels which were
significantly increased when compared to the other 3 groups (p= 4.500E-12). The CA 15-3
and ceruloplasmin levels were found to have a positive correlation Cr= 0.57). The sensivities
and specificities of both markers were similar. therefore, we propose that ceruloplasmin may
be used as a tumour marker in the follow-up of patients with breast cancer.

INTRODUCTION

Specific and highly sensitive biological markers for breast cancer, which can predict
the progression of disease. are needed 1 Classically, such markers are synthesized by the
tumor cells and released into the circulation but they mayaIso be produced by normal
tissues in response to invasion by cancer cells 2 These markers may include a variety of
enzymes, hormones and antigens but, to date, most have been used in conjunctien with
ether tests for determining the disease state, response to surgery, chemotherapy and
radiation 3
A recently discovered breast cancer antigen, CA 15-3 has been identified by
reaction with two monoclonal antibodies 4 In clinical studies S-7 , it was found to have a
higher sensitivity to breast carcinoma metastases with higher sensitivity than the most
widely used marker carcinoembryonic antigen (CEA). Also serum copper and
ceruloplasmin (an acute phase reactant synthesized in the liver) have been reported to be

Use o[ Biomarkers in Assessing Health and Envirrmmentallmpacls o[ ChemicaI

Pollu/anls. Edited by C.C. Travis. Plenum Press. New York, 1993 273
useful markers of disease activity in patients with carcinoma of the breast, lung,
gastrointestinal tract, acute leukaemias and Hodgkin's and non-Hodgkin's lymphoma 8 -10
Ceruloplasmin mayaiso be elevated in nonmalignant conditions such as pregnancy,
thyrotoxicosis. cirrhosis. and infectionsl l
In this study, we have attempted to demonstrate the value of serum ceruloplasmin as a
marker of the disease activity in patients with breast cancer and show its correlation with
CA 15-3.

MATERIALS AND METHODS

This study includes 17 female patients with benign breast diseases (BBD), 51 female
patients with histologically documented carcinoma of the breast and 18 apparently healthy
female volunteers. All the patients were from the Department of Oncology at the Hacettepe
University Hospital. The control group was clinically negative for breast disease. Their
median age was 39 years (range 25-60). The histological examination of the BBD revealed 5
fibrocystic disease. 11 fibroadenoma and 1 papillorna. Median age of this group was 24
years (range 16-51). The median age of the patients was 43 years (range 23-63).
Twenty-nine out of 51 patients with breast cancer had metastatic carcinoma or were in
relapse, with the remaining 22 being in remission. Ten of the 22 patients in remission were
not receiving therapy whilst the remaining 12 were on maintenance therapy. Local therapy
included either a radicalor modified radical mastectomy plus radiotherpy. Patients who
were in relapse or had metastases and those on maintenance therapy were given CMF
(Cyclophosphamide 100 mg/m 2 /days i.v. days 1-14, methotrexate 40 mg/m 2 i.v. days 1 and
8, and 5-Fluorouracil 600 mg/m 2 days 1-8 (CMF) together with tamoxifen 20 mg/day or
only tamoxifen 20 mg/day).
None of the patients had laboratory or clinical evidence of infection, primary
inflammatory disease, thyrotoxicosis, pregnancy, cirrhosis or proteinuria when blood
sam pies were collected. Serum ceruloplasmin and C-reactive protein levels were measured
using a radial immunodiffusion technique (Binding Site, UK) and CA 15-3 levels by an
immunoradiometric assay technique (Centocor IRMA).
Difference of the means of ceruloplasmin and CA 15-5 levels between groups were
analyzed by one-way ANOVA. Posteriori pairwise comparisons were performed by student
two-sample independent groups t test, with downward adjusted p values (significance for
these comparisons was assigned to 0.05/6=0.008, because the total number of previous
comparisons was six)12. The relationship between the ceruloplasmin and CA 15-3 levels
was analyzed using linear regression analysis. The cut-off for positivity was established by
considering the results as positive above the mean values plus two standard deviations of
control subjects. Statistical comparisons of the sensitivities and specificities were performed
by chi-square test.

RESULTS

The results of the ceruloplasmin levels were separated into 4 groups-breast cancer
patients in remission, breast cancer patients not in remission, patients with BBD and
healthy volunteers- and are shown in figure 1. The results are expressed in mean standard
error of the mean. The average ceruloplasmin level of the 29 patients with active disease
(those having metastases, in relapse or newly diagnosed with carcinoma) was 82427 mg/L
(range 525-1075), while the 22 patients in remission had an average level of 63018 mg/L
(range 525-950).
The patients with BBD had an average value of 55529 mg/L (range 350- 750), and the
control group's average value was 58417 mg/L (range 475-700).

274
 Ceruloplasmln
(lng/LJ

1000

..'.
SOO ~

.:iOO ','
-+--
!
400

200

relapse remi;;sion benign eontrol

or hrea:s't grou;:
met.sttie di:5e3.sa

Figure 1. Ceruloplasmin levels in groups

The patients not in remission were found to have significantly higher levels of
ceruloplasmin compared with the other 3 groups (p:::4,500E-12).
The difference between the patients in remission, BBD, and control group was not
statistically significant (p>0.008). All women in the 4 groups had serum C-reactive protein
concentrations in nonnallimits, that is less than 6.0 mgIL. The average CA 15-3 level for the
29 patients with active disease was 88.613.9 U/ml (range 20.2-240). while for the 22
patients in remission the average level was 18.91.1 U/ml (range 11.3-34.1). The average
CA 15-3 level in 17 patients with BBD was 15.51.5 U/ml (range 3.2-26.1), and 16.11.0
U/ml (range 10.1-26.0) in the contro! group. There was a positive correlation between the
serum ceruloplasmin and the CA 15-3 levels (r=0.57) in breast cancer patients (figure 2).
The calculations of the sensitivity and specifity were based on ceruloplasmin and CA 15-3
cut-offs of 728 mgfL and 25 U/L respectively. The sensitivities of the ceruloplasmin and CA
15-3 were 75% and 86% and the specificities were 100% and 94%, respectively. The
positive predictive value of the ceruloplasmin and CA 15-3 were 100% and 96% and the
negative predictive value were 72% and 80% respectively (table 1). The differences in these
percentages were not significant (p>0.05).

Ceruloplasmin (mg/L)
1200 - - - - - - - - - .. - -.-- ----------1

400 ,'0.570
p<0.0001
200

O~------~--------~-----
o 50 100 150 200 250
CA 15-3 level (U/mJ)

Figure 2. Relationship between serum ceruloplasmin and CA 15-3 levels

275
 Ceruloplasm1n
(Ing/l)

1000

soo ~

...
600

400
1

- ','

200

rela.pse remi:>sio>l beni"r: c~ntrol

brea:s;" g ... ot.:;:
metastc..tic ciseas~

Figurc 1. Ccruloplasmin levels in groups

The patients not in remission were found to have significant!y higher levels of
ceruloplasmin compared with the other 3 groups (p=4,500E-12).
The difference between the patients in remission, BBD, and control group was not
statistically significant (p>0.008). All women in the 4 groups had serum C-reactive protein
concentrations in normal limits, that is less than 6.0 mgfL. The average CA 15-3 level for the
29 patients with active disease \"ras 88.613.9 U/ml (range 20.2-240). while for the 22
patients in remission the average level was 18.91.1 V/mi (range 11.3-34.1). The average
CA 15-3 level in 17 patients with BBD was 15.51.5 Vlml (range 3.2-26.1), and 16.11.0
U/ml (range 10.1-26.0) in the control group. There was a positive correlation between the
serum ceruloplasmin and the CA 15-3 levels (r=0.57) in breast cancer patients (figure 2).
The calculations of the sensitivity and specifity were based on ceruloplasmin and CA 15-3
cut-offs of 728 mg/L and 25 V/L respectively. The sensitivities of the ceruloplasmin and CA
15-3 were 75% and 86% and the specificities were 100% and 94%. respectively. The
positive predictive value of the ceruloplasmin and CA 15-3 were 100% and 96% and the
negative predictive value were 72% and 80% respectively (table 1). The differences in these
percentages were not significant (p>0.05).

Ceruloplasmin (mg/L)
1200r-----~----~~~----------------------------.

1000

600

400 "0.570
p<O.OOOl
200

o I---------'----------'-----~--'-----
o 50 100 150 200 250
CA 15-3 level (U/ml)
Figure 2. Relationship between serum ceruloplasmin and CA 15-3 levels

276
Table 1. Sensitivities and specificities of ceruloplasmin and CA 15-3 in patients with
metastatic or relapse breast carcmoma
Ceruloplasmin CA 15-3
(Cut-off=728 mg/L) (Cut-off=25 Ulml)
Sensitivity 75% 86%

Specificity 100% 94%

Accuracy for positive prediction 100% 96%

Accuracy for negative prediction 72% 80%

DISCUSSION

The diagnosis and dassification of human breast cancer is mainly based upon the
morphology of the lesion. which has its own difficulties and !imitations. The tumor markers
may be useful in the assessment of the diagnosis. stage, prognosis. monitoring response to
treatment and the early detection of metastases.UO.14-16
In a study by Schapira and Schapira 17 the ceruloplasmin levels were found to be
elevated in 89% of 103 patients with carcinoma and then decreased by 35% as soon as
patients responded to treatment. These results are in accordance with those obtained in our
study. We have shown that patients with carcinoma of the breast compared to patients in
remission. BBD. and normal healthy volunteers, displayed increasing levels of
ceruloplasmin, which correlated with the progression of disease. Schapira and Schapira
have shown that the ceruloplasmin levels of patients with beast carcinoma increased 16-34
weeks before their metastases were detected.
The role ceruloplasmin plays in oncogenesis is not dear, although. it is suggested that
it may be involved in angiogenesis and neovascularization at the site of tumour growth.
Breast cancer cell lines have been found to contain ceruloplasmin mRNA whilst normal
breast cells do not express this gene l8 So. we can speculate that patients with breast
carcinoma have higher levels of ceruloplasmin than normal healthy volunteers and patients
with BBD because of an extrahepatic production proportional to breast cancer cell
proliferation.
CA 15-3 levels were also shown to be increased in 60% of patients with metastases
while in patients without metastases this rate was 30% 19. In our hospital CA 15-3 is
usually use in the follow-up of breast cancer patients. When the CA 15-3 level is increased
the ceruloplasmin level is also increased. and there is a statistical correlation between these
two parameters in breast cancer patients. As far as we know, there is no report on the
correlation between the CA 15-3 and ceruloplasmin levels. and this correlation was
demonstrated for the first time.
The increase observed in the ceruloplasmin level in breast cancer patients may be
considered to be the result of acute phase reaction but in our study we found C-reactive
protein (another acute-phase reactant) in normal limits in the whole group.
From our study, it is seen that ceruloplasmin levels are increased in breast cancer
patients and are found to correlate \\1.th CA 15-3 levels. We have shown that in the
follow-up of breast cancer patients ceruloplasmin can be used as a tumour marker. In a
further study, serial determination of ceruloplasmin and CA 15-3 levels in patients with
breast cancer would be useful for evaluating the predictive role of ceruloplasmin in this
disorder.

277
REFERENCES

1. J.Hilkens, F.Buijs, Ph. Hageman, J.Calafat, A.Sonnenberg, M.van Der Valk, Monoclonal antibodies against
human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors,
IdJCancer. 34: 197 (1990).
2. S.E.Bates, D.L.Longo, Tumor marker: Value and limitations in the management or cancer patients, Cancrr
TrratRrvirws, 12:163(1985).
3. A.C.Hollinshead, Biological markers or breast cancer: A review, CancrJ fnvest. 5(6): 501 (1987).
4. MJ.Duffy, New cancer markers, Ann C/inBioe/Jt:D}, 26: 379 (1989).
5, MJ.Duffy, A.F.Sherry, Studies on CA 15-3, a new marker in breast cancer, C/ln.SC!: 71 (Supp 15): 28
(1986).
6. D.F.Haves, V.R.Zurawski D.W.Kufe. Comparison of circulating CA 15-3 and carcinoembryonic antigen levels
in patients with breast cancer, J C/iu Onco!. 4:1542 (1986),
7. c'Tondini, D.F.Hayes. R.Gelman, T.C.Henderson, D.W.Kufe, Companson of CA 15-3 and carcinoembryonic
antigen in monitoring the c1inical course 01 patients with metastatic breast cancer, Cancer Rrs. 48:4107
(1988).
8. M.Hrgovcic, F.Tessmer, F.R.Thomas, J.F.Gamble, C.C.Shullenberger, Serum copper observation in patient.
with malignant lymphoma, Cancrr. 6:1512 (1973).
9. S.N. Sinha, E.R. Gabriel, Serum copper and zinc levels in various pathologie conditions, Am.! CYlu PatIJ.
54:570 (1970).
10. A.C.F.Margerison, J.R.Mann, Serum copper, serum ceruloplasmin, and erythrocyte sedimentation rate
measurements in children with Hodgkin's disease, Non-Hodgkin's Iymphomaand non-malignant
Iymphadenopathy, Cancer. 55:1501 (1985).
11. J.Wallach. "Interpretation or Diagnostic Tests," Little, Brown and Company, Boston/Toronto (1986).
12. B.D. Saunders, R.G.Trapp. "Basic and Clinical Biostatistics. International Edition," A Large medical book,
NorwalklConnecticut (1990).
13. W.W.Daniel. "Biostatistics: A Foundation For Analysis in The Health Sciences," John Wiley & Suns, New
York (1978).
14. T.P.Wealkes, J.P.Enteriine, J.H.Shaper, M.D.Abeluff, D.S.Ettinger, Biological marker for breast carcinoma,
Cancrr. 53:644 (1984).
15. R.E.Smith, Biochemical detection of recurrent breast cancer, Cwcrr Drtrc ~t:nl. 11 :303 (1980).
16. G.SJotti, E.Bombardieri, Circulating tumor markers in breast cancer (review), Anticancrr Res. 10:253
(1990).
17. D.Schapira, M.Schapira, Use of ceruloplasmin levels to monitor response to therapy and predict recurrence or
breast cancer, BrrllStCwcerRes Trrat. 3:221 (1983).
18. S.P.Kanapuli, H.Singh, P.Singh, A.Kumar, Ceruloplasmin gene expression in human cancer ceIIs, LlIr
Scimcr, 40:2225 (1987).
19. J .Reuesse, M.Tubiana-Hulin, AFileul-Saint-Cloud. Breast Cancer, in: "Handbook of Chemutherapy in Clinical
ncology," J .P.Droz, E.Cvitcovic, J .P.Arrnand, S.Khoury, eds., FIlS, Houston (1988).

278
 CONTIBUTORS

DR. KENT ANGER DR. PETER GOERING

Oregon Health Sciences University
CROET, L606
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Agency for Toxic Substances & PROF.MAREKJAKUBOWS~
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Departrnent of Radiobiology DR. JOHN JARRELL
University of Stockholm Dept. of Obstetrics & Gynecology
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281
 INDEX

Absorption Cadmium, 73-82, 83, 96-98, 129

of xenobiotics, 139-141 Carcinogens, 57-58
Adduets 2-acetylaminofluorene, 57-58
carboxylie acid, 54-56 exposure to genotoxic, 53-62
cysteine, 53-54 potency of inhaled formaldehyde, 42-46
DNA, 1-6, 17-30, 41-46, 53-62, 148, 212, 242 Cancer, 1-8, 17-30, 47, 65, 209-226
hemoglobin, 1-6,9-16, 17-30,41,53-62 bladder, 9-15, 210
histidine, 54 breast, 210, 265-271, 273-278
levels, 18-26 liver,96
monitoring, 19, 26 lung, 101-104,210
protein, 17-30 ovaries, 104-120,265-271
as DNA dose monitors, 58 radiogenic, 3
relationship with tumorigenesis, 58-59 prostrate, 265
terminal valine, 56 stomach, 210, 265-271
Carcinoma
Biomarkers nasal, 42-46
application to hazard identification, 32-33 Ceruloplasmin levels
application to exposure assessment, 33-34 relationship with breast cancer, 273-278
application to dose-response assessment, 34 Cesium, 255-263
behavioral, 159-168, 179-180, 183-199
neurobehavioral,229-231 Dose
biological, 83-94, 106 background, 23-26
in assessing reproduetive risks, 137-158 blood, 23-26
downstream, 39 exposure,23-26
ecological, 237-245, 247-253, in vivo, 17
plants and animals as, 255-263 lifelime, 3, 81
environmental, 63-66, 67-72, 73-82, 83-94, 106, monitoring, 18-26
240-244 response, 31-46, 53-62
tissue esterases as, 177 relationship to Manganese, 81
of exposure, 35, 81-82,177,202 target, 4-5, 23-26, 35-36, 82
to organic compounds, 83-94 Dosimeter
of effeet, 9-15, 35-36, 202, 211 DPX as, 44
with a common cause, 39 of genotoxie substances, 53-62
in urine, 75-80, 83-94, 231-233 mutation spectra as, 47-51
iconography, 36-38 DNA, 47-51
immunotoxicology,201-207 abnormal, 242
prioritization of, 240-245 as a marker of effeel, 9-15
quantitative, 34-35, 43-44 protein cross-links see DPX
of heavy metallevels, 67-72 DPX,43-46
of susceptibility, 35-36, 202
of toxicity, 95-99, 101-104 Electromyograhpic
genotoxicity, 247-253 as a biomonitor, 178-179
neurotoxieity, 159-168, 169-182, 183-199, Eleclrophile, 3, 18-23
201-207, 209-226, 227-235 genotoxic carcinogens, 54
reproductive toxicity, 105-120,121-136, 137- Endpoints
158 cytogenetic, 1
upstream, 39 in epidemiological investigations, 26
Benzene, 54-55, 83-94 multiple, 37

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Ethylating agents Pathway
as a carcinogen, 57-58 biochemical, 48
Ethylene oxide Placenta transfer, 128-132
as a carcinogen, 57-58 Pharmacokinetics, 121-136
Ethene, 23-26
Extrapolation, 31-46, 73-82, 125-126, 132 Radiation,
in vitrolin vivo, 173-174 low-LET, 2-4,
Reproduction
Formaldehyde, influence of occupational exposure, 137
carcinogenie potency of, 42-46 influence of tobacco smoke, 130-132
influence of toxicokinetics, 138-144
Gestation, 122-136 risk assessment of, 137-158
using biomarkers to assess risks, 149-155
Heavy Metals Risk Assessment, 31-46, 73-82
as a carcinogen, 67-72 components of, 183-184
exposure to, 79 definition, 144-148
of radiological situations, 255-263
Insecticides, 169-182, 229 of reproductive risks, 137-158
Ionization
of drugs, 130-131 Serum
as a biomarker of cancer, 209-226, 265-271
Kinetics, 23, 26 Serum ferritin
biokinetie properties of chemicals, 84-85 as a biomarker of lung cancer, 101-104
reaction parameters, 3 Serum FSH
toxicokinetic influence on bio markers, 90-94 as a biomarker, 113-115
toxicokinetie influence on reproduction, 138- Solvents, 83-94, 183-199, 229
144 Sperm concentrations
as a biological marker, 137-158
Lead,83
biomonitoring in the atmosphere, 67-72 Teratogenesis, 121-136
Thalidomide, 124-136
Manganese, 73-82 Tobacco smoke
Mercury, 96-98, 229 bladder cancer in smokers, 9-15
Metabolism, 143 dose-response from, 56-57
Methylating agents effects of, 8-26
as a carcinogen, 57 maternal smoking, 130-132
Model origin of protein adducts from, 20-21
pharmacokinetie, 126-136 smoker population at hazardous waste site, 218-
stochastic, 12-15 220
Mutation, 1-3 Trans-4-dimethylaminostilbene
chemical,5 as a carcinogen, 57-58
mismatch amplification mutation assay Tumor, 3, 31-46, 64, 101-104, 209-226
(MAMA), 47, 50 promotion of, 212-215
mutation spectra, 5, 47-51, 212-213 markers in effusion, 265-271
Tradescantia-Stamen-Hair-Mutation bioassay, ceruloplasmin as a marker of, 273-278
222-223, 247-253
Urethane
Neurobehavioral Evaluation System, 165-166 as a carcinogen, 57-58
Neuroendocrine effects, 183-199
Neuropathie effects, 169-182 Valproie acid, 124-136
Neurotoxicity, see Biomarkers of toxicity
Nucleophile, 3, 18-23 Xenobiotics, 201-207, 211
centers in DNA, 43 absorption of, 139-141
assessing exposure to, 230
Occupational exposure, '21-22, 80-82, 83-94 effects on stress protein, 96
affect on nervous system, 159-168 metabolism, 48
,adverse impact on reproduction, 137 placenta transfer of, 121-136
from cadmium, 73-82 produce protein synthesis patterns, 97-98
from hazardous waste site, 209-226 relationship to biomarkers, 106
Organic cOlnpounds reproductive risks from, 137-158
monitoring of exposure to, 83-94

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