Introduction

In recent years issues concerning the effects of toxic chemical wastes nuclear radiation on health have drawn
considerable attention from the general public, especial lY concerned citizenry and environmentalists.
Physicians should be aware of the effects of toxic and genotoxic agents on human health and steps they can
take to lessen the risk of exposure and toe effects on public - health. This chapter discusses the effects of toxic
metals in the environment, the basic nature of radiation and its health effects, and assessment of risks
associated with exposure to mutagenic and carcinogenic chemicals. The chapter closes with discussion of the
physician's role In responding to genetically hazardous chemicals.
Objectives
The student should be able to:
1. Discuss the concepts of toxicity and genotoxicity -- mutagenicity and carcinogenicity -- in terms of disease
causation.
2. State ways to minimize patients' direct and indirect exposure to toxic and genotoxic drugs and chemicals as
well as radiation.
3. Relate various toxic metals to the specific associated pathology.
4. Identify the basic biologic effects of ionizing radiation.
5. State the rationale on which radiation protection guides are based.
6. Discuss the nonthreshold vs. threshold models of toxicity and the Cases to which each applies.
7. Explain the risk versus benefit relationship far population, recognizing that safety of an individual is not
absolute and that any exposure carries a certain inherent risk.
8. Describe the major techniques available to detect carcinogenic and mutagenic exposures in man and in
experimental studies.
Toxic metal
Toxic metals have accumulated in our ecosystem as a result of our advanced technology. These metals have
been found in the air, water, soil, plant and animal life on land and in eater. Lead and mercury have drawn the
most publicity and concern, although these: are certainly_not the only toxic metals to which we are exposed.
Various toxic metals and the disease or syndrome they produce are given in Table 14.1 Most of the toxic
metals, such as lead, mercury, and arsenic, function by reacting with and inactivating sulfhydryl (Si) groups of
enzymes. In general, these metals inhibit neural function, causing a preponderance of adverse neurological
effect, Other metals or metallic compounds affect the lungs such as beryllium, which cause berylliosis (an
occupationally induced pneumoconiosis) and lung cancer and nickel carbonyl, which causes lung cancer.
Cadmium see. to be causative factor in hypertension in rats and is believed bY Schroeder (I) and others to be
implicated in cardiovascular disease. Pier's recent review of the role of heavy metals in health deals with some
of these In more detail (2).
The severe symptoms of lead poisoning appear in Table 14-2. Some workers, city dwellers, and ghetto children
may be under increased risk from exposure to lead. An accumulation of lead found in the blood of urban
dwellers does[ appear in suburban or rural dwellers. In some instances, the blood level is greater than 200
Vg/1, which is considered the level that first produces metabolic disturbance. Lead in the atmosphere primarily
comes from tetraethyl lead, the main antiknock ingredient in gasoline. Fortunately, the trend toward use of lead
free gasoline that has occurred in the United States has helped to lower greatly the concentration of lead in
the atmosphere. Probably the[ serious public health problem concerning lead, and indeed all the toxic metals at
this time, lies in the high Incidence of pica in children. Children, particularly of preschool age, ingest dirt,
plaster, and paint chips. The latter are particularly hazardous since ghetto areas are replete with old houses in
which indoor walls are covered with paint containing
Table 14.1
high lead content. When paint chips off, whether it is an outer or inner layer, and a child ingests a substantial
amount of it, mild or even serious lead poisoning is likely to develop. Unfortunately, at first symptoms are quite
deceptively mild and often are confused with an irritable mood, cold or other illness. One the latter stages of
poisoning develop, however, irreversible damage may result leading to encephalopaty or even death. The
answer to this problem lies in screening children, particularly those living in older areas, removal of high-lead
paints from existing homes, and a housing code that prohibits use of high-lead paint on interior galls. Federal
legislation of U.S. cities With funds during the early 19705 to enact such a program, and, Indeed, prohibited u
of high-lead paint (1% dry weight). However, these funds recently have run out, and the presence of lead paints
in older ghetto housing remains a Substantial problem.

Table 14.2
In 1953, a severe neurological disorder was first noted among persons living in the vicinity of Minamata Bay,
Japan, with 83 cases ending in either fatality or severe neurological disorder.
This clinical and pathologic syndrome was named Minamata Disease. The toxic agent finally was identified as
methyl mercuric chloride or some closely related compound, which resulted from dumping spent mercuric
chloride catalyst from a vinyl chloride plant into Minamata Bay, with possible marine bacterial action converting
the HgCl2 into CH3HgCl (3).
Since the Minamata Bay incident, organic mercurials have been found widely dispersed in the ecological food
chain in Sweden and the United States (Lake Erie). This has resulted from substantial industrial use of
mercury, as in the manufacture of vinyl chloride, paper and pulp, fungicides, medicines, etc.
In most instances, a chelating agent is used to treat heavy metal poisoning. The term chelating agent (from the
Greek word meaning "claw") refers to an organic compound that combines with metal Ion by forming two strong
covalent bonds. Thus, the chelating agent essentially removes free metal ions from the blood and Indirectly
from storage depots such as bones.
Examples of chelating agents used clinically are Ca, Na2, EDTA (calcium, sodium salt of
ethylenediaminetetraaceta) for lead, penicillamine for mercury, dimercaprol (British anti-Lewisite:BAL) for
arsenic. Chelating agents are highly toxic agents and must be administered with great caution, usually while
the patient is hospitalized.
From the physician's viewpoint, one of the most important aspects of metal toxicity is that the symptoms mimic
other diseases and conditions. For example, a recent case was diagnosed as infectious hepatitis until
hematologic and toxicologic work-up confirmed the presence of lead. Consequently, it is crucial for physicians
to look for possible occupational andnonoccupational contact with toxic metals. If any of these are suspected,
blood and/or urine samples should be submitted for analysis.
RADIATION EXPOSURE
Even though we have been in the nuclear age for more than 35 years, most people understand very little about
radiation and its effects. The horrors of Hiroshima and Nagasaki an association of radiation with death and
destruction, and the 1979 events at Three Miles Island, Pennsylvania (4), reinforced old fears. Replacing fear
with understanding is important because everyone is exposed to radiation in the forms of natural background
radiation and radiation from consumer products and diagnostic medical procedures.
The purpose of this section is to explain the basic nature of radiation and its effects.
Basic Concepts
Ionizing Radiation. Ionizing radiation refers to radiation, either electromagnetic waves or particle, having
sufficient energy to eject orbital electrons from atoms and molecules, thereby forming positive and negative ion
pairs. Ionizing radiation is produced by decay of radioactive isotopes (both artificial and natural) and by devices
such as X-ray machines and particle accelerators. Note that the term 'Ionizing radiation' does not include
ultraviolet radiation, microwaves infrared radiation, radio waves, or lasers. These are examples of non ionizing
radiation and will not be discussed at this time. Common types of radiation Include . Y radiation and X rays.
Their characteristics are listed in a general text or reference on radiological health such as that by Cember (5).
Radiation Quantities and Units. Unfortunately, two systems now are used to quantify radiation. The
conventional unite that evolved historically now are found in most journals and textbooks. The SI units flu
System International d'UnItes) have been adopted by the International Commission on Radiation Units (ICRU)
and Measurerments and presently are used by most countries in the world. The United States, however, is
behind in converting to SI units, and conventional units are used here in discussions of radiation dosimetry and
effects.
Exposure. "Exposure" is the term used to describe the intensity of X rays or y rays in air. It does not apply to
particulate radiation. The special unit of exposure is the roentgen (R), defined as:
1 R - 2.58 x 10-5 C/kg
where C - coulombs and kg - kilograms of air. The roentgen is used to describe the intensity of X or y rays, but
not particulate radiation, such as B or A particles.
Absorbed dose. Absorbed dose is the term used go describe the energy delivered To irradiated matter. The
special unit of absorbed dose Is the rad.

1 rad = 100 erg/g = 0.01 J/kg

where J = joules of energy = 10pangkat7 ergs. The rad applies to all ionizing radiation, both directly ionizing
particles (electrons, protons, alpha particles, etc.) and indirectly ionizing particles (neutrons, photons, etc.). The
rad is a more useful unit of absorbed dose than the roentgen because the rad applies universally to all radiation
-- not just X anal y rays and to all types of absorbing material -- not just air.
Dose Equivalent. For radiation protection it is important to realize certain types of radiation produce such more
damage than others. For example, a one rad dose from neutron. produces too to ten times more biological
damage than one rad from Y rays. To account for this variation, the dose Is multiplied by a term called the
.quality factor" (11), which varies with different types of radiation of the same energy (see Table 14-3).
Table 14.3
Rem is an acronym standing for roentgen - equivalent - man that refers to biological effect in humans. Because
of the variation of Q for different types of radiation, different radiations having different numbers of rd s will
correspond to 1 rem as shown in Table 14-3.
Activity, the term used to describe the amount of radioactivity, relates to the rate at which radioactive materials
decays. The special unit of activity is the curie (CI).
1 CI= 3.7 x 107 s-7
Note that activity is defined in terms of nuclear transformations per second (e.g., disintegrations) and not the
number of photons or particles emitted. Each transformation often results in emission of two or more photons
and/or particles.
Interactions with Matter
As radiation interacts with matter, energy is transferred from the radiation to the matter, the latter consisting of
atomic nuclei and extranuclear electrons. Radiation may Interact with either or both these constituents of
matter. For different types of radiation, penetrating power, as already noted, is a function of the type and energy
of the radiation as well as the nature of the absorbing medium. Actions of these radiations on matter are highly
complex and cannot be discussed in detail hero. It suffices to say the greater the radiation's energy, the more
damage will be done. In general, from an external radiation source, y-ematting isotopes present the greatest
hazard because a and a emitters cannot penetrate the skin to cause damage. Conversely, internally deposited
A emitters cause the most damage relative to internally deposited B and y emmiters.
Biological and Medical Effects of Radiation (5, 6)
Concerning cellular effects, the general biological effects resulting from interaction with ionizing radiation may
be summarized as follows:
Effects on somatic cells -- these are direct effects on the body and are of primary concern to the person
exposed to radiation.
Effects on germ cells -- these involve mutations of the chromosomes or genes in the sex cells. They represent
a potential hazard to descendants of the exposed person and are of concern to the entire society as well as to
the affected individuals and families.
Factors influencing Biological Effects. The biological effects of radiation depend on the type and energy of
radiation producing the exposure, the time of exposure, the accumulated dose, and whether it occurs at one
time (single) or in 'fractionated" exposures (intermittent). It also depends on the specific kind of tissue, its
sensitivity to radiation, and the volume of tissue exposed. Thus, exposure to 500 rads at one time probably
would be lethal, whereas the same dose given over a 20-year period probably would have little effect. In the
first case irreparable damage occurs to the cells. Table 14-4 lists typical exposures from various sources, which
gives a perspective for evaluating the magnitude of different exposures. While we do not expect students to
memorize the doses, they should know orders of dose magnitudes for different effects.

Acute Radiation Syndrome

"Radiation sickness" occurs after exposure to several hundred The syndrome, which is dose-dependent, is
characterized by nausea, fatigue, and vomiting within a few hours. Red and white blood cells increase for a day
or two and blood.
Table 14.4
platelets decrease for a few weeks. This is followed by anemia, increased susceptibility to bacterial infection
and hemorrhaging for s time that ultimately may lead to death. If the victim survives, he or she has a greater

than average chance of developing leukimia (especially in the first few years after exposure), other forms of
cancer, cardiovascular disorders, and eye cataracts. The average physician will encounter few instances of
acute radiation syndrome unless there is a nuclear catastrophe or exposure accident. The LD50 for humans
(60 days) is 400-450R, which requires bone marrow transplants and other supportive measures.
Studies of Hiroshima and Nagasaki Survivors
Ironically, out of the horrors, of Hiroshima and Nagasaki has come much of our information about the effects of
high levels of radiation, particularly concerning mutagenic and delayed effects. The Atomic Bomb Casualty
Commission, started in 1948 as cooperative venture between the National Research Council of the United
States and the National Institute of Japan, has followed the medical histories of thousands of survivors of
Hiroshima and Nagasaki atomic bombings and theIr children and concluded the following:
A-Bomb survivors have above average leukemia rates which peaked in 1951 (6 years that exposure) and were
still higher than usual in 1966.
Mortality rates for persons within 1200 m of the hypocenter in the two towns excluding effects of leukemda,
were 155 higher than in unexposed people in Japan during 1950-1960 -- a statistically significant effect.
No effect gas been found on mortality of children conceived to survivors after exposure.
Very high rates of chromosomal abnormalities have been found among survivors and their children to utero at
the time of the bombings, but not in children conceived afterward.
Radiation Sources in the Environment
Table 14-5, from the BEIR (Biological Effects of Ionizing Radiation) Report, represents the most recent
estimates for annual whole-body dose rates in the United States (8).
As Table 14-5 shows, the major contributors to the radiation dosage of the population are natural background
and medical applications (8). Of man-made radiation, the greatest fraction of dosage to the U.S. population is
from exposure to medical diagnostic procedures. Thus, "medical diagnostic radiology accounts for at least 90%
of the total man-made radiation dose to which the U. S. population is exposed. This is at least 352 of the total
radiation dose if the latter is from all sources (including natural radioactivity)." According to a recent EPA report,
the developing nuclear power industry is expected to contribute a population dose of less than IX of natural
background radiation as of 1970. The BEIR Report notes that estimated radiation dosages summarized in
Table 14-5 are merely rough estimates subject to the usual uncertainties of projection. Also, persona in high
risk groups must be considered individually.
Table 14-5
Guidelines on Dosage (8,9)
Recommended Guidelines of the Federal Radiation Council (FCR), 1960, 1961, state:
The BEM Report (8) attempts to quantitate genetic risks from radiation and offers the following findings,
recommendations and conclusions, which represent only a fraction of their discussion:
Natural background should be used as a standard for comparison. If the genetically significant exposure is kept
well below natural background, addition. consequences will neither differ in kind from chose experienced
throughout Human history, nor exceed them in quantity.
Based largely on extensive studies of Hiroshima and Nagasaki populations, a range of 20-200 rems was
assumed to be the doubling dosage for mutations. The increase to be expected among one million live-born
individuals whose ancestors had received 0.17 rem per years (5 rems per 30-year productive generation) was
estimated. it was found that for autosomal dominant traits, the current incidence of 10,000 per million live births
increased by 50-500 (to 10,050-10,500) in one generation with an equilibrium value of 10,25p-12,500 persona
affected. The increase in births of individuals with X-chromosome-linked traits also was calculated and found to
have Increased only slightly.
Diseases caused by dominant and X-chromosome-linked recessive mutations eventually will increase in
proportion to the mutation rate increase. The BEIR report suggests that for congenital anomalies and
constitutional diseases, the mutational component, i.e., fraction of incidence proportional to mutation rate, is
between 5% and 50%.
In terms of its overall contribution to ill health, the BEM report (8) assumes the mutational component to ill
health is about 200. Using this figure and a doubling dose between 20 and 200 rem, a dose of 5 rem per
generation would lead ultimately to an increase of 0.5-5.0% in all illness as a result of adverse mutational
changes.
Occupational Hazards

Uranium Mining. Uranium miners have been shown be at especially high risk for lung cancer primarily because
they inhale the decay products of radon, which is itself a disintegration product of radon, Interestingly, smoking
miners are at a substantially higher risk (10 times greater) than non smoking miners, showing the synergistic
effect of the cocacinogens -- smoking and radiation (12).
Strip-mining of phosphate (13). In 1976, the EPA and the President 's Council on Environmental Quality
ordered a sweeping, intensive study of the environmental impact of the enormous Florida phosphate industry.e
This industry, located mainly In Polk County, provides 8091 of the nation's supply and. 1/3 of the world's supply
of phosphate, which-is vital for fertilizer, chemicals, and many other purposes. Simultaneously, the EPA
announced moratorium on further expansion of seining and process until the scuds and hearings are
completed.
The known adverse environmental effects of phosphate mining are air pollution. (fluoride and burning vast
amounts of high-sulfur fossil fuels); use of vast amounts of water to wash ore, creation of high slime ponds that
may spill into rivers; water depletion, leading to subsidence and saltwater intrusion into this Florida aquifer. and
land destruction as a strip-mining of this 130,000 acres into huge, barren, gray gullies. However, the main
public health concern is that residents of the phosphate mining area are exposed to significant radiation from
radon-222, a disintegration product of radium. EPA's preliminary study in Polk County found that continuous
exposure to this radiation over a three-year period doubled local residents' chances of developing lung cancer.
The Polk County Health Department was concerned enough to have placed monitors in about 750 homes to
determine radiation levels.
Low Levels of Radiation -- Induction of Leukimia and Cancers
As noted earlier, there is a substantial controversy about how low levels of radiation will affect public health.
Specific questions include;
What is the threshold for injury to humans from radiation, and, assuming a threshold exists, are there sufficient
data on enough individuals exposed to low doses of radiation to even determine a threshold
What are the doubling doses for various mutations and cancers in humans?
Mortality from leukemia. and cancer in nuclear shipyard workers. Recent studies appear to Indicate that low
doses of radiation may result in a greater incidence of leukimia and cancer than
previously thought. Najaran and Colton (14) have studied the mortality from leukemia and cancer in nuclear
shipyard workers at Portsmouth Naval Shipyards (PNS) where nuclear submarines are repaired and refueled.
Death certificates were studied for the years 1959-1977. A total of 1,722 deaths were noted during this time.
The next of kin were contacted for 592 workers. Individuals who died under the age of 80 were classified as
nuclear workers or nonnuclear workers depending on information supplied by next of kin. Using U. S. agespecific proportionate cancer mortality for white males as a standard, the beery...I/expected ratio of leukemia
deaths was 5.6 (6 observed, 1.1 expected) among the 146 Corner nuclear workers. For all cancer death this
ratio was 1.70, The excess proportionate leukemia and cancer mortality among nuclear workers exceeds
prediction based on previous data of radiation effects in man. Information provided by past and present PAS
nuclear workers suggested total occupational dosages of 10 rem (lifetime), or approximately 0.2 rem per yea,
as measured by film badges. Yet, nuclear workers at PPS had 6 tine. proportional mortality from leukemia and
2 time mortality from all cancers expected of U.S. white males in the same age groups.

The authors recognize that a systematic error or bias may be present in the study due to
information from next of kin about classification as nuclear or nonnuclear worker. inaccuracy in
film badge readings, and other synergistic factors (smoking, asbestos exposure, industrial solvent
exposure) that would lead to greater mortality than from radiation alone. The small number of
cases of leukemia observed also leads to concern about the study's reliability. Nevertheless, the
findings suggest that present levels of FRC - recommended radiation exposure for various groups
-the general public, individual members of the general public or radiation workers -- may not, in
fact. provide sufficient protection.
Thymic irradiation and chronic myelogenous leukemia. A recent clinical report by Shimaoka and
Sokal (15) discusses two cases of positive chronic myelogenous leukemia (CML) with history of
thymic irradiation. The authors note that both patients received radiation therapy from low
voltage x-ray equipment at 2-3 months of age. Leukemia developed 18 and 22 years later, an
induction period comparable to those for leukemia caused by others radiation exposures. The
physicians note that presentation response to antileukemia therapy and the disease's clinical
Course did not differ from that of other patients.
There have been many reports of CML following radiation exposure among adult populations.
However, CML has been recorded only rarely as a complication of radiation exposure early in
life. Most followup studies of irradiated pediatric populations have detected no cases. Thus,
physicians considered it worthwhile to note that two young adults among their 65 patients with
CHL were known to have received thymic irradiation in infancy. It may be significant that in
each case the history of radiation exposure only came to light after special inquiry -- well after
diagnosis of leukemia, referral to the physician, and initial work-up.
However, this paper is a clinical case report, not an epidemiologic study. Therefore. we must
consider that CML may not have resulted from the radiation per se but from FEE administration
of radiation to the thymus, an organ playing a key role in the immune system. This report clearly
shows that long~term effects of certain medical procedures, i.e., irradiation or chemotherapy,
may not be revealed for some 20 years, roughly the induction period of leukemias and cancers.

DETECTION OF MUTAGENIC AND CARCINOGENIC CHEMICALS :

ASSESSMENT OF RISK

The carcinogenic effects of chemical exposure in man are expressed after a latent period of
months or many years. Mutagenic effects may not he observed directly for many generations.
For these reasons, several experimental techniques have been developed to detect mutagens and
carcinogens. These techniques must meet three primary objectives:

Identification of mutagenic or carcinogenic agents.

Determination of human exposure to them.
Estimation of the risks they pose.

Since no single method adequately addresses all these objectives, the current approach is to use a
battery of tests involving different levels of biological complexity that detect different kinds of
effects. Two major considerations in test design are the procedure's accuracy in duplicating man's
biological complexity and the technique's speed and economy. In general, tests using intact
animals meet the need for biological complexity better than tests using microorganisms or
cultured mammalian cells. However, animal tests are more time-consuming and expensive. With
an estimated 10,000 new chemicals developed annually, simpler tests are required for screening
and setting priorities for additional work.
Epidemiology
Human epidemiological studies are the ultimate test for human exposure and susceptibility to
carcinogens. In many cases, these studies are of great value. However, the background level of
cancer in a population often hides a small increase in tumor incidence caused by exposure to a
particular agent. Furthermore, studies involving human populations are not well controlled
because of significant individual variations in genetic makeup, diet, work habits, smoking
history, and other factors.
chemicals only if :

Consequently, epidemiological studies will detect carcinogenic

The chemicals are very potent.

A sufficiently large population is studied for a sufficiently long time after exposure.
The type of cancer produced is ordinarily very rare, i.e., if a cancer such as angiosarcoma
of the liver, which is usally very rare, occurs in many members of a relatively small
population exposed to vinyl chloride, this would strongly indicate the exposure
contributed to development of the cancer.

Detection of Carcinogens.
Animals bioassays. The most reliable system to test carcinogenic activity uses an intact mammal
as the indicator organism and tumor formation as the endpoint. To mimic exposure of large
human populations to low doses of a suspected carcinogen over a period of many years, a
relatively small number of animals (rarely more than several hundred) are exposed for the
animal's lifetime (two years for rat or mouse) to high levels of the test compound. In some cases,
this generation's progeny also are observed over a lifetime to look for effects in the F1
population. Primary weaknesses of animal lifetime studies are the uncertainties about
extrapolation of animal data to man and use of agent concentrations well above those humans
normally encounter. Nevertheless, animal lifetime studies are considered the most definitive tests
for carcinogenesis short of direct epidemiological evidence in human populations (16), despite
the fact they are expensive (about $500,000 per compound), cumbersome, and time-consuming.
Two general strategies have been adopted to meet the need for faster and more economical tests.
One strategy uses in vitro tests for carcinogenic activity based on neoplastic transformation of
cultured mammalian cells. The second uses mutagenicity tests based on the principle that
carcinogenicity and mutagenicity are positively correlated.
In vitro transformation assays. The most significant change in the neoplastic transformation of a
cell is acquisition of the ability to form tumors in a suitable host animal, but a variety of other
changes in cell morphology, growth characteristics, and biochemistry also are associated with
transformation. A number of in vitro assays now utilize these additional endpoints (17). The cell
systems commonly used at this time are primary hamster embryo cells and cell lines established
from mouse embryo fibroblasts. Treatment with many carcinogenic chemical and physical agents
can induce these lines, and, since the endpoints are easily observed, quantitative assays are
possible.

One ujor weakness of these systeas is that the calls are

fibroblastic and produce sarcomas when injected into anieaia.
Sarcomas are such less comnon in humans than carcinoaas and a
sarcoma model for cancer may be less useful than a carcinoma
eodel. A second problem is that the frequency of transformation
is such higher than would be expected for tumor induction in an
intact animal. It is possible that conditions in vitro uniquely
predispose the cells to transformation so that while some
events are not necessary in vitro. they say be required for
tueorigeneaia in vivo._More sophisticated transformation systems
are now being investigated in many laboratories, including
develop-ant of epithelial cell transformation assays.
Detection of Hutagens .
' 'i'eats with intact animals. _s_evra.l whole animal.eutegenesis..tasts .- ""Eav'e Been developed (18). One method, the dominant lethal test,
detects lethal mutations. Hale mice are treated with test agents
end then mated to several femles. The uteri of the pregnant
females are examined for early fetal death. Because the lethal
"" mutation is detected i_n the progeny of the treated animal. this
-1thod identifies eutstion in the gerainal cells.
A second procedure, the specific locus test, involves crossing
treated wild type nice with mice hosozygoua for s set of recessive
traits that result in visible markers such as spotted costs. in

the absence of mutation. the progeny would be expected to be

heterozygous for each marker and thus phenotypically like the wild
type parent. Mutations at specific loci are detected by
appearance of the recessive traits. Cytogenetic screening of
netaphase lymphocytes from bone ssrrov of treated animals also can
be used to detect damage to chronosomes or to the mitotic
apparatus.
The nomammalian organise most widely used for whole animal
eutageneais tests is a species of fruit fly, Drosoghila
melanogaster (19). The detailed understanding of this organism s
genetics, developed over several decades. makes it ideal for
various tests for both point mutation and chromosomal effects.
The only major disadvantage to its use is that it is not mamalian
and. therefore, is less valuable than sasaals for predicting risk
to humans.
WW Use of cultured eatssalian cells
to teat for mutagenesis is a great simplification over use of
intact animals but retains such of the mammalian metabolism and
genetic characteristics (20). One advantage of cell culture
methods is that human cells can be used when people could not be
exposed. Point mutations are detected in cultured cells by
selecting for resistance to certain drugs such as purine analogues
or ouabain. Cytogenetic damage is detected by examining netaphaaa

chroeoaomas in cultured cells, and use of human fibroblast or peripheral blood lymphocytes
permits observation of effects on the
human cytogenetic apparatus. The major weakness of cultured
mammalian cells as test systems is their isolation from the
metabolic activity of the intact animal.
Microbial systems. Mutagen detection methods have been developed
using many microorganisms including fungi, bacteria, and
bacteriophage. The Salmonella typhimurium system developed by
Ames, et a1. (21) is the most widely used of all mutagen
detection systems and will be the focus of this discussion. A .
series of auxotrophic histidine requiring mutants of _S__1
typhimurium have been isolated and characterized in detail. Both
A__ base substitution__and frameshift mutations are included. These
mutations have been incorporated into test strains that also
._. .cpntain mutations to increase membrane permeability and to block
n- DNA'repair. "Mutaenic agents are detected 'by their ability to "' "induce-"reversions~to histidine independence (prototrophy). ,Cells. a.
are treated and Spread on culture medium lacking histidine.
Revertant cells grow and form visible colonies while the parental
auxotrophs fail to grow.
The test poses several shortcomings. Prokaryotes may be poor
predictors of events in mammals. They are totally devoid of
the intricate absorption, compartmentalization, and excretion

patterns of mammals. Also, bacteria lack the mixed function

oxidases that are the principal mammalian mutagenactivating
enzymes. Numerous strategies have been adopted to provide
mammalian metabolic activation for both microbial and in vitro
mammalian cell systems.
Methods for Metabolic Activation of Mutagens
Mammalian liver homogenate preparations. The simplest and most
widely used activation system employs homogenates of mammalian
liver from animals treated with agents that elevate their mixed
function oxidase levels. A "standard" microsomal liver
preparation called 8-9 is used now in many laboratories (22). The
liver homogenate usually is incubated in the culture medium with
the test agent and the bacterial or mammalian cells. Major
difficulties with liver microsomal activation is that not all
activating mechanisms present in an intact animal are represented,
and the compartmentalization found in the animal is totally
absent. The greatest advantage of the liver homogenate method is
that it preserves the simplicity of the ig_11g system.
Host-mediated assay. The host-mediated assay combines the
simplicity of an in vitro system with the agent's full range of
whole animal metabolism. The test is performed by treating a
mouse with the test agent, then injecting the indicator organism
into the animal's peritoneal cavity. After an appropriate

duration of exposure, culture medium is injected i.p. and

withdrawn together with a large sample of the exposed indicator 2:8:313m'1d The banana "6
Plated and the reversion only is run
wou be for the in vitro test. 5. t himurium was the
original indicator organism used (23). Winn can.
20:? recently have been employ an indicator organism using
y ogenetic damage, mutation. and neoplastic transformation as
endpoints (20- Though a bit more complicated and lose
tepro ucible than the in vitro assay, the host-mediated assay is
more likely to reflect events occurring in the intact animal.
Bod fluid anal sis. With body fluid analysis, an intact animal
processes the test agent which is then detected in urine. blood,
or tissue extracts using standard indicator organism (25).
._ ,___VAga,1' is 99W! I? frequently. but the method can
7 be adapted to other organism. This method is simpler than the
hostmediated assay and is more indicative otw the met-bolls ..
capabilities of"an animal thanTIiv'i-Mhomogenate activation,
._-.._7Howsver, it- may produce falsenegativere'slts if' the agent is not
present in detectable amounts in the fluid tested.
A number of other activation techniques and short term bioauayn
,_ . are. either available or currently under development. Those
..___.described above are most widely used. '
Detection of Human Exposure

Once a chemical has been identified as a mutagen, two problems

remain. Estimates of the magnitude of human exposure and the
potency of the chemical must be made. Several assays are either
available or are being developed to monitor human exposure to
mutagenic agents. Some are simple adaptations of the screening
assays already described. For example, one of the best uses a
body fluid analysis is in monitoring human uginelforlmgtagintz
activity (26). Metaphase analysis ofd pertp grams: gauze
chromosomes is a sensitive technique to etec c gm 1: b.
and methods now are being dEV81Ped 0 due rep y
asuring unscheduled DNA synthesis in nonproliferating
me (27). Nondisjunctional events in germinal CEUI C5
lymphothes mi in the frequency of sperm containing two
be damned by date: ndeised chromatin associated with the Y
fluorescent Y $331125 Ato:technique now being developed may allow
chromosome) ( - in: mutations in human peripheral
ntitative detection of P0
qua 3 use alkylating agents interact with prote n
> lymPhCytes (29). eca ids their presence in the body can be
as well 38b "Sc:::lcnin::g the amount of alkylated histidine and
uantitate y e n a hemo lobin hydrolyn"
:lkylated cysteine residues present 1 8
(30). t

ions 0
1 1n inpointing 10C
Short-term bioassays are if: or "at; samples from work are"
mutagens in the environme - 1 can be tested for Juan
or near industrial facilities Iof Puncular substances 1.
the presenCe th.
activity, and if 1 m confirm or re)
nal s 8 y
suspected, direct chemical analysis may confirm or reject the cprttesence of the suspected agent
as well as provide quanuuun
a a.
While shortterm tests are becoming available to detect mutation
1 humans, we also need to consider indicators of mutation that
can be observed in the general population. Both somatic and
germinal cells are susceptible to mutagenic damage. Mutations in
somatic cells usually are not manifested in any obvious way other
than the induction of neoplasia. However, since significant lag
times between damage and tumor induction occur, and because
factors other than mutation may be involved in expression of
neoplasia, this is not a sensitive indicator of mutagenic damage.
011 the other hand mutations in germinal cells, particularly in
postmeiotic "'spermatogonial cells and 'o'ocytes within 7 weeks of
ovulation, will result in mutant zygotes and genetically defective

51- embryosr Studiesin- irradiated animals show 'that ppontaneou's

.._-_abortio.n,. .eitherv lepre-uor post-implantation,-removes~up to- 951 of
conceptuses with chromosomal abnormalities. In humans, about 151
of recognized pregnancies end in spontaneous abortion and the
incidence of chromosomal aberrations in post-implantation
abortuses is about 3671. Animal data suggest that pre-implantation
.losses are substantially higher (31). Because the majority of
chromosomal defects present in germinal cells at conception are
fatal, spontaneous abortion is thus a sensitive indicator of
mutagenic activity. Live-born children with chromosomal
abnormalities or congenital malformations also may represent
effects of mutagenic exposure. The dimensions of the problem,
which are only hinted at by the observed and unobserved frequency
of fetal loss, are very large.
A striking example of the impact of mutagenic exposure on
spontaneous abortion is seen in a study of women whose M
were exposed occupationally to vinyl chloride (32). As noted in
the chapter on occupational health, vinyl chloride is a potent
carcinogen. It is also a potent mutagen. When the reproductive
histories of wives of vinyl chloride workers were analyzed, the
age-adjusted rate of fetal death was 15.8 per 100 pregnancies.
The rate in a control group was 8.8 per 100 pregnancies.
Furthermore, the rate in the eXPOSEd group Prior 0 the

employment in vinyl chloride manufacturing was only 6.1 per 100

pregnancies. Using both parallel and retrospectve controlsi
significant increases in fetal loss occurred a ter Pater:
exposure to a known mutagen. Because males were ffexpiseln
nongenetic mechanisms -- while 9055913 " 1353 e '5 VE Y
explain the observed results than does utmn
with obstetrics, you should be
As a physuiam Perhaps invgilfg that spontaneous abortion may be
keenly aware of the pass! Y d h m 1 d/
the result of exposure to mutagenic 85 c e ca 5' a r
radiatiom Furthermore, maternal an: paternal exposures may be
equally hazardous to the health of. The fetus
Evaluation of Human Risk

Determination of an agent's potency in a human is difficult.

Intact animal tests generally provide more relevant information
for humans than short-term bioassays. Even then, variations in
human metabolism may make some individuals more susceptible to
injury than others. Several of the human genetic disorders
described in Chapter 10 clearly predispose affected individuals to
increased susceptibility to mutagenic agents (33)- It 1'
reasonable to suppose that many other more subtle variations in
genetic background may affect individual responses to mutagenic

agents.

In analyzing environmental mutagens, the bottom line is the

;::-~-_dete.r_mi_netioa_,ef .._r1s.t.-.pose.ct by an agent !0 epopuletione. Risk .
- - determination requires careful analysis of data from many 'testa.
m' l'tisWefg'hTEknFGThatmaT-i "age'ntmfs-a Eutagn or'"caFciogeh. '
He must know where it is and how much is present. If the exposed
population is small (a group of patients being treated with a
mutagenic drug or a group of industrial workers), _direct

' "determination of Eheirinternal eapssure is useful. As much as

'_ possible must be learned about metabolism of the agent in intact

animals. If human exposures to known doses of an agent have
occurred, epidemiological studies may provide useful information
if enough time has elapsed for an effect to be seen.
The ultimate conclusions about a particular chemical's impact
require a judgment that considers not only scientific evidence but
social benefits derived from use of the chemical involved. In
general, no exposure is completely free of risk. The most
constructive philosophy is to minimize the exposure, whether it is
occupational or environmental, chemical or radiation, to make the
risk "acceptable." The concept of "acceptable" risk is that risk

is accompanied by a benefit equitable to the exposed individual.

Some legislation quite clearly bans carcinogenic substances from
use in some applications. For example, the Delaney Amendment to
the Food and Drug Act bans additives from food or drugs that are
carcinogenic to animals or man. The Toxic Substance Control Act,
enacted in 1976, requires that the safety of various chemical
compounds, including industrial chemicals, be determined and
regulatory steps be taken to restrict human exposure to substances
shown to be carcinogenic or mutagenic hazards.
The Physician's Role
we must ask how physicians should respond to all of this
information. First, they should be sensitive to the existence of
genetically hazardous chemicals. They shOUXd understand that
today, chemicals are responsible for a significant percentage of
cancer incidence in the United States as well as other cauntries.
and that they probably cause a signifiCant amount of genetic
'isease as well.
At the same time physicians should realize that only a finite
number of these agents exist in the environment. Carcinogenic
chemicals enter our environment primarily as a result of human
activity. They can, largely, be removed, reduced, or avoided by
human effort as well.
With that general concept in mind, a primary care physician can

help patients avoid unnecessary exposure to carcinogens and

mutagens by being mindful of the potential sources of exposure
generated in their lifestyles. Smoking is probably the single
most carcinogenically hazardous activity. Occupational and
environmental exposures are a second significant source of
_ _EE1398i[mutpgenicrisk. ,Until now, the_.safety of workers w
inVOIVed in manufacturing chemicals has been emphasized. While
this is important, the point of manufacture may not, be the most .
difficult place to limit occupational exposures. _anufacturing._
""processeS'rn large chemical plantsusuallyare tightly controlled
and contained. The product and its intermediates remain in a
closed system and worker exposure is often low. Occupational
exposures at the point of use often are much less controlled.
Solvents and other chemicals often are used for jobs like floor .
""tilihg"uder Vfis'environmental conditions. Individuals using
them daily often work for small companies with poor safety
regulations or are self-employed or use them in hobbies. In any
case these individuals usually are poorly informed about the
genetic hazards in using these agents. The alert private
physician, mindful of potential risks that patients face and
willing to take time to counsel them, may be their only source of
information.
An area over which physicians have'greater control is the decision

to use various mutagenic drugs and other agents in caring for

their patients. Most physicians recognize risks posed by
diagnostic X rays, particularly with men and women of
child-bearing age, and they arrive at some judgment about
circumstances in which their use is Justified.
It is not so widely recognized that some commonly used
pharmaceuticals are known carcinogens and mutagens. These drugs
remain in use because their benefits appear to outweigh risks
: involved in their use. Metronidazole or Flagyl provides an
interesting example. Flagyl is used widely to treat certain
protozoan infections and TrichoEOHas Vzginalishinfec:ionst:f tb:
enitoutinar tract. It has een s own, oweve .
Earcinogenicyin mice (35) and mutaginii in bactfrziizyzziriigg52;
Bod f uid anal sis on humans n cates mu a
Perionsltreated with therapeutic doses of Flagyiag36)- T:::::;::::
like this ose a dilemma for the phys c infections, p while rarely life-threatening: caninbe czgigimeiz
uncomfortable to the Patient' The risks 0f higduielfef from a
genetic effects in Patients :9 bring temust be weighed
uncomfortable but nonthreatening situationmust be weighed carefully. Particularly in this kind of
situation, physicians
must be keenly aware of their own ability to influence patients
exposure to genetically hazardous substances.

REFERENCES

l. Schroeder HA: Municipal water and cardiovascular death rates.

JANA 195:81, 1966. .

2. Pier SM: The role of heavy metals in human health. Tex Re

3101 Med 33(1):ss1os, 1975. _J'

3. Kurland LT. Faro SN, Siedler ll: Minamata disease. Uorld

Neurol 1:370-395, 1970.

___..-__I.U_EX L_ _ Thiw 219% they _. almosL lmhnnulnnu J_V-~

Scientist April 19, I979. I s

*f 5':ts.rafim:aaeihfietn"ni'zsica? mm: England, '

'_'__"PrgaTn6;I69:''"H- '_ """" ""-"" ""' ' U "" ' '

6. Hodges L: Environmental Pollution. New York, Holt Rinehart

and Winston, 1973. r r _ > H _. V

 ""7. Ovad'i"J,'KossiRi Radiation protection program for nursing

personnel. Read before the Health Physics Society, 7th
Midyear Topical Symposium.

8. National Research Council: The Effects on Populations of

Exposure to Low Levels of Ionizing Radiation. Report of the
Advisory Committee on the Biological Effects of Ionizing
Radiations (BEIR Report), Washington DC, National Academy of
Sciences, 1970.

9. Gofman J", Tamplin AR: A proposal for at least a tenfold

reduction in the PRC guidelines for radiation exposure to the
population-atlarge. Testimony presented at Hearings of the
Joint Committee on Atomic Energy, 9lst Congress, Jan 28,
1970, GT-llloIO.

10. Pauling 1.: Genetic and somatic effects of high energy

radiation. MW 311, September 1970.
' ll. Sternglass EJ: Effects of low-level environmental radiation
on infants and children. Read before the Symposium on
' Biological Consequences of Environmental Radiation,
University of Northern Illinois, De Kalb, 111, March 20,

1971.
12. Archer VE, Wagoner JK, Lundin FE Jr: Uranium mininS and
cigarette smoking effects on man. J Occu at Med 15:20k-211,
1972. .
13. King w: Florida phosphate pollution stirs alarm. Bile1
York Times, July 24, 1976.
1" "alaran T, Colton C: Mortality from leukemia and cancer in
shipyard workers-

15' ShimaOka K. Sokal JE: Thymic irridation and chronic

myelogenous leukemia. NY State J Med 77(14):2226-2230, 1977.

16- Tomatis L: The value of long~term testing for the

implementation of primary prevention, in Hiatt HH (ed):
Ori ins of Human Cancer Cold Spring Harbor Laboratory, 1977,
pp 1339-1357.

17- Brookes P: Critical assessment of the value of in vitro cell >_

ffasf6fifi6"f3?__EYEEIEi~417#71v w carcinogenicity of
chemicals. Mutation Res 86:233-242, 1981. > V :7; I
m 18; "Russel LB, Matter-BE$ Whole-mammal mutagenicity 'tests:"
Evaluation of five methods. Mutation Res 75:279-302, 1980-

19. Hurgler FE, Sobels FH, Vogel E: Drosophlla as an assay system

~ for detecting genetic changes, in Kiley DJ et al (eds):
_:.Handbook7 of Mutagenicity Test Procedures. Amsterdam,
Elsevier, 1977, pp 335-374.

20. Howard-Flanders P: Mutagenesis in mammalian cells. Mutation

Res 86:307-327, 1981.

21. McCann J, Choi E, Yamasaki E, Ames 8N: Detection of

carcinogens as mutagens in the Salmonella/microsome test:
assay of 300 chemicals. Proc Natl Acad Sci USA 72:51355139,
1975.

22. Ames BN, Durston WE, Yamasaki E, Lee FD: Carcinogens are
mutagens: a simple test system combining liver homogenates
for activation and bacteria for detection. Proc Natl Acad
Sci USA 70:2281-2285, 1973.

23. Gabridge MG, Legator MS: A host-mediated microbial assay for

the detection of mutagenic compounds. Proc Soc Ex t Biol Med
130283183Ioz 1969.

TH Eisenstadt E

24. Le ator MS, Bueding E, Batzinger R, Connor , ,

' Fagrow MG, Ficsor, G, Hsie A, Seed J, Stafford RS: An

evaluation of the host-mediated assay and body fluid
analysis. 53535125_g 98:319374, 1982.

- he use of indirect
. Le ator MS Zimmering 5, Connor TH. T

25 ingicator systems to detect mutagenlc activity in human

subjects and experimental animals, in Hollaender A (ed):
Chemical Muta ens: Princi les and Methods for Their
Detection. New York, Plenum, 1976. pp 171-191.
l

26. Legator MS, Pullin TG, Connor TH: The isolation and detection
of mutagenic substances in body fluid of human subjects, in
Kiley BJ et al (eds): Handbook of Mutagen1c1gz Test
Procedures. [977, pp 1&9-160.

27. Peio RW, et al: Individual variation in the response of

cu tured human lymphocytes to exposure to DNA damaging

chemical agents. Marat Res 53:327-34l, 1978.

28. Kapp RN Jr, Jacobson CB: Analysis of human spermatozoa for Y

chromosomal nondisjunction. Teratogenesis Carcinog Mutagen
1:193-211,1980.

29. Albertini RJ: Studies with T-lymphocytes: an approach to

"-- --human~wmutagenicity monitoring, Vina BridgesBAret-aleds)+-Banburx Report 13: Indicators of Genotoxic Exposure, Cold
r f.... rSpring Harbor Laboratory, 1982,.pp 193-412i at- .. . H. - _

30. ot}Ea-odi'; Ehrenbergi, 'Eersahi, H'ial'iEtrm"

Evaluation of genetic risks of alkylating agents: II
Haemoglobin as a dose monitor. Mutat Res 34:1-10, 1976.

31. Vogel F, Motulsky AG (eds): Human Genetics: Hroblems and

A roaches. Berlin and New York, Springer-Verlag, 1979, pp
7477, 330-370.

32. Infante PF, Wagoner JK, Waxweiller RJ: Carcinogenic,

mutagenic and teratogenic risks associated with vinyl
chloride. Mutat Res 41:131-142, 1976.

33. Haher BM, Curren RD, Ouellette LM, McCormick JJ: Effect of
DNA repair on the frequency of mutations induced in human
cells by ultraviolet irradiation and by chemical carcinogens,
in Magee PN et al (eds): Fundamentals in Cancer Prevention.
Baltimore, University Park Press, 1976, pp 363-382.

34. Rustia M, Shubik P; Induction of lung tumors and malignant

lymphomas in mice by metronidazole. J Natl Cancer Inst
48:721-729, 1972.

35. Voogd CE, Van Der Stel JJ, Jacobs J: The mutagenic action of
nitroimidazoles, metronidazole, nimorazole, dimetridazole,
and ronidazole. Matat Res 26:483-90, 1976.

36, Connor TH, Stoeckel M, Evrard J, Legator MS: The contribution

of mcronidazone and two metabolites to the mutasenic
activity detected in urine of treated humans and with
Cancer Res 37:629-633. 1977-