Comparative study of two evaluation methods for the genotoxic

effects of environmental heavy metals on normal cells

P Virag, I Brie, ID Postescu, M Perde-Schrepler, E Fischer-Fodor, O Soritau, A Irimie and V Cernea
Prof. Dr. I. Chiricuta Cancer Institute, Cluj-Napoca, Romania
Genetic modifications caused by chronic exposure to low levels of toxic metals may activate
stress-signaling pathways, thus increasing cancer incidence among affected individuals. The aim of
this study was to evaluate the relationship between exposure to heavy metals and the incidence of
chromosomal aberrations and DNA lesions in a chronically exposed population by using specific
biomarkers. The study included 156 subjects divided into two major groups: exposed individuals
(in a heavy metal contaminated region, Maramures, Romania) and non-exposed population, as
control group (Cluj, Romania). We compared the results of two cytogenetic methods for the detection
and quantification of DNA lesions and chromosomal aberrations in normal human cells: Single Cell
Gel Electrophoresis or Comet assay and Cytokinesis Block Micronucleus assay. The methods were
performed on lymphocytes isolated from whole blood in density gradient. The basal DNA lesions and
chromosomal aberrations were evaluated, as well as the repair capacity of the supplementary lesions
induced by genotoxic agents such as ionizing radiations. Our results showed a great interindividual
variability in the basal level of the DNA lesions and chromosomal aberrations, between and within
the groups, the most affected being the heavy metals-exposed groups. Non-exposed subjects from rural
area Cluj appeared to be more susceptible to the induction of supplementary DNA lesions and
chromosomal aberrations by irradiation. The most efficient repair capacity of the radio-induced
DNA lesions was observed in the non-exposed Cluj urban group. Both cytogenetic assays (as tools
for detection of DNA lesions and chromosomal aberrations) may be used in human biomonitoring
studies as indicators of early biological effects induced by exposure to heavy metals. Toxicology and
Industrial Health 2009; 25: 253258.
Key words: chromosomal aberrations; comet assay; DNA lesions; metal carcinogenesis; micronucleus
assay
Introduction
Chronic exposure to low level of heavy metals such
as nickel, arsenic, and chromium has long been
known to increase cancer incidence among affected
individuals. Despite of well-recognized carcinogenic
potentials of heavy metals, the molecular mechan-
isms underlying their cell-transforming ability
remain poorly understood. The most recent studies
show that production of reactive oxygen species is
the main pathway in metal induced carcinogenesis,
with special emphasis on the roles of activated sig-
naling pathways, epigenetic changes, and DNA
repair process (Salnikov and Zhitkovich, 2007).
Epidemiological studies have clearly indicated
nickel compounds as human carcinogens, causing
respiratory tract malignancies in workers chroni-
cally exposed to nickel-containing dust and fumes
Toxicology and Industrial Health 2009; 25: 253258
http://tih.sagepub.com
The Author(s), 2009. 10.1177/0748233709103411
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Correspondence to: Piroska Virag, Research Department, Prof.
Dr. I. Chiricuta Cancer Institute, 400015 Republicii str., nr. 34-36,
Cluj-Napoca, Romania. Email: vpiroska@yahoo.com
(IARC, 1990). These data involve epigenetic
changes as primary events in nickel carcinogenesis
(modifications in the histone acetylation, methyla-
tion or ubiquitylation levels, DNA methylation,
activation or suppression of transcription factors)
(Karaczyn, et al., 2005; Chen, et al., 2006).
Exposure to arsenic (another environmental con-
taminant) and its compounds has been confirmed
to cause lung carcinoma, skin, respiratory system,
liver and bladder tumors (Tchounwou, et al., 2005).
Epigenetic changes, such as DNA hypomethylation
and hypermethylation, appear to be essential in
arsenic carcinogenesis, being typically observed in
cancers (Baylin and Herman, 2000).
Occupational exposure to chromium, especially
Cr(VI) compounds is a well-documented cause of
respiratory cancers (Gibb, et al., 2000), most likely
due to their great potential to induce chromosomal
damages and mutations (Reynolds, et al., 2007).
Additionally, Cr(VI) may induce Cr-DNA adducts,
which represent a key initiating event in the cancer-
inducing process (Quievryn, et al., 2003).
The aim of this study was to investigate the rela-
tionship between the exposure to heavy metals and
the incidence of DNA lesions and chromosomal
aberrations, evaluated by Single Cell Gel Electro-
phoresis or Comet assay and Cytokinesis Blocked
Micronucleus assay respectively, in a chronically
exposed population and to develop specific biomar-
kers of effect.
Subjects and methods
This study was conducted on 156 subjects divided
into two major groups: individuals exposed to dif-
ferent sources of heavy metal contamination (min-
ing industry and non-ferrous metallurgic industry)
and non-exposed population, as control group. The
study groups were the following: polluted urban
group (Baia-Mare city, Maramures district, Roma-
nia Mm urban), 40 subjects, and polluted rural
group (Recea-Tauti Magherus village, Maramures
district, Romania Mm rural), 46 subjects. The
control groups: non-polluted urban (Cluj-Napoca
city, Cluj district, Romania Cj urban), 33 sub-
jects, and non-polluted rural (Iclod village, Cluj
district, Romania Cj rural), 37 subjects, were
matched regarding age and socioeconomic status
with the study groups. Demographic information,
work history, and habits of all subjects were
collected via specific questionnaires. Informed
consent was obtained from each of the subjects
included in the study. The Ethics Committee
approved the study.
Whole blood (6 mL) was collected from each
subject in heparinized test tubes, and peripheral
mononuclear cells were isolated by centrifugation
on density gradient with Histopaque (Sigma-
Aldrich, Inc., St.Louis, MO USA). Half of the lym-
phocytes were processed as such whilst the other
half was irradiated in vitro with a Co
60
source
(Theratron 1000) at a dose of 2 Gy. Exposure
times were verified monthly (according to the
debit of the source) for DSP 100 cm, field 20 20
cm, and 0.5 cm depth. After irradiation, cells were
kept on ice to prevent repair.
Single Cell Gel Electrophoresis or Comet assay
is a highly sensitive method for the assessment of
DNA damage formation and repair both at clini-
cally relevant and low doses of irradiation. It
allows the evaluation of not only the magnitude
of the radio-induced DNA damage but also the
capacity and kinetics of the repair processes. The
technique was performed according to Tices proto-
col (Tice, et al., 2000). Briefly, non-irradiated and
half of the irradiated cells were embedded in low
melting agarose and layered onto slides precoated
with normal melting agarose. For assessment of the
repair process, the remaining irradiated cells were
submitted to Comet assay after 2 h of incubation.
The cell-slides were immersed in lyses solution to
remove all non-nucleic materials and covered with
alkaline buffer (pH 13.2) to allow DNA unwinding.
Subsequently, electrophoresis was conducted in the
same solution at field strength of 0.70.9 V/cm at
4 C (27 V300 mA). Finally, slides were neutral-
ized, fixed with absolute ethanol, stained with ethi-
dium bromide, and incubated in the dark. For the
visualization of DNA damage, an epifluorescent
microscope was used (excitation filter: 510560 nm,
barrier filter: 590 nm), and at least 200 cells were
analyzed per sample. The broken DNA fragments
migrated from the nucleus toward the anode, on a
distance and in a quantity related to the size and
number of the fragments. After labeling with the
DNA-specific fluorescent dye, the nuclear material
took a comet-like shape, with bright head and
smaller or longer tail. The extent of the DNA
Genotoxic effects of environmental heavy metals on normal cells
P Virag et al.
254
damage was assessed using Collins visual classifica-
tion method (Collins, et al., 1997). For each dose (0
and 2 Gy) and each time moment (immediately and
2 h after irradiation), a lesion score (LS) and a tail
factor (TF) were calculated according to Collins
formula.
The Cytokinesis Block Micronucleus assay is
one of the most used methods for assessing unstable
chromosomal aberrations, such as acentric chro-
mosomal fragments, which are visible in interphase
as extra nuclear corpuscles named micronuclei. The
technique, according to Fenechs protocol (Fenech,
2000), uses Cytochalasin B to block cytokinesis
process. Briefly, non-irradiated and irradiated
cells were treated with phytohemagglutinin M
(PHA) to stimulate cell division and to ensure
maximum yield of binucleated cells. Cytochalasin
B was added after 44 h of incubation, and cells
were harvested by cytocentrifugation at the end of
the 28 h incubation with Cytochalasin B. The cells
were stained with 10% Giemsa and examined at
1000 magnification. Two parameters were calcu-
lated: percent of binucleated cells with micronuclei
(%Bi+Mni) and number of micronuclei in 1000
binucleated cells (Nr.Mni/1000Bi).
Statistical processing of the experimental data
was accomplished with one-way ANOVA and
Dunnett Multiple Comparison Test (P < 0.05 sta-
tistical significance).
Results
Comet assay
Several biological parameters were evaluated based
on the LS and TF: the basal (background) level of
DNA damage, the amplitude of the supplementary
damage induced by genotoxic stress upon the
genome (-irradiation), as well as the repair process
of the radio-induced DNA lesions. LS and TF,
calculated according to Collins formula, were
expressed in relative units (UR).
As shown in Table 1, the basal (background)
level of DNA lesions (before in-vitro irradiation)
was variable, the LS values range between 35.36
UR (Cj urban group) and 80.45 UR (Mm urban
group). A similar, parallel profile was found for
the TF values, which varied between 8.44 UR
(Cj urban group) and 15.85 UR (Mm urban
group). Statistically significant higher values were
found for both parameters in Mm urban versus Cj
urban, Mm rural versus Cj rural and Mm urban
versus Mm rural groups, respectively (P < 0.0001).
In vitro irradiation of the cells induces supplement-
ary DNA damage. Our results indicate that all
values measured immediately after irradiation
were significantly higher than the basal levels
(P < 0.0001), reflecting additional DNA damages
produced by -irradiation. The magnitude of this
process ranged between 115.2 UR and 167.7 UR
for LS and between 18.11 UR and 28.92 UR for
TF in the Cj urban and Mm urban groups, respec-
tively (Table 1). Comparing the irradiated/non-
irradiated ratio values of the groups, we found sig-
nificantly higher values in Cj urban versus Cj rural
and Cj urban versus Mm urban groups (P < 0.0001)
(in the case of LS) and Cj urban and Mm urban
groups (P < 0.01) (in the case of TF parameter),
respectively (Figure 1).
The repair of radio-induced lesions was estimated
by comparing LS and TF values after 2 h of
incubation with those obtained immediately after
irradiation and with the basal levels. Thus, both
evaluated parameters showed considerable simili-
tude between the repair and basal values in the
Cj urban group: 37.46 versus 35.36 (LS) and 8.87
versus 8.44 (TF) (Table 1). In the exposed groups,
the 2 h incubation LS and TF values were higher
than the basal ones, indicating a much poorer
repair process than in the non-polluted areas.
Table 1. Mean values of the Comet assay determinations evaluated by: LS and TF [UR] in four groups
LS Basal LS 2Gy LS Repair TF Basal TF 2Gy TF Repair
Cj urban 35.36 115.2 37.46 8.44 18.11 8.87
Cj rural 44.22 118.9 54.51 10.16 19.46 11.73
Mm urban 80.45 167.7 94.27 15.85 28.92 18.30
Mm rural 59.23 154.9 68.94 12.61 25.31 14.25
LS, Lesion score; TF, Tail factor.
Genotoxic effects of environmental heavy metals on normal cells
P Virag et al.
255
Micronucleus assay
The basal and radio-induced values of the unstable
chromosomal aberrations were evaluated using the
mathematical values obtained in Micronucleus
assay: %Bi+Mni and Nr.Mni/1000BN.
The basal (background) level of the unstable
chromosomal aberrations evaluated by %Bi+Mni
ranged between 2.24 (Mm rural group) and 3.04
(Mm urban group), with an unexpectedly high
level of the parameter in the Cj rural group (3.03).
The Nr.Mni/1000BN parameter had the same ten-
dency, being even higher in the Cj rural group than
in the polluted Mm urban one: 36.63 versus 33.14
(Table 2). The statistical evaluation showed no sig-
nificant differences between these groups.
In vitro irradiation of the cells induces supplement-
ary DNA damage. All values determined immedi-
ately after irradiation were significantly higher
than the basal ones (P < 0.0001), confirming the
results obtained by comet assay regarding the addi-
tional DNA damage produced by -irradiation.
Thus, the %Bi+Mni parameter varied between
24.53 and 30.95 and the Nr.Mni/1000BN had a par-
allel evolution, ranging between 296.30 and 400.00
in Mm rural and Cj rural groups, respectively. Com-
paring the irradiated versus non-irradiated ratio
values between groups, we have not found statisti-
cally significant differences (Figure 2).
Discussion
Heavy metals and their effects on human health
have been extensively studied, although the major-
ity of the published papers reviewed the carcino-
genic effects of the acute, professional exposure to
heavy metals (Hengstler, et al., 2003; Salnikov and
Zhitkovich, 2007).
Since little is known about chronic exposure to
low doses of pollutants, the present study aimed
to evaluate the relationship between exposure to
heavy metals and the DNA lesions and chro-
mosomal aberrations in chronically exposed popu-
lation compared to non-exposed groups. Each
individual is supposed to have a specific reaction
after environmental exposure, influenced by intrin-
sic (genetic) and extrinsic components. Thus, we
found a great interindividual variability of the
basal level of DNA lesions and chromosomal aber-
rations, in accordance with the literature data
(Muller, et al., 2001; Borgmann, et al., 2002). As
it was expected, the highest level of basal DNA
lesions was observed in Mm urban group, followed
by the rural group of the same district, suggesting
that initial genomic alterations are higher in this
A B
0
1
2
3
4
Cj urban
Cj rural
Mm urban
Mm rural
I
r
r
a
d
/
n
o
n
-
i
r
r
a
d

r
a
t
i
o
Figure 1 Comet assay determinations. Irradiated/non-irradiated ratio
values: SL (a), TF (b) in the four groups. Statistical significance was
assessed by one-way ANOVA and Dunnett multiple comparison test,
***significant (P < 0.0001) for SL and **significant (P < 0.01) for TF
ratio values.
Table 2. Mean values of the Micronucleus assay determinations, evaluated by %Bi + Mni and Nr.Mni/1000 BN in four groups
Parameters Cj rural Cj urban Mm rural Mm urban
Basal level %Bi+Mni 3.03 2.57 2.24 3.04
Nr.Mni/1000BN 36.63 27.92 25.04 33.14
2 Gy %Bi+Mni 30.95 27.36 24.53 26.46
Nr.Mni/1000BN 400.00 340.70 296.30 356.70
A B
0
5
10
15
20
25
Cj urban
Cj rural
Mm urban
Mm rural
I
r
r
a
d
/
N
o
n
-
i
r
r
a
d

r
a
t
i
o
Figure 2 Micronucleus assay determinations. Irradiated/non-
irradiated ratio values: %Bi+Mni (A) and Mni/1000Bi (B) in the four
groups. Statistical significance was assessed by one-way ANOVA and
Dunnett multiple comparison test.
Genotoxic effects of environmental heavy metals on normal cells
P Virag et al.
256
area, known as being a polluted one. We found an
unexpectedly high level of spontaneous unstable
chromosomal aberrations in Cj rural group, which
has not been sustained by DNA lesions. This could
indicate a lack or a delay in the repair mechanisms
of the unstable chromosomal aberrations in a
non-exposed population to genotoxic agents or a
more pronounced sensibility to detect genetic
modifications by Comet versus Micronucleus
assay. In-vitro administration of a genotoxic agent
(-irradiation) was followed by a significant
enhancement in DNA lesions and chromosomal
alterations in all groups. The amplitude of this pro-
cess differed considerably between groups and indi-
viduals, even if the applied genotoxic agent was
identical. One of the explanations could be the
modulation of the effective irradiation dose on
DNA by some protective enzymes and metabolites
of the cellular content (Mazur, 2000). In our study,
non-exposed subjects from Cluj district, especially
urban zone, appeared to be more susceptible to the
induction of DNA lesions and chromosomal aber-
rations by irradiation. The repair capacity of DNA
is a parameter which characterizes the ability of
cells to remove lesions and recover the genome
integrity. Some studies reported correlations
between DNA repair disorders and predisposition
to diseases, including cancer (Olive, 2002). Muta-
tions in base or nucleotide excision repair genes
may influence the depth of DNA lesions induced
by irradiation (Olive, 2002; Popanda, et al., 2003),
thus the characterization of DNA repair capacity
might be useful in the identification of persons
with increased susceptibility to cancer. The most
efficient repair capacity expressed by the recovery
of LS and TF parameters was registered in Cj
urban group, the 2 h values of these parameters
practically overlapping the initial, basal ones. On
the contrary, in Mm urban group, we found the
most evident deviations from this pattern, which
sustain the existence of a great number of persons
with deficiencies of the DNA repair processes.
Comet and Micronucleus assays have been
proved to be useful in epidemiological studies, as
rapid and sensitive methods, which require a
reduced number of cells for analyses (Palyvoda,
et al., 2003; Popanda, et al., 2003). Basal and sup-
plementary genetic changes, induced by genotoxic
agents evaluated by these methods, as well as the
efficiency of intracellular repair processes evaluated
by Comet assay, might be useful as potential bio-
markers of the exposure to genotoxic agents, such
as heavy metals.
Acknowledgement
We gratefully acknowledge financial support from
the BILATHUROM, ANCS National Grant,
Romania.
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