Indian Journal of Experimental Biology
Vol 44, December 2006, pp. 997-1005
Cytogenetic effects of a mixture of selected metals following subchronic exposure
through drinking water in male rats
S H Jadhav*, S N Sarkar** & H C Tripathi†
Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, India
Received 1 May 2006; revised 11 September 2006
The current study examines the genotoxic effects of subchronic exposure via drinking water to a mixture of eight
metals (arsenic, cadmium, lead, mercury, chromium, nickel, manganese and iron) found as contaminants of water sources in
different parts of India and its possible association with oxidative stress. Male rats were exposed to the mixture at 0, 1, 10
and 100 times the mode concentration of each metal daily for 90 days. Another dose group at concentration equivalent to
maximum permissible limit (MPL) for each metal and a reference group given ip cyclophosphamide were incorporated. The
mixture at 100× level significantly increased chromosomal aberrations and micronuclei induction (2.4 folds) in bone marrow
cells and reduced the ratio of polychromatic to normochromatic erythrocytes by 25%. The mixture significantly increased
sister chromatid exchange in bone marrow (1.67 and 2.3 folds) and spleen (1.57 and 1.98 folds) cells with both 10× and
100× doses. Cyclophosphamide was more potent than the mixture in causing cytogenetic damage in these parameters. In rat
spleen, the mixture at 10× and 100× doses caused dose-dependent increase in lipid peroxidation (25.95 and 52.71%) and
decrease in the activities of superoxide dismutase (20.36 and 40.62%), catalase (18.24 and 35.50%), glutathione peroxidase
(22.33 and 36.12%) and glutathione reductase (19.22 and 31.35%) and in the level of GSH (19.76 and 35.15%). The results
suggest that the mixture induced genotoxicity in rat bone marrow and spleen cells at concentrations relatively higher than
that found in groundwater sources and the genotoxic effect could relate to induction of oxidative stress. However,
observations with lower doses indicate that additive or synergistic interactions following exposure to metal components at
MPL levels or at mode concentrations of contemporary groundwater levels in India may not result in clastogenicity in male
rats.
Keywords: Genotoxicity, Groundwater contaminants, Metal mixture, Oxidative stress, Rat
Exposure to toxic metals has become an increasingly
recognized source of illness worldwide1. Heavy
metals are ubiquitous in the environment and found in
hazardous concentrations in air, food and water1.
Exposure through food, water and occupational
sources can contribute to a spectrum of diseases1.
Groundwater contamination is a very important
source of environmental exposure to toxic metals2, 3.
It’s a global public health problem, affecting both the
industrialized and developing nations. Metal
contamination reflects natural as well as
anthropogenic sources in which many metals occur in
association with one another. Contamination of water
resources with various metals, including arsenic,
_________________
**
Correspondent author
Phone: 91-581-2300291
Fax: 91-581- 2303284
E-mail address: [email protected]
+
Since deceased
*
Present address: Department of Pharmacogenomics, School of
Pharmacy, 3307 N. Broad Street, Philadelphia PA-19104, USA
cadmium, lead, mercury, chromium, nickel,
manganese and iron, is a widespread problem in
India2-7. Metals like arsenic, chromium, cadmium,
nickel, iron and lead are carcinogenic in human and/or
animal studies8. A number of experimental and
epidemiological studies on the genotoxic effects of
heavy metals and their compounds have been
published. These demonstrate that arsenic9, 10,
cadmium11, 12, mercury13, 14, lead15, chromium16, 17,
nickel18, 19, manganese20, 21 and iron22, 23 are clastogens
inducing chromosomal aberrations, micronuclei
induction, sister chromatid exchange (SCE) and
chromosomal loss in human and animal cells.
Metal-induced oxidative stress has been shown to
cause DNA damage through the production of
reactive oxygen species (ROS) such as superoxide
anion radical (•O2–), hydrogen peroxide (H2O2) and
hydroxyl radical (OH•)24. There is considerable
evidence that ROS-mediated oxidative damage
induced by several metals is an underlying basis of
their genotoxicity10, 13, 18, 19, 24. Another important
998
INDIAN J EXP BIOL, DECEMBER 2006
mechanism of metal-induced genotoxicity has been
attributed to their ability to react with the sulfhydryl
(-SH) groups24, 25. It is known that cellular nonprotein
-SHs consist essentially of reduced glutathione
(GSH, ≈95%) and other low-molecular-weight
aminothiols such as cysteine and cysteamine. The
metal binding to -SH groups of glutathione blocks its
function as a free radical scavenger. Thus, free
radicals become available to cause DNA damage.
These mechanisms can lead to double-strand breaks
that can be visualized as the chromatid gaps and/or
give rise to chromosomal alterations such as breaks,
fragments, exchange and rearrangements26.
Current understanding of genotoxicity of these
metals is primarily based on genotoxicity studies
performed on laboratory animals exposed to a single
agent. The reported effects are seen mostly in high
levels of exposure. The human and animal
populations are ubiquitously exposed to complex
mixtures of these metals generally at much lower
levels than those routinely examined in animal studies
and the effects of any interactions between such
metals on mammalian cytogenetics is virtually
unknown. Groundwater meets 80% of India’s
drinking water requirement27. Since each hazardous
site or geologically contaminated groundwater source
has a unique set of conditions, it is practically
impossible to find a representative sample of various
sites. Hence, to simulate a worst real-life scenario, a
mixture of eight metals was designed with a relative
concentration, as recorded through literature survey,
similar to that determined in the groundwater sources
of India. The purpose of the current study was to
investigate the genotoxic potential of the mixture of
metals at environmentally realistic doses, relative to
high doses generally used in animal toxicity
experiments, and to assess whether the genotoxic
effect could be linked to cellular oxidative stress. The
genotoxic potential of the mixture was also examined
at a dose level, where each metal was incorporated in
the mixture at the drinking water maximum
permissible limit (MPL) as determined by the World
Health Organization (WHO).
Materials and Methods
Selection of metals ⎯ The mixture was formulated
to reflect the types of frequently occurring metals to
which general populations of India could conceivably
be exposed through drinking water at concentrations
above the maximum permissible limit (MPL). Data on
contamination of water resources in different parts of
India with various metals were collected through
literature survey. From the reported concentrations of
each metal, the mode concentrations of these metals
were derived by group frequency distribution. The
metals were selected on the basis of two criteria: (1)
frequency of occurrence, and (2) contamination level
above MPL in drinking water. A minimum of 15 to a
maximum of 30 reports were considered for selection
of an individual metal.
Chemicals⎯Cadmium chloride, mercuric chloride,
chromium trioxide, lead acetate and nickel chloride
were procured from E. Merck (India) Ltd., Mumbai.
Sodium arsenite, manganese chloride and ferric
chloride were purchased from Sigma Chemicals,
USA; S. D. Fine Chemicals, Mumbai and Himedia,
Mumbai, respectively. All other chemicals used in the
study were of analytical grade from Sigma Chemicals,
USA; E. Merck, Germany/India; SRL Chemicals,
India.
Mixture formulation ⎯ The mixture was prepared
following the protocol reported by Wade et al28. All
the metal salts, which are soluble in water, were
weighed into a glass bottle in amounts appropriate to
make the 100× stock. Lower doses (1× and 10×) of
the dosing solution were prepared by 10-fold serial
dilution of the 100× stock. Dosing solution for the
concentrations equivalent to the MPL (WHO) was
prepared separately. Dosing solutions were prepared
daily.
Animals and experimental protocol ⎯ Male Wistar
rats (100-120 g) procured from the Laboratory
Animal Resources Section of the Institute were
acclimatized to holding facilities for one week prior to
commencement of dosing. They were caged in pairs
in clean plastic cages containing wheat straw chips for
bedding and maintained under standard management
conditions. All animals were given standard pellet
diet (Amrut Inbred Rat and Mice Feed, Pranav Agro
Industries, Delhi) and deionized water ad libitum.
Animal care and handling were in accordance with
the Institute Animal Ethics Guidelines. Rats were
exposed daily to the mixture of metal components
through drinking water (deionized) for 90 days. Six
groups of rats comprising 15 animals each were used.
The mixture of metals and its dose levels are
presented in Table 1. Group I was given only
deionized water and served as control. Group II, the
baseline dose group (1×), was given mode
concentrations of individual metals. Groups III and IV
were, respectively, set at 10- and 100-fold
JADHAV et al.: GENOTOXICITY OF A MIXTURE OF METALS IN RATS
999
Table 1 ⎯ The mixture of metals and its dose levels for subchronic toxicity studies in male rats
Metal salt
Sodium arsenite
Cadmium chloride
Lead acetate
Mercuric chloride
Chromium trioxide
Nickel chloride
Manganese chloride
Ferric chloride
Mode concentration (ppm)a
0.200 (15)
0.055 (30)
0.120 (23)
0.044 (17)
0.180 (15)
0.100 (23)
0.560 (15)
0.700 (29)
Control
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1x
0.380
0.098
0.220
0.060
0.346
0.810
2.026
2.033
Dose levels (ppm)b
10x
3.80
0.98
2.20
0.60
3.46
8.10
20.26
20.33
100x
38.0
9.8
22.0
6.0
34.6
81.0
202.6
203.3
MPL
0.096
0.005
0.018
0.008
0.141
0.081
1.800
0.870
a
Figures in parentheses indicate the number of reports studied to derive the mode concentration of each metal. bThe baseline dose (1x)
for each chemical was adjusted to give representative elemental concentration of mode concentration and safety factors (10x and 100x)
were then applied accordingly. WHO maximum permissible limit (MPL) in drinking water was also adjusted to give representative
elemental concentration.
concentrations of those of the baseline group. Rats of
Group V received metals at concentrations equivalent
to the MPL (WHO) in drinking water (Table 1), while
rats of Group VI (Positive control) were injected i.p.
with cyclophosphamide at 20 mg/kg b. wt. in normal
sterile saline. During the course of experiment, 2 to 3
rats died in each group, including control. None of the
animals died showed any visible signs of toxicity.
However, to maintain the uniformity of sample size of
all groups, six of the surviving animals were used for
chromosomal aberration assay, while another six were
used for assessment of micronuclei induction, SCE
and oxidative stress. Rats were killed by cervical
dislocation after 24 h of the last exposure.
Assessment of chromosomal aberration ⎯
Preparation of bone marrow and assessment of
chromosomal aberration were carried out as described
by Malhi and Grover29 and Chauhan et al.30. Briefly,
24 hr prior to their sacrifice, colchicine (4 mg/kg in
water) was injected ip to arrest mitosis. After
sacrifice, both femurs were immediately dissected out
and cut at the both ends with bone snips. The marrow
was aspirated from the body of the femurs in Hank’s
balance salt solution (HBSS, pH 7.2) and cell
suspension was centrifuged for 10 min at 1000 rpm.
The pellet was resuspended in hypotonic solution of
0.56% (w/v) KCl for 30 min at 37 oC to allow osmotic
swelling of cells. The cells were centrifuged and the
pellets were resuspended in chilled Carnoy’s 3:1
methanol and glacial acetic acid fixative. After 2 hr of
fixation, the cells were centrifuged and resuspended
in the same fixative for 24 hr. The fixed cells were
then dropped on chilled and labelled slides, air dried
for 24 hr, stained with 2% Geimsa for 15-25 min,
dehydrated in 1:1 acetone and xylene mixture, pure
xylene and mounted with DPX. Fifty well spread
metaphase plates per rat were examined. Cells with
severe fragmentation of chromosomes (≥10
aberrations) were classified as pulverized and scored
singly for expressing the frequency of aberrations and
aberrant cells. Chromatid/isochromatid gaps and
endomitotic reduplication were recorded but not
included in the total of aberrations or aberrant cells.
Assessment of micronucleus induction ⎯ Isolation
of bone marrow cells and preparation of slides for
micronucleus assay were carried out following the
methods of Hayashi et al31 and Chauhan et. al30.
Briefly, marrow from femur was aspirated in the
HBSS (pH 7.2), containing EDTA and bovine serum
albumin and cell suspension was centrifuged for
10 min at 1000 rpm. The cells in the sediment were
mixed carefully. A drop of cell suspension was
smeared on clean slide, fixed with methanol for 5 min
and stained with acridine orange for 3 min at room
temperature. The slides were rinsed thrice with the
Sorensens’s buffer (pH 6.8) for 1-3 min each time,
mounted with the same buffer and sealed with Balsam
paraffin. Observations were made within a day. Prior
to scoring, slides were randomly coded so that the
scorer was unaware of the treatment group from
which each slide originated. The frequencies of
micronucleated cells were determined by scoring a
minimum of 2000 polychromatic erythrocytes (PCE)
per animal, while 200 bone marrow erythrocytes per
animal were examined to derive the ratio of PCE to
normochromatic erythrocytes (NCE).
Sister chromatid exchange (SCE) in bone marrow
and spleen cells ⎯ SCE assay was performed in bone
marrow cells and splenocytes as described by Perry
and Wolf32 and Krishna et al33. Briefly, after sacrifice
of rats, spleen and both the femurs were excised. The
1000
INDIAN J EXP BIOL, DECEMBER 2006
marrow was flushed out with Ham’s F 10 (HF-10)
medium in 5 ml sterilized vials containing HF-10
complete culture medium. Spleen was smashed with
HF-10 medium and the debris was removed. The cells
were washed with PBS containing 2% heat activated
fetal bovine serum and centrifuged at 250 × g for 6
min. The final cell suspension was cultured in 5 ml
tissue culture sterilized vials. Bone marrow and
splenocyte culture vials were incubated at 37oC in a
BOD incubator for 30 min and 40 hr, respectively.
After incubation, colchicine (30 µM) was added to
both the cultures. The cells were harvested by
decanting the contents of the vials into 15 ml
centrifuge tubes. Each vial was rinsed with 3 ml
HBSS, which was also transferred to the centrifuge
tube. The tubes were centrifuged at 285 × g for 6 min
and cells were processed for slide preparation as
described elsewhere for chromosomal aberration
assay. Staining was done according to the modified
technique of Perry and Wolf32. Slides were stained for
15 min with Hoechst 33258 (Sigma, 5 µg/ml) and
rinsed briefly in deionized water. The slides were then
mounted temporarily in citrate buffer (pH 6.8). The
cover slips were sealed with molten wax to prevent
evaporation. The same slides were then exposed to
ultraviolet light for 10 to 24 hr and examined
intermittently for differential fluorescence of the
chromatids. After removing the cover slips, slides
were incubated in distilled water for 2 hr (60°C) and
stained for 30 min in 2% Giemsa solution. Scoring
was done in 30 well differentially stained metaphases
per rat and the results were expressed as SCE/cell.
Preparation of spleen homogenate ⎯ After
sacrifice of rats, spleen was removed, cleaned and a
piece of adequate amount was stored at −80°C.
Frozen spleen samples were partially thawed and 200
mg of each sample was taken in 2 ml ice-cold normal
saline. Another 200 mg was taken in 2 ml of 0.02 M
EDTA for GSH estimation. Tissue homogenate (10%)
was prepared in ice-cold normal saline and
centrifuged for 10 min at 3000 rpm. The supernatant
was used for estimation of the following biochemical
attributes for assessment of induction of oxidative
stress in spleen.
Assay of lipid peroxidation (LPO) ⎯ LPO was
assayed by the method of Shafiq-U-Rehman34 and
expressed as nmol malondialdehyde/g wet tissue. In
brief, 1 ml of homogenate was incubated at 37°±0.5°
C for 2 hr. To each sample, 1 ml of 10% w/v
trichloroacetic acid was added. After thorough
mixing, the mixture was centrifuged at 2000 rpm for
10 min. To 1 ml of supernatant, an equal volume of
0.67% thiobarbituric acid was added and kept in
boiling water bath for 10 min. After cooling, it was
diluted with 1 ml of distilled water (DW). The
absorbance was read at 535 nm.
Determination of GSH level ⎯ GSH content was
determined by estimating free –SH groups, using 5,5/dithiobis-2-nitrobenzoic (DTNB) acid method of
Sedlak and Lindsay35 and expressed as mmol GSH/g
wet tissue. Briefly, to 1 ml supernatant, 0.8 ml of DW
and 0.2 ml of 50% trichloroacetic acid were added
and incubated at room temperature for 15 min;
centrifuged at 3000 rpm for 15 min. From this, 0.4 ml
supernatant was added to 0.8 ml of 1 M Tris buffer
(pH 8.9) followed by 0.2 ml of DTNB (0.01 M) and
absorbance was read at 412 nm within 5 min.
Determination of superoxide dismutase (SOD)
activity ⎯ SOD activity was measured by the
procedure of Madesh and Balasubramanian36. In brief,
the reaction mixture contained 0.65 ml PBS saline
(pH 7.4), 30 μl 3-(4-5-dimethylthiazol 2-xl) 2,5diphenyltetrazolium bromide (MTT; 1.25 mM), 75 μl
pyrogallol (100 μM) and 10 μl homogenate. The
mixture was incubated at room temperature for 5 min
and the reaction was stopped by adding 0.75 ml of
dimethyl sulfoxide. The absorbance was read at 570
nm against DW and the activity has been expressed as
Unit. One unit is the μg of protein required to inhibit
MTT reduction by 50%.
Determination of catalase activity ⎯ Catalase
activity was assayed by the method of Aebi37 and
expressed as k/g wet tissue. One k stands for nmol
H2O2 utilized/min. Briefly, 2 ml PBS (pH 7.4) and
10 μl homogenate were taken in a cuvette. Reaction
was started by adding 1 ml H2O2 (20 mM) and the
absorbance was recorded at every 10 sec for 1 min at
240 nm against water blank.
Determination of glutathione peroxidase (GPx)
activity ⎯ GPX activity was assayed by the method
of Paglia and Valentine38 and expressed as unit/mg
protein. One unit (U) is the nmol NADPH
utilized/min/mg protein at 25o C. The reaction mixture
contained 2.48 ml PBS (pH 7, containing 5 mM
EDTA), 0.1 ml NADPH (8.4 mM), 0.1 ml GSH
(150 mM), 0.01 ml sodium azide (112.5 mM), 4.6 U
glutathione reductase (Type III; Sigma Chemicals,
USA) and 10 μl of homogenate. The reaction was
initiated by adding 0.1 ml H2O2 (2.2 mM) to the
JADHAV et al.: GENOTOXICITY OF A MIXTURE OF METALS IN RATS
mixture containing 500-1000 μg protein. The change
in absorbance was read at 540 nm for 4 min.
Determination of glutathione reductase (GR)
activity ⎯ GR activity was measured by the method
of Goldberg and Spooner39 and expressed as unit/mg
protein. One unit is the nmol NADPH
utilized/min/mg protein at 25o C. The 3 ml of reaction
mixture contained 2.6 ml PBS (0.12 M, pH 7.2),
0.1 ml EDTA (15 mM), 0.1 ml GSSG (65.3 mM). To
this 10 μl of homogenate was added and the volume
was made up to 2.95 ml with DW. After incubation at
room temperature for 5 min, 0.05 ml of NADPH
(9.6 mM) was added. Decrease in absorbance/min
was recorded immediately at 340 nm for 3 min.
Control was run without GSSG.
Estimation of tissue protein ⎯ Protein level in the
homogenate was measured by the method of Lowry
et al.40, using bovine serum albumin as the standard.
Statistical analysis ⎯ Data were statistically
analyzed using one-way analysis of variance
(ANOVA) followed by Dunnett’s t-test to compare
the cytogenetic effects and biochemical changes
induced by different doses of the metal mixture in
bone marrow and spleen. Differences were considered
significant at the probability level of P < 0.05.
Results
Chromosomal aberrations ⎯ Chromosomal
aberrations,
viz.,
gaps,
breaks,
exchange,
pulverization and endoreduplication induced with the
different doses of the metal mixture in bone marrow
cells of rats are presented in Table 2. The mixture
increased the frequencies of chromosomal aberrations
and aberrant cells in a dose-dependent fashion
compared to the control animals (Table 2). However,
1001
the increase was significant only in rats given 100x
dose or the reference drug, cyclophosphamide. The
effect induced by cyclophosphamide was significantly
more than that of 100× dose.
Micronuclei formation ⎯ Induction of micronuclei
in bone marrow cells and ratio of PCE to erythrocytes
of rats are presented in Table 3. The mixture of metals
caused dose-related decrease in the ratio of PCE to
erythrocytes and increase in micronuclei induction.
However, significant changes in these cytogenetic end
points were observed in rats of 100× dose group. At
this dose, the PCE/erythrocyte ratio was reduced by
25% and there was 2.4-fold increase in micronuclei
induction. Effects induced by MPL, 1× and 10× doses
were not statistically significant. Cyclophosphamide
was significantly more effective than the metal mixture
in reducing the PCE/erythrocyte ratio (44.7%) and
increasing the micronuclei induction (5.47 fold).
Table 3 ⎯ Micronuclei induction in bone marrow cells of rats
exposed to the mixture of metals for 90 days through drinking
water
[Values are mean ± SE from 6 observations in each group]
Group
PCEs/200
erythrocytes
Micronuclei/2000
PCEs
Control
MPL
1×
10×
100×
Positive control
111.50 ± 7.27a
107.67 ± 7.84a
103.50 ± 9.19a
95.17 ± 6.73ab
83.50 ± 7.92b
61.67 ± 4.89c
2.16 ± 0.30a
2.50 ± 0.67a
2.83 ± 0.70ab
3.50 ± 0.84ab
5.17 ± 1.27b
11.83 ± 1.57c
MPL: Maximum permissible limit in drinking water (WHO).
Positive control: Rats were given ip cyclophosphamide at 20
mg/kg. PCE: Polychromatic erythrocytes. Values in the same
column bearing no superscript common vary significantly
(P < 0.05).
Table 2 ⎯ Chromosomal aberrations in bone marrow cells of male rats exposed to the mixture of metals for 90 days through drinking
water
Group
Control
MPL
1×
10×
100×
Positive
Control
Cells/Rat Gaps* Breaks Fragment Exchange Pulveri- Endoresation duplication
Total
Frequency of
aberrations (%)
Frequency of
aberrant cells
(%)
320/6
308/6
314/6
318/6
325/6
6 (2)
9 (4)
10 (3)
7 (2)
8 (3)
1
1
2
4
7
1
2
2
1
2
–
–
2
1
2
2
3
1
3
5
–
6
9
6
8
A
4
6
7
9
14
B
2
3
6
7
9
A
B
1.28±0.62a 0.66±0.42a
1.92±0.69a 1.00±0.44a
2.19±0.56a 1.86±0.67ab
2.82±0.42ab 2.19±0.75ab
4.31±0.62b 2.76±0.41b
1.26±0.40a
1.92±0.69ab
1.95±0.56ab
2.54±0.65ab
3.27+0.93b
310/6
12 (1)
11
2
2
6
8
21
15
6.77±0.42c
5.26±0.63c
4.80±0.78c
MPL: Maximum permissible limit in drinking water (WHO). Rats of positive control group were given ip cyclophosphamide @ 20
mg/kg. *Values in parenthesis indicate isochromatic gaps; A = Excluding endoreduplication and gaps; B = Excluding pulveristion.
Values (n = 6, mean ± SE) for frequency of aberrations (%) and aberrant cells (%) in the same column bearing no superscript common
vary significantly (P<0.05).
INDIAN J EXP BIOL, DECEMBER 2006
1002
Sister chromatid exchange (SCE) ⎯ Effects of the
mixture of metals on SCE in bone marrow and spleen
cells of rats are presented in Table 4. In both types of
the cells, effect of the mixture on SCE was dosedependent. SCEs produced by the lower exposure
levels (MPL and 1×) were not significantly different
from the control rats. But, it was significantly
increased with 10× and 100× exposure levels by 1.67
and 2.3 folds in bone marrow cells and by 1.57 and
1.98 folds in splenic cells, respectively.
Cyclophosphamide was significantly more potent than
the metal mixture in escalating the SCE in bone
marrow cells (7.41 fold) and spleen cells (6.63 fold).
Oxidative stress in spleen ⎯ Effects of the metals
mixture on oxidative stress-related indices in spleen
are presented in Table 5. The mixture at MPL and 1×
dose levels did not significantly alter any of the spleen
biochemical attributes examined. Compared to control
rats, LPO was significantly increased with 10x
(25.95%) and 100x (52.71%) doses. These two higher
doses significantly decreased all the antioxidant
parameters assayed. With these doses, the activity of
SOD was decreased by 20.36 and 40.62%, catalase by
Table 4 ⎯ Sister-chromatid exchange (SCE) frequencies in bone
marrow and spleen cells of rats exposed to the mixture of metals
for 90 days through drinking water
[Values are mean ± SE from 6 observations in each group]
Group
SCE/bone marrow cell
Control
MPL
1×
10×
100×
Positive control
a
2.15 ± 0.11
2.30 ± 0.30a
2.73 ± 0.34a
3.59 ± 0.39b
4.91 ± 0.65b
15.94 ± 1.29c
SCE/spleen cell
2.68 ± 0.27a
3.07 ± 0.28ab
3.50 ± 0.34ab
4.22 ± 0.29b
5.30 ± 0.34b
17.77 ± 0.99c
MPL: Maximum permissible limit in drinking water (WHO).
Positive control: Rats were given ip cyclophosphamide at 20
mg/kg. Values in the same column bearing no superscript
common vary significantly (P < 0.05).
18.24 and 35.50%, GPx by 22.33 and 36.12% and GR
by 19.22 and 31.35%, while the level of GSH by
19.76 and 35.15%, respectively.
Discussion
While considerable amount of research has been
devoted to the genotoxicity of single exposures to
various metals and their compounds, very less
emphasis has been given to evaluation of the
genotoxic potential of mixtures of metals. The present
results demonstrate that subchronic exposure to the
mixture of metals, found as contaminants in various
water sources of India, produces genotoxicity in rat
bone marrow and spleen cells in relatively high doses
(10× and 100×) and the cytogenetic effects were
associated with dose-dependent increase in LPO and
decrease in the enzymatic and nonenzymatic
antioxidative systems in spleen. It is interesting to
note that, in quantitative terms (% change), the impact
of the mixture on all the antioxidative attributes was
almost identical. However, no significant impact of
the MPL and 1× dose levels on cytogenetic tests and
oxidative stress paradigm suggests that the
interactions among the coexposed mixture
components in these exposure levels were not enough
to induce any chromosomal damage in bone marrow
and spleen cells, and oxidative stress in spleen. This,
in spite of the known genotoxic and oxidative stressinducing10-13, 17, 18, 24, 41 effects of these metals in
various cells, offers some support for the assumption
that derivation of safe levels of exposure from single
agent toxicity studies provides reasonable protection
from adverse effects in male animals.
Cytogenetic effects induced by the mixture in bone
marrow cells indicate its toxic implications on bone
marrow. This is also evident by the significant
reduction in PCE/erythrocytes ratio in bone marrow
cells. The mixture components such as arsenic9, 10,
cadmium11, 12, mercury13, 14, lead15, chromium16, 17,
Table 5 ⎯ Oxidative stress-related indices in spleen of rats exposed to the mixture of metals for 90 days through drinking water
[Values are mean ± SE from 6 observations in each group]
Group
Control
MPL
1×
10×
100×
Lipid peroxidation
(nmol MDA/g wet tissue)
Reduced glutathione
(mmol/g wet tissue)
21.73 ± 1.31a
23.20 ± 1.73a
24.33 ± 1.19a
27.37 ± 1.95b
33.18 ± 1.51c
0.77 ± 0.03a
0.74 ± 0.04a
0.70 ± 0.02ab
0.63 ± 0.04b
0.50 ± 0.04c
Superoxide
dismutase
(Unit)
Catalase
(k/g wet tissue)
26.36 ± 2.26a 307.02 ± 15.96a
25.39 ± 1.60a 297.42 ± 8.60a
23.70 ± 1.50ab 283.57 ± 9.37a
20.86 ± 1.27b 251.25 ± 6.63b
15.65 ± 1.36c 198.41 ± 10.38c
Glutathione
Glutathione
peroxidase
reductase
(Unit/mg protein) (Unit/mg protein)
61.98 ± 7.18a
58.99 ± 4.91ab
55.29 ± 3.14ab
48.26 ± 4.44b
39.58 ± 3.66c
146.97 ± 6.30a
138.99 ± 6.99a
130.60 ± 6.0ab
118.72 ± 6.98bc
110.63 ± 8.04c
MPL: Maximum permissible limit in drinking water (WHO). MDA: Malondialdehyde. Values in the same column bearing no superscript
common vary significantly (P < 0.05).
JADHAV et al.: GENOTOXICITY OF A MIXTURE OF METALS IN RATS
nickel18, 19, manganese20, 21 and iron22, 23 have been
reported to produce cytogenetic damage, including
chromosomal aberrations, micronuclei induction and
SCE in bone marrow and other cells. The SCE assay
is recognized as a good index of DNA damage and
can be detected at lower doses of the compounds than
those required to induce chromosomal aberrations42.
In the present study, the mixture of metals caused
significant increase in SCE with 10× and 100× doses
in both bone marrow and spleen cells. While
chromosomal aberrations and micronuclei induction
were observed with the highest dose only (100×). This
shows that the present results are in agreement with
the observation of Latt et al.42 and the SCE was more
sensitive than others assays in the current study for
detection of cytogenetic effect of the metal mixture.
Genotoxic metals can directly or indirectly damage
DNA; it means an increased risk of cancer.The toxic
metals may lower the genetic stability predominantly
by two modes of action; the induction of oxidative
DNA damage and the interaction with DNA repair
processes19, 41. Experimental studies in mammalian
cells have demonstrated that active oxygen radicals
may contribute to clastogenesis directly43 and
indirectly through the production of lipid peroxides44.
Increased
production
of
lipid
peroxide,
malondialdehyde, and decrease in antioxidative
systems, as observed in the present study, suggest that
the mixture caused generation of ROS and oxidative
stress in rat spleen. This indicates that free radicalinduced oxidative damage to DNA could be a factor
in mediating the cytogenetic changes in spleen cells
with the metal mixture. Malondialdehyde, a major end
product and biomarker of LPO, reacts with
deoxyribonucleosides to produce DNA adducts and is
documented as mutagenic in mammalian cells24,45.
Decrease in the activities of SOD, catalase and GPx
may lead to accumulation of •O2– and H2O2 in the cell.
H2O2 and •O2– themselves can cause DNA damage in
the form of chromosome breakage, rearrangement and
SCE10, 26, 46. ROS, particularly OH•, play an important
role in the genotoxicity of metals, including arsenic
and nickel in mammalian cells10, 18. SOD acts as an
antipoliferative agent, anticarcinogen and inhibitor at
initiation and promotion/transformation stage in
carcinogenesis46. Addition of the •O2– scavenging
enzyme, SOD, reduces the frequency of arseniteinduced SCEs47. GPx and catalase are important in the
inactivation of many environmental mutagens.
Catalase and GPx have been reported to reduce the
1003
arsenite-induced micronuclei in Xrs-5 cells48. Catalase
reduces the SCE levels and chromosomal
aberrations46. These enzymatic antioxidants can
prevent genetic changes by inhibiting DNA damage
induced by the reactive oxygen molecules46.
Hartwig41 reported that metals such as arsenic,
cadmium, lead, nickel and cobalt cause inhibition of
DNA repair processes at low, non-cytotoxic
concentrations of the respective metal compounds,
and concluded that even though different steps in
DNA repair are affected by the diverse metals, one
common mechanism may be the competition with
essential metal ions. Therefore, in the present study,
the observed cytogenetic effects of the metal mixture
may relate to oxidative stress-induced damage to
DNA, interference with the DNA repair process and
substitution of cellular essential metal ions.
It may be concluded that the mixture of eight heavy
metals induced genotoxicity in rat bone marrow and
spleen cells and oxidative stress in spleen at
concentrations relatively higher than that found in
groundwater sources. The oxidative stress could be
the underlying basis of the metal mixture-induced
cytogenetic effects. Results with lower doses suggest
that additive or synergistic effects of exposure to
metal contaminants at MPL levels derived by the
WHO or at concentrations representative of
contemporary Indian groundwater levels are unlikely
to induce oxidative stress and result in adverse effects
on the cytogenetics of male rats. However, it would
be important to examine the degree to which changes
in these sensitive cytogenetic end points in male rats
can predict carcinogenic potential of the mixture in
order to determine the significance of genotoxicity of
this mixture.
Acknowledgement
The Senior Research Fellowship awarded to the
first author by the Institute is gratefully
acknowledged. The authors are grateful to Dr. S. M.
Deb, Division of Animal Genetics, for assistance in
cytogenetic assays.
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