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Genotoxicity and oxidative stress in chromium-exposed tannery workers in north India

Khushboo Ambreen, Faizan Haider Khan, Smrati Bhadauria and Sudhir Kumar
Toxicol Ind Health published online 29 August 2012
DOI: 10.1177/0748233712457447

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Article
Toxicology and Industrial Health
1–10
Genotoxicity and oxidative stress in © The Author(s) 2012
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chromium-exposed tannery workers DOI: 10.1177/0748233712457447
tih.sagepub.com
in north India

Khushboo Ambreen1, Faizan Haider Khan1,

Smrati Bhadauria2 and Sudhir Kumar1

Abstract
Trivalent chromium (Cr) is an environmental contaminant, which is extensively used in tanning industries
throughout the world and causes various forms of health hazards in tannery workers. Therefore, a cross-
sectional study design was used to evaluate the DNA damage and oxidative stress condition in tannery workers
exposed to Cr in north India. The study population comprised 100 male tanners in the exposed group and 100
healthy males (no history of Cr exposure) in the comparable control group. Baseline characteristics including
age, smoking, alcohol consumption habits and duration of exposure were recorded via interviewing the sub-
jects. Blood Cr level (measured by atomic absorption spectrophotometry), DNA damage (measured by comet
assay) and oxidative stress parameters (malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase
(SOD)) were estimated in both the groups. As a result of statistical analysis, exposed group showed signifi-
cantly higher level of Cr (p < 0.0001), DNA damage (p < 0.0001), MDA (p < 0.0001), SOD (p < 0.05) and lower
level of GSH (p < 0.001) when compared with controls. Smoking, alcohol consumption habits and age had no
significant effect (p > 0.05) on DNA damage and oxidative stress parameters in both the groups. In simple and
multiple correlation analysis, DNA damage and oxidative stress parameters showed significant correlation with
Cr level and duration of exposure in exposed group. The findings of the present study revealed that chronic
occupational exposure to trivalent Cr may cause DNA damage and oxidative stress in tannery workers.

Keywords
Occupational exposure, tannery workers, trivalent chromium, DNA damage, oxidative stress

Introduction production processes of industries causes various

Hazardous nature of heavy metals is one of the key
factors that exert negative influence on human popu-
lation, at certain levels of exposure and absorption
(Ibrahim et al., 2006). Among the metals, chromium
(Cr) has been recognized as one of the widely used
hazardous metals (Cohen et al., 1993). The two most
stable forms in which Cr occurs in the environment
are trivalent and hexavalent states (Sweeney et al.,
1985). Trivalent form of Cr is extensively used as a
basic tanning agent in leather tanning industry. The
damage to the environment by the tanning industries,
which utilize major share of trivalent Cr, is becoming
an acute problem in the country and it is increasing
day by day due to rapid industrialization (Belay,
2010). The occupational exposure of the tannery
workers to the trivalent Cr used in the leather
forms of hazards (Rastogi et al., 2008). Due to the
unawareness of tannery workers, trivalent Cr enter the
body by breathing, eating and by direct cutaneous
contact and increases the risk of dermatitis, ulcers and
perforation of the nasal septum, respiratory illnesses
as well as increased lung and nasal cancer
1
Molecular and Human Genetics Laboratory, Department of
Zoology, University of Lucknow, Lucknow, India
2
Toxicology Division, Central Drug Research Institute, Lucknow,
India
Corresponding author:
Sudhir Kumar, Molecular and Human Genetics Laboratory,
Department of Zoology, University of Lucknow, Lucknow
226007, India
Email: panwarsk@yahoo.com, panwar.molgen@gmail.com

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2 Toxicology and Industrial Health
(Kornhauser et al., 2002). Therefore, trivalent Cr is
considered to be an important health risk factor for the
tannery workers. The wide use of this trivalent Cr, in
tanning industry, has elicited concern over the safety
of tannery workers.
Health hazards such as cancer, asthma, chronic
bronchitis, hypertension, chromosomal aberration,
back pains, metabolic syndrome, hemoglobin (Hb)
changes resulting from iron metabolism and DNA
detriment in lymphocytes have been reported in the
leather tannery workers (Edling et al., 1986; Iaia
et al., 2004; Veyalkin and Milyutin, 2003; Mikoczy
and Hagmar, 2005; Ory et al., 1997; Stupar et al.,
1999; Sbrana et al., 1991). According to Monteiro
Neto et al. (2010), most of the health troubles that the
leather tannery workers encounter in chronic occupa-
tional exposure condition are associated with genetic
damage. Trivalent Cr has been demonstrated to
decrease the fidelity of DNA synthesis (Snow and
Xu, 1991). Khan et al. (2012) reported that population
exposed to trivalent Cr is at high risk for health
hazards. Friedman et al. (1987) reported that trivalent
Cr induces chromosomal aberrations in human lym-
phocytes. They also suggested that trivalent Cr might
possess tumor-promoter-like properties.
Oxidative stress, caused by massive production of
lipid peroxidation products or by depletion of cells
majors antioxidant, has been proposed as one of the
possible mechanisms involved in Cr toxicity (Harris
and Shi, 2003; Pulido and Parish, 2003). In experi-
mental animals, medium- to long-term exposure to
trivalent Cr has resulted in a significant increase in the
excretion of lipid peroxidation products (Goulart
et al., 2005). Lipid peroxidation occurs as a chain
reaction initiated by excess production of free
radicals. In response to deleterious effects of free
radical-induced lipid peroxidation, cells activate
antioxidant defense mechanisms in which superoxide
dismutase (SOD), an enzymatic antioxidants and
reduced glutathione (GSH), a nonenzymatic antioxi-
dants act synergistically with one another to detoxify
the effects of lipid peroxidation (Barber and Harris,
1994; Cheung et al., 2001).
In India, leather tanning industry is one of the
oldest cottage industries, which now has taken a
predominant place in the country’s economy. There
are more than 2500 tanneries in India and nearly
80% of the tanneries are engaged in the chrome tan-
ning process by utilizing trivalent Cr (Sinha et al.,
2006). Thus, the Indian tannery workers are highly
exposed to trivalent Cr. A survey of the literature has
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revealed that adequate human data on the hazardous
nature of trivalent Cr in terms of genotoxic effects and
oxidative stress are scanty (Goulart et al., 2005;
Medeiros et al., 2003; Sellappa et al., 2011). There-
fore, the aim of the present study was to evaluate the
perilous effect of trivalent Cr with special reference to
DNA damage and oxidative stress on north Indian
tannery workers. The result of this study may be help-
ful for the early identification of hazardous effect of
Cr on tannery workers, which will be crucial to reduce
exposure and health hazard.
To the best of our knowledge, in the literature, no
investigation has been reported on north Indian tan-
nery workers to find out the involvement of trivalent
Cr in the progression of genotoxicity and oxidative
stress. In addition, the present study utilized comet
assay as a diagnostic tool to evaluate genetic damage
on tannery workers who are exposed to trivalent Cr,
although some previous studies (Cid et al., 1991;
Medeiros et al., 2003; Sellappa et al., 2011) have used
DNA protein cross-link assay, micronucleus test and
chromosomal aberration assay to explore the geno-
toxicity of trivalent Cr.
Materials and methods
Study subjects
This cross-sectional study was conducted in a total of
200 male subjects including 100 Cr-exposed tannery
male workers and 100 control males, with age ranging
15–65 years. Tannery workers who were actively
involved in the chrome tanning process and had not
been exposed to any radiation treatment for 12 months
before sampling, were recruited from outpatient clinic
at Chaudhary Ehsan Kareem Hospital, Jajmau, Kan-
pur, India. Controls were selected from the general
population of Lucknow- India, with no history of
occupational exposure to Cr and not known to be
exposed to any other environmental or occupational
carcinogens but belonged to the same age group as the
tannery workers. Similar to the exposed subjects,
those controls who had a history of exposure to any
kind of radiation treatment were excluded in this
study. Blood samples (5 ml) were collected in hepar-
inized tubes by technician strictly in accordance with
the Guidelines of Ethical and Bio-safety Committee
of University of Lucknow. Individuals participating
in the proposed study were informed about the nature
of the study and their written consents were obtained.
Each subject was asked to fill an extensive question-
naire regarding age, sex, occupation, residence,
Ambreen et al.
marital status, smoking, alcohol consumption and
duration of Cr exposure and so on.
Blood Cr analysis
Estimation of whole blood Cr was carried out accord-
ing to Granadillo et al. (1994), with slight modifica-
tion. Whole blood of 1 ml was digested with 5 ml,
3 ml and 2 ml of mixture of HNO3:HClO4 (in ratio
of 5:1), respectively, on hot plate in fume hood. The
digested sample was dissolved in 5 ml of distilled
water (DW) and filtered with Whatman filter paper
to obtain clear solution. Finally, Cr concentration was
determined by Atomic Absorption Spectrophotome-
try (AAS) with a graphite furnace and Zeeman
effect background correction (Perkin-Elmer Model
5100PC). Standard curves were included in each run,
obtained by adding known amounts of Cr standard
solution to a control biological sample. No matrix
modifier was used. Cr concentration was expressed
as microgram per liter.
Comet assay
Comet assay was carried out according to the method
of Singh et al. (1988), with slight modification. Whole
blood of 20 ml was used to perform comet assay. Fully
frosted microscopic slides were prepared in duplicate
per person. These slides were first coated with 50 ml
regular melting point agarose (1% in DW) to prepare
base layer and air dried. Base layer second coat
was prepared by placing 200 ml agarose (1% in
phosphate-buffered saline (PBS)) on slide and
covered with cover glass. After applying cover glass,
the slides were allowed to gel at 4 C for 10 min.
Coverslip was removed and second layer was
prepared by mixing 20 ml of whole blood with 80 ml
low melting point agarose (0.5% in PBS), covered
with coverslip and kept at 4 C for 10 min. Coverslip
was again removed and third layer was prepared by
placing 100 ml low melting point agarose (0.5% in
PBS) over second layer and covered with cover glass.
The coverslip was removed and the slide was
immersed in 50 ml cold lysing solution (2.5 M NaCl,
100 mM Na2EDTA, 10 mM Tris, 10% dimethyl
sulfoxide (DMSO), 1% triton-X 100) for 24 h. To
allow unwinding of DNA, the slides were placed in
alkaline electrophoresis buffer (10 N NaOH,
200 mM EDTA, DMSO) for 20 min. These slides
were further allowed to electrophoresis at 18 V
(0.7–1.0 V/cm), 300 mA for 20 min at 4 C (dim light).
After electrophoresis, slides were immersed in
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3
neutralizing solution (400 mM Tris, pH 7.5) for 5 min
and this step was repeated. Slides were then
dehydrated in absolute methanol for 5 min and kept
at room temp to dry. For staining, 75 ml of ethidium
bromide stain was applied as 4–5 small, equally
spaced droplets over the slides and cover glass was
placed. For visualization of DNA damage, slides were
observed using 40 objective fluorescent microscope
(Leica optiphase microscope equipped with a
515–560 nm excitation filter and a 590 nm barrier
filter) with a Biovis comet software.
A total of 50 individual cells were screened per
sample (25 cells from each slide). An undamaged cell
resembles an intact nucleus without a tail and a
damaged cell has the appearance of a comet. Comet
tail length (distance of DNA migration from the body
of the nuclear core) was used to evaluate the extent of
DNA damage and expressed as micrometer.
Malondialdehyde
Blood plasma malondialdehyde (MDA) concentration
was measured by the method of Ohkawa et al. (1979).
Blood plasma of 200 ml was mixed with 1 ml of 20%
acetic acid and then a 200-ml of 8% sodium dodecyl
sulphate was added subsequently (pH adjusted to 4).
It is followed by the addition of 1.5 ml of 0.8%
thiobarbutric acid and 1.1 ml of DW. Reaction
mixture was incubated in boiling water bath for 1 h.
After cooling, 3 ml of n-butanol was added. A clear
butanol fraction thus obtained was used for measuring
the absorbance at 532 nm and the results were
expressed in nanomole per milliliter.
Reduced glutathione (GSH)
Blood GSH concentration was estimated according to
the method of Beutler et al. (1963). Briefly, 200 ml of
whole blood was mixed with 1.8 ml of cold DW and
incubated for 10 min at 37 C. Followeding this,
600 ml of sulphosalicylic acid was added to the reac-
tion mixture and centrifuged at 2000 r/min for 15 min.
To 200 ml of supernatant, 400 ml of phosphate buffer
and 80 ml of 3,5-dithiobis-2-nitrobenzoic acid were
added. The absorbance of the yellow color developed
was measured at 412 nm and the results were
expressed as microgram per milliliter.
Superoxide dismutase (SOD)
SOD activity was assayed according to the method of
McCord and Fridovich (1969), with slight modification.
4 Toxicology and Industrial Health
Table 1. Demographic characteristics of study populations
Characteristics Controls
Age (years) 37.12 + 12.28
Marital status
Married (%) 61 (61.0)
Unmarried (%) 39 (39.0)
Smokera
Yes (%) 37 (37.0)
No (%) 63 (63.0)
Alcohol consumption
Yes (%) 19 (19.0)
No (%) 81 (81.0)
Duration of exposure in years – 20.56 + 5.66
a
Significant.
Briefly, 200 ml of whole blood was washed thrice with
ice cold normal saline by centrifuging three times at
10000 r/min. Red blood cells collected were hemolyzed
by adding 1.5 volume of water. The temperature was
maintained at 0–4 C by means of an ice water mixture.
Hb was then precipitated by the addition of cold ethanol
(300 ml) and chloroform (180 ml). Solutions were mixed
properly on each addition and then centrifuged. The
resulting supernatant containing SOD was taken for the
measurement of its activity. Two reaction setups were
run in parallel for SOD estimation. The tube in first
setup (experimental) received 1.2 ml (0.052 M) of
sodium pyrophosphate, 0.3 ml (186 mM) of phenazine
methosulphate , 0.3 ml (300 mM) of nitroblue tetrazo-
lium and 0.2 ml of enzyme source. The tubes in the sec-
ond setup (reference) received all the reagents
mentioned except the enzyme source. Briefly, 0.8 ml
and l ml DW was added, respectively, in both sets and
finally reactions were started simultaneously by the
addition of 0.2 ml (780 mM) of reduced nicotinamide
adenine dinucleotide (NADH) disodium salt. After an
interval of 90 s, 1 ml of glacial acetic acid was then
added to each reaction tube and absorbance was read
at 560 nm against a blank on spectrophotometer. The
SOD activity was expressed as units per gram of
hemoglobin.
Statistical analysis
The data collected were entered in Microsoft Excel
computer program and checked for any inconsistency.
The results were presented in percentages, mean and
standard deviations. The unpaired t test was used to
compare two mean values and chi-square test was
used to compare dichotomous/categorical variables.
The two-way analysis of variance was used to find out
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Tanners t and p values, w2
39.63 + 6.87 1.78, 0.08
67 (67.0) 0.78, 0.38
33 (33.0)
59 (59.0) 9.70, 0.002a
41 (41.0)
23 (23.0) 0.48, 0.49
77 (77.0)
–
the effect of smoking and alcohol consumption habits
on DNA damage and oxidative stress parameters. The
Pearson’s correlation analysis was carried out to find
out the association pattern of Cr level and duration of
exposure with DNA damage and oxidative stress. The
partial correlation was used to adjust the effect of
smoking and alcohol consumption habits on the corre-
lation pattern. p < 0.05 is being considered as statisti-
cally significant. All the analyses were carried out
using SPSS-15.0 version.
Results
Demographic characteristics
The demographic characteristics of the controls and
tannery workers (exposed group) are shown in
Table 1. A significant difference was found between
controls and exposed groups only with respect to
smoking habit. However, age, marital status and alco-
hol consumption habit showed no significant differ-
ence between controls and exposed group.
Cr concentration
The total Cr concentration in whole blood of the controls
and exposed group are illustrated in Figure 1. Exposed
group showed significantly (p < 0.0001) higher Cr
concentration (tanners: 167.58 + 23.44 mg/l) than in
controls (22.09 + 3.78 mg/l).
DNA damage
DNA damage was identified in terms of the comet tail
length in controls and exposed group (Figure 2(a)).
Exposed group showed significantly (p < 0.0001)
higher comet tail length (tanners: 23.53 + 5.27 mm)
Ambreen et al.
Figure 1. Blood Cr concentration in controls (n ¼ 100)
and tanners (n ¼ 100). Values are presented as
mean + SD. p values refer to unpaired t test between
control and exposed groups. Cr: chromium.
Figure 2. Representative photographs of DNA damage in
terms of comet tail length in different groups: (a) controls,
(b) tanners. (c) DNA damage in controls (n ¼ 100) and
tanners (n ¼ 100). Values are presented as mean + SD.
p values refer to unpaired t test between control and
exposed groups.
when compared with controls (9.27 + 2.21 mm;
(Figure 2(b)). There was no significant (p > 0.05)
effect of smoking and alcohol consumption on DNA
damage in controls as well as exposed group (Table 2).
In simple correlation analysis, DNA damage showed
significantly positive correlation with Cr
concentration (r ¼ 0.65, p < 0.01) and duration of
exposure (r ¼ 0.59, p < 0.01) in exposed group.
Whereas, no significant correlation (r ¼ 0.10,
p > 0.05) between DNA damage and Cr concentration
was observed in case of controls. DNA damage
showed no significant correlation (p > 0.05) with age
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5
in both the groups (table not shown). After adjusting
for smoking and alcohol consumption habits, there
was a significant positive correlation of DNA damage
with Cr concentration (R ¼ 0.61, p < 0.01) and dura-
tion of exposure (R ¼ 0.62, p < 0.01) in exposed group
(Table 3).
MDA concentration
Exposed group showed significantly (p < 0.0001)
higher concentration of MDA (tanners: 9.89 +
2.08 nmol/ml) than in controls (6.49 + 1.84 nmol/
ml; Figure 3). There was no significant (p > 0.05)
effect of smoking and alcohol consumption habits
on MDA concentration in controls and exposed group
(Table 2). In simple correlation analysis, MDA con-
centration showed significantly positive correlation
with Cr concentration (r ¼ 0.60, p < 0.01) and dura-
tion of exposure (r ¼ 0.53, p < 0.01) in exposed group.
Whereas, in control group, no significant correlation
(r ¼ 0.04, p > 0.05) between MDA and Cr concentra-
tion was observed. There was also no significant
correlation (p > 0.05) of MDA concentration with age
in both the groups (table not shown). After adjusting
for smoking and alcohol consumption habits, MDA
concentration showed significant positive correlation
with Cr concentration (R ¼ 0.59, p < 0.01) and duration
of exposure (R ¼ 0.54, p < 0.01) in exposed group
(Table 3).
GSH concentration
The exposed group showed significantly
(p < 0.001) lower concentration of GSH (tanners:
67.02 + 17.76 mg/ml) compared with controls
(74.38 + 11.34 mg/ml; Figure 4). There was no
significant (p > 0.05) effect of smoking and alcohol
consumption habits on GSH concentration in both
the groups (Table 2). Simple correlation analysis
showed that GSH concentration was significantly nega-
tively correlated with Cr concentration (r ¼ 0.31, p <
0.01) and duration of exposure (r ¼ 0.23, p < 0.05). No
significant correlation (r ¼ 0.07, p > 0.05) between
GSH and Cr concentration was observed in control
group. GSH concentration showed no significant corre-
lation (p > 0.05) with age in both groups (table not
shown). After adjusting for smoking and alcohol con-
sumption habits, GSH concentration showed significant
negative correlation with Cr concentration (R ¼ 0.30,
p < 0.01) and duration of exposure (R ¼ 0.22, p < 0.05)
in exposed group (Table 3).
6 Toxicology and Industrial Health

Table 2. Effects of smoking and alcohol consumption on DNA damage and oxidative stress parameters in controls and
tanners
Parameters Controls Tanners ANOVA, F and p values
DNA damage
Smoker 9.46 + 1.83 23.67 + 5.68 12.50, 0.05
Nonsmoker 9.16 + 2.41 22.07 + 5.69
Alcoholic 9.72 + 1.47 23.44 + 6.38 11.99, 0.05
Nonalcoholic 9.17 + 2.34 22.89 + 5.54
MDA
Smoker 7.22 + 1.90 10.08 + 2.10 5.71, 0.71
Nonsmoker 6.06 + 1.66 9.60 + 2.04
Alcoholic 6.36 + 2.33 9.55 + 2.62 5.05, 0.27
Nonalcoholic 6.52 + 1.72 9.98 + 1.90
GSH
Smoker 74.01 + 11.73 66.29 + 15.24 2.94, 0.34
Nonsmoker 50.00 + 3.88 68.08 + 21.02
Alcoholic 70.70 + 11.44 69.34 + 19.12 0.04, 0.88
Nonalcoholic 51.27 + 5.01 66.33 + 17.40
SOD
Smoker 76.17 + 7.51 82.26 + 7.51 0.65, 0.57
Nonsmoker 95.51 + 10.94 77.77 + 13.22
Alcoholic 78.73 + 9.11 77.48 + 12.36 0.22, 0.72
Nonalcoholic 92.12 + 12.18 81.30 + 13.01
ANOVA: analysis of variance; GSH: glutathione; MDA: malondialdehyde; SOD: superoxide dismutase.

Table 3. Simple and multiple correlation analysis taking DNA damage and oxidative stress parameters as outcomes and
Cr concentration and duration of exposure as predictorsa
DNA damage MDA GSH SOD
r R r R r R R R
Cr
Controls 0.10 0.09 0.04 0.04 0.07 0.05 0.03 0.03
Tanners 0.65b 0.61b 0.60b 0.59b 0.31b 0.30b 0.47b 0.44b
Duration of exposure
Controls – – – – – – – –
Tanners 0.59b 0.62b 0.53b 0.54b 0.23c 0.22c 0.35b 0.34c

Cr: chromium; GSH: glutathione; MDA: malondialdehyde; SOD: superoxide dismutase.

a
r indicates simple correlation and R indicates multiple correlation adjusted for smoking and alcohol consumption habits.
b
p < 0.01.
c
p < 0.05.

SOD concentration with Cr concentration (r ¼ 0.47, p < 0.01) and

SOD concentration was found to be significantly
(p < 0.05) higher in exposed (tanners: 80.42 +
12.90 U/g Hb) group when compared with controls
(76.38 + 11.34 U/g Hb; Figure 5). There was no
significant (p > 0.05) effect of smoking and alcohol
consumption habits on SOD in both the groups
(Table 2). In simple correlation analysis, SOD con-
centration showed significant positive correlation
duration of exposure (r ¼ 0.35, p < 0.01). There was
no significant correlation (p > 0.05) between SOD
concentration and age in both the groups (table not
shown). In multiple correlation analysis, a signifi-
cant positive correlation was observed for SOD con-
centration with Cr concentration (R ¼ 0.44, p < 0.01)
and duration of exposure (R ¼ 0.34, p < 0.05) in
exposed group (Table 3).

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Ambreen et al.
Figure 3. MDA concentration in controls (n ¼ 100) and
tanners (n ¼ 100). Values are presented as mean + SD.
p values refer to unpaired t test between control and
exposed groups. MDA: malondialdehyde.
Figure 4. GSH concentration in controls (n ¼ 100) and
tanners (n ¼ 100). Values are presented as mean + SD.
p values refer to unpaired t test between control and
exposed groups. GSH: glutathione.
Figure 5. SOD concentration in controls (n ¼ 100) and
tanners (n ¼ 100). Values are presented as mean + SD.
p values refer to un-paired t test between control and
exposed groups. SOD: superoxide dismutase.
Discussion
In north India, Kanpur is well known for major tan-
ning industry. Tannery workers working in these tan-
ning industries are continuously exposed to trivalent
Cr, which appear to be associated with both acute and
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7
chronic health problems. Therefore, this present study
reports the toxicological effects of trivalent Cr on tan-
nery workers, with respect to genotoxicity and oxida-
tive stress, although, trivalent Cr was initially
considered as nontoxic metal. To our knowledge, this
is the first reported study, which was conducted on
north Indian tannery workers, in order to elucidate the
evidence of hazardous effects of trivalent Cr in terms
of genotoxicity and oxidative stress.
Total blood Cr concentration was measured as an
index of Cr exposure (Rajaram et al., 1995). We found
significantly higher blood Cr concentration in
exposed group than in controls. Goulart et al. (2005)
also measured total plasma and urine Cr level in occu-
pationally exposed tannery workers and found
approximately twofold increase in plasma and urine
Cr concentration in these exposed tannery workers
in comparison with controls. Similarly, Medeiros
et al. (2003) reported a significant enhancement in the
level of Cr in urinary and plasma of tannery workers
when compared with controls. There was also a sig-
nificant elevated value of blood and urine Cr level
in exposed tannery workers compared with controls
(Rastogi et al., 2008).
Furthermore, in our study, tannery workers showed
longer duration of exposure. Changes in the cell
membrane after long-term exposure to trivalent Cr
were pointed out as a mechanism of increasing the
membrane’s permeability to trivalent Cr (Rudolf and
Cervinka, 2003), which may be a factor of higher Cr
concentration in tannery workers, under chronic
occupational exposure condition.
Comet tail length was used to quantify the DNA
damage in controls and exposed group. Mean comet
tail length was significantly higher in exposed group
when compared with controls. Medeiros et al.
(2003) observed a significant increase in DNA
damage in tannery workers compared with controls.
Sellappa et al. (2011) also reported a significant
positive genotoxic effects in relation to trivalent Cr
exposure in South Indian tannery workers. Evidence
obtained from in vitro cell-free systems shows that,
once inside the cell, trivalent Cr may directly react
with genetic material and induce DNA damage (For-
nace et al., 1981; Tsapakos and Wetterhahn, 1983). In
our study, we found no significant effect of smoking
and alcohol consumption habits on DNA damage.
Similarly, smoking and alcohol taking habits had no
significant effect on DNA damage in Cr-exposed pop-
ulation (Danadevi et al., 2004). In our study, exposed
group showed significant increase in DNA damage
8 Toxicology and Industrial Health
with increased duration of exposure. An increase in
micronucleus frequency (biomarker of DNA damage)
with an increase in the duration of exposure in tannery
workers was also observed by Sellappa et al. (2011).
Danadevi et al. (2003, 2004) reported a significant
positive correlation between DNA damage and dura-
tion of exposure. No significant correlation between
DNA damage and age was found in our study. Similar
results were also obtained by Danadevi et al. (2004).
Bukvic et al. (2001) reported a strong correlation of
DNA damage with increasing age. According to Ewis
et al. (2001), duration of exposure to the carcinogen is
the factor that should be considered rather than the
age of the worker.
The mechanism of Cr toxicity is possibly mediated
by oxidative stress, when the production of reactive
oxygen species (ROS) overwhelms antioxidant
defense mechanism (Valko et al., 2005). Excess pro-
duction of ROS induces lipid peroxidation, which
destroys cell structures, lipids, proteins and nucleic
acids (Sen et al., 2010). In our study, MDA (a stable
aldehydic products of lipid peroxidation) was found
to be significantly higher in concentration in exposed
group when compared with controls. Higher concen-
tration of MDA indicate increased rate of oxidative
stress in exposed populations. According to Harris
and Shi (2003), higher MDA concentration in
exposed group may play a key role to cause oxidative
stress induced by metals. Goulart et al. (2005)
reported that tannery workers, exposed to trivalent
Cr, showed significantly elevated concentration of
urinary MDA when compared with control values.
Trivalent Cr has been shown in vitro to be able to gen-
erate hydroxyl radicals (Shi et al., 1993) and induce
oxidative damage in the presence of hydrogen perox-
ide (Lloyd et al., 1998; Tsou et al., 1996). In our
study, there was no significant effect of smoking and
alcohol consumption habits on MDA concentration
in exposed tannery workers. We also found no sig-
nificant correlation between age and MDA concen-
tration. However, Bridges et al. (1993) describe an
age-related increase in MDA concentration, which
is influenced by smoking habit. Alcohol consump-
tion habit also induce enhanced rate of MDA
concentration (Reddy et al., 1997). In the present
study, a significant positive correlation of MDA con-
centration was observed only with Cr concentration
and duration of exposure. These findings strongly
support that higher MDA concentration in exposed
group might be due to higher chronic exposure of
Cr concentration.
Downloaded from tih.sagepub.com at St Petersburg State University on December 15, 2013
GSH is a major endogenous antioxidant, pro-
duced by the cells and plays a significant role in
protection against oxidative stress by participating
directly in the neutralization of free radicals and
reactive oxygen compounds (Scholz et al., 1989).
The GSH concentration was found to be signifi-
cantly decreased in exposed group when compared
with controls. However, Goulart et al. (2005)
observed no significant alteration in GSH concen-
tration in tannery workers exposed to trivalent Cr
compared with controls. Lenton et al. (1999) study
reported a significant decrease in intracellular anti-
oxidant GSH with increased rate of oxidative DNA
damage. The decreased value of GSH in exposed
workers may be due to its involvement in the
detoxification, inhibition of lipid peroxidation by
scavenging free radicals. GSH depletion impairs
the cell’s defense against the toxic actions of ROS
and might lead to cell injury and death (Sen, 1997).
According to Beltowski et al. (2000), the intensity
of oxidative stress is determined not only by the
free radicals production but also by antioxidants
(enzymatic and nonenzymatic) defense. In the pres-
ent study, GSH concentration showed significant
negative correlation with Cr concentration and
duration of exposure. These findings revealed that
decreased level of GSH can be as a consequence
of oxidative stress, which may be associated with
Cr exposure in chronic condition.
The activity of antioxidant enzyme SOD was found
to be significantly higher in the exposed group com-
pared with controls. SOD catalyzes the dismutation
of superoxide radicals (ROS) to less toxic hydrogen
peroxide. Thus, it also has a protective role against
free radial-induced damage and their significant
induction can be understood as an adaptive response
to oxidative stress.
Conclusion
In conclusion, the level of DNA damage measured
by comet assay was found to be significantly
increased in tannery workers exposed to trivalent
Cr. In addition, we found that the level of oxidative
stress was significantly increased in trivalent
Cr-exposed tannery workers. This is the first
reported study on tannery workers from north India,
related to the adverse effects of trivalent Cr in terms
of genetic damage and oxidative stress. However,
further studies are also needed in order to elucidate
the hazardous effects of trivalent Cr.
Ambreen et al.
Acknowledgments
The authors thank Dr Rajeev Kacker, Chaudhary Ehsan
Kareem Hospital, Jajmau, Kanpur, for providing blood
sample of tannery workers and clinical diagnosis of the
patients, Dr. K.P. Singh (Associate Professor), King
George Medical University, Lucknow, for providing blood
sample of controls and Dr Kr. P. Singh (Scientist), Indian
Institute of Toxicology Research for total Cr estimation
by atomic absorption spectrophotometry.
Funding
This research was supported by University Grant Commis-
sion (UGC F.No. 37-435/2009-SR), New Delhi, India.
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