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Increased DNA damage in blood 
cells of rat treated with lead as as-
sessed by comet assay 

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Introduction 

Lead is considered as a common occupational and 
environmental hazard throughout the world. Lead 
adversely affects the nervous, hematopoietic, endo-
crine, renal and reproductive systems of the body. Lead
-induced oxidative stress in blood and other soft tissues
has been postulated to be one of the possible mecha-
nisms of lead-induced toxic effects (Pande and Flora,
2001). In recent studies, lead has been shown to inhibit
the activities of anti-oxidant enzymes, including gluta-
thione peroxidase, catalase and superoxide dismutase
(Bolin et al., 2006; Ercal et al., 2001). Furthermore, gene-
ration of reactive oxygen species (ROS), stimulation of
lipid peroxidation and depletion of anti-oxidant reser-
ves have been postulated to be major contributors to
lead-exposure related diseases (Patrick, 2006; Silbergeld
et al., 2000). Recent evidence indicates that the lead

treatment is coupled with enzyme inhibition, fidelity of 
DNA synthesis, mutation, chromosome aberrations, 
and cancer (Johnson, 1998). 

In several human investigations, the increased levels of 
DNA damage were observed in the lead exposed 
workers using micronucleus assay or comet assay 
(Chen et al., 2006). In animal studies, lead could induce 
genotoxic damage in all organs of CD-1 mouse except 
the testicle (Valverde et al., 2002). Additionally, DNA 
damage was observed after treatment with lead in 
mouse blood cells (Devi et al., 2000) and in PC12 cells 
(Xu et al., 2006).  Poma et al. (2003) found lead acetate- 
induced micronuclei and sister chromatid exchange 
with a dose-dependent manner in human melanoma 
cells. In fact, the genotoxic effects of lead have been 
studied in a variety of systems; however the results are 
not conclusive. So, the purpose of our present study is 

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Increased DNA damage in blood cells of rat treated with lead as 
assessed by comet assay 

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Received:         30 June 2008 
Accepted:         14 July 2008 
Available Online:   15 July 2008 

DOI: 10.3329/bjp.v3i2.948 

Cite this article: 
Arif M, Kabir Y, Hassan F, Waise 
TMZ, Mazumder MEH, Rahman S. 
Increased DNA damage in blood cells 
of rat treated with lead as assessed by 
comet assay. Bangladesh J Pharmacol. 
2008; 3: 97-101. 

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A growing body of evidence suggests that oxidative stress is the key player in 
the pathogenesis of lead-induced toxicity. The present study investigated lead
-induced oxidative DNA damage, if any in rat blood cells by alkaline comet
assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and
100 mg/kg body weight for 5 days consecutively. Blood collected on day six
from sacrificed lead-treated rats was used to assess the extent of DNA dam-
age by comet assay which entailed measurement of comet length, olive tail
moment, tail DNA (%) and tail length. The results showed that treatment
with lead significantly increased DNA damage in a dose-dependent manner.
Therefore, our data suggests that lead treatment is associated with oxidative
stress-induced DNA damage in rat blood cells which could be used as an ear-
ly biomarker of lead-toxicity.

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to investigate the effect of lead on DNA damage of the 
rat blood cells and explore the correlation between the 
dose of the lead and the extent DNA damage by using 
comet assay. 

 

Materials and Methods 

Chemicals 

All chemicals used for the present study were of analy-
tical grade and purchased from Sigma, USA and Loba 
Chemie, India.  

Animals and lead administration 

Male Sprague-Dawley rats (7-8 weeks) were obtained 
from ICDDR’B (International Center for Diarrheal 
Disease and Research, Bangladesh). Rats were housed 
under standard environmental conditions and allowed 
free access to food and water for 2 weeks prior to the 
treatment. Rats of five group each were treated with 
three different lead dose levels of 25, 50, 100 mg/kg 
body weight (BW). The dose of the lead and the 
experimental paradigm of the current study were 
determined on the basis of the previous studies (Xu et 
al., 2008). Lead acetate was dissolved in saline (0.9% 
NaCl) with slightly acidic condition (as required) and 
administered intraperitoneally to animals at the doses 
of 25, 50 and 100 mg/kg body weight per day for 5 
days consecutively. The cumulative doses of lead given 
to rats were thus 125, 250 and 500 mg/kg BW. Saline 
was administered to the 5 animals of the control group 
in the same manner as in the treatment groups. 
Following five days of lead treatment, animals were 
decapitated and 1 mL of blood was collected in an 
eppendorf tube containing 100 µL of 10% EDTA. The 
blood samples were stored in a dark box at 4ºC until 
use.  

Comet assay 

The comet assay was carried out according to the 
protocol described by Tice et al. (2000) with slight 
modification. 10 µL of whole blood was suspended in 
75 µL 0.7% low melting point agarose in Phosphate 
Buffer Saline (PBS) at 37ºC and was pipetted imme-
diately into a glass microscopic slide having a layer of 
1% agarose. The slides were covered with another layer 
of agarose. Then, the slides were immersed in lysis 
solution at 4ºC for 1 hour to remove cellular mem-
branes, proteins and so forth. Slides were then placed 
in a single row in a horizontal electrophoresis tank 
containing alkaline buffer at 4ºC for 20 min for DNA 
unwinding. After 20 min, the current was switched on 
and electrophoresis was carried out at 25 V, 300 mA for 
40 min at the same temperature. Following removal of 
slides from lysis solution, the slides were washed for 
three times 5 min each with neutralization buffer. 

Subsequently the slides were visualized by silver 
staining as described by Nadin et al. (2001). All steps 
beginning with the isolation of lymphocytes were 
conducted under yellow light to minimize the 
possibility of cellular DNA damage. Slides were 
analyzed microscopically by using software called 
Computer Assay Software Project (CASP, version 
1.2.2). Threshold values of CASP parameters were 
adjusted to obtain the optimal values for our staining 
protocol. The selected parameters were: Head center 
threshold (HCT) = 0.999, Comet thresholds (CT) = 
0.005, Head threshold (HT) = 0.05, Tail threshold (TT) = 
0.05 and profile 1. The comet parameters analyzed by 
CASP were: Length of head and length of tail of comet 
(in pixels), % of DNA in the comet’s head and in the 
tail, the tail moment (arbitrary units) and olive tail 
moment (arbitrary units).  

The Olive tail moment (OTM) is [percent of DNA in the 
tail] x [distance between the center of gravity of DNA 
in the tail and that of center of gravity of DNA in the 
head in x-direction]. 

Statistical analysis 

One-way analysis of variance (ANOVA) was used for 
statistical evaluation in all experiments. Bonferroni 
posthoc test was performed for multiple group 
comparisons. Data are shown as mean ±  SEM. The 
value of p<0.05 was considered statistically significant. 

 

Results 

The alkaline comet assay showed a significant increase 
in comet length in all three treatment groups as 
compared to controls (Figure 1). As shown in Figure 
2A, treatment with lead at 25, 50 and 100 mg/kg doses 
increases the comet length by 1.8, 3 and 4 fold 
respectively.  

Olive tail moment is a commonly used marker of DNA 
damage in comet assay. OTM data shows significant 
DNA damage in lead treated rat blood cells as 
compared to the control group. Treatment with lead at 
25, 50 and 100 mg/kg doses increases the OTM by 5.4, 
11 and 22 fold respectively (Figure 2B). 

The extent of damage can be expressed by measuring 
percent of DNA present in the tail in comet assay. Dose
-dependent increase by 10, 22 and 40% of tail DNA has 
been observed following treatment with 25, 50 and 100 
mg/kg of lead respectively as compared to controls 
(Figure 2C). This data indicates that increasing concen-
tration of lead is associated with the migration of 
damaged head DNA from head to the tail region 
during electrophoresis. 

Comet tail length increased with the increase of lead 

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doses in all three different rat groups. Longest tail was 
found in 100 mg/kg group. As shown in Figure 2D, the 
mean value of tail length increased 8.5, 22.5 and 35 
times respectively in 25, 50 and 100 mg/kg lead 
treatment groups when compared with control group.  

 

Discussion 

This study was carried out to investigate the dose-
dependent effect of lead exposure on DNA damage by 
comet assay. Our data suggests that administration of 
lead is associated with DNA damage in dose-
dependent fashion. We used whole blood in our study. 
RBCs being enucleated cells, the comet images we 
obtained in this study represented only white blood 
cells, presumably lymphocytes though we didn’t isolate 
lymphocytes by traditional histopaque procedure. 
Therefore, in the present study, we examined effect of 
lead on DNA damage in white blood cells.  

The comet assay is a simple, sensitive, and rapid 
method that can be used to estimate DNA damage at 
the individual cell level through strand breaks, open 
repair sites, cross-links, and alkali labile sites caused by 
oxidative stress (Tice et al., 2000). The basis of the 
comet assay involves embedding a suspension of single 
cells in low melting point agarose on a microscope 
slide, thus creating a mini DNA agarose gel. The cells 
are lysed and then the DNA is electrophoresed. After 
silver-staining, the cells are observed under microscope 

and compared with control. In this study we found that 
cells from control rats had tightly compressed DNA 
and maintained the circular form of the normal 
nucleus, with little or no evidence of comet formation 
(Figure 1A). In contrast, cells from rats treated with 
different doses of lead acetate displayed an altered 
appearance (Figure 1B-D). The basis of this altered 
appearance is, during alkaline unwinding (at pH 13.0) 
double stranded supercoiled DNA was unwound and 
denatured. As a result, double stranded complex DNA 
became single stranded and relaxed. So while 
electrophoresis was carried out, due to electric 
attraction, fragmentized DNA (negatively charged) 
migrated towards anode leaving the intact DNA in the 
head region. If nucleus contains more damaged DNA, 
large amount of these broken DNA (which are small in 
size compared to intact DNA molecule) will migrate in 
the tail region. Thus comet like shape was formed as 
evident in Figure 1. The intensity of the comet tail 
relative to the head reflects the number of DNA breaks. 
Cells containing greater levels of DNA strand breakage 
generate comets with more intense ‘tails’ (Figure 1C 
and D). Densitometric and geometric parameters of the 
comets as determined using image analysis software 
suggest that lead administration dose-dependently 
increased the comet tail, OTM and DNA percentage in 
the tail. 

To the best of our knowledge this is the first study 
showing the extent of oxidative DNA damage 
following the administration of lead using animal 

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Figure 1: Comet images of blood cells from lead-treated rat. Following consecutive 5 days treatment with 0 (A), 25 (B), 50 (C) and 
100 (D) mg/kg lead, the extent of DNA damage was assessed by comet assay coupled with silver staining. Treatment with lead 
increases the DNA damage in a dose-dependent fashion 



 

 

model. Our results suggest that lead treatment is 
associated with oxidative stress leading to oxidative 
DNA damage. Therefore, DNA damage in the 
peripheral blood could be a suitable early bio-marker to 
monitor the environmental or occupational lead 
toxicity.  

 

Ethical Issue 

All animal experiments were carried out according to the law 
of Animal Experiments Guidelines approved by National 
Institute of Health (NIH).  

 

Acknowledgements 
This study was supported in part by grants from the 
Biotechnology Research Centre, Dhaka University, 
Bangladesh. We thank Prof. Laila N. Islam, for her support 
and encouragement in this research.  

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Figure 2: Bar diagrams showing the effect of lead on comet assay. Quantitative analysis of comet length (A), olive tail moment (B), 
tail DNA (C) and tail length (D) were carried out in rat blood cells following treatment with 0, 25, 50 and 100 mg/kg doses of lead 
for 5 consecutive days. Data are shown as mean ± SEM for separate groups of rats (n = 5). ap<0.05; bp<0.01; cp<0.001; (Bonferroni 
test) 



 

 

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Mohammad Arif (Principal contact) 
e-mail: marif567@yahoo.com 

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