Mutat Res Gen Tox En 819 (2017) 20–25

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Mutat Res Gen Tox En

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GSTP1 and XRCC1 polymorphisms and DNA damage in agricultural workers MARK
exposed to pesticides
⁎
Amal Saad-Husseina, , Magda Noshyb, Mona Tahaa, Haidan El-Shorbagyb, Eman Shahya,
Ebtesam A. Abdel-Shafya
a
National Research Centre, Egypt
b
Cairo University, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Pesticide exposure may be associated with increased risk of genotoxicity and carcinogenesis. These risks may be
Pesticide exposure affected by polymorphisms of genes for glutathione transferase-dependent metabolism of pesticides and for DNA
Comet assay repair. We studied the prevalence of GSTP1 and XRCC1 polymorphisms and their possible correlation with DNA
GSTP1 damage following prolonged pesticide exposure. DNA damage was estimated by the comet assay in peripheral
XRCC1
blood samples from 51 pesticide-exposed workers and 50 controls. GSTP1 (105) and XRCC1 (399 and 194)
Polymorphisms
genotypes were identified by restriction fragment length analysis. Individuals carrying theGSTP1 Ile-Ile or
XRCC1399 Arg-Arg genotypes showed greater DNA damage than observed for other alleles.

1. Introduction and XRCC1 genes in workers occupationally exposed to carbamate and

Pesticides are commonly used chemicals in agriculture [1]. Some
pesticides have been identified by the International Agency for Cancer
Research (IACR) as presenting risks to human health [2,3]. Carbamate
and organophosphate pesticides (OPs) [4] are among the agents that
have been linked to adverse health effects [5]and biomarkers are
available for estimating occupationally exposures to them [6,7].
Bioactivation of pesticides may give reactive metabolites that can bind
to cellular macromolecules [8].
The comet assay is a sensitive method for monitoring DNA damage/
genotoxicity [9], based on electrophoretic migration of DNA from the
nucleus [10]. Genetic variations in genes encoding enzymes involved in
xenobiotic metabolism and DNA repair may influence genotoxicity [3].
Some pesticides/metabolites are detoxified by conjugation to glu-
tathione catalyzed by glutathione transferases (GST). When such
detoxication is ineffective, toxic products may damage intracellular
molecules [5]. Several GST enzymes expressed in the liver are
polymorphic [11] and may influence the metabolism and detoxication
of pesticides [12]. Many DNA repair mechanisms mitigate the geno-
toxicity of xenobiotics [3]. XRCC1 is a DNA-repair gene that may play a
role in protection against pesticide-related genotoxicity [5]. XRC-
C1polymorphisms associated with elevated risk of DNA damage in
the pesticide-exposed population include those at exon 6 (Arg194Trp)
(C→T) and at exon 10 (Arg399Gln) (G→A) [13]. Our aim was to
evaluate association of DNA damage with polymorphisms in the GSTP1
OP pesticides in Egypt.
2. Materials and methods
2.1. Study population
We studied 51 agricultural workers occupationally exposed to
several mixtures of pesticides and 50 healthy subjects (controls); only
male subjects were studied. Both groups were recruited from the city El
Monofya, Egypt, have similar socioeconomic status, and were matched
in their ages, smoking habits, and ethnic backgrounds. The average age
of the workers was 46.3 ± 5.1 and for the controls, 45.5 ± 4.4. Ethics
committee approval was obtained from the Ethics Committee, National
Research Center, Egypt and informed consent was obtained from each
subject. Questionnaires (personal, occupational, environmental, and
medical data) were completed during a personal interview. Based on
the questionnaires, any subjects with identified occupational or envir-
onmental exposures to carcinogenic chemicals or ionizing radiation, as
well as those with a history of cancer, were excluded. Workers with
known occupational exposure to pesticides for less than 5 y were also
excluded. Individuals with identified environmental or occupational
history of exposure to pesticides were excluded from the control group.
The workers were exposed to several types of pesticides, changing
according to the weather. These pesticides included a variety of
complex components, mostly carbamates and OPs. They worked in

⁎
Corresponding author.
E-mail address: amel_h@hotmail.com (A. Saad-Hussein).

http://dx.doi.org/10.1016/j.mrgentox.2017.05.005
Received 14 January 2017; Received in revised form 25 March 2017; Accepted 5 May 2017
Available online 08 May 2017
1383-5718/ © 2017 Elsevier B.V. All rights reserved.
A. Saad-Hussein et al. Mutat Res Gen Tox En 819 (2017) 20–25

open fields. The duration of exposure to pesticides among the selected Table 1
workers was > 5 y. Workers were exposed during transportation, Demographic data of pesticide-exposed workers and controls .
storage, mixing, and field application of pesticides. There was great
Variable Control (50) Workers (51) Independent t-test
variation in the workers’ use of personal protective equipment. (mean ± SD) (mean ± SD) P-value
However, only 2% wear a mask and none wears gloves.
Consequently, inhalation, skin, and eye contact are potential routes of Age 33.4 ± 7.5 35.4 ± 11.8 0.249
No. (%) No. (%) Chi-square test
exposure.
P-value

Smoking habit
2.2. Chemicals Smokers 16 (32%) 18 (35%) 0.729
Non smokers 34 (68%) 33 (65%)
Unless specified, chemicals were of analytical grade and were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Kits for all
biochemical measurements were purchased from Bio-diagnostic Table 2
Company (Giza, Egypt)or as stated below. the most common pesticides used by agricultural workers.

Pesticide Chemical group WHO Category

2.3. Sample collection
Malathion Organophosphorus III
Chloropyrifos Organophosphorus II
A blood sample (approx. 6 ml) was collected from each participant Dimethoate Organophosphorus II
by venipuncture, then divided into three parts as follows: a) 2 ml: Carbofuran Carbamate Ib
transferred to EDTA vacutainers for DNA extraction; b) 1 ml: mixed
with 10% DMSO (final concentration) was transferred to another EDTA Ib: highly hazardous, II: moderately hazardous III: slightly hazardous, [15].
vacutainer and stored at −80 °C for comet assay analysis; c) 3 ml:
transferred to non-EDTA vacutainers, and left for 30 min at room Table 3
temperature to coagulate, and then centrifuged at 3000 rpm (specify g The difference in comet results between the pesticides-exposed workers and unexposed
controls.
force) for 10 min.
Variable Control (51) Workers (50) Kolmogorov-Smirnov
2.4. Comet assay Median Median Test
(range) (range) P value

The comet assay was performed according to the protocol of [14]. Tail length 8.50 14.59 P < 0.001
Fully frosted slides were embedded with 1% normal-melting agarose Median 1–19 2–37
and the underside of the slide was wiped to remove agarose. The slide Range
DNA% in tail 0.18 4.21 P < 0.001
was allowed to solidify at 4 °C for 10 min. After that, low-melting
Median 0.00–5.61 0.83–17.84
agarose (0.5%, 75 μl,37 °C) was mixed with an aliquot (10 μl) of whole Range
blood and smeared over the coated slide. The slides were immersed in Tail moment (μm/cell) 0.08 0.73 P < 0.001
cold lysis buffer, freshly prepared (100 mM Na2EDTA, 2.5 M NaCl, Median 0.05–1.48 0.12–1.48
10 mM Tris HCl, pH 10). 1% Triton X-100 and 10% DMSO (final Range

concentrations) were added to the lysis buffer just before immersing the
slides; then the slides in the lysis buffer jar were refrigerated overnight.
The slides were then placed for 20 min in alkaline buffer (1 mM EDTA Gene (bp) Primers
and 300 mM NaOH) (pH > 13) to allow unwinding of DNA. Electro-
phoresis was conducted at 300 mA and 25 V (∼0.74 V/cm for 25 min; GSTP1 exon5, codon105;177 F ACC CCA GGG CTC TAT GGG
the slides were drained and washed carefully with three changes of AA
neutralizing buffer (0.4 M Tris HCl, pH 7.5) for 5 min each time. The R TGA GGG CAC AAG AAG CCC
slides were dehydrated for 5 min in absolute methanol and left for CT
drying at room temperature. The whole procedure was done in dim XRCC1 exon 6, codon 194; F GCC AGG GCC CCT CCT TCA A
light to reduce DNA damage. Slides were stained with 10% ethidium 485
bromide solution and viewed under a fluorescence microscope. For R TAC CCT CAG ACC CAC GAG T
each subject, simultaneous image capture and scoring of 100 cells at XRCC1 exon10, codon 390; F CCC CAA GTA CAG CCA GGT C
400 x magnification were conducted using comet analysis software 242
(Kinetic Imaging, Ltd., Liverpool, UK). The degree of DNA damage was R TGT CCC GCT CCT CTC AGT AG
assessed according to various endpoints. Tail length, expressed in μm,
was used to estimate the extent of DNA damage distant from the
The Ile to Val substitution in GSTP1 exon 5 (codon 105) was
nucleus while% DNA in tail represents intensity of all tail pixels divided
amplified using the following conditions: denaturation at 94 °C for 30 s,
by the total intensity of all pixels in the comet. Tail moment = tail
annealing at 61 °C for 30 s, and extension at 72 °C for 30 s. The PCR
length × %DNA in tail/100.
products were digested with Alw26I for 24 h at 37 °C and then
electrophoresed on 3.5% agarose. A single fragment of 177 bp repre-
2.5. Polymerase chain reaction-Restriction fragment length polymorphism sents homozygous Ile-Ile individuals. Homozygous Val-Val individuals
(PCR-RFLP) showed 92 and 85 bp fragments and heterozygous Ile-Val individuals
showed three fragments.
2.5.1. DNA extraction Arg to Trp substitution in XRCC1exon 6 (codon 194) amplification
DNA extraction from whole blood sample was done using the was conducted at 95 °C for 30 s for denaturation, annealing at 59 °C for
Genomic DNA Purification kit (Gene JET™/Fermentas). 35 s, and extension at 72 °C for 45 s followed by digestion with PvuII.
The product was identified by gel electrophoresis using 2% agarose. A
2.5.2. Genotyping analysis fragment of 485 bp indicated homozygous Arg-Arg subjects, both 396
PCR amplification was performed using the following primers: and 89 bp fragments represent homozygous Trp-Trp subjects, and the

21
A. Saad-Hussein et al.
Fig. 1. Representative photo for comet assay A: DNA damage in the workers and B: intact DNA of control (400X).
Fig. 2. PCR product of GSTP1 exon 5 (codon 105).Lane1: the size marker (50 bp). Lane2:
represent homozygous Val-Val genotype with 92-bp and 85-bp fragments. Lanes3:
heterozygous Ile-Val genotype exhibited all the three fragments. Lane 4: Homozygous
Ile-Ile genotype as a single product fragment of 177 bp.
heterozygous Arg-Trp subjects showed all three fragments.
Arg to Gln substitution in exon 10 (codon 399) amplification was
done at 95C for 30 s for denaturation, annealing at 61 °C for 35 s, and
extension at 72 °C for 45 s. The PCR products were digested with MspI
and analyzed on 2% agarose. Homozygous Gln-Gln individuals indi-
cated a single product fragment of 242 bp, homozygous Arg-Arg
individuals showed both 148 and 94-bp fragments, and heterozygous
Arg-Gln individuals showed all of them. All digested PCR products were
analyzed in agarose gel and detected with 10 mg/ml ethidium bromide,
5 μl. Electrophoresis was conducted at 70 V (power supply Biorad,
Model 200/2.0) for 50 min and DNA fragments were visualized with a
UV transilluminator (Stratagene, USA).
2.6. Statistical analysis
Statistical analysis was performed with SPSS version 18.
Quantitative results were expressed as mean ∀standard deviation
(SD). Comparisons between the two groups were analysed by the
independent t-test. Qualitative results were expressed as number (no.)
22
Mutat Res Gen Tox En 819 (2017) 20–25
Fig. 3. PCR product of XRCC1 exon 10 (codon 399). Lane1: the size marker (100 bp).
Lane2: represent the undigested fragment of 242 bp homozygous Gln-Gln. Lanes3:
represent homozygous Arg-Arg genotype demonstrated both 148 and 94 bp fragments.
Lane 4: represents heterozygous Arg-Gln genotype that revealed all three of the
fragments.
and percent (%).Qualitative results were analyzed using Pearson’s Chi-
square (χ2) and likelihood ratio in cases where more than 25% of the
cells had expected count < 5. For skewness results, non-parametric
method two-sample Kolmogorov-Smirnov Test was used for the com-
parisons between two groups. Analysis of Variance (ANOVA) and the
post-hoc test (Bonferroni) were used to compare quantitative results
among > 2 groups. The Pearson correlation coefficient was used to
study the relationship between average tail moments and durations of
exposures. Hardy-Weinberg equilibrium was evaluated for each poly-
morphism using goodness-of-fit chi-square test for the workers and the
controls separately. All the results were tabulated and suitable figures
were illustrated. P < 0.05 was considered to be significant.
3. Results
Demographic data are shown in Table 1. There were no significant
differences in age or smoking habits between the workers and controls.
The workers were exposed to pesticides as illustrated in Table 2. The
workers included in the study reported being occupationally exposed to
pesticides for > 5 y, mean 9.8 ± 3.5 y, and mean 142 ± 81 d/y. They
perform tasks including storage, transportation, mixing, and spraying.
About 61.5% of pesticides-exposed workers reported using special
A. Saad-Hussein et al. Mutat Res Gen Tox En 819 (2017) 20–25

Table 6
Comparison of average tail moment between the different personal variables and
genotypes in workers subjects.
b c d
significant difference between each two symmetrical letters using Bonferroni test.

Variable Average tail moment (μm/ Kolmogorov-Smirnov Test P-value

cell)

N Mean ± SD

Age
< 35 24 0.74 ± 0.37 0.081
> 35 27 0.65 ± 0.22
Smoking habit
Smokers 18 0.86 ± 0.3 P < 0.05
Non smokers 33 0.65 ± 0.35
GSTP1 105
Ile-Ile 23 0.91 ± 0.34b,c P < 0.005 a

Ile-Val 24 0.60 ± 0.28 b

Val-Val 4 0.41 ± 0.19 c
XRCC1 194
Arg-Arg 45 0.72 ± 0.36 0.685
Arg-Trp 6 0.74 ± 0.29
XRCC1 399
Gln-Gln 10 0.94 ± 0.25 cd P < 0.005 a

Arg-Gln 24 0.65 ± 0.39d

Arg-Arg 17 0.54 ± 0.21 c

Fig. 4. PCR product of XRCC1 exon 6 (codon 194). Lane 1: the size marker (100 bp). significantly higher in the workers compared to controls (Table 3).
Lanes2: represent Homozygous Arg-Arg individuals reflected a single product fragment of Fig. 1 illustrates the comet assay, showing DNA damage in one worker
485 bp, Lanes3: heterozygous Arg-Trp genotype with 396-, 89-bp and 485 bp fragments. (A) and a control (B).
Concerning the PCR-RFLP results, we observed three GSTP1105
Table 4 genotypes (Fig. 2); two XRCC1 194 genotypes (Fig. 3) and three XRCC1
Distribution of different genetic polymorphism among pesticides-exposed workers 399 genotypes (Fig. 4). The prevalences of GSTP1 105, XRCC1 194 and
and control subjects.
XRCC1 399 genotypes among workers and controls are shown in
Genotype Controls (50) Workers (51) χ2test Table 4. All investigated polymorphisms for both workers and control
P value groups were in Hardy-Weinberg equilibrium (Table 5).
Table 6 summarizes the associations between average tail moment
Gene Allele No. (%) No. (%)
and the tested variables and genotypes in the pesticide-exposed work-
GSTP1 Ile- Ile 25(50%) 23(45%) 0.679a
105 ers. There was no significant difference in average tail moment between
Ile-Val 23(46%) 24(47%) the workers younger vs older than 35 y. A significant increase in tail
Val-Val 2(4%) 4(8%) moment for smoking vs non-smoking workers was seen. There was a
XRCC1 149 Arg- Arg 45(90%) 45(88%) 0.514 significant increase in average tail moment in workers with the GSTP1
Arg-Trp 5(10%) 6(12%)
XRCC1 399 Arg- Arg 8(16%) 10(20%) 0.851
Ile-Ile genotype compared with the Ile-Val and Val-Val genotypes.
Arg-Gln 26(52%) 24(47%) Concerning the XRCC1 399 polymorphism, there was a significant
Gln −Gln 16(33%) 17(33%) increase in average tail moment in the workers with Arg-Arg genotype
vs Gln-Gln and Arg-Gln genotypes. In contrast, there was no significant
a
likelihood ratio was used. difference in average tail moment between the workers with different
XRCC1 194 genotypes. Average tail moment was correlated positively
clothes during their work; in addition, 23.2% wash their work clothes with duration of exposure (d/y); r = 0.436 and p-value < 0.005
along with their family clothes. Among the workers studied, only 2% (Fig. 5).
reported wearing a mask only; 3.9%, boots and mask; 31.4%, boots
only; no-one reported wearing gloves.
Tail length, DNA% in tail and average tail moment per cell were

Table 5
Estimated and expected genotype counts for different polymorphisms in workers and controls.

Genotype Controls HWE Workers HWE

Gene Allele Observed Expected χ 2

P-Value Observed Expected χ2 P-Value

GSTP1 Ile- Ile 25 26.64 1.39 0.238 23 24.02 0.44 0.507

105 Ile-Val 23 19.71 24 21.96
Val-Val 2 3.65 4 5.02
XRCC1 149 Arg- Arg 45 45.13 0.14 0.709 45 45.18 0.19 0.655
Arg-Trp 5 4.75 6 5.65
Trp-Trp 0 0.13 0 0.18
XRCC1 399 Arg- Arg 8 8.82 0.23 0.634 10 9.49 0.03 0.771
Arg-Gln 26 24.36 24 25.02
Gln −Gln 16 16.82 17 16.49

The deviation from Hardy-Wienberg equilibrium was calculated using goodness of fit chi square test.

23
A. Saad-Hussein et al.
Fig. 5. Correlation between average tail moment (μm/cell) and duration of exposure (day/year).
4. Discussion
Some pesticides may induce oxidative stress that leads to increased
DNA damage [5]. Estimation of DNA damage related to pesticide
exposure may be achieved using the comet assay. We found a
significant increase in the average tail moment in pesticide-exposed
workers compared to controls without known exposures. Some previous
studies have shown increased DNA damage in workers exposed to
various mixtures of pesticides [16,5]. We observed a significant
increase in average tail moments of smoker workers vs non-smokers.
Others [17,18] have reported similar results. Elevated DNA damage
may result from the combined effect of smoking and pesticide exposure.
[19] observed that smoking induces oxidative stress and may lead to
higher DNA damage. However, other studies, such as [12] and [20]
reported no differences in DNA damage between smoker and non-
smoker workers occupationally exposed to OP pesticides.
The positive correlation between average tail moment and duration
of exposure (d/y) in the workers suggests that prolonged occupational
exposure increases cytogenetic damage induced by pesticides exposure.
The comet assay is a high sensitivity tool for biomonitoring the
hazardous effect of pesticides among exposed workers.
Polymorphisms in metabolism and DNA-repair genes may modulate
the extent of DNA damage in pesticide-exposed workers [3]. GSTs are
important enzymes that participate in detoxication of pesticides [21].
Our study showed that DNA damage was higher in pesticide-exposed
workers carrying the GSTP1 Ile-Ile genotype than the other two
genotypes (Ile-Val and Val-Val). These results were consistent with
previous results [3,12] that found an increase in the DNA damage in
GSTP1 Ile-Ile genotype among workers occupationally exposed to
pesticides mixtures. One study [21] found a significant association of
the GSTP1 Ile-Ile genotype with increased DNA damage, mainly in the
highly pesticide-exposed group but not in the low pesticide-exposed
group or unexposed controls. On other hands, some studies found no
association between DNA damage and different GSTP1 genotypes
[22,23]. According to [3], the GSTP1 Ile-Ile variant may have lower
enzyme activity, leading to greater accumulation of xenobiotics or their
24
Mutat Res Gen Tox En 819 (2017) 20–25
metabolites and associated with increased DNA damage.
DNA repair protects cells against genotoxicity. TheXRCC1 protein
repairs DNA strand-breaks and preserves genetic stability [13]. We
found no association between DNA damage and the XRCC1194 gene
polymorphism, but the XRCC1 399Arg-Arg genotype was associated
with elevated DNA damage in the pesticide exposed workers. These
results are similar to the study of [3], who reported an increase in DNA
damage in pesticide workers carryingXRCC1 399 Arg-Arg. XRCC1399
Arg-Arg was the least common genotype in the groups examined (16%
of controls and 20% of workers) in the present study.
5. Conclusions
In our study, pesticide-exposed workers with the GSTP1 Ile-Ile and
XRCC1399 Arg-Arg genotypes showed increased DNA damage.
Accumulation of DNA damage is a crucial step for initiation of many
cancers and assessment of DNA damage in pesticide-exposed workers
may be advisable.
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