Common polymorphisms in CYP1A1, GSTM1, GSTT1, GSTP1and XPD genes and endogenous DNA damage

Marta Wlodarczyk • Grazyna Nowicka

Received: 27 July 2011 / Accepted: 13 December 2011 / Published online: 20 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Endogenous DNA damage levels were analyzed

in relation to polymorphisms in genes encoding phase I

detoxifying enzyme—CYP1A1, phase II detoxifying

enzymes—GSTM1, GSTT1, GSTP1 and enzyme involved

in nucleotide excision repair-XPD. The study group con-

sisted of 220 healthy non-smoking volunteers; 90 men and

130 woman, 25–60 years old (44 ± 10 years). The level of

DNA damage (% DNA in tail) was evaluated by alkaline

comet assay. The genetic variants were determined by

restriction fragment length polymorphism PCR. The highest

level of DNA damage (6.7%) was found in carriers of both:

AA variant of XPD gene and M1 null variant of GSTM1

gene. The lowest level of DNA breaks (3.7%) was associ-

ated with the genotype GSTP1-AA/GSTM1 (?).

Keywords DNA damage � Comet assay � Genetic

Polymorphism � Detoxifying enzyme

Introduction

Proper transmission of genetic information in cells requires

not only accurate DNA replication but also the ability to

detect and repair both spontaneous and induced DNA

damage. The accumulation of DNA damage is a hazardous

phenomenon which may lead to development of different

pathological processes and cell death. Therefore, to maintain

genomic integrity cells possess a complex DNA damage

response mechanism and many enzymes involved in bio-

transformation of toxicants and in cellular defence against

toxicant-induced damage to the cells has been identified.

Polymorphisms of genes encoding such enzymes may sig-

nificantly modify genotoxic effects of different factors.

The P450 family enzymes mediate phase I reaction in

which xenobiotics are activated to reactive intermediate

substances. The CYP1A1 gene codes the enzyme aryl

hydrocarbon hydroxylase, which is responsible for the

metabolism of tobacco procarcinogens like PAH and aro-

matic amines [1]. The MspI (Val/Ile) polymorphism seems

to influence the activity of this enzyme [2]. It has been

recognized that the Val allele is associated with higher

catalytic enzyme activity than the Ile variant and high

levels of DNA adducts in Val allele carriers were reported

[3, 4]. However, the impact of this genetic variant on risk

of endogenous DNA damage is questionable.

Phase II enzymes are involved in the detoxification of

endogenous and exogenous electrophilic compounds. They

also protect against oxidative stress by free-radical scav-

enging [5, 6]. Polymorphisms in GST gene family,

encoding enzymes of II phase, have been associated with

increased cancer risk as well as enhanced levels of bio-

markers of genotoxicity [7]. Enzymes of GST family

conjuge electrophilic compounds with reduced glutathione

(GSH). GSTT1 and GSTM1 are involved to a certain

extent in e.g. styrene metabolism and influence the sus-

ceptibility to its toxic effects. GSTM1 is especially

involved in the metabolism of epoxides e.g. PAH epoxides

generated by cytochrome P450. GSTM1 null genotype has

been shown to be associated with higher sensitivity to

genotoxicity of tobacco smoke and enhanced cancer risk

[8, 9]. The Ile-Val substitution in GSTP1 gene reduces the

catalytic activity of the enzyme, and due to this reduction

M. Wlodarczyk (&) � G. Nowicka

Department of Nutrigenomics, National Food and Nutrition

Institute, Warsaw, Poland

e-mail: mwlodarczyk@izz.waw.pl

G. Nowicka

Department of Pharmacogenomics, Medical University

of Warsaw, Warsaw, Poland

123

Mol Biol Rep (2012) 39:5699–5704

DOI 10.1007/s11033-011-1378-x

the carriers of mutant alleles lost the capability to metab-

olize carcinogens [10].

The XPD protein is an ATP-dependent helicase, which is

a part of nucleotide excision repair system involved in

removal of UV-radiation-induced damage and chemical

adducts from DNA [11]. Mutations in XPD gene reducing

enzyme activity, result in a deficit in nucleotide excision

repair process and give rise to several human disorders

related to UV sensitivity like xeroderma pigmentosum,

Cockayne syndrome or trichothiodystrophy [12]. Most

mutations in XPD gene lead to changes in the C-terminal

third protein [13]. In addition to disease-causing mutations,

single nucleotide polymorphisms (SNPs) in the human XPD

gene have been identified. The Lys751Gln (K751Q,

rs13181) polymorphism in the exon 23 of the XPD gene, was

found to be common. It is suggested that 751Lys variant is

associated with the decreased DNA repair activity [14–17].

In the present study we analysed the relation between

polymorphisms in genes encoding enzymes of phase I

(CYP1A1), phase II (GSTM1, GSTT1, GSTP1) and DNA

repair gene (XPD) and levels of endogenous DNA damage

measured by comet assay in healthy subjects.

Methods

Study group

The study group consisted of 220 non-smoking, apparently

healthy adult volunteers: 90 men and 130 women, age

44 ± 10 years (25–60 years). All subjects were Polish

Caucasians from the Warsaw region. They underwent a

complete physical examination at the Outpatient Clinic of

Metabolic Disorders of the National Food and Nutrition

Institute. The study was conducted according to the

guidelines laid down in the Declaration of Helsinki and all

procedures involving human subjects were approved by the

Local Ethics Committee. Written informed consent was

obtained from all of the registered volunteers.

Fasting peripheral blood was collected by venipuncture

using Vacutainer tubes with heparin and EDTA as anti-

coagulants for the comet assay and gene polymorphism

analyses, respectively. Blood samples were immediately

used for comet assay analysis, while isolated DNA was

stored at -20�C until analysis.

Genotyping

DNA was isolated from a 1 ml peripheral blood lymphocyte

sample using the DNA Blood Mini Kit (A&A Biotechnol-

ogy, Gdynia, Poland) according to the manufacturer’s

protocol. The CYP1A1, GSTP1 and XPD gene polymor-

phisms was analysed using PCR amplification followed by

digestion with an appropriate restriction enzyme (restriction

fragment length polymorphism method-RFLP). Genotyping

of the polymorphisms in the GSTM1 and GSTT1 was done

by multiplex PCR, with b-globin gene as an internal control

of PCR. PCR conditions used for the analysis are shown in

Table 1.

Single-cell-gel electrophoresis

DNA integrity was evaluated by the use of alkaline single-

cell gel electrophoresis (comet assay), according to Singh

et al. [18], with some modifications [19]. Lymphocytes

were freshly isolated from 1 ml of heparinized blood by

centrifugation in a density gradient. Fifty microliters of

lymphocytes (1–3 9 105 cells/ml) were distributed with

50 ml of 2% low-melting-point agarose on a microscope

slide precoated with 0.5% normal agarose. Slides were then

immersed in a freshly prepared cold (4�C) lysis solution

(2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH

10.0–10.5, with 1% Triton X-100 added fresh) for 1 h at

4�C. After lysis, slides were placed in a horizontal gel

electrophoresis tank with fresh alkaline electrophoresis

buffer (300 mM NaOH, 1 mM Na2EDTA, pH [ 13.0) and

left in the solution for 40 min at 4�C. Electrophoresis was

conducted at 4�C for 20 min at 35 V (1 V/cm) and

Table 1 PCR-RFLP conditions

used for the analyses of the gene

polymorphisms

Tm melting temperature, bpbase pair

Polymorphism Tm

(�C)

PCR

product

(bp)

Restriction

enzyme

Alleles Bibliography

CYP1A1 Ile462Val 56 411 BseMI Val: 411 bp

Ile: 251 bp, 160 bp

[20]

GSTM1 null/? 60 215 – GSTM1(?): 215 bp [21], [19]

GSTT1 null/? 60 480 – GSTT1(?): 480 bp [21], [19]

GSTP1 Ile105Val 55 176 Alw26I A: 176 bp

G: 91 bp, 85 bp

[22]

XPD Lys751Gln 61 234 PstI A: 234 bp, 110 bp

C: 171 bp, 110 bp, 63 bp

[23]

5700 Mol Biol Rep (2012) 39:5699–5704

123

300 mA. Subsequently, slides were washed three times

with neutralizing solution (0.4 M Tris, pH 7.5), stained

with DAPI (20 lg/ml) and analyzed with a Nikon Eclipse

50i fluorescence microscope under magnification of 4009.

One hundred comets on each slide were scored by using a

Lucia Comet Assay software v.4.81 (Laboratory Imaging,

Prague, Czech Republic). All experiments were conducted

in duplicate using two blood samples taken from each

subject. Chemicals were supplied by Sigma-Aldrich, Inc.

Statistical analysis

Statistical analysis was carried out using StatsDirect sta-

tistical software (version 2.7.8). Data were expressed as

mean ± SD and values of P \ 0.05 were considered sta-

tistically significant. One way analysis of variance

(ANOVA) was used to assess the significance of the dif-

ferences of the basal DNA damage levels among the car-

riers of different genotypes and to determine effect of

combination of alleles on DNA damage levels.

Results

The frequency of genetic variants analyzed in study group

is presented in Table 2. No differences in genotype dis-

tribution between females and males were observed (data

not shown). In our group we found very low frequency of

CYP1A1 Val allele (0.05) and no ValVal homozygotes

were recognized. The occurrence of T1 null allele of

GSTT1 gene was also very low (0.09), therefore most

responders are carriers of T1 allele. Among study partici-

pants a similar frequency of M1 null and M1 positive (?)

alleles in the GSTM1 gene was observed. There were slight

differences (0.58 vs. 0.42) in the occurrence of A and C

alleles in the XPD gene locus. However, number of AA

homozygotes was twice greater then the number of CC

homozygotes, and the predominance of heterozygotes was

clearly indicated. Within the studied polymorphic forms of

GSTP1 gene, the frequency of A allele was significantly

higher than the frequency of G allele, and in the study

group domination of AA homozygotes was recorded.

The levels of basal DNA damage measured by comet

assay in relation to the polymorphisms of CYP1A1,

GSTM1, GSTT1, GSTP1 and XPD genes were analyzed

and the data are presented in Fig. 1. Highly statistically

significant differences in DNA damage between GSTM1

(?) allele carriers and GSTM1 null allele carriers were

found (P \ 0.0001).

The GSTM1 (?) allele, regardless of the presence of other

genetic variants, was associated with lower DNA damage

than GSTM1 null allele (Table 3). However, the effect of

presence of other genetic variants on DNA repairing pro-

cesses and observed levels of DNA damage can not been

disclosed. The potential impact of CYP1A1 Val, GSTT1 null

and GSTP1 A alleles can be suggested. The occurrence of

GSTT1 null allele was very low, which made impossible to

analyze the potential effect of T1 (?) allele. Simultaneous

presence of GSTM1 (?) and CYP1A1 IleVal variants was

associated with lower DNA damage than the presence of

*

** *** ****

*****

0

1

2

3

4

5

6

7

8

9

10

CYP1A1

II

CYP1A1

IV

GSTM1

(null

)

GSTM1

(+)

GSTT1 (n

ull)

GSTT1 (+

)

GSTP1 AA

GSTP1 AG

GSTP1 GG

XPD CC

XPD AC

XPD AA

DN

A d

amag

e (%

DN

A in

tai

l)Fig. 1 Genetic variants and

levels of basal DNA damage.

Arithmetic mean ± SD are

presented; P values was

calculated by one way analysis

of variance Anova: P = 0.189;

**P \ 0.0001; ***P = 0.0678;

****P = 0.4368;

*****P = 0.764

Table 2 Genotype distribution and allele frequency in the studied

population, n = 220

Genetic

polymorphism

Genotypes Genotype

distribution

n (%)

Allele

frequency

CYP1A1 IleIle 200 (91)

IleVal 20 (9) Ile 0.95

ValVal 0 (0) Val 0.05

GSTM1 M1 null 112 (51) M1 null 0. 51

M1 ? 108 (49) M1 ? 0.49

GSTT1 T1 null 20 (9) T1 null 0.09

T1 ? 200 (91) T1 ? 0.91

GSTP1 GG 22 (10)

AG 84 (38) G 0.29

AA 114 (52) A 0.71

XPD AA 68 (31)

AC 119 (54) A 0.58

CC 33 (15) C 0.42

Mol Biol Rep (2012) 39:5699–5704 5701

123

GSTM1 (?) and CYP1A1 IleIle genotypes (Table 3), indi-

cating that IleIle CYP1A1 homozygocity may be related to

disturbances in DNA repair processes. However, the low

frequency of CYP1A1 Val allele in study group did not allow

for a more profound analysis. The highest levels of DNA

damage were found in carriers of both genotype: AA geno-

type of XPD gene and M1 null variant of GSTM1 gene

(6.7%). The lowest level of DNA breaks was associated with

the genotype GSTP1-AA/GSTM1 (?) (3.7%). GSTP1/AA

homozygocity accompanied by GSTM1 (?) variant was

associated with lower DNA damage levels than GSTP1/GG

homozygocity. However, there were no differences in DNA

damage between GSTP1/AA and GSTP1/GG homozygotes

in the presence of GSTM1 null or GSTT1 null alleles.

Therefore, it may be suggested that the influence of GSTM1

and GSTT1 variants is much more stronger than that of

GSTP1 variants.

Discussion

Cells are consistently exposed to exogenous and endoge-

nous substances that may damage their DNA and cause

development of different pathological processes. To protect

and deal with DNA damage cells possess mechanisms that

allow both neutralization of harmful substances and DNA

repair. Therefore, the levels of DNA damage may be

influenced by the polymorphisms in genes encoding for

proteins involved in the metabolism of xenobiotics and

repair processes.

Most toxic compounds are activating by phase I

enzymes, such as cytochrome P450-1A1 (CYP1A1), to

become an ultimate reactive form and then may be sub-

jected to detoxification by phase II enzymes, mainly glu-

tathione S-transferase (GST). Allelic gene variants that

have impaired detoxification function may increase the rate

of genetic damage. Genetic variability in GST or CYP1A1

isoenzymes may change the balance between phase I and

phase II biotransformation and may therefore be respon-

sible for individual differences in susceptibility to ciga-

rette, alcohol, caffeine or environmental toxins.

Table 3 Differences in basal DNA damage between carriers of dif-

ferent genetic variants

Genotypes N DNA damage

(% DNA in tail)

P*

CYP1A1 IleIle/GSTM1 (?) 83 5.2 ± 2.9

CYP1A1 IleIle/GSTM1(null) 119 5.65 ± 2.03 \0.0001

CYP1A1 IleVal/GSTM1 (?) 9 4.03 ± 1.01

CYP1A1 IleVal/GSTM1 (null) 9 5.8 ± 1.3 NS

XPD AA/GSTM1 (?) 15 4.01 ± 1.1

XPD AA/GSTM1 (null) 18 6.7 ± 2.6 \0.0001

XPD AC/GSTM1 (?) 62 5.13 ± 3.1

XPD AC/GSTM1 (null) 57 5.9 ± 1.4 NS

XPD CC/GSTM1 (?) 31 4.5 ± 1.6

XPD CC/GSTM1 (null) 37 5.9 ± 1.4 \0.0001

GSTP1 AA/GSTM1(?) 4 3.7 ± 0.6

GSTP1 AA/GSTM1 (null) 18 6.4 ± 1.8 0.0088

GSTP1 AG/GSTM1 (?) 44 5.5 ± 3.9

GSTP1 AG/GSTM1 (null) 42 5.9 ± 1.4 NS

GSTP1 GG/GSTM1 (?) 46 4.4 ± 1.9

GSTP1 GG/GSTM1 (null) 66 6.1 ± 2.0 \0.0001

CYP1A1 IleIle/XPD AA 31 5.3 ± 1.7

CYP1A1 IleIle/XPD AC 105 5.6 ± 2.8

CYP1A1 IleIle/XPD CC 64 5.5 ± 2.9 NS

CYP1A1 IleVal/XPD AA 4 4.9 ± 1.7

CYP1A1 IleVal/XPD AC 9 4.1 ± 1.4

CYP1A1 IleVal/XPD CC 7 5.8 ± 1.8 NS

XPD CC/CYP1A1 IleIle 64 5.5 ± 2.95

XPD CC/CYP1A1 IleVal 7 4.7 ± 1.45 NS

XPD AC/CYP1A1 IleVal 105 5.56 ± 2.8

XPD AC/CYP1A1 IleVal 9 4.51 ± 1.4 NS

XPD AA/CYP1A1 IleIle 31 5.3 ± 1.7

XPD AA/CYP1A1 IleVal 4 4.7 ± 1.9 NS

XPD CC/GSTT1 (?) 28 5.34 ± 2.7

XPD CC/GSTT1 (null) 4 5.2 ± 1.5 NS

XPD AC/GSTT1 (?) 110 5.3 ± 2.5

XPD AC/GSTT1 (null) 9 4.0 ± 1.4 NS

XPD AA/GSTT1 (?) 60 5.2 ± 1.9

XPD AA/GSTT1 (null) 9 4.7 ± 0.8 NS

GSTT1 (?)/XPD AA 60 5.2 ± 1.9

GSTT1(?)/XPD AC 110 5.3 ± 2.5

GSTT1 (?)/XPD CC 28 5.3 ± 2.7 NS

GSTT1 (null)/XPD AA 9 4.7 ± 0.8

GSTT1 (null)/XPD AC 9 4.0 ± 1.4

GSTT1 (null)/XPD CC 4 5.2 ± 1.5 NS

GSTT1 (?)/GSTP1 AA 101 5.3 ± 2.2

GSTT1 (?)/GSTP1 AG 77 5.4 ± 3

GSTT1 (?)/GSTP1 GG 18 6.2 ± 2.1 NS

GSTT1 (null)/GSTP1 AA 11 4.2 ± 1.2

GSTT1 (null)/GSTP1 AG 9 4.5 ± 1.6

GSTT1 (null)/GSTP1 GG 4 5 ± 1.2 NS

GSTP1 AA/GSTT1 (?) 101 5.3 ± 2.2

Table 3 continued

Genotypes N DNA damage

(% DNA in tail)

P*

GSTP1 AA/GSTT1 (null) 11 4.2 ± 1.2 NS

GSTP1 AG/GSTT1 (?) 77 5.4 ± 3

GSTP1 AG/GSTT1 (null) 9 4.5 ± 1.6 NS

GSTP1 GG/GSTT1 (?) 18 6.2 ± 2.1

GSTP1 GG/GSTT1 (null) 4 5 ± 1.2 NS

Arithmetic mean ± SD are presented; *P values was calculated by

one way analysis of variance Anova

5702 Mol Biol Rep (2012) 39:5699–5704

123

Cytochrome P450 and glutathione-S transferase (GSTs)

enzymes have been shown to play a crucial role in the

carcinogen activation and detoxification. Polymorphic

variants of genes coding for cytochrome P450 enzymes

may lead to increased toxification, whereas polymorphism

in glutathione-S-transferase genes may result in impaired

or enhanced detoxification [24]. Some variants of cyto-

chrome P450 genes as well as GSTM1 and GSTT1 null

genotypes have been found to be associated with increased

risk of several cancers, including lung, breast and colon

cancer, in specific ethnic groups [25–27]. Also the poly-

morphic forms of XPD gene coding for enzyme involved in

DNA repairing, have been described to be associated with

cancer development. However, the results are inconsistent

[28, 29]. Therefore, measurement of cellular DNA damage

and its relation to occurrence of different variants of genes

encoding enzymes involved in detoxification and repairing

processes may be helpful in understanding the potential

role of these variants in cancer risk. The level of DNA

damage measured by comet assay in peripheral blood

lymphocytes is accepted as a useful marker for both the

biological effects of toxicant exposure and host DNA

repair capacity. In the present study we used comet assay to

detect basal levels of DNA damage and assess their

potential association with several genetic variants.

The frequency of XPD, GSTP1 and CYP1A1 genotypes

in our group was similar to that found in other studies on

Polish population [30–32]. Among study participants a

similar frequency of M1 null and M1 positive (?) alleles in

the GSTM1 gene was observed and it is in agreement with

our previous data [19].

In study participant the lowest basal DNA damage

(3.7%) was found in carriers of genotype GSTP1-AA/

GSTM1 (?) genotypes, while the highest (6.7%) in carriers

of AA variant in XPD gene and M1 null variant in GSTM1

gene.

Since GSTs play an important role in oxidative stress by

free radicals scavenging, deletions in GST genes resulting

in decrease of the enzymes activity could enhance the

amount of oxidative damage. The GSTM1 null and GSTT1

null genotype have been found to be associated with an

increased risk for several cancers [27, 33] and one possible

explanation of these observations could be impaired anti-

oxidant defense. In our study subjects with wild type

GSTM1 (?) seem to be better protected against DNA

damage than those with variant type (GSTM1 null); as

described by significantly lower level of basal DNA dam-

age: 4.75 versus 6.01% (P \ 0.0001). It may be hypothe-

sized that subjects having an unfavorable GSTM1 null

polymorphism may be more susceptible to oxidative DNA

damage. We didn’t find such association with respect to

GSTT1 gene, maybe due to low frequency of variant allele

(only 9% of GSTT1 null allele carriers).

The XPD polymorphisms are most extensively studied in

cancer patients, because of their influence on DNA repair

systems. A positive association between CC genotype (Gln/

Gln) and cancer development have been described [28, 32,

34–38]. However, our results did not reveal any significant

relations between variants in XPD gene and basal DNA

damage among studied healthy subjects. AA homozygosity

combined with GSTM1 (?) variant was characterized by

lower level of basal DNA damage than combined with

GSTM1 null variant: 4.01 versus 6.7% (P \ 0.0001). Also

the impact of studied CYP1A1 genetic variants as compared

to GSTM1 variants on risk of basal DNA damage does not

seem to be significant, especially that most studied subjects

were homozygotes for Ile allele (91%).

As many questions remain, further studies are needed on

larger population to clarify the influence of genetic variants

and gene–gene interactions on DNA damage response.

Such research will lead to better understanding of natural

skills of the organism to maintain genomic integrity and

lower risk of disease associated with DNA damage.

Acknowledgment This work was supported by grant number

N404042/32/0945 from the Ministry of Science and Higher

Education.

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