Mutation Research 726 (2011) 227233

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Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

Role of OGG1 Ser326Cys polymorphism and 8-oxoguanine DNA damage in risk

assessment of squamous cell carcinoma of head and neck in north Indian
population
Anil Kumar a , Mohan Chand Pant b , Hirdya Shanker Singh c , Shashi Khandelwal a,
a
CSIR-Indian Institute of Toxicology Research, Lucknow (CSIR-IITR), India
b
C.S.M. Medical University, Lucknow, India
c
Ch. Charan Singh University, Meerut, India

a r t i c l e i n f o a b s t r a c t

Article history: Squamous cell carcinoma of head and neck (SCCHN), one of the leading cancers worldwide, is most
Received 9 July 2011 prevalent in Indian sub-continent. The major risk factors involved are smoking and consumption of
Received in revised form 7 September 2011 alcohol, since they provide high free radical generating environment.
Accepted 25 September 2011
We studied 8-oxoguanine DNA-glycosylase (OGG1) Ser326Cys polymorphism in 278 SCCHN cases and
Available online 1 October 2011
278 matched controls by PCR-RFLP and observed that the variant genotype Ser/Cys exhibited an enhanced
risk of 1.7 folds (OR = 1.71, 95% CI = 1.202.93) and Cys/Cys 2.5 folds (OR = 2.55, 95% CI = 1.295.00).
Keywords:
Furthermore, we found a signicant increase in salivary cell 8-OHdG with respect to Ser/Cys and Cys/Cys
SCCHN
OGG1 polymorphism
genotypes of OGG1 in SCCHN cases, when compared to Ser/Ser and Ser/Cys genotypes of the control
8-OHdG population. Our results demonstrate that Ser326Cys variant genotype is associated with an increased
Genetic susceptibility risk of SCCHN in north India. Ser326Cys variant genotype was found to accumulate more of 8-OHdG,
Life style factors which may serve as a biomarker for early diagnosis of SCCHN.
2011 Elsevier B.V. All rights reserved.

1. Introduction
The squamous cell carcinoma of head and neck, one of the
leading cancers worldwide is most prevalent in the Indian sub-
continent. The major risk factors involved are heavy consumption
of tobacco smoking and its smokeless products such as betel nut,
gutkha, surti, khaini and pan masala. In south Asian countries, par-
ticularly in India, these addictions have entered in the life style
of majority of people [1,2]. Some of the people acquire the car-
cinogenic symptoms early and some late, and in still others the
symptoms fail to appear. Genetic susceptibility including DNA
repair polymorphic genes plays a vital role in determining cancer
incidences. Most of these cancers are preventable, if diagnosed at
early stages.
Tobacco smoke is a cocktail of 4000 chemicals, out of which
more than 55 are carcinogens and about 400 are toxic chemicals.
Nicotine, carbon monoxide, hydrogen cyanide, arsenic and DDT
are some of them. Polycyclic hydrocarbons (PAHs) and aromatic
amines are well known carcinogens that form adducts with DNA
Corresponding author at: CSIR-Indian Institute of Toxicology Research (CSIR-
IITR), P.O. Box 80, M.G. Marg, Lucknow 226001, India. Tel.: +91 522 2627586x316;
fax: +91 522 2628471.
E-mail address: skhandelwal itrc@rediffmail.com (S. Khandelwal).
[3,4]. Smoking and tobacco specic carcinogens are associated with
increase in gene mutations in several cancers including head and
neck [5]. In addition, alcohol is another factor whose consumption
appears to be associated with an increase in cancer risks, although
mechanisms are not clearly understood. Alcohol is suppose to act
like a solvent that facilitates uptake of environmental carcinogens,
especially tobacco smoke through cell membranes [6]. Smoking,
smokeless tobacco and alcohol generate free radicals that induce
oxidative and nitrative stress. They in turn may lead to oxidative
lesions in DNA. The most common of the base lesion caused by
ROS is 7,8-hydro-8-oxoguanine (8-OHdG) [3]. The latter is abun-
dant and is highly mutagenic, since it can base pair with adenine
residue, resulting in G:C to T:A transversion during replication [7].
Therefore, maintaining genome integrity seems essential in cancer
prevention and DNA repair systems are actively involved in pre-
venting insults to DNA by carcinogenic agents such as those present
in tobacco. More than 130 genes are known to be involved in DNA
repair pathways, the major pathways being nucleotide excision
repair (NER) and base excision repair (BER) [8].
Many of the DNA repair genes are highly polymorphic and have
an inuence on protein structure. They also play an imperative role
in genetic susceptibility of an individual to cancer development or
prevention. OGG1, MUTYH and MTH1 are members of base excision
repair (BER) families and are human homologous of MutM, MutY
and MutT respectively, of Escherichia coli [9,10]. These have been

1383-5718/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2011.09.015
228 A. Kumar et al. / Mutation Research 726 (2011) 227233
shown to be positively or negatively associated with cancer risk
occurrences [1113]. OGG1 along with apurinic/apyrimidinic (AP)
lyase, catalyzes the cleavage of glycosylic bond between the modi-
ed base and sugar moiety, leaving an abasic apurinic/apyrimidinic
(AP) site in DNA with a 3 -blocking end, which is successively com-
pleted by AP endonuclease (APEX), DNA polymerase and DNA
ligase III protein. The repair of 8-OHdG lesion in DNA is initiated
by OGG1 in eukaryotes and by DNA glycosylase-Fpg in bacteria.
The OGG1 protein is encoded by gene located on chromosome
3p26.2. The well known OGG1 gene polymorphism present at posi-
tion 1245 in exon 7 is a C G transversion, which results in a serine
to cysteine substitution in codon 326 (Ser326Cys). The loss of OGG1
activity in a number of model systems was shown to favor accumu-
lation of 8-hydroxy-2-deoxyguanosine and as a consequence G/C
to T/A mutations [1417].
MUTYH gene, localized on chromosome 1p34.3p32.1, encodes
a DNA glycosylase and is involved in oxidative DNA damage
repair. The enzyme excises adenine bases that are misincorporated
(instead of cytosine) opposite 8-OHdG, a major oxidative damaged
DNA lesion. Germinal mutations in the base excision repair (BER)
gene MUTYH have been described in association with predisposi-
tion to multiple colorectal adenomas and cancer [18]. The Y165C
mutation of exon 7 occurs in the pseudo helix-hairpin-helix (HhH)
motif that is thought to be involved in substrate recognition. The
G382D mutation of exon 13 occurs at a splice acceptor site and
is predicted to result in alternative splicing [19]. Biallelic loss of
MUTYH is associated with somatic G:C to T:A transversions in the
adenomatous polyposis coli (APC) gene and is thought to be respon-
sible for colorectal cancer [20], whereas Smith et al. [21] reported
high frequency of somatic K-ras gene G T transversion mutations
at codon 12 position 1 in MUTYH-associated sporadic pancreatic
cancer.
MTH1 also known as NUDT1 is localized on chromosome 7p22
and includes a polymorphic site at codon 83 in exon 4 consist-
ing of a G A transition, corresponding to Val Met substitution
[22]. MTH1 protein hydrolyzes oxidized purine nucleoside triphos-
phates, such as 8-oxo-dGTP, 8-oxo-dATP, 2-hydroxy-dATP and
2-hydroxy-rATP to monophosphates, thereby preventing misincor-
poration. The encoded protein is localized mainly in cytoplasm with
some in the mitochondria, suggesting that it is involved in san-
itization of nucleotide pools both for nuclear and mitochondrial
genomes. Met (83)-MTH1 protein is found to be more thermo labile
than that of the Val allele, in its enzyme activity and secondary
structure [23].
Bravard et al. [24] supported the idea that individuals homozy-
gous for OGG1-Cys variant could more readily accumulate
mutations under conditions of oxidative stress. Controversial
reports of OGG1-Cys326 allele are available. Some studies found
it to be associated with increased risk of developing cancers
of lung, colorectal, head and neck, prostate and orolaryngeal
[12,2529], while others showed no association in colorectal, colon,
esophageal, prostate and gastric cancer [3032].Squamous cell
carcinoma of Head and Neck is a tobacco related cancer and its inci-
dences increase with the extent of tobacco usage. OGG1Ser326Cys
polymorphism has so far been studied in gastric, esophageal and
gall bladder cancer in north Indian population [29,30] but not in
head and neck cancer. Strong association of Gln324His variant of
MUTYH gene was found in colorectal cancer of Japanese popu-
lation and on the other hand, no association in head and neck
and heptocellular and cholangiocarcinoma [33,34]. In the case of
MTH1 Val83Met polymorphism, Miyako et al. [35] demonstrated
involvement of Met83 allele in the development of type 1 dia-
betes mellitus in Japanese female population and a signicant and
strong association has also been found in lung cancer [36]. In spo-
radic Parkinsons disease, no association of MTH1 gene has been
reported [37].
Among the three oxidative DNA repair genes (OGG1, MUTYH
and MTH1), we are probably the rst to report association of OGG1
Ser326Cys polymorphism in north Indian population and its corre-
lation with oxidative DNA adduct 8-OHdG in salivary cells of SCCHN
patients.
2. Materials and methods
2.1. Subjects
The case control study involved 278 north Indian SCCHN patients and 278
geographically and racially matched healthy controls that also included visitors
of Radiotherapy Department of C.S.M. Medical University, Lucknow, but not those
related to SCCHN patients. The cases had pathologically conrmed squamous cell
carcinoma of the oral cavity, pharynx or larynx conrmed by cytological and
histopathological examinations. None of the control individuals had a personal or a
family history of malignancy. All controls were age and sex matched and as far as
possible also matched for habits. The study subjects belonging to Indo-European
linguistic group, resided in Lucknow city or neighbouring places in north India
[38]. The study was initiated following approval of the respective Human Ethi-
cal Committees of C.S.M. Medical University, Lucknow and CSIR-Indian Institute of
Toxicology Research, Lucknow. The protocol conrmed to the provisions of decla-
ration of Helsinki in 1995. Informed consent was obtained from the study subjects,
for inclusion in the study, and subject anonymity was ensured, prior to collection
of blood samples. All study subjects completed a questionnaire covering medical,
residential and occupational history, including dietary habits and family history.
The mean age of controls and cases was 50 (range 2570 years) and 52 (range
2575 years), respectively. Subjects having regular smoking habits and smoking
index (cigarettes/day 365) of more than 730 [39] and regular smokeless tobacco
chewers with chewing index more than 365 [40] (CY = frequency of tobacco chewed-
kept/day 365), were included in the category of smokers and tobacco chewers,
respectively. Less than the chewing index of 365, subjects was grouped in the cat-
egory of non-tobacco chewers. The cumulative exposure of alcohol drinking was
derived by multiplying the total yearly consumption of alcohol (in l/year) by dura-
tion in years. Those who had a cumulative exposure to alcohol of about 46 l/year
were considered as regular alcoholics [41].
2.2. Genotyping
One millilitre of blood was collected in sodium citrate (3.4%, pH 7.6) coated vials
from all the study subjects and genomic DNA was isolated from blood samples using
Qiagen mini DNA kit following the manufacturers protocol.
The quantity and purity of DNA samples were measured by Nanodrop Spec-
trophotometer (ND 1000 V.3.3) and stored at 20 C. PCR-RFLP was used for the
genotyping of OGG1 Ser326Cys, MUTYH G282D, MUTYH Y165C and MTH1 Val83Met
gene polymorphisms.
The nal volume of each PCR reaction mixture was 25 l and each reaction
tube contained 12.5 l Dream Taq Master Mix (Fermentas Life Sciences), 0.6 l of
each primer (10 pmole), 50 ng genomic DNA and 9.3 l of nuclease free water. All
PCR reactions were performed on peltier based thermal cycler (G-storm, Labmate
India). The PCR product of all the variants were visualized by electrophoresis on 1.5%
agarose gel containing 0.5 g/ml ethidium bromide. The RFLP digestion was carried
out at 37 C overnight and resolved on 2.5% agarose gel to identify the band pattern.
OGG1 Ser326Cys polymorphism was determined using the following primers:
sense 5 -ACT GTC ACT AGT CTC ACC AG-3 ; antisense 5 -TGA ATT CGG AAG GTG CTT
GGG GAA T-3 [42]. The cycling conditions were: initial denaturation at 94 C for
5 min, then 35 cycles of denaturation at 94 C for 50 s, annealing at 56 C for 30 s and
elongation at 72 C for 50 s, followed by a nal extension step at 72 C for 7 min. Ten
microlitres of 207 bp PCR product was digested with 10 units of Fnu4HI (Fermen-
tas Life Sciences). A three band pattern was observed depending on the genotype,
a single 207 bp band corresponded to Ser326Ser genotype, 207, 107 and 100 bp
bands to Ser326Cys genotype and 107 and 100 bp bands to Cys326Cys genotype.
The sequence of PCR amplied bands was conrmed commercially by a Genomics
Service Provider (Xcelris Labs Ltd, Ahmedabad, India) (Fig. 1A and B).
G382D polymorphism in MUTYH was determined using the following primers:
sense 5 -AGG GCA GTG GCA TGA GTA ACA-3 ; antisense 5 -GCT ATT CCG CTG CTC
ACT TAC CT 3 . The cycling conditions were: initial denaturation at 94 C for 4 min,
then 35 cycles of denaturation at 94 C for 40 s, annealing at 57 C for 30 s and elon-
gation at 72 C for 40 s, followed by a nal extension step at 72 C for 7 min. Ten
microlitres of 241 bp PCR product was digested with 10 units of BglII (Fermentas
Life Sciences). A three band pattern has been reported depending on the genotype, a
single 241 bp band corresponded to wild type genotype, 241,159 and 82 bp bands to
G382D heterozygous genotype and 159 and 82 bp bands to mutant genotype [34].
In a sample survey of 50 controls and 50 cases, we observed single band of 241 bp,
indicating wild type in all the subjects (Fig. 2A).
Y165C polymorphism in MUTYH was determined using the following primers:
sense 5 -GAC TGA CGG GTG ATC TCT-3 ; antisense 5 -CTG ATT GGA GTG CAA GAC 3 .
The cycling conditions were: initial denaturation at 94 C for 4 min, then 35 cycles of
denaturation at 94 C for 40 s, annealing at 56 C for 30 s and elongation at 72 C for
 A. Kumar et al. / Mutation Research 726 (2011) 227233
Fig. 1. (A) The representative PCR-RFLP analysis of OGG1 Ser326Cys polymorphism.
M = 100 bp DNA ladder, Lane 1 undigested PCR product, lane 2 Ser/Ser homozy-
gous wild genotype, lane 3 Cys/Cys homozygous mutant genotype, lane 4
Ser/Cys heterozygous genotype. (B) DNA sequence histogram revealing the indi-
cated sequence changes (arrow). In the 2nd histogram, blue line for cytosine and
black line for guanine observed in codon 326 site (marked as N), indicating the
heterozygous genotype.
40 s, followed by a nal extension step at 72 C for 7 min. Ten microlitres of 105 bp
PCR product was digested with 10 units of BvuI (Fermentas Life Sciences). A three
band pattern has been reported depending on the genotype, 105 and 83 bp bands
corresponded to wild type (Tyr165Tyr) genotype, 105, 83 and 61 bp bands to het-
erozygous (Tyr165Cys) genotype and 105 and 61 bp bands to mutant (Cys165Cys)
genotype [34]. In the pilot experiment employing 50 controls and 50 cases, we
observed 105 and 83 bp bands and 105, 83 and 61 bp bands, indicating wild type
and heterozygous genotype, respectively, in all the subjects. None of the samples
exhibited Cys165 allele (Fig. 2B).
MTH1 polymorphism was determined using the following primers: sense 5 -
CAT GGC ACC ATG CCC TGA-3 ; antisense 5 -GAG ATG GGA CCC GCA TAG-3 . The
cycling conditions were: initial denaturation at 94 C for 5 min, then 38 cycles of
denaturation at 94 C for 1 min, annealing at 61 C for 45 s and elongation at 72 C
Fig. 2. (A) The representative PCR-RFLP analysis MUTYH-G382Dpolymorphism. Lanes 15 wild genotype. M = 100 bp DNA ladder. (B) The representative PCR-RFLP analysis
MUTYH-Y165C polymorphism. Lanes 15 wild genotype, lanes 6 and 8 heterozygous genotype. M = 50 bp DNA ladder. (C) The representative PCR-RFLP analysis MTH1
Val83Met polymorphism. Lanes 13 wild genotype. M = 100 bp DNA ladder.
229
for 40 s, followed by a nal extension step at 72 C for 5 min. Ten microlitres of 247 bp
PCR product was digested with 10 units of NsiI (Fermentas Life Sciences). A three
band pattern has been reported for the three variants, a single 247 bp band corre-
sponded to Val83Val wild type, 247,150 and 97 bp bands to Val83Met heterozygous
genotype and 150 and 97 bp bands to Met83Met mutant genotype [35]. Similar to
MUTYH G382D polymorphism, we found only the wild type variant (Val83Val) in
all the 100 individuals (Fig. 2C).
2.3. Oxidative DNA adduct (8-OHdG) in salivary cells
Saliva was collected under resting condition, at least 1 h after food intake. Sub-
jects were asked to generate saliva in their mouth which was collected in a wide
mouth collection tube. They were then ltered through 100 m nylon mesh.
For DNA extraction, the samples were centrifuged at 10,000 g for 10 min and
the pellet (salivary cells) was used for DNA isolation, following the method of Miller
et al. [43] with some modications. The pellet was suspended in 3 ml of lysis buffer
(50 mM TrisHCl, 100 mM NaCl, 100 mM Na2 EDTA, 0.5% SDS adjusted to pH 8.0) and
100 l of 10% SDS and 500 l of Proteinase K (1 mg/ml in 2 mM Na2 EDTA) and further
incubated overnight at 37 C. One ml of 6 M sodium chloride was then added and
after vigorous shaking for 15 s, the samples were centrifuged at 700 g for 15 min.
Pellet was discarded and to the supernatant double volume of cold 100% ethanol was
added, to precipitate DNA. The precipitate was resuspended in 200 l of TrisHCl,
pH 8.5 and was left at 37 C to dissolve. The amount and purity of DNA was measured
by NanoDrop Spectrophotometer (ND 1000 V3.3.1).
DNA adduct (8-OHdG) was quantied using DNA Damage Quantication Kit
(BioVision, USA). Five microlitres of highly puried DNA sample (0.1 g/l) was
mixed with 5 l of aldehyde reactive probe (ARP) solution and incubated for 60 min
at 37 C, to tag apurinic (AP) sites of DNA with biotin residues. Avidinbiotin assay
was carried out according to the manual provided. 40 ARP-DNA Standard (40 ARP
sites per 105 bp) was used for the determination of AP sites in DNA samples to
ascertain the level of oxidative damage.
2.4. Statistical analysis
Genotype frequencies of OGG1 Ser326Cys among controls were determined for
Hardy Weinberg Equilibrium (HWE) using standard Chi square (2 ) test. Odds ratios
(ORs) and 95% condence intervals (95% CI) were estimated to ascertain association
of individual genotype with SCCHN risk. Multiple Logistic Regression analysis was
performed to calculate adjusted ORs for subsequent analysis of potential risk factors
like smoking, tobacco chewing and alcohol consumption. All statistical analysis was
performed with the SPSS software package (version 12.0 for windows; SPSS Chicago,
IL). The p value 0.05 was considered as signicant. Separate comparisons of 8-
OHdG contents in salivary cells with OGG1 codon326 Ser/Ser, Ser/Cys and Cys/Cys
genotypes, were conducted by using one way ANOVA (GraphPad Prism 5, CA, USA).
The false-positive report probability for the statistically signicant results was also
calculated [44].
3. Results
The present study included 278 healthy controls and equal num-
ber of head and neck cancer patients in the age group of 2570 and
2575, respectively. The median age of SCCHN cases was 52 years
and 50 years for the control group (Table 1). Most of the SCCHN
patients were critically ill and belonged to the advanced stage. The
230 A. Kumar et al. / Mutation Research 726 (2011) 227233
Table 1
Demographic variables, risk estimates and distribution of SCCHN cases by tumor
sites.
Variable Controls Cases
n (%) n (%)
Subjects 278 278
Age (mean) 50 52
Range 2570 2575
Habituates
Smokers 157 (56) 222 (80)
Non-smokers 121 (44) 56 (20)
Tobacco chewers 144 (52) 153 (55)
Non-tobacco chewers 134 (48) 125 (45)
Alcoholics 124 (45) 134 (48)
Non-alcoholics 154 (55) 144 (52)
Site of tumor
Oral cavity 155
Larynx 72
Pharynx 51
patients or controls had no family history of cancer. As evident
from Table 1, prevalence of smoking and tobacco chewing among
patients resulted in higher incidence of SCCHN.
The distribution of OGG1 genotypes in controls and patients
is shown in Table 2. The genotype frequencies in controls were
in Hardy-Weinberg equilibrium (p > 0.05). The wild type geno-
type Ser326Ser was taken as the referent category. Prevalence of
variant genotypes was enhanced in cancer patients when com-
pared to controls. A 1.7 fold increase in SCCHN risk (OR = 1.71,
95% CI = 1.202.93) was associated with Ser/Cys genotype and 2.6
fold increase (OR = 2.55, 95% CI = 1.295.00) with Cys/Cys genotype.
These risks persisted even when the data was adjusted for smok-
ing, chewing tobacco and alcohol in multivariate logistic regression
and was statistically signicant. Since we found only the wild type
variants of MUTYH G382D and MTH1 and only the wild and het-
erozygous variants of MUTYH Y165C in the pilot experiment, we
did not further pursue genotyping in rest of the samples. Marginal
risk, although statistically non-signicant, was observed with het-
erozygous variant of MUTYH Y165C (Table 2).
To investigate whether OGG1 genotype interacts with can-
cer risk modiers, i.e. smoking, tobacco chewing and alcohol
consumption, we performed stratied analyses and the data is
summarized in Table 3. Variant genotype frequency increases
in SCCHN with history of smoking, tobacco chewing and alco-
hol, when compared to their respective controls. Among smokers
with variant genotypes Ser/Cys and Cys/Cys, the risk increased
2.4 folds (OR = 2.40, 95% CI = 1.533.77) and 3.2 folds (OR = 3.15,
95% CI = 1.387.18) respectively, when compared to smoker con-
trols. Variant Ser326Cys exhibited a risk of 1.9 (OR = 1.88, 95%
CI = 1.163.05) and with Cys326Cys a risk of 3 (OR = 3.05,
95% CI = 1.327.05) for tobacco chewers, relative to their respec-
tive controls was seen. Interaction of alcohol with genotype of
OGG1 Ser326Cys genes was also analyzed and we found 1.8
folds (OR = 1.80, 95% CI = 1.066.44) increased risk for genotype
Ser326Cys and 2.6 folds with Cys326Cys (OR = 2.62, 95% CI
1.066.44).
We further analyzed the OGG1 genotype data with respect to
DNA adduct (8-OHdG) in salivary cells. The mean 8-OHdG levels in
SCCHN were observed to be 18.21 4.44 AP sites/105 bp against
11.06 8.04 AP sites/105 bp (p < 0.001), in controls. The 8-OHdG
levels in the various genotypes of OGG1 Ser326Cys in controls
and cases are shown in Table 4. On comparison of case Ser/Ser,
Ser/Cys and Cys/Cys versus control Ser/Ser, the DNA adduct levels
were found to be signicantly increased (p < 0.01). On comparisons
of case Ser/Ser, Ser/Cys and Cys/Cys versus control Ser/Cys, a sig-
nicance of p < 0.05 was observed, however, within the same test
group, a rise in 8-OHdG in the variant genotypes was apparent, but
remained statistically non-signicant.
4. Discussion
In the present study, we have evaluated the role of genetic
polymorphism of DNA Glycosylase in head and neck cancer and
observed a strong correlation of OGG1 polymorphism with SCCHN
risk. A large number of controversial studies are available of OGG1
Ser326Cys polymorphism in lung cancer: either association [12,27]
or no association [11]. Recently, it was shown to be associated with
gallbladder in Chinese [45], in north Indian population [29,46], in
head and neck cancer of Polish population [26] and type 2 Diabetes
in Mexican Americans [47]. Individuals with low OGG1 activity
have shown to exhibit an increased risk of lung and head and neck
cancers in amalgamation with tobacco smoking [48,49].
The wild allele of MTH1 observed by us, suggest no involvement
of this gene with head and neck cancer. However, some studies
report positive association of MTH1 gene with Type 1 diabetes
mellitus and colorectal cancer [35,50]. The data on 100 individu-
als suggest that the heterozygous genotype of MUTYH Y165C was
marginally associated with SCCHN (statistically non-signicant)
and we found no association in the case of G382D, where only wild
type genotype was present (Table 2). However, in Polish popula-
tion, Sliwinski et al. [51] reported MUTYH Y165C association with
the development of SCCHN.
Regarding glycosylase activity of OGG1, it is known to vary
depending on its genotypes and Cys/Cys genotype has been shown
to have lower glycosylase activity in comparison to Ser/Cys and
Ser/Ser [24]. Yeast decient of OGG1 have 50 times more G:C T:A
transversions compared to wild strain [15]. Even with advancing
age, the OGG1 activity appears to decline [52].
The potential geneenvironment interaction such as smoking,
tobacco chewing (smokeless tobacco) and alcohol play a vital role
in modulating the frequency of SCCHN. These cancer risk modi-
ers provide a powerful free radical generating environment and
an increase in 8-OHdG lesions with reduced OGG1 activity in smok-
ers is reported in a number of studies [5355]. Paz-Elizur et al. [49]
on the other hand, demonstrated no effect of smoking on OGG1
activity. The DNA glycosylase activity is also inhibited by reactive
nitrogen species [56].
In our studies, the data on gene-environment interactions illus-
trated a positive association of smoking and tobacco chewing in
SCCHN cases with OGG1 variant genotypes. These results are in
accordance with similar reports available in lung [57], head and
neck [26,58], gastric [59] and breast cancer [60]. On the contrary,
no association of smoking and OGG1 polymorphism in lung [61] and
in head and neck cancer [58] has been shown. We are perhaps the
rst to demonstrate a positive inuence of smokeless tobacco and
OGG1 polymorphism in the progression of head and neck cancer,
in north Indian population.
The role of alcohol in development of cancer remains indeci-
sive. An association of alcohol with breast, colorectal and pancreatic
cancer [6264] and no association in SCCHN [58,65], has been
demonstrated. In the present investigation, SCCHN cases consum-
ing alcohol have exhibited an 1.8 and 2.6 fold increased risk.
The probable reason could be related to the genotoxic damage
through acetaldehyde, which is the main metabolite of alcohol. Fur-
thermore, alcohol is known to activate proto-oncogenes, increase
free radical generation via cytochrome P-450 2E1 and abnormal
DNA methylation. It can suppress the immune system as well. De-
ciency of folate and nutrition also play a crucial role in enhancing
the severity of alcohol [66,67].
Signicant increase in 8-OHdG in SCCHN cases and enhanced
cancer risk by OGG1 variants suggest that the impaired repair
 A. Kumar et al. / Mutation Research 726 (2011) 227233 231

Table 2
Distribution of genotype OGG1 and MUTYH among controls (n = 278) and SCCHN cases (n = 278).

Genotype Controls Cases OR 95% CI p value

n (%) n (%)

OGG1 codon 326

Ser/Ser 169 (61) 128 (46) 1.00a
Ser/Cys 95 (34) 123 (44) 1.71 1.202.93 0.003*
Cys/Cys 14 (5) 27 (10) 2.55 1.295.00 0.007*
MUTYH codon 165
Tyr/Tyr 27 (54) 22 (44) 1.00a
Tyr/Cys 23 (46) 28 (56) 1.49 0.683.27 0.42
Cys/Cys
a
Reference group; OR: Odds ratio; 95% CI: 95% condence interval.
*
p < 0.05 is considered statistically signicant.

Table 3
Interaction of OGG1 Ser326Cys genotypes with smoking, tobacco chewing and alcohol consumption among controls (n = 278) and SCCHN cases (n = 278).

Non-smokers Smokers

Genotype Controls Cases OR 95% CI p value Controls Cases ORb 95% CI p value
n = 121 n = 56 n = 157 n = 222

Ser/Ser 62 26 1.0a 107 102 1.0a

Ser/Cys 53 27 1.22 0.642.32 0.62 42 96 2.40 1.533.77 0.001*
Cys/Cys 6 3 1.19 0.344.70 1.00 8 24 3.15 1.387.18 0.007*
Ser/Cys + Cys + Cys 59 30 1.21 0.612.40 0.63 50 120 2.52 1.613.95 0.001*

Non-tobacco chewers Tobacco chewers

Genotype Controls n = 134 Cases n = 125 OR 95% CI p value Controls n = 144 Cases n = 153 ORc 95% CI p value

Ser/Ser 88 69 1.0a 81 59 1.0a

Ser/Cys 41 49 1.52 0.912.56 0.12 54 74 1.88 1.163.05 0.014*
Cys/Cys 5 7 1.80 0.585.6 0.38 9 20 3.05 1.327.05 0.013*
Ser/Cys + Cys + Cys 46 58 1.60 0.952.73 0.07 63 94 2.05 1.263.35 0.003*

Non-alcoholics Alcoholics

Genotype Controls n = 154 Cases n = 144 OR 95% CI p value Controls n = 124 Cases n = 134 ORd 95% CI p value

Ser/Ser 101 76 1.0a 68 52 1.0a

Ser/Cys 47 57 1.61 0.992.62 0.06 48 66 1.80 1.073.02 0.027*
Cys/Cys 6 11 2.46 0.896.64 0.12 8 16 2.62 1.066.44 0.045*
Ser/Cys + Cys + Cys 53 66 1.65 1.092.72 0.07 56 82 1.92 1.133.44 0.012*
a
Reference group; OR: Odds ratio; 95% CI: 95% condence interval.
b
OR: adjusted for tobacco chewing and alcohol.
c
OR: adjusted for smoking and alcohol.
d
OR: adjusted for smoking and tobacco chewing.
*
p < 0.05 is considered statistically signicant.

activity of OGG1 may allow 8-OHdG to accumulate in the DNA
with a consequence increase in mutation rates. Janssen et al. [68]
reported that the DNA repair activity of DNA glycosylase (OGG1)
in human does not depend upon the genetic polymorphism at
Ser326Cys, whereas Chen et al. [69] reported 40% lower enzymatic
activity in lymphocyte from Cys/Cys genotype. Several studies
in vitro conrm that DNA glycosylase with Cys allele at codon
326, has less DNA repair activity [6971]. Rodent cells decient
in DNA glycosylase showing a lower activity of the Cys326 variant,
failed to provide any mechanism by which this amino acid substi-
tution affects the enzyme function [71]. Even till date, the crystal
structure available for DNA glycosylase does not include amino
acids beyond position 325. Further, Bravard et al. [24] conrmed
that lymphoblastoid cells, which carry the homozygous Cys326
allele have a 2-fold lower DNA glycosylase activity than cells with
Ser326 allele. In the present study, we found a signicant increase
in 8-OHdG with respect to Ser/Cys and Cys/Cys genotype of SCCHN,
when compared to Ser/Ser and Ser/Cys genotype of the control pop-
ulation. Occurrence of high frequency of Cys allele in smokers along
with increased 8-OHdG, may indicate suppression of DNA repair
activity of this variant, as also shown in cancers of lung, head and
neck cancer [48,49]. In this investigation, our data clearly indicates

Table 4
DNA adduct 8-OHdG in variant genotypes of OGG1.

Genotype codon 326 Controls Cases Salivary cells

n = 50 n = 50

Controls Cases
8-OHdG SD 8-OHdGa SD

Ser/Ser 29 28 9.86 4.77 15.55 7.25

Ser/Cys 15 17 9.88 4.11 17.93 6.28*
Cys/Cys 6 5 13.44 5.91 21.17 9.46*
a
AP sites/105 bp.
*
p < 0.05 is considered statistically signicant when compared to Ser/Ser genotype of control.
232 A. Kumar et al. / Mutation Research 726 (2011) 227233
the role of OGG1 Ser326Cys variant genotypes in the development
of head and neck cancer in north Indian population.
5. Conclusion
In summary, our results demonstrate that Ser326Cys polymor-
phism in OGG1 is associated with an elevated risk of SCCHN in north
Indian population. We also conrm the hypothesis that Cys326Cys
genotype appears to be more susceptible to 8-OHdG accumulation,
since variation in this genotype is known to affect its sensitivity
and DNA repair capacity.
Conict of interest statement
The authors declare that there is no conict of interest.
Acknowledgements
The authors are grateful to Director, CSIR-Indian Institute of
Toxicology Research, Lucknow, for his keen interest and sup-
port in carrying out the study. AK is thankful to UGC, New Delhi
for providing Senior Research Fellowship. Financial support from
CSIR-Network Project SIP-08 is gratefully acknowledged. CSIR-IITR
communication number: 2911.
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