Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

Mondal R, Ghosh SK. HPV infection, GSTM1-GSTT1 genotypes, mitochondrial mutations and tobacco association with oral cancer from northeast India. Head Neck Oncol. 2013 Apr 01;5(4):46.

Com

petin

g in

tere

sts:

non

e de

clar

ed. C

onfli

ct o

f int

eres

ts: n

one

decl

ared

. Al

l aut

hors

con

trib

uted

to c

once

ptio

n an

d de

sign,

man

uscr

ipt p

repa

ratio

n, re

ad a

nd a

ppro

ved

the

final

man

uscr

ipt.

Al

l aut

hors

abi

de b

y th

e As

soci

atio

n fo

r Med

ical

Eth

ics (

AME)

eth

ical

rule

s of d

isclo

sure

.

HPV infection, GSTM1-GSTT1 genotypes, mitochondrial mutations and tobacco

association with oral cancer from northeast India

R Mondal1, SK Ghosh1* 1 Biotechnology Department, Assam University Silchar-788011, Assam, India

E-mail address of each author

Rosy Mondal rosy.rose.mondal3@gmail.com

*Corresponding Author:

Prof. Sankar Kumar Ghosh

Department of Biotechnology,

Assam University, Silchar,

Pin-788011, Assam, India.

Tel: 91 9435372338

Email id- drsankarghosh@gmail.com/sankar.kumar.ghosh@aus.ac.in

ABSTRACT

Background

Northeast India has one of the world’s highest incidences of oral cancer being the most common

malignancy among head and neck cancers (HNC) approximately 30-40%. Tobacco consumption,

HPV infection and Glutathione S-transferase (GST) genes polymorphisms are the risk factors for

the development of Oral squamous cell carcinoma (OSCC). Further, mitochondrial DNA

(mtDNA) alterations are associated with various cancers, suggesting that it may be a critical

contributing factor in carcinogenesis. Here, we investigated the association of tobacco-betel quid

chewing, HPV infection, GSTM1-GSTT1 genotypes and mitochondrial D-loop mutations with

OSCC.

Materials and methods

The mutations from matched tissue samples of 35 OSCC patients with 50 control subjects were

used for PCR and direct sequencing. PCR based detection was done for high-risk HPV using a

consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism.

Results:

The results of the logistic regression analyses suggested that there was significantly increased

risk for OSCC associated of tobacco - betel quid chewing (OR, 7.07; 95% CI, 1.50-

33.32; P=0.005), HPV infection (OR, 3.03; 95% CI, 1.12-8.16; P=0.02), null genotype of

GSTM1 (OR, 3.05; 95% CI,1.23-7.56; P=0.01), GSTT1 (OR, 2.66; 95% CI,1.01-7.02;P=0.04),

both GSTM1-GSTT1 (OR, 3.93; 95% CL, 1.43-10.80; P= 0.006)and D-loop mutations (OR,

26.92; 95% CI, 5.78-125.23; P<.0001) respectively. A significant risk for mitochondrial

mutation associated with tobacco - betel quid chewing (OR, 20.83; 95% CI, 4.33-

100.21; P<0.0001), HPV infection (OR, 3.52; 95% CI, 1.29-9.53; P=0.01), null genotypes

of GSTM1 (OR, 2.89; 95% CI, 1.10-7.58; P=0.02) and both GSTM1 –GSTT1 (OR, 4.17; 95% CI,

1.27-13.69; P=0.01) respectively.

Conclusion:

We report for the first time the association of HPV infection with mtDNA mutations from India.

Large-scale studies are needed to clarify an individual’s risk to tobacco-betel quid, HPV

infection and to determine the pathological significance of these associated mtDNA somatic

mutations.

Introduction

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer and malignancy

among head and neck cancers (HNC) globally (1). India has one of the world’s highest

incidences of OSCC, which accounts for 30-40% cancers at all sites, out of which 9.4% being

oral cancer (2, 3). In Northeast India, OSCC is highest among all the states which is about 33%

(4). The factor associated with the oral cancer in this region is mainly due to consumption of

tobacco in various forms, alcohol and lack of awareness. Furthermore, smoking, alcohol use,

smokeless tobacco products, and human papillomavirus (HPV) infections are the major risk

factors for oral cavity cancer, with smoking and alcohol having synergistic effects (5, 6).

HPVs have been a prime suspect in the etiology of OSCC due to their morphological

association with squamous cell carcinomas and their ability to immortalize oral keratinocytes and

bring about transformation of epithelial cells. The global cancer incidence demonstrated that,

5.17 % of all cancers determined can be attributed to HPV according to a study initiated by

International Agency for Research in Cancer (IARC) (7). The prevalence of HPV in oral cancer

in India differs in different geographical regions within Indian subcontinent (8).

Chemical carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and

heterocyclic aromatic amines (HAAs) are implicated in oral carcinogenesis (9). These

compounds are present in tobacco smoke and also found in meat cooked on an open flame or at a

high temperature (10, 11). Metabolic activation of these compounds can lead to formation of

highly reactive mutagens that readily react with genetic material, may result in the formation of

DNA adducts, which favor cellular mutations and reactive hyperplasia in the mucosa of upper

aerodigestive tract (9, 12).

OSCC may provide an ideal tumour system to evaluate gene-environment interaction

which has been well illustrated by phase I and phase II enzymes. These two groups of enzymes

are involved in the process of biometabolization of a wide range of carcinogens from tobacco

smoke and diet, including HAAs and PAHs (12). Polymorphisms in the genes that code for these

enzymes may alter expression or function, thus increasing or decreasing the activation or

detoxication of carcinogenic compounds. The polymorphisms in combination with

environmental exposure have been hypothesized to confer a differential risk of cancer for

individuals carrying these genetic variants.

Tobacco smoke or smokeless contains various carcinogens like nitrosamines, aldehydes,

aromatic amines polycyclic aromatic hydrocarbons (PAHs) etc which are free radical forming

compounds (13). These compounds not only causes single – strand breakage in DNA but also

results in oxidation of protein thiols and lipid peroxidation, thereby triggering damage to

mtDNA. MtDNA is an easy target for oxidative DNA damage due to close proximity to reactive

oxygen species (ROS) production, the lack of protective histones proteins and the inefficient

repair mechanism as a result the mitochondrial genome has a high mutation rate, and which may

signal the genesis of cancer (14).The accumulation of ROS might also contribute to increased

nuclear gene mutagenesis (15, 16).

OSCC is a multifactorial and dynamic event in which numerous alterations contribute to

disease development. Therefore, the present study is the extended investigation of our previous

studies (17, 18). Here we examined the association of HPV infection, along with other factors,

examined in our previous reports viz. tobacco with betel nut chewing, GSTM1-GSTT1 null

genotypes and mitochondrial DNA mutations with OSCC, which are few major contributing

factors of oral cancer. Furthermore, the association of HPV infection with mitochondrial DNA

mutations was studied, as the detection and its association with mitochondrial mutations could be

coupling to other markers for OSCC associated with tobacco-betel quid chewing or smoking-

related harm and serve as biomarker in detection.

Materials and methods

Ethics Statement

The present study is approved [No: IRB/CCHRC/01/2010] by Institutional Review Board

(IRB), Cachar Cancer Hospital and Research Centre (CCHRC) (http://cacharcancerhospital.org),

Meherpur, Assam.

Sample Collection

The case–control study comprises of 35 OSCC patients (17, 18) and 50 control subjects,

and informed written consent was obtained and personal details were recorded in a questionnaire

upon interview. Information regarding age, gender, occupation and nature of consuming tobacco

habit (smoking or smokeless) and alcohol were recorded. Patients with OSCC were enrolled in

Cachar Cancer Hospital and Research Centre, the only specialized oncology centre serving the

Southern Assam. All cancers were confirmed by histopathology to be squamous cell carcinoma.

Tumours were located in the oral cavity. They were classified according to the TNM

classification and staged as recommended by the American Joint Committee on Cancer. Tumour

tissue and 250 µl of matched blood from each patient were collected at the initial diagnosis

during biopsy. All the tumour samples were diagnosed as invasive squamous cell carcinoma after

histopathological analysis using Hematoxylin and Eosin (H&E) stain. Matched blood and tumour

tissue samples were stored at -86ᵒC.

Histopathology of the tumour tissue samples

Tissue blocks were cut at the pathology laboratory sites. All H&E staining was performed

in the pathology laboratory providing the tissue specimens and using routine procedures.

DNA Isolation

For each sample, DNA was extracted from preselected regions of tumour tissue and

matched blood. The tissue samples and matched blood were digested in TES buffer and

incubated overnight at 55ºC the tissue digests. The DNA was subsequently isolated by phenol/

chloroform/ isoamylalcohol method (19) followed by ethanol precipitation and re-suspended in

TE buffer and stored at -20ºC.

PCR Amplification

Primers for D-loop amplification

The published primers used for D-loop amplification study to accomplish the objective

were: forward primer dLhuF 5’-CAGGGTCATAAAGCCTAAATAG-3’ and reverse primer

dLhuR 5’-GAGGTAAGCTACATAAACTGTG-3’ which amplify an initial 649 bp PCR

product. The PCR programme used for amplification was: initial denaturation step was done at

94ᵒC for 2 min; 30 cycles of denaturation at 94ᵒC for 30 s; annealing at 55 ᵒC for 45 s and

elongation at 72ᵒC for 90 s. The amplified product was observed in 1.5% agarose gel (17).

Multiplex PCR for GSTM1 and GSTT1

Analysis for GSTM1-GSTT1 gene polymorphism using CYP1A1 gene as internal control was

done by multiplex PCR. The forward (F) and reverse (R) primers used for the amplification

GSTT1 was F5’-TTCCTTACTGGTCCTCACATTCTC-3’ and R 5’-

TCACGGGATCATGGCCAGCA-3’, GSTM1 was F5’- GAACTCCCTGAAAAGCTAAAGC-3’

and R5’-GTTGGGCTCAAATATACGGTGG-3’, CYP1A1 was F5’

ACTGCCACTTCAGCTGTCT and R5’-GCTGCATTTGGAAGTGCTC-3’ respectively. The

PCR programme used for amplification was: initial denaturation step at 94ᵒC for 2 mins; 30

cycles of denaturation at 94ᵒC for 30s; annealing at 59ᵒC for 45s and elongation at 72ᵒC for 90s.

The amplified product was observed in 1.5% agarose gel (20).

PCR amplification for HPV detection were carried out with consensus primers

GP5+/GP6+ followed by subtype detection of HPV 16 and 18 (21, 22). Reaction mixture without

DNA template was used as a negative control and that with known DNA template was used as a

positive control which yielded PCR products of expected results. PCR amplification was carried

out with forty cycles. The PCR products were analyzed by electrophoresis on 2% agarose gel.

DNA Sequencing

The PCR products were purified using Gel Purification Kit (Qiagen, UK) and sequenced

using an ABI capillary sequencer (ABI 3500) under the service of MWGAG-BIOTECH, Bangalore,

India. The resultant DNA sequences were compared with the published reference mtDNA sequence

(NCBI accession number NC_012920 AC_000021) as well as our patients and control sequences.

Any mtDNA sequences that differed between tumour sample and its matched blood mtDNA were

scored as somatic mtDNA mutations specific to the tumour using nucleotide BLAST. All mutations

identified were confirmed by repeat PCR and sequencing.

Statistical analysis

Medians and frequencies of selected characteristics were compared between cases and

controls using the Pearson chi-square for all other categorical variables. Odds ratio (ORs) and

95% confidence interval (CI) were calculated using logistic regression analysis. P-values less

than 0.05 are considered statistically significant. P values used in this are two sided.

Results

Demographics of the study population

In order to analyze the interaction between the nature of exposure and genetic

susceptibility factors, the study population was divided into different habit groups. The

demographic characteristics of the study population are summarized in Table 1. Cases and

controls were almost similar with respect to age (P=1), gender (P=0.92), smoking habit

(P=0.84), alcohol intake (P=0.23), current vegetable intake (P=0.95), salted dry fish (P=0.82)

and fermented fish (P=0.99) respectively. However, significant differences were observed in

tobacco-betelquid intake (P=0.01) and current fruit intake (P=0.04),

Risk factors association of HPV, tobacco –betel nut chewing, GSTM1- GSTT1 and mtDNA

mutations with OSCC

The samples were primarily examined by histopathology and the HPV infected cells were

characterized by Koilocytosis, a perinuclear clearing in the squamous epithelium accompanied

by nuclear atypia. Only 14% of the cases were detected HPV positive with H&E

histopathological staining. As a result the samples which did not show any Koilocytosis were

further subjected to PCR for HPV detection using the GP5+/GP6+ primers among which 25 %

cases and 18% of controls were found to be HPV positive and were remain undetected using

H&E stain. Subsequently, the genotyping of the HPV positive samples were done using PCR for

both HPV 16 and HPV 18. We found a burden of 40% HPV positive in the case samples, and all

were HPV 18 whereas 18% HPV positive was found in controls (Figure 1A). None of the cases

have HPV 16 subtype or multiple HPV. Infection with HPV has been implicated as one of the

possible etiological factors for OSCC and in the present study, the risk of OSCC increased 3.03 -

folds (95% CI, 1.12-8.16; P=0.03) due to HPV infection (Table 2).

In Northeast India tobacco chewing is one of the customary practices. As a matter of fact,

there were 94% of cases having tobacco-betelquid chewing habit and in controls, it is 70%. The

increase risk to OSCC is 7.07 fold (95% CI, 1.50-33.32; P=0.005) among the tobacco and betel

quid chewers which is one of the major contributing factors for oral cancer.

In the study population the GST null genotype examined where it is observed that the

frequency of GSTM1 null genotype was found to be 54% in cases and 28% in controls. The

GSTT1 null genotype is 40% in cases and 20% in controls and both GSTT1 and GSTM1 null

genotype 42% in cases and 16% in controls (Figure 1B). We observed a risk of 3.05-fold to

OSCC (95% CI, 1.23-7.56, P = 0.01) due to null genotypes of GSTM1 and further the risk

increases 2.6-fold (95% CI, 1.01-7.02, P = 0.04) due to null GSTT1 (Table 2). The risk increases

further to 3.93-fold (95% CI, 1.43-10.80, P = 0.006) with both GSTM1 and GSTT1 null

genotypes.

The mtDNA mutations identified in our previous studies (17, 18) study occurred in the

hypervariable D-loop region. Different types of mutations were observed in the D-loop region

between nt 51 and 595 as the patient group were same. However, in 38% controls, we found

mutation in the D-loop region. The increase risk to OSCC is 26.92 - fold (95% CI, 5.78-125.23;

P<.0001) due to mtDNA D-loop mutations as somatic mutations in mtDNA have been

increasingly observed in human cancers and have been proposed as important oncological

biomarkers.

Risk factors association study of tobacco –betel nut chewing, HPV, GSTM1- GSTT1 and

with mtDNA D-loop mutations

The organic chemicals and toxic agents in tobacco and betel quid cause extensive damage

to human mtDNA. In our study, we observed that the risk of mtDNA mutations increased 20.83

–fold (95% CI, 4.33-100.21; P<.0001) 0001 in 58% of tobacco–betel quid chewers having

mitochondrial mutations as compared to non chewers (Table 3).

The HPV oncoproteins E6 and E7 plays an important role in accumulation of genetic

alterations that eventually lead to transformation and cancer development. We examined that,

there were, 10.6 % were HPV infected were having mtDNA mutation and 16.5% without

mtDNA mutations. Moreover, we found that the risk increases 3.52-fold of mtDNA mutations

accumulation due to presence of HPV infection (Table 3).

The frequency of GSTM1 null genotype was found to be 29.4%, GSTT1 null genotype

12.9% and both GSTT1 and GSTM1 null genotype 22.3% respectively having mtDNA mutations.

We observed a risk of 2.8-fold to mtDNA mutations (95% CI, 1.10-7.58; P=0.02) due to null

GSTM1, 2.42-fold risk due to null GSTT1 and further the risk increases 4.17-fold (95% CI, 1.27-

13.69; P=0.01) due to both null GSTM1 and GSTT1 (Table3).

Discussion

The habit of chewing tobacco and betel quid is an endemic habit throughout the Indian

subcontinent. Tobacco contains over 60 known carcinogens. Tobacco consumption by smoking

or chewing is thought to be the major etiological risk factors for the development of oral cancer

which is caused by irritation from direct contact with the mucous membranes of mouth. The

elevated number of tobacco-related OSCC cases is a major concern of this area. The reasons may

be the poor socio-economic condition and customary practice of oral consumption of tobacco in

its various forms, use of lime with betel-leaf and betel nuts, alcohol and smoking habits and also

lack of awareness. Tobacco- betel quid chewing may also increase mtDNA mutation in human

oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation

of these mutations during cell division could be important contributors to the early phase of

OSCC (23, 24). The findings of the present study well demonstrate the risk of OSCC and

mtDNA mutations to tobacco quid chewers in this region.

We report for the first time the prevalence of HPV in OSCC in Northeast India. HPV

infection, particularly, high-risk HPV is a known independent causative factor for oral cancer.

When a cell is infected with HPV, the E7 gene binds to Rb so that the Rb releases E2F and the

other proteins. This is a signal for the cell cycle to progress. As long as the E7 stays attached to

Rb, the cell cycle will continue to happen, thus causing a cycle of uncontrolled cell reproduction

(25). Similarly, viral E6 protein binds to p53, and makes it inactive. This allows the virus to take

over the cell and reproduce itself, since the virally inhibited p53 cannot stop it, or begin the

process of cell death. The repeated replication of cells with erroneous DNA information is the

beginning of malignant tumour formation. Along with blocking the cell's p53, the viral E6

protein activates telomerase, an enzyme that synthesizes the telomere repeat sequences.

Activating this enzyme maintains a repeated cell cycle that continues to produce viral cells (26).

This leads to malignancy, as the mutant cells continue uncontrolled reproduce. In the present

study, we have found significant correlation of HPV infection with mitochondrial mutation

which was also reported in a study of cervical cancer (27). Bak protein is a pro-apoptotic

member which localizes in mitochondria, and functions to induce apoptosis. Elimination of Bak

protein by HPV E6 leads to a decrease apoptosis. This E6 activity towards Bak is a key factor in

promoting the survival of HPV-infected cells which in turn facilitates the tumour development.

The detection of HPV using conventional histopathological H&E staining showing

Koilocytosis is the most common cytopathic effect and is considered by the pathologists to be

the major histopathological aspect of determination of HPV infection. However, this appearance

is not present in all infected cells and diminishes during progress from mild dysplasia to severe

dysplasia. The Koilocytosis denotes an important morphological marker for HPV infection. It is

not a precise basis for HPV diagnosis because it results in approximately 30% false-positives,

which should be considered (28). As a matter of fact, the patients remained devoid of proper

diagnosis in case of HPV infection. Molecular (PCR) tests may accurately identify different

types of HPV (of low and high cancer risks) in cells from cytological screening due to their high

sensitivities, have been the focus of attention of many studies. The advantage of this PCR based

assay, unlike the other currently available assays, is that it can report the actual genotype of the

HPV detected, rather than issue a broad based ambiguous diagnosis of HPV infection (21).

The most important risk factor for oral cancer is smoking, tobacco chewing and betel

quid. Tobacco smoke contains pyrolysis products, which are generated due to high temperatures

at the burning tip, whereas smokeless tobacco is rich in nitrosamines, PAHs aldehydes and

ketones (29). The concomitant use of betel quid leads to a 50-fold increase in reactive oxygen

species generated (30). The increased risk factor of null GSTs with accumulation of mtDNA

mutations enzyme as because possibly plays inside the mitochondrial matrix as mtDNA

protection factor regarding damage caused by reactive oxygen species. Here we have found an

risk of 2-4 folds in the patients with null GSTM1-GSTT1 along with having D-loop mutation,

which might have occurred due to ROS production by increased consumption of tobacco and

betel quid and also individual having tobacco and betel quid practice with null genotypes have

high risk of oral cancer (17, 31, 32). The distribution of these genotypes suggested a potential

influence in the incidence of OSCC. Finally, betel quid contains tender areca nuts and lime and

smokeless tobacco that have been shown to generate ROS and induce oxidative damage, and also

genetic polymorphism of certain genes can increase the risk of oral carcinogenesis as the

development of cancer is influenced by both the genetic and environmental factors.

We presented in our previous study (17, 18) mitochondrial mutation in D-loop region of

in OSCC patients from North East India. The present study investigated the D-loop region in 35

OSCC tumour tissues and matched peripheral blood to detect mutations in mtDNA which might

be related to betel quid, tobacco chewing and smoking. Our study suggests that escalated

consumption of tobacco in different forms result in increased ROS production that cause mtDNA

mutations which seems to be an important biological consequence and can also initiate or

promote oral carcinogenesis (33). In the D-loop region overall 24 mutations at different

nucleotide positions were found in OSCC patients. Whereas in the control noncancerous

subjects, the mutation found in the 19 chewers may be due to tobacco-betel quid chewing and

only two non chewers we mutation in the D-loop region. So from the observation we can say that

consumption of tobacco and quid which generates increased ROS production and in turn causing

mutation to D-loop which can eventually lead to the progression of cancer.

Conclusion

OSCC is a multifactorial and dynamic event in which numerous alterations contribute to

disease development and mitochondrial is the hallmark of cancer. Our result suggests that the

association of tobacco-betel quid chew, null GSTs genotypes, HPV infection and mutations can

be used as a possible biomarker for early detection and preventive measure of oral cancer.

Furthermore, biochemical and molecular studies will be necessary to determine the pathological

significance of these associated somatic mutations. Also PCR must be employed in combination

to histological detection for rapid, sensitive, and specific detection of HPV, thereby facilitating

early therapeutic decisions in suspected and histopathological negative cases, thus providing the

clinicians the guidance for choosing the accurate treatment in HPV infected advanced OSCC

cases.

Conflicts of interests

None declared.

Acknowledgements

Our humble acknowledgement goes to the Department of Biotechnology (DBT), Govt. of India

for providing infra-structural facilities (BT/Med/NE-SFC/2009) for conducting research on

Cancer and Cachar Cancer Hospital and Research Centre (CCHRC) for the biological samples.

References

1. Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral oncology. 2009;45(4-5):309-16. Epub 2008/09/23.

2. Bhattacharjee A, Chakraborty A, Purkaystha P. Prevalence Of Head And Neck Cancers In The North East -An Institutional Study Indian J Otolaryngol Head Neck Surg. 2006;58(1):15-9.

3. Balaram P, Nalinakumari KR, Abraham E, Balan A, Hareendran NK, Bernard HU, et al. Human papillomaviruses in 91 oral cancers from Indian betel quid chewers--high prevalence and multiplicity of infections. International journal of cancer Journal international du cancer. 1995;61(4):450-4. Epub 1995/05/16.

4. Ihsan R, Devi TR, Yadav DS, Mishra AK, Sharma J, Zomawia E, et al. Investigation on the role of p53 codon 72 polymorphism and interactions with tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk population from North East India. DNA and cell biology. 2011;30(3):163-71. Epub 2010/11/04.

5. Blot WJ, McLaughlin JK, Winn DM, Austin DF, Greenberg RS, Preston-Martin S, et al. Smoking and drinking in relation to oral and pharyngeal cancer. Cancer research. 1988;48(11):3282-7. Epub 1988/06/01.

6. Hashibe M, Brennan P, Chuang SC, Boccia S, Castellsague X, Chen C, et al. Interaction between tobacco and alcohol use and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2009;18(2):541-50. Epub 2009/02/05.

7. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. International journal of cancer Journal international du cancer. 2001;94(2):153-6. Epub 2001/10/23.

8. Shukla S, Bharti AC, Mahata S, Hussain S, Kumar R, Hedau S, et al. Infection of human papillomaviruses in cancers of different human organ sites. The Indian journal of medical research. 2009;130(3):222-33. Epub 2009/11/11.

9. Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicology and applied pharmacology. 2005;206(1):73-93. Epub 2005/06/21.

10.Felton JS, Knize MG. A meat and potato war: implications for cancer etiology. Carcinogenesis. 2006;27(12):2367-70. Epub 2006/11/04.

11.Sugimura T, Wakabayashi K, Nakagama H, Nagao M. Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. Cancer science. 2004;95(4):290-9. Epub 2004/04/10.

12.Koh WP, Nelson HH, Yuan JM, Van den Berg D, Jin A, Wang R, et al. Glutathione S-transferase (GST) gene polymorphisms, cigarette smoking and colorectal cancer risk among Chinese in Singapore. Carcinogenesis. 2011;32(10):1507-11. Epub 2011/08/02.

13.Peterson LA. Formation, repair, and genotoxic properties of bulky DNA adducts formed from tobacco-specific nitrosamines. Journal of nucleic acids. 2010;2010:11. Epub 2010/09/28.

14.Wallace DC. Mitochondria and cancer: Warburg addressed. Cold Spring Harbor symposia on quantitative biology. 2005;70:363-74. Epub 2006/07/28.

15.Ferrer MD, Sureda A, Tauler P, Palacin C, Tur JA, Pons A. Impaired lymphocyte mitochondrial antioxidant defences in variegate porphyria are accompanied by more inducible

reactive oxygen species production and DNA damage. British journal of haematology. 2010;149(5):759-67. Epub 2010/03/25.

16.Kurtz A, Lueth M, Kluwe L, Zhang T, Foster R, Mautner VF, et al. Somatic mitochondrial DNA mutations in neurofibromatosis type 1-associated tumors. Molecular cancer research : MCR. 2004;2(8):433-41. Epub 2004/08/26.

17.Mondal R, Ghosh SK, Talukdar FR, Laskar RS. Association of mitochondrial D-loop mutations with GSTM1 and GSTT1 polymorphisms in oral carcinoma: A case control study from Northeast India. Oral oncology. 2013;49(4):345-53. Epub 2012/12/26.

18.Mondal R, Ghosh SK. Accumulation of mutations over the complete mitochondrial genome in tobacco-related oral cancer from northeast India. Mitochondrial DNA. 2013. Epub 2013/01/29.

19.Ghosh SK, Mondal R. Quick diagnosis of female genital tuberculosis using multiplex fast polymerase chain reaction in Southern Assam, India. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics. 2012;118(1):72-3. Epub 2012/04/17.

20.Mondal R, Ghosh SK, Choudhury JH, Seram A, Sinha K, Hussain M, et al. Mitochondrial DNA copy number and risk of oral cancer: a report from northeast India. PloS one. 2013;8(3):e57771. Epub 2013/03/08.

21.Ghosh SK, Choudhury B, Hansa J, Mondal R, Singh M, Duttagupta S, et al. Human papillomavirus testing for suspected cervical cancer patients from Southern Assam by fast-PCR. Asian Pacific journal of cancer prevention : APJCP. 2011;12(3):749-51. Epub 2011/06/02.

22.Evans MF, Adamson CS, Simmons-Arnold L, Cooper K. Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus. BMC clinical pathology. 2005;5:10. Epub 2005/11/18.

23.Lee HC, Yin PH, Yu TN, Chang YD, Hsu WC, Kao SY, et al. Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer. Mutation research. 2001;493(1-2):67-74. Epub 2001/08/23.

24.Sharan RN, Mehrotra R, Choudhury Y, Asotra K. Association of betel nut with carcinogenesis: revisit with a clinical perspective. PloS one. 2012;7(8):e42759. Epub 2012/08/23.

25.Wiest T, Schwarz E, Enders C, Flechtenmacher C, Bosch FX. Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control. Oncogene. 2002;21(10):1510-7. Epub 2002/03/16.

26.zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nature reviews Cancer. 2002;2(5):342-50. Epub 2002/06/05.

27.Sharma H, Singh A, Sharma C, Jain SK, Singh N. Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer. Cancer cell international. 2005;5:34. Epub 2005/12/20.

28.Salvia PN, Bergo SM, Bonesso-Sabadini PI, Tagliarini EB, Hackel C, De Angelo Andrade LA. Correlation between histological criteria and human papillomavirus presence based on PCR assay in cervical biopsies. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society. 2004;14(1):126-32. Epub 2004/02/07.

29.Anantharaman D, Chaubal PM, Kannan S, Bhisey RA, Mahimkar MB. Susceptibility to oral cancer by genetic polymorphisms at CYP1A1, GSTM1 and GSTT1 loci among Indians: tobacco exposure as a risk modulator. Carcinogenesis. 2007;28(7):1455-62. Epub 2007/02/20.

30.Nair UJ, Nair J, Mathew B, Bartsch H. Glutathione S-transferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers. Carcinogenesis. 1999;20(5):743-8. Epub 1999/05/20.

31.Yadav DS, Devi TR, Ihsan R, Mishra AK, Kaushal M, Chauhan PS, et al. Polymorphisms of glutathione-S-transferase genes and the risk of aerodigestive tract cancers in the Northeast Indian population. Genetic testing and molecular biomarkers. 2010;14(5):715-23. Epub 2010/09/22.

32.Agrawal D, Gupta S, Agarwal D, Gupta OP, Agarwal M. Role of GSTM1 and GSTT1 polymorphism: susceptibility to oral submucous fibrosis in the North Indian population. Oncology. 2010;79(3-4):181-6. Epub 2011/03/02.

33.Lievre A, Blons H, Houllier AM, Laccourreye O, Brasnu D, Beaune P, et al. Clinicopathological significance of mitochondrial D-Loop mutations in head and neck carcinoma. British journal of cancer. 2006;94(5):692-7. Epub 2006/02/24.

Table Legends

Table1: Demographic characteristics of the study group.

Table 2: Odds Ratio of the major risk factors associated with OSCC.

Table 3: Odds Ratio of the major risk factors associated with mitochondrial DNA mutations.

Figure Legend

Figure 1: PCR based detection of HPV infection and GSTs polymorphism in oral cancer.

A-Bar graph showing the prevalence of HPV in OSCC patients than controls based on PCR

detection of genomic DNA isolated from oral swab and tissues. B-Showing the distribution of

GSTM1 and GSTT1 null genotypes among OSCC patients and controls based on multiplex PCR

detection of genomic DNA isolated from oral swab and tissues.

Table 1: Table 1.docx

Table1: Demographic characteristics of the study group.

a Chi square was used to examine differences.

Characteristics Subjects P value Cases (n=35) Control (n=50) Age (years)

Median

60

60

1a Gender

Male Female

25 10

35 15

0.92a

Tobacco-Betel quid Non-chewers

Chewers

2 33

15 35

0.01a

Smoking habit Non- smokers

Smokers

25 10

36 14

0.84a

Alcohol intake No-intake

Intake

31 4

38 12

0.23a

Current vegetable intake <Once per week

1–6 per week >1 per day

5 24 6

6 35 9

0.95a

Current fruit intake <Once per week

1–6 per week >1 per day

12 18 5

8 25 17

0.04a

Non-veg intake (fish) Salted Dry fish

<Once per week 1–6 per week

>1 per day Fermented fish

<Once per week 1–6 per week

>1 per day

6 17 11 5 20 9

7 29 14 7 30 13

0.82a

0.99a

Table 2: Table 2.docx

Table 2: Odds Ratio of the major risk factors associated with OSCC.

Cases/Controls OR [95% CI] P value

Tobacco-betel quid

Chewers 33/35 7.07 [1.50-33.32] 0.005

Non- chewers 2/15 1 (ref)

HPV

Absent 21/41 1 (ref)

Present 14/9 3.03[1.12-8.16] 0.02

GSTM1

Null 19/14 3.05[1.23-7.56] 0.01

Present 16/36 1(ref)

GSTT1

Null 14/10 2.66 [1.01-7.02] 0.04

Present 21/40 1 (ref)

Both GSTM1 and GSTT1

Null 15/8 3.93[1.43-10.80] 0.006

Present 20/42 1(ref)

Mt DNA mutations

Absent 2/31 26.92 [5.78-125.23] <.0001

Present 33/19 1(ref)

Table 3: Table 3.docx

Table 3: Odds Ratio of the major risk factors associated with mitochondrial DNA mutations.

‘n’ represents the number of individual out of the total number of subjects in this study , + sign

denotes the presence of genes GSTT1 and GSTM1.

Habit/HPV/Gene D-loop mutations

Odds ratio [95%CI] P Value Absence (n)

Presence (n)

Tobacco-betel quid

Chewers Non-chewers

18 15

50 2

20.83[4.33-100.21] 1(ref)

<.0001

HPV

Presence Absence

14 19

9 43

3.52[1.29-9.53] 1(ref)

0.01

GSTM1

Null +

8 25

25 27

2.89[1.10-7.58 ] 1(ref)

0.02

GSTT1

Null +

13 20

11 41

2.42[0.92-6.35] 1 (ref)

0.06

Both GSTM1 and GSTT1

Null +

4 29

19 33

4.17[1.27-13.69] 1(ref)

0.01

Figure 1: Figure 1.tif