Glutathione S-transferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers

Urmila J. Nair, Jagadeesan Nair, Babu Mathew1 and Helmut Bartsch2

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany and
1 Community Oncology, Regional Cancer Centre, Trivandrum, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oral cancer is the most common cancer in males and third most common in females in India, the main causative agent being the use of chewing tobacco with or without betel quid (BQ). However, nothing is known about the role of the host metabolic genes in oral cancer in ethnic Indian population. In this study, the prevalence of GSTM1 and GSTT1 null genotypes (GSTM1*2 and GSTT1*2) in oral premalignant leukoplakia cases and controls was ascertained in genomic DNA by a multiplex PCR technique. Biopsies taken from 98 oral leukoplakia patients and exfoliated cells from 82 healthy controls both of Indian ethnicity were analysed. GSTM1*1 (active) was present in 83% and GSTT1*1 (active) was present in 78% of all control subjects, while prevalence of GSTM1*2 and GSTT1*2 null genotypes was significantly higher among oral leukoplakia cases. The prevalence of GSTM1*2 in leukoplakia cases was 81.6% compared with 17% in controls [odds ratio (OR), 22; 95% confidence interval (CI), 10–47] and GSTT1*2 was 75.5% in the cases versus 22% in controls (OR, 11; 95% CI, 5–22). Combined null genotypes of GSTM1 and GSTT1 prevailed in 60.2% of the cases with none detected in controls. Glutathione S-transferase M1 and T1 enzymes are both known to catalyse detoxification of reactive oxygen species, lipid peroxidation products and tobacco-derived carcinogens that have been found in the saliva of BQ/tobacco chewers. Our results, still requiring confirmation by a larger study, demonstrate that the null genotypes of both GSTM1 and GSTT1 increase with high penetrance, separately or in combination, the risk for developing leukoplakia in an Indian ethnic population.

Abbreviations: BQ, betel quid; CI, confidence interval; GST, glutathione S-transferase; HNSCC, head and neck squamous cell carcinoma; LOH, loss of heterozygosity; OR, odds ratio; PAHs, polycyclic aromatic hydrocarbons; ROS, reactive oxygen species; TSNA, tobacco-specific nitrosamines.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oral cancer incidence ranks fifth in the global cancer burden (1), and a 2- to 3-fold mortality increase has been recorded in eastern and central European countries in the last 3 decades (2). In India, oral cancer, constituting 9.8% of an estimated 644 600 incident cancer cases in 1992 (3), ranks first among all cancer cases in males and is the third most common among females in many regions, with age-standardized incidence rates from 7–17/100 000 persons/year (4); the incidence rate being higher than the western rate of 3–4/100 000/year (5).

Oral cancer, a malignancy of the lip, mouth or tongue, is predominantly a squamous cell carcinoma. The prognosis is poor, and severe functional and cosmetic defects accompany its treatment. Oral cancer has been causally associated with chewing of tobacco with or without betel quid (BQ) in India and other Asian countries, whereas in western countries, cigarette smoking and heavy alcohol consumption are the main risk factors (6). Unlike tobacco smoke, which contains many carcinogenic pyrolysis products, such as polycyclic aromatic hydrocarbons (PAHs), aldehydes and nitrosamines (7), chewing of tobacco with BQ increases exposure to carcinogenic tobacco-specific nitrosamines (TSNA) [~20 µg/day in smokers (8) versus ~1000 µg/day in chewers; J.Nair, unpublished data] and to nitrosamines derived from areca nut alkaloids. Furthermore, reactive oxygen species (ROS), implicated in multistage carcinogenesis, are also generated in substantial amounts in the oral cavity during chewing (9,10). As an early sign of damage to oral mucosa, BQ/tobacco chewers often develop leukoplakia and submucous fibrosis, both well established precancerous lesions, which are accessible to diagnosis and present an important indicator for oral cancer risk. Histopathological examination of clinically diagnosed leukoplakia excludes any other definable lesion and establishes the degree of epithelial dysplasia (11). Some 2–12% of these lesions have been reported to turn malignant over several years (12), although a recent report suggests that a third of presenting patients will develop oral cancer (13).

Molecular epidemiological studies have now provided evidence that individual susceptibility to cancer is mediated by both genetic and environmental factors. The inherited differences in the effectiveness of detoxification/activation of carcinogens play a crucial role in host susceptibility. Thus, there is an urgent need for molecular markers that can predict whether a premalignant lesion will develop into an aggressive or metastasizing tumour. The µ (GSTM1) and {theta} (GSTT1) members of the glutathione S-transferase (GST) multigene family are candidate cancer-predisposing genes; they are mostly involved in the detoxification of a wide range of environmental and tobacco carcinogens, endogenously produced ROS and lipid peroxidation products, yielding excretable hydrophilic metabolites (reviewed in 14–18). The known substrates for GSTM1 include metabolically generated epoxide intermediates of benzo[a]pyrene and other PAHs (16), whereas the substrates for GSTT1 include alkyl halides, found in cigarette smoke, and lipid peroxides (16). The null genotypes that are associated with a lack of enzyme function exist at both these loci and their association with smoking-induced cancer has been investigated. Homozygous deletions of GSTM1 (GSTM1*2) have been associated with higher risk of laryngeal, lung, bladder, colon and gastrointestinal cancers (1923). The null genotype of GSTT1 (GSTT1*2) has been reported to be associated with an increased risk of brain (24) and colorectal cancer (25). Oral premalignant lesions and cancers attributable to tobacco and BQ chewing constitute a significant public health burden, particularly in India, yet no studies have investigated the impact of genetic determinants on host susceptibility to this oral disease in Indian populations. To address this question, we have established the distribution frequency of GSTT1 and GSTM1 genotypes in a cross-section of an asymptomatic Indian population and compared these with the prevalence in oral leukoplakia cases. We report here for the first time a very strong effect of GSTM1 and GSTT1 deletion polymorphisms on the risk of developing oral premalignant lesions and by inference on oral cancer in ethnic Indian BQ/tobacco chewers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
All subjects were ethnic Indians. Information on tobacco chewing, smoking and alcohol habits was obtained. Cancer of any site or any other serious disease excluded the patients from the study.

Control subjects
The allelic background prevalence in the normal asymptomatic Indian population was evaluated by analysing population controls drawn from healthy individuals visiting dental clinics for some dental procedure or healthy volunteers from Bombay, India, without any prior diagnosis of oral cancer or premalignant lesions. They were not matched for age, gender or habits with the leukoplakia cases. Each subject rinsed their mouth three times with sterile water and, thereafter, oral exfoliated cells were collected in sterile saline containing 25 mM EDTA by scraping the buccal mucosa with a fresh tooth brush. Collected samples were centrifuged, washed in saline, re-suspended and stored in saline at –80°C until use.

Cases
Subjects with clinically confirmed oral premalignant lesions, recruited for an intervention study in Trivandrum, India, were randomly selected to enter the present study as cases. The categorization of leukoplakias into homogeneous and non-homogeneous lesions was by clinical features, and the exclusion of malignancy was by histopathology of the lesions. The biopsies of oral mucosa lesions were fixed in formaldehyde, and paraffin blocks were available for our study.

DNA isolation
Genomic DNA from formaldehyde-fixed, paraffin-embedded biopsies or oral exfoliated cells was extracted using a standard method (26) with a modification to include an additional aliquot of proteinase K after 8 h incubation, and a further incubation of 18 h. A similar protocol for DNA extraction from formaldehyde-fixed, paraffin-embedded lung tissue has been reported for PCR-based genotyping of cancer susceptibility genes (27).

Genotyping assays
The homozygous null polymorphisms GSTM1*2 and GSTT1*2 were determined using a modified multiplex PCR approach for simultaneous replication of both genes for molecular analysis (28). The co-amplification of an albumin gene fragment served as an internal positive control for a successful amplification reaction. The sequences for the primer pairs were as follows: GSTM1, 5'-GAACTCCCTGAAAAGCTAAAGC-3' and 5'-GTTGGGCTCAAATATACGGTGG-3'; albumin, 5'-GCCCTCTGCTAACAAGTCCTAC-3' and 5'-GCCCTAAAAAGAAAATCGCCAATC-3'; GSTT1, 5'-TTCCTTACTGGTCCTCACATCTC-3' and 5'-TCACCGGATCATGGCCAGCC-3'.

PCR was performed in a 50 µl reaction volume containing 50 mM KCl, 2.5 mM MgCl2, 200 mM Tris–HCl (pH 8.3), 200 mM dNTPs, GSTM1 primers at 3 µg each, GSTT1 primers at 1 µg each, albumin primers at 600 ng/ml each, 50–100 ng of genomic DNA and a drop of mineral oil, placed in a preheated PCR machine at 99.9°C for 5 min. Taq DNA-Polymerase (2.5 U; Boehringer Mannheim, Germany) was added to each tube, and the PCR was performed in a Perkin-Elmer GeneAmp PCR system 2400 (Perkin Elmer, Norwalk, USA). After an initial denaturation at 95°C for 5 min, amplification was carried out for 40 cycles at 94°C for 1 min, 64°C for 1 min and 72°C for 1 min, followed by final elongation at 72°C for 7 min. The GSTM1*2 (homozygous null) was evidenced by the absence of a 215 bp fragment, the GSTT1*2 (homozygous null) was evidenced by the absence of a 480 bp fragment, and the presence of the 350 bp albumin fragment was indicative of a successful PCR (Figure 1Go). Informative PCR amplifications were observed for GSTM1 and GSTT1 in the 82 controls and 100 out of 114 cases. Two cases were further excluded from the study as complete data on them was not available. Additionally, ~30% of the samples were also screened for GSTM1 or GSTT1 using different sets of primers. GSTM1 polymorphism was determined using a three-primer based PCR assay, enabling us to amplify GSTM1 (230 bp) and GSTM4 (157 bp) genes simultaneously (29). The amplification of GSTM4 provides a positive control for each reaction. GSTT1 was also ascertained independently by the co-amplification of GSTT1 (480 bp) and CYP1A1 (312 bp) as simultaneously positive control (30). The results of these analyses were identical to those obtained by the multiplex PCR.



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1. A representative multiplex PCR analysis of GST polymorphisms. GSTM1 and GSTT1 gene PCR products resolved by agarose gel electrophoresis. Lane M, DNA molecular weight marker V (Boehringer Mannheim). Lane B, negative control. Lanes 1–8 are samples. A 350 bp DNA fragment corresponding to the albumin gene product provides an internal positive control for each reaction and can be seen in all PCR reactions. A 215 bp product is present only in those samples containing the GSTM1*1 gene while a 480 bp product is present only in those samples containing the GSTT1*1 gene.

 
The nomenclature for the polymorphisms in GSTM1 and GSTT1 genes used are as follows (31). GSTM1*1 active genotype comprises the following functional allele configurations: GSTM1*1A/*1A, GSTM1*1B/*1B, GSTM1*1A/*2 and GSTM1*1B/*2. The non-functional null or deleted allele is GSTM1*2 and the corresponding deficient phenotype was termed GSTM1 null phenotype. Similarly, GSTT1*1 represents the active genotype, while the non-functional genotype null or deleted allele was denoted as GSTT1*2.

Statistical analysis
Chi-square test used for the comparison of proportions, Fisher's exact test for the joint distribution of the genotypes and a multiple logistic regression model was calculated and conditional logistic regression analysis used to obtain odd ratios (ORs) and their 95% confidence intervals (CIs) using a statistical analysis software (SAS, Cary, NC).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The characteristics of the study population are shown in Table IGo. The age distribution was slightly different between cases and controls, the mean age being ~42 and 51 years for controls and cases, respectively. Approximately 82% of controls and 98% of cases were chewers. In India, tobacco is chewed with lime or in the form of a BQ that consists of the leaf of the betel vine (Piper betle L) wrapped around areca or betel nut (nut of Areca catechu L), slaked lime, catechu (extract of Acacia catechu L) and tobacco. The two non-chewer leukoplakia cases were heavy smokers and alcohol users. Smokers constituted 19% of controls and 24.5% of the cases. Fifty-three percent of cases and 28% of controls were alcohol drinkers. Most leukoplakia cases were in the category of heavy habituated chewers (15–20 quids/day), whereas the chewers among controls were regular but not heavy chewers (i.e. 1–2 quids after the two main meals). As the aim and design of the study was not to re-evaluate well established lifestyle risk factors for oral leukoplakia and cancer (32), no attempt was made to approximate the total amount of chewing, smoking or alcohol consumption in terms of amount per day or duration in years.


View this table:
[in this window]
[in a new window]
 
Table I. Characteristics of the study population
 
Table IIGo shows the distribution of GSTM1*2 and GSTT1*2 genotype prevalence in cases and controls. We found a highly significantly increased frequency of GSTM1 null genotype in all leukoplakia cases of 81.6%, compared with 17% in controls. Individuals with GSTM1*2 had a 22-fold higher risk of leukoplakia. The homozygous null genotype GSTT1*2 was found in 75.5% of cases and in 22% of controls, conferring an 11-fold higher risk. A total of 60.2% of the cases presented homozygous deletion of both GSTT1 and GSTM1, but none was detected among healthy control chewers, revealing a potentiation of risk in subjects with both null genotypes for oral cancer when they chew tobacco.


View this table:
[in this window]
[in a new window]
 
Table II. Number of cases and controls and OR (95% CI) of oral leukoplakia in relation to GSTM1 and GSTT1 genotypes
 
Based on clinical features, the leukoplakias were further subclassified into homogeneous and non-homogeneous leukoplakias (Table IIGo) due to the reported higher rate of malignant transformation of non-homogeneous leukoplakias (11,12). When differentiating by the type of leukoplakia, there appeared to be a non-significant increased prevalence of the GSTM1*2 in non-homogeneous cases 35/39 (89.7%) as compared with homogeneous leukoplakia cases 45/59 (76.3%). The prevalence of GSTT1*2 was 33/39 (84.6%) and 41/59 (69.5%) in non-homozygous versus homogeneous leukoplakia, respectively. However, the prevalence of both GSTM1*2 and GSTT1*2 was 74.4% in non-homozygous and significantly different from 50.8% in homogeneous leukoplakia (P = 0.0002).

When the data were analysed using logistic regression (Table IIIGo) controlling for age as a quantitative factor, gender and the influence of three major lifestyle risk factors for induction of oral cancer (i.e. tobacco chewing, smoking and alcohol consumption), a significant association with leukoplakia for both traits was observed for GSTM1*2 (OR, 22.8; 95% CI, 9.2–57) and GSTT1*2 (OR, 16.1; 95% CI, 6.5–40.3). However, when adjusting for the other genotype, higher ORs were obtained, GSTM1*2 (OR, 60.6; 95% CI, 14.6–251.2) and GSTT1*2 (OR, 46; 95% CI, 10.8–203.8) due to the fact that in this population a significant association between GSTM1 and GSTT1 genes was seen (P < 0.001). As expected, the magnitude of risk for oral leukoplakia was highest for chewing (OR, 31; P = 0.028), followed by that for alcohol abuse (OR, 12; P = 0.0005). Smoking did not emerge as a significant risk factor probably as the number of smokers was too low in our study. An apparent gender difference in risk for leukoplakias was observed with a female to male OR = 10 (P = 0.004), requiring verification, as the proportion of females and males among controls was different from our cases. The distribution of the combined genotypes of GSTM1 and GSTT1 was significantly different in cases as compared with controls (P < 0.0001). The combined null genotype of both GSTM1 and GSTT1 conferred a highly significant risk for oral leukoplakia when compared with the category of at least copy of the genes present. Thus, the GSTM1 and GSTT1 deletion genotype alone and even more in combination was demonstrated to highly predispose habitual BQ chewers to the development of oral leukoplakia, a precancerous lesion.


View this table:
[in this window]
[in a new window]
 
Table III. Conditional ORs and 95% CI from multiple logistic regression analysis of oral cancer risk factors
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ethnic differences in the prevalence of the GSTM1 null genotypes have been reported to vary between 22–35% in Africans, 38–67% in Caucasians and 33–63% in East Asian populations (33). The Pacific islanders (Oceania) have the highest reported frequency, GSTM1*2 ranges from 64% to as high as 100% in Kiribati natives (14). The GSTT1*2 genotype varies from 10–18% in Caucasians (20,24,34) to 58% in the Chinese (35). To our knowledge, this is the first report on the prevalence of these genotypes in an ethnic Indian population and among premalignant leukoplakia cases from India.

In the ethnic Indians, we observed that the prevalence of 17% (n = 82) for GSTM1*2, was relatively lower than the figure of ~50% reported in Caucasian populations, while the reported prevalence of GSTT1*2 at 23% was only marginally higher. Furthermore, we did not find any subject with null genotype for both GSTM1 and GSTT1 in 82 controls, suggesting that this combination is rare in the Indian ethnic population. The prevalence of GSTM1*2 in `Asian' Indians from Los Angeles or Malaysia was reported to be 36 or 33%, respectively (36,37). The prevalence of GSTT1*2 in Indians from Malaysia and Singapore was 16%, while the double deletion was seen in only 5% of these subjects (35). These data confirm that there are large ethnic differences in the prevalence of these polymorphic GST enzymes which are known to catalyse the detoxification of tobacco-derived carcinogens and endogenous reactive products derived from lipid peroxidation (38). We demonstrated previously that betel nut and catechu, the two major ingredients of BQ, generate ROS and 8-oxo-guanine in DNA in vitro at the alkaline pH provided by lime (39). ROS in part are responsible for the increased frequency of micronuclei observed in hamster cheek pouch upon exposure to BQ ingredients (10) and in exfoliated buccal mucosa cells of chewers (40). We also detected the formation of OH radicals in the oral cavity of subjects during chewing of BQ (9), which may cause direct damage in the oral mucosa and promote growth of initiated oral epithelial cells. The contribution of the GST supergene family to oxidative stress resistance is well established (41), and therefore the absence of one or more of the GST enzymes would result in increased ROS-mediated damage. Thus, antioxidants and free radical scavengers such as vitamins A, E and C, and beta-carotene, have widely been used as cancer chemopreventive agents to induce regression of precancerous oral lesions such as leukoplakia and erythroplakia (42).

The major cancers attributable to tobacco are those of the lung, bladder and oral cavity (7,32). Our results indicate that tobacco use to a large extent and alcohol abuse to a lesser extent are significant risk factors for oral premalignancy in concordance with the established associations between oral leukoplakia and carcinogen exposure through chewing tobacco or drinking alcohol (43). In this study, smokers constituted a low percentage of the subjects, and therefore the risk due to smoking could not be seen. Whether the risk difference according to gender seen in this study (Table IIIGo) is real, needs to be verified in a larger study, although a higher susceptibility to tobacco-associated cancers has been reported for women (44).

At the beginning of our study, there were only very few reports that related GST genotypes and oral cancer risk. The GSTM1 enzyme had been reported to be present in oral tissue (45,46), and a preliminary study (45) demonstrated that there was a relatively low fraction of oral and laryngeal cancer patients exhibiting detectable levels of GSTµ protein (28% of patients with oral/oropharyngeal cancer versus 60% in controls). Trizna et al. (47) reported an association between GSTM1*2 genotype in 42 cases and an increased risk for head and neck cancer. No association was detected between GSTM1 and/or GSTT1 genotypes and oral cancer in English Caucasians (25). Among Caucasian oral cancer patients, no association between GSTM1*2 and risk was found (48), but individuals with mutated CYP1A1 (exon 7 Ile/Val polymorphism) were at increased risk. In Taiwanese patients (49), null genotypes of GSTM1 and/or GSTT1 were associated with increased oral cancer risk, only compared with those that had both GSTM1 and GSTT1 active. Among non-chewers, only CYP2E1 c1/c2 and c2/c2 genotypes were found to confer high risk as compared with c1/c1 genotypes. Thus, from these data the influence of GSTM1/GSTT1 null genotype on oral cancer risk was not clearly demonstrated.

Based on our results, there was a significantly higher prevalence of GSTM1*2 or GSTT1*2 in all leukoplakia cases, 59% of the cases were null for both the GSTs. This figure rose to ~73% in non-homogeneous leukoplakia. The malignant transformation of the non-homogeneous lesions, involving erythroplakia and nodular leukoplakia, is known to be particularly high and reportedly ranges from 15 to 40% over a varying period of time (12). The corresponding figures for patients from Japan and Western countries ranged from 17 to 35%, respectively (50,51). BQ chewing is an ancient and socially accepted practice, the introduction of tobacco reinforced this practice and now almost all habitual chewers use BQ with tobacco. Almost every BQ/tobacco-chewing related oral malignancy is preceded by a clinically distinct premalignant stage at the site of cancer development (52). Our results revealed that genetically predisposed BQ/tobacco chewers are much more susceptible to environmental and life-style risk factors. Heavy chewers with either GSTT1*2 or GSTM1*2 genotype had an 11- to 22-fold higher risk of presenting with clinically observable leukoplakia than subjects with active GSTT1*1 or GSTM1*1. The genotype combination of GSTM1*2 and GSTT1*2 among ethnic Indians conferred a significantly high risk for oral leukoplakia and subsequently for heavy chewers to oral cancer. Because of the small study size, the absence of double deletion in the control population resulting in an OR = {infty}, does not preclude its prevalence. Therefore, assuming the value of double deletion as 1, an OR > 123 (95% CI, 16–918) was obtained. In a recent analysis (53; J.Y.Park, personal communication), the oral cancer risk in African Americans, when stratified by GSTM1*2 and high tobacco use (>20 pack-years, i.e. no. of packs of cigarettes smoked/day during 1 year), compared with GSTM1 positive genotype and <20 pack-year group, was of similarly high magnitude (OR, 112; 95% CI, 6–2001).

A genetic progression model for head and neck squamous cell carcinoma (HNSCC) to explain the field cancerization concept has been proposed, by which an entire epithelial surface is primed for neoplastic changes following prolonged carcinogen exposure leading to focal areas that progress at different rates towards invasive cancer (54,55). Microsatellite analysis in head and neck cancer for allelic loss at 10 major chromosomal loci demonstrated that the spectrum of chromosomal deletion progressively increases at each histopathological step from benign hyperplasia to dysplasia to carcinoma in situ to invasive cancer (55). GSTM1 and GSTT1 loci are not reported to be linked as individuals who are GSTT1*2 are not necessarily GSTM1*2. While there is no current evidence suggesting that GSTM1 and/or GSTT1 are lost in the pathologies of HNSCC, the question nevertheless has to be addressed whether the high incidence of these null alleles in these biopsies could be a consequence of allele loss. It has been suggested that, because amplification of chromosome 11q13 is common in the HNSCC and because GSTP1 is in the 11q13 amplicon, it is possible that the behaviour of carcinoma in situ is mediated by GSTP1 allelism (56). The GSTM1gene has been mapped to chromosome 1p13.3 and GSTT1 gene to chromosome 22q11.2. As loss of heterozygosity (LOH) has also been reported in oral cancers on chromosomes 1p11–13 (57) and chromosome 22q13 (58); gene deletion due to LOH on the specific amplicons can at present not be excluded as an additional contributory factor. Studies are underway to verify this possibility.

HNSCCs that develop in patients from India and South-East Asia frequently have abnormalities of ras oncogenes including mutations, LOH (H-ras) and amplification (K- and N-ras) in contrast to low prevalence of mutations in these ras genes in the same malignancies from Western countries (59). An increase of somatic mutations, leading to tumour formation, has been linked with GST deficiencies (33). Thus, the deficiency of both GST detoxifying enzymes in tobacco smokers and heavy chewers could lead to higher carcinogenic exposure and to multiple and progressive genetic aberrations. Our results suggest that carcinogenic intermediates derived from BQ/tobacco or generated during habitual chewing (60) are substrates for GSTT1 and GSTM1 enzymes and that oral leukoplakia risk is increased more than additively for individuals who have both GSTT1 and GSTM1 null genotypes. Our finding that the double null genotypes were significantly more frequent among the non-homogeneous leukoplakia cases is in keeping with the reported higher malignant transformation of this type of leukoplakia. These latter results need to be confirmed, given the small sample size of our study and a lack of adjustment for amount and duration of tobacco chewed.

In conclusion, our results revealed that null genotypes of GSTM1 and GSTT1, both individually and in combination, are high penetrance genetic risk factors for developing oral leukoplakia that consequently modulate the risk of developing cancerous oral lesions in habituated BQ/tobacco chewers of Indian ethnicity. Based on these data, which still require confirmation by a larger study, identification of high-risk subjects could now be explored to advise them on cessation of the habit before development of clinical symptoms of malignancy.


    Acknowledgments
 
We are grateful to Dr R.Sankaranarayanan (IARC, Lyon, France) for providing oral biopsy blocks and critical suggestions in the manuscript, and to Dr H.-R. Scherf (DKFZ) for his help in processing the oral biopsy blocks. We thank Dr A.Kopp-Schneider (DKFZ) for helping us in the statistical analysis. This work was supported by DKFZ visiting scientist award (U.J.N.).


    Notes
 
2 To whom correspondence should be addressed Email: h.bartsch{at}dkfz-heidelberg.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Parkin,D.M., Pisani,P. and Ferlay,J. (1993) Estimates of the worldwide incidence of eighteen major cancers in 1985. Int. J. Cancer, 54, 594–606.[ISI][Medline]
  2. Coleman,M.P., Esteve,J., Damiecki,P., Arslan,A. and Renard,H. (1993) Trends in Cancer Incidence and Mortality. IARC Scientific Publications No. 121, IARC, Lyon.
  3. Indian Council of Medical Research (19922) National Cancer Registry Programme: Biennial Report 1988–1989. ICMR, New Delhi, India.
  4. Parkin,D.M., Whelan,S.L., Ferlay,J., Raymond,L.A. and Young,J. (1997) Cancer Incidence in Five Continents Vol. VII. IARC Scientific Publications No. 143, IARC, Lyon.
  5. Macfarlane,G.J., Boyle,P., Evstifeeva,T.V., Robertson,C. and Scully,C. (1994) Rising trends of oral cancer mortality among males worldwide: the return of an old public health problem. Cancer Causes Control, 5, 259–265.[ISI][Medline]
  6. La Vecchia,C., Tavani,A., Franceschi,S., Levi,F., Corrao,G. and Negri,E. (1997) Epidemiology and prevention of oral cancer. Oral Oncol., 33, 302–312.[ISI][Medline]
  7. IARC (1986) Tobacco smoking. In IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 38. IARC Scientific Publications, Lyon.
  8. Hoffmann,D. and Hecht,S.S. (1985) Nicotine-derived N-nitrosamines and tobacco-related cancer: current status and future directions. Cancer Res., 45, 935–944.[ISI][Medline]
  9. Nair,U.J., Nair,J., Friesen,M.D., Bartsch,H. and Ohshima,H. (1995) Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing. Carcinogenesis, 16, 1195–1198.[Abstract]
  10. Nair,U.J., Obe,G., Friesen,M., Goldberg,M.T. and Bartsch,H. (1992) Role of lime in the generation of reactive oxygen species from betel-quid ingredients. Environ. Health Perspect., 98, 203–205.[ISI][Medline]
  11. van der Waal,I., Schepman,K.P., van der Meij,E.H. and Smeele,L.E. (1997) Oral leukoplakia: a clinicopathological review. Oral Oncol., 33, 291–301.[ISI][Medline]
  12. Sankaranarayanan,R., Mathew,B., Varghese,C., Sudhakaran,P.R., Menon,V., Jayadeep,A., Nair,M.K., Mathews,C., Mahalingam,T.R., Balaram,P. and Nair,P.P. (1997) Chemoprevention of oral leukoplakia with vitamin A and beta carotene: an assessment. Oral Oncol., 33, 231–236.[ISI][Medline]
  13. Papadimitrakopoulou,V.A., Hong,W.K., Lee,J.S., Martin,J.W., Lee,J.J., Batsakis,J.G. and Lippman,S.M. (1997) Low-dose isotretinoin versus beta-carotene to prevent oral carcinogenesis: long-term follow-up. J. Natl Cancer Inst., 89, 257–258.[Free Full Text]
  14. Board,P., Coggan,M., Johnston,P., Ross,V., Suzuki,T. and Webb,G. (1990) Genetic heterogeneity of the human glutathione transferases: a complex of gene families. Pharmacol. Ther., 48, 357–369.[ISI][Medline]
  15. Guengerich,F.P., Shimada,T., Raney,K.D., Yun,C.H., Meyer,D.J., Ketterer,B., Harris,T.M., Groopman,J.D. and Kadlubar,F.F. (1992) Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology. Environ. Health Perspect., 98, 75–80.[ISI][Medline]
  16. Hayes,J.D. and Pulford,D.J. (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol., 30, 445–600.[Abstract]
  17. Ketterer,B. and Christodoulides,L.G. (1994) Enzymology of cytosolic glutathione S-transferases. Adv. Pharmacol., 27, 37–69.[Medline]
  18. Daniel,V. (1993) Glutathione S-transferases: gene structure and regulation of expression. Crit. Rev. Biochem. Mol. Biol., 28, 173–207.[Abstract]
  19. Brockmöller,J., Kerb,R., Drakoulis,N., Staffeldt,B. and Roots,I. (1994) Glutathione S-transferase M1 and its variants A and B as host factors of bladder cancer susceptibility: a case-control study. Cancer Res., 54, 4103–4111.[Abstract]
  20. Chenevix Trench,G., Young,J., Coggan,M. and Board,P. (1995) Glutathione S-transferase M1 and T1 polymorphisms: susceptibility to colon cancer and age of onset. Carcinogenesis, 16, 1655–1657.[Abstract]
  21. Hayashi,S., Watanabe,J. and Kawajiri,K. (1992) High susceptibility to lung cancer analyzed in terms of combined genotypes of P4501A1 and Mu-class glutathione S-transferase genes. Jpn. J. Cancer Res., 83, 866–870.[ISI][Medline]
  22. Katoh,T., Nagata,N., Kuroda,Y., Itoh,H., Kawahara,A., Kuroki,N., Ookuma,R. and Bell,D.A. (1996) Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genetic polymorphism and susceptibility to gastric and colorectal adenocarcinoma. Carcinogenesis, 17, 1855–1859.[Abstract]
  23. Lafuente,A., Pujol,F., Carretero,P., Villa,J.P. and Cuchi,A. (1993) Human glutathione S-transferase mu (GST mu) deficiency as a marker for the susceptibility to bladder and larynx cancer among smokers. Cancer Lett., 68, 49–54.[ISI][Medline]
  24. Elexpuru Camiruaga,J., Buxton,N., Kandula,V. et al. (1995) Susceptibility to astrocytoma and meningioma: influence of allelism at glutathione S-transferase (GSTT1 and GSTM1) and cytochrome P-450 (CYP2D6) loci. Cancer Res., 55, 4237–4239.[Abstract]
  25. Deakin,M., Elder,J., Hendrickse,C., Peckham,D., Baldwin,D., Pantin,C., Wild,N., Leopard,P., Bell,D.A., Jones,P., Duncan,H., Brannigan,K., Alldersea,J., Fryer,A.A. and Strange,R.C. (1996) Glutathione S-transferase GSTT1 genotypes and susceptibility to cancer: studies of interactions with GSTM1 in lung, oral, gastric and colorectal cancers. Carcinogenesis, 17, 881–884.[Abstract]
  26. Volkenandt,M., Dicker,A.P., Bannerjee,D., Albino,A. and Bertino,J.R. (1993) Methods in Molecular Biology, PCR Protocols: Current Methods and Applications. Humana Press, Totowa, N.J.
  27. Blömeke,B., Bennett,W.P., Harris,C.C. and Sheilds,P.G. (1997) Serum, plasma and paraffin-embedded tissues as sources of DNA for studying cancer susceptibility genes. Carcinogenesis, 18, 1271–1275.[Abstract]
  28. Arand,M., Muhlbauer,R., Hengstler,J., Jager,E., Fuchs,J., Winkler,L. and Oesch,F. (1996) A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. Anal. Biochem., 236, 184–186.[ISI][Medline]
  29. Zhong,S., Wyllie,A.H., Barnes,D., Wolf,C.R. and Spurr,N.K. (1993) Relationship between the GSTM1 genetic polymorphism and susceptibility to bladder, breast and colon cancer. Carcinogenesis, 14, 1821–1824.[Abstract]
  30. Wiencke,J.K., Pemble,S., Ketterer,B. and Kelsey,K.T. (1995) Gene deletion of glutathione S-transferase theta: correlation with induced genetic damage and potential role in endogenous mutagenesis. Cancer Epidemiol. Biomarkers Prev., 4, 253–259.[Abstract]
  31. Garte,S.A. and Crosti,F. (1999) A nomenclature system for metabolic gene polymorphisms. In Vineis,P., Lang,M., Caporaso,N., Cuzick,J.A. and Boffetta,P. (eds) Metabolic Polymorphism and Cancer, Vol. 148. IARC Scientific Publications, Lyon, pp. 5–12.
  32. IARC (1985) Tobacco habits other than smoking; betel-quid and areca-nut chewing; and some related nitrosamines. In IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 37. IARC Scientific Publications, Lyon.
  33. Rebbeck,T.R. (1997) Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol. Biomarkers Prev., 6, 733–743.[Abstract]
  34. Nelson,H.H., Wiencke,J.K., Christiani,D.C. et al. (1995) Ethnic differences in the prevalence of the homozygous deleted genotype of glutathione S-transferase theta. Carcinogenesis, 16, 1243–1245.[Abstract]
  35. Lee,E.J., Wong,J.Y., Yeoh,P.N. and Gong,N.H. (1995) Glutathione S-transferase-theta (GSTT1) genetic polymorphism among Chinese, Malays and Indians in Singapore. Pharmacogenetics, 5, 332–334.[ISI][Medline]
  36. Lin,H.J., Han,C.Y., Bernstein,D.A., Hsiao,W., Lin,B.K. and Hardy,S. (1994) Ethnic distribution of the glutathione transferase Mu 1-1 (GSTM1) null genotype in 1473 individuals and application to bladder cancer susceptibility. Carcinogenesis, 15, 1077–1081.[Abstract]
  37. Zhao,B., Lee,E.J., Wong,J.Y., Yeoh,P.N. and Gong,N.H. (1995) Frequency of mutant CYP1A1, NAT2 and GSTM1 alleles in normal Indians and Malays. Pharmacogenetics, 5, 275–280.[ISI][Medline]
  38. Berhane,K., Widersten,M., Engstrom,A., Kozarich,J.W. and Mannervik,B. (1994) Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc. Natl Acad. Sci. USA, 91, 1480–1484.[Abstract]
  39. Nair,U.J., Floyd,R.A., Nair,J., Bussachini,V., Friesen,M. and Bartsch,H. (1987) Formation of reactive oxygen species and of 8-hydroxy- deoxyguanosine in DNA in vitro with betel quid ingredients. Chem. Biol. Interact., 63, 157–169.[ISI][Medline]
  40. Nair,U., Obe,G., Nair,J., Maru,G.B., Bhide,S.V., Pieper,R. and Bartsch,H. (1991) Evaluation of frequency of micronucleated oral mucosa cells as a marker for genotoxic damage in chewers of betel quid with or without tobacco. Mutat. Res., 261, 163–168.[ISI][Medline]
  41. Hayes,J.D. and Strange,R.C. (1995) Potential contribution of the glutathione S-transferase supergene family to resistance to oxidative stress. Free Radic. Res., 22, 193–207.[ISI][Medline]
  42. Enwonwu,C.O. and Meeks,V.I. (1995) Bionutrition and oral cancer in humans. Crit. Rev. Oral Biol. Med., 6, 5–17.[Abstract]
  43. Mao,L. (1997) Leukoplakia: molecular understanding of pre-malignant lesions and implications for clinical management. Mol. Med. Today, 3, 442–448.[ISI][Medline]
  44. Muscat,J.E., Richie,J.P.Jr, Thompson,S. and Wynder,E.L. (1996) Gender differences in smoking and risk for oral cancer. Cancer Res., 56, 5192–5197.[Abstract]
  45. Mulder,T.P., Manni,J.J., Roelofs,H.M., Peters,W.H. and Wiersma,A. (1995) Glutathione S-transferases and glutathione in human head and neck cancer. Carcinogenesis, 16, 619–624.[Abstract]
  46. Janot,F., Massaad,L., Ribrag,V., de Waziers,I., Beaune,P.H., Luboinski,B., Parise,O.Jr, Gouyette,A. and Chabot,G.G. (1993) Principal xenobiotic-metabolizing enzyme systems in human head and neck squamous cell carcinoma. Carcinogenesis, 14, 1279–1283.[Abstract]
  47. Trizna,Z., Clayman,G.L., Spitz,M.R., Briggs,K.L. and Goepfert,H. (1995) Glutathione S-transferase genotypes as risk factors for head and neck cancer. Am. J. Surg., 170, 499–501.[ISI][Medline]
  48. Park,J.Y., Muscat,J.E., Ren,Q., Schantz,S.P., Harwick,R.D., Stern,J.C., Pike,V., Richie,J.P.Jr and Lazarus,P. (1997) CYP1A1 and GSTM1 polymorphisms and oral cancer risk. Cancer Epidemiol. Biomarkers Prev., 6, 791–797.[Abstract]
  49. Hung,H.C., Chuang,J., Chien,Y.C., Chern,H.D., Chiang,C.P., Kuo,Y.S., Hildesheim,A. and Chen,C.J. (1997) Genetic polymorphisms of CYP2E1, GSTM1, and GSTT1; environmental factors and risk of oral cancer. Cancer Epidemiol. Biomarkers Prev., 6, 901–905.[Abstract]
  50. Bouquot,J.E., Weiland,L.H. and Kurland,L.T. (1988) Leukoplakia and carcinoma in situ synchronously associated with invasive oral/oropharyngeal carcinoma in Rochester, Minn., 1935–1984. Oral Surg. Oral Med. Oral Pathol., 65, 199–207.[ISI][Medline]
  51. Shibuya,H., Amagasa,T., Seto,K., Ishibashi,K., Horiuchi,J. and Suzuki,S. (1986) Leukoplakia-associated multiple carcinomas in patients with tongue carcinoma. Cancer, 57, 843–846.[ISI][Medline]
  52. WHO (1984) Control of oral cancer in developing countries. Bull. WHO 62, 817–830.[ISI][Medline]
  53. Park,J.Y., Muscat,J.E., Schantz,S.P., Stern,J.C., Richie,J.P.a. and Lazarus,P. (1998) Association between GSTM polymorphisms and risk for oral cancer in African Americans as compared with Caucasians. Proc. Am. Assoc. Cancer Res., 39, LB-23 (Abstract).
  54. Oh,Y. and Mao,L. (1997) Biomarkers in head and neck carcinoma. Curr. Opin. Oncol., 9, 247–256.[Medline]
  55. Califano,J., van der Riet,P., Westra,W., Nawroz,H., Clayman,G., Piantadosi,S., Corio,R., Lee,D., Greenberg,B., Koch,W. and Sidransky,D. (1996) Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res., 56, 2488–2492.[Abstract]
  56. Matthias,C., Bockmuhl,U., Jahnke,V., Haries,L.W., Wolf,C.R., Jones,P.W., Alldersea,J., Worrall,S.F., Hand,P., Fryer,A.A. and Strange,R.C. (1998) The glutathione S-transferase GSTP1 polymorphism: effects on susceptibility to oral/pharyngeal and laryngeal carcinomas. Pharmacogenetics, 8, 1–6.[ISI][Medline]
  57. Jin,Y., Jin,C., Wennerberg,J., Mertens,F. and Hoglund,M. (1998) Cytogenetic and fluorescence in situ hybridization characterization of chromosome 1 rearrangements in head and neck carcinomas delineate a target region for deletions within 1p11–1p13. Cancer Res., 58, 5859–5865.[Abstract]
  58. Miyakawa,A., Wang,X.L., Nakanishi,H., Imai,F.L., Shiiba,M., Miya,T., Imai,Y. and Tanzawa,H. (1998) Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13. Int. J. Oncol., 13, 705–709.[ISI][Medline]
  59. Paterson,I.C., Eveson,J.W. and Prime,S.S. (1996) Molecular changes in oral cancer may reflect aetiology and ethnic origin. Eur. J. Cancer B Oral Oncol., 32B, 150–153.
  60. Nair,J., Ohshima,H., Nair,U.J. and Bartsch,H. (1996) Endogenous formation of nitrosamines and oxidative DNA-damaging agents in tobacco users. Crit. Rev. Toxicol., 26, 149–161.[ISI][Medline]
Received October 8, 1998; revised February 24, 1999; accepted February 25, 1999.