Genetic polymorphism of drug-metabolizing enzymes and susceptibility to oral cancer

Miki Sato2, Takayuki Sato, Toshiyuki Izumo1 and Teruo Amagasa

Maxillofacial Surgery, Graduate School, Tokyo Medical and Dental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113-8549 and
1 Department of Clinical Pathology, Saitama Cancer Center, 818 Komuro, Ina-machi, Saitama 362-0806, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An individual difference in the susceptibility to chemical carcinogens is one of the most important factors in the estimate of risk of human cancer. Recently, it has been reported that genetic risk for tobacco-related cancers is associated with polymorphisms of the CYP1A1 and GSTM1 genes in terms of genotype frequencies and cigarette smoking dose. In this study, we investigated the inter-individual difference in genetically determined susceptibility to oral squamous cell carcinoma (SCC) in relation to cigarette smoking dose in a Japanese population. DNA samples were obtained from both patients and controls. We identified individuals at high risk genetically for oral SCC in terms of polymorphisms of the CYP1A1 and GSTM1 genes. This study then compared the estimated total number of cigarettes smoked by patients with those smoked by controls. In this case–control study, we estimated the odds ratios of susceptible to non-susceptible individuals. CYP1A1 genotype C and GSTM1 deficiency were frequently found among oral SCC patients. Patients with genotype C and GSTM1 deficiency contracted carcinoma after fewer cigarettes than those with other genotypes. Individuals with these two genotypes were at remarkably high risk at a low dose level of cigarette smoking. Individual differences in polymorphisms of the CYP1A1 and GSTM1 genes is one important factor in the estimate of risk of oral SCC at a low dose level of cigarette smoking.

Abbreviations: SCC, squamous cell carcinoma.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is now accepted that a large proportion of human cancers are caused by synthetic or natural chemical compounds in the environment (1). Carcinogenic risks from exposure to exogenous chemical carcinogenes depend not only on the intrinsic nature and dose of each chemical, but also may depend on inter-individual variability in sensitivity to the carcinogens (2). Most chemical carcinogens require metabolic activation by Phase I enzymes (cytochromes P-450) and detoxification by conjugation via the various Phase II enzymes (epoxide hydrolase, glutathione S-transferase, N-acetyltransferase, sulfotransferase, etc.) (3). Thus, the coordinated expression and regulation of Phase I and Phase II drug-metabolizing enzymes and their metabolic balance may be an important host factor in determining whether exposure to carcinogens results in cancer or not (46). At present, it is accepted that most of the carcinogens in our environment are activated mainly by a restricted number of P-450 species, including CYP1A1, CYP1A2, CYP2E1 and CYP3A (7,8). CYP1A1 and GSTM1 polymorphisms have been associated with an increased risk for tobacco-related diseases such as lung cancer (911). Oral cancer is also a tobacco-related disease that represents a significant problem based upon its high incidence in many parts of the world (12,13). Some studies on genetic markers for individual susceptibility to head and neck cancer have also been reported (1417). In this study, we report that polymorphisms of the CYP1A1 and glutathione S-transferase genes are associated with susceptibility to oral squamous cell carcinoma (SCC) in relation to cigarette smoking dose in a Japanese population.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental
Blood samples were obtained from 142 oral SCC patients (tongue 62, gingiva 27, buccal mucosa 24, oral floor 17 and other cases 12) and 142 healthy controls. DNA was isolated from the peripheral lymphocytes in these specimens.

Genotype identification of the CYP1A1 gene was carried out by PCR amplification followed by digestion with MspI, detecting substitution of CCGG for CTGG in the MspI site at base 264 from the additional polyadenylation [poly(A)] signal in the 3'-flanking region. Genotype A is the predominant homozygote in which the MspI site is absent at the 3'-end. A homozygous rare allele was named genotype C. Genotype B is heterozygous for both alleles (18). To determine if the MspI genotypes were associated with cancer, the frequencies of the three genotypes in oral SCC patients and healthy controls were compared. Two synthetic oligonucleotide primers (5'-TAGGAGTCTTGTCTGATGCCT-3' and 5'-CAGTGAAGAGGTGTAGCCGCT-3') were used according to the methods of Hayashi et al. (19). Thirty cycles of PCR were carried out under the following conditions: 1 min denaturation at 95°C; 1 min primer annealing at 57°C; 1 min primer extension at 72°C. The amplified fragments including the MspI site were digested with MspI for 2 h at 37°C and products were subjected to electrophoresis in 2% agarose gels.

GSTM1, one of the µ class isozymes, detoxifies benzo[a]pyrene, including the epoxide and hydroxylated forms. The GSTM1-deficient phenotype was shown to be due to a homozygous null GSTM1 gene (20). The genotypes of GSTM1 were determined by PCR according to methods described previously (9). Three oligo primers for the exons 4 and 5 region of the gene were used. Primer 1 (5'-GAAGGTGGCCTCCTCCTTGG-3') and primer 2 (5'-AATTCTGGATTGTAGCAGAT-3') could also anneal to another µ class gene (GSTM4), while primer 3 (5'-TTCTGGATTGATGCAGATCA-3') was specific for the GSTM1 gene. When the three primers were used together in a PCR assay, the single GSTM4 band (158 bp fragment) was consistently found, whereas the polymorphic 231 bp fragment could only be seen in the GSTM1-positive genome. The constant 158 bp fragment was amplified as an internal control, excluding the possibility of a false interpretation due to failure in the amplification reaction. PCR was carried out for 30 cycles under the following conditions: 1 min denaturation at 95°C; 1 min primer annealing at 50°C; 1 min primer extension at 72°C. The PCR products were subjected to electrophoresis in 2% agarose gels (Figure 1Go). The incidence of the homozygous or heterozygous allele of the complete GSTM1 gene [GSTM1(+)] or the homozygous deficient gene [GSTM1(–)] was compared between oral SCC patients and controls.



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Fig. 1. GSTM1 genotypes by PCR. Lanes 1 and 2, GSTM1(–) genome; lanes 3–5, GSTM1(+) genome.

 
Epidemiology
All of the 142 oral SCC patients were histopathologically diagnosed in the Saitama Cancer Center Hospital (78 males and 64 females). Patients consenting to participate in our study were interviewed at the time of their first admission using a standardized questionnaire concerning their cigarette and alcohol consumption history. The only exclusion criterion used was co-existing malignancies in the patients group; nine cases were missed by this criterion. They do not comprise an apparent sub-group of those with the least or most severe disease. We also collected DNA samples from 142 consenting controls who were individually matched to the patients with respect to sex and age (±1 year). The 142 age- and gender-matched controls were randomly selected from a pool of 648 apparently healthy individuals free of malignancy who were interviewed using the same questionnaire at the time of their admission for a complete physical examination to hospitals near the Saitama Cancer Center Hospital. In this study, we summarize the smoking history of the patients and controls as the estimated total number of cigarettes consumed over their lifetime until interview. To compare the two medians of cigarette consumption among patients and controls with different genotypes, the Mann–Whitney distribution was calculated to test for statistical significance. In this case–control study of 142 patients with oral SCC and matched non-cancer controls, we evaluate the relative risk for individuals with susceptible genotypes in relation to the cumulative cigarette smoking dose.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MspI genotypes of CYP1A1 and GSTM1 genotypes and oral SCC incidence
CYP1A1 genoypes A (m1/m1), B (m1/m2) and C (m2/m2) were found in 62 (43.6%), 65 (45.8%) and 15 (10.6%) individuals, respectively, among the healthy controls. This result gave a good fit to the Hardy–Weinberg equilibrium with a gene frequency of 0.665 for m1 and 0.335 for m2, in which the relative frequencies p2, 2pq and q2 of the genotypes estimated from the gene frequencies p and q must be equal to that observed. On the other hand, types A (m1/m1), B (m1/m2) and C (m2/m2) were found in 56 (39.5%), 55 (38.7%) and 31 (21.8%) oral SCC patients, respectively. The frequency of genotype C among SCC patients differed from that among healthy controls with a statistical significance of P < 0.01 ({chi}2 = 5.28, d.f. = 1). The odds ratio estimate revealed that the individuals with genotype C had an ~2.3-fold higher risk of developing cancer than those with the A genotype (95% CI ~1.1–4.7).

The frequency of GSTM1(–) in healthy controls was 64 (45.1%). The frequency of GSTM1(–) was 92 (64.8%) in oral SCC patients, which was higher than that of GSTM1(+) (P < 0.001, {chi}2 = 11.2, d.f. = 1). The odds ratio estimate revealed that the individuals with GSTM1(–) had an ~2.2-fold higher risk of developing cancer than those with GSTM1(+) (95% CI ~1.4–3.6) (Table IGo).


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Table I. Distribution of MspI genotypes of CYP1A1 and GSTM1 genotypes among oral SCC patients and healthy controls
 
Combined genotyping of the CYP1A1 and GSTM1 genes and oral SCC incidence
Six genotypes could be analyzed by combining the three MspI genotypes of CYP1A1 and the two of GSTM1 and comparing the combined frequencies between oral SCC patients and healthy controls. The frequency of the combined genotypes C and GSTM1(–) was 2.1% in healthy controls. Among oral SCC patients, the frequency of the combined genotypes C and GSTM1(–) was 14.1% (P < 0.001, {chi}2 = 12.6, d.f. = 1) (Table IIGo).


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Table II. Distribution of combined genotyping of CYP1A1 and GSTM1 genes in oral SCC patients and healthy controls
 
Cumulative cigarette dose in MspI genotypes of CYP1A1 and GSTM1 genotypes
The estimated total number of cigarettes consumed over a lifetime did not show statistical significance among the controls with genotypes A, B and C (data not shown). There are no differences in the distributions between genotypes A and B among the patients. However, the cigarette numbers for patients with genotype C was fewer than those for the other two genotypes (P < 0.001, Mann–Whitney U-test), even though age showed no differences among the patients of different genotypes. The estimated number of cigarettes smoked by the patients with GSTM1(–) was less than those with GSTM1(+) (P < 0.001, Mann-Whitney U-test). The estimated numbers of cigarettes consumed by patients with the combined genotypes C and GSTM1(–) were lower compared with those of the other combined genotypes (P < 0.001, Mann–Whitney U-test) (Table IIIGo).


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Table III. Characteristics of patients with MspI genotypes of CYP1A1 and GSTM1 genotypes
 
Relative risk associated with smoking level by MspI genotype of CYP1A1
The distribution of patients and controls by genotype for the MspI polymorphism is shown in Table IVGo. The estimated cumulative cigarette dose is divided into three categories: those that consume at least 4x105 cigarettes (a higher dose than 4x105 cigarettes is commonly regarded as a heavy smoking level), those consuming ~2–4x105 cigarettes and those consuming <2x105 cigarettes (a value at least half that of the highest group dose). Odds ratios were calculated for the MspI genotypes and cigarette dose, setting the risk of the first category with genotype A and B combined as A + B at the lowest dose level as the baseline of 1.0. The baseline of exposure could not be set as non-smokers because there were not enough non-smokers among the patient cohort. These odds ratios are designated in Table IVGo as odds ratios for genotype and dose. The relative susceptibility of genotype C compared with A + B is also estimated in Table IVGo by the other odds ratio calculated for a 2x2 table at each of the dose levels. This odds ratio was consequently equal to the ratio of the two odds ratios for genotype and dose at the same level. The susceptibility of genotype C compared with other genotypes was 4.3-fold higher (95% CI ~1.9–10.1, P < 0.001) at the lowest cigarette dose and reduced to odds ratios of 1.9 and 0.2 at the higher dose levels. The odds ratios of genotype C also decreased from 4.3 (95% CI ~1.9–10.1) to 3.1 (95% CI ~0.9–11.1) and 1.8 (95% CI ~0.2–12.9) with increased cigarette dose. On the other hand, the odds ratio of genotypes A + B increased with dose from the baseline of 1.0 to 1.6 (95% CI ~0.9–3.0) and 11.3 (95% CI ~4.2–30.8, P < 0.001). The odds ratios were not statistically significant between genotype C and other genotypes at the higher cigarette dose.


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Table IV. Relative risk associated with smoking levels by MspI genotypes of CYP1A1
 
Relative risk associated with smoking level by GSTM1 genotypes
The odds ratio of the GSTM1(–) genotype increased from 3.1 (95% CI ~1.6–5.9) to 3.9 (95% CI ~1.6–9.1) and 16.2 (95% CI ~4.3–61.0) with increased cigarette dose; this increase was statistically significant. On the other hand, the odds ratio of the GSTM1(+) genotype increased with cigarette dose from the baseline of 1.0 to 1.9 (95% CI ~0.8–4.4) and 10.8 (95% CI ~3.2–36.2). The odds ratios were not statistically significant between the GSTM1(–) and GSTM1(+) genotypes at the higher cigarette dose. The susceptibility of genotype GSTM1(–) compared with other genotypes was 3.1-fold higher (95% CI ~1.63–5.91, P < 0.001) at the lowest cigarette dose and reduced to odds ratios of 2.0 and 1.5 at the higher dose levels (Table VGo).


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Table V. Relative risk associated with smoking level by GSTM1 genotype
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The CYP1A1 and GSTM1 polymorphisms have been associated with an increased risk for smoking-related cancers such as lung, bladder and head and neck cancer (10,11,1416,2125). In this study, we investigated whether an association of oral SCC with an MspI restriction site polymorphism of the CYP1A1 gene and polymorphism of GSTM1 exists. Further, we tested whether a genetic risk for oral SCC was associated with these two polymorphisms in terms of genotype frequencies and cigarette smoking dose in a Japanese population.

The C genotype was found in 22% of oral SCC patients, a statistically significant incidence, about twice as high as that among healthy controls. This observation is consistent with a report of SCC of the lung (26). It is suggested that the C genotype confers high susceptibility to oral SCC as well as to SCC of the lung, known to be closely associated with cigarette smoking.

We then compared the incidence of at least one GSTM1 gene [GSTM1(+)] or its complete deletion [GSTM1(–)] between patients and controls. The frequency of the GSTM1(–) genotype in controls was 45% and that in patients was 65%, in good agreement with values reported previously for Japanese populations (10,11). However, two Caucasian studies have reported a lack of association between the GSTM1 polymorphism and oral cancer (15,23). This discrepancy may be ascribable to an ethnic difference in allelic frequency of the GSTM1 polymorphism or factors such as diet, alcohol consumption, smoking habit, etc.

The frequency of the combined genotypes C and GSTM1(–) was 14.1% among oral SCC patients, a statistically significant increase compared with the frequency seen among healthy controls. However, it is not certain whether these two genes work synergistically to enhance the risk of oral SCC, because there were insufficient subjects for an adequate analysis on the basis of odds ratios.

Next, estimating the cumulative cigarette dose for patients exhibiting the MspI genotypes of CYP1A1, it was found that SCC patients with genotype C had a statistically significantly lower dose than patients with genotypes A and B. Also, the estimated number of cigarettes for the patients with GSTM1(–) was fewer than for GSTM1(+) patients. It was suspected that the genotypes C and GSTM1(–) play an important role in individual differences in susceptibility to oral SCC, especially at the lowest cigarette dose level.

Also, in the present case–control study our results show that the susceptibility of patients with genotypes C and GSTM1(–) to oral SCC was remarkably higher at a lower dose level of smoking (2x105 cigarettes). Nakachi et al. stated that the lower difference in risk among the genotypes at high dose levels may reflect a dose–response relationship of the enzymatic reaction and another less likely interpretation is that individuals with genotype C have a genetically high risk of carcinoma independent of cigarette smoking (18). Although we did not have enough non-smoking patients to determine which interpretation is correct, genotype C was found among 35% of them and GSTM1(–) was found among 91% of them in this study. It is likely that the individuals with genotypes C and GSTM1(–) have a genetically high risk of carcinoma independent of cigarette smoking in the Japanese population.

To summarize, our study in a Japanese patient cohort indicates that individuals with specific polymorphisms in both the CYP1A1 and GSTM1 genes have a genetically high risk of oral SCC. This suggests that an individual difference in the susceptibility to chemical carcinogens is one of the most important considerations in the risk assessment of oral SCC.


    Acknowledgments
 
We thank Dr K.Kawajiri (Department of Biochemistry, Saitama Cancer Center Research Institute) for his technical advice; Dr S.Okabe (Department of Oral Surgery, Saitama Cancer Center Hospital) for collecting blood samples from patients; Dr K.Kino (Maxillofacial Surgery, Graduate School, Tokyo Medical and Dental University) for his statistical advice; and Prof. M.Yanagishita (Department of Biochemistry, Faculty of Dentistry, Tokyo Medical and Dental University) for his reading and editing of this manuscript.


    Notes
 
2 To whom correspondence should be addressed Email: ta-sato.os1{at}dent.tmd.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 8, 1999; revised June 18, 1999; accepted June 18, 1999.