Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62–7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09–49.7). The calculated OR for the combined genotype was 13.4 (2.92–97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36–70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Tobacco smoke and alcohol abuse play a major role in the etiology of HNSCC2(1). Other factors discussed are occupational exposures to wool dust, wood dust, mineral fibers (2, 3, 4, 5, 6), and low intake of vegetables and fruits (7). In Europe, the highest incidence rates for HNSCC are observed in the southern countries. However, the geographic distribution of incidences was not strongly correlated with the distribution of tobacco consumption (8), suggesting that other, possibly genetic, factors might modulate the risk for HNSCC.

Molecular epidemiology has begun to have an increasing impact on analyzing the complexity of gene-environment interactions at the molecular level. Whereas germ line mutations in genes of high penetration, such as APC, resulted often in a high risk for cancer per se(9), polymorphisms of genes with low penetration (i.e., genes involved in metabolisms of xenobiotics), are thought to predispose the risk of an individual if exposed to a chemical. Most environmental compounds require metabolic initiation to reactivate electrophilic intermediates before exerting their carcinogenic effects. This biotransformation is catalyzed by the cytochrome P450 mono-oxygenase system (CYP), consisting of several P450 isoenzymes in humans (10). CYP1A1 is involved in the activation of many compounds, including benzo(a)pyrene and other PAHs that are present in substances such as tobacco smoke. At least four polymorphic variants of the CYP1A1 gene have been identified (11), two of which are thought to result in increased enzyme activity (12, 13): the first is an isoleucine/valine substitution in the heme-binding region in exon 7 at nucleotide A4889G; the second is a thymine/cytosine point mutation in the 3′-noncoding region at nucleotide T6235C. The homozygotes of the variant allele of each mutation are reported to correlate with an enhanced susceptibility to smoking-induced lung cancer in a Japanese population but not in Caucasians (14, 15, 16). The isoform CYP1B1 has been demonstrated to induce metabolic activation of a variety of chemical classes of carcinogens such as arylamines, nitroaromatics, and PAHs (17). CYP1B1 catalyzes both the formation of procarcinogenic dihydrodiols of certain PAHs and their additional oxidation to ultimate carcinogenic dihydrodiolepoxide. Several polymorphisms have been identified in the coding region of the CYP1B1 gene. Most polymorphisms resulted in the formation of a truncated or nonfunctional protein. Four sense polymorphisms were found in the CYP1B1 gene: at position 48 (Arg to Gly), at position 119 (Ala to Ser), at position 432 (Val to Leu), and at position 453 (Asn to Ser; Refs. 18, 19). The Val432Leu-polymorphism was suggested to have a profound impact on the catalytic activity of the enzyme (20). An association between susceptibility to colorectal cancer and polymorphisms at residue 432 of CYP1B1 has been reported (21). In breast cancer patients, an association between CYP1B1 polymorphism and steroid receptor status has been observed (22). Similar to CYP1A1, the expression of CYP1B1 can be induced by several Ah receptor agonists, like dioxin and PAHs (23, 24).

Activated chemicals are subjected to detoxification, and the GST supergene family is an important part of the cellular defense system against chemicals with carcinogenic potential. The human GST family consists of four classes of GST isoenzymes: GSTA, GSTM, GSTT, and GSTP. Polymorphisms of the GSTM, GSTT, and GSTP classes have been shown to present an increased susceptibility to cancer. The GSTM1 polymorphism is a gene deletion providing two alleles: the null allele GSTM1*2 and the GSTM1*1. The homozygous GSTM1*2/*2 genotype expresses no GSTM1 enzyme, and ∼50% of Caucasians lack GSTM1 activity (25, 26, 27). The GSTT1 locus also has functional (GSTT1*1) and nonfunctional (GSTT1*2) alleles, and ∼20% of Caucasians lack the functional alleles (28, 29). In the GSTP1 family, four GSTP1 alleles have been identified. The wild type allele GSTP1*A differs by an A/G transition at nucleotide +313 (Val105-A114) from GSTP1*B and GSTP1*C by this transition and by a C/T transition at +341 (Val105-Val114). A GSTP1*D (Ile105-Val114) allele has been found. GSTP1 polymorphisms are thought to be associated with altered substrate specificities (30, 31). The importance of polymorphisms of the GST genes on the risk of several cancers, including skin, lung, and bladder cancers, has been intensively reviewed (26, 27, 32).

Inherent mutations of genes coding for drug-metabolizing enzymes can result in chemically induced somatic mutations of genes, which are vital for cell growth and differentiation. The p53tumor suppressor gene controls cell cycle regulation and has been identified as a vulnerable target of critical DNA damage. Loss of p53 function is attributed to uncontrolled cell proliferation and neoplastic formation. Mutations that are caused by carcinogens in cigarette smoke occur in the p53 gene early in carcinogenesis. In lung tumors, 40% of p53 gene mutations are G to T transversions, which are thought to be putative fingerprints of certain types of PAHs. Benzo(a)pyrene metabolite adducts along the p53 gene occur predominately at G positions in codons that have been found to be mutational hot spots (33, 34). An association between the frequency of tobacco-induced p53 mutations and CYP1A1 polymorphisms has been reported for patients with lung cancer. The probability of individuals homozygous for the susceptible CYP1A1 genotypes was about nine times higher than for those individuals with CYP1A1 wild type. The probability increased synergistically when susceptible CYP1A1 genotypes were combined with GSTM1*2/*2 genotypes (35).

In the current study, we investigated the interactive effects of the polymorphic variants of several phase 1 and phase 2 enzymes on the risk for HNSCC. Our main interest was focused on the putative role of CYP1B1 codon 432 polymorphism in cancer susceptibility. The data indicates an association between CYP1B1 polymorphism and HNSCC in smokers. The importance of CYP1B1 polymorphism is reflected by the high probability of susceptible genotypes for smoking-induced p53 gene mutations.

Study Group.

The patient group consisted of a total of 312 people, including 251 males with a mean age of 59.7 years and 61 females with a mean age of 59.7 years. All were histologically verified to have HNSCC. The control group consisted of 300 unrelated healthy individuals, including 176 males with a mean age of 46.7 years and 124 females with a mean age of 48.0 years, all without a history of cancer. The patients and controls were gathered between 1996 and 1998 at the Department of Oto-Rhino-Laryngology of the University of Bonn and the Medizinische Universitäts-Poliklinik, Bonn, Germany. Donors gave their informed consent, and the Ethics Committee of the Rheinische-Friedrich-Wilhelms University of Bonn approved the study. All study subjects completed a questionnaire covering medical, residential, occupational, and smoking history. For the purpose of this study, smokers were defined as individuals having smoked five or more cigarettes or pipes per day for at least 4 years during their lifetime. Nonsmokers were defined as individuals who had never smoked or who had smoked less than one pack per year.

Genotyping.

Genomic DNA was isolated from whole blood using the QIAamp Blood Maxi Kit (Qiagen, Hilden, Germany). CYP1A1 MspI and CYP1A1 IVA were genotyped by RFLP-analysis according to standard protocols (36, 37). Real-time PCR-analysis and melting-curve analysis were performed for genotyping GSTT1, GSTM1, GSTP1(38), and CYP1B1 codon 432 (39).

Tissue Sample Preparation.

A subset of 150 paraffin embedded biopsy specimens was available. For DNA extraction, 8-μm sections were taken from each paraffin block, mounted onto glass slides and air-dried overnight in an incubator at 48°C. Sections were deparaffinized with xylene and ethanol, transferred to microfuge tubes (1.5 ml; Eppendorf, Germany), and subjected to protein digestion overnight (proteinase K; Qiagen, Hilden, Germany). DNA was extracted according to Weirich et al.(40) and DNA concentration was determined photometrically.

p53 Sequence Analysis.

Primer used for amplification of exon 5–8 of the p53 gene are listed in Table 1. For specificity and sensitivity enhancement, the DNA was denaturated for 30 min at 95°C in 41.8 μl of reaction mixture containing 5 μl of 10× reaction buffer [10 mm Tris-HCl, (pH 8.3), 50 mm KCl, 1.5 mm MgCl2 and 0.001% gelatin], 34.8 μl of sterile water, and 2 μl of diluted template DNA. Afterward, the amplification mixture, consisting of 4 μl dNTPs (0.2 mm dATP, dTTP, dCTP, and dGTP), 1 μl of each primer (20 pmol), and 0.5 μl Taq-polymerase (5 units/μl; Amersham Life Science, Cleveland, OH) was added. The PCR conditions were as follows: (a) initial denaturation for 7 min at 94°C; (b) 45 cycles of denaturing for 60 s at 94°C; (c) annealing for 60 s at 60°C; (d) extension for 90 s at 72°C; and (e) final extension for 20 min at 72°C. Fragment analysis was done by horizontal polyacrylamid-gelelectrophorese on CleanGel DNA-HP 15% 36S and the DNA-Delect buffer (ETC, Kirchentellinsfurt, Germany) according to the supplier’s instructions. DNA fragments were visualized by silver staining. Sequencing of DNA-fragments was carried out according to the dye terminator method, using the oligonucleotides described in Table 1 (Qiagen, Hilden, Germany) on an automated DNA sequencer (ABI 377; Perkin-Elmer, Weiterstadt, Germany).

Nomenclature.

The following nomenclature was used to describe the different polymorphic variants. For the CYP1A1 T6235C polymorphism the genotypes were CYP1A1*1/*1 for wild type, CYP1A1*1/*2 for heterozygous, and CYP1A1*2/*2 for mutant genotypes. For the CYP1A1 Ile462Val polymorphism, the genotypes were CYP1A1*1/*1, CYP1A1*1/*3, and CYP1A1*3/*3. For the CYP1B1 Val432Leu polymorphism, the genotypes were CYP1B1*1/*1, CYP1B1*1/*2, and CYP1B1*2/*2. For the GSTP1 Ile104Val polymorphism, the genotypes were GSTP1*1/*1, GSTP1*1/*2, and GSTP1*2/*2. The GSTM1*1/*1 and GSTM1*1/*2 genotypes were referred to as GSTM1 expressors, with the GSTM1*2/*2 genotype being used to indicate the deleted variant. Similarly, the GSTT1 expressors included the genotypes GSTT1*1/*1 and GSTT1*1/*2, with the GSTT1*2/*2 genotype indicating the deleted phenotype.

Statistical Analysis.

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender. Preliminary analyses suggested the inclusion of both a linear and a quadratic term for age when comparing HNSCC patients with the control patients; whereas a model with only a linear term for age was considered to be appropriate for the subgroup analyses of HNSCC patients. The goodness-of-fit was assessed by a likelihood ratio test that compared the likelihood of the applied variable with that of an expanded model, with additional explanatory variables. This consisted of all third-order terms (i.e., cubic trend for age and the corresponding interaction terms), for the comparisons of cases with controls, and all second-order terms in the subgroup analyses (41). The results of the goodness-of-fit tests confirmed the appropriateness of the chosen model. Fisher’s exact test was used to evaluate differences in the distribution of the p53 mutation spectra between subgroups of cancer patients. All computations were carried out using the statistical software SAS, Version 6.12 (41).

Genotype Distribution in Controls and HNSCC Patients.

Inheritable traits of different phase 1 (CYP1A1 and CYP1B1) and phase 2 (GSTP1, GSTM1, and GSTT1) enzymes were analyzed in 312 patients with HNSCC and in 300 controls as host factors of cancer risk. The prevalence of different genotypes in the control population and in HNSCC patients is listed in Table 2. The observed frequencies in the control population were within the range described for Caucasians (11, 21, 26, 42). When comparing the genotype distribution between control individuals and HNSCC patients, we observed that the frequency of the mutated CYP1B1 genotypes was slightly elevated in HNSCC patients, but the difference was not significant. The genotype distribution of CYP1A1, GSTP1, GSTM1, and GSTT1 showed no significant differences between the control and cancer group (Table 2), indicating a unique distribution of the respective polymorphic alleles within the examined population.

Stratification by Smoking.

One hundred and seventy-seven members of the control group and 195 members of the HNSCC group reported having smoked cigarettes (Table 3). Genotype distribution of polymorphic phase 1 enzymes revealed a significantly higher frequency of the mutated CYP1B1 genotypes (P < 0.001) in the HNSCC group. In the HNSCC patients, 65 smokers carried the CYP1B1*2/*2 genotype (33.3%), 101 patients carried the CYP1B1*1/*2 genotype (51.8%), and only 29 patients carried the wild type CYP1B1*1/*1 genotype (14.9%); whereas, in the control group, 18.6% were homozygous, 49.2% heterozygous, and 32.3% were wild type in the described CYP1B1 polymorphism. An adjusted OR of 2.70 (1.53–4.86) was observed. Among the other polymorphic enzymes investigated, no significant difference of genotype distribution was found between smoking controls and smoking HNSCC patients (Table 3).

Analysis of Genotype Distribution in Smoking and Nonsmoking Patients and the Effect of Combined Genotypes of CYP1B1 and GSTM1, GSTT1, or GSTP1 on Risk of HNSCC.

When subgrouping the cancer patients into smokers (195 patients) and nonsmokers (117 patients), the data revealed a significantly higher frequency of CYP1B1 polymorphism in the group of smokers (P < 0.001; Table 4). The odds of smokers carrying the CYP1B1*2/*2 allele were 4.5 times higher (95% CI, 2.62–7.98) than for nonsmokers. No significant differences were found in genotype distribution of GSTM1, GSTT1, GSTP1, or CYP1A1 polymorphisms between smoking and nonsmoking patients (Table 4).

Because the polymorphism of the CYP1B1 gene seems to have an impact on the risk for HNSCC in smokers, we investigated whether combined polymorphisms of CYP1B1 and described GST genes act synergistically on the cancer risk. Tables 5, A and B, show the results of our combined genotype analyses. As shown in Table 5A, genotypes with CYP1B1*2 and GSTM1*2 alleles were more frequent in the group of smokers when compared with the group of nonsmokers, and the ORs increased synergistically with the exchange of Val to Leu in CYP1B1. The combined CYP1B1*2/*2 and GSTM1*2/*2 genotypes had an OR of 12.8 (95% CI, 4.09–49.7) compared with smokers with CYP1B1 and GSTM1 wild types (Table 5A). The combined genotype analysis of CYP1B1 and GSTP1 (Table 5B) showed a similar trend. The OR increased with the exchange of Val to Leu in the CYP1B1 and Ile to Val in the GSTP1 protein. In the group of smokers, eight patients were homozygous for the mutated CYP1B1 and GSTP1, whereas this combination was not present in the group of nonsmoking patients (Table 5B). In contrast with the results obtained by combined genotype analyses of CYP1B1 and GSTM1 or GSTP1, the GSTT1*2/*2 genotype, in combination with the polymorphic alleles of CYP1B1, seemed to attenuate the probability of smoking-induced HNSCC (Table 5A). The OR for smokers carrying the mutated CYP1B1*2 and GSTT1*2 alleles were nearly half of that which have been found for smokers having mutated CYP1B1*2 alleles and functional GSTT1 protein (Table 5,A). The value for combined CYP1B1*2/*2 and GSTT*2/*2 genotype was 13.4 (95% CI, 2.92–97.7), and the value for the genotype CYP1B1*2/*2 and GSTT1-expressor was 24.1 (95% CI, 9.36–70.5; Table 5A).

Aberrations of the p53 Gene among Smokers with HNSCC Classified by CYP1B1 Genotypes.

We also investigated the possible interaction of CYP1B1 polymorphism with an early end point of carcinogenesis, as indicated by somatic mutations of the p53 gene. Tumor DNA from 140 HNSCC patients were screened for aberrations of the p53 gene, and tumor-specific aberrations were detected in 66 cases (47.1%). The frequency of somatic mutations of the p53 gene in smokers (46 of 76) was 1.9 times higher than in nonsmokers (20 of 64). A strong association (Table 6) between aberration frequencies of the p53 gene and CYP1B1 genotypes was found for smokers. Patients with the genotype CYP1B1*1/*2 were ∼7 times more likely (OR, 7.19; 95% CI, 1.56–52.4) to show smoking-induced somatic mutations of the p53 gene than those with CYP1B1 wild type (Table 6). The OR value increased to 20 for patients with genotype CYP1B1*2/*2 (OR: 20.0; 95% CI, 3.95–160), indicating that the exchange of amino acid Val to Leu is strongly associated with the frequency of smoking-induced p53 gene mutations. The strong gene-dose-dependent effect of mutant CYP1B1 alleles on p53 mutations indicates the importance of CYP1B1 in tobacco-induced HNSCC, because no such association was found in nonsmokers (Table 6).

Effects of the Combined Genotypes of CYP1B1 and GSTM1 or GSTT1 Genes on p53 Gene Aberrations among Smokers with HNSCC.

To determine whether different genotype combinations of CYP1B1 and GSTM1 or GSTT1 affect the frequency of smoking-related p53 gene mutation, each of three genotypes of CYP1B1 gene were combined with each of the two genotypes of the GSTM1 and GSTT1 genes. The results showed that mutation frequencies of the p53 gene were lower or higher in each of the combined genotypes of CYP1B1 and GSTM1 or GSTT1 (Table 7). A mutation frequency of 80% (16 of 20) was observed for the combined CYP1B1*2/*2 and GSTM1*2/*2 genotypes. The odds of these patients having smoking-induced mutations of the p53 gene was about 26 times higher (OR, 26.8; 95% CI, 3.41–590) than those having the CYP1B1*1/*1 and GSTM1-expressor genotype. Similar results were obtained through the combined genotype analysis of CYP1B1 and GSTT1 (Table 7). The data indicates a high probability (OR, 25.8; 95% CI, 2.42–700) of p53 gene mutations for smokers with the combined CYP1B1*2/*2 and GSTT1*2/*2 genotypes. The results of this combined genotype analysis suggest a synergistic effect of CYP1B1 and GSTM1 or GSTT1 polymorphisms on the occurrence of p53 mutations. However, the low number of cases in this study must be considered.

p53 Mutational Spectrum in HNSCC.

To confirm the mutation sites and types of the p53 gene, PCR direct sequence analyses were performed. In smokers, G→T transversions were found more frequently (27%) than in nonsmokers (10%); whereas A→G mutations were more frequent in nonsmokers (25%) than in smokers (15%). The other mutations found in smokers with HNSCC were: G→A, 7%; C→T, 11%; T→C, 4%; A→T; 0%; T→A, 2%; G→C, 4%; C→A, 2%; Del, 24%; and Ins, 4%. The respective mutations in nonsmokers were: 0%, G→A; 15%, C→T; 5%, T→C; 10%, A→T; 5%, T→A; 5%, G→C; 0%, C→A; 20%, Del; and 5%, Ins. There was no correlation between the mutational spectrum of the p53 gene and CYP1B1 polymorphisms (data not shown).

This is the first study presenting data on the role of CYP1B1 codon 432 polymorphism as a susceptibility factor in smoking-related HNSCC. Genotype analyses of CYP1B1 and polymorphic GST genes revealed a significant influence of the combined polymorphisms on the vulnerability of smokers for HNSCC. Several groups have investigated the possible interference of polymorphic variants of phase 1 and phase 2 enzymes on HNSCC. Matthias et al.(43) found no differences in the distribution pattern of certain polymorphic CYP1A1 alleles or GSTT1 alleles between patients and control individuals. A moderate influence on the risk of HNSCC has been observed for GSTM1 polymorphisms. The genotype GSTM1*A/*B was found to be protective, whereas the GSTM3*A/*A genotype seemed to confer an increased risk (43, 44). Deakin et al.(45) studied the influence of GSTT1 and GSTM1 polymorphisms on the susceptibility to oral cancer. They found no association between GSTT1 or GSTM1 genotypes and susceptibility to oral cancer. Similar results have been reported for GSTM1 polymorphism (46) and for GSTM1 or GSTT1 polymorphisms (47), respectively. A small-sized study (48) revealed that the absence of GSTM1 and GSTT1 proteins confer an increased risk of HNSCC. An association between GSTP1 polymorphism and susceptibility to oral squamous cell carcinoma was found in a Japanese population (49). However, the effect was moderate (OR, 1.93; 95% CI, 1.05–3.58), and no consistent difference in the smoking status between patients and the corresponding controls could be observed. The effect of GSTP1 polymorphism on susceptibility to oral/pharyngeal and laryngeal carcinomas was also investigated previously (42). The frequency of GSTP1*A/*A was lower in the oral/pharyngeal cancer cases than in the controls. The effect was strongly correlated with the site-specific location of the carcinoma after adjustment for age and gender, but was independent of the smoking behavior of the patients. In the present study, we found no significant differences among the genotype distribution of polymorphic GSTM1, GSTT1, or GSTP1 genes between HNSCC cases and controls, and no consistent association of different GST genotypes to patient smoking behavior could be observed. Although there is a body of evidence which suggests that polymorphisms of different GST genes influence the susceptibility to cancer (especially to lung cancer), the results of the present study suggest that polymorphisms of GSTM1, GSTT1, or GSTP1 genes alone have only minor importance on the susceptibility of smokers to HNSCC.

CYP1A1 is also a candidate for susceptibility for smoking-related cancer, and several studies on a Japanese population have indicated that the mutant CYP1A1 alleles, alone or in combination with GSTM1*2/*2 genotype, contribute to an increased risk of lung cancer (35). However, the frequency of mutant CYP1A1 alleles is rare in Caucasians, and it is therefore difficult to reproduce the same results. We could not identify any effect of CYP1A1 alone or in combination with either GSTM1 or GSTT1 genotypes on the risk of HNSCC; confirming the findings of Matthias et al.(43). It is likely that the CYP1A1 gene plays only a minor role in detoxification of tobacco-derived chemicals in Caucasians. Alternatively, the effect may also be masked by the involvement of other CYP genes in these metabolisms.

The data obtained from CYP1B1 genotyping, clearly indicates an association between the susceptible genotypes of CYP1B1 and HNSCC. Smoking cases were 4.5 times more likely to carry the susceptible CYP1B1 genotype than nonsmoking cases; and the OR increased considerably when the susceptible genotypes of CYP1B1 were combined with either polymorphic GSTM1 or GSTP1 genes. Similar results have been reported for the combination of CYP1A1 polymorphisms and the GSTM1*2/*2 genotype in lung cancer (50, 51), confirming the hypothesis that imbalances in the drug metabolism system attributable to inherent genetic variations can confer a risk to individuals when exposed to chemicals. Our observation that the combination of CYP1B1 variants with the GSTT1*2/*2 genotype was underrepresented in smoking HNSCC cases is remarkable, because most studies have reported that GSTM1- or GSTT1-deficient individuals are more susceptible to the genotoxic actions of chemicals. The conclusion that there is likely an increased susceptibility of the CYP1B1*2/*2 and GSTT1-expressors is of particular biological significance because GSTT1 is involved in both the activation and inactivation of a variety of environmental and industrial chemicals, including methylene chloride, ethylene chloride, and 1,3-butadiene (52). For example, a bioactivation of haloalkanes catalyzed by GSTT1 has been described (52). Combining these haloalkanes with glutathione generates episulfonium ions, which bind to DNA. Another explanation for the lower risk of CYP1B1*2/*2 and GSTT1*2/*2-genotypes could be that individuals lacking GSTM1 or GSTT1 enzymes are thought to possess higher tissue levels of glutathione than GSTM1- or GSTT1-expressors (27). Thus, increased tissue levels of glutathione might be protective against the accumulation of reactive intermediates, including oxygen radicals, which are thought to be involved in tobacco-induced carcinogenesis.

The role of CYP1B1 in smoking-induced HNSCC carcinogenesis is reflected by the results of mutation analysis of the cancer target gene p53, which was affected significantly by the CYP1B1 genotypes. Smokers with HNSCC who have the susceptible CYP1B1*2/*2 genotype are 20 times more likely to show p53 mutations than those with the nonsusceptible CYP1B1*1/*1 genotype. This data indicates a strong and consistent association of the CYP1B1 Val432Leu polymorphism and smoking-induced p53 mutations along with a strong gene-dose-dependent effect. In addition, the findings of combined CYP1B1 genotype with either the GSTM1 or GSTT1 genotype indicate an interactive effect on p53 mutation frequency. However, because of the small number of cases and the wide range of CIs, the data has to be cautiously interpreted. In addition, the small number of cases in the p53 analysis may be the cause of the obvious discrepancies of the data in Tables 5 and 7. Whereas, in Table 5, an under-representation of the combined CYP1B1*2/*2 and GSTT1*2/*2 genotypes in smokers with HNSCC was presented, the data in Table 7 possibly indicates an interactive effect of the combined genotypes on p53 mutations. Although these problems have to be resolved by an extended study, our findings nevertheless support the hypothesis that polymorphic variants of GSTM1 or GSTT1 are moderately strong susceptibility factors for HNSCC, but may become a dominant factor in the presence of certain gene-gene combinations, as has been demonstrated for CYP1B1.

The phenotypic importance of CYP1B1 codon 432 polymorphism for metabolic activation of xenobiotics, is still unknown. Recently, it was shown that this polymorphism of CYP1B1 is associated with a 3–4-fold increase of the Km values for 2- and 4-hydroxylation of estradiol (20), whereas Hanna et al.(53) reported that the four polymorphic variants of CYP1B1 displayed 2.4–3.4-fold higher catalytic efficiencies in estrogen hydroxylation activity than the wild type enzyme. The polymorphisms at residue 432 in CYP1B1, however, had no significant influence on CYP1B1-mediated epoxidation of (−)-trans-(7R,8R)benzo(a)pyrene-7.8-dihydrodiol, deethylation of ethoxyresorufine and hydroxylation of bufuralol (20). Influence of different allelic variants of the CYP1B1 gene on the catalytic activity toward 19 procarcinogenic compounds has been investigated. It was found that the Arg48, Ser119, Leu432, and Asn 453 variants were slightly more active in catalyzing activation of PAHs, e.g., (+)- and (−)-benzo(a)pyrene-7–8-diols, 7,12-demethylbenz(a)anthracene-3,4-diol, benzo(g)chrysene-11–12-diols, and benzo(b)fluoreanthene-9–10-diol (54). In considering these data, and based on our findings, we speculate that the carcinogenic agents in cigarette smoke preferentially metabolized by polymorphic CYP1B1 seem unrelated to PAHs. It is clear that these compounds have to be identified. In addition, the phenotypic importance of CYP1B1 codon 432 polymorphism in the metabolism of xenobiotics remains to be seen.

In conclusion, we have demonstrated that the CYP1B1 Val432Leu polymorphism is an inheritable predisposing factor for smoking-induced HNSCC, and we have shown that CYP1B1 polymorphism is associated with an increased frequency of smoking-induced p53 mutations. Furthermore, we have provided preliminary data of an interactive effect of combined CYP1B1 and GST genotypes on the risk of smokers for HNSCC and on the frequencies of p53 mutations. To substantiate our findings, we are carrying out an extended molecular epidemiological study, which should provide additional clues to the importance of the combination of polymorphic variants of the CYP1B1 and GST genes as genetic determinants for HNSCC.

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2

The abbreviations used are: HNSCC, head and neck squamous cell cancer; CYP1A1, cytochrome P450 1A1; CYP1B1, cytochrome P450 1B1; PAH, polycyclic aromatic hydrocarbon; OR, odds ratio; CI, confidence interval; GST, glutathione-S-transferase.

Table 1

Primer for amplification and sequencing of p53 exon 5–8 (sequence GenBank accession no. U94788)

ExonNameLocalizationFragment size
   5′-PCR-primer  
   3′-PCR-primer  
   Forward-sequencing-primer  
   Reverse-sequencing-primer  
5′-1 12983–13006 5′-GCCGTGTTCCAGTTGCTTTATCTG-3′ 298 
 3′-B 13280–13260 5′-TGGGCAACCAGCCCTGTCGTC-3′  
 2428 13001–13020 5′-TATCTGTTCACTTGTGCCCT-3′  
5′-C 13301–13319 5′-CCTCACTGATTGCTCTTAG-3′ 186 
 3′-2 13486–13467 5′-CACTGACAACCACCCTTAAC-3′  
 2930 13302–13319 5′-CTCACTGATTGCTCTTAG-3′  
 2485 13468–13445 5′-ACCCCTCCTCCCAGAGACCCCAGT-3′  
5′-6 13974–13993 5′-CATCTTGGGCCTGTGTTATC-3′ 292 
 3′-6 14265–14243 5′-GAGTGGGAGCAGTAAGGAGATTC-3′  
 2931 13976–13993 5′-TCTTGGGCCTGTGTTATC-3′  
 2666 14199–14180 5′-GAAATCGGTAAGAGGTGGGC-3′  
5′-8 14373–14394 5′-GGAGCCTGGTTTTTTAAATGGG-3′ 308 
 3′-9 14680–14659 5′-CTAGGAAAGAGGCAAGGAAAGG-3′  
 2575 14650–14627 5′-GTGAATCTGAGGCATAACTGCACC-3′  
ExonNameLocalizationFragment size
   5′-PCR-primer  
   3′-PCR-primer  
   Forward-sequencing-primer  
   Reverse-sequencing-primer  
5′-1 12983–13006 5′-GCCGTGTTCCAGTTGCTTTATCTG-3′ 298 
 3′-B 13280–13260 5′-TGGGCAACCAGCCCTGTCGTC-3′  
 2428 13001–13020 5′-TATCTGTTCACTTGTGCCCT-3′  
5′-C 13301–13319 5′-CCTCACTGATTGCTCTTAG-3′ 186 
 3′-2 13486–13467 5′-CACTGACAACCACCCTTAAC-3′  
 2930 13302–13319 5′-CTCACTGATTGCTCTTAG-3′  
 2485 13468–13445 5′-ACCCCTCCTCCCAGAGACCCCAGT-3′  
5′-6 13974–13993 5′-CATCTTGGGCCTGTGTTATC-3′ 292 
 3′-6 14265–14243 5′-GAGTGGGAGCAGTAAGGAGATTC-3′  
 2931 13976–13993 5′-TCTTGGGCCTGTGTTATC-3′  
 2666 14199–14180 5′-GAAATCGGTAAGAGGTGGGC-3′  
5′-8 14373–14394 5′-GGAGCCTGGTTTTTTAAATGGG-3′ 308 
 3′-9 14680–14659 5′-CTAGGAAAGAGGCAAGGAAAGG-3′  
 2575 14650–14627 5′-GTGAATCTGAGGCATAACTGCACC-3′  
Table 2

Genotype distribution of the described CYP1A1, CYP1B1, GSTP1, GSTM1, and GSTT1 polymorphisms in control individuals and HNSCC patients

Control individuals (n = 300)HNSCC patients (n = 312)ORb (95% CI)P
wt/wtawt/mtmt/mtwt/wtwt/mtmt/mt
CYP1A1 81.4  17.3  1.3 79.5  19.9  0.6 0.94 (0.60–1.48)c 0.78 
T6235C             
CYP1A1 91.7  8.3  0.0 90.4  9.6  0.0 1.01 (0.54–1.91)c 0.99 
Ile462Val             
CYP1B1 36.3  46.0  17.7 25.7  50.6  23.7 1.41 (0.94–2.11)c 0.10 
Val432Leu             
GSTP1 43.3  46.0  10.7 46.5  39.1  14.4 0.78 (0.53–1.13)c 0.19 
Ile104Val             
  +d  e   +d  e    
GSTM1  51.7  48.3   46.8  53.2  1.03 (0.71–1.49) 0.89 
GSTT1  79.7  20.3   79.5  20.5  1.00 (0.64–1.60) 0.99 
Control individuals (n = 300)HNSCC patients (n = 312)ORb (95% CI)P
wt/wtawt/mtmt/mtwt/wtwt/mtmt/mt
CYP1A1 81.4  17.3  1.3 79.5  19.9  0.6 0.94 (0.60–1.48)c 0.78 
T6235C             
CYP1A1 91.7  8.3  0.0 90.4  9.6  0.0 1.01 (0.54–1.91)c 0.99 
Ile462Val             
CYP1B1 36.3  46.0  17.7 25.7  50.6  23.7 1.41 (0.94–2.11)c 0.10 
Val432Leu             
GSTP1 43.3  46.0  10.7 46.5  39.1  14.4 0.78 (0.53–1.13)c 0.19 
Ile104Val             
  +d  e   +d  e    
GSTM1  51.7  48.3   46.8  53.2  1.03 (0.71–1.49) 0.89 
GSTT1  79.7  20.3   79.5  20.5  1.00 (0.64–1.60) 0.99 
a

wt, wild type; mt, mutant.

b

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

c

Calculation for wt/wt genotype vs. wt/mt and mt/mt genotypes.

d

GSTM1- and GSTT1-expressors.

e

Deleted GSTM1 and GSTT1 genotypes.

Table 3

Genotype distribution of xenobiotic metabolizing enzymes in smoking control individuals and HNSCC patients (stratification by smoking)

Control individuals (n = 177)HNSCC patients (n = 195)ORa (95% CI)P
wt/wtwt/mtmt/mtwt/wtwt/mtmt/mt
CYP1A1 82.5  16.4  1.1 81.0  18.5  0.5 0.89 (0.49–1.60)b 0.68 
T6235C             
CYP1A1 93.2  6.8  0.0 91.8  8.2  0.0 0.93 (0.40–2.25)b 0.87 
Ile462Val             
CYP1B1 32.3  49.2  18.6 14.9  51.8  33.3 2.70 (1.53–4.86)b <0.001 
Val432Leu             
GSTP1 44.1  45.7  10.2 43.6  38.5  17.9 0.80 (0.50–1.29)b 0.37 
Ile104Val             
  +c  d   +c  d   
GSTM1  53.1  46.9   44.1  55.9  1.33 (0.83–2.13) 0.23 
GSTT1  77.6  22.4   78.9  21.1  0.94 (0.54–1.67) 0.84 
Control individuals (n = 177)HNSCC patients (n = 195)ORa (95% CI)P
wt/wtwt/mtmt/mtwt/wtwt/mtmt/mt
CYP1A1 82.5  16.4  1.1 81.0  18.5  0.5 0.89 (0.49–1.60)b 0.68 
T6235C             
CYP1A1 93.2  6.8  0.0 91.8  8.2  0.0 0.93 (0.40–2.25)b 0.87 
Ile462Val             
CYP1B1 32.3  49.2  18.6 14.9  51.8  33.3 2.70 (1.53–4.86)b <0.001 
Val432Leu             
GSTP1 44.1  45.7  10.2 43.6  38.5  17.9 0.80 (0.50–1.29)b 0.37 
Ile104Val             
  +c  d   +c  d   
GSTM1  53.1  46.9   44.1  55.9  1.33 (0.83–2.13) 0.23 
GSTT1  77.6  22.4   78.9  21.1  0.94 (0.54–1.67) 0.84 
a

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

b

Calculation for wt/wt genotype vs. wt/mt and mt/mt genotypes.

c

GSTM1- and GSTT1-expressors.

d

Deleted GSTM1 and GSTT1 genotypes.

Table 4

Genotype distribution of described XME polymorphisms in smoking and nonsmoking HNSCC patients

Nonsmokers (n = 117)Smokers (n = 195)ORa (95% CI)P
 wt/wt  wt/mt  mt/mt wt/wt  wt/mt  mt/mt   
CYP1A1 76.9  22.2  0.9 81.0  18.5  0.5 0.80 (0.45–1.43)b 0.45 
T6235C             
CYP1A1 88.0  12.0  0.0 91.8  8.2  0.0 0.65 (0.30–1.42)b 0.27 
Ile462Val             
CYP1B1 43.6  48.7  7.6 14.9  51.8  33.3 4.53 (2.62–7.98)b <0.001 
Val432Leu             
GSTP1 51.3  40.2  8.5 43.6  38.5  17.9 1.36 (0.86–2.16)b 0.19 
Ile104Val             
  +c  d   +c  d    
GSTM1  51.3  48.7   44.1  55.9  1.33 (0.84–2.11) 0.22 
GSTT1  80.3  19.7   78.9  21.1  1.09 (0.61–1.93) 0.77 
Nonsmokers (n = 117)Smokers (n = 195)ORa (95% CI)P
 wt/wt  wt/mt  mt/mt wt/wt  wt/mt  mt/mt   
CYP1A1 76.9  22.2  0.9 81.0  18.5  0.5 0.80 (0.45–1.43)b 0.45 
T6235C             
CYP1A1 88.0  12.0  0.0 91.8  8.2  0.0 0.65 (0.30–1.42)b 0.27 
Ile462Val             
CYP1B1 43.6  48.7  7.6 14.9  51.8  33.3 4.53 (2.62–7.98)b <0.001 
Val432Leu             
GSTP1 51.3  40.2  8.5 43.6  38.5  17.9 1.36 (0.86–2.16)b 0.19 
Ile104Val             
  +c  d   +c  d    
GSTM1  51.3  48.7   44.1  55.9  1.33 (0.84–2.11) 0.22 
GSTT1  80.3  19.7   78.9  21.1  1.09 (0.61–1.93) 0.77 
a

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

b

Calculation for wt/wt genotype vs. wt/mt and mt/mt genotypes.

c

GSTM1- and GSTT1-expressors.

d

Deleted GSTM1 and GSTT1 genotypes.

Table 5
A. Genotype combinations of xenobiotic metabolizing enzymes in cases (non smokers versus smokers)
GenotypesHNSCC patientsORa (95% CI)
CYP1B1 GSTM1 Nonsmokers (n = 117) Smokers (n = 195)  
*1/*1 + b 25 17 1.00c 
 d 26 12 0.72 (0.27–1.84) 
*1/*2 30 40 1.93 (0.87–4.36) 
 − 27 61 3.59 (1.64–8.08)e 
*2/*2 29 10.9 (3.57–39.3)e 
 − 36 12.8 (4.09–49.7)e 
 GSTT1    
*1/*1 +b 44 16 1.00c 
 d 13 5.45 (1.82–17.7)e 
*1/*2 43 82 5.39 (2.72–11.1)e 
 − 14 19 3.80 (1.53–9.81)e 
*2/*2 56 24.1 (9.36–70.5)e 
 − 13.4 (2.92–97.7)e 
A. Genotype combinations of xenobiotic metabolizing enzymes in cases (non smokers versus smokers)
GenotypesHNSCC patientsORa (95% CI)
CYP1B1 GSTM1 Nonsmokers (n = 117) Smokers (n = 195)  
*1/*1 + b 25 17 1.00c 
 d 26 12 0.72 (0.27–1.84) 
*1/*2 30 40 1.93 (0.87–4.36) 
 − 27 61 3.59 (1.64–8.08)e 
*2/*2 29 10.9 (3.57–39.3)e 
 − 36 12.8 (4.09–49.7)e 
 GSTT1    
*1/*1 +b 44 16 1.00c 
 d 13 5.45 (1.82–17.7)e 
*1/*2 43 82 5.39 (2.72–11.1)e 
 − 14 19 3.80 (1.53–9.81)e 
*2/*2 56 24.1 (9.36–70.5)e 
 − 13.4 (2.92–97.7)e 
B. Genotype combinations of CYP1B1 and GSTP1 in cases (non-smokers versus smokers)
CYP1B1GSTP1Nonsmokers (n = 117)Smokers (n = 195)
*1/*1 Ile/Ile 26 13 1.00c 
 Ile/Val 19 11 0.78 (0.29–2.04) 
 Val/Val 0.86 (0.21–3.36) 
*1/*2 Ile/Ile 30 39 1.63 (0.76–3.54) 
 Ile/Val 23 40 2.14 (0.98–4.79) 
 Val/Val 22 7.01 (2.20–27.6)e 
*2/*2 Ile/Ile 33 11.2 (3.62–43.6)e 
 Ile/Val 24 6.24 (2.09–21.8)e 
 Val/Val N.A.f 
B. Genotype combinations of CYP1B1 and GSTP1 in cases (non-smokers versus smokers)
CYP1B1GSTP1Nonsmokers (n = 117)Smokers (n = 195)
*1/*1 Ile/Ile 26 13 1.00c 
 Ile/Val 19 11 0.78 (0.29–2.04) 
 Val/Val 0.86 (0.21–3.36) 
*1/*2 Ile/Ile 30 39 1.63 (0.76–3.54) 
 Ile/Val 23 40 2.14 (0.98–4.79) 
 Val/Val 22 7.01 (2.20–27.6)e 
*2/*2 Ile/Ile 33 11.2 (3.62–43.6)e 
 Ile/Val 24 6.24 (2.09–21.8)e 
 Val/Val N.A.f 
a

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

b

GSTM1- and GSTT1-expressors.

c

Reference category.

d

Deleted GSTM1 and GSTT1 genotypes.

e

P < 0.01.

f

N.A., not analyzed.

Table 6

Association between p53-mutations and CYP1B1-genotypes in smokers and nonsmokers

HNSCC Patients Nonsmokers (n = 64)ORa (95% CI)HNSCC Patients Smokers (n = 76)ORa (95% CI)
Normal p53Mutant p53Normal p53Mutant p53
CYP1B1*1/*1 13 16 1.00b 10 1.00b 
CYP1B1*1/*2 24 0.10 (0.02–0.37)c 14 21 7.19 (1.56–52.4) 
CYP1B1*2/*2 N.A.d 23 20.0 (3.95–160)c 
HNSCC Patients Nonsmokers (n = 64)ORa (95% CI)HNSCC Patients Smokers (n = 76)ORa (95% CI)
Normal p53Mutant p53Normal p53Mutant p53
CYP1B1*1/*1 13 16 1.00b 10 1.00b 
CYP1B1*1/*2 24 0.10 (0.02–0.37)c 14 21 7.19 (1.56–52.4) 
CYP1B1*2/*2 N.A.d 23 20.0 (3.95–160)c 
a

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

b

Reference category.

c

P < 0.01.

d

N.A., not analyzed.

Table 7

Effect of combined genotypes of CYP1B1 and GSTM1 or CYP1B1 and GSTT1 on p53 mutation frequencies

GenotypesHNSCC Patients Smokers (n = 76)ORa (95% CI)GenotypesHNSCC Patients Smokers (n = 76)ORa (95% CI)
CYP1B1GSTM1Normal p53Mutant p53CYP1B1GSTT1Normal p53Mutant p53
*1/*1 +b 1.00c *1/*1 +b 1.00c 
 d 1.59 (0.05–53.7)  d N.A.e 
*1/*2 12.5 (1.48–283) *1/*2 12 18 7.57 (1.60–56.5) 
 − 10 13 7.23 (1.00–149)  − 5.19 (0.47–74.4) 
*2/*2 20.0 (1.91–536) *2/*2 18 19.1 (3.58–159)f 
 − 16 26.8 (3.41–590)f  − 25.8 (2.42–700) 
GenotypesHNSCC Patients Smokers (n = 76)ORa (95% CI)GenotypesHNSCC Patients Smokers (n = 76)ORa (95% CI)
CYP1B1GSTM1Normal p53Mutant p53CYP1B1GSTT1Normal p53Mutant p53
*1/*1 +b 1.00c *1/*1 +b 1.00c 
 d 1.59 (0.05–53.7)  d N.A.e 
*1/*2 12.5 (1.48–283) *1/*2 12 18 7.57 (1.60–56.5) 
 − 10 13 7.23 (1.00–149)  − 5.19 (0.47–74.4) 
*2/*2 20.0 (1.91–536) *2/*2 18 19.1 (3.58–159)f 
 − 16 26.8 (3.41–590)f  − 25.8 (2.42–700) 
a

The associations between the genotype distributions and the patients’ status were assessed by ORs and CIs that were calculated by unconditional logistic regression, adjusting for age and gender.

b

GSTM1- and GSTT1-expressors.

c

Reference category.

d

Deleted GSTM1 and GSTT1 genotypes.

e

N.A., not analyzed.

f

P < 0.01.

We thank Alexandra Bage for her skillful technical assistance.

1
Tuyns A. J., Esteve J., Raymond L., Berrino F., Benhamou E., Blanchet F., Boffetta P., Crosignani P., del Moral A., Lehmann W., et al Cancer of the larynx/hypopharynx, tobacco and alcohol: IARC international case-control study in Turin and Varese (Italy). Zaragoza and Navarra (Spain), Geneva (Switzerland) and Calvados (France).
Int. J. Cancer
,
41
:
483
-491,  
1988
.
2
Maier H., de Vries N., Weidauer H. [Occupation and cancer of the oral cavity, pharynx and larynx.].
HNO
,
38
:
271
-278,  
1990
.
3
Flanders W. D., Rothman K. J. Occupational risk for laryngeal cancer.
Am. J. Public Health
,
72
:
369
-372,  
1982
.
4
Gustavsson P., Jakobsson R., Johansson H., Lewin F., Norell S., Rutkvist L. E. Occupational exposures and squamous cell carcinoma of the oral cavity, pharynx, larynx, and oesophagus: a case-control study in Sweden.
J. Occup. Environ. Med.
,
55
:
393
-400,  
1998
.
5
Enterline P. E., Marsh G. M., Esmen N. A. Respiratory disease among workers exposed to man-made mineral fibers.
Am. Rev. Respir. Dis.
,
128
:
1
-7,  
1983
.
6
Saracci R., Simonato L., Acheson E. D., Andersen A., Bertazzi P. A., Claude J., Charnay N., Esteve J., Frentzel-Beyme R. R., Gardner M. J., et al Mortality and incidence of cancer of workers in the man made vitreous fibres producing industry: an international investigation at 13 European plants.
Br. J. Ind. Med.
,
41
:
425
-436,  
1984
.
7
Esteve J., Riboli E., Pequignot G., Terracini B., Merletti F., Crosignani P., Ascunce N., Zubiri L., Blanchet F., Raymond L., Repetto F., Tuyns A. J. Diet and cancers of the larynx and hypopharynx: the IARC multi-center study in southwestern Europe.
Cancer Causes Control
,
7
:
240
-252,  
1996
.
8
Moulin J. J., Mur J. M., Cavelier C. [Comparative epidemiology, in Europe, of cancers related to tobacco (lung, larynx, pharynx, oral cavity)].
Bull. Cancer
,
72
:
155
-158,  
1985
.
9
Caporaso N., Goldstein A. Cancer genes: single and susceptibility: exposing the difference.
Pharmacogenetics
,
5
:
59
-63,  
1995
.
10
Bartsch H., Nair U., Risch A., Rojas M., Wikman H., Alexandrov K. Genetic polymorphism of CYP genes, alone or in combination, as a risk modifier of tobacco-related cancers.
Cancer Epidemiol. Biomarkers Prev.
,
9
:
3
-28,  
2000
.
11
Cascorbi I., Brockmoller J., Roots I. A C4887A polymorphism in exon 7 of human CYP1A1: population frequency, mutation linkages, and impact on lung cancer susceptibility.
Cancer Res.
,
56
:
4965
-4969,  
1996
.
12
Puga A., Nebert D. W., McKinnon R. A., Menon A. G. Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance.
Crit. Rev. Toxicol.
,
27
:
199
-222,  
1997
.
13
Landi M. T., Bertazzi P. A., Shields P. G., Clark G., Lucier G. W., Garte S. J., Cosma G., Caporaso N. E. Association between CYP1A1 genotype, mRNA expression and enzymatic activity in humans.
Pharmacogenetics
,
4
:
242
-246,  
1994
.
14
Kawajiri K., Nakachi K., Imai K., Yoshii A., Shinoda N., Watanabe J. Identification of genetically high risk individuals to lung cancer by DNA polymorphisms of the cytochrome P450IA1 gene.
FEBS Lett.
,
263
:
131
-133,  
1990
.
15
Hirvonen A., Husgafvel-Pursiainen K., Karjalainen A., Anttila S., Vainio H. Point-mutational MspI and Ile-Val polymorphisms closely linked in the CYP1A1 gene: lack of association with susceptibility to lung cancer in a Finnish study population.
Cancer Epidemiol. Biomarkers Prev.
,
1
:
485
-489,  
1992
.
16
Hirvonen A., Husgafvel-Pursiainen K., Anttila S., Karjalainen A., Vainio H. Polymorphism in CYP1A1 and CYP2D6 genes: possible association with susceptibility to lung cancer.
Environ. Health Perspect.
,
101 (Suppl. 3)
:
109
-112,  
1993
.
17
Shimada T., Hayes C. L., Yamazaki H., Amin S., Hecht S. S., Guengerich F. P., Sutter T. R. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1.
Cancer Res.
,
56
:
2979
-2984,  
1996
.
18
Stoilov I., Akarsu A. N., Alozie I., Child A., Barsoum-Homsy M., Turacli M. E., Or M., Lewis R. A., Ozdemir N., Brice G., Aktan S. G., Chevrette L., Coca-Prados M., Sarfarazi M. Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1.
Am. J. Hum. Genet.
,
62
:
573
-584,  
1998
.
19
Bejjani B. A., Stockton D. W., Lewis R. A., Tomey K. F., Dueker D. K., Jabak M., Astle W. F., Lupski J. R. Multiple CYP1B1 mutations and incomplete penetrance in an inbred population segregating primary congenital glaucoma suggest frequent de novo events and a dominant modifier locus[Published erratum appears in Hum. Mol. Genet., 9: 1141, 2000].
Hum. Mol. Genet.
,
9
:
367
-374,  
2000
.
20
Li D. N., Seidel A., Pritchard M. P., Wolf C. R., Friedberg T. Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol.
Pharmacogenetics
,
10
:
343
-353,  
2000
.
21
Fritsche E., Bruning T., Jonkmanns C., Ko Y., Bolt H. M., Abel J. Detection of cytochrome P450 1B1 BfrI polymorphism: genotype distribution in healthy German individuals and in patients with colorectal carcinoma.
Pharmacogenetics
,
9
:
405
-408,  
1999
.
22
Bailey L. R., Roodi N., Dupont W. D., Parl F. F. Association of cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer[Published erratum appears in Cancer Res., 59: 1388, 1999].
Cancer Res.
,
58
:
5038
-5041,  
1998
.
23
Sutter T. R., Tang Y. M., Hayes C. L., Wo Y. Y., Jabs E. W., Li X., Yin H., Cody C. W., Greenlee W. F. Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2.
J. Biol. Chem.
,
269
:
13092
-13099,  
1994
.
24
Abel J., Li W., Dohr O., Vogel C., Donat S. Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice.
Arch. Toxicol.
,
70
:
510
-513,  
1996
.
25
Seidegard J., Vorachek W. R., Pero R. W., Pearson W. R. Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion.
Proc. Natl. Acad. Sci. USA
,
85
:
7293
-7297,  
1988
.
26
Strange, R. C., and Fryer, A. A. The glutathione S-transferases: influence of polymorphism on cancer susceptibility. IARC Scientific Publ. No. 148, pp. 231–49. Lyon, France: IARC, 1999.
27
Strange R. C., Jones P. W., Fryer A. A. Glutathione S-transferase.
Genetics and role in toxicology. Toxicol. Lett.
,
112–113
:
357
-363,  
2000
.
28
Schroder K. R., Hallier E., Meyer D. J., Wiebel F. A., Muller A. M., Bolt H. M. Purification and characterization of a new glutathione S-transferase, class τ, from human erythrocytes.
Arch. Toxicol.
,
70
:
559
-566,  
1996
.
29
Pemble S., Schroeder K. R., Spencer S. R., Meyer D. J., Hallier E., Bolt H. M., Ketterer B., Taylor J. B. Human glutathione S-transferase τ (GSTT1): cDNA cloning and the characterization of a genetic polymorphism.
Biochem. J.
,
300
:
271
-276,  
1994
.
30
Zimniak P., Nanduri B., Pikula S., Bandorowicz-Pikula J., Singhal S. S., Srivastava S. K., Awasthi S., Awasthi Y. C. Naturally occurring human glutathione S-transferase GSTP1–1 isoforms with isoleucine and valine in position 104 differ in enzymic properties.
Eur. J. Biochem.
,
224
:
893
-899,  
1994
.
31
Harries L. W., Stubbins M. J., Forman D., Howard G. C., Wolf C. R. Identification of genetic polymorphisms at the glutathione S-transferase π locus and association with susceptibility to bladder, testicular and prostate cancer.
Carcinogenesis (Lond.)
,
18
:
641
-644,  
1997
.
32
Hengstler J. G., Arand M., Herrero M. E., Oesch F. Polymorphisms of N-acetyltransferases, glutathione S-transferases, microsomal epoxide hydrolase and sulfotransferases: influence on cancer susceptibility.
Recent Results Cancer Res.
,
154
:
47
-85,  
1998
.
33
Hussain S. P., Harris C. C. Molecular epidemiology of human cancer.
Recent Results Cancer Res.
,
154
:
22
-36,  
1998
.
34
Bartsch H., Rojas M., Alexandrov K., Risch A. Impact of adduct determination on the assessment of cancer susceptibility.
Recent Results Cancer Res.
,
154
:
86
-96,  
1998
.
35
Nakachi K., Imai K., Hayashi S., Kawajiri K. Polymorphisms of the CYP1A1 and glutathione S-transferase genes associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population.
Cancer Res.
,
53
:
2994
-2999,  
1993
.
36
Hayashi S. I., Watanabe J., Nakachi K., Kawajiri K. PCR detection of an A/G polymorphism within exon 7 of the CYP1A1 gene.
Nucleic Acids Res.
,
19
:
4797
1991
.
37
Oyama T., Mitsudomi T., Kawamoto T., Ogami A., Osaki T., Kodama Y., Yasumoto K. Detection of CYP1A1 gene polymorphism using designed RFLP and distributions of CYP1A1 genotypes in Japanese.
Int. Arch. Occup. Environ. Health
,
67
:
253
-256,  
1995
.
38
Ko Y., Koch B., Harth V., Sachinidis A., Thier R., Vetter H., Bolt H. M., Bruning T. Rapid analysis of GSTM1, GSTT1 and GSTP1 polymorphisms using real-time polymerase chain reaction.
Pharmacogenetics
,
10
:
271
-274,  
2000
.
39
Bruning T., Abel J., Koch B., Lorenzen K., Harth V., Donat S., Sachinidis A., Vetter H., Bolt H. M., Ko Y. Real-time PCR-analysis of the cytochrome P450 1B1 codon 432-polymorphism.
Arch. Toxicol.
,
73
:
427
-430,  
1999
.
40
Weirich G., Hornauer M. A., Bruning T., Hofler H., Brauch H. Fixed archival tissue. Purify DNA and primers for good PCR yield!.
Mol. Biotechnol.
,
8
:
299
-301,  
1997
.
41
Stokes M. E., Davis C. S., Koch G. G. Logistic regression .
Categorical Data Analysis Using the SAS System
,
:
165
-213, SAS Institute, Inc. Cary, NC  
1995
.
42
Matthias C., Bockmuhl U., Jahnke V., Harries L. W., Wolf C. R., Jones P. W., Alldersea J., Worrall S. F., Hand P., Fryer A. A., Strange R. C. The glutathione S-transferase GSTP1 polymorphism: effects on susceptibility to oral/pharyngeal and laryngeal carcinomas.
Pharmacogenetics
,
8
:
1
-6,  
1998
.
43
Matthias C., Bockmuhl U., Jahnke V., Jones P. W., Hayes J. D., Alldersea J., Gilford J., Bailey L., Bath J., Worrall S. F., Hand P., Fryer A. A., Strange R. C. Polymorphism in cytochrome P450 CYP2D6, CYP1A1, CYP2E1 and glutathione S-transferase, GSTM1, GSTM3, GSTT1 and susceptibility to tobacco-related cancers: studies in upper aerodigestive tract cancers.
Pharmacogenetics
,
8
:
91
-100,  
1998
.
44
Jahnke V., Strange R., Matthias C., Fryer A. A. [Initial results of glutathione-S-transferase GSTM1 and GSTT1 genotypes and genetic predisposition for laryngeal carcinoma].
Laryngorhinootologie
,
74
:
691
-694,  
1995
.
45
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., Strange R. C. Glutathione S-transferase GSTT1 genotypes and susceptibility to cancer: studies of interactions with GSTM1 in lung, oral, gastric and colorectal cancers.
Carcinogenesis (Lond.)
,
17
:
881
-884,  
1996
.
46
Park J. Y., Muscat J. E., Ren Q., Schantz S. P., Harwick R. D., Stern J. C., Pike V., Richie J. P., Jr., Lazarus P. CYP1A1 and GSTM1 polymorphisms and oral cancer risk[Published erratum appears in Cancer Epidemiol. Biomarkers Prev., 6: 1108, 1997].
Cancer Epidemiol. Biomarkers Prev.
,
6
:
791
-797,  
1997
.
47
Oude Ophuis M. B., van Lieshout E. M., Roelofs H. M., Peters W. H., Manni J. J. Glutathione S-transferase M1, and T1, and cytochrome P4501A1 polymorphisms in relation to the risk for benign and malignant head and neck lesions.
Cancer (Phila.)
,
82
:
936
-943,  
1998
.
48
Trizna Z., Clayman G. L., Spitz M. R., Briggs K. L., Goepfert H. Glutathione s-transferase genotypes as risk factors for head and neck cancer.
Am. J. Surg.
,
170
:
499
-501,  
1995
.
49
Katoh T., Kaneko S., Takasawa S., Nagata N., Inatomi H., Ikemura K., Itoh H., Matsumoto T., Kawamoto T., Bell D. A. Human glutathione S-transferase P1 polymorphism and susceptibility to smoking related epithelial cancer; oral, lung, gastric, colorectal and urothelial cancer.
Pharmacogenetics
,
9
:
165
-169,  
1999
.
50
Kawajiri K., Nakachi K., Imai K., Watanabe J., Hayashi S. The CYP1A1 gene and cancer susceptibility.
Crit. Rev. Oncol. Hematol.
,
14
:
77
-87,  
1993
.
51
Okada T., Kawashima K., Fukushi S., Minakuchi T., Nishimura S. Association between a cytochrome P450 CYPIA1 genotype and incidence of lung cancer.
Pharmacogenetics
,
4
:
333
-340,  
1994
.
52
Guengerich F. Metabolic control of carcinogens Hengstler J. G. Oesch F. eds. .
Control Mechanisms of Carcinogenesis
,
:
12
-35, Publishing House of the Editors Mainz, Germany  
1996
.
53
Hanna I. H., Dawling S., Roodi N., Guengerich F. P., Parl F. F. Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity.
Cancer Res.
,
60
:
3440
-3444,  
2000
.
54
Shimada T., Watanabe J., Kawajiri K., Sutter T. R., Guengerich F. P., Gillam E. M., Inoue K. Catalytic properties of polymorphic human cytochrome P450 1B1 variants.
Carcinogenesis (Lond.)
,
20
:
1607
-1613,  
1999
.