Glutathione S-Transferase GSTM3 and GSTP1 Genotypes and Larynx Cancer Risk¹

GSTs³ are a family of cytosolic enzymes that are potentially important in regulating susceptibility to cancer due to their ability to metabolize reactive metabolites of carcinogens (1). Among them, GSTM1 and GSTT1 have attracted most of the interest (2), mainly because they are involved in detoxification of reactive metabolites of carcinogenic substances from tobacco smoke (3). The absence of GSTM1 and GSTT1 enzyme activities in approximately 50% and 20% of Caucasians, respectively, are due to homozygous inherited deletion in the respective genes (null genotype; Refs. 4 and 5). Results from the previous studies on larynx cancer risk associated with the GSTM1 phenotype/genotype or the GSTT1 genotype have been inconclusive, and only a moderate increase in risk, if any, was observed (6, 7, 8, 9). One of the possible reasons of the divergent findings may rely on polymorphisms of other relevant GST genes (10, 11).

Recently, polymorphisms in GSTM3 and GSTP1 loci were reported (12, 13). In the GSTM3 gene, the GSTM3*A wild type allele and the GSTM3*B variant allele have been described (12). The GSTM3*B contains a recognition motif for the YY1 transcription factor that has been postulated to regulate gene expression (14). It was suggested that GSTM3*A and GSTM3*B are expressed at different levels. Although relatively little is known about the role of GSTM3 in the metabolism of harmful agents, it is known to have overlapping substrate specificities with GSTM1 (15). The GSTM3 polymorphism could, therefore, confer different efficiencies in the metabolism of carcinogens (16). In a recent study, an increase in larynx cancer risk associated with GSTM3 AA genotype was suggested (8).

GSTP1 has been shown to detoxify active metabolites of polycyclic aromatic hydrocarbons, one of the main carcinogens of tobacco smoke (13). In the GSTP1 gene, two variant alleles, GSTP1*B and GSTP1*C, have been detected in addition to the wild type allele GSTP1*A (13). Both variant alleles have an A₃₁₃G transition, causing a change of isoleucine to Val. The specific activity and affinity for electrophilic substrates is altered in the Val variant (13). In a recent study, lack of association between polymorphism at the GSTP1 locus and larynx cancer was observed (17).

We previously reported that the GSTM1-null genotype conferred an increased risk of larynx cancer; the risk linked to GSTT1-null genotype was also increased, although not significantly (9). Additional genotyping tests were performed to examine the role of GSTM3 and GSTP1 polymorphisms in larynx cancer susceptibility.

Subjects were recruited in France from 1988–1992, and the study population has been described previously (9). In brief, cases were Caucasian patients with histologically confirmed primary squamous cell carcinoma of the larynx. A control group, frequency matched on age, sex, and hospital, consisted of all eligible Caucasian patients without previous or present malignant disease. Only regular smokers, defined as people having smoked at least five cigarettes (or cigars or pipes) per day for at least 5 years, were included. Detailed information on lifetime tobacco use and alcohol consumption was recorded during a personal interview. Participation rates were not formally recorded. Discussions with interviewers suggest that almost all eligible cases and controls agreed to participate. Blood samples from 129 larynx cancer patients and 172 controls fulfilling the above-mentioned criteria were collected in EDTA tubes and stored at −20°C. The main diseases diagnosed in the control population were rheumatological (33%), infectious and parasitic (10%), respiratory (9%), cardiovascular (8%), digestive (6%), and traumatological (6%). The main motive for admission to hospital was related to general symptoms (7.0%) for the other categories. The age distributions were quite similar in cases (55.0 years) and controls (54.9 years). Most of the study subjects were men (98% of cases and 95% of controls). Cases had a higher average daily tobacco consumption than controls (30.4 g/day versus 25.1 g/day) and a longer duration of smoking (34.6 years versus 32.2 years). The mean daily consumption of alcohol was higher in larynx cancer cases than in controls (98.1 g/day versus 77.1 g/day).

Extraction of total WBC DNA was performed using standard protocols. A multiplex PCR method was used to detect the presence or absence of GSTM1 and GSTT1 genes, as described earlier (18, 19).

The allelic variants of GSTM3 and GSTP1 genes were differentiated from the wild-type alleles by PCR-RFLP based methods, as described previously (12, 20).

ORs and 95% CIs associated with GST genotypes were calculated by unconditional logistic regression using Statistical Analysis Software, version 6.11 (21). ORs were adjusted for sex, age, and smoking- and alcohol-related variables, as described previously (9). As there were no women among glottic/subglottic larynx cancer patients, analyses in this subpopulation were restricted to men.

Gene-gene interactions and gene-smoking interactions were evaluated by the likelihood ratio test to compare the goodness of fit of the model with and without interaction term (22). To this end, the average daily consumption of tobacco and the duration of smoking were expressed as continuous or as categorical variables (22), the latter defined with two levels of smoking exposure according to the approximate median in the control population. Due to small numbers, the GSTM3 AB and BB genotypes, as well as the GSTP1 AG and GG genotypes, were combined in most analyses.

The GSTM3 and GSTP1 genotype distributions in the control population were in agreement with those predicted under the conditions of Hardy-Weinberg equilibrium (P = 0.58 and P = 0.18, respectively). The difference in the distribution of GSTM3 AA, AB, and BB genotypes between larynx cancer cases and controls was nearly significant (P <0.08; Table 1); this was explained by the lower frequency of subjects with the GSTM3 AA genotype in cases (60.5%) than in controls (72.7%). In contrast, the distribution of GSTP1 AA, AG, and GG genotypes was similar in both groups (Table 1). The frequency of the GSTM3 AA genotype was significantly higher in carriers of the GSTM1-null genotype than in those with the GSTM1-positive genotype, both among larynx cancer patients (70.4% versus 45.1%; P <0.01) and among controls (80.0% versus 64.6%; P <0.05).

The age- and sex- adjusted larynx cancer risk associated with GSTM3 (AB or BB) genotype was significantly increased (OR, 1.9; 95% CI, 1.1–3.2), whereas the OR associated with the GSTP1 (AG or GG) genotype was close to one (OR, 1.1; 95% CI, 0.7–1.7). Adjustments for smoking- and alcohol-related variables did not alter to any substantial extent the risk estimates (OR, 2.0; 95% CI, 1.1–3.4 and OR, 1.1; 95% CI, 0.7–2.0, respectively; Table 1).

Similar results were observed when analyses were restricted to men or when supraglottic and glottic/subglottic larynx cancer cases were studied separately (Table 1).

The ORs of larynx cancer linked to different GST genotype combinations are shown in Table 2. Compared with the combined GSTM3 AA- and GSTM1-positive genotype, a significant increase in risk associated with the combined GSTM3 (AB or BB) was found both in carriers of GSTM1-positive genotype (OR, 3.2; 95% CI, 1.4–7.3) and in carriers of GSTM1-null genotype (OR, 4.0; 95% CI, 1.6–10.1). Similarly, the effect of GSTM3 (AB or BB) genotype was not modified by the GSTT1 genotype. Larynx cancer risks related to GSTP1 (AG or GG) genotype were similar in carriers of GSTM1 or GSTT1 gene deletion and carriers of positive GSTM1 or GSTT1 genotypes.

No statistically significant associations were found between GSTM3 or GSTP1 genotypes and various measures of smoking exposure in the control population. The effect of GSTM3 (AB or BB) genotype on larynx cancer risk was suggested primarily among smokers with daily tobacco consumption of ≤20 g/day (OR, 3.9; 95% CI, 1.6–9.4; Table 3). The interaction test was of borderline significance when daily tobacco consumption was dichotomized (P = 0.07) and nonsignificant when it was expressed as a continuous variable (P = 0.39). This result was not substantially altered if other variables related to smoking were excluded from the model or interaction terms between duration of smoking and the genotype were introduced in the model. Larynx cancer risks associated with GST genotypes were not modified on stratification by duration of smoking or type of tobacco (black only versus black and blond tobacco; data not shown). Risk estimates were not modified on stratification by daily alcohol consumption dichotomized at the median in the control population (data not shown).

We found a 2-fold risk of larynx cancer associated with the GSTM3 (AB or BB) genotype, due to a higher frequency of the GSTM3 AB genotype and a lower frequency of GSTM3 AA genotype, in cancer patients than in controls. On the contrary, in a previous study on larynx cancer (8), a lower frequency of GSTM3 (AB or BB) genotype was found in cases than in controls. The functional consequences of the recognition site for the YY1 transcription factor in the GSTM3*B allele is yet unclear. Both negative and positive regulatory effects have been suggested (12, 16). According to our results, GSTM3*B could be considered as an at-risk allele. Thus, the role of GSTM3 gene polymorphism in susceptibility to larynx cancer is uncertain and should be further investigated.

Active metabolites of polycyclic aromatic hydrocarbons from tobacco smoke are partly detoxified by GSTP1 enzyme. Polymorphism at the GSTP1 gene could, therefore, be relevant in smoking-related cancers. In agreement with the sole previous study on larynx cancer (17), we failed to find increased risk linked to the potentially harmful GSTP1 (AG or GG) genotype. Although it was noted that isoleucine to Val substitution in the GSTP1 protein leads to changes in its catalytic properties in vitro (23), it is possible that these changes do not result in important impairment of tobacco-related carcinogen metabolism by GSTP1 in human larynx.

Although the combined GSTM3 (AB or BB) and GSTM1-null genotype conferred the highest larynx cancer risk, the ORs associated with at-risk GSTM3 or GSTP1 genotypes were similar in carriers of GSTM1- or GSTT1-positive and -null genotypes. The lack of gene-gene interactions could be due to low statistical power because of rather small numbers of individuals or could fit the hypothesis that each of GST enzymes may act independently in carcinogen metabolism.

In summary, the present data suggest that the GSTM3 (AB or BB) genotype confers an increased risk of larynx cancer. In contrast, larynx cancer risk was not associated with GSTP1 gene polymorphism. These findings need confirmation in other investigations and should give impetus to large studies on interactive effects of different GST genotypes.

We thank R. Striberni for expert technical help and C. Paoletti, M. Labbé, and C. Massoud for technical assistance. We are also indebted to the consultants and chiefs of Clinical units who allowed us to study their patients for the purpose of the study: Drs. G. Akoun, R. Arriagada, P. Baldeyrou, F. Besançon, A. Bisson, M. Bisson, F. Blanchet, F. Blanchon, A. Bouchiki, J. Brugère, C. Buffet, J. P. Camus, R. Caquet, Y. Chapuis, D. Chassagne, P. Constans, B. Dautzenberg, J. Debray, J. P. Derenne, P. Duroux, J. Fain, G. Freyss, A. Gerbaulet, Ph. Girard, J. Guerre, P. Guibout, H. Hamard, B. Housset, J. C. Imbert, F. Janot, A. Jardin, T. Le Chevalier, B. Lebeau, A. M. Leridant, Ph. Levasseur, V. G. Levy, A. Livartowski, G. Loyau, B. Luboinski, G. Mamelle, F. Mazas, P. Marandas, C. Menkes, H. Mondon, J. P. Passeron, J. Piquet, A. Rivière, M. Robillard, J. Rochemaure, R. Roy-Camille, J. C. Saltiel, G. Schwaab, J. M. Segrestaa, D. Sereni, M. Spielmann, P. Testas, G. Tobelem, and P. Vige.