Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

CYP1A1 and GSTM1 polymorphisms affect urinary 1-hydroxypyrene levels after PAH exposure.

Certain human biotransformation enzymes have been implicated in the formation and scavenging of the ultimate reactive metabolites, the diolepoxides, from polycyclic aromatic hydrocarbons (PAHs). In the present study, performed on aluminum smelter workers, we have analyzed airborne PAH, the pyrene metabolite 1-hydroxypyrene (1-OHP) in urine, and genotypes for biotransformation enzymes involved in PAH metabolism. The aim was to evaluate the correlation between external exposure and biomarkers of exposure and to investigate to what extent genetic polymorphism in metabolic enzymes can explain interindividual variation in urinary 1-OHP levels. DNA was prepared from blood samples from 98 potroom workers and 55 controls and altogether eight polymorphisms in the CYP1A1, mEH, GSTM1, GSTP1 and GSTT1 genes were analyzed. The 1-OHP excretion was found to correlate significantly (P 100-fold) and univariate and multivariate regression analyses were used to find the variables that could determine differences in excretion. The variation could, to some degree, be explained by differences in exposure to airborne particulate-associated PAHs, the use of personal respiratory protection devices, smoking habits and genetic polymorphisms in the cytochrome P450 1A1, GSTM1 and GSTT1 enzymes. The part of the variance that could be explained by differences in biotransformation genotypes seemed to be of the same order of magnitude as the variance explained by differences in exposure. In the control group as well as in the occupationally exposed group, the highest 1-OHP levels were observed in individuals carrying the CYP1A1 Ile/Val genotype who were also of the GSTM1 null genotype. The results show that urinary 1-OHP is a sensitive indicator of recent human exposure to PAHs and that it may also to some extent reflect the interindividual variation in susceptibility to PAHs.

Influence of genetic polymorphisms of biotransformation enzymes on gene mutations, strand breaks of deoxyribonucleic acid, and micronuclei in mononuclear blood cells and urinary 8-hydroxydeoxyguanosine in potroom workers exposed to polyaromatic hydrocarbons.

OBJECTIVES: Airborne exposure to polycyclic aromatic hydrocarbons (PAH) in the potroom of an aluminum reduction plant was studied in relation to genotoxic or mutagenic effects, and the possibility of host genotypes of different metabolizing enzymes modifying associations between PAH exposure and genotoxic or mutagenic response was assessed. SUBJECTS AND METHODS: Ninety-eight male potroom workers and 55 male unexposed blue-collar workers constituted the study population. Micronuclei in CD4+ and CD8+ lymphocytes, DNA (deoxyribonucleic acid) single-strand breaks, hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation frequency, and genotype for cytochrome P-4501A1, glutathione transferases M1, T1 and P1, and microsomal epoxide hydrolase were analyzed using peripheral mononuclear cells. Urine samples were collected for the analysis of 8-hydroxydeoxyguanosine. RESULTS: Micronuclei in peripheral CD4+ and CD8+ lymphocytes, DNA single-strand breaks, HPRT mutation frequency, and 8-hydroxydeoxyguanosine in urine did not differ between the potroom workers and the unexposed referents. With the exception of an observed exposure-response relationship for potroom workers with Tyr/Tyr genotype for microsomal epoxide hydrolase, between airborne PAH and CD8+ micronuclei, no correlations were found between any of the genotoxicity biomarkers and any of the exposure measures (airborne particulate PAH, airborne gas phase PAH, length of employment in the potroom, 1-hydroxypyrene in urine, or PAH-DNA adducts in peripheral lymphocytes), also when genotypes for biotransforamtion enzymes were considered. CONCLUSIONS: The results indicate that the employed biomarkers of mutagenic or genotoxic effects are not appropriate for surveillance studies of potroom workers exposed to current airborne levels of PAH. The significance of the correlation between airborne PAH and CD8+ micronuclei in Tyr/Tyr genotype subjects should be evaluated.

Characterization of a human glutathione S-transferase mu cluster containing a duplicated GSTM1 gene that causes ultrarapid enzyme activity.

The mu class glutathione S-transferase gene GSTM1 is polymorphic in humans, with approximately half of the Caucasian population being homozygous deleted for this gene. GSTM1 enzyme deficiency has been suggested to predispose people to lung and bladder cancer. Some people in a Saudi Arabian population, however, have been described previously with ultrarapid GSTM1 enzyme activity. Here we have evaluated the molecular genetic basis for this observation. Genomic DNA from two Saudi Arabian subjects exhibiting ultrarapid enzyme activity and from 13 Swedish subjects having null, one, or two GSTM1 genes were subjected to restriction fragment length polymorphism analysis using the restriction enzymes EcoRI, EcoRV, and HindIII and combinations thereof. Hybridization was carried out using a full-length GSTM1 cDNA or the 5' and 3' parts of the cDNA. The restriction mapping data revealed the presence of a GST mu cluster with two GSTM1 genes in tandem situated between the GSTM2 and GSTM5 genes. A quantitative multiplex polymerase chain reaction method, which simultaneously amplified a fragment of the GSTM1 gene and the beta-globin gene, was developed, and the genomic GSTM1 copy number was determined from the GSTM1/beta-globin ratio. This method clearly separated GSTM1 +/- subjects (ratios between 0.4 and 0.7) from GSTM1 +/+ subjects (ratios between 0.8 and 1.2). The two Saudi Arabians with ultrarapid GSTM1 activities had ratios of approximately 1.5, indicating that they carried three GSTM1 genes. These results demonstrate the existence of a novel mu class GST cluster containing a duplicated active GSTM1 gene causing ultrarapid enzyme activity.

Association between the p21 codon 31 A1 (arg) allele and lung cancer.

In previous investigations p53 polymorphisms and haplotypes have been found to be associated with different types of cancer. In this paper the codon 31 polymorphism of the p53-inducible protein p21 was studied in 144 Swedish lung cancer patients and two different control groups: 95 patients with chronic obstructive pulmonary disease (COPD) and 761 healthy controls. An increased frequency of the p21 codon 31 A1 (arg) allele was found in lung cancer patients, especially in comparison with COPD patients (p = 0.004). There was a significantly increased frequency among lung cancer patients of individuals carrying the arg allele both in comparison with COPD controls (OR = 5.2, 95% CI 1.5-18.1) and healthy controls (OR = 1.7, 95% CI = 1.0-2.9). The results of this and previous studies indicate that allelic variants of both p53 and its effector protein p21 may have an influence on lung cancer.

P53 polymorphisms and haplotypes in lung cancer.

An association between the BstU I 1-1 (Pro-Pro) genotype of the p53 codon 72 polymorphism and lung cancer has previously been reported by Kawajiri et al. A reanalysis of the data by Kawajiri et al. revealed no significant difference between patients and controls with respect to allele frequencies, and the increased frequency of BstU I 1-1 homozygotes was mostly ascribable to a deviation from the Hardy-Weinberg equilibrium. In an attempt to replicate the results by Kawajiri et al. we have studied three p53 polymorphisms (BstU I and Msp I RFLPs in exon 4 and intron 6 respectively and a 16 bp duplication in intron 3) and their haplotypes in Swedish lung cancer patients and controls. The results concerning the codon 72 polymorphism were largely negative. Thus there was no significant association between lung cancer and the BstU I 1-1 type, and only a marginal difference (P = 0.044) with respect to the BstU I allele frequency when lung cancer patients were compared with patients with chronic obstructive pulmonary disease (COPD). However, when the analysis was based on haplotype frequencies larger differences appeared and it was found that only BstU I 1 (pro) alleles linked to 16 bp 1 alleles were associated with lung cancer. Pro alleles linked to the 16 bp duplication appeared instead to confer some protection against cancer. Thus the codon 72 alleles need not be functionally involved in lung cancer, but may rather be markers in linkage disequilibrium with other cancer susceptibility sites on p53.

Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity.

Genetic polymorphism of cytochromes P450 1A1, 2D6 and 2E1: regulation and toxicological significance.

Because of important roles of cytochromes P450 in the metabolic activation of many precarcinogens, extensive research in the past has focused on the relationship between the distribution of polymorphic variants of different isozymes of P450 and cancer susceptibility. In this respect three isozymes in particular have been studied, CYP1A1, CYP2D6, and CYP2E1. Both CYP1A1 and CYP2E1 participate in the metabolism of many suspected as well as established carcinogens, whereas essentially only one carcinogenic substrate has been identified for CYP2D6. Polymorphic sites for the three CYP genes have been identified both in the open reading frame as well as in introns and the regulatory 5' region. In the present contribution we summarize the molecular epidemiological research relating CYP polymorphism to cancer susceptibility and in some cases to toxicity. An interesting polymorphism has been described on the phenotypic level for the inducibility of CYP1A1, a polymorphism that in some studies has been related to a mutation in the 3' flanking region of the CYP1A1 gene. However, the genetic basis for this polymorphism might be inherited in the genes coding for proteins responsible for the induction of CYP1A1, ie, the Ah receptor or the ARNT protein. Data on lung cancer and CYP1A1 gene polymorphism indicate that carriers of genotypes associated with CYP1A1 inducibility are at higher risk for cancer, but that, at least for Caucasians, the recognized mutations probably identify only a fraction of the inducible individuals. The amount of DNA adducts correlates well in some studies to the individual activity registered for CYP1A1. CYP2D6 phenotype and genotype have mainly been related to the incidence of lung cancer, but results from 13 different studies now show an absence of any significant correlation between these parameters. In the case of CYP2E1, some studies indicate a relationship between lung cancer and the occurrence of a rare allele, although future research is needed in order to establish a significant relationship. It is concluded that, at the present stage, none of the polymorphic sites determined in the CYP genes can yet be used as markers for increased lung cancer risk. Future research in this field might be focused on the establishment of new polymorphic sites in the CYP genes, affecting inducibility or function, and on the molecular basis for the interesting differences in CYP1A1 inducibility.

Genetic polymorphism for glutathione-S-transferase mu in asbestos cement workers.

OBJECTIVE: To investigate whether a lack of glutathione-S-transferase mu (GSTM1) activity was related to an increased risk for adverse outcome after asbestos exposure. METHODS: A study was made of 78 male former asbestos cement workers, with retrospective cohort data on exposure, radiographical findings, and lung function. Venous blood samples were obtained for the analysis of GSTM1 polymorphism by the polymerase chain reaction technique. Chest x ray films were classified according to the International Labour Organisation (ILO) 1980 classification. Vital capacity (VC) and forced expiratory volume during 1 s (FEV1) were determined. Individual estimates of asbestos exposure were calculated, and expressed as duration of exposure, average exposure intensity, and cumulative dose. Data on smoking were obtained from interviews. RESULTS: The lung function in the study group was reduced, compared with reference equations. 23% of the workers had small opacities > or = 1/0, 29% circumscribed pleural thickenings, 14% diffuse thickenings, and 12% obliterated costophrenic angles. 54% of the workers were GSTM1 deficient. They were comparable with the other workers in age, follow up time (median 30 years), and duration of exposure (median 18 years), but had a slightly higher cumulated dose (median 18 v 10 fibre-years) than the others. Neither in radiographical changes nor lung function variables were there any differences between the different GSTM1 groups. The findings were similar when smoking habits and estimated asbestos exposure were taken into account. CONCLUSIONS: We could not show that lack of GSTM1 activity was related to an increased risk for radiographical or lung function changes in a group of asbestos cement workers, followed up for a long period after the end of exposure.

Polymorphic distribution of glutathione transferase activity with methyl chloride in human blood.

Interindividual variation in the in vitro conjugation of methyl chloride with glutathione by erythrocyte glutathione transferase was investigated in 208 healthy males and females from the southern and central parts of Sweden. It was found that 11.1% of the individuals lacked this activity, whereas 46.2% had intermediate activity and 42.8% had high activity. This distribution of three phenotypes is compatible with the presence of one functional allele with a gene frequency of 0.659 and one defect allele with a gene frequency of 0.341. The proportion of non-conjugators in this Swedish material was considerably smaller than that previously found in Germany (Peter et al., Arch Toxicol 1989: 63, 351-355). The polymorphic distribution of another glutathione transferase, GST mu, was determined in the same individuals with a PCR method. No connection between the genotype for GST mu (GSTM1) and the glutatione conjugation with methyl chloride in erythrocytes was found.

Genetic susceptibility to lung cancer with special emphasis on CYP1A1 and GSTM1: a study on host factors in relation to age at onset, gender and histological cancer types.

Genetically based differences in metabolism, related to MspI restriction site and Ile-Val polymorphisms of the cytochrome P450 (CYP) 1A1 gene and the null genotype of glutathione transferase class mu (GSTM1), have been reported to be associated with lung cancer susceptibility. The present study was set up to establish the frequencies of the polymorphic genotypes of CYP1A1 and GSTM1 in Sweden, to evaluate a possible increased incidence of the genotypes associated with higher lung cancer risks among Swedish lung cancer patients and to try to make a combined risk estimate for carriers of multiple risk alleles. In a healthy control group, all under 66 years of age, 53% (174/329) of the subjects were of the GSTM1(-) genotype, while in a hospital control group 49% (39/79) carried the GSTM1(-) genotype. In the investigated lung cancer patients this genotype was found in 56% (165/296) and among those patients diagnosed before 66 years of age the deficient genotype was found in 60% (78/131). The highest proportion of the GSTM1(-) genotype was found in patients diagnosed with adenocarcinoma (63%, 29/46) and small cell carcinoma (72%, 21/29) before 66 years of age and among female squamous cell carcinoma patients (79%, 15/19). The allelic variants in CYP1A1 were equally distributed in lung cancer patients and controls. The m1/m2 and m2/m2 genotypes of the MspI site and the Ile/Val genotype were, however, slightly over-represented in squamous cell carcinoma patients. Among patients with squamous cell carcinoma diagnosed before 66 years of age the m1/m2 genotype was found in 28% (10/36), whereas the same genotype was observed in 16% (52/329) of healthy control subjects. A combined risk of squamous cell carcinoma was indicated for patients, diagnosed before 66 years of age, carrying both GSTM1(-) and m2 alleles (OR = 3.0, 95% CI = 1.2-7.2).

Aromatic DNA adducts, micronuclei and genetic polymorphism for CYP1A1 and GST1 in chimney sweeps.

Aromatic DNA adducts in total white blood cells, cytochrome P450 (CYP) class 1A1 and glutathione transferase (GST1) class mu genotypes and micronuclei in T- and B-lymphocytes were studied in 69 full-time chimney sweeps and 35 controls, all male subjects. The sweeps had a higher (22%) but statistically non-significant increase in the level of DNA adducts as compared to the controls when all individuals independent of genotype were compared. The non-inducible CYP1A1 genotype, m1/m1, lacking a MspI restriction site at the 3' end of the gene, was associated with significantly higher adduct levels in the sweeps. Among the 26 sweeps with the combined genotype m1/m1 and GST1(-), a statistically significant 60% increase in median adduct levels was observed as compared with those 14 control subjects with the corresponding genotype. Smoking also showed a significant effect on the level of adducts. The effect on DNA adducts by sweeping, smoking and genotype appeared to be additive and independent of each other. DNA adducts in sweeps were moderately but statistically significantly correlated with micronuclei in both T- and B-lymphocytes. The correlation between adduct levels and micronuclei was most marked in T-lymphocytes of individuals lacking the GST1 gene.

B- and T-lymphocyte micronuclei in chimney sweeps with respect to genetic polymorphism for CYP1A1 and GST1 (class Mu).

Epidemiological studies have shown an increased incidence of lung cancer, bladder cancer, and esophageal cancer in chimney sweeps, probably due to their exposure to PAH in soot. The work environment for sweeps has, however, improved during the last decades. It was thus important to assess whether the present exposure still may cause genotoxic effects. A further objective was to assess whether genetic polymorphisms in metabolic enzyme activities could explain some of the variation in the parameters of genotoxicity. Venous blood samples were drawn from 71 chimney sweeps and 59 control subjects. Micronuclei were analyzed in activated peripheral B- and T-lymphocytes with preserved cytoplasm. Polymorphisms for CYP1A1 and GST1 in the sweeps were analyzed by a PCR technique. The sweeps did not have higher frequencies of micronuclei in B- or T-lymphocytes than the control subjects, when allowance was made for age and smoking in a multiple regression analysis. Further, there was no association between years of active work as a sweep and any of the two micronucleus parameters. None of the sweeps had the rare CYP1A1 genotype val/val and only one individual had the m2/m2 genotype. The presence of at least one GST1 allele (GST1+) was observed in 36 subjects (51.4%). Thirteen individuals (18.6%) were of the m1/m2 or m2/m2 genotype. And among those only seven had the combined GST1- and m1/m2 genotype. No difference was observed in B- or T-lymphocyte micronucleus frequencies between sweeps with the rare CYP1A1 genotypes m1/m2, m2/m2 or ile/val compared to individuals with the m1/m1 and ile/ile genotypes. Moreover, the GST1 deficient sweeps (GST1-) did not show any altered micronucleus frequency compared to the GST1 positive sweeps. A possible reason for the lack of genotoxic effect in sweeps is the improved hygienic conditions and change in fuels, which has decreased the exposure levels for PAH. Host polymorphisms for metabolizing enzymes did not influence the micronucleus frequencies. As the sweeps did not differ from the control subjects, with respect to micronucleus frequencies, no conclusion on the importance of host polymorphisms for genotoxic risk can be drawn.