Brian F. Coles

The role of glutathione and glutathione transferases in chemical carcinogenesis.

(1990). The Role of Glutathione and Glutathione Transferases in Chemical Cardnogenesi. Critical Reviews in Biochemistry and Molecular Biology: Vol. 25, No. 1, pp. 47-70.

Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer : contribution of metabolic variation to risk

A case-control study of colorectal cancer, consisting of 157 cases and 380 controls matched by sex, ethnicity, decade of age and county of residence was performed to explore the associations between environmental exposure, metabolic polymorphisms and cancer risk. Participants were required to provide a blood sample, undergo caffeine phenotyping and complete an in-person interview that evaluated meat consumption, cooking methods and degree of doneness. A color atlas of foods cooked to different degrees of doneness was used to estimate food preparation techniques and food models were used to estimate serving portion sizes. Data was analyzed using a reference database of heterocyclic amine (HCA) exposure based on the food preferences chosen from the atlas. Data regarding individual food items cooked to different levels of doneness, as well as summary variables of foods and of food groups cooked to different degrees of doneness were also evaluated in a univariate analysis for association with colorectal cancer case status. Three measures of metabolic variation, hGSTA1 genotype, SULT1A1 genotype and the phenotype for CYP2A6 were also evaluated for possible association with colon cancer. While higher exposure to HCAs was strongly associated with colorectal cancer risk, increased consumption of five red meats cooked well done or very well done produced comparable odds ratios (OR) for colorectal cancer risk (OR=4.36, 95% CI 2.08-9.60) for the highest quartile of exposure. Similarly, individuals in the most rapid CYP2A6 phenotype quartile showed an odds ratio (OR = 4.18, 95% CI 2.03-8.90). The ORs for the low activity hGSTA1 and low activity SULT1A1 alleles were 2.0, 95% CI 1.0-3.7 and 0.6, 95% CI 0.3-1.1, respectively. Individual measures of specific HCAs provided little improvement in risk assessment over the measure of meat consumption, suggesting that exposure to other environmental or dietary carcinogens such as nitrosamines or undefined HCAs may contribute to colorectal cancer risk.

Association between a glutathione S‐transferase A1 promoter polymorphism and survival after breast cancer treatment

Glutathione S‐transferase (GST) enzymes detoxify chemotherapeutic drugs, and several studies have reported differences in survival for cancer patients who have variant genotypes for GSTP1, GSTM1 or GSTT1 enzymes. A recently described polymorphism alters hepatic expression of GSTA1, a GST with high activity in glutathione conjugation of metabolites of cyclophosphamide (CP). To consider the possible influence of the reduced‐expression GSTA1*B allele on cancer patient survival, we have conducted a pilot study of breast cancer patients treated with CP‐containing combination chemotherapy. GSTA1 genotype was determined by polymerase chain reaction and restriction fragment length polymorphism. Kaplan‐Meier methods and Cox proportional hazards models were used to evaluate survival in relation to genotype. Among 245 subjects, 35% were GSTA1*A/*A, 49% GSTA1*A/*B and 16% GSTA1*B/*B; the genotype distribution did not differ by ethnic group, age or stage at diagnosis. Among patients who had 0 or 1 GSTA1*B allele, the proportion surviving at 5 years was 0.66 (95% CI = 0.59–0.72), whereas for GSTA1*B/*B subjects the proportion was higher, 0.86 (95% CI = 0.67–0.95). Significantly reduced hazard of death was observed for GSTA1*B/*B subjects during the first 5 years after diagnosis, hazard ratio (HR) = 0.3, 95% CI = 0.1–0.8. The association varied with time, with no survival difference observed for subjects who survived beyond 5 years. These results, although based on a small study population, describe an apparent difference in survival after treatment for breast cancer according to GSTA1 genotype. Further studies should consider the possible association between the novel GSTA1*B variant and outcomes of cancer therapy.

Interindividual variation and organ-specific patterns of glutathione S-transferase alpha, mu, and pi expression in gastrointestinal tract mucosa of normal individuals

Glutathione S-transferase (GST) protein in gastrointestinal (GI) tracts of 16 organ donors, from whom all or substantial portions of the GI tract (stomach-colon) were available, was quantitated by HPLC and examined for interindividual variability/consistency of organ-specific patterns of expression. GSTP1, GSTA1, and GSTA2 were major components, and GSTM1 and GSTM3 were minor components. Consistent patterns of organ-specific expression were evident despite a high degree of interindividual variation of expression. GSTP1 was expressed throughout the GI tract and showed a decrease of expression from stomach to colon. GSTA1 and GSTA2 were expressed at high levels in duodenum and small intestine and expression decreased from proximal to distal small intestine. In contrast, GSTA1 and GSTA2 expression in colon and stomach of all subjects was low, particularly for colon where GSTA1 expression was 20- to 800-fold lower than that in corresponding small intestine. These consistent patterns of expression would suggest that compared to duodenum and small intestine, colon and to a lesser extent stomach always have low potential for GST-dependent detoxification of chemical carcinogens and are therefore at greater risk of genotoxic effects, particularly via substrates that are specific for GSTA1. This may be a factor in the greater susceptibility of stomach and colon to cancers compared to duodenum/small intestine.

Novel markers of susceptibility to carcinogens in diet: associations with colorectal cancer.

Red meats cooked at high temperatures generate mutagenic heterocyclic amines, which undergo metabolic activation by hepatic cytochrome P450 1A2 and N-acetyltransferase-2. A primary detoxification pathway involves glutathione S-transferase A1 (GSTA1), which catalyzes the reduction of the carcinogenic N-acetoxy derivative back to the parent amine. Recently, we described a polymorphism in the GSTA1 proximal promoter; the variant (GSTA1*B) allele significantly lowers enzyme expression. In a case-control study, GSTA1*B/*B genotype was associated with an increased risk of colorectal cancer, particularly among consumers of well-done meat. Dietary nitrosamines, which are bioactivated by CYP2A6, represent another potential etiologic factor for colorectal cancer. CYP2A6 converts the caffeine metabolite 1,7-dimethylxanthine (17X) to 1,7-dimethyluric acid (17U); we investigated CYP2A6 activity using the 17U/17X urinary metabolite ratio from case-control subjects who completed a caffeine phenotype assay. The distribution of CYP2A6 activity was significantly different between CRCa cases and controls, with subjects in the medium and high activity groups having an increased risk (P for trend=0.001). GSTA1 genotype and CYP2A6 phenotype should be evaluated as markers of susceptibility to dietary carcinogens in future studies.

Glutathione-S-transferase (GSTM1) Genetic Polymorphisms Do Not Affect Human Breast Cancer Risk, Regardless of Dietary Antioxidants

Glutathione-S-transferases catalyze the detoxication of carcinogen metabolites and reactive oxygen species (ROS) produced through a number of mechanisms. Glutathione-S-transferase (GST) M1 is polymorphic, and the null allele results in a lack of enzyme activity. Because there are indications that ROS may be involved in breast carcinogenesis, we sought to determine whether the GSTM1 null allele was associated with increased breast cancer, particularly among women with lower consumption of dietary sources of alpha-tocopherol, carotenoids and ascorbic acid. In a study of diet and cancer in western New York, women with primary, incident, histologically confirmed breast cancer (n = 740) and community controls (n = 810) were interviewed and an extensive food-frequency questionnaire administered. A subset of these women provided a blood specimen. DNA was extracted and genotyping performed for GSTM1. Data were available for 279 cases and 340 controls. The null allele did not increase breast cancer risk, regardless of menopausal status. There were also no differences in associations between the polymorphism and risk among lower and higher consumers of dietary sources of antioxidants or smokers and nonsmokers. These results indicate that GSTM1 genetic polymorphisms are not associated with breast cancer risk, even in an environment low in antioxidant defenses.

Expression of cytochromes P450 and glutathione S-transferases in human prostate, and the potential for activation of heterocyclic amine carcinogens via acetyl-coA-, PAPS- and ATP-dependent pathways

Dietary factors appear to be involved in the high incidence of prostate cancer in “Westernized” countries, implicating dietary carcinogens such as heterocyclic amines (HAs) in the initiation of prostate carcinogenesis. We examined 24 human prostate samples with respect to their potential for activation and detoxification of HAs and the presence of DNA adducts formed in vivo. Cytochromes P450 1B1, 3A4 and 3A5 were expressed at low levels (<0.1–6.2 pmol/mg microsomal protein). N‐Acetyltransferase (NAT) activities, using p‐aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates, were <5–5,500 and <5–43 pmol/min/mg cytosolic protein, respectively. Glutathione S‐transferases (GSTs) P1, M2 and M3 were expressed at 0.038–1.284, 0.005–0.126 and 0.010–0.270 μg/mg cytosolic protein, respectively; GSTM1 was expressed in all GSTM1‐positive samples (0.012–0.291 μg/mg cytosolic protein); and GSTA1 was expressed at low levels (<0.01–0.11 μg/mg cytosolic protein). Binding of N‐hydroxy‐PhIP to DNA in vitro occurred primarily by an AcCoA‐dependent process (<1–54 pmol/mg/DNA), PAPS‐ and ATP‐dependent binding being <1–7 pmol/mg DNA. In vivo, putative PhIP‐ or 4‐aminobiphenyl‐DNA adducts were found in 4 samples (0.4–0.8 adducts/108 bases); putative hydrophobic adducts were found in 6 samples (8–64 adducts/108 bases). Thus, the prostate appears to have low potential for N‐hydroxylation of HAs but greater potential for activation of N‐hydroxy HAs to genotoxic N‐acetoxy esters. The prostate has potential for GSTP1‐dependent detoxification of ATP‐activated N‐hydroxy‐PhIP but little potential for detoxification of N‐acetoxy‐PhIP by GSTA1. However, there were no significant correlations between expression/activities and DNA adducts formed in vitro or in vivo, DNA adducts in vivo possibly reflecting carcinogen exposure.

Expression of hGSTP1 alleles in human lung and catalytic activity of the native protein variants towards 1-chloro-2,4-dinitrobenzene, 4-vinylpyridine and (+)-anti benzo[a]pyrene-7,8-diol-9,10-oxide

The human glutathione S-transferase (GST) P1 alleles coding for Val(105) (hGSTP1*B and/or P1*C) are over- represented in lung cancer patients. However, the corresponding recombinant Val(105) protein variants tend to show higher catalytic activity than the Ile(105) variants towards bay-region diol epoxides that are thought to be etiological agents in lung cancer. We have examined 29 normal human lung samples with respect to several factors that could confound relationships between hGSTP1 allele type and cancer susceptibility, namely, inter-individual and allele-specific variation of hGSTP1 expression, and differences between the catalytic properties of the native and recombinant hGSTP1-1 variant protein products. hGSTP1 expression varied 7-fold among individuals but was independent of hGSTP1*A, P1*B or P1*C allele type. hGST subunits A1, A2, M1 and M3 were minor components, similarly variable in expression. Despite this variability of expression, the levels of hGSTP1 expression linearly correlated with those of the next most highly expressed GST, hGSTM3, even though the genes for these GSTs are on different chromosomes. Differences between the native protein variants, using 1-chloro-2,4-dinitrobenzene and (+)-anti-benzo[a]pyrene diolepoxide as substrates, were more marked than those between the recombinant variants. However, the order of differential catalytic specificity was the same for native and recombinant variants. Neither the expression of the hGSTP1 alleles nor the catalytic properties of the protein variants appears to provide a simple mechanistic rationale for the observed over-representation of the hGSTP1*B and/or 1*C alleles in lung cancer.

Effects of the ADH3, CYP2E1, and GSTP1 genetic polymorphisms on their expressions in Caucasian lung tissue.

Individual differences in lung cancer susceptibility should be considered for effective lung cancer prevention. We investigated the CYP2E1, ADH3, and GSTP1 genetic polymorphisms that biotransform xenobiotic carcinogens, and variations of their enzyme activity in Caucasian lung tissues (N=28), and found a variant distribution in pulmonary ADH and CYP2E1 activity. The ADH3*1/*1 subjects (N=8) showed significantly higher ADH activity than ADH3*2/*2 (N=3) subjects (P<0.01). On the other hand, we found a 5-fold variation in the pulmonary CYP2E1 activity using a sensitive HLPC/EC based technique. A subject with the CYP2E1-c/t allele showed 2-fold higher CYP2E1 activity than subjects with the c/c allele (N=14). GSTP1 expression comprised 83% of the total pulmonary GSTs. However, neither the GSTP1 polymorphism, nor other lifestyle factors, such as age, gender, smoking status, were found to be associated with pulmonary GST expression. In conclusion, subjects with the ADH3*1 allele showed higher ADH activity and acetaldehyde-DNA adducts in lung than other subjects; thus, the ADH3*1 allele could be considered a risk factor for lung cancer.