Autism is a behaviorally defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G>> A), transcobalamin II (TCN2 776G>> C), catechol-O-methyltransferase (COMT 472G>> A), methylenetetrahydrofolate reductase (MTHFR 677C>> T and 1298A>> C), and glutathione-S-transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.

Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N:-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 micromol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 micromol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100-116 (P < 0.01) and multicrypt foci from 52 to 27-38 (more than four crypts/focus; P < 0.05) during the post-initiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109-115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.

o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e. the neurodegenerative processes underlying Parkinson's disease and schizophrenia. The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2. The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1. Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library. Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines. In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases.

The rise in childhood asthma prevalence suggests a role for environmental factors in the etiology of this evolving epidemic; however, genetics also influence the occurrence of asthma. Glutathione S-transferase (GST) M1 may play a role in asthma and wheezing occurrence among those exposed to tobacco smoke, as it functions in pathways involved in asthma pathogenesis such as xenobiotic metabolism and antioxidant defenses. Effects of GSTM1 genotype, maternal smoking during pregnancy, and childhood environmental tobacco smoke (ETS) exposure on asthma and wheezing were investigated in 2,950 children enrolled in 4th, 7th, and 10th grade classrooms in 12 Southern California communities. The effects of in utero exposure to maternal smoking on asthma and wheezing occurrence were largely restricted to children with GSTM1 null genotype. Among GSTM1 null children, in utero exposure was associated with increased prevalence of early onset asthma (odds ratio [OR] 1.6, 95% confidence interval [CI] 1.0-2.5), asthma with current symptoms (OR 1.7, 95% CI 1.1-2.8), persistent asthma (OR 1.6, 95% CI 1.1-2.4), lifetime history of wheezing (OR 1.8, 95% CI 1.3-2.5), wheezing with exercise (OR 2.1, 95% CI 1.3-3.3), wheezing requiring medication (OR 2.2, 95% CI 1.4-3.4), and emergency room visits in the past year (OR 3.7, 95% CI 1.9-7.3). Among children with GSTM1 (+) genotype, in utero exposure was not associated with asthma or wheezing. Our findings indicate that there are important long-term effects of in utero exposure in a genetically susceptible group of children.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the "AhR gene battery." However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 mug/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid-synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal GstGsta1, as well as most Ugt and Gst isoforms in livers of mice.

Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

The A-G polymorphism at codon 104 in the glutathione S-transferase P1 (GSTP1) gene was examined in 138 male lung cancer patients and 297 healthy controls. The patients had significantly higher frequency of the GG genotype (15.9%) and a lower frequency of AA (38.4%) than the controls (9.1% and 51.5%, respectively). The level of hydrophobic DNA-adducts were determined in lung tissue from 70 current smokers. Patients with the GG genotype had a significantly higher adduct level than patients with AA (15.5 +/- 10.2 vs 7.9 +/- 5.1 per 10(8) nucleotides, P = 0.006). We also analyzed the deletion polymorphism in the GSTM1 gene in 135 male patients and 342 controls. The patients were stratified according to histology, smoking dose, age, adduct level and mutational types found in the tumors (Ki-ras and p53 genes). The results consistently indicated that the GSTM1 null genotype was associated with a slightly increased lung cancer risk. When the combined GST M1 and P1 genotypes were examined, patients with the combination null and AG or GG had significantly higher adduct levels than all other genotype combinations (P = 0.011). The distribution of combined genotypes was also significantly different in cases and controls, mainly due to increased frequency of the combination GSTM1 null and GSTP1 AG or GG among patients.

The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to elucidate the relationships among arsenic methylation capacity, body retention, and genetic polymorphisms of glutathione S-transferase (GST) M1 and T1, a total of 115 study subjects were recruited from Lanyang Basin located on the northeast coast of Taiwan. Specimens of drinking water, blood, urine, hair and toenail were collected from each study subject. Urinary inorganic and methylated arsenic were speciated by high performance liquid chromatography combined with hydride-generation atomic absorption spectrometry. Arsenic concentration in hair and toenail were quantitated by atomic absorption spectrophotometry. The polymerase chain reaction was used to determine genetic polymorphisms of GST M1 and T1. Arsenic concentrations in urine, hair, and toenail of study subjects were positively correlated with arsenic levels in their drinking water. Percentages of various arsenic species in urine (mean +/- standard error (SE) were 11.8 +/- 1.0, 26.9 +/- 1.2 and 61.3 +/- 1.4, respectively, for inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Men and women had similar arsenic methylation capability. No associations were observed between arsenic methylation capability and arsenic content in either drinking water or urine. Ratios of arsenic contents in hair and toenail to urinary arsenic content (mean +/- standard error) were 6.2 +/- 0.7 and 16.5 +/- 1.7, respectively. Genetic polymorphisms of GST M1 and T1 were significantly associated with arsenic methylation. Subjects having the null genotype of GST M1 had an increased percentage of inorganic arsenic in urine, while those with null genotype of GST T1 had an elevated percentage of DMA in urine. Arsenic contents in hair and toenail were significantly correlated with the increase in arsenic concentrations of drinking water and urine, while no significant associations were observed between arsenic contents in hair and toenail and polymorphisms of GST M1 and T1. The relationship between arsenic methylation capability and body retention was modified by genetic polymorphisms of GST M1 and T1. Arsenic contents in hair and toenail were negatively associated with MMA percentage and positively associated with DMA percentage among subjects having null genotypes of GST M1 and T1, but not among those with non-null genotypes.

Tobacco smoking and occupation are major risk factors of bladder cancer via exposure to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. Glutathione S-transferase (GST) M1, T1 and P1 are involved in the detoxification of PAH reactive metabolites. Two N-acetyltransferase isozymes, NAT2 and NAT1, have major roles in catalyzing the N-acetylation and O-acetylation of aromatic amines. Cytochrome p450 1B1 (CYP1B1) and sulfotransferase 1A1 (SULT1A1) are also involved in the metabolism of PAHs and aromatic amines. It is hypothesized that the genetic polymorphisms of these metabolic enzymes have an effect on the individual susceptibility to bladder cancer in particular by interacting with relevant environmental exposures. A hospital-based case-control study among men in Brescia, Northern Italy recruited 201 incidence cases and 214 controls from 1997-2000. Occupational exposures were blindly coded by occupational physicians. Genotyping of polymorphisms were carried out with PCR-RFLP method. Unconditional multivariate logistic regression was applied to model the association between genetic polymorphisms and bladder cancer risk. Effect modifications by age of onset, smoking and occupational exposures to PAHs and aromatic amines were evaluated. We also conducted an analysis of interaction between genetic factors. GSTM1 and GSTT1 null genotype were associated with an increased risk of bladder cancer with an odds ratio (OR) of 1.69 (95% confidence interval [CI] = 1.11-2.56) and 1.74 (95% CI = 1.02-2.95), respectively. The effect of GSTM1 null was seen particularly in heavy smokers, and there was a combined effect with occupational exposure of aromatic amines (OR = 2.77, 95% CI = 1.08-7.10). We observed a trend (p-value < 0.01) of increasing cancer risk comparing subjects with normal GSTM1 and T1 activity to subjects with one (OR = 1.82, 95% CI = 1.16-2.85) or both null genotypes (OR = 2.58, 95% CI = 1.27-5.23). NAT2 slow acetylator was associated with marginally increased risk of bladder cancer (OR = 1.50, 95% CI = 0.99-2.27), and the OR for the joint effect with occupational exposure of aromatic amines was 3.26 (95% CI = 1.06-9.95). SULT1A1 Arg213His polymorphism showed a marginal protective effect. These findings suggest that individual susceptibility to bladder cancer may be modulated by GSTM1, GSTT1 and NAT2 polymorphisms.

It has become clear that several polymorphisms of human drug-metabolizing enzymes influence an individual's susceptibility for chemical carcinogenesis. This review gives an overview on relevant polymorphisms of four families of drug-metabolizing enzymes. Rapid acetylators (with respect to N-acetyltransferase NAT2) were shown to have an increased risk of colon cancer, but a decreased risk of bladder cancer. In addition an association between a NAT1 variant allele (NAT*10, due to mutations in the polyadenylation site causing approximately two fold higher activity) and colorectal cancer among NAT2 rapid acetylators was observed, suggesting a possible interaction between NAT1 and NAT2. Glutathione S-transferases M1 and T1 (GSTM1 and GSTT1) are polymorphic due to large deletions in the structural gene. Meta-analysis of 12 case-control studies demonstrated a significant association between the homozygous deletion of GSTM1 (GSTM1-0) and lung cancer (odds ratio: 1.41; 95% CI: 1.23-1.61). Combination of GSTM1-0 with two allelic variants of cytochrome P4501A1 (CYP1A1), CYP1A1 m2/m2 and CYP1A1 Val/Val further increases the risk for lung cancer. Indirect mechanisms by which deletion of GSTM1 increases risk for lung cancer may include GSTM1-0 associated decreased expression of GST M3 and increased activity of CYP1A1 and 1A2. Combination of GST M1-0 and NAT2 slow acetylation was associated with markedly increased risk for lung cancer (odds ratio: 7.8; 95% CI: 1.4-78.7). In addition GSTM1-0 is clearly associated with bladder cancer and possibly also with colorectal, hepatocellular, gastric, esophageal (interaction with CYP1A1), head and neck as well as cutaneous cancer. In individuals with the GSTT1-0 genotype more chromosomal aberrations and sister chromatid exchanges (SCEs) were observed after exposure to 1,3-butadiene or various haloalkanes or haloalkenes. Evidence for an association between GSTT1-0 and myelodysplastic syndrome and acute lymphoblastic leukemia has been presented. A polymorphic site of GSTP1 (valine to isoleucine at codon 104) decreases activity to several carcinogenic diol epoxides and was associated with testicular, bladder and lung cancer. Microsomal expoxide hydrolase (mEH) is polymorphic due to amino acid variation at residues 113 and 139. Polymorphic variants of mEH were associated with hepatocellular cancer (His-113 allele), ovarian cancer (Tyr-113 allele) and chronic obstructive pulmonary disease (His-113 allele). Three human sulfotransferases (STs) are regulated by genetic polymorphisms (hDHEAST, hM-PST, TS PST). Since a large number of environmental mutagens are activated by STs an association with human cancer risk might be expected.

Foreign compound-metabolizing enzymes may modify the risk of chemically induced cancer. We wanted to examine enzymes with putative relevance in urinary bladder cancer using molecular genetic analyses of heritably polymorphic enzymes. Arylamine N-acetyltransferase (NAT2); glutathione S-transferases (GSTs) M1 and T1; microsomal epoxide hydrolase; and cytochrome P-450 enzymes (CYP) 1A1, 2C19, 2D6, and 2E1 were analyzed in 374 cases and in 373 controls in a hospital-based case-control study in Berlin. Slow acetylation was a significant risk factor in heavy smokers [odds ratio (OR), 2.7; 95% confidence interval (CI), 1.0-7.4], with the greatest risk noted for the allele NAT2*5B. GSTM1 deficiency was a risk factor independent of smoking and occupation (OR, 1.6; CI, 1.2-2.2). GSTT1 was associated with cancer risk in the nonsmoker subgroup (OR, 2.6; CI, 1.1-6.0). The two amino acid polymorphisms that are known in microsomal epoxide hydrolase were not associated with bladder cancer risk. CYP2D6 activity was rejected as a risk factor by phenotyping and by detailed molecular genetic analyses. CYP2C19 may have a role in bladder cancer risk, but polymorphisms in CYP1A1 and 2E1 had no statistically significant impact. Deficiencies in both NAT2 and GSTM1 failed to show significant synergistic or antagonistic interactions. In conclusion, molecular genetic analysis of a large sample specified the increased bladder cancer risk of those who are deficient in NAT2 and GSTM1; the other traits proved to be of minor impact.

BACKGROUND

It has been suggested that the genetically determined deficiency of glutathione S transferase (GST) enzymes involved in the detoxification of environmental tobacco smoke (ETS) components may contribute to the development of asthma.

METHODS

A large population of German schoolchildren (n = 3054) was genotyped for deficiencies of the GST isoforms M1 and T1. The association between GSTM1 and GSTT1 genotypes and asthma as well as atopy was investigated with respect to current and in utero ETS exposure.

RESULTS

In children lacking the GSTM1 allele who were exposed to current ETS the risk for current asthma (OR 5.5, 95% CI 1.6 to 18.6) and asthma symptoms such as wheeze ever (OR 2.8, 95% CI 1.3 to 6.0), current wheezing (OR 4.7, 95% CI 1.8 to 12.6) and shortness of breath (OR 8.9, 95% CI 2.1 to 38.4) was higher than in GSTM1 positive individuals without ETS exposure. Hints of an interaction between ETS exposure and GSTM1 deficiency were identified. In utero smoke exposure in GSTT1 deficient children was associated with significant decrements in lung function compared with GSTT1 positive children not exposed to ETS.

CONCLUSIONS

GSTM1 and GSTT1 deficiency may increase the adverse health effects of in utero and current smoke exposure.

Genes coding for the glutathione S-transferase M1 (GSTM1) and Theta 1 (GSTT1) proteins are polymorphic in humans and these genes are absent, or homozygous null, in 10-60% of different ethnic populations. These enzymes catalyze the conjugation of glutathione to numerous carcinogenic chemicals and previous epidemiologic studies have associated the null genotypes of these GST genes with higher risk of cancer. In this study the frequency of GSTM1 and GSTT1 null genotypes was determined in Japanese patients with gastric adenocarcinoma and colorectal adenocarcinoma and compared to frequencies determined in a community-based control group. The frequency of the null GSTM1 genotype in patients with gastric adenocarcinoma (56.8%) showed a statistically significant increase compared to the control group frequency (43.6%) (odds ratio (OR) = 1.70; 95% CI, 1.05-2.76). The frequency of GSTM1 null individuals was also higher among all colorectal adenocarcinoma cases, but this increase did not reach statistical significance. After grouping by tumor site, the GSTM1 null genotype was a risk factor among the subgroup with distal colorectal tumors (61.1%) (OR = 2.03; 95% CI, 1.06-3.90). No consistent difference was observed between smoking patients and corresponding controls for the frequency of the GSTM1 null genotype for either cancer, although a large risk (OR = 5.76; 95% CI 1.18-28.3) was associated with the GSTM1 null genotype in the low smoking group of gastric adenocarcinoma patients. On the other hand, no statistically significant differences were observed in the frequency of null GSTT1 genotypes in gastric (47.5%) or colorectal (48.5%) adenocarcinoma patients when compared with the control population (44.4%). These results suggest that the GSTM1 null genotype may be associated with susceptibility to gastric adenocarcinoma and distal colorectal adenocarcinoma in Japanese; however, the associations observed were relatively weak and additional studies will be needed to confirm these findings.

The effects of glutathione-S-transferase (GST) M1, GSTT1, and GSTP1 genotypes on lung function growth were investigated in 1,940 children enrolled in the Children's Health Study as fourth graders (aged 8-11 years) in two cohorts during 1993 and 1996 and were followed annually over a 4-year period. Genotypes for GSTM1 and GSTT1 and GSTP1 codon 105 variants (ile105 and val105) were determined using DNA from buccal cell specimens. We used two-level regression models to estimate the effects of GSTM1, GSTT1, and GSTP1 genotypes on the adjusted annual average lung function growth. GSTM1 null was associated with deficits in annual growth rates for FVC (-0.21%; 95% confidence interval [CI], -0.40, -0.03) and FEV(1) (-0.27%; 95% CI, -0.50, -0.04). Children who were homozygous for the GSTP1 val105 allele had slower lung function growth (FVC -0.35%; 95% CI, -0.62, -0.07; and FEV(1) -0.34%; 95% CI, -0.68, 0.00) than children with one or more ile105 alleles. Children with asthma who were homozygous for the GSTP1 val105 allele had substantially larger deficits in FVC, FEV(1), and maximal mid-expiratory flow than children without asthma. The deficits in FVC and FEV(1) growth associated with both GSTM1 null and the GSTP1 val105 allele were largest and were statistically significant in non-Hispanic white children. We conclude that GSTM1 and GSTP1 genotypes are associated with lung function growth in school children.

Mounting evidence suggests that catechol metabolites of estradiol may contribute to the development of estrogen-induced cancers. O-Methylation, catalyzed by catechol-O-methyltransferase (COMT), inactivates catechol estrogens. COMT is polymorphic in the human population, with 25% of Caucasians being homozygous for a low activity allele of the enzyme (COMT(LL)). We hypothesized that low activity COMT may be a risk factor for human breast cancer and designed a PCR-based RFLP assay to determine COMT genotype in a cohort of 112 matched, nested case-control samples. In the total study population, the odds ratios for the association of breast cancer risk with COMT(HL) and COMT(LL) genotypes were 1.30 [confidence interval (CI), 0.66-2.58] and 1.45 (CI, 0.69-3.07), respectively. Postmenopausal COMT(LL) women had a greater than 2-fold increased risk of developing breast cancer [odds ratio (OR), 2.18; CI, 0.93-5.11]. The association of COMT(LL) with the development of postmenopausal breast cancer was stronger and statistically significant in those women with a body mass index >24.47 kg/m2 (OR, 3.58; CI, 1.07-11.98). When COMT(LL) was combined with either glutathione S-transferase (GST) M1 null or with GSTP1 Ile-105-Val/Val-105-Val (intermediate/low activity, respectively) genotypes, the risk for developing postmenopausal breast cancer was also significantly increased. Our findings suggest that the allele encoding low activity COMT may be an important contributor to the postmenopausal development of breast cancer in certain women.

Oxidative stress is believed to play an important role in the pathogenesis of smoking-induced chronic obstructive pulmonary disease. We hypothesized that polymorphisms of antioxidant genes glutathione S-transferase M1 (GSTM1), GSTT1, GSTP1, and heme oxygenase-1 (HMOX1) would be associated with susceptibility to accelerated decline of lung function in smokers. We genotyped 621 subjects (299 rapid decliners [change in forced expiratory volume in 1 second (DeltaFEV(1)) = -152 +/- 2.5 ml/year] and 322 nondecliners [DeltaFEV(1) = +15 +/- 1.5 ml/year]) selected from among smokers followed for 5 years in the National Heart, Lung, and Blood Institute Lung Health Study. Because genotype frequencies were different between ethnic groups, we limited the association study to 594 whites (286 rapid decliners and 308 nondecliners). None of the genotypes studied had a statistically significant effect on decline of lung function when analyzed separately. There was an association between rapid decline of lung function and presence of all three GST polymorphisms (odds ratio [OR] = 2.83; p = 0.03). A combination of a family history of chronic obstructive pulmonary disease with GSTP1 105Ile/Ile genotype was also associated with rapid decline of lung function (OR = 2.20; p = 0.01). However, due to the multiple comparisons that were made, these associations may represent type 1 error. There was no association between HMOX1 (GT)n alleles and the rate of decline in lung function in smokers.

The genes glutathione S-transferase M1 (GSTM1) (chromosome 1p13.3) and glutathione S-transferase T1 (GSTT1) (22q11.2) code for cytosolic enzymes glutathione S-transferase (GST)-mu and GST-theta, respectively, which are involved in phase 2 metabolism. Both genes may be deleted. There is geographic and ethnic variation in genotype frequencies for both genes. In developed countries, colorectal cancer is the second most common cancer. Colorectal cancer has been inconsistently associated with polycyclic aromatic hydrocarbons in diet and tobacco. Because GST enzymes are involved in polycyclic aromatic hydrocarbon metabolism, it has been postulated that genotype may modify colorectal cancer risk associated with polycyclic aromatic hydrocarbon exposure. No consistent associations between GSTM1 or GSTT1 genotype and colorectal cancer have been observed. However, most studies have methodological limitations. Few have investigated gene-environment interactions. No interactions between GSTM1 or GSTT1 genotype and smoking and colorectal cancer risk have been reported. One polyp study suggests an interaction between GSTM1 genotype and smoking. Two studies suggest increased disease risk in subjects with high meat intake and GST nonnull genotype, contrary to the underlying hypothesis. One study suggests a strong inverse relation between colorectal adenomas and broccoli consumption, particularly in subjects who are GSTM1 null. These finding require confirmation. Methods for determining GSTM1 and GSTT1 genotype are well established. Population testing is not currently justified.

Associations between genetic polymorphisms of CYP1A1, CYP1A2, CYP2E1, GSTM1 and GSTT1 and prostate cancer (PCa) were analyzed in a case-control study of 315 individuals. The frequency of valine (Val)/valine (Val) genotypes for CYP1A1 was 11.3% in cases compared with 5.5% in controls, this polymorphism thus being associated with a significantly increased risk of PCa (odds ratio=2.4, 95% confidence interval (CI)=1.01-5.57). No links were detected between PCa and polymorphisms in other enzymes. However, the combination of CYP1A1 (Ile/Val and/or Val/Val) polymorphisms with the GSTM1 null type resulted in an OR of 2.2 (CI=1.10-4.57, 1.12-4.20, respectively). This study suggests that the CYP1A1 polymorphism and its combination with GSTM1 may be associated with PCa susceptibility in the Japanese population.

Human glutathione S-transferase (GST) M1 and T1 enzymes exhibit genetic polymorphism, with a percentage of normal individuals exhibiting a homozygous deletion of the relevant genes. We established a differential polymerase chain reaction (PCR) technique to simultaneously characterize inactivating mutations responsible for the null alleles of GSTM1 and GSTT1. Primers for GSTM1,GSTT1,and for beta-globin (as a positive control) were used to simultaneously amplify all three gene products from leukocyte DNA from 416 normal healthy human volunteers. Identical GSTM1 and CSTT1 genotypes were obtained using nine samples processed either separately or simultaneously for GSTM1 and GSTT1. The frequency of the null genotype for GSTM1 was higher in whites (114/213 or 53.5% vs 56/203 or 27.6%, p < 0.001) and for GSTT1 was higher in blacks (49/203 or 24.1% vs 32/213 or 15.0%, p = 0.019). The observed frequency of the 'double null' genotype for both GSTM1 and GSTT1 was not significantly different from that predicted if both polymorphisms were independent (p = 0.102) and did not differ by race (p = 0.120) or sex (p = 0.800). There was a higher frequency of the GSTM1 null genotype among females than males (92/202 or 45.5% vs 78/214 or 36.4%, p = 0.049). These results demonstrate that this PCR method is a simple and reliable tool to simultaneously characterize both GSTM1 and GSTT1 null genotypes.

An association between functional polymorphisms of genes resulting in decreased detoxification of carcinogens or DNA repair and aberrant promoter methylation is an attractive hypothesis in lung carcinogenesis. The genotypes at polymorphic sites of the glutathione S-transferase (GST) M1 (null/wildtype) and P1 (nucleotide 2627 A/G), myeloperoxidase (MPO) (nucleotide -463 G/A), X-ray repair cross-complementing group 1 (XRCC1) (nucleotides 26304 C/T; 28152 G/A), and NADPH quinine oxidoreductase (NQO1) (nucleotide 609 C/T) genes in 75 Chinese patients with non-small cell lung cancer (NSCLC) were characterized with polymerase chain reaction-restriction fragment length polymorphism. Results were correlated with aberrant methylation of the CDKN2A (alias p16(INK4A)), retinoic acid receptor beta (RARB), methylguanine-DNA methyltransferase (MGMT), and death-associated-protein (DAP) kinase genes in the tumors. In comparison with an age-matched control, none of the polymorphisms were associated with increased lung cancer risks. In male patients, however, the MPO -463 GG homozygous state was associated with CDKN2A (alias p16(INK4A)) methylation (odds ratio OR=3.63, 95% confidence interval CI=1.26-10.51), and the XRCC1 26304 T allele in the heterozygous/homozygous state was associated with methylation of CDKN2A (OR=6.13, 95% CI=1.55-24.16) and RARB (OR=7.67, 95% CI=1.62-36.18). In female patients, the GSTP1 G allele in the heterozygous/homozygous state was associated with RARB methylation (OR=18.0, 95% CI=0.76-427.29). These results showed that functional deficiencies in metabolic pathways that protect cells from carcinogen induced DNA damage might be linked to aberrant promoter methylation of the CDKN2A and RARB genes during lung carcinogenesis.

BACKGROUND

Cigarette smoking is associated with a twofold increased risk of pancreatic cancer. We conducted a population-based case-control study in six San Francisco Bay area counties from 1994 to 2001 to investigate associations between polymorphisms in genes for two carcinogen-metabolizing enzymes (cytochrome P450 1A1 [CYP1A1] and glutathione S-transferase [GST]), smoking, and adenocarcinoma of the exocrine pancreas.

METHODS

We used polymerase chain reaction-based methods to analyze blood samples obtained from 309 case subjects and 964 control subjects to determine their genotypes for three CYP1A1 polymorphisms (m1, m2, and m4) and for homozygous deletions of two GST genes, GSTM1 and GSTT1. Control subjects were frequency matched to case subjects by age and sex. All statistical tests were two-sided.

RESULTS

None of the genetic polymorphisms themselves affected the risk of pancreatic cancer among Caucasian study participants. However, we observed an interaction between GSTT1-null genotype and cigarette smoking among Caucasians that was more prominent among women than among men. Relative to never smokers with the GSTT1-present genotype, the age-adjusted odds ratios (ORs) of pancreatic cancer for heavy smokers with the GSTT1-null genotype were 5.0 (95% confidence interval [CI] = 1.8 to 14.5) for women and 3.2 (95% CI = 1.3 to 8.1) for men; for heavy smokers with the GSTT1-present genotype they were 2.0 (95% CI = 1.0 to 4.0) for women and 2.1 (95% CI = 1.1 to 3.9) for men. ORs for pancreatic cancer among heavy smokers with both GSTT1-null and GSTM1-null genotypes were similar in magnitude to those among heavy smokers with the GSTT1-null genotype alone. There was no evidence of an interaction between CYP1A1 polymorphisms and smoking.

CONCLUSIONS

The combination of heavy smoking and a deletion polymorphism in GSTT1 is associated with an increased risk of pancreatic cancer among Caucasians, with the associations possibly stronger in women than in men.