The role of human glutathione S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the effect of a polymorphism in hGSTA1 on colorectal cancer risk.

Food-derived heterocyclic amines (HCAs), particularly 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are implicated in the etiology of human colorectal cancer (CRC) via a process of N-oxidation followed by O-acetylation or O-sulfation to form electrophilic metabolites that react with DNA. Glutathione S-transferases (GSTs) detoxify activated carcinogen metabolites by catalysis of their reaction with GSH. However, among HCAs, only N-acetoxy-PhIP has been shown to be a substrate for the GSTs. By using a competitive DNA-binding assay, we confirm that hGSTA1-1 is an efficient catalyst of the detoxification of N-acetoxy-PhIP. Further, we show that hGSTs A2-2, P1-1, M1-1, T1-1 and T2-2 appear to have low activity towards N-acetoxy-PhIP, and that hGSTs A4-4, M2-2, M4-4 and Z1-1 appear to have no activity towards N-acetoxy-PhIP. A genetic polymorphism in the 5'-regulatory sequence of hGSTA1 has been shown to correlate with the relative and absolute levels of expression of GSTA1/GSTA2 in human liver. Examination of hGSTA1 allele frequency in 100 Caucasian CRC patients and 226 Caucasian controls demonstrated a significant over-representation of the homozygous hGSTA1*B genotype among cases compared to controls (24.0 and 13.7%, respectively, P=0.04). This corresponds to an odds ratio for risk of CRC of 2.0 (95% CI 1.0-3.7) when comparing homozygous hGSTA1*B individuals with all other genotypes. Thus, individuals who are homozygous hGSTA1*B, and who would be predicted to have the lowest levels of hGSTA1 expression in their livers, appear to be at risk of developing CRC, possibly as a result of inefficient hepatic detoxification of N-acetoxy-PhIP.

A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols.

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

Modulation of endocrine pathways by 4,4'-DDE in the deer mouse Peromyscus maniculatus.

4,4'-DDT and 4,4'-DDE are widespread environmental contaminants that cause eggshell thinning in birds, altered sex ratios in the American alligator, and changes in the anal-genital distance in rodents. These contaminants are known to cause some of their toxicity by altering steroid receptor-mediated mechanisms. However, chemical-specific alterations in the expression of hormone-metabolizing enzymes may also be a mechanism for endocrine disruption, by altering the half-life of hormones in critical tissues. Previously, we showed that 4,4'-DDE causes a dose-dependent increase in ethoxyresorufin-O-deethylase (EROD) activity, but not pentoxyresorufin-O-dealkylase (PROD) activity, in the deer mouse. In this study, we demonstrated that 4,4'-DDE elicited a corresponding increase in CYP1A protein expression but not CYP2B using Western blotting and immunoprecipitation. 4,4'-DDE-mediated changes in phase II conjugating enzymes; UDP-glucuronosyltransferase (UGT) and phenolsulfotransferase (ST), were also investigated for the first time. Prepubescent female deer mice were dosed with 4,4'-DDE by gavage on days 1 and 2, then euthanized on day 4. As anticipated, dose-dependent increases in hepatic EROD and MROD activities, but not PROD or BROD, were observed. UGT activity was monitored by incubating liver microsomes and 14C-UDP-GA with potential substrates and measuring incorporation of radioactivity into TLC-resolved glucuronides. Dose-dependent increases in conjugation were observed with p-nitrophenol (a general UGT substrate) but not testosterone. Interestingly, a biphasic dose-response curve was observed for ST activity, with a peak at the 3 mg/kg dose. Dose-dependent increases in CYP1A1 and UGT-specific immunoreactive proteins were observed, suggesting de novo synthesis as a consequence of 4,4'-DDE exposure. We also measured Phase I and II enzymes in deer mouse platelets. Preliminary results indicate that the 4,4'-DDE-induced changes in liver Phase I and II enzyme activity were similar, but not identical, to those found in platelets. These results indicate that environmentally-relevant levels of 4,4'-DDE modulate the activity and expression of CYP1A1 and phase II enzymes in the deer mouse and that certain changes may be measured non-lethally.

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

Identification of enzymes responsible for the metabolism of heme in human platelets.

The major enzymes involved in the degradation of heme were identified in human platelets. It was determined that heme oxygenase activity levels in umbilical cord blood platelets were higher, whereas biliverdin reductase activity levels were comparable with that found in platelets from adults. In membranes prepared from adenosine diphosphate-activated platelets, UDP-glucuronic acid-dependent bilirubin conjugation was detected, whereas activity was negligible in unactivated platelets and undetected in serum and heat-inactivated platelets, and in platelets prepared from umbilical cord blood. Platelet fractions were analyzed by Western blot and shown to express heme oxygenase, biliverdin reductase, and UDP-glucuronosyltransferases, and there was concordance with known developmental profiles found in other tissues. Heme oxygenase expression was higher, whereas UGT expression was lower, in neonatal compared with adult platelets. These data suggest that platelets are involved in multiple steps of heme and bilirubin metabolism and that developmental regulation of these enzymes may be similar to that in other human tissues.