Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China. (1/513)

BACKGROUND: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models. METHODS: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided. RESULTS: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682). CONCLUSIONS: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.  (+info)

Determinants of specificity for aflatoxin B1-8,9-epoxide in alpha-class glutathione S-transferases. (2/513)

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl-glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103-112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.  (+info)

Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of aspergillus flavus along a transect within the United States. (3/513)

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B1 and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 micrometer in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 micrometer in diameter. The S-strain isolates generally produced high levels of aflatoxin B1, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B1 production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B1 and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 microgram of aflatoxin B1 per ml. The percentages of isolates producing >10 microgram of aflatoxin B1 per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B1 or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.  (+info)

Potency of dietary indole-3-carbinol as a promoter of aflatoxin B1-initiated hepatocarcinogenesis: results from a 9000 animal tumor study. (4/513)

Indole-3-carbinol (I3C), a metabolite of glucobrassicin found in cruciferous vegetables, is documented as acting as a modulator of carcinogenesis and, depending on timing and dose of administration, it may promote hepatocarcinogenesis in some animal models. In this study we demonstrate that, when given post-initiation, dietary I3C promotes aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rainbow trout model at levels as low as 500 p.p.m. Trout embryos (approximately 9000) were initiated with 0, 25, 50, 100, 175 or 250 p.p.b. AFB1 by a 30 min immersion. Experimental diets containing 0, 250, 500, 750, 1000 or 1250 p.p.m. I3C were administered starting at 3 months and fish were sampled for liver tumors at 11-13 months. Promotion at the level of tumor incidence was statistically significant for all dietary levels, except 250 p.p.m. Relative potency for promotion markedly increased at dietary levels >750 p.p.m. We propose that more than one mechanism could be involved in promotion and that both estrogenic and Ah receptor-mediated pathways could be active. The estrogenicity of I3C, measured as its ability to induce vitellogenin (an estrogen biomarker in oviparous vertebrates) was evident at the lowest dietary level (250 p.p.m.), whereas CYPIA (a P450 isozyme induced through the Ah receptor pathway) was not induced until dietary levels of 1000 p.p.m. Therefore, at lower dietary levels, promotion by I3C in this model could be explained by estrogenic activities of I3C acid derivatives, as it is known that estrogens promote hepatocarcinogenesis in trout. Much stronger promotion was observed at high dietary I3C levels (1000 and 1250 p.p.m.), at which levels both CYP1A and vitellogenin were induced.  (+info)

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells. (5/513)

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.  (+info)

Divergence of West African and North American communities of Aspergillus section Flavi. (6/513)

West African Aspergillus flavus S isolates differed from North American isolates. Both produced aflatoxin B1. However, 40 and 100% of West African isolates also produced aflatoxin G1 in NH4 medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G1. This geographical and physiological divergence may influence aflatoxin management.  (+info)

Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1. (7/513)

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.  (+info)

XRCC1 polymorphisms: effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency. (8/513)

Hereditary genetic defects in DNA repair lead to increased risk of cancer. Polymorphisms in several DNA repair genes have been identified; however, the impact on repair phenotype has not been elucidated. We explored the relationship between polymorphisms in the DNA repair enzyme, XRCC1 (codons 194, 280, and 399), and genotoxic end points measured in two populations: (a) placental aflatoxin B1 DNA (AFB1-DNA) adducts in a group of Taiwanese maternity subjects (n = 120); and (b) somatic glycophorin A (GPA) variants in erythrocytes from a group of North Carolina smokers and nonsmokers (n = 59). AFB1-DNA adducts were measured by ELISA, and erythrocyte GPA variant frequency (NN and NO) was assessed in MN heterozygotes with a flow cytometric assay. XRCC1 genotypes were identified by PCR-RFLPs. The XRCC1 399Gln allele was significantly associated with higher levels of both AFB1-DNA adducts and GPA NN mutations. Individuals with the 399Gln allele were at risk for detectable adducts (odds ratio, 2.4; 95% confidence interval, 1.1-5.4; P = 0.03). GPA NN variant frequency was significantly higher in 399Gln homozygotes (19.6 x 10(-6)) than in Gln/Arg heterozygotes (11.4 x 10(-6); P < 0.05) or Arg/Arg homozygotes (10.1 x 10(-6); P = 0.01). No significant effects were observed for other XRCC1 polymorphisms. These results suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair.  (+info)

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