Inhibition of ebselen on aflatoxin B(1)-induced hepatocarcinogenesis in Fischer 344 rats.
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Aflatoxin B(1) (AFB(1)), a potent hepatocarcinogen, enhances ROS formation and causes oxidative DNA damage, which may play a role in its carcinogenicity. We have demonstrated recently that ebselen, an organic selenium compound, protects against the cytotoxicity of AFB(1) through its antioxidant capability. The present study was designed to investigate the effect of ebselen on AFB(1)-induced hepatocarcinogenesis in an animal model. Fischer 344 rats were first treated with either deionized water or ebselen (5 mg/kg, 5 days/week) via gavage for 4 weeks, then given AFB(1) (0.4 mg/kg, gavage, once a week) or AFB(1) plus ebselen (5 mg/kg, 5 days/week) for another 24 weeks. The results showed that the hepatocarcinogenicity of AFB(1) in rats was significantly reduced by ebselen treatment as indicated by a decrease in: (i) serum gamma-glutamyl transpeptidase activity; (ii) expression of mRNAs of liver alpha-fetoprotein and the placental form of glutathione S-transferase (GST-P); and (iii) the area and mean density of staining of liver GST-P foci. Ebselen treatment significantly reduced the formation of hepatic AFB(1)-DNA adducts and 8-hydroxydeoxyguanosine caused by AFB(1) exposure. These findings suggest that ebselen can inhibit the carcinogenicity of AFB(1). In addition to the reduction of AFB(1)-DNA adduct formation, the protective effect of ebselen against AFB(1)-induced oxidative DNA damage may also, at least in part, contribute to its anticarcinogenic property. (+info)
Cytochrome P450 expression and related metabolism in human buccal mucosa.
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Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies. (+info)
The chemistry and biology of aflatoxin B(1): from mutational spectrometry to carcinogenesis.
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Dietary exposure to aflatoxin B(1) (AFB(1)) is associated with an increased incidence of hepatocellular carcinoma (HCC), especially in populations in which exposure to hepatitis B virus (HBV) is a common occurrence. Most HCC samples from people living where HBV is prevalent have one striking mutational hotspot: a GC-->TA transversion at the third position of codon 249 of the p53 gene. In this review, the chemical reaction of an electrophilic derivative of aflatoxin with specific DNA sequences is examined, along with the types of mutations caused by AFB(1) and the sequence context dependence of those mutations. An attempt is made to assign the source of these mutations to specific chemical forms of AFB(1)-DNA damage. In addition, epidemiological and experimental data are examined regarding the synergistic effects of AFB(1) and HBV on HCC formation and the predominance of one hotspot GC-->TA transversion in the p53 gene of affected individuals. (+info)
Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies.
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Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer. (+info)
Hepatitis B, aflatoxin B(1), and p53 codon 249 mutation in hepatocellular carcinomas from Guangxi, People's Republic of China, and a meta-analysis of existing studies.
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The incidence of hepatocellular carcinomas (HCC) varies widely worldwide, with some of the highest incidence rates found in China. Chronic infection with the hepatitis B virus (HBV) and exposure to aflatoxins in foodstuffs are the main risk factors. A G to T transversion at codon 249 of the p53 gene (249(ser)) is commonly found in HCCs from patients in regions with dietary aflatoxin exposure. Because HBV infection is often endemic in high aflatoxin exposure areas, it is still unclear whether HBV acts as a confounder or as a synergistic partner in the development of the 249(ser) p53 mutation. Our report has two aims. First, we contribute data on HCCs from southern Guangxi, a high aflatoxin exposure area. Using DNA sequencing, we found that 36% (18 of 50) of tumors had a 249(ser) mutation. Also, 50% (30 of 60) were positive for p53 protein accumulation and 78% (28 of 36) were positive for HBV surface antigen, as detected by immunohistochemistry. Second, we present a meta-analysis, using our results along with those from 48 published studies, that examines the interrelationships among aflatoxin exposure, HBV infection, and p53 mutations in HCCs. We used a method that takes into account both within-study and study-to-study variability and found that the mean proportion of HCCs with the 249(ser) mutation was positively correlated with aflatoxin exposure (P = 0.0001). We found little evidence for an HBV-aflatoxin interaction modulating the presence of the p53 249(ser) mutation or any type of p53 mutation. (+info)
Surface binding of aflatoxin B(1) by lactic acid bacteria.
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Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B(1) complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B(1) remained bound. Nonviable bacteria retained the highest amount of aflatoxin B(1). Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B(1) from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B(1) to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B(1). Variation in temperature (4 to 37 degrees C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B(1) released. Binding of aflatoxin B(1) appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions. (+info)
Carnitine alters binding of aflatoxin to DNA and proteins in rat hepatocytes and cell-free systems.
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The objective of this study was to determine effects of L-carnitine on aflatoxin B(1) (AFB(1))-DNA adduct formation in isolated rat hepatocytes, its dose response, specificity and mode of action. All experiments were conducted in either freshly isolated rat hepatocytes or cell-free systems. There was negative linear correlation between the dosage of carnitine and formation of [(3)H]AFB(1)-DNA adducts in the hepatocytes; however, the partitioning of AFB(1) into cellular compartments was not affected by carnitine. The attenuating effect of carnitine on AFB(1)-DNA adduct formation was also present in a cell-free system, but there was lack of specificity because acetylcarnitine and gamma-aminobutyric acid (GABA) were equally effective. Carnitine appears to interfere with bioactivation of AFB(1) and binding of AFB(1)-epoxide to DNA. On the contrary, carnitine enhanced the binding of AFB(1) and its epoxide to microsomal proteins, plasma proteins and bovine serum albumin. These results indicate that carnitine diverts AFB(1)-epoxide away from DNA by promoting binding to proteins. We conclude that modulation of AFB(1) binding to proteins and DNA by carnitine alters the carcinogenic and hepatotoxic potential of AFB(1) and poses concerns about the human AFB(1)-exposure data based on the AFB(1)-albumin adduct concentrations as a biomarker. (+info)
Genetic polymorphisms of glutathione S-transferases M1 and T1 associated with susceptibility to aflatoxin-related hepatocarcinogenesis among chronic hepatitis B carriers: a nested case-control study in Taiwan.
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This study was conducted to investigate the modifying effect of glutathione S-transferase (GST) M1 and T1 polymorphisms on aflatoxin-induced hepatocarcinogenesis among chronic hepatitis B virus surface antigen (HBsAg) carriers. A total of 79 HBsAg-positive cases of hepatocellular carcinoma (HCC) diagnosed between 1991 and 1997 were identified and individually matched to one or two HBsAg-positive controls on age, gender, residence and date of recruitment from the same cancer screening cohort in Taiwan. Blood samples were tested for hepatitis B and C viral markers by enzyme immunoassay and for aflatoxin B(1) (AFB(1))-albumin adducts by competitive enzyme-linked immunosorbent assay. GSTM1 and GSTT1 genotypes were determined by PCR. There was a statistically significant relationship between detectable levels of AFB(1)-albumin adducts in serum and risk of HCC among chronic HBsAg carriers, with an adjusted odds ratio (OR) of 2.0 [95% confidence interval (CI) 1.1-3.7]. In addition, the effect of aflatoxin exposure on HCC risk was more pronounced among chronic HBsAg carriers with the GSTT1 null genotype (OR 3.7, 95% CI 1.5-9.3) than those who were non-null (OR 0.9, 95% CI 0.3-2.4). The interaction between serum AFB(1)-albumin adduct level and GSTT1 genotype was statistically significant (P = 0.03). For GSTM1 the effect of aflatoxin exposure on HCC risk in those with the null genotype was also greater (adjusted OR 2.8, 95% CI 1.0-7.8) than in those with the gene present (adjusted OR 1.8, 95% CI 0.8-4.5), but the difference was not significant (P = 0.91). Notably, when the interaction between aflatoxin exposure and GSTT1 genotype was considered, aflatoxin exposure by itself was not a significant determinant of HCC risk among chronic HBsAg carriers. These results demonstrate the importance of gene-environment interactions in the multifactorial development of HCC. (+info)