The mercapturic acid pathway metabolites of a glutathione conjugate of aflatoxin B1

A glutathione conjugate of aflatoxin B1 (AFB1) which has previously been identified as 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy aflatoxin B1 (AFB1-GSH) (E.J. Moss, D.J. Judah, M. Przybylski and G.E. Neal, Biochem. J., 210 (1983) 227-233) has been degraded in vitro to all of the intermediates of the mercapturic acid pathway (MAP) and the chromatographic and spectral characteristics of each of these compounds investigated. The cysteinylglycyl conjugate (AFB1-Cys.Gly) was prepared by incubating the AFB1-GSH conjugate with a rat hepatoma cell line rich in gamma-glutamyl-transpeptidase (GGT). Incubations of the AFB1-Cys.Gly conjugate with dipeptidase produced a metabolite, which was purified and characterized by 1H-NMR spectroscopy as 8,9-dihydro-8-(S-cysteinyl)-9-hydroxy aflatoxin B1 (AFB1-Cys). The N-acetyl derivative of the AFB1-Cys conjugate resulted from the incubation of the AFB1-GSH conjugate in vitro with isolated rat kidney cells. Mass spectral data were consistent with the compound being 8,9-dihydro-8-(S-cysteinyl-(N-acetyl))-9-hydroxy aflatoxin B1 (AFB1-Nac.Cys). A chromatographically identical compound was obtained by the chemical acetylation of AFB1-Cys.     

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.     

2-(Allylthio)pyrazine inhibition of aflatoxin B1-induced hepatocarcinogenesis in rats.

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has hepatoprotective effects against toxicants. Effect of 2-AP on hepatic tumorigenesis in association with glutathione S-transferase (GST) induction was examined in rats exposed to aflatoxin B1 (AFB1). Both AFB1-DNA adduct formation in the liver and urinary elimination of 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 (AFB1-N7-guanine) adduct were also determined. Male Sprague Dawley rats were treated with 2-AP at the daily oral doses of 10, 25 and 50 mg/kg for 16 consecutive days, during which four repeated doses of AFB1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-thirds of hepatectomy, followed by administration of phenobarbital (PB). Focal areas of hepatocellular alteration were identified after 44 days and preneoplastic foci expressing the placental form of glutathione S-transferase P (GST-P) were quantified by immunostaining of liver sections. 2-AP reduced the volume of liver occupied by GST-P foci by 65-96%. Under these experimental conditions, 2-AP treatment resulted in significant elevations in GST activity in the liver. Levels of radiolabeled AFB1 covalently bound to hepatic DNA, RNA and proteins were significantly reduced in rats treated with 2-AP for 5 days. 2-AP pretreatment also caused a 45% reduction in the urinary elimination of AFB1-N7-guanine adduct over the 24-h postdosing period. The present findings demonstrated that 2-AP exhibited protective effects against AFB1-induced hepatocarcinogenesis in rats with a marked decrease in the level of AFB1-DNA adduct. Reduction of hepatic DNA adducts might result from elevations of activity of GST, which catalyzes detoxification of the carcinogen.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

Dietary administration of 2(3)-t-butyl-4-hydroxyanisole elevates mouse liver microsome-mediated DNA binding and mutagenicity of aflatoxin B1

Administration of the phenolic antioxidant 2(3)-t-butyl-4-hydroxyanisole (BHA) to mice resulted in a 2-3-fold increase in the liver microsome catalyzed irreversible binding of aflatoxin B1 (AFB1) to calf thymus DNA and up to a 5-fold increase in the ability to induce mutations in Salmonella typhimurium TA98. Maximum induction of AFB1 binding to DNA occurred after 2 days of BHA administration whereas cytosolic glutathione S-transferase was maximally induced (6-fold) only after 10 days of BHA feeding. The induction of a new cytochrome P-450 species was indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an enhanced sensitivity to inhibition by metyrapone and alpha-naphthoflavone. Addition of control cytosol (containing glutathione S-transferase) + glutathione to control microsomes decreased AFB1 binding to DNA by 26%. However, replacement of control cytosol by BHA cytosol which contained 6 times more glutathione S-transferase only marginally enhanced the inhibition to 38%. These data suggest that BHA may exert its effect in the liver primarily through an alteration of the cytochrome P-450 dependent activation process although an increase in the conjugation of reactive metabolite may play a contributory role.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites.     

Caffeic acid phenethyl ester modulates aflatoxin B1‐induced hepatotoxicity in rats

Aflatoxin B1 (AFB1) is the most potent of the mycotoxins and is widely observed in nutrition abnormalities. There are some studies suggesting oxidative stress‐induced toxic changes on liver related to AFB1 toxicity. The aim of the present study was to evaluate whether antioxidant caffeic acid phenethyl ester (CAPE) relieves oxidative stress in AFB1‐induced liver injury in rat. Twenty‐four male rats were equally divided into three groups. The first group was used as a control. The second group received three doses of AFB1. The three doses of CAPE were given to constitute the third group with doses of AFB1. After 10 days of experiment, liver and serum samples were taken from all animals. Serum gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), glutathione s‐transferase (GST), nitric oxide (NO) and sulfhydryl values were higher in the AFB1 group than in control, whereas serum GGT, ALP, GST and NO values were decreased by in the AFB1 + CAPE group than in AFB1 group. Liver GST, total oxidant capacity, sulfhydryl, apoptosis index and ischemia‐modified albumin values were higher in the AFB1 group than in control, whereas the GST activity and apoptosis index were lower in the AFB1 + CAPE group than in the AFB1 group. There were histopathological degeneration and apoptosis in hepatocytes of AFB1 group. The findings were totally recovered by CAPE administration. In conclusion, we observed that AFB1 caused oxidative and nitrosative hepatoxicity to hepatocytes in the rat. However, CAPE induced protective effects on the AFB1‐induced hepatoxicity by modulating free radical production, biochemical values and histopathological alterations. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytotoxic effects of aflatoxin B1 and its association with cellular components in chicken embryo primary cultured cells.

We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.     

Comparison of the prevention of aflatoxin b(1)-induced genotoxicity by quercetin and quercetin pentaacetate

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B(1) (AFB(1)). We have in this report extended the observations to quercetin pentaacetate (QPA), which unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB(1) epoxidation as measured by AFB(1) binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB(1).     

Lack of effect of long-term glutathione administration on aflatoxin B1-induced hepatoma in male rats

The effect of long-term GSH administration on aflatoxin B1 (AFB1)-induced carcinogenesis in the livers of male Wistar II rats was evaluated. No significant effect of an 11 months period of reduced glutathione (GSH) administration was observed concerning both the survival curve and the incidence of liver tumors. Liver tissues of all animals were bearing tumors or nodular lesions 24 months after AFB1 treatment, regardless of GSH treatment. The capacity of the GSH conjugation system was elevated in the liver tissue of AFB1-treated animals both by an increase of GSH content and an increase of the specific activities of several GSH S-transferase isoenzymes. Likewise the specific activities of GSH related enzymes as GSSG reductase and gamma-glutamyltransferase (gamma-GT) and the activity of the GSH independent detoxication system NAD(P)H:quinone oxidoreductase were increased in the AFB1-treated livers, there was no significant effect of GSH treatment. These results demonstrate that long-term GSH treatment has no effect on the survival of AFB1-pretreated male rats on the incidence of liver tumors and on the activities of drug metabolizing systems. The hepatic detoxication capacity 24 months after AFB1 treatment is elevated.     

Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.     

An evaluation of the metabolic basis of aflatoxin B1 toxicity by using buffalo granulocytes and agranulocytes in vitro

This study was aimed at monitoring cytotoxic changes in buffalo leukocyte subpopulations exposed to aflatoxin B1 (AFB1), since no such information is available for this species. The effects of AFB1 on glutathione (GSH) S-transferase, Ca2+Mg2+ATPase and protein synthesis in leukocyte subpopulations, namely, mononuclear (MN) cells and polymorphonuclear (PMN) cells isolated from the blood of the domestic buffalo (Bos bubalis), were studied. The cells were separated by using Ficoll-Paque and incubated in the presence of AFB1. GSH S-transferase activity was found to increase in cells exposed to AFB1, but there was no difference in activity between MN and PMN cells. PMN cell ATPase activity increased after AFB1 treatment, whereas no such effect was observed in the MN cells, which showed higher basal levels of ATPase activity. In the presence of AFB1, all the cells showed significant decreases in 14C-leucine incorporation, but the MN cells showed higher 14C-leucine incorporation than the PMN cells. Nevertheless, both cell types were affected by AFB1 and participated in its detoxification. There was also an appreciable decrease in the release of myeloperoxidase by activated PMN cells in the presence of AFB1.     

Alleviation of aflatoxin B1-induced oxidative stress in HepG2 cells by volatile extract from Allii Fistulosi Bulbus

The volatile extract from Allii Fistulosi Bulbus (VEAF) was isolated by steam distillation under reduced pressure, followed by continuous liquid-liquid extraction, and its effects on aflatoxin B1 (AFB1)-induced oxidative stress were investigated in human hepatoma cells (HepG2). The main constituents of the VEAF, identified by gas chromatography/mass spectrometry, were 2-octyl-5-methyl-3(2H)-furanone, 2-hexyl-5-methyl-3(2H)-furanone, 2,5-dimethylthiophene, 3,5-diethyl-1,2,4-trithiolane and 3,4-dimethyl-2,5-dihydro-thiophene-2-one. VEAF significantly inhibited the formation of intracellular reactive oxygen species caused by AFB1 in a dose-dependent manner, concomitant with a significant decrease in the AFB1-induced cytotoxicity. VEAF pretreatment significantly reduced the levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation, whereas increased the level of reduced glutathione. The level of 8-hydroxy-2'-deoxyguanosine, a DNA oxidative stress marker, was also decreased by 49-59% with pretreatment of VEAF. With respect to the activity of AFB1 metabolizing enzymes, VEAF significantly increased the activity of glutathione S-transferase, and significantly decreased the cytochrome (CYP) P450 3A4 activity, but had a little effect on the CYP1As. These results suggest that VEAF may be selectively effective in alleviating the AFB1-induced oxidative stress, and lead to cytoprotection against AFB1 exposure.     

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.     

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.     

Enhancement of aflatoxin B1-induced hepatocarcinogenesis in rats by partial hepatectomy

The effect of enhanced cell replication induced by partial hepatectomy (PH) in aflatoxin B1 (AFB1)-induced hepatocarcinogenesis has been studied in rats of the inbred As2 strain. Animals were given 0.25 mg/kg body weight of AFB1 as a single intraperitoneal dose 24 h after PH. Non-hepatectomized animals given the same dose of AFB1 served as controls. Neoplastic nodules and hepatocellular carcinoma (HCC) were detected respectively in 100% and 90% of hepatectomized animals sacrificed between 55 and 65 weeks after AFB1 administration. None of the ten non-hepatectomized rats sacrificed at this time interval showed HCC or neoplastic nodules. On histochemical staining the tumour population was found to be heterogeneous. Thus PH resulted in enhancement of AFB1-induced hepatocarcinogenesis in rats of the AS2 strain.     

Reactivity of aflatoxin B2a antibody with aflatoxin B1-modified DNA and related metabolites.

Aflatoxin B2a (AFB2a) antiserum has been previously used in an enzyme-linked immunosorbent assay (ELISA) for the quantitation of AFB1 and AFB2a. The present investigation examined the reactivity of the antiserum toward those adducts and metabolites of AFB1 believed to play a major role in aflatoxicosis and carcinogenesis. 2,3-Dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua), the putative 2,3-(N5-formyl-2-2', 5',6'-triamino-4-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-FAPyr), 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol), AFB1-N7-Gua-modified DNA, and AFB1-FAPyr-modified DNA were prepared by in vitro incubation or chemical methods and subjected to competitive AFB2a ELISA. The antiserum showed significant reactivity with all five compounds, indicating that it had a high degree of specificity for both the cyclopentenone and the methoxy group of the parent aflatoxin molecule. Sensitivity for AFB-N7-Gua-modified DNA, AFB1-FAPyr-modified DNA, and AFB1-diol by the ELISA method was 0.1 pmol per assay. To test the applicability of immunological detection of covalent binding of AFB1 to DNA, the ELISA was compared with a conventional radioisotopic assay in two in vitro studies. The results showed that estimates of the kinetics and substrate dependence of covalent binding to calf thymus DNA in rat microsomal incubation mixtures by both methods were comparable. The broad specificity AFB2a antibody might be of considerable value in the detection of AFB1 macromolecular adducts and related metabolites in epidemiological investigations or in the diagnosis of aflatoxicosis.     

Metabolic basis of the species difference to aflatoxin B1 induced hepatotoxicity

Primary metabolism of aflatoxin B1 by the liver microsomal enzymes from a range of animal species showed both quantitative and qualitative differences. Quail was shown to have the most rapid metabolism of aflatoxin B1. The major product of metabolism in this case was found to be aflatoxin B1-8,9-dihydrodiol suggesting that the quail microsomes produced high levels of the proposed reactive intermediate aflatoxin B1-8,9-epoxide. Using this system to generate the epoxide, the ability of the cytosol prepared from each species to conjugate epoxide with reduced glutathione was investigated. Large differences in ability to conjugate were observed ranging from 0 to 72% for quail and mouse respectively. Differences in both primary and secondary metabolism of AFB1 were noted between male and female Fischer 344 rats.     

Mechanisms involved in the chemoprevention of flavonoids

Flavonoids, widespread in edible plants, have been studied extensively for their anticarcinogenic properties. However, only few studies have been done with these constituents being administered by the dietary route. In our research, the effects of feeding rats with flavone, flavanone, tangeretin, and quercetin were investigated on two steps of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis (initiation and promotion). Nonpolar flavonoids such as flavone, flavanone and tangeretin administered through the initiation period, decreased the number of -gamma-glutamyl transpeptidase-preneoplastic foci. In the same conditions of administration, quercetin, a polyhydroxylated flavonoid, showed no protective effect. Moreover, feeding rats with flavanone during the phenobarbital-induced promotion step significantly reduced the areas of placental glutathione S-transferase preneoplastic foci. Quercetin, flavone, and tangeretin, administered in the same conditions, caused no significant effect. Therefore flavanone act as an anti-initiator as well as an anti-promotor. Several mechanisms were involved in the anti-initiating effects of flavone, flavanone, and tangeretin: enhancement of enzymes involved in the detoxication of AFB1 (glutathione S-transferase, UDP-glucuronyl transferase), increase of the formation of AFB1-glutathione conjugates and inhibition of the binding of AFB1 to DNA. Although the relevance of these data to the human situation remains to be demonstrated, they confirm that several flavonoids administered by the dietary route possess promising chemoprotective effects.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.