Suppressive effect of geniposide on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats.

The effects of geniposide pretreatment on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1 hepatotoxicity in rats has been examined. For these studies, male Sprague-Dawley rats were treated with AFB1 (2 mg/kg) by i.p. administration, and the different degrees of hepatic damage were revealed by the elevations of levels of serum marker enzymes such as aspartate aminotransferase (AST), alanine amino-transferase (ALT) and gamma-glutamyltranspeptidase (gamma-GT). After pretreatment of animals with geniposide (10 mg/kg) daily for 3 consecutive days, the enzyme elevations were significantly suppressed. This suggested that the geniposide possessed chemopreventive effects on the early acute hepatic damage induced by AFB1. Under these experimental conditions, consistent elevation of the activities of glutathione S-transferase (GST) and gamma-glutamylcysteine synthetase but not glutathione peroxidase (GSH-Px) and gamma-glutamyltranspeptidase were observed. Treatment of rats with geniposide significantly lowered hepatic GSH and GSSG levels, but the ratio of GSH to GSSG was not changed. Geniposide treatment also decreased AFB1-DNA adduct formation in AFB1-treated animals. From these results, we suggest that the protective effect of geniposide on AFB1 hepatotoxicity in rats might be due to the hepatic tissues' defense mechanisms that involve the enhanced GST activity for AFB1 detoxication and induction gamma-glutamylcysteine synthetase for GSH biosynthesis.     

Modulatory effect of crocetin on aflatoxin B1 cytotoxicity and DNA adduct formation in C3H10T1/2 fibroblast cell

Crocetin is a carotenoid isolated from the seeds of Cape jasmine (Gardenia jasminoides). The cytotoxicity and DNA-adduct formation of rat microsome-activated aflatoxin B1 (AFB1) in the C3H10T1/2 cells were significantly inhibited by pretreatment of crocetin. Most significant inhibition was found at the time of 9 h after crocetin pretreatment. Under these experimental conditions, consistent elevation in the cytosolic glutathione (GSH) levels and the activities of GSH S-transferase (GST) and GSH-peroxidase (GSH-Px) were observed. Crocetin treatment also resulted in a decrease in AFB1-DNA adduct formation in vitro, while no effect of crocetin on the formation of AFB1-8,9-oxide in vitro system was detected as measured by the Trisdiol method. From these results, we suggested that the protective effect of crocetin on the AFB1-cytotoxicity in C3H10T1/2 cells might be due to the cellular defense mechanisms that elevated the cytosol GSH and the activities of GST and GSH-Px.     

Suppression of aflatoxin B1-induced hepatotoxic lesions by crocetin (a natural carotenoid)

The suppressive effects of crocetin (a natural carotenoid) on the hepatotoxic lesions induced by aflatoxin B1 (AFB1) were investigated in male Wistar rats. Rats were divided into five groups: groups I and II served as normal and solvent control respectively. Group III was given AFB1 (25 micrograms/day/rat) alone; group IV was given crocetin (0.1 mg/day/rat) alone; and group V received both AFB1 and crocetin. Rats received AFB1 and crocetin for 9 and 10 weeks respectively, and were maintained on basal diet for 35 weeks. At the end of the experiment (week 45), the incidence of liver lesions in rats of group V was significantly reduced by approximately 40% compared with group III. There were no liver lesions in rats of groups I, II and IV. A significant protective effect of crocetin on AFB1 hepatotoxicity was shown, as manifested by reduced effects on the activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase (P less than 0.01-0.001). From our previous results and present data, we suggest that the suppression of crocetin on AFB1 hepatotoxicity in the rats might be due to the defense mechanisms of hepatic tissues that elevated the GSH S-transferase activity and decreased the formation of hepatic AFB1-DNA adducts.     

Effect of butylated hydroxyanisole on in vivo and in vitro hepatic aflatoxin B1-DNA binding in rats

Butylated hydroxyanisole (BHA) pretreatment of male rats has been examined for its effect on in vivo and in vitro hepatic aflatoxin B1-DNA binding (AFB1-DNA) in these animals. No difference either in cytochrome P-450 content or microsome-mediated AFB1-DNA was observed between livers from control and treated rats. However, cytosols from treated animals showed severalfold more inhibition of microsome mediated AFB1 binding to either exogenous or endogenous DNA than cytosols from controls. Presence of 1 mM level of either trichloropropene oxide or styrene oxide partially reversed the cytosolic inhibition of binding. Intraperitoneal administration of AFB1 2h before killing produced 50% less AFB1 binding to nuclear DNA in treated than in control animals. The role of induced glutathione S-transferases in treated rats in modulating hepatic AFB1-DNA binding is discussed.     

Effect of butylated hydroxyanisole pretreatment on aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in isolated hepatocytes from rats

The effect of 2(3)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione has been examined with isolated hepatocytes and in intact rats. Young male F344 rats were fed AIN-76A diet with or without 0.75% BHA for 2 weeks. Even though there were no significant differences in either cytochrome P-450 or reduced glutathione contents, there were marked differences in AFB1 metabolism in isolated hepatocytes from these two groups. Thus, at the 33 nM AFB1 level, AFB1-DNA binding was 3-fold higher in control compared to BHA-treated hepatocytes whereas AFB1-glutathione conjugation was 5-fold higher in treated compared to controls. Even at higher AFB1 concentrations (2 and 10 microM), DNA binding was 4-6-fold higher in controls whereas thiol conjugation was 5-9-fold higher in treated compared to control hepatocytes. Addition of 0.5-1.0 mM diethylmaleate did not have any significant effect in control hepatocytes whereas its presence produced about 70-100% increase in DNA binding with 65-80% inhibition of thiol conjugation in treated hepatocytes. Addition of 1 mM styrene oxide caused 75-100% and 4-8-fold increase in AFB1-DNA binding in control and treated hepatocytes, respectively, with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-reduced glutathione conjugate. It appears that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.     

Species and sex differences of aflatoxin B1-induced glutathione S-transferase placental form in single hepatocytes.

Species and sex differences of aflatoxin B1 (AFB1)-induced glutathione S-transferase placental form (GST-P) positive single hepatocytes have been investigated 48 h after an intraperitoneal injection of AFB1 to young male and female Fischer rats (2 mg AFB1/kg body wt) and male Syrian golden hamsters (6 mg AFB1/kg body wt). The presence of GST-P positive hepatocytes was examined by the immunohistochemical method. Male rats formed three times as many AFB1-induced GST-P positive hepatocytes as females. Pretreatment of both male and female rats with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO) (4 mmol/kg body wt), 2 h and 4 h before AFB1 injection increased AFB1-induced GST-P positive hepatocytes by about 120% above the controls. Male hamsters formed several-fold less AFB1-induced GST-P positive hepatocytes than male rats. Pretreatment with BSO did not increase AFB1-induced GST-P positive hepatocytes in hamsters even though it produced an increase in hepatic necrosis. It appears that GSH and GSH S-transferases play an important role in modulating hepatic AFB1-DNA binding and AFB1-induced GST-P positive hepatocytes in rats and hamsters.     

Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation, and hepatic tumorigenesis in rats fed ethoxyquin: role of induction of glutathione S-transferases

The effects of dietary administration of ethoxyquin (EQ) on aflatoxin B1 (AFB1) metabolism, DNA adduct formation and removal, and hepatic tumorigenesis were examined in male Fischer rats. Rats were fed a semipurified diet containing 0.4% EQ for 1 wk, gavaged with 250 micrograms of AFB1 per kg 5 times a wk during the next 2 wk, and, finally, restored to the control diet 1 wk after cessation of dosing. At 4 mo, focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase. Treatment with EQ reduced by greater than 95% both area and volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Utilizing the same multiple dosing protocol, patterns of covalent modifications of DNA by AFB1 were determined. EQ produced a dramatic reduction in the binding of AFB1 to hepatic DNA: 18-fold initially and 3-fold at the end of the dosing period. Although binding was detectable at 3 and 4 mo postdosing, no effect of EQ was observed, suggesting that these persistent adducts are not of primary relevance to AFB1 carcinogenesis. Analysis of nucleic acid bases by high-performance liquid chromatography revealed no qualitative differences in adduct species between treatment groups. The inhibitory effect of EQ on AFB1 binding to DNA and tumorigenesis appears related to induction of detoxication enzymes. Rats fed 0.4% EQ for 7 days showed a 5-fold increase in hepatic cytosolic glutathione S-transferase (GST)-specific activities. Multiple molecular forms of GST were induced, and concomitant elevations in messenger RNA levels coding for the synthesis of GST subunits were observed. Correspondingly, biliary elimination of AFB1-glutathione conjugate was increased 4.5-fold in animals on the EQ diet during the first 2 h following p.o. administration of 250 micrograms of AFB1 per kg. Thus, induction by EQ of enzymes important to AFB1 detoxication, such as GST, can lead to enhanced carcinogen elimination, as well as reductions of AFB1-DNA adduct formation and subsequent expression of preneoplastic lesions, and, ultimately, neoplasia.     

A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats

The effect of phenobarbital (PB) pretreatment of rats on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation have been examined in studies in vivo and in vitro. Male Sprague-Dawley rats fed a commercial diet with 0.1% PB in their drinking water for 1 week had total wet liver weight and microsomal protein content about 27% and 38% higher, respectively, than controls. Hepatic cytochrome P-450 content, microsomal cytochrome P-450 mediated AFB1 binding to exogenous DNA and formation of hydroxy metabolites of AFB1 were also about threefold higher in PB-treated rats and cytosolic reduced glutathione S-transferase activities were about doubled. Microsome-mediated AFB1-DNA binding, when examined at 2 microM and 10 microM levels of AFB1, was inhibited two-to threefold more by cytosols of treated rats whereas AFB1-SG conjugation was two- to threefold higher by cytosols of treated rats. In reconstitution experiments with 2 microM AFB1, with intact nuclei serving as a source of endogenous DNA, addition of microsomes from either group generated a large amount of AFB1-DNA binding (68-105 pmol) and a smaller amount of AFB1-SG conjugate (12-21 pmol). The presence of cytosol from the controls reduced AFB1-DNA binding to a much lesser extent than the cytosol from the treated group whereas AFB1-SG conjugation was much higher with the cytosol from the treated group. These results are in agreement with the studies in vivo. In isolated hepatocytes at 33 nM, 2 microM and 10 microM AFB1 levels, AFB1-DNA binding was decreased 50 to 70% by prior PB-treatment whereas AFB1-SG conjugation was two- to threefold higher in treated compared to control hepatocytes. In hepatocytes, addition of 1 mM diethylmaleate increased DNA binding two- to threefold with a corresponding decrease in AFB1-SG conjugation. Addition of 1 mM styrene oxide caused 5- to 10-fold increases in AFB1-DNA binding at levels of AFB1 of 33 nM and 2 microM; but at 10 microM AFB1, increases in AFB1-DNA binding were two- to threefold. In intact rats, PB treatment reduced hepatic AFB1-DNA binding to 30% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. It appears that the induced cytosolic GSH S-transferases after PB treatment of rats plays a significant role in inhibiting hepatic AFB1-DNA binding and hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of butylated hydroxyanisole pretreatment on in vitro hepatic aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in rats

The effect of 3(2)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both in vitro hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation has been examined. For these studies, young male F344 rats were fed AIN-76 A diet with or without 0.75% BHA for 2 weeks. There were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated exogenous DNA binding to AFB1 with cytochrome P-450 from control or BHA-treated animals. There were large differences in reduced glutathione S-transferase activity with treated cytosols showing 2.5-fold higher activity than the controls. Hepatic reduced glutathione levels were 25% higher in treated than in controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate when examined at two levels of AFB1 (2 and 10 microM) and a 4-fold range of cytosolic concentrations showed that inhibition of AFB1-DNA binding was greater with cytosol from the treated compared to the controls. However, AFB1-SG conjugation was 3- to 4-fold greater in treated than in controls. Inhibition of AFB1-DNA binding by cytosol was reversed in the presence of 1 mM level of various epoxides with concomitant inhibition of AFB1-SG conjugation. In reconstitution studies with 2 microM AFB1, intact nuclei alone from either group did not yield significant amounts of either DNA binding or AFB1-SG conjugation. However, addition of microsomes from either group to these nuclei generated a large amount of AFB1-DNA binding (82-111 pmol) and a smaller amount of AFB1-SG conjugate (9-28 pmol). The presence of cytosols from the control group reduced AFB1-DNA binding to a much lesser extent than the cytosols from the treated group. However, AFB1-SG conjugation was much higher with the cytosol from treated than with the controls. These reconstitution studies with endogenous DNA show more AFB1-DNA binding with the control than with BHA-treated animals and are in agreement with the studies in vivo. It appears that induced levels of cytosolic reduced glutathione S-transferase modulate AFB1-DNA binding and AFB1 hepatocarcinogenesis.     

Comparison of aflatoxin B1-DNA binding and glutathione conjugate formation by liver preparations from rats of different ages.

The capability of the newborn rat liver to detoxify aflatoxin B1 (AFB1), a potent hepatocarcinogen is not well understood. Our present results show that immature rats are deficient in the hepatic key factors involved in biotransformation of AFB1. The activities of cytosolic glutathione S-transferases and microsomal cytochrome P-450 along with cellular glutathione (GSH) content show postnatal developmental changes. The ability of hepatic subcellular preparation from newborn rats to convert AFB1 to its reactive epoxide form, is reported for the first time in this communication. Epoxidation of [3H]AFB1 in the presence of liver microsomes from different age-groups as measured by its adduct formation to calf thymus DNA in vitro shows that newborn rats are capable of catalyzing only minimal AFB1-DNA binding compared with that of adults. Addition of cytosolic fraction of various age groups to the system suggests that young rats are less efficient in modulating the binding as compared with adults. The amount of AFB1-GSH conjugate formed is also significantly higher when adult GSH S-transferase is involved in the system. These observations show that immature liver is less efficient than a mature organ in handling a chemical carcinogen and the metabolism of AFB1 by neonatal liver differs from that in the adult.     

Grapefruit juice intake does not enhance but rather protects against aflatoxin B1-induced liver DNA damage through a reduction in hepatic CYP3A activity

Influence of grapefruit juice intake on aflatoxin B1 (AFB1)-induced liver DNA damage was examined using a Comet assay in F344 rats given 5 mg/kg AFB1 by gavage. Rats allowed free access to grapefruit juice for 5 days prior to AFB1 administration resulted in clearly reduced DNA damage in liver, to 65% of the level in rats that did not receive grapefruit juice. Furthermore, rats treated with grapefruit juice extract (100 mg/kg per os) for 5 days prior to AFB1 treatment also reduced the DNA damage to 74% of the level in rats that did not receive grapefruit juice. No significant differences in the portal blood and liver concentrations of AFB1 were observed between grapefruit juice intake rats and the controls. In an Ames assay with AFB1 using Salmonella typhimurium TA98, lower numbers of revertant colonies were detected with hepatic microsomes prepared from rats administered grapefruit juice, compared with those from control rats. Microsomal testosterone 6beta-hydroxylation was also lower with rats given grapefruit juice than with control rats. Immunoblot analyses showed a significant decrease in hepatic CYP3A content, but not CYP1A and CYP2C content, in microsomes of grapefruit juice-treated rats than in non-treated rats. No significant difference in hepatic glutathione S-transferase (GST) activity and glutathione content was observed in the two groups. GSTA5 protein was not detected in hepatic cytosol of the two groups. In microsomal systems, grapefruit juice extract inhibited AFB1-induced mutagenesis in the presence of a microsomal activation system from livers of humans as well as rats. These results suggest that grapefruit juice intake suppresses AFB1-induced liver DNA damage through inactivation of the metabolic activation potency for AFB1 in rat liver.     

Effect of beta-naphthoflavone on the metabolism of aflatoxin B1 in hamsters

The effect of beta-naphthoflavone (BNF) pretreatment of hamsters on the hepatic metabolism of aflatoxin B1 (AFB1) has been examined in studies in vitro and in vivo. Pretreatment with BNF not only increased microsomal cytochrome P-450 by 50-80% but also increased microsome-mediated AFB1 epoxidation as measured by AFB1-DNA binding 2.6 fold without significantly affecting other hydroxylations. Neither cytosolic GSH S-transferases' activities nor AFB1-GSH (AFB1-SG) conjugation were affected. In vivo, hepatic AFB1-DNA binding was also increased about 3-4-fold. These results in contrast to those observed in the rat indicate that induced species of cytochrome P-450 are primarily responsible for higher epoxidation of AFB1 in the hamster.     

Effect of purified rat and hamster hepatic glutathione S-transferases on the microsome mediated binding of aflatoxin B1 to DNA

Rat and hamster liver cytosolic glutathione (GSH) S-transferases purified by GSH-affinity chromatography have been examined for their effects on the microsome mediated binding of aflatoxin B1 (AFB1) to DNA and on the conjugation of AFB1-2,3-epoxide with GSH. Like previous studies with cytosolic preparations (Raj et al. (1984) Carcinogenesis 5, 879), our present study with purified GSH S-transferases showed 2-3-fold more inhibitory activity of AFB1-DNA binding with hamster than that with the rat. Concomitant with the inhibition of AFB1-DNA binding, increase in AFB1-GSH conjugation occurred. Subunit compositions of GSH S-transferases indicate preponderance of Yb and Ya subunits in the hamster and rat, respectively. The role of GSH S-transferases in modulating AFB1-DNA binding and AFB1 induced hepatocarcinogenesis is discussed.     

Inhibitory effect of geniposide on aflatoxin B1-induced DNA repair synthesis in primary cultured rat hepatocytes.

We have previously demonstrated that geniposide (GP) inhibits the aflatoxin B1 (AFB1) induced-hepatotoxicity and hepatic DNA binding in rats. To address the mechanism of action, the effects of GP on AFB1-induced DNA repair synthesis and AFB1 biotransformation in cultured rat hepatocytes were investigated. By evaluation of unscheduled DNA synthesis (UDS), GP reduced AFB1-induced DNA repair synthesis in a dose-dependent manner in hepatocyte cultures. GP elevates the metabolism of AFB1 and decreases the formation of AFM1. The enzyme activities of glutathione S-transferase (GST) and GSH-peroxidase (GSH-Px) in AFB1-treated hepatocyte cultures are enhanced in the presence of GP. GP reduces AFB1-induced DNA repair synthesis through an increased AFB1 detoxication metabolism. It provides one possible mechanism for the chemopreventive activity of GP.     

Changes in adult metabolism of aflatoxin B1 in rats neonatally exposed to diethylstilbestrol. Alterations in alpha-class glutathione S-transferases.

Neonatal exposure of rats to xenobiotics has been shown to produce long-term alterations in hepatic enzyme activities and in levels of DNA adducts following carcinogen exposure. We exposed newborn male rats to diethylstilbestrol (DES), pregnenolone-16 alpha-carbonitrile, 7,12-dimethylbenz[a]anthracene or phenobarbital on days 1, 3 and 5 of age. At five months of age, males were injected with 1 mg/kg of [3H]aflatoxin B1 (AFB1), killed after 2 h and examined for AF-DNA adduction in the liver. Males neonatally exposed to DES showed a 35% decrease in DNA adduction levels. Analysis of the adducted DNA bases failed to show any changes in relative proportions of individual adducts in the DES samples compared to controls. Hepatic glutathione concentrations were unchanged. However, Western blot analysis of alpha-class glutathione S-transferases (alpha GST), enzymes known to inactivate the toxic AFB1-8,9-epoxide, showed a 2-fold increase in subunit levels in the DES-treated males, suggesting that the detoxifying activity of the cytosol may have been increased. To confirm this, in vitro tests were undertaken using butylated hydroxyanisole (BHA) induced mouse microsomes to activate [3H]AFB1 in the presence of treated cytosol and GSH. Analysis of metabolites by HPLC showed that DES-treated males formed 245% of the AFB-SG conjugate relative to vehicle controls. These results indicate that neonatal DES treatment resulted in long-term changes in basal alpha GST levels and suggest that these changes were responsible for lower levels of DNA adduction following adult exposure to AFB1.     

Differential effects of butylated hydroxyanisole on metabolism of aflatoxin B1 in vitro by liver and lung microsomes

Growing epidemiological evidence points out the carcinogenic hazard of inhaled aflatoxin B1 (AFB1) to the pulmonary system. Metabolism of AFB1 by lung microsomes and its binding to calf thymus DNA are reported for the first time in this paper. In addition, the ability of dietary butylated hydroxyanisole (BHA) to modulate AFB1 adduct formation with DNA was examined. Lung microsomes from BHA-treated rats unlike those from liver caused a 50% inhibition of AFB1-DNA binding. However, pulmonary cytosolic glutathione (GSH) S-transferase activity remained unaltered. The addition of BHA-treated lung cytosol failed to produce a greater inhibition of AFB1-DNA binding than control cytosol. Microsome mediated AFB1-DNA binding was markedly inhibited (30%) by the addition of GSH alone to the incubation system. Further addition of cytosol contributed much less (10%) to the inhibition of AFB1-DNA binding. These observations together with the induction of microsomal GSH S-transferase strongly implicate the role of microsomal GSH S-transferase in the modulation of AFB1-DNA binding.     

The importance of glutathione and glutathione transferase for somatic mutations in Drosophila melanogaster induced in vivo by 1,2-dichloroethane

Two principal pathways of metabolism of the carcinogenic compoundl,2-dichloroethane (DCE) have been proposed. One is a mixedfunction oxidase dependent pathway requiring oxygen and NADPH.The other pathway depends on the presence of glutathione (GSH)and glutathione transferase (GST). The aim of this study wasto investigate the role of the latter pathway for the in vivomutagenicity of DCE in the somatic wing spot test in Drosophilamelanogaster. DCE caused a dose-dependent increase of wing spots.In order to investigate the role of cellular GSH for the mutagenicity,the level of GSH was decreased by 24 h pretreatment with buthioninesulfoximine (BSO), an efficient inhibitor of GSH synthesis.This pretreatment decreased the GSH level to 6% as comparedto the control. The pretreatment also resulted in a significantdecrease of the mutagenicity of DCE. Treat ment of the larvaewith phenobarbiturate (PB) resulted in 200% induction of cytosolicGST, and a corresponding increase in the DCE mutagenicity. Theseresults indicate that the important pathway in vivo for themutagenicity of DCE is dependent on GSH and GST. A similar experimentalprotocol was used to study interactions between aflatoxin B₁(AFB) and GSH and GST. No effect of the treatment with BSO onthe mutagenicity of AFB was observed, while pretreatment withPB caused a decrease of the mutagenicity of AFB.     

Lack of influence of butylated hydroxytoluene on modification of lung microsome mediated aflatoxin B1-DNA binding: role of pulmonary glutathione S-transferase

Phenolic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are known to inhibit tumor formation due to several chemical carcinogens including aflatoxin B1 (AFB1). Metabolic activation of AFB1 by lung microsomes and possible modification by dietary BHA was reported in an earlier communication (Allameh et al. (1988) Cancer Lett., 40, 49). Here we report the effect of dietary BHA at a high dose (0.75% for 15 days) and a low dose (0.06% for 180 days) on the activation and inactivation of AFB1 by subcellular preparations of lung. BHT at high dose alone induced hepatic cytosolic glutathione (GSH) S-transferases activity while the pulmonary enzyme was unaffected by BHT feeding. This observation was substantiated when the addition of lung cytosol from control and BHT-treated rats showed similar inhibition (50%) in the microsome mediated AFB1-DNA binding. Thus BHT appears to have little influence on the pulmonary metabolism of AFB1.     

Enhancing effect of ethanol on aflatoxin b1-induced DNA-damage in glutathione-depleted rat hepatocytes

The effect of reduced glutathione (GSH) and ethanol on aflatoxin B1 (AFB1)-induced DNA single strand breaks was studied in primary cultured hepatocytes. Buthionine sulfoximine (BSO) which decreased intracellular GSH to 13% of those of the control levels increased DNA fragmentation of AFB1-treated hepatocytes by over 17% of those without BSO. Thus, a decrease in hepatocyte GSH levels increased AFB1-induced DNA damage. Although ethanol in itself did not induce DNA damage, a combination of BSO and ethanol increased the percentage by over 23% of that with BSO only. Ethanol did not affect the amount of GSH, total cytochrome P-450 (P450), glutathione S-transferase (GST) and epoxide hydrolase (EHase) in cultured hepatocytes. However, GSH-depleted rat hepatocytes exposed to ethanol significantly increased the level of P450IIIA, which activates AFB1. The enhancing effects of ethanol in the presence of BSO are probably due to the induction of this isozyme in rat hepatocytes. The GSH-depleted hepatocytes are more susceptible to chemical carcinogens in the presence of ethanol.     

Aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation with isolated hepatocytes from rats and hamsters

Binding of aflatoxin B1 (AFB1) to DNA and AFB1-glutathione conjugation during the metabolism of AFB1 have been examined with freshly isolated hepatocytes from male Fischer rats and Syrian hamsters. Even though there was no significant difference in cytochrome P450 and glutathione contents, there were marked differences in the metabolism of AFB1 (33 nM) in hepatocytes from these two species. Thus, AFB1-DNA binding was six-fold higher in the rat than in hamster hepatocytes, whereas AFB1-glutathione conjugation was 12-fold higher in hamster than in rat hepatocytes. The addition of 0.5 mM diethylmaleate had no significant effect in rats, whereas its presence produced a nine-fold increase in AFB1-DNA binding with 85% inhibition of thiol conjugation in hamster hepatocytes. Styrene oxide (1 mM) produced 50% and 25-fold increases in AFB1-DNA binding in rat and hamster hepatocytes, respectively, with corresponding decreases in thiol conjugation. Triethyltin bromide (50 microM) inhibited both processes by 50% in rat hepatocytes, whereas it produced a nine-fold increase in AFB1-DNA binding with a concomitant decrease in thiol conjugation in hamster hepatocytes. These results suggest that glutathione S-transferases play a more significant role in modulating AFB1-DNA binding in hamster than in rat hepatocytes.