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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparative effect of dietary butylated hydroxyanisole and {beta}-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation, and carcinogenesis in rainbow trout

Butylated hydroxyanisole (BHA) and ß-naphthoflavone(BNF), both chemicals with anti-carcinogeneic properties insome experimental animals, were compared for effects on afiatoxinB1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesisin the rainbow trout. Dietary BHA had no effect on the hepatictumor incidence when fed at 0.03 or 0.3% 4 weeks prior to andduring a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, whenfed at 0.005 or 0.05% under similar conditions, significantlyreduced tumor response, which confirms previous results in trout(Nixon et al.9 Carcinogenesis, 5, 615–619, 1984). BHAfed at either 0.03 or 0.3% for 8 weeks had no post-initiationeffect on the 52 week hepatic tumor incidence of trout exposedto a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiationexposure to 0.05% BNF significantly enhanced AFB1 tumor response.The influence of dietary BHA and BNF on AFB1 metabolism andDNA adduct formation and persistence in trout were examined.A 3 week pre-treatment with 0.3% dietary BHA had no effect onin vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1,2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietaryBNF reduced hepatic AFB1-DNA adducts to 33–60% of controllevels at 0.5, 1, 2 and 4 days after AFB1 exposure. This wasaccompanied by significantly lower blood and liver levels ofAFB1 during the first 24 h after i.p. injection. Livers of BNFtrout also contained 4-fold more of the less carcinogenic metabolite,aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite withsimilar carcinogenicity as AFB1. Bile AFL-glucuronide levelswere significantly decreased in BNF-fed trout, but total bileglucuronides were significantly increased due to a 15-fold increasein aflatoxicol-M1 glucuronide. Freshly isolated hepatocytesfrom BHA-fed fish, when incubated with AFB1 for 1 h, showedno difference in levels of AFB1-DNA adducts or ratios of AFB1metabolites when compared to hepatocytes isolated from fishfed a control diet only. By contrast, dietary BNF has been previouslyshown to greatly enhance AFM1 production, reduce AFL production,and significantly reduce AFB1-DNA adduct formation in isolatedtrout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph,65, 379–385, 1984). These results indicate that dietaryBHA up to 0.3% does not alter AFB1 metabolism or DNA adductionin trout, nor does it inhibit or promote AFB1 hepatocarcinogenesisin this species. This is in contrast to anti-oxidant enhancementof AFB1-glutathione conjugation, reduction of AFB1-DNA binding,and consequent reduction of tumor response in rats. The nullresults in trout thus support enhanced glutathione conjugationas the major mechanism for BHA inhibition of AFB1 cardnogenesisin mammalian models. By contrast, BNF dietary pre-treatmentappears to inhibit AFB1 carcinogenicity in trout by enchancingglucuronide formation and elimination of the carcinogen, leadingto reduced DNA adduct formation in target tissue.     

Metabolic basis for the protective effect of the antioxidant ethoxyquin on aflatoxin B1 hepatocarcinogenesis in the rat

The effect of dietary administration of 0.5% ethoxyquin (EQ) on the in vivo induction of enzymes and effect on aflatoxin B1 (AFB1)-DNA binding in liver and the consequent in vitro metabolism of AFB1 by male Fischer F344 rat liver-derived fractions have been examined. EQ increased microsomal cytochrome P-450s, in particular those isozymes classed as phenobarbital inducible, and the in vitro rate of metabolism of AFB1. The formation of the presumed detoxified metabolites, aflatoxins M1 and Q1, was enhanced to a greater extent than was the formation of the active metabolite, aflatoxin B1-8,9 epoxide (assessed by the level of aflatoxin B1-8,9-dihydrodiol). Prolonged feeding with EQ was accompanied eventually by a reduction in the initially elevated cytochrome P-450 content, but this was not reflected in any significant decrease in the rate of AFB1 metabolism in vitro. EQ increased the glutathione S-transferase activity of the liver cytosol fractions as assessed with the model substrate 1-chloro-2,4-dinitrobenzene. The capacity of these fractions specifically to catalyze the conjugation of AFB1 with glutathione was induced to a far greater extent than was the conjugation of 1-chloro-2,4-dinitrobenzene. gamma-Glutamyl transpeptidase was induced in the periportal areas of the liver lobule. Reduced in vivo binding of [3H]AFB1 to DNA of liver and kidney was found to result from EQ treatment. It is concluded that the reduced hepatocarcinogenesis which results from feeding EQ simultaneously with AFB1 is due to the reduction in DNA-adduct formation which in turn is due at least in part to increased detoxifying metabolism in the microsomal, cytosolic, and plasma membrane compartments of the liver cells.     

Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic lipoxygenase was significantly lower than those for PHS and P450. Hepatic half-maximal binding concentrations for PHS and P450 were similar, though P450 had a much higher maximum rate than PHS and lipoxygenases. These data suggest that lipoxygenase-catalyzed AFB1 activation can occur at low AFB1 concentrations. This may be important in view of human exposure to low AFB1 concentrations and predominant lipoxygenase activity in human airway epithelial cells. When expressed per gram of tissue, renal and hepatic PHS activities and renal lipoxygenase activities for AFB1 activation were similar, and higher than the activity of pulmonary PHS, while pulmonary PHS activity for the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was similar to that in liver and lower than that in kidney.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative kinetic studies on aflatoxin B1- DNA binding and aflatoxin B1-glutathione conjugation with rat and hamster livers in vitro

Inhibition of microsome mediated aflatoxin B₁ (AFB₁) bindingto exogenous or endogenous DNA by cytosolic glutathione (GSH)S-transferases is well established from our earlier studies.Correlation between inhibition of AFB₁-DNA binding and AFB₁-GSHconjugation in vitro using rat and hamster liver subcellularfractions is elucidated in this report. Even though hamsterliver microsomes catalyzed AFB₁ binding to exogenous DNA threetimes as much as the rat, hamster cytosol inhibited AFB₁-DNAbinding catalyzed by either microsomes severalfold more thanthe rat cytosol. AFB₁ - DNA binding is found to be inverselyrelated to AFB₁-GSH conjugation at all AFB₁ concentrations (2–100µM)studied. Presence of either styrene oxide or 3,3,3-trichloropropeneoxide at 1 mM level diminished AFB₁-GSH formation in vitro confirmingsome competition by these epoxides with AFB₁-epoxide for cytosolicGSH S-transferases. In a reconstituted system with endogenousDNA, the ratio of AFB₁-GSH to AFB₁-DNA binding was found tobe 10–15 times higher with the hamster in comparison withthe rat indicating enhanced inactivation of the ultimate carcinogenicmetabolite in the hamster. These results are discussed in relationto AFB₁-DNA binding and AFB₁ hepatocardnogenicity in resistantand sensitive species.     

Effects of crocetin on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats.

The effects of crocetin pretreatment on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1 hepatotoxicity in rats has been examined. For these studies, male Wistar 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, alanine aminotransferase, lactate dehydrogenase and gamma-glutamyltranspeptidase. After pretreatment of the animals with crocetin (2 or 6 mg/kg) daily for three consecutive days, the enzyme elevations were significantly suppressed. This suggested that the crocetin possessed chemopreventive effects on the early acute hepatic damage induced by AFB1. Under these experimental conditions, consistent elevations of hepatic glutathiones (GSH) and activities of glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) were observed. Crocetin treatment also decreased AFB1-DNA adduct formation in AFB1-treated animals. From these results, we suggest that the protective effect of crocetin on AFB1 hepatotoxicity in rats might be due to the hepatic tissues' defense mechanisms that elevated the cytosol GSH and the activities of GST and GSH-Px.     

R-Goitrin- and BHA-induced modulation of aflatoxin B1, binding to DNA and biliary excretion of thiol conjugates in rats

Previous studies have shown that dietary R-goitrin is a potentinducer of hepatic glutathione S-transferase (GST) and epoxidehydrolase activities but has no effect on components of themixed function oxidase system (ethoxycoumarin O-deethylase andcytochrome P-450). In the present work effects of dietary R-goitrin(200 p.p.m.) or butylated hydroxyanisole (BHA) (7500 p.p.m.)on GST activity, binding of aflatoxin B₁, (AFB]) to DNA, invivo, and biliary excretion of thiol conjugates of AFB₁ in ratswere studied. Increases of GST activities (1.9- and 2.1-fold)were accompanied by reductions in AFB₁-DNA binding (43% and85%) and increases (1.7-and 2.2-fold) in biliary excretion ofAFB₁-thiol conjugates in R-goitrin and BHA groups, respectively.Microsomal aflatoxin 8,9-epoxidase activities were not increasedin either treatment group. The role of GST induction in thecarcinogenesis of AFB₁ and the anticarcinogenic potential ofR-goitrin are discussed.     

Mechanisms of protection against aflatoxin B(1) genotoxicity in rats treated by organosulfur compounds from garlic

Diallyl sulfide (DAS) and diallyl disulfide (DADS), two garlic constituents, were found previously to inhibit aflatoxin B(1) (AFB(1))-initiated carcinogenesis in rat liver, DADS being the most effective. In order to study the mechanisms involved in this protection, we have examined the ability of liver microsomes and cytosols from DAS- and DADS-treated rats to modulate the mutagenicity and the metabolism of AFB(1). We also examined the effects of these compounds on the expression of cytochromes P450 (CYP) and phase II enzymes known to be involved in AFB(1) metabolism. Administration of DAS (1 mmol/kg for 4 days) to rats resulted in significant inhibition of microsome-mediated mutagenicity of AFB(1), whereas DADS treatment did not alter AFB(1) mutagenicity. DAS treatment increased the metabolism of AFB(1) mainly towards the formation of AFQ(1) and AFM(1), which might account for the reduction of AFB(1) microsomal-mediated mutagenicity. DADS treatment slightly affected the oxidative metabolism of AFB(1). DAS and DADS induced CYP3A2, CYP2B1 and CYP2B2, DAS being more potent. Cytosols from DAS- and DADS-treated rats produced a significant inhibition of AFB(1)-8,9-epoxide (AFBO)-induced mutagenicity and significantly increased the cytosolic formation of AFB(1)-glutathione conjugates, DADS treatment being more effective. Western blot analysis showed that DADS is a potent inducer of glutathione S-transferase A5 (rGSTA5) and AFB(1) aldehyde reductase 1 (rAFAR1), while DAS is a weak inducer of these enzymes. Finally, we demonstrated that antibodies raised against rGSTA5 strongly reduced the antimutagenic activity of cytosols from DAS- and DADS-treated rats against AFBO. All together, these results demonstrate that DAS prevents AFB(1) mutagenicity through a dual mechanism, i.e. by modulating both the phase I and II metabolism of AFB(1), whereas DADS acts mainly by increasing the phase II metabolism of AFB(1). The induction of rGSTA5 and rAFAR1 is probably the main mechanism by which allyl sulfides give protection against AFB(1)-induced carcinogenesis.     

Glutathione conjugation of microsome-mediated and synthetic aflatoxin B1-8,9-oxide by purified glutathione S-transferases from rats.

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B1 (AFB1)-epoxide and styrene oxide has been investigated with purified GSH S-transferases (GSTs) from rats. Both styrene oxide and AFB1-epoxide were conjugated preferentially by millimicrons GSTs 3-3, 3-4 and 4-4 as compared to alpha GSTs 1-1, 1-2 and 2-2. The highest catalytic activity with styrene oxide conjugation was associated with GST 4-4. The highest catalytic activity with microsome-mediated AFB1-epoxide conjugation was observed with GST 3-3 whereas with the synthetic AFB1-epoxide conjugation was seen with GST 4-4. The catalytic activity of pi GST 7-7 was intermediate to millimicrons and alpha GSTs. It is suggested that GST 3-3 may play an important role in inactivation of AFB1-epoxide generated in vivo in the rat.     

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.     

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.     

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B(1) in human lung: differential cellular distribution and lack of significance of the GSTM1 genetic polymorphism.

Epidemiological studies suggest that aflatoxin B(1) (AFB(1)), a mycotoxin produced by certain Aspergillus species, may play a role in human respiratory cancers in occupationally-exposed individuals. AFB(1) requires bioactivation to the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH) is a critical determinant of susceptibility to AFB(1). Of the purified human GST enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB(1) exo-epoxide. The influence of the GSTM1 polymorphism on AFB(1)-GSH formation, as well as the abilities of cytosols from preparations enriched in different isolated lung cell types to conjugate AFB(1)-epoxides, were examined. In whole-lung cytosols from patients undergoing clinically indicated lobectomy, GSTM1 genotype correlated with GSTM1 phenotype as determined by [(3)H]trans-stilbene oxide conjugation: GSTM1-positive = 295 +/- 31 pmol/mg/h (n = 6); GSTM1-negative = 92.8 +/- 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast, conjugation of microsome-generated [(3)H]AFB(1)-epoxides with GSH was low and variable between patients, and did not correlate with GSTM1 genotype: GSTM1-positive = 11.9 +/- 8.1, 111 +/- 66 and 510 +/- 248 fmol/mg/h (n = 6); GSTM1-negative = 15.3 +/- 16.7, 167 +/- 225 and 540 +/- 618 fmol/mg/h (n = 4) (for 1, 10 and 100 microM [(3)H]AFB(1), respectively). GSH conjugates of AFB(1) exo-epoxide and the much less mutagenic stereoisomer AFB(1) endo-epoxide were produced in a ratio of approximately 1:1 in cytosols from both whole lung and isolated cells. Total cytosolic AFB(1)-epoxide conjugation was significantly higher in fractions enriched in alveolar type II cells (3.07 +/- 1.61 pmol/mg/h) than in unseparated lung cells (0.143 +/- 0.055 pmol/mg/h) or fractions enriched in alveolar macrophages (0. 904 +/- 0.319 pmol/mg/h; n = 4) (P < 0.05). Furthermore, AFB(1)-GSH formation and percentage of alveolar type II cells in different cell fractions were correlated (r = 0.78, P < 0.05). These results demonstrate that human lung GSTs exhibit very low conjugation activity for both AFB(1)-8,9-epoxide stereoisomers, and that this activity is heterogeneously distributed among cell types, with alveolar type II cells exhibiting relatively high activity. Of the GSTs present in human peripheral lung which contribute to AFB(1) exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most only a minor role.     

Effect of (+)-catechin, dimethyl sulfoxide and ethanol on the microsome-mediated metabolism of two hepatocarcinogens, N-nitrosodimethylamine and aflatoxin B1

Effects of catechin, a plant phenolic flavonoid, and of the commonly used organic solvents dimethyl sulfoxide (DMSO) and ethanol (EtOH) on the microsome-mediated metabolism of two hepatocarcinogens, N-nitrosodimethylamine (NDMA) and aflatoxin B1 (AFB1), are presented. Using hamster liver microsomes as a source of mixed-function oxidases, it was shown that catechin at 0.1-0.2 mM levels had no effect on the oxidation of either carcinogen. However, at 1-5 mM levels it caused a concentration-dependent inhibition (38-70%) of the formation of formaldehyde from NDMA, and at the 5 mM level it caused a 40% inhibition of AFB1-DNA binding. DMSO and EtOH totally inhibited NDMA demethylase activity but had little effect on the binding of AFB1 to DNA. These observations indicate that the mixed-function oxidases (cytochrome P450) essential for the metabolic activation of these carcinogens exhibit different sensitivities to different inhibitors.