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PubMed1998

Dietary carotenoids inhibit aflatoxin B1-induced liver preneoplastic foci and DNA damage in the rat: role of the modulation of aflatoxin B1 metabolism.

To study the effects of carotenoids on the initiation of liver carcinogenesis by aflatoxin B1 (AFB1), male weanling rats were fed beta-carotene, beta-apo-8'-carotenal, canthaxanthin, astaxanthin or lycopene (300 mg/kg diet), or an excess of vitamin A (21000 RE/kg diet), or were injected i.p. with 3-methylcholanthrene (3-MC) (6 x 20 mg/kg body wt) before and during i.p. treatment with AFB1 (2 x 1 mg/kg body wt). The rats were later submitted to 2-acetylaminofluorene treatment and partial hepatectomy, and placental glutathione S-transferase-positive liver foci were detected and quantified. The in vivo effects of carotenoids or of 3-MC on AFB1-induced liver DNA damage were evaluated using different endpoints: liver DNA single-strand breaks (SSB) induced by AFB1, and in vivo binding of [3H]AFB1 to liver DNA and plasma albumin. Finally, the modulation of AFB1 metabolism by carotenoids or by 3-MC was investigated in vitro by incubating [14C]AFB1 with liver microsomes from rats that had been fed with carotenoids or treated by 3-MC, and the metabolites formed by HPLC were analyzed. In contrast to lycopene or to an excess of vitamin A, both of which had no effect, beta-carotene, beta-apo-8'carotenal, astaxanthin and canthaxanthin, as well as 3-MC, were very efficient in reducing the number and the size of liver preneoplastic foci. In a similar way as 3-MC, the P4501A-inducer carotenoids, beta-apo-8'-carotenal astaxanthin and canthaxanthin, decreased in vivo AFB1-induced DNA SSB and the binding of AFB1 to liver DNA and plasma albumin, and increased in vitro AFB1 metabolism to aflatoxin M1, a less genotoxic metabolite. It is concluded that these carotenoids exert their protective effect through the deviation of AFB1 metabolism towards detoxication pathways. In contrast, beta-carotene did not protect hepatic DNA from AFB1-induced alterations, and caused only minor changes of AFB1 metabolism: seemingly, its protective effect against the initiation of liver preneoplastic foci by AFB1 is mediated by other mechanisms.

Aflatoxin B1↗

PubMed2000

Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver.

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.

Aflatoxin B1↗

PubMed1991

Effects on aflatoxin M1 residues in milk by addition of hydrated sodium calcium aluminosilicate to aflatoxin-contaminated diets of dairy cows.

Hydrated sodium calcium aluminosilicate (HSCAS), an anticaking agent for agricultural feeds, was added to aflatoxin (AF)-contaminated diets of 3 lactating dairy cows and evaluated for its potential to reduce aflatoxin M1 (AFM1) residues in milk. During phase I, cows were fed alternating diets that consisted of 200 micrograms of AF/kg of feed for 7 days, 0.5% HSCAS plus 200 micrograms of AF/kg of feed for 7 days, and feed with the HSCAS removed for a final 7 days. The AFM1 milk concentrations from the intervals with HSCAS added to diets were compared with those times when HSCAS was absent. The presence of 0.5% HSCAS in feed containing 200 micrograms of AF/kg reduced AFM1 secretion into the milk by an average of 0.44 micrograms/L (from pretreatment of 1.85 micrograms/L to 1.41 micrograms/L with HSCAS, a 24% reduction). Following a 10-day period of noncontaminated feed consumption and no AFM1 residues in the milk, phase II of the study was begun. The same experimental design as phase I was used, but the dosages of HSCAS and AF were changed to 1.0% and 100 micrograms/kg of feed, respectively. The addition of 1.0% HSCAS in feed containing 100 micrograms of AF/kg decreased AFM1 content in the milk by an average of 0.40 micrograms/L (from a pretreatment of 0.91 micrograms/L to 0.51 micrograms/L when HSCAS was present, a 44% reduction). These findings suggest that HSCAS, a high-affinity sorbent compound for AF in vitro, is capable of reducing the secretion of AFM1 into milk.

Aflatoxin M1↗

PubMed1982

Aflatoxin M1: in vitro preparation and comparative in vitro metabolism versus aflatoxin B1 in the rat and mouse.

A rapid, simple and relatively inexpensive biotransformation method using microsomes from 3-methylcholanthrene (3MCA) pretreated rats is described for the production of aflatoxin M1 (AFM1) is sufficient quantities for metabolism studies. A comparison is made of the metabolism of AFM1 and AFB1 by the postmitochondrial (S-10) fraction from mouse and rat hepatocytes. AFM1, in both species, is metabolized more slowly than AFB1, with its major metabolites being found in the aqueous fraction and very little associated with S-10 protein. In contrast, AFB1 is metabolized to numerous chloroform-extractable, protein bound and water-soluble metabolites. The toxicological implications of reduced protein binding and slower metabolism for AFM1 are discussed.

Aflatoxin B1↗

PubMedDate not supplied

Indole-3-carbinol and beta-naphthoflavone induction of aflatoxin B1 metabolism and cytochromes P-450 associated with bioactivation and detoxication of aflatoxin B1 in the rat.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and hepatocarcinogenic secondary metabolite of the grain mold Aspergillus flavus and related fungi. Indole-3-carbinol (I3C), found in cruciferous vegetables, can both inhibit and promote AFB1-induced carcinogenesis. We have examined the influence of dietary treatment with I3C and the well-known Ah receptor agonist beta-naphthoflavone (BNF) on the relative levels of different cytochrome P-450 (CYP) isoforms known to metabolize AFB1 in male Fischer 344 rats. After 7 days of feeding 0.2% I3C or 0.04% BNF, alone or in combination, the relative levels of hepatic CYP1A1, 1A2, 2B1/2, 2C11, and 3A were assessed by laser densitometry of Western blots. Both diets containing I3C markedly increased band densities of CYP1A1 (up to 24-fold), 1A2 (3.1-fold), and 3A1/2 (3.8-fold), and had lesser effects on the levels of 2B1/2 (1.8-fold) and no effect on CYP2C11. BNF also strongly increased band densities of CYP1A1 (12-fold) and 1A2 (2.7-fold), but had no effect on the levels of CYP2B1/2 or 3A1/2 band densities, and repressed those of CYP2C11 (2-fold). In addition, we examined the in vitro hepatic microsomal metabolism of AFB1 at 16, 124, and 512 microM substrate levels. Diets containing I3C elevated initial rates of AFM1 (a detoxication product) production 18.6- to 19.2-fold over control at 16 microM AFB1, which declined to 7.8- to 9.5-fold at 512 microM AFB1. The BNF-only diet gave similar, but less dramatic effects (5.9-fold at 16 microM AFB1, 3.5-fold at 512 microM AFB1).(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗

PubMed1983

The requirement for glutathione S-transferase in the conjugation of activated aflatoxin B1 during aflatoxin hepatocarcinogenesis in the rat.

The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.

Administration, Oral↗

PubMed1983

Interactions of aflatoxin B1 and blood components of various species in vitro: interconversion of aflatoxin B1 and aflatoxicol in the blood.

The fate of aflatoxin B1 (AFB1) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [14C]AFB1 at 37 degrees C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB1 to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB1 in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [14C]AFB1-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, high levels of radioactivity were found in both the AFB1 region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB1 and AFL showed marked interconversion of AFB1 and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB1-AFL interconversion in the blood.

Aflatoxin B1↗

PubMed1988

Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences.

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.

Aflatoxin B1↗

PubMedDate not supplied

Long term administration of low doses of mycotoxins in poultry. 2. Residues of ochratoxin A and aflatoxins in broilers and laying hens after combined administration of ochratoxin A and aflatoxin B1.

The effects of combined administration of ochratoxin A (OA) and aflatoxin B1 (AFB1) on the occurrence and the levels of residues of mycotoxins in poultry have been investigated. Male broilers and laying hens were fed from 14 days old with standard diets contaminated with 50 micrograms/kg OA and 50 micrograms/kg AFB1. Two groups of broilers and hens were withdrawn from contaminated feed at 37 and 88 days, respectively. At the time of sacrifice no significant lesions were found. Residues were compared with those found after administration of either toxin alone in former trials. Combined treatment resulted in higher content of OA in broiler livers (40 versus 5.0 micrograms/kg) and, to a lesser extent, in kidneys and skin, and of AFB1 in broiler liver and kidney (0.15 versus 0.02 microgram/kg and 0.40 versus 0.05 microgram/kg respectively). Laying hens showed smaller differences (0.20 versus 0.10 microgram/kg in liver and 0.32 versus 0.08 in kidneys). Withdrawal from treatment led to the almost complete disappearance of OA residues in broilers and in hens. These results show a synergistic effect of OA and AFB1, particularly in broilers.

Aflatoxin B1↗

PubMed1988

Mapping the binding site of aflatoxin B1 in DNA: molecular modeling of the binding sites for the N(7)-guanine adduct of aflatoxin B1 in different DNA sequences.

Aflatoxin B1 (AFB1), a potent mutagen and carcinogen, forms an adduct exclusively at the N(7) position of guanine, but the structure of this adduct in double stranded DNA is not known. Molecular modeling (using the program, PSFRODO) in conjunction with molecular mechanical calculation (using the program, AMBER) are used to assess the binding modes available to this AFB1 adduct. Two modes appear reasonable; in one the AFB1 moiety is intercalated between the base pair containing the adducted guanine and the adjacent base pair on the 5'-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA. Rotational flexibility appears feasible in the latter providing four, potential binding sites. Molecular modeling reveals that the binding sites around the reactive guanine in different sequences are not uniformly compatible for interaction with AFB1. As the sequence is changed, one particular external binding site would be expected to give a pattern of reactivities that is reasonably consistent with the observed sequence specificity of binding that AFB1 shows in its reaction with DNA (Benasutti, M., Ejadi, S., Whitlow, M. D. and Loechler, E. L. (1988) Biochemistry 27, 472-481). The AFB1 moiety is face-stacked in the major groove with its long axis approximately perpendicular to the helix axis. Favorable interactions are formed between exocyclic amino groups that project into the major groove on cytosines and adenines surrounding the reactive guanine, and oxygens in AFB1; unfavorable interactions involve van der Waals contacts between the methyl group on thymine and the AFB1 moiety. "Some of the sequence specificity of binding data can be rationalized more readily if it is assumed that 5'-GG-3' sequences adopt an A-DNA structure." Based upon molecular modeling/potential energy minimization calculation, it is difficult to predict how reactivity would change in different DNA sequences in the case of the intercalative binding mode; however, several arguments suggest that intercalation might not be favored. From these considerations a model of the structure for the transition state in reaction of AFB1 with DNA is proposed involving one particular external binding site.

Aflatoxin B1↗

PubMed1984

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) binding to 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-GSH conjugation in vitro using rat and hamster liver subcellular fractions is elucidated in this report. Even though hamster liver microsomes catalyzed AFB1 binding to exogenous DNA three times as much as the rat, hamster cytosol inhibited AFB1-DNA binding catalyzed by either microsomes severalfold more than the rat cytosol. AFB1-DNA binding is found to be inversely related to AFB1-GSH conjugation at all AFB1 concentrations (2-100 microM) studied. Presence of either styrene oxide or 3,3,3-trichloropropene oxide at 1 mM level diminished AFB1-GSH formation in vitro confirming some competition by these epoxides with AFB1-epoxide for cytosolic GSH S-transferases. In a reconstituted system with endogenous DNA, the ratio of AFB1-GSH to AFB1-DNA binding was found to be 10-15 times higher with the hamster in comparison with the rat indicating enhanced inactivation of the ultimate carcinogenic metabolite in the hamster. These results are discussed in relation to AFB1-DNA binding and AFB1 hepatocarcinogenicity in resistant and sensitive species.

Aflatoxin B1↗

PubMed1987

Effects of phenobarbital on the biliary excretion of aflatoxin P1-glucuronide and aflatoxin B1-S-glutathione in the rat.

Direct h.p.l.c. analysis of bile separated at least five major water-soluble metabolites of AFB; the two most prevalent AFB metabolites were identified as AFB-S-glutathione (AFB-GSH) and AFP1-glucuronide, which accounted for 49-57% and 4-15% of total biliary AFB metabolites, respectively. In the two hours following AFB administration, phenobarbital-treated rats eliminated 50% more AFB-derived radioactivity in bile compared with controls. No qualitative differences in the profile of biliary AFB metabolites were noted between phenobarbital-treated and control rats. However, a 90% increase in the rate of excretion of AFB-GSH was found in phenobarbital-treated animals. Phenobarbital treatment had no significant effect on the amount of AFB remaining in the liver after two hours, but decreased the amount of AFB covalently bound to hepatic DNA by 55%. When individual animals from both control and phenobarbital-treated groups were considered, the correlation between the increase in excretion of AFB-GSH and the decrease in covalent binding was significant with a correlation coefficient of 0.77. This finding is consistent with the hypothesis that induction of GSH S-transferase is responsible for the anticarcinogenic effects of phenobarbital towards AFB-induced hepatocarcinogenicity, although changes in the rate of formation of aflatoxin P1 or other biotransformation pathways may also be important.

Aflatoxin B1↗

PubMed1993

Effects of various types of aluminosilicates and aflatoxin B1 on aflatoxin toxicity, chick performance, and mineral status.

In vivo and in vitro trials were conducted to test the efficacy of four aluminosilicates (AS) (Ethacal feed component, Novasil, Perlite, and Zeobrite) to sorb aflatoxin B1 (AFB1) and alleviate aflatoxicosis in broiler chicks. Percentage sorption capacity of AS to radiolabeled AFB1 dissolved in methanol varied from 2 to 60%, whereas percentage sorption in intestinal contents varied from 0 to 40.0% according to type of AS tested. Intestinal contents alone sorbed 42% radiolabeled AFB1. Novasil and Zeobrite exhibited the highest rates of sorption (55 and 60%, respectively) in methanol. An in vivo study compared the four types of AS in combination with 0 or 2.5 ppm AFB1 fed to day-old chicks (two pens of six chicks per treatment) to 3 wk of age. Diet effects on body weight, liver lipid, bone ash, and serum Ca, P, Na, K, and Cl were measured. The AFB1 significantly decreased 2- and 3-wk body weight, and a significant interaction effect of AS and AFB1 on bird weight occurred at 2 and 3 wk of age. Three of the four AS tested alleviated the growth depression caused by AFB1. Liver lipids percentage was increased in the AFB1-treated chicks, but this effect was suppressed by three of the AS. Bone ash was not affected by AFB1 and was increased by Novasil and decreased by Ethacal. Ethacal, Novasil, Perlite, and Zeobrite all tended to decrease serum Cl, regardless of AFB1 treatment.

Adsorption↗

PubMed1991

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.

Aflatoxin B1↗

PubMed1989

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.

Aflatoxin B1↗

PubMed1986

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.

Aflatoxin B1↗

PubMed1988

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.

Aflatoxin B1↗

PubMed1981

Excretion of an aflatoxin-guanine adduct in the urine of aflatoxin B1-treated rats.

Administration of aflatoxin B1 (AFB1) to rats resulted in the urinary excretion of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1. This is the major product formed by the interaction in vivo of AFB1 with rat liver nucleic acids. The adduct was isolated from urine by the combined use of preparative and analytical high-pressure liquid chromatography and was quantitated by measurement of absorbance at 365 nm. The method allowed reproducible quantitation of adduct in urine samples from rats treated with AFB1 by i.p. injection at levels as low as 0.125 mg/kg. Application of the method to urine samples from rats given injections of AFB1 (1 mg/kg) revealed the presence of a compound chromatographically identical to authentic 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1. Spectral and chemical analysis of microgram quantities of this compound provided strong evidence that this compound is identical to authentic adduct. Measurement of this adduct in the urine of rats given injections of different doses of AFB1 showed that excretion occurs in a dose-dependent manner. Comparison of the dose-response curve for adduct excretion with that previously observed for adduct formation in rat liver DNA in vivo revealed a high degree of qualitative similarity, with the levels of adduct excreted in urine representing 30 to 40% of the levels seen initially in liver DNA.

Aflatoxin B1↗