Formation Of Aflatoxin M1 Research Articles

Inhibition of aflatoxin M1 production by bovine hepatocytes after intervention with oltipraz

It is well known that cattle ingesting aflatoxin B1 contaminated feed commodities excrete aflatoxin M1 into their milk. As aflatoxin M1 originates from hepatic metabolism, measures to prevent aflatoxin M1 formation need to be directed to either the immobilization of aflatoxin B1 in the gastrointestinal tract or the modification of hepatic metabolism of aflatoxin B1. Here we studied the influence of oltipraz and a second dithiolthione, (1,2) dithiolo (4,3‐c)‐1,2‐dithiole‐3,6 dithione (DDD) on bovine hepatic aflatoxin B1 biotransformation. Oltipraz inhibited aflatoxin B1 metabolism as no aflatoxin M1 and no aflatoxin B1‐dihydrodiol, the second metabolite found in bovine hepatocytes, was formed. DDD did not significantly inhibit aflatoxin B1 metabolism. It could be demonstrated that the inhibition of aflatoxin B1 metabolism was due to the inhibition of several cytochrome P450 enzyme activities by oltipraz. In contrast, DDD inhibited only ethoxyresorufin O‐deethylation activity. These findings suggest a high efficacy of oltipraz in inhibiting aflatoxin M1 contamination of milk from dairy cows exposed to aflatoxin B1 contaminated feeds.

A mechanism of inhibition of aflatoxin B(1) hepatocarcinogenesis by β-naphthoflavone pretreatment of rats

Pretreatment with β-naphthoflavone (BNF) inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat by stimulation of aflatoxin M1 (AFM1) hydroxylation. We have now examined the effect of BNF pretreatment of rats on AFB1-hydroxylations, AFB1- DNA binding and AFB1-glutathione (AFB1-SG) conjugation in liver in vitro, in vivo and in isolated hepatocytes. Male Fischer rats were injected i.p. with either corn oil or BNF (25 mg/kg body weight) 24 h before for in vitro and in isolated hepatocyte studies. Cytochrome P-450 contents in BNF-treated rat liver microsomes and hepatocytes were 170% and 200% of controls, respectively. At two AFB1- concentrations (2 µM and 100 µM), AFB1- epoxidation measured as AFB1-DNA binding and AFM1 hydroxylation were 60% and 300% of controls, respectively. At low AFBl level (33 nM), AFB1-binding and AFM1 formation in BNF-treated hepatocytes were 40% and 225% of controls, respectively. Formation of AFB1-SG, aflatoxin Q1 and aflatoxin P1 were not affected significantly. Hepatic nuclear AFB1-DNA binding 2 h after i.p. injection of 0.4 mg [3H]AFB1/kg body wt. in BNF-treated rats was 60% of controls. The overall data indicate that lower AFB1-DNA binding in addition to higher inactivation via AFM1 hydroxylation are responsible for the inhibition of AFB1 hepato- carcinogenesis by BNF pretreatment of rats. It is suggested that this inhibition of AFB1-DNA binding may be a direct result of lower levels of constitutive cytochromes P-450 responsible for AFB1-epoxidation.

Distribution and induction of aflatoxin B1-9a-hydroxylase activity in rat liver parenchymal and non-parenchymal cells.

Chronic administration of aflatoxin B1 (AFB1) to rats gives rise to hepatocellular and cholangiocellular carcinomas without affecting Kupffer and endothelial cells. The enzymatic conversion of AFB1 to AFB1-8,9-epoxide is the critical step in the activation of the myocotoxin, while the conversion of AFB1 to aflatoxin M1 (AFM1), catalyzed by the AFB1-9a-hydroxylase, is considered to be a detoxication route for the toxin. In the present study the distribution and inducibility of AFB1-9a-hydroxylase were analyzed in microsomes derived from freshly isolated liver parenchymal (PC) and nonparenchymal cells (i.e. Kupffer + endothelial cells, NPC). AFB1-9a-hydroxylase activity was clearly measurable in NPC and similar to that of PC. In NPC the rate of formation of AFM1 was higher (when incubating with 16 microM AFB1) than or similar (with 128 microM AFB1) to that of AFB1-8,9-epoxide, while in PC it was significantly lower. Taken together, these results suggest that the AFB1-9a-hydroxylase activity might be particularly important in NPC to protect these cells from AFB1 by converting it to a significantly less mutagenic metabolite and by reducing the amount of AFB1 available for epoxidation. Furthermore, it is shown that AFB1-9a-hydroxylase activity is inducible by phenobarbital (only in PC), 3-methylcholanthrene, isosafrole and Aroclor 1254, thus indicating that in rat liver the conversion of AFB1 to AFM1 is catalyzed by members of the cytochrome 1A and 2B families.

The model Ah-receptor agonist beta-naphthoflavone inhibits aflatoxin B1-DNA binding in vivo in rainbow trout at dietary levels that do not induce CYP1A enzymes.

beta-Naphthoflavone (BNF), a well-known Ah-receptor agonist, has been believed to inhibit aflatoxin B1 (AFB1) carcinogenesis in rats and rainbow trout primarily through induction of the cytochrome P450 1A (CYP1A) enzyme subfamily and consequent diversion of AFB1 to the less carcinogenic phase I metabolite aflatoxin M1 (AFM1). This study investigates the dose responsive effects of dietary BNF treatment on CYP1A induction. AFM1 formation, AFB1-8,9-epoxide formation and AFB1-DNA binding in the trout model. Pre-feeding diet containing 10-200 p.p.m. BNF after AFB1 i.p. injection provided dose-dependent induction of CYP1A-dependent ethoxyresorufin-O-deethylase (EROD) activity and inhibition of in vivo AFB1-DNA binding. However, most of the observable inhibition of DNA adduction (45% inhibition) had occurred at 10 p.p.m. BNF without detectable EROD induction; higher doses of BNF up to 200 p.p.m. induced EROD > 6-fold but provided only another 15% inhibition of DNA adduction in vivo. When in vitro AFB1-DNA binding was assessed using liver microsomes from trout fed 10-100 p.p.m. BNF, induced microsomal EROD activity correlated moderately with reduction of in vitro AFB1-DNA binding activity. However, BNF treatment in a low dose range (0.2-10 p.p.m.) also strongly inhibited in vivo hepatic AFB1-DNA binding (69% inhibition at 5 p.p.m. BNF in this experiment), in a dose-dependent manner, in the complete absence of detectable EROD induction. The microsomes from 5 p.p.m. BNF-treated trout had no more EROD activity than control microsomes, and no less capacity for catalyzing AFB1-DNA binding in vitro than control microsomes. Thus, the potent inhibition of hepatic AFB1-DNA binding in vivo by 5 p.p.m. BNF was a result of neither CYP1A enzyme induction nor irreversibly reduced catalytic capacity for AFB1-8,9-epoxide formation. Direct analysis of AFB1 metabolites formed in vitro by liver microsomes from trout fed 10, 100 and 500 p.p.m. BNF showed that low dietary BNF (10 p.p.m.) neither induced microsomal CYP1A-mediated AFM1 formation nor altered AFB1-8,9-epoxide formation compared to the control. By comparison, 100 and 500 p.p.m. BNF pretreatment significantly elevated microsome-catalyzed AFM1 formation in vitro (P < 0.001), and this increase was highly correlated with increased EROD activity (r2 = 0.999, P < 0.001). Upon in vitro addition, BNF was found to be a potent inhibitor of microsome-mediated AFB1-8,9-exo-epoxide formation (IC50 = 2.6 +/- 0.1 microM) and AFB1-DNA binding (inhibition constant Ki = 3.03 +/- 0.25 microM). These findings indicate that CYP1A enzyme induction can contribute modestly to BNF protection against AFB1 in this species both in vivo and in vitro at higher BNF doses, but does not do so at lower doses. Instead, enzyme inhibition by BNF against AFB1 8,9-epoxidation appears to be the predominant protective mechanism at higher BNF doses, and the sole protective mechanism at low doses, in the rainbow trout. These findings demonstrate that mechanisms of chemoprevention can change with anticarcinogen dose, and caution that even potent induction of phase I or phase II activities does not assure that pathway to be a predominant protective mechanism in vivo.

In vitro biotransformation of aflatoxin B1 (AFB1) in channel catfish liver.

The biotransformation of the dietary carcinogen aflatoxin B1 (AFB1) was examined in hepatic microsomal and cytosolic fractions from channel catfish, an aquatic species shown to be refractory to AFB1 toxicity and reported to be resistant to AFB1 hepatocarcinogenesis. Catfish liver microsomes catalyzed the in vitro oxidation of AFB1 to the reactive AFB1-8,9-epoxide (AFBO) at high substrate concentrations (128 microM AFB1) but not at low substrate concentrations (16 microM AFB1) which were more representative of environmental exposure. A similar trend was observed for the production of the hydroxylated metabolite aflatoxin M1 (AFM1). In contrast, hepatic microsomes prepared from rainbow trout, a species sensitive to AFB1 toxicity and hepatocarcinogenesis, activated AFB1 at 16 and 128 microM AFB1, with the rate of AFB1 epoxidation by trout microsomes exceeding that of catfish by more than sixfold. Treatment of channel catfish with 5,6-benzoflavone (beta NF, 20 mg/kg) resulted in a threefold increase in AFM1 formation but did not affect AFBO formation. AFB1 was rapidly reduced to aflatoxicol (AFL), a putative detoxification product of AFB1, at both low and high substrate concentrations. The rate of AFL production by channel catfish hepatic cytosol was 40- and 65-fold greater than observed for rainbow trout at 16 and 128 microM AFB1, respectively. Western blotting of catfish cytosols revealed the presence of a catfish cytosolic protein of approximately 25 kDa that displayed immunological cross-reactivity to rat GST Ya, but not to other rat or mouse alpha class GSTs with high AFBO-conjugating activity. Furthermore, catfish cytosolic GSTs did not catalyze the conjugation of AFBO with GSH. The results of these studies indicate that AFB1 is poorly oxidized by channel catfish microsomes, and suggest that the lack of microsomal AFB1 activation together with the rapid conversion of AFB1 to AFL contributes to the apparent resistance of channel catfish to AFB1 toxicity and hepatocarcinogenesis.

Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.

Effect of dietary vitamin E (alpha-tocopherol) on aflatoxin B metabolism.

Intraperitoneal injection of rats with 2 mg/kg ring labelled 14C AFB1 (spec. act. 110 mCi/mM/nmole) showed a higher level of radioactivity in the urine of test animals on diets containing 600 mg/kg vit. E 24 h after pretreatment. Analysis of the urine by chloroform extraction, thin layer chromatography and liquid scintillation counting of the various fractions showed less aflatoxin M1 (AFM1) and less unmetabolized AFB1 in test samples than in controls. Incubation of ring labelled 14C AFB1 with hepatic 10,000 g supernatant fractions, however, showed increased metabolism of AFB1 by fractions from test animals as compared with the controls. Rate of disappearance of 14C AFB1 and the consequent formation of AFM1 was greater in the test fractions than in the controls. At 30 days all test animals showed higher levels of serum vitamin E than the controls. Hepatic aniline hydroxylase and ethyl morphine N-demethylase activities of the liver fractions and blood glutathione reductase activity were greater in the tests. P-nitroanisole-O-demethylase activity was reduced while hepatic and serum reduced glutathione levels remained basically unaltered.

Studies on the formation of aflatoxin M1 in bakery products. Mycotoxins in foodstuffs. XIII.

The aflatoxinogenic moulds Aspergillus flavus NRRL 3251, A.parasiticus CMI 89717 and A.parasiticus NRRL 2999 are able to grow on various types of bread, yet, only the last fungus can produce aflatoxin M1 on these substrates. The highest yields (4 micrograms/kg) of the toxin were found on whole wheat bread at a "Säuregrad" (total acid content) of 5-6 and at an incubation temperature of 30 degrees C. The toxin levels decreased with increasing time of growth; this was probably due to a further metabolism of aflatoxin M1. A.flavus NRRL 3251 and A.parasiticus CMI 89717 grew better and produced more toxin on cake ("Rührkuchen") with aw 0.92 than on cake with lower water activity.

Metabolism of aflatoxin B1 in the isolated nuclei of rat liver.

The nuclear biotransformation of aflatoxin B1 in vitro was observed with regard to inducer specificity, pH dependency, time course, kinetics, inhibitor sensitivity, and nuclear localization, and these data were compared with those from the microsomal transformation of aflatoxin B1. The nuclei and microsomes are capable of metabolizing aflatoxin B1 into aflatoxin M1, aflatoxin Q1, and two unidentified fluorescent compounds in the presence of fortified NADPH generating system. Pretreatments of rats by 3-methylcholanthrene or polychlorinated biphenyl enhanced both the nuclear and microsomal C-9 alpha-hydroxylation of aflatoxin B1 into aflatoxin M1 and phenobarbital or polychlorinated biphenyl induced aflatoxin Q1 production. The optimal pHs for aflatoxin M1 and Q1 were 8.3 and 7.4, respectively, both in the nuclei and microsomes. Kinetic analysis revealed the Km of aflatoxin M1 formation in methylcholanthrene-induced nuclei was 9.4 x 10(-5) M, and this value was very close to that obtained with the microsomes. Inhibitor experiments revealed a high sensitivity of aflatoxin M1 formation to 7,8-benzoflavone and a low sensitivity of aflatoxin Q1 to SKF 525A. These findings and data on the detergent treatment of nuclei suggest that the nuclear cytochrome P-448 system, induced by 3-methylcholanthrene and localized in the outer membrane, catalyzes the aflatoxin M1 formation, and the cytochrome P-450 system induced by phenobarbital biotransforms aflatoxin B1 into aflatoxin Q1. Pretreatment of rats by phenobarbital was found to induce microsomal degradation or detoxication of aflatoxin B1 into water-soluble metabolites, and no such an induction was observed in the nuclei.