Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B(1) in bovine hepatocytes.

Aflatoxin B(1) (AFB(1)) biotransformation comprises cytochrome P450-mediated reactions resulting in hydroxylated and demethylated metabolites as well as AFB(1) epoxides. As the latter are highly nucleophilic, the species-specific rate of epoxidation and the ability for rapid conjugation to glutathione by glutathione S-transferase determines the individual susceptibility to AFB(1). Here we show the time- and dose-dependent rate of AFB(1)-metabolism in bovine hepatocytes. Aflatoxin M(1) (AFM(1)) is the most prominent metabolite formed within the first 2-8 hr of incubation, whereas AFB(1)-dhd is detectable in medium mainly after a prolonged incubation period. The delayed formation of AFB(1)-dhd corresponds to the cytotoxicity demonstrated by the MTT assay. alpha-Naphthoflavone and ketoconazole, inhibitors of CYP1A and CYP3A, respectively in humans, were used to evaluate the contribution of specific P450 isoenzymes in bovine biotransformation of AFB(1). Initial experiments confirmed that alpha-naphthoflavone and ketoconazole inhibited ethoxyresorufin O-deethylation and testosterone 6beta-hydroxylation also in bovine hepatocytes. Both inhibitors reduced AFM(1) and AFB(1)-dhd formation concentration dependently, suggesting that both enzyme groups contribute to the formation of these metabolites. However, the formation of AFM(1) was less inhibited by both compounds than the formation of AFB(1)-dhd.

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.

17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle.

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17alpha-ethyl-5beta-estrane-3alpha, 17beta-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.

In vitro liver models are important tools to monitor the abuse of anabolic steroids in cattle.

Current veterinary residue analysis mainly focuses on the monitoring of residues of the administered parent compound. However, it is possible that larger amounts of metabolites are excreted and that they can have a prolonged excretion period. In order to unravel specific metabolic steps and to identify possible biological markers, two in vitro liver models were used, i.e. monolayer cultures of isolated hepatocytes and liver microsomes, both prepared from liver tissue of cattle. Chostebol, boldenone, norethandrolone (NE) and ethylestrenol (EES) were used as model substrates. Results show that the metabolic profiles derived from in vitro experiments are predictive for the in vivo metabolic pathways of the steroids evaluated in this study. By means of this strategy, it is possible to identify 17 alpha-ethyl-5 beta-estrane-3 alpha,17 beta-diol (EED) as a common biological marker for NE and EES. By in vivo experiments it was shown that EED is particularly important for the detection of the abuse of NE or EES because of its high excretion levels and its prolonged presence as compared with the parent compounds or any other metabolite.

Identification of some important metabolites of boldenone in urine and feces of cattle by gas chromatography-mass spectrometry.

17 alpha-Boldenone (17 alpha-BOL) and/or 17 beta-boldenone (17 beta-BOL) appear occasionally in fecal matter of cattle. In addition to 17 alpha-BOL, a whole array of boldenone related substances can be found in the same samples. In vitro experiments with microsomal liver preparations and isolated hepatocytes combined with the excretion profiles found in urine and feces samples of in vivo experiments made it possible to identify several metabolites of 17 beta-BOL in 17 beta-BOL positive feces samples. In one animal treated with 17 beta-BOL, no 17 beta-BOL or its metabolites were present before treatment and most of these compounds disappeared gradually in time after the treatment was stopped. It is not clear what the origin is of 17 alpha-BOL and boldenone metabolites in samples screened routinely for the abuse of anabolic steroids and considered to be 'negative' because of the absence of 17 beta-BOL since other workers showed some evidence that 17 alpha-BOL can be of endogenous origin. However, in our hands, most of these 17 alpha-BOL positive samples, obtained during routinely performed screenings of cattle, contained large amounts of delta 4-androstene-3,17-dione (AED), which normally is absent from routinely screened negative samples. Furthermore, AED was absent in all samples obtained from the animals treated with 17 beta-BOL. We have no direct evidence that 17 alpha-BOL or 17 beta-BOL is of endogenous origin.