Effects of AFB1 on CYP 1A1, 1A2 and 3A6 mRNA, and P450 expression in primary culture of rabbit hepatocytes.

Although numerous studies report strong hepatic cytochrome P450 decrease during aflatoxicosis, the mechanisms involved in this decrease remain to be established. The purpose of this work is to investigate whether decreased CYP mRNA expression could explain decreased P450 expression and activity. Studies were conducted in primary cultures of rabbit hepatocytes exposed to 0.1 and 1 microM aflatoxin B1 (AFB1) incubated in the culture medium for 72 h. In order to confirm the effects of the mycotoxin, 30 microM beta-naphthoflavone or rifampicin were used as respective inductors of P450 1A1 and 1A2 or 3A6. Dose-dependent decreases of CYP mRNA expression were observed in all AFB1-treated cells; however, these decreases were not specific. Moreover, P450 expression and activity are far less decreased by the AFB1 treatment than their corresponding mRNA. Taken together, these results suggest that the specific P450 decrease observed during aflatoxicosis was not the consequence of a specific decrease of their mRNA expression.

The effects of T-2 toxin exposure on liver drug metabolizing enzymes in rabbit.

High doses of T-2 toxin are known to decrease protein synthesis and mono-oxygenase activities in rat liver. The purpose of this study was to investigate whether exposure at a low dose could alter the normal metabolism of the xenobiotic by the liver. Three doses of T-2 toxin, dissolved in olive oil, were orally and daily administered to New Zealand white rabbits for five days. At 0.5 mg/kg, three of the five animals died, whereas only a weak decrease in body weight gain and moderate signs of toxicity occurred in rabbits receiving 0.25 mg/kg/day, and the body weight increased without signs of toxicity at 0.1 mg/kg/day. At 0.25 mg/kg/day, total liver microsomal P450 content, and the activities of aminopyrine and benzphetamine N-demethylases, pentoxyresorufin O-depentylase, glutathione S-transferases accepting 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates, were decreased. By contrast, ethylmorphine and erythromycin N-demethylases, ethoxyresorufin and methoxyresorufin O-dealkylases, aniline hydroxylase, and UDP-glucuronyltransferase accepting p-nitrophenol as substrate, were unaffected. The expression of P450 1A1, 1A2, 2A1, and 2B4, but not P450 2C3 and 3A6, were also decreased, whereas microsomal conjugated dienes, fluorescent substances, and malondialdehyde contents were increased. At 0.1 mg/kg/day, neither significant effects on drug metabolizing enzymes nor microsomal oxidative damages were obtained. Taken together, these results suggest that a short exposure time to the mycotoxin would not be associated with significant changes in the normal metabolism of xenobiotics by the liver.

Cytochrome P450 decreases are correlated to increased microsomal oxidative damage in rabbit liver and primary cultures of rabbit hepatocytes exposed to AFB1.

Although numerous studies report hepatic drug metabolizing enzyme alterations during aflatoxicosis, the mechanisms involved in P450 decreases remain to be established. The purpose of this work is to investigate whether increased oxidative damage revealed by the detection of malondialdehyde (MDA), lipofuscin substances, and conjugated dienes in microsomes, could explain the decreased P450 content. Studies were conducted with two different doses of aflatoxin B1 (AFB1), both in vivo in rabbits and ex vivo in primary cultures of rabbit hepatocytes, in the presence or absence of beta-naphthoflavone or rifampicin used as respective P450 inducers. Strong negative correlations were observed between MDA and P450 contents, both in vivo and ex vivo, whereas rifampicin appears to protect the hepatocytes from oxidative damage but not AFB1 toxicity. Positive correlation were also obtained between MDA formation and lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT) or alanine amino-transferase (ALAT) releases, used as non-specific markers of AFB1 toxicity. Taken together these results suggest that the dramatic decreases of cytochrome P450 observed in vivo during aflatoxicosis could be linked, at least in part, to microsomal oxidative damage.

Glyphosate toxicosis in domestic animals: a survey from the data of the Centre National d'Informations Toxicologiques Veterinaires (CNITV).

A retrospective study was conducted of 482 glyphosate calls recorded at the Centre National d'Informations Toxicologiques Veterinaires (CNITV) of France between 1991 and 1994. Most of the calls came from veterinary practitioners (83.8%) and were related to emergencies. In the majority of calls, the CNITV did not assess clinical observations as certain or highly probable glyphosate poisoning. Only 31 cases were assessed as certain or highly probable and were linked with direct ingestion of glyphosate concentrates or sprays in 25 dogs. The symptoms were most frequently described as vomiting, hypersalivation and diarrhea; prostration and paresis were not common. Symptomatic treatment resulted in rapid recovery without sequelae.

Dose-related increase in liver heme catabolism during rabbit aflatoxicosis.

Aflatoxin B1 (AFB1) has been reported to decrease microsomal hepatic cytochrome P450 (P450) content and increase both total plasma bilirubin concentration and liver heme oxygenase activity. The purposes of this study were to determine whether liver hemoproteins contents and heme catabolizing enzymes were affected by the mycotoxin and whether these alterations were linked to hyperbilirubinemia. Male New Zealand rabbits were divided into three groups of five animals, each receiving for 5 days either arabic gum as vehicle or AFB1 at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments affected neither cytochrome b5 content nor NADPH-cytochrome reductase activity. A linear dose-dependent decrease in cytochrome P450 content and increases in both heme oxygenase and biliverdin reductase activities were observed. Bilirubin UDP-glucuronyltransferase activity was dramatically decreased at both doses, whereas cholestasis occurred only at 0.10 mg/kg. An exponential dose-dependent increase in plasma bilirubin concentration was also observed. Both the simultaneous exponential increase in bilirubinemia associated to a reduced bilirubin UDP-glucuronyltransferase activity and the absence of cholestasis at 0.05 mg/kg, suggested that the hyperbilirubinemia is more probably related to an increased heme catabolism than to an altered bile duct permeability.

In vitro interaction of AFB1 with rabbit liver monooxygenase activities.

The purpose of this study was to determine the influence of aflatoxin B1 (AFB1), incubated in vitro with rabbit liver microsomes, on some cytochrome P450-dependent monooxygenases activities. A strong competitive inhibition of the mycotoxin on aniline hydroxylation was observed. The concentration which provoked a 50% inhibition (IC50) was around 20 microM, whereas a Ki of 3 microM was determined. In contrast, only weak inhibitions of both pentoxyresorufin and ethoxyresorufin O-dealkylases (PROD and EROD) activities were obtained. They were characterized by respective IC50 of 200 and 260 microM. The inhibition was 'non competitive' for PROD activity and 'mixed' for EROD. The Ki of the reactions were respectively 177 and 510 microM. Considering the fact that AFB1 has been previously reported to decrease microsomal hepatic cytochrome P450 expression, the results obtained in this study strengthen the hypothesis that the normal metabolism of xenobiotics by the liver could be altered in AFB1 exposure.

Evaluation of an HPTLC method for the determination of strychnine and crimidine in biological samples.

In an attempt to improve the sensitivity and selectivity of thin-layer chromatographic analysis for the detection of strychnine and crimidine in biological samples, a rapid high-performance thin-layer chromatographic (HPTLC) method with densitometry is described. Fortified dog serum and stomach content samples were analyzed after extraction with chloroform. Quantitation was achieved by densitometry in the ultraviolet (UV) range (260 nm) of HPTLC silica gel 60 plates. Detection of trace levels as low as 5 ng proved feasible. Linearity was obtained over a range of 10-250-ng deposits for crimidine and 12.5-250-ng deposits for strychnine with simple or F254 plates. No interferences were observed in the UV spectra (220-380 nm) when peaks obtained with HPTLC of the standard substances and positive biological contents were scanned.

Dose-related effect of aflatoxin B1 on liver drug metabolizing enzymes in rabbit.

The effects of chronic administration of aflatoxin B1 (AFB1) on liver drug metabolism enzymes were measured in New Zealand rabbits divided into three groups of 5 animals, each receiving over 5 days either arabic gum or AFB1 in arabic gum at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments did not lead to any lethality in any of the treated groups, but the body weight gain was altered. Biochemical exploration of plasma components revealed a dose-dependent hepatotoxicity characterized by cytolysis and cholestasis. At 0.10 mg/kd/day of AFB1, significant decreases were observed in total liver microsomal cytochrome P450, several P450-dependent monooxygenase activities, all individual P450 isoenzymes levels analysed by Western-blotting and glutathione S-transferase activities. By contrast, at 0.05 mg/kg/day of AFB1, even though total cytochrome P450 was decreased by 30%, only P450 1A1 and 3A6 isoenzymes, and aniline hydroxylation, pentoxyresorufin O-depentylation, aminopyrine, erythromycin, ethylmorphine and dimethylnitrosamine N-demethylations were affected. In the same animal group, the only glutathione S-transferase accepting CDNB (1-chloro-2,4-dinitrobenzene) as substrate was decreased by 22%. UDP-glucuronyltransferase accepting p-nitrophenol as substrate was increased in both groups of animals (33-62%). The mechanisms that could contribute to the observed changes in drug metabolizing enzymes are discussed.

Reduced cytochrome P450 and increased heme oxygenase in liver during rabbit aflatoxicosis.

The administration of aflatoxin B1 (AFB1) in New Zealand rabbit for 5 days at a daily oral dose of 0.05 or 0.1 mg/kg decreased microsomal hepatic cytochrome P450 whereas a dose-dependent increase in the reduced microsomal 420 nm absorption occurred. The nature of such an absorption was then investigated. Either in vitro incubation of control microsomal proteins with AFB1 up to 800 microM and NADPH, or primary rabbit hepatocyte cultures exposure to AFB1 up to 30 microM for 24 to 72 h, failed to produce any 420 nm absorbing species, suggesting that the 420 nm absorption observed in vivo was due to an hemoprotein increase. Chemical reductions of microsomal proteins from AFB1-treated rabbits confirmed this hypothesis. Enzyme activity determinations revealed an increase in both microsomal heme oxygenase and NADPH-cytochrome c reductase activities in AFB1 treated rabbits, suggesting that the 420 nm absorption observed in vivo was related to a particular increase in heme oxygenase.

[Residues of drugs of veterinary use in food]. / Les résidus de médicaments à usage vétérinaire dans les aliments.

A rational use of veterinary drugs is one of the essential factors in the production of healthy food of animal origin, but residues may persist in such foods. These residues are present in very small amounts, and they create no public health problem as long as their toxicological significance is evaluated scientifically. This evaluation, which is made for each veterinary drug prior to applying for authorization to market, leads to the definition of tolerable residual levels as a measure designed to protect the customers. This general principle is illustrated by two examples: the risk of allergic intolerance to foods, due to traces of particularly active substances, such as penicillins; such residues may occasionally be allergenic, but they are certainly not immunogenic; the problems associated with the use of hormonal or hormone-related compounds endowed with anabolic properties. The current interdiction of these compounds does not rest on solid scientific grounds.

[Covalent fixation in vivo of trenbolone acetate and estradiol to hepatic DNA of the rat: comparative study of the fixation of tritiated molecules and post labeling with radiophosphorus]. / Fixation covalente in vivo de la trenbolone et de l'estradiol à l'ADN hépatique de rat: étude comparative de la fixation de molécules tritiées et du postmarquage au radiophosphore.

In vivo covalent binding of trenbolone and estradiol was assayed using either radiolabeled compounds or 32P-post labeling. The covalent binding index, as measured with tritiated molecules, was 2.4 for the alpha isomer of trenbolone, 5.4 for its beta isomer and 5.4 for 17-beta estradiol. Using 32P-post labeling at repeated medium doses or a single high dose did not allow any of the 3 compounds to reveal specific adducts in the background of adducts spontaneously formed in control animals. It can therefore be concluded that these steroids most probably do not have a direct genotoxic action.

[Effects of sporidesmin on liver zinc, metallothionein and cytochromes in vivo]. / Effets de la sporidesmine sur le zinc, la métallothionéine et les cytochromes hépatiques in vivo.

In vitro studies have suggested that sporidesmin hepatotoxicity may be related to thiol oxidation and generation of cytotoxic oxygen species. After a single i.p. injection of 2.8 mg/kg bw sporidesmin in guinea-pigs, hepatic and plasma zinc, hepatic metallothionein, cytochromes P-450 and b5, total glutathione and proteins (total, microsomal and cytosolic) were monitored for 21 days. The only variations observed were significant increases in liver concentrations of zinc (cytosolic and total), metallothionein, and cytochromes, which peaked on day 8 after the sporidesmin challenge (+45, 55, 50, 376 and 413%, respectively) and, except for cytochrome b5, went back to control levels before the 21st day. These results suggest that cytochromes P-450 and b5 may be involved in sporidesmin cellular damage.

Liver enzyme changes in a guinea-pig model of facial eczema (sporidesmiotoxicosis).

Sporidesmin, a hepatotoxin from Pithomyces chartarum, is responsible for facial eczema in ruminants. In an attempt to clarify the biochemical processes supporting sporidesmin toxicity and response of the liver, haematology, plasma biochemistry and liver enzyme changes were monitored for 21 days in a model for facial eczema resulting from a single intraperitoneal injection of 2.8 mg/kg BW sporidesmin to guinea pigs. Most plasma disturbances were observed 8 days after administration and accounted for starvation, liver cytolysis, and cholestasis or liver enzyme induction. Alterations of hepatic enzyme activities were intense with a maximum increase on days 2 for alkaline phosphatases (ALP) and 8 for gamma-glutamyltransferase (GGT), and a maximum decrease on day 21 for aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT). Comparison of liver and plasma enzyme changes indicates that GGT was the most reliable and significant plasma indicator of sporidesmin-associated liver alterations. Moreover, this study points out the validity of the one-dose intoxicated guinea-pig model for research on sporidesmin biochemical toxicity and pathobiology of facial eczema.

Application of whole-body autoradiography in toxicology.

Whole-body autoradiography enables the drugs and toxicants to be distributed throughout the animal. Good results are obtained with this technique. However, certain artifacts can occur that could lead to misinterpretation, and these must be known. These artifacts are described. From the metabolic point of view, autoradiography provides data on the distribution kinetics of a compound and the elimination of radioactivity in various organs. These data are a guide for quantitative research into the metabolism of a compound. From the toxicological point of view, it must be admitted that the main purpose of this technique is to reveal the sites of retention of radioactivity. Such specific organ retention could be the consequence of the activation of a minor metabolite into a very reactive compound. If this is so, it is a specific organ effect which could not be studied by other techniques and could lead the way to a more specific organ effect which could not be studied by other techniques and could lead the way to a more appropriate line of research in the study of chronic toxicity. However, it must be recalled that the fact that a compound is retained by a specific organ does not always mean that the compound exerts a toxic effect upon the said organ. With this technique, distribution study can be performed on pregnant animals, and it provides us with more data concerning the transplacental passage of radioactive metabolites. All these aspects of the technique clearly indicate that whole-body autoradiography should be insisted upon during the early stages of development of new molecules. Successive experiments could then lead to selecting the best experimental conditions for metabolic pharmacokinetics and studies in toxicology.

Gamma Glutamyl Transferase in domestic animals.

In domestic animals, Gamma Glutamyl Transferase is mainly in the kidneys, the pancreas and the intestine; its liver activity is relatively high in cows, horses, sheep and goats and very low in dogs, cats and birds. The use of plasma reference values can help to interpret the variations of serum GGT mainly in hepatobiliary diseases of cattle, sheep, goats and cholestatic disorders of dogs. Urinary GGT is a good test of kidney toxic damage.

[General schema for safety evaluation of residues of embryotoxic drugs (author's transl)]. / Schéma général d'évaluation de la sécurité des résidus de médicaments embryotoxiques.

Concerned with adapting the withdrawal time to real risks attributed to residues, the authors present a general schema of evaluation based on the metabolism-toxicity relationship. This schema takes into account: a) possible distinction between potentially toxic and atoxic metabolites for extractable residues, b) the more or less large biovailability of residues, c) the methodological evaluation difficulties of toxicity of bound residues. Without neglecting public health, this procedure leads to less constrained restrictions in use of veterinary drugs.