Biotransformation studies using rat proximal tubule cells.

The proximal tubule is the main target for nephrotoxic substances due to its specific properties, including efficient drug transport and biotransformation potential. The availability of a pure population of proximal tubule cells (PT cells) as a model to study a range of biological, pharmacological, and toxicological parameters is, therefore, of great value. A two-step PT cell-isolation procedure, based on density-gradient centrifugation, is described; this procedure can easily be introduced into each laboratory setting. The procedure routinely yields a highly pure PT cell population, comprising 20-40 × 10(6) cells, which can be used for preparation of subcellular fractions or brought into primary culture.

The effects of mebendazole on P4501A activity in rat hepatocytes and HepG2 cells. Comparison with tiabendazole and omeprazole.

Mebendazole is a benzimidazole anthelmintic widely used in veterinary and human therapy. Among benzimidazole derivatives, several drugs with inducing effect on cytochromes P450 can be found. However, the induction capacity of mebendazole on P450s has not been explored yet. In this study, the effects of mebendazole on P4501A activity was tested in primary cultures of rat hepatocytes and in human hepatoma HepG2 cell line. Two known P4501A inducers with benzimidazole structure, tiabendazole and omeprazole, were also included in the experiments with the aim of studying structure-induction relationships. After 24-, 48- and 72-h incubation of rat hepatocytes and HepG2 cells with drugs in various concentrations (0.1-100 microM), enzyme activity associated with P4501A1/2 (EROD, MROD) was measured. In addition, the P4501A1/2 protein levels in both in-vitro systems were determined by Western-blotting. Mebendazole provoked a significant increase in P4501A1/2 protein expression and P4501A activity in both in-vitro systems. Omeprazole caused a significant dose-dependent increase of P4501A activity only in HepG2 cells. Although tiabendazole treatment led to significant increase of P4501A protein level, no effect on P4501A activity was observed in either system. The results demonstrate that mebendazole possesses the ability to significantly induce P4501A. Thus, pharmacological and toxicological consequences of P4501A induction should be taken into account in human therapy. The structure-induction relationships and differences between in-vitro systems used are discussed.

The effects of benzimidazole anthelmintics on P4501A in rat hepatocytes and HepG2 cells.

Benzimidazole anthelmintics including albendazole, fenbendazole, and mebendazole are widely used in veterinary medicine. The effects of these benzimidazoles on cytochrome P4501A were investigated in primary cultures of rat hepatocytes and in the HepG2 cell line. After incubation of rat hepatocytes and HepG2 for 24-, 48-, and 72-h cells with drugs at various concentrations (0.1-50 microM), the enzyme activities associated with P4501A1/2 (7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation) were measured. The P4501A1/2 protein levels in both model systems were determined by Western blotting. Although all benzimidazoles provoked a significant increase of P4501A1/2 protein levels and P4501A activities, large differences in the induction response were found which was dependent on drug structure, concentration, and model system used. Based on the results, relationships between induction potency and structure of drug were demonstrated, as well as differences between the in vitro systems used. Therefore, pharmacological and toxicological consequences of cytochrome P4501A induction by benzimidazole drugs should be taken into account in veterinary therapy.

Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells.

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.

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.

Mutagenicity of commercial Monascus fermentation products and the role of citrinin contamination.

Pigments produced as secondary metabolites by various isolates of moulds belonging to the genus Monascus have been used traditionally as colorants in Oriental food. Modern food industry has rediscovered these moulds as promising source for natural colorants. However, recent studies evidence that one of the secondary metabolites produced by Monascus is identical in structure to the mycotoxin citrinin. Thus, a sensitive HPLC method was developed to analyse these food colorants for contamination with citrinin. The mycotoxin could be detected in all the commercial Monascus samples at concentrations varying between 0.2 to 17.1 microg/g. In addition, the mutagenicity of commercial Monascus samples applying Salmonella-microsome assay and Salmonella-hepatocyte-assay was investigated and compared to the results obtained with citrinin. Citrinin and two Monascus extracts induced a positive dose depending mutagenic response in the Salmonella-hepatocyte-assay applying strain TA-98. However, no mutagenicity could be detected in the Salmonella-microsome assay, neither with nor without S9-mix, for citrinin and Monascus extracts, applying TA-98, TA-100, TA-1535, TA-1538 and TA-97. These findings provide further evidence that citrinin requires complex cellular biotransformation to exert mutagenicity.

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.

In vitro and in vivo oxidative biotransformation in the West-African dwarf goat (Caprus hircus aegagrus): substrate activities and effects of inducers.

1. Cytochrome P450 activities in vivo and in vitro and enzyme induction by phenobarbital, beta-naphthoflavone, isoniazid and triacetyloleandomycin were investigated in the female dwarf goat. In vivo kinetics of antipyrine, sulphadimidine and caffeine were studied separately and as a combination ("cocktail'). After establishing a lack of interaction between these compounds the effects of the inducing agents were investigated. In vitro, hepatic microsomal enzyme activities and apoprotein levels were determined. 2. In the beta-naphthoflavone treated goat, the microsomal ethoxy-resorufin-O-deethylation rate was markedly increased. beta-naphthoflavone also induced caffeine plasma clearance but did not affect microsomal caffeine 1- and 3-demethylation rates. After phenobarbital treatment, caffeine plasma clearance was also increased. In contrast with beta-naphthoflavone treatment, phenobarbital treatment resulted in an increase of microsomal caffeine 1- and 3-demethylation rates. 3. Goat liver microsomes were able to hydroxylate tolbutamide, predominantly a CYP2C9 activity in man, and debrisoquine, a CYP2D activity in different species. These activities were not affected by either beta-naphthoflavone or phenobarbital. Sulphaphenazole was found to be a more potent inhibitor of tolbutamide hydroxylation than sulphadimethoxine. Quinine was a more potent inhibitor of debrisoquine hydroxylation than was quinidine. 4. As expected, the microsomal aniline-4-hydroxylation rate (CYP2E) was increased after isoniazid treatment. 5. The microsomal testosterone 6 beta-hydroxylation rate (CYP3A) was increased after phenobarbital and triacetyloleandomycin treatment. Antipyrine plasma clearance was also increased after phenobarbital treatment. 6. As cytochrome P450 activities and inducibility in the dwarf goat show many resemblances to those in man, they may be of value as a model for human biotransformation research.

Cytochrome P4502E in vivo and in vitro in the dwarf goat: effects of enzyme induction and the applicability of chlorzoxazone as marker substrate.

Cytochrome P4502E activities, inducibility and the applicability of chlorzoxazone as a marker substrate for this enzyme were investigated in female dwarf goats. Goats were treated with either isoniazid or beta-naphthoflavone. Treatment with isoniazid resulted in a 1.4 fold increase of the chlorzoxazone hydroxylation rate in hepatic microsomes. Aniline- and p-nitrophenol hydroxylation rates were increased by roughly the same extent (1.6 and 1.25 fold resp.) and increased levels of cytochrome P4502E apoproteins were found by Western blotting. Treatment with the cytochrome P4501A inducer beta-naphthoflavone resulted in a 2.5 fold induction of the in vitro chlorzoxazone hydroxylation rate, whereas the hydroxylation rates of aniline and p-nitrophenol were not induced. After treatment with isoniazid, chlorzoxazone plasma clearance was increased from 5.0 mL/min/kg to 11.0 mL/min/kg. Chlorzoxazone was almost completely excreted in the urine as conjugated hydroxy metabolites. These results do not support the hypothesis that cytochrome P4502E is of particular importance in goats, as has been suggested earlier. Furthermore, chlorzoxazone has limited value as a marker substrate for this enzyme, since cytochrome P4501A enzymes appear to play an important role in its biotransformation.

Oral bioavailability and pharmacokinetics of baquiloprim in dwarf goats.

The pharmacokinetics of baquiloprim at a dose of 8 mg kg-1 bodyweight were determined after its intravenous and intra-ruminal administration to seven healthy female dwarf goats. After intravenous injection, the plasma elimination curve showed a rapid distribution phase (mean [SD] t1/2 alpha 0.89 [0.4] hours). The mean volume of distribution at steady-state (Vdss) was 14.1 (2.7) litres kg-1 bodyweight. The mean elimination half-life (t1/2 beta) was 14.0 (2.3) hours. After intra-ruminal administration its maximum concentration in plasma (Cmax) was 0.09 (0.01 microgram ml-1 and this maximum was not reached until approximately 35 hours after administration. The systemic oral bioavailability, calculated up to 48 hours after dosing, was 33.7 (7.1) per cent. Owing to a prolonged absorption phase, the data from only four of the goats fitted reasonably to a compartmental model.