Comparative in vitro study of the biotransformation of n-alkanes by liver and small intestine microsomes from different rat strains

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Percutaneous absorption and metabolism of [14C]-ethoxycoumarin in a pig ear skin model

International audienceThe biotransformation of chemicals by the skin can be a critical determinant of systemic exposure in humans following dermal absorption. Pig ear skin, which closely resembles human skin, is a candidate ex vivo alternative model for the investigation of xenobiotics penetration and metabolism. We developed an ex vivo pig ear skin model and explored its absorption, diffusion and metabolic capabilities using the model compound 14C-ethoxycoumarin (7-EC). Experimentations were undertaken on pig ear skin explants after application of various 14C-EC doses. Diffusion was quantified as well as the production of 7-EC metabolites resulting from phases I and II enzyme activities, using radio-HPLC. After 48 h, most of the radioactivity was absorbed and was recovered in culture media (70%) or in the skin itself (10%). 7-EC metabolites were identified as 7-hydroxycoumarin (OH–C) and the corresponding sulfate (S–O–C) and glucuronide (G–O–C) conjugates. Their formation followed Michaelis–Menten kinetics with saturation reached around 100 μM of 7-EC. Results demonstrate that dermal absorption as well as phases I and II enzymatic activities of pig skin are both functional. This model should represent a valuable alternative for the study of the transdermal exposure to chemicals, combining a functional dermal barrier and active biotransformation capabilities

Disposition of benzo[c] fluorene in rats

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants and food contaminants. Some of them are mutagenic/genotoxic and have shown clear carcinogenic effects in mammals. Among the PAHs found in food commodities, benzo[c]fluorene (B[c]F) was demonstrated to be carcinogenic in rodents and the occurrence of corresponding pulmonary adducts has been demonstrated, suggesting a bioactivation pathway. B[c]F distribution and biotransformation was studied in rats given a single oral dose of radiolabelled B[c]F (0.64 or 640 μg/kg body weight). At intervals of 2, 4, 6, 8, 12, 24 and 48 h thereafter, animals were sacrificed, excreta were collected and various tissues were sampled. Radioactivity was measured in all samples before extraction with dichloromethane/methanol mixtures. Metabolic profiles were performed by radio-HPLC and metabolites were analyzed by LC–MS/MS. Results shown that B[c]F was extensively absorbed and biotransformed in rats. The metabolic balance at 48 h indicates that 8–10% of the radioactivity was eliminated in urine while 55–69% was found in faeces, depending on administered dose. The major part of fecal radioactivity corresponded to unchanged B[c]F, whereas analysis of urine samples revealed only polar metabolites (mainly glucurono- and sulfo-conjugates of mono-, di-, tri- and tetra-hydroxylated B[c]F). The highest concentrations of radioactivity in tissues were found in liver, irrespective of the administered dose, the highest values being recovered 2 h post dosing. The characterization of the metabolic pathways of BcF in rat is in progress, with the objective to identify reactive metabolites and to better understand the mechanisms of genotoxicity of this contaminant

Comparative metabolism of clenbuterol by rat and bovine liver microsomes and slices. Drug Metab Dispos 26

This paper is available online at http://www.dmd.org ABSTRACT: The metabolism of clenbuterol by liver microsomal fractions and precision-cut liver slices was studied in rats and cattle using a 14 C-labeled molecule and radio-HPLC quantitation of the resulting metabolites. 4-N-Oxidation of clenbuterol was found to be an extensive in vitro metabolic pathway in both species. Clenbuterol hydroxylamine was by far the major metabolite characterized from microsomal and slice incubation media. Trace amounts of 4-nitroclenbuterol were also detected. Another important microsomal biotransformation of clenbuterol, resulting in the production of 4-amino-3,5-dichlorobenzoic acid, was observed only when the drug was incubated with bovine liver microsomes. The corresponding glycine conjugate, namely 4-amino-3,5-dichlorohippuric acid, was detected when clenbuterol was incubated with bovine or rat liver slices. Structural characterization of the major metabolites was performed using electrospray ionization-mass spectrometry, either coupled to liquid chromatography or with direct infusion of collected samples. In addition to these compounds, only quantitatively minor metabolites were detected in bovine (but not rat) microsomal incubation media. Analysis of incubation media from liver slices also allowed the quantitation of a few additional metabolites, some of which were shown to be conjugated compounds

Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances

Metabolism and estrogenicity of benzophenone-2 and bisphenol-S in zebrafish in vitro and in vivo models

Among endocrine disruptors, benzophenones and bisphenols detected in human, in the aquatic environment and in fish, are of major concerns to human and wildlife health. Among benzophenones (UV filters) and bisphenols, benzophenone-2 (BP2) and bisphenos S (BPS, bisphenol A substitute) are hormonally active compounds that have been shown to be estrogenic and to disrupt the hormonal regulation in rat and/or fish. Several human and aquatic vertebrate bioassays have been designed over the last years with the aim to characterize the estrogeno-mimetic potential of chemicals. However, the characterization of the biotransformation capability of these biological models, which allows taking into account bioactivation/detoxification processes in effect assessment, is rarely reported. We have recently developed novel zebrafish assays used to assess the estrogenic potency of chemicals. They included ZELH zfERs cell lines based on the expression of zebrafish estrogen receptors in the hepatic cell line ZFL (ZELH zfERs), and the EASZY assay based on the expression of the Green Fluorescent Protein under the control of the cyp19a1b promoter in the zebrafish larva. They were used to investigate in a global approach, the fate and the estrogenic potency of BP2 and BPS. Metabolism of tritium-labeled chemicals was explored using radio-HPLC (metabolic profiling), and metabolite identification was investigated using biochemical tools and high resolution mass spectrometry. Whereas BP2 and BPS were found to be estrogenic in ZELH zfERs cells and in adult zebrafish, no disruption of expression of estrogeno-regulated genes specific of brain and liver in larva was observed. Regarding the BP2 and the BPS metabolism, similar metabolic profiles were shared by zebrafish in vitro and in vivo models suggesting a well conservation of metabolic capabilities. Overall, this study provides new and relevant data concerning the fate and the estrogenic activity of BP2 and BPS in novel zebrafish assays, allowing a better characterization of their respective biotransformation capabilities which is required for their use in the context of endocrine disruptors testing