Emerging mycotoxins induce hepatotoxicity in pigs' precision-cut liver slices and HepG2 cells.

Emerging mycotoxins are currently gaining more attention due to their high frequency of contamination in foods and grains. However, most data available in the literature are in vitro, with few in vivo results that prevent establishing their regulation. Beauvericin (BEA), enniatins (ENNs), emodin (EMO), apicidin (API) and aurofusarin (AFN) are emerging mycotoxins frequently found contaminating food and there is growing interest in studying their impact on the liver, a key organ in the metabolization of these components. We used an ex vivo model of precision-cut liver slices (PCLS) to verify morphological and transcriptional changes after acute exposure (4 h) to these mycotoxins. The human liver cell line HepG2 was used for comparison purposes. Most of the emerging mycotoxins were cytotoxic to the cells, except for AFN. In cells, BEA and ENNs were able to increase the expression of genes related to transcription factors, inflammation, and hepatic metabolism. In the explants, only ENN B1 led to significant changes in the morphology and expression of a few genes. Overall, our results demonstrate that BEA, ENNs, and API have the potential to be hepatotoxic.

The fate of bisphenol A, bisphenol S, and their respective glucuronide metabolites in ovarian cells.

Ovarian cells are critical for reproduction and steroidogenesis, which are functions that can be impacted by exposure to xenobiotics. As in other extra-hepatic tissues, biotransformation events may occur at the ovarian level. Such metabolic events deserve interest, notably as they may modulate the overall exposure and toxicity of xenobiotics. In this study, the comparative metabolic fate of two bisphenols was investigated in ovarian cells. Bisphenol A (BPA), a model endocrine disruptor, and its major substitute bisphenol S (BPS) were selected. Bovine granulosa cells (primary cultures) and theca explants (ex vivo tissue) were exposed for 24 hr to tritium-labeled BPA, BPS and their respective glucuronides (i.e. their major circulating forms), at concentrations consistent with low-dose exposure scenarios. Mass balance studies were performed, followed by radio-HPLC profiling. The capability of both cell compartments to biotransform BPA and BPS into their respective sulfo-conjugates was demonstrated, with sulfation being the predominant metabolic route. In theca, there was a significantly higher persistence of BPA (compared to BPS) residues over 24 hr. Moreover, only theca explants were able to deconjugate inactive BPA-glucuronide and BPS-glucuronide back into their biologically active aglycone forms. Deconjugation rates were demonstrated to be higher for BPS-G than for BPA-G. These findings raise concerns about the in situ direct release of bisphenols at the level of the ovary and demonstrate the relevance of exploring the biotransformation of bisphenols and their circulating metabolites in different ovarian cells with specific metabolic capabilities. This work also provides essential knowledge for the improved risk assessment of bisphenols.

Metabolism of versicolorin A, a genotoxic precursor of aflatoxin B1: Characterization of metabolites using in vitro production of standards.

The toxicity of mycotoxins containing bisfuranoid structures such as aflatoxin B1 (AFB1) depends largely on biotransformation processes. While the genotoxicity and mutagenicity of several bisfuranoid mycotoxins including AFB1 and sterigmatocystin have been linked to in vivo bioactivation of these molecules into reactive epoxide forms, the metabolites of genotoxic and mutagenic AFB1 precursor versicolorin A (VerA) have not yet been characterized. Because this molecule is not available commercially, our strategy was to produce a library of metabolites derived from the biotransformation of in-house purified VerA, following incubation with human liver S9 fractions, in presence of appropriate cofactors. The resulting chromatographic and mass-spectrometric data were used to identify VerA metabolites produced by intestinal cell lines as well as intestinal and liver tissues exposed ex vivo. In this way, we obtained a panel of metabolites suggesting the involvement of phase I (M + O) and phase II (glucuronide and sulfate metabolites) enzymes, the latter of which is implicated in the detoxification process. This first qualitative description of the metabolization products of VerA suggests bioactivation of the molecule into an epoxide form and provides qualitative analytic data to further conduct a precise metabolism study of VerA required for the risk assessment of this emerging mycotoxin.

Deoxynivalenol induces apoptosis and inflammation in the liver: Analysis using precision-cut liver slices.

Deoxynivalenol (DON) is one of the most common mycotoxins in cereals and their by-products. Its adverse effects on animal and human health have been extensively studied in the intestine, but little attention has been paid to another target organ for mycotoxins, the liver that is potentially exposed after intestinal absorption and enterohepatic circulation. To assess DON's toxicity in an ex vivo model structurally and physiologically closer to the whole liver, we developed a pig precision-cut liver slices (PCLS) model. PCLS contain all cell types and maintain intercellular and cell-matrix interactions, among other architectural features of the liver. The human HepG2 cell line was used for comparison. We observed that after a short exposure, DON reduced the cell viability of HepG2 cells and induced the expression of genes involved in apoptosis, inflammation and oxidative stress. When PCLS were exposed to DON, damage to the tissues was observed, with no changes in markers of liver function or injury. Exposure to the toxin also triggered liver inflammation and apoptosis, effects already observed in pigs fed DON-contaminated diets. Overall, these data demonstrate that DON had toxic effects on a liver cell line and on whole liver tissue, consistent with the effect observed during in vivo exposure. They also indicate that pig PCLS is a relevant and sensitive model to investigate the liver toxicity of food contaminants.

The pregnane X receptor drives sexually dimorphic hepatic changes in lipid and xenobiotic metabolism in response to gut microbiota in mice.

BACKGROUND: The gut microbiota-intestine-liver relationship is emerging as an important factor in multiple hepatic pathologies, but the hepatic sensors and effectors of microbial signals are not well defined. RESULTS: By comparing publicly available liver transcriptomics data from conventional vs. germ-free mice, we identified pregnane X receptor (PXR, NR1I2) transcriptional activity as strongly affected by the absence of gut microbes. Microbiota depletion using antibiotics in Pxr+/+ vs Pxr-/- C57BL/6J littermate mice followed by hepatic transcriptomics revealed that most microbiota-sensitive genes were PXR-dependent in the liver in males, but not in females. Pathway enrichment analysis suggested that microbiota-PXR interaction controlled fatty acid and xenobiotic metabolism. We confirmed that antibiotic treatment reduced liver triglyceride content and hampered xenobiotic metabolism in the liver from Pxr+/+ but not Pxr-/- male mice. CONCLUSIONS: These findings identify PXR as a hepatic effector of microbiota-derived signals that regulate the host's sexually dimorphic lipid and xenobiotic metabolisms in the liver. Thus, our results reveal a potential new mechanism for unexpected drug-drug or food-drug interactions. Video abstract.

Large-Scale Modeling Approach Reveals Functional Metabolic Shifts during Hepatic Differentiation.

Being able to explore the metabolism of broad metabolizing cells is of critical importance in many research fields. This article presents an original modeling solution combining metabolic network and omics data to identify modulated metabolic pathways and changes in metabolic functions occurring during differentiation of a human hepatic cell line (HepaRG). Our results confirm the activation of hepato-specific functionalities and newly evidence modulation of other metabolic pathways, which could not be evidenced from transcriptomic data alone. Our method takes advantage of the network structure to detect changes in metabolic pathways that do not have gene annotations and exploits flux analyses techniques to identify activated metabolic functions. Compared to the usual cell-specific metabolic network reconstruction approaches, it limits false predictions by considering several possible network configurations to represent one phenotype rather than one arbitrarily selected network. Our approach significantly enhances the comprehensive and functional assessment of cell metabolism, opening further perspectives to investigate metabolic shifts occurring within various biological contexts.

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.

Viable skin efficiently absorbs and metabolizes bisphenol A.

Skin contact has been hypothesized to contribute to human exposure to bisphenol A (BPA). We examined the diffusion and metabolism of BPA using viable skin models: human skin explants and short-term cultures of pig ear skin, an alternative model for the study of the fate of xenobiotics following contact exposure. 14C-BPA [50-800 nmol] was applied on the surface of skin models. Radioactivity distribution was measured in all skin compartments and in the diffusion cells of static cells diffusion systems. BPA and metabolites were further quantified by radio-HPLC. BPA was efficiently absorbed in short-term cultures, with no major difference between the models used in the study [viable pig ear skin: 65%; viable human explants: 46%; non-viable (previously frozen) pig skin: 58%]. BPA was extensively metabolized in viable systems only. Major BPA metabolites produced by the skin were BPA mono-glucuronide and BPA mono-sulfate, accounting together for 73% and 27% of the dose, in pig and human, respectively. In conclusion, experiments with viable skin models unequivocally demonstrate that BPA is readily absorbed and metabolized by the skin. The trans-dermal route is expected to contribute substantially to BPA exposure in human, when direct contact with BPA (free monomer) occurs.

Apple proanthocyanidins do not reduce the induction of preneoplastic lesions in the colon of rats associated with human microbiota.

Since the gut microbiota metabolizes various dietary constituents unabsorbed by the small intestine and modulates colon function, it plays an essential role in colon carcinogenesis. First, we have developed a model of human microbiota-associated rats (HMA), fed a human-type diet and injected with 1-2,dimethylhydrazine (DMH). We observed that the number and size of DMH-induced aberrant crypt foci (ACF) were significantly higher in HMA rats than in germ-free or conventional rats. Second, we used this model to assess the protective effect of an apple proanthocyanidin-rich extract (APE) on colon carcinogenesis. In this model, ACF number and multiplicity were not reduced by APE at 0.001% and 0.01% in drinking water. They were higher with APE 0.1% than with APE 0.01%. Therefore, the cross-talk between human microbiota and the colon epithelium should be taken into account in carcinogenesis models. Moreover, attention should be paid prior to using proanthocyanidin extracts as dietary supplements for humans.