Biology subjects

Ribault, C.

Publications and source records attributed to Ribault, C..

3 recordsLinked to original sources

bioRxiv2025

Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity

Primary cultures of Human Hepatocytes (PHH) are the gold standard to investigate drug-hepatotoxicity in vitro, however, large-scale studies using these primary liver cells are not possible because of the shortage in liver biopsies. HepaRG model is often considered as the closest surrogate to PHH for toxicity studies in vitro. However, differentiated HepaRG cells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both CYP2D6 and GFP. Differentiated HepaRG cells transduced with lentivirus stably expressed GFP and catalytically active human CYP2D6 enzyme at levels close to those found in high PHH metabolizers. As expected, CYP2D6 protein was found mostly located in the endoplasmic reticulum. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized in both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 catalytic activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions. However, we identified a limited set of genes such as NXF3 and TRIM63, which were up-regulated by the mRNA encoded by the lentiviral transgene. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

cell biology↗

bioRxiv2024

PPARgamma, a key modulator of metabolic reprogramming, stemness and chemoresistance associated with retrodifferentiation in human hepatocellular carcinomas

Human hepatocellular carcinomas (HCCs) with cancer stem cell (CSC) features are a subclass of therapeutically challenging cancers. We recently showed that retrodifferentiation of hepatic cancer cells into CSC-like cells leads to metabolic reprogramming and chemoresistance. The molecular mechanisms whereby differentiated cancer cells switch towards a CSC phenotype are poorly understood. By studying metabolic reprogramming associated with HCC cell plasticity, we identified an unsuspected role of peroxisome proliferator-activated receptor (PPAR){gamma} in hepatic CSC phenotype acquisition. Gene expression and metabolic analyses performed throughout cell differentiation/retrodifferentiation process of human HepaRG and HBG-BC2 HCC cells show that metabolic reprogramming in hepatic CSCs is associated with fragmented mitochondrial network, decreased respiration, de novo lipogenesis, fatty acid oxidation, but increased glycolysis and lipid storage. Mitochondrial genes downregulated in HepaRG-CSCs are also downregulated in the STEM HCC subclass. While PPAR is the main isoform in differentiated hepatic cells, we find high PPAR{gamma} expression in hepatic CSCs. Accordingly, nuclear localization of PPAR{gamma} is detected in human HCC tumors and PPAR{gamma}high/PPARlow expression is associated with the STEM HCC subclass and a poor outcome in human HCC cohorts. PPAR{gamma} silencing or/and inhibition of its target gene pyruvate dehydrogenase kinase 4 reactivates cell respiration, increases reactive oxygen species production and sensitizes hepatic CSCs to chemotherapy. Conversely, PPAR activation synergizes with chemotherapy to induce cell death. Targeting PPAR{gamma}, a key regulator of metabolic reprogramming and stemness in hepatic CSCs, or modulating the PPAR{gamma}/PPAR balance that finely tunes the differentiation/retrodifferentiation process in HCC deserves further investigation for antitumor therapy. Implications heading and statementPPAR{gamma}, a key regulator of metabolic reprogramming and stemness in hepatic CSC, reduces oxidative phosphorylation and reactive oxygen species production, therefore contributing to HCC chemoresistance.

cancer biology↗

bioRxiv2023

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

pharmacology and toxicology↗