Biology subjects

Loyer, P.

Publications and source records attributed to Loyer, P..

4 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

Insight Into the Molecular Parameters of PEI Promoting an Efficient Gene Delivery into Cells

The development of natural or synthetic polycations able to interact with nucleic acids and condense them into nanoparticles known as polyplexes, faces several unresolved challenges at the cellular level. Key issues include the intracellular trafficking of polyplexes, the endosomal escape and the release of nucleic acids into the cytosol, which are considered major bottlenecks for efficient protein expression. Here, we aim at gaining fundamental insights into the stability of polyplexes in biological media and their uptake and intracellular trafficking, while correlating data of the expression of reporter protein with both the molecular characteristics of various poly(ethylenimines) (PEI) and the physicochemical characteristics of PEI/peGFP-C3 polyplexes. For this, we chosen four samples of PEI, selected as a model polycation, with different molecular weights (Mw = 0.8, 20, 25 and 60 kg/mol) and structures (linear and branched). We found that the in vitro and in vivo stability of PEI/peGFP-C3 polyplexes, their cell internalization and transfection efficiency is dependent on the variation of polycation Mw and structure, as well as the intrinsic properties of polyplexes, such as the charge ratio (R=[N+]/[P-]). A relation between the percentage of positive cells to green fluorescent protein (GFP) and the amount of internalized nucleic acid (cyanine 5-peGFP-C3) allowed revealing the molecular characteristics of PEI promoting both higher both cell internalization and GFP expression on HEK293T cells. In the long term, the outcome of this work will be to propose guidelines to help design more effective, and less cytotoxic non-viral gene carriers with a great potential for new therapeutic applications.

biophysics↗

bioRxiv2024

The dithiocarbamate pesticides maneb and mancozeb disturb the metabolism of lipids and xenobiotics in an in vitro model of metabolic dysfunction-associated steatotic liver disease

Pesticides are increasingly recognized to be hepatotoxic but less is known about their toxicity in metabolic dysfunction-associated steatotic liver disease (MASLD). Recent investigations reported oxidative stress-induced apoptosis in differentiated hepatocyte-like HepaRG cells after a single treatment with a 7-pesticide mixture that included chlorpyrifos, dimethoate, diazinon, iprodione, imazalil, and the dithiocarbamates maneb and mancozeb. These effects were reproduced by maneb, mancozeb, or manganese chloride (MnCl2). Herein, differentiated HepaRG cells cultured for 2 weeks without (-FA) or with (+FA) a mixture of stearic and oleic acids were treated with this 7-pesticide mixture, maneb, mancozeb, or MnCl2 along the same period. While these molecules did not induce neutral lipid accumulation in -FA-HepaRG cells, they worsened steatosis in +FA-HepaRG cells. Maneb or MnCl2 impaired very low-density lipoprotein (VLDL) secretion and increased fatty acid uptake without altering mitochondrial fatty acid oxidation and de novo lipogenesis. Reduced VLDL secretion was associated with decreased mRNA levels of apolipoproteins B and C3 and microsomal triglyceride transfer protein. Zinc supplementation restored VLDL secretion, reduced fatty acid uptake and prevented the exacerbation of steatosis in +FA-HepaRG cells treated with mancozeb or MnCl2. The mixture, maneb, or MnCl2 also reduced the mRNA expression and activity of several cytochromes P450 in +FA- and -FA-HepaRG cells. This was associated with impaired biotransformation of diazinon while chlorpyrifos metabolism was unaffected. Hence, maneb, mancozeb and MnCl2 disturb the metabolism of lipids and xenobiotics in HepaRG cells, in particular in fatty acid-exposed cells. These findings could have major pathophysiological consequences in dithiocarbamate-exposed individuals with MASLD.

pharmacology and toxicology↗

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↗