In vitro modeling of human liver function is essential for assessing drug-induced toxicity, particularly in the context of metabolic dysfunction-associated steatotic liver disease (MASLD). However, standard liver organoid systems often lack xenobiotic-metabolizing enzyme activity and fail to replicate the pathological features of MASLD. Here, we present a method for generating functional, multi cell-type human liver organoids (HML) organoids from HepaRG cells, primary human macrophages, and LX-2 hepatic stellate cells. These three-dimensional (3D) organoids are cultured under defined conditions that support key hepatic functions. To mimic MASLD, we expose HML organoids to a mixture of stearic and oleic acids for 9 days, inducing steatosis and fibrogenic responses characteristic of the disease. These MASLD-HML organoids retain liver-specific functions and express key fibrosis markers. We further demonstrate the usefulness of this method to produce standardized organoids useful for the evaluation of drug-induced liver injury (DILI) through IC50 and benchmark dose calculations, particularly in the context of MASLD. Together, HML and MASLD-HML organoids provide a robust model for studying drug metabolism, toxicity, and adverse drug reactions in healthy and diseased liver states. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Culture of HepaRG cells Basic Protocol 2: Culture of LX-2 cells Basic Protocol 3: Culture of primary human unstimulated M1 macrophages Basic Protocol 4: Organoid seeding procedure 1 in agarose molds Basic Protocol 5: Organoid seeding procedure 2 in ultra-low attachment 96-well plate Alternate Protocol: Organoid seeding procedure from cryopreserved cells Basic Protocol 6: Preparation of the fatty acid mixture Basic Protocol 7: MASLD induction.

Background: Long-read, single-CpG-resolution sequencing is redefining the information-to-depth ratio in epigenomics. While conventional methylome analysis often requires high coverage, we propose a scalable pipeline designed to extract high-density regulatory logic from shallow sequencing data. Methods: By utilizing the progenitor-like HepaRG cell line as a model for liver plasticity, we validated this framework across two divergent developmental trajectories: hepatic maturation and sphere-induced retrodifferentiation. Our technical approach combines CpG-centric enrichment and regional methylation aggregation to reconstruct regulatory landscapes from sparse data. Using long-read Nanopore sequencing, we mapped the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Results: Our pipeline revealed that these trajectories are not inverse processes but engage distinct epigenetic strategies. Hepatic maturation is characterized by the accumulation of 5hmC that partially targets repressive heterochromatin (H3K9me3, H4K20me3) and pioneer factors such as FOXA2. In contrast, retrodifferentiation increases 5mC, potentially silencing adult regulators such as HNF1A via Polycomb-associated networks. In addition, aggregation-based analysis can distinguish widespread focal perturbations from a restricted subset of transcription factors that translate epigenetic changes into regional accessibility. Conclusions: This study provides a scalable computational framework for investigating cellular fate transitions, proving that high-value epigenetic insights are attainable even at reduced sequencing depths.

The human HepaRG™ cell line is the closest surrogate to primary culture of hepatocytes (PHH) for toxicology studies. However, differentiated HepaRG™ cells express low levels of the cytochrome P450 2D6 (CYP2D6) involved in the biotransformation of many drugs. Herein, progenitor HepaRG™ cells were transduced using lentiviral particles encoding both human CYP2D6 and GFP proteins. The resulting transgenic HepaRG™ cells stably expressed catalytically active CYP2D6 at levels close to those observed in PHH from rapid metabolizers and HepaSH™ hepatocytes. In CYP2D6 transgenic HepaRG™ cells, tramadol was metabolized into both N- and O-desmethyl tramadol as seen in humans while parental HepaRG™ cells produced only N-desmethyl tramadol. Following treatment with perhexiline, the CYP2D6 expressing HepaRG™ cells exhibited higher IC50 values and reduced mitochondrial damages compared to those found in parental cells. Transcriptomic analysis revealed that the expression of CYP2D6 did not significantly affect the cells’ ability to proliferate and differentiate or compromise key hepatocyte-specific functions. However, we identified a small number of genes, including NXF3 and TRIM63, which were up-regulated in transgenic cells. Using CRISPR/Cas9-mediated knockdown of GFP and/or CYP2D6 sequences, we demonstrated that NXF3 mRNA and protein inductions were triggered by the lentiviral mRNA encoding GFP and CYP2D6 rather than by genomic transgene integration. Together, these findings establish CYP2D6-transgenic HepaRG™ cells as an optimized and reliable hepatocyte-like model for studying the metabolism and toxicity of CYP2D6 substrates. Our results also support the hypothesis that the NXF3 gene may be a marker of cellular response to the expression of a lentiviral chimeric mRNA.

BACKGROUND AND AIMS:The sympathetic nervous system is involved in the progression and aggressiveness of several cancers, including hepatocellular carcinoma (HCC). However, the specific effects of sympathetic innervation and catecholamines on tumour hepatocyte behaviour remain poorly understood. In this study, we aimed to investigate the role of sympathetic fibres and catecholamines in the progression of HCC. METHODS:The distribution of sympathetic nerves was mapped in 10 human HCC samples using multiplex immunofluorescence labeling. Adrenergic receptors (ADR) and enzymes involved in catecholamine degradation were assessed in three HCC (HepaRG, BC2, Huh-7) and one hepatoblastoma (HepG2) cell lines. The impact of catecholamines on gene and protein expression and metabolic pathways was assessed in differentiated HepaRG-hepatocytes, and the results were verified in primary human hepatocyte (PHH) cultures. The clinical relevance of the findings was evaluated in two transcriptomics datasets. RESULTS:Abundant and tortuous sympathetic fibres were detected in non-tumour liver tissues adjacent to the HCC invasion front. Differentiated HepaRG-hepatocytes expressed both α1- and β2-ADR and metabolized catecholamines to normetanephrine, metanephrine, and vanillylmandelic acid. In these cells, catecholamine exposure activated inflammation, cell proliferation, and extracellular matrix remodelling pathways associated with retro-differentiation and primed epithelial-to-mesenchymal transition. The catecholamine-induced program was detected in aggressive, ECM/STEM HCC subclasses with poor patient prognosis. CONCLUSIONS:Catecholamines induce an aggressive phenotype in differentiated tumour hepatocytes localized at the invasive front, suggesting their involvement in cancer progression. Understanding the interplay between the sympathetic nervous system and HCC may offer novel therapeutic opportunities.

Pesticides are increasingly recognized to be hepatotoxic, but less is known about their toxicity in metabolic dysfunction-associated steatotic liver disease (MASLD). Herein, differentiated HepaRG cells cultured for 2 weeks without (-FA) or with (+FA) a mixture of fatty acids were treated with different pesticides, including maneb and mancozeb, during the same period. While maneb and mancozeb did not induce neutral lipid accumulation in -FA-HepaRG cells, they worsened steatosis in +FA-HepaRG cells. MnCl2 treatment reproduced these effects. Maneb or MnCl2 impaired very low-density lipoprotein (VLDL) secretion and increased fatty acid uptake. Zinc supplementation restored VLDL secretion, reduced fatty acid uptake, and prevented steatosis worsening in +FA-HepaRG cells treated with mancozeb or MnCl2. 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. These findings could have major pathophysiological consequences in dithiocarbamate-exposed individuals with MASLD.

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)γ in hepatic CSC phenotype acquisition. Gene expression and metabolic analyses performed throughout the cell differentiation/retrodifferentiation process of human HepaRG and HBG-BC2 HCC cells show that metabolic reprogramming in hepatic CSCs is associated with a fragmented mitochondrial network, decreased respiration, de novo lipogenesis, and fatty acid oxidation, but increased glycolysis and lipid storage. Mitochondrial genes downregulated in HepaRG-CSCs are also downregulated in the STEM HCC subclass. While PPARα is the main isoform in differentiated hepatic cells, we find high PPARγ expression in hepatic CSCs. Accordingly, nuclear localization of PPARγ is detected in human HCC tumors, and PPARγhigh/PPARαlow expression is associated with the STEM HCC subclass and a poor outcome in human HCC cohorts. PPARγ 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, PPARα activation synergizes with chemotherapy to induce cell death. Targeting PPARγ, a key regulator of metabolic reprogramming and stemness in hepatic CSCs, or modulating the PPARγ/PPARα balance that finely tunes the differentiation/retrodifferentiation process in HCC deserves further investigation for anti-tumor therapy.

Objective: Recently, the pig liver model perfused ex vivo using a normothermic machine perfusion (NMP) has been proposed as a suitable model to study xenobiotic metabolism and biliary excretion. The aim of our study is to describe the metabolism of NPS such as cathinones (with a focus on 4-Cl-PVP and eutylone) in blood and bile, using a normothermic perfused pig liver model. Methods: Livers (n = 4) from male large white pigs, 3-4 months of age and weighing approximately 75-80 kg, were harvested and reperfused onto an NMP (LiverAssist (R), XVIVO) using autologous whole blood at 38 degrees C. 4-ClPVP and eutylone were administered as a bolus in the circulating blood at T0 with the aim of achieving a concentration of 1 mu g/mL in the reperfusion system. The assays were carried out on plasma and bile between 0 and 120 min after cathinone administration using an targeted and untargeted approaches based on liquid chromatography coupled with high resolution mass spectrometry (Q-Exactive Thermo Scientific (R)). Results: In plasma, the concentration of 4-Cl-PVP and eutylone decreased rapidly with elimination half-lives of 4 min and 0.25 min, respectively. Their phase I and phase II metabolites were detected in plasma as early as 1 min. In bile, 4-Cl-PVP and eutylone were detected with maximum intensity between 0 and 30 min postadministration, and the main metabolites found in plasma were found in bile. Phase II derivatives showed increasing biliary excretion over time up to 120 min. Conclusion: The pig liver model perfused ex vivo using an NMP represent a promising model in pharmacotoxicology, particularly for toxicokinetic investigations of cathinones. This model may be of interest in the absence of authentic cases of cathinone consumption or other NPS consumption to identify relevant metabolites consumption markers. In addition, the possibility of collecting bile in this model represents an additional advantage for studying biliary excretion of NPS and their metabolites in forensic toxicology.

The liver's regenerative capacity is underscored by the plasticity potential of adult hepatocytes. In this context, hepatocyte-to-cholangiocyte transdifferentiation (HCT) has been ascribed with pro-regenerative functions in animal models and is a feature of end-stage human chronic liver diseases. While dampened activities of hepatocyte identity transcription factors (TFs) underlay HCT, how the cholangiocyte transcriptional program is implemented is poorly defined. Here, we identify that HCT does not involve transitioning through a hepatoblast-like transcriptional program. Furthermore, we show that HCT primarily involves induction of the archetypal transcriptional program of monopolarized epithelial cells initially repressed in hepatocytes. Indeed, HCT requires relieving H3K27me3-mediated and polycomb-dependent epigenetic silencing of epithelial TF encoding genes including Grainyhead Like Transcription Factor 2 (GRHL2). Ectopic expression of GRHL2 in hepatocytes, including in vivo in the adult mouse liver, induces epithelial genes reminiscent of those activated during HCT. Finally, GRHL2 is detected in human hepatocytes undergoing HCT as evidenced using samples from end-stage chronic liver diseases. Hence, HCT is a process chiefly characterized by induction of a conventional epithelial transcriptional program originally lacking in hepatocytes promoted by derepression of the master epithelial TF GRHL2.

Using the phage display technology, we identified a novel peptide, P11Chol, which preferentially binds to both human and rat cholangiocytes. Peptide P11Chol alignment with protein databases evidenced strong similarities with a highly conserved peptide motif from BamA/TamA-like outer membrane proteins expressed in enterobacteriaceae belonging to Pseudomonadota phylum including Photorhabdus , Providencia , Acinetobacter , Salmonella enterica and Helicobacter pylori species. In addition, we showed that Providencia stuartii bacteria were able to bind to cholangiocytes-like HepaRG cells in vitro and that P11Chol modulated this interaction suggesting the possible involvement of BamA/TamA-like outer membrane proteins in cell adhesion and/or internalization of Providencia stuartii bacteria. Using fluorescent P11Chol peptide, we next developed a flow cytometry procedure to detect and isolate rat and human liver epithelial cells from hepatic cell suspension obtained after collagenase dissociation of liver parenchyma. Three distinct P11Chol-positive rat liver epithelial cell lines (RLEC) were established, which produced functional cholangiocytes capable to form cyst-like structures in vitro and to maintain expression of specific functions in hepatocytes in coculture. The characterization of these three RLEC lines evidenced functional differences that support the concept of small and large cholangiocytes exhibiting different functional phenotypes within the intrahepatic bile tree. ### Competing Interest Statement The authors have declared no competing interest. Lebanese Association for Scientific Research (LASeR, Lebanon) Institut National de la Santé et de la Recherche Médicale (Inserm, France) Rennes Métropôle (France) Université de Bretagne Loire (UBL) Convention Industrielle de Formation par la Recherche, CIFRE n°221207A10 Convention Industrielle de Formation par la Recherche, CIFRE n°221206A10, Ministère de l’Enseignement Supérieur et de la Recherche, https://ror.org/03sjk9a61

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. ### Competing Interest Statement The authors have declared no competing interest.

The development of in vitro models that recapitulate critical liver functions is essential for accurate assessments of drug toxicity. Although liver organoids can be used for drug discovery and toxicology, they are limited by (i) the lack of expression and activity of xenobiotic-metabolizing enzymes, and (ii) the difficulty of mimicking non-alcoholic fatty liver disease (NAFLD, which influences the expression of these enzymes) in vitro. Here, we generated three-dimensional multi-cell-type liver organoids (hereafter "HML organoids") from HepaRG cells, primary human macrophages, and hepatic-stellate-cell-derived LX-2 cells. We also developed an NAFLD model by culturing HML organoids for 9 days with a mixture of stearic and oleic acids. The exposed organoids showed typical features of steatosis and expressed fibrosis markers. We subsequently used HML and NAFLD-HML organoids to model drug-induced liver injury. By estimating the IC50 and benchmark doses, we were able to improve the in vitro detection of drugs likely to be toxic in fatty livers. Thus, HML and NAFLD-HML organoids exhibited most of the liver's functions and are relevant in vitro models of drug metabolism, drug toxicity, and adverse drug event in NAFLD.

IntroductionSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. The complex pathophysiology of sepsis is associated with pro- and anti-inflammatory response, a pro-coagulant state, endothelial dysfunction and tissue hypoxia. These mechanisms lead to progressive multi organ failure. Although this is a systemic process, the pathophysiological of sepsis differs from organ to organ, and from organ to peripheral blood. Our hypothesis to explain this compartmentalization of responses is a distinct population of resident tissue macrophages, as well as a distinct migration of monocytes in target organ. Indeed, the macrophages and monocytes can start the clinical syndrome of sepsis via transcription of genes involved in inflammation. Moreover, macrophages can induce endothelial injury by release reactive oxygen species. Also hypoxia decreases expression of M1 polarization markers and increases the M2 marker. Identification of biomarkers related to specific organs (beyond the blood) will improve the understanding specific organ failure. In this way, the aim of this study was compared to the systemic inflammatory response with the lung and the liver, two organs most affected during sepsis as well as understanding the role of macrophages in this compartmentalization. For this, a murine polymicrobial sepsis model induced by caecal ligation and puncture (CLP) was used.MethodsModerate sepsis was induced by the CLP in C57BL/6 male mice (n=63), divided into 4 groups: Basal, Sham 1 day and 5 days, CLP 1 days and 5 days. Then, we analyzed histological changes, cytokine profile (by ELISA and PCR), oxidative imbalance (expression of SOD, CAT and iNOS) and polarization markers (CD86and CD206) by immunohistochemistry.ResultsRespiratory and liver failure was confirmed by histology and also by a decrease of pressure oxygen in arterial blood (PaO2) and increase of bilirubin level after 5 days of CLP. Moreover CLP induced a plasma increase in the level of TNFα, IL-6, IL-10, KC and CCL2 in the first 24hours after CLP, but with a progressive decrease at 5 days. On the other hand, our original results show that the level of some of the cytokines in the liver and the lung differ from the systemic level during sepsis. Indeed, there are increase in CCL2 and its receptor CCR2 in the lung compared to the liver, whereas in the liver rather a decrease in the expression of CX3CL1/CX3CR1, that is not altered in the lung. We were also able to highlight a more marked oxidative imbalance in the liver than in the lung. Also, we observed that CLP increases the expression of M1 CD86 and M2 CD206 markers in the liver and lung at 5 days, but with a total number of CD86-labeled cells, three times greater in the liver.ConclusionThese results support the hypothesis of a compartmentalization of the inflammatory response in sepsis, characterized by an early inflammatory response in the lung, and later chronic phase in the liver. The lung cells maybe are more resistant to having their immune functions turned off. In contrast, the liver will be more sensitive to deactivations. This compartmentalization involved the migration of monocytes: CCL2/CCR2 and CX3CL1/CX3CR1 associate with the oxidative stress.

ObjectifsLa connaissance du métabolisme des xénobiotiques et leur répartition dans l’organisme représente un challenge en toxicologie analytique et nécessite des outils et des modèles performants. Nous présentons ici un modèle de métabolisation par foie de porc perfusé ex vivo à l’aide d’une machine de perfusion normothermique (MPN) permettant de recréer les conditions physiologiques et ainsi conserver les fonctions métaboliques de l’organe. Ce modèle a été utilisé pour suivre le devenir du tramadol et de ses 5 principaux métabolites dans le sang et la bile. Cette étude a pour objectif d’évaluer la pertinence du foie de porc sur MPN comme un modèle préclinique pour étudier le métabolisme des xénobiotiques.MéthodeDes foies (n=5) de porcs mâles de race « large white », âgés de 3–4 mois et pesant environ 80kg, ont été prélevés après 1heure d’ischémie chaude suivi de 3heures en ischémie froide puis reperfusé sur une MPN (LiverAssist®, XVIVO) à l’aide de 2litres de sang total autologue à 38°C. Le cholédoque a été canulé afin de recueillir la bile. Le tramadol (100mg/L) a été administré en bolus dans le sang à T0. Les concentrations de tramadol et ses métabolites (M1 à M5) ont été mesurées à différents temps dans le sang (0 ; 1 ; 7,5 ; 15 ; 30 ; 60 ; 90 et 120minutes) et dans la bile (30 ; 60 ; 90 et 120minutes). Les dosages ont été réalisées avec une approche ciblée par chromatographie liquide (CL) couplée à la spectrométrie de masse en tandem (SM/SM (Xevo TQ-XS Waters®). Une approche non ciblée a été réalisée par CL-SM/SM haute résolution (Q-Exactive, ThermoScientific®).RésultatsDans le sang, la concentration de tramadol est en moyenne de 343ng/mL à T0 et diminue très rapidement pour être inférieure à 1ng/mL au bout de 30minutes. Son métabolite principal, le O-desmethyltramadol (M1) apparaît dès la 1ère minute dans le sang et décroît rapidement avec une concentration inférieure à 1ng/mL à 90minutes. Les autres métabolites N-desmethyl-tramadol (M2), M3, M4 et M5 ont des intensités de détection faibles et une cinétique d’élimination proche du M1 : avec un pic d’intensité à 7,5minutes avec une diminution rapide. Les dérivés glucuroconjugués (O-DMT-Gluc 426,2131m/z, O,N-demethyl-Gluc 412.1977m/z et O,N,N-Tridesmethyl-Gluc 398.1815m/z) ont une intensité importante et augmentent jusqu’à 120minutes. Dans la bile, le tramadol et ses métabolites sont détectables dès 30minutes et le restent à 60, 90 et 120minutes.ConclusionLe suivi des concentrations du tramadol et de ses métabolites nous permet de mettre en évidence une élimination très rapide du tramadol dans ce modèle de foie porcin perfusé ex vivo en condition quasi-physiologique. De plus, l’apparition rapide (dès 1minute) de métabolites montre une capacité de métabolisme très importante. Les métabolite M1 et M2 étant respectivement produits par le CYP2D6 et le CYP3A4, nous pouvons observer que ces 2 voies de métabolisation sont présentes dans ce modèle. De plus, la quantité importante de métabolites glucuronjugués montre que ce modèle possède un bon métabolisme de phase II. Enfin, la possibilité de recueil de la bile dans ce modèle représente une perspective intéressante pour l’étude du métabolisme des xénobiotiques en toxicologie médicolégale. Ce modèle semble donc métaboliquement similaire au métabolisme hépatique humain mais de façon accéléré et représente donc un modèle pertinent en toxicologie.

Supplemental Table 5 provides the lists of genes significantly deregulated between HepaRG-Spheres and HepaRG-D10 cells and between HepaRG-SP and HepaRG-D10 cells.

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.

Mucosal healing has emerged as a therapeutic goal to achieve lasting clinical remission in ulcerative colitis. Intestinal repair in response to inflammation presumably requires higher energy supplies for the restoration of intestinal barrier and physiological functions. However, epithelial energy metabolism during intestinal mucosal healing has been little studied, whereas inflammation-induced alterations have been reported in the main energy production site, the mitochondria. The aim of the present work was to assess the involvement of mitochondrial activity and the events influencing their function during spontaneous epithelial repair after colitis induction in mouse colonic crypts. The results obtained show adaptations of colonocyte metabolism during colitis to ensure maximal ATP production for supporting energetic demand by both oxidative phosphorylation and glycolysis in a context of decreased mitochondrial biogenesis and through mitochondrial function restoration during colon epithelial repair. In parallel, colitis-induced mitochondrial ROS production in colonic epithelial cells was rapidly associated with transient expression of GSH-related enzymes. Mitochondrial respiration in colonic crypts was markedly increased during both inflammatory and recovery phases despite decreased expression of several mitochondrial respiratory chain complex subunits after colitis induction. Rapid induction of mitochondrial fusion was associated with mitochondrial function restoration. Finally, in contrast with the kinetics expression of genes involved in mitochondrial oxidative metabolism and in glycolysis, the expression of glutaminase was markedly reduced in the colonic crypts both during colitis and repair phases. Overall, our data suggest that the epithelial repair after colitis induction is characterized by a rapid and transient increased capacity for mitochondrial ATP production in a context of apparent restoration of mitochondrial biogenesis and metabolic reorientation of energy production. The potential implication of energy production adaptations within colonic crypts to sustain mucosal healing in a context of altered fuel supply is discussed.