Expression and function of the urea cycle in widely-used hepatic cellular models.

The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Impact of perfluoroalkyl substances (PFAS) and PFAS mixtures on lipid metabolism in differentiated HepaRG cells as a model for human hepatocytes.

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants with various adverse health effects in humans including disruption of lipid metabolism. Aim of the present study was to elucidate the molecular mechanisms of PFAS-mediated effects on lipid metabolism in human cells. Here, we examined the impact of a number of PFAS (PFOS, PFOA, PFNA, PFDA, PFHxA, PFBA, PFHxS, PFBS, HFPO-DA, and PMPP) and of some exposure-relevant PFAS mixtures being composed of PFOS, PFOA, PFNA and PFHxS on lipid metabolism in human HepaRG cells, an in vitro model for human hepatocytes. At near cytotoxic concentrations, the selected PFAS and PFAS mixtures induced triglyceride accumulation in HepaRG cells and consistently affected the expression of marker genes for steatosis, as well as PPARα target genes and genes related to lipid and cholesterol metabolism, pointing to common molecular mechanisms of PFAS in disrupting cellular lipid and cholesterol homeostasis. PPARα activation was examined by a transactivation assay in HEK293T cells, and synergistic effects were observed for the selected PFAS mixtures at sum concentrations higher than 25 µM, whereas additivity was observed at sum concentrations lower than 25 µM. Of note, any effect observed in the in vitro assays occurred at PFAS concentrations that were at least four to five magnitudes above real-life internal exposure levels of the general population.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

In vitro screening of understudied PFAS with a focus on lipid metabolism disruption.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many industrial applications. Exposure to PFAS is associated with several health risks, including a decrease in infant birth weight, hepatoxicity, disruption of lipid metabolism, and decreased immune response. We used the in vitro cell models to screen six less studied PFAS [perfluorooctane sulfonamide (PFOSA), perfluoropentanoic acid (PFPeA), perfluoropropionic acid (PFPrA), 6:2 fluorotelomer alcohol (6:2 FTOH), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and 8:2 fluorotelomer sulfonic acid (8:2 FTSA)] for their capacity to activate nuclear receptors and to cause differential expression of genes involved in lipid metabolism. Cytotoxicity assays were run in parallel to exclude that observed differential gene expression was due to cytotoxicity. Based on the cytotoxicity assays and gene expression studies, PFOSA was shown to be more potent than other tested PFAS. PFOSA decreased the gene expression of crucial genes involved in bile acid synthesis and detoxification, cholesterol synthesis, bile acid and cholesterol transport, and lipid metabolism regulation. Except for 6:2 FTOH and 8:2 FTSA, all tested PFAS downregulated PPARA gene expression. The reporter gene assay also showed that 8:2 FTSA transactivated the farnesoid X receptor (FXR). Based on this study, PFOSA, 6:2 FTSA, and 8:2 FTSA were prioritized for further studies to confirm and understand their possible effects on hepatic lipid metabolism.

A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

Induction of human hepatic cytochrome P-450 3A4 expression by antifungal succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Suitability of human HepaRG cells and liver spheroids as in vitro model to investigate the bioactivation of the organothiophosphate pesticide parathion.

Organophosphorus compounds (OP) constitute a large group of chemicals including pesticides and nerve agents. Organothiophosphate pesticides require cytochrome P450-mediated oxidative desulphuration in the liver to form corresponding oxons, which are potent inhibitors of the enzyme acetylcholinesterase (AChE). Human HepaRG cells are a promising tool to study liver-specific functions and have been shown to maintain drug metabolizing enzymes. This research describes for the first time the in vitro metabolic activation of an organothiophosphate to its active oxon by two different HepaRG cell-based models. Monolayer cultures and liver spheroids were exposed to the model OP parathion and the quantification of the corresponding oxon was performed with an AChE inhibition assay. Our results showed a time- and dose-dependent cytochrome P450 catalyzed bioactivation and a superior metabolism capacity of the monolayer HepaRG model in comparison with the liver spheroids. Finally, HepaRG cells can be assessed as a metabolically competent cell model intermediate between cell-free preparations and intact animals and as suitable to study OP metabolism in the human liver.

Essential phospholipids impact cytokine secretion and alter lipid-metabolizing enzymes in human hepatocyte cell lines.

BACKGROUND: Essential phospholipids (EPL) are hepatoprotective. METHODS: The effects on interleukin (IL)-6 and -8 secretion and on certain lipid-metabolizing enzymes of non-cytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), polyenylphosphatidylcholine (PPC), and phosphatidylinositol (PtdIns) (both at 0.1 and 1 mg/ml), compared with untreated controls, were assessed in human hepatocyte cell lines (HepG2, HepaRG, and steatotic HepaRG). RESULTS: Lipopolysaccharide (LPS)-induced IL-6 secretion was significantly decreased in HepaRG cells by most phospholipids, and significantly increased in steatotic HepaRG cells with at least one concentration of EPL and PtdIns. LPS-induced IL-8 secretion was significantly increased in HepaRG and steatotic HepaRG cells with all phospholipids. All phospholipids significantly decreased amounts of fatty acid synthase in steatotic HepaRG cells and the amounts of acyl-CoA oxidase in HepaRG cells. Amounts of lecithin cholesterol acyltransferase were significantly decreased in HepG2 and HepaRG cells by most phospholipids, and significantly increased with 0.1 mg/ml PPC (HepaRG cells) and 1 mg/ml PtdIns (steatotic HepaRG cells). Glucose-6-phosphate dehydrogenase activity was unaffected by any phospholipid in any cell line. CONCLUSIONS: EPL, PPC, and PtdIns impacted the secretion of pro-inflammatory cytokines and affected amounts of several key lipid-metabolizing enzymes in human hepatocyte cell lines. Such changes may help liver function improvement, and provide further insights into the EPL's mechanism of action.

DMSO and Its Role in Differentiation Impact Efficacy of Human Adenovirus (HAdV) Infection in HepaRG Cells.

Differentiated HepaRG cells are popular in vitro cell models for hepatotoxicity studies. Their differentiation is usually supported by the addition of dimethyl sulfoxide (DMSO), an amphipathic solvent widely used in biomedicine, for example, in potential novel therapeutic drugs and cryopreservation of oocytes. Recent studies have demonstrated drastic effects, especially on epigenetics and extracellular matrix composition, induced by DMSO, making its postulated inert character doubtful. In this work, the influence of DMSO and DMSO-mediated modulation of differentiation on human adenovirus (HAdV) infection of HepaRG cells was investigated. We observed an increase in infectivity of HepaRG cells by HAdVs in the presence of 1% DMSO. However, this effect was dependent on the type of medium used for cell cultivation, as cells in William's E medium showed significantly stronger effects compared with those cultivated in DMEM. Using different DMSO concentrations, we proved that the impact of DMSO on infectability was dose-dependent. Infection of cells with a replication-deficient HAdV type demonstrated that the mode of action of DMSO was based on viral entry rather than on viral replication. Taken together, these results highlight the strong influence of the used cell-culture medium on the performed experiments as well as the impact of DMSO on infectivity of HepaRG cells by HAdVs. As this solvent is widely used in cell culture, those effects must be considered, especially in screening of new antiviral compounds.

Application of HepaRG cells for genotoxicity assessment: a review.

There has been growing interest in the use of human-derived metabolically competent cells for genotoxicity testing. The HepaRG cell line is considered one of the most promising cell models because it is TP53-proficient and retains many characteristics of primary human hepatocytes. In recent years, HepaRG cells, cultured in both a traditional two-dimensional (2D) format and as more advanced in-vivo-like 3D spheroids, have been employed in assays that measure different types of genetic toxicity endpoints, including DNA damage, mutations, and chromosomal damage. This review summarizes published studies that have used HepaRG cells for genotoxicity assessment, including cell model evaluation studies and risk assessment for various compounds. Both 2D and 3D HepaRG models can be adapted to several high-throughput genotoxicity assays, generating a large number of data points that facilitate quantitative benchmark concentration modeling. With further validation, HepaRG cells could serve as a unique, human-based new alternative methodology for in vitro genotoxicity testing.

BDE-47-mediated cytotoxicity via autophagy blockade in 3D HepaRG spheroids cultured in alginate microcapsules.

Polybrominated Diphenyl Ethers (PBDEs) are a major class of brominated flame retardants, and their widespread use has led them to be considered contaminants with emerging concern. PBDEs have been detected in the indoor air, house dust, food, and all environmental compartments. The congener BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) is the most prevalent, and hepatotoxicity, neurotoxicity, immunological changes, endocrine disruption, and genotoxic potential have been related to its exposure. Although the BDE-47 molecular toxicity pathway is directly related to intrinsic apoptotic cell death, the role of autophagy in BDE-47 toxicity remains unclear. In this context, three-dimensional cell culture has emerged as a good strategy for the replacement of animals in toxicological testing. Here, we used HepaRG spheroids cultured in alginate microcapsules to investigate the role of autophagy in BDE-47-mediated hepatotoxicity. We developed mature and functional HepaRG spheroids by culturing them in alginate microcapsules. Histological analysis revealed that HepaRG spheroids formed an extracellular matrix and stored glycogen. No apoptotic and/or necrotic cores were observed. BDE-47 showed concentration- and time-dependent cytotoxicity in HepaRG spheroids. In the early exposure period, BDE-47 initially disrupted mitochondrial activity and increased the formation of acid compartments that promoted the increase in autophagic activity; however, this autophagy was blocked, and long-term exposure to BDE-47 promoted efficient apoptotic cell death through autophagy blockade, as evidenced by an increased number of fragmented/condensed nuclei. Therefore, for the first time, we demonstrated BDE-47 toxicity and its cell pathway induces cell death using a three-dimensional liver cell culture, the HepaRG cell line.

A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results: We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions: Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.

Identification and Functional Characterization of Alternative Transcripts of LncRNA HNF1A-AS1 and Their Impacts on Cell Growth, Differentiation, Liver Diseases, and in Response to Drug Induction.

The long non-coding RNA (lncRNA) hepatocyte nuclear factor-1 alpha (HNF1A) antisense RNA 1 (HNF1A-AS1) is an important lncRNA for liver growth, development, cell differentiation, and drug metabolism. Like many lncRNAs, HNF1A-AS1 has multiple annotated alternative transcripts in the human genome. Several fundamental biological questions are still not solved: (1) How many transcripts really exist in biological samples, such as liver samples and liver cell lines? (2) What are the expression patterns of different alternative HNF1A-AS1 transcripts at different conditions, including during cell growth and development, after exposure to xenobiotics (such as drugs), and in disease conditions, such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD) cirrhosis, and obesity? (3) Does the siRNA used in previous studies knock down one or multiple transcripts? (4) Do different transcripts have the same or different functions for gene regulation? The presented data confirm the existence of several annotated HNF1A-AS1 transcripts in liver samples and cell lines, but also identify some new transcripts, which are not annotated in the Ensembl genome database. Expression patterns of the identified HNF1A-AS1 transcripts are highly correlated with the cell differentiation of matured hepatocyte-like cells from human embryonic stem cells (hESC), growth and differentiation of HepaRG cells, in response to rifampicin induction, and in various liver disease conditions. The expression levels of the HNF1A-AS1 transcripts are also highly correlated to the expression of cytochrome P450 enzymes, such as CYP3A4, during HepaRG growth, differentiation, and in response to rifampicin induction.

Metabolic and phenotypic changes induced by PFAS exposure in two human hepatocyte cell models.

PFAS are ubiquitous industrial chemicals with known adverse health effects, particularly on the liver. The liver, being a vital metabolic organ, is susceptible to PFAS-induced metabolic dysregulation, leading to conditions such as hepatotoxicity and metabolic disturbances. In this study, we investigated the phenotypic and metabolic responses of PFAS exposure using two hepatocyte models, HepG2 (male cell line) and HepaRG (female cell line), aiming to define phenotypic alterations, and metabolic disturbances at the metabolite and pathway levels. The PFAS mixture composition was selected based on epidemiological data, covering a broad concentration spectrum observed in diverse human populations. Phenotypic profiling by Cell Painting assay disclosed predominant effects of PFAS exposure on mitochondrial structure and function in both cell models as well as effects on F-actin, Golgi apparatus, and plasma membrane-associated measures. We employed comprehensive metabolic characterization using liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). We observed dose-dependent changes in the metabolic profiles, particularly in lipid, steroid, amino acid and sugar and carbohydrate metabolism in both cells as well as in cell media, with HepaRG cell line showing a stronger metabolic response. In cells, most of the bile acids, acylcarnitines and free fatty acids showed downregulation, while medium-chain fatty acids and carnosine were upregulated, while the cell media showed different response especially in relation to the bile acids in HepaRG cell media. Importantly, we observed also nonmonotonic response for several phenotypic features and metabolites. On the pathway level, PFAS exposure was also associated with pathways indicating oxidative stress and inflammatory responses. Taken together, our findings on PFAS-induced phenotypic and metabolic disruptions in hepatocytes shed light on potential mechanisms contributing to the broader comprehension of PFAS-related health risks.

Triclocarban induces lipid droplet accumulation and oxidative stress responses by inhibiting mitochondrial fatty acid oxidation in HepaRG cells.

Mitochondrial fatty acid oxidation (mtFAO) plays an important role in hepatic energy metabolism. Severe mtFAO injury leads to nonalcoholic fatty liver disease (NAFLD) and liver failure. Several drugs have been withdrawn owing to safety issues, such as induction of fatty liver disease through mtFAO disruption. For instance, the antimicrobial triclocarban (TCC), an environmental contaminant that was removed from the market due to its unknown safety in humans, induces NAFLD in rats and promotes hepatic FAO in mice. Therefore, there are no consistent conclusions regarding the effects of TCC on FAO and lipid droplet accumulation. We hypothesized that TCC induces lipid droplet accumulation by inhibiting mtFAO in human hepatocytes. Here, we evaluated mitochondrial respiration in HepaRG cells to investigate the effects of TCC on fatty acid-driven oxidation in cells, electron transport chain parameters, lipid droplet accumulation, and antioxidant genes. The results suggest that TCC increases oxidative stress gene expression (GCLM, p62, HO-1, and NRF2) through lipid droplet accumulation via mtFAO inhibition in HepaRG cells. The results of the present study provide further insights into the effect of TCC on human NAFLD through mtFAO inhibition, and further in vivo studies could be used to validate the mechanisms.

Legacy and alternative per- and polyfluoroalkyl substances (PFAS) alter the lipid profile of HepaRG cells.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in various industrial and consumer products. They have gained attention due to their ubiquitous occurrence in the environment and potential for adverse effects on human health, often linked to immune suppression, hepatotoxicity, and altered cholesterol metabolism. This study aimed to explore the impact of ten individual PFAS, 3 H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP/Adona), ammonium perfluoro-(2-methyl-3-oxahexanoate) (HFPO-DA/GenX), perfluorobutanoic acid (PFBA), perfluorobutanesulfonic acid (PFBS), perfluorodecanoic acid (PFDA), perfluorohexanoic acid (PFHxA), perfluorohexanesulfonate (PFHxS), perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS) on the lipid metabolism in human hepatocyte-like cells (HepaRG). These cells were exposed to different concentrations of PFAS ranging from 10 µM to 5000 µM. Lipids were extracted and analyzed using liquid chromatography coupled with mass spectrometry (LC- MS-QTOF). PFOS at 10 µM and PFOA at 25 µM increased the levels of ceramide (Cer), diacylglycerol (DAG), N-acylethanolamine (NAE), phosphatidylcholine (PC), and triacylglycerol (TAG) lipids, while PMPP/Adona, HFPO-DA/GenX, PFBA, PFBS, PFHxA, and PFHxS decreased the levels of these lipids. Furthermore, PFOA and PFOS markedly reduced the levels of palmitic acid (FA 16.0). The present study shows distinct concentration-dependent effects of PFAS on various lipid species, shedding light on the implications of PFAS for essential cellular functions. Our study revealed that the investigated legacy PFAS (PFOS, PFOA, PFBA, PFDA, PFHxA, PFHxS, and PFNA) and alternative PFAS (PMPP/Adona, HFPO-DA/GenX and PFBS) can potentially disrupt lipid homeostasis and metabolism in hepatic cells. This research offers a comprehensive insight into the impacts of legacy and alternative PFAS on lipid composition in HepaRG cells.

Cytotoxic Effects of Major and Emerging Mycotoxins on HepaRG Cells and Transcriptomic Response after Exposure of Spheroids to Enniatins B and B1.

Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays.

Non-invasive methods of monitoring Fe3O4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy.

Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.