Assessment of endocrine disruptor impacts on lipid metabolism in a fatty acid-supplemented HepaRG human hepatic cell line.

The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing worldwide. This disease encompasses several stages, from steatosis to steatohepatitis and, eventually, to fibrosis and cirrhosis. Exposure to environmental contaminants is one of the risk factors and an increasing amount of evidence points to a role for endocrine disrupting compounds (EDCs). This study assesses the impact of selected EDCs on the formation of lipid droplets, the marker for steatosis in a hepatic model. The mechanisms underlying this effect are then explored. Ten compounds were selected according to their obesogenic properties: bisphenol A, F and S, butyl-paraben, cadmium chloride, p,p'-DDE, DBP, DEHP, PFOA and PFOS. Using a 2D or 3D model, HepaRG cells were exposed to the compounds with or without fatty acid supplementation. Then, the formation of lipid droplets was quantified by an automated fluorescence-based method. The expression of genes and proteins involved in lipid metabolism and the impact on cellular respiration was analyzed. The formation of lipid droplets, which is revealed or enhanced by oleic acid supplementation, was most effectively induced by p,p'-DDE and DEHP. Experiments employing either 2D or 3D culture conditions gave similar results. Both compounds induced the expression of PLIN2. p,p'-DDE also appears to act by decreasing in fatty acid oxidation. Some EDCs were able to induce the formation of lipid droplets, in HepaRG cells, an effect which was increased after supplementation of the cells with oleic acid. A full understanding of the mechanisms of these effects will require further investigation. The novel automated detection method described here may also be useful in the future as a regulatory test for EDC risk assessment.

Inhibition by pesticides of the DJ-1/Park7 protein related to Parkinson disease.

Parkinson's disease is a severe neurodegenerative disease. Several environmental contaminants such as pesticides have been suspected to favor the appearance of this pathology. The protein DJ-1 (or Park7) protects against the development of Parkinson's disease. Thus, the possible inhibitory effects of about a hundred pesticides on human DJ-1 have been studied. We identified fifteen of them as strong inhibitors of DJ-1 with IC50 values between 0.02 and 30 µM. Thiocarbamates are particularly good inhibitors, as shown by thiram that acts as an irreversible inhibitor of an esterase activity of DJ-1 with an IC50 value of 0.02 µM. Thiram was also found as a good inhibitor of the protective activity of DJ-1 against glycation. Such inhibitory effects could be one of the various biological effects of these pesticides that may explain their involvement in the development of Parkinson's disease.

Impact of hepatitis C virus and alcohol, alone and combined, on the unfolded protein response in primary human hepatocytes.

Hepatitis C virus (HCV) infection and alcohol abuse are leading causes of chronic liver disease and frequently coexist in patients. The unfolded protein response (UPR), a cellular stress response ranging along a spectrum from cytoprotection to apoptosis commitment, has emerged as a major contributor to human diseases including liver injuries. However, the literature contains conflicting reports as to whether HCV and ethanol activate the UPR and which UPR genes are involved. Here we have used primary human hepatocytes (PHH) to reassess this issue and address combined impacts. In this physiologically relevant model, either stressor activated a chronic complete UPR. However, the levels of UPR gene induction were only modest in the case of HCV infection. Moreover, when combined to the strong stressor thapsigargin, ethanol exacerbated the activation of pro-apoptotic genes whereas HCV tended to limit the induction of key UPR genes. The UPR resulting from HCV plus ethanol was comparable to that induced by ethanol alone with the notable exception of three pro-survival genes the expressions of which were selectively enhanced by HCV. Interestingly, HCV genome replication was maintained at similar levels in PHH exposed to ethanol. In conclusion, while both HCV and alcohol activate the hepatocellular UPR, only HCV manipulates UPR signalling in the direction of a cytoprotective response, which appears as a viral strategy to spare its own replication.

Down-regulation of the expression of alcohol dehydrogenase 4 and CYP2E1 by the combination of α-endosulfan and dioxin in HepaRG human cells.

Pesticides and other persistent organic pollutants are considered as risk factors for liver diseases. We treated the human hepatic cell line HepaRG with both 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the organochlorine pesticide, α-endosulfan, to evaluate their combined impact on the expression of hepatic genes involved in alcohol metabolism. We show that the combination of the two pollutants (25nM TCDD and 10µM α-endosulfan) led to marked decreases in the amounts of both the mRNA (up to 90%) and protein (up to 60%) of ADH4 and CYP2E1. Similar results were obtained following 24h or 8days of treatment with lower concentrations of these pollutants. Experiments with siRNA and AHR agonists and antagonist demonstrated that the genomic AHR/ARNT pathway is necessary for the dioxin effect. The PXR, CAR and estrogen receptor alpha transcription factors were not modulators of the effects of α-endosulfan, as assessed by siRNA transfection. In another human hepatic cell line, HepG2, TCDD decreased the expression of ADH4 and CYP2E1 mRNAs whereas α-endosulfan had no effect on these genes. Our results demonstrate that exposure to a mixture of pollutants may deregulate hepatic metabolism.

Novel roles for AhR and ARNT in the regulation of alcohol dehydrogenases in human hepatic cells.

The mechanisms by which pollutants participate in the development of diverse pathologies are not completely understood. The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the AhR (aryl hydrocarbon receptor) signaling pathway. We previously showed that TCDD (25 nM, 30 h) decreased the expression of several alcohol metabolism enzymes (cytochrome P450 2E1, alcohol dehydrogenases ADH1, 4 and 6) in differentiated human hepatic cells (HepaRG). Here, we show that, as rapidly as 8 h after treatment (25 nM TCDD) ADH expression decreased 40 % (p < 0.05). ADH1 and 4 protein levels decreased 40 and 27 %, respectively (p < 0.05), after 72 h (25 nM TCDD). The protein half-lives were not modified by TCDD which suggests transcriptional regulation of expression. The AhR antagonist CH-223191 or AhR siRNA reduced the inhibitory effect of 25 nM TCDD on ADH1A, 4 and 6 expression 50-100 % (p < 0.05). The genomic pathway (via the AhR/ARNT complex) and not the non-genomic pathway involving c-SRC mediated these effects. Other AhR ligands (3-methylcholanthrene and PCB 126) decreased ADH1B, 4 and 6 mRNAs by more than 78 and 55 %, respectively (p < 0.01). TCDD also regulated the expression of ADH4 in the HepG2 human hepatic cell line, in primary human hepatocytes and in C57BL/6J mouse liver. In conclusion, activation of the AhR/ARNT signaling pathway by AhR ligands represents a novel mechanism for regulating the expression of ADHs. These effects may be implicated in the toxicity of AhR ligands as well as in the alteration of ethanol or retinol metabolism and may be associated further with higher risk of liver diseases or/and alcohol abuse disorders.

Exposure to metal oxide nanoparticles administered at occupationally relevant doses induces pulmonary effects in mice.

In spite of the great promises that the development of nanotechnologies can offer, concerns regarding potential adverse health effects of occupational exposure to nanoparticle (NP) is raised. We recently identified metal oxide NP in lung tissue sections of welders, located inside macrophages infiltrated in fibrous regions. This suggests a role of these NP in the lung alterations observed in welders. We therefore designed a study aimed to investigate the pulmonary effects, in mice, of repeated exposure to NP administered at occupationally relevant doses. We therefore chose four metal oxide NPs representative of those found in the welder's lungs: Fe2O3, Fe3O4, MnFe2O4 and CrOOH. These NPs were administered weekly for up to 3 months at two different doses: 5 µg, chosen as occupationally relevant to welding activity, and 50 µg, chosen as occupationally relevant to the context of an NP-manufacturing facility. Our results show that 3 month-repeated exposures to 5 µg NP induced limited pulmonary effects, characterized by the development of a mild peribronchiolar fibrosis observed for MnFe2O4 and CrOOH NP only. This fibrotic event was further extended in terms of intensity and localization after the repeated administration of 50 µg NP: all but Fe2O3 NP induced the development of peribronchiolar, perivascular and alveolar fibrosis, together with an interstitial inflammation. Our data demonstrate for the first time a potential risk for respiratory health posed by repeated exposure to NP at occupationally relevant doses. Given these results, the development of occupational exposure limits (OELs) specifically dedicated to NP exposure might therefore be an important issue to address.