ver, our information recommended that M3 and M4 renal elimination could be altered in COVID-19 sufferers with TXA2/TP Species kidney impairment. Alternatively, these metabolites may be toxic for the kidney, or may well participate to renal impairment in COVID-19 individuals, along with other components. Lastly, physique temperature in COVID-19 sufferers was negatively correlated with decreased M1 (DHCQ) and M2 (DCQ) levels. These benefits could be consistent with these from Kihara et al. (1998), reporting a decrease in CYP activity through fever, probably resulting from theInt. J. Mol. Sci. 2022, 23,11 ofaction of α adrenergic receptor list secreted pro-inflammatory cytokines [46]. Furthermore, preceding research in our laboratory have shown that hypoxia induces a switch of liver metabolism from aerobic to anaerobic glycolysis in addition to a repression of critical genes as CYP3A4 [47]. These information in the literature could also clarify the correlation discovered involving PCT, a marker of an infectious context, and PaO2 using the accumulation on the M5 metabolite (Supplementary Figure S3) [46]. Lastly, cortisol levels have been negatively correlated with HCQ. Here, we think that this context of inflammation doesn’t enable for the metabolic changes observed in vitro by steatosis alone to become observed in sufferers. This study presents various limitations. Firstly, we disclosed a modification of HCQ metabolism and toxicity in a cellular model of fatty acid overload [32,33]. Having said that, it could be exciting to perform further investigations inside a cellular model of nonalcoholic steatohepatitis (NASH) by co-culturing HepaRG cells with macrophages and stellate cells. Certainly, fatty liver can progress inside a important proportion of individuals to NASH, a diseased state that may well additional favor drug-induced hepatotoxicity through oxidative pressure and mitochondrial dysfunction [42,48]. Secondly, even though the molecular network permits for an effective exploration from the drug metabolism, it can’t be excluded that some minor metabolites weren’t detected with our methodology. Thirdly, it would be exciting to study an independent cohort of COVID-19 patients treated with HCQ, in particular to confirm our data suggesting that DCQ (M2) may favor hepatic cytolysis. All round, we showed that the HepaRG cell model coupled to molecular networking has established valuable to investigate HCQ metabolism and toxicity within the context of obesity-associated fatty liver. The improvement of cellular models relevant to other pathophysiological situations such as hypoxia or inflammation would be of particular interest to improved realize the prospective adjustments of drug metabolism and toxicity occurring in the COVID-19 population. four. Materials and Strategies four.1. Material William’s E medium was purchased from Gibco (ThermoFischer Scientific, San Jose, CA, USA). Penicillin-streptomycin was obtained from Life Technologies (Grand Island, NY, USA). Fetal Bovine Serum (FBS) was purchased from Eurobio (Courtaboeuf, France) and from HycloneTM GE Healthcare Life Sciences (Logan, UT, USA). Hydrocortisone hemisuccinate was bought from Serb (Paris, France). Dimethyl sulfoxide (DMSO), formic acid, ammonium acetate, insulin, HCQ, neutral red powder, DAPI, Nile red, and Phosphate Buffered Saline (PBS) have been obtained from Sigma-Aldrich (Saint Louis, MO, USA). Methanol, acetonitrile, and water were liquid chromatography-mass spectrometry (LC S) grade high-quality and had been purchased from Fisher Scientific UK (Loughborough, Leicestershire, UK). two,3-Bis(2-methoxy-4-nitro-5-sulfopheny)-2H-tetrazolium