Interestingly, mitochondria, nuclei, and endoplasmic reticulum remained morphologically unchanged. Cholesterol and neutral lipids (TG and cholesterol esters) were quantified by way of gas/liquid chromatography (Fig. 3). Whereas tetracycline caused no significant changes in TG content after 24 hours,

a six-fold increase was induced by a 50 μM concentration after 14 days. By contrast, GSK-3 activation cholesterol and cholesterol esters content remained unchanged. A dose-dependent increase in TG content was also observed, and cholesterol esters were slightly augmented in HepaRG cells treated by amiodarone for 14 days. In addition, phospholipids (phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, and phosphatidylinositol) were measured by way of HPLC in HepaRG cells treated with 20 μM amiodarone for 24 hours or 14 days (Fig. 4). Whereas no significant change was observed in phospholipid content after acute exposure, phosphatidylethanolamine and phosphatidylcholine levels were strongly enhanced, and sphingomyelin, phosphatidylserine, and phosphatidylinositol levels were slightly augmented after 14 days. Impairment of mitochondrial fatty acid oxidation (FAO) is considered one of the major mechanisms of liver steatosis.21 FAO was evaluated by measuring [14C]-labeled acid-soluble

β-oxidation products in HepaRG cells after 24-hour and 14-day treatments using either 20 μM tetracycline or 50 μM amiodarone (Fig. 5). A 20% diminution of FAO was observed after both acute and chronic PF-6463922 nmr amiodarone treatments, and only after chronic tetracycline exposure. To characterize gene expression changes associated with induction of phospholipidosis and steatosis, the transcriptome of HepaRG cells was analyzed after 24-hour and 14-day treatments with 20 μM amiodarone using pangenomic

oligonucleotide microarrays. Significantly modulated genes were extracted with a fold change >1.5 or <−1.5 and P ≤ 0.01 as filters. Their total numbers reached 547 and 594 with up-regulated genes representing 48% and 44%, after 24-hour and 14-day exposure, respectively (Supporting Tables 1 and 2); 176 genes were in common at the two time points. Functional analysis revealed that expression of many genes involved in the regulation of lipid metabolism (including ACOT12, ADFP, ALDH3A1, APOA2, FASN, MOGAT1, SREBP1, selleck kinase inhibitor and THRSP) or related to phospholipidosis (such as LSS, LPIN1, ASML3A, and GDPD3) was significantly altered. Various genes regulating growth/proliferation, cell death, assembly/organization, and inflammation were also substantially deregulated. To validate and complete this microarray analysis, changes in the expression of 29 genes, which are key players in lipid metabolism and/or liver-specific functions, were further examined by way of RT-qPCR in HepaRG cells exposed to several concentrations of amiodarone (5-20 μM), tetracycline (10-100 μM), and oleic acid (100-500 μM) for 24 hours or 14 days. The data are displayed in Table 2.