Notably, tetracycline was ineffective for CYP3A4 expression. Previous studies have shown that the formation of the main amiodarone Selleck ZD1839 metabolite, the dealkylated metabolite desethylamiodarone, is catalyzed by CYP3A441 and that amiodarone, but not its metabolite, is a weak inhibitor of CYP3A4-mediated activity.42 In addition to steatosis, amiodarone, like other cationic amphiphilic drugs, induced phospholipidosis, identified as intracellular lamellar inclusion bodies formed by excessive accumulation of phospholipids.
These lamellar bodies were observed in both hepatocyte-like and biliary-like HepaRG cells in agreement with the fact that phospholipidosis can be visualized in various hepatic43 and nonhepatic cell types.44 Up-regulation of the fatty acid biosynthesis-related
gene SCD suggested an enhanced synthesis of phospholipids in HepaRG cells treated with amiodarone for 24 hours. Furthermore, an induction of cholesterol synthesis, supported by overexpression of LSS, was observed, representing an indirect mechanism of phospholipidosis.36 Another gene LPIN1 was specifically overexpressed in HepaRG cells after both acute and repeat amiodarone exposure. LPIN1 encodes the phosphatidate phosphatase-1 enzyme, which converts phosphatidate to diacylglycerol. The resulting diacylglycerol serves as substrate for the synthesis of triacylglycerol as well as phosphatidylethanolamine BGJ398 and phosphatidylcholine.45 Importantly, a strong increase of phosphatidylethanolamine and phosphatidylcholine was observed in HepaRG cells treated with amiodarone for 14 days. In addition, genes involved in phospholipid degradation (GDPD3 and ASML3A) were also up-regulated after 14 days. GDPD3 and LSS were similarly found overexpressed in amiodarone-treated HepG2 cells.36, medchemexpress 46 Some of these genes (ASML3A, GDPD3,
LPL) were modulated specifically after repeat exposure with amiodarone; they likely corresponded to a defense mechanism to reduce phospholipid accumulation and therefore could represent potential biomarkers of drug-induced phospholipidosis. In conclusion, our study provides the first in vitro demonstration of drug-induced vesicular steatosis after repeat treatments. This vesicular steatosis was characterized by an excessive accumulation of TG together with the appearance of Oil Red O–stained lipid vesicles and overexpression of several genes involved in lipogenesis and droplet formation. These data provide new insight into the mechanisms of drug-induced TG accumulation in human hepatocytes and suggest that the HepaRG cell model represents a unique tool for estimating the ability of new drugs to induce steatosis and/or phospholipidosis, as well as other liver injuries, during their early development stage. This cell model should also be appropriate for investigations on steatosis reversibility as well as late steatosis stages leading to steatohepatitis.