Cytotoxic effect of 7α‐hydroxy‐4‐cholesten‐3‐one on HepG2 cells: Hypothetical role of acetaldehyde‐modified Δ4‐3‐ketosteroid‐5β‐reductase (the 37‐kd‐liver protein) in the pathogenesis of alcoholic liver injury in the rat

We recently identified ▵4-3-ketosteroid-5β-reductase as the 37 kd liver protein which is highly susceptible to acetaldehyde modification in rats continuously fed alcohol. The 5β-reductase is a key enzyme involved in bile acid synthesis. We report here that the ability to degrade 7α-hydroxy-4-cholesten-3-one (HCO) was lower in the liver cytosol of alcohol-fed rats than in control animals, suggesting an inhibition of the 5β-reductase enzyme activity by acetaldehyde modification. We also showed that HCO exhibited a time- and concentration-dependent cytotoxicity to HepG2 cells. HCO cytotoxicity was noticeable at a concentration of 2.5 μg/mL. When 10 μg/mL of HCO was added to confluent cell monolayers, 57% and 37% of cells remained viable after 24 and 48 hours of treatment. The decrease in cell viability was accompanied by an increased lactic dehydrogenase activity in the culture medium. DNA extracted from HCO-treated cells showed no evidence of DNA fragmentation when analyzed by agarose gel electrophoresis. Staining with propidium iodide showed no nuclear condensation in cells. Thus, cell death by HCO treatment was caused by necrosis and not by apoptosis. Various agents, including, serum proteins, hormones, bile acids, antioxidants, Ca++-chelators, Fe++-chelator, CYP450 inhibitor, adenylate cyclase inhibitor, protease inhibitors, and nitric oxide synthase inhibitor, did not protect against HCO cytotoxicity. We speculate that HCO concentrations may be elevated around the pericentral area in the liver after chronic alcohol ingestion, causing local cell necrosis. The release of cellular contents and protein-acetaldehyde adducts (PAAs) may activate nonparenchymal cells and provoke autoimmune reaction. Thus, the formation of the 37 kd-PAA may play an important role in the initiation of alcoholic liver injury.