Effects of Ritonavir, Lopinavir, and Alcohol on ABC Transporters and Secretion of Bile Acid and Bilirubin in Senescent Hepatocytes.

Drug- and alcohol-induced liver injury involves impaired bile acids or bilirubin secretion, but it is not known how senescence influences the secretion of hepatocytes exposed to drugs and alcohol. In this study, the toxic effects of ritonavir, lopinavir, and alcohol on hepatocyte transporters and the secretion of bile acids and bilirubin were investigated in hydrogen peroxide-induced senescent HepG2 and doxorubicin-induced senescent primary human hepatocytes. In HepG2, intracellular conjugated bilirubin increased upon senescence and extracellular conjugated bilirubin in culture medium was decreased by ritonavir and lopinavir treatment. In the primary hepatocytes, intracellular bile acids or medium bilirubin were not significantly changed upon senescence. However, intracellular bile acids were increased, and medium conjugated bilirubin were decreased in senescent primary hepatocytes treated with alcohol and the two drugs. Transcriptional expressions of adenosine triphosphate (ATP)-binding cassette (ABC) transporters (ABCB4, ABCC6, ABCB11, and ABCD3) were decreased whereas UDP-glucuronosyltransferase (UGT1A1) was increased by ritonavir and lopinavir in senescent HepG2. In senescent primary hepatocytes, expressions of ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, and ABCC6 were apparently reduced whereas UGT1A1 and the cytochrome P450 enzyme CYP7A1 were markedly increased by alcohol combined with ritonavir and lopinavir. Selective ABCC6 knockdown in the primary hepatocytes altered expressions of two senescence markers, Lamin A/C and cyclin-dependent kinase inhibitor CKI (p21), increased expressions of CYP7A1 and hydroxy methyl glutaryl-CoA reductase (HMGCR), and increased intracellular bile acids. Further, anti-cholestasis agents, ursodeoxycholic acid and glycyrrhizin, significantly ameliorated the impaired secretions of bile acids and bilirubin as well as reducing intracellular lipid accumulation and cell death caused by ritonavir, lopinavir, and alcohol in the primary hepatocytes with ABCC6 knockdown. These results indicate that senescence moderately impairs the ABC transporters of hepatocytes and secretion of bile acids or bilirubin, which become worse in the presence of the drugs and alcohol but could be improved by anti-cholestasis agents.