Effects of combined alcohol and anti-HIV drugs on cellular stress responses in primary hepatocytes and hepatic stellate and kupffer cells.

Abstract

Certain anti-HIV drugs alone or in combination are often associated with liver damages, which are frequently worsened by alcohol consumption. We previously found an endoplasmic reticulum (ER) stress mechanism for the drug- and alcohol-induced hepatic injuries in animal models and in vitro hepatocytes. However, it is unknown whether anti-HIV drugs and alcohol induce similar cellular stress responses and injuries in liver nonparenchymal cells. Primary mouse hepatocytes (PMH), kupffer cells (KC), and hepatocellular stellate cells (HSC) were freshly isolated from mouse liver and treated with DMSO, stress-inducing pharmaceutical agents, alcohol alone, or in combination with antiviral ritonavir (RIT), lopinavir (LOP), or efavirenz (EFV). Expression of cellular stress markers, protein colocalization, and cell death were analyzed with immunoblotting, immunocytochemistry, and positive double staining with Sytox green and Hoechst blue, respectively. Expression of the ER stress markers of BiP, CHOP, and SERCA and the autophagy marker LC3 was significantly changed in PMH in response to combined alcohol, RIT, and LOP, which was companied by increased cell death compared with control. In contrast, although pharmaceutical agents induced ER stress and cell death, no significant ER stress or cell death was found in KC treated with alcohol, RIT, LOP, and EFV singly or in combination. In HSC, alcohol, RIT, LOP, or EFV induced BiP, but not CHOP, SERCA, or cell death compared with vehicle control. Further in PMH, RIT and LOP or in combination with alcohol-induced dose-dependent inhibition of β-actin. Inhibition of β-actin by RIT and LOP was companied with an inhibited nuclear expression of the antioxidant response regulator Nrf2 and reduced GST downstream of Nrf2. Ascorbic acid treatment reduced the alcohol-, RIT-, and LOP-induced cell death. The data suggest for the first time that sensitivities of hepatocytes and nonparenchymal cells to alcohol and anti-HIV drugs in vitro are different in terms of cellular stress response and cell death injury. Oxidative stress mediated by Nrf2 contributes to the alcohol- and drug-induced toxicity in the hepatocytes.