Chronic Effects of Ethanol and/or Darunavir/Ritonavir on U937 Monocytic Cells: Regulation of Cytochrome P450 and Antioxidant Enzymes, Oxidative Stress, and Cytotoxicity

Background: Our recent study has shown that acute treatment with ethanol (EtOH) increases oxidative stress and cytotoxicity through cytochrome P450 2E1 (CYP2E1)-mediated pathway in U937 monocytic cells. U937 cells are derived from blood monocytes and are considered as the model system for HIV-related study. Since the prevalence of alcohol use in HIV-infected population is high, and HIV+ patients are on antiretroviral therapy (ART) soon after they are diagnosed, it is important to study the interactions between EtOH and ART in monocytes. Methods: This study examined the chronic effects of EtOH and ART (darunavir/ritonavir), alone and in combination, on expression/levels of cytochrome P450 enzymes (CYPs), antioxidant enzymes (AOEs), reactive oxygen species (ROS), and cytotoxicity in U937 cells. The mRNA and protein levels were measured using quantitative reverse transcription polymerase chain reaction and Western blot, respectively. ROS and cytotoxicity were measured using flow cytometry and cell viability assay, respectively. Results: While chronic ART treatment increased CYP2E1 protein expression by 2-fold, EtOH and EtOH+ ART increased CYP2E1 by similar to 5-fold. In contrast, ART and EtOHtreatments decreased CYP3A4 protein expression by 38 +/- 17% and 74 +/- 15%, respectively, and the combination additively decreased CYP3A4 level by 90 +/- 8%. Expressions of superoxide dismutase 1 (SOD1) and peroxiredoxin (PRDX6) were decreased by both EtOH and ART, however, the expressions of SOD2 and catalase were unaltered. These results suggested increased EtOH metabolism, increased ART accumulation, and decreased defense against ROS. Therefore, we determined the effects of EtOH and ART on ROS and cytotoxicity. While ART showed a slight increase, EtOH and EtOH+ ART displayed significant increase in ROS and cytotoxicity. Moreover, the combination showed additive effects on ROS and cytotoxicity. Conclusions: These results suggest that chronic EtOH, in the absence and presence of ART, increases ROS and cytotoxicity in monocytes, perhaps via CYP- and AOE-mediated pathways. This study has clinical implications in HIV+ alcohol users who are on ART.