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

Abstract

Background Our recent study has shown that acute treatment with ethanol 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 ethanol and ART in monocytes. Methods This study examined the chronic effects of ethanol 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 RTPCR and Western blot, respectively. ROS and cytotoxicity were measured using flow cytometry and XTT assay, respectively. Results While chronic ART treatment increased CYP2E1 protein expression by 2-fold, ethanol and ethanol+ART increased CYP2E1 by ~5-fold. In contrast, ART and ethanol treatments 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 (SOD1) and peroxiredoxin (PRDX6) were decreased by both ethanol and ART, however, the expressions of SOD2 and catalase were unaltered. These results suggested increased ethanol metabolism, increased ART accumulation, and decreased defense against ROS. Therefore, we determined the effects of ethanol and ART on ROS and cytotoxicity. While ART showed a slight increase, ethanol and ethanol+ART displayed significant increase in ROS and cytotoxicity. Moreover, the combination showed additive effects on ROS and cytotoxicity. Conclusions These results suggest that chronic ethanol, in the absence and presence of ART, increases ROS and cytotoxicity in monocytes, perhaps via CYPs and AOEs mediated pathways. This study has clinical implications in HIV+ alcohol users who are on ART.

Neuroprotection mediated by inhibition of calpain during acute viral encephalitis

“…Fifth, even direct ex vivo incubation of leukocytes with ritonavir, at concentrations similar to maximum one hour post-treatment plasma levels, only weakly impacted oxidative responses and had little to no effect on phagocytosis 47 . Sixth, direct in vitro incubation of monocytic cells with 1 μM ritonavir for 4 days did not reduce superoxide production and did not alter proliferative responses 48 . Finally, even if ritonavir therapy does alter some aspect of leukocyte infiltration or function that we have not assessed, the mice are still protected from hippocampal injury and seizures without jeopardizing viral clearance or health of the host, supporting our contention that ritonavir treatment may be a unique neuroprotective intervention during acute viral encephalitis 41 .…”

Influence of Ethanol on Darunavir Hepatic Clearance and Intracellular PK/PD in HIV-Infected Monocytes, and CYP3A4-Darunavir Interactions Using Inhibition and in Silico Binding Studies

“…Ethanol treatment exhibited a significant decrease in the apparent half-life (t 1/2 ) for DRV + RTV (Fig. 2A, (F (3,11) = 34, P = 0.003, one-way ANOVA) but not for the DRV alone ( P = 1). We observed increased trend in K value for DRV + RTV when exposed to ethanol but this increase is not statistically significant (F (3,11) = 88, P = 0.151, one-way ANOVA) when compared to DRV + RTV condition.…”

“…5). Ethanol induced HIV replication in monocytes cells appears to be via CYP2E1-mediated alcohol metabolism and resultant oxidative stress (7,11,23). Similar to 20 mM ethanol concentration, ethanol-induced increase in HIV replication was completely abolished when cells were treated with DRV or DRV + RTV at both 10 mM and 50 mM ethanol concentrations ( p < 0.05 compared to control or ethanol, student t test).…”

“…We have previously reported that ethanol induces CYP3A4 expression and alters the interaction of different PIs with CYP3A4 (9,10). In addition, a recent in vitro study revealed that ethanol and DRV/RTV regimen alone, as well as in combination alter the expression level of CYP and antioxidant proteins, which is associated with increased oxidative stress and cytotoxicity in monocytic cells (11). …”

Choosing the right pharmacotherapeutic strategy for HIV maintenance in patients with alcohol addiction

“…Further, another commonly used ARV, elvitegravir (EVG), is metabolized by CYP3A4 and a strong CYP3A4 inhibitor such as cobicistat (COBI) is coadministered as a booster to increase EVG bioavailability. Recently, Kumar group showed that the ethanol exposure decreases the intracellular levels of EVG alone and also in combination with COBI in HIV-infected macrophages [19,20]. Subsequently, co-exposing cells to EVG or EVG + COBI along with ethanol showed increased viral replication by 31% and 19%, respectively, relative to control groups that did not receive ethanol [19].…”