Benzop(a)pyrene in Cigarette Smoke Enhances HIV‐1 Replication in Monocytic Cells: Potential Role of Cytochrome P450s and Oxidative Stress

Abstract

Smoking aggravates HIV‐1 pathogenesis and leads to decreased responses to antiretroviral therapy. However, the exact mechanism by which smoking enhances HIV‐1 pathogenesis is unknown. Benzo(a)pyrene (BaP) is a major carcinogen in cigarette smoke. It requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesize that BaP is metabolized by CYP‐mediated pathway and produce reactive oxygen species (ROS). Oxidative stress resulting from increased ROS would aggravate HIV‐1 replication. Recently, we have shown that chronic exposure of BaP to U937 monocytic cells results in the significant induction of CYPs (CYP 1A1 and 3A4) and antioxidants (catalase and SOD1), and increase in the generation of ROS and cellular toxicity. We also confirmed these results in human primary macrophages. Next, we examined the chronic effect of BaP in U1 cells (HIV‐1 infected U937 monocytic cells). When U1 cells were exposed to BaP for seven days, we observed ~4 fold increase in HIV‐1 replication. In addition, there was a significant increase in the expression of CYP1A1 at mRNA level as well as there was an increase in CYP1A1 enzymatic activity. Elevated ROS and massive cell death were also observed. In order to confirm the role of CYP1A1 and 1B1 in BaP‐induced oxidative stress pathway and HIV‐1 replication, we knocked down the gene expression of CYP1A1 and 1B1 in HIV‐1 infected human primary macrophages, using SiRNA. Our results showed decrease in the viral replication in CYP1A1 and 1B1 knock‐out cells upon BaP exposure. We will determine oxidative DNA damage in untreated, and CYP1A1 and 1B1 knock‐out cells using TUNNEL assay. We are now in the process of determining the role of oxidative stress mediated through CYP pathway in HIV‐1 replication. For this, we will expose HIV‐infected primary macrophages with general and specific antioxidants, followed by exposure to BaP. Finally, we will quantify BaP metabolites in U1 cells and/or HIV‐1 infected human primary macrophages using Liquid Chromatography‐Mass Spectrometry. The results from the present work are clinically relevant as they would help to find a novel therapeutic target to improve drug therapy outcomes in HIV positive smokers.Support or Funding InformationDepartment of Pharmaceutical Sciences, University of Tennessee Health Science Center