HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

A Shah,D P Singh,S Kumar,A Kumar,S D Simon

doi:10.1038/cddis.2013.374

Abstract

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.

Highlights

(CNS) where the virus can replicate in microglia and astrocytes.[1,2] The neurotoxic effects of HIV-1 proteins, including gp[120], can be either through direct or indirect mechanisms.[3]
We demonstrate that gp[120] and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways
Reaction (Figure 7f) significantly reduced MA/gp120mediated caspase-3 activity. These findings clearly suggested that pathways involving CYP2E1, NOX family of enzymes, and FWH reaction are responsible for MA- and/or gp120-mediated apoptosis in SVGA astrocytes

Summary

Introduction

(CNS) where the virus can replicate in microglia and astrocytes.[1,2] The neurotoxic effects of HIV-1 proteins, including gp[120], can be either through direct or indirect mechanisms.[3]. Oxidative stress is suggested to have an important role in MA-mediated neurotoxicity. We have recently shown that gp[120] and MA cooperate synergistically to induce the proinflammatory cytokine IL-6 in astrocytes.[14] it is not known whether such cooperation exists at the oxidative stress level. Recent advances suggest the involvement of various cytochrome P450 (CYP) enzymes in neurotoxicity, perhaps as a result of production of reactive oxygen species (ROS) and/or reactive metabolites. The role of CYPs in drug abuse and HIV-1 neuropathogenesis is unexplored, a recent study suggests a potential role for CYP2E1 in alcoholmediated neurotoxicity.[22] NADPH oxidase (NOX) has been shown to induce ROS both independently and in association with CYP-mediated drug metabolism. We hypothesize that CYP and NOX pathways are involved in HIV- and/or MA-induced neurotoxicity

Methods

Results

Conclusion