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

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.     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.