HIV-1 gp120 and MA independently increase oxidative stress in astrocytes
Several studies have reported gp120-mediated induction of oxidative stress in astrocytes.7, 8, 23 In this study, we used SVGA astrocytic cells and transfected them with a plasmid containing a gp120 expression vector. Astrocytes were transfected for different lengths of time and ROS was measured using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (H2DC-FDA) on flow cytometer. As shown in Figure 1a, gp120 induced ROS production with a peak increase (75.2±10.6%) at 24 h compared with mock-transfected cells. To confirm the effect of exogenous gp120, we used recombinant gp120IIIB protein and obtained similar results that showed an increase in ROS production as early as 6 h. The peak ROS production (21.7±5.1%) was observed at 12 h of exposure (Figure 1b). The increase in ROS production was also found to be concentration-dependent as 2 nM gp120IIIB showed 22.1±3.2% increase compared with the control (Figure 1c).
MA is known to induce oxidative stress in various cell types in the brain mainly via dopaminergic mechanism.24, 25 To determine the effect of MA on ROS production, we treated the astrocytes with varying concentrations of MA. The results showed a concentration-dependent increase in ROS, with 500 μM showing maximal ROS (37.3±2.4%) at 24 h (Figure 1d). Furthermore, to test the effect of single dose of MA on ROS production, astrocytes were treated with 500 μM MA for various lengths of time. This dose of MA was based on the blood concentrations and tissue/serum compartmentalization as reported in literatures.26, 27, 28 Furthermore, the binge administration of MA in the range of 250 mg–1 g has been found to produce brain concentrations of MA between 164 and 776 μM.27 As expected, MA increased ROS production in a time-dependent manner and peak ROS production (29.4±3.0%) was observed at 24 h (Figure 1e).