Results

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).

HIV-1 gp120 and MA additively increase oxidative stress in astrocytes
Upon demonstrating that both MA and gp120 independently induced ROS in SVGA astrocytes; we examined whether MA and gp120 interact additively or synergistically to increase oxidative stress. We treated SVGA astrocytes with 500 μM MA and transfected with 2 μg of gp120-plasmid followed by measurement of ROS at 24 h after treatment. Clearly, astrocytes treated with both MA and gp120 showed ROS levels (107.1±12.9%) higher than either MA (41.8±4.4%) or gp120 alone (67.7±11.0%) (Figure 2a). Furthermore, two-factor ANOVA model suggested an additive effect rather than synergistic (P=0.91). To confirm the finding in primary astrocyte culture, we used 2 nM recombinant gp120IIIB protein in combination with 500 μM MA. We observed similar results in terms of ROS production in primary human fetal astrocytes using flow cytometry (5.9±2.5% for MA, 24.3±4.0% for gp120, and 54.8±13.1% for MA+gp120) (Figure 2b). Similarly, the fluorescence observed using microscopy was significantly higher when cells were treated with both gp120 and MA as compared with cells treated with either agent alone in SVGA astrocytes (Figure 2c) and primary astrocytes (Figure 2d).
As oxidative stress is known to be associated with both cell death29 and cell proliferation,30 depending on the extent of the ROS production, we analyzed the functional consequences of MA and gp120 treatments in SVGA astrocytes. Clearly, the treatment with MA and gp120 significantly increased the level of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-positive astrocytes when compared with MA or gp120 alone (Figure 2e). In parallel experiments, we quantified the level of cell death induced by MA and/or gp120 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and found that cells treated with gp120 and MA showed an additive effect and significantly higher (P≤0.01) cell death was observed (14.1±2.9%) when compared with either MA (6.8±1.5%) or gp120 (8.3±1.8%) alone (Figure 2f). These observations indicated that the oxidative stress induced by gp120 and/or MA was lethal to astrocytes and MA and gp120 additively increase the cellular toxicity.
As the combination of MA and gp120 showed increased oxidative stress, we sought to address the molecular mechanism responsible for these effects. First, we pretreated SVGA cells with 100 μM vitamin C (vit. C), a common antioxidant, followed by treatments with gp120 and MA. As expected, vit. C alone reduced basal levels of ROS production. However, it also abolished the ROS generated by either MA or gp120 alone, as well as MA+gp120 (Figure 3a). In addition, vit. C also abrogated cell death caused by MA and gp120 alone and also in combination (Figure 3b). Similarly, we also used vit. E, another canonical ROS quencher to confirm our results with vit. C. Surprisingly, TROLOX, a water-soluble form of vit. E, did not reduce ROS production (Supplementary Figure 1A). However, when we used vit. E in its native form (α-tocopherol), it reduced ROS generated by MA or gp120 alone, as well as MA+gp120 (Figure 3c). It also rescued the astrocytes from cell death (Figure 3d). Although N-acetyl cysteine (NAC) has been shown to abrogate gp120-mediated ROS production in astrocytes,23 in our studies NAC did not show any reduction in ROS production (Supplementary Figure 1B). This observation is similar to our previous report, where we showed that NAC did not reduce alcohol-mediated ROS in astrocytes.22

MA and gp120 induced the expression of CYP
As CYP has been shown to be involved in oxidative stress in many organs/tissues including the brain, we measured the mRNA expression levels of various CYPs in astrocytes treated with MA and/or gp120. Both MA and gp120 induced different isozymes of CYP at variable levels (Figure 4a). Among the isozymes induced, CYP2E1 (1.7±0.2-fold), CYP2D6 (2.3±0.3-fold), and CYP2B6 (3.2±03-fold) clearly showed additive increases (two-way ANOVA showed P-value=0.34, 0.18, and 0.84, respectively, suggesting the absence of synergy) in the levels of mRNA expression with gp120+MA when compared with either MA (nonsignificant change for CYP2E1 and CYP2D6 and 2.8±0.3-fold for CYP2B6) or gp120 (1.3±0.1-fold for CYP2E1 and 1.8±0.3-fold for CYP2D6 and nonsignificant for CYP2B6) alone (Figure 4a). These results were further confirmed at the protein level for CYP2E1 (1.3±0.1-fold for gp120+MA), CYP2D6 (1.7±0.2-fold for gp120+MA), and CYP2B6 (1.4±0.1-fold for gp120+MA) (Figure 4b). Furthermore, to confirm this phenomenon in primary astrocytes, human fetal astrocytes (HFA) were treated with gp120 IIIB and/or MA and the protein and mRNA expression levels of the CYPs were measured. Similar to SVGA astrocytes, gp120 and MA also showed additive increase in the levels of CYP2E1, CYP2B6, and CYP2D6 mRNA (Figure 4c) and protein (Figure 4d) in HFA primary cells. The analysis run for synergy using two-way ANOVA showed nonsignificant P-values, suggesting no synergistic interaction.

Role of CYP2E1 in gp120- and/or MA-mediated oxidative stress
To examine the role of CYP2E1 in oxidative stress, we pretreated astrocytes with different concentrations of diallylsulfide (DAS), a selective inhibitor of CYP2E1. Clearly, both 10 and 25 μM DAS reduced the ROS generated by either MA and/or gp120 (Figure 4e). It is of note that DAS alone showed some decrease in the basal ROS production, but its efficacy against MA- and/or gp120-mediated oxidative stress was significant and dose-dependent (P for trend=0.01). Although DAS did not significantly rescue the cell from death caused by MA alone, it significantly rescued the cell death (P≤0.01) caused by gp120 alone and MA+gp120 (Figure 4f). In parallel experiments, DAS was also found to reduce the TUNEL staining in the cells treated with MA and/or gp120 (Figure 4g). Furthermore, we also specifically knocked down CYP2E1 using siRNA in order to confirm our findings with chemical inhibitor. As expected, the knockdown of CYP2E1 resulted into reduction of ROS (Figure 4h) and it also showed reduced TUNEL staining in the cells treated with MA and/or gp120 (Figure 4i). The specific knockdown of CYP2E1 was confirmed using western blotting, which showed 75–80% knockdown of CYP2E1 (Figure 4h). These results clearly suggest the involvement of CYP2E1 in oxidative stress and cell death induced by gp120, MA, or both. We also tested fluoxetine and paroxetine, specific inhibitors for CYP2D6, and orphenadrine (OP), specific inhibitor for CYP2B6, on MA+gp120-mediated ROS generation. However, these antagonists failed to reduce the ROS formation, rather paroxetine and OP further increased oxidative stress (Supplementary Figure 2). In addition, we also specifically knocked down CYP2B6 and CYP2D6 using siRNA. However, as observed with chemical inhibitors, these siRNAs did not show any effect on ROS production (Supplementary Figure 3). Although, oxidative stress is one of the functional consequences of CYP enzyme activity, the possible role of CYP2D6 and CYP2B6 in other physiological functions cannot be disregarded.

NOX and metal chelation are involved in MA/gp120-mediated oxidative stress and cell death
The NOX family of enzymes are responsible for the transfer of electrons across biological membranes and generation of ROS.31 In addition, various NOXs also serve as essential coenzymes coupled with CYP2E1-mediated electron transfer in the generation of ROS.32 To examine the involvement of NOX in gp120±MA-mediated oxidative stress, we treated the SVGA astrocytes with various concentrations of diphenyleneiodonium (DPI), an inhibitor for NOX. DPI significantly reduced the ROS production observed with either MA or gp120 alone and MA+gp120 (Figure 5a) in a dose-dependent manner (P for trend=0.01). Furthermore, 25 nM DPI rescued the cell death induced by MA and/or gp120 (Figure 5b), thereby confirming the role of NOX in MA/gp120-mediated cell toxicity. Among the NOX family of enzymes, NOX2 and NOX4 isozymes are predominantly responsible for NOX-derived ROS in astrocytes.33, 34 Therefore, we knocked down NOX2 and NOX4 using siRNA, which also abrogated the ROS produced by MA and gp120, either alone or in combination (MA±gp120) (Figures 5c and d). Furthermore, control siRNA-transfected cells did not show any significant change in the ROS production, when compared with no-siRNA control for the respective treatment groups (P>0.3 when measured using two-way ANOVA).
Superoxides (O2•−) generated via NOX are converted into H2O2, which is further converted into tertiary effector species such as hydroxyl radical (•OH) via the Fenton–Weiss–Haber (FWH) reaction.35, 36 We therefore hypothesized that FWH reaction is a downstream mechanism of NOX-mediated ROS production. As increased expression of ferritin is an indicator of oxidative stress,37 we measured the expression of ferritin heavy chain in the cells treated with MA and/or gp120. We observed higher ferritin expressions in astrocytes treated with MA and/or gp120 than untreated controls (Figure 6a), which suggested the involvement of Fe+2↔Fe+3 cycle. Therefore, we treated astrocytes with various concentrations of deferoxamine (DFO), an inhibitor of FWH reaction, 1 h before the treatment with either MA or gp120 alone and MA+gp120. Among various doses used, 50 nM DFO was found to reduce the ROS generated by gp120 alone as well as gp120+MA (Figure 6b). Furthermore, 50 nM DFO also rescued the astrocytes from cell death induced by oxidative stress (Figure 6c). The involvement of iron cycle can also lead to increased protein carbonylation, which can further lead to apoptotic cell death.38, 39 Therefore, we measured the levels of carbonylated protein in the astrocytes treated with MA and/or gp120. As expected, we observed increased levels of protein carbonylation (Figure 6d) upon treatments. Overall, these data suggested that NOX2 and NOX4 produced superoxides, which further underwent FWH chemistry to produce peroxides, leading to increased protein carbonylation. Taken together, these resulted in increased oxidative stress and consequently cell death.

Caspase-3 is involved in MA- and/or gp120-mediated apoptosis in astrocytes
Involvement of caspase-3 is a classical mechanism of apoptosis in a variety of cells. We therefore examined the effect of MA and/or gp120 on caspase-3 protein expression and activity. Our results show increased protein expressions of caspase-3 when astrocytes were treated with MA and/or gp120. The immunoblots showed significantly increased expression of cleaved caspase-3 (17 kDa) in the cells treated with MA, gp120, and MA+gp120. However, procaspase-3 isoform (35 kDa) was slightly reduced in the cells treated with gp120+MA (Figure 7a). Furthermore, astrocytes treated with either MA or gp120 alone showed increased caspase-3 cleavage activity as compared with untreated cells. In addition, cells treated with gp120 and MA showed significantly higher (P≤0.01) caspase-3 cleavage activity as compared with either of the agents alone and the effect was found to be additive (Figure 7b). Furthermore, the inhibitors for ROS (Figure 7c), CYP2E1 (Figure 7d), NOX (Figure 7e), and FWH reaction (Figure 7f) significantly reduced MA/gp120-mediated 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.