Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells

Abstract

Males have a higher risk for developing Parkinson's disease and parkinsonism after ischemic stroke than females. Although estrogens have been shown to play a neuroprotective role in Parkinson's disease, there is little information on androgens' actions on dopamine neurons. In this study, we examined the effects of androgens under conditions of oxidative stress to determine whether androgens play a neuroprotective or neurotoxic role in dopamine neuronal function. Mitochondrial function, cell viability, intracellular calcium levels, and mitochondrial calcium influx were examined in response to androgens under both nonoxidative and oxidative stress conditions. Briefly, N27 dopaminergic cells were exposed to the oxidative stressor, hydrogen peroxide, and physiologically relevant levels of testosterone or dihydrotestosterone, applied either before or after oxidative stress exposure. Androgens, alone, increased mitochondrial function via a calcium-dependent mechanism. Androgen pretreatment protected cells from oxidative stress-induced cell death. However, treatment with androgens after the oxidative insult increased cell death, and these effects were, in part, mediated by calcium influx into the mitochondria. Interestingly, the negative effects of androgens were not blocked by either androgen or estrogen receptor antagonists. Instead, a putative membrane-associated androgen receptor was implicated. Overall, our results indicate that androgens are neuroprotective when oxidative stress levels are minimal, but when oxidative stress levels are elevated, androgens exacerbate oxidative stress damage.

Figure 1.

Androgens alter mitochondrial function. Mitochondrial membrane potentials were assayed with the fluorescent dye TMRM. Increased fluorescence is indicative of increased mitochondria membrane potentials. N27 cells were exposed to either T (10 and 100 nM) or DHT (10 nM) for 2 hours (panels A and B). After 2 hours of exposure, T and DHT significantly increased TMRM fluorescence in N27 cells compared with vehicle control. T significantly increased TMRM fluorescence compared with DHT. T exposure for 24 hours did not influence cell viability (panel C). T10, 10 nM T; T100, 100 nM T; D10, 10 nM DHT; C, vehicle control. ANOVA was followed by an LSD post hoc test. Scale bar, 10 μM. * and #, Significance compared with C and D10, respectively. Horizontal line demarcates controls normalized to 100%.

Figure 2.

Timing of hydrogen peroxide and T. Cell viability is presented as a percentage of control. Hydrogen peroxide significantly decreased cell viability. Two hours of T pretreatment significantly blocked hydrogen peroxide-induced cell death (A). Hydrogen peroxide significantly decreased cell viability. Twenty-four hours of T posttreatment significantly increased hydrogen peroxide-induced cell death in a dose-dependent manner (B). H, hydrogen peroxide; T, 100 nM T; T+H, 100 nM T pretreatment; HT, T 100 nM posttreatment; HT1, T 1 nM posttreatment; HT10, T 10 nM posttreatment; HT100, T 100 nM posttreatment; C, vehicle control. An ANOVA was followed by an LSD post hoc test. *, Significant compared with C; #, significant compared with H; **, significant compared with HT1; ***, significant compared with HT10. Horizontal line demarcates controls normalized to 100%.

Figure 3.

Oxidative stress and androgen interaction. Hydrogen peroxide significantly increased cell death. T does not alter hydrogen peroxide-induced cell death when coapplied with hydrogen peroxide (A). The antioxidant, N-acetyl-cysteine (1 mM), blocked T posttreatment and hydrogen peroxide-induced cell death (B). C, vehicle control; H, hydrogen peroxide; HT, T 100 nM posttreatment. An ANOVA was followed by an LSD post hoc test. *, Significant compared with C. Horizontal line demarcates controls normalized to 100%.

Figure 4.

The deleterious effects of T are not mediated by androgen or estrogen receptors. Neither the androgen receptor nor estrogen-α/β receptors mediate T posttreatment negative effects on cell viability, as evidenced by the lack of effect in response to the androgen receptor antagonist, flutamide (500 nM), and the estrogen receptor-α/β antagonist, ICI 182,780 (1 uM). H, hydrogen peroxide; T, 100 nM T; HT, T posttreatment; C, vehicle control. An ANOVA was followed by an LSD post hoc test. *, Significant compared with C; #, significant compared with H. Horizontal line demarcates controls normalized to 100%.

Figure 5.

T and intracellular calcium. Intracellular calcium ([Ca⁺⁺]_i) levels were measured to determine whether androgens induce calcium release from intracellular stores. FURA-2 AM-loaded N27 cells were bathed in a calcium-free HBSS buffer and basal [Ca⁺⁺]_i were determined until stable. One minute after stabilization, cells were treated with vehicle control or T. In the calcium-free buffer, baseline calcium levels were 4.5 nM and T significantly increased [Ca⁺⁺]_i to 250 nM. The addition of 2 uM ionomycin to the media was used to stimulate maximum intracellular calcium release. A paired t test was used for analysis (A). Mitochondrial function in response to Ru360 (1 uM), a mitochondrial calcium uniporter inhibitor, was used to determine whether the androgen-induced effects on mitochondrial function were mediated through calcium influx into the mitochondria. N27 cells were exposed to either T or Ru360 + T for 2 hours. Ru360 significantly blocked the androgen-induced increase in membrane potentials (B). To determine the involvement of mitochondrial calcium influx in the T posttreatment of hydrogen peroxide induced cell death, Ru360 was applied 30 minutes prior to T exposure. Ru360 significantly blocked T-induced cell death in an oxidative stress environment (C). H, hydrogen peroxide; T, 100 nM T; HT, T posttreatment; R, Ru360; RT, Ru360 + T; C, vehicle control. An ANOVA was followed by an LSD post hoc test. *, Significant compared with C; #, significant compared with C and H; **, significant compared with HT. Scale bar, 10 μM. Horizontal line demarcates controls normalized to 100%.

Figure 6.

Membrane-associated androgen receptor involvement. To examine the role of mARs, TBSA was used to bind only putative membrane androgen receptors. TBSA alone does not affect cell viability. Hydrogen peroxide significantly decreased cell viability. Twenty-four hours TBSA posttreatment significantly increased hydrogen peroxide-induced cell death (A). TBSA pretreatment for 2 hours blocked cell death induced by a 6-hour exposure to hydrogen peroxide (B). H, hydrogen peroxide; TBSA, 500 nM T conjugated to BSA; H+TBSA, TBSA posttreatment; TBSA+H, TBSA pretreatment; C, vehicle control. An ANOVA was followed by an LSD post hoc test. *, Significant compared with C; #, significant compared with H. Horizontal line demarcates controls normalized to 100%.