Effects of chronic cocaine in rat C6 astroglial cells

Abstract

Investigations with astroglial cells carry equal importance as those with neurons in drug abuse studies. The present study was aimed to investigate the effect of chronic cocaine administration on cell viability, nitric oxide (NO) production, general respiratory status of mitochondria and total protein levels in rat astroglioma cells after 24 h of treatment. In addition, the effect of cocaine was assessed for 24 h on brine shrimp larvae in order to study their sensitivity to the drug. It was observed that cocaine caused a significant dose-dependent decrease in astroglial cell viability with an LC(50) of 4.717 mM. It was found that cocaine did not induce or inhibit NO production in the cells. Evaluation of mitochondrial dehydrogenase activity in terms of formazan production in astroglial cells indicated that cocaine significantly interfered with the general respiratory status of mitochondria with an ED(50) of 6.153 mM. Furthermore, cocaine was shown to deplete the total protein levels in the cells with an ED(50) of 5.435 mM. In vivo study with brine shrimp larvae showed that these larvae were highly sensitive to cocaine with an ED(50) of 2.41 mM. In summary, our findings suggest that cocaine-induced cytotoxicity in the cells was non-specific. The cumulative effect arising from the significant loss of respiration and total cellular proteins is the cause of astroglial cell death.

Figure 1.

Effect of cocaine on astroglial cell viability. The cells were seeded in 96-well plates with complete RPMI-1640 medium containing 10% FBS and were treated with various concentrations of cocaine for 24 h. Data were represented as mean ± SEM (n=12, ^*P<0.01, highly significant in comparison to control, one-way ANOVA, Dunnett’s multiple comparison test).

Figure 2.

(A) Nitric oxide production in cocaine-treated astroglial cells. The cells were seeded in 96-well plates with complete RPMI-1640 media lacking phenol red, containing 10% FBS and treated with 2 or 3 mM cocaine for 24 h. Nitric oxide was detected with Griess reagent. Data are presented as mean ± SEM (n=15, P>0.05, insignificant in comparison to control, one-way ANOVA, Dunnett’s multiple comparison test). (B) Standard curve of sodium nitrite (25 to 400 μM).

Figure 3.

Effect of cocaine on mitochondrial respiratory status in astroglial cells. Cells were seeded in 96-well plates and treated with various concentrations of cocaine for 24 h. Data are presented as means ± SEM (n=12, ^*P<0.01, highly significant in comparison to control, one-way ANOVA, Dunnett’s multiple comparison test).

Figure 4.

Effect of cocaine on total cellular protein in astroglial cells. Cells were seeded in 96-well plates and treated with various concentrations of cocaine for 24 h. Data are presented as mean ± SEM (n=6, ^*P<0.01, highly significant in comparison to control, one-way ANOVA, Dunnett’s multiple comparison test).

Figure 5.

Effect of cocaine on brine shrimp larvae. The assay was carried out at 0, 2, 4 and 6 mM cocaine. After 24 h, the live shrimp were counted in each vial. Data are presented as mean ± SEM (n=3, ^*P<0.01, highly significant in comparison to control, one-way ANOVA, Dunnett’s multiple comparison test).