Attenuating effect of N-acetyl-L-cysteine against acute cocaine toxicity in rat C6 astroglial cells

Abstract

Astroglial cells are one of the most abundant cell types in the mammalian brain functioning in neuronal survival and in maintenance of fundamental patterns of circuitry. To date, no study has been conducted regarding the short-term impact of cocaine on these cells in cultures. The present study aimed to investigate acute cocaine (1 h) treatment on cell viability in rat C6 astroglial cells. In addition, the potential effect of N-acetyl-L-cysteine (NAC) against cocaine-induced toxicity was studied. It was observed that 1 h of acute cocaine exposure at 2, 3 and 4 mM caused a dose-dependent decrease in cell viability with an LC50 of 2.857 mM. Furthermore, cocaine treatment caused a decrease in glutathione (GSH) levels in the cells. It was found that cocaine did not exhibit pro-oxidant activity during its exposure to cells. Acute cocaine exposure did not induce nitric oxide (NO) release in the cells. A 5-point (1-5 mM) dose-response curve of NAC clearly indicated no adverse effect on astroglial cell viability. Pretreatment of cells with 5 mM NAC for 30 min, followed by its discard, and exposure to cocaine (2-4 mM) for 1 h protected cells against cytotoxicity by 90%. Treatment of cells with NAC-cocaine mixture rendered 100% protection. Further investigations revealed that the protection by NAC was through the increased GSH levels in the cells. Our results indicate that decreased GSH levels may represent one of the underlying pathologies of cell death and that antioxidant compounds which increase the GSH production could protect against cocaine-induced toxicity by promoting a pro-survival role in astroglial cells.

Figure 1

Effect of NAC on rat C6 astroglial cell viability. The cells with an initial density of 2×10⁴ were treated with various concentrations of NAC for 1 h. Data are represented as means ± SEM (n=6, ^*P>0.05, insignificant from control).

Figure 2

Morphological features of the astroglial cells. (A) Cells were treated with PBS or (B) 5 mM NAC for 1 h or (C) 2 mM cocaine for 1 h or (D) pretreated with 5 mM NAC for 30 min, followed by its discard and treatment with 4 mM cocaine for 1 h. Crystal violet-stained cells were photographed under an inverted phase contrast 1X-70 Olympus microscope with ×40 objective.

Figure 3

Scavenging activity. Various concentrations of NAC were added to 1-ml Eppendorf tubes without cells with ethanol as a solvent, and incubated in 0.1 mM 2,2-diphenyl-1-picrylhydrazyl-free radical for 30 min. Data are represented as means ± SEM (n=9, ^*P<0.01, highly significantly different from the control).

Figure 4

Determination of pro-oxidation activity of cocaine. Various concentrations of cocaine (2–4 mM) were incubated in 0.1 mM H₂DCFDA dye for 1 h at 37˚C. Data are represented as means ± SEM (n=6, ^*P>0.05, insignificant from the control)

Figure 5

(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, 3 and 4 mM cocaine for 1 h. Nitric oxide (NO) was detected with Griess reagent. Data are presented as means ± SEM (n=12, ^*P>0.05, insignificant in comparison to the control). (B) Standard curve of sodium nitrite (25–400 μM).

Figure 6

Attenuating action of NAC against cocaine-induced toxicity. Cells were pretreated with 5 mM NAC for 30 min, followed by its discard, and then treated with (A) cocaine for 1 h or (B) NAC-cocaine mixture for 1 h. Data are represented as means ± SEM (n=16), ^*P<0.01, significant in comparison the control; ^#P<0.01, significant difference between non-pretreated and pretreated cocaine-exposed cells.

Figure 7

Increased glutathione (GSH) levels of NAC pretreatment. The cells were pretreated with 5 mM NAC for 30 min, followed by its discard, and then treated with cocaine for 1 h. Data are represented as means ± SEM (n=8), ^#P<0.01, significant difference between non-pretreated and pretreated cocaine-exposed cells.