Antioxidants prevent the cytotoxicity of manganese in RBE4 cells

Manganese (Mn) is an essential trace element required for ubiquitous enzymatic reactions. Chronic overexposure to this metal may, however, promote potent neurotoxic effects. The mechanism of Mn toxicity is not well established, but several studies indicate that oxidative stress and mitochondria play major roles in the Mn-induced neurodegenerative processes that lead to dysfunction in the basal ganglia. The aim of this study was to address the toxic effects of MnCl₂ and MnSO₄ on the immortalized rat brain microvessel endothelial cell line (RBE4) and to characterize toxic mechanism associated with exposure to Mn. The cytotoxicity of Mn in RBE4 cells was evaluated using the LDH and the MTT assays. A significant increase was noted in LDH release from RBE4 cells exposed for 24 h to MnCl₂ at concentrations of 800 μM and MnSO₄ at concentrations ≥ 400 μM (p < 0.05) when compared with control unexposed cells. The MTT assay established significant decrease in cellular viability upon exposure to MnCl₂ at concentrations ≥ 100 μM and to MnSO₄ at concentrations ≥ 50 μM (p < 0.05). Thus, the cytotoxicity assays showed that the MTT assay was more sensitive than the LDH assay, suggesting that mitochondrial changes precede other toxic effects of Mn. In addition, upon exposure to MnCl₂ (200 and 800 μM), intracellular reduced glutathione (GSH) levels in RBE4 cells decreased as Mn exposure concentrations increased (p < 0.05). To confirm the oxidative hypothesis of Mn cytotoxicity, co-exposure of MnCl₂ with antioxidant agents (N-acetylcysteine [NAC] or Trolox) were carried out. The cellular viability was evaluated using the MTT assay. A significant decrease in Mn cytotoxicity was observed in co-exposed cells confirming that (1) oxidative stress plays a critical role in the mechanism of Mn toxicity, and (2) antioxidants may offer a useful therapeutic modality to reverse the aberrant effects of Mn.