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 microM and MnSO4 at concentrations > or = 400 microM (p < 0.05) when compared with control unexposed cells. The MTT assay established significant decrease in cellular viability upon exposure to MnCl2 at concentrations > or = 100 microM and to MnSO4 at concentrations > or = 50 microM (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 microM), 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.
Marreilha dos Santos
A P
AP
iMed. UL, Faculty of Pharmacy, University of Lisbon, Portugal. apsantos@ff.ul.pt
Santos
Dinamene
D
Au
Catherine
C
Milatovic
Dejan
D
Aschner
Michael
M
Batoréu
M Camila C
MC
D000111:Acetylcysteine / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002712:Chlorides / Q000633:toxicity*; D002839:Chromans / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D017895:Manganese Compounds; D051381:Rats; D013431:Sulfates / Q000633:toxicity*; D013778:Tetrazolium Salts; D013844:Thiazoles
D000975:Antioxidants; D002712:Chlorides; D002839:Chromans; D017895:Manganese Compounds; D013431:Sulfates; D013778:Tetrazolium Salts; D013844:Thiazoles; D007770:L-Lactate Dehydrogenase; C022616:thiazolyl blue; D005978:Glutathione; C025340:manganese chloride; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; C039798:manganese sulfate; D000111:Acetylcysteine