Role of glutathione on the Inhibition of DC-induced cytotoxicity
Objectives: We have previously reported that direct current (DC) induces necrotic cell death against polymorphonuclear leukocytes (PMNs) and the cytotoxic effects depend on the DC-generated metal ion and free radicals (J. Endod. 2005). The purpose of this study is to investigate how antioxidants inhibit the DC-induced cell death of PMNs.
Method: Peripheral blood PMNs were isolated by Mono-Poly density gradient centrifugation of heparinized peripheral blood. The PMNs were suspended at 2 × 106/ml in RPMI 1640 medium supplemented with 10% FBS, in the 24-well culture plate, and subjected to DC (2 mA) for 5 minutes in the presence of various antioxidants such as glutathione (GSH), N-acetyl-L-cysteine (NAC), coenzyme-Q10, vitamin C or catalase. The cytotoxic activity of DC with or without antioxidants against PMNs was evaluated by cell counting after trypan blue staining, and changes in ultrastructual morphology by transmission electron microscopy (TEM).
Results: The DC-induced cytotoxic effect was partially inhibited by GSH or NAC (p<0.05) in a dose-dependent manner, but not by vitamin C or catalase. The coenzyme-Q10 showed a little effect. The cytotoxic effect was significantly promoted by L-buthionine-(S.R)-sulfoximide (BSO) (p<0.05), inhibitor of GSH synthesis. TEM revealed that the DC induced intense vacuolization and accumulation of cellular debris, whereas normal morphology, containing a segmental nucleus, numerous azurophil granules, and specific granules in the cytoplasm, was not affected by GSH treatment.
Conclusions: These results suggest that cytotoxicity of DC was inhibited by SH-compound such as GSH and NAC. The level of intracellular GSH may be crucial to regulate the DC-induced cell death of PMNs.