Fig. 3.

Role of oxidative stress in vanadium-induced neurotoxicity in N27 dopaminergic neuronal cells. (A) Calibration curve of H₂O₂ production by polarography: H₂O₂ production was measured using an Apollo 4000 Free Radical Analyzer (WPI, Sarasota, FL) equipped with a 100-μm H₂O₂ sensor. The 100-μm H₂O₂ sensor probe was calibrated using various doses of H₂O₂ according to the manufacturer's instructions. The calibration curve was used to calculate the H₂O₂ in the control and treatment groups. (B) Vanadium induced H₂O₂ generation in N27 cells. The measurements were conducted in a 96 well plate containing N27 cells exposed to 40 μM vanadium for 4 h. Each measurement was started by insertion of the 100-μm H₂O₂ sensor probe into wells containing cells. The output signal was recorded and compared to the standard curve. (C) Effect of antioxidants on vanadium-induced neurotoxicity. N27 dopaminergic neuronal cells were co-treated with vanadium and an antioxidant solution cocktail (AO) of vitamin E, glutathione, superoxide dismutase, and catalase. The Sytox green was added to cells and then cells were observed under a Nikon inverted fluorescence microscope. The pictures were captured with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). (D) Quantitative analysis of the protective effect of AO on vanadium-induced neurotoxicity was measured by the Sytox green cytotoxicity fluorescence assay. Data represent results from four individual measurements and are expressed as mean ± S.E.M. **p<0.001 and ***p<0.01.