Oxidative stress mediated cellular responses in 4-Hydroxynonenal exposed PC12 cells

Investigations were carried out to elucidate the involvement of oxidative stress in 4-hydroxynonenal (HNE) mediated cytotoxicity in NGF induced differentiated PC12 cells. Well differentiated PC12 cells at day 8 were exposed to various concentrations (0.1–50mM) of HNE for 30 minutes-24h. Non-differentiated PC12 cells were also exposed under identical conditions and served as control. Following exposure, cytotoxicity assessment was done using standard endpoints viz., MTT, LDH release and neutral red uptake (NRU) assays. Further levels of glutathione and intracellular calcium, immunocytochemical localization of DA-D₂ receptor and HNE-protein binding were carried out using concentrations of HNE (1, 10, 25, 50mM), exerting the effect on cell growth in cytotoxicity studies. Significant cytotoxic responses were observed earliest by 2h of exposure, except for the concentration 50mM, where cytotoxicity was exerted even at 90min. HNE concentrations 10–50mM were found to be cytotoxic, 2–5mM cytostatic and 1–0.1mM non-cytotoxic. The depletion in GSH levels was statistically significant (p<0.001) since 1h of exposure and became more intensive at 2 and 4h exposures, however the exposure of 8h could not lead further depletion in GSH level. A correlation between depletion of GHS levels and increase in the intracellular Ca⁺⁺, binding of HNE with intracellular proteins and involvement of DA-D₂ receptor in HNE mediated cytotoxicity could be well established. Moreover, no significant differences in the responsiveness to HNE by NGF differentiated and non-differentiated PC12 cells could be observed. Results are suggestive the involvement of oxidative stress in HNE mediated cytotoxicity in PC12 cells.