br Materials and methods br Results br Discussion Reduced gl

Materials and methods
Results
Discussion Reduced glutathione is considered to be one of the important regulators of REDOX balance in the biological system. The cellular concentration of 4-HNE under physiological conditions ranges from 0.1 to 3μM and has no detrimental effect in the biological system except occasional apoptotic events (Srivastava et al., 2000). Interestingly, no significant effect on GSH levels has been found at physiological concentration of 4-HNE (Neely et al., 2005). In the present investigation, a dose and time dependent depletion in cellular GSH levels was observed until 4h of exposure of PC-12 Fucoidan mg to 4-HNE. Exposure for 8h however did not cause any further decrease in the levels of GSH. It has earlier been demonstrated that 4-HNE, a highly reactive compound is metabolized within 1h time (Amunom et al., 2007). However, HNE before metabolism binds to GSH and causes detrimental responses in a variety of cells (Neely et al., 2005, Amunom et al., 2007). Depletion in GSH levels following exposure of PC-12 cells to 4-HNE for 1h, observed in the present study, may be correlated with the binding of 4-HNE with GSH. The decrease in GSH levels was more marked with increase in time of exposure for 2 and 4h and might be due to triggering of cascade of events involved in oxidative stress mediated cellular injuries (Falletti and Douki, 2008). It may be possible that 4-HNE concentrations were decreased or converted into less toxic or non-toxic metabolites during 8h of incubation (Amunom et al., 2007, Yadav et al., 2008). Increase in the (Ca2+) levels in PC12 cells following exposure to 4-HNE for 1h was concentration dependent. However, changes in the (Ca2+) levels in PC12 cells following exposure to 4-HNE were not time dependent as observed in the present study. The increase in cytosolic Ca2+ may be due to net entry of Ca2+ across the plasma lemma or transient liberation of Ca2+ from intracellular stores. The early increase in total cellular Ca2+ via NMDA receptors and other pathways triggered due to the profound energy failure has also been reported in various neurodegenerative disorders (Soriano et al., 2008). 4-HNE induced neurodegeneration by elevating cytosolic Ca2+ levels are largely suggested due to activation of 2Na+/Ca2+ transporter-mediated efflux from mitochondria in experimental models of neuronal disorder (Grewer et al., 2008). Mitochondrial calcium accumulation and oxidative stress have shown to trigger the opening of a high-conductance pore in the inner mitochondrial membrane and leads to collapse the electrochemical potential for H+, thereby arresting ATP production and triggering production of reactive oxygen species (He et al., 2009, Ardón et al., 2009). Thus, our results suggest that PC12 cells could be vulnerable to increased intracellular calcium following exposure to 4-HNE. A concentration dependent increase in the 4-HNE-protein binding in PC-12 cells observed in the present study was not time dependent. The 4-HNE-protein binding might be due to depleted levels of cellular GSH and GSTP1-1 associated with oxidative stress. Association of GSH and glutathione S-transferases in detoxification of 4-HNE induced damages both in mitochondria and extra-mitochondrial compartments has been reported (Awasthi et al., 2005). 4-HNE has been shown to have strong binding with proteins and thus forms adducts. Besides the cells of neuronal origin, 4-HNE–protein binding has also been studied in cultured non-neuronal cells (Benedetti et al., 1982, Toyokuni et al., 1994). Developing fetuses have been shown to have different magnitude of binding of 4-HNE with different proteins. Role of 4-HNE in toxicity by modulating signaling pathways has been suggested (Kapphahn et al., 2006). This diversity in HNE–protein binding in different cells has been suggested due to differences in the composition of membrane phospholipids, as 4-HNE is a product of membrane lipid peroxidation (Yan and Hales, 2006). In the present study, we found that 4-HNE–protein adducts formation was predominant in cytoplasm and increased in concentration dependent manner. This increased formation of HNE–protein conjugates with increasing concentration of 4-HNE exposure was well correlated with other endpoints suggesting involvement of oxidative stress mediated damage in PC12 cells. Lower levels of cellular GSH have also been linked with increased HNE–DNA adducts formation (Yadav et al., 2008). It could possibly be an important reason for such changes as observed in the present study.