Direct evidence for antioxidant effect of Bcl-2 in PC12 rat pheochromocytoma cells

Mock-transfected PC12 rat pheochomocytoma cells and PC12 cells transfected with the bcl-2 gene, a gene associated with inhibition of apoptosis, were subjected to oxidative stress by incubation in the presence of the azo-initiator of lipid peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Extraction and chromatographic analysis by two-dimensional TLC of the major phospholipid classes showed no differences in the phospholipid composition between the mock- and bcl-2-transfected cell lines after incubation in the presence of 0.5 mM AMVN for 2 h at 37 degrees C. A method consisting of incorporation of cis-parinaric acid into the constituent membrane phospholipids before exposure to AMVN was developed to improve the sensitivity of detecting lipid peroxidation in PC12 cells. Analysis of the pattern of changes in parinaric acid-labeled phospholipids after exposure to 0.25 and 0.5 mM AMVN by HPLC showed significant oxidation of phosphatidylcholine (PC), phosphatidylethanolamine (PEA), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SPH) during a 2-h incubation. The extent of oxidation of each phospholipid class was dependent on the concentration of AMVN present up to 1 mM. Based on phospholipid fractional composition, the specific rates of PnA peroxidation in phospholipid classes were estimated. In mock-transfected PC12 cells, the order of AMVN-induced oxidation effectiveness was the same for both specific rates and relative rates: PC >> PEA > PS > SPH > PI. While a dramatic decrease in both relative and specific oxidation rates was observed for all phospholipid classes in bcl-2-transfected PC12 cells, the specific oxidation rates were higher for aminophospholipids (PEA and PS) than for other phospholipids. This suggests that antioxidant protection by bcl-2-related product(s) may be phospholipid-specific and that aminophospholipids are relatively less protected than the other phospholipids. The vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydrochromane, acted as an effective antioxidant in preventing oxidation of parinaric acid-labeled membrane phospholipids during incubation in the presence of AMVN and the extent of protection was approximately the same in both cell lines. Since, unlike the agents used to generate oxidative stress in other studies, temperature-driven generation of peroxyl radicals by AMVN is not dependent on intracellular metabolism, the results presented provide proof for antioxidant protection, rather than abrogation of radical generation afforded by bcl-2 transfection of PC12 cells.