ENZYMATIC REDUCIBILITY IN RELATION TO CYTOTOXICITY FOR VARIOUS CHOLESTEROL HYDROPEROXIDES

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is a selenoe nzyme that can catalyze the direct reduction of various membrane lipid hydroperoxides and by so doing could play a vital role in cytoprotect ion against peroxidative damage. The activity of purified testicular P HGPX on several photochemically-generated cholesterol hydroperoxide (C hOOH) species was investigated, using high-performance liquid chromato graphy with electrochemical detection for peroxide analysis and thin-l ayer chromatography with C-14-radiodetection for diol product analysis . The following ChOOH isomers were monitored: 5 alpha-OOH, 6 alpha-OOH , 6 beta-OOH (singlet oxygen adducts), and unresolved 7 alpha,7 beta-O OH (derived from 5 alpha-OOH rearrangement). Apparent first-order rate constants for GSH/PHGPX-induced peroxide loss (or diol accumulation) in Triton X-100 micelles, unilamellar liposomes, or erythrocyte ghost membranes increased in the following order: 5 alpha-OOH < 6 alpha-OOH approximate to 7 alpha,7 beta-OOH < 6 beta-OOH. A similar trend was ob served when the peroxides were incubated with Triton lysates of Se-rep lete L1210 or K562 cells, implicating PHGPX in these reactions. Consis tent with this, there was little or no ChOOH reduction if GSH was omit ted or if lysates from Se-deprived cells were used. Liposomal 5 alpha- OOH was found to be much more cytotoxic than equimolar Liposomal 6 bet a-OOH, producing a 50% loss of L1210 clonogenicity at similar to 1/5 t he concentration of the latter. Faster uptake of 5 alpha-OOH was ruled out as the basis for greater cytotoxicity, suggesting that relatively inefficient metabolism by the GSH/PHGPX system might be the reason. A s supporting evidence, it was found that cells accumulate the diol red uction product of 5 alpha-OOH more slowly than that of 6 beta-OOH duri ng incubation with tile respective peroxides. Slow detoxification coup led with rapid formation makes 5 alpha-OOH potentially the most damagi ng ChOOH to arise in cells exposed to singlet oxygen.