title: Enzymatic reducibility in relation to cytotoxicity for various cholesterol hydroperoxides.
abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is a selenoenzyme that can catalyze the direct reduction of various membrane lipid hydroperoxides and by so doing could play a vital role in cytoprotection against peroxidative damage. The activity of purified testicular PHGPX on several photochemically-generated cholesterol hydroperoxide (ChOOH) species was investigated, using high-performance liquid chromatography with electrochemical detection for peroxide analysis and thinlayer chromatography with 14C-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-OOH (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 approximately equal to 7 alpha,7 beta-OOH < 6beta-OOH. A similar trend was observed when the peroxides were incubated with Triton Iysates of Se-replete L1210 or K562 cells, implicating PHGPX in these reactions. Consistent with this, there was little or no ChOOH reduction if GSH was omitted or if lysates from Se-deprived cells were used. Liposomal 5 alpha-OOH was found to be much more cytotoxic than equimolar liposomal 6 beta-OOH, producing a 50% loss of L1210 clonogenicity at approximately 1/5 the 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. As supporting evidence, it was found that cells accumulate the diol reduction product of 5 alpha-OOH more slowly than that of 6 beta-OOH during incubation with the respective peroxides. Slow detoxification coupled with rapid formation makes 5 alpha-OOH potentially the most damaging ChOOH to arise in cells exposed to singlet oxygen.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, 53226, USA.
authors.forename: W
authors.initials: W
authors.lastname: Korytowski
authors.forename: P G
authors.initials: PG
authors.lastname: Geiger
authors.forename: A W
authors.initials: AW
authors.lastname: Girotti
mesh_terms: D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D010100:Oxygen; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D026082:Singlet Oxygen
chemical_list: D026082:Singlet Oxygen; C002843:cholesterol hydroperoxide; D002784:Cholesterol; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D010100:Oxygen