Enzymatic Reducibility in Relation to Cytotoxicity for Various Cholesterol Hydroperoxides

• Published in: Biochemistry (Easton) Vol. 35; no. 26; pp. 8670 - 8679
• Main Authors: Korytowski, Witold, Geiger, Peter G, Girotti, Albert W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02-07-1996
• Subjects: Animals; Catalysis; Cell Line; Cell Survival - drug effects; Cholesterol - analogs & derivatives; Cholesterol - metabolism; Cholesterol - toxicity; Glutathione Peroxidase - metabolism; Humans; Kinetics; Mice; Oxidation-Reduction; Oxygen; Singlet Oxygen
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960522k

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 thin-layer chromatography with 14C-radiodetection for diol product analysis. The following ChOOH isomers were monitored:  5α-OOH, 6α-OOH, 6β-OOH (singlet oxygen adducts), and unresolved 7α,7β-OOH (derived from 5α-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:  5α-OOH < 6α-OOH ≈ 7α,7β-OOH < 6β-OOH. A similar trend was observed when the peroxides were incubated with Triton lysates 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 5α-OOH was found to be much more cytotoxic than equimolar liposomal 6β-OOH, producing a 50% loss of L1210 clonogenicity at ∼1/5 the concentration of the latter. Faster uptake of 5α-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 5α-OOH more slowly than that of 6β-OOH during incubation with the respective peroxides. Slow detoxification coupled with rapid formation makes 5α-OOH potentially the most damaging ChOOH to arise in cells exposed to singlet oxygen.