PHOTODYNAMICALLY GENERATED BETA-HYDROXY-5-ALPHA-CHOLEST-6-ENE-5-HYDROPEROXIDE - TOXIC REACTIVITY IN MEMBRANES AND SUSCEPTIBILITY TO ENZYMATIC DETOXIFICATION

Singlet oxygen (O-1(2))-mediated photooxidation of cholesterol gives t hree hydroperoxide products: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hy droperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydrope roxide (6 alpha-OOH) and 3 beta-hydroxycholest-4-ene-6 beta-hydroperox ide (6 beta-OOH). These species have been compared with respect to pho togeneration rate on the one hand and susceptibility to enzymatic redu ction/detoxification on the other, using the erythrocyte ghost as a ch olesterol-containing test membrane and chloroaluminum phthalocyanine t etrasulfonate (AlPcS(4)) as a O-1(2) sensitizer. Peroxide analysis was accomplished by high-performance liquid chromatography with mercury c athode electrochemical detection (HPLC-EC[Hg]). The initial rate of 5 alpha-OOH accumulation in AlPcS(4)/light-treated ghosts was found to b e about three times greater than that of 6 alpha-OOH or 6 beta-OOH. Me mbranes irradiated in the presence of ascorbate and ferric-8-hydroxyqu inoline (Fe[HQ](2), a lipophilic iron complex) accumulated lesser amou nts of 5 alpha-OOH, 6 alpha-OOH and 6 beta-OOH but relatively large am ounts of another peroxide pair, 3 beta-hydroxycholest-5-ene-7 alpha- a nd 7 beta-hydroperoxide (7 alpha,7 beta-OOH), suggestive of iron-media ted free radical peroxidation. When photoperoxidized membranes contain ing 5 alpha-OOH, 6 alpha,6 beta-OOH and 7 alpha,7 beta-OOH (arising fr om 5 alpha-OOH rearrangement) were incubated with glutathione (GSH) an d phospholipid hydroperoxide glutathione peroxidase (PHGPX), all hydro peroxide species underwent HPLC-EC(Hg)-detectable reduction to alcohol s, the relative first order rate constants being as follows: 1.0 (5 al pha-OOH), 2.0 (7 alpha,7 beta-OOH), 2.4 (6 alpha-OOH) and 3.2 (6 beta- OOH). Relatively rapid photogeneration and slow detoxification might m ake 5 alpha-OOH more cytotoxic than the other peroxide species. To beg in investigating this possibility, we inserted 5 alpha-OOH into ghosts by transferring it from 5 alpha-OOH-containing liposomes. When expose d to Fe(HQ)2/ascorbate, these ghosts underwent GSH/PHGPX-inhibitable c hain peroxidation, as indicated by the appearance of 7 alpha,7 beta-OO H, phospholipid hydroperoxides and thiobarbituric acid reactive substa nces. Liposomal 5 alpha-OOH also exhibited a strong, Fe(HQ)(2)-enhance d, toxicity toward L1210 leukemia cells, an effect presumably mediated by damaging chain peroxidation. This appears to be the first reported example of eukaryotic cytotoxicity attributed specifically to 5 alpha -OOH.