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PHOTODYNAMICALLY GENERATED 3‐β‐HYDROXY‐5α‐CHOLEST‐6‐ENE‐5‐HYDROPEROXIDE: TOXIC REACTIVITY IN MEMBRANES and SUSCEPTIBILITY TO ENZYMATIC DETOXIFICATION
Author(s) -
Geiger Peter G.,
Korytowski Witold,
Girotti Albert W.
Publication year - 1995
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1995.tb02388.x
Subject(s) - detoxification (alternative medicine) , enzyme , chemistry , reactivity (psychology) , ene reaction , membrane , biochemistry , organic chemistry , medicine , alternative medicine , pathology
— Singlet oxygen ( 1 O 2 )‐mediated photooxidation of cholesterol gives three hydroperoxide products: 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide (5α‐OOH), 3β‐hydroxycholest‐4‐ene‐6α‐hydrope‐roxide (6α‐OOH) and 3β‐hydroxycholest‐4‐ene‐6β‐hydroperoxide (6β‐OOH). These species have been compared with respect to photogeneration rate on the one hand and susceptibility to enzymatic reduction/ detoxification on the other, using the erythrocyte ghost as a cholesterol‐containing test membrane and chloroaluminum phthalocyanine tetrasulfonate (AlPcS 4 ) as a 1 O 2 sensitizer. Peroxide analysis was accomplished by high‐performance liquid chromatography with mercury cathode electrochemical detection (HPLC‐EC[Hg]). The initial rate of 5α‐OOH accumulation in AlPcS 4 /light‐treated ghosts was found to be about three times greater than that of 6α‐OOH or 6β‐OOH. Membranes irradiated in the presence of ascorbate and ferric‐8‐hydroxyquinoline (Fe[HQ] 2 , a lipophilic iron complex) accumulated lesser amounts of 5α‐OOH, 6α‐OOH and 6β‐OOH but relatively large amounts of another peroxide pair, 3β‐hydroxycholest‐5‐ene‐7α‐ and 7β‐hydroperoxide (7α,7β‐OOH), suggestive of iron‐mediated free radical peroxidation. When photoperoxidized membranes containing 5α‐OOH, 6α,6β‐OOH and 7α,7β‐OOH (arising from 5α‐OOH rearrangement) were incubated with glutathione (GSH) and phospholipid hydroperoxide glutathione peroxidase (PHGPX), all hydroperoxide species underwent HPLC‐EC(Hg)‐detect‐able reduction to alcohols, the relative first order rate constants being as follows: 1.0 (5α‐OOH), 2.0 (7α,7β‐OOH), 2.4 (6α‐OOH) and 3.2 (6β‐OOH). Relatively rapid photogeneration and slow detoxification might make 5α‐OOH more cytotoxic than the other peroxide species. To begin investigating this possibility, we inserted 5α‐OOH into ghosts by transferring it from 5α‐OOH‐containing liposomes. When exposed to Fe(HQ) 2 /ascorbate, these ghosts underwent GSH/PHGPX‐inhibitable chain peroxidation, as indicated by the appearance of 7α,7β‐OOH, phospholipid hydroperoxides and thiobarbituric acid reactive substances. Liposomal 5α‐OOH also exhibited a strong, Fe(HQ) 2 ‐enhanced, toxicity toward LI210 leukemia cells, an effect presumably mediated by damaging chain peroxidation. This appears to be the first reported example of eukaryotic cytotoxicity attributed specifically to 5α‐OOH.