title: Lipid peroxidation in photodynamically stressed mammalian cells: use of cholesterol hydroperoxides as mechanistic reporters.
abstract: Photodynamic action of merocyanine 540, an antileukemic sensitizing dye, on murine L1210 cells results in the formation of lipid hydroperoxides and loss of cell viability. High-performance liquid chromatography with mercury cathode electrochemical detection was used for determining lipid oxidation products, including the following cholesterol-derived hydroperoxides: 5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, and unresolved 7 alpha, 7 beta-OOH. Among these species, 5 alpha-, 6 alpha-, and 6 beta-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7 alpha- and 7 beta-OOH (products of free radical reactions) became so after prolonged irradiation or during dark incubation after exposure to a light dose. These mechanistic changes were studied in a unique way by monitoring shifts in the peroxide ratio, i.e., 7-OOH/5 alpha-OOH, or 7-OOH/6-OOH. When cells (10(7)/ml) were exposed to a visible light fluence of 0.6 J/cm2 in the presence of 10 microM merocyanine 540, 7-OOH/5 alpha-OOH increased by approximately 100% after 2 h of dark incubation at 37 degrees C. The increase was much larger (approximately 250%) when cells were photooxidized after treatment with 1 microM ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 microM desferrioxamine, an avid iron chelator/redox inhibitor. Correspondingly, postirradiation formation of thiobarbituric acid-reactive material was markedly enhanced by ferric-8-hydroxyquinoline and suppressed by desferrioxamine, as was the extent of cell killing. When added to cells after a light dose, chain-breaking antioxidants such as butylated hydroxytoluene and alpha-tocopherol strongly protected against cell killing and slowed the increase in 7-OOH/5 alpha-OOH ratio. It is apparent from these results that (1) the 7-OOH/5 alpha-OOH or 7-OOH/6-OOH ratio can be used as a highly sensitive index of singlet oxygen vs. free radical dominance in photodynamically stressed cells; and (2) that postirradiation chain peroxidation plays an important role in photodynamically initiated cell killing.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: P G
authors.initials: PG
authors.lastname: Geiger
authors.forename: W
authors.initials: W
authors.lastname: Korytowski
authors.forename: F
authors.initials: F
authors.lastname: Lin
authors.forename: A W
authors.initials: AW
authors.lastname: Girotti
mesh_terms: D000818:Animals; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002470:Cell Survival; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003676:Deferoxamine / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D058085:Iron Compounds / Q000494:pharmacology; D007939:Leukemia L1210; D008027:Light; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000378:metabolism; D051379:Mice; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011744:Pyrimidinones / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D014407:Tumor Cells, Cultured
chemical_list: D005609:Free Radicals; D058085:Iron Compounds; D008054:Lipid Peroxides; D017319:Photosensitizing Agents; D011744:Pyrimidinones; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D002084:Butylated Hydroxytoluene; C003954:merocyanine dye; C002843:cholesterol hydroperoxide; D002784:Cholesterol; D003676:Deferoxamine