Lipid Peroxidation in Photodynamically Stressed Mammalian Cells: Use of Cholesterol Hydroperoxides as Mechanistic Reporters

• Published in: Free radical biology & medicine Vol. 23; no. 1; pp. 57 - 68
• Main Authors: Geiger, Peter G., Korytowski, Witold, Lin, Fubao, Girotti, Albert W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 1997
• Subjects: Animals; Butylated Hydroxytoluene - pharmacology; Cell Survival; Cholesterol - analogs & derivatives; Cholesterol - metabolism; Cholesterol hydroperoxides; Chromatography, High Pressure Liquid; Deferoxamine - pharmacology; Free radicals; Free Radicals - metabolism; iron; Iron Compounds - pharmacology; Leukemia cells; Leukemia L1210; Light; Lipid Peroxidation; Lipid Peroxides - metabolism; Mice; Photodynamic action; Photosensitizing Agents - pharmacology; Pyrimidinones - pharmacology; Reactive Oxygen Species - metabolism; Singlet oxygen; Thiobarbituric Acid Reactive Substances - analysis; Tumor Cells, Cultured; Leukemia cells; Singlet oxygen; Free radicals; Cholesterol hydroperoxides; Lipid peroxidation; iron; Photodynamic action
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/S0891-5849(96)00587-4

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 α-OOH, 6 α-OOH, 6 β-OOH, and unresolved 7 α,7 β-OOH. Among these species, 5 α-, 6 α-, and 6 β-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7 α- and 7 β-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 α-OOH, or 7-OOH/6-OOH. When cells (10 7/ml) were exposed to a visible light fluence of 0.6 J/cm 2 in the presence of 10 μM merocyanine 540, 7-OOH/5 α-OOH increased by ∼100% after 2 h of dark incubation at 37°C. The increase was much larger (∼250%) when cells were photooxidized after treatment with 1 μM ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 μM 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 α-tocopherol strongly protected against cell killing and slowed the increase in 7-OOH/5 α-OOH ratio. It is apparent from these results that (1) the 7-OOH/5 α-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. © 1997 Elsevier Science Inc.