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

1997; Elsevier BV; Volume: 23; Issue: 1 Linguagem: Inglês

10.1016/s0891-5849(96)00587-4

1873-4596

Peter Geiger, Witold Korytowski, Fubao Lin, Albert W. Girotti,

Eicosanoids and Hypertension Pharmacology

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: 5α-OOH, 6α-OOH, 6β-OOH, and unresolved 7α,7β-OOH. Among these species, 5α-, 6α-, and 6β-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas 7α- and 7β-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/5α-OOH, or 7-OOH/6-OOH. When cells (107/ml) were exposed to a visible light fluence of 0.6 J/cm2 in the presence of 10 μM merocyanine 540, 7-OOH/5α-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/5α-OOH ratio. It is apparent from these results that (1) the 7-OOH/5α-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.