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

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: image-OOH, image-OOH, image-OOH, and unresolved image,image-OOH. Among these species, image-, image-, and image-OOH (singlet oxygen adducts) were predominant in the early stages of photooxidation, whereas image- and image-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., image-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 image merocyanine 540, image-OOH increased by not, vert, similar100% after 2 h of dark incubation at 37°C. The increase was much larger (not, vert, similar250%) when cells were photooxidized after treatment with 1 image ferric-8-hydroxyquinoline, a lipophilic iron donor, whereas no increase was observed when cells were pretreated with 100 image 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 image-tocopherol strongly protected against cell killing and slowed the increase in image-OOH ratio. It is apparent from these results that (1) the image-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.