Role of hydrogen peroxide in riboflavin-sensitized photodynamic damage to cultured rat lenses.

Previous reports have differed on whether hydrogen peroxide or free radicals are the principal toxic species responsible for photodynamic damage to intact lenses cultured in medium with riboflavin, light and oxygen. To differentiate between hydrogen peroxide and free radicals, TC-199 medium containing 50 microM riboflavin was incubated in visible light prior to adding the lenses. After 4 hr the medium was placed in the dark, rat lenses, [3H]-choline and 86Rb were added, and the lenses were cultured for 4 hr. Damage to the lenses was evaluated by measuring the accumulation of choline and Rb. Lenses cultured in previously irradiated medium accumulated less choline and Rb than lenses in control medium, indicating that in the presence of light, riboflavin generates a stable factor which is toxic to lenses. The lens damage was similar to that caused by direct exposure of lenses to riboflavin and light for 4 hr. This toxicity was blocked by catalase, and was presumed to be caused by hydrogen peroxide. Because of their short lifetime in solution, photochemically generated free radicals could not have been present in the dark after the lenses were added. Rose bengal, a photosensitizer which is thought to form singlet oxygen, did not generate a stable toxic factor in the culture medium and damaged lenses only if they were present during the exposure to light. The hydrogen peroxide concentration in riboflavin-containing medium after 4 hr exposure to light was approximately 200 microM, whereas rose bengal generated less than 10 microM hydrogen peroxide.(ABSTRACT TRUNCATED AT 250 WORDS)