Ascorbate Enhances Photogeneration of Hydrogen Peroxide Mediated by the Iris Melanin[dagger]

Photochemistry and Photobiology, May/Jun 2008 by Wielgus, Albert R, Sarna, Tadeusz

Several epidemiological studies showed a correlation among the iris pigmentation, UV light exposure, and incidence of ocular melanoma and other age-related diseases in the human eye (11,30,31).

In this study, we measured the formation of H^sub 2^O^sub 2^ induced by UV and visible light that accompanied photo-oxidation of exogenous AscH- in samples of intact bovine irides, homogenates of bovine and human irides and bovine iridial melanosomes. We also analyzed the effect of iris color and the age of donors on iridial melanin photoreactivity.

MATERIALS AND METHODS

Reagents. L( )-ascorbic acid (AA) and 30% H^sub 2^O^sub 2^ were from Merck (Darmstadt, Germany), catalase (EC 1.11.1.6) from a bovine liver (Tymol free) and superoxide dismutase (SOD; EC 1.15.1.1) from bovine erythrocytes were from Sigma-Aldrich (Steinheim, Germany), sucrose was from Polskie Odczynniki Chemiczne (Gliwice, Poland), CuSO^sub 4^-5H^sub 2^O from Przedsiebiorstwo Chemiczne Odczynniki (Lublin, Poland) and sodium azide from Fisher Scientific (Schwete, Germany). All reagents were at least analytical grade and used as supplied.

Water used in all experiments was twice distilled in an all-glass apparatus. PBS was incubated with Chelex-100 (Bio-Rad Laboratories, Munich, Germany) to remove traces of heavy metal ions.

Human irides. Human eye irides were obtained from the Wisconsin Lion Eye Bank (Milwaukee, WI). For chemical analysis, each iris was pooled within one of 12 groups as described previously (12). Irides assigned to the blue and brown groups were split into four age categories in each group. Because of limited availability of the human material and low yield of melanosome isolation, we used iridial homogenates in our experiments. Combined irides of the same group were mechanically homogenized on ice in an all-glass homogenizer and suspended in PBS. The samples were stored on ice during each experiment.

Bovine irides. Bovine eyeballs were obtained from a local slaughterhouse in Krakow (Poland) and transported on ice to the laboratory. The irides were extracted as described previously (12).

Intact irides were used as a comparative material in a limited number of experiments. Before each measurement series the iris was flushed with PBS. Then, it was placed in a suitably designed thermostatic glass vessel used for irradiation with the corneal surface exposed directly to UV and light. The irides were immersed in known volume of PBS, which in some experiments contained AA at selected concentrations. In some experiments, the iris was flushed with PBS and incubated overnight in 1 mM NaN^sub 3^ isotonic solution to deactivate the tissue catalase (32). After this pretreatment, the iris was repeatedly flushed with PBS and irradiated as previously.

Iridial homogenates were prepared as described previously (12). Preincubation of the homogenates with NaN^sub 3^ was carried as follows. Selected amounts of the homogenate were centrifuged at 16 800 g for 10 min. The supernatant was replaced with the same volume of NaN^sub 3^ to obtain the final concentration of the catalase deactivator in the sample at 1 mM. After overnight incubation, the sample was centrifuged as previously. The precipitate was rinsed several times with cold PBS and suspended in such amount of the buffer to get the same volume of the homogenate as it was before the incubation.