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

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

DISCUSSION

Hydrogen peroxide photoproduction

In the mammalian eye, the iris is exposed to relatively high fluxes of solar UV and visible light. In eyes of normal individuals, without severe ocular-cutaneous albinism, excess of light that reaches the iris is absorbed and scattered by the iridial melanin. Because energy of the photons absorbed by melanin is rapidly converted into heat, no photochemical processes responsible for potential phototoxicity occur. Therefore, the pigmented ocular tissue is expected to be efficiently protected against photodamage. However, quantum yield of internal conversion and thermal relaxation of the excited melanin, although very high, is not exactly unity (17) and the residual aerobic photochemistry of melanin is responsible for small but measurable fluxes of Superoxide anion and H^sub 2^O^sub 2^ (23). Indeed, we have previously shown that under aerobic conditions, exposure of iridal homogenates to UV-VIS radiation leads to oxygen consumption and accompanying formation of O^sub 2^.^sup -^ (11). Dismutation of Superoxide anions or their reduction by the melanin functional groups is responsible for the observed accumulation of H^sub 2^O^sub 2^ (11,23,24,26). The exposure of the human iris to high intensity UV and visible light and the ability of the iridial melanin to photogenerate Superoxide anion, may explain the reported high SOD activity in the rabbit iris, which is approximately 3 and 80 times higher than in the animal retina and the lens, respectively (38).

It is worth emphasizing that in samples containing intact bovine iris, we monitored photogeneration of H^sub 2^O^sub 2^ not directly in the tissue but in the buffer in which the iris was immersed. Interestingly, the equilibrium concentration of H^sub 2^O^sub 2^ observed in our study was similar to the value of 25-35 �M determined previously in bovine AqH (9).

Although the exact cause for the observed differences in the rates of the photoformation of H^sub 2^O^sub 2^ in samples containing homogenates of human and bovine irides remains unknown, it can be hypothesized that different optical properties of the human and bovine iridial tissues, including different degree of melanin aggregation in both tissues, might play a role. It was reported that irradiation of synthetic melanin, obtained by autoxidation of dopa, led to formation of H^sub 2^O^sub 2^ that was ~5 times faster than that when more aggregated eumelanin produced enzymatically was used (39). The effect of aggregation on the efficiency of Sepia melanin to photogenerate Superoxide anion and H^sub 2^O^sub 2^ was elegantly demonstrated by the Simon's group (19,22). Thus, the observed higher rates of H^sub 2^O^sub 2^ photogeneration in homogenates of the human irides could be due to the lower aggregation state of the human iridial melanin compared with that of the bovine pigment.

Different activity of antioxidant enzymes in human and bovine iridial tissues could be an additional factor that affects the observable kinetics and maximal levels of H^sub 2^O^sub 2^ accumulation in samples of these tissues during their irradiation. The presence of catalase, Cu/Zn-SOD, basic and acidic glutathione S-transferases and glutathione peroxidases was confirmed in the rabbit (40), canine (41) and human (42) irides. It remains to be demonstrated whether or not the bovine tissue has a higher activity of SOD and catalase than the human tissue. Although results of this study are insufficient for settling the issue, they indirectly indicate that the activity of SOD and catalase might be different in both iridial tissues. Thus, the higher initial rate of H^sub 2^O^sub 2^ accumulation in the bovine homogenate, in comparison to the melanosome sample, could result from the presence of SOD that rapidly dismutates the photogenerated superoxide anion (Table 1). Endogenous catalase, on the other hand, is likely to be responsible for lower levels of the accumulating H^sub 2^O^sub 2^ in spite of higher fluxes of the photogenerated superoxide anion. Deactivation of catalase and other H^sub 2^O^sub 2^-destroying enzymes can cause accumulation of H^sub 2^O^sub 2^ at higher level in the iris and AqH. It is known that azide deactivates horseradish peroxidase (43), catalase (43-45) including the lens catalase (38), cytochrome c oxidase (46) and bovine eyes ascorbate peroxidase (47). Although azide can partially deactivate Cu/Zn-SOD (38,46) it was reported that 1 mM NaN^sub 3^ did not deactivate SOD in the rabbit lens (38). Our data show that bovine irides as well as homogenates of bovine and human irides incubated overnight with 1 mM sodium azide, exhibited higher accumulation of H^sub 2^O^sub 2^ than the nonpretreated samples. It is important to emphasize that a similar effect, i.e. an increase of H^sub 2^O^sub 2^ in the AqH, was also observed in vivo in the rabbit's eye using 3-triaminotriazole as a specific catalase inhibitor (48).