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

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

ABSTRACT

The iris in the human eye is exposed to UV and visible light transmitted by the cornea. This pigmented tissue is bathed with the aqueous humor (AqH), which contains high concentration of ascorbate. It has been postulated that the presence of ascorbate in AqH can contribute to increased photoproduction of H^sub 2^O^sub 2^ mediated by the iris melanin. In this study, we monitored generation of H^sub 2^O^sub 2^ induced by UV-VIS irradiation of bovine indes, bovine and human iris homogenates and iris melanin. Our data show that exogenous ascorbate significantly amplified the rate of H^sub 2^O^sub 2^ photoformation in all melanotic samples. Deactivation of endogenous catalase with NaN^sub 3^ in bovine irides increased the level of the accumulated H^sub 2^O^sub 2^ in the bathing solution following sample irradiation. Photoformation of H^sub 2^O^sub 2^ in samples with exogenous ascorbate was accompanied by its photo-oxidation. Both photoprocesses exhibited significant wavelength dependence. EPR spectroscopy measurements showed that ascorbyl radical is an intermediate product of the ascorbate photo-oxidation. The photoproduction of H^sub 2^O^sub 2^ and photo-oxidation of ascorbate appear to be stoichiometric processes. No significant differences in the photoreactivity of iridial melanin from donors of different age and iris color was found. We postulate that also in vivo ascorbate increases the rate of iris melanin-mediated photoformation of H^sub 2^O^sub 2^ and its steady state concentration in AqH.

INTRODUCTION

The aqueous humor (AqH) fills entirely the anterior chamber of the human eye. It is continuously exchanged at the rate 2.2-2.8 �L min^sup -1^ (1,2) supplying nutrients to the nonvascularized cornea, lens and trabecular meshwork (3). Normally, the AqH is well protected against oxidative stress that may occur in this anterior segment of the eye, by antioxidant enzymes (4), chelating proteins, amino acids (9 �M L-cysteine and L-tyrosine) and antioxidants such as 1.9-16 �M glutathione (5) and 0.8-2.5 mM ascorbate (4,6-9). Nevertheless, hydrogen peroxide (H^sub 2^O^sub 2^) is present in AqH of healthy individuals at a concentration of 20-60 �M (7,8,10). Moreover, the level of reactive oxygen species detected in AqH in some ocular diseases can be much higher. Indeed, the concentration of H^sub 2^O^sub 2^ observed in the AqH of cataract patients was 10-30 times higher than in healthy persons (5,8-10).

In the human eye, iris is an extension of the uvea. This pigmented organ is situated in the anterior chamber of the eye between the cornea and the lens (11,12) and is bathed on both its sides with AqH. The primary function of the iris is to adapt the eye to changing ambient illumination conditions (13). The iris in situ is exposed to a relatively high intensity radiation, with wavelengths longer than 295 nm, that is transmitted by the cornea and AqH at wavelengths longer than 295 nm (10,14). This pigmented tissue is visible in the eye as a colorful disk. Its color in the nonalbino eye is determined mostly by the amount of melanin (15) and spectral properties of the extracellular matrix (16). Melanin content can vary substantially depending on the iris color and the shade within the color group. The pigment amount is at least ~20% higher in brown irides in comparison to blue ones (12).

It is believed that the main role of melanin in the eye is photoprotection (17,18). Melanin absorbs UV radiation and blue light more efficiently than visible light of longer wavelengths (17,19). The absorbed radiation increases the stationary level of melanin radicals by shifting so-called comproportionation equilibrium of the quinone and hydroquinone melanin units toward semiquinones (20,21). Interaction of photoinduced melanin radicals with molecular oxygen can lead to superoxide anion (19,20,22) and H^sub 2^O^sub 2^ generation (19,20,22-24), both associated with oxygen consumption (20). Another interesting property of melanin is its ability to catalyze, in the presence of appropriate donors or acceptors, electron transfer reactions (22). We showed in our previous study (11) that the rate of oxygen photoconsumption in a sample containing melanin from human irides was ~10 times higher in the presence of exogenous ascorbate (AscH^sup -^). Simultaneously, ascorbate photo-oxidation was observed in the iridial melanin samples. Earlier studies indicated that melanosomes, isolated from bovine retinal pigment epithelium, irradiated with light in the presence of AscH^sup -^, produced H^sub 2^O^sub 2^ at higher rate than the same melanin samples without the exogenous antioxidant (25,26). The relatively high concentration of ascorbate and substantial amounts of H^sub 2^O^sub 2^ detected in the AqH of the human eye suggest that such photoprocesses can also take place in situ in the human iris and its proximity.

Absorption of short-wavelength radiation by iridial melanin increases the risk of the melanin photodamage (13,15,18). Due to the vascularization of the iris and the presence of transient metal ions in the iridial melanin structure (12), the pigment can probably undergo light-induced photobleaching (13,27). Interestingly, melanosomes from adult bovine eyes appeared lighter than those from eyes of younger donors (15). It has been demonstrated that modification of melanin structure can affect its photoreactivity (13,18,22). Importantly, an age-dependent decrease in low molecular weight antioxidants and antioxidant enzymes in the human eye tissue was observed relatively early in the middle age (20,28). Although the full biological impact of a significant depletion of antioxidant enzymes remains to be determined, it can be argued that elevated accumulation of H^sub 2^O^sub 2^ and the formation of H^sub 2^O^sub 2^-dependent oxidizing radicals will have adverse effects on normal structure and function of many biological tissues including the anterior chamber of the human eye. Conceivably such processes may lead to the lens opacification, loss of corneal endothelial cells, modification of the glycosaminoglycan secretory patterns of the trabecular meshwork cells, and other changes associated with ocular aging (29).