Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Abstract

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA-induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA-induced degradation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA-induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole-2,3,5-tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA-melanin was confirmed by direct measurements of singlet oxygen phosphorescence.

Photodegradation of Eumelanin and Pheomelanin and Its Pathophysiological Implications

“…Thus, it appears that during immediate pigment darkening an overall increase in visible absorption (with a decrease in UVA absorption) leads to a darkening of preexisting eumelanin without any change in its amount. In fact, our recent study using differential spectra of a UVA‐exposed DHICA‐melanin solution vs an unexposed solution confirms the above‐mentioned spectral changes, which involves superoxide anion production . Earlier studies of the interaction of synthetic DOPA‐melanin with oxidizing and reducing radicals generated by pulse radiolysis demonstrated that while the oxidation of melanin was accompanied by a distinct increase in its absorption in the visible region, the reduction of melanin led to its decreased absorption .…”

“…This reaction was confirmed with a synthetic DHICA‐melanin solution . Our latest study showed that singlet oxygen is generated and quenched during the UVA‐induced oxidation of DHICA‐melanin, which likely leads to cleavage of the indolequinone moiety to form free PTCA. Singlet oxygen is a highly reactive molecule that reacts with a variety of biomolecules, including aromatic amino acids and nucleotides.…”

“…The free PTCA production and the cross‐linking occur in tandem, indicating that both events result from the oxidative modification of eumelanin. In the course of the reaction, the absorption of DHICA‐melanin in the visible region (400–700 nm) increases in a UVA dose‐dependent manner . These results suggest that persistent pigment darkening is a complex oxidative process in which the eumelanin structure undergoes oxidative cleavage and cross‐linking of dihydroxyindole units simultaneously.…”

“…Furthermore, it is known that ortho ‐quinones are highly reactive and undergo Diels‐Alder cycloaddition reactions with a variety of nucleophiles . Thus, we proposed that the indolequinone moiety as an ortho ‐quinone produces an endoperoxide intermediate that gradually degrades to form free PTCA .…”

Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

“…Pyrrole derivatives identified as markers of eumelanin crosslinking were found in blue light‐exposed bovine RPE melanosomes and in human RPE cells, suggesting age‐related oxidative modifications to RPE melanin. Additionally, it has been shown that the UV‐A‐induced oxidation product of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA), indole‐5,6‐quinone‐2‐carboxylic acid, can react with ROS to form endoperoxide and other reactive intermediates . It was found that cleavage of indole‐5,6‐quinone‐2‐carboxylic acid ultimately leads to the formation of pyrrolic moieties previously implicated as markers of melanin degradation.…”