Photosensitization of Supercoiled Dna Damage by 5,6‐dihydroxyindole‐2‐carboxylic Acid, a Precursor of Eumelanin

Abstract

Abstract— The photosensitizing or photoprotecting action of 5,6‐dihydroxyindole‐2‐carboxylic acid (DICA), an intermediate in the biosynthesis of eumelanins, was investigated. Under irradiation at 313 nm, aqueous buffered solutions of DICA (22.5 μW) photosensitized the cleavage of phage φX174 DNA. The number of single strand breaks (SSB) depended on the dose of irradiation and was more important in the absence than in the presence of oxygen. In the presence of oxygen, the quantum yield of SSB was around 6′10 7 (ΦSSB) The influence of specific scavengers, such as mannitol, sodium azide or superoxide dismutase, indicated that hydroxyl radicals, superoxide anions and perhaps singlet oxygen were involved in these processes. The increase in SSB in D2O was also indicative of the participation of singlet oxygen. Comparative experiments performed with indole‐2‐carboxylic acid (IC), a dehydrox‐ylated analog of DICA, showed that this compound, although lacking a phenol group, also photosensitized DNA cleavage via a mechanism involving hydroxyl radicals. Various sources of these radicals were envisioned. Furthermore, under our conditions, DICA was not found to photoinduce the formation of DNA dimers: No increase in SSB was observed in DNA irradiated in the presence of DICA, after treatment by phage T4 endonuclease V (an enzyme that selectively cuts DNA at dimer sites), whereas, in contrast, a significant increase in SSB was detected after treatment of DNA irradiated alone. So it appears that DICA may both photosensitize DNA cleavage and reduce UV‐induced DNA dimer formation.

Absolute Rate Constants for the Quenching of Reactive Excited States by Melanin and Related 5,6-Dihydroxyindole Metabolites: Implications for Their Antioxidant Activity

“…The contrasting hypotheses and results, phototoxic (16)(17)(18)(19)(20)(21) versus photoprotective (1,6,(21)(22)(23)(24), were also given for the monomeric precursors of melanins (10), namely 5,6-dihydroxyindole (DHI) (6,16,21,24), 5,6-dihydroxyindole-2carboxylic acid (DHICA) (6,24) and 5-S-cysteinyldopa (SCD) (6,(17)(18)(19)21,23,24). The first two constitute the major monomeric building blocks of eumelanin (cf melanogenesis), the brown and dark melanin type, while pheomelanin, the reddish melanin type, incorporates SCD also (1,3,6).…”

The Soluble Eumelanin Precursor 5,6‐Dihydroxyindole‐2‐carboxylic Acid Enhances Oxidative Damage in Human Keratinocyte DNA after UVA Irradiation‡

“…The autoxidation of DHICA is unlikely to be the cause of the increase in SSB seen in this study, as unirradiated cells showed only a small increase in SSB. The photosensitizing effect of DHICA on SSB reported by Routaboul et al (14) suggests that production of H202 may indeed be involved, although to a lesser extent than other ROS. The reduction in the level of SSB in the presence of mannitol and histidine (Fig.…”

“…Unfortunately, there is a lack of experimental data on the concentration of DHICA in melanocytes and skin, so that the use of physiological concentrations remains a question of guesswork. However, the concentrations used in this study represent the lowest in the range (1-100 pM) used in other studies looking at the effects of DHICA on a variety of endpoints (14,(27)(28)(29). Furthermore, it must been noted that the the concentration of DHICA in the medium may not necessarily reflect the intracellular concentration, which will depend on the uptake of DHICA by the cell and may itself vary with extracellular concentration.…”

“…A similar finding was reported by Routaboul ef al. (14) when looking at induction of SSB by DHICA irradiated at 313 nm, although it was suggested that hydroxyl radicals were involved to a greater extent than singlet oxygen. However, any comparisons with the study by Routaboul et al (14) must take into account the cell-free conditions and comparatively high UVB (>2 MED) doses used.…”

“…Precursors of melanin that may possess similar properties to melanin are found in urine (10)(11)(12) and are thought to be present in high local concentrations in the skin (13), especially in melanocytes during melanogenesis. 5,6-Dihydroxyindole-2-carboxylic acid (DHICA), a precursor of eumelanin has been found in normal urine (10) and has been shown to photosensitize UVB-induced DNA damage in naked DNA via the generation of ROS (14). This molecule, unlike melanin, is water soluble and was therefore considered to be a useful tool to study melanogenesis-related UVA-photosensitized DNA damage in human epidermal cells.…”