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

Abstract

Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR-induced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320-400 nm). The DNA photodamage was assessed using the comet assay that detects frank single-strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2 (< 1 minimum erythemal dose). Preincubation with 0.5 microM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125-2 microM) showed this effect to be concentration dependent such that SSB increased and endonuclease III-sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA-induced photosensitization is mediated via singlet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant doses via a mechanism involving reactive oxygen species.

Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts

“…It has been shown that melanin enhances free radical generation, including reactive ROS resulting from UVA irradiation (15)(16)(17). Hence, it is possible that melanin enhances ODD.…”

“…It is well established that the major effect of UVA irradiation is the induction of reactive oxygen species (ROS) and that melanin can greatly enhance this process (15)(16)(17). It has been proposed that UVA-induced oxidative stress (OS) and oxidative DNA damage (ODD) are the two major contributors to UVA-induced melanomagenesis (18).…”

Role of the pro-survival molecule Bfl-1 in melanoma

“…However, under extreme conditions, for example harmful levels of UVA radiation, melanin can also have deleterious effects. These include toxicity 42,43 , the ability to produce reactive oxygen species (ROS) 44 and photodegradation leading to hydrogen peroxide production 45 . Pheomelanin may also have a carcinogenic effect that contributes to malignant melanoma formation as one study found that albinos with melanocytes incapable of producing melanin had a very low incidence of melanoma, with only 27 cases reported in the literature 46 .…”

Cinnamoyl-based Nrf2-activators targeting human skin cell photo-oxidative stress

“…Accumulative evidence suggests that UVA-induced photodamage and carcinogenesis of human skin occur by photo-oxidative mechanisms mediated by reactive oxygen species (ROS) [9][10][11][12][13][14]. Recent research indicates that light-driven redoxcycling of non-DNA skin chromophores (including porphyrins, riboflavin, and collagen crosslinks) acting as endogenous photosensitizers is a major source of ROS in UVA-exposed human skin [2,4,12,[14][15][16][17][18]47]. The molecular consequences downstream of light-driven ROS production on skin structural integrity, signal transduction, gene expression and ultimately tumorigenic initiation and progression are well established [6].…”