UVA-induced carbon-centred radicals in lightly pigmented cells detected using ESR spectroscopy.

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.

Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells.

Skin cancer incidence is dramatically increasing worldwide, with exposure to ultraviolet radiation (UVR) a predominant factor. The UVA component initiates oxidative stress in human skin, although its exact role in the initiation of skin cancer, particularly malignant melanoma, remains unclear and is controversial because there is evidence for a melanin-dependent mechanism in UVA-linked melanoma studies. Nonpigmented (CHL-1, A375), moderately pigmented (FM55, SKmel23), and highly pigmented (FM94, hyperpigmented FM55) human melanoma cell lines have been used to investigate UVA-induced production of reactive oxygen species using FACS analysis, at both the cellular (dihydrorhodamine-123) and the mitochondrial (MitoSOX) level, where most cellular stress is generated. For the first time, downstream mtDNA damage (utilizing a quantitative long-PCR assay) has been investigated. Using UVA, UVB, and H(2)O(2) as cellular stressors, we have explored the dual roles of melanin as a photoprotector and photosensitizer. The presence of melanin has no influence over cellular oxidative stress generation, whereas, in contrast, melanin protects against mitochondrial superoxide generation and mtDNA damage (one-way ANOVA with post hoc Tukey's analysis, P<0.001). We show that if melanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (P<0.001), providing evidence for the dual roles of melanin.

Comparable photoreactivity of hair melanosomes, eu- and pheomelanins at low concentrations: low melanin a risk factor for UVA damage and melanoma?

Melanin is known to be photoreactive and photoprotective, but its function in skin in vivo is still debated. Data is lacking of the effects of UVA irradiation on human skin melanosomes of different pigmentation, which have not been extensively degraded by isolation procedures. We have shown previously that melanosomes isolated from human oriental and black cat hair, and synthetic eumelanins, are photoreactive producing superoxide at low concentrations when exposed to UVA irradiation comparable to UK levels of sunlight. Here we investigated the UVA-irradiation of melanosomes, isolated from different colored human hair samples, using electron spin resonance spectroscopy and spin trapping. Comparable irradiation of synthetic pheomelanins synthesized from L-dopa and L-cysteine was also studied. An alkali method (5 min NaOH at 90 degrees C) could be used to isolate oriental hair melanosomes but was not suitable for auburn and blonde hair. Dithiothreitol and proteinase K resulted in melanin release from possible over-digestion of melanosomes; however, dithiothreitol and papain resulted in no melanin release and good melanosome yields with separation from residual keratin for brown, auburn and blonde hair. Melanosomes isolated by the latter method and synthetic pheomelanins were similar in UVA-photoreactivity at low concentrations, independent of hair color, and broadly comparable to synthetic melanins. Melanosome concentration at constant fluence may be more significant with respect to photodamage and UVA photocarcinogenesis (melanoma) via superoxide radical production than pigment type.

Synthetic melanin is a model for soluble natural eumelanin in UVA-photosensitised superoxide production.

Studies to UV-irradiate natural eumelanins in vitro have used insoluble pigment obtained by acid hydrolysis, which lacks melanoprotein. Eumelanin synthesised in the presence of a protein is not insoluble, and the insoluble form of melanin from acid hydrolysis may not have the same physicochemical properties as the natural pigment synthesised in vivo in the melanosome. Here we investigated radical production by three natural eumelanins exposed to solar levels of UVA; sepia melanin from Sepia officinalis, and eumelanins isolated from Oriental human and domestic cat hair. UVA irradiation of sepia melanin in solution at pH 4.5 in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave hydroperoxyl and hydroxyl radical-adducts, maximal at 0.6-2.5 mg/ml melanin concentrations. Hydroperoxyl radical production was relatively low in acetate buffer, but detected in aqueous suspensions of sepia melanin. Hair eumelanins were photoreactive with hydroperoxyl radical-adduct production at low concentrations (0.1-0.4 mg/ml melanin). Synthetic pigment after synthesis undergoes photo-oxidation (producing superoxide) at low concentrations (0.3 mg/ml) and its oxidation increases the photoreactivity at higher melanin concentrations. These findings may be physiologically relevant to the properties and function of eumelanin in vivo when it is at low concentration (found in a small proportion of Caucasian melanocytes), and suggest that synthetic melanin has the potential for the basis of a model for natural eumelanin.

An experimental and theoretical model for solar UVA-irradiation of soluble eumelanin: towards modelling UVA-photoreactions in the melanosome?

A model is developed for the UVA-irradiation of soluble eumelanin exposed to levels of irradiation comparable to sunlight. Radical production was determined in soluble dl- and l-dopa melanins exposed to solar levels of UVA, using electron spin resonance spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Steady-state concentrations of DMPO-O(2)H(.-), which increased up to 0.3 mg/ml melanin, and then declined above 0.3 mg/ml, were detected at pH 4.5. The kinetic model incorporated the photosensitizing and radical-scavenging reactions of eumelanin, and assumed semiquinone radical reduction of oxygen to be fast compared to disproportionation. The model is consistent with experimental data for melanin concentrations <0.1 mg/ml; but >0.1 mg/ml melanin is consistent only with data at raised oxygen tension. The rate-constant for reaction of the melanin semiquinone-radical and oxygen is estimated to be 10(3) mol(-1)dm(3)s(-1). In this model, where DMPO competes with melanin for HO(2)(.-), at ambient oxygen levels, eumelanin exposed to solar levels of UVA photosensitizes superoxide at concentrations <0.3 mg/ml melanin, and is increasingly stable towards oxidation when >0.3 mg/ml concentration. Eumelanin could have a negligible screening effect <0.1 mg/ml and very strong screening >1 mg/ml. This model would be biologically relevant if soluble forms of eumelanin were shown to exist in vivo, and is potentially useful for studies of the photochemistry and photophysics of eumelanin and phaeomelanin and to explore the effects of metal-ions, proteins and lipids in a model system.

Differences in production of melanin radicals by 694 nm ruby laser and UVA radiation.

BACKGROUND AND OBJECTIVES: The 694 nm ruby laser is used clinically for hair removal and the mechanism is predominantly photo thermal via melanin targeting. We investigated 694 nm laser-irradiation of human hair, and laser-irradiation of synthetic dopa melanin to establish whether photolysis and oxygen radical production is also contributory, and which may have side effects. STUDY DESIGN/MATERIALS AND METHODS: Ultraviolet-A (UVA) irradiation of melanin was used as a positive control for radical production. Laser- and UVA-irradiated hair samples, and synthetic dopa melanin in media of different viscosity, were analyzed using electron spin resonance spectroscopy, and compared. The spin trap 5,5-dimethyl- 1-pyrroline N-oxide (DMPO) was used to probe laser-irradiated dopa melanin for superoxide radical production. RESULTS: Comparable to UVA, laser-irradiation of hair increased the signal-intensity of the intrinsic melanin radical. UVA-induced radicals decay rapidly; however, laser-induced radicals decayed slowly and did not fully revert to original levels after 24 hours. Laser-induced radicals were increasingly stable with viscosity of the medium. Superoxide radicals were detected using DMPO in UVA- but not laser-irradiated synthetic dopa-melanin at pH 4.5. CONCLUSIONS: Laser-irradiation of melanin does not result in oxygen radical formation; however, a paramagnetic species, long-lived in rigid media, is detected which is worth further investigation.