Rachel M. Haywood

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 Tukeys 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.

Intensity-dependent direct solar radiation- and UVA-induced radical damage to human skin and DNA, lipids and proteins.

Skin can be exposed to high‐intensity UV‐radiation in hot countries and during sunbed use; however, the free‐radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free‐radical generation in ex vivo human skin/substitutes +/− the spin‐trap 5,5 dimethyl‐1‐pyrroline N‐oxide (DMPO) exposed to solar‐irradiation equivalent to Mediterranean sunlight. Skin‐substitutes, model DNA‐photosensitizer systems, lipids and proteins were also irradiated with low‐intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail‐keratin, tendon‐collagen, phospholipid and DNA + melanin or riboflavin. In addition to lipid‐derived (tentatively tert‐alkoxyl/acyl‐) and protein radicals detected with DMPO at lower intensities, isotropic carbon‐, additional oxygen‐ and hydrogen‐adducts were detected in solar‐irradiated skin/substitutes at higher intensities. Carbon‐adducts were detected in UVA‐irradiated human skin cells, DNA + melanin or riboflavin and soybean‐phospholipid. Anisotropic protein‐adducts, comparable to adducts in solar‐irradiated tendon‐collagen, were absent in UVA‐irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen‐adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon‐ and oxygen‐radicals) and part extracellular (protein‐ and hydrogen/H+ + e−), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid‐oxidation is direct, DNA and protein‐oxidation require photosensitisation.

Ruby laser irradiation (694 nm) of human skin biopsies: assessment by electron spin resonance spectroscopy of free radical production and oxidative stress during laser depilation.

Abstract Human skin biopsies (hair‐bearing scalp skin and non‐hair‐bearing breast skin) were treated with t‐butylhydro‐peroxide, irradiated with UV light (UVR) or irradiated with 694 nm ruby laser red light. Free‐radical production and oxidative stress were assessed with electron spin resonance spectroscopy (ESR) using the ascorbate radical as a marker. In comparison with both UVR and t‐butyl‐hydroperoxide (which readily induce the ascorbate radical in hair‐bearing and hairless skin), 694 nm red light does not result in the formation of the ascorbate radical in detectable concentrations. Spin‐trapping experiments with the spin trap 5,5‐dimethyl‐l‐pyrroline N‐oxide (DMPO) showed that while free radicals could be detected after treatment of skin with t‐butylhydroperoxide, radicals could not be trapped after laser treatment. Treatment of lasered skin (containing DMPO) with t‐bu‐tylhydroperoxide produced radical adducts as well as the ascorbate radical, demonstrating that the laser neither depletes endogenous ascorbate nor the preadministered spin trap. It is concluded that 694 nm red light does not induce oxidative stress in human skin in levels comparable either to t‐butyl hydroperoxide or UV light.

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°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.

Mitochondrial protein-linked DNA breaks perturb mitochondrial gene transcription and trigger free radical–induced DNA damage

Mitochondrial protein-linked DNA repair promotes gene transcription and protects from free radical–induced DNA damage. Breakage of one strand of DNA is the most common form of DNA damage. Most damaged DNA termini require end-processing in preparation for ligation. The importance of this step is highlighted by the association of defects in the 3′-end processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) and neurodegeneration and by the cytotoxic induction of protein-linked DNA breaks (PDBs) and oxidized nucleic acid intermediates during chemotherapy and radiotherapy. Although much is known about the repair of PDBs in the nucleus, little is known about this process in the mitochondria. We reveal that TDP1 resolves mitochondrial PDBs (mtPDBs), thereby promoting mitochondrial gene transcription. Overexpression of a toxic form of mitochondrial topoisomerase I (TOP1mt*), which generates excessive mtPDBs, results in a TDP1-dependent compensatory up-regulation of mitochondrial gene transcription. In the absence of TDP1, the imbalance in transcription of mitochondrial- and nuclear-encoded electron transport chain (ETC) subunits results in misassembly of ETC complex III. Bioenergetics profiling further reveals that TDP1 promotes oxidative phosphorylation under both basal and high energy demands. It is known that mitochondrial dysfunction results in free radical leakage and nuclear DNA damage; however, the detection of intermediates of radical damage to DNA is yet to be shown. Consequently, we report an increased accumulation of carbon-centered radicals in cells lacking TDP1, using electron spin resonance spectroscopy. Overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) reduces carbon-centered adducts and protects TDP1-deficient cells from oxidative stress. Conversely, overexpression of the amyotrophic lateral sclerosis–associated mutant SOD1G93A leads to marked sensitivity. Whereas Tdp1 knockout mice develop normally, overexpression of SOD1G93A suggests early embryonic lethality. Together, our data show that TDP1 resolves mtPDBs, thereby regulating mitochondrial gene transcription and oxygen consumption by oxidative phosphorylation, thus conferring cellular protection against reactive oxygen species–induced damage.

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: Development of an ex vivo test method

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40–65% when applied at 2 mg/cm2. SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO2-UVB filters and a novel UVA-TiO2 filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.

UVA-induced Carbon-Centered Radicals in Lightly Pigmented Cells detected using ESR Spectroscopy

Abstract 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. Graphical abstract Figure. No caption available. HighlightsCarbon radicals are detected in UVA‐irradiated cells.More carbon radicals detected in lightly melanin‐pigmented cells than cells without melanin.Increase in melanin in lightly pigmented cells decreases radical levels.Carbon radicals detected in cell nuclei, DNA‐melanin and 2′‐deoxyguanosine‐melanin.Carbon radicals likely to be nucleic acid ‐ guanine – radicals.