Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

Distinct Role of Sesn2 in Response to UVB‐Induced DNA Damage and UVA‐Induced Oxidative Stress in Melanocytes

Ultraviolet (UV) radiation, including both UVB and UVA irradiation, is the major risk factor for causing skin cancer including melanoma. Recently, we have shown that Sesn2, a member of the evolutionarily conserved stress‐inducible protein family Sestrins (Sesn), is upregulated in human melanomas as compared to melanocytes in normal human skin, suggesting an oncogenic role of Sesn2. However, the role of Sesn2 in UVB and UVA response is unknown. Here, we demonstrated that both UVB and UVA induce Sesn2 upregulation in melanocytes and melanoma cells. UVB induces Sesn2 expression through the p53 and AKT3 pathways. Sesn2 negatively regulates UVB‐induced DNA damage repair. In comparison, UVA induces Sesn2 upregulation through mitochondria but not Nrf2. Sesn2 ablation increased UVA‐induced Nrf2 induction and inhibits UVA‐induced ROS production, indicating that Sesn2 acts as an upstream regulator of Nrf2. These findings suggest previously unrecognized mechanisms in melanocyte response to UVB and UVA irradiation and potentially in melanoma formation.     

Role of UV in cutaneous melanoma

Malignant melanoma arises from epidermal melanocytes, the cells responsible for the production of the skin pigment melanin. The photoprotective role of melanin, which is transferred to neighboring keratinocytes, in UV-induced skin carcinogenesis, specifically in nonmelanoma skin cancers, has been well documented. Although melanocyte-resident melanin is expected to offer similar protection to melanocytes from UV-induced damage, UV radiation has long been suspected to have an etiologic role in cutaneous melanoma. However, nearly three decades of efforts using a variety of in vitro and in vivo models of human skin and mouse genetic models have produced conflicting data. Epidemiologic studies have also failed to establish a definitive association between UV exposure and risk of melanoma. In this review, we evaluate the dual role of the melanin pigment as a photoprotector as well as a photosensitizer and examine the evidence for association between melanin levels (constitutive and induced) and melanoma risk. We also discuss possible reasons for the lack of signature UV mutations in melanoma oncogenes known to date and potential alternative mechanisms to explain the role of UV in melanomagenesis.     

Effects and Mechanism of Nicotinamide Against UVA‐ and/or UVB‐mediated DNA Damages in Normal Melanocytes

Melanoma incidences are increasing rapidly, and ultraviolet (UV) radiation from the sun is believed to be its major contributing factor. UV exposure causes DNA damage in skin which may initiate cutaneous skin cancers including melanoma. Melanoma arises from melanocytes, the melanin‐producing skin cells, following genetic dysregulations resulting into hyperproliferative phenotype and neoplastic transformation. Both UVA and UVB exposures to the skin are believed to trigger melanocytic hyperplasia and melanomagenesis. Melanocytes by themselves are deficient in repair of oxidative DNA damage and UV‐induced photoproducts. Nicotinamide, an active form of vitamin B3 and a critical component of the human body's defense system has been shown to prevent certain cancers including nonmelanoma skin cancers. However, the mechanism of nicotinamide's protective effects is not well understood. Here, we investigated potential protective effects and mechanism of nicotinamide against UVA‐ and/or UVB‐ induced damage in normal human epidermal melanocytes. Our data demonstrated an appreciable protective effect of nicotinamide against UVA‐ and/or UVB‐ induced DNA damage in melanocytes by decreasing both cyclobutane pyrimidine dimers and 8‐hydroxy‐2′‐deoxyguanosine levels. We found that the photoprotective response of nicotinamide was associated with the activation of nucleotide excision repair genes and NRF2 signaling. Further studies are needed to validate our findings in in vivo models.     

Photobiology of ocular melanocytes and melanoma

There are two different types of ocular melanocytes and melanomas. Conjunctival melanocytes are located on the surface of the eye and are exposed to visible light and UV radiation. Recently, epidemiological studies demonstrated that sunlight plays a definite role in the occurrence of conjunctival melanoma, as it does in cutaneous melanoma. Uveal melanocytes consist of the iridal melanocytes, which are exposed to visible light and UV radiation; and the ciliary body melanocytes and choroidal melanocytes, which are not exposed to light radiation. Epidemiological studies demonstrated that sunlight may play a role in the occurrence of iridal melanoma, but may not be a major factor in the etiology of ciliary body and choroidal melanomas. Uveal melanocytes differ from epidermal melanocytes in that epidermal melanocytes respond to UV radiation and skin color becomes darker after exposure to sunlight; but uveal melanocytes do not respond to UV radiation and the iris color remains stable after exposure to sunlight. Recently, in vitro studies indicate that this phenomenon is determined both by cellular factors and environmental factors.     

Oxidative Stress and Genotoxicity in Melanoma Induction: Impact on Repair Rather Than Formation of DNA Damage?

Keratinocytes and melanocytes, two cutaneous cell types located within the epidermis, are the origin of most skin cancers, namely carcinomas and melanomas. These two types of tumors differ in many ways. First, carcinomas are almost 10 times more frequent than melanomas. In addition, the affected cellular pathways, the mutated genes and the metastatic properties of the tumors are not the same. This review addresses another specificity of melanomas: the role of photo-oxidative stress. UVA efficiently produces reactive oxygen species in melanocytes, which results in more frequent oxidatively generated DNA lesions than in other cell types. The question of the respective contribution of UVB-induced pyrimidine dimers and UVA-mediated oxidatively generated lesions to mutagenesis in melanoma remains open. Recent results based on next-generation sequencing techniques strongly suggest that the mutational signature associated with pyrimidine dimers is overwhelming in melanomas like in skin carcinomas. UVA-induced oxidative stress may yet be indirectly linked to the genotoxic pathways involved in melanoma through its ability to hamper DNA repair activities.     

DNA Damage, Repair and Photoadaptation in a Xiphophorus Fish Hybrid

Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F₁ hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.     

Comparative Action Spectrum for Ultraviolet Light Killing of Mouse Melanocytes from Different Genetic Coat Color Backgrounds

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony-forming ability after irradiation. The cell lines were wild-type black (melan-a) and the mutants brown (melan-b) and albino (melan-c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200–280 Dm), 82.3% output at 254 nm, TL01 (UVB, 280–320 nm), 64.2% at 310–311 nm, FS20, broadband with peak output at 312 nm and Alisun-S (UVA, 320–400 nm), broadband with peak output at 350–354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild-type melan-a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan-c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan-b was more sensitive than melan-a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6–4 photoproducts) produced in melan-a than in melan-c cells by UVC, UVB and FS20 lamps. Unlike melan-c, melan-a and melan-b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin-like centers. The contribution of pheome-lanin was higher in melan-b than in melan-a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild-type melan-a melanocytes were well melanized and ellipsoidal, whereas those of melan-b melanocytes tended to be spherical. In the albino line (melan-c) the melanocytes contained only early-stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.     

Molecular responses to stress induced in normal human caucasian melanocytes in culture by exposure to simulated solar UV

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300-400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV-B and 110 kJ/m2 UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2-M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320-400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight-induced genotoxicity and maybe to melanocyte transformation.     

Melanin content of cultured human melanocytes and UV-induced cytotoxicity

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.     

Induction of primary cutaneous melanomas in C3H mice by combined treatment with ultraviolet radiation, ethanol and aloe emodin

The role of ultraviolet (UV) radiation in the induction of nonmelanoma skin cancer is widely accepted, although its precise contribution to the development of primary cutaneous melanoma skin cancer requires further definition. We found that painting aloe emodin, a trihydroxyanthraquinone from Aloe barbadensis, in ethyl alcohol vehicle on the skin of mice in conjunction with exposure to UVB (280-320 nm) radiation results in the development of melanin-containing skin tumors. C3H/HeN mice were treated thrice weekly with aloe emodin in a 25% ethanol in water vehicle and exposed to 15 kJ/m2 UV radiation. Neither ethanol vehicle nor aloe emodin alone induced skin tumors in the absence of UV radiation. In two separate experiments, 20-30% of the mice treated with a combination of UV radiation and ethanol vehicle and 50-67% of the UV-irradiated animals given aloe emodin in ethanol vehicle developed primary cutaneous melanin-containing tumors. The diagnosis of melanoma was established using Fontana silver stain for melanin; these tumors were negative for vimentin and keratin. Melanin-containing melanosomes were observed by transmission electron microscopy in tumors diagnosed as melanomas. Although the mechanism of carcinogenesis in these mice is currently unknown, our findings have led to the development of the first facile murine model for the induction of primary melanoma. This model has the potential to clarify the role of UV radiation in the etiology of malignant melanoma.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

Modulation of Melanogenesis and Antioxidant Status of Melanocytes in Response to Phototoxic Action of Doxycycline

Doxycycline is a commonly used tetracycline antibiotic showing the broad spectrum of antibacterial action. However, the use of this antibiotic is often connected with the risk of phototoxic reactions that lead to various skin disorders. One of the factors influencing the photosensitivity reactions is the melanin content in melanocytes. In this study, the impact of doxycycline and UVA irradiation on cell viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn‐DP) was examined. The exposure of cells to doxycycline and UVA radiation resulted in concentration‐dependent loss in melanocytes viability and induced melanin biosynthesis. Significant changes were stated in cellular antioxidant enzymes activity: SOD, CAT and GPx, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain the mechanisms of phototoxic reactions that occur in normal human epidermal melanocytes in vivo after exposure of skin to doxycycline and UVA radiation.     

In Vivo Optical Properties of Melanocytic Skin Lesions: Common Nevi, Dysplastic Nevi and Malignant Melanoma

We present an in vivo study of the optical properties of common nevi, dysplastic nevi and malignant melanoma skin lesions in human subjects. Reflectance spectra were measured on 1379 skin lesions, in the visible and near-infrared spectral regions, using a spectral imaging system, in a clinical setting. Analysis of the data using a reflectance model revealed differences between the optical properties of melanin present in nevi and melanoma lesions. These differences, which are in agreement with our previous observations on average reflectance spectra, may be potentially useful for the noninvasive characterization of pigmented skin lesions and the early diagnosis of melanoma.     

The Endogenous Tryptophan‐derived Photoproduct 6‐formylindolo[3,2‐b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo

UV‐chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6‐formylindolo[3,2‐b]carbazole (FICZ), a tryptophan‐derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol^?1 cm^?1), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC‐25, HaCaT‐ras II‐4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm^?2) and FICZ (≥10 nm ), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg‐sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 “high‐risk” mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV‐induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.     

In vivo SPECTROPHOTOMETRIC EVALUATION OF NEOPLASTIC AND NON-NEOPLASTIC SKIN PIGMENTED LESIONS–I. REFLECTANCE MEASUREMENTS

Reflectance spectrophotometry from 400 to 800 nm on different cutaneous pigmented lesions, including primary and metastatic malignant melanoma, pigmented nevi, lentigo and seborrhoeic keratosis, has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of the different lesions manifest dissimilar patterns, particularly in the near IR region. Comparison of reflectance of nevi with that of malignant melanomas results in a highly significant difference (P less than 10(-6)) between the two samples. Though interpretation of the spectra remains difficult as a result of the complexity of the optical processes of scattering and absorption, our results suggest that a detailed analysis of the reflectance spectrum may give clinically useful information, and could be utilized as an aid in clinical diagnosis of cutaneous pigmented lesions, especially where malignant melanoma is concerned.     

The Autophagy Receptor Adaptor p62 is Up‐regulated by UVA Radiation in Melanocytes and in Melanoma Cells

UVA (315–400 nm) is the most abundant form of UV radiation in sunlight and indoor tanning beds. However, much remains to be understood about the regulation of the UVA damage response in melanocytes and melanoma. Here, we show that UVA , but not the shorter waveband UVB (280–315 nm), up‐regulates adaptor protein p62 in an Nrf2‐ and reactive oxygen species (ROS )‐dependent manner, suggesting a UVA ‐specific effect on p62 regulation. UVA ‐induced p62 up‐regulation was inhibited by a mitochondria‐targeted antioxidant or Nrf2 knockdown. In addition, p62 knockdown inhibited UVA ‐induced ROS production and Nrf2 up‐regulation. We also report here a novel regulatory feedback loop between p62 and PTEN in melanoma cells. PTEN overexpression reduced p62 protein levels, and p62 knockdown increased PTEN protein levels. As compared with normal human skin, p62 was up‐regulated in human nevus, malignant melanoma and metastatic melanoma. Furthermore, p62 was up‐regulated in melanoma cells relative to normal human epidermal melanocytes, independent of their BRAF or NRAS mutation status. Our results demonstrated that UVA up‐regulates p62 and induces a p62‐Nrf2 positive feedback loop to counteract oxidative stress. Additionally, p62 forms a feedback loop with PTEN in melanoma cells, suggesting p62 functions as an oncogene in UVA ‐associated melanoma development and progression.     

Melanin from epidermal human melanocytes: Study by pyrolytic GC/MS

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation.     

Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.     

Determination of wavelength-specific UV protection factors of sunscreens in intact skin by EPR measurement of UV-induced reactive melanin radical

There remains an unmet need for skin tissue-based assays for the measurement of the UVA protection and efficacy of sunscreens. Here we describe development of a novel electron paramagnetic resonance assay that uses the photogeneration of reactive melanin radical as a measure of UV light penetration to melanocytes in situ in skin. We have used areas of focal melanocytic hyperplasia in the skin of Monodelphis domestica to model the human nevus. We show that we are able to use this assay to determine the monochromatic protection factors (mPF) of research and commercial sunscreens at specific narrow wavebands of UVB, UVA and blue visible light. Both commercial sunscreens, a sun protection factor (SPF) 4 and an SPF 30 product, had mPFs in the UVB range that correlated well with their claimed SPF. However, their mPF in the UVA ranges were only about one-third of claimed SPF. This technique can be used to design and assay sunscreens with optimally balanced UVA and UVB protection.