DNA Damage In Melanocytes Research Articles

Furocoumarins potentiate UVA-induced DNA damage in skin melanocytes

Epidemiological studies have found that high citrus fruit consumption was associated with higher risk of skin cancer. Citrus fruits and some vegetables contain furocoumarins, which may interact with ultraviolet radiation to induce skin cancer. We aimed to determine the effects of two furocoumarins, including 8-methoxypsoralen (8-MOP) and 6′,7′-dihydroxybergamottin (DHB), on UVA-induced DNA damage in human epidermal melanocytes, the origin of melanoma. Our hypothesis was that these dietary furocoumarins increase UVA-induced DNA damage in melanocytes, compared to cells exposed to UV alone. We incubated melanocytes with 8-MOP or DHB, followed by exposure to physiological doses of UVA radiation. We used Western blots to quantify the UVA-induced DNA damage measured by the fraction of phosphorylated histone variant H2AX (γH2AX), which is a marker of DNA damage, relative to total H2AX (γH2AX/H2AX) in the presence or absence of furocoumarins. To quantify the UVA-induced change in γH2AX/H2AX, we calculated the UVA:Control ratio as the ratio of γH2AX/H2AX in UVA-exposed cells to that in cells without UVA (control). The mean UVA:Control ratios were borderline significantly higher for cells treated with 8-MOP and significantly higher for cells treated with DHB, compared to that of untreated cells. This study suggests that furocoumarins (particularly 8-MOP and DHB) enhance UVA-induced DNA damage in melanocytes, which is a potential novel mechanism for citrus and furocoumarins to elevate the risk of skin cancer.

8-methoxypsoralen Potentiates Solar UV-Induced DNA Damage of Skin Melanocytes

ObjectivesThere is little research on how diet may influence skin cancer. 8-methoxypsoralen (8-MOP) is a photocarcinogenic dietary furocoumarin that exists in parsley, parsnip, and celery. Phototherapy that uses 8-MOP and ultraviolet radiation (UVR) exposure to treat skin disorders is known to induce skin cancer. However, the biological mechanism of its carcinogenicity is not understood. We aimed to determine whether 8-MOP affects the extent of UVA-induced DNA damage in human epidermal melanocytes, the origin of melanoma, the most fatal type of skin cancer. MethodsOxidative DNA damage leads to the phosphorylation of H2AX (a member of the histone H2A family), generating γ-H2AX. We used γ-H2AX as a marker for DNA damage in human epidermal melanocytes untreated or treated with 100 μM 8-MOP for 12 hours and exposed or not to 3J/cm2 UVA. Each of the 4 groups were analyzed by western blots using H2AX and γ-H2AX antibodies. We used ImageJ/FIJI software for densitometric analysis to quantify the band intensities. The intensities of γ-H2AX bands were divided by that of H2AX bands (γ-H2AX/total H2AX) to measure the amount of DNA damage. To measure how 8-MOP interacts with UV to affect DNA damage, the UV: Control ratios for untreated and 8-MOP treated cells were calculated as the ratio of γ-H2AX/total H2AX in cells exposed to UV to that of cells without UV. T-tests were performed to calculate the significances of differences between γ-H2AX/total H2AX and between UV: Control ratios of the 4 groups. ResultsThe mean UV: Control ratio for 8-MOP treated cells was borderline significantly higher than the mean UV: Control ratio for untreated cells (p = 0.07), suggesting that 8-MOP interacts with UV to enhance DNA damage. When cells were treated with 8-MOP and exposed to UV, γ-H2AX/total H2AX increased compared to untreated cells exposed to UV, although the increase was not statistically significant (p = 0.20). Conclusions8-MOP potentiates UVA-induced DNA damage in melanocytes, which could be one of the mechanisms how 8-MOP and UV exposure induce skin cancer. Other dietary furocoumarins in fruits and vegetables should be evaluated in terms of the effect on DNA damage. Funding SourcesBrown Cancer Center.

Acetyl zingerone: An efficacious multifunctional ingredient for continued protection against ongoing DNA damage in melanocytes after sun exposure ends

ObjectiveRecent research has shown that significant levels of cyclobutane pyrimidine dimers (CPDs) in DNA continue to form in melanocytes for several hours in the dark after exposure to ultraviolet radiation (UVR) ends. We document the utility of a new multifunctional ingredient, 3‐(4‐hydroxy, 3‐methoxybenzyl)‐pentane‐2,4‐dione (INCI acetyl zingerone (AZ)), to protect melanocytes against CPD formation after UVR exposure ends.MethodsThe use of AZ as an intervention to reduce CPD formation after irradiation was assessed in vitro by comparing kinetic profiles of CPD formation for several hours after irradiation in cells that were untreated or treated with AZ immediately after irradiation. Multifunctional performance of AZ as an antioxidant, quencher and scavenger was established using industry‐standard in vitro chemical assays, and then, its efficacy in a more biological assay was confirmed by its in vitro ability to reduce intracellular levels of reactive oxygen species (ROS) in keratinocytes exposed to UVA radiation. Molecular photostability was assessed in solution during exposure to solar‐simulated UVR and compared with the conventional antioxidant α‐tocopherol.ResultsEven when added immediately after irradiation, AZ significantly inhibited ongoing formation of CPDs in melanocytes after exposure to UVA. Incubation with AZ before irradiation decreased intracellular levels of UVA‐induced ROS formation in keratinocytes. Compared with α‐tocopherol, the molecular structure of AZ endows it with significantly better photostability and efficacy to neutralize free radicals (∙OH, ∙OOH), physically quench singlet oxygen (1O2) and scavenge peroxynitrite (ONOO−).ConclusionThese results designate AZ as a new type of multifunctional ingredient with strong potential to extend photoprotection of traditional sunscreens and daily skincare products over the first few hours after sun exposure ends.

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.

Roles of UVA radiation and DNA damage responses in melanoma pathogenesis.

The growing incidence of melanoma is a serious public health issue that merits a thorough understanding of potential causative risk factors, which includes exposure to ultraviolet radiation (UVR). Though UVR has been classified as a complete carcinogen and has long been recognized for its ability to damage genomic DNA through both direct and indirect means, the precise mechanisms by which the UVA and UVB components of UVR contribute to the pathogenesis of melanoma have not been clearly defined. In this review, we therefore highlight recent studies that have addressed roles for UVA radiation in the generation of DNA damage and in modulating the subsequent cellular responses to DNA damage in melanocytes, which are the cell type that gives rise to melanoma. Recent research suggests that UVA not only contributes to the direct formation of DNA lesions but also impairs the removal of UV photoproducts from genomic DNA through oxidation and damage to DNA repair proteins. Moreover, the melanocyte microenvironment within the epidermis of the skin is also expected to impact melanomagenesis, and we therefore discuss several paracrine signaling pathways that have been shown to impact the DNA damage response in UV-irradiated melanocytes. Lastly, we examine how alterations to the immune microenvironment by UVA-associated DNA damage responses may contribute to melanoma development. Thus, there appear to be multiple avenues by which UVA may elevate the risk of melanoma. Protective strategies against excess exposure to UVA wavelengths of light therefore have the potential to decrease the incidence of melanoma. Environ. Mol. Mutagen. 59:438-460, 2018. © 2018 Wiley Periodicals, Inc.

Melanoma and nonmelanoma skin cancer chemoprevention: Arole for nicotinamide?

Ultraviolet radiation (UVR) causes DNA damage in melanocytes by producing photolesions such as cyclobutane pyrimidine dimers and 8-oxo-7-hydrodeoxyguanosine. The production of reactive oxygen species by UVR also induces inflammatory cytokines that, together with the inherent immunosuppressive properties of UVR, propagate carcinogenesis. Nicotinamide (Vitamin B3 ) enhances DNA repair, modulates the inflammatory environment produced by UVR, and reduces UV-induced immunosuppression. As nicotinamide reduces the incidence of actinic keratoses and nonmelanoma skin cancers in high-risk individuals and enhances repair of DNA damage in melanocytes, it is a promising agent for the chemoprevention of melanoma in high-risk populations.

Ultraviolets A et dommages de l’ADN : leur place dans la cancérogenèse cutanée

Skin cancer is the most common human malignancy, and sunlight exposure is known to play a role in its genesis. Ultraviolet B (UVB) (300-320 nm) has long been considered responsible for the skin damage underlying these cancers, whereas the toxicity of UVA (320-400 nm) has been largely overlooked The intimate mechanisms of photocarcinogenicity remain poorly understood, but UV-induced DNA damage appears to be a major initiating event. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts (6-4PPs) are the main dimeric lesions induced by UVB, whereas the genotoxic effects of UVA have long been attributed to oxidative damage, the main lesion being the oxidized base 8-oxo-7,8dihydroguanine (8-oxoGua). However; powerful new techniques for analyzing DNA damage (the Comet assay, and especially HPLC-MSIMS) have demonstrated that UVA irradiation mainly triggers the formation of CPDs, especially CPD-TT both in cell models and in total human skin. A direct photochemical process is currently thought to account for CPD induction by UVA. The multilayer structure of the epidermis protects against UVB-induced dipyrimidine lesions in total skin but offers only weak protection against UVA. In addition, repair efficiency is undermined by UVA. CPDs, the main DNA lesions induced by UVA in total skin (which is more permeable to UVA), are inefficiently repaired CPDs have strong mutagenic potential, and recent studies clearly show that CPDs, rather than 8-Oxo-Gua, are the main mutagenic photoproducts induced by UVA. The UV signature of induced mutations is characterized by transitions from C to T or CC to TT in dipyrimidine sequences. These mutations target the p53, patched 1 and SMO genes in carcinomas, and the PTEN RAC1, PPP6C, STK19 and PPP6C genes in melanomas of exposed skin. UVA also mainly induces CPDs in melanocytes, in amounts similar to those observed in keratinocytes, demonstrating that melanin does not prevent CPD formation. In contrast, UVA induces far more abundant 8-oxo-Gua production in melanocytes than in keratinocytes. Thus, under UVA irradiation, oxidative stress contributes more to DNA damage in melanocytes than in keratinocytes. In addition, baseline oxidative damage (in the absence of UVA) is already higher in melanocytes. The photosensitizer may be melanin itself. This is supported by a recent study based on a murine model, in which melanoma induction was shown to require both UVA and the presence of melanin in melanocytes, and is associated with oxidative damage to DNA. Conversely, UVB was found to initiate melanoma through a direct, pigment-independent pathway. Thus, two wavelength-dependent pathways can induce melanoma, with melanin playing an unexpected role. Constitutive pigmentation is very effective in preventing UV-induced damage, and a clear correlation can thus be found between, on the one hand, the amount of CPD TT produced by both UVB and UVA and, on the other hand, the minimum erythematous dose and the phototype. Melanin is thus a two-facetted molecule, protecting the skin when its synthesis is complete and when melanosomes take on their nucleus-protective geometric configuration in keratinocytes, but having a pro-oxidant action when only partially polymerized and exposed to UV Repeated exposure of volunteer skin shows that a tan induced by UVB provides little protection against DNA damage caused by subsequent exposure, while tanning with UVA provides no protection at all. Yet both UVB and UVA provoke DNA damage. All these recent data highlight the potential role of UVA in skin carcinogenesis, and reinforce epidemiological studies showing an increased risk of melanoma among users of tanning lamps, particularly young women. The decision by the International Agency for Research on Cancer to classify UVA and tanning devices as group 1 carcinogens, and the opinion issued by the French National Academy of Medicine on tanning booths, therefore appear to be fully justified. The use of tanning salons should be permanently banned

Melanocytes and keratinocytes have distinct and shared responses to ultraviolet radiation and arsenic

The rise of melanoma incidence in the United States is a growing public health concern. A limited number of epidemiology studies suggest an association between arsenic levels and melanoma risk. Arsenic acts as a co-carcinogen with ultraviolet radiation (UVR) for the development of squamous cell carcinoma and proposed mechanisms include generation of oxidative stress by arsenic and UVR and inhibition of UVR-induced DNA repair by arsenic. In this study, we investigate similarities and differences in response to arsenic and UVR in keratinocytes and melanocytes. Normal melanocytes are markedly more resistant to UVR-induced cytotoxicity than normal keratinocytes, but both cell types are equally sensitive to arsenite. Melanocytes were more resistant to arsenite and UVR stimulation of superoxide production than keratinocytes, but the concentration of arsenite necessary to inhibit the activity of the DNA repair protein poly(ADP-ribose)polymerase and enhance retention of UVR-induced DNA damage was essentially equivalent in both cell types. These findings suggest that although melanocytes are less sensitive than keratinocytes to initial UVR-mediated DNA damage, both of these important target cells in the skin share a mechanism related to arsenic inhibition of DNA repair. These findings suggest that concurrent chronic arsenic exposure could promote retention of unrepaired DNA damage in melanocytes and act as a co-carcinogen in melanoma.

The role of keratinocyte‐derived α‐melanocyte‐stimulating hormone and endothelin 1 signaling in a murine melanoma‐susceptible model

Nuclear receptor RXRα is a member of steroid hormone retinoid‐X‐receptor family. Mice with epidermal deletion of RXRα (RXRαep−/−) develop melanoma‐like tumors upon chemical carcinogen treatment. This suggests that distinct non‐cell autonomous molecular events mediated by RXRα in keratinocytes may regulate melanoma progression.Melanocyte homeostasis in skin is largely influenced by signals from neighboring keratinocytes, autocrine signals, and environmental factors, such as ultraviolet radiation (UVR). UVR affects melanocytes directly by damaging the DNA and indirectly by stimulating the synthesis of keratinocyte‐derived factors, such as endothelin 1 (EDN1) and α‐melanocyte stimulating hormone (α‐MSH).Pharmacological inhibition of EDN1 signaling enhances UV‐induced DNA damage in melanocytes, but it does not affect the proliferation of keratinocytes, melanocytes, and dermal cells in control (RXRαL2/L2) and RXRαep−/− mice. In contrast, inhibition of α‐MSH signaling reduces UV‐induced DNA damage in all skin cell types. Moreover, it also leads to minimal increase in proliferation of dermal cells, but not of keratinocytes and melanocytes in the skin of RXRαL2/L2 and RXRαep−/− mice.This research will help to evaluate strategies for preventing and/or curing UV‐induced melanoma by using a combination of antagonists and/or agonists of keratinocyte‐derived factors.Grant Funding Source: NIEHS grant R01ES016629 (to AI)

The specificity of UVA-induced DNA damage in human melanocytes

Exposure to solar UV radiation is the origin of most skin cancers, including deadly melanomas. Melanomas are quite different from keratinocyte-derived tumours and exhibit a different mutation spectrum in the activated oncogenes, possibly arising from a different class of DNA damage. In addition, some data suggest a role for UVA radiation in melanomagenesis. To get further insight into the molecular mechanisms underlying induction of melanoma, we quantified a series of UV-induced DNA damage in primary cultures of normal human melanocytes. The results were compared with those obtained in keratinocytes from the same donors. In the UVB range, the frequency and the distribution of pyrimidine dimers was the same in melanocytes and keratinocytes. UVA was also found to produce thymine cyclobutane dimer as the major DNA lesion with an equal efficiency in both cell types. In contrast, following UVA-irradiation a large difference was found for the yield of 8-oxo-7,8-dihydroguanine; the level of this product was 2.2-fold higher in melanocytes than in keratinocytes. The comet assay showed that the induction of strand breaks was equally efficient in both cell types but that the yield of Fpg-sensitive sites was larger in melanocytes. Our data show that, upon UVA irradiation, oxidative lesions contribute to a larger extent to DNA damage in melanocytes than in keratinocytes. We also observed that the basal level of oxidative lesions was higher in the melanocytes, in agreement with a higher oxidative stress that may be due to the production of melanin. The bulk of these results, combined with qPCR and cell survival data, may explain some of the differences in mutation spectrum and target genes between melanomas and carcinomas arising from keratinocytes.

The eyes have it

In his book, ‘The Astonishing Hypothesis’, Francis Crick describes a simple experiment to demonstrate that while both eyes see, the brain interprets information from only one eye at a time. A small mirror is used to direct the gaze of one eye to an uninteresting surface such as a blank wall, while the other eye focuses on a face ahead of the subject. The brain now sees the face, not the wall. By moving a hand transiently across the field of vision of the misdirected eye, the brain’s attention is redirected by the movement towards the wall. The result is that the subject sees a composite image, frequently just a pair of eyes staring out from a blank wall. The effect is very striking, but it also draws attention to the brain’s priorities: the face is dominant over the wall, and movement is dominant over the face. But the fact that the eyes remain even though the rest of the face is erased by the movement across the field of vision, suggests that eyes hold a special place in the brain’s attention. It is perhaps surprising therefore that the genetics of eye pigmentation has attracted less attention than that of skin or hair. In this issue, Rick Sturm and Mats Larsson redress the balance by reviewing the genetic basis for the variations in iris pigmentation. In particular, they highlight an SNP in an intron of a gene upstream from OCA2 that may account for much of the blue/brown eye colour variation by affecting the regulation of OCA2 expression. Eye colour variations reflect the genetic variation affecting genes implicated in the manufacture of the pigment melanin in melanosomes, specialised lysosome-related organelles. Over the years significant progress has been made in understanding both the genetics and the chemistry behind melanin production, yet many key features of melanin remain to be understood, not least the structure of the polymer. In fact, even using the term melanin is a simplification as birds and mammals make two chemically distinct types of melanin, the black/brown eumelanin and the yellow/reddish brown pheomelanin. The review from Sho Ito and John Simon covers the process of ‘mixed’ melanogenesis, reflecting natural systems in which a combination of pheo- and eumelanin is produced and highlights several outstanding questions relating the presence of combinations of different pigment types within the melanosome. One major role for melanosomes in humans is in photo-protection where pigment transferred from melanocytes to keratinocytes affords protection against UV-induced DNA damage. How this is achieved and regulated is an important issue given the alarming increase in incidence of melanoma. One area that is attracting increasing attention is the role of MC1R in photo protection. This receptor signals primarily via the cAMP pathway and is activated in skin in response to keratinocyte-derived MSH. Increased MC1R signalling in melanocytes leads to elevated pigmentation and transcription of genes implicated in melanin synthesis and consequently contributes to the tanning response. Evidence is also accumulating (see Song et al in the next issue of PCMR for example), that MC1R signalling also facilitates repair of DNA damage. One long-time ambition is to induce a natural tanning response and photoprotection without exposure to UV irradiation, and to this end Zalfa Abdel-Malek et al report the use of short MSH-related peptides that can stimulate the MC1R receptor and reduce UV-induced DNA damage in melanocytes. In fact the use of MSH-related compounds for therapeutic purposes is already bringing clinical benefits to patients with specific photo-sensitivities in the form of Alfamelanotide. However, the dangers of unregulated use by the public of compounds such as ‘Melanotan’ to achieve tanning without UV exposure is highlighted in the letter from Veronique de Marmol et al.

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.

Comparison of Melanocytes and Keratinocytes in Ultraviolet-Induced DNA Damage per Minimum Erythema Dose Sunlight: Applicability of Ultraviolet Action Spectra for Risk Estimates

Action spectra are being used in risk estimates for ultraviolet (UV) damage. The purpose of our investigation was to compare the susceptibility of cultured melanocytes and keratinocytes to UV-induced DNA damage per minimum erythema dose (MED) and to determine whether the predictions made with action spectra agree with the damage actually induced. Genetic damage was measured as the number of T4-endonuclease V-sensitive sites (ESS). Predictions made with the action spectrum for the induction of DNA damage in melanocytes after irradiation with sunlight and a solar simulator were 15.9 and 13.2 ESS per 10(8) daltons per MED, respectively; with the action spectrum for the induction of DNA damage in keratinocytes the predictions were 12.1 and 9.8 ESS per 10(8) daltons per MED, respectively. To determine the actual damage per MED, cultured cells were irradiated with sunlight or a solar simulator, and MED was determined with an erythema UV meter. The induction of DNA damage in melanocytes after sunlight and solar simulator irradiation was 8.01 and 6.7 ESS per 10(8) daltons per MED, respectively, and in keratinocytes 7.49 and 7.12 ESS per 10(8) daltons per MED, respectively. This was considered to be in agreement with the predicted data. The use of action spectra for risk estimates in melanocytes appears justified.