UV-generated free radicals (FR) in skin: their prevention by sunscreens and their induction by self-tanning agents

In the past few years, the cellular effects of ultraviolet (UV) irradiation induced in skin have become increasingly recognized. Indeed, it is now well known that UV irradiation induces structural and cellular changes in all the compartments of skin tissue. The generation of reactive oxygen species (ROS) is the first and immediate consequence of UV exposure and therefore the quantitative determination of free radical reactions in the skin during UV radiation is of primary importance for the understanding of dermatological photodamage. The RSF method (radical sun protection factor) herein presented, based on electron spin resonance spectroscopy (ESR), enables the measurement of free radical reactions in skin biopsies directly during UV radiation. The amount of free radicals varies with UV doses and can be standardized by varying UV irradiance or exposure time. The RSF method allows the determination of the protective effect of UV filters and sunscreens as well as the radical induction capacity of self-tanning agents as dihydroxyacetone (DHA). The reaction of the reducing sugars used in self-tanning products and amino acids in the skin layer (Maillard reaction) leads to the formation of Amadori products that generate free radicals during UV irradiation. Using the RSF method three different self-tanning agents were analyzed and it was found, that in DHA-treated skin more than 180% additional radicals were generated during sun exposure with respect to untreated skin. For this reason the exposure duration in the sun must be shortened when self-tanners are used and photoaging processes are accelerated.     

Changes in ultraviolet absorbance and hence in protective efficacy against lipid peroxidation of organic sunscreens after UVA irradiation

Owing to the spectral distribution of solar UV, the UVA component of sunlight is now believed to be the main cause of photoaging and photocarcinogenesis and is much more effective than UVB in inducing peroxidative damage. Consequently, most skin care cosmetic products now include UVA filters in their formulations along with UVB filters. These modern sunscreens should provide and maintain their initial absorbance, hence protection, throughout the entire period of exposure to sunlight. However, not all UVA and UVB filters are sufficiently photostable. In this study, we examine the correlation between the photochemical degradation of sunscreen agents under UVA irradiation, with particular reference to the UVA-absorber 4-tert-butyl-4'-methoxydibenzoylmethane, alone and in combination with other organic UV filters (2-ethylhexyl 4 methoxycinnamate and 2-ethylhexyl 2-cyano-3,3-diphenylacrylate) and their ability to prevent UVA-induced lipid peroxidation. Since antioxidants are also added to formulations to deactivate free radicals generated during UVA exposure, vitamin E and the synthetic antioxidant, bis(2,2,6,6-tetramethyl-1-oxyl-piperidine-4-yl)sebacate, a nitroxide derivative, were also included in this study. By using simple in vitro tests, the results show that a decrease in spectral absorbance of the UV filters correlates in most cases with increased UVA-induced lipid peroxidation; this depends on the specific UV absorber analysed and also on whether they are alone or in combination. Furthermore, the combined presence or absence of antioxidants has a profound effect on this oxidative event. In particular, the nitroxide appears to be a more efficient photo-antioxidant than vitamin E. Similar experiments were also performed under natural sunlight and the results obtained did not differ substantially from those performed under UVA. The results presented and discussed in this work may help in understanding the effects of UVA/UVB absorbers and antioxidants upon the level of UV-induced ROS generated under UVA exposure and in natural sunlight which could be relevant for improving the photoprotection and efficacy of skin care cosmetic formulations.     

The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair

Throughout the body, melanin is a homogenous biological polymer containing a population of intrinsic, semiquinone-like radicals. Additional extrinsic free radicals are reversibly photo-generated by UV and visible light. Melanin photochemistry, particularly the formation and decay of extrinsic radicals, has been the subject of numerous electron spin resonance (ESR) spectroscopy studies. Several melanin monomers exist, and the predominant monomer in a melanin polymer depends on its location within an organism. In skin and hair, melanin differs in content of eumelanin or pheomelanin. Its bioradical character and its susceptibility to UV irradiation makes melanin an excellent indicator for UV-related processes in both skin and hair. The existence of melanin in skin is strongly correlated with the prevention against free radicals/ROS generated by UV radiation. Especially in the skin melanin (mainly eumelanin) ensures the only natural UV protection by eliminating the generated free radicals/ROS. Melanin in hair can be used as a free radical detector for evaluating the efficacy of hair care products. The aim of this study was to investigate the suitability of melanin as protector of skin against UV generated free radicals and as free radical indicator in hair.     

Radical protection by sunscreens in the infrared spectral range

One essential reason for skin ageing is the formation of free radicals by excessive or unprotected sun exposure. Recently, free radical generation in skin has been shown to appear not only after irradiation in the UV wavelength range but also in the infrared (IR) spectral range. Sunscreens are known to protect against radicals generated by UV radiation; however, no data exist for those generated by IR radiation. This paper has investigated four different, commercially available sunscreens and one COLIPA standard with regard to radical formation in the skin after IR irradiation, using electron paramagnetic resonance spectroscopy. The use of sunscreens has led to reduced amounts of radicals compared to untreated skin. Furthermore, absorption and scattering properties and the radical protection factor of the formulations were determined to investigate their influence on the radical protection of the skin. None of these formulations contained an optical absorber in the IR range. The protection efficiency of the sunscreens was shown as being induced by the high scattering properties of the sunscreens, as well as the antioxidants contained in the formulations.     

Photostabilization of butyl methoxydibenzoylmethane (Avobenzone) and ethylhexyl methoxycinnamate by bis-ethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S), a new UV broadband filter

It is now well documented that chronic UVA exposure induces damage to human skin. Therefore, modern sunscreens should not only provide protection from both UVB and UVA radiation but also maintain this protection during the entire period of exposure to the sun. UVA filters, however, are rare and not sufficiently photostable. We investigated the effect of the introduction of a new UV filter, bis-ethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S), in oil in water sunscreen formulations on the photostability of butyl methoxydibenzoylmethane (Avobenzone [AVB]) after irradiation with an optically filtered Xenon arc source (UV irradiance adjusted at 1 mean effective dose [MED]/min). With spectrophotometrical methods to assess the sun protection factor (SPF) and UVA ratio and chromatographical methods to determine the amount of UV filters recovered after irradiation we showed that Tinosorb S prevented the photodegradation of AVB in a concentration-dependent way, leading to a sustained SPF and UVA ratio even after irradiation with doses of up to 30 MED. Since AVB was shown to destabilize ethylhexyl methoxycinnamate (EHM) we tested the effect of Tinosorb S in sunscreens containing this UV filter combination. Here too Tinosorb S showed photoprotective properties toward both UV filters. Thus, Tinosorb S can be used successfully to improve the photostability and efficiency of sunscreens containing AVB and EHM.     

Human Hair as a Natural Sun Protection Agent: A Quantitative Study

The rising incidence of skin cancers attributable to excessive sun exposure has become a major health concern worldwide. While numerous studies have analyzed the sun protective effect of sunscreens, clothing and antioxidants, none to date have measured the photoprotective effect of hair, despite clinical evidence that individuals with balding or thinning hair are at greater risk of skin lesions that can progress to cancer, hence the recommendation to use hats or umbrellas. We analyzed the level of protection offered by hair according to hair density, thickness and color using the spectral transmittance and corrected for relative erythema effectiveness. Our results show that hair provides a barrier against both UVB and UVA radiation which is significantly increased with respect to the hair density, thickness and the presence of melanins. This is the first study to quantify sun protection factor offered by hair, namely hair ultraviolet protection factor (HUPF). We believe that hair should be recognized as an important natural sun barrier in the prevention of UV‐induced skin cancers.     

Evaluation of the protective effect of sunscreens on in vitro reconstructed human skin exposed to UVB or UVA irradiation

We have previously shown that skin reconstructed in vitro is a useful model to study the effects of UVB and UVA exposure. Wavelength-specific biological damage has been identified such as the formation of sunburn cells (SBC) and pyrimidine dimers after UVB irradiation and alterations of dermal fibroblasts after UVA exposure. These specific effects were selected to evaluate the protection afforded by two sunscreens after topical application on the skin surface. Simplified formulations having different absorption spectra but similar sun protection factors were used. One contained a classical UVB absorber, 2-ethylhexyl-p-methoxycinnamate. The other contained a broad-spectrum absorber called Mexoryl SX, characterized by its strong absorbing potency in the UVA range. Both filters were used at 5% in a simple water/oil vehicle. The evaluation of photoprotection on in vitro reconstructed skin revealed good efficiency for both preparations in preventing UVB-induced damage, as shown by SBC counting and pyrimidine dimer immunostaining. By contrast, only the Mexoryl SX-containing preparation was able to efficiently prevent UVA-specific damage such as dermal fibroblast disappearance. Our data further support the fact that skin reconstructed in vitro is a reliable system to evaluate the photoprotection provided by different sunscreens against specific UVB and UVA biological damage.     

UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.     

Photostability of UV Absorber Systems in Sunscreens†

Sunscreens are used to protect the human skin against harmful UV radiation. Today there is a trend toward higher sun protection factors (SPF) and better UVA protection. Methods for the assessment of SPF and UVA protection involve irradiation of the product, and the photostability properties of the sunscreen have an influence on its performance. Sunscreens often contain more than one UV filter. Thus it is important to understand the photostability properties of the complete system. The filter combinations used may exhibit destabilizing, stabilizing or inert interactions. For that reason, besides assessment of the properties of the single filters, photostabilities of binary filter combinations are investigated. Destabilization occurs when two UV absorbers undergo a chemical reaction after absorption of UV radiation. Stabilization may be achieved when the optical density of the system is very high, giving rise to a self‐protection effect of the sunscreen film. Photounstable UV absorbers may be additionally stabilized by employing triplet quenchers. Being aware of these mechanisms and applying them for specific UV filter combinations can help in designing efficient sunscreens.     

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.     

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.     

Measurements of the Protective Effect of Topically Applied Sunscreens using in vitro Three-dimensional Dermal and Skin Equivalents

Abstract— For preventing or minimizing acute and chronic skin damage caused by UV radiation, the use of sunscreens is probably the most important measure. To screen the protective efficacy of new sunscreen molecules or formulations against UV rays, we evaluated as in vitro testing methods the use of two three-dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen-glycosaminoglycans-chitosan matrix populated by normal human fibroblasts. The SE is comprised of a fully differentiated epidermis realized by seeding keratinocytes onto the DE. In this study, we demonstrated that the DE and SE models react to the deleterious effects of UVA and UVB. Then, we extended our research to the evaluation of their usefulness for photoprotection trials. Sunscreen agents (Euso-lex 8020 and 6300) and commercially available sunscreens (chemical and physical filter formulations) that protect the skin against either UVA or UVB were evaluated. The tested products were applied (n = 6) topically (10 μL) and incubated for 30 min prior to irradiation over a range of UVA (0-50 J/cm²) or UVB (0-5 J/cm²). The photoprotection provided by the tested sunscreen molecules and formulations was evaluated by measurement of residual cellular viability 24 h postirradiation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) test and assessment of the inflammation response by interleukin-la release assay. When sunscreens were applied prior to UV exposure, a higher residual cellular viability versus control was obtained, demonstrating the photoprotective effects of the tested products. These in vitro models could be used for screening tests to evaluate the protective effects of sunscreen molecules and formulations, especially for UVA trials because there is a lack of consensus for an in vivo method.     

Hydrochlorothiazide Enhances UVA‐Induced DNA Damage

The UVA is currently thought to be carcinogenic because, similar to UVB, it induces the formation of cyclobutane pyrimidine dimers (CPDs). Various drugs have been reported to cause photosensitive drug eruptions as an adverse effect. Although the precise mechanism of photosensitive drug eruption remains to be elucidated, it is generally accepted that free radicals and other reactive molecules generated via UV‐irradiated drugs play important roles in the pathogenesis of photosensitive drug eruptions. The waveband of concern for photo‐reactive drugs is UVA‐visible light, but some extend into the UVB region. We tested whether photosensitive drugs could enhance CPD formation after UVA exposure by using isolated DNA in the presence of several reported photosensitive drugs using high‐performance liquid chromatography. We found that the diuretic agent hydrochlorothiazide (HCT) significantly enhanced the production of TT dimers over a wide range of UVA. Furthermore, we investigated whether UVA plus HCT could enhance CPD production in xeroderma pigmentosum model mice defective in nucleotide excision repair. Immunofluorescence studies showed that CPD formation in the skin significantly increased after 365 nm narrow‐band UVA irradiation in the presence of HCT, compared with that in wild‐type mice. HCT could be used with caution because of its enhancement of UVA‐induced DNA damage.     

Near-ultraviolet photolysis of beta-phenylpyruvic acid generates free radicals and results in DNA damage

Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.     

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.     

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.     

Radiation sources providing increased UVA/UVB ratios induce photoprotection dependent on the UVA dose in hairless mice

In studies involving mice in which doses of UVA (320-400 nm) and UVB (290-320 nm) radiation were administered alone or combined sequentially, we observed a protective effect of UVA against UVB-induced erythema/edema and systemic suppression of contact hypersensitivity. The UVA immunoprotection was mediated by the induction of the stress enzyme heme oxygenase-1 (HO-1) in the skin, protection of the cutaneous Th1 cytokines interferon-gamma (IFN-gamma) and IL-12 and inhibition of the UVB-induced expression of the Th2 cytokine IL-10. In this study, we seek evidence for an immunological waveband interaction when UVA and UVB are administered concurrently to hairless mice as occurs during sunlight exposure in humans. A series of spectra providing varying ratios of UVA/UVB were developed, with the UVA ratio increased to approximately 3.5 times the UVA component in solar simulated UV (SSUV). We report that progressively increasing the UVA component of the radiation while maintaining a constant UVB dose resulted in a reduction of both the erythema/edema reaction and the degree of systemic immunosuppression, as measured as contact hypersensitivity. The UVA-enhanced immunoprotection was abrogated in mice treated with a specific HO enzyme inhibitor. UVA-enhanced radiation also upregulated the expression of cutaneous IFN-gamma and IL-12 and inhibited expression of both IL-6 and IL-10, compared with the activity of SSUV. The results were consistent with the previously characterized mechanisms of photoprotection by the UVA waveband alone and suggest that the UVA component of solar UV may have beneficial properties for humans.     

Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice

The UVB (280-315 nm)- and UVA1 (340-400 nm)-induced migration of Langerhans cells (LC) from the epidermis and accumulation of dendritic cells (DC) in the lymph nodes draining the exposed skin site of C3H/HeN mice have been investigated. One minimum erythemal dose (MED) of UVB (1.5 kJ/m2) and of UVA1 (500 kJ/m2) were chosen, which have been shown previously to suppress delayed hypersensitivity (DTH). UVB irradiation resulted in a reduction in epidermal LC numbers, local to the site of the exposure, which was most apparent 12 h after exposure, but, in contrast, UVA1 had no significant effect even at 72 h after exposure. UVA1 did not exert any protection against the UVB-mediated depletion in LC numbers. The reduction in local LC following UVB exposure was prevented by systemic (intraperitoneal) treatment of mice with neutralising antibodies to either tumor necrosis factor (TNF)-alpha or interleukin (IL)-beta 2 h prior to the irradiation. It has been reported previously that UVB exposure caused an increase in the number of dendritic cells (DC) in the lymph nodes draining the irradiated skin site. In the present study we have shown that UVA1 had a similar effect. Pretreatment of the mice with neutralising antibodies to IL-1beta (by intraperitoneal injection) substantially inhibited DC accumulation induced by both UV regimens. However, anti-TNF-alpha antibodies affected only the UVB-induced increase, and did not alter the elevation in DC numbers observed following UVA1 exposure. These results indicate that UVB causes the migration of LC from the epidermis and an accumulation of DC in the draining lymph nodes by a mechanism that requires both TNF-alpha and IL-1beta. In contrast, UVAI does not cause LC migration from the epidermis and the accumulation of DC in the draining lymph nodes observed following UVA1 exposure requires IL-1beta, but not TNF-alpha. It is likely therefore that UVA1 acts through a different mechanism from UVB and may target a cutaneous antigen presenting cell other than LC, such as the dermal DC.     

Assessment of the photoprotection properties of sunscreens by chromatographic measurement of DNA damage in skin explants

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens.     

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non-radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB-induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.