Ultraviolet Spectral Energy Differences Affect the Ability of Sunscreen Lotions to Prevent Ultraviolet-Radiation-Induced Immunosuppression*

Acute exposure to UV radiation causes immunosuppression of contact hypersensitivity (CH) responses. Past studies conducted with unfiltered sunlamps emitting nonsolar spectrum UV power (wavelengths below 295 nm) or using excessive UV doses have suggested sunscreens may not prevent UV-induced immunosuppression in mice. This study was thus designed to evaluate critically the effects of different UV energy spectra on the immune protection capacity of sunscreen lotions. Minimum immune suppression doses (MISD), i.e. the lowest UV dose to cause~50% suppression of the CH response to dinitrofluorobenzene in C3H mice, were established for three artificial UV sources. The MISD for each UV source was 0.25 kJ/m² for unfiltered FS20 sunlamps (FS), 0.90 kJ/m² for Kodacel-filtered FS20 sunlamps (KFS), which do not emit UV power at wavelengths <290 nm, and 1.35 kJ/m² for a 1000 W filtered xenon arc lamp solar simulator. Using MISD as baseline, sunscreens with labeled sun protection factors (SPF) of 4, 8, 15 and 30 were tested with each UV source to establish their relative immune protection factors. The immune protection factor of each sunscreen exceeded its labeled SPF in tests conducted with the solar simulator, which has a UV power spectrum (295–400 nm) similar to that of sunlight. Conversely, sunscreen immune protection factors were significantly less than the labeled SPF in tests conducted with FS and KFS. Comparison of the immunosuppression effectiveness spectra showed that relatively small amounts of nonsolar spectrum UV energy, i.e. UVC (200–290 nm) and/or shorter wavelength UVB (between 290 and 295 nm), produced by FS and KFS contributes significantly to the induction of immunosuppression. For example, 36.3% and 3.5% of the total immunosuppressive UV energy from FS and KFS, respectively, lies below 295 nm. Sunscreen absorption spectra showed that transmission of immunosuppressive UV energy below 295 nm for FS was at least eight-fold higher than that for KFS. Compared to the solar simulator UV spectrum the transmission of nonsolar immunosuppressive UV energy through sunscreens was >15-fold higher for FS and ≥1.5-fold higher for KFS. These data demonstrate that relevant evaluations of sunscreen immune protection can only be obtained when tests are conducted with UV sources that produce UV power spectra similar to that of sunlight and UV doses are employed that are based on established MISD.     

Isoflavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation

Isoflavones derived from many edible plants have been reported to possess significant antioxidant, estrogenic and tyrosine kinase inhibitory activity. Genistein has been found previously to provide protection from oxidative damage induced by UV radiation both in vitro and following dietary administration. We have therefore examined the potential of a number of isoflavones from red clover (Trifolium pratense) and some metabolically related compounds to offer protection from UV irradiation in hairless mice by topical application after UV exposure. We show that whereas the primary isoflavones, daidzein, biochanin A and formononetin, were inactive, 20 microM lotions of genistein and the metabolites equol, isoequol and the related derivative dehydroequol had powerful potential to reduce the inflammatory edema reaction and the suppression of contact hypersensitivity induced by moderate doses of solar-simulated UV radiation. For equol the protection was concentration dependent and 5 microM equol markedly reduced the UV-induced inflammation but abrogated the UV-induced immunosuppression. Equol protected similarly from immunosuppression induced by the putative epidermal mediator, cis-urocanic acid (UCA), indicating a potential mechanism of action involving inactivation of this UV-photoproduct. Since immunosuppression induced by both UV radiation and by cis-UCA appears to be an oxidant-dependent response our observations support the actions of these topically applied isoflavones and their metabolites as antioxidants. They also indicate that lotions containing equol, unlike topical UV sunscreens, more readily protect the immune system from photosuppression than from the inflammation of the sunburn reaction, even when applied after exposure, and thus such compounds may have a future role as sun-protective cosmetic ingredients.     

p53 tumor suppressor gene: a critical molecular target for UV induction and prevention of skin cancer

The relationship between exposure to UV radiation and development of skin cancer has been well established. Several studies have shown that UVB induces unique mutations (C-->T and CC-->TT transitions) in the p53 tumor suppressor gene that are not commonly induced by other carcinogens. Our studies have demonstrated that UV-induced mouse skin cancers contain p53 mutations at a high frequency and that these mutations can be detected in UV-irradiated mouse skin well before the appearance of skin tumors. This observation suggested that it might be possible to use p53 mutations as a biologic endpoint for testing the efficacy of sunscreens in photoprotection studies. Indeed, application of SPF 15 sunscreens to mouse skin before each UVB irradiation resulted in reduction in the number of p53 mutations. Because p53 mutations represent an early essential step in photocarcinogenesis, these results imply that inhibition of this event may protect against skin cancer development. This hypothesis was confirmed by our finding that sunscreens used in p53 mutation inhibition experiments also protected mice against UVB-induced skin cancer.     

EFFECT OF DIETARY LIPID ON UV LIGHT CARCINOGENESIS IN THE HAIRLESS MOUSE

Abstract— Isocaloric feeding of diets varying in lipid content to albino hairless mice has shown that their susceptibility to skin tumorigenesis induced by simulated solar UV light was not affected by the level of polyunsaturated fat, 5% or 20%. However a qualitative effect of dietary lipid was demonstrated. Mice fed 20% saturated fat were almost completely protected from UV tumorigenesis when compared with mice fed 20% polyunsaturated fat. Multiple latent tumours were detected in the saturated fat-fed mice by subsequent dietary replenishment, suggesting that a requirement for dietary unsaturated fat exists for the promotion stage of UV-induced skin carcinogenesis.     

Interaction of UVB-absorbing sunscreen ingredients with cutaneous molecules may alter photoimmune protection.

Studies of the photoimmunoprotective properties of sunscreens have produced disparate results. In this study in hairless mice, we compared two UVB absorbers, 2-ethylhexyl-p-methoxycinnamate (2-EHMC) and octyl-N-dimethyl-p-aminobenzoate (o-PABA), individually formulated in a common base lotion with a sunburn protection factor of 6. We measured their capacity to protect against suppression of the contact hypersensitivity (CHS) induced by three daily exposures of the dorsum to 6x the minimal erythemal/edematous dose (MED) of solar-simulated UV radiation (SSUV), in comparison with base lotion-treated mice exposed to 3 x 1 MED of SSUV. All treatments produced a similar minimal erythema. CHS was equally suppressed in mice irradiated through o-PABA and base lotion, but the suppression was significantly reduced in mice irradiated through 2-EHMC. Neither UVB absorber inhibited the epidermal photoisomerization to the immunosuppressive mediator, cis-urocanic acid. However, when mice were treated with exogenous cis-urocanic acid topically on the dorsum, but not when injected subcutaneously on the abdomen, suppression of CHS was observed in o-PABA- and base lotion-treated mice, but not in 2-EHMC-treated mice. Thus, the enhanced immunoprotection in mice irradiated through 2-EHMC apparently resulted from the direct inactivation of epidermal cis-urocanic acid by 2-EHMC. We conclude that comparative assessment of photoimmunoprotection by UV absorbers requires SSUV, erythemally matched exposures and consideration of potential interactions with cutaneous molecules.     

Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced by UV radiation alone or with a chemical cocarcinogen

Isoflavones derived from many edible plants, such as genistein from the soybean, have well-documented antioxidant and estrogenic activity but may also be anticarcinogenic. In this study, we examined the potential of the isoflavone equol [(S)-4',7-dihydroxyisoflavane] to protect from skin carcinogenesis in the hairless mouse. Daily topical applications of equol lotions significantly protected against skin carcinogenesis induced by chronic exposure to solar-simulated UV radiation (SSUV) or by topical treatment with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or by the combined cocarcinogenic treatment of DMBA followed by chronic SSUV. Monitoring of tumor development for 40 weeks showed significantly delayed tumor appearance and reduced tumor multiplicity in all equol-treated groups. In mice treated with either SSUV or DMBA + SSUV, equol significantly reduced the proportion of tumors progressing from benign papillomas to malignant squamous cell carcinoma (SCC) by 33-58% and reduced the average diameter of SCC by 71-82%. In a short-term study, equol dose dependently inhibited the SSUV induction of the tumor promotion biomarker enzyme, ornithine decarboxylase, in the skin, suggesting the anticarcinogenic activity of equol may be attributed to its inhibition of the tumor promotion phase of carcinogenesis.     

Alternative methods to evaluate the protective ability of sunscreen against photo-genotoxicity

Numerous epidemiological investigations show that sunlight is carcinogenic to humans and that the use of sunscreen may be effective in decreasing the risk of skin cancer. The biological activity of a sunscreen is evaluated by its ability to protect human skin from erythema as represented by a Sun Protection Factor (SPF). We propose that the sunscreen's protective effect against sunlight-induced genotoxicity, including mutation, should also be taken into account. In this study we examined the protective ability of sunscreens against natural sunlight and UV-induced genotoxicity in Drosophila somatic cells. We prepared three kinds of sunscreen samples, each with an SPF value of 20, 40 or 60 and compared their protective activities with commercial sunscreens. When a sunscreen of SPF 20, 40 or 60 was pasted on the plastic cover of a petri dish in which Drosophila larvae were exposed to the sun or UV lamps, genotoxicity decreased as the SPF of the sunscreen increased, relative to levels of genotoxicity observed in samples without sunscreen. However, the protective abilities of sunscreens were unexpectedly not so different from each other. To reveal the relationship between the protective activity of sunscreen and the wavelength of light with which larvae were irradiated through the sunscreen, we measured the transmittance of light through the petri dish cover on which the sunscreen was pasted. Effective protection was demonstrated by removing components of light whose wavelengths were below 315 nm. We suggest, that the measurement of anti-genotoxic activity and the determination of the wavelengths of light transmitted through the sunscreen should be an alternative method for evaluating the effectiveness of a sunscreen.     

UV-induced immunosuppression in the balance

Around 1980, experiments with hairless mice showed us that UV-induced actinic keratoses (AK) and ensuing skin carcinomas did not arise independently: the rate of occurrence in one skin area was increased considerably if AKs had already been induced separately in another distant skin area, i.e. a systemic effect. The ground laying work of Margaret Kripke in the 1970s provided a fitting explanation: UV-induced immunosuppression and tolerance toward the UV-induced tumors. From Kripke's work a new discipline arose: "Photoimmunology." Enormous strides were made in exploring and expanding the effects from UV carcinogenesis to infectious diseases, and in elucidating the mechanisms involved. Stemming from concerns about a depletion of the ozone layer and the general impact of ambient UV radiation, the groups I worked in and closely collaborated with explored the anticipated adverse effects of UV-induced immunosuppression on healthy individuals. An important turning point was brought about in 1992 when the group of Kevin Cooper reported that immunosuppression could be induced by UV exposure in virtually all human subjects tested, suggesting that this is a normal and sound physiological reaction to UV exposure. This reaction could actually protect us from illicit immune responses against our UV-exposed skin, such as observed in idiopathic polymorphic light eruption. This premise has fruitfully rekindled the research on this common "sun allergy," affecting to widely varying degrees about one in five Europeans with indoor professions.     

Measurement of in vivo sunscreen immune protection factors in humans

This study investigates the level of protection provided by sunscreens against solar-simulated UV radiation-induced immunosuppression in humans. The in vivo immune protection factors (IPF) of two broad-spectrum sunscreens were determined by assessing their ability to prevent UV-induced suppression of nickel contact hypersensitivity (CHS) in 15 nickel-allergic volunteers. Each volunteer was irradiated on unprotected skin of the back with different doses of UV daily for 4 days. Multiples of these UV doses were concurrently delivered to sunscreen-treated sites on the contralateral back. Nickel patches were then applied to both irradiated sites and adjacent, unirradiated control sites. Nickel-induced erythema at each site was measured 72 h later with a reflectance spectrometer. Comparison of the nickel reactions of irradiated and unirradiated skin revealed linear UV dose-responses for immunosuppression in both unprotected and sunscreen-treated skin. The minimum level of immunosuppression that can be reliably detected with this method is 20%. Therefore, the UV dose that reduces mean nickel CHS by 20% is the minimal immune suppression dose (MISD). Sunscreen IPF were determined by dividing the mean MISD of sunscreen-treated skin by that of unprotected skin. The sunscreens, with sun protection factors of 9 and 24, had IPF of 6.5 and > 25, respectively.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Evaluation of an Economical Sunlamp that Emits a Near Solar UV Power Spectrum for Conducting Photoimmunological and Sunscreen Immune Protection Studies

Expense and inconvenience have restricted the use of the filtered xenon are lamp (solar simulator) as a UV source for conducting large-scale animal studies. Because sunscreen immunoprotective levels are significantly affected by the UV power spectrum of the source it is imperative that a solar simulating source be used for accurate measurements of sunscreen protection levels that are relevant to human UV exposures from sunlight. However, relatively inexpensive sunlamps, e. g. the UVA-340, that emit a UV power spectrum similar to that of a solar simulator are available. Unlike FS-type UVB sunlamps, which have a significant amount of effective immunosuppressive nonsolar UV energy at wavelengths below 295 nm, the immunosuppression effectiveness spectrum of UVA-340 sunlamps was nearly identical to that of a solar simulator. The purpose of this study was to evaluate this sunlamp for conducting photoimmunological and sunscreen immune protection studies. Groups of C3H mice were exposed to a range of UVA-340 sunlamp doses (0.25 KJ/m² to 20.0 KJ/m²) to establish a dose-response curve and determine the minimum immune suppression dose (MISD) for induction of local-type suppression of contact hypersensitivity (CH). The MISD, defined as the lowest UV dose given to produce ～50% suppression of the CH response in mice, was determined to be 1.0 kJ/m² for UVA-340 sunlamps. Immune protection tests on four marketed sunscreen lotions (sun protection factors [SPF] 4, 8, 15 and 30) were then conducted with UVA-340 sunlamps using MISD as the endpoint. The immune protection factors for these sunscreens were equivalent to the level of protection predicated by their labeled SPF. These results are similar to those we have previously obtained using a solar simulator. We conclude from these data that the immunosuppressive effects of UVA-340 sunlamps are similar to those of a solar simulator; however, further studies are needed to determine if UVA-340, or similar, sunlamps are a viable alternative to the solar simulator for conducting large-scale animal experiments that require a relevant UV solar spectrum.     

SUNSCREENS WITH BROAD-SPECTRUM ABSORPTION DECREASE THE trans TO cis PHOTOISOMERIZATION OF UROCANIC ACID IN THE HUMAN stratum corneum AFTER MULTIPLE UV LIGHT EXPOSURES

Abstract The trans to cis photoisomerization of urocanic acid (UCA) in skin is considered to play an important role in the mechanism of immunosuppression. We have investigated the effects of skin type and various sunscreens with low sun protection factor (SPF) on the UV-induced cis -UCA formation in human skin after exposure to artificial IJV light. The rate of cis -UCA formation depends little on the skin type and is reduced by topical application of sunscreens. The rate of cis -UCA formation decreases with increasing SPF and only broad-spectrum, highly protective sunscreens offer protection against the UV-induced formation of cis -UCA, which accumulates in the stratum corneum after multiple UV exposures. A theoretical approach to estimate the distribution of cis -UCA after irradiation indicates that this compound may diffuse into the deeper layers of the epidermis with D ∼ 10⁻¹⁷ m²/s, and that its elimination from the stratum corneum is mainly due to desquamation.     

Unexpected photolysis of the sunscreen octinoxate in the presence of the sunscreen avobenzone

A major concern raised about photostability studies of sunscreen products is that the photodegradation of sunscreens does not readily translate into changes in product performance. This study examines the correlation between photochemical degradation of sunscreen agents and changes in protection provided by sunscreen films. Films of a commercial sunscreen product containing avobenzone, oxybenzone and octinoxate were irradiated using a fluorescent UV-A phototherapy lamp with additional UV-B blocking filter. Periodically, during irradiation the transmittances of the films were measured and samples collected for chemical analysis of the sunscreen agents using high-performance liquid chromatography techniques. The results show that UV-induced changes in UV transmittance of sunscreen films correlate with changes in concentration of sunscreen agents. In a parallel experiment, we also irradiated a thin film of the same product in the cavity of an electron spin resonance (ESR) spectrometer. We report the concomitant photolysis of avobenzone and octinoxate that predominates over expected E/Z photoisomerization and that irradiation of a film of this product produced free radicals detected by ESR spectroscopy that persisted even after exposure had ended.     

Protection from inflammation, immunosuppression and carcinogenesis induced by UV radiation in mice by topical Pycnogenol

Pycnogenol is a standardized extract of the bark of the French maritime pine, Pinus pinaster Ait., that has multiple biological effects, including antioxidant, anti-inflammatory and anticarcinogenic properties. This study describes the effect of topical application of lotions containing Pycnogenol to Skh:hr hairless mice undergoing minimally inflammatory daily exposures to solar-simulated UV radiation (SSUV). We report that concentrations of Pycnogenol of 0.05-0.2% applied to the irradiated dorsal skin immediately after exposure resulted in dose-dependent reduction of the inflammatory sunburn reaction, measured as its edema component. When mice received three consecutive daily exposures of minimally edematous SSUV, their ability to raise a contact hypersensitivity (CHS) reaction was suppressed by 54%. Pycnogenol lotions applied postirradiation reduced this immunosuppression to 22% (0.05% Pycnogenol) and 13% (0.1% Pycnogenol). Furthermore, when CHS was suppressed by 71% with exogenous treatment with cis-urocanic acid, the putative epidermal mediator of photoimmunosuppression, 0.2% Pycnogenol lotion reduced the immunosuppression to 18%. Chronic exposure to SSUV on 5 days/week for 10 weeks induced skin tumors from 11 weeks in both control mice and in mice receiving daily applications of 0.05% Pycnogenol, but tumor appearance was significantly delayed until 20 weeks in mice receiving 0.2% Pycnogenol. Furthermore, whereas 100% of control mice had at least one tumor by 30 weeks, and mice treated with 0.05% Pycnogenol by 33 weeks, the maximum tumor prevalence in mice treated with 0.2% Pycnogenol was significantly reduced to 85%, with some mice remaining tumor free. Average tumor multiplicity was also significantly reduced by 0.2% Pycnogenol, from 5.2 in control mice to 3.5 at 35 weeks. Thus, topical Pycnogenol offered significant and dose-dependent protection from SSUV-induced acute inflammation, immunosuppression and carcinogenesis, when applied to the skin after daily irradiation. Pycnogenol, therefore, in addition to its recognized health benefits in other organs, appears to have potential in providing photoprotection for humans in a complementary role with sunscreens, having demonstrable activity when applied to the skin after, rather than before, UV exposure.     

No adaptation to UV-induced immunosuppression and DNA damage following exposure of mice to chronic UV-exposure

It is well known that ultraviolet (UV) radiation induces erythema, immunosuppression and carcinogenesis. We hypothesized that chronic exposure to solar UV radiation induces adaptation that eventually prevents the suppression of acquired immunity. We studied adaptation for UV-induced immunosuppression after chronic exposure of mice to a suberythemal dose of solar simulated radiation (SSR) with Cleo Natural lamps, and subsequent exposure to an immunosuppressive dose of solar or UVB radiation (TL12). After UV dosing, the mice were sensitized and challenged with either diphenylcyclopropenone (DPCP) or picryl chloride (PCl). To assess the adaptation induced by solar simulated radiation, we measured the proliferative response and cytokine production of skin-draining lymph node cells after immunization to DPCP, the contact hypersensitivity (CHS) response to PCl, and thymine-thymine (T-T) cyclobutane dimers in the skin of mice. After induction of immunosuppression by SSR or by TL12 lamps, the proliferative response of draining lymph node cells after challenge with DPCP, or the CHS after challenge with PCl, showed significant suppression of the immune response. Chronic irradiation from SSR preceding the immunosuppressive dose of UV failed to restore the suppressed immune response. Reduced lipopolysaccharide-triggered cytokine production (of IL-12p40, IFN-gamma, IL-6 and TNF-alpha) by draining lymph node cells of mice sensitized and challenged with DPCP indicated that no adaptation is induced. In addition, the mice were not protected from T-T dimer DNA damage after chronic solar irradiation. Our studies reveal no evidence that chronic exposure to low doses of SSR induces adaptation to UV-induced suppression of acquired immunity.     

Biochemical Composition and Antioxidant Properties of Lavandula angustifolia Miller Essential Oil are Shielded by Propolis Against UV Radiations

UV radiations are principal causes of skin cancer and aging. Suntan creams were developed to protect epidermis and derma layers against photodegradation and photooxidation. The addition of antioxidant plant extracts (i.e. essential oil) to sunscreens is habitually performed, to increase their UV protective effects and to contrast pro‐radical and cytotoxic compounds present in these solutions. According to these observations, in the present work, the alteration of chemical composition and bioactive properties of Lavandula angustifolia Miller essential oil, exposed to UV light, was investigated. UV induced a significant deterioration of lavender oil biochemical profile. Moreover, the antioxidant activity of this solution, in in vitro tests and directly on B16‐F10 melanoma cells, greatly decreased after UV treatment. Our results also showed that essential oil was shielded from UV stress by propolis addition. Even after UV treatment, bee glue highly protected lavender oil secondary metabolites from degradation and also preserved their antiradical properties, both in in vitro antioxidant assays and in cell oxidative damage evaluations. This research proposed propolis as highly efficient UV protective and antiradical additive for sunscreens, cosmetics and alimentary or pharmaceutical products containing plant extracts.     

Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d-/- mice

We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)-induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d-/- mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d-/- mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d-/- than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d-/- mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.     

Epidermal reconstructs: a new tool to study topical and systemic photoprotective molecules

In this study, we compared the effects of sunscreens and antioxidants on reconstructed epidermis made with or without melanocytes 24 h after UVB, UVA or UVA+B irradiation. For this purpose, we studied sunburn cells and cyclobutane pyrimidine dimer formation, protein and lipid oxidation, catalase and superoxide dismutase activities and vitamin E levels. Topical sunscreens protected against direct cell death and thymine dimer formation whereas their protective effect against protein and lipid oxidation and antioxidant depletion was less marked partly due to the difficulty of spreading the cream. Antioxidant molecules protected against direct cell death and protein oxidation but not against thymine dimer formation. Since topical sunscreens and systemic antioxidant protected the skin differently, we speculate that their association might protect more efficiently against UV-induced damage. This model is relevant to study systemic molecules but is less practical, due to the technical limitations of studying topical molecules.