Switching from healthy to unhealthy oxidative stress - does the radical type can be used as an indicator?

Ultraviolet (UV) radiation leads to the formation of free radicals, which may cause immunological modulations, skin aging or skin cancer. Sunlight exposure in the UVA region according to CIE 85 promotes almost 46% of radical formation in skin. A critical radical concentration characterized by the inversion of the domination of primary ROS (reactive oxygen species) to an excess of secondary LOS (lipid oxygen species) is proven for the spectral regions UV and or VIS light and is intended to be a marker for an imbalance in the redox system, which can no longer compensate harmful effects. To investigate whether this transition point is also universally valid for one spectral region, the radical formation during and after targeted UVA in situ-irradiation at 365 ± 5 nm and three different irradiances (31, 94 and 244 mW/cm2) was investigated in ex vivo porcine skin using x-band electron paramagnetic resonance (EPR) spectroscopy. The quantification was performed with the spin probe 3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl (PCA), the spin trap 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO) was used to characterize the radical species. Furthermore, the viability of the skin cells after irradiation was controlled by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, skin integrity was examined by histological analysis. A significant dose dependence in the radical formation is given at higher irradiance. The transition point was detected in the range of 0.5 MED after irradiation with the highest irradiance. From this point on the proportion of LOS increases with increasing dose and the proportion of ROS decreases. After switching off the UVA irradiation no further quantitative changes were detected, but rapid changes in the radical pattern were observed demonstrating the importance of in situ irradiation during the use of spin traps. Heat-pre-stressed skin showed more LOS than ROS already at the beginning of the irradiation, leading to the assumption that the transition point to the distress-level has already been reached. In summary, a postulated transition point could be verified for the UVA spectral region using only one spin trap combined with in-situ irradiation. A certain degree of stress is necessary to detect an inversion of the ratio of ROS to LOS. This reversal indicates an imbalance in the redox status. However, at low intensities no changes at all in radical pattern appeared over time (dose), probably it can be compensated by adaptation processes of the skin.

Skin type differences in solar-simulated radiation-induced oxidative stress.

BACKGROUND: Human skin protects the body from external damage, pathogens and oxidative stress factors such as ultraviolet (UV) radiation. Excessive exposure to UV radiation can lead to increased production of free radicals and hence to skin damage such as inflammation, premature skin ageing and skin cancer. Besides UV, the visible and near infrared (NIR) regions are also a source of radical production. Half of all free radicals are induced by the visible + NIR region of the solar spectrum in people with skin types I-III, but data on the effects in people with skin types IV-VI are missing. OBJECTIVES: This in vivo pilot study addressed the distribution of radical production in skin types IV and V during irradiation in the UV, visible and NIR spectral regions, comparing the first results with those of skin type II. METHODS: The measurements were performed in vivo using L-band electron paramagnetic resonance spectroscopy and the spin probe PCA. RESULTS: In skin types IV-V most radicals were induced in the visible + NIR region, followed by the NIR and UV regions of the sun spectrum. Significantly (P ≤ 0·05) more radicals were induced in skin types IV-V than in type II during NIR irradiation, whereas skin types IV-V exhibited significantly less UV-induced radicals (P ≤ 0·01) than skin type II. CONCLUSIONS: All spectral regions (UV, visible and NIR) cause free radical formation in skin types II and IV-V. After 4 min of solar-simulated exposure (UV-NIR), the radical formation in skin types IV-V is 60% of that in skin type II. Therefore people with darker skin types also need solar protection.

Multicenter methodology comparison of the FDA and ISO standard for measurement of in vitro UVA protection of sunscreen products.

In vitro standard methods are available and accepted worldwide to assess UVA protection of sunscreen products. Though, harmonisation of methods has made progress in the last decade, still two differing methods - one by FDA the other by ISO - are in use. In a multicentre study including 9 centres in Germany, 4 different commercial sunscreen products were assessed using both methods to discover their similarities and differences. UVA protection factor and Critical Wavelength were detected at various substrate type (sandblasted versus moulded PMMA plates), at different surface roughness of the plates as well as at different product application dose using two different irradiation spectra. Results: The strongest influence on UVA protection factor results from the surface roughness of the plates. Depending on the roughness (accepted range of 2 to 7 µm in the FDA method) a variability in the UVA protection factor of up to 25% was observed, while the much narrower definition of plate roughness by ISO (4.5 to 5.2 µm) had no relevant influence on the test results. Sandblasted plates in our assessment led to higher UVA protection factors and produced less scattered results compared to moulded plates. These differences were not pronounced. Application dose and spectra of the irradiation source were of negligible influence on UVA protection factor results for the investigated UV-filter combinations. The UVA protection factor which is the endpoint of the ISO method was found to be a parameter with a high potential to differentiate among different test products. The endpoint of the FDA method - the Critical Wavelength - was found to be an unambitious endpoint. Insensitivity to all described modifications of the method was observed. All investigated products performed similar and passed the Critical Wavelength criteria independent of method and parameters.

[New aspects in the development of sunscreening agents]. / Neue Aspekte bei der Entwicklung von Sonnenschutzmitteln.

Sunlight is indispensable for life. Skin aging is determined not only by genetic factors but also by the influence of solar radiation which in all its spectral regions induces free radicals in the human skin. Below a critical concentration, free radicals are important for signaling processes in the human body. Exceeding a critical threshold value (FRTV), however, free radicals lead to cellular damage which, in turn, can induce skin aging, immunosuppression and even skin cancer. Recently it could be shown that 50% of the free radicals induced in the human skin by solar radiation were caused by the visible and infrared spectral regions. Taking into consideration that people who use sunscreen stay considerably longer in the sun than unprotected people, their skin could be exposed to free radical formation in the visible and infrared spectral regions, which far exceeds the critical threshold. Filter substances like those used in sunscreens for skin protection in the ultraviolet region are not available for the visible and infrared spectral regions. Using the natural protection mechanisms of the human skin as examples, however, the protective effect of sunscreens can be considerably enhanced by the addition of scattering and reflective pigments, such as titanium dioxide, as well as of antioxidants which neutralize the free radicals. Currently the focus of sunscreen development is shifting from mere UV protection to protection covering the entire solar spectrum.

Uptake of antioxidants by natural nutrition and supplementation: pros and cons from the dermatological point of view.

The pros and cons of the systemic and topical application of antioxidant substances are a subject of intense discussion among experts, with resulting confusion for consumers and producers. The objective of the present article is to clarify the various uncertainties relating to the use of antioxidant substances in dermatology. Whereas inappropriate application of antioxidant substances (concerning their concentration and composition) might induce harmful effects, the consumer will definitively benefit from physiological concentrations and compositions of antioxidants. The most suitable method is the consumption of natural antioxidants in the form of fruit and vegetables, for example. In addition, the skin, which also accumulates antioxidant substances, may profit from a sufficient antioxidative level, as damage induced by sun radiation in addition to skin aging is reduced.

In vitro sun protection factor: still a challenge with no final answer.

In the past, several attempts have been made to develop in vitro methods for determining protection against UV radiation. To date however, there is no broadly accepted method. Various known and unknown parameters influence the transmission measurements of scattering films, such as the multifaceted compositions of sunscreens, the technical limitations of measurement devices as well as the difficulty to apply very thin films of sunscreen in a reproducible manner throughout different laboratories. In vitro data were measured in this multicenter study to compare possible methodologies and strategies for an in vitro approach to the sun protection factor (SPF). This publication will not present a final in vitro SPF test method, but it will point out which technical side effects may influence such a method. Influential factors such as the quality of spectrophotometer used, the amount of product applied, pretreatment of samples, time and temperature of equilibration, size of the measured surface, the application process or the calculation on the basis of standardized data are presented and discussed. Finally, a reduction of the standard deviations within single laboratories could be realized for in vitro SPF testing, but no improvement of the interlaboratory comparison was obtained. The development of a valid and reliable SPF in vitro test still remains a challenge, and further work is necessary to develop a satisfactory method.

Radical production by infrared A irradiation in human tissue.

The influence of the ultraviolet (UV) irradiation of the sun on the formation of free radicals in human skin is well investigated. Up to now, only small amounts of data are available stating that infrared (IR) irradiation can produce free radicals in the skin. In the present study, the formation of free radicals in human skin, subsequent to IRA irradiation (600-1,500 nm), has been demonstrated by means of two different methods. Firstly, the radical formation was detected indirectly by the degradation of the cutaneous carotenoid antioxidants beta-carotene and lycopene, which was investigated in vivo by resonance Raman spectroscopic measurements. Secondly, the direct observation of produced radicals subsequent to IRA irradiation of the skin was performed in vitro by electron paramagnetic resonance spectroscopy. Taking into account the results of the present study and previous UV light studies, it can be expected that also solar irradiation in the visible spectral range will produce free radicals in the human skin. Therefore, the current sun protection strategies should be reconsidered. Furthermore, it was shown in the present study that the side effect in the form of radical formation could be significantly reduced by increasing the protection system of the human organism in form of the antioxidant network.

[UV, visible and infrared light. Which wavelengths produce oxidative stress in human skin?]. / UV, sichtbares Licht, Infrarot. Welche Wellenlängen produzieren oxidativen Stress in menschlicher Haut?

Experimental evidence suggests that the creation of free radicals--mainly reactive oxygen species (ROS)--is the common photobiological answer to the skin-sunlight interaction. The free radical action spectrum (wavelength dependency) for ultraviolet and visible light (280-700 nm) has been determined by quantitative ESR spectroscopy. Visible light produces around 50% of the total oxidative stress caused by sunlight. Reactive species like *O(-)(2), *OH and *CHR are generated by visible light. The amount of ROS correlates with the visible light intensity (illuminance). We demonstrated the creation of excess free radicals by near-infrared light (NIR, 700-1600 nm). Free radical generation does not depend exclusively on the NIR irradiance, but also on the NIR initiated skin temperature increase. The temperature dependence follows the physiological fever curve. Our results indicate that the complex biological system skin creates the same type of free radicals over the entire active solar spectrum. This general response will make it possible to define the beneficial or deleterious action of sunlight on human skin by introduction of a free radical threshold value.

The missing link--light-induced (280-1,600 nm) free radical formation in human skin.

The recent European recommendation on the efficacy of sunscreen products requests now a minimum ratio of UVA/UVB protection. However, the visible and the infrared (IR) parts of the sun spectrum have received little attention concerning their possible contribution to skin damage. A common biophysical answer for the different wavelengths of the sun spectrum can be found in the creation of excess free radicals - mainly reactive oxygen species (ROS). Thanks to electron spin resonance spectroscopy applied to skin biopsies, we determined for the first time the free radical action spectrum covering UV and visible light (280-700 nm). Convolution of the action spectrum with sunlight spectral irradiance showed that 50% of the total skin oxidative burden was generated by visible light. Creation of ROS by visible light was experimentally confirmed by varying the illuminance of a spotlight. We also evidenced the creation of excess free radicals by near-IR radiation. In that case, free radical generation does not depend exclusively on the dose, but also on the skin temperature increase initiated by near-IR light. Some phenomena which are still unclear, such as the question about the deleterious or beneficial role of sunlight, are reviewed, implying the research on new protection strategies for the prevention of skin cancer.

Influence of microparticles on the homogeneity of distribution of topically applied substances.

The uppermost layer of the skin--the stratum corneum--represents a barrier of the human organism to the environment. It prevents the penetration of substances coming into contact with the skin into the human body. In cases of strong exposure, the skin has to be protected additionally by barrier creams. In the present study, the influence of microparticles on the homogeneity of distribution of the protection cream on the skin was investigated by laser scanning microscopy. The protection cream contained antioxidant substances with a high radical protection factor for the prevention of palmar-plantar erythema often occurring during chemotherapy with doxorubicin. In this case, the chemotherapeutic substance comes out with the sweat onto the skin surface, from which it penetrates into the skin like topically applied. It was found that particles increase the homogeneity of distribution and, thus, the protection efficacy is significantly increased. The obtained results are important, not only for the prevention of side effects during chemotherapy, but also for the development and application of sunscreens and all types of topically applied drugs and cosmetics, which should form an efficient protection film on the skin.

In vitro assessment of water resistance of sun care products: a reproducible and optimized in vitro test method.

The aim of the study was to develop a simple reproducible and reliable in vitro water resistance (WR) method to assess the sun care products. This paper is the result of a scientific collaboration between seven different international industrial laboratories and testing institutes. The same group has already achieved an in vitro protocol for the sun protection factor (SPF) determination [1]. The in vitro WR of sunscreens was tested by applying the same principle as in vivo, which determines the percentage of retention of sunscreen products by assessing the SPF before and after water immersion. Special care was taken to study the parameters influencing the WR and the possibility to follow the kinetics of sunscreen retention during water immersion. The influence of different water qualities has been tested, and osmosed water (1-3 microS cm(-1)) was chosen for the main ring study. Measurement was carried out after 5, 20 and 40 min of immersion. Histograms of selected products demonstrate the percentage of WR at all measuring times and centres, and the regression coefficient to the in vivo determination was shown and statistical calculations clearly demonstrate the reproducibility of the results between the different evaluation centres. The presented method is a practical, convenient and relevant tool for WR screening of sun care and skin care products. It even has the potential to be the starting point for the replacement of the in vivo method in future.

Effect of supplemented and topically applied antioxidant substances on human tissue.

Systemic and topical application of antioxidant substances for the medical treatment and prophylaxis of many diseases as well as additional protection of the skin against the destructive action of free radicals and other reactive species has become very popular during the past years. Stimulated by the positive results of a fruit and vegetable diet in supporting medical treatment and in cosmetics, artificial and extracted antioxidant substances have been broadly applied. Surprisingly, not only positive but also strong negative results have been obtained by different authors. According to study reports artificial and extracted antioxidant substances support different kinds of medical therapies, if they are applied in mixtures of different compounds at low concentration levels. In the case of the application of high concentration of some single compounds, side effects were often observed. Regarding skin treatment by systemically applied antioxidant substances for cosmetic purposes, positive cosmetic effects as well as no effects, but almost no side effects, apart from a number of allergic reactions, were reported. One reason for this seems to be the lower concentration of systemically applied antioxidant substances in comparison with a medical application. Topical application of antioxidant substances is closely related to cosmetic treatment for skin protection and anti-aging. Positive results were also obtained in this case. The present review is an attempt to classify and summarize the published literature concerning the efficiency of action of systemic and topical applications of antioxidant substances, such as carotenoids and vitamins, on human organism and especially on the skin. The available literature on this topic is very extensive and the results are often contradictory. Nevertheless, there are some clear tendencies concerning systemic and topical application of antioxidant substances in medicine and cosmetics, and we summarize them in the present paper.

Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products.

The aim of this study was to evaluate the interest of a new three-dimensional epithelial model cultivated from human corneal cells to replace animal testing in the assessment of eye tolerance. To this end, 65 formulated cosmetic products and 36 chemicals were tested by means of this in vitro model using a simplified toxicokinetic approach. The chemicals were selected from the ECETOC data bank and the EC/HO International validation study list. Very satisfactory results were obtained in terms of concordance with the Draize test data for the formulated cosmetic products. Moreover, the response of the corneal model appeared predictive of human ocular response clinically observed by ophthalmologists. The in vitro scores for the chemicals tested strongly correlated with their respective scores in vivo. For all the compounds tested, the response of the corneal model to irritants was similar regardless of their chemical structure, suggesting a good robustness of the prediction model proposed. We concluded that this new three-dimensional epithelial model, developed from human corneal cells, could be promising for the prediction of eye irritation induced by chemicals and complex formulated products, and that these two types of materials should be tested using a similar protocol. A simple shortening of the exposure period was required for the chemicals assumed to be more aggressively irritant to the epithelial tissues than the cosmetic formulae.

Integrated sun protection factor: a new sun protection factor based on free radicals generated by UV irradiation.

The present work uses the initial step of the whole cascade of biological effects in the skin, the creation of free radicals by means of UVA/UVB radiation, to develop a total sun protection factor. Until now, existing in vivo indices have not been fully satisfying: SPF only reflects protection from UVB light, and persistent pigment darkening is restricted to the UVA part of the sun spectrum. The quantitative measurement of free radicals generated in human skin biopsies by means of electron spin resonance X-band spectroscopy allows to determine a new total SPF. This new sun protection index covers all UVA/UVB wavelengths taking into account their effects in the epidermis as well as the dermis. Use of skin biopsies avoids exposure of human volunteers to potentially harmful radiations. The new index is always practically equal or lower than the in vivo SPF depending on the level of a product's UVA/UVB photoprotection balance. With this, we propose to name this new protection index 'integrated sun protection factor'.

Sunscreen application at the beach.

BACKGROUND: The sun protection factor (SPF) of sunscreens is determined after application of a standard amount. The European Cosmetic Toiletry and Perfumery Association (COLIPA) standard amount is 2 mg/cm(2). Real-life application of sunscreen is probably less than this. AIM: To determine the amount of sunscreen present on the skin of people at the beach. METHODS: Volunteers at the beach were selected randomly and were not aware of being tested for the adequacy of their sunscreen application. All volunteers had applied sunscreen. Application had been more than 30 min before testing (sometimes up to 4 h earlier). The amounts of sunscreen applied to different body sites were determined quantitatively by tape stripping. Actual amounts of sunscreen applied were compared with the COLIPA standard. Also, sunscreen containing a fluorescent dye was applied to the skin of volunteers in a laboratory setting. The distribution of sunscreen application was visualized by UVA photography in a darkened room. RESULTS: Sixty volunteers, 33 males and 27 females, aged 17-68 years (median 32 years), were recruited at the beach. Sunscreen coverage was inadequate at all body sites. Coverage at various body sites differed greatly. Most volunteers had applied 10% or less of the COLIPA standard amount to all body sites assessed. The best protected areas were the upper arm and décolleté but, even in these areas, most volunteers had only applied 10% of the COLIPA standard amount. The worst protected areas were the ears and top of the feet. The back was typically badly protected if treated by the volunteers themselves. The back was better protected if another person had applied the sunscreen. In the laboratory, the fluorescent dye-containing sunscreen showed the same pattern of sunscreen application as at the beach. CONCLUSIONS: In real life, at the beach, very little sunscreen remains present on the skin.

Efficiency of a continuous height distribution model of sunscreen film geometry to predict a realistic sun protection factor.

Irregularities in the geometry of sunscreen films spread on rough areas, like skin, is often presented as being the main cause of the degree of UV absorption achieved by the UV filters that are inside. Until now, only the step film, a model invented by O'Neill, was simple enough to calculate UV data close to in vitro experimental data, after determination of a limited number of fraction areas with their corresponding thickness. However, such models are obviously too simple to represent a real situation. In the present work, more complex distributions of film thickness were calculated, with an infinite number of individual heights. Realistic models were achieved via a probability function. The consequences for UV absorption were deduced, and the calculated UV data were compared to experimental in vitro data on sunscreen products measured after being spread on a roughened PMMA substrate. The latter substrate was previously selected for its ability to achieve a good correlation with in vivo SPF. An optimized version of the continuous mathematical model was finally determined in order to achieve UV curves, similar in shape and intensity to the experimental ones. The latter model can be used to predict realistic SPF values.

Electron spin resonance detection of UVA-induced free radicals.

Free radicals generated during UV irradiation of human skin biopsies were measured with electron spin resonance spectroscopy by using spin traps. The generation of hydroxyl and lipid radicals in skin is mainly caused by the UVA part of the solar spectrum.

Characterization of the barrier function in a reconstituted human epidermis cultivated in chemically defined medium.

The aim of the study was to characterize and assess the maturation process of the barrier function in a reconstituted human epidermis (REp) cultivated in a chemically defined medium. For this purpose histo-morphological analysis, percutaneous absorption studies and non-invasive measurements were performed. In order to understand the time course of the barrier development, REp cultivated from the same pool of normal human keratinocytes were harvested and measured after several increasing periods of exposure at the air-liquid interface. From these results we concluded that a maturation period of at least 16 days at the air-liquid interface was required for an optimal barrier function development of REp. At this time point, the permeability of the skin cultures for caffeine (CAF) was 20-25-fold higher than that of normal human skin (NHS) biopsies. Non-invasive measurements of skin water content, transepidermal water loss (TEWL) and pH were achieved after the same period of time. Results showed that the stratum corneum (SC) of REp was slightly more hydrated than that of ex vivo and in vivo, human skin. TEWL was slightly higher through REp than through NHS, and the pH of the REp models was very close to that of in vivo, normal human skin. In the latter part, assuming that this type of model could be routinely used, we quantified over a 1-year period the barrier function variability of this skin culture model between batches. The satisfactory results obtained with a 20% coefficient of variation indicated that this REp model has a consistent and reproducible barrier function. This leads us to suggest that the skin model might be considered an alternative membrane to normal human skin for permeation screening tests.

Sunscreen in vitro spectroscopy: application to UVA protection assessment and correlation with in vivo persistent pigment darkening.

In the present study, we have described an in vitro spectroscopic method to evaluate the sunscreen products for UVA sun protection factor. The roughened PMMA plates have been used as a transparent substrate on to which the test product is spread. The UVA protection factors have been deduced from the UV-transmittance data measured in the UVA area. In order to be as close as possible to the in vivo protection factors, issued from the PPD end-point, the treated polymethylmethacrylate (PMMA) plates are submitted to different UV-irradiation doses, before the measurement. The correlation in vitro/in vivo is poor when the sunscreens are not irradiated. A UV dose of about 2 minimal pigmenting dose (MPD) is enough to achieve a good correlation between in vitro and in vivo data issued from the 13 tested sunscreens. These results are consistent with the fact that the photostability of sunscreens is challenged during an in vivo PPD test.

Formulation and interest of a decorative emulsifier-free O/W emulsion with a positive zeta potential.

The aim of this study was to formulate an emulsifier-free foundation with a positive zeta potential. This formulation was achieved through a comparative study carried out on 16 cationic hydrogels. Measurements of the cationic activity of those gels were undertaken. Sixteen emulsions, including 0.4% of cationic gel, were formulated and the zeta potential of each was quantified. No correlation between those two measurements could be established. Pigments introduced in the emulsion to obtain a foundation slightly reduced the zeta potential value of the formula. The skin coloration induced by the application of the formula with a positive electric charge was found more stable than that of classical and transfer-proof formulae. Moreover, this formula displayed a higher hydrating potential compared to transfer-proof formulae.