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Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin's response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm 2 and a UV lamp at 3 mJ/cm 2 . Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana-Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes., (© 2024. The Author(s).)

The inactivation of multi resistant pathogens is an important clinical need. One approach is UV-C irradiation, which was previously not possible in vivo due to cytotoxicity. Recently, far UV-C irradiation at λ < 240 nm was successfully used on skin with negligible damage. A potential application site is the nasal vestibule, where MRSA accumulates and cannot be treated using antiseptics. We irradiated 3D mucosa models and excised human mucosa with 222 and 233 nm far UV-C in comparison to 254 nm and broadband UV-B. Eradication efficiency was evaluated by counting colony forming units; irritation potential was evaluated by hen's egg-chorioallantoic membrane assay and trans epithelial electrical resistance; cell viability was assessed by MTT. DNA damage and cell protective mechanisms were evaluated immunohistopathologically. On mucosa models, MRSA reduced by ≈ 5 log 10 for 60 mJ/cm 2 irradiation at 233 nm. A slightly increased cell viability was observed after 24 h. Lower doses showed lower irritation potential than the positive controls or commercial mouthwash, while 80 mJ/cm 2 had strong irritation potential. DNA damage occurred only superficially and decreased after 24 h. On excised human mucosa, < 10% of keratinocytes were affected after 150 mJ/cm 2 222 nm or 60 mJ/cm 2 233 nm., (© 2023. The Author(s).)

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)-the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed., Competing Interests: The authors declare no conflict of interest.

A newly developed UVC LED source with an emission wavelength of 233 nm was proved on bactericidal efficacy and skin tolerability. The bactericidal efficacy was qualitatively analysed using blood agar test. Subsequently, quantitative analyses were performed on germ carrier tests using the MRSA strain DSM11822, the MSSA strain DSM799, S. epidermidis DSM1798 with various soil loads. Additionally, the compatibility of the germicidal radiation doses on excised human skin and reconstructed human epidermis was proved. Cell viability, DNA damage and production of radicals were assessed in comparison to typical UVC radiation from discharge lamps (222 nm, 254 nm) and UVB (280-380 nm) radiation for clinical assessment. At a dose of 40 mJ/cm 2 , the 233 nm light source reduced the viable microorganisms by a log 10 reduction (LR) of 5 log 10 levels if no soil load was present. Mucin and protein containing soil loads diminished the effect to an LR of 1.5-3.3. A salt solution representing artificial sweat (pH 8.4) had only minor effects on the reduction. The viability of the skin models was not reduced and the DNA damage was far below the damage evoked by 0.1 UVB minimal erythema dose, which can be regarded as safe. Furthermore, the induced damage vanished after 24 h. Irradiation on four consecutive days also did not evoke DNA damage. The radical formation was far lower than 20 min outdoor visible light would cause, which is classified as low radical load and can be compensated by the antioxidant defence system., (© 2022. The Author(s).)

The exposure to sun radiation is indispensable to our health; however, a long-term and high exposure could lead to cell damage, erythema, premature skin aging, and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals which may induce oxidative stress at elevated concentrations. Different skin models, such as porcine-, murine-, human- ex vivo skin, reconstructed human skin (RHS) and human skin in vivo, were investigated during and after irradiation using X- and L-band EPR spectroscopy within different spectral regions (UVC to NIR). The amount of radical formation was quantified with the spin probe PCA and the radical types were measured ex vivo with the spin trap DMPO. The radiation dose influences the types of radicals formed in the skin. While reactive oxygen species (ROS) are always pronounced at low doses, there is an increase in lipid oxygen species (LOS) at high doses. Furthermore, the radical types arise independent from the irradiation wavelength, whereas the general amount of radical formation differs with the irradiation wavelength. Heat pre-stressed porcine skin already starts with higher LOS values. Thus, the radical type ratio might be an indicator of stress and the reversal of ROS/LOS constitutes the point where positive stress turns into negative stress.Compared to light skin types, darker types produce less radicals in the ultraviolet, similar amounts in the visible and higher ones in the infrared spectral region, rendering skin type-specific sun protection a necessity., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2021.)

Multiresistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause serious postoperative infections. A skin tolerant far-UVC (< 240 nm) irradiation system for their inactivation is presented here. It uses UVC LEDs in combination with a spectral filter and provides a peak wavelength of 233 nm, with a full width at half maximum of 12 nm, and an irradiance of 44 µW/cm 2 . MRSA bacteria in different concentrations on blood agar plates were inactivated with irradiation doses in the range of 15-40 mJ/cm 2 . Porcine skin irradiated with a dose of 40 mJ/cm 2 at 233 nm showed only 3.7% CPD and 2.3% 6-4PP DNA damage. Corresponding irradiation at 254 nm caused 15-30 times higher damage. Thus, the skin damage caused by the disinfectant doses is so small that it can be expected to be compensated by the skin's natural repair mechanisms. LED-based far-UVC lamps could therefore soon be used in everyday clinical practice to eradicate multiresistant pathogens directly on humans., (© 2021. The Author(s).)

The daily consumption of tobacco products leads to a boost in free radical production in tissues, promoting the risk for malignancies, metabolic alterations and chronic-inflammatory diseases. This study aimed to broaden the knowledge of the status of the antioxidative (AO) system in the skin, compared to the blood, of healthy appearing smokers. Both, the basic status compared to non-smokers and the short-term impact of controlled cigarette consumption in smokers were analyzed. Our study showed that the basic level of the AO system of smokers significantly differed from that of non-smokers. As determined by resonant Raman spectroscopy (RRS), the levels of exogenous AOs were decreased in both, the skin, in vivo (β-carotene and lycopene), and blood plasma (β-carotene only). In contrast, the levels of glutathione (GSH), the prototypical endogenous AO, which were analyzed by fluorimetric assays in cutaneous tape strips and blood plasma, were increased in the skin, although unchanged in the blood of smokers. Elevated cutaneous GSH levels were reflected by an elevated overall radical scavenging activity in the skin, as quantified by non-invasive electron paramagnetic resonance (EPR) spectroscopy. Analysis of the expression of selected stress-associated genes in blood immune cells by quantitative RT-PCR in subgroups of non-smokers and smokers additionally demonstrated the downregulation of AKR1C2 in smokers, and its negative correlation with blood plasma levels of the protective immune mediator interleukin-22, assessed by the ELISA technique. Controlled cigarette consumption did not alter exogenous or endogenous AOs in the skin of smokers, but decreased lycopene levels in blood plasma. Moreover, there was a decline in blood IL-22 levels, while no relevant response of blood cell gene expressions was found after the considered short time. Our data therefore demonstrate a strengthened endogenous AO status in the skin of smokers, which may indicate a long-term adaptation to chronic oxidative stress in this specific organ. While this effect was not clearly visible in the blood, this compartment seems to be useful as an immediate indicator of the body's AO consumption. Moreover, decreased levels of AKR1C2, which we show for the first time to be expressed in immune cells, may be a candidate marker for long-term smoking. In addition, this study demonstrates that the rate constant of a spin probe decline determined by EPR spectroscopy mainly represents the endogenous AO status of a tissue., Competing Interests: The authors declare no conflict of interest.

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications.

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis is still not fully understood. Since inflammatory processes correlate with oxidative stress, the redox status may play a key role in AD. In this study, electron paramagnetic resonance (EPR) spectroscopy was mainly used to investigate the redox status in normal and inflammatory skin equivalents mimicking characteristics of AD in vitro using EPR spin probes (TEMPO, PCA) and a spin trap (DMPO). The total antioxidant status in the hydrophilic and lipophilic compartments of skin (microenvironment) showed no differences between the skin equivalents. In the inflammatory skin equivalents, a decreased glutathione concentration in the epidermis and an increased metabolic radical production could be observed compared to normal skin equivalents. The induction of external stress by simulated solar irradiation (UVB-NIR) resulted in the same amount and type of radicals in normal and inflammatory skin equivalents. For the first time, the antioxidant and oxidant status of inflammatory in vitro skin equivalents was analyzed by EPR to elucidate their redox status using different methods which focus on various microenvironments. Our investigations suggested that the redox status in atopic skin could be different, but this should be investigated more comprehensively, because the results can vary depending on the used methods and where the investigations take place., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The skin is exposed to many stress factors which, in turn, can promote a shift of the antioxidant (AO) network towards the prooxidative side, supporting the development of various skin disorders. A balanced diet, in combination with a healthy lifestyle could reduce oxidative stress. Carotenoids are essential nonenzymatic AOs and main components of the exogenous AO system. To examine the interdependence between endogenous and exogenous AOs, secondary keratinocytes (HaCaT) were treated with various Beta (β-)-carotene concentrations with subsequent stress treatment by moderate irradiation (700-2000 nm). To facilitate the uptake of β-carotene, an innovative nanocrystal solution was used. Cell viability assay was applied to HaCaT cells to evaluate suitable concentration of β-carotene, whereby the uptake was measured by resonant Raman spectroscopy. The redox status was determined before and after supplementation with two selected β-carotene concentrations (0.02 and 0.1 μg/ml) and irradiation. Reactive oxygen species (ROS) were measured by electron paramagnetic resonance spectroscopy and the AO glutathione (GSH) by a fluorescent-based assay for evaluating the endogenous redox status. An increase of ROS and a reduction of GSH after irradiation was observed. Interestingly, the applied β-carotene, already induce oxidative stress. Nevertheless, an effective protection against irradiation could be observed for the lower dose. The high dose turned pro-oxidative., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the last decade, cutaneous carotenoid measurements have become increasingly popular, as carotenoids were found to be a biomarker of nutrition rich in fruits and vegetables, permitting monitoring of the influence of various stress factors. For such measurements, in addition to the specific and selective resonance Raman spectroscopy (RRS), newly developed low expensive small and mobile sensors that are based on spatially resolved reflectance spectroscopy (SRRS) are used for cutaneous carotenoid measurements. Human volunteers of different age exhibiting skin types I to III were investigated using RRS and two SRRS-based sensors to determine the influence of these parameters on the measuring results. In two studies on volunteers of either the same age or skin type, however, the respective other parameter being varied and no significant influences of age or skin type could be detected. Furthermore, the kinetic changes resulting from the intake and discontinued intake of a vegetable extract rich in carotenoids showed a good correlation among the three sensors and with the detected blood carotenoids. This illustrates that the SRRS-based sensors and RRS device provide reliable cutaneous carotenoid values independent of age and skin types I to III of the volunteers.