Skin Responses to Micro Scale Field Size of Solar‐Simulated Radiation – Preliminary Evaluation by Reflectance Confocal Microscopy in vivo

Erythema and pigment responses of human skin following an acute exposure to ultraviolet radiation (UVR) are frequently used to determine the photosensitivity of the skin. In this study we investigated the responses of the skin to a micro‐scale area of UVR exposure (MiR) and compared the responses to a macro‐scale area of exposure (MaR). Ten human volunteers were tested with solar‐simulated radiation on their upper arm or back using a beam size of 8 mm and 0.2 mm in diameter. The fluence required to produce a minimally perceptible erythema (MED) using the MiR was found to be higher than that for the MaR. The erythema response extended beyond the exposed area and this became pronounced when the beam size was microscopic. Reflectance confocal microscopy in vivo revealed that MiR induced cellular alterations within a confined area of smaller dimensions than the area of exposure. Pigment responses were confined within the areas of cellular damage. The erythema expression of exposed skin recovered faster for the sites receiving MiR even when the applied fluence was higher than the MED for the MaR. Through the use of MiR we were able to visualize spatially dissimilar skin responses of erythema and pigmentation suggesting different cellular mechanisms.     

Salt water bathing prior to UVB irradiation leads to a decrease of the minimal erythema dose and an increased erythema index without affecting skin pigmentation

The combination of salt water baths and solar radiation is known as an effective treatment for patients with psoriasis and atopic dermatitis. To determine whether increased susceptibility to UVB radiation may contribute to this therapeutic effect we have studied the effect of bathing the skin in salt water prior to UVB irradiation. Twelve subjects were phototested on the volar aspects of their forearms with increasing doses of UVB radiation. One forearm was exposed to 5% salt water prior to irradiation. The minimal erythema dose (MED) was determined and the erythema index and skin pigmentation were assessed by photometric measurement. The combination of salt water bath and irradiation yielded a significant decrease of the MED when compared to UVB alone (median 90 mJ/cm2 vs 130 mJ/cm2, P < 0.01). Analysis of variance showed a significant influence of salt water bath on erythema (P < 0.05) but not on skin pigmentation. Within the MED test area the erythema index of the salt water exposed forearms was elevated significantly (P < 0.05) while skin pigmentation was not affected. Thus, bathing the skin in salt water leads to a decreased threshold level for the elicitation of UVB-induced erythema and a selective increase of the erythemal response. This sensitization to the effects of shortwave UVB radiation may increase immunosuppressive effects of UVB radiation and may lead to an increased efficacy of UVB phototherapy. However, there is also an increased sunburn risk when salt water baths are followed by exposure to UV radiation.     

Isomerization of urocanic acid after ultraviolet radiation is influenced by skin pigmentation

Exposure to ultraviolet (UV) radiation may induce erythema, DNA damage and suppression of immune responses. Melanin pigmentation offers protection against the first two of these effects, but immunosuppression seems to occur irrespective of the subject's pigmentation. Cis-urocanic acid (cis-UCA), produced by isomerization of trans-UCA in the stratum corneum on UV exposure, initiates some of the immunomodulatory effects of UV radiation. In the present study the relationship between skin pigmentation and UCA isomerization has been examined in 28 healthy individuals of skin types I-IV. Pigmentation is measured in five areas of not recently exposed back skin before irradiation with 0, 0.45, 0.9, 1.8 and 3.6 standard erythema dose (SED) of filtered broadband UV-B (1 SED = 10 mJ cm-2 at 298 nm). The concentration of UCA isomers is measured immediately after the irradiation. With 3.6 SED, the relative production of cis-UCA is close to the maximum obtainable, irrespective of skin type. A significant negative correlation is found between pigmentation and relative production of cis-UCA at 0.45 and 1.8 SED, and between pigmentation and absolute production of cis-UCA at 0.45 SED. At doses of 0.45 and 0.9 SED the relative and absolute production of cis-UCA are higher in the group with skin types I and II when compared with the group with skin types III and IV. The higher isomerization in the lightly pigmented subjects than in the more pigmented ones may indicate that people with fair skin are at a relatively higher risk of immunosuppression when exposed to low doses of UV radiation.     

Adaptation of the human skin by chronic solar-simulating UV irradiation prevents ultraviolet-B irradiation-induced rise in serum C-reactive protein levels

Exposure of the skin to UV radiation induces local inflammation. We hypothesized that inflammation induced by erythemal UV-B irradiation could elevate levels of serum C-reactive protein (CRP) and that suberythemal repeating doses of solar-simulating UV radiation (SSR) would produce photoadaptation to such inflammation. Separation-free high-sensitivity assays of CRP show an increase by 42% (P = 0.046) in CRP concentrations in healthy human subjects 24 h after a 3 minimal erythemal dose (MED) dose of UV-B delivered onto a 100 cm2 skin area. Preceding daily suberythemal doses of whole-body SSR for 10 or 30 consecutive days completely prevented the CRP increase. UV-B-induced skin erythema was partially attenuated by 30 preceding days of SSR only (P = 0.00066). After 10 daily SSR doses, the mean baseline CRP concentrations (0.24 +/- 0.21 mg/L) declined by 35% (P = 0.018). Using high-sensitivity analysis of serum CRP as the endpoint marker for cutaneous inflammation, we show that acute exposure of even a relatively small skin area to erythemal UV-B induces skin inflammation detectable also at the systemic level and that photoadaptation by preceding repeating suberythemal doses of SSR reduces signs of inflammation. Our data complement the view given by previous studies in that local photoadaptation also has systemic manifestations.     

Erythemal UV Radiation on Days with Low UV Index Values—an Analysis of Data from the German Solar UV Monitoring Network over a Ten‐year Period

According to the World Health Organization and partner organizations, no protection against ultraviolet (UV) radiation is required on days with “low” values (i.e., values <3) of the Global Solar Ultraviolet Index (UVI). Erythemal irradiance (E_er) data of such days were analyzed to evaluate this claim. Measurements from 9 stations of the German solar UV monitoring network from 2007 to 2016 yielded 14,431 daily E_er time series of low UVI days. Erythemal doses for certain fixed time intervals—acquired from measurements on horizontal planes—were compared with the average minimal erythemal dose (MED) of skin phototype II. Doses from days with rounded UVI values of 0 were insufficient to induce erythema and even on days with rounded UVI values of 1 doses exceeding 1 MED of skin type II could only be acquired under very specific circumstances of prolonged exposure. Conversely, sun exposure on days with rounded UVI values of 2 can indeed provide doses sufficient to induce erythema in skin type II after two hours around noon. In conclusion, our analyses do not support the claim of harmlessness currently associated with the entire low UVI exposure category in public guidance on interpretation of the UVI.     

Suppression of UVB-induced cutaneous erythema by a previous UVB exposure

People who vacation in sunny places are exposed to the sun on multiple occasions at least on a daily basis. The clinical assessment of sun exposure is erythema in the first 48 h after exposure and pigmentation at times greater than 3-5 days. The purpose of this investigations was to determine the extent to which consecutive erythemogenic exposures result in additive erythema responses. Studies were conducted in which volunteers were first exposed to a graded series of fluences of UVB radiation and then on subsequent days (1-3 days) the same sites along with the surrounding unexposed skin were challenged with varying fluences of UVB radiation. The erythema reactions were assessed clinically and were objectively documented with diffuse reflectance spectroscopy. The sites that received two exposures always showed a reduced erythema response compared to a single erythemogenic exposure. The suppression of erythema was more pronounced when the second exposure was given 48 h after the first. The erythema suppression was maximal when the first exposure was at 1.3 minimum erythema dose (MED). The pigment response to the first exposure was completely suppressed for fluences less than 1.5 MED. We thus provide evidence for a decoupling of the classical sequence of erythema-pigmentation response. We also show that the erythema induced by a second exposure may be substantially suppressed by an earlier exposure, and that this cannot be due to melanin photoprotection or due to substantial thickening of the stratum corneum. We propose that the cause may be some diffusible element of yet unknown origin.     

Diffuse Reflectance Spectroscopy as a Reliable Means of Comparing Ultraviolet Radiation‐induced Erythema in Extreme Skin Colors

Erythema is widely considered an indicator of skin cancer susceptibility, but assessments are challenging in black skin because melanin can mask erythema under traditional visual and advanced objective Commission Internationale de l'Eclairage (CIE) L *a *b * assessments. Using spectral measurements (400–700 nm) from a spectrophotometer, an algorithm was developed to measure erythema in white Caucasians (n = 9) and black West Africans (n = 11) 19–24 h postsolar simulated radiation (SSR) exposures to the volar forearm. The derived spectrum achieved showed a strong maximum peak for hemoglobin at 580 nm and a linear slope between 650 and 700 nm for melanin absorption, as reported by other authors. Absorption by hemoglobin at 580 nm was used as a proxy for erythema, and melanin was quantified between 650 and 700 nm. Our algorithm corrected the erythema measurements for stray specular (mirror‐like) reflection and the melanin‐masking effect. A linear relationship between SSR exposure and erythema was evident (p < 0.0001 for white and black skin), and white skin is 8.4 times more responsive to SSR compared to black skin. The prediction of ultraviolet radiation sensitivity is vital in both clinical and investigative dermatology especially in the determination of starting phototherapy doses. Our methodology allows for the accurate assessment of erythema independent of constitutive pigmentation.     

Mitigating Photosensitivity of Erythropoietic Protoporphyria Patients by an Agonistic Analog of α‐Melanocyte Stimulating Hormone†

Erythropoietic protoporphyria (EPP) is a rare hereditary disorder characterized by dermal accumulation of the photosensitizer protoporphyrin IX. Following sunlight exposure, the resulting photosensitivity is manifested first as pain, later as erythema, edema and dermal lesions. Afamelanotide (Nle⁴‐d‐Phe⁷‐α‐MSH), a synthetic analog of α‐melanocyte stimulating hormone and agonist of the melanocortin‐1‐receptor, promotes melanin synthesis, increasing skin pigmentation. This study examines the efficacy of afamelanotide in preventing symptoms in patients with EPP. A sustained‐release subcutaneous implant of 20 mg afamelanotide was administered twice, with a 60‐day interval to five EPP patients. Therapeutic efficacy was assessed by a photoprovocation test using standardized white light irradiation, melanin density (MD) determination and daily recording of sunlight exposure and symptoms. From Day 30 to Day 120 tolerance to photoprovocation significantly increased compared with baseline (P = 0.007) and skin MD was significantly higher than that recorded at baseline (P = 0.004). Except for two low‐grade pain episodes, patients recorded no phototoxic events past Day 4 of treatment. Tolerance to natural sunlight was up to 24 times longer than prior to therapy. The findings demonstrate beneficial effects of afamelanotide in patients with EPP. Due to the limited number of patients enrolled and the design being an open‐label study, confirmation by a large‐scale trial is required.     

Constitutive and Relative Facultative Skin Pigmentation among Victorian Children Including Comparison of Two Visual Skin Charts for Determining Constitutive Melanin Density

Our aim was to examine the association between ethnicity, phenotype, sun behavior and other characteristics, and constitutive and relative facultative skin pigmentation. A total of 191 participants were recruited, with a mean age of 7.6 years (SD 3.4), during 2009–2011 from Maternal and Child Health Centres (MCHC) and schools in Melbourne, Australia. Parental questionnaire data were obtained on sun behavior and examination consisted of noting the child's natural skin, hair and eye color, ethnicity, nevi count and spectrophotometric melanin density (MD). Constitutive skin pigmentation was estimated from buttock MD. Relative facultative skin pigmentation was estimated by hand compared with buttock absorption. Ethnicity, hair color and skin color were associated with constitutive and facultative skin pigmentation on univariate analysis. Higher ambient ultraviolet radiation (UVR) in the past month, greater freckling, greater nevi and increased sun exposure over the past year were related to darker facultative skin pigmentation. Sun exposure over the life course was not. The two skin charts accounted for 39.7% and 21.4% of buttock MD, respectively. Relative facultative skin pigmentation is associated with recent UVR levels, not life‐course sun exposure. Relative facultative skin pigmentation may not be a useful measure of sun exposure over the early life course. Skin color charts can be used to assess constitutive skin pigmentation if spectrophotometry is not available.     

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP-13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar-simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP-13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP-13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.     

Molecular responses to stress induced in normal human caucasian melanocytes in culture by exposure to simulated solar UV

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300-400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV-B and 110 kJ/m2 UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2-M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320-400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight-induced genotoxicity and maybe to melanocyte transformation.     

Ultrasound measurements of skin thickness after UV exposure: a feasibility study

High-frequency ultrasound images were used to measure the thickness of the dermis and epidermis of four human subjects. These measurements were performed before and after a single exposure to ultraviolet radiation (UV). Doses ranging from 0.5 to 3 minimal erythema doses (MED) were delivered to the skin of the back of four human subjects, and thickness measurements were made over a period of 16 days. We found: (1) exposures > or = 2 MED caused a 10-30% increase in the thickness of the dermis-epidermis layer; (2) the thickening response was not always in direct proportion to the UV dose; (3) maximum thickening response time was 48 h for the 2.8-3.0 MED exposure levels; (4) "diffusion" or spreading of the thickening response to neighboring areas occurred in some cases, as far as 4 cm from the exposed region (center-to-center), with changes ranging from 12% to 17%; (5) decreased thickness of the dermis-epidermis layer of up to 12% was observed for 3 out of 4 of the subjects.     

Objective measurement of minimal erythema and melanogenic doses using natural and solar-simulated light

Very little information exists on the amount of natural and artificial UV light required to cause sunburn and tanning in individuals with very pale skin who are at the greatest risk of developing skin cancer. We have investigated minimal erythema dose (MED) and minimal melanogenic dose (MMD) in a group of 31 volunteers with Fitzpatrick skin types I and II using an Oriel 1000 W xenon arc solar simulator and natural sunlight in Sydney, Australia. We measured the erythemal and melanogenic responses using conventional visual scoring, a chromameter and an erythema meter. We found that the average MED measured visually using the artificial UV source was 68.7 +/- 3.3 mJ/cm2 (3.4 +/- 0.2 standard erythema doses [SED]), which was significantly different from the MED of sunlight, which was 93.6 +/- 5.6 mJ/cm2 (P < 0.001) (11.7 +/- 0.7 SED). We also found significant correlations between the solar-simulated MED values, the melanin index (erythema meter) and the L* function (chromameter). The average MMD (obtained in 16 volunteers only) using solar-simulated light was 85.6 +/- 4.9 mJ/cm2, which was significantly less than that measured with natural sunlight (118.3 +/- 8.6 mJ/cm2; P < 0.05). We mathematically modeled the data for both the chromameter and the erythema meter to see if we were able to obtain a more objective measure of MED and differentiation between skin types. Using this model, we were able to detect erythemal responses using the erythema index function of the erythema meter and the a* function of the chromameter at lower UV doses than either the standard visual or COLIPA methods.     

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.     

UV-induced unscheduled DNA synthesis in human skin: dose response, correlation with erythema, time course and split dose exposure in vivo

Unscheduled DNA synthesis (UDS) has been shown to be saturated above a threshold dose of UV-C in human fibroblasts in vitro. We have investigated by autoradiography whether a similar saturation occurs in human skin in vivo with UV-B and whether this phenomenon correlates with the erythemal response. In addition, we determined the time course of UDS at 24 h after exposure and the effect of dual exposures separated by 24 h. The dose-response curve was established by exposure to 1/16, 1/8, 1/4, 1/2, 1, 2, 3, 4 and 6 MEDs UV-B. For the time-course study, areas exposed to 1/2 and 2 MEDs were biopsied after 1, 3, 6, 12 and 24 h. Autoradiography was performed in vitro. The dose-response curve showed a significant increase in UDS from 1/16 to 1 minimal erythema dose (MED), whereas no significant difference was observed between 1 MED and the higher UV-B doses tested. The 24 h time sequence revealed a gradual decrease in UDS activity. The 1/2 MED curve declined more rapidly and reached the zero-level between 12 h and 24 h, whereas about 50% of the initial UDS value was still retained 24 h after 2 MEDs. The dual-dose study revealed that a second hit of fractions of the MED resulted in lower levels of UDS than induced by these fractions alone in previously untreated areas. UDS increases with the erythemal dose between 1/16 and 1 MED. It reaches a plateau after 1 MED and cannot be increased by doses up to 6 MEDs, suggesting a saturation of excision repair in vivo. Time course studies support such a saturation phenomenon. The failure to increase significantly UDS by a second irradiation 24 h after the first exposure needs further clarification. Since persistence of DNA lesions may lead to an accumulation after repeated exposures, additional mechanisms other than excision repair may protect human skin by error-free removal of possibly mutagenic sites. Photoreactivation may be important in this respect.     

Solar-simulated Ultraviolet Irradiation Induces Selective Influx of CD4+ T Lymphocytes in Normal Human Skin

Abstract— The proportion and composition of the human cutaneous CD3+ T lymphocyte population was determined in situ following a single exposure to physiological, erythema-inducing doses of simulated solar radiation, mainly consisting of UV radiation. Biopsies were taken 1, 2 and 7 days after local irradiation of normal volunteers with 1,2 and 4 MED by a xenonarc lamp and immunohistochemistry was performed on cryostat sections. Ultraviolet radiation caused an initial decrease of intraepidermal CD3+ T-cell numbers or even could lead to T-cell depletion 24 and 48 h postirradiation, and this was followed by an infiltration of T cells in the epidermis as determined 1 week after UV exposure. The number of dermal CD3+ T ceDs was increased 24 h after irradiation, reached a maximum at 48 h and subsequently declined at day 7, though remained significantly higher than the unirradiated control Double staining demonstrated that the CD3+ T cells, which immigrated into the (epi)dermis upon UV exposure, coexpressed CD4 but not CD8. Therefore the CD4/CD8 ratio in skin was markedly increased during the first week upon UV exposure. Our time course study shows that UV radiation affects die T-cell population within human skin by depleting the majority of epidermal T cells and initiating a selective influx of CD4+ T cells.     

Occupational UV Exposure of Environmental Agents in Valencia,Spain

Excessive exposure to ultraviolet radiation (UVR) is considered the most important environmental risk factor in the development of melanoma and skin cancer. Outdoor workers are among those with the highest risk from exposure to solar UVR, as their daily activities constantly expose them to this radiation source. A study was carried out in Valencia, Spain, in summer 2012 and involved a group of 11 workers for a period of six 2‐day recordings. Sensitive spore‐film filter‐type personal dosimeters (VioSpor) were used to measure erythemal UVR received by environmental agents in the course of their daily work. Median 2‐day UV exposure was 6.2 standard erythema dose (SED), with 1 SED defined as effective 100 J m^?2 when weighted with the Commission Internationale de L′Eclairage's (CIE) erythemal response function. These workers were found to receive a median of 8.3% total daily ambient ultraviolet erythemal radiation. Comparison with the occupational UV exposure limit showed that the subjects had received an erythemal UV dose in excess of occupational guidelines, indicating that protective measures against this risk are highly advisable.     

Ultraviolet B radiation induces upregulation of calcitonin gene-related peptide levels in human Finn chamber skin samples

One of the neuropeptides that plays a role in UVB-induced immunomodulation is calcitonin gene-related peptide (CGRP), as demonstrated in several animal studies. CGRP can be detected in human skin, but effects of UVB exposure on CGRP levels in human skin are not known. We determined CGRP levels in human Finn chamber skin samples of 15 UVB-irradiated and 10 control volunteers. Filter samples were collected prior to and immediately after a UVB exposure protocol (5 consecutive days, with one personally determined minimal erythema dose (MED(jp)) per day). CGRP levels in filter samples were determined using a commercially available radioimmunoassay kit. CGRP could be detected in the filter samples and volunteers showed statistically significantly increased levels after UVB exposure. In addition, the CGRP levels of UVB-exposed volunteers were positively correlated with the dose of UVB in J/m(2) that they received on 5 consecutive days. In other words, higher UVB doses resulted in higher CGRP levels. In summary, CGRP, a mediator in UVB-induced immunomodulation, could be detected in human Finn chamber skin samples, and was significantly increased after UVB exposure. The CGRP level appeared to depend on the amount of UVB the volunteers received.     

A comparative pilot study on ultraviolet-induced skin changes assessed by noninvasive imaging techniques in vivo

The effects of acute and chronic ultraviolet (UV) on the morphology of human skin have been extensively studied ex vivo by means of histological investigations. However, innovative skin imaging techniques enable visualization of micromorphological structures in vivo. We aimed to perform a correlation study evaluating in vivo dose and time dependent skin changes following solar-simulated irradiation using noninvasive techniques such as optical coherence tomography (OCT) and confocal laser scanning microscopy (CLSM). The forearms of 10 healthy subjects were exposed to 1 minimal erythema dose (MED) and 3 MED of solar-simulated radiation. Noninvasive measurements were performed before and 24 h and 72 h after UV exposures. We demonstrate definite OCT and CLSM findings obtained from UV-exposed skin, including an increase in epidermal thickness (hyperproliferation, acanthosis), a reduction in dermal reflectivity (dermal edema), an increase in brightness of the basal layer (pigmentation), and an increase in vessel diameter within the dermal papillae (vasodilatation). A moderate to strong linear association between the methods employed was observed. In conclusion, noninvasive high-resolution imaging techniques such as OCT and CLSM may be promising tools for photobiological studies aimed at assessing photoadaptive and/or phototoxic processes in vivo. However, larger studies are needed to demonstrate the applicability of the findings presented in this pilot study.