Solar-simulated Radiation Exposure Research Articles

O06 Naïve human keratinocytes initiate a chromatin-protective mechanism in response to ultraviolet radiation through the upregulation of histone deacetylase 5

Abstract Introduction and aims During normal development and throughout life, epigenetic changes arise in response to an individuals’ behaviour and environmental exposures. Histone deacetylases (HDACs) are chromatin-modifying enzymes that regulate gene expression and their activity and expression in primary human keratinocytes alters with increasing age. Solar ultraviolet radiation (UVR) is a major causative factor of skin ageing, but how this factor affects HDAC expression and activity is largely unknown. We aimed to determine how UVR exposure affects HDAC expression and activity in primary human keratinocytes. Methods Normal human epidermal keratinocytes (NHEKs) from juvenile (aged 1–6 years; n = 3), middle-aged (56–61 years; n = 3) and aged (83–85 years; n = 3) donors were exposed to solar-simulated radiation (SSR) and HDAC abundance and activity were assayed. Results Exposure to SSR significantly increased HDAC activity in primary NHEKs irrespective of donor age (P < 0.05). However, Western blotting for HDAC abundance revealed that there was a differential response to SSR exposure across age groups; juvenile NHEKs expressed significantly more HDAC5 in response to SSR (P < 0.05) than cells from middle-aged or aged donors. Furthermore, following SSR exposure of juvenile NHEKs, the cellular localization of HDAC5 was redistributed to the nucleus from the cytoplasm. Dual immunofluorescence staining revealed that those NHEKs with nuclear HDAC5 expression were also positive for p16INK4a, a robust marker of cellular senescence (P < 0.01). Conclusions Our Results suggest that naïve human keratinocytes are responsive to UVR exposure and appear to initiate a chromatin-protective mechanism through the upregulation of HDAC5 to prevent gene expression of damaged DNA.

289 Oral administration of table grapes has a photoprotective effect on the sunburn response and on cyclobutane pyrimidine dimers in humans in vivo

288 Dietary green tea catechins protect dermal elasic fibers from UV-induced remodeling N Charoenchon, LE Rhodes, A Nicolaou, G Williamson, MD Farrar and RE Watson 1 The University of Manchester, Manchester, United Kingdom and 2 The University of Leeds, Leeds, United Kingdom Cutaneous photoaging is associated with dermal elastic fiber remodeling; daily photoprotection is therefore essential to prevent ultraviolet (UV)-induced skin damage. Green tea (C sinensis), rich in polyphenol catechins (GTC), have been reported to have significant skin benefits in vitro and in vivo. We have performed a double-blind randomised controlled trial to assess whether systemic GTC supplementation can protect dermal elastic fibers from acute UV exposure. Healthy white Caucasians (n1⁄450; 18-65 years) were randomized (1:1) to 12 weeks daily oral supplement (1080mg GTC with 100mg viatmin C) or placebo (maltodextrin). Biopsies were taken from skin exposed to 3xMED of solar simulated radiation (SSR) and unexposed skin at baseline and post-supplementation; compliance was confirmed by urinary GTC metabolite analysis. Dermal elastic fibers were identified via histology and immunohistochemistry, their distribution assessed in the papillary dermis (from the dermal-epidermal junction to a depth of 100mm) by image analysis. Differences between UV-exposed and unexposed skin were analysed by paired t-test. A total of 44 subjects completed the study, being compliant with supplementation and providing all skin samples. At baseline, SSR induced a significant overall loss of elastic fiber components (P1⁄4 0.001), and specifically fibulins-2 and -5. Post-supplementation, UV-mediated loss of fibulin-5 remained significant in the placebo group with mean (SD) % fiber area 19.4 (3.8) and 17.7 (4.1) in unexposed and UV-exposed skin resectively (P1⁄40.01), whilst in the active group fibulin-5 was protected with % fiber area 18.1 (4.0) and 17.1 (2.7) in unexposed and UV-exposed skin respectively (P1⁄4 0.30). Hence, acute SSR exposure results in elastic fiber remodeling similar to that observed in chronically photoaged skin. Furthermore, in a randomized control trial, dietary GTC protected fibulin-5 microfibrils in the papillary dermis from UV-mediated degradation. 289 Oral administration of table grapes has a photoprotective effect on the sunburn response and on cyclobutane pyrimidine dimers in humans in vivo AS Oak, R Shafi, S Katiyar, AT Slominski, W Cantrell, C Wang, RK Srivastava, C Li, M Athar and C Elmets Dermatology, University of Alabama at Birmingham, Birmingham, AL Table grapes contain proanthocyanidins and resveratrol which have been shown to have photoprotective properties in animal models. The purpose of this study was to determine whether oral administration of grapes had a similar effect in humans. Eleven healthy human volunteers (Fitzpatrick skin phototypes I-III, aged 19-46 and without any significant comorbidities) were recruited for the study (uncontrolled longitudinal study). The minimal erythema dose (MED) on the inner aspect of the forearm was determined using a broadband UVB light source. Skin biopsies were also performed at baseline from a sun protected area on the hip and from the 1 MED treated site on the forearm. Volunteers were then instructed to take 25 grams of grape powder three times per day for 2 weeks. At the end of the treatment period, the MED was again performed. In addition, skin biopsies were repeated at the end of two weeks in 9 of the 11 subjects after taking the grape powder. The biopsies were processed and stained for cyclobutane pyrimidine dimers (CPDs). The mean MED SEM increased in subjects from 205 30 J/m to 346 59 J/m, after taking the grape powder, which was statistically significant (paired t-test, p1⁄40.05). Oral administration of grape powder also inhibited CPD formation. There was a 32% decline in CPDs in pretreatment vs. post-grape treatment biopsies from the MED skin site (pretreatment: 32.6 5.2%, posttreatment: 23.1 4.1%, p1⁄40.005). Oral administration of grape powder did not have a significant effect on the number of CPDs in the unirradiated skin sites (pretreatment: 2.7 .9%, posttreatment: 3.8 .9%, p1⁄4.15). These studies demonstrate that oral administration of grapes has a photoprotective effect on the MED and in attenuating CPDs in human skin, and validate the findings from preclinical studies.

A new hair follicle-derived human epidermal model for the evaluation of sunscreen genoprotection

Induction of skin cancer is the most deleterious effect of excessive exposure to sunlight. Accurate evaluation of sunscreens to protect the genome is thus of major importance. In particular, the ability of suncare products to prevent the formation of DNA damage should be evaluated more directly since the Sun Protection Factor is only related to erythema induction. For this purpose, we developed an in vitro approach using a recently characterized reconstituted human epidermis (RHE) model engineered from hair follicle. The relevance of this skin substitute in terms of UV-induced genotoxicity was compared to ex vivo explants exposed to solar-simulated radiation (SSR). The yield of bipyrimidine photoproducts, their rate of repair, and the induction of apoptosis were very similar in both types of skin samples. In order to evaluate the protection afforded by sunscreen against DNA damage, bipyrimidine photoproducts were quantified in tissue models following SSR exposure in the presence or absence of a SPF50+ formula. A rather high DNA protection factor of approximately 20 was found in RHE, very similar to that determined for explants. Thus, RHE is a good surrogate to human skin, and also a convenient and useful tool for investigation of the genoprotection of sunscreens.

Comparative analysis of solar radiation‐induced cellular damage between ex vivo porcine skin organ culture and in vitro reconstructed human epidermis

Reconstructed human epidermis models (RHE) constitute an innovative alternative to study phototoxicity and photoprotection in the cosmetic industry. However, little information is currently available concerning the harmful effects of solar-simulated radiation (SSR) in these in vitro skin models. In this study, the phototoxic effects of a single acute SSR dose of 275 kJ m(-2) were evaluated in a validated RHE model (from SkinEthic), and were compared with those obtained from an ex vivo skin organ culture recently developed from domestic pig ears. The RHE model was well differentiated in vitro and released a significant level of the cytosolic enzymes lactate dehydrogenase (LDH) and extracellular signal-related kinase 2 (ERK2) protein in the culture medium 24 h after SSR exposure. The SSR-induced cytotoxicity was related to the formation of sunburn cells and the appearance of DNA damage (thymine dimer and DNA fragmentation) in keratinocytes. Interestingly, these DNA alterations were associated with the activation of the caspase-3 protease, mainly in the basal layers of the epidermis. In addition, the RHE model responses were comparable with porcine skin following solar irradiation, and none of the above cellular responses was observed in non-irradiated skin models. Finally, topical application of a broad-spectrum UVB + A sunscreen formulation efficiently protected both the RHE and pig skin against the deleterious effects of SSR. Thus, both RHE and ex vivo pig skin organ culture models are complementary tools in the assessment of SSR-induced DNA damage and apoptosis, and they may be used to evaluate the photoprotective capacity of cosmetic formulations.

Sunscreens containing the broad‐spectrum UVA absorber, Mexoryl® SX, prevent the cutaneous detrimental effects of UV exposure: a review of clinical study results

UVA exposure of human skin mainly produces reactive oxygen species (ROS) leading to DNA, cell and tissue damage. It alters immune function, pigmentation and it is certainly responsible for a large part of photoaging changes. Moreover UVA is implicated in the etiology of several photodermatoses. As a consequence, to provide adequate protection, sunscreens or skin care products for daily use protective products need UVA absorbers combined with UVB ones. To assess the efficacy of sunscreens containing a broad-spectrum UVA absorber the Mexoryls SX or ecamsule and to compare formulations with and without it through a large number of clinical studies in human volunteers and patients. The following assessments were conducted: *Prevention of excessive pigmentation induced by UV exposure in Caucasian and Asian skins using a method that measures pigmentation protection factors (PPF). *Efficacy against DNA damage by measurement of pyrimidine dimer formation and p53 protein accumulation. *Protection of immune system using delayed type hypersensitivity (DTH) reactions to recall antigens, isomerization of urocanic acid (UCA), alteration of Langerhans cells (LC) density, morphology and function. *Reduction of epidermal and dermal alterations induced by repeated UVA or UV solar simulated radiation (SSR) using histology or immunohistology. *Prevention of the polymorphous light eruption (PMLE) in patients prone to develop this disease. Mexoryls SX-containing formulations showed a dose-dependent level of protection against pigmentation. For a same sun protection factor (SPF) the higher the UVA protection was, the higher was the PPF. Pyrimidine dimer formation and p53 accumulation were significantly reduced by formulations with Mexoryls SX. In the studies looking at the suppression of DTH reactions to recall antigens by the different UV spectra, the LC alterations and the cis UCA formation, Mexoryls SX formulations always showed a higher protective potency than sunscreen without it even when the protection against erythema was similar (products with same SPF). Mexoryls SX formulations also prevented or significantly decreased to minimal, ferritin, tenascin and lysozyme expression induced by repeated UVA or SSR exposure. It also reduced the enhancement of collagenase 2 mRNA expression induced by SSR exposure. Finally PMLE study demonstrated that UVA protection was essential for the prevention of this photodermatose. Mexoryls SX formulated in sunscreens or daily use products have been shown to be an effective UV absorber, leading to an increased efficacy of these products against a large number of biological damage induced by UVA, SSR or sun exposure.

Increased cyclooxygenase expression and thymine dimer formation after repeated exposures of humans to low doses of solar simulated radiation

The impact of repeated doses of solar simulated radiation (SSR) has not been evaluated, particularly to determine if photoadaptation and photoprotection develop over time. In this study, erythema, pigmentation, cyclooxygenase (COX)-1 and 2 expression and thymine dimer (dTT) formation were evaluated in the skin of irradiated subjects of phototype II or III. Groups of 7-10 volunteers were whole-body irradiated with a low dose of SSR on each of 10 consecutive days followed by a single erythemal ultraviolet B (UVB) dose on a small body area, or irradiated only with the single erythemal UVB dose on a small body area, or irradiated with the low dose of SSR on each of 30 consecutive days, or were unirradiated. Erythema and pigmentation were measured 24 h after the final SSR or UVB, and skin biopsies collected for the assessment of COX(+) cells and dTT(+) nuclei. The repeated SSR exposures induced a small increase in pigmentation without erythema, and were slightly protective against the erythemal effects of the subsequent high UVB dose. The number of COX-1(+) and 2(+) cells increased as a result of 10-days SSR and rose still further after 30-days SSR, indicating that photoadaptation had not developed. The SSR exposures did not result in any protection against the further increase in COX-1 and 2 expression caused by the erythemal UVB dose. In contrast, for dTT formation, the repeated SSR exposures led to a limited degree of both photoadaptation and photoprotection.

Elicitation of contact hypersensitivity after repeated suberythemal exposures of humans to solar simulated radiation: number of epidermal Langerhans cells

Ultraviolet radiation suppresses contact hypersensitivity (CHS). The role of epidermal Langerhans cells (LCs, CD1a(+)) in the elicitation phase of CHS is uncertain. To assess the effect of low-doses of solar simulated radiation (SSR) on LC numbers at the CHS elicitation site. 3 groups (each about 30 volunteers) were whole-body irradiated with suberythemal SSR on 2, 10 or 30 consecutive days before sensitization with diphenylcyclopropenone. Another group was not irradiated. Elicitation of CHS took place 3 weeks later with subsequent evaluation by visual scoring and spongiosis grade. CD1a(+) cells in the epidermis from the elicitation site were counted. No difference in CHS intensity between the unirradiated controls and all 3 irradiated groups was found, but a significant negative correlation between the spongiosis grade and the number of SSR exposures was shown. The number of epidermal CD1a(+) cells in the 10- and 30-day groups was reduced compared with the unirradiated group, and the 30-day group had significantly fewer than the 10-day group. Low daily doses of SSR induce suppression of CHS, leading to depletion of LCs at the CHS elicitation site. The effect on the CHS and LCs is cumulative, indicating that photoadaptation for these parameters does not develop over the 30 day irradiation period.

Adaptation to the UV-induced suppression of phagocytic activity in murine peritoneal macrophages following chronic exposure to solar simulated radiation

Exposure of certain strains of mice to ultraviolet radiation (UVR) is known to suppress both local and systemic immune responses, including a reduction in the phagocytic activity of peritoneal macrophages. However, in many instances, the immunological effects have been observed following a single or a limited number of doses of UVR from sources containing a higher proportion of UVB than that emitted by the sun. The first aim of the present study was to establish whether a single exposure of C3H/HeN mice to solar simulated radiation (SSR) suppressed the ability of the peritoneal macrophages to phagocytose opsonised sheep red blood cells. The mice were irradiated with SSR from Cleo Natural lamps and a single dose of 31.9 J cm(-2) was found to be the minimal dose for significant suppression of macrophage phagocytic activity. Such a dose did not modulate the surface expression of I-A(k), CD11b, CD86 or FcgammaRII/III (CD32/16) on the macrophages. The second aim was to assess whether repeated SSR exposures with a dose below the minimal immunosuppressive dose affected macrophage activity and, if so, to test for photoadaptation by repeated exposures followed by a single, normally immunosuppressive dose of SSR, and then assaying the macrophage activity. Groups of mice were irradiated on each of 2, 10 and 30 days with 14.9 J cm(-2) SSR, followed in some instances by a single additional exposure of 31.9 J cm(-2) on the same day as the last irradiation. The phagocytic activity of the peritoneal macrophages was tested 24 h later. It was reduced by 32%, 18% and 4% respectively after 2, 10 and 30 repeated exposures to SSR, and by 39%, 21% and 7% respectively after 2, 10 and 30 repeated exposures plus the additional higher dose at the end. Thus, although the macrophage activity was initially suppressed by the SSR, photoadaptation of this immune parameter occurred following repeated exposures.

Matrix Metalloproteinase‐1 Production Observed After Solar‐Simulated Radiation Exposure is Assumed by Dermal Fibroblasts but Involves a Paracrine Activation Through Epidermal Keratinocytes¶

ABSTRACTChronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix‐degrading proteases. Among them, matrix metalloproteinase‐1 (MMP‐1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar‐simulated radiation (SSR) on MMP‐1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast‐populated dermal equivalent led to an increase in MMP‐1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP‐1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP‐1 in response to SSR exposure. However, when conditioned medium from SSR‐exposed keratinocytes was added to human fibroblasts in culture, MMP‐1 production was induced. Altogether, these data show that MMP‐1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.

Matrix metalloproteinase-1 production observed after solar-simulated radiation exposure is assumed by dermal fibroblasts but involves a paracrine activation through epidermal keratinocytes.

Chronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix-degrading proteases. Among them, matrix metalloproteinase-1 (MMP-1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar-simulated radiation (SSR) on MMP-1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast-populated dermal equivalent led to an increase in MMP-1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP-1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP-1 in response to SSR exposure. However, when conditioned medium from SSR-exposed keratinocytes was added to human fibroblasts in culture, MMP-1 production was induced. Altogether, these data show that MMP-1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.

Effects of solar-simulated radiation dose fractionation on CD1a+ Langerhans cells and CD11b+ macrophages in human skin.

There are few human studies investigating the immunosuppressive effects of exposure to solar-simulated radiation (SSR) and its relationship with sunburn/erythema, and few comparative data on the importance of SSR exposure regimens. To evaluate whether SSR-induced erythema is a reliable end-point for assessing damage to antigen-presenting cells (APCs) in human skin. We compared the relationship between SSR-induced erythema and alterations in epidermal CD1a+ Langerhans cells (LCs) and CD11b+ macrophages in human volunteers after single exposures to 0, 0.5, 1, 2 or 3 minimal erythema doses (MED). We also investigated whether SSR exposure leads to an accumulation or accommodation of the same end-points by comparing the effects of a relatively low cumulative SSR dose (3 MED) given in varying daily dose fractions (4 x 0.75 MED, 2 x 1.5 MED and 1 x 3 MED). Single SSR exposures induced a dose-dependent increase in erythema. CD1a+ LCs remaining in the irradiated epidermis showed a dose-dependent increase in cell size and altered morphology. Significant depletion of CD1a+ LCs and presence of CD11b+ macrophages only occurred in sites irradiated with 2 MED and 3 MED. Dose fractionation had no effect on the final erythemal response but the 4 x 0.75 MED and 1 x 3 MED protocols were better tolerated than 2 x 1.5 MED for alterations in CD1a+ LC and CD11b+ cell numbers. In contrast, dose fractionation protected against alterations in CD1a+ LC morphology or cell size. We found that erythema is a poor indicator of alterations in epidermal APCs and that dose fractionation is an important parameter in the immunological effects of ultraviolet radiation.

Ultraviolet Radiation Induced Apoptosis in Human Skin In Vivo

Sunburn cells, having many characteristics of apoptotic cells, appear in human skin after exposure to UVB. Timecourses and dose responses for solar simulated radiation (SSR)-induced sunburn cells in human volunteers of skin type II have been determined. For the time-course, two groups of volunteers were exposed to two minimal erythema doses (MED) of SSR. Punch biopsies were obtained from Group 1 immediately, 3, 6, 12, 18 and 24 h after SSR exposure and Group 2 were biopsied immediately, 18, 24, 36, 48 and 72 h after exposure. For the dose-response (Group 3), biopsies were taken 24 h after SSR exposure to 0, 0.25, 0.5, 1, 2 and 3 MED. Sections were stained with H&E and also using TUNEL and analysed by light microscopy. Results show a dose-dependent appearance of SBC after SSR exposure. The time point for maximum SBC counts with both H&E staining and TUNEL staining lie between 24 and 36 h.

Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: Evaluation and modification of risk of human use in an albino mouse skin model

The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle ( i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5–6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.