Narrow‐band UVB‐induced Externalization of Selected Nuclear Antigens in Keratinocytes: Implications for Lupus Erythematosus Pathogenesis†

The aim of this study was to analyze whether sera obtained from patients with lupus erythematosus (LE) react with membrane structures found on keratinocytes irradiated with narrow‐band ultraviolet B (NB‐UVB). We applied atomic force microscopy (AFM) to visualize cell surface structures expressing nuclear antigens upon apoptosis following NB‐UVB irradiation. Immortalized human keratinocytes (HaCaT) were cultured under standard conditions, irradiated with 800 mJ cm^?2 NB‐UVB light and imaged by AFM mounted on an inverted optical microscope. It was observed that NB‐UVB irradiation provoked significant alterations of the keratinocyte morphology and led to the membrane expression of antigens recognized by anti‐La and anti‐Ro 60 kDa sera but not by antidouble‐strand DNA sera. The presence of La and Ro 60 kDa antigens on keratinocyte surfaces after NB‐UVB irradiation was limited mainly to the small bleb‐like protrusions found on the keratinocytes by AFM. A closer investigation by AFM also revealed that some structures positively stained with anti‐Ro 60 kDa serum were also located submembranously. We hypothesize that the externalization of some nuclear antigens because of NB‐UVB exposure might be responsible for exacerbation of skin symptoms in patients suffering from LE.     

Epidermal cis-urocanic acid levels correlate with lower specific cellular immune responses after hepatitis B vaccination of ultraviolet B-exposed humans

Urocanic acid (UCA) is a major UV-absorbing chromophore in the epidermis and has been suggested to act as one of the initiators of UV-induced immunosuppression. cis-UCA, the isomer from UCA that is formed upon UV exposure, has been shown to impair some cellular immune responses. cis-UCA levels were determined in a study in which the influence of ultraviolet B (UVB) exposure on immune responses after hepatitis B vaccination in human volunteers was established. A significant increase in cis-UCA levels was found in the skin of UVB-exposed volunteers compared with controls. cis-UCA levels, calculated as the percentage of the total UCA amount, in UVB-exposed volunteers correlated significantly with the cumulative UVB dose received in 5 consecutive days, i.e. the higher the UVB dose (J/m2), the higher the cis-UCA levels (until a cis-UCA plateau was reached in the so-called photostationary state). Correlations between skin cis-UCA levels and immune responses were determined, and they revealed no statistically significant correlations among lymphocyte proliferation responses after either mitogenic stimulation or stimulation with recall antigens. No correlation was found between cis-UCA levels and hepatitis B-specific antibody titers. However, we found a statistically significant negative correlation between cis-UCA levels and hepatitis B-specific lymphocyte proliferation responses when volunteers were irradiated with UVB before hepatitis B vaccination. In other words, volunteers with high cis-UCA levels caused by UVB exposure showed lower cellular immune responses against hepatitis B antigen after hepatitis B vaccination.     

UVB Radiation Affects Growth,Reproduction and Tissue Structure of Daphnia magna Across Several Temperatures

We examined the effects of daily exposure to UVB on growth, reproduction and histological characteristics of Daphnia magna over two generations at 20, 22, 25 and 30°C. Animals were exposed to 16 h of UVA and photosynthetically active radiation daily. Treated animals received 6 h of UVB during the light phase. Parental (P) generation growth and reproduction was impaired by exposure to UVB at all temperatures, with the poorest production at 30°C. First brood size decreased with UVB exposure; it was lowest at 30°C. Although F1 length at birth increased with P generation age, F1 produced by UVB‐exposed mothers were smaller at all temperatures. The F1 generation was followed at 20 and 25°C; at both temperatures UVB exposure reduced F1 growth and reproduction. F1 growth and F2 production were lowest when both P and F1 generations were exposed to UVB. UVB exposure damaged ovarian and gut tissue at both 25 and 30°C; the consequences of this exposure were more severe at 30°C. The observed tissue damage may relate directly to the UVB‐induced impairment of growth and reproduction. This study provides new insights into the effects of UVB on an important component of the pelagic zooplankton.     

Endogenous Retinoic Acid Required to Maintain the Epidermis Following Ultraviolet Light Exposure in SKH‐1 Hairless Mice

Ultraviolet light B (UVB) exposure induces cutaneous squamous cell carcinoma (cSCC), one of the most prevalent human cancers. Reoccurrence of cSCC in high‐risk patients is prevented by oral retinoids. But oral retinoid treatment causes significant side effects; and patients develop retinoid resistance. Exactly how retinoids prevent UVB‐induced cSCC is currently not well understood. Retinoid resistance blocks mechanistic studies in the leading mouse model of cSCC, the UVB‐exposed SKH‐1 hairless mouse. To begin to understand the role of retinoids in UVB‐induced cSCC we first examined the localization pattern of key retinoid metabolism proteins by immunohistochemistry 48 h after UVB treatment of female SKH‐1 mice. We next inhibited retinoic acid (RA) synthesis immediately after UVB exposure. Acute UVB increased RA synthesis, signaling and degradation proteins in the stratum granulosum. Some of these proteins changed their localization; while other proteins just increased in intensity. In contrast, acute UVB reduced the retinoid storage protein lectin:retinol acyltransferase (LRAT) in the epidermis. Inhibiting RA synthesis disrupted the epidermis and impaired differentiation. These data suggest that repair of the epidermis after acute UVB exposure requires endogenous RA synthesis.     

A Review of Sunscreen Safety and Efficacy

The use of sunscreen products has been advocated by many health care practitioners as a means to reduce skin damage produced by ultraviolet radiation (UVR) from sunlight. There is a need to better understand the efficacy and safety of sunscreen products given this ongoing campaign encouraging their use. The approach used to establish sunscreen efficacy, sun protection factor (SPF), is a useful assessment of primarily UVB (290–320 nm) filters. The SPF test, however, does not adequately assess the complete photoprotective profile of sunscreens specifically against long wavelength UVAI (340–400 nm). Moreover, to date, there is no singular, agreed upon method for evaluating UVA efficacy despite the immediate and seemingly urgent consumer need to develop sunscreen products that provide broad-spectrum UVB and UVA photoprotection. With regard to the safety of UVB and UVA filters, the current list of commonly used organic and inorganic sunscreens has favorable toxico-logical profiles based on acute, subchronic and chronic animal or human studies. Further, in most studies, sunscreens have been shown to prevent the damaging effects of UVR exposure. Thus, based on this review of currently available data, it is concluded that sunscreen ingredients or products do not pose a human health concern. Further, the regular use of appropriate broad-spectrum sunscreen products could have a significant and favorable impact on public health as part of an overall strategy to reduce UVR exposure.     

Phosphorylation of Histone H2AX Generated by Linear Alkylbenzene Sulfonates and its Suppression by UVB Exposure

We previously demonstrated that the nonionic surfactants, nonylphenol polyethoxylates (NPEOs) induced the phosphorylation of histone H2AX (γ‐H2AX), accompanied by DNA double‐strand breaks (DSBs), and that exposure to ultraviolet (UV) degraded NPEOs, which sometimes enhanced their DNA‐damaging ability. In this study, we showed that linear alkylbenzene sulfonates (LAS), general anion surfactants, also generated DSBs with γ‐H2AX, and this ability was attenuated by UVB exposure. In the human breast adenocarcinoma cell line, MCF‐7, γ‐H2AX was generated in a dose‐dependent manner immediately after cells were treated with LAS, and this was attributed to the formation of DSBs and was independent of cell cycle phases. The ability to generate γ‐H2AX was markedly reduced in LAS exposed to UVB. HPLC analysis revealed that LAS were a mixture of various alkyl chain lengths, the peaks of which were detected at individual retention times. UVB evenly decreased all peaks of LAS, without migration of peaks to other retention times, which indicated that UVB may degrade the benzene ring of LAS, but did not shorten the alkyl chains. UVB is an important environmental factor in the degradation of LAS exhibiting the ability to induce DSBs, the most serious type of DNA damage.     

Sunshine is an Important Determinant of Vitamin D Status Even Among High‐dose Supplement Users: Secondary Analysis of a Randomized Controlled Trial in Crohn's Disease Patients

Sunshine is considered to be the most important source of vitamin D. Due to an increased risk of skin cancer, sun avoidance is advised, but this directly contributes to the high prevalence of vitamin D deficiency. The simple solution is to advise vitamin D supplementation. The aim of this study was to examine the absolute and relative contribution of sunshine and supplementation to vitamin status. This study was a secondary analysis of an RCT of 92 Crohn's disease patients in remission (49% female, median age = 44). Participants were randomized to 2000 IU day^?1 of vitamin D3 or placebo for 1 year, with 25‐hydroxyvitamin D (25(OH)D) being measured at baseline and every 4 months. Based on participant's place of residence, daily ambient UVB dose at wavelengths that can induce vitamin D synthesis (D‐UVB) was obtained. Cumulative and weighted ambient D‐UVB (cw‐D‐UVB) exposure prior to each blood draw was calculated for each participant. Linear regression analysis and multilevel modeling were used to examine the association between UVB exposure, supplementation and 25(OH)D concentration. There was considerable annual variation in D‐UVB, cw‐D‐UVB and 25(OH)D. Both supplementation and cw‐D‐UVB were found to be strongly associated with 25(OH)D: in multilevel model, an increase of approximately 6 nmol L^?1 for every 100 kJ m^?2 in cw‐D‐UVB was found, among those receiving placebo and supplementation (P < 0.0001). Treatment was associated with increase of 23 nmol L^?1 (P < 0.0001). Sunshine is an important determinant of 25(OH)D concentration, even in those who are taking high‐dose vitamin D supplements and reside at a higher mid‐latitude location.     

Reduced Levels of Tissue Inhibitors of Metalloproteinases in UVB‐Irradiated Corneal Epithelium

Tissue inhibitors of metalloproteinases (TIMPs) are the major endogenous regulators of metalloproteinase activity in tissues. TIMPs are able to inhibit activity of all known matrix metalloproteinases (MMPs) and thus participate in controlling extracellular matrix synthesis and degradation. We showed previously elevated expressions of MMPs in the rabbit corneal epithelium upon UVB exposure and suggested that these enzymes might be involved in corneal destruction caused by excessive proteolysis. The aim of this study was to investigate TIMPs in the corneal epithelium after UV irradiation using immunohistochemical and biochemical methods. We found that as compared to control rabbit corneas where relatively high levels of TIMPs were present in the epithelium, repeated irradiation of the cornea with UVB rays (not with UVA rays of similar doses) significantly decreased TIMPs in corneal epithelial cells. The results of this study point to the suggestion that the decrease in TIMPs in the corneal epithelium after UVB irradiation contributes to increased proteolytic activity of MMPs in UVB‐irradiated corneal epithelium found previously.     

UV radiation may explain intercity difference for cataract in A-bomb survivors

Accurate assessment of risk factors is important for the evaluation of radiation-induced ocular lens damage. Our previous study identified a significant city difference between Hiroshima and Nagasaki atomic-bomb survivors in terms of cataract prevalence, prompting further analysis. This study analyzed the sites of lens opacities and used model fitting that incorporated the variable impact of UV on the eye, based on the hypothesis that the city difference in the prevalence of cataract was due to differences in UV radiation between the two cities. The results suggested that cataracts among Nagasaki residents were more frequently located at the inferior nasal portion of the lens compared to cataracts in Hiroshima residents, with no ionizing radiation-specific localization observed. Based on the angles of incidence, UV was suggested as a possible cause of the city difference. We therefore analyzed models of city differences in terms of UVA and UVB levels. The UVB model provided a better fit than the UVA model, suggesting that UVB might account for the city difference. The current study implicated the geographic location of the subject, the investigation period, and outdoor activities as potentially important surrogate factors for UVB influence in radiation-induced cataract. In addition, the superior temporal portion of the lens seemed the most suitable for evaluating the effects of ionizing radiation because of the lesser amount of UVB interference at that site.     

Light‐mediated DNA Repair Prevents UVB‐induced Cell Cycle Arrest in Embryos of the Crustacean Macrobrachium olfersi

High levels of ultraviolet‐B (UVB) radiation can negatively affect aquatic animals. Macrobrachium olfersi is a prawn that lives in clear freshwaters and during the breeding season, females carry eggs in an external brood pouch. Therefore, we hypothesize that eggs are also exposed to environmental UVB radiation. The aim of this study was to investigate whether UVB radiation induces DNA damage and compromises cell cycle in embryos of M. olfersi. In laboratory, UVB irradiance (310 mW. cm^?2) that embryos receive in the natural environment was simulated. After irradiation, embryos were kept under different light conditions in order to recognize the presence of cell repair. UVB radiation induces DNA damage, specifically thymine dimers. After 48 h of UVB exposure, a significant decrease in the level of these dimers was observed in embryos kept under visible light while it remained constant in the dark. Moreover, under visible light and darkness, a decrease in proliferation was observed after 48 h of irradiation. An increase in PCNA expression and decrease in p53 expression were observed after, respectively, 1 and 48 h of exposure. Our results showed that UVB radiation disturbs the cell cycle and induces DNA damage in M. olfersi embryos. However, under visible light these embryos showed successful DNA repair.     

In Vitro Investigations on the Effect of Dermal Fibroblasts on Keratinocyte Responses to Ultraviolet B Radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280–320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB‐induced damage. To investigate these processes, established two and three‐dimensional culture models were utilized to study the impact of fibroblast–keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase‐3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast‐produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation‐induced damage.     

N4‐Methylation of Cytosine Drastically Favors the Formation of (6‐4) Photoproducts in a TCG Context

Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5‐methylcytosine (5mC), some bacterial species contain in their genome N⁴‐methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV‐induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6‐4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine.     

Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice

The UVB (280-315 nm)- and UVA1 (340-400 nm)-induced migration of Langerhans cells (LC) from the epidermis and accumulation of dendritic cells (DC) in the lymph nodes draining the exposed skin site of C3H/HeN mice have been investigated. One minimum erythemal dose (MED) of UVB (1.5 kJ/m2) and of UVA1 (500 kJ/m2) were chosen, which have been shown previously to suppress delayed hypersensitivity (DTH). UVB irradiation resulted in a reduction in epidermal LC numbers, local to the site of the exposure, which was most apparent 12 h after exposure, but, in contrast, UVA1 had no significant effect even at 72 h after exposure. UVA1 did not exert any protection against the UVB-mediated depletion in LC numbers. The reduction in local LC following UVB exposure was prevented by systemic (intraperitoneal) treatment of mice with neutralising antibodies to either tumor necrosis factor (TNF)-alpha or interleukin (IL)-beta 2 h prior to the irradiation. It has been reported previously that UVB exposure caused an increase in the number of dendritic cells (DC) in the lymph nodes draining the irradiated skin site. In the present study we have shown that UVA1 had a similar effect. Pretreatment of the mice with neutralising antibodies to IL-1beta (by intraperitoneal injection) substantially inhibited DC accumulation induced by both UV regimens. However, anti-TNF-alpha antibodies affected only the UVB-induced increase, and did not alter the elevation in DC numbers observed following UVA1 exposure. These results indicate that UVB causes the migration of LC from the epidermis and an accumulation of DC in the draining lymph nodes by a mechanism that requires both TNF-alpha and IL-1beta. In contrast, UVAI does not cause LC migration from the epidermis and the accumulation of DC in the draining lymph nodes observed following UVA1 exposure requires IL-1beta, but not TNF-alpha. It is likely therefore that UVA1 acts through a different mechanism from UVB and may target a cutaneous antigen presenting cell other than LC, such as the dermal DC.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

Dietary Feeding of Opuntia Humifusa Inhibits UVB Radiation‐Induced Carcinogenesis by Reducing Inflammation and Proliferation in Hairless Mouse Model

It has been validated that ultraviolet B (UVB) irradiation induced both squamous and basal cell carcinomas, as a tumor initiator and promoter. Opuntia humifusa is a member of the Cactaceae family which has been demonstrated in our previous study to have a chemopreventive effect in 7, 12‐dimethylbenz[a]anthracene and 12‐O‐tetradecanoylphorbol‐13‐acetate induced skin carcinogenesis models. Therefore, this study was designed to determine the protective effects of O.humifusa against photocarcinogenesis. O. humifusa was administrated to mice as a dietary feeding, following exposure to UVB radiation (180 mJ/cm²) twice a week of 30 weeks for skin tumor development in hairless mice. Dietary O.humifusa inhibited UVB‐induced epidermal hyperplasia, infiltration of leukocytes, level of myeloperoxidase and the levels of proinflammatory cytokines, tumor necrosis factor‐ α (TNF‐α), interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6), in UVB exposed skin. Also, O.humifusa significantly inhibited both protein and mRNA expression level of cyclooxygenase‐2 (COX‐2), nitric oxide synthase (iNOS), proliferating cell nuclear antigen (PCNA) and cyclin D1 compared to the non‐O.humifusa treated group. Collectively, these results suggest that O.humifusa could inhibit photocarcinogenesis in mouse skin and that protective effect is associated with the inhibition of not only UVB‐induced inflammatory responses involving COX‐2, iNOS and proinflammatory cytokines, but also the down‐regulation of UVB‐induced cellular proliferation.     

Kinetic Evidence for the Influence of Subsurface Oxygen on Palladium Surfaces Towards CO Oxidation at High Temperatures

Transient state kinetics of the catalytic oxidation of CO with O₂ on Pd‐surfaces has been measured under isothermal conditions by using a molecular beam approach. Systematic studies were carried out as a function of reaction temperature and CO+O₂ composition. With sufficient kinetic evidence, we have demonstrated the positive influence of subsurface oxygen towards CO‐adsorption and oxidation to CO₂ at high temperatures (600–900 K) on Pd‐surfaces, and the likely electronic nature of the surface changes with oxygen in the subsurface. These studies also provide a direct proof for CO‐adsorption with a significantly reactive sticking coefficient at high temperatures on Pd‐surfaces exhibiting a significant subsurface O‐coverage.     

UVB-INDUCED SUPPRESSION OF THE MIXED EPIDERMAL CELL LYMPHOCYTE REACTION IS CRITICALLY DEPENDENT ON IRRADIANCE

The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the effects of ultraviolet B (UVB) radiation on the skin immune system. In UVB experiments dosimetry is very important. The influence of irradiance on the MECLR was studied in vitro using Philips FS40 lamps with variable UV intensities. Irradiation of isolated epidermal cells with high irradiance impaired the alloactivating capacity more than irradiation with low irradiance. In vivo, the influence of long-term UVB exposure on the MECLR was studied by treating normal healthy volunteers with suberythemagenic doses of UVB thrice weekly during 4 weeks. The first set of experiments, using low irradiance Sylvania UV-21 F75/85 W lamps, resulted in a decrease of MECLR responses of 83.1%. In the second set of experiments performed a year later, employing an identical protocol except for the use of high irradiance Waldmann UV-21 F85/100 W lamps, an increase of MECLR responses of 99.7% was observed. Volunteers of both sets of experiments received equal doses of UVB. In conclusion, this study shows that in vitro UVB-induced suppression of the MECLR is critically dependent on irradiance and therefore might explain contradictory results described in the literature. The in vivo data suggest that, comparable to the in vitro experiments, irradiance may influence the effects of UVB irradiation in vivo. Further experiments should prove whether this is indeed the case.     

An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB‐induced Melanoma

Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB‐inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor‐bearing and tumor‐free fish were given a challenge UVB dose and (6–4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter‐individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter‐individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.