Photoprotection by antioxidants against UVB-radiation-induced damage in pig skin organ culture

Topically applied antioxidants constitute an important group of protective agents against skin damage induced by ultraviolet radiation. The current study was performed to investigate whether a recently developed ex vivo pig skin model was suitable for short-term studies of the mechanism(s) of UVB-radiation-induced skin damage; the protective effect of topical application of alpha-tocopherol, l-ascorbic acid, alpha-lipoic acid, glutathione ethylester and N-acetylcysteine was tested. Increasing doses of the antioxidants were applied topically on ex vivo pig skin explants and allowed to penetrate for 60 min. Epidermal antioxidant bioavailability was measured before and 60 min after exposure to an ultraviolet B (UVB) radiation of 7.5 kJ/m2. Cell viability (trypan blue dye exclusion) and apoptosis were measured 48 h later in isolated keratinocytes. UVB-radiation-induced epidermal lipid peroxidation was determined immediately after exposure of the skin to a UVB dose of 28 kJ/m2. All antioxidants tested became bioavailable in pig skin epidermis, and none of them were depleted after UVB-radiation exposure. Increasing doses of the antioxidants tested decreased UVB-radiation-induced cell death and apoptosis. The highest doses of antioxidants prevented UVB-radiation-induced lipid peroxidation; alpha-lipoic acid only tended to decrease lipid peroxidation. In conclusion, a single topical dose of the above antioxidants on ex vivo pig skin can reduce UVB-radiation-induced oxidative stress and lipid peroxidation and thereby reduce apoptotic stimuli and cell death. Furthermore, the ex vivo pig skin model was a useful tool for testing compounds for their antioxidant activity.     

Repeated small perturbation approach reveals transcriptomic steady states

The study of biological systems dynamics requires elucidation of the transitions of steady states. A "small perturbation" approach can provide important information on the "steady state" of a biological system. In our experiments, small perturbations were generated by applying a series of repeating small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using cDNA microarrays. Repeated small doses (10 J/m2) of ultraviolet B (UVB) exposure modulated the expression profiles of two groups of genes in opposite directions. The genes that were up-regulated have functions mainly associated with anti-proliferation/anti-mitogenesis/apoptosis, and the genes that were down-regulated were mainly related to proliferation/mitogenesis/anti-apoptosis. For both groups of genes, repetition of the small doses of UVB caused an immediate response followed by relaxation between successive small perturbations. This cyclic pattern was suppressed when large doses (233 or 582.5 J/m2) of UVB were applied. Our method and results contribute to a foundation for computational systems biology, which implicitly uses the concept of steady state.     

In situ biological dose mapping estimates the radiation burden delivered to 'spared' tissue between synchrotron X-ray microbeam radiotherapy tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to 'spared' tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30-40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies.     

Ultraviolet radiation-induced erythema in human skin

We have evaluated UVR-induced erythema in previously unexposed buttock skin of volunteers of skin types I, II, III, and IV. Studies were done with solar-simulated radiation (SSR), UVB, and UVAI and we determined the just perceptible minimal erythema dose (MED) and, in some cases, quantified erythema with a reflectance device. The results show that there is a trend for increased SSR MED with skin type, with the MED of skin type IV being approximately twice that of skin type I, a smaller difference than one might have expected. However, there is a very considerable overlap of MED between skin types which shows that MED is a very poor indictor of skin type. Quantitative dose-response and time course studies with SSR and UVAI showed broadly similar responses when comparable MED-based exposures were given. We used our data to test the new concept of the standard erythema dose (SED) with two different erythema action spectra, and confirmed that the SED approach works with the different UVR sources that we studied.     

Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.     

Novel anti-inflammatory peptides as cosmeceutical peptides

Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E₂ is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE₂ synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm²), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24 h. The erythema index (EI) was measured before and 24 h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p = 0.041 and p = 0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.     

Carotenoid supplementation reduces erythema in human skin after simulated solar radiation exposure

Excessive exposure to solar radiation, especially ultraviolet A (UVA: 320-400 nm) and ultraviolet B (UVB: 290-320 nm) radiation, may induce UV-carcinogenesis and erythema in the skin. Although the protective effects of carotenoids against skin lesions are still unclear, beta-carotene has been proposed as an oral sun protectant. The purpose of this study was to determine the magnitude of the protective effects of oral alpha- and beta-carotene supplementation for 24 weeks on UVA- and UVB-induced erythema in humans. While being exposed to UVA and UVB radiation, 22 subjects (11 men and 11 women) were supplemented with natural carotenoids for 24 weeks. Each day for the first 8 weeks, subjects were given 30 mg of natural carotenoids containing 29.4 mg of beta-carotene, 0.36 mg of alpha-carotene, and traces of other carotenoids in vegetable oil. The natural carotenoid dose was progressively raised by 30-mg increments, at every 8 weeks, from 30 mg to 90 mg. Small areas (1 cm2) of the skin were exposed to increasing doses of UV light (16-42 mJ/cm2) to determine the minimal erythema dose (MED). MED was defined as a uniform pink color with well-defined borders. MED readings were obtained by visual inspection 24 hr postirradiation. Blood samples taken during supplementation were used to determine alpha- and beta-carotene serum levels and for a lipid peroxidation analysis. During natural carotenoid supplementation, the MED of solar simulator radiation increased significantly (P<0.05). After 24 weeks of supplementation, serum beta-carotene levels were increased from 0.22 microg/ml (95% CI; 0.16-0.27) to 1.72 microg/ml (95% CI;1.61-1.83). Similarly, alpha-carotene serum levels increased from 0.07 microg/ml (95% CI;0.048-0.092) to 0.36 microg/ml (95% CI; 0.32-0.40). Serum lipid peroxidation was significantly (P<0.05) inhibited in a dose-dependent manner during natural carotenoid supplementation. The present data suggest that supplementation with natural carotenoids may partially protect human skin from UVA- and UVB-induced erythema, although the magnitude of the protective effect is modest.     

Pigmentation effects of solar-simulated radiation as compared with UVA and UVB radiation

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre-existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2-week repetitive exposure of human skin to solar-simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin-related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5-S-cysteinyldopa (5SCD) were elevated about 4-fold in SSR- or UVB-exposed skin compared with UVA-exposed or control skin. Levels of protein-bound form of 5SCD tended to be higher in SSR- or UVB-exposed skin than in UVA-exposed or control skin. Total levels of 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR- than in UVB-exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.     

Interactions between the UVA and UVB wavebands relevant for immune function and carcinogenesis

Abstract

Exposure of the skin of mice and men to increasing doses of UV radiation causes erythema, chronic hyperplasia, mutation, accelerated photo-ageing and photocarcinogenesis. Moderate exposure also suppresses T cell-mediated immune function, a defect which is a prerequisite for the promotion or outgrowth phase of the UV-initiated tumour. This immunosuppressed state is accompanied by dysregulated cutaneous cytokine patterns, particularly a relative deficit of Th1-type cytokines like interleukin (IL)-12 and interferon-γ (IFN-γ). The cutaneous photoreceptor for the immunosuppression may be either, or both, epidermal DNA or urocanic acid (UCA). Naturally occurring trans-UCA photo-isomerises in the stratum corneum and epidermis to cis-UCA, which has local and systemic immunosuppressive properties. The action spectrum for the photo-immunosuppression is maximal in the UVB (280–320 nm) waveband. However, longer wavelength UVA (320–400 nm), which interacts with skin predominantly via oxidative reactions, is not immunosuppressive at environmental exposure doses and, unexpectedly, as we have demonstrated in several strains of mice and opossums, can provide protection from UVB immunosuppression.¹     

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.     

A New Model of Biodosimetry to Integrate Low and High Doses

Biological dosimetry, that is the estimation of the dose of an exposure to ionizing radiation by a biological parameter, is a very important tool in cases of radiation accidents. The score of dicentric chromosomes, considered to be the most accurate method for biological dosimetry, for low LET radiation and up to 5 Gy, fits very well to a linear-quadratic model of dose-effect curve assuming the Poisson distribution. The accuracy of this estimation raises difficulties for doses over 5 Gy, the highest dose of the majority of dose-effect curves used in biological dosimetry. At doses over 5 Gy most cells show difficulties in reaching mitosis and cannot be used to score dicentric chromosomes. In the present study with the treatment of lymphocyte cultures with caffeine and the standardization of the culture time, metaphases for doses up to 25 Gy have been analyzed. Here we present a new model for biological dosimetry, which includes a Gompertz-type function as the dose response, and also takes into account the underdispersion of aberration-among-cell distribution. The new model allows the estimation of doses of exposures to ionizing radiation of up to 25 Gy. Moreover, the model is more effective in estimating whole and partial body exposures than the classical method based on linear and linear-quadratic functions, suggesting their effectiveness and great potential to be used after high dose exposures of radiation.     

Alteration of the immune response to Mycobacterium bovis BCG in mice exposed chronically to low doses of UV radiation

BALB/c mice were exposed on shaved dorsal skin to 1 minimal erythemal dose (MED) of UVB radiation (2.25 kJ/m2) from a bank of six FS-40 sunlamps three times per week. The total number of irradiations ranged from 1 to 27. At regular intervals, groups of mice were injected in the left hind foot pad with 1 x 10(6) live mycobacteria (Mycobacterium bovis BCG) 3 days after the last UVB exposure. The mice were tested 21 and 42 days after infection for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli by injecting PPD into the right hind foot pad and measuring the foot pad swelling 24 hr later. The course of infection was followed by assessing the number of bacterial colony forming units in the lymph node draining the site of BCG infection and the spleen. Mice exposed from 1 to 15 times to 1 MED of UV radiation showed a significant suppression in their DTH response to PPD compared with the unirradiated mice. At the same time, the number of bacterial colony-forming units in the lymph node and spleen of the UV-irradiated mice was greater than in control mice. With continued exposure to UVB, however, the DTH response recovered to a normal level, and there was no longer an increase in the number of viable bacteria in the lymphoid organs. These results indicate that early in the course of chronic UV irradiation, mice were impaired in their ability to mount a DTH response to BCG and to clear these bacteria from their lymphoid organs; later the mice recovered from these effects of UV, with continued treatment. A dose-response study using single doses of UV radiation indicated that a dose of 2.7 kJ/m2 suppressed the DTH response by 50%. Thus, exposure of mice to a single or multiple low doses of UV radiation prior to infection can interfere with systemic immunity to mycobacteria.     

Differential Effects of Doses and Forms of Dietary Selenium on Immune Cell Numbers in the Skin of Ultraviolet-irradiated and Unirradiated Mice

The effect of three different doses of dietary l-selenomethionine (SM) and sodium selenite (SS) on skin selenium (Se) content, glutathione peroxidase (GPx) activity, Langerhans cell (LC) and mast cell numbers in ultraviolet radiation-B (UVB)-irradiated and unirradiated C3H/HeN mice was determined. After weaning, groups of mice were given Se-deficient, Se-adequate, or Se-high diets. Six weeks later, some animals in each group were exposed to a single UVB dose (acute), while others were exposed three times weekly for the following 40 weeks (chronic). The skin Se content and GPx activity increased in all the Se-supplemented groups, and the latter was not altered by UVB exposure. Generally, the Se-containing diets caused an increase in LC numbers at 6 weeks and a further rise at 40 weeks, but did not prevent the loss induced by acute or chronic UVB radiation. Skin mast cell numbers were highest in animals fed the Se-deficient diet after 6 and 40 weeks. Acute and chronic UVB radiation decreased the mast cell number and dietary Se did not prevent the reduction. While the present study shows that Se plays an important role in governing the number of LCs and mast cells in the skin, no protective effect against the immunomodulating properties of UVB radiation on these cell types was observed. However, this conclusion may only apply to the experimental conditions chosen, and additional studies at different Se dosages and reduced intensities of chronic UVB exposure are required to confirm the results.     

LC/ESI-MS analysis of two elastin cross-links, desmosine and isodesmosine, and their radiation-induced degradation products

In this work, the effect of Fenton reaction on two elastin cross-linked amino acids, desmosine (DES) and isodesmosine (IDE), in the absence or presence of different wavelength radiations generated from artificial sources has been evaluated using LC/ESI-MS. Irradiation as well as incubation of DES or IDE solutions in the presence of Fe(2+) and H(2)O(2) resulted in products with m/z 497.1 and 481.1 for [M+H](+). A strongly dose-dependent degradation of both amino acids was observed upon exposure to UVB at doses ranging from 0 to 3 J/cm(2) and a moderate dose-dependent degradation upon exposure to UVA at doses 10 times higher than that of UVB. A significant time-dependent degradation of DES and IDE was also observed upon exposure of these amino acids to a lamp emitting visible light similar to sunlight. Exposure of both amino acids to IR radiation (520 W) for 8 h did not cause significant degradation.     

Relationship between skin response to ultraviolet exposure and skin color type

Sun exposure is responsible for detrimental damage ranging from sunburn to photoaging and skin cancer. This damage is likely to be influenced by constitutive pigmentation. The relationship between ultraviolet (UV) sensitivity and skin color type was analyzed on 42 ex vivo skin samples objectively classified from light to dark skin, based on their values of individual typology angle (ITA) determined by colorimetric parameters. The biologically efficient dose (BED) was determined for each sample by quantifying sunburn cells after exposure to increasing doses of UV solar-simulated radiation. Typical UV-induced biologic markers, other than erythema, such as DNA damage, apoptosis and p53 accumulation, were analyzed. A statistically significant correlation was found between ITA and BED and, ITA and DNA damage. Interestingly, DNA lesions were distributed throughout the whole epidermal layers and the uppermost dermal cells in light, intermediate and tanned skin while they were restricted to suprabasal epidermal layers in brown or dark skin. Our data support, at the cellular level, the relationship between UV sensitivity and skin color type. They emphasize the impact of DNA damage accumulation in basal layer in relation to the prevalence of skin cancer.     

Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.     

Keratinocytes exposed to ultraviolet radiation reveal three down-regulated genes with potential function in differentiation and cell cycle control

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.     

Photoprotective Potential of Penta-O-Galloyl-β-DGlucose by Targeting NF-κB and MAPK Signaling in UVB Radiation-Induced Human Dermal Fibroblasts and Mouse Skin

Exposure of the skin to ultraviolet radiation can cause skin damage with various pathological changes including inflammation. In the present study, we identified the skin-protective activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (pentagalloyl glucose, PGG) in ultraviolet B (UVB) radiation-induced human dermal fibroblasts and mouse skin. PGG exhibited antioxidant activity with regard to intracellular reactive oxygen species (ROS) generation as well as ROS and reactive nitrogen species (RNS) scavenging. Furthermore, PGG exhibited anti-inflammatory activity, inhibiting the activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, resulting in inhibition of the expression of pro-inflammatory mediators. Topical application of PGG followed by chronic exposure to UVB radiation in the dorsal skin of hairless mice resulted in a significant decrease in the progression of inflammatory skin damages, leading to inhibited activation of NF-κB signaling and expression of pro-inflammatory mediators. The present study demonstrated that PGG protected from skin damage induced by UVB radiation, and thus, may be a potential candidate for the prevention of environmental stimuli-induced inflammatory skin damage.     

Central corneal thickness considered an index of corneal hydration of the UVB irradiated rabbit cornea as influenced by UVB absorber

UVB radiation from sunlight induces an acute corneal inflammation, photokeratitis, accompanied by changes in corneal hydration. We employed a method of ultrasonic pachymetry for daily examination of central corneal thickness as an index of corneal hydration of the rabbit cornea repeatedly irradiated by UVB radiation (312 nm, daily dose of 0.25 J/cm(2) during three or four days) as influenced by UVB absorber (actinoquinol combined with hyaluronic acid) dropped on the ocular surface during irradiation. One day after the third irradiation procedure the animals were sacrificed and corneas examined immuno-histochemically for peroxynitrite formation, a marker of oxidative damage, the antioxidant aldehyde dehydrogenase 3A1 and endothelial nitric oxide synthase, an enzyme generated nitric oxide. Results show that UV absorber combined with hyaluronic acid protected the cornea against UVB-induced changes in corneal thickness and microscopical disturbances to the cornea (both seen after buffered saline application) until the fourth experimental day. These UVB doses are equivalent to a daily exposure of 2.5 hrs of the human cornea to solar UVB radiation for three consecutive days. It is suggested that actinoquinol/ hyaluronic acid drops might be helpful for the human eye in the defence against photooxidative and other oxidative processes.