Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.     

Wavelength dependence of histological, physical, and visible changes in chronically UV-irradiated hairless mouse skin

Albino hairless mice (Skh: HR-1) exposed chronically to sub-erythemal doses of UV radiation display physical, visible and histological alterations. Using narrow bandwidth radiation covering the UV radiation spectrum from 280-380 nm, the wavelength dependence of these alterations was determined. The wavelength dependence spectra indicate that for all but one parameter measured (skin sagging), UV-B radiation is considerably more efficient than UV-A radiation in producing changes in the skin. However, in natural sunlight there is considerably more UV-A than UV-B radiation, providing the potential for UV-A to have a larger contribution to skin damage than UV-B. This argues in favor of using broad spectrum photoprotective agents to shield the skin adequately from UV-induced aging. The spectra were also used to develop potential associations among events by determining which events occur at similar wavelengths. There seems to be a correspondence between mouse visible skin wrinking (UV-B event) and two histological events: increase in glycosaminoglycans and alteration in collagen. There was no obvious correspondence among UV-A-induced events.     

ULTRAVIOLET-INDUCED DNA DAMAGE AND ITS PHOTOREPAIR IN TAIL FIN CELLS OF THE MEDAKA, Oryzias lutipes

Abstract— To study UV-induced damage and its photorepair in vivo , endonuclease-sensitive sites (ESS) induced by UV irradiation in the DNA of the tail fin of the Medaka, Oryzias latipes . were examined. Isolated fins were irradiated with UV-B, but no ESS were detected after increasing doses of UV-B up to 500 J/m². Then fins were irradiated with UV-B and fin cells were dissociated by treatment with trypsin and EDTA. Ultraviolet-B-induced ESS were detected in the epithelial cells near the tissue surface but not in the inner cells. The capacity for photo repair of both inner and outer epithelial cells and cells in fin ray was the same and was higher than that for a line of cultured cells, OL32.     

p53 induction in normal human skin in vitro following exposure to solar simulated UV and UV-B irradiation.

Exposure of normal human breast skin ex vivo to physiological levels of UV-B and solar simulated UV results in a UV dose- and time-dependent increase in epidermal p53, as determined by PAGE analysis. Peak p53 levels are detected 12 to 24 h post irradiation with UV-B (470-1410 mJ cm-2) and solar simulated UV (5-12 minimal erythema dose (MED) equivalents). Irradiation with an FS20 UV-B lamp, contaminated with UV-A and UV-C (74-1111 mJ cm-2), also induces peak levels after 12 h incubation at 37 degrees C but these levels persist to 36 h post UV irradiation. In all cases p53 levels start to return to normal by 48 h culture. A significant positive correlation is demonstrated between UV-B dose (47-1645 mJ cm-2) and p53 level (p < 0.01, R > 0.977) in explants cultured for 24 h at 37 degrees C post irradiation. The FS20 induces a 'UV-B' dose-dependent increase in p53 to a maximum from 370 to 1111 mJ cm-2. Similarly, solar simulated UV induces a plateau of peak p53 induction between 5 and 15 MED equivalents. Immunohistochemical analysis using microwave retrieval on 5 microns sections shows the same pattern of p53 staining with UV-B and solar UV insult, but proves unreliable as a method of quantification. These results suggest that the skin explant model may be a useful tool in the evaluation of UV-induced epidermal cell damage, providing a valuable alternative to in vivo studies.     

COMPARATIVE POTENCY OF DIFFERENT UV SOURCES IN REDUCING THE DENSITY AND ANTIGEN-PRESENTING CAPACITY OF LANGERHANS CELLS IN C3H MICE

Abstract Although broadband UV-B irradiation has been shown to induce selective immunosuppression in a variety of experimental systems, the wavelength dependence of the immunomodulation and the initial events in the skin remain unclear. In the present study three UV lamps were used at suberythermal doses on C3H mice: a conventional broadband UV-B source (270–350 nm), a narrowband UV-B source (311–312 nm) and a UV-A source (320400 nm). Their effects on the photoisomerization of the naturally occumng trans- isomer of urocanic acid (UCA) to cis- UCA, on the density of Langerhans cells and on the ability of epidermal cells to stimulate allogeneic lymphocytes in the mixed skin lymphocyte reaction (MSLR) were ascertained. Broadband UV-B irradiation was more efficient than narrowband UV-B at reducing the density and function of Langerhans cells, while UV-A irradiation was least effective. These changes were most pronounced immediately following irradiation, were dose dependent and were only detected in UV-exposed areas of skin. There was a close correlation between the UV-induced reduction in Langerhans cell density and the formation of cis -UCA in the epidermis. This correlation was not detected between the reduction in the MSLR response following UV irradiation in vivo and cis-UCA formation.     

Studies of dielectric properties of mammalian tissues after gamma-irradiation

In vitro dielectric measurements (relative permeability and conductivity) of excised liver, kidney, cardiac muscle, spleen and eye of rabbits, were carried out at frequencies of 1–250 kHz and at room temperature. These were done before, immediately and 7 days after gamma irradiation at doses 1–5 Gy. The obtained results indicated significant increase in both relative permitivity and conductivity of tissues at higher doses immediately after irradiation. After 7 days, the changes showed some recovery, more obvious at lower doses. These changes in dielectric properties, after irradiation, may reflect the particular biological organization of each tissue and some mechanisms of radiation damage to these tissues particular to cell membrane, counter-ion polarization associated with intrinsic membrane charges and conductive transport in extracellular medium. This may help to elucidate the mechanisms of variation of dielectric properties of different tissues under the effect of radiation.     

EFFECTS OF UV IRRADIATION ON A LIVING SKIN EQUIVALENT

Abstract— The Living Skin Equivalent (LSE™) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280–400 nm) or UV-A (320–400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R₅₀ or UV-A₅₀) in different lots of LSE were 0.053 ± 0.021 J/cm² (n = 29) and 11.6 ± 4.9 J/cm² (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-α, tumor necrosis factor-α and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of U V radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.     

Biological weighting function for the mortality of Boeckella gracilipes (Copepoda, Crustacea) derived from experiments with natural solar radiation

We performed in situ experiments during the austral summer of 1998 to quantify the mortality of the fresh-water copepod Boeckella gracilipes as a function of the UV dose. The copepods were exposed to solar radiation at the water-surface for approximately 24-34 h. Long-pass cut-off filters (Schott) were used in the exposure experiments. UV radiation and PAR were measured with an IL-1700 (International Light Inc.) and a PUV-500 radiometer (Biospherical Instruments Inc.). A biological weighting function for UV-induced mortality was calculated by fitting a model based on a logistic curve. Our results show that UV damage in this species is strongly wavelength- and dose-dependent. B. gracilipes was highly vulnerable to both UV-B (290-320 nm) and UV-A radiation (< 360 nm). The shape of the BWF obtained for B. gracilipes resembles more closely the action spectra (AS) for UV-induced erythema, than the AS for naked DNA.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis

Ultraviolet B (UV-B) radiation is a modality widely used for the treatment of different skin diseases. One of the major mechanisms of UV-B immunosuppression in this treatment modality is thought to be an apoptosis-inducing effect on T cells infiltrating the skin. We examined the T-cell apoptosis-induction capacities of four different UV light sources, with and without UV filters. The xenon chloride (XeCl) laser proved to be the strongest apoptosis inducer. The use of a phtalic acid filter eliminated UV radiation almost completely below 300 nm, which resulted in a severe decrease in the apoptosis-inducing capacity of different UV-B sources. Using the results of the measurements with polychromatic UV light sources, the wavelength dependence of UV-B light for the induction of T-cell apoptosis was also determined. The regression line of the action spectrum demonstrated a continuous decrease from 290 to 311 nm. The apoptosis-inducing capacity of the XeCl laser was almost four times higher than the calculated value according to the action spectrum, which might be attributed to the high irradiance of the laser as compared with nonlaser light sources.     

Using ultra-weak photon emission to determine the effect of oligomeric proanthocyanidins on oxidative stress of human skin

Exposure of skin to ultraviolet (UV) radiation triggers oxidative stress in skin tissue that can lead to erythema, early skin aging or even cancer. It is suggested that oligomeric proanthocyanidins (OPCs), phytonutrients that belong to the polyphenol family have an anti-oxidant/anti-inflammatory activity on the skin. Measuring ultra-weak photon emission (UPE) is a non-invasive, fairly-sensitive and convenient technique for continuously monitoring oxidative stress. The present study was undertaken to confirm anti-oxidant activity of the specific OPCs cream formulation in human skin by measuring UPE of skin. In the present study 25 healthy female subjects participated. As a baseline measurement of skin, UPE was recorded from the dorsal surface of the subjects’ hands before (spontaneous UPE) and after exposure to UV (UV-induced UPE). The effects of the OPCs cream on spontaneous and UV-induced UPE were measured using a fractionated UV exposure protocol. UV exposure resulted in an increase in UPE from both hands. Repeat UV exposure also resulted in a long-term increase of spontaneous UPE. This is likely due to depletion of anti-oxidant capacity of skin resulting in sensitization of skin to UV. It was assessed by measuring spontaneous UPE at 80 min after each UV exposure. Application of the OPCs cream immediately after UV exposure resulted in a significant (approx. 30%) decrease in UV-induced UPE. Topical OPCs cream application also reduced sensitization of skin to UV following repeated UV exposure (i.e., reduced long-term increase in spontaneous UPE). This study indicates that the specific OPCs cream formulation significantly decreases UV-induced oxidative stress in human skin based on UPE measurement. It therefore suggests that regular use of this OPCs cream might protect skin from harmful effects of UV.     

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.     

UV-induced changes in pigment content and light penetration in the fruticose lichen Cladonia arbuscula ssp. mitis

The response of the lichen, Cladonia arbuscula (Wallr.) Flot. ssp. mitis (Sandst.) Ruoss to enhanced UV-B (280-315 nm) radiation was investigated with respect to: (a) changes in phenolic content; (b) differential pigment accumulation under visible and UV radiation with increasing distance from thallus apices; and (c) the internal distribution of UV-B radiation within the thallus measured with quartz optical fibres. In a short-term experiment, lichens were exposed for 7 days in a growth chamber to visible light with or without additional UV-B radiation. For a longer term experiment, lichens were grown outdoors under both natural UV radiation, and supplemental UV-A (315-400 nm)+UV-B provided by lamps. Controls were placed under filters that removed the radiation below 290 nm from the natural sunlight. The concentration of total phenolic compounds was measured spectrophotometrically at the termination of the experiments, in different parts of the lichen podetia. UV-exposed lichens showed increased accumulation of phenolics compared to those not grown under UV. At the termination of the long-term experiment, fibre optic measurements of the penetration of radiation into lichen thallus reflected the influence of growth under UV radiation, whereby UV was more strongly attenuated as compared to that in lichens not exposed to enhanced levels of UV-B radiation. Results indicated that in Cladonia, UV-B radiation induces the accumulation of phenolic compounds that may have a protective role. In addition, the morphological distribution of phenolic compounds was different under visible and supplemental UV-B radiation. Internal radiation measurements served to visualise the attenuation of radiation with thallus depth for different wavelengths in the UV-B waveband.     

How do UV Photomorphogenic Responses Confer Water Stress Tolerance?¶†

Although ultraviolet-B (UV-B) radiation is potentially harmful, it is an important component of terrestrial radiation to which plants have been exposed since invading land. Since then, plants have evolved mechanisms to avoid and repair UV radiation damage; therefore, it is not surprising that photomorphogenic responses to UV-B are often assumed to be adaptations to harmful radiation. This presupposes that the function of the observed responses is to prevent UV damage. It has been hypothesized that, as with blue light, UV-B provides a signal important for normal plant development and might be perceived within developing plants through nondestructive processes, perhaps through UV-specific signal perception mechanisms. UV signal perception can lead to photomorphogenic responses that may confer adaptive advantages under conditions associated with high-light environments, such as water stress. Plant responses to UV radiation in this regard include changes in leaf area, leaf thickness, stomatal density, photosynthetic pigment production and altered stem elongation and branching patterns. Such responses may lead to altered transpiration rates and water-use efficiencies. For example, we found that the cumulative effect of ambient UV-B radiation upon stomatal density and conductance can lead to altered water-use efficiencies. In field settings, UV might more properly be viewed as a photomorphogenic signal than as a stressor. Hence, it might be insufficient to attempt to fully evaluate the adaptive roles of plant responses to UV-B cues upon stress tolerance by the simultaneous application of UV and drought stress during development. We propose that rather than examining a plant's response to combinations of stressors one might also examine how a plant's response to UV induces tolerance to subsequently applied stresses.     

Effects of UV-B radiation on the Patagonian Jaborosa magellanica Brisben

Treatment of Jaborosa magellanica with artificial UV-B radiation caused changes in plant growth, plant chemistry and increase DNA polymorphisms. Spectrophotometric analysis showed that UV-B radiation decreases the chlorophylls content, and increases the amount of UV-B absorbing compounds (e.g., phenylpropanoids). Other UV-induced alterations include reduction in leaf area, alterations in plant architecture, and DNA damage. Using random primers and PCR amplification procedure, a high degree of polymorphism was detected when treated plants were compared to non-irradiated plants. These biochemical changes may be interpreted as plant response to UV-B radiation stress and as an indicator of DNA damage.     

Inflammatory doses of UV may not be necessary for skin carcinogenesis

The UV wavelengths in sunlight are the main cause of skin cancer in humans. Sunlight causes gene mutations, immunosuppression and, at higher doses, inflammation. While it is clear that immunosuppression and gene mutations are essential biologic events via which UV causes skin cancer, the requirement for UV-induced inflammation is less certain. Both the UVB (290-320 nm) and UVA (320-400 nm) wavebands within sunlight can cause skin cancer, gene mutations and immunosuppression. However, UVB, but not UVA, at realistic doses can cause inflammation, and UVB induces skin cancer, immunosuppression and gene mutations at doses much lower than those required to cause inflammation. Inflammation enhances skin carcinogenesis, but may not be UV induced, and inflammatory mediators at doses too low to cause inflammation may be required. UV-induced mutations can cause epidermal cells to make proinflammatory factors or to induce them in the surrounding stroma, creating an oxidizing environment in which additional oncogenic mutations are likely to take place, even in the absence of UV. Our hypothesis is therefore that subinflammatory doses of both UVA and UVB cause benign skin tumors. One of the effects of sunlight-induced mutations may be the production of inflammatory mediators that enhance carcinogenesis.     

Ultraviolet-B radiation causes an upregulation of survivin in human keratinocytes and mouse skin

Understanding of the mechanism of ultraviolet (UV)-mediated cutaneous damages is far from complete. The cancer-specific expression of Survivin, a member of the inhibitor of apoptosis family of proteins, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a target for cancer treatment. This study was designed to investigate the modulation of Survivin during UV response, both in vitro and in vivo. We used UV-B-mediated damages in normal human epidermal keratinocytes (NHEK) cells as an in vitro model and SKH-1 hairless mouse model for the in vivo studies. For in vitro studies, NHEK were treated with UV-B and samples were processed at 5, 15, 30 min, 1, 3, 6, 12 and 24 h after treatment. Our data demonstrated that UV-B exposure (50 mJ/cm2) to NHEK resulted in a significant upregulation in Survivin messenger RNA (mRNA) and protein levels. We also observed that UV-B exposure to NHEK resulted in significant (1) decrease in Smac/DIABLO and (2) increase in p53. For in vivo studies, the SKH-1 hairless mice were subjected to a single exposure of UV-B (180 mJ/cm2), and samples were processed at 3, 6, 12 and 24 h after UV-B exposure. UV-B treatment resulted in a significant increase in protein or mRNA levels (or both) of Survivin, phospho-Survivin and p53 and a concomitant decrease in Smac/DIABLO in mouse skin. This study demonstrated, for the first time, the involvement of Survivin (and the associated events) in UV-B response in vitro and in vivo in experimental models regarded to have relevance to human situations.     

DYES AND DYE MIXTURES USEFUL FOR GENERATION OF UV IN A FLASHLAMP DRIVEN TUNABLE DYE LASER

Abstract— We have used a flashlamp driven tunable dye laser combined with angle tuned frequency doubling crystals for producing UV-B radiation for action spectra studies of various organisms. Optimum UV-B power generation is needed to provide biologically effective doses at wavelengths greater than 300 nm. Optimizing power will also serve to lengthen the lifetime of dyes and other laser components at shorter wavelengths where UV-B output is more than adequate. While much information is available on dyes and dye performance from manufacturers, little information is available on the use of dyes and dye mixtures for providing the continuous high power spectrum of wavelengths necessary for biological UV action spectroscopy. We have examined a number of dyes and dye mixtures for optimal laser performance at wavelengths from 260 to 330 nm. The dyes and dye mixtures discussed here provide adequate power output in the UV-B wavelength range and have allowed us to perform numerous UV-B action spectra studies using the tunable dye laser.     

Influence of UV radiation on four freshwater invertebrates

Laboratory tests confirmed a negative and variable response of the following four species to artificial UV radiation: Cypridopsis vidua, an ostracode; Chironomus riparius, a midge larvae; Hyalella azteca, an amphipod; and Daphnia magna, a daphnid. Severe damage occurred at UV-B irradiance ranging from 50 to 80% of incident summer values. Under constant exposure to UV and photosynthetically active radiation (PAR) the acute lethal response was recorded at 0.3, 0.8, 0.8 and 4.9 W m-2 UV-B for D. magna, H. azteca, C. riparius and C. vidua, respectively. Sublethal UV-B damage to invertebrates included impaired movement, partial paralysis, changes in pigmentation and altered water balance (bloating). A series of UV-B, UV-A and PAR treatments, applied separately and in combination, revealed a positive role for both UV-A and PAR in slowing down UV-B damage. Mean lethal concentration values of the species typically more tolerant to UV and PAR (Cypridopsis, Chironomus) decreased conspicuously when both UV-A and PAR were eliminated. For UV-B-sensitive species (Hyalella, Daphnia) these differences were notably smaller. We suggest that this gradation of sensitivity among the tested species demonstrates potential differences in repairing mechanisms which seem to work more efficiently for ostracodes and chironomids than for amphipods and daphnids. Manipulations with a cellulose acetate filter showed that lower range UV-B (280-290 nm), produced by FS-40 lamps, may cause excessive UV damage to invertebrates.