PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

UNSCHEDULED DNA SYNTHESIS: A QUANTITATIVE INDICATOR OF RESIDUAL IMMUNODETECTABLE PYRIMIDINE DIMERS IN HUMAN FIBROBLASTS AFTER ULTRAVIOLET-B IRRADIATION

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.     

Protection of photosystem II against UV-A and UV-B radiation in the cyanobacterium Plectonema boryanum: the role of growth temperature and growth irradiance

Plectonema boryanum UTEX 485 cells were grown at 29 degrees C and 150 mumol m-2 s-1 photosynthetically active radiation (PAR) and exposed to PAR combined with ultraviolet-A radiation (UV-A) at 15 degrees C. This induced a time-dependent inhibition of photosystem II (PSII) photochemistry measured as a decrease of the chlorophyll a fluorescence ratio, Fv/Fm, to 50% after 2 h of UV-A treatment compared to nontreated control cells. Exposure of the same cells to PAR combined with UV-A + ultraviolet-B radiation (UV-B) caused only a 30% inhibition of PSII photochemistry relative to nontreated cells. In contrast, UV-A and UV-A + UV-B irradiation of cells cultured at 15 degrees C and 150 mumol m-2 s-1 had minimal effects on the Fv/Fm values. However, cells grown at 15 degrees C and lower PAR irradiance (6 mumol m-2 s-1) exhibited similar inhibition patterns of PSII photochemistry as control cells. The decreased sensitivity of PSII photochemistry of P. boryanum grown at 15 degrees C and 150 mumol m-2 s-1 to subsequent exposure to UV radiation relative to either control cells or cells grown at low temperature but low irradiance was correlated with the following: (1) a reduced efficiency of energy transfer to PSII reaction centers; (2) higher levels of a carotenoid tentatively identified as myxoxanthophyll; (3) the accumulation of scytonemin and mycosporine amino acids; and (4) the accumulation of ATP-dependent caseinolytic proteases. Thus, acclimation of P. boryanum at low temperature and moderate irradiance appears to confer significant resistance to UV-induced photoinhibition of PSII. The role of excitation pressure in the induction of this resistance to UV radiation is discussed.     

Sensitivity of the early life stages of macroalgae from the Northern Hemisphere to ultraviolet radiation

The reproductive cells of macroalgae are regarded as the life history stages most susceptible to various environmental stresses, including UV radiation (UVR). UVR is proposed to determine the upper depth distribution limit of macroalgae on the shore. These hypotheses were tested by UV-exposure experiments, using spores and young thalli of the eulittoral Rhodophyceae Mastocarpus stellatus and Chondrus crispus and various sublittoral brown macroalgae (Phaeophyceae) with different depth distribution from Helgoland (German Bight) and Spitsbergen (Arctic). In spores, the degree of UV-induced inhibition of photosynthesis is lower in eulittoral species and higher in sublittoral species. After UV stress, recovery of photosynthetic capacity is faster in eulittoral compared to sublittoral species. DNA damage is lowest while repair of DNA damage is highest in eulittoral compared to sublittoral species. When the negative impact of UVR prevails, spore germination is inhibited. This is observed in deep water kelp species whereas the same UVR doses do not inhibit germination of shallow water kelp species. A potential acclimation mechanism to increase UV tolerance of brown algal spores is the species-specific ability to increase the content of UV-absorbing phlorotannins in response to UV-exposure. Growth rates of young Mastocarpus and Chondrus gametophytes exposed to experimental doses of UVR are not affected while growth rates of all young kelp sporophytes exposed to UVR are significantly lowered. Furthermore, morphological UV damage in Laminaria ochroleuca includes tissue deformation, lesion, blistering and thickening of the meristematic part of the lamina. The sensitivity of young sporophytes to DNA damage is correlated with thallus thickness and their optical characteristics. Growth rate is an integrative parameter of all physiological processes in juvenile plants. UV inhibition of growth may affect the upper distribution depth limit of adult life history stages. Juveniles possess several mechanisms to minimize UVR damage and, hence, are less sensitive but at the expense of growth. The species-specific susceptibility of the early life stages of macroalgae to UVR plays an important role for the determination of zonation patterns and probably also for shaping up community structure.     

Comparative photobiology of growth responses to two UV-B wavebands and UV-C in dim-red-light- and white-light-grown cucumber (Cucumis sativus) seedlings: physiological evidence for photoreactivation

We examined the influence of short-term exposures of different UV wavebands on the elongation and phototropic curvature of hypocotyls of cucumbers (Cucumis sativus L.) grown in white light (WL) and dim red light (DRL). We evaluated (1) whether different wavebands within the ultraviolet B (UV-B) region elicit different responses; (2) the hypocotyl elongation response elicited by ultraviolet C (UV-C); (3) whether irradiation with blue light-enriched white light (B/WL) given simultaneous with UV-B treatments reversed the effect of UV in a manner indicative of photoreactivation; and (4) whether responses in WL-grown plants were similar to those grown in DRL. Responses to brief (1-100 min) irradiations with three different UV wavebands all induced inhibition of elongation measured after 24 h. When WL-grown seedlings were irradiated with light containing proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm), inhibition of hypocotyl elongation was induced at a threshold of 0.5 kJ m(-2), whereas exposure to UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation at a threshold of 1.6 kJ m(-2). The UV-C treatment induced reduction in elongation at a threshold of <0.01 kJ m(-2) for DRL-grown plants and <0.03 kJ m(-2) for WL-grown plants. B/WL caused 50% reversal of the short-wavelength UV-B-induced inhibition of elongation in DRL-grown seedlings but did not reverse the effect of long-wavelength UV-B. B/WL caused 30% reversal of the UV-C-induced inhibition of elongation in WL-grown seedlings but did not affect the response to short-wavelength UV-B. Short-wavelength UV-B also induced positive phototropic curvature in both types of seedlings, and this was reversed 60% or completely in DRL-grown and WL-grown seedlings, respectively. The similarity of responses between the etiolated (DRL-grown) and de-etiolated (WL-grown) seedlings indicates that the short-wavelength specific response may be relevant to natural light environments, and the apparent photoreactivation implicates DNA damage as the sensory mechanism for the response.     

Comparison of changes in metal toxicity following exposure of water with high dissolved organic carbon content to solar, UV-B and UV-A radiation

This study examines the effects of natural solar radiation on the metal-binding capacity of dissolved organic matter (DOM). Newington Bog water (35.5 mg L⁻¹ dissolved organic carbon [DOC]) was irradiated for 20 days under UV-B lamps in the laboratory and under natural solar radiation. In the presence of irradiated DOM, IC₅₀ (contaminant concentration required to reduce algal growth by 50%) was significantly decreased with UV-B treatment for four metals: Pb, 64%; Cu, 63%; Ni, 35% and Cd, 40%. Solar radiation also significantly decreased IC₅₀ of Pb (58%) and Cu (49%), DOC concentration (11%), DOM fluorescence (DOMFL, 33%) and DOC-specific UV absorbance. Further experiments on Raisin River water (20.7 mg DOC L⁻¹) exposed to 20 days of artificial UVA and UV-B radiation produced significant decreases in IC₅₀ for Cu (48%) with UV-A and for Pb (43%) with UV-B. DOC concentration was decreased 20% by UV-B and 24% by UV-A. DOMFL decreased 51.5% in the first 5 days of UV-A exposure, an effect that was not observed with the UV-B treatment. The UV-A treatment decreased UV absorbance more at longer wavelengths and over a broader wavelength band than did the UV-B treatment. Change in toxicity with UV irradiation was inconsistent among the metals tested in this study, indicating that some organic metal-binding ligands were more quickly removed or altered than others. The DOM remaining after irradiation appears to be qualitatively different from the unirradiated DOM. The much greater irradiance of UV-A makes its contribution to the removal and/or alteration of DOM at least as important as the influence of higher energy UV-B.     

VIABILITY OF THE SPORES FORMED AFTER UV IRRADIATION IN DICTYOSTELIUM DISCOIDEUM

Both abilities of germination of spores formed after UV irradiation and of growth of amoeboid cells emerged from the spores were studied on two kinds of Dictyostelium discoideum strains, NC-4 and ys-13.
An inhibition of germination was observed on the spores of ys -13 when formed after UV irradiation, while no inhibition was detected on the ability of germination of spores of NC-4. The amoeboid cells of ys -13 emerged from the spores showed a heavy delay of growth, although no delay of growth was detected even on the amoeboid cells of NC-4 emerged from the spores formed after UV irradiation. The strain of NC-4 must repair UV lesions fully before spore formation, while the spores of ys-13 must keep some UV lesions unrepaired and send them to the next generation of amoeboid cells. The characters of UV lesion inheritable through the spores to the next amoeboid cells in ys-13 were discussed.     

Antioxidant defenses and DNA damage induced by UV-A and UV-B radiation in the crab Chasmagnathus granulata (Decapoda, Brachyura)

The photoprotector role of pigment dispersion in the melanophores of the crab, Chasmagnathus granulata, against DNA and oxidative damages caused by UV-A and UV-B was investigated. Intact and eyestalkless crabs were used. In eyestalkless crabs, the dorsal epidermis of the cephalothorax (dispersed melanophores) and the epidermis of pereiopods (aggregated melanophores) were analyzed. Intact crabs showed only dispersed melanophores in the two epidermis. Antioxidant enzymes activity and lipoperoxidation content were analyzed after UV-A (2.5 J/cm2) or UV-B (8.6 J/cm2) irradiation. DNA damage was analyzed by single cell electrophoresis (comet) assay, after exposure to UV-B (8.6 J/cm2). UV-A radiation increased the glutatione-S-transferase activity in the pereiopods epidermis of eyestalkless crabs (P<0.05). UV-B radiation induced DNA damage in the dorsal epidermis of eyestalkless crabs (P<0.05). In pereiopod epidermis of eyestalkless crabs, there was no significant difference between control and UV-B-exposed crabs. In the pereiopods epidermis of eyestalkless, the control group showed higher scores of DNA damage and approximately 50% of cellular viability. Because in eyestalkless and irradiated crabs the cellular viability was approximately 5%, it was not possible to observe nuclei for determination of DNA damage. The findings show that melanophores can play a role in the defense against harmful effects of a momentary exposure to UV radiation.     

SURVIVAL, SPORE FORMATION AND EXCISION REPAIR OF UV-IRRADIATED DEVELOPING CELLS OF DICTYOSTELIUM DISCOIDEUM NC-4

Abstract— The synchronously developing aggregates of the cellular slime mold, Dictyostelium discoideum NC-4, were disaggregated into individual cells and irradiated with 254 nm UV light at preaggregation (0h), late interphase (6h), late aggregation (12 h), and preculmination (18 h). When assayed for replica-tive ability (colony formation), the developing cells at 0, 6, 12, and 18h showed the same sensitivity as vegetative cells; the 10% survival dose (D₁₀) was 160 J/m². The spores were more sensitive, with D₁₀ of 70 J/m². Excision repair of the nuclear DNA of the developing cells was studied by alkaline sucrose gradients. UV-induced single-strand breakage and rejoining of the DNA occurred to the same final extent in the cells from the 0, 6, 12 and 18 h stages of development, but a longer time was required for the completion of rejoining at the later stages (for example, at 54 J/m², 6.6 h for preculmination cells, 3.3 h for preaggregation cells). When the cells irradiated at various stages were required to redevelop, as measured by the relative numbers of spores produced, their sensitivity for completing this development increased the later the stage from which they were taken. The D₁₀s for spore production were 200, 130, 100 and 70 J/m² for cells at the 0, 6, 12 and 18 h stages, respectively. The fractional viability among the spores that appeared after this treatment was the same independent of the stage at which the cells were irradiated; the D₁₀ for this viability was 160 J/m², the same as if the cells had been plated immediately with no intervening developmental sequence. We conclude that DNA excision repair as related to replicative ability is retained at all stages of development; however, development seems independent of replicative ability and depends upon DNA and/or non-DNA damage in a more complex way.     

PYRIMIDINE DIMER FORMATION AND GERMINATION OF UV-IRRADIATED SPORES OF DICTYOSTELIUM DISCOIDEUMNC–4 ANDys–13

Abstract— Survival, UV-photoproducts and germination of UV-irradiated spores of Dictyostelium discoi-deum were studied on two strains,NC–4 andys–13. The spores ofNC–4 are about 35 times more resistant to UV thanys–13 spores at 10% survival. Pyrimidine dimers were formed in UV-irradiated spores in both strains. No photoproducts other than pyrimidine dimers were detected. The formation of pyrimidine dimers in spores was about 2% in both strains at 800 J/m₂. In the germination of spores, the conversion of spores into swollen spores was not affected by UV in both strains, but the emergence of amoebae from the swollen spores was suppressed, which was more distinctive inys–13 spores than inNC–4 spores. The emerged amoebae from the UV-irradiatedNC–4 spores were viable, while those from theys–13 spores were inviable even when they succeeded in emergence.     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

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.     

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.     

Effects of UVB irradiance on conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae: a study of reciprocity and recovery

We tested the effects of irradiances of 920 and 1200 mW m-2 (weighted irradiance) on the conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae. The conidia were exposed to the two irradiances for 1, 2, 4, 6, 7 or 8 h. Increased exposure decreased relative percent culturability. The inactivation provoked by the irradiance of 1200 mW m-2 was higher than for the 920 mW m-2, with a reduction in the 50% lethal time (LT50) from 6 h 40 min to 4 h 26 min. Reciprocity was not observed when conidia in water suspension and germinants in different stages of the germinative process were exposed to a 17.3 kJ m-2 total dose at both irradiance levels. Although nonreciprocity was observed in all situations, its magnitude varied as a function of metabolic state and/or cell-cycle phase in which the conidia were at the exposure time. The least difference between the effects of the two irradiance levels was observed when nongerminating conidia in suspension were exposed, and the greatest was observed when conidia were exposed during an advanced germination phase. Doses of 6.6 and 17.3 kJ m-2 supplied through the two irradiance levels delayed the germination of the surviving conidia. At both doses, delay was greater during exposure to the higher irradiance. Nonreciprocity was higher for the 17.3 kJ m-2 dose. Nonreciprocity magnitude, in addition to depending on the conidial physiological state, also depended on dose. The results demonstrate the importance of evaluating the impact of the increase in irradiance during the different stages of the fungal life cycle, especially during the stages which are more sensitive to UV, and not simply in dormant conidia.     

Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B-induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B-mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10-40 microg/mL) for 24 h before UV-B (40 mJ/cm(2)) exposure dose dependently inhibited UV-B-mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 microg/mL) inhibited UV-B-mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B-mediated activation of nuclear factor kappa B (NF-kappaB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated degradation and phosphorylation of IkappaBalpha and activation of IKKalpha. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated nuclear translocation and phosphorylation of NF-kappaB/p65 at Ser(536). Taken together, our data shows that PFE protects against the adverse effects of UV-B radiation by inhibiting UV-B-induced modulations of NF-kappaB and MAPK pathways and provides a molecular basis for the photochemopreventive effects of PFE.     

Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA

Double-stranded, covalently closed, supercoiled circular DNA from phage fd (replicative form) was irradiated with increasing doses of UV light at 254 nm, 290 nm, 313 nm and 365 nm, and subjected to electrophoresis on agarose slab gels. Increasing the doses of UV light at 254 and 290 nm promotes a smooth reduction in the electrophoretic mobility of the sample, as would be expected if the major effect of light at these two wavelengths were to induce the formation of photoproducts leading to the unwinding of the double strand. At high doses, UV light at 290 nm introduces single-strand breaks (1.2 kJ m-2 per nick per million phosphodiester bonds). UV light at 313 nm promotes an abrupt change in the electrophoretic mobility, as would be expected if the effect of this wavelength were to induce single-strand breaks, leading to the transformation of the supercoiled molecules in their relaxed form (23 kJ m-2 in order to introduce one nick per million phosphodiester bonds). UV light at 365 nm also promotes single-strand breaks in DNA (140 kJ m-2 per nick per million phosphodiester bonds).     

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.     

LONG-TERM EFFECTS OF A SINGLE DOSE OF ULTRAVIOLET-B ON ALBINO RABBIT CORNEA-I. in vivo ANALYSES

Both eyes of female albino rabbits (1.9 kg) were exposed to a single dose of UV-B (300 +/- 9 nm; 0.125 J/cm2 total dose) between 13.30 and 15.00 h. The average irradiance was 209 +/- 4 microW/cm2 delivered over 612 +/- 13 s. At various time periods thereafter (every 12 h for 3 days, 6, 7, 14, 28, 42, 56, 112, 224 and 336 days post-irradiation), the animals were subjected to a full slit lamp examination to evaluate the status of the cornea and the anterior segment along with optical or ultrasonic pachometry of central corneal thickness. The results were compared with studies on age-matched rabbits over the same time period. In response to the UV-B irradiation, the corneas showed a modest edema (20% increase in central corneal thickness) that peaked at 48 h. Nearly normal central corneal thickness returned in 6 days and followed by a secondary very slight swelling (less than 5%) that resolved by 14 days. The edema was accompanied by keratitis over the same period. Thereafter, both control and UV-B irradiated corneas progressively increased in thickness with age. Biomicroscopy also revealed the appearance of granular opacities in the corneal epithelium that peaked at 72-96 h and resolved over 28 days. In addition, very small microdot opacities of the corneal epithelium were present in the UV-B irradiated corneas that reached maximum at 72 h but persisted to some degree throughout the evaluation period. Biomicroscopy also revealed a progressive disruption of the homogeneous nature of the corneal stroma by the appearance of large 'bread crumb'-like opacities that started at 72 h and was still present at the end of the evaluation period. These results suggest that long-term evaluation of the cornea is important after acute UV-B exposure and indicate that acute exposure to UV-R can produce corneal changes resembling those reported following chronic exposure to UV-R-rich environments.     

Induction of DNA strand breaks and DNA-protein cross-links in normal human skin fibroblasts following exposure to 254 nm UV radiation

Levels of DNA strand breaks and DNA-protein cross-links (DPCs) were measured using the alkaline elution assay in normal human skin fibroblasts irradiated with 0-200 J m-2 of 254 nm UV radiation and incubated for 0-24 h. On incubation, the yields of both single-strand breaks (SSBs) and DPCs increased with similar kinetics and remained elevated. In addition, when SSBs were measured under conditions in which DPCs were not eliminated by treatment with proteinase K, a measurable yield of SSBs could not be detected. Hence, the SSBs that form in the UV-irradiated cells following incubation appear to be associated with the DPCs.     

Circadian rhythms of resistance to UV-C and UV-B radiation in Euglena as related to "escape from light" and "resistance to light"

Radiation-induced stress, either from visible or UV light, is strongest at midday. We found that, in the absence of stress or time cues, Euglena gracilis Z was the most resistant to UV-C and UV-B at subjective midday, whether judged from immediate or reproductive survival. The circadian UV-resistance rhythms were free-running in stationary cultures under 1-h light/1-h dark cycles or continuous darkness, indicating that cell-cycle dependent DNA susceptibility to UV was not involved. We moreover examined what was the primary cause of the circadian UV resistance, estimated as the immediate cell survival. The half-maximal lethal dose (LD(50)) of UV-C at subjective midday (the most resistant phase) was 156 J/m(2), which is approximately 3-fold that at subjective midnight. The same was true for UV-B, except the LD(50) was approximately 13-fold that of UV-C. Temperature during UV irradiation had little effect, indicating that survival was not mediated via enzymatic reactions. Non-enzymatic antioxidants were added 5 min before UV irradiation. Dimethylsulfoxide (a hydroxyl radical scavenger) increased survival after UV-B, but had little effect after UV-C; conversely, sodium ascorbate increased survival after UV-C, but not after UV-B. These findings suggest that circadian rhythms of resistance to UVs involve a common mechanism for maximizing non-enzymatic antioxidative capacity at subjective midday, but the specific antioxidants differ.