Photobiological effects of UVA and UVB light in zebrafish embryos: evidence for a competent photorepair system

The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6-7, 12, and 24h post-fertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebrafish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD(50) of 850 J/cm(2)) to UVA, confirming their suitability for photoactivation and photorepair studies.     

Effects of Light Environment During Culture on UV-lnduced Cyclobutyl Pyrimidine Dimers and Their Photorepair in Rice (Oryza sativa L.)

Abstract— We examined the effects of a light environment during culture of rice plants ( Oryza sativa ) on the steady-state cyclobutyl pyrimidine dimer (CPD) level, CPD induction by challenge UVB exposure and the ability to photorepair CPD. The steady-state CPD level in plants grown under visible radiation with supplemental UVB radiation in a growth chamber was several times higher than in plants grown without supplemental UVB radiation, whereas in outdoor-grown plants, it was not enhanced by supplemental UVB radiation. The susceptibility to CPD induction by challenge UVB exposure was highest in dark-grown plants and decreased with increasing irradiance of visible radiation at low and high levels and outdoors. Chronic UVB radiation reduced the susceptibility to UV-induced CPD in plants grown both indoors and outdoors. There was a significant negative correlation between CPD levels induced by challenge UVB exposure and the content of UV-absorbing compounds. The UV-induced CPD could be reduced by subsequent blue radiation in all samples except in dark-grown seedlings. The higher the irradiance of visible radiation in the culture, the greater the ability to photorepair CPD. Chronic UVB radiation did not increase the ability to photorepair CPD.     

Developmental Responses of Amphibians to Solar and Artificial UVB Sources: A Comparative Study

Abstract— Many amphibian species, in widely scattered locations, currently show population declines and/or reductions in range, but other amphibian species show no such declines. There is no known single cause for these declines. Differential sensitivity to UVB radiation among species might be one contributing factor. We have focused on amphibian eggs, potentially the most UVB-sensitive stage, and compared their resistance to UVB components of sunlight with their levels of photolyase, typically the most important enzyme for repair of the major UV photoproducts in DNA, cyclobutane pyrimidine dimers. Photolyase varied 100-fold among eggs/oocytes of 10 species. Among three species–Hyla regilla, Rana cascadae, and Bufo boreas–for which resistance of eggs to solar UVB irradiance in their natural locations was measured, hatching success correlated strongly with photolyase. Two additional species, Rana aurora and Ambystoma gracile, now show similar correlations. Among the low-egg-photolyase species, R. cascadae and B. boreas are showing declines, and the status of A. gracile is not known. Of the two high-photolyase species, populations of H. regilla remain robust, but populations of R. aurora are showing declines. To determine whether levels of photolyase or other repair activities are affected by solar exposures during amphibian development, we have initiated an extended study of H. regilla and R. cascadae, and of Xenopus laevis, laboratory-reared specimens of which previously showed very low photolyase levels. Hyla regilla and R. cascadae tadpoles are being reared to maturity in laboratories supplemented with modest levels of UV light or light filtered to remove UVB wavelengths. Young X. laevis females are being reared indoors and outdoors. Initial observations reveal severe effects of both UVA and UVB light on H. regilla and R. cascadae tadpoles and metamorphs, including developmental abnormalities and high mortalities. Assays of photolyase levels in the skins of young animals roughly parallel previous egg/oocyte photolyase measurements for all three species.     

Diel Cycles of DNA Damage and Repair in Eggs and Larvae of Northern Anchovy, Engraulis mordax, Exposed to Solar Ultraviolet Radiation

Abstract— Damage from UVB radiation (280–320 nm) in the form of cyclobutane pyrimidine dimers (CPD) in DNA and the capacity for their repair were measured in newly spawned eggs and yolk-sac larvae of northern anchovy, Engraulis mordax, exposed to natural diel cycles of sunlight. The CPD were measured by a newly developed chemiluminescent immunoblot assay capable of measuring CPD in samples as small as 50 ng DNA. Eggs and yolk-sac larvae exposed to full irradiance levels died. At lower dose rates, equivalent to deeper more natural locations in the water column, there was a diel cycle of dimer concentration that tracked solar intensity. This diel cycle was due to the interaction of damage and repair processes. Repair of CPD in anchovy eggs and larvae could be attributed to true photodependent repair that could be stopped by moving samples into the dark. The CPD present at sunset remained until the following morning. The diel cycles of damage and repair were maintained over at least 4 days without a long-term upward or downward trend in dimer concentration. This indicates that at the UVB doses used for these experiments, there was no long-term accumulation of CPD nor an induction of increased repair capacity. Unhatched embryos spawned in the dark also exhibited a strong photorepair response, suggesting that photolyase expression was innate and not dependent on previous light exposure. The diel cycle observed here indicates that, at least for northern anchovy, the CPD concentration at the time of sampling is a good indicator of dose rate but a poor indicator of cumulative dose (i.e. late afternoon samples have the highest cumulative dose but relatively low CPD concentrations). The CPD immunoassay described here has the required sensitivity for measuring DNA damage in wild populations of ichthyoplankton exposed to natural sunlight. These results will guide the collection and interpretation of field data on natural levels of CPD in wild larvae collected at different depths and times of the day.     

DNA光复活作用机理的研究进展*

"环丁烷型嘧啶二聚体(Pyr< > Pyr) 是太阳光中紫外线造成DNA 损伤的主要光化学产物。DNA 光复活酶(或称光解酶) 能够利用可见光裂解二聚体的环丁烷环而修复DNA。本文对DNA 光复活过程中的光解酶对Pyr< > Pyr 的识别和光催化Pyr< > Pyr 裂解反应进行了综述, 介绍了DNA 光解酶的结构、DNA 的主要UV 光化学产物。较详尽地评述了国际上在光解酶催化二聚体裂解的途径以及模型研究方面的最新进展, 并预测了该领域的发展前景。     

Photoreactivation in Paramecium tetraurelia under conditions of various degrees of ozone layer depletion

Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2 x s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2 x s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels.     

CONJUGATED DIENE FORMATION PROMOTED BY TRIPLET ACETONE ACTING UPON ARACHIDONIC ACID

Abstract— Post UV-B(280–320 nm) exposure to UV-A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossum Monodelphis domestica [Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica. Photochem. Photobiol . 40 ,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000-fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE-cellulose and DNA-cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 m M . The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.     

Sublethal exposure to UV radiation affects respiration rates of the freshwater cladoceran Daphnia catawba

We examined the effects of UV radiation (UVR) on metabolic rates of the freshwater cladoceran Daphnia catawba. We exposed D. catawba to UVB for 12 h in a lamp phototron at levels of 2.08 and 4.16 kJ m(-2) both with and without concomitant exposure to UVA and visible photorepair radiation (PRR). We also included a group that received PRR only and a dark control group. Respiration rates were measured for 6 h following exposure. Respiration rates increased by 31.8% relative to the dark control at the lowest level of UVB stress (2.08 kJ m(-2) UVB with PRR), whereas respiration was inhibited by 70.3% at the highest stress level (4.16 kJ m(-2) UVB without PRR). Survival rates in the group that received PRR only and the group exposed to 2.08 kJ m(-2) and PRR were not significantly different from that in the control group; however, the survival rate was reduced for all other UVR exposures. We hypothesize that enhanced respiration rates reflect energetic costs related to repair of cellular components damaged by sublethal levels of UVR. Increases in respiration rate of the magnitude we found in our experiment could significantly reduce energetic reserves available for growth and reproduction, especially in cases where these costs are incurred repeatedly during a series of days with high levels of UVR.     

UVA-induced photo recovery during early zebrafish embryogenesis

DNA photorepair has been widely studied in simple aquatic organisms that live in the marine environment, but is less understood in more complex species that live in freshwater. In the present study, we evaluated UVA-induced DNA photo recovery in embryonic stages of zebrafish, Danio rerio, a freshwater model species. Evaluation of UVB exposure and UVA photo recovery of zebrafish embryos revealed different UVB tolerances and capacities for UVA photo recovery at different stages of development. Effective UVA photo recovery was observed at 3h post-fertilization (hpf), 6-7 hpf, and 12 hpf, but not in the early cleavage stage (2-32 cells). UVA photo recovery was most effective during the gastrula stage (6-7 hpf) of development, and less effective at earlier stages (e.g., 3 hpf) or later stages (e.g., 12 hpf). Embryos at the cleavage stage of development were found to be tolerant to extreme levels of UVB exposure, and possible mechanisms were discussed. For embryos at 6-7 hpf, examination of time window (or delay of UVA exposure) that would still permit recovery from UVB exposure suggested a short time period of 2h. The transgenic fli-1 zebrafish with fluorescent vascular structure was used to show that embryos with normal morphological appearance could exhibit a disrupted vascular patterning, suggesting that this endpoint could provide a sensitive tool for detection of UV damage.     

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.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

The Effect of Cold Stress on UVB Injury in Mouse Skin and Cultured Keratinocytes

Abstract— The effect of cold stress on skin damage caused by UVB irradiation was investigated both in vivo and in vitro. Ear skin of mice that had been exposed to cold stress at 0°C for 20 min and at 5°C for 24 h was exposed to UVB radiation. Sunburn cell production was less in mice exposed to the lower temperature. In addition, the effect of cold stress on the survival rate of UVB-irradiated rat keratinocytes was examined in a cytotoxicity test, with the results showing that keratinocytes exposed to cold stress of 0°C had a higher survival rate than control cells. To pursue a promising clue for explaining the result, we examined metallothionein (MT) production in rat keratinocytes that had been exposed to cold stress at 0°C. Microfluorometric quantification showed a positive correlation between the time course and the intensity of immunofluorescence for MT, indicating that the molecule is inducible by exposure to cold stress in our experimental system. These results suggest that epidermal cells that have been exposed to cold stress maintain a higher resistance to UV radiation than nonexposed controls in vivo and in vitro , and that MT with radical-scavenging activity might contribute, at least in part, to photoprotection against UVB-induced oxidative damage in mammalian skin.     

Effect of cyclobutane pyrimidine dimers on apoptosis induced by different wavelengths of UV.

Ultraviolet radiation within three different wavelength ranges, UVA (340-400 nm), UVB (290-320 nm) or UVC (200-290 nm), was shown to induce apoptosis in OCP13 cells, derived from the medaka fish. Morphological changes such as cell shrinkage and a decrease in the number of nucleoli appeared 4 h after UVA, UVB or UVC irradiation, although with different relative efficiencies. Doses required to induce apoptosis with similar efficiencies were about 2500-fold higher for UVA and 10-fold higher for UVB than for UVC. The following phenomena occurred after UVA irradiation but not after UVB or UVC irradiation. (1) Ultraviolet-A-induced cell detachment occurred with or without cycloheximide pretreatment. (2) Cells attached to plastic showed morphological changes such as rounding up of nuclei without a change in the cell distribution. (3) Morphological changes after UVA irradiation could not be evaded by photorepair treatment. (4) Morphological changes did not occur in cells attached to glass coverslips but only those in plastic dishes. (5) Apoptosis occurred without detectable increase of caspase-3-like activity. (6) Morphological changes were inhibited by N-acetylcysteine, a scavenger of active oxygen species. These results suggest the existence of two different pathways leading to apoptosis, one for long- (UVA) and the other for short- (UVB or UVC) wavelength radiation.     

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.     

Depth-dependent Effects of Ultraviolet Radiation on Survivorship,Oxidative Stress and DNA Damage in Sea Urchin (Strongylocentrotus droebachiensis) Embryos from the Gulf of Maine

A field experiment was conducted on the early embryos of the green sea urchin Strongylocentrotus droebachiensis at different depths in the Gulf of Maine (GOM) to assess the effects of UV radiation (UVR: 300–400 nm) on survivorship, oxidative stress and DNA damage. Embryos experimentally placed at 1 m were exposed to UVB (300–320 nm) where a significant decrease in survivorship was observed as well as significant increases in the activity of the antioxidant enzyme superoxide dismutase and DNA damage. DNA damage includes both cyclobutane pyrimidine dimer photoproducts from direct exposure to UVA (320–400 nm) and indirect DNA damage associated with the production of reactive oxygen species. All embryos had equivalent concentrations of the UVR-absorbing compounds known as mycosporine-like amino acids and despite the fact that these compounds absorb primarily in the UVA portion of the spectrum they did not provide protection for embryos from DNA damage in the field at depths less than 5 m. DNA damage and survivorship of green sea urchin embryos in the GOM was directly related to the optical properties of the water column and the differential attenuation of UVB and UVA wavelengths.     

The Effect of Chronic Exposure to Artificial UVB Radiation on the Survival and Reproduction of Daphnia magna Across Two Generations

We examined the effects of daily (chronic) exposure to artificial UVB radiation on the survival and reproduction of Daphnia   magna over two generations. Control and experimental animals in each generation (parental and F1) were exposed to 16 h of UVA radiation and photosynthetically active radiation daily. In addition, experimental animals were exposed to 6 h of UVB during the middle of the light period. Survival and reproduction were followed for 12 days for each individual. Survival and production of F1 were significantly lower in the UVB exposed parental generation Daphnia than in controls. F1 exposure to UVB significantly decreased F1 survival and reproduction. Reproduction was lowest in UVB exposed F1 animals whose parents were also exposed to UVB. Adverse effects of UVB on offspring production may be magnified in successive generations suggesting that short-term experiments could underestimate the impact of increased UVB exposure on populations.     

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides

Application of Aloe barbadensis poly/oligosaccharides to UV-irradiated skin prevents photosuppression of delayed-type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant-derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 micrograms tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xyloglucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)-10 observed in UV-irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL-10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL-10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV-activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.     

Interspecific variation in UV defense mechanisms among temperate freshwater fishes

An important step in predicting the effects of future increases in UV radiation (UVR) is to evaluate the mechanisms that organisms use to prevent and repair DNA damage and determine how those mechanisms influence UVR sensitivity. Damage is prevented to varying degrees through photoprotection and repaired via two main pathways: nucleotide excision repair and photoenzymatic repair. At present, little is known about the generality or similarity of these defenses among temperate freshwater fishes. We used laboratory experiments to compare UVR defense mechanisms among five freshwater fish species representing four families and three orders. Purified DNA, freeze-killed larvae and live larvae were exposed to UVB radiation for 12 h in the presence or absence of photorepair radiation. After exposure, we quantified frequencies of cyclobutane pyrimidine dimers in each exposure treatment. All five species used photoprotection and proportional decreases in dimer frequency were similar for all species. Evidence of excision repair was also found for all species but proportional decreases in photoproduct frequencies varied among species. Finally, evidence of photoenzymatic repair was found for only two of the five species.