The role of the spectral sensitivity curve in the selection of relevant biological dosimeters for solar UV monitoring

To estimate the risk of enhanced UV-B radiation due to stratospheric ozone depletion, phage T7 and uracil thin-layer biological dosimeters have been developed, which weight the UV irradiance according to induced DNA damage. To study the molecular basis of the biological effects observed after UV irradiation, the spectral sensitivity curves of the two dosimeters and induction of the two major DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts ((6-4)PDs), in phage T7 have been determined for polychromatic UV sources. CPDs and (6-4)PDs are determined by lesion-specific monoclonal antibodies in an immunodotblot assay. Phage T7 and uracil biological dosimeters together with a Robertson-Berger (RB) meter have been used for monitoring environmental radiation from the polar region to the equator. The biologically effective dose (BED) established with the three different dosimeters increases according to the changes in the solar angle and ozone column, but the degree of the change differs significantly. The results can be explained based on the different spectral sensitivities of the dosimeters. A possible method for determining the trend of the increase in the biological risk due to ozone depletion is suggested.     

Ambient Solar Radiation-Induced Photodamage in Marine Bacterioplankton

Abstract— There has been much recent concern about the effects of increased UV radiation at certain locations on the earth's surface. There have been extensive studies of ultraviolet radiation effects on phytoplankton and primary production, yet the effects of UVB upon bacterioplankton have been largely overlooked. Bacteria play a central role in the cycling of nutrients and energy flow to higher trophic levels, serving as both mineralizers and secondary producers that are consumed by higher organisms. We have begun to investigate the induction of DNA photodamage by UVB in marine planktonic communities using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers in samples collected from the northern Gulf of Mexico. DNA damage in the bacterioplankton size-fraction (< 0.8 μ.m) was greater than in the larger eukaryotic size fraction (>0.8 μm <120 μm) in 9 of 10 samples. Diel patterns of dimer accumulation and repair were observed in surface waters over a 48 h period in the bacterioplankton size fraction and in the larger eukaryotic plankton size fraction. Depth profiles of DNA damage in the bacterioplankton size fraction appear to be dependent on surface water mixing. Damage was greatest in surface waters, decreased with depth and could be detected to 10 m in calm seas. No net accumulation of damage was observed in moderate seas, even at the surface. Solar radiation was found to inhibit significantly both ³H-thymidine and ¹⁴C-leucine incorporation. Ultraviolet B was responsible for approximately half of the total inhibition of ³H-thymidine incorporation, UVA contributing the other half of the inhibition. The vast majority of ¹⁴C-leucine incorporation inhibition was due to UVB, suggesting that protein synthesis is less affected by UVA. The results demonstrate that direct measures of DNA damage can be made of indigenous planktonic communities and that bacterioplankton are highly susceptible to UVB damage and may serve as a more sensitive indicator of UVR stress than other microorganisms.     

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

An immunoassay based on CE–LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL⁻¹) of DNA under a low UVB fluence of 65 J m⁻² for CPDs or 195 J m⁻² for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.     

Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m²). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.     

Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 10(3) and 10(5) times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176-180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.     

DETECTION OF UVR-INDUCED DNA DAMAGE IN MOUSE EPIDERMIS in vivo USING ALKALINE ELUTION

Abstract— Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320–400 nm) or indirectly by UVB (280–320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kj/m² (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1 -β-D-arabinofuranosylcytosine were administered (intraperi-toneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kj/m² (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kj/m² (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.     

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra‐S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6‐4 pyrimidine–pyrimidone photoproducts was highest in DNA from UVC‐irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA–UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8‐oxo‐7,8‐dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.     

Ultraviolet Light‐Induced Cyclobutane Pyrimidine Dimers in Rabbit Eyes

Sunlight exposure of the eye leads to pathologies including photokeratitis, cortical cataracts, pterygium, actinic conjunctivitis and age‐related macular degeneration. It is well established that exposure to ultraviolet (UV) radiations leads to DNA damage, mainly cyclobutane pyrimidine dimers (CPDs). CPD formation is the principal factor involved in skin cancer. However, the exact mechanism by which sunlight induces ocular pathologies is not well understood. To shed light on this issue, we quantified the CPD formation onto DNA of rabbit ocular cells following UVB exposure. We found that CPDs were induced only in the structures of the ocular anterior chamber (cornea, iris and lens) and were more concentrated in the corneal epithelium. Residual UVB that pass through the cornea are completely absorbed by the anterior layers of the iris. CPDs were also detected in the central portion of the lens that is not protected by the iris (pupil). By determining the UV‐induced DNA damage formation in eyes, we showed that anterior ocular structures are a reliable physical barrier that protects the subjacent structures from the toxic effects of UV. Although the corneal epithelium is the structure where most of the CPDs were detected, no cancer is related to solar exposure.     

Temperature-dependent formation and photorepair of DNA damage induced by UV-B radiation in suspension-cultured tobacco cells

Two photoproducts of DNA damage, i.e. cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), induced by UV-B radiation in suspension-cultured tobacco cells were quantified by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. CPDs and 6-4PPs were induced in tobacco cells by UV-B radiation. Photorepair of CPDs was faster than that of 6-4PPs. UV-B radiation induces formation of CPDs and 6-4PPs even at 0 degrees C, but low temperature significantly decreases the UV-B-induced (in contrast to UV-C-induced) formation of CPDs and 6-4PPs. Low temperature also retarded the removal of CPDs and 6-4PPs under white light, and almost no photorepair of CPDs and 6-4PPs was detected at 0 degrees C. When purified DNA from tobacco cells grown in darkness was irradiated with UV-B, formation of CPDs and 6-4PPs took place at the same speed at different temperatures. It indicated that formation of CPDs and 6-4PPs induced by UV-B was temperature-independent in a non-cellular system. Based on our results for suspension-cultured tobacco cells, not only the photorepair but also UV-B-induced formation of CPDs and 6-4PPs are temperature-dependent.     

Pyrimidine (6-4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR

UVC irradiation of genomic DNA induces two main types of potentially mutagenic base modifications: cyclobutane pyrimidine dimers (CPDs) and the less frequent (15-30% of CPD levels) pyrimidine (6-4) pyrimidone photoproducts (6-4PP). Ligation-mediated PCR (LMPCR), a genomic sequencing technique, allows CPD mapping at nucleotide resolution following irradiation with sublethal doses of UVB or UVC for most cell types. In contrast, a dose of 80 J/m(2) of UVC that is lethal for the majority of cell types is necessary to map 6-4PP by the LMPCR technique. This compromises the use of LMPCR to study the repair of 6-4PP. To date, no other techniques have been developed to study 6-4PP repair at nucleotide resolution. We have therefore adapted a recently developed technique for the mapping of 6-4PP: terminal transferase-dependent PCR (TDPCR). TDPCR is in many ways similar to LMPCR. This technique is more sensitive and allows the mapping of 6-4PP at UVC doses as low as 10 J/m(2) in genomic DNA and in living cells.     

Photoacclimation to long-term ultraviolet radiation exposure of natural sub-Antarctic phytoplankton communities: Fixed surface incubations versus mixed mesocosms

Solar UVB radiation (280-320 nm) is known to have detrimental effects on marine phytoplankton. Associated with the seasonal ozone hole in Antarctica, stratospheric ozone depletion occasionally influences the sub-Antarctic (Beagle Channel, Argentina) region, enhancing levels of UVB. The primary objective of this work was to study the effects of several (i.e. 6-10) days of exposure to UVB on the taxonomic composition and photosynthetic inhibition of local phytoplankton communities. For different light treatments, fixed-depth incubations placed in an outdoors water tank were compared with incubations in 1900 L mesocosms, where vertical mixing was present. Phytoplankton growth was inhibited by UV radiation (UVR) in fixed-depth experiments but not in the mixed mesocosms. Under fixed and mixed conditions alike, photosynthesis was significantly inhibited by UVB at the beginning of the experiment but no longer after several days of exposure, suggesting that cells had acclimated to radiation conditions. There was a change in species composition in response to UVR exposure in both experiments, which likely explained acclimation. In the community exposed to fixed conditions this change was from a phytoflagellate-dominated assemblage to a community with high relative abundance of diatoms after 6 days of exposure. UVA was responsible for most of the observed growth inhibition; however, the reduction in photosynthesis was produced by UVB. The reasons behind this variability in responses to UVR are associated with species-specific sensitivity and acclimation, and the previous light history of cells. In the community exposed in mesocosms, an assemblage codominated by phytoflagellates and diatoms was observed at the beginning of the experiments. After 10 days of exposure, green algae (Eutreptiella sp.) had increased, and phytoflagellates were the dominant group. The synthesis of mycosporine-like amino acids (MAAs), antioxidant enzymes and photosynthetic antenna pigments, in relation to repair and protection processes, may explain the reduced inhibition of both growth and photosynthesis that was observed in the phytoplankton community after several days of exposure. For environments such as the Beagle Channel seasonally exposed to the ozone hole, the results obtained from the fixed-depth experiments show that species can cope with UVR by means of MAA synthesis, while mixing would primarily promote a change in species composition and defense strategies.     

The Evaluation of Noninvasive Measurements of Erythema as a Potential Surrogate for DNA Damage in Repetitively UV‐exposed Human Skin

Erythema (i.e. visible redness) and DNA damage caused by ultraviolet radiation (UVR) in human skin have similar action spectra and show good correlation after a single exposure to UVR. We explored the potential to use instrumental assessments of erythema as a surrogate for DNA damage after repeated exposures to UVR. We exposed 40 human subjects to three different exposure schedules using two different UVR sources. Cyclobutane‐pyrimidine dimers (CPDs) in skin biopsies were measured by immunofluorescence, and erythema was assessed by both the Erythemal Index (EI) and the Oxy‐hemoglobin (Oxy‐Hb) content. Surprisingly, the skin with the highest cumulative dose ended up with the lowest level of DNA damage, and with the least erythema, as assessed by Oxy‐Hb (but not EI) 24 h after the last UV exposure. Although the level of CPDs, on average, paralleled Oxy‐Hb (R² = 0.80–0.94, P = 0.03–0.11), the correlation did not hold for the pooled individual measurements (R² = 0.009, P = 0.37) due to potential individual differences in UV‐induced photoadaptation. We suggest that the methodology may be optimized to improve the correlation between DNA damage level and erythema to enable noninvasive risk assessment based on erythema/Oxy‐Hb content for individual human subjects.     

Interleukin‐17 Mediated Inflammatory Responses Are Required for Ultraviolet Radiation‐Induced Immune Suppression

Ultraviolet radiation (UVR) induces immunosuppression and is a major factor for development of skin cancer. Numerous efforts have been made to determine mechanisms for UVR‐induced immunosuppression and to develop strategies for prevention and treatment of UVR‐induced cancers. In the current study, we use IL‐17 receptor (IL‐17R) deficient mice to examine whether IL‐17 mediated responses have a role in UVB (290–320)‐induced immunosuppression of contact hypersensitivity responses. Results demonstrate that IL‐17 mediated responses are required for UVB‐induced immunosuppression of contact hypersensitivity responses. The systemic immune suppression and development of regulatory T cells are inhibited in UVB‐treated IL‐17R deficient mice compared to wild‐type animals. The deficiency in IL‐17R inhibits the infiltration and development of a tolerogenic myeloid cell population in UVB‐treated skin, which expresses CD11b and Gr‐1 and produces reactive oxygen species. We speculate that the development of the tolerogenic myeloid cells is dependent on IL‐17‐induced chemokines and inflammatory mediators in UVB‐treated skin. The inhibition of the tolerogenic myeloid cells may be attributed to the suppression of regulatory T cells in UVR‐treated IL‐17R^?/? mice. The findings may be exploited to new strategies for prevention and treatment of UVR‐induced skin diseases and cancers.     

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.     

Long-term Effects of 222-nm ultraviolet radiation C Sterilizing Lamps on Mice Susceptible to Ultraviolet Radiation

Germicidal lamps that emit primarily 254 nm ultraviolet radiation (UV) are routinely utilized for surface sterilization but cannot be used for human skin because they cause genotoxicity. As an alternative, 222-nm UVC has been reported to exert sterilizing ability comparable to that of 254-nm UVC without producing cyclobutane pyrimidine dimers (CPDs), the major DNA lesions caused by UV. However, there has been no clear evidence for safety in chronic exposure to skin, particularly with respect to carcinogenesis. We therefore investigated the long-term effects of 222-nm UVC on skin using a highly photocarcinogenic phenotype mice that lack xeroderma pigmentosum complementation group A (Xpa-) gene, which is involved in repairing of CPDs. CPDs formation was recognized only uppermost layer of epidermis even with high dose of 222-nm UVC exposure. No tumors were observed in Xpa-knockout mice and wild-type mice by repetitive irradiation with 222-nm UVC, using a protocol which had shown to produce tumor in Xpa-knockout mice irradiated with broad-band UVB. Furthermore, erythema and ear swelling were not observed in both genotype mice following 222-nm UVC exposure. Our data suggest that 222-nm UVC lamps can be safely used for sterilizing human skin as far as the perspective of skin cancer development.     

Resistance of the genome of Escherichia coli and Listeria monocytogenes to irradiation evaluated by the induction of cyclobutane pyrimidine dimers and 6-4 photoproducts using gamma and UV-C radiations

The effect of gamma and UV-C irradiation on the production of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs) in DNA was investigated to compare the natural resistance of the genome of a Gram-positive bacterium and a Gram-negative bacterium against irradiation. Solution of pure DNA and bacterial strains Listeria monocytogenes and Escherichia coli were irradiated using gamma and UV-C rays. Extracted DNA from bacteria and pure DNA samples were then analysed by ELISA using anti-CPDs and anti-6-4 PPs monoclonal antibodies. The results show that gamma rays, as well as UV-C rays, induce the formation of CPDs and 6-4 PPs in DNA. During UV-C irradiation, the three samples showed a difference in their sensitivity against formation of CPDs (P≤0.05). Pure DNA was the most sensitive while the genome of L. monocytogenes was the most resistant. Also during UV-C irradiation, the genome of L. monocytogenes was the only one to show a significant resistance against formation of 6-4 PPs (P≤0.05). During gamma irradiation, for both types of lesion, pure DNA and the genome of E. coli did not show significant difference in their sensitivity (P>0.05) while the genome of L. monocytogenes showed a resistance against formation of CPDs and 6-4 PPs.     

Effect of UVB Radiation on Utilization of Inorganic Nitrogen by Antarctic Microalgae

Abstract— The impact of UVB (280-315 nm) radiation (WG 305) on uptake of ¹⁵N-ammonium and ¹⁵N-nitrate of marine phytoplankton from station 219 (47°W, 61.5°S) and sea ice-algae from station 265 (22.6°W, 73.29°S) was studied during the Polarstern Cruise (EPOS III, Leg 3) to the Weddell Sea, Antarctica 1989. Uptake rates of ¹⁵NH₄⁺ were higher and more affected by UVB radiation than those of ¹⁵N0₃-. Pool sizes of the main amino acids changed in response to the used inorganic nitrogen source and UV exposure. Pools of glutamine, serine and glycine decreased, whereas those of alanine, asparagine and glutamate increased after UVB irradiation. The ¹⁵N-incorporation into the amino acids was reduced as a result of UVB exposure of phytoplankton and ice algae. Results are discussed with reference to an inhibitory effect on the enzymes of both carbon and nitrogen metabolism as well as to adaptation strategies.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

The relationship between UVB screening and cytoprotection by microcorpuscular ZnO or ascorbate against DNA photodamage and membrane injuries in keratinocytes by oxidative stress.

Decreased cell viability and increased formation of cyclobutane-type pyrimidine dimers (CPDs) in DNA of UVB-irradiated keratinocytes were shown to be appreciably restored by the addition of w/o emulsion of microcorpuscular zinc oxide (mcZnO) with a corpuscle diameter of 0.15 microm. The cytoprotection was exerted only by 20 wt/wt% mcZnO at levels equivalent to 40- to 100-microm-thick emulsion layers, which screened 90-92% of the incident UVB. However, protection was not seen by mcZnO below 20-microm thickness, which, unexpectedly, screened 79% of the incident radiation. This suggests that thorough UVB screening is necessary for cytoprotection. This may be attributable to involvement of intracellular reactive oxygen species (ROS) secondarily generated from UVB-irradiated mcZnO. Intracellular ROS was increased in mcZnO-added cells in a time-dependent manner even after UVB irradiation, contrasting with reduction of intracellular ROS in ascorbic acid-added cells. UVB-induced disruption of cell membrane integrity was reduced by mcZnO at 100-microm thickness, equivalent to the addition of ascorbic acid of 50 microM. Thus, mcZnO was thought to be cytoprotective through reductions of intracellular ROS generation, CPD formation and cell membrane disintegration when added so abundantly so as to achieve UVB-screening more than 90%.     

Molecular response to ultraviolet radiation exposure in fish embryos: implications for survival and morphological development

UVR exposure is known to cause developmental defects in a variety of organisms including aquatic species but little is known about the underlying molecular mechanisms. In this work we used zebrafish (Danio rerio) embryos as a model system to characterize the UVR effects on fish species. Larval viability was measured for embryos exposed to several UVR spectral treatments by using a solar simulator lamp and an array of UV cutoff filters under controlled conditions in the laboratory. Survival rate and occurrence of development abnormalities, mainly caudal (posterior) notochord bending/torsion, were seriously affected in UV-exposed larvae reaching values of 53% and 72%, respectively, compared with non-UV-exposed larvae after 6 days postfertilization (dpf). In order to elucidate the molecular mechanisms involved, a matricellular glycoprotein named osteonectin and the expression of a DNA-repair related gene, p53, were studied in relation to UVR exposure. The results indicate that osteonectin and p53 expression were increased under UVR exposure due to wavelengths shorter than 335 nm (i.e. mainly UVB) and 350 nm (i.e. short UVA and UVB), respectively. Furthermore, parallel experiments with microinjections of osteonectin-capped RNA showed that malformations induced by osteonectin overexpression were similar to those observed after a UVR exposure. Consequently this study shows a potential role of osteonectin in morphological deformities induced by solar UV radiation in zebrafish embryos.