Simultaneous Irradiation with Different Wavelengths of Ultraviolet Light has Synergistic Bactericidal Effect on Vibrio parahaemolyticus

Ultraviolet (UV) irradiation is an increasingly used method of water disinfection. UV rays can be classified by wavelength into UVA (320–400 nm), UVB (280‐320 nm), and UVC (<280 nm). We previously developed UVA sterilization equipment with a UVA light‐emitting diode (LED). The aim of this study was to establish a new water disinfection procedure using the combined irradiation of the UVA‐LED and another UV wavelength. An oxidative DNA product, 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG), increased after irradiation by UVA‐LED alone, and the level of cyclobutane pyrimidine dimers (CPDs) was increased by UVC alone in Vibrio parahaemolyticus. Although sequential irradiation of UVA‐LED and UVC‐induced additional bactericidal effects, simultaneous irradiation with UVA‐LED and UVC‐induced bactericidal synergistic effects. The 8‐OHdG and CPDs production showed no differences between sequential and simultaneous irradiation. Interestingly, the recovery of CPDs was delayed by simultaneous irradiation. The synergistic effect was absent in SOS response‐deficient mutants, such as the recA and lexA strains. Because recA‐ and lexA‐mediated SOS responses have crucial roles in a DNA repair pathway, the synergistic bactericidal effect produced by the simultaneous irradiation could depend on the suppression of the CPDs repair. The simultaneous irradiation of UVA‐LED and UVC is a candidate new procedure for effective water disinfection.     

Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 10(2) and 10(5) more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

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.     

Comparative Action Spectrum for Ultraviolet Light Killing of Mouse Melanocytes from Different Genetic Coat Color Backgrounds

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony-forming ability after irradiation. The cell lines were wild-type black (melan-a) and the mutants brown (melan-b) and albino (melan-c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200–280 Dm), 82.3% output at 254 nm, TL01 (UVB, 280–320 nm), 64.2% at 310–311 nm, FS20, broadband with peak output at 312 nm and Alisun-S (UVA, 320–400 nm), broadband with peak output at 350–354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild-type melan-a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan-c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan-b was more sensitive than melan-a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6–4 photoproducts) produced in melan-a than in melan-c cells by UVC, UVB and FS20 lamps. Unlike melan-c, melan-a and melan-b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin-like centers. The contribution of pheome-lanin was higher in melan-b than in melan-a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild-type melan-a melanocytes were well melanized and ellipsoidal, whereas those of melan-b melanocytes tended to be spherical. In the albino line (melan-c) the melanocytes contained only early-stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.     

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.     

ACTIVATION OF NF-KB IN HUMAN SKIN FIBROBLASTS BY THE OXIDATIVE STRESS GENERATED BY UVA RADIATION

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)-KB by oxidant stress generated via the UVA (320–380nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF-KB that bound to its recognition sequence in DNA. The UVA radiation-dependent activation of NF-KB in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation-dependent activation of NF-KB appeared to be correlated with membrane damage, and activation could be prevented by a-tocopherol and butylated hydroxytol-uene, agents that inhibited UVA radiation-dependent peroxidation of cell membrane lipids. The activation of NF-KB by the DNA damaging agents UVC (200–290nm) and UVB (290–320nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation-dependent activation of NF-KB over all wavelength ranges examined.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF PHOTOREACTIVATION

Abstract— Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light ( Λ < 300 nm). The enzyme requires near UV and visible wavelengths (300 < Λ < 600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non-enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non-enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE from E. coli reveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme and dimer.     

Photodynamic therapy resistant human colon carcinoma HT29 cells show cross-resistance to UVA but not UVC light

The isolation of photodynamic therapy (PDT)-resistant HT29 human colon adenocarcinoma cells has been reported previously. These PDT-resistant variants show increased expression of the Hsp27 and BNip3 proteins and a decreased expression of mutant p53 protein compared with parental HT29 cells. Because mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. In the present report, we examined the colony survival of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells to ultraviolet light (UV) treatment. The HT29 PDT-resistant variants showed cross-resistance to long-wavelength UVA (320-400 nm) but not to short-wavelength UVC (200-280 nm) light. Cell sensitivity to UVA or UVC was then correlated with Hsp27, BNip3 and mutant p53 protein levels in the PDT-resistant variants as well as in several clonal variants of HT29 cells that express different levels of Hsp27, BNip3 and mutant p53. We show that increased expression of Hsp27 and BNip3 and decreased expression of mutant p53 correlated with increased resistance to UVA. In contrast, increased expression of Hsp27 and BNip3 correlated with increased sensitivity to UVC, whereas increased expression of mutant p53 showed no significant correlation with sensitivity to UVC. These results suggest that the PDT-resistant HT29 cell variants are differentially sensitized to UVA compared with UVC due, in part at least, through the altered expression levels of BNip3, Hsp27 and mutant p53.     

Molecular assessment of UVC radiation-induced DNA damage repair in the stromatolitic halophilic archaeon, Halococcus hamelinensis

The halophilic archaeon Halococcus hamelinensis was isolated from living stromatolites in Shark Bay, Western Australia, that are known to be exposed to extreme conditions of salinity, desiccation, and UV radiation. Modern stromatolites are considered analogues of very early life on Earth and thus inhabitants of modern stromatolites, and Hcc. hamelinensis in particular, are excellent candidates to examine responses to high UV radiation. This organism was exposed to high dosages (up to 500 J/m(2)) of standard germicidal UVC (254 nm) radiation and overall responses such as survival, thymine-thymine cyclobutane pyrimidine dimer formation, and DNA repair have been assessed. Results show that Hcc. hamelinensis is able to survive high UVC radiation dosages and that intact cells give an increased level of DNA protection over purified DNA. The organism was screened for the bacterial-like nucleotide excision repair (NER) genes uvrA, uvrB, uvrC, as well as for the photolyase phr2 gene. All four genes were discovered and changes in the expression levels of those genes during repair in either light or dark were investigated by means of quantitative Real-Time (qRT) PCR. The data obtained and presented in this study show that the uvrA, uvrB, and uvrC genes were up-regulated during both repair conditions. The photolyase phr2 was not induced during dark repair, yet showed a 20-fold increase during repair in light conditions. The data presented is the first molecular study of different repair mechanisms in the genus Halococcus following exposure to high UVC radiation levels.     

Somatic-cell mutation induced by UVA and monochromatic UV radiation in repair-proficient and -deficient Drosophila melanogaster

Near-ultraviolet light (UVA: 320-400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair-deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 +/- 1.5 spots/wing/kJ vs 0.3 +/- 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 +/- 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 +/- 2.9 x 10(-3) vs 3.1 +/- 1.1 x 10(-3) spots/wing/kJ; at 340 nm, 3.5 +/- 0.9 x 10(-3) vs 2.0 +/- 0.7 x 10(-3). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.     

Genotoxicity of visible light (400–800 nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400–800 nm) and ultraviolet A (UVA)/visible (315–800 nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8 J/cm², respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1 mM), ERT (0.5 mM) and mannitol (1.5 mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.     

Photoreactivation of UV-lnactivated Spores of Trichoderma harzianum

Abstract— Photoreactivation in the filamentous soil fungus Trichoderma harzianum is of interest because its blue, UVA photoreceptors (cryptochromes) may share homology with DNA photolyases. Furthermore, this organism antagonizes, by mycoparasitism, a number of soil-borne pathogens. Photoreactivation is thus important as one of the factors that may contribute to survival in the field. Exposure of asexually produced spores (conidia) to UVC inhibits germination. Nongerminating spores either do not swell or are inhibited later in germination, swelling but failing to put out a germ tube. Both types of inhibition can be reversed by photoreactivation with visible and UVA (320-400 nm) light, restoring high germination percentages. Conidia of mutants lacking the normal greenish pigmentation are more sensitive to UVC (200-280 nm) than wild-type conidia but photoreactivation still occurs. The action spectrum for photoreactivation indicates that T. harzianum has a DNA photolyase with a pterin as second chromophore. The most effective wavelengths for photoreactivation correspond to valleys, rather than peaks, in the action spectrum for photoinduction of sporulation. Furthermore, mutants with defects in photoinduction of sporulation ( dimY ) are not defective in photoreactivation. Induction of sporulation and DNA photorepair, while sharing parts of the blue/UVA spectrum, are different, by spectroscopic, kinetic and genetic criteria.     

UVB-induced Cyclobutane Pyrimidine Dimer Frequency Correlates with Skin Cancer Mutational Hotspots in p53

Abstract— Ultraviolet light has been identified as the major carcinogen in skin cancer and the p53 tumor suppressor gene is a major target for UV-induced mutations. The mutations are probably caused by unrepaired UV-induced cyclobutane pyrimidine dimers (CPD) and possibly by the less frequent pyrimidine (6-4) pyrimidone photoproducts. While hot spots for p53 mutations in human nonmela-noma skin tumors correspond quite well to slow spots for CPD repair in cultured cells irradiated with the model mutagen 254 nm UVC (which is not present in terrestrial sunlight), they do not all coincide with sequences that are initially frequently damaged by 254 nm UVC. Using LMPCR (ligation-mediated polymerase chain reaction), we show that environmentally relevant UVB light induces CPD at CC and PyrmC positions much more frequently than does UVC light, and that all eight skin cancer hot spots in p53 are also hot spots for UVB-induced CPD. Our results show that methylation of dipyrimidine sites (PyrmCpG) is associated with an increase rate of CPD formation upon UVB irradiation. Consequently, DNA methylation may increase the mutagenic potential of UVB and explains that several p53 mutation hot spots are found at PyrmCpG. The distribution patterns of CPD formation and the photofootprint patterns found along exons 5 and 6 of p53 gene are suggestive of DNA folding into nucleosomes.     

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non-radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB-induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.     

SPECTRAL DEPENDENCE OF UV-INDUCED IMMEDIATE AND DELAYED APOPTOSIS: THE ROLE OF MEMBRANE AND DNA DAMAGE

Abstract— The phototoxicity of each waveband region of UV radiation (UVR), i.e., UVA (32CM100 nm), UVB (290–320 nm) and UVC (200–290 nm), was correlated with an apoptotic mechanism using equilethal doses (10% survival) on murine lymphoma L5178Y-R cells. Apoptosis was qualitatively monitored for DNA "ladder" formation (multiples of 200 base pair units) using agarose gel electrophoresis, while the percentages of apoptotic and membrane-permeabilized cells were quantified over a postexposure time course using flow cytometry. The UVA1 radiation (340–400 nm) induced both an immediate (<4 h) and a delayed (>20 h) apoptotic mechanism, while UVB or UVC radiation induced only the delayed mechanism. The role of membrane damage was examined using a lipophilic free-radical scavenger, vitamin E. Immediate apoptosis and membrane permeability increased in a UVA1 dose-dependent manner, both of which were reduced by vitamin E. However, vitamin E had little effect on UVR-induced delayed apoptosis. In contrast, the DNA damaging agents 2,4- and 2,6-diaminotoluene exclusively induced delayed apoptosis. Thus, immediate apoptosis can be initiated by UVA 1-induced membrane damage, while delayed apoptosis can be initiated by DNA damage. Moreover, the results suggest that immediate and delayed apoptosis are two independent mechanisms that exist beyond the realm of photobiology.     

ALPHA POLYMERASE INVOLVEMENT IN EXCISION REPAIR OF DAMAGE INDUCED BY SOLAR RADIATION AT DEFINED WAVELENGTHS IN HUMAN FIBROBLASTS

Abstract Cultured fibroblasts derived from normal human skin have been irradiated at a series of monochromatic wavelengths throughout the ultraviolet region and exposed to the specific α polymerase inhibitor, aphidicolin (1 μg/m l , 2 days) prior to assay for colony forming ability. Repair of 75-80% of the lethal damage induced by UVC (254 nm) or UVB (302 nm, 313 nm) radiation is inhibited by aphidicolin suggesting that such damage is repaired by a common α polymerase dependent pathway. Exposure to aphidicolin after irradiation at longer UVA (334 nm, 365 nm) or a visible (405 nm) wavelength leads to slight protection from inactivation implying that the processing of damage induced in this wavelength region is quite distinct from that occurring at the shorter wavelengths and does not involve α polymerase.     

Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400-800nm) and ultraviolet A (UVA)/visible (315-800nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8J/cm(2), respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.