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.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

THE VASCULAR RESPONSE OF HUMAN SKIN TO ULTRAVIOLET RADIATION

The vascular response of human skin to 300 nm (UV-B) and 254 nm (UV-C) ultraviolet radiation was assessed using the reflectance measurement of erythema and the technique of laser Doppler velocimetry. For both wavelengths, the increase in measured Doppler blood flux varied with the increase in erythema in a quadratic manner predicted by a simple model based on the principles of fluid mechanics. This suggests that the mean red blood cell velocity increases significantly in areas of UV-B and UV-C erythema. No qualitative difference in response to these two wavelengths was demonstrated, suggesting that the same blood vessels are involved in the causation of both UV-B and UV-C erythema.     

Removing UV-A and UV-C radiation from UV-B fluorescent lamp emissions. Differences in the inhibition of photosynthesis in the marine alga Dunaliella tertiolecta using chromate versus cellulose acetate-polyester filters

Ultraviolet-B (UV-B; 280-320 nm)-emitting lamps unavoidably emit ultraviolet-A (UV-A; 320-400 nm) and ultraviolet-C (UV-C; <280 nm) radiation. Short-wavelength-blocking filters are generally used to limit the wave bands of UV under investigation. The widespread use of such filters means that all exposures to UV-B radiation will have a significant UV-A component. Therefore, the physiological effects unique to UV-B exposure are difficult to clearly isolate. This study presents a method to remove the UV-A and UV-C "contamination" using a liquid potassium chromate (K(2)CrO(4)) filter, thus allowing more direct assessment of the effects of UV-B exposure. Cultures of the green marine alga Dunaliella tertiolecta were grown in the absence of UV radiation. Sunlamps supplied the UV radiation for a 24 h exposure (solar radiation was not used in this study). The UV radiation was filtered either by the standard method (i.e. cellulose acetate (CA) with polyester = Mylar controls) or by a liquid filter of potassium chromate. Photosynthetic responses were compared. Major decreases in the ratio of variable to maximal fluorescence in dark-adapted cells and photosynthetic capacity were observed in CA-filtered cultures, whereas no change was observed in cells exposed to the same UV-B flux with the UV-A removed by K(2)CrO(4). The use of a CA filter with a Mylar control does not link results unequivocally to UV-B radiation. Such results should be interpreted with caution.     

RELATIVE INDUCTION OF CYCLOBUTANE DIMERS and CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro and in vivo WITH ULTRAVIOLET-C and ULTRAVIOLET-B LIGHT

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.     

COMPARATIVE STUDIES ON THE LETHAL, MUTAGENIC, AND RECOMBINOGENIC EFFECTS OF ULTRAVIOLET-A,-B,-C, AND VISIBLE LIGHT WITH AND WITHOUT 8-METHOXYPSORALEN IN Saccharomyces cerevisiae

Abstract
Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8MOP) with UV-A or visible light were studied in the haploid strain XV185–14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and horn+ reverse mutations was measured in strain XV185–14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185–14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185–14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.     

UV PHOTOBIOLOGY OF THE NEMATODE Caenorhabditis elegans: ACTION SPECTRA, ABSENCE OF PHOTOREACTIVATION and EFFECTS OF CAFFEINE

Action spectra for UV inactivation of reproduction in Caenorhabditis elegans have been obtained for strains N2 (wild-type) and rad-3 (radiation-sensitive). Use of a dye laser radiation source, providing high intensity in a narrow wavelength band, has permitted more detail (14 wavelengths between 260 and 320 nm) than available in the spectra for other multicellular organisms. Overall sensitivity of N2 is similar to that of wild-type Escherichia coli; that of rad-3 is 30-fold higher between 265 and 310 nm; relative sensitivity decreases above 310 nm but also seems to increase for irradiation below 265 nm. Tests for photoreactivation and for modification of survival by post-irradiation treatment with caffeine were negative.     

Physiological Responses of Scenedesmus quadricauda (Chlorophyceae) to UV-A and UV-C Light

Despite intensive research focused on the effects of UV-B, deeper metabolic responses to UV-A and UV-C are still scarce. Besides, especially microalgal species had to develop efficient protective features in comparison with tissue structure of vascular plants. We exposed axenic cultures of Scenedesmus quadricauda (Chlorophyceae) to UV-A (366 nm) and UV-C (254 nm) light over 1 h. Both wavelengths stimulated increase in soluble proteins, superoxide radical and hydrogen peroxide, but had a nonsignificant effect on cell viability. Within 17 detected free amino acids, five (including proline) increased in response to UV-A while only aspartic acid and histidine increased in UV-C treatment. Total soluble phenols and flavonoids were influenced neither by UV-A nor by UV-C while selected flavonols (quercetin and kaempferol) decreased in UV-A and were not detected in UV-C treatment. Benzoic acid derivatives increased preferentially after UV-A illumination (vanillic acid and vanillin) while cinnamic derivatives (caffeic, chlorogenic and p-coumaric acids) decreased in both UV-A and UV-C. It is concluded that UV light stimulated oxidative stress while exposure time was not sufficient to stimulate larger changes in phenolic metabolites. Present findings in the context of available data and with emphasis on phenolics in algae are discussed.     

Polymer-based triphenyl tetrazolium chloride films for ultraviolet radiation monitoring

Ultraviolet (UV) radiation monitoring films were prepared from solutions of polymers (polyvinyl, alcohol, PVA, or polyvinyl butyral, PVB), containing triphenyl tetrazolium chloride dye (TTC). These films have a pronounced response to the main UV radiation spectral regions [UV-A (400–320 nm), UV-B (320–280 nm), and UV-C (280–180 nm)] showing different sensitivities. PVA/TTC film has its maximum sensitivity in the UV-A region, while PVB/TTC film has its maximum sensitivity in the UV-C region. Both films have almost the same sensitivity in the UV-B region. The radiation-induced colour change is analysed spectrophotometrically at the maximum of the visible absorption band peaking at 492 nm wavelength. The measurement uncertainty of estimating ultraviolet radiation energy incident per unit area on the films is found to be about 3.5% (1 σ). The study of the effect of radiance exposure, incident wavelength, and storage conditions have been carried out to characterise the use of these films for actinometric monitoring artificial ultraviolet radiation sources which are used for medical and industrial applications.     

THE ROLE OF SOLAR UV-B IN GROWTH REGULATION OF CRESS (Lepidium sativum L.) SEEDLINGS

Abstract— Action spectra for growth reduction within the 260 nm to 305 nm waveband were measured for hypocotyls and roots of young etiolated cress ( Lepidium sativum ) seedlings. The action spectra show increasing photon effectiveness with decreasing wavelength and resemble those due to DNA damage. Using short term irradiations, photoreceptors absorbing in the visible range were found to be without influence. As no photoreactivation could be found and the seedlings showed no outward signs of damage, this growth effect may be due to a UV photoreceptor. A modeling calculation was carried out to estimate the effectiveness of solar UV-B on this response both under present conditions and under reduced ozone levels. Even under present conditions, solar UV-B could be involved in regulating growth during the period immediately after germination.     

The effect of the short wavelength ultraviolet radiation. An extension of biological dosimetry to the UV-C range

Polycrystalline uracil thin layer can be used as biological dosimeter for assessing exposure to UV radiation. The dimerization and reversion efficiency of the ultraviolet radiation in the UV-B and the UV-C range were quantified on polycrystalline uracil thin layers irradiated with quasi-monochromatic radiation using interference filters of 10nm bandwidth. The dimer formation and monomerization (reversion) dose-effect relations were determined by optical spectroscopy. The decrease of the OD value of the uracil thin layer at 288 nm was taken as a measure of the dimer formation, while the increase of the OD of a completely irradiated (until reaching the saturation level) uracil layer was taken as the sign of the monomerization. The two processes in the UV-B and the UV-C range take place simultaneously, the individual characterization of the dimerization efficiency was performed from the initial slope of the dimerization dose-effect function and an action spectrum for dimerization was constructed in the UV-C range too. The reversion efficiency was found to be practically the same with all of the investigated wavelengths: 200 nm, 210 nm, 220 nm, 230 nm, 240 nm The possible biological relevance of the reversion of dimers are discussed.     

Transmission of UV-radiation through human epidermal layers as a factor influencing the minimal erythema dose

Abstract— The extent to which transmission of human Caucasian epidermis and stratum corneum influences the Minimal Erythema Dose (MED) was examined for UV-B and UV-C.
Transmission correlated well with variations in MED for UV-B and UV-C that exist between individuals, as measured on the skin of the lower back and anterior upper leg.
Regional differences of the MED that occur within the same individual between different parts of the body, were related less well to differences in transmission. For UV-B, the difference in transmission of stratum corneum and epidermis between upper leg and lower back was on the average too small to completely account for the difference in MED UV-B. Other parameters, therefore, have to be involved in determining such regional variations in MED UV-B. For UV-C, on average, the difference in transmission of stratum corneum was smaller and of epidermis larger than the difference in MED UV-C between upper leg and lower back, though the deviations were not significant.
A series of UV-B irradiations of the lower back resulted in an increase in MED UV-B and MED UV-C, which was paralleled by a decrease in transmission of stratum corneum and epidermis. The average decrease in UV-B transmission of stratum corneum was too small and that of epidermis somewhat too large to account for the average increase in MED UV-B. The average decrease in UV-C transmission of stratum corneum was about as large as the average increase in MED UV-C. Consequences of these observations for the location of the primary reactions leading to erythema are discussed.
The relationship between log MED UV-C and log MED UV-B was confirmed to be approximately linear.     

COMPARATIVE STUDIES ON THE LETHAL, MUTAGENIC, AND RECOMBINOGENIC EFFECTS OF ULTRAVIOLET-A,-B,-C, AND VISIBLE LIGHT WITH AND WITHOUT 8-METHOXYPSORALEN IN Saccharomyces cerevisiae

Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.     

GENETIC EFFECTS OF UV-B: MUTAGENICITY OF 308 nm LIGHT IN CHINESE HAMSTER V79 CELLS

Abstract —Chinese hamster V79 cells were irradiated with 254 nm (UV-C) and 308 nm (UV-B) light, emitted by a germicidal lamp and an excimer laser, respectively. Induction of mutations at two distinct genetic loci was measured by selecting colonies resistant to 6-thioguanine or to ouabain. Unlike 6-thioguanine resistance which can be presumed to be due to many different types of genetic damage, mutation to ouabain resistance seems to result from base-pair substitution events only. Much higher doses of 308 than of 254 nm radiation are required to induce equivalent numbers of mutants. However, induction of cell inactivation and 6-thioguanine resistant mutations with the two UV sources appears to be correlated, suggesting that a common mechanism, perhaps involving the induction of pyrimidine-containing dimers, is involved. The frequency of ouabain resistant mutants per lethal event is on the other hand much higher after irradiation with the 308 nm light. This latter finding further defines a part of the UV-B spectral region which seems to induce a unique kind of DNA damage which specifically results in base-pair substitution events. Action spectra studies therefore appear necessary in the definition of the mutagenic effects of UV-B radiations in mammalian cells.     

A DYE LASER SOURCE OF MONOCHROMATIC UV-B and UV-C RADIATIONS FOR BIOLOGICAL ACTION SPECTROSCOPY

Abstract We have used a flashlamp driven tunable dye laser as a radiation source for observing UV-C and UV-B action spectra of two eukaryotic microorganisms. The general nature of the irradiation system and various operating parameters are described. The laser produces the high power output required for UV-B action spectroscopy; it is tunable throughout the UV-B range with the very narrow bandwidth required for high resolution action spectra. The peak power density does not appear to reach the threshold of 'two photon' biological effects, a concern when using pulsed lasers for action spectroscopy. The laser is somewhat more expensive and considerably more difficult to operate compared with arc-monochrometer systems; it is generally equal or greater in UV-B power and has distinct advantages in tunability and bandwidth of the spectral output.     

Safety Evaluation of Far-UV-C Irradiation to Epithelial Basal Cells in the Corneal Limbus

Basal cells in the corneal limbus play an important role in the turnover cycle because they are the source of all cells that constitute the corneal epithelium. We examined the penetration depth of ultraviolet (UV) light in the corneal limbus and assessed the safety of Far-UV-C on stem cells in the basal area of the corneal limbus. Rats were irradiated with UV at peaks of 207, 222, 235, 254 and 311 nm while under anesthesia. The UV penetration depth in the rat corneal limbal epithelium was wavelength dependent: 311 nm UV-B and 254 nm UV-C reached the basal cells of the epithelium, and 235 nm radiation reached the middle area; however, 207 and 222 nm UV-C reached only the superficial layer of the epithelium. Porcine cornea, which is similar to the human eye in size and structure, were irradiated with 222 and 254 nm UV-C. As in rats, 222 nm UV-C reached only the superficial layer of the porcine corneal limbal epithelium. These results indicate that Far-UV-C, such as radiation of wavelengths of 207 and 222 nm, could not reach corneal epithelial stem cells, i.e. the cells remained intact. It is unlikely that the turnover of the corneal epithelium is obstructed or disrupted by exposure to Far-UV-C.     

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.     

Role of white light in reversing UV-B-mediated effects in the N2-fixing cyanobacterium Anabaena BT2

The effects of various irradiances of artificial UV-B (280-315 nm) in the presence or absence of visible light (photosynthetically active radiation) on growth, survival, 14CO2 uptake and ribulose 1,5-bisphosphate carboxylase (RuBISCO) activity were studied in the N2-fixing cyanobacterium Anabaena BT2. We tested the hypothesis whether or not visible radiation offers any protection against UV-B-induced deleterious effects on growth and photosynthesis in Anabaena BT2. Attempts were also made to determine the irradiances of UV-B where inhibitory effects could be mitigated by simultaneous irradiation with visible light. Exposure of cultures to 0.2 W m(-2) or higher irradiance of UV-B caused inhibition of growth and survival and growth ceased above 1.0 W m(-2). 14CO uptake and RuBISCO activity were found to be more sensitive to UV-B and around 60% reduction in 14CO2 uptake and RuBISCO activity occurred after exposure of cultures to 0.4 W m(-2) for 1 h. However, growth, 14CO2 uptake and RuBISCO activity were nearly normal when UV-B (0.4 W m(-2)) and visible light (14.4 W m(-2)) were given simultaneously. Blue radiation (450 nm) was found to be the most effective in photoreactivation against UV-B, better than UV-A or any other light wavelength band. Our results demonstrate that the studied cyanobacterium possesses active photoreactivation mechanism(s) against UV-B-mediated damage which in turn probably allow survival under natural conditions in spite of being continuously exposed to the UV-B component present in the solar radiation. Continued growth of many algae and cyanobacteria in the presence of intense solar UV-B radiation under natural conditions seems to be due to the active role of photoreactivation.