THE WAVELENGTH DEPENDENCE OF THE EFFECT OF 8-METHOXYPSORALEN PLUS ULTRAVIOLET RADIATION ON THE INDUCTION OF LATENT SIMIAN VIRUS 40 FROM A MAMMALIAN CELL

Abstract— The effect of 8-methoxypsoralen (8-MOP) plus ultraviolet radiation (UV) of different wavelengths in the region 238–365 nm on the induction of SV40 from SV40-transformed Syrian hamster kidney cells was investigated. Results indicate that 8-MOP + UV treatment activates as much as 1000-fold more virus than UV alone at wavelengths in the region 302–365 nm. At wavelengths below 302 nm, 8-MOP addition to cells prior to irradiation shows little, if any, effect. A wavelength dependence for this viral induction is presented.     

THE WAVELENGTH DEPENDENCE OF HERPES SIMPLEX VIRUS INACTIVATION BY ULTRAVIOLET RADIATION

The effect of different wavelengths of ultraviolet (UV) radiation on Herpes simplex virus when assayed on mammalian cells (measured by plaque forming ability) was investigated. The wavelength dependence of viral inactivation was obtained for 11 different wavelengths over the region 238–297 nm. The resulting action spectrum does not closely follow the absorption spectrum of either nucleic acid or protein. The most effective wavelengths for viral inactivation are over the region 260–280 nm.     

THE EXCITATION OF 8-METHOXYPSORALEN WITH VISIBLE LIGHT: REVERSED PHASE HPLC QUANTITATION OF MONOADDUCTS and CROSS-LINKS

Abstract— The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA,320–400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1000 ng/mL) and 419 nm light (up to 12 J/cm²). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.     

MULTIPLE EFFECTS OF UV RADIATION (265–330 NM) ON FUNGAL SPORE EMERGENCE

We have investigated the influence of narrow-band UV radiation, 265–330 nm. on germination of spores of the fungus Cladosporium cucumerinum Ellis and Arth., using a Xe arc lamp and filters. Reciprocity of time and dose rate was demonstrated when fungal spores were subjected to UV radiation at 325 nm but failed to hold at 265 nm. Based on these findings, data on fluence response, and partial action spectra, we propose that there are two biologically active sites in this organism that are affected by radiation between 265 and 330 nm and that might be influenced by changes in the stratospheric ozone layer: a short-wave-sensitive site (265–295 nm) and a long-wave-sensitive site (300–330 nm). Data obtained with narrow-band interference filters confirmed previous reports of damage to nucleic acid from UV at 265–295 nm and in addition demonstrated significant inhibition by UV at 300–320 nm. Further studies of the 300 330 nm portion of the spectrum, using combinations of plastic and glass filters, showed that the influence of UV radiation in this region was primarily to produce a non-photoreactivable delay in germ-tube emergence.     

MUTAGENICITY OF MONOADDUCTS AND CROSS-LINKS INDUCED IN ASPERGILLUS NIDULANS BY 8-METHOXYPSORALEN PLUS 365 NM RADIATION

Abstract— 8-Methoxypsoralen plus 365 nm radiation induces mutation at the methionine supressor loci of Aspergillus inhibitor-deficient conidia at low doses of near-UV radiation with one-hit kinetics and at higher near-UV radiation doses with two-hit kinetics. These results and others suggest that both monoadducts and cross-links, formed by 8-methoxypsoralen and DNA upon exposure to UV radiation, are capable of inducing mutation. Evidence is also presented that induced furocoumarin cross-links are responsible for the inactivation of the Aspergillus conidium.     

PHOTOREACTIVATION OF ICR 2A FROG CELLS AFTER EXPOSURE TO MONOCHROMATIC ULTRAVIOLET RADIATION IN THE 252–313 nm RANGE

Abstract— Exposure of ICR 2A frog cells to photoreactivating light after treatment with monochromatic ultraviolet (UV) radiation in the 252–313 nm range resulted in an increase in survival with similar photoreactivable sectors for each of the wavelengths tested. As photoreactivating enzyme is specific for the repair of pyrimidine dimers in DNA, these findings support the hypothesis that these are critical lesions responsible for killing of cells exposed to UV radiation in this wavelength range. The action spectra for cell killing and production of UV-endonuclease sensitive sites were similar to the DNA absorption spectrum though not identical. Because the number of endonuclease sensitive sites is a reflection of the yield of pyrimidine dimers, these data also suggest that the induction of dimers in DNA by UV radiation in the 252–313 nm range is the principal event leading to cell death.     

Measurement of UVA Exposure to Solar Radiation

Exposure to solar UVA (320–400 nm) radiation can damage DNA and lead to skin disorders. Conventional dosim-etry using a single piece of polysulfone or diglycol carbonate (CR-39) cannot provide accurate measurement of the biologically effective irradiance for erythema for the UVA waveband. A package employing four dosimeters (polysulfone, nalidixic acid, 8-methoxypsoralen and phe-nothiazine) has been shown to be effective for use as a spectrum evaluator for evaluating the UVA source spectrum. In Brisbane, on a horizontal position, the spectrum evaluator requires about 5 min exposure in summer and about 20 min in winter. This amounts to about 10 mJ cm-² of erythemal UV radiation.     

NEAR ULTRAVIOLET INACTIVATION STUDIES ONESCHERICHIA COLI TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASE

Abstract— –The response of two pyridoxal-phosphate-requiring enzymes of E. coli, tryptophanase and tryptophan synthetase, to near UV light (320–400 nm) has been studied. Tryptophanase is inactivated both in vivo and in vitro, but tryptophan synthetase is resistant to near UV under both conditions. This shows that near UV inactivation is not general for pyridoxal-phosphate-requiring enzymes. Substrate protection against light inactivation is demonstrated for tryptophanase. It is furthermore shown that pyridoxal phosphate is required for inactivation of this enzyme. However, the action spectrum for this inactivation does not coincide with the absorption spectrum of tryptophanase or of pyridoxal phosphate.     

Action spectra for photoinduced inactivation of bacteriophage T7 sensitized by 8-methoxypsoralen and angelicin

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.     

AN ACTION SPECTRUM FOR LETHAL PHOTOSENSITIZATION OF Candida albicans BY 8-MOP AFTER LOW-DOSE BROAD-BAND UV-A IRRADIATION; AN ACTION SPECTRUM FOR 8-MOP 4′,5′- MONOADDUCTS?

An action spectrum was obtained for lethal photosensitization of Candida albicans that had been pre-irradiated with a sub-lethal low dose of broad-band UV-A after incubation with 8-methoxypsoralen and then washed to remove any non-photobound 8-methoxypsoralen. The resultant spectrum with a peak in the 320-335 nm region was similar to that obtained by a conventional method (single irradiation only) and also to the absorption spectrum of the 4',5'-monoadduct. These data support the hypothesis that the chromophore for lethal photosensitization by 8-methoxypsoralen is the 8-methoxypsoralen/DNA 4',5'-monoadduct rather than 8-methoxypsoralen itself.     

An unusual case of photoreactivation observed in an insect egg (Smittia Spec., chironomidae, diptera).

Abstract— An action spectrum for inactivation by ultraviolet (UV) radiation of Smittia eggs during intravitelline cleavage was established, taking into account wavelength-dependent shielding of the effective targets. Under the assumption of a random distribution of the effective targets in the egg, the action spectrum displayed only one very distinct peak at 295 nm. The eggs were photoreactivable with an action spectrum similar but not identical to that found for direct photoreactivation (PR) in E. coli Indirect PR seems not involved because light was effective only after but not before UV. Temperature dependence and dose rate saturation could not be observed. The photoreactivable sector (PRS) was 0.75 after UV inactivation at 295 nm but only 0.32 after UV inactivation at 265 nm. Initial PR rates were highest after 295 nm and lowest after 265 nm. During migration of cleavage nuclei into the periplasm, when the shielding of nuclei by yolk material decreases by an order of magnitude, no corresponding increase in the sensitivity of the eggs to UV was observed. After inactivation at the blastoderm stage, when the nuclei are no longer shielded by yolk material, the PRS was also high (0.79) after UV of 295 nm but again lower (0.59) after 265 nm. These data are difficult to understand within the conceptual framework of light-dependent enzymatic splitting of UV-induced pyrimidine dimers in nucleic acids. Yet this type of PR seems to play a vital role in the survival of Smittia eggs under sunlight without need for pigmentation or shading.     

Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.     

PHOTOSENSITIZING EFFECTS OF 8-METHOXYPSORALEN ON THE SKIN OF HAIRLESS MICE—I. FORMATION OF INTERSTRAND CROSS-LINKS IN EPIDERMAL DNA

Abstract— The photomediated induction of interstrand cross-links by 8-methoxypsoralen has been measured in the epidermal DNA of hairless mice. Equivalent efficiencies for cross-link induction were determined for HRS/J/Anl and SKH: hairless-1 mice. A wavelength dependence on the relative efficiency of cross-link induction was observed; a broad spectrum light source, 300–400 nm, was approximately 5 times more effective in cross-link formation than a 365 nm light source. Repeated exposure to 8-methoxypsoralen followed by ultraviolet light, 5 times a week for 6 weeks, altered epidermal thickness and resulted in a decreased efficiency for DNA cross-link formation.     

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV-irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8-hydroxy-10-methyl-5-deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8-hydroxy-5-deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.     

REPAIR OF 313-NM INDUCED LESIONS AND PHOTOPROTECTION IN YEAST CANDIDA GUILLIERMONDII

Abstract. The present communication is concerned with the effects of near-UV radiation (300–380 nm) on yeast Candida guilliermondii. It was found that certain doses of 313 nm irradiation caused inactivation of the yeast which was exhibited in a way different from the lethal action of far-UV radiation. It was also found that the cells inactivated by 313 nm are capable of recovering vitality, if incubated for some time in a non-nutrient medium. The yeast inactivated by far-UV radiation also proved to be capable of recovering, though to a lesser degree. Both 334 nm radiation and non-lethal doses at 313 nm induced the photoprotective effect against far-UV damage. The effect was exhibited if there was a certain time interval (2–4 h) between the exposures to photoprotective light and subsequent far-UV radiation. Within this time interval the extent of photoprotection was dependent on temperature.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

ULTRAVIOLET ACTION SPECTRA FOR PHOTOBIOLOGICAL EFFECTS IN CULTURED HUMAN LENS EPITHELIAL CELLS

Abstract— The action spectrum for cell killing by UV radiation in human lens epithelial (HLE) cells is not known. Here we report the action spectrum in the 297–365 nm region in cultured HLE cells with an extended lifespan (HLE B-3 cells) and define their usefulness as a model system for photobiological studies. Cells were irradiated with monochromatic radiation at 297, 302, 313, 325, 334 and 365 nm. Cell survival was determined using a clonogenic assay. Analysis of survival curves showed that radiation at 297 nm was six times more effective in cell killing than 302 nm radiation; 297 nm radiation was more than 260, 590, 1400 and 3000 times as effective in cell killing as 313, 325, 334 and 365 nm radiation, respectively. The action spectrum was similar in shape to that for other human epithelial cell lines and rabbit lens epithelial cells. The effect of UV radiation on crystallin synthesis was also determined at different wavelengths. To determine whether exposure to UV radiation affects the synthesis of β-crystallin, cells were exposed to sublethal fluences of UV radiation at 302 and 313 nm, labeled with [³⁵S]methionine and the newly synthesized βY-crystallin was analyzed by immunoprecipitation and western blotting using an antibody to β-crystallin. The results show a decrease in crystallin synthesis in HLE cells irradiated at 302 and 313 nm at fluences causing low cytotoxicity. The effect of radiation on membrane perturbation was determined by measuring enhancement of synthesis of prostaglandin E₂ (PGE₂). Synthesis of PGE₂ occurs at all UV wavelengths tested in the 297–365 nm region. The slope of the PGE₂ response curves was higher than that of cell killing curves in cultured HLE cells. These data show that cultured HLE cells with extended lifespan are a suitable system for investigating photobiological responses of cells to UV radiation.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

ENHANCEMENT OF UV-INDUCED SKIN CARCINOGENESIS BY AZATHIOPRINE: ROLE OF PHOTOCHEMICAL SENSITISATION

Abstract The phototoxicity of various drugs (chlorpromazine (CPZ), metronidazole (MET), 8-methoxypsoralen (8-MOP), azathioprine (AZA) and the azathioprine metabolites, 6-mercaptopurine (6-MP), methylnitrothio-imidazole (MNTI), methylnitroimidazole (MNI)) was determined in hairless (Skh-hrl) mice exposed to a light source with broad emission (290–700 nm). Chlorpromazine, MET, 8-MOP, AZA, MNI and 6-MP were phototoxic, as determined by the oedematous response of skin, at doses comparable to those used clinically.
The effects of long-term drug therapy and UV radiation on skin carcinogenesis were then determined. Chlorpromazine and MET, and to a lesser extent AZA, MNTI and 6-MP, enhanced photocarcinogen-esis. In each case, both the tumour yield and the proportion of malignant:benign skin tumours were increased. The results of this study imply that prolonged treatment of mice with low-level phototoxins predisposes to photocarcinogenesis.     

ULTRAVIOLET RADIATION IN ANTARCTICA: INHIBITION OF PRIMARY PRODUCTION

With the seasonal formation of the ozone hole over Antarctica, there is much concern regarding the effects of increased solar UV-B radiation (280–320 nm) on the marine ecosystem in the Southern Ocean. In situ incubations of natural phytoplankton assemblages in antarctic waters indicate that under normal ozone conditions UV-B radiation is responsible for a loss of approximately 4.9% of primary production in the euphotic zone, whereas UV radiation with wavelengths between 320 and 360 nm causes a loss of approximately 6.2%. When combined with data on the action spectrum for photoinhibition by UV radiation, our data suggest that the enhanced fluence of UV-B radiation under a well-developed ozone hole (150 Dobson units) would decrease daily primary productivity by an additional amount of 3.8%. Calculations that take into consideration the extent and duration of low stratospheric ozone concentrations during September to November indicate that the decrease in total annual primary production in antarctic waters due to enhanced UV-B radiation would be 0.20%.