SKIN DAMAGE IN ADULT AMPHIBIANS AFTER CHRONIC EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The effects of repeated UV exposure on the skin of the European crested newt, Triturus cristatus carnifex , have been investigated. The animals were irradiated 3 times per week with a Westing-house FS40T12 fluorescent sun lamp (wavelength spectrum 275–350 nm). Two groups of animals received the same total fluence of 1.3 × 10⁵ J/m² in single fluences of either 1570 J/m² (group A) or 9430 J/m² (group C), and one group received a total fluence of 2.6 × 10⁵ J/m² in single fluences of 4710 J/m² (group B). All the animals were killed 7 months after the first UV exposure, but at different intervals after the last exposure. Striking epidermal hyperplasia was found in the newts irradiated at the lower fluence rate (group A). In the animals given the higher total fluence (group B), the most prominent skin changes were dermal fibrosis and irregular thinning and thickening of the epidermis. No significant skin changes were found in group C., in which if there had been UV lesions, they had been repaired during the 5 month interval between the last irradiation and the killing of the animals. No skin tumors developed in any experimental group.     

THE CYTOTOXIC AND MUTAGENIC EFFECTS OF UVA RADIATION ON L5178Y MOUSE LYMPHOMA CELLS

Abstract— A broad-band UVA source that emits primarily350–400 nm radiation and no measurable radiation below 340 nm was used to test toxicity and mutagenicity at the thymidine kinase locus in L5178Y, subclone 3.7.2C (TK⁺/^-) mouse lymphoma cells. Cells were exposed to a fluence of 0 to 80 × 10⁴ J/m². The relationship between UVA fluence and survival was found to have a shoulder region followed by an exponential decrease in survival at higher fluence levels. An exposure-dependent increase in mutation was observed with increasing fluences from 0 to about 60 × 10⁴ J/m². An approximately 3- to 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells was seen at a fluence that resulted in 90% cell killing. We conclude that UVA radiation is a mutagen in the L5178Y mouse lymphoma cells used in this study.     

INTERACTION OF 8-METHOXYPSORALEN AND NEAR-UV LIGHT CAUSES MUTATION AND CYTOTOXICITY IN MAMMALIAN CELLS

Abstract The interaction of near-UV light and a photosensitizer, 8-methoxypsoralen (8-MOP), was studied in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase system; cell survival (cloning efficiency) and mutation induction (resistance to 6-thioguanine) were quantified. Exposure of cells to either 8-MOP up to 20 μg/m l (93 μ M ) or near–UV light up to 40000 J/m² had no effect on either survival or mutation frequency. Preincubation of cells with 8–MOP from 5 to 120 min prior to irradiation with various fluences did not affect cell survival or mutation frequency. Survival decreased and mutation frequency increased linearly when either the 8-MOP concentration or fluence was increased while the other factor was held as a constant. Mutation frequency appears to show reciprocity relative to the product of 8-MOP concentration times fluence of near–UV light [(μg/m l )·(J/m²)] throughout a range apparently limited by high cell lethality. The observed pooled data on mutation, f (x), as a function of (μg/m l )·(J/m²), x , fit a linear dose–response line, f (x) = (34.2 + 0.05 x ) × 10^-6. Cell survival, however, does not appear to exhibit such reciprocity.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

FREQUENCY OF ULTRAVIOLET RADIATION-INDUCED MUTATION AT THE hprt LOCUS IN REPAIR-PROFICIENT MURINE FIBROBLASTS TRANSFECTED WITH THE denV GENE OF BACTERIOPHAGE T4

Abstract— Thc frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts. Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m²UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers. Control cells were somewhat more resistant than denV-transfected cells to killing by UVR. The average frequency of spontancous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10⁶ surviving cells, respectively; there was no statistically significant difference between control and dcnV-transfected cells. However, after exposure to 75 or 150 J/m² UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance. After exposure to a fluence of 75 J/m², the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per lo^h surviving cells for control cells and 92 mutant colonies for denV-transfectcd cells; after 150 J/m², control cells had 205 6-TG-resistant colonies per 10⁶ cells, while dmV-transfected cclls had 61 mutant colonies. These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

THE EFFECTS OF FAR ULTRAVIOLET RADIATION ON RESPIRATION AND CATION ACCUMULATION BY ISOLATED MITOCHONDRIA OF PHASEOLUS VULGARIS

Abstract. The respiration rates and respiratory control ratios of isolated bean mitochondria have been measured following exposure to 0, 150, 300 and 900 J/m² of far UV radiation (190–300 nm) from a mercury vapour light source with 90% total radiant intensity at 254 nm. Loss of respiratory control occurred at 150 J/m² and inhibition of respiration was significant at the highest exposure dosage. The uptake of both ⁴⁵Ca and ⁸⁵Sr have been measured following a 10min incubation of isolated mitochondria with 2 m M cation. Significant decreases in cation accumulation were observed following exposure to 900 J/m². The effect seemed to be associated with loss of active transport of the ions as a result of respiratory uncoupling or reduced electron transport. There was no significant effect of storage on respiration or ion transport nor was there any indirect effect of irradiated suspending medium on mitochondria.     

CYTOTOXICITY AND GENOTOXICITY OF UVA IRRADIATION IN CHINESE HAMSTER OVARY CELLS MEASURED BY SPECIFIC LOCUS MUTATIONS, SISTER CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS

Abstract— The increasing use of artificial UVA (320-400 nm) suntanning devices has brought attention to possible hazardous effects of UVA. In contrast with earlier studies, several groups recently have described that UVA possibly is mutagenic. In this paper we evaluate the genotoxic properties of broad band UVA using CHO cells and three different assays: specific locus (HGPRT) mutations, chromosome aberrations, and sister chromatid exchanges (SCEs). The UVA-source was an UVASUN 2000 S (Mutzhas), emitting UVA above 340 nm. The survival curve of the cells exhibited a shoulder up to 200 kJ/m², that was followed by exponential killing at higher fluences. Mutations were induced linearly in the fluence range from 0-200 kJ/m² ( P < 0.001) to a level seven fold higher than the spontaneous, followed by a decrease at fluences above 300 kJ/m². Over the total range of tested fluences (0-300 kJ/m²) a linear dose-response relationship was observed for UVA-induced SCEs ( P < 0.001). A significantly higher percentage of the cells showed chromosomes with aberrations at the higher levels of exposure (200, 300 and 400 kJ/m²), but no dose response was demonstrated. Our results confirm recent findings showing that UVA is mutagenic in mammalian cells and suggest that UVA exposure may contribute to the total burden of genetic damage caused by exposure to ultraviolet light.     

THE PHOTODYNAMIC EFFECT OF HEMATOPORPHYRIN ON DNA

Abstract— Breakage of DNA in vitro and inside E. colt cells has been determined after exposure to monochromatic 365 nm light in the presence of 10 µM hematoporphyrin. When measured by alkaline sucrose sedimentation, the yields of breaks were 1.4 × 10^-12 per dalton and per J/m² for Col El-DNA in vitro and 5.9 × 10^-3 per dalton and per J/m² for superinfecting phage Λ DNA inside E. coli cells made permeable by toluene. No breaks were found by neutral sucrose sedimentation, demonstrating that the lesions represent alkali-labile bonds. The majority of the alkali-labile bonds were induced by singlet oxygen, as evidenced by the several-fold higher yield obtained in D₂O-containing buffer.     

Alteration of uracil-DNA glycosylase activity by uracil dimers in DNA

Abstract The formation of colonies in solid medium was used as a criterion of viability to determine the effect of ultraviolet radiation on Trichomonas vaginalis. Both viability (colony) counts and total cell (hemocytometer) counts were used to estimate physiological ages of cell populations to be irradiated. Washed-cell suspensions in 0.6% saline were exposed to far- (254 nm) and near-UV (300–400 nm) radiation and dose-response survival curves were constructed from colony counts. The effect of far-UV was found to be independent of growth phase with the D₀ for exponential, early stationary, and late stationary cells 2.6, 2.7, and 2.7 J/m², respectively. Survival to near-UV increased with the age of cells with the estimated D₅₀ being 216 J/m² for exponential cells, 1360 J/m² for early stationary cells, and 4200 J/m² for late stationary cells. Exponential cells of Trichomonas gallinae irradiated with near-UV had a D₅₀ of 340 J/m². T. vaginalis is highly sensitive to far-UV relative to protozoa. T. vaginalis and T. gallinae are highly sensitive to near-UV relative to other microorganisms.     

PHOTOBIOLOGICAL ACTIVITY OF MARMESIN (5-B-HYDROXYISOPROPYL-4–5 DIHYDROFUROCOUMARIN) IN CHINESE HAMSTER V79 CELLS

Abstract— Marmesin was isolated from the medicinal plant, Afraegle paniculata. Its cytotoxicity and mutagenicity in Chinese hamster V79 cells when sensitized to near ultraviolet (NUV) and long wavelength ultraviolet light or black light (BL) were assayed.
Marmesin was extremely cytotoxic in the dark. This cytotoxicity was photoenhanced in NUV and BL; the photoenhanced lethality being higher in NUV than in BL. The LD₅₀ of marmesin under NUV and BL photosensitization were 0.002 μ M and (0.012 μ M ), respectively. In the absence of NUV and BL, marmesin's LD₅₀ was 0.013 μ M . NUV and BL without marmesin were not significantly cytotoxic at the fluence rates of 0.29 W/m² and 4.2 W/m², respectively, for up to 20 min. In contrast to the observed high cytotoxicity of marmesin, its mutagenicity at the HGPRT locus (A_zG^r) was weak. The implication of this result in the high incidence of skin cancer in Nigeria in which A. paniculata is used as a medicinal plant is discussed.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

NEAR-UV INDUCTION OF SISTER CHROMATID EXCHANGES UNDER AEROBIC AND ANAEROBIC CONDITIONS

Abstract— The induction of sister chromatid exchanges (SCE) and cell sensitivity in mouse myeloma cells (66.2 subclone of MPC11) by irradiation with monochromatic near-UV (365 nm) light were studied under aerobic and anaerobic conditions. Sister chromatid exchanges were studied using the fluorescence-plus-Giemsa technique, and sensitivity was determined by the ability of irradiated and nonirradiated control cells to form colonies in soft agar. Cells were found to be 16 times more sensitive to near-UV light under aerobic exposure, producing an F₃₇ value of 7 × 10⁴ J/m² compared to the F₃₇ value of 11.5 × 10⁵ J/m² under anaerobic conditions. The induction of SCE was also 12 times more efficient for aerobic irradiation than for anaerobic irradiation. The data suggest that the SCE-inducing potential of DNA lesions differs when near-UV irradiation is performed in the presence or absence of air. In addition, the DNA lesions responsible for lethality and also those lesions leading to SCE induction may differ under the two irradiation conditions.     

In vitro Fluence Rate Effects in Photodynamic Reactions with AIPcS4 as Sensitizer

Abstract— It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AlPcS₄) is more efficient in photosensi-tization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m² as compared to 500 W/m². Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo , does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explanation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.     

ANALYSIS OF MULTIPLE PHOTORECEPTOR PIGMENTS FOR PHOTOTROPISM IN A MUTANT OF Arabidopsis thaliana

Abstract
The shape of the fluence-response relationship for the phototropic response of the JK224 strain of Arabidopsis thaliana depends on the fluence rate and wavelength of the actinic light. At low fluence rate (0.1 μmol m^-2s^-1), the response to 450-nm light is characterized by a single maximum at about 9 μmol m^-2. At higher fluence rate (0.4 μmol m^-2s^-1), the response shows two maxima, at 4.5 and 9 μmol m^-2. The response to 510-nm light shows a single maximum at 4.5 μmol m^-2. Unilateral preirradiation with high fluence rate (25 μmol m^-2s^-1) 510-nm light eliminates the maximum at 4.5 μmol m^-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 μmol m^-2 is unaffected. Based on these results, it is concluded that a single photoreceptor pigment has been altered in the JK224 strain of Arabidopsis thaliana.     

In Vivo Fluence Rate Effect in Photodynamic Therapy of Early Cancers with Tetra(m-hydroxyphenyl)chlorin

Abstract— Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra( m -hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm² delivered at fluence rates of 15 and 150 mW/cm². Similar tests were also performed at 514 nm with a fluence of 80 J/cm² delivered at fluence rates ranging from 25 to 125 mW/cm². At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

Suppression of Delayed-type Hypersensitivity and Hemolysis Induced by Previously Photooxidized Psoralen: Effect of Fluence Rate and Psoralen Concentration

Abstract— The kinetics of the formation of biologically active psoralen photooxidation (POP) products were analyzed by the biological effects produced. Effects of the UV light fluence rate and psoralen concentration during the preir-radiation were investigated to assess the yield of POP products, which were active in vivo (inducing suppression of delayed-type hypersensitivity [DTH] reaction to sheep red blood cells) and in vitro (altering the human erythrocyte membrane permeability). It was shown that the reciprocity law of the irradiation fluence rate and time was not valid in the case of POP-induced hemolysis and DTH suppression. Immunosuppressive POP products were more efficiently formed at low fluence rate (20.8 W/m²), whereas POP hemolysins were more efficiently produced at a high fluence rate (180 W/m²) of UV light. The yield of immunosuppressive POP products was enhanced in dilute psoralen solutions, while the POP hemolysins yield increased with increasing psoralen concentration. A kinetic scheme for psoralen photoproduct formation was proposed. Kinetic analysis showed that a labile intermediate was produced as the result of excitation of psoralen. This intermediate was either converted to a stable immunosuppressive POP product, or two intermediates combined to form a POP hemolysin. It is proposed that PUVA therapy conditions are more favorable for the formation of immunosuppressive rather than membrane-damaging psoralen photooxidation products.     

THE ENHANCEMENT OF PHYTOCHROME ACTION ON BARLEY LEAF UNFOLDING BY INTERMITTENT LIGHT

Abstract. The phytochrome controlled unfolding of etiolated barley leaves proceeds best if orange light (0.6 J/m²/s; 625 ± 25 nm) is given as two appropriately timed short pulses rather than as a single longer light period.