THE ACTION OF ULTRAVIOLET RADIATION ON THE CELL PROLIFERATION OF TWO-CELL MOUSE EMBRYOS

Abstract— The two-cell mouse embryo has a unique cell cycle of a short DNA synthesis (S) phase and an extremely long post-DNA synthesis (G₂) phase. An attempt was made to investigate the radiation biology of the long G₂ phase using UV radiation as a probe. Two cell mouse embryos, at various positions in the cell cycle, were UV-irradiated in phosphate-buffered saline. The embryos were cultured for a few hours to 3 days to assay for their cell proliferative characteristics. The embryos were most sensitive to the killing action of UV radiation in the late G₂ phase. The embryos divided more than two times after low UV fluences before dying and experienced G₂ phase delays.
These results can be contrasted to the situation in somatic cells, in which the action of UV radiation is S phase selective. One possibility is that the target for the action of UV radiation is different in two-cell mouse embryos from that in somatic cells and that the target is similar to that for X-ray effects.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: SEDIMENTATION STUDIES WITH THE USE OF 5''-BROMODEOXYURIDINE PHOTOLYSIS

Abstract Relative to their L5178Y-S counterparts, L5178Y-R cells have an impaired capacity to form patches in DNA after exposure to UVC radiation. The photolysis of 5'-bromodeoxyuridine (BrdUrd) incorporated into DNA was used to estimate the number of 'repair patches'formed in response to a 254 nm UV (UVC) exposure. L5178Y-S cells, typical of rodent cell lines, formed a small number of patches in exposed DNA (1-2 patches per 1 times 10⁸ dalton during a 6 h recovery after an exposure of 20 J/m²). In contrast, DNA extracted from L5178Y-R cells exposed to UVC and subsequently incubated with BrdUrd for 6 h showed no evidence of BrdUrd incorporation indicating no capacity to form sites of repair (fewer than 0.5 sites of BrdUrd incorporation per 1 times 10⁸ dalton). Moreover, in L5178Y-R cells high fluences of UVC caused an extensive DNA degradation. Such degradation was not observed in L5178Y-S cells during the 24-h post-exposure period. These results are consistent with the notion that L5178Y-R cells have a reduced capacity to repair DNA damage induced by UVC radiation.     

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.     

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.     

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.     

SYNERGISTIC INTERACTION BETWEEN THE TRYPTOPHAN PHOTOPRODUCTS AND NEAR-ULTRAVIOLET LIGHT IN AMOEBA

Abstract. Both S- and G₂-phase cells of amoeba are found to be sensitized by coirradiation of cells with Tryptophan (Trp). At a fluence of 1.5 times 10⁵ Jm^-2 to which G₂ phase cells are resistant, they show a drastic (8-fold) increase in sensitivity when irradiated in 0.5% Trp solution. Freshly irradiated Trp solution is found to be lethal to G₂ cells that have received a sublethal fluence of near-UV light. Irradiated Trp, however, does not kill unirradiated amoeba.
Nuclear transplantation experiments have shown that the lethality of cells when coirradiated with Trp is due to damage located predominantly in the cytoplasm. It is suggested that sublethal fluence of near-UV light makes the cells "leaky" to the toxic photooxidation products of Trp solution.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated 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.     

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.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

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.     

POSTIRRADIATION PROPERTIES OF A UV-SENSITIVE VARIANT OF CHO

Abstract A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation (D₀ of 0.3 J/m² as compared to 3.2 J/m² for the wild type). 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.     

EFFICIENT PHOTODYNAMIC ACTION OF VICTORIA BLUE BO AGAINST THE HUMAN LEUKEMIC CELL LINES K-562 AND TF-1

Abstract— Photodynamic induced cytotoxicity by Victoria blue BO (VB-BO), merocyanine 540 (MC540), Nile blue A (NB) and 4-tetrasulfonatophenyl-porphyrin (4-TSPP) has been studied on two human leukemic cell lines: K-562 and TF-1. Cells were incubated with dyes and irradiated with different doses of white light. Cell survival was assessed by propidium iodide (PI) staining using flow cytometry analysis. Concentrations of 5 x 10 ⁸ M VB-BO were found to kill 75% of cells, and a concentration of 1 × 10⁻⁷ M induced more than 99% of cell killing. To obtain the same cytotoxic level, the presence of 2.6 × 10⁻⁵ M of MCS40 during irradiation was needed. Under the conditions used, NB was ineflective as a photosensitizer, although uptake studies showed that this dye was taken by the cells in much greater amounts than any other studied dye. Cell cycle distribution of TF-1 cells, surviving MC540 or VB-BO photoscnsitization has bccn studied by flow cytometry analysis after staining with Hoechst 33342 and PI. It was found that cells in G₁ phase were slightly more resistant toward MCS40– and VB-BO-mediated photosensitization than cells in other phascs of the ccll cycle     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.     

INHIBITION OF DNA SYNTHESIS BY PSORALEN-INDUCED LESIONS IN XERODERMA PIGMENTOSUM AND FANCONI''S ANEMIA FIBROBLASTS

Abstract— Exposure of human cells to psoralens and near-UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8-Methoxypsoralen (8-MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8-MOP and 7500 J/m² of near-UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near-UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8-MOP plus near-UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8-MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.     

PHOTOSENSITIZATION OF HUMAN DIPLOID CELL CULTURES BY INTRACELLULAR FLAVINS AND PROTECTION BY ANTIOXIDANTS

Abstract— The damaging effects of near ultraviolet and visible light on WI-38 human diploid lung fibroblasts were investigated. WI-38 cells in culture were killed by light doses ranging from 2 to 10 × 10³ W/m² h. There was an inverse correlation between culture age, i.e. population doubling level and photosensitivity. However, this effect could not be related to capacity for DNA synthesis and cell division.
Flavins were clearly implicated as endogenous photosensitizers, and antioxidants such as d, l -α-tocopherol (vitamin E), BHT and ascorbic acid were found to afford the cells protection from light damage. Furthermore, products of lipid peroxidation could be detected in cell homogenates irradiated in the presence of ribofiavin.     

PRIOR EXPOSURE OF HUMAN CELLS TO NEAR UV-RADIATION GIVES A DECREASE IN THE AMOUNT OF THE UNSCHEDULED DNA-SYNTHESIS INDUCED BY FAR UV-RADIATION

Pretreatment of human cells with near UV radiation (UVA) in fluences exceeding 5 × 10⁴ Jm⁻² caused a decrease in the amount of the unscheduled DNA synthesis induced by far UV radiation (UVC). The DNA repair synthesis, as measured by the incorporation of [³H] -thymidine, is reduced by nearly a factor of 2 for a UVA radiation exposure of 1.5 × 10⁵ Jm⁻². Since solar UVA fluence rate is rather independent of latitude, this figure corresponds to a UVA exposure time of 50-60 min from noon sunlight in the summer time.     

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K⁺-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m²). ATPase activity measured in the absence of K⁺ and K⁺-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m².