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%.     

INHIBITION OF THE IMMUNE RESPONSE TO ALLOANTIGEN IN THE RAT BY EXPOSURE TO ULTRAVIOLET RADIATION

Exposure of mice to ultraviolet radiation (UV) followed by alloantigen sensitization can suppress the immune response to that alloantigen. In order to assess the applicability of using UV-induced immunosuppression in organ transplantation, the effectiveness of UV in prolonging the survival of vascularized organ allografts must be determined. Because, for technical reasons, rats are better suited than mice for such experiments, we first wanted to determine whether UV suppresses the immune response of inbred rats to alloantigens. The data presented here demonstrate that exposure of rats to UV (115-129 kJ/m2) prior to alloantigenic sensitization decreases the mixed lymphocyte response to alloantigen. The depression of the proliferative response to alloantigen was selective in that spleen cells from the UV-treated rats could respond to mitogenic stimulation. In contrast to previous results with mice, suppressor cells could not be demonstrated in the spleens of the UV-treated rats. In addition, UV treatment after sensitization inhibited the response to alloantigen. These data suggest that treatment of the recipient with UV before or after alloantigenic sensitization may provide a novel method of inhibiting immune responses to allogeneic antigens.     

PSORALEN PLUS ULTRAVIOLET RADIATION-INDUCED INHIBITION OF DNA SYNTHESIS AND VIABILITY IN HUMAN LYMPHOID CELLS IN VITRO*

Abstract— 8-Methoxypsoralen (8-MOP) plus high intensity long wavelength ultraviolet radiation (UV-A) is presently being used to induce remissions of psoriasis and mycosis fungoides. Previous studies demonstrated inhibition of DNA synthesis in circulating leukocytes from some patients during this therapy. The present study is designed to determine whether conditions of 8-MOP concentration and UV-A exposure attained during therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (³HTdR) incorporation and cell growth in suspension culture following UV-A exposure alone or with therapeutic concentrations of 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblas-toid cell lines. UV-A exposure alone induced a dose-dependent inhibition of HTdR incorporation in both types of lymphoid cells (3000 J/m² resulted in 77% of control ³HTdR incorporation). Pre-incubation with 0.1 μg/m/ 8-MOP before UV-A exposure induced a significantly greater inhibition of ³HTdR incorporation (3000 J/m² resulted in 61% of control ³HTdR incorporation). Greater inhibition of ³HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0μg/mC 8-MOP (3000 J/m² resulted in 41% of control) but not in the lymphocytes (3000 J/m² resulted in 63% of control). The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,000 J/m²) but preincubation with 0.1 μg/mC 8-MOP resulted in 40% survival after 3000 J/m² (D₃₇ approximately 3000 J/m²) and preincubation with 1.0 μg/ 8-MOP resulted in 0.6% survival after 3000 J/,² (D₃₇ approximately 800 J/m²). This study suggests that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. The long term consequences of such damage remain to be determined.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

INHIBITION OF NUCLEIC ACID SYNTHESIS IN CELLS EXPOSED TO 200 MICROMETER RADIATION FROM THE FREE ELECTRON LASER

Vertebrate tissue culture cells were exposed to 200 um wavelength radiation (1CO W/cm²) from a free electron laser (FEL) of the electrostatic generator type. Cell cultures demonstrated no morphological alterations but a statistically significant (P .05) proportion of the cells exhibited inhibition of DNA synthesis. This study demonstrates the first biological effects of the FEL and the feasibility of performing biological investigations with this device.     

EFFECTS OF ULTRAVIOLET RADIATION ON THE MITOTIC CYCLE AND DNA, RNA AND PROTEIN SYNTHESIS IN MAMMALIAN EPIDERMIS IN VIVO

Abstract— Acute effects of ultraviolet radiation on the mitotic cycle and macromolecular synthesis were investigated on hairless mouse epidermis in vivo. Colcemid was used to arrest mitoses in metaphase and thus allow more accurate mitotic counts. The radioactive tracers, TdR-³H, cytidine-³H, and the amino acids, histidine-³H and methionine-³H were used to examine DNA, RNA and protein synthesis, respectively. Using these techniques, we found that wavelengths shorter than 320 nm markedly inhibited mitosis, increased the basal cell turnover time and depressed DNA, RNA and protein synthesis within the first few hours post-irradiation. By 24hr, recovery and acceleration of these functions were in progress, reaching a peak by 48–72 hr and persisting though to a lesser degree for 7 days. This stage of acceleration was associated with epidermal hyperplasia and most likely represented post-injury cell renewal.     

THE INACTIVATION OF RIBONUCLEIC ACID FROM TOBACCO MOSAIC VIRUS BY ULTRAVIOLET RADIATION AT DIFFERENT WAVE-LENGTHS

Abstract— Although both thymine and uracil can form similar dimers, exposing RNA of tobacco mosaic virus lo ultraviolet radiation of different wavelengths did not reproduce any of the phenomena that implicate dimerization of thymine residues as a major cause of the inactivation of a bacterial transforming DNA. If uracil residues dimerize at all in the irradiated RNA, such dimerization either does not affect infectivity or is not photoreversible in the same way as dirnerization of thymine residues in DNA. Unlike inactivation of the transforming DNA, inactivation of the virus-RNA seems to be a function of the amount of absorbed radiation energy, irrespective of the wave-length within the range 285 to 230 mμ and irrespective of a change in the wave-length during irradiation.     

INHIBITION OF 80 kDa PROTEIN PHOSPHORYLATION BY SHORT-WAVELENGTH UV LIGHT IN NIH 3T3 CELLS

The exposure of NIH 3T3 fibroblast cells to 254 nm UV radiation resulted in a temporary depression of DNA synthesis and inhibition of 80 kDa protein phosphorylation. This inhibition of protein phosphorylation was correlated with decreased protein kinase C activity in the membrane fractions of UV-damaged cells. The inositol tnphosphate contents measured, by the competitive binding assay using bovine adrenal binding protein, showed 80% reduction in the fibroblasts treated with 15 J/m² of UV light. The intracellular diacylglycerol concentration was also markedly reduced in UV-damaged cells. The results suggest that UV light causes acute reductions of inositol triphosphate and diacylglycerol contents in cells along with decreases in membrane protein kinase C activity, which leads to the inhibition of phosphorylation of an acidic protein of 80 kDa.     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

ULTRAVIOLET LIGHT INDUCES DOUBLE-STRAND BREAKS IN DNA OF CULTURED HUMAN P3 CELLS AS MEASURED BY NEUTRAL FILTER ELUTION

Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.     

STUDY OF PYRIMIDINE DIMERS IN MAMMALIAN CELLS SURVIVING LOW DOSES OF ULTRAVIOLET RADIATION*

Abstract— Ultraviolet-induced pyrimidine dimers were not found to be excided from the DNA of Chinese hamster cells in small oligounucleotides. At doses whereby many cells survive the radiation, the dimers were still associates with the large polynucleotides even after 48 hr of postirradiation incubation.     

EXPOSURE TO LONG WAVELENGTH ULTRAVIOLET RADIATION DECREASES PROCESSING OF LOW DENSITY LIPOPROTEIN BY CULTURED HUMAN FIBROBLASTS

Exposure of MRC5 human fibroblasts to UVA radiation (365 nm) resulted in a dose-dependent decrease in low density lipoprotein (LDL) uptake and degradation by cells. Following a 25 J/cm² irradiation dose, about 45% and 70% reduction in ¹²⁵I-LDL uptake and degradation were observed, respectively. Under the same conditions, the ¹⁴C-sucrose uptake was also decreased to about the same extent as LDL uptake. Cell pretreatment with the antioxidants vitamin E and vitamin C did not prevent the UVA-induced fall in LDL degradation. These results point to the possible effects of UVA radiation on receptor-mediated and nonspecific uptake of exogenous molecules. With special regard to the alterations in receptor-mediated processing of exogenous ligands, such a phenomenon could be of importance in UVA-induced skin degenerative processes.     

IN ACTIVATION OF A STRAIN OF TOBACCO NECROSIS VIRUS AND OF THE RNA ISOLATED FROM IT, BY ULTRAVIOLET RADIATION OF DIFFERENT WAVE-LENGTHS

Abstract— A train of tobacco necrosis virus (TNV) and infective nucleic acid isolated from it (TNV-RNA) are equally susceptible to inactivation by U.V. radiation at all wave-lengths tested (230-290 m_μ) and can be photoreactivated to the same extent by exposing inoculated host plants to daylight. The shape of the action spectrum for inactivation by U.V. of TNV and of TNV-RNA follows that of the absorption spectrum of TNV-RNA. Thus, unlike the RNA of tobacco mosaic virus, the RNA of TNV behaves in all these respects in the same way irrespective of whether it is inside or outside the virus particle. To inactivate TNV or TNV-RNA to 50 per cent of their original infectivities, each mg of RNA must absorb about 0.27 joules of radiation energy of any wave-length between 230 and 290 mp, which corresponds to a quantum yield of about 0.65 ×10^-3 at 260 mμ.     

INDUCTION OF DNA–PROTEIN CROSS-LINKING IN CHINESE HAMSTER CELLS BY MONOCHROMATIC 365 AND 405 NM ULTRAVIOLET LIGHT

Abstract— The survival, the induction of DNA-protein cross-linking, and the number of T4-endonuclease sensitive sites were measured in Chinese hamster cells that had been irradiated with 365 and 405 nm monochromatic light. The survival measurements show that cells are somewhat less sensitive to 405 nm light than to 365 nm light. The difference is expressed predominantly in the shoulder widths of the survival curves, whereas the slopes of the two curves are about the same. Induction of pyrimidine dimers, as indicated by the number of endonuclease-sensitive sites, after exposures that produce about 10% survival is very low at 365 nm (˜ 4 endonuclease sites per 2 × 10⁸ daltons), while no dimers are detected at 405 nm. In contrast, DNA-protein cross-links are induced rather effectively at either wavelength even after exposures that result in a relatively high survival (60-20%). Our measurements support the conclusion that lethality in mammalian cells after irradiations with 365 or 405 nm light is caused by a nondimer damage, possibly DNA-protein cross-links.     

CORRELATION BETWEEN ENDOGENOUS GLUTATHIONE CONTENT AND SENSITIVITY OF CULTURED HUMAN SKIN CELLS TO RADIATION AT DEFINED WAVELENGTHS IN THE SOLAR ULTRAVIOLET RANGE

Abstract— Glutathione depletion of cultured human skin fibroblasts by treatment with buthionine-S,R-sulfoximine (BSO) sensitises them to radiation at a series of defined wavelengths throughout the solar UV range. We now show that there is a close quantitative correlation between cellular glutathione content (as depleted by BSO) and sensitivity to radiation at 365 nm. A weaker correlation is observed when cells are depleted of glutathione using diethylmaleimide. Both fibroblasts and epidermal keratinocytes derived from the same foreskin biopsy are sensitised to radiation at 313 nm by glutathione depletion. However, the keratinocytes are sensitised to a much lesser extent, an observation which agrees quantitatively with the higher residual levels of cellular glutathione remaining after maximum depletion by BSO (approximately 25% for the keratinocytes vs less than 5% for the fibroblasts). At low to intermediate fluence levels, 10 mM cysteamine present during irradiation at 302 nm is able to almost completely reverse the sensitising effects of glutathione depletion suggesting that the endogenous thiol protects against radiation at this wavelength by a free radical scavenging mechanism. At 313 nm, the sensitisation is not reversed by cysteamine suggesting that glutathione plays a more specific role in protection against radiation at longer wavelengths. Xeroderma pigmentosum group A fibroblasts (excision deficient) are also sensitised to radiation at 313 and 365 nm by depletion of glutathione but since the sensitization is less than that observed for the normal strain, we cannot conclude that glutathione protects against a sector of DNA damage susceptible to excision repair. The results provide further evidence that endogenous glutathione is involved in protecting human skin cells against a wide range of solar radiation damage and suggest that while free radical scavenging is involved at the shortest wavelength (302 nm) tested, a more specific role of glutathione is involved in protection against radiation at longer wavelengths.