OXYGEN-DEPENDENCE OF NEAR UV (365 NM) LETHALITY AND THE INTERACTION OF NEAR UV AND X-RAYS IN TWO MAMMALIAN CELL LINES

Abstract— The colony-forming ability of Chinese hamster cells (V-79) and HeLa cells has been measured after near-ultraviolet (UV) irradiation, predominantly at 365 nm. To avoid the production of toxic photoproducts, cells were irradiated in an inorganic buffer rather than in tissue culture medium. Under these circumstances near-UV lethality was strongly oxygen-dependent. Both cell lines were approximately 10⁴ times more sensitive to 254 nm irradiation than to 365 nm radiation when irradiated aerobically. Pretreatment with 6 times 10⁵ Jm^-2 365 nm radiation sensitised the HeLa, but not the V-79 cell line to subsequent X-irradiation. Pretreatment of cells with 17 Jm^-2 254 nm radiation, a dose calculated to produce twenty times more pyrimidine dimers than the 365 nm dose, produced only slight sensitisa-tion to X-rays. It is suggested that the sensitisation to X-rays seen in the HeLa cells after 365 nm treatment is not the result of lesions induced in DNA by the near-UV radiation, but may reflect the disruption of DNA-repair systems.     

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.     

LONG WAVELENGTH UV RADIATION AFFECTS CHEMILUMINESCENCE OF HUMAN POLYMORPHONUCLEAR LEUCOCYTES

Abstract— Luminol-enhanced chemiluminescence in suspensions of human polymorphonuclear leucocytes stimulated with the chemotactic oligopeptide formyl-methionyl-leucyl-phenylalanine was increased by exposure of the cells to long wavelength UV radiation (mainly320–400 nm). Leucocytes treated with 0.3 × 10⁴ J m^-2 of UV responded with doubled peak values, and 4 × 10⁴ J m^-2 lead to a five-fold increase in peak chemiluminescence, as compared with non-exposed cells. Supernatants isolated from irradiated leucocytes contained increased amounts of both myeloperoxidase and lactate dehydrogenase and showed higher chemiluminescence values, when compared with supernatants from sham-irradiated cells. The results suggest that leucocyte degranulation contributes to the inflammatory properties of long wavelength UV.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

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.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

SENSITIVITIES TO MONOCHROMATIC 254-nm AND 365-nm RADIATION OF CLOSELY RELATED STRAINS OF Saccharomyces cerevisiae WITH DIFFERING REPAIR CAPABILITIES

Abstract— Sensitivity to monochromatic 254- and 365-nm radiation was compared in closely related yeast strains with defects in one or more of the excision-repair ( rad1 ), error-prone repair ( rad18 ), or recombinational-repair ( rad51 ) pathways. At 254 nm, mutants defective in a single repair pathway exhibited slight to moderate UV sensitivity; those defective in two separate pathways were somewhat more UV sensitive, while triple mutants defective in all three pathways exhibited extreme UV sensitivity with a lethal event corresponding to 0.05 J m⁻². Repair defects also rendered mutants sensitive to 365-nm radiation; strains with single defects exhibited slight sensitivity, mutants with two defective pathways were more sensitive, and triple mutants exhibited maximal sensitivity with a lethal event corresponding to 2.4 times 10⁴ J m⁻². In the triple mutant ( rad1, rad18, rad51 ) at both 254 and 365 nm, the dose per lethal event was almost identical with comparable values in a repair-deficient double mutant ( uvrA, recA ) of Escherichia coli. In the E. coli mutant pyrimidine dimers are believed to be the primary cause of lethality at both wavelengths. Evidence for dimer involvement in the yeast mutant was obtained by demonstrating that lethality at both 254 and 365 nm was photoreactivated by light at 405 nm.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

SYNERGISTIC LETHAL ACTION OF ULTRAVIOLET-VIOLET RADIATIONS AND MILD HEAT IN ESCHERICHIA COLI

Abstract— The lethal interaction of far ultraviolet (254nm), near ultraviolet (334 and 365nm) and violet visible (405nm) radiation treatment with mild heat treatment was studied. Except at 254nm, a strong positive radiation dose-dependent interaction (synergism) was always observed. The efficiency of sensitisation to heat, as a function of dose at each wavelength, was found to be directly correlated with the dose necessary to eliminate the shoulder from the survival curve of a repair proficient strain but was apparently unrelated to the relative near-ultraviolet sensitivities of a repair deficient strain. The interaction was independent of the order of treatments. A radiation dose of 10⁶ Jm^-2 at 365nm slightly sensitised a cell population to 45°C incubation (normally non-lethal) and strongly sensitised the cells to 48°C treatment (normally 80 percent survival after 2 hours). It is proposed that in addition to DNA damage, both heat treatment and near ultraviolet treatment interfere with DNA recovery mechanisms so that the combination of the two agents inevitably leads to a strong positive interaction.     

PHOTO-OXIDATION EFFECTS ON β-HYDROXYBUTYRATE DEHYDROGENASE: STUDIES OF MEMBRANE FRAGMENTS AND INTACT MITOCHONDRIA

The influence of singlet oxygen (¹O₂), generated by red light irradiation of oxygenated suspensions containing aluminium phthalocyanine sulphonate, on the membrane bound enzyme β-hydroxybutyrate dehydrogenase was investigated. The inactivation rates were measured using a spectrophotometry assay which involves disruption of the mitochondria. A novel NMR assay was also used to measure the activity of the enzyme in intact mitochondria. Relatively high inactivation rates of around 10⁹ M ⁻¹ s⁻¹ were observed in H₂O buffer, and rates in D₂O were a factor of 1.7 faster. Significant differences in enzyme inactivation rates by ¹O₂ were observed, not only between disrupted and intact mitochondria but also between the NMR assay results and the spectrophotometric assay results. The results indicate the value of a specific assay which does not require the disruption of the biological system.     

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.     

PHOTOLYSIS OF PHOSPHODIESTER BONDS IN PLASMID DNA BY HIGH INTENSITY UV LASER IRRADIATION

Abstract— The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 times 10^-5 until a maximum value of 4.5 times 10^-4 is attained at intensities of 10¹¹ W m^-2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m^-2). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed.     

PROTECTION OF ESCHERICHIA COLI CELLS AGAINST THE LETHAL EFFECTS OF ULTRAVIOLET AND X IRRADIATION BY PRIOR X IRRADIATION: A GENETIC AND PHYSIOLOGICAL STUDY

Abstract— When log phase cells of wild-type E. coli K-12 were maintained in growth medium after X irradiation, they became progressively more resistant to a subsequent exposure to UV or X radiation. The time to achieve maximum resistance was about 60 min. The uvrB, uvrD, polA and certain exrA strains (W3110 background) also demonstrated this X ray-induced resistance to subsequent UV or X irradiation but recA, recB, lex (AB1157 or W3110 backgrounds) and other exrA strains (AB1157 background) did not. The resistance induced in wild-type, uvrB and uvrD cells was characterized by the production or enhancement of a shoulder on the survival curves obtained for the second irradiation, while the resistance induced in the W3110 exrA strains was expressed only as a change in slope. The induction of resistance in the W3110 exrA strain was not inhibited by the presence of chloramphenicol, but that in the wild-type cells appeared to be. The production or enhancement of a shoulder on the survival curves of the rec ⁺ lex ⁺ exr ⁺ cells is consistent with the concept of the radiation induction of repair enzymes. Alternative explanations, however, are discussed.     

THE INTERACTION OF A GENE (nur) CONTROLLING NEAR-UV SENSITIVITY AND THE polA1 GENE IN STRAINS OF E. COLI K12

Abstract— When stationary cell populations of the Escherichia coli W3110 strain and the polA1 mutant (p3478) derived directly from it were compared for their sensitivity to near-UV (NUV, 300–400 nm) inactivation, the polA1 strain proved to be more sensitive. By appropriate matings and transductions, four essentially isogenic strains have been developed which carry all four possible combinations of genes conferring far-UV (FUV, 200-300 nm) sensitivity ( polA1 vs. polA ⁺) and NUV sensitivity ( nur vs. nur ⁺). Stationary cells of strains carrying either the polA1 or polA ⁺ allele in combination with the nur allele are indistinguishable in their sensitivity to NUV inactivation and are equivalent in their NUV sensitivity to the original polA1 mutant strain (p3478). With the two strains carrying the nur ⁺ allele, stationary cell populations of the polA1 strain are clearly more sensitive to NUV inactivation than is the polA ⁺ strain. The NUV sensitizing effect of the polA1 mutation in a nur ⁺ genetic background is about the same as that of the nur mutation at the 0.37 survival level. This may mean that the polA1 and nur mutations sensitize E. coli stationary cell populations to NUV inactivation by a common mechanism.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.     

SENSITIZATION OF ULTRAVIOLET-IRRADIATED Escherichia coli K-12 BY DIFFERENT AGARS: INHIBITION OF A rec AND exr GENE-DEPENDENT BRANCH OF THE uvr GENE-DEPENDENT EXCISION-REPAIR PROCESS

Abstract— A plaque assay for adenovirus 2 on normal human fibroblasts has been developed and used to measure the survival of ultraviolet-irradiated virus on six human fibroblast cell lines. When four xeroderma pigmentosum cell lines were used as viral hosts, an average of one lethal event per virus in the viral population was made with 10, 15, 62, and 78 J m^-2 respectively, while using two normal cell lines as hosts, 197 and 205 J m^-2 were required to inflict the same damage. These differences are attributed to the known repair deficiency of xeroderma pigmentosum cells, and are discussed in the light of previous data obtained using other animal viruses.     

THE ACTION SPECTRUM AND DOSE RESPONSE STUDIES OF UNSCHEDULED DNA SYNTHESIS IN NORMAL HUMAN FIBROBLASTS

Abstract— Excision repair of DNA damage by UV has been assessed in normal human fibroblasts in culture by measuring unscheduled DNA synthesis. Dose response experiments indicated that the same chromophore was involved in UV-induced damage and excision repair at three different wavelengths between 260 and 300 nm. Action spectra for unscheduled DNA synthesis were determined at wavelengths between 260 and 320 nm 30 min after irradiation using 2 doses of UV, 100 J m^-2and 10Jm^-2. Experiments at the lower dose were carried out because it appeared that repair was saturated with the higher dose at 260 and 280 nm. To explore this part of the spectrum further, experiments were performed with different doses at 260 and 280 nm and unscheduled DNA synthesis assessed 30 min and 24 h after irradiation. At 24 hr after irradiation a significantly greater amount of unscheduled DNA synthesis occurred at 280 nm. It is suggested, therefore, that both DNA and protein are concerned in the absorption of UV which leads to DNA damage and excision repair.     

AN INSTRUMENT FOR ACTION SPECTRUM STUDIES IN DERMATOLOGY

Abstract— An instrument designed for convenient determination of action spectra for cutaneous photo-responses in man and experimental animals is described. Light from 450 W Xe lamp is dispersed by a concave holographic grating. The spectrum from 244 to 616 nm is projected as a planar strip (2 times 17 cm) intercepted by a grid with 31 ports. The bandwidth at each port is 12 nm and the size of the port increases from about 4 × 4 mm to 6 × 8 mm from the low to high wavelength limits, respectively. Typical fluence rates in quanta m^-2 s^-1 are 4.0 times 10¹⁹ at 298 nm, 16 times 10¹⁹ at 394nm and 22 times 10¹⁹ at 538 nm. Responses due to delayed erythema in normal skin and to musk ambrette photoallergy and solar urticaria in patients skin have been elicited with this instrument.