FORMATION BY HYDROGEN PEROXIDE OR 254 nm RADIATION OF A NEAR-UV CHROMOPHORE FROM PEPTIDE-BOUND CYSTEINE

Abstract Treatment of glutathione or N-acetylcysteinamide in water with hydrogen peroxide, or with 254 nm radiation together with molecular oxygen, results in the formation of a near-UV chromophore having maximal absorption at 305 nm. From examination of related compounds, it is apparent that the N-acylcysteinamide residue is the key element required for generation of the 305 nm chromophore. The structure of this near-UV chromophore is stable to base but unstable in aqueous acid, is relatively sensitive to oxidation by hydrogen peroxide but is only very slowly reduced by sodium borohydride and displays good thermal stability at 50°C.     

DNA STRAND BREAKS IN MAMMALIAN CELLS EXPOSED TO LIGHT IN THE PRESENCE OF RIBOFLAVIN AND TRYPTOPHAN

Abstract— When mammalian cells were exposed to visible-fluorescent light or near-UV light in the medium containing riboflavin and L-tryptophan, single-strand breaks appeared in their DNA. This did not occur if either riboflavin or tryptophan was omitted from the medium. The same effect was observed when cells were added to the pre-irradiated medium, indicating that a stable photoproduct was responsible. The induced DNA lesions were shown to be equally repairable in both excision proficient and defective (xeroderma pigmentosum) human cell lines. The active photoproduct formed was shown to be hydrogen peroxide. The possible relationship between these results and the near-UV induced killing of mammalian cells is discussed.     

DEPENDENCE ON STAGE OF GROWTH OF MAMMALIAN CELL SENSITIVITY TO NEAR-UV IRRADIATION*

Abstract The variable sensitivity of exponentially growing mouse myeloma cells (66.2 subclone of MPC11) to monochromatic near-UV (365 nm) radiation was studied by determination of the relative cloning efficiency (i.e. survival) of cells in soft agar. Maximum sensitivity of the cells to near-UV light was found during the middle and late stages of exponential growth when the survival was only one-tenth of that during the early stage of growth. In addition, the shapes of near-UV survival curves changed with the stage of the cells irradiated. These data indicate that stages of growth can substantially alter the response of mammalian cells to near-UV radiation. These data further suggest the importance of testing the response of the cells throughout the growth employed in the experimental procedure.     

EFFECT OF FAR-UV AND NEAR-UV RADIATION ON THE CELL SURFACE CHARGE OF THE PROTOZOAN

Abstract— Cell electrophoresis was used to detect the effect of far-UV or near-UV radiation on the cell surface charge of the pathogenic protozoan Tritrichomonas foerus . Either far-UV or near-UV radiation interfered with the surface charge of T. foetus at fluences which inhibited cell growth by 50%. Both UV-radiations induced a significant decrease on surface charge of T. foetus , as evaluated by measurement of its electrophoretic mobility (EPM). Determinations of EPM of protozoa in solution of low ionic strength indicated that the decrease in the EPM induced by far-UV is much less pronounced than that observed for near-UV or control cells.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

NEAR-UV-INDUCED BREAKS IN PHAGE DNA: SENSITIZATION BY HYDROGEN PEROXIDE (A TRYPTOPHAN PHOTOPRODUCT)

Abstract— Near-UV irradiation of l -tryptophan yields a large number of photoproducts. When this mixture is added to recombinationless ( rec ) mutants of bacteria, the cells are killed. The most toxic component of tryptophan photoproducts has been identified as hydrogen peroxide (H₂O₂). We now report that both tryptophan photoproducts and H₂O₂ sensitize phage DNA to near-UV radiation resulting in enhanced killing as well as enhanced DNA breakages. We conclude that the in situ production of H₂O₂ via tryptophan photolysis may be an important biological event.     

Patterns of protein synthesis in a growth delay mutant (nuv) of Escherichia coli after treatment by near-UV radiation or hydrogen peroxide

When Escherichia coli cells are stressed by hydrogen peroxide (H2O2), synthesis of a large number of proteins is repressed, while several other proteins are induced. Since there is evidence that some lethal effects of near-UV (NUV) radiation may be directly or indirectly due to hydrogen peroxide generated by NUV light, treatment of cells with NUV radiation or H2O2 might be expected to repress and induce the same set of proteins. In this study, we compared the effects of H2O2 and NUV irradiation on patterns of protein induction and/or repression which were separate from the 4-thiouridine-dependent response using growth delay mutants (nuv). Concentrating initially on the proteins that ceased synthesis following NUV irradiation in an nuv mutant, we observed that these were not the same as those that ceased synthesis following H2O2 treatment. Inspection of two-dimensional polyacrylamide gel electrophoresis proteins indicated that NUV irradiation repressed synthesis of a different set of proteins, although there was some overlap between the two (45%). It was also observed that the new proteins which appeared after each of the two treatments were different. This suggests that the induction and/or repression of new proteins following NUV irradiation is not triggered solely via oxidative stress, although there is some overlap between the proteins that are induced or repressed following the two treatments.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

Effect of the Irradiation Conditions on the Yield of Hydrogen Peroxide in Frozen Aqueous Solutions of Adenine Derivatives

Russian Journal of Physical Chemistry A - Comparative results are presented from detecting hydrogen peroxide (H2O2) in aqueous solutions of adenine derivatives irradiated at 77 K in the near-UV...     

PHOTOMIMETIC EFFECT OF SEROTONIN ON YEAST CELLS IRRADIATED BY FAR-UV RADIATION

Abstract— The effect of serotonin on the survival of far-UV irradiated cells of the yeast Candida guillier-mondii was studied. Serotonin was found to have a photomimetic property. Preincubation of cells with serotonin results in protection against far-UV inactivation, whereas the post-radiation treatment with serotonin causes a potentiation of far-UV lethality. Both effects are similar to those produced by near-UV (334 nm) radiation. The observations provide support to the idea advanced by us previously that photosynthesized serotonin is the underlying cause of the two effects of near-UV radiation, photo-protection and potentiation of far-UV lethality. Experiments with an excision-deficient strain of the yeast Saccharomyces cerevisiae suggest that the effect of serotonin is by its binding to DNA.     

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.     

HYDROGEN PEROXIDE MEDIATES UV-INDUCED IMPAIRMENT OF ANTIGEN PRESENTATION IN A MURINE EPIDERMAL-DERIVED DENDRITIC CELL LINE

Abstract— Ultraviolet-B (290–320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4* T cell clones as responders, was inhibited significantly (>50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50–100 J/m²). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules ( e.g. Ia, CD54, CD1 la and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 miM H₂0₂ or f-butyl H₂0₂ mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

EARLY EFFECTS OF NEAR-ULTRAVIOLET RADIATION ON YEAST CELLS: CHANGES IN CELL SIZE

Exponentially growing yeast cells showed a drastic shrinkage when irradiated in water suspension (but not in buffer) with broad-band near-UV radiation. This was clearly seen soon after irradiation in size-distribution curves measured by a Coulter counter. This early shrinkage was observed at a near-UV fluence where the survival enters a region of exponential decline after a large shoulder. We further observed the formation of a distinct band in a density-gradient-centrifugation profile, presumably corresponding to the near-UV-affected cells. A survival test showed that the cells making up this band were dead.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

POSSIBLE INVOLVEMENT OF MEMBRANE DAMAGE IN THE IN ACTIVATION BY BROAD-BAND NEAR-UV RADIATION IN Saccharomyces cerevisiae CELLS

Multiple cellular effects of near-UV radiation (300-380 nm) on inactivation, disruption of the permeability barrier and induction of gene conversion at the trp 5 locus were simultaneously measured in the same culture of a diploid strain of the yeast Saccharomyces cerevisiae in order to assess the critical lethal damage. Inactivation of exponential phase cells in water appeared to be closely related to the disruption of the permeability barrier. Inactivation and membrane damage were remarkably oxygen dependent, whereas the induction of genetic changes was very low and dependent much less on oxygen. The dependence on the temperature for inactivation and membrane damage was both low, conforming with the expectation that the processes are mainly photochemical and not enzymatic. These features are very contrasted with the characteristics of far-UV radiation effects. Possible involvement of membrane damage in near-UV inactivation of exponential phase yeast cells is discussed.     

Visible light induced photocatalytic reaction of rhodamine B dye via 12-tungstosilicic acid in water

Using 12-tungstosilicic acid (SiW124-) as the catalyst, rhodamine B (RhB) dye in an aerated aqueous solution can undergo an effective photocatalytic stepwise N-deethylation process under visible light irradiation, and dioxygen is reduced to hydrogen peroxide by the reducedSiW12 4-. This provides the potential for moving polyoxometalate-based photocatalytic processes from the near-UV into the visible region of the spectrum.     

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.     

RESISTANCE OF PIGMENTED HUMAN CELLS TO KILLING BY SUNLIGHT AND OXYGEN RADICALS

Solar irradiation of a panel of human cell lines revealed three phenomena relevant to understanding the biological role of melanin; a heavily melanised melanoma line (MM418) was considerably more resistant to solar killing compared with HeLa and amelanotic melanoma cells of similar size and DNA content; MM418 cells were also resistant to killing by artificial UVB and by hydrogen peroxide generated in situ with extracellular glucose oxidase; and no difference in survival between the cell lines was found using 254 nm UV or gamma radiation. MM418 cells were resistant to sunlight when irradiated as attached monolayers but not when irradiated in suspension. Further studies showed that resistance to solar radiation in MM418 cells was not due to less DNA damage, as judged by inhibition of semiconservative DNA synthesis, or to enhanced constitutive or induced repair determined by reactivation of irradiated adenovirus. These results indicate that melanisation protects human cells from solar UVB in vitro and that the mechanism is associated with protection from hydrogen peroxide-type damage rather than direct shielding of DNA.     

REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.