Jennifer G. Peak

INDUCTION OF DIRECT AND INDIRECT SINGLE‐STRAND BREAKS IN HUMAN CELL DNA BY FAR‐ AND NEAR‐ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single‐strand breaks (SSBs, frank breaks plus alkali‐labile sites) in human P3 teratoma cells in culture by monochromatic 254‐, 270‐, 290‐, 313‐, 334‐, 365‐, and 405‐nm radiation is described. The cells were held at +0.5d̀C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D2O environment (compared with the normal H2O environment) did not alter the rate of induction of SSBs by 290‐nm radiation, whereas the D2O environment enhanced the induction of SSBs by 365‐ and 405‐nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs. wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far‐UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near‐UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365‐nm radiation.

ULTRAVIOLET ACTION SPECTRA FOR DNA DIMER INDUCTION, LETHALITY, AND MUTAGENESIS IN Escherichia coli WITH EMPHASIS ON THE UVB REGION

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high‐pressure Hg‐Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.

Comparison of initial yields of DNA-to-protein crosslinks and single-strand breaks induced in cultured human cells by far- and near-ultraviolet light, blue light and X-rays

The initial yields of DNA-to-protein crosslinks (dpc) caused by ionizing and nonionizing radiations were compared, with emphasis upon values within the biological dose ranges (D0). Induction of dpc in cold (0-0.5 degrees C) human P3 teratocarcinoma cells was measured by using alkaline elution techniques after exposure to monochromatic UVC (254 nm), UVB (313 nm), UVA (365 and 405 nm), and blue light (434 nm). UVC and UVB light induced detectable numbers (about 100 dpc per cell per D0). Monochromatic UVA radiations produced yields about 8 times higher than UVC or UVB (for 365 nm, about 1500 dpc per cell per D0) Similar results at low doses were obtained for measurements of single-strand breaks induced by the different radiations. The action spectra for dpc were closely similar. The biological significance of these relatively high numbers of DNA lesions caused by environmental nonionizing radiation that readily penetrates into human skin is not understood.

SINGLE‐STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract— The induction of single‐strand breaks (alkali‐labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far‐UV (below 320 nm) component and a minor near‐UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near‐UV (above 320 nm) wavelengths faster than the wild‐type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali‐labile bonds induced by 365‐nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali‐labile bonds, probably apurinic and apyrimidinic sites.

Induction of DNA-protein crosslinks in human cells by ultraviolet and visible radiations: action spectrum

Abstract— DNA‐protein crosslinking was induced in cultured human P3 teratocarcinoma cells by irradiation with monochromatic radiation with wavelengths in the range254–434 nm (far‐UV, near‐UV, and blue light). Wavelength 545 nm green light did not induce these crosslinks, using the method of alkaline elution of the DNA from membrane filters. The action spectrum for the formation of DNA‐protein crosslinks revealed two maxima, one in the far‐UV spectrum that closely coincided with the relative spectrum of DNA at 254 and 290 nm, and one in the visible light spectrum at 405 nm, which has no counterpart in the DNA spectrum. The primary events for the formation of DNA‐protein crosslinks by such long‐wavelength radiation probably involve photosensitizers. This dual mechanism for DNA‐protein crosslink formation is in strong contrast to the single mechanism for pyrimidine dimer formation in DNA, which apparently has no component in the visible light spectrum.

The effects of the ultraviolet wavelengths of radiation present in sunlight on human cells in vitro.

This yearly review is intended to supplement and complement similar reviews appearing in the past two years (Kantor, 1985; Gange and Rosen, 1986; Peak and Peak, 1986a). Kantor’s review, “Effects of Sunlight on Mammalian Cells,” was limited to manuscripts that appeared in 1984. Gange and Rosen’s “UVA Effects on Mammalian Skin and Cells” was limited to work published in 1985; Peak and Peak’s “DNA-to-Protein Crosslinking and Backbone Breaks Caused by Farand Near-Ultraviolet and Visible Radiation in Mammalian Cells” considered DNA damages only. Although this review limits itself to papers known to us that either appeared or were in press from January 1986 through July 1987, necessary limited references to earlier work are included. Other reviews relating to this topic that have appeared since 1984 include “UV-Carcinogenesis” (van der Leun, 1984), “Effect of UV Light on Humans” (Grieter and Gschnait, 1984), and “Photoimmunology” (Morison, 1984). Our review includes only studies meeting specific qualifications: (1) The experimental radiation source contained at least some portion of the ultraviolet radiation (UV) present in the solar spectrum reaching the surface of the earth, i.e. 29WOO nm. (2) Only studies that involved human cells were considered. (3) Only in vitro work (cells in culture) was included, with the exception of a small section on studies of human epidermis. (4) Effects mediated by endogenous photosensitizers were considered. A few selected papers that deviated from the above conditions were reviewed, however, because they contributed directly to the objectives of the review. Much of the motivation for focusing on studies of the effects of the wavelength region referred to in this review as the “solar UV wavelength region,”* is provided by three observations. First, although the many studies concerning the wavelength region

Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation

The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either γ radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher). Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against y‐radiation‐induced ssb (single‐strand breaks—relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH. Here we show that these quenchers also protect against 365‐nm‐induced ssb. The ratios for protection against 365‐nm induced DNA ssb in isolated B. subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH. In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.

SINGLET OXYGEN INDUCES FRANK STRAND BREAKS AS WELL AS ALKALI- AND PIPERIDINE-LABILE SITES IN SUPERCOILED PLASMID DNA

Abstract

DNA BREAKAGE CAUSED BY 334‐nm ULTRAVIOLET LIGHT IS ENHANCED BY NATURALLY OCCURRING NUCLEIC ACID COMPONENTS AND NUCLEOTIDE COENZYMES

Abstract— The induction of breaks in DNA in vitro caused by 334‐nm UV radíation is enhanced by the following compounds (fluence enhancement factors and concentrations used in parentheses): 4‐thiouridine (6.9, 1 mM), 5‐methylamino‐2‐thiouridine (7.5, 1 mM), 2‐thiouracil (41.0, 1 mM), riboflavin (14.4.0.1 mM), and the oxidized (6.8, 1 mM) and reduced (3.4, 1 mM) forms of nicotinamide adenine dinucleotide. Anoxia and diazobicyclo(2.2.2)octane reduce the number of DNA breaks caused by 334‐nm radiation plus 4‐thiouridine by 70 and 76%, respectively.

Different (direct and indirect) mechanisms for the induction of DNA-protein crosslinks in human cells by far- and near-ultraviolet radiations (290 and 405 nm)

Abstract— Apparent DNA‐protein crosslinking induced by monochromatic 290 and 405 nm Tadiations was measured in cultured human P3 teratocarcinoma cells with DNA alkaline elution techniques. The rates of the induction of crosslinks by 290 nm radiation were the same when the cells were irradiated either aerobically or anaerobically or when the cells were in an H2O or D2O aqueous environment. With 405 nm radiation, anaerobic irradiation reduced the induction of the crosslinks (dose modifying factor is about 0.2), and about twice as many crosslinks were observed when the cells were irradiated in an environment of D2O rather than H2O. The results are consistent with the hypothesis that far‐UV radiation induces DNA‐protein crosslinks by a direct mechanism, whereas near‐UV radiation induces crosslinks via indirect photodynamic photosensitizations in which unidentified cellular endogenous photosensitizers and reactive species of oxygen are used.