Meyrick J. 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.

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra.

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.

PHOTOSENSITIZED PRODUCTION OF SUPEROXIDE ANION BY MONOCHROMATIC (290–405 nm) ULTRAVIOLET IRRADIATION OF NADH and NADPH COENZYMES

Abstract— The reduced pyridine coenzymes NADPH and NADH produced superoxide anion(“CK”) from ground state molecular oxygen when irradiated by ultraviolet (UV) radiation extending from 290 to 405 nm as detected by cytochrome c reduction. Superoxide dismutase (SOD), but not catalase or heat‐inactivated SOD, decreased the amount of cytochrome c reduced, indicating that O2− was responsible for the reduction of cytochrome c. Decreased oxygen tension during irradiation also inhibited production of O2−. Quantum yields for the production of the anion were in the region of 10−7 to 10−9 mol per photon. These data indicate that NADH and NADPH can act as type II photosensitizers of both far‐and near‐UV radiation, and that the deleterious biological effects of exposure to these radiations such as erythema and dermal carcinogenesis may be mediated at least in part through the generation of O2−.

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.

Superoxide anion is generated from cellular metabolites by solar radiation and its components

Several endogenous cellular constituents were tested for their ability to produce superoxide anion (O2-) from ground-state molecular oxygen upon irradiation by solar radiation. The pyridine cofactors NADPH and NADH, riboflavin, and the nucleosides 2-thiouracil and 4-thiouridine were found to sensitize the transmission of photon energy from solar radiation and monochromatic radiation (290, 334, 365, and 405 nm) to oxygen, resulting in O2- formation, as detected by superoxide dismutase-inhibitable cytochrome c reduction. Quantum yields for the production of O2- indicate that NADPH is the most efficient and riboflavin the least efficient of the compounds tested. These data indicate that endogenous compounds may participate in the production of O2- by solar radiation and imply that O2- may play a role in sunlight-induced erythema and dermal carcinogenesis.

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.

Effects of UV radiation on cells

UV radiation interacts with mammalian cells in a very complex manner, although DNA appears to be the main chromophore. Recent literature within this field is reviewed. The review is concentrated on the following main topics: Chromophores for UV action, photoproducts in DNA, repair of UV-induced DNA damage, wavelength interactions, inactivation, mutagenesis, transformation and protection mechanisms against UV damage.

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