P-AMINOBENZOIC ACID CAN SENSITIZE THE FORMATION OF PYRIMIDINE DIMERS IN DNA: DIRECT CHEMICAL EVIDENCE

Thymine-containing photoproducts with chromatographic properties similar to those of cyclobutyl pyrimidine dimers can be formed in [³H]-thymine-labeled DNA in solution by 313 nm ultraviolet radiation in the presence of para-aminobenzoic acid (PABA), a compound used in sunscreen preparations. In the absence of PABA, similar fluences of 313 nm radiation do not produce significant numbers of these photoproducts. The thymine-containing photoproducts can be reversed by 254 nm radiation so that the tritium label migrates with the mobility of thymine monomer, a behavior characteristic of thymine-containing cyclobutyl pyrimidine dimers. This result supports previous, but less direct, data from other laboratories indicating that PABA can sensitize dimer formation in the DNA of bacterial and mammalian cells.     

PYRIMIDINE DIMERS INDUCED IN ESCHERICHIA COLI DNA BY ULTRAVIOLET RADIATION PRESENT IN SUNLIGHT

Abstract— Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine–thymine dimers (C < > T) were more frequent than thymine dimers (T < > T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295–315 nm), C < > T are probably more important than T < > T.     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are discussed.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

DNA DAMAGE and ITS REPAIR IN Dictyostelium discoideum IRRADIATED BY HEALTH LAMP LIGHT (UV- B)

Irradiation by health lamp (HL) light (280–320 nm) more efficiently induced cell killing and mutation in a radiation sensitive mutant (TW8) of Dictyostelium discoideum as compared with the parental wild-type strain (NC4). This light as well as a germicidal lamp-light (254 nm) produced pyrimidine dimers. The dimers were removed from DNA molecules by excision repair in NC4, but more slowly in TW8. It is suggested that pyrimidine dimers are the main DNA damage caused by HL light in D. discoideum , and that this results in cell killing and induced mutation.     

LONGWAVE ULTRAVIOLET RADIATION (UVA)-INDUCED ALTERATION OF EPIDERMAL DNA SYNTHESIS

Abstract— Longwave ultraviolet radiation(UVA–320–400nm) is known to induce inflammation, pigmentation and tumor production in mammalian skin. The mechanisms by which such radiation induces these biologic phenomena are poorly defined. In an effort to broaden our knowledge in this area, we examined the effect of UVA on DNA biosynthesis in Hartley strain albino guinea pig skin. The animals were irradiated with selected doses of solar simulated UVA, and DNA was assayed by [³H]thymidine incorporation into epidermal DNA by autoradiography. These studies revealed that UVA inhibited DNA synthesis in a dose dependent manner between 40 and 80 J cm^-2 at 3 h post-irradiation. This inhibition was followed by a stimulation of synthesis at 5 h and a second inhibition/ stimulation at 8 and 24 h, respectively. Although the mechanism of alteration is undefined, our data suggest that UVA has profound effects on DNA biosynthesis in mammalian epidermis.     

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.     

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.     

FORMATION OF 5-HYDROXYMETHYLCYTOSINE-CONTAINING PYRIMIDINE DIMERS IN UV-IRRADIATED BACTERIOPHAGE T4 DNA

Cyclobutyl pyrimidine dimers composed of 5-hydroxymethylcytosine and thymine (5HMC>T dimer for a mutant of T4 ( denV ) that is unable to excise pyrimidine dimers from its DNA. The ability of 5HMC to form dimers suggests that other modified pyrimidines such as 5-methylcytosine can participate in dimer formation, particularly at the UV wavelengths in sunlight likely to be responsible for the induction of skin cancer.     

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.     

PHOTOREVERSIBILITY OF UV-INDUCED THYMINE DIMERS AND ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS

Abstract— Irradiation of synchronously dividing 16-cell embryos of a sea-urchin ( Hemicentrotus pul-cherrimus ) with 200 J m⁻² of UV light (254 nm) resulted in the complete inhibition of normal pluteus-larva formation when the embryos were cultured in the dark after UV-irradiation. Illumination of the UV-irradiated embryos with visible light (11 W m⁻²) for 1 h immediately after the UV-irradiation reversed the abnormal morphogenesis. Measurement of thymine dimers indicates that the degree of UV-induced abnormal morphogenesis is greatly correlated with the amount of thymine dimers in the DNA of the embryos. The degree of the photoreversal decreased with an increase in the interval between UV-irradiation and exposure to visible light. Visible light was ineffective as to the reversibility of both thymine dimers and the abnormal morphogenesis at 60 min after the UV-irradiation, when the UV-irradiated 16-cell embryos entered the next cell cycle.     

SUNLIGHT-INDUCED PYRIMIDINE DIMERS IN HUMAN SKIN FIBROBLASTS IN COMPARISON WITH DIMERIZATION AFTER ARTIFICIAL UV-IRRADIATION

Abstract— We compared artificial UV-sources such as germicidal- or sun-lamps with summer noon sunlight in Switzerland for selective efficiency in the induction of pyrimidine dimers in the DNA of human cells. In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer densities (T-T) by high pressure liquid chromatography in cultures of xeroderma pigmentosum cells of group A. Using far-UV light from a germicidal lamp, we found a rate of formation per Jirr² for C-T and T-T of 0.0019% and 0.0024%, respectively, of the total thymine radioactivity in hydrolysates of [³H]thymidine labeled cells. After irradiation with an unfiltered sunlamp we measured a rate of formation of 0.0005% per Jm-² both for C-T and T-T, based on the sunlamp emission of 297 ±4 nm wavelength. Utilization of Kodacel- or Mylar-filters lowered the rate of dimerization by a factor of 2 and 60, respectively. One hour of irradiation with noon summer sunlight induced 0.038 ±0.012% C-T and 0.036 ±0.011% T-T. This extent of dimer production is equivalent to 15 Jm^-2 of far-UV exposure at 254 nm.     

THE AVAILABILITY OF SOLAR RADIATION BELOW 290 nm AND ITS IMPORTANCE IN PHOTOMODIFICATION OF POLYMERS

Abstract— On a percentage basis, ozone is a very minor component of the atmosphere; at STP it would make a layer only about 2 mm thick. On almost every other basis (biological, meteorological, paleontological, photochemical, etc.) it is a major component, due mainly to the tremendous reduction in solar ultraviolet flux which it causes in the 220–290 nm region. Since no data are available for Λ < 285 nm, a rational basis for estimating the flux reaching the earth's surface in this region is discussed. Variations in ozone concentration are of great importance, and it is possible to have more radiation with Λ < 270 nm fall on a surface in one extreme day than in several years of typical days. Often, persons involved in studies of polymer degradation by sunlight mention that a negligible fraction (1 ppm) of the radiant flux reaching the earth's surface is associated with wavelengths below 290 nm and infer that studies at shorter wavelengths will not be of much practical value. Such inferences are questionable for at least two reasons. (1) The quantum flux density below 290 nm is about 10¹⁶ photons cm^-2 month^-1, so that considerable long-term damage is possible since most of the flux will be absorbed in a layer only a few microns thick. (2) Even if solar radiation below 290 nm were completely absent, the existence of correlations between absorption peaks in the near ultraviolet and visible, and in the infrared with ionization potentials typically 6–12 eV or 200-100 nm) is evidence that we may expect studies in the ultraviolet and extreme ultraviolet (EUV) to provide important clues to the problem of improving the resistance of polymers to sunlight.     

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.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

IMPACT OF UV-B RADIATION UPON ESTUARINE MICROCOSMS

Abstract— Twelve flow-through estuarine microcosms were exposed daily to four different levels of UV-B radiation (290–320. nm)(1.57 ± 10², 6.43 ± 10³, 6.86 ± 10³ and 7.61 ± 10³ J·m_-2d⁻¹) in addition to a natural level of visible solar radiation (380-800. nm). The parameters studied over a four week period were phytoplankton community composition, plankton biomass (ash-free dry weight), chlorophyll a concentration and primary productivity (radiocarbon uptake). With increased exposure to UV-B radiation there was an obvious alteration of the community composition. Daily exposure to enhanced levels of UV-B radiation also depressed the biomass, the chlorophyll a concentration and the radiocarbon uptake of samples from the ecosystems.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

DO PYRIMIDINE DIMER YIELDS CORRELATE WITH ERYTHEMA INDUCTION IN HUMAN SKIN IRRADIATED in situ WITH ULTRAVIOLET LIGHT (275–365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad-band UV-B (290-320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV-B sensitivity (low minimal erythema dose) than in subjects of low UV-B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34-36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275-365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.     

SURVIVAL AND PYRIMIDINE DIMERS IN CULTURED FISH CELLS EXPOSED TO CONCURRENT SUN LAMP ULTRAVIOLET AND PHOTOREACTIVATING RADIATIONS

Abstract— Cultured fishcells(RBCF–1 line) were irradiated with filtered sun lamp ultraviolet (SL-UV; > 280 nm) together with or followed by illumination with daylight(DL) radiation (> 350 nm). The colony forming ability of the cells decreased with increasing fluence of SL-UV. Concurrent exposure of cells to SL-UV and DL, however, increased survival relative to exposure to SL-UV alone. The photoreactivable fraction reached 0.52 at22–25_‡C. By using a constant fluence modification factor of 86, the shape of dose-survival curve was found to be almost the same for 254 nm and SL-UV. In parallel with photoreactivation of cell survival, changes in the numbers of pyrimidine dimers in permeabilized cell DNA and in extracted total DNA were determined by measurements of endonuclease-sensitive sites (ESS). The yield of ESS in both DNA's increased almost linearly with increasing SL-UV fluence, although the yield in extracted DNA was about double of that in permeabilized cell DNA. The yield of ESS per unit fluence by 254 nm was about 70-fold greater than SL-UV. The fraction of cells inactivated per ESS was almost the same for 254 UV and SL-UV. In SL-UV-irradiated cells, the photoreactivable fractions in terms of ESS were _‡ 10% higher in extracted DNA than in the DNA of permeabilized cells and also were higher when DL was administered separately after SL-UV-irradiation. When irradiated cells were exposed to DL at 0_‡C, the photoreactivable fractions of both DNAs were appreciably less, indicating that the photoreactivation of ESS was enzymatic. These results support the suggestion that the mechanism for cell killing, mainly formation of pyrimidine dimers, by SL-UV is the same as that by 254 UV.