PHOTOREACTIVATION OF ULTRAVIOLET IRRADIATED NON-DIVIDING POPULATIONS OF ICR 2A FROG CELLS

Abstract— Ultraviolet (UV) irradiation of non-dividing populations of ICR 2A frog cells led to their detachment from the surface of the culture dish and eventual lysis. Exposure of the cells to photoreactivating light after UV irradiation prevented cell killing and was accompanied by a loss of endonuclease sensitive sites from DNA. This photoreversal did not take place when the cells were exposed at 4°C to photoreactivating light indicating that the reversal was the result of photoenzymatic repair. As the action of photoreactivating enzyme is specific for the repair of pyrimidine dimers in DNA, these results suggest that pyrimidine dimers in DNA are the critical lesions leading to the death of non-dividing populations of UV irradiated cells.     

IRRADIATION OF CIRCULAR DNA WITH 254 nm RADIATION OR SENSITIZATION IN THE PRESENCE OF Ag+ EVIDENCE FOR UNWINDING BY PHOTOPRODUCTS OTHER THAN PYRIMIDINE DIMERS

Abstract— Structural alterations of DNA irradiated with UV light were analyzed by the agarose gel technique. Relaxed, circular pAT 153 DNA molecules were sensitized by broad band radiation with a maximum at 313 nm in the presence of silver ions or irradiated with 254 nm light in buffer only. In both cases the electrophoretic mobility of DNA topoisomers was altered as a linear function of UV exposure. For DNA irradiated in the sensitized reaction the unwinding angle per site sensitive to Micrococcus luteus pyrimidine dimer endonuclease was found tobe–11.4°. This value is significantly smaller thanthe–14.3° already known for DNA topoisomers irradiated with 254 nm light. The irradiated DNAs were a very good substrate for the Escherichia coli photoreactivating enzyme (PRE). However, the photoenzymic removal of all sites sensitive to the endonuclease specific for pyrimidine dimers was not coupled to a full restoration of the original electrophoretic mobility. Thirty and 23% of the unwinding were still present in the photoreactivated topoisomers and the unwinding angles per pyrimidine dimer were then recalculatedas–10.1°and–8.7° for DNAs irradiated with 254 nm and sensitized, respectively. The limited difference between these two values could result from the different base composition of the pyrimidine dimers generated in the conditions of irradiation used. These results show that the tertiary structure of DNA is measureably altered by UV photodamages other than pyrimidine dimers.     

PHOTOREACTIVATION OF ICR 2A FROG CELLS EXPOSED TO SOLAR UV WAVELENGTHS

Abstract Exposure of ICR 2A frog cells to photoreactivating light (PRL) following irradiation with a fluorescent sun lamp (FSL) resulted in an enhancement in survival compared with FSL-irradiated cells incubated in the dark. Hence, pyrimidine dimers played a role in the killing of cells exposed to the UV produced by this source. However, when the light was passed through a series of filters to remove increasing segments of the wavelength region shorter than 320 nm, the effect of the PRL progressively decreased, demonstrating that non-dimer photoproducts play an increasingly important role in the killing of cells exposed to wavelengths approaching 320 nm. Cells were also exposed to 313 nm UV produced by a monochromator and it was found, once again, that the effectiveness of the PRL treatment depended on the filter the beam was passed through. These results indicate that for both FSL-produced UV and 313 nm UV emitted by a monochromator, that the critical photoproducts induced within the cell depend on the filter used in conjunction with the UV source.     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

Incandescent lamps can produce pyrimidine dimers in DNA

DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.     

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.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

XERODERMA PIGMENTOSUM FIBROBLASTS INCLUDING CELLS FROM XP VARIANTS ARE ABNORMALLY SENSITIVE TO THE MUTAGENIC AND CYTOTOXIC ACTION OF BROAD SPECTRUM SIMULATED SUNLIGHT

Abstract— The cytotoxic and mutagenic effects of broad spectrum simulated sunlight, as delivered by a Westinghouse Sun Lamp FS 20 filtered to eliminate wavelengths below 290 nm, were determined in diploid human skin fibroblasts which differ in their ability to repair pyrimidine dimers, and compared with results obtained with UV 254 nm radiation. The cell strains tested included normal fibroblasts; excision repair-deficient xeroderma pigmentosum (XP) cells from patients XP12BE (complementation group A). XP7BE (group D). and XP2BI (group G): and an XP variant patient (XP4BE) whose cells excise pyrimidinc dimers at a normal rate, but exhibit abnormal replication of DNA containing unexcised lesions. Cytotoxicity was assayed from loss of colony-forming ability. The group A cells were most sensitive to the killing effect of the Sun Lamp; the group D and G cells were slightly less sensitive; the XP variant cells showed intermediate sensitivity; and normal cells were most resistant. When the Sun Lamp survival curves for the group A, group D, the XP variant and normal cells were compared with their respective UV 254 nm survival curves, the relationships between the strains were virtually identical (i. e. the curves were related by a constant fluence modification factor). suggesting a common lesion for cell killing. The marker for mutagenesis was resistance to 6-thioguanine. The group A XP cells proved most sensitive to mutations induced by the simulated sunlight: the variant cells were intermediate; and the normal cells were the most resistant. Again, when the curves for mutations induced in these cell strains by simulated sunlight were compared with their respective 254 nm UV mutation curves, these were related by a constant fluence modification factor. suggesting a common lesion for mutagenesis. These results. taken together with published data indicating that at equicytotoxic levels of UV254 nm radiation and the filtered Sun Lamp. the number of pyrimidine dimers in the DNA of XP12BE cells was equal. support the hypothesis that the dimer is the lesion principally involved in both effects. Our data also support the hypothesis that mutations are involved in the sunlight-induced skin cancer of XP patients.     

Photorepair of Ultraviolet Radiation (UVR)-lnduced Pyrimidine Dimers in Lens Epithelial DNA of Monodelphis domestica

Abstract— The repair of UV radiation-induced pyrimidine dimers has been measured in lens epithelial DNA of the marsupial Monodelphis domestica using a pyrimidine dimerspecific endonuclease from Micrococcus luteus. Approximately 40% of the initially induced dimers were repaired during 90 min exposures to photoreactivating light. This capacity of the lens epithelium to photorepair pyrimidine dimers may provide a means with which to determine whether pyrimidine dimers in lens epithelial DNA are involved in UV radiation-induced pathologic changes of the lens.     

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.     

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.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

DESTRUCTION OF PHOTOREACTIVATING ENZYME BY 365 nm RADIATION*

Abstract— Following the observation that in vivo photoreactivation of 365-nm-induced pyrimidine dimers could not be observed chemically, a study was made of the inactivation of photoreactivating enzyme activity by this near-ultraviolet wavelength. It was observed that: (1) Dimers induced in extracted bacterial DNA by 365 nm radiation are completely photoreactivable and are monomerized as an exponential function of the photoreactivation time. (2) Photoreactivability of 254-nm-induced damage in Escherichia coli B/r Hcr is progressively destroyed in vivo as a function of the dose of 365 nm radiation. (3) The ability of the yeast photoreactivating enzyme to monomerize dimers induced at 365 nm in bacterial DNA is destroyed in vitro as a function of the dose of 365 nm radiation, and at a rate comparable to killing of E. coli. These results are consistent with biological measurements which indicate that photoreactivability of ultraviolet (near and far) lethal damage is reduced by exposure of the bacteria to 365 nm radiation.     

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 CHROMOSOME ABERRATIONS IN ICR 2A FROG CELLS EXPOSED TO265–313 nm MONOCHROMATIC ULTRAVIOLET WAVELENGTHS and PHOTOREACTIVATING LIGHT

Abstract— Exposure of ICR 2A cells to either 265, 289, 302 or 313 nm monochromatic UV wavelengths caused the induction of chromosome aberrations with chromatid gaps and breaks being the most common type of aberration detected. Treatment of U V-irradiated cells with photoreactivating light (PRL) resulted in a lower yield of aberrations demonstrating that pyrimidine dimers are involved in the formation of chromosome aberrations induced by the UV wavelengths tested. However, the decrease in the level of aberrations resulting from PRL treatment of 313 nm-irradiated cells was significantly less than for the other wavelengths indicating that non-dimer photoproducts may have played an important additional role in the induction of chromosome aberrations by this UV wavelength.     

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations

—Action spectra for UV-induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254-365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254-313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.     

RESTRICTION ENZYME CLEAVAGE OF UV-IRRADIATED DNA: A COMPARISON OF FAR-UV AND MID-UV WAVELENGTHS

UV-irradiated DNA is less susceptible to restriction by Type II endonucleases than unirradiated DNA presumably due to photolesions formed in the recognition sites. Previous reported studies have used 254 nm radiation or 313 nm plus acetophenone, both treatments which introduce pyrimidine dimers in preference to other photolesions. To assess the effect of a longer wavelength, at which the ratio of pyrimidine dimer formation to the formation of other photolesions is reduced, two different DNAs were irradiated with UV of either 254 or 313 nm and restricted with suitable restriction endonucleases. Restriction patterns were analysed for novel fragments resulting from UV-induced alteration of enzyme recognition sites. EcoRI restriction of 254 nm irradiated lambda DNA produced six novel bands, only three of which were observed following restriction of 313 nm irradiated lambda. These three represented the largest fragments resulting from single site blocks. Novel fragments involving adjacent site blocks observed at 254 nm were not found with 313 nm radiation. Comparison of 254 nm irradiated pSV₂gpt to that irradiated at 313 nm, both restricted with Dral, revealed a more complex pattern. Although all sites were singly blocked by radiation of both wavelengths, multiple site blocks produced by 313 nm radiation did not occur in the order predicted by the 254 nm radiation dose response. These data suggest that certain sites in pSV₂gpt may be more refractory to multiple site blocks than others when irradiated at 313 nm.     

CONJUGATED DIENE FORMATION PROMOTED BY TRIPLET ACETONE ACTING UPON ARACHIDONIC ACID

Abstract— Post UV-B(280–320 nm) exposure to UV-A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossum Monodelphis domestica [Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica. Photochem. Photobiol . 40 ,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000-fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE-cellulose and DNA-cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 m M . The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.     

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light

Abstract—The influence of photoreactivating light on the fate of UV-induced DNA damage has been measured in the epidermis of hairless mice using damage-specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence-dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV-induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.