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.     

STUDY OF PYRIMIDINE DIMERS IN MAMMALIAN CELLS SURVIVING LOW DOSES OF ULTRAVIOLET RADIATION*

Abstract— Ultraviolet-induced pyrimidine dimers were not found to be excided from the DNA of Chinese hamster cells in small oligounucleotides. At doses whereby many cells survive the radiation, the dimers were still associates with the large polynucleotides even after 48 hr of postirradiation incubation.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

CORRELATION BETWEEN INACTIVATION OF HUMAN CELLS and NUMBERS OF PYRIMIDINE DIMERS INDUCED BY A SUN LAMP and 254 nm RADIATION

Abstract— Nondividing human fibroblasts are inactivated by radiation from a source (a Westinghouse sun lamp) that simulates the UV spectrum of sunlight. Survival curves determined for a DNA excision repair-proficient and a repair-deficient strain (XP12BE) are related to those determined using germicidal light (254 nm) by constant fluence modification factors. In addition, the same fraction of XP12BE cells are killed per pyrimidine dimer by 254 nm and sun lamp light. These results, when related to other survival and photoreactivation studies, suggest that the mechanism for inactivation of nondividing human cells by sun lamp light is the same as that by 254 nm and that pyrimidine dimers are the major responsible photolesion. Repair reverses some of the lethal effects of this light. We suggest that these conclusions apply to sunlight-irradiated skin cells in vivo.     

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.     

USE OF METABOLIC INHIBITORS TO INVESTIGATE THE EXCISION REPAIR OF PYRIMIDINE DIMERS AND NON-DIMER DNA DAMAGES INDUCED IN HUMAN AND ICR 2A FROG CELLS BY SOLAR ULTRAVIOLET RADIATION

Abstract— ICR 2A frog and normal human skin fibroblasts were exposed to either 5 J/m² of 254 nm UV or 50 kJ/m² of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1–β-D-arabinofuranosyl cytosine (ara C) for 0–20 min (human fibroblasts) or 0–4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-f-old higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV.     

EFFICIENCY OF REPAIR OF PYRIMIDINE DIMERS AND PSORALEN MONOADDUCTS IN NORMAL AND XERODERMA PIGMENTOSUM HUMAN CELLS

Abstract —Repair of DNA damage produced by ultraviolet light or 5-methylisopsoralen in normal and xeroderma pigmentosum human cells involves many similar steps. Aphidicolin and cytosine arabinoside block repair of both kinds of damage with similar efficiency, indicating that DNA polymerase a has a major role in repair for these lesions. In xeroderma pigmentosum cells of various complementation groups, the relative efficiency of excision repair for both ultraviolet- and 5-methylisopsoralen-induced damage was group A < C < D, indicating a close resemblance between both kinds of lesions in relation to the repair deficiencies in these groups. At high doses, the maximum rate of repair of damage by ultraviolet light was about twice that for methylisopsoralen damage, possibly because ultraviolet-induced damage forms a substrate that is more readily recognized and excised than that of the psoralen adducts. Differences in the structural distortions to DNA caused by these kinds of damage could be detected using single strand specific nucleases which excised dimers but not 5-MIP adducts from double strand DNA.     

ENHANCEMENT OF PHOTOREPAIR OF ULTRAVIOLET-INDUCED PYRIMIDINE DIMERS BY PREILLUMINATION WITH FLUORESCENT LIGHT IN THE GOLDFISH CELL LINE. THE RELATIONSHIP BETWEEN SURVIVAL AND YIELD OF PYRIMIDINE DIMERS

The enhancement of photorepair of UV-induced pyrimidine dimers by preillumination with fluorescent light, previously reported with RBCF-1 cells derived from caudal fin of a goldfish, was studied in terms of clonogenic ability and yields of dimers. In the logarithmic growth phase, the ability of photorepair increased with the time after preillumination, reached a maximum at 8 h, and gradually declined. At 8 h, the dose decrement with the photorepair-treatment for 20 min at 7.5 J/m2 UV increased by preillumination for 1 h from 1.6 to 3.1 J/m2 in terms of restoration of survival and from 1.2 to 4.3 J/m2 in terms of the disappearance of dimers. Incubation of the preilluminated cells in the medium containing cycloheximide (0.5 microgram/mL) after preillumination until UV-irradiation diminished their enhancement of photorepair. In the density-inhibited state, the ability of photorepair was higher than in the log phase, and it was hardly enhanced by preillumination.     

DNA DAMAGE AND REPAIR IN HUMAN CELLS EXPOSED TO SUNLIGHT

Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (> 320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful.     

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET-IRRADIATED CHINESE HAMSTER CELLS

Abstract —As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer-containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer-containing DNA is replicated in ultraviolet-irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.     

EARLY ALTERATIONS OF ACTIN IN CULTURED HUMAN KERATINOCYTES AND FIBROBLASTS EXPOSED TO LONG-WAVELENGTH RADIATIONS. POSSIBLE INVOLVEMENT IN THE UVA-INDUCED PERTURBATIONS OF ENDOCYTOTIC PROCESSES

Abstract Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320-400 nm) radiations markedly decreased the actin reactivity with fluorescein-labeled phalloidin. This indicates a change in the degree of poly merization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA-induced inhibition of specific and nonspecific endocytotic processes.     

QUANTIFICATION OF PYRIMIDINE DIMERS AND APURINIC SITES IN DNAs OF UNIFORM LENGTH

Abstract— A new simple in vitro assay for the determination of pyrimidine dimers and/or apurinic/apyrimidinic sites in non-radioactive DNA has been developed. In this procedure, DNA substrates of uniform length-which may be supercoiled, partially relaxed, relaxed or linear-are treated with agents which produce specific single strand nicks at the site of the lesion. The number of lesions per molecule can be expressed as a function of the amount of single-stranded molecules left intact after the specific nicking treatment. Unreacted molecules, which retain the original uniform length, are separated from the other smaller reaction products by electrophoresis on an alkaline agarose gel. In the case of circular molecules, the substrate is linearized by the use of an appropriate restriction endonuclease before loading on the gel. The amount of intact DNA molecules is obtained by integrating the corresponding peak of absorption after densitometric scan of the negative of the gel picture. This assay can detect up to eight damaged sites per duplex molecule. This method could be particularly useful when dealing with mixtures of DNA with different degrees of supercoiling or for comparisons between linear and circular DNA substrates.     

TRANSFORMATION OF BALB 3T3 CELLS EXPOSED TO A GERMICIDAL UV LAMP AND A SUNLAMP

Abstract— The effect of germicidal UV and sunlamp exposure on direct and simian virus-40 (SV-40) transformation of Balb 3T3 cells was studied. Transformation was determined by the ability of transformed cells to grow as clones in agar. Radiation from these lamps enhanced direct transformation, and enhanced viral transformation to approximately the same degree. Enhanced transformation was seen with exposures of light that caused no measurable cell killing, which suggests that the induction of new transformants is involved rather than the selection of pre-existing transformants. Induction is also suggested by post-irradiation growth kinetics experiments.     

INHIBITION OF PROTEIN SYNTHESIS IN CULTURED TOBACCO CELLS BY ULTRAVIOLET RADIATION

Abstract— Ultraviolet (254 nm) irradiation of liquid-cultured tobacco cells strongly and quickly inhibited their ability to incorporate labeled amino acids into protein. An incident dose of only 388 J/m² reduced incorporation to 37 per cent of the original rate. The effect on amino acid incorporation did not seem to depend on inhibition of amino acid uptake, inhibition of the supply of nucleoside triphosphates, or inhibition of the supply of messenger RNA to cytoplasmic ribosomes.     

PHOTOREACTIVATION OF PYRIMIDINE DIMERS IN DNA FROM THYROID CELLS OF THE TELEOST, POECILIA FORMOSA

Abstract— We have developed and used a simple technique to estimate the quantity of pyrimidine dimers in unlabeled cellular DNA. DNA is extracted from cells, treated with an endonuclease specific for dimers, and its molecular weight estimated by its electrophoretic mobility on alkaline agarose slab gels. The technique is used to show that cells from thyroid tissue of the fish Poecilia formosa have photoreactivating activity towards dimers in the cellular DNA.