Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histitine and repair.

Abstract— The action spectrum for the oxygen-independent inactivation of native transforming DNA from Haemophilus influenzae with near-UV radiation revealed a shoulder beginning at 334 and extending to 460 nm. The presence of 0.2 M histidine during irradiation produced a small increase in inactivation at 254, 290 and 313 nm, a large increase at 334 nm and a decrease in inactivation at 365, 405 and 460 nm. Photoreactivation did not reverse the DNA damage produced at pH 7.0 at 334, 365, 405 and 460 nm, but did reactivate the DNA after irradiation at 254, 290 and 313 nm. The inactivation of DNA irradiated at 254, 290 and 313 nm was considerably greater when the transforming ability was assayed in an excision-defective mutant compared with the wild type, although DNA irradiated at 334, 365, 405 and 460 nm showed smaller differences. These results suggest that the oxygen-independent inactivation of H. influenzae DNA at pH 7 by irradiation at 334, 365, 405 and 460 nm is caused by lesions other than pyrimidine dimers.     

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

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 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.     

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.     

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.     

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 +0.5C 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.     

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.     

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.     

DNA excision-repair ability of embryonic fibroblasts measured by UDS and thymine dimer excorporation in four inbred mice strains

Differences in the excision-repair capability of embryonic fibroblasts of four inbred strains of mice following various degrees of UV irradiation were assessed. Two methods of determination were used: (1) the incorporation of 3H-thymidine during unscheduled DNA synthesis (UDS) as measured by an autoradiographic technique; (2) the rate of excision of thymine dimers (TT) in the acid-insoluble fraction of the cellular DNA as determined by a dimerspecific radioimmuno-assay. Based on UDS, the repair rates of the four strains could be ranked in decreasing order as follows: DBA/2 (DB); C57BL/6J (B6); AKR/N (AK); CBA/J (CB). The calculated rate for DBA/2 (DB) is approximately twice that of CBA/J (CB). The determination of the TT excision rate indicates that 72 h after irradiation a maximum of 50% of the original UV-induced dimers in the DB strain could be repaired. In the three remaining strains the relatively reduced repair rates of 15% - 40% did not differ significantly.     

Unrepaired cyclobutane pyrimidine dimers do not prevent proliferation of UV-B-irradiated cultured human fibroblasts

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.     

ALPHA POLYMERASE INVOLVEMENT IN EXCISION REPAIR OF DAMAGE INDUCED BY SOLAR RADIATION AT DEFINED WAVELENGTHS IN HUMAN FIBROBLASTS

Abstract Cultured fibroblasts derived from normal human skin have been irradiated at a series of monochromatic wavelengths throughout the ultraviolet region and exposed to the specific α polymerase inhibitor, aphidicolin (1 μg/m l , 2 days) prior to assay for colony forming ability. Repair of 75-80% of the lethal damage induced by UVC (254 nm) or UVB (302 nm, 313 nm) radiation is inhibited by aphidicolin suggesting that such damage is repaired by a common α polymerase dependent pathway. Exposure to aphidicolin after irradiation at longer UVA (334 nm, 365 nm) or a visible (405 nm) wavelength leads to slight protection from inactivation implying that the processing of damage induced in this wavelength region is quite distinct from that occurring at the shorter wavelengths and does not involve α polymerase.     

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.     

DAMAGE BY SOLAR RADIATION AT DEFINED WAVELENGTHS. INVOLVEMENT OF INDUCIBLE REPAIR SYSTEMS

Abstract The susceptibility of bacteriophage damaged by solar-ultraviolet (UV, 290-380 nm) radiations at denned wavelengths and by radiation at a visible wavelength (405 nm) to the Weigle reactivation system induced by far-UV (254 nm) irradiation of the host cell has been studied in a repair competent strain of Escherichia coli . The sector of inducible repair diminishes with wavelength, being very small after 313 nm irradiation and absent after irradiation at longer wavelengths. However, irradiation of bacteria at wavelengths as long as 313 nm induces a bacteriophage reactivation system as effectively as radiation at 254 nm in both the repair competent and an excision deficient host cell. At longer wavelengths pre-irradiation of the repair competent host cell enhances reactivation of 254 nm irradiated bacteriophage but the reactivation is smaller and the process quite distinct from that induced in the 254-313 nm region. We conclude that, with increasing wavelength, damage induced by solar UV radiations becomes increasingly less susceptible to repair systems induced by far-UV (pyrimidine dimers) and that this type of inducible repair system is no longer induced by wavelengths longer than 313 nm.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

INCREASED LEVELS OF UNSCHEDULED DNA SYNTHESIS IN UV-IRRADIATED HUMAN FIBROBLASTS PRETREATED WITH SODIUM BUTYRATE

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 m M results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m². The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine.     

Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA

Double-stranded, covalently closed, supercoiled circular DNA from phage fd (replicative form) was irradiated with increasing doses of UV light at 254 nm, 290 nm, 313 nm and 365 nm, and subjected to electrophoresis on agarose slab gels. Increasing the doses of UV light at 254 and 290 nm promotes a smooth reduction in the electrophoretic mobility of the sample, as would be expected if the major effect of light at these two wavelengths were to induce the formation of photoproducts leading to the unwinding of the double strand. At high doses, UV light at 290 nm introduces single-strand breaks (1.2 kJ m-2 per nick per million phosphodiester bonds). UV light at 313 nm promotes an abrupt change in the electrophoretic mobility, as would be expected if the effect of this wavelength were to induce single-strand breaks, leading to the transformation of the supercoiled molecules in their relaxed form (23 kJ m-2 in order to introduce one nick per million phosphodiester bonds). UV light at 365 nm also promotes single-strand breaks in DNA (140 kJ m-2 per nick per million phosphodiester bonds).     

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.     

Comparative photobiology of growth responses to two UV-B wavebands and UV-C in dim-red-light- and white-light-grown cucumber (Cucumis sativus) seedlings: physiological evidence for photoreactivation

We examined the influence of short-term exposures of different UV wavebands on the elongation and phototropic curvature of hypocotyls of cucumbers (Cucumis sativus L.) grown in white light (WL) and dim red light (DRL). We evaluated (1) whether different wavebands within the ultraviolet B (UV-B) region elicit different responses; (2) the hypocotyl elongation response elicited by ultraviolet C (UV-C); (3) whether irradiation with blue light-enriched white light (B/WL) given simultaneous with UV-B treatments reversed the effect of UV in a manner indicative of photoreactivation; and (4) whether responses in WL-grown plants were similar to those grown in DRL. Responses to brief (1-100 min) irradiations with three different UV wavebands all induced inhibition of elongation measured after 24 h. When WL-grown seedlings were irradiated with light containing proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm), inhibition of hypocotyl elongation was induced at a threshold of 0.5 kJ m(-2), whereas exposure to UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation at a threshold of 1.6 kJ m(-2). The UV-C treatment induced reduction in elongation at a threshold of <0.01 kJ m(-2) for DRL-grown plants and <0.03 kJ m(-2) for WL-grown plants. B/WL caused 50% reversal of the short-wavelength UV-B-induced inhibition of elongation in DRL-grown seedlings but did not reverse the effect of long-wavelength UV-B. B/WL caused 30% reversal of the UV-C-induced inhibition of elongation in WL-grown seedlings but did not affect the response to short-wavelength UV-B. Short-wavelength UV-B also induced positive phototropic curvature in both types of seedlings, and this was reversed 60% or completely in DRL-grown and WL-grown seedlings, respectively. The similarity of responses between the etiolated (DRL-grown) and de-etiolated (WL-grown) seedlings indicates that the short-wavelength specific response may be relevant to natural light environments, and the apparent photoreactivation implicates DNA damage as the sensory mechanism for the response.     

Application of the biotin-dUTP chromosome labelling technique to study the role of 5-bromo-2'-deoxyuridine in the formation of UV-induced sister chromatid exchanges in CHO cells

The role of 5-bromo-2'-deoxyuridine (BrdU) in the formation of sister chromatid exchanges (SCEs) in cells exposed to UV radiation was studied. Cells were unifilarily labelled (labelling of one strand of chromosomal DNA) with BrdU or biotin-16-2'-deoxyuridine (biotin-dU) and irradiated in G(1) phase of the cell cycle either with 254 nm, which is absorbed by all nucleobases including bromouracil (BrU) or with 313 nm radiation, which is predominantly absorbed by the BrU moiety. Elevated SCE frequencies were observed in cells irradiated at 254 nm (1.2 and 3.0 J m(-2)) which were pre-labelled with BrdU or biotin-dU. Following irradiation at 313 nm (38 and 96 J m(-2)) a statistically elevated SCE frequency was observed in cells pre-labelled with BrdU but not with biotin-dU. In cells pre-labelled with BrdU, UV-radiation at 254 nm was 50-80 times more effective in inducing SCEs than that at 313 nm. This result can be accounted for by the fact that in BrdU-DNA the cross-section for uracilyl radical and bromine atom formation is approximately 100-fold higher at 254 nm than that at 313 nm. Upon irradiation at 254 nm, BrdU had a strong sensitising effect on SCE induction: the SCE frequencies observed in cells pre-labelled with BrdU are approximately 6 times higher than in cells pre-labelled with biotin-dU. From this it follows that BrdU-induced damage is responsible for more than 80% of the SCEs formed in UV irradiated cells unifilarily labelled with BrdU. Based on photochemical considerations and the fact that chemical agents which form DNA interstrand cross-links are among the most potent inducers of SCEs, we propose that an interstrand cross-link may be the major lesion leading to SCEs in BrdU-labelled cells.     

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.