1 distribution and repair of bipyrimidine photoproducts in solar uv-irradiated mammalian cells ...

JBC Papers in Press. Published on May 25, 2000 as Manuscript M001450200

DISTRIBUTION AND REPAIR OF BIPYRIMIDINE PHOTOPRODUCTS IN SOLAR UV-IRRADIATED MAMMALIAN CELLS. POSSIBLE ROLE OF DEWAR PHOTOPRODUCTS IN SOLAR MUTAGENESIS †

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Daniel PERDIZ , Pál GRÓF , Mauro MEZZINA , Osamu NIKAIDO , ‡

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Ethel MOUSTACCHI , Evelyne SAGE *

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CNRS UMR 218, LRC-1 du CEA, Institut Curie - Recherche, F-75248 Paris Cedex 05,

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Institute of Biophysics and Radiation Biology, Semmelweis University of Medicine, H-1444 Budapest, Hungary ¶

CNRS URA 1923, Généthon III – CNRS, F-91002 Evry, France

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Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan # present address : CNRS UMR 2027, Institut Curie, Centre Universitaire, Bat 110, F-91405 Orsay, France Corresponding author: Evelyne Sage, Fax: (33) 1 69 86 94 29 ; Email: [email protected]

† This work was supported by grants from Ligue Nationale Française contre le Cancer, Institut Curie (Genotoxicology program). DP is recipient of a fellowship from MENRT and from Académie Nationale de Médecine; PG and ES are on a joint CNRS/Hungarian Academy of Sciences program.

1 Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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France

Key words: ultraviolet radiation, sunlight, UVA, bipyrimidine photoproducts, cyclobutane pyrimidine

dimers,

(6-4)

photoproducts,

Dewar

photoproducts,

DNA

repair,

photomutagenesis.

Abbreviations: CPD, cyclobutane pyrimidine dimers; (6-4)PP, pyrimidine (6-4) pyrimidone photoproducts;

DewarPP,

Dewar

photoproducts;

8-oxodGuo,

8-oxo-7,8-dihydro-2'-

deoxyguanosine; SSL, simulated solar light; NER, nucleotide excision repair; TCR, transcription-coupled repair; GGR, global genomic repair; IDB, ImmunoDotBlot.

Dr. P. Hughes and Y. Cohen for critical reading of the manuscript.

running title: solar UV induction and repair of bipyrimidine photoproducts

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Acknowledgments: We are grateful to Yvette Rolland for excellent technical assistance and to

ABSTRACT In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP) and their Dewar valence photoisomers (DewarPP), was examined in Chinese hamster ovary cells irradiated with UVC, UVB, UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immunodotblot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses,

of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0.12:0.014 for UVB and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently and at the same rate as CPD. In contrast, removal of (6-4) photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that beside CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.

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DewarPP were more efficiently produced by SSL than by UVB (ratios of DewarPP to (6-4)PP

INTRODUCTION Overwhelming evidence associates the steadily increasing incidence of skin cancer with an increased exposure to the UV components of sunlight (1). The UV induction of DNA damage is unambiguously an essential step in photocarcinogenesis (2). In the mutated p53 tumor suppressor gene of skin cancer, the majority of mutations harbor the “UV mutational signature”, i.e. C → T transitions and CC → TT tandem double mutations, which occur at sites where major DNA photoproducts are formed (3, 4). The photolesions that are readily produced at these sites by UVC (254 nm) and UVB (280-320 nm), such as cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP), are

photoproducts

(DewarPP),

which

are

valence

isomers

of

(6-4)PP

formed

via

photoisomerisation at wavelengths around 320 nm (6, 7), have received much less attention to date. Until recently, the investigation of DNA photolesions has been predominantly conducted using UVC 254 nm or UVB. However, wavelengths lower than 290 nm do not reach the Earth’s surface due to absorption by the stratosphere. Consequently, the human population is mostly exposed to longer wavelength UVB (λ > 295 nm; 8) and UVA (320-400 nm) radiation, which constitutes about 5% and 95%, respectively, of the solar spectrum. The relatively weak UVB component is believed to be responsible for most of the biological effects of sunlight, which are mediated by direct absorption of UVB by DNA. Since UVA is poorly absorbed by DNA, its genotoxic effect has been attributed to indirect photosensitizing reactions (9, 10). At the DNA level, photosensitization induces oxidative damage either by charge transfer from excited endogenous chromophores, or by reactions with reactive oxygen species (ROS) that are generated at these wavelengths. Indeed, photoinduced oxidative DNA modifications, such as single strand breaks, DNA-protein crosslinks, alkali-labile sites, 8-oxo-

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thought to represent the predominant forms of premutagenic damage (5). The Dewar

7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) have been observed (11 for review, 12-15). However, the direct effect of UVA has only recently been described (14-16). In mammalian cells, UV-induced bipyrimidine photoproducts are removed via nucleotide excision repair (NER), either by transcription-coupled repair (TCR) or global genomic repair (GGR), while oxidative photolesions and single strand breaks are most likely eliminated by base excision repair (17). The repair kinetics and efficiencies of UV-induced damage vary considerably from one type of photolesion to another, and between species, such as rodent and human. For example, (6-4)PP are rapidly and efficiently removed in both organisms, probably via GGR, whereas CPD are repaired rather slowly and incompletely

The mutational specificities of UVC, UVB, and UVA radiation, and simulated solar light (SSL) were previously determined at the aprt locus in Chinese Hamster Ovary (CHO) cells that were proficient or deficient in DNA repair (18-20). Briefly, it was found that 1) in the NER-proficient cell line, GC → AT transitions and CC →TT tandem double mutations were the major types of events following SSL exposure, whereas the former type of mutation largely predominated following UVB irradiation; 2) while GC → AT transitions still contributed up to 27% of the changes after UVA exposure, they were no longer preponderant; 3) in contrast, in the NER-deficient cell line, GC → AT transitions, all of which occurred at bipyrimidine sites, represented a large proportion of the mutational events induced by UVA. Altogether, these results suggested the formation of bipyrimidine photoproducts by UVA radiation and a role for such damage in UVA-induced mutagenesis. Indeed, we later observed a significant production of CPD in plasmid DNA irradiated with UVA (16). In order to better understand the relative contribution of the different UV components to solar mutagenesis, we previously examined their respective DNA damage profiles. In particular, we investigated the relative spectral effectiveness in the induction of CPD and 8-

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(17).

oxodGuo. We showed that CPD are formed at least as frequently as 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodGuo) by UVA radiation (15). Here, using specific antibodies, we determine the yield of three bipyrimidine photoproducts, CPD, (6-4)PP and DewarPP in the genomic DNA of CHO cells exposed to physiologically relevant doses of UVC, UVB, UVA and SSL. An estimation of the levels of these three types of photolesions was previously obtained by immunostaining cells irradiated with UVC, broad- or narrow- band UVB, or natural sunlight with monoclonal antibodies similar to those used here (21-23). Clingen and col. (22) assigned the increase in arbitrary grey scale values to the induction of antibody binding sites and estimated the relative production of (6-4)PP and DewarPP by assuming an

account the very different affinities of the three antibodies for their respective substrates, nor the possibility of multimeric forms of the immunoglobulins. In addition, comparison of the photolesion induction by various UV lamps was made possible only by expressing the fluence rates as dimer-equivalent fluences. In contrast to estimating relative values, determination of the absolute amount of different damage due to UV radiation allows a comparison of the biological effectivenesses of two or more types of photobiological action, e.g. induction of CPD and other types of photolesion. This is not the case for the determination of relative lesion induction, even if it can provide a precise action spectrum for a given photobiological effect. In this report, we describe the results of a method that allows a determination of the absolute amounts of these photoproducts. This method relies on the calibration of the immunochemical signals for each antibody using specific DNA repair enzymes. We present the first absolute determination of all three bipyrimidine photoproducts using one detection method. Our data show that the distribution of the bipyrimidine photolesions varies greatly depending on the wavelength region considered, and we demonstrate that UVA radiation produces significant amounts of CPD at doses comparable to those of human exposure. In

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equal luminescence signal for all three antibodies. However, their data neither took into

addition, the relatively high production of DewarPP by SSL observed in this study, suggests that DewarPP may be a biologically relevant photolesion. However, the cytotoxic and mutagenic properties of this damage are not well established mainly due to the difficulties in detecting DewarPP. In this respect, we analyzed the capacity of CHO cells to remove DewarPP, as well as CPD and (6-4)PP, using the same immunological approach. Furthermore, in order to investigate the possible effects of other types of lesions, i.e. DNA strand breaks and oxidative DNA damage, on the repair of the bipyrimidine photoproducts, CHO cells were irradiated with either UVB, UVA radiation or SSL. We demonstrate that DewarPP are removed with similar kinetics and efficiency as CPD, although they are valence

similarity between the responses of DewarPP and CPD suggests that DewarPP may contribute significantly to the mutagenesis that occurs after exposure to sunlight.

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isomers of (6-4)PP. In contrast, the removal of (6-4)PP is almost complete within 6 h. The

EXPERIMENTAL PROCEDURES Irradiation sources UVC irradiation was performed using a germicidal lamp emitting primarily at 254 nm. Broad band UVB irradiation (290-320 nm) was carried out with a set of six 15 W fluorescent tubes (Vilber Lourmat, Torcy, France) having a spectral irradiance very similar to that of FS20 lamps. The incident light was filtered through a Schott WG 305 cut-off filter (thickness 2mm) which efficiently blocks contaminating wavelengths below 290 nm. Polychromatic UVA radiation was obtained from an Osram Ultramed 400-W gas discharge lamp, emitting through a 2 cm water layer held in pyrex-glass, and through a Schott WG345 filter (thickness

(320-400 nm) including 0.05% of UVA2 wavelengths (320-340 nm), 39.2% of visible light and less than 5x10-4% UVB (280-320 nm). The simulated solar light (SSL) was produced by a 2500W xenon compact arc lamp (Conrad-Hanovia Inc., Newark, NJ) and passed through a Schott WG320 filter (thickness 3mm). The incident light was composed of 0.8% UVB, 6% UVA, 44.5% visible light, 48.7% infrared and less than 10-5 % UVC. The proportions of UVB, UVA, visible light and infrared in natural terrestrial sunlight are approximately 0.3%, 5.1%, 62.7% and 31.9% respectively. Emission spectra after filtration were previously presented (13, 16, 18). Percentage spectral irradiance values, which were normalized for the UV region (290-400 nm) only, are given in the results section. UVC fluence rate (0.16 J.m -2.s-1) was measured with a Latarjet dosimeter and the irradiation time did not exceed 4 min. UVB (12.5 J.m-2.s-1) and UVA (135 J.m-2.s-1) fluence rates were measured with a radiometer VLX 3W equiped with interferential filters (Vilber Lourmat, Torcy, France); the exposure time did not exceed 15 min and 120 min, respectively. According to a YSI Kettering 65A thermopile (Yellow Spring Instruments, OH) the fluence rate for SSL was 1250 J.m-2.s-1. Irradiation lasted no longer than 45min. The UV wavelengths

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3mm) and an anticaloric KG1 filter. The delivered radiation comprised 60.8% UVA radiation

contributed to approximately 6.8% of the total energy measured with the thermopile. Irradiation of CHO cells and genomic DNA isolation CHO cell lines AT3-2 and UVL9 (kindly provided by G.Adair, University of Texas, Smithville, USA), which are respectively proficient and deficient in nucleotide excision repair (mutated in ERCC1 gene), were used. Cells were routinely grown in alpha-minimal essential medium containing 10% fetal calf serum (Gibco) and 16 µg/ml gentallin. Two to four hours prior to irradiation, 10 7 cells were seeded on 60 mm dishes (Costar). Cells were then washed and irradiated with UVB, UVA and SSL in phosphate-buffered saline (PBS) on ice to prevent repair during exposure. Irradiation with UVC was performed at room temperature, since short

For photolesion quantification, NER- cells were used to avoid repair during long irradiation periods. After irradiation, cells, maintained on ice, were immediately scrapped from the dishes into ice-cold PBS buffer and pellets were stored at -20°C until use. For repair experiments, confluent NER+ cells received either 1 kJ.m-2 of UVB, 1000 kJ.m-2 of UVA or 4500 kJ.m -2 of SSL (equivalent to 306 kJ.m-2 of UV energy, a conversion that was used throughout the experiments described here). The duration of irradiation corresponded to 160 sec, 120 min and 45 min, respectively. To study the repair of the DewarPP after UVB exposure, a dose of 5 kJ.m -2 was given to ensure sufficient induction of this photoproduct. In order to get enough DNA for the immuno dot-blot assay, one dish per repair time was used. Immediately after irradiation, cells were scrapped into cold PBS buffer and one half was pelleted and stored at –20°C for the determination of the photolesion at time t = 0, whereas the other half was allowed to undergo repair for a set time in fresh medium at 37°C. At times t = 2, 4, 6 and 24 h after irradiation, cells were harvested, washed, pelleted and stored at -20°C. At the repair time of 24 h, cellular growth was not observed. For DNA extraction, cells were lysed for 1 h at 37°C in lysis buffer (20 mM Tris-HCl

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exposures were required.

pH8, 20 mM NaCl, 20 mM EDTA and 0.5% SDS). Samples were then incubated 4 h at 37°C with 100 µg/ml of RNase A, 5 U/ml of RNase T1, and overnight at 37°C with 10 µl of proteinase K (25 mg/ml, Boehringer). DNA was purified by two extractions with phenol and chloroform/isoamyl alcohol (25:24:1 v/v/v) and further precipitated by the cold ethanol procedure. The amount of DNA was determined spectrophotometrically (Shimadzu UV-160A spectrophotometer) on the basis of its absorbance at 260 nm. Detection of the bipyrimidine photoproducts by ImmunoDotBlot assay Cyclobutane pyrimidine dimers, (6-4)PP and DewarPP were detected by using TDM-2, 64M-2 and DEM-1 monoclonal antibodies, respectively (24-25). For ImmunoDotBlot (IDB)

was loaded (Hybri-dot manifold, Life Technologies, Bethesda, MD, USA) on nitrocellulose membrane (0.2µm, BA83 Schleicher & Schuell, Dassel, Germany). After blotting, the dots were rinsed twice with 100 µl of PBS. Membranes were saturated overnight at 4°C in PBS containing 5% of non-fat dry milk (nfm) and 0.1% tween 20 (Sigma) and, then, incubated for 1 h at 37°C with TDM-2, 64M-2 or DEM-1 antibody (dilution 1/1000, 1/250 and 1/1000 respectively, in 0.5% nfm-0.1% tween20-PBS). After extensive washing with 0.5% nfm-0.1% tween20-PBS (nfm-TPBS), membranes were incubated 1h at room temperature with a 1/2000 dilution of a second antimouse horseradish peroxidase-conjugated antibody (Calbag, San Francisco, CA, USA) in nfm-TPBS buffer. Blots were then washed extensively with nfmTPBS buffer and peroxidase activity was revealed with the enhanced chemiluminescence blotting detection system (RPN2106, ECL ™, Amersham, UK). Membranes were immediately exposed to X-ray films (Kodak XAR) for different times depending on the antibody (for a given antibody, the same exposure time was always used). Relative luminescence intensity was determined using a Biocom image analyser and Macroautorag software (Biocom, Les Ulis, France).

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analysis (slightly modified from 26), a triplicate of 500 ng of heat-denatured DNA per dot

In repair experiments, a streptavidin/biotin system was used to increase the luminescence signal for detection of the (6-4)PP after UVB. In this case, after incubation with 64M-2 antibodies in TPBS, membranes were washed extensively with TPBS and incubated 1h at room temperature with biotinylated second antibody (1/5000 in TPBS, Calbag, San Francisco, CA, USA). Next, membranes were washed three times for 15 min with TPBS, incubated 30 min with streptavidin-peroxidase solution (1/5000 in TPBS) and further processed as described above. For each repair time within a given experiment, the percentage of remaining lesions was deduced by comparing the decrease in the luminescence intensity with the luminescence at time t = 0h using the same set of irradiated cells. The fraction of

follows : two series of irradiation experiments and two IDB assays per experiment were performed and each sample was dotted in duplicate. Quantitative determination of CPD in plasmid DNA by plasmid relaxation and ImmunoDotBlot assays The pZ189 plasmid DNA (5500 bp) was UVB-irradiated on ice in 10 mM Na-phosphate buffer pH 7.5 at doses ranging between 0.5 and 5 kJ.m-2. The number of CPD per plasmid was determined by measuring the conversion of irradiated supercoiled plasmid (form I) DNA to the open circular (form II) following digestion with the pyrimidine dimer-specific enzyme T4 endonuclease V (DenV protein, obtained from Applied Genetics, New York, and from Dr. J. Brouwer, Leiden University, The Netherlands). Briefly, 150 ng of pZ189 DNA was incubated for 30 min at 37°C in 10 µl of reaction buffer (50 mM KH 2PO4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA) with or without 0.2 µg of DenV protein. Samples were then electrophoresed through a 0.8% agarose gel in the presence of ethidium bromide. Photographic negatives of the gels were scanned using a Biocom image analyser. The number of enzyme sensitive sites (ESS) per plasmid as a function of the dose was calculated from the

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damage remaining at various times was expressed as a mean value of 8 determinations as

Poisson distribution and corrected for differential binding of ethidium bromide to supercoiled versus relaxed DNA. CPD were also detected by TDM-2 antibody in irradiated plasmids after linearization and denaturation (triplicates of 500 ng) using the IDB assay as described above. The linearization procedure ensured complete denaturation of the plasmid molecules. The luminescence intensity was determined as a function of the dose. Finally, a calibration curve, which represented the luminescence intensity as a function of CPD/kbp, was obtained and used to calculate the number of CPD in the genomic DNA of irradiated cells. Quantitative determination of (6-4)PP in plasmid DNA by plasmid relaxation and

400 ng of pCAT plasmid DNA (4610pb, Promega, Madison, WI, USA) was subjected to 10 to 40 J.m -2 of UVC radiation. CPD were removed under UVA illumination by incubation of (250 ng) irradiated DNA for 1 h in 50 mM Tris-HCl pH7.4, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, in the presence of 30 ng of photolyase from Escherichia coli (a generous gift from Dr. A. Sancar, University of Chapel Hill, NC, USA). The UVA source used was a HPW 125 Philips lamp, which emits mainly at 365 nm; a dose of 50 kJ.m-2 was necessary for complete photoreversion of CPD. An aliquot of 100 ng of the treated plasmid was then digested with DenV protein to check for the completion of the photoreversion. After photoreversion of CPD, remaining photolesions (essentially (6-4)PP) were detected by UvrABC endonuclease. Briefly, DNA (125 ng) was incubated 5 min at 37°C with 0.75 pmoles of UvrA protein and 1.8 pmole of UvrB protein in reaction buffer (50 mM Tris-HCl pH7.5, 10 mM MgCl2, 85 mM KCl, 1 mM DTT and 2 mM ATP). Then, 0.75 pmole of UvrC protein was added for 30 min at 37°C and the reaction was stopped by adding 0.05% SDS and heating for 5 min at 65°C. The samples were then electrophoresed, stained, photographed and analyzed as described above. The number of (6-4)PP per kbp could therefore be established as

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ImmunoDotBlot assays

a function of the UVC doses, which were chosen to produce less than one (6-4)PP per plasmid molecule. At each step a control reaction was performed on irradiated or unirradiated plasmid DNA in the absence of the protein. In the case of UvrABC digestion, a mock reaction was also carried out in the presence of only two proteins. In parallel, (6-4)PP were also detected in UVC-irradiated plasmid by monoclonal 64M2 antibody by using the IDB assay, as described above. Similarly, the luminescence intensity obtained with 64M-2 antibody was determined as a function of the UVC dose, and a calibration curve (luminescence intensity as a function of (6-4)PP/kbp) was established. Quantitative determination of DewarPP by photoreversion of (6-4)PP

kJ.m-2 was used as substrate. Triplicates of 500 ng of DNA per dose were then subjected to IDB using 64M-2 and DEM-1 antibodies. The exact number of initial (6-4)PP per kbp was calculated from the calibration curve obtained using the 64M-2 antibody. In order to convert (6-4)PP into their DewarPP isomers, the remaining DNA was re-irradiated with a single dose (450 kJ.m-2) of UVA radiation (Osram Ultramed 400-W gas discharge lamp without cut-off filters; thus, at least 0.3% of the emitted photons were in the range of 295-316 nm, see above). This DNA was again subjected to IDB with the two antibodies. The number of (6-4)PP that disappeared following isomerization was deduced from the calibration curve for the 64M-2 antibody luminescence signal. Since one DewarPP is assumed to result from the photoisomerization of one (6-4)PP at wavelengths around 320 nm (6, 27), the number of (64)PP that disappeared during the UVA re-irradiation step corresponded to the number of DewarPP produced. For each initial UVB dose, the increase in the luminescence intensity obtained with the DEM-1 antibody after the photoisomerization process was correlated with the number of DewarPP per kbp, thus establishing a calibration curve for the DEM-1 luminescence signal.

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Genomic DNA from cells exposed to UVB radiation at doses ranging from 2 to 10

Cell survival For cell survival determination, the CHO repair-proficient and deficient cells were irradiated by UVB radiation (0 – 5 kJ.m-2 ) or SSL (0 – 306 kJ.m -2) in PBS in 60 mm petri dishes as described above, washed with PBS and trypsinized. Aliquots of 102 to 106 cells were seeded in triplicate into 60 or 100 mm dishes. After 10 days of incubation in growth medium, colonies were stained with methylene blue in methanol and counted. Survival was calculated as the ratio of the cloning efficiencies of irradiated over unirradiated cells x 100. The surviving fraction was plotted versus the amount of each of the three photoproducts. To calculate the correlation coefficient, all the experimental points obtained with each source of

was determined. The correlation between damage induction and cell survival was considered as statistically significant when p 0.05), and resemble those of CPD. The repair rate of DewarPP follows a two step process. The repair rates of CPD and DewarPP (figure 6 A and C) do not exhibit significant

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after UVA (15) and are rapidly repaired) does not interfere with the repair of CPD. The CPD

differences. Relationship between induction of bipyrimidine photoproducts and cytotoxicity Since the role of each of the three bipyrimidine photoproducts in UV-induced lethality may vary depending on the cell type (22), we examined the response of the repair-proficient CHO AT3-2 cells. Survival, assayed by determining clonogenic efficiencies, was plotted as a function of the amount of CPD, (6-4)PP and DewarPP produced by UVB radiation and SSL, which produce significant amounts of all three lesions (figure 7 A, B and C). Inserts in figure 7 show that cell killing was well correlated with the formation of CPD (r = -0.71, p < 0.05) and also of (6-4)PP (r = -0.68, p < 0.05). Since we observed that 90% of (6-4)PP were

clonogenic efficiency of the irradiated cells is questionable. This discrepancy may be due to the parallel induction of CPD and (6-4)PP (32). However, high correlation coefficients for CPD and (6-4)PP were observed while investigating the role of these lesions in the lethality of the repair-deficient UVL9 cell line upon UVB and SSL irradiation. The r values were -0.85 and -0.77 (p < 0.05) for CPD and (6-4)PP, respectively. In the absence of repair, the lethality at a given dose of radiation depends on the intrinsic cytotoxicity of the lesion and its amount. Consequently, both lesions seem to be cytotoxic. There was no statistically significant correlation between the induction of DewarPP +

and the lethality of irradiated NER cells (r = -0.25, p > 0.05), whereas a statistically -

significant but loose correlation was observed in irradiated NER cells (r = - 0.64, p = 0.05). +

This weak (or null for NER ) correlation may not be surprising considering the low production of DewarPP in comparison to CPD (table 1), at least in the case of UVB exposure (ratio 1:70). DewarPP formation depends on the formation of (6-4)PP, but the action spectra of these lesions are very different (see in the discussion). The LD10 values (amount of -

damage leading to 10% survival, or lethal dose) calculated for SSL-irradiated NER cells are 21

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removed within 6 h after UV exposure, the contribution of this lesion to the loss of the

0.12 CPD, 0.015 (6-4)PP and 0.006 DewarPP per kbp. This apparently suggests that the former photolesion is the least cytotoxic. However, the ratios between the LD10 values for both the NER+ and NER- cell lines follow exactly the same ratios for the induction of these lesions upon SSL exposure. It seems that the lethality is governed by the extent of each lesion and not only by the intrinsic cytotoxicity of these lesions. These data clearly demonstrate that quantitative analysis of damage induction is important for our understanding of how each photoproduct contributes to biological repercussions of solar UV radiation.

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DISCUSSION We quantitatively analyzed the induction of the three bipyrimidine photoproducts, i.e. CPD, (6-4)PP and DewarPP, in CHO cells exposed to the different UV components of solar radiation. The present study provides information about the absolute amounts of three photolesions produced by various regions of the UV spectrum and about how efficiently these types of DNA damage are produced by certain wavelengths. Furthermore, the repair rate of each of these photolesions was also examined at the genomic level. The yield of each of the bipyrimidine photoproducts was assessed using previously described monoclonal antibodies, TDM-2, 64M-2, DEM-1 (24, 25). These and similar

detection of these photolesions and to study their repair as well (7, 21-23, 26, 33-36). The TDM-2 antibody, specific for CPD, binds preferentially to TT dimers, recognizes CT dimers and binds to a lesser extent to CC and TC dimers (24). TC (6-4)PP (37) and expectedly TC DewarPP predominate among these types of photoproducts and are ideal substrates for the 64M-2 and DEM-1 antibodies, respectively, but these antibodies can also recognize TT derivatives (25). It thus appears that these antibodies are suitable for providing a good estimate of the yields of formation of these photolesions. In accord with earlier observations (21-24, 34, 35), we found that they were sensitive enough to detect the photoproducts induced at physiologically relevant doses of radiation, i.e. those that gave low toxicity in cultured cells or those of the normal human environment. However, in contrast to other studies (22, 38), the detection method reported here allowed us to examine for the induction of all three bipyrimidine photoproducts within the same dose range and for each of the UV sources. Importantly, the three photolesions could be quantified in the same irradiated DNA sample.

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monoclonal or polyclonal antibodies have already been largely used for the in vitro or in situ

CPD and UVA radiation, role in solar mutagenesis The yields of CPD formed by UVC and UVB radiation shown in table 1 are in excellent agreement with those reported in the literature (33, 39-43). In addition, we observed induction of CPD by UVA radiation in mammalian cells (figure 3). This confirms our previous observation using plasmid DNA (16), and is in agreement with other studies (14, 42, 44). We further demonstrated that CPD are produced mostly by UVA1 wavelengths in the interval between 345-400 nm, which corresponds to the spectral irradiance of our UVA source (table 2). The yield of CPD produced by UVA follows exactly the DNA absorption at these wavelengths (45) and Setlow’ s action spectrum (46). Thus, compared to UVC, 105 higher

these UVA doses are still biologically relevant since they correspond to a few hours sunlight exposure at zenith in summer (16). Similarly, (6-4)PP and DewarPP can also be produced by UVA radiation and detected at trace levels (Perdiz and Sage, unpublished data). Interestingly, we show in table 2 that about 10% of CPD induced by SSL irradiation was formed by the UVA component. This is an additional argument indicating that UVA radiation may partly contribute to the DNA damage induced by sunlight, and consequently, UVA may participate to solar light mutagenesis more than previously expected. Indeed, a majority of the mutations induced by sunlight and found in skin tumors (3, 47, 48) are GC to AT transitions and tandem double CC to TT mutations, all of which can be attributed to bipyrimidine photoproducts (mainly to CPD in repair-proficient non-XP individuals). Furthermore, the mutational specificity of UVA comprises a large amount of GC to AT transition located at bipyrimidine sites (19, 49). This class of mutation is over-represented (65% of all events) in excision repair deficient cells (20). Such base changes are likely to be due to the CPD produced by UVA.

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doses of UVA are necessary to produce the same amount of CPD. Despite this difference,

DewarPP, a major lesion induced by sunlight The yield of (6-4)PP formation after UVC radiation (table 1) is in good agreement with that reported in (41) (no value available either for UVB or DewarPP). We observed that (64)PP were 200 times less efficiently formed by UVB than by UVC, which is in accordance with the published action spectrum for the induction of CPD and (6-4)PP (32). DewarPP were not formed after UVC radiation at biologically relevant doses (21, and this report). However, they were produced by broad-band UVB (22, and this report) and, moreover, were more extensively induced by natural (21) or simulated sunlight (this report), reflecting the maximal absorption of (6-4)PP which lies between 310 and 340 nm (6, 30).

UVC, 1:0.12:0.014 for UVB and 1:0.18:0.06 for SSL. This illustrates that the distribution of the different classes of photolesion varies depending on the UV source, as already observed (21, 22, 35), and clearly demonstrates that the biological effects of sunlight cannot be deduced solely on the basis of studies with UVC. Furthermore, DewarPP was formed with high yield, making it the third major photolesion induced by SSL (about ten times more frequent than 8oxodGuo (15) or cytosine hydrates (43)). This indicates that this lesion may contribute in a previously unsuspected manner to the effects produced by sunlight exposure. Differences in kinetics and efficiencies of CPD, DewarPP and (6-4)PP removal All three lesions are most likely eliminated by the NER process (29), although the repair mechanism of DewarPP has not been elucidated in mammalian cells. We first observed that the rate of removal of all of these photolesions did not depend on the source of irradiation. This indicates that the presence of relatively large amounts of single strand breaks and 8-oxodGuo or other oxidized bases in UVA- or SSL- irradiated cells (12-15, 38) does not impair the processing of bipyrimidine photoproducts by the cellular machinery. On the other hand, SSB and oxidative damage are processed by repair pathways other than NER, i.e. base

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The comparative rates of the formation of CPD, (6-4)PP and DewarPP were 1:0.25 for

excision repair (50). In human cells, it has been shown that most strand breaks and a large proportion of oxidized bases are repaired within 1 h and 4 h, respectively (38, 51, 52). The rate of removal of base damage is similar to that observed for (6-4)PP. The repair rate of CPD has been extensively studied in both rodent and human cells. We observed that at most 20% of CPD was eliminated after a repair time of 6 h and 25 to 50% after 24 h. This is within the range of published repair times for global genomic repair in cultured rodent cells or rodent skin under similar conditions of irradiation (40, 53, 54). In contrast, 70% of CPD are removed by TCR from expressed genes after 24 h (55). In accordance with the literature (56), we report fast and efficient removal of (6-4)PP in CHO

in human cells, 26, 38, 57, 58). In contrast, only 50-60% of (6-4)PP were lost from active genes within the 6 h period following exposure of CHO cells to 30-40 J.m-2 of UVC (41, 55). Hairless mouse epidermis was even less efficient in removing (6-4)PP after treatment of the animals with a unique UVB dose (54). Furthermore, a chronic low-dose of UVB exposure significantly reduced the excision repair of both CPD and (6-4)PP in mice (36). DewarPP have received much less attention than other forms of DNA damage. Indeed there is only one report dealing with its repair rate (38). We found that the repair rate of DewarPP was very similar to that of CPD, but much lower than that of (6-4)PP. After a repair time of 6 h, CHO cells did not remove more than 15% to 30% of DewarPP, and the repair efficiency was no more than 20% to 50% after 24 h. Using radioimmunoassay, Rosenstein & Mitchell (38) observed a slightly faster repair rate of DewarPP in human skin fibroblasts than in the rodent cells described here. This is in agreement with reported differences in the repair kinetics between the two species (17). A conformational basis for the differences in the repair rates of the three bipyrimidine photoproducts may be tentatively considered. Most likely, the recognition of the photolesion

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cells after UVB and SSL, i.e. 90% removal within the 6 h period following irradiation (vs 3 h

by NER proteins, which bind to sites of DNA damage, is the rate-limiting step in the repair process. Most of the investigations report greater DNA unwinding by (6-4)PP and DewarPP than by cis-syn CPD, and a smaller distortion of DNA by DewarPP than by (6-4)PP (59-61). This is in agreement with the 10 fold higher rate of excision of (6-4)PP over CPD by E. coli UvrABC excinuclease (29) and human cell extracts (62) and with the relative affinities of DNA damage binding proteins, E. coli UvrA and human UV-damaged DNA binding (UVDDB) protein for (6-4)PP, DewarPP and CPD (10, 4-2.8, 1, respectively; 63). However, DewarPP are excised at the same rate as (6-4)PP by UvrABC excinuclease (29), and at the same rate and extent as CPD in CHO cells (figure 6 A and C). The fast removal of (6-4)PP by

(6-4)PP, whereas UV-DDB activity, which is absent from certain xeroderma pigmentosum E groups and hamster cells (64), may be required for targeting CPD and DewarPP for GGR. It is tempting to speculate that DewarPP, as well as CPD, are efficiently repaired by TCR but not by GGR, and that (6-4)PP are mainly repaired by GGR. Another possibility could be that, due to the distorsion of the DNA helix at the DewarPP site, the DewarPP is still able to bind XPC/hHR23B and other associated proteins, but with lower affinity compared to the affinity of these proteins for (6-4)PP. Thus, according to this hypothesis, the DewarPP lesion would be repaired by GGR, but with slow kinetics. Biological relevance of DewarPP The relative contribution of CPD and (6-4)PP to UVC- and solar UV- induced mutagenesis has been a matter of debate for almost 20 years (65). Studies on site specific mutagenesis in E. coli and yeast has helped to elucidate the intrinsic mutagenicity of the three bipyrimidine photoproducts (66-69). In short, (6-4)PP and DewarPP are more mutagenic than the corresponding CPD. In eucaryotic cells, it is likely that these lesions produce mutations via error-prone bypass by DNA polymerase ζ lacking proofreading activity (70, 71 for review). 27

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GGR may be due to the tight binding of XPC/hHR23B to the highly distorded region carrying

As our results show that DewarPP are no longer quantitatively negligible compared to CPD lesions, especially at TC sites, it is legitimate to question about biological contribution of this type of lesion to solar UV-mutagenesis. Considering the fast and efficient repair of (64)PP and the slow and inefficient removal of CPD and DewarPP, most of the mutations that we observed in the repair-proficient CHO cells after SSL (18) would appear to be due to CPD and DewarPP. Indeed, it has been previously emphasized that (6-4)PP are mainly responsible for the mutations in repair-deficient, but not in repair-proficient cells (5). The SSL-induced mutation spectrum in CHO cells was particularly marked by an increase in tandem double CC to TT events; such mutations are very rare after UVC (18-20). At such sites, CPD are readily

know the intrinsic mutagenicity of CC CPD in mammalian cells, and based on the fact that TT (6-4)PP and TT DewarPP, but not TT CPD, produced some tandem double mutations (68), it is tempting to suggest that at least part of the CC to TT double events occurring after irradiation of CHO cells with SSL are due to DewarPP. Interestingly, such mutational events have also been recovered in the mutated p53 gene of skin tumors from normal and XP individuals exposed to sunlight (3, 4). The determination of the yield of formation of these photolesions by the different UV components of solar light should help to estimate the risks associated with exposure to environmental sunlight or artificial sources such as sunbeds.

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formed after SSL, representing about 20% of the total CPD content (72). Since we do not

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LEGENDS OF FIGURES

Figure 1: Calibration curves for the antibody luminescence signals. (A) TDM-2, (B) 64M-2 and (C) DEM-1 antibodies. Each value represents the mean+/-SEM of three independent experiments. Figure 2: Strategy for the quantification of Dewar photoproducts. Figure 3: Quantification of Cyclobutane Pyrimidine Dimers in DNA of CHO cells exposed to UVC, UVB, SSL and UVA radiation. Two independent series of cells were used for each type of radiation. Two IDB experiments were performed for each DNA sample,

were compared using a three-way analysis of variance (dose, irradiation experiments, IDB) with three repeated measurements for one factor (IDB). Dose-effect on DNA damage formation was considered as statistically significant when p < 0.05. Figure 4: Quantification of Pyrimidine (6-4) Pyrimidone in the DNA of CHO cells exposed to UVC, UVB and SSL radiation. Irradiation experiments and data analysis were performed as described in the legend of figure 3. Figure 5: Quantification of Dewar photoproducts in DNA of CHO cells exposed to UVB and SSL radiation. Irradiation experiments and data analysis were performed as described in the legend of figure 3.

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which was loaded in triplicate. Each point represents the mean+/-SEM of 12 values. Means

Figure 6: Repair of CPD (A), (6-4)PP (B) and DewarPP (C) in CHO cells. Cells were analysed at different times after receiving 1 kJ.m -2 (A and B) or 5 kJ.m -2 (C) of UVB (lozenge), 1000 kJ.m-2 of UVA (circle) and 306 kJ.m -2 of SSL (square). Each data point is the mean value calculated from 4 IDB assays from two independent irradiation experiments, in which samples were dotted in duplicate on membrane. Bars represent the standard error of the mean values. Inserts shows representative IDB assays on genomic DNA from UVB- (A and B) or -SSL (C) irradiated cells. Figure 7: Correlation between CPD (A), (6-4)PP (B) and DewarPP (C) induction and the survival of NER-proficient AT3-2 cells irradiated by UVB (lozenge) and SSL (square).

are considered to be a single population, and the corresponding regression line is obtained. r2 is the square of the correlation coefficient from the linear regression. The correlation is statistically significant (p < 0.05) for CPD and (6-4)PP.

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Bars represent the standard errors of the mean values. In inserts, all the experimental points

Table 1 : Yields of formation of CPD, (6-4)PP and DewarPP in the DNA of cells exposed to UV radiation (#)

Lesion / kbp / J.m-2

Ratio

(6-4)PP

DewarPP (6-4)/CPD

Dewar/CPD

Dewar/(6-4)

UVC

2.2x10-2

5.3x10-3

nd

1:4

UVB

2.0x10-4

2.4x10-5

2.9x10-6

1:8

1:70

1:8

UVA

3.0x10-7

nd

nd

SSL*

2.1x10-6

3.7x10-7

1.3x10-7

1:6

1:16

1:3

# The yields were calculated from the slope of the regression lines given in figures 3, 4, 5. For UVC- and UVB- induced CPD and (6-4)PP only the linear parts of the curves were used.

* The yields of photoproducts were calculated taking into account only the UV region (6.8%) of the whole emitted radiation.

Nd , not detectable at the physiological doses used

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CPD

Table 2 : Physical characteristics (% irradiance) and calculated biological effectiveness (% CPD yield) of the UV sources used (*)

Source Region UVB

UVA

SSL

Irradiance (%)

38.8

0.0077

4.6

CPD yield (%)

99.6

11.5

89.1

Irradiance (%)

61.2

100

95.4

CPD yield (%)

0.4

88.5

10.9

Irradiance (%)

43.9

0.7

24.2

CPD yield (%)

0.4

1.8

8.2

Irradiance (%)

19.4

99.3

71.8

CPD yield (%)

0

86.7

3.5

UVB (290-320 nm)

UVA2 (320-345 nm)

UVA1 (345-400 nm)

* The details of the calculations have been extensively described by Douki et al. (15) Values of spectral irradiances for the various intervals are based on measurements of the spectral irradiances in 1 nm steps for the given UV source. CPD spectral effectiveness was calculated on the basis of the action spectrum for CPD formation from 14.

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UVA (320-400 nm)

Table 3: Relative spectral effectiveness for CPD formation

* Source

UVB

UVA

SSL

**Irradiance UVB/UVA

0.635

7.68x10-5

0.048

#

CPD UVB/UVA

257

0.13

8.2

Enhancement factor

406

1695

169

##

*UVB, UVA and SSL correspond to the UV sources used in the experiments, and as specified in the Materials and Methods.

400 nm (UVA) for the different UV sources. # Ratio of CPD yields due to UVB versus UVA region. The CPD yields were calculated as the products of the measured spectral distribution of the given source and the CPD’s spectral sensitivity for the interval considered. ## The enhancement factor represents the ratios of CPD yields normalized by the irradiance ratios. It highlights the relative importance of the UVB component of a lamp’s emission spectrum for CPD induction.

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** Ratio of the sum of irradiances between 290-320 nm (UVB) to those between 320-

Table 4 : Number of bipyrimidine photoproducts (per kbp) immediately after irradiation and at t = 24 h repair time

Photoproducts CPD

Photoproducts per kbp T=0h T=24h

Radiation -2

UVB (1 kJ.m ) -2

SSL (306 kJ.m ) -2

UVA (1000 kJ.m ) (6-4)PP

-2

UVB (1 kJ.m ) -2

SSL (306 kJ.m ) DewarPP

-2

UVB (5 kJ.m ) -2

SSL (306 kJ.m )

% of remaining damage

0.22

0.15

68

0.67

0.34

51

0.25

0.19

76

0.024

0

0

0.11

0.004

4

0.015

0.007

47

0.0306

0.024

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39

A

30

40

2

Luminescence Intensity

15 10 5 0 0

0.5

2

1

1.5

2

2.5

R = 0.978

30 25

(a.u.)

20

B

y = 46.231x

35

R = 0.9894

25

20 15 10 5 0 0

CPD/kbp

0.1

0.2

0.3

0.4

0.5

0.6

0.7

(6-4)PP/kbp

C

25 y = 273.58x 2

R = 0.9225

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15

(a.u.)

Luminescence Intensity

(a.u.)

Luminescence Intensity

y = 12.992x

10 5 0 0

0.02

0.04

0.06

0.08

DewarPP/kbp

Fig. 1

40

UVB CHO cells

Photoisomerization UVA (320nm) 1st 64M-2 IgG

IDB DEM-1 IgG 2nd IDB

L.I.

64M-2 IgG

DEM-1 IgG

1

L.I.

L.I.

(6-4)PP/kbp

L.I. (6-4)PP/kbp

2

2

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1

1 (6-4)PP isomerized = 1 DewarPP formed

(6-4)PP/kbp

1 =

(6-4)PP/kbp

2

(DewarPP/kbp) formed by photoisomerization



L.I.

2

-

L.I.

1

Fig. 2

41

p