The influence of the wavelength of ultraviolet radiation on survival, mutation induction and DNA repair in irradiated chinese hamster cells

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Mutation Research, 72 ( 1 9 8 0 ) 4 9 1 - - 5 0 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

THE INFLUENCE OF THE WAVELENGTH OF U L T R A V I O L E T RADIATION ON SURVIVAL, MUTATION INDUCTION AND DNA REPAIR IN IRRADIATED CHINESE HAMSTER CELLS

B. Z E L L E , R.J. R E Y N O L D S *, M.J. K O T T E N H A G E N , A. S C H U I T E a n d P.H.M. L O H M A N

Medical Biological Laboratory TNO, Lange Kleiweg 139, P.O. Box 45, 2280 AA Rijswijk Z.H. (The Netherlands) ( R e c e i v e d 28 F e b r u a r y 1 9 8 0 ) ( R e v i s i o n received 7 May 1 9 8 0 ) ( A c c e p t e d 12 M a y 1 9 8 0 )

Summary Chinese hamster ovary cells were used to compare the c y t o t o x i c i t y and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with t h a t of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse "Sun L a m p " ) , of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal n u m b e r of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation. The numbers of dimers induced were determined by a biochemical m e t h o d detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers. On the basis of induced dimers, more cells were killed by > 3 1 0 - n m UV than by > 2 9 0 - n m UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5--6 times as m a n y m u t a n t s were induced per dimer by > 3 1 0 - n m UV than by > 2 9 0 - n m UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were n o t caused by differences in repair of dimers: cells with an equal n u m b e r of dimers induced by either 254-nm or near-UV * Present address: Department of Physiology, Harvard University, S c h o o l o f Public Health, Boston, MA 02115 (U.S.A.). Abbreviations: BUdR, 5-bromodeoxyuridine; FUdR, 5-fluorodeoxyuridine; PBS, phosphate-buffered saline; PRE, photoreaetivating enzyme; TCA, trichloroacetic acid; TdR, thymidine; TG, 64hioguanine.

492 showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical a m o u n t of repair replication. The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.

The action of ultraviolet radiation on mammalian cells in culture is usually studied with low-pressure mercury lamps ("germicidal lamps") as the laboratory source of "far-UV". The radiation emitted is fairly monochromatic, and most of the radiant energy is emitted at 254 nm. Because of the strong absorbance of DNA at this wavelength, lesions are readily formed, the cyclobutyl pyrimidine dimer being the predominant p h o t o p r o d u c t (Setlow, 1968). For this reason, germicidal lamps are excellent tools for the induction of pyrimidine dimers. The relevance of results obtained from studies with germicidal lamps in understanding the effects of the UV region of sunlight can be questioned, however, as wavelengths below 290 nm do not normally reach the earth's surface (Parrish et al., 1978). Several investigators have shown that differences exist between the biological effects of far-UV and of UV of longer wavelengths ( " n e a r - U V " ) , with regard to survival and mutation induction (Todd et al., 1968; Coohill et al., 1977; Hsie et al., 1977; Jacobson et al., 1978; R o t h m a n and Setlow, 1979). In these studies, UV doses were compared on the basis of emitted or absorbed energy, whereas the relation between the biological effects and the number of pyrimidine dimers has been given little attention. In the investigation with cells from the Chinese hamster ovary (CHO) reported here we compared the induction of dimers by far-UV and near-UV sources and their removal by excision repair, and we correlated the cytotoxicity and the mutagenicity of the different types of UV with the number of dimers induced by the radiation. In these studies 3 forms of radiation were compared, namely the normal 254-nm far-UV and radiation emitted by a "Sun L a m p " so filtered that either UV < 2 9 0 nm or < 3 1 0 nm was removed. Dimers were quantitated by using specific endonucleases from Micrococcus lu teus that produce single-strand incisions near pyrimidine dimers (Paterson et al., 1973). Part of these studies has been presented previously (Reynolds and Lohman, 1978). Materials and methods Chemicals

Ham's F10 medium, b u t without hypoxanthine or thymidine, newborn calf serum and foetal calf serum were obtained from Flow laboratories (Great Britain). Hypoxanthine was obtained from Schuchardt (Germany), and L-glutamine from BDH Chemicals (Great Britain). Sodium-penicillin G and streptomycin were obtained from Gist-Brocades (The Netherlands). Phenol, tetra-sodium pyrophosphate, trichloroacetic acid, NaC1, KC1, Na2HPO4" 12H20, K2HPO4 and KH2PO4 were supplied b y Merck (Germany), as reagent-grade chemicals. NaI was supplied by Lamers and Indemans (The Netherlands), and 2-mercaptoethanol by Fluka (Switzerland). 5-Bromodeoxyuridine, 5-fluorodeoxyuridine,

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6-thioguanine and ethidium bromide were purchased from Sigma Chemical (U.S.A.), hydroxyurea and calf-thymus DNA (Na salt) from Nutritional Biochemicals, (U.S.A.). [3H]Thymidine and [14C]thymidine were obtained from The Radiochemical Centre, Amersham (Great Britain). Cell c u l t u r e

Monolayer cultures of cells from Chinese hamster ovary (CHO), kindly donated by Drs. R.E. Meyn and R.M. Humphrey, were grown in Ham's F10 medium, supplemented with 15% newborn calf serum (or with 15% foetal calf serum in experiments on the removal of UV-endonuclease-susceptible sites), 30 pM hypoxanthine, 1 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 pg/ml). The absence of mycoplasma contamination was periodically checked by the method of Peden (1975). Cells to be used for experiments were seeded in tissue-culture dishes and incubated at 37°C in a humidified 5% CO2 atmosphere. Radiation sources

Far-UV radiation, predominantly at 254 nm, was provided by a single 15-W, low-pressure mercury, germicidal lamp (Philips TUV lamp). The incident dose rate of the 254-nm radiation was 0.39 W - m -2 as determined by chemical actinometry according to the procedure of Parker and Hatchard (Calvert and Pitts, 1966); the value was corrected for the contribution of radiation of longer wavelengths on the basis of the relative intensities given by the manufacturer. Near-UV radiation was provided by two 20-W, "Sun-light" fluorescent lamps (Westinghouse FS20 "Sun Lamp") at a distance of 32 cm from the samples. In all experiments, near-UV radiation was filtered, either through the plastic lids of tissue-culture dishes, 6 cm diameter (Greiner; the lids were all from the same

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F i g . 1. E m i s s i o n s p e c t r a o f t h e f l u o r e s c e n t " S u n L a m p " ( W e s t i n g h o u s e F S 2 0 ) filtered e i t h e r b y the lid o f a tissue-culture dish (Greiner, 6 cm diameter) ( ) o r I C I M e l i n e x t y p e 4 4 2 f i l m ( 2 3 /~m t h i c k ) (..... ), and t r a n s m i s s i o n o f t h e filters u s e d ( . . . . . and ...... , resp.). T h e e m i s s i o n s p e c t r a w e r e c a l c u l a t e d f r o m t h e t r a n s m i s s i o n s p e c t r a m e a s u r e d w i t h a B e c k m a n A c t a III s p e c t r o p h o t o m e t e r , and t h e l a m p ' s u n f i l t e r e d s p e c t r u m as given b y t h e m a n u f a c t u r e r .

494 batch, transmission curves vary from batch to batch), to remove wavelengths below 290 nm, or through a sheet of polyethylene terephthalate film (Melinex, ICI, type 442, thickness 23 ttm) to remove wavelengths below 310 nm. Incident dose rates for the 2 types of radiation were 2.5 and 2.2 W • m -2, resp., as determined by a calibrated thermopile (Eppley Laboratories, U.S.A.) which was shielded against the infrared radiation emitted by the current limiter of the "Sun L a m p " . The resulting spectra are presented in Fig. 1. Spectra were calculated as the product of the filters' transmission spectra (also shown in Fig. 1) and the emission spectrum of the "Sun L a m p " (taken from the Westinghouse technical bulletin).

Assay of UV-endonuclease-susceptible sites (UV-endo sites) To label cellular DNA, about 2 × 10 s CHO cells were seeded on 6-cm tissueculture dishes (Greiner), grown in non-radioactive medium for 24 h and then in medium containing 0.25 pCi/ml [3H]TdR (17 Ci/mmole) for 24 h. Before irradiation the cells were washed free of medium with 2 ml of phosphate-buffered saline (PBS), consisting of 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.14 M NaC1 and 2.7 mM KC1, and then covered with 2 ml of this solution. Irradiations were from above and through the PBS layer. After irradiation, cells to receive further incubations were covered with fresh, non-radioactive medium and incubated at 37°C. At different times thereafter, up to 32 h, cells still in monolayer were rinsed twice with PBS and stored at --70°C before DNA isolation and quantitation of UV-endo sites. UV-endo sites were assayed by a procedure similar to that of Paterson et al. (1973) recently described in detail by Zelle and L o h m a n (1979). Briefly, DNA purified from CHO cell lysates by phenol extraction and dialysed against the endonuclease reaction buffer was divided into 2 fractions and incubated with or w i t h o u t UV endonucleases from Micrococcus luteus. The numbers of UVendo sites were calculated from the resulting DNA molecular weight distributions as determined by' sedimentation through calibrated alkaline sucrose gradients.

Photoreactivation of dimers in vitro Photoreactivating enzyme (PRE), purified from the actinomycete Streptomyces griseus, was the generous gift of Dr. A.P.M. Eker (Eker and FichtingerSchepman, 1975). DNA was extracted from either irradiated or unirradiated cells by a procedure identical with that followed for UV-endo site analysis up to the dialysis step. The aqueous layer containing the DNA obtained after phenol extraction was dialysed overnight against 0.01 M NaC1, 0.01 M KH2PO4/ K2HPO4 at pH 7.0 and 4 ° C. The reaction mixture contained 125 pl DNA solution, 7.5 pl tRNA (dissolved at 1 mg/ml), 100 pl PRE buffer (125 pl when PRE was n o t to be added) consisting of 0.01 M NaC1, 0.1% (w/v) bovine serum albumin, 5 mM 2-mercaptoethanol, 0.01 M KH2POJK2HPO4 at pH 7.0. The samples to which PRE was to be added received 25 pl of PRE solution made by diluting stock-PRE 1 : 12 in PRE buffer. Before this addition all samples were pre-warmed to 37°C. The samples to be incubated for 0 min were then put in the dark and in ice, and thereafter the enzyme was added to all samples meant to receive it. The sam-

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ples with longer incubation periods were incubated at 37°C, with or w i t h o u t PRE and with or w i t h o u t exposure to white fluorescent light, filtered through window glass 8 m m thick for 2.5--5 min. In the experiments with far-UV-irradiated DNA the PRE was added before the pre-warming period at 37°C, and exposure time to the white fluorescent light was shortened to 1 or 2.5 min. Reactions were stopped by putting the samples in the dark and in ice. DNA was re-extracted with phenol and processed as described for the determination of UV-endo sites.

Chromatographic analysis of thymine-containing dimers CHO cells were incubated for 3 days in medium containing [3H]TdR (1 pCi/ ml; 17 Ci/mmole). The cells were thoroughly washed with PBS and exposed to far-UV or to near-UV > 2 9 0 nm. DNA was isolated according to the procedure of the assay for UV-endo sites, and was precipitated by adding 1.5 ml of icecold 20% trichloroacetic acid (TCA) to 1 ml of DNA solution. The precipitate was centrifuged at 3 0 0 0 g and dried. Hydrolysis and chromatography were done as described by Carrier and Setlow (1971). The a m o u n t of thymine-containing dimers present in each sample was expressed as a percentage of the total detected [3H]activity on the chromatogram.

DNA repair replication 3 days before irradiation, about l 0 s cells were seeded onto 6-cm culture dishes (Greiner) in 3 ml F10 medium and incubated at 37 ° C. 2 h before irradiation, the medium was replaced by 3 ml fresh medium containing 5-bromodeoxyuridine (BUdR; 6.5 /aM) and 5-fluorodeoxyuridine (FUdR; 1 pM). Subsequently the cells were washed twice with PBS, and UV irradiations were done through a thin layer of PBS (2 ml/plate) covering the cells. The PBS was replaced by 3 ml medium containing 4 mM h y d r o x y u r e a , 6.5 pM BUdR, 1 pM FUdR and [3H]TdR (10 pCi/ml; 17 Ci/mmole). The cells were incubated in this medium for 3 h at 37°C. Then the cells were incubated for 1 h in the same medium but w i t h o u t the radioactive DNA precursor ("chase"). After completion of the whole procedure the medium was removed from the dishes and the cells were frozen at --70°C, until the DNA was isolated and analyzed by NaI density-gradient centrifugation by a modified m e t h o d of L o h m a n et al. (1973). In this m e t h o d 2 isopycnic bandings of DNA were performed on guidance of ethidium bromide present in the gradient, which forms a fluorescent complex with DNA. After the first run in NaI gradients the DNA band, having the normal buoyant density of Chinese hamster DNA, was collected and mixed with a known a m o u n t of 14C-labelled M. luteus DNA. This mixture was rebanded in a second run. (The M. luteus DNA, which has a higher density than the normal Chinese hamster DNA, served as an internal standard in the second NaI gradient for determining the absolute a m o u n t of DNA in each gradient.) After the second centrifugation the gradient was scanned directly in a fluorimeter for measuring the fluorescence of both the Chinese hamster and the M. luteus DNA. After scanning of the tube the gradient was fractioned into about 16 fractions of about 0.5 ml each, to which was added 0.5 ml of a solution containing calf-thymus DNA (200 pg/ml) and 2 ml 10% (w/v) TCA in

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0.01 M sodium pyrophosphate. The fractions were kept in ice for about 2.5 h and subsequently filtered through glass-fibre filters type GF/C (Whatman). The filters were washed with cold 5% (w/v) TCA and with cold ethanol and dried, and the radioactivity was measured as described by L o h m a n et al. (1973). The procedures of these authors were also followed for the expression of the results as disintegrations per minute per microgram of parental DNA.

Survival determinations Cells were trypsinized, counted in a h a e m o c y t o m e t e r , serially diluted and plated on 6-cm dishes. The dilutions were chosen so as to result in 100--300 clones per dish; for each experiment and dose 4 dishes were used. The dishes were allowed to stand for 4 h at 37 ° C to permit a t t a c h m e n t of the cells to the surface. The medium was then removed, the cells were rinsed twice with PBS and irradiated through a thin layer of PBS (2 ml/dish). After the irradiation, fresh m e d i u m was added and the cells were incubated for 7 days, after which the clones were scored. Mutation induction Mutation to 6-thioguanine (TG) resistance was detected as described by Van Zeeland and Simons (1976). After irradiation with either far-UV or near-UV (>290 nm or > 3 1 0 nm), cells were allowed expression times of 8 days before being replated in selective medium which contained TG (5 pg/ml). For each experiment and UV dose, ten 9 ~ m dishes were inoculated after the expression period, each with 10 s cells. Resistant colonies were counted after 7 days of growth in the selective medium. Simultaneously the plating efficiencies were determined by inoculating four 6-cm dishes, each with 250 cells, which were incubated in non-selective medium. Results

Induction of UV-endonuclease-susceptible sites Typical dose--response curves for the induction of UV-endonuclease-susceptible sites (UV-endo sites) by t h e 3 types of UV are presented in Fig. 2. For all 3 types the number of UV-induced sites increased linearly with the dose, i.e. with the exposure time. On the basis of incident energy, near-UV induced sites much less efficiently than 254-nm UV; induction of similar numbers of sites therefore required considerably longer exposure times, namely 15-fold longer for near-UV > 2 9 0 nm and 3800-fold longer for near-UV > 3 1 0 nm. For a proper comparison of the efficiencies with which the UV-endo sites are induced, the differences in exposure time have to be corrected for the differences in dose rate. The rates were 0.39, 2.5 and 2.2 W • m - : , resp., for 254, > 2 9 0 and > 3 1 0 nm. The incident energy dependence for the induction of UVendo sites in CHO cells calculated from the data a m o u n t e d to 0.026 sites per 106 mol. wt. of DNA induced per J . m -2 for 254-nm UV, 0.27 × 10 -3 for > 2 9 0 - n m UV and 1.2 × 10 .6 for > 3 1 0 - n m UV. Thus, per unit of incident energy, the far-UV source was 96-fold more efficient in the induction of UVendo sites than our near-UV > 2 9 0 - n m source and 22 000-fold more efficient than our near-UV > 3 1 0 - n m source.

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F i g . 2. I n d u c t i o n o f U V - e n d o s i t e s i n t h e D N A o f C H O c e l l s b y i r r a d i a t i o n w i t h far- a n d n e a r - U V . T h e U V d o s e is g i v e n i n a r b i t r a r y u n i t s , p r o p o r t i o n a l t o t h e d u r a t i o n o f t h e i r r a d i a t i o n , e , 2 5 4 - n m U V , 1 u n i t = 4 sec o f i r r a d i a t i o n ; D > 2 9 0 - n m U V , 1 u n i t = 1 r a i n ; o, > 3 1 0 - n m U V . 1 u n i t = 1 0 0 r a i n . E a c h p o i n t is t h e average of 2 or 3 independent determinations; vertical bars indicate either the standard deviation or the distance between 2 values.

UV-endonuclease substrate specificity For the interpretation of the results obtained with the M. luteus endonucleases it appeared of paramount importance to establish whether this m e t h o d really exclusively measures pyrimidine dimers or whether other lesions in UVirradiated DNA were also assayed. To investigate the substrate specificity of the M. lu teus UV-endonuclease the susceptibility of UV-endo sites to photoreactivation in vitro was examined, as an extension of our studies on sites induced by 254-nm UV in h u m a n cells (Paterson et al., 1973). CHO cells grown in the presence of [3H]TdR were exposed either to near- or to far-UV for durations calculated to induce comparable numbers of UV-endo sites. A far-UV exposure time of 12 sec, corresponding to 4.7 J • m -2, was used to induce about 0.12 UV-endo sites per 106 mol. wt. of DNA. A near-UV > 2 9 0 - n m exposure time of 180 sec and a near-UV > 310-nm exposure time of 800 min were used to induce a similar number of sites. DNA extracted from the irradiated cells was exposed to photoreactivating light in the presence of photoreactivating enzyme (PRE) purified from Streptomyces griseus (Eker and Fichtinger-Schepman, 1975), re-extracted with phenol and assayed for the presence of UV-endo sites. More than 90% of the UV-endo sites induced either by far-UV, near-UV > 2 9 0 nm or near-UV > 3 1 0 nm were photoreactivable (Fig. 3). In the 3 Expts. depicted in Fig. 3 the rate of photoreactivation appeared to be higher with the far-UV irradiated DNA (note the difference in the scales of the abscissa). This was because far-UV-irradiated DNA was pre-incubated with PRE, allowing the enzyme to bind to the dimers. In later experiments with near-UV-irradiated DNA this pre-incubation was o m i t t e d because it was feared t h a t pre-incubation might lead to a partial inactivation of PRE, although control experiments for comparing the 2 methods gave identical results. Incubation of the DNA with PRE in the dark, or exposure of the DNA to photoreactivating light in the absence of PRE had no effect on the number of UV-endo sites detected. Because cyclobutyl pyrimidine dimers are the only UV-induced lesions

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photoreoctivotion period (rain) Fig. 3. E n z y m i c p h o t o r e a c t i v a t i o n o f U V - e n d o sites in v i t r o . P h e n o l - e x t r a c t e d D N A was f r o m u n i r r a d i a t e d , f a x - U V - i r r a d i a t e d ( p a n e l A), n e a r - U V ~ 2 9 0 - n m - i r r a d i a t e d ( p a n e l B) o r n e a r - U V ) 3 1 0 - n m - i r r a d i a t e d ( p a n e l C) C H O cells. T h e D N A was t h e n i n c u b a t e d at 3 7 ° C w i t h or w i t h o u t p h o t o r e a c t i v a t i n g e n z y m e ( P R E ) a n d w i t h o r w i t h o u t e x p o s u r e to w h i t e f l u o r e s c e n t light ( P R L ) b e f o r e e x t r a c t i o n w i t h p h e n o l a n d U V - e n d o site d e t e r m i n a t i o n . U n i r r a d i a t e d cells, A D © ; i r r a d i a t e d cells, A m, o. + P R E , - - P R L ( ~ A ) . - - P R E , + P R L ( D N ) ; + P R E , + P R L ( o , o ) . U V doses w e r e 4.7 J - m - 2 , 4 7 0 J . m -2 a n d 106 k J . m - 2 , resp., f o r t h e 3 t y p e s o f r a d i a t i o n .

known to be susceptible to enzymic photoreactivation (Wulff and Rupert, 1962; Cook, 1967; Setlow, 1967; Patrick and Harm, 1973) these experiments indicate that most, if n o t all, UV-endo sites induced either by far-UV, near-UV > 2 9 0 nm or near-UV > 3 1 0 nm are equivalent to pyrimidine dimers in DNA. Close examination of Fig. 3 will reveal that small numbers of UV-endo sites were found even in the unirradiated DNA. UV-endo sites were found in unirradiated DNA only when radioactively 3H-labelled DNA was held at --70 ° C for at least several days before extraction and analysis for sites. This "non-specific" endonucleolytic activity was probably the result of 7-endonuclease activity present in crude extracts of M. luteus (Paterson and Setlow, 1972) which probably attacks lesions produced by radioisotopic decay in the 3H-labelled DNA during storage at --70°C (W.L. Carrier, personal communication). For the purpose of quantitating UV-endo sites, unirradiated samples were run with all experiments, and all other data were corrected for the numbers of "non-specific" sites detected. The photoreactivation experiments indicate that at least 90% of the sites detected by the UV-endonuclease preparation used are equivalent to pyrimidine dimers. The possibility remained, however, that the enzyme does n o t recognize all dimers, b u t only a fraction of them. Because, for our studies, it was essential to be certain that all pyrimidine dimers were detected, we checked this possibility by comparing the number of UV-endo sites induced in the D N A per unit energy with the number of dimers that could be detected chromatographically. For the chromatographic analysis, CHO cells were grown in the presence of [3H]TdR and were exposed either to far-UV at doses ranging from 29 to 59 J • m -2, or to near-UV > 2 9 0 nm at doses ranging from 2.6 to 6.8 kJ • m -2. DNA was isolated, hydrolysed and subjected to chromatography. The

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Fig. 4. I n d u c t i o n o f t h y m i n e - c o n t a i n i n g p h o t o p r o d u c t s , i d e n t i f i e d as p y r i m i d i n e d i m e r s b y c h r o m a t o g r a p h y , in t h e D N A o f C l i O cells b y i r r a d i a t i o n w i t h f a r - U V a n d n c a x - U V . T h e U V d o s e is given in arbit r a r y u n i t s , p r o p o r t i o n a l t o t h e d u r a t i o n o f t h e i r r a d i a t i o n , e , 2 5 4 - n m , 1 u n i t = 4 sec of r a d i a t i o n ; D, > 2 9 0 - r i m U V , 1 u n i t is 1 m i n . F o r e a c h dose of f a r - U V t h e results of 2 E x p t s . are given. F o r n e a r - U V e a c h p o i n t is t h e a v e r a g e o f 3 E x p t s . ( e r r o r bars r e p r e s e n t s t a n d a r d d e v i a t i o n s ) , w i t h t h e e x c e p t i o n of the result after 45 rain, which was d e t e r m i n e d once.

results were expressed as the fraction of the t h y m i n e molecules that were found to be incorporated into thymine-containing dimers. Fig. 4 shows the linear relationship between the incorporation of t h y m i n e in these photoproducts and the UV dose. The fraction of t h y m i n e molecules converted into thymine-containing dimers is 36 × 10 -6 per J • m -2 for far-UV and 0.27 X 10 -6 per J • m -2 for our source of near-UV > 2 9 0 nm. The ratio of the 3 types of dimer, namely t h y m i n e - - t h y m i n e (T--T), thym i n e - c y t o s i n e (T--C) and cytosine--cytosine (C--C), in the DNA of UV-irradiated mammalian cells differs from t h a t in the DNA of UV-irradiated bacteria. This might be due to the difference in substrate, naked DNA in bacteria and chromatin in mammalian cells, but also to differences in the GC/AT ratio ( R o t h m a n and Setlow, 1979). The ratio T--T/T--C/C--C in the DNA of HeLa cells is 60/26/14 after far-UV and 43.5/43.5/13 after near-UV {W.L. Carrier, personal communication). From these ratios it can be calculated that 1 thymine molecule f o u n d in a p h o t o p r o d u c t represents 0.685 dimer after far-UV and 0.766 dimer after near-UV. In mammalian DNA about 30% of the nucleotides is thymine, so 106 mol. wt. of DNA contains 976 thymines. On the basis of these data we calculate that, per 106 mol. wt. of DNA in mammalian cells, far-UV will induce 24 X 10 -3 pyrimidine dimers per J • m -2, and o u r near-UV > 2 9 0 nm 0.20 × 10 -3. These values compare very well with the experimentally determined numbers of UV-endo sites induced per 106 mol. wt., which were 26 × 10 -3 and 0.27 X 10 -3, resp. The conclusion appears warranted, therefore, that the UV-endo sites detected with our procedures are fully representative for the pyrimidine dimers present in DNA.

Post-irradiation disappearance of UV-endonuclease-sensitive sites CHO cells are capable of excision repair of pyrimidine dimers (Meyn et al., 1974). For the intended comparison of the biological effects of the 3 types of

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F i g . 5. D i s a p p e a r a n c e o f U V - e n d o s i t e s i n C H O c e l l s d u r i n g i n c u b a t i o n a f t e r i r r a d i a t i o n w i t h f a r - U V (o), n e a r - U V > 2 9 0 n m ( o ) o r n e a r - U V > 3 1 0 n m (o). F a r - U V d o s e s w e r e 1 . 2 ( p a n e l A ) a n d 2 . 9 ( p a n e l B) J - m - 2 , a n d n e a r - U V e x p o s u r e s w e r e c h o s e n so as t o r e s u l t i n t h e i n d u c t i o n o f c o m p a r a b l e n u m b e r s o f U V - e n d o s i t e s . N e a r - U V > 2 9 0 - n m d o s e s w e r e 9 6 a n d 2 4 0 J • m - 2 i n p a n e l s A a n d B, r e s p . , a n d t h e n e a r U V > 3 1 0 - r i m d o s e w a s 2 6 k J • m -2 ( p a n e l A ) . E a c h p o i n t is t h e a v e r a g e o f 3 i n d e p e n d e n t d e t e r m i n a t i o n s .

UV, it was o f imp or t a nc e t o know w h e t h e r dimers induced by far- or by nearUV are repaired with equal efficiency, or w het her differences in additionally induced lesions affect the repair system. This was investigated by examination of the decrease in the n u m b e r of UV-endo sites during a post-irradiation incubation o f the cells at 37 ° C. Experiments with far- and near-UV were p e r f o r m e d simultaneously, and doses were chosen so t hat comparisons could be made after the induction of similar numbers of UV-endo sites. F or these studies, 2 doses of UV were used, the lower one inducing a b o u t 0.03 sites per 106 mol. wt. of DNA, and the higher one 2.5 times as many. For 254-nm UV the doses were 1.2 and 2.9 J • m -2, resp., and for > 2 9 0 - n m UV 96 and 240 J . m -2. For > 3 1 0 - n m UV, however, only the lower dose could be given (26 kJ • m - : ) , because an irradiation inducing 0.075 sites per 106 mol. wt. o f DNA would have required an exposure o f a b o u t 8 h. The results are shown in Fig. 5A (lower doses) and Fig. 5B (higher doses). In all cases a t i m e - d e p e n d e n t r e d u c t i o n in the n u m b e r of detectable sites was f o u n d . The time course and the e x t e n t of this reduct i on were the same, irrespective o f w h e t h e r far- or near-UV was used, when a comparable n u m b e r of sites was induced. After the lower dose, 30% fewer sites were det ect ed 24 h after the irradiation, whereas after the higher dose a decrease of a b o u t 20% was observed over this period. The e x t e n t of dimer removal is in agreement with results obtained by Meyn et al. (1974) in the same cell line after 254-nm UV irradiation.

DNA-repair replication As a second criterion t hat could be used to d e t e c t possible differences in repair o f lesions induced by either far- or near-UV, DNA-repair replication was

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measured. For these determinations the incorporation of [3H]TdR into nonreplicating DNA during the first 3 h after irradiation was assayed, by a m e t h o d with NaI density-gradient centrifugation. Various UV doses were given that ranged between 0 and 20 J • m-2 of far-UV and 0 and 2.7 kJ • m -: of near-UV > 2 9 0 nm; in both cases the numbers of UV-endo sites induced per 104 mol. wt. of DNA ranged between 0 and 0.5. Irradiations with near-UV > 3 1 0 nm were not included in these experiments as the exposure times would have had to be extremely long. The results of duplicate experiments are shown in Fig. 6. From this we can conclude that there was no clear difference in repair replication in CHO cells irradiated with either far- or near-UV > 2 9 0 nm.

Cytotoxicity of far- and near-UV radiations To determine the relative cytotoxicities of far- and near-UV the colonyforming ability of CHO cells was examined after various radiation exposures. Fig. 7 shows the results for far-UV and near-UV > 2 9 0 nm. In Fig. 7A these results are presented as functions of the incident energies. Clearly, per unit of incident energy, far-UV is much more cytotoxic. Although n o t visible, owing to the scale used, the far-UV survival curve, too, is characterized by a well-defined shoulder region at low doses and by an exponential decline at higher doses; when plotted on appropriate scales, the 2 curves are rather similar in shape (as can be judged from Fig. 7B). For both curves, a D37 as well as a Do can be given. For far-UV the D37 (the dose killing 63% of the cells) a m o u n t e d to 10 J • m-2; the Do (the additional dose required to lower the survival by 63%

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Fig. 7. S u r v i v a l o f C H O cells i r r a d i a t e d w i t h f a r - U V (o) or n e a r - U V > 2 9 0 n m ( a ) , e x p r e s s e d as f u n c t i o n o f U V d o s e ( p a n e l A ) o r o f n u m b e r o f U V - e n d o sites i n d u c e d ( p a n e l B). E a c h p o i n t is t h e a v e r a g e o f 3 or more Expts. F i g . 8. S u r v i v a l o f C H O cells i r r a d i a t e d w i t h f a r - U V (o), n e a r - U V > 2 9 0 nm (~) or n e a r - U V > 3 1 0 a m (©), e x p r e s s e d as f u n c t i o n o f n u m b e r o f U V - e n d o s i t e s i n d u c e d . E a c h p o i n t is t h e a v e r a g e o f 3 or m o r e e x p t s .

in the exponential section of the curve) was 2.4 J • m -2. The values for near-UV > 2 9 0 nm were 700 and 130 J • m -2, resp. In Fig. 7B the colony-forming abilities of far-UV and near-UV >290-nm-irradiated cells are replotted as functions of the numbers of UV-endo sites induced. When compared in this way, CHO cells appear more sensitive to near-UV >290nm radiation than to far-UV. Expressed in UV-endo sites per 106 mol. wt. of DNA the D37 and Do values are, for far-UV 0.26 and 0.062, for near-UV > 2 9 0 nm 0.19 and 0.035, resp. Survival after irradiation with > 310-nm UV was also examined. For practical reasons, irradiation periods had to be limited to maximally 200 min, so only the survival range between 100 and 70% could be studied. By extrapolation, the D37 was calculated to be as high as 46 kJ • m -2. The results did not allow the determination of Do. Fig. 8 shows the relation between survival and the number of UV-endo sites measured in these preparations. For comparison the corresponding parts of the curves in Fig. 7B have been included. It is obvious that the > 3 1 0 - n m UV -- although less lethal per unit dose of energy -- was by far the most harmful on the basis of cell killing per induced pyrimidine dimer. When expressed in this manner the extrapolated D37 value is 0.055 UV-endo sites per 106 mol. wt. of DNA. Because the experiments with > 3 1 0 - n m UV required irradiation periods up to more than 3 h, as compared with 1 or 10 min for the other 2 UV sources, the possible occurrence of artifacts had to be considered. For that reason a number of control experiments was performed. With each survival experiment it was checked whether 200-min incubation with PBS itself influenced the plating efficiency of the cells in comparison with an incubation of 10 min or

503

less: the plating efficiencies were equal and ranged from 70 to 90% among the various experiments. It was also checked whether the combination of irradiation and long incubation in PBS influenced survival, for example by having an effect on the cell cycle. To this end, cells were irradiated with a dose of >290nm UV that under normal conditions resulted in 24% survival. For this dose a 6.5-min irradiation period was required. Various set-ups were tested: after irradiation, cells were immediately incubated with medium, or t h e y were incubated with PBS for 150 min and then with medium; t h e y were first incubated with PBS for 150 min and then irradiated, or they were given half of the UV doses before a 150-min incubation with PBS and the second half thereafter. In all cases the s~rne survival was obtained.

Mutation induction by far- and near-UV radiations To examine the relative mutagenicities of far- and near-UV radiations, the abilities of these radiations to induce resistance to 6-thioguanine (TG) in CHO cells were studied. Normal CHO cells are sensitive to TG, and resistance arises through forward mutations in the structural gene for hypoxanthine--guanine phosphoribosyltransferase (HPRTase) (Van Zeeland and Simons, 1976; O'Neill et al., 1977). To allow complete expression of the mutations, our procedure included an expression time of 8 days, as suggested by Van Zeeland and Simons {1976), before the cells were incubated in medium with TG. The numbers of TG-resistant clones were corrected for the corresponding plating efficiencies. In Fig. 9A the induced m u t a t i o n frequencies have been plotted as functions

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of the total incident energies. All 3 radiation sources induced mutations in proportion to dose, the slopes of the dose--response curves corresponding to 3.3, 0 . 0 4 2 and 0.95 X 10 -3 mutations per 10 s surviving cells per J • m -2 for farUV, near-UV > 2 9 0 nm and near-UV > 3 1 0 nm, resp. Thus, as was found for the loss of colony-forming ability, per unit o f incident energy far-UV is much more effective than near-UV in the induction of mutations. In Fig. 9B the mutation frequencies have been replotted as functions of the number of UV-endo sites induced. The numbers o f mutations per 10 s surviving

TABLE 1 COMPARISON SOURCES

OF T H E B I O C H E M I C A L AND B I O L O G I C A L E F F E C T S IN CHO CELLS OF 3 UV

Property

(unit)

254-nm UV

>290-rim UV a

>310-rim UV a

Essb dimers c D37 D37 DO DO IMF e IMF

(perJ.m -2) (per J - m - 2 ) (J • m - 2 ) (ESS) (J • m -2) (ESS) (per J • m -2) ( p e r ESS)

26 X 10 -3 24 X 10 -3 10 0.26 2.4 0.062 3.3 130

0 . 2 7 X 10 -3 0 . 2 0 × 10 -3 700 0.19 130 0.035 0.042 150

1.2 X 10 -6 n.d. d 46 × 103 0.055 n.d. n.d. 0 . 9 5 × 10 -3 790

a

b

c d e

T h e i n d i c a t i o n s ~ 2 9 0 - n m U V a n d ~ 3 1 0 - n m U V r e f e r t o t h e U V s o u r c e s u s e d in t h e s e studies. A cons i d e r a b l e f r a c t i o n o f t h e e n e r g y w a s e m i t t e d a t w a v e l e n g t h s well a b o v e t h e l o w e r limits; p r o b a b l y t h e s e w a v e l e n g t h s did n o t c o n t r i b u t e s i g n i f i c a n t l y t o t h e i n d u c t i o n o f p y r i m i d i n e d i m e r s a n d - - possibly - - of s o m e other p h o t o p r o d u c t s . Consequently, the ratios b e t w e e n the effects observed with near-UV and the d o s e o f i n c i d e n t e n e r g y h a v e t o b e c o n s i d e r e d w i t h c a u t i o n . T h e v a l u e s given are valid f o r t h e i r r a d i a t i o n c o n d i t i o n s d e s c r i b e d in this p a p e r ; o t h e r U V s o u r c e s m a y y i e l d d i f f e r e n t ratios. ESS, U V - e n d o sites i n d u c e d p e r 105 m o l . w t . o f D N A . I n d u c e d p e r 105 tool. w t , o f D N A ( c l ~ o m a t o g r a p h i c i d e n t i f i c a t i o n ) . n.d., n o t d e t e r m i n e d . I M F , i n d u c e d m u t a t i o n f r e q u e n c y , i.e. n u m b e r o f T G - r e s i s t a n t m u t a n t s i n d u c e d p e r 105 survivors.

505 cells expected to occur when 1 site per 106 mol. wt. of D N A is induced, are 130, 150 and 800 for far-UV, near-UV > 2 9 0 nm and near-UV > 3 1 0 nm, resp. Per UV-endo site induced, near-UV > 2 9 0 nm is at least as mutagenic as far-UV, whereas near-UV > 3 1 0 nm is markedly more mutagenic. The survival and mutation induction results of Figs. 7, 8 and 9 are combined in Fig. 10, where the number of induced mutants is plotted as function of survival. From this graph it is clear that no distinct differences exist in the relations between mutagenicity and c y t o t o x i c i t y of the 3 sources of UV.

Survey of the results A compilation of the various effects determined for the 3 types of UV is given in Table 1. Discussion We have investigated the induction in DNA of CHO cells of the predominant UV lesion, the cyclobutyl pyrimidine dimer, by 3 different UV sources, namely the c o m m o n l y used 254-nm far-UV and 2 sources of near-UV. The near-UV was a continuous spectrum radiation in the UV-wavelength region up to a b o u t 370 nm, with lower cut-off limits at 290 and 310 nm, resp. All 3 types of radiation induced M. luteus UV-endonuclease-sensitive sites in direct proportionality to the dose. These sites could be removed from the DNA for more than 90% by treatment with a purified photoreactivating enzyme in vitro. This strongly suggests that the sites recognized by the M. luteus extract are the pyrimidine dimers induced b y UV. However, the specificity of photolyases or photoreactivating enzymes towards the monomerization of cyclobutyl pyrimidine dimers is not definitively proven; so far no other DNA p h o t o p r o d u c t s have been shown to be enzymically photoreactivable. (For a review, see Setlow, 1967.) On the other hand, supporting evidence that UV-endo sites are equivalent to pyrimidine dimers has n o w been obtained by our comparison with the chromatographically determined numbers of dimers (see also Setlow et al., 1975). We feel rather confident, therefore, that the UV-endo sites measured in our experiments represent true pyrimidine dimers. The remaining of a small fraction of the induced UV-endo sites after several minutes of treatment with photoreactivating light in the presence of DNA-photolyase is in agreement with results of Cook and Worthy (1972) and Childs et al. (1978), that a b o u t 10% of the initially induced pyrimidine dimers (measured chromatographically or as T4-UV-endo sites, resp.) could n o t be photoreactivated. Dimer induction has been reported for wavelengths up to 365 nm (Tyrell, 1973; Webb et al., 1976), b u t as in these experiments broad-spectrum UV sources were used from which the 365-nm radiation was selected by use of a m o n o c h r o m a t o r and filters that cut off below 310 nm, it might be possible that the dimers measured were induced by wavelengths just above the cut-off wavelength of 310 nm. Nevertheless, these results agree with our finding that dimers can be induced by wavelengths above 310 nm. The enzymic dimer assay has the great advantage that it is sufficiently sensitive to allow the determination of dimers induced by non-lethal doses of UV,

506 and even to follow their post-irradiation removal, whereas chromatographic methods of dimer analysis after an acidic hydrolysis of DNA are rather insensitive and need higher doses. We were able to study the removal of dimers in vivo after doses t h a t would allow more than 70% of the cells to survive. It is known that UV induces a variety of lesions in addition to the dimers, of which have been identified cytosine hydrate, t h y m i n e - - t h y m i n e adducts and their dehydration product (Murphy, 1975), single-strand breaks, alkali-labile bonds (Elkind and Han, 1978), the spore-photoproduct 5-thyminyl-5,6-dihydrothymine (Varghese, 1970), and DNA--protein cross-links (Smith, 1962). Furthermore, DNA of cells irradiated with near-UV during incubation in medium can be damaged by p h o t o p r o d u c t s from components of the medium, e.g. t r y p t o p h a n , riboflavine and tyrosine (Nixon and Wang, 1977). In our experiments, we tried to prevent these damages by thoroughly washing the cells with, and irradiation while incubated in, phosphate-buffered saline. But the other photoproducts mentioned above will also have occurred in our preparation. It appears likely that most of these p h o t o p r o d u c t s have different action spectra (e.g., see Cerutti and Natrawali, 1980). It is to be expected that the ratios between the various products formed, including pyrimidine dimers, vary with the wavelength of the UV with which the cells are irradiated. Consequently, for each of the 3 types of UV studied, a differently composed set of lesions will be induced. It would not have been surprising, therefore, if this had affected the repair processes in such a manner that the rate of dimer removal had been found to depend on the wavelength of irradiation. However, the time courses for the disappearance of dimer sites in far- or near-UV irradiated cells were indistinguishable from each other when comparable numbers of sites had been induced. Evidently, the other UV-lesions do n o t interfere with the removal of dimers. As a second approach to study the repair of lesions induced in the DNA by the various UV sources, we determined DNA-repair replication. This technique measures the incorporation of DNA precursors in parental DNA as the result of excision-repair processes during the 3-h post-irradiation incubation period studied. The a m o u n t of repair replication depends both on the number of excised photoproducts and on the size of the repair patches. Lesions involved in repair processes t h a t give short patches, e.g. single-strand breaks, will not be detected by this assay unless they form a substantial part of the total population of lesions being repaired. When repair replication was used as the criterion, no difference was observed in the response of CHO cells whether irradiated by far-UV or near-UV > 2 9 0 nm if the same number of dimers was initially induced. So no evidence could be obtained for non-dimer lesions being involved in repair pathways, although it must be assumed t h a t these lesions are induced at different ratios to dimers by UV of different wavelengths. The occurrence of repair of these lesions by processes n o t involving resynthesis of DNA is, of course, not excluded by this result. Neither of the biochemical assays used revealed any difference in excision repair of the DNA of CHO cells damaged by irradiation with either far- or nearUV, when comparable a m o u n t s of dimers were induced. With respect to the biological effects of the radiations used we compared their c y t o t o x i c i t y and mutagenicity. Survival studies with mammalian cells in culture after irradiation with UV of various wavelengths have been reported

507

(Todd et al., 1968; Hsie et al., 1977; Elkind et al., 1978; Jacobson et al., 1978}, b u t comparisons were made only on the basis of UV dose and the shape of the survival curves. In most cases, similarities between the survival curves were observed, b u t Elkind et al. {1978) noticed differences when they compared the effects of germicidal and sun lamps. We used the number of UV-endo sites induced b y far-UV, near-UV > 2 9 0 nm and near-UV > 3 1 0 nm, rather than the dose of incident energy, to compare the biological effects of these types of radiation. In our opinion, comparison on the basis of energy was less interesting because our near-UV sources emit a continuous spectrum with wavelength regions that give a significant contribution to the emitted energy b u t that are of no importance, presumably, for the induction of specific biological effects. By relating the effects to one well-known and well identifiable lesion, the pyrimidine dimer, we expected to obtain information on the relative importance of the dimer for the biological consequences of the irradiation and on the possible contribution of other damage. This approach appeared to be fruitful. Clear differences were observed in the c y t o t o x i c i t y , lethality per dimer being lowest after far-UV and highest after near-UV > 3 1 0 nm. The conclusion must be that dimers are n o t the only lethal lesions induced b y near-UV radiation. This is in accord with the observations of Elkind et al. (1978). The mutagenic effects of the 3 types of radiation varied greatly with the dose, 254-nm being far more mutagenic per unit of incident energy than near-UV. But when the mutation frequency was compared on the basis of an equal number of induced dimers, far-UV showed the lowest mutagenic effect and > 3 1 0 nm UV the strongest, the same order as with the cytotoxicity. Per induced dimer, near-UV > 3 1 0 nm was 5--6 times more mutagenic than the other 2 radiations. This stresses the probability that lesions other than pyvimidine dimers play a significant role in the biological effects of near-ultraviolet radiations. When mutation frequency is expressed as a function of survival, the 3 types of radiation are comparable, indicating that the lesions that contribute to the lethality are equally important for the induction of mutations. Our results demonstrate that wavelength regions of UV that are emitted by our natural source of UV, the sun, b u t also b y the various types of solaria presently enjoying a still increasing popularity, induce damage in cellular DNA that is both c y t o t o x i c and mutagenic. Although relatively high doses of these radiations are needed for the induction of a substantial a m o u n t of damage, the effects should n o t be neglected in view of their rather widespread occurrence. Little is k n o w n a b o u t the specific nature of the harmful DNA lesions caused by UV of long wavelengths. They may or m a y n o t be identical with {some of} the lesions induced, next to the pyrimidine dimer, by far-UV. For the understanding of the biological effects of the UV sources to which humans are normally exposed, and especially for the understanding of the relationship between DNA damage and the clinical s y m p t o m s observed in patients with xeroderma pigmentosum, it is necessary to use near-UV sources in experiments with human cells. Our data indicate that with far-UV the possibly also induced "near-UV lesions" are totally overshadowed with regard to their deleterious biological effects by the much more effectively induced pyrimidine dimers.

508

Acknowledgements This work was supported by Euratom grant contract EUR 200-76 BIO N. We thank Drs. F. Berends and D. Bootsma for reviewing the manuscript. References Calvert, J.G., and J.N. Pitts (1966) Photochemistry, Wiley, New York, pp. 783--786. C a r r i e r , W . L . , a n d R . B . S e t l o w ( 1 9 7 1 ) T h e e x c i s i o n o f p y r i m i d i n e d i m e r s ( T h e d e t e c t i o n o f d i m e r s in s m a l l a m o u n t s ) , in: S.P. C o l o w i c k a n d N . O . K a p l a n ( E d s . ) , M e t h o d s in E n z y m o l o g y , Vol. X X I , A c a d e m i c Press, N e w Y o r k , p p . 2 3 0 - - 2 3 7 . C e r u t t i , P . A . , a n d M. N e t r a w a l i ( 1 9 8 0 ) F o r m a t i o n a n d r e p a i r o f D N A d a m a g e i n d u c e d b y i n d i r e c t a c t i o n o f u l t r a v i o l e t l i g h t in n o r m a l a n d x e r o d e r m a p i g m e n t o s u m s k i n f i b r o b l a s t s , P r o c . 6 t h I n t . C o n g r e s s , Tokyo. C h i l d s , J . D . , M.C. P a t e r s o n , B.P. S m i t h a n d N . E . G e n t n e r ( 1 9 7 8 ) E v i d e n c e f o r a n e a r - U V - i n d u c e d p h o t o p r o d u c t o f 5 - h y d r o x y m e t h y l c y t o s i n e in b a c t e r i o p h a g e T 4 t h a t c a n b e r e c o g n i z e d b y e n d o n u c l e a s e V, Mol. G e n . G e n e t . , 1 6 7 , 1 0 5 - - 1 1 2 . C o o h i U , T . P . , S.P. M o o r e a n d S. D r a k e ( 1 9 7 7 ) T h e w a v e l e n g t h d e p e n d e n c e o f u l t r a v i o l e t i n a c t i v a t i o n o f h o s t c a p a c i t y in a m a m m a l i a n cell-virus s y s t e m , P h o t o c h e m . P h o t o b i o l . , 2 6 , 3 8 7 - - 3 9 1 . C o o k , J . S . ( 1 9 6 7 ) D i r e c t d e m o n s t r a t i o n o f t h e m o n o m e r i z a t i o n o f t h y m i n e - c o n t a i n i n g d i m e r s in U V - i r r a d i a t e d D N A b y y e a s t p h o t o r e a c t i v a t i n g e n z y m e a n d l i g h t , P h o t o c h e m . P h o t o b i o l . , 6, 9 7 - - 1 0 1 . Cook, J.S., and T.E. Worthy (1972) Deoxyribonucleic acid photoreactivating enzyme, A quantitative chemical assay and estimation of molecular weight of one yeast enzyme, Biochemistry, 11,388--393. Eker, A.P.M., and A.M.J. Fichtinger-Schepman (1975) Studies on a DNA photoreactivating enzyme from S t r e p t o m y c e s griseus, II. P u r i f i c a t i o n o f t h e e n z y m e , B i o c h i m . B i o p h y s . A c t a , 3 7 8 , 5 4 - - 6 3 . E l k i n d , M.M., a n d A. H a n ( 1 9 7 8 ) D N A - s i n g l e - s t r a n d l e s i o n s d u e t o " s u n l i g h t " a n d U V light: a c o m p a r i s o n o f t h e i r i n d u c t i o n in C h i n e s e h a m s t e r cells a n d h u m a n cells, a n d t h e i r f a t e in C h i n e s e h a m s t e r cells, Photochem. Photobiol., 27,717--724. E l k i n d , M.M., A. H a n a n d C.M. C h a n g - L i u ( 1 9 7 8 ) " S u n l i g h t " - i n d u c e d m a m m a l i a n cell killing: a c o m p a r a tive s t u d y o f u l t r a v i o l e t a n d n e a r - u l t r a v i o l e t i n a c t i v a t i o n , P h o t o c h e m . P h o t o b i o l . , 2 7 , 7 0 9 - - 7 1 5 . Hsie, A . W . , A . P . Li a n d R . M a c h a n o f f ( 1 9 7 7 ) A f l u e n c e r e s p o n s e s t u d y o f l e t h a l i t y a n d m u t a g e n i c i t y o f w h i t e , b l a c k a n d b l u e f l u o r e s c e n t light, s u n l a m p a n d s u n l i g h t i r r a d i a t i o n i n C h i n e s e h a m s t e r o v a r y cells, M u t a t i o n R e s . , 4 5 , 3 3 3 - - 3 4 2 . J a c o b s o n , E . D . , K. Krell, M . J . D e m p s e y , M . H . L u g o , O. E l l i n g s o n a n d C.W. H e n c h II ( 1 9 7 8 ) T o x i c i t y a n d m u t a g e n i c i t y o f r a d i a t i o n f r o m f l u o r e s c e n t l a m p s a n d a s u n l a m p in L S 1 7 8 Y m o u s e l y m p h o m a cells, M u t a t i o n Res. 5 1 , 6 1 - - 7 5 . L o h m a n , P . H . M . , W . J . Kleijer, M . L . S l u y t e r a n d I . A . A . M a t t h i j s ( 1 9 7 3 ) R e p a i r r e p l i c a t i o n in h u m a n cells s t u d i e d b y s o d i u m i o d i d e i s o p y e n i c c e n t r i f u g a t i o n o f D N A in a f i x e d a n g l e r o t o r , A n a l . B i o c h e m . , 54, 178--187. M e y n , R . E . , D . L . V i z a r d , R . R . H e w i t t a n d R . M . H u m p h r e y ( 1 9 7 4 ) F a t e o f p y r i m i d i n e d i m e r s in t h e D N A o f u l t r a v i o l e t - i r r a d i a t e d C h i n e s e h a m s t e r cells, P h o t o c h e m . P h o t o b i o l . , 2 0 , 2 2 1 - - 2 2 6 . M u r p h y , T.M. ( 1 9 7 5 ) N u c l e i c a c i d s : i n t e r a c t i o n w i t h s o l a r U V r a d i a t i o n , C u r r . T o p . R a d i a t . R e s . , 1 0 , 199--228. N i x o n , B . T . , a n d R . J . W a n g ( 1 9 7 7 ) F o r m a t i o n o f p h o t o p r o d u e t s l e t h a l f o r h u m a n cells in c u l t u r e b y d a y light fluorescent light and bilirubin light, Photochem. Photobiol., 26,589--593. O ' N e i l l , P., P . A . B r i m e r , R . M a c h a n o f f , G.P. H i r s c h a n d A . W . Hsie ( 1 9 7 7 ) A q u a n t i t a t i v e a s s a y o f m u t a t i o n i n d u c t i o n a t t h e h y p o x a n t h i n e - - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e l o c u s in C h i n e s e h a m s t e r o v a r y cells ( C H O / H G P R T s y s t e m ) : d e v e l o p m e n t a n d d e f i n i t i o n o f t h e s y s t e m , M u t a t i o n R e s . , 4 5 , 9 1 - 101. P a r r i s h , J . A . , R . R o x A n d e r s o n , F. U r b a c h a n d D. P i t t s ( 1 9 7 8 ) S o u r c e s o f U V - A , in: U V - A , B i o l o g i c a l E f f e c t s o f U l t r a v i o l e t R a d i a t i o n w i t h E m p h a s i s o n H u m a n R e s p o n s e s t o L o n g w a v e U l t r a v i o l e t , Plenum, New York, pp. 7--36. Paterson, M.C., and R.B. Setlow (1972) Endonucleolytic activity from Micrococcus luteus that acts on v - r a y i n d u c e d d a m a g e in p l a s m i d D N A o f Escherichia coli m i n i c e i l s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 69, 2927--2931. P a t e r s o n , M.C., P . H . M . L o h m a n a n d M . L . S l u y t e r ( 1 9 7 3 ) Use o f a n U V - e n d o n u c l e a s e f r o m M i c r o c o c c u s l u t e u s t o m o n i t o r t h e p r o g r e s s o f D N A r e p a i r in U V - i r r a d i a t e d h u m a n cells, M u t a t i o n Res., 19, 2 4 5 - 256. P a t r i c k , M . H . , a n d H. H a r m ( 1 9 7 3 ) S u b s t r a t e s p e c i f i c i t y o f a b a c t e r i a l U V - e n d o n u c l e a s e a n d t h e o v e r l a p w i t h in v i t r o p h o t o e n z y m a t i c r e p a i r , P h o t o c h e m . P h o t o b i o l . , 1 8 , 3 7 1 - - 3 8 6 . P e d e n , K.W.C. ( 1 9 7 5 ) A r a p i d a n d s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m y c o p l a s m a a n d o t h e r i n t r a c e l l u l a r contaminants, Experientia, 31, 1111--1112.

509 R e y n o l d s , R . J . , a n d P . H . M . L o h m a n ( 1 9 7 8 ) M i c r o c o c c u s l u t e u s U V - e n d o n u c l e a s e - s e n s i t i v e sites i n far- a n d n e a r - U V - i r r a d i a t e d C h i n e s e h a m s t e r o v a r y cells, in: P.C. H a n a w a l t , E.C. F r i e d b e r g a n d C . F . F o x ( E d s . ) , D N A R e p a i r M e c h a n i s m s , A c a d e m i c Press, N e w Y o r k , p p . 2 7 - - 3 0 . R o t h m a n , R . H . , a n d R . B . S e t l o w ( 1 9 7 9 ) A n a c t i o n s p e c t r u m f o r cell killing a n d p y r i m i d i n e d i m e r f o r m a t i o n in C h i n e s e h a m s t e r V - 7 9 cells, P h o t o c h e m . P h o t o b i o l . , 2 0 , 5 7 - - 6 1 . S e t l o w , J . K . ( 1 9 6 7 ) T h e e f f e c t s o f u l t r a v i o l e t r a d i a t i o n a n d p h o t o r e a c t i v a t i o n , in: M. F l o r k i n a n d E . H . S t o t z ( E d s . ) , C o m p r e h e n s i v e B i o c h e m i s t r y , V o l . 2 7 , Elsevier, A m s t e r d a m , p p . 1 5 7 - - 2 0 9 . S e t l o w , R . B . ( 1 9 6 8 ) P h o t o p r o d u c t s in D N A i r r a d i a t e d i n vivo, P h o t o c h e m . P h o t o b i o l . , 7 , 6 4 3 - - 6 4 9 . S e t l o w , R . B . , W.L. C a r r i e r a n d J. S t e w a r t ( 1 9 7 5 ) E n d o n u c l e a s e sensitive sites in U V - i r r a d i a t e d D N A , Biophys. J., 15,194a. Smith, K.C. (1962) Dose-dependent decrease in extractibility of deoxyribonucleic acid (DNA) from bact e r i a f o U o w i n g i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t o r w i t h visible l i g h t p l u s d y e , B i o e h e m . B i o p h y s . Res. Commun., 8,157--163. T o d d , P., T.P. C o o h i l l a n d J . A . M a h o n e y ( 1 9 6 8 ) R e s p o n s e s o f c u l t u r e d C h i n e s e h a m s t e r cells t o u l t r a v i o l e t light of different wavelengths, Radiation Res., 35, 390--400. T y r e l l , R . M . ( 1 9 7 3 ) I n d u c t i o n o f p y r i m i d i n e d i m e r s in b a c t e r i a l D N A b y 3 6 5 n m r a d i a t i o n , P h o t o c h e m . Photobiol., 17,69--73. V a r g h e s e , A . J . ( 1 9 7 0 ) 5 - T h y m i n y l - 5 , 6 - d i h y d r o t h y m i n e f r o m D N A i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t , Bioc h e m . B i o p h y s . Res. C o m m u n . , 3 8 , 4 8 4 - - 4 9 0 . W e b b , R . B . , M.S. B r o w n a n d R . M . T y r e l l ( 1 9 7 6 ) L e t h a l e f f e c t s o f p y r i m i d i n e d i m e r s i n d u c e d a t 3 6 5 n m in s t r a i n s o f E. coli d i f f e r i n g i n r e p a i r c a p a b i l i t y , M u t a t i o n R e s . , 3 7 , 1 6 3 - - 1 7 2 . W u l f f , D . L . , a n d C.S. R u p e r t ( 1 9 6 2 ) D i s a p p e a r a n c e o f t h y m i n e p h o t o d i m e r in u l t r a v i o l e t i r r a d i a t e d D N A u p o n t r e a t m e n t w i t h a p h o t o r e a e t i v a t i n g e n z y m e f r o m b a k e r ' s y e a s t , B i o c h e m . B i o p h y s . Res. C o m mun., 7,237--240. Zeeland, A.A. van, and J.W.I.M. Simons (1976) Linear dose--response relationships after prolonged e x p r e s s i o n t i m e s in V 7 9 C h i n e s e h a m s t e r cells, M u t a t i o n R e s . , 3 5 , 1 2 9 - - 1 3 8 . Zelle, B., a n d P . H . M . L o h m a n ( 1 9 7 9 ) R e p a i r o f U V - e n d o n u c l e a s e - s u s c e p t i b l e sites in t h e 7 c o m p l e m e n t a tion groups of xeroderma pigmentosum A through G, Mutation Res., 62, 363--368.