377
Mutation Research, 43 (1977) 377--385 © Elsevier/North-Holland Biomedical Press
CHROMOSOME DAMAGE IN CHINESE HAMSTER CELLS SENSITIZED TO N E A R - U L T R A V I O L E T LIGHT BY P S O R A L E N AND ANGELICIN
M.J. ASHWOOD-SMITH ,1, E.L. GRANT 1 J.A. HEDDLE **2 and G.B. FRIEDMAN 3
1 Departments of Biology and 3 Physics, University of Victoria, Victoria, B.C. (Canada V8W 2Y2) and the 2 Department of Biology, York University, Downsview, Ontario (Canada M3J 1P3) (Received November 1st, 1976) (Revision received January 7th, 1977) (Accepted January 13th, 1976)
Summary The clastogenic effect of furocoumarins psoralen and angelicin in the presence of near-UV (320--380 nm) differs greatly, as do their modes of interaction with DNA. Psoralen, which requires only one-fifth as much light energy to produce the same lethal effect as angelicin at equimolar concentrations, is able to cross-link DNA whereas angelicin cannot. The frequency of micronuclei which arise from chromosomal fragments shows the same differential effect as lethality. Indeed aberrations account for much or all of the lethality observed. Metaphase analysis at comparable aberration frequencies revealed that angelicin and psoralen both induce chromatid deletions and a wide spectrum of chromatid exchanges. These data show that both cross-links and monoadducts to the DNA can result in chromosomal aberrations. The relative contributions of cross-links and monoadducts to chromosomal aberrations still remain to be determined. It it n o t e w o r t h y that extensive chromosomal damage is induced in mammalian cells b y the combination of psoralen and near-UV, a treatment which is currently widely used in the therapy of psoriasis.
Introduction Furocoumarins or psoralens (Fig. 1) sensitize bacteria, viruses, and eucaryotic cells to near-ultraviolet light (320--380 nm, near-UV) by covalently binding to pyrimidine bases as m o n o a d d u c t s and producing DNA cross-links through diadducts [2,3,5]. The resulting lethal and mutagenic effects have been sum* Supported by t h e ** Supported by t h e
National Research Council of National Cancer Institute.
Canada.
378 marized and a tentative mechanism of action proposed in which the DNA-psoralen monoadducts are significant not only as pre-requisites for cross-links but also as unique lethal and mutagenic lesions [2]. Considering the clinical use of psoralen plus light therapy in treatment of vitiligo [6] and, more recently, psoriasis [12,15] it is surprising that there exists only a single report of chromosome aberrations produced in mammalian cells by photo-sensitization with furocoumarins [13]. Peripheral blood lymphocytes from patients with Fanconi's anemia and normal adults were tested for susceptibility to chromosome breakage by the traditional method of examining mitotic chromosomes at metaphase [131. In comparison to this traditional method of scoring chromosome aberrations, the micronucleus test is evolving as a simple, effective and rapid means of screening chemicals for mutagenic action [9,14]. After telophase, micronuclei form in the cytoplasm from acentric fragments that were left behind at anaphase as the centric elements moved towards the spindle poles. Since the crosslinking agent mitomycin C produces micronuclei [11], and since mitomycin C and 8-methoxypsoralen cause chromosome aberrations in mammalian cells [13], psoralen plus near-UV treatment was expected to produce micronuclei.
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Fig. 1. M o l e c u l a r s t r u c t u r e of the r e p r e s e n t a t i v e f u r o c o u m a r i n s , p s o r a l e n and angelicin ( i s o p s o r a l e n ) . Fig. 2. P r o j e c t i o n of (A) a p s o r a l e n m o l e c u l e , a n d (B,C) angelicin m o l e c u l e s b e t w e e n the c o m p l e m e n t a r y s t r a n d s o f t h e D N A helix at the site o f t w o o p p o s i n g t h y m i n e residues ( f r o m ref. [ 5 ] ) . B o t h t h e 3,4- a n d 4 ' , 5 ' - d o u b l e b o n d s of p s o r a l e n can p h o t o r e a c t w i t h the 5 , 6 - d o u b l e b o n d s of t h e t h y m i n e s w i t h little dist o r t i o n o f the D N A s t n a c t u r e . B o t h t h e s e d o u b l e b o n d s are n e c e s s a r y for light i n d u c e d cross-linking. Angelicin lacks t h e o r i e n t a t i o n n e c e s s a r y for cross-linking. M o n o a d d u c t i o n o c c u r s a n d e v i d e n c e i n d i c a t e s t h a t the 4 ' , 5 ' - d o u b l e b o n d (C) r e a c t s m o r e rapidly t h a n the 3 , 4 - b o n d [ 5 ] . T h e i n t e r a c t i o n o f p s o r a l e n a n d o t h e r a n a l o g u e s w i t h nucleic acids e x a m i n e d b y e t h i d i u m f l u o r e s c e n c e assay also suggests this p r e f e r e n c e . (Lown, personal c o m m u n i c a t i o n ) .
379 This present study was designed to test this expectation and to determine the incidence o f micronuclei per cell induced by psoralen and near-UV. On the basis of preliminary results a letter advocating caution in the therapeutic use of psoralens was published [1]. Comparison with X-ray damage served as a reference point or positive control [9]. In addition, the mutagenic and lethal effects of DNA monoadducts alone were examined by the use of angelicin, the angular furocoumarin which is structurally incapable of cross-linking nearly opposing pyrimidine bases [5] (Fig. 2). Materials and methods Cell line and culture Serially propagated Chinese hamster ovary fibroblasts designated as Puck's clone A (CHA cells) were grown in Eagle's minimal essential medium, with Earle's balanced salt solution (Grand Island Biological Co.). The medium was supplemented with 10% (v/v) fetal calf serum (Grand Island Biological Co.), 2 mM L-glutamine and 50 pg/ml kanamycin sulfate (Sigma Chemical Co.) and buffered at pH 7.2 with 0.05 M tricine (N-tris ( h y d r o x y m e t h y l ) m e t h y l glycine; Sigma Chemical Co.) and 0.09% (w/v) sodium bicarbonate. The complete medium is referred to as CTM. Monolayers of CHA cells were grown at 37°C on the surface of disposable plastic tissue culture flasks (Falcon). For all experiments cells were collected, prior to confluence, by treatment for approx. 5 min at 37°C with 0.25% (w/v) trypsin (Difco Bacto-trypsin) in a Ca 2+, Mg 2+ free balanced salt solution. Addition of an equal volume of CTM stopped the trypsinization and the resulting cell suspension was centrifuged at 75 g for 3 min. The pellet was resuspended in fresh CTM to a working concentration of 1 X 106 cells/ml. Cell survival Colony forming ability after 10 days incubation was the measure of cells survival. For counting, colonies were stained 30 min with 0.1% (w/v) methylene blue. After considering plating efficiency of approx. 60% and the treatment to be given (i.e. X-irradiation, psoralen or angelicin photosensitization), appropriate numbers of cells were plated in 35 mm (for X-rays) or 60 mm disposable plastic tissue culture dishes (Falcon) with 5 ml CTM and incubated for 2 h at 37°C to allow for cell attachment prior to photosensitization or X-irradiation. Monolayers of cells covered with 0.5 to 1.0 ml of CTM were irradiated at 24°C for periods of time up to 3 h (in the case of the larger X-ray doses). Following treatment, additional CTM was added to give a total of 3 ml and incubation for micronucleus detection or colony forming ability then commenced. X-irradiation A Picker Zephyr therapy unit, 0.35 m m Cu H.V.L. (2 m m A1 and 0.25 mm Cu added) was used at 10 mA and a nominal 120 kV. An output correction for voltage variation was applied at 2.2%/V and a conversion factor of 0.94 rad/ roentgen used. Irradiation was done in air at 16 cm focal to CTM surface distance using an
380 open 5 cm diameter field. The dose rate was 127 rad/min, backscatter being provided by a 30 X 30 × 5 cm deep wax base. The absorbed central axis dose was taken to be at 91.5% of the surface dose, corresponding to 5 mm of liquid. At the periphery of the 35 mm dish, the corresponding d ep th dose was then some 86%. An overall accuracy of 5% was used for the final absorbed dose along the central axis to take into account time setting errors, voltage fluctuations and o u t p u t calibration uncertainty. Photosensitization The light source m o u n t e d 9 cm from the target, was two parallel black light bulbs (General Electric Co. F20T12.BLB) which emitted 13.4 J/m 2 (measured by chemical a c t i n o m e t r y ) mostly between 320 and 380 nm. Solutions of psoralen or angelicin, 1.72 mg/ml dissolved in 95% ethanol, or ethanol alone, were diluted in CTM w i t h o u t serum to final concentrations of 1.85 X 10 -4 M (34.4 pg/ml) and 2% ethanol. After cell attachment, CTM was replaced by 3 ml of medium plus the appropriate photosensitizing agent. Irradiation began after 10 min exposure to the chemicals; cells were then rinsed with 0.85% saline and 5 ml CTM added for continued incubation. Micronucleus test 5 × 10 ~ cells were plated per dish for detection of c h r o m o s o m e damage. To provide time for the chromosomal damage to be expressed as micronuclei, samples were taken at 0, 18, 29, 42 and 53 h post-irradiation. At these times cells were trypsinized as described, resuspended in 5 ml 1% (w/v) trisodium citrate, left 15 min and repelleted. Cells were then resuspended in 1 ml freshly prepared Carnoy's fixative (3 parts e t h a n o l : 1 part glacial acetic acid) and when dispersed made up to 5 ml with fixative. After 5 min the suspended cells were again centrifuged and this final pellet was resuspended in 0.1 and 0.3 ml fixative depending on its size. Four slides per t r e a t m e n t were made immediately, allowed to d r y in air for several hours then stained 5 min in Gurr's new improved Giemsa diluted 1 : 10 with dilute (0.07 M) PO4 buffer, pH 7.0, rinsed with distilled water to decolourize the cytoplasm and air dried again. A minim u m of 2000 cells were scored for each dose at each expression time. Results are expressed as micronuclei per 100 cells rather than the percentage of cells with micronuclei; there is little difference in this way of presentation as the n u m b er of cells with more than 1 micronucleus is small and follows a Poisson distribution. Chromosome aberrations To examine the actual types of c h r o m o s o m e damage induced by the various treatments, 10 -6 M colcemid was added for 2 h after 25 h expression time at the D30 level. Slides were prepared as described above and 100 arrested metaphases scored for aberrations. Results
CHA cells exposed to various doses of near-ultraviolet light alone or in the presence of 2% ethanol showed no loss of viability as measured by the ability
381
to form visible colonies; furocoumarins dissolved in ethanol, in the dark were w i t h o u t effect. However, irradiation in the presence of psoralen or angelicin resulted in cell death (Fig. 3). Given equimolar concentrations, psoralen was found at the Dl0 level, to be 4.6 times more effective than angelicin as a lethal agent. This difference probably arises from the ability of psoralen, due to its molecular configuration, to photoreact with the 5,6 o~ o~ I-
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Fig. 3. Survival of C H A cells t r e a t e d w i t h 1 . 8 5 × 10-4 M p s o r a l e n ( o ) o r 1 . 8 5 × 10-4 M angelicin (,~) as a f u n c t i o n o f e x p o s u r e to n e a r - u l t r a v i o l e t light. E t h a n o l a l o n e , f u r o c o u m a r i n s dissolved in e t h a n o l or light a l o n e h a d n o l e t h a l e f f e c t s . E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f f o u r s e p a r a t e dishes c o u n t e d a f t e r t e n d a y s p o s t - i r r a d i a t i o n i n c u b a t i o n at 3 7 ° C . Bars i n d i c a t e s t a n d a r d d e v i a t i o n . Fig. 4. T h e i n c i d e n c e of C H A cells w i t h m i c r o n u c l e i at zero to 53 h a f t e r t r e a t m e n t w i t h p s o r a l e n ( 1 . 8 5 × 1 0 -4 M) a n d v a r i o u s d o s e s of n e a x - u l t r a v i o l e t light. A t l e a s t 2 0 0 0 ceils w e r e s c o r e d p e r p o i n t ; b a c k g r o u n d n u m b e r s h a v e b e e n s u b t r a c t e d . T r e a t m e n t w i t h e t h a n o l , e t h a n o l a n d light, or light a l o n e gave results c o m p ~ a b l e t o c o n t ~ o | b a c k g r o u n d v a l u e s , p s o r a l e n w i t h o u t l i g h t h a d n o e f f e c t ; ( i ) 1 3 4 J / m 2 ; (~) 2 1 0 J / m 2 ; ( o ) 281 J / m 2 ; a n d ( e ) 3 6 2 J / m 2. Bars i n d i c a t e s t a n d a r d d e v i a t i o n .
382 post-irradiation cell cycle was, in fact, a b o u t 29 h. Ben-Hur and Elkind [3] also indicated that psoralen treated cells, as judged by dose fractionation experiments, had a long refractory period. For comparison Fig. 6 illustrates survival of CHA cells exposed to X-rays and Fig. 7 shows the corresponding incidence of micronuclei. Following any X-ray dose, the first mitosis (up to 29 h expression time) resulted in the scoring of a maximum number of micronuclei per 100 cells. Further cell division caused continuous though declining production of micronuclei due to fragments which initially failed to produce micronuclei. The declining production of micronuclei coupled with cell death or inability to divide caused a dilution of cells containing micronuclei and resulted in a decrease in micronuclei per 100 cells scored at expression times greater than 29 h. Angelicin damage, with doses of 804 J/m 2 or less, showed a pattern similar to X-rays (Fig. 5). At higher near-UV exposures the peak incidence of micronuclei appeared to be delayed (42 h for 1072 J/m 2) and at the highest doses no decline in production occurred up to 53 h. Psoralen damage resulted in similarly delayed expression and an apparent continuing increase in the number of micronuclei per 100 cells up to 53 h expression time (Fig. 4). Evidently the CHA cells responded to X-ray damage as expected, so differences in results observed with the psoralen must be due to differences in the way in which the lesions are expressed or repaired rather than simply a function of the t y p e of cells examined. Plotting the percent survival against the number of micronuclei per cell after
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Fig. 5. T h e i n c i d e n c e o f C H A cells w i t h m i c r o n u c l e i at z e r o to 53 h a f t e r t r e a t m e n t w i t h a n g e l i c i n ( 1 . 8 5 X 10 - 4 M) a n d v a r i o u s d o s e s o f n e a r - u l t x a v i o l e t light. A t l e a s t 2 0 0 0 cells w e r e s c o r e d p e r p o i n t ; c o n t r o l b a c k g r o u n d n u m b e r s h a v e b e e n s u b t r a c t e d . T r e a t m e n t w i t h e t h a n o l , e t h a n o l a n d l i g h t or l i g h t a l o n e gave r e s u l t s c o m p a r a b l e t o c o n t r o l b a c k g r o u n d v a l u e s ; a n g e l i c i n w i t h o u t l i g h t h a d n o e f f e c t . ( a ) 2 6 8 J / m 2 ; (~) 536 J / m 2 ; ( e ) 8 0 4 J / m 2 ; (0) 1 0 7 2 J / m 2 ; ( v ) 1 3 4 0 J / m 2 . B a r s i n d i c a t e s t a n d a r d d e v i a t i o n . Fig. 6. S u r v i v a l o f C H A cells as a f u n c t i o n o f e x p o s u r e t o X - i r r a d i a t i o n . E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f f o u r s e p a r a t e d i s h e s c o u n t e d a f t e r t e n d a y s p o s t - i r r a d i a t i o n i n c u b a t i o n at 3 7 ° C . B a r s i n d i c a t e s t a n d a r d d e v i a t i o n . I r r a d i a t i o n s c a r r i e d o u t in air.
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one division (29 h expression time) the number of micronuclei at 29 h in the furocoumarin treated cells rather than the maximum micronucleus frequency was used, as quantitative problems associated with the possible replication of some micronucleus fragments were thus avoided) enabled comparison of the effects of damage incurred by psoralen and angelicin photosensitization. Fig. 8 shows that, given this expression time, angelicin and psoralen produced comparable numbers of micronuclei at comparable survivals despite the differences in cross-linking ability and resultant difference in lethal action. The slope of a line through the psoralen and angelicin data points corresponded to 10.7 lethal hits per micronucleus (see Discussion). The X-ray results were slightly higher but generally lay within the expected theoretical limits of 0.1 to 0.2 micronucleus per lethal hit. Cells (at least 150 cells per point per treatment) examined in the first posttreatment mitosis after psoralen or angelicin photosensitization possessed all types of chromatid aberrations. Discussion This investigation reports the induction of micronuclei by psoralen plus nearUV treatment in mammalian cells. Matter and Grauwiler [11] found that compounds reported by them to be positive in the micronucleus test had previously been shown mutagenic in mice. Similarly, psoralen-light treatment has recently been found tumorigenic in mice [7]. While a negative result in the micronucleus test need not mean lack of mutagenicity [11] a positive result demands that caution be exercised in the use of a particular regime. The treatment of psoria-
384 sis by UV and psoralens [12,15] should, in the light of our results, be viewed with some degree of caution [1]. Micronuclei, indicators of chromosome breaks, may represent only a small fraction of the actual alterations induced in chromosomal DNA by chemicals or irradiation [10,11]. The data presented here show that both angelicin and psoralen can produce chromosomal damage in the presence of near-UV b u t that the efficiencies differ. Since angelicin plus near-UV can produce chromosomal damage although only monoadducts to the DNA are formed (no cross-links), monoadducts must lead to chromosomal damage. Since psoralen plus near-UV is five times as effective as angelicin plus near-UV at producing chromosomal damage the cross-links formed by psoralen may be more likely than monoadducts to result in chromosomal aberrations. However there remains a degree of uncertainty as Ben-Hur and Elkind [3] q u o t e as an upper limit a ratio of monoadducts to DNA crosslinks of 7.8 for 4,5',8-trimethylpsoralen in Chinese hamster cells. This ratio is, according to Ben-Hut and Elkmd [3] an overestimate and may n o t be applicable to other furocoumarins. Recent work (Lown, personal communication) on phage ~ DNA, in vitro, in which m o n o a d d u c t formation was measured by ethidium bromide fluorescence analysis indicated that the rate of m o n o a d d u c t formation was twice as fast for psoralen compared with angelicin. Until direct measurements of angelicin and psoralen m o n o a d d u c t formation, in vivo, are made, it will not be possible to evaluate exactly the contributions of the various types of photochemical lesions to the biological actions of psoralen. Thus both m o n o a d d u c t s and cross-links lead to chromosomal damage b u t with differing probabilities. This unequal response could be the result of partly or completely separate repair pathways for the two lesions or to differing efficiencies of a single repair system for these lesions. Our data on chromosomal aberrations and lethality produced by these treatments suggests that at least a large fraction and possibly all of the cell lethality may be due to chromosomal breakage. We have used micronuclei observed after the first mitosis (30 h) as the criterion of chromosomal damage. The expected relationship between cell lethality and the number of micronuclei observed at the first mitosis depends upon two factors: {1) the fraction of cell lethality caused b y chromosomal breakage and (2) the fraction of chromosomal damage expressed as micronuclei. This fraction of the cell killing that is attributable to chromosomal breakage can be calculated by taking account of the known rates of formation of micronuclei from chromosomal fragments and the fraction of lethal aberrations that result in fragments. The second factor is needed because fragments frequently do not form micronuclei b u t rather are swept into daughter nuclei [4]. Measurements on chromatid fragments in Chinese hamster ovary cells suggest that the proportion of fragments that become micronuclei is 0.15 per cell division (Heddle, unpublished results). The first factor is needed because chromatid translocations (symmetrical chromatid interchanges) do not give fragments when rejoining is complete, b u t as a result of random centromere segregation [8 ] will produce a lethal duplication/deficiency half the time. Assuming that symmetrical and asymmetrical exchanges are produced in equal numbers, then for these aberrations fragments will be formed in 2/3 of the lethal aberrations, i.e. there will be 1.5 lethal events/fragments from chromatid exchanges. Thus the number o f lethal events per micronucleus at the first
385 mitosis produced from chromatid exchanges can be estimated as 1.5 lethal events × 1 fragment = 10 lethal events fragment 0.15 micronuclei micronucleus The other c o m m o n aberrations that are lethal events all give micronuclei, so that the number of lethal events per micronucleus for these aberrations is one. Thus, the more there are of these, the more the average number of lethal events/fragments approaches one. At this maximum value, there will be 1/0.15 = 7 lethal events/micronucleus. In fact the regression of In survival (S) versus micronuclei at 30 h (the time at which about one post-treatment mitosis has occurred) is Survival = 1.22 p-10.7 where p = frequency of micronuclei (r = - 0 . 9 1 4 ) This is quite close to the predicted value which means that chromosomal aberrations could be responsible for virtually all of the cell lethality. There may, however, be some lethality arising by other mechanisms because (a) the value of 0.15 micronuclei per fragment is an experimental value subject to to some uncertainty, and (b) cytological examination of the cells in their first post-treatment mitosis has shown that all types of chromatid aberrations are produced by both angelicin and psoralen and thus that the maximum value of 10 lethal events per micronucleus will not be reached. It is clear, however, that much and possibly all of the lethality can be accounted for by chromosome breakage. References 1 A s h w o o d - S m i t h , M.J. a n d E. G r a n t , C h r o m o s o m e d a m a g e p r o d u c e d b y p s o r a l e n a n d u l t r a v i o l e t light, Brit. Med. J., 1 ( 1 9 7 6 ) 3 4 2 . 2 A s h w o o d - S m i t h , M . J . a n d E. G r a n t , C o n v e r s i o n o f p s o r a i e n D N A m o n o a d d u c t s in E. coli t o i n t e r s t r a n d D N A c r o s s - l i n k s b y n e a r ultraviolet light ( 3 2 0 - - 3 6 0 rim). I n a b i l i t y o f a n g e l i c i n t o f o r m crosslinks, i n vivo, E x p e r i e n t i a , in press. 3 B e n - H u r , E. a n d M.M. E l k i n d , D N A c r o s s - l i n k i n g i n C h i n e s e h a m s t e r cells e x p o s e d t o n e a r - u l t r a v i o l e t light in the p r e s e n c e o f 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n , B i o c h i m . B i o p h y s . A c t a , 3 3 1 ( 1 9 7 3 ) 1 8 1 - - 1 9 3 . 4 C a r r a n o , A . V . a n d J . H . H e d d l e , T h e f a t e o f c h r o m o s o m e a b e r r a t i o n s . J. T h e o r . Biol., 3 8 ( 1 9 7 3 ) 2 8 9 - 304. 5 D a l l ' A c q u a , F., S. M a r c i a n i , L. C i a v a t t a a n d G. R o d i g h i e r o , F o r m a t i o n o f i n t e r s t r a n d c r o s s - l i n k i n g in p h o t o r e a c t i o n s b e t w e e n f u r o e o u m a r i n s a n d D N A , Z. N a t u r f o r s c h . , 26 ( 1 9 7 1 ) 5 6 1 - - 5 6 9 . 6 F i t z p a t r i c k , T . B . , K . A . A n n d t , A . M . El M o f t y a n d M . A . P a t h a k , H y d r o q u i n o n e a n d p s o r a l e n s in the t h e r a p y o f h y p e r m e l a m o s i s a n d vitiligo, A r c h . D e r m a t o l . , 9 3 ( 1 9 6 6 ) 5 8 9 - - 6 0 0 . 7 G r e b e , D . D . , T h e t u m o r i g e n i c e f f e c t s o f p s o r a l e n a n d u l t r a v i o l e t light, P r o c . A m . A s s o c . C a n c e r R e s . , 16 (1975) 117. 8 H e d d l e , J . A . , S. W o l f f , D. Whissell a n d J . E . Cleaver, D i s t r i b u t i o n o f c h r o m a t i d s a t m i t o s i s , S c i e n c e , 158 (1967) 929--931. 9 H e d d l e , J . A . , A r a p i d in vivo t e s t f o r c h r o m o s o m e d a m a g e , M u t a t i o n R e s . , 1 8 ( 1 9 7 3 ) 1 8 7 - - 1 9 0 . 10 Latt, S.A. Sister chromatid exchanges, indices of human chromosome damage and repair: detection b y f l u o r e s c e n c e a n d i n d u c t i o n b y m i t o m y c i n C, P r o c . N a t l . A c a d . Sci. U . S . A . , 7 1 ( 1 9 7 4 ) 3 1 6 2 - - 3 1 6 6 . 11 M a t t e r , B.E. a n d J . G r a u w i l e r , M i c r o n u c l e i i n m o u s e b o n e - m a r r o w cells. A s i m p l e in vivo m o d e l f o r the e v a l u a t i o n o f d r u g i n d u c e d c h r o m o s o m a l a b e r r a t i o n s , M u t a t i o n Res., 2 3 ( 1 9 7 4 ) 2 3 9 - - 2 4 9 . 1 2 M e d i c a l P o s t ( E d i t . ) 1 1 , N o . 26 ( 1 9 7 5 ) 1. 13 S a s a k i , M.S. a n d H. T o n o m u r a , A h i g h s u s c e p t i b i l i t y o f F a n c o n i ' s a n e m i a t o c h r o m o s o m e b r e a k a g e b y DNA cross-linking agents, Cancer Res., 33 (1973) 1829--1835. 1 4 S c h m i d , W., T h e m i c r o n u c l e u s t e s t , M u t a t i o n R e s . , 3 1 ( 1 9 7 3 ) 9 - - 1 5 . 1 5 W e b s t e r , G., C o m b i n e d 8 - m e t h o x y p s o r a i e n a n d b l a c k l i g h t t h e r a p y o f p s o r i a s i s , Brit. J . D e r m a t . , 9 0 (1974) 317--323.
Mutation Research, 43 (1977) 377--385 © Elsevier/North-Holland Biomedical Press
CHROMOSOME DAMAGE IN CHINESE HAMSTER CELLS SENSITIZED TO N E A R - U L T R A V I O L E T LIGHT BY P S O R A L E N AND ANGELICIN
M.J. ASHWOOD-SMITH ,1, E.L. GRANT 1 J.A. HEDDLE **2 and G.B. FRIEDMAN 3
1 Departments of Biology and 3 Physics, University of Victoria, Victoria, B.C. (Canada V8W 2Y2) and the 2 Department of Biology, York University, Downsview, Ontario (Canada M3J 1P3) (Received November 1st, 1976) (Revision received January 7th, 1977) (Accepted January 13th, 1976)
Summary The clastogenic effect of furocoumarins psoralen and angelicin in the presence of near-UV (320--380 nm) differs greatly, as do their modes of interaction with DNA. Psoralen, which requires only one-fifth as much light energy to produce the same lethal effect as angelicin at equimolar concentrations, is able to cross-link DNA whereas angelicin cannot. The frequency of micronuclei which arise from chromosomal fragments shows the same differential effect as lethality. Indeed aberrations account for much or all of the lethality observed. Metaphase analysis at comparable aberration frequencies revealed that angelicin and psoralen both induce chromatid deletions and a wide spectrum of chromatid exchanges. These data show that both cross-links and monoadducts to the DNA can result in chromosomal aberrations. The relative contributions of cross-links and monoadducts to chromosomal aberrations still remain to be determined. It it n o t e w o r t h y that extensive chromosomal damage is induced in mammalian cells b y the combination of psoralen and near-UV, a treatment which is currently widely used in the therapy of psoriasis.
Introduction Furocoumarins or psoralens (Fig. 1) sensitize bacteria, viruses, and eucaryotic cells to near-ultraviolet light (320--380 nm, near-UV) by covalently binding to pyrimidine bases as m o n o a d d u c t s and producing DNA cross-links through diadducts [2,3,5]. The resulting lethal and mutagenic effects have been sum* Supported by t h e ** Supported by t h e
National Research Council of National Cancer Institute.
Canada.
378 marized and a tentative mechanism of action proposed in which the DNA-psoralen monoadducts are significant not only as pre-requisites for cross-links but also as unique lethal and mutagenic lesions [2]. Considering the clinical use of psoralen plus light therapy in treatment of vitiligo [6] and, more recently, psoriasis [12,15] it is surprising that there exists only a single report of chromosome aberrations produced in mammalian cells by photo-sensitization with furocoumarins [13]. Peripheral blood lymphocytes from patients with Fanconi's anemia and normal adults were tested for susceptibility to chromosome breakage by the traditional method of examining mitotic chromosomes at metaphase [131. In comparison to this traditional method of scoring chromosome aberrations, the micronucleus test is evolving as a simple, effective and rapid means of screening chemicals for mutagenic action [9,14]. After telophase, micronuclei form in the cytoplasm from acentric fragments that were left behind at anaphase as the centric elements moved towards the spindle poles. Since the crosslinking agent mitomycin C produces micronuclei [11], and since mitomycin C and 8-methoxypsoralen cause chromosome aberrations in mammalian cells [13], psoralen plus near-UV treatment was expected to produce micronuclei.
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Fig. 1. M o l e c u l a r s t r u c t u r e of the r e p r e s e n t a t i v e f u r o c o u m a r i n s , p s o r a l e n and angelicin ( i s o p s o r a l e n ) . Fig. 2. P r o j e c t i o n of (A) a p s o r a l e n m o l e c u l e , a n d (B,C) angelicin m o l e c u l e s b e t w e e n the c o m p l e m e n t a r y s t r a n d s o f t h e D N A helix at the site o f t w o o p p o s i n g t h y m i n e residues ( f r o m ref. [ 5 ] ) . B o t h t h e 3,4- a n d 4 ' , 5 ' - d o u b l e b o n d s of p s o r a l e n can p h o t o r e a c t w i t h the 5 , 6 - d o u b l e b o n d s of t h e t h y m i n e s w i t h little dist o r t i o n o f the D N A s t n a c t u r e . B o t h t h e s e d o u b l e b o n d s are n e c e s s a r y for light i n d u c e d cross-linking. Angelicin lacks t h e o r i e n t a t i o n n e c e s s a r y for cross-linking. M o n o a d d u c t i o n o c c u r s a n d e v i d e n c e i n d i c a t e s t h a t the 4 ' , 5 ' - d o u b l e b o n d (C) r e a c t s m o r e rapidly t h a n the 3 , 4 - b o n d [ 5 ] . T h e i n t e r a c t i o n o f p s o r a l e n a n d o t h e r a n a l o g u e s w i t h nucleic acids e x a m i n e d b y e t h i d i u m f l u o r e s c e n c e assay also suggests this p r e f e r e n c e . (Lown, personal c o m m u n i c a t i o n ) .
379 This present study was designed to test this expectation and to determine the incidence o f micronuclei per cell induced by psoralen and near-UV. On the basis of preliminary results a letter advocating caution in the therapeutic use of psoralens was published [1]. Comparison with X-ray damage served as a reference point or positive control [9]. In addition, the mutagenic and lethal effects of DNA monoadducts alone were examined by the use of angelicin, the angular furocoumarin which is structurally incapable of cross-linking nearly opposing pyrimidine bases [5] (Fig. 2). Materials and methods Cell line and culture Serially propagated Chinese hamster ovary fibroblasts designated as Puck's clone A (CHA cells) were grown in Eagle's minimal essential medium, with Earle's balanced salt solution (Grand Island Biological Co.). The medium was supplemented with 10% (v/v) fetal calf serum (Grand Island Biological Co.), 2 mM L-glutamine and 50 pg/ml kanamycin sulfate (Sigma Chemical Co.) and buffered at pH 7.2 with 0.05 M tricine (N-tris ( h y d r o x y m e t h y l ) m e t h y l glycine; Sigma Chemical Co.) and 0.09% (w/v) sodium bicarbonate. The complete medium is referred to as CTM. Monolayers of CHA cells were grown at 37°C on the surface of disposable plastic tissue culture flasks (Falcon). For all experiments cells were collected, prior to confluence, by treatment for approx. 5 min at 37°C with 0.25% (w/v) trypsin (Difco Bacto-trypsin) in a Ca 2+, Mg 2+ free balanced salt solution. Addition of an equal volume of CTM stopped the trypsinization and the resulting cell suspension was centrifuged at 75 g for 3 min. The pellet was resuspended in fresh CTM to a working concentration of 1 X 106 cells/ml. Cell survival Colony forming ability after 10 days incubation was the measure of cells survival. For counting, colonies were stained 30 min with 0.1% (w/v) methylene blue. After considering plating efficiency of approx. 60% and the treatment to be given (i.e. X-irradiation, psoralen or angelicin photosensitization), appropriate numbers of cells were plated in 35 mm (for X-rays) or 60 mm disposable plastic tissue culture dishes (Falcon) with 5 ml CTM and incubated for 2 h at 37°C to allow for cell attachment prior to photosensitization or X-irradiation. Monolayers of cells covered with 0.5 to 1.0 ml of CTM were irradiated at 24°C for periods of time up to 3 h (in the case of the larger X-ray doses). Following treatment, additional CTM was added to give a total of 3 ml and incubation for micronucleus detection or colony forming ability then commenced. X-irradiation A Picker Zephyr therapy unit, 0.35 m m Cu H.V.L. (2 m m A1 and 0.25 mm Cu added) was used at 10 mA and a nominal 120 kV. An output correction for voltage variation was applied at 2.2%/V and a conversion factor of 0.94 rad/ roentgen used. Irradiation was done in air at 16 cm focal to CTM surface distance using an
380 open 5 cm diameter field. The dose rate was 127 rad/min, backscatter being provided by a 30 X 30 × 5 cm deep wax base. The absorbed central axis dose was taken to be at 91.5% of the surface dose, corresponding to 5 mm of liquid. At the periphery of the 35 mm dish, the corresponding d ep th dose was then some 86%. An overall accuracy of 5% was used for the final absorbed dose along the central axis to take into account time setting errors, voltage fluctuations and o u t p u t calibration uncertainty. Photosensitization The light source m o u n t e d 9 cm from the target, was two parallel black light bulbs (General Electric Co. F20T12.BLB) which emitted 13.4 J/m 2 (measured by chemical a c t i n o m e t r y ) mostly between 320 and 380 nm. Solutions of psoralen or angelicin, 1.72 mg/ml dissolved in 95% ethanol, or ethanol alone, were diluted in CTM w i t h o u t serum to final concentrations of 1.85 X 10 -4 M (34.4 pg/ml) and 2% ethanol. After cell attachment, CTM was replaced by 3 ml of medium plus the appropriate photosensitizing agent. Irradiation began after 10 min exposure to the chemicals; cells were then rinsed with 0.85% saline and 5 ml CTM added for continued incubation. Micronucleus test 5 × 10 ~ cells were plated per dish for detection of c h r o m o s o m e damage. To provide time for the chromosomal damage to be expressed as micronuclei, samples were taken at 0, 18, 29, 42 and 53 h post-irradiation. At these times cells were trypsinized as described, resuspended in 5 ml 1% (w/v) trisodium citrate, left 15 min and repelleted. Cells were then resuspended in 1 ml freshly prepared Carnoy's fixative (3 parts e t h a n o l : 1 part glacial acetic acid) and when dispersed made up to 5 ml with fixative. After 5 min the suspended cells were again centrifuged and this final pellet was resuspended in 0.1 and 0.3 ml fixative depending on its size. Four slides per t r e a t m e n t were made immediately, allowed to d r y in air for several hours then stained 5 min in Gurr's new improved Giemsa diluted 1 : 10 with dilute (0.07 M) PO4 buffer, pH 7.0, rinsed with distilled water to decolourize the cytoplasm and air dried again. A minim u m of 2000 cells were scored for each dose at each expression time. Results are expressed as micronuclei per 100 cells rather than the percentage of cells with micronuclei; there is little difference in this way of presentation as the n u m b er of cells with more than 1 micronucleus is small and follows a Poisson distribution. Chromosome aberrations To examine the actual types of c h r o m o s o m e damage induced by the various treatments, 10 -6 M colcemid was added for 2 h after 25 h expression time at the D30 level. Slides were prepared as described above and 100 arrested metaphases scored for aberrations. Results
CHA cells exposed to various doses of near-ultraviolet light alone or in the presence of 2% ethanol showed no loss of viability as measured by the ability
381
to form visible colonies; furocoumarins dissolved in ethanol, in the dark were w i t h o u t effect. However, irradiation in the presence of psoralen or angelicin resulted in cell death (Fig. 3). Given equimolar concentrations, psoralen was found at the Dl0 level, to be 4.6 times more effective than angelicin as a lethal agent. This difference probably arises from the ability of psoralen, due to its molecular configuration, to photoreact with the 5,6 o~ o~ I-
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382 post-irradiation cell cycle was, in fact, a b o u t 29 h. Ben-Hur and Elkind [3] also indicated that psoralen treated cells, as judged by dose fractionation experiments, had a long refractory period. For comparison Fig. 6 illustrates survival of CHA cells exposed to X-rays and Fig. 7 shows the corresponding incidence of micronuclei. Following any X-ray dose, the first mitosis (up to 29 h expression time) resulted in the scoring of a maximum number of micronuclei per 100 cells. Further cell division caused continuous though declining production of micronuclei due to fragments which initially failed to produce micronuclei. The declining production of micronuclei coupled with cell death or inability to divide caused a dilution of cells containing micronuclei and resulted in a decrease in micronuclei per 100 cells scored at expression times greater than 29 h. Angelicin damage, with doses of 804 J/m 2 or less, showed a pattern similar to X-rays (Fig. 5). At higher near-UV exposures the peak incidence of micronuclei appeared to be delayed (42 h for 1072 J/m 2) and at the highest doses no decline in production occurred up to 53 h. Psoralen damage resulted in similarly delayed expression and an apparent continuing increase in the number of micronuclei per 100 cells up to 53 h expression time (Fig. 4). Evidently the CHA cells responded to X-ray damage as expected, so differences in results observed with the psoralen must be due to differences in the way in which the lesions are expressed or repaired rather than simply a function of the t y p e of cells examined. Plotting the percent survival against the number of micronuclei per cell after
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Fig. 5. T h e i n c i d e n c e o f C H A cells w i t h m i c r o n u c l e i at z e r o to 53 h a f t e r t r e a t m e n t w i t h a n g e l i c i n ( 1 . 8 5 X 10 - 4 M) a n d v a r i o u s d o s e s o f n e a r - u l t x a v i o l e t light. A t l e a s t 2 0 0 0 cells w e r e s c o r e d p e r p o i n t ; c o n t r o l b a c k g r o u n d n u m b e r s h a v e b e e n s u b t r a c t e d . T r e a t m e n t w i t h e t h a n o l , e t h a n o l a n d l i g h t or l i g h t a l o n e gave r e s u l t s c o m p a r a b l e t o c o n t r o l b a c k g r o u n d v a l u e s ; a n g e l i c i n w i t h o u t l i g h t h a d n o e f f e c t . ( a ) 2 6 8 J / m 2 ; (~) 536 J / m 2 ; ( e ) 8 0 4 J / m 2 ; (0) 1 0 7 2 J / m 2 ; ( v ) 1 3 4 0 J / m 2 . B a r s i n d i c a t e s t a n d a r d d e v i a t i o n . Fig. 6. S u r v i v a l o f C H A cells as a f u n c t i o n o f e x p o s u r e t o X - i r r a d i a t i o n . E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f f o u r s e p a r a t e d i s h e s c o u n t e d a f t e r t e n d a y s p o s t - i r r a d i a t i o n i n c u b a t i o n at 3 7 ° C . B a r s i n d i c a t e s t a n d a r d d e v i a t i o n . I r r a d i a t i o n s c a r r i e d o u t in air.
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Fig. 7. T h e i n c i d e n c e o f C H A cells w i t h m i c r o n u c l e i at zero to 53 h a f t e r X - i r r a d i a t i o n . A t least 2 0 0 0 cells w e r e s c o r e d p e r p o i n t , c o n t r o l b a c k g r o u n d n u n b e r s h a v e b e e n s u b t r a c t e d . (m) 52 rad; ( v ) 1 0 4 rad; (o) 2 5 7 rad; a n d (~) 5 1 5 rad. Bars i n d i c a t e s t a n d a r d d e v i a t i o n . Fig. 8. Survival o f C H A cells as a f u n c t i o n o f the i n c i d e n c e of m i c r o n u e l e i p e r cell a f t e r 29 h e x p r e s s i o n t i m e . C o n t r o l b a c k g r o u n d values h a v e b e e n s u b t r a c t e d . (m) Psoralen; (©) angelicin; (@) X-rays. T h e s h a d e d r e g i o n s h o w s t h e a r e a w h e r e N ( t h e n u m b e r of m i c r o n u c l e i p e r l e t h a l h i t ) e q u a l s 0.1 t o 0.2 w h i c h is, t h e o r e t i c a l l y , w h e r e the results w o u l d be e x p e c t e d to lie.
one division (29 h expression time) the number of micronuclei at 29 h in the furocoumarin treated cells rather than the maximum micronucleus frequency was used, as quantitative problems associated with the possible replication of some micronucleus fragments were thus avoided) enabled comparison of the effects of damage incurred by psoralen and angelicin photosensitization. Fig. 8 shows that, given this expression time, angelicin and psoralen produced comparable numbers of micronuclei at comparable survivals despite the differences in cross-linking ability and resultant difference in lethal action. The slope of a line through the psoralen and angelicin data points corresponded to 10.7 lethal hits per micronucleus (see Discussion). The X-ray results were slightly higher but generally lay within the expected theoretical limits of 0.1 to 0.2 micronucleus per lethal hit. Cells (at least 150 cells per point per treatment) examined in the first posttreatment mitosis after psoralen or angelicin photosensitization possessed all types of chromatid aberrations. Discussion This investigation reports the induction of micronuclei by psoralen plus nearUV treatment in mammalian cells. Matter and Grauwiler [11] found that compounds reported by them to be positive in the micronucleus test had previously been shown mutagenic in mice. Similarly, psoralen-light treatment has recently been found tumorigenic in mice [7]. While a negative result in the micronucleus test need not mean lack of mutagenicity [11] a positive result demands that caution be exercised in the use of a particular regime. The treatment of psoria-
384 sis by UV and psoralens [12,15] should, in the light of our results, be viewed with some degree of caution [1]. Micronuclei, indicators of chromosome breaks, may represent only a small fraction of the actual alterations induced in chromosomal DNA by chemicals or irradiation [10,11]. The data presented here show that both angelicin and psoralen can produce chromosomal damage in the presence of near-UV b u t that the efficiencies differ. Since angelicin plus near-UV can produce chromosomal damage although only monoadducts to the DNA are formed (no cross-links), monoadducts must lead to chromosomal damage. Since psoralen plus near-UV is five times as effective as angelicin plus near-UV at producing chromosomal damage the cross-links formed by psoralen may be more likely than monoadducts to result in chromosomal aberrations. However there remains a degree of uncertainty as Ben-Hur and Elkind [3] q u o t e as an upper limit a ratio of monoadducts to DNA crosslinks of 7.8 for 4,5',8-trimethylpsoralen in Chinese hamster cells. This ratio is, according to Ben-Hut and Elkmd [3] an overestimate and may n o t be applicable to other furocoumarins. Recent work (Lown, personal communication) on phage ~ DNA, in vitro, in which m o n o a d d u c t formation was measured by ethidium bromide fluorescence analysis indicated that the rate of m o n o a d d u c t formation was twice as fast for psoralen compared with angelicin. Until direct measurements of angelicin and psoralen m o n o a d d u c t formation, in vivo, are made, it will not be possible to evaluate exactly the contributions of the various types of photochemical lesions to the biological actions of psoralen. Thus both m o n o a d d u c t s and cross-links lead to chromosomal damage b u t with differing probabilities. This unequal response could be the result of partly or completely separate repair pathways for the two lesions or to differing efficiencies of a single repair system for these lesions. Our data on chromosomal aberrations and lethality produced by these treatments suggests that at least a large fraction and possibly all of the cell lethality may be due to chromosomal breakage. We have used micronuclei observed after the first mitosis (30 h) as the criterion of chromosomal damage. The expected relationship between cell lethality and the number of micronuclei observed at the first mitosis depends upon two factors: {1) the fraction of cell lethality caused b y chromosomal breakage and (2) the fraction of chromosomal damage expressed as micronuclei. This fraction of the cell killing that is attributable to chromosomal breakage can be calculated by taking account of the known rates of formation of micronuclei from chromosomal fragments and the fraction of lethal aberrations that result in fragments. The second factor is needed because fragments frequently do not form micronuclei b u t rather are swept into daughter nuclei [4]. Measurements on chromatid fragments in Chinese hamster ovary cells suggest that the proportion of fragments that become micronuclei is 0.15 per cell division (Heddle, unpublished results). The first factor is needed because chromatid translocations (symmetrical chromatid interchanges) do not give fragments when rejoining is complete, b u t as a result of random centromere segregation [8 ] will produce a lethal duplication/deficiency half the time. Assuming that symmetrical and asymmetrical exchanges are produced in equal numbers, then for these aberrations fragments will be formed in 2/3 of the lethal aberrations, i.e. there will be 1.5 lethal events/fragments from chromatid exchanges. Thus the number o f lethal events per micronucleus at the first
385 mitosis produced from chromatid exchanges can be estimated as 1.5 lethal events × 1 fragment = 10 lethal events fragment 0.15 micronuclei micronucleus The other c o m m o n aberrations that are lethal events all give micronuclei, so that the number of lethal events per micronucleus for these aberrations is one. Thus, the more there are of these, the more the average number of lethal events/fragments approaches one. At this maximum value, there will be 1/0.15 = 7 lethal events/micronucleus. In fact the regression of In survival (S) versus micronuclei at 30 h (the time at which about one post-treatment mitosis has occurred) is Survival = 1.22 p-10.7 where p = frequency of micronuclei (r = - 0 . 9 1 4 ) This is quite close to the predicted value which means that chromosomal aberrations could be responsible for virtually all of the cell lethality. There may, however, be some lethality arising by other mechanisms because (a) the value of 0.15 micronuclei per fragment is an experimental value subject to to some uncertainty, and (b) cytological examination of the cells in their first post-treatment mitosis has shown that all types of chromatid aberrations are produced by both angelicin and psoralen and thus that the maximum value of 10 lethal events per micronucleus will not be reached. It is clear, however, that much and possibly all of the lethality can be accounted for by chromosome breakage. References 1 A s h w o o d - S m i t h , M.J. a n d E. G r a n t , C h r o m o s o m e d a m a g e p r o d u c e d b y p s o r a l e n a n d u l t r a v i o l e t light, Brit. Med. J., 1 ( 1 9 7 6 ) 3 4 2 . 2 A s h w o o d - S m i t h , M . J . a n d E. G r a n t , C o n v e r s i o n o f p s o r a i e n D N A m o n o a d d u c t s in E. coli t o i n t e r s t r a n d D N A c r o s s - l i n k s b y n e a r ultraviolet light ( 3 2 0 - - 3 6 0 rim). I n a b i l i t y o f a n g e l i c i n t o f o r m crosslinks, i n vivo, E x p e r i e n t i a , in press. 3 B e n - H u r , E. a n d M.M. E l k i n d , D N A c r o s s - l i n k i n g i n C h i n e s e h a m s t e r cells e x p o s e d t o n e a r - u l t r a v i o l e t light in the p r e s e n c e o f 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n , B i o c h i m . B i o p h y s . A c t a , 3 3 1 ( 1 9 7 3 ) 1 8 1 - - 1 9 3 . 4 C a r r a n o , A . V . a n d J . H . H e d d l e , T h e f a t e o f c h r o m o s o m e a b e r r a t i o n s . J. T h e o r . Biol., 3 8 ( 1 9 7 3 ) 2 8 9 - 304. 5 D a l l ' A c q u a , F., S. M a r c i a n i , L. C i a v a t t a a n d G. R o d i g h i e r o , F o r m a t i o n o f i n t e r s t r a n d c r o s s - l i n k i n g in p h o t o r e a c t i o n s b e t w e e n f u r o e o u m a r i n s a n d D N A , Z. N a t u r f o r s c h . , 26 ( 1 9 7 1 ) 5 6 1 - - 5 6 9 . 6 F i t z p a t r i c k , T . B . , K . A . A n n d t , A . M . El M o f t y a n d M . A . P a t h a k , H y d r o q u i n o n e a n d p s o r a l e n s in the t h e r a p y o f h y p e r m e l a m o s i s a n d vitiligo, A r c h . D e r m a t o l . , 9 3 ( 1 9 6 6 ) 5 8 9 - - 6 0 0 . 7 G r e b e , D . D . , T h e t u m o r i g e n i c e f f e c t s o f p s o r a l e n a n d u l t r a v i o l e t light, P r o c . A m . A s s o c . C a n c e r R e s . , 16 (1975) 117. 8 H e d d l e , J . A . , S. W o l f f , D. Whissell a n d J . E . Cleaver, D i s t r i b u t i o n o f c h r o m a t i d s a t m i t o s i s , S c i e n c e , 158 (1967) 929--931. 9 H e d d l e , J . A . , A r a p i d in vivo t e s t f o r c h r o m o s o m e d a m a g e , M u t a t i o n R e s . , 1 8 ( 1 9 7 3 ) 1 8 7 - - 1 9 0 . 10 Latt, S.A. Sister chromatid exchanges, indices of human chromosome damage and repair: detection b y f l u o r e s c e n c e a n d i n d u c t i o n b y m i t o m y c i n C, P r o c . N a t l . A c a d . Sci. U . S . A . , 7 1 ( 1 9 7 4 ) 3 1 6 2 - - 3 1 6 6 . 11 M a t t e r , B.E. a n d J . G r a u w i l e r , M i c r o n u c l e i i n m o u s e b o n e - m a r r o w cells. A s i m p l e in vivo m o d e l f o r the e v a l u a t i o n o f d r u g i n d u c e d c h r o m o s o m a l a b e r r a t i o n s , M u t a t i o n Res., 2 3 ( 1 9 7 4 ) 2 3 9 - - 2 4 9 . 1 2 M e d i c a l P o s t ( E d i t . ) 1 1 , N o . 26 ( 1 9 7 5 ) 1. 13 S a s a k i , M.S. a n d H. T o n o m u r a , A h i g h s u s c e p t i b i l i t y o f F a n c o n i ' s a n e m i a t o c h r o m o s o m e b r e a k a g e b y DNA cross-linking agents, Cancer Res., 33 (1973) 1829--1835. 1 4 S c h m i d , W., T h e m i c r o n u c l e u s t e s t , M u t a t i o n R e s . , 3 1 ( 1 9 7 3 ) 9 - - 1 5 . 1 5 W e b s t e r , G., C o m b i n e d 8 - m e t h o x y p s o r a i e n a n d b l a c k l i g h t t h e r a p y o f p s o r i a s i s , Brit. J . D e r m a t . , 9 0 (1974) 317--323.
