419
Mutation Research, 51 ( 1 9 7 8 ) 4 1 9 - - 4 2 5 © 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
CAFFEINE INHIBITION OF PREREPLICATION REPAIR OF MITOMYCIN C-INDUCED DNA DAMAGE IN HUMAN PERIPHERAL LYMPHOCYTES *
Y U T A K A ISHII ** a n d M I C H A E L A B E N D E R
Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) (Received 29 N o v e m b e r 1 9 7 7 ) (Revision received 7 M a r c h 1 9 7 8 ) ( A c c e p t e d 8 March 1 9 7 8 )
Summary Caffeine increases the number of sister-chromatid exchanges (SCE) induced by mitomycin C (MMC) in human peripheral lymphocytes in culture. This enhancement decreases when the treated cells are held in medium before phytohemagglutinin (PHA) stimulation, or when caffeine is added to cultures some time after PHA stimulation but prior to DNA synthesis. There thus appears to be a caffeine-sensitive prereplication repair system, presumably an excision mechanism, capable of repairing a fraction of the MMC-induced DNA lesions. Introduction In mammalian cells caffeine is well known to inhibit postreplication repair of DNA lesions [ 5,11,12,23--25,39,40]. Nevertheless, the details of the biological effects of caffeine on mammalian cells are unclear. It has been reported that caffeine enhances mutagenesis by ultraviolet light and chemicals [1,31,32], transformation by chemical carcinogens [7], the induction of chromosomal aberrations by UV and chemicals [3,10,14,19,20,29--32,41] and the induction of SCE by psoralin and UV [42]. On the other hand, there are also reports that caffeine suppresses UV mutagenesis [9,38,39], chemical transformation [16] and the induction of SCE by both UV and chemicals [17,18], and even that it has no influence on the induction of SCE by chemicals [30]. In bacteria, especially Escherichia coli, the details of caffeine effects are less * Research supported by the U.S. Department of Energy. ** Present address: Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 (Japan)
Abbreviations: BrdU, 5obromodeoxyuridine; MMC, mitomycin C; PHA, phytohemagglutinin; SCE, sister-cbxomatid exchange(s).
420 confusing. Caffeine inhibits excision-repair [2,28,33,36,37], and enhances UV mutagenesis in repair-proficient cells, while suppressing it in excision-repairdeficient cells [4,26,27,35,36,43,45]. Recently Kondo [21] concluded that caffeine suppresses error-free excision-repair at low doses and suppresses errorprone postreplication-repair at high doses in repair-proficient cells, b u t can suppress only the error-prone postreplication form of repair in excision-repairdeficient cells. It is possible that an effect on an excision-repair system might explain some of the somewhat conflicting reports on caffeine effects in mammalian cells. We here report the results of experiments showing that human peripheral lymphocytes possess a caffeine-sensitive prereplication repair mechanism which can remove, presumably b y excision, some b u t not all of the MMC-induced lesions that give rise to SCE. Materials and methods
Cell culture and treatment 0.5 ml of whole, heparinized human peripheral blood was used for each culture, which contained 10 ml of Eagle's minimal essential medium (Gibco) containing 20% foetal calf serum (Gibco). Cultures were treated with 150 ng/ml of MMC (Sigma) for 4 h at 37°C in a 5% CO2 atmosphere. The cells were then washed twice in fresh medium to remove free MMC and resuspended in 10 ml of medium to which 0.25 ml of PHA (Gibco) and 5-bromodeoxyuridine (BrdU; Calbiochem, final concentration 25 pM) were added. Caffeine (Calbiochem) was added at the times and levels noted. Cultures were incubated in 5% CO2 at 37°C for 72 h in the dark. 2 h before harvesting the cells 0.15 ml of colcemid (Gibco) was added to each culture. Slide preparation Cultures were centrifuged and resuspended in 75 mM KCI hypotonic solution for 12 min, fixed in 3 : 1 methanol--acetic acid for 30 rain, twice washed with fresh fixative, d r o p p e d onto glass slides and air dried. Staining was done essentially according to Goto et al. [13]. Slides were stained for 10 min in 10 -4 M Hoechst 33258 (a generous gift of Dr. H. Loewe of Farbwerke Hoechst), immersed in water and m o u n t e d in pH 7.0 citrate-phosphate buffer (0.174 M Na2HPO4 and 0.013 M citrate). These slides were illuminated with two 15-W black light tubes (Westinghouse) for a b o u t 20 min following which they were stained in 2% Giemsa (Harleco) in 0.05 M phosphate buffer at pH 6.8 for 20 min. Upon removal from the stain the slides were soaked in two changes of acetone, one of 1 : 1 acetone--toluene, two of toluene, and then air dried. Cover slips were m o u n t e d with Permount (Fisher). Scoring SCE were scored in all of the chromosomes of 20 intact metaphase spreads for each experimental point. Results and discussion The results of an experiment to determine the effect of various concentrations of caffeine on SCE yield are shown in Fig. 1. N o t only did the caffeine
421 I
I
]
I
J
80
7O ._J .J w
,-r i,i (3. LmJ (.9 Z
60
.50
T (..) X W O t'O n." -crj
40
30
nr"
W I--
2O
I0
0 0
I
I
50
I00 CAFFEINE
I 200
I 300
(/J.g/ml)
Fig. 1. E f f e c t o f c a f f e i n e o n " s p o n t a n e o u s " a n d M M C - i n d u c e d S C E . Cells w e r e t r e a t e d w i t h M M C at 1 5 0 n g / m l f o r 4 h , w a s h e d , a n d t h e n c u l t u r e d i n t h e p z e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f c a f f e i n e in t h e medium.
itself induce some SCE, but it clearly interacted synergistically to produce large increases in the yields of MMC-induced SCE. The higher doses of caffeine evidently produced cell-cycle delay, for there were fewer second metaphase figures in these samples. For this reason a caffeine concentration of 200 pg/ml was used for the rest of the experiments. Speculating that the caffeine enhancement might, as in E. coli, result from inhibition of a prereplication excision form of D N A repair, we did a typical "liquid holding" experiment. After MMC treatment, cells were washed and then held in fresh medium without PHA for various times before incubation with PHA, BrdU and, where appropriate, caffeine. The results are presented in Fig. 2. There is clearly no effect of holding time on the SCE yields in cells which were not MMC-treated, cultured either with or without caffeine, nor in cells treated with MMC and cultured in absence of caffeine. On the other hand, the yield of MMC-induced SCE clearly decreases during the first 18 h of liquid holding in cells cultured in the presence of caffeine: after 18 h the addition of caffeine no longer resulted in any increase over that induced by the caffeine alone (i.e., over additivity). These results may be interpreted as confirming the existence
422 I
F
T
70
_I .J l.lJ o n,hl O.q') hi (.9 Z ,,::[ "r 0 X bJ
60
+CAF
50
a
\ +MMC
/
40 -CAF
r-,
30 o -r w" hi b"
20 +CAF
\ /
IO
-MMC
- CAF
I
0
0
6
I
I
18
30
HOLDING TIME BEFORE STIMULATION
48
(h )
Fig. 2. Effect o f "liquid h o l d i n g " t i m e o n SCE frequencies. A f t e r 4 h in the presence (circles) or absence (triangles) o f MMC ( 1 5 0 n g / m l ) , cells w e r e w a s h e d and t h e n held, u n s t i m u l a t e d , in fresh m e d i u m w i t h o u t P H A o r B r d U for the i n d i c a t e d times prior t o the a d d i t i o n o f P H A a n d BrdU w i t h (closed s y m b o l s ) or w i t h o u t ( o p e n s y m b o l s ) caffeine.
of the caffeine-sensitive DNA-repair mechanism in these cells; one which is clearly pre-replicational, since human lymphocytes are in a Go state until after PHA stimulation, and do not synthesize any D N A during at least the first 12 h following stimulation. Our result appears in conflict with that recently reported by Palitti and Bechetti [ 3 0 ] , w h o found that 10 -3 M caffeine failed to increase the induction of SCE in MMC-treated Chinese hamster cells. However, since rodent cells have only a poor prereplication excision system for UV-induced pyrimidine dimers, it may be that Chinese hamster cells are deficient in their ability to excise MMC-induced lesions as well. A caffeine enhancement of SCE induction in h u m a n lymphocytes by psoralin plus U V light,which like M M C causes D N A strand--strand crosslinks,has recently been reported by Waksvik, Brogger and Stene [42]. While their remits do not establish that this was a prereplication effect, it is possible that at least part of their caffeine effect was on a repair system similar to or the same as that affected in the present experiments. A possible explanation for the lack of any change in MMC-induced S C E yield with liquid holding in cellscultured in the absence of caffeine is that the repair system already effects the m a x i m u m repair possible during the pre-DNA synthesis phase of the PHA-stimulated portion of the incubation period. Fig. 3
423 70
.J ..J hi (_:, E: LLI {3...
LIJ (.9 Z "tO X ILl El I"-
I
I
I
60
+CAF
50
Q
\ +MMC
/
40
-CAF 30
0 3:: {Z: I,I I'--
+CAF
20 A
\ /
-MMC
I0 - CAF
0 0
I
t
I
6
12
18
TIME
AFTER
STIMULATION
24
(h )
Fig. 3. E f f e c t o f c u l t u r e t i m e prior t o a d d i t i o n o f c a f f e i n e o n SCE y i e l d s . A f t e r 4 h in the p r e s e n c e ( c i r c l e s ) or a b s e n c e (triangles) o f M M C , cells w e r e w a s h e d and t h e n c u l t u r e d in the p r e s e n c e o f P H A and B r d U . C a f f e i n e w a s a d d e d t o s o m e o f t h e c u l t u r e s ( c l o s e d s y m b o l s ) at t h e i n d i c a t e d t i m e s , b u t n o t t o others (open symbols).
shows the results of an experiment which confirms this explanation. Following treatment with MMC, cells were washed twice and then incubated in fresh medium containing PHA and BrdU. Caffeine was added after various incubation times. During the first 12 culture hours the frequency of MMC-induced SCE decreased as a function of increasing delay in the addition of the caffeine; after 12 h no further effect was noted. The plateau of SCE frequency appears to approximate the sum of the yields from MMC alone and from caffeine alone reasonably well. The experiments presented in Figs. 2 and 3 both suggest that this caffeinesensitive repair system "saturates", and is only capable of repairing a fraction o f those MMC-induced DNA lesions capable of inducing SCE. This is in agreement with our observation, presented elsewhere [15], of third cycle, Go MMC-induced yields of SCE compatible with the persistence through at least three cell cycles of all of the D N A lesions capable o f inducing SCE that were present at the beginning of the first S phase. Why some of the MMC lesions are repairable by the prereplication system while others are not is not obvious; it could result from there being several types of lesion or from lesions being located in chromatin more or less accessable to the repair system. Recently Day [6] showed that caffeine inhibited host-cell reactivation of UV-irradiated adenovirus in human cells and inhibited excision-repair. Very
424
similar results were obtained for UV-induced mutation in E. coil (Y. Ishii, unpublished data). After UV irradiation, the bacteria were held in phosphate buffer supplemented with glucose for various times before being plated on growth medium with caffeine or with acriflavine, another inhibitor of excisionrepair [ 8 , 3 4 , 4 4 ] . The UV-induced mutation frequency was decreased in a manner similar to Fig. 2 (the same effect has also been reported for MMCinduced mutations and acriflavine [22] ). Another UV experiment gave results similar to those of the experiment illustrated in Fig. 3. UV-irradiated E. coli were cultured in growth medium for various times and then plated onto agar medium with caffeine or with acriflavine. The frequency of UV-induced mutations decreased as the interval prior to caffeine or acriflavine increased, during the UV-induced growth delay period. It thus appears that the possession of caffeine-sensitive prereplication DNA-repair systems is a property of many types of cells, and that it influences the effectiveness of a variety of mutagenic and carcinogenic agents. References 1 Arlett, C.F., and S.A. Harcourt, The i n d u c t i o n of 8-azaguanine-resistant m u t a n t s in cultured Chinese h a m s t e r ceils b y ultraviolet light: the effect of changes in post-irradiation conditions, Mut a t i on Res., 14 (1972) 431---437. 2 Bendigkeit, H.E., and P.C. Hanawalt, Caffeine i n h i b i t i o n of DNA repair replication in Eschertchta colt, Bacteriological Proceedings 1968, Abstracts of the 6 8 t h A nnua l Meeting, p. 36. 3 Brogger, A., Caffeine-induced e n h a n c e m e n t of c h r o m o s o m e damage in h u m a n l y m p h o c y t e s treated with m e t h y l m e t h a n e s u l p h o n a t e , m i t o m y e i n C and X-rays, Mut a t i on Res., 23 (1974) 353--360. 4 Clarke, C.H., Caffeine- and a m i n o acid-effects u p o n try + revertant yield in UV4rradiated hcr+ and hcrm u t a n t s of E. colt B/r, Mol. Gen. Genet., 99 (1967) 97--108. 5 Cleaver, J.E., and G.H. Thomas, Single strand i n t e r r u p t i o n s in DNA and the effects of caffeine in Chinese h a m s t e r cells irradiated w i t h ultraviolet Hght, Biochem. Biophys. Res. Commun., 36 (1969) 203--208. 6 Day I n , R.S., Caffeine i n h i b i t i o n of the repair of ultraviolet-irradiated adenovirus in h u m a n cells, M u t a t i o n Res., 33 (1975) 321--326. 7 Donovan, P~I., and J.A. DiPaolo, Caffeine e n h a n c e m e n t of chemical carcinogen-induced transformat i o n of cultured Syrian hamster ceils, Cancer Res., 34 (1974) 2720--2727. 8 Feiner, R.R., and R. Hill, Effect of basic dyes on host-cell reactivation of ultra violet-irradiated phage, Nature (London), 200 (1963) 2 9 1 - - 2 9 3 . 9 Fo x, M., The effect of p o s t - t r e a t m e n t w i t h caffeine on survival and UV-induced m u t a t i o n frequencies in Chinese h amster and mouse l y m p h o m a cells in vitro, M u t a t i o n Res., 24 (1974) 187--204. 10 Fre',', J.V., an d S. Venitt, C h r o m o s o m e damage in the bone ma rrow of mice t r e a t e d w i t h the me t hyl ating agents m e t h y l m e t h a n e s u l p h o n a t e and N-methyl-N-nitrosourea in t he presence or absence of caffeine, and its relationship w i t h t h y m o m a i n d u c t i o n , Mut a t i on Res., 30 (1975) 89--96. 11 Fujiwara, Y., Characteristics of DNA synthesis following ultraviolet light irradiation in mouse L cells. Postreplication repair, Exp. Cell Res., 75 (1972) 483---489. 12 Fujiwaxa, Y., and T. K o n d o , Caffeine-sermitive repair of ultraviolet light-damaged DNA of mouse L cells, Biochem. Biophys. Res. C o m m u n . , 47 (1972) 552--564. 13 Goto, K., T. A k e m a t s u , H. Shimazu and T. Sugiyama, Simple differential Giemsa staining of sister c h r o m a t i d s after t r e a t m e n t w i t h photosensitive dyes and exposure to light and the me c ha ni s m of staining, C h r o m o s o m a , 53 (1975) 223--230. 14 Hartley-Asp, B., and B.A. Kihiman, Caffeine, caffeine derivatives and c h r o m o s o m a l aberration, IV. Synergism b e t w e e n m i t o m y c i n C end caffeine in Chinese hamster cells, Hereditas, 69 (1971) 326--326. 15 Ishii, Y., and M. A Bender, Factors influencing the frequency of m i t o m y c i n C-induced sister-chrom a t i d exchanges in 5 - b r o m o d e o x y u r i d i n e - s u b s t i t u t e d h u m a n l y m p h o c y t e s in culture, Mutation Res., 51 (1978) 411--416. 16 Kakunaga, T., Caffeine inhibits cell t r a n s f o r m a t i o n by 4-nitroqutnoline-l-oxide, Nature (London), 259 (1975) 248--250. 17 Kato, H., I n d u c t i o n of sister c h r o m a t i d exchanges by UV light a nd its i n h i b i t i o n by caffeine, Exp. Cell Res., 82 (1973) 383--390.
425 1 8 K a t o , H . , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s b y c h e m i c a l m u t a g e n s a n d its p o s s i b l e r e l e v a n c e t o D N A r e p a i r , E x p . Cell R e s . , 8 5 ( 1 9 7 4 ) 2 3 9 - - 2 4 7 . 1 9 K i h l m a n , B . A . , S. S t u r e l i d , B. H a r t l e y - A s p a n d K . N i l s s o n , C a f f e i n e p o t e n t i a t i o n o f t h e c h r o m o s o m e d a m a g e p r o d u c e d in b e a n r o o t t i p s a n d in C h i n e s e h a m s t e r cells b y v a r i o u s c h e m i c a l a n d p h y s i c a l a g e n t s , M u t a t i o n R e s . , 17 ( 1 9 7 3 ) 2 7 1 - - 2 7 5 . 2 0 K i h l m a n , B . A . , S. S t u r e l i d , B. H a r t l e y o A s p a n d K . N i l s s o n , T h e e n h a n c e m e n t b y c a f f e i n e o f t h e freq u e n c i e s o f c h r o m o s o m a l a b e r r a t i o n s i n d u c e d in p l a n t a n d a n i m a l cells b y c h e m i c a l a n d p h y s i c a l agents, Mutation Res., 26 (1974) 105--122. 21 K o n d o , S., M i s r e p a i r m o d e l f o r m u t a g e n e s i s a n d c a r c i n o g e n e s i s , i n : P . N . M a g e e et al. (Eds.), F u n d a m e n t a l s in C a n c e r P r e v e n t i o n , U n i v . o f T o k y o Press, T o k y o / U n i v . P a r k Press, B a l t i m o r e , 1 9 7 6 , p p . 417--429. 2 2 K o n d o , S., H . I c h i k a w a , K . I w o a n d T. K a t o , B a s e - c h a n g e m u t a g e n e s i s a n d p r o p b a g e i n d u c t i o n in s t r a i n s o f E s c h e r i c h i a coli w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , G e n e t i c s , 6 6 ( 1 9 7 0 ) 1 8 7 - - 2 1 7 . 2 3 L e h m a n n , A . R . , E f f e c t o f c a f f e i n e o n D N A s y n t h e s i s in m a m m a l i a n cells, B i o p h y s . J., 1 2 ( 1 9 7 2 ) 1316--1325. 2 4 L e h m a n n , A . R . , a n d S. K i r k - B e l l , P o s t - r e p l i c a t i o n r e p a i r o f D N A in u l t r a v i o l e t - i r r a d i a t e d m a m m a l i a n cells, N o g a p s in D N A s y n t h e s i z e d l a t e a f t e r u l t r a v i o l e t i r r a d i a t i o n , E u r . J. B i o c h e m . , 31 ( 1 9 7 2 ) 4 3 8 - 445. 2 5 L e h m a n n , A . R . , a n d S. K i r k - B e l l , E f f e c t s o f c a f f e i n e a n d t h e o p h y l l i n e o n D N A s y n t h e s i s i n u n i r r a d i a t e d a n d U V - i r r a d i a t e d m a m m a l i a n cells, M u t a t i o n R e s . , 2 6 ( 1 9 7 4 ) 7 3 - - 8 2 . 2 6 L i e b , M., E n h a n c e m e n t o f u l t r a v i o l e t - i n d u c e d m u t a t i o n i n b a c t e r i a b y c a f f e i n e , Z. V e r e r b u n g s i . , 9 2 (1961) 416--429. 27 L u m b , J . R . , A . S . S i d e r o p o u l o s a n d D . M . S h a n k e l , I n h i b i t i o n o f d a r k r e p a i r o f u l t r a v i o l e t d a m a g e in D N A b y c a f f e i n e a n d 8 - c h l o r o c a f f e i n e , K i n e t i c s o f i n h i b i t i o n , Mol. G e n . G e n e t . , 1 0 2 ( 1 9 6 8 ) 1 0 8 - 111. 2 8 M e t z g e r , K . , O n t h e d a r k r e a e t i v a t i o n m e c h a n i s m in u l t r a v i o l e t i r r a d i a t e d b a c t e r i a , B i o e h e m . B i o p h y s . Res. Commun., 15 (1964) 101--109. 2 9 N i l s s o n , K . , a n d A . R . L e h m a n n , T h e e f f e c t o f m e t h y l a t e d o x y p u r i n e s o n t h e size o f n e w l y - s y n t h e s i z e d D N A a n d o n t h e p r o d u c t i o n o f c h r o m o s o m e a b e r r a t i o n s a f t e r U V i r r a d i a t i o n in C h i n e s e h a m s t e r cells, Mutation Res., 30 (1975) 255--266. 30 Palitti, F., and A. Becchetti, Effect of caffeine on sister-chromatid exchanges and chromosomal aberr a t i o n s i n d u c e d b y m u t a g e n s i n 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 . , 4 5 ( 1 9 7 7 ) 1 5 7 - - 1 5 9 . 31 R o b e r t s , J . J . , a n d J . E . S t u r r o c k , E n h a n c e m e n t b y c a f f e i n e o f N - m e t h y l - N o n i t r o s o u r e a i n d u c e d m u t a t i o n s a n d c h r o m o s o m e a b e r r a t i o n s i n 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 . , 2 0 ( 1 9 7 3 ) 2 4 3 - - 2 5 5 . 3 2 R o b e r t s , J~I., J . E . S t u r o c k a n d K . N . W a r d , T h e e n h a n c e m e n t b y c a f f e i n e of a l k y l a t i o n - i n d u c e d cell d e a t h , m u t a t i o n s a n d c h r o m o s o m a l a b e r r a t i o n s in C h i n e s e h a m s t e r cells, as a r e s u l t o f i n h i b i t i o n o f post-replication DNA repair, Mutation Res., 26 (1974) 129--143. 3 3 S a u e r b i e r , W., I n h i b i t i o n o f h o s t cell r e a c t i v a t i o n in p h a g e T1 b y c a f f e i n e , B i o c h e m . B i o p h y s . Res. Commun, 14 (1964) 340--346. 3 4 S e t l o w , R . B . , P h y s i c a l c h a n g e s in m u t a g e n e s i s , J . Cell. C o m p . P h y s i o l . , 6 4 ( S u p p l . 1) ( 1 9 6 4 ) 5 1 - - 6 8 . 3 5 S h a n k e l , D . M . , " M u t a t i o n a l s y n e r g i s m " o f u l t r a v i o l e t l i g h t a n d c a f f e i n e in E s c h e r i c h i a coli, J. B a c teriol., 84 (1962) 410---415. 36 Sideropoulos, A.S., and D.M. Shankel, Mechanism of caffeine enhancement of mutations induced by s u b l e t h a l u l t r a v i o l e t d o s a g e s , J. B a e t e r i o l . , 9 6 ( 1 9 6 8 ) 1 9 8 - - 2 0 4 . 37 S h i m a d a , K . , a n d Y . T a k a g i , T h e e f f e c t o f c a f f e i n e o n t h e r e p a i r o f u l t r a v i o l e t - d a m a g e d D N A i n b a c teria, Biochim. Biophys. Acta, 145 (1967) 763--770. 3 8 T r o s k o , J . E . , a n d E . H . Y . C h u , E f f e c t s o f c a f f e i n e o n t h e i n d u c t i o n o f m u t a t i o n s in C h i n e s e h a m s t e r cells b y u l t r a v i o l e t l i g h t , M u t a t i o n R e s . , 1 2 ( 1 9 7 1 ) 3 3 7 - - 3 4 0 . 39 Trosko, J.E., and E.H.Y. Chu, Inhibition of repair of UV-damaged DNA by caffeine and mutation 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, C h e m . - B i o l . I n t e r a c t . , 6 ( 1 9 7 3 ) 3 1 7 - - 3 3 2 . 4 0 T r o s k o , J . E . , P. F r a n k , E . H . Y . C h u a n d J . E . B e c k e r , C a f f e i n e i n h i b i t i o n o f p o s t r e p l i e a t i o n r e p a i r o f N - a c e t o x y - 2 - a c e t y l a m i n o f l u o r e n e - d a m a g e d D N A i n C h i n e s e h a m s t e r cells, C a n c e r R e s . , 3 3 ( 1 9 7 3 ) 2444--2449. 41 V a n d e n B e r g , H . W . , a n d J . J . R o b e r t s , P o s t - r e p l i c a t i o n r e p a i r o f D N A in C h i n e s e h a m s t e r cells t r e a t e d with cis-platinum(II) diammine dichloride, Enhancement of toxicity and chromosome damage by caffeine, Mutation Res., 33 (1975) 279--284. 4 2 W a k s v i k , H . , A . B r o g g e r a n d J . S t e n e , P s o r a l i n / U V A t r e a t m e n t a n d c h r o m o s o m e s , I. A b e r r a t i o n s a n d sister c h r o m a t i d e x c h a n g e in h u m a n l y m p h o c y t e s in v i t r o a n d s y n e r g i s m w i t h c a f f e i n e , H u m a n G e n e t . , 38 (1977) 195--207. 4 3 W i t k i n , E . M . , P o s t - i r r a d i a t i o n m e t a b o l i s m a n d t h e t i m i n g o f u l t r a v i o l e t - i n d u c e d m u t a t i o n s in b a c t e r i a , P r o c . 1 0 t h I n t e r n . C o n g r . o f G e n e t . , V o l . 1, M o n t r e a l , C a n a d a , 1 9 5 8 , p p . 2 8 0 - - 2 9 9 . 44 Witkin, E.M., Modification of mutagenesis initiated by ultraviolet light through post-treatment of bact e r i a w i t h b a s i c d y e s , J . Cell. C o m p . P h y s i o l . , 5 8 ( S u p p l . 1) ( 1 9 6 1 ) 1 3 5 - - 1 4 4 . 45 Witkin, E.M., and E.L. Farcluharson, Enehancement and diminution of ultraviolet-light-initiated mutagenesis b y p o s t - t r e a t m e n t w i t h c a f f e i n e in E s c h e r i c h i a eoU, i n : G . E . W . W o i s t e n h o l m e a n d M. O ' C o n n o r ( E d s . ) , M u t a t i o n as C e l l u l a r P r o c e s s , C i b a F o u n d . S y r u p . 1 9 6 9 , p p . 3 6 - - 4 9 .
Mutation Research, 51 ( 1 9 7 8 ) 4 1 9 - - 4 2 5 © 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
CAFFEINE INHIBITION OF PREREPLICATION REPAIR OF MITOMYCIN C-INDUCED DNA DAMAGE IN HUMAN PERIPHERAL LYMPHOCYTES *
Y U T A K A ISHII ** a n d M I C H A E L A B E N D E R
Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) (Received 29 N o v e m b e r 1 9 7 7 ) (Revision received 7 M a r c h 1 9 7 8 ) ( A c c e p t e d 8 March 1 9 7 8 )
Summary Caffeine increases the number of sister-chromatid exchanges (SCE) induced by mitomycin C (MMC) in human peripheral lymphocytes in culture. This enhancement decreases when the treated cells are held in medium before phytohemagglutinin (PHA) stimulation, or when caffeine is added to cultures some time after PHA stimulation but prior to DNA synthesis. There thus appears to be a caffeine-sensitive prereplication repair system, presumably an excision mechanism, capable of repairing a fraction of the MMC-induced DNA lesions. Introduction In mammalian cells caffeine is well known to inhibit postreplication repair of DNA lesions [ 5,11,12,23--25,39,40]. Nevertheless, the details of the biological effects of caffeine on mammalian cells are unclear. It has been reported that caffeine enhances mutagenesis by ultraviolet light and chemicals [1,31,32], transformation by chemical carcinogens [7], the induction of chromosomal aberrations by UV and chemicals [3,10,14,19,20,29--32,41] and the induction of SCE by psoralin and UV [42]. On the other hand, there are also reports that caffeine suppresses UV mutagenesis [9,38,39], chemical transformation [16] and the induction of SCE by both UV and chemicals [17,18], and even that it has no influence on the induction of SCE by chemicals [30]. In bacteria, especially Escherichia coli, the details of caffeine effects are less * Research supported by the U.S. Department of Energy. ** Present address: Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 (Japan)
Abbreviations: BrdU, 5obromodeoxyuridine; MMC, mitomycin C; PHA, phytohemagglutinin; SCE, sister-cbxomatid exchange(s).
420 confusing. Caffeine inhibits excision-repair [2,28,33,36,37], and enhances UV mutagenesis in repair-proficient cells, while suppressing it in excision-repairdeficient cells [4,26,27,35,36,43,45]. Recently Kondo [21] concluded that caffeine suppresses error-free excision-repair at low doses and suppresses errorprone postreplication-repair at high doses in repair-proficient cells, b u t can suppress only the error-prone postreplication form of repair in excision-repairdeficient cells. It is possible that an effect on an excision-repair system might explain some of the somewhat conflicting reports on caffeine effects in mammalian cells. We here report the results of experiments showing that human peripheral lymphocytes possess a caffeine-sensitive prereplication repair mechanism which can remove, presumably b y excision, some b u t not all of the MMC-induced lesions that give rise to SCE. Materials and methods
Cell culture and treatment 0.5 ml of whole, heparinized human peripheral blood was used for each culture, which contained 10 ml of Eagle's minimal essential medium (Gibco) containing 20% foetal calf serum (Gibco). Cultures were treated with 150 ng/ml of MMC (Sigma) for 4 h at 37°C in a 5% CO2 atmosphere. The cells were then washed twice in fresh medium to remove free MMC and resuspended in 10 ml of medium to which 0.25 ml of PHA (Gibco) and 5-bromodeoxyuridine (BrdU; Calbiochem, final concentration 25 pM) were added. Caffeine (Calbiochem) was added at the times and levels noted. Cultures were incubated in 5% CO2 at 37°C for 72 h in the dark. 2 h before harvesting the cells 0.15 ml of colcemid (Gibco) was added to each culture. Slide preparation Cultures were centrifuged and resuspended in 75 mM KCI hypotonic solution for 12 min, fixed in 3 : 1 methanol--acetic acid for 30 rain, twice washed with fresh fixative, d r o p p e d onto glass slides and air dried. Staining was done essentially according to Goto et al. [13]. Slides were stained for 10 min in 10 -4 M Hoechst 33258 (a generous gift of Dr. H. Loewe of Farbwerke Hoechst), immersed in water and m o u n t e d in pH 7.0 citrate-phosphate buffer (0.174 M Na2HPO4 and 0.013 M citrate). These slides were illuminated with two 15-W black light tubes (Westinghouse) for a b o u t 20 min following which they were stained in 2% Giemsa (Harleco) in 0.05 M phosphate buffer at pH 6.8 for 20 min. Upon removal from the stain the slides were soaked in two changes of acetone, one of 1 : 1 acetone--toluene, two of toluene, and then air dried. Cover slips were m o u n t e d with Permount (Fisher). Scoring SCE were scored in all of the chromosomes of 20 intact metaphase spreads for each experimental point. Results and discussion The results of an experiment to determine the effect of various concentrations of caffeine on SCE yield are shown in Fig. 1. N o t only did the caffeine
421 I
I
]
I
J
80
7O ._J .J w
,-r i,i (3. LmJ (.9 Z
60
.50
T (..) X W O t'O n." -crj
40
30
nr"
W I--
2O
I0
0 0
I
I
50
I00 CAFFEINE
I 200
I 300
(/J.g/ml)
Fig. 1. E f f e c t o f c a f f e i n e o n " s p o n t a n e o u s " a n d M M C - i n d u c e d S C E . Cells w e r e t r e a t e d w i t h M M C at 1 5 0 n g / m l f o r 4 h , w a s h e d , a n d t h e n c u l t u r e d i n t h e p z e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f c a f f e i n e in t h e medium.
itself induce some SCE, but it clearly interacted synergistically to produce large increases in the yields of MMC-induced SCE. The higher doses of caffeine evidently produced cell-cycle delay, for there were fewer second metaphase figures in these samples. For this reason a caffeine concentration of 200 pg/ml was used for the rest of the experiments. Speculating that the caffeine enhancement might, as in E. coli, result from inhibition of a prereplication excision form of D N A repair, we did a typical "liquid holding" experiment. After MMC treatment, cells were washed and then held in fresh medium without PHA for various times before incubation with PHA, BrdU and, where appropriate, caffeine. The results are presented in Fig. 2. There is clearly no effect of holding time on the SCE yields in cells which were not MMC-treated, cultured either with or without caffeine, nor in cells treated with MMC and cultured in absence of caffeine. On the other hand, the yield of MMC-induced SCE clearly decreases during the first 18 h of liquid holding in cells cultured in the presence of caffeine: after 18 h the addition of caffeine no longer resulted in any increase over that induced by the caffeine alone (i.e., over additivity). These results may be interpreted as confirming the existence
422 I
F
T
70
_I .J l.lJ o n,hl O.q') hi (.9 Z ,,::[ "r 0 X bJ
60
+CAF
50
a
\ +MMC
/
40 -CAF
r-,
30 o -r w" hi b"
20 +CAF
\ /
IO
-MMC
- CAF
I
0
0
6
I
I
18
30
HOLDING TIME BEFORE STIMULATION
48
(h )
Fig. 2. Effect o f "liquid h o l d i n g " t i m e o n SCE frequencies. A f t e r 4 h in the presence (circles) or absence (triangles) o f MMC ( 1 5 0 n g / m l ) , cells w e r e w a s h e d and t h e n held, u n s t i m u l a t e d , in fresh m e d i u m w i t h o u t P H A o r B r d U for the i n d i c a t e d times prior t o the a d d i t i o n o f P H A a n d BrdU w i t h (closed s y m b o l s ) or w i t h o u t ( o p e n s y m b o l s ) caffeine.
of the caffeine-sensitive DNA-repair mechanism in these cells; one which is clearly pre-replicational, since human lymphocytes are in a Go state until after PHA stimulation, and do not synthesize any D N A during at least the first 12 h following stimulation. Our result appears in conflict with that recently reported by Palitti and Bechetti [ 3 0 ] , w h o found that 10 -3 M caffeine failed to increase the induction of SCE in MMC-treated Chinese hamster cells. However, since rodent cells have only a poor prereplication excision system for UV-induced pyrimidine dimers, it may be that Chinese hamster cells are deficient in their ability to excise MMC-induced lesions as well. A caffeine enhancement of SCE induction in h u m a n lymphocytes by psoralin plus U V light,which like M M C causes D N A strand--strand crosslinks,has recently been reported by Waksvik, Brogger and Stene [42]. While their remits do not establish that this was a prereplication effect, it is possible that at least part of their caffeine effect was on a repair system similar to or the same as that affected in the present experiments. A possible explanation for the lack of any change in MMC-induced S C E yield with liquid holding in cellscultured in the absence of caffeine is that the repair system already effects the m a x i m u m repair possible during the pre-DNA synthesis phase of the PHA-stimulated portion of the incubation period. Fig. 3
423 70
.J ..J hi (_:, E: LLI {3...
LIJ (.9 Z "tO X ILl El I"-
I
I
I
60
+CAF
50
Q
\ +MMC
/
40
-CAF 30
0 3:: {Z: I,I I'--
+CAF
20 A
\ /
-MMC
I0 - CAF
0 0
I
t
I
6
12
18
TIME
AFTER
STIMULATION
24
(h )
Fig. 3. E f f e c t o f c u l t u r e t i m e prior t o a d d i t i o n o f c a f f e i n e o n SCE y i e l d s . A f t e r 4 h in the p r e s e n c e ( c i r c l e s ) or a b s e n c e (triangles) o f M M C , cells w e r e w a s h e d and t h e n c u l t u r e d in the p r e s e n c e o f P H A and B r d U . C a f f e i n e w a s a d d e d t o s o m e o f t h e c u l t u r e s ( c l o s e d s y m b o l s ) at t h e i n d i c a t e d t i m e s , b u t n o t t o others (open symbols).
shows the results of an experiment which confirms this explanation. Following treatment with MMC, cells were washed twice and then incubated in fresh medium containing PHA and BrdU. Caffeine was added after various incubation times. During the first 12 culture hours the frequency of MMC-induced SCE decreased as a function of increasing delay in the addition of the caffeine; after 12 h no further effect was noted. The plateau of SCE frequency appears to approximate the sum of the yields from MMC alone and from caffeine alone reasonably well. The experiments presented in Figs. 2 and 3 both suggest that this caffeinesensitive repair system "saturates", and is only capable of repairing a fraction o f those MMC-induced DNA lesions capable of inducing SCE. This is in agreement with our observation, presented elsewhere [15], of third cycle, Go MMC-induced yields of SCE compatible with the persistence through at least three cell cycles of all of the D N A lesions capable o f inducing SCE that were present at the beginning of the first S phase. Why some of the MMC lesions are repairable by the prereplication system while others are not is not obvious; it could result from there being several types of lesion or from lesions being located in chromatin more or less accessable to the repair system. Recently Day [6] showed that caffeine inhibited host-cell reactivation of UV-irradiated adenovirus in human cells and inhibited excision-repair. Very
424
similar results were obtained for UV-induced mutation in E. coil (Y. Ishii, unpublished data). After UV irradiation, the bacteria were held in phosphate buffer supplemented with glucose for various times before being plated on growth medium with caffeine or with acriflavine, another inhibitor of excisionrepair [ 8 , 3 4 , 4 4 ] . The UV-induced mutation frequency was decreased in a manner similar to Fig. 2 (the same effect has also been reported for MMCinduced mutations and acriflavine [22] ). Another UV experiment gave results similar to those of the experiment illustrated in Fig. 3. UV-irradiated E. coli were cultured in growth medium for various times and then plated onto agar medium with caffeine or with acriflavine. The frequency of UV-induced mutations decreased as the interval prior to caffeine or acriflavine increased, during the UV-induced growth delay period. It thus appears that the possession of caffeine-sensitive prereplication DNA-repair systems is a property of many types of cells, and that it influences the effectiveness of a variety of mutagenic and carcinogenic agents. References 1 Arlett, C.F., and S.A. Harcourt, The i n d u c t i o n of 8-azaguanine-resistant m u t a n t s in cultured Chinese h a m s t e r ceils b y ultraviolet light: the effect of changes in post-irradiation conditions, Mut a t i on Res., 14 (1972) 431---437. 2 Bendigkeit, H.E., and P.C. Hanawalt, Caffeine i n h i b i t i o n of DNA repair replication in Eschertchta colt, Bacteriological Proceedings 1968, Abstracts of the 6 8 t h A nnua l Meeting, p. 36. 3 Brogger, A., Caffeine-induced e n h a n c e m e n t of c h r o m o s o m e damage in h u m a n l y m p h o c y t e s treated with m e t h y l m e t h a n e s u l p h o n a t e , m i t o m y e i n C and X-rays, Mut a t i on Res., 23 (1974) 353--360. 4 Clarke, C.H., Caffeine- and a m i n o acid-effects u p o n try + revertant yield in UV4rradiated hcr+ and hcrm u t a n t s of E. colt B/r, Mol. Gen. Genet., 99 (1967) 97--108. 5 Cleaver, J.E., and G.H. Thomas, Single strand i n t e r r u p t i o n s in DNA and the effects of caffeine in Chinese h a m s t e r cells irradiated w i t h ultraviolet Hght, Biochem. Biophys. Res. Commun., 36 (1969) 203--208. 6 Day I n , R.S., Caffeine i n h i b i t i o n of the repair of ultraviolet-irradiated adenovirus in h u m a n cells, M u t a t i o n Res., 33 (1975) 321--326. 7 Donovan, P~I., and J.A. DiPaolo, Caffeine e n h a n c e m e n t of chemical carcinogen-induced transformat i o n of cultured Syrian hamster ceils, Cancer Res., 34 (1974) 2720--2727. 8 Feiner, R.R., and R. Hill, Effect of basic dyes on host-cell reactivation of ultra violet-irradiated phage, Nature (London), 200 (1963) 2 9 1 - - 2 9 3 . 9 Fo x, M., The effect of p o s t - t r e a t m e n t w i t h caffeine on survival and UV-induced m u t a t i o n frequencies in Chinese h amster and mouse l y m p h o m a cells in vitro, M u t a t i o n Res., 24 (1974) 187--204. 10 Fre',', J.V., an d S. Venitt, C h r o m o s o m e damage in the bone ma rrow of mice t r e a t e d w i t h the me t hyl ating agents m e t h y l m e t h a n e s u l p h o n a t e and N-methyl-N-nitrosourea in t he presence or absence of caffeine, and its relationship w i t h t h y m o m a i n d u c t i o n , Mut a t i on Res., 30 (1975) 89--96. 11 Fujiwara, Y., Characteristics of DNA synthesis following ultraviolet light irradiation in mouse L cells. Postreplication repair, Exp. Cell Res., 75 (1972) 483---489. 12 Fujiwaxa, Y., and T. K o n d o , Caffeine-sermitive repair of ultraviolet light-damaged DNA of mouse L cells, Biochem. Biophys. Res. C o m m u n . , 47 (1972) 552--564. 13 Goto, K., T. A k e m a t s u , H. Shimazu and T. Sugiyama, Simple differential Giemsa staining of sister c h r o m a t i d s after t r e a t m e n t w i t h photosensitive dyes and exposure to light and the me c ha ni s m of staining, C h r o m o s o m a , 53 (1975) 223--230. 14 Hartley-Asp, B., and B.A. Kihiman, Caffeine, caffeine derivatives and c h r o m o s o m a l aberration, IV. Synergism b e t w e e n m i t o m y c i n C end caffeine in Chinese hamster cells, Hereditas, 69 (1971) 326--326. 15 Ishii, Y., and M. A Bender, Factors influencing the frequency of m i t o m y c i n C-induced sister-chrom a t i d exchanges in 5 - b r o m o d e o x y u r i d i n e - s u b s t i t u t e d h u m a n l y m p h o c y t e s in culture, Mutation Res., 51 (1978) 411--416. 16 Kakunaga, T., Caffeine inhibits cell t r a n s f o r m a t i o n by 4-nitroqutnoline-l-oxide, Nature (London), 259 (1975) 248--250. 17 Kato, H., I n d u c t i o n of sister c h r o m a t i d exchanges by UV light a nd its i n h i b i t i o n by caffeine, Exp. Cell Res., 82 (1973) 383--390.
425 1 8 K a t o , H . , I n d u c t i o n o f sister c h r o m a t i d e x c h a n g e s b y c h e m i c a l m u t a g e n s a n d its p o s s i b l e r e l e v a n c e t o D N A r e p a i r , E x p . Cell R e s . , 8 5 ( 1 9 7 4 ) 2 3 9 - - 2 4 7 . 1 9 K i h l m a n , B . A . , S. S t u r e l i d , B. H a r t l e y - A s p a n d K . N i l s s o n , C a f f e i n e p o t e n t i a t i o n o f t h e c h r o m o s o m e d a m a g e p r o d u c e d in b e a n r o o t t i p s a n d in C h i n e s e h a m s t e r cells b y v a r i o u s c h e m i c a l a n d p h y s i c a l a g e n t s , M u t a t i o n R e s . , 17 ( 1 9 7 3 ) 2 7 1 - - 2 7 5 . 2 0 K i h l m a n , B . A . , S. S t u r e l i d , B. H a r t l e y o A s p a n d K . N i l s s o n , T h e e n h a n c e m e n t b y c a f f e i n e o f t h e freq u e n c i e s o f c h r o m o s o m a l a b e r r a t i o n s i n d u c e d in p l a n t a n d a n i m a l cells b y c h e m i c a l a n d p h y s i c a l agents, Mutation Res., 26 (1974) 105--122. 21 K o n d o , S., M i s r e p a i r m o d e l f o r m u t a g e n e s i s a n d c a r c i n o g e n e s i s , i n : P . N . M a g e e et al. (Eds.), F u n d a m e n t a l s in C a n c e r P r e v e n t i o n , U n i v . o f T o k y o Press, T o k y o / U n i v . P a r k Press, B a l t i m o r e , 1 9 7 6 , p p . 417--429. 2 2 K o n d o , S., H . I c h i k a w a , K . I w o a n d T. K a t o , B a s e - c h a n g e m u t a g e n e s i s a n d p r o p b a g e i n d u c t i o n in s t r a i n s o f E s c h e r i c h i a coli w i t h d i f f e r e n t D N A r e p a i r c a p a c i t i e s , G e n e t i c s , 6 6 ( 1 9 7 0 ) 1 8 7 - - 2 1 7 . 2 3 L e h m a n n , A . R . , E f f e c t o f c a f f e i n e o n D N A s y n t h e s i s in m a m m a l i a n cells, B i o p h y s . J., 1 2 ( 1 9 7 2 ) 1316--1325. 2 4 L e h m a n n , A . R . , a n d S. K i r k - B e l l , P o s t - r e p l i c a t i o n r e p a i r o f D N A in u l t r a v i o l e t - i r r a d i a t e d m a m m a l i a n cells, N o g a p s in D N A s y n t h e s i z e d l a t e a f t e r u l t r a v i o l e t i r r a d i a t i o n , E u r . J. B i o c h e m . , 31 ( 1 9 7 2 ) 4 3 8 - 445. 2 5 L e h m a n n , A . R . , a n d S. K i r k - B e l l , E f f e c t s o f c a f f e i n e a n d t h e o p h y l l i n e o n D N A s y n t h e s i s i n u n i r r a d i a t e d a n d U V - i r r a d i a t e d m a m m a l i a n cells, M u t a t i o n R e s . , 2 6 ( 1 9 7 4 ) 7 3 - - 8 2 . 2 6 L i e b , M., E n h a n c e m e n t o f u l t r a v i o l e t - i n d u c e d m u t a t i o n i n b a c t e r i a b y c a f f e i n e , Z. V e r e r b u n g s i . , 9 2 (1961) 416--429. 27 L u m b , J . R . , A . S . S i d e r o p o u l o s a n d D . M . S h a n k e l , I n h i b i t i o n o f d a r k r e p a i r o f u l t r a v i o l e t d a m a g e in D N A b y c a f f e i n e a n d 8 - c h l o r o c a f f e i n e , K i n e t i c s o f i n h i b i t i o n , Mol. G e n . G e n e t . , 1 0 2 ( 1 9 6 8 ) 1 0 8 - 111. 2 8 M e t z g e r , K . , O n t h e d a r k r e a e t i v a t i o n m e c h a n i s m in u l t r a v i o l e t i r r a d i a t e d b a c t e r i a , B i o e h e m . B i o p h y s . Res. Commun., 15 (1964) 101--109. 2 9 N i l s s o n , K . , a n d A . R . L e h m a n n , T h e e f f e c t o f m e t h y l a t e d o x y p u r i n e s o n t h e size o f n e w l y - s y n t h e s i z e d D N A a n d o n t h e p r o d u c t i o n o f c h r o m o s o m e a b e r r a t i o n s a f t e r U V i r r a d i a t i o n in C h i n e s e h a m s t e r cells, Mutation Res., 30 (1975) 255--266. 30 Palitti, F., and A. Becchetti, Effect of caffeine on sister-chromatid exchanges and chromosomal aberr a t i o n s i n d u c e d b y m u t a g e n s i n 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 . , 4 5 ( 1 9 7 7 ) 1 5 7 - - 1 5 9 . 31 R o b e r t s , J . J . , a n d J . E . S t u r r o c k , E n h a n c e m e n t b y c a f f e i n e o f N - m e t h y l - N o n i t r o s o u r e a i n d u c e d m u t a t i o n s a n d c h r o m o s o m e a b e r r a t i o n s i n 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 . , 2 0 ( 1 9 7 3 ) 2 4 3 - - 2 5 5 . 3 2 R o b e r t s , J~I., J . E . S t u r o c k a n d K . N . W a r d , T h e e n h a n c e m e n t b y c a f f e i n e of a l k y l a t i o n - i n d u c e d cell d e a t h , m u t a t i o n s a n d c h r o m o s o m a l a b e r r a t i o n s in C h i n e s e h a m s t e r cells, as a r e s u l t o f i n h i b i t i o n o f post-replication DNA repair, Mutation Res., 26 (1974) 129--143. 3 3 S a u e r b i e r , W., I n h i b i t i o n o f h o s t cell r e a c t i v a t i o n in p h a g e T1 b y c a f f e i n e , B i o c h e m . B i o p h y s . Res. Commun, 14 (1964) 340--346. 3 4 S e t l o w , R . B . , P h y s i c a l c h a n g e s in m u t a g e n e s i s , J . Cell. C o m p . P h y s i o l . , 6 4 ( S u p p l . 1) ( 1 9 6 4 ) 5 1 - - 6 8 . 3 5 S h a n k e l , D . M . , " M u t a t i o n a l s y n e r g i s m " o f u l t r a v i o l e t l i g h t a n d c a f f e i n e in E s c h e r i c h i a coli, J. B a c teriol., 84 (1962) 410---415. 36 Sideropoulos, A.S., and D.M. Shankel, Mechanism of caffeine enhancement of mutations induced by s u b l e t h a l u l t r a v i o l e t d o s a g e s , J. B a e t e r i o l . , 9 6 ( 1 9 6 8 ) 1 9 8 - - 2 0 4 . 37 S h i m a d a , K . , a n d Y . T a k a g i , T h e e f f e c t o f c a f f e i n e o n t h e r e p a i r o f u l t r a v i o l e t - d a m a g e d D N A i n b a c teria, Biochim. Biophys. Acta, 145 (1967) 763--770. 3 8 T r o s k o , J . E . , a n d E . H . Y . C h u , E f f e c t s o f c a f f e i n e o n t h e i n d u c t i o n o f m u t a t i o n s in C h i n e s e h a m s t e r cells b y u l t r a v i o l e t l i g h t , M u t a t i o n R e s . , 1 2 ( 1 9 7 1 ) 3 3 7 - - 3 4 0 . 39 Trosko, J.E., and E.H.Y. Chu, Inhibition of repair of UV-damaged DNA by caffeine and mutation 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, C h e m . - B i o l . I n t e r a c t . , 6 ( 1 9 7 3 ) 3 1 7 - - 3 3 2 . 4 0 T r o s k o , J . E . , P. F r a n k , E . H . Y . C h u a n d J . E . B e c k e r , C a f f e i n e i n h i b i t i o n o f p o s t r e p l i e a t i o n r e p a i r o f N - a c e t o x y - 2 - a c e t y l a m i n o f l u o r e n e - d a m a g e d D N A i n C h i n e s e h a m s t e r cells, C a n c e r R e s . , 3 3 ( 1 9 7 3 ) 2444--2449. 41 V a n d e n B e r g , H . W . , a n d J . J . R o b e r t s , P o s t - r e p l i c a t i o n r e p a i r o f D N A in C h i n e s e h a m s t e r cells t r e a t e d with cis-platinum(II) diammine dichloride, Enhancement of toxicity and chromosome damage by caffeine, Mutation Res., 33 (1975) 279--284. 4 2 W a k s v i k , H . , A . B r o g g e r a n d J . S t e n e , P s o r a l i n / U V A t r e a t m e n t a n d c h r o m o s o m e s , I. A b e r r a t i o n s a n d sister c h r o m a t i d e x c h a n g e in h u m a n l y m p h o c y t e s in v i t r o a n d s y n e r g i s m w i t h c a f f e i n e , H u m a n G e n e t . , 38 (1977) 195--207. 4 3 W i t k i n , E . M . , P o s t - i r r a d i a t i o n m e t a b o l i s m a n d t h e t i m i n g o f u l t r a v i o l e t - i n d u c e d m u t a t i o n s in b a c t e r i a , P r o c . 1 0 t h I n t e r n . C o n g r . o f G e n e t . , V o l . 1, M o n t r e a l , C a n a d a , 1 9 5 8 , p p . 2 8 0 - - 2 9 9 . 44 Witkin, E.M., Modification of mutagenesis initiated by ultraviolet light through post-treatment of bact e r i a w i t h b a s i c d y e s , J . Cell. C o m p . P h y s i o l . , 5 8 ( S u p p l . 1) ( 1 9 6 1 ) 1 3 5 - - 1 4 4 . 45 Witkin, E.M., and E.L. Farcluharson, Enehancement and diminution of ultraviolet-light-initiated mutagenesis b y p o s t - t r e a t m e n t w i t h c a f f e i n e in E s c h e r i c h i a eoU, i n : G . E . W . W o i s t e n h o l m e a n d M. O ' C o n n o r ( E d s . ) , M u t a t i o n as C e l l u l a r P r o c e s s , C i b a F o u n d . S y r u p . 1 9 6 9 , p p . 3 6 - - 4 9 .
