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Synergistic effect of the carcinogen 4-nitroquinoline 1-oxide and the mutagen caffeine on mammalian cells. White, James Franklin 1971

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THE SYNERGISTIC EFFECT OF THE CARCINOGEN 4-NITROQUINOLINE I-OXIDE AND THE MUTAGEN CAFFEINE ON MAMMALIAN CELLS by JAMES FRANKLIN WHITE B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1969 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o f t h e R e q u i r e m e n t s f o r t h e Degree o f MASTER OF SCIENCE i n G e n e t i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h C o l u m b i a J u n e , 1971 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r ee t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t he Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f GENETICS The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date JUNE 23 , 1971 i ABSTRACT Numerous s t u d i e s on b a c t e r i a l s y s t ems have shown t h a t c e l l s u r v i v a l f o l l o w i n g e x p o s u r e t o v a r i o u s mutagens i s g r e a t l y i n f l u e n c e d by t h e c a p a c i t y o f t h e c e l l s t o r e p a i r t h e i n d u c e d DNA l e s i o n s . C a f f e i n e ( 1 - 3 - 7 -t r i m e t h y I x a n t h i n e ) , has been shown t o r e d u c e t h i s r e p a i r - c a p a c i t y t h e r e b y p r o d u c i n g m u t a g e n i c , and c y t o t o x i c e f f e c t s . The q u e s t i o n was r a i s e d as t o w h e t h e r o r n o t c a f f e i n e m i g h t r e d u c e t h e r e p a i r - c a p a c i t y o f mammalian c e l l s by i n t e r a c t i n g w i t h t h e DNA l e s i o n s p r o d u c e d by e i t h e r t h e c a r c i n o g e n i c , and o n c o g e n i c 4 - n i t r o q u i n o I i n e l - o x i d e , o r u l t r a v i o l e t l i g h t (UV) i r r a d i a t i o n . T h i s was o f i n t e r e s t due t o t h e p o t e n t i a l a p p l i c a t i o n s t h a t s y n e r g i s t i c r e l a t i o n s h i p s may h o l d f o r c h e m o t h e r a p y . An e s t a b l i s h e d l i n e o f S y r i a n - h a m s t e r c e l l s ( BHK -21 , c l o n e 1 3 ) , was used t h r o u g h o u t t h e e x p e r i m e n t s . A r g i n i n e d e p r i v a t i o n was emp loyed t o a r r e s t t h e c u l t u r e d c e l l s a t G | - p h a s e . The m u t a g e n i c 4NQ0, UV, and c a f f e i n e were a p p l i e d t o t h e s e n o n - d i v i d i n g c e l l s . The c a f f e i n e e x p o s u r e was a t v a r y i n g t i m e i n t e r v a l s p r i o r t o , d u r i n g , and a f t e r 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s ( u n s c h e d u l e d DNA s y n t h e s i s ) . S i m i l a r l y , c a f f e i n e was added t o c e l l s i m m e d i a t e l y f o l l o w i n g U V - i n d u c e d D N A - r e p a i r s y n t h e s i s . E x p o s u r e o f BHK-21 c e l l s t o c a f f e i n e , and 4NQ0 a ppea red t o r e d u c e t h e i r c o l o n y - f o r m i n g a b i l i t y t o a g r e a t e r e x t e n t t h a n when t h e c e l l s were e xpo sed t o e i t h e r c h e m i c a l a l o n e . The a d d i t i o n o f c a f f e i n e comb ined w i t h 4NQ0 t o c e l l s , i n G j - p h a s e , d i d n o t a p p e a r t o s i g n i f i c a n t l y i n f l u e n c e t h e i r r a t e o f f l o w i n t o S - p h a s e , o r t h r o u g h t o m e t a p h a s e . The e f f e c t o f c a f f e i n e on 4NQ0- ( b u t n o t UV- ) i n d u c e d D N A - r e p a i r s y n t h e s i s , u s i n g i s o t o p e l a b e l l i n g , and a u t o r a d i o g r a p h y s u g g e s t e d a r e d u c t i o n i n t h e amount o f D N A - r e p a i r s y n t h e s i s . T h i s r e d u c t i o n a p p e a r e d t o be dependen t on t h e c a f f e i n e c o n c e n t r a t i o n , and on t h e t i m e o f e x p o s u r e o f t h e c e l l s t o t h i s c h e m i c a l . An i n i t i a l i n q u i r y was made i n t o w h e t h e r o r n o t t h i s s y n e r g i s t i c e f f e c t between c a f f e i n e and 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s c o u l d be d e t e c t e d i n human l y m p h o c y t e s i n v i t r o . However , v e r y low l e v e l s o f 4NQ0 - i nduced DNA-r e p a i r s y n t h e s i s were o b s e r v e d i n t h e s e c e l l s . I t was t h e r e f o r e n o t c o n d u c i v e f o r t h e a u t o r a d i o g r a p h i c a n a l y s i s o f D N A - r e p a i r s y n t h e s i s f o l l o w i n g 4NQ0 e x p o s u r e . A model e x p l a i n i n g t h e a p p a r e n t c a f f e i n e s u p p r e s s i o n o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s i s p o s t u l a t e d . The p o t e n t i a l m e d i c a l i m p l i c a t i o n s a r e d i s c u s s e d . i i i ACKNOWLEDGEMENTS The work i n t h i s t h e s i s was p e r f o r m e d under t h e s u p e r v i s i o n o f D r . H.F. S t i c h , C a n c e r R e s e a r c h C e n t e r , U n i v e r s i t y o f B r i t i s h C o l u m b i a whose d i r e c t i o n and a d v i c e i s g r e a t l y a p p r e c i a t e d . I wou ld l i k e t o e s p e c i a l l y t h a n k M r s . S t i c h whose e x c e l l e n t t e c h n i c a l a s s i s t a n c e and a d v i c e i s a l s o g r a t e f u l l y a c k n o w l e d g e d . F i n a l l y , I t h a n k D r . R .L . N o b l e f o r p r o v i d i n g t h e n e c e s s a r y s p a c e and e q u i p m e n t . i V TABLE OF CONTENTS Page ABSTRACT j ACKNOWLEDGEMENTS i i i TABLE OF CONTENTS i v L I ST OF TABLES v i L I ST OF FIGURES v i i INTRODUCTION , I A. C a f f e i n e M u t a g e n i c i t y I B. S y n e r g i s t i c E f f e c t s 2 1. R e s u l t s w i t h C a f f e i n e and U l t r a v i o l e t L i g h t ( U V ) 2 2 . R e s u l t s w i t h C a f f e i n e and O t h e r A g e n t s 4 C. C l i n i c a l and E x p e r i m e n t a l C o n s i d e r a t i o n s 4 PROBLEM 6 EXPERIMENTAL 7 A. Methods and M a t e r i a l s 7 1. C e l I T ype s 7 2 . T i s s u e C u l t u r e 7 3. C h e m i c a l and P h y s i c a l Mutagens 8 4 . U n s c h e d u l e d DNA S y n t h e s i s 9 5. A u t o r a d i o g r a p h i c A n a l y s i s II 6. C e l I S u r v i v a l 12 7. CeI I C y c I e 13 8. U n s c h e d u l e d DNA s y n t h e s i s i n Human L ymphocy te s 14 a 9. E v a l u a t i o n o f E x p e r i m e n t s 14 b B. R e s u l t s 15 I. DNA R e p a i r S y n t h e s i s Induced by 4NQ0 i n t h e P r e s e n c e o r Ab sence o f C a f f e i n e 15 2 . DNA R e p a i r S y n t h e s i s Induced by UV i n t h e P r e s e n c e o r Ab sence o f C a f f e i n e 18 3. S u r v i v a l 20 4 . C e l I C y c l e A n a l y s i s 24 5. DNA R e p a i r S y n t h e s i s i n Human Lymphocy te s i n v i t r o 27 DISCUSSION 29 A. S p e c u l a t i o n and O u t l o o k 35 BIBLIOGRAPHY 39 L IST OF TABLES e No, The e f f e c t o f c a f f e i n e and 4NQ0 on t h e p r o g r e s s i o n o f c e l l s f r o m G j - p h a s e i n t o S - p h a s e , The e f f e c t o f c a f f e i n e and 4NQ0 on t h e f l o w o f c e l l s f r o m G| -pha se t o m e t a p h a s e . A model f o r e x c i s i o n r e p a i r . L IST OF FIGURES F i g u r e 1. The e f f e c t o f c a f f e i n e on 4NQ0 - i nduced DNA-r e p a i r s y n t h e s i s . 2 . The t i m e - d e p e n d e n t e f f e c t o f c a f f e i n e on 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s . 3 . The e f f e c t o f c a f f e i n e on U V - i n d u c e d DNA-r e p a i r s y n t h e s i s . 4 . The e f f e c t o f c a f f e i n e , and 4NQ0 on t h e c o l o n y - f o r m i n g a b i l i t y o f BHK-21 c e l l s . 5. The e f f e c t o f c a f f e i n e on t h e c o l o n y - f o r m i n g a b i I i t y o f BHK-21 c e l I s . 6. The e f f e c t o f c a f f e i n e p r e - t r e a t m e n t on c e l l c o I o n y - f o r m i ng ab i M t y . 7. D N A - r e p a i r s y n t h e s i s i n human l y m p h o c y t e s i n v i t r o . INTRODUCTION The q u e s t i o n o f t h e m u t a g e n i c i t y o f c a f f e i n e ( 1 - 3 - 7 - t r i m e t h y I x a n t h i n e ) , a r o s e f r o m t h e f e a r t h a t , due t o i t s s i m i l a r i t y t o t h e n a t u r a l p u r i n e n u c l e o s i d e s , i t m i g h t be s u b s t i t u t e d f o r t h e s e ba se s i n t o DNA. Numerous s t u d i e s on v i r u s e s , b a c t e r i a , p l a n t s , i n s e c t s , and mammals have p r oduced an a c c u m u l a t i o n o f f a c t s and p r e d i c t i o n s . W i t h r e s p e c t t o t h e l a t t e r , c a f f e i n e has been c i t e d as one o f t h e most dange rou s mutagens consumed by man ( 3 8 ) . A. C a f f e i n e M u t a g e n i c i t y C a f f e i n e p o s s e s s e s b o t h a l e t h a l , and m u t a g e n i c e f f e c t i n c e r t a i n s t r a i n s o f b a c t e r i a ( 9 , 1 0 , 2 0 , 2 3 , 4 5 ) , and i n c r e a s e s t h e m u t a t i o n r a t e i n a s c o m y c e t e s Oph i o s t o m a ( 1 8 ) . S t u d i e s w i t h h i g h e r p l a n t s , ( V i c i a f a b a , AI I i urn c e p a , and P i sum s a t i v u m ) , have shown t h a t c a f f e i n e c a u s e s a s e v e r e m i t o t i c d e p r e s s i o n , i n h i b i t s f o r m a t i o n o f t h e c e l l w a l l r e s u l t i n g i n p o l y p l o i d y , and i n d u c e s c h r o m o s o m e . a b e r r a t i o n s ( 3 2 , 3 3 , 3 4 ) . S i m i l a r l y , a r e d u c t i o n i n t h e m i t o t i c r a t e i n c u l t u r e s o f f e t a l human k i d n e y c e l l s has been d e s c r i b e d f o l l o w i n g 8 - e t h o x y c a f f e i n e t r e a t m e n t ( 1 6 ) . Chromosome a b e r r a t i o n s have a l s o been o b s e r v e d i n human l e u k o c y t e s a f t e r t h e i r e x p o s u r e t o 8 - e t h o x y c a f f e i n e , as w e l l as i n c a f f e i n e t r e a t e d HeLa c e l l s ( 2 9 , 4 6 ) . However , c a f f e i n e m u t a g e n i c i t y has no t been c l e a r l y d e m o n s t r a t e d i n v i v o . R e p o r t s t h a t c a f f e i n e i n d u c e s s e x - l i n k e d r e c e s s i v e l e t h a l m u t a t i o n s i n D r o s o p h i l a ( 3 , 4 7 ) , have been b a l a n c e d by c o n t r a d i c t o r y f i n d i n g s ( 2 , 7 2 ) . A t t e m p t s t o e v a l u a t e t h e e f f e c t o f c a f f e i n e , on t h e r a t e o f n o n - d i s j u n c t i o n i n D r o s o p h i l a , have a l s o p r o d u c e d b o t h p o s i t i v e , and n e g a t i v e r e s u l t s ( 7 , 4 3 ) . 2. R e p o r t s t h a t c a f f e i n e can i nduce chromosome b r e a k a g e , and l o s s o f t h e X-chromosome i n bo th male and f e m a l e Drosoph i I a , howeve r , a p p e a r more c o n s i s t e n t ( 7 , 4 2 ) . E x p e r i m e n t s w i t h m ice have a t t e m p t e d t o d e f i n e t h e g e n e t i c e f f e c t o f c a f f e i n e on t h e p r o d u c t i o n o f r e c e s s i v e , and dominan t l e t h a l m u t a t i o n s ( 1 , 1 4 , 3 8 , 4 0 ) . The se s t u d i e s have p r o d u c e d p r i m a r i l y n e g a t i v e r e s u l t s , a l t h o u g h i n one c a s e , t h e p r egnancy r a t e o f f e m a l e m i ce mated t o c a f f e i n e t r e a t e d male m i ce was r e d u c e d ( 1 4 ) . B. S y n e r g i s t i c E f f e c t s I. R e s u l t s w i t h c a f f e i n e and u l t r a v i o l e t l i g h t (UV) T h e r e a r e t h r e e main r e p a i r mechan i sms , known i n b a c t e r i a , w h i c h p romote s u r v i v a l a f t e r U V - e x p o s u r e : p h o t o r e a c t i v a t i o n ( 3 0 , 3 1 ) , e x c i s i o n r e p a i r ( 5 , 5 7 ) , and p o s t r e p l i c a t i o n (recomb i n a t i o n a I r e p a i r ) , ( 2 7 , 3 7 , 5 4 , 6 7 ) . S t u d i e s have f a i l ed t o d e m o n s t r a t e t h a t c a f f e i n e i n h i b i t s p h o t o r e a c t i v a t i o n o f U V - i n d u c e d p y r i m i d i n e d i m e r s i n s t r a i n s o f E. c o I i ( 3 9 , 4 1 ) . However , e a r l y e x p e r i m e n t s showed t h a t c a f f e i n e i n c r e a s e d t h e l e t h a l , and m u t a g e n i c e f f e c t o f c e r t a i n UV - i r r a d i a t e d s t r a i n s o f E_. co I i . T h i s o c c u r r e d when n o n -t o x i c , and n o n - m u t a g e n i c c o n c e n t r a t i o n s o f c a f f e i n e were added t o t h e p o s t -i r r a d i a t i o n p l a t i n g medium ( 3 9 , 5 9 , 6 3 ) . L a t e r e x p e r i m e n t s d e m o n s t r a t e d t h a t t h i s m u t a t i o n a l (and l e t h a l ) s y n e r g i s m was o n l y e v i d e n t i n b a c t e r i a l s t r a i n s c a p a b l e o f e x c i s i o n r e p a i r ( H c r + ) , and no t i n s t r a i n s w i t h a d e f e c t i v e e x c i s i o n r e p a i r (Her ) mechani sm ( 6 , 4 1 , 6 0 ) . F u r t h e r m o r e , f r om t h e r e s u l t s , o f one o f t h e s e e x p e r i m e n t s , i t was c o n c l u d e d t h a t c a f f e i n e a f f e c t s e x c i s i o n r e p a i r by i n h i b i t i n g t h e e x c i s i o n o f U V - i n d u c e d t h y m i n e d i m e r s ( 6 0 ) . C a f f e i n e has a l s o been shown t o i n h i b i t h o s t c e l l r e a c t i v a t i o n (HCR) o f U V - i r r a d i a t e d b a c t e r i o p h a g e s , T j , and A ( 5 5 , 71 ) as w e l l as m u t a t i o n a l f r e q u e n c y d e c l i n e w h i c h has been d e s c r i b e d as a s p e c i a l f o r m of e x c i s i o n r e p a i r ( 6 5 , 6 6 , 6 8 ) . 3. An i n v e s t i g a t i o n o f t h e e f f e c t o f c a f f e i n e on some U V - i r r a d i a t e d ( H c r ~ ) s t r a i n s o f JE. c o l i has shown t h a t c a f f e i n e s l i g h t l y i n c r e a s e s t h e l e t h a l i t y ( b u t n o t t o t h e e x t e n t t h a t i t does i n H c r + s t r a i n s ) , and r e d u c e s t h e m u t a t i o n r a t e (6,60,69). T h i s a n t i m u t a g e n i c e f f e c t o f c a f f e i n e was i n t e r p r e t e d i n t e r m s o f t h e p o s t - r e p l i c a t i o n ( r e c o m b i n a t i o n a I ) r e p a i r mechan i sm i n JE. c o l i (27,37,54,67). I t was p o s t u l a t e d t h a t t h i s r e p a i r mechan i sm p romotes s u r v i v a l by c l o s i n g t h e gaps p r o d u c e d when u n e x c i s e d d i m e r s pas s t h r o u g h t h e r e p l i c a t i n g p o i n t (54,67,69). T h i s r e p a i r i s a p p a r e n t l y f a c i l i t a t e d by some f o rm o f r e c o m b i n a t i o n between c omp l emen ta r y d a u g h t e r s t r a n d s , bo th o f w h i c h may c o n t a i n g a p s , b u t a t d i f f e r e n t l e v e l s (54). F u r t h e r m o r e , e r r o r s a r e p r o d u c e d i n t h e DNA o f JE. c o l i s t r a i n s c a r r y i n g t h e w i l d t y p e gene p r o d u c t , f o r r e c o m -b i n a t i o n , d u r i n g t h i s r e c o m b i n a t i o n a I r e p a i r . Thu s , i t was p o s t u l a t e d t h a t c a f f e i n e r e d u c e s t h e e f f i c i e n c y o f t h i s gap r e p a i r t h e r e b y p r o m o t i n g c e l l l e t h a l i t y , b u t i n c r e a s e s t h e a c c u r a c y o f such r e p a i r t h e r e b y r e d u c i n g t h e number o f i n t r o d u c e d e r r o r s (67). A l e t h a l s y n e r g i s m has been r e p o r t e d between c a f f e i n e , and UV i n mouse L c e l l s (51). When t h e s e c e l l s were i n c u b a t e d i n n o n - t o x i c c o n c e n t r a t i o n s o f c a f f e i n e , f o l l o w i n g t h e i r e x p o s u r e t o UV, a r e d u c t i o n i n t h e i r c o l o n y - f o r m i n g a b i l i t y (CFA) was o b s e r v e d . T h i s r e d u c t i o n i n CFA was n o t e v i d e n t u n l e s s c a f f e i n e was p r e s e n t when t h e c e l l s p a s s ed t h r o u g h t h e i r f i r s t S - p h a s e , f o l l o w i n g t h e i r i r r a d i a t i o n . C o n s e q u e n t l y , a c a f f e i n e s e n s i t i v e r e p a i r mechani sm w h i c h o p e r a t e s d u r i n g S -pha se i n mouse L c e l l s was p o s t u l a t e d (11,12). C l e a v e r , however , c o u l d no t show a c a f f e i n e e f f e c t on U V - i n d u c e d u n s c h e d u l e d DNA s y n t h e s i s , o r r e p a i r r e p l i c a t i o n i n HeLa c e l l s (8 )• F u r t h e r m o r e , c a f f e i n e d i d n o t a f f e c t U V - i n d u c e d u n s c h e d u l e d DNA s y n t h e s i s i n human l ymphocy t e s i n v i t r o (15). 4. 2 . R e s u l t s w i t h c a f f e i n e and o t h e r a g e n t s A l t h o u g h c a f f e i n e enhance s U V - m u t a g e n e s i s i n b a c t e r i a l s t r a i n s o f E_. c o l i , i t does n o t , i n t h e s e s t r a i n s , i n c r e a s e t h e f r e q u e n c y o f X - r a y i n d u c e d m u t a t i o n s ( 1 3 ) . C a f f e i n e a l s o has no e f f e c t on t h e CFA o f X - i r r a d i a t e d mouse L c e l l s ( 5 1 ) . However , a l e t h a l s y n e r g i s m has been r e p o r t e d w i t r c a f f e i n e , and m i t o m y c i n C i n t h e s e c e l l s . T h i s r e p o r t shows t h a t a r e d u c t i o n i n t h e CFA o f m i t o m y c i n C t r e a t e d mouse L c e l l s o c c u r s when t h e y a r e i n c u b a t e d i m m e d i a t e l y f o l l o w i n g t r e a t m e n t i n n o n - t o x i c c o n c e n t r a t i o n s o f c a f f e i n e ( 5 2 ) . In v i v o e x p e r i m e n t s w i t h m i ce have f a i l e d t o d e m o n s t r a t e any m u t a t i o n a l s y n e r g i s m , between c a f f e i n e , and X - r a y s , a s d e t e r m i n e d by t h e i n d u c t i o n o f dom inan t l e t h a l m u t a t i o n s . A r e d u c t i o n i n t h e p regnancy r a t e o f f e m a l e s mated t o t r e a t e d ma le m i c e , howeve r , was o b s e r v e d ( 1 4 ) . C. C l i n i c a l and E x p e r i m e n t a l C o n s i d e r a t i o n s I t has been s u g g e s t e d t h a t t h e e x t e n s i o n o f i d e a s , and i n f o r m a t i o n , c o n c e r n i n g r e p a i r mechan i sms , and t h e i r i n h i b i t o r s f r om m i c r o b i a l s y s t ems t o e u k a r y o t i c s y s t ems i s i n v a l i d ( 5 8 ) . Thus t h e e v i d e n c e f o r c a f f e i n e i n d u c e d l e t h a l i t y , and m u t a g e n e s i s i n b a c t e r i a c a n n o t be d i r e c t l y e x t r a p o l a t e d t o man. F u r t h e r m o r e , s i n c e c a f f e i n e m u t a g e n e s i s has n o t been c l e a r l y d e m o n s t r a t e d i n v i v o ( I ) , t h e t y p e o f f o r e b o d i n g w a r n i n g as m e n t i o n e d a t t h e b e g i n n i n g o f t h i s i n t r o d u c t i o n wou ld a p p e a r t o be u n j u s t i f i e d . H o w e v e r , c e r t a i n c h a r a c t e r i s t i c s o f c a f f e i n e may w a r r a n t f u r t h e r r e s e a r c h i n t o t h i s a r e a : i t i s h i g h l y w a t e r - s o l u b l e , an a q u a - l i q u o r does n o t e x i s t ( 4 ) , i t r e a d i l y p a s s e s t h r o u g h t h e p l a c e n t a l b a r r i e r t o f e t a l t i s s u e s ( 2 1 ) , and i t i s f ound i n v a r i o u s s o f t d r i n k s , c o f f e e and a w i d e r ange o f m e d i c i n e s ( I ) . These c h a r a c t e r i s t i c s c o u p l e d w i t h t h e c o n s i d e r a t i o n t h a t l e t h a l s y n e r g i s i m has been o b s e r v e d between c a f f e i n e , and o t h e r a g e n t s i n a t l e a s t one mammalian t i s s u e c u l t u r e s y s t e m may s u g g e s t t h e i m p o r t a n c e o f c a f f e i n e t o man ( 5 1 , 5 2 ) . 5. F o r i n s t a n c e , i t has been s u g g e s t e d t h a t c a f f e i n e may be a f a c t o r i n t h e p r o d u c t i o n o f T u r n e r s ' Syndrome ( 7 ) , and t h a t m i t o m y c i n C t r e a t m e n t s may be made more e f f e c t i v e by t h e i n t r o d u c t i o n o f c a f f e i n e ( 5 2 ) . PROBLEM The c o n c e p t o f m u t a t i o n a l , and l e t h a l s y n e r g i s m , a s d e s c r i b e d i n m i c r o - o r g a n i s m s , may have i m p o r t a n c e i n c l i n i c a l m e d i c i n e . D e l e t e r i o u s m u t a t i o n s c o u l d be p r o d u c e d i n h i g h e r o r g a n i s m s , i n c l u d i n g man, by t h e s y n e r g i s t i c a c t i o n o f s e v e r a l weak mutagens consumed i n m i n u t e amount s . F u r t h e r m o r e , a l e t h a l s y n e r g i s m between t w o , o r mo re , c h e m i c a l s , o r p h y s i c a a g e n t s may have p r a c t i c a l a p p l i c a t i o n s i n t h e t r e a t m e n t o f n e o p l a s t i c c e l l s The p o s s i b i l i t y o f a s y n e r g i s t i c r e l a t i o n s h i p between c a f f e i n e , and 4NQ0, and between c a f f e i n e and u l t r a v i o l e t l i g h t ( U V ) , was i n v e s t i g a t e d i n an i n v i t r o s y s t e m . C u l t u r e d BHK-21 c e l l s were e xpo sed t o 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 a t d i f f e r e n t t i m e s p r i o r t o , d u r i n g , and a f t e r 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s . S i m i l a r l y , t h e s e c e l l s were expo sed t o c a f f e i n e i m m e d i a t e l y f o l l o w i n g U V - i n d u c e d D N A - r e p a i r s y n t h e s i s . The e f f e c t o f c a f f e i n e on D N A - r e p a i r s y n t h e s i s i n d u c e d by t h e s e a g e n t s was examined by u s i n g i s o t o p e l a b e l l i n g comb ined w i t h a u t o r a d i o g r a p h y . The c o l o n y - f o r m i n g a b i l i t y ( C F A ) , o f c e l l s t r e a t e d w i t h c a f f e i n e , 4NQ0, and a c o m b i n a t i o n o f c a f f e i n e p l u s 4NQ0, was a l s o i n v e s t i g a t e d . An i n q u i r y i n t o t h e p o t e n t i a l s y n e r g i s t i c i n f l u e n c e o f c a f f e i n e , and 4NQ0 on t h e f l o w o f c e l l s f r om t h e G| -pha se i n t o S - p h a s e , and t h r o u g h t o metaphase was p u r s u e d . The p o s s i b i l i t y o f a p p l y i n g some o f t h e above p r o c e d u r e s t o human l ymphocy te s i n v i t r o , was i n v e s t i g a t e d . 7 . EXPERIMENTAL A. Methods and M a t e r i a l s 1. C e l I t y p e s An e s t a b l i s h e d S y r i a n - h a m s t e r c e l l l i n e (BHK-21 , c l o n e 1 3 ) , was used i n t h e f o l l o w i n g e x p e r i m e n t s . T h i s c e l l l i n e i s e a s i e r t o c l o n e t h a n p r i m a r y h a m s t e r c e l l s , and i t s chromosomes a r e s i m i l a r t o t h o s e o f t h e l a t t e r , w i t h r e s p e c t t o p l o i d y . I s o l a t e d human l ymphocy te s were a l s o emp loyed i n t h i s wo rk . 2 . T i s s u e C u l t u r e (a ) BHK-21 e e l Is C e l l s were grown i n 32 o z . p r e s c r i p t i o n b o t t l e s , a t 37°C , i n E a g l e ' s M i n i m a l E s s e n t i a l Medium (MEM) w i t h H a n k ' s s a l t s and s o d i u m b i c a r -b o n a t e (4 mIs/800 mis MEM). The medium was s u p p l e m e n t e d w i t h I 0% f e t a l b o v i n e ( c a l f ) s e r u m , p e n i c i l l i n (100 u n i t s / m l . ) s t r e p t o m y c i n (100 m i c r o -g r a m s / m l ) , \% Kanamyc in and \% F u n g i z o n e . When t h e c e l l s had grown i n t o a m o n o l a y e r t h e y were removed w i t h t r y p s i n (1 .25 /0 , c e n t r i f u g e d t w i c e , and s eeded e i t h e r o n t o c o v e r - s l i p s ( i n L e i g h t o n t u b e s ) , o r i n t o 60 x 15 mm d i a m e t e r p l a s t i c p e t r i d i s h e s ( F a l c o n P a s t i e s , O x n a r d , C a l i f o r n i a ) . L e i g h t o n t u b e c u l t u r e s were i n c u b a t e d i n MEM a t 37°C f o r 2 t o 3 d a y s . P e t r i d i s h c u l t u r e s , i n MEM w i t h a d d i t i o n a l s od i um b i c a r b o n a t e added (12 m l s/800 mi s MEM), were i n c u b a t e d f o r t h e same l e n g t h o f t i m e i n a h u m i d i f i e d i n c u b a t o r ( 37°C ) s t a f f e d w i t h 5% CO2 and 95% a i r . In b o t h c a s e s , f o l l o w i n g t h e i n c u b a t i o n p e r i o d t h e c e l l s were washed w i t h MEM d e f i c i e n t i n a r g i n i n e (ADM). They were t h e n i n c u b a t e d f o r an a d d i t i o n a l 2 days i n ADM s u p p l e m e n t e d w i t h o n l y 5% c a l f s e rum. I n c u b a t i o n i n ADM f o r 2 t o 3 days a r r e s t s t h e e e l Is a t t h e G , - p h a s e o f t h e e e l I c y c l e , and t h e a d d i t i o n o f a r g i n i n e t o t h e medium (MEM) s t i m u l a t e s t h e s e c e l l s i n t o d i v i s i o n ( 1 7 , 6 1 ) . 8. (b ) Lymphocy te s A vo lume o f venous b l o o d (15 m i s ) was o b t a i n e d f r om a h e a l t h y v o l u n t e e r , and m ixed w i t h 0 .04 mis (400 u n i t s ) , o f pheno l f r e e h e p a r i n ( F i s h e r S c i e n t i f i c C o . , F a i r l a w n , N . J . ) . I t was t h e n i n c u b a t e d a t an a n g l e o f 6 0 ° ( a t 37°c) f o r 45 m i n , o r u n t i l most o f t h e r e d b l o o d c e l l s had s e t t l e d . The vo lume o f t h e l e u k o c y t e l a y e r was t h e n r e c o r d e d , removed, and p l a c e d i n t h e t o p o f a c o t t o n packed c o l u m n . The co lumn was t h e n i n c u b a t e d a t 37°C f o r 30 m i n . A t t h e end o f t h i s p e r i o d a vo lume o f MEM t w i c e t h a t o f t h e o r i g i n a l vo lume o f l e u k o c y t e s , was pou red i n t o t h e t o p o f t h e c o l u m n , and t h e e l u a t e was c o l l e c t e d a t t h e o p p o s i t e e n d . The e l u a t e was c e n t r i f u g e d , t h e s u p e r n a t a n t was removed, and t h e . p e l l e t was r e s u s p e n d e d i n MEM. 3 . C h e m i c a l and P h y s i c a l Mutagens (a ) 4 - n i t r o q u i n o l i n e l - o x i d e (4NQ0) An a c c u r a t e l y m e a s u r e d - g u a n t i t y ( 0 . 0038 gms) o f 4NQ0 (Dauch i P u r e C h e m i c a l C o . , Tokyo ) was d i s s o l v e d i n 0 .4 mis o f 100$ e t h a n o l . A vo lume o f 20 mis o f d i s t i l l e d w a t e r was t h e n added t o p r o d u c e a s t o c k s o l u t i o n o f 1x10 ^M c o n c e n t r a t i o n . T h i s s o l u t i o n was s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n , and s t o r e d i n t h e f r i d g e . W o r k i n g s o l u t i o n s o f — ^  mm "J mm "J I x1 0 , 4x10 , and 8x10 M c o n c e n t r a t i o n were p r e p a r e d by d i l u t i n g t h e s t o c k s o l u t i o n w i t h t h e a p p r o p r i a t e vo lume o f ADM. (b) C a f f e i n e ( I - 3 - 7 - t r i m e t h y I x a n t h i n e ) A p r e c i s e l y measured amount ( 0 . 3884 gms) , o f c a f f e i n e (Eas tman O r g a n i c C h e m i c a l s , R o c h e s t e r , N.Y. ) was d i s s o l v e d i n 20 mi s o f d i s t i l l e d w a t e r . T h i s p r o d u c e d a 1x10 ' M c o n c e n t r a t i o n s t o c k s o l u t i o n . 9. T h i s s o l u t i o n was s t e r i l i z e d by p a s s i n g i t t h r o u g h a m i l l i p o r e f i l t e r . I t - 3 - 4 was s t o r e d a t room t e m p e r a t u r e . W o r k i n g s o l u t i o n s o f 4x10 t o 2 .5x10 M c o n c e n t r a t i o n were p r e p a r e d by d i l u t i n g t h e s t o c k s o l u t i o n w i t h t h e a p p r o p r i a t e vo lume o f ADM. ( c ) U l t r a v i o l e t l i g h t (UV) U V was a d m i n i s t e r e d by i r r a d i a t i o n w i t h a S y l v a n i a g e r m i c i d a l lamp (G|^T8) a t a d i s t a n c e o f 7 i n c h e s . T h i s p r o d u c e d a dose o f 30 e r g s / 2 mm / s e c as measured by a U V - l i g h t m e t e r ( S c i e n t i f i c A p p a r a t u s , Thomas C o . , P h i l a d e l p h i a ) . The UV-I i g h t me te r was c a l i b r a t e d a g a i n s t E. c o l i s u r v i v a l r a t e s f o l l o w i n g U V - i r r a d i a t i o n by D r . J . Kemp (S imon F r a s e r U n i v e r s i t y , B u r n a b y , B . C . , ( S t i c h , p e r s o n a l c o m m u n i c a t i o n ) . 4 . U n s c h e d u l e d DNA S y n t h e s i s ( a ) Gene r a I U n s c h e d u l e d DNA s y n t h e s i s ( 5 0 ) , w h i c h w i l l be r e f e r r e d t o as D N A - r e p a i r s y n t h e s i s , has been shown t o . b e i n d u c e d i n mammalian c e l l s l i n e s , and s t r a i n s by v a r i o u s a g e n t s i n c l u d i n g U V ( 5 0 , 5 1 ) . 4NQ0 ( 2 5 , 61 ) m e t h y l methane s u l p h a t e ( 2 4 ) , and X - r a y s ( 2 2 ) . The o n c o g e n i c , and m u t a g e n i c 4NQ0 i n d u c e s D N A - r e p a i r s y n t h e s i s i n S y r i a n - h a m s t e r c e l l s t h r o u g h o u t t h e c e l l c y c l e ( 6 1 ) . The amount o f i n d u c e d D N A - r e p a i r s y n t h e s i s can be enumera ted by c o u n t i n g t h e number o f s i l v e r g r a i n s o v e r t h e n u c l e i o r chromosomes o f t h e s e c e l l s f o l l o w i n g i s o t o p e l a b e l l i n g , and a u t o r a d i o g r a p h y ( 6 1 ) . (b ) 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s i n t h e p r e s e n c e o r ab s ence o f c a f f e i n e  L e i g h t o n t u b e c u l t u r e s o f BHK-21 c e l l s w h i c h had been e x p o s e d - 4 - 3 t o ADM f o r 48 h r s , were e x p o s e d t o c a f f e i n e ( 2 . 5 x10 t o 4x10 M) f o r 6 , 3 , and z e r o h r s , p r i o r t o t h e comb ined a d d i t i o n o f 4NQ0 (1x10 M) , and c a f f e i n e - 4 - 3 ( 2 . 5 x 1 0 t o 4x10 M). The c u l t u r e s were i n c u b a t e d i n t h e s e 4 N Q 0 - c a f f e i n e 10. s o l u t i o n s f o r 1.5 h o u r s . These s o l u t i o n s were t h e n washed o u t , and - 4 - 3 r e p l a c e d w i t h f r e s h c a f f e i n e s o l u t i o n s ( 2 . 5 x10 t o 4x10 M) t o w h i c h was added a s u f f i c i e n t vo lume o f "^H-TdR ( S chwa r z B i o r e s e a r c h , O r a n g e b e r g , N .Y . , 17.9 c u r i e s / m i I I i m o l e ) , t o p r oduce a f i n a l c o n c e n t r a t i o n o f 10 m i c r o c u r i e s / m l . The c u l t u r e s were i n c u b a t e d f o r 1.5 h r s i n t h e s e c a f f e i ne-"^H-TdR s o l u t i o n s . In o t h e r e x p e r i m e n t s t h e c u l t u r e s were e x p o s e d t o 4NQ0 (1x10 ^M) f o r 1.5 h r s , - 4 - 3 wa shed , and t r e a t e d w i t h c a f f e i n e ( 2 . 5 x10 t o 4x10 M) f o r 1.5 h o u r s . F o l l o w i n g t h i s i n c u b a t i o n p e r i o d t h e c a f f e i n e s o l u t i o n s were washed o u t , and r e p l a c e d w i t h f r e s h c a f f e i n e s o l u t i o n s o f t h e same c o n c e n t r a t i o n s , b u t t o w h i c h was added ^H-TdR ( f i n a l c o n c e n t r a t i o n 10 m i c r o c u r i e s / m l ) . The c u l t u r e s were i n c u b a t e d i n t h e s e s o l u t i o n s f o r 1.5 h o u r s . T h r o u g h o u t t h i s e n t i r e s e r i e s t h e c o n t r o l s c o n s i s t e d o f c u l t u r e s o n l y t r e a t e d w i t h 4NQ0 (1x10 ^ M ) . E x p e r i m e n t s c o m p a r i n g d i f f e r e n t c e l l t r e a t m e n t s were a l w a y s c a r r i e d o u t a t t h e same t i m e and under i d e n t i c a l c o n d i t i o n s . ( c ) U V - i n d u c e d D N A - r e p a i r s y n t h e s i s i n t h e p r e s e n c e o r a b s e n c e o f c a f f e i n e  L e i g h t o n t u b e c u l t u r e s o f BHK-21 c e l l s , w h i c h had been e xpo sed t o ADM f o r 48 h r s , were washed i n pho spha te b u f f e r , and p l a c e d i n p l a s t i c p e t r i d i s h e s . The c o v e r - s l i p s were t h e n c o v e r e d w i t h a f i l m o f b u f f e r and i m m e d i a t e l y i r r a d i a t e d w i t h UV f o r 30 s e c a t a d i s t a n c e o f 7 i n c h e s . T h i s 2 p r o d u c e d a t o t a l UV dosage o f 900 ergs/mm . F o l l o w i n g i r r a d i a t i o n t h e c u l t u r e s were i n c u b a t e d f o r 1.5 h r s , i n new L e i g h t o n t u b e s c o n t a i n i n g c a f f e i n e - 4 - 3 ( 2 . 5 x 1 0 t o 4x10 M ) . Upon t h e c o m p l e t i o n o f t h i s i n c u b a t i o n t h e c a f f e i n e s o l u t i o n s were washed o u t , and r e p l a c e d w i t h c a f f e i n e s o l u t i o n s o f t h e same c o n c e n t r a t i o n w i t h "^H-TdR ( f i n a l c o n c e n t r a t i o n 10 m i c r o c u r i e s / m l ) . The c u l t u r e s were i n c u b a t e d i n t h e s e s o l u t i o n s f o r 1.5 h o u r s . In c o n t r o l e x p e r i m e n t s U V - i r r a d i a t e d c u l t u r e s were no t e x p o s e d t o c a f f e i n e , o t h e r w i s e t h e y were t r e a t e d i n e x a c t l y t h e same way as t h e c a f f e i n e t r e a t e d c u l t u r e s . 11. 5. A u t o r a d i o g r a p h i c A n a l y s i s ( a ) F i x a t i o n and s t a i n i n g o f c e l l s In a l l c a s e s f o l l o w i n g t h e l a b e l l i n g p e r i o d , t h e c o v e r - s l i p s were washed (4x ) w i t h H a n k ' s b a l a n c e d s a l t s o l u t i o n , and t h e n t r e a t e d f o r 10 min w i t h \% s od i um c i t r a t e . The c o v e r - s l i p s c u l t u r e s were t h e n f i x e d by p l a c i n g them s u c c e s s i v e l y i n t o f o u r s o l u t i o n s (10 m i n . e a ) , o f C a r n o y s ( a l c o h o l - a c e t i c a c i d , 3 : 1 ) . The f o u r changes i n f i x a t i v e f a c i l i t a t e d remova l o f u n i n c o r p o r a t e d ^H-TdR. A f t e r f i x a t i o n t h e c o v e r - s I i p c u l t u r e s were a i r d r i e d , and t o a l l o w e a s e i n h a n d l i n g were mounted ( c e l l s i d e up) on m i c r o s c o p e s l i d e s u s i n g parawax ( A m e r i c a n Can C o . , Neenah, W i s c o n s i n ) , as an a d h e s i v e . The c e l l s were t h e n s t a i n e d f o r 20 min i n 2% o r c e i n -a c e t i c a c i d {50%) and p l a c e d s u c c e s s i v e l y i n t o t h e f o l l o w i n g s o l u t i o n s : two change s o f 20$ e t h a n o l ( each 10 m i n ) , two changes o f \Q% e t h a n o l ( e ach 10 m i n ) , and f i v e changes o f d i s t i l l e d w a t e r (each 10 m i n ) . The c e l l s were t h e n a i r d r i e d , and p r e p a r e d f o r a u t o r a d i o g r a p h y . (b ) P r e p a r a t i o n o f a u t o r a d i o g r a p h s A f t e r t h e c o v e r - s l i p s c u l t u r e s had d r i e d t h e c e l l s were c o a t e d , i n d a r k , by d i p p i n g t h e s l i d e s i n t o a u t o r a d i o g r a p h i c e m u l s i o n (Kodak : N u c l e a r T r a c k E m u l s i o n ) w h i c h had been h e a t e d t o 4 2 ° C . F o l l o w i n g an hou r d r y i n g p e r i o d , t h e s l i d e s were p l a c e d i n l i g h t - t i g h t boxes w i t h a d e h y d r a t i n g a g e n t ( D r i e r i t e ) , and e x p o s e d a t 4°C f o r 10 t o H d a y s . A f t e r e x p o s u r e t h e s l i d e s were d e v e l o p e d w i t h Kodak D—19 f o r 2 m i n , a t 2 0 ° C , f i x e d f o r 10 m i n , a t t h e same t e m p e r a t u r e , i n Kodak R a p i d F i x , and washed i n r u n n i n g w a t e r ( 20°C) f o r I h o u r . The s l i d e s were t h e n d e h y d r a t e d by p l a c i n g them s u c c e s s i v e l y i n t o t h e f o l l o w i n g s o l u t i o n s : 95% e t h a n o l ( 1 . 5 m i n ) , b u t a n o l ( 1 . 5 m i n ) , b u t a n o I : x y I o I ( 1 : 1 , 1.5 m i n ) , and x y l o l (2 change s each 5 m i n ) . The s l i d e s were t h e n c o v e r e d w i t h c o v e r - s l i p s u s i n g DPX (BHH R e a g e n t ) . The a u t o r a d i o g r a p h s were exam ined under t h e l i g h t m i c r o s c o p e ( l O O O x ) . S i n c e BHK-21 c u l t u r e s c o n t a i n c e l l s w i t h v a r i o u s d e g r e e s o f p l o i d y , g r a i n s were c o u n t e d o n l y o v e r t h e n u c l e i o f t h e s m a l l e s t c e l l s i n each a u t o r a d i o g r a m . 6. CeI I S u r v i va I P e t r i d i s h c u l t u r e s o f BHK-21 c e l l s w h i c h had been i n c u b a t e d f o r 48 h r s , i n ADM were t r e a t e d f o r 1.5 h r s , w i t h 4NQ0 ( 8 x l O ~ 7 M ) e i t h e r a f t e r - 4 - 3 a 3 h r , c a f f e i n e ( 2 . 5 x 1 0 t o 2x10 M) e x p o s u r e , o r w i t h o u t s uch an e x p o s u r e . F o l l o w i n g t h i s i n c u b a t i o n p e r i o d t h e 4NQ0 s o l u t i o n was washed - 4 - 3 o u t and r e p l a c e d w i t h c a f f e i n e ( 2 . 5 x 1 0 t o 2x10 M) f o r 15 h o u r s . In a v a r i a t i o n o f t h e above e x p e r i m e n t , f o l l o w i n g t h e 1.5 h r s , 4NQ0 e x p o s u r e t h e c e l l s were i n c u b a t e d i n ADM f o r 10 h r s , and t h e n e x p o s e d t o c a f f e i n e f o r 15 h o u r s . In a l l c a s e s , a f t e r t h e c a f f e i n e i n c u b a t i o n p e r i o d t h e c e l l s were removed w i t h t r y p s i n ( 1 . 2 5 $ ) , c e n t r i f u g e d t w i c e , s u spended i n MEM, and s eeded i n t o 60x15 mm p l a s t i c p e t r i d i s h e s (200 c e l l s / d i s h ) . The p e t r i d i s h e s were t h e n i n c u b a t e d a t 3 7 ° , i n a h u m i d i f i e d i n c u b a t o r (5$ C 0 9 95% a i r ) f o r 10 t o 12 d a y s . C o n t r o l s c o n s i s t e d o f c u l t u r e s e xpo sed o n l y t o 4NQ0 o r o n l y t o c a f f e i n e . A f t e r t h e 10 t o 12 days i n c u b a t i o n p e r i o d t h e c e l l s were f i x e d i n C a r n o y s ( f o r 10 m i n ) . They were t h e n washed t w i c e : once i n 10% e t h a n o l , and once i n d i s t i l l e d w a t e r . F o l l o w i n g t h i s t h e c e l l s were s t a i n e d w i t h 2% t o l u i d i n e b l u e ( f o r 5 m i n ) , r i n s e d t w i c e w i t h d i s t i l l e d w a t e r , and a i r d r i e d . The e f f e c t o f 4NQ0 and c a f f e i n e on t h e c o l o n y f o r m i n g a b i l i t y (CFA) was d e t e r m i n e d by c o u n t i n g t h e number o f c l o n e s p e r p e t r i d i s h . F o u r r e p l i c a t e p l a t e s were used f o r each e x p e r i m e n t a l t r e a t m e n t , and t h e r e s u l t s were a v e r a g e d . 7. C e l I C y c l e (a ) G| -pha se t o S - pha se c o n v e r s i o n L e i g h t o n t u b e c u l t u r e s o f BHK-21 c e l l s , w h i c h had been e x p o s e d t o ADM f o r 48 h r s , were t r e a t e d f o r 1.5 h r s , w i t h a comb ined s o l u t i o n o f 4NQ0 ( 8 x l O " 7 M ) , and c a f f e i n e ( 2 . 5 x l 0 ~ 4 t o 2 x l O _ 3 M ) . A f t e r t h i s s o l u t i o n had been washed o u t , i t was r e p l a c e d w i t h a f r e s h s o l u t i o n o f c a f f e i n e o f t h e same c o n c e n t r a t i o n f o r 1.5 h o u r s . Then , t h e c o v e r - s l i p s were r i n s e d , and p l a c e d i n t o new L e i g h t o n t u b e s c o n t a i n i n g MEM t h e r e b y t r i g g e r i n g t h e c e l l s i n t o d i v i s i o n . T h i s medium was t h e n r e p l a c e d w i t h medium c o n t a i n i n g 3 H - T d R ( f i n a l c o n e . 10 m i c r o c u r i e s / m l ) 7.5 h r s , and 13.5 h r s , l a t e r . The c u l t u r e s were f i x e d 1.5 h r s , f o l l o w i n g t h e r e s p e c t i v e a d d i t i o n o f 3 H - T d R . The c o n t r o l s , i n t h i s s e r i e s , c o n s i s t e d o f c e l l s t r e a t e d w i t h o n l y c a f f e i n e , o r 4NQ0, o r w i t h o u t e i t h e r o f t h e s e a g e n t s . The p r o c e d u r e f o r t h e f i x a t i o n and s t r a i n i n g o f c e l l s as w e l l as f o r t h e p r e p a r a t i o n o f a u t o r a d i o g r a p h s was t h e same as b e f o r e ( s ee METHODS AND MATERIALS 5 a , 5 b ) . The f r e q u e n c y o f c e l l s p a s s i n g i n t o S - pha se was d e t e r m i n e d by c o u n t i n g t h e number o f h e a v i l y - l a b e l l e d c e l l s i n each a u t o r a d i o g r a p h . T h i s v a l u e was e x p r e s s e d as a p e r c e n t a g e o f t h e l i g h t l y l a b e l l e d c e l l s . (b ) The f l o w o f c e l l s f r o m G|-phase t o metaphase L e i g h t o n tube: c u I t u r e s o f BHK-21 c e l l s a r r e s t e d a t G| -phase - 4 - 3 by ADM were i n c u b a t e d i n c a f f e i n e ( 2 . 5 x10 t o 2x10 M) t o g e t h e r w i t h 4NQ0 (4x10 7 t o 8x10 7 M) f o r 1.5 h o u r s . A f t e r . t h i s i n c u b a t i o n i n t e r v a l t h e - 4 - 3 s o l u t i o n s were washed o u t , and r e p l a c e d w i t h c a f f e i n e ( 2 . 5 x10 t o 2x10 M) f o r an a d d i t i o n a l 1.5 h o u r s . When t h i s i n c u b a t i o n p e r i o d was c o m p l e t e d t h e c o v e r - s l i p c u l t u r e s were t r a n s f e r r e d i n t o new L e i g h t o n t u b e s , and t r i g g e r e d i n t o d i v i s i o n by t h e a d d i t i o n o f a r g i n i n e (MEM). I d e n t i c a l l y t r e a t e d c e l l c u l t u r e s were t h e n i n c u b a t e d f o r 20 o r 24 h r s , p r i o r t o t h e a d d i t i o n o f 0 .06 mis o f 0 . 01$ c o l c h i c i n e . C o l c h i c i n e a r r e s t s c e l l s a t metaphase ( 1 9 , 2 8 , 4 4 ) . A f t e r a 6 h r , e x p o s u r e t o c o l c h i c i n e t h e c o v e r - s l i p c u l t u r e s were p l a c e d i n 1$ s od i um c i t r a t e ( f o r 10 m i n ) , f i x e d i n C a r n o y s ( f o r 10 m i n ) , a i r d r i e d , s t a i n e d ( f o r 4 m i n ) , i n 2$ o r c e i n -a c e t i c a c i d ( 5 0 $ ) , d e h y d r a t e d , and mounted , c e l l s i d e down on m i c r o s c o p e s l i d e s ( s ee METHODS AND MATERIAL 5 a ) . C o n t r o l s c o n s i s t e d o f c u l t u r e s t r e a t e d o n l y w i t h c a f f e i n e , o r 4NQ0, o r w i t h o u t any c a f f e i n e / 4 N Q 0 t r e a t m e n t . The f r e q u e n c y o f c e l l s p a s s i n g f r om G|-phase t o metaphase was enumera ted by c o u n t i n g t h e number o f metaphase p l a t e s i n each c e l l s a m p l e . T h i s v a l u e was e x p r e s s e d as a p e r c e n t a g e o f t h e c e l l s n o t a t me tapha se . C o u n t i n g was done unde r t h e l i g h t m i c r o s c o p e ( l O O O x ) . 8. U n s c h e d u l e d DNA S y n t h e s i s i n Human Lymphocy te s The s u s p e n s i o n o f l ymphocy t e s i n MEM was e x p o s e d t o 4NQ0 - 6 3 ( I x 1 0 M) t o g e t h e r w i t h H-TdR ( f i n a l c o n e . 10 m i c r o c u r i e s / m l ) , f o r 1.5 h o u r s . F o l l o w i n g t h i s t r e a t m e n t , t h e c e l l s were washed t w i c e w i t h H a n k ' s b a l a n c e d s a l t s o l u t i o n , and su spended i n 1$ s od i um c i t r a t e f o r 10-15 m i n u t e s . A t t h e end o f t h i s i n t e r v a l C a r n o y s f i x a t i v e was added t o t h e s u s p e n s i o n w h i c h was t h e n c e n t r i f u g e d . The c e l l s were r e s u s p e n d e d i n C a r n o y s , and smears were made on s t a n d a r d m i c r o s c o p e s l i d e s . The c e l l s were t h e n s t a i n e d and p r e p a r e d f o r a u t o r a d i o g r a p h i c a n a l y s i s as b e f o r e ( s ee METHODS AND MATERIALS 5 a , 5 b ) . 14 b. 9. E v a l u a t i o n o f E x p e r i m e n t s (a ) U n s c h e d u l e d D N A - r e p a i r e x p e r i m e n t s The g r a i n s o v e r t h e n u c l e i o f a t l e a s t 50 c e l l s p e r samp le were c o u n t e d i n t h e s e e x p e r i m e n t s . A minimum o f 3 r e p l i c a t e e x p e r i m e n t s f o r e a ch s e r i e s was p e r f o r m e d , and t h e r e s u l t s s how ing t h e t y p i c a l t r e n d a r e p r e s e n t e d under t h e s e c t i o n RESULTS. ( b ) CeI I eye Ie The e f f e c t o f c a f f e i n e and 4NQ0 on t h e f l o w o f c e l l s f r o m G j - p h a s e t o S - p h a s e , and t o metaphase was d e t e r m i n e d by s c r e e n i n g a t l e a s t 500 c e l l s f o r h e a v i l y l a b e l l e d S -phase c e l l s , and a minimum o f 1000 c e l l s f o r metaphase p l a t e s r e s p e c t i v e l y . The p r o g r e s s i o n t o S -pha se e x p e r i m e n t s was r e p e a t e d t w i c e , and t h e f l o w t o metaphase e x p e r i m e n t was r e p e a t e d t h r e e t i m e s . ( c ) Su r v i va I In t h e s e e x p e r i m e n t s e ach s e r i e s was r e p e a t e d a t l e a s t t w i c e . W i t h i n each s e r i e s each e x p e r i m e n t a l t r e a t m e n t was r e p l i c a t e d f o u r t i m e s . (d) D N A - r e p a i r i n human l y m p h o c y t e s N i n e t e e n s e r i e s o f e x p e r i m e n t s were p e r f o r m e d w i t h 4NQ0 and human l y m p h o c y t e s . V a r i o u s m o d i f i c a t i o n s were made i n some o f t h e s e e x p e r i m e n t s i n an a t t e m p t t o i n c r e a s e t h e amount o f 4NQ0 - i nduced u n s c h e d u l e d D N A - r e p a i r s y n t h e s i s . The r e s u l t s , f r o m one o f t h e s e s e r i e s r e p r e s e n t t h e t y p i c a l t r e n d , and a r e p r e s e n t e d unde r t h e s e c t i o n RESULTS. T h i s s e r i e s was r e p e a t e d t h r e e t i m e s . B. RESULTS DNA R e p a i r S y n t h e s i s Induced by 4NQ0 i n t h e P r e s e n c e o r  Ab sence o f C a f f e i n e When L e i g h t o n t u b e c u l t u r e s , a r r e s t e d a t G j - p h a s e by ADM,were - 4 - 3 e xpo sed t o c a f f e i n e ( 2 . 5 x10 t o 4x10 M) f o r 6 , o r , 3 h r s p r i o r t o t h e i r e x p o s u r e f o r 1.5 h r s t o t h e same c o n c e n t r a t i o n s o f c a f f e i n e comb ined w i t h 4NQ0 (1x10 ^M) , t h e r e a p p e a r e d t o be a s i g n i f i c a n t v a r i a t i o n i n t h e number o f g r a i n s o b s e r v e d o v e r t h e n u c l e i o f t h e s e c e l l s ( F i g u r e I ) . T h i s v a r i a t i o n a p p e a r e d t o be dependen t on t h e c a f f e i n e c o n c e n t r a t i o n . When t h e c e l l s were expo sed t o i n c r e a s i n g c o n c e n t r a t i o n s o f c a f f e i n e f e w e r g r a i n s were o b s e r v e d o v e r t h e i r n u c l e i . T h i s c a f f e i n e s u p p r e s s i o n o f g r a i n s d i d no t a p p e a r t o v a r y s i g n i f i c a n t l y between c e l l s t r e a t e d w i t h c a f f e i n e f o r 6 h r s compared t o 3 h r s p r i o r t o t h e a d d i t i o n o f c a f f e i n e and 4NQ0 ( F i g u r e I ) . - 4 - 3 F u r t h e r m o r e , c e l l s e xpo sed t o c a f f e i n e ( 2 . 5 x10 t o 4x10 M) t o g e t h e r w i t h 4NQ0 ( Ix10 6 M ) f o r 1.5 h r s , b u t no t p r e v i o u s l y t r e a t e d w i t h c a f f e i n e , showed a s i m i l a r c a f f e i n e dependen t s u p p r e s s i o n o f g r a i n s o v e r t h e i r n u c l e i , ( F i g u r e 2 B ) . However , when t h e c e l l s were t r e a t e d w i t h c a f f e i n e ( 2 . 5 x 1 0 - 4 t o 4 x l 0 ~ 3 M ) , a f t e r a 1.5 h r 4NQ0 ( l x l 0 " 6 M ) e x p o s u r e , l i t t l e v a r i a t i o n i n t h e number o f g r a i n s o v e r t h e i r n u c l e i was o b s e r v e d ( F i g u r e 2 A ) . 16. 20-U " 1 1 1 1 1 | — i 4I0" 3 2I0~ 3 NO - 3 5I0" 4 2.5I0'4 0 M CAFFEINE CONC. FIGURE I. The e f f e c t o f c a f f e i n e on 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s . BHK-21 c e l l s were t r e a t e d w i t h c a f f e i n e f o r 6 h r s • • , o r 3 h r s • 0 , p r i o r t o a 1.5 h r e x p o s u r e t o c a f f e i n e combined w i t h 4NQ0 CI - 10 ^ M) . The c e l l s were t h e n t r e a t e d f o r 1.5 h r s w i t h c a f f e i n e t o g e t h e r w i t h 3 H - T d R ( f i n a l c o n e . 10 u C i / m l ) . The b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n f r om t h e mean. 17. 0' 1 • 1 1 I r 4 I 0 " 3 2 I 0 " 3 IIO" 3 5 ICT 4 2.5-I0"4 OM. CAFFEINE CONC. FIGURE 2. The t i m e - d e p e n d e n t e f f e c t o f c a f f e i n e on 4NQ0 - i nduced D N A - r e p a i r A s y n t h e s i s . ' C e l l s e x p o s e d f o r 1.5 h r s s u c c e s s i v e l y t o 4NQ0 ( 1 * 1 0 ^ M ) , c a f f e i n e , and c a f f e i n e w i t h ^H-TdR ( f i n a l c o n e . 10 u C i / m l ) , B • . ' C e l l s e xpo sed f o r 1.5 h r s t o c a f f e i n e comb ined w i t h 4NQ0 ( I - 1 0 ^ M ) , p r i o r t o e x p o s u r e f o r 1.5 h r s t o c a f f e i n e w i t h "Vl-TdR ( f i n a l c o n e . 10 u C i / m l ) , » » . The b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n f r om t h e mean. DNA R e p a i r S y n t h e s i s Induced by UV i n t h e P r e s e n c e and Ab sence  o f C a f f e i n e From t h e a u t o r a d i o g r a p h s o f c e l l s a r r e s t e d a t G j - p h a s e by 2 ADM f o l l o w e d by e x p o s u r e t o UV (900 ergs/mm ), and t r e a t m e n t w i t h - 4 c a f f e i n e ( 2 . 5x10 M) f o r 3 h r s , i t seemed t h a t t h e r e was l i t t l e v a r i a t i o n i n t h e number o f g r a i n s o b s e r v e d o v e r t h e n u c l e i o f t h e s e e e l Is ( F i g u r e 3 ) . 19. 20-18-5 10-< o 8-d 2 6-4-2-0l , . , r , 4».I0" 3 2-I0" 3 l-IO"3 5^I0"4 2.5-I0"4 OM. CAFFE INE CONC. FIGURE 3. The e f f e c t o f c a f f e i n e on U V - i n d u c e d D N A - r e p a i r s y n t h e s i s . The c e l l s were t r e a t e d w i t h c a f f e i n e f o r 1.5 h r s f o l l o w i n g UV-2 e x p o s u r e (900 ergs/mm ). The c e l l s were t h e n e x p o s e d t o c a f f e i n e w i t h ^H-TdR ( f i n a l c o n e . 10 y C i / m l ) . The b a r s r e p r e s e n t one s t a n d a r d d e v i a t i o n f r om t h e mean. S u r v i v a I When c e l l s a r r e s t e d a t G| -pha se by ADM were t r e a t e d f o r 1.5 h r s w i t h 4NQ0 ( 8 x l O ~ 7 M ) , t h e n e xpo sed t o c a f f e i n e ( 2 . 5 x 1 0 ~ 4 t o 4 x l O _ 3 M ) f o r 15 h r s , t h e i r c o l o n y - f o r m i n g a b i l i t y a p p e a r e d t o be s e v e r e l y r e d u c e d . F u r t h e r m o r e , t h i s r e d u c t i o n i n CFA was most p r onounced a t t h e h i g h e r c a f f e i n e c o n c e n t r a t i o n s ( F i g u r e 4 A ) . When t h e c e l l s were i n c u b a t e d i n ADM f o r 10 h r s a f t e r t h e 4NQ0 e x p o s u r e ( 1 . 5 h r s , - 7 - 4 - 3 8x10 M ) , and t h e n i n c u b a t e d i n c a f f e i n e ( 2 . 5 x 1 0 t o 4x10 M) f o r 15 h r s , o n l y a s l i g h t e f f e c t on t h e i r CFA was o b s e r v e d ( F i g u r e 4 B ) . S i m i l a r l y , o n l y a s l i g h t e f f e c t on CFA was o b s e r v e d , when c e l l s were t r e a t e d o n l y t o c a f f e i n e , b u t under i d e n t i c a l c o n d i t i o n s a s t h e c e l l s t r e a t e d w i t h c a f f e i n e i m m e d i a t e l y a f t e r o r 10 h r s f o l l o w i n g 4NQ0 e x p o s u r e ( F i g u r e 5 ) . The. CFA o f c e l l s e x p o s e d t o c a f f e i n e ( 2 . 5 x 1 0 ~ 4 t o 2 x l 0 ~ 3 M ) f o r 3 h r s , t r e a t e d w i t h 4NQ0 (8x10 7 M ) f o r 1.5 h r s and i n c u b a t e d f o r - 4 - 3 15 h r s i n c a f f e i n e ( 2 . 5 x10 t o 2 x l 0 M ) , was r e d u c e d . T h i s r e d u c t i o n d i d n o t a p p e a r t o v a r y s i g n i f i c a n t l y compared t o c e l l s n o t p r e v i o u s l y e xpo sed t o c a f f e i n e , bu t o t h e r w i s e t r e a t e d t h e same ( F i g u r e 6 ) . 21. 100-9 0 80 -CAFFEINE CONC. FIGURE 4 . The e f f e c t o f c a f f e i n e , and 4NQ0 on t h e c o l o n y - f o r m i n g a b i l i t y o f BHK-21 c e l l s . A - C e l l s e xpo sed t o 4NQ0 ( 8 * I 0 ~ 7 M ) , f o r 1.5 h r s , i n c u b a t e d w i t h c a f f e i n e f o r 15 h r s , and p l a t e d i n MEM, • • . C e l l s s i m i l a r l y e xpo sed t o 4NQ0, i n c u b a t e d i n ADM f o r 10 h r s , i n c u b a t e d w i t h c a f f e i n e f o r 15 h r s , and p l a t e d i n MEM, # — — # . The v a l u e s a r e c o r r e c t e d f o r t h e e f f e c t o f c a f f e i n e a l o n e on c e l l CFA ( s ee F IG . 5 ) . 22. 60 < 50 o 40 30 20 10 2.510 -4 510 -4 110 - 3 2-10 - 3 M. CAFFEINE CONC. FIGURE 5. The e f f e c t o f c a f f e i n e on t h e c o l o n y - f o r m i n g a b i l i t y o f BHK-21 c e l l s . The c e l l s were e xpo sed t o c a f f e i n e f o r 15 h r s unde r i d e n t i c a l c o n d i t i o n s t o c e l l s e xpo sed t o c a f f e i n e , and 4NQ0. A ( , Bj, a r e t h e r e s p e c t i v e c o n t r o l s f o r A, B, o f F I G . 4 . 23 . 2I0~ 3 MCT 3 CAFFEINE 5I0~ 4 CONCENTRATION M. FIGURE 6 . The e f f e c t o f c a f f e i n e p r e - t r e a t m e n t on c e l l c o l o n y - f o r m i n g a b i l i t y . C e l l s t r e a t e d w i t h c a f f e i n e f o r 3 h r s , exposed t o 4NQ0 ( 8 * 1 M ) f o r 90 m i n , i n c u b a t e d w i t h c a f f e i n e f o r 15 h r s , were p l a t e d i n MEM,fm%- B . C o n t r o l c e l l s were no t p r e - t r e a t e d w i t h c a f f e i n e o t h e r w i s e t h e y were t r e a t e d e x a c t l y as a b o v e , . The b a r s r e p r e s e n t t h e r ange o f c l o n e s i n 4 r e p l i c a t e p l a t e s . CeI I e y e l e ana I y s i s ( a ) The e f f e c t o f c a f f e i n e and/o r 4NQ0 on t h e f l o w o f c e l l s  f r o m G j - p h a s e i n t o S - p h a s e . ( T a b l e I) - 4 When c e l l s were t r e a t e d a t G| -phase w i t h c a f f e i n e (5x10 t o 2x10 3 M ) , comb ined w i t h 4NQ0 (8x10 7 M ) , t h e i r p r o g r e s s i o n i n t o S - pha se d i d no t a p p e a r t o be a f f e c t e d compared t o u n t r e a t e d c e l l s . F u r t h e r m o r e , n e i t h e r c a f f e i n e , no r 4NQ0 t r e a t m e n t a l o n e , a p p e a r e d t o i n f l u e n c e c e l l p r o g r e s s i o n f r o m G j - p h a s e t o S - p h a s e . (b) The e f f e c t o f c a f f e i n e , and/o r 4NQ0 on t h e f l o w o f c e l l s f r o m G j - p h a s e t o m e t a p h a s e . ( T a b l e 2 ) . - 4 C e l l s , i n t h e G j - p h a s e , were t r e a t e d w i t h c a f f e i n e (5x10 t o 2 x l 0 ~ 3 M ) t o g e t h e r w i t h 4NQ0 ( 4 x 1 0 ~ 7 , 8 x l 0 ~ 7 M ) , o r w i t h e i t h e r a g e n t a l o n e . The i n f l u e n c e o f t h i s t r e a t m e n t on t h e f I ow o f ce I Is t o metapha se i s i l l u s t r a t e d by t h e f o l l o w i n g : ( i ) w i t h i n c r e a s i n g c a f f e i n e c o n c e n t r a t i o n : no n o t i c e a b l e t r e n d ( i i ) w i t h i n c r e a s i n g 4NQ0 c o n c e n t r a t i o n s : s l i g h t m i t o t i c i nh i b i t i on ( i i i ) w i t h 4NQ0, and i n c r e a s i n g c a f f e i n e c o n c e n t r a t i o n s : no def i n i t e t r e n d ( i v ) w i t h c a f f e i n e , and i n c r e a s i n g 4NQ0 c o n c e n t r a t i o n s : m i t o t i c i n h i b i t i o n I t a p p e a r s t h a t t h e f l o w o f c e l l s , t r e a t e d a t G . - p h a s e w i t h 4NQ0 25. t o m e t a p h a s e , i s r e d u c e d . T h e r e f o r e , t h e s l i g h t m i t o t i c i n h i b i t i o n seen w i t h c a f f e i n e and 4NQ0 i s p r o b a b l y due t o t h e i n f l u e n c e o f t h e I a f t e r chemi c a I . CAFFEINE CONC. 2 - I 0 ~ 3 1•I0~ 3 - 4 5-10 * 0 M 8 - I 0 " 7 M 6.7 5.1 8.9 8.3 * 0 9.1 10.1 8.7 10.5 TABLE I. The e f f e c t o f c a f f e i n e , and 4NQ0 on t h e p r o g r e s s i o n o f c e l l s f r om G j - p h a s e i n t o S - p h a s e . The v a l u e s a r e t h e number o f S - pha se n u c l e i / s a m p l e c e l l p o p u l a t i o n ( e x p r e s s e d i n p e r c e n t ) . * C o n t r o l = u n t r e a t e d c e l l s . 2 - 1 0 " 3 CAFFEINE 1• I0~ 3 CONC. - 4 5-10 0 M 8- 10*" 7 M 5.4 0 .24 1 .4 2 .5 4 - I 0 " 7 M 10.4 13.7 6 .5 6 . 5 0 12.9 16.6 17.8 14. 1 TABLE 2 . The e f f e c t o f c a f f e i n e , and 4NQ0 on t h e f l o w o f c e l l s f r om G j - p h a s e t o me tapha se . The v a l u e s a r e t h e number o f metaphase p l a t e s / s a m p l e c e l l p o p u l a t i o n ( e x p r e s s e d i n p e r c e n t ) . * C o n t r o I = u n t r e a t e d c e l l s . 27 . 5. DNA R e p a i r S y n t h e s i s i n Human Lymphocy te s I t a p p e a r e d t h a t human l y m p h o c y t e s , i n v i t r o , show o n l y low l e v e l s o f D N A - r e p a i r s y n t h e s i s f o l l o w i n g e x p o s u r e t o 4NQ0 f o r 1.5 h o u r s . F u r t h e r m o r e , a l a r g e v a r i a b i l i t y i n t h e amount o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s was o b s e r v e d i n t h e s e c e l l s ( F i g u r e 7 ) . 28. lOO-i 30-co _i _ J L J O U -O g 20-< L U O cn L U a. 10-12 13 ' 14 4 5 6 7 8 9 10 II No. GRAINS/NUCLEUS FIGURE 7. D N A - r e p a i r s y n t h e s i s i n human l ymphocy te s i n v i t r o . The h i s t o g r a m shows t h e d i s t r i b u t i o n o f g r a i n s o v e r t h e n u c l e i o f c e l l s w h i c h had been t r e a t e d f o r 1.5 h r s w i t h 4NQ0 ( | - I 0 ~ 6 M) comb ined w i t h ^H-TdR ( f i n a l c o n e . 10 y C i / m l ) . DISCUSSION D N A - r e p a i r s y n t h e s i s has been i n d u c e d by 4NQ0 i n normal and n e o p l a s t i c human c e l l s as w e l l a s S y r i a n - h a m s t e r c e l l s ( 4 8 , 6 1 ) . The e x p e r i m e n t s i n t h i s t h e s i s i n d i c a t e t h a t D N A - r e p a i r i n a S y r i a n -hams te r c e l l l i n e (BHK-21 , c l o n e 13 ) , a r r e s t e d a t G| -phase and e x p o s e d t o t h e h i g h l y o n c o g e n i c 4NQ0, i s s u p p r e s s e d by c a f f e i n e . T h i s e f f e c t a p p e a r s t o be dependen t on t h e t i m e o f c a f f e i n e a d d i t i o n ( i . e . , e i t h e r p r i o r t o , o r d u r i n g t h e i n d u c t i o n o f DNA-r e p a i r s y n t h e s i s ) , and on t h e c o n c e n t r a t i o n o f c a f f e i n e . A t i m e s t u d y o f t h e d u r a t i o n o f 4NQ0 - i nduced D N A - r e p a i r i n S y r i a n - h a m s t e r c e l l s d e m o n s t r a t e d t h a t r e p a i r s y n t h e s i s was i n i t i a t e d a l m o s t i m m e d i a t e l y f o l l o w i n g t h e a d d i t i o n o f 4NQ0, and r e a c h e s a maximum e f f i c i e n c y between 1.5 h r s t o 4 h r s l a t e r . A l s o , t h e m a j o r amount o f t h e r e p a i r was shown t o be c o m p l e t e d by 8 t o 10 h r s f o l l o w i n g 4NQ0 e x p o s u r e a l t h o u g h low l e v e l s o f r e p a i r s y n t h e s i s c o n t i n u e d f o r a p r o l o n g e d p e r i o d ( 6 1 ) . The a p p a r e n t s u p p r e s s i o n o f 4NQ0 - i nduced D N A - r e p a i r by c a f f e i n e c o u l d be i n t e r p r e t e d i n t e r m s o f t h e a f o r e m e n t i o n e d t i m e s t u d y , and t h e e x c i s i o n - r e p a i r model o f " c u t and p a t c h " as p r o p o s e d by S e t low ( 5 8 ) , f o r t h e r e p a i r mechani sm i n b a c t e r i a . ( T a b l e 3 ) . F o r i n s t a n c e , c a f f e i n e may be i n h i b i t i n g one o f t h e enzymes ( i . e . an e n d o n u c l e a s e , e x o n u c l e a s e , DNA p o l y m e r e a s e , o r I i g a s e ) w h i c h i s p o s t u l a t e d t o be r e s p o n s i b l e f o r t h e r e p a i r o f DNA ( 5 8 ) . ^ / L E S I O N — X X INCISION AND EXCISION _ X X DEGRADATION RESYNTHESIS REJOINING TABLE 3. A MODEL FOR EXCISION REPAIR ( m o d i f i e d f rom R. B. S e t low, 1967 ) . S i n c e t h e d u r a t i o n o f 4NQ0 - i nduced D N A - r e p a i r c o n t i n u e s f o r a t l e a s t 10 h r s ( 6 1 ) , and y e t c a f f e i n e has l i t t l e e f f e c t on D N A - r e p a i r ( F i g . 2 A ) , when added 1.5 h r s a f t e r t h e a d d i t i o n o f 4NQ0, i t c o u l d be p o s t u l a t e d t h a t c a f f e i n e i s a f f e c t i n g one o f t h e e a r l y s t a g e s i n t h e r e p a i r o f DNA. Thu s , c a f f e i n e , when p r e s e n t d u r i n g t h e i n d u c t i o n o f damage, may be a b l e t o i n h i b i t one o f t h e enzymes n e c e s s a r y f o r e x c i s i o n . F u r t h e r -mo re , i t c o u l d be p o s t u l a t e d , t h a t i f c a f f e i n e i s no t added w i t h i n 1.5 h r s f o l l o w i n g t h e i n i t i a l i n d u c t i o n o f DNA l e s i o n s t h a t enough t i m e has e l a p s e d f o r t h e m a j o r amount o f t h e s e , l e s i o n s t o be e x c i s e d . T h i s h y p o t h e s i s f o r t h e t i m e dependen t e f f e c t o f c a f f e i n e , on t h e s u p p r e s s i o n o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s , n e c e s s i t a t e s t h a t c a f f e i n e does i n f a c t i n t e r a c t w i t h enzymes . I n f o r m a t i o n e x i s t s w h i c h s u g g e s t s t h a t c a f f e i n e e x e r t s i t s l e t h a l and m u t a g e n i c e f f e c t i n U V - i r r a d i a t e d b a c t e r i a by a f f e c t i n g t h e enzymes n e c e s s a r y f o r t h e r e p a i r o f t h e b a c t e r i a l DNA r a t h e r t h a n by d i r e c t l y a f f e c t i n g t h e DNA i t s e l f . S i n c e c a f f e i n e does n o t a f f e c t p h o t o r e a c t i v a t i on o f U V - i r r a d i a t e d s t r a i n s o f E. Co I i , i t has been p o s t u l a t e d t h a t c a f f e i n e does no t b i n d t o E^ . c o l i DNA ( 4 1 , 5 5 ) . In f a c t , an i n v i t r o s t u d y has d e m o n s t r a t e d t h a t i s o l a t e d E_. c o l i DNA does no t b i n d w i t h c a f f e i n e ( 3 9 ) . A l s o , t h e r e s u l t s f r o m one s t u d y show t h a t c a f f e i n e i s no t b a s e - s u b s t i t u t e d i n t o t h e DNA o f E_. c o l i ( 3 5 ) . C a f f e i n e has been shown howeve r , t o a f f e c t t h e t h e r m a l d e -n a t u r a t i o n p r o f i l e o f DNA. Thus i t was s u g g e s t e d t h a t i t m i g h t i n t e r a c t w i t h d e n a t u r e d s i n g l e s t r a n d DNA ( 2 2 ) . 32 . O t h e r s t u d i e s w i t h E_. c o l i have d e m o n s t r a t e d t h a t c a f f e i n e i n h i b i t s some, b u t no t a l l , o f t h e p u r i n e n u c l e o s i d e p h o s p h o r y I a s e s o f b o t h t h e r i b o s e , and d e o x y r i b o s e t y p e s ( 3 6 ) . As w e l l , c a f f e i n e has been shown t o i n h i b i t t h e U V - i n d u c e d d e g r a d a t i o n o f phage DNA, i n i t s h o s t b a c t e r i u m ( 5 3 ) . C a f f e i n e a l s o a f f e c t s m u s c l e p o l y m e r a s e s i n mammalian ce I Is J_n v i t r o ( 7 0 ) . D N A - r e p a i r s y n t h e s i s has been i n d u c e d i n n o r m a l , and n e o p l a s t i c human c e l l s , as w e l l a s i n S y r i a n - h a m s t e r , and mouse L c e l l s f o l l o w i n g U V - e x p o s u r e ( 4 8 , 5 0 , 6 1 ) . The e x p e r i m e n t s i n t h i s t h e s i s showed t h a t c a f f e i n e does n o t a p p e a r t o s u p p r e s s UV (900 ergs/mm ) , - i n d u c e d DNA-r e p a i r s y n t h e s i s i n BHK-21 c e l l s . C l e a v e r , (8) has r e p o r t e d s i m i l a r 2 r e s u l t s f o r t h e e f f e c t o f c a f f e i n e on t h e UV (1000 ergs/mm ) , - i n d u c e d D N A - r e p a i r s y n t h e s i s i n H e l a c e l l s . S i n c e c a f f e i n e a p p e a r s t o s u p p r e s s 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s , t h e above r e s u l t s a p p e a r i n c o n s i s t e n t . T h i s f o l l o w s becau se 4NQ0 and UV have been shown t o p o s s e s s a s i m i l a r p a t t e r n w i t h r e g a r d s t o r e p a i r , a t l e a s t i n b a c t e r i a ( 3 7 ) . However , i n one s t u d y , i t was c o n c l u d e d t h a t t h e b a c t e r i a l d a r k r e p a i r mechani sm ( e x c i s i o n r e p a i r ) , c o u l d r e p a i r U V - i n d u c e d , b u t no t 4 - h y d r o x y a m i n o q u i n o l i n e I - o x i d e ( t h e r e d u c t i o n p r o d u c t o f 4 N Q 0 ) - i n d u c e d damage i n phage DNA. T h i s mechani sm c o u l d r e p a i r damage i n d u c e d by e i t h e r a g e n t i n t h e h o s t b a c t e r i a ( 7 1 ) . F u r t h e r m o r e , a n o t h e r s t u d y has shown t h a t t h e r e a r e s t r i k i n g d i f f e r e n c e s i n t h e s e n s i t i v i t y t o UV and 4NQ0 i n t h r e e mammalian c e l l l i n e s . I t was c o n c l u d e d f r om t h i s s t u d y , t h a t t h e e f f e c t o f UV, and 4NQ0 on c e l l s i s d i f f e r e n t , a s w e l l as t h e p r o c e s s o f r e a c t i v a t i o n f o r t h e damage p r o d u c e d by t h e s e a g e n t s ( 2 5 ) . I t has been r e p o r t e d t h a t t h e l e t h a l and m u t a g e n i c enhancement by c a f f e i n e on U V - i r r a d i a t e d E . c o l i s t r a i n s i s most p r onounced a t low UV d o s e s , and may no t be o b s e r v e d a t a I I, a t do se s above 500 o r 2 1000 ergs/mm ( 6 , 1 3 , 6 3 , 6 4 ) . S i n c e t h e r a t e o f p y r i m i d i n e d i m e r p r o d u c t i o n i s dose dependen t ( 5 6 ) , i t has been s u g g e s t e d t h a t t h e e f f e c t o f c a f f e i n e i s more p r onounced a t t h e l ower UV do se s becau se i t i s a t t h e s e e x p o s u r e s t h a t e x c i s i o n i s most r a p i d and e f f i c i e n t ( 6 8 ) . W i t h more i n t e n s e U V - i r r a d i a t i o n t h e r a t e o f e x c i s i o n may a l r e a d y be so s l o w , t h a t t h e e f f e c t o f c a f f e i n e i s n e g l i g i b l e ( 6 8 ) . The same phenomenon m i g h t a c c o u n t f o r t h e a f o r e m e n t i o n e d r e s u l t s w i t h r e s p e c t t o a l a c k o f c a f f e i n e s u p p r e s s i o n o f U V - i n d u c e d DNA-r e p a i r s y n t h e s i s i n BHK-21 c e l l s a s w e l l a s i n He I a c e l l s . When BHK-21 c e l l s ( i n ADM) were t r e a t e d w i t h 4NQ0 f o r 1.5 h r s , i n c u b a t e d i n c a f f e i n e f o r 15 h r s and p l a t e d i n MEM, a s i g n i f i c a n t r e d u c t i o n i n t h e i r CFA was o b s e r v e d . T h i s r e d u c t i o n was more p r o n o u n c e d a t t h e h i g h e r c a f f e i n e c o n c e n t r a t i o n s , and i t d i d no t a p p e a r t o be a t t r i b u t a b l e t o t h e e f f e c t o f c a f f e i n e a l o n e ( F i g s . 4 A , 5 ) . Thu s , i t a ppea red t h a t a l e t h a l s y n e r g i s m e x i s t s between c a f f e i n e , and 4NQ0 i n BHK-21 c e l l s . I t i s t e m p t i n g t o draw a c o r r e l a t i o n between t h i s c a f f e i n e s y n e r g i s m , and t h e s u p p r e s s i o n o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s by c a f f e i n e ( F i g s . I , 2B ) . Such a c o r r e l a t i o n , does no t n e c e s s a r i l y i m p l y t h a t a s u p p r e s s e d r e p a i r mechan i sm p r o d u c e d c e l l d e a t h . I t i s o f i n t e r e s t , however , t h a t when BHK-21 c e l l s were i n c u b a t e d f o r 15 h r s i n c a f f e i n e 10 h r s f o l l o w i n g t h e i r e x p o s u r e t o 4NQ0, no o r l i t t l e r e d u c t i o n i n t h e i r CFA was o b s e r v e d ( F i g . 4 B ) . T h i s i n t e r v a l o f t i m e (10 h r s ) c o r r e l a t e s w i t h t h e t i m e n e c e s s a r y f o r t h e r e p a i r o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s ( 6 1 ) . Such a t i m e - d e p e n d e n t e f f e c t o f c a f f e i n e has a l s o been d e m o n s t r a t e d i n s t r a i n s o f £ . c o l ? . When c a f f e i n e was added t o t h e s e b a c t e r i a l s t r a i n s a t i n c r e a s i n g t i m e i n t e r v a l s f o l l o w i n g t h e i r e x p o s u r e t o UV, an i n c r e a s e i n s u r v i v a l o c c u r r e d ( 4 1 ) . F u r t h e r m o r e , when c a f f e i n e was added t o t h e s e c u l t u r e s 5 min a f t e r t h e i r e x p o s u r e t o UV, a l o s s o f 50% o f t h e r e c o v e r a b l e m u t a n t s o c c u r r e d ; i f a 20 m in i n t e r v a l between U V - e x p o s u r e , and c a f f e i n e t r e a t m e n t was emp loyed no m u t a n t s were r e c o v e r e d ( 3 9 ) . T h i s t i m e dependen t e f f e c t o f c a f f e i n e was i n t e r p r e t e d as r e s u l t i n g f rom a c a f f e i n e i n h i b i t i o n o f t h e e n z y m a t i c r e p a i r mechan i sm i n b a c t e r i a (J39). When U V - i r r a d i a t e d mouse L c e l l s were i n c u b a t e d i n t h e ab sence o f c a f f e i n e p r i o r t o p l a t i n g them i n i t s p r e s e n c e , no r e d u c t i o n i n s u r v i v a l was o b s e r v e d ( 5 1 ) . I t was p o s t u l a t e d t h a t t h e a b s e n c e o f c a f f e i n e a l l o w e d an S -pha se r e p a i r mechan i sm t i m e t o r e p a i r t h e i n d u c e d damage, whereas c a f f e i n e p r e s e n t e d i m m e d i a t e l y f o l l o w i n g U V - e x p o s u r e c o u l d i n h i b i t t h i s r e p a i r mechan i sm p r o d u c i n g an i n c r e a s e i n c e l l l e t h a l i t y ( 1 1 , 1 2 , 5 1 ) . The e f f e c t o f c a f f e i n e combined w i t h 4NQ0 d i d no t a p p e a r t o i n h i b i t t h e f l o w of c e l l s f r o m G| -phase t o S -pha se compared t o u n t r e a t e d c e l l s . F u r t h e r m o r e , n e i t h e r c a f f e i n e nor 4NQ0 added i n d i v i d u a l l y , a p p e a r e d t o s u p p r e s s t h e f l o w of c e l l s i n t o S - p h a s e . I t has been r e p o r t e d t h a t c a f f e i n e can c a u s e a s l i g h t d e l a y i n t h e p r o g r e s s i o n o f mouse L c e l l s f r o m G j - p h a s e t o S - p h a s e . However , 2 low e x p o s u r e s o f UV (100 ergs/mm ) d i d no t i n c r e a s e t h i s e f f e c t ( I I ) . When BHK-21 c e l l s were e xpo sed t o 4NQ0 t h e r e a p p e a r e d t o be a s l i g h t i n h i b i t o r y e f f e c t on t h e f l o w o f t h e s e c e l l s t o m e t a p h a s e . C a f f e i n e d i d no t a p p e a r t o i n c r e a s e t h i s phenomenon. F u r t h e r m o r e , c a f f e i n e by i t s e l f d i d no t a p p e a r t o i n h i b i t t h e f l o w o f c e l l t o m e t a p h a s e . I t h a s , howeve r , been r e p o r t e d t h a t c a f f e i n e has a s i i g h t e f f e c t on t h e p r o g r e s s i o n o f mouse L c e l l s t o metaphase ( 1 1 , 1 2 ) . T h i s e f f e c t was n o t i n c r e a s e d by e x p o s u r e o f t h e c e l l s t o UV p r i o r t o i n c u b a t i n g them i n c a f f e i n e ( 1 2 ) . T h u s , a s y n e r g i s t i c r e l a t i o n s h i p does no t a p p e a r t o o c c u r between c a f f e i n e and e i t h e r 4NQ0 o r UV, w i t h r e s p e c t t o t h e f i r s t c e l l p r o g r e s s i o n f r om G| -phase t o m e t a p h a s e . A. S p e c u l a t i o n and O u t l o o k C o n s i d e r i n g t h e r e s u l t s o f t h e p r e s e n t e d e x p e r i m e n t s , i t a p p e a r s t h a t c a f f e i n e c an s u p p r e s s 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s . T h i s s u p p r e s s i o n may be r e s p o n s i b l e f o r t h e r e d u c t i o n i n s u r v i v a l o f c e l l s e x p o s e d t o 4NQ0, and c a f f e i n e . T h i s l o s s o f c e l l v i a b i l i t y does n o t a p p e a r t o o c c u r d u r i n g S - p h a s e , G ^ - p h a s e , p r o p h a s e o r me tapha se . I t i s l i k e l y , bu t u n p r o v e n , t h a t t h e mechan i sm r e s p o n s i b l e f o r t h e c a f f e i n e s u p p r e s s i o n o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s , i s t h e same as t h a t w h i c h p r o d u c e s c e l l l e t h a l i t y . I n f o r m a t i o n on t h i s may be g a i n e d by e x a m i n i n g t h e e f f e c t o f c a f f e i n e on t h e r e p a i r o f DNA l e s i o n s i n d u c e d by a l k y l a t i n g a g e n t s such as m e t h y l methane s u l f o n a t e , n i t r o g e n m u s t a r d , o r c y c l o p h o s p h a m i d e s . These a g e n t s p r o d u c e s i n g l e - s t r a n d b r e a k s i n DNA t h e r e b y e l i m i n a t i n g t h e r e q u i r e m e n t f o r an e x c i s i o n enzyme ( 8 a , 4 9 , 6 2 , 6 7 ) . If c a f f e i n e does n o t i n c r e a s e t h e e f f e c t o f ' t h e s e a l k y l a t i n g a g e n t s on t h e i n d u c t i o n o f DNA damage, and c e l l s u r v i v a l t h e n t h i s wou ld r e i n f o r c e t h e s u g g e s t i o n t h a t t h e i n c r e a s e i n c e l l l e t h a l i t y i n 4NQ0 t r e a t e d , and c a f f e i n e expo sed c e l l s i s c a u s e d by an i n t e r f e r e n c e o f t h e e x c i s i o n mechan i sm i n t h e D N A - r e p a i r p r o c e s s . I t was hoped t h a t t h e a p p a r e n t s y n e r g i s t i c r e l a t i o n s h i p between c a f f e i n e , and 4NQ0, i n BHK-21 c e l l s , c o u l d be d e m o n s t r a t e d i n a human l y m p h o c y t e s i n v i t r o . However , due t o t h e low l e v e l s o f 4NQ0 - i nduced D N A - r e p a i r s y n t h e s i s t h e s e c e l l s were no t c o n d u c i v e f o r a u t o r a d i o g r a p h i c ana Iys i s . The f o l l o w i n g mechan i sm i s p r o p o s e d t o a c c o u n t f o r t h e s y n e r g i s t i c r e l a t i o n s h i p between c a f f e i n e , and 4NQ0 as o b s e r v e d i n BHK-21 c e l l s . I t i s p o s t u l a t e d t h a t : 37 . 1. 4NQ0 i n d u c e s l e s i o n s i n DNA i n s i t u ( 2 6 ) . 2 . The e x c i s i o n o f t h e s e l e s i o n s , by t h e comb ined e f f e c t o f an endonuc I e a s e , and e x o n u c l e a s e , o c c u r s v e r y r a p i d l y f o l l o w i n g t h e i r i n d u c t i o n . 3. A m a j o r amount o f t h e damaged s i n g l e s t r a n d s equence s i s e x c i s e d w i t h i n a r e l a t i v e l y s h o r t t i m e ( i . e . 1.5 h r s ) . E x c i s i o n t o a much s m a l l e r d e g r e e seems t o c o n t i n u e f o r t h e d u r a t i o n o f D N A - r e p a i r s y n t h e s i s . 4 . The t i m e n e c e s s a r y f o r t h e c o m p l e t i o n o f D N A - r e p a i r i s d ependen t on t h e r a t e - l i m i t i n g s t e p o f one o f t h e o t h e r enzymes ( i . e . a DNA p o l y m e r a s e o r l i g a s e ) . 5. When c a f f e i n e i s p r e s e n t e d d u r i n g t h e i n d u c t i o n o f DNA l e s i o n s by 4NQ0, i t i s c a p a b l e o f i n h i b i t i n g t h e e x c i s i o n enzyme t h e r e b y s u p p r e s s i n g t h e amount o f DNA^ r e p a i r . 6. When c a f f e i n e i s added 1.5 h r s a f t e r t h e a d d i t i o n o f 4NQ0 a m a j o r amount o f t h e e x c i s i o n has a l r e a d y o c c u r r e d . The r e s i d u a l e x c i s i o n g o i n g on a t t h i s t i m e i s no t s u f f i c i e n t l y a f f e c t e d , by a 3 h r e x p o s u r e t o c a f f e i n e t o be r e f l e c t e d i n a r e d u c e d u n s c h e d u l e d DNA s y n t h e s i s . 7 . When c a f f e i n e i s added 1.5 h r s a f t e r t h e e x p o s u r e t o 4NQ0 and a l l o w e d t o r e m a i n f o r a f u r t h e r 15. h r s , a s i g -n i f i c a n t r e d u c t i o n i n c e l l s u r v i v a l o c c u r s . T h i s e f f e c t i s p r o b a b l y due t o t h e i n h i b i t i o n o f t h e r e s i d u a l amounts o f e x c i s i o n i n t h e D N A - r e p a i r p r o c e s s , by t h e p r o l o n g e d e x p o s u r e t o c a f f e i n e . 8. When c a f f e i n e i s added 10 h r s a f t e r 4NQ0 e x p o s u r e and a l l o w e d t o r ema i n f o r a f u r t h e r 15 h r s , no r e d u c t i o n i n c e l l s u r v i v a l o c c u r s becau se t h e m a j o r amount o f t h e DNA r e p a i r , i n most o f t h e c e l l s , has been c o m p l e t e d . I t i s s u g g e s t e d t h a t t h e c o n c e p t o f l e t h a l s y n e r g i s m between two a g e n t s , a s i l l u s t r a t e d i n t h i s t h e s i s , may have some s i g n i f i c a n c e i n c l i n i c a l m e d i c i n e . If c a f f e i n e c o u l d i n t e r f e r e w i t h r e p a i r p r o c e s s e s f o l l o w i n g x - r a y i r r a d i a t i o n , t h e n l ower do se s o f x -i r r a d i a t i o n c o u l d p r o d u c e t h e d e s i r e d e f f e c t s , t h e r e b y i n c r e a s i n g t h e e f f i c i e n c y o f t h e t r e a t m e n t , and r e d u c i n g t h e s i d e e f f e c t s . S i m i l a r i l y , a c omb ined a p p l i c a t i o n o f c a f f e i n e w i t h v a r i o u s o t h e r t h e r a p e u t i c a g e n t s , w h i c h a f f e c t DNA, m i g h t i n c r e a s e t h e e f f i c i e n c y o f s uch a g e n t s . I t has a l r e a d y been s u g g e s t e d t h a t c a f f e i n e m i g h t i n c r e a s e t h e e f f e c t i v e -ne s s o f m i t o m y c i n C t r e a t m e n t ( 5 2 ) . However , w h e t h e r c a f f e i n e i s a s u i t a b l e a g e n t f o r t h i s p u r p o s e i s q u e s t i o n a b l e . The e f f e c t i v e c o n c e n -t r a t i o n o f c a f f e i n e a p p e a r t o o h i g h . They m i g h t p r o d u c e a s e v e r e t o x i c e f f e c t i f i n j e c t e d i n t o mammals i n c l u d i n g man. One t h i n g t o c o n s i d e r , i s t h a t i f c a f f e i n e i s i n j e c t e d i n t o a n i m a l s , i t s e f f e c t i v e c o n c e n t r a t i o n i n t h e t i s s u e s w i l l depend on t h e i r w a t e r c o n t e n t . Hence , i t may have p o t e n t i a l i n t h e t r e a t m e n t o f n e o p l a s t i c c o n d i t i o n s i n s uch o r g a n s as t h e k i d n e y s . The s e a r c h f o r a l e s s t o x i c a g e n t c a p a b l e o f s u p r e s s i n g r e p a i r p r o c e s s e s must be c o n t i n u e d . 39 . BIBLIOGRAPHY ( I) A d l e r , L.D. The p r o b l e m o f c a f f e i n e m u t a g e n i c i t y i n','Chemi c a I M u t a g e n e s i s i n Mammals and M a n " . Voge l F. and G. R o h r b o r n e d s . N.Y. , S p r i n g e r - V e r l a g . pp . 383 -403 ( 1 9 7 0 ) . (2 ) A l d e r s o n , T. and A . H . Khan. C a f f e i n e i nduced m u t a g e n e s i s i n D r o s o p h i l a . N a t u r e ( L o n d . ) 2 15 : 1080-1081 ( 1 9 6 7 ) . (3 ) And rew, L .E . The m u t a g e n i c a c t i v i t y o f c a f f e i n e i n D r o s o p h i l a . Am. N a t u r a l i s t 4 3 : 135-138 ( 1 9 5 9 ) . (4 ) A x e l r o d , J . and J . R e i c h e n t h a l . The f a t e o f c a f f e i n e i n man and a method f o r i t s e s t i m a t i o n i n b i o l o g i c a l m a t e r i a l . J . P h a r m a c o l , e x p . T h e r . 107: 519 -523 ( 1 9 5 3 ) . (5 ) B o y c e , R.P. and P. H o w a r d - F l a n d e r s . R e l e a s e o f U l t r a v i o l e t L i g h t -i n d u c e d t h y m i n e d i m e r s f r om DNA i n E. c o l i K -12 . P r o c . N a t l . A c a d . S c i . U .S . , 5 1 : 293 -300 ( 1 9 6 4 ) . (6) C l a r k , C .H . C a f f e i n e and amino a c i d - e f f e c t s upon t r y + r e v e r t a n t y i e l d i n U V - i r r a d i a t e d h c r + and h e r m u t a n t s o f E. C o l ? . B/r . Mo I. Gen . G e n e t . , 9 9 : 97 -108 ( 1 9 6 7 ) . (7) C l a r k , A .M. and E .G. C l a r k . The g e n e t i c e f f e c t s o f c a f f e i n e i n D r o s o p h i l a M e l a n o g a s t e r . M u t a t i o n R e s . , 6: 227 -234 ( 1 9 6 8 ) . (8 ) C l e a v e r , J . E . R e p a i r r e p l i c a t i o n o f mammalian c e l l D N A : e f f e c t s o f compounds t h a t i n h i b i t DNA s y n t h e s i s o r d a r k r e p a i r . R a d i a t i o n R e s . , 37 : 334 -348 ( 1 9 6 9 ) . 40 . (8a) D e c k e r , D .G. , Mus sey , E. , M a l k a s i a n , G .D . , and C . E . J o h n s o n . C y c l o p h o s p h a m i d e i n t h e t r e a t m e n t o f O v a r i a n c a n c e r . C l i n e . O b s t e t . G y n e c , I I : 382 -400 ( 1 9 6 8 ) . (9 ) Demerec, M . , W a l l a c e , B. and E.M. W i t k i n . The gene . C a r n e g i e I n s t . Wash. Y e a r Book, 47 : 169-176 ( 1 948 ) . / ( 1 0 ) ; Demerec, M . , B e r t a n i , G. and J . F l i n t . A s u r v e y o f c h e m i c a l s f o r m u t a g e n i c a c t i o n i n E_. c o l i . Am. 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