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Relationships between Mutagen-induced DNA repair, chromosome aberrations and sister chromatid exchanges MacRae, W. Donald 1978

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R e l a t i o n s h i p s between Mutagen-induced DNA R e p a i r , Chromosome A b e r r a t i o n s and S i s t e r C h r o m a t i d Exchanges by W. Dona Id MacRae B . S c . , U n i v e r s i t y o f V i - c t o r i a , 1974 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 l m e n t o f t h e Requirements o f t h e Degree o f MASTER OF SCIENCE , . i n THE FACULTY OF GRADUATE STUDIES (Genetics) 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 re q u i red 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 Columbia May, 1978 (c) w. Donald MacRae, 1978. In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thesis fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of Medical Genetics The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date September 11. 1978. i ABSTRACT The i n d u c t i o n of s i s t e r c h r o m a t i d e x c hanges(SCEs).and chromosome a b e r r a t i o n s by mutagenic a g e n t s was examined i n normal human and xeroderma pigmentosum ceI Is and C h i n e s e hamster ovary(CHO) c e l l s . The e f f e c t s o f t h e a l t e r a t i o n o f 5'-bromodeoxyuridine concen-t r a t i o n on t h e spontaneous and mutagen-induced SCE f r e q u e n c y , t h e t i m e of t r e a t m e n t and s a m p l i n g , a n d t h e d u r a t i o n of mutagen t r e a t m e n t were each c o n s i d e r e d w i t h r e s p e c t t o t h e a p p l i c a b i l i t y o f t h e t e c h -nique, as a t e s t f o r d e t e c t i n g m u tagenic and c a r c i n o g e n i c a g e n t s . SCE i n d u c t i o n by U.V. I i g h t , 4 - n i t r o q u i n o I i n e 1-oxide and N-methyl-N ' - n i t r o - n i t r o s o g u a n i d i n e i n xeroderma pigmentosum and normal human c e l l s was s t u d i e d as a p o s s i b l e means o f e x a m i n i n g t h e r e l a t i o n s h i p s between u n s c h e d u l e d DNA s y n t h e s i s , c h r o m o s o m e a b e r r a t i o n s and SCEs. Xeroderma pigmentosum c e l l s were more r e s p o n s i v e t h a n normal c e l l s w i t h r e s p e c t t o SCE f o r m a t i o n a f t e r t r e a t m e n t w i t h each o f t h e s e m u t a g e n i c a g e n t s . To f u r t h e r probe t h e r e l a t i o n s h i p s between SCEs,chromosome a b e r -r a t i o n s , D N A r e p a i r and c e l l s u r v i v a l , t h e r e s p o n s e of c e l l s a r r e s t e d by a m i n o - a c i d d e p r i v a t i o n t o mutagen t r e a t m e n t was examined.CHO c e l l s were a r r e s t e d by a r g i n i n e d e p r i v a t i o n and t r e a t e d w i t h mutagen.They were found t o be m a r k e d l y more s e n s i t i v e t h a n d i v i d i n g ceI I s , s h o w i n g a reduced s u r v i v a l and an e l e v a t e d f r e q u e n c y of chromosome a b e r r a t i o n s . No removal of t h o s e l e s i o n s which produced chromosome a b e r r a t i o n s was d e t e c t e d a f t e r p o s t - t r e a t m e n t i n c u b a t i o n i n a r g j n i n e d e f i c i e n t mediurn.U.V.-induced SCEs,on t h e o t h e r h a n d , c o u l d be reduced i n number by h o l d i n g t h e c e l l s i n a r g i n i n e d e f i c i e n t medium f o r 24 hours a f t e r i r r a d i a t i o n . T h i s phenomenon,which was i n t e r p r e t e d as t h e removal of SCE-producing l e s i o n s , w a s not o b s e r v e d a f t e r t r e a t -ment w i t h e i t h e r o f t h e a l k y l a t i n g a g e n t s N - m e t h y I - N ' - n i t r o - n i t r o s o -g u a n i d i n e o r m i t o m y c i n C.The i m p l i c a t i o n s o f t h e s e f i n d i n g s w i t h r e s p e c t t o mammalian DNA r e p a i r mechanisms a r e ' d i s c u s s e d . ACKNOWLEDGEMENTS I am i n d e b t e d t o Dr. H.F. S t i c h f o r h i s s u p e r v i s i o n and h e l p f u l c r i t i c i s m s c o n c e r n i n g t h i s work. The f i n a n c i a l s u p p o r t o f t h e B.C. Non-Animal Research F o u n d a t i o n i s g r a t e f u l l y acknowledged. I t i s a l s o my good f o r t u n e and p l e a s u r e t o e x t e n d t h a n k s t o a l l members of t h e group f o r t h e i r s u p p o r t and good company. FIGURES AND ILLUSTRATIONS F i g u r e 1 . C y t o I o g i c a I consequences of a s i n g l e s t r a n d exchange o c c u r r i n g a f t e r t h e f i r s t S-phase a f t e r i n c o r p o r a t i o n o f I abe I'. F i g u r e 2.Spontaneous SCE f r e q u e n c i e s of CHO and human c e l l s grown i n t h e p r e s e n c e o f d i f f e r e n t BrdU c o n c e n t r a t i o n s . F i g u r e 3.Spontaneous and MMC-induced SCE f r e q u e n c y i n human c e l l s grown i n d i f f e r e n t c o n c e n t r a t i o n s of BrdU. F i g u r e 4 . E f f e c t o f s a m p l i n g t i m e and t r e a t m e n t t i m e on t h e MMC-induced SCE f r e q u e n c y • i n human c e l l s . F i g u r e 5.Dose-response c u r v e f o r SCEs induced i n human c e l l s by MMC. F i g u r e 6.Dose-response c u r v e f o r SCEs formed i n : CHO ceI Is i n resp o n s e t o a c u t e and c h r o n i c t r e a t m e n t s w i t h MNNG. F i g u r e 7.Dose-response c u r v e s f o r SCEs induced in. human c e l l s by t h e chromate compounds i ^ C ^ O y and K ^ C r O ^ F i g u r e 8.Comparison o f do s e - r e s p o n s e c u r v e s f o r d e c r e a s e d s u r v i v a l and SCEs induced i n CHO c e l l s by U.V. l i g h t . F i g u r e 9.Dose-response c u r v e f o r SCEs induced i n normal human and X P E e e l Is by U.V. I i g h t . F i g u r e 1 0 . I n d u c t i o n of SCEs i n normal human and XPp c e l l s by 4NQ0 and M.NNG, F i g u r e 11.The o n s e t o f S-phase i n CHO c e l l s r e l e a s e d from a r g i n i n e d e p r i v a t i o n . F i g u r e 12.The o n s e t of m i t o s i s i n CHO c e l l s r e l e a s e d from a r g i n i n e d e p r i v a t i o n . F i g u r e 13.The e f f e c t o f maintenance of CHO c e l l s i n ADM upon c e l l s u r v i v a I . F i g u r e 1 4 . I n d u c t i o n o f chromosome a b e r r a t i o n s by U.V. i n ADM and I DM*-arrested c e l l s . F i g u r e 1 5 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n d i v i d i n g CHO 46 e e l Is by U.V. I i g h t . F i g u r e 1 6 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM-arrested and d i v i d i n g CHO c e l l s by MNNG. F i g u r e 24.U.V. induced chromosome a b e r r a t i o n s i n ID M - a r r e s t e d CHO c e l l s w i t h and w i t h o u t a 24 hour p e r i o d o f p o s t t r e a t m e n t i n c u b a t i o n i n I DM. 49 F i g u r e 1 7 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM-arrested 50 CHO e e l Is by MMC. F'gure 1 8 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM-arrested 51 and d i v i d i n g CHO c e l l s by AD. F i g u r e 19.Comparison o f s u r v i v a l o f ADM-arrested and d i v i d i n g 55 c e l l s a f t e r U.V. i r r a d i a t i o n . F i g u r e 2 0 . E f f e c t o f U.V. i r r a d i a t i o n (2 erg-mm 2) on t h e 58 chromosome a b e r r a t i o n f r e q u e n c y o f A D M - a r r e s t e d • a n d ' d i v i d i n q CHO c e I t s F i g u r e 2 1 . I n d u c t i o n o f SCEs i n ADM-arrested c e l l s a t i n t e r v a l s 60 b e f o r e and a f t e r t h e o n s e t o f S-phase by U.V. Iight,MMC and MNNG. F i g u r e 2 2 . S u r v i v a l , o f ADM-arrested CHO c e l l s a f t e r i r r a d i a t i o n 61 w i t h U.V. I i g h t . F i g u r e 23.Recovery o f ADM-arrested XP^ c e l l s t r e a t e d w i t h MNNG. 63 64 P l a t e 1 . H a r l e q u i n s t a i n e d metaphase o f CHO c e l l . 65 P l a t e 2 . H a r l e q u i n s t a i n e d metaphase o f CHO c e l l showing 65 m u l t i p l e chromosome a b e r r a t i o n s and i n c r e a s e d SCE f r e q u e n c y . P l a t e 3.Morphology of ADM-arrested CHO c e l l s . 66 P l a t e 4.Morphology of d i v i d i n g CHO c e l l s . 66 P l a t e 5.Chromosome a b e r r a t i o n s induced i n ADM-arrested CHO 67 ee l I by U.V. I i g h t . P l a t e 6 . M i c r o n u c l e i induced i n ADM-arrested CHO c e l l s by U.V. 67 l i g h t . ' LIST OF TABLES T a b l e I. Optimum Is^HPO^ i n c u b a t i o n t e m p e r a t u r e s f o r v a r i o u s BrdU 19 c o n c e n t r a t i o n s i n human and CHO c e l l s . T a b l e I I . Types of chromosome a b e r r a t i o n s s c o r e d . 21 T a b l e I I I . C o m p i l a t i o n of mean spontaneous SCE f r e q u e n c i e s o b t a i n e d 26 in a v a r i e t y of e x p e r i m e n t s under s l i g h t l y d i f f e r e n t c o n d i t i o n s . T a b l e IV. I n c i d e n c e of SCEs and chromosome a b e r r a t i o n s i n CHO c e l l s 44 r e l e a s e d from ADM-arrest a t v a r i o u s t i m e s . T a b l e V. Chromosome a b e r r a t i o n s induced i n CHO c e l l s a r r e s t e d i n 47 ADM f o r 7 days and i r r a d i a t e d w i t h U.V. 2 hours p r i o r t o r e l e a s e from ADM. T a b l e V I . SCE i n d u c t i o n i n ADM-arrested and d i v i d i n g CHO c e l l s by 52 U.V., MNNG and AD. T a b l e V l l . Chromosome a b e r r a t i o n s and SCEs induced i n ADM-arrested 54 CHO e e l I s . T a b l e V I I I . Chromosome a b e r r a t i o n s induced i n ADM-arrested CHO c e l l s 57 by U.V., MNNG and MMC. v i i TABLE OF CONTENTS INTRODUCT ION MATERIALS AND METHODS 10 I. T i ssue C u I t u r e 10 a M e d i a 10 b) CeI Is 11 c) C u I t u r e Methods 1T I I . Mutagen T r e a t m e n t 14 I N . C y t o l o g i c a l P r e p a r a t i o n 16 IV. Label I i n g Methods 17 a) R a d i o a c t i v e l a b e l l i n g 17 b) BrdU 18 V. A n a l y s i s 19 a M i t o t i c r a t e 20 b) Chromosome a b e r r a t i o n s 20 c) S i s t e r c h r o m a t i d exchanges 22 d) C o l o n y f o r m a t i o n 22 e) i n c o r p o r a t i o n o f ^ H - t h y m i d i n e 22 RESULTS 24 I. P r e l i m i n a r y S t u d i e s 24 a) E f f e c t of BrdU c o n c e n t r a t i o n 24 b) E f f e c t o f s a m p l i n g and t r e a t m e n t t i m e s 25 c) E f f e c t o f d u r a t i o n o f t r e a t m e n t 30 I I . The I n d u c t i o n o f SCEs i n X P E and Normal C e l l s 34 I I I . R e l e a s e o f C e l I s from ADM/I DM 37 a) C h a r a c t e r i z a t i o n o f ADM-mediated a r r e s t and r e l e a s e 37 b) l n d u c t i o n of chromosome a b e r r a t i o n s 44 c) I n d u c t i o n of SCEs 52 d) S u r v i v a l 54 V i i i e) R e p a i r i n ADM-arrested c e l l s 56 f ) O t h e r a r r e s t e d c e l l systems 59 DISCUSSION a ) T e c l h n i c a l commentary 68 b.)SCE i n d u c t i o n i n XP c e l l s 72 c) Recovery of e e l Is from ADM 75 d) R e p a i r o f chromosome and S C E - i n d u c i n g l e s i o n s 80 SUMMARY 86 BIBLIOGRAPHY 88 APPENDICES 1 95 2. 3 96 97 4 97 i x .ABBREVIATIONS USED AD - a d r i amyc i n ( d o x o r u b i c i n) ADM - a r g i n i n e d e f i c i e n t medium BrdU -5'-bromodeoxyuri d i ne CHO - C h i n e s e hamster o v a r y FBS - f e t a l b o v i n e serum 1 DM - i s o l e u c i n e d e f i c i e n t medium MEM -minimum e s s e n t i a l medium MMC - M i t o m y c i n C MNNG -N-methyl-N'-ni t r o - n i t r o s o g u a n i di ne 4NQ0 T - 4 - n i t r o q u i nol i n e - l - o x i de SCE - s i s t e r c h r o m a t i d exchange UDS ^ u n s c h e d u l e d DNA s y n t h e s i s U.V. - u l t r a v i o l e t XP -xeroderma pigmentosum 1 INTRODUCTION The f i r s t c y t o l o g i c a l e v i d e n c e f o r t h e e x i s t e n c e of exchanges between newly d u p l i c a t e d s i s t e r c h r o m a t i d s was p r e s e n t e d o v e r t w e n t y y e a r s ago by J.H.TayI o r ( 1 9 5 8 ) who examined t r i t i a t e d t h y m i d i n e l a b e l l e d Bellevalia chromosomes.Later i t was found t h a t t h e i n c o r p -o r a t e d t r i t i u m was i t s e l f e f f e c t i v e i n i n d u c i n g an i n c r e a s e d s i s t e r c h r o m a t i d exchange(SCE) I eve I ( M a r i n and P r e s c o t t , 1 9 6 4 ; G i b s o n and P r e s c o t t , 1 9 7 2 ) and t h e q u e s t i o n of whether t h e r e i s a r e a l spontaneous i n c i d e n c e o f SCEs has not y e t been s e t t Ied.Subsequent work r e v e a l e d t h a t many a g e n t s which damage mamma I i a n .DNA a l s o i n d uce S C E s ( K a t o , 1974a).With t h e development of an improved l a b e l l i n g method u t i l i z i n g t h e t h y m i d i n e analogue 5 ' - b r o m o - 2 1 - d e o x y u r i d i n e ( B r d U ) ( L a t t , 1 9 7 3 ) , many ag e n t s were i d e n t i f i e d as b e i n g c a p a b l e of i n d u c i n g SCEs.These i n c l u d e t h e p h y s i c a l a g e n t s X - r a d i a t i o n ( M a r i n and P r e s c o t t , 1 9 6 4 ; P e r r y and E v a n s , 1 9 7 5 ) , y - r a d i a t i o n ( S o Iomon and Bob row,1975) and U.V. I ight(Kato,1974a;Rommelaere et al < -1973).A v a r i e t y o f ' c h e m i c a l a g e n t s have a l s o been examined and many of t h e m u t a g e n i c / c a r c i n o g e n i c chem-i c a l s found t o be p o t e n t i n d u c e r s of SCEs.Chemicais r e p r e s e n t a t i v e o f t h e b i f u n c t i o n a l and m o n o f u n c t i o n a I a l k y l a t i n g a g e n t s ( L a t t , 1 9 7 3 ; K a t o , 1974a;Perry and Evans,1975;So Iomon and Bob r o w , 1 9 7 5 ) , i n t e r c a I a t i n g a g e n t s ( K a t o , 1 9 7 4 a ; P e r r y and E v a n s , 1 9 7 5 ) , m y c o t o x i n s ( T a k e h i s a and W o l f f , 1 9 7 7 ) , a m i n o f I u o r e n e d e r i v a t i v e s ( T a k e h i s a and Wo I f f , 1 9 7 7 ; N a t a r a j a n et al. ,1976) and a r o m a t i c h y d r o c a r b o n s ( R u d i g e r et al. 1976) a l l produce 2 marked i n c r e a s e s i n SCE f r e q u e n c y . The p r e s e n t e v i d e n c e s u g g e s t s t h a t a g e n t s w h i c h i n d u c e SCEs a l s o induce chromosome a b e r r a t i o n s , a I though t h e c o n v e r s e i s not n e c e s s a r i l y t r u e ( K a t o , 1 9 7 7 b ) . T h e y i e l d o f chromosome a b e r r a t i o n s does not c o m p l e t e -ly p a r a l l e l t h e y i e l d o f SCEs a l t h o u g h t h e m a j o r i t y of a c t i v e a g e n t s a ppear t o induce s i g n i f i c a n t , a n d o f t e n d r a m a t i c i n c r e a s e s a t doses t o o low t o y i e l d s i g n i f i c a n t numbers of chromosome a b e r r a t i o n s C P e r r y and Evans,1975).The extreme s e n s i t i v i t y o f t h e r e s p o n s e and t h e ease and p r e c i s i o n w i t h which SCEs may be s c o r e d a r e i m p o r t a n t a d v a n t a g e s in a p p l y i n g t h i s e n d p o i n t as an a s s a y of m u t a g e n i c / c a r c i n o g e n i c a c t i v i t y . In vitro a c t i v a t i o n of p r e c a r c i n o g e n s u s i n g l i v e r microsomal m a t e r i a l has been s u c c e s s f u l l y a p p l i e d w i t h SCE ana I y-s i s (Taken i sa and W o l f f , 1 9 7 7 : S t e t k a and Wo I f f , 1 9 7 8 ; N a t a r a j a n et al.3 1976),aI Iowing t h e maximum pos-s i b l e l a t i t u d e i n s c r e e n i n g f o r m u t a g e n s / c a r c i n o g e n s by in vitro methods.Indeed,SCE i n d u c t i o n i s c u r r e n t l y r e c o g n i z e d as t h e most s e n s -i t i v e i n d i c a t o r of a g e n t s which damage mammalian DNA(Wolff et al.,\911). A s i g n i f i c a n t drawback i n u s i n g t h i s e n d p o i n t as an i n d i c a t o r of m u t a g e n i c / c a r c i n o g e n i c a c t i v i t y i s t h a t n e i t h e r t h e m o l e c u l a r mechanism no r t h e b i o l o g i c a l s i g n i f i c a n c e of t h e phenomenon have been c l a r i f i e d . Thus,one i s f o r c e d t o r e l y upon t h e e x t e n s i v e a n a l y s i s o f known compounds i n o r d e r t o a s s e s s t h e p r o b a b i l i t y o f a c h i e v i n g e i t h e r f a l s e n e g a t i v e o r f a l s e p o s i t i v e r e s u l t s . 3 Many wo r k e r s have drawn a t t e n t i o n t o t h e s i m i l a r i t i e s between the b e h a v i o r of SCEs and a r e c o m b i n a t i o n : r e p a i r ' m e c h a n i s m ( K a t o , 1 9 7 4 a ; W o l f f et al. j, 1974; S a s a k i , 1977) .There a r e p r e s e n t l y t h r e e r e c o g n i z e d enzyme-mediated mechanisms by w h i c h mammalian DNA can be r e p a i r e d : p h o t o r e a c t i v a t i o n , e x c i s i o n r e p a i r and p o s t - r e p I i c a t i o n r e p a i r . T h i s l a t t e r may i n v o l v e a r e c o m b i n a t i o n a I p r o c e s s . P h o t o r e a c t i v a t i on i s r e s p o n s i b l e f o r t h e c l e a v i n g of .11.V.-induced p y r i m i d i n e dimers and i s dependent upon i n c i d e n t r a d i a n t energy ( R u p e r t et al. j 1 9 7 2 ) . T h i s a c t i v i t y has been c l e a r l y d e m o n s t r a t e d i n non-mammalian c e l l s w h i l e i t s e x i s t e n c e i n mamma I i a n ceI I s- has"been d e t e c t e d o n l y by some i n v e s t i g a t o r s ( S u t h e r I and,1975). E x c i s i o n r e p a i r r e f e r s t o t h e removal of damaged DNA and i t s r e s y n t h e s i s on t h e undamaged complementary s t r a n d by a non-semicon-s e r v a t i v e m e c h a n i s m . l t has been i n t e n s i v e l y s t u d i e d i n both b a c t e r i a l and mammalian c e l l s by e x a m i n i n g e i t h e r t h e removal of damage such as t hymine dimers(Regan et al.,\96B) o r r a d i o a c t i v e I y l a b e l l e d c a r c i n o g e n -DNA a d d u c t s ( I g e n a g a e t 'aZ. j1974) o r t h e r e s y n t h e s i s of e x c i s e d r e g i o n s of t h e genome.Resynthesis has been s t u d i e d by: a) UnscheduIed DNA s y n t h e s i s ( U D S ) : t h e i n c o r p o r a t i o n o f r a d i o a c t i v e p r e c u r s o r s i n t o t h e DNA of c e l l s not u n d e r g o i n g s e m i c o n -s e r v a t i v e DNA rep I i c a t i o n ( R a s m u s s e n and P a i n t e r , 1 9 6 4 ) , b) R e p a i r rep I i c a t i o n . L i k e U D S ,this t e c h n i q u e measures t h e i n c o r p o r a t i o n o f new n u c l e o t i d e s i n t o t h e p a r e n t a l damaged DNA.The p o s s i b i l i t y t h a t t h i s i s due t o s t i m u l a t e d s e m i c o n s e r v a t i v e r e p -4 I i c a t i o n , h o w e v e r i s excIuded(Rasmussen and P a i n t e r , 1 9 6 6 ) . c ) B r d U Photo I y s i s . T h e i n c o r p o r a t i o n of new n u c l e o t i d e s can a l s o be measured i f BrdU,which i s s e n s i t i v e t o r u p t u r e by 313 nm I i g h t , i s used as a l a b e l (Regan et al._, 1971 ) .The e x t e n t of r u p t u r e w i l l be dependent upon t h e e x t e n t of BrdU uptake which w i l l depend upon t h e number of l e s i o n s r e p a i r e d . The n a t u r e of t h e enzymes r e s p o n s i b l e f o r e x c i s i o n r e p a i r in mammalian c e l l s i s now under s t u d y and e n d o n u c l e a s e a c t i v i t y s p e c i f i c f o r X-ray damaged DNA(Brent,1973) and U.V.-damaged DNACKapIan,19750 have been i d e n t i f i e d . D e s p i t e t h e w e l l e s t a b l i s h e d i mportance of e x c i s i o n r e p a i r , i t has not been p o s s i b l e t o o b t a i n a good c o r r e l a t i o n between a c e l l l i n e ' s c a p a c i t y t o c a r r y o u t t h i s r e p a i r , u p o n e x p o s u r e t o damaging agent-,and s u r v i v a l , t h e u l t i m a t e measure of a s u c c e s s f u l r e p a i r mech-an i sm(Bertram et al. , 1974a; B e r t r a m et al., 1974b; R o b e r t s et al, ,1971b; R o b e r t s , 1 9 7 2 ; S h a e f f e r and M e r t z , 1 9 7 1 ) . T h i s d i f f i c u l t y has been p a r t -i a l l y overcome by t h e d e t e c t i o n o f a t h i r d r e p a i r mechanism a c t i n g upon D N A . F o l l o w i n g t h e r e p l i c a t i o n o f U.V.-damaged DNA,it i s p o s s i b l e t o d e t e c t gaps i n t h e d a u g h t e r s t r a n d s which c o r r e s p o n d w i t h t h e number of dimers formed(Meyn and Humphrey,1971).These gaps a r e sub-s e q u e n t l y f i l l e d by t h e p r o c e s s known as p o s t - r e p I i c a t ion . r e p a i r (Lehmann,1972).In b a c t e r i a , g a p f i l l i n g i n v o l v e s t h e r e p l a c e m e n t of a p p r o x i m a t e l y 10^ n u c I e o t i d e s ( H o w a r d - F I a n d e r s , 1 9 6 8 ) and mechanisms f o r t h e exchange o f s i n g l e DNA s t r a n d s between s i s t e r c h r o m a t i d s have been proposedCRupp et al.., 197J) .The o b s e r v a t i o n t h a t t h y m i n e dimers 5 induced i n p a r e n t a l DNA a r e t r a n s f e r r e d t o d a u g h t e r s t r a n d s upon rep I i c a t i o n ( G a n e s a n , 1 9 7 4 ) i n d i c a t e s t h a t some form o f r e c o m b i n a t i o n i s t a k i ng p i ace. Whereas t h e e v i d e n c e f o r r e c o m b i n a t i o n i n b a c t e r i a i s c o n s i s t e n t w i t h proposed mechanisms i n v o l v i n g s i n g l e s t r a n d exchanges,no p a r a l l e l w i t h mammalian c e l l s i s y e t e v i d e n t C K a t o , 1 9 7 7 b ) . F u r t h e r m o r e , n o c y t o -l o g i c a l e v i d e n c e f o r t h e e x i s t e n c e o f s i n g l e s t r a n d exchanges has been f o r t h c o m i n g . A s i n g l e s t r a n d exchange o c u r r i n g i n t h e f i r s t c e l l c y c l e a f t e r a d d i t i o n o f a l a b e l compound s h o u l d r e s u l t i n an u n l a b e l -e d segment a p p e a r i n g i n one c h r o m a t i d a t t h e subsequent m i t o s i s ( F i g u r e l ) . T h i s uneven l a b e l l i n g p a t t e r n would be p r e s e r v e d a t t h e second mi t o s i s. Ta'y I o r ' s f a i l u r e t o o b s e r v e such l a b e l l i n g p a t t e r n s (1958) l e d him t o c o n c l u d e t h a t s i s t e r c h r o m a t i d exhanges were t h e r e s u l t o f d o u b l e , b u t not s i n g l e s t r a n d exchanges.A I though t h e o c c u r -rence o f ' t r i t i a t e d t h y m i d i n e i n o p p o s i t e r e g i o n s o f s i s t e r c h r o m a t i d s ( i s o IabeI Iing) was l a t e r r e p o r t e d , t h e b e t t e r r e s o l u t i o n p e r m i t t e d w i t h t h e BrdU l a b e l l i n g method de m o n s t r a t e d t h a t t h e s e i s o l a b e l l e d r e g i o n s w e r e , i n f a c t , t w o s i s t e r c h r o m a t i d exchanges e x i s t i n g i n c l o s e p r o x i m i t y ( K a t o , 1 9 7 4 b ) . F u r t h e r e v i d e n c e f o r t h e c o n n e c t i o n between s i s t e r c h r o m a t i d exchanges and DNA r e p a i r p r o c e s s e s was o b t a i n e d by s t u d y i n g c e l l s from i n d i v i d u a l s w i t h g e n e t i c d i s e a s e s i n v o l v i n g a d e f e c t i n some a s p e c t o f DNA r e p a i r o r m e t a b o l i s m . l t was f i r s t o b s e r v e d t h a t Bloom's syndrome, c e l l s p o s s e s s a g r e a t l y e l e v a t e d spontaneous SCE f r e q u e n c y F i g u r e 1 . C y t o I o g i c a I consequences of a s i n g l e s t r a n d exchange : c u r r i n g a f t e r t h e f i r s t S-phase a f t e r i n c o r p o r a t i o n of l a b e l (45© ) 7 ( C h a g a n t i et al.3 1974).Bloom's syndrome i s a g e n e t i c d i s e a s e i n v o l v i n g v a r i o u s symptoms a s s o c i a t e d w i t h growth r e t a r d a t i o n . L y m p h o c y t e s and f i b r o b l a s t s from a f f e c t e d i n d i v i d u a l s p o s s e s s an e l e v a t e d spontaneous f r e q u e n c y of chromosome a b e r r a t i o n s , p r i m a r i l y i n v o l v i n g exchanges between homologous chromosomes(German,1974).Fanconi 's anemia and a t a x i a t e l a n g i e c t a s i a a r e two o t h e r g e n e t i c d i s e a s e s i n w h i c h t h e spontaneous f r e q u e n c y of chromosome a b e r r a t i o n s i s h i g h ( B l o o m et a Z . j 1 9 6 6 ) , I n c e l l s o f such a f f e c t e d i n d i v i d u a I s , h o w e v e r , t h e spontaneous SCE r a t e i s normal ( B a r t r a m et al., 1 9 7 6 ; S p e r I i n g , 1 9 7 5 ) . F a n c o n i 's anemia c e l l s , w h i c h appear t o be d e f e c t i v e i n some s t e p o f t h e e x c i s i o n r e p a i r o f U.V.-induced t h y m i d i n e d i m e r s ( P o o n et al., 1974),however,do not respond t o c h e m i c a l t r e a t m e n t w i t h e l e v a t e d SCE f r e q u e n c i e s ( L a t t et al., 1975). What emerges from t h e s e d i s p a r a t e l i n e s o f e v i d e n c e i s a view of t h e s i s t e r c h r o m a t i d exchange as a phenomenon i n t i m a t e l y i n v o l v e d i n DNA r e p a i r p r o c e s s e s . T h e r e l a t i o n s h i p between t h e SCE and u n s c h e d u l e d DNA s y n t h e s i s , p o s t - r e p I i c a t i o n r e p a i r and t h e chromosome a b e r r a t i o n i s , however,as y e t o b s c u r e . l t remains t o be e s t a b l i s h e d : i ) what t y p e o f l e s i o n s a r e r e s p o n s i b l e f o r i n d u c i n g t h e SCE, i i ) what f a c t o r s r e g u l a t e t h e f o r m a t i o n and perhaps t h e m o d i f i c a t i o n o r removal of t h e s e l e s i o n s and t h e i r t r a n s f o r m a t i o n i n t o t h e SCE, i i i ) what a r e t h e m o l e c u l a r mechanisms f o r SCE f o r m a t i o n , a n d i v ) what i s t h e b i o l o g i c a l s i g n i f i c a n c e o f t h e SCE: i . e . does i t r e p r e s e n t some a s p e c t of DNA damage o r DNA r e p a i r ; d o e s i t c o n f e r s u r v i v a l 8 v a I u e , r e d u c e v i a b i l i t y o r perhaps r e p r e s e n t a m u t a t i o n a l e v e n t ? T h i s r e s e a r c h has a t w o f o l d p u r p o s e - I t s f i r s t aim i s t o e v a l u a t e t h e v a l i d i t y of t h e t e c h n i q u e of SCE a n a l y s i s as a s e n s i t i v e i n d i c a t o r of t h e a c t i o n of m u t a g e n i c / c a r c i n o g e n i c a g e n t s . M o s t w o r k e r s i n t h i s a r e a have used a c h r o n i c e x p o s u r e t o c h e m i c a l t o induce e l e v a t e d SCE l e v e l s . S u c h an approach t a k e s no a c c o u n t of t h e p o s s i b i l i t y t h a t t h e c h e m i c a l may be a c t i v e a t a s p e c i f i c s t a g e i n t h e c e l l c y c l e o r t h a t i t may be s t a b l e i n t h e c u l t u r e f o r o n l y a s h o r t p e r i o d o f t i m e . T r e a t m e n t s of long and s h o r t d u r a t i o n a r e compared i n an a t t e m p t t o c l a r i f y t h i s q u e s t i o n . F u r t h e r m o r e , m a n y s t u d i e s have been c o n d u c t e d o n l y o v e r l i m i t e d dose ranges.The i n d u c t i o n of SCEs by u l t r a v i o l e t r a d i a t i o n was examined o v e r a wide dose range and compared w i t h c e l l s u r v i v a l . The p r i m a r y g o a l of t h i s work,however,was t o e l u c i d a t e t h e n a t u r e o f t h e l e s i o n s r e s p o n s i b l e f o r t h e i n d u c t i o n of t h e SCE.The s p e c i f i c q u e s t i o n a d d r e s s e d was:what happens t o t h e l e s i o n s between t h e t i m e o f t h e i r f o r m a t i o n and t h e t i m e of t h e i r t r a n s f o r m a t i o n i n t o SCEs,pre-sumably d u r i n g t h e S-phase o f t h e c e l l c y c l e . T h i s q u e s t i o n was a p p r o a c h e d i n two w a y s : u s i n g XP c e l l s and c e l l s a r r e s t e d i n t h e n o n - p r o l i f e r a t i n g s t a t e by d e p r i v a t i o n of t h e e s s e n t i a l amino a c i d a r g i n i n e . In t h e f i r s t case,XP and normal c e l l s were c o m p a r e d . A c c o r d i n g t o . t h e p r o t o c o l u s e d , c e l l s were t r e a t e d w i t h c h e m i c a l p r i o r t o t h e a d d i t i o n of B r d U . S i n c e h a r l e q u i n s t a i n e d c e l l s have n e c e s s a r i l y undergone two S-phases i n t h e p r e s e n c e of. BrdU,such c e l l s w i l l have been exposed t o c h e m i c a l a t some p o i n t p r i o r t o S-phase,In t h i s l a b e l l i n g scheme,SCEs 9 c o u l d p o s s i b l y o c c u r a t any t i m e between t h e f i r s t S-phase i n BrdU and th e t i m e o f h a r v e s t i n g . O t h e r e v i d e n c e s t r o n g l y s u p p o r t s t h e i d e a t h a t t h e p r o c e s s t a k e s p l a c e d u r i n g S-phase,perhaps a t t h e t i m e t h e f o r k of r e p l i c a t i n g DNA t r a v e r s e s t h e Iesion(Kato,1974d; Wo I f f et al.3197A). The e a r l i e s t p o s s i b l e t i m e t h a t a l e s i o n c o u l d be c o n v e r t e d i n t o an SCE i s t h u s when passed by t h e r e p l i c a t i n g f o r k d u r i n g t h e f i r s t S-phase a f t e r a d d i t i o n o f BrdU. In t h i s t r e a t m e n t regimen,then,SCEs w i l l be t h e r e s u l t o f l e s i o n s which have s u r v i v e d t h e u n d e f i n e d t i m e p e r i o d between e x p o s u r e t o mutagen and SCE f o r m a t i o n d u r i n g S-phase,DNA damage e x i s t i n g d u r i n g t h i s t i m e s h o u l d be s u b j e c t t o r e p a i r by unscheduled DNA s y n t h e s i s -(UDS).By comparing t h e b e h a v i o u r o f normal and XP c e l l s d e f i c i e n t i n UDS i t s h o u l d be p o s s i b l e t o examine t h e e f f e c t o f UDS upon l e s i o n s which a r e r e s p o n s i b l e f o r t h e i n d u c t i o n o f SCEs. The second approach i n v o l v e d v a r y i n g t h e t i m e between e x p o s u r e t o mutagen and t h e o n s e t o f S-phase.This was a c c o m p l i s h e d by i n d u c i n g a s t a t e o f p r o l i f e r a t i v e a r r e s t i n c e l l s by d e p r i v a t i o n o f t h e e s s e n t i a l amino a c i d a r g i n i n e . R e t u r n i n g t h e c e l l s t o normal growth medium causes a g r a d u a l o n s e t o f DNA s y n t h e s i s and a r e s u m p t i o n o f a c t i v e proI i f - . e r a t i o n . T h e t i m e o f t r e a t m e n t can t h e n be v a r i e d w i t h r e s p e c t t o t h e t i m e o f o n s e t o f DNA s y n t h e s i s , i n t h i s way a l t e r i n g t h e t i m e t h a t t h e mutagen-induced l e s i o n s e x i s t i n c e l l s p r i o r t o S-phase. 10 MATERIALS AND METHODS I. T i s s u e C u I t u r e a) Media E a g l e ' s Minimum E s s e n t i a l Medi.urn(MEM).Powdered minimum e s s e n t i a l medium(Grand I s l a n d B i o l o g i c a l C o m p a n y , B e r k e I e y , C a I i f o r n i a ) was r e c o n s t i t u t e d w i t h d e - i o n i z e d d i s t i l l e d w a t e r and s t e r i l i z e d by passage t h r o u g h 0.22 m i c r o n m i l l i p o r e f i I t e r ( M i I I i p o r e F i l t e r C o r p o r a t i o n , M a s s . ) . A r g i n i n e and I s o l e u c i n e D e f i c i e n t Medium(ADM and IDM).Arg i n i n e and i s o l e u c i n e d e f i c i e n t media were p r e p a r e d by o m i t t i n g e i t h e r amino a c i d from t h e f o r m u l a f o r E a g l e ' s MEM.The e s s e n t i a l amino a c i d s ( G e n e r a I B i o c h e m i c a I s , C h a g r i n F a l l s , O h i o ) were f i r s t weighed and d i s s o l v e d as d e s c r i b e d i n A ppendix 2 . N o n - e s s e n t i a I amino a c i d s and v i t a m i n s were added i n t h e form of a 100X l i q u i d c o n c e n t r a t e ( F l o w L a b o r a t o r i e s , I n g I e w o o d , C a I i f o r n i a ) . T h e r e s u l t i n g 2X c o n c e n t r a t e of medium was s t o r e d a t 4°C u n t i l c o m b i n a t i o n w i t h serum. Ant i b i o t i c s . A t t h e t i m e of i t ' s p r e p a r a t i o n , c u I t u r e medium was supplemented w i t h 100 u n i t s / m l p e n i c i l l i n G ( G e n e ral B i o c h e m i c a I s ) and 100 ug/ml s t r e p t o m y c i n suI f a t e ( G e n e r a I B i o c h e m i c a I s ) . I m m e d i a t e Iy p r i o r t o use,100 ug/ml Kanamycin(Grand I s l a n d B i o l o g i c a l Company, Grand I s l a n d , N . Y . ) was added. Sodium b i c a r b o n a t e . A s t o c k s o l u t i o n of 1% sodium b i c a r b o n a t e ( F i s h e r S c i e n t i f i c Company,Fair Lawn,New J e r s e y ) was s t e r i l i z e d by 11 f i I t r a t i o n . E a c h 800 ml f l a s k o f medium was supplemented w i t h 10-12 ml of b i c a r b o n a t e s o l u t i o n i m m e d i a t e l y p r i o r t o use. F e t a l B o v i n e Serum(FBS).FetaI b o v i n e s e r u m C M i c r o b i o I o g i c a I A s s o c i a t e s , B e t h e s d a , M a r y I and) was s t o r e d a t -20^C and heat i n a c t i v a t e d (56^C f o r 30 m i n u t e s ) p r i o r t o use.Serum was added t o each f l a s k of medium as i t was p r e p a r e d d i r e c t l y p r i o r t o use. T r y p s i n.A 2.5 % s o l u t i o n o f t r y p s i n ( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n ) -i n .Hank's-.BSS (Append i x 1) was p r e p a r e d , d i s s o I ved by s t i r r i n g , and c e n t r i f u g e d a t 1800 rpm f o r 10 minutes.The s u p e r n a t a n t was f i l t e r e d f i r s t t h r o u g h Whatman No. 1 f i l t e r p aper and t h e n t h r o u g h a s t e r i l e 0.22 uM mi I I i pore f i I t e r ( M i I I i pore Corp.).Kanamyci n(100 yg/mI) and F u n g i z o n e ( G r a n d I s l a n d B i o I o g i c a I ( G i b c o ) ) ( 2 . 5 ug/ml) were th e n added. b) C e l I s 'Human s k i n f i b r o b I a s t s . H u m a n f i b r o b l a s t s t r a i n s were o b t a i n e d from e x p l a n t s of s k i n punch b i o p s y m a t e r i a l from v o l u n t a r y d o n o r s . C e l l s from normal young a d u l t s ( 2 0 - 2 5 y e a r s o l d ) and an i n d i v i d u a l a f f e c t e d w i t h t h e g e n e t i c d i s e a s e xeroderma pigmentosum(XP) were ut i I i zed. C h i n e s e hamster ovary(CHO) e e l Is.The e s t a b l i s h e d l i n e of C h i n e s e hamster o v a r y f i b r o b l a s t s was o b t a i n e d from Dr. L. S k a r s g a a r d , V a n c o u v e r . I t i s a p s e u d o d i p I o i d l i n e w i t h a modal number of 21 chromosomes. c) C u l t u r e Methods i ) S t o c k c u I t u r e s . S t o c k c u l t u r e s were m a i n t a i n e d i n 75 cm^ p l a s t i c 12 t i s s u e c u l t u r e f I a s k s ( F a I con P l a s t i c s , L o s A n g e I e s , C a I i f o r n i a ) . H u m a n c e l l s were grown i n MEM supplemented w i t h 15% FBS and s u b c u l t u r e d 1/2 o r 1/3 e v e r y 1-2 weeks.CHO c e l l s were m a i n t a i n e d i n MEM + 5% FBS and s u b c u l t u r e d 1/50 o r 1/100 a t weekly i n t e r v a l s . I n each c a s e , s u b -c u l t u r i n g c o n s i s t e d o f a 5 minute i n c u b a t i o n o f t h e c e l l s w i t h a 2.5$ t r y p s i n s o l u t i o n f o l l o w e d by d i s p e r s a l o f t h e c e l l s i n normal medium. Human c e l l s r e q u i r e s c r a p i n g w i t h a r u b b e r p o l i c e m a n w h i l e CHO c e l l s can be d i s p e r s e d by v i g o r o u s a g i t a t i o n o f t h e c u l t u r e v e s s e I . C u I t u r e s were m a i n t a i n e d a t 37°C i n a i r c o n t a i n i n g 5% CO2 and s a t u r a t e d w i t h w a t e r . i i ) E x p e r i m e n t a I c u I t u r e s . E x p e r i m e n t s were c a r r i e d o u t upon c e l l s g r o w i n g e i t h e r on 22 mm2 g l a s s c o v e r s I i p s ( C o r n i n g G l a s s W o r k s , C o r n i n g , New Y o r k ) i n 35 mm p l a s t i c p e t r i d i s h e s ( F a I con) o r i n 5 cm p l a s t i c d i s p o s a b l e p e t r i , p I a t e s (Fa I c o n ) . A c t i ve I y p r o l i f e r a t i n g c e l l s and t h o s e a r r e s t e d i n t h e G] phase by d e p r i v a t i o n o f e i t h e r a r g i n i n e o r i s o l e u c i n e were used. D i v i d i n g ceI I s : C u I t u r e s o f d i v i d i n g c e l l s were p r e p a r e d by f i r s t t r y p s i n i z i n g a s t o c k c u l t u r e f o r 5 minutes and d i s p e r s i n g t h e c e l l s i n normal medium.After p e r f o r m i n g t h e a p p r o p r i a t e di I u t i o n s , t h e c e l l s 2 were added i n 2 ml t o 35 mm p e t r i d i s h e s . M i t o t i c f i g u r e s c o u l d be o b s e r v e d a f t e r a p p r o x i m a t e l y 24 hours f o r human c e l l s and 12 hours f o r CHO e e l I s . Human f i b r o b l a s t s were seeded a t a d e n s i t y of 30,000-50,000 c e l l s p e r pI a t e . E x p e r i m e n t s i n v o l v i n g s a m p l i n g f o r SCEs and chromosome a b e r r a t i o n s c o u l d be begun 3 and 4 d a y s ( r e s p e c t i v e I y ) a f t e r s e e d i n g . D i f f i c u I t i e s were e n c o u n t e r e d i n o b t a i n i n g good c y t o g e n e t i c p r e p a r a t i o n s of c u l t u r e s o f h i g h c e l l d e n s i t y and s u f f i c i e n t m i t o t i c c e l l s f o r a n a l y s i s i n c u l t u r e s o f low c e l l d e n s i t y . C o n s i s t e n t c e l l s e e d i n g was t h e r e f o r e o f t h e utmost i m p o r t a n c e . CHO c e l l s were seeded a t a d e n s i t y o f e i t h e r 60,000 c e l l s p e r p l a t e f o r SCE e x p e r i m e n t s o r 80,000 c e l l s p e r p l a t e f o r chromosome a b e r r a t i o n e x p e r i m e n t s . I n each c a s e , t h e e x p e r i m e n t was begun on t h e f o I low i ng day. A r r e s t e d e e l I s : Human c e l l s were in d u c e d t o undergo r e p l i c a t i v e a r r e s t by t r a n s f e r r i n g a d i v i d i n g c u l t u r e t o medium d e f i c i e n t i n t h e e s s e n t i a l amino a c i d a r g i n i n e ( A D M ) . E x p e r i m e n t s were begun upon t h e s e c e l l s a f t e r 4 days i n ADM. CHO c e l l s were a r r e s t e d l e s s r e a d i l y by a r g i n i n e d e p r i v a t i o n . A s u i t a b l e p r o t o c o l was d e v e l o p e d whereby 50,000-60,000 c e l l s p e r p l a t e were seeded i n ADM supplemented w i t h 2% F B S . A f t e r a t t a c h m e n t , t h e c e l l s underwent a b r i e f r e p l i c a t i v e p e r i o d p r i o r t o a r r e s t . E x p e r i m e n t s were begun 5 days a f t e r s eeding.CeI Is seeded i n medium d e f i c i e n t i n t h e e s s e n t i a l amino a c i d i s o l e u c i n e behaved s i m i l a r l y . S u r v j v a l e x p e r i m e n t s : C I one f o r m i n g a b i l i t y was used as an in d e x of c e l l s u r v i v a I . D i v i d i n g CHO c e l l s were h a r v e s t e d by t r y p s i n i z a t i o n , suspended i n Hank's BSS and seeded i n 0.1 ml a l i q u o t s i n t o 5 cm 14 p l a s t i c p e t r i - p I a t e s ( F a I con) f o r a f i n a l d e n s i t y of 5,000-6,000 c e . l l s p e r p l a t e . T h r e e ml of t h e a p p r o p r i a t e growth medium was t h e n a d d e d . D i v i d i n g c e l l s were t r e a t e d a f t e r a l l o w i n g 6 hours f o r a t t a c h -. ment and i n c u b a t e d i n MEM+ 15% F B S . A r r e s t e d c e l l s were i n c u b a t e d i n ADM + 2% FBS f o r 5 days p r i o r t o t r e a t m e n t . T h i s a l l o w e d s u f f i c i e n t t i m e f o r most c e l l s t o undergo one d i v i s i o n and f o r t h e d a u g h t e r c e l l s formed t o s e p a r a t e and become a r r e s t e d . I I. Mutagen Treatment a) U l t r a - v i o l e t i r r a d i a t i o n C u l t u r e s were p r e p a r e d f o r U.V. i r r a d i a t i o n by r e p l a c e m e n t of t h e medium w i t h phosphate b u f f e r e d s a l i n e ( P B S ) s o l u t i o n ( s e e A ppendix 3 ) . The 35 mm and 5 cm p e t r i d i s h e s r e c e i v e d 1 and 3 ml r e s p e c t i v e l y . _? -1 C e l l s were i r r a d i a t e d a t an i n c i d e n t dose of 1 erg-mm z s e c d e l i v e r e d by a S y l v a n i a g e r m i c i d a l lamp(G15T8),at 20 ^C and r e t u r n e d t o growth medium. b) Chemical t r e a t m e n t 4 - n i t r o q u i n o I ine 1 - o x i d e ( 4 N Q 0 ) ( D a i i c h i Pure Chemical Company, Tokyo) i s not r e a d i l y s o l u b l e i n w a t e r . T h e r e f o r e , 0 . 4 ml of 100% e t h a n o l was f i r s t added t o t h e weighed sample f o l l o w e d by 9.6 ml of t i s s u e c u l t u r e medium. N - m e t h y I - N ' - n i t r o - n i t r o s o g u a n i d i n e ( M N N G ) ( A I d r i c h B i o c h e m i c a l Company,Inc.,Mi Iwaukee,Wisconsin) was d i s s o l v e d i n 10 ml of s t e r i l e d i s t i l l e d w a t e r and heated t o a p p r o x i m a t e l y 50 ^C w i t h v i g o r o u s a g i t a t i o n . 15 M i t o m y c i n C(MMC)(Sigma Chemical C o.,St. L o u i s , M i s s o u r i ) . w h i c h i s prepared as a powdered m i x t u r e w i t h 96% NaCl t o i n c r e a s e i t s s o l u b i l -i t y , w a s s i m p l y d i s s o l v e d i n d i s t i l l e d w a t e r . A d r i a m y c i n ( A D ) ( A d r i a L a b o r a t o r i e s o f C a n a d a , L t d . , M i s s a u g a , O n t a r i o ) i s combined i n a m i x t u r e w i t h 80$ d e x t r o s e and was d i s s o l v e d in. w a t e r . The chromate compounds r ^ C ^ O y and I^CrO^ ( F i s h e r ) were d i s s o l v e d i n w a t e r and t h e r e s u l t i n g pH n e u t r a l i z e d . Each o f t h e s t o c k s o l u t i o n s of c h e m i c a l were d i l u t e d i n medium supplemented w i t h 2,2.5 o r 15 % FBS.MEM,ADM.or Hank's BSS was used as mediurn,depending upon t h e e x p e r i m e n t b e i n g u n d e r t a k e n . D i I u t i o n s were such t h a t t h e s t o c k s o l u t i o n was d i l u t e d a t l e a s t 100 t i m e s by medium b e f o r e b e i n g used t o t r e a t c e l l s . C h e m i c a l t r e a t m e n t s c o n s i s t e d o f a 1-3.5 hour i n c u b a t i o n of c e l l s w i t h t h e c h e m i c a l s o l u t i o n a t 37 ^ C . A f t e r t r e a t m e n t , c u I t u r e s were c l e a n s e d of c h e m i c a l by e i t h e r washing t h e p e t r i , d i s h w i t h 2 ml of medium p r i o r t o a d d i t i o n o f normal t i s s u e c u l t u r e medium,or by t r a n s -f e r r i n g t h e g l a s s c o v e r s I i p , w i t h a t t a c h e d e e l I s , t o a p e t r i d i s h - c o n -t a i n i n g normal medium a f t e r f i r s t r i n s i n g i t t w i c e i n f r e s h medium. In t h e c a s e of c h r o n i c c h e m i c a l t r e a t m e n t s , t h e chemical was i n c l u d e d i n t h e growth medium(MEM + 15 % FBS) f o r t h e d u r a t i o n of BrdU l a b e l l i n g ( a p p r o x i m a t e l y 24 hours f o r CHO c e l l s and 48 hours f o r human c e l l s ) . 16 I I I .Cyto l o g i ca I P r e p a r a t i o n Reagents Co Ich i c i ne(BDH ChemicaIs,Ltd.,PooIe,EngI and).A 4 mg/ml s o l u t i o n in d i s t i l l e d w a t e r was p r e p a r e d and s t o r e d a t 4 ^C f o r no more than two w e e k s . C u I t u r e s were t r e a t e d by a d d i n g 0 . 0 1 ml o f t h i s s o l u t i o n t o 2 ml of c u l t u r e medium f o r a f i n a l c o l c h i c i n e c o n c e n t r a t i o n of . 2 0 ug/ml. MqCIQ(Fi s h e r ) . A 0 . 0 1 5 M s o l u t i o n i n d i s t i l l e d w a t e r was used as a h y p o t o n i c t r e a t m e n t . F i x a t i v e . M e t h a n o I and a c e t i c a c i d were combined 3/1 by volume. A c e t o - o r c e i n.A 2 % s o l u t i o n of a c e t o - o r c e i n s t a i n ( B D H C h e m i c a l s ) was p r e p a r e d by r e f l u x i n g i n a c e t i c a c i d f o r 6 hours.The s u s p e n s i o n was f i l t e r e d w i t h Whatman # 1 paper p r i o r t o use. GJ_emsa_, (Sigma) .A s t o c k s o l u t i o n was p r e p a r e d by d i s s o l v i n g 7 . 5 g of Giemsa powder i n 5 0 0 ml of g l y c e r o l by h e a t i n g a t 5 6 *^ C f o r 2 h o u r s . A f t e r t h e a d d i t i o n of 5 0 0 ml o f m e t h a n o l , t h e s o l u t i o n was aged f o r . 1 week and f i l t e r e d t h r o u g h Whatman # 1 paper.A w o r k i n g Giemsa s o l u t i o n was p r e p a r e d as a 2 % s o l u t i o n of t h e s t o c k s o l u t i o n w i t h Sorensens b u f f e r ( s e e A ppendix 4 ) . b ) T e c h n i q u e s S u r v i v a l e x p e r i m e n t s . C u I t u r e s were dec a n t e d and f i x a t i v e added. A f t e r 10 m i n u t e s , t h e p l a t e s were r i n s e d i n d i s t i 1 l e d w a t e r and a i r -d r i e d . T h e y were t h e n s t a i n e d f o r 10 m i n u t e s w i t h a 1% Giemsa s o l u t i o n i n d i s t i l l e d w a t e r . F i n a I l y t h e y were r i n s e d and a i r - d r i e d . Chromosome prepar a t i o n s . H u m a n c e l l c u l t u r e s were t r e a t e d w i t h 2 0 17 yg/ml of c o l c h i c i n e f o r 4 hours.CHO c e l l s were t r e a t e d w i t h t h e same s o l u t i o n f o r 2 h o u r s . C e l l s were h a r v e s t e d by t r a n s f e r r i n g t h e g l a s s c o v e r s I i p ,w i t h a t t a c h e d e e l I s , t o 2 ml of 0.015 M M g C ^ s o l u t i o n . A f t e r 10-12 m i n u t e s of h y p o t o n i c t r e a t m e n t , t h e c e l l s were s l o w l y f i x e d by t h e d r o p w i s e a d d i t i o n o f a p p r o x i m a t e l y 0.5 ml of f i x a t i v e . The c o v e r s l i p s were th e n t r a n s f e r r e d t o 2 ml of f i x a t i v e . A f t e r 10 m i n u t e s , t h e y were removed,dipped i n 100$ methanol and a i r - d r i e d . Chromosome p r e p a r a t i o n s were s t a i n e d w i t h 2% a c e t o - o r c e i n , d e h y d r a t e d by immersion i n e t h a n o I , b u t a n o I , b u t a n o I / x y I o I and x y l o l and mounted i n P e r m o u n t ( F i s h e r ) . S t a i n i n g of t h e chromosomes t o o b t a i n t h e h a r l e q u i n p a t t e r n i s d e s c r i b e d below i n S e c t i o n IV. IV.LabeI I i ng Methods a) R a d i o a c t i v e l a b e l l i n g DNA s y n t h e s i s was m o n i t o r e d by t h e i n c o r p o r a t i o n o f t r i t i a t e d t h y m i d i n e ( ^ H - T d R ) . A n aqueous s o l u t i o n o f ^H-TdR(New En g l a n d N u c l e a r , Boston,Mass.) of s p e c i f i c a c t i v i t y 20 Ci/mmol c o n t a i n i n g 1 mCi/ml of r a d i o a c t i v i t y ( i n 0.0122 mg/ml t h y m i d i n e ) was d i l u t e d t o a f i n a l w o r k i n g s o l u t i o n o f 1 yCi / m l i n t h e a p p r o p r i a t e t i s s u e c u l t u r e mediurn.CuItures were i n c u b a t e d i n t h i s s o l u t i o n f o r f i x e d t i m e i n t e r v a l s . C e l l s were h a r v e s t e d by immersing t h e c o v e r s I i p s , w i t h a t t a c h e d e e l I s , i n 0.015 M M g C ^ f o r 10 mi n u t e s , f i x a t i ve f o r 10 minutes and a i r d r y i n g . 18 Dry c o v e r s l i p s were m o u n t e d ( c e l l s i d e up) t o g l a s s m i c r o s c o p e s I i d e s ( C a n a d i a n L a b o r a t o r y S u p p l i e s ) u s i n g m e l t e d p a r a f f i n wax.The c e l l s were th e n r e h y d r a t e d by p a s s i n g t h e s l i d e s t h r o u g h 100% e t h a n o l , 50% e t h a n o l , 2 0 % e t h a n o l and two changes of d i s t i l l e d w a t e r , t h e n a i r - d r i e d . T h e s e s l i d e s were th e n c o a t e d w i t h Kodak NTB-3 n u c l e a r t r a c k emu I s i o n , w h i c h had been heated t o 43°C i n a w a t e r bath and a i r -d r i e d . A M m a n i p u l a t i o n s were performed i n c o m p l e t e darkness.The s l i d e s were s t o r e d a t 4°C i n l i g h t - t i g h t boxes w i t h added d e s i c a n t ( C a S 0 4 ) f o r 7 days. The s l i d e s were p r o c e s s e d a t 18°C in Kodak D-19 d e v e l o p e r ( 3 mi nutes ), d i s t i I I ed w a t e r s t o p bath,Kodak f i x e r U O m i n u t e s ) , a n d hypo-c l e a r i n g a g e n t ( 1 . 5 m i n u t e s ) . A f t e r 1/2-1 hour i n r u n n i n g w a t e r a t 18°C t h e y were a i r - d r i e d . F i n a l l y , t h e s l i d e s were s t a i n e d i n 2% a c e t o - o r c e i n and d e h y d r a t e d in 100% e t h a n o l , b u t a n o l , b u t a n o l / x y l o l and x y l o l and mounted i n Permount by o v e r l a y i n g a g l a s s c o v e r s l i p . b )BrdU H a r l e q u i n s t a i n e d chromosomes were o b t a i n e d a f t e r t h e growth of c e l l s i n medium c o n t a i n i n g BrdU.A 2 mM s t o c k s o l u t i o n o f BrdU(Sigma) i n d i s t i I l e d w a t e r was p r e p a r e d and s t e r i I i z e d by m i l l i p o r e ( 0 . 2 2 uM pore s i z e : M i I I i p o r e ) f i I t r a t i o n . C e I Is were grown i n medium c o n t a i n i n g a number of BrdU c o n c e n t r a t i o n s r a n g i n g between 2.5 and 80 uM. Chromosome p r e p a r a t i o n s were o b t a i n e d as d e s c r i b e d a b o v e . A f t e r 19 s t o r i n g t h e s l i d e s f o r a t l e a s t one day a f t e r f i x a t i o n , t h e y were t r e a t e d as f o I Iows: i ) i n c u b a t i o n i n 0.75 M I^^HPO^ s o l u t i o n f o r 2.5 minutes.The i n c u b a t i o n t e m p e r a t u r e v a r i e d depending upon t h e c e l l : t y p e and t h e c o n c e n t r a t i o n o f BrdU usedCsee T a b l e I b e l o w ) . CELL TYPE CONCENTRATION BrdU(uM) human CHO 2.5 5 10 20 40 85 83 81 79 77 82 80 78 76 74 T a b l e I. Optimum Na2HP0^ i n c u b a t i o n t e m p e r a t u r e s f o r v a r i o u s BrdU c o n c e n t r a t i o n s i n human and CHO c e l l s . i i ) t h r e e r i n s e s i n d i s t i l l e d w a t e r , i i i ) s t a i n i n g i n a 2% s o l u t i o n o f Giemsa i n Sorensens b u f f e r f o r 10 m i n u t e s . i v ) r i n s i n g i n d i s t i l l e d w a t e r and a i r - d r y i n g . The s l i d e s were th e n mounted i n Permount. V . A n a l y s i s F i v e p a r a m e t e r s o f c e l l s g r o w i n g i n c u l t u r e were u t i l i z e d d u r i n g t h i s s t u d y : m i t o t i c rate,chromosome a b e r r a t i o n s , s i s t e r c h r o m a t i d e x c h a n g e s , c o I o n y f o r m a t i o n and t h e uptake o f ^H-thymidine.The meaning of t h e s e s t a t i s t i c s and t h e means by which t h e y were g a t h e r e d and 20 o r g a n i z e d i s o u t l i n e d below. a) M i t o t i c r a t e A h i g h m i t o t i c " r a t e i s p r e s e n t i n h e a l t h y p r o l i f e r a t i n g c e l l c u l t u r e s . A low r a t e i s i n d i c a t i v e o f t h e p r e s e n c e of t o x i c m a t e r i a l s w h i c h i n h i b i t c e l l growth o r t h e absence o f n u t r i e n t s r e q u i r e d f o r pro I i f e r a t i o n . M i t o t i c f i g u r e s were c o u n t e d under 400X m a g n i f i c a t i o n . T h e number of o r c e i n - s t a i n e d n u c l e i p e r f i e l d was f i r s t e n u m e r a t e d , f o l l o w e d by t h e number of m i t o t i c f i g u r e s . F i e I d s were s e l e c t e d a t random from 2 each o f t h e f o u r q u a d r a n t s o f t h e 22 mm c o v e r s I i p s . S u f f i c i e n t f i e l d s were c o u n t e d t o p r o v i d e a sample of more'than 10^ e e l Is.The p e r c e n t a g e of m i t o t i c c e l l s was c a l c u l a t e d from t h e t o t a l c e l l s counted.No a c c o u n t was t a k e n o f t h e d u r a t i o n of c o I c h i c i n e - m e d i a t e d metaphase b l o c k , w h i c h d i r e c t l y i n f l u e n c e s t h e m i t o t i c r a t e . T h i s time,however,was kept c o n s t a n t w i t h i n each e x p e r i m e n t ( a p p r o x i m a t e l y 2 hours f o r CHO c e l l s ; 4 hours f o r human ceI I s ) . b) Chromosome a b e r r a t i o n s Chromosome a b e r r a t i o n s a r e complex a n a t o m i c a I t e r a t i o n s , v i s i b I e i n c e l l s i n t h e metaphase s t a t e , w h i c h a p p e a r t o r e s u l t from some form of damage t o t h e D N A - p r o t e i n complex of t h e n u c I e u s . T h e i r a n a l y s i s i s r e l a t i v e l y c o m p I i c a t e d . I n g e n e r a l , t w o t y p e s of a b e r r a t i o n s a r e r e c o g n i z e d : t h o s e which i n v o l v e a break o r exchange a t t h e same l o c u s of two c h r o m a t i d s such t h a t t h e y a r e r e a l i g n e d i n an i d e n t i c a l f a s h i o n (chromosome t y p e ) and t h o s e i n which t h e two p a i r e d c h r o m a t i d s a r e a f f e c t e d d i f f e r e n t l y ( t h e c h r o m a t i d t y p e ) . T h e f o l l o w i n g scheme was 21 u t i l i z e d i n c a t e g o r i z i n g t h e o b s e r v e d chromosome c o n f i g u r a t i o n s . TYPE OF ABERRATION CHROMOSOME CHROMAT1D n © 1 I /A break r i n g d i c e n t r i c b r eak exchange gap comp 1 e t e : i ncomp1ete T a b l e I I . Types of chromosome a b e r r a t i o n s s c o r e d . D i p l o i d metaphase p l a t e s composed of m o d e r a t e l y condensed chromosomes, which were s p r e a d s u f f i c i e n t l y w e l l t h a t o n l y s m a l l segments o v e r l a p p e d , were s e l e c t e d and viewed under 1000X m a g n i f i c a t i o n . T h e number o f each t y p e o f a b e r r a t i o n p r e s e n t was recorded.The most commonly used s t a t i s t i c d e r i v e d from t h i s d a t a was t h e mean number of any of t h e s e t y p e s of a b e r r a t i o n s ( g a p s e x c l u d e d ) p e r metaphase e e l I.Table V p r e s e n t s c o m p l e t e d a t a o f t h i s s o r t f o r U.V. induced chromosome a b e r r a t i o n s . S t a n d a r d e r r o r s of t h e mean(SEM) were c a l c u l a t e d r o u t i n e l y f o r t h e mean number o f a b e r r a t i o n s p e r ceI I.In genera I,between 40 and 50 metaphase c e l l s were a n a l y s e d per datum,the r e s u l t i n g SEM b e i n g found t o be a c c e p t a b l y Iow.Throughout t h i s t h e s i s , t h e b a r s p r e s e n t on c u r v e s d e s c r i b i n g chromosome a b e r r a t i o n s r e f e r t o t h e SEM. The p e r c e n t a g e of c e l l s w i t h a t l e a s t one chromosome a b e r r a t i o n was a l s o u t i l i z e d i n s e v e r a l i n s t a n c e s . R e f e r e n c e t o T a b l e V and F i g u r e 22 24 i l l u s t r a t e s t h a t t h i s p a r a m eter a g r e e s r e l a t i v e l y w e l l w i t h t h e mean number of a b e r r a t i o n s p e r c e l l . c ) S i s t e r c h r o m a t i d exchanges A n a l y s i s o f h a r l e q u i n - s t a i n e d chromosomes f o r SCEs i s a r e l a t i v e l y s t r a i g h t f o r w a r d procedure.WeI I s p r e a d d i p l o i d metaphase p l a t e s w i t h a c l e a r l y d i f f e r e n t i a t e d h a r l e q u i n s t a i n i n g p a t t e r n were s e l e c t e d f o r s c o r i n g and o b s e r v e d under 1000X m a g n i f i c a t i o n . D i s c r e t e r e v e r s a l s i n th e o p p o s i t e s t a i n i n g o f t h e two c h r o m a t i d s of t h e s e chromosomes were e a s i l y v i s i b I e ( P I a t e 1).These c o n f i g u r a t i o n s were r e c o r d e d as t h e number o f SCEs p r e s e n t i n each metaphase p l a t e . I n g e n e ra I,between 25 and 50 metaphase c e l l s were a n a l y s e d . T h e SEM was c a l c u l a t e d and p l o t t e d a l o n g w i t h t h e mean number o f SCEs p e r metaphase p l a t e . d) C o l o n y f o r m a t i o n C o l o n i e s o f CHO c e l l s c o n s i s t i n g o f more t h a n a p p r o x i m a t e l y 25 c e l l s were v i s i b l e w i t h o u t m a g n i f i c a t ion.These were s c o r e d i n t h i s manner and t h e t o t a l number of c o l o n i e s p e r p e t r i p l a t e r e c o r d e d . E x p e r i m e n t s were c o n d u c t e d i n d u p l i c a t e o r t r i p l i c a t e and t h e mean number o f c o l o n i e s p er p l a t e and t h e s t a n d a r d d e v i a t i o n o f t h e two o r t h r e e f i g u r e s c a I c u I a t e d . T h e b a r s s u r r o u n d i n g each p o i n t on c u r v e s d e s c r i b i n g c o l o n y f o r m a t i o n r e p r e s e n t t h e s t a n d a r d d e v i a t - j o n . e) I n c o r p o r a t i o n o f ^ H - t h y m i d i n e DNA s y n t h e t i c a c t i v i t y i n S-phase c e l l s was measured by t h e i n c o r p o r a t i o n o f "'H-thym i d i n e . A u t o r a d i ograph i c p r e p a r a t i o n s o f l a b e l l e d c e l l c u l t u r e s were examined a t 400X m a g n i f i c a t i o n . N u c I e i were s c o r e d as S-phase i f t h e d e n s i t y o f o v e r l a y i n g s i l v e r g r a i n s v i s i b l e a t t h m a g n i f i c a t i o n was n o t i c e a b l y g r e a t e r t h a n t h e background l e v e l . A t l e a s t 10"* c e l l s were o b s e r v e d f o r each datum and t h e p e r c e n t a g e of S-phase c e l l s c a l c u l a t e d . RESULTS At t h e t i m e of commencement of t h i s w o r k , i t was known t h a t SCEs c o u l d be induced i n s e v e r a l ' t y p e s of c e l l s by a v a r i e t y of t r e a t m e n t p r o t o c o l s w i t h s e v e r a l m u tagenic c h e m i c a l s and p h y s i c a l a g e n t s . l t was t h e r e f o r e n e c e s s a r y t o f i r s t e s t a b l i s h some b a s i c p a rameters of t h e systems b e i n g u t i l i z e d h e r e . T h i s i s d e s c r i b e d below i n S e c t i o n I . S e c t i o n II d e a l s w i t h t h e d i f f e r e n t i a l r e s p o n s e o f XP and normal c e l l s t o SCE i n d u c t i o n by mutagens.The f i n a l s e c t i o n d e s c r i b e s a system d e v e l o p e d t o s t u d y t h e r o l e of SCEs and chromosome a b e r r a t i o n s i n DNA r e p a i r and c e l l s u r v i v a l . I PRELIMINARY STUDIES The BrdU l a b e l l i n g method i s based upon t h e t h e o r y t h a t c e l l s grown i n medium c o n t a i n i n g BrdU i n e x c e s s f o r two c e l l c y c l e s w i l l c o n t a i n DNA which i s u n i f i M a r l y l a b e l l e d i n one d a u g h t e r s t r a n d and b i f i M a r l y l a b e l l e d in t h e o t h e r . T h i s d i f f e r e n c e i n t h e s i s t e r c h r o m a t i d s i n second m i t o s i s c e l l s may t h e n be d i s t i n g u i s h e d by a d i f f e r e n t i a l s t a i n i n g p r o c e d u r e ( P I a t e s 1-2). a) E f f e c t of BrdU c o n c e n t r a t i o n The SCE r a t e has been shown t o be dependent upon t h e c o n c e n t r a t i o n of BrdU c o n t a i n e d i n t h e growth mediurn.ConfI i c t i n g r e s u l t s have been p u b l i s h e d by d i f f e r e n t w o r k e r s ( K a t o , 1 9 7 4 b ; W o I f f and P e r r y , 1 9 7 4 ) . Because v a r i a t i o n s i n c u l t u r e c o n d i t i o n s and changes induced by mutagen t r e a t m e n t c o u l d w e l l a f f e c t t h e uptake of B r d U , i t was i m p o r t a n t t o e s t a b l i s h t h e range o f v a r i a t i o n i n spontaneous SCE f r e q u e n c y a t t r i b u t i b l e t o changes i n BrdU c o n c e n t r a t i o n . T h e spontaneous f r e q u e n c i e s i n t h e CHO and human c e l l s used f o r t h e s e e x p e r i m e n t s were examined o v e r t h e c o n c e n t r a t i o n range 2.5-80 uMCFigure 2 ) . The mean spontaneous f r e q u e n c i e s range between 10.2 and 10.6 f o r CHO c e l l s and 7.5 and 9.4 f o r human e e l Is.These v a l u e s a r e w i t h i n t h e range o f v a r i a t i o n o b s e r v e d i n c o n t r o l c u l t u r e s from a v a r i e t y o f e x p e r i m e n t s ( T a b l e II I) and i n d i c a t e : t h a t the. spontaneous .SCE•fre-quency i s independent o f BrdU c o n c e n t r a t i o n o v e r t h e range o f c o n c e n t r a t i o n s t e s t e d . In o r d e r t o examine t h e e x t e n t o f p o s s i b l e s y n e r g i s t i c i n t e r a c t i o n between BrdU and t h e mutagenic agent used t o t r e a t t h e ceI Is,MMC-induced SCEs were' examined i n c e l l s grown i n d i f f e r e n t c o n c e n t r a t i o n s of BrdU. F i g u r e 3 shows no s i g n i f i c a n t d i f f e r e n c e i n t h e i n c r e a s e d f r e q u e n c y of SCEs induced by t r e a t m e n t w i t h 10 ^ M MMC a t c o n c e n t r a t i o n s o f BrdU r a n g i n g between 5 and 40 uM. b ) E f f e c t o f s a m p l i n g and t r e a t m e n t t i m e s To examine t h e e f f e c t o f t r e a t m e n t t i m e and s a m p l i n g t i m e upon t h e f r e q u e n c y of MMC-induced SCEs,non-synchronous c u l t u r e s o f human c e l l s were t r e a t e d p r i o r t o , a n d 24 hours a f t e r t h e a d d i t i o n o f BrdU,and sampled a t 4 hour i n t e r v a Is.The SCE f r e q u e n c i e s o f c e l l s t r e a t e d i n such a way,which were i n t h e i r second m i t o s i s a f t e r BrdU a d d i t i o n ( M 2 c e l l s ) i s p r e s e n t e d i n F i g u r e 4.Dose-dependent i n c r e a s e s 26 EXPERIMENT SCE FREQUENCY(STANDARD ERROR OF MEAN) CONCENTRATION BrdU(yM) HUMAN CELLS CHO CELLS. 1 3 8(0 3) .5 6 3(0 6) . 2 9 7(0 5) 5 9 8(0 4) 9 8(0 5) 8 0(0 6) 3 5 3(0 3 ) X P F . 20 5 9(0 4) h 4 5 0(0 4) 10 5 7(0 5 ) X P E 5 12.6(0.6) 20 10.8(0.5) 6 7 8(0 4) 5 7 3 5(0 2) 5 8 4 6(0 3) 5 9 7 5(0 4) 5 8 8(0 4) 10 7 6(0 5) 20 7 9( 0 4) 40 9 4(0 4) 80 10 6 2(0 6) 5 5 8(0 5) 6 7(0 4) 11 8.7(0.4) 20 12 10.6(0.5) 2.5 10.3(0.4) 5 10.2(0.5) 10 10.6(0.5) 20 10.2(0.4) 40 13 9.2(0.6) 20 14 7.1(0.5) 20 .15 7.5(0.3) 20 16 . 10.3(0.6) 20 17 11.8(0.5) 20 18 11.5(0.5) 20 19 9.6(0.6) 20 T a b l e I I I-.Compi l a t i o n of mean spontaneous SCE f r e q u e n c i e s o b t a i n e d i n a v a r i e t y o f e x p e r i m e n t s under s l i g h t l y d i f f e r e n t c u l t u r e c o n d i t i o n s . BrdU c o n c e n t r a t i o n ( y M ) F i g u r e 2.Spontaneous SCE f r e q u e n c i e s of CHO and human c e l l s grown i n p r e s e n c e of d i f f e r e n t BrdU c o n c e n t r a t i o h s . C H O c e l l s were grown f o r 24 hours,human c e l l s f o r 48 hours i n MEM + 15% FBS i n t h e p r e s e n c e o f BrdU. 28 1 1 1 1 1 0 2 0 30 40 BrdU C o n c e n t r a t i o n (uM) F i g u r e 3.Spontaneous and MMC-induced SCE f r e q u e n c y i n human c e l l s grown i n d i f f e r e n t BrdU c o n c e n t r a t i o n s . M M C - t r e a t e d c e l l s were exposed t o a TO - 7 M s o l u t i o n o f c h e m i c a l i n MEM+2.5$FBS p r i o r t o a d d i t i o n o f BrdU. 29 F i g u r e 4 . E f f e c t of s a m p l i n g t i m e and t r e a t m e n t t i m e on t h e MMC-induced SCE frequency.One t r e a t m e n t p r o t o c o l c a l l e d f o r a 2.5 h MMC t r e a t m e n t p r i o r t o a d d i t i o n of BrdU( );the o t h e r i n v o l v e d . a s i m i l a r t r e a t m e n t of c e l l s which had been i n c u b a t e d i n 5 uM. BrdU f o r 24 h( • ) . C e l l s used were human f i b r o b l a s t s . 30 in SCE f r e q u e n c i e s a r e e v i d e n t a t a l l s a m p l i n g t i m e s . T h e s e t i m e s r e p r e s e n t o n l y t h o s e a t which a s i g n i f i c a n t p r o p o r t i o n of M2 c e l l s a r e p r e s e n t , P r i o r t o 44 hours a f t e r BrdU a d d i t i o n , m o s t metaphase c e l l s a r e s t i l l i n t h e i r f i r s t m i t o s i s ( M l ) and t h e h a r l e q u i n p a t t e r n i s not e v i d e n t . A f t e r 64 hours,most metaphases p r e s e n t a r e t h o s e of t h i r d d i v i s i o n ( M 3 ) ceI Is.A I though an e l e v a t i o n i n SCE f r e q u e n c y i s d e t e c t i b l e i n a l l samples where M2 c e l l s were s c o r e d , t h e r e i s c o n s i d e r a b l e v a r i a t i o n i n t h e MMC-induced SCE f r e q u e n c y amongst t h e v a r i o u s samples.Both t r e a t m e n t regimens produce a peak SCE f r e q u e n c y between 48 and 60 hours a f t e r BrdU a d d i t i o n , w i t h t h a t o f t h e p r e - t r e a t m e n t c e i l s o c c u r r i n g s l i g h t l y e a r l i e r t h a n i n t h e c a s e of t h e p o s t - t r e a t m e n t . T h e maximum and minimum f r e q u e n c i e s f o r both p r o t o c o l s v a r y by a f a c t o r o f a p p r o x i m a t e l y 1.5.A d o s e - r e s p o n s e c u r v e f o r p o s t - t r e a t m e n t w i t h MMC i s p r e s e n t e d i n F i g u r e 5 f o r purposes of c o m p a r i s o n , c) E f f e c t o f d u r a t i o n of t r e a t m e n t Most o f t h e c h e m i c a l t r e a t m e n t s employed i n t h i s s t u d y were o f between 1 and 3 hours d u r a t i o n . T h i s p r o t o c o l was g e n e r a l l y s u f f i c i e n t t o produce a marked re s p o n s e a t low d o s e s . T h i s was not always t h e c a s e , however,and F i g u r e s 6 and 7 i l l u s t r a t e t h e c o n t r a s t i n g b e h a v i o u r of. t h e a g e n t s MNNG and chromate.MNNG t r e a t m e n t s of 2 and 24 hours produce s i m i l a r d o s e - r e s p o n s e c u r v e s C F i g u r e 6).The c h r o n i c t r e a t m e n t , i n f a c t , p r o d u c e s a s l i g h t l y l e s s e r y i e l d o f S C E s . T h i s i s perhaps due t o t h e h i g h e r serum c o n c e n t r a t i o n i n t h e t r e a t m e n t medium and i t s 31 i b 9 10 8 10 7 - c o n t r o l C o n c e n t r a t i o n [ M D F i g u r e 5, Dose-response c u r v e f o r SCEs induced i n human c e l l s by MMC.Cells were grown f o r 30 hours i n 5 yM B r d U , t r e a t e d f o r 2.5 hours w i t h MMC i n MEM + 2 . 5 %FBS and r e t u r n e d t o MEM + 15 %FBS + 5 yM BrdU f o r f o r 20.5 hours.The t o t a l t i m e between BrdU a d d i t i o n and h a r v e s t i n g was 53 h o u r s . c o n t r o l C o n c e n t r a t i o n CMD F i g u r e 6.Dose-response c u r v e f o r SCEs formed i n CHO c e l l s in response to a c u t e and c h r o n i c t r e a t m e n t s w i t h MNNG.The a c u t e t r e a t m e n t c o n s i s t e d o f a 1 h t r e a t m e n t o f MNNG i n MEM+2% F B S , f o l l o w e d bv a 24 h i n c u b a t i o n i n MEM+15$FBS +20uM BrdU.The c h r o n i c t r e a t m e n t i n v o l v e d i n c u b a t i o n o f c e l l s i n MEM+15%FBS+20uM BrdU+MNNG f o r 24 h CO 5-\ . 1 7 T ~ I — f l 6 e 1(5' l(56 control C O N C E N T R A T I O N S ) F i g u r e 7.Dose-response c u r v e f o r SCEs induced i n human c e l l s by t h e chromate compounds ^ C ^ O y C ) and I^CrC^C ) . C h r o n i c t r e a t m e n t c o n s i s t e d o f a 48 h e x p o s u r e t o c h e m i c a l in MEMt15$FBS+ 5uM BrdU.Acute t r e a t m e n t i n v o l v e d a 2 h e x p o s u r e t o c h e m i c a l i n MEM +2.5$FBS p r i o r t o a d d i t i o n of BrdU. 34 i n t e r a c t i o n w i t h MNNG.In t h e case o f t h e chromate compounds I^CrO^ and I ^ C r ^ O y , h o w e v e r , c h r o n i c ' e x p o s u r e r e s u l t s i n a c o n s i d e r a b l y g r e a t e r SCE y i e l d t h a n a 2-hour p r e - e x p o s u r e ( F i g u r e 7 ) . d) SCE i n d u c t i o n and c e l l s u r v i v a l The v i a b i l i t y o f m u t a g e n - t r e a t e d c e l l s showing e l e v a t e d SCE f r e q u e n c i e s i s of i n t e r e s t b oth from a t h e o r e t i c a l and a p r a c t i c a l s t a n d p o i n t . U . V . - i n d u c e d SCEs were examined o v e r a wide dose range. C l o n e - f o r m i n g c a p a c i t y o f s i m i l a r l y t r e a t e d c e l l s was used as a measure of c e l l v i a b i I i t y . F i g u r e 8 i l l u s t r a t e s t h a t CHO c e l l s can undergo a p p r o x i m a t e l y a 5 - f o l d i n c r e a s e i n SCE f r e q u e n c y b e f o r e t h e c l o n e - f o r m i n g c a p a c i t y i s markedly reduced.The SCE f r e q u e n c y i n c r e a s e s a p p r o x i m a t e l y l i n e a r l y w i t h U.V. dose t o a maximum o f s l i g h t l y more tha n 70 S C E s ' D e r e e l I , a f t e r w hich i t drops s h a r p l y and approaches t h e c o n t r o l l e v e l . T h e U.V. dose i n d u c i n g t h e maximum number of SCEs i s a p p r o x i m a t e l y t h a t a t w h i c h t h e s u r v i v a l ' b e g i n s t o d e c r e a s e m a r k e d l y . II THE INDUCTION OF SCEs IN XP E AND NORMAL CELLS The r e s p o n s e s o f XP and normal c e l l s were compared w i t h r e s p e c t t o U.V. i r r a d i a t i o n and 4HQ0 and MNNG t r e a t m e n t .A I I work was c o n d u c t e d u s i n g a p a r t i c u l a r XP e e l I strain ( X P r ; ) i n which U.V.-induced UDS was a p p r o x i m a t e l y 2']% t h a t of normal ce I I s (Lam, 1977). F i g u r e 9 p r e s e n t s t h e d o s e - r e s p o n s e r e l a t i o n s h i p s f o r U.V.-indu c e d SCEs i n normal and XP F e e l Is.A d e t e c t i b l e i n c r e a s e i n SCE 1 2 0 U.V. D o s e ( e r g / m m 2 ) F i g u r e 8.Comparison o f do s e - r e s p o n s e c u r v e s f o r d e c r e a s e d s u r v i v a l and SCEs induced i n CHO c e l l s by U.V. I i g h t . C e l l s were i r r a d i a t e d i m m e d i a t e l y p r i o r t o - t h e a d d i t i o n o f BrdU and sampled a f t e r 24 h f o r SCEs and 5 days f o r s u r v i v a l . 36 F i g u r e 9. Dose-response c u r v e f o r SCEs induced i n normal human and X P E c e l l s by U.V. l i g h t . C e l l s were i r r a d i a t e d i m m e d i a t e l y p r i o r t o t h e a d d i t i o n o f BrdU and sampled 52 h t h e r e a f t e r . f r e q u e n c y i s e v i d e n t i n XP^ c e l l s a t a U.V. dose as low as 1 erg-mm - 2. At 2 erg-mm~ 2,the f r e q u e n c y i s doubled in X P E , b u t unchanged i n normal human f i brob l.asts.The SCE y i e l d r e a c h e s a maximum in X P E c e l l s a t 8 erg-mm - 2 and. t h e n , I i k e t h a t o f CHO e e l I s , d r o p s s h a r p l y . XPj- e e l Is a r e a l s o more s e n s i t i v e t o t r e a t m e n t w i t h t h e m u t a g e n i c / c a r c i n o g e n i c c h e m i c a l s 4NQ0 and MNNGCFigure 10).4NQ0 in d u c e s a d o u b I i of SCE f r e q u e n c y a t 10"~? M.The y i e l d o f SCEs i s o n l y s l i g h t l y , b u t c o n s i s t e n t l y h i g h e r i n X P E than normal ceI I s . T r e a t m e n t w i t h 10" 5 M MNNG,however,induces a 6 - f o l d i n c r e a s e in SCE f r e q u e n c y i n X P E c e l l s and a 4 - f o l d i n c r e a s e i n normal c e l l s . I I I RELEASE OF CELLS FROM ADM/I DM In an a t t e m p t t o s t u d y t h e i n t e r a c t i o n o f u n s c h e d u l e d DNA s y n t h e s i s w i t h t h e i n d u c t i o n o f s i s t e r c h r o m a t i d exchanges,and t h e c e l l c y c l e s p e c i f i c i t y o f SCE i n d u c t i o n , a model s y s t e m was c o n s t r u c t e d . T h i s was based upon t h e o b s e r v a t i o n t h a t c e l l s can be a r r e s t e d i n t h e i r rep I i c a t i v e c y c l e by d e p r i v a t i o n o f an e s s e n t i a l amino a c i d ( E v e r h a r t , 1 9 7 2 ; T o b e y , 1 9 7 3 ; W e i s s f e I d and Rouse , 1 9 7 7 ) . T h i s a r r e s t does n o t i n t e r f e r e w i t h t h e c e l l s ' a b i l i t y t o p e r f o r m u n s c h e d u l e d DNA s y n t h e s i s ( U D S ) ( S t i c h et al.j 1973).Pro I i f e r a t i v e a r r e s t o f CHO c e l l s m e d i a t e d by a r g i n i n e and i s o l e u c i n e d e p r i v a t i o n was used most e x t e n s i v e l y i n t h i s s t u d y . a) C h a r a c t e r i s t i c s o f ADM-mediated a r r e s t and r e l e a s e A f t e r . a b r i e f s e r i e s o f t r i a l s i n which s e e d i n g . p r o t o c o I s and 38 1 1 1 1 1 1 i61 0 1 0 9 io8 1 0 7 1 0 G io5 I- c o n t r o l C o n c e n t r a t i o n [ M 3 F i g u r e 10. I nduct i on of SCEs i n normal human and XPrr c e l l s by 4NQ0 and MNNG.Cells were t r e a t e d w i t h c h e m i c a l in MEM+2.5#FBS f o r 1.5 h,30 h a f t e r t h e a d d i t i o n o f 10uM BrdU.Samples were t a k e n 54 h a f t e r BrdU a d d i t i o n ( n o r m a I c e l l s = — . — ; XP F c e l l s = ). 39 degree o f s u p p l e m e n t a t i o n of ADM w i t h FBS were v a r i e d , a f i n a l g e n e r a l p r o t o c o l was - s e t t l e d . u p o n . T h i s c o n s i s t e d of s u s p e n d i n g t r y p s i n i z e d CHO c e l l s from a d i v i d i n g c u l t u r e i n ADM + 2% F B S . A f t e r s e e d i n g , t h e s e c e l l s a t t a c h e d and underwent l i m i t e d pro I i f e r a t i o n . U p o n c e s s a t i o n of p r o l i f e r a t i o n , t h e c e l l s adopted a c h a r a c t e r i s t i c morphology q u i t e d i f f e r e n t from t h a t o f d i v i d i n g ceI I s . A D M - a r r e s t e d c e l l s were s t e l l a t e i n appearance,sometimes w i t h long s l e n d e r c y t o p l a s m i c p r o c e s s e s ( P I a t e 3 ) . I D M - a r r e s t e d c e l l s assumed a s i m i l a r m orphoIogy.CytopIasmic i n c I us i o n s , v i s i b I e by phase m i c r o s c o p y , a I so became p r o m i n e n t ( P l a t e 3 ) . In c o n t r a s t , a d i v i d i n g c u l t u r e o f CHO cel.Is c o n s i s t e d o f s p i n d l e -shaped o r s p h e r i c a l b o d i e s not u s u a l l y p o s s e s s i n g p r o m i n e n t i n c l u s i o n s ( P l a t e 4 0 . A f t e r 5 days i n ADM +2% FBS,DNA s y n t h e s i s was d e t e c t i b l e by a u t o r a d i o g r a p h y i n few e r t h a n 1% of c e l l s , i ) r e c o v e r y o f DNA s y n t h e t i c a c t i v i t y If ADM-arrested c e l l s a r e r e t u r n e d t o complete growth medium(MEM+ 15% FBS) t h e c e l l s resume DNA s y n t h e s i s a f t e r a b r i e f l a g p e r i o d . T h e m a j o r i t y o f c e l l s e n t e r S-phase between 6 and 8 hours a f t e r r e l e a s e from ADM and,by 14 hours a f t e r r e l e a s e , 1 0 0 % of c e l l s have i n c o r p o r -a t e d 3 H - T d R ( F i g u r e 1.1). i U r e c o v e r y o f m i t o t i c a c t i v i t y M i t o t i c f i g u r e s were v i s i b l e i n t h e c u l t u r e s 18 hours a f t e r ADM re I ease ( F i gure .12).The f r e q u e n c y o f m i t o s e s g r a d u a l l y i n c r e a s e d t h e r e a f t e r t o a r a t e o f a p p r o x i m a t e l y 2% c o l l e c t e d o v e r a 2 hour p e r i o d , 2 4 hours a f t e r ADM-release. F i g u r e 11.The o n s e t of S-phase i n CHO c e l l s r e l e a s e d from a r g i n i n e d e p r i v a t i o n . T h e i n c o r p o r a t i o n o f 3 H - t h y m i d i n e ( 1 . uCi/mI) i n t o t h e n u c l e u s , a s d e t e c t e d by a u t o r a d i o g r a p h y , w a s used t o measure DNA s y n t h e s i s . Ce I I s were l a b e l l e d c o n t i n u o u s l y a f t e r r e l e a s e from ADM. F i g u r e 12.The o n s e t of m i t o s i s i n CHO c e l l s r e l e a s e d from a r g i n i n e d e p r i v a t i o n . 100-i 80H 60-40H 20H 0.4 _L_ 0 2 4 6 8 10 12 14 16 Time after release Chi Time after release Ch"} 42 i i i ) e f f e e t o f maintenance i n ADM f o r ex t e n d e d t i m e p e r i o d s The e f f e c t o f p r o l o n g i n g t h e d u r a t i o n o f ADM a r r e s t . w a s examined f i r s t w i t h r e s p e c t t o s u r v i v a I . F i g u r e 13 shows t h a t c l o n e - f o r m i n g c a p a c i t y f a l l s o f f r a p i d l y from t h e t i m e of s e e d i n g i n ADM.Between 100 and 200 hours a f t e r s e e d i n g , s u r v i v a I remains r e l a t i v e l y c o n s t a n t a t a p p r o x i m a t e l y 20$ and drops o f f g r a d u a l l y t h e r e a f t e r . S i n e e a l l e x p e r i m e n t s were performed on c e l l s m a i n t a i n e d i n ADM f o r between 5 and 7 d a y s , t h e s u r v i v a l o f t h e s e c e l l s would be e x p e c t e d t o a l s o be reduced t o some e x t e n t . I f s i m i l a r c o n d i t i o n s p e r s i s t e d i n c u l t u r e s of h i g h e r c e l l d e n s i t y , t h e r e s u l t s o f t h e s u r v i v a l e x p e r i m e n t would i n d i c a t e a f a i r l y c o n s t a n t s u r v i v a l o v e r ' t h e p e r i o d between 5 and 7 days i n ADM. To d e t e r m i n e whether t h e r e d u c t i o n i n v i a b i I i t y of e e l Is i n ADM had any b e a r i n g upon e i t h e r o f t h e i n d i c e s under s t u d y , t h e spontaneous i n c i d e n c e o f SCEs and chromosome a b e r r a t i o n s i n c e l l s a r r e s t e d f o r v a r i o u s p e r i o d s o f t i m e was examined(Tab Ie IV).No s i g n i f i c a n t i n c r e a s e i n SCE o r chromosome a b e r r a t i o n f r e q u e n c y was o b s e r v e d up t o 9 days a f t e r ADM tr e a t m e n t . O n Iy a t 18 days a f t e r ADM t r e a t m e n t was any s i g -n i f i c a n t d i f f e r e n c e d e t e c t e d . T h e spontaneous chromosome a b e r r a t i o n f r e q u e n c y was a p p r o x i m a t e l y d o u b l e d w h i l e t h e SCE f r e q u e n c y was not s i g n i f i c a n t l y a f f e c t e d ( T a b l e I V ) . 43 Hours in ADM F i g u r e 13.The e f f e c t o f m a i n t e n a n c e of CHO c e l l s i n ADM upon c e l l s u r v i v a I . C l o n e - f o r m i n g a b i l i t y was used as an index of s u r v i v a l 44 TIME IN ADM(days) SCE PER METAPHASE (S.E.M.) % CELLS WITH CHROMOSOME ABERRATIONS 5 10.6(0.3) 3 6 •' 11.3(0.4) 3 7 11.1(0.5) 4 . 8 10.3(0.6) 3 9 11.4(0.5) 5 18 12.7(0.3) 12 T a b l e IV. I n c i d e n c e o f SCEs and chromosome a b e r r a t i o n s i n CHO c e l l s r e l e a s e d from ADM-arrest a t v a r i o u s t i m e s . b) I n d u c t i o n o f chromosome a b e r r a t i o n s i ) U.V. I t was o b s e r v e d t h a t A DM-arrested c e l l s were p a r t i c u l a r l y s e n s i t i v e w i t h r e s p e c t t o t h e i n d u c t i o n o f chromosome a b e r r a t i o n s ( P l a t e 5> and m i c r o n u c l e i ( P l a t e 6).The d o s e - r e s p o n s e r e l a t i o n s h i p s of chromosome a b e r r a t i o n s induced i n c e l l s a r r e s t e d i n ADM and I DM f o r s i x days a r e compared i n F i g u r e 14.The c u r v e s have q u i t e d i s t i n c t a p p e a r a n c e s . T h a t f o r ADM-treated c e l l s r i s e s s h a r p l y and p l a t e a u s a t a p p r o x i m a t e l y 4 a b e r r a t i o n s p e r e e l I,There i s a much more g r a d u a l i n c r e a s e i n t h e number o f a b e r r a t i o n s g e n e r a t e d i n d i v i d i n g c e l l s t o a maximum y i e l d o f a p p r o x i m a t e l y 3 a b e r r a t i o n s p e r c e l l f o l l o w e d by a d e c l i n e a t h i g h U.V. d o s e s ( F i g u r e 15).An i n c r e a s e i n a b e r r a t i o n f r e q u e n c y i n ADM-arrested c e l l s i s d e t e c t i b l e w i t h as l i t t l e as 0.5 erg*mm i r r a d i a t i o n : a p p r o x i m a t e Iy 1/100th t h a t r e q u i r e d t o e l i c i t a d e t e c t i b l e r e s p o n s e i n d i v i d i n g c e l l s ( F i g u r e s 14 and 1 5 ) . F i g u r e 14. I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM and I DM a r r e s t e d CHO c e l l s by U.V. l i g h t . C e l l s were r e l e a s e d from a r r e s t i m m e d i a t e l y a f t e r i r r a d i a t i o n by a d d i t i o n of MEM + 15$ FBS and sampled 20 hours t h e r e a f t e r . 46 F i g u r e 15. I n d u c t i o n o f chromosome a b e r r a t i o n s i n d i v i d i n g CHO c e l l s by U.V. I i g h t . N o n - s y n c h r o n o u s c u l t u r e s o f d i v i d i n g CHO c e l l s were i r r a d i a t e d and sampled 10,13,22 and 24 hours l a t e r . 47 i i ) A b e r r a t i o n s p e r c e l l vs % c e l l s w i t h a b e r r a t i o n s Chromosome a b e r r a t i o n s a r e e x p r e s s e d t h r o u g h o u t as t h e mean number o f a b e r r a t i o n s p e r c e l l . T h i s i s i n o r d e r t o f a c i l i t a t e a more d i r e c t c o m p a rison of t h e y i e l d o f a b e r r a t i o n s w i t h t h a t of SCEs.In a l l c a s e s , t h e a b e r r a t i o n s were d i s t r i b u t e d t h r o u g h o u t t h e c e l l pop-u l a t i o n and e x p r e s s i n g t h e damage as t h e per c e n t of c e l l s w i t h a chromosome a b e r r a t i o n d i d not a l t e r t h e n a t u r e of t h e r e s u l t s . A c o m p a r i s o n of t h e s e two i n d i c e s , a I o n g w i t h t h e t y p e s of chromosome damage o b s e r v e d , i s o f f e r e d , i n T a b l e V. U.V. DOSE ; ( e r g / mm ) TYPES OF ABERRATIONS(PER 100 CELLS) ABs/ CELL (SEM) % CELLS w i t h ABs BREAKS RINGS DICENT. BREAKS EXCHANGES GAPS COMPL. INCOMP. 0 0 1 0 2 0 1 2, .04(.01) 4 0.5 0 2 18 14 14 56 30 1.9(.17) .70 1 0 2 16 . 10 32 84 62 2.7(.3) 76 2 2 10 .18 36 38 64 60 3.1(.3) 80 4 0 2 •8 28 34 74 50 2.6(.3) 74 8 2 6 12 28 16 84 48. 2.7(.3) 78 16 0 4 22 20 30 130 10C 3.9(A) 76 28 0 8 10 16 32 72 38 2.6(.3) 78 T a b l e V.Chromosome a b e r r a t i o n s induced i n CHO c e l l s a r r e s t e d i n ADM f o r 7 days and i r r a d i a t e d w i t h U.V. 2 hours p r i o r t o ADM re I ease. i i i ) C hemical t r e a t m e n t of ADM-arrested c e l l s The extreme s e n s i t i v i t y o f A DM-arrested ce I Is w i t h r e s p e c t t o chromosome a b e r r a t i o n s was a l s o o b s e r v e d i n c e l l s t r e a t e d w i t h c h e m i c a l 48 mutagens.MNNG was found t o cause a dose-dependent i n c r e a s e i n chromosome a b e r r a t i o n s o v e r a c o n c e n t r a t i o n range w h i c h had no n o t i c e a b l e a f f e c t upon d i v i d i n g e e l I s ( F i g u r e ' 1 6 ) . I n t h i s e x p e r i m e n t , d i v i d i n g c e l l s were s y n c h r o n i z e d by s e l e c t i o n of m i t o t i c c e l l s by s h a k i n g ( T e r a s i m a and Tolmach,1961).More t h a n 90% of ceI Is s e I e c t e d by t h i s t e c h n i q u e c o u l d be seen t o be i n m i t o s i s 15 minutes a f t e r s e e d i ng.Ce I Is were t r e a t e d 2 and 6 hours a f t e r p l a t i - n g when DNA s y n t h e s i s was d e t e c t e d i n 3 and 67 % o f c e l l s r e s p e c t i v e l y by i n c o r p o r a t i o n of ^H-TdR(1 yCi/ m l f o r 0.5 h o u r ) . N e i t h e r of t h e s e t r e a t m e n t regimens produced a d e t e c t i b l e i n c r e a s e i n a b e r r a t i o n f r e q u e n c y a t t h e doses t e s t e d ( F i g u r e 16.). As i n t h e c a s e of U.V.,the chromosome a b e r r a t i o n s induced i n A DM-arrested c e l l s by MNNG were p r i m a r i l y of t h e c h r o m a t i d exchange type.The f r e q u e n c i e s of c h r o m a t i d b r e a k s and exchanges a r e i n c l u d e d i n F i gure 16. MMC and A d r i a m y c i n ( A D ) a l s o were found t o induce l a r g e numbers o f chromosome a b e r r a t i o n s a t low c o n c e n t r a t i o n s i n ADM-arrested c e l l s . Both were of s i m i l a r p o t e n c y , i n d u c i n g a p p r o x i m a t e l y 0.3 a b e r r a t i o n s p e r ceI I a t a dose of 10"^ M(Fi gures 11 and 18 ).Wh i Ie t h e maxi mum number of a b e r r a t i o n s induced by MMC b e g i n s t o p l a t e a u a t 4 a b e r r a t i o n s p e r e e l I,AD induced a maximum of o n l y 0.5 a b e r r a t i o n s per c e l l . A n i n c r e a s e d a b e r r a t i o n f r e q u e n c y induced i n d i v i d i n g c e l l s by AD was a l s o d e t e c t e d . C e I Is t r e a t e d w h i l e i n S-phase were more s e n s i t i v e M N N G C o n c e n t r a t i o n C J J M ] F i g u r e 1 6 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM-arrested and d i v i d i n g CHO c e l l s by MNNG.Cells i n ADM were s y n c h r o n i z e d by s e l e c t i o n o f m i t o t i c c e l l s by s h a k i n g and t r e a t e d 2 and 6 h a f t e r p l a t i n g t o a c c o m p l i s h G] and S phase t r e a t m e n t r e s p e c t i v e I y . C e I Is were h a r v e s t e d 13 h a f t e r pI a t i n g . F i g u r e s i n b r a c k e t s i n d i c a t e t h e numbers o f c h r o m a t i d exchanges and c h r o m a t i d b r e a k s ( r e s p e c t i v e I y ) s c o r e d p e r 100 metaphases. 50 4-1 Q) U L CO a ui c g ra L r_ 5 < 0 20 40 60 80 M M C ConijunLi-BLionCpM } 100 F i g u r e 1 7 . I n d u c t i o n o f chromosome a b e r r a t i o n s i n ADM-arrested CHO c e l l s by MMC.Cells were t r e a t e d f o r 2 h p r i o r t o r e l e a s e and sampled a f t e r 24 h . F i g u r e s i n b r a c k e t s i n d i c a t e t h e numbers' o f c h r o m a t i d exchanges and c h r o m a t i d b r e a k s C r e s p e c t ! v e l y ) s c o r e d p er 100 metaphases. 51 20 40 60 80 100 A D C o n c e n c r a c i o n C p M D F i g u r e 18.. I n d u c t i o n of chromosome a b e r r a t i o n s i n A DM-arrested and d i v i d i n g CHO c e l l s by A D . A r r e s t e d c e l l s w e r e . t r e a t e d f o r 2 h, r e t u r n e d t o ADM f o r 24 h and sampled 24 h a f t e r r e l e a s e / D i v i d i n g c e l l s were t r e a t e d i n t h e S and G ] phase 2 and 6 h a f t e r p l a t i n g ( r e s p e c t i v e l y ) . 52 than t h o s e i n G^.The y i e l d o f a b e r r a t i o n s g e n e r a t e d in ADM-arrested c e l l s was markedly g r e a t e r than t h a t induced in e i t h e r S o r G p t r e a t e d ceI I s ( F i g u r e 18). c) I n d u c t i o n o f SCEs D e s p i t e t h e marked s e n s i t i v i t y of ADM-arrested CHO e e l I s , w i t h r e s p e c t t o t h e i n d u c t i o n o f chromosome a b e r r a t i o n s , n o marked d i f f e r -ence i n SCE y i e l d s was o b s e r v e d w i t h any o f t h e f o u r a g e n t s examined. T a b l e VI i n c l u d e s t h e SCE f r e q u e n c y induced i n ADM-a r r e s t e d and d i v i d i n g c e l l s by U.V.,MNNG and AD. AGENT DOSE MEAN SCE PER METAPHASE • (M o r erg-mm~2) ADM ARRESTED(condi t i o n s ) DI V IDING(con d i t i on s) 0 10.3(0.6) 10.6(0.3) 5' 21.6(0.6) 10.3(0.6)(non-syn U.V. 20 36.7(2.3) (+ 11 h o u r s ) 20.0(0.9) p r e -40 55.4(3.0) 42.3(0.9) l a b e l ) 60 no r e c o v e r y 71.0(2.9) 0 1 1.2(0.5) 10.9(0.5) 3.7X10-7 44.2(2.0) (0 h o u r s ) 3 6 . 4 ( 1 . 4 ) ( n o n - s y n MNNG 1.1X10-6 81.8(3.2) 78.8(2.1) p r e -3.3X10" 6 no r e c o v e r y 114.5(5.5) l a b e l ) 7 . 4 X 1 0 - 1 0 10.5(0.5) 9.6(0.6) AD 3.7X10-9 10.4(0.7) ( + 6 ho u r s ) 1 0 . 8 ( 0 . 4 ) ( s y n 1.8X10-8 1 1.3(0.9) 12.5(0.5) S-phase) 9.2X10-8 16.9(1.2) 20.1(0.8) T a b l e V!. SCE i n d u c t i o n i n ADM-arrested and d i v i d i n g c e l l s by U.V., MNNG and AD. B r a c k e t e d f i g u r e s i n column headed " c o n d i t i o n s " r e f e r t o the c o n d i t i o n o f t h e e e l I s , w i t h r e s p e c t t o t h e c e l l c y c l e , a t t h e t i m e o f t r e a t m e n t ; + h o u r s i n d i c a t e s t h e t i m e o f t r e a t m e n t a f t e r r e l e a s e from ADM; non-synchronous c u I t u r e s ( n o n - s y n ) were t r e a t e d p r i o r t o a d d i t i o n o f BrdU; s y n c h r o n o u s ( s y n ) c u l t u r e s were t r e a t e d d u r i n g S-phase. 53 In t h e case of U.V t r e a t m e n t , t h e ADM-arrested c e l l s produced a s l i g h t l y g r e a t e r y i e l d of SCEs th a n t h e d i v i d i n g c e l l s . . B a s e d upon t h e a b i l i t y of CHO c e l l s t o remove U.V.-induced ,SCE-producing l e s i o n s ( d i s c u s s e d below under s e c t i o n (e) )' t h i s i s r e a d i l y e x p l a i n e d , s i n c e t h e ADM-arrested c e l l s were t r e a t e d w h i l e u n d e r g o i n g DNA s y n t h e s i s . T h e d i v i d i n g e e l Is,on t h e o t h e r hand,were t r e a t e d b e f o r e t h e a d d i t i o n of BrdU,and t h u s p r i o r t o S - p h a s e . A d r i a m y c i n i n d u c e d s l i g h t l y more SCEs i n t h e d i v i d i n g t h a n i n t h e ADM-arrested e e l I s . T h i s a l s o may be e x p l a i n e d by t h e f a c t t h a t o n l y t h e f o r m e r were i n S-phase when t r e a t e d . T h e d i v i d i n g c e l l s had been s y n c h r o n i z e d and were t r e a t e d w h i l e u n d e r g o i n g DNA s y n t h e s i s : t h e ADM-arrested c e l l s were t r e a t e d 6 hours a f t e r ADM r e l e a s e a t a t i m e when o n l y 12% of c e l l s had i n i t i a t e d DNA s y n t h e s i s ( F i g u r e 1 1 ) . The y i e l d of chromosome a b e r r a t i o n s and SCEs induced i n ADM-a r r e s t e d c e l l s i n t h e phase i m m e d i a t e l y p r i o r t o t h e second m i t o s i s i n BrdU was a l s o e x a m i n e d . T h i s was a c c o m p l i s h e d by a l l o w i n g d i v i d i n g CHO c e l l s t o come t o a p r o l i f e r a t i v e a r r e s t i n ADM supplemented w i t h 20 uM B r d U . C u I t u r e s a r r e s t e d i n t h i s way were t r e a t e d w i t h U.V. and sampled 24 hours a f t e r r e l e a s e from ADM;i.e.at t h e f i r s t m i t o s i s a f t e r r e l e a s e . C e l I s h a r v e s t e d a t t h i s t i m e d i s p l a y e d M2 and M3 l a b e l l i n g p a t t e r n s i n a r a t i o of a p p r o x i m a t e l y 1:2, i n d i c a t i n g t h a t t h e m a j o r i t y of c e l l s had undergone two d i v i s i o n s i n ADM p r i o r t o a r r e s t ; t h e r e m a i n d e r o n l y one.Only M2 c e l l s were a n a l y s e d f o r SCEs and a b e r r a t i o n s . 54 The r e s u l t s a r e p r e s e n t e d below i n T a b l e V l l . U.V. DOSE ( e r g / mm2) CHROMOSOME ABERRATIONS PER 100 CELLS SCE/MET. (SEM) CHROM. AB.PER MET(SEM) CHROMOSOME CHROMATID BREAKS RINGS DICENT. BREAKS EXCHANGE 0 1 0 0 2 1 1 0 2 15 89. 6 69 15.5(.9) 15.8C.7) 0.21(.05) 1.7(.3) T a b l e VI I.Chromosome a b e r r a t i o n s and SCEs induced i n ADM-arrested CHO c e l l s . C e l l s were l a b e l l e d w i t h 20 yM BrdU p r i o r t o a r r e s t , i r r a d i a t e d and .sampled upon d i v i d i n g once a f t e r r e l e a s e . The f r e q u e n c y o f SCEs i n t r e a t e d and u n t r e a t e d c e l l s i s s i m i l a r , w h i l e t h a t o f chromosome a b e r r a t i o n s i n t h e s e M2 c e l l s i s c o n s i d e r a b l y h i g h e r i n t h e i r r a d i a t e d e e l Is.The spontaneous i n c i d e n c e of SCEs and chromosome a b e r r a t i o n s a r e both s l i g h t l y h i g h , p r o b a b I y a r i s i n g a s . a r e s u l t o f t h e BrdU l a b e l r e m a i n i n g i n t h e c e l l s ' DNA o v e r t h e 6 day ADM a r r e s t p e r i o d , d) S u r v i v a l C e l l s a r r e s t e d f o r 6 days i n ADM were compared t o d i v i d i n g ceI Is w i t h r e s p e c t f o e e l I s u r v i v a l a f t e r U.V. i r r a d i a t i o n ( F i gure 19). ADM-arrested c e l l s a r e a p p r o x i m a t e l y 3X more s e n s i t i v e t h a n d i v i d i n g c e l l s as measured by c o l o n y f o r m i n g a b i l i t y . I n a d d i t i o n , t h e ADM-a r r e s t e d c e l l s showed no d e t e c t i b l e p l a t e a u a t low d o s e s , h e l d t o be i n d i c a t i v e o f a r e p a i r c a p a c i t y ( K i h I man,1977). 55 100 ID > > C O a r r e s t e d 30 'I. T " 60 \ d i v i d i n g I 90 1 2 0 1 U.V. Dose (ergs/mm ) F i g u r e 19. Comparison o f s u r v i v a l o f ADM-arrested and d i v i d i n g c e l l s a f t e r U.V. i r r a d i a t i o n . A r r e s t e d c e l l s were r e l e a s e d i m m e d i a t e l y a f t e r i r r a d i a t i o n by t h e a d d i t i o n o f MEM + 15% FBS. 56 e) R e p a i r i n ADM-arrested c e l l s The p o s s i b i I i t y t h a t mutagen-induced damage r e s u l t i n g i n SCEs o r chromosome a b e r r a t i o n s may be r e p a i r e d by c e l l s m a i n t a i n e d i n ADM was i n v e s t i g a t e d . i ) Chromosome a b e r r a t i o n s Chromosome a b e r r a t i o n s induced by U.V.,MNNG and MMC a t v a r i o u s t i m e s b e f o r e and a f t e r r e l e a s e from ADM a r e summarized i n T a b l e V I M . The r e s u l t s o f t h e s e e x p e r i m e n t s i n d i c a t e a t r e n d o f d e c r e a s i n g chromosome a b e r r a t i o n f r e q u e n c y t h e l o n g e r t h e h o l d i n g t i m e i n ADM a f t e r t r e a t m e n t . Subsequent e x p e r i m e n t s f a . i l e d t o p r o v i d e r e p r o d u c i b l e r e s u l t s and an e x p e r i m e n t i n v o l v i n g r e p e a t e d s a m p l i n g was c a r r i e d o u t . T h i s i s p r e s e n t e d i n F i g u r e 2 0 - C e l l s h e l d i n ADM f o r 24 hours a f t e r U.V. t r e a t m e n t and t h o s e r e l e a s e d from ADM d i r e c t l y a f t e r t r e a t -ment show s i m i l a r i n c r e a s e s i n chromosome a b e r r a t i o n f r e q u e n c y a t t h e s i x samples t a k e n . I n both c a s e s , t h e m i t o s e s which appear f i r s t i n t h e r e c o v e r i n g c u l t u r e ( 2 4 hours a f t e r ADM r e l e a s e ) a r e t h e most s e v e r e l y a f f e c t e d . T h e chromosome a b e r r a t i o n f r e q u e n c y f a l l s o f f r a p i d l y t o a l e v e l a p p r o x i m a t e l y 4 t i m e s t h a t o f t h e spontaneous one.Whereas 2 e r g -mm induced a s i g n i f i c a n t i n c r e a s e i n t h e chromosome a b e r r a t i o n f r e q u e n c y o f ADM-arrested c e I I s , d i v i d i n g c e l l s d i d not show a d e t e c t -i b l e r e s p o n s e . AGENT DOSE(erg-mm 2 . TREATMENT TIME ABERRATIONS/CELL or M) ( r e l a t i v e t o ADM (S.E.M.) release) 2 -24 0.81(0.25) - 8 2.08(0.30) - 2 1.82(0.27) 0 1.80(0.27) 5 -24 1.36(0.26) - -8 2.12(0.30) - 2 2.40(0.35) 0 2.52(0.29) U.V. 10 -24 1 .92(0.27) - 8 2.82(0.35) - 2 2.94(0.33) 0 3.43(0.34) 15 -24 1 .94(0.28) - 8 2.42(0.35) - 2 3.00(0.33) 0 4.67(0.41 ) MNNG 8X10" 7 -24 0.98(0.22) - 8 1 .96(0.25) - 2 2.10(0.27) 0 2.32(0.28) 10" 7 -24 1 .30(0.24) - 8 1.80(0.26) Q - 2 2.74(0.28) MMC 3.3X10 0 -24 0.70(0.14) - 8 0.85(0.19) - 2 0.86(0.14) 1.1X10"8 -24 0.54(0.12) - 8 0.53(0.11) - 2 0.82(0.15) Table VI 1.1-.Chromosome aber r a t i o n s induced in ADM ar r e s t e d CHO c e l l s by U.V.,MNNG and MMC.Cells were maintained in ADM f o r 6 days t r e a t e d 24,8,2 and immediately p r i o r to release and sampled 24 hours t h e r e a f t e r . 58 1.5 1.0-4 CD u L_ CD C L CO c o ro !_ 1_ CD -O. < 0.5-t i m e a f t e r i r r a d i a t i o n (h) F i g u r e 20. . E f f e c t o f U.V. i r r a d i a t i o n (2 erg-mrrf ) on chromosome a b e r r a t i o n f r e q u e n c y o f ADM-arrested and d i v i d i n g CHO c e l l s . ADM-a r r e s t e d c e l l s were r e l e a s e d from ADM by a d d i t i o n o f MEM + 15$ FBS e i t h e r i mmed i'ate \y (O O) o r 24 h (•-•-•) a f t e r i r r a d i a t i o n . D i v i d i n g c e l l s (• •) were m a i n t a i n e d i n MEM + 15$ FBS t h r o u g h o u t t h e e x p e r i m e n t , 59 i i ) SCEs U.V,,MNNG and MMC were a l s o examined f o r t h e i r a b i l i t y t o induce SCEs p r i o r t o ADM-reI e a s e . F i g u r e 21 r e v e a l s t h r e e d i f f e r e n t p a t t e r n s o f SCE i n d u c t i o n w i t h r e s p e c t t o t h e t i m e of t r e a t m e n t . U.V. in d u c e s t h e g r e a t e s t number o f SCEs when t h e c e l l s a r e t r e a t e d w h i l e u n d e r g o i n g S - p h a s e . P o s t - t r e a t m e n t i n c u b a t i o n of i r r a d i a t e d c e l l s i n ADM c l e a r l y has r e s u l t e d i n an e f f e c t i v e d e c r e a s e i n t h e SCE yield.MNNG i n d u c e s s i m i l a r SCE y i e l d s when t r e a t e d 24 hours and i m m e d i a t e l y p r i o r t o ADM-reI ease.When t h e c e l l s a r e t r e a t e d a f t e r t h e o n s e t of S-phase,however,the SCE f r e q u e n c y drops by a f a c t o r o f a p p r o x i m a t e l y 1/2.MMC,on t h e o t h e r h a n d , i n d u c e s s i m i l a r numbers of SCEs a t each of t h e t i m e s t r e a t e d , i i i ) S u r v i vaI The e f f e c t o f a 24 hour p o s t - t r e a t m e n t i n c u b a t i o n p e r i o d i n ADM upon c e l l s u r v i v a l was examined a f t e r U.V. i r r a d i a t i o n . F i g u r e 22 i l l u s t r a t e s t h a t an ADM h o l d i n g p e r i o d d i d not i n c r e a s e t h e a b i l i t y o f a r r e s t e d c e l l s t o r e c o v e r and form c o l o n i e s . T h e v i a b i l i t y o f t h e s e c e l l s w a s , i n f a c t , s I i g h t I y r e d u c e d , f ) O t h e r a r r e s t e d c e l l s ystems In o r d e r t o r o u g h l y a s c e s s t o what e x t e n t a r g i n i n e d e p r i v a t i o n i n CHO c e l l s r e f l e c t s t h e g e n e r a l model of a n o n - p r o l i f e r a t i n g c e l l , two o t h e r a r r e s t e d c e l l systems were examined. 60 control -24 0 * 5 '8 .11 TREATMENT TIME: RELATIVE TO ADM RELEASE(h) F i g u r e 2 1 . I n d u c t i o n o f SCEs i n ADM-arrested c e l l s a t i n t e r v a l s b e f o r e and a f t e r o n s e t o f S-phase by U.V. light,MMC and MNNG.CuItures were i r r a d i a t e d w i t h 5 O ,20HD and 40 HI erg-mm - 2 and t r e a t e d w i t h 1 0 - 7 D and 1 0 ~ 6 fflM MMC and MNNG i n blank' s~ BSS+2$FBS f o r 1 h . C e l l s were r e l e a s e d w i t h MEM+15$ FBS+20 pM BrdU and sampled 36 h a f t e r re I ease.Pane I I shows t h e o n s e t o f S-phase i n c e l l s r e l e a s e d from ADM. 61 F i g u r e 22. S u r v i v a l o f ADM-arrested CHO c e l l s a f t e r i r r a d i a t i o n w i t h U.V. l i g h t . C u l t u r e s were r e l e a s e d e i t h e r i m m e d i a t e l y a f t e r i r r a d i a t i o n by t h e a d d i t i o n o f MEM + 15$ F B S ( " — • ) , o r a f t e r a 24 hour p o s t - t r e a t m e n t i n c u b a t i o n i n ADM(o---a). 62 i ) A D M - a r r e s t e d human c e l l s The xeroderma pigmentosum s t r a i n XP^ was examined w i t h r e s p e c t t o r e c o v e r y a f t e r t r e a t m e n t of ADM-arrested c e l l s w i t h 4NQ0 and MNNG. M i t o t i c f i g u r e s a r e v i s i b l e i n human c e l l s r e c o v e r i n g from ADM d e p r i v a t i o n 30 hours a f t e r r e t u r n t o normal medium(Figure 2 3 ) . I n t h e case o f CHO c e l l s , m i t o s i s i s d e t e c t i b l e 18 hours a f t e r r e t u r n t o normal med i um(Fi gure 1 1 ). Some m i t o t i c r e c o v e r y was d e t e c t e d i n XPp; -5 c e l l s t r e a t e d w i t h 3X10 M MNNG,while normal r e c o v e r y was o b s e r v e d a t lower d o s e s ( F i g u r e 2 3 ) . I n t h e case of 4N00,no m i t o t i c r e c o v e r y -5 -Pi was o b s e r v e d o v e r t h e c o n c e n t r a t i o n range 3X10 t o 3X10 M.No i n c r e a s e d f r e q u e n c y of chromosome a b e r r a t i o n s was d e t e c t i b l e i n t h e MNNG t r e a t e d e e l l s ( F i g u r e 2 3 ) . i i ) I D M - a r r e s t e d CHO c e l l s CHO c e l l s a r r e s t e d by d e p r i v a t i o n o f t h e e s s e n t i a l amino a c i d i s o l e u c i n e were examined w i t h r e s p e c t t o t h e i r s e n s i t i v i t y t o U.V. CHO c e l l s r e c o v e r from I DM d e p r i v a t i o n c o n s i d e r a b l y more r a p i d l y t h a n e i t h e r A DM-arrested CHO c e l l s o r ADM-a r r e s t e d human c e l l s . A s few as 6 hours a f t e r r e l e a s e from I DM,a- d e t e c t i b l e m i t o t i c r a t e was e v i d e n t . A l t h o u g h not as s e n s i t i v e t o U.V. as ADM-arrested c e l l s , I D M a r r e s t e d c e l l s were markedly more s e n s i t i v e t h a n d i v i d i n g c e l l s ( F i g u r e 1 4 ) . The e f f e c t of a 24 hour p o s t - t r e a t m e n t I DM i n c u b a t i o n p e r i o d upon t h e f r e q u e n c y o f chromosome a b e r r a t i o n s was e x a m i n e d ( F i g u r e 2 4 ) . W h i l e t h e . two c u l t u r e s r e c o v e r e d d i f f e r e n t I y , t h e r e was no marked d i f f e r e n c e i n th e chromosome damage produced i n c e l l s d i v i d i n g o v e r t h e p e r i o d between 6 and 36 hours a f t e r ADM r e l e a s e . 5 C 5 . 4 ) 2 0 3 0 4 0 5 0 T i m e a f t e r r e l e a s e C h l F i g u r e 23.Recovery of ADM-arrested XPc c e l l s t r e a t e d w i t h MNNG. C e l l s were t r e a t e d w i t h MNNG i n MEM+2,5$ FBS f o r 3.5 h i m m e d i a t e l y p r i o r t o r e l e a s e from ADM.Bracketed f i g u r e s i n d i c a t e t h e p e r c e n t of m i t o t i c c e l l s w i t h chromosome aberratipns.MNNG c o n c e n t r a t i o n s a r e as f o l lows: a • , I O - 4 M ; o -O , 3X10"^ M ; A A , 10 M ; • « c o n t r o I . 64 —j— r — j 7 -6 12 18 24 30 36 Time a f t e r r e l e a s e ( h ) F i g u r e 24. U.V.-induced chromosome a b e r r a t i o n s i n I D M - a r r e s t e d CHO c e l l s w i t h ( 0 — - O ) and without(» — • ) a 24 hour p e r i o d o f p o s t t r e a t m e n t i n c u b a t i o n i n I DM(50 erg•mm 2), 65 P l a t e 1 . H a r l e q u i n s t a i n e d metaphase o f CHO e e l I ( a f t e r i r r a d i a t i o n w i t h 15 erg-mm""2 of U.V. l i g h t ) , P l a t e 2 , H a r l e q u i n s t a i n e d metaphase of CHO c e l l showing m u l t i p l e chromosome a b e r r a t i o n s and i n c r e a s e d SCE frequency(ADM a r r e s t e d c e l l t r e a t e d w i t h 3 X10" 7 MNNG). X P l a t e 3.Morphology of ADM-arrested CHO c e I I s , C u I t u r e has been i n c u b a t e d i n ADM f o r 6 days. P l a t e 4.Morphology of d i v i d i n g CHO c e l l s . P l a t e 6.Micro-nuclei induced i n ADM-arrested c e l l s by U.V. l i g h t (15 erg"mm" ). 68 DISCUSSION a) T e c h n i c a l commentary In genera I , s i s t e r c h r o m a t i d exchanges v i s u a l i z e d i n t h e B r d U - i n c o r p o r a t e d h a r l e q u i n chromosomes,a re a s e n s i t i v e and p r e c i s e measure o f t h e a c t i o n o f many m u t a g e n i c / c a r c i n o g e n i c agents.The t e c h n i q u e s used produced p r e p a r a t i o n s i n which SCEs were r e a d i l y s c o r e d ( P I a t e s 1 - 2 ) . I n c r e a s e s i n SCE f r e q u e n c y were induced in an a p p r o x i m a t e l y l i n e a r l y dose-dependent manner by t h e mutagenic agents t e s t e d . T h e v a r i a t i o n i n t h e number of SCEs p e r metaphase o f m u t a g e n - t r e a t e d c e l l s i s s m a l l and c o u n t s of as few as 15-20 c e l l s can produce a r e l i a b l e datum.A mi n o r s h o r t c o m i n g i s t h a t t h e spon-taneous SCE l e v e l shows s m a l l , b u t s i g n i f i c a n t v a r i a t i o n s between e x p e r i m e n t s ( T a b Ie I I I).Thus i n c r e a s e s i n SCE f r e q u e n c y • I ess t h a n t w i c e t h e spontaneous l e v e l c a n n o t be c o n c l u s i v e l y a t t r i b u t e d t o an e x p e r i m e n t a l f a c t o r . T h e cause of t h e v a r i a t i o n , a I though n o t known,is presumed t o be r e l a t e d t o s l i g h t u n c o n t r o l l e d v a r i a t i o n s i n c u l t u r e c o n d i t i o n s . S u c h f a c t o r s as i ) g e n e r a l p r o l i f e r a t i v e c a p a c i t y o f t h e c e l l s , i i ) m i n o r v a r i a t i o n s in i n c u b a t i o n t e m p e r a t u r e and h u m i d i t y and i i i ) t h e s o u r c e o r b a t c h o f f e t a l b o v i n e serum may be r e s p o n s i b I e . V a r i a t i o n s of t h i s s o r t have been r e p o r t e d p r e v i o u s l y ( T a k e h i s a and Wo I f f , 1 9 7 7 ) . S i g n i f i c a n t minor v a r i a t i o n s i n spontaneous SCE f r e q u e n c y have been a t t r i b u t e d t o d i f f e r e n c e s in serum l o t s ( K a t o and' Sandberg,1977a). 69 SCE f r e q u e n c y was shown t o be independent of BrdU c o n c e n t r a t i o n o v e r a 1 6 - f o l d range i n c o n c e n t r a t i o n ( F i g u r e 1).Independence p e r -s i s t e d when th e SCE i n c i d e n c e was i n c r e a s e d by t r e a t m e n t w i t h t h e drug MMCCFigure 2).The independence o f SCE f r e q u e n c y and BrdU c o n c e n t r a t i o n e f f e c t i v e l y e l i m i n a t e s t h e p o s s i b i l i t y t h a t s m a l l v a r i a t i o n s i n t h e e x t e n t o f BrdU i n c o r p o r a t i o n , p e r h a p s induced by chemical, t r e a t m e n t , cou I d a l t e r t h e SCE f r e q u e n c y . Vary i ng a c c o u n t s o f t h e e f f e c t s o f BrdU c o n c e n t r a t i o n on SCE f r e q u e n c y e x i s t i n t h e I i t e r a t u r e . W h i le W o l f f and P e r r y ( 1 9 7 4 ) - r e p o r t a s t e e p r i s e in SCE f r e q u e n c y between 0.25 and 1 u M and a g r a d u a l i n c r e a s e a t h i g h e r c o n c e n t r a t i o n s , K a t o C 1 9 7 4 a ) o b s e r v e d a c o n s t a n t l e v e l between 0.2 and 8 u M . W h e t h e r o r n o t BrdU i s r e s p o n s i b l e f o r t h e i n d u c t i o n o f S C E s , i t i s c l e a r t h a t , w i t h t h e c o n c e n t r a t i o n s and c u l t u r e c o n d i t i o n s used h e r e , t h e l a b e l compound does not m a r k e d l y a l t e r t h e SCE l e v e l . SCE i n d u c t i o n has r e c e n t l y been added t o t h a t group o f in vitro methods which produce an i n d e x o f m u t a g e n i c / c a r c i n o g e n i c a c t i v i t y ( P e r r y and Evans,1975).One s i g n i f i c a n t advantage i s t h e extreme s e n s i t i v i t y o f t h e r e s p o n s e , S i g n i f i c a n t , a n d o f t e n ' d r a m a t i c i n c r e a s e s i n SCE f r e q u e n c y induced by mutagens can be d e t e c t e d a t c o n c e n t r a t -i o n s n o t markedly r e d u c i n g v i a b i l i t y . I n a l l c a s e s , t h e t r e a t e d c e l l has s u r v i v e d two c e l l c y c l e s . S t a g e s p e c i f i c i t y o f mutagen a c t i o n i s an i m p o r t a n t c o n c e r n h e r e , s i n c e t r e a t e d c e l l s must t r a v e r s e 2 S,2 Q>2> 2 M and a t l e a s t 1 G 1 phase between t h e t i m e o f BrdU a d d i t i o n and 70 samp I i n g . T h u s , f o r s t a g e s p e c i f i c a g e n t s , t h e optimum re s p o n s e may be dependent upon which o f t h e s e seven s t a g e s t h e c e l l s a r e in when t r e a t e d . S i m i I a r SCE y i e l d s were produced when MMC was added p r i o r t o and 24 hours a f t e r BrdU a d d i t i o n ( F i g u r e 3).The s a m p l i n g t i m e a t which t h e maximum res p o n s e was g e n e r a t e d , h o w e v e r , d i f f e r e d s l i g h t l y f o r t h e two t r e a t m e n t p r o t o c o I s . F o r t h e s i n g l e agent t e s t e d , s l i g h t d i f f e r e n c e s i n t h e p a t t e r n o f SCE i n d u c t i o n r e s u l t e d from d i f f e r e n t t r e a t m e n t p r o t o c o l s . A l s o o f imp o r t a n c e i s t h e . d u r a t i o n o f e x p o s u r e t o c h e m i c a l a g e n t . I n g e n e r a l , t h e g o a l , a t l e a s t w i t t v r e s p e c t t o d e t e c t i o n , s h o u I d be t o a c h i e v e t h e g r e a t e s t p o s s i b l e damage t o DNA w i t h t h e l e a s t amount o f t o x i c i t y . I n o r d e r t o reduce t o x i c i t y , a n d t o b e s t q u a n t i f y t h e e x p o s u r e dose,a c h e m i c a l t r e a t m e n t o f t h e o r d e r o f s e v e r a l hours i s f a v o u r e d . D a t a has been p r e s e n t e d on t h e compounds chromate and MNNG(Figures 6 and 7 ).Whereas t h e d u r a t i o n o f MNNG e x p o s u r e does n o t seem t o be of i m p o r t a n c e , o n l y a 48 h ex p o s u r e o f human c e l l s t o chromate i n d u c e s a marked i n c r e a s e i n SCE fre q u e n c y . T h e p r o d u c t -i o n o f damage•by chromate,as d e t e c t e d by UDS,has been shown t o r e l y upon some t i m e - d e p e n d e n t m e c h a n i s m ( W h i t i n g and Stich,1978).MNNG,on th e o t h e r h a n d , e x e r t s i t s e f f e c t upon c e l l s i n t h e f i r s t t h r e e hours of exposure(Law ley and Th a t c h e r , 1 9 7 0 ) . T h e s I i g h t l y • l e s s e r r e s p o n s e o f c e l l s t o c h r o n i c e x p o s u r e o f MNNG may be due t o i t s i n t e r a c t i o n w i t h t h e h i g h e r serum l e v e l s p r e s e n t ( 1 5 % FBS. vs 2% FBS). 71 N o t w i t h s t a n d i n g t h e advantages of a t r e a t m e n t o f s h o r t d u r a t i o n , a c h r o n i c e x p o s u r e t o c h e m i c a l may be more e f f i c i e n t i n t h e r o u t i n e d e t e c t i o n o f ' m u t a g e n i c a c t i v i t y a t low c o n c e n t r a t i o n s . The k i n e t i c s of SCE i n d u c t i o n a t low and moderate doses appear t o be q u i t e s t r a i g h t f o r w a r d . T h e s i t u a t i o n becomes more complex a t doses where t h e c e l l s ' v i a b i l i t y i s markedly reduced.Three d i s t i n c t p a t t e r n s of r esponse were e v i d e n t : i ) C h e m i c a l l y t r e a t e d human c e l l s r e a c h e d a maximum SCE f r e q u e n c y of 30-40 SCEs p e r e e l I.At h i g h e r c o n c e n t r a t i o n s , m i t o t i c c e l l s were n o t present.CHO c e l l s t r e a t e d w i t h chromate a l s o behaved i n t h i s way (MacRae and S t i c h , 1 9 7 8 ) . i i ) CHO c e l l s t r e a t e d w i t h t h e a l k y l a t i n g a g e n t s MMC o r MNNG a c h i e v e d a maximum SCE f r e q u e n c y of 120-140 p e r e e l I.Due t o t h e d i f f i c u l t y e n c o u n t e r e d i n d e t e c t i n g two SCEs o c c u r r i n g in c l o s e p r o x i m i t y , t h e a c t u a l f r e q u e n c y c o u l d c o n c e i v a b l y have been c o n s i d e r -a b l y g r e a t e r . A t h i g h e r d o s e s , t h e chromosomes of M2 c e l l s were ex-t e n s i v e l y broken and sometimes c o m p l e t e l y f r a g m e n t e d . i i i ) U.V. r a d i a t i o n produced a maximum SCE f r e q u e n c y of a p prox-i m a t e l y 70 p e r CHO c e l l and 25 p e r human f i b rob I a s t ( F i gures :8 and 9 ) . A t h i g h e r d o s e s , t h e SCE f r e q u e n c y d e c r e a s e d and i n CHO c e l l s r e a c h e d t h e spontaneous I eve I ( F i g u r e 9 ) . 1 1 i s n o t known whether t h i s e f f e c t r e p r e s e n t s a s u p p r e s s i o n of t h e mechanisms whereby SCEs are formed which i s m e diated by some phenomenom a s s o c i a t e d w i t h t h e a d m i n i s t r a t i o n 72 of h i g h U.V. d o s e s , o r s i m p l y t h e s e l e c t i v e e l i m i n a t i o n o f s e v e r e l y a f f e c t e d c e l l s from t h e p o p u l a t i o n as t h e r e s u l t o f reduced v i ab i I i t y . ' I t appears t h a t each agent reduces v i a b i l i t y a t a dose s l i g h t l y d i f f e r e n t t o t h a t which produces t h e maximum y i e l d o f SCEs.Chromate and U.V. seem more e f f e c t i v e i n r e d u c i n g v i a b i I i t y ( r e I a t i v e t o t h e i r a b i l i t y t o i nduce SCEs) t h a n a r e MMC and MNNG.The independence o f t h e i n d u c t i o n o f l e t h a l damage and SCEs s u g g e s t s t h a t SCEs a r e not t h e m s e l v e s 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 v i a b i I i t y . R e d u c t i o n i n v i a b i I i t y ( r e I a t i v e t o SCE i n d u c t i o n ) a l s o seems t o be dependent upon t h e c e l l l i n e used. In t h e r o u t i n e d e t e c t i o n of m u t a g e n s / c a r c i n o g e n s i t i s o f p r a c t i c a l i m p o r t a n c e t o use a e e l I,such'as t h e CHO l i n e , i n which t h e maximum y i e l d o f SCEs induced by most a g e n t s i s h i g h . b) SCE i n d u c t i o n i n X P E e e l Is The q u e s t i o n n a t u r a l l y a r i s e s w h e t h e r SCEs a r e t h e r e s u l t o f a p r o c e s s r e f l e c t i n g DNA damage o r r e p a i r . M o d e r a t e i n c r e a s e s i n SCE f r e q u e n c y appear t o r e p r e s e n t a s u b l e t h a l e f f e c t , a I though t h e l o n g -t e r m e f f e c t s on c e l l f u n c t i o n s have not been examined.The maximum SCE f r e q u e n c y i s induced a t a dose which reduces s u r v i v a l a l t h o u g h SCEs do n o t appear t o be d i r e c t l y r e s p o n s i b l e f o r r e d u c i n g v i a b i l i t y . In t h e case o f r e p a i r s y n t h e s i s , i t i s p o s s i b l e t o c o r r e l a t e t h e low l e v e l s found a f t e r t r e a t m e n t w i t h i n h i b i t o r s ( F o x , 1 9 7 4 ; M o u t o n , 1 9 7 2 ) 73 o r i n XP ce I Is(W i I I i ams and Li t t I e , 1 9 7 7 ) w i t h reduced v i ab i I i t y . T h i s p r o v i d e s e v i d e n c e t h a t an a c t u a l r e p a i r p r o c e s s i s in o p e r a t i o n . No s p e c i f i c i n h i b i t o r s o f SCE f o r m a t i o n a r e y e t known and t h e r e s p o n s e of c e l l s w i t h a mutant .genotype are o f h e i g h t e n e d i m p o r t a n c e . T h r e e o b s e r v a t i o n s c o n c e r n i n g t h e response- o f mutant c e l l t y p e s may be made: i ) B l o o m ' s syndrome c e l l s have an e l e v a t e d spontaneous SCE r a t e (5-10X n o r m a l ) ( C h a g a n t i et al., 1974). i i ) F a n c o n i ' s anemia c e l l s produce l e s s t h a n o n e - h a l f as many SCEs as normal c e l l s when t r e a t e d w i t h MMC ( L a t t . al.3\915). t i l ) X P c e l l s produce an SCE f r e q u e n c y 2-4X t h a t o f normal c e l l s when t r e a t e d w i t h U.V l i g h t C D e W e e r d - K a s t e l e i n et al.,\911) o r v a r i o u s a l k y l a t i n g a g e n t s ( W o l f f et al.3\911). R e c e n t l y i t has been shown t h a t a t a x i a t e l a n g i e c t a s i a c e l l s produce i n c r e a s e s i n SCE f r e q u e n c y s i m i l a r t o normal c e l l s when t r e a t e d w i t h e i t h e r MMC,EMS o r AD(Gal loway,1977).Bloom's syndrome e e l I s , I i k e normal ceI I s , r e s p o n d t o MMC t r e a t m e n t by an i n c r e a s e i n t h e SCE f r e q u e n c y ( S h i r a i s h i and Sandberg,1978). These o b s e r v a t i o n s do n o t , u n f o r t u n a t e Iy,shed l i g h t on t h e r o l e o f SCEs i n DNA r e p a i r mechanisms . a f f e c t i n g c e l l s u r v i v a I . F a n c o n i ' s anemia c e l l s a r e s e n s i t i v e t o t r e a t m e n t w i t h MMC.The reduced SCE y i e l d i n d u c e d i n t h e s e c e l l s by MMC has t h e r e f o r e been i n t e r p r e t e d as b e i n g due t o a reduced r e p a i r c a p a c i t y . T h e l i m i t e d number o f 74 doses examined l e a v e s doubts,however,as t o whether t h i s r e f l e c t s o n l y a g e n e r a l t o x i c i t y . The d a t a p r e s e n t e d here a g r e e s wi.th t h a t o f W o l f f et al, (1977) and De Weerd K a s t e l e i n et aZ,(1977),XP c e l l s a r e more r e s p o n s i v e t h a n normal c e l l s w i t h r e s p e c t t o SCE f o r m a t i o n when t r e a t e d w i t h U.V. l i g h t ( F i g u r e 8),MNNG o r 4NQ0(Figure 9),These r e s u l t s d i f f e r s l i g h t l y from t h o s e o f W o l f f et al, (1977) i n t h e magnitude o f t h e d i f f e r e n c e i n resp o n s e o f XP and.normal c e l l s . X P c e l l s were a p p r o x i m a t e l y 4 and 2 - f o l d more s e n s i t i v e t o MNNG and 4NQ0 r e s p e c t i v e I y , a c c o r d i n g t o t h e i r work and t h e a b s o l u t e numbers o f SCEs o b s e r v e d a t t h e h i g h e r c h e m i c a l c o n c e n t -r a t i o n s t e s t e d were c o n s i d e r a b l y l a r g e r than t h o s e o b s e r v e d i n t h i s s t u d y . T h i s d i f f e r e n c e may r e f l e c t t h e n a t u r e o f t h e c e l l s used.Those w o r k e r s used c e l l l-ines which had been t r a n s f o r m e d by SV40 v i r u s w h i l e t h e s e e x p e r i m e n t s were c o n d u c t e d u s i n g s t r a i n s o f dermal f i b r o b l a s t s . "XP c e l l s a r e d e f i c i e n t i n t h e e x c i s i o n o f damage produced by U.V. r a d i a t i o n and 4NQ0 and respond as normal c e l l s t o MNNG t r e a t m e n t ( S t i c h and San, 1 9 7 0 ; S t i c h et al,, 1 9 7 3 ) . C o r r e s p o n d i n g I y , y i e I d s o f chromosome a b e r r a t i o n s g r e a t e r t h a n t h o s e o f normal c e l l s a r e induced i n X P ' c e l l s o n l y by U.V. l i g h t and 4NQ0.The s i m p l e s t e x p l a n a t i o n of t h i s s i t u a t i o n i s t h a t chromosome a b e r r a t i o n s r e s u l t from t h e p r e s e n c e o f l e s i o n s which have n o t been removed by e x c i s i o n r e p a i r . I n t h e case o f SCEs,however, no d i s t i n c t i o n between t h e two c l a s s e s o f c h e m i c a l s i s e v i d e n t . X P c e l l s a r e most s e n s i t i v e t o U . V . , s l i g h t l y l e s s so t o MNNG and even l e s s so t o ^NQO.It appears t h a t t h o s e l e s i o n s w h i c h induce chromosome a b e r r a t i o n s a r e d i s t i n c t from t h o s e t h a t induce S C E s . I f t h i s i s t r u e , t h e n XP c e l l s may a l s o be d e f e c t i v e i n t h e e x c i s i o n o f some MNNG-induced l e s i o n s . T h i s s o r t o f l e s i o n presumably does not l e a d t o t h e f o r m a t i o n o f chromosome a b e r r a t i o n s . S i n c e MNNG-treated XP and normal c e l l s show s i m i l a r s u r v i v a l c u r v e s ( S a n , 1 9 7 2 ) , i t appears t h a t the. l e s i o n does not induce s u f f i c i e n t damage t o markedly a l t e r v i a b i l i t y , I n k e e p i n g w i t h t h i s view i s t h e r e c e n t i n t e r e s t i n g o b s e r v a t i o n t h a t XP c e l l s a r e d e f e c t i v e i n t h e remova of DNA damage induced by m o n o f u n c t i o n a I a l k y l a t i n g a g e n t s ( G o t h - G o I d s t e i n J977).The major u n e x c i s e d s p e c i e s appears t o be 0 6 - a I k y I g u a n i n e . c) Recovery o f c e l l s from ADM The a d u l t body i s c o m p r i s e d p r e d o m i n a n t l y o f n o n - p r o l i f e r a t i n g e e l Is.As a consequence,many c a n c e r s may be e x p e c t e d t o a r i s e as a p o p u l a t i o n o f d i v i d i n g c e l l s which were t r a n s f o r m e d as a r e s u l t o f e v e n t s o c c u r r i n g i n a n o n - d i v i d i n g c e l l . l t i s e x p e c t e d t h a t t h e p o t e n t i a l o f c e l l s t o r e p a i r c a r c i n o g e n - i n d u c e d damage w h i l e i n t h e p r e - r e p I i c a t i v e s t a t e , a n d t h e t i m e between e x p o s u r e t o damage and i n i t i a t i o n o f c e l l d i v i s i o n would be m a j o r f a c t o r s i n f l u e n c i n g t h e p r o c e s s . T h e i m p o r t a n c e o f t h i s a s p e c t o f c a r c i n o g e n e s i s i s s u p p o r t e d by s e v e r a l l i n e s o f e x p e r i m e n t a l e v i d e n c e . Tumour i n d u c t i o n i n mouse s k i n i s r e l a t e d t o t h e f r a c t i o n o f c e l l s u n d e r g o i n g d i v i s i o n a t t h e t i m e o f t r e a t m e n t . T h e tumour y i e l d can be i n c r e a s e d by t r e a t m e n t w i t h agents which s t i m u l a t e c e l l d i v i s i o n p r i o r t o t r e a t m e n t ( F r e i and Harnoso,1967) o r reduced by t r e a t m e n t w i t h c h e m i c a l s which i n h i b i t DNA s y n t h e s i s a f t e r c a r c i n -ogen t r e a t m e n t ( B a t e s et al. .,1968). E t h y I n i t r o s o u r e a i s known t o pro d u c e b r a i n tumours upon f e t a l e x p o s u r e b u t i s not c a r c i n o g e n i c t o t h e a d u l t b r a i n ( D r u c k e r y , 1 9 7 0 ) . D i m e t h y I n i t r o s a m i n e has been shown t o produce tumours-more e f f e c t i v e l y i n r e g e n e r a t i n g t h a n normal I i v e r ( C r a d d o c k , 197.1 ;Craddock, 76 1973;Capps et al., 1973;Pound et aZ.,,1973). One o f t h e o b j e c t i v e s o f t h i s work was t o s t u d y t h e i n d u c t i o n of SCEs and chromosome a b e r r a t i o n s i n n o n - p r o l i f e r a t i n g e e l I s . G i v e n a s y s t e m i n which i t i s p o s s i b l e t o induce a r r e s t e d c e l l s t o undergo d i v i s i o n , o n e can v a r y t h e t i m e between t r e a t m e n t w i t h mutagen and r e s u m p t i o n o f r e p l i c a t i v e a c t i v i t y . I n t h i s w a y , i t i s p o s s i b l e t o s t u d y t h e e f f e c t o f r e p a i r a c t i v i t i e s which o p e r a t e i n t h e n o n - p r o l i f e r a t i n g c e l l upon t h o s e i n d i c e s of damage which a r e r e v e a l e d i n t h e d i v i d i n g ceI I. D e p r i v a t i o n o f t h e e s s e n t i a l amino a c i d a r g i n i n e was chosen as a means of a r r e s t i n g c e l l s i n a p r e - r e p I i c a t i v e s t a t e . D i s t i n e t d i f f e r e n c e s i n t h e b e h a v i o u r of a r g i n i n e a r r e s t e d CHO c e l l s and d i v i d i n g CHO c e l l s i n t h e p r e - r e p I i c a t i v e , o r G-| s t a g e , however, were d e t e c t e d . E a c h o f t h e mutagenic a g e n t s examined induced many more chromosome a b e r r a t i o n s i n t h e c e l l s d e p r i v e d of a r g i n i n e ( F i g u r e s 34,16,17,18),A s t u d y of s u r v i v a l a f t e r U.V. i r r a d i a t i o n showed t h a t A D M - a r r e s t e d CHO c e l l s were a p p r o x i m a t e l y 3 t i m e s more s e n s i t i v e t h a n d i v i d i n g c e l l s ( F i g u r e 1 9 ) . I n c o n t r a s t , s i m i I a r y i e l d s o f SCEs were induced i n a r g i n i n e d e p r i v e d c e l l s d a b l e V I ) . In o r d e r t o e v a l u a t e how s p e c i f i c was t h e phenomenon of s e n s -i t i z a t i o n o f a r g i n i n e a r r e s t e d e e l Is,human c e l l s a r r e s t e d by a r g i n i n e d e p r i v a t i o n and CHO c e l l s a r r e s t e d by i s o l e u c i n e d e p r i v a t i o n were e x a m i n e d . N e i t h e r proved t o be as s e n s i t i v e as ADM-arrested CHO ce! I s , a l t h o u g h t h e I D M - a r r e s t e d c e l l s were more s e n s i t i v e t h a n 77 d i v i d i n g ceI I s ( F i g u r e s 14 and 2 4 ) . A l t h o u g h t h e s e c o m p a r i t i v e s t u d i e s a r e l i m i t e d , t h e y s u g g e s t t h a t t h e s e n s i t i z a t i o n phenomenon o b s e r v e d i s s p e c i f i c f o r t h e c e l l ! t y p e and,to some e x t e n t , t o t h e amino a c i d d e f i c i e n t i n t h e growth medium. Work c a r r i e d o u t on d e n s i t y i n h i b i t e d C h i n e s e hamster c e l l s ( l i n e HA1) s u p p o r t s t h e o b s e r v a t i o n t h a t n u t r i t i o n a l f a c t o r s a f f e c t t h a t t y p e of r e p a i r o f p o t e n t i a l l y l e t h a l l e s i o n s which t a k e s p l a c e p r i o r t o t h e o n s e t o f DNA s y n t h e s i s ( H a h n , 1 9 7 5 ) . L e s i o n s i n d u c e d by U.V.-I i k e ag e n t s a r e p a r t i c u l a r l y a f f e c t e d . F r o m t h e p r e s e n t s t u d y , i t appears t h a t t h e e s s e n t i a l amino a c i d a r g i n i n e i s an e x t r e m e l y i m p o r t a n t n u t r i t i o n a l f a c t o r o f t h i s s o r t . A r g i n i n e i s one of t h e most abundant amino a c i d s i n t h e h i s t o n e p r o t e i n s , r e p r e s e n t i n g a p p r o x i m a t e l y .13$ of t h e a r g i n i n e - r i c h h i s t o n e s I I I and I V U o h n s,1971 ) . S i n c e t h e s e h i s t o n e s appear' t o p l a y a major-r o l e i n m a i n t a i n i n g t h e s t r u c t u r a l i n t e g r i t y o f c h r o m a t i n ( E v a n s , 1 9 7 7 ) , i t i s not s u r p r i s i n g t h a t a r g i n i n e d e p r i v a t i o n has an e f f e c t upon chromosome s t r u c t u r e . One i m p o r t a n t q u e s t i o n emerges i n t h i s r e g a r d : i s t h e s e n s i t i v i t y o f ADM-arrested CHO c e l l s t h e r e s u l t o f an i n c r e a s i n g l y e f f i c i e n t i n d u c t i o n o f damaging l e s i o n s o r a reduced e f f i c i e n c y o f t h e i r r e p a i r i U n f o r t u n a t e Iy a d e f i n i t i v e r e s p o n s e i s not p o s s i b l e . T h e d i f f e r i n g b e h a v i o u r of chromosome a b e r r a t i o n s and SCEs s u g g e s t s e i t h e r t h a t a ) s i m i l a r numbers of a b e r r a t i o n and S C E - i n d u c i n g l e s i o n s a r e produced i n ADM-arrested and d i v i d i n g c e l l s and a r g i n i n e d e p r i v a t i o n a f f e c t s o n l y t h a t mechanism wh i.ch produces chromosome a b e r r a t i o n s , o r b)more chromosome a b e r r a t i o n - p r o d u c i n g than SCE-p r o d u c i n g l e s i o n s a r e induced i n ADM-arrested c e l l s compared t o d i v i d i n g ceI I s . I n t h i s c a s e , t h e mechanisms p r o d u c i n g SCEs and chromosome a b e r r a t i o n s would both be u n a f f e c t e d by a r g i n i n e • d e p r i v a t i o n . P o s s i b i l i t y (b) i m p l i e s t h a t a r g i n i n e d e p r i v a t i o n a l t e r s t h a t c e l l u l a r t a r g e t o f mutagens which i s r e s p o n s i b l e o n l y f o r t h e f o r m a t i o n o f chromosome a b e r r a t i o n s . T h e h i s t o n e p r o t e i n s a r e a p o s s i b l e c a n d i d a t e . A c c o r d i n g t o t h e c u r r e n t view of c h r o m a t i n s t r u c t u r e , t h e a r g i n i n e - r i c h h i s t o n e s I I I and IV form t h e c e n t r a l c o r e o f t h e nucleosome,the b a s i c u n i t o f chromat i n (Carrieri n i -O t e r o , 1 9 7 6 ) . I n such a p o s i t i o n , t h e y c o u l d w e l l p l a y a c e n t r a l r o l e i n m a i n t a i n i n g chromosome s t r u c t u r e d u r i n g t h o s e m e t a b o l i c p r o c e s s e s , s u c h as DNA r e p a i r and rep I i c a t i o n , w h i c h a l t e r chromosome s t r u c t u r e ( C I e a v e r , 1 9 7 4 ) . By t h e same r e a s o n i n g , a r g i n i n e d e p r i v a t i o n may a f f e c t t h e r e p a i r p r o c e s s e s d i r e c t I y , w i t h o n l y t h e mechanism l e a d i n g t o form-a t i o n o f chromosome a b e r r a t i o n s b e i n g f a v o u r e d . The e x p l a n a t i o n f a v o u r e d here i s t h a t a c e r t a i n l e v e l o f a r g i n i n e i s r e q u i r e d d u r i n g S-phase f o r t h e o p e r a t i o n o f some 79 r e p a i r process.When a l t e r e d , t h i s r e s u l t s i n t h e p r o d u c t i o n of chromosome a b e r r a t i o n s . T h e S-phase r e c o v e r y o f CHO c e l l s from a r g i n i n e d e p r i v a t i o n may be.so r a p i d as t o p e r m i t DNA s y n t h e s i s to. p r o c e e d a t a r g i n i n e l e v e l s which a r e below t h i s l i m i t . S o m e s u p p o r t of t h i s h y p o t h e s i s i s p r o v i d e d by t h e i n c i d e n c e o f a b e r r a t i o n s in c e l l s d i v i d i n g a t d i f f e r e n t t i m e s a f t e r ADM-reI e a s e ( F i g u r e 2 0 ).The h i g h e s t a b e r r a t i o n f r e q u e n c y o c c u r s i n t h o s e c e l l s which d i v i d e f i r s t a f t e r ADM' r e l e a s e . I t s u b s e q u e n t l y f a l l s r a p i d I y . T h o s e c e l l s r e c o v e r i n g f i r s t would be most l i k e l y t o have e n t e r e d S-phase f i r s t and t h u s t o have had l e s s t i m e f o r rep I e n i s h m e n t o f t h e a r g i n i n e p o o l . The l i t e r a t u r e c o n t a i n s o n l y scant, r e f e r e n c e s t o t h e r o l e of p r o t e i n s in DNA r e p a i r mechanisms.Few a u t h o r s have r e c o g n i z e d t h a t t h e p r o t e i n component of t h e c h r o m a t i n may w e l l be a f a c t o r which modulates DNA damage and r e p a i r phenomena(CIeaver,1974;KihI man,1977). Only two e x p e r i m e n t a l o b s e r v a t i o n s which s u p p o r t t h i s view a r e p r e s e n t -ly known.Chu(1965) has r e p o r t e d t h a t i ) t h e a c t i o n s p e c t r u m f o r U.V.-induced chromosome damage r e v e a l s s i g n i f i c a n t a c t i v i t y a t t r i b -u t e le t o a b s o r b t i o n of energy by p r o t e i n s and i i ) t h e chromosome a b e r r a t i o n f r e q u e n c y of U . V . - i r r a d i a t e d c e l l s can be g r e a t l y enhanced by p o s t - t r e a t m e n t i n c u b a t i o n w i t h an i n h i b i t o r of p r o t e i n s y n t h e s i s . These o b s e r v a t i o n s c l e a r l y o u t l i n e t h e importance of p r o t e i n s t o p r o -c e s s e s a f f e c t i n g both damage and r e p a i r of c h r o m a t i n . One o t h e r p o s s i b l e e x p l a n a t i o n f o r t h e r a p i d d e c l i n e i n chromo-some a b e r r a t i o n f r e q u e n c i e s w i t h a d v a n c i n g s a m p l i n g t i m e s i s worthy 80 of m e n t i o n . l t i s p o s s i b l e t h a t t h e c e l l s a r r e s t e d i n ADM are a non-u n i f o r m p o p u l a t i o n w i t h r e s p e c t t o s e n s i t i v i t y t o DNA damage.A I though o t h e r w o r k e r s have assumed t h a t a r g i n i n e - d e p r i v e d c e l l s a r e a r r e s t e d in a G^,or p r e - r e p I i c a t i v e s t a g e ( F r e e d and S c h a t z , 1 9 6 9 ) , i t i s d i f -f i c u l t t o e l i m i n a t e t h e p o s s i b i l i t y t h a t a s m a l l r e s i d u a l amount o f DNA s y n t h e s i s i s t a k i n g p l a c e i n a t l e a s t some e e l Is.The s i t e s of DNA s y n t h e s i s c o u l d be p a r t i c u l a r l y s u s c e p t i b l e t o damage by mutagens. A l i m i t e d amount of DNA s y n t h e s i s has been d e t e c t e d i n CHO c e l l s m a i n t a i n e d in ADM f o r 48 hours by some i n v e s t i g a t o r s ( W e i s s f e I d and Rouse,1977).In t h e p r e s e n t s t u d y , n o more t h a n 0.5% of CHO c e l l s m a i n t a i n e d in ADM + 2% FBS f o r 6 days showed i n c o r p o r a t i o n o f d e t e c t -i b l e 3H-TdR o v e r a 2 hour p e r i o d . l t i s u n I i k e l y , h o w e v e r , t h a t l i m i t e d n u c l e o t i d e i n c o r p o r a t i o n o c c u r r i n g a t a v e r y slow r a t e would be de-t e c t e d by t h e methods used.The q u e s t i o n o f l i m i t e d l e v e l s o f DNA s y n t h e s i s t h u s remains a v e r y d i f f i c u l t one t o a d d r e s s e x p e r i m e n t a l l y . d) R e p a i r . o f chromosome and SCE p r o d u c i n g l e s i o n s Whatever t h e mechanism of ADM-mediated sens i t i v i ty,. i t i s c l e a r t h a t t h e p o s t t r e a t m e n t i n c u b a t i o n o f c e l l s i n ADM had l i t t l e e f f e c t upon t h e y i e l d of chromosome a b e r r a t i o n s . A I though i t a p p e a r s , f r o m t h e r e s u l t s p r e s e n t e d i n T a b l e V I I I , t h a t some r e m o v a l ( o r " r e p a i r " ) o f chromosome a b e r r a t i o n - p r o d u c i n g l e s i o n s was o c c u r r i n g , t h i s i s p r o b -a b l y a t t r i b u t i b l e t o s a m p l i n g e r r o r . S i n c e t h e f r e q u e n c i e s r e p o r t e d a r e based upon o n l y a s i n g l e sample t i m e ( 2 4 h a f t e r r e l e a s e , from ADM), 81 t h e t o t a l y i e l d of chromosome a b e r r a t i o n s produced i n t h e r e c o v e r i n g c e l l s may not be a d e q u a t e l y r e p r e s e n t e d . A more e x t e n s i v e s t u d y o f U.V.-induced chromosome a b e r r a t i o n s produced i n A D M - a r r e s t e d ( F i g u r e 20) and I DM a r r e s t e d c e l l s ( F i g u r e 24) shows no i n d i c a t i o n of any r e d u c t i o n i n chromosome a b e r r a t i o n y i e l d a f t e r p o s t - i r r a d i a t ion i n c u b a t i o n in t h e n o n - p r o l i f e r a t i n g s t a t e . T h i s i s i n agreement w i t h t h e s u r v i v a l d a t a which a l s o shows no e v i -dence o f a r e p a i r a c t i v i t y . ( F i g u r e 19). The r e p a i r c a p a c i t y of n o n - p r o l i f e r a t i n g c e l l s has been b e s t c h a r a c t e r i z e d w i t h r e s p e c t t o X-ray induced I e s i o n s . S i g n i f i c a n t i n -c r e a s e s in s u r v i v a l o f i r r a d i a t e d c u l t u r e s can be o b s e r v e d a f t e r m aintenance in t h e s t a t i o n a r y p h a s e ( a c h i e v e d by d e n s i t y - d e p e n d e n t i n h i b i t i o n of growth) f o r v a r i o u s t i m e s p r i o r t o s u b c u I t u r i n g . T h e phenomenon has been termed p o t e n t i a l l y l e t h a l damage r e p a i r ( P L D R ) ( L i t t I e , 1 9 7 3 ) . I t has been shown t o be dependent upon c e l l t o c e l l c o n t a c t and some f a c t o r p r e s e n t i n t h e medium of s t a t i o n a r y phase cu l t u r e s ( L i t t l e , 1 9 7 3 ) . U.V.-induced damage has a l s o been examined i n t h i s way (Hahn, 1975). Recovery from U.V has a has a ha I f - I i f e o f a p p rox-i m a t e l y 20 h o urs and i s p a r t i c u l a r l y s e n s i t i v e t o t h e n u t r i t i o n a l s t a t u s o f t h e c e l l s both b e f o r e and a f t e r . i r r a d i a t ion.The a l k y l a t i n g agent- MMS behaved s i m i l a r l y t o U.V.(Hahn,1975).In view of t h e im-p o r t a n c e of n u t r i t i o n a l f a c t o r s i n U.V and a l k y l a t i n g a g e n t - i n d u c e d PLDR,the absence of r e c o v e r y in ADM-arrested c e l l s i s not s u r p r i s i n g . In c o n t r a s t t o U.V.-induced chromosome a b e r r a t i o n s , U.V.-induced 82 SCEs were o b s e r v e d t o be reduced i n number by p o s t - i r r a d i a t i o n m a intenance of c e l l s in ADM(Figure 2 ) ) . N e i t h e r o f t h e a l k y l a t i n g a g ents showed t h i s b e h a v i o u r . I t has been p r e v i o u s l y been r e p o r t e d t h a t U.V.-induced SCEs can be p h o t o r e a c t i v a t e d i n c e l l s p o s s e s s i n g t h e n e c e s s a r y enzyme (.Kato,1974c).This s u g g e s t s t h a t a p h o t o r e a c t i v a t i b I e p h o t o p r o d u c t , p o s s i b l y t h e t h y m i d i n e d i m e r , i s r e s p o n s i b l e f o r i n d u c i n g SCEs.These d a t a a r e i n agreement w i t h t h i s i n t e r p r e t a t i o n . C H O c e l l s p o s s e s s t h e a b i I i t y , a I though I i m i t e d , t o ' e x c i s e p y r i m i d i n e dimers from t h e i r DNA(KihIman,1977).UDS,accepted as a measure of e x c i s i o n r e p a i r a c t i v -i t y , c a n r e a d i l y be d e t e c t e d in ADM-arrested CHO c e l l s o v e r t h e dose range 20-60 erg*mm (Lam,persona I communicat ion).SCEs a r e most e f f i c -i e n t l y i n d u c e d i n t h e S-phase of t h e e e l I c y c l e by t h i s a g e n t . T h i s i s in agreement w i t h t h e f i n d i n g s of o t h e r s ( K a t o , 1 9 7 4 d ; W o I f f et al. 3 1974) and i s i n t e r p r e t e d as r e f l e c t i n g t h e a b i l i t y of t h e c e l l s t o remove U.V.-induced l e s i o n s p r i o r t o t h e i r r e p l i c a t i o n d u r i n g S-phase. An a l t e r n a t e e x p l a n a t i o n o f t h e s e r e s u I t s , h o w e v e r , w a r r a n t s c o n -s i d e r a t i o n . I t i s known t h a t BrdU,when i n c o r p o r a t e d i n t o t h e c e l l s ' DNA i n c r e a s e s r a d i o s e n s i t i v i t y f o r v i s i b I e ( B e n d e r et al,,1972),U.V. and i o n i z i n g r a d i a t i o n ( D j o r d j e v i c and Szyba1 s k i , 1 9 6 0 ) . I n t h e e x p e r -im e n t a l p r o t o c o l f o l l o w e d ( F i g u r e 21) some samples were i r r a d i a t e d a f t e r i n t r o d u c t i o n o f BrdU i n t o t h e medium.lt i s t h e r e f o r e p o s s i b l e * t h a t h i g h l e v e l s o f SCEs induced i n S-phase c e l l s were t h e r e s u l t o f some i n t e r a c t i o n of BrdU a l r e a d y i n c o r p o r a t e d i n t o DNA and t h e U.V. r a d i a t i o n , U n f o r t u n a t e I y , i t was not p o s s i b l e t o t e s t t h i s p o s s i b i l i t y , t h e o n l y a l t e r n a t i v e b e i n g t o u t i l i z e a l a b e l ( s u c h as 3 H - t h y m i d i n e ) 83 which would n o t , i t s e I f , i n t e r a c t w i t h U.V. l i g h t . With t h i s d i f f i c u l t y i n m i n d , i t i s s t i l l p o s s i b l e t o examine t h e r e s u l t s .from F i g u r e 21 and t o c o m p a r e t h e SCEs induced, by U.V. both 24 hours b e f o r e and i m m e d i a t e l y p r i o r t o r e l e a s e from ADM.In both i n s t a n c e s , B r d U was added t o t h e medium o n l y a f t e r i r r a d i a t i o n . F o r each o f t h e doses examined,the samples which were i n c u b a t e d i n ADM f o r 24 hours a f t e r i r r a d i a t i o n show lower SCE l e v e l s . T h i s s u p p o r t s t h e view t h a t U.V.-induced l e s i o n s which r e s u l t i n SCEs can be removed by non-p r o l i f e r a t i n g c e l l s m a i n t a i n e d i n ADM. In c o n t r a s t , a 24 hour p e r i o d o f p o s t - t r e a t m e n t i n c u b a t i o n i n ADM had no e f f e c t upon t h e SCE l e v e l s induced by e i t h e r MMC o r MNNG(Figure 2J ) . T h e s i m p l e s t e x p l a n a t i o n o f t h i s o b s e r v a t i o n i s t h a t S C E - p r o d u c i n g l e s i o n s were not removed d u r i n g t h e 24 hours t h e c e l l s were i n c u b a t e d i n ADM.MMC i s a b i f u n c t i o n a l a l k y l a t i n g a gent and ind u c e s i n t e r s t r a n d c o y a l e n t a d d u c t s i n DNAClyer and SzybaI s k i , 1 9 6 4 ) . 1 1 has been proposed t h a t t h i s s o r t of l e s i o n i s not r e a d i l y removed by t h e e x c i s i o n r e p a i r pathway and remains;'' i n t h e DNA u n t i l S-phase, t o become m a n i f e s t as an S C E ( S c h a f e r , 1 9 7 7 ) . T h i s d a t a would s u p p o r t t h i s i d e a were i t not f o r t h e f a c t t h a t s i m i l a r r e s u l t s were o b t a i n e d f o r MNNG.This c h e m i c a l induces p r i m a r i l y : monoadducts i n t h e form o f 7-methyIguanine,3-methyIadenine and 0 ^ m e t h y I g u a n i n e ( R o b e r t s et al., 1971 a).Of t h e s e , o n Iy 0 -methyl g u a n i n e i s known t o p e r s i s t i n DNA f o r e x t e n d e d p e r i o d s of t i m e ( L o v e I e s s , 1 9 6 9 ; Lawley,1970).The l o n g - l i v e d l e s i o n s i nduced by MNNG t h e r e f o r e do not appear to be t h e r e s u l t o f i n t e r s t r a n d DNA c r o s s l i n k s . SCE y i e l d s induced a f t e r r e l e a s e from ADM d i f f e r s f o r t h e a g e n t s 84 MMC and MNNG(Figure 2 1 ) . W h i l e t h e same number of SCEs were induced by MMC a t v a r i o u s t i m e s a f t e r release,MNNG induced fewer SCEs when t h e c e l l s were t r e a t e d 8 and 1] hours a f t e r r e l e a s e from ADM,i.e. a f t e r t h e o n s e t o f S-phase,This unexpected d i f f e r e n c e i n b e h a v i o u r of t h e two a g e n t s i s not r e a d i l y e x p l a i n a b l e . l t i n d i c a t e s , h o w e v e r , t h a t t h e p r o c e s s of SCE i n d u c t i o n may be more complex t h a n a n t i c i p a t e d . The r e s u l t s o b t a i n e d w i t h A DM-arrested c e l l s d i f f e r s l i g h t l y from t h o s e o b t a i n e d w i t h XP e e l I s . B o t h systems a r e i n a c c o r d w i t h t h e c o n c e p t t h a t UVy, i n d u c e s l e s i o n s which a r e removed l e s s r e a d i l y i n XP c e l l s t h a n i n normal human c e l l s and ADM-arrested e e l Is.These l e s i o n s would r e s u l t i n SCEs when p r e s e n t i n c e l l s u n d e r g o i n g S-phase. In t h e c a s e of MNNG t r e a t m e n t , h o w e v e r , t h e two systems behave d i f f e r e n t l y . Fewer SCEs a r e induced i n normal human t h a n XP ceI I s , s u g g e s t i n g t h a t t h e normal c e l l s p o s s e s s t h e a b i l i t y t o remove MNNG-induced l e s i o n s w h i c h r e s u l t i n S C E s . T h i s a b i l i t y was not d e m o n s t r a t e d by ADM-arrested CHO e e l Is,where t h e l e s i o n s appeared t o have s u r v i v e d 24 hours of p o s t -t r e a t m e n t i n c u b a t i o n i n ADM.In view of t h e d i f f e r e n t r e p a i r c a p a c i t i e s of d i v i d i n g and ADM-arrested e e l I s , i t i s o f i n t e r e s t t o know how c l o s e l y each system resembles t h e b e h a v i o u r of c e l l s e x i s t i n g in vivo. 85 SUMMARY T h i s work c o n f i r m s t h e o b s e r v a t i o n o f o t h e r s ( P e r r y and Evans, 1975) t h a t SCEs a r e u s e f u l i n d i c a t o r s o f t h e a c t i o n o f m u t a g e n i c a g e n t s . I n a d d i t i o n t o t h e h i g h l e v e l o f s e n s i t i v i t y a c h i e v a b l e w i t h t h i s t e c h n i q u e , t w o f u r t h e r advantages can be r e c o g n i z e d : i ) d u e t o t h e f a c t t h a t c e l l s must undergo a t l e a s t two d i v i s i o n s b e f o r e p r o d u c i n g h a r I e q u i n - s t a i n e d . c h r o m o s o m e s , t h e e f f e c t o f e x p o s u r e t o s l i g h t l y o r 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 h e m i c a l s o v e r long p e r i o d s o f t i m e may be a s c e s s e d . T h i s may a l l o w t h e d e t e c t -ion o f an agent which i s both m u t a g e n i c and t o x i c o v e r t h e same c o n c e n t r a t i o n range. i i ) t h e l e s i o n s p r o d u c i n g SCEs ap p e a r t o be r e l a t i v e l y l o n g - l i v e d and n o t a f f e c t e d m a r k e d l y by UDS.Their s t u d y may t h e r e f o r e p r o v i d e new i n f o r m a t i o n on t h e a c t i o n o f m u t a g e n i c a g e n t s . A l t h o u g h t h e l e v e l s o f UDS i n d u c e d i n XP and normal c e l l s by U.V. and-4NQ0 v a r i e s r o u g h l y i n v e r s e l y w i t h t h e y i e l d o f chromosome a b e r r a t i o n s and SCEs,no c o r r e l a t i o n i s e v i d e n t i n t h e c a s e o f damage induced by MNNG.This s u g g e s t s t h a t XP and normal c e l l s respond d i f f e r e n t l y t o MNNG and t h a t XP c e l l s may be d e f i c i e n t i n t h e r e p a i r of a t l e a s t p a r t o f t h e damage induced by t h i s a g e n t . An e x a m i n a t i o n of r e p a i r a c t i v i t i e s i n c e l l s a r r e s t e d by d e p r i v a t i o n o f an e s s e n t i a l amino a c i d has shown t h a t : i ) t h e e s t a b l i s h e d C h i n e s e hamster c e l l l ine,CHO, r e a c t s 86 t o a r g i n i n e d e p r i v a t i o n by p r o d u c i n g a d r a m a t i c a l l y h i g h e r i n c i d e n c e .of mutagen-induced chromosome a b e r r a t i o n s a f t e r r e c o v e r y t h a n d i v i d i ng ceI I s , i i ) t h e i n c i d e n c e o f SCEs i n A D M - a r r e s t e d CHO c e l l s i s s i m i l a r - t o t h a t i n d i v i d i n g c e l l s , i i i ) n o r e p a i r of t h o s e U.V.-induced l e s i o n s which produce chromosome a b e r r a t i o n s a f t e r r e c o v e r y was d e t e c t e d i n ADM-arrested c e l l s i v ) o f t h e t h r e e a g e n t s examined,U.V.,MMC and MNNG, o n l y U.V. in d u c e d S C E - p r o d u c i n g Iesions- which c o u l d be removed i n ADM-arrested c e l l s . From t h e s e r e s u l t s , i t a ppears t h a t SCEs a r e t h e r e s u l t o f r e l a t i v e l y l o n g - l i v e d l e s i o n s induced by mutagenic c h e m i c a I s . S u c h l e s i o n s may not be removed.by an e x c i s i o n r e p a i r mechanism. A l t h o u g h an e l e v a t e d SCE f r e q u e n c y i s u s u a l l y a s s o c i a t e d w i t h an i n c r e a s e d i n c i d e n c e o f chromosome a b e r r a t i o n s ( K a t o , 1 9 7 7 a ) , no c o r r e l a t i o n between t h e two phenomena was e v i d e n t from t h e r e s u l t s o f t h i s study.Any common pathway i n t h e p r o c e s s e s by which SCEs and chromosome a b e r r a t i o n s a r e formed does not appear t o be a f f e c t e d by amino a c i d d e p r i v a t i o n . 87 BIBLIOGRAPHY Bartram,C.R.,T.Koske-WestphaI and E . P a s s a r g e . 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M o i . B i o l . , 5 t h . p 174(1971). Shafer,D.A.Rep I i c a t i o n bypass model o f s i s t e r c h r o m a t i d exchanges and i m p l i c a t i o n s f o r Bloom's syndrome and F a n c o n i ' s anemia.Hum.Genet.39: 177( 1977)'. S h i r a i s h i , Y . and A . A . S a n d b e r g . E f f e c t s o f m i t o m y c i n C on s i s t e r c h r o m a t i d exchange i n normal and Bloom's syndrome ceI I s . M u t a t . R e s . 4 9 : 2 3 3 ( 1 9 7 8 ) . Solomon,E. and M . B o b r o w . S i s t e r c h r o m a t i d exchanges-a s e n s i t i v e assay of age n t s damaging human chromosomes.Mutat.Res.30:273(1975). Sperling,K.,R.D.Wegner,H.Riehm-ei al. Frequency and d i s t r i b u t i o n o f s i s t e r c h r o m a t i d exchanges i n a case o f F a n c o n i ' s anemia.Humangenetik 27:227(1975). Stetka,D.G. and S.. Wo I f f . S i s t e r c h r o m a t i d exchange.-'as an assay f o r g e n e t i c damage induced by mutagen^carcinogens. I I.In vitro t e s t f o r compounds r e q u i r i n g m e t a b o l i c a c t i v a t i o n . M u t a t . R e s . 4 1 : 3 4 3 ( 1 9 7 8 ) . S t i c h , H . F . and R.H.C. San.DNA r e p a i r and c h r o m a t i d a n o m a l i e s i n mammalian c e l l s exposed t o 4 - n i t r o q u i n o I i n e - 1 - o x i d e . M u t a t . R e s . 1 0 : 389(1970). S t i c h , H . F . , W . S t i c h and R.H.C.San.Chromosome a b e r r a t i o n s i n xeroderma pigmentosum c e l l s exposed t o t h e c a r c i n o g e n s , 4 - n i t r o q u i n o I i n e - 1 -o x i de and N-methyI-N'-n i t r o - n i t r o s o g u a n i d i n e . P r o c . S o c . E x p . B i o l . M e d . 142:114(1973). S u t h e r I a n d , B . M . P h o t o r e a c t i v a t i o n i n animal e e l I s . L i f e S c i . 1 6 :1 ( 1 9 7 5 ) . T a k e h i s a , S . and S . W o l f f . I n d u c t i o n o f s i s t e r c h r o m a t i d exchanges i n C h i n e s e hamster c e l l s by c a r c i n o g e n i c mutagens r e q u i r i n g m e t a b o l i c a c t i v a t i o n . M utat. Res. 45 :263( 1977~) . Tay l o r , J ,H. S i s t e r c h r o m a t i d exchanges i n t r i t i urm- I abe I I ed chromosomes. G e n e t i c s 43:515(1958). Terasima,T. and L.J.To Imach.Changes i n X-ray s e n s i t i v i t y o f HeLa c e l l s d u r i n g t h e d i v i s i o n c y c l e . N a t u r e 190:1210(1961). T o b e y , R . A . P r o d u c t i o n and c h a r a c t e r i z a t i o n o f mammalian c e l l s r e v e r s i b l y a r r e s t e d i n G-| by growth i n i so I e u c i ne-def i c i e n t med i urn. Methods i n C e l I . B i o l . 6 : 6 7 ( 1 9 7 3 ) . W e i s s f r e Id,A.S. and H.Rouse.Continued i n i t i a t i o n o f DNA s y n t h e s i s i n a r g i n i n e - d e p r i v e d C h i n e s e hamster o v a r y e e l I s . J . C e l l B i o l . 7 3 : 2 0 0 ( 1 9 7 7 ) . W h i t i n g , R . F . and H.F. S t i c h . I n d u c t i o n by chromate o f DNA damage and r e p a i r in c u l t u r e d human ceI I s . C h e m . - B i o I . I n t e r a c t i o n s ( t o be sub-• m l t t e d . Wi I Iiams,J.R. and J . B . L i t t I e . D N A r e p a i r i n c u l t u r e d mammalian c e l l s . I n G r o w t h , N u t r i t i o n and M e t a b o l i s m o f C e l l s i n C u l t u r e . V o l I I I(eds G .H.Rothblat and V . J . C r i s t o f a Io)Academic Press,New Y o r k ( 1 9 7 7 ) . W o l f f , S . and P . P e r r y . D i f f e r e n t i a I Giemsa s t a i n i n g of s i s t e r c h r o m a t i d s and t h e s t u d y of s i s t e r c h r o m a t i d exchanges w i t h o u t a u t o r a d i o g r a p h y . . Chromosoma 48 : 3 4 1 ( 1 9 7 4 ) . Wo I f f , S . , J . B o d y c o t e and R . B . P a i n t e r . S i s t e r c h r o m a t i d exchanges induced i n C h i n e s e hamster c e l l s by U.V. i r r a d i a t i o n o f d i f f e r e n t s t a g e s of t h e ceI I ' c y c l e : t h e n e c e s s i t y f o r c e l l s t o pass t h r o u g h S.Mutat.Res.25:73 (1974). . W o l f f , S . , B . R o d i n and J . E . C l e a v e r . S i s t e r c h r o m a t i d exchanges i n d u c e d by m u t a g e n i c c a r c i n o g e n s i n normal and xeroderma pigmentosum c e l l s . N a t u r e 265:347(1977). 95 APPENDIX 1 Hank's B a l a n c e d S a l t S o l u t i o n 1) NaCl KCI MgS0 4-7H 20 Na 2HP04 K H 2 P 0 4 g I u c o s e i n 800 ml d i s t i l l e d w a t e r 2) C a C I 2 i n 100 ml d i s t i l l e d w a t e r 3) Phenol red co m b i n i n g t h e s e , a d j u s t i n g t h e and making up t o a f i n a l volume of 1 wei g h t ( g ) • 80 ' 4 1 0.48 0.60-10 1.4 0. 1 pH t o 7.0 w i t h 0.05 N NaOH, l i t e r makes a 10X s o l u t i o n . APPENDIX 2 A r g i n i n e D e f i c i e n t and I s o l e u c i n e D e f i c i e n t Medium(ADM and I DM) 1) E s s e n t i a l amino a c i d s weight(mg) L-h i s t i d i ne 310 L - I e u c i ne 520 L- Iys i ne 580 L- i so Ieuc i ne 5 2 0 ( i n ADM o n l y ) L-arg i n i ne 5 2 0 ( i n I DM o n l y ) L-meth i on i ne 150 L-phenyI a I an i ne 320 L - t h r e o n i ne 480 L - t r y p t o p h a n 100 L - v a l i n e 460 d i s s o I v e d i n 100 ml of Hank' s BSS L - t y r o s i ne 360 d i s s o I v e d i n 100 ml of 0.1 N HCI L - c y s t i ne 240 d i s s o I v e d i n 100 ml of 0.1 N HCI L-gIutami ne 2.92 d i s s o l v e d i n 100 ml of Hank' s BSS 2) N o n - e s s e n t i a l amino a c i d s L - a l a n i n e 89 L - a s p a r a g i n e 150 L - a s p a r t i c a c i d 133 L-gIutami c a c i d 147 L - p r o I i ne 115 L - s e r i n e 105 g l y c i n e 75 d i s s o l v e d i n 100 ml of Hank's BSS In l i e u o f t h i s m i x t u r e , a p r e p a r e d s o l u t i o n o f 100X n o n - e s s e n t i a amino a c i d s ( F l o w L a b o r a t o r i e s ) was added. 3) V i t a m i n s foI i c ac i d 10 c h o I i ne chI o r i de 100 n i c o t i nami de 100 i - i nos i t o I 200 p y r i doxaI 100 r i b o f I a v i n 10 D-Ca-pantothenate 100 t h i a m i n e HCI 100 d i s s o l v e d i n 100 ml of Hank's BSS In l i e u of t h i s m i x t u r e , a p r e p a r e d s o l u t i o n of 100X v i t a m i n s (Flow L a b o r a t o r i e s ) was added. Each s o l u t i o n , a I o n g w i t h 995 ml of 1 OX Hank's B S S , i s combined and made up t o 10 I i t e r s . T h i s i s s t e r i l i z e d by passage t h r o u g h a 0.22 uM mi I I i pore f i I t e r ( M i I I i pore) . APPENDIX 3 Phosphate B u f f e r e d S a l i n e ( P B S ) w e i g h t ( g ) NaCl 8.0 KCI 0.2 Na-^HPO, 1.15 K H 2 P O 4 - 0.2 d i s s o l v e i n 1 I of d i s t i l l e d w a t e r . APPENDIX 4 SORENSEN'S BUFFER (pH of 6.8) w e i g h t ( g ) K H 2 P 0 4 66.3 N a 2 H P 0 4 13.8 make up t o 1 I w i t h d i s t i l l e d w a t e r f o r a 10X s o l u t i o n . Di I u t e p r i o r t o use. PUBLICATIONS: MacRae, W.D., R.F. Whiting and H.F. Stich, Sister chromatid exchanges induced in cultured mammalian ce l l s by chromate, Chemico-Biological Interactions( accepted for publication). V MacRae, W.D., E.A. MacKinnon and H.F. Stich, The fate of U.V.-induced lesions affecting SCEs, chromosome aberrations and survival of CHO c e l l s arrested by deprivation of arginine, Chromosoma( accepted for publication). MacRae, W.D., E.A. MacKinnon and H.F. Stich, Effects of arginine deprivation upon chromosome aberrations, SCEs and survival of CHO c e l l s treated with mutagenic agents, Mutation Research ( submitted). MacRae, W.D., E.A. MacKinnon and H.F. Stich, The induction of sister chromatid exchanges and chromosome aberrations in CHO c e l l s arrested in the c e l l cycle by arginine deprivation, In Vitro ( submitted). MacRae, W.D. and H.F. Stich, Induction of sister chromatid exchanges in Chinese hamster ovary c e l l s by hydrazine, dimethylhydrazine and isoniazidj the effects of metal catalysis, hydrogen peroxide and the duration of exposure( to be submitted). MacRae, W.D. and H.F. Stich, Induction of sister chromatid exchanges in Chinese hamster ovary c e l l s by the thi o l compounds cysteine, cysteamine and glutathione( to be submitted). MacRae, W.D. and H.F. Stich, Induction of sister chromatid exchanges in Chinese hamster ovary,-cells by the reducing agents b i s u l f i t e arid ascorbic acid( to be submitted). 

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