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The role of metabolic activation of chemical precarcinogens in the induction of DNA damage and repair… Laishes, Brian Anthony 1974

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THE ROLE OF METABOLIC ACTIVATION OF CHEMICAL PRECARCINOGENS IN THE INDUCTION OF DNA DAMAGE AND REPAIR IN MAMMALIAN CELLS by BRIAN ANTHONY LAISHES B . S c . ( H o n . ) , C a r l e t o n U n i v e r s i t y , 1969 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f G e n e t i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BR IT ISH COLUMBIA November, 1974 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 shal l make it f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th i s 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 G e n e + i c s The Univers i ty of B r i t i s h Columbia Vancouver 8. Canada Date November 15, 1974 i ABSTRACT I t has been p r o p o s e d t h a t DNA a l t e r a t i o n s may be i n v o l v e d i n t h e " e a r l y " s t a g e s o f c h e m i c a l c a r c i n o g e n e s i s . The r e l a t i o n s h i p between m e t a b o l i c a c t i v a t i o n o f c h e m i c a l p r e c a r c i n o g e n s , DNA damage i n d u c e d by r e a c t i v e c a r c i n o g e n i c m e t a b o l i t e s , and r e p a i r o f t h e damaged DNA was t h e r e f o r e i n v e s t i g a t e d i n c u l t u r e d mammalian c e l l s and i n t h e i n t a c t a n i m a l . A t t e n t i o n was a l s o f o c u s e d on t h e r e l a t i o n s h i p o f t h e s e e v e n t s t o o r g a n s p e c i f i c i t y o f t umor i n d u c t i o n by c h e m i c a l p r e c a r c i n o g e n s . S y n t h e t i c a l l y p r e p a r e d m e t a b o l i t e s o f t h e p r e c a r c i n o g e n 2 - a c e t y I a m i n o f I u o r e n e (AAF) - t h e p r o x i m a t e c a r c i n o g e n N - h y d r o x y -2 - a c e t y I a m i n o f I u o r e n e ( N - h y d r o x y - A A F ) , and t h e u l t i m a t e c a r c i n o g e n N - a c e t o x y - 2 - a c e t y I a m i n o f I u o r e n e ( N - a c e t o x y - A A F ) - were used t o i n v e s t i g a t e t h e r e l a t i o n s h i p between t h e s t a g e o f m e t a b o l i c a c t i v a t i o n and t h e e x t e n t o f DNA damage i n d u c e d i n c u l t u r e d human f i b r o b l a s t s . As e s t i m a t e d by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e , t h e e x t e n t o f DNA damage i n d u c e d by e q u i m o l a r doses o f AAF and i t s m e t a b o l i t e s , f o l l o w e d t h e o r d e r : N - a c e t o x y - A A F > N - h yd roxy -AAF > AAF. T h i s p a r a l l e l l e d t h e o r d e r o f t h e l e v e l s o f DNA r e p a i r s y n t h e s i s , e s t i m a t e d by t h e u n s c h e d u l e d 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 ( 3 H - T d R ) . U n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR may, t h e r e f o r e , be a r e f l e c t i o n o f DNA damage, t h e amount o f w h i c h , i n t u r n , may be d e t e r m i n e d by t h e r e a c t i v i t y o f t h e c a r c i n o g e n i c m e t a b o l i t e t o w h i c h t h e c e l l s a r e e x p o s e d . A t e c h n i q u e o f J_n v i t r o s imu I a t i on o f j_n_ v i v o metabo l i sm o f p r e c a r c i n o g e n s p r o v i d e d a w o r k a b l e s y s tem i n w h i c h t h e r o l e o f m e t a b o l i c a c t i v a t i o n o f p r e c a r c i n o g e n s i n t h e i n d u c t i o n o f DNA i i damage and r e p a i r , chromosome a b e r r a t i o n s , and c e l l l e t h a l i t y c o u l d be s t u d i e d w i t h c u l t u r e s o f human " r e c e p t o r " c e l l s . When t h e p r e c a r c i n o g e n s d i m e t h y I n i t r o s a m i n e (DMN), a f l a t o x i n B l , and s t e r i g m a t o c y s t i n were " a c t i v a t e d " , by c o m b i n a t i o n w i t h NADPH-dependen t a c t i v a t i o n s y s t ems c o n t a i n i n g t h e p o s t - m i t o c h o n d r i a I s u p e r n a t a n t (9S) f r a c t i o n o f mouse l i v e r homogenate, t h e i r a b i l i t y t o i n d u c e h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s , chromosome a b e r r a t i o n s , and c e l l l e t h a l i t y was i n c r e a s e d . The r e s u l t s o f a l k a l i n e s u c r o s e g r a d i e n t s t u d i e s i n d i c a t e d t h a t DNA damage i nduced by r e a c t i v e m e t a b o l i t e s may a c c o u n t f o r t h e s e b i o l o g i c a l r e s p o n s e s . D i f f e r e n t s u b c e l l u l a r f r a c t i o n s o f mouse, r a t , and duck l i v e r s were f ound c a p a b l e o f p o t e n t i a t i n g t h e a b i l i t y o f a f l a t o x i n Bl t o i n d u c e h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s . H i gh l e v e l s o f DNA r e p a i r s y n t h e s i s were a l s o i n d u c e d by a f l a t o x i n Bl o r s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h t h e 9S f r a c t i o n f r om l i v e r , k i d n e y , o r lung o f s e v e r a l a n i m a l s p e c i e s . T h i s i m p l i e s t h a t c i r c u l a t i n g a f l a t o x i n Bl o r s t e r i g m a t o c y s t i n i n v i v o may be c o n v e r t e d t o DNA-damaging m e t a b o l i t e s i n l u n g , k i d n e y , and l i v e r o f t h e s e a n i m a l s . I t i s p r o p o s e d , t h e r e f o r e , t h a t t h e " h e p a t o c a r c i n o g e n s " a f l a t o x i n B l and s t e r i g m a t o c y s t i n s h o u l d have t h e c a p a c i t y t o i n d u c e t umo r s i n o r g a n s o t h e r t h a n t h e I i v e r . M y c o t o x i n s t h a t c o n t a i n an i s o l a t e d v i n y l e t h e r d o u b l e bond ( a f l a t o x i n s B l , G l , a f l a t o x i c o l , and s t e r i g m a t o c y s t i n ) , when " a c t i v a t e d " , i n d u c e d h i g h e r l e v e l s o f DNA r e p a i r s y n t h e s i s t h a n m y c o t o x i n s l a c k i n g t h i s m o l e c u l a r f e a t u r e ( a f l a t o x i n s B2 and G 2 ) . The a c t i v i t y o f " a c t i v a t e d " a f l a t o x i n s B l , G l , B2 , and G2 i n t h i s c i i i i n v i t r o s y s t em p a r a l l e l l e d t h e i r a c t i v i t y as c a r c i n o g e n s . R e p a i r - d e f i c i e n t x e r ode rma p igmentosum (XP) c e l l s r e spond w i t h l ower l e v e l s o f u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , as com-p a r e d w i t h normal c e l l s , f o l l o w i n g e x p o s u r e t o U V - r a d i a t i o n and numerous c h e m i c a l c a r c i n o g e n s . A l k a l i n e s u c r o s e g r a d i e n t s t u d i e s , i n c o m b i n a t i o n w i t h s t u d i e s o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR by human c e l l s expo sed t o 4 - n i t r o q u i n o I i n e I - o x i d e (4NQ0), d e m o n s t r a t e d t h a t t h e r e p a i r o f 4NQ0 - i nduced DNA damage may e n t a i l t h e " r e s y n t h e s i s " o f e x c i s e d DNA s t r a n d segments and t h e " r e j o i n i n g " o f s m a l l DNA s t r a n d s . A c o m p a r a t i v e l y s l o w r e t u r n o f t h e s e d i m e n t a -t i o n p r o f i l e s o f 4NQ0-damaged XP-DNA t o w a r d s t h e p o s i t i o n o f c o n t r o l XP-DNA i n a l k a l i n e s u c r o s e g r a d i e n t s , may r e f l e c t s l o w e r r a t e s o f r e p a i r o f 4NQ0 - i nduced damage i n XP c e l l s t h a n i n normal c e l l s . T r e a t m e n t w i t h " a c t i v a t e d " DMN and a f l a t o x i n B l r e s u l t e d i n t h e i n d u c t i o n o f l ower l e v e l s o f DNA r e p a i r s y n t h e s i s , h i g h e r f r e q u e n c i e s o f chromosome a b e r r a t i o n s , and h i g h e r l e v e l s o f c e l l l e t h a l i t y i n XP c e l l s t h a n i n s i m i l a r l y t r e a t e d normal c e l l s . The r e s u l t s o b -t a i n e d w i t h XP c e l l s i n d i c a t e t h a t d e f e c t i v e o r i n c o m p l e t e DNA r e p a i r s y n t h e s i s may p l a y a r o l e i n c a r c i n o g e n e s i s i n d u c e d by t h e s e p r e c a r c i nogens . The p o s s i b l e r o l e o f m e t a b o l i c a c t i v a t i o n and DNA damage and r e p a i r i n o r g a n - s p e c i f i c tumor i n d u c t i o n was i n v e s t i g a t e d i n v i v o . The e x t e n t o f DNA damage and p a t t e r n s o f r e p a i r i n l u n g , k i d n e y , and l i v e r o f m i c e , a f t e r s i n g l e s u b c u t a n e o u s i n j e c t i o n s o f 4NQ0 o r DMN, were d e m o n s t r a t e d by an a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e . The e x t e n t o f DNA damage a p p e a r e d maximal a t 4 h p o s t - i n j e c t i o n . A t t h i s t i m e , t h e e x t e n t o f DNA damage f rom 4NQ0 f o l l o w e d t h e o r d e r : lung > k i d n e y > l i v e r , w h i l e t h a t f r om DMN f o l l o w e d t h e o r d e r : i V l i v e r > k i d n e y and l u n g . The l e v e l s o f DNA damage may r e f l e c t d i f f e r e n c e s i n t h e d i s t r i b u t i o n and m e t a b o l i c a c t i v a t i o n o f t h e p r e c a r c i n o g e n s i n t h e s e o r g a n s . R e p a i r o f i n d u c e d DNA damage was e v i d e n t a t l a t e r s a m p l i n g t i m e s and was no t c o m p l e t e i n t h e t i s s u e s w h i c h had s u s t a i n e d t h e h i g h e s t l e v e l s o f DNA damage. T h i s o r g a n s p e c i f i c i t y o f DNA damage seems t o c o r r e l a t e w i t h l e v e l s o f DNA r e p a i r s y n t h e s i s , e s t i m a t e d by t h e i n c o r p o r a t i o n s o f 3 H - T d R , and w i t h s i t e s o f t umor f o r m a t i o n . Tumor i n d u c t i o n by c h e m i c a l p r e c a r c i n o g e n s may be i n f l u e n c e d by m u l t i p l e f a c t o r s t h a t c o n t r o l " e a r l y " s t a g e s o f c h e m i c a l c a r c i n o g e n e s i s such as m e t a b o l i c a c t i v a t i o n o f p r e -c a r c i n o g e n s , b i n d i n g t o DNA, DNA damage, and DNA r e p a i r . The c e n t r a l h y p o t h e s i s i s t h a t h i g h l e v e l s o f DNA damage i n d u c e d by r e a c t i v e m e t a b o l i t e s o f p r e c a r c i n o g e n s i n v i v o , r e q u i r e h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s and r e s u l t i n t h e p o s s i b l e o c c u r r e n c e o f d e f e c t i v e o r i n c o m p l e t e DNA r e p a i r . T h i s may l e a d t o n e o p l a s t i c t r a n s f o r m a t i o n t h r o u g h a l t e r a t i o n s o f t h e i n f o r m a t i o n a l c o n t e n t o f DNA. V TABLE OF CONTENTS Page ABSTRACT . i TABLE OF CONTENTS . v L IST OF TABLES . . . . . - x i L I ST OF FIGURES x i i i ACKNOWLEDGEMENTS . . x x i i ABBREVIATIONS . . . . . . x x i i i INTRODUCTI ON . . . I EVIDENCE FROM CANCER EPIDEMIOLOGY I CHEMICAL CARCINOGENESIS IN HUMANS . 3 EXPERIMENTAL CARCINOGENESIS 7 PHARMACOLOGY OF CHEMICAL CARCINOGENS 8 (a) D i s t r i b u t i o n o f A p p l i e d C h e m i c a l s . . . . 8 (b) B i o t r a n s f o r m a t i o n o f F o r e i g n Compounds i n t h e An ima l Body . II ( c ) B i o t r a n s f o r m a t i o n o f C h e m i c a l P r e c a r c i n o g e n s . II ( i ) A r o m a t i c Amines 12 ( i i ) N i t r o q u i n o l i n e N -Ox ide s 15 ( i i i ) H y d r o c a r b o n s 16 ( i v ) N i t r o s a m i n e s 17 ( v ) A f l a t o x i n s 19 INTERACTION OF CHEMICAL CARCINOGENS WITH CELLULAR MACROMOLECULES 22 ROLE OF CHEMICAL-DNA INTERACTIONS IN CARCINOGENESIS . . 25 DNA REPAIR AND CARCINOGENESIS . . . . . . . 26 ORGANOTROPIC CHEMICAL CARCINOGENS 34 v i Page OBJ ECTiVES 36 MATERIALS AND METHODS 40 MATERIALS 40 METHODS 43 ESTIMATION OF IN VITRO DNA DAMAGE BY ALKALINE SUCROSE GRADIENT CENTRIFUGATI ON 43 ASG Type I 43 ASG Type 2 44 ASG Type 3 . . . ' 45 ACTIVATION OF CHEMICAL PRECARCINOGENS IN VITRO . . . 46 (a ) P r e p a r a t i o n o f P o s t - M i t o c h o n d r i a l F r a c t i o n (9S F r a c t i o n ) o f T i s s u e Homogenates . . . . 46 (b) P r e p a r a t i o n o f 105,000 x g F r a c t i o n s o f L i v e r Homogenates : S u p e r n a t a n t ( I 05S ) and P e l l e t t ( I 05P ) . • 47 ( c ) P r e p a r a t i o n o f A c t i v a t i o n M i x t u r e s and T r e a t m e n t o f C e l l C u l t u r e s 48 (d) P r o t e i n D e t e r m i n a t i o n 49 (e ) A n a l y s i s o f Me thano l E x t r a c t s o f A c t i v a t i o n M i x t u r e s by UV S p e c t r o s c o p y 49 ( f ) A n a l y s i s o f C h l o r o f o r m E x t r a c t s o f A c t i v a t i o n M i x t u r e s by T h i n - L a y e r Ch romatog raphy . . . 50 (g) S y n t h e s i s o f A f l a t o x i c o l 50 TECHNIQUE FOR CULTURING HUMAN FIBROBLASTS FROM SKIN BIOPSIES 50 IN VITRO BIOLOGICAL END-POINTS OF ANALYSIS . . . . 51 (a) E s t i m a t i o n o f DNA R e p a i r S y n t h e s i s i n C u l t u r e d Human Ce l I s 51 (b) A n a l y s i s o f Chromosome A b e r r a t i o n s i n C u l t u r e d Ce I I s 53 ( c ) A n a l y s i s o f C e l l S u r v i v a l i n v i t r o . . . . 53 v i i Page (d) T r e a t m e n t s o f C e l l C u l t u r e s w i t h Sma l l Vo lumes . 54 ANALYSIS OF IN VIVO DNA DAMAGE 55 (a) L abe l I i ng o f DNA _i_n_ v i v o . . . . ' . . 55 (b) L a b e l l i n g o f P r o t e i n i n v i v o . . . . . 56 ( c ) A d m i n i s t r a t i o n o f C h e m i c a l C a r c i n o g e n s . . . 56 (d) E s t i m a t i o n o f DNA Damage i n L i v e r . . . . 56 S p a t u l a - S q u a s h T e c h n i q u e 56 Dounce H o m o g e n i z a t i o n . . . . . . 57 (e ) E s t i m a t i o n o f DNA Damage i n K i d n e y . . . . 57 S p a t u l a - S q u a s h T e c h n i q u e 57 Dounce H o m o g e n i z a t i o n 57 ( f ) E s t i m a t i o n o f DNA Damage i n Lung . . . . 58 M i n c i n g T e c h n i q u e . 58 Dounce H o m o g e n i z a t i o n 59 (g) DNA D e t e r m i n a t i o n . . . . . . . . 59 (h) H i s t o l o g i c a l P r e p a r a t i o n s . . . . . . 59 RESULTS AND DISCUSSION 61 SECTION I DNA DAMAGE AND REPAIR IN CULTURED MAMMALIAN CELLS EXPOSED TO PRECARCINOGENS AND SYNTHETIC CARCINOGENIC METABOLITES 61 RESULTS . . . . . . 61 (a) ASG Type I 62 ( i ) Speed o f C e n t r i f u g a t i o n . . . . . . 62 ( i i ) Number o f C e l l s L a y e r e d . . . . . 65 ( i i i ) L y s i s C o n d i t i o n s . . . . . . 67 ( i v ) E x t e n t o f P r o t e i n and L i p i d C o n t a m i n a t i o n o f Sed imented DNA 70 v i i i Page ( v ) A p p l i c a t i o n s o f ASG Type I . . . 7 3 (b) ASG Type 2 78 ( i ) T e c h n i c a l Deve lopment . . . . . 78 ( i i ) A p p l i c a t i o n s o f ASG Type 2 . . . 81 ( c ) ASG Type 3 97 DISCUSSION OF SECTION I 102 SECTION 2 DNA DAMAGE AND REPAIR IN CULTURED MAMMALIAN CELLS EXPOSED TO PRECARCINOGENS WITH CONCOMITANT ACTIVATION IN VITRO 113 RESULTS 113 (a) D i m e t h y I n i t r o s a m i n e (DMN) „ . . . . . 1 1 4 ( i ) DNA R e p a i r S y n t h e s i s 114 ( i i ) DNA Damage 119 ( i i i ) DNA R e p a i r S y n t h e s i s i n Normal and XP C© E l s • • • o • • • * • I27 ( i v ) Chromosome A b e r r a t i o n s o f Normal and XP C e l l s , . . 1 2 9 (v ) C l o n e - f o r m i n g C a p a c i t y o f Normal and XP C e l l s . . . • 132 (b) A f l a t o x i n B l and S t e r i g m a t o c y s t i n . . . . 134 ( i ) DNA R e p a i r S y n t h e s i s 134 ( i i ) DNA Damage . 142 ( i i i ) DNA R e p a i r S y n t h e s i s i n Normal and XP Ce I I s . . 147 ( i v ) Chromosome A b e r r a t i o n s o f Normal and XP Ce I I s 151 (v ) C l o n e - f o r m i n g C a p a c i t y o f Normal and XP Ce I I s 161 ( c ) S e l e c t i o n o f A c t i v a t i o n M i x t u r e s . . . . 166 ( i ) P r e p a r a t i o n s f r om V a r i o u s O rgans o f D i f f e r e n t A n i m a l s 166 i x Page ( i i ) P r e p a r a t i o n s f r om V a r i o u s S u b c e l l u l a r F r a c t i o n s o f L i v e r Homogenates . . . 1 6 7 DISCUSSION OF SECTION 2 178 SECTION 3 DNA DAMAGE AND REPAIR IN MAMMALIAN CELLS IN VIVO 190 RESULTS 190 (a) T e c h n i c a l Deve lopment . 1 9 1 (b) A n a l y s i s o f DNA Damage and R e p a i r i n v i v o . . 2 1 1 ( i ) 4NQ0 - i nduced DNA Damage and R e p a i r i n v i v o 212 ( i i ) DMN- induced DNA Damage and R e p a i r i n v i vo 224 DISCUSSION OF SECTION 3 . . . . . . . . . 240 SUMMARY . 2 5 2 S e c t i o n I 252 S e c t i o n 2 252 S e c t i o n 3 . . . 2 5 6 GENERAL DISCUSSION . . . . 259 The P o s s i b l e R o l e o f M e t a b o l i c A c t i v a t i o n o f P r e c a r c i n o g e n s i n t h e I n d u c t i o n o f DNA Damage and R e p a i r i n Mammal ian C e l l s 261 The P o s s i b l e R o l e o f DNA Damage and R e p a i r i n C a r c i n o g e n e s i s 263 The P o s s i b l e L i n k Between M e t a b o l i c A c t i v a t i o n , DNA Damage and R e p a i r , and C a r c i n o g e n e s i s . . . 264 The P o s s i b l e R o l e o f M e t a b o l i c A c t i v a t i o n and DNA Damage and R e p a i r i n t h e O r g a n - S p e c i f i c i t y o f Tumor I n d u c t i o n • 266 P o s s i b l e V a r i a b l e s I n f l u e n c i n g N e o p l a s t i c T r a n s f o r m a t i o n Induced by P r e c a r c i n o g e n s 271 X Page P e r s p e c t i v e s 275 BIBLIOGRAPHY 277 x i L I ST OF TABLES Tab Ie Page 1 Death r a t e pe r 100,000 p o p u l a t i o n f o r m a l i g n a n t neop la sms i n 1964-65 . . . I 2 R e l a t i v e s t a n d a r d i z e d m o r t a l i t y r a t i o s f o r c a n c e r a t v a r i o u s s i t e s . M o r t a l i t y i n m i g r a n t and s t a t i o n a r y p o p u l a t i o n s 2 3 Summary o f . m e t h o d s a v a i l a b l e t o e s t i m a t e DNA damage and r e p a i r i n mammalian c e l l s 31 4 The l e v e l o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o b l a s t s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f DMN, w i t h and w i t h o u t an a c t i v a t i o n s y s t em . . . . 116 5 L e v e l s o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o b l a s t s exposed t o DMN a l o n e and t o DMN i n c o m b i n a t i o n w i t h c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s tems • . . 120 6 F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a -t i o n s i n c u l t u r e d normal and XPe c e l l s ' f o l l o w i n g e x -p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f DMN w i t h and w i t h -o u t an a c t i v a t i o n s y s t e m 130 7 L e v e l s o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o -b l a s t s e xpo sed t o a f l a t o x i n B l o r s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s tems 135 8 F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a -t i o n s i n c u l t u r e d normal and XPe c e l l s f o l l o w i n g e x -p o s u r e t o a f l a t o x i n B l . w i t h and w i t h o u t an a c t i v a t i o n s y s t em 152 T a b l e 9 F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal and XPe c e l l s f o l l o w -i n g e x p o s u r e t o a f l a t o x i n Bl i n c o m b i n a t i o n w i t h c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s tems 10 F requency o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal and XPe c e l l s f o l l o w -ing e x p o s u r e t o s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s tems 11 The a c t i v a t i o n c a p a c i t y o f 9S f r a c t i o n s f r om v a r i o u s o r g an s o f d i f f e r e n t a n i m a l s . Normal human c e l l s e xpo sed t o a f l a t o x i n Bl and s t e r i g m a t o c y s t i n i n c om-b i n a t i o n w i t h a c t i v a t i o n s y s t ems . . 12 G r o s s m i c r o s c o p i c a p p e a r a n c e o f homogenates o f mouse o r g a n s p r e p a r e d w i t h a Dounce t i s s u e - g r i n d e r 13 E s t i m a t i o n o f t h e r e l a t i v e amounts o f l u n g , k i d n e y , and l i v e r DNA l a y e r e d p e r g r a d i e n t . . . . 14 Methods o f t i s s u e p r e p a r a t i o n used f o r t h e e s t i m a t i o n o f DNA damage by a l k a l i n e s u c r o s e g r a d i e n t c e n t r i -f u g a t i o n . . . . . . . . . x i i Page 159 160 168 193 210 243 x i i i L IST OF FIGURES F i g u r e Page 1 S t r u c t u r e s o f t h e a f l a t o x i n s and s t e r i g m a t o c y s t i n . 5 2 S t r u c t u r e o f 2 - a c e t y I a m i n o f I u o r e n e (AAF) . . . 6 3 S t r u c t u r e o f 4 - n i t r o q u i n o l i n e l - o x i d e (4NQ0) . . 7 4 S c h e m a t i c r e p r e s e n t a t i o n o f t h e p o s s i b l e r o u t e s f o l l o w e d by a c h e m i c a l c a r c i n o g e n a d m i n i s t e r e d t o an an ima l e i t h e r e n t e r a l l y o r p a r e n t e r a l l y . . . 10 5 P r o p o s e d b i o t r a n s f o r m a t i o n o f AAF 13 6 P r o p o s e d b i o t r a n s f o r m a t i o n o f 4NQ0 16 7 P r o p o s e d b i o t r a n s f o r m a t i o n o f b e n z ( a ) a n t h r a c e n e . . 17 8 P r o p o s e d b i o t r a n s f o r m a t i o n o f DMN 19 9 Some r o u t e s o f m e t a b o l i s m o f a f l a t o x i n B l 22 10 G e n e r a l i z e d scheme f o r t h e m e t a b o l i s m o f c h e m i c a l p r e c a r c i n o g e n s t o f o rm r e a c t i v e e I e c t r o p h i I i c metabo l i t e s 23 11 C o v a l e n t b o n d i n g o f a me thy l g r oup t o t h e N-7 p o s i t i o n o f g u a n i n e 24 12 A s c h e m a t i c model f o r e x c i s i o n r e p a i r . . . . 29 13 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f DNA f rom human s k i n f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a -t i o n a t 35 ,000 r e v . / m i n f o r 3 .5 h a t 2 0°C . . . . 64 14 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om human s k i n f i b r o -b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 2 0 , 000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. D i f f e r e n t numbers o f e e l Is l a y e r e d p e r g r a d i e n t 64 15 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 2 0 , 000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. D i f f e r e n t number o f e e l Is l a y e r e d . . . 64 x i v F i g u r e Page 16 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom normal and XPe f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 2 0 , 000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. . . . . . . 69 17 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom normal and XPe f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 2 0 , 000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. 69 18 S e d i m e n t a t i o n p r o f i l e s o f DNA and p r o t e i n r e l e a s e d f r om normal human f i b r o b l a s t s . .72 19 S e d i m e n t a t i o n p r o f i l e s o f DNA and p r o t e i n r e l e a s e d f r om normal c e l l s f o l l o w i n g e x p o s u r e t o N - a c e t o x y - A A F o r 4NQ0 72 20 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human f i b r o b l a s t s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f 4NQ0 . . . 74 21 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om human f i b r o b l a s t s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f N - a c e t o x y - A A F . 77 22 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om human f i b r o b l a s t s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f N - h y d r o x y - A A F and o f AAF . . . 77 23 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om human f i b r o b l a s t s ( 8 - 10 x I 0 3 e e l I s ) 80 24 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om 8-10 x I 0 3 XPe f i b r o b l a s t s . 80 25 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om normal human c e l l s expo sed t o 2 x I 0 ~ 6 M 4NQ0 and 0 , 12, and 24 h p o s t -t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM 83 26 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om XPe c e l l s e x p o s e d t o 2 x I 0 ~ 5 M 4NQ0 and 0 , 12, and 24 h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h ' MEM 83 XV F i g u r e Page 27 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om normal human c e l l s e xpo sed t o 5 x I 0 ~ 6 M 4NQ0 and 0 , 12, and 24 h p o s t -t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM 86 28 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om XPe c e l l s e xpo sed t o 5 x I 0 ~ 6 M 4NQ0 and 0 , 12, and 24 h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM 86 29 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o 5 x 1 0 ~ 6 M 4NQ0 f o r : (A) 3 h, and (B) I .5 h 92 30 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r o m normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o 5 x 1 0 " 6 M 4NQ0 f o r : (A) 0 .5 h , (B) 18 m i n , and (C) 6 min . . . 94 31 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human c e l l s f o l l o w i n g l - m i n e x p o s u r e o f t h e c e l l s u s p e n s i o n s t o 4NQ0: (A) normal c e l l s , and (B) XPe c e l l s . . . 96 32 S e d i m e n t a t i o n p r o f i l e s o f DNA f r o m normal human c e l l s f o l l o w i n g no t r e a t m e n t . C e n t r i f u g a t i o n a t 25 ,000 r e v . / m i n f o r 0 .5 h a t 2 0 ° C . . . . . . 101 33 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom normal and XPe c e l l s f o l l o w i n g 0 .5 h e x p o s u r e s t o 4NQ0. C e n t r i -f u g a t i o n a t 25 ,000 r e v . / m i n f o r 0 .5 h a t 2 0 ° C . . 101 34 The dependence o f DNA r e p a i r s y n t h e s i s on t h e m o l e c u l -a r a l t e r a t i o n s i n d u c e d i n DNA by r e a c t i v e c a r c i n o g e n i c metabo I i t e s 112 35 E f f e c t o f d u r a t i o n o f e x p o s u r e t o DMN, w i t h and w i t h -o u t c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , on t h e l e v e l o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b rob I a s t s 117 36 L e v e l o f DNA r e p a i r s y n t h e s i s o f human c e l l s e x p o s e d t o DMN p l u s an a c t i v a t i o n s y s t em c o n t a i n i n g v a r i o u s amounts o f NADPH . . 1 2 1 XV i F i g u r e Page 37 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human f i b r o b l a s t s e xpo sed t o MEM, a c t i v a t i o n s y s t e m o n l y , DMN p l u s a c t i v a t i o n s y s t em c o n t a i n i n g h e a t - t r e a t e d 9S f r a c t i o n , DMN p l u s a c t i v a t i o n s y s t e m w i t h o u t NADPH, o r DMN a I one . . . 124 38 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human f i b r o b l a s t s expo sed t o d i f f e r e n t c o n c e n t r a t i o n s o f DMN i n c o m b i -n a t i o n w i t h an a c t i v a t i o n s y s t em . 124 39 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om human c e l l s e x -posed t o DMN p l u s an a c t i v a t i o n s y s t e m w i t h p o s t -t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM f o r : 0 , 4 , and 12 h 126 40 L e v e l o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f DMN w i t h and w i t h o u t an a c t i v a t i o n s y s t e m „ . . , . .. 128 41 The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f DMN w i t h and w i t h o u t an a c t i v a t i o n s y s t em 133 42 The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal human c e l l s e xpo sed t o a f l a t o x i n Bl p l u s an a c t i v a t i o n s y s t e m c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f NADP . 138 43 E f f e c t o f d u r a t i o n o f e x p o s u r e t o a f l a t o x i n B l , w i t h and w i t h o u t an a c t i v a t i o n s y s t em p r e s e n t , on t h e l e v e l o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human ce I I s . . . • . . 139 44 The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n s B l , G l , B 2 , and G2 , i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em 140 F i g u r e 45 The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal c e l l s exposed t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i c o l a l o n e and i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t ems c o n -t a i n i n g t h e 9S f r a c t i o n o f d u c k , r a t , o r mouse I i v e r 46 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f rom c u l t u r e d human c e l l s expo sed t o MEM, a c t i v a t i o n s y s t e m o n l y , a f l a t o x i n Bl a l o n e , a f l a t o x i n Bl p l u s an a c t i v a t i o n s y s t e m , and DMN p l u s an a c t i v a t i o n s y s t e m 47 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom human c e l l s e xpo sed t o s t e r i g m a t o c y s t i n w i t h and w i t h o u t an a c t i v a t i o n s y s t em p r e s e n t 48 The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s expo sed t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l a l o n e and i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em 49 The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g -m a t o c y s t i n a l o n e and i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em . . . ..• . . . 50 F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a -t i o n s i n normal human and XPe c e l l s f o l l o w i n g e x -p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl w i t h and w i t h o u t an a c t i v a t i o n s y s t em w i t h added NADPH 51 F r equency o f metaphase p l a t e s w i t h chromosome a b e r r a -t i o n s i n normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl i n c o m b i n a t i o n w i t h an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t em xv i i Page 143 145 145 149 150 156 xv i i i F i g u r e Page 52 The c l o n e - f o r m i n g c a p a c i t y o f normal and XPe c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl w i t h and w i t h o u t an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s tem p r e s e n t 162 53 The c l o n e - f o r m i n g c a p a c i t y o f normal and XPe c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl w i t h and w i t h o u t an a c t i v a t i o n s y s t em c o n t a i n i n g added NADPH ' . . 163 54 The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g m a t o c y s t i n w i t h and w i t h o u t an a c t i v a t i o n s y s t e m d e s i g n e d t o g e n e r a t e NADPH 165 55 The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l i n c o m b i n a t i o n w i t h N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t ems c o n t a i n i n g t h e 9S f r a c t i o n o f : (A) mouse l i v e r , (B) r a t l i v e r , and (C) duck l i v e r . 171 56 The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t ems c o n t a i n i n g t h e I05P f r a c t i o n o f : (A) mouse l i v e r , (B) r a t l i v e r , and (C) duck l i v e r . . . 173 57 The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems c o n t a i n i n g t h e I05S f r a c t i o n o f : (A) mouse l i v e r , (B) r a t l i v e r , and (C) duck l i v e r . . . 175 58 The p o s s i b l e l i n k between m e t a b o l i c a c t i v a t i o n , e a r l y DNA a l t e r a t i o n s , and b i o l o g i c a l r e s p o n s e s a t t h e m o l e c u l a r , c h r o m o s o m a l , and c e l l u l a r l e v e l s . . . 179 x i x F i g u r e Page 59 The p o s s i b l e l i n k between e a r l y DNA a l t e r a t i o n s , DNA r e p a i r , and n e o p l a s t i c t r a n s f o r m a t i o n . . . . 184 60 A p r o p o s e d s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p between t h e p r e s e n c e o f an i s o l a t e d v i n y l e t h e r d o u b l e bond and t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n c u l t u r e d human c e l l s by a f l a t o x i n s , a f l a t o x i c o l , and s t e r i g m a t o c y s t i n - w i t h an a c t i v a t i o n s y s t e m p r e s e n t . . . . . . . 187 61 A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om u n t r e a t e d mouse t i s s u e s f o l l o w i n g Dounce h o m o g e n i z a t i o n : (A) l u n g , (B) k i d n e y , and (C) l i v e r 196 62 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om a 4 N Q 0 - t r e a t e d mouse and an u n t r e a t e d mouse. T i s s u e s were sub-: j e c t e d t o Dounce h o m o g e n i z a t i o n : (A) l u n g , (B) k i d n e y , and (C) l i v e r 199 63 S e d i m e n t a t i o n p r o f i l e s o f DNA f r om t i s s u e s o f m i c e k i l l e d 4 h and 16 h f o l l o w i n g 4NQ0 a d m i n i s t r a t i o n : (A) lung (Dounce h o m o g e n i z a t i o n ) , (B) k i d n e y ( s p a t u l a - s q u a s h ) , and (C) l i v e r ( s p a t u l a - s q u a s h ) . . 203 64 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom d i f f e r e n t w e i g h t s o f mouse lung t i s s u e p r e p a r e d by t h e m i n c i n g t e c h -n i q u e : (A) 0.5-1 mg, 1-2 mg, and (B) 2-3 mg, 4 -5 mg. 208 65 S e d i m e n t a t i o n p r o f i l e s o f DNA f rom t h e l ung s o f two 4 N Q 0 - t r e a t e d m i c e . T i s s u e was p r e p a r e d by t h e m i n c i n g t e c h n i q u e 4 h a f t e r t h e a d m i n i s t r a t i o n o f 4NQ0 . . 208 66 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om mouse t i s s u e s 4 h a f t e r t h e a d m i n i s t r a t i o n o f 20 mg 4NQ0/kg body w e i g h t : (A) lung ( m i n c i n g t e c h n i q u e ) , (B) k i d -ney ( s p a t u l a - s q u a s h ) , and l i v e r ( s p a t u l a - s q u a s h ) . . 214 X X F i g u r e Page 67 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om mouse t i s s u e s 4 h a f t e r t h e a d m i n i s t r a t i o n o f 40 mg o r 80 mg 4NQ0/kg body w e i g h t . T i s s u e s were p r o c e s s e d as i n F i g u r e 66 : (A) l u n g , (B) k i d n e y , and (C) l i v e r ' . • . • 216 68 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f rom mouse t i s s u e s a t 4 h and 16 h p o s t - t r e a t m e n t w i t h 4NQ0. T i s s u e s were p r o c e s s e d as i n F i g u r e 66 : (A) l u n g , (B) k i d n e y , and (C) l i v e r . . . . . . . 221 69 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f rom mouse t i s s u e s 4 h a f t e r t h e a d m i n i s t r a t i o n o f I mg and 8 mg DMN/kg body w e i g h t . T i s s u e s were p r o c e s s e d as i n F i g u r e 66 : (A) l u n g , (B) k i d n e y , and (C) l i v e r . 226 70 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f rom mouse t i s s u e s 4 h a f t e r t h e a d m i n i s t r a t i o n o f 100 mg and 500 mg DMN/kg body w e i g h t . T i s s u e s were p r o -c e s s e d as i n F i g u r e 66 : (A) l u n g , (B) k i d n e y , and (C) I i v e r 228 71 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om mouse t i s s u e s a t 4 , 8 , and 12 h p o s t - t r e a t m e n t w i t h DMN. T i s s u e s were p r o c e s s e d as i n F i g u r e 6 6 : (A) l u n g , (B) k i d n e y , and (C) l i v e r 232 72 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f rom mouse t i s s u e s a t 6 days p o s t - t r e a t m e n t w i t h DMN. T i s s u e s were p r o c e s s e d as i n F i g u r e 66 : (A) l u n g , (B) k i d -ne y , and (C) l i v e r . 234 73 S e d i m e n t a t i o n p r o f i l e s o f DNA r e l e a s e d f r om mouse t i s s u e s a t 2 weeks p o s t - t r e a t m e n t w i t h DMN. T i s s u e s were p r o c e s s e d as i n F i g u r e 6 6 : (A) l u n g , (B) k i d n e y , and (C) l i v e r 236 XX i F i g u r e Page 74 The dependence o f DNA r e p a i r s y n t h e s i s on t h e m o l e c u l a r a l t e r a t i o n s i nduced i n DNA i n v i v o by r e a c t i v e c a r c i n o g e n i c m e t a b o l i t e s o f p r e c a r c i n o g e n s . 250 75 Chem i ca l c a r c i n o g e n e s i s d e s c r i b e d i n t h r e e p h a s e s , d e m o n s t r a t i n g a l i n k between m e t a b o l i c a c t i v a t i o n , DNA damage and r e p a i r , and n e o p l a s t i c t r a n s f o r m a -t i o n 266 76 E a r l y pha se s o f c h e m i c a l c a r c i n o g e n e s i s and t h e f a c t o r s w h i c h may i n f l u e n c e p r o g r e s s i o n f r om one phase t o a n o t h e r 272 xx i i ACKNOWLEDGEMENTS T h e r e i s no q u e s t i o n t h a t I f o und t h i s a r e a o f r e s e a r c h t o be most i n t e r e s t i n g and r e l e v a n t and I t h a n k my r e s e a r c h s u p e r v i s o r , Dr. H.F. S t i c h , f o r i n t r o d u c i n g me t o t h e t o p i c . I am t r u l y g r a t e f u l f o r D r . S t i c h ' s e f f o r t s i n p r o v i d i n g a w e l l - r o u n d e d t r a i n i n g w i t h s t i m u l a t i n g d i s c u s s i o n s and c o n t i n u a l s u p p o r t . As f e l l o w s t u d e n t s , R i c h a r d , D o r o t h e e , Peggy , P a t , P a u l , J i m , and B r u c e p r o v i d e d c o u n t l e s s good t i m e s w i t h many p r o v o c a t i v e d i s c u s s i o n s o v e r t o n s o f Dim Sum l u n c h . The t e c h n i c a l a s s i s t a n c e o f M r s . S t i c h and C h a r l o t t e was w i t h o u t equa l and g r e a t l y a p p r e c i a t e d . I t h a n k D r s . B r u ce Dunn and G l e n n Slemmer who p r o v i d e d c r i t i c a l comments and h e l p f u l d i s c u s s i o n d u r i n g t h e w r i t i n g o f t h e t h e s i s . F o r h i s s u p p o r t , I t h a n k D r . R.L. N o b l e , D i r e c t o r o f t h e C a n c e r R e s e a r c h C e n t r e , UBC, where t h i s r e s e a r c h was c o n d u c t e d . I am g r a t e f u l t o D r . E. F a r b e r and c o - w o r k e r s , F e l s R e s e a r c h I n s t i t u t e , P h i l a d e l p h i a , f o r t h e i r g r a d i e n t t e c h n i q u e d e s i g n e d f o r i n v i v o s t u d i e s , and t o J i m K o r o p a t n i c k f o r h i s a s s i s t a n c e w i t h t h e i n v i v o e x p e r i m e n t s . The s u p p o r t o f t h i s r e s e a r c h by t h e N a t i o n a l C a n c e r I n s t i t u t e o f Canada , i n t h e fo rm o f g r a n t s t o D r . S t i c h and a p e r s o n a l R e s e a r c h F e l l o w s h i p , i s g r a t e f u l l y a c k n o w l e d g e d . xx i i i ABBREVIATIONS C h e m i c a l C a r c i n o g e n s AAF - 2 - a c e t y I a m i n o f I u o r e n e A f l a . B I - a f l a t o x i n Bl 4AQ0 - 4 - a m i n o q u i n o I i n e I - o x i d e BA - b e n z ( a ) a n t h r a c e n e B A - e p o x i d e - e p o x i d e o f b e n z ( a ) a n t h r a c e n e BP - b e n z ( a ) p y r e n e DMN - d i m e t h y I n i t r o s a m i n e N - a c e t o x y - A A F - N - a c e t o x y - 2 - a c e t y I am i n o f I u o r e n e N -hyd roxy -AAF - N - h y d r o x y - 2 - a c e t y I a m i n o f I u o r e n e 4NQ0 - 4 - n i t r o q u i n o I i n e I - o x i d e MCA - m e t h y I c h o l a n t h r e n e M C A - e p o x i d e - e p o x i d e o f m e t h y I c h o l a n t h r e n e S u b c e l l u l a r F r a c t i o n s 9S - p o s t - m i t o c h o n d r i a I ( 9 , 000 x g) s u p e r n a t a n t , " c r u d e m i c r o s o m e s " I05P - 105,000 x g p e l l e t t , "washed m i c r o s o m e s " I05S - 105,000 x g s u p e r n a t a n t , c y t o s o l , " s o l u b l e f r a c t i o n " M i s c e l l a n e o u s Terms ADM - a r g i n i n e - d e f i c i e n t medium ASG - a l k a l i n e s u c r o s e g r a d i e n t E D T A / s a l i n e b u f f e r - 0 .024 M EDTA/0.075 M NaC I , pH 7.5 G6P - g I u c o s e - 6 - p h o s p h a t e 3 H-TdR - t r i t i a t e d t h y m i d i n e ( t h y m i d i n e - m e t h y I - 3 H ) i . p . - i n t r a p e r i t o n e a l MEM - E a g l e ' s m i n i m a l e s s e n t i a l medium NADP - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( o x i d i z e d f o rm) xx i v NADPH - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e pho spha te ( r e d u c e d fo rm) PBS - D u l b e c c o ' s p ho spha te b u f f e r e d s a l i n e P B S / s u c r o s e - 0 .25 M s u c r o s e i n p h o s p h a t e - b u f f e r e d s a l i n e , pH 7.5 s u b c u . - s u b c u t a n e o u s TLC - t h i n - l a y e r c h r oma tog r aphy UV - u I t r a - v i o l e t XP - x e r ode rma p igmentosum XPe - xe rode rma p i gmentosum, Edmonton p a t i e n t A l l o t h e r a b b r e v i a t i o n s and n o m e n c l a t u r e were used i n a c c o r d a n c e w i t h t h e p o l i c y o f t h e B i o c h e m i c a l J o u r n a l ( B i o chem. J . (1972) 126, 1 -19) . I INTRODUCTION EVIDENCE FROM CANCER EPIDEMIOLOGY M o r t a l i t y s t a t i s t i c s , c o m p i l e d i n t h e U n i t e d S t a t e s o v e r t h e p a s t s e v e n t y y e a r s , i n d i c a t e t h a t c a n c e r has moved f r om s i x t h t o s econd p l a c e as a d i s e a s e c a u s e o f d e a t h (U.S . D e p t . o f H.E.W., 1973) . These r e p o r t s have a l s o shown a s t e a d y i n -c r e a s e i n c a n c e r i n c i d e n c e and m o r t a l i t y i n v i r t u a l l y e v e r y age g r o u p , and i m p l y t h a t t h e r i s e i n t umor i n c i d e n c e c o u l d be a t t r i b u t e d t o an a c c u m u l a t i o n o f c a r c i n o g e n s i n t h e e n v i r -onment. The i n c i d e n c e o f common c a n c e r s i n ma l e s and f e m a l e s o f v a r i o u s p o p u l a t i o n s i s shown i n T a b l e I ( Seg i e_t a j _ . , 1969 ) . T a b l e I. Death r a t e p e r 100,000 p o p u l a t i o n f o r m a l i g n a n t neop la sms i n 1964-65. ( A b r i d g e d f r om Seg i e t a l . , 1969) M a l e Fema1e Stomach 1 n t e s t i ne L i v e r B r e a s t U t e r u s U.S. w h i t e 9 14 5 22 10 U.S. n o n - w h i t e 18 1 1 7 20 23 E n g l a n d , Wales 23 12 3 24 10 J apan 69 3 15 4 14 The s t r i k i n g d i f f e r e n c e s i n i n c i d e n c e s a r e o b v i o u s , f o r e x a m p l e , 2 between s tomach c a n c e r i n U.S. ma l e s and J a p a n e s e m a l e s . Such d i f f e r e n c e s c o u l d have t h e i r o r i g i n s i n g e n e t i c o r e n v i r -onmenta l f a c t o r s . Tha t c a n c e r e t i o l o g y s h o u l d f o c u s on e n v i r o n m e n t a l s t i m u l i has been r e i n f o r c e d t o a g r e a t e x t e n t by e p i d e m i o l o g i c s t u d i e s o f m i g r a n t p o p u l a t i o n s . These s u r v e y s have d e m o n s t r a t e d t h a t c a n c e r p a t t e r n s i n i m m i g r a n t s t e n d t o assume t h o s e o f t h e h o s t c o u n t r y , r a t h e r t h a n t h o s e o f t h e c o u n t r y o f o r i g i n ( H a e n s z e l , 1961; S t a s z e w s k i and H a e n s z e l , 1965; Haen s ze l and K u r i h a r a , 1968) . F o r e x a m p l e , a s t u d y o f t h e i n c i d e n c e o f c a n c e r s a t s e v e r a l s i t e s i n U.S. w h i t e m a l e s , J a p a n e s e m a l e s , and t h e N i s e i , who a r e s e c o n d - g e n e r a t i o n J a p a n e s e i m m i g r a n t s i n N o r t h A m e r i c a , d e m o n s t r a t e d t h a t t h e N i s e i had i n c i d e n c e s a p p r o x i m a t e l y midway between t h o s e o f t h e J a p a n e s e and U.S. ma le s ( T a b l e 2 ) ( H a e n s z e l and K u r i h a r a , 1968). T h i s o b s e r v a t i o n no t o n l y l e a d s t o T a b l e 2. R e l a t i v e s t a n d a r d i z e d m o r t a l i t y r a t i o s f o r c a n c e r a t v a r i o u s s i t e s . M o r t a l i t y i n m i g r a n t and s t a t i o n a r y p o p u l a t i o n s . ( A b r i d g e d f r om Haen s ze l and K u r i h a r a , 1968) J a p a n e s e N i s e i U.S. w h i t e s Stomach 100 38 17 1 n t e s t i nes 100 290 490 Lung, b r onchu s 100 170 320 t h e f i r m c o n c l u s i o n t h a t t h e m a j o r f a c t o r s d e t e r m i n i n g t h e s e 3 i n c i d e n c e s a r e e n v i r o n m e n t a l r a t h e r t h a n g e n e t i c , bu t i n f e r s t h a t t h e s e f a c t o r s a r e p r i m a r i l y c h e m i c a l i n n a t u r e . F u r t h e r m o r e , t h e i d e n t i f i c a t i o n o f o c c u p a t i o n a l c a n c e r s , such as 2 - naph thy I a m i n e - i n d u c e d b l a d d e r c a n c e r ( B o n s e r , 1967) , and i a t r o g e n i c c a n c e r s , s uch as t h o s e i n d u c e d by a l k y l -a t i n g d rugs (Roe, 1966) , have i n d i c a t e d t h a t g e n e t i c f a c t o r s a r e r e l a t i v e l y i n s i g n i f i c a n t . E s t i m a t e s o f t h e p r o p o r t i o n o f human c a n c e r s w i t h a p o s s i b l e c h e m i c a l e t i o l o g y run as h i g h as 9 0 $ ( H i g g i n s o n , 1969; Boy l a n d , 1969) . CHEMICAL CARCINOGENESIS IN HUMANS D u r i n g t h e p a s t f i f t y y e a r s , s t u d i e s i n t h e f i e l d o f c h e m i c a l c a r c i n o g e n e s i s have c o n t i n u a l l y r e v e a l e d new c h e m i c a l a g e n t s c a p a b l e o f i n d u c i n g t umor s i n many a n i m a l s p e c i e s . Such a l i s t o f c h e m i c a l s i s f a r t o o long t o p e r m i t even a c u r s o r y s u r v e y ( r e v i e w e d by : Heupe r , 1966; M i l l e r , 1970; Magee, 1972; I .A.R.C. Monog raph , 1972) . However , i t i s i n s t r u c t i v e t o d e v e l o p a scheme whereby a l l such c h e m i c a l s can be r e l a t e d t o c a r c i n o g e n e s i s i n humans. By u s i n g t h e c h e m i c a l i n d u c t i o n o f c a n c e r i n humans as a b a s e l i n e , t h r e e m a j o r c a t e g o r i e s o f c h e m i c a l s can be d e s c r i b e d , a l l o f w h i c h c o n t a i n c h e m i c a l s t h a t a r e d e f i n e d as c a r c i n o g e n s on t h e b a s i s o f l a b o r a t o r y t e s t s : I. E s t a b l i s h e d c a r c i n o g e n s i n c l u d e t h o s e c h e m i c a l s f o r w h i c h m e d i c a l , e p i d e m i o l o g i c , p a t h o l o g i c , and e x p e r i m e n t a l e v i d e n c e i s s u b s t a n t i a l enough t o e s t a b l i s h c a u s a l r e l a t i o n s between human e x p o s u r e s t o t h e s e a g e n t s and t h e s u b s e q u e n t A deve l opment o f c a n c e r s . I n c l u d e d as e s t a b l i s h e d c h e m i c a l c a r -c i n o g e n i c s t i m u l i i n humans a r e no t o n l y pu re c h e m i c a l s such as 2 - naph thy I am ine , 4 - a m i n o b i p h e n y I , and n i c k e l , bu t a l s o v a r i e d c r u d e c h e m i c a l m i x t u r e s s uch as s o o t , c o a l t a r s , p e t r o l e u m r e s i d u e s , and a r o m a t i c amines ( r e v i e w e d by : Heupe r , 1966; H i g g i n s o n , 1969) . These a g e n t s were i d e n t i f i e d t h r o u g h t h e o b s e r v a t i o n o f h i g h r i s k g r o u p s t h a t were l o c a t e d m a i n l y i n o c c u p a t i o n a l s e t t i n g s - a f e a t u r e w h i c h a l l o w e d p r e v e n t a t i v e measures t o be e x e r c i s e d w i t h r e l a t i v e e a s e . 2 . S u s p e c t e d c a r c i n o g e n s i n c l u d e t h o s e c h e m i c a l s f o r w h i c h human e x p o s u r e i s u n e q u i v o c a l l y e s t a b l i s h e d , but where t h e v a r i o u s l i n e s o f e v i d e n c e do no t w a r r a n t t h e e s t a b l i s h -ment o f a c a u s a l r e l a t i o n between human e x p o s u r e s t o t h e a g e n t s and t h e s u b s e q u e n t deve l opmen t o f c a n c e r s . A g r o w i n g l i s t o f s u s p e c t e d c a r c i n o g e n s i n c l u d e s such a g e n t s as b e r y l l i u m , i r o n o x i d e , and p y r o l y s i s p r o d u c t s o f : c a r b o n , p e t r o l e u m , wood, and t o b a c c o . M o r e o v e r , w i t h i n t h e p a s t f i f t e e n y e a r s , a v a s t a r r a y o f n a t u r a l l y - o c c u r r i n g c h e m i c a l s has become s u s p e c t enough t o p r o v o k e s e r i o u s c o n s i d e r a t i o n o f t h e h y p o t h e s i s t h a t t h e m a j o r i t y o f human c a n c e r s a r e i nduced by e n v i r o n m e n t a l c h e m i c a l s . F o r e x a m p l e , m y c o t o x i n s , w h i c h a r e t o x i c mold m e t a b o l i t e s , a r e c u r r e n t l y unde r e x t e n s i v e s c r u t i n y s i n c e t h e d i s c o v e r y o f a f l a t o x i n s f o l l o w i n g mass o u t b r e a k s o f p o i s o n i n g i n p o u l t r y ( r e v i e w e d by : I .A.R.C. Monog raph , 1972; Wogan, l 9 7 3 ) ( F i g u r e I ) . The t remendous i m p o r t a n c e o f a f l a t o x i n s as p o t e n t i a l p u b l i c h e a l t h h a z a r d s became c l e a r when i t was r e c o g n i z e d t h a t t h e s e t o x i c a g e n t s were p r o d u c e d by A spe r g i I I us f I a v u s , an u b i q u i t o u s f ungu s 5 c l o s e l y l i n k e d t o f o o d s p o i l a g e . AFLATOXIN Bl AFLATOXIN B2 STERIGMATOCYSTIN F i g u r e I. S t r u c t u r e s o f t h e a f l a t o x i n s and s t e r i g m a t o c y s t i n . A f l a t o x i n B l , a f l a t o x i n G l , and s t e r i g m a t o c y s t i n have been known t o c o n t a m i n a t e human f o o d s ( K r a y b i l l and S h a p i r o , 1969; P u r c h a s e and Van de r W a t t , 1970; Shank e t aj_. , 1972; Wogan, 1973). W i t h t h e knowledge t h a t t h e l i v e r i s t h e main t a r g e t t i s s u e i n many a n i m a l s p e c i e s f o r bo th t h e t o x i c and c a r c i n o g e n i c a c t i o n s o f t h e a f l a t o x i n s ( Wogan and Newberne, 1967; Newberne and B u t l e r , 1969; Wogan, 1973) , t h e p o s s i b l e r o l e s o f t h e a f l a -t o x i n s and s t e r i g m a t o c y s t i n a r e b e i n g i n v e s t i g a t e d w i t h r e g a r d t o t h e e t i o l o g y o f human l i v e r c a n c e r i n c e r t a i n r e g i o n s o f t h e w o r l d ( O e t t l e , 1965; Wogan, 1968; Shank e t al_. , 1972; Wogan, 1973) . D i r e c t e v i d e n c e t o s u p p o r t t h e h y p o t h e s i s , t h a t 6 a f l a t o x i n s may be i n v o l v e d as c a u s a t i v e a g e n t s i n human l i v e r c a n c e r ( O e t t l e , 1965) , i s l i m i t e d and m a i n l y d e r i v e s f r om s t u d i e s i n A f r i c a and A s i a where d a t a on i n c i d e n c e and d i s t r i b u t i o n o f p r i m a r y l i v e r c a n c e r c o r r e l a t e s w i t h t h e f r e q u e n c y o f a f l a t o x i n c o n t a m i n a t i o n i n f o o d s . F u r t h e r m o r e , t h e d a t a have s t i m u l a t e d more i n v e s t i g a t i o n o f t h e p u b l i c h e a l t h h a z a r d imposed by o t h e r m y c o t o x i n s . F o r e x a m p l e , t h e p r o d u c t i o n o f t h e f u n g a l t o x i n l u t e o s k y r i n on " y e l l o w e d r i c e " has been i m p l i c a t e d i n t h e i n -d u c t i o n o f l i v e r t umor s i n A s i a n s ( r e v i e w e d by M i l l e r , 1973) . 3. P o t e n t i a l c a r c i n o g e n s i n c l u d e t h o s e c h e m i c a l s f o r w h i c h human e x p o s u r e i s no t e s t a b l i s h e d , b u t where l a b o r -a t o r y t e s t i n g has r i g o r o u s l y d e m o n s t r a t e d t h e c a r c i n o g e n i c i t y o f t h e t e s t c h e m i c a l . The m a j o r i t y o f p o t e n t i a l c a r c i n o g e n s a r e a c t u a l l y s y n t h e t i c a l l y - d e r i v e d c h e m i c a l s and a r e g e n e r a l l y no t e n c o u n t e r e d n a t u r a l l y . An examp le i s 2 - a c e t y I a m i n o f I u o r e n e (AAF, F i g u r e 2 ) , a s y n t h e t i c a r o m a t i c amine o r i g i n a l l y p a t e n t e d as a p r i n c i p l e i n g r e d i e n t f o r an i n s e c t i c i d e . F o l l o w i n g t h e l a b o r a t o r y t e s t s w h i c h r e v e a l e d t h e c a r c i n o g e n i c p r o p e r t y o f AAF, t h e c h e m i c a l was i m m e d i a t e l y banned f r om t h e m a r k e t . A d i f f e r e n t c l a s s o f s y n t h e t i c compounds, w i t h s t r u c t u r e s ba sed H N - C - C H o II J • F i g u r e 2. S t r u c t u r e o f 2 - a c e t y I a m i n o f I u o r e n e (AAF) 7 on t h e p a r e n t compound, 4 - n i t r o q u i n o l i n e l - o x i d e ( F i g u r e 3 ) , show v a r y i n g d e g r e e s o f c a r c i n o g e n i c i t y . A l t h o u g h d i s c o n t i n u e d as an e f f i c i e n t f u n g i c i d e , 4 - n i t r o q u i n o I i n e l - o x i d e and i t s i s omer s and d e r i v a t i v e s p r o v i d e a s upe rb s e r i e s o f e x p e r i m e n t a l c h e m i c a l c a r c i n o g e n s . T h i s i s n o t o n l y a t t r i b u t a b l e t o t h e v a r y i n g deg ree s o f c a r c i n o g e n i c i t y , b u t a l s o t o t h e h i g h s o l -u b i l i t i e s i n p h y s i o l o g i c s o l u t i o n s as w e l l as t h e c o m p a r a t i v e l y s i m p l e s t r u c t u r e s o f t h e compounds. • F i g u r e 3. S t r u c t u r e o f 4 - n i t r o q u i n o I i n e l - o x i d e (4NQ0) EXPERIMENTAL CARCINOGENESIS A l t h o u g h t h e l i s t o f p o t e n t i a I c h e m i c a l c a r c i n o g e n s i s i ndeed l o n g , i t i s i m p o r t a n t t o n o t e t h a t t h e use o f t h e I a b e I " c a r c i n o g e n " d e r i v e s f r om t h e a b i l i t y o f t h e t e s t c h e m i c a l t o i n duce a tumor i n a l a b o r a t o r y a n i m a l under a s t a n d a r d s e t of c o n d i t i o n s ( I . A . R . C . Monog raph , 1972) . The f i r s t chem-i c a l l y i nduced t u m o r , i n t h e l a b o r a t o r y , was a c h i e v e d by Yamagiwa and I c h i k awa i n 1915 by a p p l y i n g c o a l t a r on t h e e a r s o f r a b b i t s (Yamagiwa and I c h i k a w a , 1915) . By r e p e a t e d 8 a p p l i c a t i o n , t h e s e i n v e s t i g a t o r s i n d u c e d m a l i g n a n t s k i n t u m o r s , o r e p i t h e l i o m a s . C u r r e n t r e p o r t s o f c a r c i n o g e n i c i t y t e s t r e s u l t s a r e c a r e f u l t o s p e c i f y t h e r o u t e o f a d m i n i s t r a t i o n o f t h e c h e m i c a l s i n c e compounds can be i n j e c t e d s u b c u t a n e o u s I y , i n t r a p e r i t o n e a I l y , o r i n t r a m u s c u l a r l y , o r even f e d o r i n h a l e d as w e l l as b e i n g a p p l i e d t o t h e s k i n . A l t h o u g h t h e e x p e r i m e n t a l c h e m i c a l s may p r o d u c e t umor s a t s e v e r a l s i t e s ( W e i s b u r g e r and W e i s b u r g e r , - 1967 ) , i t i s more common t o o b s e r v e some deg ree o f o r g a n - s p e c i f i c i t y f o r t umor i n d u c t i o n , c a l l e d " o r g a n o t r o p y " , w h i c h can be d r a s t i c a l l y a l t e r e d by t h e r o u t e o f a d m i n i s t r a t i o n ( S t e w a r t , 1967; W e i s b u r g e r and W e i s b u r g e r , 1967) . PHARMACOLOGY OF CHEMICAL CARCINOGENS (a ) D i s t r i b u t i o n o f A p p l i e d C h e m i c a l s The r o u t e o f a d m i n i s t r a t i o n o f t e s t c h e m i c a l s c an be o f paramount i m p o r t a n c e i n e s t a b l i s h i n g t h e i r m e t a b o l i c f a t e . T h i s becomes p a r t i c u l a r l y a p p a r e n t when t h e a p p l i e d c h e m i c a l i s r a p i d l y m e t a b o l i z e d i n t h e l i v e r s i n c e e n t e r a l a d m i n i s t r a t i o n a l l o w s a h i g h e r c o n c e n t r a t i o n o f i n i t i a l s u b s t a n c e t o r e a c h t h e l i v e r v i a t h e h e p a t i c - p o r t a l s y s t e m . On t h e o t h e r hand , where b i o t r a n s f o r m a t i o n i s a s s o c i a t e d w i t h " d e t o x i c a t ! o n " , c h e m i c a l s w i l l be more s l o w l y d e t o x i f i e d a f t e r p a r e n t e r a l admi n i s t r a t i o n . In o r d e r t o c l a r i f y t h e i n t e r r e l a t i o n s between t h e p r o c e s s e s o f e n t r y , d i s t r i b u t i o n , and e x c r e t i o n o f c h e m i c a l c a r c i n o g e n s in mammals, t h e v a r i o u s r o u t e s t h a t can be f o l l o w e d 9 by an a d m i n i s t e r e d c h e m i c a l a r e o u t l i n e d i n F i g u r e 4 . F o r e x a m p l e , a d o r s a l s u b c u t a n e o u s i n j e c t i o n o f a c h e m i c a l wou l d a l l o w t h e f o l l o w i n g pathways t o e v o l v e : 1. The c h e m i c a l w o u l d e n t e r t h e b l o o d s t r e a m and c i r c u l a t e d i r e c t l y t o a l l t h e body t i s s u e s , i n c l u d i n g t h e k i dneys and I i v e r . 2 . If t h e compound i s h i g h l y p o l a r , t h e k i d n e y s c o u l d e x t r a c t much o f t h e c h e m i c a l d i r e c t l y f o r e x c r e t i o n . 3. The c h e m i c a l c o u l d be m e t a b o l i z e d i n t h e l i v e r , and t h e n : (a ) be e x c r e t e d i n t h e b i l e t o t h e g a s t r o - i n t e s t i n a I ( G . I . ) t r a c t where f u r t h e r r e a c t i o n s m i g h t o c c u r , o r (b) be e x c r e t e d i n t h e b i l e t o t h e G . I . t r a c t o n l y t o be r e a b s o r b e d and r e t u r n t o t h e l i v e r and t i s s u e s f o r p o s s i b l e f u r t h e r m e t a b o l i s m , o r ( c ) c i r c u l a t e f r om t h e l i v e r d i r e c t l y t o t h e t i s s u e s f o r p o s s i b l e r e a b s o r p t i o n o r p o s s i b l e e x c r e t i o n . S i m i l a r r o u t e s can be e n v i s a g e d f o r i . p . o r o r a l a d m i n i s t r a t i o n o f a c h e m i c a l a n d , u l t i m a t e l y , a p r o c e s s o f m o b i l e e q u i l i b r i u m i s s e t up. T h i s i n v o l v e s i n t e r r e l a t e d , i n t e r d e p e n d e n t , and c o n t i n u o u s p r o c e s s e s i n wh ich t h e compound-i s c i r c u l a t e d , a b s o r b e d by t i s s u e s , r e l e a s e d , and r e - c i r c u l a t e d . The d i s t r i b u t i o n f i n a l l y a p p r o a c h e s c e s s a t i o n when t h e compound i s c o m p l e t e l y e l i m i n a t e d f rom t h e body , u s u a l l y p a r t l y b i o t r a n s -f o r m e d . f e e d i n g s u b c u . T I S S U E S kB L O O D L I V E R KIDNEYS! B L A D D E R S T O M A C H t i. p. 1 S M A L L I N T E S T I N E C O L O N R E C T U M V F i g u r e 4 . S c h e m a t i c r e p r e s e n t a t i o n o f t h e p o s s i b l e r o u t e s f o l l o w e d by a c h e m i c a l c a r c i n o g e n a d m i n i s t e r e d t o an a n i m a l e i t h e r e n t e r a l l y ( f e e d i n g ) o r p a r e n t e r a l l y ( i . p . = i n t r a -p e r i t o n e a l , s u b c u . = s u b c u t a n e o u s ) ( Adap ted f rom W e i s b u r g e r , 1972 ) . 11 (b) B i o t r a n s f o r m a t i o n o f F o r e i g n Compounds i n t h e An ima l Body In b road t e r m s , a f o r e i g n compound w i t h i n t h e a n i m a l body has t h r e e p o s s i b l e f a t e s : 1. I t c o u l d be e x c r e t e d e n t i r e l y unchanged - w h i c h i s t r u e f o r many h i g h l y p o l a r compounds, o r 2 . I t c o u l d unde rgo s p o n t a n e o u s r e a c t i o n s t o p r o d u c t s w i t h o u t t h e m e d i a t i o n o f enzymes , o r 3. I t c o u l d be t r a n s f o r m e d t o p r o d u c t s w i t h t h e m e d i a t i o n o f enzymes . B i o c h e m i c a l l y , t h e m e t a b o l i s m d e s c r i b e d i n f a t e #3 can be g r ouped i n t o f o u r b a s i c t y p e s o f r e a c t i o n s : ( a ) o x i d a t i o n s , (b) r e d u c t i o n s , ( c ) h y d r o l y s e s , and ( d ) c o n j u g a t i o n s ( r e v i e w e d by H u t s o n , 1970) . P h y s i o l o g i c a l l y , i t s h o u l d be e m p h a s i z e d t h a t t h e s e r e a c t i o n s can p o s s i b l y o c c u r t o v a r y i n g d e g r e e s t h r o u g h o u t a l l t i s s u e s o f t h e body ( r e v i e w e d by Hu t son , I 9 7 0 ) . M o r e o v e r , m u l t i - s t e p t r a n s f o r m a t i o n s can p o s s i b l y o c c u r a t one o r more l o c a l e s ( r e v i e w e d by : H u t s o n , 1 9 7 0 ; I r v i n g , [970; M i l l e r , 1970 ) . F o r e x a m p l e , an o x i d a t i o n p r o d u c t fo rmed i n t h e l i v e r m i g h t s u b s e q u e n t l y undergo c o n j u g a t i o n i n t h e i n t e s t i n e ( I r v i n g , 1970) . A l l p o s s i b l e p e r m u t a t i o n s and c o m b i n a t i o n s must be c o n s i d e r e d when e v a l u a t i n g t h e m e t a b o l i c f a t e o f f o r e i g n compounds. ( c ) B i o t r a n s f o r m a t i o n o f C h e m i c a l P r e c a r c i n o g e n s In c h e m i c a l c a r c i n o g e n e s i s , t h e t e r m " p r e c a r c i n o g e n " r e f e r s t o a c h e m i c a l t h a t , upon i n j e c t i o n i n t o a mammalian body , i s m e t a b o l i z e d t o one o r more p r o d u c t s w h i c h t h e n r e a c t w i t h t i s s u e m a c r o m o l e c u l e s and r e s u l t i n t h e p r o d u c t i o n o f a n e o -p l a sm ( r e v i e w e d by M i l l e r and M i l l e r , 1969 a , b ; M i l l e r , 1970) . In o t h e r w o r d s , t h e c h e m i s t r y o f t h e p r e c a r c i n o g e n i s such t h a t t h e compound i s u n r e a c t i v e t o w a r d s c e l l u l a r m a c r o m o l e c u l e s , under p h y s i o l o g i c c o n d i t i o n s , u n t i l t r a n s f o r m e d t o an a c t i v e m e t a b o I i t e . I t i s e s s e n t i a l t o s u r v e y some c l a s s i c e xamp le s o f t h e b i o t r a n s f o r m a t i o n s o f c h e m i c a l p r e c a r c i n o g e n s i n o r d e r t o d e m o n s t r a t e t h e f u l l i m p l i c a t i o n s o f m e t a b o l i s m i n c h e m i c a l c a r c i n o g e n e s i s . C e r t a i n c h e m i c a l c a r c i n o g e n s , such as s y n t h e t i c N - m e t h y I - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) a r e s p o n t a n e o u s l y r e a c t i v e t o w a r d s c e l l u l a r m a c r o m o l e c u l e s and show h i g h d e g r e e s o f r e a c t i v i t y w i t h DNA, f o r e x a m p l e , even s o l u t i o n i n v i t r o ( M c C a l l a , 1968) . On t h e o t h e r h a n d , most o f t h e s y n t h e t i c and n a t u r a l l y - o c c u r r i n g c h e m i c a l c a r c i n o g e n s , such as t h e p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s , r e q u i r e m e t a b o l i c c o n v e r s i o n t o r e a c t i v e i n t e r m e d i a t e s p r i o r t o e n g a g i n g i n c h e m i c a l r e a c t i o n s w i t h DNA. The c a r c i n o g e n s employed i n t h e s t u d i e s r e p o r t e d h e r e i n f a l l i n t o t h e l a t t e r c a t e g o r y and t h e r e f o r e t h e m a j o r c o n c e p t s c o n c e r n i n g t h e i r m e t a b o l i c a c t i v a t i o n w i l l be s u r v e y e d . ( i ) A r o m a t i c Amines A c l a s s i c examp le o f a p r e c a r c i n o g e n i s 2 - a c e t y I a m i n o -f I u o r e n e ( A A F ) , t h e m e t a b o l i s m o f w h i c h was l a r g e l y worked o u t i n t h e l a b o r a t o r y o f James and E l i z a b e t h M i l l e r ( r e v i e w e d by M i l l e r and M i l l e r , 1969 a,b ; M i l l e r , 1970). 13 • C — C H j II o Precarcinogen AAF. N-hydroxy-AAF Proximate Carcinogen N-acetoxy-AAF Ultimate Carcinogen F i g u r e 5. P r o p o s e d b i o t r a n s f o r m a t i o n o f AAF (Adap ted f r om M i l l e r , The m e t a b o l i t e o f AAF, N - h y d r o x y - 2 - a c e t y I a m i n o f I u o r e n e ( N -h y d r o x y - A A F ) , was f i r s t i s o l a t e d f r om t h e u r i n e o f r a t s t h a t were f e d AAF by Cramer and t h e M i l l e r s i n I960 (Cramer e t a I., I 9 60 ) . The t e r m " p r o x i m a t e c a r c i n o g e n " was c o i n e d by t h e M i l l e r s t o d e s c r i b e t h e N - h y d r o x y I a t e d fo rms o f c a r c i n o g e n i c a m i n e s . The t e r m d e s i g n a t e s s u b s t a n c e s t h a t a r e more c l o s e l y r e l a t e d t o t h e a c t u a l f o rms o f t h e c a r c i n o g e n s t h a n a r e t h e p a r e n t am ine s . In f a c t , s y n t h e t i c a l l y p r e p a r e d N -hyd roxy compounds have been d e m o n s t r a t e d t o be more c a r c i n o g e n i c t h a n t h e i r p a r e n t compounds ( r e v i e w e d by M i l l e r and M i l l e r 1969 a , b ; M i l l e r , f o rm hyd roxy I amine d e r i v a t i v e s i s an o x i d a t i v e r e a c t i o n t h a t i s c a t a l y s e d by t h e m i c r o s o m a l f r a c t i o n s d e r i v e d f rom l i v e r , l u n g , and bI a d d e r mucosa ( U e h l e k e , 1966) . O x i d a t i o n , i n g e n e r a l , 1970 ) . 1970) . The N - h y d r o x y I a t i o n r e a c t i o n o f a r o m a t i c amines t o 14 i s a common pathway o f m e t a b o l i s m o f f o r e i g n o r g a n i c compounds. A l t h o u g h many o x y g e n a s e s a r e p r e s e n t i n most t i s s u e s , t h e e n d o -p l a s m i c r e t i c u l u m o f c e r t a i n c e l l s , e x p e c i a l l y t h e h e p a t i c pa renchyma l c e l l s , c o n t a i n s a comp lex s e t o f enzymes w h i c h c a t a l y z e t h e h yd roxy I a t i on o f o r g a n i c compounds. W i t h m o l e c u l a r oxygen and an e l e c t r o n d o n o r , NADPH, b e i n g r e q u i r e d f o r t h e r e a c t i o n , a g e n e r a l r e a c t i o n may be w r i t t e n a s : R -CH 3 + NADPH + H + + 0 2 - R-CH 2 0H + NADP + + H 2 0 One o f t h e atoms o f oxygen i s i n c o r p o r a t e d i n t o t h e s u b s t r a t e R - C H 3 , and t h e o t h e r atom o x i d i z e s NADPH and i s r educed t o w a t e r . W i t h r e s p e c t t o t h i s mechan i sm, t h e s e enzymes have been t e rmed " m i x e d f u n c t i o n o x i d a s e s " (Mason, 1957) a n d , more c o n c i s e l y , " m o n o - o x y g e n a s e s " ( H a y a i s h i , 1969 ) . A l t h o u g h n o t a t y p i c a l mono-oxygenase enzyme i n a s t r i c t s e n s e , t h e N -hydroxy I a t i n g enzyme does r e q u i r e NADPH and o x y g e n . Such o x i d a t i o n s , c a t a l y z e d by enzymes i n t h e e n d o p l a s m i c r e t i c u l u m , have been c h a r a c t e r i z e d a t v a r i o u s l e v e l s : t h e i n t a c t a n i m a l , p e r f u s e d o r g a n s o r t i s s u e s l i c e s , and m i c r o s o m e s . F u r t h e r m o r e , t h e many t y p e s o f m i c r o s oma l o x i d a t i o n r e a c t i o n s , as w e l l as c h a r a c t e r i s t i c s o f t h e m i c r o s o m a l e l e c t r o n t r a n s p o r t c h a i n , have been summar i zed ( r e v i e w e d by H u t s o n , 1970) . A f u r t h e r m e t a b o l i c s t e p i n t h e b i o t r a n s f o r m a t i o n o f AAF, namely t h e e s t e r i f i c a t i o n o f N - h y d r o x y - A A F , i s b e l i e v e d t o o c c u r i n v i v o i n r a t l i v e r w i t h t h e f o r m a t i o n o f a s u l f a t e o r g l u c u r o n i d e e s t e r ( I r v i n g , 1972) . T y p i c a l c o n j u g a t i o n r e a c t i o n s , i n e s s e n c e , i n v o l v e t h e c h e m i c a l l i n k i n g o f f o r e i g n compounds and m e t a b o l i t e s w i t h endogenous s u b s t r a t e s t o f o rm t h e s o - c a l l e d c o n j u g a t e s . In f a c t , a f i r m c o n n e c t i o n between i n v i t r o r e a c t i v i t y and i n v i vo c a r c i n o g e n i c i t y c o u l d o n l y be e s t a b l i s h e d f o r AAF when t h e n e w l y - a d d e d h yd roxy I g r oup was e s t e r i f i e d ( F i g u r e 5 ) . In o t h e r w o r d s , o n l y t h e " u l t i m a t e " c a r c i n o g e n , N - a c e t o x y - A A F is. a b l e t o b i n d , f o r e x a m p l e , w i t h g u a n o s i n e i n v i t r o and t h e s e same, b i n d i n g p r o d u c t s a r e i s o l a t e d f r o m r a t l i v e r i n v i v o a f t e r f e e d i n g w i t h AAF. M o r e o v e r , c o n j u g a t e s o f N -hyd roxy compounds a r e t h e o n l y known m e t a b o l i c a I l y - f o r m e d c o n j u g a t e s t h a t d i s p l a y chemica I r e a c t i v i t y , w i t h o u t enzyme m e d i a t i o n , w i t h t i s s u e macromoLecu Ies l e a d i n g t o t h e f o r m a t t o n o f c o v a I e n t bonds unde r p h y s i o l o g i c c o n d i t i o n s ( r e v i e w e d by: M i l l e r and M i l l e r , 1969 a „ b ; M i l l e r , 1970; I r v i n g , I 97Q) . ( i i ) N i t r o q u i n o I i ne N -Ox ide s E x i s t i n g i n a m o s t h i g h I y o x M i z e d s t a t e , 4N.Q0 c o n s t i t u t e s a w i d e l y - s t u d i e d example, o f a c a r c i n o g e n t h a t i s e n z y m a t i c a I Iy r educed t o i t s . p r o x i m a t e c a r c i n o g e n i c f o r m ("Figure 6 ) , 4 - h yd r o x yam i noqu i no I i n e l - o x - i de (4HAQ0) ( Sug imura e t a l 1.965; Kawa.zoe e t a j _ . , 1972 ) . T h e r e has been c o n s i d e r a b l e s p e c u l a t i o n , based on s o l i d c h e m i c a l d a t a , as t o t h e s ub sequen t r e a c t i v e s t e p s of 4HAQ0 i n v i vo as w e l l as p r o p o s a l s f o r d i r e c t a c t i o n o f 4N.Q0 i n i n d u c i n g a l t e r a t i o n s i n ce l . I u l a r macromo lecu l e s ( Pau l and Montgomery , 1971; Kawazoe e t a I., 1972; Matsuyama and N a g a t a , 1972 ) . W i t h e x p e r i m e n t a l @viidance s t r o n g l y i n f a v o u r o f t h e c o n v e r s i o n o f 4N.Q0 to . 4HAQ0 as an i n i t i a l m e t a b o l i c s t e p , 16 F i g u r e 6. P r o p o s e d b i o t r a n s f o r m a t i o n o f 4NQ0 (Adap ted f r om Kawazoe e_t a j _ . , 1972) . i t i s becoming i n c r e a s i n g l y a p p r o p r i a t e t o r e f e r t o 4NQ0 as a " p r e c a r c i n o g e n " . ( i i i ) H y d r o c a r b o n s The g e n e r a t i o n o f e I e c t r o p h i I i c c h e m i c a l s p e c i e s f r om a r o m a t i c h y d r o c a r b o n c a r c i n o g e n s has been d e m o n s t r a t e d t o o c c u r v i a e p o x i d e i n t e r m e d i a t e s ( Se l k i r k et_ a j _ . , 1971; G r o v e r e t a I., 1972) fo rmed by o x i d a t i o n o f t h e K - r e g i o n d o u b l e bond , a p r o c e s s m e d i a t e d by l i v e r enzymes ( F i g u r e 7 ) . By c o m p a r i s o n w i t h s y n t h e t i c e p o x i d e s , i t has been s u g g e s t e d t h a t t h e h i g h l y r e a c t i v e e p o x i d e i n t e r m e d i a t e s a r e t h e u l t i m a t e c a r c i n o g e n i c f o rms o f t h e h y d r o c a r b o n s ( r e v i e w e d by H e i d e I b e r g e r , 1973) . 17 Carbonium ion Ultimate Carcinogen F i g u r e 7. P r o p o s e d b i o t r a n s f o r m a t i o n o f b e n z ( a ) a n t h r a c e n e (Adap ted f r om H e i d e l b e r g e r , 1973) . ( i v ) N i t r o s a m i n e s The p r e c e d i n g e xamp le s o f known b i o t r a n s f o r m a t i o n s o f p r e c a r c i n o g e n s a r e c l a s s i c m i l e s t o n e s i n t h e t h e o r e t i c a l deve l opment o f c h e m i c a l c a r c i n o g e n e s i s m a i n l y becau se a number o f p r o p o s e d m e t a b o l i t e s , c o n t a i n e d i n t h e schemes o f m e t a b o l i c c o n v e r s i o n , a r e amenab le t o s y n t h e t i c p r e p a r a t i o n . However , t h e r e e x i s t numerous c l a s s e s o f c h e m i c a l c a r c i n o g e n s (a) whose m e t a b o l i c t r a n s f o r m a t i o n s have been o n l y p a r t i a l l y d e l i n e a t e d , w i t h few s t e p s v e r i f i e d by e x p e r i m e n t a l r e s u l t s , and (b) t h a t g i v e r i s e t o p r opo sed a c t i v e m e t a b o l i t e s t h a t , because o f c h e m i c a l i n s t a b i l i t y , have e i t h e r been i m p o s s i b l e t o s y n t h e s i z e o r i m -p o s s i b l e t o s t o r e w i t h t h e t e c h n i q u e s a v a i l a b l e t o d a t e . The n i t r o s a m i n e s , f o r e x a m p l e , c o n s t i t u t e no t o n l y a v e r i t a b l e a r s e n a l o f e x p e r i m e n t a l c h e m i c a l c a r c i n o g e n s , b u t a l s o a l a r g e c l a s s o f compounds (more t h a n 100 known c a r c i n o g e n i c n i t r o s a m i n e s ) some o f w h i c h a r e r o u t i n e l y d e t e c t e d i n t h e e n v i r -onment ( H e d l e r and M a r q u a r d t , 1968; r e v i e w e d i n I .A .R .C. Monog raph , 1972) . To d a t e , t h e b i o t r a n s f o r m a t i o n mechanisms o f t h e n i t r o s -ami nes r ema i n l a r g e l y s p e c u l a t i v e becau se o f t h e i n s t a b i l i t y o f t h e m e t a b o l i t e s . The s i m p l e s t compound i n t h e c l a s s o f d i a l k y I — n i t r o s a m i n e s , d i m e t h y I n i t r o s a m i n e (DMN), i s m e t a b o l i z e d i n v i v o ( H e a t h , 1962) and succumbs t o o x i d a t i v e N - d e a I k y l a t i o n i n v i t r o w i t h r a t l i v e r m i c r o s o m a l p r e p a r a t i o n s ( M i z r a k i and Emmelot , 1962) . F u r t h e r m o r e , a c o m p a r a t i v e i n v i t r o s t u d y showed t h a t DMN i s m e t a b o l i z e d i n human l i v e r s l i c e s a t abou t t h e 'same r a t e as. i n r a t l i v e r s l i c e s (Montesano and Magee, 1970) . S t e p w i s e ( F i g u r e 8 ) , t h e g e n e r a l pathway f o r t h e c o n -v e r s i o n o f d i m e t h y l n i t r o s a m i n e i n t o h i g h l y r e a c t i v e a l k y l a t i n g a g e n t s b e g i n s w i t h an a - h y d r o x y l a t i o n o f one o f t h e m e t h y l g r o u p s , w h i c h i s f o l l o w e d by i t s h y d r o l y t i c c l e a v a g e t o f o r m m o n o m e t h y l -n i t r o s a m i n e . I t has been p r opo sed t h a t t h e m o n o m e t h y I n i t r o s a m i n e c o n v e r t s s p o n t a n e o u s l y , v i a t h e d i a z o h y d r o x i d e , t o d i a z o m e t h a n e w h i c h y i e l d s t h e methy l c a r b o n i u m i o n as t h e u l t i m a t e r e a c t i v e e l e c t r o p h i l e (Magee and H u l t i n , 1972; Magee and F a r b e r , 1972 ) . On t h e o t h e r hand , i t has been d e m o n s t r a t e d t h a t t h e f o r m a t i o n o f m e t h y l a t e d g u a n i n e i n v i v o i s a t r a n s m e t h y l a t i o n r e a c t i o n w h i c h a r g u e s a g a i n s t t h e d i a z o m e t h a n e i n t e r m e d i a t e ( L i j i n s k y , a t a j _ . , 1968 ) . The f a c t r ema in s t h a t t h e b i o t r a n s f o r m a t i o n i n t e r m e d i a t e s o f DMN 19 m e t a b o l i s m a r e u n s t a b l e and t h e r e f o r e d i f f i c u l t , i f no t i m p o s s i b l e , t o s t u d y r i g o r o u s l y i n v i t r o . CHq CH VN-0 e n z Y m a + i c • \ - N = 0 h y d r ° ' Y s i s • CH 3 -N-N=0 / o x i d a t i o n • I 2 OH M o n o m e t h y l -D i m e t h y l - n i t r o s a m i n e n i t r o s a m i ne ( P r e c a r c i n o g e n ) ^ +CH 3 4 CH 3 -N sN < CH 3-N=N-OH ( U l t i m a t e D i a z o - D i a z o h y d r o x i d e C a r c i n o g e n ) methane F i g u r e 8. P r o p o s e d b i o t r a n s f o r m a t i o n of DMN (Adap ted f r o m Magee and F a r b e r , 1962) (v ) A f l a t o x i ns T h i s b r i e f s u r v e y o f t h e b i o t r a n s f o r m a t i o n mechanisms o f m a j o r c l a s s e s o f c h e m i c a l c a r c i n o g e n s wou ld be i n c o m p l e t e w i t h -o u t h i g h l i g h t i n g t h e c u r r e n t p r o p o s a l s c o n c e r n i n g t h e m e t a b o l i s m o f t h e a f l a t o x i ns . Based on c a r c i n o g e n i c i t y t e s t s i n t h e r a t , a f l a t o x i n Bl i s t h e most p o t e n t h e p a t o c a r c i n o g e n known (Newberne and B u t l e r , 1969) . The c a r c i n o g e n i c i t y o f a f l a t o x i n B l i n t h e l i v e r v a r i e s c o n s i d e r a b l y f r om s p e c i e s t o s p e c i e s and y e t t h e l i v e r r e m a i n s t h e p r i m e t a r g e t f o r bo th t h e t o x i c and c a r c i n o g e n i c a c t i o n s o f a f l a t o x i n Bl (Wogan and Newberne, 1967; Newberne and B u t l e r , 1969; r e v i e w e d by : Wogan, 1973; I .A.R.C. Monog raph , 1972) . A f l a t o x i n Bl i n d u c e s tumor s l e s s f r e q u e n t l y a t s i t e s o t h e r t h a n t h e l i v e r ( r e v i e w e d by: Wogan, 1973) , can p r o d u c e sa rcomas a t t h e s i t e o f s u b c u . i n j e c t i o n ( D i c k e n s and J o n e s , 1965; Wogan e t a j _ . , 1971 ) , and can i n d u c e s k i n t umor s i n m i ce by s k i n - p a i n t i n g w i t h a f l a t o x i n B l f o l l o w e d by a c r o t o n o i l p r o m o t e r ( L i n d e n f e l s e r e t a I., 1974) . Weak, n o n c o v a l e n t i n t e r a c t i o n s have been d e s c r i b e d between a f l a t o x i n Bl and i s o l a t e d c e l l u l a r macromoI ecu Ies i n non -e n z y m a t i c j_n_ v j j f r o s y s tems (Sporn et_ a j _ . , 1966; C l i f f o r d and Ree s , 1967; Wogan, 1973) and y e t s t r o n g , c o v a l e n t i n t e r a c t i o n s have been s u g g e s t e d by t h e f o r m a t i o n o f n u c l e i c a c i d - and p r o t e i n - b o u n d r a d i o a c t i v i t y a f t e r t h e a d m i n i s t r a t i o n o f 3 H - l a b e l l e d a f l a t o x i n Bl i n v i v o ( L i j i n s k y e t a j _ . , 1970 ) . ' F u r t h e r m o r e , o n l y c h r o m a t i n f r o m t h e l i v e r s o f a f l a t o x i n B l - t r e a t e d r a t s has i m p a i r e d t e m -p l a t e a c t i v i t y f o r RNA p o l y m e r a s e w h i l e t h e d i r e c t a d d i t i o n o f a f l a t o x i n Bl t o i n v i t r o s y s t ems had no e f f e c t on t h e t e m p l a t e a c t i v i t y o f c h r o m a t i n (Edwards and Wogan, 1970) . F i n a l l y , two s t r a i n s o f S a l m o n e l l a t y p h i m u r i u m h i s t i d i n e a u x o t r o p h s were k i l l e d when i n c u b a t e d w i t h a f l a t o x i n Bl and a r a t l i v e r homogenate c o n -t a i n i n g l i v e r m ic ro somes and c o - f a c t o r s n e c e s s a r y f o r m i c r o s o m a l m i x e d - f u n c t i o n o x i d a s e a c t i v i t y . E i t h e r component a l o n e p r o d u c e d no l e t h a l i t y and t h i s s u g g e s t e d t h a t a f l a t o x i n Bl had been c o n -v e r t e d t o a f o rm t h a t cau sed l e t h a l i t y i n t h e b a c t e r i u m ( G a r n e r e t aj_. , 1971: G a r n e r e_t aj_. , 1972) . T h u s , t h e metaboI i c c o n -v e r s i o n o f a f l a t o x i n Bl t o r e a c t i v e i n t e r m e d i a t e s i n v i v o i s s u g g e s t e d . In v i e w o f t h e p o t e n t c a r c i n o g e n i c and t o x i c p r o p e r t i e s o f a f l a t o x i n Bl ( F i g u r e I ) , d i s c u s s i o n o f t h e m e t a b o l i t e s t r u c -t u r e s w i l l be l i m i t e d t o t h i s compound and i t s m e t a b o l i t e s ( F i g u r e 21 9 ) . A f l a t o x i n Ml has been i s o l a t e d f r om t h e u r i n e o f many a n i m a l s p e c i e s , i n c l u d i n g human, f o l l o w i n g i n g e s t i o n o f a f l a t o x i n Bl (Wogan, 1973) . A f l a t o x i n PI has been i s o l a t e d f r om t h e u r i n e o f r h e s u s monkeys f e d a f l a t o x i n B l (Wogan, 1973 ) . A f l a t o x i n B2a and a f l a t o x i c o l ( D e t r o y and H e s s e l t i n e , 1970) have been i d e n t i f i e d as p r o d u c t s o f m i c r o s o m a l enzyme m e t a b o l i s m i n v i t r o ( P a t t e r s o n and R o b e r t s , 1970; P a t t e r s o n and R o b e r t s , 1972 ) . I t has been empha s i zed t h a t t h e s e m e t a b o l i t e s do no t n e c e s s a r i l y r e p r e s e n t t e r m i n a l m e t a b o l i t e s o f a f l a t o x i n Bl s i n c e t h e y t h e m s e l v e s a r e c a p a b l e o f f u r t h e r m e t a b o l i c r e a c t i o n by o t h e r r o u t e s ( P a t t e r s o n and R o b e r t s , 1972) . The p r o p o s e d u n t i m a t e c a r c i n o g e n i c f o rm of a f l a t o x i n Bl i s t h e e p o x i d e ( F i g u r e 9 ) ( S c h o e n t a I , 1970; G a r n e r a t a_k, 1972) . To d a t e , t h e e p o x i d e o f a f l a t o x i n Bl has e l u d e d i s o l a t i o n f rom e n z y m a t i c o r c h e m i c a l s y n t h e s e s (Swensen, e t a I., 1974) . F i g u r e 9. Some r o u t e s o f m e t a b o l i s m o f a f l a t o x i n Bl ( A d a p t e d f rom Wogan, 1973) . INTERACTION OF CHEMICAL CARCINOGENS WITH CELLULAR MACROMOLECULES The f o r e g o i n g s u r v e y o f b i o t r a n s f o r m a t i o n mechanisms can be g e n e r a l i z e d by n o t i n g t h a t an e I e c t r o p h i I i c s p e c i e s i s h y p o t h e s i z e d as t h e u l t i m a t e r e a c t a n t i n e v e r y c a s e ( F i g u r e 10) ( M i l l e r and M i l l e r . 1969 a . b : M i l l e r . 1970) . F u r t h e r m o r e , when 23 t h e e s t e r o f AAF, N - a c e t o x y - A A F , f o r e x a m p l e , i s r e f e r r e d t o as t h e u l t i m a t e c a r c i n o g e n i c f o r m , i t i s u n d e r s t o o d t h a t t h e t e r m P r e c a r c i nogen S t e p s i n M e t a b o I i c T r a n s f o r m a t i o n P r o x i m a t e C a r c i nogen E l e c t r o p h i I i c S p e c i e s as U l t i m a t e C a r c i n o g e n i c Form e . g . Ca rbon ium i o n : + C H 3 F i g u r e 10. G e n e r a l i z e d scheme f o r t h e m e t a b o l i s m o f c h e m i c a l p r e c a r c i n o g e n s t o f o rm r e a c t i v e e I e c t r o p h i I i c m e t a b o l i t e s . i n c l u d e s t h e s u b s e q u e n t t r a n s i e n t e I e c t r o p h i I i c s p e c i e s t h a t i m m e d i a t e l y p r e c e d e s t h e r e a c t i o n between t h e c h e m i c a l and t h e e e l I u I a r n u c l e o p h i l e . G e n e r a l l y , i t i s t h e s e e l e c t r o p h i I i c s p e c i e s o f t h e p r e c a r c i n o g e n s t h a t seek o u t e l e c t r o n - d e n s e c e n t e r s , i n t h e com-p o n e n t s o f c e l l u l a r m a c r o m o l e c u l e s , w i t h w h i c h t o f o rm c o v a l e n t bonds . Such c e n t e r s a r i s e i n p r o t e i n and i n t h e n u c l e i c a c i d s , RNA and DNA. However, o n l y i n t e r a c t i o n s w i t h DNA w i l l be d i s c u s s e d f u r t h e r . A l t h o u g h t h e n i t r o g e n atom a t t h e 7 - p o s i t i o n o f g u a n i n e a p p e a r s t o be t h e most r e a c t i v e s i t e o f a l l t h e DNA bases t o w a r d a l k y l a t i n g a g e n t s ( Ba rdo s e t a j _ . , 1965; B o y l a n d , 1969) and i s s t e r i c a l l y a v a i l a b l e s i n c e i t i s s i t u a t e d i n t h e w ide g r o o v e o f t h e DNA d o u b l e h e l i x ( R e i n e r and Zamenhof, 1957 ) , i t i s by no means t h e o n l y s i t e o f a t t a c k by v a r i o u s c a r c i n o g e n s ( F i g u r e I I ) . 24 In f a c t , t h e r e a r e a c t u a l l y few p o s i t i o n s o f g u a n i n e , a d e n i n e , and c y t o s i n e t h a t have not been r e p o r t e d t o be a t t a c k e d by c h e m i c a l c a r c i nogens J_n_ v i vo o r j_n_ v i t r o ( r e v i e w e d by: Mi I I e r and Mi I I e r , 1971; I r v i n g , 1973; Sarma e t a I., 1974) . s u g a r F i g u r e I I. C o v a l e n t b o n d i n g o f a methy l g roup t o t h e N-7 p o s i t i o n o f g u a n i n e . In a d d i t i o n , t h e r e a r e numerous p o s s i b i l i t i e s o f n o n -c o v a l e n t i n t e r a c t i o n s o f c a r c i n o g e n s w i t h DNA. These t a k e t h e fo rm o f : (1) i n t e r c a l a t i o n , r e s u l t i n g f r om i n s e r t i o n o f a r i g i d , p l a n a r c a r c i n o g e n between base p a i r s i n t h e DNA d o u b l e h e l i x , and (2) a d l i n e a t i o n , o r e x t e r n a l b i n d i n g o f t h e c a r c i n o g e n t o t h e bases p e r p e n d i c u l a r t o t h e p l a n e s o f t h e base p a i r s ( A r c o s and A r g u s , 1968) . I n t e r c a l a t i o n mode l s have been p r o p o s e d t o e x p l a i n o b -s e r v e d f r a m e - s h i f t m u t a t i o n s ( Le rman , 1964) and t o a c c o u n t f o r t h e c a r c i n o g e n i c a c t i o n o f numerous p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s ( B o y l a n d and G r e e n , 1962; I r v i n g , 1973; Sarma e t a j _ . , 1974) . E x t e r n a l b i n d i n g by c h a r g e - t r a n s f e r complex f o r m a t i o n s , an i n t e r a c t i o n t h a t 25 i s much weaker t h a n t h a t o f i n t e r c a l a t i o n , has been p r o p o s e d f o r p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s ( A r c o s and A r g u s , 1968 ) , a f l a -t o x i n s ( I r v i n g , 1973) , and 4 -n i troq-u i no I i ne I - o x i d e (Ma I k i n and Z a h a l s k y , 1966; Pau l and Montgomery , 1971) . These n o n c o v a l e n t i n t e r a c t i o n s o f c a r c i n o g e n s w i t h DNA e x h i b i t o n l y weak b i n d i n g f o r c e s and so t h e r e i s no f i r m e v i d e n c e f o r t h i s t y p e o f b i n d i n g i n v i v o ( r e v i e w e d by I r v i n g , 1973) . ROLE OF CHEMICAL-DNA INTERACTIONS IN CARCINOGENESIS The model s y s t e m o f e x p e r i m e n t a l I i v e r c a r c i n o g e n e s i s has p r o v i d e d some i n s i g h t i n t o t h e r e l a t i o n s h i p s between t h e s o -c a l l e d " i n i t i a t i o n " s t a g e o f c h e m i c a l c a r c i n o g e n e s i s and t h e s u b -s e q u e n t s t e p s l e a d i n g up t o t h e o n s e t o f l i v e r c a n c e r . S i n c e t h e o r i g i n a l d i s c o v e r y o f a model f o r l i v e r c a r c i n o g e n e s i s by S a s a k i and Y o s h i d a ( S a s a k i and Y o s h i d a , 1935 ) , t h e r e a l i z a t i o n has d e -v e l o p e d t h a t l i v e r c a n c e r i s a m u l t i s t e p p r o c e s s i n w h i c h new c e l l p o p u l a t i o n s appea r t o p l a y an i m p o r t a n t r o l e . The t e r m " i n i t i a t i o n " r e f e r s t o an e a r l y , r a p i d s t e p ( h o u r s t o day s ) i n t h e l i v e r and t h e s u b s e q u e n t s t e p s o f unknown number, i n v o l v i n g t h e a p p e a r a n c e o f new c e l l p o p u l a t i o n s , have been r e f e r r e d t o as " n e o p l a s t i c d e v e l o p m e n t " ( F o u l d s , 1969) o r , more p r e c i s e l y , " n e o p l a s t i c c e l l u l a r e v o l u t i o n " ( F a r b e r , 1973) . T h e r e i s an e c l i p s e p e r i o d o f s e v e r a l weeks f o l l o w i n g r e c o v e r y f r om i n i t i a l c e l l damage, r e s u l t i n g f r om a s i n g l e i n -j e c t i o n o f t h e h e p a t o c a r c i n o g e n d i m e t h y I n i t r o s a m i n e , d u r i n g p a r t i a l h e p a t e c t o m y , u n t i l t h e a p p e a r a n c e o f p r o g r e s s i v e c e l l u l a r 26 a l t e r a t i o n s l e a d i n g t o l i v e r c a n c e r ( r e v i e w e d by F a r b e r e t a I., 1974) . The re i s , t h e r e f o r e , a need f o r some l o n g - t e r m memory i n t h e i n i t i a l l e s i o n o r l e s i o n s a n d , on t h e b a s i s o f c u r r e n t c o n -c e p t s o f c e l l u l a r h e r e d i t y , DNA s t a n d s o u t as t h e s i m p l e s t c e l l u l a r mac romo lecu Ie t o h a n d l e c o n c e p t u a l l y . In t h i s c o n t e x t , t h e r e f o r e , t h e t e r m " i n i t i a t i o n " r e f e r s t o an e a r l y m o l e c u l a r damage t o DNA. G e n e r a l l y , t h e s o - c a l l e d " s o m a t i c m u t a t i o n t h e o r y " o f c h e m i c a l c a r c i n o g e n e s i s ( r e v i e w e d by F o u l d s , 1.969) p r o p o s e s t h a t t h e c h e m i c a l c a r c i n o g e n i n t e r a c t s w i t h t h e c e l l u l a r DNA i n s uch a way as t o a l t e r i t s n u c l e o t i d e sequence so as t o r e s u l t i n a n o n l e t h a l p e r p e t u a t e d c h a n g e . The p r e c i s e mechani sm by w h i c h t h i s permanent DNA damage o c c u r s i s unknown. DNA REPAIR AND CARCINOGENESIS The p o s s i b l e mechan i sms f o r t h e i n d u c t i o n o f a permanent o r " f i x e d " a l t e r a t i o n i n t h e p r i m a r y sequence o f DNA can o n l y be f u l l y r e a l i z e d when t h e c u r r e n t mechan i sms of DNA r e p a i r a r e c o n -s i d e r e d . F o l l o w i n g t h e i n i t i a l i n t e r a c t i o n s between c h e m i c a l c a r -c i n o g e n s and c e l l u l a r DNA, s u b s e q u e n t m o l e c u l a r e v e n t s can i n c l u d e a d e p u r i n a t i o n o f DNA, f o l l o w e d by d i s r u p t i o n o f t h e s u g a r - p h o s p h a t e backbone . T h i s r e a c t i o n o c c u r s a f t e r t r e a t m e n t w i t h c e r t a i n a l k y l a t i n g a g e n t s ( e . g . n i t r o g e n m u s t a r d ) known t o c o v a l e n t l y bond an a IkyI g roup t o p u r i n e bases a n d , t o a l e s s e r e x t e n t , p y r i m i d i n e ba ses i n DNA ( L a w l e y , 1966) . A p a r t f rom t h i s " s p o n t a n e o u s " h y d r o -l y t i c c h a i n f i s s i o n o f n u c l e i c a c i d s , an enzyme-med i a ted removal 27 o f a l k y l a t e d DNA m o i e t i e s f r om DNA m o l e c u l e s has been p r o p o s e d ( C r a t h o r n and R o b e r t s , 1966; S t r a u s s a t a\_., 1968; Ikegami e t a I . , 1970) . T h i s remova l o f damaged DNA segments i s t h o u g h t t o be a component o f a p r o c e s s o f DNA r e p a i r , a d e v i c e whereby t h e c e l l a t t e m p t s t o r e g a i n normal f u n c t i o n . The phenomenon o f DNA r e p a i r has been i n v e s t i g a t e d i n b a c t e r i a , c u l t u r e d c e l l s , and i n t a c t a n i m a l s f o l l o w i n g e x p o s u r e t o r a d i a t i o n bo th u l t r a v i o l e t and i o n -i z i n g , and c h e m i c a l c a r c i n o g e n s ( r e v i e w e d by : P a i n t e r , 1971; S t i c h and L a i s h e s , 1973; I r v i n g , 1973; Lohman and Boo t sma , 1974) . The e s s e n t i a l f e a t u r e s o f e x c i s i o n - r e p a i r a r e shown i n F i g u r e 12. A l t h o u g h o r i g i n a l l y d e s c r i b e d f o r t h e r e p a i r o f UV-i nduced DNA damage, t h e e x c i s i o n - r e p a i r s y s t em m i g h t w e l l a p p l y t o t h e r e p a i r o f c h e m i c a l - D N A b i n d i n g s i t e s . The b i n d i n g o f c h e m i c a l s , c o v a l e n t l y o r n o n c o v a I e n t I y , t o DNA c o u l d r e s u l t i n a l o c a l i z e d s t r u c t u r a l d i s t o r t i o n o f t h e DNA h e l i x (Pau l and Montgomery , 1971) w h i c h , as i n t h e c a s e o f U V - i n d u c e d p y r i m i d i n e d i m e r s , c o u l d s e r v e as a r e c o g n i t i o n s i t e f o r e n d o n u c l e a s e a t t a c k . " S p o n t a n e o u s " h y d r o -l y t i c c h a i n f i s s i o n o f DNA c o u l d r e s u l t i n b y p a s s i n g t h e e n d o -n u c l e a s e s t e p and t h e p r o c e s s wou ld s i m p l y c o n t i n u e a t t h e p o l y -merase s t e p . W i t h r e g a r d t o t h e i m p o r t a n c e o f " c o i n c i d e n t s i n g l e -s t r a n d b r e a k s " , i . e . d o u b l e - s t r a n d b r e a k s , i t i s a p p a r e n t t h a t t h e r e s y n t h e s i s o f t h e new s i n g l e - s t r a n d s e gmen t s , as i n F i g u r e 12, i s e n t i r e l y dependent on t h e e x i s t e n c e o f t h e i n t a c t , comp lementa r y DNA s t r a n d t o a c t as a t e m p l a t e . If t h i s r e m a i n i n g t e m p l a t e were damaged o r b r o k e n , t h e n a f a i t h f u l copy d u r i n g r e p a i r s y n t h e s i s wouId be un I i k e I y . F u r t h e r m o r e , t h e i n i t i a t i o n s t e p o f c a r c i n o g e n e s i s , as 28 F i g u r e 12. A s c h e m a t i c model f o r e x c i s i o n r e p a i r (Adap ted f r om K e l l y , e t a L , 1969; B o y l e , 1972; S t i c h and L a i s h e s , 1973) 1. A DNA d o u b l e h e l i x , e i t h e r c o n t a i n i n g UV damage (D) o r bound chem-i c a l ( C ) . 2 . (a) The r e g i o n a d j a c e n t and on t h e 5 ' s i d e o f t h e damage i s a t t a c k e d by an e n d o n u c l e a s e and n i c k e d . (b) Spontaneous h y d r o l y t i c c h a i n f i s s i o n o f t h e DNA i s p o s s i b l e w i t h c e r t a i n bound c h e m i c a l s . 3. A DNA p o l y m e r a s e b i n d s t o t h e DNA a t t h e n i c k e d s i t e and b e g i n s r e -p a i r p o l y m e r i z a t i o n o f t h e damaged s t r a n d - u s i n g t h e comp lementa r y s t r a n d as a t e m p l a t e . 4. When t h e n e x t h yd rogen -bonded base p a i r i s r e a c h e d , a p h o s p h o d i e s t e r bond i s h y d r o l y z e d and t h e damaged o r c h e m i c a l l y - b o u n d DNA m o i e t y i s r e l e a s e d i n a s h o r t o l i g o n u c l e o t i d e . 5. The p o l y m e r a s e and h y d r o l y t i c a c t i v i t i e s may c o n t i n u e . 6. The p o l y m e r a s e i s f i n a l l y r e p l a c e d by a p o l y n u c l e o t i d e I i g a s e w h i c h r e s t o r e s t h e i n t e g r i t y of- t h e p h o s p h o d i e s t e r backbone o f DNA. 29 5' 3 ' L o c a l D i s t o r t i o n C = B o u n d C h e m i c a l C / D = U V - l n d u c e d D i m e r B\ 3 ' 5' E n d o n u c 2 a C l e a s e ^ / ^ " " ^ S p o n t a n e o u s C h a i n C F i s s i o n " " - - - ^ . - ^ ^ ^ 2 b D N A P o l y m e r a s e ^ ^ ^ • t P o l y n u c l e o t i d e L i g a s e d e s c r i b e d f o r t h e l i v e r m o d e l , can now be v i ewed as i n v o l v i n g a permanent damage t o DNA a r i s i n g t h r o u g h i n a d e q u a t e o r f a u l t y r e p a i r ( r e v i e w e d by F a r b e r e t a j _ . , 1974) . The s t u d y o f c a r c i n o g e n e s i s i n v i t r o has a l l o w e d a more c a r e f u l i s o l a t i o n and d i s s e c t i o n o f t h e p r o c e s s e s i n v o l v e d i n r a d i a t i o n - and chemica I I y - i n d u c e d DNA damage and r e p a i r . The methods o f e s t i m a t i n g DNA r e p a i r have been summar ized ( P a i n t e r , 1971; S t i c h and L a i s h e s , 1973) and a r e o u t l i n e d i n T a b l e 3. An e f f e c t i v e a n a l y s i s o f t h e u n s c h e d u l e d 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 i s a c c o m p l i s h e d u s i n g t h e t e c h n i q u e o f a u t o -r a d i o g r a p h y (Rasmussen and P a i n t e r , 1964,1966; S t i c h e t a I., 1970, 1972, 1973) . The p r o c e d u r e u s u a l l y i n v o l v e s t h e a u t o r a d i o g r a p h i c d e t e c t i o n o f 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 i n t o t h e n u c l e i o f c e l l s , w i t h normal DNA r e p l i c a t i o n a s s o c i a t e d w i t h chromosomal d u p l i c a t i o n 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 , p r i o r t o e x p o s u r e t o t h e c a r c i n o g e n . The u t i l i t y o f t h i s t e c h n i q u e l i e s i n t h e f a c t s t h a t : (a) t h e c y t o l o g i c l o c a t i o n o f i n c o r p o r a t e d t h y m i d i n e can be i m m e d i a t e l y l o c a t e d ; (b) t h e l e v e l o f r e p a i r s y n t h e s i s i n d i f f e r e n t c e l l t y p e s , o f v a r i o u s d e g r e e s o f p l o i d y , can be s t u d i e d ; ( c ) t h o s e c e l l s t h a t e s caped t h e DNA r e p l i c a t i o n b l o c k i n g a g e n t s , and so c o n t i n u e d t o i n c o r p o r a t e t r i t i a t e d t h y -m i d i n e d u r i n g s e m i c o n s e r v a t i v e DNA r e p l i c a t i o n , can be l o c a t e d ; (d) a low l e v e l o f u n s c h e d u l e d 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 i n few c e l l s can be d e t e c t e d e a s i l y . DNA r e p a i r s y n t h e s i s has been s u c c e s s f u l l y a p p l i e d t o i n d i c a t e DNA changes i nduced by p h y s i c a l (Rasmussen and P a i n t e r , 1964, 1966; P a i n t e r and C l e a v e r , 1969) o r c h e m i c a l ( S t i c h e t a I., 31 T a b l e 3. Summary o f methods a v a i l a b l e t o e s t i m a t e DNA damage and r e p a i r i n mammalian c e l l s . METHODS FOR "D IRECT" ESTIMATION OF DNA DAMAGE 1. A l k a l i n e and n e u t r a l s u c r o s e g r a d i e n t d e t e c t i o n o f DNA f r a g -m e n t a t i o n (McGrath and W i l l i a m s , 1966; L e t t e t a k , 1967; L a i s h e s and S t i c h , 1973a; S t i c h and L a i s h e s , 1973; P a i n t e r , 1971 ). 2 . B i o c h e m i c a l d e t e c t i o n o f e xpo sed ends o f b r oken DNA m o l e c u l e s (Da I rymp I e e t a j _ . , 1969 a , b ) . 3. Lo s s o f l a b e l l e d a l k y l a t i n g a g e n t s f r om DNA ( C r a t h o r n and R o b e r t s , 1966) . 4. E n z y m a t i c a s s a y s o f damaged DNA s i t e s ( r e v i e w e d by Lohman and Boot sma, 1974) . METHODS FOR " IND IRECT" ESTIMATION OF DNA DAMAGE  I n c o r p o r a t i o n o f p y r i m i d i n e a n a l o g s d u r i n g r e s y n t h e s i s o f e x c i s e d segments o f DNA: 1. T r i t i a t e d t h y m i d i n e i n c o r -p o r a t i o n b y : (a) a u t o r a d i o g r a p h y (Rasmussen and P a i n t e r , 1964, 1966; P a i n t e r and C l e a v e r , 1969; S t i c h e t a I. , 1970, 1972, 1973) . (b) l i q u i d s c i n t i l l a t i o n c o u n t i n g (Evans and Norman, 1968; L i e b e r m a n e t a I., 1972; Bu rk e t a j _ . , 1972 ) . 2. B r o m o d e o x y u r i d i n e i n c o r p o r a t i o n d e t e c t e d b y : (a) p h o t o l y s i s and a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n (Regan e t a j _ . , I 97 I ) . (b) bouyan t d e n s i t y c e s i u m c h l o r i d e g r a d i e n t c e n t r i f u g a t i o n (Rasmussen and P a i n t e r , 1964, 1966; P a i n t e r and C l e a v e r , 1968 ) . 32 1970, 1972, 1973; L i ebe rman e_t a l . , 1972) a g e n t s i n mammalian c e l l s . C a r c i n o g e n i c i s omer s and d e r i v a t i v e s r e l a t e d t o 4NQ0 and 4 - n i t r o p y r i d i n e l - o x i d e were a c t i v e i n t r i g g e r i n g DNA r e p a i r s y n t h e s i s ( S t i c h et_ a j _ . , 1970) . However , o n l y t h e s y n t h e t i c (K -r e g i o n ) e p o x i d e s o f b e n z ( a ) a n t h r a c e n e (BA) and m e t h y I c h o I a n t h r e n e (MCA), and no t t h e p a r e n t compounds, BA and MCA, were a b l e t o e voke t h e r e s p o n s e o f DNA r e p a i r s y n t h e s i s ( S t i c h and S an , 1973) . I t i s o f i n t e r e s t t o no te t h a t B A - e p o x i d e and MCA -epox i de have been p r o p o s e d a s t h e u l t i m a t e c a r c i n o g e n i c f o r m o f t h e p a r e n t h y d r o -c a r b o n " p r e c a r c i nogen s " (Boy I and 1969; S e l k i r k e_t a j _ . , 1971; G r o v e r e t a I., 1972) . S i m i l a r l y , not t h e p r e c a r c i n o g e n AAF, bu t o n l y t h e p r o x i m a t e c a r c i n o g e n N -hyd roxy -AAF was a b l e t o i n d u c e h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n mammalian c e l l s ( S t i c h e t a I., 1972; L i ebe rman e t a I., 1972 ) . T h i s c o r r e l a t i o n has been d u p l i c a t e d w i t h s y n t h e t i c p r o x i m a t e and u l t i m a t e c a r c i n o g e n i c f o rms o f o t h e r c l a s s e s o f a r o m a t i c amines w i t h d i f f e r e n t a r y l g r o u p s : s t i l b e n e and b i p h e n y l ( S t i c h e_t a j _ . , 1973) . P r o m i n e n t among t h e many p o s s i b l e mechanisms o f m e t a -b o l i c a c t i v a t i o n o f t h e s y n t h e t i c q u i n o l i n e N - o x i d e s i s t h e i n i t i a l r e d u c t i o n o f t h e n i t r o g roup ( r e v i e w e d by Kawazoe e t a l . , 1972 ) . Such an e n z y m i c r e d u c t i o n may be n e c e s s a r y t o e voke t h e o b s e r v e d r e s p o n s e o f DNA r e p a i r s y n t h e s i s i n human c e l l c u l t u r e s . However , many c a r c i n o g e n s , such as AAF and BA a p p a r e n t l y r e q u i r e o x i d a t i v e m e t a b o l i s m p r i o r t o e x e r t i n g t h e i r e f f e c t on t h e human c e l l c u l t u r e s ( S t i ch e_t a I., 1973) . If s y n t h e t i c mode l s o f t h e p r opo sed o x i d a t i v e m e t a b o l i t e s such as N - a c e t o x y - A A F and B A - e p o x i d e , were no t a v a i l -a b l e , s t u d i e s o f DNA damage and r e p a i r r e l a t e d t o t h e i n v i v o 33 b e h a v i o u r o f t h e s e compounds wou ld l i k e l y be d i f f i c u l t t o e x e c u t e i n v i t r o w i t h human f i b r o b l a s t c u l t u r e s . However , t h e r e r ema in s a l a r g e number o f c h e m i c a l c a r c i n o g e n s o f v a r i o u s s t r u c t u r a l t y p e s t h a t have m e t a b o l i c p r o d u c t s t h a t a r e e s s e n t i a l l y unknown. In most c a s e s t h i s i s b e cau se i s o l a t i o n o f t h e a c t i v e m e t a b o l i t e s i s i m p o s s i b l e w i t h t h e t e c h -n i q u e s a v a i l a b l e t o d a t e . T h e r e f o r e , i t seemed e s s e n t i a l t o d e v e l o p a method whereby c h e m i c a l p r e c a r c i n o g e n s c o u l d be m ixed w i t h m e t a b o I i c a I Iy a c t i v e t i s s u e e x t r a c t s w i t h c o n c o m i t a n t e x p o s u r e o f human c e l l " r e c e p t o r " c u l t u r e s . The method o f c h o i c e was t o comb ine c h e m i c a l p r e -c a r c i n o g e n s w i t h a p o s t - m i t o c h o n d r i a I s u p e r n a t a n t f r a c t i o n o f a n i m a l l i v e r homogenate, f o r t i f i e d w i t h NADPH. F o r e x a m p l e , M a i l i n g ( M a i l i n g , 1971) r e p o r t e d t h a t d i m e t h y I n i t r o s a m i n e , o n l y when m i xed w i t h a c o m p l e t e a c t i v a t i o n s y s t em ( c o n t a i n i n g a mouse l i v e r p o s t - m i t o c h o n d r i a I s u p e r n a t a n t f r a c t i o n , f o r t i f i e d w i t h NADPH) e x e r t e d a p ronounced t o x i c e f f e c t on S a l m o n e l l a t y p h i m u r i u m and a l s o enhanced t h e m u t a g e n i c r e s p o n s e o f t h e b a c t e r i a by i n d u c i n g a l a r g e number o f r e v e r t a n t s . An i n v i t r o s y s t e m , c a p a b l e o f s i m u l a t i n g i n v i v o m e t a b o l i c a c t i v a t i o n , would p e r m i t s t u d y o f t h e c a p a b i l i t i e s o f many unknown and u n s t a b l e m e t a b o l i t e s o f p r e c a r c i n o g e n s t o i n d u c e DNA damage and r e p a i r i n c u l t u r e d c e l l s . F u r t h e r m o r e , t h e p o s s i b l e r o l e o f DNA damage and r e p a i r i n c a r c i n o g e n e s i s , i n v o l v i n g a c t i v e m e t a b o l i t e s g e n e r a t e d f rom p r e c a r c i n o g e n s , c o u l d be i n v e s t i g a t e d by t h e use o f r e p a i r - d e f i c i e n t c u l t u r e d f i b r o b l a s t s d e r i v e d f rom p a t i e n t s a f f l i c t e d w i t h xe rode rma p igmentosum (XP) ( C l e a v e r 34 1970 a , b ; Burk e t a j _ . , 1972) , as " r e c e p t o r " c e l l s . E xpo su re o f normal and r e p a i r - d e f i c i e n t human c e l l s t o p r e c a r c i n o g e n s , i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t e m s , c o u l d r e v e a l i n f o r m a t i o n r e g a r d i n g t h e b i o l o g i c a l i m p l i c a t i o n s o f DNA damage and r e p a i r i n d u c e d by many unknown and u n s t a b l e m e t a b o l i t e s o f p r e c a r c i n o g e n s . T h i s c o u l d be a c c o m p l i s h e d by o b s e r v i n g t h e l e v e l s o f i n d u c e d DNA damage and r e p a i r a t t h e m o l e c u l a r and chromosomal l e v e l s , w i t h a t t e n t i o n a l s o f o c u s e d on c y t o t o x i c e f f e c t s . ORGANOTROPIC CHEMICAL CARCINOGENS The r o l e o f m e t a b o l i c a c t i v a t i o n i n t h e o b s e r v e d t i s s u e -s p e c i f i c i t y o f tumor i n d u c t i o n by c e r t a i n c h e m i c a l c a r c i n o g e n s i s s t i l l a m a t t e r o f c o n j e c t u r e ( M i l l e r , 1970; Magee, 1972) . F u r t h e r -more , a t t e m p t s t o r e l a t e t h e t i s s u e - s p e c i f i c i t y o f t umor i n d u c t i o n t o l e v e l s o f a l k y l a t i o n ( e . g . Magee, 1972) and b i n d i n g o f c a r c i n o -gens ( L i j i n s k y e t a j _ . , 1970) have f a i l e d . I t i s t h e r e f o r e a r e a s o n -a b l e c o n t e n t i o n t h a t a l t h o u g h l e v e l s o f c a r c i n o g e n b i n d i n g t o DNA i n t a r g e t and n o n - t a r g e t t i s s u e s may no t be r e l a t e d t o t h e t i s s u e - s p e c i f i c i t y o f t u m o r - i n d u c t i o n , t h e i m p o r t a n t q u e s t i o n may be t h e a b i l i t y o f t h e c a r c i n o g e n t o e voke h i g h l e v e l s o f d e t e c t -a b l e DNA damage and r e p a i r i n some t i s s u e s and no t i n o t h e r s . I t t h e r e f o r e seemed a p p r o p r i a t e t o d e s i g n t e c h n i q u e s f o r e s t i m a t i n g l e v e l s o f a c t u a l DNA damage and r e p a i r , i n t h e i n t a c t a n i m a l , t h a t o c c u r f o l l o w i n g c a r c i n o g e n a d m i n i s t r a t i o n . The t e c h n i q u e s f o r t h e a n a l y s i s o f DNA damage i n v i vo s h o u l d be amen-a b l e t o c o m p a r i s o n s o f bo th t a r g e t and n o n - t a r g e t t i s s u e s u s i n g 35 v a r i o u s c h e m i c a l c a r c i n o g e n s e x h i b i t i n g a h i g h d e g r e e o f o r g a n o -t r o p y i n t h e t e s t a n i m a l . Recen t s t u d i e s have r e v e a l e d h i g h l e v e l s o f DNA damage i n r a t l i v e r w i t h i n hou r s a f t e r t h e a d m i n i s t r a t i o n o f many c h e m i c a l s known t o be h e p a t o c a r c i nogens (Cox et_ aj_. , 1973; Damjanov e t a I . , 1973) . I t was d e c i d e d t o adap t t h e t e c h n i q u e emp loyed by Cox e t  a I. f o r use w i t h l i v e r and o t h e r t i s s u e s i n m i c e . In t h i s manner , a s e a r c h c o u l d be c a r r i e d o u t f o r a c o r r e l a t i o n between t h e s i t e o f i n d u c e d DNA damage and s i t e s o f tumor i n d u c t i o n by c h e m i c a l c a r c i n o g e n s e x h i b i t i n g v a r i o u s d e g r e e s o f o r g a n o t r o p y i n m i c e . In a d d i t i o n , w i t h t h e a s s u m p t i o n t h a t i n v i t r o m e t a b o I i c a c t i v a t i o n s y s t e m s , e m p l o y i n g t i s s u e e x t r a c t s , a r e r e l a t e d t o a c t u a l m e t a b o l i c b e h a v i o u r i n v i v o , t h e i n v e s t i g a t i o n o f t h e r e l a -t i o n s h i p between DNA damage, s i t e o f tumor i n d u c t i o n , and s i t e o f m e t a b o l i c a c t i v a t i o n may be c a r r i e d o u t i n v i t r o w i t h c u l t u r e d human c e l l s employed as " r e c e p t o r s " . A g a i n , a t t e n t i o n was no t t o be f o c u s e d on t h e a b i l i t y o f t h e a c t i v a t i o n s y s t ems t o p o t e n t i a t e t h e l e v e l o f b i n d i n g o f c a r c i n o g e n s t o DNA b u t r a t h e r on t h e a b i l i t y o f t h e a c t i v a t i o n s y s t ems t o p o t e n t i a t e t h e DNA damaging c a p a c i t y o f c e r t a i n p r e c a r c i n o g e n s . W i t h t h e a s s u m p t i o n t h a t t h e r e s p o n s e s o f t h e human c e l l " r e c e p t o r s " a r e i n d i c a t i v e o f t h e r e s p o n s e s o f t h e c o n s t i t u e n t c e l l s o f v a r i o u s t i s s u e s i n v i v o , a p o s s i b l e r e l a t i o n s h i p between t h e s i t e o f t umor i n d u c t i o n and t h e s i t e o f DNA damage may be o b s e r v e d by s e l e c t i n g e x t r a c t s f r o m v a r i o u s t a r g e t and n o n - t a r g e t t i s s u e s . 36 OBJECTIVES The human p o p u l a t i o n i s e xpo sed t o unknown amounts o f c a r c i n o g e n i c c h e m i c a l s 1 t h a t may be man-made, as i n t h e c a s e o f c e r t a i n a r o m a t i c amines ( r e v i e w e d by M i l l e r , 1 973 ) , o r n a t u r a l l y -o c c u r r i n g , as t y p i f i e d by t h e g r o w i n g l i s t o f c a r c i n o g e n i c c h e m i c a l s , such as a f l a t o x i n s , t h a t a r e p r o d u c e d by mou lds ( r e v i e w e d by Wogan, 1973; M i l l e r , 1973 ) . S t u d i e s o f t h e c h e m i c a l r e a c t i v i t i e s o f t h e s e c a r c i n o -g e n i c c h e m i c a l s w i t h i s o l a t e d c e l l u l a r macromoI ecu Ies u n d e r p h y s i o l o g i c c o n d i t i o n s ' have r e v e a l e d t h a t a l a r g e m a j o r i t y o f t h e compounds a r e d i s t i n c t l y u n r e a c t i v e ( e . g . M i l l e r , 1970 ) . On t h e o t h e r hand , i n v e s t i g a t i o n s have r e v e a l e d t h a t many o f t h e same c a r c i n o g e n s e x h i b i t an a b i l i t y t o b i n d t o c e l l u l a r macromoI ecu Ies i n t h e i n t a c t an ima l ( e . g . M i l l e r , 1970 ) . The se o b s e r v a t i o n s i m p l y t h a t t h e c h e m i c a l b o n d i n g i n v i v o i s n o t a r e s u l t o f d i r e c t a c t i o n o f t h e " c a r c i n o g e n s " , b u t i n f a c t o c c u r s t h r o u g h t h e a c t i o n o f a m e t a b o l i t e o f t h e " p r e c a r c i n o g e n " . The r o l e o f m e t a b o l i c a c t i v a t i o n i n t h e f i n a l c h e m i c a l -r e a c t i v e p o t e n t i a l o f c a r c i n o g e n s has o n l y begun t o c l a r i f y f o r c e r t a i n c l a s s e s o f t h e s e compounds ( r e v i e w e d by M i l l e r , 1970, 1973 ) , and i t i s now r e c o g n i z e d t h a t t h e r e a c t i v e f o rms o f t h e s e s t r u c t u r e s a r e e l e c t r o p h i I i c ( M i l l e r , 1970) . However , t h e p o s i t i o n o f t h e s e u l t i m a t e c e l l u l a r i n t e r a c t i o n s w i t h t h e r e a c t i v e c a r c i n o g e n m e t a b o -l i t e s i n t h e o v e r a l l scheme o f c a n c e r i n d u c t i o n r ema i n s o b s c u r e . I. The t e r m " c a r c i n o g e n i c c h e m i c a l s " w i l l r e f e r t o n o n - v i r a l and n o n - r a d i o a c t i v e c a r c i n o g e n i c c h e m i c a l s . 37 The most a t t r a c t i v e h y p o t h e s i s f o c u s e s on t h e i n t e r a c t i o n o f c a r -c i n o g e n m e t a b o l i t e s w i t h c e l l u l a r DNA i n such a manner as t o i n d u c e a f i x e d a l t e r a t i o n i n t h e i n f o r m a t i o n a l c o n t e n t o f t h e genome - t h e r e s u l t o f w h i c h l e a d s t o n e o p l a s t i c t r a n s f o r m a t i o n ( e . g . F a r b e r e t a J L , 1974) . I t i s t h e r e f o r e a p p a r e n t t h a t s t u d i e s d e s i g n e d t o demon-s t r a t e a b i o l o g i c a l r e s p o n s e t o c h e m i c a l p r e c a r c i n o g e n e x p o s u r e must n e c e s s a r i l y c o n s i d e r t h e s t a t u s o f m e t a b o l i c a c t i v a t i o n o f t h e t e s t c h e m i c a l s . The s t u d i e s h e r e i n w i l l a t t e m p t t o l i n k t h e phenomenon o f m e t a b o l i c a c t i v a t i o n w i t h t h e i n d u c t i o n o f DNA damage and r e p a i r i n bo th human c e l l s i n v i t r o , and m i c e i n v i v o . I t i s p e r t i n e n t t o t h e d e s i g n o f i n v e s t i g a t i o n s o f DNA damage and r e p a i r , c o n d u c t e d w i t h c u l t u r e d f i b r o b l a s t s , t h a t t h e t r e a t e d c e l l s a r e expo sed t o r e a c t i v e m e t a b o l i t e s o f t h e p r e c a r c i n o -g en s . These s t u d i e s w i l l employ t h r e e s o u r c e s o f r e a c t i v e p r e c a r -c i nogen m e t a b o l i t e s : 1. M e t a b o l i t e s g e n e r a t e d s y n t h e t i c a l l y f r om t h e p r e c a r -c i n o g e n , e . g . N -hyd roxy -AAF and N - a c e t o x y - A A F ( e . g . M i l l e r and M i l l e r , 1 9 6 9 a , b ) . 2. C h e m i c a l s t h a t a r e a p p a r e n t l y m e t a b o l i z e d by t h e c u l t u r e d f i b r o b l a s t s , e . g . 4NQ0 (Kawazoe et_ a i l_ . , 1972 ) . 3. M e t a b o l i t e s g e n e r a t e d i n v i t r o by e x p o s i n g p r e c a r c i n o g e n s t o t i s s u e e x t r a c t s ( e . g . G a r n e r e t a l . , 1972 ) . Two o b j e c t i v e s o f t h e s t u d i e s c o n d u c t e d i n v i t r o w i t h human f i b r o b l a s t s r e q u i r e a p p l i c a t i o n s o f t h e b i o p h y s i c a l t e c h n i q u e o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . F i r s t l y , t h e e x t e n t o f f r a g m e n t a t i o n o f human DNA, e s t i m a t e d by a l k a l i n e s u c r o s e g r a d i e n t 38 c e n t r i f u g a t i o n , w i l l be compared w i t h t h e l e v e l s o f DNA r e p a i r s y n t h e s i s , e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , f o l l o w i n g e x p o s u r e o f human c e l l s t o s y n t h e t i c a l l y - d e r i v e d m e t a b o l i t e s o f t h e p r e c a r c i n o g e n 2 - a c e t y I a m i n o f I u o r e n e . S e c o n d l y , t h e t i m e - c o u r s e o f r e p a i r o f DNA damage i n normal and r e p a i r - d e f i c i e n t XP c e l l s , e s t i m a t e d by c o m p a r a t i v e v e l o c i t y s e d i m e n t a t i o n o f c o n t r o l and t r e a t e d DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s , w i l l be compared w i t h t h e l e v e l s o f DNA r e p a i r s y n t h e s i s , e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , f o l l o w i n g e x p o s u r e t o 4NQ0. In o r d e r t o a s s e s s t h e e f f e c t o f m e t a b o l i t e s o f t h e m a j o r i t y o f p r e c a r c i n o g e n s , whose a c t i v e p r o d u c t s a r e no t a v a i l a b l e f rom s o u r c e s #1 o r #2, an i n v i t r o model s y s t em o f m e t a b o l i c a c t i v a t i o n o f c h e m i c a l p r e c a r c i n o g e n s w i l l be d e v e l o p e d . T h i s s y s t e m w i l l i n v o l v e m i x i n g p r e c a r c i n o g e n s w i t h f o r t i f i e d t i s s u e e x t r a c t s ( a c t i v a t i o n s y s t e m s ) w i t h c o n c o m i t a n t e x p o s u r e o f human " r e c e p t o r " c e l l c u l t u r e s . F o l l o w i n g e x p o s u r e s o f bo t h normal human c e l l s and r e p a i r - d e f i c i e n t human c e l l s t o t h e p r e c a r c i n o g e n s i n c o m b i n a t i o n w i t h t h e a c t i v a t i o n s y s t e m , t h e f o l l o w i n g b i o l o g i c a l r e s p o n s e s w i l l be a s s e s s e d : (a) DNA r e p a i r s y n t h e s i s , (b ) c h r omo -some a b e r r a t i o n s , and ( c ) e e l I s u r v i v a l . F u r t h e r m o r e , t h e e x t e n t o f DNA damage i n d u c e d by e x p o s u r e t o t h e a c t i v a t i o n s y s t e m , w i l l be e s t i m a t e d by u t i l i z i n g a t e c h n i q u e o f v e l o c i t y s e d i m e n t a t i o n o f DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s . The a c t i v a t i o n c a p a c i t y o f t i s s u e e x t r a c t s d e r i v e d f rom l i v e r , k i d n e y , and lung o f v a r i o u s a n i m a l s p e c i e s w i l l be compared by e s t i m a t i n g t h e l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal human c e l l s i n d u c e d by e x p o s u r e t o p r e c a r c i n o g e n s i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems c o n t a i n i n g t h e e x t r a c t s . 39 The a c t i v a t i o n c a p a c i t y o f v a r i o u s s u b c e l l u l a r f r a c t i o n s o f l i v e r s f rom d i f f e r e n t a n i m a l s p e c i e s w i l l be e s t i m a t e d by c o m p a r i n g t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n normal human and r e p a i r - d e f i c i e n t XP c e l l s f o l l o w i n g e x p o s u r e t o t h e p r e c a r -c i n o g e n a f l a t o x i n B l a l o n e , and i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems c o n t a i n i n g t h e v a r i o u s l i v e r homogenate f r a c t i o n s . The a b i l i t y o f " a c t i v a t e d " a f l a t o x i n s B l , B 2 , G l , and G2 , s t e r i g m a t o c y s t i n , and a f l a t o x i c o l , a p r o d u c t o f r e d u c t i v e m e t a b o l i s m o f a f l a t o x i n B l , t o i n d u c e DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s w i l l be d e t e r m i n e d w i t h a t t e n t i o n f o c u s e d on p o s s i b l e r e l a t i o n s h i p s between c h e m i c a l s t r u c t u r e , l e v e l s o f DNA r e p a i r s y n t h e s i s , and c a r c i n o g e n i c i t y . S t u d i e s o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage i n v ? vo w i l l be f a c i I i t a t e d by t h e a p p l i c a t i o n o f a t e c h n i q u e o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n f o r t h e s i m u I a t a n e o u s e s t i m a t i o n o f DNA f r a g m e n t a t i o n i n mouse l i v e r , k i d n e y , and lung f o l l o w i n g e x p o s u r e t o c a r c i n o g e n s i n v i v o . The q u e s t i o n o f o r g a n s p e c i f i c i t y o f c h e m i c a l c a r c i n o g e n - i n d u c e d tumor f o r m a t i o n w i l l be i n v e s t i g a t e d by e m p l o y i n g c h e m i c a l c a r c i n o g e n s t h a t e x h i b i t o r g a n - s p e c i f i c t umor i n d u c t i o n , e . g . 4NQ0 and DMN. The t i m e - c o u r s e o f r e p a i r o f DNA damage i n mouse l i v e r , k i d n e y , and lung w i l l be e s t i m a t e d by c o m p a r a t i v e v e l o c i t y s e d i m e n t a t i o n o f DNA, f r om t r e a t e d and u n t r e a t e d a n i m a l s , f o l l o w i n g s i n g l e s u b -c u t a n e o u s i n j e c t i o n s o f 4NQ0 o r DMN. In t h i s manner , a t t e m p t s w i l l be made t o c l a r i f y t h e r e l a t i o n s h i p s between t u m o r - i n d u c t i o n , m e t a b o l i c a c t i v a t i o n , and DNA damage and r e p a i r i n d u c e d i n mouse l i v e r , k i d n e y , and lung by 4NQ0 and DMN. 40 MATERIALS AND METHODS MATERIALS  Chemicals Nicotinamide adenine d inuc leo t ide phosphate (ox id ized and reduced forms), and gIucose-6-phosphate (monosodium s a l t ) were purchased from the Sigma Chemical Company, St. Lou i s , Mo. Sucrose, EDTA (e thy lened iaminetet raacet i c a c i d ) , sodium c h l o r i d e , sodium hydroxide and other common reagents were obtained from the F i sher Chemical Company, Vancouver, B.C. For the preparat ion of s c i n t i l l a t i o n f l u i d , 2,5-d iphenyloxazole (PPO) and l , 4 - d i ( - 2 ( 5 -phenyloxazoyI))-benzene (POPOP) were purchased from Kent Labora-t o r i e s , Vancouver, B.C. "Aquaso l " premixed s c i n t i l l a t i o n f l u i d was a product of the New England Nuclear Co rpora t i on , Do rva l , P.O. C o l c h i c i n e was purchased from B r i t i s h Drug Houses L t d . , Poo le, England. Sodium borohydride was purchased from the Sigma Chemical Company, St. Lou i s , Mo. Radiochemicals Thymidine-methyl - 3 H ( s p e c i f i c a c t i v i t y 20 Ci/mmol), thymidine-2- 1 1 *C ( s p e c i f i c a c t i v i t y >50 mCi/mmol), L - l euc i ne -4 ,5 - 3 H ( s p e c i f i c a c t i v i t y 36 Ci/mmol), cho l ine-methy I - 3 H ( s p e c i f i c a c t i v i t y 1.0 Ci/mmol), and l l 4 C-mixed amino ac id mixture ( s p e c i f i c a c t i v i t y of component amino ac ids ranged: 129-414 mCi/mmol) were obta ined from the New England Nuclear Corpora t i on , Dorva l , P.O. 41 C h e m i c a l C a r c i n o g e n s D i m e t h y l n i t r o s a m i n e (DMN) was p u r c h a s e d f r om K & K L a b o r a t o r i e s , P l a i n v i e w , N.Y. A f l a t o x i n s B l , B2 , G l , G2 , and s t e r i g m a t o c y s t i n were p u r c h a s e d f r om Makor C h e m i c a l s , J e r u s a l e m , I s r a e l . D r . Y. Kawazoe, N a t i o n a l C a n c e r C e n t r e R e s e a r c h I n s t i t u t e , T o k y o , k i n d l y p r o v i d e d h i g h l y p u r i f i e d s amp le s o f 4 - n i t r o q u i n o l i n e l - o x i d e (4NQ0) and 4 - a m i n o q u i n o I i n e l - o x i d e (4AQ0). D r . James A. M i l l e r , M c A r d l e L a b o r a t o r y f o r C a n c e r R e s e a r c h , U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , W i s c o n s i n , k i n d l y p r o v i d e d p u r i f i e d s amp le s o f 2 - a c e t y I a m i n o f i u o r e n e ( A A F ) , N - h y d r o x y - 2 - a c e t y I a m i n o f I u o r e n e ( N - h y d r o x y - A A F ) , and N - a c e t o x y - 2 - a c e t y I a m i n o f I u o r e n e ( N - a c e t o x y -A A F ) . M a t e r i a Is M i l l i p o r e f i l t e r s ( 2 . 5 cm, po re s i z e 0 .45 y) and M i l l i -p o r e f i l t e r h o l d e r s (model XXIO 025 03) were p u r c h a s e d f r om t h e M i l l i p o r e F i l t e r C o r p o r a t i o n , M o n t r e a l , P.Q. S i l i c a Ge l t h i n -l a y e r c h r oma tog r aphy p l a t e s (20 cm x 20 cm, s i l i c a g e l on p l a s t i c b a c k i n g s : Eastman "Ch romagram" Shee t # 6060) were o b t a i n e d f r om Eastman Kodak Company, R o c h e s t e r , N.Y. T i s s u e C u l t u r e S u p p l i e s E a g l e ' s m i n i m a l e s s e n t i a l medium (MEM), f e t a l c a l f se rum, k a n a m y c i n , f u n g i z o n e , p e n i c i l l i n , and s t r e p t o m y c i n were o b t a i n e d f r om t h e Grand I s l a n d B i o l o g i c a l Company, O a k l a n d , C a l i f . G l a s s c o v e r s l i p s were p u r c h a s e d f r om C o r n i n g G l a s s Work s , C o r n i n g , N.Y. P e t r i d i s h e s were o b t a i n e d f r om F a l c o n P l a s t i c s t h r o u g h F i s h e r S c i e n t i f i c Company, V a n c o u v e r , B.C. E x p e r i m e n t a l A n i m a l s A l l e x p e r i m e n t a l a n i m a l s were o b t a i n e d f rom t h e An imal U n i t , F a c u l t y o f M e d i c i n e , U n i v e r s i t y o f B r i t i s h C o l u m b i a . A n i m a l s were not i n b r e d and i n c l u d e d : Sw i s s m i c e ( o r i g i n : Connaught L a b o r a t o r i e s , W i l l o w d a l e , O n t a r i o ) , W i s t a r r a t s ( o r i g i n : B i o - b r e e d i n g L a b o r a t o r i e s , O t t a w a , . O n t a r i o ) , New Z e a l a n d Wh i t e r a b b i t s , M u s c o v i t e d u c k s , and S y r i a n h a m s t e r s . M i c e and r a t s m a i n t a i n e d d u r i n g e x p e r i m e n t a t i o n were f e d a s t a n d a r d P u r i n a Lab Chow and w a t e r ad l i b i t u m and s u b j e c t e d t o I2 -h I i g h t c y c l e s . 43 METHODS ESTIMATION OF IN VITRO DNA DAMAGE BY ALKALINE SUCROSE GRADIENT  CENTRIFUGATION The a l k a l i n e s u c r o s e g r a d i e n t (ASG) t e c h n i q u e s emp loyed i n t h e s e s t u d i e s o f DNA damage i n d u c e d i n c u l t u r e d mammalian c e l l s a r e m o d i f i c a t i o n s o f t h e t e c h n i q u e r e p o r t e d by M c G r a t h and W i l l i a m s (1966) and L e t t e_t aj_. ( 1 9 6 7 ) . S imi I a r mod i f i c a t i o n s o f t h e McGra th -Wi I I iams t e c h n i q u e have been a p p l i e d i n numerous i n v e s t i g a t i o n s o f DNA damage i n d u c e d by i o n i z i n g r a d i a t i o n ( e . g . McBurney et_ a j _ . , 1971) and c h e m i c a l c a r c i n o g e n s ( e . g . Andoh and T o s h i n o r i , 1972) . ASG Type I C u l t u r e s o f human d i p l o i d f i b r o b l a s t s were grown i n 3.5 cm d i a . p l a s t i c P e t r i d i s h e s ( w i t h o u t c o v e r s l i p s ) . The r e p l i -c a t i n g c e l l s were grown f o r 20 -24 h i n MEM c o n t a i n i n g 2-4 y C i / m l 3 H - T d R , f o l l o w e d by a 3-4 h i n c u b a t i o n i n n o n r a d i o a c t i v e MEM t o a l l o w f o r t h e i n c o r p o r a t i o n o f r e s i d u a l i n t r a c e l l u l a r a c i d - s o l u b l e r a d i o a c t i v i t y . To l a b e l DNA w i t h ^ C , c e l l s were grown i n 1 1 + C -TdR (0.1 y C i / m l ) i n MEM f o r 20 -24 h, f o l l o w e d by a 3-4 h i n c u b a t i o n i n n o n r a d i o a c t i v e MEM t o a l l o w f o r t h e i n c o r p o r a t i o n o f r e s i d u a l i n t r a -c e l l u l a r a c i d - s o l u b l e r a d i o a c t i v i t y . To l a b e l l i p i d componen t s , c e l l s were grown w i t h 3 H - c h o l i n e (I y C i / m l ) f o r 20 -24 h. P r o t e i n was l a b e l l e d by g r o w i n g c e l l s i n 3 H - l e u c i n e (I y C i / m l ) f o r 20 -24 h. 44 F o l l o w i n g e x p o s u r e t o t h e c h e m i c a l s a t 3 7° C, t h e c u l t u r e s were washed 3 t i m e s w i t h PBS (pH 7 . 0 ) , and s c r a p e d f r om t h e P e t r i d i s h e s i n t o v e r s e n e s o l u t i o n ( 0 .05$ EDTA i n PBS, pH 7 . 0 ) . The c e l l s u s p e n s i o n s were g e n t l y v o r t e x e d t o d i s p e r s e any a g g r e g a t e s . The vo lume o f each s u s p e n s i o n was a d j u s t e d t o g i v e a p p r o x i m a t e l y 1-2 x I 0 5 c e l l s / m l . A 0 . 2 ml p o r t i o n o f t h e c e l l s u s p e n s i o n ( abou t 2-4 x I 0 4 c e l l s c o n t a i n i n g abou t 3-6 x I 0 3 3 H - c o u n t s / m i n ) was p i p e t t e d on t o p o f a 0 .7 ml o v e r l a y ( 1 . 0 M NaOH - 0.01 M EDTA) on 12.3 ml o f a 10-30% l i n e a r s u c r o s e g r a d i e n t made up i n 0 . 3 M NaOH, 0.01 M EDTA, and 0 .5 M NaCI i n a n i t r o c e l l u l o s e c e n t r i f u g e t u b e . A f t e r a 30 -min l y s i s p e r i o d a t room t e m p e r a t u r e ( 2 5 ° C ) , t h e g r a d i e n t s were c e n t r i f u g e d i n b u c k e t s o f an SW-40 r o t o r on model L2 -65B u I t r a c e n t r i f u g e (Beckman I n s t r u m e n t s I n c . , P a l o A l t o , C a l i f . ) a t 20 ,000 r e v . / m i n and 2 0 ° C f o r 7 .5 h. F o l l o w i n g c e n t r i f u g a t i o n , a p p r o x i m a t e l y 30 s e q u e n t i a l f r a c t i o n s o f 15 d r o p s each were c o l l e c t e d f r o m t h e bo t tom o f p i e r c e d t u b e s , p r e c i p i t a t e d w i t h 8 -10% t r i c h l o r o a c e t i c a c i d ( T C A ) , and c o l l e c t e d on M i I 11 p o r e n i t r o c e l l u l o s e membrane f i l t e r s . A c i d - s o l u b l e r a d i o a c t i v i t y was removed by wa sh i ng t h e f i l t e r s w i t h 5-8% TCA. The a c i d - i n s o I u b I e r a d i o a c t i v i t y was c o u n t e d on e i t h e r a P a c k a r d T r i - C a r b Model 3314 o r a N u c l e a r - C h i c a g o M a r k ' I I A l i q u i d s c i n t i l l a t i o n s p e c t r o p h o t o m e t e r . T4 phage , used as a r e f e r e n c e , c o n t a i n e d 3 2 P - l a b e l l e d DNA, and was p r e p a r e d by D r . R. Mi I l e r , Depa r tment o f M i c r o b i o l o g y , w i t h i n I h p r i o r t o l a y e r i n g on t h e g r a d i e n t s . ASG Type 2 The p r o c e d u r e f o l l o w e d f o r e x p e r i m e n t s c o n d u c t e d w i t h 45 ASG Type 2 was i d e n t i c a l t o t h a t o f ASG Type I, t h e o n l y change b e i n g t h a t f e w e r c e l l s were l a y e r e d p e r g r a d i e n t : 8 -10 x I 0 3 c e l l s i n s t e a d o f 2 -4 x I 01* c e l l s . ASG Type 3 The t e c h n i q u e f o l l o w e d f o r e x p e r i m e n t s i n v o l v i n g ASG Type 3 was a d a p t e d f r om t h e ASG t e c h n i q u e d e v e l o p e d by Cox e t a I. (1973) i n t h e l a b o r a t o r y o f D r . E. F a r b e r f o r t h e e s t i m a t i o n o f DNA damage i n d u c e d i n r a t l i v e r i n v i v o . F o l l o w i n g c h e m i c a l t r e a t m e n t , t h e c e l l c u l t u r e s were r i n s e d 4 t i m e s w i t h c o l d EDTA/NaCI b u f f e r , v o r t e x e d l i g h t l y t o d i s p e r s e a g g r e g a t e s , p e l l e t t e d (2 m i n , 1000 x g ) , and r e s u s -pended i n s u f f i c i e n t EDTA/NaCI b u f f e r ( 100 -300 y l ) t o g i v e 5-10 x \0h e e l Is p e r 50 p i . An a I i q u o t ( 50 -250 y l c o n t a i n i n g about 1-5 x I 0 5 c e l l s ) o f t h e c e l l s u s p e n s i o n was added t o an o v e r l a y ( 0 . 3 m l ) o f a l y s i n g s o l u t i o n (0 .3 M NaC I , 0 .03 M EDTA, 0.1 M T r i s - H C I b u f f e r , and 0 .5% sod ium d o d e c y l s u l f a t e , pH 10) on t o p o f a l i n e a r a l k a l i n e s u c r o s e g r a d i e n t ( 5 - 2 0 % , 5 m l ) c o n t a i n i n g 0 .9 M NaCI and 0 .3 M NaOH, and p r e p a r e d o v e r a I ml c u s h i o n o f 2 .3 M s u c r o s e . An a d d i t i o n a l a l i q u o t o f 0 . 1 - 0 . 3 ml l y s i n g s o l u t i o n was added t o t h e t o p o f t h e e e l Is and t h e t u b e was f i l l e d w i t h m i n e r a l o i l . A f t e r a 10-min l y s i s p e r i o d a t room t e m p e r a t u r e ( 2 5 ° ) , t h e g r a d i e n t s were c e n t r i f u g e d i n b u c k e t s o f an SW-40 r o t o r on model L2 -65B u l t r a c e n t r i f u g e (Beckman I n s t r u m e n t s I n c . , P a l o A l t o , C a l i f . ) a t 25 ,000 r e v / m i n and 2 0 ° C f o r 30 m i n . F o l l o w i n g c e n t r i f u g a t i o n , a p p r o x i m a t e l y 15 s e q u e n t i a l f r a c t i o n s o f 16 d r o p s each were c o l l e c t e d f rom t h e bo t tom o f 46 p i e r c e d t u b e s , and a s s a y e d f o r r a d i o a c t i v i t y as d e s c r i b e d f o r ASG Type I. ACTIVATION OF CHEMICAL PRECARCINOGENS IN VITRO (a) P r e p a r a t i o n o f P o s t - M i t o c h o n d r i a l F r a c t i o n (9S F r a c t i o n ) o f  T i s s u e Homogenates A n i m a l s were k i l l e d by d e c a p i t a t i o n , e x s a n g u i n a t e d , and i m m e d i a t e l y d i s s e c t e d . A l l s u b s e q u e n t s t e p s were c o n d u c t e d w i t h s o l u t i o n s and c o n t a i n e r s m a i n t a i n e d a t 4 °C . L i v e r s , k i d n e y s , and l ung s were removed, t r immed o f a d h e r i n g c o n n e c t i v e t i s s u e , and p l a c e d i n t o s e p a r a t e b e a k e r s c o n t a i n i n g P B S / s u c r o s e b u f f e r (PBS, w i t h c a l c i u m and magnesium i o n s , c o n t a i n i n g 0 .25 M s u c r o s e , pH 7 . 5 ) . Organs o f t h e same t y p e were p o o l e d i n t h e same c o n t a i n e r o f P B S / s u c r o s e b u f f e r where t h e o r g a n s were wa shed , dabbed on a b s o r b e n t t i s s u e , and q u i c k l y t r a n s f e r r e d t o s e p a r a t e b e a k e r s o f f r e s h P B S / s u c r o s e b u f f e r such t h a t 6 g o f each o r g a n were c o n t a i n e d i n 10 ml b u f f e r . The o r g a n s were m inced w i t h s c i s s o r s and t h e c o n t e n t s o f each b e a k e r were t r a n s f e r r e d t o h o m o g e n i z a t i o n v e s s e l s . The t i s s u e s were t h e n homogen ized by 10 t o 12 u p - a n d -down s t r o k e s o f a l o o s e - f i t t i n g P o t t e r - E l v e j h e m Homogen i ze r o p e r a t i n g a t 1000 r e v . / m i n . The homogenate was c e n t r i f u g e d a t 9 ,000 x g f o r 10 min ( a t 4 U C . ) i n a p r e - c o o l e d r o t o r (Beckman, Type 40) and n i t r o c e l l u l o s e c e n t r i f u g e t u b e s . The r e s u l t i n g p o s t - m i t o c h o n d r i a l s u p e r n a t a n t f r a c t i o n s ( t o be r e f e r r e d t o as "9S f r a c t i o n s " ) , d e r i v e d f r om each t y p e o f o r g a n , were p o o l e d , 47 m i xed t h o r o u g h l y t o e n s u r e homogene i t y o f each b a t c h , and t h e n d i s t r i b u t e d i n measured a l i q u o t s t o p o l y p r o p y l e n e t u b e s , c a p p e d , and i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n . The t u b e s were s t o r e d i n a Revco f r e e z e r a t - 7 1 ° C. (b) P r e p a r a t i o n o f I 05 ,000xg F r a c t i o n s o f L i v e r Homogenates :  S u p e r n a t a n t ( I 05S ) and P e l l e t ( I 05P ) Fo r f u r t h e r f r a c t i o n a t i o n o f t h e 9S f r a c t i o n , t h e s u p e r n a t a n t f r o m t h e 9 ,000xg c e n t r i f u g a t i o n was c e n t r i f u g e d a t I 05 ,000xg f o r one hour ( a t 4 ° C ) . The s u p e r n a t a n t (105S f r a c t i o n ) was removed w i t h c a r e t o a v o i d c o n t a m i n a t i o n by s e d i m e n t e d m a t e r i a l , a l l o c a t e d t o p o l y p r o p y l e n e t u b e s , and i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n . The t u b e s were s t o r e d i n a Revco f r e e z e r a t - 7 1 ° C. The p e l l e t was r e s u s p e n d e d i n a vo lume o f f r e s h P B S / s u c r o s e b u f f e r by g e n t l e m i x i n g , d i r e c t l y i n t h e n i t r o c e l l u l o s e c e n t r i f u g e t u b e , w i t h a s m a l l - b o r e t e f l o n homogen i z e r ( P o t t e r -El v e j hem) o p e r a t i n g a t low s p e e d . The s u s p e n s i o n was c e n t r i f u g e d a g a i n a t I 05 ,000xg f o r I h, t h e s u p e r n a t a n t " w a s h " was d i s c a r d e d , and t h e p e l l e t was a g a i n r e s u s p e n d e d i n f r e s h P B S / s u c r o s e b u f f e r . F o l l o w i n g c e n t r i f u g a t i o n a t I 05 ,000xg f o r I h, t h e p e l l e t r e s u l t i n g f rom t h e second wash was r e s u s p e n d e d i n t h e o r i g i n a l vo lume o f P B S / s u c r o s e b u f f e r t o c o n s t i t u t e a s u s p e n s i o n o f "washed m i c r o s o m e s " ( I 05P f r a c t i o n ) . The s u s p e n s i o n was a l l o c a t e d t o p o l y p r o p y l e n e t u b e s and i m m e d i a t e l y f r o z e n i n l i q u i d n i t r o g e n . The I05S and I05P f r a c t i o n s were s t o r e d i n a Revco f r e e z e r a t - 7 1 ° C. 48 ( c ) P r e p a r a t i o n o f A c t i v a t i o n M i x t u r e s and T r e a t m e n t o f C e l l  C u I t u r e s F o r e x p o s u r e t o a p r e c a r c i n o g e n i n a c o m p l e t e a c t i v a t i o n s y s t e m , each c e l l c u l t u r e was t r e a t e d w i t h a 1:1 m i x t u r e o f t h e a c t i v a t i o n s y s tem ( A . S . ) and a s o l u t i o n o f t h e p r e c a r c i n o g e n . The A . S . was p r e p a r e d by d i s s o l v i n g , f o r each c e l l c u l t u r e t o be t e e a t e d , 4 . 0 ymo le s NADPH o r NADP, 25 ymo le s M g C I 2 > and 20 ymo le s 66P i n 0.4 ml o f 9S f r a c t i o n ( thawed i m m e d i a t e l y p r i o r t o u s e ) , c o n t a i n i n g e x t r a c t f r om a p p r o x i m a t e l y 240 mg o f o r g a n w e t - w e i g h t , and a d j u s t i n g t h e pH t o 7 .2 w i t h 0.1 N NaOH. The c h e m i c a l p r e c a r c i n o g e n s were d i s s o l v e d i n d i m e t h y l s u l f o x i d e (DMSO) and d i l u t e d w i t h MEM (ADM was used i n e x p e r i m e n t s m e a s u r i n g DNA r e p a i r s y n t h e s i s , and P B S / s u c r o s e was u sed i n p r e p a r a t i o n s d e s t i n e d f o r UV s p e c t r o g r a p h i c a n a l y s i s ) . F u r t h e r d i l u t i o n s were made w i t h MEM ( o r ADM, o r P B S / s u c r o s e b u f f e r ) . Mos t o f t e n , t h e p r e p a r a t i o n s i n v o l v e d abou t 2 mg q u a n t i t i e s o f c h e m i c a l s , d i s s o l v e d i n 0 .2 ml DMSO, and d i l u t e d w i t h aqueous medium t o g i v e 10 ml o f a 10 3 M c o n c e n t r a t i o n . F o r t r e a t m e n t o f c o n t r o l p r e p a r a t i o n s , i d e n t i c a l d i l u t i o n s o f DMSO a l o n e were p r e p a r e d . S i n c e t h e a c t i v a t i o n s y s t em and p r e c a r c i n o g e n s o l u t i o n were m i xed 1:1, s o l u t i o n s o f p r e c a r c i n o g e n s were p r e p a r e d a t t w i c e t h e d e s i r e d c o n c e n t r a t i o n s . Equa l vo lumes o f p r e c a r c i n o g e n s o l u t i o n and a c t i v a t i o n s y s tem ( u s u a l l y 0 .5 ml each ) were m i x e d , v o r t e x e d q u i c k l y , and t h e r e s u l t i n g a c t i v a t i o n m i x t u r e was added t o c e l l c u l t u r e s w i t h i n 49 5 s o f m i x i n g . The c u l t u r e s were t h e n i n c u b a t e d a t 37 C. i n an a tmosphe re o f a i r w i t h o u t f u r t h e r a g i t a t i o n . (d) P r o t e i n D e t e r m i n a t i o n The c o n c e n t r a t i o n o f p r o t e i n in each b a t c h o f 9S , I 05S, and I05P f r a c t i o n s was d e t e r m i n e d by t h e F o l i n - C i o c a I t e a u method , u s i n g s t a n d a r d p r o c e d u r e s . The r e s p o n s e o f t h e a s s a y was s t a n d a r d i z e d f o r each d e t e r m i n a t i o n , u s i n g f r e s h l y - p r e p a r e d s t a n d a r d s o f b o v i n e serum a l b u m i n . Volumes o f b a t c h e s , when n e c e s s a r y , were a d j u s t e d p r i o r t o use t o g i v e equa l c o n c e n t r a t i o n s o f p r o t e i n . (e ) A n a l y s i s o f Me thano l E x t r a c t s o f A c t i v a t i o n M i x t u r e s by  UV S p e c t r o s c o p y A c t i v a t i o n m i x t u r e s d e s t i n e d f o r UV s p e c t r o g r a p h i c a n a l y s i s were p r e p a r e d w i t h c h e m i c a l s d i s s o l v e d i n P B S / s u c r o s e b u f f e r t o a v o i d s p e c t r a l i n t e r f e r e n c e by t h e pheno l r e d i n d i c a t o r p r e s e n t i n MEM o r ADM. An a l i q u o t ( 0 . 8 m l ) o f a c t i v a t i o n m i x t u r e was added t o an e q u a l vo lume o f m e t h a n o l , s a t u r a t e d w i t h sod ium s u l f a t e , v o r t e x e d , and c e n t r i f u g e d i m m e d i a t e l y a t 2 ,000 x g f o r 3 min t o remove t h e p r o t e i n p r e c i p i t a t e . A I ml p o r t i o n o f t h e s u p e r n a t a n t was t h e n removed and s canned f rom 300 nm t o 400 nm on a Ca ry Model II s p e c t r o p h o t o m e t e r ( e . g . P a t t e r s o n and R o b e r t s , 1972) . The r e f e r e n c e c e l l c o n t a i n e d a methano l i c e x t r a c t o f an i d e n t i c a l p r e p a r a t i o n c o n t a i n i n g no c h e m i c a l . In i n s t a n c e s where a c t i v a t i o n m i x t u r e s were removed d i r e c t l y f r om c e l l c u l t u r e s f o r UV s p e c t r o s c o p y , t h e c e l l c u l t u r e s 50 were washed w i t h P B S / s u c r o s e b u f f e r p r i o r t o t r e a t m e n t . ( f ) A n a l y s i s o f C h l o r o f o r m E x t r a c t s o f A c t i v a t i o n M i x t u r e s by  T h i n - L a y e r Ch romatog raphy M e t h a n o l i c e x t r a c t s o f a c t i v a t i o n m i x t u r e s c o n t a i n i n g a f l a t o x i n B l were e x t r a c t e d w i t h equa l vo l umes o f c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was t h e n c o n c e n t r a t e d and s p o t t e d on S i l i c a G e l t h i n - l a y e r p l a t e s , d e v e l o p e d w i t h e t h y l a c e t a t e : c h l o r o f o r m ( 2 : 1 ) , and a n a l y z e d u n d e r UV l i g h t ( G a r n e r e t a I., 1972) . (g) S y n t h e s i s o f A f l a t o x i c o l The t e c h n i q u e o f G a r n e r e t a I . ( 1972 ) was f o l l o w e d . B r i e f l y , a f l a t o x i n B l was d i s s o l v e d i n c h l o r o f o r m and r e a c t e d w i t h s od ium b o r o h y d r i d e i n i s o p r o p y l a l c o h o l f o r abou t 90 min a t 2 5 ° C. The r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f d i l u t e a c e t i c a c i d and t h e p r o d u c t s were e x t r a c t e d w i t h c h l o r o f o r m . The c h l o r o f o r m e x t r a c t was s t r e a k e d on t h i n - l a y e r p l a t e s ( s i l i c a g e l ) and d e v e l o p e d w i t h e t h y l a c e t a t e : c h l o r o -f o rm ( 2 : 1 ) . A f l u o r e s c e n t band a t Rf 0 .54 was e l u t e d w i t h methano l a n d , on t h e b a s i s o f T L C , UV, and mass s p e c t r a l p r o p e r t i e s , a p p e a r e d i d e n t i c a l w i t h a f l a t o x i c o l ( G a r n e r e t a L , 1972 ) . TECHNIQUE FOR CULTURING HUMAN FIBROBLASTS FROM SKIN BIOPSIES S k i n - p u n c h b i o p s i e s were t a k e n f rom t h e f o r e a r m o f an XP p a t i e n t ( f e m a l e , I 8 - y e a r s - o I d , M . T . , Edmonton) and c o n t r o l 51 p e r s o n s o f c o m p a r a b l e age . The s k i n p i e c e was t e a s e d i n t o t i n y f r a g m e n t s w h i c h were s a n d w i c h e d between g l a s s c o v e r s l i p s and i n c u b a t e d i n MEM (15 -20% f e t a l c a l f serum) f o r 2 - 3 weeks a t 3 7 ° C i n a i n c u b a t o r . G rowth medium was changed e v e r y t h i r d day . When f i b r o b l a s t s a p p e a r e d t o m i g r a t e f rom t h e t i s s u e f r a g m e n t s , t h e " s a n d w i c h " was opened and t h e t i s s u e f r a g m e n t s were removed l e a v i n g a p a r t i a l m o n o l a y e r o f f i b r o b l a s t s on t h e c o v e r s l i p . These i n d i v i d u a l c o v e r s l i p s were a g a i n i n c u b a t e d as a b o v e , a l l o w -ing t h e f i b r o b l a s t s t o grow up i n t o a m o n o l a y e r . S u b s e q u e n t l y , t h e f i b r o b l a s t s were s u b c u l t u r e d by s t a n d a r d t e c h n i q u e s . The c u l t u r e s were k e p t i n p l a s t i c P e t r i d i s h e s and m a i n t a i n e d i n a p l a t e a u phase a t 3 7 ° C i n a C 0 2 i n c u b a t o r . The c e l l c u l t u r e s u sed t h r o u g h o u t t h e s e s t u d i e s were o f t r a n s f e r p a s s a ge s 3 t o 6. The c u l t u r e s were r o u t i n e l y m a i n t a i n e d i n E a g l e ' s MEM, s u p p l e m e n t e d w i t h \5% f e t a l c a l f serum and a n t i -b i o t i c s ( p e n i c i l l i n 200 U / m l , s t r e p t o m y c i n 40 y g / m l ) . IN VITRO BIOLOGICAL END-POINTS OF ANALYSIS (a) E s t i m a t i o n o f DNA R e p a i r S y n t h e s i s i n C u l t u r e d Human C e l l s C u l t u r e s o f human d i p l o i d f i b r o b l a s t s were grown e i t h e r i n L e i g h t o n t u b e s ( c o n t a i n i n g 10 x 35 mm g l a s s c o v e r s l i p s ) o r 3.5 cm d i a m e t e r p l a s t i c P e t r i d i s h e s ( c o n t a i n i n g 22 x 22 mm g l a s s c o v e r s l i p s ). When t h e r e p l i c a t i n g c e l l c u l t u r e s were e s t i m a t e d t o be abou t 50 -70% c o n f l u e n t , normal DNA r e p l i c a t i o n a s s o c i a t e d w i t h t h e d u p l i c a t i o n o f chromosomes was 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 ( S t i c h e t aj_. , 1970 ) . In o r d e r t o r e d u c e t h e l e v e l o f a r g i n i n e , t h e c o v e r s l i p s were removed, r i n s e d i n a r g i n i n e - f r e e MEM (ADM) and 52 t r an s fe r red into new Leighton tubes or Pet r i dishes conta in ing ADM and only 5% fe ta l ca l f serum. Fol lowing 1.5 days in ADM, the m i t o t i c rate and frequency of c e l l s in S-phase dropped below 0.1 % of those in normal c e l l cu l tu re s (S t ich et aj_., 1970). The cu l tu re s were usua l l y kept in ADM for 2.5-3.5 days before t r e a t -ment and es t imat ion of DNA repa i r s yn thes i s . Fo l lowing exposure t o the chemicals (d i s so lved in ADM), the c e l l cu l tu re s were r insed 3 times with ADM and then incubated at 37° C. with ADM conta in ing 10 yCi/ml 3H-TdR. Fol lowing the p u l s e - l a b e l i n g with 3H-TdR (3 h ) , the c e l l c u l t u r e s were prepared f o r autoradiography in the fo l l ow ing manner: r insed (3 t imes) with Hank's balanced s a l t s o l u t i o n , t rea ted with \% sodium c i t r a t e s o l u t i on f o r 10 min, f i xed in al coho l - ace t i c ac id (3:1)- f o r 10 min, washed 3 t imes (10 min each) in f resh f i x a t i o n s o l u t i o n , and a i r - d r i e d . The c o v e r s l i p s were attached to microscope s l i d e s with melted p a r a f f i n wax, r insed through PBS bu f fe r (pH 7.0) and 4 changes of d i s t i l l e d water, dipped into Kodak NTB-3 emulsion (d i l u ted 1:1 with d i s t i l l e d water ) , allowed to a i r - d r y f o r 30 min, s tored a t 4° C. in l i g h t - t i g h t boxes f o r 14 days, and developed by standard photographic procedures. The autoradiograms were s ta ined f o r 20 min in 2% o r c e i n - a c e t i c ac id (50%), and dehydrated by t r a n s f e r through : 100% e thano l , butano l , butano l / xylene (1:1), and two changes of xy lene. The autoradiograms were mounted in permount. The g ra ins appearing over d i p l o i d c e l l nuc le i in the autoradiographic preparat ions were counted. Gra in counts were r e s t r i c t e d to small interphase nuc le i and were e a s i l y 53 d i s t i n g u i s h a b l e f rom t h e o c c a s s i o n a l n u c l e u s t h a t e s c a p e d t h e b l o c k o f a r g i n i n e - d e p r i v a t i o n and a p p e a r e d as e x t r e m e l y h e a v i l y l a b e l l e d n u c l e i . A t l e a s t 30 n u c l e i , a t random l o c a t i o n s t h r o u g h o u t t h e e n t i r e c o v e r s l i p c u l t u r e , were a n a l y z e d f o r g r a i n number. When t h e g r a i n number a p p e a r e d be low 10 g r a i n s / n u c l e u s , a t l e a s t 100 n u c l e i were s c o r e d . (b) A n a l y s i s o f Chromosome A b e r r a t i o n s i n C u l t u r e d C e l l s C u l t u r e s o f human d i p l o i d f i b r o b l a s t s were grown i n 3 .5 cm d i a m e t e r p l a s t i c P e t r i d i s h e s ( c o n t a i n i n g 22 x 22 mm g l a s s c o v e r s l i p s ) . R e p l i c a t i n g c u l t u r e s , o f a b o u t 50 -70% c o n f l u e n c y , were s u b j e c t e d t o c h e m i c a l t r e a t m e n t and r i n s e d t h r e e t i m e s w i t h MEM (no serum) b e f o r e i n c u b a t i o n w i t h f r e s h MEM a t 3 7 ° C. F o r t h e e s t i m a t i o n o f chromosome a b e r r a t i o n s , d i v i d i n g c e l l s were a r r e s t e d a t metaphase w i t h c o l c h i c i n e ( 0 . 06 ml o f 0 . 001% c o l c h i c i n e s o l u t i o n p e r ml o f MEM) w h i c h was a p p l i e d 5 h p r i o r t o s a m p l i n g . The c o v e r s l i p c u l t u r e s were p r e t r e a t e d f o r 10 min i n 1% sod ium c i t r a t e s o l u t i o n , f i x e d i n a l c o h o l - a c e t i c a c i d ( 3 : 1 ) f o r 10 m i n , a i r - d r i e d , s t a i n e d i n 2% o r c e i n - a c e t i c a c i d ( 5 0 % ) , d e h y d r a t e d t h r o u g h 100% e t h a n o l , b u t a n o l , b u t a n o I / x y I e n e ( 1 : 1 ) , x y l e n e , and mounted w i t h pe rmount . F o r each s a m p l e , about 150 w e l l - s p r e a d metaphase p l a t e s were a n a l y s e d f o r c h r o m a t i d b r e a k s , e x c h a n g e s , and f r a g m e n t a t i o n . ( c ) A n a l y s i s o f C e l l S u r v i v a l In V i t r o Two t h o u s a n d human d i p l o i d f i b r o b l a s t s were seeded i n 54 5 cm diameter p l a s t i c Pe t r i d ishes (without c o v e r s l i p s ) and exposed 14 h l a te r to the chemicals . Fol lowing chemical treatment, the cu l tu re s were r insed three times and incubated in f resh MEM which was changed every three days. On the n inth day post - t reatment, the cu l tu re s were f i xed with e t h a n o l - a c e t i c ac id (3:1) , and s ta ined with \% t o l u i d i n e blue for 20 min, r insed with water, and a i r - d r i e d . The c lones conta in ing 50 c e l l s or more were counted under a regular d i s s ec t i n g microscope. U sua l l y , three to s i x cu l t u re s were averaged f o r each concentrat ion of t e s t chemica l . (d) Treatment of C e l l Cu l tures with Small Volumes A technique was developed whereby c u l t u r e s of- human f i b r o b l a s t s , grown on 22 mm square cove r s l i p s in 3.5 cm diameter Pe t r i d i shes , could be exposed t o volumes of carcinogen so lu t i ons as small as 50 y l . In a s t e r i l e hood (preferab ly not a blow-out hood to avoid drying the c u l t u r e s ) , a s e r i e s of new Pe t r i dishes (3.5 cm diameter) were placed beside the c e l l c u l t u r e s . S t e r i l e forceps were used to remove c o v e r s l i p s , dab them on s t e r i l e t i s s u e to remove excess medium, and p lace them in new Pet r i d i shes . Each c o v e r s l i p was sealed to the bottom of the Pe t r i d ish by gent ly press ing down in the centre of the c o v e r s l i p with the s t e r i l e forceps . Treatment so lu t i on s (e.g. 50 y l ) were added immediately to the centre of each c o v e r s l i p . With no aqueous media present in the new Pet r i d i s h , the treatment s o l u t i o n remained on top of the c o v e r s l i p . Treatments were f o r less than 45 min and * A de ta i l ed s t a t i s t i c a l eva luat ion of c lon ing data and gra in counts appears in the Ph.D. thes i s of Richard H.C. San, Un i ve r s i t y of B.C. 55 were h a l t e d by t h e a d d i t i o n o f 2 ml o f wash medium w i t h s u b s e q u e n t wa sh i n g i n t h e u s ua l manner. C u l t u r e s t r e a t e d i n t h i s manner c o u l d be p r o c e s s e d f o r t h e e s t i m a t i o n o f DNA r e p a i r s y n t h e s i s , f r e q u e n c y o f chromosome a b e r r a t i o n s , o r c e l l s u r v i v a l . F o r c e l l s u r v i v a l s t u d i e s w i t h c e l l s on 22 mm s q u a r e c o v e r s l i p s , about 500 c e l l s were seeded p e r 3.5 cm d i a m e t e r P e t r i d i s h and p r o c e s s e d i n t h e u s u a l way. ANALYSIS OF IN V IVO DNA DAMAGE (a) L a b e l l i n g o f DNA i n v i v o F o r s i m u l t a n e o u s l a b e l l i n g o f l i v e r , k i d n e y , and lung DNA, i n f a n t Sw i s s m i c e were i n j e c t e d s u b c u t a n e o u s l y w i t h s t o c k ^H-TdR. L i t t e r s i z e s were m a i n t a i n e d a t 8 o r 9 m i c e and r u n t s were d i s c a r d e d . The i n j e c t i o n s commenced when t h e m i c e were 2 -o r 3 - d a y s - o l d and c o n t i n u e d e v e r y s e cond day f o r a t o t a l o f 7 i n j e c t i o n s o f 25 y C i (25 y l ) e a c h . A gas c h r o m a t o g r a p h s y r i n g e , c a l i b r a t e d i n m i c r o l i t r e s and e q u i p p e d w i t h a l ong n e e d l e was u s e d . To a v o i d l e a k a g e , t h e s k i n o f v e r y s m a l l m i c e was p i n c h e d up and t h e n e e d l e was i n s e r t e d d o r s a I - p o s t e r i o r , and pushed under t h e l o o s e s k i n such t h a t 25 y l was d e p o s i t e d i n t h e neck r e g i o n o f t h e mouse. The n e e d l e was w i t h d r a w n and t h e s k i n h e l d t o g e t h e r f o r a few s e cond s t o e n s u r e a s e a l . M i c e were used when 4 - 6 months o l d . 56 (b) L a b e l l i n g o f P r o t e i n i n v i v o The l a b e l l i n g method o f Cox e_t_ a_L (1973) was m o d i f i e d f o r use w i t h m i c e . Young a d u l t Sw i s s m i c e , u s u a l l y c o n t a i n i n g 3 14 H - l a b e l l ed DNA, were i n j e c t e d w i t h 0.1 ml C -amino a c i d m i x t u r e (25 p C i ) e v e r y 4 h f o r a t o t a l o f 100 uC i g i v e n i n 4 i n j e c t i o n s . The m i c e were used 4 - 6 h a f t e r t h e l a s t i n j e c t i o n . ( c ) A d m i n i s t r a t i o n o f Chem i ca l C a r c i n o g e n s C h e m i c a l c a r c i n o g e n s were i n j e c t e d s u b c u t a n e o u s Iy i n e i t h e r DMSO v e h i c l e (0.1 ml p e r 25 g mouse) o r 0 .9% NaCI (0.1 ml p e r 25 g mouse ) . (d ) E s t i m a t i o n o f DNA Damage i n L i v e r Spatu I a - Squash T e c h n i q u e The method o f Cox e t a I. (1973) was e s s e n t i a I l y f o l l o w e d . The a n i m a l s were k i l l e d by c e r v i c a l d i s l o c a t i o n w i t h d e c a p i t a t i o n t o a l l o w adequa te e x s a n g u i n a t i o n . The l i v e r was removed q u i c k l y , s t r i p p e d o f c o n n e c t i v e t i s s u e and g a l l b l a d d e r , and washed i n i c e - c o l d E D T A / s a l i n e b u f f e r . The l i v e r was dabbed on a t i s s u e and p l a c e d i n a P e t r i d i s h bo t tom w h i c h was on t o p o f c r u s h e d i c e . I c e - c o l d E D T A / s a l i n e was added (I m l/g l i v e r ) and t h e l i v e r was m e t h o d i c a l l y s q u a s h e d , w i t h t h e spoon p o r t i o n o f a " s p o o n u l a " s p a t u l a , f o r 3-5 m i n . The e n t i r e homogenate was t r a n s f e r r e d t o a s m a l l , c o l d c e n t r i f u g e t u b e . C e l l a g g r e g a t e s and t i s s u e f r a g m e n t s were spun down a t 1,000 x g f o r 30 s i n p r e - c o o l e d c e n t r i f u g e b u c k e t s . A d i l u t e d a l i q u o t o f t h e s u p e r n a t a n t was u sed t o e s t i m a t e 57 the c e l l concentra t ion in a hemacytometer chamber. A l k a l i n e sucrose grad ients were prepared I h ahead of time according to Cox et_ aj_. (1973) and over-1 a id with 0.3 ml of lys ing s o l u t i on immediately p r i o r t o layer ing the c e l l suspension. About 10-50 yl of c e l l suspension, conta in ing about I x I0 5 c e l l s , was rou t i ne l y layered. Fol lowing a 30 min l y s i s p e r i o d , another a l i -quot of lys ing s o l u t i on (0.3 ml) was added gent ly down the wal ls of the cen t r i f u ge tube. The grad ients were o v e r l a i d with mineral o i l , and cent r i fuged according to Cox et a I. (1973). F rac t ions of 16 drops each were c o l l e c t e d and analyzed as descr ibed f o r ASG Type I (Page 43). Dounce Homogenization A Dounce t i s s u e - g r i n d e r (e.g. B e l l c o # 1984-00040) with a " l a r g e " c learance pe s t l e was used. A l l steps were c a r r i e d out at o 4 C and are o u t l i n e d in Table 12. (e) Est imation of DNA Damage in Kidney Spatula-Squash Technique The method of prepar ing k idney, fo r a l k a l i n e sucrose grad ient a n a l y s i s , by the spatula-squash technique was i den t i ca l to that descr ibed fo r l i v e r . Dounce Homogenization The method descr ibed f o r l i v e r preparat ions was a l so used f o r kidney (Table 12). 58 ( f ) Est imat ion of DNA Damage in Lung Mincing Technique The medial lobe of the r i gh t lung was removed from a f re sh l y k i l l e d , exsanguinated mouse, and placed in i c e - c o l d EDTA/sal ine bu f fe r in a p l a s t i c Pe t r i d i sh . The Pe t r i d i sh was kept on top of crushed ice throughout the procedure. A c o l d , sharp (new) razor blade was used f o r each prepar -a t i o n . The an te r i o r t i p of the lung lobe was cut o f f (about 2 mm) and another s l i c e was removed as a rectangu lar shape of 2-3 mm in width. Watch-maker tweezers were used to' handle the t i s s u e p iece which was 4 mg wet-weight. The 4 mg lung p iece was cut to produce p ieces of 1-2 mg wet-weight. Each p iece was p laced in separate regions of a large Pe t r i d i sh which was on top of crushed i c e , and 10 pi of i c e - c o l d EDTA/sal ine was added t o each p i e c e . Each 1-2 mg p iece was minced with the razor blade by chopping s t r a i g h t up-and-down while turn ing the Pe t r i d i sh with the o ther hand. The t i s s u e was qu i ck l y s t ruck about 100-130 times with the blade u n t i l a l l p ieces were s u f f i c i e n t l y fragmented to pass through the bore of a 10 yl Corning d i sposab le micro-sampling pi pet (e.g. cat.# 7099-S). The mi need fragments were drawn into a 20 yl or 50 yl sampling pi pe t , and layered on the a l k a l i n e sucrose gradients as descr ibed f o r l i v e r c e l l suspensions. The region of the Pe t r i d i s h , where the mincing was c a r r i e d ou t , was subsequently washed twice with 10 yl por t ions of EDTA/sal ine bu f fe r and the two washes were layered on the grad ient as w e l l . 59 F o l l o w i n g a 25 -m in l y s i s p e r i o d , t h e t i p o f a f i r e -f u s e d 20 uI m i c r o - s a m p l i n g p i p e t was i n s e r t e d j u s t unde r t h e s u r f a c e o f t h e l y s i n g s o l u t i o n , a g a i n s t t h e w a l l o f t h e c e n t r i f u g e t u b e . The t u b e was t h e n g e n t l y r o t a t e d a b o u t 4 t i m e s t o remove a i r b u b b l e s t h a t o r i g i n a t e d i n t h e lung f r a g -ments and c o l l e c t e d on t h e s u r f a c e o f t h e l y s i n g s o l u t i o n . A n o t h e r 0 .3 ml o f l y s i n g s o l u t i o n was added down t h e w a l l s o f t h e c e n t r i f u g e t u b e and t h e g r a d i e n t s were c e n t r i f u g e d f o l l o w i n g a t o t a l o f 30 min o f l y s i s . C e n t r i f u g a t i o n and a n a l y s i s o f f r a c t i o n s were as d e s c r i b e d f o r t h e e s t i m a t i o n s o f I i v e r DNA damage. Pounce H o m o g e n i z a t i o n The method o f ' h o m o g e n i z i n g lung w i t h a Dounce t i s s u e - g r i n d e r was i d e n t i c a l t o t h a t emp loyed f o r l i v e r and k i d n e y p r e p a r a t i o n s and i s e s s e n t i a l l y d e s c r i b e d i n T a b l e 12. (g) DNA D e t e r m i n a t i o n The amount o f DNA l a y e r e d / g r a d i e n t was e s t i m a t e d f r o m a l i q u o t s o f c e l l s u s p e n s i o n s and t i s s u e f r a g m e n t s o f l i v e r , k i d n e y , and lung by t h e s t a n d a r d d i p h e n y l a m i n e d e t e r m i n a t i o n o f DNA c o n c e n t r a t i o n . (h) H i s t o l o g i c a l P r e p a r a t i o n s P i e c e s o f l i v e r , k i d n e y , o r l ung t i s s u e were f i x e d i n a c e t i c a c i d : e t h a n o l ( 1 : 3 ) f o r 4 -6 h and t h e n p l a c e d i n 70% e t h a n o l o v e r n i g h t . D e h y d r a t i o n , c l e a r i n g , embedd i n g , s e c t i o n i and s t a i n i n g ( h e m a t o x y I i n / e o s ! n ) were by s t a n d a r d t e c h n i q u e s . 61 RESULTS AND DISCUSSION SECTION I DNA DAMAGE AND REPAIR IN CULTURED MAMMALIAN CELLS EXPOSED  TO PRECARCINOGENS AND SYNTHETIC CARCINOGENIC METABOLITES RESULTS C u l t u r e d human c e l l s have been shown t o e x h i b i t DNA r e p a i r s y n t h e s i s f o l l o w i n g t r e a t m e n t w i t h . c h e m i c a I c a r c i n o g e n s . The l e v e l s o f DNA r e p a i r s y n t h e s i s were commonly e s t i m a t e d by a b i o a s s a y i n v o l v i n g t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( r e v i e w e d by S t i c h and L a i s h e s , 1973) . However , t h e mechanism o f DNA r e p a i r s y n t h e s i s i n mammalian c e l l s i s c o m p l e x , p o o r l y u n d e r s t o o d , and t h e r e f o r e r e q u i r e s i n v e s t i g a t i o n by d i f f e r e n t t e c h n i q u e s . The o b s e r v a t i o n o f c h e m i c a l c a r c i n o g e n - i n d u c e d u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR i n t o mammalian c e l l s r e f l e c t s t h e " r e s y n t h e s i s " o f e x c i s e d r e g i o n s o f damaged DNA. On t h e o t h e r hand , t h e b i o p h y s i c a l t e c h n i q u e o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f f e r s t h e a d v a n t a g e o f a d i r e c t d e m o n s t r a t i o n o f a l t e r e d s e d i m e n t a t i o n p r o p e r t i e s o f c h e m i c a l c a r c i n o g e n - t r e a t e d DNA, p r e s u m a b l y a r i s i n g t h r o u g h t h e f r a g m e n t a t i o n o f t h e DNA i n t o s m a l l e r p i e c e s . S p e c i f i c a l l y , t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e i n v o l v e s t h e v e l o c i t y s e d i m e n t a t i o n o f DNA, e x t r a c t e d f r o m e i t h e r t r e a t e d o r u n t r e a t e d human c e l l s , t h r o u g h a c o n t i n u o u s l i n e a r g r a d i e n t o f a l k a l i n e s u c r o s e . If m e a n i n g f u l e s t i m a t e s o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage were t o be o b t a i n e d w i t h t h e a l k a l i n e s u c r o s e g r a d i e n t method , i t was e s s e n t i a l t o employ a t e c h n i q u e t h a t wou ld r educe t h e amount o f damage i nduced i n DNA by m a n i p u l a t i o n d u r i n g - e x t r a c t i o n 62 and h a n d l i n g . T h i s was a c c o m p l i s h e d by u s i n g m o d i f i c a t i o n s o f t h e M c G r a t h and W i l l i a m s t e c h n i q u e (McGra th and W i l l i a m s , 1966 ) , s i m i l a r t o t h a t f i r s t emp loyed by L e t t et_ aj_. (1967) f o r mammalian c e l l s In t h i s manner, e x t e n s i v e m e c h a n i c a I - s h e a r damage o f DNA was a v o i d e d by l y s i n g c e l l s i n a l y s i n g s o l u t i o n o v e r l a y p l a c e d d i r e c t l y on t o p o f t h e g r a d i e n t . Th ree a l k a l i n e s u c r o s e g r a d i e n t (ASG) t e c h n i q u e s were d e v e l o p e d i n t h e s e s t u d i e s , a l l o f w h i c h i n v o l v e d m o d i f i c a t i o n s o f t h e McGra th and W i l l i a m s t e c h n i q u e : (a) ASG Type I, (b) ASG Type 2 , and ( c ) ASG Type 3. S i n c e t h e r e s u l t s and i n t e r p r e t a t i o n s o f t h e a n a l y s i s depend on t h e m o d i f i c a t i o n o f t h e b a s i c M c G r a t h -W i l l i a m s t e c h n i q u e e m p l o y e d , t h e t e c h n i c a l d e v e l o p m e n t o f t h e t h r e e ASG t e c h n i q u e s w i l l be d e s c r i b e d t o g e t h e r w i t h t h e a p p l i c a t i o n s o f e a c h . (a ) ASG Type I ( i ) Speed o f C e n t r i f u g a t i o n I t was d e c i d e d t o employ a common " h i g h s a l t " a l k a l i n e g r a d i e n t base (McBurney e_t a j _ . , 1971) and t o u t i l i z e a 10-30% l i n e a r s u c r o s e g r a d i e n t d e s i g n e d t o f i l l t h e n i t r o c e l l u l o s e c e n t r i -f u ge t u b e s o f an SW-40 s w i n g i n g - b u c k e t r o t o r o f a Beckman L2 -65B u I t r a c e n t r i f u g e . To b e g i n c h o o s i n g a speed o f c e n t r i f u g a t i o n , an e x p e r -iment was c a r r i e d o u t whereby 3 H-DNA, r e l e a s e d f r om abou t 2 x lO 1* u n t r e a t e d ( c o n t r o l ) human s k i n f i b r o b l a s t s , was s e d i m e n t e d t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s . P r e l i m i n a r y r un s a t 35 ,000 r e v . / m i n 63 F i g u r e 13. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om 2 x I 0 4 human s k i n f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 35 ,000 r e v . / m i n f o r 3.5 h a t 2 0 ° C. D u p l i c a t e r u n s . F i g u r e 14. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i I e s o f 3H-DNA r e l e a s e d f r om human s k i n f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 2 0 , 000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. The number o f c e l l s l a y e r e d p e r g r a d i e n t : 6-7 x IO 4 ( • — » ) , 4 -5 x I 0 4 ( O — O ) , 2 -3 x 10k (•—•). F i g u r e 15. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om human s k i n f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. The number o f c e l l s l a y e r e d p e r g r a d i e n t : 12-15 x I 0 3 ( © — • ) , 12-15 x I 0 3 ( 0 - - 0 ) , 8 -10 x I 0 3 (• — • ) . . il 13 v \ °**... " A o • - 0 " X ! ° — i — 10 so F R A C T I O N N U M B E R i — S E D I M E N T A T I O N 14 — I — I O - 1 — 3 0 ao F R A C T I O N N U M B E R 1 — S E D I M E N T A T I O N 15 ri X I O — i — so F R A C T I O N N U M B E R — S E D I M E N T A T I O N I 3 0 65 p o s i t i o n e d a peak o f DNA r a d i o a c t i v i t y w i t h i n t h e l ower h a l f o f t h e g r a d i e n t a f t e r 3.5 h o f c e n t r i f u g a t i o n . Sub sequen t r e p e a t s o f t h i s e x p e r i m e n t p r o d u c e d n o n - r e p r o d u c i b l e s e d i m e n t a t i o n p r o -f i l e s o f DNA r a d i o a c t i v i t y , e xamp le s of w h i c h a r e d e p i c t e d i n F i g u r e 13. The p r o f i l e s e x h i b i t e d bo th e r r a t i c p o s i t i o n i n g o f peaks o f DNA r a d i o a c t i v i t y and f r e q u e n t s h a r p p e a k s , r a t h e r t h a n p r o f i l e s r e s e m b l i n g a more normal d i s t r i b u t i o n o f DNA f r a g m e n t s . I t was t h e r e f o r e d e c i d e d t o r e d u c e t h e speed o f c e n t r i -f u g a t i o n t o 20 ,000 r e v . / m i n i n an e f f o r t t o s t a b i l i z e t h e p o s i -t i o n i n g o f t h e peaks o f DNA r a d i o a c t i v i t y . E x p e r i m e n t s were d e s i g n e d t o e s t a b l i s h an a p p r o p r i a t e d u r a t i o n o f c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n such t h a t c o n t r o l DNA p r o f i l e s wou ld a p p e a r w i t h i n t h e bo t tom h a l f o f t h e g r a d i e n t . P r o f i l e s s i m i l a r t o t h o s e i n F i g u r e 14 were o b t a i n e d w i t h 2 x I 0 4 l a y e r e d c e l l s a f t e r 7.5 h o f c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n a t 2 0 ° C. These p r o f i l e s were h i g h l y r e p r o d u c i b l e i n 25 o u t o f 26 c o n s e c u t i v e e x p e r i m e n t s , d i d no t p r e s e n t s h a r p peaks as i n t h e p r o f i l e s o f F i g u r e 13, and showed " b i m o d a l t a i l i n g " i n 3 o u t o f 26 r u n s . ( i i ) Number o f C e l l s L a y e r e d W i t h a c e n t r i f u g a t i o n speed s e t a t 20 ,000 r e v . / m i n f o r a d u r a t i o n o f 7 .5 h, i t was n e c e s s a r y t o d e t e r m i n e t h e e f f e c t o f t h e number o f c e l l s l a y e r e d on t h e morpho logy o f t h e s e d i m e n t a t i o n p r o f i l e and t h e l o c a t i o n o f t h e peak of DNA r a d i o a c t i v i t y w i t h i n t h e g r a d i e n t . 66 E x p e r i m e n t s were c a r r i e d o u t i n wh i ch g r a d i e n t s were c e n t r i f u g e d s i m u l t a n e o u s l y , each c o n t a i n i n g DNA r e l e a s e d f r om a d i f f e r e n t number o f c e l l s : 6-7 x I 0 \ 4 -5 x \0k, 2 -3 x I 0 4 , 12-15 x I 0 3 , and 8-10 x I 0 3 e e l I s . W i t h 2 -3 x \0k and 4 -5 x I 0 4 l a y e r e d c e l l s , a p r o m i n e n t peak o f DNA r a d i o a c t i v i t y appea red a t f r a c t i o n 5 ( F i g u r e 14 ) . I n c r e a s i n g t h e c e l l number l a y e r e d t o 6 -7 x 101* had t h e e f f e c t o f mov ing t h i s peak f u r t h e r down t h e g r a d i e n t , t o w a r d s t h e b o t t o m , a t f r a c t i o n 3 ( F i g u r e 14) . When t h e c e l l number l a y e r e d was d e -c r e a s e d t o 12-15 x I 0 3 , an uneven , b imoda l p r o f i l e a p p e a r e d w i t h a new peak o f DNA r a d i o a c t i v i t y o c c u r r i n g a t abou t f r a c t i o n 10 ( F i g u r e 15 ) . F u r t h e r r e d u c t i o n o f t h e c e l l number l a y e r e d t o 8-10 x I 0 3 enhanced t h e amount o f DNA r a d i o a c t i v i t y a p p e a r i n g i n t h e r e g i o n o f f r a c t i o n 10 w i t h a d i s t i n c t 3 H-DNA peak a t f r a c t i o n I I ( F i g u r e 15 ) . W i t h 2-4 x \0h c e l l s l a y e r e d p e r g r a d i e n t - t o be r e -f e r r e d t o a s ASG Type I - a peak o f c o n t r o l DNA r a d i o a c t i v i t y was r o u t i n e l y o b s e r v e d a t f r a c t i o n 5. The s i m i l a r i t y between ASG s e d i m e n t a t i o n p r o f i l e s o b t a i n e d w i t h 3 H-DNA r e l e a s e d f rom e i t h e r normal human s k i n f i b r o b l a s t s o r xe rode rma p igmentosum (XP) s k i n f i b r o b l a s t s i s i l l u s t r a t e d i n F i g u r e 16. About one o u t o f e i g h t c o n t r o l p r o f i l e s e x h i b i t e d s l i g h t t a i l i n g t h a t i s s u g g e s t i v e o f t h e b imoda l p r o f i l e s o b t a i n e d w i t h 10-15 x I 0 3 c e l l s l a y e r e d . T h i s t a i l i n g e f f e c t was o b t a i n e d w i t h bo th normal and XP c e l l s ( F i g u r e ' 17 ) . The p o s i t i o n o f s e d i m e n t a t i o n o f T4 phage DNA ma r ke r i s i l l u s t r a t e d i n F i g u r e 16. T h i s o b s e r v a t i o n i n d i c a t e d t h a t t h e S 67 v a l u e o f t h e c o n t r o l DNA peak a t f r a c t i o n 5 i s a bou t 150. F o l l o w i n g g r a d i e n t c a l i b r a t i o n w i t h T4 phage , t h e m o l e c u l a r w e i g h t o f t h e c o n t r o l DNA peak , a t f r a c t i o n 5, was e s t i m a t e d t o be 4 .4 x I O 8 , u s i n g t h e r e l a t i o n s h i p o f S t u d i e r ( 1 9 6 5 ) . R e s e r v a t i o n s c o n c e r n i n g t h e use o f m o l e c u l a r w e i g h t c a l c u l a t i o n s w i l l be d i s c u s s e d . ( i i i ) L y s i s C o n d i t i o n s D i f f i c u l t y w i t h c o n s i s t e n c y i n p o s i t i o n i n g c o n t r o l DNA s e d i m e n t a t i o n p r o f i l e s has been c o u n t e r e d by e m p l o y i n g v a r i o u s , methods d e s i g n e d t o p a r t i a l l y deg rade c o n t r o l DNA i n o r d e r t o enhance d e n a t u r a t i o n and r e d u c e t h e " g e I - a g g r e g a t e " c h a r a c t e r o f t h e s e d i m e n t i n g DNA: 1. " z e r o d o s e " amounts o f i o n i z i n g r a d i a t i o n , e i t h e r X - r a y s ( e . g . L e t t e t a I . , 1967; McBurney e t a_l_., 1971) o r y - r a y s ( e . g . Whee l e r e t a j _ . , 1974 ) , a d m i n i s t e r e d t o c e l l s p r i o r t o l a y e r i n g , o r 2. r i g o r o u s l y s i s c o n d i t i o n s o f e i t h e r l ong d u r a t i o n o r h i g h a l k a l i n i t y (e.g. . Moroson and F u r l a n , 1969 ) . I t was d e c i d e d t o f o l l o w t h e e x p e r i e n c e o f Moroson and F u r l a n (1969) and f i x a l y s i s p e r i o d a t a c o n v e n i e n t d u r a t i o n o f 30 min b u t employ a r e l a t i v e l y s t r o n g c o n c e n t r a t i o n o f NaOH (I M) i n t h e l y s i n g s o l u t i o n . W i t h t h i s s t r o n g a l k a l i n e l y s i n g s o l u t i o n , no e f f e c t o f 0 .3% sod ium dodecy I s u l f a t e on c o n t r o l o r t r e a t e d DNA p r o f i l e s was o b s e r v e d . T h e r e f o r e , no d e t e r g e n t was i n c l u d e d i n t h e l y s i n g s o l u t i o n . The f a c t t h a t t h e s t r o n g a l k a l i n e l y s i s c o n d i t i o n s m i g h t have been i n d u c i n g a c e r t a i n amount o f b a s e - c a t a l y z e d h y d r o l y s i s o f DNA 68 F i g u r e 16. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom normal human f i b r o b l a s t s (•—•) and XPe f i b r o b l a s t s ( O — O ) f o l l o w i n g c e n t r i f u g a t i on a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. The number o f c e l l s l a y e r e d p e r g r a d i e n t : 2-4 x IO 4 . The a r r o w i n d i c a t e s t h e p o s i t i o n o f s e d i m e n t a t i o n o f T4 phage DNA m a r k e r . F i g u r e 17. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom normal human f i b r o b l a s t s (•—•) and XPe f i b r o b l a s t s ( O — O ) f o l l o w i n g c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. The number o f c e l l s l a y e r e d p e r g r a d i e n t : 2 -4 x I O 4 . 69 F R A C T I O N N U M B E R S E D I M E N T A T I O N 70 was s u g g e s t e d by t h e f a c t t h a t l o n g e r l y s i s p e r i o d s o f 60 min o r more p r oduced a s h i f t o f t h e peak of DNA r a d i o a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t . T h i s i n d i c a t e d t h a t DNA f r a g m e n t a t i o n was o c c u r r i n g d u r i n g t h e l o n g e r l y s i s p e r i o d s . ( i v ) E x t e n t o f P r o t e i n and l i p i d C o n t a m i n a t i o n o f Sed imen ted DNA The e x t e n t o f p r o t e i n c o n t a m i n a t i o n o f t h e s e d i m e n t e d DNA r a d i o a c t i v i t y i n ASG Type I g r a d i e n t s , was d e t e r m i n e d by l a b e l l i n g o f DNA w i t h 3 H - l e u c i n e w i t h s i m u l t a n e o u s l a b e l l i n g o f DNA w i t h ^ C - t h y m i d i n e . S e d i m e n t a t i o n p r o f i l e s were a n a l y z e d f o l l o w i n g 30 -m in . l y s i s and c e n t r i f u g a t i o n o f t h e DNA and p r o t e i n r a d i o a c t i v i t y t h r o u g h a l k a l i n e g r a d i e n t s a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. The r e s u l t i n g s e d i m e n t a t i o n p r o f i l e s r e v e a l e d t h a t t h e m a j o r i t y o f t h e a c i d - p r e c i p i t a b Ie p r o t e i n r a d i o a c t i v i t y banded nea r t h e t o p o f t h e g r a d i e n t w i t h up t o \% o f t h e t o t a l 3 H - p r o t e i n c o u n t s a p p e a r i n g w i t h t h e p e l l e t t e d 1 4 C - D N A and no d e t e c t a b l e 3 H - p r o t e i n c o u n t s c o - s e d i m e n t i ng w i t h t h e peak o f c o n t r o l 1 I + C-DNA ( F i g u r e 18) . P r o f i l e s , s i m i l a r t o t h o s e i n F i g u r e 18, were o b t a i n e d w i t h d o u b I e - I a b e I I e d XPe c e l l s and w i t h bo th normal and XPe c e l l s when l i p i d s were l a b e l l e d w i t h 3 H - c h o l i n e . D u p l i c a t e e x p e r i m e n t s , i n v o l v i n g d o u b I e - I a b e l I i ng w i t h 1 4 C - t h y m i d i n e and e i t h e r 3 H - l e u c i n e o r 3 H - c h o I i n e , were c a r r i e d o u t w i t h bo th normal and XPe c e l l c u l t u r e s w h i c h were expo sed t o t h e c a r c i n o g e n s N - a c e t o x y - A A F o r 4NQ0 j u s t p r i o r t o l a y e r i n g . The r e s u l t i n g s e d i m e n t a t i o n p r o f i l e s r e v e a l e d t h a t no d e t e c t a b l e 3 H - p r o t e i n 71 F i g u r e 18. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f l l *C-DNA (• — • ) and 3 H - p r o t e i n ( O — O ) r e l e a s e d f rom 2-4 x lO 1* normal human f i b r o -b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. S e d i m e n t a t i o n p r o f i l e s o f l t f C-DNA and 3 H - p r o t e i n r e l e a s e d f rom XPe f i b r o b l a s t s were s u p e r i m p o s a b I e on t h o s e d e r i v e d f rom t h e normal c e l l s . F i g u r e 19. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 1 4 C - D N A (•—•) and 3 H - p r o t e i n (O—O) r e l e a s e d f rom 2-4 x 101* normal c e l l s f o l l o w i n g I h e x p o s u r e t o I x 10 4 M N - a c e t o x y - A A F , and l l + C-DNA (•--•) and 3 H - p r o t e i n ( s u p e r imposab I e on 3 H - p r o t e i n p r o f i l e ( O — O ) ) • re I e a s ed f r om 2-4 x 10k normal e e l Is f o l l o w i n g I h e x p o s u r e t o 5 x 10 6 M 4NQ0. C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. 72 18 m h 2 3 0 (J J <• H 0 h z 111 o a Ul a V \ aa •'*' ^—n I O — i — 3 Q F R A C T I O N N U M B E R I — S E D I M E N T A T I O N i o H s H ) I O 2 Q 3 0 F R A C T I O N N U M B E R S E D I M E N T A T I O N 73 o r 3 H — l i p i d r a d i o a c t i v i t y c o - s e d i m e n t e d w i t h t h e peaks o f f r a g m e n t e d l t f C-DNA and t h a t a l l o f t h e d e t e c t a b l e p r o t e i n o r l i p i d r a d i o -a c t i v i t y a p p e a r e d t o band a t t h e t o p o f t h e g r a d i e n t ( F i g u r e 19) . The l a c k o f d e t e c t a b l e 3 H - p r o t e i n o r 3 H — l i p i d r a d i o -a c t i v i t y c o - s e d i m e n t i n g w i t h e i t h e r c o n t r o l ^ C - D N A peaks o r f r a g -mented l l t C-DNA p e a k s , f o l l o w i n g c a r c i n o g e n e x p o s u r e , i n d i c a t e d t h a t t h e a l k a l i n e l y s i n g s o l u t i o n was e f f i c i e n t i n r e l e a s i n g s e d -imented 1 1 + C-DNA f r om g r o s s c o n t a m i n a t i o n by c e l l u l a r p r o t e i n and l i p i d a c i d - p r e c i p i t a b Ie mac romo lecu I e s . ( v ) A p p l i c a t i o n s o f ASG Type I The u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R was u sed t o e s t i m a t e t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n c u l t u r e d human c e l l s e xpo sed t o AAF and i t s m e t a b o l i t e s ( S t i c h e t a j _ . , 1972) . The r e s u l t s showed t h a t no d e t e c t a b l e DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s f o l l o w e d e x p o s u r e t o 2 x I O - 5 M ' AAF , a low l e v e l o f r e p a i r s y n t h e s i s f o l l o w e d e x p o s u r e t o 2 x I O - 5 N - h y d r o x y -AAF, and a h i g h l e v e l o f r e p a i r s y n t h e s i s f o l l o w e d e x p o s u r e t o 2 x I O - 5 M N - a c e t o x y - A A F . In t h e f o l l o w i n g e x p e r i m e n t s , t h e r e l a t i v e DNA damage i n d u c e d i n human s k i n f i b r o b l a s t s by e x p o s u r e t o AAF, N - h y d r o x y - A A F , and N - a c e t o x y - A A F was d e m o n s t r a t e d by m e a s u r i n g t h e s e d i m e n t a t i o n o f DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s . The e x t e n t o f DNA damage was i n d i c a t e d by t h e amount o f s h i f t o f t h e peaks o f t r e a t e d 3 H-DNA t o w a r d s t h e t o p o f t h e a l k a l i n e g r a d i e n t s . I n i t i a l e x p e r i m e n t s i n v o l v e d t r e a t m e n t o f t h e c e l l c u l t u r e s w i t h 4NQ0 t o c o r r e l a t e t h e d a t a w i t h p r e v i o u s s t u d i e s o f j 74 F i g u r e 20. A I k a I i n e , s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom 2 -4 x \0k human s k i n f i b r o b l a s t s e xpo sed t o v a r i o u s c o n c e n t r a t i o n s o f 4NQ0. The 4NQ0 do se s g i v e n t o t h e c e l l s : c o n t r o l (• — • ) , 4 x I 0 ~ 7 M ( O - - 0 ) , 5 x I 0 ~ 7 M ( • • — • ) , and I x I 0 ~ 6 M (• — • ) . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. 75 c h e m i c a l l y i n d u c e d DNA damage e m p l o y i n g a l k a l i n e s u c r o s e g r a d i e n t s (Ho r i kawa e t aj_. , 1970; Andoh and T o s h i n o r i , 1972) . C u l t u r e s were e xpo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f 4NQ0, r a n g i n g f rom 4 x I O - 7 M t o I x I O - 6 M, and s u b j e c t e d t o a n a l y s i s by ASG Type I . E xpo su re o f t h e c e l l c u l t u r e s t o a minimum c o n c e n t r a t i o n o f 4 x I O - 7 M 4NQ0 was r e q u i r e d t o i n duce a s h i f t o f t h e peak o f 3 H -DNA f r om t h e p o s i t i o n o f t h e c o n t r o l DNA peak t o w a r d s t h e t o p o f t h e g r a d i e n t . T r e a t m e n t s w i t h h i g h e r c o n c e n t r a t i o n s o f 4NQ0 i n d u c e d g r e a t e r s h i f t s o f peaks o f t r e a t e d 3 H-DNA t o w a r d s t h e t o p o f t h e g r a d i e n t ( F i g u r e 2 0 ) , whereas no a l t e r a t i o n f rom t h e c o n t r o l s e d i m e n t a t i o n p r o f i l e was o b s e r v e d a t 4NQ0 c o n c e n t r a t i o n s be low 4 x I O - 7 M. No a l t e r a t i o n f r o m t h e p o s i t i o n o f t h e c o n t r o l s e d -i m e n t a t i o n p r o f i l e was o b s e r v e d when c e l l s were l y s e d i n t h e u s ua l o v e r l a y c o n t a i n i n g c o n c e n t r a t i o n s o f 4NQ0 up t o I x I 0 ~ 3 M. E xpo su r e o f t h e c e l l c u l t u r e s f o r 30 min t o a minimum c o n c e n t r a t i o n o f 2 x I O - 5 M N - a c e t o x y - A A F , w i t h s u b s e q u e n t a n a l y s i s by ASG Type I, p r o d u c e d a s h i f t f r om t h e c o n t r o l peak ( F i g u r e 2 1 ) . G r e a t e r s h i f t s . o f 3H-DNA peaks t o w a r d s t h e t o p o f t h e g r a d i e n t o c c u r r e d w i t h i n c r e a s e s i n c o n c e n t r a t i o n o f N - a c e t o x y - A A F a p p l i e d t o t h e c e l l c u l t u r e s ( F i g u r e 2 1 ) . N o ' s h i f t f rom t h e c o n t r o l p r o f i l e , t o w a r d s t h e t o p o f t h e g r a d i e n t , was d i s c e r n e d f o l l o w i n g 30 -m in e x p o s u r e s o f t h e c e l l c u l t u r e s t o e i t h e r N -hyd roxy -AAF o r AAF a t c o n c e n t r a t i o n s as h i g h as 4 x IO"" 3 M ( F i g u r e 2 2 ) . DNA r e p a i r s y n t h e s i s , e s t i m a t e d by t h e u n s c h e d u l e d 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 , o c c u r r e d t o an a p p r e c i a b l e e x t e n t f o l l o w i n g 76 F i g u r e 2 1 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom 2 -4 x IO1* human s k i n f i b r o b l a s t s exposed t o v a r i o u s c o n -c e n t r a t i o n s o f N - a c e t o x y - A A F . The N - a c e t o x y - A A F do se s g i v e n t o t h e c e l l s : c o n t r o l ( • — • ) , 2 x I 0 ~ 5 M ( O - - O ) , 3 x I0~ 5 M ( • — • ) , 4 x I 0 ~ 5 M ( a — • ) , and 5 x I 0 ~ 5 M ( A - - A ) . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. F i g u r e 22 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom 2-4 x IO1* human s k i n f i b r o b l a s t s e xpo sed t o v a r i o u s c o n -c e n t r a t i o n s o f N -hyd roxy -AAF and o f AAF. The N -hyd roxy -AAF do se s g i v e n t o t h e e e l I s : 2 x I 0 ~ 5 M t o 4 x I 0 ~ 3 M, 30 -m in e x p o s u r e ( • — • ) ; 2 x I 0 ~ 5 M, 5-h e x p o s u r e (••-••); 5 x 10 5 M, 5-h e x p o s u r e ( • — • ) . The AAF doses g i v e n t o t h e e e l Is ranged f r om 2 x 10 5 M t o 4 x 10 3 M, 30 -m in o r 5-h e x p o s u r e ( O - - O ) . The 4 x 10 3 M c o n c e n t r a t i o n s were g r e a t e r t h a n t h e s o l u b i l i t i e s o f N -hyd roxy -AAF and AAF i n MEM a t 3 7 ° C. The c o n t r o l s e d i m e n t a t i o n p r o f i l e s ( no t shown) s u p e r i m p o s e d t h e p r o f i l e (••—•). C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n - f o r 7.5 h a t 2 0 ° C. 77 a 5 h e x p o s u r e t o N -hyd roxy -AAF a l t h o u g h a t a much l ower l e v e l t h a n t h a t o b s e r v e d f o l l o w i n g N - a c e t o x y - A A F e x p o s u r e s a t c o n c e n t r a t i o n s o f 2 x I O - 5 M ( S t i c h e_t a\_., 1972) . T h e r e f o r e , e x p e r i m e n t s were c o n d u c t e d whereby human c e l l c u l t u r e s were expo sed t o v a r i o u s c o n c e n t r a t i o n s o f N - hyd roxy -AAF and o f AAF, r a n g i n g f rom 2 x I O - 5 t o 4 x I O - 3 M, f o r 5 h b e f o r e ASG a n a l y s i s was e x e c u t e d . The r e s u l t s o f t h e 5 h e x p o s u r e s i n d i c a t e d t h a t a minimum o f 2 x I 0 ~ 5 M N -hyd roxy -AAF was r e q u i r e d t o i n d u c e a s h i f t o f t h e t r e a t e d DNA p r o f i l e f r om t h e c o n t r o l p r o f i l e t o w a r d s t h e t o p o f t h e g r a d i e n t . G r e a t e r s h i f t s , i n d i c a t i n g g r e a t e r DNA f r a g m e n t a t i o n , o c c u r r e d w i t h i n c r e a s e s i n c o n c e n t r a t i o n o f N -hyd roxy -AAF a p p l i e d t o t h e c e l l c u l t u r e s f o r 5 h, a s t y p i f i e d by t h e p r o f i l e r e s u l t i n g f rom 5 x I 0 ~ 5 M N -hyd roxy -AAF e x p o s u r e s ( F i g u r e 2 2 ) . No a l t e r a t i o n f r om t h e p o s i t i o n o f t h e c o n t r o l s e d i -m e n t a t i o n p r o f i l e was o b s e r v e d when c e l l s were l y s e d i n t h e u sua l manner but w i t h t h e l y s i n g s o l u t i o n c o n t a i n i n g up t o I x I 0 ~ 3 M AAF o r N - h y d r o x y - A A F . (b) ASG Type 2 ( i ) T e c h n i c a l Deve lopment I t was o b s e r v e d ( F i g u r e s 14 and 15) t h a t t h e amount o f pe l l e t t e d DNA r a d i o a c t i v i t y d e c r e a s e d f rom 11-25% o f t o t a l DNA r a d i o a c t i v i t y e n c o u n t e r e d w i t h 2-4 x IO 4 c e l l s l a y e r e d , t o 5-9% o f t o t a l DNA r a d i o a c t i v i t y , e n c o u n t e r e d w i t h 8-10 x IO 3 c e l l s l a y e r e d . T h e r e f o r e , by r e d u c i n g t h e c e l l number l a y e r e d t o 8-10 x IO 3 f r om 2-4 x I O 4 , emp loyed i n ASG Type I, an a t t e m p t was made t o r e d u c e t h e amount o f pe l l e t t e d DNA r a d i o a c t i v i t y such t h a t a t l e a s t 91% o f t h e t o t a l DNA r a d i o a c t i v i t y wou ld be p o s i t i o n e d w i t h i n t h e 79 F i g u r e 23_ A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom 8-10 x IO 3 normal human c e l l s f o l l o w i n g c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. D u p l i c a t e r u n s . The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . F i g u r e 24. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om 8-10 x I 0 3 XPe f i b r o b l a s t s f o l l o w i n g c e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. D u p l i c a t e r u n s . The h o r i z o n t a l b a r , i n t h e same p o s i t i o n as t h a t i n F i g u r e 2 3 , i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . 80 I 1 r-O I O so F R A C T I O N N U M B E R — S E D I M E N T A T I O N 81 g r a d i e n t as n o n - p e l l e t t e d DNA r a d i o a c t i v i t y . The a l k a l i n e g r a d i e n t b a s e , l y s i s c o n d i t i o n s and c e n t r i -f u g a t i o n p a r a m e t e r s were i d e n t i c a l t o t h o s e d e s c r i b e d f o r ASG Type I. On l y t h e number o f l a y e r e d c e l l s was r educed t o 8-10 x IO 3 i n t h e deve l opmen t o f ASG Type 2. S u c c e s s i v e r un s w i t h 8-10 x IO 3 normal s k i n f i b r o b l a s t s l a y e r e d p e r g r a d i e n t d e m o n s t r a t e d a v a r i a b i l i t y o f t h e p o s i t i o n o f t h e peak o f DNA r a d i o a c t i v i t y . Two normal c o n t r o l p r o f i l e s , e x h i b i t i n g peaks w i t h i n t h e range of f r a c t i o n s II t o 14 i n c l u s i v e , were r e p r e s e n t a t i v e o f t h o s e c o n s i s t e n t l y e n c o u n t e r e d f o r ASG Type 2 ( F i g u r e 2 3 ) . S i m i l a r r e s u l t s were o b t a i n e d w i t h 8-10 x IO 3 XPe c e l l s l a y e r e d pe r g r a d i e n t . Two sample XPe c o n t r o l p r o f i l e s , e x h i b i t i n g peak s w i t h i n t h e r ange of f r a c t i o n s II t o 14 ( F i g u r e 24) were s i m i l a r t o t h e p r o f i l e s o f 3 H-DNA f rom normal c e l l s ( F i g u r e 2 3 ) . In o r d e r t o a c c o u n t f o r t h e v a r i a b i l i t i e s i n c o n t r o l p r o f i l e s , e v e r y run i n c l u d e d a t l e a s t one c o n t r o l g r a d i e n t t o p e r m i t d i r e c t c o m p a r i s o n s t o be made w i t h i n each r u n . ( i i ) A p p l i c a t i o n s o f ASG Type 2 The d i s c o v e r y t h a t c e l l s o f p a t i e n t s a f f l i c t e d w i t h xe rode rma p igmentosum (XP) a r e d e f i c i e n t i n t h e r e p a i r o f DNA damage i n d u c e d by u l t r a v i o l e t r a d i a t i o n ( r e v i e w e d by C l e a v e r , 1970 b) l ed t o t h e d i s c o v e r y t h a t c e l l s d e r i v e d f r om t h e s e p a t i e n t s a l s o have a r educed c a p a c i t y t o e x h i b i t DNA r e p a i r s y n t h e s i s f o l l o w i n g e x p o s u r e t o v a r i o u s c h e m i c a l c a r c i n o g e n s ( r e v i e w e d by S t i c h and L a i s h e s , 1973; S t i c h e_t a_l_., 1973 ) . Reduced l e v e l s o f 82 F i g u r e 25. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom 8-10 x IO 3 normal human f i b r o b l a s t s f o l l o w i n g no t r e a t -ment ( c o n t r o l ) ( • — • ) , and a I . 5 -h e x p o s u r e t o 2 x 10 6 M 4NQ0 w i t h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM: 0 h ( • — • ) , 12 h (•••••), and 24 h ( O - - O ) . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. F i g u r e 26 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om 8-10 x I 0 3 XPe f i b r o b l a s t s f o l l o w i n g no t r e a t m e n t ( c o n t r o l ) ( • — • ) , and a 1.5-h e x p o s u r e t o 2 x 10 6 M 4NQ0 w i t h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM: 0 h ( • — • ) , 12 h ( • — • ) , and 24 h ( O - - O ) . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. 83 84 DNA r e p a i r s y n t h e s i s i n XP c e l l s , e s t i m a t e d by t h e u n s c h e d u l e d 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 , was f i r s t d e m o n s t r a t e d f o l l o w i n g e x p o s u r e t o 4NQ0 ( S t i c h and San , 1971) . These o b s e r v a t i o n s were t h e n e x t e n d e d t o o t h e r c l a s s e s o f c a r c i n o g e n s : t h e a r o m a t i c am ine s ( S t i c h e t a L , 1972; L i ebe rman e t a j _ . , 1972; S e t l o w e t a l . , 1972) and t h e p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s ( S t i c h and San , 1973) . The q u e s t i o n a r o s e as t o whe the r t h e low l e v e l s o f u n s c h e d u l e d i n c o r p o r a t i o n o f H-TdR i n c h e m i c a l c a r c i n o g e n - t r e a t e d XP c e l l s r e f l e c t e d a low r a t e o f r e j o i n i n g o r r e f o r m a t i o n o f damaged DNA s t r a n d s i n XP c e l l s compared t o normal c e l l s . I t was d e c i d e d t o use ASG Type 2 t o compare t h e e x t e n t o f DNA damage i nduced by 4NQ0 i n normal and XP c e l l s a t v a r i o u s t i m e s p o s t - t r e a t m e n t . E x p e r i m e n t s were c o n d u c t e d i n w h i c h c u l t u r e s o f normal human c e l l s and c u l t u r e s o f XP c e l l s were expo sed t o 2 x I0~ e M 4NQ0 f o r 1.5 h (a c o n c e n t r a t i o n and e x p o s u r e t i m e c o m p a r a b l e t o t h o s e emp loyed by S t i c h and San ( 1971 ) ) and t h e n i n c u b a t e d i n f r e s h g r o w t h medium f o r 0 , 12, and 24 h. The s e d i m e n t a t i o n p r o f i l e s o f H-DNA r e l e a s e d f rom normal c e l l s , w h i c h were s u b j e c t e d t o 2 x I 0 ~ 6 M 4NQ0 t r e a t m e n t and 12 h 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 , e x h i b i t e d a s h i f t o f DNA r a d i o a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t f r om t h e p o s i t i o n o f c o n t r o l DNA peaks ( F i g u r e 2 5 ) . S i n c e t h i s s h i f t was no t as g r e a t as t h a t e x -3 h i b i t e d by H-DNA r e l e a s e d f rom t r e a t e d c e l l s w i t h no p o s t - t r e a t -ment i n c u b a t i o n , t h e " r e t u r n " o f t h e s e d i m e n t a t i o n p r o f i l e s t o w a r d s t h e c o n t r o l r e g i o n i n d i c a t e d t h a t a r e p a i r o f DNA damage was o c c u r -r i n g . The s e d i m e n t a t i o n p r o f i l e s , r e s u l t i n g f rom 24 h o f p o s t -85 F i g u r e 27 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r om 8-10 x IO 3 normal human f i b r o b l a s t s f o l l o w i n g no t r e a t -ment ( c o n t r o l ) ( • — • ) , and a I . 5 -h e x p o s u r e t o 5 x 10 6 M 4NQ0 w i t h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM: 0 h ( a — o ) , 12 h (••--•), and 24 h ( 0 - - 0 ) . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. F i g u r e 28 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r o m 8-10 x I 0 3 XPe f i b r o b l a s t s f o l l o w i n g no t r e a t m e n t ( c o n t r o l ) (••—•), and a 1.5-h e x p o s u r e t o 5 x 10 6 M 4NQ0 w i t h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM: 0 h ( • — • ) , 12 h ( • — • ) , and 24 h ( O - - O ) . C e n t r i f u g a t i o n a t 20,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. 86 I 1 r-O I O 2 0 F R A C T I O N N U M B E R — S E D I M E N T A T I O N 87 t r e a t m e n t i n c u b a t i o n o f normal c e l l s , e x h i b i t e d no d e t e c t a b l e s h i f t o f DNA r a d i o a c t i v i t y t o w a r d s t h e t op o f t h e g r a d i e n t , f r om t h e p o s i t i o n o f c o n t r o l DNA peaks ( F i g u r e 2 5 ) . T h i s o b s e r v a t i o n i n d i c a t e d t h a t t h e DNA damage p r o d u c e d i n normal c e l l s by 2 x I0~ 6 M 4NQ0 was e s s e n t i a l l y r e p a i r e d by 24 h, as e s t i m a t e d by t h i s t e c h n i q u e . The s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom XPe c e l l s , w h i c h were s u b j e c t e d t o 2 x 10 6 M 4NQ0 t r e a t m e n t and 12 h 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 , a l s o e x h i b i t e d a s h i f t o f DNA r a d i o -a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t f r o m t h e p o s i t i o n o f c o n t r o l DNA peaks ( F i g u r e 2 6 ) . As o b s e r v e d w i t h normal c e l l s , t h i s s h i f t was not as p r onounced as t h a t r e s u l t i n g f r om no p o s t -t r e a t m e n t i n c u b a t i o n , and so t h e r e t u r n o f t h e s e d i m e n t a t i o n p r o -f i l e s t o w a r d s t h e c o n t r o l r e g i o n i n d i c a t e d t h a t a r e p a i r o f t h e DNA damage was a l s o o c c u r r i n g i n XPe c e l l s by 12 h p o s t - t r e a t m e n t . However , t h e s e d i m e n t a t i o n p r o f i l e s r e s u l t i n g f rom 24 h 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 o f XPe c e l l s a l s o e x h i b i t e d a s h i f t o f DNA r a d i o a c t i v i t y , " t o w a r d s t h e t o p o f t h e g r a d i e n t , f r om t h e p o s i t i o n o f c o n t r o l 3H-DNA peaks ( F i g u r e 2 6 ) . T h i s o b s e r v a t i o n i n d i c a t e d t h a t , u n l i k e normal c e l l s , XPe c e l l s were no t a b l e t o c o m p l e t e l y r e p a i r t h e DNA damage, as e s t i m a t e d by t h i s t e c h n i q u e , by 24 h. E x p e r i m e n t s c o n d u c t e d w i t h a h i g h e r 4NQ0 c o n c e n t r a t i o n , 5 x I0~ 6 M 4NQ0 a p p l i e d t o normal and XPe c e l l s f o r 1.5 h , p r o d u c e d s i m i l a r r e s u l t s f o l l o w i n g 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 f r e s h g r owth medium f o r 0, 12, and 24 h. The s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom normal c e l l s , w h i c h were s u b j e c t e d t o 5 x I0~ 6 M 4NQ0 t r e a t m e n t and 12 h 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 , e x h i b i t e d a 88 s h i f t o f DNA r a d i o a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t f r om t h e p o s i t i o n o f s e d i m e n t a t i o n o f c o n t r o l 3H-DNA peaks ( F i g u r e 2 7 ) . T h i s s h i f t was not as g r e a t as t h a t e x h i b i t e d by 3H-DNA r e l e a s e d f rom t r e a t e d c e l l s w i t h no p o s t - t r e a t m e n t i n c u b a t i o n , and so c o n s t i t u t e d a r e t u r n o f t h e s e d i m e n t a t i o n p r o f i l e t o w a r d s t h e c o n t r o l r e g i o n . T h i s r e t u r n appea red t o be c o m p l e t e f o l l o w i n g 24 h o f p o s t - t r e a t -ment i n c u b a t i o n i n f r e s h MEM; t h e s e d i m e n t a t i o n p r o f i l e was e s s e n t i a l l y s u p e r i m p o s a b l e on t h a t o f t h e c o n t r o l . T h i s o b s e r v a t i o n i n d i c a t e d t h a t t h e DNA damage p r o d u c e d i n normal c e l l s by 5 x I 0 ~ 6 M 4NQ0 i s l a r g e l y r e p a i r e d by 24 h, as e s t i m a t e d by t h i s t e c h n i q u e . The s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om XPe c e l l s , w h i c h were a l s o s u b j e c t e d t o 5 x I O " 6 M 4NQ0 t r e a t m e n t and 12 h 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 , e x h i b i t e d a s h i f t o f DNA r a d i o -a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t f r om t h e p o s i t i o n o f c o n t r o l 3H-DNA peaks ( F i g u r e 2 8 ) . As o b s e r v e d w i t h normal c e l l s , t h i s s h i f t was no t as p r onounced as t h a t r e s u l t i n g f r o m no p o s t - t r e a t m e n t i n c u b a t i o n , and so t h e r e t u r n o f t h e s e d i m e n t a t i o n p r o f i l e s t o -wards t h e c o n t r o l r e g i o n i n d i c a t e d t h a t a r e p a i r o f t h e 4NQ0-i n d u c e d DNA damage was a l s o o c c u r r i n g i n XPe c e l l s by 12 h p o s t -t r e a t m e n t . However , t h e s e d i m e n t a t i o n p r o f i l e s r e s u l t i n g f rom 24 h 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 o f XPe c e l l s a l s o e x h i b i t e d a c o n s i d e r a b l e s h i f t o f DNA r a d i o a c t i v i t y t o w a r d s t h e t o p o f t h e g r a d i e n t f r om t h e p o s i t i o n o f c o n t r o l DNA peaks ( F i g u r e 2 8 ) . T h i s o b s e r v a t i o n i n d i c a t e d t h a t a c o n s i d e r a b l e p r o p o r t i o n o f DNA damage, i n d u c e d by 5 x I O - 6 M 4NQ0 i n XPe c e l l s and e s t i m a t e d by t h i s t e c h n i q u e , r ema ined f o l l o w i n g 24 h 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 . 89 Th ree c o n s i s t e n t o b s e r v a t i o n s appea red f rom t h e s e ASG s t u d i e s o f 4NQ0- i nduced DNA damage and r e p a i r i n normal and XPe ceI I s : 1. F o l l o w i n g t r e a t m e n t w i t h e i t h e r 2 x I 0 ~ 6 M 4NQ0 o r 5 x I O - 6 M 4NQ0 and a 24 h p e r i o d of p o s t - t r e a t m e n t i n c u b a t i o n , s e d i m e n t a t i o n p r o f i l e s o f H-DNA f r om normal human c e l l s a p p r o -x i m a t e d t h e p o s i t i o n i n g o f t h e c o n t r o l 3H-DNA p r o f i l e s . T h e r e -f o r e , t h e r e p a i r o f t h e 4NQ0 - i nduced DNA f r a g m e n t a t i o n i n normal c e l l s a ppea red l a r g e l y c o m p l e t e a t 24 h p o s t - t r e a t m e n t . 2. In c o n t r a s t , f o l l o w i n g t r e a t m e n t w i t h e i t h e r 2 x I O " 6 M 4NQ0 o r 5 x I 0 - 6 M 4NQ0 and a 24 h 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 , s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA f rom XPe c e l l s d i d no t a p p r o x i m a t e t h e p o s i t i o n i n g o f c o n t r o l 3H-DNA p r o f i l e s . The o b s e r v e d s h i f t s o f t h e t r e a t e d XPe 3 H-DNA, f rom t h e p o s i t i o n o f c o n t r o l H-DNA pea.ks, were somewhat g r e a t e r a t t h e h i g h e r c o n -c e n t r a t i o n o f 4NQ0. T h e r e f o r e , t h e . r e p a i r o f t h e 4NQ0 - i nduced DNA f r a g m e n t a t i o n i n XPe c e l l s d i d no t appea r t o be c o m p l e t e by 24 h. 3. In a l l ASG e x p e r i m e n t s c o n d u c t e d w i t h 1.5 h e x p o s u r e s o f normal and XPe c e l l s t o 4NQ0, t h e r e s u l t i n g peaks o f s e d i m e n t e d 3 H-DNA f r om t r e a t e d XPe c e l l s c o n s i s t e n t l y d e m o n s t r a t e d a g r e a t e r s h i f t t o w a r d s t h e t op o f t h e g r a d i e n t t h a n t h e c o r r e s p o n d i n g peaks o f H-DNA p r o f i l e f r om t r e a t e d normal c e l l s . Fo r e x a m p l e , t h e peak o f t h e 3 H-DNA p r o f i l e f r om normal c e l l s t r e a t e d w i t h 5 x I O - 6 M 4NQ0 f o r 1.5 h, a p p e a r s a t f r a c t i o n 21 ( F i g u r e 2 7 ) . In c o n t r a s t , t h e peak o f t h e 3 H-DNA p r o f i l e o f XPe c e l l s t r e a t e d i n t h e same manner , a p p e a r s a t f r a c t i o n 24 ( F i g u r e 2 8 ) . T h i s p a r t i c u l a r 90 b e h a v i o u r was o b s e r v e d i n a l l e x p e r i m e n t s i n v o l v i n g 1.5 h e x -p o s u r e s o f normal and XPe c e l l s t o 4NQ0, w i t h o u t e x c e p t i o n , and d i d n o t a p p e a r t o be r e l a t e d t o t h e v a r i a b i l i t y e n c o u n t e r e d w i t h t h e c o n t r o l p r o f i l e s . C o n t r o l g r a d i e n t s , c o n t a i n i n g u n t r e a t e d 3H-DNA f r om t h e two c e l l t y p e s , were r o u t i n e l y r u n t o g e t h e r w i t h t h e g r a d i e n t s c o n t a i n i n g t h e t r e a t e d p r e p a r a t i o n s . The o b s e r v a t i o n s d e s c r i b e d as p o i n t #3 above i n d i c a t e t h a t i d e n t i c a l 1.5 h t r e a t m e n t s o f XPe and norma l c e l l c u l t u r e s w i t h 4NQ0, p r o d u c e d g r e a t e r DNA damage i n XPe c u l t u r e s , as i n d i c a t e d by t h e ASG a n a l y s e s . In o r d e r t o d e t e r m i n e w h e t h e r t h e s e d i f f e r e n c e s o b s e r v e d a t 1.5 h were a r e f l e c t i o n o f a r e d u c e d r a t e o f r e p a i r i n XPe c e l l s , e x p e r i m e n t s were c o n d u c t e d i n w h i c h normal and XPe c e l l c u l t u r e ' s were s u b j e c t e d t o 5 x I O - 6 M 4N00 t r e a t m e n t s f o r v a r i o u s t i m e s : 3 h , 1.5 h, 0 . 5 h, 18 m i n , and 6 m i n , and i m m e d i a t e l y s u b j e c t e d t o ASG a n a l y s i s u s i n g ASG Type 2 . The r e s u l t s o f t h i s s e r i e s o f e x p e r i m e n t s d e m o n s t r a t e d a c o n s i s t e n t l y g r e a t e r s h i f t o f t h e 3 H-DNA s e d i m e n t a t i o n p r o f i l e o f t r e a t e d XPe c e l l s , t o w a r d s t h e t o p o f t h e g r a d i e n t , t h a n t h a t o f t r e a t e d normal c e l l s f o l l o w i n g e x p o s u r e o f b o t h c u l t u r e s t o 5 x I O " 6 M 4NQ0 f o r 3 h C F i g u r e 2 9 A ) , 1.5 h ( F i g u r e 2 9 B ) , 0 .5 h ( F i g u r e 3 0 A ) , and 18 m i n ( F i g u r e 3 0 B ) . The d i f f e r e n c e s between t h e p o s i t i o n i n g o f t h e 3H-DNA s e d i m e n t a t i o n p r o f i l e s f r o m t r e a t e d normal and XPe c e l l s was l e s s p r o n o u n c e d f o l l o w i n g 6 -m in e x p o s u r e s t o 5 x I O - 6 M 4NQ0 ( F i g u r e 3 0 C ) . F u r t h e r r e d u c t i o n s i n e x p o s u r e t i m e s were deemed n e c e s s a r y t o v e r i f y w h e t h e r o r no t e x p o s u r e t i m e s o f t h e o r d e r o f one m i n u t e 91 F i g u r e 2 9 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r o m 8-10 x IO 3 normal c e l l s ( • — © ) and XPe c e l l s ( 0 - - - 0 ) f o l l o w i n g e x p o s u r e t o 5 x 10 6 M 4NQ0 f o r : (A) 3 h (B) I.5 h The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA peaks o m i t t e d f o r c l a r i t y . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7 .5 h a t 2 0 ° C. 92 S E D I M E N T A T I O N 93 F i g u r e 3 0 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom 8-10 x IO 3 normal c e l l s (•—•) and XPe c e l l s ( O — O ) f o l l o w i n g e x p o s u r e t o 5 x 10 6 M 4NQ0 f o r : (A) 0 .5 h (B) 18 min (C) 6 m in The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA peaks o m i t t e d f o r c l a r i t y . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. 94 F R A C T I O N N U M B E R — S E D I M E N T A T I O N .a I i 1 r-o 10 20 30 F R A C T I O N N U M B E R — S E D I M E N T A T I O N F R A C T I O N N U M B E R • 4 — S E D I M E N T A T I O N 95. F i g u r e 3 1 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom human c e l l s f o l l o w i n g one -m in e x p o s u r e o f t h e c e l l s u s -p e n s i o n s ( i n v e r s e n e s o l u t i o n : 0 .05% EDTA i n PBS) t o : no t r e a t m e n t ( • — • ) , I x 1 0 - 5 M 4NQ0 ( O — O ) , and I x 10 - l+ M 4 NQO (•••••). (A) Norma I ceI Is (B) XPe e e l Is The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . C e n t r i f u g a t i o n a t 20 ,000 r e v . / m i n f o r 7.5 h a t 2 0 ° C. P E R C E N T O F T O T A L 3 H C O U N T S Ul • P E R C E N T O F T O T A L 3 H C O U N T S 97 would p r o d u c e g r e a t e r s h i f t s i n JH-DNA s e d i m e n t a t i o n p r o f i l e s f r om 4 N Q 0 - t r e a t e d XPe c e l l s . In o r d e r t o a c c o m p l i s h t h i s , c e l l c u l t u r e s were h a r v e s t e d i n PBS b u f f e r t o p r o d u c e s u s p e n s i o n s o f s i n g l e - c e l l s , m i xed a t " 0 " t i m e w i t h 4NQ0 d i s s o l v e d i n b u f f e r , drawn up a g r a d i e n t - l o a d i n g p i p e t t e , and q u i c k l y l a y e r e d a t t h e l - m i n mark No s h i f t i n 3H-DNA s e d i m e n t a t i o n p r o f i l e s , t o w a r d s t h e t o p o f t h e g r a d i e n t f r om t h e p o s i t i o n o f c o n t r o l 3H-DNA p e a k s , c o u l d be d e t e c t e d f o l l o w i n g t r e a t m e n t o f e i t h e r normal c e l l s ( F i g u r e 3 IA) o r XPe c e l l s ( F i g u r e 3 IB ) w i t h I x I O " 5 M 4NQ0 f o r I m i n . I n c r e a s i n g t h e 4NQ0 t o I x IO" 1* M, and t r e a t i n g c e l l s u s -p e n s i o n s f o r I m i n , r e s u l t e d i n a marked s h i f t i n t h e 3H-DNA s e d i m e n t a t i o n p r o f i l e s f r om t h e p o s i t i o n o f c o n t r o l 3 H-DNA p e a k s , w i t h e i t h e r normal ( F i g u r e 3 IA) o r XPe ( F i g u r e 3 IB ) c e l l s u s p e n s i o n s . The g r e a t e r s h i f t s o f 3H-DNA s e d i m e n t a t i o n p r o f i l e s f r om t r e a t e d XPe c e l l s , t o w a r d s t h e t o p o f t h e g r a d i e n t s , compared t o t h o s e f r o m t r e a t e d normal c e l l s , was no l o n g e r e v i d e n t f o l l o w i n g s h o r t - t e r m 4NQ0 e x p o s u r e s c o n d u c t e d i n t h i s manner . The r e s u l t s , o f t h e s h o r t - t e r m 6 -m in and l - m i n 4NQ0 t r e a t m e n t s o f t h e normal and XPe c u l t u r e s o r s u s p e n s i o n s , i n d i c a t e d t h a t t h e amount o f DNA damage i n d u c e d i n normal and XPe c e l l c u l t u r e s , a s e s t i m a t e d by ASG a n a l y s i s , was s i m i l a r f o l l o w i n g s h o r t e r e x -p o s u r e s and was no t g r e a t e r i n XPe c e i l s a s ' r e p e a t e d Iy o b s e r v e d f o l l o w i n g 4NQ0 t r e a t m e n t s o f 18 min t o 3 h, i n c l u s i v e . ( c ) ASG Type 3 One o f t h e most d i f f i c u l t p e r i m e n t a l c h e m i c a l c a r c i n o g e n e s i s , p r o b l e m s e n c o u n t e r e d i n e x -p a r t i c u l a r l y s i n c e t h e a d v e n t 98 o f more f a c i l e methods o f t i s s u e c u l t u r e , has been t h e l a c k o f t e c h n i q u e s d e s i g n e d t o b r i d g e t h e gap between i n v i t r o and _i_n_ v i v o s t u d i e s . Few t e c h n i q u e s a r e a v a i l a b l e t h a t a l l o w dua l a p p l i c a t i o n , t o s t u d i e s , f o r e x a m p l e , o f c h e m i c a l l y i n d u c e d DNA damage and r e p a i r i n bo th c u l t u r e d c e l l s and t h e i n t a c t a n i m a l . I t was f o r t h i s r e a s o n t h a t t h e a l k a l i n e s u c r o s e g r a d i e n t method , a m o d i f i c a t i o n o f t h e McGra th -Wi I I iams t e c h n i q u e (McGrath and W i l l i a m s , 1966) d e s i g n e d by Cox e t aj_. (1973) f o r t h e e s t i m a t i o n o f DNA damage and r e p a i r i n r a t l i v e r i n v i v o (Damjanov e_t a_l_., 1973 ) , was a d a p t e d f o r use w i t h c u l t u r e d human c e l l s . The t e c h n i c a l a s p e c t s o f t h e a d a p t a t i o n o f t h e a l k a l i n e g r a d i e n t t e c h n i q u e - t o be d e s i g n a t e d as ASG Type 3 - were s i m p l e and i n v o l v e d o n l y an a d j u s t m e n t o f t h e c e l l number l a y e r e d p e r g r a d i e n t . I t was f ound t h a t " i n t a c t " c o n t r o l 3 H-DNA s e d i m e n t e d on t o a 2 .3 M s u c r o s e c u s h i o n p l a c e d a t t h e bot tom o f t h e g r a d i e n t , f o l l o w i n g a 10-min l y s i s p e r i o d a t room t e m p e r a t u r e w i t h a b o u t I x IO 5 c e l l s l a y e r e d pe r g r a d i e n t ( F i g u r e 3 2 ) . C o n t r o l 3H-DNA peaks f rom c u l t u r e d human c e l l s most o f t e n appea red a t o r nea r t h e t o p o f t h e 2 .3 M c u s h i o n o f s u c r o s e a t f r a c t i o n s 2 o r 3 , w i t h an S v a l u e o f abou t 180 (Damjanov e t a I., 1973) . Peaks o f 3H-DNA a r i s i n g w i t h i n t h e c o n t r o l r e g i o n , i n d i c a t e d by t h e h o r i z o n t a l b a r ( F i g u r e s 3 2 , 3 3 ) , were d e f i n e d as i n t a c t DNA. The v a r i a b i l i t y o f c o n t r o l 3 H-DNA p e a k s , a l t h o u g h d e m o n s t r a t e d f o r normal c e l l s , a l s o a p p l i e d f o r XPe c e l l s ( F i g u r e 3 2 ) . No d e t e c t a b l e p r o t e i n o r l i p i d r a d i o a c t i v i t y was l o c a t e d i n t h e peaks o f c o n t r o l DNA r a d i o a c t i v i t y f o l l o w i n g a n a l y s i s by ASG Type 3 o f d o u b l e -l a b e l l e d c e l l c u l t u r e s ( w i t h l l + C - t h y m i d i ne and e i t h e r 3 H - l e u c i n e 99 o r 3 H - c h o I i n e ) . The r e s u l t s i n d i c a t e d t h a t c o n t r o l 3H-DNA p e a k s , f r e e f rom g r o s s c o n t a m i n a t i o n by p r o t e i n - o r l i p i d , c o u l d be r e p r o d u c i b l y o b t a i n e d by l a y e r i n g abou t I x IO 5 c e l l s on a l k a l i n e g r a d i e n t s of ASG Type 3. Fo l l ow ing e x p o s u r e t o 5 x I O - 5 M 4NQ0 f o r 0 .5 h, t h e 4NQ0 - i nduced DNA damage i n normal and XPe c e l l s was e s t i m a t e d by ASG Type 3 ( F i g u r e 3 3 ) . The r e s u l t i n g s h i f t s i n t h e s e d i m e n t a t i o n p r o f i l e s o f normal and XPe c e l l s f r om t h e p o s i t i o n o f c o n t r o l DNA peaks was i n d i c a t i v e o f DNA damage i nduced i n t h e DNA o f t h e c e l l s by t h e 4NQ0 t r e a t m e n t . F u r t h e r a p p l i c a t i o n s o f ASG Type 3 i n v o l v i n g o t h e r c l a s s e s o f c h e m i c a l c a r c i n o g e n s w i l l be d i s c u s s e d i n l a t e r s e c t i o n s . 100 F i g u r e 3 2 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r om normal c e l l s s u b j e c t e d t o no t r e a t m e n t . ' D u p l i c a t e r u n s . C o n t r o l p r o f i l e s o f 3 H-DNA r e l e a s e d f r om XPe c e l l s s u p e r i m p o s e d t h e p r o f i l e s i l l u s t r a t e d . The h o r i z o n t a l ba r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . The a r r o w i n d i c a t e s t h e p o s i t i o n o f s e d i m e n t a t i o n o f T4 phage DNA m a r k e r . C e n t r i f u g a t i o n a t 25 ,000 r e v . / m i n f o r 0 .5 h a t 2 0 ° C. F i g u r e 3 3 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f dH-DNA r e l e a s e d f rom normal (•—•) and XPe (O—-O) c e l l s f o l l o w i n g 0.5 h e x p o s u r e s t o 5 x 10 6 M 4NQ0. The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . C e n t r i f u g a t i o n a t 25 ,000 r e v . / m i n f o r 0 .5 h a t 2 0 ° C. 101 DISCUSSION OF SECTION I 102 F o l l o w i n g t h e deve l opmen t o f t e c h n i q u e s t o d i f f e r e n t i a t e between DNA r e p a i r s y n t h e s i s and DNA r e p l i c a t i o n a s s o c i a t e d w i t h t h e d u p l i c a t i o n o f chromosomes (Rasmussen and P a i n t e r , 1 964 , 1966 ) , S t i c h and c o - w o r k e r s d e m o n s t r a t e d t h e a b i l i t y o f c h e m i c a l c a r c i n o g e n s t o i n duce DNA r e p a i r s y n t h e s i s i n c u l t u r e d mammalian c e l l s ( e . g . S t i c h e t aj_. , 1970,1972; S t i c h and S a n , 1973) . The b i o a s s a y s f o r DNA r e p a i r s y n t h e s i s i n v o l v e d e s t i m a t i o n s o f t h e l e v e l s o f u n s c h e d u l e d i n c o r p o r a -t i o n o f 3 H - T d R , a p r o c e s s w h i c h o c c u r s d u r i n g t h e " r e s y n t h e s i s " o f e x -c i s e d r e g i o n s c o n t a i n i n g damaged DNA ( F i g u r e 12 ) . T h e r e f o r e , i t was assumed t h a t l e v e l s o f DNA r e p a i r s y n t h e s i s , i n d u c e d by t r e a t m e n t w i t h c h e m i c a l c a r c i n o g e n s , were a r e f l e c t i o n o f t h e amount o f DNA damage  i n d u c e d by t h e c h e m i c a l c a r c i n o g e n s . I t was d e c i d e d t o t e s t t h i s a s s u m p t i o n by e m p l o y i n g t h e b i o -p h y s i c a l t e c h n i q u e o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . T h i s t e c h n i q u e i n v o l v e s t h e v e l o c i t y s e d i m e n t a t i o n o f DNA, e x t r a c t e d f r om c h e m i c a l c a r c i n o g e n - t r e a t e d and u n t r e a t e d human c e l l s , t h r o u g h a c o n -t i n u o u s l i n e a r g r a d i e n t o f a l k a l i n e s u c r o s e . W i t h t h i s t e c h n i q u e , f r a g m e n t e d DNA a p p e a r s t o s e d i m e n t s l o w e r t h a n i n t a c t DNA and t h e e x -t e n t o f f r a g m e n t a t i o n i s e s t i m a t e d by c o m p a r i n g t h e s e d i m e n t a t i o n p r o f i l e s o f DNA d e r i v e d f rom bo th t r e a t e d and u n t r e a t e d c e l l c u l t u r e s . T h e r e f o r e , when v e l o c i t y s e d i m e n t a t i o n o f DNA f r om c h e m i c a l c a r c i n o g e n -t r e a t e d c e l l s p r o d u c e s peak s o f DNA t o w a r d s t h e t o p o f t h e g r a d i e n t s , whereas u n t r e a t e d DNA a p p e a r s as peak s t o w a r d s t h e bo t tom o f t h e g r a d i e n t s , t h e r e s u l t s i n d i c a t e t h a t f r a g m e n t a t i o n o f t h e DNA was i n d u c e d by t h e t r e a t m e n t . The e x t e n t o f DNA f r a g m e n t a t i o n may i n t u r n be i n t e r p r e t e d as a r e f l e c t i o n o f t h e l e v e l o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage. S i m i l a r l y , a t i m e - d e p e n d e n t " r e t u r n " o f t h e 103 s e d i m e n t a t i o n p r o f i l e o f t r e a t e d DNA from upper r e g i o n s o f t h e g r a d i e n t t o w a r d s t h e p o s i t i o n o f u n t r e a t e d DNA s e d i m e n t a t i o n p r o f i l e s , i n t h e l ower r e g i o n s o f t h e g r a d i e n t , has i m p o r t a n t i m p l i c a t i o n s . T h i s b e h a v i o u r may be i n t e r p r e t e d as a p r o d u c t i o n o f l a r g e r DNA p i e c e s f r om s m a l l e r DNA p i e c e s by a p r o c e s s of r e j o i n i n g o f damaged DNA  s t r a n d s d u r i n g r e p a i r o f DNA damage. C e r t a i n a r o m a t i c a m i n e s , a m i d e s , and am inoazo dyes a r e c a r c i n o g e n i c , g e n e r a l l y i n d u c i n g t umor s i n t i s s u e s d i s t a n t f r om t h e s i t e s o f a d m i n i s t r a t i o n ( r e v i e w e d by M i l l e r , 1970 ) . The p r o p o s a l t h a t t h e s e c a r c i n o g e n s may be e n z y m a t i c a I I y a c t i v a t e d i n t h e t i s s u e s i n w h i c h t h e y i nduce neop la sms l e d t o t h e e s t a b l i s h m e n t o f N - h y d r o x y l a -t i o n f o l l o w e d by N - h y d r o x y - e s t e r i f i c a t i on as t h e l i k e l y i n i t i a l and u l t i m a t e r e a c t i o n s i n t h e m e t a b o l i c a c t i v a t i o n o f c a r c i n o g e n i c a r o m a t i c amines and amides ( r e v i e w e d by M i l l e r , 1970 ) . S t u d i e s o f DNA r e p a i r s y n t h e s i s ( S t i c h e t a I., 1972) u t i l i z i n g e s t i m a t i o n s o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , r e v e a l e d t h a t e q u i m o l a r c o n c e n t r a t i o n s o f t h e p r e c a r c i n o g e n AAF and i t s p r o x i m a t e ( N - h y d r o x y - A A F ) and u l t i m a t e ( N - a c e t o x y - A A F ) m e t a b o l i t e s t r i g g e r e d v a r i o u s l e v e l s o f DNA r e p a i r s y n t h e s i s , i n c u l t u r e d human f i b r o b l a s t s , t h a t f o l l o w e d t h e o r d e r : N - a c e t o x y - A A F > N -hyd roxy -AAF > AAF The d a t a r e g a r d i n g t h e v a r i o u s l e v e l s o f DNA r e p a i r s y n t h e s i s t h a t were i n d u c e d by AAF and i t s s y n t h e t i c m e t a b o l i t e s , l e d t o t h e a s s u m p t i o n t h a t t h e o b s e r v e d l e v e l s o f u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR i n c o r p o r a t i o n were r e f l e c t i o n s o f t h e e x t e n t o f DNA damage i n d u c e d by AAF and i t s s y n t h e t i c m e t a b o l i t e s . A l k a l i n e s u c r o s e g r a d i e n t s t u d i e s , u s i n g ASG Type I, r e v e a l e d t h a t N - a c e t o x y - A A F r a p i d l y i n d u c e d f r a g m e n t a t i o n o f t h e human DNA, whereas l o n g e r e x p o s u r e s t o N - h y d r o x y -AAF were r e q u i r e d t o p r o d u c e d e t e c t a b l e DNA f r a g m e n t a t i o n . No d e t e c t -104 a b l e l e v e l s of DNA f r a g m e n t a t i o n were i nduced by AAF. These s t u d i e s i n d i c a t e d t h a t t h e e x t e n t o f DNA damage i n d u c e d by AAF and i t s s y n t h e t i c m e t a b o l i t e s f o l l o w e d t h e same o r d e r as t h a t o f t h e l e v e l s o f DNA r e p a i r s y n t h e s i s . Hence , on' t h e b a s i s o f t h e a l k a l i n e s u c r o s e g r a d i e n t s t u d i e s , t h e a s s u m p t i o n t h a t t h e l e v e l s of u n s c h e d u l e d  i n c o r p o r a t i o n o f 3 H-TdR were r e f l e c t i o n s of t h e amount o f DNA  damage i nduced by AAF and i t s s y n t h e t i c m e t a b o l i t e s a p p e a r s t o be v a I i d . The c a r c i n o g e n i c a r o m a t i c amine AAF and i t s p r o x i m a t e m e t a b o l i t e N - h yd roxy -AAF f a i l t o r e a c t r e a d i l y w i t h mammalian t i s s u e components a t p h y s i o l o g i c a l pH. However , t h e s y n t h e t i c e s t e r N - a c e t o x y - A A F , one o f t h e s u i t a b l e c a n d i d a t e s f o r t h e u l t i m a t e c a r c i n o g e n i c m e t a b o l i t e o f AAF, does e x h i b i t h i g h r e a c t i v i t y t o w a r d s mammalian DNA, RNA, and p r o t e i n bo th i n v i v o and i n v i t r o ( r e v i e w e d by M i l l e r , 1970) . The c a p a c i t y o f t h e u l t i m a t e c a r c i n o g e n N - a c e t o x y -AAF t o r e a d i l y i n d u c e DNA damage i n c u l t u r e d human f i b r o b l a s t s , as e s t i m a t e d by t h e s e d i m e n t a t i o n o f DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s , c o n c u r s w i t h t h e r e p o r t s o f h i g h r e a c t i v i t y o f o n l y  N - a c e t o x y - A A F , and no t t h e p r o x i m a t e c a r c i n o g e n N -hyd roxy -AAF o r  t h e p r e c a r c i n o g e n AAF, t o c e l l u l a r macromoI ecu Ies i n v i t r o ( r e v i e w e d by Mi I l e r , 1970) . DNA r e p a i r s y n t h e s i s s t u d i e s r e v e a l e d a c o n c e n t r a t i o n d e -pendence o f t h e l e v e l s o f u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR i n t o human c e l l s , i n d u c e d by t h e m e t a b o l i t e s of AAF and by*4NQ0. G e n e r a l l y , i n c r e a s i n g t h e c o n c e n t r a t i o n s o f c h e m i c a l c a r c i n o g e n t o w h i c h t h e c e l l s were e x p o s e d , r e s u l t e d i n i n c r e a s i n g l e v e l s o f u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( r e v i e w e d by S t i c h and L a i s h e s , 1973; S t i c h et_ a j _ . , 1973) . 105 A n a l y s e s e m p l o y i n g ASG Type I d e m o n s t r a t e d t h a t e x p o s u r e o f c u l t u r e d human c e l l s t o i n c r e a s i n g c o n c e n t r a t i o n s o f t h e m e t a b o l i t e s of AAF o r of 4NQ0, r e s u l t e d i n i n c r e a s i n g s h i f t s o f s e d i m e n t a t i o n p r o f i l e s t o w a r d s t h e t o p o f t h e g r a d i e n t s . Thu s , a c o n c e n t r a t i o n dependence  o f t h e l e v e l o f u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR may be a r e f l e c t i o n  o f l e v e l s o f DNA damage w h i c h a r e g o v e r n e d , i n t u r n , by t h e amount o f  c h e m i c a l c a r c i n o g e n t o w h i c h t h e c e l l i s e x p o s e d . S t u d i e s o f DNA r e p a i r s y n t h e s i s , u t i l i z i n g t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , were c o n d u c t e d w i t h c u l t u r e d human f i b r o -b l a s t s d e r i v e d f rom bo th normal i n d i v i d u a l s and p a t i e n t s a f f l i c t e d w i t h xe rode rma p igmentosum (XP) ( S t i c h and S an , 1971) . The d i s c o v e r y t h a t low l e v e l s o f DNA r e p a i r s y n t h e s i s were t r i g g e r e d i n XP c e l l s , compared w i t h normal c e l l s , f o l l o w i n g e x p o s u r e o f t h e c e l l c u l t u r e s t o v a r i o u s doses o f 4NQ0, s u g g e s t e d t h a t : ( a ) t h e o b s e r v e d i n c o r p o r a -t i o n o f 3 H-TdR i n t o bo th c e l l t y p e s may be r e f l e c t i n g a c o m p l e t e r e p a i r o f t h e damaged DNA, i n c l u d i n g t h e " l i g a t i o n " o f t h e newly s y n t h e s i z e d " p a t c h " o f DNA i n t o t h e p a r e n t a l s t r a n d ( F i g u r e 1 2 ) , and (b) t h e c o m p a r a t i v e l y low l e v e l s o f i n c o r p o r a t i o n o f 3 H-TdR i n t o XP c e l l s was r e f l e c t i n g s l o w e r r a t e s o f r e p a i r o f 4NQ0 - i nduced DNA damage i n XP c e l l s compared w i t h normal c e l l s . The a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e , ASG Type 2 , was emp loyed t o i n v e s t i g a t e t h e t i m e - d e p e n d e n t r e t u r n o f f r a g m e n t e d DNA  t o w a r d s t h e p o s i t i o n o f s e d i m e n t a t i o n of u n t r e a t e d DNA. The c o n s t i t u e n t DNA o f normal and XP c e l l s , f o l l o w i n g e x p o s u r e t o 4NQ0, was c e n t r i f u g e d t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s a t v a r i o u s t i m e s p o s t - t r e a t m e n t . F i r s t l y , t h e r e s u l t s r e v e a l e d t h a t t h e s e d i m e n t a t i o n p r o f i l e s o f 4NQ0 - i nduced f r a g m e n t e d DNA, i n bo th c e l l t y p e s , r e t u r n e d t o w a r d s t h e p o s i t i o n o f u n t r e a t e d DNA peaks d u r i n g p o s t - t r e a t m e n t i n c u b a t i o n s . I t 106 was t h e r e f o r e i n d i c a t e d t h a t 4NQ0- i nduced DNA damage was b e i n g r e p a i r e d i n bo th c e l l t y p e s and t h a t t h e r e p a i r may have i n v o l v e d t h e " l i g a t i o n " o r " r e j o i n i n g " o f s m a l l e r DNA s t r a n d s t o p r oduce l a r g e r DNA s t r a n d s w i t h h i g h e r s e d i m e n t a t i o n v e l o c i t i e s . T h e r e f o r e , i t a p p e a r s t h a t t h e " r e s y n t h e s i s " o f e x c i s e d DNA r e g i o n s - i n d i c a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR - may be o c c u r r i n g i n c o n j u n c t i o n w i t h t h e " r e j o i n i n g " o f s m a l l e r DNA s t r a n d s t o p r oduce l a r g e r DNA s t r a n d s ( F i g u r e 12) . S e c o n d l y , t h e r e s u l t s r e v e a l e d t h a t t h e r e t u r n o f t h e s e d i m e n t a t i o n p r o f i l e o f f r a g m e n t e d DNA, d e r i v e d f r om 4 N Q 0 - t r e a t e d c e l l s , t o w a r d s t h e p o s i t i o n o f u n t r e a t e d DNA peaks was s l o w e r t h a n i n normal c e l l s f o l l o w i n g s i m i l a r t r e a t m e n t . I t was t h e r e f o r e i n -d i c a t e d t h a t DNA.damage, i n d u c e d by e q u i m o l a r c o n c e n t r a t i o n s o f 4NQ0 i n normal and XP c e l l s , was r e p a i r e d f a s t e r i n normal c e l l s t h a n i n XP c e l l s . The p r o p o s a l has been pu t f o r t h t h a t t h e r e s p o n s e o f XP c e l l s , t o 4NQ0 t r e a t m e n t , w i t h l owe r l e v e l s o f i n c o r p o r a t i o n o f 3 H-TdR t h a n o b s e r v e d i n s i m i l a r l y t r e a t e d normal c e l l s , may be a r e f l e c t i o n o f t h e i n a b i l i t y o f XP c e l l s t o e x c i s e 4NQ0 - i nduced damaged DNA m o i e t i e s a s q u i c k l y as normal c e l l s ( S t i c h and San , 1971) . If t h e XP c e l l s e x h i b i t a d e f e c t a t t h e e x c i s i o n s t e p o f t h e e x c i s i o n r e p a i r p r o c e s s ( F i g u r e 12 ) , t h e n i t i s l i k e l y t h a t t h e d e f e c t w i l l be d e t e c t e d w i t h t e c h n i q u e s d e s i g n e d t o r e v e a l any s t e p s o f t h e p r o c e s s s ub sequen t t o t h e e x c i s i o n s t e p . S p e c i f i c a l l y , a d e f e c t i v e e x c i s i o n s t e p s h o u l d be d e t e c t a b l e bo th a t t h e " r e s y n t h e s i s " s t e p ( i n c o r p o r a t i o n o f 3 H-TdR) and a t t h e l a t e r " l i g a t i o n " s t e p ( a l k a l i n e g r a d i e n t s ) . These r e s u l t s i n d i c a t e d t h a t t h e r e may be a d e f e c t i n t h e r e p a i r o f 4NQ0- i nduced DNA damage i n XP c e l l s , p o s s i b l y a t t h e e x c i s i o n s t e p , t h a t i s m a n i f e s t e d by s l o w e r r a t e s o f " r e j o i n i n g " o f s m a l l DNA s t r a n d s t o p r o d u c e l a r g e r DNA s t r a n d s . 107 I t i s o f i n t e r e s t t o no te t h e o b s e r v a t i o n s o f R o b b i n s and c o - w o r k e r s who o b s e r v e d t h a t UV-damaged XP l ymphocy te s e x h i b i t e d l ong d u r a t i o n s o f 3 H-TdR i n c o r p o r a t i o n i n v i t r o u n t i I t h e same t o t a l amount o f 3 H-TdR was i n c o r p o r a t e d as i n normal l ymphocy te s ( r e v i e w e d by R o b b i n s , 1974) . In o t h e r w o r d s , a l t h o u g h t h e r a t e o f DNA r e p a i r i s s l o w e r i n XP c e l l s , i t i s p o s s i b l e t h a t l o n g e r d u r a t i o n s o f r e p a i r a l l o w t h e same o v e r a l l amount o f r e p a i r t o be r e a c h e d i n XP c e l l s as compared t o t h a t i n normal c e l l s . The r e s u l t s o f s h o r t e r e x p o s u r e s o f c u l t u r e d normal and XP c e l l s t o 4NQ0 r e v e a l e d t h a t d i f f e r e n c e s i n s e d i m e n t a t i o n v e l o c i t y between normal and XP DNA o c c u r r e d f o l l o w i n g d u r a t i o n s o f e x p o s u r e s o f a t l e a s t 6 m i n u t e s . The r e s u l t s i n d i c a t e d t h a t g r e a t e r 4 N Q 0 - i n -duced DNA damage o c c u r r e d i n XP c e l l s , compared w i t h normal c e l l s , d u r i n g e x p o s u r e s t o 4NQ0 o f 6 - m i n u t e s d u r a t i o n o r more . These r e s u l t s can be i n t e r p r e t e d w i t h t h e a s s u m p t i o n t h a t 4NQ0-bound s i t e s on DNA o r 4NQ0-damaged s i t e s on DNA may be a l k a l i n e l a b i l e . Then i t i s r e a s o n a b l e t o i n f e r f r om t h e s e o b s e r v a t i o n s t h a t (a) DNA r e p a i r s y n ^ t h e s i s i s i n p r o g r e s s i n bo th c e l l t y p e s d u r i n g 4NQ0 e x p o s u r e s o f a t l e a s t 6 m i n u t e s , and (b) a t l e a s t a 6 - m i n u t e e x p o s u r e t o 4NQ0 i s r e q u i r e d i n o r d e r t h a t t h e r a t e o f " r e m o v a l " o f 4NQ0 - i nduced damaged (a I k a I i - 1 a b i I e ) s i t e s i n t h e DNA o f normal c e l l s becomes s u f f i c i e n t l y g r e a t e r t h a n t h e r a t e o f " r e m o v a l " i n t h e DNA o f XP c e l l s , t o be d e t e c t a b l e by t h i s t e c h n i q u e . In t h i s manner , a b u i l d - u p o f 4NQ0-i n d u c e d damaged ( a I k a I i - I a b i I e ) s i t e s may o c c u r i n t h e DNA o f XP c e l l s . T h i s b u i l d - u p may be e x p r e s s e d by s l o w e r s e d i m e n t a t i o n v e l o c i t i e s ( g r e a t e r f r a g m e n t a t i o n ) o f t h e t r e a t e d XP DNA compared t o t h e t r e a t e d normal DNA. 108 The t h r e e t e c h n i q u e s o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i -f u g a t i o n (ASG Types I, 2 , and 3 ) , d e s c r i b e d h e r e i n , a r e a l l m o d i f i c a -t i o n s o f t h e t e c h n i q u e o f McGra th and W i l l i a m s (1966) i n t h a t mechan i ca I  s h e a r i n g of DNA was r e d u c e d by l y s i n g t h e c e l l s d i r e c t l y on t o p o f t h e  g r a d i e n t s . The i n t r o d u c t i o n o f t h e t h r e e ASG t e c h n i q u e s e m p h a s i z e s t h e g e n e r a l v e r s a t i l i t y o f t h e m e t h o d , w i t h c e r t a i n a d v a n t a g e s o f f e r e d by some t e c h n i c a l v a r i a t i o n s and not o t h e r s . F o r e x a m p l e , ASG Type 3 has been a d a p t e d .to a n a l y s e s o f DNA damage and r e p a i r i n bo th i n v i t r o ( L a i s h e s and S t i c h , 1973a) and i n v i vo (Cox a t a l . , 1973) s y s t e m s . I t I s i m p o r t a n t t o c o n s i d e r t h e p o s s i b l e m o l e c u l a r i n t e r - p r e t a t i o n s o f t h e s h i f t s i n s e d i m e n t a t i o n p r o f i l e s t h a t a r e d e t e c t e d f o l l o w i n g v e l o c i t y s e d i m e n t a t i o n o f DNA, r e l e a s e d f r o m c h e m i c a l c a r c i n o g e n - t r e a t e d c e l l s , t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s . I n f o r m a t i o n i s no t y e t a v a i l a b l e t o p e r m i t a f u l l u n d e r s t a n d i n g , a t t h e m o l e c u l a r l e v e l , o f t h e c au se and n a t u r e o f t h e i n d i v i d u a l  c h e m i c a l c a r c i n o g e n - i n d u c e d l e s i o n i n DNA. F o l l o w i n g t r e a t m e n t o f a c u l t u r e d mammalian c e l l w i t h a c a r c i n o g e n i c a l k y l a t i n g a g e n t , f o r e x a m p l e , t h e p o s s i b l e r o u t e s by w h i c h a D N A - s t r a n d b r e a k may a r i s e , d u r i n g a l k a l i n e l y s i s and s e d i m e n t a t i o n o f t h e c o n s t i t u e n t DNA t h r o u g h an a l k a l i n e s u c r o s e g r a d i e n t , i n c l u d e : 1. Enzyme ( e n d o n u c l e a s e ) - m e d i a t e d s t r a n d b r e a k s t h a t o c c u r when an e n d o n u c l e a s e r e c o g n i z e s a - chemica l c a r c i n o g e n - b o u n d DNA m o i e t y and n i c k s t h e DNA s t r a n d as p a r t o f a r e p a i r p r o c e s s ( F i g u r e 12) ( C r a t h o r n and R o b e r t s , 1966; S t r a u s s e t a j _ . , 1968; Van L a n c k e r and Tomura, 1974b). 2. Spon taneous d e p u r i n a t i o n of c h e m i c a l c a r c i n o g e n - b o u n d DNA ( L a w l e y , 1966; S t r a u s s and H i l l , 1970) w i t h s u b s e q u e n t h y d r o l y s i s o f t h e s u g a r - p h o s p h a t e c h a i n ( F i g u r e 12) ( Law ley a t aj_. , 1969; L i n d a h l 109 and A n d e r s o n , 1972) . 3. Spon taneous d e p u r i n a t i o n o f c h e m i c a l c a r c i n o g e n - b o u n d DNA ( L a w l e y , 1966; S t r a u s s and H i l l , 1970) w i t h s u b s e q u e n t a l k a l i -p romoted h y d r o l y s i s o f t h e s u g a r - p h o s p h a t e c h a i n ( L a w l e y , 1966; S t r a u s s e_t aj_. , 1968; L i n d a h l and A n d e r s o n , 1972) . 4 . A I k a I i - p r o m o t e d h y d r o l y s i s o f t h e i n t e r m e d i a t e t r i e s t e r s , p r o d u c e d as a r e s u l t o f a l k y l a t e d p h o s p h a t e g r o u p s , w i t h r e s u l t i n g s c i s s i o n o f t h e s u g a r - p h o s p h a t e backbone ( F r e e s e , 1969; W a l k e r and E w a r t , 1973) . 5. A l k a l i n e h y d r o l y s i s enhanced by c h e m i c a l c a r c i n o g e n c o n t a i n e d w i t h i n t h e l y s i n g l a y e r ( e . g . A b e l s o n and Penman, 1973; P a l c i c and S k a r s g a r d , p e r s o n a l c o m m u n i c a t i o n ) . The t e r m s "DNA damage" and "DNA f r a g m e n t a t i o n " a r e , however ' , b e i n g u sed t o r e f e r c o l l e c t i v e l y t o DNA b r e a k s a r i s i n g by one o r more o f t h e f o r e g o i n g p o s s i b i l i t i e s . No a t t e m p t s have been made t o i n t e r p r e t t h e o b s e r v a t i o n s o f t h e s e a l k a l i n e s u c r o s e g r a d i e n t s t u d i e s i n t e r m s o f q u a n t i t a t i v e  DNA m o l e c u l a r w e i g h t changes f o r t h r e e r e a s o n s , a l l o f w h i c h may be i n t e r r e l a t e d t o v a r y i n g d e g r e e s . F i r s t l y , anomalous s e d i m e n t a t i o n i n a l k a l i n e s u c r o s e g r a d i e n t s has been r e p o r t e d f o r l a r g e mammalian DNA m o l e c u l e s w h i c h were t h o u g h t t o r e p r e s e n t f u l l y - d e n a t u r e d s i n g l e s t r a n d s o f DNA (McBurney e t a j _ . , I 971 ;. Ormerod and Lehmann, I 971 a , b ; El k i n d , 1971) . However, more r e c e n t s t u d i e s have d e m o n s t r a t e d t h e appea r ance o f d o u b l e - s t r a n d e d mammalian DNA even w i t h s t r o n g a l k a l i n e d e n a t u r i n g c o n d i t i o n s , and t h e r e f o r e s u g g e s t t h a t t h e p r e s e n c e o f u n d e n a t u r e d m o l e c u l e s may be p a r t l y r e s p o n s i b l e f o r s e d i m e n t a t i o n a n o m a l i e s (S impson e_t a j _ . , 1973; J o l l e y and O rmerod , 1974) . S e c o n d l y , t h e a s s o c i a t i o n o f DNA w i t h t r a c e c o n t a m i n a n t s may a c c o u n t , i n p a r t , I 10 f o r some o f t h e changes o b s e r v e d i n t h e s e d i m e n t a t i o n v e l o c i t y o f mammalian DNA i n a l k a l i n e s u c r o s e g r a d i e n t s ( e . g . Ormerod and Lehmann, 1971b). No C o n t a m i n a t i n g p r o t e i n o r l i p i d , under t h e c o n d i t i o n s d e s c r i b e d h e r e i n , c o u l d be d e t e c t e d i n t h e c o n t r o l o r t r e a t e d DNA peaks l o c a t e d w i t h i n t h e g r a d i e n t ( e . g . F i g u r e s 18 and 19) . However, t h e s e o b s e r v a t i o n s do no t p r o v e t h a t peaks o f DNA, r e s u l t i n g f r om ASG a n a l y s e s c o n d u c t e d i n t h i s manner , a r e f r e e f rom t r a c e c o n t a m i n a t i o n s by c e l l u l a r p r o t e i n o r l i p i d a t l e v e l s be low t h o s e d e t e c t a b l e under t h e e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d . T h i r d l y , changes i n s e d i m e n t a t i o n p r o p e r t i e s have been a t t r i b u t e d t o a l t e r a t i o n s o f t e r t i a r y c o n f o r m a t i o n a l o n e , w i t h o u t c o n c o m i t a n t changes i n m o l e c u l a r w e i g h t , i n s t u d i e s w i t h t h e i n t a c t , h i g h l y - f o l d e d chromosome o f E s c h e r i c h i a c o l i ( S t o n i n g t o n and P e t t i J o h n , 1971; Worce l and B u r g i , 1972) . I t i s t h e r e f o r e f e a s i b l e t h a t c o n f o r m a t i o n a l changes i n t h e t e r t i a r y s t r u c t u r e o f mammalian DNA may p a r t l y a c c o u n t f o r t h e o b s e r v e d changes i n s e d i m e n t a t i o n v e l o c i t y . Hence , t h e f a c t o r s o f i n c o m p l e t e a l k a l i n e d e n a t u r a t i o n  o f DNA, t r a c e c o n t a m i n a t i o n o f DNA, o r v a r i a t i o n s i n t e r t i a r y  c o n f o r m a t i o n o f DNA, s u g g e s t t h a t c a u t i o n s h o u l d be e x c e r c i s e d when i n t e r p r e t i n g changes i n DNA s e d i m e n t a t i o n v e l o c i t y , i n a l k a l i n e s u c r o s e g r a d i e n t s , s o l e l y on t h e b a s i s o f changes i n DNA m o l e c u l a r w e i g h t . In a d d i t i o n , one o r more o f t h e s e t h r e e f a c t o r s may a c c o u n t f o r (a) t h e n o n - r e p r o d u c i b l e , e r r a t i c s e d i m e n t a t i o n p r o f i l e s o f human DNA i n a l k a l i n e s u c r o s e g r a d i e n t s c e n t r i f u g e d a t h i g h r o t o r s p e e d s , and (b) t h e b imoda l p r o f i l e s and b imoda l " t a i l i n g " o f t h e s e d i m e n t i n g human DNA. W i t h r e g a r d t o t h e p o s s i b i l i t i e s o f m e t a b o I i sm of t h e • I l l p r e c a r c i n o g e n s 4NQ0 and AAF by t h e c u l t u r e d human f i b r o b l a s t s , i t i s i n t e r e s t i n g t o n o t e t h a t s h o r t e x p o s u r e s t o l e s s t h a n m i c r o m o l a r c o n c e n t r a t i o n s o f 4NQ0 were c a p a b l e o f i n d u c i n g e x t e n s i v e DNA damage, as e s t i m a t e d by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e , whe rea s AAF f a i l e d t o i n d u c e any d e t e c t a b l e DNA damage a t a l l c o n c e n t r a t i o n s t e s t e d . One a t t r a c t i v e e x p l a n a t i o n f o r t h i s d i f f e r e n c e i n v o l v e s t h e a s s u m p t i o n t h a t l i t t l e , i f a n y , AAF was c o n v e r t e d ( m e t a b o l i z e d ) by t h e human f i b r o b l a s t s t o a DNA-damaging a g e n t ( s uch as N - a c e t o x y - A A F ) w h i l e 4NQ0 was r a p i d l y c o n v e r t e d t o f o rms c a p a b l e o f i n d u c i n g DNA darrage d e t e c t a b l e by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e e m p l o y e d . Such a d i f f e r e n c e i n m e t a b o l i c c o n v e r s i o n i s n o t s u r p r i s i n g when t h e d i f f e r e n c e s i n t h e c h e m i s t r y o f t h e p r o p o s e d b i o t r a n s f o r m a t i o n a r e c o n s i d e r e d . The h i g h l y - o x i d i z e d 4NQ0 m o l e c u l e p r o b a b l y unde r goe s e n z y m i c r e d u c t i o n t o 4HAQ0, p r i o r t o damag ing i n t e r a c t i o n s w i t h . DNA ( r e v i e w e d by Kawazoe e t a j _ . , 1 9 7 2 ) , whe rea s AAF l i k e l y r e q u i r e s e n z y m i c o x i d a t i o n t o t h e N - h y d r o x y d e r i v a t i v e w i t h s u b s e q u e n t N - h yd r o x y e s t e r i f i c a t i o n s t i l l a n e c e s s a r y s t e p p r i o r t o h i g h r e a c t i v i t y w i t h DNA ( r e v i e w e d by M i l l e r , 1 970 ) . The DNA damage o b s e r v e d f o l l o w i n g l o n g e r e x p o s u r e s t o N - h y d r o x y - A A F i n f e r s t h a t t h e human f i b r o b l a s t s may e i t h e r (a ) a c c u m u l a t e some f o rm o f d i r e c t N - h y d r o x y - A A F - i n d u c e d DNA damage, o r (b ) s l o w l y m e t a b o l i z e N - h y d r o x y - A A F t o a DNA-damaging a g e n t s u c h a s N - a c e t o x y - A A F , w i t h a c o n c o m i t a n t b u i l d - u p o f DNA damage t h a t i s d e t e c t a b l e by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e e m p l o y e d . Compa r i s on s between t h e r e s p o n s e s o f normal and XPe c e l l s f o l l o w i n g t r e a t m e n t w i t h a c h e m i c a l c a r c i n o g e n , i n g e n e r a l , r e s t s on t h e a s s u m p t i o n t h a t t h e c h e m i c a l c a r c i n o g e n i s t a k e n i n t o t h e two c e l l t y p e s a t an i d e n t i c a l r a t e . T h i s a s s u m p t i o n r e m a i n s t o be I 12 t e s t e d i n d e t a i l w i t h , f o r e x a m p l e , t r a c e r s t u d i e s u s i n g l a b e l l e d c a r c i n o g e n s . However , t h e r e s u l t s (pages 96 and 97) d e m o n s t r a t i n g e q u a l 4NQ0 - i nduced DNA damage i n normal and XPe c e l l s , f o l l o w i n g a l - m i n e x p o s u r e t o 4NQ0, s u g g e s t s t h a t t h e up t ake o f t h e c h e m i c a l c a r c i n o g e n i n t o normal and XPe c e l l s may we I I be equa I . The p o s s i b l e l i n k between DNA r e p a i r s y n t h e s i s and t h e m o l e c u l a r changes i n d u c e d i n DNA by r e a c t i v e fo rms o f p r e c a r c i n o g e n s i s o u t l i n e d i n F i g u r e 34 . The r e l a t i o n s h i p o b s e r v e d between t h e M e t a b o l i c B i n d i n g DNA ^ R e p a i r o f A c t i v a t i o n o f w t o DNA Damage DNA Damage P r e c a r c i n o g e n s F i g u r e 34 . The dependence o f DNA r e p a i r s y n t h e s i s on t h e m o l e c u l a r a l t e r a t i o n s i n d u c e d i n DNA by r e a c t i v e c a r c i n o g e n i c m e t a b o l i t e s . e x t e n t o f DNA damage ( e s t i m a t e d by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e ) ( L a i s h e s and S t i c h , 1 973b ) , t h e l e v e l o f DNA r e p a i r s y n t h e s i s ( e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) ( S t i c h e t a j _ . , 1 972 ) , and t h e p a r t i c u l a r m e t a b o l i t e o f AAF w i t h w h i c h t h e c e l l s were t r e a t e d ( r e v i e w e d by M i l l e r , 1 970 ) , i s an e xamp le o f how t h e l e v e l s o f DNA damage and r e p a i r a r e i n f a c t g o v e r n e d by t h e m e t a b o l i c s t a t e o f t h e c h e m i c a l c a r c i n o g e n . I 13 SECTION 2 DNA DAMAGE AND REPAIR IN CULTURED MAMMALIAN CELLS EXPOSED  TO PRECARCINOGENS WITH CONCOMITANT ACTIVATION IN VITRO RESULTS E x p e r i m e n t s d e s i g n e d t o d e t e r m i n e w h e t h e r s y n t h e t i c m e t a b o l i t e s o f t h e p r e c a r c i n o g e n AAF were a b l e t o i n d u c e DNA damage, e s t i m a t e d by a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , and DNA r e p a i r , e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , i n c u l t u r e d human c e l l s d e m o n s t r a t e d t h a t t h e l e v e l s o f DNA damage and r e p a i r were h i g h e r f o l l o w i n g e x p o s u r e t o t h e m e t a b o l i t e s and n o t t o t h e p r e c a r c i n o g e n i t s e l f ( L a i s h e s and S t i c h , 1973b; S t i c h e t a I., 1972) . In f a c t , AAF and each o f i t s m e t a b o l i t e s i n d u c e d d i f f e r e n t l e v e l s o f DNA damage and r e p a i r t h a t f o l l o w e d t h e o r d e r : u l t i m a t e c a r c i n o g e n p r o x i m a t e c a r c i n o g e n _ p r e c a r c i n o g e n ( N - a c e t o x y - A A F ) ( N - h y d r o x y - A A F ) (AAF) No m e a s u r a b l e DNA damage and r e p a i r i n d u c e d by t h e p r e c a r c i n o g e n AAF was a l s o c h a r a c t e r i s t i c o f t h e r e s p o n s e s i n d u c e d by e x p o s u r e o f human c e l l c u l t u r e s t o a v a r i e t y o f o t h e r p r e c a r c i n o g e n s ( r e v i e w e d by S t i c h and L a i s h e s , 1973 ) . Many o f t h e s e c h e m i c a l s a r e i n f a c t p o t e n t h e p a t o c a r c i n o g e n s w i t h unknown and u n s t a b l e m e t a b o l i t e s w h i c h , t h e r e f o r e , a r e no t amenable t o s y n t h e t i c p r e p a r a t i o n . If t h e e f f e c t s o f t h e s e " e v a s i v e " a c t i v e m e t a b o l i t e s on t h e genome o f human c e l l s were t o be d e t e r m i n e d , i t was n e c e s s a r y t o d e v e l o p a s y s t e m whereby human " r e c e p t o r " c e l l s c o u l d be expo sed t o such t r a n s i e n t m e t a b o l i t e s g e n e r a t e d i n v i ' t r o . The method o f c h o i c e was t o adap t an i n v i t r o m e t a b o I i c s y s t e m , e m p l o y i n g t i s s u e e x t r a c t s f o r t i f i e d w i t h n e c e s s a r y c o - f a c t o r s , t o t h e g e n e r a t i o n o f m e t a b o l i t e s o f p r e c a r c i n o g e n s I 14 w i t h c o n c o m i t a n t e x p o s u r e t o human c e l l " r e c e p t o r " c u l t u r e s . E xpo su r e o f t h e human c e l l s t o p r e c a r c i n o g e n s , m ixed w i t h c o m p l e t e " a c t i v a t i o n s y s t e m s " , a l l o w e d an e s t i m a t i o n o f t h e l e v e l s o f DNA damage and r e p a i r , f r e q u e n c y o f chromosome a b e r r a t i o n s , and c e l l l e t h a l i t y i n d u c e d by r e a c t i v e m e t a b o l i t e s g e n e r a t e d i n v i t r o . ( a ) D i m e t h I y n i t r o s a m i n e (DMN) The p o t e n t h e p a t o c a r c i n o g e n , DMN, was s e l e c t e d f o r i n v i t r o a c t i v a t i o n e x p e r i m e n t s becau se o f i t s p o t e n t c a r c i n o g e n i c p r o p e r t i e s , i t s p o s s i b l e s i g n i f i c a n c e i n human c a n c e r i n d u c t i o n , i t s p o s i t i o n as t h e " p a r e n t " compound o f a v a s t c o l l e c t i o n o f n i t r o s a m i n e c a r c i n o g e n s , and i n d i c a t i o n s t h a t DMN i s a p r e c a r c i n o g e n t h a t r e q u i r e s m e t a b o l i c c o n v e r s i o n t o a r e a c t i v e m e t a b o l i t e i n v i v o i n o r d e r t o e x e r t i t s c a r c i n o g e n i c e f f e c t s ( r e v i e w e d i n I .A .R .C. Monog raph , 1972) . F o l l o w i n g e x p o s u r e o f c u l t u r e d human f i b r o b l a s t s t o DMN a l o n e and t o DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , c o n t a i n i n g t h e p o s t - m i t o c h o n d r i a I s u p e r n a t a n t (9S) f r a c t i o n o f mouse l i v e r and added NADPH, v a r i o u s s t u d i e s were c a r r i e d o u t . As w e l l as e s t i m a t i n g DNA damage and r e p a i r b y . t h e b i o p h y s i c a l t e c h n i q u e o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , t h r e e b i o l o g i c a l e n d - p o i n t s were e s t i m a t e d a t t h e m o l e c u l a r , c h r omosoma l , and c e l l u l a r l e v e l s . R e s p e c t i v e l y , t h e s e b i o l o g i c a l e n d - p o i n t s w e r e : t h e l e v e l o f D N A . r e p a i r s y n t h e s i s , 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 , and t h e s e n s i t i v i t y o f t h e c e l l s t o a l e t h a l e f f e c t . ( i ) DNA Repa i r S y n t h e s i s E x p e r i m e n t s were d e s i g n e d whereby m o n o l a y e r c u l t u r e s o f I 15 human c e l l s , w i t h DNA rep I i c a t i o n t e m p o r a r i l y b l o c k e d by a r g i n i n e -d e p r i v a t i o n , were e x p o s e d f o r v a r i o u s t i m e s t o 5 x I 0 ~ 2 M DMN a l o n e and t o 5 x I O - 2 M DMN p l u s an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH. F o l l o w i n g t h e s e t r e a t m e n t s , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n t h e human c e l l s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 3 5 ) . 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 d e m o n s t r a t e d t h a t a p r onounced i n c r e a s e i n t h e l e v e l o f DNA r e p a i r s y n t h e s i s o c c u r r e d o n l y i n t h o s e c e l l s e xpo sed t o DMN p l u s t h e a c t i v a t i o n s y s t e m , and t h a t peak a c t i v i t y , e s t i m a t e d i n t h i s manner , o c c u r r e d f o l l o w i n g 60 -m in e x p o s u r e s . The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f DMN, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , on t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s was i n v e s t i g a t e d . F o l l o w i n g 60 -m in e x p o s u r e s o f t h e c e l l c u l t u r e s t o c o n c e n t r a t i o n s o f DMN r a n g i n g f rom 5 x 10 1 1 M t o I x I O - 1 M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( T a b l e 4 ) . The r e s u l t s d e m o n s t r a t e d t h a t t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were h i g h e s t when DMN was comb ined w i t h an a c t i v a t i o n s y s t e m , and were dependent on t h e c o n c e n t r a t i o n o f DMN t o w h i c h t h e c u l t u r e s were e x p o s e d . The l e v e l s o f DNA r e p a i r s y n t h e s i s , i n r e -sponse t o e x p o s u r e s t o DMN a l o n e , i n c r e a s e d w i t h i n c r e a s i n g c o n -c e n t r a t i o n s o f DMN t o a peak o f a c t i v i t y a t I x I O " 1 M DMN. The l e v e l s o f DNA r e p a i r s y n t h e s i s , i n r e s p o n s e t o e x p o s u r e s t o DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , a l s o i n c r e a s e d w i t h i n c r e a s -ing c o n c e n t r a t i o n s o f DMN t o a peak o f a c t i v i t y a t 5 x I 0 ~ 2 M. A s l i g h t d e c r e a s e i n a c t i v i t y was o b s e r v e d f o l l o w i n g t r e a t m e n t w i t h T a b l e 4 . The l e v e l o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o b l a s t s e xpo sed f o r 60 min t o v a r i o u s c o n c e n t r a t i o n s o f DMN a l o n e , and t o v a r i o u s c o n c e n t r a t i o n s o f DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH. ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n -s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) . CONCENTRATI ON _ D . . . . . , r . „ ._ OF DMN (M) GRAINS/NUCLEUS (a) (b) 5.0 x 10 4 5.0 x I O " 3 7.5 x I O - 3 I.0 x I O - 2 2.5 x I 0 " 2 5.0 x I O " 2 7.5 x I O " 2 I.0 x I 0 " 1 3.6 0.1 8.4 0.1 14.2 0 .2 16.8 0 .2 17.6 0 . 3 2 1 . 3 0 .5 19.3 0 .6 17.2 0.7 (a) DMN p l u s c o m p l e t e a c t i v a t i o n s y s t em (b) DMN a l o n e 117 30A • 11 _ i u • z z < LI CD 2 5 2 0 1 5 M 1 Q 5 H / A' — i — 2 0 — I — 40 6 Q — I — S O I 1 D O -A -r 1 2 0 E X P O S U R E T I M E ( m i n u t e s ) F i g u r e 35 . E f f e c t o f d u r a t i o n o f e x p o s u r e t o 5 x I O " 2 M DMN a l o n e : run I (O — O ) , run 2 ( A — A ) , and t o 5 x I O " 2 M DMN p l u s an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH: run I ( • • — • ) , run 2 ( A — - A ) , on t h e l e v e l o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) . I 18 DMN a t c o n c e n t r a t i o n s above 5 x I 0 ~ 2 M. E x p e r i m e n t s were d e s i g n e d t o r e v e a l t h e e f f e c t o f c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , combined w i t h 5 x I O - 2 M DMN, on t h e l e v e l s o f DNA r e p a i r s y n t h e s i s -i nduced i n normal c e l l s . F o l l o w -ing a 60 -m in e x p o s u r e t o DMN i n c o m b i n a t i o n w i t h t h e c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( T a b l e 5 ) . The r e s u l t s d e m o n s t r a t e d t h a t a h i g h l e v e l o f DNA r e p a i r s y n t h e s i s was i n d u c e d o n l y when DMN was i n c u b a t e d w i t h t h e a c t i v a t i o n s y s tem c o n t a i n i n g NADPH. The e x c l u s i o n o f M g C I ? and G6P d i d no t r e d u c e t h e l e v e l s o f DNA r e p a i r s y n t h e s i s as d i d t h e e x c l u s i o n o f NADPH. H i gh l e v e l s o f DNA r e p a i r s y n t h e s i s were a l s o dependent on t h e p r e s e n c e o f a h e a t - l a b i l e ( a t 100° C f o r 10 m in ) component o f t h e mouse l i v e r 9S f r a c t i o n . DMN a l o n e i n d u c e d low l e v e l s o f DNA r e p a i r s y n t h e s i s . Low l e v e l s o f DNA r e p a i r s y n t h e s i s were o b s e r v e d when c e l l s were expo sed t o DMN mixed w i t h an a c t i v a t i o n s y s t e m f o r t i f i e d w i t h NADP i n s t e a d o f NADPH. The p o s s i b i l i t y t h a t DMN, a t t h e c o n c e n t r a t i o n s t e s t e d , i n h i b i t s t h e g e n e r a t i o n o f t h e r e q u i r e d amounts o f NADPH f r om NADP v i a t h e p h o s p h o g I u c o n a t e pathway was no t i n v e s t i g a t e d . The s l i g h t d rop i n a c t i v i t y o b s e r v e d ( F i g u r e 35) f o l l o w i n g e x p o s u r e f o r 120 min r a i s e d t h e q u e s t i o n o f p o s s i b l e t o x i c e f f e c t s o f t h e a c t i v a t i o n m i x t u r e a g a i n s t t h e DNA r e p a i r mechanism of t h e c e l l s . To o b t a i n f u r t h e r i n f o r m a t i o n on t h e p r o b l e m , c e l l c u l t u r e s were e x p o s e d t o 900 e rg s/mm 2 U V - l i g h t ( S t i c h and S a n , 1970) and s u b s e q u e n t -l y i n c u b a t e d w i t h t h e c o m p l e t e a c t i v a t i o n s y s t e m . As i n d i c a t e d i n T a b l e 5, t h e c e l l s e x h i b i t e d no d e t e c t a b l e i n h i b i t i o n o f DNA r e p a i r s y n t h e s i s by e x p o s u r e t o t h e c o m p l e t e a c t i v a t i o n s y s t em f o r up t o I 19 I 20 m i n . DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s was used as an e n d - p o i n t t o a s s e s s t h e e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f t h e c o - f a c t o r , NADPH, on t h e l e v e l o f a c t i v a t i o n o f DMN. An e x p e r i m e n t 'was c a r r i e d o u t i n wh i ch t h e c e l l c u l t u r e s were expo sed f o r 60 min t o 5 x I O - 2 M DMN p l u s v a r i o u s c o n c e n t r a t i o n s o f NADPH, r a n g i n g f r o m 0 mM t o 12.5 mM, c o n t a i n e d i n c o m p l e t e a c t i v a t i o n s y s t e m s . The l e v e l s o f e n s u i n g DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 3 6 ) . The r e s u l t s i n d i c a t e t h a t maximum i n d u c t i o n o f DNA r e p a i r s y n t h e s i s o c c u r r e d f o l l o w i n g e x p o s u r e t o DMN p l u s t h e a c t i v a t i o n s y s t em c o n t a i n i n g a t l e a s t 4 mM NADPH. T h i s c o n c e n t r a t i o n o f NADPH was employed i n a l l s u b s e q u e n t e x p e r i m e n t s i n v o l v i n g DMN a c t i v a t i o n . ( i i ) DNA Damage In o r d e r t o d e t e r m i n e w h e t h e r DNA damage, i n d u c e d by DMN, c o u l d be d e t e c t e d by b i o p h y s i c a l me thod s , e x p e r i m e n t s were d e s i g n e d whereby bo th t r e a t e d and u n t r e a t e d c e l l c u l t u r e s were p r o c e s s e d f o r e s t i m a t i o n o f t h e e x t e n t o f DNA f r a g m e n t a t i o n a n a l y z e d by a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n . C u l t u r e s o f r e p l i c a t i n g human f i b r o b l a s t s were l a b e l l e d w i t h 3 H-TdR and t r e a t e d f o r 60 min w i t h (a) a c o m p l e t e a c t i v a t i o n s y s t em c o n t a i n i n g mouse l i v e r 9S f r a c t i o n and NADPH, G6P, and M g C I 2 , (b) 5 x 10 2 M DMN i n c o m b i n a t i o n - w i t h an a c t i v a t i o n s y s t e m c o n -t a i n i n g h e a t - t r e a t e d ( 1 0 0 ° C f o r 10 m in ) mouse l i v e r 9S f r a c t i o n t o w h i c h NADPH, G6P, and MgC I 2 were a d d e d , ( c ) 5 x I 0 ~ 2 M DMN i n 120 T a b l e 5. L e v e l s o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o -b l a s t s e xpo sed f o r 60 min t o 5 x I O - 2 M DMN. C e l l s were t r e a t e d w i t h DMN a l o n e and i n c o m b i n a t i o n w i t h c o m p l e t e ( A . S . ) and i n c o m p l e t e a c t i v a t i o n s y s t e m s . The 9S f r a c t i o n o f mouse l i v e r was u sed i n t h e a c t i v a t i o n s y s t e m s . The d a t a was o b t a i n e d by t h e a u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR. TREATMENT GRAINS/NUCLEUS DMN a l o n e 0 .5 DMN + A . S . 2 3 . 2 DMN + A . S . : w i t h o u t NADPH 1 .0 u s i n g NADP 2 . 0 w i t h o u t MgC12 22 .5 w i t h o u t G6P 20 . 0 w i t h o u t 9S 0 .0 w i t h h e a t e d 9S 1 .5 A . S . o n l y 0 .0 UV (900 e rgs/mm 2 ) 85 .5 UV (900 e rg s/mm 2 ) p l u s A . S . (120 min) 85 .7 (a) 100° C f o r 10 min 121 2 5 h S 12 mM N A D P H C O N C E N T R A T I O N F i g u r e 36. The l e v e l o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human c e l l s e xpo sed f o r I h ( O — O ) and 2 h (• — • ) t o 5 x I O - 2 M DMN p l u s an a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f mouse I i v e r and v a r i o u s c o n c e n t r a t i o n s o f NADPH. The amounts o f t h e o t h e r c o - f a c t o r s , G6P and M g C I 2 , were n o t v a r i e d . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r -p o r a t i o n o f 3 H - T d R ) . 122 c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g mouse l i v e r 9S f r a c t i o n , G6P, M g C I 2 , w i t h no NADPH ,p re sen t , (d) 5 x 1 0 " 2 M DMN w i t h no a c t i v a t i o n s y s t e m , and (e) MEM. The c e l l s were t h e n p r o -c e s s e d f o r t h e e s t i m a t i o n o f t h e ex- tent o f DNA f r a g m e n t a t i o n a n a l y z e d by v e l o c i t y s e d i m e n t a t i o n o f 3 H-DNA, r e l e a s e d f rom t r e a t -ed and u n t r e a t e d c e l l s , t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g -u r e 3 7 ) . The r e s u l t s o f t h e a l k a l i n e s u c r o s e g r a d i e n t a n a l y s e s r e v e a l e d no s h i f t i n t h e s e d i m e n t a t i o n p r o f i l e o f 3 H-DNA t o r e g i o n s o f g r e a t e r DNA f r a g m e n t a t i o n , t o w a r d s t h e t o p o f t h e g r a d i e n t , f o l l o w i n g e x p o s u r e t o e i t h e r (a ) t h e a c t i v a t i o n s y s t e m o n l y , (b ) 5 x 10 2 M DMN mixed w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g a h e a t - t r e a t e d 9S f r a c t i o n , ( c ) 5 x I O - 2 M DMN m ixed w i t h an a c t i v a t i o n s y s t em w i t h o u t NADPH f o r t i f i c a t i o n , and (d) 5 x I 0 ~ 2 M DMN a l o n e ( F i g u r e 3 7 ) . C e l l c u l t u r e s were expo sed f o r 60 min t o v a r i o u s c o n -c e n t r a t i o n s o f DMN, r a n g i n g f r om 5 x I 0 ~ 3 M t o I x I O - 1 M, i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g mouse l i v e r 9S f r a c t i o n and NADPH, G6P, and M g C I 2 . The c e l l s were t h e n s u b j e c t e d t o t h e a l k a l i n e s u c r o s e g r a d i e n t a n a l y t i c a l p r o c e d u r e ( F i g u r e 3 8 ) . The r e s u l t s showed t h a t a s h i f t In t h e s e d i m e n t a t i o n p r o -f i l e o f 3 H-DNA t o w a r d s t h e t o p o f t h e g r a d i e n t o c c u r r e d f o l l o w i n g e x p o s u r e t o 5 x I O - 3 M DMN i n c u b a t e d t o g e t h e r w i t h t h e c o m p l e t e a c t i v a t i o n s y s t e m . A g r e a t e r s h i f t r e s u l t e d when t h e DMN c o n c e n t r a -t i o n , m i xed w i t h t h e c o m p l e t e a c t i v a t i o n s y s t e m , was i n c r e a s e d t o 5 x I 0 ~ 2 M ( F i g u r e 3 8 ) . I n c r e a s i n g t h e DMN c o n c e n t r a t i o n t o 7.5 x I O - 2 M o r t o I x I 0 _ 1 M t o g e t h e r w i t h t h e c o m p l e t e a c t i v a t i o n s y s t e m , d i d no t i n c r e a s e t h e s h i f t o f t h e s e d i m e n t a t i o n 123 F i g u r e 37. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom c u l t u r e d human f i b r o b l a s t s e x p o s e d f o r 60 min t o : MEM ( c o n t r o l ) ( • — • ) , a c t i v a t i o n s y s tem o n l y , o r 5 x I 0 ~ 2 M DMN p l u s a c t i v a t i o n s y s t e m c o n t a i n i n g h e a t - t r e a t e d ( 1 0 0 ° C, 10 m in ) 9S f r a c t i o n , o r 5 x I O " 2 M DMN p l u s a c t i v a t i o n s y s t e m w i t h o u t NADPH, o r 5 x I O - 2 M DMN a l o n e ( s u p e r i m p o s a b l e p r o f i l e s ) ( O - — O ) . A c o m p l e t e a c t i v a t i o n s y s t em c o n t a i n e d t h e 9S f r a c t i o n o f mouse l i v e r , NADPH, G6P, and MgCI2- The h o r i z o n -t a l b a r d e s i g n a t e s t h e p o s i t i o n o f c o n t r o l DNA peaks (ASG Type 3 ) . F i g u r e 38 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r o m c u l t u r e d human c e l l s e x p o s e d f o r 60 m in t o : MEM ( c o n t r o l ) (• — • ) , 5 x I 0 ~ 3 M DMN p l u s a c o m p l e t e a c t i v a t i o n s y s tem ( • — - o ) , and 5 x I O " 2 M DMN p l u s a c o m p l e t e a c t i v a t i o n s y s tem ( O - - O ) . T r e a t m e n t w i t h 5 x I 0 ~ 2 M DMN p l u s a c o m p l e t e a c t i v a t i o n s y s t em f o r 30 min d i d no t a l t e r t h e p o s i t i o n o f t h e s e d i m e n t a t i o n p r o f i l e f r o m t h a t r e s u l t i n g f r om t h e 60 -m in e x p o s u r e . The a c t i v a t i o n s y s tem c o n t a i n e d mouse l i v e r 9S f r a c t i o n , NADPH, G6P , and MgCl2» The h o r i z o n t a l b a r d e s i g n a t e s t h e p o s i t i o n o f c o n t r o l DNA peaks (ASG Type 3 ) . 125 p r o f i l e any f u r t h e r t o w a r d s t h e t o p o f t h e g r a d i e n t ( no t i l l u s t r a -t e d ). AI s o , t h e exc I u s i o n o f e i t h e r G6P o r MgCl2 d i d no t a f f e c t t h e s h i f t o f t h e s e d i m e n t a t i o n p r o f i l e i n d u c e d by e x p o s u r e t o 5 x I 0 ~ 2 M DMN i n c o m b i n a t i o n w i t h t h e a c t i v a t i o n s y s t em c o n -t a i n i n g mouse l i v e r 9S f r a c t i o n and NADPH ( n o t i l l u s t r a t e d ) . The f o r e g o i n g r e s u l t s o f t h e a l k a l i n e s u c r o s e g r a d i e n t a n a l y s e s ( F i g u r e s 37 and 38) i n d i c a t e d t h a t DNA f r a g m e n t a t i o n i n c u l t u r e d human c e l l s , d e t e c t a b l e by t h i s t e c h n i q u e , was i nduced by a 60 -m in e x p o s u r e t o DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o m p r i s i n g a mouse l i v e r 9S f r a c t i o n , NADPH, G6P, and M g C I 2 . The i n d u c t i o n o f d e t e c t a b l e f r a g m e n t a t i o n appea red t o be (a ) dependent on t h e combined p r e s e n c e o f DMN, NADPH, and a h e a t - l a b i l e com-ponent o f t h e mouse l i v e r 9S f r a c t i o n , (b) u n a f f e c t e d by t h e e x -c l u s i o n o f G6P o r MgC l £ f r om t h e a c t i v a t i o n p r e p a r a t i o n c o n t a i n i n g mouse l i v e r 9S f r a c t i o n and NADPH, and ( c ) dependent on t h e c o n -c e n t r a t i o n o f DMN i n t h e r ange o f 5 x 1 0 " 3 M t o 5 x 1 0 " 2 M , com-b i n e d w i t h t h e a c t i v a t i o n s y s t e m , w i t h no i n c r e a s e i n DNA f r a g m e n t -a t i o n i n d u c e d by do se s o f 7.5 x 1 0 ~ 2 M and I x I O " 1 M DMN, c o n t a i n e d i n t h e a c t i v a t i o n s y s t e m . I t i s i n t e r e s t i n g t o no te t h a t t h e s e o b s e r v a t i o n s d e m o n s t r a t e d t h a t DMN- induced DNA damage i n human c e l l s , e s t i m a t e d by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e , was dependent on t h e p r e s e n c e o f t h e same f a c t o r s r e q u i r e d t o evoke h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( T a b l e 5 ) . An e x p e r i m e n t was d e s i g n e d whereby c u l t u r e s o f human f i b r o -b l a s t s , l a b e l l e d w i t h 3 H - T d R , were t r e a t e d f o r 60 min w i t h 5 x I O - 2 M DMN m i x e d w i t h a c o m p l e t e a c t i v a t i o n s y s t e m , c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r p l u s c o - f a c t o r s . F o l l o w i n g remova l o f t h e I 1 F i g u r e 39 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom normal human c e l l s e xpo sed f o r 60 min t o 5 x I 0 ~ 2 M DMN p l u s an a c t i v a t i o n s y s t em and s u b j e c t e d t o 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 f r e s h MEM f o r : 0 h ( • - — • ) , 4 h and 12 h ( B — - n ) ( s upe r imposab I e on c o n t r o l p r o f i l e w h i c h i s no t shown) . The a c t i v a t i o n s y s t e m c o n t a i n e d mouse l i v e r 9S f r a c t i o n and added NADPH. The h o r i z o n t a l b a r d e s i g n a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . (ASG Type 3 ) . 127 c a r c i n o g e n p r e p a r a t i o n , t h e c e l l c u l t u r e s were s u b j e c t e d t o v a r i o u s p e r i o d s ( r a n g i n g f r o m 0 h t o 12 h) 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 f r e s h g rowth medium p r i o r t o t h e e s t i m a t i o n o f t h e e x t e n t o f DNA f r a g m e n t a t i o n by v e l o c i t y s e d i m e n t a t i o n o f t h e c o n s t i t u e n t 3H-DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g u r e 3 9 ) . The s e d i m e n t a t i o n p r o f i l e s , o b t a i n e d f o l l o w i n g t h e p e r i o d s o f 4 h and 12 h p o s t - t r e a t m e n t i n c u b a t i o n , r e v e a l e d a r e t u r n o f t h e s e d i m e n t a t i o n p r o f i l e s t o w a r d s t h e c o n t r o l DNA r e g i o n . S p e c i f i c a l l y , a 4 h p o s t - t r e a t m e n t i n c u b a t i o n r e s u l t e d i n , a b imoda l s e d i m e n t a t i o n p r o f i l e t h a t p a r t i a l l y o v e r l a p p e d t h e c o n t r o l r e g i o n , whereas a 12 h p o s t - t r e a t m e n t i n c u b a t i o n r e s u l t e d i n a s e d i m e n t a t i o n p r o f i l e t h a t was s u p e r i m p o s a b I e on a c o n t r o l s e d i m e n t a t i o n p r o f i l e . These r e s u l t s i n d i c a t e d t h a t a r e p a i r o f DNA damage, e s t i m a t e d by t h i s t e c h n i q u e f o l l o w i n g t r e a t m e n t w i t h DMN i n c o m b i n a -t i o n w i t h an a c t i v a t i o n s y s t e m , was o c c u r r i n g d u r i n g t h e p o s t - t r e a t -ment o f t h e c e l l c u l t u r e s and a p p e a r s t o be c o m p l e t e by 12 h p o s t -t r e a t m e n t . ( i i i ) DNA R e p a i r S y n t h e s i s i n Normal and XP C e l l s The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f DMN, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , on t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n normal and r e p a i r - d e f i c i e n t (XPe) human c e l l s was i n v e s t i g a t e d . F o l l o w i n g 60 -m in e x p o s u r e s o f t h e c e l l c u l t u r e s t o c o n c e n t r a t i o n s o f DMN r a n g i n g f r o m 5 x I O " 3 M t o I x I O - 1 M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were e s t i -mated by t h e u n s c h e d u l e d i n c o r p o r a t i on o f 3 H-TdR (F i g u r e 4 0 ) . The r e s u l t s showed t h a t t h e l e v e l s of DNA r e p a i r s y n t h e s i s 128 I O ~ 2 I O ]w\ C O N C E N T R A T I O N F i g u r e 40 . The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal (C) and XPe c e l l s expo sed f o r 60 min t o v a r i o u s c o n c e n t r a t i o n s o f DMN a l o n e (normal c e l l s : w-a , XPe c e l l s : • - • • • ) , and t o v a r i o u s c o n c e n t r a t i o n s o f DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH (normal c e l l s : © — ©, XPe c e l l s : O — O ) . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) . 129 were h i g h e s t i n bo th c e l l t y p e s when DMN was combined w i t h an a c t i v a - ' t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH. A peak o f a c t i v i t y was p r o d u c e d when t h e c o n c e n t r a t i o n o f DMN was 5 x IO - 2 M, w i t h h i g h e r DMN c o n c e n t r a t i o n s r e s u l t i n g i n a s l i g h t drop i n a c t i v i t y . The l e v e l s o f DNA r e p a i r s y n t h e s i s were l owe r i n XPe c e l l s , compared w i t h normal c e l l s , f o l l o w i n g e x p o s u r e t o DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . The low l e v e l s o f DNA r e p a i r s y n t h e s i s o b s e r v e d w i t h bo th c e l l t y p e s f o l l o w i n g e x -p o s u r e t o DMN a l o n e a p p e a r e d t o i n c r e a s e s l i g h t l y w i t h i n c r e a s i n g DMN c o n c e n t r a t i o n and were maximal f o l l o w i n g e x p o s u r e t o I x IO - 1 M DMN a l o n e . F o r e x a m p l e , t r e a t m e n t w i t h I x IO - 1 M DMN a l o n e r e -s u l t e d i n l e v e l s o f DNA r e p a i r s y n t h e s i s o f 0.7 g r a i n s / n u c l e u s i n normal c e l l s , and 0.2 g r a i n s / n u c l e u s i n XPe c e l l s . ( i v ) Chromosome A b e r r a t i o n s o f Normal and XP C e l l s The q u e s t i o n s a r o s e as t o w h e t h e r damage c o u l d be e x p r e s s e d a t t h e chromosome l e v e l , by e x p o s u r e o f t h e c e l l c u l t u r e s t o DMN, and w h e t h e r any e f f e c t c o u l d be d e t e c t e d i n t h e f r e q u e n c y o f c h r o m -osome a b e r r a t i o n s i n d u c e d by e x p o s u r e o f t h e c e l l c u l t u r e s t o a c o m b i n a t i o n o f DMN and an a c t i v a t i o n s y s t e m . An e x p e r i m e n t was d e s i g n e d whereby c u l t u r e s o f normal and XPe c e l l s were expo sed f o r 60 min t o c o n c e n t r a t i o n s o f DMN r a n g i n g f rom I x I0~2 M t o I x I 0 _ 1 M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m . C u l t u r e s t r e a t e d w i t h t h e a c t i v a t i o n s y s tem a l o n e were used as c o n t r o l s . F o l l o w i n g t r e a t m e n t , t h e c u l t u r e s were s u b j e c t e d t o e i t h e r 20- , 30-, o r 40-h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h g r owth medium. The c u l t u r e s were samp led a t t h e end o f t he p o s t - t r e a t m e n t i n c u b a t i o n s and t h e p e r c e n t o f metaphase p l a t e s c o n t a i n i n g chromosome a b e r r a t i o n s 130 T a b l e 6. F r equency o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal (C) c e l l s and XPe c e l l s f o l l o w i n g e x p o s u r e f o r 60 min t o c o n c e n t r a t i o n s o f DMN r a n g i n g f r o m I x I O - 2 M t o I x I O - 1 M. The c e l l s were expo sed t o DMN a l o n e , o r t o DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em ( A . S . ) , and t o an A . S . o n l y . The A . S . c o n t a i n e d t h e 9S f r a c t i o n o f mouse \ i v e r and added NADPH. The c u l t u r e s were s amp led a t 40 h p o s t - t r e a t m e n t . PERCENT METAPHASE PLATES WITH CHROMOSOME ABERRATIONS CONCENTRATION OF DMN (M) DMN A l o n e DMN + A . S . C XPe C XPe 1 x I O " 1 0 2 0 .5 20 5 x I O " 2 0 0 0 6 1 x I O " 2 0 0 0 0 A . S . A l o n e 0 0 C o n t r o l 0 0 131 was e s t i m a t e d ( T a b l e 6 ) . On l y d e f i n i t e c h r o m a t i d o r i s o - c h r o m a t i d b r e a k s w i t h d i s l o c a t e d d i s t a l f r a g m e n t s , and s i n g l e and m u l t i p l e exchange f i g u r e s were i n c l u d e d i n t h e c o u n t s . The r e s u l t s o f t h i s e x p e r i m e n t d e m o n s t r a t e d t h a t h i g h e r f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s were d e t e c t e d i n bo th normal and XPe c e l l s f o l l o w i n g e x p o s u r e s t o DMN p l u s an a c t i v a t i o n s y s t e m , compared t o t h e f r e q u e n c i e s o f chromosome a b e r r a t i o n s d e t e c t e d f o l l o w i n g e x p o s u r e s t o e i t h e r DMN o r t h e a c t i v a t i o n s y s t em a l o n e . F r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s above t h e c o n t r o l l e v e l s were no t d e t e c t e d f o l l o w i n g e i t h e r 2 0 - o r 30 -h p o s t - t r e a t m e n t i n c u b a t i o n s ( n o t s hown) . A f r e q u e n c y o f chromosome a b e r r a t i o n s above c o n t r o l l e v e l s was no t d e t e c t e d i n normal c e l l s e xpo sed t o DMN a l o n e , o r i n normal and XPe c e l l s expo sed t o t h e a c t i v a t i o n s y s t em o n l y . In XPe c e l l s e xpo sed t o DMN a l o n e (I x I O - 1 M ) , 2% o f t h e metaphase p l a t e s a n a l y z e d c o n t a i n e d chromosome a b e r r a t i o n s . The f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n XPe c e l l s were c o n s i s t e n t l y h i g h e r t h a n t h o s e o b -s e r v e d i n normal c e l l s and a c h i e v e d t h e h i g h e s t l e v e l s f o l l o w i n g e x p o s u r e t o I x I O - 1 M DMN, bo th w i t h and w i t h o u t an a c t i v a t i o n s y s t e m . F o r e x a m p l e , a low f r e q u e n c y o f chromosome a b e r r a t i o n s (0.5%) was o b s e r v e d i n normal c e l l s e xpo sed t o I x I O - 1 M DMN -p l u s an a c t i v a t i o n s y s t e m , whereas XPe c e l l s , s u b j e c t e d t o t h e same t r e a t m e n t , e x h i b i t e d 20% metaphase p l a t e s w i t h chromosome a b e r r a t i o n s . T h u s , t h e f r e q u e n c i e s o f chromosome a b e r r a t i o n s o b s e r v e d i n normal and XPe c e l l s were h i g h e s t f o l l o w i n g t r e a t m e n t w i t h t h e h i g h e s t c o n c e n t r a t i o n o f DMN t e s t e d (I x I O - 1 M) i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , w i t h s a m p l i n g c o n d u c t e d a t 40 h p o s t -t r e a t m e n t . (v ) C l o n e - f o r m i n g C a p a c i t y o f Normal and XP C e l l s 132 In o r d e r t o d e t e r m i n e t h e l e t h a l e f f e c t o f a c t i v a t e d and n o n - a c t i v a t e d DMN, t h e c l o n e - f o r m i n g c a p a c i t i e s o f normal and XPe c e l l s were e s t i m a t e d f o l l o w i n g 60 -m in e x p o s u r e s t o e i t h e r DMN a l o n e , o r DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH ( F i g u r e 4 1 ) . The r e s u l t s d e m o n s t r a t e d a p o t e n t i a t i o n o f t h e l e t h a l e f f e c t o f DMN by c o m b i n i n g DMN w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . The s e n s i t i v i t y o f XPe c e l l s t o w a r d s t h e l e t h a l e f f e c t o f a c t i v a t e d DMN a p p e a r e d t o be g r e a t e r t h a n t h a t o f t h e normal c e l l s . 133 A I O O so n 6 0 \ C ( D M N » A . S . ) I O O S O \ I X P B I O M N > A S J : C O N C E N T R A T I O N C O N C E N T R A T I O N F i g u r e 4 1 . The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s e xpo sed f o r 60 min t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f r o m 5 x IO - 1 * M t o I x I O - 1 M) o f DMN a l o n e ( • — • ) and- t o v a r i o u s c o n c e n t r a t i o n s o f DMN i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH ( 0 - - 0 ) : (A) Normal e e l Is (B) XPe c e l l s The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . 134 (b) A f l a t o x i n B l and S t e r i g m a t o c y s t i n Because o f t h e e x t r e m e l y p o t e n t c a r c i n o g e n i c p r o p e r t i e s o f a f l a t o x i n Bl (Wogan and Newberne, 1967; Newberne and B u t l e r , 1969; Wogan, 1973 ) , i t s u b i q u i t o u s n a t u r e (Wogan, 1968, 1973; M i l l e r , 1973 ) , i t s p o s s i b l e r e l e v a n c e t o human c a n c e r s (Wogan, 1973a,b ; M i l l e r , 1973) , and i n d i c a t i o n s t h a t a f l a t o x i n B l i s a p r e c a r c i n o g e n t h a t r e q u i r e s m e t a b o l i c c o n v e r s i o n t o a r e a c t i v e m e t a b o l i t e i n v i v o ( G a r n e r e t a [ . , 1971, 1972; G a r n e r , 1972; G u r t o o and Dave , 1973; Ames e t a_l_., 1973; P a t t e r s o n and R o b e r t s , 1972; Wogan, 1973 ) , t h i s f u n g a l m e t a b o l i t e was s e l e c t e d f o r a s s a y i n t h e i n v i t r o a c t i v a t i o n e x p e r i m e n t s . E l a b o r a t e d by a w i d e r v a r i e t y o f f u n g i t h a n a r e t h e a f l a t o x i n s , and s t r u c t u r a l l y r e l a t e d t o t h e a f l a t o x i n s , t h e h e p a t o -c a r c i n o g e n s t e r i g m a t o c y s t i n was a l s o s e l e c t e d f o r s t u d y ( P u r c h a s e and Van de r W a t t , 1970; M i l l e r , 1973; Wogan, 1973 ) . ( i ) DNA R e p a i r S y n t h e s i s E x p e r i m e n t s were d e s i g n e d t o r e v e a l t h e e f f e c t o f c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , comb ined w i t h a f l a t o x i n B l (6 x l O - 5 M) o r s t e r i g m a t o c y s t i n (6 x I 0 ~ 5 M ) , on t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n normal c e l l s . F o l l o w i n g 30 -m in e x p o -s u r e t o t h e c h e m i c a l s i n c o m b i n a t i o n w i t h t h e c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( T a b l e 7 ) . Two a c t i v a t i o n s y s tems were t e s t e d . F i r s t l y , d i r e c t a d d i t i o n of t h e c o - f a c t o r NADPH t o t h e a c t i v a t i o n m i x t u r e r e s u l t e d i n h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s , when combined w i t h e i t h e r a f l a t o x i n Bl o r s t e r i g m a t o c y s t i n w i t h c o n c o m i t a n t e x p o s u r e o f t h e c e l l c u l t u r e s . 0 135 T a b l e 7. L e v e l s o f DNA r e p a i r s y n t h e s i s o f c u l t u r e d human f i b r o -b l a s t s e xpo sed f o r 30 min t o 6 x I 0 ~ 5 M a f l a t o x i n B l , o r 6 x I O " 5 M s t e r i g m a t o c y s t i n . C e l l s were t r e a t e d w i t h t h e c a r c i n o g e n s a l o n e and i n c o m b i n a t i o n w i t h c o m p l e t e ( A . S . ) and i n c o m p l e t e a c t i v a t i o n s y s t e m s . The 9S f r a c t i o n o f mouse l i v e r was u sed i n t h e a c t i v a t i o n s y s t e m s . The d a t a , e x p r e s s e d as g r a i n s p e r n u c l e u s , were o b t a i n e d by t h e a u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R . STERIGMATOCYSTIN AFLATOXIN B l NADPH 3 NADP b NADPH 3 NADP b C a r c i n o g e n a l o n e 2 .4 - 0.4 _ C a r c i n o g e n + A . S . 3 9 . 1 40 .0 15.1 15.5 C a r c i n o g e n + A . S . : w i t h o u t NADPH/NADP 0 . 0 - 0 .0 -wi t h o u t M g C I 2 39.1 3 8 . 9 16.3 15.2 w i t h o u t G6P 37 .6 4 .6 16.8 0 .3 w i t h o u t 9S 0 .5 0.4 0 .2 0 .0 w i t h hea ted 9 S C 5.2 4 . 0 0.4 0 .3 A . S . o n l y 0 . 0 0 .0 - -(a) The A . S . i n t h e s e p r e p a r a t i o n s were f o r t i f i e d w i t h NADPH. (b) The A . S . i n t h e s e p r e p a r a t i o n s were f o r t i f i e d w i t h NADP. ( c ) 100° C f o r 10 m i n . 136 The e x c l u s i o n o f M g C I 2 and G6P d i d no t r e d u c e t h e l e v e l s o f DNA r e -p a i r s y n t h e s i s as d i d t h e e x c l u s i o n o f NADPH. H i g h l e v e l s o f DNA r e p a i r s y n t h e s i s were dependent on t h e p r e s e n c e o f a h e a t - l a b i l e (100 C f o r 10 m in ) component o f t h e mouse l i v e r 9S f r a c t i o n . The c a r c i n o g e n s a l o n e i n d u c e d low l e v e l s o f DNA r e p a i r s y n t h e s i s . The second a c t i v a t i o n s y s t e m , i n w h i c h t h e r e d u c e d c o -f a c t o r NADPH i s g e n e r a t e d f r om t h e o x i d i z e d c o - f a c t o r NADP, p o t e n t i -a t e d h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s when comb ined w i t h e i t h e r a f l a t o x i n B l o r s t e r i g m a t o c y s t i n w i t h c o n c o m i t a n t e x p o s u r e o f t h e c e l l c u l t u r e s . The a d d i t i o n o f G6P as w e l l as NADP was n e c e s s a r y f o r t h e i n d u c t i o n o f h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s whereas t h e a d d i t i o n o f MgC I 2 d i d no t enhance t h e s e a c t i v i t i e s . M o r e o v e r , t h e e x p r e s s i o n o f h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n t h e c e l l s was dependen t on t h e p r e s e n c e o f a h e a t - l a b i l e component o f t h e 9S f r a c t i o n . The a c t i v a t i o n s y s t e m a l o n e i n d u c e d no d e t e c t a b l e DNA r e p a i r s y n t h e s i s . These o b s e r v a t i o n s i n d i c a t e d t h a t t h e p r e s e n c e o f (a) t h e c o - f a c t o r NADPH, o r t h e c o - f a c t o r NADP p l u s t h e c o - f a c t o r G6P, and (b) t h e mouse l i v e r 9S f r a c t i o n c o n t a i n i n g a component t h a t i s h e a t -l a b i l e a t 100 C (10 m i n ) , a r e e s s e n t i a l i n g r e d i e n t s f o r t h e a c t i -v a t i o n s y s t em t h a t , i n c o m b i n a t i o n w i t h a f l a t o x i n B l o r s t e r i g m a t o -c y s t i n , p r o d u c e d h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n t h e human ceI I s . DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s was u sed as an e n d - p o i n t t o a s s e s s t h e e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f t h e c o - f a c t o r NADP on t h e l e v e l o f a c t i v a t i o n o f a f l a t o x i n B l . An e x p e r i m e n t was e x e c u t e d whereby c e l l c u l t u r e s were e x p o s e d f o r 30 min t o 6 x 10 5 M a f l a t o x i n Bl p l u s v a r i o u s c o n c e n t r a t i o n s o f NADP, 137 r a n g i n g f r om 0.25 mM t o 8 mM, c o n t a i n e d i n c o m p l e t e a c t i v a t i o n s y s t e m s . The l e v e l s o f e n s u i n g DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n -s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 4 2 ) . The r e s u l t s i n d i c a t e d t h a t maximum i n d u c t i o n o f DNA r e p a i r s y n t h e s i s o c c u r r e d f o l l o w i n g e x p o s u r e t o a f l a t o x i n B l p l u s t h e a c t i -v a t i o n s y s t em c o n t a i n i n g a t l e a s t 4 mM NADP. T h i s c o n c e n t r a t i o n o f NADP, w h i c h i s c o m p a r a b l e w i t h t h a t r e c e n t l y emp loyed by Ames and c o - w o r k e r s i n a b a c t e r i a l a s s a y s y s t em (Ames e t a I., 1973 ) , was employed i n a l l s u b s e q u e n t e x p e r i m e n t s i n v o l v i n g a f l a t o x i n Bl o r . s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t e m s . E x p e r i m e n t s were d e s i g n e d whereby m o n o l a y e r c u l t u r e s o f human c e l l s were e xpo sed f o r v a r i o u s t i m e s t o 6 x I O - 5 M a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t e m . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f t h e human c e l l s were s u b s e q u e n t l y e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r -a t i o n o f 3 H-TdR ( F i g u r e 4 3 ) . In t h i s e x p e r i m e n t , a marked i n c r e a s e i n t h e l e v e l o f DNA r e p a i r s y n t h e s i s o c c u r r e d o n l y i n t h o s e c e l l s e x p o s e d t o a f l a t o x i n B l p l u s t h e c o m p l e t e a c t i v a t i o n s y s t e m w i t h peak a c t i v i t y o c c u r r i n g f o l l o w i n g 30 -m in e x p o s u r e s . The c a p a c i t y o f t h e h i g h l y o n c o g e n i c a f l a t o x i n B l t o e l i c i t a DNA r e p a i r s y n t h e s i s was compared w i t h t h a t o f t h e m o d e r a t e l y o n c o g e n i c a f l a t o x i n G l and t h e w e a k l y o n c o g e n i c a f l a t o x i n s B2 and G2 (Wogan e t a_L , 1971; Bu t I e r e t a j _ . , 1969; E p s t e i n e t a ]_. , 1969 ) . Human c e l l c u l t u r e s were e xpo sed f o r 30 min t o each o f t h e s e com-pound s , a t c o n c e n t r a t i o n s r a n g i n g f r om 1.2 x I 0 ~ 5 M t o 5 x I O - 4 M, w i t h and w i t h o u t a c t i v a t i o n s y s t e m s . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f t h e human c e l l s were t h e n e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 4 4 ) . 138 F i g u r e 42 . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal human c e l l s e xpo sed f o r 30 min t o 6 x I 0 ~ 5 M a f l a t o x i n Bl p l u s an NADPH-g e n e r a t i n g a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse I i v e r and v a r i o u s c o n c e n t r a t i o n s o f NADP. The amounts o f t h e o t h e r c o - f a c t o r s , G6P and M g C I 2 , were no t v a r i e d . ( A u t o r a d i o g r a p h -i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) 139 F i g u r e 43 . E f f e c t o f d u r a t i o n o f e x p o s u r e t o 6 x I 0 ~ s M a f l a t o x i n B l a l o n e ( • • — • ) , and t o 6 x I 0 ~ 5 M a f l a t o x i n Bl p l u s an NADPH - gene r a t -i ng a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r ( • — • • , O - — O ) , on t h e l e v e l o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) C O N C E N T R A T I O N F i g u r e 44. The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal c e l l e x p o s e d f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l ( • • — • ) , a f l a t o x i n Gl ( 0 - - 0 ) , a f l a t o x i n B2 (vu . - *7 ) , and a f l a t o x i n G2 (•—••) i n c o m b i n a t i o n w i t h an NADPH-generat i ng a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o -g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) 141 A f l a t o x i n B l , w i t h o u t " a c t i v a t i o n " , i n d u c e d low l e v e l s o f DNA r e p a i r s y n t h e s i s i n human c e l l s ( no t i l l u s t r a t e d ) a t t h e f o l -l ow i n g c o n c e n t r a t i o n s : 5 x IO - 1* M ( 0 . 7 g r a i n s / n u c l e u s ) , 1.2 x I 0 _ l + M ( 0 . 5 g r a i n s / n u c l e u s ) , 6 x I O " 5 M ( 0 . 3 g r a i n s / n u c l e u s ) , and 1.2 x I 0 ~ 5 M (0.1 g r a i n s / n u c l e u s ) . No DNA r e p a i r s y n t h e s i s was d e t e c t e d i n t h e human c e l l s f o l l o w i n g e x p o s u r e t o a f l a t o x i n s G l , B2 , and G2 w i t h o u t a c t i v a t i o n . The r e s u l t s o f e x p o s u r e s o f t h e human c e l l s t o t h e c h e m i c a l compounds i n c o m b i n a t i o n w i t h an a c t i -v a t i o n s y s t em r e v e a l e d t h a t t h e h i g h e s t l e v e l s o f DNA r e p a i r s y n -t h e s i s were i nduced by t h e h i g h l y o n c o g e n i c a f l a t o x i n B l , l owe r l e v e l s o f DNA r e p a i r s y n t h e s i s were i n d u c e d by t h e m o d e r a t e l y o n -c o g e n i c a f l a t o x i n G l , and no d e t e c t a b l e DNA r e p a i r s y n t h e s i s was i nduced by t h e w e a k l y o n c o g e n i c d e r i v a t i v e s B2 and G2 . I t was o f i n t e r e s t t o d e t e r m i n e w h e t h e r e x p o s u r e t o a f l a t o x i c o I , a r e d u c t i v e m e t a b o l i t e o f a f l a t o x i n B l ( D e t r o y and H e s s e l t i n e , 1970; P a t t e r s o n and R o b e r t s , 1972 ) , c o u l d i n d u c e DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . To an swer t h i s q u e s t i o n , a f l a t o x i c o l was s y n t h e s i z e d by sod ium b o r o h y d r i d e r e -d u c t i o n o f a f l a t o x i n B l , e x t r a c t e d w i t h c h l o r o f o r m , p u r i f i e d on s i l i c a - g e l t h i n l a y e r s , and i d e n t i f i e d on t h e b a s i s o f T LC , UV, and mass s p e c t r a l p r o p e r t i e s ( G a r n e r e t a l _ . , I 9 72 ) ( page 5 0 ) . An e x p e r i m e n t was t h e n d e s i g n e d whereby human c e l l c u l t u r e s were expo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i c o l , r a n g i n g f r om 1.2 x I O - 5 M t o 5 x I 0 _ l t M, and t o v a r i -ous c o n c e n t r a t i o n s of a f l a t o x i c o l , r a n g i n g f r om I.2 x I 0 ~ 5 M t o 5 x I 0 _ l + M, i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t ems c o n t a i n i n g t h e 9S f r a c t i o n f r om duck l i v e r , o r r a t l i v e r , o r mouse l i v e r . The l e v e l s o f i nduced DNA r e p a i r s y n t h e s i s were t h e n m o n i t o r e d by t h e 142 u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 4 5 ) . The r e s u l t s d e m o n s t r a t e d t h a t a f l a t o x i c o l by i t s e l f was i n e f f e c t i v e , a t a l l c o n c e n t r a t i o n s t e s t e d , i n e v o k i n g d e t e c t a b l e l e v e l s o f DNA r e p a i r s y n t h e s i s i n human c e l l s . H i gh l e v e l s o f DNA r e p a i r s y n t h e s i s were i n d u c e d i n t h e human c e l l s o n l y when expo sed t o a f l a t o x i c o l i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t ems c o n t a i n i n g t h e 9S f r a c t i o n o f d u c k , r a t , o r mouse I i v e r . ( i i ) DNA Damage C u l t u r e s o f r e p l i c a t i n g normal human f i b r o b l a s t s were l a b e l l e d w i t h 3 H-TdR and t r e a t e d f o r 30 m in w i t h a f l a t o x i n B l a t c o n c e n t r a t i o n s r a n g i n g f rom 3 x I 0 ~ 5 M t o 5 x I 0 - 1 + M, w i t h and w i t h o u t an NADPH -gene ra t i n g a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . The c e l l s were t h e n p r o c e s s e d f o r t h e e s t i m a t i o n o f t h e e x t e n t o f DNA f r a g m e n t a t i o n a n a l y z e d by v e l o c i t y s e d i m e n t a t i o n o f 3 H-DNA, r e l e a s e d f r om t h e t r e a t e d and u n t r e a t e d c e l l s , t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g u r e 4 6 ) . The r e s u l t i n g s e d i m e n t a t i o n p r o f i l e s r e v e a l e d no s h i f t o f 3 H-DNA t o w a r d s t h e t o p o f t h e g r a d i e n t , i n d u c e d by e x p o s u r e t o 5 x 10 4 M a f l a t o x i n Bl a l o n e . The c o n c e n t r a t i o n o f 5 x 1 0 - 1 + M a f l a t o x i n Bl was t h e h i g h e s t c o n c e n t r a t i o n t e s t e d and s i n c e l o w e r c o n c e n t r a t i o n s ( 2 . 5 x I O - 4 M and l o w e r ) , w i t h o r w i t h o u t an a c t i -v a t i o n s y s t e m , a l s o p r o d u c e d s e d i m e n t a t i o n p r o f i l e s s u p e r i m p o s a b l e on c o n t r o l p r o f i l e s , t h e s e a r e no t i l l u s t r a t e d . A c o n c e n t r a t i o n o f 5 x IO" 1* M a f l a t o x i n B l , i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , was r e q u i r e d t o i n duce a d e t e c t a b l e s h i f t o f 3 H-DNA s e d i m e n t a t i o n p r o f i l e t o w a r d s t h e t o p o f t h e g r a d i e n t . The a p p e a r a n c e o f t h e peak C O N C E N T R A T I O N F i g u r e 4 5 . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal e e l e x p o s e d f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s of a f l a t o x i c o l a l o n e ( A — A ) and i n c o m b i n a t i o n w i t h an NADPH-generat i ng a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f duck (Musco -v i t e ) l i v e r ( • — • ) , r a t ( W i s t a r ) l i v e r ( 0 - - 0 ) , o r mouse ( S w i s s ) l i v e r ( • • — • ) . The a b s c i s s a i s c a l i b r a t e d i n l o g -a r i t h m i c u n i t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n of t h e u n s c h e d -u l e d i n c o r p o r a t i o n o f 3 H - T d R ) 144 Figure 46. A l ka l i ne sucrose gradient sedimentation p r o f i l e s of 3H-DNA released from cu l tured human f i b r o b l a s t s exposed f o r 30 min to : MEM (contro l ) ( • — • ) ; a c t i v a t i o n system on l y , or 5 x I O - 4 M a f l a t o x i n Bl alone (superimposable p r o f i l e s ) (O -—O) ; 5 x 10 4 M a f l a t o x i n Bl plus a complete a c t i v a t i o n system (•-•-••); 5 x I0~ 2 M DMN plus a complete a c t i v a t i o n system ( A — A ) : only the 3H-DNA contained in f r a c t i o n s 7 to 15 are shown f o r c l a r i t y . The a c t i v a t i o n system contained the 9S f r a c t i o n of mouse l i v e r . The hor izonta l bar designates the pos i t i on of control DNA peaks (ASG Type 3 ) . Figure 47. A l k a l i n e sucrose gradient sedimentation p r o f i l e s of 3H-DNA released from cu l tu red human f i b r o b l a s t s exposed f o r 30 min t o : MEM (contro l ) ( • — • ) , 5 x I O - 4 M s ter i gmatocys t in alone (O-—O), 5 x IO"1* M s ter igmatocys t in p lus an a c t i v a t i o n system (•••«••). The a c t i v a t i o n system contained the 9S f r a c t i o n of mouse l i v e r . The hor izonta l bar designates the p o s i t i o n of control DNA peaks (ASG Type 3). 145 o f 3H-DNA a t f r a c t i o n 4 i n d i c a t e d t h a t DNA f r a g m e n t a t i o n , as d e m o n s t r a t e d by t h i s t e c h n i q u e , was i nduced by 5 x I 0 _ l + M a f l a -t o x i n B l o n l y when t h e c a r c i n o g e n was combined w i t h an a c t i v a t i o n s y s t e m . T r ea tmen t w i t h 5 x IO - 1 * M DMN, i n c o m b i n a t i o n w i t h t h e same a c t i v a t i o n s y s t em (NADPH a d d e d ) , i n d u c e d a marked s h i f t i n s e d i m e n t a t i o n p r o f i l e o f 3H-DNA w i t h a peak o f DNA r a d i o a c t i v i t y a p p e a r i n g a t f r a c t i o n 10. The a c t i v i t y o f t h e a c t i v a t i o n s y s t em was c h e c k e d i n t h i s manner . C u l t u r e s o f normal human f i b r o b l a s t s c o n t a i n i n g 3H-DNA were t r e a t e d w i t h s t e r i g m a t o c y s t i n a t c o n c e n t r a t i o n s r a n g i n g f r om 3 x I O - 5 M t o 5 x \0~k M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m . The c e l l s were t h e n a n a l y z e d by v e l o c i t y s e d i m e n t a t i o n o f 3 H-DNA t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g u r e 4 7 ) . The r e s u l t i n g s e d i m e n t a t i o n p r o f i l e s r e v e a l e d a s h i f t t o w a r d s t h e t o p o f t h e g r a d i e n t i nduced by 5 x I 0 - 1 + M s t e r i g m a t o -c y s t i n a l o n e o r i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . A l t h o u g h b o t h s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA d e r i v e d f r o m t r e a t e d c u l t u r e s e x h i b i t e d peaks o f 3H-DNA a t f r a c t i o n 5 , t h e p r o f i l e r e s u l t i n g f rom t r e a t m e n t w i t h s t e r i g m a t o c y s t i n p l u s an a c t i v a t i o n s y s t e m was b r o a d e r w i t h a d i s t i n c t t a i l i n g o f 3H-DNA t o w a r d s t h e t o p o f t h e g r a d i e n t . T h i s o b s e r v a t i o n i n d i c a t e d t h a t t h a t g r e a t e r DNA f r a g m e n t a t i o n o c c u r r e d when c e l l s were t r e a t e d w i t h 5 x 10 M s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . The c o n c e n t r a t i o n of 5 x I O - 4 M s t e r i g m a t o c y s t i n was t h e h i g h e s t c o n c e n t r a t i o n t e s t e d , and s i n c e l o w e r c o n c e n t r a t i o n s ( 2 . 5 x I 0 - I + M and l o w e r ) , w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , p r o d u c e d s e d i m e n t a t i o n p r o f i l e s s u p e r i m p o s a b I e on c o n t r o l p r o f i l e s , t h e s e a r e n o t i l l u s t r a t e d . T r e a t m e n t o f c e l l c u l t u r e s w i t h DMN p l u s 147 an a c t i v a t i o n s y s tem (NADPH added) was used as a c o n t r o l t o c heck t h e a c t i v i t y o f t h e a c t i v a t i o n s y s t em and 3H-DNA r e l e a s e d f r om t h e s e c u l t u r e s e x h i b i t e d a p r o f i l e as i n F i g u r e 4 6 , a l t h o u g h n o t i l l u s t r a t e d a g a i n i n F i g u r e 47 . ( i i i ) DNA R e p a i r S y n t h e s i s i n Normal and XP C e l l s The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l , w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , on t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n normal and r e p a i r - d e f i c i e n t (XPe) human c e l l s was i n v e s t i g a t e d . F o l l o w i n g 30 min e x p o s u r e s o f c u l t u r e d normal and XPe f i b r o b l a s t s t o c o n c e n t r a t i o n s o f a f l a t o x i n B l r a n g i n g f rom 1.2 x I 0 - 6 M t o 5 x I O - 4 M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were e s t i m a t e d by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 4 8 ) . The r e s u l t s d e m o n s t r a t e d t h a t t h e l e v e l s o f DNA r e p a i r s y n -t h e s i s were h i g h e s t i n bo th c e l l t y p e s when a f l a t o x i n Bl was combined w i t h an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t e m , c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . A peak o f a c t i v i t y was p r o d u c e d when t h e c o n c e n t r a t i o n s o f a f l a t o x i n Bl r anged f r om 6 x I 0 ~ 5 M t o 1.2 x I O - 4 M, w i t h h i g h e r a f l a t o x i n Bl c o n c e n t r a t i o n s r e s u l t i n g i n a s l i g h t d rop i n a c t i v i t y . F u r t h e r m o r e , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were r educed i n XPe c e l l s , compared w i t h normal c e l l s , f o l l o w i n g e x p o s u r e t o a f l a t o x i n Bl i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . The low l e v e l s o f DNA r e p a i r s y n t h e s i s o b s e r v e d w i t h bo th c e l l t y p e s f o l l o w i n g e x p o s u r e t o a f l a t o x i n B l a l o n e a p p e a r e d t o i n c r e a s e s l i g h t l y w i t h i n c r e a s i n g c o n c e n t r a t i o n s and were max imal f o l l o w i n g e x p o s u r e t o 5 x I O - 4 M a f l a t o x i n Bl a l o n e . F o r e x a m p l e , 148 f o l l o w i n g t r e a t m e n t s w i t h 5 x I O - 4 M a f l a t o x i n B l , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were o b s e r v e d t o be 0.6 g r a i n s / n u c l e u s i n normal c e i l s and 0 .2 g r a i n s / n u c l e u s i n XPe c e l l s . C u l t u r e s o f normal and XPe c e l l s were expo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g m a t o c y s t i n , r a n g i n g f r o m 3 x I O " 6 M t o 5 x \0~h M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m . The l e v e l s o f e n s u i n g DNA r e p a i r s y n t h e s i s were r e v e a l e d by e s -t i m a t i n g t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H-TdR ( F i g u r e 4 9 ) . The r e s u l t s d e m o n s t r a t e d t h a t t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were h i g h e s t i n bo th c e l l t y p e s f o l l o w i n g e x p o s u r e t o s t e r i g m a t o c y s t i n i n . c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . The l e v e l s o f DNA r e p a i r s y n t h e s i s i n bo th c e l l t y p e s , f o l l o w i n g e x -po su r e t o s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m , i n c r e a s e d w i t h i n c r e a s i n g s t e r i g m a t o c y s t i n c o n c e n t r a t i o n s w i t h t h e h i g h e s t a c t i v i t y o b s e r v e d w i t h t h e h i g h e s t s t e r i g m a t o c y s t i n c o n c e n -t r a t i o n . Low l e v e l s o f DNA r e p a i r s y n t h e s i s were o b s e r v e d i n XPe c e l l s , compared w i t h normal c e l l s , f o l l o w i n g e x p o s u r e o f bo th c e l l t y p e s t o s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m . The low l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d by e x p o s u r e t o s t e r i g -m a t o c y s t i n a l o n e appea red t o i n c r e a s e s l i g h t l y w i t h i n c r e a s i n g c o n c e n -t r a t i o n s o f s t e r i g m a t o c y s t i n and were maximal f o l l o w i n g e x p o s u r e t o 5 x I O - 4 M s t e r i g m a t o c y s t i n a l o n e . Fo r e x a m p l e , f o l l o w i n g e x p o s u r e s t o 5 x I 0 - I f ' M s t e r i g m a t o c y s t i n , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s were o b s e r v e d t o be 3.0 g r a i n s / n u c l e u s i n normal c e l l s , and 1.5 g r a i n s / n u c l e u s i n XPe c e l l s . 149 2 0 n C O N C E N T R A T I O N F i g u r e 48 . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal (C) and XPe e e l Is e xpo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l a l o n e (normal e e l I s : o««««0, XPe c e l l s : • — - • ) , and t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l i n c o m b i n a t i o n w i t h an NADPH - gene r a t i n g a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r (normal c e l l s : • — • , XPe c e l l s : • — o ) . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o -g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) . 150 F i g u r e 49 . The l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal (C) and XPe c e l l s e xpo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g m a t o c y s t i n a l o n e (normal c e I I s : XPe c e l l s : a — — • ) , and t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g m a t o c y s t i n , i n c o m b i n a -t i o n w i t h an NADPH - gene r a t i n g a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f mouse I i v e r (normal e e l I s : o — " O , XPe e e l I s : • — — • ) . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) 151 ( i v ) Chromosome A b e r r a t i o n s o f Normal and XP C e l l s I t was o f i n t e r e s t t o d e t e r m i n e w h e t h e r damage c o u l d be evoked a t t h e chromosome l e v e l by e x p o s u r e o f t h e c e l l c u l t u r e s t o a f l a t o x i n Bl and what e f f e c t , i f a n y , c o u l d be d e t e c t e d i n 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 by e x p o s i n g c e l l c u l t u r e s t o a c o m b i n a t i o n of a f l a t o x i n B l and an a c t i v a t i o n s y s t e m . An e x p e r i m e n t was d e s i g n e d whereby c u l t u r e s o f normal and XPe c e l l s were e xpo sed t o 6 x 1 0 " 5 M a f l a t o x i n B l , w i t h and w i t h -o u t an a c t i v a t i o n s y s t e m . C u l t u r e s e x p o s e d t o t h e a c t i v a t i o n s y s t em a l o n e were u sed as c o n t r o l s . The t r e a t m e n t s were c o n d u c t e d f o r 5 , 10, 2 0 , and 30 min and t h e n t h e c u l t u r e s were s u b j e c t e d t o e i t h e r 2 5 - , 3 0 - , o r 40 -h p o s t - t r e a t m e n t i n c u b a t i o n s i n f r e s h MEM. The c u l t u r e s were s amp led a t t h e end o f 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 s and t h e p e r c e n t o f metaphase p l a t e s c o n t a i n i n g chromosome a b e r r a t i o n s was e s t i m a t e d ( T a b l e 8 ) . O n l y d e f i n i t e c h r o m a t i d o r i s o - c h r o m a t i d b r eak s w i t h d i s l o c a t e d d i s t a l f r a g m e n t s , and s i n g l e and m u l t i p l e exchange f i g u r e s were i n c l u d e d i n t h e c o u n t s . The r e s u l t s o f t h i s e x p e r i m e n t showed t h a t h i g h e r f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s were d e t e c t e d i n bo th normal and XPe c e l l s f o l l o w i n g e x p o s u r e t o a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t e m , compared w i t h t h e f r e q u e n c i e s o f chromosome a b e r r a t i o n s d e t e c t e d f o l l o w i n g e x p o s u r e s t o e i t h e r a f l a t o x i n Bl o r t h e a c t i v a t i o n s y s tem a l o n e . A low f r e q u e n c y o f chromosome a b e r r a t i o n s ( abou t 3% o f metaphase p l a t e s ) was i n d u c e d i n XPe c e l l s expo sed t o a f l a t o x i n B l f o r 5 , 10, 2 0 , and 30 min ( s a m p l i n g t i m e : 30 h ) . A f r e q u e n c y o f chromosome a b e r r a t i o n s above c o n t r o l l e v e l s was n o t d e t e c t e d i n normal c e l l s e x p o s e d t o a f l a t o x i n Bl a l o n e , o r i n normal 152 Table 8. Frequency of metaphase p la tes with chromosome aberrat ions in cu l tured normal human c e l l s (C) and XPe c e l l s fo l lowing exposure f o r periods of 5 min to 30 min to 6 x I0~5 M a f l a t o x i n Bl a lone, to 6 x I0~5 M a f l a tox in Bl in combination with an a c t i v a t i o n system (A.S. ) , and to an A.S. on ly . The a c t i v a t i o n system contained the 9S f r a c t i o n of mouse I iver and was designed to generate NADPH. The cu l tures were sampled at 25 h, 30 h, and 40 h post - treatment. PERCENT METAPHASE PLATES WITH CHROMOSOME ABERRATIONS SAMPLING DURATION OF EXPOSURE (min) Af la BI+A.S. Af la Bl Alone A.S. Alone Control TIME ( h ) a C XPe C XPe C XPe C XPe 25 5 0 10 0 0 0 0 0 0 10 0 14 0 4 0 0 0 0 20 0 25 0 3 0 0 0 0 30 8 75 0 3 0 0 0 0 30 5 9 32 0 3 0 0 0 0 10 6 47 0 3 0 0 0 0 20 17 100 0 3 0 0 0 0 30 16 100 0 3 0 0 0 0 40 5 4 29 0 2 0 0 0 0 10 4 57 0 3 0 0 0 0 20 6 83 0 2 0 0 0 0 30 7 85 0 3 0 0 0 0 (a) Sampling time is expressed in hours (h) post - treatment. 153 and XPe c e l l s expo sed t o t h e a c t i v a t i o n s y s t em o n l y . A marked i n c r e a s e i n t h e f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n XPe c e l l s was o b s e r v e d w i t h l o n g e r e x p o s u r e t i m e s t o a f l a t o x i n Bl p l u s an a c t i v a t i o n s y s t e m . A n a l y s i s o f t h e XPe s amp le s c o l l e c t e d a t 25 h p o s t - t r e a t m e n t r e v e a l e d an i n c r e a s e i n f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s f r o m 10%, f o l l o w i n g a 5 -min t r e a t m e n t , t o 75%, f o l l o w i n g a 30 -m in t r e a t m e n t . An i n c r e a s e i n f r e q u e n c y o f chromosome a b e r r a t i o n s f r om 32% , f o l l o w i n g a 5 -min t r e a t m e n t , t o 100%, f o l l o w i n g b o t h 20 -m in and 30 -m in t r e a t m e n t s , was r e v e a l e d i n s amp le s c o l l e c t e d a t 30 h p o s t - t r e a t m e n t . A n a l y s i s o f s amp le s c o l l e c t e d 10 h l a t e r , a t 40 h p o s t - t r e a t m e n t , r e v e a l e d a s i m i l a r i n c r e a s e i n 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 f r om 29%, f o l l o w i n g a 5 -min t r e a t m e n t , t o 85% f o l l o w i n g a 30 -m in t r e a t m e n t . The f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n normal c e l l s e xpo sed t o a f l a t o x i n Bl p l u s an a c t i v a t i o n s y s t em were c o m p a r a t i v e l y l owe r t h a n t h o s e In XPe c e l l s , and a c h i e v e d t h e h i g h e s t l e v e l s f o l l o w i n g 2 0 - and 30 -m in t r e a t m e n t s (17% and 16% o f metaphase p l a t e s , r e s p e c t i v e l y ) , sampled a t 30 h p o s t - t r e a t m e n t . A n a l y s i s o f s amp le s c o l l e c t e d a t 25 h p o s t - t r e a t m e n t r e v e a l e d an i n c r e a s e i n f r e q u e n c y o f chromosome a b e r r a t i o n s f r om 0%, f o l l o w i n g 5 - , 1 0 - , and 20 -m in t r e a t m e n t s , t o 8% f o l l o w i n g a 30 -m in t r e a t m e n t . A n a l y s i s o f s amp le s c o l l e c t e d a t 30 h p o s t - t r e a t m e n t r e v e a l e d an i n c r e a s e i n f r e q u e n c y o f chromosome a b e r r a t i o n s f r o m 9% ( 5 -m in t r e a t m e n t ) t o 16% ( 30 -m in t r e a t m e n t ) . A s l i g h t i n c r e a s e i n f r e q u e n c y o f chromosome a b e r r a t i o n s f r om 4% ( 5 - o r 10-min t r e a t m e n t s ) t o 7% ( 30 -min t r e a t m e n t ) was a l s o r e v e a l e d i n s amp le s c o l l e c t e d a t 40 h 154 p o s t - t r e a t m e n t . To d e t e r m i n e t h e e f f e c t o f a f l a t o x i n Bl c o n c e n t r a t i o n on t h e f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n -duced i n normal and XPe c e l l s , an e x p e r i m e n t was e x e c u t e d i n w h i c h normal and XPe c e l l s were e xpo sed f o r 30 min t o v a r i o u s c o n c e n t r a -t i o n s o f a f l a t o x i n B l , r a n g i n g f r o m 6 x I O " 6 M t o I .2 x IO - 1* M, w i t h and w i t h o u t an a c t i v a t i o n s y s t e m . Two a c t i v a t i o n s y s t ems were e m p l o y e d : one t o w h i c h NADPH was added ( F i g u r e 5 0 ) , and one i n w h i c h NADPH was g e n e r a t e d ( F i g u r e 5 1 ) . The r e s u l t s o f t h e e x p e r i m e n t r e v e a l e d t h a t t h e f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n bo th normal and XPe c e l l s were h i g h e r f o l l o w i n g combined t r e a t m e n t w i t h a f l a t o x i n Bl and an a c t i v a t i o n s y s t e m t h a n when t r e a t m e n t s were c o n d u c t e d w i t h a f l a t o x i n B l a l o n e . The f r e q u e n c i e s o f chromosome a b e r r a t i o n s were c o n s i s t e n t l y h i g h e r i n p o p u l a t i o n s o f XPe c e l l s compared t o t h e f r e q u e n c i e s o f chromosome a b e r r a t i o n s i n p o p u l a t i o n s o f normal c e l l s f o l l o w i n g c o m p a r a b l e t r e a t m e n t s . F o l l o w i n g t r e a t m e n t w i t h a f l a t o x i n B l a l o n e , t h e f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a -t i o n s i n XPe c e l l s r e a c h e d 9% w i t h 1.2 x 10"^ M a f l a t o x i n B l , t h e h i g h e s t c o n c e n t r a t i o n t e s t e d , whereas t h e f r e q u e n c y i n normal c e l l s , t r e a t e d i n t h e same manner , n e v e r r o s e above 2% ( F i g u r e 5 0 ) . F o l l o w -i n g t r e a t m e n t w i t h a f l a t o x i n Bl p l u s an a c t i v a t i o n s y s t e m , 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 i n XPe c e l l s e x h i b i t e d b a s i c a l l y t h e same p a t t e r n w i t h b o t h t y p e s o f a c t i v a t i o n s y s t ems ( F i g u r e s 50 and 51) and i n c r e a s e d p r o g r e s s i v e l y t o peak v a l u e s w i t h t h e two h i g h e s t c o n c e n -t r a t i o n s o f a f l a t o x i n B l : 88% (6 x I O - 5 M a f l a t o x i n B l ) , 88% ( 1 . 2 x IO - 1* M a f l a t o x i n Bl ) w i t h t h e a c t i v a t i o n s y s t em c o n t a i n i n g added NADPH ( F i g u r e 5 0 ) ; 93% (6 x I 0 " 5 M a f l a t o x i n B l ) , 100% 155 F i g u r e 50. F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal human c e l l s , f o l l o w i n g 30 -m in e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f rom 6 x I O - 6 M t o 1.2 x I 0 _ l + M) o f e i t h e r a f l a t o x i n B l a l o n e ( a — • ) o r a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t em ( • — • ) , and i n XPe c e l l s , f o l l o w i n g 30 -m in e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f r om 6 x I O - 6 M t o 1.2 x I O - 4 M ) ' o f e i t h e r a f l a t o x i n Bl a l o n e ( • - - • ) o r a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t em ( O — - O ) . The c u l t u r e s were sampled a t 30 h p o s t - t r e a t m e n t . The a c t i v a t i o n s y s tem c o n t a i n e d t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH. The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . F i g u r e 5 1 . F r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal c e l l s ( • — • ) and XPe c e l l s ( 0 - — O ) f o l l o w i n g 30 -m in e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l ( r a n g i n g f rom 6 x I 0 ~ 6 M t o 1.2 x I O - 4 M) p l u s an a c t i v a t i o n s y s t e m . The c u l t u r e s were sampled a t 30 h p o s t - t r e a t m e n t . The a c t i v a t i o n s y s tem was d e s i g n e d t o g e n e r a t e NADPH and c o n t a i n e d t h e 9S f r a c t i o n o f mouse l i v e r . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . CONCENTRATION ( 1 . 2 x 10 4 M a f l a t o x i n B l ) w i t h t h e a c t i v a t i o n s y s t em t h a t was g e n e r a t i n g NADPH ( F i g u r e 5 1 ) . On t h e o t h e r hand , t h e f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n normal c e l l s e x h i b i t e d peak v a l u e s i n t h e range o f 20 -22% when t r e a t e d w i t h t h e two h i g h e s t c o n c e n t r a t i o n s o f a f l a t o x i n B l , combined w i t h e i t h e r o f t h e a c t i v a t i o n s y s t e m s . Normal and XPe c e l l s a l s o d i f f e r e d i n t h e number o f chromosome a b e r r a t i o n s p e r chromosome comp lement . F o r e x a m p l e , t h e a v e r a g e number o f c h r o m a t i d b r e a k s p e r k a r y o t y p e o f c o n t r o l and XPe c e l l s , f o l l o w i n g e x p o s u r e t o 1.2 x I O - 4 M a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t e m , were 1.6 and 5.9 r e s p e c t i v e l y . An e x p e r i m e n t was d e s i g n e d t o r e v e a l t h e e f f e c t o f 30 -m in e x p o s u r e s t o c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , comb ined w i t h a f l a t o x i n Bl (6 x I O - 5 M ) , on 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 i n d u c e d i n normal and XPe c e l l s ( T a b l e 9 ) . The h i g h e s t f r e q u e n c y o f metaphase p l a t e s w i t h c h r o m o -some a b e r r a t i o n s i n b o t h normal and XPe c e l l s , o c c u r r e d f o l l o w i n g e x p o s u r e t o a f l a t o x i n Bl i n c o m b i n a t i o n w i t h t h e c o m p l e t e a c t i -v a t i o n s y s t em and was r educed by t h e e x c l u s i o n o f G6P f r o m t h e a c t i v a t i o n m i x t u r e . A low f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s was i n d u c e d i n XPe c e l l s by a f l a t o x i n B l a l o n e o r i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g no 9S f r a c t i o n o r no NADP. A low f r e q u e n c y o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s was i nduced i n normal c e l l s by a f l a t o x i n B l a l o n e o r i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g no 9S f r a c t i o n . No chromosome a b e r r a t i o n s were d e t e c t e d i n normal c e l l s t r e a t e d w i t h a f l a t o x i n B l i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em l a c k i n g NADP. 158 A s i m i l a r e x p e r i m e n t was d e s i g n e d t o r e v e a l t h e e f f e c t o f 3 0 - m i n e x p o s u r e s t o c o m p l e t e and i n c o m p l e t e a c t i v a t i o n s y s t e m s , combined w i t h s t e r i g m a t o c y s t i n (6 x I O - 6 M ) , on 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 i nduced i n normal and XPe c e l l s ( T a b l e 10 ) . Two a c t i v a t i o n s y s tems were u s e d . F i r s t l y , d i r e c t a d d i t i o n o f t h e c o - f a c t o r NADPH t o t h e a c t i v a t i o n m i x t u r e ( d e s i g n a t e d as "NADPH a d d e d " ) r e s u l t e d i n h i g h f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n p o p u l a t i o n s o f bo t h c e l l t y p e s , when combined w i t h s t e r i g m a t o c y s t i n w i t h c o n c o m i t a n t e x p o s u r e t o t h e c e l l c u l t u r e s . The e x c l u s i o n o f MgCI 2 and G6P d i d n o t p r o d u c e a marked r e d u c t i o n i n 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 i n e i t h e r c e l l t y p e . However , low f r e q u e n c i e s o f metaphase p l a t e s w i t h c h r o m o -some a b e r r a t i o n s were i n d u c e d i n XPe c e l l s by e x p o s u r e t o t h e a c t i -v a t i o n s y s t em a l o n e and by e x p o s u r e t o s t e r i g m a t o c y s t i n a l o n e o r i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em e x c l u d i n g t h e 9S f r a c t i o n o r NADP. The f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n d u c e d i n XPe c e l l s f o l l o w i n g e x p o s u r e t o s t e r i g m a t o c y s t i n a l o n e was m a r k e d l y r educed bu t s t i l l was c o n s i d e r a b l y g r e a t e r t h a n t h e f r e q u e n c i e s o f chromosome a b e r r a t i o n s e voked i n normal c e l l s e x -posed t o s t e r i g m a t o c y s t i n a l o n e . S e c o n d l y , t h e a c t i v a t i o n s y s t e m i n w h i c h t h e c o - f a c t o r NADPH was g e n e r a t e d f rom added NADP ( d e s i g n a t e d as "NADPH g e n e r a t i n g " ) r e s u l t e d i n h i g h f r e q u e n c i e s o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n p o p u l a t i o n s o f bo th c e l l t y p e s , when comb ined w i t h s t e r i g m a t o c y s t i n w i t h c o n c o m i t a n t e x p o s u r e t o t h e c e l l c u l t u r e s . The a d d i t i o n o f G6P was n e c e s s a r y f o r t h e i n d u c t i o n o f h i g h f r e -q u e n c i e s o f chromosome a b e r r a t i o n s . The e x c l u s i o n o f M g C I 2 d i d no t 159 T a b l e 9. F r equency o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal (C) and XPe c e l l s f o l l o w i n g e x p o s u r e t o 6 x I O " 5 M a f l a t o x i n B l . The c e l l s were t r e a t e d w i t h a f l a t o x i n Bl a l o n e o r w i t h a f l a t o x i n Bl i n c o m b i n a t i o n w i t h c o m p l e t e ( A . S . ) and i n c o m -p l e t e a c t i v a t i o n s y s t e m s . The 9S f r a c t i o n o f mouse l i v e r was u sed and t h e a c t i v a t i o n s y s t e m s , where i n d i c a t e d , were d e s i g n e d t o g e n e r a t e NADPH. S amp l i n g t i m e : 30 h p o s t - t r e a t m e n t . PERCENT METAPHASE PLATES WITH CHROMOSOME ABERRATIONS. TREATMENT C XPe A f l a t o x i n Bl a l o n e I 3 y A f l a t o x i n B l + A . S . 12 81 A f l a t o x i n Bl + A . S . : w i t h o u t NADP 0 I w i t h o u t M g C I 2 10 70 w i t h o u t G6P 3 10 w i t h o u t 9S I 3 A . S . o n l y 0 0 .4 160 T a b l e 10. F r equency o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d normal (C) and XPe c e l l s f o l l o w i n g e x p o s u r e t o 6 x I 0 ~ 6 M s t e r i g m a t o c y s t i n . The c e l l s were t r e a t e d w i t h s t e r i g m a t o c y s t i n a l o n e o r w i t h s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h c o m p l e t e ( A . S . ) and i n c o m p l e t e a c t i v a t i o n s y s t e m s . The a c t i v a t i o n s y s t ems u sed t h e 9S f r a c t i o n o f mouse l i v e r a n d , where i n d i c a t e d , were e i t h e r d e -s i g n e d t o g e n e r a t e NADPH o r c o n t a i n e d added NADPH. S a m p l i n g t i m e : 30 h p o s t - t r e a t m e n t . PERCENT METAPHASE PLATES WITH CHROMOSOME ABERRATIONS NADPH NADPH ADDED GENERATING TREATMENT C XPe C XPe S t e r i g m a t o c y s t i n a l o n e 0 1 1 — _ S t e r i g m a t o c y s t i n + A . S . 40 70 50 77 S t e r i g m a t o c y s t i n + A . S . : w i t h o u t NADP/NADPH 0 3 - -w i t h o u t M g C I 2 50 58 40 54 w i t h o u t G6P 38 61 3 6 w i t h o u t 9S 1 8 0 5 A . S . o n l y 0 0.5 0 0 .4 161 m a r k e d l y r e d u c e 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 . Low f r e q u e n c i e s o f metaphase p l a t e s wijfh chromosome a b e r r a t i o n s were i n d u c e d i n XPe c e l l s by e x p o s u r e t o t h e a c t i v a t i o n s y s t em a l o n e o r by s t e r i g m a t o c y s t i n i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em e x c l u d i n g t h e 9S f r a c t i o n . I t i s i n t e r e s t i n g t o n o t e t h a t t h e f r e q u e n c i e s o f m e t a -phase p l a t e s w i t h chromosome a b e r r a t i o n s i n d u c e d i n normal and XPe c e l l s , by e x p o s u r e t o s t e r i g m a t o c y s t i n p l u s an a c t i v a t i o n s y s t e m , d i d no t e x h i b i t t h e p ronounced d i f f e r e n c e e n c o u n t e r e d f o l l o w i n g e x p o s u r e o f t h e two c e l l t y p e s t o a f l a t o x i n B l p l u s an a c t i v a t i o n s y s t e m . (v ) C l o n e - f o r m i n g C a p a c i t y o f Normal and XP C e l l s In o r d e r t o d e t e r m i n e t h e l e t h a l e f f e c t o f a c t i v a t e d and n o n - a c t i v a t e d a f l a t o x i n B l , t h e c l o n e - f o r m i n g c a p a c i t i e s o f normal and XPe c e l l s were e s t i m a t e d f o l l o w i n g 30 -m in e x p o s u r e t o e i t h e r a f l a t o x i n Bl a l o n e , o r a f l a t o x i n B l i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and d e s i g n e d t o g e n e r a t e NADPH ( F i g u r e 5 2 ) . The r e s u l t s showed a p o t e n t i a t i o n o f t h e l e t h a l e f f e c t o f a f l a t o x i n Bl by c o m b i n i n g t h e compound w i t h an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . The s e n s i t i v i t y o f XPe c e l l s t o w a r d s t h e l e t h a l e f f e c t o f a c t i v a t e d a f l a t o x i n B l was c o n s i d e r a b l y g r e a t e r t h a n t h a t o f t h e normal c e l l s . The c l o n e - f o r m i n g c a p a c i t y o f normal and XPe c e l l s was e s t i m a t e d f o l l o w i n g 3 0 -m in e x p o s u r e t o e i t h e r a f l a t o x i n B l a l o n e , o r a f l a t o x i n Bl i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH ( F i g u r e 5 3 ) . 162 CONCENTRATION F i g u r e 52 . The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s e x p o s e d f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f r o m 3 x I O - 6 M t o 1.2 x IO" 1* M) o f a f l a t o x i n B l a l o n e : normal c e l l s ( • « — • ) , XPe c e l l s ( a - - a ) , and t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l ( r a n g i n g f rom 3 x I O - 6 M t o 6 x I 0 ~ 5 M) i n c o m b i n a t i o n w i t h ah N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t e m c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r : normal c e l l s ( o — - O ) , XPe c e l l s ( • — - • ) . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . 163 CONCENTRATION F i g u r e 53 . The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s e xpo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f r om 3 x I 0 ~ 6 M t o 1.2 x 10 - l + M) o f a f l a t o x i n B l a l o n e : normal c e l l s (• — • ) , XPe c e l l s (•——•) , and t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l ( r a n g i n g f rom 3 x I 0 ~ 6 M t o 6 x I 0 ~ 5 M) i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH: normal c e l l s (O — O ) , XPe c e l l s ( • - - • ) . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . 164 The r e s u l t s d e m o n s t r a t e d a p o t e n t i a t i o n o f t h e l e t h a l e f f e c t o f a f l a t o x i n Bl by c o m b i n i n g t h e compound w i t h an a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and added NADPH. A g a i n , i t was a p p a r e n t t h a t t h e s e n s i t i v i t y o f XPe c e l l s t o w a r d s t h e l e t h a l e f f e c t o f a c t i v a t e d a f l a t o x i n Bl was c o n s i d e r a b l y g r e a t e r t h a n t h a t o f t h e normal c e l l s . A c o m p a r i s o n o f t h e c l o n e - f o r m i n g c a p a c i t i e s o f normal and XPe c e l l s , f o l l o w i n g e x p o s u r e t o a f l a t o x i n B l i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems e i t h e r d e s i g n e d t o g e n e r a t e NADPH ( F i g u r e 5 2 ) , o r c o n t a i n i n g added NADPH ( F i g u r e 5 3 ) , r e v e a l e d s i m i l a r l e v e l s o f s e n s i t i v i t i e s t o l e t h a l f a c t o r s g e n e r a t e d by e i t h e r s y s t e m . To d e t e r m i n e t h e l e t h a l e f f e c t o f a c t i v a t e d and n o n -a c t i v a t e d s t e r i g m a t o c y s t i n , t h e c l o n e - f o r m i n g c a p a c i t y o f normal c e l l s was e s t i m a t e d f o l l o w i n g a 30 -m in e x p o s u r e t o e i t h e r s t e r i g -m a t o c y s t i n a l o n e , o r i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s t e m c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r and d e s i g n e d t o g e n e r a t e NADPH ( F i g u r e 5 4 ) . The r e s u l t s d e m o n s t r a t e d a g r e a t p o t e n t i a t i o n o f t h e l e t h a l e f f e c t o f s t e r i g m a t o c y s t i n by c o m b i n i n g t h e compound w i t h an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t e m c o n t a i n i n g t h e 9S f r a c t i o n o f mouse I i v e r . I— C O N C E N T R A T I O N F i g u r e 54 . The c l o n e - f o r m i n g c a p a c i t y o f human c e l l s expo sed f o r 30 min t o v a r i o u s c o n c e n t r a t i o n s ( r a n g i n g f r o m 3 x I O - 7 M t o 1.2 x I O - 4 M) o f s t e r i g m a t o c y s t i n a l o n e ( # — • ) , and t o v a r i o u s c o n c e n t r a t i o n s o f s t e r i g m a t o c y s t i n ( r a n g i n g f r o m 3 x I 0 ~ 7 M t o 6 x I O " 6 M) p i us an N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r ( O — o ) . The a b s c i s s a i s c a l i -b r a t e d i n l o g a r i t h m i c u n i t s . ( c ) S e l e c t i o n o f A c t i v a t i o n M i x t u r e s 166 The i n d u c t i o n o f h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n human c e l l s was a c h i e v e d f o l l o w i n g e x p o s u r e t o p r e c a r c i n o g e n s i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r homogenate. I t was o f i n t e r e s t t o d e t e r m i n e w h e t h e r t h e e n d - p o i n t o f DNA r e p a i r s y n t h e s i s , i n d u c e d i n human c e l l s , was an e x c l u s i v e phenomenon t r i g g e r e d o n l y by e x p o s u r e o f t h e c e l l s t o t h e c o m b i n a t i o n o f p r e c a r c i n o g e n s w i t h a mouse l i v e r 9S f r a c t i o n . C o u l d t h e e n d - p o i n t be a c h i e v e d by e x p o s u r e o f c e l l c u l t u r e s t o p r e c a r c i n o g e n s m i xed w i t h a c t i v a t i o n s y s t ems c o n t a i n i n g t i s s u e f r a c t i o n s f r o m t h e l i v e r o f o t h e r a n i m a l s p e c i e s ? Is t h e r e s p o n s e o f DNA r e p a i r s y n t h e s i s r e s t r i c t e d t o i n d u c t i o n by c o n c o m i t a n t e x -p o s u r e s t o p r e c a r c i n o g e n s m i xed w i t h a c t i v a t i o n s y s t e m s d e r i v e d o n l y f r om l i v e r ? Can s u b c e l l u l a r l i v e r f r a c t i o n s o t h e r t h a n t h e 9S f r a c t i o n be u t i l i z e d i n a c t i v a t i o n s y s tems t o i n d u c e , i n c o m b i n a -t i o n w i t h p r e c a r c i n o g e n s , h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s i n human c e l l s ? The e x p e r i m e n t s t o f o l l o w were d e s i g n e d t o an swer t h e s e q u e s t i o n s . ( i ) P r e p a r a t i o n s f r om V a r i o u s Organs o f D i f f e r e n t A n i m a l s E x p e r i m e n t s were o r g a n i z e d t o a s s e s s t h e a c t i v a t i o n c a p -a c i t y o f 9S f r a c t i o n s d e r i v e d f rom v a r i o u s o r g a n s o f d i f f e r e n t a n i -ma l s t o i n d u c e , i n c o m b i n a t i o n w i t h e i t h e r a f l a t o x i n Bl o r s t e r i g -m a t o c y s t i n , DNA r e p a i r s y n t h e s i s i n human c e l l s . The l e v e l s o f DNA r e p a i r s y n t h e s i s i n human c e l l c u l t u r e s were e s t i m a t e d by t h e u n -s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , f o l l o w i n g 30 -m in e x p o s u r e t o 167 e i t h e r a f l a t o x i n B l (6 x IO""5 M ) , o r s t e r i g m a t o c y s t i n (6 x I O - 5 M ) , m ixed w i t h a c t i v a t i o n p r e p a r a t i o n s c o n t a i n i n g 9S f r a c t i o n s d e r i v e d f rom homogenates o f l i v e r , k i d n e y , o r l u n g , f r om t h e f o l l o w i n g a n i m a l s : hamste r ( S y r i a n ) , mouse ( S w i s s ) , r a b b i t (New Z e a l a n d W h i t e ) , duck ( M u s c o v i t e ) , t r o u t ( R a i n b o w ) , and f l o u n d e r ( S t a r r y ) . O n l y t h e l i v e r 9S f r a c t i o n s were p r e p a r e d f r o m i t h e t r o u t o r f l o u n d e r . The r e s u l t s o f one e x p e r i m e n t a r e i l l u s t r a t e d i n T a b l e I I . The h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s o b s e r v e d i n t h e c e l l s e xpo sed t o e i t h e r a f l a t o x i n Bl o r s t e r i g m a t o c y s t i n i n c o m b i -n a t i o n w i t h any o f t h e a c t i v a t i o n p r e p a r a t i o n s , a r e i n d i c a t i v e o f t h e h i g h a c t i v a t i o n c a p a c i t y o f each o f t h e 9S f r a c t i o n s d e r i v e d f rom t h e l i v e r , k i d n e y , o r lung o f t h e d i f f e r e n t a n i m a l s . I t i s , t h e r e f o r e , e v i d e n t t h a t t h e i n d u c t i o n o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human c e l l s i s n o t an e x c l u s i v e phenomenon t r i g g e r e d o n l y by t h e combined a p p l i c a t i o n o f a f l a t o x i n B l o r s t e r i g m a t o c y s t i n w i t h a mouse l i v e r 9S f r a c t i o n . ( i i ) P r e p a r a t i o n s f r om V a r i o u s S u b c e l l u l a r F r a c t i o n s o f L i v e r  Homogenates In o r d e r t o i n v e s t i g a t e t h e i n t r a c e l l u l a r l o c a l i z a t i o n o f t h e " a c t i v a t i o n c o m p o n e n t s " n e c e s s a r y t o p o t e n t i a t e t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n c u l t u r e d human c e l l s by a f l a t o x i n B l , a c t i v a t i o n sy s tems were p r e p a r e d f rom d i f f e r e n t s u b c e l l u l a r f r a c t i o n s o f t h e l i v e r o f d i f f e r e n t a n i m a l s . S p e c i f i c a l l y , t h e l i v e r s o f m i c e ( S w i s s ) , r a t s ( W i s t a r ) , and duck s ( M u s c o v i t e ) were homogen ized and f r a c t i o n a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n i n t o t h r e e s u b c e l l u l a r f r a c t i o n s : t h e 9S f r a c t i o n ( p o s t - m i t o c h o n d r i a I s u p e r n a t a n t ) , t h e I05P f r a c t i o n (washed m i c r o s o m e s ) , and t h e I05S f r a c t i o n ( c y t o s o l ) . 168 Tab Ie I I . The a c t i v a t i o n c a p a c i t y o f 9S f r a c t i o n s f r om v a r i o u s o r g an s o f d i f f e r e n t a n i m a l s . Normal human f i b r o b l a s t s were e xpo sed (0 .5 h) t o 6 x I O " 5 M a f l a t o x i n Bl o r t o 6 x I O " 5 M s t e r i g m a t o -c y s t i n . The c a r c i n o g e n s were comb ined w i t h a c t i v a t i o n s y s t ems d e s i g n e d t o g e n e r a t e NADPH. ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) DNA REPAIR SYNTHESIS (GRAINS/NUCLEUS) AFLATOXIN B l STERIGMATOCYSTIN Hamster L i v e r 24 .7 36 . 1 ( S y r i an) K i d n e y 21 .3 4 3 . 6 Lung 20 . 6 20 .4 Mouse L i v e r 13.3 16.4 (Swi s s ) Ki dney 1 1.8 16.3 Lung 12.2 14.0 R a b b i t L i v e r 27 .6 27 .7 (New Z e a l a n d Ki dney Wh i t e ) 9 .6 15.0 Lung 13.0 24.1 Duck L i v e r 38 .5 4 3 . 7 ( M u s c o v i t e ) Ki dney 22 .3 -33 . 7 Lung 27 .4 18.7 T r o u t L i v e r 2 3 . 0 25 . 0 (Ra i nbow) F1ounder L i v e r 2 0 . 0 2 3 . 0 ( S t a r r y ) No A c t i v a t i o n P r e p a r a t i o n 0 .2 1 . 1 169 The vo l umes o f t h e p r e p a r a t i o n s were a d j u s t e d , when n e c e s s a r y , t o g i v e e q u i v a l e n t p r o t e i n c o n c e n t r a t i o n s d o w r y m e t h o d ) . A c t i v a t i o n p r e p a r a t i o n s , d e s i g n e d t o g e n e r a t e NADPH, were p r e p a r e d f r om each l i v e r f r a c t i o n o f each a n i m a l . The a c t i v a t i o n c a p a c i t y o f each f r a c t i o n was e s t i m a t e d by m i x i n g each p r e p a r a t i o n w i t h v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l w i t h c o n c o m i t a n t e x p o s u r e o f human c e l l c u l t u r e s . S u b s e q u e n t l y , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s o f t h e c e l l s were a s s e s s e d as i n d i c a t o r s o f t h e a c t i v a t i o n c a p a c i t y o f t h e v a r i o u s s u b c e l l u l a r f r a c t i o n s . I t was d e c i d e d t o comb ine t h e i n v e s t i g a t i o n o f t h e a c t i v a t i o n c a p a c i t i e s o f t h e s u b c e l l u l a r f r a c t i o n s w i t h an i n v e s t i g a t i o n o f t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n normal and XPe c e l l s . The a n a l y s i s was c a r r i e d o u t by e s t i m a t i n g t h e l e v e l s o f DNA r e p a i r s y n t h e s i s o f normal and XPe c e l l s by t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , f o l l o w i n g 0 . 5 - h e x p o s u r e t o v a r i o u s c o n -c e n t r a t i o n s o f a f l a t o x i n B l , r a n g i n g f r om 1.2 x I O " 6 M t o 5 x I O - 4 M, i n c o m b i n a t i o n w i t h t h r e e t y p e s o f a c t i v a t i o n s y s t e m s : ( I ) c o n t a i n -i n g t h e 9S l i v e r f r a c t i o n f rom mouse, r a t , o r duck ( F i g u r e 5 5 ) , (2) c o n t a i n i n g t h e I05P l i v e r f r a c t i o n f r o m mouse, r a t , o r duck ( F i g u r e 5 6 ) , and (3) c o n t a i n i n g t h e I05S I i v e r ' f r a c t i o n f r om mouse, r a t , o r duck ( F i g u r e 5 7 ) . O v e r a l l , t h e r e s u l t s ( F i g u r e s 5 5 , 5 6 , and 57) d e m o n s t r a t e d , t h a t , w i t h t h e e x c e p t i o n o f t h e I05S f r a c t i o n o f r a t l i v e r , a l l t h e s u b c e l l u l a r f r a c t i o n s had t h e c a p a c i t y t o " a c t i v a t e " a f l a t o x i n B l , as i n d i c a t e d by t h e l e v e l s o f DNA r e p a i r s y n t h e s i s d e t e c t e d i n normal and XPe c e l l s . No DNA r e p a i r s y n t h e s i s c o u l d be d e t e c t e d i n normal and XPe c e l l s expo sed t o a f l a t o x i n B l , a t t h e c o n c e n t r a t i o n s t e s t e d , 170 F i g u r e 55 . The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal (C) c e l l s ( • — • ) and XPe c e l l s ( O - —O) f o l l o w i n g e x p o s u r e t o v a r i o u s c o n -c e n t r a t i o n s o f a f l a t o x i n Bl i n c o m b i n a t i o n w i t h N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t ems c o n t a i n i n g t h e 9S f r a c t i o n o f : (A) mouse ( S w i s s ) I i v e r (B) r a t ( W i s t a r ) I i v e r (C) duck ( M u s c o v i t e ) l i v e r The i l l u s t r a t i o n , i n g r a p h ( C ) , o f 160 g r a i n s / n u c l e u s i n d u c e d i n normal c e l l s by 2 .5 x I 0 - I t M a f l a t o x i n Bl , i n c o m b i n a t i o n w i t h an a c t i v a t i o n s y s tem c o n t a i n i n g t h e 9S f r a c t i o n o f duck l i v e r , i s an a p p r o x i m a t i o n s i n c e g r a i n c o u n t s a t t h i s d e n s i t y a r e no t r e l i a b l e . F o r t h e same r e a s o n , t h e r e s u l t s o f t h e e x p o s u r e t o 5 x 10 4 M a f l a t o x i n Bl c o u l d no t be a n a l y z e d . The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) . 171 C O N C E N T R A T I O N 172 F i g u r e 56. The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal (C) c e l l s (••—•) and XPe c e l l s ( O — - O ) f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n B l i n c o m b i n a t i o n w i t h NADPH-gener -a t i n g a c t i v a t i o n s y s tems c o n t a i n i n g t h e I05P f r a c t i o n o f : (A) mouse I i v e r (B) r a t I i v e r (C) duck I i v e r The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o -g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) C O N C E N T R A T I O N 174 F i g u r e 57 . The l e v e l o f DNA r e p a i r s y n t h e s i s o f normal (C) c e l l s ( • — • ) and XPe c e l l s ( O - —o) f o l l o w i n g e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a f l a t o x i n Bl i n c o m b i n a t i o n w i t h N A D P H - g e n e r a t i n g a c t i v a t i o n s y s t ems c o n t a i n i n g t h e I05S f r a c t i o n o f : (A) mouse I i v e r (B) r a t I i v e r (C) duck I i v e r The a b s c i s s a i s c a l i b r a t e d i n l o g a r i t h m i c u n i t s . ( A u t o r a d i o g r a p h i c d e t e c t i o n o f t h e u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R ) C O N C E N T R A T I O N 176 when combined w i t h t h e I05S f r a c t i o n o f r a t l i v e r . In a l l c a s e s where DNA r e p a i r s y n t h e s i s was d e t e c t e d , h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s were i n d u c e d i n normal c e l l s , whereas XPe c e l l s e x h i b i t e d l e v e l s o f DNA r e p a i r s y n t h e s i s t h a t were l o w e r t h a n t h e l e v e l s o b s e r v e d i n normal c e l l s . G e n e r a l l y , i n bo th c e l l t y p e s , t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f a f l a t o x i n B l . A c t i v a t i o n m i x t u r e s c o n t a i n i n g a f l a t o x i n B l and t h e I05S l i v e r f r a c t i o n s were s e l e c t e d f o r a s s a y by p h y s i c a l methods . U l t r a v i o l e t s p e c t r o s c o p y o f methano l e x t r a c t s ( P a t t e r s o n and R o b e r t s , 1972) o f t h e combined a f l a t o x i n B l / a c t i v a t i o n s y s t e m , f o l l o w i n g i n c u b a t i o n f o r 30 min a t 3 7 ° C , r e v e a l e d a maximum a b s o r p t i o n a t X = 330 nm o n l y when a c t i v a t i o n s y s t ems c o n t a i n e d t h e I 05S f r a c t i o n o f duck l i v e r . The a b s o r p t i o n a t 330 nm, w i t h a s i m u l t a n e o u s r e d u c t i o n i n a b s o r p t i o n a t 363 nm, i s i n d i c a t i v e o f c o n v e r s i o n o f a f l a t o x i n B l (X = 363 nm) t o a r educed m e t a b -max. o l i t e , a f l a t o x i c o l (X = 330 nm) ( D e t r o y and H e s s e l t i n e , 1970; max. P a t t e r s o n and R o b e r t s , 1972) . No s h i f t f r om X 363 nm, i n d i c a t i v e . max. o f unchanged a f l a t o x i n B l , was d e t e c t e d i n t h e UV s p e c t r a o f methano l e x t r a c t s of a c t i v a t i o n m i x t u r e s c o n t a i n i n g a f l a t o x i n B l i n c o m b i n a -t i o n w i t h r a t l i v e r I05S f r a c t i o n . When mouse l i v e r I05S f r a c t i o n was e m p l o y e d , a v e r y s l i g h t s h o u l d e r a t 330 nm, w i t h X 363 nm ITI9X a ( a f l a t o x i n B l ) , was d e t e c t e d o n l y o c c a s i o n a l l y and n o t r e p r o d u c i b I y . T h i n - l a y e r c h r o m o a t o g r a p h y (TLC) o f a c h l o r o f o r m e x t r a c t o f t h e combined. a f I a t o x i n B l / a c t i v a t i o n s y s t e m , f o l l o w i n g i n c u b a t i o n f o r 30 m in a t 3 7 ° C r e v e a l e d a f l u o r e s c e n t s p o t o f Rf 0 . 5 5 , i n d i c a t i v e o f a f l a t o x i c o l ( G a r n e r e t a j _ . , 1972) ( R f o f a f l a t o x i n B l : 0 . 4 0 ) , o n l y when a c t i v a t i o n s y s tems c o n t a i n e d t h e I05S f r a c t i o n o f duck l i v e r . When mouse o r r a t l i v e r was s u b s t i t u t e d as a s o u r c e o f t h e I 05S f r a c t i o n , no f l u o r e s c e n t s p o t o f Rf 0 .55 was d e t e c t e d . I t i s o f i n t e r e s t t o no te t h a t a l t h o u g h a f l a t o x i c o l a p p e a r s t o be p r o d u c e d by t h e duck l i v e r I05S f r a c t i o n , e x p o s u r e o f c u l t u r e d human c e l l s t o s y n t h e t i c a l l y p r e p a r e d a f l a t o x i c o l r e s u l t e d i n h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s o n l y when t h e a f l a t o x i c o l was combined w i t h a 9S f r a c t i o n o f mouse, r a t , o r duck l i v e r s ( F i g u r e 4 5 ) . 178 DISCUSSION OF SECTION 2 I t i s a x i o m a t i c t h a t c h e m i c a l c a r c i n o g e n s must i n t e r a c t w i t h c e l l u l a r m o l e c u l e s i n a manner n e c e s s a r y t o p r o d u c e n e o p l a s t i c t r a n s -f o r m a t i o n . In t h i s c o n t e x t , i t has been o b s e r v e d t h a t DMN ( r e v i e w e d by Magee, 1972) and a f l a t o x i n Bl ( L i j i n s k y e t a I., 1970) b i n d s t r o n g l y t o DNA i n t h e i n t a c t an ima l whereas a f l a t o x i n B l , f o r e x a m p l e , e x h i b i t s o n l y a s m a l l amount o f weak b i n d i n g t o DNA i n v i t r o . These o b s e r v a t i o n s have been i n t e r p r e t e d t o i n d i c a t e t h a t t h e p r e -c a r c i nogens have been metabo l i c a l l y c o n v e r t e d _[n_ v i v o t o metabo l i t e s t h a t a r e h i g h l y r e a c t i v e w i t h DNA and t h e r e f o r e bond c o v a l e n t l y t o t h e m a c r o m o l e c u l e ( r e v i e w e d by M i l l e r , 1970, 1973) . F o r e x a m p l e , N - a c e t o x y - A A F , a s y n t h e t i c a l l y p r e p a r e d , u l t i m a t e c a r c i n o g e n i c m e t a b o l i t e o f AAF, e x h i b i t s p o t e n t r e a c t i v i t y t o w a r d s c e l l u l a r mo I ecu I e s . I t was t h e r e f o r e e l e c t e d t o d e s i g n a s i m u l a t i o n o f i n v i v o c o n d i t i o n s o f p r e c a r c i n o g e n m e t a b o l i s m such t h a t t h e b i o l o g i c a l r e -s pon se s o f human ee l I c u l t u r e s t o DNA damage i n d u c e d by m e t a b o l i t e s c o u l d be e s t i m a t e d . C o n c o m i t a n t e x p o s u r e o f c e l l c u l t u r e s t o t h e p r e c a r c i n o g e n s i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s t ems wou ld h o p e -f u l l y " c a p t u r e " t h e DNA-damaging e f f e c t s o f u n s t a b l e m e t a b o l i t e s t h a t h a ve , so f a r , evaded i s o l a t i o n . The l e v e l s o f DNA r e p a i r s y n t h e s i s , f r e q u e n c y o f c h r o m o -some a b e r r a t i o n s , and c l o n e - f o r m i n g c a p a c i t y o f c u l t u r e d human c e l l s were e s t i m a t e d f o l l o w i n g e x p o s u r e t o t h e p r e c a r c i n o g e n s DMN, and t h e s t r u c t u r a l l y - r e l a t e d m y c o t o x i n s , a f l a t o x i n Bl and s t e r i g m a t o c y s t i n . The e x p o s u r e s t o t h e p r e c a r c i n o g e n s were c o n d u c t e d w i t h and w i t h o u t c o n c o m i t a n t m e t a b o l i c a c t i v a t i o n i n y i t r o . These b i o l o g i c a l r e s p o n s e s 179 t o t r e a t m e n t w i t h t h e c h e m i c a l c a r c i n o g e n s may be e x p r e s s i o n s o f DNA a l t e r a t i o n s t h a t r e s u l t i n an u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , damage a t t h e chromosome l e v e l , and a l e t h a l a c c u m u l a t i o n o f DNA l e s i o n s . The r e s u l t s c l e a r l y d e m o n s t r a t e a marked p o t e n t i a t i o n  o f t h e a b i l i t y o f DMN, a f l a t o x i n B l , and s t e r i g m a t o c y s t i n t o  t r i g g e r h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s , chromosome a b e r r a t i o n s , and c e l l l e t h a l i t y , when comb ined w i t h a c t i v a t i o n s y s t ems c o n t a i n - i ng t h e 9S f r a c t i o n o f mouse l i v e r . E s t i m a t e s o f DNA f r a g m e n t a t i o n by a l k a l i n e s u c r o s e g r a d i e n t a n a l y s e s , r e v e a l e d t h a t g r e a t e r DNA damage was i n d u c e d i n human c e l l s by DMN, a f l a t o x i n B l , and s t e r i g m a t o c y s t i n when combined w i t h an a c t i v a t i o n p r e p a r a t i o n f rom mouse l i v e r . The DNA-damaging e f f e c t was more p ronounced f o r a c t i v a t e d DMN t h a n w i t h a c t i v a t e d a f l a t o x i n B l and s t e r i g m a t o c y s t i n . N e v e r t h e l e s s , t h e r e s u l t s i n d i c a t e t h a t t h e  e x t e n t o f e a r l y DNA damage may be l i n k e d t o t h e b i o l o g i c a l r e s p o n s e s  o f h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s , chromosome a b e r r a t i o n s , and  e e l I l e t h a l i t y - a l l o f w h i c h may be c o n t r o l l e d a t an " e a r l y " s t a g e by t h e m e t a b o l i c f o rm o f t h e c h e m i c a l c a r c i n o g e n s ( F i g u r e 5 8 ) . R e p a i r o f ^ C h r o m o s o m e ^ C e l I DNA Damage A b e r r a t i o n s Death (Gene M u t a t i o n s ) F i g u r e 58. The p o s s i b l e l i n k between m e t a b o l i c a c t i v a t i o n , e a r l y DNA a l t e r a t i o n s , and b i o l o g i c a l r e s p o n s e s a t t h e m o l e c u l a r , c h r omosoma l , and c e l l u l a r l e v e l s . M e t a b o l i c D . .. . i • .. , _ ^ B i nd i ng w D N A A c t i v a t i o n o f n M A • • n o . t o DNA Damaqe P r e c a r c i n o g e n s a 180 The d i f f e r e n c e between t h e e x t e n t o f DNA damage, e s t i m a t e d on a l k a l i n e s u c r o s e g r a d i e n t s , i n d u c e d i n human c e l l s by a c t i v a t e d a f l a t o x i n Bl and s t e r i g m a t o c y s t i n i n v i t r o on t h e one hand , and t h a t i n d u c e d by a c t i v a t e d DMN i n v i t r o on t h e o t h e r ( L a i s h e s and S t i c h , 1973b) , can be r a t i o n a l i z e d , among o t h e r p o s s i b i l i t i e s , on t h e b a s i s o f d i f f e r e n t amounts o f a I k a I i - I a b i I e s i t e s p o s s i b l y i n -duced i n DNA by t h e two d i f f e r e n t s t r u c t u r a l t y p e s o f c h e m i c a l s . The i n d u c t i o n o f a I k a I i - 1 a b i I e s i t e s i s t h o u g h t t o c au se a v a s t o v e r - e s t i m a t i o n o f a c t u a l D N A - s t r a n d r u p t u r e i n d u c e d i n v i v o ( L a w l e y , 1966; S t r a u s s and H i l l , 1970; L i n d a h l and A n d e r s o n , 1972) . F u r t h e r m o r e , a I k a I i n e d e n a t u r a t i o n c o u l d be d r a s t i c a l l y i n f l u e n c e d by r e l a t i v e l y few i n t e r - s t r a n d c r o s s - l i n k s p o s s i b l y i n d u c e d i n DNA by t h e m y c o t o x i n s and t h e i r m e t a b o l i t e s . C r o s s - 1 i n k s wou ld a f f e c t t h e s e d i m e n t a t i o n p r o p e r t i e s o f t h e t r e a t e d DNA ( J o l l e y and O rmerod , 1974; S impson e t a j _ . , 1973 ) . In t h i s c o n t e x t , i t i s w o r t h w h i l e t o draw a t t e n t i o n t o o t h e r i n v e s t i g a t i o n s t h a t have a l s o d e t e c t e d o n l y low l e v e l s o f DNA f r a g m e n t a t i o n i n d u c e d by h i g h c o n c e n t r a t i o n s o f a f l a t o x i n Bl b o t h i n human c e l l s i n v i t r o w i t h o u t a c t i v a t i o n (Umeda e t a j _ . , 1972 ) , and i n r a t I i v e r e e l I s i n v i vo ( K e e f e and Edwa rd s , 1974 ) . G e n e r a l l y , i t may be u s e f u l t o c o n s i d e r f r om t h i s and o t h e r s t u d i e s , t h e c o m p a r a t i v e l y s m a l l amount o f a f l a -t o x i n B l - i n d u c e d DNA damage d e t e c t a b l e on a l k a l i n e s u c r o s e g r a d i e n t s on t h e one hand , and t h e e x t r e m e c a r c i n o g e n i c p o t e n c y o f a f l a t o x i n B l , on t h e o t h e r . T h i s o b s e r v a t i o n s u g g e s t s t h a t a r e a s o n a b l e amount  o f c a u t i o n s h o u l d be e x e r c i s e d when c o m p a r i n g t h e e x t e n t o f DNA damage, d e t e c t e d e x c l u s i v e l y by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h -n i q u e , w i t h t h e c a r c i n o g e n i c c a p a c i t y o f t h i s g roup o f compounds. 181 I t i s i n t e r e s t i n g t h a t t h e r e p a i r o f t h e DNA damage, i n d u c e d by t r e a t m e n t o f human e e l I c u l t u r e s w i t h a c t i v a t e d DMN and e s t i m a t e d by t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e , was f a i r l y r a p i d and appea red t o be c o m p l e t e by 12 h. T h i s o b s e r v a -t i o n i s i n s h a r p c o n t r a s t w i t h t h e t ' ime r e q u i r e d (2 weeks o r more) f o r t h e c o m p l e t i o n o f t h e r e p a i r o f DNA damage f o l l o w i n g DMN a d -m i n i s t r a t i o n t o m i c e ( L a i s h e s and S t i c h , u n p u b l i s h e d o b s e r v a t i o n s ) and r a t s (Damjanov a t a j _ . , 1973 ) . The r e a s o n f o r t h i s d i f f e r e n c e i s no t known. The b imoda l s e d i m e n t a t i o n p r o f i l e s o b s e r v e d d u r i n g t h e s h o r t e r r e p a i r p e r i o d s o f a b o u t 4 h p o s t - t r e a t m e n t a r e , howeve r , c o m p a r a b l e t o t h e b imoda l p r o f i l e s f r e q u e n t l y o b s e r v e d i n i n v i v o s t u d i e s ( L a i s h e s and S t i c h , u n p u b l i s h e d o b s e r v a t i o n s ; Damjanov a t a j _ . , 1973) and imp l y t h a t one p o p u l a t i o n o f DNA f r a g m e n t s i s b e i n g r e p a i r e d a t a f a s t e r r a t e t h a n a second p o p u l a t i o n o f DNA f r a g m e n t s ( e . g . Damjanov e t a j _ . , 1973 ) . Many a p p r o a c h e s have been t a k e n t o u n c o v e r t h e m o l e c u l a r mechani sm o f t h e c a r c i n o g e n i c a c t i o n o f t h e most p o t e n t h e p a t o -c a r c i n o g e n , a f l a t o x i n B l . A t t e n t i o n has been f o c u s e d on t h e i n t e r -a c t i o n o f a f l a t o x i n Bl w i t h e v e r y c l a s s o f c e l l u l a r macromoI ecu Ies ( r e v i e w e d by G o l d b l a t t , 1969; Wogan, 1973 ) . A l t h o u g h s t r o n g b i n d i n g o f a f l a t o x i n Bl t o DNA has been d e m o n s t r a t e d bo th i n v i v o ( L i j i n s k y e_t a\_., 1970) and i n v i t r o . , o n l y w i t h a c t i v a t i o n ( G a r n e r , 1973; G u r t o o e t a j _ . , 1974 ) , a r gument s have been p r e s e n t e d a g a i n s t t h e r o l e o f a f I a t o x i n - i n d u c e d DNA a l t e r a t i o n s i n a f l a t o x i n c a r c i n o -g e n e s i s ( W i l l i a m s and R a b i n , 1971; K e e f e and Edward s , 1974 ) . In t h e s t u d i e s d e s c r i b e d h e r e i n , howeve r , a t t e n t i o n has been f o c u s e d on t h r e e b i o l o g i c a l r e s p o n s e s : DNA r e p a i r s y n t h e s i s , chromosome a b e r r a t i o n s , and c e l l s u r v i v a l w h i c h were e s t i m a t e d w i t h r e p a i r -182 d e f i c i e n t XP c e l l s as w e l l as w i t h normal human c e l l s f o l l o w i n g e x p o s u r e t o " a c t i v a t e d " and " n o n - a c t i v a t e d " p r e c a r c i n o g e n s . The f o l l o w i n g r e l a t i o n s h i p s were o b s e r v e d f r om c o m p a r i s o n s o f t h e r e s p o n s e s o f XP c e l l s w i t h normal c e l l s : XP c e l l s e x h i b i t e d low  l e v e l s o f DNA r e p a i r s y n t h e s i s , h i g h f r e q u e n c i e s o f chromosome  a b e r r a t i o n s , and an e l e v a t e d s e n s i t i v i t y t o w a r d s a c y t o t o x i c e f f e c t . In v i e w o f t h e p r o f o u n d s u s c e p t i b i l i t y o f t h e XP p a t i e n t t o t h e c a r c i n o g e n i c e f f e c t o f U V - r a d i a t i o n damage, and t h e r e d u c e d a b i l i t y o f t h e f i b r o b l a s t s d e r i v e d f r o m t h e p a t i e n t t o r e p a i r e i t h e r UV-i n d u c e d o r c h e m i c a l c a r c i n o g e n - i n d u c e d damage t o DNA ( e . g . C l e a v e r , 1970a ,b ; S t i c h and S a n , 1971 ) , t h e s e o b s e r v a t i o n s have i m p o r t a n t i m p l i c a t i o n s . The c o m p a r a t i v e l y low r e s p o n s e o f DNA r e p a i r s y n t h e s i s i n d u c e d by a c t i v a t e d a f l a t o x i n Bl i n XP c e l l s , s u g g e s t s t h a t XP c e l l s have a low c a p a c i t y t o cope w i t h a f l a t o x i n B l - i n d u c e d DNA l e s i o n s . Hence , t h e r o l e o f DNA r e p a i r i n a f l a t o x i n Bl c a r c i n o g e n e s i s i s  imp I i c a t e d . T h i s i m p l i c a t i o n , t o g e t h e r w i t h t h e d r a m a t i c c h r omo -some-damaging e f f e c t o f a c t i v a t e d a f l a t o x i n Bl i n XP c e l l s , s u g g e s t s a r e - e v a l u a t i o n o f t h e r o l e o f a f l a t o x i n B l - i n d u c e d DNA a l t e r a t i o n s i n t h e c o n s i d e r a t i o n s o f t h e p o s s i b l e mechan i sms o f c a n c e r i n d u c t i o n by a f I a t o x i n B l . S i m i l a r a rgument s can be r a i s e d f o r t h e n e c e s s a r y c o n s i d e r -a t i o n o f DMN- induced DNA a l t e r a t i o n s and DNA r e p a i r i n t h e f r a m e -work o f mechanisms p u t f o r t h t o e x p l a i n DMN c a r c i n o g e n e s i s . I t i s o f i n t e r e s t t o no te t h a t t h e r e l a t i o n s h i p between low l e v e l s o f DNA r e p a i r s y n t h e s i s i n XP c e l l s , h i g h f r e q u e n c i e s o f damage t o chromosomes i n XP c e l l s , and i n c r e a s e d s e n s i t i v i t y o f XP c e l l s t o l e t h a l e f f e c t s has been o b s e r v e d f o l l o w i n g t r e a t m e n t s w i t h many s y n t h e t i c a l l y p r e p a r e d m e t a b o l i t e s o f p r e c a r c i n o g e n s ; f o r e x a m p l e , 183 N-acetoxy-AAF (St ich et aj_., 1972) and metabol i tes of o ther aromatic amines (St ich et a_l_., 1973) as well as epoxides of BA and methy l -cholanthrene (S t ich and San, 1973). The in v i t r o metabol ic a c t i -vat ion of precarc inogens, during exposure of human c e l l c u l t u r e s , has now permitted study of the r e l a t i o n s h i p s between these b i o -log i ca l responses with precarcinogens tha t do not have s t ab le carc inogen ic metabo l i tes . The most a t t r a c t i v e general hypothesis put fo r th to connect these b i o l o g i c a l responses po ints out the p o s s i b i l i t y that chemical carc inogen- induced DNA les ions in XP c e l l s , f o r example, may not be repa i red soon enough such tha t repa i r Is  complete p r i o r to the onset of DNA r e p l i c a t i o n as soc ia ted with chromosome dup l i c a t i on and c e l l d i v i s i o n . In o ther words, the r e su l t of " incomplete" DNA r e p a i r is the express ion of damage at the chromosome level a r i s i n g from DNA synthes i s using DNA molecules with s trand breaks or a l t e r e d components (e.g . bound alky I groups) as a template (St ich and San, 1973; Strauss et a l . , 1972; S t i ch and La i shes, 1973). Entry in to c e l l d i v i s i o n with unrepa i red , damaged DNA molecules may a l so lead to c e l l death due to m u l t i p l e In ter -rupt ions in the newly synthes ized DNA s t rands . Such a phenomenon has been observed in bac te r i a (reviewed by W i t k i n , 1969) but hard evidence to support t h i s idea is s t i l l lacking f o r mammalian c e l l systems. However, an integra l involvement of l a rger chromosome aberrat ions and the observed loss of c lon ing capac i ty cannot be ru led out (reviewed by S t i ch and La i shes ) . The low leve l s of DNA r e p a i r synthes i s observed in XP c e l l s impl ies that there may be a connection between DNA r e p a i r and neo-p l a s t i c transformat ion - a l l pos s ib ly being r e f l e c t i o n s of e a r l y DNA a l t e r a t i o n s c o n t r o l l e d by the metabol ic form of the chemical carc inogen (Figure 59). 184 Ce l I Death Chromosome A b e r r a t i o n s N e o p I a s t i c T r a n s f o r m -a t i o n F i g u r e 59. The p o s s i b l e l i n k between e a r l y DNA a l t e r a t i o n s , DNA r e p a i r , and n e o p l a s t i c t r a n s f o r m a t i o n . Low l e v e l s o f DNA r e p a i r s y n t h e s i s were o b s e r v e d i n XP c e l l s , compared w i t h normal c e l l s , expo sed t o a f l a t o x i n B l i n c o m b i n a t i o n w i t h a c t i v a t i o n s y s tems c o n t a i n i n g d i f f e r e n t s u b c e l l u l a r f r a c t i o n s (9S , I05P, I05S f r a c t i o n s ) f r o m mouse, r a t , and duck l i v e r s . I t i s i n t e r e s t i n g t h a t a l l o f t h e s u b c e l l u l a r f r a c t i o n s t e s t e d , w i t h t h e e x c e p t i o n o f t h e I05S f r a c t i o n o f r a t l i v e r , were a b l e t o evoke d e t e c t a b l e l e v e l s o f DNA r e p a i r s y n t h e s i s . W i t h t h e a s s u m p t i o n t h a t d i f f e r e n t s u b c e l l u l a r f r a c t i o n s r e p r e s e n t t h e a c t u a l s u b c e l l u l a r d i s t r i b u t i o n s o f a c t i v a t i o n enzymes , i t has been p r o p o s e d t h a t d i f f e r e n t m e t a b o l i c r o u t e s f o r p r e c a r c i n o g e n s a r e l o c a t e d i n d i f f e r e n t s u b c e l l u l a r l o c a l e s ( e . g . s o l u b l e c y t o s o l enzymes , v e r s u s membrane-bound m i c r o s o m a l enzymes) ( e . g . P a t t e r s o n and R o b e r t s , 1972 ) . I f t h i s i s t h e c a s e f o r i n t r a c e l l u l a r a f l a t o x i n B l m e t a b o l i s m , t h e n i t a p p e a r s t h e m e t a b o l i t e s o f a f l a t o x i n B l , c a p a b l e o f t r i g g e r i n g DNA r e p a i r s y n t h e s i s , a r e p o s s i b l y b e i n g p r o d u c e d by d i f f e r e n t s u b c e l l u l a r f r a c t i o n s o f mouse, r a t , and duck l i v e r s . T h i s s u g g e s t s t h a t e i t h e r one s p e c i f i c m e t a b o l i t e , c a p a b l e o f i n d u c i n g DNA l e s i o n s t h a t r e s u l t i n DNA r e p a i r s y n t h e s i s , i s b e i n g p r o d u c e d by d i f f e r e n t s u b c e l l u l a r f r a c t i o n s , o r d i f f e r e n t DNA-damaging m e t a b o l i t e s a r e b e i n g p r o d u c e d by t h e d i f f e r e n t s u b c e l l u l a r f r a c t i o n s . When t h e low l e v e l s o f DNA M e t a b o I i c A c t i v a t i o n o f • P r e c a r c i nogens B i nd i ng " t o DNA ' JDNA ^ Damage' J tepa i r o f "DNA Damage" r e p a i r s y n t h e s i s o b s e r v e d i n XP c e l l s , compared w i t h normal c e l l s , a r e a l s o c o n s i d e r e d i n t h e above r e a s o n i n g , i t s u g g e s t s t h a t a f l a t o x i n B l - i n d u c e d DNA a l t e r a t i o n s may p l a y a b road r o l e i n a f l a t o x i n Bl c a r c i n o g e n e s i s i n d i f f e r e n t a n i m a l s , f o l l o w i n g m e t a b o l i c c o n v e r s i o n o f t h e p a r e n t compound t o one o r more a c t i v e m e t a b o l i t e s by a v a r i e t y o f p o s s i b l e s u b c e l l u l a r r o u t e s . I t i s i n t e r e s t i n g t h a t t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n c u l t u r e d human c e l l s f o l l o w i n g e x p o s u r e t o a c t i v a t i o n s y s tems c o n t a i n i n g h i g h l y o n c o g e n i c a f l a t o x i n Bl , o r m o d e r a t e l y o n c o g e n i c a f l a t o x i n G l , o r t h e w e a k l y o n c o g e n i c a f l a t o x i n s B2 and G2 , p a r a l l e l l e d t h e l e v e l s o f b i n d i n g t o DNA o b s e r v e d i n v i t r o ( G u r t o o e t a j _ . , 1974 ) . A l s o r e l a t e d t o t h e l e v e l s o f DNA r e p a i r s y n t h e s i s , were t h e l e v e l s o f o n c o g e n i c i t y o f t h e compounds i n e x p e r i m e n t a l s y s tems ( r e v i e w e d by Wogan, 1973) . A f l a t o x i n s B l and G l c o n t a i n t h e 2 , 3 - d o u b l e b o n d , whereas a f l a t o x i n s B2 and G2 do n o t . The h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s , o b s e r v e d w i t h a c t i v a t e d a f l a t o x i n B l and G l , a r e t h e r e f o r e c o n s i s t e n t w i t h t h e p r o p o s a l s t h a t t h e u l t i m a t e c a r c i n o g e n i c m e t a b o l i t e o f a f l a t o x i n Bl may be t h e 2 , 3 - e p o x i d e ( S c h o e n f a l , 1970; G a r n e r a t a ]_ . , 1972 ) . M o r e o v e r , s t e r i g m a t o c y s t i n i s s t r u c t u r a l l y r e l a t e d t o t h e a f l a t o x i n s and a l s o c o n t a i n s t h e i s o l a t e d v i n y l e t h e r ( 2 , 3 - ) d o u b l e bond . I t i s i n t e r e s t i n g t h a t , on t h e b a s i s o f t h i s s t r u c t u r a l f e a t u r e , t h e 2 , 3 - e p o x i d e c o u l d a l s o be h y p o t h e s i z e d as t h e u l t i m a t e c a r c i n o g e n i c m e t a b o l i t e o f s t e r i g m a t o c y s t i n . I t was o b s e r v e d t h a t a c t i v a t e d s t e r i g m a t o c y s t i n i nduced h i g h l e v e l s ' o f DNA r e p a i r s y n t h e s i s i n c u I t u r e d e e l I s . F u r t h e r e v i d e n c e f o r t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p was g a i n e d f rom t h e i n v e s t i g a t i o n o f t h e a b i l i t y o f t h e r e d u c e d 186 m e t a b o l i t e o f a f l a t o x i n B l , a f l a t o x i c o l , t o i n d u c e DNA r e p a i r s y n -t h e s i s i n human c e l l s . T h i s compound, l i k e t h e a f l a t o x i n s B l and G l , c o n t a i n s a 2 , 3 - d o u b l e bond and i n d u c e d h i g h l e v e l s o f DNA r e p a i r s y n t h e s i s o n l y when combined w i t h a c t i v a t i o n s y s t ems c o n t a i n i n g t h e 9S f r a c t i o n o f mouse, r a t , o r duck l i v e r s . T h i s o b s e r v a t i o n s u g g e s t s t h a t a l t h o u g h UV and TLC e v i d e n c e i n d i c a t e d t h e f o r m a t i o n o f a f l a t o x i c o l f r om a c o m b i n a t i o n o f a f l a t o x i n Bl w i t h an a c t i v a t i o n s y s t em c o n t a i n i n g duck l i v e r I05S f r a c t i o n ( P a t t e r s o n and R o b e r t s , 1972) , t h e r e i s p r o b a b l y f u r t h e r m e t a b o l i c c o n v e r s i o n o f a f l a t o x i c o l b e f o r e DNA r e p a i r s y n t h e s i s i s t r i g g e r e d . I t i s i n t e r e s t i n g t h a t a f l a t o x i c o l , o n l y when a c t i v a t e d , e x h i b i t e d t o x i c i t y f o r S a l m o n e l l a t y p h i murium a t l e v e l s a p p r o a c h i n g t h o s e e x h i b i t e d by a f l a t o x i n s B l , G l , and s t e r i g m a t o c y s t i n ( G a r n e r e t a I., 1971, 1972) . The s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s between t h e  p r e s e n c e o f t h e 2 , 3 - d o u b l e bond and t h e i n d u c t i o n o f h i g h l e v e l s  o f DNA r e p a i r s y n t h e s i s a r e summar ized i n F i g u r e 60 . I t has been r e p e a t e d l y s u g g e s t e d t h a t an o r g a n o t r o p i c e f f e c t o f a p r e c a r c i n o g e n may be a t t r i b u t e d t o i t s o r g a n - s p e c i f i c a c t i v a t i o n . C o n s i d e r i n g t h e p r e d o m i n a n t h e p a t o t o x i c and h e p a t o -c a r c i n o g e n i c e f f e c t o f a f l a t o x i n Bl and s t e r i g m a t o c y s t i n , an e x c e p t i o n a l l y h i g h a c t i v a t i o n p o t e n t i a l o f l i v e r f r a c t i o n s , compared t o k i d n e y and lung f r a c t i o n s , s h o u l d be e x p e c t e d . The r e s u l t s , howeve r , r e v e a l e d no g r e a t d i f f e r e n c e s i n t h e a c t i v a t i o n c a p a c i t i e s o f S9 f r a c t i o n s d e r i v e d f r om l i v e r , k i d n e y , o r l u n g . In a d d i t i o n , t h e r e were o n l y s m a l l v a r i a t i o n s between t h e a c t i v a t i o n c a p a c i t i e s o f t h e S9 l i v e r f r a c t i o n s o f d i f f e r e n t s p e c i e s ( S y r i a n h a m s t e r , S w i s s mouse, New Z e a l a n d W h i t e r a b b i t , M u s c o v i t e d u c k , Ra inbow t r o u t , S t a r r y f l o u n d e r ) a l t h o u g h t h e i r s e n s i t i v i t y t o w a r d s t h e 187 d) ct) Aflatoxin Bl Aflatoxin Gl Aflatoxicol Sterigmatocystin IGH LEVELS OF DNA REPAIR SYNTHESI  LOW EPOXIDE CI] d] Aflatoxin B2 Aflatoxin G2 F i g u r e 60 . A p r o p o s e d s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p between t h e p r e s e n c e o f an i s o l a t e d v i n y l e t h e r d o u b l e bond ( p o t e n t i a l e p o x i d e f o r m a t i o n ) and t h e l e v e l s o f DNA r e p a i r s y n t h e s i s i n d u c e d i n c u l t u r e d human f i b r o b l a s t s by a f l a t o x i n s , a f l a t o x i c o l , and s t e r i g -m a t o c y s t i n , w i t h c o n c o m i t a n t a c t i v a t i o n by a N A D P H - g e n e r a t i n g s y s t em c o n t a i n i n g t h e 9S f r a c t i o n o f mouse l i v e r . 188 c a r c i n o g e n i c e f f e c t o f a f l a t o x i n B l seems t o d i f f e r (Wogan, 1973 ) . W i t h t h e a s s u m p t i o n t h a t t h e i s o l a t e d t i s s u e f r a c t i o n s employed i n t h i s s t u d y r e f l e c t t h e a c t i v a t i o n b e h a v i o u r o f t h e l i v i n g c e l l s i n t h e i n t a c t a n i m a l , t h e r e s u l t s can be i n t e r p r e t e d as an i n d i -c a t i o n t h a t s e v e r a l t i s s u e s have t h e c a p a c i t y t o m e t a b o I i c a I Iy  a c t i v a t e a f l a t o x i n B l . V a r i o u s l i n e s o f e v i d e n c e l e nd s u p p o r t t o t h i s s p e c u l a t i o n . F o r e x a m p l e , s i m i l a r l e v e l s o f t r i t i a t e d a f l a t o x i n Bl were f ound bound t o DNA i n v a r i o u s o r g a n s o f r a t s up t o 8 weeks f o l l o w i n g a s i n g l e i n j e c t i o n o f t h e compound ( L i j i n s k y erf a j _ . , 1970) . No c o n n e c t i o n appea red t o e x i s t between t h e i n t e r a c t i o n o f t h e a f l a t o x i n s and t h e i r m e t a b o l i t e s w i t h DNA, and o r g a n - s p e c i f i c c a r c i n o g e n e s i s . F u r t h e r m o r e , t h e i n d u c t i o n o f m a l i g n a n t sa rcomas and f i b r o s a r c o m a s was a c h i e v e d i n r a t s f o l l o w i n g s u b c u t a n e o u s i n j e c t i o n o f a f l a t o x i n B l and s t e r i g m a t o c y s t i n . ( D i c k e n s a t a j _ . , 1966) . A l s o , s k i n tumor s were i n d u c e d i n m i c e by p a i n t i n g w i t h a f l a t o x i n Bl f o l l o w e d by p r o m o t e r s ( L i n d e n f e l s e r a f a_L , 1974 ) , and h i g h l e v e l s o f k i d n e y t umo r s have been o b s e r v e d i n W i s t a r r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f a f l a t o x i n B l ( E p s t e i n a t a j _ . , 1969 ) . F i n a l l y , e ven c u l t u r e d human c e l l s e x h i b i t a l ow, b u t d e t e c t a b l e l e v e l o f DNA r e p a i r s y n t h e s i s and chromosome a b e r r a t i o n s when exposed t o n o n - a c t i v a t e d a f l a t o x i n B l o r s t e r i g -m a t o c y s t i n ( e . g . T a b l e s 7 , 9 , 1 0 ) . W i t h t h e a s s u m p t i o n t h a t t h e l e v e l s o f DNA r e p a i r s y n t h e s i s o b s e r v e d i n v i t r o , w i t h a f l a t o x i n Bl a c t i v a t i o n by f r a c t i o n s f r o m d i f f e r e n t t i s s u e s , a r e a r e f l e c t i o n o f i n v i v o e v e n t s , t h e p o s s i b i . l i t y a r i s e s t h a t such DNA a l t e r a t i o n s a r e i n d u c e d by a f l a t o x i n Bl i n v a r i o u s t i s s u e s i n v i v o . Hence , i t i s r e a s o n a b l e t o s p e c u l a t e t h a t a f l a t o x i n B l - i n d u c e d tumor s t h a t d e v e l o p i n any one p a r t i c u l a r t i s s u e and no t i n a n o t h e r , may a r i s e as a 189 r e s u l t o f some e x p r e s s i o n o f t h e i n i t i a l i n d u c t i o n o f DNA a l t e r a t i o n s  and DNA r e p a i r s y n t h e s i s by r e a c t i v e c a r c i n o g e n i c m e t a b o l i t e s . One such p a r a m e t e r g o v e r n i n g t h e t i s s u e - s p e c i f i c i t y o f a f l a t o x i n B l -i n d u c e d tumor f o r m a t i o n may be t h e i n a b i l i t y o f one o r more i n d i v i d u a l c e l l s o f t h e s u s c e p t i b l e t i s s u e t o r e p a i r DNA a l t e r a t i o n s i n an e r r o r - f r e e manner ( e . g . L a w l e y , 1968; r e v i e w e d by F a r b e r e t a I. , 1973; S t i c h and L a i s h e s , 1973) . 190 SECTION 3 DNA DAMAGE' AND REPAIR IN MAMMALIAN CELLS IN VIVO  RESULTS One of t h e most i n t r i g u i n g q u e s t i o n s i n c h e m i c a l c a r c i n o -g e n e s i s c o n c e r n s t h e r o l e o f DNA damage and r e p a i r i n n e o p l a s t i c t r a n s f o r m a t i o n . One o f t h e d i f f i c u l t i e s t h a t i s i n h e r e n t i n s t u d i e s o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage and r e p a i r c o n -d u c t e d w i t h mammalian c e l l c u l t u r e s , c o n c e r n s t h e a c q u i s i t i o n o f m e t a b o l i t e s o f p r e c a r c i n o g e n s t h a t a r e i d e n t i c a l t o t h o s e g e n e r a t e d i n v i v o . In t h i s r e s p e c t , s t u d i e s o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage and r e p a i r c o n d u c t e d w i t h t h e i n t a c t a n i m a l o f f e r a d i s t i n c t advan tage o v e r c e l l c u l t u r e s y s t e m s . On t h e o t h e r h a n d , s t u d i e s o f DNA damage and r e p a i r c o n d u c t e d w i t h t h e i n t a c t a n i m a l a r e more t e c h n i c a l l y d i f f i c u l t t o e x e c u t e . A combined i n v i v o / i n v i t r o t e c h n i q u e has been r e c e n t l y d e v e l o p e d t o d e m o n s t r a t e a c o r r e l a t i o n between t h e s i t e o f t umor i n d u c t i o n and t h e s i t e o f DNA r e p a i r s y n t h e s i s f o l l o w i n g s i n g l e -doses o f e i t h e r o f t h e p r e c a r c i n o g e n s 4NQ0 o r DMN ( S t i c h and K i e s e r , 1974 ) . These s t u d i e s i m p l i e d t h a t o r g a n - s p e c i f i c u n s c h e d u l e d i n c o r p o r a t i o n o f 3 H - T d R , f o l l o w i n g s i n g l e - d o s e a d m i n i s t r a t i o n o f 4NQ0 o r DMN, may be a r e f l e c t i o n of e a r l y DNA a l t e r a t i o n s i n d u c e d by t h e m e t a b o l i t e s o f t h e s e p r e c a r c i n o g e n s . I t was , t h e r e f o r e , d e s i r a b l e t o employ a t e c h n i q u e t h a t wou ld p e r m i t an e s t i m a t i o n o f t h e e x t e n t o f e a r l y DNA damage i n v i v o and t h e p a t t e r n o f r e p a i r o f t h e DNA damage i n v i v o . M o r e b v e r , a d a p t a t i o n o f such a t e c h n i q u e t o v a r i o u s o r g a n s , s t u d i e d s i m u l t a n e o u s l y , wou ld p e r m i t a c o m p a r i s o n t o be made 191 between t h e s i t e o f DNA damage, t h e p a t t e r n s o f DNA r e p a i r , and t h e s i t e o f tumor i n d u c t i o n . In o r d e r t o pu r sue c o m p a r a t i v e s t u d i e s o f c h e m i c a l c a r c i n o g e n - i n d u c e d DNA damage and r e p a i r i n v i v o , i t was d e c i d e d t o adap t a r e c e n t m o d i f i c a t i o n o f t h e a l k a l i n e s u c r o s e g r a d i e n t t e c h n i q u e (Cox e t a_L , 1973) f o r a s i m u l t a n e o u s e s t i m a -t i o n o f DNA damage i n v a r i o u s mouse o r g an s f o l l o w i n g s i n g l e - d o s e a d m i n i s t r a t i o n o f 4NQ0 o r DMN i n v i v o . (a) T e c h n i c a l Deve lopment The key t o o b t a i n i n g r a p i d l y s e d i m e n t i n g DNA f r om l i v e r , under t h e c o n d i t i o n s o f a l k a l i n e s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o u t l i n e d by Cox and c o - w o r k e r s (Cox e t a I., 1973) was t o p r o c u r e c e l l s u s p e n s i o n s by g e n t l y s q u a s h i n g t h e o r g a n , i n b u f f e r , w i t h t h e b l u n t end o f a s p a t u l a . These i n v e s t i g a t o r s o b s e r v e d t h a t DNA f r om l i v e r homogenate, p r e p a r e d w i t h a P o t t e r - E I v e h j e m homogen i zen , banded n e a r t h e t o p o f t h e g r a d i e n t s i n d i c a t i n g t h a t t h e DNA had been f r a g m e n t e d by t h e h o m o g e n i z a t i o n p r o c e d u r e . T h e r e f o r e , an a t t e m p t was made t o p r e p a r e e e l I s u s p e n s i o n s f r om v a r i o u s mouse o r g a n s - some o f w h i c h had t e x t u r e s t h a t were no t amenab le t o g e n t l e s p a t u l a - s q u a s h i n g - u s i n g a m a n u a l , l a r g e - c l e a r a n c e Dounce t i s s u e - g r i n d e r . When g e n t l e hand o p e r a t i o n i s e m p l o y e d , t h e Dounce h o m o g e n i z a t i o n t e c h n i q u e may be c l a s s e d , w i t h r e g a r d t o t h e i n t e n s i t y o f t i s s u e - d i s r u p t i o n , somewhere between t h e s p a t u l a -squash and P o t t e r - E I v e h j e m t e c h n i q u e s . M i c r o s c o p i c e x a m i n a t i o n o f t h e homogenate s , w i t h and w i t h o u t s t a i n i n g , i n d i c a t e d t h a t s u s p e n s i o n s o f s i n g l e c e l l s 2 c o u l d be d e r i v e d 2 . As d e s c r i b e d by Cox e t aj_. ( 1 9 7 3 ) , t h e t e r m s " s u s p e n s i o n s o f f r om homogenates p r e p a r e d w i t h a h a n d - o p e r a t e d Dounce t i s s u e - g r i n d -e r ( T a b l e 12 ) . C o m p a r a t i v e l y h i g h c o n c e n t r a t i o n s o f s i n g l e c e l l s were o b s e r v e d i n p r e p a r a t i o n s o b t a i n e d f r om l i v e r , k i d n e y , l u n g , and s p l e e n , whereas low c o n c e n t r a t i o n s o f s i n g l e c e l l s ( t h e m a j o r i t y o f c e l l s pe l l e t t e d as a g g r e g a t e s ) were o b s e r v e d i n p r e -p a r a t i o n s f r om t h ymus , u r i n a r y b l a d d e r , t e s t e s ( m o s t l y s p e r m a t o -z o a ) , and b r a i n . H o m o g e n i z a t i o n o f s u b m a x i l l a r y g l a n d , c a r d i a c o r p y l o r i c s t o m a c h , and s m a l l o r l a r g e i n t e s t i n e , p r o d u c e d m o s t l y e e l I a g g r e g a t e s . A t t e n t i o n was f o c u s e d , howeve r , on t h e t a s k o f o b t a i n i n g , r e p r o d u c i b l y , i n t a c t DNA s i m u l t a n e o u s l y f r om homogenates o f l u n g , k i d n e y , and l i v e r f r om t h e same mouse i n o r d e r t h a t DNA damage i n d u c e d by c h e m i c a l c a r c i n o g e n s i n v i v o cou Id be compared i n t h e s e o r g a n s . The c r i t e r i o n f o r d e s i g n a t i n g c o n t r o l DNA as " i n t a c t " was e s t a b l i s h e d as t h e a b i l i t y o f t h e DNA t o s e d i m e n t t o a c o n t r o l r e g i o n ( e . g . F i g u r e 61) o f a l k a l i n e s u c r o s e g r a d i e n t s unde r t h e c o n d i t i o n s d e s c r i b e d by Cox e t aj_. ( 1 9 7 3 ) . In o r d e r t o d e t e c t m i c r o g r a m q u a n t i t i e s o f DNA r e l e a s e d f rom mouse l u n g , k i d n e y , and l i v e r , l i t t e r s o f i n f a n t S w i s s m i c e were a d m i n i s t e r e d 3 H-TdR on an i n j e c t i o n s c h e d u l e encompa s s i n g t h e f i r s t 14 days o f l i f e . The m i c e were t h e n u sed f o r e x p e r i -m e n t a t i o n when t h e y were 4 t o 6 months o f a ge . To a s s e s s t h e f e a s i b i l i t y o f e m p l o y i n g t h e Dounce t i s s u e -g r i n d e r t o o b t a i n c e l l s u s p e n s i o n s f o r a l k a l i n e s u c r o s e g r a d i e n t a n a l y s e s , an e x p e r i m e n t was d e s i g n e d whereby t h e l u n g s , k i d n e y s , s i n g l e c e l l s " and " s i n g l e c e l l s u s p e n s i o n s " r e f e r t o p r e p a r a t i o n s c o n t a i n i n g bo th i n t a c t c e l l s and i n t a c t n u c l e i w i t h l i t t l e a t t a c h e d c y t o p l a s m . 193 T a b l e 12. G r o s s m i c r o s c o p i c a p p e a r a n c e o f homogenates o f mouse o r g a n s p r e p a r e d w i t h a Dounce t i s s u e - g r i n d e r . ORGAN 3 APPEARANCE OF HOMOGENATE BEFORE CENTRIFUGATION b APPEARANCE OF SUPERNATANT L i v e r c d s i n g l e c e l l s , some a g g r e g a t e s s i n g l e e e l Is Ki dney s i n g l e c e l l s , some a g g r e g a t e s s i n g l e e e l 1s Lung s i n g l e c e l l s , some a g g r e g a t e s s i n g l e e e l 1s S p l e e n s i n g l e c e l l s , some a g g r e g a t e s s i ng1e ce1 1 s Thymus s i n g l e c e l l s , some a g g r e g a t e s few s i ng1e e e l 1 s U r i n a r y B l a d d e r s i n g l e c e l l s , some a g g r e g a t e s few s i n g 1 e e e l 1s T e s t e s few s i n g l e c e l l s , m o s t l y sperm s p e r m a t o z o a B r a i n few c e l l s o r a g g r e g a t e s few s i n g l e e e l Is Submax i11a ry G l a n d a g g r e g a t e s few s i n g l e e e l 1s C a r d i a c Stomach a g g r e g a t e s few s i n g l e e e l 1s P y l o r i c Stomach a g g r e g a t e s few s i ng1e ce1 1 s Sma11 1 n t e s t i ne a g g r e g a t e s few s i n g l e e e l 1s La rge I n t e s t i n e a g g r e g a t e s few s i n g l e e e l 1s (a ) A w e t - w e i g h t o f a b o u t 50 -100 mg o f each o r g a n was homogen i zed i n 0.5-1 ml o f E D T A / s a l i n e b u f f e r . (b ) F o l l o w i n g h o m o g e n i z a t i o n w i t h 6 f u l l t u r n s o f t h e p e s t l e o f a Dounce t i s s u e - g r i n d e r , t h e homogenate was c e n t r i f u g e d a t 1000 x g f o r 30 s . An a l i q u o t o f homogenate was o b s e r v e d i n a h e m a c y t o -mete r chamber and a smear p r e p a r a t i o n was f i x e d w i t h a c e t i c a c i d -e t h a n o l ( 1 : 3 ) and s t a i n e d w i t h h e m a t o x y I i n - e o s i n . ( c ) The t e r m " s i n g l e c e l l s " , as d e f i n e d by Cox et_ aj_. ( 1 9 7 3 ) , i n -c l u d e s i n t a c t n u c l e i w i t h l i t t l e a d h e r i n g ' c y t o p I asm. The o c c a s i o n a l a p p e a r a n c e o f a g g r e g a t e s o f 2 -4 c e l l s i s d i s r e g a r d e d . (d) The t e r m " a g g r e g a t e s " r e f e r s t o c l u m p s o f 5 o r more c e l l s . 194 and l i v e r s f r o m two young a d u l t ma l e m i c e , p r e l a b e l l e d w i t h 3 H - T d R 3 were homogen ized i n E D T A / s a l i n e b u f f e r w i t h a Dounce t i s s u e - g r i n d e r . The o r g a n s f r o m one mouse were homogen ized w i t h 6 f u l l t u r n s ^ o f t h e homogen i ze r p e s t l e and t h e o r g a n s f rom t h e o t h e r mouse were homo-g e n i z e d w i t h 12 f u l l t u r n s o f t h e homogen i ze r p e s t l e . F o l l o w i n g t h e removal o f c e l l a g g r e g a t e s , a l i q u o t s o f t h e c e l l s u s p e n s i o n s ( c o n t a i n i n g a b o u t I x I O 5 c e l l s ) were l a y e r e d on t o p o f a l k a l i n e s u c r o s e g r a d i e n t s w h i c h were t h e n c e n t r i f u g e d ( F i g u r e 6 1 ) . The r e s u l t i n g s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA e x h i b i t e d peaks o f DNA r a d i o a c t i v i t y i n t h e c o n t r o l r e g i o n 5 when c e l l s were d e r i v e d f r om a l l t h r e e o r g a n s - l u n g , k i d n e y , and l i v e r - by homo-g e n i z a t i o n w i t h 6 f u l l t u r n s o f t h e Dounce h o m o g e n i z e r p e s t l e . However , more 3 H-DNA a p p e a r e d above t h e c o n t r o l r e g i o n i n t h e l i v e r DNA p r o f i l e t h a n i n t h e k i d n e y o r lung p r o f i l e s . T h i s i n d i c a t e s t h a t t h e r e was more f r a g m e n t e d DNA i n d u c e d i n t h e l i v e r t h a n i n e i t h e r t h e k i d n e y o r lung by 6 f u l l t u r n s o f t h e Dounce h o m o g e n i z e r . F o l l o w i n g h o m o g e n i z a t i o n w i t h 12 f u l l t u r n s o f t h e Dounce homo-g e n i z e r p e s t l e , c e l l s d e r i v e d f r om a l l t h r e e o r g a n s r e l e a s e d 3 H-DNA t h a t r e s u l t e d i n peak s o f DNA r a d i o a c t i v i t y l o c a t e d above t h e c o n t r o l r e g i o n . . These r e s u l t s i n d i c a t e t h a t t h e DNA o f a l l t h r e e 3 . A l l e x p e r i m e n t s i n t h i s s e r i e s o f i n v i v o e x p e r i m e n t s were c o n -d u c t e d w i t h m i c e p r e l a b e l l e d w i t h ^JT-TdR. 4 . The t e r m " f u l l t u r n " r e f e r s t o one 3 6 0 ° r o t a t i o n o f t h e Dounce h o m o g e n i z e r p e s t l e . 5 . F r a c t i o n s I t o 4 , i n c l u s i v e , a r e d e s i g n a t e d t h e " c o n t r o l r e g i o n " o f t h e s e g r a d i e n t s ( i n d i c a t e d by a h o r i z o n t a l b a r w i t h e v e r y p r o -f i l e ) becau se t h e 3 H-DNA peak was most o f t e n l o c a t e d i n f r a c t i o n s 2 and 3 when d e r i v e d f r om u n t r e a t e d ( c o n t r o l ) m i c e . O c c a s i o n a l l y ( abou t I o u t o f 20 r u n s ) t h i s peak a p p e a r e d i n f r a c t i o n s I o r 4 . The 2 .3 M s u c r o s e c u s h i o n i s c o n t a i n e d i n f r a c t i o n s I and 2. 195 F i g u r e 61. A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r om e e l I s u s p e n s i o n s d e r i v e d f r om u n t r e a t e d mouse t i s s u e s f o l l o w i n g h o m o g e n i z a t i o n w i t h a Dounce t i s s u e -g r i n d e r o p e r a t e d w i t h 6 f u l l t u r n s o f t h e p e s t l e (9 — •) and 12 f u l l t u r n s o f t h e p e s t l e ( o - - o ) . (A) Lung (B) K i d n e y (C) L i v e r The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . 196 L I V E R o r g a n s s t u d i e d - l u n g , k i d n e y , and l i v e r - was f r a g m e n t e d by homo-g e n i z a t i o n w i t h 12 f u l l t u r n s o f t h e p e s t l e . M i c r o s c o p i c e x a m i n a -t i o n o f t h e homogenates r e v e a l e d c h a r a c t e r i s t i c s s i m i l a r t o t h o s e d e s c r i b e d i n T a b l e 12 f o r l u n g , k i d n e y , and l i v e r . A l t h o u g h t h e s e d i m e n t a t i o n b e h a v i o u r o f t h e DNA was d i f f e r e n t , depend i n g on whe the r h o m o g e n i z a t i o n was c o n d u c t e d w i t h 6 o r 12 f u l l t u r n s o f t h e p e s t l e , no d i f f e r e n c e was o b s e r v e d between t h e a p p e a r a n c e o f homogenates p r e p a r e d by 6 o r 12 f u l l t u r n s o f t h e Dounce homogen i z e r p e s t l e . The se r e s u l t s d e m o n s t r a t e t h a t h o m o g e n i z a t i o n o f mouse l u n g , k i d n e y , o r l i v e r by 12 f u l l t u r n s o f t h e Dounce homogen i z e r p e s t l e , compared w i t h h o m o g e n i z a t i o n by 6 f u l l t u r n s o f t h e p e s t l e , i n c r e a s e d t h e amount o f f r a g m e n t e d 3 H-DNA d e r i v e d f r o m a l l t h r e e o r g a n s , a s e s t i m a t e d by c e n t r i f u g a t i o n t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s . The Dounce h o m o g e n i z a t i o n method was a p p l i e d t o a n a l y z e t h e e f f e c t o f 4NQ0 on t h e DNA o f mouse l u n g , k i d n e y , and l i v e r i n  v i v o . The l u n g s , k i d n e y s , and l i v e r s f r om two m i c e , k i l l e d 4 h a f t e r t h e a d m i n i s t r a t i o n o f 80 mg 4NQ0/kg body w e i g h t t o one mouse and s o l v e n t v e h i c l e (DMSO) a l o n e t o t h e o t h e r ( c o n t r o l ) mouse, were homogen ized s e p a r a t e l y w i t h o n l y 6 f u l l t u r n s o f t h e homogen i z e r p e s t l e . The c o n s t i t u e n t 3H-DNA was s e d i m e n t e d t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g u r e 6 2 ) . The s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f rom lung and k i d n e y c e l l s , d e r i v e d f rom t h e u n t r e a t e d ( c o n t r o l ) mouse c o n -t a i n e d p r o m i n e n t peaks o f 3H-DNA i n t h e c o n t r o l r e g i o n s . L i v e r p r e p a r a t i o n s p r o d u c e d e r r a t i c p r o f i l e s . The p r o f i l e o f c o n t r o l 198 F i g u r e 62 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f rom c e l l s u s p e n s i o n s d e r i v e d f r om t i s s u e s o f an u n t r e a t e d ( c o n t r o l ) mouse ( • — • ) and a 4 N Q 0 - t r e a t e d mouse ( O - - O ) . 4NQ0 i n 0.1 ml DM SO was i n j e c t e d s u b c u t a n e o u s I y a t 80 mg/kg body w e i g h t . The c o n t r o l a n i m a l r e c e i v e d 0.1 ml DMSO a l o n e . The m i c e were k i l l e d 4 h a f t e r t h e a d m i n i s t r a t i o n o f 4NQ0 i n DMSO o r DMSO a l o n e . C e l l s u s p e n s i o n s were o b t a i n e d f rom t i s s u e s homogen ized i n a Dounce t i s s u e - g r i n d e r w i t h o n l y 6 f u l l t u r n s o f t h e p e s t l e . (A) Lung (B) K i dney (C) L i v e r The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . L U N G 200 l i v e r 3H-DNA c o n s i s t e d o f a peak o f p e l l e t t e d 3 H-DNA, amoun t i ng t o abou t 15% o f t h e t o t a l 3 H - c o u n t s , w i t h most o f t h e r e m a i n i n g 3H-DNA a p p e a r i n g above t h e c o n t r o l r e g i o n t o w a r d s t h e t o p o f t h e g r a d i e n t . The l o c a t i o n s o f t h e bands o f 3H-DNA r e l e a s e d f rom e i t h e r lung o r k i d n e y p r e p a r a t i o n s d e r i v e d f rom a 4 N Q 0 - t r e a t e d mouse, were s h i f t e d t o w a r d s t h e t o p o f t h e g r a d i e n t s , above t h e c o n t r o l p r o f i l e s . T h i s s h i f t o f 3H-DNA was g r e a t e r f o r lung t h a n f o r k i d n e y p r e p a r a t i o n s . A c o m p a r i s o n o f t h e . s e d i m e n t a t i o n p r o f i l e o f 3H-DNA r e l e a s e d f rom t h e l i v e r p r e p a r a t i o n d e r i v e d f rom t h e 4 N Q 0 - t r e a t e d mouse, w i t h t h e 3H-DNA p r o f i l e r e s u l t i n g f rom t h e c o n t r o l l i v e r p r e p a r a t i o n , r e v e a l e d a r e d u c t i o n i n t h e amount o f p e l l e t t e d DNA r a d i o a c t i v i t y ( f r om 15% t o 2%) and a s l i g h t s h i f t o f t h e r e m a i n i n g DNA r a d i o -a c t i v i t y t o w a r d s t h e bo t tom o f t h e g r a d i e n t , be low t h e c o n t r o l p r o f i I e . The r e s u l t s i n d i c a t e d t h a t f r a g m e n t a t i o n o f 3 H-DNA i n v i v o was i n d u c e d by t r e a t m e n t o f a mouse w i t h 4NQ0. The 4NQ0 t r e a t m e n t i n d u c e d more f r a g m e n t a t i o n i n 3 H-DNA d e r i v e d f rom lung t h a n i n 3H-DNA d e r i v e d f r om k i d n e y . The l o c a t i o n o f t h e c o n t r o l p r o f i l e o f l i v e r 3H-DNA s u g g e s t s t h a t some f r a g m e n t a t i o n o f l i v e r DNA was i n d u c e d by t h e h o m o g e n i z a t i o n p r o c e d u r e . Because o f t h e p o s i t i o n o f t h e c o n t r o l p r o f i l e o f l i v e r DNA r a d i o a c t i v i t y , l i t t l e i n f o r m a t i o n can be g a t h e r -ed f rom a c o m p a r i s o n o f t h e 3H-DNA p r o f i l e s r e l e a s e d f rom t h e l i v e r s o f t r e a t e d and u n t r e a t e d m i c e . I t i s a p p a r e n t , h oweve r , t h a t no s h i f t i n t h e l i v e r 3H-DNA c o n t r o l p r o f i l e , t o w a r d s t h e t o p o f t h e g r a d i e n t , was i n d u c e d by t h e 4NQ0 t r e a t m e n t . Because o f t h e e r r a t i c c o n t r o l p r o f i l e s and a p p a r e n t f r a g m e n t a t i o n o f 3 H - D N A ' r e I e a s ed f r om u n t r e a t e d l i v e r p r e p a r a t i o n s f o l l o w i n g h o m o g e n i z a t i o n w i t h a Dounce t i s s u e - g r i n d e r ( F i g u r e s 61 and 6 2 ) , t h e e x p e r i m e n t a l a p p r o a c h was m o d i f i e d . An e x p e r i m e n t was e x e c u t e d i n w h i c h lung was t h e o n l y mouse o r g a n t h a t was homogen ized w i t h t h e Dounce t i s s u e - g r i n d e r , whereas k i d n e y and l i v e r were p r o c e s s e d w i t h t h e s p a t u l a - s q u a s h t e c h n i q u e . As a s i n g l e , s u b c u t a n e o u s i n j e c t i o n , 4NQ0 was a d m i n i s t e r e d t o two m i c e w h i c h , a l o n g w i t h c o n t r o l s , were k i l l e d a t 4 h and 16 h p o s t - t r e a t m e n t . C e l l s u s p e n s i o n s f r o m l u n g , k i d n e y , and l i v e r were i m m e d i a t e l y p r e p a r e d and t h e c o n s t i t u e n t 3H-DNA was s e d i -mented t h r o u g h a l k a l i n e s u c r o s e g r a d i e n t s ( F i g u r e 6 3 ) . The s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA, r e l e a s e d f r om c e l l s d e r i v e d f rom l u n g , k i d n e y , o r l i v e r o f two u n t r e a t e d ( c o n t r o l ) m i c e , c o n t a i n e d p r o m i n e n t peaks o f 3H-DNA i n t h e c o n t r o l r e g i o n s o f t h e g r a d i e n t s . The s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA, r e l e a s e d f rom lung and k i d n e y p r e p a r a t i o n s o f t h e 4 N Q 0 - t r e a t e d mouse k i l l e d 4 h p o s t - t r e a t m e n t , e x h i b i t e d d e f i n i t i v e s h i f t s t o w a r d s t h e t o p o f t h e g r a d i e n t s , above t h e c o n t r o l DNA p e a k s . The s h i f t was g r e a t e r f o r l ung 3 H-DNA . than f o r k i d n e y 3 H-DNA. The s h i f t was n o t a s d e c i s i v e f o r l i v e r 3 H-DNA r e l e a s e d f r o m t h e 4 N Q 0 - t r e a t e d mouse a t 4 h p o s t - t r e a t m e n t , a l t h o u g h a s l i g h t " t a i l i n g " o f l i v e r 3H-DNA was o b s e r v e d above t h e c o n t r o l DNA a t f r a c t i o n s 5 and 6. A t 16 h f o l l o w i n g 4NQ0 i n j e c t i o n t h e s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA, r e -l e a s e d f r om t h e lung p r e p a r a t i o n , e x h i b i t e d a b imoda l a p p e a r a n c e w i t h one peak of 3H-DNA above t h e c o n t r o l r e g i o n t o w a r d s t h e t o p o f t h e g r a d i e n t . The 3H-DNA r e l e a s e d f r om t h e k i d n e y p r e p a r a t i o n , p r o c e s s e d a t 16 h f o l l o w i n g 4NQ0 t r e a t m e n t , e x h i b i t e d a peak w i t h i n t h e c o n t r o l r e g i o n and a " t a i l i n g " o f 3H-DNA above t h e c o n t r o l 202 F i g u r e 63 . A l k a l i n e s u c r o s e g r a d i e n t s e d i m e n t a t i o n p r o f i l e s o f 3 H-DNA r e l e a s e d f r om t i s s u e s o f u n t r e a t e d ( c o n t r o l ) m ice - ( a — • ) and t i s s u e s f r o m m i c e k i l l e d 4 h (•- —a ) and 16 h ( A — - » A ) f o l -l o w i n g 4NQ0 a d m i n i s t r a t i o n . 4NQ0 i n DMSO was i n j e c t e d s u b c u -t a n e o u s I y a t 80 mg/kg body w e i g h t . C o n t r o l s r e c e i v e d DMSO a l o n e . C e l l s u s p e n s i o n s were p r e p a r e d f r om t h e t i s s u e s by d i f f e r e n t method s : (A) Lung ( p r o c e s s e d by t h e Dounce h o m o g e n i z a t i o n t e c h n i q u e ) (B) K i dney ( p r o c e s s e d by t h e s p a t u l a - s q u a s h t e c h n i q u e ) (C) L i v e r ( p r o c e s s e d by t h e s p a t u l a - s q u a s h t e c h n i q u e ) One s e t o f c o n t r o l p r o f i l e s i s i l l u s t r a t e d . The h o r i z o n t a l b a r i n d i c a t e s t h e p o s i t i o n o f c o n t r o l DNA p e a k s . S E D I M E N T A T I O N 204 p r o f i l e a t f r a c t i o n s 5 and 6. No s h i f t o f DNA r a d i o a c t i v i t y t o -wards t h e t o p o f t h e g r a d i e n t , f r om t h e p o s i t i o n o f c o n t r o l 3 H-DNA, was o b s e r v e d i n t h e p r o f i l e o f 3 H-DNA r e l e a s e d f r om t h e l i v e r p r e p a r a t i o n p r o c e s s e d a t 16 h a f t e r t h e 4NQ0 t r e a t m e n t . The r e s u l t s d e m o n s t r a t e d t h a t f r a g m e n t a t i o n o f 3 H-DNA, ' was i n d u c e d by 4NQ0 t r e a t m e n t i n v i v o . L e s s f r a g m e n t a t i o n o f 3 H-DNA, r e l e a s e d f rom lung o r k i d n e y p r e p a r a t i o n s , was d i s c e r n e d a t 16 h f o l l o w i n g 4NQ0 t r e a t m e n t t h a n a t 4 h p o s t - t r e a t m e n t , w h i c h i n d i c a t e d t h a t a r e p a i r o f t h e 4NQ0 - i nduced f r a g m e n t e d DNA had o c c u r r e d i n mouse lung and k i d n e y i n v i v o . The s m a l l amount o f f r a g m e n t e d l i v e r DNA, d e t e c t e d as a " t a i l i n g " o f t r e a t e d l i v e r 3 H-DNA (4 h) above t h e c o n t r o l DNA peak was not d e t e c t e d i n t h e p r o f i l e o f l i v e r 3 H-DNA o b s e r v e d a t 16 h p o s t - t r e a t m e n t . These r e s u l t s showed t h a t a r e p a i r o f t h e l i v e r DNA damage, d e t e c t e d a t 4 h p o s t - t r e a t m e n t , was c o m p l e t e d by 16 h p o s t - t r e a t m e n t . However , a c o m p a r i s o n o f k i d n e y and lung p r e p a r a t i o n s was s u b j e c t t o c r i t i c i s m s i n c e homogenates o f t h e s e two o r g a n s were no t p r o c e s s e d i n t h e same manner: k i d n e y p r e p a r a t i o n s were d e r i v e d by t h e s p a t u l a - s q u a s h t e c h n i q u e , whereas lung was s u b j e c t e d t o h o m o g e n i z a t i o n w i t h a Dounce t i s s u e - g r i n d e r . The f a c t t h a t f r a g m e n t e d c o n t r o l DNA f r om l i v e r was o b t a i n e d f o l l o w i n g homogen-i z a t i o n w i t h a Dounce t i s s u e - g r i n d e r ( F i g u r e s 61 and 6 2 ) , whereas i n t a c t DNA was o b t a i n e d f o l l o w i n g s p a t u l a - s q u a s h i n g ( F i g u r e 6 3 ) , i n d i c a t e s t h a t t h e Dounce h o m o g e n i z a t i o n t e c h n i q u e was a l t e r i n g t h e s e d i m e n t a t i o n b e h a v i o u r o f t h e DNA - a p p a r e n t l y by m e c h a n i c a l i n d u c t i o n o f DNA f r a g m e n t a t i o n . The q u e s t i o n t h e n a r i s e s as t o what e x t e n t t h e o b s e r v e d f r a g m e n t a t i o n o f lung DNA can be a t t r i b -u t e d t o t h e 4NQ0 t r e a t m e n t . Does 4NQ0 t r e a t m e n t r e n d e r t h e lung 205 t i s s u e more s u s c e p t i b l e t o m e c h a n i c a l d i s r u p t i o n , and t h e r e b y enhance t h e p r o p o r t i o n o f f r a g m e n t e d DNA i nduced by t h e m e c h a n i c a l s h e a r i n g f o r c e s o f t h e Dounce h o m o g e n i z a t i o n p r o c e d u r e ? An a l t e r n a t i v e method o f o b t a i n i n g lung p r e p a r a t i o n s , more c o m p a r a b l e t o t h e g e n t l e s p a t u l a - s q u a s h me thod , was needed i n o r d e r t o a l l o w m e a n i n g f u l c o m p a r i s o n s t o be made between l e v e l s o f DNA damage and DNA r e p a i r i n l u n g , k i d n e y , and l i v e r f o l l o w i n g t h e i n v i v o a p p l i c a t i o n o f c h e m i c a l c a r c i n o g e n s . I n v e s t i g a t i o n s r e v e a l e d t h a t mouse lung t i s s u e f l o a t e d r e a d i l y on t o p o f t h e a l k a l i n e l y s i n g s o l u t i o n t h a t was emp loyed as an o v e r l a y on t h e a l k a l i n e s u c r o s e g r a d i e n t s . W i t h a s h a r p r a z o r b l a d e , f r e s h , u n t r e a t e d mouse lung t i s s u e was p r e p a r e d as f r a g m e n t s - o f many d i f f e r e n t s i z e s and i t was d i s c o v e r e d t h a t t h e t i s s u e f l o a t e d on t h e a l k a l i n e l y s i n g s o l u t i o n r e g a r d l e s s o f t h e s i z e o f t h e l ung t i s s u e p i e c e . Sma l l l ung p i e c e s , l e s s t h a n I mm t h i c k , were o b s e r v e d t o s i n k s l o w l y i n t h e l y s i n g s o l u t i o n a f t e r f l o a t i n g f o r abou t 30 min a t room t e m p e r a t u r e . V e r y s m a l l a i r b u b b l e s a p p e a r e d n e a r t h e s u r f a c e as t h e p i e c e s s e t t l e d . T i s s u e f r a g m e n t s f rom l i v e r and k i d n e y , p r e p a r e d i n t h e same manner and s u b j e c t e d t o s i m i l a r t r i a l s a s t h e lung p i e c e s , were o b s e r v e d t o s i n k i m m e d i a t e l y and r a p i d l y . The o b v i o u s i n t e r p r e t a t i o n o f t h e s e o b s e r v a t i o n s was t h a t lung t i s s u e c o n t a i n e d s u f f i c i e n t a l v e o l a r a i r t o f l o a t t h e t i s s u e f r a g m e n t s on t h e a l k a l i n e l y s i n g s o l u t i o n . A g r a d u a l p e n e t r a t i o n o f t h e a l k a l i n e l y s i n g s o l u t i o n i n t o t h e lung t i s s u e ( a b o u t 30 m i n ) , r e s u l t e d i n t h e l y s i s o f t h e c o n s t i t u e n t c e l l s , t h e l o s s o f some t r a p p e d a l v e o l a r a i r ( o b s e r v e d as f l o a t i n g b u b b l e s ) , w i t h a s u b s e q u e n t l o s s o f buoyancy o f t h e t i s s u e f r a gmen t and d e s c e n t i n t h e a l k a l i n e l y s i n g s o l u t i o n . When t h e e x p e r i m e n t s were c o n d u c t e d w i t h t h e a l k a l i n e l y s i n g s o l u t i o n p l a c e d as an o v e r l a y on t h e a l k a l i n e s u c r o s e g r a d i e n t s , s i n k i n g lung f r a g m e n t s were h a l t e d a t t h e g r a d i e n t / o v e r l a y i n t e r f a c e whereas s i n k i n g l i v e r o r k i d n e y f r a g m e n t s c o n t i n u e d t o d rop t h r o u g h t h e g r a d i e n t . To d e t e r m i n e t h e e f f e c t o f d i f f e r e n t amounts o f lung t i s s u e on t h e s e d i m e n t a t i o n b e h a v i o u r o f lung DNA i n a l k a l i n e s u c r o s e g r a d i e n t s , an e x p e r i m e n t was c a r r i e d o u t w i t h d i f f e r e n t s i z e s o f t i s s u e p i e c e s ( w e t - w e i g h t s ) : 0.5-1 mg, 1-2 mg, 2 -3 mg, and 4-5 mg, w h i c h were c u t f r o m t h e r i g h t m e d i a l l obe o f t h e lung o f an u n t r e a t e d mouse. Each p i e c e was s u b s e q u e n t l y chopped w i t h a s h a r p r a z o r b l a d e u n t i l a l l p o r t i o n s o f each m inced p i e c e c o u l d pa s s t h r o u g h t h e bo re o f a s t a n d a r d C o r n i n g 10 uI m i c r o - s a m p l i n g p i p e t t e . The f r a g m e n t s d e r i v e d f r om each s i z e o f lung f r a g m e n t were l a y e r e d s e p a r a t e l y on t h e l y s i n g s o l u t i o n o v e r l a y on t o p o f a l k a l i n e s u c r o s e g r a d i e n t s . F o l l o w i n g a 30 -m in l y s i s p e r i o d and removal o f t h e a i r b u b b l e s r e l e a s e d f r om t h e f r a g m e n t s , t h e g r a d i e n t s were c e n t r i f u g e d ( F i g u r e 6 4 ) . F o l l o w i n g c e n t r i f u g a t i o n , t h e t i s s u e " f r a g m e n t s , l e a c h e d o f i n t a c t c e l l s , were l o c a t e d i n f r a c t i o n s two o r t h r e e . The s e d i m e n t a t i o n p r o f i l e s o f 3H-DNA r e l e a s e d f r om t h e lung f r a g m e n t s c o n t a i n e d p r o m i n e n t peaks o f 3 H-DNA i n t h e c o n t r o l r e g i o n w i t h s i m i l a r p r o f i l e s o b t a i n e d w i t h d i f f e r e n t w e t - w e i g h t s i z e s o f lung t i