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A short term in vivo bioassay for the organ specificity of carcinogens Koropatnick, D. James 1978

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A SHORT TERM IN VIVO BIOASSAY FOR THE ORGAN SPECIFICITY OF CARCINOGENS by D. JAMES KOROPATNICK B.Sc, University of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Genetics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1978 (§) D. James Koropatnick, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . (fau&fics Pragma The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 D A T E QCMSA Q j m ABSTRACT The p o s s i b i l i t y o f u s 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 a n a l y s i s o f t h e d i g e s t i v e t r a c t t i s s u e o f mice t o i n v e s t i g a t e t h e c a r c i n o g e n i c p o t e n t i a l o f o r g a n o t r o p i c compounds was examined. Young S w i s s mice were i n j e c t e d w i t h 3H-Td'R t o l a b e l the DNA o f t h e e p i t h e l i a l c e l l s o f t h e d i g e s t i v e t r a c t . T h i r t y h l a t e r t h e y were f o r c e - f e d c a r c i n o g e n i c o r non-c a r c i n o g e n i c c h e m i c a l s . T i s s u e samples were t a k e n f o u r h p o s t - t r e a t m e n t and h y d r o l y z e d on t o p of the a l k a l i n e s u c r o s e g r a d i e n t . 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 i n d i c a t e d t h a t : (1) b o t h c u l t u r e d human f i b r o b l a s t s and e p i t h e l i a l c e l l s o f t h e g a s t r o i n t e s t i n a l s y s t e m show a 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 a f t e r t r e a t m e n t w i t h t h e c a r c i n o g e n MNNG t h a t i s t a k e n t o i n d i c a t e r e p a i r ; (2)- t h e c a r c i n o g e n 4 - n i t r o q u i n o l i n e 1 - o x i d e (4NQ0) and 6-methyl 4NQ0 cause DNA f r a g m e n t a t i o n i n t h e e p i t h e l i a l c e l l s o f t h e g a s t r o i n t e s t i n a l s y s t e m w h i l e t h e n o n - c a r c i n o g e n 6NQ0 l a c k s t h i s c a p a c i t y ; (3) 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 2AAF ca u s e d DNA f r a g m e n t a t i o n i n esophagus and stomach c e l l s w h i l e t h e p r e c a r c i n o g e n 2AAF p r o d u c e d no s i g n i f i c a n t e f f e c t ; (4) o n l y t h e c a r c i n o g e n i c n i t r o s a t i o n p r o d u c t s o f m e t h y l g u a n i d i n e damaged the DNA o f g a s t r i c e p i t h e l i a l c e l l s ; (5) t h e p r e c a r c i n o g e n s 2AAF and DMN p r o d u c e d DNA f r a g m e n t a t i o n i n t h e main t a r g e t o r g a n - t h e l i v e r -b u t had l i t t l e e f f e c t on t h e e p i t h e l i a l c e l l s o f t h e stomach; and (6) e x t r a c t s o f t h e c a r c i n o g e n i c p l a n t pteridium aquilinvjn ( b r a c k e n f e r n ) showed an o r g a n o t r o p i c DNA-fragmenting a b i l i t y in vivo and in vitro t h a t c o r r e s p o n d e d t o i t s . o r g a n - s p e c i f i c tumour i n d u c t i o n i n c a t t l e and r a t s . T r e a t m e n t w i t h h e a t a p p e a r s t o d r a s t i c a l l y r e d u c e t h e DNA-fragmenting a b i l i t y o f th e p l a n t . The r e s u l t s s u g g e s t t h a t t h e a p p l i c a t i o n o f t h 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 o t h e e p i t h e l i a l c e l l s o f esophagus, stomach and l i v e r o f p r e - l a b e l l e d ( 3H-TdR) and f o r c e - f e d young mice i n c o r p o r a t e s t h e adva n t a g e s o f in vitro s h o r t term b i o a s s a y s w i t h t h e c o m p l e t e n e s s o f t e s t s u s i n g whole mammals. . TABLE OF CONTENTS Abstract Table of Contents L i s t of Tables L i s t of Figures . A cknow1e d g em e n t s. Introduction . Carcinogenic Process DNA Rep ait-Chemical Carcinogens Primary or Ultimate Carcinogen Secondary or Procarcinogens Cocarcinogens. . Aromatic Amines . Nitrogen Compounds . D i a l k y l a m i n e s . . . . Ni t r o s a t a b l e Compounds. Ni t r o q u i n o l i n e N-Oxides Complex Mixtures. Organ S p e c i f i c i t y . Mat e r i a l s and Methods . Chemicals. . Radionuclides Chemical Carcinogens N i t r o s a t i o n of Methylguanidine Extraction of Bracken Fern Experimental Animals Human C e l l Cultures Administration of Chemicals A l k a l i n e Sucrose Gradient Analysis of DNA Damage Results  In V i t r o . In Vivo  Incorporation of Labelled Precursor into DNA Control Sedimentation P r o f i l e s . . . . Testing of Carcinogenic and Non-carcinogenic Compounds DNA Repair . . . Precarcinogens and Ultimate Carcinogens D i f f e r e n t i a l E f f e c t on Ga s t r i c E p i t h e l i a l C e l l s and L i v e r C e l l s . .' Nitrosated Compounds Complex Mixtures ." . • i v LIST OF TABLES TABLE PAGE Procarcinogenic Compounds 10 Carcinogenic A c t i v i t y of Chemicals 11 LIST OF FIGURES FIGURE PAGE 1 P r o p o s e d E x c i s i o n R e p a i r Pathway . . . . . . . . . . 0 2 DNA S e d i m e n t a t i o n P r o f i l e s o f C o n t r o l Human S k i n F i b r o b l a s t s 3 DNA S e d i m e n t a t i o n P r o f i l e s o f 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 E x p o s u r e t o MNNG 4 R e p a i r o f Human S k i n F i b r o b l a s t s Over 30 h f o l l o w i n g ^ E x p o s u r e t o MNNG 5 DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse G a s t r i c „> E p i t h e l i a l C e l l s ( C o n t r o l s ) . . . - . . •. .' . . . . . 6 DMA S e d i m e n t a t i o n P r o f i l e s o f Mouse C a r d i a c Stomach E p i t h e l i a l 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 and ,„ 6-methyl 4NQ0 7 DNA S e d i m e n t a t i o n P r o f i l e of Mouse C a r d i a c Stomach E p i t h e l i a l C e l l s F o l l o w i n g E x p o s u r e t o 3-methyl 4N00 8 D N A . S e d i m e n t a t i o n P r o f i l e of Mouse C a r d i a c Stomach , , LL IX E p i t h e l i a l C e l l s F o l l o w i n g E x p o s u r e t o 6N00. . . . 9 DNA S e d i m e n t a t i o n P r o f i l e o f Mouse P y l o r i c Stomach . E p i t h e l i a l C e l l s f o l l o w i n g E x p o s u r e t o 6NQ0. . . . 9A R e p a i r o f Mouse E p i t h e l i a l C e l l s Over 30 h f o l l o w i n g E x p o s u r e t o MNNG 4 0 10 DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse G a s t r i c E p i t h e l i a l and L i v e r C e l l s f o l l o w i n g E x p o s u r e t o 2AAF : . . . . . . . . . . . . 4 5 11 DNA S e d i m e n t a t i o n . P r o f i l e s o f Mouse G a s t r i c E p i t h e l i a l and L i v e r 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 2AAF . . . 48 12 DNA S e d i m e n t a t i o n P r o f i l e s of Mouse C a r d i a c Stomach E p i t h e l i a l and L i v e r 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 51 13 DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse C a r d i a c Stomach E p i t h e l i a l and L i v e r C e l l s F o l l o w i n g Exposure, t o MNNG 51 14 DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse C a r d i a c Stomach E p i t h e l i a l C e l l s F o l l o w i n g E x p o s u r e t o MG-HC1 o r t h e N i t r o s a t i o n P r o d u c t s o f MG-HC.l 53 PAGE DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse P y l o r i c Stomach and E s o p h a g e a l E p i t h e l i a l 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 N i t r o s a t i o n P r o d u c t s o f MG-HC1 . . DNA S e d i m e n t a t i o n P r o f i l e s o f C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g E x p o s u r e t o Four D i f f e r e n t E x t r a c t s o f B r a c k e n F e r n . . . DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse C a r d i a c Stomach E p i t h e l i a l C e l l s F o l l o w i n g E x p o s u r e t o Four D i f f e r e n t E x t r a c t s o f B r a c k e n F e r n DNA S e d i m e n t a t i o n P r o f i l e s o f Mouse E p i t h e l i a l C e l l s f r o m V a r i o u s G a s t r o i n t e s t i n a l A r e a s F o l l o w i n g E x p o s u r e t o a DNA-fragmenting B r a c k e n F e r n E x t r a c t 53 56 5 ACKN OWLEDGEMENTS I thank my research supervisor, Dr. H.E'. S t i c h , for h i s stimulating i n t e r e s t and help i n the preparation of t h i s t h e s i s . Without h i s nice balance of patience (to allow thought) and encouragement (to promote speed) i t would never have been done. Thanks also to my fellow researchers Bob, Lan, Richard, Paul, Brian and Charlee. They were t a c t f u l enough to pretend that the odour of the. mice didn't matter. I thank Dr. R.L. Noble, Director of the Cancer Research Centre, U.B.C, f o r h i s support. The help of the National Cancer.Institute of Canada, i n the form of grants to Dr. H.F.. S t i c h , i s g r a t e f u l l y acknowledged. INTRODUCTION Among th e a g e n t s t h a t may o r do cause tumours t o a r i s e i n t h e human p o p u l a t i o n , c h e m i c a l s are i m p l i c a t e d as among th e most i m p o r t a n t . I t i s e s t i m a t e d t h a t 70-90% o f cancers, a r e t h e r e s u l t o f e n v i r o n m e n t a l causes ( H i g g i n s o n , 1972; Wynder and M a b u c h i , 1 972). I f such f a c t o r s can be i d e n t i f i e d and e l i m i n a t e d , measures may be t a k e n t o 'minimise t h e i r i m p a c t . As l o n g ago as 1775 a B r i t i s h s u rgeon named P e r c i v a l P o t t d i s c o v e r e d t h a t chimney sweeps e x h i b i t c a n c e r o f t h e s c r o t u m , a p p a r e n t l y as a r e s u l t o f c o n t i n u a l e x p o s u r e to s o o t and t a r ( P o t t , 1 7 7 5 ) . H y g i e n i c measures t a k e n t o remove s o o t f r o m c l o t h i n g ( t a k e n i n Sweden by t h e Sweeper's G u i l d , b u t n o t i n B r i t a i n ) d r a m a t i c a l l y l o w e r e d the i n c i d e n c e o f t h e s e tumours. A n o t h e r c l a s s i c example o f e n v i r o n m e n t a l l y c a u s e d c a n c e r s i s the development of n a s o p h a r y n g e a l tumours by employees i n f a c t o r i e s where the r e f i n i n g o f n i c k e l by a now o b s o l e t e p r o c e s s t o o k p l a c e ( D o l l , 1 9 7 0 ) . I n the s y n t h e t i c dye i n d u s t r y , c a n c e r o f t h e u r i n a r y b l a d d e r was l i n k e d , t o e x p o s u r e t o c e r t a i n a r o m a t i c amines and r e l a t e d c h e m i c a l s (e.g.j 2-naphthylam.ine) i n a s i g n i f i c a n t p r o p o r t i o n o f t h o s e c o n t a m i n a t e d ( F r i e d e l l , 1969; H a r p e r , 1969). These a r e examples of o c c u p a t i o n a l c a n c e r s . However, the t o t a l i n c i d e n c e of c a n c e r s u g g e s t s t h a t t h e s e o c c u p a t i o n a l c a n c e r s r e p r e s e n t o n l y a s m a l l p r o p o r t i o n of t o t a l w o r l d tumours ( S i l v e r b e r g and H o l l e b , 1974; E c k a r d t , 1972). The r e a l c a n c e r p r o b l e m l i e s i n t h e g e n e s i s o f t h o s e tumours w h i c h a f f e c t m i l l i o n s o f p e o p l e - g a s t r i c c a n c e r i n Japan and c e r t a i n p a r t s oJ E u r o p e , l i v e r c a n c e r i n A f r i c a , and c o l o r e c t a l , b r e a s t and p r o s t a t e c a n c e r i n t h e W e s t e r n w o r l d ( e s p e c i a l l y A n g l o - S a x o n c o u n t r i e s ) . I n o r d e r t o d e t e c t and e l i m i n a t e t h e causes o f t h e s e w i d e s p r e a d tumours, e p i d e m i o l o g i c a l s t u d i e s may p r o v i d e a c l u e t o t h e d i s c o v e r y of s u s p i c i o u s a g e n t s and c i r c u m s t a n c e s t h a t can g i v e r i s e t o c a n c e r s . Then the p r e c i s e n a t u r e o f t h a t 2 agent or c i r c u m s t a n c e must be d e t e r m i n e d by u s i n g i t t o produce tumours i n an a n i m a l u s e d as a model (even though the same agent m i g h t n o t produce, the same r e s u l t i n man). B i o a s s a y s a r e the methods by w h i c h t h i s purpose i s c a r r i e d o u t . They a r e s p e c i f i c a l l y d e s i g n e d t o d e t e c t c a r c i n o g e n i c c h e m i c a l s , n o t o n l y as a p r o c e d u r e f o r d e t e r m i n i n g t h e cause of e s t a b l i s h e d tumours, b u t as a method f o r a v o i d i n g t h e s p r e a d o f c a r c i n o g e n i c r i s k by t h e i n t r o d u c t i o n of new c h e m i c a l s w h i c h may have a p o t e n t i a l f o r i n i t i a t i n g human c a n c e r . The p o w e r f u l c a r c i n o g e n 4 - n i t r o q u i n o l i n e - l - o x i d e (4NQ0) was d e v e l o p e d f o r use as a f u n g i c i d e b e f o r e i t was d i s c o v e r e d t o cause l u n g tumours i n t e s t a n i m a l s (Kawazoe, et al. 3 1967) and the r i s k o f i n a d v e r t e n t l y c a u s i n g tumours by m e d i c a l use o f t h i s c h e m i c a l was a v o i d e d by making i t s c a r c i n o g e n i c . p o t e n t i a l known. T h e r e f o r e , e v a l u a t i n g t h e c a r c i n o g e n i c p o t e n t i a l o f e n v i r o n m e n t a l c h e m i c a l s c o n c e r n s i t s e l f w i t h two t y p e s o f a g e n t s : s y n t h e t i c c h e m i c a l s e i t h e r p r e s e n t l y o r p o t e n t i a l l y i n use i n t h e human e n v i r o n m e n t , 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 s w h i c h may be r e s p o n s i b l e f o r e x i s t i n g c a n c e r s . B i o a s s a y systems must be made as s e n s i t i v e , r e l i a b l e , and s p e c i f i c as p o s s i b l e i n t h e d e t e c t i o n of c a r c i n o g e n i c i t y i n c h e m i c a l s . They must a l s o be e c o n o m i c a l , f a s t , and as f o o l p r o o f as p o s s i b l e ( e i t h e r i n t h e m s e l v e s o r by c r o s s - c h e c k i n g w i t h o t h e r b i o a s s a y s ) . CARCINOGENIC PROCESS A s e r i e s of complex i n d i v i d u a l r e a c t i o n s and p r o c e s s e s a r e t h o u g h t t o l e a d t o t h e f i n a l overt, c a n c e r i n man and a n i m a l s t h a t have t h e p r i m a r y a p p l i c a t i o n o f e n v i r o n m e n t a l c h e m i c a l s as t h e i r cause. Each of t h e s e r e a c t i o n s and c o n d i t i o n s may be s u b j e c t t o c o n t r o l by a number o f p o s s i b l e m o d i f y i n g f a c t o r s : 1) Upon a p p l i c a t i o n o f t h e c h e m i c a l c a r c i n o g e n , 3 i t may be a c t i v a t e d t o an u l t i m a t e c a r c i n o g e n . T h i s r e a c t i o n , may be m o d i f i e d by b i o c h e m i c a l d e t o x i f i c a t i o n and e l i m i n a t i o n r e a c t i o n s . 2) The u l t i m a t e c a r c i n o g e n may react, w i t h r e c e p t o r s i n t he c e l l . I t i s . p o s t u l a t e d t h a t t h e r e l e v a n t r e c e p t o r i s DNA, a l t h o u g h RNA and p r o t e i n r e c e p t o r s have n o t been, r u l e d o u t . T h i s i n t e r a c t i o n i s s u b j e c t t o s t e r e o c h e m i c a l c o n d i t i o n s and c o m p e t i t i v e , i n h i b i t i o n t h a t are. n o t y e t w e l l d e f i n e d . The a l t e r e d r e l e v a n t m a c r o m o l e c u l e {e.g., DNA) may then be r e p a i r e d and r e s t o r e d by r e p a i r enzyme systems whose, s u s c e p t i b i l i t y t o e r r o r may p l a y a s i g n i f i c a n t p a r t i n a l t e r i n g t h e c e l l r e c e p t o r r e s p o n s i b l e f o r c o n v e r s i o n t o t h e tumour s t a t e . 3) D u p l i c a t i o n of t h e abnormal r e c e p t o r so t h a t i t may be s u b s e q u e n t l y immune t o t h e o p e r a t i o n of r e p a i r s y s t e m s . 4) M u l t i p l i c a t i o n o f t h e c e l l s c o n t a i n i n g t h e abnormal r e c e p t o r as l a t e n t tumour c e l l s . 5) Growth o f l a t e n t c e l l s t o form.a w e l l - d i f f e r e n t i a t e d tumour. 6) C o n v e r s i o n o f t h e tumour by p r o g r e s s i o n t o an in d e p e n d e n t u n d i f f e r e n t i a t e d tumour. The f i r s t two s t e p s o u t l i n e d above - b i o t r a n s f o r m a t i o n of p r e c a r c i n o g e n s t o u l t i m a t e c a r c i n o g e n s , f o l l o w e d by a t t a c k o f c e l l u l a r o r m o l e c u l a r r e c e p t o r s - may 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 l e c t r o p h l l e 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 case ( M i l l e r , 1 9 7 0 ) . These e l e c t r o p h i l i c s p e c i e s w i l l b i n d g e n e r a l l y t o e l e c t r o n - r i c h c e n t e r s . These c e n t e r s a r e found i n p r o t e i n s and n u c l e i c a c i d s , i n c l u d i n g b o t h DNA and RJNA. The r e a c t i o n o f e l e c t r o p h i l e s w i t h e l e c t r o n -r i c h c e n t e r s r e s u l t s i n c o v a l e n t b o n d i n g and, u l t i m a t e l y , an abnormal r e c e p t o r i n the c e l l . W i t h r e s p e c t t o DNA a l k y l a t i o n t he most r e a c t i v e s i t e . & f a l l t h e DNA bas e s appears t o be 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 gua n i n e ( t h e p r i n c i p a l a l k y l a t i n g s i t e o f MNRG) 4 a l t h o u g h i t i s o n l y one o f many s i t e s a v a i l a b l e f o r a l k y l a t i o n . O t h e r p o s i t i o n s i m p l i c a t e d as b e i n g p a r t i c u l a r l y s u s p e c t i n c a r c i n o g e n e s i s a r e the 6 - p o s i t i o n oxygen and 3 - p o s i t i o n n i t r o g e n of g u a n i n e , the I , 3 and 7 - p o s . i t i o n n i t r o g e n s o f a.denine, and t h e n i t r o g e n a t t h e 3 - p o s i t i o n o f c y t o s i n e . I n f a c t , a l m o s t a l l p o s s i b l e alkylat.able s i t e s a r e s u s p e c t s i n c e these s i t e s have been r e p o r t e d t o be a t t a c k e d , by c a r c i n o g e n s in vivo and in vitro ( r e v i e w e d by Sa.rma, et at., 1974: L a w l e y , 1976). I n a d d i t i o n t o a l k y l a t i o n o f DNA, i n t e r c a l a t i o n . ( 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 a d l i n e a t i o n ( e x t e r n a l b i n d i n g o f c h a r g e - t r a n s f e r complexes t o b a s e s 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 ) may o c c u r . I n s p i t e o f the l a c k o f s t r e n g t h o f a d l i n e a t i o n , i t has been p r o p o s e d as a model f o r i n t e r f e r e n c e w i t h DNA by 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 , 1 9 6 8 ) , a f l a t o x i n s ( I r v i n g , 1973) and 4NQ0 ( P a u l and Montgomery, .1971). B o t h n o n c o v a l e n t i n t e r a c t i o n s a r e much weaker t h a n c o v a l e n t l i n k a g e and t h i s . h a s p r e v e n t e d the development o f f i r m e v i d e n c e f o r t h e r e l e v a n c e o f n o n c o v a l e n t b i n d i n g . in vivo ( r e v i e w e d by L a w l e y , 1976). The model s y s t e m d e v e l o p e d t o e x p l a i n l i v e r c a r c i n o g e n e s i s p r o v i d e s some i n s i g h t i n t o t he e v e n t s subsequent t o DNA-chemical i n t e r a c t i o n t h a t l e a d f i n a l l y t o c a n c e r . The " i n i t i a t i o n " p r o c e s s , o r e a r l y r a p i d e v e n t s o c c u r r i n g i n hours o r d a y s , l e a d s to t h e appearance o f new c e l l p o p u l a t i o n s and has been r e f e r r e d t o as " 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 , 1 9 7 3 ) . When a s i n g l e dose o f d i m e t h y l n i t r o s a m i n e (DMN) i s a d m i n i s t e r e d d u r i n g l i v e r c e l l d i v i s i o n f o l l o w i n g p a r t i a l h epatectomy, 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 between r e c o v e r y f r o m i n i t i a l - c e l l damage u n t i l t h e appearance o f c e l l u l a r a l t e r a t i o n s l e a d i n g t o p r o g r e s s i v e c e l l p o p u l a t i o n changes and f i n a l l y 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 , et o.l., 1 9 7 4 ) . S i n c e a l i n k must be f o u n d between t h e i n i t i a l damage t o t h e c e l l and f i n a l a ppearance o f t h e tumour, DNA has been proposed, as t h e r e l e v a n t c e l l u l a r a c c e p t o r o f c a r c i n o g e n s . S p e c i f i c a l l y , 5 t h e s o m a t i c m u t a t i o n t h e o r y o f c a r c i n o g e n e s i s p r o p o s e s that: c h e m i c a l c a r c i n o g e n s a r e a c c e p t e d by c e l l u l a r DNA i n s u c h a way as t o produce a n o n l e t h a l , h e r i t a b l e DNA damage e x p r e s s e d u l t i m a t e l y as a tumour when such c e l l s a r e v i e w e d as a group ( r e v i e w e d by F o u l d s , 1969). DNA REPAIR When an i n t e r a c t i o n o c c u r s between c h e m i c a l c a r c i n o g e n s and DNA t h e r e may be a d i r e c t c h e m i c a l d e p u r i n a t i o n t h a t l e a d s t o d i s r u p t i o n o f the s u g a r phosphate backbone and t h u s s i n g l e -s t r a n d b r e a k s . T h i s can o c c u r a f t e r t r e a t m e n t w i t h a l k y l a t i n g a g e n t s t h a t a l t e r c o v a l e n t and h y d r o p h o b i c b o n d i n g c h a r a c t e r i s t i c s o f b a s e s {e.g., n i t r o g e n m u s t a r d ) . These a g e n t s b i n d a l k y l groups t o p u r i n e b a s e s f o r t h e most p a r t , b u t t h e y w i l l a l s o a l k y l a t e p y r i m i d i n e s ( L a w l e y , 1976). However, t h i s " s p o n t a n e o u s " h y d r o l y s i s o f DNA c h a i n s i s n o t t h e o n l y method f o r s t r a n d b r e a k a g e . Enzyme-mediated r e m o v a l of a l k y l a t e d bases of DNA has a l s o been proposed. ( I k e g a m i , et at.3 1970). T h i s c e l l - m e d i a t e d e x c i s i o n of damaged DNA segments i s t h o u g h t t o be p a r t o f a p r o c e s s o f DNA r e p a i r - an a t t e m p t by the. c e l l t o m a i n t a i n t r u e DNA c o p i e s f o r immediate, use. and t r a n s m i s s i o n t o d a u g h t e r c e l l s . DNA r e p a i r may o c c u r by a v a r i e t y o f methods. P h o t o r e a c t i v a t i o n r e p a i r r e q u i r e s a s i n g l e enzyme t o b i n d and p h o t o c a t a l y t i c a l l y c l e a v e thymine d i m e r s . P o s t - r e p l i c a t i o n i ~ e p a i r i n c l u d e s a l l t h o s e p r o c e s s e s whereby e r r o r s i n DNA a r e r e p a i r e d a f t e r c e l l ! r e p l i c a t i o n has t a k e n p l a c e and damaged a r e a s i n DNA s t r a n d s have-been b y p a s s e d by t h e r e p l i c a t i o n enzymes i n s u c h a way as t o l e a v e gaps i n DNA s t r a n d s . T h i s p r o c e s s i s l a r g e l y t h e o r e t i c a l i n mammalian c e l l s , and i n c l u d e s damage b y p a s s t h a t may t a k e p l a c e by means o f DNA r e c o m b i n a t i o n ( r e v i e w e d by H a n a w a l t , 1975). E x c i s i o n r e p a i r may o c c u r a c c o r d i n g t o the mechanism p r o p o s e d i n F i g u r e 1. T h i s . i s a p r o c e s s by w h i c h t h e c e l l r e p a i r s damage t o DNA by r e m o v i n g t h e . a f f e c t e d p o r t i o n and r e p l a c i n g i t w i t h Figure 1 distortion caused by bound chemical a) damage - spec i f i c endonuclease incision, or b) spontaneous hydrolytic f i s s ion i •it JX. o) DNA po lymerase fills gap \ , b) 5 ' exonuclease degrades d i s tor ted DNA segment polynucleotide l igase reforms 5 ' ond 3 1 end* repl icat ion daughter strands —*• — — 7 th e c o r r e c t s t r u c t u r e b e f o r e u n d e r g o i n g d i v i s i o n . The b i n d i n g o f c h e m i c a l t o DNA ( e i t h e r c o v a l e n t l y o r n o n - c o v a l e n t l y ) may l e a d t o l o c a l d i s t o r t i o n t h a t can s e r v e as a 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 , or as a p o s i t i o n f o r " s p o n t a n e o u s " h y d r o l y t i c f i s s i o n of t h e DNA c h a i n ( P a u l and Montgomery, 1971). However, where coincident s i n g l e s t r a n d b r e a k s t a k e p l a c e a f a i t h f u l r e c o n s t r u c t i o n o f t h e o r i g i n a l s t r u c t u r e i s u n l i k e l y , due t o t h e absence o f an i n t a c t complementary s t r a n d a c t i n g as a t e m p l a t e f o r p o l y m e r a s e a c t i v i t y . . E x c i s i o n r e p a i r may be m o n i t o r e d i n c e l l s by a v a r i e t y o f methods. D e n s i t y l a b e l l i n g by r a d i o a c t i v e 5 - b r o m o u r a c i l o r o t h e r d e n s i t y l a b e l s ( 1 3 C , 1 5 N , ; o r 3H) and o b s e r v a t i o n o f ne w l y s y n t h e s i z e d DNA a t the. p a r e n t a l buoyant d e n s i t y i n a C s C l e q u i l i b r i u m d e n s i t y g r a d i e n t i s one method (Hanawa.lt and Copper, 1971). U n s c h e d u l e d s y n t h e s i s o f t h e DNA o f c e l l s r a d i o a c t i v e l y l a b e l l e d w i t h thymine w h i l e out of S-phase has been t a k e n t o mean r e p a i r s y n t h e s i s (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 , et al., 1970, 1973, 1 9 7 7 ) . The f i n a l r e j o i n i n g s t e p o f e x c i s i o n r e p a i r as w e l l . a s the i n i t i a l , i n c i s i o n o r h y d r o l y t i c c l e a v a g e s t e p c a n be m o n i t o r e d by e x a m i n i n g the s i n g l e - s t r a n d m o l e c u l a r weight, d i s t r i b u t i o n o f 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 zone s e d i m e n t a t i o n (McGrath and W i l l i a m s , 1966: L e t t , et afl., 1967; L a i s h a s and S t i c h , 1973; S t i c h and L a i s h e s , 1973; K o r o p a t n i c k , et al., 1975; A b a n o b i , et al,, 1977). T h i s procedure, w i l l r e g i s t e r b r e a k s i n t h e a l k a l i - l a b i l e l i n k a g e s i n t h e backbone o f t h e DNA s t r u c t u r e , a l t h o u g h DNA i s s t a b i l i z e d i n t h i s r e s p e c t due t o a l a c k o f 2 ' - h y d r o x y ! groups on t h e r i b o s e . However, t h e r e j o i n i n g o f b r e a k s does n o t n e c e s s a r i l y mean t h a t d i s t o r t i o n s have been removed o r t h a t the o r i g i n a l n u c l e o t i d e sequence o f t h e DNA has been r e s t o r e d . A l s o , d e g r a d a t i o n c f a l a r g e amount o f DNA may unmask a s m a l l amount o f r e s i d u a l h i g h m o l e c u l a r w e i g h t DNA t o g i v e the f a l s e - i m p r e s s i o n t h a t gaps have been r e p a i r e d . 8 CHEMICAL CARCINOGENS C h e m i c a l c a r c i n o g e n s may be d i v i d e d i n t o t h r e e m a j o r t y p e s a c c o r d i n g t o c u r r e n t v i e w s : p r i m a r y o r u l t i m a t e c a r c i n o g e n s , s e c o n d a r y o r p r o c a r c i n o g e n s , o r c o c a r c i n o g e n s ( p r o m o t i n g a g e n t s and f a c t o r s ) . PRIMARY OR ULTIMATE CARCINOGENS These c h e m i c a l s owe t h e i r b i o l o g i c a l r e a c t i v i t y t o the c h e m i c a l p r o p e r t i e s i n t r i n s i c t o them when t h e y e n t e r the o r g a n i s m . They can i n t e r a c t d i r e c t l y w i t h t i s s u e s and c e l l components t o y i e l d m o d i f i e d m a c r o m o l e c u l e s w h i c h may l e a d t o t h e g e n e s i s o f p r e n e o p l a s t i c c e l l s . These a r e c h a r a c t e r i s t i o f changes l e a d i n g t o t r a n s f o r m e d c a n c e r c e l l s . A l k y l a t i n g a g e n t s o f v a r i o u s t y p e s a r e examples o f t h e s e k i n d s o f c a r c i n o g e n s , i n c l u d i n g n i t r o g e n s and s u l p h u r m u s t a r d s , s u l p h o n i c e s t e r s and s u l t o n e s , e t h y l e n e i m i n e s and i m i d e s , s t r a i n e d o r a,3-unsaturate.d l a c t o n e s , e p o x i d e s , p e r o x i d e s and c h l o r o a l k y l e t h e r s ( M i l l e r , 1 9 7 0 ) . They a r e i n t h e i r f i n a l r e a c t i v e form as a d m i n i s t e r e d and g e n e r a l l y t a k e p a r t i n .S^2 ( s u b s t i t u t i o n , n u c l e o p h i l i c , b i m o l e c u l a r ) r e a c t i o n s i n w h i c h an a l k y l a t i n g e l e c t r o p h i l e combines w i t h a n u c l e o p h i l e i n t h e c e l l u l a r t a r g e t t i s s u e s o r m a c r o m o l e c u l e s ( P r i c e , et a.l.j 1969; R o s s , 1962; S h a p i r o , 1969). They a r e f r e q u e n t l y n o t s t r o n g c a r c i n o g e n s , and may r e q u i r e m u l t i p l e l a r g e doses a t l o c a l t i s s u e s i t e s t o e x h i b i t c a r c i n o g e n i c i t y i n t e s t a n i m a l s . T h i s , presumably,• i s due t o the p r e s e n c e o f i n t e r f e r i n g n u c l e o p h i l e s ( s u c h as w a t e r and p r o t e i n ) w h i c h may d i s p o s e o f much o f t h e e l e c t r o p h i l e b e f o r e i t s e n t r y i n t o t a r g e t c e l l s o r r e a c t i o n w i t h t a r g e t t i s s u e s ( M i l l e r , 1969; M i l l e r , 1970). A s m a l l group o f i n o r g a n i c c h e m i c a l s s u c h as b e r y l l i u m , cadmium, .cobalt', . n i c k e l , l e a d , manganese, and chromium a r e , i n t h e i r i o n i c f o r m s , e l e c t r o p h i l e s and have been shown to be c a r c i n o g e n i c ( F o r s t and Haro, 1969; C l a y s o n , 1 9 6 2 ) . G e n e r a l l y , t h a t c l a s s o f c h e m i c a l s t h a t a r e c 9 e i e c t r o p h i l i c r e a g e n t s , and p o s s i b l y t h o s e w i t h t h e a b i l i t y t o form f r e e r a d i c a l s , c o m p r i s e t h e u l t i m a t e c a r c i n o g e n s . SECONDARY OR PROCARCINOGENS D i f f e r i n g w i d e l y i n s t r u c t u r e and c h a r g e , most carcinogenic c h e m i c a l s f a l l i n t o t h i s . c l a s s ( T a b l e 1 ) . They i n c l u d e i n d i v i d u a l c h e m i c a l s s y n t h e s i z e d by man and complex m i x t u r e s t h a t o c c u r n a t u r a l l y . They a r e o f t e n c h e m i c a l l y and b i o c h e m i c a l l y i n e r t ( w i t h r e s p e c t t o t a r g e t m a c r o m o l e c u l e s r e q u i r e d f o r c a r c i n o g e n e s i s ) and spontaneous o r h o s t m e d i a t e d and c o n t r o l l e d a c t i v a t i o n r e a c t i o n s a r e r e q u i r e d t o c o n v e r t p r o c a r c i n o g e n s t o t h e i r u l t i m a t e l y r e a c t i v e s p e c i e s . . When t h e s e p r o c a r c i n o g e n s a r e spontaneously c o n v e r t e d t o p r i m a r y c a r c i n o g e n s by h y d r o l y s i s t h e y may e x h i b i t a c t i v i t y i n a b r o a d range o f t a r g e t s p e c i e s and organs due t o t h e s i m p l i c i t y and u n i v e r s a l i t y o f t h e i r " a c t i v a t i o n " . On t h e o t h e r hand, where s p e c i f i c h o s t - c o n t r o l l e d b i o c h e m i c a l a c t i v a t i o n i s r e q u i r e d t h e r e may be g r e a t d i v e r s i t y i n a c t i v i t y f rom o r g a n t o o r g a n , i n d i v i d u a l t o i n d i v i d u a l , o r s p e c i e s t o s p e c i e s . A c t i v a t i o n ay depend on some s p e c i f i c enzyme, s y s t e m ( M i l l e r and M i l l e r , 197.1; F a r b e r , 1973; W e i s b u r g e r , 1973; W e i s b u r g e r and Weisburger,. 1973; G r o v e r , e t al., 1974; Oesch, 1972; Sugimura and K a w a c h i , 1973). T h i s s u p p l i e s a p o s s i b l e e x p l a n a t i o n f o r t h e o b s e r v a t i o n t h a t a c h e m i c a l which i s c a r c i n o g e n i c i n some systems may show no c a r c i n o g e n i c a c t i v i t y a t a l l i n o t h e r s p e c i e s where t h e r e q u i r e d a c t i v a t i n g enzyme o r enzymes a r e a b s e n t . COCARCINOGENS These a re age n t s t h a t a r e u n a b l e t o produce tumours by t h e m s e l v e s b u t w i l l p o t e n t i a t e , t he a c t i o n o f u l t i m a t e o r . p r o c a r c i n o g e n s . Complex m i x t u r e s s u c h as t o b a c c o smoke a r e now t h o u g h t t o c o n t a i n large, amounts o f c o c a r c i n o g e n s , b u t r a t h e r s m a l l r e l a t i v e amounts o f p r o c a r c i n o g e n s (Wynder and m 10 T a b l . e _ l P r o c a r c i n o g e n l c compounds P o l y n u c l e a r a r o m a t i c and h e t e r o c y c l i c , h y d r o c a r b o n s A r o m a t i c and h e t e r o c y c l i c amines and azo dyes N i t r o a r y l - and f u r a n - d e r i v a t i v e s N i t r o s a m i n e s , -amides, - u r e a s , -carbamates A l k y l t r i a z e n e s , d i a l k y l h y d r a z i n e s C y c a s i n , s a f r o l e , e t h i o i a i n e A c e t a m i d e , t h i o a m i d e s C h l o r i n a t e d h y d r o c a r b o n s A f l a t o x i n s , m y c o t o x i n s P y r r o l i z i d i n e a l k a l o i d s B r a c k e n f e r n .Carbamates ( u r e t h a n ) ( a d a p t e d f r o m W e i s b u r g e r , 1976) 11 T a b l e 2 C a r c i n o g e n A n i m a l P r i n c ip_aJ.^_s_it e o f t x nn o x 11: f o r m a t i o n MNNG 4NQ0, 6-methyl 4NQ0 3-methyl 4NQ0 6NQ0 2 AAF, N - a c e t o x y 2AAF mm MMU r a t mouse S.G. h a m s t e r r a b b i t , dog r a t , mouse h a m s t e r , g u i n e a P i g r a t , mouse m i c e , r a t s r a t mouse S.G. hamster r a b b i t r a t , mouse g l a n d u l a r stomach f o r e s t o m a c h i n t e s t i n e . , S.C. i n t e s t i n e , f o r e s t o n i a c h s k i n ( s i t e o f i n j e c t i o n ) ' g l a n d u l a r stomach i n t e s t i n e l u n g stomach, i n t e s t i n e l u n g , l u n g adenomas, l e u k e m i a , stomach, i n t e s t i n e • . no tumours b r e a s t , l i v e r , b l a d d e r r e n a l p e l v i s , a c o u s t i c d u c t c o l o n , l u n g , p a n c r e a s b l a d d e r , kidney-l i v e r , k i d n e y , n a s a l c a v i t i e s . l i v e r , l u n g , k i d n e y l i v e r , n a s a l c a v i t i e s , l i v e r c e n t r a l and p e r i p h e r a l n e r v o u s s y s t e m , i n t e s t i n e k i d n e y , f o r e s t o m a c h , g l a n d u l a r stomach, s k i n and annexes, j a w , b l a d d e r , u t e r u s , v a g i n a , l u n g , l i v e r , p h a r y n x , esophagus, t r a c h e b r o n c h i , o r a l c a v i t y , p a n c r e a s , e a r d u c t s B r a c k e n f e r n c a t t l e , r a t s l o w e r i l e u m , u r i n a r y b l a d d e r Hoffmann, 1967; Van Burner'., et al., 1973; S a f f i o t t i , 1969). The b e s t known c o c a r c l n o g e n i s a c r o t o n o i l , t h e e x t r a c t of. c r o t o n r e s i n . I t promotes mouse s k i n tumour f o r m a t i o n a f t e r the a p p l i c a t i o n o f a c a r c i n o g e n i c 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 such as 2-methylcholanthre.ne ( H e c k e r , 1971; B o u t w e l l , 1974; S i v a k and Van Durnen, 1971). AROMATIC AMINES An example o f an . u l t i m a t e c a r c i n o g e n i s t h e a r o m a t i c amine N-acetoxy - 2 a c e t y l a m i n o f l u o r e n e (N-acetoxy-2AAF). T h i s i s t he N - h y d r o x y l a t i o n and sub s e q u e n t e s t e r i f i c a t i o n p r o d u c t o f the p r o c a r c i n o g e n 2 - a c e t y l a m l n o f l u o r e n e (2AAF)(Cramer, et al., 1960; M i l l e r , et al., 1961; L a i s h e s and S t i c h , 1973). O i the 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 2AAF, N~acetoxy-2AAF shows h i g h r e a c t i v i t y toward mammalian DNA, RNA and p r o t e i n b o t h in vivo and 13 in vitro ( M i l l e r and M i l l e r , 1 9 6 9 ) . An o x i d a t i o n r e a c t i o n 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 found i n l i v e r , l u n g and b l a d d e r m u c o s a l c e l l s ( U e h l e k e , 1966) i s r e s p o n s i b l e f o r t h e N-hydro-x y l 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 produce hydroxy3.amine d e r i v a t i v e s s u c h as t h e c a r c i n o g e n i c I n t e r m e d i a t e N-hydroxy-2AAF. Most t i s s u e s c o n t a i n o x ygenases, b u t c e r t a i n c e l l s ( h e p a t i c p a r e n c h y m a l c e l l s a r e an example) c o n t a i n i n t h e i r e n d o p l a s m i c r e t i c u l u m t h e complete pathway o f enzymes r e s p o n s i b l e f o r t h e h y d r o x y l a t i o n o f o r g a n i c compounds. The g e n e r a l h y d r o x y l a t i o n r e a c t i o n may be w r i t t e n a s : R-CH 3 + NADPH 4- H + + 0 2 ->• R-CH2<3H + NADP + + H 20 The r e s p o n s i b l e enzymes have been termed "mixed f u n c t i o n o x y g enases".(Mason, 1957) and, more r e c e n t l y , "mono-oxygenases" H a . y a i s h i , 1969) and r e q u i r e NADPH and oxygen. They have been i d e n t i f i e d i n i n t a c t a n i m a l s , p e r f u s e d o r g a n s , t i s s u e s l i c e s and m i c r o s o m e s , and the many t y p e s o f o x i d a t i o n r e a c t i o n s and e l e c t r o n t r a n s p o r t c h a i n c h a r a c t e r i s t i c s have been summarized ( r e v i e w e d by H u t s o n , 1970). However, t h e N ~ h y d r o x y l a t i o n p r o d u c t o f 2AAF has v e r y l i t t l e a b i l i t y t o b i n d n u c l e i c a c i d s in vitro ( I r v i n g , et al., 1969) and a f i r m c o n n e c t i o n between in vitro r e a c t i v i t y and in vivo c a r c i n o g e n i c i t y c o u l d be e s t a b l i s h e d f o r 2AAF o n l y when the h y d r o x y l group o f N-hydroxy 2AAF was e s t e r i f i e d . The " 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 2AAF, i s a b l e t o b i n d , f o r example, t o g u a n i n e in vitro, w h i l e 2AAF and N-hydroxy 2AAF c a n n o t . N u c l e i c a c i d b i n d i n g p r o d u c t s i s o l a t e d f r o m t h e in vitro s y s t e m have a l s o been i s o l a t e d f r o m t h e l i v e r s o f r a t s p r e v i o u s l y f e d 2AAF ( r e v i e w e d by M i l l e r , 1970). NITROGEN COMPOUNDS A n o t h e r d i r e c t l y a c t i n g c a r c i n o g e n i s t h e N - m e t h y l -N - n i t r o s o compound N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) w h i c h c a u s e s tumours i n r a t s , m i c e , h a m s t e r s , r a b b i t s and dogs. 14 , H C 3 \ M — NO / = C \ MNNG NHN0 2 I t w i l l alkylate the macromolecular constituents of target tissues, presumably by spontaneous N-demethylation and tautomerization In neutral aqueous media to produce transient methyldiazohydroxide (a source of methyldlazonium and methyl-carbonium ions which can alkylate macromolecules). MNNG w i l l also react with t h i o l s to l i b e r a t e methylating species, which i s almost cert a i n l y a mode of activation in vivo (reviewed by Lawley, 1976). DIALKYLAMINES The N-nitroso dialkylamines are- a large class of compounds, some of which are routinely detected i n the environment. (Hedler and Marquardt, 1968). More than 100 carcinogenic nitrosamines have been i d e n t i f i e d that can alkylate the macromolecular .constituents of target tissues. The simplest of these compounds, i s dimethylnitrosamine (DMN) which i s metabolized in vivo by a mechanism delineated by Heath i n 1962. He showed that DMN undergoes N-demethylation to y i e l d N-nitrosomethylamine and tautomerization to produce methyldiazohydroxide, which i s a possible source of methyldizonium and methy1carbonium ions -the same a l k y l a t i n g species produced by MNNG (see a b o v e ) . I n th i s case, however, DNM w i l l not. react spontaneously and requires enzymic N-demethylation i n order to be activated. Thus, i t i s a procarcinogen rather than an ultimately carcinogenic compound. Unfortunately the enzymatically produced metabolites of DMN have so far been d i f f i c u l t to prepare s y n t h e t i c a l l y , as we l l as i s o l a t e and store for s p e c i f i c analysis and testing. Therefore, the i d e n t i f i c a t i o n of relevant biotransformation 15 3KC enzymic oxidation D M N '6 alkyfafion of «s -target macromolecutes CH3h hydrolysis CH 3 CH 3N 2 4-mechanisms remains largely speculative due to the i n s t a b i l i t y of the metabolites (Laishes, 1974). DMN i s a potent l i v e r carcinogen i n the r a t , and i s metabolized i n human l i v e r s l i c e s at close to the same rate as i n rat l i v e r s l i c e s (Montesano and Magee, 1970). NITROSATABLE COMPOUNDS Another example of the nitrosodialkylamines are the nitrosation products of methylguanidine, a naturally occurring compound which, when nitrosated, w i l l produce at least two carcinogenic and mutagenic metabolites i n a mixture of products. Methylnitrosoguanidine (MNNG) and raethylnitrosourea (MNU) are these compounds (Endo, et dl. , 1974; Kapeller-Adler, 1930; Komarrow, 1929). Compounds such, as methylguanidine are of 3 H C\ ' ' N — N O - MMJ / o==~-c • \ l H 2 importance to man, since«(like many other n a t u r a l l y occurring or man-made substances) they can be nitrosated i n the acid 16 c o n d i t i o n s f o u n d i n t h e human stomach (Endo, e t d l . , 1974; Endo and T a k a h a s h i , 1973; Lane and B a i l e y , 1973) o r a t n e u t r a l pH by a l i m e n t a r y b a c t e r i a (Hawksworth and H i l l , 1971) . Because o f t h e u b i q u i t o u s n a t u r e o f t h e s e n i t r o s a t a b l e compounds t h e y have, been i m p l i c a t e d i n t h e g e n e s i s o f human c a r c i n o m a s (Sanders and S c h w e i n s b e r g , 1972; Lo and S t i c h , 1975). NITROQTJIN01,INE N-OXIDES Among t h e p r o c a r c i n o g e n s a r e t h e n i t r o q u i n o l i n e N-o x i d e s , o f w h i c h 4 - n i t r o q u i n o l i n e N - o x i d e (4NQ0) i s a good example. I t must be e n z y m a t i c a l l y r e d u c e d t o i t s u l t i m a t e c a r c i n o g e n i c s t a t e . , 4 - h y d r o x y a m i n o q u i n o l i n e 1 - o x i d e (4IIAQ0) i n o r d e r t o r e a c t w i t h n u c l e i c a c i d (Kawazoe, et al., 1972). 4WQ0 ' reducfioit macromoSecuie binding Because o f i t s h i g h l y o x i d i z e d s t a t e , 4NQ0 has a low de g r e e o f e l e c t r o p h i l i c r e a c t i v i t y , so t h a t i t does not r e a c t c o v a l e n t l y w i t h DNA in vitro. However, i t may i n t e r c a l a t e and w i l l c e r t a i n l y p h y s i c a l l y b i n d t o DNA w i t h o u t a c t i v a t i o n . I n t h i s c a s e 4NQ0 i s a s s o c i a t e d w i t h p u r i n e r a t h e r t h a n p y r i m i d i n e b a s e s . On t h e other hand, 4N00 i n j e c t e d i n t o r a t s b e a r i n g t r a n s p l a n t a b l e hepatoma c e l l s i n a s c i t e s form i s found c o v a l e n t l y bound t o the DNA o f t h o s e c e l l s . T h e r e f o r e , w h i l e 4NQ0 can become a s s o c i a t e d w i t h DNA whether i t i s e n z y m i c a l l y t r a n s f o r m e d or n o t , t h e o v e r a l l e v i d e n c e s u g g e s t s t h a t c o n v e r s i o n , t o 4HAQ0 i s n e c e s s a r y • ( r e v i e w e d by C l a y s o n and G a r n e r , 1976). I t may be t h a t t h e f a i l u r e o f 6NQ0 and 3-methyl 4NQ0 t o be good c a r c i n o g e n i c compounds i n a n i m a l s i s because o f t h e i r i n a b i l i t y t o be e n z y m a t i c a l l y c o n v e r t e d t o t h i s u l t i m a t e form. (Kawazoe, et a l . , 1967). COMPLEX MIXTURES A l t h o u g h most l a b o r a t o r y t e s t i n g i s done w i t h c h e m i c a l s i n as p u r e a s t a t e as can be o b t a i n e d , most c a r c i n o g e n s i n man's e n v i r o n m e n t e x i s t as p a r t s o f complex m i x t u r e s , and a r e exposed t o h im as s u c h . A good example o f t h e s e a r e c a r c i n o g e n s o f p l a n t o r i g i n - c y c a s i n f r o m c y c a d n u t s , s a f r o l e from s a s s a f r a s , and, i n p a r t i c u l a r , t h e c a r c i n o g e n i c p l a n t pteridium aquilinum, or b r a c k e n f e r n . By t h e l a t e 1 9 t h c e n t u r y t he l e t h a l p r o p e r t i e s o f t h i s p l a n t were s c i e n t i f i c a l l y r e c o g n i z e d ( S t o r i e r , .1893; Almond, 1894). The e a r l i e s t i n t i m a t i o n of a c a r c i n o g e n i c l i n k w i t h t h e p l a n t came i n 1960 (von R o s e n b e r g e r and Heeschen, 1960) who d e s c r i b e d h e m a t u r i a and p o l y p f o r m a t i o n i n u r i n a r y b l a d d e r mucosa o f b r a c k e n - f e d c a t t l e . Work w i t h r a t s and m i c e , c o r r o b o r a t e d by g e o g r a p h i c a l and e p i d e m i o l o g i c a l d a t a w i t h T u r k i s h b r a c k e n - f e d c a t t l e (Pamukcu, 1 9 6 3 ) , has shown t h a t t h e c a r c i n o g e n i c agent o r a g e n t s i n b r a c k e n f e r n cause a d e n o c a r c i n o m a s o f t h e i n t e s t i n a l mucosa p r e d o m i n a n t l y i n t h e i l e u m and the u r i n a r y b l a d d e r (Evans and Mason, 196.5; Evans and Widdop, 1966). Young a n i m a l s a r e p a r t i c u l a r l y s u s c e p t i b l e ( E v a n s , 1968^ 1969, 1972). C h e m i c a l s t u d i e s i n v o l v i n g t h e e x t r a c t i o n o f t h e p l a n t w i t h b o t h o r g a n i c and i n o r g a n i c s o l v e n t s have v a r i o u s l y a s c r i b e d the c a i ' c i n o g e n i c e f f e c t o f b r a c k e n f e r n t o o r g a n i c a c i d s (Wang, et al., 1 9 7 3 ) , i n d a n o n e s (Kuroyanage, et al., 1 9 7 4 ) , p t e r o q u i l i n (Kwasn iewsk i , 1 9 5 5 ) , s h i k i m i c a c i d (Evans and Osman, 1974) 18 and t a n n i n (Wang, et al., 1976). D e f i n i t e i d e n t i f i c a t i o n o f the c a r c i n o g e n i c o r p r o c a r c i n o g e n i c agents on b r a c k e n f e r n has y e t t o t a k e p l a c e . The b r a c k e n c a r c i n o g e n may o r may n o t be ha z a r d o u s t o humans, b u t t h e r e a r e p o s s i b i l i t i e s f o r danger w h i c h s h o u l d n o t be o v e r l o o k e d . The c o n t a m i n a t i o n o f m i l k and d a i r y p r o d u c t s o f c a t t l e t h a t a r e f e d b r a c k e n f o d d e r , o r c o n t a m i n a t i o n o f t h e w a t e r s u p p l y a r e t h e most l i k e l y p o s s i b i l i t i e s . B r a c k e n b e e r i s brewed i n some i s o l a t e d a r e a s o f Norway and S i b e r i a , b u t one s u s p e c t s t h a t t h i s e x p o s u r e i s somewhat l i m i t e d ( H a r r i n g t o n , 1967). One u n e x p l a i n e d , b u t v e r y i n t e r e s t i n g , o b s e r v a t i o n i s t h a t t h e r e i s a marked r e g i o n a l p r e v a l e n c e o f l i p , mouth, esophagus and stomach c a n c e r i n s o u t h e r n Wales i n comparison, w i t h t h e r e s t o f t h e U n i t e d Kingdom (Evans, 1976). Farmers a re p a r t i c u l a r l y v u l n e r a b l e . I f c o n t a m i n a t i o n of. cow's m i l k i s r e l e v a n t t h e n t h e m a r g i n a l f a r m i n g and f r e e range p r a . c t i c e s c h a r a c t e r i s t i c o f much Welsh f a r m i n g may be p a r t i c u l a r l y s i g n i f i c a n t . I n J a p a n , xtfhich s h a r e s t o p p l a c e i n t h e w o r l d for. i n c i d e n c e of stomach c a n c e r , immature b r a c k e n f e r n , o r warabi, a r e c o n s i d e r e d a d e l i c a c y when s t e e p e d i n h o t w a t e r and sodium b i c a r b o n a t e and t h e n s e r v e d c o l d . There i s a n a r k e d r e g i o n a l d i s t r i b u t i o n o f t h e s e stomach tumours i n Ja p a n . The same vegetab le i s s e r v e d i n e a s t e r n and western. Canada as " f i d d l e h e a d g r e e n s " . . ORGAN SPECIFICITY C h e m i c a l c a r c i n o g e n s a r e a l m o s t e x c l u s i v e l y s p e c i f i c f o r v a r i o u s o r g a n s . F o r example, 4NQ0 causes p r i m a r i l y l u n g tumours (Kawazoe, et al. , 1969) and DMN l i v e r tumours i n mice (Magee, .1972). D i i n e t h y l h y d r a z i n e i n d u c e s c o l o n i c tumours i n mice ( K a n a g a l i n g a m and B a l i s , 1975) and b r a c k e n f e r n i l e u m and b l a d d e r tumours i n cattle'•;('?amukcu, 1955). There a re s e v e r a l p o s s i b l e mechanisms t o e x p l a i n the o b s e r v e d o r g a n o t r o p y : 1) The d i s t r i b u t i o n o f e n v i r o n m e n t a l l y a v a i l a b l e c a r c i n o g e n may v a r y from t i s s u e , t o t i s s u e . I n t h i s way, t h e overwhelming m a j o r i t y o f tumours caused by t o b a c c o smoke w i l l o c c u r i n t h e l u n g , where smoke a r r i v e s f i r s t . S i m i l a r l y , d i r e c t l y -a c t i n g c a r c i n o g e n s cause s k i n turnouts a t t h e s i t e , o f a p p l i c a t i o n , and i n g e s t e d compounds may cause g a s t r o - i n t e s t i n a l t r a c t tumours b e c a u s e t h i s i s t h e t i s s u e f o r s t encountered, by t h e c a r c i n o g e n , 2) The a c t i v a t i o n o f p r o c a r c i n o g e u i c . compounds may v a r y from t i s s u e t o t i s s u e . C e r t a i n o rgans c o n t a i n h i g h e r l e v e l s o f the. P-450 cytochrome f r a c t i o n t h a t c a r r i e s out t h e o x i d a t i o n r e s p o n s i b l e f o r a c t i v a t i o n o f m u t a g e n i c and c a r c i n o g e n i c p o l y c y c l i c h y d r o c a r b o n s {e.g.., l i v e r , k i d n e y ) ( r e v i e w e d by H e i d e l b e r g e r , 1 9 7 6 ) . These organs may produce h i g h e r l e v e l s of u l t i m a t e c a r c i n o g e n s : , and t h e r e f o r e e x h i b i t g r e a t e r s u s c e p t i b i l i t y t o tumours. 3) The i n a c t i 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 s may v a r y f r o m o r g a n t o o r g a n . I f a c t i v a t e d c a r c i n o g e n s a r e c i r c u l a t e d t h r o u g h o u t t h e body, t h o s e o r g a n s w h i c h may i n a c t i v a t e t h e c h e m i c a l e f f i c i e n t l y may be l e s s s u s c e p t i b l e t o tumour i n d u c t i o n . 4) The r e p a i r o f damage i n d u c e d by c a r c i n o g e n s may v a r y between o r g a n s . I t has been o b s e r v e d t h a t r e p a i r o f damage, caused t o DNA i n one p o r t i o n o f t h e d i g e s t i v e t r a c t i s s l o w e r : t h a n r e p a i r o f damage i n f l i c t e d i n a n o t h e r p a r t (Kanagalingam. and. B a l i s , 1 9 7 5 ) . 5) The p r o l i f e r a t i v e r a t e s o f c e l l s i n d i f f e r e n t o r g a n s may c o n t r i b u t e t o t h e i n i t i a t i o n . o f tumours, s i n c e damage t o c r i t i c a l m a c r o m o l e c u l e s may be " f i x e d " more e a s i l y i n r a p i d l y d i v i d i n g t i s s u e s where t h e r e i s l e s s t i m e . t o r e p a i r damage i n f l i c t e d by exogenous a g e n t s and enough d i v i s i o n s t o e x h i b i t t h e p r o l i f e r a t i o n o f c e l l t y p e s c h a r a c t e r i s t i c , o f t h e 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 i n t h e f i r s t s t a g e o f tumour f o r m a t i o n . F o r example, e t h y l n i t r o s o u r e a i s n o n - c a r c i n o g e n i c t o a d u l t b r a i n b u t i s c a r c i n o g e n i c t o f e t a l b r a i n ( D r u c k r e y , et al. s 1970) and d i m e t h y l n i t r o s a m i n e , g i v e n as a s i n g l e d ose, i s c a r c i n o g e n i c t o p a r t i a l l y h e p a t e c t o m i z e d l i v e r o r l i v e r p r e t r e a t e d w i t h a s i n g l e dose o f c a r b o n t e t r a c h l o r i d e , b u t n o t t o n o r m a l l i v e r ( C r a d d o c k , 1971; Pound, et al., 1 9 7 3 ) . 20 6) G e n e t i c p r e d i l e c t i o n f o r tumours i n c e r t a i n c e l l t y p e s m i g h t cause exogenous f a c t o r s to produce tumours s p e c i f i c a l l y i n t h o s e c e l l t y p e s i n a f f e c t e d i n d i v i d u a l s . . Those, a f f e c t e d b y xeroderma pigmentosum-, F a n c o n i ' s anemia and Bloom's syndrome show h i g h l e v e l s o f tumours o f the s k i n (Poon, et al-., 1974) and ataxia telangsotasia p a t i e n t s have a h i g h l e v e l o f ne r v o u s \ d e t e r i o r a t i o n as w e l l as stomach and o t h e r GI tumours. \ The r o l e s o f these, p r o c e s s e s i n o r g a n - s p e c i f i c i t y o f • tumour i n d u c t i o n 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 (Magee, 1972). A l s o , a t t e m p t s t o r e l a t e s i t e s o f a l k y l a t i o n o f DNA t o o r g a n o t r o p y have mat w i t h f a i l u r e ( L i j i n s k y , et al., 1970). S i n c e tumour s p e c i f i c i t y may n o t be r e l a t e d t o t h e s i t e o f b i n d i n g o f c h e m i c a l s .. to DNA, i t seems r e a s o n a b l e t h a t t h e i m p o r t a n t . i n i t i a t i n g e f f e c t m i g h t be the amount o f m e a s u r a b l e DNA damage and r e p a i r t h a t o c c u r s i n some t i s s u e s and n o t i n o t h e r s . A v a r i e t y o f s h o r t term b i o a s s a y s have been d e v e l o p e d t o a s s e s s t h e h a z a r d t h e s e c h e m i c a l s pose t o humans. In vitro t e s t s i n c l u d e t r a n s f o r m a t i o n o f c e l l c u l t u r e s ( D i p a o l o and N e l s o n , 1973; H e i d e l b e r g e r , 1974; K a t s u t a and T a k o t a , 1 9 7 2 ) , m u t a g e n i c i t y t e s t s ( r e v i e w e d by W e i s b u r g e r , 1975; H o l l a n d e r , 1 9 7 1 ) , p r e s e n c e o f f o e t a l p r o t e i n s o r a n t i g e n s ( K r o e s , et o.i., 1973; Nechaud and U r i e l , 1 9 7 3 ) , t h e m i c r o n u c l e u s t e s t ( H e d d l e , 1 9 7 3 ) , and a u t o r a d i o g r a p h i c a s s a y f o r DNA r e p a i r ( S t i c h and San, 1973; C l e a v e r , 1973). In vivo t e s t i n g has been much more d i f f i c u l t o wing t o t h e c o m p l e x i t y o f t h e system, b u t work has been done t o meeisure the amount o f DNA damage and r e p a i r t h a t o c c u r s i n t a r g e t organs;;-in vivo. H i g h l e v e l s o f DNA damage have been shown t o o c c u r a f t e r the a d - m i n i s t r a t i o n o f c h e m i c a l s known t o be h e p a t o c a r c i n o g e n s (Cox, et al., 1973; Damjanov, et al, 1973; L a i s he s , et al., 1975; Abanob'i;, et al., 1977). I t seems r e a s o n a b l e t o use o b s e r v a t i o n o f DNA damage as a c r i t e r i o n f o r o r g a n -s p e c i f i c i t y o f c a r c i n o g e n a c t i o n I n t h e v a r i o u s t i s s u e s o f m i c e . C o r r e l a t i o n between t h e s i t e s o f i n d u c e d DNA damage and p r e v i o u s l y i n v e s t i g a t e d tumour p r o d u c t i o n c o u l d be i n v e s t i g a t e d t o determine, what t h e l i n k between t h e two i s . 21, MATERIALS. AND METHODS CHEMICALS S u c r o s e , EDTA ( e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ) , sodium c h l o r i d e , sodium h y d r o x i d e and o t h e r common r e a g e n t s were o b t a i n e d f r o m . t h e F i s h e r C h e m i c a l Company, V a n c o u v e r , B.C. 2 , 5 - d i p h e n y l o x a z o l e (PRO) and l , 4 - d i ( 2 ( 5 - p h e n y l o x . a z o y l ) ) -benzene (POPOP) f o r p r e p a r a t i o n of s c i n t i l l a t i o n f l u i d x^ere p u r c h a s e d f r o m K e n t L a b o r a t o r i e s , . V a n c o u v e r , B.C. RADIONUCLIDES -T h y m i d i n e - m e t h y l - 3 H ( s p e c i f i c a c t i v i t y 20 Ci/mmole) was o b t a i n e d f r o m t h e New. E n g l a n d N u c l e a r C o r p o r a t i o n , D o r v a l , P.Q. CHEMICAL CARCINOGENS Dime thy I n . i t r o s a m i n e (DMN) was p u r c h a s e d from.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. 4 - n i t r o q u i n o l i n e l ~ o x i d e (4NQO) , 6 - n i C r o q u . i n o l i n e 1 - o x i d e , and 3-methyl 4-NQ0 were p u r c h a s e d f r o m t h e D a i i c h i P u r e Chemical, Company, Tokyo, J a p a n . N-a i e t h y 1 - N * - n i t r o - N - n i t r o s o g u a n i d i n e was p u r c h a s e d from t h e A l d r . i c h C h e m i c a l Company, M i l w a u k e e , W i s c o n s i n . 2 - a c e t y l -a m i n o f l u o r e n e (2AAF) and N-acetoxy-2AAF were k i n d l y p r o v i d e d b y 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 Cancer R e s e a r c h , M a d i s o n , W i s c o n s i n . NITR0SATI.0N OF METHYLGUANIDINE The n i t r o s a t i o n p r o c e d u r e was a m o d i f i c a t i o n o f t h a t employed by Endo, et dl., 1973. 218 mgm (2 mmole) o f anhydrous m e t h y l g u a n i d i n e h y d r o c h l o r i d e (MG-H01)(Sigma C h e m i c a l Co.) 22 was d i s s o l v e d i n 2 ml o f d i s t i l l e d w a t e r . 414 mgm (6 minole) o f sodium n i t r i t e was d i s s o l v e d i n a n o t h e r 2 m l o f d i s t i l l e d w a t e r . 0.2 ml (0.2 mmole) o f t h e MG-HCl, 0.2 ml (0.6 mmole) o f t h e sodium n i t r i t e , 0.1 ml o f 10 N H C l and 0.5 ml o f . d i s t i l l e d w a t e r were mixed i n a 9.5 X 1.5 cm t e s t t u b e and i n c u b a t e d a t 37° C f o r 1 h.. Than, the pK- o f t h e s o l u t i o n was a d j u s t e d t o 7.0 w i t h 10 M NaOH and the. volume t o 2 ml w i t h . . . d i s t i l l e d w a t e r . T h i s was t h e 0.1 M s t o c k s o l u t i o n o f n i t r o s a t i o n p r o d u c t s o f MG-HCI (Lo and S t l - c h , • 1975) . EXTRACTION Off BRACKEN FERN The upper 10-20 cm. of-young, b r a c k e n f e r n p l a n t s (pteridiurn aquilinum). t h a t had a t t a i n e d a, t o t a l h e i g h t , of. , a p p r o x i m a t e l y 30-40 cm were c o l l e c t e d f r o m the immediate s u r r o u n d i n g s o f t h e James A. Mather b u i l d i n g on t h 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 campus. They were h a r v e s t e d i n m i d - J u n e . The l e a v e s were n o t y e t p r e s e n t and t h e heads o f the. f e r n s were f o l d e d i n t h e c h a r a c t e r i s t i c " f i d d l e h e a d " shape (warabi i n J a p a n e s e ) . No at t e m p t was made t o . d i s t i n g u i s h between t h e s e v e r a l s u b - s p e c i e s o f f e r n . The c o l l e c t e d p l a n t s were e x t r a c t e d i n 4 ways: 1) C o l d w a t e r e x t r a c t i o n i ) The p l a n t s were m i x e d w i t h d i s t i l l e d e t h y l a c e t a t e (0.67 gm o f p l a n t p e r ml e t h y l a c e t a t e ) i n an O s t e . r i z e r b l e n d e r u n t i l a f i n e p uree was formed (30-60 s e c a t top s p e e d ) . The s o l i d phase was a l l o w e d t o s e t t l e f o r s e v e r a l seconds and t h e r e l a t i v e l y c l e a r green, s u p e r n a t a n t was f i l t e r e d t h r o u g h a d o u b l e l a y e r o f c h e e s e c l o t h and t h e n v a c u u m - f i l t e r e d t h r o u g h C e l i t e ( d i a t o m a c e o u s earth,. 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.). i i ) The f i l t e r e d s u p e r n a t a n t was washed w i t h an e q u a l volume o f w a t e r and the w a t e r phase r e t a i n e d . T h i s was t h e w a t e r e x t r a c t o f t h e e t h y l a c e t a t e e x t r a c t ( e x t r a c t no. 1 ) . 23 2) C o l d e t h y l a l c o h o l , e x t r a c t i o n i ) The p l a n t s were e x t r a c t e d i n an O s t e r i z e r b l e n d e r (0.67 gm p l a n t p e r m l o f d i s t i l l e d e t h y l a l c o h o l ) b y t h e same, method as t h a t employed f o r e x t r a c t no. 1. i i ) The s u p e r n a t a n t was f i l t e r e d t h r o u g h c h e e s e c l o t h and C e l i t e . T h i s was t h e c o l d e t h a n o l e x t r a c t ( e x t r a c t no. 2 ) . . 3) Sodium b i c a r b o n a t e / h o t w a t e r e x t r a c t i o n i ) 0.03 gm sodium b i c a r b o n a t e p e r gm f r e s h p l a n t , was s p r i n k l e d o v e r t h e f r e s h b r a c k e n fern , s h o o t s . i i . ) F r e s h l y b o i l e d d i s t i l l e d w a t e r was p o u r e d over t h i s (4 m l p e r gm f r e s h p l a n t ) and . l e t s t a n d f o r .10 min. . . i i i ) The brown sweet s m e l l i n g i n f u s i o n was p o u r e d o f f and r e t a i n e d . T h i s was t h e sodium b i c a r b o n a t e . / h o t w a t e r e x t r a c t ( e x t r a c t no. 4 ) . 4) E t h y l a l c o h o l e x t r a c t i o n o f r e s i d u e f r o m e x t r a c t no. 3 i ) The e x t r a c t e d p l a n t from p r o c e s s no 3 {i.e., t h e p l a n t s t h a t were e x t r a c t e d u s i n g h o t w a t e r and sodium b i c a r b o n a t e ) were washed 4 t i m e s , u s i n g 8 l i t r e s o f f r e s h , c o l d t a p w a t e r e a c h t i m e ( i n V a n c o u v e r t h i s i s " s o f t " w a t e r , w i t h r e l a t i v e l y -few m i n e r a l s o l u t e s ) . A f i f t h a l i q u o t o f 8 l i t r e s was added and l e t s t a n d overnight.. T h i s p r o c e d u r e was dona i n a s t a i n l e s s s t e e l b u c k e t . i i ) The f i f t h wash was p o u r e d o f f and t h e p l a n t s e x t r a c t e d w i t h d i s t i l l e d e t h y l a l c o h o l as i n e x t r a c t i o n p r o c e d u r e no. 2 {i.e., 0.67 gm p l a n t p e r m l d i s t i l l e d e t h y l a l c o h o l i n an O s t e r i z e r b l e n d e r ) . i i i ) The r e s u l t i n g s u p e r n a t a n t , f i l t e r e d t h r o u g h d o u b l e d c h e e s e c l o t h and C e l i t e , was t h e e t h y l a l c o h o l e x t r a c t o f h o t w a t e r / sodium b i c a r b o n a t e e x t r a c t e d f e r n ( e x t r a c t no. 4) The w a t e r - d i s s o l v e d p r o d u c t s o f e x t r a c t i o n p r o c e d u r e s 1 t o 4 were d i v i d e d i n t o a l i q u o t s o f a maximum of 250 m l , f r o z e n i n l i q u i d n i t r o g e n and l y o p h i l i z e d under l o w p r e s s u r e u n t i l d r y n e s s ( t h i s t o o k , a t most, 2 d a y s ) . The r e s u l t i n g t a r r y powders were g r e e n i s h - b l a c k and s o l u b l e i n w a t e r . EXPERIMENTAL ANIMALS . O u t b r e d , 2 month o l d , male S w i s s m i c e were o b t a i n e d . f r o m t h e A n i m a l 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 ( 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 ) . They were 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 on a d i e t o f standard P u r i n a Lab Chow and w a t e r ad li-bi-ttim, and were s u b j e c t e d t o a . 12 h l i g h t c y c l e . HUMAN CELL CULTURES " . S k i n punch b i o p s i e s were t a k e n .from t h e forearm, o f a 22 y e a r o l d C a u c a s i a n f e m a l e . The s k i n p i e c e was t e a s e d i n t o m i n u t e f r a g m e n t s w i t h s y r i n g e n e e d l e s and the p i e c e s sandwiched between g l a s s c o v e r s i i p s and i n c u b a t e d i n MEM . ( m i n i m a l e s s e n t i a l medium w i t h 12-20% f e t a l c a l f serum) f o r 2 t o 3 weeks a t 37° i n a CG2 i n c u b a t o r . Growth 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 began t o m i g r a t e f r o m t h e t i s s u e f r a g m e n t s t h e c o v e r s l i p s were opened and g r o s s t i s s u e f r a g m e n t s 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 s . These were i n c u b a t e d as above u n t i l t he f i b r o b l a s t s became, a m o n o l a y e r , a t w h i c h p o i n t t h e c e l l 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 . C u l t u r e s were m a i n t a i n e d , i n a p l a t e a u phase, a t 37° C i n a CO^ i n c u b a t o r i n p l a s t i c P e t r i d i s h e s . T r a n s f e r p a s s a g e s 3 t o 6 were used f o r a l l e x p e r i m e n t s . The c u l t u r e s were r o u t i n e l m a i n t a i n e d i n E a g l e ' s MEM, supplemented w i t h 15% f e t a l c a l f serum and a n t i b i o t i c s (200 u n i t s p e n i c i l l i n / m l , 40 microgm s t r e p t o m y c i n / m l ) . 25 In o r d e r t o o b t a i n c e l l 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 s e d i m e n t a t i o n a n a l y s i s , a p p r o x i m a t e l y 60,000 c e l l s were seeded i n 5 cm 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 ) and a l l o w e d t o grow t o confluenc.y (7-9 d a y s ) . The c e l l s were u s e d i m m e d i a t e l y . ADMINISTRATION OF CHEMICALS The c a r c i n o g e n i c and n o n - c a r c i n o g e n i c . c o m p o u n d s were f o r c e - f e d b y e s o p h a g e a l i n t u b a t i o n (under e t h e r a n a e s t h e s i a ) . i n a v e h i c l e o f H^OtDMSO (dimethy.lsulphoxlde.) (1:1) i n a t o t a l volume o f 0.1 ml. w i t h a 1 ml t u b e r c u l i n s y r i n g e . A f t e r 4 h 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 and e x s a n g u i n a t i o n . and t i s s u e samples t a k e n . F o r t i s s u e c u l t u r e s , c h e m i c a l s were d i s s o l v e d i n MEM ( 2 . 5 % f e t a l c a l f serum) and added i n a volume of 5 ml t o t h e c u l t u r e d f i b r o b l a s t s . They were l e f t f o r from 30 min t o 2 h, a t w h i c h t i m e t h e media was removed and t h e c e l l s used. ALKALINE .SUCROSE GRADIENT ANALYSIS OF DNA DAMAGE a ) L a b e l l i n g o f g a s t r i c e p i t h e l i u m and l i v e r t i s s u e DNA: Young S w i s s m i c e were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 5 X 10 C i (0.05 ml) of 3H-TdR ( s p e c i f i c a c t i v i t y , 20 C i / mmole) to l a b e l t h e DNA of e p i t h e l i a l c e l l s o f t h e esophagus, stomach and l i v e r . A f t e r 20 h t h e m i c e were i n j e c t e d i n t rape r i tone-al l y w i t h 0.5 m l s t e r i l i s e d 0.9% N a C l t o cause them t o e x c r e t e as- ' much f r e e % l a b e l as p o s s i b l e f r o m t h e t i s s u e s . The c a r c i n o g e n s and n o n - c a r c i n o g e n s were a p p l i e d 30 h a f t e r t he i n j e c t i o n o f 3H-TdR. b) L a b e l l i n g o f c u l t u r e d human f i b r o b l a s t DNA: When 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 grown i n 5 cm 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 were 80 t o 90% f u l l , medium was removed, and 5 ml o f MEM (15% f e t a l , c a l f serum) c o n t a i n i n g 2 m i c r o C i / m l (2 m i c r o l i t e r s o f 20 C i / mmole s p e c i f i c a c t i v i t y 3H-TdR p e r ml o f MEM) was added. When the p l a t e s were 26 c o n f l u e n t (24 t o 48 h l a t e r ) t h e l a b e l l e d medium was remove, t h e c e l l s were r i n s e d 3 t i m e s w i t h s t e r i l e , 37° c EDTA / s a l i n e b u f f e r , and s u b j e c t e d immediate ly t o t h e c h e m i c a l s o f i n t e r e s t . c ) P r e p a r a t i o n o f l i v e r and gas t r o - l n t e s t i n a l e p i t h f t l i a l t i s s u e : The method of Cox, et al.} (1973) was f o l l o w e d i n a l l . o f t h e e s s e n t i a l s . 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 and d e c a p i t a t i o n t o a l l o w e x s a n g u i n a t i o n . The l i v e r was removed, 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 . EDTA / s a l i n e b u f f e r (0.024 M EDTA / 0.075 M N a C l , pH 7.4). The l i v e r was dabbed on t i s s u e t o d r y and p l a c e d i n a P e t r i d i s h k e p t on c r u s h e d i c e . 1 m l ice-'Cold EDTA / s a l i n e b u f f e r was added f o r e v e r y gram o f l i v e r (wet w e i g h t ) and the. l i v e r squashed w i t h a s p a t u l a (3 t o 5 m i n ) . The homogenate, w i t h o u t l a r g e p i e c e s , 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 1000 X g f o r 30 s e c i n p r e - c o o l e d c e n t r i f u g e t u b e s . A d i l u t e d . a l i q u o t o f s u p e r n a t a n t was us e d t o e s t i m a t e t he c e l l c o n c e n t r a t i o n 5 6 i n a hemacytometer chamber. 5 X 10 t o 1 X 10 were r o u t i n e l y u s e d i n e a c h a l k a l i n e s u c r o s e g r a d i e n t (ASG) s e d i m e n t a t i o n t u b e . P i e c e s of esophagus, c a r d i a c an.d p y l o r i c stomach, duodenum, and i n t e s t i n e ( i n c l u d i n g s u r f a c e e p i t h e l i u m , t h e musoulax'is mucosae and submucosal l a y e r ) were removed and r i n s e d q u i c k l y i n i c e - c o l d EDTA / s a l i n e b u f f e r t o r e m o v e . f e c a l or g a s t r i c c o n t e n t s and dabbed d r y . A c o l d , new r a z o r b l a d e was u s e d t o remove a 1.0 t o 1.5 mgm p i e c e of t i s s u e ( s l i c e d p e r p e n d i c u l a r to t h e d i r e c t i o n of t h e s h e e t of t i s s u e ) . 10 m i c r o 1 of i c e c o l d b u f f e r was added and t h e r a z o r b l a d e was us e d t o mince the. p i e c e by c h o p p i n g up and down 100 to'ISO t i m e s w h i l e t u r n i n g t he P e t r i d i s h w i t h t h e o t h e r hand. T i s s u e p i e c e s s h o u l d be a b l e to p a s s t h r o u g h t h e b o r e of a 10 raicrol C o r n i n g d i s p o s a b l e m i c r o - s a m p l i n g p i p e t . The t i s s u e was t h e n ready f o r l a y e r i n g . 27 d) P r e p a r a t i o n _of c u l t u r e d human f i b r o b l a s t s : F i b r o b l a s t s were exposed t o c h e m i c a l s f o r fr o m one-h a l f t o two h o u r s , a t w h i c h t i m e t h e c h e m i c a l was removed and the c e l l s washed .3 t i m e s w i t h i c e - c o l d EDTA / s a l i n e b u f f e r . 0.5 m l of c o l d EDTA / s a l i n e b u f f e r was added and. the c e l l s were s c r u b b e d away f r o m the d i s h , w i t h a r u b b e r p o l i c e m a n . The 0.5 ml c e l l s u s p e n s i o n was p l a c e d i n a 3 ml c e n t r i f u g e tube and spun a t 2600 r.p.m- i n a c l i n i c a l c e n t r i f u g e f o r 5 min,. The c e l l - f r e e , s u p e r n a t a n t was removed and d i s c a r d e d . 100 m i c r o 1 c o l d EDTA / s a l i n e b u f f e r was added and t h e c e l l s , were k e p t on an i c e - b e d i n p r e p a r a t i o n , for. l a y e r i n g , on g r a d i e n t s . 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 , s e d i m e n t a t i o n : G r a d i e n t s were p r e p a r e d an hou r b e f o r e use-, a c c o r d i n g t o t h e method o f Cox, et at. 3 1973. I n t o 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 (Beckman I n s t r u m e n t Co., Van c o u v e r , B.C.) was l a i d , i n s u c c e s s i o n : 1 m l o f 2.3 M s u c r o s e ; 5-20% a l k a l i n e s u c r o s e g r a d i e n t (0.9 M NaCl,. 0.3 M NaOH); 0.3 ml l y s i n g s o l u t i o n (0.3 M N a C l , 0.03 M EDTA, 0-1 M t r - i s - H C l , 0.5% sodium d o d e c y l s u l p h a t e ( S D S ) ) ; 5 X 1 0 5 - I X 1 0 6 c e l l s o r i n t a c t c e l l n u c l e i i n a volume n o t e x c e e d i n g 50 m i c r o ! ; 0.3 m l l y s i n g s o l u t i o n ; i s o - o c t a n e i t o w i t h i n 0.5 cm of t h e top o f t h e t u b a . G r a d i e n t s were p l a c e d i n the b u c k e t s o f a Beckman 5W40 u l t r a c e n t r i f u g e . r o t o r and spun a t 77,561 X g a t an av e r a g e r a d i u s o f 11.10 cm (25,000 r..p'..mi.) f o r 30 min a t 20° C w i t h the. brake o f f i n a Beckman L2B u l t r a c e n t r i f u g e . F i f t e e n s e q u e n t i a l f r a c t i o n s were t a k e n f r o m t h e bottoms o f t h e 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 (TCA) and. c o l l e c t e d on 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 w a s h i n g the. f i l t e r s w i t h 8-10% TCA and e t h a n o ! . A c i d i n s o l u b l e r a d i o a c t i v i t y was counted, by i m m e r s i n g t h e d r i e d f i l t e r s i n t o l u e n e s c i n t i l l a t i o n f l u i d (3 1 t o l u e n e , 12 gm PPO, 0.3 gm 28 POPOP) and c o u n t i n g f o r 10 min p e r v i a l on the N u c l e a r Chicago Mark 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 . f) 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 SH l a b e l i n c e l l u l a r DNA: 1) M i c e were i n j e c t e d w i t h 50 m i c r o C i % - T d R sub-c u t a n e o u s l y , as d e s c r i b e d above. A f t e r 30 h t h e a n i m a l s were s a c r i f i c e d and samples o f l i v e r , esophagus, c a r d i a c and p y l o r i c , stomach, duodenum,, c o l o n , j e j e u n u m , u r i n a r y b l a d d e r and i l e u m were e x c i s e d , washed i n . i c e - c o l d EDTA / s a l i n e b u f f e r , and d e h y d r a t e d , c l e a r e d , embedded, s e c t i o n e d , and s t a i n e d ( h e m a t o x y l i n / e o s i n ) by s t a n d a r d t e c h n i q u e s . 2) S l i d e s w i t h t i s s u e s l i c e s were d i p p e d i n Kodak NTB-3 e m u l s i o n ( d i l u t e d 1:1 w i t h d i s t i l l e d w a t e r ) , a l l o w e d t o a i r d r y f o r 30 m i n , s t o r e d a t 4° C i n l i g h t - t i g h t boxes f o r 14 d a y s , and d e v e l o p e d by s t a n d a r d p h o t o g r a p h i c p r o c e d u r e s . 29 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 l c a r c i n o g e n s . T h i s has been done by o b s e r v a t i o n 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 by a u t o r a d i o g r a p h y o f the- c e l l s ( r e v i e w e d b y S t i c h , et a t . , 1977) . U n f o r t u n a t e l y , t h i s , r e p a i r mechanism 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 r a t h e r t h a n a s s a y i n g . f o r t h e damaged r e g i o n s t h e m s e l v e s . W i t h t h i s i n m i n d , i t was a t t e m p t e d t o show DNA r e p a i r by means o f a decrease i n t h e number o f s i n g l e - s t r a n d b r e a k s o v e r a p e r i o d o f t i m e . The 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 s e d i m e n t a t i o n has t h e a d v a n t a g e o f b e i n g 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, pr e s u m a b l y b e c a u s e of 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 . 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 i n v o l v e s 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 c o n t i n u o u s l y i n c r e a s i n g 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 . IN VITRO When human f i b r o b l a s t s , were, r u n on the g r a d i e n t t h e s e d i m e n t a t i o n p r o f i l e i l l u s t r a t e d i n F i g . 2 was o b s e r v e d . When t h e c e l l s were, i n c u b a t e d f o r o n e - h a l f h w i t h MNNG — 6 (5 X 10 M) i n 2.5% MEM, t h e s e d i m e n t a t i o n p r o f i l e s h i f t e d f r o m t h e c o n t r o l l e v e l t o t h a t i l l u s t r a t e d i n F i g . 3 . T h i s d e m o n s t r a t e s a d e c r e a s e i n m o l e c u l a r w e i g h t s i m i l a r t o a d e c r e a s e o b s e r v e d p r e v i o u s l y w i t h a v a r i e t y o f c a r c i n o g e n s (4NQ0, N - a c e t o x y 2AAF, n i t r o s a t i o n p r o d u c t s ) . However, when, t h e MNNG was removed a f t e r o n e - h a l f hour and t h e c e l l s m a i n t a i n e d w i t h 10% MEM a t 37° C f o r up t o 30 h, t h e r e s u l t s shown i n F i g . 4 were, o b s e r v e d . DNA sedimented n e a r t h e t o p of th e g r a d i e n t u n t i l a p p r o x i m a t e l y 16 rto 18 h. f o l l o w i n g c a r c i n o g e n t r e a t m e n t . Then, f r o m 18 t o 30 h t h e DNA F i g u r e 2 DNA s e d i m e n t a t i o n , p r o f i l e , o f c u l t u r e d human f i b r o b l a s t s c e n t r i f u g e d . t h r o u g h a 5-20% a l k a l i n e s u c r o s e g r a d i e n t . No c a r -c i n o g e n s were a p p l i e d . F i g u r e 3 DNA s e d i m e n t a t i o n p r o f i l e o f c u l t u r e d human f i b r o b l a s t s -6 p r e t r e a t e d f o r 30 min w i t h 5 X 10 M MNNG i n 2.5% MEM. 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 r i m a r y s e d i m e n t a t i o n , peak o f DNA from c o n t r o l c e l l s . % OF T O T A L 3 H C O U N T S F i g u r e 4 DNA s e d i m e n t a t i o n p r o f i l e s o f c u l t u r e d human f i b r o b l a s t s —ft t r e a t e d f o r 30 min w i t h 5 X 10 M MNNG i n 2.5% MEM and t h e n m a i n t a i n e d i n 10% MEM f o r : (A) 0 h r , (B) 12 h r , (C) 18 hr,. (D) 30 h r The. h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peaks o DNA f r o m c o n t r o l c e l l s . Lo LO g r a d u a l l y began t o sediment a t c l o s e t o c o n t r o l l e v e l s , a l t h o u g h the a p p a r e n t m o l e c u l a r w e i g h , n e v e r q u i t e reached t h a t o f n o r m a l u n t r e a t e d c e l l s o v e r t h e t i m e p e r i o d chosen. IN VIVO INTO DNA When a u t o r a d i o g r a p h e d s l i c e s o f g a s t r i c , e s o p h a g e a l , d u o d e n a l , u r i n a r y b l a d d e r , and c o l o n i c t i s s u e t h a t had been p u l s e - l a b e l l e d w i t h 50 m i c r o C i 3H-TdR i n vivo, I t was o b s e r v e d t h a t t h e c e l l s i n c o r p o r a t i n g l a b e l were t h e c e l l s o f t h e o v e r l y i n g e p i t h e l i a l l a y e r o f t h o s e t i s s u e s . O t h e r c e l l t y p e s i n t h e g a s t r i c t r a c t appear t o i n c o r p o r a t e l a b e l a t a much l o w e r r a t e t h a n t h e . . f a s t - p r o l i f e r a t i n g e p i t h e l i a l c e l l s . T r i t i u m c o u n t s r e c o v e r e d f r o m g r o s s 1 t o 2 mgm samples o f esophagus 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 were t y p i c a l l y o f t h e o r d e r o f 50 t o 100 d i s i n t e g r a t i o n s p e r m i n u t e (dpm) a f t e r s u b t r a c t i o n o f b a c k g r o u n d c o u n t s , w h i l e s i m i l a r l y t r e a t e d 1 t o 2 mgm samples o f o t h e r g a s t r i c t r a c t t i s s u e s y i e l d e d 150 t o 200 dpm a f t e r s u b t r a c t i o n o f b a c k g r o u n d c o u n t s . CONTROL SEDIMENTATION PROFILES When 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 was a p p l i e d t o esophagus, stomach and c o l o n i c t i s s u e s , s e d i m e n t a t i o n p r o f i l e s s u c h as t h o s e i n F i g . 5. were o b s e r v e d . G a s t r i c t r a c t c e l l s o f a n i m a l s f o r c e - f e d t h e H 0:DMS0 (1:1) v e h i c l e . ( 0 . 1 m l p e r mouse) c o n s i s t e n t l y gave s e d i m e n t a t i o n p r o f i l e s taken, t o i n d i c a t e no DNA damage ( F i g . 5 ) . On a l l r u n s , whether t r e a t e d o r u n t r e a t e d w i t h c h e m i c a l c a r c i n o g e n s , t h e g r a d i e n t s e x h i b i t e d a s l i g h t amount o f t i s s u e d e b r i s j u s t above t h e 2.3 M c u s h i o n a f t e r s e d i m e n t a t i o n . I t m i g h t be a r g u e d t h a t t h i s d e t r i t u s t e n d s t o sed i m e n t damaged DNA t o g i v e f a l s e c o n t r o l p e a k s , b u t t h e r e was l i t t l e o r no b i n d i n g o f 3I-I-TdR l a b e l t o h e a v i l y s e d i m e n t i n g 35 Figure. 5 DNA s e d i m e n t a t i o n p r o f i l e s o f .Swiss mease g a s t r i c t r a c t e p i t h e l i a l c e l l s de-rived from.*' . -{VV) • cardiac•-stxjfflasri-r, • (B) .py±crrxcr•' stomach.-. (C) esoph a g u s , (D) duciien.ura, (H) • de s c e n d i n g c o l o a * and (F) u r i n a r y b l a d d e r . • No c a r c i n o g e n s were -.applied t o the. ©ice-: % OF TOTAL C O U N T S 37 S E D I M E N T A T I O N 33 f r a g m e n t s o f mouse l u n g ( L a i s b . e s , et al., 1975). TESTING OF CARCINOGENIC AND NDN-CABCINOCBNIC COMPOUNDS The DNA-damaging c a p a c i t y o f h i g h l y o n c o g e n i c and non-o n c o g e n i c 4NQO d e r i v a t i v e s was e s t i m a t e d by f o r c e - f e e d i n g r a t s w i t h v a r i o u s n i t r o q u i u o l i n e d e r i v a t i v e s 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 and comparing 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 f o l l o w i n g c e n t r i f u g a t i o u 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 . . A p p l i c a t i o n o f t h e s t r o n g l y c a r c i n o g e n i c compounds. 4NQ0 (1 X 1 0 ~ 4 gm / gm body w e i g h t ) and 6-methyl 4NQ0 (1 X -4 10 gm. / gm body w e i g h t ) s h i f t s 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 DNA r e l e a s e d b y c a r d i a c a n d . p y l o r i c stomach t i s s u e and esophagus t o t h e r i g h t , i n d i c a t i n g s e v e r e DNA f r a g m e n t a t i o n i n the squamous e p i t h e l i a l c e l l s ( F i g . 6 ) . . The weakly, c a r c i n o g e n i c 3 - i n e t h y l 4NQ0, 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 , e l i c i t e d v e r y l i t t l e , damage ( F i g . 7). A p p l i c a t i o n o f t h e n o n - c a r c i n o g e n i c i s o m e r 6NQ0 (2 X 10 ^ gm / gm body w e i g h t ) y i e l d e d a s e d i m e n t a t i o n p r o f i l e ( F i g 8) s i m i l a r t o t h a t o f c o n t r o l s , as d i d e p i t h e l i u m o f esophagus and p y l o r i c stomach ( F i g . 9). DNA REPAIR M i c e f o r c e - f e d t h e c a r c i n o g e n . MNNG (.35 micrograms p e r gram mouse) and a s s a y e d f o r DNA damage/in cabd iac stomach e p i t h e l i a l c e i l s f r o m 4 t o 30 h f o l l o w i n g showed a 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 f r o m f r a g m e n t e d t o c l o s e t o c o n t r o l l e v e l s ( F i g . 9A), a l t h o u g h c o n t r o l l e v e l were n o t r e a c h e d i n t h e p e r i o d o f t i m e o f i n v e s t i g a t i o n . PRECARCIN0GENS AND ULTIMATE CARCINOGENS • M i c e ' f o r c e - f e d t h e p r e c a r c l n o g e n 2AAF showed no s i g n i f i c a n t change 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 DNA r e l e a s e d f r o m t h e c a r d i a c o r p y l o r i c p a r t o f t h e stomach ( F i g , 1 0 ) . However, mice t r e a t e d w i t h t h e u l t i m a t e c a r c i n o g e n N-acetoxy 2AAF i n c o n c e n t r a t i o n s e q u i m o l a r t o t h a t o f 2AAF {v.e. , 1.1 X 10 gm./ gm body w e i g h t ) showed 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 i n d i c a t e d DNA damage ( F i g . 1 1 ) . 39 F i g . 10 DNA sedimentation p r o f i l e s of cardiac stomach e p i t h e l i a l c e l l s from Swiss mice force^fed. 35 micrograms MNNG per gram of mouse, and assayed at A)'. 4 h, B) 12 h, C) 18 h, and D) 30 h following administration. The h o r i z o n t a l bars i n d i c a t e the primary sedimentation peak of DNA from control c e l l s , % OF TOTAL 3 H COUNTS Figure 6 DNA sedimentation p r o f i l e s of cardiac stomach e p i t h e l i a l c e l l s from, mice force-fed 1 X 10 1 gm/gm body weight of: (A) 4NQ0, or (B) 6-methyl 4NQ0. Mice were s a c r i f i c e d 4 hr after administration. The horizontal bars indicate the primary sedimentation peaks of DNA from control c e l l s . Figure 7 DNA sedimentation p r o f i l e of cardiac stomach e p i t h e l i a l -4 c e l l s from mice force-fed 1 X 10 gm/gm body weight of 3-methyl 4NQ0. Mice were s a c r i f i c e d 4 hr a f t e r administration. The h o r i z o n t a l bars indicate, the primary sedimentation peaks of DNA from co n t r o l c e l l s . % O F T O T A L C O U N T S Figure 8 DNA sedimentation p r o f i l e of cardiac stomach e p i t h e l i a l -4 c e l l s derived from Swiss mice force-fed 2 X 10 gm/gm body weight of 6NQ0. Mice, were s a c r i f i c e d 4 hr after administration* the horizontal bar indicates the primary sedimentation peak of DNA from control c e l l s . Figure 9 ^ DNA sedimentation p r o f i l e of p y l o r i c stomach e p i t h e l i a l -4 c e l l s derived from Swiss mice force-fed 2 X 10 gm/gm body weight of 6NQ0. Mice were s a c r i f i c e d 4 hr after administration. The horizontal bar indicates the primary sedimentation peak of DNA from control c e l l s . % O F T O T A L 3 H C O U N T S o ro o w o o o F i g u r e 10 DNA s e d i m e n t a t i o n p r o f i l e s o f c e l l s f rom S w i s s m i c e -4 f o r c e - f e d 8 X. 10 gm/gm body w e i g h t o f 2AAF. (A) c a r d i a c stomach e p i t h e l i u m , (B) p y l o r i c stomach e p i t h e l i u m , and. (C) l i v e r . The mice were s a c r i f i c e d 4 h r a f t e r a d m i n i s t r a t i o n . The h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peak of DN o f t i s s u e s from c o n t r o l m i c e . % O F T O T A L ° H C O U N T S F i g u r e 11 DNA s e d i m e n t a t i o n p r o f i l e s o f c e l l s of S w i s s m i c e f o r c e -f e d 1.01 X 10 ~* gm/gm body w e i g h t of N - a c e t o x y 2AAF. (A) c a r d i a c stomach e p i t h e l i u m , and (B) l i v e r . M i c e were s a c r/if i c e d 4 h r a f t e r a d m i n i s t r a t i o n . The h o r i z o n t a l b a r s i n d i c a t e , t h e p r i m a r y s e d i m e n t a t i o n peaks of DNA o f t i s s u e s from c o n t r o l m i c e . 48 30 i i — i 20 H o o 1 0 A \ A A 0 — —1 1 1 0 15 -J, H 301 5— Li. o-P 20 0s" 10 0 / 0 T 10 S E D I M E N T A T I O N B \ \ \ 9 \ / —» 15 DIFFERENTIA!, EFFECT .ON GASTRIC EPITHELIAL; CELLS AND LIVER CELLS I t was found that application of the 3H-TdR aliquot s u f f i c i e n t to l a b e l gastric e p i t h e l i a l c e l l s of 2 month old Swiss mice (50 microCi) also l a b e l l e d some l i v e r c e l l s i n the same. 30 h period. These mice were not p a r t i a l l y hepatectomized as indicated i n the method of Cox, et at., 1975. The t o t a l o 5 recoverable, acid-precipitable 3H-labelled moiety from 10 l i v e r c e l l s (counted with a hemacytometer) was 60 c.p.m. over background, as compared to 150 to 200 c.p.m. over background for similar g a s t r i c tract tissue. The simultaneous l a b e l l i n g of the DNA. of l i v e r and gastric tract e p i t h e l i a l c e l l s permitted investigation of the action of precarcinogens and carcinogens of two tissues which d i f f e r i n t h e i r s u s c e p t i b i l i t y to DNA fragmentation by the same carcinogen. An organ-specific effect becomes apparent.if the action of the p r e c a r c i n o g e n 2AAF . i s compared with, that, of the ultimate carcinogen N-acetoxy 2AAF.(Fig. 10 and 11). Both compounds induce DNA fragmentation i n the l i v e r but only the d i r e c t l y active N-acetoxy 2AAF appears to damage the e p i t h e l i a l c e l l s of stomach or esophagus. Similar r e s u l t s were obtained with the precarcinogen DMN and the a l k y l a t i n g carcinogen MNNG. Application of DMN (1.5 X 10 ~* gm / mg body weight) by esophageal intubation resulted i n sedimentation p r o f i l e s s i m i l a r to controls for c e l l s of the cardiac stomach, but samples of l i v e r from the same mouse gave, sedimentation p r o f i l e s i n d i c a t i n g DNA damage (Fig. 12). The d i r e c t l y active. MNNG produces DNA fragmentation i n the stomach as w e l l as i n the l i v e r (Fig. 13). NITROSATED COMPOUNDS Gastric c e l l s of mice force-fed the mutagenic and carcinogenic products of methyl guanidine nitrosated by reaction . in a c i d i c solution showed DNA fragmentation of cardiac stomach 50 F i g u r e 12 DNA s e d i m e n t a t i o n p r o f i l e s o f c e l l s f r o m Swiss mice f o r c e -f e d 1.5 X 10 ^ gm.gm body w e i g h t DMN. (A) c a r d i a c stomach e p i t h e l i u m , and (B) l i v e r . M i c e were s a c r i f i c e d 4 h r a f t e r a d m i n i -s t r a t i o n . The h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peaks o f DNA of t i s s u e s from c o n t r o l m i c e . F i g u r e 13 DNA s e d i m e n t a t i o n p r o f i l e s o f c e l l s f rom S w i s s m i c e f o r c e - f e d 3.5 X 10 ^ gm/gm body w e i g h t MNNG. (A) c a r d i a c stomach e p i t h e l i u m , and (B) l i v e r . M i c e were s a c r i f i c e d 4 h r a f t e r a d m i n i s t r a t i o n . The h o r i z o n t a l bars'; i n d i c a t e t h e p r i m a r y s e d i -m e n t a t i o n peaks of DNA o f t i s s u e s from c o n t r o l m i c e . S E D I M E N T A T I O N F i g u r e 14 DNA s e d i m e n t a t i f f l a - ^ r o f i l e a o f c a r d i a c stomach e p i t h e l i a l c e l l s o f S w i s s m i c a f o r c e . - f e d (A) 2.6 X 10 ^ gm/gra body w e i g h t o f MG-HC1, o r (B) t h e n i f e r o s a t i o n produc.t o f 4.4 X 10 " gm/gm body xveight o f MG-HC1. M i c e were s a c r i f i c e d 4 h r a f t e r a d m i n i s t r a -t i o n . The h o r i z o n t a l b a r s i n d i c a t e the p r i m a r y s e d i m e n t a t i o n peaks o f DNA of t i s s u e s , f r o m c o n t r o l m i c e . / I I F i g u r e 15 j DNA s e d i m e n t a t i o n p r o f i l e s o f c e l l s o f S w i s s m i c e f o r c e -f e d t h e n i t ro s a t ion p r o d u c t o f 4.4 X 10 gm/gm body w e i g h t o f MG-HC1. (A) p y l o r i c stomach e p i t h e l i u m , and (B) e s o p h a g e a l e p i t h e -l i u m . M i c e were s a c r i f i c e d 4 h r a f t e r a d m i n i s t r a t i o n . The h o r i z b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peaks o f DNA of t i s s u e s f r o m c o n t r o l m i c e . on Ul to c e l l s 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 method ( F i g . 1 4 ) . The n o n - n i t r o s a t e d p r e c u r s o r , M G-HC1showed no d e t e c t a b l e a b i l i t y t o change s e d i m e n t a t i o n v e l o c i t y o f DNA r e l e a s e d f r o m c a r c i a c stomach c e l l s . The MG-HC1 was t r e a t e d w i t h a c i d and n e u t r a l i z e d i n t h e manner d e s c r i b e d by Lo and S t i c h ( 1 9 7 5 ) . S i m i l a r r e s u l t s w e r e . o b t a i n e d f r o m p y l o r i c stomach and esophagus removed f r o m t h e same mice ( F i g . 1 5 ) . COMPLEX MIXTUTd'S W h i l e t h e s c i e n t i s t l i k e s t o t e s t compounds i n as p u r e a c o n d i t i o n as he can o b t a i n them, most c a r c i n o g e n s , t h a t . a p p e a r i n man's e n v i r o n m e n t a r e i n complex m i x t u r e s and he i s exposed t o them i n t h a t form. As•an example of t h i s p a t t e r n , p l a n t c a r c i n o g e n s a r e a g e n e r a l example, and a s p e c i f i c one i s t h e o r g a n o t r o p i c c a r c i n o g e n i c p l a n t b r a c k e n f e r n (pteridium aquilinurn) . The e x t r a c t s o f t h i s p l a n t were d i s s o l v e d i n MEM and a p p l i e d - t o c u l t u r e d human f i b r o b l a s t s f o r a p e r i o d o f 30 min ( F i g . 16) o r d i s s o l v e d i n 0.9% N a C l i n d i s t i l l e d w a t e r and f o r c e - f e d t o % - l a b e l l e d m i c e by e s o p h a g e a l i n t u b a t i o n under e t h e r • a n e s t h e s i a ( F i g . 1 7 ) . A f t e r 30 m i n f o r c u l t u r e d f i b r o b l a s t s o r 4 h f o r mouse t i s s u e s samples were t a k e n and a n a l y z e d f o r DNA f r a g m e n t a t i o n . . 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 i n d i c a t e t h a t e x t r a c t s ! and 2 have a DNA-damaging c a p a c i t y , and t h a t b r a c k e n f e r n . s h o o t s t r e a t e d w i t h b o i l i n g w a t e r and sodium b i c a r b o n a t e l o s e a g r e a t d e a l o f t h e i r d e t e c t a b l e D N A -fragmenting a b i l i t y . When g a s t r o i n t e s t i n a l samples o f mic e f o r c e - f e d e x t r a c t n o . l (4 mgm p e r gm mouse) were t a k e n 8 h a f t e r i n t u b a t i o n , DNA f r a g m e n t a t i o n appeared-.to be most pronounced i n c a r d i a c stomach, l o w e r i l e u m and u r i n a r y b l a d d e r ( F i g . 1 8 ) , c o r r e l a t i n g w e l l w i t h o b s e r v a t i o n s o f tumour s p e c i f i c i t y i n c a t t l e and r a t s ( H i r o n o , et al.3 1970). F i g u r e 16 DNA s e d i m e n t a t i o n p r o f i l e s o f c u l t u r e d human f i b r o b l a s t s t r e a t e d f o r 30 m i n w i t h (A) 30 mgm/ral o f b r a c k e n f e r n e x t r a c t no. 1 ( c o l d w a t e r e x t r a c t ) , o r (B) 30 mgm/ral. of. e x t r a c t no. 3 (sodium b i c a r b o n a t e / h c t w a t e r e x t r a c t ) } , o r (C) 30 mgm/ml o f e x t r a c t no. 2 ( c o l d e t h y l a l c o h o l e x t r a c t ) , o r (D) 30 mgm/ml o f e x t r a c t no. 4 ( e t h y l a l c o h o l e x t r a c t of sodium b i c a r b o n a t e / h o t w a t e r e x t r a c t e d p l a n t ) . A l l e x t r a c t s were d i s s o l v e d i n 2.5% MEM. The h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peaks o f DNA of t i s s u e s f r o m c o n t r o l m i c e . . % O F T O T A L 3 H C O U N T S F i g u r e 17 DNA s e d i m e n t a t i o n p r o f i l e s o f c a r d i a c stomach e p i t h e l i u m o f S w i s s m i c e f o r c e - f e d (A) 4 mgm/gm mouse o f b r a c k e n f e r n e x t r a c t n o . l ( c o l d w a t e r e x t r a c t ) , o r (B) 4 mgm/gm mouse of e x t r a c t no. 3 (sodium b i c a r b o n a t e / h o t w a t e r e x t r a c t o f b r a c k e n f e r n ) , o r (C) 4 mgm/gm mouse o f e x t r a c t no. 2 ( c o l d e t h y l a l c o h o l e x t r a c t ) , o r (D) 4 mgm/gm mouse o f e x t r a c t no. 4 ( e t h y l a l c o h o l e x t r a c t o f sodium b i c a r b o n a t e / h o t w a t e r e x t r a c t e d p l a n t ) . M i c e were s a c r i f i c e d 4 h r a f t e r a d m i n i s t r a t i o n . The h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n peaks o f DNA o f t i s s u e s from c o n t r o l m i c e . % O F T O T A L H C O U N T S F i g u r e 18 DNA s e d i m e n t a t i o n p r o f i l e s o f g a s t r o i n t e s t i n a l c e l l s f r o m S w i s s mice f o r c e - f e d 4 mgm/ml of l y o p h i l i z e d c o l d e t h a n o l e x t r a c t o f b r a c k e n f e r n d i s s o l v e d i n 0.5 ml of d i s t i l l e d w a t e r D u p l i c a t e r u n s a r e i l l u s t r a t e d f o r each t i s s u e . Mice were s a c r i f i c e d . 8 h r a f t e r a d m i n i s t r a t i o n . The h o r i z o n t a l b a r s i n d i c a t e t h e p r i m a r y s e d i m e n t a t i o n r e g i o n o f DNA o f t i s s u e s f r o m c o n t r o l m i c e . S E D I M E N T A T I O N 61 DISCUSSION I n t h i s s t u d y t h e p r a c t i c a l i t y o f u s i n g DNA f r a g m e n t a t i o n as a s h o r t - t e r m b i o a s s a y t o stuQy t h e a b i l i t y o f c h e m i c a l s t o damage m a c r o m o l e c u l e s i n t h e c e l l was i n v e s t i g a t e d . I n t h e p a s t DNA f r a g m e n t a t i o n a s s a y s have been u s e d p r i m a r i l y on human o r a n i m a l c e l l s c u l t u r e d in vitro., as an a l t e r n a t i v e more r e l e v a n t . t o t h e human s y s t e m t h a n t h e " c l a s s i c a l " r o d e n t tumour i n c i d e n c e t e s t s . I n t h e c u l t u r e d c e l l s y s t e m i t has been shown, t h a t c a r c i n o g e n s may i n d u c e f r a g m e n t a t i o n and t h a t an i n c r e a s e o f a p p a r e n t DNA m o l e c u l a r w e i g h t from damaged t o . n e a r - c o n t r o l l e v e l s o v e r 30 h o u r s o c c u r s , s u g g e s t i n g r e p a i r o f t h e c a r c i n o g e n - i n d u c e d l e s i o n s . T h a t r e p a i r t a k e s p l a c e a t a l l a l s o s u g g e s t s t h a t t h e s e l e s i o n s a r e n o t t h e t y p e t h a t l e a d o n l y t o c e l l d e a t h , b u t r a t h e r t h o s e t h a t can l e a d t o m u t a t i o n s t h a t m i g h t u l t i m a t e l y bej e x p r e s s e d as tumours in vivo. However, t h e s e f a s t in vitro t e s t s cannot c o m p l e t e l y r e p l a c e the s l o w e r and more c o s t l j ? r o d e n t tumour i n c i d e n c e t e s t s , s i n c e s t u d i e s e m p l o y i n g c u l t u r e d c e l l s cannot be e a s i l y a d d r e s s e d t o t h e p r o b l e m s o f m e t a b o l i c a c t i v a t i o n and i n a c t i v a t i o n , t h e complex i n t e r a c t i o n o f c a r c i n o g e n s w i t h numerous i n t r a -and e x t r a c e l l u l a r compounds and t h e m u l t i p l i c i t y o f c e l l t y p e s t h a t may l e a d t o t h e o b s e r v e d o r g a n o t r o p y o f many c a r c i n o g e n s . I t w o u l d be h i g h l y d e s i r a b l e t o have an in vivo t e s t t h a y c o u l d be used t o i n v e s t i g a t e t h e i n i t i a t i o n s t e p of c a r c i n o g e n e s i s , b u t does n o t i g n o r e t h e process of c a r c i n o g e n e s i s by o b s e r v i n g o n l y t h e d i r e c t i n c i d e n c e o f tumours i n t h e way t h a t c l a s s i c a l t e s t s do. I n t h e f i n a l . a n a l y s i s t h e s e r o d e n t t e s t s must be done t o s a y d e f i n i t e l y whether a compound can. cause a tumour, b u t a f a s t , i n e x p e n s i v e i n t e r m e d i a t e in vivo a s s a y would be u s e f u l t o " p r e s c r e e n " compounds t o b r i n g t h o s e w i t h w h i c h s u s p i c i o n l i e s down t o a manageable number, and t o i n v e s t i g a t e t h e v a r i o u s p r o c e s s e s and m o d i f y i n g f a c t o r s of tumour i n i t i a t i o n . I n s h o r t , a r e l i a b l e i n d i c a t o r t h a t can be measured w i t h i n 6 a s h o r t p e r i o d of a p p l i c a t i o n o f t h e c a r c i n o g e n i s needed. T h i s s t u d y has i n v e s t i g a t e d t h e use o f DNA f r a g m e n t a t i o n , measured by a chenge i n t h e v e l o c i t y o f s e d i m e n t a t i o n on an a l k a l i n e s u c r o s e g r a d i e n t , a s one o f t h e s e s h o r t - t e r m b i o a s s a y s t o d e t e r m i n e t h e in vivo a c t i o n o f c a r c i n o g e n s . . The f r a g i n e n t a t i o n o f DNA (one o f the presumed . . i n i t i a t i n g s t e p s i n c a r c i n o g e n e s i s ) may be a s s a y e d f o r w i t h i n a few h o u r s f o l l o w i n g t h e a p p l i c a t i o n o f c a r c i n o g e n s o r p r e c a r c i n o g e n s , and can be com p l e t e d w i t h i n a two day p e r i o d . The a c t i o n o f c a r c i n o g e n s i n f r a g m e n t i n g DNA has a l r e a d y been employed by o t h e r s w o r k i n g in vivo f o r l i v e r , l u n g , k i d n e y and j e j e u n u m o f r a t s o r m i c e . However, a l l o f t h e s e t e s t s have employed t h e l a b e l l i n g o f DNA w i t h H-TdR over a two week p e r i o d i n t h e i n f a n c y o f t h e t e s t a n i m a l s , f o l l o w e d by a s i x week w a i t i n g p e r i o d f o r t h e m i c e t o r e a c h a d u l t h o o d . . P a r t i a l h e patectomy o r nephrectomy may a l s o be u s e d i n o r d e r t o i n d u c e renewed growth of n o r m a l l y m e t a p l a s t i c a l l y dormant t i s s u e , b u t t h i s i s r e s t r i c t e d t o t i s s u e s c a p a b l e o f r e g e n e r a t i o n . The l o n g p r e l a b e l l i n g p e r i o d r e q u i r e d by t h e s e methods may be a v o i d e d by c h o o s i n g g a s t r i c e p i t h e l i a l c e l l s as a t e s t s y s t e m . The.se c o n t i n u e d i v i d i n g i n t o t h e a d u l t h o o d o f t h e a n i m a l , w i t h t h e a v e r a g e t u r n o v e r t i m e o f t h e c e l l s being. 20. t o 40 . h o u r s . These t i s s u e s have an advantage i n t h e i r use o v e r f i b r o b l a s t s i n . t h a t t h e y a r e e p i t h e l i a l and a r e t h e c e l l s f r o m w h i c h turriours normal!}' a r i s e . A l s o , t h e y grow i n a s h e e t over a b a s a l l a y e r t h a t i s much l e s s l i k e l y t o i n c o r p o r a t e l a b e l . T h e r e f o r e , e p i t h e l i a l c e l l s may be. s e l e c t i v e l y chosen as a r e l a t i v e l y homogeneous p o p u l a t i o n . f o r s t u d y . As a t e s t f o r the e f f e c t i v e n e s s o f u s i n g g a s t r i c e p i t h e l i a l c e l l DNA f r a g m e n t a t i o n as a s c r e e n i n g a s s a y f o r t h e c a r c i n o g e n i c i t y o f c e r t a i n c h e m i c a l s , i t was shown t h a t : 1) S e d i m e n t a t i o n , p r o f i l e s o f the DNA of c e l l s t r e a t e d w i t h s t r o n g c a r c i n o g e n s (4NQ0, 6-rneChyl 4NQ0) s h i f t e d f a r t o t h e r i g h t ( f r a g m e n t e d DNA), b u t t h e n o n - c a r c i n o g e n 6NQ0 was u n a b l e t o fragment g a s t r i c c e l l DNA, even a t h i g h e r c o n c e n t r a t i o n s . The weak c a r c i n o g e n 3-methyl 4NQ0 was a b l e t o fragment. DNA o n l y p a r t i a l l y . T h i s i n d i c a t e s t h a t 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 , w h i c h a r e an i n d i c a t o r o f DNA damage in vivo, may a l s o a c c u r a t e l y r e f l e c t , t h e c a r c i n o g e n i c i t y o f c h e m i c a l s . 2) The 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 ' 2AAF i s a b l e t o i n d u c e DNA f r a g m e n t a t i o n i n g a s t r i c e p i t h e l i a l c e l l s w h i l e i t s p r e c u r s o r 2AAF i s u n a b l e t o do so 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 S i m i l a r l y , t h e u l t i m a t e c a r c i n o g e n DMN was a b l e t o damage t h e DNA o f l i v e r ( i t s . m a i n t a r g e t o rgan i n t h e f o r m a t i o n o f t u m o u r s ) , b u t d i d n o t e l i c i t damage i n t h e e p i t h e l i a l c e l l s o f t he g a s t r i c t r a c t , where p r i m a r y tumours a r e n o t n o r m a l l y i n d u c e d i n t e s t a n i m a l s . T h e . r e a s o n f o r t h i s d i f f e r e n c e i n c a p a c i t y t o a l l o w DNA f r a g m e n t a t i o n may be one o r more o f s e v e r a l - d i f f e r e n c e s i n a b i l i t y t o aotivo.te t h e p r e c a r c i n o g e n inactivate t h e a c t i v a t e d c a r c i n o g e n , o r t h e - p r e s e n c e o f o t h e r m o d i f y i n g f a c t o r s - b u t t h e i n t e r e s t i n g o b s e r v a t i o n i s t h a t the or g a n s p e c i f i c i t y t h a t i s e v i d e n t i n in vivo c a r c i n o g e n i c i t y t e s t s may be p a r a l l e l e d i n t h e s h o r t t e r m by an a s s a y t h a t can be c o m p l e t e d i n h o u r s o r da y s . 3) When MG and i t s n i t r o s a t i o n . p r o d u c t s were f e d t o th e m i c e , o n l y t h e c a r c i n o g e n i c n i t r o s a t i o n p r o d u c t s of t h e MG were a b l e t o damage t h e DNA oE g a s t r i c e p i t h e l i a l c e l l s . P r e c u r s o r s and c a r c i n o g e n i c p r o d u c t s may a l s o be d i f f e r e n t i a t e d u s i n g t h i s technique.. 4) When complex m i x t u r e s a r e f e d t o t h e m i c e , t h e s i t e s o f or g a n DNA damage c o r r e s p o n d t o t h e s i t e s o f tumour i n c i d e n c e o b s e r v e d e p i d e m i o l o g i c a l l y . I n t h e case, o f b r a c k e n f e r n s h o o t s t h e i l e u m and u r i n a r y b l a d d e r showed t h e g r e a t e s t amount o f DNA f r a g m e n t a t i o n . T h i s . i s ; a l s o .-where tumours ap p e a r e d i n mice: f e d d r i e d , m i l l e d b r a c k e n f e r n and i n c a t t l e g r a z e d on f r e s h b r a c k e n f e r n . Also., t he f e r n t h a t was t r e a t e d w i t h b o i l i n g w a t e r and sodium b i c a r b o n a t e p r i o r t o a p p l i c a t i o n t o human c e l l s in vitro o r mouse' g a s t r i c c e l l s in vivo showed 64 a l a r g e d e c r e a s e i n a b i l i t y t o fragment DNA. Heat appears' I t o be a b l e t o i n a c t i v a t e t he DNA f r a g m e n t i n g p r o p e r t i e s o f t h e b r a c k e n f e r n e x t r a c t . . \ F r a g m e n t a t i o n o f DNA (as a s s a y e d by a l k a l i n e sucrose 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 ) may be used i n a s c e r t a i n i n g t h e t a r g e t o r g a n s o f o r g a n o t r o p i c c a r c i n o g e n s . o r p r e c a r c i n o g e n s . . A good c o r r e l a t i o n between t h e s i t e o f g r e a t e s t DNA damage . ( L a i s h e s , et al.3 1975) and DNA r e p a i r ( S t i c h and K i e s e r , 1974) has a l r e a d y . b e e n o b s e r v e d . . I n s u p p o r t of t h i s i t may now be p o i n t e d out t h a t 4NQ0 and. MNNG cause e p i t h e l i a l c e l l damage i n t h e stomach (where tumours•appear when t h e s e c a r c i n o g e n s a r e a p p l i e d , t o mice) and t h e p r e c a r c i n o g e n s 2AAF and DMN cause. DNA f r a g m e n t a t i o n m a i n l y i n t h e l i v e r , a l s o . t h e s i t e o f tumour f o r m a t i o n . S i n c e an assess m e n t o f DNA f r a g m e n t a t i o n a l o n e may i n d i c a t e o n l y t h e p r i m a r y i n i t i a t i o n , s t e p i n c a r c i n o g e n e s i s an in vivo assay, o f DNA repair i s ; d e s i r a b l e . K i e s e r and S t i c h (1974) have a c c o m p l i s h e d t h i s f o r l i v e r and kidney,, and Cox, et al.j (1.973) have a s s a y e d f o r DNA. r e p a i r in vivo b y m o n i t o r i n g t h e r e c o v e r y o f l i v e r DNA t o l a r g e r s i z e s f o l l o w i n g f r a g m e n t a t i o n w i t h DMN. C u l t u r e d human f i b r o b l a s t s in vitro o r g a s t r i c e p i t h e l i a l c e l l s in vivo a l s o appear t o i n c r e a s e 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 t h e i r DNA f o l l o w i n g f r a g m e n t a t i o n by a p p l i c a t i o n o f MNNG. R e p a i r b e g i n s t o t a k e p l a c e a t 16 t o 18 h o u r s f o l l o w i n g a d m i n i s t r a t i o n . o f c h e m i c a l , and DNA a c h i e v e s a maximum r a t e o f s e d i m e n t a t i o n f o l l o w i n g 24 t o 30 h o u r s o f r e p a i r . U s i n g t h e r e p a i r , o f g a s t r i c c e l l DNA a f t e r damage by MNHG as a c o n t r o l , i t i s e a s y t o f o r e s e e t h e use o f r e c o v e r y o f DNA s e d i m e n t a t i o n v e l o c i t y as an a s s a y f o r v a r i a t i o n s i n r e p a i r b o t h i n rate and extent when i n d i v i d u a l o r g a n s o r v a r i o u s chemica ls a r e t e s t e d . I f v a r i a b i l i t y i n e i t h e r damage o r r e p a i r o f DNA (measured by a r e c o v e r y o f s e d i m e n t a t i o n v e l o c i t y a f t e r damage) f i n d s a p a r a l l e l i n i n c i d e n c e o f tumours, measured i n r o d e n t t e s t s o r by human e p i d e m i o l o g i c a l d a t a , t h e n v a l u a b l e i n f o r m a t i o n 65 might- be g l e a n e d c o n c e r n i n g t h e i m p o r t a n c e of DNA damage o r r e p a i r i n tumour i n i t i a t i o n . A l t h o u g h a s h i f t i n DNA s e d i m e n t a t i o n p r o f i l e s t o i n d i c a t e damage a f t e r t r e a t m e n t w i t h c a r c i n o g e n s may be used as an i n d i c a t o r o f o r g a n o t r o p y , c a r e must be e x e r c i s e d i n t h e i n t e r p r e t a t i o n o f such d a t a . 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 do n o t p r o d u c e i n f o r m a t i o n on t h e c e l l u l a r l e v e l , b u t on t h e m o l e c u l a r l e v e l . T h e r e f o r e , , o t h e r t e s t s t h a t p r o d u c e i n f o r m a t i o n a t t h e l e v e l o f t h e c e l l must be employed to c o r -r o b o r a t e and a m p l i f y r e s u l t s . One p r o f i t a b l e way o f d o i n g t h i s a p p e a r s t o be by u s i n g a u t o r a d i o g r a p h i c a n a l y s i s of DNA r e p a i r s y n t h e s i s , t o a l l o w r e c o g n i t i o n o f t h e e x a c t c e l l t y p e t h a t i s damaged. • T h i s may .help p r o v e o r d i s p r o v e t h e i d e a t h a t c a r c i n o g e n - i n d u c e d DNA l e s i o n s a r e r e s t r i c t e d t o t a r g e t c e l l s , and t i s s u e s . The use of DNA f r a g m e n t a t i o n in vivo, as measured 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 , a p p ears t o be a p r o m i s i n g method f o r d e t e r m i n i n g o r g a n o t r o p y by m e a s u r i n g b o t h DNA damage and r e p a i r . The use of l i v e r t i s s u e and g a s t r i c e p i t h e l i a l t i s s u e combines t h e c o n v e n i e n c e of .in vitvo s h o r t terra b i o a s s a y s w i t h t h e c o m p l e t e n e s s o f the- m e t a b o l i c s y s t e m o f t h e whole mammal. LITERATURE CITED A b a n o b i , S.E., Popp, J.A,, Chang, S.K., H a r r i n g t o n , G.W., L o t l i k a r , P.D., H a d j i o l o v , D., L e v i t t . , M., Raf a l a k s h i n , . S., Sarma, D.S.R., J . N a t ' l . C a n c e r I n s t . 5 8 ( 2 ) : 263-270 (1977) Almond, N., J . Comp. P a t h o l . Ti 165 (1894) A r c o s , J.C., A r g u s , M.F., Advan. ' Cancer Res. 11: 305 (1968) B o u t w e l l , R.K., C r i t . Rev. T o x i c o l . 38: 419-443 (1974) C l a yson, D.B., C h e m i c a l C a r c i n o g e n e s i s , L i t t l e , Brown and Co. B o s t o n , 1962 C l a y s o n , D.B. , G a r n e r , R.C., i n : C h e m i c a l C a r c i n o g e n s , . (C.A. S e a r l e , ed.) ACS Monograph 173, pp.. 431-437 "(1976) C l e a v e r , J . E . , R a d l a t . Res. 57: 207-227 (1973) Cox, R. , Damjanov, I . , Abanobe, S.E.., Sarma, D.S.R. , Cancer Res. 33: 2114-2121 (1973) Craddock, V.M., J . N a t ' l . C a n c e r I n s t . 47_: 899-907 (1971) Cramer,. J.W. , M i l l e r , J.A-, M i l l e r , E.G.,- J . B i o l . Chem. 235: 885-888 (1960) Damjanov, I . , Cox, R., Sarma, S.D.R., F a r b e r , E., Cancer Res. 33: 2122 (1973) D i p a o l o , J.A., N e l s o n , R.L., A r c h P a t h o l . 95: 380-385 (1973) D o l l , R., Morgan, L.G. , S p e i z e r , F.E.., B r i t . J . Cancer 24: 623 (1970) D r u c k r e y , H., Schagen, B., I v a n k o v i c , S., Z. K r e h s f o r s c h u n g 2 4 : 141-161, (1970) E c k a r d t , R.E., E n v i r o n m e n t and C a n c e r , W i l l i a m s and W i l k i n s , B a l t i m o r e , (1972) Endo, H., T a k a h a s h i , K., Biochem. B i o p h y s . Res. Commun, 54: 1384-1392 (1973) Endo, H. , T a k a h a s h i , K. , A o y a g i , H. , Gann 65_: 45-54 (1974) Endo, H., T a k a h a s h i , K., K i n o s h i t a , N. Baba, T., N a t u r e 245: 325-326 (1973) E v a n s , I.A., Widdop, B., B r i t . Emp. Cane. Campaign f o r Res. Ann. R e p o r t , p.377 (.1966) 67 Evans, l . A . , Cancer Res. _28: 2252 (1968) E v a n s , l.A.., O ncology 1970: P r o c . I n t . Cancer Congress 10*"^ 5: 178 (1972) Evans, I.A., i n : C h e m i c a l C a r c i n o g e n s (ACS Monograph 173) (C>E. S e a r l e , e d . ) , A m e r i c a n Chemical. S o c i e t y , W a s h i n g t o n , D.C., pp. 696-697, (1976) E v a n s , l . A . , Mason, J . , N a t u r e (London) 194: 584 (1965) E v a n s , l.A.., Osman, M.A. , N a t u r e (London) 250: 348-349 (1974) F a r b e r , E., C u r r . Res. O n c o l . Lect.. 95. (1973) F a r b e r , E., D.S.R. Sarma, R a j a l a k s h m i , S., S h i n o z u k a , H. i n : P r i n c i p l e s o f L i v e r D i s e a s e ( B e c k e r , E.F., e d . ) , M a r c e l D e k k e r , New Y o r k (1974) F o u l d s , L. , N e o p l a s t i c Development, Academic. Press,. London (1969) F r i e d e l l , G.H., J . N a t ' l Cancer I n s t . 43: 215 (1969) F u r s t , A., H a r o , R,T., i n : The J e r u s a l e m Symposia on Quantum C h e m i s t r y and B i o c h e m i s t r y - P h y s i c o c h e m i c a l Mechanisms o f C a r c i n o g e n e s i s , v o l . 1, pp 310-320, J e r u s a l e m : I s r a e l Academy o f S c i e n c e s and H u m a n i t i e s (1969) G r o v e r , P.L., Hewer, A., Sims, P., Biochem. P h a r m a c o l . 23: 323 (1974) H a n a w a l t , P., i n : M o l e c u l a r Mechanisms f o r R e p a i r o f DNA, pp. 421-437, (P. H a n a w a l t , R..B. S e t l o w , e d s . ) , Plenum P r e s s , New Y o r k (1975) Hawksworth, G. , H i l l , M.J., Biochem. J . P r o c . 122: 28-29 (1971) H a y a i s h i , 0., Ann. Rev. Biochem. 38i: 21 (1969) H e a t h , D.F., Biochem. J . 85: 72 (1962) H e c k e r , E., Methods i n Cancer Res. 6: 439 (1971) H e d d l e , J.A., M u t a t . Res. 18: 187-190 (1973) H e d l e r , L ; , M a r q u a r d t , P., Fd. Cosmet. T o x i c o l . J5: 341 (1968) H e i d e l b e r g e r , C , Cancer ( s u p p l e m e n t ) 4 0 ( 1 ) : 430-433 (1974) H e i d e l b e r g e r , C , i n ; C a r c i n o g e n e s i s - a comprehensive s u r v e y , ( R . I . F r e u d e n t h a l , P. J o n e s , e d s . ) , p . l , Raven P r e s s , Nex<r Y o r k (1976) H i g g i n s o n , J . , Environment: and Cancer, (R.L. C l a r k , ed.) > W i l l i a m s and W i l k i n s , B a l t i m o r e (1972) H i r o n o , I . , S h i b u y a , C., F u s h i m i , K. Haga, M., J . N a t ' l . C a n c e r I n s t . 45: 179-188 (1970) H o l l a n d e r , C.F., Exp. G e r o n t o l . .5: ,313-321 (1971) H a r p e r , W.C., O c c u p a t i o n a l and E n v i r o n m e n t a l Cancers o f t h e U r i n a r y System, Y a l e U n i v e r s i t y , New Haven and London (1969) H u t s o n , D.K., i n : F o r e i g n Compound M e t a b o l i s m i n Mammals, v o l . 1, (E.D. Hathaway, e d . ) , p. 314, The Chemical, S o c i e t y , London (1974) I k e g a m i , S., Namoto, N., Sugimura, T., C h e m - B i o l . I n t e r a c t i o n s 1: 321 (1970) I r v i n g , C.C., i n : Methods i n . C a n c e r Res., (H. B u s c h , ed.) 8: 189, ( 1 9 7 3 ) , Academic P r e s s , New York, I r v i n g , C C , Veazey, R.A., B i o c h i m . P i o p h y s . A c t a 179: 189-198 (1969) K a n a g a l i n g a m , K., B a l i s , M.E., Cancer 36: 2364-2372 (1975) ( c o n f e r e n c e s upplement) K a p e l l e r - A d l e r , R., K r a e l , J , , Biochem. Z. 221: 437-460 (1930) K a t s u t a , H. T a k o t a , T., J . N a t ' l . Cancer I n s t . 49: 1563-1576 (1972) Kawazoe, Y., A r a k i , M. , Huang, G.F., i n : T o p i c 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 , (W. N a k a h a r a , et_ a^l. , e d s . ) , U n i v . o f Tokyo P r e s s , Tokyo, p . l , (1972) Kawazoe, y., D a h a r a , N., A r a k i , M., Tamura, M., Gann 60: 617 (1969) Kawazoe, Y., T a c h i b a n a , M., A o k i , K., N a k a h a r a , W., Biochem. P h a r m a c o l . 16: 631-636 (1967) K i e s e r , D., S t i c h , H.F., P r o c . Soc. Exp. B i o l . Med. 145: 1339-1342 (1974) Komarrow, S.A., Biochem. Z. 211: 326-351 (1929) K r o e s , R., W i l l i a m s , G.M,, W e i s b u r g e r , J.H., C a n c e r Res. 33: 613-617 (1973) K o r o p a t n i c k , D.J., S t i c h , H.F., I n t . J . Cancer 17: 765-772 (1976) 69 Kuroyanage, M., Fukuoka, M. , Y o s h i h i r a , K. , Chem. Pharm. B u l l (Tokyo) 22: 1087 (1972) K w a s n l e w s k i , V., (L.) Kuhn. A r c h . Pharm. (Weinheim) 288: 307-311 (1955) L a i s h e s , B.A., PhD. T h e s i s , U n i v . o f B.C. (1974) L a i s h e s , B.A., K o r o p a t n i c k , D.J., S t i c h , H.F., P r o c . Soc. Exp. B i o l . Med. 149: 978-982 (1975) L a i s h e s , B.A.., S t i c h , H.F., Can. J . o f Biochem. 5 1 ( 7 ) : 990-994 (1973) L a n e , R.P. , B a i l e y , . M.E. , Food Cosmet. T o x i c o l . 11: 851-854 (1973) L a w l e y , P.D., i n : C h e m i c a l C a r c i n o g e n s , ( S e a r l e , C.E. , ed.)., A m e r i c a n C h e m i c a l S o c i e t y , W a s h i n g t o n , D.C, p. 101 (1976) L e t t , J.T., C a l d w e l l , I . , Dean, C . J . , A l e x a n d e r , P., N a t u r e 214: 790 (1967) L i j i n s k y , W., L e e , K.Y., G a l l a g h e r , C.H., Cancer Res. 30: 2280 (1970) L o , L.W. , S t i c h , I-I.F. , M u t a t i o n Res. 30: 397-406 (1975) M c G r a t h , R.A., W i l l i a m s , R.W., N a t u r e 212; 534 (1966) Magee, P.N., i n : T o p i c 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 (W. N a k a h a r a , S. Takayama, T. Sugimura, S. Odashima, e d s . ) , p . l , U n i v e r s i t y o f Tokyo P r e s s , Tokyo (1972) Mason, H.S., Adv. Enzymol. 19: 79 (1957) M i l l e r , J.A., Cancer Res. 30: 559-576 (1970) M i l l e r , E., Ann. N.Y. Acad. S c i . 163: 731-750 (1.969) M i l l e r , J.A., M i l l e r , E.G., i n : P h y s i c o - c h e m i c a l Mechanisms o f C a r c i n o g e n e s i s , (E.D. Bergmann, B. P u l l m a n , e d s . ) , J e r u s a l e m , p. 237, (1969) M i l l e r , J.A. , M i l l e r , E.G., J . Nat'.'L Cancer I n s t . 47: V (1971) M i l l e r , E.C., M i l l e r , J.A., Hartmann, H.A. , Cancer Res. 21: 815-824 (1961) Montesano, R. Magee, P.N. , N a t u r e 228: 173 (1970) Oesch, F., X e n o b i o t i c a _3: 305 (1972) P a i n t e r , R.B., C l e a v e r , J.E. , R a c l i a t . Res. 37_: 451 (1969) Pamukcu, A.M., Zent.ra.lbl. V e t e r i n a e r m e d . 2: 409-429 (1955) Pamukcu, A.M., Ann. N.Y. Acad. S c i . 108: 938 (1963) P a u l , J.S.., Montgomery, P.O'B. , L o u i s , J.B., Cancer Res. 31: 413 (1971) Poon, P.K., O ' B r i e n , R.L., P a r k e r , J.W., N a t u r e 250: 223-225 (1974) P o t t , P., C h i r u r g i c a l Works 5>: 63 (1775) Pound, A.W., Lawson, T.A., H o r n , L., B r i t . J . Cancer 27: 451-459 (1973) P r i c e , C.C., Gaucher, G.M. , Koneur, P., Shibakawa,.. R., Sowa, J.R., Yamaguchi, M.„ Arm. N.Y. Acad. S c i . 163: 593-598 (1969) Rasmussen, R.E., P a i n t e r , R.B., N a t u r e 203: 1360 (1964) Rasmussen, R.E., P a i n t e r , R.B., J . C e l l B i o l . _29: 11 (1966) R o s s , W.C. , B i o l o g i c a l A l k y l a t i n g A g e n t s , B u t t e r w o r t h , London, (1962) S a f f i o t t i , U., P r o g . Exp. Tumour Res. 11: 302 (1969) S a n d e r s , J . , S c h w e i n s b e r g , F., IARC S c i e n t i f i c P u b l . _3: 97-103 (1972) Sarma, D.S.R., R a j a l a k s m i , S. F a r b e r , E., i n : P r i n c i p l e s o f L i v e r D i s e a s e ( F.F. B e c k e r , ed.),. M a r c e l Dekker,. New Y o r k (1974) S h a p i r o , R., Ann. N.Y. Acad. S c i . 163: 624-630 (1969) • S i l v e r b e r g , E., A . I . H o l l e b , A . I . , CA, A Cancer J . C l i n . 24: 2 (1974) S i v a k , A., Van Durnen, B.L., C h e m ^ B i o l , I n t e r a c t . 3: 401 (1971) S t i c h H.F., K i e s e r , D., P r o c . Soc. Exp. B i o l , and Med. 145: 1339-1342 (1974) S t i c h , H.F., L a i s h e s , B.A., i n : P a t h o b i o l o g y A n n u a l , 1973, (H.L. I o a c h i m , e d . ) , M e r e d i t h C o r p o r a t i o n , New Y o r k , New Youk, (1973) S t i c h , H.F., San, R.H.C., Mut Res. 10: 389-404 (1970) S t i c h , H.F., San, R.H.C., P r o c . Soc. Exp. B i o l . Med. 142: 155 (1973) S t i c h , H.F., L a i s h e s , B.A., Can. J . Biochem: 5 1 ( 7 ) : 990-994 (1973) S t i c h . , H.F., San, R.H.C., Lam, P . , ' K o r o p a t n i c k , J . , L o , L., i n : O r i g i n s o f Human C a n c e r , C o l d S p r i n g Harbour L a b o r a t o r y , pp. 1499-1511 (1977) S t o r i e r , D.N. , J . Comp. P a t h o l . . j5: 276 (1893) S u g i m u r a , T., K a w a c h i , T., Methods i n Cancer Res. 7_: 245 (1973) U e h l e k e , H., L i f e Sc. 5_: 1489 (1966) Van Duuren, B.L., K a t z , C , G o l d s c h r a i d t , B.M., J . N a t ' l C a n c e r I n s t . 51-2: 703 (1973) Von R o s e n b e r g e r , G., Heeschen, W., Deut. T i e r a r t z l . Wochschr. _67: 201 (1960) Wang, C.Y., C h i u , C.W., Painukcu, A.M., B r y a n , G.T., J . N a t ' l C a n c e r I n s t . 5 6 ( 1 ) : 33-36 (1976) Wang, C.Y., Pamukcu, A.M., B r y a n , G.T., Phytoche.mistry 12: 2298-2299 (1973) W e i s b u r g e r , J.H., Cancer M e d i c i n e , H o l l a n d and F r e i , e d s . , Le a and F e b i g e r , P h i l a d e l p h i a (1973) W e i s b u r g e r , J.H., W e i s b u r g e r , E.K., Pharm. Rev. 25: 1 (1973) W e i s b u r g e r , J.H., P r o c . Soc. Exp. B i o l . Med. 1 4 8 ( 4 ) : 1119-1121 (1975) Wynder, E.L., Hoffmann, D., Tobacco and Tobacco Smoke, Academic P r e s s , New Y o r k (1967) Wynder, E.L., M a b u c h i , K., P r e v . Med. 1: 300 (1972) 

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