@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Medical Genetics, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Koropatnick, D. James"@en ; dcterms:issued "2010-02-23T20:15:17Z"@en, "1978"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "The possibility of using alkaline sucrose gradient analysis of the digestive tract tissue of mice to investigate the carcinogenic potential of organotropic compounds was examined. Young Swiss mice were injected with ³H-TdR to label the DNA of the epithelial cells of the digestive tract. Thirty h later they were force-fed carcinogenic or non-carcinogenic chemicals. Tissue samples were taken four h post-treatment and hydrolyzed on top of the alkaline sucrose gradient. Shifts in sedimentation profiles indicated that: (1) both cultured human fibroblasts and epithelial cells of the gastrointestinal system show a shift in sedimentation profile after treatment with the carcinogen MNNG that is taken to indicate repair; (2)- the carcinogen 4-nitroquinoline 1-oxide (4NQO) and 6-methyl 4NQO cause DNA fragmentation in the epithelial cells of the gastrointestinal system while the non-carcinogen 6NQO lacks this capacity; (3) the ultimate carcinogen N-acetoxy 2AAF caused DNA fragmentation in esophagus and stomach cells while the precarcinogen 2AAF produced no significant effect; (4) only the carcinogenic nitrosation products of methylguanidine damaged the DNA of gastric epithelial cells; (5) the precarcinogens 2AAF and DMN produced DNA fragmentation in the main target organ -the liver -but had little effect on the epithelial cells of the stomach; and (6) extracts of the carcinogenic plant pteridium aquilinvjn (bracken fern) showed an organotropic DNA-fragmenting ability in vivo and in vitro that corresponded to its .organ-specific tumour induction in cattle and rats. Treatment with heat appears to drastically reduce the DNA-fragmenting ability of the plant. The results suggest that the application of the sucrose gradient technique to the epithelial cells of esophagus, stomach and liver of pre-labelled (³H-TdR) and force-fed young mice incorporates the advantages of in vitro short term bioassays with the completeness of tests using whole mammals."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/20816?expand=metadata"@en ; skos:note "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. 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Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "A short term in vivo bioassay for the organ specificity of carcinogens"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/20816"@en .