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Experimental Approaches Towards the Biochemical Analysis of Chemical Carcinogenesis BRIAN A . LAISHES McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI 53706
The purpose of this presentation is to highlight developments in chemical carcinogenesis research that are directed towards understanding the development of malignancy at the molecular level. Because of the breadth of this topic, a high degree of selection has been necessary in order to adhere to space limitations, and, unfortunately, many important studies could not be included. An effort has been made to present selected studies that give a historical perspective to certain research developments and to include studies that exemplify efforts to delineate the truly complex biology of cancer development. Early Epidemiologic Data Early studies that represent the beginnings of our knowledge of chemical carcinogenesis were reviewed briefly by E.C. Miller (1). The first of these studies, in 1761, was by the physician John Hill of London, England, who reported on the development of nasal cancer as a consequence of excessive use of tobacco snuff (2). Percival Pott, a surgeon in London, reported on the unusually high incidence of cancer of the skin of the scrotum of young men who had worked as chimney sweeps in their childhood (3). The first preventive measures against chemically induced cancer in humans arose 3 years later through the Danish chimney sweepers' guild urging its members to take daily baths (4). During the f o l l o w i n g century, further observations o f higher i n c i d e n c e s o f s p e c i f i c c a n c e r s o f t h e s k i n and u r i n a r y b l a d d e r were r e p o r t e d i n i n d i v i d u a l s w i t h p a r t i c u l a r p r i o r c h e m i c a l exposures (1). I t was n o t u n t i l t h e 1930's t h a t t h e e p i d e m i o l o g i c d a t a were m i r r o r e d b y d e f i n i t i v e l a b o r a t o r y d a t a o n t h e c a r c i n o g e n i c i t y o f pure c h e m i c a l s f o r e x p e r i m e n t a l animals.
0097-6156/81/0160-0197$05.00/0 ©
1981 American Chemical Society
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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THE PESTICIDE CHEMIST AND MODERN TOXICOLOGY
L a b o r a t o r y M o d e l s and
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C a n c e r o f t h e s k i n became t h e f i r s t e x p e r i m e n t a l m o d e l o f c h e m i c a l l y i n d u c e d c a n c e r i n 1915 when Yamagiwa and Ichikawa, i n J a p a n , i n d u c e d s k i n c a r c i n o m a s i n the e a r s o f r a b b i t s by repeated t o p i c a l a p p l i c a t i o n s of c o a l tar for long periods (see 5). I n 1918, T s u t s u i i n d u c e d s k i n c a n c e r i n m i c e w i t h t a r s and, i n 1922, P a s s e y i n d u c e d s k i n c a n c e r i n m i c e w i t h ether e x t r a c t s of t a r s [5). The a c t i v e m o l e c u l e s r e s p o n s i b l e f o r t h e i n d u c t i o n o f s k i n c a n c e r w i t h t a r s and t a r e x t r a c t s became t h e o b j e c t s o f numerous i n v e s t i g a t i o n s . One i m p o r t a n t l e a d was uncovered by H i e g e r , who showed t h a t t h e f l u o r e s c e n c e s p e c t r a o f p r o d u c t s f r o m t h e c a r c i n o g e n i c t a r s and o f s y n t h e t i c b e n z [ a ] a n t h r a c e n e d e r i v a t i v e s were s i m i l a r (6») . T h u s , i n 1930, Kennaway and Hieger demonstrated the c a r c i n o g e n i c i t y o f d i b e n z [ a , h ] a n t h r a c e n e , t h e f i r s t p u r e , s y n t h e t i c c a r c i n o g e n (Ί)· The c a r c i n o g e n i c h y d r o c a r b o n , b e n z o [ a j p y r e n e , was s o o n i s o l a t e d f r o m c o a l t a r by Cook, H e w e t t , and H i e g e r {&). Cancer o f the l i v e r , the f i r s t e x p e r i m e n t a l v i s c e r a l c a n c e r , became a m o d e l o f c h e m i c a l l y i n d u c e d c a n c e r i n 1933 when Y o s h i d a i n d u c e d l i v e r t u m o r s i n r a t s and m i c e w i t h o r a l a d m i n i s t r a t i o n s of o-aminoazotoluene (2 ,3-dimethyl-4-aminoazobenzene) (9). o - A m i n o a z o t o l u e n e i s a d e r i v a t i v e o f t h e a z o dye s c a r l e t r e d ( 1 - [ 4 - ( o - t o l y l a z o ) - o - t o l y l a z o ] - 2 - n a p h t h o l ) , w h i c h was used by F i s c h e r i n 1906 t o i n d u c e p r o l i f e r a t i v e l e s i o n s i n t h e s k i n of r a b b i t s (10). The s k i n l e s i o n s i n d u c e d by F i s c h e r d i d n o t become f r a n k c a n c e r s , h o w e v e r , and r e g r e s s e d when t h e a p p l i c a t i o n s o f s c a r l e t r e d were s t o p p e d . 1
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 i n t r o d u c e d a s an e x p e r i m e n t a l m o d e l i n 1938 when H u e p e r , W i l e y , and W o l f e i n d u c e d c a n c e r i n t h e u r i n a r y b l a d d e r o f d o g s by f e e d i n g them 2 - n a p h t h y l amine (11). R e g a r d i n g t h e i n d u c t i o n o f c a n c e r by p u r e c h e m i c a l s , i t i s n o t e w o r t h y t h a t i n 1932 L a c a s s a g n e i n d u c e d mammary c a n c e r s i n m a l e m i c e by e s t r o n e t r e a t m e n t , t h u s p i o n e e r i n g an e x p e r i m e n t a l model f o r hormone-induced tumors (12). Initiation-Promotion The t w o - s t e p , i n i t i a t i o n - p r o m o t i o n c o n c e p t was f i r s t c o n c e i v e d by Rous and c o - w o r k e r s a b o u t 40 y e a r s ago (13,_14) , and t h i s concept c o n t i n u e s to p l a y a prominent r o l e i n experimental d e s i g n s p r o b i n g the b i o l o g y o f the cancer d i s e a s e p r o c e s s . T h e s e i n v e s t i g a t o r s s t u d i e d t h e r o l e s o f i r r i t a t i o n and t h e s t i m u l a t i o n o f c e l l d i v i s i o n on the i n d u c t i o n o f tumors i n r a b b i t e a r s p r e v i o u s l y t r e a t e d w i t h c o a l t a r . H o l e s were p u n c h e d i n the r a b b i t s e a r s w i t h a c o r k b o r e r , and i t was f o u n d t h a t t u m o r s a p p e a r e d a l o n g t h e e d g e o f t h e wound. The d i s c o v e r y by B e r e n b l u m o f t h e c o c a r c i n o g e n i c p r o p e r t i e s o f c r o t o n o i l 1
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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( w i t h b e n z o [ a ] p y r e n e ) (15,16) l e d t o t h e r e m a r k a b l e d i s c o v e r y by M o t t r a m t h a t b e n z o [ a j p y r e n e need be a p p l i e d o n l y o n c e t o i n d u c e t u m o r s when i t was f o l l o w e d by r e p e t i t i v e a p p l i c a t i o n s o f c r o t o n o i l ( 1 7 ) . I t was d e m o n s t r a t e d t h a t t h e d o s e o f t h e i n i t i a t i n g a g e n t d e t e r m i n e s t h e e v e n t u a l tumor y i e l d , that the p r o m o t i n g a g e n t d e t e r m i n e s t h e d u r a t i o n o f t h e l a t e n t p e r i o d (18), and t h a t t h e i n i t i a t i o n s t e p i s i r r e v e r s i b l e ( 1 9 ) . Reversi n g t h e o r d e r o f t r e a t m e n t , by a d m i n i s t e r i n g c r o t o n o i l f o r many weeks f o l l o w e d by a s i n g l e d o s e o f b e n z o [ a j p y r e n e , r e s u l t e d i n no t u m o r s ( 2 0 ) . B o u t w e l l d e m o n s t r a t e d t h a t d o s e s o f c r o t o n o i l t h a t were e i t h e r t o o s m a l l o r t o o w i d e l y s e p a r a t e d r e s u l t e d i n no p r o m o t i o n , t h u s d o c u m e n t i n g t h e r e v e r s i b i l i t y o f t h e e f f e c t o f tumor p r o m o t e r s ( 2 1 ) . The g e n e r a l i t y o f t h e i n i t i a t i o n - p r o m o t i o n , t w o - s t e p s y s t e m i s remarkable. Armuth a n d B e r e n b l u m h a v e e x t e n d e d t h e s y s t e m t o mouse l i v e r a n d l u n g , u s i n g d i m e t h y l n i t r o s a m i n e a s i n i t i a t o r (22); t o r a t mammary g l a n d , u s i n g 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e as i n i t i a t o r (23); and t o a system o f two-stage t r a n s p l a c e n t a l l i v e r c a r c i n o g e n e s i s i n C57BL/6 m i c e (2£,2j5). Transplacental, i n i t i a t i o n - p r o m o t i o n e x p e r i m e n t s were r e p o r t e d by G o e r t t l e r a n d L o e h r k e , who t r e a t e d m i c e p r e n a t a l l y b y i n j e c t i n g t h e i n i t i a t ing agents 7,12-dimethylbenz[a]anthracene o r e t h y l carbamate i n t o t h e p r e g n a n t m o t h e r ( 2 6 ) ; t h e o f f s p r i n g , when t r e a t e d b e t w e e n t h e a g e s o f 12 a n d 26 weeks w i t h t o p i c a l a p p l i c a t i o n s o f t h e tumor p r o m o t e r 1 2 - 0 - t e t r a d e c a n o y l - p h o r b o l - 1 3 - a c e t a t e ( T P A ) , e x h i b i t e d tumors i n s k i n a n d i n o t h e r o r g a n s . Tumor p r o m o t i o n a c t i v i t y h a s b e e n d e m o n s t r a t e d f o r a v a r i e t y of agents i n v a r i o u s organs: b u t y l a t e d h y d r o x y t o l u e n e (BHT) i n mouse l u n g ( 2 7 ) , b i l e a c i d s i n c o l o n (28) , s a c c h a r i n a n d c y c l a m a t e i n r a t u r i n a r y b l a d d e r ( 2 9 ) , TPA i n a n in v i v o - i n v i t r o r a t t r a c h e a model ( 3 £ , 3 1 ) , and phénobarbital (32-36) and p o l y c h l o r i n a t e d b i p h e n y l s (34,37^.38) i n r a t l i v e r . The b a s i c p r i n c i p l e s o f t h e w e l l - k n o w n t w o - s t e p i n i t i a t i o n p r o m o t i o n system a r e o u t l i n e d i n F i g u r e 1 (39). The q u a l i t a t i v e d i f f e r e n c e s i n the responses o f the target t i s s u e t o i n i t i a t i n g or p r o m o t i n g a g e n t s a r e r e m a r k a b l e i n t h a t i n i t i a t i n g a g e n t s , w h i c h a r e o f t e n c o m p l e t e c a r c i n o g e n s a t h i g h e r d o s e s , c a n be a d m i n i s t e r e d i n l o w d o s e s t h a t do n o t p r o d u c e t u m o r s (21,39.) · S i m i l a r l y , m u l t i p l e doses o f promoting agents t h a t a r e n o t c o m p l e t e c a r c i n o g e n s i n d u c e e s s e n t i a l l y no t u m o r s , w h e r e a s h i g h i n c i d e n c e s o f t u m o r s a r i s e when t h e s e same d o s e s o f i n i t i a t ing and promoting agents a r e a d m i n i s t e r e d i n sequence. Reversing the o r d e r o f e x p o s u r e a b o l i s h e s t h e s y n e r g i s m . F i n a l l y , there i s l i t t l e o r no r e c o v e r y f r o m t h e e f f e c t s o f i n i t i a t i n g a g e n t s (19,4£) , w h e r e a s t i s s u e s c a n r e c o v e r f r o m t h e e f f e c t s o f p r o m o t i n g a g e n t s (21^,41) . R e c e n t s t u d i e s w i t h t h e mouse s k i n t u m o r i g e n e s i s m o d e l h a v e revealed f a s c i n a t i n g q u a n t i t a t i v e d i f f e r e n c e s i n the response of the t a r g e t t i s s u e t o i n i t i a t i n g o r promoting agents. As r e p o r t e d b y B o u t w e l l , t h e tumor r e s p o n s e i n d u c e d b y r e p e t i t i v e
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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a p p l i c a t i o n o f 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e (DMBA) a l o n e r e q u i r e s a b o u t 10 t i m e s a s much DMBA a s t h a t r e q u i r e d when DMBA ( i n i t i a t i o n ) i s f o l l o w e d by r e p e t i t i v e a p p l i c a t i o n s o f t h e tumor p r o m o t e r TPA (42). Cocarcinogenesis C o c a r c i n o g e n e s i s (15,43) was d i s c o v e r e d b e f o r e t h e twostage c o n c e p t o f i n i t i a t i o n - p r o m o t i o n ( j ^ , ] ^ , 1 6 , Γ7,18) . Co c a r c i n o g e n e s i s has been t h e s u b j e c t o f r e c e n t r e v i e w s (40,44,45) and a l s o o f some c o n f u s i o n . Boutwell presented a clear d e f i n i tion: C o c a r c i n o g e n e s i s denotes the s i t u a t i o n i n which a second f a c t o r ( c o c a r c i n o g e n ) , when i n t r o d u c e d t o g e t h e r w i t h t h e c a r c i n o gen, i n c r e a s e s the r e s p o n s e t o the c a r c i n o g e n . The t e r m c o c a r c i n o g e n e s i s has no i m p l i c a t i o n o f d e n o t i n g a s p e c i f i c s t e p i n tumor d e v e l o p m e n t ( 2 1 ) . The d i s t i n c t i o n b e t w e e n c a r c i n o g e n e s i s and tumor p r o m o t i o n i s n o t a l w a y s c l e a r . On t h e o t h e r h a n d , some i n v e s t i g a t o r s have c l e a r l y d e m o n s t r a t e d t h a t some c o c a r c i n o g e n s a r e n o t tumor p r o m o t e r s and, c o n v e r s e l y , t h a t some tumor p r o m o t e r s a r e n o t c o c a r c i n o g e n s (4
