Chemical Properties of Ultimate Carcinogenic Metabolites of

14 Chemical Properties of Ultimate Carcinogenic Metabolites of Arylamines and Arylamides

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FRED F. KADLUBAR and FREDERICK A. BELAND National Center for Toxicological Research, Jefferson, AR 72079

A number of arylamines and arylamides are carcinogenic in a variety of tissues of several species including the urinary bladder of man. These compounds undergo metabolic activation to ultimate carcinogens through a number of enzymatic and nonenzymatic pathways. In this review, these activation mechanisms are considered in detail and their relative con tribution to the observed carcinogenicity of these compounds is discussed. The metabolism o f c a r c i n o g e n i c a r y l a m i n e s and a r y l a m i d e s r e s u l t s i n a broad spectrum o f r e a c t i v e , e l e c t r o p h i l i c m e t a b o l i t e s t h a t form c o v a l e n t adducts w i t h c e l l u l a r c o n s t i t u e n t s . These a c t i v a t i o n p a t h ways a r e summarized i n F i g u r e I . Arylamides and p r i m a r y a r y l a m i n e s are r e a d i l y i n t e r c o n v e r t e d by N - a c e t y l t r a n s f e r a s e s and N-deacetyl a s e s (reviewed i n 1) and they a r e i n i t i a l l y a c t i v a t e d by cytochrome P-450- and f l a v i n - c o n t a i n i n g monooxygenases t o form N-hydroxy a r y l amides and N-hydroxy a r y l a m i n e s , r e s p e c t i v e l y (reviewed i n 2_). These N-hydroxy m e t a b o l i t e s , which can a l s o be i n t e r c o n v e r t e d by enzymatic N - d e a c e t y l a t i o n / N - a c e t y l a t i o n (1_), a r e proximate c a r c i n o g e n s s i n c e they a r e g e n e r a l l y more c a r c i n o g e n i c and mutagenic than t h e i r parent compounds. F u r t h e r enzymatic o r non-enzymatic p r o c e s s e s lead to u l t i m a t e c a r c i n o g e n s , which a r e u s u a l l y d e f i n e d by t h e i r e l e c t r o p h i l i c r e a c t i v i t y w i t h n u c l e i c acids or p r o t e i n s 03). N-Hydroxy a r y l a m i d e s a r e c o n v e r t e d t o u l t i m a t e carcinogens through conjugation with s u l f u r i c , a c e t i c or g l u c u r o n i c acids (reviewed i n J ^ , 4 ) . S u l f u r i c a c i d c o n j u g a t i o n i s c a t a l y z e d by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulf©trans f e r a s e s and y i e l d s N - s u l f o n y l o x y a r y l a m i d e s ( I ) ; w h i l e N-acetoxy a r y l a m i d e s ( I I ) a r e formed through nonenzymatic e s t e r i f i c a t i o n w i t h a c e t y l coenzyme A o r by a p e r o x i d a s e - m e d i a t e d , one-electron oxida t i o n and s u b s e q u e n t d i s m u t a t i o n o f a n i t r o x y l r a d i c a l (5^,6^). N - G l u c u r o n y l o x y a r y l a m i d e s ( i l l ) a r e a l s o formed by enzymatic con j u g a t i o n and they can undergo subsequent N - d e a c e t y l a t i o n t o N - g l u c u r o n y l o x y a r y l a m i n e s ( I V ) . An a d d i t i o n a l pathway by which N-hydroxy a r y l a m i d e s a r e a c t i v a t e d i s through an i n t r a m o l e c u l a r rearrangement to N-acetoxy arylamines ( V ) w h i c h i s c a t a l y z e d by c y t o s o l i c N,0-acyltransferases. This chapter not subject to U.S. copyright. Published 1985, American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


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Arylamines and Arylamides

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N-Hydroxy a r y l a m i n e s a r e a l s o c o n v e r t e d t o N-acetoxy a r y l a m i n e s ( V ) , b u t a p p a r e n t l y by an a c e t y l coenzyme A-dependent enzymatic O-esterification 0,8). S i m i l a r l y , N-sulfonyloxy arylamines ( V I ) are thought t o a r i s e by a PAPS-dependent enzymatic O - s u l f o n y l a t i o n o f N-hydroxy a r y l a m i n e s ( 9 , 1 0 ) ; w h i l e 0 - s e r y l o r 0 - p r o l y l e s t e r s ( V I I ) a r e formed by t h e i r c o r r e s p o n d i n g aminoacyl tRNA s y n t h e t a s e s i n a ATP-dependent r e a c t i o n ( 1 1 , 1 2 ) . N-Hydroxy a r y l a m i n e s r e a d i l y form g l u c u r o n i d e c o n j u g a t e s , b u t i n c o n t r a s t t o the N-hydroxy a r y l a m i d e s , these are N - g l u c u r o n i d e s which are u n r e a c t i v e and s t a b l e a t n e u t r a l pH. The N - g l u c u r o n i d e s a r e r e a d i l y t r a n s p o r t e d t o t h e lumens o f t h e u r i n a r y b l a d d e r and i n t e s t i n e where they can be h y d r o l y z e d t o the f r e e N-hydroxy a r y l a m i n e s by m i l d l y a c i d i c u r i n e o r by i n t e s t i n a l b a c t e r i a l 3 - g l u c u r o n i d a s e s ( 1 3 , 1 4 ) . Non-enzymatic a c t i v a t i o n o f N-hydroxy a r y l a m i n e s c a n occur i n an a c i d i c environment by p r o t o n a t i o n (15,16) o f t h e N-hydroxy group ( V I I I ) as w e l l as by a i r o x i d a t i o n ( r e v i e w e d i n 17) t o a nitrosoarene (IX). A l t e r n a t i v e m e t a b o l i c p a t h w a y s i n v o l v e r i n g - o x i d a t i o n and peroxidation of arylamines. Although r i n g - o x i d a t i o n i s g e n e r a l l y c o n s i d e r e d a d e t o x i f i c a t i o n r e a c t i o n , an e l e c t r o p h i l i c iminoquinone (X) can be formed by a secondary o x i d a t i o n o f t h e aminophenol m e t a b o l i t e ( J ^ 8 , J _ 9 ) . L a s t l y , r e a c t i v e imines ( X I ) can be formed from the p r i m a r y a r y l a m i n e s by p e r o x i d a s e - c a t a l y z e d r e a c t i o n s t h a t i n v o l v e free r a d i c a l intermediates (reviewed i n 20). Only a l i m i t e d number o f a c t i v a t i o n pathways appear t o be a v a i l a b l e t o N-methyl a r y l a m i n e s . F o l l o w i n g enzymatic N - h y d r o x y l a t i o n t o secondary N-hydroxy a r y l a m i n e s (21,22), these compounds a r e c o n v e r t e d i n t o r e a c t i v e e l e c t r o p h i l e s through enzymatic e s t e r i f i c a t i o n (9) t o N - s u l f o n y l o x y - N - m e t h y l a r y l a m i n e s ( X I I ) o r by f u r t h e r oxidation to N-arylnitrones (XIII). I n t h i s r e v i e w , t h e c h e m i c a l p r o p e r t i e s o f these e l e c t r o p h i l i c m e t a b o l i t e s ( I - X I I I ) a r e d i s c u s s e d i n terms o f t h e i r m e t a b o l i c f o r m a t i o n and r e a c t i v i t y w i t h n u c l e o p h i l e s , s o l v o l y s i s and redox c h a r a c t e r i s t i c s , r e a c t i o n mechanisms, and t h e i r r o l e as u l t i m a t e carcinogenic metabolites. N-Sulfonyloxy Arylamides ( i ) The m e t a b o l i c f o r m a t i o n o f N - s u l f o n y l o x y - N - a c e t y l - 2 - a m i n o f l u o r e n e (N-sulfonyloxy-AAF) and i t s observed electrophilic reactivity, p r o v i d e d the f i r s t e v i d e n c e f o r t h e importance o f enzymatic con j u g a t i o n r e a c t i o n s i n chemical c a r c i n o g e n e s i s (23,24). This r e a c t i o n was shown t o be c a t a l y z e d by PAPS-dependent s u l f o t r a n s f e r a s e s t h a t a r e l o c a t e d p r e d o m i n a n t l y i n l i v e r c y t o s o l and has been s u b s e q u e n t l y demonstrated f o r N-hydroxy a r y l a m i d e m e t a b o l i t e s o f s e v e r a l other carcinogens, i n c l u d i n g N-acety1-4-aminobipheny1 (AABP), b e n z i d i n e , N-acetyl-2-aminophenanthrene and p h e n a c e t i n . Accordingly, thecontribution of this metabolic activation pathway t o t h e f o r m a t i o n o f c o v a l e n t l y - b o u n d adducts o f a r y l a m i d e s w i t h c e l l u l a r p r o t e i n s and n u c l e i c a c i d s has been t h e s u b j e c t o f numerous i n v e s t i g a t i o n s , and has been reviewed e x t e n s i v e l y by Mulder (25). From t h e s e and more r e c e n t d a t a (4,26,27) i t i s a p p a r e n t , p a r t i c u l a r l y i n t h e case o f N-hydroxy-AAF TN-OH-AAF), t h a t _in v i v o formation of reactive N-sulfonyloxy derivatives i s primarily



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r e s p o n s i b l e f o r the c a r c i n o g e n adducts w i t h h e p a t i c p r o t e i n , RNA, DNA and g l u t a t h i o n e (GSH) t h a t r e t a i n the N - a c e t y l group. With N-OH-AAF, f o r e x a m p l e , t h e s e N - a c e t y l a t e d a d d u c t s a c c o u n t f o r 70-80%, 60-80%, and 15-30% of the t o t a l b i n d i n g to r a t l i v e r p r o t e i n , RNA, and DNA, r e s p e c t i v e l y (28,30); and GSH-AAF adducts e x c r e t e d i n the b i l e account f o r about 10% of the dose g i v e n ( 2 6 ) . I n comparable s t u d i e s w i t h N-hydroxy-AABP (N-OH-AABP), N - a c e t y l a t e d adducts r e p r e s e n t about 10% and 20% of the RNA and DNA b i n d i n g , r e s p e c t i v e l y (3C0 ; and N - a c e t y l a t e d adducts d e r i v e d from 4 ' - f l u o r o N-OH-AABP and N , N ' - d i a c e t y l b e n z i d i n e amount t o 10-20% of the t o t a l DNA-bound p r o d u c t s (_3jL^,_32_). I n c o n t r a s t , o n l y d e a c e t y l a t e d adducts are d e t e c t a b l e i n r a t h e p a t i c DNA a f t e r a d m i n i s t r a t i o n of N - a c e t y l 4 - a m i n o s t i l b e n e (33) or N - a c e t y l - 7 - f l u o r o - 2 - a m i n o f l u o r e n e ( 3 4 ) , both of which induce tumors i n the l i v e r and o t h e r t i s s u e s . S i m i l a r l y , o n l y d e a c e t y l a t e d DNA adducts are found i n r a t l i v e r a f t e r treatment w i t h the e x t r a h e p a t i c a r y l a m i d e c a r c i n o g e n , N-acetyl-2-aminophenanthrene ( 3 5 ) , or w i t h the N - h y d r o x y - N - a c e t y l d e r i v a t i v e of the c o l o n carcinogen, 3,2'-dimethyl-4-aminobiphenyl (36). S t r u c t u r a l i d e n t i f i c a t i o n of the N - a c e t y l a t e d adducts found i n v i v o has shown t h a t b i n d i n g t o p r o t e i n or GSH i n v o l v e s p r e d o m i n a n t l y o r t h o - r i n g s u b s t i t u t i o n of the a r y l a m i d e w i t h the s u l f u r atom i n m e t h i o n i n e or c y s t e i n e , r e s p e c t i v e l y . I n c o n t r a s t , a r y l a m i d e b i n d i n g t o n u c l e i c a c i d s in. v i v o i n v o l v e s both ^ - s u b s t i t u t i o n at the C-8 p o s i t i o n of guanine and o r t h o - r i n g s u b s t i t u t i o n w i t h the e x o c y c l i c N atom of guanine (26,29-31,37,38). S e v e r a l s y n t h e t i c N - s u l f o n y l o x y a r y l a m i d e s have been prepared i n o r d e r t o compare t h e i r r e a c t i v i t y w i t h n u c l e o p h i l e s t o t h a t observed i n v i v o and i n i n v i t r o m e t a b o l i c systems. S y n t h e t i c N - s u l f o n y l o x y AAF r e a c t s a p p r e c i a b l y w i t h b o t h p r o t e i n or m e t h i o n i n e t o g i v e h i g h y i e l d s of ortho-methylmercapto d e r i v a t i v e s t h a t are i d e n t i c a l to those formed jLn v i v o . S i m i l a r l y , m e t h i o n i n e has been shown to t r a p 65-85% of N - s u l f o n y l o x y - A A F generated i n i n c u b a t i o n s c o n t a i n i n g PAPS, N-OH-AAF, and h e p a t i c c y t o s o l i c sulf©transferase ( 9 ) . NS u l f o n y l o x y - A A F a l s o r e a c t s w i t h GSH Ln v i t r o t o g i v e 1-, 3-, 4-, and 7-AAF r i n g - s u b s t i t u t e d g l u t a t h i o n - S - y l adducts ( 3 9 ) , of which two ( 1 - , 3-) are major b i l i a r y m e t a b o l i t e s ( 2 6 ) . N-(Guanosin-8-yl)AAF, a major i n v i v o adduct w i t h h e p a t i c RNA, can be prepared by r e a c t i o n of guanosine w i t h N - s u l f o n y l o x y - A A F or by _in v i t r o s u l f o t r a n s f e r a s e a c t i v a t i o n of N-OH-AAF i n the presence of RNA or guano s i n e (40_). R e a c t i o n of N - s u l f onyloxy-AAF w i t h DNA y i e l d s both N-(deoxyguanosin-8-yl)-AAF and 3 - ( d e o x y g u a n o s i n - N - y l ) - A A F , which are i d e n t i c a l t o the N - a c e t y l a t e d adducts found _in v i v o (30,41). However, a s i m i l a r r e a c t i o n w i t h deoxyguanosine i n an aqueous medium g i v e s o n l y the C 8 - s u b s t i t u t e d p r o d u c t ; w h i l e both C8- and ^ - s u b s t i t u t e d adducts can be prepared by r e a c t i o n of N - s u l f o n y l o x y - A A F w i t h deoxyguanosine i n anhydrous d i m e t h y l s u l f o x i d e / t r i e t h y l a m i n e ( 4 1 ) . Though much l e s s r e a c t i v e than N - s u l f o n y l o x y - A A F , N-sulfonyloxy e s t e r s of N-OH-AABP and i t s 4 ' - f l u o r o d e r i v a t i v e have been prepared and shown t o r e a c t w i t h m e t h i o n i n e t o g i v e o r t h o - s u b s t i t u t e d m e t h y l mercapto a r y l a m i d e s and w i t h DNA t o g i v e C8- and N - s u b s t i t u t e d d e o x y g u a n o s i n e - a r y l a m i d e adducts (reviewed i n 4 2 ) . A g a i n , o n l y C8s u b s t i t u t e d guanine d e r i v a t i v e s are o b t a i n e d on r e a c t i o n of N - s u l fonyloxy-AABP w i t h deoxyguanosine, guanosine, or RNA. N-Sulfonyloxy-N-acetyl-2-aminophenanthrene has been prepared and shown to r e a c t t o a l i m i t e d e x t e n t w i t h m e t h i o n i n e , deoxyguanosine and deoxy2

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KADLUBAR AND BELAND

adenosine t o g i v e 1-methvlmercapto, N - ( d e o x y g u a n o s i n - 8 - y l ) , and l-(deoxyadenosin-N -yl) derivatives, respectively. Metabolic f o r m a t i o n o f N - s u l f o n y l o x y p h e n a c e t i n has a l s o been proposed s i n c e hepatic sulfotransferase-catalyzed activation o f N-hydroxy p h e n a c e t i n l e a d s t o t h e f o r m a t i o n o f adducts w i t h p r o t e i n , n u c l e i c a c i d s and GSH (25,43). From these s t u d i e s and those i n v o l v i n g N-acetoxy arylamides ( v i d e i n f r a ) , i t i s c l e a r t h a t any proposed r e a c t i o n mechanism must account f o r the a b i l i t y o f d i f f e r e n t n u c l e o p h i l e s t o d i r e c t sub s t i t u t i o n t o the _N-, o r t h o - and m e t a - r i n g p o s i t i o n s o f t h e a r y l a m i d e and should be c o n s i s t e n t w i t h r e a c t i o n k i n e t i c s and w i t h s o l v o l y s i s or rearrangement p r o d u c t s found i n t h e r e a c t i o n medium. In this r e g a r d , s t u d i e s w i t h model compounds such as m e t h a n e s u l f o n a t e e s t e r s of N-hydroxy a c e t a n i l i d e s (44,45) and N - s u l f o n y l o x y a c e t a n i l i d e s (46) have been p a r t i c u l a r l y u s e f u l . These data i n d i c a t e t h a t r e a c t i v e N - s u l f o n y l o x y d e r i v a t i v e s undergo h e t e r o l y t i c cleavage o f t h e N-0 bond t o form an i n t i m a t e i o n p a i r c o n s i s t i n g o f a p a r t i a l l y d e l o c a l i z e d s i n g l e t n i t r e n i u m / c a r b e n i u m c a t i o n and t h e s u l f a t e a n i o n ( F i g u r e 2 ) , as o r i g i n a l l y proposed by S c r i b n e r e t a l . (47) and more r e c e n t l y supported by m o l e c u l a r o r b i t a l c a l c u l a t i o n s ( 4 8 ) . Collapse of t h e i o n p a i r by i n t e r n a l r e t u r n r e s u l t s i n an o - s u l f onyloxy a c e t a n i l i d e w h i l e reducing agents convert i t to the parent acetanilide. E v i d e n c e has a l s o been p r e s e n t e d t h a t h y d r o l y s i s o f the i o n p a i r may proceed through an imine i n t e r m e d i a t e which would account f o r met a- and p o s s i b l y ^ - s u b s t i t u t e d products (45,46). In a d d i t i o n , e a r l i e r s t u d i e s w i t h the m e t a b o l i c a l l y generated N - s u l fonyloxy ester of phenacetin (£-ethoxyacetanilide) i n d i c a t e t h a t N - a c e t y l benzoquinone imine i s formed as a r e a c t i v e i n t e r m e d i a t e (49). R e c e n t l y , t h e d e c o m p o s i t i o n o f N - s u l f o n y l o x y - A A F under aqueous c o n d i t i o n s has been f u r t h e r examined and appears t o be c o n s i s t e n t w i t h t h i s o v e r a l l mechanism ( 5 0 ) . That i s , t h e major products appear t o be 1- and 3 - s u l f onyloxy-AAF w i t h s m a l l amounts o f AAF, 4-hydroxy-AAF, and a dimer formed by a d d i t i o n o f t h e e l e c t r o p h i l e onto t h e aromatic r i n g o f another AAF m o l e c u l e ( 5 1 ) . Furthermore, the r e l a t i v e y i e l d s o f AAF c o u l d be i n c r e a s e d by a d d i t i o n o f t h e r e d u c i n g agent, a s c o r b i c a c i d ( 5 2 ) . The involvement o f t h e n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e anion p a i r as t h e major e l e c t r o p h i l i c r e a c t a n t from a r y l a m i d e carcinogens i s a l s o c o n s i s t e n t w i t h t h e nature o f the p r o d u c t s formed w i t h c e l l u l a r n u c l e o p h i l e s ( v i d e supra) and i s i n a c c o r d w i t h t h e P e a r s o n h a r d / s o f t acid-base concept o f e l e c t r o p h i l i c s u b s t i t u t i o n ( 5 3 ) . Thus, o r t h o - s u b s t i t u t i o n o f t h e a r y l a m i n e i s f a v o r e d by s o f t n u c l e o p h i l e s (RSCH , RSH, RNH ) w h i c h tend t o advance i o n p a i r s e p a r a t i o n r e s u l t i n g i n g r e a t e r charge d e r e a l i z a t i o n i n t h e a r o m a t i c ring ( 4 8 ) ; w h i l e ^ - s u b s t i t u t i o n i s f a v o r e d w i t h hard n u c l e o p h i l e s t h a t a t t a c k a t i g h t i o n p a i r w i t h a p o s i t i v e n i t r o g e n c e n t e r ( 5 4 ) . Both types o f s u b s t i t u t i o n r e p r e s e n t an S I r e a c t i o n mechanism w h i c h i s determined by t h e s t r e n g t h o f t h e s u l f a t e l e a v i n g group, a l o n g w i t h f o r m a t i o n o f t h e i o n p a i r whose o v e r a l l r e a c t i v i t y o r s e l e c t i v i t y (N v s . o r t h o s u b s t i t u t i o n ) can be i n f l u e n c e d by changes i n r e a c t i o n medium and by t h e nature o f t h e n u c l e o p h i l e (41,42,55). Although metabolically-formed N-sulfonyloxy arylamides are s t r o n g e l e c t r o p h i l e s , b i n d t o c e l l u l a r macromolecules, and have l o n g been c o n s i d e r e d u l t i m a t e c a r c i n o g e n s , t h e i r p r e c i s e r o l e i n a r y l 6


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R e a c t i o n Mechanism f o r N - S u l f o n y l o x y A r y l a m i d e s ( I ) . Ac, a c e t y l ; RSCH , m e t h i o n i n e ; RSH, g l u t a t h i o n e o r c y s t e i n e ; RNH , N - g u a n i n e and/or N - a d e n i n e - n u c l e o s i d e s , - n u c l e o t i d e s , o r - n u c l e i c a c i d s ; RCH, C 8 - g u a n i n e - n u c l e o s i d e s , - n u c l e o t i d e s , o r - n u c l e i c a c i d s , o r C7-AAF. 3

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amide t u m o r i g e n e s i s i s n o t c e r t a i n . F o r example, N - s u l f o n y l o x y - A A F i s n o t a d i r e c t - a c t i n g , l o c a l c a r c i n o g e n ( 5 6 ) , even though i t i s highly toxic (57). I t i s mutagenic when r e a c t e d w i t h p u r i f i e d _B. s u b t i l i s t r a n s f o r m i n g DNA ( 5 8 ) , b u t does n o t serve as a d i r e c t - a c t i n g mutagen i n the IS. typhimurium t e s t system (51,52,59,60) . D u r i n g c h r o n i c a d m i n i s t r a t i o n o f a c a r c i n o g e n i c dose o f AAF, h e p a t i c s u l f o t r a n s f e r a s e a c t i v i t y i s g r e a t l y d i m i n i s h e d ( 6 1 ) and d e a c e t y l a t e d arylamine-DNA adducts e v e n t u a l l y account f o r 97-100% o f t h e t o t a l adducts ( 6 2 ) . I n a d d i t i o n , e x t r a h e p a t i c t i s s u e s which have l i t t l e or no sulf©transferase a c t i v i t y and c o n t a i n o n l y d e a c e t y l a t e d a d d u c t s , a r e a l s o s u s c e p t i b l e t o AAF o r N-OH-AAF c a r c i n o g e n e s i s (63,64). However, s e n s i t i v i t y t o h e p a t i c tumor i n d u c t i o n by AAF c o r r e l a t e s w e l l w i t h h e p a t i c s u l f a t e a v a i l a b i l i t y and w i t h s e x , s t r a i n , and s p e c i e s d i f f e r e n c e s i n h e p a t i c sulf©transferase l e v e l s (reviewed i n 4^,25^). Thus, i t has been proposed t h a t N - s u l f o n y l o x y a r y l a m i d e s may n o t be r e s p o n s i b l e f o r i n i t i a t i n g h e p a t i c tumorigenes i s , b u t may r a t h e r s e r v e t o promote f i x a t i o n o f an i n i t i a t i n g l e s i o n through a c y t o t o x i c response t h a t induces c e l l r e p l i c a t i o n ( 25^, 6 0 ) . N-Acetoxy A r y l a m i d e s ( I I ) N-Acetoxy a r y l a m i d e s have been w i d e l y used as s y n t h e t i c models t o study e l e c t r o p h i l i c r e a c t i v i t y w i t h c e l l u l a r c o n s t i t u e n t s and they y i e l d r e a c t i o n p r o d u c t s s i m i l a r t o those observed w i t h t h e N - s u l f o n y l o x y e s t e r s . F u r t h e r m o r e , s i n c e they a r e h i g h l y c a r c i n o g e n i c a t l o c a l s i t e s o f a p p l i c a t i o n (56,65,66) they have a l s o been regarded as u l t i m a t e c a r c i n o g e n s (47jT~ However, t h e N-acetoxy e s t e r s a r e g e n e r a l l y l e s s r e a c t i v e than t h e c o r r e s p o n d i n g s u l f o n y l o x y d e r i v a t i v e s , they e x h i b i t much l o n g e r h a l f - l i v e s i n aqueous s o l u t i o n (41,55,57,67-71) and t h e i r r e a c t i o n mechanism i s d e c i d e d l y more complex. They r e a c t , a t l e a s t i n p a r t by an S ^ l mechanism i n v o l v i n g i o n p a i r f o r m a t i o n s i m i l a r t o t h a t shown i n F i g u r e 2. This i s supported by: a) t h e i r lower e l e c t r o p h i l i c r e a c t i v i t y and s e l e c t i v i t y i n comparison t o N - s u l f onyloxy e s t e r s which i s due t o t h e d e c r e a s e d s t r e n g t h and h a r d n e s s o f t h e a c e t a t e l e a v i n g g r o u p (41,55,67,72); b) t h e i r thermal rearrangement t o o r t h o - a c e t o x y a r y l amides ( 6 9 ) ; c ) t h e i r f a c i l e r e d u c t i o n t o t h e parent arylamide (68,73); ~d) t h e i r c o n v e r s i o n t o r e a c t i v e imines (45,74); and e) t h e i r r e a c t i v i t y w i t h n u c l e o p h i l e s t o g i v e N^-, o r t h o - and metas u b s t i t u t e d p r o d u c t s (42,75). Y e t h e t e r o l y t i c c l e a v a g e a t t h e N-0 bond must occur t o o n l y a minor e x t e n t because u n l i k e N - s u l f o n y l o x y a r y l a m i d e s (46,50), N-acetoxy a r y l a m i d e s have been shown t o undergo p r e f e r e n t i a l l y cleavage o f e s t e r l i n k a g e t o form a hydroxamate a n i o n and presumably an a c e t y l c a t i o n which would account f o r the observed a c e t y l a t i o n o f l y s i n e i n p r o t e i n s and r i b o s e i n n u c l e i c a c i d s (66,72,76,77). E v i d e n c e has a l s o been p r e s e n t e d that N-acyloxy a r y l a m i d e s may decompose h o m o l y t i c a l l y t o y i e l d f r e e r a d i c a l s t h a t c o u l d a r y l a m i d a t e DNA bases and a l s o r e s u l t i n DNA-protein c r o s s l i n k s (78-81). However, i n v i e w o f t h e r e l a t i v e s t a b i l i t y o f Nacetoxy a r y l a m i d e s i n aqueous media, t h e i r r a p i d r e a c t i o n w i t h added n u c l e o p h i l e s , and t h e f a i l u r e t o d e t e c t r a c e m i z a t i o n t o N - [ ( ( ^ - 0 ) acetoxy]-arylamides on prolonged incubation of N-[(carbonyl- 0)a c e t o x y ] - a r y l a m i d e s i n t h e absence o f n u c l e o p h i l e s ( 8 2 ) , i t appears t h a t an S^2 r e a c t i o n i ^ y o l v i n ^ b | ^ ^ e c | ^ displacement o f a c e t a t e l 8

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or hydroxamate by t h e a t t a c k i n g n u c l e o p h i l e r e p r e s e n t s a more p r o b a b l e mechanism t o account f o r t h e major a r y l a m i d a t e d o r a c e t y l a t e d p r o d u c t s o b t a i n e d (27,75,76,82). The r o l e o f N - a c e t o x y a r y l a m i d e s as m e t a b o l i c a l l y f o r m e d u l t i m a t e c a r c i n o g e n s in v i v o a l s o appears t o be l i m i t e d . Their enzymatic f o r m a t i o n v i a p e r o x i d a t i o n o f N-hydroxy a r y l a m i d e s can be e x c l u d e d s i n c e t i s s u e s c o n t a i n i n g h i g h l e v e l s o f p e r o x i d a s e s such as the r a t mammary g l a n d (83) and t h e dog u r i n a r y b l a d d e r (84) do n o t form a c e t y l a t e d carcinogen-DNA adducts in v i v o ( 6 3 ) . T h e i r non enzymatic f o r m a t i o n by r e a c t i o n o f a c e t y l coenzyme A w i t h N-hydroxy a r y l a m i d e s (6^) cannot be e x c l u d e d ; however, even i f formed, t h e i r d i r e c t r e a c t i o n w i t h c e l l u l a r DNA appears u n l i k e l y as treatment o f c u l t u r e d c e l l s w i t h s y n t h e t i c N-acetoxy AAF (85,86) r e s u l t s p r i m a r i l y i n d e a c e t y l a t e d arylamine-DNA a d d u c t s , a p p a r e n t l y due t o r a p i d N - d e a c e t y l a t i o n t o form t h e r e a c t i v e N-acetoxy a r y l a m i n e ( V ) . N-Glucuronyloxy

A r y l a m i d e s ( I I I ) and A r y l a m i n e s ( I V )

M e t a b o l i c c o n j u g a t i o n o f N-hydroxy a r y l a m i d e s t o form N - g l u c u r o n y l oxy e t h e r s ( i l l ) r e p r e s e n t s a major pathway f o r b i l i a r y and u r i n a r y e x c r e t i o n o f a r o m a t i c amine c a r c i n o g e n s (87,88). W h i l e these c o n j u gates a r e g e n e r a l l y c o n s i d e r e d t o be s t a b l e d e t o x i f i c a t i o n p r o d u c t s , the N - g l u c u r o n y l o x y d e r i v a t i v e s o f AAF, N - a c e t y l - 4 - a m i n o s t i l b e n e , p h e n a c e t i n , b u t n o t o f AABP o r N-acetyl-2-aminophenanthrene, have been shown t o r e a c t s l o w l y e i t h e r w i t h p r o t e i n , n u c l e i c a c i d s , o r t h e i r c o n s t i t u e n t s (89-91). S i n c e r e a c t i o n o f N-glucuronyloxy-AAF w i t h m e t h i o n i n e and g u a n o s i n e y i e l d s o r t h o - m e t h y l m e r c a p t o and N - ( g u a n o s i n - 8 - y l ) d e r i v a t i v e s (89) , r e s p e c t i v e l y , a r e a c t i o n mechanism i n v o l v i n g f o r m a t i o n o f a n i t r e n i u m / c a r b e n i u m c a t i o n g l u c u r o n y l l a c t o n a t e a n i o n p a i r can be envisaged ( F i g u r e 3, path a ) . S t u d i e s on the mechanism o f d e c o m p o s i t i o n o f N - g l u c u r o n y l o x y phenac e t i n (92) a r e c o n s i s t e n t w i t h t h i s h y p o t h e s i s as o r t h o - g l u c u r o n y l o x y p h e n a c e t i n was the major rearrangement p r o d u c t , and evidence f o r an imine i n t e r m e d i a t e (45,92) l e a d i n g t o a m e t a - s u b s t i t u t e d d e r i v a t i v e and t o N - a c e t y l benzoquinone imine and i t s r e a c t i o n p r o d u c t s was obtained. The r e d u c t i o n p r o d u c t , p h e n a c e t i n , was a l s o o b t a i n e d a l t h o u g h i t s f o r m a t i o n was n o t i n c r e a s e d by a s c o r b a t e . However, an i n t e r n a l redox p r o c e s s y i e l d i n g p h e n a c e t i n and s a c c h a r i c a c i d i s plausible. C o n v e r s i o n o f N-glucuronyloxy-AAF t o an N - g l u c u r o n y l o x y a r y l amine ( I V ) has a l s o been demonstrated ( F i g u r e 3, paths b and c ) . T h i s can occur s p o n t a n e o u s l y a t a l k a l i n e pH by m i g r a t i o n o f t h e N - a c e t y l group t o t h e 2 ' - h y d r o x y l o f t h e g l u c u r o n y l moiety (93) o r i n t i s s u e s by enzymatic N - d e a c e t y l a t i o n ( 9 4 ) . N-Glucuronyloxy-2a m i n o f l u o r e n e (AF) i s h i g h l y e l e c t r o p h i l i c , d i r e c t l y mutagenic, and r e a c t s w i t h n u c l e i c a c i d s and w i t h m e t h i o n i n e and guanosine (and 5 ' - g u a n y l i c a c i d ) t o g i v e t h e c o r r e s p o n d i n g ortho-methylmercapto and N-(guan-8-yl) d e r i v a t i v e s ($9_>95_,j>6_), presumably v i a an S 1 mecha nism. I n t e r e s t i n g l y , enzymatic f o r m a t i o n o f N - g l u c u r o n y l o x y a r y l a mines by d i r e c t O - g l u c u r o n i d a t i o n o f N-hydroxy a r y l a m i n e s does n o t appear t o o c c u r , as o n l y s t a b l e N-hydroxy a r y l a m i n e N - g l u c u r o n i d e s are o b t a i n e d i n i n v i t r o h e p a t i c microsomal i n c u b a t i o n s ( 1 6 ) . N - G l u c u r o n y l o x y a r y l a m i d e s do n o t appear t o be i m p o r t a n t i n h e p a t o c a r c i n o g e n e s i s as t h e i r i n c r e a s e d m e t a b o l i c f o r m a t i o n does n o t result i n i n c r e a s e d h e p a t i c macromolecular binding (4,25). N



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F i g u r e 3. R e a c t i o n Mechanism f o r N - G l u c u r o n y l o x y A r y l a m i d e s ( I I I ) and A r y l a m i n e s ( I V ) . AC.; a c e t y l ; R S C H , m e t h i o n i n e ; RCH, C8-guanine-nucleosides, - n u c l e o t i d e s , o r - n u c l e i c a c i d s . Pathways a, b , and c are d i s c u s s e d i n the t e x t . 3



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P r o l o n g e d r e s i d e n c e i n the i n t e s t i n e o r u r i n a r y b l a d d e r lumen c o u l d a l l o w time f o r s i g n i f i c a n t r e a c t i o n w i t h t i s s u e components; however, N-glucuronyloxy-AAF was o n l y weakly c a r c i n o g e n i c a t l o c a l subcu taneous s i t e s o f a p p l i c a t i o n ( 8 9 ) . Enzymatic d e a c e t y l a t i o n t o N - g l u c u r o n y l o x y - A F has been d e t e c t e d i n h e p a t i c t i s s u e but t h i s a c t i v i t y i n d i f f e r e n t s p e c i e s does n o t c o r r e l a t e w i t h t h e i r r e l a t i v e s u s c e p t i b i l i t y t o AAF h e p a t o c a r c i n o g e n e s i s ( 9 4 ) . On the o t h e r hand, the a l k a l i n e pH-induced c o n v e r s i o n t o a r e a c t i v e d e r i v a t i v e may p l a y an important r o l e i n u r i n a r y b l a d d e r c a r c i n o g e n e s i s (87) by AAF and o t h e r a r y l a m i d e s i n those s p e c i e s o r i n d i v i d u a l s where normal u r i n e pH i s a l k a l i n e ( e . g . normal r a b b i t u r i n e pH i s 8.5-9.0). N-Acetoxy A r y l a m i n e s (V) E a r l y s t u d i e s on t h e jLn v i t r o m e t a b o l i c a c t i v a t i o n o f c a r c i n o g e n i c N-hydroxy a r y l a m i n e s i n d i c a t e d t h a t N-acetoxy a r y l a m i n e s (V) a r e formed as h i g h l y r e a c t i v e i n t e r m e d i a t e s t h a t y i e l d adducts w i t h p r o t e i n s and n u c l e i c a c i d s (40,97). W i t h N-hydroxy a r y l a m i d e s as s u b s t r a t e s , an enzyme mechanism i n v o l v i n g i n t r a m o l e c u l a r N , 0 - a c e t y l t r a n s f e r was proposed ( 9 8 ) ; w h i l e an i n t e r m o l e c u l a r p r o c e s s c o u l d be demonstrated u s i n g N-hydroxy a r y l a m i n e s as s u b s t r a t e s and N-hydroxy a r y l a m i d e s as a c e t y l donors (^?). S i n c e t h a t time, t h i s a c y l t r a n s f e r a s e has been e x t e n s i v e l y c h a r a c t e r i z e d (J_) and p u r i f i e d t o homo g e n e i t y from h e p a t i c and e x t r a h e p a t i c t i s s u e s o f s e v e r a l s p e c i e s (reviewed i n 100). More r e c e n t l y , Flammang e_t a l . (7,101) have shown t h a t a c e t y l coenzyme A can serve e f f e c t i v e l y as an a c e t y l donor f o r t h i s enzyme, c a t a l y z i n g the apparent d i r e c t O - a c e t y l a t i o n of s e v e r a l c a r c i n o g e n i c N-hydroxy a r y l a m i n e s . Because o f t h e i r i n s t a b i l i t y and h i g h r e a c t i v i t y , s y n t h e t i c N-acetoxy a r y l a m i n e s have never been i s o l a t e d (97,99). However, NMR s p e c t r a l evidence f o r the e x i s t e n c e o f N - a c e t o x y - 4 - a m i n o q u i n o l i n e 1-oxide has been o b t a i n e d (102,103); and N-acetoxy-4-aminoazobenzene (104), N-acetoxy-2-amino-6-methyldipyrido [l,2-a:3 ,2 -d]imidazole (N-acetoxy-Glu-P-1); r e f . 1 0 5 ) , and N-acetoxy-3-amino-l-methyl-5Hp y r i d o [ 4 , 3 - b ] i n d o l e (N-acetoxy-Trp-P-2; r e f . 106) have been prepared as i n t e r m e d i a t e s and then r e a c t e d w i t h n u c l e o s i d e s o r n u c l e i c a c i d s t o a f f o r d N-(guan-8-yl) p r o d u c t s . I n each o f these c a s e s , a more s t a b l e imino tautomer can e x i s t ( F i g u r e 4 ) . S i m i l a r attempts a t p r e p a r a t i o n o f N-acetoxy-4-aminobiphenyl have n o t been s u c c e s s f u l (107); however, N - a c e t o x y - N - t r i f l u o r o a c e t y l - 4 - a m i n o b i p h e n y l has been prepared and shown t o r e a c t r a p i d l y i n aqueous b u f f e r w i t h guanosine (or 5 - g u a n y l i c acid) to give N-(guan-8-yl)-4-aminobiphenyl d e r i v a t i v e s , a p p a r e n t l y by s e q u e n t i a l d e t r i f l u o r o a c e t y l a t i o n and g e n e r a t i o n o f an e l e c t r o p h i l i c N-acetoxy a r y l a m i n e (108). Evidence f o r t h e f o r m a t i o n o f o t h e r N-acetoxy a r y l a m i n e s _iri s i t u has been o b t a i n e d by treatment o f N-hydroxy a r y l a m i n e s w i t h a c e t i c anhydride i n b u f f e r e d aqueous s o l u t i o n s c o n t a i n i n g N - a c e t y l m e t h i o n i n e which yielded the corresponding ortho-methylmercapto a r y l a m i n e s ( 9 7 ) . W i t h i n v i t r o m e t a b o l i c a c t i v a t i o n systems, e n z y m a t i c a l l y generated N-acetoxy arylamines have a l s o b e e n shown t o r e a c t with N - a c e t y l m e t h i o n i n e o r 2-mercaptoethanol t o y i e l d ortho-aIkylmereapto a r y l a m i n e s (68,97,99) and w i t h n u c l e o s i d e s o r n u c l e i c a c i d s t o g i v e N - ( g u a n - 8 - y l ) - and o r t h o - ( g u a n - N - y 1 ) - a r y l a m i n e s (97,101,103,104). From t h e i r h i g h r e a c t i v i t y and n u c l e o p h i l i c s e l e c t i v i t y , i t seems l i k e l y t h a t N-acetoxy a r y l a m i n e s r e a d i l y undergo h e t e r o l y t i c f

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4. R e a c t i o n M e c h a n i s m f o r N - A c e t o x y A r y l a m i n e s ( V ) . A c , a c e t y l ; RSCH m e t h i o n i n e ; RNH , N - g u a n i n e - n u c l e o s i d e s , - n u c l e o t i d e s , o r - n u c l e i c a c i d s ; RCH, C8-guanine-nucleosides, -nucleotides, or - n u c l e i c a c i d s . Pathways and h e t e r o l y t i c c l e a v a g e s a and b a r e d i s c u s s e d i n t h e t e x t . Dashed arrows i n d i c a t e proposed pathways. 2

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c l e a v a g e t o form s i n g l e t n i t r e n i u m / c a r b e n i u m c a t i o n - a c e t a t e anion p a i r s ( F i g u r e 4, path a ) . N u c l e o p h i l i c a t t a c k by RSCH , RNH , o r RCH would then g i v e t h e observed o r t h o - and ^ - s u b s t i t u t e d p r o d u c t s . Under a c i d i c c o n d i t i o n s , h y d r o l y s i s o f N - a c e t o x y - 4 - a m i n o q u i n o l i n e 1-oxide t o 4 - h y d r o x y a m i n o q u i n o l i n e - l - o x i d e ( F i g u r e 4, path b) has a l s o been observed (102). A l t h o u g h t h e i d e n t i f i c a t i o n o f decompo s i t i o n p r o d u c t s o f c h e m i c a l l y o r e n z y m a t i c a l l y - g e n e r a t e d N-acetoxy a r y l a m i n e s i n n e u t r a l aqueous s o l u t i o n has n o t been r e p o r t e d , model s t u d i e s w i t h N-benzoyloxy-4-aminophenanthrene (109) suggest t h a t i n t e r n a l rearrangement t o an o r t h o - a c e t o x y a r y l a m i n e and an N-hydroxy a r y l a c e t a m i d e should occur ( F i g u r e 4, dashed a r r o w s ) . The l a t t e r c o n v e r s i o n has i m p o r t a n t i m p l i c a t i o n s f o r enzyme mechanisms. Thus, f o r N-hydroxy a r y l a m i d e N , 0 - a c e t y l t r a n s f e r a s e , c o n v e r s i o n t o an N-acetoxy a r y l a m i n e and i n t e r n a l r e t u r n t o an N-hydroxy a r y l a c e t a mide r e p r e s e n t s a c y c l i c p r o c e s s w h i c h would t e r m i n a t e upon a d d i t i o n of a n u c l e o p h i l e and may be r e s p o n s i b l e f o r t h e s u i c i d e i n a c t i v a t i o n of t h e enzyme ( 9 9 ) . F o r N-hydroxy a r y l a m i n e O - a c e t y l a s e , t h e rearrangement o f t h e i n i t i a l N-acetoxy a r y l a m i n e i n t e r m e d i a t e t o an N-hydroxy a r y l a c e t a m i d e product r e p r e s e n t s an o v e r a l l enzymatic N - a c e t y l a t i o n o f an N-hydroxy a r y l a m i n e , which i s a well-documented m e t a b o l i c pathway f o r a r o m a t i c amines (1_). An i m p o r t a n t r o l e f o r N-acetoxy a r y l a m i n e s as u l t i m a t e c h e m i c a l c a r c i n o g e n s seems l i k e l y i n v i e w o f t h e i r h i g h r e a c t i v i t y , t h e wide t i s s u e and s p e c i e s d i s t r i b u t i o n (7,98) o f enzyme(s) t h a t c a t a l y z e t h e i r f o r m a t i o n , and t h e p r e v a l e n c e o f n o n - a c e t y l a t e d arylamine-DNA adducts i n c a r c i n o g e n - t a r g e t t i s s u e s (110). In addition, synthetic N - a c e t o x y - 4 - a m i n o q u i n o l i n e - l - a c e t a t e , which generates t h e acetoxy a r y l a m i n e on r e a c t i o n w i t h t h i o l s (102,103), i s h i g h l y c a r c i n o g e n i c at s i t e s o f a p p l i c a t i o n ( 1 1 1 ) . R e c e n t l y , S a i t o et_ a l . ( 8 ) have shown t h a t t h e N-hydroxy m e t a b o l i t e s o f t h e mutagenic h e t e r o c y c l i c amines, Trp-P-2 and G l u - P - 1 , a r e m e t a b o l i c a l l y a c t i v a t e d t o u l t i m a t e mutagens by an a c e t y l coenzyme A-dependent enzyme p r e s e n t w i t h i n t h e t e s t b a c t e r i u m , as o r i g i n a l l y proposed by S a k a i £t a l . (112) and McCoy e_t a l . (113). Thus, m e t a b o l i c f o r m a t i o n o f N-acetoxy a r y l a m i n e s would appear a major pathway f o r both m u t a t i o n i n d u c t i o n and i n i t i a t i o n o f c a r c i n o g e n e s i s .


3

2

N-Sulfonyloxy Arylamines ( V I ) F o r c e r t a i n c a r c i n o g e n i c p r i m a r y N-hydroxy a r y l a m i n e s , m e t a b o l i c 0- s u l f o n y l a t i o n t o a r e a c t i v e e s t e r has been demonstrated. With r a t h e p a t i c sulf©transferase p r e p a r a t i o n s , the PAPS-dependent a c t i v a t i o n of N-hydroxy d e r i v a t i v e s o f 4-aminobiphenyl, 4-aminoazobenzene, 1- naphthylamine, and 2-naphthylamine y i e l d e d e l e c t r o p h i l i c i n t e r mediates t h a t formed adducts w i t h m e t h i o n i n e o r n u c l e i c a c i d s ; w h i l e N-hydroxy-4-aminostilbene, N-hydroxy-3,2'-dimethyl-4-aminobiphenyl, N-hydroxy-N -acetybenzidine and N-hydroxy-AF were n o t a c t i v a t e d i n t h i s i n v i t r o system (9,37,101,114). By comparison, mouse h e p a t i c sulf©transferase has r e c e n t l y been shown t o c a t a l y z e t h e a c t i v a t i o n of both N-hydroxy-AF (10) and N-hydroxy-4-aminoazobenzene (115) t o i n t e r m e d i a t e s t h a t r e a c t w i t h guanosine t o y i e l d N-(guan-8-yl) products. L i k e t h e N-acetoxy a r y l a m i n e s , a r e a c t i o n mechanism f o r N - s u l f o n y l o x y e s t e r s would be expected t o i n v o l v e f o r m a t i o n o f a n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e a n i o n p a i r which then r e a c t s w i t h 1



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m e t h i o n i n e i n p r o t e i n s and guanine ( o r adenine) i n n u c l e i c a c i d s t o g i v e o r t h o - and N - s u b s t i t u t e d p r o d u c t s ( F i g u r e 5 ) . I n t e r e s t i n g l y , the s i m p l e e l e c t r o p h i l i c e s t e r , h y d r o x y l a m i n e - O - s u l f o n i c a c i d , a l s o r e a c t s w i t h guanosine under a c i d i c c o n d i t i o n s t o g i v e t h e C8-subs t i t u t e d araino-guanosine a p p a r e n t l y by a s i m i l a r mechanism ( 1 1 6 ) . Furthermore, t h i s mechanism i s c o n s i s t e n t w i t h d e c o m p o s i t i o n p r o d u c t s i d e n t i f i e d from sulf©transferase i n c u b a t i o n s o f N-hydroxy-2naphthylamine (9) and w i t h rearrangement p r o d u c t s observed w i t h s y n t h e t i c a l l y - p r e p a r e d N - s u l f o n y l o x y a n i l i n e and N - s u l f o n y l o x y - 2 naphthylamine (117). That i s , t h e o r t h o - s u l f o n y l o x y a r y l a m i n e was a major product and t h i s c o u l d a r i s e by i n t e r n a l r e t u r n upon c o l l a p s e of t h e i o n p a i r ( o f . N - s u l f o n y l o x y a r y l a m i d e s ) . N-Hydroxy-N-sulfonyl-2-naphthylamine may a l s o be f o r m e d as an i n t e r m e d i a t e rearrangement p r o d u c t as i t has been r e p o r t e d (117,118) t o decompose to ortho-sulfonyloxy-2-naphthylamine and 2 - a m i n o - l - n a p h t h o l , t h e l a t t e r o f which was a l s o d e t e c t e d i n t h e sulf©transferase i n c u b a t i o n w i t h N-hydroxy-2-naphthylamine ( 9 ) . The e l e c t r o p h i l i c i o n p a i r a l s o appeared t o undergo a f a c i l e r e d u c t i o n t o 2-naphthylamine. I n t h i s m e t a b o l i c a c t i v a t i o n system, t h i s proceeded a t t h e expense o f N-hydroxy-2-naphthylamine, w h i c h was o x i d i z e d t o 2,2'-azoxynaphthal e n e ; however, o t h e r r e d u c i n g agents may serve t h i s purpose Jjri v i v o and e f f e c t i v e l y d e t o x i f y t h e r e a c t i v e e s t e r . S i m i l a r redox p r o c e s ses c o u l d occur w i t h N-acetoxy a r y l a m i n e s and o t h e r p r i m a r y a r y l amine O-esters b u t t h i s has n o t y e t been i n v e s t i g a t e d . The r o l e o f N - s u l f o n y l o x y a r y l a m i n e s as u l t i m a t e c a r c i n o g e n s appears t o be l i m i t e d . F o r N-hydroxy-2-naphthylamine, c o n v e r s i o n by r a t h e p a t i c sulf©transferase t o a N - s u l f o n y l o x y m e t a b o l i t e r e s u l t s p r i m a r i l y i n d e c o m p o s i t i o n t o 2-amino-l-naphthol and 1 - s u l f o n y l o x y 2-naphthylamine which a r e a l s o major u r i n a r y m e t a b o l i t e s ; and r e a c t i o n w i t h added n u c l e o p h i l e s i s v e r y low, which suggests an o v e r a l l d e t o x i f i c a t i o n process (9,17). However, f o r 4-aminoazobenzene and N-hydroxy-AAF, which a r e p o t e n t hepatocarcinogens i n t h e newborn mouse, evidence has been p r e s e n t e d t h a t s t r o n g l y i m p l i c a t e s t h e i r N - s u l f o n y l o x y a r y l a m i n e e s t e r s as u l t i m a t e h e p a t o c a r c i n o g e n s i n t h i s s p e c i e s (10,104). T h i s i n c l u d e s the i n h i b i t i o n o f arylamine-DNA adduct f o r m a t i o n and t u m o r i g e n e s i s by t h e s u l f o t r a n s f e r a s e i n h i b i t o r p e n t a c h l o r o p h e n o l , t h e reduced tumor i n c i d e n c e i n brachyraorphic mice t h a t a r e d e f i c i e n t i n PAPS b i o s y n t h e s i s (10,115), and t h e r e l a t i v e l y low O - a c e t y l t r a n s f e r a s e a c t i v i t y of mouse l i v e r f o r N-hydroxy-4aminoazobenzene and N-OH-AF (7,114,115). O-Seryl

( O - P r o l y l ) E s t e r s ( V I I ) o f N-Hydroxy

Arylamines

The f o r m a t i o n o f 0 - s e r y l o r 0 - p r o l y l e s t e r s ( F i g u r e 1) o f c e r t a i n Nhydroxy a r y l a m i n e s has been i n f e r r e d from the o b s e r v a t i o n s t h a t h i g h l y r e a c t i v e i n t e r m e d i a t e s can be generated _in v i t r o by i n c u b a t i o n w i t h ATP, s e r i n e o r p r o l i n e , and t h e c o r r e s p o n d i n g aminoacyl tRNA s y n t h e t a s e s (11,12,119). F o r example, a c t i v a t i o n o f N-hydroxy4 - a m i n o q u i n o l i n e - l - o x i d e (119 ,120) , N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) t o n u c l e i c acid-bound p r o d u c t s was dem o n s t r a t e d u s i n g s e r y l - t R N A s y n t h e t a s e from y e a s t o r r a t a s c i t e s hepatoma c e l l s . More r e c e n t l y , h e p a t i c c y t o s o l i c p r o l y l - , b u t n o t s e r y l - , tRNA s y n t h e t a s e was shown t o a c t i v a t e N-hydroxy-Trp-P-2 ( 1 2 ) ; however, no a c t i v a t i o n was d e t e c t a b l e f o r t h e N-hydroxy metab o l i t e s o f AF, 3 , 2 - d i m e t h y l - 4 - a m i n o b i p h e n y l , or N -acetylbenzidine (122). ,

1



F i g u r e 5. 3

2

2

R e a c t i o n Mechanism f o r N - S u l f o n y l o x y A r y l a m i n e s ( V I ) . R S C H , m e t h i o n i n e ; R N H » N - g u a n i n e - and -adeninen u c l e o s i d e s o r - n u c l e i c a c i d s ; RCH, C 8 - g u a n i n e - n u c l e o s i d e s or - n u c l e i c a c i d s . The dashed arrow i n d i c a t e s a proposed pathway.

OH


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6

The i d e n t i f i c a t i o n o f C 8 - g u a n y l and N - a d e n y l a d d u c t s o f 4-aminoquinoline-l-oxide (102,103) i n DNA m o d i f i e d by t h e m e t a b o l i c a l l y - g e n e r a t e d 0 - s e r y l e s t e r and t h e s i m i l a r i t y o f t h e adduct p r o f i l e w i t h t h a t o b t a i n e d on r e a c t i o n o f DNA w i t h N-acetoxy-4-arainoq u i n o l i n e - 1 - o x i d e s u g g e s t an e l e c t r o p h i l i c r e a c t i o n m e c h a n i s m s i m i l a r t o t h a t f o r t h e N-acetoxy o r N - s u l f o n y l o x y arylamines ( F i g u r e s 4 and 5 ) . However, N - s e r y l o x y o r N - p r o l y l o x y arylamines have n o t been s y n t h e s i z e d and t h e d e c o m p o s i t i o n products of the e s t e r s generated i n v i t r o have n o t y e t been s t u d i e d . A l t h o u g h aminoacyl-tRNA s y n t h e t a s e s a r e n e c e s s a r y f o r p r o t e i n s y n t h e s i s i n a l l t i s s u e s , t h e i r importance i n c h e m i c a l c a r c i n o genesis i s d i f f i c u l t to assess. M u t a t i o n i n d u c t i o n by t h i s pathway has been s t u d i e d e x t e n s i v e l y ( 1 2 3 ) , y e t m e t a b o l i c a c t i v a t i o n i n a c a r c i n o g e n - t a r g e t t i s s u e has n o t been demonstrated. The o n l y e x c e p t i o n i s h e p a t i c p r o l y l - t R N A s y n t h e t a s e a c t i v a t i o n o f N-hydroxy-TrpP-2; however, h e p a t i c O - a c e t y l a t i o n o f t h i s s u b s t r a t e a l s o o c c u r s t o an a p p r e c i a b l e e x t e n t ( 1 2 ) . F u r t h e r i n v e s t i g a t i o n s i n v o l v i n g the use o f s p e c i f i c enzyme i n h i b i t o r s would be h e l p f u l i n a d d r e s s i n g t h i s problem. P r o t o n a t i o n o f N-Hydroxy A r y l a m i n e s

(VIII)

The f o r m a t i o n o f O-protonated N-hydroxy a r y l a m i n e s ( F i g u r e 6) under a c i d i c c o n d i t i o n s has been w e l l documented as an i n t e r m e d i a t e s t e p i n t h e Bamberger rearrangement t o form aminophenols and o t h e r o r t h o or p a r a - s u b s t i t u t e d p r o d u c t s (124-128). From a b s o r p t i o n s p e c t r a l d a t a i n v o l v i n g p r o t o n a t i o n e q u i l i b r i a ( 1 2 8 ) , t h e exchange e x p e r i ments o f [ 0]H20 i n t o p r o d u c t s o r s t a r t i n g m a t e r i a l (126,127), and from s t u d i e s o f r e a c t i o n k i n e t i c s (125,128), t h e p r o t o n a t e d h y d r o x y l a m i n e s appear t o be r e l a t i v e l y s t a b l e s p e c i e s whose r e a r r a n g e ment proceeds by an S ^ l mechanism w i t h e l i m i n a t i o n o f water as t h e rate-determining step. The e l e c t r o p h i l i c n a t u r e o f t h i s i n t e r m e d i a t e was i n i t i a l l y c o n s i d e r e d by H e l l e r e_t a l . ( 1 2 5 ) ; w h i l e K r i e k (15), who proposed t h a t e l i m i n a t i o n o f water r e s u l t e d i n a h i g h l y e l e c t r o p h i l i c a r y l n i t r e n i u m i o n , f i r s t demonstrated r e a c t i o n s w i t h biological nucleophiles. Since t h a t t i m e , t h e r e a c t i o n o f c a r c i n o g e n i c N-hydroxy a r y l a m i n e s w i t h n u c l e i c a c i d s under m i l d l y a c i d i c c o n d i t i o n s has been shown t o be an e f f e c t i v e procedure f o r p r e p a r a t i o n and i d e n t i f i c a t i o n o f a r y l a m i n e - n u c l e o s i d e adducts and b o t h o r t h o - and N - s u b s t i t u t e d p r o d u c t s have been o b t a i n e d (reviewed i n 110). These i n c l u d e o_-(guan-N - y l ) , o_-(guan-0 - y l ) , o_-(aden-N y l ) , N-(guan-8-yl), and N - ( a d e n - 8 - y l ) a d d u c t s . Of t h e s e , t h e N( g u a n - 8 - y l ) d e r i v a t i v e s have u s u a l l y been t h e major r e a c t i o n product. I n c o n t r a s t t o the r e a c t i v i t y o f N - s u l f o n y l o x y and N-acetoxy e s t e r s o f a r y l a m i d e s and a r y l a m i n e s , t h e r e l a t i v e r e a c t i v i t y o f p r o t o n a t e d N-hydroxy a r y l a m i n e s w i t h n u c l e o p h i l e s g e n e r a l l y d e c r e a s e s i n t h e o r d e r : DNA > d e n a t u r e d DNA > rRNA = p r o t e i n > tRNA » n u c l e o t i d e s s n u c l e o s i d e s s m e t h i o n i n e s GSH (2,13-17,30,36,40,127,129, 130). F u r t h e r m o r e , t h e r a t e o f r e a c t i o n w i t h DNA was found t o be not o n l y f i r s t o r d e r w i t h r e s p e c t t o N-hydroxy a r y l a m i n e concen t r a t i o n , b u t a l s o f i r s t o r d e r w i t h r e s p e c t t o DNA c o n c e n t r a t i o n (127,129,131). These d a t a suggested t h a t t h e r e a c t i o n mechanism was e i t h e r S 2 o r S I w i t h t h e i n v o l v e m e n t o f an i n t e r m e d i a t e i n t h e N N XT


356


r a t e - d e t e r m i n i n g step ( 1 3 2 ) . I n v i e w o f the r e l a t i v e l y slow e x change ( 4 0 % / h r ) o f [ 0 ] H 0 i n t o N - h y d r o x y - l - n a p h t h y l a m i n e a t pH 5, the slower c o n v e r s i o n o f N - h y d r o x y - l - n a p h t h y l a m i n e t o aminonaphthols ( 1 % / h r ) , i t s r a p i d r e a c t i o n w i t h DNA. ( 2 5 % / h r ) , and t h e e s t a b l i s h e d S ^ l r e a c t i o n mechanism f o r the Bamberger r e a c t i o n , a p a r t i a l l y d e l o c a l i z e d h y d r a t e d n i t r e n i u m / c a r b e n i u m i o n i n t e r m e d i a t e ( F i g u r e 6) was proposed (127,132,133). T h i s i n t e r m e d i a t e i s analogous t o an i n t i m a t e i o n p a i r formed under n e u t r a l c o n d i t i o n s as d e s c r i b e d f o r the e l e c t r o p h i l i c O - e s t e r s o f N-hydroxy a r y l a m i d e s and a r y l a m i n e s (vide supra). Thus, the o v e r a l l r e a c t i v i t y and s e l e c t i v i t y ( N - v s . r i n g s u b s t i t u t i o n ) o f p r o t o n a t e d N-hydroxy a r y l a m i n e s should be d e t e r mined by a b i l i t y o f the n u c l e o p h i l e t o d e s o l v a t e the h y d r a t e d i o n , t o d e l o c a l i z e f u r t h e r t h e p o s i t i v e c h a r g e , and t o r e s u l t i n product f o r m a t i o n (132,134). Such a mechanism i s c o n s i s t e n t w i t h the p r e f e r e n t i a l formation o f N - s u b s t i t u t e d products from r e a c t i o n w i t h n u c l e i c a c i d s (110) and from s o l v o l y s i s o f N-hydroxy a r y l a m i n e s i n b e n z e n e / t r i f l u o r o a c e t i c a c i d (135); w h i l e a r y l r i n g - s u b s t i t u t e d products a r e p r e f e r e n t i a l l y o b t a i n e d on s o l v o l y s i s o f N-hydroxy arylamines (135) o r 1 - n a p h t h y l a z i d e (136) i n b e n z e n e / t r i f l u o r o m e t h a n e s u l f o n i c a c i d . A l t e r n a t i v e l y , upon d e s o l v a t i o n , a t r u e i o n p a i r c o u l d be formed between a n e g a t i v e l y charged n u c l e o p h i l e o r c a t a l y s t and t h e n i t r e n i u m / c a r b e n i u m c a t i o n , which c o u l d c o l l a p s e t o the product o r undergo i n t e r n a l rearrangement. However, t h e l a t t e r mechanism seems improbable s i n c e , u n l i k e t h e e l e c t r o p h i l i c O - e s t e r s , the r e a c t i v i t y o f p r o t o n a t e d N-hydroxy a r y l a m i n e s w i t h DNA i s u n a f f e c t e d by r e d u c i n g agents and t h e i r r e a c t i o n w i t h s t r o n g , low molecular-weight nucleophiles such as 4 - ( _ p - n i t r o b e n z y l ) p y r i d i n e cannot be d e t e c t e d (127,129,131). The e x c e p t i o n a l r e a c t i v i t y o f DNA f o r p r o t o n a t e d N-hydroxy a r y l a m i n e s can be r a t i o n a l i z e d by a t l e a s t two mechanisms. First, i n t e r c a l a t i o n o f the e l e c t r o p h i l i c i n t e r m e d i a t e between DNA bases c o u l d s t e r i c a l l y a s s i s t i n d e s o l v a t i o n and i n d i r e c t i n g t h e e l e c t r o p h i l i c c e n t e r o f the c a r c i n o g e n over the n u c l e o p h i l i c r e g i o n o f the DNA base. T h i s seems u n l i k e l y , however, as p r e t r e a t m e n t o f DNA w i t h c i s - P t , which decreased t h e DNA contour l e n g t h by 50%, f a i l e d t o reduce t h e r e a c t i v i t y o f N - h y d r o x y - l - n a p h t h y l a m i n e f o r t h e DNA (137). A second p o s s i b i l i t y i n v o l v e s an e l e c t r o s t a t i c a t t r a c t i o n between t h e e l e c t r o p h i l e and t h e phosphate backbone o f t h e DNA ( 7 7 ) . T h i s seems more p r o b a b l e s i n c e e i t h e r h i g h i o n i c s t r e n g t h o r s t o i c h i o m e t r i c ( t o DNA-P) amounts o f Mg s t r o n g l y i n h i b i t DNA adduct f o r m a t i o n (77,137). I n a d d i t i o n , e v i d e n c e has been presented t h a t N-hydroxy arylamine-DNA/RNA p h o s p h o t r i e s t e r s may be formed which induce s t r a n d breaks (137,138) and c o u l d serve as a c a t a l y s t f o r d e s o l v a t i o n and subsequent adduct f o r m a t i o n . The importance o f p r o t o n a t e d N-hydroxy a r y l a m i n e s as u l t i m a t e c a r c i n o g e n s has been suggested f o r some time (28,40,139). From s t u d i e s on t h e i r r e a c t i v i t y w i t h n u c l e i c a c i d s a t d i f f e r e n t pH's (2,15,16,63,130,131), the pK f o r p r o t o n a t i o n o f t h e N-hydroxy group appears t o be between pH 5 and 6; t h u s , a s i g n i f i c a n t p r o p o r t i o n (1-10%) o f t h e N-hydroxy d e r i v a t i v e e x i s t s as t h e p r o t o n a t e d form even under n e u t r a l c o n d i t i o n s . T h i s would account f o r the s i g n i f i c a n t l e v e l s o f c o v a l e n t m o d i f i c a t i o n o f DNA observed i n v i t r o by r e a c t i o n w i t h N-hydroxy a r y l a m i n e s a t n e u t r a l pH. C o n s e q u e n t l y , i t has been proposed t h a t in_ v i v o f o r m a t i o n o f n o n - a c e t y l a t e d aryl l 8


2

+

a



14.

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amine-DNA adducts may a r i s e , a t l e a s t i n p a r t , by t h e d i r e c t r e a c t i o n with protonated N-hydroxy arylamines (2,28,139) . This h y p o t h e s i s i s f u r t h e r supported by t h e o b s e r v a t i o n t h a t s y n t h e t i c o r m e t a b o l i c a l l y - g e n e r a t e d N-OH-AF r e a c t s a p p r e c i a b l y w i t h DNA i n i s o l a t e d l i v e r n u c l e i t o y i e l d d e t e c t a b l e l e v e l s o f N-(deoxyguanosin8-yl)-AF ( 2 ) . T h i s i s c o n s i s t e n t w i t h t h e h i g h c o n c e n t r a t i o n o f DNA w i t h i n c e l l n u c l e i ( c a . 50 mg/ml) and w i t h the f i r s t order r e l a t i o n between r e a c t i o n r a t e s and DNA c o n c e n t r a t i o n s . P r o t o n a t e d N-hydroxy a r y l a m i n e s have a l s o been proposed t o be u l t i m a t e c a r c i n o g e n s f o r t h e u r i n a r y b l a d d e r (16,17,140,141) s i n c e u r i n e pH i s s l i g h t l y a c i d i c i n a number o f s p e c i e s (14,142). F u r t h e r m o r e , p h a r m a c o k i n e t i c s t u d i e s have shown t h a t i n c r e a s e d u r i n e a c i d i t y and decreased frequency o f u r i n a t i o n a r e p r e d i c t i v e o f r e l a t i v e s p e c i e s s u s c e p t i b i l i t y t o u r i n a r y b l a d d e r c a r c i n o g e n e s i s (142); and n e o p l a s t i c t r a n s f o r m a t i o n o f c u l t u r e d human f i b r o b l a s t s by N-hydroxy a r y l a m i n e s i s g r e a t l y enhanced by i n c u b a t i o n a t pH 5 as compared t o pH 7 ( 1 4 3 ) . Nitrosoarenes (IX) N i t r o s o a r e n e s a r e r e a d i l y f o r m e d by t h e o x i d a t i o n o f p r i m a r y N-hydroxy a r y l a m i n e s and s e v e r a l mechanisms appear t o be i n v o l v e d . These i n c l u d e : 1) t h e m e t a l - c a t a l y z e d o x i d a t i o n / r e d u c t i o n t o n i t r o soarenes, azoxyarenes and a r y l a m i n e s (144); 2) t h e 0 - d e p e n d e n t , m e t a l - c a t a l y z e d o x i d a t i o n t o n i t r o s o a r e n e s (145); 3) t h e 0 - d e p e n d e n t , hemoglobin-mediated c o - o x i d a t i o n t o n i t r o s o a r e n e s and methem o g l o b i n (146); and 4) t h e 0 -dependent c o n v e r s i o n o f N-hydroxy arylamines to nitrosoarenes, nitrosophenols and n i t r o a r e n e s (147,148) . Each of these processes can i n v o l v e i n t e r m e d i a t e n i t r o x i d e r a d i c a l s , s u p e r o x i d e a n i o n r a d i c a l s , hydrogen p e r o x i d e and h y d r o x y l r a d i c a l s , a l l o f which have been observed i n model systems (149,151). Although these r a d i c a l s a r e e l e c t r o p h i l i c and have been suggested t o r e s u l t i n DNA damage (151,152), a c a u s a l r e l a t i o n s h i p has n o t y e t been e s t a b l i s h e d . N i t r o s o a r e n e s , on t h e o t h e r hand, a r e r e a d i l y formed i n i n v i t r o m e t a b o l i c i n c u b a t i o n s (2,153) and have been shown t o r e a c t c o v a l e n t l y w i t h l i p i d s (154), p r o t e i n s (28,155) and GSH (17,156-159). N i t r o s o a r e n e s a r e a l s o r e a d i l y reduced t o N-hydroxy a r y l a m i n e s by a s c o r b i c a c i d (17,160) and by reduced p y r i d i n e n u c l e o t i d e s (9,161) . The mechanism o f r e a c t i o n o f n i t r o s o a r e n e s w i t h GSH has been s t u d i e d e x t e n s i v e l y and i s known t o i n v o l v e an a d d i t i o n r e a c t i o n w i t h the t h i o l group t o form an N-hydroxy-N-(glutathion-S-yl)a r y l a m i n e adduct. T h i s i n t e r m e d i a t e can r e a r r a n g e t o an N - ( g l u t a t h i o n - S - y l ) - a r y l a m i n e S-oxide o r can be reduced t o an N-hydroxy a r y l a m i n e o r an N - ( g l u t a t h i o n - S - y l ) - a r y l a r a i n e ( F i g u r e 7 ) . I t i s i n t e r e s t i n g t o note t h a t 4-aminobiphenyl has r e c e n t l y been r e p o r t e d to form h i g h l e v e l s o f a hemoglobin adduct ( 5 % o f t h e dose) t h a t appears t o a r i s e by a d d i t i o n o f 4 - n i t r o s o b i p h e n y l t o a c y s t e i n y l s u l f h y d r y l group i n t h e p r o t e i n , forming an N-S l i n k a g e ( 1 6 2 ) . B i n d i n g o f n i t r o s o a r e n e s t o n u c l e i c a c i d s has been suggested ( 4 3 , 163), b u t n e g a t i v e r e s u l t s were o b t a i n e d i n subsequent s t u d i e s (40,159). Thus, t h e r o l e o f n i t r o s o a r e n e s as u l t i m a t e c a r c i n o g e n s per se seems u n l i k e l y , a l t h o u g h m o d i f i c a t i o n o f a c r i t i c a l c e l l u l a r p r o t e i n cannot be e x c l u d e d . A r o l e f o r n i t r o s o a r e n e s i n a r y l a m i n e c a r c i n o g e n e s i s has been 2

2

2



358

OH

-

OH

/

2


©

8 ^

N 8

(H O)

2

2

OH

V+RCH

+ RNH (ROHK '

(ROH) —%

^ - N H

^ - N H

2

©

H

K

2

NHR

CR -OR )

(-OR)

F i g u r e 6. R e a c t i o n Mechanism and F o r m a t i o n o f P r o t o n a t e d N - H y d r o x y Arylamines ( V I I I ) . R N H , N - g u a n i n e - and N - a d e n i n e nucleic a c i d s ; ROH, 0 - g u a n i n e - n u c l e i c a c i d s ; RCH, C8-guanine- and C 8 - a d e n i n e - n u c l e i c a c i d s . 2

8

2

6

OH + RSH SR

+ 2e"

+ 2e

fe

H + RSH

I N—SR

i

OH

F i g u r e 7. R e a c t i o n Mechanism f o r N i t r o s o a r e n e s ( I X ) , RSH, g l u t a t h i o n e o r c y s t e i n e .


t SR

14.

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359

suggested t o be due t o t h e i r f a c i l e i n t e r c o n v e r s i o n w i t h N-hydroxy a r y l a m i n e s by o x i d a t i o n and r e d u c t i o n and t h e i r r a p i d d e t o x i f i c a t i o n by r e a c t i o n w i t h GSH ( 1 5 9 ) . C o n s e q u e n t l y , a d d i t i o n o f a s c o r b i c a c i d s i g n i f i c a n t l y increased 2-nitrosofluorene mutagenicity (160); whereas, a d d i t i o n o f GSH s t r o n g l y i n h i b i t e d mutagenic a c t i v i t y (164) and GSH d e p l e t i o n h a s r e s u l t e d i n i n c r e a s e d DNA damage i n h e p a t o c y t e s by N-OH-AF ( 1 6 5 ) .


Iminoquinones (X) and D i i m i n e s ( X I ) The f o r m a t i o n o f iminoquinones (166,167) and d i i m i n e s (20,168) as i n t e r m e d i a t e s i n t h e o x i d a t i o n o f aminophenols and a r y l d i a m i n e s has been w e l l e s t a b l i s h e d . These i n t e r m e d i a t e s r e a d i l y undergo a d d i t i o n r e a c t i o n s w i t h n u c l e o p h i l e s t o y i e l d N-, o r t h o - , o r m e t a - s u b s t i t u t e d p r o d u c t s ( F i g u r e 8 ) . F o r example, 2 - a m i n o - l - n a p h t h o l , w h i c h has l o n g been suggested as a p r o x i m a t e carcinogenic metabolite of 2-naph t h y lamine ( 1 6 9 ) , i s r e a d i l y o x i d i z e d i n a i r o r by cytochrome c_ t o 2-imino-l-naphthoquinone. T h i s iminoquinone i s e l e c t r o p h i l i c and can b i n d c o v a l e n t l y t o p r o t e i n and DNA, can undergo r e a c t i o n w i t h aryl-NH2 groups t o g i v e m e t a - s u b s t i t u t e d p r o d u c t s , o r can h y d r o l y z e t o form 2-amino-l,4-naphthoquinone (19,166,167,170-172). In this r e g a r d , t h e major r e a c t i o n p r o d u c t o f 2-imino-l-naphthoquinone with DNA has been r e c e n t l y i d e n t i f i e d as 4-(deoxyguanosin-N - y l ) - 2 - a m i n o 1,4-naphthoquinoneimine ( 1 9 ) . D i i m i n e s a r e formed d i r e c t l y by p e r o x i d a t i v e m e t a b o l i s m o f aryldiamines. F o r example, 4 , 4 ' - d i i m i n o b i p h e n y l (or benzidined i m i n e ) , a p r o d u c t o f b e n z i d i n e p e r o x i d a t i o n whose f o r m a t i o n i n v o l v e s a c a t i o n r a d i c a l i n t e r m e d i a t e (20,168), r e a d i l y b i n d s t o p r o t e i n and n u c l e i c a c i d (173,174). This diimine a l s o reacts with i t s e l f t o form, an azo d^mer (20) o r r e a c t s w i t h GSH t o g i v e an o r t h o - s u b s t i t u t e d g l u t a t h i o n - S - y l c o n j u g a t e ( 1 7 5 ) , w i t h phenols t o g i v e an N - s u b s t i t u t e d i n d o d y e ( 1 7 6 ) , and w i t h DNA t o g i v e N-(deoxyguanosin-8-yl)-benzidine (174). Other s i m i l a r l y r e a c t i v e i m i n e s and iminoquinones have been shown t o be formed i n b i o l o g i c a l systems, n o t a b l y N-acetyl-jr-benzoquinone i m i n e , w h i c h has been i d e n t i f i e d as t h e major h e p a t o t o x i c m e t a b o l i t e o f acetaminophen and phenacetin (reviewed i n 91). Over t h e l a s t few y e a r s , t h e s i g n i f i c a n c e o f these i n t e r m e d i a t e s as u l t i m a t e c a r c i n o g e n s has r e c e i v e d new impetus s i n c e p r o s t a g l a n d i n H s y n t h a s e , a mammalian p e r o x i d a s e which i s w i d e l y d i s t r i b u t e d i n e x t r a h e p a t i c t i s s u e s ( 1 7 7 ) , can mediate t h e c o o x i d a t i o n o f s e v e r a l carcinogenic arylamines t o intermediates that bind c o v a l e n t l y t o p r o t e i n and n u c l e i c a c i d (20,168,178,179). For the u r i n a r y bladder c a r c i n o g e n , 2-naphthy1amine, t h e f o r m a t i o n o f 2 - a r a i n o - l - n a p h t h o l and i t s subsequent o x i d a t i o n t o 2-imino-l-naphthoquinone have been shown t o be p r i m a r i l y r e s p o n s i b l e f o r DNA b i n d i n g i n t h e i i i v i t r o p e r o x i dase system ( 1 8 0 ) . Furthermore, about 20-30% o f t h e 2 - n a p h t h y l amine-DNA adducts formed i n t h e dog u r i n a r y b l a d d e r , which c o n t a i n s h i g h l e v e l s o f p r o s t a g l a n d i n H synthase ( 8 4 ) , appears t o be d e r i v e d from the a d d i t i o n r e a c t i o n o f 2-imino-l-naphthoquinone (19). For b e n z i d i n e , another u r i n a r y b l a d d e r c a r c i n o g e n , t h e major b e n z i d i n e DNA adduct formed i n t h e p r o s t a g l a n d i n H synthase-mediated r e a c t i o n and i n t h e u r i n a r y b l a d d e r o f dogs g i v e n b e n z i d i n e was shown t o be i d e n t i c a l t o t h e N - ( g u a n - 8 - y l ) d e r i v a t i v e t h a t was p r e p a r e d by r e a c t i o n w i t h s y n t h e t i c 4,4'-diiminobiphenyl (174). Recently, the



360


F i g u r e 8.

R e a c t i o n Mechanisms f o r Iminoquinones (X) and I m i n e s ( X I ) . RNH , N - g u a n i n e - n u c l e i c acids or arylamines; RCH, C8guanine-nucleic a c i d s o r _ D _ - s u b s t i t u t e d p h e n o l s ; RSH, glutathione or cysteine. 2

2


14.

KADLUBAR AND BELAND


361


p e r o x i d a t i v e m e t a b o l i s m o f AF has been c a r e f u l l y s t u d i e d and found t o r e s u l t i n t h e f o r m a t i o n o f a " h e a d - t o - t a i l " dimer, 2-aminod i f l u o r e n y l a m i n e , whose f u r t h e r o x i d a t i o n t o a r e a c t i v e d i i m i n e may be r e s p o n s i b l e f o r macromolecular b i n d i n g (181-183). However, f o r each o f these c a r c i n o g e n s , t h e r e i s a l s o good evidence t h a t e l e c t r o p h i l i c r a d i c a l c a t i o n s (20,150,168,182) can be produced and t h a t these may y i e l d c o v a l e n t adducts w i t h p r o t e i n and n u c l e i c a c i d s . Further s t u d i e s on t h e i d e n t i f i c a t i o n o f these adducts should p r o v i d e u s e f u l i n f o r m a t i o n on the r o l e o f r a d i c a l i n t e r m e d i a t e s i n arylamine carcinogenesis. N - S u l f o n y l o x y ( X I I ) and N - A r y l n i t r o n e N-Methyl A r y l a m i n e s

( X I I I ) D e r i v a t i v e s of

Although several N-methyl-substituted a r y l a m i n e s have been shown t o be c a r c i n o g e n i c (184-186), m e t a b o l i c a c t i v a t i o n pathways have been i n v e s t i g a t e d p r i m a r i l y f o r the h e p a t o c a r c i n o g e n i c aminoazo dyes, N-methyl-4-aminoazobenzene (MAB) and i t s 3'-methyl d e r i v a t i v e (9,21, 22,187,188). N-Hydroxy-N-methyl a r y l a m i n e s a r e g e n e r a l l y regarded as p r o x 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 s (22,187,189) and have been shown t o be c o n v e r t e d t o e l e c t r o p h i l i c N - s u l f o n y l o x y d e r i v a t i v e s by h e p a t i c s u l f o t r a n s f e r a s e s (9,187) o r t o r e a c t i v e N - a r y l n i t r o n e s by a i r oxidation (21). M e t a b o l i c a l l y - f o r m e d N-sulfonyloxy-MAB was found t o r e a c t w i t h m e t h i o n i n e , g u a n o s i n e , and GSH t o g i v e o r t h o - m e t h y l m e r c a p t o , g u a n - 8 - y l , and o r t h o - g l u t a t h i o n - S - y l p r o d u c t s (9,190); and these were t h e same major adducts found i n v i v o i n r a t h e p a t i c p r o t e i n , n u c l e i c a c i d , and b i l e , r e s p e c t i v e l y , a f t e r MAB a d m i n i s t r a t i o n (191-193) . T h e s e s t u d i e s were a i d e d by t h e a v a i l a b i l i t y o f s y n t h e t i c N-benzoyloxy e s t e r s which show s i m i l a r r e a c t i v i t y toward n u c l e o p h i l e s and a r e p o t e n t , d i r e c t - a c t i n g c a r c i n o g e n s and mutagens (57,194,195). A d d i t i o n a l experiments have shown t h a t s u b s t i t u t e d guan-N - y l and aden-N - y l d e r i v a t i v e s a r e a l s o formed i n DNA a f t e r r e a c t i o n Ln v i t r o w i t h N-benzoyloxy-MAB and a f t e r d o s i n g w i t h MAB i n vivo (196-198) , which suggests a similar reactivity for metabolically-formed N-sulfonyloxy esters. Thus, l i k e the N - s u l f o n y l o x y e s t e r s o f a r y l a m i d e s and o f p r i m a r y a r y l a m i n e s ( F i g u r e s 2 and 5 ) , a r e a c t i o n mechanism f o r N - s u l f o n y l oxy-N-methyl a r y l a m i n e s i s expected t o i n v o l v e f o r m a t i o n of a n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e a n i o n p a i r which r e a c t s t o g i v e b o t h N- o r r i n g - s u b s t i t u t e d p r o d u c t s , depending on t h e s o f t n e s s o r hardness o f t h e n u c l e o p h i l e ( F i g u r e 9 ) . R e c e n t l y , N-sulfonyloxy-MAB was prepared s y n t h e t i c a l l y and i t s s o l v o l y s i s and r e a c t i o n w i t h GSH was examined ( 1 9 9 ) . I n a d d i t i o n t o t h e expected r i n g - s u b s t i t u t e d g l u t a t h i o n - S - y l a d d u c t s , a g l u t a t h i o n - S - m e t h y l e n e c o n j u g a t e was obtained. T h i s suggests t h a t i n t e r n a l d e c o m p o s i t i o n o f t h e i n t i m a t e i o n p a i r i n v o l v e s l o s s o f s u l f u r i c a c i d and f o r m a t i o n o f a methimine ( F i g u r e 9 ) , w h i c h can h y d r o l y z e t o formaldehyde and the p r i m a r y a r y l a m i n e o r c a n r e a c t w i t h GSH v i a a Mannich c o n d e n s a t i o n t o y i e l d the g l u t a t h i o n - S - m e t h y l e n e p r o d u c t ( 2 0 0 ) . I n i n v i t r o N-hydroxy-MAB s u l f o t r a n s f e r a s e - a c t i v a t i n g systems, N-sulfonyloxy-MAB a l s o appears t o undergo r a p i d r e d u c t i o n t o MAB ( F i g u r e 9) w i t h t h e concomitant o x i d a t i o n o f N-hydroxy-MAB t o the N - a r y l n i t r o n e (9K The o x i d i z i n g p r o p e r t i e s o f t h e N - s u l f o n y l o x y MAB i o n p a i r i s c o n s i s t e n t w i t h r e s u l t s o b t a i n e d f o r t h e p r i m a r y 2

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Chemical Properties of Ultimate Carcinogenic Metabolites of

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