Carcinogen-DNA Binding - American Chemical Society

15 Carcinogen-DNA Binding A Probe for Metabolic Activation In Vivo and In Vitro C. ANITA H. BIGGER and ANTHONY DIPPLE

Downloaded by YORK UNIV on September 21, 2016 | http://pubs.acs.org Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch015

LBI-Basic Research Program, Chemical Carcinogenesis Program, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 Electrophilic metabolites of chemical carcinogens are believed to be responsible for initiation of the carcinogenic process. These metabolites are generally too reactive to permit direct measurement in biological systems, but their formation can be conveniently monitored through examination of their DNA reaction products. Using this approach, we have found that in mouse embryo cells in vitro or in mouse skin in vivo 7,12-dimethylbenz[a] anthracene (DMBA) binds to deoxyadenosine and deoxyguanosine residues in DNA through metabolically generated syn and anti bay region dihydrodiol epoxides. However, in subcellular systems, i.e. liver 9000 x g supernatants or microsomal preparations, other reactive metabolites, including the 5,6-epoxide, are primarily responsible for DNA binding, indicating that these systems are not good models for the in vivo metabolic activation of DMBA. DMBA-DNA binding was also studied in mouse skin in the presence of inhibitors of tumor initiation by DMBA and in mouse strains of different susceptibiities to DMBA. Tumor inhibition was in some cases, but not all, correlated with an alteration in DMBA-DNA binding, suggesting that in order to initiate the carcinogenic process, DMBA may be required, in addition to binding covalently to DNA, to interact in some other way with target cells. Chemical carcinogens are not generally directly acting per se but are metabolically activated to electrophilic derivatives which are believed to be the actual initiators of the carcinogenic process (1). The electrophilic derivatives are extremely reactive and it is difficult, if not impossible, to monitor their production directly in biological systems. However, their formation can be monitored by analyzing the products of their reaction with DNA. This approach was used historically very successfully in studies of 0097-6156/85/0277-0187$06.50/0 © 1985 American Chemical Society

Finley and Schwass; Xenobiotic Metabolism: Nutritional Effects ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


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the a c t i v a t i o n of c e r t a i n p o l y c y c l i c aromatic hydrocarbon c a r c i n o gens. For example, i n 1973, the work o f B a i r d et: a l • (2) c a s t doubt on the then p o p u l a r t h e o r y ( 3 ) t h a t the K - r e g i o n e p o x i d e m e t a b o l i t e s ( F i g u r e 1) o f 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 were u l t i m a t e c a r c i n o g e n s by showing t h a t the K - r e g i o n e p o x i d e o f 7m e t h y l b e n z [ j i ] a n t h r a c e n e was not the m e t a b o l i t e b i n d i n g t o DNA i n mammalian c e l l s . In t h a t same y e a r , Borgen e t a l . ( 4 ) demonstrated t h a t a d i h y d r o d i o l m e t a b o l i t e o f b e n z o [ a J p y r e n e was a p r e c u r s o r t o DNA-binding i n an jLn v i t r o m i c r o s o m e - c a t a l y z e d DNA b i n d i n g system. As a consequence o f t h e s e s t u d i e s , Sims jet a l . ( 5 ) p r o p o s e d t h a t t h e D N A - r e a c t i v e and c a r c i n o g e n i c m e t a b o l i t e was a v i c i n a l d i h y d r o d i o l epoxide (Figure 1). They s u b s t a n t i a t e d t h i s by showing t h a t the DNA-binding p r o d u c t s formed i n c e l l s t r e a t e d w i t h b e n z o [ a ] p y r e n e were c h r o m a t o g r a p h i c a l l y i d e n t i c a l to t h o s e formed by a l l o w i n g a c r u d e p r e p a r a t i o n o f the s y n t h e t i c d i h y d r o d i o l e p o x i d e o f t h i s compound to r e a c t ixi v i t r o w i t h DNA. The d i h y d r o d i o l e p o x i d e theory of a c t i v a t i o n of p o l y c y c l i c aromatic hydrocarbons was f u r t h e r r e f i n e d by J e r i n a and D a l y (6^) who, t h r o u g h s t r u c t u r e a c t i v i t y s t u d i e s , p r o p o s e d t h a t the most r e a c t i v e v i c i n a l d i h y d r o d i o l e p o x i d e would have the e p o x i d e a d j a c e n t to a bay r e g i o n , e.g. t h e a r e a between p o s i t i o n s 1 and 12 o f 7,12-dimethylbenz[aJanthracene (DMBA) ( F i g u r e 1 ) . T h i s t h e o r y has been l a r g e l y s u b s t a n t i a t e d by the c a r c i n o g e n i c a c t i v i t i e s o f a p p r o p r i a t e m e t a b o l i t e s ( 7 ) . These s t u d i e s demonstrated t h a t DNA-binding can be a r e l i a b l e probe of metabolic a c t i v a t i o n . In c o n t r a s t t o s t u d i e s o f metabol i t e s p e r se, which u s u a l l y i n v o l v e l a r g e numbers o f m e t a b o l i t e i n t e r m e d i a t e s , DNA-binding m o n i t o r s o n l y c h e m i c a l l y r e a c t i v e metabolites. A l s o , i f t h e r e i s no s e l e c t i v e r e p a i r o f s p e c i f i c a d d u c t s , DNA-binding m o n i t o r s the c u m u l a t i v e p r o d u c t i o n o f m e t a b o l i t e s o v e r time, w h i l e d i r e c t measurement o f m e t a b o l i t e s c a n show the m e t a b o l i t e spectrum o n l y a t the time o b s e r v e d . T h i s can be p a r t i c u l a r l y c r i t i c a l f o r s t u d i e s o f a c t i v a t i o n o f complex c h e m i c a l s s u c h as 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 whose p r i m a r y m e t a b o l i t e s a r e s u b j e c t t o s e c o n d a r y and t e r t i a r y m e t a b o l i s m ( 8 ) . F o r t h e s e r e a s o n s , we have used DNA-binding as a probe o f m e t a b o l i c a c t i v a t i o n o f the 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 c a r c i n ogen DMBA. T h i s c a r c i n o g e n has not been s t u d i e d as e x t e n s i v e l y as c e r t a i n o t h e r p o l y c y c l i c a r o m a t i c h y d r o c a r b o n c a r c i n o g e n s because s y n t h e s i s o f the p r o p o s e d u l t i m a t e c a r c i n o g e n i c m e t a b o l i t e , the bay r e g i o n d i h y d r o d i o l e p o x i d e , has not been a c h i e v e d . However, we chose to study t h i s c a r c i n o g e n , and c o n t i n u e t o do so, because i t i s the most p o t e n t o f the 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 and a n a l y s i s o f i t s a c t i v a t i o n may r e v e a l more r e a d i l y t h a n t h a t o f weaker c a r c i n o g e n s the c e l l u l a r i n t e r a c t i o n s s p e c i f i c to i n i t i a t i o n o f the c a r c i n o g e n i c p r o c e s s . M a t e r i a l s and Methods DNA was i s o l a t e d from mouse s k i n (9,10) and c e l l s i n c u l t u r e ( 1 1 13) exposed t o [ % ] - o r [ ^ C ] - D M B A , o r r e c o v e r e d from m i x t u r e s c o n t a i n i n g c a l f thymus DNA, [ 3 H ] - D M B A , c o f a c t o r s and e i t h e r A r o c l o r - i n d u c e d r o d e n t l i v e r microsomes (14-17), S-9 f r a c t i o n (16-18) or 12 h D M B A - i n d u c e d (100 nmoles i n 0.1 ml benzene) mouse


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15.

F i g u r e 1. M e t a b o l i c pathways f u n c t i o n o x i d a s e ; EH, e p o x i d e

f o r a c t i v a t i o n o f DMBA. hydrase.

MFO, mixed


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s k i n e p i d e r m a l homogenate ( 1 9 ) , In some e x p e r i m e n t s , mouse s k i n was p r e t r e a t e d (5 min) w i t h v a r i o u s i n h i b i t o r s b e f o r e [%]-DMBA treatment (10), A f t e r p u r i f i c a t i o n , t h e DNA was e n z y m a t i c a l l y h y d r o l y z e d t o D M B A - d e o x y r i b o n u c l e o s i d e a d d u c t s (20) and a n a l y z e d by Sephadex LH-20 column chromatography ( 2 1 ) , where DMBA-ndeoxyribo n u c l e o s i d e a d d u c t s e l u t e a f t e r 250 m l o f e l u a n t has c l e a r e d t h e column, and by h i g h p r e s s u r e l i q u i d chromatography ( 9 , 2 0 ) ,


Results

and D i s c u s s i o n

I d e n t i f i c a t i o n o f DNA-Reactive M e t a b o l i t e s G e n e r a t e d i n a T a r g e t T i s s u e , Mouse S k i n , I n V i v o . Our i n i t i a l s t u d i e s f o c u s e d on a c t i v a t i o n o f DMBA i n mouse embryo c e l l s i n c u l t u r e because o f t h e ease of i s o l a t i o n o f s u f f i c i e n t DNA f o r adduct c h a r a c t e r i z a t i o n . The c e l l s were exposed t o DMBA and t h e i s o l a t e d DNA e n z y m a t i c a l l y hydrolyzed to deoxyribonucleosides. DMBA-deoxyribonucleoside a d d u c t s were c h a r a c t e r i z e d by f l u o r e s c e n c e measurements (11,22), by p h o t o s e n s i t i v i t y s t u d i e s (12) and by column chromatography (23,24). These s t u d i e s p r o v i d e d e v i d e n c e t h a t t h e D N A - r e a c t i v e m e t a b o l i t e g e n e r a t e d i n t h e s e c e l l s i s a bay r e g i o n d i h y d r o d i o l epoxide. The e n z y m a t i c s t e p s i n t h i s a c t i v a t i o n pathway ( F i g u r e 1) i n v o l v e o x i d a t i o n o f DMBA by mixed f u n c t i o n o x i d a s e s t o a 3,4e p o x i d e which i s c o n v e r t e d by e p o x i d e h y d r a s e t o a 3 , 4 - d i h y d r o diol. T h i s i s , i n t u r n , o x i d i z e d by mixed f u n c t i o n o x i d a s e s t o the d i h y d r o d i o l e p o x i d e . C h a r a c t e r i z a t i o n o f t h e mouse embryo c e l l DMBA-DNA a d d u c t s a l l o w e d t h e use o f cochromatography t o i n v e s t i g a t e t h e DMBA-DNA a d d u c t s formed i n a t a r g e t t i s s u e , mouse s k i n . Tritium-labeled DMBA was p a i n t e d on the shaved backs o f mice and c a r b o n - 1 4 - l a b e l e d DMBA was used t o t r e a t mouse embryo c e l l s i n c u l t u r e . A f t e r i s o l a t i o n , mouse embryo c e l l and mouse s k i n DMBA-DNA were e n z y m a t i c a l l y d i g e s t e d and the D M B A - d e o x y r i b o n u c l e o s i d e a d d u c t s a n a l y z e d by Sephadex LH-20 column chromatography ( 1 5 ) . As c a n be seen i n F i g u r e 2, t h e mouse s k i n and mouse embryo c e l l a d d u c t s e x h i b i t i d e n t i c a l chromatographic behavior. T h i s i n d i c a t e s t h a t a c t i v a t i o n i n mouse s k i n a l s o p r o c e e d s through t h e bay r e g i o n dihydrodiol-epoxide pathway. These r e s u l t s a r e i n agreement w i t h r e s u l t s o b t a i n e d t h r o u g h d i f f e r e n t approaches i n o t h e r l a b o r a t o r i e s . V i g n y et_ a l . (25) found t h e f l u o r e s c e n c e spectrum o f mouse s k i n DMBA-DNA c o n s i s t e n t w i t h r e a c t i o n o f t h e DNA w i t h a bay r e g i o n d i h y d r o d i o l e p o x i d e o f DMBA. The bay r e g i o n d i h y d r o d i o l e p o x i d e o f DMBA has n o t y e t been c h e m i c a l l y s y n t h e s i z e d , so i t has n o t been p o s s i b l e t o compare b i o l o g i c a l l y a c t i v a t e d DMBA-DNA a d d u c t s w i t h an a u t h e n t i c s t a n d a r d . However, Cooper e t a l . (26) f o u n d t h a t t h e p r o d u c t s o f r e a c t i o n o f the m - c h l o r o p e r o x y b e n z o i c a c i d - o x i d i z e d 3 , 4 ^ d i h y d r o d i o l o f DMBA w i t h DNA gave DMBA-DNA a d d u c t s which cochromatographed w i t h those o b t a i n e d from DMBA-treated mouse s k i n . In a d d i t i o n , t h e 3,4-dihyd r o d i o l o f DMBA ( p r e c u r s o r o f t h e bay r e g i o n d i h y d r o d i o l e p o x i d e ) i s a v e r y p o t e n t c a r c i n o g e n i n i n i t i a t i o n - p r o m o t i o n t e s t s on mouse s k i n s u g g e s t i n g t h a t the D N A - r e a c t i v e m e t a b o l i t e i n mouse s k i n i s a l s o t h e u l t i m a t e c a r c i n o g e n (27-29).



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Volume (ml) F i g u r e 2. Sephadex LH-20 column chromatography o f DMBA-deoxyribon u c l e o s i d e a d d u c t s formed by e n z y m a t i c d i g e s t i o n o f DNA from mouse embryo c e l l s exposed t o [ ^C]-DMBA (0,2 ug/ml) f o r 24 h (•-•) and o f DNA from t h e s k i n o f f e m a l e NIH Swiss mice t r e a t e d w i t h [ H]-DMBA (10 ug/mouse) f o r 24 h ( 0 - 0 ) . The arrow denotes the p o s i t i o n o f e l u t i o n o f an added u v - a b s o r b i n g marker 4 - ( p - n i t r o b e n z y l ) p y r i d i n e . "Reproduced w i t h p e r m i s s i o n f r o m R e f . 15. C o p y r i g h t 1980, I R L Press". 3



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XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

Comparison o f t h e A c t i v a t i o n P r o v i d e d by T i s s u e Homogenates, I n t a c t C e l l u l a r Systems, and T a r g e t T i s s u e , Short term t e s t s f o r d e t e c 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 commonly r e l y on t h e i n c l u s i o n o f s u b c e l l u l a r homogenates, such as a r a t l i v e r S-9 f r a c t i o n , t o p r o v i d e m e t a b o l i c a c t i v a t i o n o f t e s t c h e m i c a l s ( 3 0 ) , The j u s t i f i c a t i o n f o r t h e u s e o f homogenates seems t o r e s t on an assumption t h a t they r e p r o d u c e t a r g e t t i s s u e a c t i v a t i o n . We d e c i d e d t o t e s t t h a t assumption by examining t h e D N A - r e a c t i v e m e t a b o l i t e s g e n e r a t e d by homogenate 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. In our i n i t i a l s t u d y ( 1 4 ) , we compared t h e DMBA-DNA adducts formed i n t h e p r e s e n c e o f r a t - l i v e r microsomes and c a l f thymus DNA w i t h those formed i n mouse s k i n in v i v o and w i t h t h o s e formed by r e a c t i o n o f the c h e m i c a l l y s y n t h e s i z e d K - r e g i o n e p o x i d e ( F i g u r e 1) o f DMBA w i t h c a l f thymus DNA, F i g u r e 3 shows t h e Sephadex LH-20 column c h r o m a t o g r a p h i c p r o f i l e s f o r t h e s e DMBA-deoxyribonucleoside a d ducts. The m i c r o s o m a l l y - g e n e r a t e d a d d u c t s were n o t c h r o m a t o g r a p h i c a l l y i d e n t i c a l w i t h the mouse s k i n a d d u c t s b u t i n s t e a d e l u t e d i n c o i n c i d e n c e w i t h t h e K - r e g i o n epoxide-^derived a d d u c t s . This s u g g e s t e d t h a t r a t l i v e r homogenates a r e n o t good models f o r i n vivo a c t i v a t i o n . We examined a c t i v a t i o n by o t h e r s u b c e l l u l a r 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 under v a r i o u s c o n d i t i o n s . F i g u r e 4 shows t h e e f f e c t o f a 1 0 - f o l d d i f f e r e n c e i n s u b s t r a t e c o n c e n t r a t i o n on the DMBA-DNA a d d u c t s g e n e r a t e d by r a t l i v e r S9 f r a c t i o n ( 1 8 ) . At a h i g h s u b s t r a t e c o n c e n t r a t i o n ( F i g u r e 4b), a l a r g e p r o p o r t i o n o f t h e a d d u c t s was d e r i v e d from the K - r e g i o n e p o x i d e o f DMBA (peaks D and E ) p l u s a d d u c t s d e r i v e d from an u n i d e n t i f i e d m e t a b o l i t e (peak B). However, when t h e c o n c e n t r a t i o n of s u b s t r a t e was lowered 1 0 - f o l d , t h e Sephadex LH-20 column c h r o m a t o g r a p h i c p r o f i l e changed d r a m a t i c a l l y ( F i g u r e 4 a ) . Peaks A and C now p r e d o m i n a t e d . We have shown t h a t t h e e l u t i o n o f peak C c o i n c i d e s w i t h e l u t i o n o f the mouse embryo c e l l bay r e g i o n d i h y d r o d i o l e p o x i d e a d d u c t s ( 1 8 ) , w h i l e peak A e l u t e s a t t h e same p o s i t i o n i n t h e g r a d i e n t as a d d u c t s i d e n t i f i e d a s 7-hydroxymethyl12-methylbenz[a_]anthracene-bay r e g i o n d i h y d r o d i o l epoxide-

Carcinogen-DNA Binding - American Chemical Society

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