Hydroperoxide-Dependent Oxygenation of ... - ACS Publications

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Downloaded by UNIV OF MICHIGAN ANN ARBOR on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

Hydroperoxide-Dependent Oxygenation of Polycyclic Aromatic Hydrocarbons and Their Metabolites LAWRENCE J. MARNETT Department of Chemistry, Wayne State University, Detroit, MI 48202

Fatty acid hydroperoxides in the presence of heme complexes and heme proteins oxidize benzo(a)pyrene and 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to quinones and diol epoxides, respectively. The oxidizing agent is a peroxyl radical derived from the fatty acid hydroperoxide but not a higher oxidation state of a mammalian peroxidase. The stereochemistry of (±)-BP-dihydrodiol epoxidation is distinct from that catalyzed by mixed-function oxidases, which provides a convenient method for discriminating the contributions of the two systems to BP-7,8-dihydrodiol metabolism in cell homogenates, cell or organ culture. Using this method, epoxidation of BP-7,8-dihydrodiol has been detected during prostaglandin biosynthesis, l i p i d peroxidation, and xenobiotic oxygenation. Fatty acid hydroperoxide-dependent oxidation constitutes a novel pathway for metabolic activation of polycyclic hydrocarbons and other carcinogens which has widespread potential in vivo significance. Oxidation is intimately linked to the activation of polycyclic aromatic hydrocarbons (PAH) to carcinogens (1-3?. Oxidation of PAH in animals and man is enzyme-catalyzed and is a response to the introduction of foreign compounds into the cellular environment. The most intensively studied enzyme of PAH oxidation is cytochrome P-450, which is a mixed-function oxidase that receives its electrons from NADPH via a one or two component electron transport chain (_1) . Some forms of this enzyme play a major role in systemic metabolism of PAH (4). However, there are numerous examples of carcinogens that require metabolic activation, including PAH, that induce cancer in tissues with low mixed-function oxidase activity (_5) . In order to comprehensively evaluate the metabolic activation of PAH, one must consider a l l cellular pathways for their oxidative activation. Peroxidases have been implicated in carcinogenesis by PAH, aromatic amines, and estrogens inter alia (6-9). These enzymes catalyze the reduction of hydrogen peroxide and organic 0097-6156/85/0283-0307S06.00/0 © 1985 American Chemical Society

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

308

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

h y d r o p e r o x i d e s and u s e a wide v a r i e t y o f compounds a s r e d u c i n g a g e n t s ( E q u a t i o n 1 ) . Important o b s e r v a t i o n s on t h e o x i d a t i o n o f PAH


ROOH + DH

2

+ ROH + D + H 0 2

(1)

by p e r o x i d a s e s have been made by C a v a l i e r i and Rogan and a r e d e s c r i b e d i n t h e i r c h a p t e r i n t h i s volume and elsewhere (6^) . T e n y e a r s ago we r e p o r t e d t h a t b e n z o ( a ) p y r e n e (BP) i s o x i d i z e d d u r i n g the o x y g e n a t i o n o f a r a c h i d o n i c a c i d by p r o s t a g l a n d i n H (PGH) s y n t h a s e (10). PGH s y n t h a s e i s a w i d e l y d i s t r i b u t e d enzyme o f p o l y u n s a t u r a t e d f a t t y a c i d m e t a b o l i s m t h a t p o s s e s s e s a p e r o x i d a s e a c t i v i t y and g e n e r a t e s h y d r o p e r o x y e n d o p e r o x i d e s as i n i t i a l p r o d u c t s o f f a t t y a c i d o x y g e n a t i o n (11-13). Its principal function i s to biosynthesize PGH2i t h e e n d o p e r o x i d e i n t e r m e d i a t e o f p r o s t a g l a n d i n and thromboxane b i o s y n t h e s i s ( F i g u r e 1) (11,14). The o t h e r enzyme o f u n s a t u r a t e d f a t t y a c i d o x y g e n a t i o n i s l i p o x y g e n a s e (15^ . I t oxygenates u n s a t u r a t e d f a t t y a c i d s t o hydroperoxides t h a t a r e reduced t o a l c o h o l s o r c o n v e r t e d t o l e u k o t r i e n e s ( F i g u r e 1) ( 1 6 ) . These two enzymes, PGH s y n t h a s e and l i p o x y g e n a s e , r e p r e s e n t t h e p r i n c i p a l s o u r c e s o f o r g a n i c h y d r o p e r o x i d e s i n mammalian t i s s u e ( 1 7 ) . Our i n v e s t i g a t i o n s o f t h e o x i d a t i o n o f PAH by t h e h y d r o p e r o x i d e p r o d u c t s o f PGH s y n t h a s e and l i p o x y g e n a s e c a t a l y s i s i n d i c a t e t h a t t h i s pathway c a n g e n e r a t e u l t i m a t e c a r c i n o g e n i c forms o f PAH and t h a t t h e mechanisms o f o x i d a t i o n a r e d i s t i n c t from t h o s e o f c l a s s i c p e r o x i d a s e - c a t a l y z e d o x i d a tion. F a t t y a c i d hydroperoxide-dependent o x i d a t i o n , t h e r e f o r e , r e p r e s e n t s a n o v e l pathway f o r t h e m e t a b o l i c a c t i v a t i o n o f PAH. Benzo(a)pyrene

Oxidation

I n c u b a t i o n o f BP w i t h a r a c h i d o n i c a c i d and ram s e m i n a l v e s i c l e m i c r o somes, a r i c h s o u r c e o f PGH s y n t h a s e , p r o d u c e s 1,6-, 3,6-, and 6,12q u i n o n e s as t h e e x c l u s i v e p r o d u c t s o f o x i d a t i o n ( F i g u r e 2) ( 1 8 ) . These a r e t h e same quinones t h a t a r e formed when 6-hydroxy-BP i s o x i d i z e d by a i r o r microsomes ( 1 9 ) . However, t h e r e i s no d e f i n i t i v e e v i d e n c e t h a t 6-hydroxy-BP i s an i n t e r m e d i a t e i n t h e i r f o r m a t i o n by PGH s y n t h a s e . Among a l l o f t h e s t a b l e m e t a b o l i t e s o f BP, t h e q u i n o n e s a r e d i s t i n c t i v e because, u n l i k e p h e n o l s and d i h y d r o d i o l s , t h e y a r e n o t d e r i v e d from arene o x i d e s . Thus, arene o x i d e s do n o t appear t o be p r o d u c t s o f BP o x i d a t i o n by PGH s y n t h a s e (19,20). P o t e n t i n h i b i t i o n o f PGH synthase-dependent BP o x i d a t i o n by a n t i o x i dants suggests t h a t t h e quinones are p r o d u c t s o f f r e e r a d i c a l r e a c t i o n s (18) . A d d i t i o n o f RNA o r DNA p r i o r t o o x i d a t i o n o f BP by PGH s y n t h a s e r e s u l t s i n s u b s t a n t i a l n u c l e i c a c i d b i n d i n g (17,21). Addition of RNA f i v e m i n u t e s a f t e r i n i t i a t i o n o f o x i d a t i o n l e a d s t o no c o v a l e n t b i n d i n g ( 1 7 ) . T h i s i m p l i e s t h a t t h e quinones do n o t b i n d t o n u c l e i c a c i d b u t r a t h e r a s h o r t - l i v e d i n t e r m e d i a t e i n t h e i r format i o n does. A r a c h i d o n i c a c i d o x y g e n a t i o n i n ram s e m i n a l v e s i c l e microsomes i s complete w i t h i n two min, which s u g g e s t s t h a t t h e r e a c t i v e i n t e r m e d i a t e i s g e n e r a t e d c o n c u r r e n t l y w i t h PGH2« The s t r u c t u r e s o f t h e n u c l e i c a c i d a d d u c t s have n o t been e l u c i d a t e d so the i d e n t i t y o f t h e r e a c t i v e i n t e r m e d i a t e i s unknown. Despite the high l e v e l o f nucleic a c i d binding that i s evident, no mutagenic s p e c i e s c a n be d e t e c t e d when BP i s i n c u b a t e d w i t h ram


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Thromtoxano

Pathways o f o x y g e n a t i o n o f u n s a t u r a t e d f a t t y a c i d s i n animal t i s s u e .

6,12F i g u r e 2.

P r o d u c t s o f BP o x i d a t i o n b y a r a c h i d o n i c a c i d s e m i n a l v e s i c l e microsomes.

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POLYCYCLIC HYDROCARBONS AND

CARCINOGENESIS

s e m i n a l v e s i c l e microsomes and a r a c h i d o n i c a c i d i n t h e p r e s e n c e o f Salmonella typhimurium strains. The p r e s e n c e o f t h e Salmonella s t r a i n s and n u t r i e n t b r o t h i n t h e i n c u b a t i o n s does n o t i n h i b i t quinone f o r m a t i o n . F u r t h e r m o r e , one o f t h e s t r a i n s employed, TA98, has been r e p o r t e d t o d e t e c t 6-hydroxy-BP as a mutagen (23_) . I t may be t h a t t h e i n t e r m e d i a t e r e s p o n s i b l e f o r n u c l e i c a c i d b i n d i n g i s t o o u n s t a b l e t o s u r v i v e t r a n s i t a c r o s s t h e b a c t e r i a l c e l l w a l l and membrane. A l t e r n a t i v e l y , t h e i n t e r m e d i a t e may b i n d t o DNA b u t n o t induce mutation. T h i s i s u n l i k e l y because t h e g e n e r a t i o n o f b u l k y a d d u c t s on a DNA m o l e c u l e u s u a l l y r e s u l t s i n m u t a t i o n . Although some adducts o f p o l y c y c l i c h y d r o c a r b o n s t o DNA appear t o be more mutagenic than o t h e r s , t h e d i f f e r e n c e s a r e n o t g r e a t e r than an o r d e r o f magnitude (24,25). T h u s , i t i s u n l i k e l y t h a t i f adducts a r e formed they a r e n o t m u t a g e n i c . Two o t h e r p o l y c y c l i c h y d r o c a r b o n s , 3 - m e t h y l c h o l a n t h r e n e and 7,12-dimethylbenzanthracene are oxidized during arachidonate metabolism (21,26). Hydroxymethyl compounds t h a t do n o t a r i s e from arene o x i d e s appear t o be t h e p r o d u c t s formed from 7 , 1 2 - d i m e t h y l benzanthracene . 7,8-Dihydroxy-7,8-Dihydrobenzo(a)pyrene

Oxidation

In c o n t r a s t t o t h e r e s u l t s w i t h BP, i n c u b a t i o n o f B P - 7 , 8 - d i h y d r o d i o l w i t h ram s e m i n a l v e s i c l e microsomes and a r a c h i d o n a t e g e n e r a t e s a s p e c i e s t h a t i s s t r o n g l y mutagenic t o Salmonella s t r a i n s TA98 and TA100 ( F i g u r e 3) ( 2 2 ) . F o r m a t i o n o f t h e mutagen i s i n h i b i t e d by i n d o m e t h a c i n i n d i c a t i n g t h e i n v o l v e m e n t o f PGH s y n t h a s e . Similar e x p e r i m e n t s w i t h B P - 4 , 5 - d i h y d r o d i o l and B P - 9 , 1 0 - d i h y d r o d i o l do n o t g e n e r a t e p o t e n t mutagens, which s u g g e s t s t h a t a c t i v a t i o n i s s p e c i f i c f o r t h e p r e c u r s o r o f t h e b a y - r e g i o n d i o l e p o x i d e (22). The o b v i o u s i n t e r p r e t a t i o n o f t h e s e e x p e r i m e n t s i s t h a t PGH s y n t h a s e c a t a l y z e s the e p o x i d a t i o n o f B P - 7 , 8 - d i h y d r o d i o l t o the u l t i m a t e c a r c i n o g e n BP-diol epoxide. To c o n f i r m t h i s we i d e n t i f i e d t h e p r o d u c t s o f BP7 , 8 - d i h y d r o d i o l o x i d a t i o n (27,28). T h e o r e t i c a l l y , t h e epoxide oxygen c a n be i n t r o d u c e d from e i t h e r s i d e o f t h e m o l e c u l e g i v i n g r i s e to syn- o r a n t i - d i o l epoxides. Each epoxide h y d r o l y z e s r a p i d l y t o a m i x t u r e o f c i s and t r a n s t e t r a h y d r o t e t r a o l s ( F i g u r e 4 ) . When i n c u b a t i o n s o f B P - 7 , 8 - d i h y d r o d i o l and PGH s y n t h a s e a r e a l l o w e d t o p r o c e e d f o r 15 min, two p r o d u c t s a r e o b t a i n e d t h a t we i d e n t i f i e d as t h e c i s and t r a n s t e t r a o l s d e r i v e d from t h e a n t i - d i o l e p o x i d e (27,28,29). H y d r o l y s i s p r o d u c t s o f t h e s y n - d i o l e p o x i d e were n o t d e t e c t e d . When i n c u b a t i o n s were t e r m i n a t e d a f t e r 3 min, a new p r o d u c t was d e t e c t e d t h a t we i d e n t i f i e d as a m e t h y l e t h e r t h a t i s formed by m e t h a n o l y s i s o f t h e a n t i - d i o l e p o x i d e ( E q u a t i o n 2) ( 2 9 ) , T h i s r e a c t i o n can o n l y have o c c u r r e d a f t e r t e r m i n a t i o n o f t h e r e a c t i o n because t h e r e was no methanol i n t h e i n c u b a t i o n m i x t u r e . A d d i t i o n a l experiments confirmed t h a t methanolysis occurs d u r i n g chromatography ( r e v e r s e p h a s e , methanol-water g r a d i e n t s ) . The d e t e c t i o n o f t h e m e t h y l e t h e r i s i m p o r t a n t because i t c o n f i r m s t h a t a d i o l e p o x i d e i s g e n e r a t e d , s u r v i v e s s o l v e n t e x t r a c t i o n , and t h e n undergoes s o l v o l y s i s on t h e HPLC column. T h i s p r o v i d e s d i r e c t e v i d e n c e f o r t h e f o r m a t i o n o f t h e a n t i - d i o l e p o x i d e as a p r o d u c t o f PGH synthase-dependent c o o x i d a t i o n o f B P - 7 , 8 - d i h y d r o d i o l . The c o r r e l a t i o n o f the rate o f BP-7,8-dihydrodiol o x i d a t i o n , a n t i - d i o l e p o x i d e f o r m a t i o n , and mutagen g e n e r a t i o n a r e shown i n F i g u r e 5 (30).


12.


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Oxygenation


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F i g u r e 3.

F i g u r e 4.

Induction of mutation i n typhimurium TA98 by BP-7,8d i h y d r o d i o l , a r a c h i d o n i c a c i d , and ram s e m i n a l v e s i c l e microsomes. C o n c e n t r a t i o n dependence on B P - 7 , 8 - d i h y d r o diol. (Reproduced w i t h p e r m i s s i o n from Ref. 22. C o p y r i g h t 1978 Academic.)

D i o l epoxide products o f BP-7,8-dihydrodiol t h e i r hydrolysis products.

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Comparison o f the time course o f PGH2 b i o s y n t h e s i s , BP7 , 8 - d i h y d r o d i o l metabolism, and g e n e r a t i o n o f a mutagen from B P - 7 , 8 - d i h y d r o d i o l by RSVM. (Reproduced w i t h p e r m i s s i o n from Ref. 30. C o p y r i g h t 1982 M a r c e l Dekker.)



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313

Oxygenation

Further support f o r e p o x i d a t i o n o f BP-7,8-dihydrodiol t o the a n t i - d i o l e p o x i d e i s t h e i d e n t i f i c a t i o n o f RNA and DNA a d d u c t s formed as a r e s u l t o f i n c u b a t i o n o f B P - 7 , 8 - d i h y d r o d i o l , PGH s y n t h a s e , and p o l y g u a n y l i c a c i d o r DNA (30,31). Following digestion of the nucleic a c i d , t h e major g u a n o s i n e and deoxyguanosine a d d u c t s were i d e n t i f i e d as a r i s i n g by a d d i t i o n o f t h e e x o c y c l i c amino group o f g u a n o s i n e t o the b e n z y l i c c a r b o n o f t h e a n t i - d i o l e p o x i d e ( T a b l e I) (.31) . These experiments a l s o d e f i n e d the s t e r e o c h e m i s t r y o f e p o x i d a t i o n . Both enantiomers o f B P - 7 , 8 - d i h y d r o d i o l are e p o x i d i z e d a t equal r a t e s t o e n a n t i o m e r s o f t h e a n t i - d i o l e p o x i d e . The d i r e c t i o n o f oxygen i n t r o d u c t i o n i s from t h e same s i d e o f t h e m o l e c u l e as t h e h y d r o x y l group a t carbon-8 o f B P - 7 , 8 - d i h y d r o d i o l . Table I .

R e l a t i v e Y i e l d s o f D i a s t e r e o m e r i c A d d u c t s From A n t i - d i o l E p o x i d e P l u s P o l y g u a n y l i c A c i d Compared t o A d d u c t s Genera t e d D u r i n g M e t a b o l i s m o f B P - 7 , 8 - d i h y d r o d i o l by Ram S e m i n a l V e s i c l e s i n the Presence o f A r a c h i d o n i c A c i d

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When t h e 7 , 8 - h y d r o x y l groups a r e m i s s i n g , e p o x i d e i n t r o d u c t i o n o c c u r s from b o t h s i d e s o f t h e p y r e n e r i n g . Thus 7 , 8 - d i h y d r o b e n z o ( a j pyrene i s c o o x i d i z e d by PGH s y n t h a s e t o a p o t e n t mutagen t h a t i s i d e n t i f i e d by p r o d u c t and n u c l e i c a c i d b i n d i n g s t u d i e s as 9,10-epoxy7 , 8 , 9 , 1 0 - t e t r a h y d r o b e n z o ( a ) p y r e n e ( E q u a t i o n 3) (32J . The s t r u c t u r e s o f t h e g u a n o s i n e a d d u c t s formed i n i n c u b a t i o n s c o n t a i n i n g p o l y g u a n y l i c a c i d i n d i c a t e t h a t e q u a l amounts o f e p o x i d e a r e formed by i n t r o d u c t i o n o f oxygen from above and below t h e p l a n e o f t h e p y r e n e r i n g (Equation 3 ) . These f i n d i n g s i n d i c a t e t h a t PGH s y n t h a s e i n t h e p r e s e n c e o f a r a c h i d o n a t e c a n c a t a l y z e t h e t e r m i n a l a c t i v a t i o n s t e p i n BP c a r c i n o g e n e s i s and t h a t t h e r e a c t i o n may be g e n e r a l f o r d i h y d r o d i o l metabol i t e s o f p o l y c y c l i c hydrocarbons. G u t h r i e e_t. a l . have shown t h a t PGH s y n t h a s e c a t a l y z e s t h e a c t i v a t i o n o f c h r y s e n e and b e n z a n t h r a c e n e d i h y d r o d i o l s t o p o t e n t mutagens ( 3 3 ) . As i n t h e c a s e w i t h BP, o n l y the d i h y d r o d i o l t h a t i s a p r e c u r s o r t o bay r e g i o n d i o l e p o x i d e s i s activated. We have r e c e n t l y shown t h a t 3 , 4 - d i h y d r o x y - 3 , 4 - d i h y d r o b e n z o ( a ) a n t h r a c e n e i s o x i d i z e d by PGH s y n t h a s e t o t e t r a h y d r o t e t r a o l s d e r i v e d from t h e a n t i - d i o l e p o x i d e ( E q u a t i o n 4) ( 3 4 ) . N a t u r e o f O x i d a n t s G e n e r a t e d From F a t t y A c i d

Hydroperoxides

PGH s y n t h a s e c o n t a i n s two h e m e - r e q u i r i n g a c t i v i t i e s ( 1 3 ) . The c y c l o oxygenase component oxygenates a r a c h i d o n i c a c i d t o t h e h y d r o p e r o x y e n d o p e r o x i d e , PGG2, and t h e p e r o x i d a s e component r e d u c e s PGG t o t h e hydroxy e n d o p e r o x i d e , PGH2. The c y c l o o x y g e n a s e i s i n h i b i t e d by nons t e r o i d a l a n t i i n f l a m m a t o r y agents such as a s p i r i n and i n d o m e t h a c i n , 2



314


but t h e p e r o x i d a s e i s n o t (35,36) . Both components a r e c o n t a i n e d on the same 70,000 D a l t o n p r o t e i n ( 1 3 ) . The p r e s e n c e o f a p e r o x i dase as an i n t e g r a l component o f PGH s y n t h a s e i m p l i e s t h a t h y d r o p e r o x i d e - d e p e n d e n t o x i d a t i o n s are c a t a l y z e d by t h i s component (37_) . As a f i r s t a p p r o x i m a t i o n one m i g h t e x p e c t t h a t the mechanisms o f t h e s e o x i d a t i o n s would be analogous t o t h o s e o f o t h e r heme p e r o x i dases. E x t e n s i v e s t u d i e s have e s t a b l i s h e d t h a t the c a t a l y t i c c y c l e f o r the r e d u c t i o n o f h y d r o p e r o x i d e s by h o r s e r a d i s h p e r o x i d a s e i s t h e one d e p i c t e d i n F i g u r e 6 (38) . The r e s t i n g enzyme i n t e r a c t s w i t h t h e p e r o x i d e t o form an e n z y m e - s u b s t r a t e complex t h a t decomposes t o a l c o h o l and an i r o n - o x o complex t h a t i s two o x i d i z i n g e q u i v a l e n t s above the r e s t i n g s t a t e o f t h e enzyme. F o r c a t a l y t i c t u r n o v e r t o o c c u r the i r o n - o x o complex must be r e d u c e d . The two e l e c t r o n s a r e f u r n i s h e d by r e d u c i n g s u b s t r a t e s e i t h e r by e l e c t r o n t r a n s f e r from s u b s t r a t e t o enzyme o r by oxygen t r a n s f e r from enzyme t o s u b s t r a t e . S u b s t r a t e o x i d a t i o n by the i r o n - o x o complex s u p p o r t s c o n t i n u o u s h y d r o p e r o x i d e r e d u c t i o n . When e i t h e r r e d u c i n g s u b s t r a t e o r h y d r o peroxide i s exhausted, the c a t a l y t i c c y c l e stops. We have d e v e l o p e d an a s s a y t o i d e n t i f y p e r o x i d a s e r e d u c i n g subs t r a t e s based on t h e i r a b i l i t y t o s t i m u l a t e r e d u c t i o n o f 1-hydrop e r o x y - 5 - p h e n y l — 4 - p e n t e n e ( E q u a t i o n 5) ( 3 9 ) . The h y d r o p e r o x i d e i s i n c u b a t e d w i t h l i m i t i n g c o n c e n t r a t i o n s o f p e r o x i d a s e i n the p r e s e n c e o r absence o f a p o t e n t i a l r e d u c i n g s u b s t r a t e . I n the absence o f r e d u c t a n t c a t a l y t i c r e d u c t i o n cannot o c c u r and n e g l i g i b l e q u a n t i t i e s o f a l c o h o l a r e p r o d u c e d (the h y d r o p e r o x i d e and a l c o h o l are q u a n t i t a t e d a f t e r s e p a r a t i o n by HPLC). I n the p r e s e n c e o f a good r e d u c i n g s u b s t r a t e c a t a l y t i c t u r n o v e r o c c u r s and q u a n t i t i e s o f a l c o h o l a r e produced t h a t are s t o i c h i o m e t r i c w i t h reducing s u b s t r a t e o x i d i z e d . The a s s a y appears t o be g e n e r a l f o r a l l p l a n t and a n i m a l , heme and non-heme, p e r o x i d a s e s . One can rank the r e l a t i v e e f f i c a c y o f reducing substrates u s i n g t h i s assay. A r o m a t i c amines, p h e n o l s , c a t e c h o l s , 8 - d i c a r b o n y l s , n i t r o g e n h e t e r o c y c l e s , and a r o m a t i c s u l f i d e s a r e good t o e x c e l l e n t r e d u c i n g s u b s t r a t e s (39^) . In c o n t r a s t , p o l y c y c l i c h y d r o c a r b o n s and d i h y d r o d i o l m e t a b o l i t e s o f PAH a r e v e r y poor t o n o n - r e d u c i n g compounds. Because BP and B P - 7 , 8 - d i h y d r o d i o l do not s t i m u l a t e h y d r o p e r o x i d e r e d u c t i o n they cannot be o x i d i z e d by h i g h e r o x i d a t i o n s t a t e s o f the p e r o x i d a s e ( i r o n - o x o complexes) The c o n c e n t r a t i o n s o f h y d r o p e r o x i d e , PGH s y n t h a s e , and BP o r BP-7,8d i h y d r o d i o l a r e analogous t o t h o s e i n which BP o r B P - 7 , 8 - d i h y d r o d i o l o x i d a t i o n can be d e t e c t e d i n ram s e m i n a l v e s i c l e microsomes. T h e r e f o r e , we c o n c l u d e t h a t t h e o x i d i z i n g agent t h a t c o n v e r t s BP t o q u i n o n e s o r B P - 7 , 8 - d i h y d r o d i o l t o d i o l e p o x i d e s i s n o t an i r o n - o x o intermediate of peroxidase turnover. S u p p o r t f o r t h i s c o n c l u s i o n i s p r o v i d e d by the h y d r o p e r o x i d e s p e c i f i c i t y o f BP o x i d a t i o n . The scheme p r e s e n t e d i n F i g u r e 6 r e q u i r e s t h a t the same o x i d i z i n g agent i s g e n e r a t e d by r e a c t i o n o f 2 ° 2 r perox> a c i d s , o r a l k y l h y d r o p e r o x i d e s w i t h t h e p e r o x i d a s e . O x i d a t i o n o f any compound by the i r o n - o x o i n t e r m e d i a t e s s h o u l d be s u p p o r t e d by any h y d r o p e r o x i d e t h a t i s r e d u c e d by the p e r o x i d a s e . T h i s i s c l e a r l y not the case f o r o x i d a t i o n o f BP by ram s e m i n a l v e s i c l e microsomes as t h e d a t a i n F i g u r e 7 i l l u s t r a t e . Quinone f o r m a t i o n i s s u p p o r t e d by f a t t y a c i d h y d r o p e r o x i d e s b u t v e r y p o o r l y o r not a t a l l by s i m p l e a l k y l h y d r o p e r o x i d e s o r H 0 2 . The f a c t t h a t H

2



12.

MARNETT


Oxygenation

F i g u r e 6. C a t a l y t i c c y c l e o f h o r s e r a d i s h

peroxidase.


316


PGG


2

somes on t h e c o n c e n t r a t i o n o f d i f f e r e n t h y d r o p e r o x i d e s . A b b r e v i a t i o n s used a r e 20:4, a r a c h i d o n i c a c i d ; 15-HPEA, 1 5 - h y d r o p e r o x y - e i c o s a t e t r a e n o i c a c i d ; t-BuOOH, t - b u t y l hydroperoxide. The s t r u c t u r e i s PGG i s g i v e n i n F i g u r e 1. 2


12.

MARNETT


Oxygenation

317

H2O2 does n o t s u p p o r t o x i d a t i o n i s e s p e c i a l l y s i g n i f i c a n t because the same c o n c e n t r a t i o n s o f H 0 2 s u p p o r t v i g o r o u s o x i d a t i o n o f r e d u c i n g s u b s t r a t e s such as a r o m a t i c amines and p h e n y l b u t a z o n e . T h e r e f o r e , we c o n c l u d e t h a t BP and B P - 7 , 8 - d i h y d r o d i o l a r e o x i d i z e d by a s p e c i e s t h a t i s n o t a f u n c t i o n a l i n t e r m e d i a t e o f p e r o x i d a s e catalysis. The o x i d i z i n g agent t h a t i s r e s p o n s i b l e f o r t h e o x y g e n a t i o n o f BP and B P - 7 , 8 - d i h y d r o d i o l appears t o be a f r e e r a d i c a l . Reaction o f f a t t y a c i d h y d r o p e r o x i d e s w i t h m e t a l complexes g e n e r a t e s a l k o x y l and p e r o x y l r a d i c a l s t h a t can o x i d i z e o r g a n i c m o l e c u l e s (40-43). Incub a t i o n o f f a t t y a c i d hydroperoxides with c e r t a i n hemeproteins o r t h e i r p r o s t h e t i c group, h e m a t i n , c a u s e s o x i d a t i o n o f BP t o q u i n o n e s and B P - 7 , 8 - d i h y d r o d i o l t o d i o l e p o x i d e s (17,40). I n the case o f BP7 , 8 - d i h y d r o d i o l e p o x i d a t i o n , t h e s o u r c e o f t h e e p o x i d e oxygen i s m o l e c u l a r oxygen; e p o x i d a t i o n i s p o t e n t l y i n h i b i t e d by a n t i o x i d a n t s , and e p o x i d a t i o n i s s u p p o r t e d by u n s a t u r a t e d b u t n o t s a t u r a t e d f a t t y a c i d hydroperoxides (Table I I ) ( 4 0 , 4 4 ) . These o b s e r v a t i o n s a r e analogous t o t h e r e s u l t s o f m i c r o s o m a l i n c u b a t i o n s and a r e c o n s i s t e n t w i t h a f r e e r a d i c a l mechanism o f h y d r o p e r o x i d e - d e p e n d e n t e p o x i dation. BP o x i d a t i o n t o q u i n o n e s o c c u r s d u r i n g a u t o x i d a t i o n o f l i p i d s i n i t i a t e d by enzymes o r y - i a d i a t i o n (45,46) .


2

r r

In t h e case o f t h e h e m a t i n - c a t a l y z e d r e a c t i o n we have p r o p o s e d t h a t p e r o x y l r a d i c a l s a r e t h e e p o x i d i z i n g a g e n t s ( 4 0 ) . The mechanism i s i l l u s t r a t e d i n F i g u r e 8. Hematin r e d u c e s t h e h y d r o p e r o x i d e t o an a l k o x y l r a d i c a l t h a t c y c l i z e s t o t h e a d j a c e n t d o u b l e bond. The i n c i p i e n t c a r b o n - c e n t e r e d r a d i c a l c o u p l e s w i t h O2 t o form a p e r o x y l r a d i c a l t h a t we p r o p o s e e p o x i d i z e s B P - 7 , 8 - d i h y d r o d i o l . P e r o x y l r a d i c a l s a r e well-known i n c h e m i c a l systems t o e p o x i d i z e i s o l a t e d d o u b l e bonds such as t h e 9 , 1 0 - d o u b l e bond o f B P - 7 , 8 - d i h y d r o d i o l ( E q u a t i o n 6) ( 4 7 ) . However, t h e y have been l a r g e l y i g n o r e d as p o t e n t i a l o x i d i z i n g a g e n t s i n b i o c h e m i c a l systems a l t h o u g h t h e i r h a l f - l i v e s ( 0 . 1 - 1 0 sec) s u g g e s t they c a n s e r v e as d i f f u s i b l e , s e l e c t i v e o x i d a n t s (48) . The mechanism o u t l i n e d i n F i g u r e 8 i s c o n s i s t e n t w i t h a l l o f t h e e x p e r i m e n t a l o b s e r v a t i o n s and e x p l a i n s t h e r e q u i r e m e n t f o r a d o u b l e bond i n t h e v i c i n i t y o f t h e h y d r o p e r o x i d e (Table I I ) . The a b i l i t y o f p e r o x y l r a d i c a l s t o e p o x i d i z e d o u b l e bonds appears t o depend upon t h e a b i l i t y o f t h e p e r o x y l r a d i c a l o l e f i n adduct t o s t a b i l i z e t h e c a r b o n - c e n t e r e d r a d i c a l . Thus, 3,4dihydroxy-3,4-dihydrobenzo(a)anthracene i s o x i d i z e d t o 1/6 t h e e x t e n t o f B P - 7 , 8 - d i h y d r o d i o l and a f l a t o x i n B i i s e p o x i d i z e d t o o n l y a s l i g h t e x t e n t (34,49). Peroxyl r a d i c a l s are the species t h a t propagate a u t o x i d a t i o n o f the u n s a t u r a t e d f a t t y a c i d r e s i d u e s o f p h o s p h o l i p i d s (50). In addit i o n , p e r o x y l r a d i c a l s a r e i n t e r m e d i a t e s i n the metabolism o f c e r t a i n d r u g s s u c h as p h e n y l b u t a z o n e ( 5 1 ) . E p o x i d a t i o n o f BP-7,8d i h y d r o d i o l has been d e t e c t e d d u r i n g l i p i d p e r o x i d a t i o n i n d u c e d i n r a t l i v e r microsomes by a s c o r b a t e o r NADPH and d u r i n g t h e p e r o x i d a t i c o x i d a t i o n o f p h e n y l b u t a z o n e (52,53) . These f i n d i n g s suggest t h a t peroxyl radical-mediated epoxidation o f BP-7,8-dihydrodiol i s general and may s e r v e as t h e p r o t o t y p e f o r s i m i l a r e p o x i d a t i o n s o f o t h e r o l e f i n s i n a v a r i e t y o f b i o c h e m i c a l systems. In addition, peroxyl radical-dependent epoxidation of BP-7,8-dihydrodiol e x h i b i t s the same s t e r e o c h e m i s t r y as t h e a r a c h i d o n i c a c i d - s t i m u l a t e d e p o x i d a t i o n by ram s e m i n a l v e s i c l e microsomes. T h i s n o t o n l y p r o v i d e s a d d i t i o n a l



Table I I ,

E p o x i d a t i o n o f U n s a t u r a t e d and S a t u r a t e d F a t t y A c i d Hydroperoxides 0

HYDROPEROXIDE

2

UPTAKE


(*M)

n

" l6 33 C

H

7 , 8 - D I O L OXIDATION

VI (ttM/ain.)

0.1610.02

0 0 H

A ^ X C O O H 6 I ±3

6.510.6

160116

12.310.9

160113

1211.3

16015

1211.6

00H

00H

H00

/=V=W

C 0 0 H

\=A7 vv 00 H / V ^ / V

/

C

0

0

C

3

M

y v v w 00H

/

N

^ ^ C 0 0 C H

3

6511

7.011.1

240115

1611.6

00H

/ \

/

^ s / V

/

C

0

0

C

H

3

0OH


MARNETT


Oxygenation


12.


319

320


e v i d e n c e t h a t the o x i d i z i n g agent i n the enzymatic r e a c t i o n i s a p e r o x y l r a d i c a l b u t a l s o s u g g e s t s t h a t the s t e r e o c h e m i s t r y o f BP7 , 8 - d i h y d r o d i o l o x i d a t i o n i s an i m p o r t a n t and g e n e r a l d i a g n o s t i c probe t o d i f f e r e n t i a t e e p o x i d a t i o n by m i x e d - f u n c t i o n o x i d a s e s and by peroxyl r a d i c a l s .


S i g n i f i c a n c e o f F a t t y A c i d Hydroperoxide-Dependent PAH

Oxidation

What i s the s i g n i f i c a n c e o f a r a c h i d o n i c a c i d - d e p e n d e n t x e n o b i o t i c metabolism? E x p e r i m e n t s d e s c r i b e d above f i r m l y e s t a b l i s h t h a t i t can cause m e t a b o l i c a c t i v a t i o n in vitro, Dihydrodiol metabolites of p o l y c y c l i c hydrocarbons are o x i d i z e d t o d i o l epoxides t h a t r e p r e s e n t the u l t i m a t e c a r c i n o g e n i c forms o f the p a r e n t h y d r o c a r b o n s . Intere s t i n g l y , o n l y d i h y d r o d i o l s t h a t form bay r e g i o n d i o l e p o x i d e s a r e a c t i v a t e d by PGH s y n t h a s e ; no a c t i v a t i o n o f o t h e r PAH d i h y d r o d i o l s occurs. A r a c h i d o n a t e - d e p e n d e n t c o o x i d a t i o n i s e s s e n t i a l l y an a c t i v a t i o n pathway s p e c i f i c f o r g e n e r a t i o n o f b a y - r e g i o n d i o l e p o x i d e s . Work d e s c r i b e d e l s e w h e r e i n d i c a t e s t h a t a r o m a t i c amines can a l s o be o x i d i z e d t o mutagenic d e r i v a t i v e s a l t h o u g h the i d e n t i t y o f the mutag e n i c d e r i v a t i v e i s , a t p r e s e n t , u n c e r t a i n (54). Is i t p o s s i b l e t c q u a n t i t a t e the r e l a t i v e c o n t r i b u t i o n o f h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a t i o n o f PAH in vitro, i n c e l l s and o r g a n s , and in vivo? Adding a r a c h i d o n i c a c i d o r NADPH t o s u p p o r t o x i d a t i o n i i i vitro g i v e s a good estimate of o x i d a t i v e p o t e n t i a l but i t s r e l a t i o n to c e l l u l a r o x i d a t i o n i s n o t s t r a i g h t f o r w a r d . L i k e w i s e , " s p e c i f i c " i n h i b i t o r s can be h e l p f u l i n in vitro e x p e r i m e n t s but t h e i r use can be compromised i n c e l l u l a r , o r g a n i s m a l , o r in vivo e x p e r i m e n t s by o v e r l a p p i n g s p e c i f i c i t i e s o r a l t e r e d p o t e n c i e s . F o r example, many compounds t h a t i n h i b i t l i p o x y g e n a s e a c t i v i t y a t low c o n c e n t r a t i o n i n m i c r o s o m a l o r c y t o p l a s m i c f r a c t i o n s a r e i n e f f e c t i v e when t h e y a r e employed i n c e l l u l a r experiments. The r e a s o n f o r the d i f f e r e n t i a l e f f e c t i s unc l e a r b u t the i m p l i c a t i o n f o r the use o f such compounds as in vivo i n h i b i t o r s i s obvious. A p o t e n t i a l l y p o w e r f u l probe f o r s o r t i n g o u t the c o n t r i b u t i o n of h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t p o l y c y c l i c hydrocarbon o x i d a t i o n i s stereochemistry. F i g u r e 9 summ a r i z e s the s t e r e o c h e m i c a l d i f f e r e n c e s i n e p o x i d a t i o n o f (±)-BP-7,8d i h y d r o d i o l by h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e dependent pathways (31,55,56). The (-)-enantiomer o f BP-7,8d i h y d r o d i o l i s c o n v e r t e d p r i m a r i l y t o the ( + ) - a n t i - d i o l epoxide by b o t h pathways whereas the (+)-enantiomer o f B P - 7 , 8 - d i h y d r o d i o l i s c o n v e r t e d p r i m a r i l y t o the ( - ) - a n t i - d i o l e p o x i d e by h y d r o p e r o x i d e dependent o x i d a t i o n and t o the ( + ) - s y n - d i o l e p o x i d e by m i x e d - f u n c t i o n oxidases. The s t e r e o c h e m i c a l c o u r s e o f o x i d a t i o n by cytochrome P-450 isoenzymes was f i r s t e l u c i d a t e d f o r t h e m e t h y c h o l a n t h r e n e i n d u c i b l e form but we have d e t e c t e d the same s t e r e o c h e m i c a l p r o f i l e u s i n g r a t l i v e r microsomes from c o n t r o l , p h e n o b a r b i t a l - , o r m e t h y l c h o l a n t h r e n e - i n d u c e d a n i m a l s (32). The o n l y d i f f e r e n c e between the microsomal p r e p a r a t i o n s i s the r a t e of o x i d a t i o n . The f i n d i n g s summarized i n F i g u r e 9 p r o v i d e a p r a c t i c a l d i a g n o s t i c t o o l f o r d i s t i n g u i s h i n g the two r o u t e s o f o x i d a t i o n . R e a c t i o n s can be p e r f o r m e d w i t h c e l l u l a r o r s u b c e l l u l a r p r e p a r a t i o n s and ( ± ) - o r ( + ) - B P - 7 , 8 - d i h y d r o d i o l and the t e t r a o l h y d r o l y s i s


12.

MARNETT


Oxygenation

321


(•>-ANTI

(•>-8YN

-ANTI

F i g u r e 9.

©

-

MIXED-FUNCTION 0XIDA8E DEPENDENT

(D

- P E R O X I D E - M E T A L DEPENDENT

S t e r e o c h e m i c a l d i f f e r e n c e s between f a t t y a c i d h y d r o p e r o x i d e - and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a t i o n of (±)-BP-7,8-dihydrodiol.



322

POLYCYCLIC HYDROCARBONS AND

CARCINOGENESIS

p r o d u c t s o f the d i o l e p o x i d e s s e p a r a t e d by HPLC and q u a n t i t a t e d (57). When the s u b s t r a t e i s (±)-BP-7,8-dihydrodiol an a n t i / s y n r a t i o i n e x c e s s o f 2.5 i s seen f o r p e r o x i d e - d e p e n d e n t o x i d a t i o n and an a n t i / s y n r a t i o o f 1 f o r m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a tion. When the s u b s t r a t e i s ( + ) - B P - 7 , 8 - d i h y d r o d i o l the a n t i / s y n r a t i o f o r the mixed-function oxidase-dependent r e a c t i o n decreases t o MD.3. The t e n f o l d d i f f e r e n c e i n the a n t i / s y n r a t i o between p e r o x i d e - and cytochrome P-450-dependent e p o x i d a t i o n makes i t an e x t r e m e l y s e n s i t i v e i n d i c a t o r o f the pathway o f o x i d a t i o n . We have e x p l o i t e d i t t o demonstrate t h a t l i p i d p e r o x i d a t i o n i n r a t l i v e r microsomes c a u s e s e p o x i d a t i o n ( 5 2 ) . By u s i n g (+)-BP-7,8-dihydrodiol, we have been a b l e t o d i s t i n g u i s h e p o x i d a t i o n caused by NADPHdependent l i p i d p e r o x i d a t i o n i n m e t h y l c h o l a n t h r e n e - i n d u c e d r a t l i v e r microsomes (52). These microsomes c o n t a i n an e x t r e m e l y a c t i v e c y t o chrome P-450 toward B P - 7 , 8 - d i h y d r o d i o l but i t i s p o s s i b l e to d i f f e r e n t i a t e the c o n t r i b u t i o n o f l i p i d p e r o x i d a t i o n t o e p o x i d a t i o n by d e t e r m i n i n g the y i e l d o f t e t r a o l s from the ( - ) - a n t i - d i o l e p o x i d e . A l t h o u g h i t has been s u s p e c t e d f o r some time t h a t l i p i d p e r o x i d a t i o n c o u l d cause x e n o b i o t i c o x i d a t i o n i n t h e p r e s e n c e o f an a c t i v e c y t o chrome P-450, our s t u d i e s o f B P - 7 , 8 - d i h y d r o d i o l oxidation provided the f i r s t c l e a r c u t d e m o n s t r a t i o n o f i t . S t e r e o c h e m i s t r y has a l s o been employed t o d e t e c t a r a c h i d o n i c a c i d - d e p e n d e n t B P - 7 , 8 - d i h y d r o d i o l e p o x i d a t i o n i n c u l t u r e d hamster t r a c h e a (58). These examples i l l u s t r a t e the power o f such s t e r e o c h e m i c a l p r o b e s . PGH s y n t h a s e and t h e r e l a t e d enzyme l i p o x y g e n a s e occupy a p o s i t i o n a t t h e i n t e r f a c e o f p e r o x i d a s e c h e m i s t r y and f r e e r a d i c a l c h e m i s t r y and can c l e a r l y t r i g g e r m e t a b o l i c a c t i v a t i o n by b o t h mechanisms. The p e r o x i d a s e pathway a c t i v a t e s compounds such as d i e t h y l s t i l b e s t r o l and a r o m a t i c amines whereas the f r e e r a d i c a l pathway a c t i v a t e s p o l y c y c l i c h y d r o c a r b o n s (59). B o t h pathways r e q u i r e s y n t h e s i s of hydroperoxide i n order to t r i g g e r o x i d a t i o n . The r a t e - l i m i t i n g s t e p i n h y d r o p e r o x i d e s y n t h e s i s i s r e l e a s e o f a r a c h i d o n i c a c i d from p h o s p h o l i p i d s t o r a g e (60,61). Release i s c a t a l y z e d by p h o s p h o l i p a s e s and i s s t i m u l a t e d by a q e n t s t h a t a c t a t the c e l l s u r f a c e such as hormones, i o n o p h o r e s , tumor p r o m o t e r s , e t c (62). Arachidonic acid-dependent c o o x i d a t i o n i s , t h e r e f o r e , a pathway t h a t l i n k s e v e n t s a t t h e c e l l s u r f a c e t o i n t r a c e l l u l a r o x i dation of xenobiotics. I t i s a l s o a model f o r o x i d a t i o n o f xenob i o t i c s by o t h e r p e r o x i d a s e s and by f r e e r a d i c a l s . T h e r e a r e few r e p o r t s o f x e n o b i o t i c m e t a b o l i s m by p e r o x y l o r a l k o x y l f r e e r a d i c a l s b u t the p o t e n t i a l i s enormous. U n s a t u r a t e d f a t t y a c i d s are p r e s e n t i n a l l c e l l s t o some e x t e n t and, i n f a c t , a r e q u i t e abundant i n most cells. F o r example, v e n t r i c u l a r m y o c a r d i a l muscle c o n t a i n s 14.1 ymol l i n o l e i c and a r a c h i d o n i c a c i d s p e r gram wet w e i g h t t i s s u e (63). B o t h f a t t y a c i d s a r e q u i t e s u s c e p t i b l e t o l i p i d p e r o x i d a t i o n which generates peroxvl r a d i c a l s capable of o x i d i z i n g c e r t a i n x e n o b i o t i c s , e.g., B P - 7 , 8 - d i h y d r o d i o l . I n h i b i t i o n of l i p i d peroxidation i s o b v i o u s l y a t a s k t h a t must be c o n s t a n t l y p e r f o r m e d by c e l l s t o p r e v e n t t i s s u e d e s t r u c t i o n and x e n o b i o t i c m e t a b o l i s m . The t u r n o v e r o f o n l y 0.1% o f the u n s a t u r a t e d f a t t y a c i d r e s i d u e s o f c e l l s c o u l d g e n e r a t e a v e r y s i g n i f i c a n t amount o f p e r o x y l r a d i c a l s i n s i d e membrane r e g i o n s o f c e l l s where many x e n o b i o t i c s arp d i s s o l v e d . M e t a b o l i s m o f a r o m a t i c amines and B P - 7 , 8 - d i h y d r o d i o l has been d e t e c t e d d u r i n g a r a c h i d o n a t e o x y g e n a t i o n i n i n t a c t c e l l s and i n c u l t u r e d t r a c h e a (64,58). Exogenous a r a c h i d o n a t e was added t o



12.

MARNETT


Oxygenation

323

s t i m u l a t e h y d r o p e r o x i d e s y n t h e s i s and c o o x y g e n a t i o n i n most o f t h e studies. R e c e n t l y , though, Amstad and C e r u t t i r e p o r t e d t h a t t h e l e v e l s o f a f l a t o x i n B^-DNA a d d u c t s formed i n C3H i O l S f i b r o b l a s t s were d e c r e a s e d by t r e a t m e n t o f t h e c e l l s w i t h i n d o m e t h a c i n o r e i c o s a t e t r a y n o i c a c i d , i n h i b i t o r s o f a r a c h i d o n a t e o x y g e n a t i o n ( 6 5 ) . They concluded that a s i g n i f i c a n t f r a c t i o n o f t o t a l a f l a t o x i n e p o x i d a t i o n by i O T ^ c e l l s o c c u r s as a r e s u l t o f a r a c h i d o n a t e - d e p e n d e n t cooxygenation. T h i s i m p l i e s t h a t c o o x y g e n a t i o n t a k e s p l a c e i n c e l l s and t h a t i t i s t r i g g e r e d by r e l e a s e o f a r a c h i d o n a t e from endogenous s t o r e s . To what e x t e n t does c o o x y g e n a t i o n o c c u r in vivo and i s i t important i n chemical c a r c i n o g e n e s i s ? This i s a very d i f f i c u l t q u e s t i o n t o answer a t t h e p r e s e n t t i m e . Recent r e s u l t s demonstrate t h a t a r o m a t i c amines and d i a m i n e s c a n be c o o x i d i z e d in vivo (66,67). In t h e case o f 3-napthylamine i t i s e s t i m a t e d t h a t 30% o f t h e a d d u c t s t h a t form t o DNA i n t h e dog b l a d d e r , a t a r g e t o r g a n f o r n a p t h y l a m i n e c a r c i n o g e n e s i s , a r i s e as a r e s u l t o f a r a c h i d o n a t e - d e p e n d e n t c o o x i d a t i o n ( 6 6 ) . T h i s c o n c l u s i o n i s based on t h e d e t e c t i o n o f u n i q u e p e r o x i d a s e a d d u c t s t c DNA t h a t a r e s t r u c t u r a l l y d i s t i n c t from mixedf u n c t i o n oxidase-generated adducts. In contrast, pretreatment o f A/HeJ mice w i t h a s p i r i n o r i n d o m e t h a c i n does n o t lower t h e l e v e l s o f DNA a d d u c t s formed from BP i n l u n g n o r does i t reduce t h e i n c i d e n c e o f l u n g neoplasms i n d u c e d by BP ( 6 8 ) . C o n t r o l e x p e r i m e n t s i n d i c a t e t h a t a s p i r i n t r e a t m e n t a b o l i s h e s PGH s y n t h a s e a c t i v i t y in vivo (68). T h i s s u g g e s t s t h a t PGH synthase-dependent c o o x i d a t i o n does n o t p l a y a r o l e i n l u n g t u m o r i g e n e s i s by b e n z o ( a ) p y r e n e i n t h e adenoma model. T h i s may be r e l a t e d t o t h e h i g h l e v e l s o f t h e endogenous a n t i o x i d a n t , v i t a m i n E , i n r o d e n t l u n g ( 6 9 ) . However, a d m i n i s t r a t i o n o f a s p i r i n t o g u i n e a p i g s does n o t lower t h e l e v e l s o f p r o t e i n o r DNA a d d u c t s formed from BP i n s e v e r a l d i f f e r e n t t i s s u e s , so t h e l e v e l s o f v i t a m i n E may n o t be a d e t e r m i n a n t o f BP c o o x i d a t i o n ( 7 0 ) . The t i s s u e d i s t r i b u t i o n o f PGH s y n t h a s e s u g g e s t s t h a t i t does n o t p l a y a major r o l e i n s y s t e m i c d r u g m e t a b o l i s m because most o f t h e t i s s u e s where i t i s p r e s e n t i n h i g h c o n c e n t r a t i o n do n o t r e c e i v e a s i g n i f i c a n t p r o p o r t i o n o f c a r d i a c o u t p u t (12) . However, s e v e r a l o f t h e s e t i s s u e s , e.g., k i d n e y and u t e r u s , a r e t a r g e t o r g a n s f o r carcinogens that require metabolic a c t i v a t i o n . In oraer t o detect arachidonate-dependent metabolic a c t i v a t i o n i n these t i s s u e s , i t w i l l be n e c e s s a r y t o d e v e l o p u n i q u e and s p e c i f i c p r o b e s . I f s y s t e m i c m e t a b o l i s m o f a g i v e n compound p r o c e e d s w i t h a unique p a t t e r n o f s t e r e o c h e m i s t r y (e.g., BP-7,8-dihydrodiol) o r produces u n i q u e DNA a d d u c t s (3-napthylamine) t h e n i t s h o u l d be p o s s i b l e t o q u a n t i t a t e t h e e x t e n t t o which u n i q u e s t e r e o i s o m e r s o r DNA a d d u c t s a r e formed. In vitro studies d e f i n e these d i s t i n c t i v e features o f c o o x i d a t i v e m e t a b o l i s m and g u i d e t h e i n t e l l i g e n t d e s i g n o f c r i t i c a l experiments. H o p e f u l l y , by u s i n g s u c h d i a g n o s t i c p r o b e s i t w i l l be p o s s i b l e t o p r o v i d e q u a n t i t a t i v e answers t o q u e s t i o n s about t h e e x t e n t t o which c o o x i d a t i o n o f p o l y c y c l i c h y d r o c a r b o n s and o t h e r c a r c i n o g e n s o c c u r s in vivo. Acknowledgments T h i s r e s e a r c h h a s been g e n e r o u s l y s u p p o r t e d by g r a n t s from t h e American Cancer S o c i e t y (BC244) and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h (GM23642). LJM i s a r e c i p i e n t o f an American Cancer S o c i e t y F a c u l t y R e s e a r c h Award (FRA243).



324


Literature Cited 1. Conney, A. H. Cancer Res. 1982, 42, 4875-4917. 2. Harvey, R. G. Acc. Chem. Res. 1981, 14, 218-26. 3. Sims, P.; Grover, P.L. In "Polycyclic Hydrocarbons and Cancer"; Gelboin, H.V.; Ts'o, P. O. P., Eds.; Academic: New York, NY, 1978; Vol. 1, pp. 117-81. 4. Levin, W.; Lu, A. Y. H.; Ryan, D.; Wood, A. W.; Kapitulnik, J.; West, S.; Huang, M.-T.; Conney, A. H.; Thakker, D. R.; Holder, G.; Yagi, H.; Jerina, D. M. In "Origins of Human Cancer"; Hiatt, H. H.; Watson, J. D.; Winsten, J. A., Eds.; Cold Spring Harbor: Cold Spring Harbor, 1977; pp. 659-82. 5. Rydstrom, J.; Montelius, J.; Bengtsson, M. "Extrahepatic Drug Metabolism and Chemical Carcinogenesis"; Elsevier: New York, 1983. 6. Cavalieri, E. L.; Rogan, E. G. In "Free Radicals in Biology"; Pryor, W. A., Ed.; Academic: New York, 1984; Vol. 6, pp. 323-69. 7. Bartsch, H.; Hecker, E. Biochim. Biophys. Acta 1971, 237, 56778. 8. Floyd, R. A.; Soong, L. M.; Culver, P. L. Cancer Res. 1976, 36, 1510-19. 9. Metzler, M.; McLachlan, J. A. Biochem. Biophys. Res. Comm. 1978, 85, 874-84. 10. Marnett, L. J.; Wlodawer, P.; Samuelsson, B. J. Biol. Chem. 1975, 250, 8510-17. 11. Hamberg, M.; Svensson, J.; Wakabayashi, T.; Samuelsson, B. Proc. Natl. Acad. Sci. USA 1974, 71, 345-49. 12. Christ, E. J.; Van Dorp, D. A. Biochim. Biophys. Acta 1972, 270, 537-45. 13. Ohiki, S.; Ogino, N.; Yamamoto, S.; Hayaishi, O. J. Biol. Chem. 1979, 254, 839-46. 14. Nugteren, D. H.; Hazelhof, E. Biochim. Biophys. Acta 1973, 326, 448-61. 15. Hamberg, M.; Samuelsson, B. Proc. Natl. Acad. Sci. USA 1974, 71, 3400-04. 16. Samuelsson, B. Science (Washington, D.C.) 1983, 220, 56895775. 17. Marnett, L. J.; Reed, G. A. Biochemistry 1979, 18, 2923-29. 18. Marnett, L. J.; Reed, G. A.; Johnson, J. T. Biochem. Biophys. Res. Comm. 1977, 79, 569-76. 19. Lorentzen, R. J.; Caspary, W. J.; Lesko, S. A.; Ts'o, P.O.P. Biochemistry 1975, 14, 3970-77. 20. Lesko, S.; Caspary, W.; Lorentzen, R.; Ts'o, P.O.P. Biochemistry 1975, 14, 3978-84. 21. Sivarajah, K.; Anderson, M. W.; Eling, T. Life Sci. 1978, 23, 2571-78. 22. Marnett, L. J.; Reed, G. A.; Dennison, D. J. Biochem. Biophys. Res. Comm. 1978, 82, 210-16. 23. Wislocki, P. G.; Wood, A. W.; CHang, R. L.; Levin, W.; Yagi, H.; Hernandez,O.;Dansette, P. M.; Jerina, D. M.; Conney, A. H. Cancer Res. 1976, 36, 3350-57. 24. Fahl, W. E.; Scarpelli, D.; Gill, K. Cancer Res. 1981, 41, 3400-06. 25. Brooks, P.; Osborne, M. R.; Carcinogenesis 1982, 3, 1223-26. 26. Reed, G. A., unpublished data. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


12. MARNETT Hydroperoxide-Dependent Oxygenation

325

27. Marnett, L. J.; Johnson, J. T.; Bienkowski, M. J. FEBS Letts. 1979, 106, 13-16. 28. Sivarajah, K.; Mukhtar, H.; Eling, T. FEBS Letts. 1979, 106, 17-20. 29. Marnett, L. J.; Bienkowski, M. J. Biochem. Biophys. Res. Comm. 1980, 96, 639-47. 30. Marnett, L. J.; Panthananickal, A.; Reed, G. A. Drug Metab. Rev. 1982, 13, 235-47. 31. Panthananickal, A.; Marnett, L. J. Chem. Biol. Interact. 1981, 33, 239-52. 32. Panthananickal, A.; Weller, P.; Marnett, L. J. J. Biol. Chem. 1983, 258, 4411-18. 33. Guthrie, J.; Robertson, I. G.C.;Zeiger, E.; Boyd, J. A.; Eling, T. E. Cancer Res. 1982, 42, 1620-23. 34. Dix, T. A.; Buck, J.; Marnett, L. J., manuscript in preparation. 35. Egan, R. W.; Gale, P. H.; Baptista, E. M.; Kennicott, K. L.; VandenHeuvel, W. J. A.; Walker, R. W.; Fagerness, P. E.; Kuehl, F. A., Jr. J. Biol. Chem. 1981, 256, 7352-61. 36. Marnett, L. J.; Siedlik, P. H.; Fung, L. W.-M. J. Biol. Chem. 1982, 257, 6957-64. 37. Pagels, W. R.; Sachs, R. J.; Marnett, L. J.; Dewitt, D. L.; Day, J. S.; Smith, W. L. J. Biol. Chem. 1983, 258, 6517-23. 38. Dunford, H. B. Coord. Chem. Rev. 1976, 19, 187-251. 39. Weller, P.; Markey,C.;Marnett, L., manuscript in preparation. 40. Dix, T. A.; Marnett, L. J. J. Amer. Chem. Soc. 1981, 103, 6744-46. 41. Gardner, H. W.; Eskins, K.; Grams, G. W.; Inglett, G. E. Lipids 1972, 7, 324-34. 42. Gardner, H. W.; Weisleder, D.; Kleiman, R. Lipids 1978, 13, 246-52. 43. Hamberg, M. Lipids 1975, 10, 87-92. 44. Dix, T. A.; Fontana, R.; Panthananickal, A.; Marnett, L. J., submitted for publication. 45. Morgenstern, R.; DePierre, J. W.; Lind, C.; Guthenberg, C.; Mannervik, B.; Ernster, L. Biochem. Biophys. Res. Comm. 1981, 99, 682-90. 46. Gower, J. D.; Wills, E. D. Carcinogenesis 1984, 5, 1183-89. 47. Mayo, R. Acc. Chem. Res. 1968, 1, 193-201. 48. Pryor, W. A. In "Free Radicals in Biology and Aging"; Armstrong, D., Ed.; Raven: New York, in press. 49. Battista, J. R.; Marnett, L. J. Carcinogenesis, submitted. 50. Porter, N. A. Met. Enzymol. 1984, 105, 273-82. 51. Marnett, L. J. In "Free Radicals in Biology"; Pryor, W. A., Ed.; Academic: New York, 1984; Vol. 6, pp. 63-94. 52. Dix, T. A.; Marnett, L. J. Science 1983, 221, 77-79. 53. Reed, G. A.; Brooks, E. A.; Eling, T. E. J. Biol. Chem. 1984, 259, 5591-95. 54. Robertson, I. G. C.; Sivarajah, K.; Eling, T. E.; Zeiger, E. Cancer Res. 1983, 43, 476-80. 55. Thakker, D. R.; Yagi, H.; Akagi, H.; Koreeda, M.; Lu, A. Y. H.; Levin, W.; Wood, A. W.; Conney, A. H.; Jerina, D. M. Chem. Biol. Interact. 1977, 16, 281-300.



326


56. Deutsch, J.; Vatsis, K. P.; Coon, M.; Leutz, J.C.;Gelboin, H. V. Mol. Pharmacol. 1979, 14, 1011-18. 57. Dix, T. A.; Marnett, L. J. Met. Enzymol. 1984, 105, 347-52. 58. Reed, G. A.; Grafstrom, R.C.;Krauss, R. S.; Autrup, H.; Eling, T. E. Carcinogenesis 1984, 5, 955-60. 59. Marnett, L. J.; Eling, T. E. In "Reviews in Biochemical Toxicology"; Hodgson, E.; Bend, J. R.; Philpot, R. M., Eds.; Elsevier/North Holland: New York, 1983; Vol. 5, pp. 135-72. 60. Lands, W. E. M.; Samuelsson, B. Biochim. Biophys. Acta 1968, 164, 426-29. 61. Vonkeman, H.; Van Dorp, D. A. Biochim. Biophys. Acta 1968, 164, 430-32. 62. Galli, C.; Galli, G.; Porcellati, G. "Advances in Prostaglandin and Thromboxane Research"; Raven: New York, 1978; Vol. 3. 63. Fletcher, R. Lipids 1972, 7, 728-732. 64. Wong, P. K.; Hampton, M. J.; Floyd, R. A. In "Prostaglandins and Cancer: First International Conference"; Powles, T. J.; Bockman, R. S.; Honn, K. V.; Ramwell, P., Eds.; Liss: New York, 1982; pp. 167-79. 65. Amstad, P.; Cerutti, P. Biochem. Biophys. Res. Comm. 1983, 112, 1034-40. 66. Yamazoe, Y.; Miller, D. W.; Gupta, R.C.;Zenser, T. V.; Weis, C. C.; Kadlubar, F. F. Proc. Amer. Assoc. Cancer Res. 1984, 25, 91. 67. Zenser, T. V.; Mattammal, M. B.; Brown, W.W.;Davis, B. B. Kidney International 1979, 16, 688-94. 68. Adriaenssens, P. I.; Sivarajah, K.; Boorman, G. A.; Eling, T. E.; Anderson, M. W. Cancer Res. 1983, 43, 4762-67. 69. Kornbrust, D. J.; Mavis, R. D. Lipids 1980, 15, 315-22. 70. Garattini, E.; Coccia, P.; Romano, M.; Jiritano, L.; Noseda, A.; Salmona, M. Cancer Res. 1984, 44, 5150-5155. RECEIVED May 13, 1985


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