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UBC Theses and Dissertations

The binding of benzo[a]pyrene to rat liver protein and nucleic acids in vivo Gontovnick, Larry Stuart 1978

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THE BINDING OF BENZO[A]PYRENE TO RAT LIVER PROTEIN AND NUCLEIC ACIDS IN VIVO by LARRY STUART GONTOVNICK B . S c , M c G i l l U n i v e r s i t y , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES i n THE FACULTY OF PHARMACEUTICAL SCIENCES DIVISION OF PHARMACOLOGY AND TOXICOLOGY We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA January , 1978 Larry S tuar t Gontovnick , 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department o f S ^ ^ ^ x ^ ^ y ^ ^ V w ^ C v X ^ CSV ^ S ^ ^ N ^ Q L ^ v ^ i O ^ l . The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ( p ^ AQr\q>. i i ABSTRACT In the p r e s e n t s t u d y r a t s were p r e t r e a t e d w i t h a g e n t s t h a t a r e known to a f f e c t the a c t i v i t i e s o f the b e n z o [ a ] p y r e n e (BP) m e t a b o l i z i n g enzymes in v i t r o . A l s o , agents wh ich a r e known t o a l t e r the l e v e l s o f h e p a t i c g l u t a t h i o n e were u s e d . These e x p e r i m e n t s were c a r r i e d out in o r d e r to d e t e r m i n e the e f f e c t s o f enzyme i n d u c t i o n , enzyme i n h i b i t i o n , and g l u t a t h i o n e l e v e l s on the degree o f c o v a l e n t b i n d i n g o f BP t o l i v e r m a c r o m o l e c u l e s in v i v o . In a d d i t i o n the r o l e s o f a r y l h y d r o c a r b o n h y d r o x y l a s e (AHH) and e p o x i d e h y d r a t a s e (EH) can be s t u d i e d in t h i s way. S i n c e the degree o f c o v a l e n t b i n d i n g 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 (PAH's ) and t h e i r t u m o r - i n i t i a t i n g a b i l i t y have been shown t o c o r r e l a t e ( 1 3 ) , the f a c t o r s wh ich govern the e x t e n t o f c o v a l e n t b i n d i n g a r e o f major i m p o r t a n c e . 3 When H-BP was a d m i n i s t e r e d i n t r a p e r i t o n e a11 y t o male W i s t a r r a t s , a c e r t a i n amount o f the compound was bound i r r e v e r s i b l y t o l i v e r m a c r o m o l e c u l e s . The d e g r e e o f i r r e v e r s i b l e b i n d i n g was found t o be dependent on both the dose o f BP a d m i n i s t e r e d and the t ime a f t e r i t s i n j e c t i o n . The d e g r e e o f b i n d i n g was found t o be l i n e a r l y dependent on t h e dose o f BP between t h e range o f 0.125 and 12.5 u m o l e s , t h e r e b y s u g g e s t i n g t h a t the m e t a b o l i z i n g pathways o f BP were not s a t u r a t e d a t t h e s e l e v e l s . The s t u d y showed the maximum l e v e l o f b i n d i n g to o c c u r a t 12 t o 18 hours a f t e r 1.25y mole o f BP, and t h i s f e l l to 60% o f maximum by ^8 h o u r s . The BP dosage o f 1.25y mole was employed t h r o u g h o u t the s t u d y . The r e s u l t s showed t h a t p r e t r e a t i n g r a t s w i t h SKF 525 - A s i g n i f i c a n t l y d e c r e a s e d the l e v e l o f i r r e v e r s i b l y bound BP f rom c o n t r o l l e v e l s by about 30%. The d e c r e a s e in b i n d i n g a f t e r SKF 525 - A t r e a t m e n t i s in a g r e e m e n t w ' t h the e v i d e n c e t h a t the cy tochrome P-k50 enzymes a r e r e s p o n s i b l e f o r the a c t i v a t i o n o f BP t o r e a c t i v e i n t e r m e d i a t e s , and t h a t they can be i n h i b i t e d by t h i s i i i compound (42). SKF 5 2 5 - A at 35 mg/kg, a dose wh ich p r o d u c e s i n h i b i t i o n o f AHH in v i t ro (7*0 d i d not d e c r e a s e the b i n d i n g o f BP in v i v o . SKF 5 2 5 - A at 50 mg/kg o r h i g h e r was r e q u i r e d t o p r o d u c e a d e c r e a s e in b i n d i n g , i n d i c a t i n g the n e c e s s i t y t o r e a c h a h i g h e r e f f e c t i v e h e p a t i c c o n c e n t r a t i o n o f SKF 525~A t o i n h i b i t the i r r e v e r s i b l e b i n d i n g o f BP in v i v o , in c o n t r a s t t o a l e s s e r amount o f SKF 525"A r e q u i r e d t o i n h i b i t BP h y d r o x y l a s e in v i t r o . O r a l methadone p r e t r e a t m e n t f a i l e d t o a l t e r the l e v e l o f BP b i n d i n g t o l i v e r macromolecu 1es. Methadone was found to i n c r e a s e h e p a t i c e p o x i d e h y d r a -t a s e by 212% in male W i s t a r r a t s (66), but in t h e p r e s e n t s t u d y t h i s d i d not have any i n f l u e n c e on the d e g r e e o f b i n d i n g o f BP in v i v o . 3 - M e t h y1c h o l a n t h r e n e (3~MC) p r e t r e a t m e n t was found t o s i g n i f i c a n t l y d e c r e a s e the l e v e l o f i r r e v e r s i b l y bound BP f rom c o n t r o l by about 30%. The p o s s i b l e c a u s e s o f t h i s 3"MC induced d e c r e a s e in b i n d i n g a r e d i s c u s s e d . One p o s s i b i l i t y i s t h a t t h e 3"MC induced d e c r e a s e c o u l d be due t o an a l t e r a t i o n in the pathways o f BP m e t a b o l i s m . The e x i s t e n c e in the l i v e r o f v a r i o u s forms o f cy toch rome P-450 (39) a l o n g w i t h the e v i d e n c e t h a t 3~MC i nduces a s p e c t r a l l y d i s t i n c t c y t o c h r o m e P-hk8 (40,58) c o u l d s u g g e s t t h a t 3 -MC a l t e r s BP m e t a b o l i s m t o s i t e s on the BP m o l e c u l e t h a t p r o d u c e l e s s r e a c t i v e i n t e r m e d i a t e s , and t h e r e b y d e c r e a s e s the d e g r e e o f b i n d i n g . N e i t h e r x y s t e i n e nor d i e t h y l m a l e a t e p r e t r e a t m e n t a l t e r e d the l e v e l o f i r r e v e r s i b l y bound BP f rom c o n t r o l . The d a t a o b t a i n e d f rom t h e s e e x p e r i m e n t s can be e x p l a i n e d by one o r more o f the f o l l o w i n g mechan isms : no s i g n i f i c a n t d e p l e t i o n o f g l u t a t h i o n e by BP o c c u r s , t h e r e i s a l a c k o f a g l u t a t h i o n e t h r e s h o l d l e v e l f o r b i n d i n g t o t a k e p l a c e , o r t h e r e i s a d r a s t i c a l l y d i f f e r e n t r o l e f o r g l u t a t h i o n e than i t s r o l e in the p r o t e c t i o n o f h e p a t i c m a c r o m o l e c u l e s f rom a l k y l a t i o n by a c t i v e m e t a b o l i t e s o f a c e t a m i n o p h e n . The e n z y m e - m e d i a t e d b i n d i n g o f BP t o l i v e r macromolecu 1es in v i t r o and i v : i t s i n h i b i t i o n by methadone, SKF 525 _ A, 3_MC, g l u t a t h i o n e , and cys t e i n e was demonstrated and the relevance of these f i n d i n g s towards the present experiments was discussed. Throughout the study a second population of animals showed binding of BP that was both q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t from the f i r s t . The percentage of animals that f e l l i n t o t h i s 2nd population was 19% (^ 6 out of 250) of a l l the animals used in the study. Dr. G.D. Bellward ( Supervisor ) TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION METABOLIC ACTIVATION OF PAH 1s TO EPOXIDES THE PATHWAYS OF BP METABOLISM FACTORS THAT INFLUENCE THE RATE AND PATHWAYS OF METABOLISM OF THE PAH1 EFFECT OF ENZYME INDUCTION AND INHIBITION ON THE BINDING OF lAH's IN VIVO THE OBJECTIVES OF THE PRESENT STUDY MATERIALS AND METHODS CHEMICALS ANIMALS ANIMAL TREATMENTS FOR IN VIVO BINDING EXPERIMENTS LIVER PREPARATION DETERMINATION OF IRREVERSIBLE BINDING OF 3 H - B P IN VIVO DETERMINATION OF IRREVERSIBLE BINDING OF 3 H - B P IN VITRO  RESULTS CONTROL EXPERIMENTS IRREVERSIBLE BINDING OF 3 H - B P IN VITRO EFFECT OF IN VITRO ADDITIONS ON THE IRREVERSIBLE BINDING OF 3 H-BP RELATIONSHIP BETWEEN IRREVERSIBLE BINDING IN VIVO AND DOSE OF BP IRREVERSIBLE BINDING IN VIVO WITH TIME EFFECT OF PRETREATMENTS ON THE IRREVERSIBLE BINDING OF 3 H - B P IN VIVO,  DISCUSSION SUMMARY AND CONCLUSIONS BIBLIOGRAPHY V I LIST OF TABLES T a b l e Page 3 C o n d i t i o n s f o r t h e i r r e v e r s i b l e b i n d i n g o f H -benzo [a ]py rene t o ra t l i v e r 10,000 x g s u p e r n a t a n t m a c r o m o l e c u l e s in v i t r o 29 II The e f f e c t o f v a r i e d l e v e l s o f the N A D P H - g e n e r a t i n g sys tem 3 c o f a c t o r s on t h e i r r e v e r s i b l e b i n d i n g o f H -benzo [a ]py rene 30 in v i t r o . I l l The e f f e c t o f c y s t e i n e and d i e t h y l m a l e a t e t r e a t m e n t s on t h e 3 d e g r e e o f i r r e v e r s i b l e b i n d i n g o f H - b e n z o [ a j p y r e n e t o r a t 77 l i v e r macromolecu 1es in v i v o . V I I LIST OF FIGURES F i g u r e Page 1 P o s s i b l e mechanisms o f c a r c i n o g e n e s i s by the u l t i m a t e e l e c t r o p h i 1 i c r e a c t a n t s d e r i v e d f rom c h e m i c a l c a r c i n o g e n s o r p r e c a r c i n o g e n s 2 2 B e n z o [ a ] p y r e n e m e t a b o l i s m 7 3 R e a c t i o n s o f b e n z o [ a ] p y r e n e by cy tochrome P -450 mixed f u n c t i o n o x y g e n a s e s , e p o x i d e h y d r a t a s e , g l u t a t h i o n e t r a n s f e r a s e , and o t h e r 9 c o n j u g a t i n g enzymes h C o n t r o l e x p e r i m e n t s ; 20 3 5 The e f f e c t o f i n c u b a t i o n t ime on the i r r e v e r s i b l e b i n d i n g o f H-b e n z o f a ] p y r e n e in v i t ro 22 6 The e f f e c t o f p r o t e i n c o n c e n t r a t i o n on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [ a ] p y r e n e in v i t r o 2k 7 The e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on the i r r e v e r s i b l e b i n d i n g o f 3|H-benzo[ a] py rene in v i t ro 27 8 The e f f e c t o f methadone on the i r r e v e r s i b l e b i n d i n g o f H-b e n z o [ a ] p y r e n e in v i t ro 32 3 9 The e f f e c t o f SKF 525 "A on the i r r e v e r s i b l e b i n d i n g o f H-b e n z o [ a ] p y r e n e in v i t r o 3k 3 10 The e f f e c t o f g l u t a t h i o n e on the i r r e v e r s i b l e b i n d i n g o f H-b e n z o [ a ] p y r e n e in v i t ro 36 3 11 The e f f e c t o f c y s t e i n e on the i r r e v e r s i b l e b i n d i n g o f H-b e n z o [ a ] p y r e n e in v i t r o 38 12 The e f f e c t o f 3~ m e t h y1c h o l a n t h r e n e on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [ a ] p y r e n e in v i t r o kO 3 13 The e f f e c t o f dose on the i r r e v e r s i b l e b i n d i n g o f H - b e n z o [ a ] p y r e n e t o t o t a l l i v e r p r o t e i n and n u c l e i c a c i d s in v i vo e x p r e s s e d as k3 pmoles bound/mg p r o t e i n 3 ]k The e f f e c t o f dose on the i r r e v e r s i b l e b i n d i n g o f H - b e n z o [ a ] p y r e n e t o r a t l i v e r macromolecu 1es in v i v o e x p r e s s e d as pmoles bound/gram kS wet we igh t o f l i v e r 3 15 The d o s e - i r r e v e r s i b l e b i n d i n g c u r v e o f H - b e n z o [ a ] p y r e n e in the second p o p u l a t i o n o f r a t s e x p r e s s e d as pmoles bound/mg p r o t e i n k~] 3 16 The d o s e - i r r e v e r s i b 1 e b i n d i n g c u r v e o f H - b e n z o [ a ] p y r e n e in the second p o p u l a t i o n o f r a t s e x p r e s s e d as pmoles bound/gram wet kS we igh t o f 1 i ve r 17 The e f f e c t o f methadone t r e a t m e n t on the i r r e v e r s i b l e b i n d i n g o f 3 H - b e n z o [ a ] p y r e n e in v i v o e x p r e s s e d as pmoles bound/gram wet 51 we igh t o f 1 i v e r v i i i F i gure Page 18 The e f f e c t o f methadone t r e a t m e n t on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [ a ]py rene in v i v o e x p r e s s e d as pmoles bound/mg p r o t e i n 53 19 The e f f e c t o f methadone t r e a t m e n t s on t h e i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [ a ] p y r e n e in v i v o in the second p o p u l a t i o n o r r a t s 55 e x p r e s s e d as pmoles bound/gram wet w e i g h t o f l i v e r 20 The e f f e c t o f methadone t r e a t m e n t on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [a ]py rene in v i v o in the second p o p u l a t i o n o f r a t s 57 e x p r e s s e d as pmoles bound/mg p r o t e i n . 21 The e f f e c t o f c o r n o i l and 3~methy1cholanthrene t r e a t m e n t s on t h e i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [a ]py rene in v i v o e x p r e s s e d 60 as pmoles bound/gram wet w e i g h t o f l i v e r 22 The e f f e c t o f c o r n o i l and 3~methy1cholanthrene t r e a t m e n t s on t h e i r r e v e r s i b l e b i n d i n g o f 3r|-benzo [a Jpyrene in v i v o e x p r e s s e d 62 as pmoles bound/mg p r o t e i n 23 The e f f e c t o f c o r n o i l and 3~methy1cholanthrene t r e a t m e n t s on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [aJpy rene in v i v o in the second p o p u l a t i o n o f r a t s e x p r e s s e d as pmoles bound/gram wer we igh t 66 o f 1 iver 2k The e f f e c t o f c o r n o i l and 3~methy1cholanthrene t r e a t m e n t s on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [aJpyrene in v i v o in the 68 second p o p u l a t i o n o f r a t s e x p r e s s e d as pmoles bound/mg p r o t e i n 25 A c o m p a r i s o n o f the e f f e c t s o f c o r n o i l and 3 ~ m e t h y l c h o l a n t h r e n e (3"MC) t r e a t m e n t s on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [a ]pyrene in v i vo in a n i m a l s i n j e c t e d w i t h the compound at e i t h e r 2k o r k8 70 hours a f t e r the l a s t dose o f e i t h e r c o r n o i l o r 3~MC 26 A c o m p a r i s o n o f the e f f e c t s o f c o r n o i l and 3 ~ m e t h y l c h o l a n t h r e n e (3~MC) t r e a t m e n t s on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [a ]pyrene 72 in v i v o in the second p o p u l a t i o n o f r a t s i n j e c t e d w i t h the compound at e i t h e r 2k o r 48 hours a f t e r the l a s t dose o f e i t h e r c o r n o i l o r 3~MC 27 The e f f e c t o f SKF 525~A t r e a t m e n t on the i r r e v e r s i b l e b i n d i n g o f ^ H - b e n z o [a ]pyrene in v i vo 7k i x . L IST OF ABBREVIATIONS AHH a r y l h y d r o c a r b o n h y d r o x y l a s e BP b e n z o t a ] p y r e n e dpm d i s i n t e g r a t i o n s per m i n u t e EH e p o x i d e h y d r a t a s e GSHT g l u t a t h i o n e - S - t r a n s f e r a s e i . p . i n t r a p e r i t o n e a l l y i . v . i n t r a v e n o u s l y 3-MC 3 - m e t h y 1 c h o l a n t h r e n e NADP + n i o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e NADPH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ( reduced) PAH 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 . E . M . the s t a n d a r d e r r o r o f the mean TCA t r i c h l o r o a c e t i c a c i d X . ACKNOWLEDGEMENTS l a m d e e p l y g r a t e f u l t o D r . G . D . B e l 1 ward f o r her g u i d a n c e and encouragement t h r o u g h o u t t h i s s t u d y . I would l i k e to thank my commit tee members ( D r s . J . H . M c N e i l l , J . G . S i n c l a i r , B . D . R o u f o g a 1 i s , and S . K a t z ) f o r t h e i r c o n s t r u c t i v e c r i t i c i s m o f t h i s m a n u s c r i p t . I am i n d e b t e d to Maureen O t t e n f o r s t a r t i n g me o f f on the r i g h t f o o t in the l a b , and to S tephen Ng f o r h i s t e c h n i c a l a s s i s t a n c e . F i n a l l y , a s p e c i a l thanks to my fami 1 y , c o l 1 e g u e s , a n d c l o s e f r i e n d s f o r t h e i r moral s u p p o r t and u n d e r s t a n d i n g , w h i c h was g r e a t l y a p p r e c i a t e d . 1. INTRODUCTION Chemica l c a r c i n o g e n e s i s was f i r s t documented by the B r i t i s h su rgeon S i r P e r c i v a l P o t t , in 1775, who c a l l e d a t t e n t i o n t o the h i g h i n c i d e n c e o f s c r o t a l c a n c e r in the chimney sweeps o f London and a t t r i b u t e d t h i s to t h e i r c o n s t a n t c o n t a c t w i t h c o a l t a r and s o o t (1). F o l l o w i n g t h i s o b s e r v a t i o n by about 150 y e a r s was the i s o l a t i o n o f a p d 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 (PAH) , b e n z o [ a ] p y r e n e ( B P ) , f rom c o a l t a r and the d e m o n s t r a t i o n t h a t i t p roduced c a n c e r on the s k i n o f mice (2). 12 1 T r 6 5 B e n z o [ a ] p y r e n e BP, the most commonly found PAH, i s a p o w e r f u l c a r c i n o g e n wh ich i s p r e s e n t in t o b a c c o smoke (3), i s a c o n t a m i n a n t o f the urban e n v i r o n m e n t (k), and is s u s p e c t e d o f c o n t r i b u t i n g t o the i n c r e a s i n g i n c i d e n c e o f c a n c e r o f the r e s p i r a -t o r y t r a c t in man (5). METABOLIC ACTIVATION OF PAH'S TO EPOXIDES Many c l a s s e s o f c h e m i c a l c a r c i n o g e n s become c o v a l e n t l y bound t o DNA, RNA and p r o t e i n s o f the c e l l s in t a r g e t t i s s u e s (6). It has been known s i n c e 1951 t h a t c a r c i n o g e n i c PAH 1 s a r e c o v a l e n t l y bound to mouse s k i n p r o t e i n s (7), mouse s k i n DNA (8), and t o DNA, RNA, and p r o t e i n s o f t r a n s f o r m a b l e rodent c e l l s in c u l t u r e (9). In 1969, i t was d i s c o v e r e d t h a t P A H ' s , wh ich d i d not b i n d c o v a l e n t l y t o DNA in the t e s t t u b e , were bound c o v a l e n t l y to DNA a f t e r i n c u b a t i o n o f the PAH i n the p r e s e n c e o f e n d o p l a s m i c r e t i c u l u m ( m i c r o s o m e s ) and NADPH (10). T h e s e e x p e r i m e n t s showed t h e r e q u i r e m e n t f o r e n z y m a t i c a c t i v a t i o n in o r d e r f o r P A H ' s t o become c o v a l e n t l y bound t o DNA. More r e c e n t l y , a c t i v a t i o n o f BP by the n u c l e a r e n v e l o p e has a l s o been r e p o r t e d (11). The c o v a l e n t i n t e r a c t i o n d e s c r i b e d i s thought t o be c r i t i c a l t o the c a r c i n o g e n i c p r o c e s s . The a c t i v a t e d o r u l t i m a t e c a r c i n o g e n s a r e u s u a l l y e l e c t r o p h i 1 i c 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 n u c l e o p h i l i c s i t e s o f v i t a l m a c r o -m o l e c u l e s in c e l l s , t h e r e b y i n i t i a t i n g the p r o c e s s o f c a r c i n o g e n e s i s . T h i s has been i n t e r p r e t e d g e n e r a l l y to mean t h a t the c r u c i a l , c a u s a t i v e , c h e m i c a l e v e n t i s a two - e l e c t r o n r e a c t i o n r e s u l t i n g in a s t a b l e , c o v a l e n t bond between the c a r c i n o g e n and t h e s u s p e c t e d t a r g e t m o l e c u l e , DNA (12). A p r e l i m i n a r y s t u d y i n d i c a t e d a c o r r e l a t i o n between t h e tumor - i n i t i a t i n g a b i l i t y o f s e v e r a l h y d r o c a r b o n s and t h e i r a b i l i t y t o b i n d c o v a l e n t l y t o DNA (13)- Thus the d e g r e e o f b i n d i n g o f a c a r c i n o g e n i s o f u l t i m a t e i m p o r t a n c e . F i g u r e 1 shows the b a s i c h y p o t h e s i s . PRECAR.CINOGENS METABOLISM ARC I X CARCINOGENIC ELECTROPHILIC REACTANTS ( ULTIMATE CARCINOGENS ) R-A S N 1 t °+ + A-« + NUCLEOPHILES IN CRITICAL ' CELLULAR TARGETS: BASES IN NUCLEIC ACIDS AMINO AC I OS IN PROTEINS OTHER CELLULAR COMPONENTS ALTERED NUCLEIC ACIDS OR PROTEINS OR BOTH ~1 * GENETIC EFFECTS EPIGENETIC EFFECTS DIRECT: MUTATIONS * * > > > > V M / / ' CHANGE IN GENOME EXPRESSION INDIRECT: ACTIVATION OF \ \ / SELECTION OF LATENT VIRUS ^ ^ I*' ' T U M 0 R C E U S NEOPLASIA' F i g u r e 1. P o s s i b l e mechanisms o f c a r c i n o g e n e s i s by the u l t i m a t e c a r c i n o g e n i c e l e c t r o p h i 1 i c r e a c t a n t s d e r i v e d f rom c h e m i c a l c a r c i n o g e n s o r p r e c a r c i n o g e n s ( 3-B o y l a n d , in 1950, s u g g e s t e d t h a t e p o x i d e s were formed as i n t e r m e d i a t e s in the m e t a b o l i s m o f PAH 's and t h a t t h e s e i n t e r m e d i a t e s were i n v o l v e d in the c a r c i n o g e n i c a c t i v i t y shown by many h y d r o c a r b o n s (15)- L a t e r work i n d i c a t e d (a) t h a t e p o x i d e s were formed as m e t a b o l i t e s o f h y d r o c a r b o n s in model systems in v i t r o ( 1 6 ) , (b) t h a t t h e s e compounds c o u l d b i n d c o v a l e n t l y 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 ( 1 7 ) , and (c) t h a t many o f them were b i o l o g i c a l l y a c t i v e and c o u l d induce m u t a t i o n s in mammalian c e l l s ( 1 8 ) , b a c t e r i a (1.9), and b a c t e r i o p h a g e (20), m a l i g n a n t t r a n s f o r m a t i o n in rodent c e l l s in c u l t u r e (21), and c a n c e r in e x p e r i m e n t a l a n i m a l s (22). The i n c u b a t i o n o f BP w i t h the m i c r o s o m a l f r a c t i o n , o x y g e n , and NADPH p roduced the K - r e g i o n e p o x i d e , b e n z o [ a ] p y r e n e 4,5 - oxide (23). The K - r e g i o n e p o x i d e o f BP r e a c t e d c o v a l e n t l y w i t h n u c l e i c a c i d s in v i t r o (24), was shown t o be m u t a g e n i c in b a c t e r i a l (25) and mammalian (18) c e l l s , and c o u l d t r a n s f o r m v a r i o u s c e l l s in c u l t u r e (26). K - r e g i o n e p o x i d e s were t e s t e d in c e l l c u l t u r e and found t o be more e f f e c t i v e in c a u s i n g n e o p l a s t i c t r a n s f o r m a t i o n than were the p a r e n t h y d r o c a r b o n s (27). However , a t t e m p t s to d e m o n s t r a t e t h e i r i n c r e a s e d c a r c i n o g e n i c p o t e n t i a l in v i v o were u n s u c c e s s f u l and BP-4,5 - oxide was found to be a weak c a r c i n o g e n in c o m p a r i s o n to BP (28). B e n z o [ a ] p y r e n e 4,5 _oxide T h e r e Is i n c r e a s i n g e v i d e n c e t h a t the K - r e g i o n e p o x i d e o f BP i s not the majo r r e a c t i v e i n t e r m e d i a t e in v i v o (29). S t u d i e s have s u g g e s t e d t h a t the m a j o r r e a c t i v e i n t e r m e d i a t e s in v i v o a r e the 7 , 8 - d i h y d r o x y - 9 , 1 0 - e p o x y ~ 7 , 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 d i o l e p o x i d e m e t a b o l i t e s (30). Two i somers o f t h i s compound have been s y n t h e s i z e d (31). HO-' ( ± ) -7 6 ,8a -d i h y d r o x y - 9 6,10a-epoxy-7 , 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 ( D i o l E p o x i d e 1) 9 1 0 > o o 8 HO' ( ± ) -7 f i , 8 a - d i h y d r o x y - 9 a , 10a , epoxy -7 , 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 ( D i o l E p o x i d e 2) It has been shown t h a t the d i o l e p o x i d e s o f BP r e a c t w i t h DNA t o g i v e a p r o d u c t w h i c h i s c h r o m a t o g r a p h i c a l l y i n d i s t i n g u i s h a b l e f rom t h a t o b t a i n e d by i n c u b a t i n g hamste r embryo c e l l s w i t h BP (32). It has a l s o been shown t h a t the f l u o r e s c e n c e s p e c t r u m o f the BP d i o l - e p o x i d e c o n j u g a t e w i t h DNA r e s e m b l e s t h a t o f the DNA c o n j u g a t e s f rom B P - t r e a t e d mouse s k i n (33) and f rom i n t e r a c t i o n w i t h B P - 7 , 8 - d i h y d r o d i o l m e d i a t e d by r a t l i v e r mic rosomes (34). G r o v e r and Sims showed t h a t the h y d r o c a r b o n - d e o x y r i b o n u c l e o s i d e p r o d u c t s formed in B P -t r e a t e d human b r o n c h i a l mucosa and mouse s k i n were i n d i s t i n g u i s h a b l e f rom t h o s e t h a t were fo rmed when 7 , 8 - d i h y d r o - 7 , 8 - d i h y d r o x y - B P - 9 , 1 0 - o x i d e r e a c t e d w i t h DNA in s o l u t i o n . The same h y d r o c a r b o n - D N A p r o d u c t s were a l s o found in h y d r o l y s a t e s o f DNA f rom 'mouse s k i n t r e a t e d w i t h 7 , 8 - d i h y d r o - 7 , 8 - d i h y d r o x y - B P (35)- L e v i n s t u d i e d mouse s k i n tumour d e v e l o p m e n t w i t h BP and the 4 , 5 " , 7 , 8 - , and 9.10- BP 5. e p o x i d e s . He found t h a t o f t h e s e e p o x i d e s s t u d i e d , o n l y BP-7,8 - ox ide was h i g h l y c a r c i n o g e n i c , a l t h o u g h l e s s c a r c i n o g e n i c than BP (36). A d d i t i o n a l s t u d i e s r e v e a l e d t h a t B P-7,8d i h y d r o d i o l was c o n s i d e r a b l y more c a r c i n o g e n i c than BP-7,8 - o x ide (37)- T h e r e f o r e , the two m e t a b o l i c p r e c u r s o r s o f the d i o l e p o x i d e s a r e p o t e n t c a r c i n o g e n s . Both d i o l e p o x i d e 1 and d i o l e p o x i d e 2 were found t o be h i g h l y m u t a g e n i c toward b a c t e r i a l and mammalian c e l l s (38). Weibe l , u s i n g d i f f e r e n t forms o f c y t o c h r o m e P-kSO f rom s o l u b i l i z e d r a b b i t l i v e r microsomes showed o x y g e n a t i o n o f BP at d i f f e r e n t p o s i t i o n s d e p e n d i n g on the form o f c y t o c h r o m e P-k50 u s e d . He a l s o showed t h a t some forms p roduced d i o l s and o t h e r s d i d not (39). T h e r e f o r e , the s p e c i f i c i t i e s o f d i f f e r e n t forms o f c y t o c h r o m e P-kSO may channe l BP m e t a b o l i s m i n t o the v a r i o u s a c t i v a t i o n and d e t o x i f i c a t i o n pathways and t h e r e b y h e l p d e t e r m i n e the c a r c i n o g e n i c a c t i v i t y o f t h e s e compounds. It s h o u l d a l s o be noted t h a t the PAH's induce t h e f o r m a t i o n o f a c y t o c h r o m e s p e c t r a l l y d i f f e r e n t f rom cy tochrome P-450, namely c y t o c h r o m e P-448 (kO). A l s o , Oesch has shown t h a t the a d d i t i o n o f homogeneous e p o x i d e h y d r a t a s e (EH) has a d i f f e r e n t e f f e c t on BP m u t a g e n i c i t y i f BP i s a c t i v a t e d by m i c r o s o m a l p r e p a r a t i o n s f rom 3~ m e t h y l -c h o l a n t h r e n e (3 - M C ) - p r e t r e a t e d mice ( 4 l ) . T h u s , i t a p p e a r s t h a t t h e r e l a t i v e f o r m a t i o n o f one o r more e p o x i d e s in v i v o w i l l depend on the t ypes and q u a n t i t i e s o f t h e v a r i o u s cy tochromes p r e s e n t a t the s i t e o f m e t a b o l i s m . S p e c i e s and t i s s u e d i f f e r e n c e s in the pathways o f BP m e t a b o l i s m and l e v e l s o f m e t a b o l i t e s produced c o u l d a l s o be e x p l a i n e d by the d i f f e r e n t forms o f c y t o c h r o m e P-^50 p r e s e n t . 6 . THE PATHWAYS OF BP METABOLISM It i s now e s t a b l i s h e d t h a t the i n i t i a l s t e p in the m e t a b o l i s m o f PAH 1 s i s c a r r i e d out by the "mixed f u n c t i o n o x y g e n a s e s " , enzymes t h a t a r e NADPH-dependent and c a t a l y z e the i n c o r p o r a t i o n o f m o l e c u l a r oxygen i n t o the s u b -s t r a t e m o l e c u l e s ( 4 2 ) . The oxygenases a r e i n v o l v e d in the m e t a b o l i s m o f most f o r e i g n compounds i n c l u d i n g d rugs and i n s e c t i c i d e s as we l1 as in the m e t a b o l i s m o f many endogenous s u b s t a n c e s such as s t e r o i d s ( 4 3 ) . Figure- . 2 shows the known r o u t e s o f BP m e t a b o l i s m ( 4 4 ) . The sequence o f enzyme a c t i o n can be o u t l i n e d as f o l l o w s . The i n i t i a l o x y g e n a t i o n i s c a t a l y z e d by the mixed f unct ion oxyg~eriaies',' wh ich c o n t a i n m u l t i p l e forms o f cy tochrome P - 4 5 0 . T h i s r e s u l t s in the f o r m a t i o n o f e p o x i d e s a t the 4 , 5 ; 7 , 8 ; and 9 , 1 0 p o s i t i o n s . The most s t a b l e o f t h e s e , the 4 , 5 ~ o x i d e , has been i s o l a t e d under in v i t r o cond i t i ons and the 7 , 8 - and 9 , 1 0 - o x i d e s a r e l i k e l y i n t e r m e d i a t e s ( 4 5 ) . T h e i r f o r m a t i o n can be deduced f rom t h e p r e s e n c e o f a l l t h r e e o f the c o r r e s p o n d i n g d i h y d r o d i o 1 s . These a r e formed by the a c t i o n o f e p o x i d e h y d r a -t a s e on the o x i d e i n t e r m e d i a t e s ( 4 6 ) . E v i d e n c e o f t h i s mechanism i s the f i n d i n g that , an e p o x i d e h y d r a s e i n h i b i t o r , 1 , 1 , 1 - t r i c h 1 o r o p r o p y 1 e n e o x i d e , e l i m i n a t e s d i h y d r o d i o l f o r m a t i o n ( 4 6 ) , and the r e a d d i t i o n o f a p a r t i a l l y p u r i f i e d e p o x i d e h y d r a t a s e r e s u l t s in the a p p e a r a n c e o f the d i h y d r o d i o l s ( 4 7 ) . P h e n o l s and q u i n o n e s can e i t h e r be formed n o n - e n z y m a t i c a 1 1 y f rom the o x i d e i n t e r m e d i a t e o r be the r e s u l t o f a d i r e c t o x y g e n a t i o n independent o f e p o x i d e i ntermed i a c y . The mixed f u n c t i o n o x y g e n a s e , any 1 h y d r o c a r b o n h y d r o x y l a s e (AHH), i s the p r i m a r y c a t a l y t i c a t t a c k on the P A H ' s . Enzyme p u r i f i c a t i o n s t u d i e s have shown t h a t t h i s sys tem is composed o f m u l t i p l e forms o f the enzyme ( 3 9 ) . The v a r i o u s forms o f cy toch rome P - 4 5 0 have d i f f e r e n t c a t a l y t i c a c t i v i t y w i t h r e s p e c t t o the f o r m a t i o n o f the v a r i o u s BP m e t a b o l i t e s . The e x p e r i m e n t a l 7-0 I ! CONJUGATES • BOUND MACROMOLECULES DNA RNA PROTEIN F i g u r e 2. B e n z o [ a ] p y r e n e m e t a b o l i s m . Drawn S t r u c t u r e s a r e m e t a b o l i t e s t h a t have been i s o l a t e d and c h a r a c t e r i z e d , w i t h the e x c e p t i o n o f the 9,10- and 7,8 - epox ides , whose p r e s e n c e i s c o n f i r m e d by e n z y m a t i c d i o l p r o d u c t s and n o n e n z y m a t i c pheno l f o r m a t i o n . B r a c k e t e d f i g u r e s a r e p o s s i b l e o r p r o b a b l e m e t a b o l i t e s not y e t c o n f i r m e d , (hk) 8. e v i d e n c e s u g g e s t s t h a t each fo rm o f the cy tochrome P-450 may have s e p a r a t e s i t e p r e f e r e n c e s on the BP m o l e c u l e , wh ich r e s u l t s in o x y g e n a t i o n a t d i f f e r e n t p o s i t i o n s . Thus the s p e c i f i c form o f the c y t o c h r o m e may r e l a t e t o the d e t o x -i f i c a t i o n o r a c t i v a t i o n pathways o f m e t a b o l i s m . A second major enzyme sys tem i n v o l v e d in PAH m e t a b o l i s m i s e p o x i d e h y d r a -t a s e . Hydrocarbon e p o x i d e s a r e m e t a b o l i z e d by the e p o x i d e h y d r a t a s e p r e s e n t in c e l l s o f many t i s s u e s in the body . The enzymes appear t o be l o c a t e d e x c l u -s i v e l y on the smooth e n d o p l a s m i c r e t i c u l u m o f c e l l s and a r e t h e r e f o r e found in the m i c r o s o m a l f r a c t i o n s when c e l l u l a r components a r e s e p a r a t e d by c e n t r i f u g a t i o n . The e p o x i d e h y d r a s e i s l o c a t e d in c l o s e p r o x i m i t y t o o n l y some forms o f c y t o -chrome P-450 (39, 48) . . U n l i k e the mic rosomal o x y g e n a s e , the e p o x i d e h y d r a t a s e s do not r e q u i r e NADPH o r , as f a r as i s known, any o t h e r c o f a c t o r (48). T h i s enzyme has been a s s a y e d u s i n g both s t y r e n e o x i d e and BP-4,5 -oxide as s u b s t r a t e s . K a p i t u l n i k , u s i n g human l i v e r e p o x i d e h y d r a t a s e showed t h a t the r a t e s o f h y d r a -t i o n o f the 4,5_,7,8-, and 9,10-oxides were s i m i l a r and t h a t the s t r o n g c o r r e l a -t i o n (r> 0.95) between the h y d r a t i o n o f t h e s e and o t h e r e p o x i d e s and the h y d r a t i o n o f s t y r e n e o x i d e i n d i c a t e d the p r e s e n c e o f a s i n g l e e p o x i d e h y d r a t a s e (49). He a l s o s u g g e s t e d t h a t a s i n g l e e p o x i d e c o u l d be p r e d i c t i v e o f the c a p a c i t y t o h y d r a t e e p o x i d e s f rom a . l a r g e number o f f o r e i g n c h e m i c a l s . A n o t h e r enzyme sys tem i n v o l v e d in BP m e t a b o l i s m i s the g 1 u t a t h iori 'e- 's -t r a n s f e r a s e (GSHT) , wh ich c o n v e r t s the e p o x i d e i n t e r m e d i a t e s to w a t e r - s o l u b l e c o n j u g a t e s (50). T h i s enzyme i s p r e s e n t in the s o l u b l e f r a c t i o n o f ra t l i v e r p r e p a r a t i o n s and c a t a l y z e s the r e a c t i o n s o f g l u t a t h i o n e , an endogenous t r i p e p t i d e , w i t h c e r t a i n o r g a n i c s u b s t r a t e s . N o n - e n z y m a t i c c o n j u g a t i o n o f g l u t a t h i o n e has a l s o been d e m o n s t r a t e d (51). It has been shown in v i t r o t h a t n e i t h e r c y s t e i n e , N - a c e t y1 c y s t e i n e nor o x i d i z e d g l u t a t h i o n e c o u l d be s u b s t i t u t e d f o r reduced g l u t a t h i o n e t o p r o d u c e c o n j u g a t e s o f 3,4 - d i ch1oron i t ro -benzene (52). The g l u t a t h i o n e c o n j u g a t e o f the d i o l - e p o x i d e o f b e n z a n t h r a c e n e has been i s o l a t e d (53) In a d d i t i o n , t h e r e a r e r e p o r t s o f the p r e s e n c e o f d i f f e r e n t BP c o n j u g a t e s i n u r i n e and b i l e . It has been s u g g e s t e d t h a t t h e s e may be g l u c u r o n i d e s a n d / o r s u l f a t e s (54). BP-3 - y l h y d r o g e n su lphate ( the s u l p h a t e e s t e r o f 3~hydroxy-BP) has been i d e n t i f i e d f rom m e t a b o l i t e s p r o d u c e d by b o t h r a t l i v e r 105,000 x g s u p e r n a t a n t and human, h a m s t e r and r a t l u n g c u l t u r e s (55). It has a l s o been r e p o r t e d t h a t a s e r i e s o f h y d r o x y l a t e d m e t a b o l i t e s o f BP i n c l u d i n g p h e n o l s , d i h y d r o d i o l s and e p o x i d e s f o r m g l u c u r o n i d e c o n j u g a t e s in the p r e s e n c e o f UDP-y l u c u r o n i c a c i d and mic rosomes (5&)• The c o n j u g a t i o n o f BP-4,5~oxide, h o w e v e r , may be v i a the d i h y d r o d i o l w h i c h i s fo rmed by BP-4,5~oxide h y d r a t a s e , a n o t h e r m i c r o s o m a l enzyme (56). F i g u r e 3 shows t h e r e a c t i o n s o f BP by c y t o c h r o m e P-kSO mixed f u n c t i o n o x y g e n a s e s , e p o x i d e h y d r a t a s e , GSHT, and o t h e r c o n j u g a t i n g enzymes (57). 0(04.-EPOXIDES CYT P-ISO I HYORATASE - BP-OXIDES » - DIHYDROOIOU SSH-TRANSFERASE F i g u r e 3. R e a c t i o n s o f b e n z o [ a ] p y r e n e by c y t o c h r o m e P-450 mixed f u n c t i o n o x y g e n a s e s , e p o x i d e h y d r a t a s e , GSH t r a n s f e r a s e , and o t h e r c o n j u g a t i n g enzymes (57). 10. FACTORS THAT INFLUENCE THE RATE AND PATHWAYS OF METABOLISM OF THE PAH'S When a n i m a l s a r e t r e a t e d w i t h any o n e . o f a l a r g e number o f o r g a n i c compounds, the l e v e l s o f the m i c r o s o m a l o x y g e n a s e s in the l i v e r and in many o t h e r o r g a n s , such as l u n g , k i d n e y , s k i n . a n d s m a l l i n t e s t i n e a r e r a i s e d (58). The e f f e c t s o f p r e t r e a t i n g a n i m a l s w i t h enzyme i n d u c e r s on the in v i t r o h e p a t i c m e t a b o l i s m o f PAH 's was f i r s t d e s c r i b e d by Conney e t a l . (59), who t r e a t e d r a t s w i t h PAH 's and showed i n c r e a s e s in the l e v e l o f h e p a t i c AHH (BP h y d r o x y l a s e ) . Compounds o t h e r than PAH's w i l l a l s o induce t h e o x y g e n a s e . They i n c l u d e compounds w i t h such d i v e r s e s t r u c t u r e s as p h e n o b a r b i t a l , a m i n o p y r e n e , c h l o r d a n e , and r i f a m p i n (60, 61, 62). The compound w i d e l y used as an i n h i b i t o r o f AHH i s SKF 525-A (63). T h i s compound when a d m i n i s t e r e d i n t r a p e r i t o n e a l1 y i n t o r a t s p r o d u c e s a marked d e c r e a s e in h e p a t i c AHH a c t i v i t y . B e n z o f l a v o n e i s a w i d e l y employed compound t h a t w i l l i n h i b i t AHH in v i t r o (64). E p o x i d e h y d r a s e i s a l s o i n d u c i b l e by p r e t r e a t m e n t w i t h p h e n o b a r b i t a l and P A H ' s (48). I n d u c t i o n , however , i s s m a l l as compared w i t h the c o r r e s p o n d i n g i n c r e a s e s in the l e v e l s o f AHH. A l a r g e number o f compounds have been d e s c r i b e d t h a t w i l l i n h i b i t the h y d r a t i o n o f s t y r e n e o x i d e in v i t r o . Examples o f i n h i b i t o r s o f t h i s enzyme a r e c y c l o h e x a n e o x i d e and 3,4 -d ihydronaphtha1ene (65)- B e l l w a r d e t a l . showed t h a t in male W i s t a r r a t s t r e a t e d w i t h methadone t h e r e was an i n c r e a s e in h e p a t i c e p o x i d e h y d r a t a s e a c t i v i t y w i t h o u t an i n c r e a s e in the h e p a t i c AHH a c t i v i t y (66). T h i s marked t h e f i r s t c a s e o f an agent s p e c i f i c a l l y i n c r e a s -ing e p o x i d e h y d r a t a s e ^ a c t i v i t y w i t h o u t a f f e c t i n g AHH. PAH 's and p h e n o b a r b i t a l t r e a t m e n t a r e known t o i n d u c e the GSHT a c t i v i t y in the r a t l i v e r (51). The r o l e o f g l u t a t h i o n e has been s t u d i e d e x t e n s i v e l y by M i t c h e l l and c o - w o r k e r s and t h e i r d a t a i n d i c a t e t h a t in a n i m a l s g l u t a t h i o n e i s e s s e n t i a l f o r the p r o t e c t i o n o f t h i o l and o t h e r nucleoph i . l i c g roups in p r o t e i n s and" 11. n u c l e i c a c i d s f rom a v a r i e t y o f t o x i c d rug m e t a b o l i t e s and o t h e r a l k y l a t i n g a g e n t s (67). These w o r k e r s have p o s t u l a t e d t h a t the h e p a t o t o x i c i t y o f a g e n t s such as a c e t o m i n a p h e n , i s o n i a z i d , bromobenzene and f u r o s e m i d e c o u l d be due t o m e t a b o l i c a c t i v a t i o n o f t h e s e compounds t o r e a c t i v e i n t e r m e d i a t e s wh ich b i n d c o v a l e n t l y to h e p a t i c m a c r o m o l e c u l e s , thus l e a d i n g t o c e l l n e c r o s i s (68). The e f f e c t s o f enzyme i n d u c t i o n , enzyme i n h i b i t i o n , and the r o l e o f g l u t a t h i o n e on the c o v a l e n t b i n d i n g o f t h e s e a g e n t s to 1 i ver m a c r o m o l e c u l e s in v i v o has been s t u d i e d t h o r o u g h l y (68). The i m p o r t a n c e o f enzyme i n d u c t i o n , enzyme i n h i b i t i o n and g l u t a t h i o n e on the m e t a b o l i s m and b i n d i n g o f PAH 1 s in v i t r o have been we;ll documented . Recent s t u d i e s on the m e t a b o l i s m o f BP by h e p a t i c mic rosomal f r a c t i o n s f rom normal and 3~MC t r e a t e d r a t s have shown t h a t the amounts o f the n o n - K - r e g i o n m e t a b o l i t e s , the 7,8- and the 9,10- d i h y d r o d i o l s were i n c r e a s e d t o g r e a t e r e x t e n t s than a r e the p h e n o l s and the K - r e g i o n d i h y d r o d i o l when l i v e r s f rom 3 - M C - t r e a t e d a n i m a l s were used (69). L i v e r microsomes f rom 3"MC p r e t r e a t e d r a t s m a r k e d l y i n c r e a s e d the b i n d i n g o f BP to c e l l u l a r m a c r o m o l e c u l e s (70). It has a l s o been shown t h a t e p o x i d e h y d r a t a s e i n h i b i t o r s p r e s e n t in the i n c u b a t i o n tube w i l l a l s o i n c r e a s e the b i n d i n g o f BP t o c e l l u l a r macromolecu 1es (46). K ing showed t h a t g l u t a t h i o n e added to the i n c u b a t i o n tube m a r k e d l y d e c r e a s e d the b i n d i n g o f BP t o added DNA (70). T h e r e f o r e , the e n z y m a t i c a c t i v i t i e s o f AHH, e p o x i d e h y d r a t a s e and GSHT, a l o n g w i t h the g l u t a t h i o n e l e v e l s in the c e l l c o u l d p l a y i m p o r t a n t r o l e s in the m e t a b o l i s m and b i n d i n g o f BP t o c e l l u l a r m a c r o m o l e c u l e s in v i v o . The f a c t o r s wh ich w i l l d e t e r m i n e the d e g r e e o f b i n d i n g o f a g i v e n e p o x i d e t o c e l l u l a r m a c r o m o l e c u l e s in v i v o w i l l be (a) i t s r a t e o f f o r m a t i o n , (b) i t s r a t e o f e n z y m a t i c c o n v e r s i o n i n t o the r e l a t e d d i h y d r o d i o l , (c) i t s r a t e o f e n z y m a t i c and n o n - e n z y m a t i c c o n v e r s i o n i n t o the r e l a t e d g l u t a t h i o n e c o n j u g a t e , 12. and (d) t h e r a t e a t wh ich i t w i l l a l k y l a t e c e l l u l a r macromolecu 1es. A n o t h e r impor tant f a c t o r , ment ioned b e f o r e , i s the r e l a t i v e abundances o f the v a r i o u s t ypes o f c y t o c h r o m e P-450, wh ich w i l l g o v e r n wh ich s i t e on the BP m o l e c u l e w i l l undergo o x i d a t i o n . EFFECT OF ENZYME INDUCTION AND INHIBITION ON THE BINDING OF PAH's IN VIVO. S i n c e the b i n d i n g o f PAH's i s thought to be the p r ime event in the c a r c i n o g e n i c p r o c e s s , t h e f a c t o r s t h a t a f f e c t the l e v e l s o f b i n d i n g o f PAH's in v i v o a r e o f ext reme i m p o r t a n c e . U n f o r t u n a t e l y , l i t t l e work in t h i s a r e a has been c a r r i e d o u t . The m e t a b o l i s m and c o v a l e n t b i n d i n g o f BP has been d e t e r m i n e d in i s o l a t e d p e r f u s e d lungs o f sham- and c i g a r e t t e smoked-exposed and 3"MC p r e t r e a t e d male W i s t a r r a t s (71). A f t e r i n t r a t r a c h e a l i n s t i l l a t i o n 3 o f H - B P , t h e amount o f c o v a l e n t l y bound r a d i o a c t i v i t y was s i g n i f i c a n t l y i n c r e a s e d in the lungs o f a n i m a l s exposed t o c i g a r e t t e smoke and in the lungs o f 3"MC t r e a t e d a n i m a l s compared t o the c o r r e s p o n d i n g sham-exposed a n i m a l s . The t o t a l amount o f d i h y d r o d i o l s formed in the lung o f smoke -exposed r a t s was 5 t i m e s g r e a t e r than in the s h a m - t r e a t e d a n i m a l s , w i t h the 4 , 5 " , 7 , 8 - , and 9 , 1 0 - d i h y d r o d i o l s each showing 5 " f o l d i n c r e a s e s . The amount o f 3~hydroxy-BP was a l s o i n c r e a s e d . THE OBJECTIVES OF THE PRESENT STUDY In t h e p r e s e n t s t u d y we a r e u t i l i z i n g the male W i s t a r r a t l i v e r as our model sys tem t o s t u d y the e f f e c t o f enzyme i n d u c t i o n , enzyme i n h i b i t i o n and g l u t a t h i o n e l e v e l s on the b i n d i n g o f BP t o h e p a t i c macromo1 ecu 1es in v i v o , a f t e r i t s i . p . a d m i n i s t r a t i o n . The l i v e r i s employed in t h i s s t u d y f o r the f o l l o w i n g r e a s o n s , F i r s t l y , the e n z y m a t i c e f f e c t s in v i t ro o f the i n d u c e r s and i n h i b i t o r s o f the BP m e t a b o l i z i n g enzymes t h a t we a r e u s i n g have been s t u d i e d most e x t e n s i v e l y in the l i v e r . S e c o n d l y , the o p p o r t u n i t y to s t u d y the e f f e c t o f s e l e c t i v e i n d u c t i o n o f e p o x i d e h y d r a t a s e by methadone is l i m i t e d 13, t o i t s s i t e o f i n d u c t i o n , the l i v e r ; and t h i r d l y , h e p a t i c g l u t a t h i o n e f u n c t i o n and a g e n t s wh ich a f f e c t i t s f u n c t i o n have been w e l l documented in t h i s t i s s u e (72, 73). The c a r c i n o g e n i c a c t i v i t y o f BP i s thought to be due t o the c o v a l e n t r e a c t i o n o f m e t a b o l i c a l l y formed e p o x i d e s w i t h t a r g e t m a c r o m o l e c u l e s such as DNA, RNA, and p r o t e i n (12). We a r e u t i l i z . i n g a g e n t s , wh ich have been shown in v i t r o t o a f f e c t the pathways i n v o l v e d in t h e f a t e o f t h e s e e p o x i d e s , as t o o l s to s t u d y the f a c t o r s t h a t i n f l u e n c e the i n-, v i vo b i n d i n g o f BP. SKF 525"A has been shown to d e c r e a s e BP h y d r o x y l a s e , the mono-oxygenase r e s p o n s i b l e f o r t h e f o r m a t i o n o f e p o x i d e i n t e r m e d i a t e s (74). Methadone p r e t r e a t m e n t showed a s e l e c t i v e i n c r e a s e in e p o x i d e h y d r a t a s e a c t i v i t y , the enzyme r e s p o n s i b l e f o r the f u r t h e r t r a n s f o r m a t i o n o f e p o x i d e s t o d i h y d r o d i o l s (66). 3~MC i n c r e a s e d the a c t i v i t y o f B P - h y d r o x y 1 a s e as w e l l as induced the f o r m a t i o n o f a s p e c t r a l l y d i f f e r e n t c y t o c h r o m e , namely cy tochrome P-448 (40, 58). It a l s o i n c r e a s e d EH and GSHT a c t i v i t i e s , but t o a lower d e g r e e than the i n c r e a s e in AHH a c t i v i t y (,4 8, 51). C y s t e i n e i s a p r e c u r s o r in the b i o s y n t h e s i s o f g l u t a t h i o n e (73) and d i e t h y l ma 1eate"dep1etes h e p a t i c g l u t a t h i o n e l e v e l s (72). T h e r e f o r e , b y . u s i n g t h e s e a g e n t s we can s t u d y the r o l e s o f AHH, EH and g l u t a t h i o n e on the b i n d i n g o f BP in v i v o . 14. MATERIALS AND METHODS  CHEMICALS: 3-H-BP ( g e n e r a l l y l a b e l l e d ; s p e c i f i c a c t i v i t y 8 .3 and 40 Ci/mmole) was obta ined from Amersham c o r p o r a t i o n , (Toronto, O n t . ) . Methadone'HCl was obta ined through the Canadian Department of Heal th and W e l f a r e . SKF 525 - A ( ( 2 - d i e t h y l - a m i n o e t h y l - 2 , 2 - d i p h e n y l v a l e r a t e - H C 1 ) was a g i f t from Smi th , K l i n e and French L a b s . , (Mont rea l , Que . ) . 3"MC, d i e t h y l maleate and BP were s u p p l i e d by Eastman Organic Chemica ls , (Rochester , N . Y . ) ; M g C ^ ' b ^ O by B r i t i s h Drug House, (Toronto, O n t . ) ; NADP, g l u c o s e - 6 - p h o s p h a t e , g lucose -6 -phosphate dehydrogenase, g l u t a t h i o n e (reduced) and bovine serum albumin by Sigma Chemical C o . , (S t . L o u i s , M o . ) ; L - ( + ) - c y s t e i n e h y d r o c h l o r i d e by F i s h e r S c i e n t i f i c C o . , ( F a i r Lawn, N . J . ) . A l l other reagents and s o l v e n t s were the best a v a i l a b l e commercial grades . The p u r i t y of the ^H-BP was 98% as shown by t h i n - l a y e r chromatography on s i l i c a g e l , (Eastman, Rochester , N.Y.) us ing two so l ven t systems (benzene-hexane, 1:15 and benzene -e thano l , 9 : 1 ) . The n o n - l a b e l l e d BP was d i s s o l v e d in benzene, f i l t e r e d and r e c r y s t a l 1 i z e d from c o l d methanol . ANIMALS: Wis ta r r a t s , males (200-300gm)»obtained l o c a l l y ( U n i v e r s i t y of B r i t i s h Columbia) were employed throughout the s tudy . For each exper iment , c o n t r o l and t r e a t e d rats were matched fo r age and weight w i t h minimal v a r i a t i o n . The a n i m a l s , 4 per cage, were a l lowed to e q u i l i b r a t e fo r at l e a s t one week under c o n t r o l l e d c o n d i t i o n s (22°C, l i g h t s on 0600 to 2000 h o u r s ) . 'Lobund' grade corn -cob bedding , (Paxton P rocess ing L t d . , Paxton , 111.) was used. Pur ina Lab Chow and tap water were s u p p l i e d ad 1i b i turn, unless o therwise s t a t e d . 15. ANIMAL TREATMENTS FOR IN VIVO BINDING EXPERIMENTS 3H-BP d i l u t e d w i t h n o n - l a b e l l e d BP was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y (1.25 y m o l e 3H-BP, 125 WCi in 0.5 m l , c o r n o i1 ) , The dose o f 3 H - B P was a l t e r e d by v a r y i n g the amount o f n o n - l a b e l l e d BP, the volume and q u a n t i t y o f r a d i o a c t i v i t y i n j e c t e d r e m a i n i n g c o n s t a n t . A n i m a l s were s a c r i f i c e d a t the i n d i c a t e d t imes a f t e r 3 H - B P . 3~MC in c o r n o i l was i n j e c t e d i . p . a t 20 mg/kg f o r 2 c o n s e c u t i v e d a y s . C o n t r o l a n i m a l s r e c e i v e d c o r n o i l o n l y . A n i m a l s were a d m i n i s t e r e d 3 H - B P 2k hours a f t e r the l a s t 3_MC i n j e c t i o n , u n l e s s o t h e r w i s e s t a t e d . SKF 525-A ( s i n g l e dose a t 35 mg/kg, 50 mg/kg o r 75 mg/kg) was g i v e n i . p . in s a l i n e . C o n t r o l a n i m a l s r e c e i v e d s a l i n e o n l y . 3 H - B P was a d m i n i s t e r e d 3 hours a f t e r SKF 525-A. D i e t h y l m a l e a t e in c o r n o i l (0.6 m l/kg i . p . ) was i n j e c t e d 30 m i n u t e s b e f o r e a d m i n i s t r a t i o n o f H -BP . C o n t r o l a n i m a l s r e c e i v e d c o r n o i l o n l y . L - ( + ) - c y s t e i n e * H C l in s a l i n e (150 mg/kg i . p . ) was a d m i n i s t e r e d 5 m inutes •3 b e f o r e H - B P . C o n t r o l a n i m a l s r e c e i v e d s a l i n e a l o n e . The p r e t r e a t m e n t s o l u t i o n s were a d m i n i s t e r e d a t a volume to animal we i g h t r a t i o o f a p p r o x i m a t e ! y 2.3 ml / k g . Methadone 'HC l was d i s s o l v e d in the d r i n k i n g w a t e r . The dosage o f methadone was i n c r e a s e d once a week, u n t i l k weeks-,' as they d e v e l o p e d a t o l e r a n c e t o the d r u g . A l l methadone doses g i v e n as mg/kg a r e f o r a 2k hour p e r i o d . A n i m a l s were m a i n t a i n e d on methadone up u n t i l the t ime o f s a c r i f i c e in o r d e r t o p r e v e n t the deve lopment o f a b s t i n e n c e symptoms. L lVER PREPARATION A l l a n i m a l s were s t u n n e d by a b low on the h e a d , k i l l e d by d e c a p i t a t i o n and b l e d . The l i v e r s were then p e r f u s e d w i t h i c e - c o l d 1.15% KC1 in s i t u and 16. p l a c e d i m m e d i a t e l y jn the i c e - c o l d KC1 s o l u t i o n , DETERMINATION OF IRREVERSIBLE BINDING OF 3 H - B P IN VIVO Q u a n t i t a t i o n o f the i r r e v e r s i b l e b i n d i n g o f J H - B P to t o t a l l i v e r p r o t e i n and n u c l e i c a c i d s , a f t e r i t s i . p . i n j e c t i o n i n t o r a t s , was d e t e r m i n e d a c c o r d i n g t o the method o f Jo 1 low e t a l . ( 75 ) . F i v e grams o f l i v e r t i s s u e were minced and homogenized in 20 ml o f i c e - c o l d 1.15% KC1 , u s i n g a m o t o r - d r i v e n P o t t e r -El vehjem h o m o g e n i z e r . D u p l i c a t e 1 ml a l i q u o t s o f e a c h homogenate , e q u i v a l e n t t o 0.25 gm o f 1 i v e r , were then added t o 2 ml: o f 0.9 M t r i c h l o r o a c e t i c a c i d ( TCA) , in d i s p o s a b l e c u l t u r e t u b e s , to p r e c i p i t a t e the p r o t e i n and n u c l e i c a c i d s . The tubes were then v o r t e x e d and c e n t r i f u g e d ( I n t e r n a t i o n a l . c e n t r i f u g e , s i z e 2, model EXD) at room t e m p e r a t u r e , a t 1,000 x g , f o r 10 min . The s u p e r -n a t a n t was d i s c a r d e d and the p r o t e i n resuspended in 3 ml o f 0.6 M^  TCA, u s i n g a v o r t e x s h a k e r and s t a i n l e s s s t e e l s p a t u l a to improve m i x i n g . The tubes were c e n t r i f u g e d a g a i n a t 1,000 x g f o r 3 min and the s u p e r n a t a n t was d i s c a r d e d . The p r o t e i n was resuspended in 0.6 M^  TCA t w i c e more and a f t e r the s u p e r n a t a n t was d i s c a r d e d on the l a s t 0.6 M^  TCA w a s h , the p r o t e i n was r e s u s p e n d e d ~<kn 3 ml o f 80% m e t h a n o l , mixed w i t h a s p a t u l a , as a b o v e , c e n t r i f u g e d a t 1,000 x g f o r 3 min and the s u p e r n a t a n t d i s c a r d e d . The p r o c e d u r e was r e p e a t e d u n t i l no f u r t h e r r a d i o a c t i v i t y c o u l d be removed. F u r t h e r wash ings w i t h a c e t o n e , c h l o r o f o r m and e t h e r f a i l e d to remove a d d i t i o n a l r a d i o a c t i v i t y . The e x t r a c t e d p r o t e i n was then d i s s o l v e d i n 5 : ml o f 1N NaOH and a 0.5 ml a l i q u o t , e q u i v a l e n t t o 0.025 gm o f l i v e r , was taken f rom each sample and added a l o n g w i t h 0.5 ml o f 1.2 N HCl t o 15 ml o f B i o f l u o r s c i n t i l l a t i o n f l u i d , (New E n g l a n d N u c l e a r , D o r v a l , Que . ) and c o u n t e d in a S e a r l e Mark I I I l i q u i d s c i n t i l l a t i o n c o u n t e r . The 1.2 N HCl was needed t o d e c r e a s e chemi11uminescence in the samples (76 )• R a d i o a c t i v i t y was c o r r e c t e d f o r background and f o r q u e n c h i n g . Quench s t a n d a r d s 17. were p r e p a r e d u s i n g H - t o l u e n e , (New .Eng land N u c l e a r , Dorva l Q u e . ) , B i o f l u o r and a c e t o n e as the q u e n c h i n g a g e n t . The v i a l s were s e a l e d w i t h wax. The c o u n t i n g e f f i c i e n c y f o r t r i t i u m in the l i q u i d s c i n t i l l a t i o n c o u n t e r ranged f rom kd% t o k8%. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the method o f S u t h e r l a n d e_t_ aj_. (77 ) as m o d i f i e d by Robson et_ a_l_. (78 ) , u s i n g c r y s t a l l i n e b o v i n e serum a l b u m i n as the p r o t e i n s t a n d a r d . DETERMINATION OF IRREVERSIBLE BINDING OF 3 H - B P IN VITRO 3 For q u a n t i t a t i o n o f the i r r e v e r s i b l e b i n d i n g o f H-BP in v i t r o , 5 grams o f l i v e r t i s s u e were minced and homogenized in 20 m i s . o f i c e - c o l d 1.15% KC1, f o r 1 m i n . 15 s e e s - ' , u s i n g a m o t o r - d r i v e n g l a s s - t e f l o n h o m o g e n i z e r . The homogenate was then c e n t r i f u g e d , ( i n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e , Type B-20) a t 4°C, a t 10,000 x g , f o r 10 m i n s . and the s u p e r n a t a n t d i l u t e d 1:5 w i t h t h e KC1 s o l u t i o n , and t h i s was used as the enzyme s u s p e n s i o n , u n l e s s i n d i c a t e d o t h e r w i s e . The i n c u b a t i o n was done in a t o t a l o f 5 ml-., c o n t a i n i n g t h e f - G l lowing : j . 1 m l . o f enzyme p r e p a r a t i o n , e q u i v a l e n t to 0.05 gift o f l i v e r , the N A D P H - g e n e r a t i n g s y s t e m in 3-9 mlVo"foJ0.<'2- ft fph'oVp'ha'fe'e^ 12.5 u m o l e s o f NADP + , 6.25 u m o l e s o f MgCl^ and 2.0 u n i t s o f g1ucose-6 -phosphate 3 6 d e h y d r o g e n a s e ) and 1.015 u m o l e s o f H-BP (2.221 x 10 dpm) in 50 u1 o f a c e t o n e . Drug a d d i t i o n s were made p r i o r to the i n c u b a t i o n . SKF 525~A, m e t h a d o n e - H C 1 , c y s t e i n e - H C l or g l u t a t i o n e ( reduced) was added in 50 LI 1 o f 0.2 M phosphate b u f f e r . C o n t r o l tubes r e c e i v e d 50 u1 o f the b u f f e r . 3"MC was added in 50 y1 o f amyl a l c o h o l and the c o n t r o l tubes f o r the 3~MC e x p e r i m e n t s a l s o r e c e i v e d 50 y1 o f amyl a l c o h o l . The b l a n k s c o n t a i n e d 1 ml o f enzyme s u s p e n s i o n b o i l e d f o r 10 min.v , a t 100°C. B l a n k s wh ich c o n t a i n e d b o i l e d enzyme and the h i g h e s t c o n c e n t r a t i o n o f 18. the d rug s t u d i e d were a l s o u s e d . The tubes were p r e i n c u b a t e d f o r 1 m i n - , the enzyme s u s p e n s i o n added at 0 t ime and the tubes i n c u b a t e d f o r 50 m i n . : Both the p r e i n c u b a t i o n and i n c u b a t i o n were c a r r i e d o u t a t 37°C i n a s h a k i n g w a t e r b a t h . The r e a c t i o n s were s t o p p e d a t t h e d e s i r e d t ime by a d d i n g 1 ml o f 0.9 TCA. The p r e c i p i t a t e d p r o t e i n and n u c l e i c a c i d s were v o r t e x e d and c e n t r i f u g e d f o r 10 min[ at room t e m p e r a t u r e a t 1,000 x g . The s u p e r n a t a n t was d i s c a r d e d and the p r e c i p i t a t e e x t r a c t e d t w i c e w i t h 0.6 M TCA, 3 t imes w i t h 80% m e t h a n o l , 3 t imes w i t h a c e t o n e and a t l e a s t 3 t imes w i t h e t h e r . E x t r a c t i o n s were c a r r i e d o u t by a d d i n g 3 ml o f the a p p r o p r i a t e s o l v e n t t o the p r e c i p i t a t e , v o r t e x i n g , c e n t r i f u g i n g a t room t e m p e r a t u r e a t 1,000 x g f o r 3 min. . and p o u r i n g o f f the s u p e r n a t a n t . It was found t h a t by f u r t h e r e x t r a c t i o n s no a d d i t i o n a l r a d i o a c t i v i t y c o u l d be removed f rom the p r e c i p i t a t e . The p r e c i p i t a t e was then d i s s o l v e d in 5 m l o f 1N_Na0H. A 0.5 ml a l i q u o t , e q u i v a l e n t to 0.005 gm 1 i v e r , and 0.5 ml . o f 1.2 N_ HCl were added to 15 ml o f B i o f l u o r and c o u n t e d as p r e v i o u s l y d e s c r i b e d . A l l c a l c u l a t i o n s were p e r f o r m e d on a Wang 600 c a l c u l a t o r . The s t u d e n t ' s t - t e s t f o r u n p a i r e d sample means was used f o r a l l c o m p a r i s o n s . The l e v e l o f s i g n i f i c a n c e chosen was a t p<0.05- The l i n e a r r e g r e s s i o n a n a l y s i s was p e r f o r m e d and the c o r r e l a t i o n c o e f f i c i e n t f o r the r e g r e s s i o n l i n e c a l c u l a t e d when a l i n e a r r e l a t i o n s h i p was s t a t i s t i c a l l y t e s t e d . 19. RESULTS CONTROL EXPERIMENTS Figure,-"4 shows the degree o f i r r e v e r s i b l e b i n d i n g in three d i f f e r e n t c o n t r o l exper iments . In the f i r s t experiment animals were s a c r i f i c e d immediately 3 a f t e r H-BP a d m i n i s t r a t i o n . The l e v e l of b i n d i n g was found to be about 3 3 pmoles/gram wet weight of l i v e r . In the second experiment H-BP (192.5 pmoles) or 770 pmoles/gram wet weight o f l i v e r ; 40,000 dpm) in 100 y l of corn o i l was added to the TCA p r e c i p i t a t e d a l i q u o t of a t y p i c a l l i v e r homogenate. Th is 3 q u a n t i t y of H-BP corresponded to the average amount of r a d i o a c t i v i t y found in a 1 ml a l i q u o t o f l i v e r homogenates of 4 corn o i l p r e t r e a t e d f a t s - i n j e c t e d 3 3 w i t h H-BP and s a c r i f i c e d 13 hours l a t e r . The amount of H-BP i r r e v e r s i b l y bound to t o t a l l i v e r p r o t e i n and n u c l e i c a c i d s a f t e r the e x t r a c t i o n methods was found to be 16 pmoles/gram wet weight o f l i v e r . In the t h i r d experiment 3 H-BP in 200 y l corn o i l was added to a t y p i c a l l i v e r homogenate' and 1 m l -a l i q u o t s c o n t a i n i n g 192.5 pmoles of H-BP (40,000 dpm) or 770 pmoles/gram wet weight of l i v e r were p r e c i p i t a t e d in TCA. A f t e r the e x t r a c t i o n procedure the 3 l e v e l of i r r e v e r s i b l y bound H-BP was found to be 8 pmoles/gram wet weight of l i v e r . The i r r e v e r s i b l e b ind ing in those corn o i l t r e a t e d animals was 276 pmoles/gram wet weight o f l i v e r . IRREVERSIBLE BINDING OF 3 H-BP IN VITRO ''H-BP (1.015 y mole, 2.221 x 10 dpm) was incubated at 37 C fo r va ry ing lengths o f time in the presence of an NADPH-generating system and the 10,000 x g 1 i v e r . s u p e r n a t a n t . F igure 5 i n d i c a t e s that l i n e a r i t y of b ind ing to rat l i v e r p r o t e i n and n u c l e i c ac ids was a f u n c t i o n of i n c u b a t i o n t ime . The c o r r e l a t i o n c o e f f i c i e n t of the reg ress ion l i n e was 0.974. Dependence of the b ind ing on p r o t e i n c o n c e n t r a t i o n i s shown in f i g u r e 6. The b i n d i n g was l i n e a r up to the 20. F igure 4. N o n - s p e c i f i c i r r e v e r s i b l e b i n d i n g . A: four ra ts were s a c r i f i c e d immediately a f t e r 3|-|-benzo [a]pyrene (3H-BP) i n j e c t i o n (1.25 umole : 125 y C i ) in 0.5 ml,, corn o i l . B: four 1 ml a l i q u o t s from each o f two l i v e r homogenates were p r e c i p i t a t e d in t r i c h l o r o a c e t i c a c i d (TCA) and 3H-BP added (192.5 y m o l e ; 4 0 , 0 0 0 dpm) in 100 y l corn o i l . C: 3H-BP was added to two separate l i v e r homogenates; four 1 ml a l i q u o t s c o n t a i n i n g 192.5 ymole 3H-BP ( 4 0 , 0 0 0 dpm) were taken from each homogenate and p r e c i p i t a t e d in TCA. Values represent i r r e v e r s i b l e b i n d i n g in the e x t r a c t e d pel l e t s . 3 H - B P B O U N D l e s / g r a m wet w e i g h t o f !tver±s.e.m.(n) _ i « a — i o i W 22. F i g u r e 5 . The e f f e c t o f i n c u b a t i o n t ime on the i r r e v e r s i b l e b i n d i n g o f 3n - benzo[a ]pyrene ( 3 H - B P ) in v i t r o . R e a c t i o n tubes c o n t a i n i n g 3H-BP (1.015 y m o l e ; 2.221 x 10 dpm), the N A D P H -g e n e r a t i n g sys tem and the 10,000 x g s u p e r n a t a n t e q u i v a l e n t to 0.05 gram o f l i v e r were i n c u b a t e d a t 37°C f o r v a r y i n g p e r i o d s o f t i m e . B l a n k s c o n t a i n e d b o i l e d enzyme. V a l u e s a r e c o r r e c t e d f o r n o n - s p e c i f i c b i n d i n g and a r e the a v e r a g e s o f d u p l i c a t e s . The c o r r e l a t i o n c o e f f i c i e n t o f the r e g r e s s i o n 1i ne was 0.974• 2k. Figure &. The e f f e c t of prote in concentrat ion on the i r r e v e r s i b l e binding of -*H-benzo[a]pyrene (3H-BP) in v i t r o . & React ion tubes containing 3H-BP (1.015 ymole; 2.221 x 10^ dpm), the NADPH - generating system and the 10,000 x gy. supernatant were incubated at 37^ fo r 50 mini,. Blanks contained bo i led enzyme. Values are corrected for n o n - s p e c i f i c b inding and are the averages of d u p l i c a t e s . 26.^  10,000 x g s u p e r n a t a n t p r o t e i n c o n t e n t e q u i v a l e n t to 0.05 gram wet w e i g h t o f l i v e r . The c o r r e l a t i o n c o e f f i c i e n t o f a r e g r e s s i o n l i n e f rom the o r i g i n to the a b s c i s s a v a l u e o f 0.05 had a c o r r e l a t i o n c o e f f i c i e n t o f 0.989- F i g u r e 7 shows the e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on the b i n d i n g r e a c t i o n . S u b s e q u e n t l y , 3 a l l r e a c t i o n s were c a r r i e d o u t f o r 50 m i n u t e s , c o n t a i n i n g 1.015 u m o l e H-BP (2.221 x 10^  dpm) and a d i l u t e d 10,000 x g s u p e r n a t a n t e q u i v a l e n t to 0.05 gram wet w e i g h t o f l i v e r . To d e t e r m i n e the e n z y m a t i c n a t u r e o f the b i n d i n g r e a c t i o n , e x p e r i m e n t s were c a r r i e d out under a tmosphere and w i t h o u t the N A D P H - g e n e r a t i n g s y s t e m . These r e s u l t s a r e shown in T a b l e I. The p r e s e n c e o f a ^ a tmosphere d e c r e a s e d the b i n d i n g by 75% o f i t s c o n t r o l v a l u e . D e l e t i n g the N A D P H - g e n e r a t i n g sys tem reduced t h e b i n d i n g t o k0% o f i t s c o n t r o l v a l u e . T a b l e II shows the e f f e c t s o f i n c r e a s i n g some o r a l l o f the c o f a c t o r s o f N A D P H - g e n e r a t i n g s y s t e m . I n c r e a s i n g the amount o f g1ucose-6 -phosphate , g1ucose-6 -phosphate d e h y d r o g e n a s e , NADP + o r a l l o f t h e s e c o f a c t o r s a t once showed no s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e o f BP b i n d i n g f rom c o n t r o l . A f t e r the e x t r a c t i o n p r o c e d u r e s , r a d i o a c t i v i t y was p r e s e n t in the b o i l e d enzyme b l a n k s . A h i g h e r l e v e l o f r a d i o a c t i v i t y was o b s e r v e d i f a c t i v e enzyme, w i t h o u t t h e N A D P H - g e n e r a t i n g sys tem a d d e d , was used as b l a n k s . B o i l e d p r o t e i n b l a n k s done under o r w i t h o u t the N A D P H - g e n e r a t i n g s y s t e m showed the same l e v e l o f r a d i o a c t i v i t y as t h e b o i l e d enzyme b l a n k s done under normal c o n d i t i o n s . 3 The i r r e v e r s i b l e b i n d i n g o f H-BP to 10,000 x g s u p e r n a t a n t p r o t e i n . ' and n u c l e i c a c i d s r e f e r s o n l y t o enzyme-med ia ted ~, b i n d i n g . A l l v a l u e s have been c o r r e c t e d f o r n o n - s p e c i f i c o r p h y s i c a l b i n d i n g . The p h y s i c a l b i n d i n g was 3 shown to i n c r e a s e w i t h i n c r e a s i n g amounts o f e i t h e r H-BP o r p r o t e i n . 27. F i g u r e '7. The e f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on the i r r e v e r s i b l e b i n d i n g o f 3H -benzo[a]pyrene (3H-BP) in v i t r o . R e a c t i o n tubes c o n t a i n i n g ^ H -B P a t v a r i o u s c o n c e n t r a t i o n s , the N A D P H - g e n e r a t i n g sys tem and the 10,000 x g s u p e r n a t a n t e q u i v a l e n t to 0.05 gram o f l i v e r were i n c u b a t e d a t 37°C f o r 50 m i n ? . B l a n k s c o n t a i n e d b o i l e d enzyme. V a l u e s a r e c o r r e c t e d f o r n o n - s p e c i f i c b i n d i n g and a r e the a v e r a g e s o f dup1 i c a t e s . 3H-BP BOUND n m o l e s / g r a m w e t w e i g h t o f l i v e r / m i n 29. TABLE il Conditions f o r the i r r e v e r s i b l e binding of H-benzo[a]pyrene PH-BP) to rat l i v e r 10,000 x g supernatant macromolecules in v i t r o . CONDITION DPMC DPM MINUS BLANK IRREVERSIBLY BOUND JH-BP nm^'Mss/gram wet weight of 1iver/minute blank 617 c o n t r o l 0 1273 atmosphere 781 0 656 164 0 1.199 0.299* blank c o n t r o l ' -NADPH 508 1331 848 0 823 340 0 1.504 0.622 3 The r a d i o a c t i v i t y found in the f i n a l a l i q u o t (average of d u p l i c a t e s ) k The blanks contained 1 ml?, b o i l e d enzyme suspension. C The controls.-.eonita.'in£dtftj3ec#!QPp4§$er.rjea'et&§(\,:iw,x$u<rr ea s44§§§.r|jfeed-in 'methods1' 30. TABLE I I The e f f e c t o f v a r i e d l e v e l s o f the N A D P H - g e n e r a t i n g s y s t e m c o f a c t o r s 3 3 on the i r r e v e r s i b l e b i n d i n g o f H - b e n z o [ a ] p y r e n e ( H-BP) in v i t r o . CONDITION DPM 3 DPM MINUS BLANK IRREVERSIBLY BOUND 3 H - B P nmoles/gram wet weight , of liver/minute b 1 ank 590 0 0 c o n t r o l C 1571 981 1 .793 5 x g1u c o s e-6 - p h o s p h a t e 1619 1029 1 .881 5 x g l u c o s e-6 - p h o s p h a t e 1527 937 1 .713 d e h y d r o g e n a s e 5 x NADP + 1535 945 1 .727 5 x a l1 o f t h e above 1275 685 1 .258 a The r a d i o a c t i v i t y found in the f i n a l a l i q u o t (ave rage o f d u p l i c a t e s ) , k The b l a n k s c o n t a i n e d 1 ml b o i l e d enzyme s u s p e n s i o n . c The c o n t r o l s c o n t a i n e d the c o m p l e t e r e a c t i o n m i x t u r e as d e s c r i b e d in ' m e t h o d s ' . 31. . EFFECT OF IN VITRO ADDITIONS ON THE IRREVERSIBLE BINDING OF 3 H - B P 3 The e f f e c t o f methadone on the i r r e v e r s i b l e b i n d i n g o f H-BP in v i t r o i s -h -3 -2 shown in f i g u r e y. Methadone c o n c e n t r a t i o n s o f 10 M^ , 10 M_ and 10 M_ d e c r e a s e d the d e g r e e o f b i n d i n g f rom c o n t r o l by k0%, 60% and 43%, r e s p e c t i v e l y . Lower c o n c e n t r a t i o n s had no e f f e c t . -5 -k SKF 525-A a t c o n c e n t r a t i o n s o f 10 M^  and 10 ^ d e c r e a s e d the l e v e l o f b i n d i n g f rom c o n t r o l by 26% and 37%, r e s p e c t i v e l y , as o b s e r v e d in f i g u r e % . A g a i n , lower c o n c e n t r a t i o n s had no e f f e c t . -2 G l u t a t h i o n e added to the r e a c t i o n m i x t u r e a t a c o n c e n t r a t i o n o f 10 M_ 3 d e c r e a s e d the i r r e v e r s i b l e b i n d i n g o f H-BP f rom c o n t r o l by 27% as shown in -2 f i g u r e 10. A t c o n c e n t r a t i o n s lower than 10 ( ^ g l u t a t h i o n e had no e f f e c t . F i g u r e Blshows t h a t c y s t e i n e e x h i b i t s an i n h i b i t i o n o f the i r r e v e r s i b l e 3 -2 b i n d i n g o f H-BP f rom c o n t r o l by 53% a t a c o n c e n t r a t i o n o f 10 M_. C o n c e n t r a t i o n s -2 of c y s t e i n e in the r e a c t i o n m i x t u r e below 10 M^  had no e f f e c t on the c o n t r o l l e v e l o f b i n d i n g . 3-MC added t o the r e a c t i o n m i x t u r e a l s o i n h i b i t e d the i r r e v e r s i b l e b i n d i n g 3 : -5 -k o f H -BP. F i g u r e ^ shows the d e g r e e o f i n h i b i t i o n f rom c o n t r o l a t 10 M_, 10 M_ _ o and 10 H o f 3"MC. The p e r c e n t i n h i b i t i o n a t t h e s e doses a r e 27%,' 44% and 44%, r e s p e c t i v e l y . It s h o u l d be noted t h a t none o f the compounds used in v i t r o , a t the h i g h e s t c o n c e n t r a t i o n u s e d , ;'a^l\ei:e^d: the l e v e l o f n o n - s p e c i f i c b i n d i n g o f 3 3 H-BP. The i n h i b i t i o n i s the d e c r e a s e in the e n z y m e - m e d i a t e d b i n d i n g o f H -BP. RELATIONSHIP BETWEEN IRREVERSIBLE BINDING IN VIVO AND DOSE OF BP 3 H - B P (0.139, 1.25, 5-93 o r 12.5 Mmole i . p . ; 125 y C i ) in 0.5 ml c o r n o i1 was a d m i n i s t e r e d to normal r a t s . The a n i m a l s were k i l l e d 2k hours l a t e r and the l i v e r s examined f o r i r r e v e r s i b l y bound r a d i o a c t i v i t y . As shown in f i g u r e 13, 32. F i g u r e 8. The e f f e c t o f methadone on the i r r e v e r s i b l e b i n d i n g o f 3rl -benzo a py rene (^H-BP) in v i t r o . R e a c t i o n tubes c o n t a i n i n g 3H-BP (1.015 y m o l e ; 2.221 x 10 dpm) , the NADPH - g e n e r a t i n g s y s t e m , the 10,000 x g s u p e r n a t a n t e q u i v a l e n t to a 0 .05 gram o f l i v e r and v a r i o u s c o n c e n t r a -t i o n s o f methadone were i n c u b a t e d at 37°C f o r 50 m i n . - . B l a n k s c o n t a i n e d b o i l e d enzyme. V a l u e s a r e c o r r e c t e d f o r n o n - s p e c i f i c b i n d i n g and a r e the a v e r a g e s o f 6 tubes (3 s e p a r a t e e x p e r i m e n t s ) . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e f rom c o n t r o l at p<0.05 (n=6). 33-3H-BP BOUND n m o l e s / g r a m w e t w e i g h t o f l i v e r / m i n - s.e.m. O -* M — — i — — — i — cr 34. F igure 9. The e f f e c t of SKF 525-A on the i r r e v e r s i b l e b i n d i n g of 3 H-benzo[a]pyrene ( 3 H-BP ) in v i t r o . React ion tubes c o n t a i n i n g 3 H - B P (1.015 umole ; 2.221 x 106 dpm), the NADPH-generating system, the 10,000 x g supernatant e q u i v a l e n t to 0.05 gram of l i v e r and var ious c o n c e n t r a t i o n s o f SKF 525-A were incubated at 37°C f o r 50 m i n ; . Blanks conta ined b o i l e d enzyme. Values are c o r r e c t e d f o r non-s p e c i f i c b i n d i n g and are the averages o f 6 tubes (3 separate exper iments ) . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p<0.05 (n=6). 35. H-BP BOUND nmoles/gram wet weight of liver/min cr c rO Oi" to «> tn S3 Ln > °A" n O 3 ft (D 3 —• a ^ o* I S _ w o-r cn on in 36. F igure VO. The e f f e c t of g l u t a t h i o n e on the i r r e v e r s i b l e b ind ing of 3H-benzo[a]pyrene (3H-BP ) in v i t r o . React ion tubes c o n t a i n i n g 3H-BP (1.015 U mole; 2.221 x 1 0 b dpm) ," the NADPH-generat ing sys tem, the 10,000 x g supernatant e q u i v a l e n t to 0 .05 gram of l i v e r and va r ious c o n c e n t r a t i o n s of g l u t a t h i o n e were incubated at 37°C fo r 50 mins . Blanks conta ined b o i l e d enzyme. Values are c o r r e c t e d fo r n o n - s p e c i f i c b ind ing and are the averages of 6 tubes (3 separate exper iments ) . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p<0.05 (n=6). 37-38. Figure-BI. . The e f f e c t o f c y s t e i n e on the i r r e v e r s i b l e b i n d i n g of 3rl -benzo[a]pyrene (3H-BP) in v i t r o . React ion tubes c o n t a i n i n g 3H-BP (1.015 y m o l e ; 2.221 x 10 6 dpm), the NADPH-generating system, the 10,000 x g supernatant e q u i v a l e n t to 0 .05 gram of l i v e r and va r ious c o n c e n t r a t i o n s of c y s t e i n e were incubated at 37°C f o r 50 miri;s. Blanks conta ined b o i l e d enzyme. Values are co r rec ted fo r non-s p e c i f i c b i n d i n g and are the averages o f 6 tubes (3 separate exper iments ) . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p<0.05 (n=6). 39. ho. F igure .132. The e f f e c t of 3 - methy l cho lan th rene (3 -MC) on the i r r e v e r s i b l e b i n d i n g of 3|-j-benzo[a]pyrene (3H-BP) in v i t r o . React ion tubes c o n t a i n i n g 3H-BP (1.015 Umole; 2.221 x 10° dpm), the NADPH-genera t ing system, the 10,000 x g supernatant equ i va len t to 0 .05 gram of l i v e r and va r ious c o n c e n t r a t i o n s of 3 -MC were incubated at 37°C fo r 50 mins'. Blanks conta ined b o i l e d enzyme. Values are c o r r e c t e d f o r n o n - s p e c i f i c b i n d i n g and are the averages of 6 tubes (3 separate exper iments ) . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p<0.05 (n=6). 41. I 9 I * 4 2 . the degree of b i n d i n g to l i v e r t i s s u e , expressed on a per mg. p r o t e i n b a s i s , was l i n e a r over the e n t i r e dose range s t u d i e d . The degree of b ind ing to l i v e r t i s s u e expressed on a per gram wet weight of l i v e r b a s i s was a l s o l i n e a r over the e n t i r e dose range s t u d i e d , as observed in figure-1,4. A second popu la t ion of animals a l s o showed a l i n e a r d o s e - b i n d i n g r e l a t i o n s h i p which was markedly lower than the f i r s t as shown in f i g u r e s IS and ) 6 - The second p o p u l a t i o n represented 25.6% (n=10) o f the t o t a l number of animals used f o r the dose b ind ing study (n=39)« The c o r r e l a t i o n c o e f f i c i e n t s f o r a l l 4 curves were g reate r than 0.999. It was found that the v a r i a t i o n in animal weight (200 - - ' 300gm) had no e f f e c t on the degree of i r r e v e r s i b l e b i n d i n g of H-BP. The dose 3 of H-BP used in a l l subsequent in v i v o s t u d i e s was 1.25ymole. IRREVERSIBLE BINDING IN VIVO WITH TIME 3 A f t e r the a d m i n i s t r a t i o n of H-BP (1.25 ymole i . p . ; 125 u C i ) in 0.5 ml corn o i l to normal r a t s , the amount of i r r e v e r s i b l y bound r a d i o l a b e l l e d m a t e r i a l was determined at va r ious time i n t e r v a l s , as shown in f i g u r e s I J and 1^8 (tap water c o n t r o l s ) . The i r r e v e r s i b l e b i n d i n g increased to a maximum of about 4 0 0 pmoles/gram wet weight of l i v e r or 2 . 7 4 pmoles/mg. p r o t e i n by 18 hours and f e l l to about 200 pmoles/gram wet weight of l i v e r or 1.25 pmoles/mg. p r o t e i n by 4 8 hours . A second popu la t ion of ra ts showed a complete ly d i f f e r e n t t i m e -b i n d i n g r e l a t i o n s h i p , as observed in f i g u r e s . H(9 and 2 0 (tap water c o n t r o l s ) . At 1i hours the i r r e v e r s i b l e b i n d i n g was at a l e v e l of almost 500 pmoles/gram wet weight of l i v e r or 3-5 pmoles/mg. p r o t e i n and f e l l to about 6 0 pmoles/gram wet weight of l i v e r or 0 . 4 pmoles/mg; p r o t e i n by 2 4 hours . Th is second p o p u l a t i o n represented 15-4% (n=6) o f the t o t a l number of animals used f o r the t i m e - b i n d i n g S i t udy (n=39). 4 3 . F igure ']Q. The e f f e c t of dose on the i r r e v e r s i b l e b i n d i n g of 3H-benzo[a]pyrene (3H-BP) to t o t a l rat l i v e r p r o t e i n and n u c l e i c a c i d s in v i v o expressed as pmoles bound/mg. p r o t e i n . Rats i n j e c t e d w i t h v a r i o u s doses of ^H-BP a d m i n i s t r a t i o n and t h e i r l i v e r s examined fo r i r r e v e r s i b l e b i n d i n g . The c o r r e l a t i o n c o e f f i c i e n t o f the r e g r e s s i o n l i n e was 0 . 9 9 9 -kk. 3 H B P BOUND pmoles/mg proteint s.e.m.(n) us. F igure 14. The e f f e c t of dose on the i r r e v e r s i b l e b i n d i n g o f 3 H-benzo[a]pyrene ( 3 H - B P ) t o . r a t l i v e r macromolecules in v i v o expressed as pmoles/ bound/gram wet weight of l i v e r . Rats i n j e c t e d w i t h va r ious doses of 3 H - B P in 0 . 5 ml,;?, corn o i l were s a c r i f i c e d 2 4 hours a f t e r 3 H - B P a d m i n i s t r a t i o n and t h e i r l i v e r s examined fo r i r r e v e r s i b l e b i n d i n g . The c o r r e l a t i o n c o e f f i c i e n t of the r e g r e s s i o n l i n e was 0 . 9 9 9 -he. 3H~BP BOUND p m o l e s / g r a m wet weight of l i v e r l s.e.m.(n) hi. F igure U5- The dose v s . i r r e v e r s i b l e b ind ing curve of 3H-benzo[a]pyrene (3H-BP) in the second p o p u l a t i o n of r a t s expressed as pmoles bound/mg.> p r o t e i n . Rats i n j e c t e d w i t h va r ious doses of 3H-BP a d m i n i s t r a t i o n and t h e i r l i v e r s examined fo r i r r e v e r s i b l e b i n d i n g . The c o r r e l a t i o n c o e f f i c i e n t of the r e g r e s s i o n l i n e was 0.999. 48. 3H-BP BOUND pmoles/mg protein±s.e.m.(n) CO Q_ O t/> fl> CO I 3 o_ F igure 16. The dose v s . i r r e v e r s i b l e b ind ing curve of 3|-|-benzo[a]pyrene (3H-BP) in the second p o p u l a t i o n of ra ts expressed as pmoles bound/gram wet weight of l i v e r . Rats i n j e c t e d w i t h va r ious doses of 3H-BP in 0.5 ml corn o i l were s a c r i f i c e d 2k hours a f t e r 3H-BP a d m i n i s t r a t i o n and t h e i r l i v e r s examined fo r i r r e v e r s i b l e b i n d i n g . The c o r r e l a t i o n c o e f f i c i e n t of the r e g r e s s i o n l i n e was 0.999-50. H-BP BOUND pmoles/gram wet weight of liver i s.e.m.(n) O O ro W O O o o O O o o Oi O O O o 5 1 . F i g u r e 17- The e f f e c t of methadone t reatment on the i r r e v e r s i b l e b i n d i n g of 3H -benzo[ajpyrene (3H-BP) in v i v o expressed as pmoles bound/ gram wet weight of l i v e r . Rats i n j e c t e d w i t h 3H-BP (1.25 umole; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes •af te r 3H-BP a d m i n i s t r a t i o n . Q tap water c o n t r o l s - — - H — — — methadone t r e a t e d No s i g n i f i c a n t d i f f e r e n c e from c o n t r o l was seen at any of the t imes s t u d i e d ( s t u d e n t ' s t - t e s t , l e v e l of s i g n i f i c a n c e a t p<0.05). 52. 53. F.gure 18. The e f f e c t of methadone t reatment on the i r r e v e r s i b l e b i n d i n g of ^H-benzoLaJpyrene (*H-BP) in v i v o expressed as pmoles bound/ mg p r o t e i n . Rats i n j e c t e d w i t h 3H-BP (l.25nmole; 1 2 5 u C i ) in 0.5 m] corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r 3H-BP A r l m i n i c -J- -» +- T tap water c o n t r o l s methadone t r e a t e d No s i g n i f i c a n t d i f f e r e n c e from c o n t r o l was seen at any of the t imes s t u d i e d ( s t u d e n t ' s t - t e s t , l e v e l o f s i g n i f i c a n c e at p<0.05) 54. 3H-BP B O U N D p m o l e s / m g p r o t e i n ! s.e.m.(n) O -»• l O to i _ _ i i i 55. Figure 19. The e f f e c t of methadone treatment on the i r r e v e r s i b l e binding of 3rl-benzo[a]pyrene ( 3H-BP) in v i v o in the second population of ra t s expressed as pmoles bound/gram wet weight of l i v e r . Rats i n j e c t e d w i t h 3H-BP ( l . 2 5 u m o l e ; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at various times a f t e r 3H-BP a d m i n i s t r a t i o n . © tap water c o n t r o l s • — — II — — . methadone t r e a t e d 56. 57. F i g u r e 20. The e f f e c t of methadone t reatment on the i r r e v e r s i b l e b i n d i n g of 3H -benzo[a]pyrene ( 3 H - B P) i n v i v o in the second p o p u l a t i o n of r a t s expressed as pmoles bound/mg p r o t e i n . Rats i n j e c t e d w i t h ^ H - B P (1.25 y m o l e ; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r 3 H - B P a d m i n i s t r a t i o n . tap water c o n t r o l s methadone t r e a t e d J 59. EFFECT OF PRETREATMENTS ON THE IRREVERSIBLE BINDING OF JH-SP IN VIVO Methadone was added to the d r i n k i n g water of a s e r i e s of ra ts in the f o l l o w i n g amounts: 0.25 mg/ml f o r 1 week, 0.50 mg/ml the second week, 0.75 mg/ml the t h i r d week, and 1.0 mg/ml f o r the f o u r t h week. The dose of methadone ingested per animal averaged 5 0 , 80, 95 and 115 mg/kg/day dur ing the four weeks. No evidence of withdrawal symptoms, such as acute loss of weight or wet shakes was observed. Th is t rea tment , at a s i m i l a r dose schedule to the one above, has p r e v i o u s l y been shown to increase epoxide hydrase a c t i v i t y in male ra ts to 212% of c o n t r o l s w i t h no change in a r y l hydrocarbon hydroxy lase a c t i v i t y (66). Pa i red groups of animals t r e a t e d w i t h methadone as above showed induc t ion of EH w i t h no changes in AHH a c t i v i t y throughout t h i s study as monitored in t h i s l a b o r a t o r y . 3 H-BP (1.25 ymole i . p . ; 125 yC i ) in 0.5 ml corn o i l was admin is te red to the methadone p r e t r e a t e d animals and they were s a c r i f i c e d at e i t h e r 3, 6, 12, 3 18, 2k o r k8 hours a f t e r H-BP. The degree of i r r e v e r s i b l e b ind ing in the methadone p re t rea ted animals was not s i g n i f i c a n t l y d i f f e r e n t from the tap water c o n t r o l s at any of the times s t a t e d , as shown in f i g u r e s 17 and 18 (methadone t reated] A second p o p u l a t i o n of animals p r e t r e a t e d w i t h methadone had a t i m e - b i n d i n g curve markedly d i f f e r e n t from the f i r s t , as observed in f i g u r e s 19 and 20 (methadone t r e a t e d ) . Th is second p o p u l a t i o n of animals represented 27-5% (n=l4) of a l l the animals p re t rea ted w i t h methadone (n=5l). Groups of k animals were p r e t r e a t e d w i t h 3"MC (20 mg/kg i . p . f o r 2 c o n s e c u t i v e days) in corn o i l , admin is te red H-BP (1.25 pmole i . p . ; 125 yC i ) in 0.5 ml corn o i l 2k hours a f t e r the l a s t dose of 3~MC and s a c r i f i c e d at 3 v a r i o u s t imes a f t e r H-BP. Contro l animals rece ived corn o i l wi thout 3"MC. F igures 21 and 22 show the t i m e - b i n d i n g curves f o r these two sets of an imals . The i r r e v e r s i b l e b ind ing in ra ts p r e t r e a t e d w i t h corn o i l rose to a leve l of 275 pmoles/gram wet weight of l i v e r or 2.1 pmoles/mg p r o t e i n by 13 hours and remained at about that l e v e l to k2 hours a f t e r H-BP a d m i n i s t r a t i o n . In ra ts 60. F i g u r e 21. The e f f e c t o f corn o i l and 3 _ m e t h y l c h o l a n t h r e n e (3_MC) t reatments on the i r r e v e r s i b l e b i n d i n g of 3 H-benzo [a ]py rene (3R-BP) in v i v o expressed as pmoles bound/gram wet weight of l i v e r . Rats i n j e c t e d w i t h 3H-BP ( l . 2 5 u m o l e ; 125 p C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r 3H-BP a d m i n i s t r a t i o n . O corn o i l t r e a t e d • • — — 3"MC t r e a t e d A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from corn o i l t r e a t e d animals a t p<0.05-62. F i g u r e 22. The e f f e c t of corn o i l and 3 - methy lcho lan th rene (3 -MC) t reatments on the i r r e v e r s i b l e b i n d i n g of 3 H-benzo [a ]py rene ( 3H-BP) in v i v o expressed as pmoles bound/mg. p r o t e i n . Rats i n j e c t e d w i t h 3H-BP (1.25 y m o l e ; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r 3 H - B P a d m i n i s t r a t i o n . O corn o i l t r e a t e d • — - Q - — 3-MC t r e a t e d A s t e r i s k s i n d i c a t e an imals at p<0.05. s i g n i f i c a n t d i f f e r e n c e from corn o i l t r e a t e d 64. pre t rea ted w i t h 3~MC in corn o i l , i r r e v e r s i b l y bound H-BP reached a l e v e l of 200 pmoles/gram wet weight of l i v e r or 1.5 pmoles/mg p r o t e i n by 24 hours and mainta ined that l e v e l through to 42 hours . The leve l of i r r e v e r s i b l e b ind ing in the 3"MC t r e a t e d ra ts was s i g n i f i c a n t l y lower that that of the corn o i l t r e a t e d rats at 8, 13, 24 and 42 hours a f t e r 3 H - B P . Th is represented a 30% decrease from the c o n t r o l l e v e l s at each of the times p r e v i o u s l y mentioned. The t i m e - b i n d i n g curve of the corn o i l t r e a t e d animals was both q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t from that of t h e . tap -water c o n t r o l a n i m a l s . The maximum leve l of i r r e v e r s i b l e b i n d i n g observed in the corn o i l t rea ted animals was 72.5% of the h ighest l eve l observed in the tap water c o n t r o l an imals . A decrease to 50% of the maximum l e v e l by 48 hours was observed in the tap water animals w h i l e the maximum l e v e l was e s s e n t i a l l y maintained to 42 hours in the corn o i l t r e a t e d r a t s . A second popu la t ion of corn o i l and 3~MC t r e a t e d ra ts d i s p l a y e d t i m e -b ind ing r e l a t i o n s h i p s shown in f i g u r e s 23 and 24. Th is second group represented 12.1% (n=33) and 22.2% (n=4) of the t o t a l number of 3"MC (n=33) and corn o i l (n=18) t r e a t e d r a t s , r e s p e c t i v e l y . The b ind ing was h ighest at 1 hour and f e l l to 15% of t h i s va lue by 42 hours . One group of animals p re t rea ted w i t h 3~MC (20 mg/kg i . p . f o r 2 consecut i ve •3 days) in corn o i l and another w i t h corn o i l a lone were admin is te red H-BP (1.25 p'mole i . p . ; 125 y C i) in 0.5 ml corn o i l 48 hours a f t e r the l a s t 3~MC or corn o i l i n j e c t i o n . F igure 25 shows the degree of b ind ing at 24 hours a f t e r •3 H-BP in animals i n j e c t e d w i t h t h i s compound at e i t h e r 24 or 48 hours a f t e r the l a s t dose of 3~MC. There was no s t a t i s t i c a l d i f f e r e n c e in the degree of 3 i r r e v e r s i b l e b ind ing between the two corn o i l t reated groups ( H-BP 24 hours and 48 hours a f t e r the l a s t dose of corn o i l ) . There was a l s o no s t a t i s t i c a l d i f f e r e n c e between the two 3_MC t reated groups ( H-BP 24 hours and 48 hours a f t e r the l a s t dose of 3"MC). However the 3 -MC treatment decreased s i g n i f i c a n t l y 65. the b ind ing by 25% and 23% of the corn o i l c o n t r o l l e v e l s in animals admin is te red H-BP 2k and 48 hours a f t e r 3"MC, r e s p e c t i v e l y . F igu re 26 shows the degree of i r r e v e r s i b l e b ind ing observed in 30% (n=6) of the ra ts that were admin is te red e i t h e r corn o i l or 3 -MC and s a c r i f i c e d 2k hours 3 a f t e r H-BP (n=20). These animals e x h i b i t e d markedly lower b ind ing l e v e l s than animals in the f i r s t p o p u l a t i o n . SKF 525-A (35 mg/kg, 50 mg/kg or 75 mg/kg i . p . ) in s a l i n e was admin is te red 3 to r a t s , and c o n t r o l animals were i n j e c t e d w i th s a l i n e a l o n e . H-BP (1.25 pmole i . p . ; 125 viCi) in 0.5 ml corn o i l was admin is te red 3 hours a f t e r SKF 525 -A and some animals s a c r i f i c e d at 3 hours and others at 2k hours a f t e r the H-BP. The e f f e c t of SKF 525_A on the l e v e l of i r r e v e r s i b l e b ind ing i s shown in f i g u r e 27. SKF 525-A at 35 mg/kg had no e f f e c t on the l e v e l of b ind ing at 3 and 2k hours 3 a f t e r H-BP as compared to the s a l i n e c o n t r o l s . However, SKF 525"A at 50 mg/kg 3 and at 75 mg/kg decreased the b i n d i n g from c o n t r o l at 3 hours a f t e r ..H-BP. by 3 27% and 31%, r e s p e c t i v e l y and at 2k hours a f t e r H-BP by 29% and 34%, r e s p e c t i v e l y . There was no s t a t i s t i c a l d i f f e r e n c e in the l e v e l s of b ind ing between the s a l i n e 3 pre t rea ted and tap water c o n t r o l animals at e i t h e r 3 or 24 hours a f t e r H-BP. A second p o p u l a t i o n of animals d i s p l a y e d l e v e l s of b ind ing shown in f i g u r e 27. About 12% (n=7) of a l l animals used in the SKF 525-A study (n=58) f e l l i n to the second p o p u l a t i o n . These l e v e l s were markedly lower than the r e s p e c t i v e l e v e l s in f i r s t p o p u l a t i o n a n i m a l s . Table 111 shows the e f f e c t of c y s t e i n e and d i e t h y l maleate t reatments on 3 the i r r e v e r s i b l e b ind ing of H-BP. Cys te ine (150 mg/kg) in s a l i n e admin is te red 3 i . p . 5 minutes before H-BP produced no s i g n i f i c a n t change in b ind ing at e i t h e r 3 3 or 24 hours a f t e r H-BP a d m i n i s t r a t i o n . The s a l i n e c o n t r o l va lues were the same as the s a l i n e and tap water c o n t r o l s p r e v i o u s l y mentioned (F igures 17 and 27). D i e t h y l maleate (0.6 ml/kg) in corn o i l admin is te red i . p . 30 minutes 3 before H-BP a l s o produced no s i g n i f i c a n t change in b ind ing at 24 hours a f t e r 3 ' H-BP a d m i n i s t r a t i o n . A g a i n , the corn o i l c o n t r o l va lues were the same as 66. F igure 23- The e f f e c t of corn o i l and 3 -methy lcho lanthrene (3-MC) t reatments on the i r r e v e r s i b l e b i n d i n g of J H - b e n z o [ a ] p y r e n e ( 3H-BP ) in v i v o in the second p o p u l a t i o n of r a t s expressed as pmoles bound/gram wet weight of l i v e r . Rats i n j e c t e d w i t h 3 H - B P (1.25 ymo le ; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r 3 H - B P a d m i n i s t r a t i o n . O -H corn o i l t r e a t e d 3-MC t r e a t e d 68. F igure 2k. The e f f e c t of corn o i l and 3 -methy l cho lan th rene (3 _MC) t reatments on the i r r e v e r s i b l e b i n d i n g o f 3 H - b e n z o [ a ] p y r e n e ( 3 H - B P ) in v i v o i n the second p o p u l a t i o n of r a t s expressed as pmoles bound/mg p r o t e i n . Rats i n j e c t e d w i t h 3 H - B P ( l . 2 5 y m o l e ; 125 y C i ) in 0.5 ml corn o i l were s a c r i f i c e d at v a r i o u s t imes a f t e r ^ H - B P a d m i n i s t r a t i o n . Q corn o i l t reatment — — * 3_MC treatment 70. Figure 25. A comparison o f the e f f e c t s of corn o i l and 3 _methylcholanthrene (3 _MC) treatments on the i r r e v e r s i b l e binding of 3H-benzo[aIpyrene (3H-BP) in vi.vo in animals i n j e c t e d with the compound at e i t h e r 2k 6rj48 hours a f t e r the l a s t dose of e i t h e r corn o i l or 3_MC. A l l r a t s received 3H-BP . (1.25 y m o l e ; 125 y C i ) in, 0.5ml i v corn o i l and were s a c r i f i c e d 2k hours l a t e r . A and B. Rats r e c e i v i n g 3 H - B P 2k hours a f t e r the l a s t dose of corn o i l or 3-MC. C and D. Rats r e c e i v i n g 3H-BP kB h o u r s . a f t e r the l a s t dose of corn o i l or 3-MC. A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from corn o i l treated animals at p<0.05. 71. H-BP BOUND p m o l e s / g r a m wet w e i g h t of l i v e r ±s.e.m.(n) O O O O __L_ ca o o S + * 5 r—j-H e H H CO L. T 1 1 1 " 1 1 — i r (uj'iu'O's; u|940jd Buj/saioiud aNnoa da-H c 72. F igure '2;6C. A comparison of the e f f e c t s o f corn o i l and 3 -methyIcholanthrene (3_MC) t reatments on the i r r e v e r s i b l e b i n d i n g of 3n -benzo[a]pyrene (3H-BP) in v i vo in the second p o p u l a t i o n of ra ts i n j e c t e d w i t h the compound at e i t h e r 2k o r k8 hours a f t e r the l a s t dose of e i t h e r corn o i l or 3-MC. A l l r a t s rece ived 3H-BP (1.25 ymole; 125 uC i ) in 0.5 m l ; corn o i l and were s a c r i f i c e d 2k hours l a t e r . A and B. Rats r e c e i v i n g 3H-BP 2k hours a f t e r the l a s t dose of corn o i l or 3-MC. C and D. Rats r e c e i v i n g 3H-BP k8 hours a f t e r the l a s t dose of corn o i l or 3-MC. 73. H-BP BOUND pmoles/gram wet weight of liver - s.e.m.(n) O i • - . 0 — —• co I n CO I 00 2.S CO 5 co 1 o o o o to o • 4^  T " o -1 o CO , ( U ) l i r a ' s + u i 9 4 0 j d Biu/sa | O i u d QNnoa da-H 74. F igure 2<7; The e f f e c t of SKF 525 -A treatment on the i r r e v e r s i b l e b ind ing of 3H-benzo[ajpyrene (3H -BP) in v i v o . A l l animals were i n j e c t e d w i t h 3H-BP ( l .25umole; 125 p C i ) in 0.5 ml . corn o i l : 3 hours a f t e r SKF 525-A or s a l i n e . A. I r r e v e r s i b l e b i n d i n g in ra ts s a c r i f i c e d 3 hours a f t e r ^ H - B P . B . I r r e v e r s i b l e b ind ing in r a t s s a c r i f i c e d 2 4 hours a f t e r 3 H - B P . C. I r r e v e r s i b l e b ind ing in the second p o p u l a t i o n of r a t s s a c r i f i c e d 2 4 hours a f t e r 3 H - B P . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from s a l i n e t r e a t e d animals at p<0.05. 75. 3 H - B P BOUND ( pmoles/gram wet weight of li ver I s.e.m o o O O w o o S T * •Jf JH-BPB0UND pmoles/gram wet weight of l iver! s.e.m.(n) ro £> en i ca o o < 1 1 I 1 1 1 1 n £ -rt £ Ln tn «J i 76. p r e v i o u s l y reported (F igures 18 and 2 2 ) . Table III a l s o shows the l e v e l of b i n d i n g obta ined in 2nd p o p u l a t i o n r a t s . About 13% (n=2) o f the animals used in the c y s t e i n e and d i e t h y l maleate study (n=l6) f e l l i n to the 2nd p o p u l a t i o n . Throughout a l l the s t u d i e s combined, the t o t a l percent of animals f a l l i n g in to the second p o p u l a t i o n was 19% (46 out of 250 a n i m a l s ) . 77. TABLE I I I The e f f e c t of c y s t e i n e and d i e t h y l maleate t reatments on the degree of i r r e v e r s i b l e b i n d i n g of H-benzo[a]pyrene ( H-BP) in v i v o . TREATMENT IRREVERSIBLY BOUND 3 H - B P a pmoles/gram wet weight of l i v e r s a l i n e c o n t r o l 0 • a c y s t e i n e corn o i1 c o n t r o l d i e t h y l m a l e a t e 3 sa1i ne c o n t r o 1 C 4. • c c y s t e i n e 329.5 ± 14.96 (4) 376.3 ± 53.83 (3) n' s-97 (1)b 275.7 ± 15.24 (3) 147 ± (1)b 288.3 ± 28.1 (4) n- s-212.9 ± 20.7 (4) 260.6 ± 9.37 (3) n- s-48.7 ± ( l ) b a The data represents the mean ± S . E . M . (n) of n r a t s s a c r i f i c e d 24 hours a f t e r H-BP a d m i n i s t r a t i o n (1.25 ymo le ; 125 yCi) in 0.5 ml corn o i l . b Values obta ined in the second popu la t ion of r a t s . C The data represents the mean ± S . E . M . (n) of n ra ts s a c r i f i c e d 3 hours a f t e r H-BP a d m i n i s t r a t i o n (1.25 ymo le ; .125 y C i ) in 0.5 ml corn o i l . n - S * Mean not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l at p<0.05-78. DISCUSSION The present study was c a r r i e d out to determine the r o l e s of AHH, EH, and g l u t a t h i o n e on the i r r e v e r s i b l e b ind ing of BP to t o t a l l i v e r p r o t e i n and n u c l e i c ac ids in v i v o . When ^H-BP was i n j e c t e d i . p . i n to male Wistar r a t s , a small amount of the compound was bound i r r e v e r s i b l y to l i v e r macromolecu 1es. The degree of i r r e v e r s i b l e b i n d i n g was found to be dependent on both the dose o f BP admin is te red (F igures 1.3 a n d . 14) and the time a f t e r i t s i n j e c t i o n (F igures 17 and 18, tapwater ) . The r e s u l t s show that p r e t r e a t i n g r a t s w i t h SKF 525 _ A s i g n i f i c a n t l y decreased the l e v e l of i r r e v e r s i b l y bound BP from c o n t r o l (F igure 2 7 ) . In a d d i t i o n , p r e t r e a t i n g r a t s w i t h 3 -MC a l s o s i g n i f i c a n t l y decreased the l e v e l of i r r e v e r s i b l y bound BP from c o n t r o l (F igures 21 and 2 2 ) . However, o r a l methadone pretreatment f a i l e d to a l t e r the l e v e l of b i n d i n g to l i v e r macro-molecules (F igures 17 and 1 8 , methadone), and n e i t h e r c y s t e i n e nor d i e t h y l maleate pretreatment a l t e r e d the l e v e l of i r r e v e r s i b l y bound BP from c o n t r o l (Table I I I ) . In the present study i r r e v e r s i b l e b i n d i n g was c h a r a c t e r i z e d as f o l l o w s . 3 The f r a c t i o n of the admin is te red dose of H-BP which could hot be removed from the TCA p r e c i p i t a t e d l i v e r homogenate by r e p e t i t i v e so l vent e x t r a c t i o n was termed i r r e v e r s i b l y bound BP. The cova lent nature of the BP-macromolecular complexes has been w e l l e s t a b l i s h e d (79) and numerous authors have r e f e r r e d to t h i s non -ex t rac tab1e r a d i o a c t i v i t y as c o v a l e n t l y bound ( 8 0 ) . To f u r t h e r e s t a b l i s h the i r r e v e r s i b i l i t y of the unextractab1e BP and the e f f i c a c y o f the 3 e x t r a c t i o n t e c h n i q u e , H-BP was added to a t y p i c a l rat l i v e r homogenate e i t h e r before or a f t e r TCA p r e c i p i t a t i o n , as d e s c r i b e d in Methods. The amount of r a d i o a c t i v i t y added corresponded to that found in l i v e r homogenates, of corn o i l t r e a t e d r a t s s a c r i f i c e d 13 hours a f t e r 3 H - B P , that y i e l d e d 276 pmoles of H-BP/gram wet weight of l i v e r (F igure 2 1 ) . A f t e r the e x t r a c t i o n techniques 79. 3 the. r a d i o a c t i v i t y remaining corresponded to 16 pmoles o f H-BP/gram wet weight 3 3 of l i v e r when H-BP was added a f t e r TCA p r e c i p i t a t i o n and 8 pmoles o f H-BP/ 3 gram wet weight of l i v e r when H-BP was added before TCA p r e c i p i t a t i o n (F igure 3 4 ) . These va lues corresponded to about 5% and 3% of the 276 pmoles of H-BP bound/gram wet weight of l i v e r , r e s p e c t i v e l y . It was a l s o found that in ra ts 3 3 s a c r i f i c e d immediately a f t e r H-BP a d m i n i s t r a t i o n , on ly 3 pmoles of H-BP/gram wet weight of l i v e r was i r r e v e r s i b l y bound (F igure 4 ) . T h e r e f o r e , the e f f i c a c y of the e x t r a c t i o n techniques was e s t a b l i s h e d and thus any r a d i o a c t i v i t y that cou ld not be removed by these methods was r e f e r r e d to as i r r e v e r s i b l y bound. Prodi and coworkers (8l) c a r r i e d out a study showing the leve l of b i n d i n g of BP w i t h t ime a f t e r i t s i . p . i n j e c t i o n in to female Wis ta r r a t s . The dose i n j e c t e d was 1.25 ymole and they observed the b ind ing of BP at 22, 70, and 168 hours a f t e r i t s a d m i n i s t r a t i o n . The b ind ing to t o t a l h e p a t i c p r o t e i n and n u c l e i c a c i d s was found to be h ighest at 22 hours a f t e r BP, f a l l i n g to 70% and 37% of maximum by 70 and 168 h o u r s , r e s p e c t i v e l y . Trie/did not repor t l e v e l s o f b i n d i n g at any t ime l e s s than 22 hours . J o l l o w and coworkers (75) showed the cova lent b i n d i n g of acetaminophen, a p o t e n t i a l h e p a t o t o x i n , to l i v e r p r o t e i n and n u c l e i c a c i d s , w i t h t ime a f t e r i t s i . p . a d m i n i s t r a t i o n to mice. The. b ind ing rose sharp l y to a maximum by 5 hours and f e l l to 25% o f t h i s va lue by 2k hours . He a l s o showed tha t the g r e a t e s t c o n c e n t r a t i o n of acetaminophen in the l i v e r was reached in 2 hours . T h e r e f o r e , the maximum l e v e l of b i n d i n g occur red a f t e r the h ighest c o n c e n t r a t i o n of acetaminophen was reached in the l i v e r . Delwaide (82) showed that BP i n j e c t e d i . p . in to ra ts reached the maximum l e v e l s in the l i v e r by k to 6 hours , depending on the amount of BP a d m i n i s t e r e d . The present study showed the g r e a t e s t l e v e l of b i n d i n g to occur at 12-18 hours a f t e r BP a d m i n i s t r a t i o n , and t h i s f e l l to 60% of maximum by 48 hours (F igures 17 and 18) . 80. Corn o i l t reatment was found to decrease both the maximum l e v e l of BP b ind ing and the r a t e by which i t reached t h i s l e v e l . It was a l s o observed that t h i s h ighest l e v e l was e s s e n t i a l l y mainta ined to 42 hours (F igures '21 and 22, corn o i l ) . The e f f e c t of corn o i l on the b ind ing of BP at 24 hours a f t e r i t s a d m i n i s t r a t i o n was the same when corn o i l was admin is te red at e i t h e r 3» 2k or 48 hours before BP (Table III and F igure 25, corn o i l ) . It appeared that corn o i l had an e f f e c t on the a b s o r p t i o n of BP from the i . p . c a v i t y . Therefore i t was e s s e n t i a l that a l l animals p r e t r e a t e d w i t h agents that were d i s s o l v e d in corn o i l had animals that were t r e a t e d w i t h the corn o i l v e h i c l e a lone as t h e i r c o n t r o l s . S a l i n e pretreatment -was found not to a l t e r the b i nd i ng o f BP. The degree of b i n d i n g was found to be l i n e a r l y dependent on the dose of BP between the range of 0.125 and 12.5 pmoles (F igures .1 3< and T 4) Th is would imply that the enzymatic pathways i n v o l v i n g BP metabol ism were not s a t u r a t e d . The dose chosen f o r the in v i vo experiments was 1.25 ymole . The b ind ing of BP was expressed on a per mg p r o t e i n and per gram wet weight of l i v e r b a s i s f o r the methadone and 3"MC exper iments . This was c a r r i e d out s i n c e agents which induce microsomal enzyme a c t i v i t y a re sometimes known to induce microsomal p r o t e i n and t o t a l l i v e r p r o t e i n (83). In a l l of the experiments performed the p r o t e i n content of the e x t r a c t e d p e l l e t d i d not d i f f e r fo r any t reatment . The enzyme r e s p o n s i b l e f o r the fo rmat ion of the i r r e v e r s i b l y bound i n t e r -mediates of BP, the BP e p o x i d e s , is a cytochrome P-kSO mixed f u n c t i o n o x i d a s e , namely BP hydroxy lase (39). The decrease in the i r r e v e r s i b l e b i n d i n g of BP by SKF 525-A and by 3"MC cou ld be due to t h e i r i n h i b i t i o n of BP h y d r o x y l a s e , thereby dec reas ing the amount of epoxides produced. SKF 525_A has been shown to i n h i b i t ra t h e p a t i c AHH when admin is te red i . p . before s a c r i f i c e (74) and when added to the incubat ion tube ( 8 4 ) . 3-MC i s a l s o a s u b s t r a t e fo r AHH (85) 81. and i t s presence in the l i v e r could i n h i b i t the metabol ism of BP. Thus, the i r r e v e r s i b l e b ind ing of BP to l i v e r macromolecul es was assayed i n vi t r o and the e f f e c t s of these agents on the i ri v i t ro b i nd i ng of BP was s t u d i e d . In the present s t u d y , :the assay was c a r r i e d out us ing the 10,000 x g l i v e r supernatant and the NADPH-generating system. A f t e r i n c u b a t i o n , the T C A - p r e c i p i t a t e d p r o t e i n was washed e x t e n s i v e l y and the degree of i r r e v e r s i b l e b i n d i n g q u a n t i t a t e d . There was a s i g n i f i c a n t amount of BP bound to the l i v e r macromolecules when b o i l e d enzyme blanks were used. Using a c t i v e enzyme produced an a d d i t i o n a l amount of b i n d i n g . Th is inc rease cou ld be reduced by e i t h e r the d e l e t i o n of the NADPH-generating system or incubat ion under N^ atmosphere. T h e r e f o r e , the enzymatic nature and the cytochrome P-450 dependency of t h i s b ind ing was e s t a b l i s h e d . The non-enzymatic b i n d i n g , which was not i n f l u e n c e d by N^ or NADPH, was s u b t r a c t e d from the t o t a l b ind ing to y i e l d enzymatic b i n d i n g . Non-enzymatic b i n d i n g has been d i s c u s s e d in d e t a i l by G r i l l i (86). The enzymatic b i n d i n g was shown to be l i n e a r w i t h incubat ion time (F igure .5) and p r o t e i n content (F igure 6) f o r our assay c o n d i t i o n s . A l l the drugs t e s t e d decreased the enzymatic b i n d i n g , but not the non-enzymatic b ind ing of BP to l i v e r macromolecules in v i t r o . SKF 525-A, at a c o n c e n t r a t i o n of 10 ^M_, produced a s i g n i f i c a n t decrease in the b ind ing of BP (F igure 9). It has been reported that the SKF 525_A c o n c e n t r a t i o n in the l i v e r w i t h i n 2 hours a f t e r i . p . i n j e c t i o n of 50 mg/kg of the compound may be „ - r .. - -4 roughly est imated to be in the range of 10-65 yg/gm or 0 , 3 to 1 , 7 x 1.0 M. It was a l s o reported that " a f t e r the if.;p. i n j e c t i o n of 80 mg/kg of SKF 525"A to -4 • r a t s , the c o n c e n t r a t i o n in the l i v e r was 68 ug/,gm or 1.8 x 10 M l hour a f t e r the i n j e c t i o n and the c o n c e n t r a t i o n was found to decrease as a f u n c t i o n of time (84). It was shown that SKF 525-A at 35 mg/kg i . p . 3 hours before s a c r i f i c e decreased s i g n i f i c a n t l y the a c t i v i t y of AHH in v i t r o (74). In the 82. present study doses of 50 mg/kg and 75 mg/kg 3 hours before BP were needed to s i g n i f i c a n t l y decrease the i r r e v e r s i b l e b ind ing from c o n t r o l (F igure 27). A l s o , i t was found that SKF 525~A at 35 mg/kg i . p . d i d not a l t e r s i g n i f i c a n t l y the degree of b i n d i n g . Thus, a dose of 35 mg/kg i . p . apparent l y d id not produce s u f f i c i e n t c o n c e n t r a t i o n s of SKF 525 -A in the l i v e r to decrease cytochrome P-450 a c t i v i t y s u f f i c i e n t l y to i n h i b i t BP b i n d i n g . In t h i s study doses of at l e a s t 50 mg/kg i . p . were necessary to produce i n h i b i t i o n of AHH in v i v o and thus reduce the metabol ism of BP and i t s b ind ing to hepat i c t i s s u e . The present data i n d i c a t e that higher l e v e l s o f SKF 525_A must be obta ined in the l i v e r to i n h i b i t BP b i n d i n g in v i v o , in c o n t r a s t to the l e s s e r amount of SKF 525 -A necessary to i n h i b i t the metabolism of BP in v i t r o . Th is could suggest that the enzyme r e s p o n s i b l e f o r the format ion of 3 _0H BP in v i t r o is not the same enzyme r e s p o n s i b l e f o r the product ion of the t i s s u e bound m e t a b o l i t e s in v i v o , and that the a f f i n i t i e s of these enzymes fo r SKF 525_A are d i f f e r e n t . It should a l s o be noted that the d i f f e r e n c e could be a r e s u l t of the in v i t r o p r e p a r a t i o n and assay of the enzyme. In the present s t u d y , 3_MC a l s o decreased s i g n i f i c a n t l y the enzymatic b ind ing of BP in v i t r o (F igure 12). It has been shown that 3-MC w i l l i n h i b i t hepat i c AHH in v i t r o from 3 -MC induced r a t s , but not from untreated rats (87). The c o n c e n t r a t i o n of 3 -MC used to i n h i b i t AHH in the s t u d i e s was 1.87 x 10 ^M_. We found that 3~MC at a c o n c e n t r a t i o n of 10 ^M_wi l l i n h i b i t the enzymatic b ind ing of BP in v i t ro . Th is decrease in b ind ing appears to oppose the r e s u l t s obta ined in the study c i t e d , s i n c e we used untreated r a t s . AHH is s tud ied in v i t r o by q u a n t i t a t i n g the format ion of 3 - 0H BP, i t s major m e t a b o l i t e , from / BP. As p r e v i o u s l y s t a t e d , 3-MC w i l l not i n h i b i t the fo rmat ion of 3 -0H BP in untreated a n i m a l s , but t h i s does not i n d i c a t e that the other pathways of BP metabol ism w i l l be a f f e c t e d , most prominent ly the k,5 and 7,8 regions on the BP nuc leus . Thomas and Fur long (38) showed that in 3"MC induced rat l i v e r m i c r o -somes the format ion of the macromolecu 1e-bound spec ies of BP is a major r e a c t i o n in the in v i t ro system, o c c u r r i n g at a ra te approx imate ly 60% that of the format ion of BP phenols . T h e r e f o r e , i t i s suggested that the decrease in 83. b ind ing in v i t ro cou ld be due to the i n h i b i t i o n of the a c t i v a t i o n pathways that are r e s p o n s i b l e f o r the metabol ism of BP to r e a c t i v e i n t e r m e d i a t e s . BP was admin is te red 24 hours a f t e r 3 _MC, which was i n j e c t e d i . p . f o r 2 consecut i ve days at a dose of 20 mg/kg/day. This treatment produced a 30% decrease in b ind ing from c o n t r o l (F igures 21 and 22). It could be argued that the r e s i d u a l amount of 3-MC present in the l i v e r 2k hours a f t e r the l a s t 3 -MC i n j e c t i o n cou ld be caus ing a d i r e c t i n h i b i t i o n of the metabo l i c a c t i v a t i o n of BP. Th is i n h i b i t i o n was shown in v i t r o (F igure 12). T h e r e f o r e , an experiment was c a r r i e d out in which Bp W a s admin is te red 48 hours a f t e r the l a s t 3_MC i n j e c t i o n . As shown in f i g u r e 25, there was no d i f f e r e n c e in the percent change from c o n t r o l at 2k hours a f t e r BP between the two 3~MC groups. Th is would suggest that the decrease in b i n d i n g seen w i t h 3~MC t reated animals i s not due to the d i r e c t i n h i b i t i o n of BP hydroxy lase by r e s i d u a l 3~MC. In the present s t u d i e s methadone treatment produced no s i g n i f i c a n t change in the degree of BP b i n d i n g in v i v o , but produced a k0% and 60% decrease in -k -3 b i n d i n g from c o n t r o l at c o n c e n t r a t i o n s in the incubat ion tube of 10 M^  and 10 H_, r e s p e c t i v e l y (F igure -8;). Methadone was a l s o found to i n h i b i t BP hydroxy lase -k in v i t ro at a c o n c e n t r a t i o n of 5 x 10 M^  (66). However, methadone was admin is te red v i a the d r i n k i n g water and the amount of methadone l i k e l y to be present in the 1 i v e r r t i s s u e of t rea ted animals i s in the range of 10 ^M (89). It should be noted that methadone was found to inc rease h e p a t i c epoxide hydratase by 212% in male r a t s (66) but that the same methadone treatment appeared not to have any i n f l u e n c e on the degree of b i n d i n g of BP in v i v o , ( F i g u r e s 18 and 19). 3~MC treatment a l s o induces h e p a t i c epoxide hydratase a c t i v i t y (48) and t h i s cou ld be the cause of the decrease b ind ing of BP in 3"MC t rea ted a n i m a l s . There i s i n c r e a s i n g ev idence f o r a s i n g l e epoxide hydratase tha t c a t a l y z e s the hyd ra t ion of a wide number of s u b s t r a t e s i n c l u d i n g s ty rene ox ide and BP 4 , 5 - o x i d e (90). 8k. It has been shown that in 9 human l i v e r samples s ty rene ox ide hydratase a c t i v i t y produced c o r r e l a t i o n s of g r e a t e r than 0.95 when p l o t t e d aga ins t the h y d r a t i o n of e i t h e r the k,5~, 7,8- o r 9,10-epoxide o f BP(k3). T h e r e f o r e , i t is l o g i c a l to assume that i n d u c t i o n of the hyd ra t ion of s ty rene ox ide w i l l produce a s i m i l a r inc rease in the h yd r a t ion of any o f the BP epox ides . There-f o r e , i t does not seem l i k e l y that 3~MC alone could decrease BP b i n d i n g due to i t s inc rease in EH a c t i v i t y wi thout methadone showing a s i m i l a r e f f e c t . Prev ious s t u d i e s have shown that g l u t a t h i o n e is e s s e n t i a l fo r the p r o t e c t -ion of th ioH and other n u c l e o p h i l i c groups in animal t i s s u e s from a v a r i e t y of t o x i c drug m e t a b o l i t e s (91). It has been shown that when the dose of a c e t a -minophen i s s u f f i c i e n t l y la rge to dep le te h e p a t i c g l u t a t h i o n e in the form of mercaptur ic a c i d c o n j u g a t e s , the m e t a b o l i t e can no longer be d e t o x i f i e d by t h i s pathway and t h e r e f o r e a r y l a t e s o ther c e l l u l a r n u c l e o p h i l e s (67). S i m i l a r r e l a t i o n s h i p s have been found between h e p a t i c g l u t a t h i o n e and h e p a t i c cova len t b ind ing of a t o x i c m e t a b o l i t e of another h e p a t o t o x i n , bromobenzene (92). G l u t a t h i o n e conjugates of BP have been i d e n t i f i e d and g l u t a t h i o n e has been shown in v i t r o to form conjugates w i t h BP epoxides both enzymatica11y and non-enzymatica11y (50). In the present study we examined the r o l e of g l u t a t h i o n e on the b ind ing of BP both in v i vo and in v i t r o . G l u t a t h i o n e had no e f f e c t on the b i n d i n g of BP to l i v e r macromolecules in v i t ro u n t i l a c o n c e n t r a t i o n of 10 f4 (F igure 10). It was a l s o found that c y s t e i n e i n h i b i t e d the b i n d i n g of BP -2 at a c o n c e n t r a t i o n o f 10 M_ (F igure 1:1). Ne i ther c y s t e i n e nor d i e t h y l maleate pret reatments a l t e r e d the b ind ing of BP in v i v o (Table I I I ) . King et a 1. (70) showed that g l u t a t h i o n e at a c o n c e n t r a t i o n in the incubat ion mixture of 3 mM i n h i b i t e d the b i n d i n g of BP and BP~7,8-diol to DNA. Both c y s t e i n e and g l u t a --k th ione at c o n c e n t r a t i o n s of 5 x 10 M^  i n h i b i t e d the b ind ing of acetaminophen to h e p a t i c macromolecules in v i t r o . (93) • Thus, g l u t a t h i o n e and c y s t e i n e are 85. capable of i n h i b i t i n g the b i n d i n g of BP to h e p a t i c macromolecules in v i t r o , s i m i l a r to i n h i b i t i o n of acetaminophen b i n d i n g , a t a c o n c e n t r a t i o n that i s c l o s e to the est imated i n t r a c e l l u l a r c o n c e n t r a t i o n of g l u t a t h i o n e , 3mM (9-0. The ro le of g l u t a t h i o n e in the b ind ing of BP to hepat i c macromolecules appears to be d r a m a t i c a l l y d i f f e r e n t than i t s p r o t e c t i v e r o l e fo r c e r t a i n h e p a t o t o x i n s , such as acetaminophen. M i t c h e l l showed that c o v a l e n t b i n d i n g of acetaminophen to h e p a t i c macromolecules in v i vo occur red only w i t h doses of acetaminophen that caused a 70% or more d e p l e t i o n of l i v e r g l u t a t h i o n e (67). No s i g n i f i c a n t b ind ing occur red u n t i l 70% o f the h e p a t i c g l u t a t h i o n e was d e p l e t e d . It was a l s o shown that d e p l e t i n g h e p a t i c g l u t a t h i o n e w i t h d i e t h y l maleate produced a marked inc rease in the b i n d i n g of acetaminophen w i t h h e p a t i c t i s s u e . Converse l y , treatment w i th c y s t e i n e decreased the b i n d i n g . C y s t e i n e , a g l u t a t h i o n e precursor (73), i s known to r e s t o r e the hepat ic s t o r e s of g l u t a t h i o n e (95) when they have been dep le ted by acetaminophen and i t i s thought tha t by t h i s mechanism and by i t s a b i l i t y to form conjugates w i th acetaminophen, c y s t e i n e can decrease the b ind ing of acetaminophen m e t a b o l i t e s . In the present s t u d y , p r e t r e a t i n g wi th d i e t h y l maleate d i d not a l t e r the b ind ing of BP. P r e t r e a t i n g w i t h c y s t e i n e f a i l e d to a l t e r the b i n d i n g of BP, and the b ind ing of BP versus dose , in the range of 0.13 to 1 2 . 5 M m o l e was l i n e a r thus i n d i c a t i n g the absence of a t h r e s h o l d dose f o r b i n d i n g . The c y s t e i n e experiment and the low doses of BP used i n d i c a t e d that BP at these doses d i d not d e p l e t e h e p a t i c g l u t a t h i o n e . If BP was bound to h e p a t i c macromolecules because i t was d e p l e t i n g h e p a t i c g l u t a t h i o n e , c y s t e i n e treatment should have decreased the b ind ing and d i e t h y l maleate treatment should have increased the b i n d i n g . T h e r e f o r e , t h e r e appears to be no p r o t e c t i v e r o l e of g lu tath ione fo r BP b i n d i n g by the same mechanism as i t p r o t e c t s the l i v e r from acetaminophen a c t i v e m e t a b o l i t e s . The present data suggest that at low doses of BP there is i n s i g n i f i c a n t a l t e r a t i o n in g l u a t h i o n e l e v e l s and that even a f t e r d e p l e t i o n of 86. 70% of the h e p a t i c g l u t a t h i o n e , there i s s u f f i c i e n t g l u t a t h i o n e present to conjugate w i t h BP. S ince g l u t a t h i o n e conjugates of BP epoxides are known to e x i s t the presence of g l u t a t h i o n e is necessary . But w i t h low doses of c a r c i n -ogens, the amount necessary would be f a r l e s s than the amount requi red to d e t o x i f y drugs which are taken in t h e r a p e u t i c q u a n t i t i e s . A second p o s s i b l e mechanism fo r g l u t a t h i o n e should be c o n s i d e r e d . The c o n t r o l l i n g f a c t o r in the fo rmat ion of g l u t a t h i o n e conjugates of BP could be the .GSHT 'instead of the c o n c e n t r a t i o n of h e p a t i c g l u t a t h i o n e . GSHT c a t a l y z e s the con jugat ion of g l u t a t h i o n e w i t h BP epoxides (50). 3_MC has been shown to increase the con jugat ion o f epoxides by induc ing GSHT a c t i v i t y (51). The decrease in b ind ing w i t h 3_MC could be due to the i n d u c t i o n of these enzymes. U n f o r t u n a t e l y , we do not have, at t h i s t i m e , a compound which s e l e c t i v e l y induces or i n h i b i t s t h i s enzyme and t h e r e f o r e a decrease in BP b ind ing due to an increase in GSHT a c t i v i t y by 3_MC remains a poss i b i 1 i t y . The 3"MC induced decrease in BP b i n d i n g could a l s o be due to an a l t e r a t i o n in the pathways in BP metabol ism. Zampaglione (96) and coworkers found that 3-MC induced p r o t e c t i o n from bromobenzene's h e p a t o t o x i c i t y . SKF 525~A a l s o b locked bromobenzene-induced l i v e r n e c r o s i s , but i t b locked bromobenzene metabol ism in v i v o . 3~MC d i d not a l t e r the rate of bromobenzene metabol ism in v i v o . It was found that there was an increase in b romopheny ld ihydrod io l , bromocatechol and 2-bromophenol e x c r e t i o n . It was suggested that 3 -MC i n d u c t i o n may d i r e c t bromobenzene metabol ism in to a comparat i ve l y nontox ic pathway. Wiebel (39) showed the metabol ism of BP by d i f f e r e n t forms of cytochrome P-450 from r a b b i t l i v e r . He found that the r e l a t i v e amounts of BP m e t a b o l i t e s formed by v a r i o u s cytochrome p r e p a r a t i o n s and by l i v e r microsomes were d r a m a t i c a l l y d i f f e r e n t . He t h e r e f o r e s t a t e d that s i n c e the mixed f u n c t i o n ox idases were 87-engaged in both the a c t i v a t i o n and d e t o x i f i c a t i o n of PAH's , d i f f e r e n c e s in the r e l a t i v e d i s t r i b u t i o n of the m u l t i p l e forms of cytochrome P - 4 5 0 might be a key f a c t o r in determin ing the s u s c e p t i b i l i t y of t i s s u e s , i n d i v i d u a l s , and spec ies to the c y t o t o x i c and c a r c i n o g e n i c a c t i o n of PAH's . K i n o s h i t a (97), us ing l i v e r microsomes from c o n t r o l and 3~MC t r e a t e d r a t s showed an increase in the o v e r a l l metabol ism of BP w i t h microsomes from 3"MC t r e a t e d a n i m a l s , but more i m p o r t a n t l y , showed a la rge r inc rease in metabol ism at the n o n - K - r e g i o n of BP versus the K - r e g i o n . Thus, the e x i s t a n c e in the l i v e r of va r ious forms of cytochrome P - 4 5 0 , a long w i t h the evidence that 3"MC induces a s p e c t r a l l y d i s t i n c t cytochrome P-kkQ {kO, 58), could suggest that 3_MC a l t e r s BP metabol ism towards pathways that produce less r e a c t i v e i n t e r m e d i a t e s , and thereby decrease the degree of b i n d i n g . In i s o l a t e d per fused lungs Cohen (71) showed that 3~MC increased the b i n d i n g of BP to lung t i s s u e . A dose of 2 nmole BP was i n s t i l l e d i n t r a t r a c h e a l l y and the n o n r e c i r c u l a t i n g p e r f u s a t e was c o l l e c t e d . The increase in b i n d i n g in lung t i s s u e i s o p p o s i t e to the r e s u l t s obta ined in the present exper iments , which showed a decrease in h e p a t i c b ind ing of BP in 3"MC t r e a t e d r a t s . Th is d i f f e r e n c e could l i e in the dose of BP used in the two separate exper iments . It i s p o s s i b l e that 2 nmoles admin is te red i n t r a t r a c h e a 11y could be s a t u r a t i n g the cytochrome P-450 enzyme u n i t s at the s i t e s of metabol ism. Induct ion by 3-MC, shown to inc rease BP-hydroxy lase a c t i v i t y by i n c r e a s i n g the amount of enzyme present (98) cou ld t h e r e f o r e increase the fo rmat ion of r e a c t i v e metabo l -i t e s in the 3_MC t r e a t e d r a t s . In the present exper iment , BP i s admin is tered i .p . . and , at any g iven t i m e , the amount of BP that passes through the l i v e r , which conta ins a 2 0 - f o l d g rea te r q u a n t i t y of cytochrome P-^50 than the lung (99), might not s a t u r a t e the enzyme s i t e s . Thus, in t h i s c a s e , 3 -MC i n d u c t i o n would not a f f e c t the ra te of metabol ism, but instead a f f e c t the pathways of metabol ism, i . e . , induce the format ion of cytochromes w i t h d i f f e r e n t s i t e 88. preferences on the BP molecule that leads to the product ion of l e s s r e a c t i v e i ntermed i a t e s . Furthermore, i n d u c t i o n by 3_MC in the lung versus the l i v e r a f t e r i t s i . p . a d m i n i s t r a t i o n has been s tud ied (100). It was shown that lower doses of 3 - MC, which produced i n d u c t i o n of AHH in the l u n g , d i d not a f f e c t the a c t i v i t y of t h i s enzyme in the l i v e r . The f o l d - i n d u c t i o n of the enzyme was shown to be much h igher in the lung than in the l i v e r . This is f u r t h e r ev idence fo r the e x i s t a n c e of d i f f e r e n t forms of cytochrome P-kSO w i t h i n and amongst v a r i o u s organs and evidence f o r d i f f e r e n c e s in t h e i r r e l a t i v e i n d u c i b i 1 i t y . Throughout the study a second popu la t ion of animals showed b ind ing of BP that was both q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t from the f i r s t . The percentage of animals that f e l l i n to t h i s 2nd p o p u l a t i o n was 19% (46 out of 250 a n i m a l s ) . There is no mention of t h i s phenomenon in the l i t e r a t u r e and the causes of t h i s d i f f e r e n c e is unknown at t h i s po in t in t ime . The t i m e -b ind ing curve in t h i s 2nd p o p u l a t i o n of animals (F igures 19 and 2 0 , tap water) is s i m i l a r to the k i n e t i c s of the plasma drug c o n c e n t r a t i o n w i t h t ime a f t e r i t s i . v . a d m i n i s t r a t i o n . Whether t h i s o b s e r v a t i o n of two d i s t i n c t animal p o p u l -a t i o n s is due to a d i f f e r e n c e in a b s o r p t i o n , d i s t r i b u t i o n , or metabol ism between the two groups is at present h i g h l y s p e c u l a t i v e . Sex, spec ies and s t r a i n v a r i a t i o n s in drug metabol ism are commonly known (101) . C e r t a i n s t r a i n s of mice show an inc rease in hepat i c AHH a c t i v i t y w i t h 3_MC t rea tment , whereas AHH in c e r t a i n other s t r a i n s are non - induc ib1e w i t h 3_MC (102). Again a g e n e t i c d i f f e r e n c e between the present s t u d y ' s two groups in t h e i r a b i l i t y to metabo l i ze BP is a l s o s p e c u l a t i v e . One p o s s i b l e mechanism to account fo r the o b s e r v a t i o n s found is that the degree of b ind ing of BP to l i v e r macromo1 ecu 1es in v i v o a f t e r i t s i . p . a d m i n i s t r a t i o n into ra ts appears to depend s t r o n g l y on the r e l a t i v e abundance of the v a r i o u s forms 89. of cytochrome P - 4 5 0 p resent . The decrease in b ind ing w i t h 3 - MC t reatment could be due to the format ion of d i f f e r e n t cytochromes which a l t e r the metabolism of BP to pathways which produce less r e a c t i v e i n t e r m e d i a t e s . The decrease in b ind ing a f t e r SKF 5 2 5 ~ A t reatment is in agreement w i th the evidence that the cytochrome P - 4 5 0 dependent enzymes are r e s p o n s i b l e f o r the a c t i v a t i o n of B P . t o r e a c t i v e in termediates and that these enzymes can be i n h i b i t e d by SKF 5 2 5 - A ( 4 2 ) . The lack of a r o l e fo r g l u t a t h i o n e regard ing the b i n d i n g of BP i s d r a m a t i c a l l y d i f f e r e n t from i t s r o l e in the p r o t e c t i o n of acetaminophen hepat i c b i n d i n g , and is probably due to a p h y s i o l o g i c a l excess of t h i s s u l f h y d r y l compound as compared to the small q u a n t i t y of p o l y c y c l i c aromat ic hydrocarbon. In order to c l a r i f y which parameters c o n t r o l the a c t i v a t i o n and b ind ing of BP to c e l l u l a r macromolecules a number of experiments are necessary . I s o l a t i o n of the v a r i o u s forms of h e p a t i c and lung cytochrome P - 4 5 0 and a study of the k i n e t i c s of BP metabol ism and b ind ing w i t h these enzymes would be of major i n t e r e s t in l i g h t of the present exper iments . Secondly , repeat ing the 3"MG experiments in lung t i s s u e could g i v e a b e t t e r comparison w i t h the s t u d i e s of Cohen ( 7 1 ) . F i n a l l y , s t u d i e s us ing h igher doses of BP to see i f the r e s u l t s of treatment are d i f f e r e n t at doses of BP that s a t u r a t e the h e p a t i c cytochrome P - 4 5 0 enzymes would g i ve s t ronger support to the d i s c u s s i o n of the present d a t a . 90. SUMMARY AND CONCLUSIONS 1. The hepatic i r r e v e r s i b l e binding of JH-BP in v i v o , administered i . p . , was found to be both dose and time dependent. 2. The degree of binding was shown to be l i n e a r l y dependent on the dose of BP administered between 0.125 and 12.5 ymole BP. 3- The maximum l e v e l of binding occurred at 12 to 18 hours a f t e r BP administra-t i o n and f e l l to 60% of maximum by 48 hours. 4. SKF 525-A, an agent which i n h i b i t s AHH in v i t r o (74), decreased the i r r e -v e r s i b l e binding of BP to t o t a l l i v e r p r o t e i n and n u c l e i c acids in vivo to about 70% of c o n t r o l . 5. 3-MC, an agent which induces AHH and the formation of a s p e c t r a l l y d i s t i n c t form of cytochrome P-450, namely cytochrome P-448 (40, 58), decreased the i r r e v e r s i b l e binding of BP to l i v e r macromolecules in vivo by 30% from control l e v e l s . 6. Cysteine, a precursor of g l u t a t h i o n e (73), and d i e t h y l maleate, which depletes hepatic g l u t a t h i o n e (72), had no s i g n i f i c a n t e f f e c t on the l e v e l of i r r e v e r s i b l y bound BP from c o n t r o l . 7. Methadone, which was shown to s e l e c t i v e l y increase EH a c t i v i t y (66) a l s o had no e f f e c t on the l e v e l of BP i r r e v e r s i b l y bound to l i v e r macromolecules in v i v o . 8. Cysteine (10 3M), g l u t a t h i o n e (10" 2M), 3~MC (10 _ 5M), SKF 525-A (10 _ 5M), and methadone (10 M), a l l s i g n i f i c a n t l y decreased the binding of BP in v i t r o to 10,000 x g. l i v e r supernatant macromolecules. Lower concentrations had no s i g n i f i c a n t e f f e c t . 9- A second population of animals e x h i b i t e d binding of BP that was both q u a l i t a t i v e l y and q u a n t i t a t i v e l y d i f f e r e n t from the f i r s t . The 2nd popula-t i o n represented 19% (46 out of 250) of the t o t a l number of animals used in the study. 91 • These r e s u l t s i n d i c a t e that the in v i v o i r r e v e r s i b l e b ind ing of 3 H-BP to rat h e p a t i c t i s s u e i s mainly dependent on the a c t i v i t y of the cytochrome P-450 mixed f u n c t i o n ox idase enzyme system. Increased l e v e l s of EH, or changes in g l u t a t h i o n e l e v e l s do not appear r a t e - c o n t r o l l i n g . 92. j3 I BL [ OGRAPHY (1.) 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