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Effects of 1-α- acetylmethadol on neonatal androgen induced imprinting in the rat Finlayson, Malcolm John Paul 1980

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EFFECTS (DF 1-oc- ACETYLMETHADOL ON NEONATAL ANDROGEN INDUCED IMPRINTING IN THE RAT by MALCOLM JOHN PAUL FINLAYSON B.A., San J o s e S t a t e U n i v e r s i t y 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n o f P h a r m a c o l o g y and T o x i c o l o g y i n t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF June 1 MALCOLM JOHN PAUL BRITISH COLUMBIA 980 FINLAYSON, 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i . ABSTRACT I t i s w e l l known t h a t an age and s e x d e p e n d e n t d i f f e r e n c e e x i s t s f o r t h e h e p a t i c m i c r o s o m a l m e t a b o l i s m o f c e r t a i n compounds i n t h e r a t . W i t h c e r t a i n enzymes t h i s d i f f e r e n c e has been shown t o be d e p e n d e n t on t h e p r e s e n c e o f t e s t o s t e r o n e i n t h e male n e o n a t e g i v i n g r i s e t o t h e t e r m n e o n a t a l a n d r o g e n i n d u c e d i m p r i n t i n g . Removal o f t e s t o s t e r o n e f r o m t h e male n e o n a t e w i l l r e s u l t i n t h e r e d u c t i o n o r a b o l i t i o n o f i m p r i n t -i n g and o f t h e e x p r e s s i o n o f t h e sex d i f f e r e n c e a t p u b e r t y . S i n c e o p i a t e s a r e known t o r e d u c e p l a s m a t e s t o s t e r o n e l e v e l s and have i n h i b i t o r y e f f e c t s on t h e p r o s t a t e g l a n d and s e m i n a l v e s i c l e , we have p o s t u l a t e d t h a t in. u t e r • and n e o n a t a l e x p o s u r e t o o p i a t e s w i l l i n h i b i t i m p r i n t i n g as e x p r e s s e d by a d e c r e a s e i n t h e s e x d e p e n d e n t h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s . To t e s t t h i s h y p o t h e s i s l - o < - a c e t y l m e t h a ' d o l - H C l (LAAM) was a d m i n i s t e r e d v i a t h e d r i n k i n g w a t e r t o f e m a l e W i s t a r r a t s t h r o u g h o u t p r e g n a n c y and l a c t a t i o n . The dose was i n c r e a s e d t h r o u g h o u t p r e g n a n c y s u c h t h a t an a v e r a g e maximum o f 1 . 1 5 - 0 . 1 0 mg/kg/d ay was a c h i e v e d d u r i n g t h e t h i r d t r i m e s t e r . C o n t r o l s were p a i r f e d and w a t e r e d . Pups were not removed t o s u r r o g a t e dams as t h e n e o n a t a l p e r i o d i s l i k e l y t o be i m p o r t a n t t o i m p r i n t i n g . On day 2 1 pupa'were weaned f r o m LAAM and c o n t r o l dams, and s e p a r a t e d as t o t h e i r s e x and e x p e r i m e n t a l g r o u p s . A l l pups r e c e i v e d l a b chow and w a t e r a_d l i b i t u m u n t i l t h e y r e a c h e d m a t u r i t y ( d a y s 9 0 - 1 2 0 ) . The a p p a r e n t Km, Vmax, and b a s a l s p e c i f i c a c t i v i t i e s were d e t e r m i n e d f o r a m i n o p y r i n e N - d e m e t h y l a s e ( A P ) , e t h y l m o r p h i n e l\l-demethylase ( E T ) , a n i l i n e h y d r o x y l a s e ( A N ) , and b e n z o ( a ) p y r e n e h y d r o x y l a s e (BP) f r o m t h e h e p a t i c m i c r o s o m a l f r a c t i o n o f t h e m a t u r e male and f e m a l e o f f s p r i n g . D i f f e r e n c e s were c o n s i d e r e d s i g n i f i c a n t a t p "\D.05 u s i n g a t w o - t a i l e d S t u d e n t ' s t - t e s t . The dose o f LAAM a d m i n i s t e r e d d i d n o t p r o d u c e any n e o n a t a l w i t h d r a w a l o r i n h i b i -t i o n o f d e v e l o p m e n t o f t h e pups. I n t h e a d u l t m a l e , i n . u t e r o and n e o n a t a l e x p o s u r e t o LAAM r e s u l t e d i n d e c r e a s e s i n Km f o r AP, Vmax f o r AN and b a s a l s p e c i f i c a c t i v i t y f o r ET, and i n c r e a s e s i n t h e Km, Vmax and b a s a l s p e c i f i c a c t i v i t y f o r BP. F o r t h e f e m a l e LAAM p r o d u c e d an i n c r e a s e i n Km f o r AP and a d e c r e a s e i n t h e Vmax f p r ET and BP as w e l l as a d e c r e a s e i n t h e b a s a l s p e c i f i c a c t i v i t y o f BP. These r e s u l t s show t h a t 'i n . u t e r o and n e o n a t a l e x p o s u r e t o LAAM a f f e c t s c e r t a i n h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s e x p r e s s e d i n t h e mature r a t . F u r t h e r m o r e , - t h e s e o b s e r v a t i o n s a r e c o n s i s t e n t w i t h i n h i b i t o r y e f f e c t s o f LAAM on i m p r i n t i n g r e s u l t i n g i n a change i n t h e p r o f i l e o f c y t o c h r o m e s P-45D p r e s e n t i n t h e h e p a t i c m i c r o s o m a l f r a c t i o n . G a i l D. B e l l w a r d , . Ph.D. S u p e r v i s o r i v . TABLE OF CONTENTS Page ABSTRACT i i LIST DF TABLES v i LIST DF FIGURES v i i LIST DF ABBREVIATIONS v i i i ACKNOWLEDGEMENT i x INTRODUCTION ' 1 C l i n i c a l S i g n i f i c a n c e o f 1- - A c e t y l m e t h a d o l . 1 E f f e c t o f O p i a t e s on O f f s p r i n g 2 1- - A c e t y l m e t h a d o l M e t a b o l i s m J_r_ V i v o . . . . 4 E f f e c t o f O p i a t e s on M e t a b o l i s m _I_n V i t r o . . . 5 B a s i s f o r Sex Dependent D i f f e r e n c e s 9 An d r o g e n I n d u c e d I m p r i n t i n g 12 M a i n t e n a n c e o f I m p r i n t i n g 14 Summary 1 B MATERIALS AND METHODS 21 C h e m i c a l s and R e a g e n t s 21 A n i m a l s 22 T r e a t m e n t 22 H e p a t i c M i c r o s o m a l P r e p a r a t i o n 23 Enzyme A s s a y s 24 RESULTS 30 Dose o f 1- - A c e t y l m e t h a d o l 30 G r o s s E f f e c t s o f 1- - A c e t y l m e t h a d o l . 3 1 E f f e c t o f 1- - A c e t y l m e t h a d o l on H e p a t i c M i c r o s o m a l Monooxygenase K i n e t i c P a r a m e t e r s , A p p a r e n t Km and Vmax 37 Page E f f e c t o f l - o c - A c e t y l m e t h a d o l on H e p a t i c M i c r o s o m a l Monooxygenase B a s a l S p e c i f i c A c t i v i t y 50 S t a t i s t i c a l C a l c u l a t i o n s 57 DISCUSSION ' 5B SUMMARY AND CONCLUSIONS 77 REFERENCES .' BO L I 5 T OF TABLES Page I Dosage s c h e d u l e o f 1 - c X - A c e t y l m e t h a d o l . . . . . 23 I I A v e r a g e dose o f 1 - o C - A c e t y l m e t h a d o l (mg/kg/d) p e r dam d u r i n g g e s t a t i o n I I I G e n e r a l b i r t h and d e v e l o p m e n t a l s t a t i s t i c s f o r c o n t r o l and 1 - o t - A c e t y l m e t h a d o l o f f s p r i n g . . . 32 IV E f f e c t o f 1 - c x - A c e t y l m e t h a d o l on t h e g r o w t h r a t e o f male and f e m a l e pups and p a i r weaned m a l e s 36 V E f f e c t o f l - o ( - A c e t y l m e t h a d o l on t h e a p p a r e n t Km and Vmax o f a m i n o p y r i n e N - d e m e t h y l a s e , e t h y l m o r p h i n e N - d e m e t h y l a s e , a n i l i n e h y d r o x y l a s e and b e n z o ( a ) p y r e n e h y d r o x y l a s e . . . 49 VI E f f e c t o f 1 - o C - A c e t y l m e t h a d o l on t h e b a s a l s p e c i f i c a c t i v i t i e s o f a m i n o p y r i n e N - d e m e t h y l a s e , e t h y l m o r p h i n e N - d e m e t h y l a s e , a n i l i n e h y d r o x y l a s e : and b e n z o ( a ) p y r e n e h y d r o x y l a s e . 56 V I I S t a t i s t i c a l c o m p a r i s o n o f t h e e f f e c t s o f 1 - t t - A c e t y l m e t h a d o l on k i n e t i c p a r a m e t e r s , a p p a r e n t Km, Vmax and b a s a l s p e c i f i c a c t i v i t y o f a m i n o p y r i n e N - d e m e t h y l a s e , e t h y l m o r p h i n e N - d e m e t h y l a s e , a n i l i n e h y d r o x y l a s e and b e n z o ( a ) p y r e n e h y d r o x y l a s e . . . 57 V I I I Summary o f h o r m o n a l f l u c t u a t i o n s p r o d u c e d by v a r i o u s m a n i p u l a t i o n s 72 IX Summary o f r e s u l t s 78 v i i . L I S T OF FIGURES Page Pathway f o r t h e i_n v i v o : N - d e m e t h . y l a t i o n o f l - c X - A c e t ' y l m e t h a d o l 6 A l t e r n a t i v e i n ' v i v o m e t a b o l i c pathways f o r 1 - c x - A c e t y l m e t h a d o l 7 E f f e c t o f l - o c " - A c e t y l m e t h a d o l on t h e g r o w t h r a t e o f male and f e m a l e pups and p a i r weaned m a l e s 34 E f f e c t o f 1 - o c - A c e t y l m e t h a d o l on t h e a p p a r e n t Km and Vmax o f a m i n o p y r i n e N - d e m e t h y l a s e i n male and f e m a l e r a t s . . . . 38 E f f e c t o f l - o f - A c e t y l m e t h a d o l on t h e a p p a r e n t Km and Vmax o f e t h y l m o r p h i n e N - d e m e t h y l a s e i n male and f e m a l e r a t s . . . . 41 E f f e c t o f l - o c - A c e t y l m e t h a d o l on t h e a p p a r e n t Km and Vmax o f a n i l i n e h y d r o x y l a s e i n male and f e m a l e r a t s 44 E f f e c t o f 1 - o t - A c e t y l m e t h a d o l on t h e a p p a r e n t Km and Vmax o f b e n z o ( a ) p y r e n e h y d r o x y l a s e i n male and f e m a l e r a t s 47 E f f e c t o f 1 - o c - A c e t y l m e t h a d o l on t h e b a s a l s p e c i f i c a c t i v i t y o f a m i n o p y r i n e N - d e m e t h y l a s e and e t h y l m o r p h i n e N - d e m e t h y l a s e i n male and f e m a l e r a t s . . . . 51 E f f e c t o f 1 - o i - A c e t y l m e t h a d o l on t h e b a s a l s p e c i f i c a c t i v i t y o f a n i l i n e h y d r o x y l a s e and b e n z o ( a ) p y r e n e h y d r o x y l a s e i n male and f e m a l e r a t s 53 v i i i . LIS T OF ABBREVIATIONS LAAM - l - i X - a c e t y l m e t h a d o l - H C l AP - a m i n o p y r i n e N - d e m e t h y l a s e ET - e t h y l m o r p h i n e N - d e m e t h y l a s e AN - a n i l i n e h y d r o x y l a s e BP - b e n z o ( a ) p y r e n e h y d r o x y l a s e LH - l u t e i n i z i n g hormone F 5 H - f o l l i c l e s t i m u l a t i n g hormone B 5 A - b a s a l s p e c i f i c a c t i v i t y LH-RH - l u t e i n i z i n g hormone r e l e a s i n g hormone G-6-P - g l u c o s e - 6 - p h o s p h a t e ACKNOWLEDGEMENT I am d e e p l y g r a t e f u l t o Dr. G a i l D. B e l l w a r d f o r h e r i n s i g h t and g u i d a n c e p r o v i d e d t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . T h i s s t u d y was s u p p o r t e d by a g r a n t f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada t o Dr. G.D. B e l l w a r d . INTRODUCTION LAAM CL I N I C A L SIGNIFICANCE l - * C - A c e t y l m e t h a d o l (LAAM), a s y n t h e t i c c o n g e n e r o f methadone ( S p e c t e r e_t a l _ . , 1 9 4 9 ) , i s an o p i a t e w h i c h l i k e methadone can s u b s t i t u t e f o r m o r p h i n e o r h e r o i n on t h e o p i a t e r e c e p t o r s s u c h as t h o s e i n t h e l i m b i c s y s t e m ( G o l d s t e i n 1 9 7 5 ) , The c r o s s t o l e r a n c e t h a t e x i s t s between t h e s e o p i a t e s a l l o w s f o r t h e use o f m e t h a d o n e - H C l i n p l a c e o f h e r o i n w i t h o u t t h e p r e c i p i t a t i o n o f w i t h d r a w a l symptoms. Methadone as s u c h has been used i n m a i n t e n a n c e programs f o r t h e r e h a b i l i t a t i o n o f h e r o i n a d d i c t s ( J a f f e and 5 e n a y , 1 9 7 1 ) . LAAM has r e c e n t l y been s u g g e s t e d as an a l t e r n a t i v e t o methadone i n t h e s e m a i n t e n a n c e p r o g r a m s . U n l i k e methadone, however, LAAM w i l l b l o c k w i t h d r a w a l symptoms f r o m 48-72 h o u r s ( J a f f e and Se n a y , 1 971 , Zaks e_t a l . , 1 972 ) , and i s r e p o r t e d n o t t o p r o d u c e t h e n a r c o t i c r u s h t h a t methadone does ( G o l d s t e i n , 1 9 7 5 ) . T h i s i s not t o i m p l y t h a t LAAM i s s u p e r i o r t o methadone f o r t h e r e h a b i l i t a t i o n o f o p i a t e a d d i c t s . T h e r e a r e i n s t a n c e s when LAAM w o u l d n o t be e m p l o y e d , f o r example when s u p e r v i s i o n i s r e q u i r e d ( Z a k s e_t a_l . , 1 972 ). N e v e r t h e l e s s , LAAM does have c e r t a i n a d v a n t a g e s . W h i l e r e c e i v i n g LAAM t h e r a p y t h e a d d i c t s d a i l y l i v i n g p a t t e r n i s n o t p r e d i c a t e d by t h e c l i n i c . He i s n o t r e q u i r e d t o be i n t h e p r o x i m i t y o f t h e c l i n i c a t some p o i n t i n any one 24 hour p e r i o d , t h u s e n a b l i n g him t o h o l d a j o b t h a t m i g h t 2. r e q u i r e a t t e n d a n c e a t t h e c l i n i c a t an i n c o n v e n i e n t t i m e . T h e r e i s a g r e a t p s y c h o l o g i c a l a d v a n t a g e i n h e r e n t i n LAAM m a i n t e n a n c e as w e l l . The s i m p l e f a c t t h a t t h e a d d i c t i s n o t i n g e s t i n g a dose o f e u p h o r i c d r u g each day W i l l remove t h e abuse p a t t e r n f r o m h i s l i f e s t y l e m a k i n g t h e p s y c h o l o g i c a l a s p e c t o f w i t h d r a w a l e a s i e r t o d e a l w i t h . A n o t h e r p o s i t i v e a s p e c t t o t h e use o f LAAM i s t h a t p a t i e n t s a r e a b l e t o p a s s t h e weekend w i t h o u t a " t a k e home" d o s e . Such i s n o t t h e c a s e w i t h methadone. Thus i l l i c i t use o f t h e d r u g and a c c i d e n t a l p o i s o n i n g s a r e a v o i d e d ( J a f f e and 5 e n a y , 1 9 7 1 ) . However, one s i t u a t i o n w h i c h may be d i f f i c u l t t o a v o i d i s t h a t o f t h e p r e g n a n t m a i n t e n a n c e p a t i e n t . EFFECT OF OPIATES ON OFFSPRING T h e r e a r e c o n s e q u e n c e s p r o d u c e d by ( p r e n a t a l ) e x p o s u r e t o o p i a t e d r u g s . F o r e x a m p l e , p r o g e n y o f o p i a t e a d d i c t e d women show an i n c r e a s e d i n c i d e n c e o f low b i r t h w e i g h t and n e o n a t a l c o m p l i c a t i o n s , s u c h as n a r c o t i c w i t h d r a w a l and r e s p i r a t o r y d i s t r e s s ( S t i m m e l and Adamsons ,1 9 7 6 ) . E f f e c t s o f o p i a t e t h e r a p y a r e e a s i l y d e m o n s t r a b l e w i t h a n i m a l s t u d i e s . C h a n d l e r e t a_l.,(1 975) r e p o r t t h a t f o r t h e r a t , methadone, 25 mg/kg/day o r a l l y t h r o u g h o u t g e s t a t i o n (21 d a y s ) , w i l l r e s u l t i n a d e c r e a s e i n f e t a l g r o w t h , i n t e r f e r e n c e w i t h t r i g g e r i n g o f p a r t u r i t i o n and p a s s i b l e m a l f o r m a t i o n s . O f f s p r i n g o f m i c e i n j e c t e d s u b c u t a n e o u s l y w i t h m e t h a d o n e - H C l o r m o r p h i n e s u l f a t e t w i c e d a i l y f o r f i v e days p r i o r t o m a t i n g (maximum dose 20 mg/kg and 240 mg/kg r e s p e c t i v e l y ) t o d r u g n a i v e m a l e s were o b s e r v e d t o 3. have d e l a y e d p i n n a u n f o l d i n g and eye o p e n i n g as compared t o c o n t r o l s . A l s o n o t e d was a p r o n o u n c e d d e c r e a s e i n - t h e h o p p i n g and r e a r i n g b e h a v i o r among male pups o f o p i a t e t r e a t e d dams ( F r i e d l e r ,1 977) . S m i t h e t a l , , ( 1 9 7 7 ) have r e p o r t e d t h a t n e o n a t a l m i c e i n j e c t e d s u b c u t a n e o u s l y w i t h methadone o r LAAM e x h i b i t e d a r e t a r d e d g r o w t h r a t e . T h i s r e t a r d a t i o n c o u l d be i n h i b i t e d by t h e n a r c o t i c a n t a g o n i s t , n a l o x o n e , w h i c h was w i t h o u t e f f e c t on w e i g h t g a i n i t s e l f . Pace and c o - w o r k e r s (1974) have shown t h a t r a t s t r e a t e d o r a l l y w i t h methadone 30 mg/kg on days 8-1 4 o f g e s t a t i o n show a s i g n i f i c a n t d e c r e a s e i n a v e r a g e pup w e i g h t and a v e r a g e l i t t e r s i z e a t w e a n i n g when compared t o a c o n t r o l g r o u p . F u r t h e r m o r e , o n l y 2 9 % o f t h o s e l i v e - b o r n pups s u r v i v e d p a s t 48 h o u r s as compared t o B0% f o r c o n t r o l . S i n c e LAAM i s ' r e p o r t e d t o c r o s s t h e p l a c e n t a and i s p r e s e n t i n m i l k ( M c G i n t y and F o r d , 1 9 7 6 ) p r e - and n e o n a t a l a n i m a l s a r e s u s c e p t i b l e t o e x p o s u r e d u r i n g g e s t a t i o n and l a c t a t i o n . T h i s i s o f g r e a t i m p o r t a n c e i n humans when c o n s i d e r i n g t h a t many o f t h e p a t i e n t s i n v o l v e d i n m a i n t e n a n c e p r o g r a m s a r e women o f c h i l d b e a r i n g a ge. S h o u l d t h e s e women c h o o s e t o g i v e b i r t h t o and n u r s e c h i l d r e n , n e o n a t a l c o m p l i c a t i o n s may be a v o i d e d by c a r e f u l management o f m a t e r n a l m a i n t e n a n c e . A c u t e w i t h d r a w a l o f t h e p r e g n a n t woman may p l a c e s t r e s s on t h e f e t u s as t h e f e t u s may w i t h d r a w s i m u l t a n e o u s l y . A l s o o p i a t e w i t h d r a w a l d u r i n g t h e f i r s t t r i m e s t e r may i n d u c e a b o r t i o n w h i l e d r u g w i t h d r a w a l d u r i n g t h e t h i r d t r i m e s t e r may i n d u c e p r e m a t u r e 4. l a b o u r ( F i n n e g a n , 1 9 7 8 ) . Flore i m p o r t a n t l y , o p i a t e s may a l s o e l i c i t l o n g t e r m , f a r -r e a c h i n g , s u b t l e e f f e c t s on o f f s p r i n g w h i c h have n o t p r e v i o u s l y been d e s c r i b e d . These e f f e c t s may be p r o d u c e d by LAAM o r one o r more o f i t s m e t a b o l i t e s . I t i s t h e r e f o r e o f i n t e r e s t t o examine t h e m e t a b o l i s m o f LAAM. LAAM METABOLISM I IM VIVO LAAM i s m e t a b o l i z e d i n v i v o t o a c t i v e m e t a b o l i t e s by t h e m i x e d f u n c t i o n o x i d a s e s y s t e m . LAAM i s N - d e m e t h y l a t e d f i r s t t o n o r a c e t y l m e t h a d o l (NAM) and t h e n t o d i n o r a c e t y l m e t h a d o l (NNAM) F i g . 1) (McMahon e_t a l ' . , 1 965; and B i l l i n g s e t a l . , 1 9 7 3 ) . Jjn v i v o t h e r a t e o f t h e f i r s t N - d e m e t h y l a t i o n i s a b o u t t h r e e t i m e s t h a t o f t h e s e c o n d (McMahon e_t al__., 1 965 ). The a f f e c t o f b o t h NAM and NNAM i s a b o u t 15 t i m e s t h a t o f LAAM f o r d e p r e s s i o n o f e l e c t r i c a l l y - i n d u c e d t w i t c h o f g u i n e a p i g i l e u m ( N i c k a n d e r e_t a l . , 1 9 7 4 ) . LAAM i s d i s t r i b u t e d t h r o u g h o u t t h e body (McMahon e_t a _ l . , 1 965; Sung and Way, 1 954) and i s e x c r e t e d m a i n l y i n t h e f e c e s , u r i n e and b i l e (McMahon e_t a _ l . , 1965; M i s r a and M u l e , 1 9 7 5 ) . B o t h o p t i c a l i s o m e r s o f LAAM p o s s e s s a n a l g e s i c p r o p e r t i e s ; however, o < - d - a c e t y l m e t h a d o l i s more 3 c o m p e t i t i v e t h a n < x - l - a c e t y l m e t h a d o l f o r b i n d i n g o f H - l i g a n d s t o o p i a t e r e c e p t o r s . However, t h e N - d e m e t h y l a t i o n o f o6-d-a c e t y l m e t h a d o l d e c r e a s e s i t s b i n d i n g a f f i n i t y w h i l e N - d e m e t h y l a -t i o n o f o e - l - a c e t y l m e t h a d o l i n c r e a s e s t h e b i n d i n g a f f i n i t y ( H o r n g e_t a l _ . , 1 9 7 6 ) . T h i s may e x p l a i n t h e s h o r t e r d u r a t i o n o f a c t i o n o f t h e d e x t r o r o t a t o r y f o r m . LAAM can a l s o undergo h y d r o l y s i s t o m e t h a d o l w h i c h can t h e n u n d e r g o an o x i d a t i o n t o 5. methadone, o r an I M - d e m e t h y l a t i o n t o n o r m e t h a d o l . Methadone i s t h e n N - d e m e t h y l a t e d and u n d e r g o e s i m m e d i a t e c y c l i z a t i o n t o 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l - 2 - e t h y l i d e n e - p y r r o l i d i n e ( F i g . 2 ) . I t s h o u l d a l s o be n o t e d t h a t i n , v i v o o p i a t e s may i n c r e a s e t h e r a t e o f t h e i r own m e t a b o l i s m . M a s t e n and c o - w o r k e r s (1978) have shown t h a t o r a l a d m i n i s t r a t i o n o f methadone t o mice r e s u l t e d i n an i n d u c t i o n o f h e p a t i c m i c r o s o m a l m i x e d f u n c t i o n o x i d a s e s y s t e m (FIFO). A t w o - f o l d i n c r e a s e i n t h e N - d e m e t h y l a s e a c t i v i t y was c o r r e l a t e d w i t h an i n c r e a s e i n m i c r o s o m a l p r o t e i n c o n c e n t r a t i o n and a d e c r e a s e i n p h e n o b a r b i t a l - i n d u c e d s l e e p t i m e . C h r o n i c o r a l d o s e s o f methadone have a l s o been shown t o s i g n i f i c a n t l y d e c r e a s e t h e h a l f l i f e o f t h e d r u g i n r a t s , f u r t h e r s u g g e s t i n g an i n c r e a s e d r a t e o f m e t a b o l i s m . However, c h r o n i c a d m i n i s t r a t i o n o f methadone and m o r p h i n e i n t r a p e r i t o n -e a l l y ( i . p . ) seems t o have no e f f e c t , ( A l v a r e s and Kappa, 1972; P e t e r s , 1973) o r t o c a u s e a d e c r e a s e ( C o c h i n and A x e l r o d , 1 9 5 9 ) i n h e p a t i c N - d e m e t h y l a s e a c t i v i t y . EFFECTS OF OPIATES ON METABOLISM IN VITRO Kato ert a l , . , (1 970a) have exam i n e d t h e e f f e c t s o f m o r p h i n e on h e p a t i c m i c r o s o m a l s t e r o i d m e t a b o l i s m . S i n c e o p i a t e s do a p p e a r t o a f f e c t t h e h e p a t i c MFO s y s t e m , t h e y may p r o d u c e i n d i r e c t e f f e c t s on t h e p r i m a r y a c t i v i t y o f t h e s y s t e m , ( e . g . m e t a b o l i s m o f s t e r o i d s and x e n o b i o t i c s u b s t r a t e s ) . These i n v e s t i g a t o r s r e p o r t t h a t m o r p h i n e a d m i n i s t r a t i o n , i . p . , r e s u l t s i n a s i g n i f i c a n t d e c r e a s e i n t h e c o n t e n t o f MFO c y t o c h r o m e B-450, t h e t e r m i n a l o x i d a s e o f t h e MFO s y s t e m , i n male b u t n o t f e m a l e 6. F i g u r e 1 . Pathway f o r t h e I_n V i v o N - d e m e t h y l a t i o n o f LAAM O-CO-Oi, C r u L A A M ^CHrCH-NC/ N - d e m e t h y l a t i o n O-CO-CH3 i n , ^ xx, o-ocx - d e m e t h y l a t i o n O OCXH3 rCH, N N A M 7. F i g u r e 2 . A l t e r n a t i v e I_n V i v o M e t a b o l i c Pathway f o r LAAM LAAM _| hydrolysis l,5-dimethyl-3,3-diphenyh 2-«thylidene-pyrrolidene a. r a t s . F u r t h e r m o r e , p r o g e s t e r o n e and t e s t o s t e r o n e h y d r o x y l a t i o n was d e c r e a s e d i n o p i a t e t r e a t e d m a l es b u t n o t f e m a l e s . Work i n t h i s l a b o r a t o r y has p r o d u c e d i n t e r e s t i n g r e s u l t s w i t h r e s p e c t t o i n d u c t i o n o f t h e h e p a t i c m i c r o s o m a l m i x e d f u n c t i o n o x i d a s e b e n z o ( a ) p y r e n e h y d r o x y l a s e (BP) f o r t h e m e t a b o l i s m o f b e n z o ( a ) p y r e n e . When r a t s were a d m i n i s t e r e d LAAM o r a l l y v i a d r i n k i n g w a t e r , h e p a t i c b e n z o ( a ) p y r e n e h y d r o x y l -ase a c t i v i t y was i n c r e a s e d i n f e m a l e s , whereas m a l e s e x h i b i t e d a s l i g h t i n h i b i t i o n o f a c t i v i t y ( G o n t o v n i c k and B e l l w a r d , m a n u s c r i p t i n p r e s s ) . C h r o n i c a d m i n i s t r a t i o n o f methadone, v i a d r i n k i n g w a t e r , p r o d u c e d r e s u l t s s i m i l a r t o t h a t s e e n w i t h LAAM. Female r a t s e x h i b i t e d an i n c r e a s e i n BP a c t i v i t y w i t h no change e x p r e s s e d by m a l e s ( G o n t o v n i c k e_t a _ l . , 1 9 7 9 ) . K a t o and c o - w o r k e r s (1970b) have r e p o r t e d t h a t c h r o n i c i n t r a p e r i t o n -e a l a d m i n i s t r a t i o n o f m o r p h i n e p r o d u c e d a s i g n i f i c a n t d e c r e a s e i n t h e N - d e m e t h y l a t i o n o f a m i n o p y r i n e i n male but n o t f e m a l e r a t s . M a s r y e_t a _ l . , (1 974 ) have documented b o t h t e m p o r a l and sex r e l a t e d d i f f e r e n c e s i n t h e m e t a b o l i s m o f v a r i o u s s u b s t r a t e s . U s i n g h e p a t i c p r e p a r a t i o n s f r o m r a t s r a n g i n g i n d e v e l o p m e n t f r o m p r e p u b e r t a l t o a d u l t a n i m a l s i t was shown t h a t e n z y m a t i c N - d e m e t h y l a s e a c t i v i t i e s o f t h e t y p e I b i n d i n g s u b s t r a t e s , ( A m a x 3B3-390nm and A m i n 41B-427nm by d i f f e r e n c e s p e c t r a a n a l y s i s ) , e t h y l m o r p h i n e and a m i n o p y r i n e were i n c r e a s e d i n m a l e s r e l a t i v e t o f e m a l e s as a f u n c t i o n o f age. On t h e o t h e r hand, 9. t h e h y d r o x y l a s e a c t i v i t y o f t h e t y p e I I b i n d i n g c o m p o u n d , ( Amax 4 2 5 - 4 3 5 n m a n d Tvmin 3 9 0 - 4 0 5 n m b y d i f f e r e n c e s p e c t r a a n a l y s i s ) , a n i l i n e was f o u n d t o d e c r e a s e w i t h a g e w i t h no s e x d i f f e r e n c e s . T h i s g r o u p h a s a l s o n o t e d t h a t a s t h e a g e o f t h e m a l e i n c r e a s e s , t h e Km f o r t h e h e p a t i c m i c r o s o m a l N - d e m e t h y l a -t i o n o f e t h y l m o r p h i n e . d e c r e a s e s , w h e r e a s t h e Km i n t h e f e m a l e d o e s n o t c h a n g e . I n 1 9 7 6 D e s c h ( 1 9 7 6 ) r e p o r t e d t h a t no d i f f e r e n c e i n BP a c t i v i t y was p r e s e n t up t o d a y 5 o f l i f e b e t w e e n m a l e a n d f e m a l e r a t s . H o w e v e r , a f t e r d a y 90 o f l i f e BP a c t i v i t i e s o f m a l e s w e r e i n c r e a s e d - r e l a t i v e t o f e m a l e s . B A S I S FOR SEX DEPENDENT D I F F E R E N C E S , S e x a n d t e m p o r a l d i f f e r e n c e s h a v e b e e n a t t r i b u t e d t o h o r m o n a l f a c t o r s . M a s r y a n d M a n n e r i n g ( 1 9 7 4 ) h a v e d e m o n s t r a t e d t h a t c a s t r a t i o n o r e s t r o g e n t r e a t m e n t o f a d u l t m a l e r a t s w i l l d e c r e a s e e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y a n d c y t o c h r o m e P -450 l e v e l s a n d i n c r e a s e t h e Km t o w a r d s f e m a l e v a l u e s . A d m i n i s t r a t i o n o f t e s t o s t e r o n e t o c a s t r a t e d m a l e s a n d n o r m a l f e m a l e s r e s u l t e d i n a n i n c r e a s e i n e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y a n d a d e c r e a s e i n t h e Km t o w a r d s t h o s e o f a d u l t m a l e v a l u e s ( M a s r y a n d M a n n e r i n g , 1 9 7 4 b ) . I n c o n j u n c t i o n w i t h t h i s , i t h a s b e e n w e l l d o c u m e n t e d t h a t o p i a t e s r e d u c e p l a s m a t e s t o s t e r o n e l e v e l s ( A z i z i e_t al. , 1 9 7 3 ; M e n d e l s o n e_t a _ l . , 1 9 7 5 ; M e n d e l s o n e_t a _ l . , 1 9 7 6 ; C i c e r o e_t a l . , 1 9 7 4 ) , i n human a d u l t m a l e s . I t was a l s o n o t e d t h a t m o r p h i n e d e c r e a s e d t h e w e i g h t s o f t h e p r o s t a t e g l a n d s a n d s e m i n a l v e s i c l e s •1.0. and d e c r e a s e d t h e s e c r e t o r y a c t i v i t y o f t h e s e m i n a l v e s i c l e s i n r a t s ( C i c e r o , e t a l . , 1 974; 51adek e_t a l . , 1 9 7 4 ) . S l a d e k and c o - w o r k e r s (1974) have d e m o n s t r a t e d t h e e f f e c t s o f m o r p h i n e on immature and m a t u r e male and f e m a l e r a t s . These s t u d i e s have shown t h a t m o r p h i n e d e c r e a s e d t h e m e t a b o l i s m o f t h e t y p e I compounds e t h y l m o r p h i n e and h e x o b a r b i t a l and t h e c y t o c h r o m e P-450 l e v e l s - i n human a d u l t m a l e s o n l y . The a d m i n i s t r a t i o n o f m o r p h i n e had no e f f e c t i n a d u l t f e m a l e o r immature male and f e m a l e r a t s . M o r p h i n e was shown t o have no e f f e c t on t h e m e t a b o l i s m o f a n i l i n e under any o f t h e s e c o n d i t i o n s . I t was f u r t h e r shown t h a t when a d u l t f e m a l e r a t s were t r e a t e d w i t h t e s t o s t e r o n e t h e m e t a b o l i s m o f e t h y l m o r p h i n e and h e x o b a r b i t a l i n c r e a s e d . W i t h t h e s u b s e q u e n t a d d i t i o n o f m o r p h i n e , however, t h i s i n c r e a s e was r e v e r s e d . T h i s t r e a t m e n t had no e f f e c t on a n i l i n e h y d r o x y l a s e a c t i v i t y ( M a s r y and Mannering,- 1 9 7 4 ) . K a t o e_t a _ l . , (1 970a) have d e m o n s t r a t e d t h a t a d m i n i s t r a t i o n o f m o r p h i n e w i l l r e s u l t i n . a s i g n i f i c a n t d e c r e a s e i n t h e h y d r o x y l -a t i o n o f t e s t o s t e r o n e and p r o g e s t e r o n e as w e l l as t h e c y t o c h r o m e P-450 c o n t e n t i n male r a t s b u t n o t i n f e m a l e r a t s . F u r t h e r m o r e , K a t o e_t a l _ . , (1971) have shown t h a t a d m i n i s t r a t i o n o f m o r p h i n e i . p , d e c r e a s e d t h e h y d r o x y l a t i o n o f h e x o b a r b i t a l i n t h e male r a t t o t h a t o f t h e f e m a l e v a l u e , w hereas m o r p h i n e was w i t h o u t e f f e c t on h e x o b a r b i t a l m e t a b o l i s m i n t h e f e m a l e r a t . R e s u l t s f r o m o u r l a b o r a t o r y have d e m o n s t r a t e d t h a t h e p a t i c b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y was r e d u c e d by c a s t r a t i o n o f male r a t s w hereas o v a r i e c t o m y had no e f f e c t on t h e f e m a l e l e v e l s •-. 11. ( G o n t o v n i c k e_t a _ l . , 1 9 7 9 ) . A d m i n i s t r a t i o n o f methadone t o s h a m - o p e r a t e d m a l e s d i d n o t s i g n i f i c a n t l y i n c r e a s e b e n z o ( a ) - -p y r e n e h y d r o x y l a s e a c t i v i t y , b u t a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y was s e e n when methadone was g i v e n t o s h a m - o p e r a t e d f e m a l e s , o v a r i e c t o m i z e d f e m a l e s and c a s t r a t e d m a l e s ( G o n t o v n i c k e t a l . , 1 9 7 9 ) . These s e x and age d e p e n d e n t d i f f e r e n c e s have a l s o been d e s c r i b e d f o r t h e m e t a b o l i s m o f s t e r o i d s u b s t r a t e s i n t h e r a t . E i n a r s s o n e_t a _ l . , (1 973) have shown, f o r e x a m p l e , t h a t male r a t s e x h i b i t a g r e a t e r a c t i v i t y f o r t h e 2 / 5 - h y d r o x y l a t i o n o f 5oC-androstane-3<*, 1 7 / ^ - d i o l , 6 / C ? - h y d r o x y l a t i o n o f 4 - a n d r o s t a n e - 3 , 1 7 - d i o n e and 4 - p r e g n e n e - 3 , 2 0 - d i o n e , and 1 B - h y d r o x y l a t i o n o f 5o< - a n d r o s t a n e - 3 oc f 1 ~]^3 - d i o l . They a l s o r e p o r t e d t h a t c a s t r a t i o n o f t h e male w o u l d c o m p l e t e l y a b o l i s h t h e s e s e x d i f f e r e n c e s , and t h a t a d m i n i s t r a t i o n o f t e s t o s t e r o n e w o u l d s t i m u l a t e enzymes o f t h i s g r o u p . E i n a r s s o n and c o - w o r k e r s (1973) a l s o s t u d i e d a s e c o n d group o f h e p a t i c m i c r o s o m a l s t e r o i d a c t i v i t i e s . These were t h e 2 o c -h y d r o x y l a t i o n o f 4-pr e g n e n e - 3 , 2 0 - d i o n e , and 5 c ^ - a n d r o s t a n e - 3 cX . 1 7 / f - d i o l , t h e 16°<-hydroxylation o f 4 - a n d r o s t e n e - 3 , 1 7 - d i o n e , 1 6 t h e /\ ~^19 s ^ e r ° i c ' e p o x i d a t i o n o f 4, 1 6 - a n d r o s t a d i e n - 3 - o n e , and t h e 1 7 / 5 - h y d r o x y s t e r o i d r e d u c t i o n o f 4 - a n d r o s t e n e - 3 , 1 7 - d i o n e . These a c t i v i t i e s were g e n e r a l l y g r e a t e r i n male t h a n f e m a l e r a t s . O n l y c a s t r a t i o n o f t h e n e o n a t e w o u l d remoye t h e s e x d i f f e r e n c e s i n t h e s e a c t i v i t i e s . P o s t p u b e r t a l c a s t r a t i o n r e s u l t e d i n a p a r t i a l d e c r e a s e t o w a r d s t h a t o f t h e f e m a l e v a l u e . However, 12. w i t h r e s p e c t t o t h e f i r s t g r o up o'f enzymes, c a s t r a t i o n o f e i t h e r t h e n e o n a t a l o r p o s t p u b e r t a l male was e q u a l l y e f f e c t i v e . ANDROGEN INDUCED IMPRINTING Chung e_t a_l . , (1 975 ) have r e p o r t e d t h a t male r a t s c a s t r a t e d a t b i r t h w i l l r e s p o n d l e s s t o a n d r o g e n s t i m u l a t i o n as a d u l t s , (as measured by t h e m e t a b o l i s m o f a m i n o p y r e n e , e t h y l m o r p h i n e and h e x o b a r b i t a l ) t h a n i f e x p o s e d t o a n d r o g e n s as a n e o n a t e . F u r t h e r m o r e , t h e Km f o r t h e N - d e m e t h y l a t i o n o f e t h y l m o r p h i n e was compared f o r v a r i o u s g o n a d a l s t a t e s . In t h e same s t u d y Chung n o t e d t h a t t h e Km f o r a d u l t m a l e s , c a s t r a t e d a t b i r t h , was s i m i l a r t o t h a t o f n o r m a l a d u l t f e m a l e s . I t was a l s o r e p o r t e d t h a t a d u l t m a l e s c a s t r a t e d a t b i r t h and n e o n a t a l l y t r e a t e d w i t h a n d r o g e n s were s i m i l a r t o a d u l t m a l e s c a s t r a t e d a t day 20 o f l i f e . The Km r e p o r t e d f o r t h e s e l a t t e r two gr o u p s was e q u a t e d t o t h o s e f o r n o r m a l a d u l t m a l e s , and t o a d u l t m a l es c a s t r a t e d a t b i r t h and p o s t p u b e r t a l l y t r e a t e d w i t h a n d r o g e n s . Denef and DeMoor (1972) have shown t h a t c a s t r a t i o n o f male r a t s a t b i r t h w i l l remove t h e s e x u a l d i f f e r e n t i a t i o n of C o r t i s o l m e t a b o l i s m . However, i f t h e male was c a s t r a t e d on day 10 o f l i f e , t h e m e t a b o l i s m o f C o r t i s o l d i f f e r e n t i a t e d n o r m a l l y . These i n v e s t i g a t o r s have a l s o d e m o n s t r a t e d t h a t a s i n g l e a d m i n i s t r a t i o n o f t e s t o s t e r o n e on day 1 o f l i f e t o t h e c a s t r a t e d n e o n a t e , male o r f e m a l e , w o u l d r e s u l t i n a m a s c u l i n i z a t i o n o f C o r t i s o l m e t a b o l i s m i n t h e a d u l t a n i m a l (Denef and DeMoor, 1 9 6 8 ) . F u r t h e r e v i d e n c e t h a t t h i s s e x u a l d i f f e r e n t i a t i o n t o a c h a r a c t e r -i s t i c m a s c u l i n e t y p e o f m e t a b o l i s m was due t o androgen, e x p o s u r e 13. o f t h e n e o n a t e s , was d e m o n s t r a t e d u s i n g t h e a n d r o g e n a n t a g o n i s t c y p r o t e r o n e a c e t a t e . C y p r o t e r o n e was a b l e t o p r e v e n t t h e m a s c u l i n i z i n g e f f e c t s o f t e s t o s t e r o n e i n t h e f e m a l e r a t and i n h i b i t t h e n o r m a l d i f f e r e n t i a t e d m e t a b o l i c p a t t e r n i n t h e male (Denef and DeMoor, 1 9 7 2 ) . From t h e s e s t u d i e s i t was c o n c l u d e d t h a t t h e e a r l i e r t h e a n i m a l i s i s o l a t e d f r o m a n d r o g e n expos.ure t h e more - f e m i n i z e d t h e e n z y m a t i c p a t t e r n w i l l be. F u r t h e r m o r e , i t was s u g g e s t e d t h a t i n i t i a l l y t h e mammalian l i v e r i s f e m i n i n e and t h a t t h e c h a r a c t e r i s t i c male e n z y m a t i c r e s p o n s e s f i r s t s e e n a f t e r p u b e r t y a r e p r e d e t e r m i n e d by some, t e s t i c u l a r f a c t o r n e o n a t a l l y (DeMoor and Denef, 1968; G u s t a f s s o n and S t e n b e r g , 1 9 7 4 a ) . The e f f e c t o f a n d r o g e n e x p o s u r e d u r i n g t h e n e o n a t a l p e r i o d r e s u l t i n g i n d i f f e r e n t i a t i o n a t m a t u r i t y i s r e f e r r e d t o as n e o n a t a l a n d r o g e n i n d u c e d i m p r i n t i n g ( E i n a r s s o n e_t a l . , 1 973; Chung, 1 9 7 4 ) . G u s t a f s s o n and S t e n b e r g (1974a) have shown t h a t 1 7 - d i o n e f o r e x a m p l e ) , t h i s i m p r i n t i n g i s i r r e v e r s i b l e . T h a t i s , m a l e s c a s t r a t e d n e o n a t a l l y do n o t r e s p o n d t o t e s t o s t e r o n e t r e a t m e n t as a d u l t s . U s i n g o t h e r s t e r o i d s u b s t r a t e s t h e s e i n v e s t i g a t o r s have a l s o shown t h a t m a l e s c a s t r a t e d a t b i r t h r e s p o n d e d l e s s t o t e s t o s t e r o n e s t i m u l a t i o n as a d u l t s t h a n d i d a n i m a l s c a s t r a t e d a t day 14 o f l i f e f u r t h e r i l l u s t r a t i n g t h e i r r e v e r s i b l e n a t u r e o f n e o n a t a l i m p r i n t i n g . T h i s s t u d y s u g g e s t s t h a t a c r i t i c a l t i m e f o r i m p r i n t i n g may e x i s t . Chung (1974) has d e t e r m i n e d t h i s c r i t i c a l t i m e f o r t h e i m p r i n t i n g o f two s e x f o r c e r t a i n enzymes, 14. d e p e n d e n t c h a r a c t e r i s t i c s o f e t h y l m o r p h i n e N - d e m e t h y l a s e , t h e Km and Vmax, as 2 t o 4 days and 12 t o 14 days r e s p e c t i v e l y . T h i s may c o i n c i d e w i t h t h e e x p r e s s i o n o f t e s t o s t e r o n e f r o m t h e L e y d i g c e l l s i n t h e t e s t i s p r i o r t o t h e i r a c t i v a t i o n d u r i n g p u b e r t y . MAINTENANCE OF IMPRINTING Ther e a r e many f a c t o r s t h a t a p p e a r t o a f f e c t t h e m a i n -t e n a n c e o f a n d r o g e n i n d u c e d i m p r i n t i n g i n t h e a d u l t , and a l l a r e a s s o c i a t e d w i t h e n d o c r i n e f u n c t i o n . K a t o e_t_ a l . , (1 965; 1971); have r e p o r t e d t h a t a d r e n a l e c t o m y w i l l d e c r e a s e t h e h e p a t i c m i c r o s o m a l m e t a b o l i s m o f h e x o b a r b i t a l and a m i n o p y r i n e , b u t n o t a n i l i n e i n male r a t s . A d r e n a l e c t o m y was shown t o have no e f f e c t on t h e s e p a r a m e t e r s i n f e m a l e r a t s . The p i t u i t a r y hormones a d r e n o c o r t i c o t r o p i c hormone (ACTH) and g r o w t h hormone (GH) have been shown t o e l i c i t s i m i l a r e f f e c t s ( K a t o e_t a l _ . , 1 965j<-. W i l s o n , 1 9 7 0 ) . A d r e n a l e c t o m y , however, d i d not a p p e a r t o a f f e c t t h e a p p a r e n t Km o f e t h y l m o r p h i n e N - d e m e t h y l a s e i n a d u l t male r a t s , n or was t h e Km a f f e c t e d by c a s t r a t i o n o r t h y r o p a r a t h y r o i d -ectomy (Chung, 1 9 7 4 ) . Chung has f u r t h e r r e p o r t e d t h a t f o r male r a t s , o n l y h y p o p h y s e c t o m y p r o d u c e d a s i g n i f i c a n t change i n t h e Km o f e t h y l m o r p h i n e N - d e m e t h y l a t i o n , w h i l e f e m a l e s were n o t a f f e c t e d . T h i s i s i n c o n t r a s t t o M a s r y and c o - w o r k e r s (1974a) who r e p o r t e d t h a t t h e Km was i n c r e a s e d by c a s t r a t i o n . C o l b y e_t a_l.,(1 973) have d e m o n s t r a t e d t h a t f o l l o w i n g t h e h y p o p h y s e c t o m y o f c a s t r a t e d a d u l t male r a t s t h e h e p a t i c m e t a b o l i s m o f c o r t i c o s t e r o n e f a i l e d t o r e s p o n d t o s t i m u l a t i o n by t e s t o s t e r o n e . A l s o Kramer e_t a_ l . , (1 975 ) have d e m o n s t r a t e d 15. t h a t - t h e e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y c o u l d n o t be r e s t o r e d by t e s t o s t e r o n e a d m i n i s t r a t i o n t o c a s t r a t e d hypo-p h y s e c t o m i z e d r a t s o f e i t h e r s e x . G u s t a f s s o n and S t e n b e r g (1974b) have a l s o shown t h a t t h e m a s c u l i n i z i n g e f f e c t s o f t e s t o s t e r o n e on h e p a t i c s t e r o i d m e t a b o l i s m c o u l d n o t be d e m o n s t r a t e d i n h y p o p h y s e c t o m i z e d r a t s . F u r t h e r m o r e , hypo-p h y s e c t o m y has been d e s c r i b e d as l e a d i n g t o an o v e r a l l m a s c u l i n -i z a t i o n o f m e t a b o l i s m i n f e m a l e r a t s , and a f e m i n i z a t i o n o f h e p a t i c s t e r o i d m e t a b o l i s m i n m a l e s ( G u s t a f s s o n and S t e n b e r g , 1 9 7 4 b ) . T h i s i n d i c a t e s t h e p o s s i b l e e x i s t e n c e o f h y p o p h y s e a l f a c t o r ( s ) n e c e s s a r y f o r t h e e x p r e s s i o n o f t e s t o s t e r o n e on h e p a t i c m e t a b o l i s m . G u s t a f s s o n and S t e n b e r g (1976a) e x a m i n e d t h e e f f e c t s o f autonomous p i t u i t a r y i m p l a n t s on h e p a t i c s t e r o i d m e t a b o l i s m . P i t u i t a r i e s f r o m f e m a l e r a t s i m p l a n t e d u n d e r t h e r e n a l c a p s u l e i n c a s t r a t e d h y p o p h y s e c t o m i z e d m a l e s p r o d u c e d f e m i n i z a t i o n o f m e t a b o l i s m w h i c h was n o n r e s p o n s i v e t o t e s t o s t e r o n e a d m i n i s t r a -t i o n . Denef (1974) has shown t h a t h y p o p h y s e c t o m y w i l l remove t h e s e x u a l d i f f e r e n t i a t i o n o f t e s t o s t e r o n e m e t a b o l i s m i n t h e r a t and t h a t i m p l a n t a t i o n o f autonomous p i t u i t a r i e s , f r o m male o r f e m a l e d o n o r s , w o u l d f e m i n i z e h e p a t i c t e s t o s t e r o n e m e t a b o l i s m o f g o n a d e c t o m i z e d h y p o p h y s e c t o m i z e d male r a t s . B u r k e and c o -w o r k e r s (1978) s t u d i e d t h e e f f e c t o f a d u l t male and f e m a l e p i t u i t a r y i m p l a n t s i n p r e p u b e r t a l male r a t s . S i n c e t h e r e c i p i e n t s o f t h e i m p l a n t s were n o t s e x u a l l y d i f f e r e n t i a t e d w i t h r e s p e c t t o m e t a b o l i s m a t t h e t i m e o f i m p l a n t a t i o n i t was n o t 16. r e q u i r e d t h e y b e . h y p o p h y s e c t o m i z e d . The r e s u l t s i n d i c a t e d t h a t t h e r e were e s s e n t i a l l y no d i f f e r e n c e s between t h e male and f e m a l e p i t u i t a r y i m p l a n t . R e g a r d l e s s o f t h e s e x u a l o r i g i n o f t h e i m p l a n t a f e m i n i z a t i o n o f h e p a t i c x e n o b i o t i c m e t a b o l i s m r e s u l t e d . E x p e r i m e n t a l e v i d e n c e t o t h i s p o i n t s u g g e s t s t h a t a f e m i n i z i n g f a c t o r i s r e l e a s e d f r o m t h e p i t u i t a r y i n t h e f e m a l e r a t and t h a t , i n t h e m a l e , t h i s r e l e a s e i s i n h i b i t e d by hypo-t h a l a m i c r e l e a s e i n h i b i t o r y f a c t o r ( s ) . T h a t t h e f e m i n i z i n g f a c t o r i s one o t h e r t h a n p r i m a r y p i t u i t a r y hormones was shown by G u s t a f s s o n e_t a _ l . , (1 9 7 5 a ) . H y p o p h y s e a l e x t r a c t s f r o m a d u l t male and f e m a l e r a t s were a d m i n i s t e r e d t o hepatoma c e l l t i s s u e c u l t u r e s . The f e m a l e e x t r a c t was shown 'to f e m i n i z e t h e g o n a d a l l y r e g u l a t e d i m p r i n t e d s t e r o i d m e t a b o l i z i n g enzyme 5oc r e d u c t a s e , w h e r e a s male e x t r a c t had no e f f e c t . C o n c u r r e n t a n a l y s i s o f o t h e r p u r i f i e d p i t u i t a r y hormones, l u t e i n i z i n g hormone (LH) and p r o l a c t i n , f a i l e d t o p r o d u c e a s i g n i f i c a n t i n c r e a s e i n 5 « r e d u c t a s e a c t i v i t y when a d m i n i s t e r e d t o hepatoma t i s s u e c e l l c u l t u r e i n p h y s i o l o g i c a l c o n c e n t r a t i o n . However, when t h y r o i d s t i m u l a t i n g hormone and f o l l i c l e s t i m u l a t i n g hormone (F5H) were i n t r o d u c e d , a n e a r t w o - f o l d i n c r e a s e i n a c t i v i t y w-as n o t e d b u t n o t d i s c u s s e d 'fry t h e s e i n v e s t i g a t o r s . K ramer's group (1977) have shown t h a t t h e a d m i n i s t r a t i o n o f e i t h e r LH sor F5H t o c a s t r a t e d male r a t s w i l l r e s u l t i n an i n c r e a s e i n t h e r a t e o f e t h y l m o r p h i n e N - d e m e t h y l a t i o n but t h e r a t e s o f a m i n o p y r i n e and a n i l i n e m e t a b o l i s m were n o t c h a n g e d . 17. In f e m a l e s , however, F5H i n c r e a s e d e t h y l m o r p h i n e and amino-p y r i n e m e t a b o l i s m w h e r e a s LH o n l y i n c r e a s e d t h e m e t a b o l i s m o f e t h y l m o r p h i n e . N e i t h e r F5H o r LH a f f e c t e d t h e m e t a b o l i c r a t e o f a n i l i n e m e t a b o l i s m i n f e m a l e s . Kramer s u g g e s t e d t h a t LH and FSH have t h e s e e f f e c t s on h e p a t i c m i x e d f u n c t i o n o x i d a s e s i n d e p e n d e n t o f g o n a d a l hormones. I t has a l s o been r e p o r t e d t h a t FSH a c t s t o i n c r e a s e t h e a c t i v i t y o f a n d r o g e n - d e p e n d e n t enzymes and n o t a n d r o g e n - i n d e p e n d e n t enzymes ( G u s t a f s s o n and S t e n b e r g , 1 9 7 5 b ) . T h i s i s c o n s i s t e n t w i t h f i n d i n g s t h a t e t h y l m o r p h i n e and a m i n o p y r i n e m e t a b o l i s m a r e i n c r e a s e d by t e s t o s t e r o n e whereas h y d r o x y l a t i o n o f a n i l i n e i s i n d e p e n d e n t o f i t s e f f e c t s as d i s c u s s e d e a r l i e r . To d e t e r m i n e w h e t h e r t h e h y p o t h a l a m u s was u l t i m a t e l y r e s p o n s -i b l e f o r r e g u l a t i o n o f t h e s e c r e t i o n o f f e m i n i z i n g f a c t o r f r o m t h e p i t u i t a r y , G u s t a f s s o n e_t a _ l . , (1 976b) p r o d u c e d e l e c t r o l y t i c l e s i o n s i n v a r i o u s a r e a s o f t h e h y p o t h a l a m u s and e x a m i n e d t h e e f f e c t s . The r e s u l t was an o v e r a l l f e m i n i z a t i o n o f male m e t a b o l -i s m and no s i g n i f i c a n t e f f e c t s on f e m a l e s . I t was p o s t u l a t e d t h e r e f o r e , t h a t t h e f e m i n i z i n g f a c t o r s e c r e t e d by t h e f e m a l e p i t u i t a r y , i s i n h i b i t e d by a h y p o t h a l a m i c r e l e a s e i n h i b i t o r y f a c t o r i n t h e m a l e . T h i s i n h i b i t o r y f a c t o r , w h i c h i s n o t p r e s e n t i n t h e f e m a l e , may be a r e s u l t o f n e o n a t a l t e s t i c u l a r a n d r o g e n - i m p r i n t i n g o f t h e male h y p o t h a l a m u s c a u s i n g i t t o d i f f e r e n t i a t e a t p u b e r t y and e x p r e s s a m a s c u l i n e m e t a b o l i c p a t t e r n . 1 8 . S U M M A R Y I t has been e s t a b l i s h e d t h a t sex d i f f e r e n c e s e x i s t f o r t h e h e p a t i c m i c r o s o m a l m e t a b o l i s m o f v a r i o u s x e n o b i o t i c s and s t e r o i d s i n t h e r a t . T h i s d i f f e r e n t i a t i o n o f m e t a b o l i s m i s due t o a n e o n a t a l t e s t i c u l a r a n d r o g e n i c f a c t o r a f f e c t i n g t h e male t o d e v e l o p a c h a r a c t e r i s t i c m e t a b o l i c p a t t e r n p o s t - p u b e r t a l l y . T h i s p r o c e s s has been t e r m e d a n d r o g e n - i n d u c e d i m p r i n t i n g . The h y p o p h y s e a l - h y p o t h a l a m i c a x i s i s n e c e s s a r y f o r t h e m a i n t e n a n c e o f a n d r o g e n i m p r i n t i n g and t h e d i f f e r e n t i a t e d m e t a b o l i s m t h a t f o l l o w s . Removal o f a n d r o g e n e x p o s u r e w i l l a b o l i s h d i f f e r e n t i a -t i o n a t p u b e r t y as d e m o n s t r a t e d by c a s t r a t i o n and c y p r o t e r o n e a c e t a t e s t u d i e s . In c e r t a i n c a s e s t h e r e m o v a l o f a n d r o g e n s f r o m t h e n e o n a t e w i l l p r o d u c e i r r e v e r s i b l e e f f e c t s on m e t a b o l i s m t h a t l e a d t o a l a c k o f r e s p o n s e i n t h e a d u l t male f o l l o w i n g t e s t o s -t e r o n e s t i m u l a t i o n a t m a t u r i t y . R e s t o r a t i o n o f m e t a b o l i c d i f f e r e n t i a t i o n may be a c c o m p l i s h e d by t e s t o s t e r o n e a d m i n i s t r a -t i o n a t t h e c r u c i a l n e o n a t a l t i m e p e r i o d . The a d u l t a n i m a l may r e s p o n d t o t e s t o s t e r o n e by e x h i b i t i n g a male m e t a b o l i c p a t t e r n i n some c a s e s ; i r r e v e r s i b l e l o s s o f o r d e c r e a s e i n d i f f e r e n t i a -t i o n can o c c u r i n o t h e r i n s t a n c e s . The f a c t t h a t o p i a t e s a r e c a p a b l e o f c r o s s i n g t h e p l a c e n t a l b a r r i e r , and a r e f o u n d i n m i l k , r e s u l t s i n n e o n a t e s e x h i b i t i n g w i t h d r a w a l symptoms. In a d d i t i o n o p i a t e s a r e known t o r e d u c e p l a s m a t e s t o s t e r o n e l e v e l s . I t i s t h e r e f o r e p o s s i b l e t h a t c h r o n i c o p i a t e t h e r a p y d u r i n g p r e g n a n c y and l a c t a t i o n may a f f e c t a n d r o g e n - i n d u c e d i m p r i n t i n g s i m i l a r t o t h a t s e e n w i t h c a s t r a t i o n 1 9 . and a n t i a n d r o g e n t r e a t m e n t . The l o n g e r a c t i n g o p i a t e s p r e s e n t l y a v a i l a b l e have p r o m i s e i n t h e m a i n t e n a n c e o f h e r o i n a d d i c t s . The use o f t h e s e o p i a t e s by f e m a l e a d d i c t s , however, may p r e s e n t a p r o b l e m . S i n c e many women on m a i n t e n a n c e programs a r e o f c h i l d b e a r i n g age i t i s o f o b v i o u s i n t e r e s t b o t h s c i e n t i f i c a l l y and c l i n i c a l l y t o d e t e r m i n e t h e p o s s i b l e ' e f f e c t s o f LAAM on a n d r o g e n - i n d u c e d i m p r i n t i n g . We p r o p o s e t h e r e f o r e t h a t i n . u t e r o and n e o n a t a l e x p o s u r e t o t h e o p i a t e LAAM may r e s u l t i n an i n h i b i t i o n o f i m p r i n t i n g , s i m i l a r t o t h a t s e e n by c a s t r a t i o n , as e x p r e s s e d by a d e c r e a s e i n s e x d e p e n d e n t enzyme a c t i v i t i e s o f t h e a d u l t male r a t . To t e s t t h i s h y p o t h e s i s t h e k i n e t i c p a r a m e t e r s ; a p p a r e n t Km, Vmax, and b a s a l s p e c i f i c a c t i v i t y w i l l be m o n i t o r e d f o r t h e h e p a t i c m i c r o s o m a l c y t o c h r o m e P-450 s e x d e p e n d e n t a c t i v i t i e s : a m i n o -p y r i n e N - d e m e t h y l a s e ( A P ) , e t h y l m o r p h i n e N - d e m e t h y l a s e ( E T ) , and b e n z o ( a ) p y r e n e h y d r o x y l a s e ( B P ) . These a c t i v i t i e s w i l l be compared w i t h t h a t o f a n i l i n e h y d r o x y l a s e .(AN) w h i c h as p r e v i o u s -l y m e n t i o n e d i s n o t s e x d e p e n d e n t , and w i l l s e r v e as a c o n t r o l . I f LAAM i n g e s t e d by t h e dam d e c r e a s e s f e t a l and n e o n a t a l a n d r o g e n s s u f f i c i e n t l y t o i n h i b i t i m p r i n t i n g i t w o u l d r e s u l t i n one o r more o f t h e f o l l o w i n g : a d e c r e a s e i n t h e s e x d e p e n d e n t a c t i v i t i e s , a d e c r e a s e i n Vmax and an i n c r e a s e i n Km i n t h e male a t m a t u r i t y w i t h no change i n a n i l i n e h y d r o x y l a s e l e v e l s . Enzyma-t i c p a r a m e t e r s r e s u l t i n g f r o m h e p a t i c m i c r o s o m a l f r a c t i o n o f f e m a l e r a t s a r e n o t e x p e c t e d t o show any d e v i a t i o n f r o m c o n t r o l . On t h e o t h e r hand, i f no i r r e v e r s i b l e e f f e c t s on i m p r i n t i n g o c c u r , t h e a c t i v i t i e s o f t h e s e x d e p e n d e n t enzymes i n t h e a d u l t male w i l l be s i g n i f i c a n t l y h i g h e r t h a n f e m a l e s and n o t d i f f e r e n t f r o m c o n t r o l . 21 MATERIALS AND METHODS  CHEMICALS AND REAGENTS A c e t y l a c e t o n e , b e n z o ( a ) p y r e n e (BP) ( r e c r y s t a l l i z e d f r o m m e t h a n o l f o l l o w i n g d i s s o l u t i o n i n benzene .)f b o v i n e serum a l b u m i n , c a l c i u m a c e t a t e , g l u c o s e - 6 - p h o s p h a t e ( G 6 P ) , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (G6PD)., 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 ( r e d u c e d f o r m ) (NADH), 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 ( r e d u c e d f o r m ) (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 (NADP), q u i n i n e s u l f a t e , s e m i c a r b a z i d e HC1, and t r i s m a base were o b t a i n e d f r o m Sigma C h e m i c a l Co. ( S t . L o u i s , Mo.). T r i c h o l o r a c e t i c a c i d (TCA) was o b t a i n e d f r o m J.T. B a k e r C h e m i c a l Co. ( P h i l l i p s b u r g , l \ l . J . ) . l - 0 6 - A c e t y l m e t h a d o l - H C l l e v o r o t a t o r y (LAAM) was o b t a i n e d f r o m M a l l i n c k r o d t C h e m i c a l Works ( S t . L o u i s , Mo.) as was ammonium a c e t a t e , f o r m a l d e h y d e , ' s o d i u m h y d r o x i d e and s o d i u m p h o s p h a t e d i b a s i c . A n i l i n e HC1, and p a r a - a m i n o p h e n o l HC1 were o b t a i n e d f r o m Eastman Kodak Co. ( R o c h e s t e r , N.Y.). A m i n o p y r i n e was o b t a i n e d f r o m M a t h e s o n , Coleman and B e l l ( C i n c i n n a t i , O h i o ) . E t h y l m o r p h i n e HC1, magnesium c h l o r i d e , p o t a s s i u m c h l o r i d e and z i n c s u l f a t e were s u p p l i e d by B r i t i s h Drug House Pharma-c e u t i c a l s ( T o r o n t o , O n t . ) . Sodium . c a r b o n a t e , p h e n o l r e a g e n t , p o t a s s i u m p h o s p h a t e m o n o b a s i c were s u p p l i e d by F i s h e r S c i e n t i f i c Co. ( F a i r Lawn, N. J . ) . A l l o t h e r c h e m i c a l s were o f t h e b e s t r e a g e n t g r a d e a v a i l a b l e . 22. ANIMALS W i s t a r r a t s were o b t a i n e d f r o m C a n a d i a n B r e e d i n g Farms ( M o n t r e a l , Q u e b e c ) . A l l a n i m a l s were i s o l a t e d i n a s e p a r a t e (R) a n i m a l room on Lobund b e d d i n g ( P a x t o n P r o c e s s i n g L t d . P a x t o n , -1 1 1 . ) , and a l l o w e d t o e q u i l i b r a t e f o r 5-7 days un d e r c o n t r o l l e d t e m p e r a t u r e (22°C) and l i g h t i n g (on 06Q0-20DQ h r s . ) . I n d i v i d u a l m a l e s (200-22Dg) were p a i r e d o v e r n i g h t w i t h two f e m a l e s (18D-200g).. The f o l l o w i n g m o r n i n g v a g i n a l smears were e x a m i n e d by l i g h t m i c r o s c o p y t o d e t e r m i n e i f c o p u l a t i o n had o c c u r r e d . Those f e m a l e s t e s t i n g p o s i t i v e were s e p a r a t e d two p e r cage i n t o two g r o u p s , e x p e r i m e n t a l and c o n t r o l . TREATMENT E x p e r i m e n t a l a n i m a l s r e c e i v e d l a b chow ( P u r i n a L a b o r a t o r y chow, R a l s t o n P u r i n a o f Canada L t d . , W o odstock, Ont.) and l - c x - a c e t y l m e t h a d o l (LAAM) d i s s o l v e d i n t a p w a t e r ad. l i b i t u m . The dosage o f LAAM was i n c r e a s e d s u c h t h a t a maximum was a c h i e v e d d u r i n g t h e t h i r d t r i m e s t e r o f p r e g n a n c y as i n d i c a t e d i n T a b l e I . T h i s dosage s c h e d u l e had p r e v i o u s l y been d e t e r m i n e d n o t t o c a u s e r e s o r p t i o n o f t h e f e t u s , (Ng, 1 9 7 8 ) . C o n t r o l a n i m a l s were p a i r f e d and w a t e r e d . 23 Day o f P r e g n a n c y 0-4 4-8 i-wea n i n g TABLE I A v e r a g e C o n c e n t r a t i o n o f LAAM i n t h e d r i n k i n g w a t e r (mg/ml)  0 . 0025 0.005 0.01 0 A v e r a g e Dose o f LAAM (mq/kq/day) 0.385 0.70 1 . 36 At t h e end o f t h e t h i r d t r i m e s t e r o f p r e g n a n c y t h e f e m a l e s were i s o l a t e d one p e r c a g e , and c o n t i n u e d t o r e c e i v e LAAM u n t i l t h e pups were weaned. F o l l o w i n g b i r t h , pups were n o t t r a n s f e r r e d t o s u r r o g a t e m o t h e r s as LAAM i s f o u n d i n m i l k and t h e n e o n a t a l p e r i o d i s c r i t i c a l t o i m p r i n t i n g . When pups were weaned t h e y were s e p a r a t e d as t o s e x and t r e a t m e n t g r o u p . A l l a n i m a l s were w e i g h e d once p e r week o r when t r e a t m e n t was a l t e r e d . A n i m a l s o f b o t h g r o u p s were a l l o w e d t o m a t u r e n o r m a l l y , t o 90-120 days o f age, r e c e i v i n g l a b chow and w a t e r a_d l i b i t u m . E x p e r i m e n t s w i t h c o n t r o l and e x p e r i m e n t a l g r o u p s were c a r r i e d o u t w i t h i n 48 h r s . o f each o t h e r u s i n g t h e h e p a t i c m i c r o s o m a l f r a c t i o n . HEPATIC MICROSOMAL PREPARATION A n i m a l s were s a c r i f i c e d between 0900-1000 h r s . They were s t u n n e d by a blow t o t h e head, d e c a p i t a t e d and b l e d . B l o o d was c o l l e c t e d i n h e p a r i n i z e d t u b e s and p l a s m a was s e p a r a t e d by c e n t r i f u g a t i o n and f r o z e n f o r s u b s e q u e n t d e t e r m i n a t i o n o f p l a s m a t e s t o s t e r o n e l e v e l s . The i n f e r i o r vena c a v a were c u t 24. and l i v e r s were p e r f u s e d w i t h 20ml o f i c e c o l d KC1 1.15% v i a t h e p o r t a l v e i n . L i v e r s were e x c i s e d i n t o i c e c o l d KC1 1.15%, b l o t t e d d r y and w e i g h e d . Two 5 gram s a m p l e s o f each l i v e r were h o m o g e n i z e d s e p a r a t e l y i n 20ml o f KC1 1.15$ ( i c e c o l d ) u s i n g a P o t t e r E l v e j h e m h o m o g e n i z e r . The homogenates were c e n t r i f u g e d a t 10,000xg i n an IEC model B-20 c e n t r i f u g e f o r 10 m i n u t e s a t 4°C. L i p i d r e m a i n i n g a t t h e t o p o f t h e s u p e r n a t a n t was removed and t h e d u p l i c a t e s u p e r n a t a n t s were c o m b i n e d . From t h e p o o l e d d u p l i c a t e s 8ml a l i q u o t s were t r a n s f e r r e d i n t o e a ch o f t h r e e u l t r a c e n t r i f u g e t u b e s r e s p e c t i v e l y . The s a m p l e s were c e n t r i f u g e d a t 100,000xg f o r 65 m i n u t e s i n a Beckman L5-50 u l t r a c e n t r i f u g e a t 4°C w i t h a t y p e 65 f i x e d a n g l e r o t o r . F o l l o w i n g c e n t r i f u g a t i o n t h e s u p e r n a t a n t was d i s c a r d e d and each p e l l e t was washed w i t h 3 v o l u m e s o f c o l d 0.1M N a l ^ P O ^ b u f f e r pH 7.2, and r e s u s p e n d e d i n 4ml o f t h e same b u f f e r . T h i s p r o c e d u r e was c a r r i e d o u t on i c e . The t r i p l i c a t e s were c o m b i n e d a t t h i s p o i n t t o f o r m one m i c r o s o m a l p o o l p e r e x c i s e d r a t l i v e r . The m i c r o s o m a l enzyme s u s p e n s i o n s were k e p t on i c e f o r t h e d u r a t i o n o f t h e a s s a y s , ( n e v e r l o n g e r t h a n 4 h o u r s ) , t h e n f r o z e n f o r l a t e r p r o t e i n d e t e r m i n a t i o n . ENZYME ASSAYS H e p a t i c m i c r o s o m a l p a r a m e t e r s were used t o c y t o c h r o m e P-450 d e p e n d e n t e n z y m a t i c m o n i t o r t h e e f f e c t s o f LAAM on n e o n a t a l 25. a n d r o g e n i n d u c e d i m p r i n t i n g . The enzyme a s s a y s used were t h e s e x d e p e n d e n t a c t i v i t i e s , b e n z o ( a ) p y r e n e h y d r o x y l a s e ( B P ) , a m i n o p y r i n e N - d e m e t h y l a s e ( A P ) , e t h y l m o r p h i n e N - d e m e t h y l a s e ( E T ) , and t h e s e x i n d e p e n d e n t a c t i v i t y a n i l i n e h y d r o x y l a s e ( A N ) . A l l a s s a y s have been p r e v i o u s l y d e t e r m i n e d i n o u r l a b o r a t o r y t o be l i n e a r w i t h t i m e and enzyme c o n c e n t r a t i o n , and s a t u r a t e d w i t h r e s p e c t t o t h e c o - f a c t o r and m a x i m a l s u b s t r a t e c o n c e n t r a -t i o n u s e d . A l l s a m p l e s were a s s a y e d i n d u p l i c a t e . The maximum v e l o c i t y (Vmax) and Km. were d e t e r m i n e d f o r each e n z y m a t i c a c t i v i t y u s i n g t h e E a d i e - H o f s t e e e q u a t i o n and l i n e a r r e g r e s s i o n a n a l y s i s o f f i v e s u b s t r a t e c o n c e n t r a t i o n s . I t i s o f g r e a t i m p o r t a n c e t h a t t h e s u b s t r a t e c o n c e n t r a t i o n s a r e c h o s e n t o encompass not o n l y t h e r a n g e o f a c t i v i t i e s by r e c t a n g u l a r h y p e r b o l i c a n a l y s i s b u t as w e l l be r e l a t i v e l y e q u i -d i s t a n t w i t h m i n i m a l s c a t t e r u s i n g t h e E a d i e - H o f s t e e e q u a t i o n . S t a t i s t i c a l d i f f e r e n c e s were t e s t e d u s i n g a t w o - t a i l e d S t u d e n t s t - t e s t . D i f f e r e n c e s were c o n s i d e r e d s i g n i f i c a n t a t p< 0.05. AMINOPYRINE N-DEMETHYLASE AND ETHYLMORPHINE N-DEMETHYLASE The N - d e m e t h y l a t i o n s o f a m i n o p y r i n e and e t h y l m o r p h i n e were d e t e r m i n e d by t h e methods o f M a s r y and c o - w o r k e r s ( 1 9 7 4 a ) , and LaDu e_t a l . , (1971) r e s p e c t i v e l y . The a c t i v i t i e s o f t h e enzymes were measured by t h e amount o f f o r m a l d e h y d e p r o d u c e d w h i c h was q u a n t i t a t e d a c c o r d i n g t o t h e method o f Nash (1953) u s i n g f o r m a l d e h y d e as a s t a n d a r d . A c t i v i t y was r e p o r t e d as mmoles f o r m a l d e h y d e p r o d u c e d p e r m i n u t e p e r m i l l i g r a m p r o t e i n . The f i n a l volume o f t h e i n c u b a t i o n m i x t u r e (1.5 ml) c o n t a i n e d 0.2 ml enzyme s u s p e n s i o n , 6.26^/<rnale MgCl^* 11.15 ^ttmole s e m i c a r b a z i d e , 1 .0y«mole NADP, 1 0 p i n o l e 0-6-P, 5 u n i t s o f G-6-P d e h y d r o g e n a s e , 0.581 mmole TRIS b u f f e r pH7.5, 0.1 ml d i s t i l l e d H 20 and e i t h e r 20, 4.0, 2.0, 0.2, o r 0.08/^moles o f a m i n o p y r i n e i n 100yul d i s t i l l e d h^O o r , 225, 1 1 2.5, 56 .25, 15.0, o r 7.5^/^moles o f e t h y l m o r p h i n e i n 1 OO^^l o f d i s t i l l e d H^O t o d e t e r m i n e k i n e t i c c o n s t a n t s . The enzyme s u s p e n s i o n was p r e i n c u b a t e d f o r . one m i n u t e and f i f t e e n s e c o n d s a t 37°C, a t w h i c h t i m e t h e a p p r o p r i a t e s u b s t r a t e ( a m i n o p y r i n e o r e t h y l -m o r p h i n e ) and s u b s t r a t e c o n c e n t r a t i o n were added t o t h e i n c u b a t i o n m i x t u r e . B o t h enzyme b l a n k s and s u b s t r a t e b l a n k s were u s e d . The i n c u b a t i o n t i m e s were 10 m i n u t e s f o r male t i s s u e and 20 m i n u t e s f o r f e m a l e t i s s u e . The r e a c t i o n was s t o p p e d w i t h t h e a d d i t i o n o f 0.5 ml o f c o l d ; z i n c s u l f a t e b%. The s a m p l e s were m i x e d and p l a c e d on i c e f o r f i v e m i n u t e s a t w h i c h t i m e 0.5 ml o f s a t u r a t e d Ba(OH),-, was added t o e a c h s a m p l e and m i x e d a g a i n . The r e s u l t i n g s u s p e n s i o n was c e n t r i f u g e d f o r 10 m i n u t e s a t low s p e e d . A 1.5 ml a l i q u o t o f t h e s u p e r n a t a n t was removed and added t o 0.5 ml o f D o u b l e Nash r e a g e n t and i n c u b a t e d f o r 15 o m i n u t e s a t 60 C. Samples were a l l o w e d t o c o o l f o r f i v e m i n u t e s b e f o r e t h e a b s o r b a n c e was m easured a t 412 nm w i t h a P e r k i n - E l m e r . model 124 d o u b l e beam s p e c t r o p h o t o m e t e r . 27. ANILINE HYDROXYLASE A n i l i n e h y d r o x y l a s e a c t i v i t y was d e t e r m i n e d by t h e method o f I m a i e_t a _ l . , ( 1 9 6 6 ) . A c t i v i t y was a s s e s s e d c o l o r i m e t r i c a l l y as t h e d e v e l o p m e n t o f p a r a - a m i n o p h e n o l r e l a t i v e t o an a u t h e n t i c s t a n d a r d . A c t i v i t y was e x p r e s s e d as mmoles p a r a - a m i n o p h e n o l p r o d u c e d p e r m i n u t e p e r m i l l i g r a m o f p r o t e i n . The f i n a l volume o f t h e i n c u b a t i o n m i x t u r e (2ml) c o n t a i n e d 0.25 ml NaK^PO^ 0.1IYI pH 7.2, 0.25 ml enzyme s u s p e n s i o n , 2 . 5 / ^ m o l e NADP, 5^/<(moleG-6-P , 2.5 u n i t s o f G-6-P d e h y d r o g e n a s e , 12.5^/tmole K l g C l 2 , 0.94 ml NaK 2P0' 4 0.1M. pH 7.4 and e i t h e r 2.5, 0.25, 0.025, 0.01, 0.005^<moles o f a n i l i n e i n 0.5 ml d i s t i l l e d H^O- The enzyme p r e p a r a t i o n was p r e i n c u b a t e d f o r one m i n u t e and f i f t e e n o s e c o n d s a t 37 C b e f o r e t h e a d d i t i o n o f t h e a n i l i n e . F o l l o w i n g t h e a d d i t i o n o f a n i l i n e t h e i n c u b a t i o n was c o n t i n u e d f o r a f u r t h e r 15 m i n u t e s a t w h i c h t i m e t h e r e a c t i o n was t e r m i n a t e d by t h e a d d i t i o n o f 1.0 ml TCA 20$. Enzyme and s u b s t r a t e were added t o t h e a p p r o p r i a t e b l a n k s a t t h i s p o i n t . The r e s u l t i n g s u s p e n s i o n was c e n t r i f u g e d a t low s p e e d f o r 10 m i n u t e s . A 1,0ml a l i q u o t o f t h e s u p e r n a t a n t was added t o 0.5ml N a 2 C 0 3 10$ w i t h 1.0ml p h e n o l 2$ i n 0.2N NaOH. o The s a m p l e s were m i x e d and i n c u b a t e d a t 37 C f o r 30 m i n u t e s f o r c o l o r d e v e l o p m e n t . F o l l o w i n g t h e i n c u b a t i o n t h e s a m p l e s were a l l o w e d t o c o o l f o r f i v e m i n u t e s and a b s o r b a n c e was measured a t 640 nm w i t h a P e r k i n - E l m e r model 124 d o u b l e beam s p e c t r o -p h o t o m e t e r . 28 . BENZO(A)PYRENE HYDROXYLASE H e p a t i c b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y was d e t e r m i n e d a c c o r d i n g t o t h e method o f N e b e r t and G e l b o i n (1968) as m o d i f i e d by A l v a r e s ( 1 9 7 0 ) . The amount o f 3 - h y d r o x y b e n z o ( a ) p y r e n e p r o d u c e d was q u a n t i t a t i v e l y d e t e r m i n e d f l u o r o m e t r i c a l l y as mmoles p e r m i n u t e p e r mg o f p r o t e i n . Q u i n i n e s u l f a t e was used as t h e f l u r o m e t r i c s t a n d a r d . A s t a n d a r d c u r v e r e l a t i n g t h e f l u o r e s c e n c e o f q u i n i n e and 3 - h y d r o x y b e n z o ( a ) p y r e n e had been p r e v i o u s l y d e t e r m i n e d i n t h i s l a b o r a t o r y . The f i n a l volume o f t h e i n c u b a t i o n m i x t u r e (1.0ml) c o n t a i n e d 0.36/^mole NADH, 0.36 y&mole NADPH, 3yUmole M g C l ^ , 8.7 nmole b o v i n e serum a l b u m i n , 150yul enzyme p r e p a r a t i o n , 25yumole T r i s b u f f e r pH 7.5, and e i t h e r 80, 10, 7.5, 5, o r 2.5 nmoles o f b e n z o ( a ) p y r e n e i n 4 0 ^ ( 1 o f a c e t o n e f o r d e t e r m i n a t i o n o f k i n e t i c c o n s t a n t s . E x c e p t f o r enzyme b l a n k s , t h e enzyme p r e p a r a t i o n was p r e i n c u b a t e d f o r one m i n u t e and f i f t e e n s e c o n d s w i t h t h e i n c u b a t i o n m i x t u r e . At t h i s p o i n t BP i n t h e a p p r o p r i a t e c o n c e n t r a t i o n was added and t h e i n c u b a t i o n was c o n t i n u e d f o r an a d d i t i o n a l 2.5 m i n u t e s . The r e a c t i o n was t e r m i n a t e d by t h e a d d i t i o n o f 1.0ml o f i c e c o l d a c e t o n e f o l l o w e d by 3.3ml o f he x a n e , and t h e s a m p l e s were m i x e d . Enzyme o r s u b s t r a t e was added t o t h e a p p r o p r i a t e b l a n k s a t t h i s t i m e . Samples were m i x e d a g a i n and a l l o w e d t o s e p a r a t e . A 2.0 ml a l i q u o t o f t h e s u p e r n a t a n t was t r a n s f e r r e d t o 4.0 ml o f 1.ON NaOH and m i x e d . The s a m p l e s were f r o z e n i n a d r y - i c e a c e t o n e b a t h , t h e hexane phase was d i s c a r d e d and t h e aqueous NaOH phase thawed 29. i n a w a t e r b a t h . F l u o r e s c e n c e was measured w i t h a T u r n e r S p e c t r o f l u o r o m e t e r M odel 430, w i t h e x c i t a t i o n and e m i s s i o n w a v e l e n g t h s s e t a t 396nm and 522nm r e s p e c t i v e l y . The i n s t r u m e n t was z e r o e d w i t h 1.ON NaOH and c a l i b r a t e d w i t h q u i n i n e s u l f a t e 0.1g i n 1.01 H 2 5 0 4 0.1N. D u r i n g t h e c a l c u l a t i o n o f r e s u l t s f r o m t h e b e n z o ( a ) p y r e n e h y d r o x y l a s e a s s a y , t h e c o m p u t e r l i n e a r r e g r e s s i o n a n a l y s i s o f t h e E a d i e - H o f s t e e e q u a t i o n d i d n o t a d e q u a t e l y d e s c r i b e t h e r e c t a n g u l a r h y p e r b o l i c p l o t . As s u c h , k i n e t i c d a t a f o r b e n z o ( a ) p y r e n e h y d r o x y l a s e was t a k e n d i r e c t l y f r o m t h e i n d i v i d u a l r e c t a n g u l a r h y p e r b o l i c p l o t s o f t h e d a t a . PROTEIN DETERMINATION P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d by t h e method o f S u t h e r l a n d e_t a_ l . , (1 949) "as m o d i f i e d by Robson and c o - w o r k e r s (1 968) . 30 RESULTS DOSE OF LAAM The a v e r a g e dose o f LAAM (mg/kg/day) each dam r e c e i v e d d u r i n g g e s t a t i o n i s p r e s e n t e d i n T a b l e I I . The a v e r a g e dose a t t h e t e r m i n a t i o n o f p r e g n a n c y , 1.20 - 0.06 mg/kg/day, d i d n o t e x c e e d t h e maximum s e t f o r t h i n t h e d o s i n g s c h e d u l e . A d m i n i s t r a -t i o n o f LAAM was c o n t i n u e d p o s t p a r t u m t h r o u g h o u t l a c t a t i o n . A l t h o u g h t h e volume o f LAAM i n g e s t e d by each dam was r e c o r d e d ( d a t a n o t shown) t h e amount o f d r u g each pup r e c e i v e d w o u l d be v e r y d i f f i c u l t t o a s s e s s . As p r e v i o u s l y m e n t i o n e d LAAM c r o s s e s t h e p l a c e n t a l b a r r i e r and i s s e c r e t e d i n m i l k . However, t h e p o s i t i o n o f t h e pup ±n_ u t e r o and t h e h i e r a r c h y o f t h e l i t t e r mates as w e l l as t h e number o f pups p e r l i t t e r have b e a r i n g on t h e n u r s i n g p o s i t i o n and u l t i m a t e l y on t h e amount o f m i l k consamed. As s u c h , q u a n t i t a t i o n o f t h e dose o f LAAM t h a t each pup was e x p o s e d t o i n . u t e r o and n e o n a t a l l y was n o t p o s s i b l e . 31 TABLE I I A v e r a qe dose o f LAAM (mg/kg/day) - 5EM t h a t e ach dam r e c e i v e d on days 4, 8, 14 and 20 o f g e s t a t i o n . Day A v e r a g e Dose o f o f LAAM p e r Dam G e s t a t i o n (mq/kq/day) - 5EM. 4 0.26 ± D.D1 8 0.51 - 0.03 14 1.10 ± 0.09 2D 1 .20 - 0.D6 GRD55 EFFECTS OF LAAM A d e s c r i p t i o n o f b i r t h s t a t i s t i c s a r e p r e s e n t e d i n T a b l e I I I . T h i s s t u d y i n c l u d e d 96 c o n t r o l and 140 LAAM o f f s p r i n g . As c a n be s e e n , LAAM d i d not a f f e c t t h e number o f pups p e r l i t t e r (10.8 - 0.8 v e r s u s 11.9 - 1.1 f o r c o n t r o l ) n o r d i d i't r e s u l t i n any change i n t h e mean days t o eye o p e n i n g o f t h e pups (15.5 i 0.4 days f o r LAAM o f f s p r i n g as compared t o 15.6 - 0.4 days f o r c o n t r o l p u p s ) . A l s o r e p o r t e d i n t h i s t a b l e a r e t h e p e r c e n t a g e o f s t i l l b i r t h s o b s e r v e d p e r t o t a l pups p e r g r o u p . The v a l u e s were 1.1$ and 2.1$ f o r c o n t r o l and LAAM r e s p e c t i v e l y . However, u n l e s s a c o u n t o f t h e pups i s t a k e n a t t h e t i m e o f b i r t h i t i s d i f f i c u l t t o be c o n f i d e n t t h a t t h e numbers o f s t i l l -b i r t h s a n d / o r a n o m a l i e s o b s e r v e d i s a c c u r a t e as t h e dam w i l l o f t e n consume s t i l l b o r n o r d e f o r m e d o f f s p r i n g . We a l s o o b s e r v e d one LAAM pup, a p p a r e n t l y h e a l t h y w i t h t h e e x c e p t i o n o f t h e r i g h t h i n d l i m b w h i c h t e r m i n a t e d i n n e c r o t i c t i s s u e above t h e f o o t . TABLE I I I ; GENERAL BIRTH &. DEVELOPMENT STATISTICS FOR LAAM AND CONTROL OFFSPRING Group Number o f Pups Number o f pups p e r L i t t e r - SEM (n) Number o f pups S t i l l b o r n P e r c e n t S t i l l b o r n Days t o Eye O p e n i n g p e r L i t t e r ± SEM (n) C o n t r o l 96 11.9 - 1.1 (B) 1 1 .1 15.5 ± 0.4 (8) . LAAM 1 40 10.8 ± 0.8 (13) 3 2 - 1 15.6 - 0.4 (13) rv)' Whether t h i s was a d e f e c t o f d e v e l o p m e n t o r w h e t h e r t h e f o o t was s e v e r e d c o u l d n o t be d e t e r m i n e d . F u r t h e r we n o t e d o n l y one a b n o r m a l p r e g n a n c y . A f e m a l e t e s t i n g p o s i t i v e t o ' v a g i n a l smear and r e c e i v i n g LAAM d e v e l o p e d s i g n s o f l a b o u r on day 25 o f g e s t a t i o n . B l e e d i n g was o b s e r v e d b u t no pups were d e l i v e r e d E x a m i n a t i o n o f t h e u t e r u s r e v e a l e d s e v e n buds a l o n g w i t h a l a r g p l a c e n t a . These were t h e o n l y r e s o r p t i o n s n o t e d i n t h i s s t u d y . W i t h r e s p e c t t o f u r t h e r d e v e l o p m e n t , LAAM d i d n o t i n h i b i t g r o w t h r a t e as i n d i c a t e d i n F i g u r e 3a &. b. The a v e r a g e w e i g h t o f t h e pups b o t h male and f e m a l e on days 7, 14 and 21 were 14.18 i 1.46 gm, 25.85 - 2.41 gm, 35.65 - 4.83 gm, r e s p e c t i v e l y f o r c o n t r o l and 12.45 - 1.56 gm, 28.30 ± 1.82 gm, 47.9 - 4.0 gm r e s p e c t i v e l y f o r LAAM ( T a b l e I V a ) . Pups were n o t w e i g h e d on day 1 as p r e v i o u s work i n t h e l a b o r a t o r y has s u g g e s t e d t h a t h a n d l i n g may have c o n t r i b u t e d t o f a t a l i t y . H a n d l i n g t h e r e f o r e was k e p t t o a minimum. The g r o w t h r a t e o f t h e m a l e s f o l l o w i n g w e a n i n g a r e i l l u s t r a t e d i n F i g u r e 3b. The a v e r a g e w e i g h t o f t h e male o f f s p r i n g on days 27, 34, and 41 were 75.70 - 4.69 gm, 117.64 - 5.61 gm, 187.19 - 2.44 gm, r e s p e c t i v e l y f o r c o n t r o l and 91.37 - 8.07 gm, 133.60 - 9.36 gm, 199.65 ± 6.20 gm r e s p e c t i v e l y f o r male LAAM o f f s p r i n g . I n a l l LAAM d i d n o t p r o d u c e any s i g n i f i c a n t e f f e c t s on p h y s i c a l d e v e l o p m e n t o f o f f s p r i n g e x p o s e d . T h i s was n o t u n e x p e c t e d as t h e dose o f LAAM used d i d n o t r e s u l t i n any s e d a t i o n o f t h e dams n o r were any pups o b s e r v e d t o e x h i b i t w i t h d r a w a l symptoms p o s t p a r t u m . F u r t h e r m o r e , t h e d o s i n g r e g i m e n o f LAAM was s e l e c t e d , b a s e d on 34. F i g u r e 3a. Mean wt (gm) o f pups p e r l i t t e r - 5EM f r o m day 7 t o day 21, (n) # o f l i t t e r s , (C) C o n t r o l , (L) LAAM e x p o s e d , (M) M a l e , (F) F e m a l e . F i g u r e 3b. Mean wt (gm) o f male r a t s p e r group p a i r e d weaned m a l e s - 5EM f r o m day 27 t o day 4 1 , (n) # o f g r o u p s , (C) C o n t r o l , (L) LAAM e x p o s e d , (M) M a l e , (F) F e m a l e . a Growth Rato-pupt b Growth Rato-maUt ••V97 I I ? M I t f 41 TABLE, IV a &. b. EFFECT DF LAAM ON THE GROWTH RATE OF MALE AND FEMALE PUPS AS COMPARED TO CONTROL (a) MeanVw-t : o f pups p e r l i t t e r , gm - SEM (n) l i t t e r (male and f e m a l e ) day 7 1 4 ' 21 C o n t r o l '14.18 - 1 .46 (8) 25,85 - 2.47 (8) 35 . 63--i 4.83 (8) LAAM 12.45 ± 1.56 (13) 28.30 t 1.82 (13) 47.90 - 4.00 (13) (b) Mean wt o f male pups ( p o s t w e a n i n g ) gm - SEM (n) g r o u p s day 27 34 41 C o n t r o l 75.70 - A.69 (6) 117.64 ± 5.61 (6) 187.19 - 2.44 (6) LAAM 91 .37 - 8.07 (7) 133.60 ± 9.36 (7) 199.65 ± 6.20 (7) 37. p r i o r s t u d i e s i n t h e l a b o r a t o r y , as n o t t o r e s u l t i n r e s o r p t i o n s o r s t i l l b i r t h s . T h i s was c o n f i r m e d by t h e low i n c i d e n c e o f b o t h and t h u s , g r o s s n e o n a t a l e f f e c t s o f o p i a t e s were n o t a n t i c i p a t e d a t t h e s e d o s e s . These r e s u l t s a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d by Zagon e_t a _ l . , (1 977) who d e m o n s t r a t e d t h a t low d o s e s o f m o r p h i n e t h r o u g h o u t g e s t a t i o n and l a c t a t i o n p r o d u c e d no i n h i b i t i o n o f d e v e l o p m e n t . EFFECT OF LAAM DM HEPATIC MICROSOMAL M0N00XYGENA5E KINETIC  PARAMETERS. APPARENT KM &, VMAX The a p p a r e n t Km f o r t h e N - d e m e t h y l a t i o n o f a m i n o p y r i n e i n t h e a d u l t male r a t e x p o s e d in_ u t e r o and n e o n a t a l l y t o LAAM (LAAM male F i g u r e 4 ) , d e c r e a s e d s i g n i f i c a n t l y f r o m a c o n t r o l v a l u e o f 0.138 - 0.02 mM t o 0.066 - 0.01 mM s i m i l a r t o t h a t l o f t h e c o n t r o l f e m a l e v a l u e o f 0.074 - 0.007 mM ( T a b l e Va) . T h i s r e s u l t ,.did n ot a g r e e w i t h o u r o r i g i n a l h y p o t h e s i s , b u t i s c o n s i s t e n t w i t h a f e m i n i z a t i o n o f t h e a c t i v i t y . E x p o s u r e t o LAAM r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n t h e f e m a l e Km ( F i g u r e 4) t o 0.22 - 0.05 mM ( T a b l e Va) . T h i s p a r t i c u l a r e f f e c t had n o t been p r e d i c t e d . F u r t h e r m o r e , as shown i n F i g u r e 4, LAAM d i d n o t p r o d u c e any s i g n i f i c a n t c h a n g e s i n t h e Vmax o f e i t h e r male o r f e m a l e a l t h o u g h t h e r e was a s l i g h t t e n d e n c y t o w a r d s a d e c r e a s e i n t h e LAAM t r e a t e d m a l e . However, we have c o n f i r m e d t h e s e x dependence o f t h i s a c t i v i t y as t h e c o n t r o l male Vmax, + - 1 - 1 3 (2.08 - 0.21 mmoles min mg X 10 ), was c o n s i d e r a b l y g r e a t e r F i g u r e 4 . E f f e c t o f i_n u t e r o and n e o n a t a l e x p o s u r e t o LAAM on t h e a p p a r e n t Km (mM) - 1 - 1 3 and Vmax (mmole min mg X 1 • ) o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s : A m i n o p y r i n e N - d e m e t h y l a s e . (C) C o n t r o l , (L) LAAM e x p o s e d , (M) M a l e , (F) Female. * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , S t u d e n t ' s t - t e s t , t w o - t a i l e d , p <0.05. - 40. + -1 t h a n t h a t o f t h e c o n t r o l f e m a l e , (0.83 - 0.08 mmoles min mg" 1 X 1 0 3 ) . In u t e r o and n e o n a t a l e x p o s u r e t o LAAM d i d n o t r e s u l t i n a s i g n i f i c a n t change i n Km f o r e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y i n male o r f e m a l e r a t s ( F i g u r e 5 ) . I n a d d i t i o n LAAM d i d n o t c a u s e a s i g n i f i c a n t change i n t h e male Vmax b u t d i d , however, d e c r e a s e t h e f e m a l e Vmax f r o m 0.57 - 0.04 t o 0.42 - 0.02 - 1 - 1 3 mmoles min mg X 10 ( T a b l e V b ) . As w i t h a m i n o p y r i n e N - d e m e t h y l -ase we have d e m o n s t r a t e d t h e s e x de p e n d e n t d i f f e r e n c e f o r e t h y l m o r p h i n e N - d e m e t h y l a s e . The c o n t r o l male Vmax (1.195 --1 -1 3 0.08 mmoles min mg X 10 ) was s i g n i f i c a n t l y g r e a t e r t h a n t h a t o b s e r v e d f o r t h e c o n t r o l f e m a l e (0.57 - 0.04 mmoles min -'' -1 3 mg X 10 ). These r e s u l t s a r e c o n s i s t e n t w i t h t h e s e x d i f f e r e n c e f o r Km and Vmax o f e t h y l m o r p h i n e N - d e m e t h y l a t i o n documented by M a s r y and c o - w o r k e r s ( 1 9 7 4 a ) . The e f f e c t s o f LAAM on t h e Km and Vmax f o r t h e s e x i n d e p e n d e n t a c t i v i t y a n i l i n e h y d r o x y l a s e a r e shown i n F i g u r e 6. 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 d i f f e r e n c e s were s e e n f o r Km i n LAAM male and f e m a l e g r o u p s as compared t o t h e i r r e s p e c t i v e c o n t r o l s . In t h e c a s e o f t h e LAAM m a l e , h o w e v e r , t h e d e c r e a s e i n Km p r o d u c e d by LAAM ( f r o m 0.049 - 0.007 t o 0.028 ± 0.004 mM) ( T a b l e Vc) a l t h o u g h n o t s i g n i f i c a n t u s i n g a t w o - t a i l e d Student's t - t e s t a t ..the 0.05 l e v e l o f s i g n i f i c a n c e , ' had a o n e - t a i l e d t e s t been e m p l o y e d , t h e d i f f e r e n c e s w o u l d be s i g n i f i c a n t . In u t e r o and n e o n a t a l e x p o s u r e t o LAAM d i d s i g n i f i c a n t l y r e d u c e t h e Vmax o f t h e male f r o m a c o n t r o l v a l u e o f 1.06 i 0.03 mmoles min" 1mg~ 1 X 1 0 3 t o 0.75 ± 0.08 mmoles min~ 1mg~ 1 X 1 0 3 F i g u r e 5 . E f f e c t o f i_n u t e r o and n e o n a t a l e x p o s u r e t o LAAM on t h e a p p a r e n t Km (mM) and Vma - 1 - 1 3 (mmoles min mg X ID ) o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s : E t h y l m o r p h i n e N - d e m e t h y l a s e . (C) C o n t r o l , (L) LAAM e x p o s e d , (M) M a l e , (F) F e m a l e . * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , S t u d e n t ' s t - t e s t , t w o - t a i l e d , p < 0.05. 4 3 . below t h a t o f t h e c o n t r o l f e m a l e v a l u e 0.88 - 0.07 mmoles - 1 - 1 3 min mg X 10 ) ( T a b l e V c ) . No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d f o r LAAM e f f e c t s on t h e f e m a l e Vmax o f a n i l i n e h y d r o x y l a s e . These r e s u l t s do n o t , however, s u p p o r t t h e sex i n d e p e n d e n c e r e p o r t e d f o r a n i l i n e h y d r o x y l a s e . A l t h o u g h no d i f f e r e n c e was n o t e d f o r t h e Km between male and f e m a l e , a s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d f o r t h e Vmax. The c o n t r o l male Vmax was s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l f e m a l e v a l u e ; 1.06 - 0.03 mmoles min ''mg ^ X 1 0 3 and 0.88 - 0.07 mmoles - 1 - 1 3 min mg X 10 r e s p e c t i v e l y , w h i c h i s n o t c o n s i s t e n t w i t h a r e p o r t by Masry and c o - w o r k e r s ( 1 9 7 4 a ) . However, t h i s a p p a r e n t sex dependence o f a n i l i n e h y d r o x y l a s e Vmax i s i n a g r e e m e n t w i t h r e p o r t s by S k e t t and G u s t a f s s o n ( 1 9 7 9 ) . I t s h o u l d be, however, n o t e d t h a t t h e d i f f e r e n c e s a r e m i n o r as compared t o t h e o t h e r enzymes, p a r t i c u l a r l y b e n z o ( a ) p y r e n e h y d r o x y l a s e . As you c a n s e e i n F i g u r e 7, LAAM p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n t h e Km f o r b e n z o ( a ) p y r e n e h y d r o x y l a s e i n t h e m a l e , ( f r o m a c o n t r o l v a l u e o f 4.75 - 0 . 5 2 ^ ^ t o 6 .66 - 0.34^M) ( T a b l e Vd) s u c h t h a t i t a p p r o a c h e s t h e v a l u e o b t a i n e d f o r t h e c o n t r o l f e m a l e (9.25 - 0.56 / M ) . The Km f o r t h e LAAM f e m a l e d i d n o t e x h i b i t any s t a t i s t i c a l l y s i g n i f i c a n t c h a n g e . W i t h r e g a r d t o t h e Vmax, LAAM p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n t h e + - 1 - 1 3 male o v e r c o n t r o l , 1.49 - 0.05 mmoles min mg X 10 and + - 1 - 1 3 1 .27 - O.OB mmoles min mg X 10 r e s p e c t i v e l y . I n a d d i t i o n t h e Vmax f o r t h e LAAM f e m a l e (0.09 - 0.009 mmoles min~ 1mg~ 1 X 1 0 3 ^ was shown t o d e c r e a s e ( F i g u r e 7) s i g n i f i c a n t l y from c o n t r o l (0.13 - 0.004 mmoles min" 1mg~ 1 X 1Q 3) ( T a b l e V d ) . These F i g u r e 6. E f f e c t o f ini u t e r o and n e o n a t a l e x p o s u r e t o LAAM on t h e a p p a r e n t Km (mM) and Vma -1 -1 3 (mmoles min mg X 10 ) o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s : A n i l i n e H y d r o x y l a s e . (C) C o n t r o l , ( L) LAAM e x p o s e d , (M) M a l e , (F) Female. * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , S t u d e n t ' s t - t e s t , t w o - t a i l e d , p < 0.05 . 46. r e s u l t s were c o n s i s t e n t w i t h v a l u e s p r e v i o u s l y o b t a i n e d i n t h i s l a b o r a t o r y ( G o n t o v n i c k e_t a _ l . , 1 9 7 9 ) . F i g u r e 7. E f f e c t o f in_ u t e r o and n e o n a t a l e x p o s u r e t o LAAM on t h e a p p a r e n t Km ( ^ t M) and Vina - 1 - 1 3 (mmoles min mg X 10 ) o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s : B e n z o ( a ) p y r e n e H y d r o x y l a s e . (C) C o n t r o l , (L)LAAM e x p o s e d , (M) M a l e , (F) Female. * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , 5 t u d e n t ' s t - t e s t ,tw.Q-t a i l e d , p < 0.05. 49. TABLE V: EFFECT OF LAAM DN THE APPARENT KM AND VMAX FOR AMINOPYRINE &. ETHYLMORPHINE N-DEMETHYLASE AND ANILINE 8, BENZO(A) PYRENE HYDROXYLASE IN MALE AND FEMALE RATS a) A m i n o p y r i n e N - d e m e t h y l a s e Group Km (mM) C o n t r o l male LAAM male C o n t r o l f e m a l e LAAM f e m a l e ±SEM 0.138 - 0.02 (10) 0.066 i 0.01 ( 8 ) * 0.074 ± 0.007 (7) 0.220 - 0.05 ( 1 1 ) * -1 •1 Vmax (mmoles min mg X 10 - 5EM (n) 2.08 - 0.21 (9) 1%59 - 0.19 (8) 01.8 3 - 0.08 (9-)' 0.83 - 0.04 (11) b) E t h y l m o r p h i n e N - d e m e t h y l a s e Group C o n t r o l male LAAM male C o n t r o l f e m a l e LAAM f e m a l e Km (mM) -SEM (n) 3.81 ± 0.58 (10) 3.29 - 0.64 (11) 5 . 74 . i • . 7-1 (11) 7.36 ± 1.06 (8) -1 -1 3 Vmax (mmoles min mg X 10 ) - SEM- (n) + 195 - 0.08 (10) 0.99 - 0.07 (1 3) 0.57 - 0.04 (10) 0.42 - 0.02 ( 7 ) * c) A n i l i n e H y d r o x y l a s e Group Km (mM) C o n t r o l male LAAM male C o n t r o l f e m a l e LAAM f e m a l e + 5EM (n) 0.049 - 0.007 (15) 0.028 - 0.004 '(B) 0.033 - 0.010 (9) 0.043 - 0.014 (7) -1 -1 3, Vmax (mmoles min mg X 10 ' - SEM (n) 1 .06 i 0.03 (15) -0.75 - 0.08 ( 8 ) * " 0.88 - 0.07 (10) 0.96 - 0.06 (8)' d) B e n z o ( a ) p y r e n e H y d r o x y l a s e Group Km {yuM) + -SEM (n) + 4.75 - 0.52 (7) 6.66 ± 0.34 ( 8 ) * 9.25 ± 0.56 (8) -1 -1 3 Vmax (mmoles min mg X 10 C o n t r o l male LAAM male C o n t r o l f e m a l e LAAM f e m a l e 11.78 - 1.63 (8) * s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l - 5EM (n) 1 .27 - 0.08 (7) 1 .49 - 0.05 ( 8 ) * 0.13 - 0.004 (8) ^0.09 p < 0.05 + 0.009 ( 8 ) * 50. EFFECTS OF LAAM ON HEPATIC MICROSOMAL M0N00XYGENA5E BASAL S P E C I F I C A C T I V I T I E S . The e f f e c t o f LAAM on t h e b a s a l s p e c i f i c a c t i v i t i e s , t h a t i s t h e s p e c i f i c a c t i v i t y a t maximum s u b s t r a t e c o n c e n t r a t i o n , f o r t h e N - d e m e t h y l a t i o n o f a m i n o p y r i n e and e t h y l m o r p h i n e a r e shown i n F i g u r e 8a &. b. A l t h o u g h i n . u t e r o and n e o n a t a l e x p o s u r e t o LAAM p r o d u c e d no e f f e c t on t h e b a s a l s p e c i f i c a c t i v i t y f o r a m i n o p y r i n e N - d e m e t h y l a s e i n e i t h e r t h e a d u l t male o r f e m a l e r a t , t h e c o n t r o l male v a l u e was g r e a t e r t h a n t h e c o n t r o l f e m a l e , + - 1 - 1 3 + • -1 2,78 - 0.27 mmoles min mg X 10 and' 0.87 T 0.01 mmoles min - 1 3 mg X 10 r e s p e c t i v e l y ( T a b l e V i a ) . However, f o r e t h y l m o r p h i n e N - d e m e t h y l a s e a s i g n i f i c a n t r e d u c t i o n f r o m a c o n t r o l v a l u e o f + - 1 - 1 3 + -1 1.35 - 0.08 mmoles min mg X 10 t o 1.04 - 0.07 mmoles min - 1 3 mg X 10 was o b s e r v e d f o r t h e LAAM male ( T a b l e V I b ) . T h i s d e c r e a s e was t o w a r d s t h a t , o f t h e c o n t r o l f e m a l e ' l e v e l (0.57 --1 -1 3 0.05 mmoles min mg X 10 ). The s e x d e p e n d e n t d i f f e r e n c e s f o r a m i n o p y r i n e and e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t i e s a r e i n agreement w i t h p r e v i o u s o b s e r v a t i o n s ( M a s r y e_t a _ l . , 1 974a; Chung, 1 9 7 4 ) . The b a s a l s p e c i f i c a c t i v i t y o f a n i l i n e h y d r o x y l a s e and b e n z o ( a ) p y r e n e h y d r o x y l a s e a r e p r e s e n t e d i n F i g u r e 9a & b. As p r e d i c t e d LAAM d i d n o t e x e r t an e f f e c t on a n i l i n e h y d r o x y l a s e a c t i v i t y i n e i t h e r male o r f e m a l e r a t s . However, t h e q u e s t i o n o f t h e non-sex dependence o f t h i s a c t i v i t y i s r a i s e d as t h e - 1 - 1 3 c o n t r o l male v a l u e 1,08 - 0.03 mmoles min mg X 10 was s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l f e m a l e 0.89 - .0.05 mmoles - 1 - 1 3 ' min mg X 10 ( T a b l e V i c ) . T h i s i s n o t c o n s i s t e n t w i t h r e p o r t s F i g u r e 8, E f f e c t o f i n . u t e r o and n e o n a t a l e x p o s u r e t o LAAM on the b a s a l s p e c i f i c a c t i v i ( z e r o o r d e r k i n e t i c s w i t h r e s p e c t t o s u b s t r a t e c o n e . ) , o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s . - 1 - 1 3 (a) A m i n o p y r i n e N - d e m e t h y l a s e (mmoles min mg X 10 ) -1 -1 ' 3 (b) E t h y l m o r p h i n e N- d e m e t h y l a s e (mmoles min mg X 10 ) (C) C o n t r o l , (L)LAAM e x p o s e d , (M) M a l e , (F) Female. / * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , 5 t u d e n t ' s t - t e s t ,t'.W]6-t a i 1 e d, p <0.05. F i g u r e 9. E f f e c t o f ijn u t e r o and n e o n a t a l e x p o s u r e t o LAAM on t h e b a s a l s p e c i f i c a c t i v i t y , ( z e r o o r d e r k i n e t i c s w i t h r e s p e c t t o s u b s t r a t e c o n e . ) , o f h e p a t i c m i c r o s o m a l enzymes i n a d u l t (90-120 d) male and f e m a l e r a t s . - 1 - 1 3 (a) A n i l i n e H y d r o x y l a s e (mmoles min mg X 10 ) - 1 - 1 3 (b) B e n z o ( a ) p y r e n e H y d r o x y l a s e (mmoles min mg X 10 ) (C) C o n t r o l , ( L) LAAM e x p o s e d , (M) M a l e , (F) Female. * i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , S t u d e n t ' s t - t e s t , t w o - t a i l e d , p < 0.05. 55 . by M a s r y and c o - w o r k e r s (1974a) i n r e g a r d t o t h e Vmax f o r a n i l i n e h y d r o x y l a s e , The b a s a l s p e c i f i c a c t i v i t i e s f o r b e n z o ( a ) p y r e n e h y d r o x y l a s e e x h i b i t e d a s e x d e p e n d e n t d i f f e r e n c e ( F i g u r e ,9b) as a n t i c i p a t e d . However, t h e i n c r e a s e i n a c t i v i t y f r o m 1.27 '- •.•B mmoles min mg - 1 X.1FJ 3 t o 1.49 - 0.05 mmoles min~ 1mg~ 1 X1 0 3 .(Table VIb) p r o d u c e d by i n . u t e r o and n e o n a t a l e x p o s u r e t o LAAM i n t h e male was n o t e x p e c t e d . T h i s s e e m i n g l y p a r a d o x i c a l e f f e c t w i l l be d e a l t w i t h - i n t h e d i s c u s s i o n . E x p o s u r e t o LAAM p r o d u c e d a s i g n i f i c a n t d e c r e a s e i n b a s a l s p e c i f i c a c t i v i t y o f t h e f e m a l e f r o m 0.13 - 0.004 mmoles m i n ~ 1 m g " 1 . x 1 Q 3 t o 0.09 - 0.01 mmoles min" 1mg" 1 X 1 0 3 ( T a b l e V I d ) . The v a l u e s p r e s e n t i n T a b l e ' V I I i l l u s t r a t e t h e l e v e l s a t w h i c h t h e ch a n g e s s e e n a r e s i g n i f i c a n t f o r a l l a c t i v i t i e s and p a r a m e t e r s m e a s u r e d . I t i s r e a d i l y a p p a r e n t t h a t had t h e h y p o t h e s i s been t e s t e d a t p <0.10 o r had a o n e - t a i l e d t - t e s t been u s e d , many o f t h e cha n g e s a p p e a r i n g s i g n i f i c a n t l y d i f f e r e n t w o u l d have t e s t e d s o . 56 . TABLE V I : EFFECT DF LAAM ON BASAL S P E C I F I C A C T I V I T I E S OF AMINOPYRINE AND ETHYLMORPHINE N-DEMETHYLASE AND ANILINE AND BENZO(A)PYRENO HYDROXYLASE (mmoles min~ 1mg~ 1 X 1 0 3 ) AT MAXIMUM SUBSTRATE CONCENTRATION IN MALE AND FEMALE RATS. a) A m i n o p y r i n e N - d e m e t h y l a s e Group male f e m a l e S u b s t r a t e c o n e , (mM) 1 3 .33 13.33 C o n t r o l i S E M (n) 2.78 ± 0.27 (8) 0.87 - 0.10 (9) LAAM ±SEM (n) 2.49 -• 0.25 (-8) 1 .04 ± 0.03 (9) b) E t h y l m o r p h i n e N - d e m e t h y l a s e Group male f e m a l e S u b s t r a t e cone, (mM) 150 1 50 C o n t r o l i S E M (n.) 1.35 ± 0.08 (8 ) 0.57 - 0.05 (8) 0.51 LAAM i S E M (n) 1 .04 ± 0.07 ( 1 2 ) * + 0.04 (9) A n i l i n e H y d r o x y l a s e Group male f e m a l e S u b s t r a t e cone (mM.) 1 .25 1 .25 C o n t r o l i S E M (n) LAAM i S E M (n) 1.08 - 0.03 (15) 1.14 i 0.04 (8) 0.89 - 0.05 (10) 1.07 - 0.08 (9) d) B e n z o ( a ) p y r e n e H y d r o x y l a s e Group male f e m a l e S u b s t r a t e c o n e . (mM) 0 .08 0.08 C o n t r o l i S E M (n) LAAM i SEM (n) 1.27 - 0.08 (7) 1.49 - 0.05 ( 8 ) * 0.13 - 0.004 (8) 0.09 ? 0.01 ( 8 ) * + s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l @ p <0.05 TABLE V I I : STATISTICAL CALCULATIONS FOR THE COMPARISON OF THE EFFECTS OF LAAM ON APPARENT KM, VMAX AND BASAL S P E C I F I C A C T I V I T I E S FOR AMINOPYRINE AND ETHYL-MORPHINE N-DEMETHYLASE, AND ANILINE AND BENZO(A) PYRENE HYDROXYLASE IN MALE AND FEMALE RATS. Enzyme Sex (M,F) P a r a m e t e r (Km,Vmax,BSA) S i g n i f i c a n c e ( t w o - t a i l e d ) (§ p< : c a l c t t a b AP AP AP AP M F M F Km • Km Vmax Vmax 0.01* 0.01* 0.20 N.S. 2.9770 2:-. 9501 1 . 71 30 2.9208 2.9208 1.3410 ET ET ET ET M F M F Km Km Vmax Vmax 0.60 0.30 0.10 0.02* 0.5 98 0 1.3210 1.9290 2.9398 0.5333 1.0690 1.7207 2.6020 AN AN AN AN M F M F Km Km Vmax Vmax 0.10 0.60 0.001 * 0.50 2.0783 0.5980 4.3860 0.8420 1.7207 0.5366 3.8190 0.6901 BP BP BP BP M F M F Km Km Vmax Vmax 0.01* 0.20 0.05* ••• 0.00.5* 3.1 510 1 .4680 2.3970 4.0610 3.0123 1 .3450 2.1604 3.3257 AP AP M F B5A BSA 0.50 0.20 0.7881 1.6283 0.6924 1.3370 ET ET M F , B5A BSA 0.02* 0.90 2.8740 0.1400 2.5520 0.1278 AN AN M F BSA BSA 0.30 0.10 1.1899 1.9505 1.0630 1.7396 BP BP M F BSA BSA 0.001* 0.005* 6.8644 4.0614 4.2210 3.3257 58 . DISCUSSION The e f f e c t s o f in_ u t e r o and n e o n a t a l e x p o s u r e t o t h e o p i a t e 1-©(-acetylmethadol on c e r t a i n h e p a t i c m i c r o s o m a l mono-o x y g e n a s e a c t i v i t i e s i n t h e a d u l t r a t have been p r e s e n t e d . These e f f e c t s c a n be c o n s i d e r e d a permanent a l t e r a t i o n i n t h e m e t a b o l i c p r o f i l e o f t h e s e a n i m a l s . T h a t i s , a n i m a l s were removed f r o m e x p o s u r e t o LAAM on day 21 o f l i f e and were a l l o w e d t o d e v e l o p n o r m a l l y r e c e i v i n g f o o d and w a t e r ad. l i b i t u m , u n t i l d ays 90-120. T h e r e f o r e , a t t h e minimum, t h e s e a n i m a l s were no t e x p o s e d t o LAAM f o r a p p r o x i m a t e l y 70 d a y s . T h i s f a c t must be k e p t i n mind when c o n s i d e r a t i o n o f e f f e c t s and mechanisms a r e u n d e r t a k e n . The p r e s e n t s t u d y i s n o t e x a m i n i n g t h e t r a n s i e n t e f f e c t s o f LAAM on d r u g m e t a b o l i z i n g enzymes, b u t r a t h e r t h e l o n g t e r m permanent changes t h a t have been p r o d u c e d . W i t h t h e dosage l e v e l s e mployed no o b s e r v a t i o n s o f n e o n a t a l w i t h d r a w a l , dam s e d a t i o n o r i n h i b i t i o n o f n e o n a t a l d e v e l o p m e n t were n o t e d ; t h e r e f o r e t h i s s t u d y was d e s i g n e d t o d e t e c t o n l y s u b t l e c h a n g e s i n m e t a b o l i s m . In t h i s l i g h t t h e r e s u l t s p r e s e n t e d a r e v e r y s i g n i f i c a n t . However, one must e x e r t c a u t i o n when i n t e r p r e t i n g t h e s e r e s u l t s . F o r e x a m p l e , t h e h y p o t h e s i s t h a t t h i s s t u d y was b a s e d on, t h a t i s , n e o n a t a l a n d r o g e n i n d u c e d i m p r i n t i n g , has been documented f o r t h e most p a r t by s t u d i e s i n v o l v i n g c a s t r a t i o n a n d / o r h y p o p h y s e c t o m y . In b o t h i n s t a n c e s s o m e t h i n g i s b e i n g removed f r o m t h e a n i m a l and t h e r e s u l t i n g e f f e c t s n o t e d . I n t h e p r e s e n t s t u d y we a r e a d m i n i s t e r i n g an ex'-ogenous d r u g d i r e c t l y t o t h e a n i m a l t o e f f e c t a c h a n g e . 59 The d r u g i t s e l f may be e x e r t i n g a number o f a c t i o n s w h i c h e i t h e r d i r e c t l y o r i n d i r e c t l y a l t e r t h e p a r a m e t e r s m e a s u r e d . F u r t h e r m o r e , t h e s e p a r a m e t e r s a r e not b e i n g measured i n a p u r i f i e d s y s t e m and i f one e n z y m a t i c pathway i s r e d u c e d a n o t h e r may become more i m p o r t a n t . Thus, changes i n t h e Km and Vmax o f a p a r t i c u l a r enzyme a c t i v i t y c a n n o t be i n t e r p r e t e d as changes o n l y i n a s p e c i f i c enzyme i t s e l f w i t h any v a l i d i t y . The " a p p a r e n t Km" used i n t h i s s t u d y i s d e f i n e d as t h a t s u b s t r a t e c o n c e n t r a t i o n n e c e s s a r y t o r e a c h one h a l f maximum v e l o c i t y of" t h e r e a c t i o n . T h i s Km i s used t o a s s i s t i n t h e d e s c r i p t i o n of" t h e k i n e t i c s c u r v e . The "Km" does n o t r e p r e s e n t t h e a f f i n i t y o f t h e e n z y m e - s u b s t r a t e c o m p l e x nor i s i t t h e d i s s o c i a t i o n c o n s t a n t f o r t h e same r e a c t i o n between e x p e r i m e n t a l g r o u p s . We a r e u n a b l t o c o r r e c t t h e Km f o r t h e i n i t i a l r a t e o f r e a c t i o n as t h e a v e r a g s u b s t r a t e c o n c e n t r a t i o n and t h e amount o f s u b s t r a t e b e i n g m e t a b o l i z e d by t h e p a r t i c u l a r enzyme a r e unknown. T h i s i s due t o t h e i n h e r e n t u n c e r t a i n t y o f t h e i n v o l v e m e n t o f o t h e r pathways and t h e i r a c t i o n s on b o t h t h e a v a i l a b i l i t y o f s u b s t r a t e f o r m e t a b o l i s m and t h e p r o d u c t f o r q u a n t i t a t i o n . I t i s f o r t h e s e r e a s o n s t h a t r e l a t i v e c h anges o n l y a r e exam i n e d and c o n c l u s i o n s a r e drawn f r o m t h e s e changes i n l i g h t o f t h e p r e c e d i n g u n c e r t a i n t i e s . Based on t h e h y p o t h e s i s t h a n an i n h i b i t i o n o f i m p r i n t i n g , p r o d u c e d by a d e p r e s s i o n o f t e s t o s t e r o n e l e v e l s i n t h e male n e o n a t e , w o u l d be e x p r e s s e d by p r e d i c t a b l e s h i f t s o f t h e k i n e t i c p a r a m e t e r s o f c e r t a i n h e p a t i c m i c r o s o m a l a c t i v i t i e s , t h e : a h t i c i -60. p a t e d e f f e c t s o f LAAM were b o r n e i n some, b u t n o t a l l , c a s e s . I t was e x p e c t e d t h a t had an i n h i b i t i o n o f i m p r i n t i n g been p r o d u c e d t h e n an i n c r e a s e i n Km w o u l d r e s u l t f o r male e t h y l -m o r p h i n e N - d e m e t h y l a s e a c t i v i t y . T h i s p r e d i c t i o n was b a s e d on c a s t r a t i o n s t u d i e s by Chung (1 974; 1 975) and M a s r y e_t a l . , (1 974a) who o b s e r v e d an i n c r e a s e i n Km and d e c r e a s e s i n Vmax and s p e c i f i c a c t i v i t i e s o f e t h y l m o r p h i n e N - d e m e t h y l a s e i n c a s t r a t e d male r a t s s u c h t h a t t h e v a l u e a p p r o a c h e d t h a t o f a c o n t r o l f e m a l e . The r e s u l t s f o r t h e Km and Vmax o f t h e LAAM male d i d n o t show any s i g n i f i c a n t change due t o .exposure t o LAAM ( F i g u r e 5 ) . However, a s i g n i f i c a n t d e c r e a s e i n t h e b a s a l s p e c i f i c a c t i v i t y o f t h i s enzyme was o b s e r v e d f o r t h e LAAM male ( F i g u r e B b ) . I t was c o n c l u d e d t h e r e f o r e , t h a t t h e r e g u l a r dosage o f LAAM as o u t l i n e d was n o t s u f f i c i e n t t o r e d u c e t e s t o s t e r o n e l e v e l s t o t h e e x t e n t s e e n by c a s t r a t i o n , however, t h i s i s n o t t o s a y t h a t an i n h i b i t i o n o f i m p r i n t i n g d i d n o t o c c u r . The r e s u l t s o f t h e b a s a l s p e c i f i c a c t i v i t i e s a r e i n d i c a t i v e o f a s u b t l e i n h i b i t i o n . One o f t h e a p p a r e n t p a r a d o x e s e n c o u n t e r e d d e a l t w i t h t h e s h i f t i n t h e Km f o r a m i n o p y r i n e N - d e m e t h y l a s e . We had p r e d i c t e d LAAM t o p r o d u c e an i n c r e a s e i n Km . i n t h e m a l e . T h i s p r e d i c t i o n a r o s e f r o m d a t a i n v o l v i n g t h e Km f o r N - d e m e t h y l a t i o n o f e t h y l m o r p h i n e i n t h e r a t as none was a v a i l a b l e f o r a m i n o p y r i n e . I t was e x p e c t e d t h a t t h e a d u l t male Km w o u l d be l e s s t h a n t h a t o f t h e f e m a l e (as i s t h e c a s e w i t h e t h y l m o r p h i n e N - d e m e t h y l a s e ) and t h a t an i m p r i n t i n g i n h i b i t i o n p r o d u c e d by LAAM w o u l d i n c r e a s e 61 . t h e male Km t o t h a t o f t h e c o n t r o l f e m a l e . However, as can be s e e n i n F i g u r e 4 , t h e Km f o r t h e c o n t r o l male was g r e a t e r t h a n t h a t f o r t h e c o n t r o l f e m a l e , and f u r t h e r m o r e LAAM p r o d u c e d a s h i f t ( d e c r e a s e ) i n t h e male t o t h e l e v e l o f t h e c o n t r o l f e m a l e . Thus, even t h o u g h t h e s h i f t i n t h e Km was t h e r e v e r s e o f t h a t a n t i c i p a t e d , i t was n o n e t h e l e s s i n a f e m i n i z e d d i r e c t i o n . We o b s e r v e d no o t h e r s i g n i f i c a n t c h anges i n a m i n o p y r i n e e n z y m a t i c p a r a m e t e r s i n t h e male due t o LAAM. • f i n t e r e s t a r e t h e e f f e c t s o f LAAM on N - d e m e t h y l a s e a c t i v i t i e s o f t h e f e m a l e s . The h y p o t h e s i s , b a s e d on c a s t r a t i o n s t u d i e s , p r e d i c t s t h a t an i n h i b i t i o n o f i m p r i n t i n g w i l l n o t r e s u l t i n changes i n t h e k i n e t i c p a r a m e t e r s o f f e m a l e r a t s . I t was c l e a r l y shown ( F i g u r e 4 ) , however, t h a t LAAM p r o d u c e d a t w o - f o l d i n c r e a s e i n Km f o r a m i n o p y r i n e N - d e m e t h y l a s e a c t i v i t y i n t h e f e m a l e . ( I t i s i n t e r e s t i n g t h a t s u c h a l a r g e change i n Km d i d not p r o d u c e even a s l i g h t f l u c t u a t i o n i n Vmax.) F u r t h e r m o r e , as i l l u s t r a t e d i n F i g u r e 5, LAAM -decreased t h e e t h y l m o r p h i n e N - d e m e t h y l a t i o n Vmax o f t h e f e m a l e c o n t r a r y t o what t h e o r i g i n a l h y p o t h e s i s w o u l d have p r e d i c t e d . To r e i t e r a t e , when t h e h y p o t h e s i s was c o n s t r u c t e d p r e d i c t i o n s f o r N - d e m e t h y l a s e r e s p o n s e were ba s e d on s t u d i e s t h a t e mployed e t h y l m o r p h i n e . I t was c o n c l u d e d f r o m t h e s e s t u d i e s ( M a s r y e_t a _ l . , 1974a; Kramer e t a l . , 1977; Chung, 1974; Chung e t a l . , 1975) t h a t an i n h i b i t i o n ..of i m p r i n t i n g w o u l d n o t a l t e r t h e Km o r t h e b a s a l s p e c i f i c a c t i v i t y f o r e t h y l m o r p h i n e N - d e m e t h y l a s e i n t h e f e m a l e r a t . T h i s , i n f a c t , was t h e c a s e as shown i n F i g u r e s 3 62. and 6. However, as no o t h e r v a l u e s were a v a i l a b l e we p r e d i c t e d t h a t an i n h i b i t i o n o f i m p r i n t i n g w o u l d n o t a f f e c t t h e Km o f a m i n o p y r i n e N - d e m e t h y l a t i o n n o r t h e Vmax f o r e i t h e r a m i n o p y r i n e o r e t h y l m o r p h i n e i n t h e f e m a l e as w e l l . T h e r e f o r e , t h e changes t h a t were s e e n i n t h e s e p a r a m e t e r s do n o t r e f u t e p r e v i o u s l y p u b l i s h e d d a t a upon w h i c h t h e i m p r i n t i n g h y p o t h e s i s i s c o n s t r u c t e d b e c a u s e s u c h e x p e r i m e n t s had n o t p r e v i o u s l y been c a r r i e d o u t . However, t h e y do i n t r o d u c e an a p p a r e n t c o m p l i c a t i o n i n t h a t t h e o r i g i n a l h y p o t h e s i s i n v o k e d o n l y changes i n t e s t o s t e r o n e l e v e l s i n t h e male n e o n a t e , w i t h no ch a n g e s e x p e c t e d i n t h e f e m a l e . F u r t h e r m o r e , we o b s e r v e d a d e c r e a s e i n t h e Vmax f o r a n i l i n e h y d r o x y l a s e i n t h e LAAM male ( F i g u r e 6 ) . T h i s d e c r e a s e was no t a n t i c i p a t e d as a n i l i n e h y d r o x y l a s e has p r e v i o u s l y been r e p o r t e d t o be a s e x i n d e p e n d e n t a c t i v i t y ( M a s r y e_t a _ l . , 1 974a; Kramer e_t aj_ . , 1 9 7 5 ) . T h i s c o n c l u s i o n was b a s e d on s t u d i e s w h i c h d e m o n s t r a t e d t h a t - c a s t r a t i o n w o u l d n o t a l t e r t h e b a s a l s p e c i f i c a c t i v i t y o f t h e . e n z y m e . We p o s t u l a t e d t h e r e f o r e , t h a t an i n h i b i t i o n o f a n d r o g e n i n d u c e d i m p r i n t i n g w o u l d n o t p r o d u c e a change i n t h e k i n e t i c p a r a m e t e r s o f t h i s a c t i v i t y . I n as much as t h e b a s a l s p e c i f i c a c t i v i t y o f t h e male was n o t e f f e c t e d by e x p o s u r e t o LAAM o u r p o s t u l a t e was c o n f i r m e d ( F i g u r e 9 d ) . However, t h e d e c r e a s e i n t h e Km ( w h i c h n e a r l y a c h i e v e d s i g n i f i c a n c e a t p<(0.05) and t h e d e c r e a s e i n Vmax f r o m c o n t r o l f o r t h e LAAM male r a i s e d q u e s t i o n s as t o t h e s e x - i n d e p e n d e n c e o f t h i s a c t i v i t y ( F i g u r e 6 ) . Our r e s u l t s d e m o n s t r a t e a s i g n i f i c a n t d i f f e r e n c e between c o n t r o l male and f e m a l e "for t h e 63. h y d r o x y l a t i o n o f a n i l i n e , w h i c h i s c o n s i s t e n t w i t h t h e enzyme, a n i l i n e h y d r o x y l a s e , b e i n g a s e x d e p e n d e n t a c t i v i t y as s t a t e d by S k e t t and G u s t a f s s o n (1 9 7 9 ) . I t s h o u l d be s t a t e d , • however, t h a t t h e d i f f e r e n c e s a r e s m a l l as compared t o t h e o t h e r enzymes m e a s u r e d . W i t h r e s p e c t t o b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y we o b s e r v e d an i n c r e a s e d Km f o r t h e LAAM male r e l a t i v e t o c o n t r o l ( F i g u r e 7 ) . T h i s i n c r e a s e was i n t h e d i r e c t i o n o f t h e c o n t r o l f e m a l e v a l u e . LAAM was not shown t o p r o d u c e any s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on t h e Km o f t h e f e m a l e ( F i g u r e 7 ) . These r e s u l t s a r e c o n s i s t e n t w i t h o u r p r e d i c t i o n f o r an i n h i b i t i o n o f i m p r i n t i n g p r o d u c e d by LAAM. I n c o n t r a s t , a s u p r i s i n g f i n d -i n g was t h a t o f t h e e f f e c t o f LAAM on t h e Vmax and b a s a l s p e c i f i c a c t i v i t y i n t h e m a l e . E x p o s u r e t o LAAM r e s u l t e d i n an i n c r e a s e i n b e n z o ( a ) p y r e n e h y d r o x y l a t i o n ( F i g u r e 7, 9 b ) . T h i s i s a s e e m i n g l y p a r a d o x i c a l s i t u a t i o n . On one hand we have a d r u g w h i c h , up t o t h i s p o i n t , a p p e a r s t o be i n h i b i t i n g i m p r i n t i n g as e x p r e s s e d by a f e m i n i z a t i o n o f s e x d e p e n d e n t a c t i v i t i e s i n t h e m a l e , y e t t h e r e s u l t s c l e a r l y d e m o n s t r a t e an i n c r e a s e i n a c t i v i t y f o r an o b v i o u s l y s e x d e p e n d e n t enzyme ( F i g u r e 7, 9 b ) , w h i c h has been shown t o be r e d u c e d i n t h e male by c a s t r a t i o n ( G o n t o v n i c k e_t a _ l . , 1 979 ). However, we a r e d e a l i n g w i t h a s u b s t r a t e i n t h i s c a s e t h a t has a g r e a t e r a f f i n i t y f o r a f o r m o f c y t o c h r o m e s P-450 d i f f e r e n t f r o m t h a t o f t h e o t h e r s u b s t r a t e s e m p l o y e d i n t h i s s t u d y . S p e c i f i c a l l y a m i n o p y r i n e , a n i l i n e and e t h y l m o r p h i n e a r e 64.. m e t a b o l i z e d by t h e p h e n o b a r b i t a l i n d u c e d f o r m o f c y t o c h r o m e , c y t o c h r o m e P-450; w h e r e a s , b e n z o ( a ) p y r e n e i s a s u b s t r a t e o f t h e 3 - m e t h y l c h o l a n t h r e n e (3MC) i n d u c e d f o r m , c y t o c h r o m e . P^-450 o r c y t o c h r o m e P-448 , C y t o c h r o m e s P-450 d e f i n e a group o f heme p r o t e i n s o f w h i c h 20 f o r m s have been i d e n t i f i e d t o d a t e ( G u e n g e r i c h , 1 9 7 9 ) . These f o r m s have s e l e c t i v e s u b s t r a t e s p e c i f i c i t i e s . Ryan and c o -w o r k e r s (1979) have d e m o n s t r a t e d t h e s u b s t r a t e a f f i n i t y f o r t h e f o r m o f P-450 i n d u c e d by 3MC (P-448) t o be t w o - f o l d g r e a t e r f o r t h e P-448 s u b s t r a t e benzo (a) p y r e n e t h a n f o r the. P-450 s u b -s t r a t e a n i l i n e . F u r t h e r m o r e , N e b e r t e_t a l . , (1 972) have shown t h a t w hereas b o t h 3MC and p h e n o b a r b i t a l i n c r e a s e t o t a l c y t o c h r o m e s P-450 t w o - f o l d , 3MC w i l l i n c r e a s e b e n z o ( a ) p y r e n e h y d r o x y l a t i o n e i g h t - f o l d and p h e n o b a r b i t a l o n l y t w o - f o l d ' . 5 i m i l a r r e s u l t s have been d e m o n s t r a t e d by F a h l and c o - w o r k e r s ( 1 9 7 8 ) . T h e r e f o r e , s l i g h t c h a n g e s i n t h e s p e c i f i c P-450's p r e s e n t i n t h e m i c r o s o m a l f r a c t i o n can r e s u l t i n d r a m a t i c changes i n t h e end p o i n t m e t a b o l -i s m , T h e r e f o r e i t i s o u r c o n c l u s i o n t h a t t h e e f f e c t s s e e n a r e c o n s i s t e n t w i t h an a l t e r a t i o n o f n o r m a l i m p r i n t i n g p r o d u c e d by LAAM r e s u l t i n g i n a change i n t h e p r o f i l e o f c y t o c h r o m e s P-450 p r e s e n t i n t h e h e p a t i c m i c r o s o m a l f r a c t i o n . T h i s change i n p r o f i l e o f P-450 i s a m a n i f e s t a t i o n o f an a l t e r e d g e n e t i c e x p r e s s i o n l e a d i n g u l t i m a t e l y t o a d i f f e r e n t m e t a b o l i c p a t t e r n . T h a t i s n o t t o s a y t h a t t h e t o t a l amount o f P-450 has chan g e d n e c e s s a r i l y , b u t t h e r a t i o o f f o r m s o f P-450 t h a t c o n s t i t u t e 65. t h e t o t a l ( p r o f i l e ) have c h a n g e d . The i m p l i c a t i o n s o f t h i s s t u d y a r e f a r - r e a c h i n g . We have d e m o n s t r a t e d t h a t a low dose a d m i n i s t r a t i o n o f an o p i a t e a n a l g e s i c (LAAM) t o r a t s d u r i n g g e s t a t i o n and l a c t a t i o n w i l l p e r m a n e n t l y a l t e r t h e m e t a b o l i c p a t t e r n ' o f t h e o f f s p r i n g . I t w o u l d be o f g r e a t i n t e r e s t t o examine t h e e f f e c t s o f i n d u c e r s and i n h i b i t o r s o f t h e P-450 s y s t e m i n t h e s e o f f s p r i n g t o f u r t h e r c h a r a c t e r i z e t h i s a l t e r e d m e t a b o l i s m . I t w o u l d be o f e q u a l , i f n o t g r e a t e r , i m p o r t a n c e t o examine t h e " r e s p o n s e o f t h e s e o f f s p r i n g t o p r e c a r c i n o g e n s w h i c h a r e m e t a b o l i z e d t h r o u g h r e -a c t i v e i n t e r m e d i a t e s by t h e P-450 s y s t e m t h u s d e m o n s t r a t i n g w h e t h e r o r n o t t h e o f f s p r i n g e x p r e s s i n g t h e a l t e r e d m e t a b o l i c p a t t e r n were p r e d i s p o s e d o r more s u s c e p t i b l e t o b i n d i n g o f r e a c t i v e i n t e r m e d i a t e s ' t o g e n e t i c m a t e r i a l . F o r s i m i l a r r e a s o n s i t w o u l d be o f i n t e r e s t t o c o n d u c t m u t a g e n i c i t y s t u d i e s a s w e l l . P r i o r t o p o s t u l a t i o n o f m e c h a n i s m ( s ) c o n s i s t e n t w i t h t h e o b s e r v a t i o n s , we a g a i n must d e l i n e a t e t h e d i f f e r e n c e s i n t h e d e s c r i p t i o n o f i m p r i n t i n g . As d e s c r i b e d e a r l i e r , i m p r i n t i n g i s d e r i v e d f r o m . s t u d i e s on s t e r o i d m e t a b o l i c p a t t e r n s and d r u g m e t a b o l i c p a t t e r n s . The q u e s t i o n s a s k e d by i n v e s t i g a t o r s i n e a c h o f t h e s e a r e a s a l t h o u g h s i m i l a r a r e n o t e x a c t l y t h e same. The c o n s i d e r a b l e amount o f work done by G u s t a f s s o n and S t e n b e r g and c o - w o r k e r s (1974a;1974b; 1974c) and DeMoor and Denef (1968; 1972; 1974) have d e m o n s t r a t e d t h a t r e m o v a l o f a n d r o g e n s f r o m t h e male n e o n a t a l r a t w i l l p r o d u c e a f e m a l e m e t a b o l i c p a t t e r n 66. f o r s t e r o i d m e t a b o l i s m a t m a t u r i t y . F u r t h e r , t h e y have f o u n d t h a t a d m i n i s t r a t i o n o f a s i n g l e dose o f t e s t o s t e r o n e on day 1 o f l i f e t o t h e s e c a s t r a t e d male r a t s w i l l r e s u l t i n d e v e l o p m e n t o f a r e l a t i v e l y n o r m a l male m e t a b o l i c p a t t e r n a t m a t u r i t y . I n a d d i t i o n , G u s t a f s s o n ' s group ( 1 9 7 4 c ) has shown t h a t when t h e male n e o n a t a l r a t i s c a s t r a t e d i t w i l l r e s p o n d l e s s a t m a t u r i t y t o t e s t o s t e r o n e s t i m u l a t i o n o f c e r t a i n s t e r o i d e n z y m a t i c a c t i v i t i e s t h a n male n e o n a t e s e x p o s e d t o a n d r o g e n . T h i s i s t h e s e c o n d d e s c r i p t i o n o f i m p r i n t i n g w h i c h i s f o r t h e most p a r t u s e d by i n v e s t i g a t o r s i n d r u g m e t a b o l i s m . Kato and G i l l e t t e ( 1 9 6 5 ) , M a s r y and M a n n e r i n g ( 1 9 7 4 b ) , Chung and c o - w o r k e r s (1975) examine p r i m a r i l y r e s p o n s e o f d r u g . m e t a b o l i z i n g enzymes t o t e s t o s t e r o n e s t i m u l a t i o n i n v a r i o u s g o n a d a l s t a t e s , and n o t how t h e m e t a b o l i c p a t t e r n i s a l t e r e d by t h e p r e s e n c e o r a b s e n c e o f t e s t o s t e r o n e i n t h e n e o n a t a l r a t a l o n e . Our h y p o t h e s i s p r o p o s e s t h a t LAAM w i l l r e d u c e t e s t o s t e r o n e t o t h e e x t e n t t h a t an i n h i b i t i o n o f i m p r i n t i n g w i l l r e s u l t . T h i s i n h i b i t i o n w i l l be e x p r e s s e d as a r e d u c t i o n o f t h e s e x d e p e n d e n t h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g e n z y m a t i c a c t i v i -t i e s . Thus we w o u l d p o s t u l a t e a f e m i n i z a t i o n i n t h e a d u l t male w i t h no ch a n g e s i n a c t i v i t i e s f o r t h e f e m a l e n o r t h e s e x i n d e p e n d e n t a c t i v i t i e s i n e i t h e r s e x . In o t h e r w o r d s , we w o u l d e x p e c t t o see e f f e c t s s i m i l a r t o t h o s e d e m o n s t r a t e d by c a s t r a -t i o n on t h e same s y s t e m , a s s u m i n g LAAM i s p r e s e n t i n s u f f i c i e n t c o n c e n t r a t i o n t o r e d u c e t e s t o s t e r o n e . In some c a s e s o u r r e s u l t s c o n f o r m t o t h i s h y p o t h e s i s . We 67. have d e m o n s t r a t e d t h a t in_ u t e r o and n e o n a t a l e x p o s u r e o f t h e male t o LAAM has p r o d u c e d a f e m i n i z a t i o n o f t h e a p p a r e n t Km f o r a m i n o p y r i n e N - d e m e t h y l a s e (0.138 t 0.02 t o 0.066 - 0.01mM) ( F i g u r e 4) and b e n z o ( a ) p y r e n e h y d r o x y l a s e (4.75 - 0.52 t o 6.66 - 0.34yuM) ( F i g ure 7) as w e l l as t h e b a s a l s p e c i f i c a c t i v i t y f o r e t h y l m o r p h i n e N - d e m e t h y l a s e (1 .35 i 0.08 t o + - 1 - 1 3 1.04 - 0.07 mmoles min mg X 10 ) ( F i g u r e 8 b ) . I n f u r t h e r s u p p o r t o f t h e h y p o t h e s i s f e m a l e s e x h i b i t e d no change i n : Vmax f o r a m i n o p y r i n e N - d e m e t h y l a s e (0.83 - 0.08 t o 0.83 - 0.04 - 1 - 1 3 mmoles min mg X 10 ) ( F i g u r e 4) and a n i l i n e h y d r o x y l a s e (0.88 i 0.07 t o 0.96 - 0.06.mmoles m i n " mg X 10 ) ( F i g u r e 6 ) ; Km f o r e t h y l m o r p h i n e N - d e m e t h y l a s e (5.74 - 0.71 t o 7.36 i 1.06mM) ( F i g u r e 5 ) , a n i l i n e h y d r o x y l a s e (0.033 - 0.01 t o 0.043 - 0.014mM) ( F i g u r e 6 ) , and b e n z o ( a ) p y r e n e h y d r o x y l a s e (9.25 - 0.56 t o 1 1 . 78 - 1 .63yM) ( F i g u r e 7 ) ; and b a s a l s p e c i f i c a c t i v i t y f o r a m i n o p y r i n e N - d e m e t h y l a s e (0.87 - 0.10 t o 1.04 - 0.03 mmoles - 1 - 1 3 + min mg X 10 ) ( F i g u r e 8 a ) , e t h y l m o r p h i n e N - d e m e t h y l a s e (0.57 -+ - 1 - 1 3 0,05 t o 0.51 - 0.04 mmoles min mg X 10 ) ( F i g u r e 8b) and a n i l i n e h y d r o x y l a s e (0.89-0.05 t o 1.07 - 0.08 mmoles min ,mg 3 X 10 ) ( F i g u r e 9 a ) . I n a d d i t i o n LAAM d i d n o t p r o d u c e any e f f e c t on t h e b a s a l s p e c i f i c a c t i v i t y (a s e x i n d e p e n d e n t a c t i v i t y ) o f a n i l i n e h y d r o x y l a s e i n t h e male (1.08 - 0.03 t o 1.14 - 0.04 mmoles - 1 - 1 3 min mg X 10 ) ( F i g u r e 9 a ) . I t s h o u l d be n o t e d t h a t no change i n t h e k i n e t i c p a r a m e t e r s o f t h e f e m a l e and s e x i n d e p e n d e n t a c t i v i t i e s i n t h e male a r e i m p o r t a n t ( a l t h o u g h n o t t o t h e same d e g r e e as a f e m i n i z a t i o n o f male s e x de p e n d e n t a c t i v i t i e s ) 68. b e c a u s e t h e y a r e c o n s i s t e n t w i t h p o s t u l a t i o n s b a s e d on o u r h y p o t h e s i s , However, n o t a l l t h e r e s u l t s r e p o r t e d i n t h - i s s t u d y c o n f o r m t o t h e o r i g i n a l h y p o t h e s i s . I_n u t e r o and n e o n a t a l e x p o s u r e t o LAAM p r o d u c e d an i n c r e a s e i n t h e a p p a r e n t Km i n t h e f e m a l e f o r a m i n o p y r i n e N - d e m e t h y l a s e (0.074 ± 0.007 t o 0.22 - 0.05 mM). ( F i g u r e 4) and a d e c r e a s e i n Vmax f o r e t h y l m o r p h i n e N - d e m e t h y l -ase (0.57 - 0.04 t o 0.42 ± 0.02 mmoles m i n - 1 m g " 1 X 1 0 3 ) ( F i g u r e 5) and b e n z o ( a ) p y r e n e h y d r o x y l a s e (0.13 - 0.004 t o 0.09 - 0.009 -1 -1 3 mmoles min mg X 10 ) ( F i g u r e 7 ) . (The b a s a l s p e c i f i c a c t i v i t y f o r b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y i n t h e f e m a l e e x h i b i t e d s i m i l a r e f f e c t s . ) F u r t h e r m o r e , o u r p o s t u l a t i o n f o r t h e e f f e c t s o f LAAM i n t h e male were n o t t o t a l l y b o r n e o u t . No i n c r e a s e i n a p p a r e n t Km f o r e t h y l m o r p h i n e N - d e m e t h y l a s e (3.81 - 0.58 t o 3.29 - 0.64 mM ) ( F i g u r e 5) o r d e c r e a s e o f Vmax (2,08 i 0.21 t o 1.59 - 0.19 mmoles - 1 - 1 3 • + min mg X 10 ) ( F i g u r e 4) o r b a s a l s p e c i f i c a c t i v i t y (2.78 ''-+ - 1 - 1 3 0,27 t o 2.49 - 0.25 mmoles min mg X 10 ) ( F i g u r e 8a) f o r a m i n o p y r i n e N - d e m e t h y l a s e were o b s e r v e d . Based on c a s t r a t i o n s t u d i e s by Chung ( 1 9 7 5 ) , Kramer and c o - w o r k e r s ( 1 9 7 5 ) , and M a s r y and M a n n e r i n g (1974b) l e a d i n g t o f e m i n i z a t i o n , t h e s e k i n e t i c p a r a m e t e r s were a n t i c i p a t e d . I t i s p o s s i b l e t h a t t h e dose o f LAAM employed was n o t s u f f i c i e n t t o p r o d u c e t h e e x p e c t e d e f f e c t s . T h i s r e a s o n i n g , however, does n o t e x p l a i n t h e e f f e c t s .LAAM p r o d u c e d on f e m a l e k i n e t i c p a r a m e t e r s n o r t h e d i s c r e p a n c y ob-s e r v e d w i t h r e s p e c t t o t h e h y d r o x y l a t i o n o f b e n z o ( a ) p y r e n e i n t h e m a l e . 69. F i g u r e 7 c l e a r l y i n d i c a t e s an i n c r e a s e i n Vrnax (1 .27 -0.08 t o 1.49 - 0.05 mmoles min mg X 10 ) f o r b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y i n t h e LAAM m a l e . A s i m i l a r e f f e c t was p r o d u c e d by LAAM on t h e b a s a l s p e c i f i c a c t i v i t y o f b e n z o ( a ) p y r e n e i n t h e male as shown i n F i g u r e 9b. These r e s u l t s are. i n d i r e c t c o n t r a s t t o r e p o r t s o f t e s t o s t e r o n e dependence o f b e n z o ( a p y r e n e h y d r o x y l a s e by Kramer and c o - w o r k e r s (1979) and G o n t o v n i c e t a l , , ( 1 9 7 9 ) . B o t h s t u d i e s d e m o n s t r a t e d t h a t c a s t r a t i o n o f t h male r a t p r o d u c e d a s i g n i f i c a n t d e c r e a s e i n b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t y . F u r t h e r , Kramer's g r o u p was a b l e t o show t e s t o s t e r o n e r e v e r s a l o f a c t i v i t i e s r e d u c e d by c a s t r a t i o n . I n t h e s e s t u d i e s , however, (and o t h e r s Kato and G i l l e t t e , 1965; M a s r y and M a n n e r i n g , 1 9 7 4 b ) male r a t s were n o t c a s t r a t e d p r i o r t o t h e c r i t i c a l t i m e e s t a b l i s h e d f o r i m p r i n t i n g , i . e . t h e f i r s t days o f l i f e (Chung e_t a l . , 1 975; G u s t a f s s o n and S t e n b e r g , 1974c; Denef and DeMoor, 1 9 6 8 ) . Thus t h e r a t s were i m p r i n t e d b e f o r e c a s t r a t i o n and c o u l d r e s p o n d t o t e s t o s t e r o n e s t i m u l a t i o n o f t h e s e x d e p e n d e n t a c t i v i t i e s . Chung and c o - w o r k e r s (1975) and Chung (1977) have shown t h a t t h e e a r l i e r t h e male i s c a s t r a t e d t h e l e s s i t w i l l r e s p o n d t o t e s t o s t e r o n e s t i m u l a t i o n o f s e x d e p e n d e n t m e t a b o l i s m o f e t h y l m o r p h i n e and a m i n o p y r i n e . T h i s c a s e i s more a p p l i c a b l e t o o u r s i t u a t i o n as i t d e a l s w i t h t h e r e m o v a l o f t e s t o s t e r o n e f r o m t h e n e o n a t e . In t h i s s t u d y (Chung e_t a _ l . , 1 975) t h e i n v e s t i g a t o r s d e m o n s t r a t e d e t h y l m o r p h i n e N - d e m e t h y l a s e as a more s e n s i t i v e measure o f t e s t o s t e r o n e e x p o s u r e whereas a m i n o -70. p y r i n e N - d e m e t h y l a s e was o n l y m a r g i n a l l y a f f e c t e d by t e s t o s -t e r o n e r e m o v a l o r r e a d m i n i s t r a t i o n i n t h e male r a t . We, however, d i d n o t o b s e r v e any i n c r e a s e d s e n s i t i v i t y t o t h e e f f e c t s o f LAAM i n t h e male on e t h y l m o r p h i n e o v e r a m i n o p y r i n e N - d e m e t h y l -ase a c t i v i t i e s . I t becomes a p p a r e n t t h a t a d i f f e r e n t mechanism a c t i n g i n d e p e n d e n t l y o r i n c o n j u n c t i o n w i t h an i n h i b i t i o n o f a n d r o g e n d e p e n d e n t i m p r i n t i n g may d e s c r i b e t h e o b s e r v e d e f f e c t s o f -LAAM. T h a t i s n o t t o s a y t h a t i m p r i n t i n g i s r u l e d o u t n o r t h a t i t i s w h o l l y r e s p o n s i b l e f o r t h e r e s u l t s r e p o r t e d i n t h i s s t u d y . S e v e r a l q u e s t i o n s must be a n s w e r e d b e f o r e t h e r o l e o f i m p r i n t i n g can be d e c i d e d . I f LAAM were i n f a c t a c t i n g i n p a r t t o i n h i b i t a n d r o g e n - d e p e n d e n t i m p r i n t i n g t h e n one w o u l d e x p e c t t h a t : one, a dose r e s p o n s e r e l a t i o n s h i p w o u l d be d e m o n s t r a b l e , e.g. a t i n c r e a s e d d o s e s o f LAAM we w o u l d see g r e a t e r f e m i n i z i n g e f f e c t s ; two, t h a t t e s t o s t e r o n e l e v e l s i n t h e n e o n a t e on day 1 o f l i f e w o u l d be d e c r e a s e d i n a dose d e p e n d e n t f a s h i o n ; and t h r e e , t h a t t e s t o s t e r o n e a d m i n i s t r a t i o n d u r i n g t h e c r i t i c a l n e o n a t a l p e r i o d w o u l d r e v e r s e t h e n e o n a t a l e f f e c t s o f LAAM s e e n a t m a t u r i t y . I f t h e s e p o i n t s , p a r t i c u l a r l y t h e t h i r d p o i n t , c o u l d n o t be demon-s t r a t e d t h e n LAAM w o u l d u n d o u b t e d l y be a c t i n g v i a a n o t h e r mechanism. Ba s e d on t h e p r e l i m i n a r y o b s e r v a t i o n s i n t h i s s t u d y we c a n n o t d i s c o u n t t h e e x i s t e n c e o f a n o t h e r mechanism. The e l u c i d a t i o n o f t h i s mechanism w o u l d be e x t r e m e l y d i f f i c u l t , as so many v a r i a b l e s a r e p r e s e n t w h i c h have n o t t h e m s e l v e s been 71 . d e f i n i t i v e l y c h a r a c t e r i z e d . I t i s known, f o r e x a m p l e , t h a t f o r t e s t o s t e r o n e t o p r o d u c e i t s e f f e c t s , t h e n o r m a l s t e r o i d m e t a b o l i c pathway and a f u n c t i o n a l t e s t o s t e r o n e r e c e p t o r must b o t h be p r e s e n t , o t h e r w i s e a , f e m a l e m e t a b o l i c p a t t e r n ( B a r d i n e t a l . , 1 975) w i l l r e s u l t a l o n g w i t h t h e e x p r e s s i o n o f some fo r m o f p s e u d o h e r m a p h r o d i t i s m ( B a r d i n e_t a _ l . , 1 975; Walsh e_t a l . , 1 9 7 4 ) . The m a j o r i t y o f t h e s t u d i e s d e s c r i b i n g i m p r i n t i n g employ c a s t r a t i o n o f t h e n e o n a t e . T e s t o s t e r o n e l e v e l s a r e n o r m a l l y d e c r e a s i n g i n t h e male n e o n a t a l r a t m a i n t a i n e d a t a n o r m a l f e m a l e l e v e l u n t i l p u b e r t y ( F o r e s t , 1 9 7 9 ) . I t w o u l d be o f i n t e r e s t t o measure t e s t o s t e r o n e l e v e l s i n t h e c a s t r a t e d n e o n a t e r e l a t i v e t o c o n t r o l as t h e l e v e l s o f t e s t o s t e r o n e d u r i n g t h e c r i t i c a l p e r i o d f o r i m p r i n t i n g have n o t as y e t been c o r r e l a t e d w i t h m e t a b o l i c e f f e c t s a t m a t u r i t y . T h i s , h o w e v e r , s h o u l d n o t be t h e o n l y p a r a m e t e r s t u d i e d as c a s t r a t i o n p r o d u c e s many more e f f e c t s t h a n s i m p l e d e c r e a s e i n serum t e s t o s t e r o n e l e v e l s . C i c e r o e_t a _ l . , (1 979) have shown t h a t c a s t r a t i o n p r o d u c e s an i n c r e a s e i n LH l e v e l s i n t h e male r a t w h i c h are' t e s t o s t e r o n e r e v e r s i b l e . ( T a b l e V I I I ) . F u r t h e r , t h i s g r o up has d e m o n s t r a t e d , t h a t t h e t e s t o s t e r o n e r e v e r s a l o f LH l e v e l s i s b l o c k e d by n a l o x o n e , s u g g e s t i n g a r o l e f o r endogenous o p i o i d s i n t h e n e g a t i v e f e e d b a c k o f LH by t e s t o s t e r o n e . I n s u p p o r t o f t h i s , Hahn and F i s h m a n (1979) have shown t h a t c a s t r a t i o n r e s u l t s i n an i n c r e a s e i n o p i a t e r e c e p t o r p o p u l a t i o n i n t h e r a t b r a i n t h a t can be b l o c k e d by a d m i n i s t r a t i o n o f t e s t o s t e r o n e t o t h e c a s t r a t e d 72. TABLE V I I I SUMMARY OF HORMONAL RESPONSE TO VARIOUS TREATMENTS (se e T e x t f o r d e t a i l s ) TREATMENT HORMONAL 5TATUS RELATIVE TO NORMAL MALE RAT OR TO PREVIOUS TREATMENTS IN VIVO C a s t r a t i o n O p i a t e s N a l o x o n e C a s t r a t i o n + T e s t o s t e r o n e C a s t r a t i o n + T e s t o s t e r o n e + N a l o x o n e LH I TESTOSTERONE PROLACTIN • • IN VITRO N a l o x o n e LH-RH LH-RH + N a l o x o n e LH-RH + T e s t o s t e r o n e LH-RH + T e s t o s t e r o n e + N a l o x o n e • • • 73. m a l e . To d e t e r m i n e t h e l e v e l a t w h i c h t h i s r e g u l a t i o n may o c c u r , ' C i c e r o ' s group ( 1 9 8 0 ) , u s i n g p i t u i t a r y s l i c e s , f o u n d t h a t n a l o x o n e d i d n o t i n t e r f e r e w i t h d i h y d r o t e s t o s t e r o n e ' s i n h i b i -t i o n o f LH-RH s t i m u l a t e d i n c r e a s e s i n LH l e v e l s . F u r t h e r m o r e , o p i a t e a n t a g o n i s t s and a n d r o g e n s t e r o i d s were shown n o t t o compete f o r b i n d i n g i n t h e h y p o t h a l a m u s . They c o n c l u d e d , t h e r e -f o r e , t h a t o p i a t e a n t a g o n i s t s do n o t i n c r e a s e serum LH i n t h e male r a t by a d i r e c t t e s t o s t e r o n e d e p e n d e n t mechanism b u t r a t h e r i n h i b i t an endogenous o p i o i d r e s p o n s i b l e f o r r e g u l a t i o n . I t c a n a l s o be p o s t u l a t e d f r o m t h e i r r e s u l t s t h a t t h i s i n d i r e c t mechanism does n o t a c t a t t h e h y p o p h y s e a l l e v e l v i a LH-RH ( n a l o x o n e was i n e f f e c t i v e i n b l o c k i n g d i h y d r o t e s t o s t e r o n e ' s e f f e c t on LH-RH). I t must be remembered t h a t t h e s e were i n  v i t r o s t u d i e s , however. The meaning o f i_n v i v o h y p o t h a l a m i c LH-RH c o n t e n t i s q u e s t i o n a b l e when r e l a t i n g i t t o p e r i p h e r a l LH c o n c e n t r a t i o n . C h i a p p a and F i n k (1972) have shown t h a t p i t u i t a r y c o n t e n t o f LH and F5H i n c r e a s e s i n t h e male and f e m a l e r a t w i t h a ge. T h i s i n c r e a s e i s p a r a l l e l l e d by an i n c r e a s e i n h y p o t h a l a m i c LH-RH c o n c e n t r a t i o n i n r a t s o f b o t h s e x e s . However, p l a s m a c o n c e n t r a t i o n s o f LH d i d n o t e x h i b i t t h e i n c r e a s e s i n e i t h e r s e x t h a t were o b s e r v e d f o r p i t u i t a r y and h y p o t h a l a m i c c o n t e n t . P l a s m a F5H, ( r e l e a s e d by LH-RH as w e l l ) , d i s p l a y e d an i n c r e a s e t o a maximum by day 8 o f l i f e , f o l l o w e d by a de-c r e a s e , i n t h e f e m a l e . I n c o n t r a s t , t h e male showed a s l o w s t e a d y i n c r e a s e i n p l a s m a F5H u n t i l day 50 o f l i f e a f t e r w h i c h l e v e l s d e c l i n e d . I t has a l s o been s u g g e s t e d t h a t endogenous 74. o p i o i d s p a r t i c i p a t e i n t h e r e g u l a t i o n o f p r o l a c t i n l e v e l s , ( G o l d e_t a _ l . , 1 9 7 9 ) . G o l d and c o - w o r k e r s (1 979) have demon-s t r a t e d a dose d e p e n d e n t n a l o x o n e i n h i b i t i o n o f serum p r o l a c t i n l e v e l s i n monkeys. M o r p h i n e was shown t o i n c r e a s e p r o l a c t i n i n a dose d e p e n d e n t manner. T h i s e f f e c t i s b e l i e v e d t o be due t o a r e l e a s e o f p r o l a c t i n f r o m i n h i b i t o r r e g u l a t i o n by dopamine i n t h e median eminance ( F e r l a n d e_t aJL. , 1 977) t h r o u g h t h e b i n d i n g o f o p i a t e r e c e p t o r s i n t h e h y p o p h y s e a l - h y p o t h a l a m i c a x i s . T h i s i s o f i n t e r e s t as B e l a n g e r and c o - w o r k e r s (1979) r e p o r t p r o l a c t i n p a r t i c i p a t e s i n t h e "down r e g u l a t i o n " o f t e s t i c u l a r LH r e c e p t o r s i n d u c e d by LH-RH a g o n i s t s . T h e r e i s r e a s o n t o b e l i e v e , t h e r e f o r e , t h a t o p i a t e s and a n d r o g e n r e g u l a -t i o n a r e i n t e r - r e l a t e d by an i n d i r e c t pathway p o s s i b l y i n v o l v -i n g f e e d b a c k t o one o r more o f t h e hormones a n d / o r r e c e p t o r s d i s c u s s e d . DeGree : f i and c o - w o r k e r s (1 980) u s i n g o v a r i a n and p i t u i t a r y g r a f t s i n a d u l t and n e o n a t a l l y c a s t r a t e d m a l e s have shown t h a t n e o n a t a l e x p o s u r e t o t e s t i c u l a r ' a n d r o g e n s p r e v e n t s t h e a d u l t r a t f r o m r e l e a s i n g p r o l a c t i n i n a s u r g e - l i k e f a s h i o n n e c e s s a r y f o r o v u l a t i o n . F u r t h e r m o r e , L o r e n z e n and c o - w o r k e r s (1980) have s u g g e s t e d t h a t p r o l a c t i n e i t h e r d i r e c t l y o r i n d i r e c t l y i n h i b i t s g o n a d o t r o p i n r e l e a s e i n male r a t s . In a d d i t i o n , B a r k e y e t a l . , (1979) have d e m o n s t r a t e d t h a t c a s t r a t i o n d e c r e a s e d p r o -l a c t i n r e c e p t o r s i n t h e a c c e s s o r y s e x o r g a n s o f a d u l t male r a t s . The l e v e l s o f t h e s e r e c e p t o r s were i n c r e a s e d i n t h e l i v e r by s i m i l a r t r e a t m e n t . T e s t o s t e r o n e a d m i n i s t r a t i o n t o t h e s e 75. c a s t r a t e d m a l e s r e d u c e d t h e b i n d i n g i n t h e p r o s t a t e and s e m i n a l v e s i c l e t o t h a t o f a t e s t o s t e r o n e t r e a t e d n o r m a l a d u l t male r a t . A l s o t e s t o s t e r o n e was r e p o r t e d t o d e c r e a s e p r o l a c t i n b i n d i n g t o t e s t i c u l a r homogenate. These w o r k e r s c o n c l u d e d t h a t t e s t o s t e r o n e i s r e q u i r e d f o r t h e m a i n t e n a n c e o f p r o l a c t i n r e c e p t o r s i n a c c e s s o r y s e x g l a n d s , and e x e r t s a n e g a t i v e e f f e c t on p r o l a c t i n r e c e p t o r s i n t h e t e s t i s and t h e l i v e r . Thus t e s t o s t e r o n e r e g u l a t i o n may depend on a v e r y c o m p l e x i n t e r p l a y between p r o l a c t i n , F5H, and LH and t h e i r r e g u l a t o r y m e c h a n i s m s . I f LAAM i s i n h i b i t i n g i m p r i n t i n g i t may be a c t i n g a t any one o r more o f t h e s e p o i n t s e i t h e r t o r e d u c e t e s t o s t e r o n e l e v e l s d u r i n g t h e c r i t i c a l f i r s t days o f l i f e , o r v i a some o t h e r as y e t unknown mechanism. F o r i n s t a n c e , LAAM may be a c t i n g a t t h e l e v e l o f t h e h o r m o n a l p e p t i d a s e s w h i c h have been p o s t u l a t e d as t h e u l t i m a t e r e g u l a t o r y p o i n t i n h o r m o n a l f e e d b a c k mechanisms ( G r i f f it.bs and K e l l y , 1 9 7 9 ) . S t u d i e s t o d a t e do n o t i n d i c a t e an i m p o r t a n t r o l e f o r F5H i n t h e m e d i a t i o n o f i m p r i n t i n g . F5H a c t s i n c o n j u n c t i o n w i t h LH i n t h e t e s t i s m a k i n g LH a more p r o b a b l e c a n d i d a t e f o r r e g u l a -t i o n o f i m p r i n t i n g . LH i s t h e h y p o p h y s e a l hormone t h a t d i r e c t -l y r e g u l a t e s t e s t o s t e r o n e l e v e l s . LH i t s e l f i s un d e r c o n t r o l o f LH-RH f r o m t h e h y p o t h a l a m u s . One may e n v i s i o n a r o l e f o r LH-RH i n t h e m e d i a t i o n o f i m p r i n t i n g , however, i_n v i v o s t u d i e s d e m o n s t r a t e d no s e x d i f f e r e n c e t o be p r e s e n t on h y p o t h a l a m i c LH-RH l e v e l s w h i c h w o u l d n o t be e x p e c t e d i f LH-RH were m a i n -t a i n i n g a s e x d e p e n d e n t d i f f e r e n c e . The same p o i n t c a n be"made 76. f o r LH, no s e x d i f f e r e n c e was n o t e d f o r h y p o p h y s e a l o r p l a s m a l e v e l s w i t h age"'in t h e r a t . T h i s as w i t h LH-RH t e n d s t o r u l e o u t LH'as a n i e d i a t o r o f i m p r i n t i n g . T e s t o s t e r o n e u n d o u b t e d l y p l a y s an i m p o r t a n t r o l e i n i m p r i n t i n g . I t s p r e s e n c e i s r e -q u i r e d f o r e x p r e s s i o n o f t h e s e x d i f f e r e n c e s , and t h e l e v e l o f o p i a t e and p r o l a c t i n r e c e p t o r s i n v a r i o u s t i s s u e s . Hypo-p h y s e c t o m y s t u d i e s d e s c r i b e d e a r l i e r s u g g e s t t h e h y p o p h y s e a l -h y p o t h a l a m i c a x i s t o be t h e p o i n t o f m e d i a t i o n o f i m p r i n t i n g . However, i t i s o f i n t e r e s t t h a t c a s t r a t i o n and o p i a t e s p r o d u c e a s i m i l a r e f f e c t on t e s t o s t e r o n e l e v e l s b u t r e s u l t i n o p p o s i t e c h a n g e s i n LH c o n c e n t r a t i o n ( T a b l e V I I I ) . S i n c e o u r h y p o t h e s i s i s b a s e d on c a s t r a t i o n s t u d i e s , t h e s e d i f f e r e n c e s w i t h r e s p e c t t o LH may e x p l a i n t h e d i s c r e p a n c i e s we have o b s e r v e d ( e . g . t h e i n c r e a s e i n BP a c t i v i t y i n t h e LAAM m a l e ) . Thus, more t h a n one mechanism may be i n v o l v e d w i t h t h e i n h i b i t i o n o f i m p r i n t i n g p r o d u c e d by LAAM, one LH d e p e n d e n t and t h e o t h e r t e s t o s t e r o n e d e p e n d e n t . F i n a l l y , p r o l a c t i n i s l i n k e d t o o p i a t e , g o n a d o t r o p i n and s e x s t e r o i d f u n c t i o n . U n f o r t u n a t e l y d a t a a r e n o t a v a i l a b l e f o r s p e c u l a t i o n on t h e i m p o r t a n c e o f t h e i n t e r p l a y o f p r o l a c t i n w i t h t h i s s y s t e m o r t h e r e l a t i o n t o a n d r o g e n i m p r i n t i n g . The s t u d i e s f r o m w h i c h p r o l a c t i n i n f o r m a t i o n has been e x t r a c t e d do n o t d e a l w i t h o u r h y p o t h e s i s p e r se and t h u s s p e c u l a t i o n i s u n w a r r a n t e d . I t w o u l d be o f i n t e r e s t t o measure t h e f l u c t u a t i o n s i n g o n a d o t r o p i n s and t h e i r c o r r e s p o n d i n g r e c e p t o r l e v e l s w i t h age and p h y s i o l o g i c a l s t a t u s ( i . e . c a s t r a t i o n , h y p o p h y s e c t o m y , e t c . ) 77. and c o r r e l a t e t h i s t o m e t a b o l i c p a t t e r n s t h a t r e s u l t i n t h e a d u l t . I f h o r m o n a l f l u c t u a t i o n s a r e p r o d u c e d i t w o u l d be s i g n i f i c a n t as w e l l t o d e t e r m i n e t h e r e v e r s i b i l i t y o f t h e i r e f f e c t s by r e p l a c e m e n t t h e r a p y . However, one must be c a u t i o u s as t o t h e s i g n i f i c a n c e o f r e p l a c e m e n t t h e r a p y ; i t i s non-p h y s i o l o g i c and may have no t a r g e t o r g a n f r o m w h i c h t o e l i c i t a r e s p o n s e . T h i s i s s e e n i n t h e c a s e o f g o n a d o t r o p i n a d m i n i s t r a -t i o n t o c a s t r a t e d o r h y p o p h y s e c t o m i z e d a n i m a l s ' where no t a r g e t o r g a n i s p r e s e n t and f i n e f e e d b a c k i n t e r a c t i o n i s l o s t . A n o t h e r p o s s i b l e mechanism, n o t y e t c o n s i d e r e d , t o e x p l a i n o u r o b s e r v a t i o n s i s t h a t o f a d i r e c t t o x i c i n t e r a c t i o n p r o d u c e d by LAAM a t t h e c e l l u l a r l e v e l , i . e . , a t e r a t o g e n i c e f f e c t . T h a t i s , a permanent a l t e r a t i o n o f g e n e t i c e x p r e s s i o n due t o t h e b i n d i n g o f a compound t o g e n e t i c m a t e r i a l d u r i n g d i f f e r e n t i a t i o n and d e v e l o p m e n t c o u l d o c c u r . I n t h i s c a s e , however, t h e e x p r e s -s i o n w o u l d n o t be a g r o s s c o n g e n i t a l m a l f o r m a t i o n b u t r a t h e r a s u b t l e change i n m e t a b o l i c p a t t e r n due t o t h e p r e s e n c e o f LAAM i n u t e r o . SUMMARY AND CONCLUSIONS In u t e r o and n e o n a t a l e x p o s u r e t o LAAM r e s u l t s i n a permanent a l t e r a t i o n o f h e p a t i c m i c r o s o m a l c y t o c h r o m e P-450 • de p e n d e n t enzymes as measured by cha n g e s i n a p p a r e n t Km, Vmax, and b a s a l s p e c i f i c a c t i v i t y f o r a m i n o p y r i n e and e t h y l m o r p h i n e N - d e m e t h y l a s e and a n i l i n e and b e n z o ( a ) p y r e n e h y d r o x y l a s e i n t h e a d u l t r a t , A summary o f t h e s e e f f e c t s f o l l o w s : 78. TABLE IX A EFFECTS OF LAAM THAT SUPPORT AN INHIBITION OF ANDROGEN INDUCED IMPRINTING B EFFECTS OF LAAM THAT DO NOT SUPPORT AN INHIBITION OF ANDROGEN INDUCED IMPRINTING Lc? Km AP| »-C L? Km A P f — - C o Lo* Km B P t — ^ C L? Vmax E T | — • N o t p r e d i c t e d Lo* BSA E T | — • C Lo* Vmax BPt II II Lcf Vmax AN*—*-C Lo* BSA B P t — it II L? Vmax B P j — - " II II L9 BSA B P I — II II L9 Vmax AP: L9 Km ET: L9 Km AN : L ? Vmax AN: L ? Km BP: L ? BSA AP: L9 BSA ET: No change f r o m C Lo* Km ET: Lo* Vmax AP: Lo* BSA AP: Lo" Vmax ET: s h o u l d have changed L = A d u l t male o r f e m a l e r a t s e x p o s e d t o LAAM i n . u t e r o and n e o n a t a l l y C = A d u l t male o r f e m a l e c o n t r o l r a t s o* = A d u l t male r a t $ = A d u l t f e m a l e r a t t = I n c r e a s e | = D e c r e a s e E f f e c t a p p r o a c h e s 79. The e f f e c t s i n column A a r e c o n s i s t e n t w i t h an i n h i b i t i o n o f i m p r i n t i n g i n t h e male n e o n a t e p r o d u c e d by LAAM as a r e s u l t o f d e c r e a s e d e x p o s u r e t o a n d r o g e n d u r i n g a c r i t i c a l p e r i o d r e s u l t i n g i n a change i n t h e p r o f i l e o f c y t o c h r o m e s P-45D p r e s e n t i n t h e h e p a t i c m i c r o s o m a l f r a c t i o n . The r e s u l t s i n column B do n o t s u p p o r t t h i s h y p o t h e s i s , and must be i n t e r p r e t e d t o i n d i c a t e t h e p r e s e n c e o f a n o t h e r mechanism, e i t h e r a l o n e o r i n c o n j u n c t i o n w i t h c h a n g e s i n a n d r o g e n - i n d u c e d i m p r i n t i n g . 80. REFERENCES A l v a r e s , A.P., S c h i l l i n g , G., G a r b u t , A., and Kuntzman, R., S t u d i e s on t h e H y d r o x y l a t i o n o f 3 , 4 - B e n z p y r e n e by H e p a t i c M i c r o s o m e s , B i o c h e m . P h a r m a c o l . 1_9: 1 449-1 455, 1 970. A l v a r e s , A.P., and Kappas, A., I n f l u e n c e o f P h e n o b a r b i t a l on t h e M e t a b o l i s m and A n a l g e s i c E f f e c t o f Methadone i n R a t s , J . Lab. C l i n . Med. 7_9: 439- 4 5 1 , 1 972. 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