UBC Theses and Dissertations

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UBC Theses and Dissertations

Isoniazid hepatotoxicity Zhou, Ting 1990

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I S O N I A Z I D H E P A T O T O X I C I T Y by TING ZHOU M.D., Shanghai Medical University, 1982 THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES PHARMACOLOGY AND THERAPEUTICS We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 19 9 0 (c) Ting Zhou, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology & Therapeutics The University of British Columbia Vancouver, Canada Date March 26, 1990 DE-6 (2/88) ABSTRACT I s o n i a z i d ( I N H ) , one o f t h e m o s t e f f e c t i v e a g e n t s i n t h e t r e a t m e n t a n d p r o p h y l a x i s o f t u b e r c u l o s i s a s s o c i a t e d w i t h a m i l d i n c r e a s e i n l i v e r t r a n s a m i n a s e s i n up t o 20% o f t r e a t e d p a t i e n t s a n d s e v e r e h e p a t o t o x i c i t y i n up t o 2% o f t r e a t e d p a t i e n t s . S i n c e t h e 1 9 7 0 ' s , a number o f s t u d i e s h a v e b e e n r e p o r t e d on t h e m e c h a n i s m o f I N H - i n d u c e d h e p a t o t o x i c i t y . T h e o r i g i n a l s t u d i e s i n r a t s s u g g e s t e d t h a t t h e h e p a t o t o x i c e f f e c t o f INH was d u e t o t h e m i c r o s o m a l m e t a b o l i s m o f a c e t y l h y d r a z i n e ( A c H z , a m e t a b o l i t e o f INH) t o a r e a c t i v e i n t e r m e d i a t e . M o r e r e c e n t l y o t h e r w o r k e r s h a v e b e e n u n a b l e t o r e p r o d u c e t h e o r i g i n a l r e s u l t s i n t h e r a t , o r o t h e r m e c h a n i s t i c m o d e l s h a v e b e e n p r o p o s e d . T h e r e f o r e , i t i s n e c e s s a r y t o e s t a b l i s h a r e p r o d u c i b l e a n i m a l m o d e l w h i c h r e s e m b l e s INH h e p a t o t o x i c i t y i n t h e human b e i n g . I n t h e s e e x p e r i m e n t s , , m a l e New Z e a l a n d w h i t e r a b b i t s w e r e u s e d , a n d d i v i d e d i n t o 12 t r e a t m e n t g r o u p s o f 6-8 r a b b i t s e a c h . S e r u m a r g i n i n o s u c c i n a t e l y a s e ( A S A L ) l e v e l s a n d h i s t o l o g i c a l c h a n g e s i n l i v e r s l i d e s w e r e c h o s e n as i n d i c e s o f h e p a t o t o x i c i t y . A f t e r t h e d e t e r m i n a t i o n o f a c e t y l a t o r p h e n o t y p e f o r e a c h r a b b i t , INH a n d i t s m e t a b o l i t e s , a c e t y l i s o n i a z i d ( A c I N H ) , h y d r a z i n e (Hz) a n d A c H z w e r e a d m i n i s t e r e d i n a t w o - d a y r e g i m e n o r a l l y o r s u b c u t a n e o u s l y . T h e r e s u l t s showed t h a t t h e s e r u m A S A L l e v e l i n r a b b i t s i s a s e n s i t i v e a n d s p e c i f i c enzyme m a r k e r w h i c h p a r a l l e l s t h e i n c i d e n c e o f h e p a t i c n e c r o s i s s e e n on h i s t o l o g y . T h e s e r u m A S A L c o n t r o l v a l u e s 4 . 3 + 2 . 6 (SD) T a k a h a r a u n i t s w e r e m a i n t a i n e d u n t i l a b o u t 24 h r s a f t e r t h e f i r s t c h a l l e n g e o f INH i n t h e t w o - d a y r e g i m e n ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s . c ) ; p e a k v a l u e s o f up t o 2674 T a k a h a r a u n i t s o c c u r r e d a t a b o u t 72 h r s . No s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e t o x i c i t y o f INH g i v e n o r a l l y a n d s u b c u t a n e o u s l y was d e t e c t e d . P h e n o b a r b i t a l (PB) ( 0 . 1 m g / k g x 3 d , i » p » ) p r e t r e a t m e n t i n c r e a s e d t h e e l e v a t i o n o f s e r u m A S A L l e v e l c a u s e d by INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , p . o . ) s i g n i f i c a n t l y ( p < 0 . 0 5 , F t e s t ) c o m p a r e d w i t h t h e g r o u p w i t h o u t PB p r e t r e a t m e n t . T h e 65 e x p e r i m e n t a l r a b b i t s w e r e c l a s s i f i e d i n t o p o p u l a t i o n s o f a c e t y l a t o r p h e n o t y p e b y m e a s u r i n g t h e i r a c e t y l a t i o n r a t e o f s u l f a m e t h a z i n e ( S M Z ) : f a s t a c e t y l a t o r s w i t h a SMZ t 1 / 2 o f 1 2 . 8 + 4 . 4 (SD) (n=54) a n d s l o w a c e t y l a t o r s w i t h a SMZ t 1 / 2 o f 5 0 . 3 + 1 0 . 4 (SD) ( n = l l ) . Among t h e r a b b i t s c h a l l e n g e d w i t h INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , p . o . o r s . c . ) w i t h o r w i t h o u t PB p r e t r e a t m e n t , no c o r r e l a t i o n was f o u n d b e t w e e n t h e p e a k s e r u m A S A L v a l u e s a n d a c e t y l a t i o n r a t e r e p r e s e n t e d by SMZ ^1/2 ( r = 0 . 0 5 , n = 1 8 ) . T h i s l a c k o f t h i s c o r r e l a t i o n was a l s o p r e s e n t i n r a b b i t s c h a l l e n g e d w i t h A c I N H a n d H z . Among INH a n d i t s m e t a b o l i t e s , A c I N H , Hz a n d A c H z , Hz i s t h e m o s t p o t e n t h e p a t o t o x i n . I t s e f f e c t i s d o s e - d e p e n d e n t i i i o v e r t h e d o s e r a n g e ( 0 . 1 0 + 3 x 0 . 0 7 , 0 . 1 4 + 3 x 0 . 1 0 a n d 0 . 1 9 + 3 x 0 . 1 4 m m o l / k g / d x 2 d , p . o . ) . A c H z ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s . c ) , p r o d u c e d no s i g n i f i c a n t h e p a t o t o x i c e f f e c t , w h i c h i s c o n t r a d i c t o r y t o t h e r e s u l t s r e p o r t e d by o t h e r a u t h o r s . A c I N H ( 0 . 2 8 + 3 x 0 . 2 0 m m o l / k g / d x 2 d , s . c , 0 . 4 2 + 3 x 0 . 3 0 m m o l / k g / d x 2 d , s . c . o r p . o . ) h a d a i n t e r m e d i a t e h e p a t o t o x i c e f f e c t w h i c h i s s i m i l a r t o t h a t o f I N H . T h e r e s u l t s showed t h a t (1) t h e r a b b i t i s a r e p r o d u c i b l e a n i m a l m o d e l f o r s t u d y i n g INH h e p a t o t o x i c i t y ; (2) t h e r e l e a s e o f A S A L t o s e r u m a n d p a t h o l o g i c a l c h a n g e s r e s e m b l e t h a t s e e n i n human b e i n g s ; (3) t h e h e p a t o t o x i c i t y o f INH i s p o t e n t i a t e d by PB p r e t r e a t m e n t w h i c h i s i n a c c o r d a n c e w i t h t h e e v i d e n c e i n human b e i n g s o f a n i n c r e a s e d r i s k i n t h e p r e s e n c e o f m i c r o s o m a l enzyme i n d u c e r s ; (4) t h e a c e t y l a t i o n r a t e d o e s n o t a f f e c t t h e h e p a t o t o x i c i t y o f I N H ; (5) among t h e m e t a b o l i t e s t e s t e d , Hz was m o s t p o t e n t . T h e s e d a t a i n d i c a t e t h a t t h e h y p o t h e s i s t h a t INH h e p a t o t o x i c i t y i s due t o m i c r o s o m a l m e t a b o l i s m o f A c H z i s p r o b a b l y i n c o r r e c t . We t h i n k t h a t i t i s m o r e l i k e l y t h a t Hz i s r e s p o n s i b l e . F u r t h e r s t u d i e s a r e r e q u i r e d t o e l u c i d a t e t h e e x a c t m e c h a n i s m i n t h e r a b b i t m o d e l . i v TABLE OF CONTENTS CHAPTER P a g e A B S T R A C T i i L I S T OF T A B L E S v i i L I S T OF F I G U R E S v i i i ACKNOWLEDGEMENT x A B B R E V I A T I O N S x i 1 INTRODUCTION 1 1 .1 G E N E R A L CONCEPT OF INH 1 1 . 1 . 1 CHEMISTRY 1 1 . 1 . 2 T H E R A P E U T I C S USES 3 1 . 1 . 3 MECHANISMS OF A C T I O N S 5 1 . 1 . 4 BIOTRANSFORMATION 7 1 . 1 . 5 PHARMACOKINETICS 11 1 . 1 . 6 A D V E R S E E F F E C T S 12 1.2 H E P A T O T O X I C I T Y OF INH AND I T S M E T A B O L I T E S 13 1 . 2 . 1 INH 14 1 . 2 . 2 A c I N H 16 1 . 2 . 3 A c H z 17 1 . 2 . 4 Hz 19 1 .3 F E A T U R E S OF I N H - I N D U C E D H E P A T O T O X I C I T Y 22 1 . 3 . 1 ENZYME INDUCTION OR I N H I B I T I O N 22 1 . 3 . 2 ANIMAL S P E C I E S 25 1 . 3 . 3 A C E T Y L A T O R PHENOTYPE 2 7 1.4 SERUM A R G I N I N O S U C C I N A T E L Y A S E AS A MARKER ENZYME IN H E P A T O T O X I C STUDY 31 1 .5 THE O B J E C T I V E S OF OUR S T U D I E S 32 1 . 5 . 1 H Y P O T H E S I S 32 1 . 5 . 2 TO E S T A B L I S H SERUM A S A L AS A MARKER ENZYME IN THE R A B B I T MODEL 33 1 . 5 . 3 TO COMPARE THE H E P A T O T O X I C I T Y OF INH AND I T S M E T A B O L I T E S 34 1 . 5 . 4 TO C L A R I F Y THE L I N K BETWEEN A C E T Y L A T I O N R A T E AND I N H - I N D U C E D H E P A T O T O X I C I T Y 34 1 . 5 . 5 TO C L A R I F Y THE R E L A T I O N S H I P BETWEEN THE A C T I V I T Y OF MICROSOMAL O X I D A S E AND I N H - I N D U C E D H E P A T O T O X I C I T Y 35 v T A B L E OF CONTENTS ( C o n t i n u e d ) C H A P T E R P a g e 2 M A T E R I A L S AND METHODS 35 2 . 1 S U B J E C T 35 2 . 2 M A T E R I A L S 35 2 . 3 METHODS 36 2 . 3 . 1 ANIMAL TREATMENT 36 2 . 3 . 2 PHENOTYPING 37 2 . 3 . 3 A D M I N I S T R A T I O N 39 2 . 3 . 4 A S A L MEASUREMENT 41 2 . 4 H I S T O L O G I C A L STUDY 44 2 . 4 . 1 T I S S U E F I X A T I O N 44 2 . 4 . 2 H - E S T A I N E D S L I D E S 4 5 2 . 4 . 3 O I L RED "O" S T A I N E D S L I D E S 45 2 . 5 S T A T I S T I C A L METHODS 45 3 R E S U L T 4 6 3 . 1 E F F E C T S OF INH AND I T S M E T A B O L I T E S ON E L E V A T I O N OF SERUM A S A L L E V E L 4 6 3 . 1 . 1 T I M E COURSE OF THE E L E V A T I O N OF SERUM A S A L L E V E L A F T E R INH A D M I N I S T R A T I O N 4 6 3 . 1 . 2 THE E F F E C T OF PB ON I N H - I N D U C E D H E P A T O T O X I C I T Y 4 9 3 . 1 . 3 I N F L U E N C E OF D I F F E R E N T A D M I N I S T R A T I O N ROUTES 4 9 3 . 1 . 4 E F F E C T OF H z , A c I N H AND A c H z ON E L E V A T I O N OF SERUM A S A L L E V E L 52 3 . 1 . 5 THE R E L A T I O N S H I P BETWEEN THE E L E V A T I O N OF A S A L L E V E L AND THE A C E T Y L A T O R R A T E 58 3 . 2 E F F E C T S OF INH AND I T S M E T A B O L I T E S ON P A T H O L O G I C A L CHANGES 60 3 . 3 THE R E L A T I O N S H I P BETWEEN E L E V A T I O N OF SERUM A S A L L E V E L AND H E P A T I C NECROSIS 62 4 D I S C U S S I O N 71 B I B L I O G R A P H Y 81 v i LIST OF TABLES T A B L E P a g e T a b l e 1 S e r u m A S A L r e s p o n s e t o d r u g c h a l l e n g e s 4 7 T a b l e 2 T h e p a r a m e t e r s o f S M Z - a c e t y l a t o r p h e n o t y p e i n r a b b i t s 58 v i i LIST OF FIGURES P a g e F i g u r e 1 C h e m i c a l s t r u c t u r e s o f INH a n d i t s m a j o r m e t a b o l i t e s 4 2 M e t a b o l i c p a t h w a y s o f INH 9 3 m e t a b o l i c p a t h w a y s o f A c H z 18 4 M e t a b o l i c p a t h w a y s o f Hz 21 5 T i m e c o u r s e o f s e r u m A S A L r e l e a s e f o l l o w i n g c h a l l e n g e w i t h INH 4 8 6 S e r u m A S A L l e v e l s o b t a i n e d f o l l o w i n g o r a l c h a l l e n g e w i t h INH i n t h e r a b b i t s w i t h o r w i t h o u t PB p r e t r e a t m e n t 50 7 S e r u m A S A L l e v e l s i n r a b b i t s o r a l l y a n d s u b c u t a n e o u s l y a d m i n i s t e r e d w i t h INH 51 8 S e r u m A S A L l e v e l s i n r a b b i t s o r a l l y a d m i n i s t e r e d w i t h d i f f e r e n t d o s e s o f Hz 53 9 S e r u m A S A L l e v e l s i n r a b b i t s o r a l l y a d m i n i s t e r e d w i t h INH a n d Hz 54 10 S e r u m A S A L l e v e l s i n r a b b i t s s u b c u t a n e o u s l y a d m i n i s t e r e d w i t h two d o s e s o f A c I N H 55 11 S e r u m A S A L l e v e l s i n r a b b i t s o r a l l y a d m i n i s t e r e d w i t h INH a n d A c I N H 5 6 12 S e r u m A S A L l e v e l s i n r a b b i t s a d m i n i s t e r e d w i t h INH a n d A c H z 57 13 P l o t o f t h e p l a s m a A c S M Z / S M Z + A c S M Z r a t i o a t 20 m i n u t e s a f t e r i n t r a v e n o u s i n j e c t i o n o f SMZ a g a i n s t SMZ h a l f - l i f e 59 14 T h e r e l a t i o n s h i p o f SMZ h a l f - l i f e t o t h e p e a k s e r u m A S A L v a l u e i n t h e r a b b i t s c h a l l e n g e d w i t h INH 61 15 l i v e r t i s s u e w i t h H - E s t a i n f r o m c o n t r o l r a b b i t 63 v i i i L I S T OF F I G U R E S ( C o n t i n u e d ) F i g u r e P a g e 16 V a c u o l a t e d d e g e n e r a t i o n i n r a b b i t l i v e r t i s s u e 64 17 F o c a l n e c r o s i s i n r a b b i t l i v e r t i s s u e 65 18 S u b - m a s s i v e n e c r o s i s i n r a b b i t l i v e r t i s s u e 66 19 l i v e r t i s s u e w i t h O i l R e d "0" s t a i n f r o m c o n t r o l r a b b i t 67 20 V a r i o u s d e g r e e s o f f a t t y m e t a m o r p h o s i s i n r a b b i t s l i v e r t i s s u e s 68 21 T h e r e l a t i o n s h i p b e t w e e n t h e i n c i d e n c e o f n e c r o s i s a n d p e a k s e r u m A S A L v a l u e s 72 i x ACKNOWLEDGEMENTS T h e a u t h o r i s g r a t e f u l f o r t h e e n c o u r a g e m e n t a n d a d v i c e r e c e i v e d f r o m t h e members o f t h e D e p a r t m e n t o f P h a r m a c o l o g y a n d T h e r a p e u t i c s 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 . P a r t i c u l a r t h a n k s a r e d u e t o D r . R . A . W a l l , D r . J . M . W r i g h t a n d D r . F . A b b o t t o f h e r S u p e r v i s o r y C o m m i t t e e . I n a d d i t i o n t h e a u t h o r e x p r e s s h e r a p p r e c i a t i o n f o r t h e d e d i c a t i o n a n d v a l u a b l e r e s e a r c h a d v i c e a n d f i n a n c i a l s p o n s o r s h i p o f h e r s u p e r v i s o r , D r . R . A . W a l l a n d D r . J . M . W r i g h t . S i n c e r e t h a n k s a r e due t o M r . S . Adams f o r h i s e x p e r t t e c h n i c a l a s s i s t a n c e . T h a n k s m u s t a l s o be e x t e n d e d t o t h e s e c r e t a r i a l s t a f f o f t h e d e p a r t m e n t , J a n e l l e Swetnam, E l a i n e J a n , a n d M a r g a r e t Wong f o r t h e i r a s s i s t a n c e w h i c h f a c i l i t a t e d t h e c o m p l e t i o n o f t h i s t h e s i s . F i n a l l y , t h e a u t h o r o f f e r s h e r s i n c e r e s t g r a t i t u d e t o h e r p a r e n t s a n d h e r h u s b a n d f o r t h e i r l o v e , u n d e r s t a n d i n g a n d e n c o u r a g e m e n t . x LIST OF ABBREVIATIONS AcHz a c e t y l h y d r a z i n e AcINH a c e t y l i s o n i c o t i n y l h y d r a z i n e AcSMZ a c e t y l s u l f a m e t h a z i n e ASAL a r g i n i n o s u c c i n a t e l y a s e BNPP b i s - p a r a - n i t r o p h e n y l phosphate DiAcHz d i a c e t y l h y d r a z i n e HPLC h i g h performance l i q u i d chromatography i . p. 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 INA i s o n i c o t i n i c a c i d INH i s o n i c o t i n y l h y d r a z i n e p.o. o r a l a d m i n i s t r a t i o n PB p h e n o b a r b i t a l RMP r i f a m p i c i n s .c. subcutaneous a d m i n i s t r a t i o n SMZ s u l f a m e t h a z i n e t l / 2 a p p a rent h a l f - l i f e x i Z H O U , T . 1 1 INTRODUCTION 1.1 G E N E R A L CONCEPT OF INH I s o n i a z i d ( i s o n i c o t i n y l h y d r a z i n e , I N H , i s o n i c o t i n i c a c i d h y d r a z i d e , p y r i d i n e - 4 - c a r b o x y l i c a c i d h y d r a z i d e ) i s one o f t h e m o s t a c t i v e a g e n t s a g a i n s t m y c o b a c t e r i u m t u b e r c u l o s i s . S i n c e i t s i n t r o d u c t i o n i n 1952 ( B e r n e t e i n e t al.), INH h a s r e m a i n e d as t h e m a j o r d r u g f o r t h e t r e a t m e n t a n d t h e p r o p h y l a x i s o f t u b e r c u l o s i s ( A l b a n o a n d T o m a s i , 1987 ; Thomas et al., 1 9 8 1 ) . I t s p h a r m a c o l o g i c a l a c t i o n s h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d . I n r e c e n t y e a r s t h e r e has b e e n r e n e w e d i n t e r e s t i n i t s m e t a b o l i s m d u e t o t h e d i s c o v e r y t h a t i t c a u s e s h e p a t o t o x i c i t y i n a m i n o r i t y o f human s u b j e c t s ( M a d d r e y a n d B o i t n o t t , 1973; B l a c k e t al., 1 9 7 5 ) . S t u d i e s on I N H - i n d u c e d h e p a t o t o x i c i t y h a v e b e e n c a r r i e d o n i n t h i s l a b o r a t o r y i n r e c e n t y e a r s . T h e s t u d i e s r e p o r t e d i n t h i s t h e s i s a r e a s e r i e s o f e x p e r i m e n t s i n w h i c h t h e h e p a t o t o x i c i t y o f INH a n d some o f i t s m a j o r m e t a b o l i t e s w e r e t e s t e d in vivo u s i n g b i o c h e m i c a l a n d p a t h o l o g i c a l m e t h o d s . Z H O U , T . 2 1 . 1 . 1 C H E M I S T R Y INH i s t h e h y d r a z i d e o f i s o n i c o t i n i c a c i d , w h i c h has t h e e m p i r i c a l f o r m u l a C g H 7 N 3 0 ( m o l e c u l a r w e i g h t 1 3 7 . 2 ) . I t s s o l u b i l i t y i n w a t e r i s 13% a t 2 2 ° C a n d 25% a t 3 7 - ° C . I t i s 20% s o l u b l e i n b o i l i n g m e t h a n o l a n d e t h a n o l a n d o n l y a b o u t 2.5% s o l u b l e i n m e t h a n o l a n d e t h a n o l a t 2 0 ° C . INH i s p o o r l y s o l u b l e i n o r g a n i c s o l v e n t s i m m i s c i b l e w i t h w a t e r . I t r e a c t s e a s i l y w i t h a c e t o n e , f o r m i n g a s o l u b l e h y d r a z o n e . T h e d i s c o v e r y o f INH was somewhat f o r t u i t o u s . I t was f i r s t p r e p a r e d by M e y e r a n d M a l l y (1912) a t t h e G e r m a n C h a r l e s U n i v e r s i t y i n P r a g u e f r o m e t h y l i s o n i c o t i n a t e a n d h y d r a z i n e h y d r a t e . F o r 38 y e a r s no one saw a n y r e a s o n t o s t u d y INH f o r i t s s u i t a b i l i t y a s a n a n t i t u b e r c u l o u s . F u r t h e r m o r e , G a r d n e r ( 1 9 5 1 ) , w h i l e s t u d y i n g t h e c h e m i c a l l y v e r y s i m i l a r p r o d u c t o b t a i n e d f r o m t h e r e a c t i o n o f h y d r o x y l a m i n e w i t h e t h y l i s o n i c o t i n a t e f a i l e d t o o b s e r v e a n y t u b e r c u l o s t a t i c a c t i v i t y . T h e a n t i t u b e r c u l o u s e f f e c t o f n i c o t i n i c a c i d a m i d e d i s c o v e r e d by C h o r i n e (1945) l e d F o x t o s t a r t a s e a r c h f o r a n t i t u b e r c u l o u s a g e n t s among p y r i d i n e d e r i v a t i v e s . One o f t h e s e p y r i d i n e d e r i v a t i v e s was t h i o s e m i c a r b a z o n e o f p y r i d i n e - 4 - a l d e h y d e ( B e h n i s c h e t al., 1 9 4 8 ; L e v a d i t i e t al., 1 9 5 1 ; F o x , 1 9 5 1 ; L e v a d i t i e t al., 1 9 5 2 ) . T h e s t a r t i n g m a t e r i a l f o r t h i s s y n t h e s i s was t h e m e t h y l e s t e r o f i s o n i c o t i n i c a c i d , a n d t h e f i r s t i n t e r m e d i a t e was I N H . I n t e r m s o f t u b e r c u l o s t a t i c a c t i v i t y , INH was f o u n d t o be m o r e Z H O U , T . 3 a c t i v e t h a n a n y p r e v i o u s l y known s u b s t a n c e a n d c o u l d be m o d i f i e d i n many ways w i t h o u t a b o l i s h i n g i t s a c t i v i t y ( F o x , 1 9 5 3 ) . INH f o r m s c o m p l e x e s w i t h many h e a v y m e t a l s a l t s . I t r e d u c e s a m m o n i a c a l s i l v e r s a l t s o l u t i o n s y i e l d i n g s i l v e r e d m i r r o r s . I t r e a c t s e a s i l y w i t h a l d e h y d e s a n d k e t o n e s t o g i v e h y d r a z o n e s a s i s a l s o t h e c a s e w i t h p h y s i o l o g i c a l a n d f o o d c o n s t i t u e n t s s u c h as g l u c o s e , p y r u v i c a c i d , a n d o x a l a c e t i c a c i d . T h e s e r e a c t i o n p r o d u c t s a r e e n c o u n t e r e d as t y p i c a l m e t a b o l i t e s o f I N H , a s w e l l a s a c e t y l i s o n i c o t i n o y l - h y d r a z i n e ( A c I N H ) , d i i s o n i c o t i n o y l h y d r a z i n e , N - i s o n i c o t i n o y l g l y c i n e ( i s o n i c o t i n u r i c a c i d ) , a n d i s o n i c o t i n i c a c i d ( I N A ) . A t t e m p t s h a v e b e e n made t o m o d i f y INH c h e m i c a l l y t o o b t a i n INH d e r i v a t i v e s w i t h i m p r o v e d p h a r m a c o k i n e t i c , t o x i c o l o g i c a l , a n d a n t i t u b e r c u l o u s p r o p e r t i e s , e s p e c i a l l y f o r c o m b a t t i n g t h e I N H - r e s i s t a n t m y c o b a c t e r i a . A l t h o u g h some d e r i v a t i v e s do a p p r o a c h INH i n a c t i v i t y a n d e x h i b i t one a d v a n t a g e o r a n o t h e r , o n t h e w h o l e INH i s s u p e r i o r t o t h e m a l l i n a c t i v i t y . T h e c h e m i c a l s t r u c t u r a l f o r m u l a o f INH a n d i t s m a j o r m e t a b o l i t e s a r e shown i n F i g . 1. 1 . 1 . 2 T H E R A P E U T I C S USES INH i s s t i l l c o n s i d e r e d t o be t h e p r i m a r y d r u g f o r t h e c h e m o t h e r a p y o f t u b e r c u l o s i s , a n d a l l p a t i e n t s w i t h d i s e a s e c a u s e d b y I N H - s e n s i t i v e s t r a i n s o f t h e t u b e r c l e b a c i l l u s Z H O U , T . 4 o II C-NH-NH 2 Isoniazid (INH) o o II II C-NH-NH-C-CH3 Acetylisoniazid (AcINH) H 2 N-NH 2 Hydrazine (Hz) Isonicotinic Acid (INA) o II C H 3 - C -NH-NH 2 Acetylhydrazine (AcHz) o o II II C H 3 - C - N H - N H - C - C H 3 Diacetylhydrazine (DiAcHz) F i g . 1. C h e m i c a l s t r u c t u r e s o f INH a n d i t s m a j o r m e t a b o l i t e s . Z H O U , T . 5 s h o u l d r e c e i v e t h e d r u g i f t h e y c a n t o l e r a t e i t ( M a n d e l l a n d S a n d e , 1 9 8 5 ) . INH has t h e t h e r a p e u t i c a d v a n t a g e o f n o t h a v i n g a c t i v i t y a g a i n s t m o s t o t h e r b a c t e r i a . INH d o e s h o w e v e r h a v e o t h e r p h a r m a c o l o g i c a l e f f e c t s . One o f t h e s e i s i t s a b i l i t y t o i n h i b i t y - a m i n o b u t y r i c a c i d (GABA) a m i n o t r a n s f e r a s e a n d r a i s e c o n c e n t r a t i o n s o f GABA i n b r a i n a n d c e r e b r o s p i n a l f l u i d . B a s e d on t h i s a c t i o n , INH has b e e n t e s t e d f o r i t s t h e r a p e u t i c v a l u e s i n s e v e r a l c e n t r a l n e r v e s y s t e m d i s o r d e r s , e . g . , H u n t i n g t o n d i s e a s e ( P e r r y e t al., 1 9 7 9 ; P e r r y e t al., 1 9 8 2 ) , t r e m o r s a s s o c i a t e d w i t h m u l t i p l e s c l e r o s i s ( S a b r a e t al. , 1982 ; D u q u e t t e , e t al., 1 9 8 5 ; H a l l e t t e t al., 1985; F r a n c i s e t al., 1 9 8 6 ; B o z e k e t al., 1 9 8 7 ) , a n d P a r k i n s o n ' s d i s e a s e ( G e r s h a n i k e t al., 1988 ) . R e c e n t l y , INH h a s a l s o b e e n e x a m i n e d f o r i t s e f f e c t i n t h e t h e r a p y o f r h e u m a t o i d a r t h r i t i s when a d m i n i s t e r e d w i t h r i f a m p i c i n ( M c C o n k e y a n d S i t u r a y a k e , 1 9 8 8 ) . 1 . 1 . 3 MECHANISMS OF A C T I O N S W h i l e t h e e x a c t m e c h a n i s m b y w h i c h INH i s e f f e c t i v e i n t h e t r e a t m e n t o f t u b e r c u l o s i s i s u n k n o w n , t h e r e a r e s e v e r a l h y p o t h e s e s . T h e s e i n c l u d e e f f e c t s o n l i p i d s , n u c l e i c a c i d b i o s y n t h e s i s , a n d g l y c o l y s i s ( H e r m a n a n d W e b e r , 1 9 8 0 ) . T a k a y a m a e t a l . ( 1975 ) s u g g e s t e d a p r i m a r y a c t i o n o f INH t o i n h i b i t t h e b i o s y n t h e s i s o f m y c o l i c a c i d , a n i m p o r t a n t Z H O U , T . 6 c o n s t i t u e n t o f t h e m y c o b a c t e r i a l c e l l w a l l . Low c o n c e n t r a t i o n s o f t h e d r u g may p r e v e n t e l o n g a t i o n o f t h e v e r y - l o n g - c h a i n f a t t y a c i d p r e c u r s o r o f t h e m o l e c u l e . S i n c e m y c o l i c a c i d s a r e u n i q u e t o m y c o b a c t e r i a , t h i s a c t i o n w o u l d e x p l a i n t h e h i g h d e g r e e o f s e l e c t i v i t y o f t h e a n t i m i c r o b i a l a c t i v i t y o f I N H . E x p o s u r e t o INH l e a d s t o a l o s s o f a c i d f a s t n e s s a n d a d e c r e a s e i n t h e q u a n t i t y o f m e t h a n o l -e x t r a c t a b l e l i p i d o f t h e m i c r o o r g a n i s m s . O n l y I N H - s e n s i t i v e t u b e r c l e b a c i l l i t a k e up t h e d r u g . T h i s u p t a k e a p p e a r s t o be a n a c t i v e p r o c e s s , a l t h o u g h m o s t o f t h e d r u g w i t h i n t h e b a c i l l i i s t h e i s o n i c o t i n i c a c i d m e t a b o l i t e ( J e n n e a n d B e g g s , 1 9 7 3 ) . I n t h e t r e a t m e n t o f H u n t i n g t o n d i s e a s e , when l a r g e d o s e s o f INH a r e g i v e n t o a n i m a l s , b r a i n GABA c o n t e n t i n c r e a s e s ( P e r r y a n d H a n s e n , 1973; P e r r y e t al., 1 9 7 4 ) . T h i s r e s u l t was s u g g e s t e d t o be due t o t h e i n h i b i t i o n o f G A B A -a m i n o t r a n s f e r a s e , t h e f i r s t o f two s e q u e n t i a l enzymes t h a t d e g r a d e GABA i n t h e b r a i n . I n H u n t i n g t o n d i s e a s e , t h e r e i s a m a r k e d l o s s o f s m a l l n e u r o n s i n t h e c a u d a t e n u c l e u s a n d p u t a m e n , a s s o c i a t e d w i t h p r o l i f e r a t i o n o f a s t r o c y t e s , a n d c o n s i d e r a b l e l o s s o f n e u r o n s i n t h e c e r e b r a l c o r t e x . Many o f t h e n e u r o n s t h a t d i e o f f p r o b a b l y b e l o n g t o a p o p u l a t i o n o f c e l l s t h a t u t i l i z e GABA as a n i n h i b i t o r y n e u r o t r a n s m i t t e r . B i o c h e m i c a l a n a l y s e s h a v e shown a m a r k e d d e c r e a s e i n t h e c o n t e n t o f GABA i n t h e c a u d a t e n u c l e u s , p u t a m e n , g l o b u s p a l l i d u s , a n d s u b s t a n t i a n i g r a o f H u n t i n g t o n d i s e a s e p a t i e n t s , a n d a c o r r e s p o n d i n g d e c r e a s e i n t h e a c t i v i t y o f Z H O U , T . 7 t h e enzyme t h a t s y n t h e s i z e s G A B A , g l u t a m i c a c i d d e c a r b o x y l a s e ( G A D ) . H o w e v e r , i n a d o u b l e - b l i n d c l i n i c a l t r i a l ( P e r r y e t al., 1982) INH m a r k e d l y i n c r e a s e d c e r e b r o s p i n a l f l u i d GABA c o n c e n t r a t i o n s , b u t t h e r e was a l a c k o f c l i n i c a l i m p r o v e m e n t i n m o s t H u n t i n g t o n d i s e a s e p a t i e n t s . I n r e c e n t y e a r s , INH has b e e n e x a m i n e d f o r i t s u s e f u l n e s s a g a i n s t a c t i o n t r e m o r a s s o c i a t e d w i t h m u l t i p l e s c l e r o s i s . T h e e a r l y s t u d i e s g a v e c o n t r a d i c t o r y r e s u l t s t h a t w e r e d u e , i n p a r t , t o t h e m e c h a n i s m o f e v a l u a t i o n , w h i c h was s u b j e c t t o p a t i e n t a n d o b s e r v e r b i a s . T h e l a t e r s t u d i e s u s e d t e c h n i q u e s s u c h as p o l a r i z e d l i g h t g o n i o m e t r y ( F r a n c i s e t al., 1986) a n d t r e m o g r a m s ( B o z e k e t al., 1987) t o e v a l u a t e t h e e f f e c t i v e n e s s o f I N H . P o l a r i z e d l i g h t g o n i o m e t r y d e m o n s t r a t e d a two t o t h r e e - f o l d r e d u c t i o n o f t r e m o r when s t a n d a r d m e t h o d s o f c l i n i c a l a s s e s s m e n t showed o n l y m a r g i n a l i m p r o v e m e n t . B o z e k ' s g r o u p c o n c l u d e d t h a t , a l t h o u g h INH o n l y s i g n i f i c a n t l y i m p r o v e s a p r o p o r t i o n o f p a t i e n t s , a c l i n i c a l t r i a l i s w a r r a n t e d i n m u l t i p l e s c l e r o s i s p a t i e n t s w i t h d e b i l i t a t i n g p o s t u r a l t r e m o r . 1 . 1 . 4 BIOTRANSFORMATION S i n c e t h e i n t r o d u c t i o n o f INH f o r t h e t r e a t m e n t o f t u b e r c u l o s i s i n t h e e a r l y 1 9 5 0 ' s , t h e r e h a s b e e n a g r e a t d e a l o f w o r k d o n e t o e l u c i d a t e i t s m e t a b o l i c p a t h w a y , p a r t i c u l a r l y a s i t r e l a t e s t o I N H - i n d u c e d l i v e r i n j u r y . The Z H O U , T . 8 r e l e v a n t a s p e c t s o f t h e c u r r e n t l y a c c e p t e d scheme o f t h e m e t a b o l i c f a t e o f INH a r e shown i n F i g . 2 . T h e f i r s t p u b l i s h e d i d e n t i f i c a t i o n o f INH m e t a b o l i t e s o c c u r r e d i n t h e same y e a r a s t h e i n t r o d u c t i o n o f t h e d r u g , when s t u d i e s i n d o g s i d e n t i f i e d INA as a m a j o r m e t a b o l i t e ( K e l l y e t al., 1 9 5 2 ) . T h i s was f o l l o w e d b y t h e f i r s t s t u d i e s i n man ( C u t h b e r t s o n e t al., 1 9 5 3 ) , i n w h i c h INA a n d i s o n i c o t i n o y l g l y c i n e a s w e l l a s INH i t s e l f w e r e i d e n t i f i e d i n u r i n e . By t h e m i d 1 9 6 0 ' s i t h a d b e e n e s t a b l i s h e d t h a t a c e t y l a t i o n i s t h e m a j o r r o u t e o f INH i n a c t i v a t i o n i n m a n . T h e enzyme r e s p o n s i b l e f o r a c e t y l a t i o n i s l i v e r N - a c e t y l t r a n s f e r a s e ( J e n n e , 1 9 6 5 ) . T h e p r o d u c t i o n o f A c I N H b y t h i s p a t h w a y was f o u n d t o be b i - m o d a l l y d i s t r i b u t e d a l l o w i n g t h e c l a s s i f i c a t i o n o f i n d i v i d u a l s i n t o r a p i d a n d s l o w a c e t y l a t o r s ( P e t e r s e t al., 1 9 6 5 ) . I t h a s b e e n e s t a b l i s h e d t h a t t h e s e d i f f e r e n c e s i n a c e t y l a t i o n c a p a c i t y a r e u n d e r g e n e t i c c o n t r o l . T h e i n v o l v e d g e n e d e t e r m i n e s t h e amount o f N - a c e t y l t r a n s f e r a s e p r e s e n t i n t h e l i v e r . I n t h e m i d 1 9 7 0 ' s much w o r k was d o n e o n t h e m e t a b o l i s m o f I N H . D e t a i l e d p h a r m a c o k i n e t i c s t u d i e s u n d e r t a k e n on a s l o w a n d a r a p i d a c e t y l a t o r o f INH e n a b l e d a p p r o x i m a t e f i r s t - o r d e r r a t e c o n s t a n t s t o be c a l c u l a t e d f o r t h e m e t a b o l i c p r o c e s s e s i n v o l v e d i n t h e c o n v e r s i o n o f INH t o A c I N H , I N A , i s o n i c o t i n y l g l y c i n e , a c e t y l h y d r a z i n e ( A c H z ) , a n d d i a c e t y l h y d r a z i n e ( D i A c H z ) , a n d f o r t h e e x c r e t i o n o f INH a n d t h e m e t a b o l i t e s i n t h e u r i n e . S t u d i e s o f t h e m e t a b o l i s m o f Z H O U , T . 9 CONHNH* > INH-HYDRAZONES i A c H z I N H - G l y C H 3 CONHNH 2 C H 3 CONHNHOH I I I I V C H 3 C O N - N H I I I K 11 C H 3 C * A C E T Y L F R E E R A D I C A L ACETYL TRANSFERASE CH 3CONHNHCOCH 3 DiAcHz hypothet ical toxicat ion pathway F i g . 2. M e t a b o l i c p a t h w a y s o f i s o n i a z i d ( I N H ) . Z H O U , T . 10 INH s h o w e d t h a t t h e r a p i d a c e t y l a t o r s a c e t y l a t e d INH 5-6 t i m e s m o r e r a p i d l y t h a n t h e s l o w a c e t y l a t o r s - L a t e r i t was d e m o n s t r a t e d t h a t a c i d - l a b i l e h y d r a z o n e s a r e a l s o f o r m e d ( M i t c h e l l e t al., 1 9 7 5 b ) , a n d t h a t some INH i s h y d r o l y z e d d i r e c t l y t o INA a n d h y d r a z i n e ( H z ) . T h e m a j o r m e t a b o l i c r o u t e f o r t h e f o r m a t i o n o f INA f r o m INH i s v i a A c I N H . A c I N H i s c l e a v e d t o INA a n d A c H z b y a n a m i d a s e , a n d A c H z may t h e n be a c e t y l a t e d p o l y m o r p h i c a l l y by N - a c e t y l t r a n s f e r a s e t o D i A c H z i n a m a n n e r a n a l o g o u s t o t h e a c e t y l a t i o n o f I N H . I n d i v i d u a l s d i f f e r e d i n t h e i r a b i l i t y t o c o n j u g a t e I N A w i t h g l y c i n e , a n d t h e s e d i f f e r e n c e s w e r e u n r e l a t e d t o t h e r a t e s a t w h i c h INH was a c e t y l a t e d . T h e c o n j u g a t i o n o f INA w i t h g l y c i n e a n d t h e a c e t y l a t i o n o f INH a p p e a r e d t o be p a r t i a l l y s a t u r a t e d in vivo a f t e r t h e a d m i n i s t r a t i o n o f d o s e s o f as l i t t l e a s 250 mg o f e i t h e r c o m p o u n d ( G o r d o n e t al., 1975) . A c H z was p o s t u l a t e d t o be t h e m e t a b o l i t e m o s t l i k e l y r e s p o n s i b l e f o r t h e h e p a t i c i n j u r y . A c H z was d e m o n s t r a t e d t o b e e l i m i n a t e d f r o m t h e b o d y b y a t l e a s t t h r e e r o u t e s ( T i m b r e l l e t al., 1977 ) . F i r s t , A c H z i s e x c r e t e d i n t h e u r i n e a s f r e e A c H z a n d as t h e a - o x o g l u t a r i c a c i d a n d p y r u v i c a c i d h y d r a z o n e s . S e c o n d , i t i s a c e t y l a t e d t o D i A c H z , w h i c h i s e x c r e t e d i n t h e u r i n e . T h i r d , t h e A c H z i s e l i m i n a t e d v i a m e t a b o l i s m by t h e h e p a t i c m i c r o s o m a l e n z y m e s y s t e m , a p a t h w a y t h a t i s t h o u g h t t o p r o d u c e t h e r e a c t i v e i n t e r m e d i a t e w h i c h i s r e s p o n s i b l e f o r t h e h e p a t o t o x i c i t y ( M i t c h e l l e t a l . , 1976; N e l s o n et al., 1976b). A s shown i n F i g . 2 , Z H O U , T . 11 N - a c e t y l t r a n s f e r a s e , a m i d a s e a n d 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 p l a y v e r y i m p o r t a n t r o l e s i n d i f f e r e n t s t e p s i n t h e m e t a b o l i c p a t h w a y s o f I N H . 1 . 1 . 5 P H A R M A C O K I N E T I C S INH i s r a p i d l y a b s o r b e d b o t h o r a l l y a n d p a r e n t e r a l l y . T h e a d m i n i s t r a t i o n o f t h e r a p e u t i c d o s e s (5 m g / k g / d ) r e s u l t s i n p e a k p l a s m a c o n c e n t r a t i o n s o f 3 - 5 ^ g / m l w i t h i n 1-2 h r s . INH d i f f u s e s r e a d i l y i n t o a l l b o d y f l u i d s a n d t i s s u e s . T h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n i s 0 . 6 7 + 0 . 1 5 1 / k g . T h e r e i s no a p p r e c i a b l e b i n d i n g t o p r o t e i n i n t h e p l a s m a . INH a n d m e t a b o l i t e s a r e p r i m a r i l y e x c r e t e d i n u r i n e . T h e c o n c e n t r a t i o n i n t h e c e n t r a l n e r v o u s s y s t e m a n d c e r e b r o s p i n a l f l u i d i s a b o u t o n e - f i f t h o f t h e p l a s m a l e v e l . T h e i n t r a c e l l u l a r a n d e x t r a c e l l u l a r l e v e l s a r e s i m i l a r . T h e a v e r a g e c o n c e n t r a t i o n o f a c t i v e INH i n t h e p l a s m a o f r a p i d a c e t y l a t o r s i s a b o u t o n e - t h i r d t o o n e - h a l f o f t h a t i n s l o w a c e t y l a t o r s . T h e a v e r a g e h a l f - l i f e o f INH i n r a p i d a c e t y l a t o r s i s l e s s t h a n 1 .5 h r s , w h e r e a s i n s l o w a c e t y l a t o r s t h e h a l f - l i f e i s 3 h r s . A b o u t 55% o f w h i t e a n d b l a c k p e r s o n s i n t h e USA a r e s l o w a c e t y l a t o r s , w h e r e a s 10% E s k i m o s , O r i e n t a l s , a n d a b o u t 30% N a t i v e A m e r i c a n s a r e s l o w a c e t y l a t o r s . Z H O U , T . 12 1 . 1 . 6 A D V E R S E E F F E C T S 1) . A l l e r g i c R e a c t i o n s : f e v e r , d e r m a t i t i s , a n g i i t i s a n d h e p a t i t i s a r e o c c a s i o n a l l y s e e n . 2) . I d i o s y n c r a t i c r e a c t i o n s : I N H - i n d u c e d l u p u s e r y t h r e m a t o s u s , r h e u m a t i c - l i k e s y n d r o m e s , v a s c u l i t i s a n d v a r i o u s h e m a t o l o g i c d i s o r d e r s s u c h as a g r a n u l o c y t o s i s ( M e h r o t r a a n d G u p t a , 1 9 7 3 ) , e o s i n o p h i l i a , t h r o m b o c y t o p e n i a , a n e m i a ( R o b i n s o n a n d F o a d i , 1969; B o t t o m l e y , 1982; M c C u r d y a n d D o n o h o e , 1 9 6 6 ) , a n d p u r e r e d c e l l a p l a s i a (Goodman a n d B l o c k , 1964; C l a i b o r n e a n d D u t t , 1 9 8 4 ) . I n 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 d e f i c i e n c y , INH may c a u s e h e m o l y s i s . 3) . D i r e c t T o x i c i t y : T h e m o s t common t o x i c e f f e c t s ( 1 0 -20%) a r e o n t h e p e r i p h e r a l a n d c e n t r a l n e r v o u s s y s t e m s . T h e s e h a v e b e e n a t t r i b u t e d t o a r e l a t i v e p y r i d o x i n e d e f i c i e n c y , p e r h a p s r e s u l t i n g f r o m c o m p e t i t i o n o f INH w i t h p y r i d o x a l p h o s p h a t e f o r a n enzyme ( a p o t r y p t o p h a n a s e ) . T h e s e t o x i c r e a c t i o n s i n c l u d e p e r i p h e r a l n e u r i t i s , i n s o m n i a , r e s t l e s s n e s s , m u s c l e t w i t c h i n g , d i z z i n e s s , a t a x i a , p a r e s t h e s i a , s t u p o r , u r i n a r y r e t e n t i o n , a n d e v e n o p t i c a t r o p h y , c o n v u l s i o n s t o x i c e n c e p h a l o p h t h y a n d m e n t a l a b n o r m a l i t i e s s u c h as e u p h o r i a , i m p a i r m e n t o f memory , s e p a r a t i o n o f i d e a s a n d r e a l i t y , l o s s o f s e l f - c o n t r o l , f l o r i d p s y c h o s e s a n d p s y c h o t i c e p i s o d e s ( M a n d e l l a n d S a n d e , 1 9 8 5 ) . M o s t o f t h e s e c o m p l i c a t i o n s c a n be p r e v e n t e d b y t h e a d m i n i s t r a t i o n o f p y r i d o x i n e . A c c i d e n t a l INH o v e r d o s e c a n be Z H O U , T . 13 t r e a t e d w i t h i n t r a v e n o u s p y r i d o x i n e i n a m o u n t s e q u i v a l e n t t o t h e INH i n g e s t e d . H e p a t o t o x i c i t y i s one o f m o s t s e r i o u s a n d e x t e n s i v e l y s t u d i e d a d v e r s e e f f e c t s o f I N H . T h e m e c h a n i s m o f I N H - i n d u c e d h e p a t o t o x i c i t y i s t h e t o p i c o f t h i s t h e s i s ; m o r e d e t a i l s a b o u t t h e r e l a t e d s t u d i e s w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . 1.2 H E P A T O T O X I C I T Y OF INH AND I T S M E T A B O L I T E S W i t h p r o l o n g e d u s e on a l a r g e s c a l e i n c h e m o p r o p h y l a x i s , INH h a s b e e n a s s o c i a t e d w i t h h e p a t o t o x i c i t y ; t h e i m p l i c a t i o n o f INH as a c a u s e o f c h r o n i c a c t i v e h e p a t i t i s a n d s u b a c u t e h e p a t i c n e c r o s i s has b e e n a c c e p t e d o n l y s i n c e 1 9 6 9 . A b n o r m a l l i v e r f u n c t i o n t e s t s , c l i n i c a l j a u n d i c e , s e v e r e a n d m u l t i l o b u l a r n e c r o s i s h a v e b e e n o b s e r v e d . R i s k o f INH h e p a t o t o x i c i t y i n c r e a s e s w i t h i n c r e a s i n g a g e . I t o c c u r s r a r e l y u n d e r age 2 0 , i n 1.5% o f t h o s e b e t w e e n 30 a n d 5 0 , a n d i n 2.5% o f o l d e r p e r s o n s . T h e r i s k o f h e p a t o t o x i c i t y s i g n i f i c a n t l y i n f l u e n c e s t h e d e c i s i o n f o r t h e p r o p h y l a c t i c u s e o f I N H . I n up t o 20% o f p a t i e n t s t a k i n g INH w i t h s m a l l r i s e i n a s p a r t a t e a m i n o t r a n s f e r a s e (AST) i s o b s e r v e d . I n many o f t h e s e p a t i e n t s INH c a n be c o n t i n u e d ( M i t c h e l l e t al., 1 9 7 5 a ) . S e v e r e INH h e p a t o t o x i c i t y n e c e s s i t a t e s p r o m p t d i s c o n t i n u a t i o n o f t h e d r u g , a s a number o f f a t a l i n t o x i c a t i o n c a s e s h a v e b e e n r e p o r t e d ( B l a c k e t al., 1 9 7 5 ) . Z H O U , T . 14 I t was f i r s t s u g g e s t e d i n 1974 t h a t t h e m e t a b o l i t e r e s p o n s i b l e f o r t h e l i v e r , n e c r o s i s was A c I N H ( S n o d g r a s s et al., 1 9 7 4 ) . L a t e r , e v i d e n c e f r o m a r a t m o d e l i n d i c a t e d t h a t t h e h e p a t o c e l l u l a r i n j u r y may be d u e t o t h e m e t a b o l i c a c t i v a t i o n o f A c H z , t h e f u r t h e r m e t a b o l i t e o f A c I N H , t o a r e a c t i v e i n t e r m e d i a t e ( N e l s o n e t al., 1 9 7 6 b ; M i t c h e l l e t al., 1976; T i m b r e l l e t al., 1 9 8 0 ) . A number o f i n v e s t i g a t o r s h a v e t r i e d b u t h a v e n o t b e e n a b l e t o r e p r o d u c e t h e r a t m o d e l o f A c H z h e p a t o t o x i c i t y ( W r i g h t e t al., 1 9 8 6 b ; T i m b r e l l J . A . , p e r s o n a l c o m m u n i c a t i o n ; J o l y J . G . , p e r s o n a l c o m m u n i c a t i o n ) . H z , a n o t h e r m e t a b o l i t e o f I N H , was r e c e n t l y i m p l i c a t e d as a c a u s e o f h e p a t i c n e c r o s i s i n t h e r a b b i t (Noda e t al., 1 9 8 3 ) . T h e r e f o r e , t h e m e c h a n i s m o f I N H - i n d u c e d h e p a t o t o x i c i t y r e m a i n s u n c l e a r . 1 . 2 . 1 INH A l t h o u g h INH i s w e l l known t o c a u s e t o x i c h e p a t i t i s i n man ( D i c k i n s o n e t al., 1981 ; G i r l i n g , 1 9 8 2 ) , t h e r e i s l i t t l e e v i d e n c e t h a t INH i t s e l f i s h e p a t o t o x i c . M o s t s t u d i e s s u g g e s t t h a t m e t a b o l i t e s a r e r e s p o n s i b l e f o r t h e l i v e r t o x i c i t y . H o w e v e r , INH h a s p o t e n t i a l e f f e c t s on t h e m e t a b o l i s m o f i t s m e t a b o l i t e s ; t h e r e b y i t c o u l d i n t e r f e r e w i t h t h e i r h e p a t o t o x i c i t y . I t was shown i n h u m a n s , when INH was a d m i n i s t e r e d w i t h l a b e l e d A c H z ( ^ - A c H z ) , t h e r a t e o f e l i m i n a t i o n o f A c H z was c o n s i s t e n t l y l o w e r t h a n i n t h e a b s e n c e o f I N H , a n d t h e Z H O U , T . 15 u r i n a r y e x c r e t i o n o f D i A c H z a n d t h e r a t i o o f D i A c H z / A c H z i n u r i n e d e c r e a s e d ( P e r e t t i e t al., 1 9 8 7 ) . T h e same r e s u l t was o b t a i n e d f r o m a r a t m o d e l a s w e l l . When INH was c o -a d m i n i s t e r e d w i t h - ^ C - A c H z t o r a t s , t h e r a t i o o f D i A c H z t o A c H z e x c r e t e d i n t h e u r i n e was s i g n i f i c a n t l y l o w e r t h a n when A c H z was a d m i n i s t e r e d a l o n e ( T i m b r e l l a n d W r i g h t , 197 9; T i m b r e l l , 1 9 7 9 ) . T h e s e s t u d i e s i n d i c a t e t h a t INH a n d i t s m e t a b o l i t e s i n h i b i t t h e a c e t y l a t i o n o f A c H z i n r a t s a n d h u m a n s . T h e i n h i b i t i o n o f t h i s d e t o x i f i c a t i o n p a t h w a y c o u l d l e a d t o a n i n c r e a s e i n t h e h e p a t o t o x i c i t y o f A c H z . I n a p a t h o l o g i c a l s t u d y , h o w e v e r , t h e r e s u l t s showed t h a t INH r e d u c e d t h e h e p a t o t o x i c i t y o f A c H z i n p h e n o b a r b i t a l ( P B ) - p r e t r e a t e d r a t s when b o t h a g e n t s w e r e a d m i n i s t e r e d s i m u l t a n e o u s l y ( B a h r i e t al., 1 9 8 1 ) . T h i s may be due t o t h e i n h i b i t i o n o f c y t o c h r o m e P - 4 5 0 m e d i a t e d a c t i v a t i o n o f A c H z b y INH ( T i m b r e l l a n d W r i g h t , 1979; T i m b r e l l , 1 9 7 9 ) . INH i n h i b i t s l i v e r m i c r o s o m a l e n z y m e s i n man ( K u t t e t al., 1 9 6 8 ) . T h i s i n h i b i t i o n o f A c H z m e t a b o l i s m b y INH c o u l d h a v e i m p o r t a n t i m p l i c a t i o n s i n t h e in vivo t o x i c i t y o f I N H : t h e h i g h e r t h e p l a s m a l e v e l o f I N H , t h e l e s s t o x i c w o u l d be t h e A c H z p r o d u c e d f r o m i t . T h i s may e x p l a i n i n p a r t why l a r g e s i n g l e d o s e s o f INH do n o t p r o d u c e l i v e r n e c r o s i s i n P B -p r e t r e a t e d a n i m a l s w h e r e a s s e v e r a l s m a l l e r d o s e s c a u s e d m i l d t o x i c i t y ( M i t c h e l l e t al., 1 9 7 6 ) . A l l I N H - i n d u c e d l i v e r damage may n o t be t h e c o n s e q u e n c e Z H O U , T . 16 o f a m e t a b o l i c t o x i c m e c h a n i s m . H e i s e y et al (1980) r e p o r t e d t h a t INH i n d u c e d h e p a t i t i s i s d u e t o a n a l l e r g i c m e c h a n i s m i n g u i n e a p i g s . I t was f o u n d t h a t INH a n d I N A , i t s m e t a b o l i t e , c o u l d c a u s e a c u t a n e o u s d e l a y e d - t y p e h y p e r s e n s i t i v i t y r e a c t i o n i n g u i n e a p i g a n d t h a t t h e h e p a t i t i s a f t e r c h r o n i c a d m i n i s t r a t i o n o f INH p r o b a b l y was a n a l l e r g i c r e a c t i o n t o b o t h INH a n d I N A . I n t h e same e x p e r i m e n t , e o s i n o p h i l i a a n d e o s i n o p h i l s p r e s e n t i n h e p a t i c l e s i o n s s u p p o r t t h i s s u g g e s t i o n . 1 . 2 . 2 A c I N H A c I N H , a f i r s t m a j o r m e t a b o l i t e o f I N H , i s f o r m e d by a c e t y l a t i o n o f I N H . I t was d e m o n s t r a t e d t h a t A c I N H c a u s e d h e p a t i c n e c r o s i s i n r a t s ( S n o d g r a s s e t al., 1974; M i t c h e l l e t al., 1 9 7 6 ) , a n d t h e n e c r o s i s was g r e a t l y p o t e n t i a t e d by P B . T h e s e r e s u l t s s u g g e s t e d t h a t A c I N H i s a h e p a t o t o x i n . H o w e v e r , e v i d e n c e t h a t A c H z , t h e p r i m a r y m e t a b o l i t e o f A c I N H i s t h e t o x i n f o l l o w s : (1) A c H z i s much more p o t e n t i n h e p a t o t o x i c i t y t h a n A c I N H i t s e l f ; (2) n e c r o s i s c a u s e d by A c I N H c a n be p r e v e n t e d b y p r e t r e a t m e n t w i t h b i s - p a r a -n i t r o p h e n y l p h o s p h a t e ( B N P P ) , w h i c h i n h i b i t s h y d r o l y s i s o f A c I N H t o A c H z ; (3) BNPP h a s no e f f e c t o n A c H z - i n d u c e d n e c r o s i s . Z H O U , T . 17 1 . 2 . 3 A c H z Some i n v e s t i g a t o r s p o s t u l a t e d t h a t I N H - r e l a t e d l i v e r damage i s c a u s e d by t h e m e t a b o l i t e , A c H z , f o r m e d by m e t a b o l i c h y d r o l y s i s o f A c I N H , t h e m a j o r m e t a b o l i t e o f INH ( M i t c h e l l e t al., 1 9 7 5 b ; N e l s o n e t al., 1 9 7 6 b ) . S t u d i e s i n humans ( T i m b r e l l e t al., 1977) h a v e c o n f i r m e d t h a t A c H z i s a m e t a b o l i t e o f INH i n m a n . S t u d i e s w i t h r a t l i v e r m i c r o s o m e s h a v e i n d i c a t e d t h a t m e t a b o l i c o x i d a t i o n o f A c H z by t h e m i c r o s o m a l P - 4 5 0 enzyme s y s t e m l e a d s t o a r e a c t i v e a c y l a t i n g a g e n t t h a t c o v a l e n t l y b i n d s t o m i c r o s o m a l p r o t e i n ( F i g . 3) ( N e l s o n e t al., 1976b; M i t c h e l l e t al., 1 9 7 6 ) . S t u d i e s i n r a t in vivo h a v e d e m o n s t r a t e d t h a t A c H z b i n d s c o v a l e n t l y t o r a t l i v e r p r o t e i n a n d t o t h e p r o t e i n o f o t h e r o r g a n s as w e l l . T h e c o v a l e n t b i n d i n g t o m a c r o m o l e c u l e s i n t h e l i v e r , b u t n o t t o t h o s e o f o t h e r o r g a n s , was i n c r e a s e d by p r e t r e a t m e n t w i t h P B . A c H z d i d n o t p r o d u c e d e t e c t a b l e l e s i o n s i n o r g a n s o t h e r t h a n t h e l i v e r , d e s p i t e t h e o c c u r r e n c e o f c o v a l e n t b i n d i n g t o p r o t e i n i n t h o s e o r g a n s . T h i s r e s u l t may s u g g e s t t h a t o n l y a c r i t i c a l f r a c t i o n o f t h e c o v a l e n t b i n d i n g t o s p e c i f i c s u b c e l l u l a r c o m p o n e n t s i s r e l a t e d t o t h e s u b s e q u e n t d e v e l o p m e n t o f t i s s u e l e s i o n s (Woodward a n d T i m b r e l l , 1 9 8 4 ) . H i s t o l o g i c a l s t u d y d e m o n s t r a t e d t h a t A c H z c a u s e s a d o s e - d e p e n d e n t c e n t r i l o b u l a r h e p a t i c n e c r o s i s i n P B - p r e t r e a t e d r a t s . - B a l l o o n i n g d e g e n e r a t i o n a n d i n f i l t r a t i o n by i n f l a m m a t o r y c e l l s w e r e c o n f i n e d t o t h e c e n t r i l o b u l a r a r e a i n t h e same a n i m a l s ZHOU, T . 18 o CH,CNHNH a Acetylhydrazine 0 CH.CNHNHOH Postulated Microsomal Pathway - H , 0 O CH,CN=NH O i CH,C * or CH.C* " I I O i -> CH.CNHNHCCH, Diacetylhydrazine O CH.CNHN-R a - O x o a c i d Hydrazones NH t NH t Hydrazine -> C H . C - O H - ^ C O , A c e t a t e Covalent Binding ^ Hepat ic to Macromo lecu le s Nec ro s i s F i g . 3 . Metabolic pathways of acetylhydrazine (AcHz). Z H O U , T . 19 ( B a h r i e t al., 1 9 8 1 ) . T h e p o s t u l a t e d m e t a b o l i c p a t h w a y i n v o l v e s a c e t y l a t i o n o f INH t o A c I N H f o l l o w e d b y c l e a v a g e t o A c H z , w h i c h u n d e r g o e s o x i d a t i o n t o y i e l d t h e r e a c t i v e a c e t y l g r o u p ( T i m b r e l l e t al., 1 9 8 0 ) . 1.2.4 Hz Hz i s a t o x i c ( B a c k e t al., 1 9 7 8 ) , c a r c i n o g e n i c ( T o t h , 1 9 8 0 ) , a n d m u t a g e n i c ( K i m b a l l , 1977) c o m p o u n d w h i c h i s a l s o a n i m p o r t a n t c h e m i c a l i n t e r m e d i a t e , a c o n s t i t u e n t o f r o c k e t f u e l , a n d a m e t a b o l i t e o f c e r t a i n d r u g s ( T i m b r e l l a n d H a r l a n d , 1979 ; B l a i r e t al., 1 9 8 0 ) . A l t h o u g h numerous b i o c h e m i c a l s t u d i e s h a v e b e e n c a r r i e d o u t o n Hz t o x i c i t y , t h e f a t e o f Hz in vivo a n d t h e m e c h a n i s m o f Hz h e p a t o t o x i c i t y a r e s t i l l l a r g e l y u n k n o w n . Hz h a s b e e n d e m o n s t r a t e d t o c a u s e a d o s e - r e l a t e d i n c r e a s e i n l i v e r t r i g l y c e r i d e s a n d i n l i v e r w e i g h t a n d t o c a u s e a d e c r e a s e i n h e p a t i c g l u t a t h i o n e . T h e a c c u m u l a t i o n o f l i p i d d r o p l e t s a n d t h e s w e l l i n g o f m i t r o c h o n d r i a w e r e d e t e c t a b l e b y e l e c t r o n m i c r o s c o p y 30 m i n a f t e r a d m i n i s t r a t i o n o f Hz ( S c a l e s a n d T i m b r e l l , 1 9 8 2 ) . U s i n g a m e a s u r e m e n t o f t h e i n c r e a s e i n l i v e r w e i g h t w h i c h m i r r o r s t h e h e p a t i c a c c u m u l a t i o n o f t r i g l y c e r i d e s t o a s s e s s t h e t o x i c i t y o f h y d r a z i n e , i t was d e m o n s t r a t e d t h a t p r e t r e a t m e n t o f a n i m a l s w i t h PB o r p i p e r o n y l b u t o x i d e r e s p e c t i v e l y d e c r e a s e s a n d i n c r e a s e s t h e h e p a t o t o x i c i t y . P r i o r d e p l e t i o n o f h e p a t i c g l u t a t h i o n e b y a d m i n i s t r a t i o n o f d i e t h y l m a l e a t e ZHOU, T . 20 h a d no e f f e c t o n t h e t o x i c i t y ( T i m b r e l l e t al., 1 9 8 2 ) . In vitro s t u d i e s i n d i c a t e d t h a t Hz i s m e t a b o l i z e d b y r a t l i v e r m i c r o s o m a l e n z y m e s . S t u d i e s o f t h e m e t a b o l i s m o f Hz h a v e i n d i c a t e d t h a t a c e t y l a t i o n t a k e s p l a c e t o A c H z a n d D i A c H z b u t o n l y t o a l i m i t e d e x t e n t ( W r i g h t a n d T i m b r e l l , 197 8; P e r r y e t al., 1 9 8 1 ) . A c H z i s p o t e n t i a l l y h e p a t o t o x i c a n d D i A c H z i s n o t h e p a t o t o x i c . M i c r o s o m a l e n z y m e - m e d i a t e d o x i d a t i v e m e t a b o l i s m may l e a d t o t h e p r o d u c t i o n o f n i t r o g e n , p e r h a p s v i a t h e d i i m i d e i n t e r m e d i a t e ; t h e r e i s e v i d e n c e t h a t e x p i r e d n i t r o g e n i s i n d e e d a m a j o r m e t a b o l i t e o f Hz i n m i c e in vivo ( N e l s o n a n d G o r d o n , 1 9 8 0 ) . A n a l t e r n a t i v e r o u t e o f m e t a b o l i s m i s t h e f o r m a t i o n o f h y d r a z o n e s , p a r t i c u l a r l y t h e p y r u v a t e a z i n e ( F i g . 4 ) . H o w e v e r , P y r u v a t e a z i n e i s v e r y much l e s s t o x i c t h a n Hz i t s e l f on a m o l a r b a s i s . T h e r e f o r e t h e f o r m a t i o n o f t h e h y d r a z o n e o f Hz w i t h p y r u v a t e seems t o be a d e t o x i c a t i o n r e a c t i o n . T h e s e a n d o t h e r r e s u l t s s u g g e s t t h a t a l t h o u g h Hz i s m e t a b o l i z e d v i a s e v e r a l r o u t e s , t h e h e p a t o t o x i c i t y may w e l l be due t o t h e p a r e n t c o m p o u n d r a t h e r t h a n a m e t a b o l i t e ( T i m b r e l l e t al., 1 9 8 2 ) . N o d a e t al p r o v e d t h a t t h e m a i n m e t a b o l i c p a t h w a y o f Hz i s o x i d a t i o n i n c o m p a r i s o n t o a c e t y l a t i o n (Noda e t al., 1983; N o d a e t al., 1 9 8 5 ) . T h e y i m p l i c a t e t h i s m e t a b o l i c p a t h w a y as t h e m a i n r e s p o n s i b i l i t y f o r t h e h e p a t o t o x i c i t y o f I N H . T h e e f f e c t s o f r i f a m p i c i n (RMP) on INH m e t a b o l i s m i n r a b b i t s w e r e u s e d as e v i d e n c e (Noda e t al., 1 9 8 3 ) : a f t e r t h e RMP p r e t r e a t m e n t , no r e m a r k a b l e c h a n g e s w e r e o b s e r v e d i n t h e ZHOU, T. 21 N H J - N H J Hydrazine O II C H , - C ~ N H - N H 1 Acetylhydrazine o o II II C H , C - N H - N H - C - C H , Diacetylhydrazine C H S C H S I I C = N - N = C l l C O O H C O O H Pyruvate Azine H A N » N H - O H H 2 N - N = 0 H N = N H or C o v a l e n t binding to l iver p ro te in I Liver necrosis F i g . 4. Metabolic pathways of hydrazine (Hz). Z H O U , T . 22 p l a s m a l e v e l s o f INH o r t h e m e t a b o l i t e s , A c I N H , A c H z a n d D i A c H z . P l a s m a l e v e l s o f Hz w e r e r e d u c e d . H i s t o l o g i c a l s t u d i e s d e m o n s t r a t e d t h a t Hz c a u s e d m o r e r e m a r k a b l e h e p a t i c n e c r o s i s i n r a b b i t s p r e t r e a t e d w i t h RMP t h a n i n n o n - R M P - p r e t r e a t e d r a b b i t s . T h e s e o b s e r v a t i o n s s u g g e s t t h a t Hz i s a k e y i n t e r m e d i a t e o f I N H - i n d u c e d h e p a t o t o x i c i t y t h r o u g h b i o t r a n s f o r m a t i o n t o a r e a c t i v e i n t e r m e d i a t e by m i c r o s o m a l o x i d a t i o n . 1 .3 F E A T U R E S OF I N H - I N D U C E D H E P A T O T O X I C I T Y 1 . 3 . 1 ENZYME INDUCTION OR I N H I B I T I O N F i g . 2 shows t h e r e a r e s e v e r a l m e t a b o l i c p a t h w a y s o f I N H : t h e f o r m a t i o n o f INH h y d r a z o n e s , a c e t y l a t i o n t o A c I N H w h i c h u n d e r g o e s f u r t h e r h y d r o l y s i s t o A c H z a n d I N A , o r d i r e c t h y d r o l y s i s t o Hz a n d I N A . M e t a b o l i c s t u d i e s w i t h A c H z h a v e i n d i c a t e d t h a t t h e r e a r e a l s o s e v e r a l p a t h w a y s o f m e t a b o l i s m o t h e r t h a n t h e m i c r o s o m a l e n z y m e - m e d i a t e d p a t h w a y : t h e f o r m a t i o n o f h y d r a z o n e s , a c e t y l a t i o n t o D i A c H z , a n d p o s s i b l y h y d r o l y s i s t o a c e t a t e a n d Hz ( s e e F i g . 3) ( W r i g h t a n d T i m b r e l l , 1978; T i m b r e l l e t a l . , 1 9 8 0 ) . The m e t a b o l i c p a t h w a y s o f H z , a s m e n t i o n e d a b o v e , a t l e a s t i n c l u d e a c e t y l a t i o n a n d o x i d a t i o n . T h e r e l a t i v e i m p o r t a n c e o f t h e s e p a t h w a y s w i l l be c r u c i a l t o t h e d e v e l o p m e n t o f t h e t o x i c i t y . T h e a c t i v i t i e s o f t h e e n z y m e s , i . e . , a c e t y l t r a n s f e r a s e , a m i d a s e , a n d Z H O U , T . 23 m i c r o s o m a l e n z y m e s a r e c r i t i c a l i n t h e s e p a t h w a y s ; a n y f a c t o r w h i c h c a n i n d u c e o r i n h i b i t t h e s e e n z y m e s may a l t e r t h e m e t a b o l i s m o f INH a n d t h e r e b y a f f e c t I N H - i n d u c e d h e p a t o t o x i c i t y . I t h a s b e e n shown t h a t I N H , t h e p a r e n t d r u g f r o m w h i c h A c H z i s f o r m e d in v i v o , may i n t e r a c t w i t h A c H z . T h u s , in v i v o , c o a d m i n i s t e r e d INH i n h i b i t s t h e a c e t y l a t i o n o f A c H z i n r a t s ( W r i g h t a n d T i m b r e l l , 1978) w h e r e a s in v i t r o INH i n h i b i t s t h e m i c r o s o m a l e n z y m e - m e d i a t e d c o v a l e n t b i n d i n g o f A c H z t o p r o t e i n ( T i m b r e l l a n d W r i g h t , 1 9 7 9 ) . F r o m F i g . 3 i t c a n b e a p p r e c i a t e d t h a t i n h i b i t i o n o f a c e t y l a t i o n o f A c H z c o u l d d i v e r t more A c H z t h r o u g h t h e t o x i c p a t h w a y a n d t h e r e f o r e i n c r e a s e t o x i c i t y . C o n v e r s e l y , i n h i b i t i o n o f t h e m i c r o s o m a l e n z y m e - m e d i a t e d p a t h w a y w o u l d r e d u c e t h e t o x i c i t y . RMP i s a n a g e n t w h i c h i s commonly c o a d m i n i s t e r e d w i t h INH i n t h e t r e a t m e n t o f t u b e r c u l o s i s . I t h a s b e e n s u g g e s t e d t h a t t h i s c o m b i n a t i o n c a u s e s more s e r i o u s h e p a t i t i s t h a n t h e a d m i n i s t r a t i o n o f INH a l o n e ( P e s s a y r e e t al., 1 9 7 7 ) . H o w e v e r , r e v i e w o f t h e l i t e r a t u r e i s m o s t l y a g a i n s t RMP i n c r e a s i n g t h e i n c i d e n c e o f INH h e p a t o t o x i c i t y . I n a n i m a l s RMP i s a n u n p r e d i c t a b l e i n d u c e r , w i t h l a r g e s p e c i e s d i f f e r e n c e s ( v a n d e n B r o e k e t al, 1 9 8 0 ) , e . g . , i t i s d e m o n s t r a t e d t o i n d u c e c y t o c h r o m e P - 4 5 0 i n m i c e ( P e s s a y r e a n d M a z e l , 197 6; H e u b e l a n d N e t t e r , 197 9) a n d i n r a b b i t s (Thomas a n d Z e i t z , 1980; N o d a e t al., 1983; W h i t e h o u s e e t al., 1 9 8 5 ) , b u t n o t g u i n e a p i g s ( v a n d e n B r o e k e t al., Z H O U , T . 24 1 9 8 0 ) . R a t s h a v e shown d i f f e r e n t r e s u l t s , e . g . , i n d u c t i o n , r e d u c t i o n a n d no s i g n i f i c a n t c h a n g e o f c y t o c h r o m e P - 4 5 0 ( P e s s a y r e a n d M a z e l , 1976; v a n d e n B r o e k e t al., 1980; B a h r i e t al., 1 9 8 1 ; P i r i o u e t al., 1 9 8 3 ) . A f t e r a d m i n i s t r a t i o n o f INH t o r a t s , t h e s e r u m c o n c e n t r a t i o n s o f Hz i n RMP o r P B -p r e t r e a t e d g r o u p s w e r e l o w e r t h a n t h o s e i n t h e c o n t r o l g r o u p , w h i l e t h e c o n c e n t r a t i o n s o f A c H z w e r e u n a l t e r e d . I n e a c h o f t h e p r e t r e a t e d g r o u p s a p r o n o u n c e d i n c r e a s e i n t h e o x i d a t i v e e l i m i n a t i o n r a t e o f Hz was o b s e r v e d (Noda e t al., 1 9 8 5 ) . A s m e n t i o n e d a b o v e h i s t o l o g i c a l s t u d i e s p r o v e d g r e a t e r h e p a t i c n e c r o s i s c a u s e d b y Hz i n r a b b i t s p r e t r e a t e d w i t h RMP t h a n i n t h e c o n t r o l r a b b i t s . I n humans RMP i s a p o t e n t i n d u c e r o f c y t o c h r o m e P - 4 5 0 , b u t many s t u d i e s showed no e v i d e n c e t h a t g i v i n g RMP w i t h INH i n c r e a s e s t h e r i s k o f h e p a t i t i s (Remmer H . e t al., 197 3; G i r l i n g , 1978; G i r l i n g , 1982; T i m b r e l l e t al., 1 9 8 5 ) . A m i d a s e w h i c h h y d r o l y z e s INH t o INA a n d Hz a n d A c I N H t o INA a n d A c I N H a l s o p l a y s a n i m p o r t a n t r o l e i n INH m e t a b o l i s m . I t h a s b e e n shown t h a t t h i s enzyme i s i n h i b i t e d b y A c I N H , t h e m a i n m e t a b o l i t e o f I N H . A m i d a s e i s a l s o s t r o n g l y i n d u c e d i n r a t s b y PB a n d RMP, r e s p e c t i v e l y (Sendo e t al., 1 9 8 4 ) . T h e r e f o r e , s t u d i e s i n w h i c h i n d u c t i o n w i t h t h e s e a g e n t s i n c r e a s e s t o x i c i t y m u s t be i n t e r p r e t e d w i t h c a u t i o n . M i c r o s o m a l enzyme i n d u c e r s h a v e n o t b e e n shown t o i n d u c e N - a c e t y l t r a n s f e r a s e a c t i v i t y . H y d r o c o r t i s o n e has b e e n r e p o r t e d t o c a u s e h y p e r t r o p h y o f t h e h e p a t i c t i s s u e Z H O U , T . 25 r e s u l t i n g i n a g r e a t e r a c e t y l t r a n s f e r a s e a c t i v i t y ( R e e v e s e t al., 1 9 8 7 ) . L a r g e d o s e s o f h y d r o c o r t i s o n e i n d u c e s u l f a m e t h a z i n e (SMZ) a c e t y l a t i o n i n b o t h " f a s t " a n d "s low" a c e t y l a t o r r a b b i t s ( S o u i c h a n d C o u r t e a u , 1 9 8 1 ) . 1 . 3 . 2 ANIMAL S P E C I E S I n t h e s e a r c h f o r a s u i t a b l e a n i m a l m o d e l i t h a s b e e n f o u n d t h a t t h e r a t , mouse a n d g u i n e a p i g a r e a l l l e s s s u s c e p t i b l e t o INH i n d u c e d h e p a t o t o x i c i t y t h a n man (Thomas e t al., 1 9 8 1 ) . No s t u d i e s h a v e shown h i s t o l o g i c a l o r b i o c h e m i c a l i n d i c a t i o n o f n e c r o s i s i n t h e r a t a r i s i n g f r o m c h a l l e n g e w i t h INH i t s e l f . A c c o r d i n g t o B a h r i a n d T i m b r e l l ( 1 9 8 1 ) , n e c r o s i s c a n o n l y be o b s e r v e d i n r a t s t r e a t e d w i t h PB b e f o r e c h a l l e n g e w i t h A c H z . C o v a l e n t m o d i f i c a t i o n o f l i v e r m a c r o m o l e c u l e s h a s b e e n d e m o n s t r a t e d b y t r e a t m e n t o f r a t s ( o r r a t l i v e r m i c r o s o m e s ) w i t h A c I N H o r A c H z w i t h o r w i t h o u t PB p r e t r e a t m e n t , b u t t h e r e l a t i o n s h i p b e t w e e n h i s t o l o g i c a l l y d e m o n s t r a t e d l i v e r n e c r o s i s a n d c o v a l e n t b i n d i n g d o e s n o t a p p e a r t o be p a r a l l e l (Woodward a n d T i m b r e l l 1984 ) . A n o t h e r p r o b l e m w i t h t h e r a t m o d e l i s t h e i n a b i l i t y i n t h i s l a b o r a t o r y a n d o t h e r s t o e v e r p r o d u c e a n y e v i d e n c e o f n e c r o s i s i n t h e r a t ( r e g a r d l e s s o f s t r a i n ) w i t h I N H , A c I N H , A c H z o r Hz i n n a i v e o r PB p r e t r e a t e d a n i m a l s . F u r t h e r m o r e , n e i t h e r t h i s l a b o r a t o r y n o r a n y p r e v i o u s s t u d y h a s r e p o r t e d s i g n i f i c a n t r e l e a s e i n t h e r a t o f t h e m a r k e r enzymes o f Z H O U , T . 26 n e c r o s i s , i . e . , a s p a r t a t e a m i n o t r a n s f e r a s e ( A S T ) a n d a l a n i n e a m i n o t r a n s f e r a s e ( A L T ) . D i s s a t i s f a c t i o n w i t h t h e r a t i n e x p e r i m e n t a l s t u d i e s o f INH h e p a t o t o x i c i t y l e d t o i n v e s t i g a t i o n o f t h e r a b b i t r e s p o n s e t o INH b y W h i t e h o u s e e t al. (1978) a n d o u r l a b o r a t o r y . T h e s e s t u d i e s s u g g e s t e d t h a t INH t r e a t m e n t d i d c a u s e l i v e r damage r e s u l t i n g i n r e l e a s e o f s i g n i f i c a n t a m o u n t s o f A S T a n d A L T i n t o t h e s e r u m i n t h e r a b b i t . By s t u d y i n g t h e m e t a b o l i s m o f - ^ C - A c I N H b y t h e r a t a n d r a b b i t , Thomas e t al. (1984) i n v e s t i g a t e d t h e m e c h a n i s m f o r t h e g r e a t e r s e n s i t i v i t y t o I N H - i n d u c e d h e p a t o t o x i c i t y o f t h e r a b b i t c o m p a r e d t o t h e r a t ; t h e r e s u l t s s u g g e s t e d t h a t h i g h e r a m i d a s e a c t i v i t y i n t h e r a b b i t c o m p a r e d t o t h e r a t l e a d s t o g r e a t e r c o n v e r s i o n o f A c I N H t o A c H z w h i c h may t h e r e f o r e l e a d t o g r e a t e r h e p a t o t o x i c i t y . N o d a e t al. (1983) d e m o n s t r a t e d h i s t o l o g i c a l l y t h a t Hz c a u s e d l i v e r c e n t r i l o b u l a r n e c r o s i s i n r a b b i t s . T h e y showed Hz was f o r m e d f r o m INH i n r a b b i t . F u r t h e r m o r e , N - a c e t y l t r a n s f e r a s e a c t i v i t y i s p o l y m o r p h i c a l l y i n h e r i t e d i n t h e human b u t n o t i n t h e r a t , m o u s e , o r g u i n e a p i g . T h e r a b b i t , h o w e v e r , shows s i m i l a r g e n e t i c a l l y d e t e r m i n e d N - a c e t y l t r a n s f e r a s e a c t i v i t y t o t h e human ( W h i t e h o u s e e t al., 1 9 7 8 ) , a n d t h e a b s e n c e o f N - a c e t y l t r a n f e r a s e i n l i v e r o f s l o w a c e t y l a t o r r a b b i t s was d e m o n s t r a t e d t o be d u e t o a gene d e l e t i o n ( B l u m e t al., 1 9 8 9 ) , t h e r e f o r e t h e r a b b i t p r o v i d e s a b e t t e r m o d e l f o r t h e s t u d y o f h e p a t o t o x i c i t y i n t h e human. Z H O U , T . 27 1 . 3 . 3 A C E T Y L A T O R PHENOTYPE ACETYLATOR PHENOTYPE IN HUMAN AND RABBIT S h o r t l y a f t e r t h e i n t r o d u c t i o n o f I N H , i t was f o u n d t h a t t h e r e e x i s t e d u n u s u a l l y l a r g e p e r s o n - t o - p e r s o n d i f f e r e n c e s i n t h e c o n v e r s i o n o f t h e p a r e n t d r u g t o i n a c t i v e m e t a b o l i t e s . S u b s e q u e n t s t u d i e s showed t h e s e d i f f e r e n c e s t o be r e p r o d u c i b l e , s t a b l e , i n d i v i d u a l c h a r a c t e r i s t i c s t h a t a r e m a n i f e s t w h e t h e r t h e d r u g i s m e a s u r e d i n b l o o d o r u r i n e a n d a f t e r e i t h e r o r a l o r i n t r a v e n o u s a d m i n i s t r a t i o n . B r o a d e r i n v e s t i g a t i o n s o f l a r g e p o p u l a t i o n s o f t u b e r c u l o s i s p a t i e n t s h e a l t h y s u b j e c t s , i n c l u d i n g f a m i l y s t u d i e s w i t h i d e n t i c a l a n d f r a t e r n a l t w i n s h a v e c l e a r l y e s t a b l i s h e d t h a t a c e t y l a t i o n a b i l i t y i s u n d e r g e n e t i c c o n t r o l . T h e same p h e n o m e n o n o f i n h e r i t e d d i f f e r e n c e s i n d r u g m e t a b o l i s m was f o u n d i n t h e r a b b i t . I t h a s b e e n shown t h a t m a m m a l i a n t i s s u e s c o n t a i n a v a r i e t y o f N - a c e t y l a t i n g e n z y m e s t h a t c a t a l y z e t h e t r a n s f e r o f t h e a c e t y l g r o u p f r o m a c e t y l -c o e n z y m e A t o d r u g s . One o f t h e s e e n z y m e s , INH N -a c e t y l t r a n s f e r a s e , i s p r e s e n t i n t h e l i v e r a n d d u o d e n a l m u c o s a i n man a n d t h e r a b b i t (Weber e t al., 1 9 7 5 ) . In vivo a n d in vitro s t u d i e s o f t h e g e n e t i c c o n t r o l o f s u l f a d i a z i n e a n d INH m e t a b o l i s m i n t h e r a b b i t s u g g e s t e d t h a t SD a n d INH a c e t y l a t i o n r a t e may be c o n t r o l l e d b y a s i m p l e M e n d e l i a n a u t o s o m a l c h a r a c t e r i s t i c s , w i t h r a p i d a c e t y l a t i o n d o m i n a n t Z H O U , T . 28 t o s l o w ( F r y m o y e r a n d J a c o x , 1 9 6 3 a ; F r y m o y e r a n d J a c o x , 1 9 6 3 b ) . T h e d i f f e r e n t amounts o f a c e t y l a s e a c t i v i t y c o r r e s p o n d t o t h e h o m o z y g o u s r e c e s s i v e , h e t e r o z y g o u s a n d h o m o z y g o u s d o m i n a n t c o n d i t i o n s , r e s p e c t i v e l y , e . g . , s l o w a c e t y l a t o r s a r e h o m o z y g o u s r e c e s s i v e ; r a p i d a c e t y l a t o r s a r e d o m i n a n t RR o r R r . T h e s e a n d e a r l i e r s t u d i e s e s t a b l i s h e d t h e u s e o f t h e t e r m s " r a p i d , " a n d "s low" a c e t y l a t o r s t o d e s i g n a t e v a r y i n g c a p a c i t i e s t o a c e t y l a t e INH a n d o t h e r d r u g s ( P e t e r s e t al., 1 9 6 5 ) . I t was i n i t i a l l y r e p o r t e d t h a t r a p i d a c e t y l a t o r s a r e a t g r e a t e r r i s k o f INH h e p a t o t o x i c i t y p r e s u m a b l y b e c a u s e t h e y p r o d u c e m o r e A c I N H a n d t h u s A c H z . T h e d i s c o v e r y t h a t A c H z i s a l s o a c e t y l a t e d by t h e g e n e t i c a l l y d e t e r m i n e d N - a c e t y l t r a n s f e r a s e c o m p l i c a t e d t h e p i c t u r e . I n s t u d i e s c o m p a r i n g u r i n a r y r e c o v e r y o f m e t a b o l i t e s o f INH i n r a p i d a n d s l o w a c e t y l a t o r s , r a p i d a c e t y l a t o r s e x c r e t e more A c I N H a n d D i A c H z t h a n s l o w a c e t y l a t o r s ; t h e amount o f A c H z was s i m i l a r i n b o t h p h e n o t y p e s . EFFECT OF ACETYLATOR PHENOTYPE ON INH METABOLISM When p l a s m a c o n c e n t r a t i o n s o f INH a n d i t s m e t a b o l i t e s , A c I N H , A c H z a n d D i A c H z , w e r e m e a s u r e d i n h e a l t h y human Z H O U , T . 29 s u b j e c t s a f t e r t h e i n g e s t i o n o f I N H , t h e a r e a s u n d e r t h e p l a s m a c o n c e n t r a t i o n - t i m e c u r v e o f A c I N H a n d D i A c H z w e r e g r e a t e r i n r a p i d a c e t y l a t o r s o f I N H . I n c o n t r a s t , t h e a r e a u n d e r t h e p l a s m a c o n c e n t r a t i o n - t i m e c u r v e o f A c H z , t h e p o s t u l a t e d p r e c u r s o r o f a t o x i c m e t a b o l i t e f o r m e d f r o m I N H , was g r e a t e r i n s l o w a c e t y l a t o r s . T h i s o c c u r r e d e v e n t h o u g h r a p i d a c e t y l a t o r s g e n e r a t e d more A c I N H a n d t h u s more A c H z f r o m I N H , b e c a u s e t h e r a p i d a c e t y l a t o r s a l s o a c e t y l a t e d A c H z f a s t e r t o D i A c H z t h a n d i d t h e s l o w a c e t y l a t o r s . Due t o t h i s c o m p l e x r e l a t i o n s h i p b e t w e e n a r e a s u n d e r t h e p l a s m a c o n c e n t r a t i o n - t i m e c u r v e o f A c H z a n d t h e r a t e o f INH a c e t y l a t i o n ( i . e . , a f a s t e r r a t e o f f o r m a t i o n o f A c H z i s a c c o m p a n i e d b y a f a s t e r c l e a r a n c e t o D i A c H z ) , t h e r a t e o f a c e t y l a t i o n o f INH m i n i m a l l y i n f l u e n c e s t h e e x p o s u r e o f m o s t p a t i e n t s t o A c H z . T h i s p h a r m a c o k i n e t i c a n a l y s i s h a s a l s o shown t h a t t h e p l a s m a h a l f - l i f e o f A c H z i s a b o u t f i v e t i m e s l o n g e r t h a n t h a t o f I N H , a n d t h u s r e p e a t e d d o s e s o f INH s h o u l d l e a d t o a n a c c u m u l a t i o n o f A c H z i n t h e s l o w e s t a c e t y l a t o r s i n w h i c h t h e p l a s m a h a l f - l i f e o f A c H z i s 2 0 - p l u s h r s . F u r t h e r m o r e , a l a r g e d o s e (10 m g / k g ) o f INH l e a d s t o s a t u r a t i o n o f a c e t y l a t i o n , w h i c h may be e s p e c i a l l y c o n t r i b u t o r y t o a n a c c u m u l a t i o n o f A c H z i n s l o w a c e t y l a t o r s b y d e c r e a s i n g c l e a r a n c e t o D i A c H z ( L a u t e r b u r g e t al., 1 9 8 5 b ) . Z H O U , T . 30 EFFECT OF ACETYLATOR PHENOTYPE ON INH-INDUCED HEPATOTOXICITY S t u d i e s i n man t o d e t e r m i n e w h e t h e r t h o s e g e n e t i c a l l y c l a s s i f i e d as f a s t a c e t y l a t o r s a r e more o r l e s s s u s c e p t i b l e t o I N H - i n d u c e d h e p a t o t o x i c i t y h a v e p r o d u c e d c o n t r a d i c t o r y r e s u l t s . M i t c h e l l e t al. (1975b) c o n c l u d e d t h a t r a p i d a c e t y l a t o r s a r e a t more r i s k b u t m o r e r e c e n t p r o s p e c t i v e s t u d i e s ( D i c k i n s o n e t al., 1981 ; M u s c h e t al., 1982; L a u t e r b u r g e t a i . , 1985a) h a v e f o u n d t h e r e v e r s e . M i t c h e l l ' s g r o u p h a v e p r e s e n t e d more r e c e n t e v i d e n c e t h a t s l o w a c e t y l a t o r s a r e a t g r e a t e r r i s k . O f i n t e r e s t i s t h e f a c t t h a t t h e y f a i l e d t o r e f e r t o t h e i r p r e v i o u s p a p e r d e m o n s t r a t i n g t h e o p p o s i t e . I n a n e x p e r i m e n t (Thomas e t al., 1987) w h e r e ^ C - A c I N H was a d m i n i s t e r e d o r a l l y t o r a b b i t s a t a d o s e o f 200 m g / k g , i t was f o u n d t h a t c o v a l e n t b i n d i n g o f t o h e p a t i c p r o t e i n i n c r e a s e d as t h e a c e t y l a t i o n r a t e d e c r e a s e d . I n p l a s m a a n d u r i n e , A c H z l e v e l s w e r e n e g a t i v e l y c o r r e l a t e d w i t h a c e t y l a t i o n r a t e a n d D i A c H z l e v e l s w e r e p o s i t i v e l y c o r r e l a t e d . I f b i n d i n g i s r e s p o n s i b l e f o r I N H - i n d u c e d h e p a t o t o x i c i t y , t h i s i s a v i t a l new o b s e r v a t i o n . H o w e v e r , i n t h e s e s t u d i e s A c I N H a n d n o t INH i t s e l f was a d m i n i s t e r e d . When INH i s g i v e n , b i n d i n g o f t h e a c e t y l m o i e t y c a n n o t be t e s t e d as i t i s e n d o g e n o u s l y d e r i v e d . A f t e r a d m i n i s t r a t i o n o f INH t h e r a t e o f A c I N H f o r m a t i o n i s d e t e r m i n e d by t h e r a t e Z H O U , T . 31 o f a c e t y l a t i o n ; t h i s w i l l t e n d t o c o u n t e r a c t t h e e f f e c t s s e e n i n t h i s s t u d y . L e v e l s o f t h e o t h e r h e p a t o t o x i c m e t a b o l i t e , H z , w e r e m e a s u r e d i n s e r u m i n c l i n i c a l s t u d i e s . I t was f o u n d t h a t h y d r a z i n e c o n c e n t r a t i o n s a r e h i g h e r i n human s l o w a c e t y l a t o r s t h a n f a s t a c e t y l a t o r s when INH i s a d m i n i s t e r e d ( B l a i r e t al., 1985; S a r m a e t al., 1 9 8 6 ) . I t i s i m p o r t a n t t o be a w a r e t h a t f a c t o r s o t h e r t h a n c o v a l e n t b i n d i n g o f t h e a c e t y l m o i e t y o f A c H z may be i n v o l v e d i n h e p a t o t o x i c i t y o f I N H . 1.4 SERUM A R G I N I N O S U C C I N A T E L Y A S E AS A MARKER ENZYME  I N H E P A T O T O X I C STUDY A r g i n i n o s u c c i n i c a c i d l y a s e ( A S A L ) i s a n enzyme o f t h e u r e a c y c l e w h i c h h a s b e e n shown t o e x i s t i n m a m m a l i a n k i d n e y a n d l i v e r . T h e a c t i o n o f A S A L i s t o a c t o n a r g i n i n o s u c c i n i c a c i d , t h u s l i b e r a t i n g a r g i n i n e a n d f u m a r i c a c i d . A r g i n i n e i s i n t u r n a c t e d u p o n by a r g i n a s e t o f o r m u r e a i n t h e u r e a c y c l e ( T a k a h a r a a n d N a t e l s o n , 1 9 6 7 ) . T h e r e a s o n t h a t s e r u m A S A L was be c h o s e n t o m o n i t o r h e p a t o t o x i c i t y s temmed f r o m c l i n i c a l l i t e r a t u r e w h i c h s u g g e s t e d t h a t r e l e a s e o f t h i s enzyme i n t o p l a s m a i s a more s e n s i t i v e a n d s p e c i f i c i n d i c a t o r o f l i v e r damage i n man t h a n Z H O U , T . 32 s e r u m A S T o r A L T . T h e l a t t e r two h a v e b e e n w e l l e s t a b l i s h e d as s e n s i t i v e i n d i c a t o r s o f h e p a t o c e l l u l a r damage ( C a m p a n i n i e t al., 1970; S i m s a n d R a u t a n e n , 1 9 7 5 ) . A c o m p a r i s o n b e t w e e n r e l e a s e o f A S A L a n d r e l e a s e o f A S T a n d A L T t o f o u r d r u g t r e a t m e n t s showed s i g n i f i c a n t c o r r e l a t i o n b e t w e e n l o g A S A L a n d l o g A S T ( r = 0 . 8 1 6 ) a n d b e t w e e n l o g A S A L a n d l o g A L T ( r = 0 . 8 7 9 ) ( W a l l e t al., u n p u b l i s h e d d a t a ) . H o w e v e r , t h e r e l a t i o n s h i p b e t w e e n A S A L a n d p a t h o l o g i c a l n e c r o s i s h a s n o t b e e n e s t a b l i s h e d . 1 .5 THE O B J E C T I V E S OF OUR S T U D I E S 1 . 5 . 1 H Y P O T H E S I S (1) I N H - i n d u c e d h e p a t o t o x i c i t y may be c a u s e d by one o f i t s m e t a b o l i t e s r a t h e r t h a n t h e p a r e n t d r u g i t s e l f . I n t h i s c a s e , t h e p o t e n c y o f t h e t o x i c i t y s h o u l d be g r e a t e r u s i n g t h e m e t a b o l i t e t h a n w i t h I N H . (2) A n y f a c t o r w h i c h h a s an e f f e c t o n t h e t o x i c INH m e t a b o l i c p a t h w a y , s u c h as t h e i n d u c t i o n o f t h e t o x i c p a t h w a y o r i n c r e a s e d f o r m a t i o n o f t h e t o x i c m e t a b o l i t e may a l t e r t h e INH h e p a t o t o x i c i t y . T h e a i m o f t h e s e s t u d i e s was t o t e s t t h e h e p a t o t o x i c p o t e n c y o f INH a n d i t s m e t a b o l i t e s i n a r a b b i t m o d e l , a n d e v a l u a t e t h e r o l e o f t h e m i c r o s o m a l e n z y m e s a n d r a t e o f a c e t y l a t i o n o n t h i s t o x i c i t y . Z H O U , T . 33 1 . 5 . 2 TO E S T A B L I S H SERUM A S A L AS A MARKER ENZYME I N THE  R A B B I T MODEL T h e r a b b i t was c h o s e n as t h e e x p e r i m e n t a l m o d e l f o r t h e f o l l o w i n g r e a s o n s : 1) i t h a s b e e n shown t o e x h i b i t e l e v a t i o n s i n s e r u m A S T a n d A L T enzymes when s u b j e c t e d t o c h r o n i c INH c h a l l e n g e ( M a r t i n e s e t al., 1 9 8 0 ) . 2) INH has b e e n d e m o n s t r a t e d t o p r o d u c e h e p a t i c n e c r o s i s , a n d o t h e r m e t a b o l i c c h a n g e s i n t h i s m o d e l (Thomas e t al., 1981; W h i t e h o u s e e t al., 1983; Thomas e t al, 1 9 8 4 ) . 3) a c e t y l a t i o n r a t e i s g e n e t i c a l l y d e t e r m i n e d i n t h e r a b b i t , i n a m a n n e r s i m i l a r t o h u m a n s . A S A L was c h o s e n as t h e b i o c h e m i c a l i n d i c a t o r f o r l i v e r damage i n t h i s s t u d y . I t was m e a s u r e d b e f o r e a n d a f t e r d r u g c h a l l e n g e f o r e a c h e x p e r i m e n t a l a n i m a l . I n o r d e r t o e s t a b l i s h t h e v a l i d i t y o f m e a s u r i n g p l a s m a A S A L l e v e l s as a means o f a s s e s s i n g h e p a t o t o x i c i t y i n t h e r a b b i t , we n e e d t o e s t a b l i s h a r e l a t i o n s h i p b e t w e e n s e r u m A S A L l e v e l s a n d h e p a t i c n e c r o s i s i n t h e r a b b i t m o d e l b y d o i n g h i s t o l o g i c a l e x a m i n a t i o n o f t h e l i v e r . T h u s , i n some r a b b i t s b o t h b i o c h e m i c a l m e a s u r e m e n t a n d h i s t o l o g i c a l e x a m i n a t i o n w e r e c o m p a r e d t o e s t i m a t e t h e v a l i d i t y f o r A S A L b e i n g u s e d as a m a r k e r enzyme o f l i v e r n e c r o s i s . Z H O U , T . 34 1 . 5 . 3 TO COMPARE THE H E P A T O T O X I C I T Y OF INH AND I T S  M E T A B O L I T E S T h e r e l a t i v e p o t e n c y o f t o x i c i t y o f INH a n d i t s m e t a b o l i t e s i s a n i m p o r t a n t s t e p i n t h e u n d e r s t a n d i n g o f t h e m e c h a n i s m o f I N H - i n d u c e d h e p a t o t o x i c i t y . By c o m p a r i n g t h e e l e v a t i o n o f s e r u m A S A L l e v e l , t h e d i f f e r e n c e o f h e p a t o t o x i c i t y b e t w e e n t h e same m o l a r d o s e s o f INH a n d some o f i t s m e t a b o l i t e s , i . e . A c I N H , A c H z a n d H z , was t e s t e d . I n a d d i t i o n , INH was a d m i n i s t e r e d o r a l l y o r s u b c u t a n e o u s l y t o t e s t w h e t h e r d i r e c t d e l i v e r y o f INH t o t h e l i v e r b y t h e p o r t a l c i r c u l a t i o n a f t e r o r a l a d m i n i s t r a t i o n a f f e c t e d t h e t o x i c i t y . 1 . 5 . 4 TO C L A R I F Y THE L I N K BETWEEN A C E T Y L A T I O N R A T E AND I N H - INDUCED H E P A T O T O X I C I T Y C o n t r a d i c t o r y r e s u l t s a b o u t t h e r e l a t i o n s h i p b e t w e e n t h e a c e t y l a t i o n r a t e a n d I N H - i n d u c e d h e p a t o t o x i c i t y h a s b e e n r e p o r t e d b y d i f f e r e n t a u t h o r s ( M i t c h e l l e t al., 1975b; D i c k i n s o n e t al., 1981 ; M u s c h e t al., 1982; L a u t e r b u r g e t al., 1 9 8 5 a ; Thomas e t al., 1 9 8 7 ) . A n a n a l y s i s o f t h e r e l a t i o n s h i p b e t w e e n a c e t y l a t i o n r a t e a n d t h e e l e v a t i o n o f s e r u m A S A L l e v e l i n t h e r a b b i t c h a l l e n g e d w i t h INH o r i t s m e t a b o l i t e s was u n d e r t a k e n t o c l a r i f y a n y l i n k b e t w e e n N - a c e t y l t r a n s f e r a s e a c t i v i t y a n d h e p a t o t o x i c i t y . Z H O U , T . 35 1 . 5 . 5 TO C L A R I F Y THE R E L A T I O N S H I P BETWEEN THE A C T I V I T Y OF  MICROSOMAL O X I D A S E AND I N H - I N D U C E D H E P A T O T O X I C I T Y PB i s a w e l l known l i v e r m i c r o s o m a l o x i d a s e i n d u c e r . I n t h i s e x p e r i m e n t , INH was a d m i n i s t e r e d w i t h a n d w i t h o u t PB p r e t r e a t m e n t t o t e s t t h e e f f e c t o f i n d u c t i o n o f m i c r o s o m a l e n z y m e s o n t h e h e p a t o t o x i c i t y o f I N H . 2 M A T E R I A L S AND METHODS 2 . 1 S U B J E C T M a l e New Z e a l a n d w h i t e r a b b i t s ( 2 - 3 kg) w e r e o b t a i n e d f r o m t h e A n i m a l C a r e U n i t o f 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 . 2 . 2 M A T E R I A L S : I s o n i c o t i n i c a c i d h y d r a z i d e a n d p e n t o b a r b i t o n e s o d i u m w e r e o b t a i n e d f r o m B r i t i s h D r u g H o u s e C h e m i c a l s C a n a d a L t d . H y d r a z i n i u m s u l p h a t e was p u r c h a s e d f r o m F i s h e r S c i e n t i f i c . A l d r i c h C h e m i c a l C o . was t h e s u p p l i e r o f a c e t y l h y d r a z i n e . S u l f a m e t h a z i n e (SMZ) r e q u i r e d f o r d e t e r m i n a t i o n o f SMZ a c e t y l a t o r p h e n o t y p e was o b t a i n e d f r o m M a t t h e i s o n , C o l e m a n a n d B e l l C o . A c e t y l i s o n i c o t i n i c a c i d h y d r a z i d e was s e m i -s y n t h e s i z e d i n t h i s l a b o r a t o r y f r o m m a t e r i a l s s u p p l i e d by Z H O U , T . 36 B r i t i s h D r u g H o u s e C h e m i c a l s C a n a d a L t d , a c c o r d i n g t o t h e m e t h o d o f v o n S a s s e n e t al. ( 1 9 8 5 ) , w h e r e i n INH was r e a c t e d w i t h a f o u r - f o l d e x c e s s o f a c e t i c a c i d a n h y d r i d e a t room t e m p e r a t u r e f o r 1 .5 h r s w i t h c o n t i n u o u s s t i r r i n g , a n d t h e s y n t h e s i z e d p r o d u c t was r e c r y s t a l l i z e d i n m e t h a n o l - d i e t h y l e t h e r ( 1 : 4 ) ; h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) a n a l y s i s o f t h e A c I N H showed t h a t c o n t a m i n a t i o n b y I N H , A c H z , o r Hz was b e l o w t h e l e v e l o f d e t e c t i o n ( c a . 0 . 2 - 0 . 4 % a t t h e d e t e c t o r s e n s i t i v i t y u s e d ) . R e a g e n t s f o r t h e A S A L a s s a y a c c o r d i n g t o C a m p a n i n i e t al. (1970) w e r e f r o m f o l l o w i n g s o u r c e s : a s o l u t i o n o f b a r i u m a r g i n i n o s u c c i n a t e ( S i g m a C h e m i c a l C o . ) was c o n v e r t e d t o a s o l u t i o n o f t h e s o d i u m s a l t b y a d m i x t u r e w i t h s o d i u m s u l f a t e a n d c e n t r i f u g a t i o n ; 2 , 4 - d i c h l o r o - l - n a p h t h o l ( S i g m a C h e m i c a l C o . ) was r e c r y s t a l l i s e d i n h e p t a n e a c c o r d i n g t o p r o c e d u r e s o u t l i n e d b y C a m p a n i n i , a n d t h e o t h e r r e a g e n t s r e q u i r e d f o r t h e A S A L a s s a y w e r e a l l o f r e a g e n t g r a d e o b t a i n e d f r o m l o c a l c h e m i c a l s u p p l y h o u s e s . 2 . 3 METHODS 2 . 3 . 1 A N I M A L TREATMENT R a b b i t s w e r e h o u s e d i n s t a i n l e s s s t e e l c a g e s w i t h f r e e a c c e s s t o P u r i n a l a b o r a t o r y chow a n d w a t e r . T h e a c e t y l a t i o n p h e n o t y p e o f e a c h t e s t a n i m a l was d e t e r m i n e d b e f o r e Z H O U , T . 37 h e p a t o t o x i c c h a l l e n g e by HPLC a n a l y s i s f o l l o w i n g a d m i n i s t r a t i o n o f S M Z . R a b b i t s w e r e a s s i g n e d i n t o 12 t r e a t m e n t g r o u p s o f 6-8 r a b b i t s e a c h . T h e y w e r e g r o u p s o f (1) INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , o r a l l y ( p . o . ) ) ; (2) INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s u b c u t a n e o u s l y , ( s . c . ) ) ; (3) INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , p . o . ) f o l l o w i n g PB p r e t r e a t m e n t ( 0 . 1 m m o l / k g x 3 d , i n t r a p e r i t o n e a l l y ( i . p . ) ) ; (4) INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s . c . ) f o l l o w i n g PB p r e t r e a t m e n t ( 0 . 1 m m o l / k g x 3 d , i . p . ) ; (5) A c I N H ( 0 . 2 8 + 3 x 0 . 2 0 m m o l / k g / d x 2 d , s . c ) ; (6) A c I N H ( 0 . 4 2 + 3 x 0 . 3 0 m m o l / k g / d x 2 d , s . c ) ; (7) A c I N H ( 0 . 4 2 + 3 x 0 . 3 0 m m o l / k g / d x 2 d , p . o . ) ; (8) A c H z ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s . c ) ; (9) Hz ( 0 . 1 0 + 3 x 0 . 0 7 m m o l / k g / d x 2 d , p . o . ) ; (10) Hz ( 0 . 1 4 + 3 x 0 . 1 0 m m o l / k g / d x 2 d , p . o . ) , (11) Hz ( 0 . 1 9 + 3 x 0 . 1 4 m m o l / k g / d x 2 d , p . o . ) a n d (12) two r a b b i t s , r a n d o m l y c h o s e n as c o n t r o l a n i m a l s f o r h i s t o l o g i c a l s t u d i e s , w e r e g i v e n s a l i n e s u b c u t a n e o u s l y i n t h e same r e g i m e n as t h e d r u g t r e a t m e n t i n o t h e r g r o u p s . 2 . 3 . 2 PHENOTYPING SAMPLING R a b b i t was p l a c e d i n t o a r e s t r a i n i n g f r a m e a n d p r e p a r e d f o r c a n n u l i z a t i o n t o t a k e t h e b l o o d s a m p l e s - t h e h a i r a r o u n d t h e l a t e r a l e a r v e i n was s h a v e d o f f , a n d t h e e a r v e i n was t h e n p r i c k e d w i t h a n e e d l e . A 22 g a u g e j e l c o c a t h e t e r was h e p a r i n i z e d a n d t h e n i m p l a n t e d i n t o t h e v e i n a n d Z H O U , T . 38 s e c u r e d . I n p r e p a r a t i o n f o r t h e c o l l e c t i o n o f b l o o d s a m p l e , a l l t h e E p p e n d o r f t u b e s w e r e l a b e l e d a n d 1 m l s y r i n g e s w e r e h e p a r i n i z e d . A f t e r a b l a n k s a m p l e was t a k e n , a d o s e o f 10 m g / k g SMZ f r o m a s t o c k s o l u t i o n was a d m i n i s t e r e d t h r o u g h t h e o p p o s i t e e a r v e i n . S t a r t i n g f r o m t i m e 0 , t h e t i m e o f a d m i n i s t r a t i o n o f t h e d r u g , a p p r o x i m a t e l y 0 . 5 m l o f b l o o d w e r e w i t h d r a w n i n t o t h e h e p a r i n i z e d s y r i n g e s f r o m t h e i n s e r t e d c a t h e t e r a t t i m e s o f a p p r o x i m a t e l y 5 , 1 0 , 15 , 2 0 , 2 5 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 , 8 0 , 9 0 , 1 0 5 , 1 2 0 , 1 3 5 , 1 5 0 , 180 m i n u t e s p o s t - i n j e c t i o n . T h e c o l l e c t e d b l o o d was t h e n e j e c t e d i n t o m a r k e d e p p e n d o r f t u b e s a n d 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 f o r t h e a n a l y s i s o f a c e t y l s u l f a m e t h a z i n e ( A c S M Z ) a n d S M Z . EXTRACTION SMZ a n d AcSMZ w e r e e x t r a c t e d f r o m t h e p l a s m a s a m p l e s t h r o u g h t h e f o l l o w i n g s t e p s : 1. 100 fil o f p l a s m a was w i t h d r a w n a n d p l a c e d i n t o a s e c o n d E p p e n d o r f t u b e a l o n g w i t h 100 pi o f a c e t o n i t r i l e , t o d e n a t u r e t h e p r o t e i n , a n d a l l o w e d t o s i t f o r a t l e a s t 2 m i n . 2 . 200 pi o f d i s t i l l e d w a t e r was a d d e d t o t h e t u b e , t h e n m i x e d o n a v o r t e x m i x e r . 3 . E p p e n d o r f t u b e s c o n t a i n i n g t h e m i x t u r e was c e n t r i f u g e d (5000 rpm) f o r two m i n . 4 . A p p r o x i m a t e l y 200 jul s u p e r n a t e was r e m o v e d f r o m t h e e p p e n d o r f t u b e s a n d t r a n s f e r r e d t o a g l a s s v i a l . Z H O U , T . 39 A s p e c i f i e d v o l u m e o f t h e s u p e r n a t e i n t h e g l a s s v i a l was i n j e c t e d i n t o t h e HPLC t o d e t e r m i n e t h e c o n c e n t r a t i o n o f SMZ a n d AcSMZ i n t h e p l a s m a . ANALYSIS OF SAMPLES T h e s a m p l e s w e r e a n a l y z e d b y H P L C . T h e c o n d i t i o n s u n d e r w h i c h t h e HPLC a n a l y s e s w e r e r u n w e r e a s f o l l o w s : t h e HPLC c o l u m n was p a c k e d w i t h O D S - 2 ; t h e s o l v e n t was a 0 . 0 1 M pH 5 . 0 N a A c , 15% MeOH, 8% MeCN s o l u t i o n a t a f l o w r a t e o f 1 m l / m i n u t e ; t h e a b s o r b a n c e was s e t a t 254 nm a n d 10 mV w i t h 0 . 0 8 as a f u l l s c a l e d e f l e c t i o n r e a d i n g ; a n d t h e p a p e r f e e d was s e t a t a s p e e d o f 2 . 5 m m / m i n . T h e c o n c e n t r a t i o n s w e r e d e t e r m i n e d b y c o m p a r i s o n o f t h e p e a k h e i g h t s o b t a i n e d w i t h t h o s e o b t a i n e d f r o m a 0 . 0 1 m g / m l s t a n d a r d s o l u t i o n o f SMZ a n d A c S M Z . CALCULATION F r o m p l o t t i n g t h e c o n c e n t r a t i o n a g a i n s t t i m e , t h e K e ( f i r s t o r d e r e l i m i n a t i o n r a t e c o n s t a n t ) was c a l c u l a t e d f o r e a c h r a b b i t r e s p e c t i v e l y . T h e h a l f - l i f e ("^1/2) w a s c a l c u l a t e d f r o m t - ^ 2 = i n 2 / K e * T h e r a t i ° o f A c S M Z / S M Z + A c S M Z was a l s o c a l c u l a t e d f o r t h e 20 m i n s a m p l e . 2 . 3 . 3 A D M I N I S T R A T I O N INH a n d i t s m e t a b o l i t e s w e r e g i v e n o r a l l y a n d s u b c u t a n e o u s l y ; PB was i n j e c t e d i n t r a p e r i t o n e a l l y . Z H O U , T . 4 0 O r a l a d m i n i s t r a t i o n was d o n e o n a n i m a l s p r o t e c t e d by a p p r o p r i a t e r e s t r a i n i n g f r a m e s . 10% s u c r o s e was u s e d as t h e s o l v e n t i n t h e d r u g p r e p a r a t i o n f o r o r a l a d m i n i s t r a t i o n . T h e s o l u t i o n was g i v e n v i a a s y r i n g e p l a c e d i n t o t h e m o u t h o f t h e r a b b i t f o l l o w e d b y 1 m l o f 5% d e x t r o s e t o e n s u r e c o m p l e t e i n g e s t i o n . S u b c u t a n e o u s a d m i n i s t r a t i o n o f d r u g i n n o r m a l s a l i n e was p e r f o r m e d b y i n j e c t i o n u n d e r a f o l d o f g e n t l y p i n c h e d s k i n b e t w e e n t h e two s h o u l d e r b l a d e s . I n j e c t i o n a t t h i s l o c a t i o n a p p e a r e d n o t t o d i s c o m f o r t t h e r a b b i t . I n t r a p e r i t o n e a l i n j e c t i o n r e q u i r e d m a n u a l p o s i t i o n i n g o f t h e r a b b i t t o e x p o s e i t s u n d e r b e l l y f o r i n j e c t i o n . Two g r o u p s o f r a b b i t s w e r e p r e t r e a t e d w i t h PB w h i c h was d i s s o l v e d i n p h y s i o l o g i c a l s a l i n e a n d g i v e n a t 0 . 1 0 m m o l / k g / d o n t h e t h r e e d a y s p r i o r t o i n i t i a t i o n o f t h e INH c h a l l e n g e . T h i s d o s e o f PB h a s b e e n shown t o be e f f e c t i v e i n c a u s i n g i n d u c t i o n o f c y t o c h r o m e P - 4 50 i n t h e r a b b i t ( W h i t e h o u s e e t a i . , 1 9 8 5 ) . E a c h o f t h e two d a y s o f t h e INH o r m e t a b o l i t e s b e g a n w i t h a m o r n i n g l o a d i n g d o s e o f one t h i r d o f t h e d a i l y t o t a l f o l l o w e d b y t h r e e s u b s e q u e n t i n j e c t i o n s o f two n i n t h s o f t h e t o t a l 3 , 6 , a n d 9 h o u r s l a t e r . D u r i n g t h e two d a y s o f d o s i n g t h e o r a l t r e a t m e n t g r o u p s w e r e a l l o w e d w a t e r a d libitum b u t n o t f o o d . A l l r a b b i t s w e r e c a r e f u l l y o b s e r v e d f o r a n y s i g n s Z H O U , T . 41 o f d i s t r e s s d u r i n g t h e 15 h r s f o l l o w i n g t h e m o r n i n g d o s e . I n t h e e v e n t o f s i g n s o f d e h y d r a t i o n 5% s t e r i l e d e x t r o s e s o l u t i o n was a d m i n i s t e r e d s u b c u t a n e o u s l y o r o r a l l y t o r e s u s c i t a t e t h e a n i m a l . 2 . 3 . 4 A S A L MEASUREMENT SAMPLING B l o o d s a m p l e s ( a b o u t 0 . 7 - 1 m l ) w e r e t a k e n by h e p a r i n i z e d s y r i n g e m a i n l y f r o m t h e l a t e r a l e a r v e i n a n d o c c a s i o n a l l y , f r o m t h e c e n t r a l e a r a r t e r y when b l o o d s a m p l e s c o u l d n o t b e o b t a i n e d f r o m t h e v e i n . I n some e x t r e m e c a s e s t h e v e i n was p a r t i c u l a r l y v a s o a c t i v e a n d t h u s , t h e t o p i c a l a p p l i c a t i o n o f a v e r y s m a l l amount o f x y l e n e was n e c e s s a r y t o d i l a t e t h e v e s s e l s . T h e x y l e n e was i m m e d i a t e l y r e m o v e d w i t h a l c o h o l f o l l o w i n g b l o o d s a m p l i n g . T h e 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 a n d a s s a y e d w i t h i n 24 h r s ( s t o r e d a t - 2 0 ° C ) . F o r e a c h r a b b i t , b l o o d s a m p l e s w e r e t a k e n b e f o r e a n d d u r i n g t h e 5 d a y p e r i o d f o l l o w i n g i n i t i a t i o n o f e a c h d r u g c h a l l e n g e . ASAL ANALYSIS S e r u m A S A L l e v e l s w e r e d e t e r m i n e d b y f o l l o w i n g t h e p r o c e d u r e o u t l i n e d b y C a m p a n i n i e t al ( 1 9 7 0 ) : Z H O U , T . 42 Substrate. M i x 4 m l o f (a) w i t h 80 m l o f (b) a n d 40 m l o f ( c ) . (a ) S o d i u m a r g i n i n o s u c c i n a t e 20 m m o l / 1 . (b) P h o s p h a t e b u f f e r , 0 . 2 M , pH 7 . 0 . ( c ) E t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) s o l u t i o n , 0 . 2 M , pH 7 . 0 Dichloronaphthol Solution. E q u a l v o l u m e s o f (a) a n d (b) a r e m i x e d o n t h e d a y o f t h e t e s t . (a) T h y m i n e s o l u t i o n . 2 g o f t h y m i n e i s d i s s o l v e d i n 10% NaOH a n d d i l u t e d t o 100 m l w i t h 10% N a O H . K e e p r e f r i g e r a t e d . (b) D i c h l o r o n a p h t h o l . 60 mg o f p u r i f i e d 2 , 4 - d i c h l o r o - l -n a p h t h o l i s d i s s o l v e d i n 95% e t h a n o l a n d d i l u t e d t o 100 m l w i t h 95% e t h a n o l . K e e p r e f r i g e r a t e d . NaOCl solution. D i l u t e 12 m l o f c o m m e r c i a l C l o r o x (5.25% N a O C l ) t o 100 m l w i t h w a t e r . Arginine standard (1 m m o l / 1 ) . D i s s o l v e 1 7 . 4 2 mg o f a r g i n i n e i n 0 . 1 N HC1 a n d d i l u t e t o 100 m l w i t h 0 . 1 N H C 1 . K e e p r e f r i g e r a t e d . L a b e l two t u b e s as unknown a n d control. T o e a c h t u b e a d d 0 . 3 m l s u b s t r a t e . T o t h e unknown t u b e a d d 0 . 1 m l o f s e r u m . M i x a n d i n c u b a t e f o r 1 h r a t 3 7 ° C , t h e n a d d 0 . 1 m l o f t h e same s e r u m t o t h e control t u b e a n d m i x . P r o m p t l y a d d 0 .2 m l o f t r i c h l o r o a c e t i c a c i d s o l u t i o n (20 g / 1 0 0 m l ) t o b o t h t u b e s , m i x , a n d l e t s t a n d f o r 5 m i n . C e n t r i f u g e b o t h Z H O U , T . 43 t u b e s f o r 10 m i n . 0 . 5 m l o f t h e s u p e r n a t e n t i s t r a n s f e r e e ! t o a s e c o n d t e s t t u b e w h i c h i s i m m e r s e d i n a n i c e b a t h . A d d t o t h e c o n t e n t s o f e a c h t u b e , m i x i n g a f t e r e a c h a d d i t i o n , 0 . 1 m l o f 10% N a O H , 0 . 2 5 m l o f d i c h l o r o n a p h t h o l s o l u t i o n , a n d 0 . 1 m l o f N a O C l s o l u t i o n . L e t s t a n d i n i c e b a t h f o r 15 m i n a n d r e a d a t 515 nm on a n L K B c o l o r i m e t e r . T h e d i f f e r e n c e i n a b s o r b a n c e o f t h e unknown t u b e a n d t h e control t u b e i s c o m p a r e d w i t h t h e s t a n d a r d t o o b t a i n t h e A S A L u n i t s . T h e 20 u n i t s s t a n d a r d was p r e p a r e d w i t h A S A L m e a s u r e m e n t o f s e r u m s a m p l e s e a c h t i m e . T o t h e standard t u b e a d d 0 . 7 5 m l o f p h o s p h a t e b u f f e r , 0 . 0 5 m l o f a r g i n i n e s t a n d a r d a n d 0 .2 0 m l o f w a t e r a n d m i x . T o t h e t u b e t h e n a d d 0 . 5 m l o f t r i c h l o r o a c e t i c a c i d s o l u t i o n (20 g / 1 0 0 m l ) . T h e c o l o r i s d e v e l o p e d as f o r t h e s e r a o n a 0 . 5 m l a l i q u o t f r o m t h e t u b e . I n t h i s t h e s i s , s e r u m A S A L a c t i v i t y was c a l c u l a t e d i n T a k a h a r a u n i t ( T a k a h a r a a n d N a t e l s o n , 1 9 6 7 ) , w h i c h i s e q u i v a l e n t t o m i c r o m o l e s o f a r g i n i n o - s u c c i n a t e d i g e s t e d / 1 0 0 m l o f s e r u m / h r . A s i g n i f i c a n t h e p a t o t o x i c r e s p o n s e was a s s i g n e d when two s u c c e s s i v e s e r u m A S A L l e v e l s r o s e t o a v a l u e e x c e e d i n g t h e u p p e r 9 9 t h l o g n o r m a l p e r c e n t i l e (12 T a k a h a r a u n i t s ) . Z H O U , T . 44 2 . 4 H I S T O L O G I C A L STUDY H i s t o l o g i c a l e x a m i n a t i o n s w e r e c o n d u c t e d o n r a b b i t s f r o m g r o u p s o f P B / I N H ( p . o . ) , P B / I N H ( s . c ) , INH ( p . o . ) , INH ( s . c ) , A c I N H ( s . c ) , a n d Hz ( h i g h d o s e , p . o . ) , r e s p e c t i v e l y . F o r t h e h i s t o l o g i c a l s t u d y , a n i m a l s w e r e s a c r i f i c e d b y a b l o w t o t h e b a c k o f t h e h e a d a t t h e f i f t h d a y e x c e p t f o r a few r a b b i t s a t t h e t h i r d o r f o u r t h d a y b e c a u s e o f d i s t r e s s a n d w e a k n e s s . T h e i r l i v e r wedges were o b t a i n e d f o r t i s s u e p r e p a r a t i o n s . A l l h i s t o l o g i c a l s l i d e s w e r e e x a m i n e d a n d r e p o r t e d by a p a t h o l o g i s t who i s b l i n d t o t h e t r e a t m e n t g r o u p s o r t h e c o r r e s p o n d i n g s e r u m A S A L v a l u e s . 2 . 4 . 1 T I S S U E F I X A T I O N F o r e a c h r a b b i t , t h r e e 1 mm t h i c k s l i c e s (one f r o m e a c h l o b e ) w e r e r e m o v e d f r o m t h e l i v e r f o r H e m a t o x y l i n - E o s i n ( H - E ) s t a i n i n g s l i d e p r e p a r a t i o n s . T h e s e s l i c e s w e r e p l a c e d i n B o u i n ' s f i x a t i v e s o l u t i o n f o r 1 h r t h a n m a i n t a i n e d i n 70% e t h a n o l . A n o t h e r t h r e e 5 mm t h i c k s l i c e s ( a l s o one f r o m e a c h l o b e ) w e r e t a k e n f o r o i l r e d "o" s t a i n i n g s l i d e p r e p a r a t i o n s a n d t h e y w e r e f i x e d i n 10% f o r m a l i n s o l u t i o n . Z H O U , T . 45 2 . 4 . 2 H - E S T A I N E D S L I D E S T h e s l i c e s m a i n t a i n e d i n e t h a n o l w e r e s e n t t o t h e D e p a r t m e n t o f P a t h o l o g y f o r s l i d e p r e p a r a t i o n . T h e H - E s t a i n e d s l i d e s w e r e e x a m i n e d f o r t h e g e n e r a l p a t h o l o g i c a l c h a n g e s s u c h as i n f l a m m a t o r y c e l l i n f i l t r a t i o n , v a r i o u s d e g e n e r a t i o n s , a n d h e p a t i c n e c r o s i s . 2 . 4 . 3 O I L RED "0" S T A I N E D S L I D E S T h e s e s l i d e s w e r e a l s o made by same d e p a r t m e n t as a b o v e . F a t t y a c c u m u l a t i o n i n t h e h e p a t o c y t e s c a n be s t a i n e d b y o i l r e d "o" s t a i n i n g . 2 . 5 S T A T I S T I C A L METHOD T h e v a l u e s o f s e r u m A S A L l e v e l a r e r e p r e s e n t e d as mean p l u s a n d m i n u s s t a n d a r d e r r o r ( S E ) . I n o r d e r t o c o m p a r e t h e h e p a t o t o x i c i t y among d i f f e r e n t t r e a t m e n t g r o u p s , s t a t i s t i c a l a n a l y s i s h a s b e e n made o n t h e s e r u m A S A L l e v e l s o f e a c h g r o u p u s i n g t w o - f a c t o r a n a l y s i s o f v a r i a n c e ( M o n t g o m e r y , 1 9 8 4 ) , p < 0 . 0 5 was a c c e p t e d as s i g n i f i c a n t l e v e l . Z H O U , T . 46 3 R E S U L T S 3 . 1 E F F E C T S OF INH AND I T S M E T A B O L I T E S ON E L E V A T I O N OF  SERUM A S A L L E V E L S e r u m A S A L l e v e l s w e r e m e a s u r e d i n a l l r a b b i t s b e f o r e a d m i n i s t r a t i o n o f a n y d r u g . S u m m a r i z i n g t h e v a l u e s f r o m a l l r a b b i t s e r u m s a m p l e s ( n = 8 7 ) , t h e mean c o n t r o l v a l u e was 4 .24 T a k a h a r a u n i t s , w i t h a s t a n d a r d d e v i a t i o n (SD) o f 2 . 5 8 , a n d a s t a n d a r d e r r o r (SE) o f 0 . 2 8 . T h e s e c o n t r o l v a l u e s w e r e u s e d t o a s s e s s t h e e f f e c t s o f INH a n d i t s m e t a b o l i t e s . T h e h e p a t o t o x i c r e s p o n s e t o t h e v a r i o u s d r u g t r e a t m e n t s , d e t e r m i n e d as t h e r e l e a s e o f A S A L i n t o t h e s e r u m , was c o n s i d e r e d s i g n i f i c a n t ( c r i t e r i o n I , T a b l e 1) i f t h e r e w e r e a t l e a s t two v a l u e s i n c r e a s e d t o v a l u e s g r e a t e r t h a n t h e u p p e r 9 9 t h l o g n o r m a l p e r c e n t i l e . A l e s s s e v e r e c r i t e r i o n ( I I ) was a l s o u s e d , i . e . , a t l e a s t two v a l u e s s h o w i n g a d o u b l i n g o f t h e n o r m a l p o p u l a t i o n mean v a l u e w i t h i n t h e u s u a l 30 -96 h r t i m e f r a m e f o r r e s p o n s e ( s e e T a b l e 1 ) . 3 . 1 . 1 T I M E COURSE OF THE E L E V A T I O N OF SERUM A S A L L E V E L  A F T E R INH A D M I N I S T R A T I O N P r e v i o u s e x p e r i m e n t s i n t h i s l a b o r a t o r y showed t h a t a s i n g l e d o s e o f INH d i d n o t c a u s e a r i s e i n s e r u m A S A L , when h i g h d o s e s l e d t o d e a t h o f t h e r a b b i t s d u e t o n e u r o t o x i c i t y ZHOU, T. 47 (Wall et al, unpublished data). In a multiple dose regimen, INH, administrated e i t h e r o r a l l y or subcutaneously, caused a remarkable r i s e i n serum ASAL l e v e l i n most animals. The co n t r o l ASAL l e v e l s ranged between 3-10 Takahara units i n these groups. A f t e r INH was administrated, the serum ASAL le v e l s were stable for the f i r s t 24 hrs, and then rose quickly to a peak values at about 72 hrs a f t e r the f i r s t dose, followed by a decline. F i g . 5 represents the time course of the ele v a t i o n of serum ASAL l e v e l from two rabbits treated with INH. Table 1. Serum ASAL response to drug challenges ADMINIS- TOTAL* NUMBER FRACTIONAL DRUG TRATION REGIMEN DOSE OF RESPONSE (%) ROUTE (mmol/kg) (mmol/kg) ANIMALS I** n * * * INH s.c. 0.36 + 3 x 0.26 2.28 7 57. 1 85.7 INH p.o. 0.36 + 3 x 0.26 2.28 8 62.5 87.5 PB i . p. 0.1 x3 d 0.30 6 INH s.c. 0.36 + 3 x 0.26 2.28 100.0 100.0 PB i . p. 0.1 x : 3 d 0.30 8 • INH p.o. 0.36 + 3 x 0.26 2.28 87.5 100.0 AcINH s.c. 0.28 + 3 x 0.20 1.76 8 25.0 75.0 AcINH s.c. 0.42 + 3 x 0.30 2.64 8 50.0 75.0 AcINH p.o. 0.42 + 3 x 0.30 2.64 8 50.0 75.0 AcHz s.c. 0.36 + 3 x 0.26 2.28 6 0.0 16.6 Hz p.o. 0.10 + 3 x 0.07 0.59 6 0.0 16.6 Hz p.o. 0.14 + 3 x 0.10 0.89 6 33.3 83.3 Hz p.o. 0.19 + 3 x 0.14 1.19 8 100.0 100.0 * t o t a l dose expressed over 2 days ** I: two measurements of ASAL >12 Takahara units ( c f . p.43) *** I I : two measurements of ASAL >8 Takahara units ZHOU, T. 4 8 F i g . 5. Time course of serum ASAL release followrng 9 challenge with INH (0.73 imol/kg/dx2 d s.c.). Rabbit A, slow acetylator; rabbit B, f a s t acetylator. Z H O U , T . 49 3 . 1 . 2 THE E F F E C T OF PB ON I N H - I N D U C E D H E P A T O T O X I C I T Y I n t h e g r o u p o f t h e r a b b i t s p r e t r e a t e d w i t h PB ( 0 . 1 m m o l / k g / d x 3 d , i . p . ) , a w e l l known m i c r o s o m a l enzyme i n d u c e r , t h e e l e v a t i o n o f s e r u m A S A L l e v e l s a f t e r a d m i n i s t r a t i o n o f INH 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 i n t h e g r o u p n o t p r e t r e a t e d w i t h PB ( F i g . 6 ) , s u g g e s t i n g t h a t PB e n h a n c e d t h e h e p a t o t o x i c i t y o f I N H . 3 . 1 . 3 I N F L U E N C E OF D I F F E R E N T A D M I N I S T R A T I O N ROUTES I n o r d e r t o t e s t t h e i n f l u e n c e o f d i f f e r e n t a d m i n i s t r a t i o n r o u t e s , INH was g i v e n i n t h e same d o s e v i a t h e o r a l a n d s u b c u t a n e o u s r o u t e . T h e c o m p a r i s o n b e t w e e n t h e two r o u t e s was d o n e i n b o t h PB p r e t r e a t e d r a b b i t s a n d n o n -p r e t r e a t e d r a b b i t s . T h e r e s u l t s showed t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n s e r u m A S A L l e v e l s b e t w e e n t h e g r o u p s o f r a b b i t s t r e a t e d by t h e two a d m i n i s t r a t i o n r o u t e s . F i g . 7 shows t h a t t h e e l e v a t i o n o f A S A L l e v e l s c a u s e d by INH i n o r a l g r o u p was a l m o s t t h e same as t h a t i n s u b c u t a n e o u s g r o u p i n t e r m s o f t h e p e a k v a l u e s a n d p e a k t i m e ( b o t h g r o u p s w e r e p r e t r e a t e d w i t h PB) . A c I N H was a l s o a d m i n i s t e r e d v i a t h e o r a l a n d s u b c u t a n e o u s r o u t e s a n d no s i g n i f i c a n t d i f f e r e n c e was f o u n d . ZHOU, T. 50 F i g . 6 . Serum ASAL le v e l s obtained following o r a l challenge with INH (0.36+3X0.26 mmol/kg/dx2d) i n rabbits with (n=8) or without (n=8) PB pretreatment (0.1 mmol/kg/dx3d, i . p . ) . A s i g n i f i c a n t difference i s found between the two groups (p<0.05, F t e s t ) . ZHOU, T. 51 F i g . 7 . Serum ASAL l e v e l s i n rabbits obtained following o r a l (n=8) and subcutaneous (n=6) administration of INH (0.36+3x0.26 mmol/kg/dx2d) with PB pretreatment (0.1 mmol/kg/dx3d, i . p . ) . In the subcutaneous group the l a s t two points are not ca l c u l a t e d because two rabbits died before these samples. There i s no s i g n i f i c a n t difference between the two groups (p>0.05, F t e s t ) . Z H O U , T . 52 3 . 1 . 4 E F F E C T OF H z , A c I N H AND A c H z ON E L E V A T I O N OF SERUM  A S A L L E V E L Among t h e m e t a b o l i t e s o f I N H , Hz was t h e a g e n t t h a t h a d t h e m o s t p o t e n t e f f e c t o n t h e e l e v a t i o n o f s e r u m A S A L l e v e l . F i g . 8 i l l u s t r a t e s t h e d o s e - r e s p o n s e r e l a t i o n s h i p o f Hz on s e r u m A S A L l e v e l s . A t t h e l o w e s t d o s e ( 0 . 6 2 m m o l / k g ) , t h e r e was a l m o s t no c h a n g e i n A S A L l e v e l s . D o u b l i n g t h e d o s e ( 1 . 2 4 m m o l / k g ) m a n i f e s t e d a d r a m a t i c r i s e i n s e r u m A S A L l e v e l s , w h i l e a n i n t e r m e d i a t e d o s e o f Hz ( 0 . 9 3 m m o l / k g ) h a d l e s s e f f e c t . T h e e l e v a t i o n o f s e r u m A S A L c a u s e d b y t h e h i g h e s t d o s e o f Hz 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 b y I N H , a l t h o u g h t h e m o l a r d o s e o f Hz ( h i g h d o s e ) was o n l y a p p r o x i m a t e l y h a l f o f t h e d o s e o f INH ( F i g . 9 ) . A m o l a r d o s e o f Hz e q u a l t o t h a t o f INH c a u s e d i m m e d i a t e m o r t a l i t y i n t h e r a b b i t s . M e a n w h i l e , b o t h h i g h d o s e a n d l o w d o s e o f A c I N H c a u s e d some e l e v a t i o n o f s e r u m A S A L l e v e l s ( F i g . 1 0 ) . When A c I N H was c o m p a r e d t o t h e p a r e n t d r u g , I N H , t h e d i f f e r e n c e i n h e p a t o t o x i c i t y b e t w e e n t h e s e two a g e n t s was n o t s i g n i f i c a n t ( F i g . 1 1 ) . A c H z i n a d o s e e q u i m o l a r t o INH ( 0 . 3 6 + 3 x 0 . 2 6 m m o l / k g / d x 2 d , s . c . ) d i d n o t s i g n i f i c a n t l y c h a n g e s e r u m A S A L l e v e l s d u r i n g t h e t i m e o f 90 h r s a f t e r t h e a g e n t was a d m i n i s t e r e d ( F i g . 1 2 ) . ZHOU, T. 53 _ O Hz low dose Time (post 1st administration, h) F i g . 8. Serum ASAL le v e l s i n rabbits o r a l l y administered with d i f f e r e n t doses of Hz (low dose 0.10+3x0.07 mmol/kg/dx2d, n=6; middle dose 0.14+3x0.10 mmol/kg/dx2d, n=6; high dose 0.19+3x0.14 mmol/kg/dx2d, n=8). In the high dose group the l a s t two points are not calcula t e d because three rabbits died before these samples. S i g n i f i c a n t differences are found among these three groups (p<0.05, F test) and between high dose and middle dose groups and middle dose and low dose groups, res p e c t i v e l y (p<0.05, Duncan 1s multiple range t e s t ) . ZHOU, T F i g . 9 . Serum ASAL l e v e l s i n rabbits o r a l l y administered with INH (0.36+3x0.26 mmol/kg/dx2d, n=8) and Hz (0.19+3x0.14 mmol/kg/dx2d, n=8) . In the Hz group the l a s t two points are not calcula t e d because three rabbits died before these samples. S i g n i f i c a n t difference i s found between the two groups (p<0.05, F t e s t ) . Z H O U , T . 55 0 20 40 60 Time (post 1st admin is t ra t ion , h) 80 F i g . 1 0 . S e r u m A S A L l e v e l s i n r a b b i t s s u b c u t a n e o u s l y c h a l l e n g e d w i t h two d o s e s o f A c I N H ( l o w d o s e 0 . 2 8 + 3 x 0 . 2 0 m m o l / k g / d x 2 d , n=8; h i g h d o s e 0 . 4 2 + 3 x 0 . 3 0 m m o l / k g / d x 2 d , n=8) . S i g n i f i c a n t d i f f e r e n c e i s f o u n d b e t w e e n t h e two g r o u p s ( p < 0 . 0 5 , F t e s t ) . ZHOU, T. 56 T i m e ( p o s t 1 s t a d m i n i s t r a t i o n , h ) F i g . 11. Serum ASAL le v e l s i n rabbits o r a l l y administered with INH (0.36+3x0.26 mmol/kg/dx2d, n=8) and AcINH (0.42+3x0.30 mmol/kg/dx2d, n=8). There i s no s i g n i f i c a n t d ifference between the two groups (p>0.05, F t e s t ) . ZHOU, T. 57 T i m e ( P o s t 1 s t a d m i n i s t r a t i o n , h ) F i g . 12. Serum ASAL le v e l s i n rabbits administered with INH (0.36+3x0.26 mmol/kg/dx2d, p.o., n=8) and AcHz (0.36+3x0.26 mmol/kg/dx2d, s . c , n=6) . A s i g n i f i c a n t difference i s found between the two groups (p<0.05, F t e s t ) . Z H O U , T . 58 3 . 1 . 5 THE R E L A T I O N S H I P BETWEEN THE E L E V A T I O N OF SERUM A S A L  L E V E L AND THE A C E T Y L A T O R R A T E OF I N D I V I D U A L ANIMALS T h e r a t e o f a c e t y l a t i o n i n r a b b i t s i s g e n e t i c a l l y c o n t r o l l e d . I t i s c o n f i r m e d by many a u t h o r s t h a t r a b b i t s c a n b e c l a s s i f i e d as s l o w o r r a p i d a c e t y l a t o r s ( F r y m o y e r a n d J a c o x , 1 9 6 3 a ; F r y m o y e r a n d J a c o x , 1 9 6 3 b ; W e b e r e t al., 1976; B l u m e t a i . , 1989) SMZ h a l f - l i f e a n d t h e p l a s m a A c S M Z / S M Z + A c S M Z r a t i o a t 20 m i n u t e s a f t e r i n t r a v e n o u s i n j e c t i o n o f 10 m g / k g o f SMZ w e r e m e a s u r e d i n o u r e x p e r i m e n t a l a n i m a l s . T h e y a r e u s e d as t h e i n d i c e s t o d e t e r m i n e t h e a c e t y l a t i o n r a t e o f t h e r a b b i t s . F i g . 1 3 . shows t h e r e i s a s t r i k i n g b i - m o d a l d i s t r i b u t i o n o f t h e a c e t y l a t i o n r a t e i n t h i s p o p u l a t i o n o f r a b b i t s , i . e . , one g r o u p w e r e p h e n o t y p e d r a p i d a c e t y l a t o r s a n d a n o t h e r g r o u p w e r e p h e n o t y p e d s l o w a c e t y l a t o r s ( s e e T a b l e 2 ) . T a b l e 2 . T h e p a r a m e t e r s o f S M Z - a c e t y l a t o r p h e n o t y p e i n r a b b i t s . A c e t y l a t o r N SMZ h a l f -• l i f e (min) A c S M Z / S M Z + A c S M Z (%)* g r o u p mean+SE r a n g e mean+SE r a n g e R a p i d 54 1 2 . 7 + 0 . 6 5 . 9 - 2 8 . 0 8 4 . 3 + 1 . 3 5 0 - 1 0 0 S l o w 11 5 0 . 3 + 3 . 1 3 9 . 0 - 7 7 . 2 2 8 . 5 + 0 . 7 25 -34 T h e r a t e was c a l c u l a t e d a t 20 m i n u t e s a f t e r SMZ i n j e c t i o n . ZHOU, T. 59 F i g . 13. Plot of the plasma AcSMZ/SMZ+AcSMZ r a t i o at 2 0 minutes a f t e r intravenous i n j e c t i o n of SMZ (10 mg/kg) against SMZ h a l f - l i f e . Each point represents the two determinations i n each animal. Note there i s a s t r i k i n g bi-modal d i s t r i b u t i o n of the acetylation rate i n t h i s population of rabbits. The group i n the upper l e f t were phenotyped rapid acetylators and the group i n the lower r i g h t were phenotyped slow acetylators (refer to Table 2 ) . Z H O U , T . 60 Many a u t h o r s h a v e s u g g e s t e d t h a t t h e a c e t y l a t o r r a t e a f f e c t s t h e h e p a t o t o x i c i t y o f I N H . T h e e v i d e n c e i s h o w e v e r c o n t r a d i c t o r y . I n t h i s s t u d y , i n a g r o u p o f r a b b i t s c h a l l e n g e d w i t h INH w i t h o r w i t h o u t PB p r e t r e a t m e n t , i n e i t h e r o r a l o r s u b c u t a n e o u s a d m i n i s t r a t i o n r o u t e , t h e d e g r e e o f h e p a t o t o x i c i t y a s s e s s e d by t h e p e a k s e r u m A S A L l e v e l s was c o r r e l a t e d w i t h t h e a c e t y l a t o r p h e n o t y p e . T h e r e l a t i o n s h i p b e t w e e n t h e p e a k s e r u m A S A L v a l u e s a n d a c e t y l a t i o n r a t e ( r e p r e s e n t e d b y SMZ ^1/2) i - s s u m m a r i z e d i n F i g . 14 . T h e r e s u l t s showed no c o r r e l a t i o n b e t w e e n t h e s e two p a r a m e t e r s ( c o r r e l a t i o n c o e f f i c i e n t ( r ) = 0 . 0 5 ) . A s i m i l a r l a c k o f c o r r e l a t i o n was f o u n d i n t h e r a b b i t s c h a l l e n g e d w i t h A c I N H a n d H z . 3 . 2 E F F E C T S OF INH AND I T S M E T A B O L I T E S ON P A T H O L O G I C A L  CHANGES H i s t o l o g i c a l s t u d i e s w e r e c a r r i e d o u t o n t h e l i v e r o f t h e r a b b i t s c h a l l e n g e d w i t h A c I N H , Hz a n d INH w i t h o r w i t h o u t PB p r e t r e a t m e n t . A l l o f t h e s e t h r e e c h e m i c a l s c a u s e d v a c u o l a r d e g e n e r a t i o n i n t h e h e p a t i c t i s s u e o f m o s t e x p e r i m e n t a l ZHOU, T. 61 5000 1000 c D O o o 100 r=0.05 < CO < 10 0 20 40 60 SMZ T 1 / 2 (min) 80 F i g . 14. The r e l a t i o n s h i p of SMZ h a l f - l i f e to the peak serum ASAL value i n rabbits challenged with INH (0.36+3x0.26 mmol/kg/dx2d) o r a l l y or subcutaneously, with or without PB (0.1 mmol/kg/dx3d, i.p.) pretreatment. Each point represents the two determinations i n each animal. Z H O U , T . 62 a n i m a l s a n d c a u s e d some d e g r e e o f n e c r o s i s i n a f r a c t i o n o f t h e a n i m a l s . C o m p a r e d w i t h t h e c o n t r o l h e p a t i c t i s s u e s a m p l e s ( F i g . 1 5 ) , h i s t o l o g i c a l e x a m i n a t i o n o n t h e H e m a t o x y l i n - E o s i n s t a i n e d l i v e r t i s s u e s l i c e s r e v e a l e d s w o l l e n p a r e n c h y m a l c e l l s a n d l a r g e v a c u o l a t e d r e g i o n s i n t h e c e n t e r o f t h e l i v e r l o b u l e ( F i g . 1 6 ) . Some s l i c e s made f r o m t h e r a b b i t w i t h a m i l d e l e v a t i o n o f s e r u m A S A L showed s m a l l f o c a l n e c r o s i s ( F i g . 1 7 ) . T h e s l i c e s made f r o m some r a b b i t s whose s e r u m A S A L r o s e t o a n e x t r e m e l y h i g h l e v e l showed s u b - m a s s i v e n e c r o s i s . T h e n e c r o s i s i n v o l v e d c e n t r a l a n d m i d d l e z o n e s o f t h e l o b u l e , a n d t h e a r r a n g e m e n t o f t h e p a r e n c h y m a a r o u n d t h e c e n t r a l v e i n was d i s r u p t e d ( F i g . 1 8 ) . O i l r e d "0" s t a i n e d s l i c e s w e r e made t o show f a t t y a c c u m u l a t i o n i n t h e h e p a t o c y t e s . C o m p a r e d w i t h t h e s l i c e s f r o m c o n t r o l r a b b i t ( F i g . 1 9 ) , v a r i o u s d e g r e e s o f h e p a t o c y t e f a t t y m e t a m o r p h o s i s ( F i g . 20) w e r e d e m o n s t r a t e d i n a l m o s t a l l o f t h e t i s s u e s l i c e s made f r o m t h e r a b b i t s c h a l l e n g e d w i t h INH o r i t s m e t a b o l i t e s . 3 . 3 THE R E L A T I O N S H I P BETWEEN E L E V A T I O N OF SERUM A S A L L E V E L  AND H E P A T I C NECROSIS I n t h i s s t u d y , h i s t o l o g i c a l e x a m i n a t i o n o f l i v e r s l i c e s was c o n d u c t e d i n s i x o u t o f t h e e l e v e n t r e a t m e n t g r o u p s ( n = 4 5 ) . T h e r e l a t i o n s h i p b e t w e e n p e a k s e r u m A S A L l e v e l s a n d t h e d e g r e e o f l i v e r damage was e x p l o r e d . Among 45 a n i m a l s , no h e p a t i c n e c r o s i s was o b s e r v e d i n 6 r a b b i t s c h a l l e n g e d F i g . 15. L i v e r t i s s u e from a c o n t r o l r a b b i t t r e a t e d with s a l i n e (1 mlx4/dx2d, s . c ) . (a) H-E s t a i n , x l O . (b) H-E s t a i n , xlOO. Z H O U , T . 64 (b) F i g . 16. L i v e r t i s s u e f r o m a r a b b i t w i t h a p e a k A S A L v a l u e o f 466 T a k a h a r a u n i t s a f t e r a d m i n i s t r a t i o n o f Hz ( 0 . 1 9 + 3 x 0 . 1 0 m m o l / k g / d x 2 d , p . o . ) . (a ) M a s s i v e v a c u o l a t i o n o f l i v e r c e l l s . A r o u n d t h e c e n t r a l v e i n s t h e r e i s a b r o a d a r e a o f d i s i n t e g r a t e d v a c u o l a t e d l i v e r c e l l s . O u t s i d e t h e z o n e n u m e r o u s s w o l l e n l i v e r c e l l s c a n be s e e n ( H - E s t a i n x 4 0 ) . (b) V a c u o l a t e d l i v e r c e l l s ( H - E s t a i n , x 4 0 0 ) . ZHOU, T. 65 (b) F i g . 17. L i v e r t i s s u e from the r a b b i t with a peak ASAL va l u e of 73.6 Takahara u n i t s a f t e r a d m i n i s t r a t i o n of Hz (0.19+3x0.10/kg/dx2d, p.o.). (a) F o c a l necrosis with inflammatory c e l l s ( H - E s t a i n , xlOO). (b) N e c r o t i c l i v e r c e l l s i n the f o c a l n e c r o s i s area ( H - E s t a i n , x400). ZHOU, T. 66 (b) Fi g . 18. Liver tissue from the rabbit with a peak serum ASAL value of 2674 Takahara units a f t e r administration of INH (0.36+3x0.26 mmol/kg/dx2d, p.o.) with PB pretreatment (0.1 mmol/kg/dx3d, i . p . ) . (a) Sub-massive necrosis of l i v e r c e l l s . Necrosis involves contral and middle zones. Vacuolated l i v e r c e l l s are around necrotic zones (H-E s t a i n , x40). (b) Necrosis of l i v e r c e l l s (H-E s t a i n , xlOO). Z H O U , T . 67 F i g . 19. L i v e r t i s s u e w i t h o i l r e d "0" s t a i n i n g f r o m t h e c o n t r o l r a b b i t . No v i s i b l e f a t t y m e t a m o r p h o s i s i s s e e n i n t h e s l i c e ( x 4 0 ) . ZHOU, T 20. L i v e r t i ssues with o i l red "0" s ta in ing from d i f f e r e n t rabbits showing various degrees of fa t ty metamorphosis. (a) L i v e r t i s sue from the rabb i t with a peak serum ASAL value of 30.6 Takahara units a f ter adminis trat ion of INH ( 0 . 36+3x0.26/kg/dx2d, p . o . ) . Dense, large droplets of fa t ty metamorphosis were found around c e n t r a l veins (x40). ZHOU, T. 69 F i g . 20. (b) L i v e r t i s s u e from the r a b b i t w i t h a peak serum ASAL value of 466 Takahara u n i t s a f t e r a d m i n i s t r a t i o n of Hz (0.19+3x0.10 mmol/kg/dx2d, p.o.). V a r i o u s s i z e d r o p l e t s of f a t t y metamorphosis i n v o l v e s a l l zones of the l o b u l e (x40). ZHOU, T. 7 0 (d) F i g . 20. (c) L i v e r t i s s u e from the r a b b i t w i t h a peak serum ASAL value of 611.5 Takahara u n i t s a f t e r a d m i n i s t r a t i o n of INH (0.36+3x0.26 mmol/kg/dx2d, p.o.). F a t t y metamorphosis of moderate degree i n v o l v e s c e n t r a l and middle zones (x40). (d) Same l i v e r s l i c e as (c) showing f a t t y d r o p l e t s of v a r i o u s s i z e i n l i v e r c e l l s (x400). Z H O U , T . 71 w i t h INH o r A c I N H who e x h i b i t e d no s i g n i f i c a n t r i s e i n A S A L l e v e l s . I n c o n t r a s t , i n 39 r a b b i t s w h i c h h a d a s i g n i f i c a n t r e l e a s e o f A S A L , 2 7 w e r e d e m o n s t r a t e d t o h a v e v a r i o u s d e g r e e s o f n e c r o s i s . F u r t h e r m o r e , we d i v i d e d t h e p e a k s e r u m A S A L v a l u e s i n t o g r o u p s u s i n g d i f f e r e n t r a n k , a n d t h e r e l a t i o n s h i p b e t w e e n t h e p e a k s e r u m A S A L l e v e l a n d t h e p r e s e n c e o f h e p a t i c n e c r o s i s i n t h e e x p e r i m e n t a l a n i m a l i s shown i n F i g . 2 1 . T h e c o m p a r i s o n o f t h e e l e v a t i o n o f s e r u m A S A L l e v e l s a n d t h e p r e s e n c e o r a b s e n c e o f h e p a t i c n e c r o s i s i n d i c a t e s t h a t t h e p e a k s e r u m A S A L v a l u e s c a n be u s e d as a m e a s u r e o f t h e i n c i d e n c e o f n e c r o s i s i n t h e r a b b i t i n some d e g r e e . H o w e v e r , we a t t e m p t e d t o q u a n t i t a t e t h e h e p a t i c n e c r o s i s t o f u r t h e r c l a r i f y t h i s r e l a t i o n s h i p , b u t a p a r a l l e l r e l a t i o n s h i p b e t w e e n t h e d e g r e e o f n e c r o s i s a n d t h e p e a k s e r u m A S A L v a l u e s c o u l d n o t be e s t a b l i s h e d ( s e e d i s c u s s i o n ) . 4 D I S C U S S I O N I n o r d e r t o i n v e s t i g a t e t h e m e c h a n i s m o f INH t o x i c i t y i n h u m a n s , i t i s n e c e s s a r y t o e s t a b l i s h a n a n i m a l m o d e l w h i c h r e s e m b l e s a s c l o s e l y a s p o s s i b l e t h e h e p a t o t o x i c e f f e c t s e e n i n h u m a n s . Among t h e t e s t e d a n i m a l s p e c i e s , i t h a s b e e n v e r y d i f f i c u l t o r i m p o s s i b l e t o o b t a i n ZHOU, T. 72 1 0 0 r 8 0 CD o .52 *co o o CD 6 0 4 0 2 0 -0 - 1 2 n = 6 S e r u m A S A L ( T a k a h a r a u n i t ) F i g . 2 1 . The r e l a t i o n s h i p between the incidence of necrosis i n s l i c e s and peak serum ASAL values i n the rabbits challenged by INH, AcINH and Hz o r a l l y or subcutaneously, with or without PB pretreatment (refer to t e x t ) . Necrosis rate i s the f r a c t i o n of animals sampled i n the ASAL ranges quoted which shows h i s t o l o g i c a l l y demonstrable hepatic necrosis. Z H O U , T . 73 r e p r o d u c i b l e h e p a t o t o x i c i t y w i t h INH i n t h e r a t , mouse a n d g u i n e a p i g (Thomas e t a l . , 1981 ; Woodward a n d T i m b r e l l , 1984; W r i g h t e t a l . , 1 9 8 6 b ) . W h i t e h o u s e e t al ( 1978) showed t h a t a d m i n i s t r a t i o n o f INH c a u s e d s i g n i f i c a n t r e l e a s e o f A S T a n d A L T i n t o s e r u m i n r a b b i t s . N o d a e t a l . ( 1983) f u r t h e r r e p o r t e d t h a t INH t r e a t m e n t c a u s e d b o t h f a t t y l i v e r a n d c e n t r i l o b u l a r n e c r o s i s h i s t o l o g i c a l l y i n r a b b i t s . T h e d a t a i n t h i s t h e s i s c o n f i r m s t h e v a l u e o f t h e r a b b i t as a m o d e l f o r INH h e p a t o t o x i c i t y b o t h b i o c h e m i c a l l y a n d h i s t o l o g i c a l l y . I t was f o u n d t h a t INH t r e a t m e n t c a u s e d a n e l e v a t i o n o f s e r u m A S A L l e v e l s a b o u t 24 h r s a f t e r t h e f i r s t a d m i n i s t r a t i o n , w i t h p e a k v a l u e s o f up t o 2000 u n i t s c o m p a r e d w i t h t h e c o n t r o l v a l u e s o f 4 . 3 + 2 . 6 (SD) u n i t s . I t was a l s o f o u n d t h a t INH t r e a t m e n t c a u s e d s i g n i f i c a n t h i s t o l o g i c a l c h a n g e s by l i g h t m i c r o s c o p y , i n c l u d i n g f a t t y l i v e r , v a c u o l a t i v e d e g r a d a t i o n a n d n e c r o s i s . B a s e d o n t h e s e d a t a , s e r u m A S A L l e v e l c o u l d be c o n s i d e r e d a v e r y s e n s i t i v e i n d i c a t o r o f l i v e r damage; a l l o f t h e a n i m a l s w h i c h h a d n e c r o s i s o n h i s t o l o g y d i s p l a y e d a n e l e v a t i o n i n s e r u m A S A L l e v e l t o some e x t e n t . I n t h e r a b b i t s w h e r e t h e s e r u m A S A L l e v e l was e l e v a t e d w i t h no e v i d e n c e o f h e p a t i c n e c r o s i s , t h e e x p l a n a t i o n c o u l d be as f o l l o w i n g : 1 ) . T h e l i m i t a t i o n o f s a m p l i n g : f o r e a c h r a b b i t l i v e r , o n l y a s m a l l f r a c t i o n o f t i s s u e was t a k e n f o r h i s t o l o g i c a l e x a m i n a t i o n , i . e . , t h e Z H O U , T . 74 l i v e r damage may n o t be homogeneous a n d n e c r o s i s may h a v e b e e n p r e s e n t i n a r e a s t h a t w e r e n o t s a m p l e d . 2 ) . T h e enzyme r e l e a s e o c c u r r e d b e f o r e m o r p h o l o g i c a l c h a n g e : i n g e n e r a l n e c r o s i s o n l y e v i d e n t o n h i s t o l o g i c a l e x a m i n a t i o n 8 h r s a f t e r i t s o c c u r r e n c e . Some e x p e r i m e n t a l a n i m a l s w e r e s a c r i f i c e d s o o n a f t e r t h e r i s e o f A S A L l e v e l b e c a u s e o f d i s t r e s s a n d w e a k n e s s . I n t h e s e c a s e s , t h e l a c k o f e v i d e n c e f o r n e c r o s i s was u n d e r s t a n d a b l e . 3 ) . A S A L m i g h t be r e l e a s e d i n t o s e r u m f r o m c e l l s a s a r e s u l t o f damage a n d r e p a i r m e c h a n i s m s i n c e l l s , e . g . , a t t h e s t a g e o f f a t t y i n f i l t r a t i o n a n d h e p a t i c s t e a t o s i s . R e p o r t s by W h i t e h o u s e e t al. (1982) a n d M c K e n n i s e t al. (1985) h a v e d e m o n s t r a t e d t h a t INH a n d i t s d e r i v a t i v e s c a n a l s o i n d u c e f a t t y i n f i l t r a t i o n a n d h e p a t i c s t e a t o s i s i n r a b b i t s . I n t h i s s t u d y , v a c u o l a t i o n was f o u n d i n H - E s t a i n e d s l i d e s f r o m a l m o s t a l l o f t h e e x p e r i m e n t a l a n i m a l s . O i l r e d "o" s t a i n e d s l i d e s p r o v e d t h a t f a t t y d e g e n e r a t i o n was p r e s e n t t o v a r y i n g d e g r e e i n a l l e x p e r i m e n t a l a n i m a l s . T h e e x p l a n a t i o n f o r t h e d i f f e r e n t s u s c e p t i b i l i t y t o INH h e p a t o t o x i c i t y among t h e a n i m a l s p e c i e s i s , p r o b a b l y , t h a t t h e p r e s e n c e o f l a r g e i n t e r s p e c i e s d i f f e r e n c e s i n t h e k i n e t i c s o f t h e m e t a b o l i c enzymes i n v o l v e d ( W h i t e h o u s e e t a l . , 1 9 8 3 ) . We h a v e d e m o n s t r a t e d t h a t INH c a u s e d a h e p a t o t o x i c e f f e c t a n d t h a t PB i n d u c t i o n d o e s e n h a n c e t h e h e p a t o t o x i c i t y Z H O U , T . 75 o f INH i n r a b b i t s . T h i s i s c o n s i s t e n t w i t h d a t a i n humans w h e r e v a r i o u s i n d u c i n g a g e n t s h a v e b e e n i m p l i c a t e d as i n c r e a s i n g t h e r i s k o f INH h e p a t o t o x i c i t y ( P e s s a y r e a n d M a z e l , 1976; W r i g h t e t al., 1982; W r i g h t e t al., 1984; B a r b a r e e t al., 1986). T h i s r e s u l t i s a l s o c o n s i s t e n t w i t h t h e p r o p o s a l s o f o t h e r a u t h o r s u s i n g t h e r a t m o d e l . A l t h o u g h Thomas e t al. (1987) s u g g e s t e d t h a t t h e r o l e o f t h e m i c r o s o m a l o x i d a s e i n t h e r a b b i t may n o t be a s i m p o r t a n t i n m e d i a t i n g I N H - i n d u c e d h e p a t o t o x i c i t y a s i t i s i n t h e r a t , PB may be a c t i n g b y i n d u c t i o n o f P - 4 5 0 e n z y m e s , l e a d i n g t o t h e f o r m a t i o n o f a r e a c t i v e i n t e r m e d i a t e . H o w e v e r , PB i s known t o i n d u c e many e n z y m e s a n d i t c o u l d be e n h a n c i n g t o x i c i t y by some o t h e r m e c h a n i s m s s u c h as i n d u c t i o n o f a m i d a s e a c t i v i t y ( S e n d o e t al., 1984). INH h e p a t o t o x i c i t y was n o t s i g n i f i c a n t l y d i f f e r e n t when INH was a d m i n i s t e r e d b y two r o u t e s , o r a l a n d s u b c u t a n e o u s . T h i s r e s u l t s u g g e s t s t h a t t h e b i o a v a i l a b i l i t y o f INH a d m i n i s t e r e d o r a l l y i s n e a r l y c o m p l e t e a n d t h a t INH h e p a t o t o x i c i t y i s n o t e n h a n c e d as a r e s u l t o f t h e t o t a l d o s e g o i n g t o t h e l i v e r v i a t h e p o r t a l c i r c u l a t i o n a f t e r o r a l a d m i n i s t r a t i o n . A l t h o u g h i t was w i d e l y s u g g e s t e d t h a t a c e t y l a t o r r a t e a f f e c t s t h e h e p a t o t o x i c i t y o f INH i n m a n , t h e d a t a i s c o n t r o v e r s i a l : some r e p o r t s s u g g e s t t h a t r a p i d a c e t y l a t o r s a r e a t g r e a t e r r i s k ( M i t c h e l l e t al., 1975b) a n d o t h e r s Z H O U , T . 76 s u g g e s t t h a t s l o w a c e t y l a t o r s a r e a t g r e a t e r r i s k ( D i c k i n s o n e t al., 1 9 8 1 ; M u s c h e t al., 1982; L a u t e r b u r g e t al., 1985a). T h e r e s u l t s f r o m t h i s e x p e r i m e n t s u p p o r t n e i t h e r p r o p o s i t i o n . We saw no o b v i o u s r e l a t i o n s h i p b e t w e e n t-^/2 o f SMZ (as a n i n d e x o f a c e t y l a t i o n ) a n d s e r u m A S A L i n t h e r a b b i t s c h a l l e n g e d w i t h INH ( F i g . 1 4 ) , s u g g e s t i n g t h a t r a t e o f a c e t y l a t i o n i s n o t c r i t i c a l t o t h e m e c h a n i s m o f I N H -i n d u c e d h e p a t o t o x i c i t y . Two h y p o t h e t i c a l m e c h a n i s m s w h i c h w o u l d be c o n s i s t e n t a r e as f o l l o w s . 1) T h e h e p a t o t o x i c i t y o f INH i s m a i n l y d u e t o t h e m e t a b o l i t e , Hz ( s e e b e l o w ) , a n d t h e a c e t y l a t e d m e t a b o l i t e o f H z , A c H z , i s a l s o c o n s i d e r e d as a h e p a t o t o x i n , s o t h a t t h e i n f l u e n c e o f a c e t y l a t o r r a t e i s l i m i t e d . 2) I n f o l l o w i n g t h e t h e o r y p r o p o s e d b y M i t c h e l l e t al ( 1 9 7 6 ) , t h e h e p a t o t o x i c s p e c i e s i s f o r m e d f r o m a s e r i e s o f enzyme c a t a l y s e d r e a c t i o n s w h i c h c a u s e t h e s t e p w i s e m e t a b o l i s m o f INH t o A c I N H t o A c H z ( t h e e s s e n t i a l p r e c u r s o r m o l e c u l e t o t h e t o x i c s p e c i e s ) , a n d a c e t y l a t i n g t o D i A c H z i s a d e t o x i f i c a t i o n m e t a b o l i c p a t h w a y o f A c H z . T h e r e f o r e , N - a c e t y l t r a n s f e r a s e p a r t i c i p a t e s b o t h p r o d u c t i o n a n d e l i m i n a t i o n o f A c H z . A c c o r d i n g t o t h i s t h e o r y , when INH was a d m i n i s t e r e d , f a s t a c e t y l a t o r t e n d e d t o g e n e r a t e more A c I N H , t h u s , m o r e A c H z ; a n i n c r e a s e d h e p a t o t o x i c i t y c o u l d be e x p e c t e d a l t h o u g h t h e e l i m i n a t i o n o f A c H z v i a D i A c H z c o u l d a l s o b e i n c r e a s e d . When A c I N H o r A c H z was c h a l l e n g e d , Z H O U , T . 77 h o w e v e r , a c e t y l a t i o n was o n l y r e l a t e d t o t h e e l i m i n a t i o n b u t n o t p r o d u c t i o n o f A c H z , i . e . , f a s t a c e t y l a t o r o n l y t e n d e d t o g e n e r a t e m o r e D i A c H z f r o m A c H z ; t h e r e b y , t h e h e p a t o t o x i c i t y s h o u l d be d e c r e a s e d . A c H z i s t h e h e p a t o t o x i c m e t a b o l i t e b u t t h e a c e t y l a t o r r a t e d e t e r m i n e s b o t h t h e p r o d u c t i o n a n d i n a c t i v a t i o n o f A c H z ; t h e e n d r e s u l t i n t h e r a b b i t m o d e l i s a n e q u i v a l e n t e x p o s u r e t o A c H z i n b o t h p h e n o t y p e s . C o m p a r i s o n o f t h e h e p a t o t o x i c i t y o f INH a n d i t s m e t a b o l i t e s , A c I N H , A c H z a n d H z , d e m o n s t r a t e d t h a t A c H z h a d no h e p a t o t o x i c e f f e c t i n e q u i m o l a r d o s e s ; Hz was s i g n i f i c a n t l y more h e p a t o t o x i c t h a n INH a n d was t h e m o s t t o x i c among t h e f o u r c h e m i c a l s t e s t e d , a n d t h e h e p a t o t o x i c p o t e n c y o f A c I N H was s i m i l a r t o I N H . How d o t h e s e r e s u l t s r e l a t e t o p r e v i o u s s t u d i e s ? M i t c h e l l e t al (1975) s u g g e s t e d t h a t I N H - i n d u c e d h e p a t o t o x i c i t y r e s u l t s f r o m t h e p r o d u c t i o n o f A c H z i n t h e l i v e r a n d i t s f u r t h e r m e t a b o l i s m t o a r e a c t i v e a c y l a t i n g i n t e r m e d i a t e . I n t h e e a r l y p u b l i s h e d d a t a u s i n g t h e r a t m o d e l a p r o g r e s s i v e i n c r e a s e i n t o x i c i t y a l o n g t h e s e r i e s I N H - A c I N H - A c H z i s e i t h e r shown o r i n f e r r e d ( N e l s o n e t al., 1976; T i m b r e l l e t al., 1 9 8 0 ) . T h e i n a b i l i t y o f o t h e r g r o u p s t o r e p r o d u c e t h e r a t m o d e l c a l l s i t i n t o q u e s t i o n . L i k e w i s e t h e d a t a f r o m o u r o b s e r v a t i o n shown i n T a b l e 1 a n d F i g . 11 a n d F i g . 12 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 . T h e d a t a o f T a b l e 1 d o e s n o t p r o v i d e e v i d e n c e t h a t t h e h e p a t o t o x i c i t y o f Z H O U , T . 78 A c I N H i s h i g h e r t h a n t h a t o f INH i n r a b b i t s . T h i s r e s u l t was s i m i l a r t o o u r p r e v i o u s s t u d i e s i n w h i c h i t a p p e a r s t h a t A c I N H a n d INH a r e a p p r o x i m a t e l y e q u i t o x i c o n a m m o l / k g / d a y b a s i s . S i n c e A c I N H o n l y h a s same , i f n o t l o w e r , h e p a t o t o x i c p o t e n c y as INH d o s e , A c I N H seems n o t t o be a a c c e p t a b l e m a j o r i n t e r m e d i a t e o f I N H - i n d u c e d h e p a t o t o x i c i t y . A c H z was n o t f o u n d t o be h e p a t o t o x i c i n t h i s s t u d y . T h e a d m i n i s t r a t i o n i n same m o l a r d o s e i n t h e same r e g i m e n as f o r INH d i d n o t l e a d t o a s i g n i f i c a n t r e l e a s e o f A S A L . T h i s r e s u l t w o u l d t e n d t o d e n y t h e m a j o r r o l e o f A c H z i n I N H -i n d u c e d h e p a t o t o x i c i t y p r o p o s e d b y p r e v i o u s a u t h o r s . Thomas e t al.(1984) p r o p o s e d t h a t h i g h e r a m i d a s e a c t i v i t y i n t h e r a b b i t c o m p a r e d t o t h e r a t l e a d s t o f a s t e r c o n v e r s i o n o f A c I N H t o A c H z w h i c h i n t u r n l e a d s t o g r e a t e r c o v a l e n t b i n d i n g a n d h e p a t o t o x i c i t y . T h e r e s u l t s f r o m o u r p r e v i o u s a n d p r e s e n t s t u d i e s do i n d i c a t e t h a t r a b b i t i s more s e n s i t i v e t o I N H - i n d u c e d h e p a t o t o x i c i t y , b u t o u r d a t a c a n n o t s u p p o r t t h a t t h i s i s d u e t o a h i g h e r A c H z l e v e l . M e a n w h i l e , o u r d a t a a r e i n c o n s i s t e n t w i t h a n o t h e r s u g g e s t i o n p r o p o s e d b y Thomas e t al. (1987) t h a t i n t h e r a b b i t c o v a l e n t b i n d i n g t o h e p a t i c p r o t e i n was more d e p e n d e n t o n t h e a c e t y l a t i o n r a t e t h a n on i n d u c t i o n o f m i c r o s o m a l o x i d a s e . O u r s t u d i e s show t h a t I N H - i n d u c e d h e p a t o t o x i c i t y i s i n c r e a s e d b y t h e i n d u c i n g a g e n t , P B , b u t i s n o t r e l a t e d w i t h t h e a c e t y l a t o r r a t e . Z H O U , T . 79 N o d a e t al. (1983) s u g g e s t e d t h a t H z , b u t n o t A c I N H o r A c H z , i s t h e k e y i n t e r m e d i a t e o f I N H - i n d u c e d h e p a t o t o x i c i t y i n a r a b b i t m o d e l . H z , a p r o v e n m e t a b o l i t e o f INH i n r a b b i t (Thomas e t al., 1 9 8 1 ) , d i d l e a d t o a d o s e - r e l a t e d r e l e a s e o f A S A L , i n t h e p r e s e n t s t u d y ( F i g . 8 ) . I n t h e h i g h d o s e g r o u p g i v e n h a l f o f t h e m o l a r d o s e o f I N H , h e p a t i c n e c r o s i s was f o u n d i n a l l e i g h t a n i m a l s . A s m e n t i o n e d a b o v e , a l t h o u g h a c e t y l a t i o n o f Hz t o A c H z may t a k e p l a c e , t h i s o c c u r s t o o n l y a l i m i t e d e x t e n t ( W r i g h t a n d T i m b r e l l , 1978; P e r r y e t al., 1 9 8 1 ) . I n t h i s e x p e r i m e n t , e v e n t h e h i g h d o s e o f Hz was o n l y h a l f o f t h e m o l a r d o s e o f A c H z ; t h e r e f o r e , i t i s i m p o s s i b l e t h a t H z - i n d u c e d h e p a t o t o x i c i t y i s a r e s u l t o f c o n v e r s i o n o f Hz t o A c H z . T h i s r e s u l t s u p p o r t t h e v i e w t h a t Hz i s m o r e l i k e l y t h e m e t a b o l i t e o f INH w h i c h a c c o u n t s f o r h e p a t o t o x i c i t y (Thomas e t al., 1981 ; N o d a e t al., 1 9 8 3 ) . T h e d a t a p r e s e n t e d i n t h i s t h e s i s a r e c o n s i s t e n t w i t h t h e r e s u l t s p r o v i d e d by W r i g h t e t al. ( 1 9 8 6 a ) . I n t h a t s t u d y i t was d e m o n s t r a t e d t h a t Hz was t h e m o s t t o x i c o f t h e f o u r c h e m i c a l s i . e . , I N H , A c I N H , A c H z a n d Hz t e s t e d i n i s o l a t e d r a t h e p a t o c y t e s , a n d t h a t PB p r e t r e a t m e n t p o t e n t i a t e s t h e Hz a n d A c H z i n d u c e d h e p a t o t o x i c i t y . I n s u m m a r y , t h e r e s u l t s i n t h i s t h e s i s show t h a t t h e r a b b i t i s a r e p r o d u c i b l e a n i m a l m o d e l t o s t u d y t h e h e p a t o t o x i c e f f e c t o f I N H . T h i s m o d e l r e s e m b l e s ZHOU, T. 80 h e p a t o t o x i c i t y i n t h e human b e i n g : 1) I t r e q u i r e s m u l t i p l e doses o f INH t o produce t o x i c i t y . 2) The e a r l i e s t m a n i f e s t a t i o n i s a r e l e a s e o f l i v e r enzymes, e.g., ASAL, i n t o serum. 3) The h i s t o l o g i c a l changes i n l i v e r o f n e c r o s i s a r e s i m i l a r t o t h a t o c c u r r e d i n t h e humans and 4) The e f f e c t i s p o t e n t i a t e d by i n d u c i n g a g e n t s . I n t h e r a b b i t model we found t h a t t h e r a t e o f a c e t y l a t i o n d i d not a f f e c t t h e h e p a t o t o x i c e f f e c t o f INH. U s i n g t h e r a b b i t model we found t h a t among t h e INH m e t a b o l i t e s , Hz was t h e most p o t e n t h e p a t o t o x i n and t h a t AcHz was t h e l e a s t p o t e n t i f i t had any t o x i c e f f e c t a t a l l . These d a t a i n d i c a t e t h a t INH h e p a t o t o x i c i t y i s p r o b a b l y not due t o m i c r o s o m a l m e t a b o l i s m of AcHz, but i s more l i k e l y due t o Hz. We c o n c l u d e t h a t t h e mechanism o f INH h e p a t o t o x i c i t y needs t o be r e e v a l u a t e d , and t h a t t h e r a b b i t model w i l l l i k e l y p rove v a l u a b l e i n e l u c i d a t i n g t h e mechanism. Z H O U , T . 81 B I B L I O G R A P H Y A l b a n o E . a n d T o m a s i A . ( 1 9 8 7 ) . S p i n t r a p p i n g o f f r e e r a d i c a l i n t e r m e d i a t e s p r o d u c e d d u r i n g t h e m e t a b o l i s m o f i s o n i a z i d a n d i p r o n i a z i d i n i s o l a t e d h e p a t o c y t e s . B i o c h e m . P h a r m a c o l . 3_6: 2 9 1 3 - 2 9 2 0 . B a c k K . C . , C a r t e r V . L . a n d Thomas A . A . ( 1 9 7 8 ) . O c c u p a t i o n a l h a z a r d s o f m i s s i l e o p e r a t i o n s w i t h s p e c i a l r e g a r d t o t h e h y d r a z i n e p r o p e l l a n t s . A v i a t . S p a c e E n v i r o n . M e d . 4 9 : 5 9 1 - 5 9 8 . B a h r i A . K . , C h i a n g C . S . a n d T i m b r e l l J . A . ( 1 9 8 1 ) . A c e t y l h y d r a z i n e h e p a t o t o x i c i t y . T o x i c o l . A p p l . P h a r m a c o l . 6_0: 5 6 1 - 5 6 9 . B a r b a r e J . C , L a l l e m e n t P . Y . , V o r h a u e r W. a n d V e y s s i e r P . ( 1 9 8 6 ) . H e p a t o t o x i c i t e de 1 ' i s o n i a z i d e : i n f l u e n c e de l a c a r b a m a z e p i n e ? G a s t r o e n t e r o l o g i e C l i n i q u e e t B i o l o g i q u e 10: 5 2 3 - 5 2 4 B e h n i s c h R . , M i e t z s c h F . a n d S c h m i d t H . ( 1 9 4 8 ) . V e r f a h r e n z u r h e r s t e l l u n g v o n T h i o s e m i c a r b a z o n e n . DBP 9 2 7 : 5 0 5 . B e r n s t e i n J . , L o t t W . A . , S t e i n b e r g B . A . , a n d Y a l e H . L . ( 1 9 5 2 ) . C h e m o t h e r a p y o f e x p e r i m e n t a l t u b e r c u l o s i s : V . i s o n i c o t i n i c a c i d h y d r a z i d e ( n y d r a z i d ) a n d r e l a t e d c o m p o u n d s . Am. R e v . T u b e r c . 6_5: 375 -364 . B l a c k M . , M i t c h e l l J . R . , Zimmerman H . J . , I s h a k K . G . a n d E p l e r G . R . ( 1 9 7 5 ) . I s o n i a z i d h e p a t i t i s i n 114 p a t i e n t s . G a s t r o e n t e r o l o g y 6_9: 2 8 9 - 3 0 2 . B l a i r I . A . , T i n o c o R . M . , B r o d i e M . J . , C l a r e R . A . , D o l l e r y C . T . , T i m b r e l l J . A . a n d B e e v e r I . A . ( 1 9 8 5 ) . P l a s m a h y d r a z i n e c o n c e n t r a t i o n s i n man a f t e r i s o n i a z i d a n d h y d r a l a z i n e a d m i n i s t r a t i o n . Hum. T o x i c o l . 4_: 1 9 5 - 2 0 2 . B l a i r I . A . , C l a r e R . A . , M a n s i l l a - T i n o c o R . a n d T i m b r e l l J . A . ( 1 9 8 0 ) . T h e a n a l y s i s o f h y d r a z i n e i n p l a s m a . A b s t r . W o r l d C o n f . C l i n . P h a r m a c o l . T h e r . 0 8 0 6 . B l u m M . , G r a n t D . M . , D e m i e r r e A . a n d M e y e r U . A . ( 1 9 8 9 ) . N - a c e t y l a t i o n p h a r m a c o g e n e t i c s : a gene d e l e t i o n c a u s e s a b s e n c e o f a r y l a m i n e N - a c e t y l t r a n s f e r a s e i n l i v e r o f s l o w a c e t y l a t o r r a b b i t s . P r o c . N a t l . A c a d . S c i . USA 86: 9 5 5 4 - 9 5 5 7 . Z H O U , T . 82 B o z e k C . B . , K a s t r u k o f f L . E . , W r i g h t J . M . , P e r r y T . L . a n d L a r s e n T . A . ( 1 9 8 7 ) . A c o n t r o l l e d t r i a l o f i s o n i a z i d t h e r a p y f o r a c t i o n t r e m o r i n m u l t i p l e s c l e r o s i s . J . N e u r o l . 2 3 4 : 3 6 - 3 9 . C a m p a n i n i R . Z . , T a p i a R . A . , S a r n a t W. a n d N a t e l s o n S . ( 1 9 7 0 ) . E v a l u a t i o n o f s e r u m a r g i n i n o s u c c i n a t e l y a s e ( A S A L ) c o n c e n t r a t i o n s as a n i n d e x t o p a r e n c h y m a l l i v e r d i s e r s e . C l i n . C h e m . 16_i 4 4 - 5 3 . C h o r i n e V . ( 1 9 4 5 ) . A c t i o n de 1 ' a m i d e n i c o t i n i q u e s u r l e s b a c i l l e s d u g e n r e M y c o b a c t e r i u m . C . R . A c a d . S c i . ( P a r i s ) 2 2 0 : 1 5 0 - 1 5 1 . C l a i b o r n e R . A . a n d D u t t A . K . ( 1 9 8 5 ) . I s o n i a z i d - i n d u c e d p u r e r e d c e l l a p l a s i a . Am. R e v . R e s p i r . D i s . 1 3 1 : 9 4 7 - 9 4 9 C u t h b e r t s o n W . F . J . , I r e l a n d D . M . a n d W o l f f W. ( 1 9 5 3 ) . D e t e c t i o n a n d i d e n t i f i c a t i o n o f some m e t a b o l i t e s o f i s o n i c o t i n i c a c i d h y d r a z i d e ( i s o n i a z i d ) i n human u r i n e . J . B i o c h e m . 5 5 : 6 6 9 - 6 7 1 . D i c k i n s o n D . S . , B a i l e y W . C . , H i r s c h o w i t z B . I . S o o n g S . - J . , E i d u s L . a n d H o d g k i n M . M . ( 1 9 8 1 ) . R i s k f a c t o r s f o r i s o n i a z i d (INH) i n d u c e d l i v e r d y s f u n c t i o n . J C l i n . G a s t r o e n t e r o l o g y 3_: 2 7 1 - 2 7 9 . D r u c k r e y H . ( 1 9 7 3 ) . S p e c i f i c c a r c i n o g e n i c a n d t e r a t o g e n i c e f f e c t s o f i n d i r e c t a l k y l a t i n g m e t h y l a n d e t h y l c o m p o u n d s , a n d t h e i r d e p e n d e n c y o n s t a g e s o f o n t o g e n i c d e v e l o p m e n t . X e n o b i o t i c a _3: 2 7 1 - 3 0 3 . D u q u e t t e P . , P l e i n e s J . a n d d u S o u i c h P . ( 1 9 8 5 ) , I s o n i a z i d f o r t r e m o r i n m u l t i p l e s c l e r o s i s : a c o n t r o l l e d s t u d y . N e u r o l o g y 3 5 : 1 7 7 2 - 1 7 7 5 . F o x H . H . ( 1 9 5 3 ) . T h e c h e m i c a l a t t a c k o n t u b e r c u l o s i s . T r a n s . N . Y . A c a d . S c i . 15: 2 3 4 - 2 4 2 . F r a n c i s D . A . , G r u n d y D . a n d H e r o n J . R . ( 1 9 8 6 ) . T h e r e s p o n s e t o i s o n i a z i d o f a c t i o n t r e m o r i n m u l t i p l e s c l e r o s i s a n d i t s a s s e s s m e n t u s i n g p o l a r i s e d l i g h t g o n i o m e t r y . J . N e u r o l . N e u r o s u r g e r y P s y c h i a t r y 4j?: 8 7 - 8 9 . F r y m o y e r J . W . a n d J a c o x R . F . ( 1 9 6 3 a ) . I n v e s t i g a t i o n o f t h e g e n e t i c c o n t r o l o f s u l f a d i a z i n e a n d i s o n i a z i d m e t a b o l i s m i n t h e r a b b i t . J . L a b . C l i n . M e d . 6_2: 8 9 1 -9 0 4 . F r y m o y e r J . W . a n d J a c o x R . F . ( 1 9 6 3 b ) . 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D e v e l o p . B i o c h e m . L 3 : 2 3 5 - 2 3 8 . W e b e r W . W . , M i c e l i J . N . , H e a r s e D . J . a n d Drummond G . S . ( 1 9 7 6 ) . N - a c e t y l a t i o n o f d r u g s ; P h a r m a c o g e n e t i c s t u d i e s i n r a b b i t s s e l e c t e d f o r t h e i r a c e t y l a t o r c h a r a c t e r i s t i c s . D r u g M e t a b . D i s p o s . 4: 9 4 - 1 0 1 . W h i t e h o u s e L . W . , I v e r s o n F . a n d Wong L . T . ( 1 9 8 5 ) . E f f e c t s o f r i f a m p i n p r e t r e a t m e n t o n h e p a t i c p a r a m e t e r s i n t h e r a b b i t . T o x i c o l . L e t t . 2 4 : 1 3 1 - 1 3 6 . W h i t e h o u s e L . W . , T r y p h o n a s L . , P a u l C . J . , S o l o m o n r a j G . , Thomas B . H . a n d Wong L . T . ( 1 9 8 3 ) . I s o n i a z i d - i n d u c e d h e p a t i c s t e a t o s i s i n r a b b i t s : a n e x p l a n a t i o n f o r s u s c e p t i b i l i t y a n d i t s a n t a g o n i s m by p y r i d o x i n e h y d r o c h l o r i d e . C a n . J . P h y s i o l . P h a r m a c o l . 6_1: 4 7 8 - 4 8 7 . Z H O U , T . 89 W h i t e h o u s e L . W . , T r y p h o n a s L . , Thomas B . H . a n d P a u l C P . ( 1 9 7 8 ) . I s o n i a z i d t o x i c i t y a l o n e a n d i n c o m b i n a t i o n w i t h e t h a n o l i n t h e r a b b i t . I : p a t h o l o g i c a n d b i o c h e m i c a l f a c t o r s . ( A b s t r . ) T o x i c o l . A p p l . P h a r m a c o l . 4 5 : 3 5 1 . Woodward K . N . a n d T i m b r e l l J . A . ( 1 9 8 4 ) . A c e t y l h y d r a z i n e h e p a t o t o x i c i t y : t h e r o l e o f c o v a l e n t b i n d i n g . T o x i c o l o g y 3_0: 65 -74 W r i g h t J . M . a n d T i m b r e l l J . A . ( 1 9 7 8 ) . F a c t o r s a f f e c t i n g t h e m e t a b o l i s m o f C - a c e t y l h y d r a z i n e i n r a t s . D r u g M e t a b . D i s p o s . 6_: 5 6 1 - 5 6 6 . W r i g h t J . M . K a s s G . E . N . a n d O r r e n i u s S . ( 1 9 8 6 a ) . T h e c y t o t o x i c i t y o f i s o n i a z i d a n d m e t a b o l i t e s s t u d i e d i n I s o l a t e d r a t h e p a t o c y t e s . ( A b s t r . ) A c t a P h a r m a c o l . T o x i c o l . 59_ ( s u p p l . 5 ) : 2 2 1 . W r i g h t J . M . , N g a i H . , Adams S . , B a i n A . a n d W a l l R . A . ( 1 9 8 6 b ) . L a c k o f h e p a t o t o t x i c i t y o f a c e t y l h y d r a z i n e i n r o d e n t s . ( A b s t r . ) A c t a P h a r m a c o l . T o x i c o l . _59 ( s u p p l . 5) : 2 2 1 . W r i g h t J . M . , S t o k e s E . F . a n d Sweeney V . ( 1 9 8 2 ) . I s o n i a z i d -i n d u c e d c a r b a m a z e p i n e t o x i c i t y a n d v i c e v e r s a : a d o u b l e d r u g i n t e r a c t i o n . New E n g . J . M e d . 3 0 7 : 1 3 2 5 - 1 3 2 7 . W r i g h t J . M . , W a l l R . A . a n d Adams S . P . ( 1 9 8 4 ) . C a r b a m a z e p i n e -i n d u c e d i s o n i a z i d h e p a t o t o x i c i t y : a s e r i o u s d r u g i n t e r a c t i o n . C l i n . I n v e s t . M e d . 1_ ( s u p p l . 3 9 ) . 

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