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Isoniazid hepatotoxicity Zhou, Ting 1990

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ISONIAZID  HEPATOTOXICITY  by TING ZHOU M.D.,  Shanghai M e d i c a l U n i v e r s i t y ,  1982  THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES PHARMACOLOGY AND  THERAPEUTICS  We accept t h i s t h e s i s as conforming t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA March 19 9 0 (c) T i n g  Zhou, 1990  In  presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  publication of  and study.  thesis for scholarly by  his  or  her  the  requirements that the  I further agree  purposes  representatives.  may be It  this thesis for financial gain shall not  Department of  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  March 26, 1990  advanced  Library shall make it  granted  by the  understood be  Pharmacology & T h e r a p e u t i c s  an  that permission for extensive  is  permission.  for  head  that  allowed without  of  my  copying  or  my written  ABSTRACT Isoniazid the  (INH),  treatment  with  a  mild  and  one  the  prophylaxis  increase  in  treated  patients  and  treated  patients.  Since  been  of  reported  liver  severe the  on  most  of  effective  agents  tuberculosis  associated  transaminases  in  up  hepatotoxicity  in  up  1970's,  the  a  number o f  mechanism  in  to  20%  of  2%  of  to  studies  of  have  INH-induced  hepatotoxicity. The  original  hepatotoxic metabolism a  effect of  reactive  been other is  to  necessary  were  and  these  establish  a  and  of  metabolites, were  divided  Serum  to  recently  been  results  into  human  New  12  changes  in  phenotype  for  After each  acetylisoniazid administered  in  a  lyase  slides the rabbit,  (AcINH), two-day  INH)  the  to  have  rat,  or  Therefore,  it  model  which  being.  treatment  liver  of  workers  animal  Zealand  argininosuccinate  the  microsomal  in  proposed.  the  male  the  other  reproducible in  that  a metabolite  original  hepatotoxicity.  subcutaneously.  suggested  due  (AcHz,  have  experiments,,  each.  acetylator  AcHz  to  models  rats  was  More  reproduce the  histological  indices  INH  INH h e p a t o t o x i c i t y  used,  rabbits  in  acetylhydrazine  mechanistic  In  of  intermediate.  unable  resembles  studies  white  rabbits  groups  of  (ASAL) were  6-8  levels  chosen  as  determination  of  INH  and  its  hydrazine  (Hz)  and  regimen  orally  or  The is  a  the  results  showed  sensitive  and  incidence  serum  ASAL  of  control until  INH  two-day  peak 72  the  values  of  up  serum ASAL  enzyme  seen  4.3+2.6  24  hrs  (SD)  after  (0.36+3x0.26  to  Takahara  in  which  on  rabbits  parallels  histology.  Takahara  the  regimen 2674  level  marker  necrosis  values  about  the  first  units  were  challenge  mmol/kg/dx2d,  units  The  occurred  of  s . c ) ;  at  about  hrs. No  given  significant  orally  (PB)  and  (0.1 of  the  significantly  acetylation  rate  SMZ t  with  a  SMZ t  challenged or  between  /  1  1  2  /  2  with  of  the  correlation  was  SMZ ^1/2 also  of  INH  Phenobarbital increased  INH  (p<0.05,  (SD)  F  were  the  (0.36+3x0.26  test)  compared  ASAL  and  (n=ll).  values  acetylators  slow  acetylators  Among  n=18).  in  the  p.o.  correlation and  their  fast  mmol/kg/dx2d, no  into  measuring  (SMZ):  (SD)  (r=0.05, present  classified  by  (n=54)  pretreatment,  serum  by  phenotype  INH ( 0 . 3 6 + 3 x 0 . 2 6  peak by  caused  rabbits  50.3+10.4  PB  detected.  sulfamethazine  12.8+4.4  of  without  represented  AcINH  acetylator of  toxicity  pretreatment.  experimental of  a  level  PB  the  pretreatment  p.o.)  65  with  i»p»)  was  ASAL  populations  with  subcutaneously  group without  The  between  serum  mmol/kg/dx2d, with  difference  mg/kgx3d,  elevation  rabbits or  was  s.c.) found  acetylation This  rabbits  lack  of  challenged  rate this with  and H z . Among  is  specific hepatic  maintained in  that  the  most  INH a n d potent  its  metabolites,  hepatotoxin. Its i i i  AcINH, effect  Hz is  and  AcHz,  Hz  dose-dependent  over  the  dose  0.19+3x0.14  range  mmol/kg/dx2d,  mmol/kg/dx2d,  s . c ) ,  effect,  is  other  (0.10+3x0.07,  which  produced  contradictory  authors.  AcINH  mmol/kg/dx2d,  hepatotoxic  effect  results  animal  model  release  of  that  seen  potentiated the  rate  does  of  by  is the  serum  INH  and  tested,  due  incorrect.  in  of  an  is  most  that  studies rabbit  iv  a  reproducible (2)  changes  in  (4) of  the  resemble of  INH  accordance risk  the  is  with  in  the  acetylation  INH;  (5)  among  potent.  the  microsomal  the  is  s . c ,  INH.  increased  inducers;  that  We t h i n k  Further  mechanism  which  Hz was  to  of  by  intermediate  hepatotoxicity  hepatotoxicity  indicate is  that  a  hepatotoxicity;  the  PB p r e t r e a t m e n t  the  had  pathological  (3)  reported  mmol/kg/dx2d,  p.o.)  the  hepatotoxic  results  rabbit  enzyme  data  the  (1)  microsomal  responsible. exact  that  affect  to  to  beings  not  significant  or  and  (0.36+3x0.26  no  similar  human  hepatotoxicity probably  is  studying  to  in  metabolites These  s.c.  human b e i n g s ;  evidence  presence  the  for  ASAL in  which  showed  AcHz  (0.28+3x0.20  0.42+3x0.30  The  p.o.).  0.14+3x0.10  it are  hypothesis metabolism is  more  of  likely  required  model.  that  to  INH  AcHz  is  that  Hz  elucidate  TABLE OF CONTENTS  CHAPTER  Page  ABSTRACT L I S T OF T A B L E S L I S T OF F I G U R E S ACKNOWLEDGEMENT ABBREVIATIONS  i i vii v i i i x xi  1  INTRODUCTION  1  1.1  G E N E R A L C O N C E P T OF INH 1.1.1 CHEMISTRY 1.1.2 THERAPEUTICS USES 1.1.3 MECHANISMS OF A C T I O N S 1.1.4 BIOTRANSFORMATION 1.1.5 PHARMACOKINETICS 1.1.6 ADVERSE E F F E C T S  1.2  HEPATOTOXICITY 1.2.1 INH 1.2.2 AcINH 1.2.3 AcHz 1.2.4 Hz  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 I N D U C T I O N OR I N H I B I T I O N 1.3.2 ANIMAL S P E C I E S 1.3.3 A C E T Y L A T O R PHENOTYPE  1.4  1.5  OF  INH  AND  1 1 3 5 7 11 12 ITS  METABOLITES  SERUM A R G I N I N O S U C C I N A T E L Y A S E AS ENZYME I N H E P A T O T O X I C STUDY  22 22 25 27  A MARKER  T H E O B J E C T I V E S OF OUR S T U D I E S 1.5.1 HYPOTHESIS 1.5.2 TO E S T A B L I S H SERUM A S A L A S A MARKER ENZYME I N T H E R A B B I T MODEL 1.5.3 TO COMPARE T H E 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 1.5.4 TO C L A R I F Y T H E 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 1.5.5 TO C L A R I F Y T H E R E L A T I O N S H I P BETWEEN T H E 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  v  13 14 16 17 19  31 32 32 33 34 34  35  TABLE  OF CONTENTS  (Continued)  CHAPTER  2  Page  MATERIALS  AND METHODS  35  2.1  SUBJECT  35  2.2  MATERIALS  35  2.3  METHODS  36  2.3.1 2.3.2 2.3.3 2.3.4  ANIMAL TREATMENT PHENOTYPING ADMINISTRATION A S A L MEASUREMENT  2 . 4 H I S T O L O G I C A L STUDY 2.4.1 TISSUE FIXATION 2.4.2 H - E STAINED SLIDES 2.4.3 O I L RED " O " S T A I N E D 2.5  3  STATISTICAL  36 37 39 41  SLIDES  METHODS  44 44 45 45 45  RESULT  46  3.1  E F F E C T S O F I N H 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 O F SERUM A S A L L E V E L 3.1.1 T I M E COURSE O F T H E E L E V A T I O N O F 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 3.1.2 T H E E F F E C T OF P B ON I N H - I N D U C E D HEPATOTOXICITY 3.1.3 INFLUENCE OF D I F F E R E N T ADMINISTRATION ROUTES 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 O F SERUM A S A L L E V E L 3.1.5 T H E R E L A T I O N S H I P BETWEEN T H E E L E V A T I O N OF A S A L L E V E L AND T H E A C E T Y L A T O R R A T E  3.2  3.3  4  E F F E C T S O F I N H 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 THE RELATIONSHIP  BETWEEN E L E V A T I O N O F  SERUM A S A L  AND H E P A T I C  LEVEL  DISCUSSION  BIBLIOGRAPHY  NECROSIS  46 46 49 49 52 58  60  62  71  vi  81  LIST OF TABLES  TABLE  Table  Page  1  Serum  ASAL  response  Table 2 The p a r a m e t e r s in rabbits  of  to  drug  challenges  SMZ-acetylator  vii  phenotype  47  58  LIST OF FIGURES  Figure  1  Page  Chemical structures metabolites  of  INH a n d  its  major 4  2  Metabolic  pathways  of  INH  9  3  metabolic  pathways  of  AcHz  18  4  Metabolic  pathways  of  Hz  21  5  Time c o u r s e of challenge with  6  7  8  9  10  11  12  13  14  15  serum ASAL INH  release  following 48  Serum ASAL l e v e l s o b t a i n e d f o l l o w i n g oral 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  Serum ASAL l e v e l s i n r a b b i t s o r a l l y and subcutaneously a d m i n i s t e r e d w i t h INH  51  Serum ASAL l e v e l s administered with  in rabbits orally d i f f e r e n t doses of  53  Serum ASAL l e v e l s administered with  in rabbits I N H a n d Hz  Serum ASAL l e v e l s administered with  in rabbits subcutaneously two d o s e s o f AcINH  55  Serum ASAL l e v e l s administered with  in rabbits orally INH a n d A c I N H  56  Serum ASAL l e v e l s w i t h INH a n d A c H z  in  Hz  orally 54  rabbits  administered 57  P l o t o f t h e p l a s m a AcSMZ/SMZ+AcSMZ 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  The r e l a t i o n s h i p serum ASAL v a l u e w i t h INH  61  l i v e r tissue with control rabbit  of in  SMZ h a l f - l i f e t o t h e peak the r a b b i t s challenged  H-E s t a i n  from 63  v i i i  LIST  OF F I G U R E S  (Continued)  Figure  Page  16  Vacuolated  17  Focal  18  Sub-massive  19  liver tissue from c o n t r o l  20  21  degeneration  necrosis  Various rabbits  in  in  rabbit  necrosis  in  liver rabbit  w i t h O i l Red rabbit  degrees of f a t t y liver tissues  rabbit  "0"  liver  tissue  tissue liver  64 65  tissue  66  stain 67  metamorphosis  in 68  The r e l a t i o n s h i p between the i n c i d e n c e necrosis and peak serum ASAL v a l u e s  ix  of 72  ACKNOWLEDGEMENTS  The received and  author from  Wright  the  thanks  and D r .  In  addition and  sponsorship  of  Wright. expert  the  Wong  this  due of  author  R.  her  Supervisory  research  are  Dr.  due  A.  Wall,  to  Columbia. Dr.  appreciation  A.  and  Wall  Mr.  J .  M.  Committee.  advice R.  advice  Pharmacology  British  Dr.  her  of  and  S.  for  the  financial  and  Dr.  Adams  J . M.  for  his  assistance.  must  also  department, their  of  to  supervisor,  thanks  Department  express  valuable  technical  for  University  the  the  encouragement  at  Abbott  her  the  of  the  Sincere  Thanks  for  members  are  F.  dedication  of  grateful  Therapeutics  Particular  of  is  be  Janelle  assistance  extended  to  Swetnam, which  the  Elaine  secretarial Jan,  facilitated  and  the  staff  Margaret  completion  thesis.  Finally,  the and  author  her  parents  her  and  encouragement.  offers  husband  for  x  her  sincerest  their  love,  gratitude  to  understanding  LIST OF ABBREVIATIONS AcHz  acetylhydrazine  AcINH  acetylisonicotinyl  AcSMZ  acetyl  ASAL  argininosuccinate  BNPP  bis-para-nitrophenyl  DiAcHz  diacetylhydrazine  HPLC  high  i . p.  intraperitoneal  INA  isonicotinic  acid  INH  isonicotinyl  hydrazine  p.o.  oral  PB  phenobarbital  RMP  rifampicin  s.c.  subcutaneous  SMZ  sulfamethazine  t  l/2  hydrazine  sulfamethazine lyase phosphate  performance l i q u i d  chromatography  administration  administration  apparent  administration  half-life  xi  ZHOU,  T.  1  INTRODUCTION  1  1.1  GENERAL  CONCEPT  Isoniazid  OF INH  (isonicotinyl  hydrazine,  INH,  acid  hydrazide, pyridine-4-carboxylic acid  of  the  most  tuberculosis.  Since  INH h a s  al.), and  the  1987;  prophylaxis  extensively renewed  that  it  subjects  in  in  this  1981).  in  Its  its  In  are  hepatotoxicity  of  tested  using  a  for  (Albano  recent  m e t a b o l i s m due in 1973;  a  hepatotoxicity  recent  series  I N H a n d some  (Bernetein the and  of of  years.  years to  The  et have  Tomasi,  experiments its  major  of  been  human  1975). carried reported  which  metabolites  b i o c h e m i c a l and p a t h o l o g i c a l  has  discovery  al.,  in  have  there  the  studies  et  treatment  minority  Black  one  mycobacterium  1952  drug  is  pharmacological actions  and B o i t n o t t ,  laboratory in  vivo  major  in  tuberculosis  INH-induced  thesis  in  the  hepatotoxicity  (Maddrey  this  as  hydrazide)  against  introduction  of  interest  on  agents  investigated.  causes  Studies on  its  remained  T h o m a s et al.,  been been  active  isonicotinic  the were  methods.  ZHOU,  1.1.1  is  the  empirical  solubility 20%  2  CHEMISTRY  INH the  T.  2.5%  water  in  soluble  soluble easily  in  first  in  Charles  Furthermore,  hydroxylamine  The  by  Chorine  isonicotinic terms  of  years  137.2).  Its  37-°C.  and  It  only  INH i s  water.  is  about poorly  It  reacts  fortuitous.  (1912)  no  has  hydrazone.  ethyl  as  at  saw  an  isonicotinate  the  any  was  German and  reason  to  antituberculous.  studying from  It  isonicotinate  one  while  1951; this acid,  effect  (1945)  the  the  failed  among  chemically  reaction  of  observe  any  to  was et  tuberculostatic  the  to  al., was  first  activity,  start  acid a  amide  search  derivatives.  for  One  of  thiosemicarbazone  al.,  et  synthesis and  Fox  nicotinic  pyridine  (Behnisch Levaditi  of  led  derivatives  pyridine-4-aldehyde  for  from  obtained  agents  pyridine  material  with  which  activity.  antituberculous  Fox,  Mally  (1951),  antituberculous  discovered  1951;  and  at  20°C.  somewhat  suitability  ethyl  at  soluble  was  38  product with  a  25%  ethanol  ethanol  Prague  For  Gardner  tuberculostatic  these  in  and  acid,  weight  2 2 ° C and  immiscible  INH  its  similar  and  Meyer  hydrate. for  at  forming  by  (molecular  methanol  of  University  INH  13%  solvents  discovery  hydrazine  very  is  acetone,  isonicotinic  7  methanol  prepared  of  CgH N30  boiling  organic  with  The  study  formula  in  soluble  hydrazide  1948;  Levaditi  1952). the  methyl  intermediate INH was  The  al.,  starting ester  was  found  et  of  to  of  INH. be  In  more  ZHOU,  active  than  modified  any  in  previously  many  ways  known  without  substance  abolishing  its  and  T.  could  activity  3  be  (Fox,  1953). INH  forms  reduces  complexes  ammoniacal  mirrors.  It  silver  reacts  hydrazones  as  also  constituents  such  as  acid.  reaction  metabolites  of  (AcINH),  obtain  acid), have  INH  derivatives advantage  with  as  the  do  or  and  on  made  to  give  and  food  oxalacetic as  typical  N-isonicotinoylglycine acid  modify  with  the  to  acetylisonicotinoyl-hydrazine  INH-resistant  another,  and  It  silvered  and ketones  acid,  isonicotinic  INH  yielding  encountered  improved  antituberculous  approach  salts.  physiological  pyruvic are  metal  (INA).  INH  chemically  pharmacokinetic,  properties,  mycobacteria.  in  activity  whole  INH i s  to  and  especially  Although  some  exhibit  one  superior  to  them  activity.  The  chemical  metabolites  are  structural  shown  THERAPEUTICS  INH  is  s t i l l  chemotherapy caused  well  been  and  combatting  1.1.2  case  products  derivatives  toxicological,  in  aldehydes  diisonicotinoylhydrazine,  Attempts  all  with  the  heavy  solutions  glucose,  I N H , as  (isonicotinuric  for  many  salt  easily  is  These  with  by  of  in  Fig.  formula  of  INH  and  its  major  1.  USES  considered  tuberculosis,  INH-sensitive  to  be  and  strains  the all of  primary  drug  patients  with  the  tubercle  for  the  disease bacillus  ZHOU,  o  II C-NH-NH  2  o  o  II  II  C-NH-NH-C-CH3  Isoniazid (INH)  Acetylisoniazid (AcINH)  H N-NH 2  o  2  Hydrazine (Hz)  II C H - C-NH-NH 3  2  Acetylhydrazine (AcHz)  o  o  II  II  CH3-C-NH-NH-C-CH3 Diacetylhydrazine (DiAcHz)  Isonicotinic Acid (INA)  Fig.  1. C h e m i c a l its  major  structures  of  metabolites.  INH a n d  T.  4  ZHOU,  should  receive  Sande,  1985).  having  activity  INH One  of  brain been  al.,  against  most  however its  for  ability  1979;  multiple  its  sclerosis  1985;  Hallett  al.,  1987),  et  al.,  and  pharmacological  fluid.  e.g.,  (Sabra  Based  on  this  values  in  Huntington  et  al.  ,  1982 ;  Francis  Parkinson's  et  acid  GABA  in  action,  INH h a s  several  central  (Perry  associated Duquette,  al.,  disease  not  effects.  of  disease  tremors  and  of  y-aminobutyric  concentrations  1982),  1985;  advantage  inhibit  5  (Mandell  other  raise  al.,  it  bacteria.  therapeutic  et  tolerate  other  to  and  disorders, Perry  can  therapeutic  have  cerebrospinal  system  they  the  is  tested  if  has  aminotransferase and  nerve  drug  INH  does  these  (GABA)  the  T.  with  et  1986;  al.,  Bozek  (Gershanik  et  et  et al.,  1988 ) . Recently, the  therapy  rifampicin  1.1.3  of  (McConkey  the  treatment  hypotheses.  of  et  inhibit  the  examined  arthritis  and S i t u r a y a k e ,  mechanism  tuberculosis  These and al.  been  when  for  its  effect  administered  in with  1988).  OF ACTIONS  exact  biosynthesis, Takayama  also  rheumatoid  MECHANISMS  While the  INH has  include  by is  biosynthesis  of  on  (Herman  suggested  INH i s  unknown,  effects  glycolysis  (1975)  which  a  lipids, and  primary  mycolic  there  effective are  action  acid,  several  nucleic Weber,  an  of  in  acid 1980).  INH  to  important  ZHOU,  constituent  of  concentrations  the  of  very-long-chain mycolic  acids  explain  the  activity  and  tubercle  Beggs,  of take  the  doses  the of  to  INH  in  the  the to  drug.  although  This  most  isonicotinic  a  quantity  microorganisms.  of  acid  Low of  the  molecule.  this  of  leads  the  wall.  elongation  of  selectivity  up t h e  result  treatment  INH  increases  are  Only  the  Since  action  would  antimicrobial loss of  uptake  of  acid  methanol-  INH-sensitive appears  drug  metabolite  marked  suggested  GABA  in  loss  putamen,  that  loss that  of die  utilize  Biochemical  analyses  content  GABA  patients,  to  of  and and  in  be  brain.  to  of  two  GABA  the  to  be  within  the  (Jenne  and  as  when  al.,  sequential  the  caudate  in  the  of  cerebral  an  inhibitory  shown caudate nigra  corresponding  to  a  marked  nucleus, of  decrease  This  of  GABA-  enzymes  that  there  is  a  nucleus  and  astrocytes,  and  cortex. a  Many  of  population  of  neurotransmitter. decrease putamen,  Huntington in  content  1974).  Huntington disease, in  large  GABA  inhibition  probably belong  have the  et  proliferation  neurons off  brain  Perry  due  neurons  substantia a  1973;  In  with  disease,  animals,  first  small  associated  neurons  to  the  the  of  considerable  pallidus,  Huntington  ( P e r r y and Hansen,  was  degrade  of  given  aminotransferase,  cells  prevent  mycobacteria,  of  the  cell  6  1973).  In  the  to  decrease  process,  is  may  precursor  Exposure  a  bacilli  bacilli  acid  degree  lipid  active  drug  unique  INH.  extractable  an  are  of  fastness  the  fatty  high  mycobacterial  T.  the  in  the  globus disease  activity  of  ZHOU,  the  enzyme  that  decarboxylase trial  (GAD).  (Perry  of  However,  et  cerebrospinal lack  synthesizes  al.,  fluid  clinical  GABA,  in  1982)  GABA  glutamic  7  acid  a  double-blind  clinical  INH  markedly  increased  concentrations,  improvement  T.  in  but  most  there  Huntington  was  a  disease  patients. In  recent  usefulness sclerosis. were  due,  subject  against  part,  patient  techniques  such  al.,  1986)  and  the  effectiveness  demonstrated standard  a  improvement.  trial  is  of to  of  improves  a  in  multiple  which  was  studies  used  (Francis  al.,  to  assessment  1987) light  of  et  evaluate goniometry  of  showed  that,  proportion  that  goniometry  reduction  group concluded  results  later  Polarized  three-fold  clinical  postural  et  its  multiple  evaluation, The  light  (Bozek  INH.  with  contradictory  bias.  for  tremor  when  only  marginal  although  INH o n l y  patients,  sclerosis  a  clinical  patients  with  tremor.  BIOTRANSFORMATION  Since  the  tuberculosis deal  tremograms  examined  associated  mechanism of  polarized  warranted  debilitating  1.1.4  as  been  gave  and o b s e r v e r  Bozek's  significantly  the  has  tremor  studies  to  two  methods  INH  action  The e a r l y in  to  years,  of  in  work  particularly  as  introduction the done it  early to  of  INH  1950's,  elucidate  relates  to  for  the  treatment  there  has  been  its  metabolic  INH-induced l i v e r  a  of  great  pathway,  injury.  The  ZHOU,  relevant  aspects  metabolic  fate  of  the  of  INH  published  identification  same  as  year  dogs  the  INA  This  was  (Cuthbertson  et  in  Fig.  INH m e t a b o l i t e s the  major  followed  drug,  the  as  in  of  when  first in  studies  (Kelly  which  the in  et  studies  INH i t s e l f  the  The  occurred  first  1953), well  2.  metabolite  by  al., as  scheme  8  al.,  in  man  INA  were  and  identified  urine. By  the  acetylation The  mid is  the  enzyme  by  this  pathway  allowing slow  the  are  under  amount  of  In of slow  route  of  to  these  mid  Detailed a  et  al.,  The  present  m u c h w o r k was  acetylator  rate  constants  processes  involved  metabolites  in  (DiAcHz), the  urine.  in  the  done  INH  to  be  in  the  and  gene  Studies  has  been  determines  the  liver.  on  the  metabolism  undertaken  enabled  conversion  excretion of  and  capacity  the  on  a  approximate  calculated  the  AcINH  rapid  It  acetylhydrazine for  man. liver  of  acetylation  studies  of  INA, i s o n i c o t i n y l g l y c i n e ,  diacetylhydrazine  in  in  distributed  into  1965).  involved  pharmacokinetic  rapid  production  individuals  that  is  bi-modally  differences  control.  1970's  The  be  of  (Peters  established  acetylation  1965).  found  been  INH i n a c t i v a t i o n  for  N-acetyltransferase  first-order  AcINH,  major  classification  genetic  and  metabolic  had  was  that  the  INH.  it  (Jenne,  acetylators  established  1960's  responsible  N-acetyltransferase  the  a  accepted  shown  of  as  isonicotinoylglycine in  are  i n t r o d u c t i o n of  identified  1952).  currently  T.  for of  INH  (AcHz), of  the to and  INH a n d  metabolism  of  ZHOU,  CONHNH*  >  i  C H CONHNH 3  INH-HYDRAZONES  C H CONHNHOH  2  3  I I I  AcHz  I  ACETYL TRANSFERASE CH CONHNHCOCH 3  V  CH3CON-NH I I I  K  3  DiAcHz  11  INH-Gly  CH C* 3  ACETYL FREE RADICAL  hypothetical  Fig.  2.  Metabolic  toxication  pathways  pathway  of  isoniazid  (INH).  T.  9  ZHOU,  INH  showed  times  that  more  the  rapidly  rapid  than  acetylators  the  slow  acetylated  acetylators-  demonstrated  that  acid-labile  hydrazones  (Mitchell  al.,  1975b),  that  et  directly route is  to  for  the  cleaved  be  INA  to  in  glycine, at  glycine  little  these  INH  was  the  in  saturated  by  an  analogous  differed  and  INA f r o m  by  their  acetylation after  250  mg  the of  the  were The  of  is  via and  it  was  formed  hydrolyzed metabolic  AcINH. AcHz  AcINH  may  acetylation to  conjugate  unrelated  to  conjugation  INH a p p e a r e d  to  administration  of  either  5-6  then  N-acetyltransferase  ability  differences  INH  also  major  INH i s  amidase,  to  acetylated.  vivo  as  in  INH  The  10  Later  are  some  (Hz).  polymorphically manner  and  which  of  INA and AcHz  a  Individuals  hydrazine  formation  acetylated  DiAcHz  and  and  T.  compound  of  INH.  INA  with  the  of  to  rates  INA  be  with  partially  doses  (Gordon  of  et  as al.,  1975) . AcHz  was  responsible be  for  the  eliminated  (Timbrell urine acid is  postulated  as  et  metabolism pathway which  al.,  by  that is  in  is  the  and  as  the  Nelson  the  et  to for  is  Third,  AcHz  by  at  was  is  acetylated the  AcHz  microsomal  the  the  most  three  acid to is  and  the  pyruvic which  eliminated  reactive  As  in  DiAcHz,  enzyme  shown  to  routes  excreted  hepatotoxicity  1976b).  likely  demonstrated  least  AcHz  produce  al.,  metabolite  a-oxoglutaric  hepatic  thought  the  First,  it  urine.  responsible  1976;  body  Second,  the  be  injury.  1977 ) .  AcHz  hydrazones.  excreted  hepatic  from al.,  free  to  system,  via a  intermediate (Mitchell  in  Fig.  et  2,  ZHOU,  N-acetyltransferase, oxidases  play  metabolic  1.1.5  in INH  pathways  is  peak  rapidly  plasma  diffuses  microsomal  roles  in  mixed  different  function  steps  in  the  INH.  of  and  is  about The  acetylators  is  acetylators  the  persons  in  of  in in  is  USA a r e and  about  3  of  slow 30%  the  hrs. The  There INH  the  of  hrs,  is and  plasma of  INH  55%  acetylators,  of  that  in  in in  of  and  level.  similar.  whereas  About  Native  system  plasma  are  one-half  hrs.  1/kg.  plasma.  levels  1.5  1-2  tissues.  nervous  half-life  than  and  results  urine.  INH i n to  within  the  one-fifth  one-third  parenterally.  mg/kg/d)  ^g/ml  central  active  half-life  (5  0.67+0.15  extracellular  less  Orientals,  acetylators.  about  average  the  is  excreted the  and  fluids  protein  in  is  concentration  acetylators.  Eskimos,  to  3-5  body  distribution  fluid  orally doses  of  a l l  primarily  intracellular  black  into  concentration  acetylators  both  therapeutic  binding  are  cerebrospinal  average  of  readily  appreciable  The  absorbed  concentrations  volume  metabolites  The  of  administration  apparent no  important  and  11  PHARMACOKINETICS  INH The  very  amidase  T.  The rapid slow rapid slow  white  and  whereas  10%  Americans  are  slow  ZHOU,  1.1.6  ADVERSE  1) .  are  2) .  occasionally  erythrematosus,  anemia and  and  (Robinson  Donohoe,  Block,  3) . 20%) These  and  Direct  are  on  have  pyridoxal  Dutt,  The most and  from  include muscle  paresthesia,  convulsions such  of  ideas  florid  psychoses  1985).  Most  of  administration  and  these of  toxic as and  reality,  pyridoxine.  effects  pyridoxine of  INH  and  can  and  Accidental  of  optic mental memory,  self-control,  (Mandell be  These  ataxia, even  impairment of  with  insomnia,  dizziness,  loss  (10-  systems.  neuritis,  episodes  complications  hemolysis.  nervous  encephalophthy  and  glucose-6-  (apotryptophanase).  retention,  euphoria,  psychotic  In  competition  peripheral  urinary  McCurdy  (Goodman  relative  twitching,  stupor,  abnormalities  enzyme  1984).  central  resulting an  1982;  common t o x i c  perhaps  and  thrombocytopenia,  aplasia  a  lupus  agranulocytosis  I N H may c a u s e  to  for  as  cell  attributed  restlessness,  separation  red  been  phosphate  and  vasculitis  Bottomley,  deficiency,  peripheral  reactions  atrophy,  and  Toxicity:  the  1969;  pure  Claiborne  such  eosinophilia,  Foadi,  dehydrogenase  deficiency,  toxic  1966),  1964;  phosphate  1973),  and  angiitis  INH-induced  syndromes,  disorders  Gupta,  dermatitis,  reactions:  rheumatic-like  hematologic  (Mehrotra  fever,  seen.  Idiosyncratic  various  12  EFFECTS  A l l e r g i c Reactions:  hepatitis  T.  and  prevented  INH o v e r d o s e  Sande, by can  the be  ZHOU,  treated the  with  INH  intravenous  adverse  the  is  one  effects  hepatotoxicity about  in  amounts  13  equivalent  to  ingested.  Hepatotoxicity studied  pyridoxine  T.  is  of  the  related  of  most  and  INH. The mechanism  topic  studies  serious  of  will  this  be  of  thesis;  discussed  extensively INH-induced  more  in  details  the  following  section.  1.2  HEPATOTOXICITY  With  O F I N H AND I T S  prolonged  chemoprophylaxis, hepatotoxicity; active  INH  the  hepatitis only  clinical  jaundice,  since  observed.  increasing those  between The  decision patients  Severe  risk  of  hepatotoxicity  for  the  prophylactic INH  be  INH  intoxication  in  is  use  of  cases  have  been  of  has  with  in  1.5%  influences  rise  reported  a  up in  et  to  of  al.,  number et  the  20%  of  aspartate  many  (Black  of  persons.  al.,  these 1975a).  necessitates as  tests,  increases  In  In  been  have  20,  INH.  (Mitchell  chronic  necrosis  older  observed.  drug,  in with  function  age  of  small  continued  the  liver  significantly  hepatotoxicity of  cause  necrosis  under  2.5%  with  (AST)  can  discontinuation  and  a  hepatotoxicity  and  INH  50,  INH as  multilobular  rarely  scale  associated  hepatic  and  INH  occurs  taking  of  Abnormal  30  aminotransferase patients  of  large  been  subacute  severe  It  a  has  1969.  Risk  age.  on  implication  and  accepted  been  use  METABOLITES  prompt of  fatal 1975).  ZHOU,  It  was  first  responsible  for  al.,  Later,  1974).  the  suggested  the  evidence  reactive  of  AcHz,  the  intermediate  1976;  Timbrell  et  have  tried  but  not  of  AcHz  Hz, a  another  cause  1983).  of  1.2.1  1986b;  I N H , was in  the  Mitchell  the  a et  rat  model  Timbrell J . A . ,  communication).  recently rabbit  mechanism  to  investigators  reproduce  al.,  that  implicated  (Noda  of  et  al.,  INH-induced  unclear.  INH  INH i s  (Dickinson  evidence  et  that  suggest  al.,  INH  that  well  metabolism  of  with  hepatotoxicity.  It labeled  was AcHz  is  metabolites  its  shown (  consistently  INH  are has  humans,  ^-AcHz), lower  toxic  1982),  responsible  thereby  in  the  of  is  Most for  it  could  absence  studies liver on  of  INH,  the  interfere  administered  elimination  in  little  the  effects  I N H was  rate  hepatitis  there  hepatotoxic.  when  the  than  cause  potential  metabolites;  in  to  Girling,  itself  However,  their  known  1981;  toxicity.  was  of  the  remains  of  et  metabolic  AcINH,  1976b;  J . G . , personal  necrosis  Therefore,  Although man  Joly  the of  A number to  metabolite  indicated  to  al.,  able  metabolite  hepatic  hepatotoxicity  et  (Wright et  communication;  due  14  (Snodgrass  model  metabolite  1980).  been  the  AcINH  rat  be  further  al.,  hepatotoxicity  personal  as  have  may  (Nelson  al.,  was  from a  injury  that  1974  liver, necrosis  hepatocellular  activation  in  T.  of and  with AcHz the  ZHOU,  urinary urine  excretion  decreased  obtained  AcHz AcHz  was  a  rat  with  excreted  DiAcHz  (Peretti  from  administered  of  in  -^C-AcHz the  urine  1979).  These  inhibit  humans.  The  inhibition  lead  to  an  increase  In  a  pathological  INH r e d u c e d  the  (PB)-pretreated  inhibition by  INH  inhibits 1968).  liver  This  important higher AcHz  the  toxicity All  when  both  P-450  implications  in  the  from of  it.  INH  animals  do  not  ( M i t c h e l l et  in  AcHz  1979;  vivo  the  may  several  al.,  1976).  INH-induced l i v e r  damage  may  administered be  due  by  liver  of  AcHz INH  et  of  al.,  would  part  doses  the  have  INH: be  why  necrosis  be  the  INH c o u l d  toxic in  to  1979).  (Kutt  toxicity  not  could  were  man  smaller  and  showed  activation  explain  produce  rats  results  may  less  its  phenobarbital  metabolism in  and  AcHz.  Timbrell, in  197 9 ;  in  This  mediated  to  when  pathway  the  agents  INH,  This  whereas  of  enzymes AcHz  of  than  INH  AcHz  co-  DiAcHz  Wright,  in was  was  lower  that  of  1981).  al.,  of  of  detoxification  Wright,  level  ratio  and  however,  result  INH  hepatotoxicity  microsomal  plasma  the  same  When  indicate  hepatotoxicity  and  The  15  DiAcHz/AcHz  significantly  study,  et  of  (Timbrell  of  doses  pretreated  rats,  inhibition  produced  single  to  this  cytochrome  (Timbrell  well.  acetylation  the  (Bahri  of  as  was  of  rats  simultaneously  ratio  1987).  studies  the  in  the  al.,  alone  metabolites  that  et  model  administered  Timbrell,  and  T.  the the  large in  caused  PBmild  consequence  ZHOU,  of  a metabolic  that in  toxic  INH i n d u c e d guinea  hepatitis  pigs.  metabolite,  It  an  experiment, lesions  1.2.2  reaction  a  acetylation  and  allergic INH  INH  its  and  that  the  INH p r o b a b l y  and  INA.  eosinophils  INA,  delayed-type  pig of  reported mechanism  and  cutaneous guinea  both  (1980)  In  present  the in  was same  hepatic  suggestion.  first  of  necrosis  et  1976),  al.,  These  However, the  can  be  nitrophenyl AcINH  to  necrosis.  It  the  of  INH,  is  demonstrated  that  AcINH  (Snodgrass  necrosis  suggested that  AcHz,  follows: than  (3)  the  AcINH  is  has  a  no  Mitchell by  hepatotoxin.  metabolite  of  AcINH  more  potent  in  necrosis  caused  by  pretreatment which  by  caused  potentiated  is  much (2)  formed  1974;  greatly  AcINH  itself; by  al.,  primary  AcHz  (BNPP), BNPP  et  was  that  (1)  prevented  phosphate  AcHz;  metabolite  was  rats  and  evidence  hepatotoxicity AcINH  in  results  toxin  major  INH.  hepatic  is  an  that  a in  al  16  AcINH  AcINH,  PB.  to  this  to  et  administration  eosinophilia  support  due  cause  chronic  Heisey  found  reaction  after  allergic  is  was  could  hypersensitivity hepatitis  mechanism.  T.  with  inhibits effect  on  bis-para-  hydrolysis  of  AcHz-induced  ZHOU,  1.2.3  damage  investigators  is  caused hydrolysis  (Mitchell  et  humans  metabolite have  of  microsomal agent  (Nelson  et  enzyme  rat  in vivo  have  rat  liver  protein  The  covalent  well.  not  to  lesions  occurrence result  covalent related  PB.  to  demonstrated  leads  to  to  did  in  causes  only  protein  a  infiltration  confined  the  centrilobular  liver,  the  of  the  components  is  tissue  lesions study  centrilobular  rats.  the  by  organs.  Histological  in  as  detectable  those  -  inflammatory  area  the  despite  dose-dependent  by  organs  fraction  of  PB-pretreated  and  to  increased  subcellular  1984).  acylating  covalently  in  in  a critical  the  in  liver,  development  by  Studies  produce  the  to  degeneration to  not  a  3)  other  was  is  (Fig.  1976).  of  in  microsomes  protein  binds  organs,  specific  AcHz  AcHz  INH  AcHz  reactive  al.,  of  AcHz  macromolecules  binding  Timbrell,  a  protein  than  and  necrosis  that  AcHz  subsequent  that  et  other  the  to  of  by  Studies  liver  microsomal  to  that  1976b).  oxidation  the  other  suggest  binding  (Woodward  hepatic  of  covalent  may  to  metabolite  rat  liver  formed  confirmed that with  Mitchell  binding  organs of  binds  and  with  in  Studies  AcHz,  al.,  have  demonstrated  those  pretreatment  1977)  system  1976b;  al.,  INH-related  major  et  metabolic  covalently  et  the  Nelson  man.  that  metabolite,  AcINH,  al.,  that  P-450  that  of  INH i n  indicated  the  1975b;  al.,  (Timbrell  postulated  by  metabolic  This  17  AcHz  Some  but  T.  Ballooning cells  same  were  animals  ZHOU, T .  O i  ->  CH.CNHNHCCH, Diacetylhydrazine  o CH,CNHNH  a  Acetylhydrazine  O CH.CNHN-R a  -Oxoacid  Hydrazones  0 CH.CNHNHOH NH NH t  t  Hydrazine -H,0  Postulated  O  Microsomal  ->  CH,CN=NH  Acetate  Pathway  CH,C* I  CH.C-OH-^CO,  O i or C H . C *  " I  Covalent Binding to Macromolecules  F i g . 3 . M e t a b o l i c pathways o f a c e t y l h y d r a z i n e  ^  Hepatic Necrosis  (AcHz).  18  ZHOU,  (Bahri  et  involves  al.,  1981).  acetylation  of  AcHz,  which undergoes  group  (Timbrell  1.2.4  1980),  is  a  and  Harland,  Blair  studies  a  in  by  the  decrease  detectable  pathway  cleavage  to  reactive  acetyl  carcinogenic  (Toth,  and  of  measurement  the  toxicity  of  animals  decreases hepatic  increase  and  PB  increases  glutathione  of was  or  the  to and  and in  also rocket  (Timbrell  on  Hz  and  numerous toxicity,  mechanism  of  Hz  unknown. cause in  a  liver  of  liver  dose-related and  to  The a c c u m u l a t i o n  of  weight  mitrochondria 30  Timbrell,  min 1982).  weight  triglycerides demonstrated  piperonyl  the by  out  is  of  Although  microscopy  accumulation  with  and  swelling  it  drugs  glutathione.  (Scales  hydrazine,  constituent  carried  largely  the  Hz  a  1980).  vivo  electron  administration  hepatic  al.,  hepatic  compound w h i c h  certain  triglycerides  by  of  1977)  demonstrated  in  droplets  1978),  been  s t i l l  been liver  et  in  are  al.,  of  have  Hz  has  increase  of  followed  yield  intermediate,  1979;  of  et  (Kimball,  metabolite  fate  Hz  of  (Back  chemical  hepatotoxicity  the  to  metabolic  1980).  a  biochemical  lipid  AcINH  oxidation  and mutagenic  fuel,  cause  INH t o  al.,  toxic  important  the  postulated  19  Hz  Hz  an  et  The  T.  that  butoxide  hepatotoxicity.  a d m i n i s t r a t i o n of  after Using  which to  were  a  mirrors  assess  the  pretreatment respectively  Prior  depletion  diethyl  maleate  ZHOU,  had  no  effect  on  studies  vitro  microsomal  of  the  takes  extent AcHz  lead  place  the  intermediate;  nitrogen  is  (Nelson  and  much the be  indeed  is  the  that  Noda  pathway INH.  et  as  The  in et  4).  the  of  is  rat  liver  Hz  of  on  hydrazone  of  molar  metabolized  via  be  due  to  et  al.,  the  1985).  the  other  parent  the very  seems  to  suggest  routes,  compound  the  rather  1982).  for  rifampicin  used  as  evidence  RMP p r e t r e a t m e n t ,  no  remarkable changes  were  of  Therefore  results  several  implicate  main r e s p o n s i b i l i t y  vivo  is  pyruvate  acetylation  They  in  route  azine  main metabolic  to  expired  basis.  Hz w i t h and  the  particularly  Pyruvate  These  not  via  mice  alternative  a  al.,  is  that in  a  metabolism  perhaps  Hz  to  et  DiAcHz  oxidative  hydrazones,  itself  comparison  effects  and  that  only  Perry  evidence  An  of  but  197 8;  of  rabbits  by  In  indicated  DiAcHz  nitrogen,  However,  (Timbrell  al.,  and  1980).  al p r o v e d t h a t  oxidation  1983;  1982).  al.,  have  hepatotoxic  reaction. is  Hz  major metabolite  may w e l l  a metabolite Noda  is  Hz  of  Timbrell,  formation  the  detoxication  hepatotoxicity than  a  than  of  although  and  there  (Fig.  toxic  formation a  et  metabolized  enzyme-mediated  Gordon,  azine  less  is  AcHz  production  diimide  metabolism  to  potentially  Microsomal  to  pyruvate  Hz  metabolism  (Wright  is  hepatotoxic. may  that  (Timbrell  20  enzymes.  acetylation  1981).  toxicity  indicated  Studies  limited  the  T.  (RMP)  (Noda et  pathway (Noda  this  of  et  Hz  al.,  metabolic  the  hepatotoxicity  of  on  INH m e t a b o l i s m  in  al., were  1983):  after  observed  in  the the  ZHOU, T.  NHJ-NHJ Hydrazine  O II CH,-C~NH-NH  CH  1  I  S  C H  C=N-N=C  Acetylhydrazine  l  COOH Pyruvate  o o II II CH,C-NH-NH-C-CH,  I  H N»NH-OH A  S  l  COOH  H N-N=0 2  HN=NH  Azine  or  Diacetylhydrazine  C o v a l e n t binding to liver protein  Liver  Fig.  4.  I  necrosis  M e t a b o l i c pathways o f h y d r a z i n e ( H z ) .  21  ZHOU,  plasma  levels  DiAcHz.  of  INH o r  Plasma  studies  levels  demonstrated  necrosis  in  is  a  key  through  1.3.1  2  the  These  I N D U C T I O N OR  shows  undergoes  direct  hydrolysis indicated  metabolism  of  and  metabolic  the  to  remarkable  with  RMP  observations  a  and  hepatic  than  in  suggest  that  hepatotoxicity  reactive  intermediate  by  than  of and  are the  metabolic  pathways  acetylation to  AcHz  several  microsomal  and  1978; Hz,  Timbrell as  et  mentioned  INA, with  or AcHz of  enzyme-mediated  to  acetate  of  AcINH  pathways  acetylation Hz  to  (see  al.,  DiAcHz, Fig.  1980).  above,  at  3) The  least  oxidation.  importance  i.e.,  also  to  and  hydrazones,  development  enzymes,  several  hydrolysis  there  Timbrell,  the  INHIBITION  INH h y d r a z o n e s ,  hydrolysis  relative  HEPATOTOXICITY  Hz a n d I N A . M e t a b o l i c s t u d i e s  that  acetylation  to  of  further  pathways  The crucial  more  AcHz  Histological  INH-induced  to  are  formation of  possibly  include  there  other  the  (Wright  of  rabbits.  formation  pathway:  reduced.  pretreated  OF INH-INDUCED  which  and  Hz c a u s e d  intermediate  ENZYME  Fig.  have  that  were  AcINH,  22  oxidation.  FEATURES  INH:  Hz  biotransformation  microsomal  1.3  metabolites,  of  rabbits  non-RMP-pretreated Hz  the  T.  of  of  these  pathways  the  toxicity.  acetyltransferase,  The  will  be  activities  amidase,  and  ZHOU,  microsomal factor the  enzymes  which  are  critical  can induce  metabolism  of  or  INH  in  inhibit  and  these  these  thereby  T.  23  pathways;  enzymes affect  any  may  alter  INH-induced  hepatotoxicity. It AcHz  has been  is  formed  shown in  vivo,  vivo,  coadministered  rats  (Wright  inhibits AcHz can  the  to  could  and  therefore  that  more  1978)  through  the  o f AcHz  pathway  INH of  Fig. 3  i t  From  of  AcHz  pathway  and  inhibition  would  in  binding  acetylation  Conversely,  in  vitro  covalent  toxic  which  Thus,  in  1979). of  enzyme-mediated  AcHz.  whereas  inhibition  toxicity.  from  the acetylation  and Wright,  AcHz  drug  with  enzyme-mediated  (Timbrell  increase  microsomal  may i n t e r a c t  Timbrell,  appreciated divert  INH, the parent  INH i n h i b i t s  microsomal  protein  be  that  of  the  reduce  the  toxicity. RMP INH that  is  i n the this  an agent treatment  However,  of  review  increasing is  the an  to  al.,  197 6 ;  but  of  It  is  et  inducer, et  al,  Noda  et  pigs  with  P-450  al., (van  mice and  1983; den  RMP  animals species  e.g.,  in  197 9)  In  the  1977).  against  large  1980),  than  al.,  mostly  with  suggested  hepatitis  INH h e p a t o t o x i c i t y .  and Netter,  guinea  has been  (Pessayre  cytochrome  1980;  coadministered  serious  literature  Heubel  not  more  Broek  induce  and Z e i t z ,  1985),  the  den  commonly  alone  unpredictable  demonstrated  (Thomas  causes  incidence  (van  Mazel,  is  tuberculosis.  INH  of  differences  and  of  combination  administration  RMP  which  i t  is  (Pessayre in  rabbits  Whitehouse Broek  et  et al.,  ZHOU,  1980).  Rats  reduction  and  (Pessayre et  al.,  INH  have  group,  the  the  oxidative  the  greater  hepatic  In but  humans  many  the  Girling,  and  by  AcINH,  of  It  main  strongly  induced  in  et  1984).  these  agents  increases  RMP o r the  control  increase  In  in  (Noda  the  et  studies  rabbits  of PB-  unaltered.  of  al.,  proved  pretreated  cytochrome  giving  (Remmer  H.  INH t o an  et  al.,  INA a n d  this  of  PB a n d  al.,  197 3 ;  Hz a n d A c I N H role is  in  in  inhibited  Amidase  toxicity  must  inducers  have  which be  to INH  is  also  RMP, r e s p e c t i v e l y  studies  INH  1985).  enzyme  INH.  P-4 50,  RMP w i t h  et  important  that  metabolite  Therefore,  in  that  Timbrell  shown  by  in  were  in  inducer  evidence  1982;  rats  Bahri  rabbits.  plays  been  1980;  observed  Hz  hepatitis  also has  by  potent no  Hz  pronounced  Hz was  caused  hydrolyzes  the  al.,  a  of  AcHz  P-450  administration  histological  control  showed  which  of  above  Girling,  AcINH  metabolism.  the  risk  1978;  Amidase INA  rate  al.,  those  of a  et  24  induction,  cytochrome  After  than  groups  RMP i s  studies  increases  lower  necrosis  in  Broek  1983).  concentrations  mentioned  RMP t h a n  den  e.g.,  of  concentrations  were  elimination  As  results,  change  van  al.,  pretreated  1985).  with  et  serum  groups  while  of  1976;  Piriou  rats,  different  significant  and M a z e l ,  pretreated  each  no  1981;  to  shown  T.  (Sendo  induction  with  interpreted  with  caution. Microsomal induce  enzyme  N-acetyltransferase  reported  to  cause  activity.  hypertrophy  of  not  been  Hydrocortisone the  hepatic  shown has  to been  tissue  ZHOU,  resulting al.,  in  a  greater  1987).  Large  sulfamethazine acetylator  1.3.2  rabbits  found  the  et  al.,  (1981), PB  rats  without  liver  PB  does  Timbrell  1984 ) .  AcHz  or  neither  in Hz this  significant  "slow"  "fast"  and  1981).  of  necrosis  only  not  be  the in  been  microsomes) but  appear  liver to  the  be  others  rat  (regardless or  laboratory release  in  to  of  the  any rat  rats  is  produce strain)  the  or from  Timbrell  treated  with  modification  of  by  treatment  of  or  AcHz  or  and  with  between covalent  (Woodward  the any with  animals.  previous of  and  necrosis  model  ever  (Thomas  relationship  PB p r e t r e a t e d nor  in  parallel  rat  less  arising  Bahri  AcINH  the  been  all  man  rat  Covalent  with  are  has  histological  the to  it  than  demonstrated  and  naive  in  observed  AcHz.  problem with  pig  shown  According  with has  model  guinea  have  demonstrated  laboratory  necrosis  both  hepatotoxicity  pretreatment,  binding  Another  et  induce  animal  and  studies  can  challenge  histologically  this  induced  INH i t s e l f .  necrosis  rat  suitable  mouse  No  macromolecules (or  (Reeves  hydrocortisone  and C o u r t e a u ,  a  indication  with  before  liver  INH  1981).  challenge  for  rat,  to  biochemical  in  activity  25  SPECIES  the  susceptible  of  acetylation  (Souich  search  that  doses  (SMZ)  ANIMAL  In  acetyltransferase  T.  and  inability  in  evidence  of  INH,  AcINH,  Furthermore,  study marker  has  reported  enzymes  of  ZHOU,  necrosis,  i.e.,  aspartate  aminotransferase  INH  (AST) and  with  the  rat  hepatotoxicity  led  to  investigation  to  laboratory. cause  INH  amounts  of  studying Thomas  AST  et  al.  sensitivity compared  higher  amidase to  lead  polymorphically  human  in  pig.  therefore  the  rabbit.  the  rat  and  mechanism  results  for  the  of  the  suggested  compared  AcINH  to  AcHz  Noda et Hz  rabbits.  to  that  the  rat  which  may  al.  (1983)  caused  They  By  rabbit,  hepatotoxicity  that  liver  showed  Hz  be the  human  rabbit,  al., liver  due  to  rabbit  hepatotoxicity  the  activity but  not  however,  N-acetyltransferase  et in  in  The  determined  to  did  in  the  rabbit  of  INH t r e a t m e n t  significant  hepatotoxicity.  inherited  (Whitehouse  of  the  our  was  rabbit.  N-acetyltranferase  1989),  the  rabbit  of  serum by  the  of  and  N-acetyltransferase  guinea  demonstrated  in  necrosis  INH i n  genetically  rat;  greater  Furthermore,  or  alanine  studies  (1978)  release  INH-induced  the  of  al. that  -^C-AcINH  histologically  centrilobular  mouse,  in the  conversion  to  et  investigated  activity  demonstrated  from  of  to  to  greater  therefore  formed  ALT into  (1984)  experimental  suggested  resulting  metabolism  rabbit  leads  studies  and  in  Whitehouse  damage  the  greater  by  These  liver  26  (ALT).  Dissatisfaction  response  study  aminotransferase  T.  in  1978), of a  slow gene  provides the  and  shows  activity the  acetylator deletion a  human.  in  better  is  the  rat,  similar to  the  absence  of  rabbits  was  (Blum model  et for  al., the  ZHOU,  1.3.3  ACETYLATOR  ACETYLATOR  that  after  there  differences metabolites. be  the  after  whether  either  and  subjects,  fraternal  acetylation  in  tissues  inherited  the  rabbit.  catalyze  the  coenzyme  A  a  mucosa  in  man  and  in  vitro  and  INH m e t a b o l i s m  acetylation autosomal  and  studies  rate  family  in  may  differences  blood  or  studies  under  genetic  differences  are  urine  and  has  of  been  of  rabbit of  the  the be  characteristics,  identical  control. drug  that  The  metabolism  shown  that  from  these the  (Weber genetic  rabbit  group  et  al.,  control  suggested  controlled  by  rapid  a  INH  and  that  N-  duodenal  1975). of  was  acetyl-  enzymes, liver  same  mammalian  acetyl  in  with  with  enzymes  of  present  patients  N-acetylating  the One  Broader  established  in  to  that  tuberculosis  clearly  variety  the  in  found  inactive  characteristics  have  It  is  these  to  of  drugs.  acetyltransferase,  drug  populations  transfer to  was  person-to-person  parent  showed  it  administration.  is  of  INH,  intravenous  twins  contain  the  measured  including  ability  phenomenon found  large  is  of  large  individual  drug or  of  of  studies  stable,  oral  investigations healthy  unusually  conversion  the  RABBIT  introduction  Subsequent  reproducible,  manifest  the  existed in  27  PHENOTYPE  PHENOTYPE IN HUMAN AND  Shortly  T.  In  vivo  sulfadiazine  that  SD  simple  acetylation  and  INH  Mendelian dominant  ZHOU,  to  slow  (Frymoyer  1963b).  The  and  different  correspond  to  homozygous  dominant  acetylators  the  are  dominant  RR o r  use  the  of  designate drugs  It  R r . These  was  et  al.,  risk  INH h e p a t o t o x i c i t y  produce  more AcINH  and thus  also  acetylated  by  N-acetyltransferase urinary  and  slow  and  DiAcHz  similar  EFFECT  in  slow  slow  acetylators  are  studies  established  the  "slow"  acetylators  to  INH  and  other  of  rapid  rapid  acetylators  presumably  because that  genetically the  acetylators  acetylators;  the  at  they  AcHz  is  determined  picture.  metabolites  are  of  In INH  excrete amount  studies in  rapid  more  AcINH  of  AcHz  was  phenotypes.  plasma  AcHz  and  e.g.,  The d i s c o v e r y  the  recovery  OF ACETYLATOR  When AcINH,  both  AcHz.  complicated  acetylators, than  activity  rapid  acetylate  reported that  greater  comparing  to  Jacox,  1965).  initially of  and  28  heterozygous  respectively,  and e a r l i e r  capacities  and  acetylase  recessive,  recessive;  "rapid,"  Frymoyer  of  conditions,  homozygous  varying  1963a;  amounts  homozygous  terms  (Peters  Jacox,  T.  and  PHENOTYPE ON INH METABOLISM  concentrations DiAcHz,  were  of  INH a n d  measured  its  in  metabolites, healthy  human  ZHOU,  subjects plasma  after  under  in  rapid  the  from  in  acetylators  faster  to  complex  of  slow  DiAcHz  a  the than  did  acetylation  (i.e.,  a  accompanied  by  acetylation  of  that  should  lead  acetylators  leads  to  by  rate  of  in  of an  INH,  the  Furthermore,  to  an  and  of  large  acetylation,  accumulation  clearance  to  of  Due  (10  which AcHz  DiAcHz  in  may  is  the  rate  of  of  most  has five  the of  also times  of  AcHz  be  INH  slowest  mg/kg)  slow  INH  AcHz  doses  half-life dose  of  of  about  in  this  plasma  rate  analysis is  to  the  exposure  AcHz  plasma a  AcHz  the  the  though  acetylated  repeated  of  the  AcHz  DiAcHz),  AcHz  thus  accumulation  which  saturation  of  AcHz,  more  formation  to  area  from INH,  thus  under  and  were  the  formed  also  pharmacokinetic  half-life  of  and  areas  clearance  This  plasma  to  decreasing  1985b).  faster  that  hrs.  contributory  faster  the  DiAcHz  acetylators.  AcHz  under  contrast,  AcINH  slow  29  o c c u r r e d even  acetylators  of  and  curve  INH m i n i m a l l y i n f l u e n c e s  the  than  20-plus  a  AcHz.  longer  In  This  more  the  areas  metabolite  between  curve  shown  toxic  rapid  concentration-time  to  the  AcINH  INH.  acetylators.  relationship  patients  of  of  generated  INH, because  INH,  concentration-time  precursor  greater  of  curve  acetylators  plasma  postulated  rapid  ingestion  concentration-time  greater  was  the  T.  of  is INH  especially acetylators  (Lauterburg  et  al.,  ZHOU,  EFFECT  OF ACETYLATOR  Studies classified to  in  as  fast  studies  group  are  they  In  the  urine,  When  studies INH i s  tested  as  INH t h e  this AcINH  is  rate  that  recent et  1982;  al.,  Mitchell's that  interest their  rapid  prospective  reverse.  is  slow  the  previous  is  is  a  of  not of  where  dose to  of  fact paper  levels  new  In  were  mg/kg, protein  plasma  for  with  INH-induced  itself  was  the  acetyl  moiety  After  and  positively  INH  formation is  200  correlated  observation.  derived.  ^C-AcINH  hepatic  decreased.  responsible  a vital  binding  AcINH  at  rate  DiAcHz  and  1987)  al.,  negatively  endogenously of  et  were  and  given,  it  to  binding  binding  hepatotoxicity,  contradictory  evidence  Of  rabbits  acetylation  rate If  to  covalent  levels  correlated.  these  (Thomas  orally  the  acetylation  susceptible  Musch  recent  refer  less  concluded  found the  risk.  genetically  produced  1981;  have  HEPATOTOXICITY  those  or  more  30  opposite.  found that  AcHz  but  more  to  more  have  al.,  greater  experiment  as  are  risk  1985a)  failed  an  increased  et  at  administered was  more  whether  (1975b)  al.  presented  demonstrating  of  at  a i . ,  have  acetylators  was  et  (Dickinson et  determine  hepatotoxicity  are  Lauterburg  that  to  acetylators  Mitchell  acetylators  it  man  INH-induced  results.  PHENOTYPE ON INH-INDUCED  T.  However,  in  administered. can  not  be  administration  d e t e r m i n e d by t h e  rate  ZHOU,  of  acetylation;  seen  in  this  Levels measured  be  of  the  serum  other in  than  to  counteract  hepatotoxic  clinical  fast  the  al.,  1985;  aware  that  factors  moiety  of  are  Sarma  AcHz  when  al.,  other  than  be  It  higher  et  may  metabolite,  studies.  acetylators  et  acetyl  tend  concentrations  acetylators (Blair  will  31  effects  study.  in  hydrazine  this  T.  in  It  covalent in  were  found  that  human  INH i s  1986).  involved  was  Hz,  slow  administered  is  important  binding  of  to the  hepatotoxicity  of  INH.  1.4  SERUM IN  ARGININOSUCCINATE  HEPATOTOXIC  and  cycle liver.  acid, in  thus  turn  cycle  which The  (Takahara The  acid  has  been  action  of  lyase shown  ASAL  liberating arginine  acted  reason  hepatotoxicity  A S A MARKER  ENZYME  STUDY  Argininosuccinic urea  LYASE  upon  by  serum  stemmed  suggested  that  release  sensitive  and s p e c i f i c  to  is  and  act  an  enzyme  the  in  mammalian k i d n e y  on  argininosuccinic  fumaric acid.  to  of  form  urea  Arginine in  the  is  urea  1967). ASAL  from of  is  exist to  arginase  and N a t e l s o n , that  (ASAL)  this  was  be  chosen  clinical enzyme  i n d i c a t o r of  monitor  literature  into  liver  to  plasma  damage  in  is  which a  more  man  than  ZHOU,  serum AST or  A L T . The  as  sensitive  indicators  et  al.,  1970;  release  of  ASAL  treatments and  log  AST  et  between  ASAL  1975).  of  and  al.,  THE OBJECTIVES  1.5.1  well  (Campanini  A comparison ALT to  correlation log  unpublished and  damage  AST and  between  established  four  drug  log  ASAL  between ASAL  and  data).  pathological  between  log  ALT  However,  the  necrosis  has  not  O F OUR S T U D I E S  HYPOTHESIS  (1)  INH-induced  metabolites  case, the  release  (r=0.816)  been  32  established.  1.5  its  have  hepatocellular  significant  (Wall  relationship  of  two  and Rautanen,  and  showed  (r=0.879)  been  Sims  latter  T.  the  effect  on  of  the  potency evaluate  aim of  acetylation  the with  of  INH  toxic  these  role on t h i s  its of  the  parent  INH.  (2)  metabolic or  caused  drug  studies  was  metabolites  by  itself. be  one In  greater  factor  pathway,  increased  INH  which such  of this  using has as  formation of  an the the  hepatotoxicity. to in  microsomal  toxicity.  be  should Any  the  the  may  toxicity  pathway  may a l t e r  INH a n d  the  than  toxic  metabolite The  of  than  the  induction toxic  rather  potency  metabolite  hepatotoxicity  test a  the  rabbit  enzymes  hepatotoxic model, and  rate  and of  ZHOU,  1.5.2  TO E S T A B L I S H RABBIT  The  was  following  reasons:  elevations  in  been  INH  Whitehouse  et  to  ASAL in  means  of  was  this  been  et  hepatic  Thomas  et in  the  rabbit,  has  other 1981;  al.,  3)  to  INH  and  et  1984).  exhibit  2)  necrosis,  al,  the  subjected  1980).  (Thomas  for  to  when  al.,  model  model  shown  A L T enzymes  produce  the  the  It  each  of  of  in  the  the  acetylation in  compared  estimate of  between  rabbit  and  the  liver  before  plasma  in  a  manner  necrosis.  ASAL  serum  ASAL  by in  doing some  for  ASAL  for after  In  rabbit,  histological  validity  and  the  model  Thus,  indicator  animal.  measuring  liver.  measurement  enzyme  measured  hepatotoxicity  biochemical to  was  relationship  necrosis  biochemical  experimental  validity  assessing a  as  study.  for  examination  marker  experimental  has  determined  chosen  this  establish hepatic  IN THE  humans.  challenge establish  it  1983;  genetically  similar  the  (Martines  in  al.,  as  AST and  to  changes  damage  1)  challenge  demonstrated  is  chosen  serum  metabolic  rate  A S A MARKER ENZYME  33  MODEL  rabbit  chronic  SERUM A S A L  T.  liver drug  order levels we  to as  need  levels  a to  and  histological rabbits  both  examination  were  being  used  as  a  ZHOU,  1.5.3  T O COMPARE T H E H E P A T O T O X I C I T Y  T.  34  O F I N H AND I T S  METABOLITES  The  relative  metabolites mechanism By  is  of  an  INH-induced  of  INH  and  was  tested.  of  subcutaneously  affected  1.5.4  test  by  relationship serum  ASAL  metabolites  whether  the  i.e.  was  ASAL  its the  same AcINH,  level,  delivery  after  oral  the  molar doses AcHz  administered  direct  circulation  of  and  orally INH t o  of Hz, or the  administration  BETWEEN A C E T Y L A T I O N R A T E AND I N H -  results  rate  1981;  al.,  Thomas between level was  in  the  et  authors et  al.,  1987).  acetylation the  rabbit  activity  relationship  hepatotoxicity  (Mitchell  Musch  undertaken  N-acetyltransferase  about  and INH-induced  different  et  1985a;  and  HEPATOTOXICITY  acetylation  al.,  INH  understanding of  serum  between  INH  THE LINK  Contradictory  Dickinson  of  toxicity.  TO C L A R I F Y  reported  the  of  metabolites,  portal  INDUCED  the  in  elevation  addition, to  the the  the  its  In  toxicity  hepatotoxicity.  hepatotoxicity  some  by  of  important step  comparing  difference  liver  potency  to and  1982;  al.,  rate  et  al.,  analysis  and  the  clarify  of  INH o r  link  hepatotoxicity.  been  1975b;  elevation  with  any  has  Lauterburg  An  challenged  between  et the of its  between  ZHOU,  1.5.5  TO C L A R I F Y THE R E L A T I O N S H I P MICROSOMAL O X I D A S E  PB this  is  a well  experiment,  pretreatment enzymes  2  2.1  on  the  microsomal  administered effect  hepatotoxicity  of  of  HEPATOTOXICITY  oxidase with  inducer.  In  without  PB  and  induction  of  microsomal  INH.  M A T E R I A L S AND METHODS  SUBJECT  Male from  test  35  BETWEEN T H E A C T I V I T Y O F  AND I N H - I N D U C E D  liver  I N H was  to  the  known  T.  the  New  Zealand  Animal  white  Care  rabbits  (2-3  of  University  Unit  the  kg)  were  obtained  of  British  Columbia.  2.2  MATERIALS:  Isonicotinic were  obtained  Hydrazinium Aldrich  Bell  Co. (SMZ)  phenotype Co.  synthesized  Acetyl in  this  hydrazide  British  sulphate  Sulfamethazine  and  from  Chemical  acetylator  acid  was was  Drug  and House  purchased the  from  for  obtained  isonicotinic laboratory  Chemicals  supplier  required was  pentobarbitone  acid  Ltd.  Scientific.  acetylhydrazine.  determination  from  from  Canada  Fisher of  sodium  of  Mattheison, hydrazide  materials  SMZ  Coleman  was  semi-  supplied  by  ZHOU,  British method with  Drug of  a  House  von  Chemicals  Sassen  four-fold for  synthesized  product  (1:4);  1.5  high  analysis  of  AcHz,  Hz was  at  or  the  assay  a  by  by  the  assay  ASAL  chemical  2.3  stirring,  and  liquid that  level  used).  Reagents  of  barium  converted  to  with  in  Campanini, were  supply  heptane and  all  of  the  reagent  a  INH,  the  ASAL  were  from  solution  of  the  sulfate  (Sigma  and  Chemical Co.) to  reagents  grade  (HPLC)  argininosuccinate  according other  the  0.2-0.4%  for  sodium  2,4-dichloro-l-naphthol  room  by  (ca.  (1970)  al.  the  reacted  chromatography contamination  et  36  methanol-diethyl  detection  solution was  in  I N H was  of  admixture  recrystallised  outlined  continuous  Campanini  Co.)  centrifugation; was  the  to  sources:  salt  at  sensitivity  Chemical  sodium  anhydride  showed  below  according  (Sigma  acid  recrystallized  AcINH  to  acetic  with  was  according  wherein  of  hrs  Ltd,  (1985),  performance  the  detector  following  al.  excess  temperature  ether  et  Canada  T.  procedures required  obtained  from  for  local  houses.  METHODS  2.3.1  ANIMAL  Rabbits access  to  phenotype  TREATMENT  were  Purina of  housed  in  laboratory  each  test  stainless chow  and  animal  steel water.  was  cages The  with  free  acetylation  determined  before  ZHOU,  hepatotoxic  challenge  administration Rabbits rabbits  by  HPLC  analysis  were  each.  assigned  They  into  were  orally  12  groups  treatment  of  (p.o.));  (1)  INH  (2)  (s.c.));  mmol/kg/dx2d,  p.o.)  PB p r e t r e a t m e n t  following  intraperitoneally  mmol/kg/dx2d,  (6)  s.c.) i.p.);  AcINH  (0.42+3x0.30  (10)  Hz  (0.14+3x0.10  chosen  given  drug  treatment  as  control  saline  (8)  AcHz  and  animals  (7)  AcINH  (0.36+3x0.26  (0.10+3x0.07  p.o.)  (0.1  mmol/kg/dx2d,  s . c ) ;  mmol/kg/dx2d,  subcutaneously  i n other  pretreatment  mmol/kg/dx2d,  Hz  mmol/kg  (0.36+3x0.26  (0.28+3x0.20  p.o.);  (9)  mmol/kg/dx2d,  were  2.3.2  AcINH  (0.42+3x0.30  s . c ) ;  (0.19+3x0.14 randomly  (5)  (0.36+3x0.26 (0.1  INH  PB  6-8  (0.36+3x0.26  INH  (4)  following  mmol/kg/dx2d,  mmol/kg/dx2d, p.o.);  (i.p.));  (3)  of  (0.36+3x0.26  INH  subcutaneously,  s . c ) ;  following  groups  mmol/kg/dx2d,  mmol/kgx3d,  37  o f SMZ.  mmol/kg/dx2d,  x3d,  T.  mmol/kg/dx2d, p.o.),  Hz  two  rabbits,  for histological  studies,  in  (12)  (11)  the  same  regimen  as  the  groups.  PHENOTYPING  SAMPLING Rabbit for  was  placed  cannulization  around was  was  the  then  lateral  pricked  heparinized  to  into take  ear vein with  a  and  then  a restraining the was  needle.  blood shaved A  22  implanted  frame  and prepared  samples off,  and the  gauge into  -  jelco the  the ear  hair vein  catheter vein  and  ZHOU,  secured. all  the  In  preparation  Eppendorf  heparinized. 10  mg/kg  the  SMZ f r o m  of  catheter  25,  40,  minutes into  blank  stock  the  50,  60,  70,  post-injection.  marked  centrifugation (AcSMZ)  for  1 ml  sample  was  taken,  was  of  90,  120,  blood  plasma  analysis  of  time  ml  of  blood the  15,  20,  150,  180  135,  was  then  ejected  separated  acetyl  of  from 10,  was  of  through  the  5,  were  dose  syringes  105,  and  a  0.5  approximately  80,  syringes  0,  approximately  tubes  the  time  38  sample,  administered  from  The c o l l e c t e d  eppendorf  blood  and  heparinized  times  of  labeled  Starting  the  at  collection  solution  drug,  into  inserted  the  were  vein.  withdrawn  30,  a a  ear  administration were  tubes  After  opposite  for  T.  by  sulfamethazine  and SMZ.  EXTRACTION SMZ through  the  1. second  and  then  the  pi on  a  plasma  tube  of  tubes  and  from  the  plasma  samples  withdrawn  with  allowed  100  pi  to  sit  water  was  and of  placed  into  acetonitrile,  for  at  added  least to  the  a to  2 min. tube,  mixer.  tubes  rpm)  Approximately  eppendorf  along  vortex  (5000  was  distilled  Eppendorf  centrifuged  extracted  steps:  protein,  200  3.  4.  fil o f  Eppendorf  mixed  were  following  100  denature 2.  AcSMZ  for 200  containing two  mixture  was  min.  jul s u p e r n a t e  and t r a n s f e r r e d  the  to  a  was  glass  removed vial.  from  the  ZHOU,  A  specified  was  injected  SMZ  a n d AcSMZ  ANALYSIS  the  HPLC  was  NaAc,  15%  ml/minute; as  was  a  set  and  the  the  HPLC t o  the  supernate  determine  in  the  the  glass  vial  concentration  of  plasma.  at  were  analyzed  analyses  packed with MeOH, the  a  8%  were  MeCN  scale speed  by  2.5  from  a  set  follows: was  at  a  The  peak  mg/ml  a  the  rate  nm a n d and  under HPLC  0 . 0 1 M pH  flow  254  reading;  the  0.01  as  at  mm/min.  of  The c o n d i t i o n s  solvent  solution  deflection of  HPLC.  the  was  comparison  obtained  by  r u n were  ODS-2;  absorbance  full  determined those  in  samples  column  0.08  into  of  39  OF SAMPLES  The which  volume  T.  10  the  of  standard  paper  obtained  solution  1  mV w i t h  concentrations  heights  5.0  feed were with  of  SMZ  the  K  AcSMZ.  CALCULATION From (first each  plotting  order  was  also  2.3.3  concentration  elimination  rabbit  calculated  the  from  rate  respectively. t-^2  calculated  =  i  for  n  2  /  the  constant) The  K e  *  20  against  T  h  e  min  was  time,  calculated  half-life r  a  t  i °  o  f  ("^1/2)  e  for w  a  s  AcSMZ/SMZ+AcSMZ  sample.  ADMINISTRATION  INH  and  subcutaneously;  its  metabolites  P B was  injected  were  given  orally  intraperitoneally.  and  ZHOU,  Oral  administration  appropriate solvent  in  solution the  restraining the  was  followed  by  between  the  location  the  of  two  1  ml  animals  sucrose oral  5%  protected  by  as  the  administration.  The  placed  of  to  on  was  into  used  the  dextrose  challenge. causing  injection  expose  This  were  three dose  Each  et  of  a i . , the  morning  mouth to  of  ensure  three  total  6,  and  the  oral  not  food.  A l l  days  been  gently  manual for  and  to  pinched at  this  positioning  injection.  Two  PB  which  was  given  at  0.10  with  saline  of  dose  subsequent  9 hours  treatment  of  saline  rabbit.  required  prior  normal  Injection  the  initiation  shown  cytochrome  loading  by  fold  underbelly  PB h a s  in  to  P - 4 50  be  of  the  INH  effective  in  the  in  rabbit  1985).  two  followed 3,  of  a  pretreated  days  of  induction  (Whitehouse  its  drug  blades.  discomfort  physiological the  of  under  shoulder  not  to  in  mmol/kg/d  a  syringe  injection  rabbits  dissolved  with  10%  administration  appeared  rabbit  groups  a  by  Intraperitoneal of  on  40  ingestion.  performed  skin  frames.  via  Subcutaneous was  done  preparation for  given  rabbit  complete  drug  was  T.  of  were  one  INH o r third  injections  later.  groups  rabbits  the  were  During  the  allowed  carefully  of  metabolites of  the  two two  water  observed  daily  ninths days ad  of  libitum  for  any  began total of  the  dosing but signs  ZHOU,  of  distress  the  during  event  of  solution  the  15  signs  was  of  following  the  2.3.4  MEASUREMENT  the  dehydration  administered  resuscitate  ASAL  hrs  T.  morning dose.  5%  sterile  subcutaneously  or  41  In  dextrose orally  to  taken  by  animal.  SAMPLING Blood  samples  heparinized  syringe  occasionally, could the  not  vein  be  dilate  with  of the  a  (stored  at  -20°C).  and  during  ASAL  the  central from  very  the  The  the  ear  vein.  In  was  vein  blood  some  extreme  thus,  xylene  the  was  The  assayed blood  following  samples cases topical  removed  plasma  within  samples  and  necessary  immediately  sampling.  period  ear  when  and  xylene  and  were  lateral  artery  rabbit,  day  ml)  of  blood  For each 5  1  amount  centrifugation  the  -  vasoactive  small  following  by  drug  from  vessels.  separated  each  mainly  particularly  alcohol  before  0.7  obtained  was  application to  from  (about  was  24  were  hrs taken  initiation  of  challenge.  ANALYSIS Serum  procedure  ASAL  levels  outlined  by  were  Campanini  determined et  al  by  (1970):  following  the  ZHOU,  Mix  Substrate. of  4 ml  of  (a)  with  80  ml  of  (b)  T.  and  42  40  ml  (c).  0.2  (a)  Sodium a r g i n i n o s u c c i n a t e  (b)  Phosphate  (c)  Ethylenediamine  M , pH  buffer,  mixed (a)  10%  mmol/1.  M , pH  7.0.  tetraacetic  acid  (EDTA)  solution,  7.0  Dichloronaphthol are  0.2  20  on  the  day  Thymine  NaOH  and  Equal  Solution. of  the  of  (a)  and  (b)  dissolved  in  test.  solution.  diluted  volumes  2  to  g  of  100  thymine  ml  is  with  10%  NaOH.  Keep  refrigerated. (b) naphthol with  Dichloronaphthol. is  dissolved  95% e t h a n o l . NaOCl  (5.25%  to  Arginine  Keep  in  0.3  serum. the ml  95%  ethanol  Dilute  100  purified  2,4-dichloro-l-  and  diluted  to  of  commercial  100  ml  refrigerated.  ml w i t h (1  standard 0.1  mg o f  N HC1 a n d  12  ml  Clorox  water. mmol/1).  dilute  Dissolve  to  100  ml  17.42  with  mg  0.1  of  N HC1.  refrigerated. Label  add  Keep  solution.  NaOCl)  arginine  in  60  ml  tubes,  tubes  serum  to  the  and  let  and  unknown To  incubate  trichloroacetic mix,  as  substrate.  Mix and  same of  two  the  for  unknown  1 hr  at  tube  acid  solution  stand  for  tube  37°C,  control  (20 min.  add  then  and mix.  5  To  control.  each 0.1  add  g/100  ml)  ml  0.1  Promptly  tube  ml  add to  Centrifuge  of of 0.2 both both  ZHOU,  tubes a  for  10  second  the  test  ml  of  and  0.1  min  and r e a d  add  0.75  ml  color  is  immersed  of  solution. nm o n  units serum  as  ice  after  stand  tube  to  samples  ice  the  the  time.  sera  on  ml  To the  solution a  (20 0.5  for  15  difference tube  is  units.  To the  and mix.  to  solution,  control  0.05  to  addition,  The  ASAL  Add  bath  prepared  buffer,  acid  bath.  each  in  the  was  each  water  for  and  obtain  standard  trichloroacetic developed  an  transferee!  dichloronaphthol  Let  unknown  of  is  an LKB c o l o r i m e t e r .  phosphate  0.2 0 ml  in  mixing  ml  standard  of  supernatent  tube,  0.25  the  the  the  43  with  ASAL  standard  tube  of  arginine  tube g/100  ml  then  add  ml).  The  aliquot  from  tube. In  this  Takahara  of  to  micromoles  when  A  two  exceeding  Takahara  serum  (Takahara  serum/hr.  assigned value  thesis,  unit  equivalent ml  is  515  of  and  of  which each  of  ml  standard  the  at  20  measurement  of  NaOCl  with  The  ml  NaOH,  of  absorbance  compared  0.5  of  10%  ml  0.5  tube  contents  0.1  in  min.  T.  units).  ASAL and  of  Natelson,  significant  upper  was  calculated  1967),  arginino-succinate  successive  the  activity  hepatotoxic  serum  99th  log  ASAL  is  digested/100 response  levels  normal  which  in  rose  percentile  was to  a (12  ZHOU,  2.4  from  groups  ( s . c ) ,  of  AcINH  sacrificed day  examinations  PB/INH  respectively.  obtained All  a  the  blow  for  of  a  tissue  who  (H-E)  were  is  staining  Bouin's  ethanol. lobe)  fixative  taken  were  for  fixed  the  were  to  p.o.),  animals  head  third  INH  at or  Their  liver  examined  and  were  the  fifth  fourth wedges  day were  the  treatment  from  1 mm t h i c k the  liver  preparations. solution  in  for  5 mm t h i c k  oil  reported groups  by  a  or  the  from  each  values.  three  Another three  were  they  slide  at  dose,  study, the  rabbits  INH ( p . o . ) ,  (high  of  weakness.  blind  rabbit,  removed  Hz  back  slides  FIXATION  lobe)  ( s . c ) ,  on  preparations.  2.4.1  each  the  and  serum ASAL  For  conducted  histological  corresponding  TISSUE  PB/INH  rabbits  histological  pathologist  were  and  to  few  distress  for  (p.o.),  ( s . c ) , For  by  except  because  and  44  H I S T O L O G I C A L STUDY  Histological  in  T.  red  10%  "o"  slices for  Hematoxylin-Eosin  These  slices  1 hr than slices staining  formalin  (one  were  maintained  (also slide  solution.  one  placed in  from  70% each  preparations  ZHOU,  2.4.2  H - E STAINED  The  slices  Department stained  of  changes  as  degenerations,  2.4.3  Fatty  by  red  oil  plus  slide  examined  for  were  were  sent  preparation.  the  cell  general  to  the  The  H-E  pathological  infiltration,  various  necrosis.  STAINED  SLIDES  also  accumulation  "o"  ethanol  for  and h e p a t i c  slides  above.  in  inflammatory  O I L RED "0"  These  2.5  maintained  were  such  45  SLIDES  Pathology  slides  T.  in  made the  by  same  hepatocytes  department can  be  as  stained  staining.  S T A T I S T I C A L METHOD  The  values  and  minus  hepatotoxicity analysis  has  group  using  1984),  p<0.05  of  serum  standard among been  level  are  error  (SE).  In  different  made  two-factor was  ASAL  on  the  as  order  treatment  analysis  accepted  represented  serum of  to  groups, ASAL  significant  compare  level.  mean the  statistical  levels  variance  as  of  each  (Montgomery,  ZHOU,  3  46  RESULTS  3.1  EFFECTS  O F I N H AND I T S M E T A B O L I T E S  SERUM A S A L  LEVEL  Serum  levels  ASAL  administration rabbit  serum  Takahara a  T.  to  there  was  the  within  least  upper  a the  was  usual  of  These  INH a n d  log  30-96  of  to  at  2.58,  and  values  I,  were  drug  into  Table  values  the 1)  if  greater  A  less  severe  least  two  values  population for  4.24  ASAL  percentile.  frame  all  was  various  (criterion  normal  hr time  the  i.e.,  from  value of  before  metabolites.  increased  used, the  values  (SD)  its  release  normal  of  rabbits  control  to  the  values  also  doubling  deviation  0.28.  as  two  a l l  mean c o n t r o l  significant  99th  (II)  the  response  determined  at  criterion showing  of  in  Summarizing the  standard  effects  considered  were  than  a  hepatotoxic  treatments, serum,  drug.  (SE)  the  measured  (n=87),  with  error  assess  The  any  samples  units,  standard  used  of  were  ON E L E V A T I O N O F  response  mean  value  (see  Table  1).  3.1.1  TIME  COURSE  AFTER  high  dose  doses  OF SERUM A S A L  LEVEL  INH A D M I N I S T R A T I O N  Previous single  OF THE E L E V A T I O N  of led  experiments INH d i d to  death  not of  in  this  cause the  laboratory a  rise  rabbits  in due  showed  that  serum ASAL, to  a  when  neurotoxicity  ZHOU, T. (Wall et al, u n p u b l i s h e d d a t a ) . In a m u l t i p l e dose  47  regimen,  INH, a d m i n i s t r a t e d e i t h e r o r a l l y o r s u b c u t a n e o u s l y , caused a remarkable control these  ASAL  were  quickly  i n serum  levels  groups.  levels  dose,  rise  ASAL  ranged  After  f o r the f i r s t  by  a  decline.  24  a n i m a l s . The  Takahara  administrated,  t o a peak v a l u e s a t about  followed  i n most  between 3-10  INH was  stable  level  units i n  t h e serum ASAL  h r s , and  then  rose  72 h r s a f t e r  the  first  Fig. 5  r e p r e s e n t s t h e time  c o u r s e o f t h e e l e v a t i o n of serum ASAL l e v e l from two r a b b i t s t r e a t e d w i t h INH.  T a b l e 1. Serum ASAL response t o drug c h a l l e n g e s  DRUG  ADMINISTRATION ROUTE  TOTAL* REGIMEN DOSE (mmol/kg) (mmol/kg)  NUMBER FRACTIONAL OF RESPONSE (%) 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 INH  i . p. s.c.  0.1 x3 d 0.30 0.36 + 3 x 0.26 2.28  100.0  100.0  PB • INH  i . p. p.o.  0.1 x :3 d 0.36 + 3 x 0.26  0.30 2.28  8  87.5  100.0  AcINH AcINH AcINH  s.c. s.c. p.o.  0.28 + 3 x 0.20 0.42 + 3 x 0.30 0.42 + 3 x 0.30  1.76 2.64 2.64  8 8 8  25.0 50.0 50.0  75.0 75.0 75.0  AcHz  s.c.  0.36 + 3 x 0.26  2.28  6  0.0  16.6  Hz Hz Hz  p.o. p.o. p.o.  0.10 + 3 x 0.07 0.14 + 3 x 0.10 0.19 + 3 x 0.14  0.59 0.89 1.19  6 6 8  0.0 33.3 100.0  16.6 83.3 100.0  6  * t o t a l dose e x p r e s s e d over 2 days ** I : two measurements o f ASAL >12 Takahara u n i t s ( c f . p.43) *** I I : two measurements o f ASAL >8 Takahara u n i t s  ZHOU, T.  Fig. 9  5. Time c o u r s e of serum ASAL r e l e a s e followrng c h a l l e n g e w i t h INH (0.73 i m o l / k g / d x 2 s.c.). Rabbit A, slow a c e t y l a t o r ; r a b b i t B, fast acetylator. d  48  ZHOU,  3.1.2  T H E E F F E C T O F PB ON I N H - I N D U C E D  In  the  group  mmol/kg/dx3d, the  elevation  INH  was  i.p.), of  with  of  the  oral  and  two  routes  to  of  Fig.  shows  in  oral  group  done  rabbits that  the  was  terms  of  the  The in  in  that  by  the  the  (0.1  inducer, of  group  not  PB e n h a n c e d  the  the  as  AcINH  subcutaneous  different  same  dose  via  between  the  rabbits  and  there  levels  levels that  and peak was  also  routes  and  in time  no  between  the  caused  routes. b y INH  subcutaneous (both  groups  administered no  non-  was  administration  same  PB) .  oral  ASAL  ASAL  values  of  the  showed  of  peak  the  ROUTES  comparison  two  the  found.  in  The  serum  elevation  almost  given  results  with  was  that  PB p r e t r e a t e d  pretreated  difference  PB  administration  influence  route.  were  and  with  enzyme  ADMINISTRATION  both  treated  group  in  in  difference  groups  than  I N H was  subcutaneous  rabbits.  significant  after  suggesting  test  routes,  was  pretreated  6),  OF D I F F E R E N T  order  administration  pretreated  levels  greater  (Fig.  HEPATOTOXICITY  known m i c r o s o m a l  ASAL  49  INH.  INFLUENCE  In  rabbits  a well  serum  PB  hepatotoxicity  7  the  significantly  pretreated  3.1.3  of  T.  via  significant  ZHOU, T.  Fig.  6 . Serum ASAL 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 (0.36+3X0.26 mmol/kg/dx2d) i n r a b b i t s w i t h (n=8) o r without (n=8) PB pretreatment (0.1 mmol/kg/dx3d, i.p.). A s i g n i f i c a n t d i f f e r e n c e i s found between t h e two groups (p<0.05, F t e s t ) .  50  ZHOU, T.  Fig.  7. Serum ASAL levels i n rabbits obtained following oral (n=8) and subcutaneous (n=6) a d m i n i s t r a t i o n of INH (0.36+3x0.26 mmol/kg/dx2d) w i t h PB pretreatment (0.1 mmol/kg/dx3d, i.p.). In the subcutaneous group the l a s t two p o i n t s are not c a l c u l a t e d because two r a b b i t s died b e f o r e these samples. There i s no s i g n i f i c a n t d i f f e r e n c e between t h e two groups (p>0.05, F test).  51  ZHOU,  3.1.4  E F F E C T OF H z , A c I N H ASAL  Among the  most  Fig.  8  metabolites  potent  effect  illustrates  serum ASAL  levels.  was  no  mmol/kg) while effect. dose  change  The  of  the  in a  elevation  Hz  was  the  Hz e q u a l  ASAL  O F SERUM  dose  Hz  serum  of  that  the  of  dose  that  serum ASAL  (0.62  in  greater  of  ASAL  by  than  (high  Hz  levels,  the  dose)  (1.24  had  that  on  there  dose  mmol/kg)  caused  Hz  the  had  level.  mmol/kg),  serum  (0.93  ASAL  of  agent  Doubling  rise  dose  of  dose  dramatic of  the  relationship  levels.  significantly  half  to  elevation  lowest  of  molar  approximately  I N H , Hz was  dose-response  intermediate  although  of  At  of  on t h e  the  manifested  an  ON E L E V A T I O N  52  LEVEL  the  almost  AND A c H z  T.  less  highest by  INH,  was  of  INH ( F i g .  9).  INH c a u s e d  immediate  mortality  only  A molar  dose  in  the  rabbits.  some was  Meanwhile,  both  elevation  of  compared  to  hepatotoxicity (Fig.  high  serum the  dose ASAL  parent  between  these  and  low  levels drug, two  dose (Fig.  INH,  agents  of  AcINH  10).  the was  caused  When  AcINH  difference not  in  significant  11). AcHz  in  a  mmol/kg/dx2d, levels  s.c.)  during  administered  the  (Fig.  dose did time 12).  equimolar not of  to  significantly 90  hrs  INH change  after  the  (0.36+3x0.26 serum  ASAL  agent  was  ZHOU, T.  _  O Hz low dose  Time (post 1st administration, h)  Fig.  8. Serum ASAL levels i n rabbits orally 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 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 t h e h i g h dose group t h e l a s t two p o i n t s a r e not c a l c u l a t e d because t h r e e r a b b i t s d i e d b e f o r e these samples. S i g n i f i c a n t d i f f e r e n c e s a r e found among t h e s e t h r e e groups (p<0.05, F t e s t ) and between h i g h dose and middle dose groups and middle dose and low dose groups, respectively (p<0.05, Duncan s m u l t i p l e range t e s t ) . 1  53  ZHOU, T  Fig.  9. Serum ASAL levels i n rabbits orally administered with INH (0.36+3x0.26 mmol/kg/dx2d, n=8) and Hz (0.19+3x0.14 mmol/kg/dx2d, n=8) . In t h e Hz group t h e l a s t two p o i n t s are not c a l c u l a t e d because t h r e e r a b b i t s d i e d b e f o r e these samples. S i g n i f i c a n t d i f f e r e n c e i s found between t h e two groups (p<0.05, F t e s t ) .  ZHOU,  0  20  40  60  Time (post 1st a d m i n i s t r a t i o n , h)  Fig.  1 0 . Serum ASAL levels subcutaneously challenged with AcINH (low dose 0.28+3x0.20 n=8; high dose 0.42+3x0.30 n=8) . Significant difference between t h e two g r o u p s (p<0.05,  in rabbits two d o s e s o f mmol/kg/dx2d, mmol/kg/dx2d, is found F test).  T.  55  80  ZHOU, T.  Time  Fig.  (post  1st a d m i n i s t r a t i o n , h)  11. Serum ASAL levels i n rabbits orally 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 i f f e r e n c e between the two groups (p>0.05, F test).  56  ZHOU, T.  Time  Fig.  (Post  1st administration,  h)  12. Serum ASAL 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 (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 significant difference is found between the two groups (p<0.05, F t e s t ) .  57  ZHOU,  3.1.5  THE RELATIONSHIP LEVEL  The  be  of  It  Blum  confirmed  as  1963a; et  acetylation  is  classified  Jacox,  slow  1989)  AcSMZ/SMZ+AcSMZ  ratio  of  10  experimental determine shows  group were  is  a  rate  in  this  slow  N  authors  that  1963b;  are  of  population  of  the  plasma  intravenous  the  in  see  to  Fig.  13.  of  the  i.e.,  one  rabbits,  Table  another  group  2).  SMZ-acetylator phenotype  SMZ h a l f -• l i f e (min) mean+SE range  our  indices  rabbits.  and  1976;  al.,  distribution  acetylators (  and  measured as  bi-modal  (Frymoyer  after  the  ANIMALS  can  and  used  ASAL  rabbits  Weber et  were  58  genetically  acetylators  SMZ  rate  rapid  of  is  minutes  acetylators  The parameters  Acetylator group  of  They  rabbits  half-life  20  striking  phenotyped  phenotyped  T a b l e 2. rabbits.  at  acetylation  there  were  rapid  SMZ  mg/kg  animals.  the  acetylation  or  b y many  O F SERUM  OF I N D I V I D U A L  in  Frymoyer and Jacox,  a i . ,  injection  THE ELEVATION  AND T H E A C E T Y L A T O R R A T E  rate  controlled.  BETWEEN  T.  in  AcSMZ/SMZ+AcSMZ (%)* mean+SE range  Rapid  54  12.7+0.6  5.9-28.0  84.3+1.3  50-100  Slow  11  50.3+3.1  39.0-77.2  28.5+0.7  25-34  was  calculated  The  rate  at  20  minutes  after  SMZ i n j e c t i o n .  ZHOU, T. 59  Fig.  13. P l o t o f t h e plasma AcSMZ/SMZ+AcSMZ r a t i o a t 2 0 minutes 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 of SMZ (10 mg/kg) a g a i n s t SMZ h a l f - l i f e . Each p o i n t represents t h e two d e t e r m i n a t i o n s i n each a n i m a l . Note t h e r e 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 a c e t y l a t i o n rate i n t h i s p o p u l a t i o n of r a b b i t s . The group i n t h e upper l e f t were phenotyped r a p i d a c e t y l a t o r s and t h e group i n t h e lower r i g h t were phenotyped slow acetylators ( r e f e r t o Table 2 ) .  ZHOU,  Many affects  authors  the  challenged  of  or  results  the  by  showed  (correlation correlation  3.2  assessed  peak  (represented  with  or  by  was  the  ASAL  found  in  is  group PB  i-  peak  (r) the  The  and  between =  rabbits  in  these  0.05).  rabbits  the  A  in  degree  levels  was  relationship  acetylation  summarized  s  rate  pretreatment,  phenotype.  60  however  of  serum ASAL  values  correlation  coefficient  evidence  a  without  acetylator  administration route,  SMZ ^1/2) no  the  The  in  acetylator  serum  that  INH.  study,  subcutaneous  with  the  of  this  INH  hepatotoxicity  correlated  and  In  with  oral  between  suggested  hepatotoxicity  contradictory.  either  have  T.  Fig. two  rate  14.  The  parameters  similar  challenged  lack  with  of  AcINH  Hz.  EFFECTS  O F I N H 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  Histological the  rabbits  without All  PB  studies  challenged  were with  carried AcINH,  out Hz  on and  the  liver  of  INH  with  or  pretreatment. of  degeneration  these in  the  three hepatic  chemicals tissue  of  caused most  vacuolar  experimental  ZHOU, T.  61  5000 1000 c D O  o  r=0.05  100  o  <  10  CO  < 0  20  40 SMZ  Fig.  T1/2  60 (min)  14. The r e l a t i o n s h i p of SMZ h a l f - l i f e t o the peak serum ASAL v a l u e i n r a b b i t s c h a l l e n g e d w i t h 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 p o i n t represents the two determinations i n each animal.  80  ZHOU,  animals the  and  caused  animals.  samples  some  15),  Hematoxylin-Eosin  the from  the  small  rabbits showed and  of  the  rabbit  focal  whose  Oil  red  control  fatty all  3.3  the  INH o r  of  the  "0"  rabbit  tissue  In  (n=45).  The  the  degree  no  hepatic  and  vein  slices  (Fig.  19),  (Fig.  an  the  was  20) made  from  involved  made  to with  degrees  some level  central  arrangement  Compared  were  showed  high  disrupted  were  various  made  in made  ASAL  extremely  necrosis  hepatocytes.  of  the  (Fig.  18).  show  fatty  the  of  slices  hepatocyte  demonstrated  in  the  challenged  from  in  BETWEEN E L E V A T I O N  rabbits  almost  O F SERUM A S A L  LEVEL  NECROSIS  histological six  out  relationship of  lobule,  slices  study,  conducted  to  slices  serum  slices  revealed regions  Some  of  the  metabolites.  HEPATIC  this  The  The  central  the  its  17). rose  stained  16).  of  tissue on  slices  vacuolated  (Fig.  62  fraction  hepatic  tissue  elevation  necrosis.  THE RELATIONSHIP AND  was  ASAL  metamorphosis  of  with  serum  in  mild  a  examination  large  lobule  (Fig.  around the  accumulation from  a  and  in  control  liver  cells  with  zones  parenchyma  the  liver  sub-massive  necrosis  histological  necrosis  middle  with  stained  parenchymal  center  of  Compared  (Fig.  swollen  degree  T.  liver  necrosis  of  was  the  between  damage  was  examination eleven  peak  observed  in  6  liver  treatment  serum  explored.  of  ASAL  Among rabbits  slices groups  levels 45  and  animals, challenged  Fig.  15. L i v e r t i s s u e f r o m a c o n t r o l r a b b i t treated with saline (1 mlx4/dx2d, s . c ) . (a) H-E s t a i n , x l O . (b) H-E s t a i n , x l O O .  ZHOU,  T.  (b)  Fig.  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 value of 466 Takahara units after a d m i n i s t r a t i o n o f Hz (0.19+3x0.10mmol/kg/dx2d, 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 cells. Around the c e n t r a l veins there i s a broad area of disintegrated vacuolated liver cells. O u t s i d e t h e zone numerous s w o l l e n l i v e r cells can be seen (H-E s t a i n x40). (b) Vacuolated liver cells (H-E s t a i n , x400).  64  ZHOU, T.  (b) Fig.  17. L i v e r t i s s u e f r o m t h e r a b b i t w i t h a p e a k ASAL value of 73.6 Takahara units after administration of Hz (0.19+3x0.10/kg/dx2d, p.o.). (a) F o c a l n e c r o s i s w i t h inflammatory cells (H-E s t a i n , xlOO). (b) N e c r o t i c liver c e l l s i n the f o c a l necrosis area (H-E stain, x400).  65  ZHOU, T.  (b) Fig.  18. L i v e r t i s s u e from the r a b b i t with a peak serum ASAL v a l u e of 2674 Takahara u n i t s 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 n e c r o s i s of l i v e r c e l l s . N e c r o s i s i n v o l v e s c o n t r a l and middle zones. Vacuolated liver cells are around n e c r o t i c zones (H-E s t a i n , x40). (b) N e c r o s i s of l i v e r c e l l s (H-E s t a i n , xlOO).  66  Z H O U ,  Fig.  19. L i v e r tissue with oil red "0" staining from the control rabbit. No visible fatty metamorphosis i s seen i n the s l i c e (x40).  T .  67  ZHOU, T  20. L i v e r t i s s u e s with o i l red "0" s t a i n i n g from d i f f e r e n t r a b b i t s showing v a r i o u s degrees of f a t t y metamorphosis. (a) 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 30.6 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 INH ( 0 . 36+3x0.26/kg/dx2d, p.o.). Dense, large droplets of fatty metamorphosis were found around c e n t r a l v e i n s (x40).  ZHOU, T.  Fig.  20. (b) L i v e r t i s s u e f r o m t h e r a b b i t w i t h a peak serum ASAL v a l u e o f 466 T a k a h a r a u n i t s after administration 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 o f f a t t y metamorphosis i n v o l v e s a l l zones o f t h e l o b u l e (x40).  69  ZHOU, T.  (d) Fig.  20. ( c ) L i v e r t i s s u e f r o m t h e r a b b i t w i t h a peak serum ASAL v a l u e o f 611.5 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 INH (0.36+3x0.26 mmol/kg/dx2d, p . o . ) . F a t t y metamorphosis o f moderate degree i n v o l v e s c e n t r a l a n d m i d d l e zones ( x 4 0 ) . (d) Same liver slice as (c) droplets of various s i z e i n l i v e r  showing fatty cells (x400).  70  ZHOU,  with  INH o r  levels.  In  release  ASAL, necrosis.  values  into  of  shown  Fig.  measure  that  of  degree.  parallel peak  ASAL  in  the  or  of  between  values  ASAL  the  absence  in  could  the  of  various  peak  the  level  and  the  animal of  can  be  not  of  be  is  serum  necrosis used  rabbit  as  in  the  a  some  hepatic  relationship,  degree  serum  and  hepatic  the  ASAL  rank,  quantitate  this the  have  elevation  values  necrosis  clarify  to  in  71  significant  experimental  of  to  rise a  divided  serum  attempted  further  relationship  serum  peak  serum ASAL  incidence  had  different  comparison  peak  we  we  using  presence  the  the  to  The  which  demonstrated  necrosis  the  However,  necrosis  were  the  significant  rabbits  groups  21.  and  39  no  Furthermore,  hepatic  levels  indicates  in  between  presence in  exhibited  27  of  relationship  ASAL  who  contrast,  of  degrees ASAL  AcINH  T.  but  necrosis  and  established  a the (see  discussion).  4  DISCUSSION  In in  order  humans,  to  it  which  resembles  effect  seen  has  been  in  is  investigate necessary as  closely  humans.  very  Among  difficult  the to  mechanism establish  as  possible  the  tested  or  of an the  animal  impossible  INH  toxicity  animal  model  hepatotoxic species, to  it  obtain  ZHOU, T.  100  r  80  CD  o  .52 *co o o  60  40  CD 20  - 1 2 n = 6  0 S e r u m  Fig.  ASAL  (Takahara  unit)  2 1 . The r e l a t i o n s h i p between the i n c i d e n c e o f n e c r o s i s i n s l i c e s and peak serum ASAL v a l u e s i n t h e r a b b i t s c h a l l e n g e d by INH, AcINH and Hz o r a l l y o r subcutaneously, w i t h o r w i t h o u t PB pretreatment ( r e f e r t o t e x t ) . N e c r o s i s r a t e i s the f r a c t i o n o f animals sampled i n t h e ASAL ranges quoted which shows histologically demonstrable h e p a t i c n e c r o s i s .  72  ZHOU,  reproducible guinea 1984; that and  pig  hepatotoxicity (Thomas  Wright  et  al. ,  administration ALT into  reported  that  INH  this  for  al.,  of in  INH  in  rabbits.  treatment  the  Noda  et  et  caused  value  hepatotoxicity  rat,  al  al.  ( 1978)  showed  release  o f AST  ( 1983) fatty  in  rabbits.  the  both  and  Timbrell,  both  of  73  mouse  and  significant  histologically  confirms  the  Woodward  Whitehouse  INH c a u s e d  necrosis  thesis  INH  1981;  1986b).  serum  centrilobular in  et  with  T.  further  liver  rabbit  and  The  as  a  data model  biochemically  and  histologically. It  was  serum  found  ASAL  was  with  also  data,  indicator necrosis to  level  was  ASAL  control that  was  by  each  taken  light  with be  no  as  rabbit for  up  and  be  4.3+2.6  the  of an  the  evidence following: liver,  histological  a  very  animals  1).  The a  units.  It  significant  on  fatty these  sensitive which  had  in  serum  ASAL  the  serum  ASAL  hepatic  only  units  Based  where  of  2000  including  elevation  rabbits  first  (SD)  necrosis.  of  the  caused  considered  all  elevation  to  microscopy,  displayed In  of  of  an  after  treatment  damage;  extent.  could  for  values  could  histology  caused  hrs  values  INH  level  elevated  sampling:  24  degradation  liver  some  explanation  peak  changes  of on  level  tissue  the  vacuolative serum  about  with  found  histological liver,  INH t r e a t m e n t  levels  administration, compared  that  necrosis,  the  limitation  of  small  examination,  fraction  of  i.e.,  the  ZHOU,  liver been  damage present  release  in  after  before  was  serum  from  mechanisms infiltration  INH  and  and  hepatic  was  found  in  H-E  animals.  cases, 3).  at  in  al.  can  slides  O i l red  "o"  present  8  hrs were  because  of  of  evidence  might  be  released  damage  and  repair  the  stage  of  fatty  have  this  from  by W h i t e h o u s e demonstrated  fatty study,  slides  varying  et  that  infiltration  almost  stained to  general  lack  Reports  In  enzyme  animals  level  the  induce  rabbits.  in  have  of  (1985)  also  The  examination  ASAL  result  may  74  vacuolation a l l  of  the  proved  that  degree  in  all  animals.  hepatotoxicity  among  the  of  presence  al.,  et  2).  change:  ASAL  steatosis.  was  explanation  of  a  stained  degeneration  kinetics  of  e.g.,  derivatives  experimental  these  cells,  steatosis  sampled.  experimental  rise  as  and McKennis  experimental  The  In  cells  necrosis  histological Some  and h e p a t i c  its  not  understandable.  in  and  morphological  the  and weakness.  (1982)  were  on  after  necrosis  fatty  that  evident  soon  al.  homogeneous  occurrence.  sacrificed distress  be  areas  only  its  into  not  occurred  necrosis  for  may  T.  the  for the  the  different  animal  large  species  interspecies  metabolic  susceptibility  enzymes  is,  probably,  differences  involved  to  in  (Whitehouse  INH that the et  1983). We  effect  have  demonstrated  and t h a t  that  PB i n d u c t i o n d o e s  INH  caused  enhance  the  a  hepatotoxic  hepatotoxicity  ZHOU,  of  INH i n  where  rabbits.  various  increasing  1976;  Barbare  et  the  microsomal mediating may  be  INH  Wright  et  al.,  of  other  in  by  to  many e n z y m e s  induce other  INH INH  was  This  a  reactive  al.,  using  may  as  is  in  of  of  the  the  PB  PB  to  the  is  known  toxicity  amidase  in  rat,  leading  enhancing  induction  with  important  However,  be  1984;  Although  role  enzymes,  could  as  model.  be  it  al.,  and  consistent  the  not as  P-450  et  rat  that  as  (Pessayre  also  the  humans  by  activity  1984).  administered  was  by  suggests orally  hepatotoxicity to  is  in  75  implicated  Wright  intermediate.  such  hepatotoxicity  administered  of  and i t  mechanisms  result  going  result  rabbit  induction  data  been  1982;  INH-induced hepatotoxicity  of  et  have  suggested  the  with  hepatotoxicity  authors  (1987)  oxidase  acting  (Sendo  This  1986).  formation  some  agents  of  al.  et  consistent  risk  al.,  proposals  Thomas  is  inducing  the  Mazel,  This  T.  is  the  not  liver  not  two  routes,  that is  significantly  the  via  the  as  and  complete a  result  portal  of  when  subcutaneous.  bioavailability  nearly  enhanced  oral  different  of  INH  and  that  INH  the  total  dose  circulation  after  oral  acetylator  rate  administration. Although affects  the  it  at  widely  hepatotoxicity  controversial: are  was  greater  some risk  reports  suggested of  INH  suggest  (Mitchell  et  that in  that al.,  man, rapid 1975b)  the  data  is  acetylators and  others  ZHOU,  suggest  that  et  1981;  al.,  The  slow  acetylators  Musch  results  et  from  proposition.  We s a w  SMZ  index  (as  an  rabbits of  challenged  acetylation  induced would  be  not  The  is  influence  of  2)  (see  also  the  enzyme  catalysed of  to  toxic  detoxification  elimination  of  administered, more  expected also  be  increased.  in  the  that  rate  of  INH-  mechanisms  mainly  which  due  to  the  metabolite  hepatotoxin,  proposed is  formed  AcHz  by  so  that  Mitchell  from  cause  of the  (the  pathway  to  elimination AcINH  to  AcHz.  production when  generate  or  AcHz AcHz  DiAcHz  via was  is  Therefore, and  I N H was  more  hepatotoxicity of  of  precursor  theory,  to  series  essential  both  tended  al  stepwise  of  this  a  et  the  and a c e t y l a t i n g  increased  When  of  ASAL  acetylated  which  to  acetylator  the  t-^/2  limited.  theory  According  an  between  mechanism  is  participates  AcHz;  although  a  is  metabolic  AcHz. fast  as  species),  N-acetyltransferase  INH  species  AcINH  the  and the  reactions  INH t o  the  of  rate  hepatotoxic  neither  suggesting  hypothetical  considered  the  1985a).  follows.  below),  following  (1976),  molecule  as  acetylator  In  metabolism  are  to  al.,  serum  76  (Dickinson  support  and 14),  critical Two  et  relationship  (Fig.  hepatotoxicity Hz  AcHz,  thus,  INH  risk  Lauterburg  acetylation)  with  is  greater  experiment  obvious  of  consistent  metabolite,  a  no  at  1982;  this  hepatotoxicity.  1)  Hz,  al.,  are  T.  AcINH,  could  DiAcHz  be  could  challenged,  ZHOU,  however, not  acetylation  production of  generate should the  more  be  AcHz,  AcHz  is  rate  of  metabolites,  of  AcHz  of  the  AcINH,  AcHz  more  was  these  Mitchell  relate  to  from  liver  further  metabolism  intermediate. model  a  In  the  progressive is  either  1976;  et  al.,  to  reproduce  the  data  and  Fig.  Table  the  from  rat  our  12  do  1 does  not  1980).  not  support  provide  and  model  is  INH  and  its  that  AcHz  had  Hz  was  the  most  doses; and  and  was  the  previous  to  hepatotoxic  studies?  that  a  The  into  this  evidence  in  of  the  the  question. and  rat  series et  other  1  the  acylating  (Nelson  Table  the  in  using  al., groups  Likewise Fig.  11  data  of  hepatotoxicity  of  hypothesis. that  AcHz  along  inability  shown  of  data  inferred  it  INH-induced  reactive  toxicity  or  calls  observation  production rabbit  production  in  shown  model  INH  published  increase  INH-AcINH-AcHz Timbrell  the  early  but  of  suggested  results  its  metabolite  INH.  hepatotoxicity and  to  phenotypes.  tested,  (1975)  al  the  tended  hepatotoxicity  demonstrated  than  s i m i l a r to  results  et  in  equimolar  chemicals  only  the  both  Hz,  hepatotoxic  four  AcINH  both  in  in  but  hepatotoxic  result  and  elimination  the  hepatotoxicity  effect  the  do  to  exposure  significantly among  end  the  thereby,  the  the  to  77  acetylator  determines  AcHz;  hepatotoxic  How  fast  decreased.  Comparison  potency  i.e.,  AcHz;  equivalent  toxic  related  from  acetylator  no  only  DiAcHz  inactivation an  was  T.  The  ZHOU,  AcINH  is  higher  similar AcINH  to  our  and  basis.  major  as  AcINH INH  found  INH  only  dose,  to  administration did  result  not  in  activity  of  conversion  molar dose  deny  previous  and p r e s e n t  not  support  Meanwhile,  and  AcHz  this  our data  are  al.  binding  to  protein  Our  increased the  hepatic  studies  by t h e  acetylator  than  show  lower,  hepatotoxic  be  on  that  (1987)  a  acceptable  in  to  of  induction  leads leads  in  more of  INH-induced PB, but  This INH-  to to  greater from is  our  data  higher  AcHz  another the  faster  rabbit  but  rabbit  dependent microsomal  not  our more can  level.  suggestion covalent on  the  oxidase.  hepatotoxicity is  for  amidase  results  that  with  as  in  higher  rat  The  The  ASAL.  AcHz  was  authors.  turn  a  that  of  that  the  was  regimen  release role  AcHz  study.  same  indicate  inducing agent,  rate.  the  inconsistent  by Thomas e t  rate  to  due  proposed  acetylation  in  that  mmol/kg/day  hepatotoxicity, is  was  a  this  hepatotoxicity. do  result  appears  on  to  in  which  studies  78  hepatotoxicity.  proposed  INH-induced that  not  major  compared  to  binding  to  the  it  p r o p o s e d by p r e v i o u s  rabbit  AcINH  not  significant  al.(1984)  covalent  sensitive  seems  same  to  of  if  hepatotoxic  tend  the  same,  This  which  equitoxic  be  a  rabbits.  in  INH-induced  to  et  in  has  AcINH  hepatotoxicity  Thomas  INH i n  studies  lead  would  induced  of  approximately  intermediate  not  that  previous  INH a r e  Since  potency  than  T.  related  is with  ZHOU,  Noda  et  al.  (1983)  is  the  key  intermediate  AcHz, in  a  rabbit  (Thomas  et  ASAL,  in  given  half  found  in  the  a l l  a  al.,  1981). half  of  In  more  the it  results was  take and  Hz t o  AcHz.  the  (Thomas  al., in  that  and t h a t  AcHz  induced  In  summary,  of  et  Hz was AcHz  1978;  high  dose  is  al. the and  et  are  to  Perry  et  of  a  Hz  was  it  is  result  of  the  view  that  accounts  for  1983).  al.,  consistent  (1986a).  In  with  that  most  toxic  of  Hz  tested  in  PB p r e t r e a t m e n t  was  occurs  therefore,  Noda  group  although  this  support  thesis  dose  of  necrosis  INH w h i c h  1981;  this  INH, AcINH,  and  result  metabolite  presented  place,  AcHz;  or  rabbit  release  above,  hepatotoxicity  This  et  hepatic  the  AcINH  INH i n  high  Timbrell,  of  hepatocytes,  hepatotoxic  mentioned  dose  p r o v i d e d by W r i g h t  a  As  molar  Hz-induced  the  INH,  may  (Wright  In  79  hepatotoxicity  of  of  the  i.e.,  is  8).  even  rat  rabbit  AcHz  not  a dose-related  experiment,  demonstrated  chemicals  to  this  likely  data  to  but  INH-induced  (Fig.  dose  Hz,  metabolite  lead  animals.  extent  hepatotoxicity The  molar  Hz  that of  did  that  of  a proven  study  eight  of  conversion is  the  limited  impossible  Hz  present  of  only  Hz,  1981),  al.,  acetylation  only  model.  suggested  T.  study  the  four  isolated  potentiates  the  Hz  hepatotoxicity.  the  results  reproducible effect  of  in  this  animal INH.  thesis model  This  show to  model  that  study  the the  resembles  ZHOU, T. hepatotoxicity doses  of  i n t h e human b e i n g :  INH  manifestation  to  produce  is a  release  1)  I t requires  toxicity. of  liver  2)  is  s i m i l a r t o that occurred potentiated  found  that  by  the  inducing  rate  of  agents.  that  the  hepatotoxin toxic  of necrosis  I n t h e r a b b i t model  acetylation  e f f e c t o f INH. U s i n g INH  ASAL,  i n t h e humans a n d 4) The e f f e c t  hepatotoxic among  earliest  e.g.,  i n t o s e r u m . 3) The 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 are  multiple  The  enzymes,  d i d not  affect  t h e r a b b i t m o d e l we  metabolites,  Hz  was  the  most  effect  at  a l l . These  i s probably  data  indicate  n o t due t o m i c r o s o m a l  o f A c H z , b u t i s more l i k e l y  the found  potent  due t o Hz. We  that  rabbit  model  e l u c i d a t i n g t h e mechanism.  will  likely  INH  metabolism  conclude that the  m e c h a n i s m o f INH h e p a t o t o x i c i t y n e e d s t o be r e e v a l u a t e d , the  we  a n d t h a t AcHz was t h e l e a s t p o t e n t i f i t h a d any  hepatotoxicity  that  80  prove  valuable  and in  ZHOU,  T.  81  BIBLIOGRAPHY  Albano E. and Tomasi A. (1987). Spin trapping of free radical intermediates produced during the metabolism of isoniazid and iproniazid in isolated hepatocytes. 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