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Genetic control of the teratogenic response to acetazolamide in mice Biddle, Frederick Gordon 1973

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GENETIC CONTROL OF THE TERATOGENIC RESPONSE TO ACETAZOLAMIDE IN MICE  by  FREDERICK GORDON BIDDLE B.Sc,  University of Windsor, 1966  M.Sc, University of Windsor, 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the F i e l d of Genetics  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA February, 1973  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the  requirements  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y  s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and  by h i s r e p r e s e n t a t i v e s .  be g r a n t e d by  the Head of my  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not written  permission.  Department o f  Medical Genetics  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  February  10,  Columbia  1973-  thesis  Department  I t i s u n d e r s t o o d t h a t c o p y i n g or  that  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s f o r s c h o l a r l y purposes may  for  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT Acetazolamide i s a potent c a r b o n i c and  a known t e r a t o g e n .  postaxial ectrodactyly  anhydrase i n h i b i t o r  In mice, i t Induces a s p e c i f i c of the r i g h t f o r e l i m b ;  the  l e f t forelimb  are  produced by t h i s drug except f e t a l death w i t h h i g h The  i s also affected.  occasionally  No o t h e r malformations doses.  SWV s t r a i n of mice was found t o be r e s i s t a n t t o any  teratogenic  or embryotoxic a c t i o n of a c e t a z o l a m i d e .  Very  high doses r e s u l t e d i n death of the SWV dam. The  teratogenic  a c t i o n and dose-response r e l a t i o n s h i p  of acetazolamide were documented f o r the s e n s i t i v e CBA/J strain.  Mid-day 10 was found t o be the embryo s e n s i t i v e  stage f o r the i n d u c t i o n of f o r e l i m b W i t h an a p p r o p r i a t e  ectrodactyly.  a c e t a z o l a m i d e dosage regime of two  i n j e c t i o n s on day 10 of pregnancy, a g e n e t i c made of the t e r a t o g e n i c resistance the  a n a l y s i s was  s e n s i t i v i t y of the CBA/J s t r a i n and  of the SWV s t r a i n .  Reciprocal  crosses  between  two demonstrated t h a t r e s i s t a n c e was dominant t o  sensitivity.  No a f f e c t e d SWV.CBA  hybrids  were found i n  SWV dams b u t a low frequency of e c t r o d a c t y l y was found among CBA.SWV F to s m a l l  1  hybrids  i n CBA/J dams.  T h i s d i s p a r i t y may be due  sample s i z e of the t r e a t e d SWV.CBA F^ h y b r i d s and  was n o t considered  t o be a d i f f e r e n t i a l  influence  A backcross program was s e t up u s i n g  of the dams.  the CBA/J dam  throughout.  Both a b i o m e t r l c a l a n a l y s i s and an examination  of the mean f r e q u e n c i e s suggested t h a t three controlled  of e c t r o d a c t y l y i n each  independently segregating  p r e d i c t e d BC^ g e n e r a t i o n  inbred  loci  the t r a i t of embryonic s e n s i t i v i t y t o the  t e r a t o g e n i c a c t i o n of a c e t a z o l a m l d e .  achieved  generation  From t h i s model, the  mean frequency of e c t r o d a c t y l y was  i n a f u r t h e r b r e e d i n g program.  Also,  the CBA/J  s t r a i n frequency of e c t r o d a c t y l y was recovered  with  two BG2 s i r e s and t h i s suggested t h a t the CBA/J genotype had been  recovered. I t was found t h a t i n d u c t i o n of e c t r o d a c t y l y and f e t a l  death were two separate a c t i o n s The  of a c e t a z o l a m l d e .  embryo t r a n s f e r technique was used i n a p r e l i m i n a r y  i n v e s t i g a t i o n of the CBA/J and SWV acetazolamlde.  s t r a i n responses t o  I t was demonstrated that the t e r a t o g e n i c  environment does e x i s t i n the SWV dam a f t e r a c e t a z o l a m i d e a d m i n i s t r a t i o n and t h a t r e s i s t a n c e of t h i s s t r a i n i s a property The  of the embryo. s e n s i t i v i t y and r e s i s t a n c e of the CBA/J and SWV  s t r a i n s remained w i t h dlchlorphenamide, another inducing  c a r b o n i c anhydrase I n h i b i t o r .  SWV may have a g e n e r a l The other  ectrodactyly  I t i s suggested  r e s i s t a n c e t o t h i s .class of  r e s u l t s of t h i s study a r e d i s c u s s e d  that  teratogens.  i n the l i g h t of  i n v e s t i g a t i o n s of the mechanism of a c e t a z o l a m i d e  teratogenesls  as w e l l as i n the context  genetic v a r i a t i o n i n teratogenic ill  of other  responses.  s t u d i e s of  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  ix  LIST OF APPENDICES  x  ACKNOWLEDGMENTS  xi  Chapter I. II.  III.  1  INTRODUCTION  10  GENERAL MATERIALS AND METHODS 1.  Mice:  2.  Time of pregnancy  source and maintainance  10 11  3. Source of teratogenic carbonic anhydrase inhibitors  11  TERATOGENIC ACTION OF ACETAZOLAMIDE IN CBA/J AND SWV  12  Materials and Methods  13  1.  Acetazolamide administration  13  2.  Examination of f e t a l malformations  13  Results and Discussion 1. 2.  3.  ,  14  Preliminary study of the time of action of acetazolamide i n CBA/J and SWV  14  Dose-response of CBA/J and SWV to acetazolamide administered a t 10 A.M. and k P.M. on day 10 of pregnancy  17  C r i t i c a l time of action of a single i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamlde i n CBA/J iv  20  4.  Range and d i s t r i b u t i o n  of d i g i t a l 24  anomalies found i n CBA/J  25  Conclusion IV.  RESPONSE TO ACETAZOLAMIDE OP RECIPROCAL HYBRID EMBRYOS FROM CROSSES BETWEEN CBA/J AND SWV  V.  26  M a t e r i a l s and Methods  26  R e s u l t s and D i s c u s s i o n ANALYSIS OF THE GENETIC CONTROL OF EMBRYONIC SENSITIVITY TO THE TERATOGENIC ACTION OF  26  ACETAZOLAMIDE  29  M a t e r i a l s and Methods  29 29  1.  Breeding  2.  A d m i n i s t r a t i o n of acetazolamide  program and  31  f e t a l examination 3.  VI.  data  31  R e s u l t s and D i s c u s s i o n 1. B i o m e t r i c a l e s t i m a t i o n of the number of l o c i i n v o l v e d w i t h acetazolamide teratogenic s e n s i t i v i t y 2 . E s t i m a t i o n of the number of l o c i u s i n g the g e n e r a t i o n mean f r e q u e n c i e s of ectrodactyly  33  Conclusion  45  TEST OF THE THREE-LOCUS MODEL  46  M a t e r i a l s and Methods  46  1.  VII.  G e n e t i c a n a l y s i s of b r e e d i n g  Breeding  program and t e r a t o g e n i c t e s t i n g  33 40  46  R e s u l t s and D i s c u s s i o n  46  Conclusion EMBRYO TRA.NSFER EXPERIMENTS TO INVESTIGATE THE ACETAZOLAMIDE RESPONSE OF THE CBA/J AND SWV STRAINS  55  v  56  57  M a t e r i a l s and Methods 1.  Embryo t r a n s f e r procedure  57  2.  Developmental s t a g i n g of t r a n s f e r r e d embryos  57  A d m i n i s t r a t i o n of acetazolamide t o dams a f t e r embryo t r a n s f e r  58  3.  58  R e s u l t s and D i s c u s s i o n V I I I . RESPONSE OF THE CBA/J AND SWV STRAINS TO DICHLORPHENAMIDE  63  M a t e r i a l s and Methods  64  1. 2.  P r e p a r a t i o n of dichlorphenamide s o l u t i o n for injection A d m i n i s t r a t i o n of dichlorphenamide and  64  f e t a l examination  64 64  R e s u l t s and D i s c u s s i o n  67  IX. GENERAL DISCUSSION 1.  S t r a i n variations i n teratogenic  2„  responses G e n e t i c model f o r a c e t a z o l a m i d e - i n d u c e d ectrodactyly  3.  67 73  Biochemical basis f o r s t r a i n differences i n response  to acetazolamide  79 82  X. SUMMARY BIBLIOGRAPHY  85  APPENDICES  89  vi  LIST OP TABLES Table  Page  1.  P r e l i m i n a r y Study of Time of A c t i o n of Acetazolamide i n CBA/J and SWV  15  2.  Dose E f f e c t of Acetazolamide. i n CBA/J and SWV Administered a t 10 A.M. and 4 P.M. on Day 10  18  3.  Time of A c t i o n of a S i n g l e I n t r a p e r i t o n e a l I n j e c t i o n of Acetazolamide i n CBA/J  21  Types and D i s t r i b u t i o n of Acetazolamide-Induced E c t r o d a c t y l y Found i n the Time of A c t i o n Study w i t h CBA/J Using a S i n g l e I n t r a p e r i t o n e a l I n j e c t i o n  23  5.  E f f e c t of Acetazolamide on R e c i p r o c a l F between CBA/J and SWV  28  6.  CBA/J Progeny Data from CBA/J Males Mated to CBA/J Females  34  F]_ Progeny Data from SWV Males Mated to CBA/J Females  35  8.  BCi Progeny Data from F^ Males Mated to CBA/J Females  36  9.  BC2 Progeny Data from BC^ Males Mated to CBA/J Females  37  Means and Variances of CBA/J, SWV, Fj. and BC^ S i r e s Scores Using the Weighted A r c s i n e Transformation and the C a l c u l a t i o n of the Number of L o c i C o n t r o l l i n g the Response to Acetazolamide  41  P r o b a b i l i t y that a Fetus i s G e n o t y p i c a l l y l i k e CBA/J i n the Backcross Study Considering 1, 2, 3 and 4 Independently Segregating L o c i  43  12.  BC3 Progeny Data from BC Females  48  13.  T h e o r e t i c a l and Observed Generation Means f o r E c t r o d a c t y l y Based on Three-Locus Model i n Backcross Study w i t h CBA/J Dams  4.  7.  10.  11.  2  1  Hybrids  Males Mated to CBA/J  vil  51  14.  Development of Embryos In Normal Matlngs and Embryo T r a n s f e r s Determined by Somite Number  59  15.  Summary of Embryo T r a n s f e r s w i t h CBA/J and SWV S t r a i n s and Treatment w i t h Acetazolamide  61  16.  Effect  66  17.  of Dichlorphenamide on CBA/J and SWV  C o r t i s o n e - I n d u c e d C l e f t P a l a t e i n Progeny of > B a c k c r o s s Matings w i t h the A and C 5 7 B L S t r a i n s  vlli  71  LIST OF FIGURES Figure 1.  Page  P e r c e n t e c t r o d a c t y l y Induced i n CBA/J by acetazolamide a d m i n i s t e r e d a t d i f f e r e n t g e s t a t i o n a l times and 500 mg/kg p e r I n j e c t i o n  16  2.  Dose e f f e c t of acetazolamlde In CBA/J and SWV a d m i n i s t e r e d a t 10 A.M. and 4 P.M. on day 10  19  3.  Time of a c t i o n of a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide i n CBA/J  22  4. ' Mating scheme to i n v e s t i g a t e the g e n e t i c s s e n s i t i v i t y to the t e r a t o g e n i c a c t i o n of acetazolamide 5.  6.  7.  8.  of the 30  Means and d i s t r i b u t i o n s of the p e r c e n t e c t r o d a c t y l y scores of the CBA/J, SWV, F« and BC-L s i r e s  39  Comparison of the observed mean p e r c e n t e c t r o d a c t y l y of the f e t a l g e n e r a t i o n s w i t h the p r o b a b i l i t y that a generation i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n f o r 1, 2, 3 and 4 l o c i  44  Means and p e r c e n t e c t r o d a c t y l y scores of the CBA/J, SWV, F i , B C i and BC2 s i r e s p l o t t e d a g a i n s t the p r o b a b i l i t y t h a t the f e t a l g e n e r a t i o n i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n based on the 3 - l o c u s model  49  Means and d i s t r i b u t i o n s of the mean weighted a r c s i n e scores of the CBA/J, SWV, F^, BC-j_ and B C s i r e s p l o t t e d a g a i n s t the p r o b a b i l i t y t h a t the f e t a l g e n e r a t i o n i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n based on the 3 - l o c u s model  50  ix  2  LIST OP APPENDICES Appendix I.  p a g e  D i s t r i b u t i o n of Acetazolamide-Induced D i g i t a l Anomalies i n the CBA/J S t r a i n  x  89  ACKNOWLEDGMENTS I would l i k e  to thank the f o l l o w i n g members of my t h e s i s  committee a t the U n i v e r s i t y of B r i t i s h Columbia support and guidance project:  d u r i n g the course of t h i s  for their thesis  D r . J . R. M i l l e r , D i v i s i o n of M e d i c a l G e n e t i c s ;  Dr. C. W. Roberts, Department of P o u l t r y S c i e n c e ; D r . H. F. S t i c h , Cancer Research  Centre.  I would l i k e t o thank my f e l l o w student, A l a n C. P e t e r s o n , f o r the many hours  t h a t we spent d i s c u s s i n g and  u s i n g the p r e i m p l a n t a t l o n mouse embryo techniques f o r v a r i o u s s t u d i e s i n g e n e t i c s and t e r a t o l o g y and f o r the o p p o r t u n i t y t o use  these techniques i n the p r e l i m i n a r y i n v e s t i g a t i o n of the  s t r a i n v a r i a t i o n s i n response  to acetazolamide.  I thank the f o l l o w i n g people, without whose c o o p e r a t i o n , t h i s t h e s i s would n o t be complete: (1)  Dr. W. M. Layton, Department of Anatomy-Cytology, Dartmouth M e d i c a l S c h o o l , Hanover, New Hampshire, f o r h i s many l o n g l e t t e r s of e n t h u s i a s t i c d i s c u s s i o n c o n c e r n i n g the t e r a t o l o g y of acetazolamide and f o r making a v a i l a b l e t o me s e v e r a l of h i s u n p u b l i s h e d s t u d i e s ;  (2)  Dr. K. Takano, S e n i o r S c i e n t i s t , Takeda Chemical I n d u s t r i e s L t d . , T a k a t s u k i , Japan, f o r h i s d i s c u s s i o n s d u r i n g the e a r l y stages of t h i s t h e s i s w h i l e he was a v i s i t i n g s c i e n t i s t w i t h D r . M i l l e r ;  (3)  Dr. M. C. Green, The Jackson L a b o r a t o r y , B a r Harbor, Maine, f o r making a v a i l a b l e t o me the r e s u l t s of a p r e l i m i n a r y i n v e s t i g a t i o n of the response t o acetazolamide of s e v e r a l mouse s t r a i n s and a c o n f i r m a t i o n of the response of the CBA/J s t r a i n ; xi  (4)  Dr. W. D. Dorian, D i r e c t o r , Medical Research Department, Merck Sharp and Dohme (Canada) L t d . , Pointe C l a i r e , Quebec, f o r h i s generous g i f t of dichlorphenamidej  (5)  Dr. J . E . Baer, Director of Drug Metabolism, Merck Sharp Dohme Research Laboratories, West Point, Pennsylvania, f o r his instructions f o r the solution of dichlorphenamide. I acknowledge the support of Medical Research Council  Grant No. MT-1062 to Dr. J . R. M i l l e r and the assistance of a Medical Research Council Studentship from 1968 to 1972.  xii  CHAPTER I INTRODUCTION Acetazolamide (or Dlamox  v  ;  )  i s a potent  carbonic  anhydrase i n h i b i t o r which has been used c l i n i c a l l y i n human p a t i e n t s s i n c e 1953.  diuretic  (1965a) found i t to be  Layton and  t e r a t o g e n i c i n r a t s and  malformation i s a r e d u c t i o n d e f o r m i t y , localized  i n the p o s t a x l a l s u r f a c e  predominantly the r i g h t of the right and  forelimb.  teratogenic l e s i o n sidedness  of the The  mice.  The  forelimb,  peculiar localization  i n the f o r e l i m b w i t h a predominant  i s unique i n both t e r a t o g e n i c a l l y Induced There appears to  be no developmental b a s i s f o r t h i s one-sided  SWV  Hallesy  usually ectrodactyly,  spontaneously o c c u r r i n g m a l f o r m a t i o n s .  The  as a  present  study r e p o r t s the  localization.  complete r e s i s t a n c e of  the  s t r a i n of mice to the t e r a t o g e n i c a c t i o n of  acetazolamide, a d e f i n i t i o n  of t h i s r e s i s t a n c e by  w i t h a s e n s i t i v e s t r a i n CBA/J, and t h i s r e s i s t a n c e i n a breeding CBA/J s t r a i n .  a g e n e t i c a n a l y s i s of  program w i t h  E v i d e n c e i s presented  model of i n h e r i t a n c e , w i t h  the s e n s i t i v e  to support  r e s i s t a n c e to t'he  of these s t u d i e s were presented  meeting of the T e r a t o l o g y  Society  Maren, Mayer and Wadworth (1954), 1  to the  ( B i d d l e and  a three  gene  teratogenic  a c t i o n of acetazolamide b e i n g a dominant t r a i t . results  comparison  Preliminary 12th  annual  Miller,  1972).  i n an e a r l y r e p o r t  on  2  the pharmacology of acetazolamlde, experimence and  from p e r s o n a l  i t appeared to have no  s t a t e d , from  communications from o t h e r s ,  obvious e f f e c t on the  dogs.  Maren  the f i n d i n g s of L a y t o n and H a l l e s y (1965a)  on the b a s i s t h a t c a r b o n i c anhydrase i n h i b i t i o n i s the known f u n c t i o n of acetazolamide  only  by Maren, 1967)  (reviewed  maximal p h y s i o l o g i c a l c a r b o n i c anhydrase i n h i b i t i o n can achieved  w i t h doses lower than those  Hallesy. was  that  embryonic  development of r a t s , guinea p i g s , r a b b i t s and (1965) d i s p u t e d  their  used by L a y t o n  and be  and  P o s s i b l y , some o t h e r unknown f u n c t i o n of the drug  responsible f o r i t s teratogenicity. L a y t o n and H a l l e s y (1965b) r e b u t t e d Maren's (1965)  arguments by  s t a t i n g t h a t the h i g h dose of  acetazolamide  administered  i n the d i e t might be r e q u i r e d f o r a  sustained  enzyme i n h i b i t i o n d u r i n g the embryo s e n s i t i v e p e r i o d .  If  the t e r a t o g e n i c a c t i o n i s d i r e c t l y on the embryo, t h i s  high  dose l e v e l i n the dam  might be  e s s e n t i a l to m a i n t a i n  t e r a t o g e n i c l e v e l of the drug i n the embryo.  They suggested  t h a t s t r u c t u r a l l y d i f f e r e n t c a r b o n i c anhydrase should be  screened  and,  i f they produced  a  inhibitors  similar-anomalies,  the c a u s a t i v e r o l e of c a r b o n i c anhydrase i n h i b i t i o n i n the acetazolamide In 1967,  t e r a t o g e n e s l s would be more  secure.  H a l l e s y and L a y t o n found t h a t dichlorphenamide,  a s t r u c t u r a l l y d i f f e r e n t c a r b o n i c anhydrase administered acetazolamide Wilson,  inhibitor,  i n the d i e t of r a t s , a l s o produced type  of f o r e l i m b anomalies.  And  the in  1968,  Maren, Takano and E l l i s o n found t h a t ethoxzolamide,  3 s t r u c t u r a l l y d i f f e r e n t from both acetazolamide and dichlorphenamide, malformations  produced  the acetazolamide  type f o r e l i m b  i n rats.  W i l s o n e t a l . (1968) i n v e s t i g a t e d more thoroughly the time of a c t i o n of acetazolamide and found i n j e c t i o n s of 1,000 produced  mg/kg ( d i v i d e d  that  subcutaneous  i n t o 2 i n j e c t i o n s p e r day)  the t e r a t o g e n i c l e s i o n on day 10 and 11.  the e a r l y f o r e l i m b stage of r a t embryos.  This i s  When t h i s h i g h dose  was a d m i n i s t e r e d on day 10, a few extreme cases of a d a c t y l y , m l c r o m e l i a and a m e l i a of the r i g h t f o r e l i m b w i t h severe e c t r o d a c t y l y of the l e f t f o r e l i m b were found. sidedness of the d e f e c t s t i l l  remained.  co-workers were n o t a b l e t o demonstrate, assay, c a r b o n i c anhydrase day  The r i g h t  W i l s o n and h i s by standard  a c t i v i t y i n the r a t embryo  enzyme until  13 which i s much beyond the limb s e n s i t i v e p e r i o d of  day 10 and 11.  However, they c o u l d d e t e c t a c t i v i t y i n the  a l l a n t o i c membrane and p l a c e n t a l a t e on day 11. the t e r a t o g e n i c a c t i o n of c a r b o n i c anhydrase the embryo was s t i l l  Therefore,  inhibition in  questioned.  Takano, Yokota and Nagata (1971) succeeded i n demonstrating in  the presence  of c a r b o n i c anhydrase  activity  the f o r e - and hindllmb buds of the r a t embryo by  h i s t o c h e m i c a l assays of s e c t i o n s of the limb buds u s i n g l i g h t and e l e c t r o n microscopy.  With standard enzyme  assay,  such as t h a t used by W i l s o n e t a l . (1968), of whole embryo p r e p a r a t i o n s , the enzyme a c t i v i t y was p r o b a b l y masked by a l a r g e amount of enzyme-free t i s s u e .  The a c t i v i t y  was  4  present on day 11 i n the sections of a l l limb buds and was concentrated i n the area between the epidermis and the underlying mesenchyme. of  I t was also l o c a l i z e d on the surface  some i n t e r n a l mesenchymal c e l l s .  There appeared to be  more a c t i v i t y i n the forelimb than i n the hindlimb but this was not q u a n t i t a t i v e l y determined.  I f 500 mg/kg of  acetazolamide was administered to the dam 1 hour before i t was k i l l e d , the carbonic anhydrase a c t i v i t y i n the embryonic limb buds was greatly diminished or completely i n h i b i t e d . This suggested that the embryo could be the primary to  the teratogenic action of acetazolamide.  responder  However, the  r i g h t sidedness of the defect and the immunity of the hindlimbs were s t i l l unexplainable. Layton (1971) demonstrated that hamsters also responded to  the teratogenic action of acetazolamide i n the same  manner as rats and mice 0  With single i n t r a p e r i t o n e a l  i n j e c t i o n s , the time of action was c r i t i c a l l y defined on late day 8 f o r this species. This i s the time a t which the forelimb bud f i r s t appears i n the hamster.  However, i n  hamsters both forelimbs were affected equally and most of the malformations were b i l a t e r a l . to  There was s t i l l a postaxlal  preaxial pattern to the severity of the, reduction  deformity and administration of acetazolamide l a t e r i n the sensitive period resulted i n more severe ectrodactyly.  In  a d d i t i o n , a few hindlimbs showed a reduction deformity but most of these were of the preaxial surface and of the l e f t hindlimb.  These hindlimb anomalies occurred l a t e i n the  5 s e n s i t i v e p e r i o d and c o i n c i d e d w i t h the l a t e r appearance of the h l n d l i m b  bud.  V i c k e r s (1972) r e p o r t e d an independent acetazolamlde  teratogenesis i n rats.  examination  He presented the  e x t e n s i v e whole-mount and h i s t o l o g i c a l examinations many v a r i a t i o n s of the f o r e l i m b anomalies  of first  of the  i n mature f e t u s e s .  Suzuki and Takano (1969) e s t a b l i s h e d that day 9 of pregnancy w i t h ICR-JCL mice was f o r acetazolamide-induced o b s e r v a t i o n and acetazolamide  the s e n s i t i v e embryonic  e c t r o d a c t y l y i n the mouse.  stage  This  the v a r i a t i o n s of the time of a c t i o n of  t h a t are found w i t h d i f f e r e n t s t r a i n s of mice  w i l l be d i s c u s s e d l a t e r i n the p r e s e n t study. In a s e a r c h f o r the p h y s i o l o g i c a l mechanism of acetazolamlde  t e r a t o g e n e s i s i n r a t s , Maren and E l l i s o n were  a b l e to d e f i n e two a c t i o n s of the drug which were r e q u i s i t e s f o r t e r a t o g e n e s i s (Maren and E l l i s o n , E l l i s o n and Maren, 1972a, 1972b).  1972a, 1972b,  1972c;  Respiratory acidosis,  potassium d e p l e t i o n and m e t a b o l i c a c i d o s i s of the dam,  which  are the p h y s i o l o g i c a l e f f e c t s of the e l e c t r o l y t e l o s s due c a r b o n i c anhydrase  I n h i b i t i o n , were  as c a u s a t i v e a c t i o n s .  themselves  Only c a r b o n i c anhydrase  which cause r e n a l l o s s of potassium produced l e s i o n of a c e t a z o l a m i d e .  Those i n h i b i t o r s  could be made t e r a t o g e n i c by other drugs  ruled  to  out  inhibitors  the t e r a t o g e n i c which do not  ( t h e o p h y l l i n e ) or  d i e t a r y r e s t r i c t i o n s which cause potassium d e f i c i e n c y of the dam.  These drugs  not produce  or potassium d e f i c i e n c y by themselves  the a c e t a z o l a m i d e - t y p e  limb  anomalies.  do  6 C o n v e r s e l y , p o t a s s i u m - r e t a i n i n g drugs  ( t r i a m t e r e n e or  a m l l o r i d e ) or potassium supplementation i n c o n j u n c t i o n w i t h acetazolamide reduced  the frequency and s e v e r i t y of the  d e f e c t s o r p r o v i d e d complete l e v e l s of potassium  protection.  However, e x c e s s i v e  or p o t a s s i u m - r e t a i n i n g drugs caused an  i n c r e a s e i n the frequency and s e v e r i t y of the a c e t a z o l a m i d e defects.  T h e r e f o r e , both c a r b o n i c anhydrase  a potassium Imbalance, a c t i o n s to produce  i n h i b i t i o n and  a t l e a s t i n the dam, a r e n e c e s s a r y  the a c e t a z o l a m i d e - t y p e l e s i o n .  There was  a s u g g e s t i o n t h a t t h i s was o c c u r r i n g i n the embryo  because  term f e t u s e s t h a t had been t r e a t e d w i t h a c e t a z o l a m i d e i n u t e r o and expressed the limb anomalies had a s i g n i f i c a n t l y h i g h e r l i v e r potassium c o n c e n t r a t i o n than t h e i r normal mates and, i n t u r n , these were both h i g h e r than  litter  control  fetuses. Experiments w i t h i n t r a u t e r i n e a d m i n i s t r a t i o n of acetazolamide suggested t h a t the s i t e of t e r a t o g e n i c a c t i o n of t h i s and r e l a t e d c a r b o n i c anhydrase the embryonic  unit  inhibitors i s within  (embryo p r o p e r p l u s embyronic  membranes).  S c o t t (1970) a d m i n i s t e r e d s m a l l q u a n t i t i e s of acetazolamide to d i f f e r e n t i n t r a u t e r i n e s i t e s of day 11 pregnant  rats.  More of the t y p i c a l acetazolamide malformations were  produced  when i t was a d m i n i s t e r e d c l o s e to the embryo, i n o r near the d e c l d u a c a p s u l a r i s and/or y o l k sac and the extraembryonic coelom, than when i t was d e p o s i t e d f u r t h e r away i n the d e c i d u a b a s a l i s and/or malformations  chorioallantoic placenta.  No  o c c u r r e d i n embryos a t c o n t r o l o r n o n - i n j e c t e d  7 sites. site was  Thus, the maternal  organism does not appear to be  of primary a c t i o n of t h i s t e r a t o g e n . a systemic e f f e c t of the dam,  expected a t n o n - i n j e c t e d s i t e s .  If teratogenesis  a f f e c t e d embryos would be The g r e a t e r number and more  s e v e r e l y a f f e c t e d embryos found when the drug was i n s i t e s c l o s e r to the embryo suggested a c t i o n of acetazolamide  deposited  t h a t the t e r a t o g e n i c  i s d i r e c t l y on the embryo.  I n t r a u t e r i n e a d m i n i s t r a t i o n of acetazolamide undertaken  w i t h hamsters by S t o r c h and Layton  p e r s o n a l communication). to the u t e r i n e lumenal acetazolamide-type  the  In t h i s case, l t was  was 1972,  (Layton,  administered  spaces between embryos.  limb malformations were found  More of the i n embryos  near the s i t e of a p p l i c a t i o n than i n o t h e r p a r t s of the u t e r u s which suggested responder  t h a t the embryonic u n i t i s the  to the t e r a t o g e n i c a c t i o n of the drug.  acetazolamide was  primary  If  administered i n t r a p e r i t o n e a l l y a t a  t e r a t o g e n i c dose l e v e l and a m l l o r i d e , a drug which b l o c k s potassium  e x c r e t i o n , was  a d m i n i s t e r e d i n t r a l u m e n a l l y to the  u t e r u s , the number of malformed embryos i n the a m i l o r i d e t r e a t e d horn was  reduced.  T h i s agreed w i t h the h y p o t h e s i s  t h a t the a c t i o n of a c e t a z o l a m l d e and  the  resulting  p h y s i o l o g i c a l changes r e q u i r e d to c r e a t e the t e r a t o g e n i c environment  i s a t the l e v e l of the embryo or embryonic u n i t .  To undertake SWV  the p r e s e n t study of the r e s i s t a n c e of the  s t r a i n of mice to the t e r a t o g e n i c a c t i o n of a c e t a z o l a m i d e ,  i t was  n e c e s s a r y to d e f i n e the t e r a t o g e n i c parameters  sensitive strain.  In t h i s case, the CBA/J s t r a i n was  for a chosen.  8 The behaviour of the SWV s t r a i n was then examined i n the l i g h t of t h i s i n f o r m a t i o n (Chapter I I I ) . The  response  of the r e c i p r o c a l h y b r i d embryos from  c r o s s e s between SWV and CBA/J to a c e t a z o l a m i d e , a d m i n i s t e r e d a t the a p p r o p r i a t e time and dosage, was examined. found  I t was  t h a t embryonic r e s i s t a n c e t o the t e r a t o g e n i c a c t i o n i s ,  dominant t o s e n s i t i v i t y  (Chapter I V ) .  a p p r o p r i a t e b r e e d i n g scheme —  T h i s l e d to an  a repeated b a c k c r o s s program  to the s e n s i t i v e CBA/J s t r a i n dam' —  which would d i s c r i m i n a t e  between a s i n g l e gene and a more complex, p o l y g e n i c mode of i n h e r i t a n c e and, i f the i n h e r i t a n c e of s e n s i t i v i t y t o acetazolamide was p o l y g e n i c , i t would permit an e s t i m a t i o n of the number of g e n e t i c f a c t o r s i n v o l v e d i n t h i s t r a i t .  A  t h r e e - l o c u s model was suggested by two separate methods of a n a l y s i s of the b r e e d i n g d a t a (Chapter V) and, w i t h i n the l i m i t a t i o n s d i s c u s s e d , t h i s was confirmed by f u r t h e r b r e e d i n g t e s t s of a s e g r e g a t i n g g e n e r a t i o n (Chapter V I ) . S t u d i e s on the "acetazolamide  syndrome" i n the  l i t e r a t u r e , d i s c u s s e d p r e v i o u s l y , i n d i c a t e d t h a t the t e r a t o g e n i c a c t i o n was a t the l e v e l of the embryo or embryonic membranes.  An attempt  was made t o p a r t i t i o n the  embryo and dam by embryo t r a n s f e r s between- the CBA/J and SWV strains.  T e r a t o g e n i c s e n s i t i v i t y and r e s i s t a n c e a r e  p r o p e r t i e s of the embryonic u n i t The  response  (Chapter V I I ) .  of the two s t r a i n s to dichlorphenamide, a  s t r u c t u r a l l y d i f f e r e n t c a r b o n i c anhydrase examined.  The response  i n h i b i t o r , was  of these two s t r a i n s w i t h  dichlorphenamide i s the same as with acetazolamide (Chapter V I I I ) .  CHAPTER II GENERAL MATERIALS AND METHODS 1.  Mice:  source and malntalnance.  The mice used i n t h i s study were Inbred strains of Mus musculus and were maintained i n the Zoology Vivarium at the University of B r i t i s h Columbia.  SWV  i s an inbred s t r a i n  developed and maintained by Dr. J . R. M i l l e r and i t was at the  generation of inbreeding at the time of t h i s study.  CBA/J i s another inbred s t r a i n .  At the s t a r t of t h i s  p r o j e c t , CBA/J mice were from an inbreeding colony a t generation F g _ 1 Q i n t h i s laboratory; however, they originated from the Jackson Laboratory (Bar Harbor, Maine) at inbreeding generation F i j g ^ i ^ '  L a  ^  e r  ^  n  the p r o j e c t , the CBA/J mice  were purchased d i r e c t l y from the Jackson Laboratory. There are no spontaneously occurring limb malformations in either the SWV  or CBA/J s t r a i n .  The mice were maintained on an 18 hour l i g h t period ( 6 A.M.  to 12 midnight) and a 6 hour dark period.  fed Purina Laboratory Chow and water ad  They were  libitum.  Females used f o r timed matlngs were caged i n groups of 4 per cage and males were caged s i n g l y .  Following timed  matings, pregnant females were caged i n groups of 2 or 3.  10  11 2  .  Timing of pregnancy. Females w i t h v a g i n a l s i g n s of p r o e s t r u s were p l a c e d  s i n g l y w i t h a male a t the end copulation plug, i n d i c a t i n g  Source a.  mating,  was  found  the  If a following  d e s i g n a t e d as day 0 of pregnancy.  morning, t h i s day was 3.  of the l i g h t p e r i o d .  of t e r a t o g e n i c c a r b o n i c anhydrase  inhibitors.  Acetazolamlde.  The acetazolamide used was a c e t a z o l a m i d e , Diamox  v  p a r e n t e r a l sodium  , manufactured  by the L e d e r l e  P r o d u c t s Department of Gyanamid of Canada, L t d . , M o n t r e a l . I t i s s u p p l i e d i n 500 hydroxide  mg v i a l s w i t h s u f f i c i e n t sodium  to s o l u b i l i z e i t i n d i s t i l l e d water w i t h a  r e s u l t i n g pH of 9 . 2 .  The acetazolamide was  obtained from  the  Pharmacy of the Vancouver G e n e r a l H o s p i t a l . b.  Dichlorphenamide.  Dichlorphenamide f o r human use)  i s an i n v e s t i g a t i o n a l  of Merck and Co.,  through the c o u r t e s y of Dr. W.  new  I n c . and was  drug  (not  supplied  D. D o r i a n , D i r e c t o r of the  M e d i c a l Research Department of Merck, Sharp and Dohme Canada Ltd.  CHAPTER I I I TERATOGENIC ACTION OF ACETAZOLAMIDE IN CBA/J AND SWV The  t e r a t o g e n e s i s of acetazolamide  d i e t method throughout H a l l e s y (1965&)*  i n mice by the drug-  pregnancy was presented by Layton and  They used Manor Farms mice which a r e a  commercially a v a i l a b l e and g e n e t i c a l l y heterogeneous stock.  albino  The most f r e q u e n t d e f o r m i t y was the absence of  d i g i t V of the r i g h t f o r e l i m b . A time of a c t i o n study w i t h acetazolamide was made by Suzuki and Takano (1969; ICR-JCL mice.  Takano, p e r s o n a l communication) w i t h  T h i s i s a random-bred s t o c k of mice maintained  by the Japanese Cancer L a b o r a t o r y and d e r i v e d from the ICR stocks i n the U n i t e d S t a t e s .  With two subcutaneous  i n j e c t i o n s of 500 mg/kg a d m i n i s t e r e d a t 10 A.M. and 4 P.M., Suzuki and Takano found t h a t day 9 was the s e n s i t i v e day f o r s i n g l e day treatment In  i n t h i s stock of mice.  the p r e s e n t study of the acetazolamide  the SWV s t r a i n ,  r e s i s t a n c e of  the time of t e r a t o g e n i c a c t i o n was  r e i n v e s t i g a t e d f o r the s e n s i t i v e CBA/J s t r a i n s i n c e day 9 was found n o t to be the t e r a t o g e n i c a l l y s e n s i t i v e day. An i n t r a p e r i t o n e a l route f o r a d m i n i s t r a t i o n of acetazolamide was p r e f e r r e d r a t h e r than the subcutaneous route of Suzuki and Takano (1969) because a n e c r o t i c epidermal l e s i o n was produced  by the l a t t e r route that was u n a e s t h e t i c and made 12  13  the t r e a t e d animals i r r i t a b l e and d i f f i c u l t  t o handle.  With m u l t i p l e and s i n g l e dose a d m i n i s t r a t i o n of a c e t a z o l a m i d e , day 10 i s the s e n s i t i v e day f o r the CBA/J s t r a i n and, w i t h i n the dosage l e v e l s employed, the SWV i s r e s i s t a n t t o any t e r a t o g e n i c o r embryotoxic  strain  a c t i o n of t h i s  drug. M a t e r i a l s and Methods 1.  Acetazolamide a d m i n i s t r a t i o n . fry)  The a c e t a z o l a m i d e , p h a r m a c e u t i c a l Diamox^  ' preweighed  to 500 mg p e r v i a l w i t h sodium hydroxide, was d i s s o l v e d w i t h 5 ml of s t e r i l e d i s t i l l e d The acetazolamide  water.  s o l u t i o n was i n j e c t e d  i n t o the dam u s i n g a 0.25  lntraperitoneally  ml t u b e r c u l i n s y r i n g e and a 26  gauge, \ i n c h (0.46 x 13 mm) d i s p o s a b l e n e e d l e .  The times o f  a d m i n i s t r a t i o n d u r i n g pregnancy and the dosages p e r k i l o g r a m body weight  o f the dam a t the time of i n j e c t i o n a r e g i v e n i n  the p r e s e n t a t i o n of the r e s u l t s .  The f o l l o w i n g a r e the  volumes a d m i n i s t e r e d per gram mouse and the c o r r e s p o n d i n g mg/kg dosages:  500  mg/kg; All  0.0025  0.0075  ml/g =  ml/g =  750  250  mg/kg;  mg/kg;  not occur spontaneously Examination All  0.010  ml/g =  ml/g =  1,000  mg/kg.  c o n t r o l l i t t e r s were u n t r e a t e d s i n c e the  a c e t a z o l a m l d e - l n d u c e d malformations  2.  0.005  of f e t a l  a r e so s p e c i f i c and do  i n the s t r a i n s of mice  used.  malformations.  pregnancies were terminated on day 1 8 .  The dams  1 4  were k i l l e d by c e r v i c a l d i s l o c a t i o n , the u t e r i were removed and t h e i r contents recorded.  Resorptions were defined as any  trace of resorbing embryonic or placental tissue and a l l dead fetuses, regardless of limb condition, were c l a s s i f i e d as resorptions.  The number of embryo implantations was defined  as the sum of the number of resorptions and l i v e fetuses. Live fetuses (spontaneously moving) were recorded as to limb condition and other external malformations.  A fetus with any  limb reduction deformity of the "acetazolamlde type" was defined as having ectrodactyly. Results and Discussion 1.  Preliminary study of the time of action of acetazolamide in CBA/J and SWV. The r e s u l t s of the preliminary study of the time of  teratogenic action of acetazolamlde (500 mg/kg per i n j e c t i o n ) in the CBA/J and SWV strains are presented i n Table 1.  The  1 0 A.M. and 4 P.M. times f o r treatment were used f o r convenience and were the times used by Suzuki and Takano ( 1 9 6 9 )  i n t h e i r study of ICR-JCL mice.  Ectrodactyly was  induced by acetazolamlde i n CBA/J but not i n SWV. The change with time i n the frequency of ectrodactyly i n CBA/J, using the multiple dosage regime of Table 1 , i s graphically presented i n Figure 1 . Two i n j e c t i o n s of 500 mg/kg (at 1 0 A'.M. and 4 P.M.) on each of days 9 t 1 0 and 1 1 of pregnancy resulted i n 73% ectrodactyly i n l i v e fetuses  TABLE 1 Preliminary Study of Time of Action of Acetazolamide i n CBA/J and SWV  Times of treatment*  Affected litters  Resorptions/ implants  Ectrodactyly/ l i v e fetuses  No.  %  No.  0/4  9/33  27  0/24  0  2 . day 10 (10 AM, 4 PM)  V5  7/35  20  9/28  32  3c day 10 (10 AM, 4 PM) day 11 (10 AM)  4/4  5/30  17  8/25  32  2/7  9/62  15  3/53  6  2/4  1V25  56  8/11  73  0/4  9/63  14  0/54  0  CBA/J  -  1. day 9  (10 AM, 4 PM)  4„ day 10 (4 PM)  day 11 (10 AM)  5 . day 9 (10 AM, 4 PM) day 10 (10 AM, 4 PM) day 11 (10 AM, 4 PM)  •  SWV 1. day 9 (10 AM, 4 PM) day 10 (10 AM, 4 PM) day 11 (10 AM, 4 PM)  * Treatment = 500 mg/kg per i n j e c t i o n administered  ihtraperitoneally.  16  80"  t  I  J  i  70-  40-  < CO  1 I * 30x TJ  o o  ~o Tj  20-  c d) (J  °" 10-  o-  1 1 day 9  day 10 Time  FIGURE 1:  during  day 11 pregnancy  Percent ectrodactyly induced i n CBA/J by acetazolamide administered at d i f f e r e n t gestational times and 500 mg/kg per i n j e c t i o n . Arrows indicate time of i n j e c t i o n and l i n e s connecting arrows designate treated groups.  but also a high frequency of resorption ( 5 6 ^ ) .  Two  injections on day 9 at t h i s dosage did not r e s u l t i n ectrodactyly.  This i s contrary to the. findings of Suzuki and  Takano (1969) who found day 9 to be the most sensitive day with this dosage regime f o r ICR-JCL mice. day 10 resulted i n ^2% ectrodactyly.  Two i n j e c t i o n s on  Adding another  i n j e c t i o n a t 10 A.M. on day 11 did not change the frequency of ectrodactyly.  A single i n j e c t i o n a t 4 P.M. on day 10  followed by another at 10 A.M. on day 11 resulted i n a lower frequency of ectrodactyly ( 6 % ) .  Therefore, day 10 was  inferred to be the sensitive day f o r the acetazolamide induction of ectrodactyly i n the CBA/J s t r a i n of mice. 2.  Dose-response of CBA/J and SWV to acetazolamide administered a t 10 A.M. and 4 P.M. on day 10 of pregnancy. An examination was made of the dose-response to  acetazolamide i n CBA/J and SWV using the method of two i n j e c t i o n s a t 10 A.M. and 4 P.M. on day 10.  The data are  presented i n Table 2 and g r a p h i c a l l y displayed i n Figure 2 . The data f o r 500 mg/kg (2 doses on day 10) f o r CBA/J are repeated from Table 1. In CBA/J, the frequencies of both ectrodactyly and resorption increased with increased dose.  In SWV, no  ectrodactyly was found and there was no change i n the control resorption frequency.  However, 3 out of 8 SWV dams died  a f t e r treatment with the highest dose.  No death a f t e r  treatment occurred i n any of the CBA/J dams.  18 TABLE 2 Dose E f f e c t of Acetazolamide In CBA/J and SWV Administered at 10 A.M. and 4 P.M. on Day 10  Dose/ injection (mg/kg)  Affected litters  Resorptions/ implants No.  %  Ectrodactyly/ l i v e fetuses No.  %  CBA/J  1. 0  0/7  8/62  13  0/54  0  2. 500  4/5  7/35  20  9/28  32  3. 750  9/10  23/85  27  29/62  47  4. 1,000  4/4  18/36  50  11/18  61  1. 0  0/13  18/173  10  0/156  0  2. 750  0/11  15/162  9  0/147  0  3. l.ooo  o/5*  8/75  11  0/67  0  SWV  * 3/8 treated SWV dams died a f t e r treatment with 1,000 mg/kg per i n j e c t i o n .  19  FIGURE 2:  Dose e f f e c t of acetazolamide In CBA/J and a d m i n i s t e r e d a t 10 A.M. and 4 P.M. on day  SWV 10.  20 From these observations, i t was concluded that the SWV s t r a i n was r e s i s t a n t to any teratogenic or embryotoxlc action of acetazolamide. 3.  Critical  time of a c t i o n of a single i n t r a p e r i t o n e a l  i n j e c t i o n of acetazolamlde i n CBA/J. A c r i t i c a l evaluation of the time of teratogenic action of a single 1,000 mg/kg, i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide was made with the CBA/J s t r a i n . presented i n Table 3 and Figure 3.  This i s  From the graphical data  in Figure 3. the most sensitive time to a single i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide appeared to be mid-day 10. This time agrees well with the appearance of the forelimb bud i n mice.  I t also agrees with the c r i t i c a l  evaluation of the limb sensitive period f o r hamster embryos (Layton, 1971) allowing f o r differences i n the rates of embryonic development of mice and hamsters. There i s no explanation f o r the drop i n resorption frequency at 4 P.M. on day 10 (Figure 3 ) .  I t may be due to  sampling error; however, i t may prove i n t e r e s t i n g f o r l a t e r re-evaluation. The d i s t r i b u t i o n of limb anomalies found i n the CBA/J fetuses with ectrodactyly i n Table 3 Is presented i n Table 4. The predominance of affected r i g h t forellmbs was obvious. There was also an Indication that, when acetazolamide was administered l a t e r i n the limb sensitive stage, the r i g h t  21 TABLE 3 Time of A c t i o n of a S i n g l e I n t r a p e r i t o n e a l I n j e c t i o n of Acetazolamide  Time of treatment*  Affected litters  i n CBA/J  Resorptions/ implants  Ectrodactyly/ l i v e fetuses  No.  %  No.  %  1/8  18/64  28  1A6  2  4 AM  2/10  27/85  32  3/58  5  3. day 10 - 10 AM  5/10  26/81  32  9/55  16  4. day 10 -  5/9  12/77  16  9/65  14  2/10  24/86  28  3/62  5  1. day 9 2  0  day  - 10 PM  10 -  4 PM  5. day 10 - 10 PM  * Treatment i s a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of 1,000 mg/kg.  3CH  20H  10-  oday 9  day 10  10 PM  4 AM  1  1  day 10 10 A M  day 10 4 PM  Time during pregnancy of a single i.p. injection  FIGURE 3:  day 10 10 PM (1000 rngy^kg)  Time of a c t i o n of a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide i n CBA/J.  23 TABLE 4 Types and D i s t r i b u t i o n of Acetazolamide-Induced Ectrodactyly Found i n the Time of Action Study with CBA/J Using a Single Intraperitoneal Injection  Time of injection  Type of ectrodactyly  Number Female  1. day 9  - 10 AM  1. R  -  ii. R  -  3. day 10 - 10 AM  i. R  -  4. day 10 -  i. R  2. day 10 -  4 AM  4 PM  i. R  ii. R iii. R L iv. R  5. day 10 - 10 AM  1. R 11. R L  —  -  Male . T o t a l  V absent  0  1  V abnormal  1  0  V absent  1  1  V absent V abnormal  5 0  V absent  1 0  V absent V absent  2  0  I V , V absent  1  l  V absent  1  1  V absent V absent  1  0  2k  forelimb ectrodactyly became more severe and some l e f t forelimb involvement occurred. This increase i n severity of 1  the ectrodactyly agrees with Layton s (1971) findings with hamsters. 4.  Range and d i s t r i b u t i o n of d i g i t a l anomalies found i n CBA/J. The range of acetazolamide-lnduced forelimb anomalies  was extensive.  The largest treated sample of CBA/J embryos  i n t h i s study was made to e s t a b l i s h the CBA/J s t r a i n response frequency f o r use i n subsequent Interpretation of the genetic analysis (Table 6, Chapter V ) .  Five CBA/J s i r e s were mated  to a number of CBA/J females and the dams were treated with 750 mg/kg a t 10 A.M. and k P.M. on day 10.  There were no  obvious differences In the range of anomalies between the 5 s i r e groups and the fetuses were pooled f o r examination of the limbs.  The range and d i s t r i b u t i o n of d i g i t a l anomalies  found i n these CBA/J fetuses are presented i n Appendix I . I t was c l e a r from Appendix I that the most common type of ectrodactyly was an absence of d i g i t V of the r i g h t forelimb.  There was a predominance of r i g h t sidedness that  confirms the general findings of other studies using mice and rats and discussed In Chapter I.  There were a few cases i n  which the l e f t side was the predominant s i d e .  The s p e c i f i c  l o c a l i z a t i o n of the acetazolamide teratogenic l e s i o n remains the most i n t r i g u i n g embryological mystery of t h i s teratogen.  25 Conclusion The CBA/J s t r a i n responds acetazolamlde t h i s drug.  appears  i n the manner expected from o t h e r s t u d i e s w i t h  The  predominantly  to the t e r a t o g e n i c a c t i o n of  e c t r o d a c t y l y i s of the f o r e l i m b  of the r i g h t s i d e .  and  However, the SWV  strain  to be completely r e s i s t a n t to the t e r a t o g e n i c  embryotoxic  a c t i o n s of a c e t a z o l a m i d e .  No e c t r o d a c t y l y  and was  induced d u r i n g pregnancy and no change i n the c o n t r o l r e s o r p t i o n frequency was Administration  found.  of acetazolamide  l a t e r i n the limb  s e n s i t i v e p e r i o d of the s e n s i t i v e CBA/J s t r a i n produces a more severe e c t r o d a c t y l y of the r i g h t f o r e l i m b w i t h some l e f t forelimb  involvement.  CHAPTER IV RESPONSE TO ACETAZOLAMIDE OF RECIPROCAL Fj_ HYBRID EMBRYOS FROM CROSSES BETWEEN CBA/J AND The d i f f e r e n c e In the responses  SWV  of the CBA/J and SWV  s t r a i n s to the t e r a t o g e n i c a c t i o n of acetazolamide i s dramatic.  CBA/J i s s e n s i t i v e w h i l e SWV  resistant.  The response  i s completely  o f the r e c i p r o c a l h y b r i d embryos  was i n v e s t i g a t e d t o i n i t i a t e  the g e n e t i c a n a l y s i s of t h i s  trait. M a t e r i a l s and Methods R e c i p r o c a l c r o s s e s were made between the CBA/J and SWV strains.  The dams were t r e a t e d w i t h acetazolamide  on day 10  of pregnancy w i t h two i n t r a p e r i t o n e a l i n j e c t i o n s ( a t 10 A.M. and 4 P.M.)  of 750 mg/kg p e r i n j e c t i o n .  These times were the  most s e n s i t i v e f o r the CBA/J embryo w i t h the m u l t i p l e i n j e c t i o n regime and t h i s dosage d i d n o t r e s u l t i n too g r e a t an i n c r e a s e i n the normal r e s o r p t i o n frequency  (Chapter I I I ) .  The f e t u s e s were examined on day i d as d e s c r i b e d i n Chapter I I I .  F o r the d e s i g n a t i o n of f e t a l o r i g i n , the s t r a i n  of the dam i s l i s t e d  first.  R e s u l t s and D i s c u s s i o n The responses of the r e c i p r o c a l F^ h y b r i d embryos  26  from  27  crosses "between CBA/J and SWV are presented i n Table 5. In the sample of treated SWV.CBA  embryos, there was no  induced ectrodactyly and the resorption frequency was e s s e n t i a l l y that of the control l i t t e r s .  In the sample of  treated CBA.SWV F^ embryos, 1 out of 117 l i v e fetuses exhibited induced ectrodactyly and the resorption frequency was of the control l e v e l . Comparison of these r e c i p r o c a l F^ hybrid embryo responses with those of the CBA/J and SWV Inbred strains i n Chapter I I I indicated that embryonic resistance to the teratogenic action of acetazolamlde could be considered to be a dominant t r a i t and s e n s i t i v i t y to be a recessive t r a i t . The f a c t that 1 affected CBA.SWV F 1 fetus was found while no affected SWV.CBA F^ fetuses were observed cannot be taken to be a r e c i p r o c a l cross difference or evidence f o r a dam e f f e c t .  The difference between 1 and 0 affected with  these sample sizes was unanalysable.  The responses of the  two types of F^ embryos were assumed to be equal and e s s e n t i a l l y zero.  Therefore, a backcross program using the  sensitive CBA/J s t r a i n should quickly recover genes f o r embryonic s e n s i t i v i t y to the teratogenic action of acetazolamide.  TABLE 5 E f f e c t of Acetazolamlde on Reciprocal F. Hybrids between CBA/J and SWV  Affected litters  Mating Dam  X  Sire  Resorptions/ implants  Ectrodactyly/ l i v e fetuses  No.  %  No.  %  0/104  0  0/41  0  1.  SWV  X  CBA/J (treated)*  0/8  10/114  9  2.  SWV  X  CBA/J. ( c o n t r o l ) * *  0/3  6/47  13  3.  CBA/J  X  SWV  (treated)  1/14  12/129  9  1/117  1  4.  CBA/J  X  SWV  (control)  0/4  5/32  16  0/28  0  * treated = 750 mg/kg, i n t r a p e r i t o n e a l l y , at 10 A.M. and 4 P.M. on day 10. ** control = no treatment.  CHAPTER V ANALYSIS OF THE GENETIC CONTROL OF EMBRYONIC SENSITIVITY TO THE TERATOGENIC ACTION OF ACETAZOLAMIDE Embryonic resistance to the teratogenic action of acetazolamide i s a dominant t r a i t .  A backcross breeding  program was used to recover genes f o r s e n s i t i v i t y .  The  breeding system and some of the approaches to the analyses of the data were modeled on the i n v e s t i g a t i o n of the genetic v a r i a t i o n i n mice to the teratogenicity of 5 - f l u o r o u r a c i l by Dagg, Schlager and Doerr (1966). The data c o l l e c t e d from crosses with CBA/J and SWV  in  the preceedlng sections of t h i s thesis were not included i n the following a n a l y s i s . Materials and Methods 1.  Breeding program. The backcross program i s i l l u s t r a t e d i n Figure 4.  crosses were made to the CBA/J dam  All  to keep the maternal  e f f e c t ( i f any) constant i n each generation. F^ and BC^ males were c o l l e c t e d . 5 SWV  Then 5 CBA/J males,  males, 6 F^ males and 12 BC^ males were each mated to  several CBA/J females.  The CBA/J dams were subsequently  treated with acetazolamide at the CBA/J limb sensitive time 29  30-  CBA/J male  —CZZ  SWV  CBA/J female  male  F^ male  7 BCj_ male  FIGURE 4:  Mating scheme to I n v e s t i g a t e the g e n e t i c s s e n s i t i v i t y to the t e r a t o g e n i c a c t i o n of acetazolamlde.  of the  and the fetuses from terminated pregnancies were scored f o r the presence or absence of induced ectrodactyly.  Since the  contribution of the CBA/J dam i n each mating was constant, the f e t a l scores were used to g e n e t i c a l l y define t h e i r respective s i r e s . 2.  Administration of acetazolamide and f e t a l examination. Acetazolamide was administered i n t r a p e r i t o n e a l l y to  the pregnant CBA/J dams on day 10 at 10 A.M. and 4 P.M. at 750 mg/kg per i n j e c t i o n .  Pregnancies were terminated on  day 18 and uterine contents were examined as described previously. 3.  Genetic analysis of breeding data. For each s i r e , a mean frequency of ectrodactyly was  calculated from the t o t a l number of i t s l i v e progeny. The mean frequency of ectrodactyly f o r the s i r e s of each cross and backcross generation was calculated by averaging the mean frequencies of the s i r e s of each generation. In a d d i t i o n , the ectrodactyly frequency data were transformed f o r a s t a t i s t i c a l evaluation of the data.  With  binomial data, i n this case the presence or absence of ectrodactyly, the means and variances are related and not independent.  Following the approach of Dagg eit a l . (1966),  an arcsine transformation of the frequency data was used. In the present study, the Freeman-Tukey arcsine transformation f o r binomial d i s t r i b u t i o n s and small samples  32 (n<50)  was used.  Tables f o r this  p r e s e n t e d by M o s t e l l e r and Youtz used i n c o n j u n c t i o n  transformation  (1961).  w i t h the a r c s i n e  (see Snedecor and Cochran,  were  These t a b l e s can be  t a b l e s d e r i v e d by B l i s s  19^7, pp. 569-571) f o r l a r g e  samples (n > 5 0 ) . F o r each l i t t e r  from each s i r e , an a r c s i n e v a l u e (x^)  was found from the above-mentioned t a b l e s of the FreemanTukey a r c s i n e  transformation.  T h i s was based on the number  of l i v e f e t u s e s w i t h e c t r o d a c t y l y and the t o t a l number of l i v e fetuses i n that l i t t e r . number of l i v e f e t u s e s  T h i s was then weighted by the  ( k ^ i n that l i t t e r .  F o r each s i r e ,  a mean weighted a r c s i n e v a l u e was c a l c u l a t e d from the sum of the weighted a r c s i n e d i v i d e d by the t o t a l number of l i v e f e t u s e s from t h a t s i r e .  That i s  mean weighted a r c s i n e = ^ ( k ^ x ^ )  where k^ i s the number of f e t u s e s i n the _ith l i t t e r and x^ i s the Freeman-Tukey a r c s i n e of the r a t i o  of the number of  f e t u s e s w i t h e c t r o d a c t y l y t o the t o t a l number of f e t u s e s i n that  litter. Means and v a r i a n c e s  of each s i r e g e n e r a t i o n  were  c a l c u l a t e d from the average of the mean weighted a r c s i n e s of the  s i r e s of t h a t g e n e r a t i o n .  No f u r t h e r w e i g h t i n g of the  d a t a was made. The number of g e n e t i c f a c t o r s i n v o l v e d sensitivity  i n the t r a i t of  t o the t e r a t o g e n i c a c t i o n of a c e t a z o l a m i d e was  c a l c u l a t e d from the formula d e r i v e d by Wright  (1934).  The  3 3  formula employs the means and strain  sires,  the F^  variances  s i r e s and  the BC^  of the sires.  two The  Freeman-Tukey a r c s i n e transformed data f o r these were used.  Wright's  Rf  16 (V C  , "  B  generations  formula i s  ( 1 9 3 4 )  n =  inbred  V  E)  where n = number of l o c i  (or segregation  index),  R = phenotypic range of the two p a r e n t a l s t r a i n s (In t h i s case, i t i s the d i f f e r e n c e between the means of the CBA/J and SWV s i r e s . ) , V ^ B C  and V An  = variance  of the BC  = an estimate variance.  a c e t a z o l a m i d e was  sires,  of the non-genetic or environment  independent estimate  i n v o l v e d i n the t r a i t  1  of the number of g e n e t i c f a c t o r s  of t e r a t o g e n i c s e n s i t i v i t y  made u s i n g  the g e n e r a t i o n  to  means of  the  untransformed e c t r o d a c t y l y frequency d a t a .  The  assumptions  made i n t h i s a n a l y s i s are d i s c u s s e d below.  The  f i n d i n g s of  the two  approaches to t h i s a n a l y s i s of the number of  f a c t o r s are  compared. R e s u l t s and  1.  Blometrlcal estimation  Discussion  of the number of l o c i  with acetazolamlde teratogenic The BC^ 8  involved  sensitivity.  r e l e v a n t progeny d a t a f o r the CBA/J, SWV,  s i r e s mated to CBA/J females are l i s t e d and  genetic  9 respectively.  embryo i m p l a n t a t i o n s ,  i n Tables  and 6,  7,  These t a b l e s i n c l u d e the number of  the number of r e s o r p t i o n s , the  percent  34 TABLE 6 CBA/J Progeny Data from CBA/J Males Mated to CBA/J  CBA/J male No.  Resorptions/ implants No.  %  Females  Ectrodactyly/ l i v e fetuses No.  %  mean weighted arcsine  1.  713  8/45  17.8  17/37  45-9  41.99  2.  714  16/46  34.8  15/30  50.0  45.31  3.  715  12/48  25.0  18/36  50.0  43.46  4.  725  1/44  2.3  23/43  54.5  46.86  5.  726  20/46  43.5  14/26  53.8  47.16  50.6  44.96  Means  24.7  Summary: 1. Mean p e r c e n t r e s o r p t i o n  24.7  2. Mean (Xa/) percent e c t r o d a c t y l y  50.6  3. Mean  44.96  (X)  4. Variance  of mean weighted a r c s i n e s (V) of mean weighted a r c s i n e s  4.91  35 TABLE 7 F  1  Progeny Data from SWV Males Mated to CBA/J  male No.  Implants No.  %  No.  Females  live  fetuses  %  mean weighted arcsine  1.  1393  14/56  25.0  0/42  0  10o23  2.  1446  3/45  6.7  0/42  0  9.48  3.  1463  6/62  9.7  2/56  3.6  12.48  4.  1582  9/52  17.3  1/43  2.3  12.64  5.  1825  10/51  19.6  0/41  0  10.88  Means  15.7  1.2  11.14  Summary: 1.  Mean p e r c e n t  2.  Mean (X^) percent  3.  Mean (X) of mean weighted a r c s i n e s  4. Variance  resorption ectrodactyly  (V) of mean weighted a r c s i n e s  15.7 1.2 11.14 1.92  3 6  T A B L E  BG  Progeny Data from F  male No.  1  8  Males Mated to CBA/J  Implants No.  %  No.  6/50  Females  live  fetuses  %  mean weighted arcsine  1 2 . 0  19.02  1 / 4 5  2 . 2  1 1 . 8 6  25.0  0 / 3 6  0  1 0 . 8 3  8 / 4 0  2 0 . 0  3/32  9 . 4  1 8 . 5 9  25  8 / 6 8  1 1 . 8  3/60  5 . 0  1 4 . 5 7  26  7/60  1 1 . 7  5 / 5 3  9 . 4  1 8 . 1 7  1 .  1 3  7 / 5 7  1 2 . 3  2 .  2 2  2 / 4 7  4 . 3  3 .  23  4 .  2 4  5 .  6 .  1 2 / 4 8  1 4 . 2  Means  6 . 3  1 5 . 5 1  Summary: 1.  Mean percent  2.  Mean ( X ^ )  3.  Mean ( X ) of mean weighted a r c s i n e s  4.  Variance  resorption,.  percent  ectrodactyly  (V) of mean weighted a r c s i n e s  1 4 . 2 6 . 3 15.51 1 3 . 0 0  37 TABLE 9  BC0 Progeny Data from BC, Males Mated to CBA/J Females  BCj, male No.  Ectrodactyly/ l i v e fetuses  Resorptions/ implants No.  mean weighted arcsine  No.  1.  35  13/45  28.9  11/32  34.4  34.91  2.  36  13/49  26.5  6/36  16.7  24.24  3.  37  23/54  42.6  10/31  32.3  33.97  4.  42  11/50  22.0  5/39  12.8  22.98  5.  48  16/68  23.5  15/52  28.8  32.48  6.  49  16/66  24.2  18/50  36.0  36.98  7.  50  6/52  11.5  6/46  13.0  21.62  8.  51  8/47  17.0  9/39  23.1  29.70  9.  52  13/56  23.1  7/43  16.3  25.47  10.  53  9/70  12.9  13/61  21.3  28.35  11.  68  6/48  12.5  9/42  21.4  27.66  12.  69  5/52  9-6  4/47  8.5  18.11  22.1  28.04  Means  21.2  Summary: 1. Mean  , 21.2  2. Mean  , 22.1  3. Mean (X*) of mean weighted a r c s i n e s .  ,  28.04  4. Variance (V) of  .  33.77  38 resorption, the number of l i v e progeny, the number of progeny with ectrodactyly, the percent ectrodactyly score f o r l i v e fetuses and mean weighted Freeman-Tukey arcsine transformed ectrodactyly score f o r each s i r e i n each generation.  Also  included are the mean frequency or mean percentage of resorption, the mean frequency of ectrodactyly and the mean and variance of the mean weighted Freeman-Tukey arcsine scores f o r each generation of s i r e s . A discussion of the resorption response to acetazolamide i n this backcross program i s deferred to Chapter V I . The frequency d i s t r i b u t i o n of ectrodactyly i n the progeny of the CBA/J, SWV, F^ and BC^ s i r e s i s i l l u s t r a t e d i n a  Figure 5* I f  single gene determined the embryonic  response to acetazolamide, a blmodal d i s t r i b u t i o n would be expected i n the ectrodactyly scores of the BC^ s i r e s .  Some  would behave l i k e the F^ s i r e s and the r e s t would behave l i k e the CBA/J inbred s t r a i n s i r e s .  This was not the case and a  single gene hypothesis was r e j e c t e d . Wright's (1934) formula f o r the estimation of the number of segregating genetic factors or l o c i involved i n a t r a i t i s predicated on the assumption that the genes have equal e f f e c t and that they are independently segregating, that i s they are not l i n k e d . Further discussions of the formula and i t s derivations can be found i n Wright (1934, 1968) and Falconer (I960, pp. 217-219). The c a l c u l a t i o n s f o r two estimates  of the number of l o c i  Involved i n the t r a i t of teratogenic s e n s i t i v i t y to  60  0®  50H  ©0 ©  E o 40H  © © ©  < CO  U  ^ 30-  © © ©©  u D "D  £ 20" o  OJ  ©©  c  ©©  CJ  \J 1_  © ©©  CO  °- 10-  ©  ©  © ©  ©  —<  ©  CBA/J  BC]  ©®© l  SWV  Fl  Sires  FIGURE 5:  Means and d i s t r i b u t i o n s of the percent e c t r o d a c t y l y scores of the CBA/J, SWV, F sires.  1 1  and BC  4 0  acetazolamide,  1  using Wright s (1934) formula, are outlined i n  Table 10. One estimate was n = 3.44.  This was made using the  variance of the Fji s i r e s as an estimate of the environmental variance.  This approach was suggested by Wright (1934).  Excluding sex-linked t r a i t s , the T?^ sires are g e n e t i c a l l y heterogeneous but they are a l s o g e n e t i c a l l y homogeneous. Therefore, any v a r i a t i o n they e x h i b i t w i l l be due to environment or chance. Another estimate was n = 2.63.  This was made using an  average of the CBA/J, SWV and F^ s i r e variances as an estimate of the environmental variance.  This method was  used by Dagg e_t a l . (1966) and i s a l s o v a l i d since these three groups of s i r e s , although they are g e n e t i c a l l y d i f f e r e n t , are each g e n e t i c a l l y homogeneous. These two blometrlcal approaches suggested that there are three l o c i (2.63 -- 3.44) which control embryonic s e n s i t i v i t y to the teratogenic action of acetazolamide. 2. Estimation of the number of l o c i using the generation mean frequencies of ectrodactyly. Since the number of l o c i c o n t r o l l i n g ^ t h e t r a i t of teratogenic s e n s i t i v i t y was estimated to be r e l a t i v e l y small, another method of d e r i v i n g this estimate was t r i e d .  I t made  use of the generation mean frequencies of ectrodactyly as the genetic t r a i t and, n a i v e l y , i t can be referred to as gene counting.  41 TABLE  10  Means and V a r i a n c e s of CBA/J, SWV,  F^, and BC^ S i r e  Scores  U s i n g the Weighted A r c s i n e T r a n s f o r m a t i o n and the Calculation  of the Number of L o c i  Controlling  the Response t o Acetazolamlde  Sire  Mean  Variance  CBA/J  44.96  4.91  SWV  11.14  1.92  F  15.51  13.00  t  BC,  28.04  33.77  E s t i m a t i o n of number of l o c i u s i n g Wright's  (1934)  formula: 1.  n = 3.44, u s i n g the F^ s i r e v a r i a n c e t o estimate Vg.  2.  n = 2.63, u s i n g the average  of the CBA/J,  SWV,  and F. s i r e v a r i a n c e s t o estimate V„ l £J  42 Teratogenic s e n s i t i v i t y to acetazolamide i s recessive to r e s i s t a n c e .  On the basis of one locus, the p r o b a b i l i t i e s  that the progeny from the SWV,  F^ and BC^  s i r e s are  g e n e t i c a l l y l i k e the CBA/J inbred s t r a i n are 0, 0.50  and  0.75. r e s p e c t i v e l y . I f several independently segregating l o c i are involved and the CBA/J and SWV  a l l e l e s at each  locus are recessive and dominant, r e s p e c t i v e l y , these 11  p r o b a b i l i t i e s (p^) become ( p ^ , where n i s the number of loci.  Therefore, the product of the mean frequency of  ectrodactyly found i n the CBA/J inbred s t r a i n (50.6#, Table 6) and the p r o b a b i l i t y that a generation i s l i k e the CBA/J inbred s t r a i n becomes the expected generation mean frequency of ectrodactyly. The p r o b a b i l i t i e s that a generation i s l i k e the CBA/J Inbred s t r a i n and the respective expected generation mean frequencies of ectrodactyly from these c a l c u l a t i o n s f o r 1, 2, 3 and 4 l o c i are l i s t e d i n Table 11. A t h i r d backcross generation using BCg  s i r e s i s a l s o included since t h i s  information w i l l be important i n Chapter V I . Figure 6 i s a graphical comparison of the observed generation mean frequencies of ectrodactyly and  the  p r o b a b i l i t y that that generation i s l i k e the CBA/J s t r a i n for  1, 2, 3 and 4 l o c i .  straight l i n e .  The data f o r three l o c i f i t a  The expected generation means from the  three-locus model (Table 11) f o r the F^ s i r e s (BC^ progeny) and the BC 1  s i r e s (BC 2 progeny) agreed with those found i n  Tables 8 and 9»  This estimate  of three l o c i was  i n complete  TABLE 11 P r o b a b i l i t y that a Fetus i s Genotypically l i k e CBA/J i n the Backcross Study Considering 1, 2, 3 and 4 Independently Segregating L o c i  Fetus  F  l  1-locus model  Sire  2-locus model  0  F  0.50  (25.3)*  0.25  0.75  (37.8)  0.563 (28.5)  0.422 (21.4)  0.316 (16.0)  0.875 (44„3)  0.766 (38.8)  0.670 (33.9)  0.586 (29.7)  BC 2 BC^  BC 2  0  4-locus model  SWV l BC 1  0  3-locus model  (12.7)  0.125  0 (6.3)  0.063  (3.2)  * Numbers i n parentheses are the expected percentages of ectrodactyly f o r each f e t a l generation f o r the four genetic models and are based on 50.6$ ectrodactyly found i n the inbred (homozygous) CBA/J s t r a i n . The models assume that resistance i s dominant to sensitivity.  60 1- locus 2 - locus  T 1.0  1  Probability  FIGURE 6:  1  0.8  0.6 that  fetal  1  1  0.4 generation  r 0  0.2 is  like  CBA/j  Comparison of the observed mean percent e c t r o d a c t y l y of the f e t a l g e n e r a t i o n s w i t h the p r o b a b i l i t y that a generation i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n f o r 1, 2 , 3 and 4 l o c i .  45  agreement w i t h t h a t made w i t h the b i o m e t r i c a l approach. This three-locus  model a l s o p r e d i c t s t h a t the mean  frequency of e c t r o d a c t y l y from B C  2  s i r e s b a c k c r o s s e d t o CBA/J  females ( t h a t i s BC^ progeny) w i l l be 33.9%  (Table  11).  This  w i l l be t e s t e d i n Chapter V I . I f three  independently segregating  loci  c o n t r o l the  t r a i t of embryonic s e n s i t i v i t y to the t e r a t o g e n i c a c t i o n of a c e t a z o l a m i d e , i t can be p r e d i c t e d  that 1/8  should behave l i k e the CBA/J s i r e s . (see F i g u r e  and be  T h i s was n o t found  5 ) . however, only 12 BC^ s i r e s were sampled and  t h i s absence may be due simply distribution  of the BC^ s i r e s  of BC^ s i r e scores  to sampling e r r o r .  The  covered t h a t of the F^ s i r e s  extended beyond toward t h a t of the CBA/J s i r e s as would expected w i t h t h i s g e n e t i c model.  The r e c o v e r y  of a BC^  s i r e w i t h a CBA/J s i r e b r e e d i n g response w i l l be one way to t e s t t h i s model i n f u t u r e  studies.  Conclusion Two d i f f e r e n t  approaches to the g e n e t i c a n a l y s i s of  the b r e e d i n g data suggested t h a t three  independently  segregating  loci  i n the homozygous r e c e s s i v e c o n d i t i o n a r e  responsible  f o r the t r a i t of t e r a t o g e n i c  s e n s i t i v i t y to  acetazolamide of the CBA/J embryo when compared w i t h the SWV s t r a i n .  One method of a n a l y s i s p r e d i c t e d  backcross generation, design,  not included  t h a t the t h i r d  i n the e x p e r i m e n t a l  would have a mean frequency of e c t r o d a c t y l y of 33. 9%.  CHAPTER VI TEST OF THE THREE-LOCUS MODEL Two approaches to the genetic analysis of the breeding program with CBA/J and SW  suggested a three-locus model f o r  the inheritance of embryonic s e n s i t i v i t y to the teratogenic action of acetazolamide with resistance being a dominant trait.  From t h i s hypothesis, the generation mean of the  t h i r d backcross generation, that i s the mean of the BC^ s i r e ectrodactyly scores, was predicted to be  33*9%»  This  prediction was tested i n a breeding program. Materials and Methods 1.  Breeding program and teratogenic t e s t i n g . Thirteen BC^ males were selected at random from  backcrosses of 5 BC^ s i r e s to CBA/J females.  These BCg males  were each mated to several d i f f e r e n t CBA/J females.  The  CBA/J dams were injected i n t r a p e r l t o n e a l l y with acetazolamide at 750 mg/kg per i n j e c t i o n a t 10 A.M. and 4 P.M. on day 10 of pregnancy.  The pregnancies were terminated on day 18 and the  uterine contents examined. Results and Discussion The ectrodactyly scores f o r the 13 BC? s i r e s are l i s t e d  46  47 i n Table 12.  The observed mean frequency of ectrodactyly i n  BC^ progeny from these BC 2 s i r e s was  33,1%.  This agreed very  favorably with the predicted mean of 33.9$ (Chapter V) derived from the three-locus model. The d i s t r i b u t i o n of the BC 2 s i r e s ' percent ectrodactyly scores i s plotted i n Figure 7, and that of the mean weighted Freeman-Tukey arcsine scores i s plotted i n Figure 8, against the p r o b a b i l i t y  that the f e t a l generation  (BC^) i s l i k e the  CBA/J inbred s t r a i n from the three-locus model. distributions  f o r the CBA/J, SWV,  The relevant  and BC^ s i r e s are  Included f o r comparison. From the arcsine scores of the CBA/J s i r e s , the range of the CBA/J phenotype can be defined as the mean arcsine of the CBA/J s i r e s plus and minus two standard d e v i a t i o n s . 44.96 + 4.43 (or 40.53 — 49.39).  This was  Two BC 2 s i r e s f e l l within  t h i s range (BC 2 males # 91 and # 102). CBA/J s t r a i n phenotype was recovered.  Therefore, the The f a c t that the  arcsine score f o r BC 2 s i r e # 91 (^9.97) Is s l i g h t l y greater than the upper l i m i t of the a r b i t r a r i l y defined range of the CBA/J s i r e s (49.39) cannot be considered meaningful u n t i l further breeding studies have investigated this The  observation.  t h e o r e t i c a l l y expected and observed generation means  f o r the SWV, F^, BC^ and BC 2 s i r e s are summarized i n Table 13*  The calculations have been made with the three-  locus model and the mean frequency of 50.6$ ectrodactyly from the CBA/J s i r e s .  The t h e o r e t i c a l l y expected arcsine scores  i n t h i s table were taken from the arcsine tables of B l i s s  48 TABLE 12 BCo  Progeny Data from B C ? Males Mated  3 male No. B G  Resorptions/ implants  t o C B A / J Females  Ectrodactyly/ l i v e fetuses  No.  %  No.  mean weighted arcsine  %  1.  89  12/39  30.8  3/27  llol  19.49  2.  90  16/52  30.8  14/36  38.9  38.46  3.  91*  8/57  14.0  29/49  59.2  49.97  4.  92  10/54  18.5  11/44  25.0  28.67  5.  93  19/52  36.5  10/33  30.3  34.65  6.  94  8/49  16.3  14/41  34.1  36.69  7.  95  14/55  25.5  15/41  36.6  35.99  8.  96  22/54  40.7  11/32  34.4  36.04  9.  97  9/47  19.1  9/38  23.7  30.20  10.  98  11/53  20.8  15/42  35.7  34.46  11.  99  7/52  13.5  13/45  28.9  33.32  12.  101  7/51  13.7  8/44  19.8  25.97  13.  102*  10/52  19.2  23/42  54.8  48.95  33.1  34.84  Means  23.0  Summary: 1. Mean  ,.  23.0  2. Mean  ..  33.1  3.  Mean (X) of mean weighted a r c s i n e s .  * E c t r o d a c t y l y scores the C B A / J males.  ..  34.84  of two BG£ males a r e i n the range o f  49  60-  0 0  © 50-  0 ©  © 40-  0  E o  <  u  1 0  ©© ©©  © 0 ©  © ©  30-  0  © ©  © ©O  ©  20D ~D  ®0  O  k.  U CD  c  <D  ©0  ©  10"  © ©©  ©  U k.  <D  ©  Q_  oFetus: Sire:  0 ©  © ©  T  1.0 CBA/J CBA/J Probability  FIGURE 7:  0.670 BC BC 3  that  fetal  T  0.422 BC BC]  0.125 BCi 1  2  2  generation  0©©  F  is  like  I  0  h  SWV  CBA/j  Means and percent e c t r o d a c t y l y scores of the CBA/J, SWV, Fj_, BGj_ and B G s i r e s p l o t t e d a g a i n s t the p r o b a b i l i t y that, the f e t a l g e n e r a t i o n i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n based on the 3-locus model. 2  50  60-  0  * 50E  ©  D  © ©  < CQ  u 40-  0  © © © © © © © © © © ©  0§ 00©  © © ©  £ 30o "O O  o 20-  ©  ®8  0  ©  a> OJ c  00 0 0 0  © ©  10-  "55  1  0.670  1.0 Fetus: Sire:  BC3 BC 2  CBA/J CBA/J Probability  FIGURE 8:  that  fetal  1 0.422  0.125  BC 2 BC]  BCi  generation  SWV  is  like  CBA/j  Means and d i s t r i b u t i o n s of the mean weighted a r c s i n e scores of the CBA/J, SWV, Fj_, BC;, and BC£ s i r e s p l o t t e d a g a i n s t the p r o b a b i l i t y that the f e t a l g e n e r a t i o n i s g e n o t y p i c a l l y l i k e the CBA/J s t r a i n based on the 3 - l o c u s model.  TABLE 13 Theoretical and Observed Generation Means f o r Ectrodactyly Based on Three-Locus Model i n Backcross Study with CBA/J Dams  Sire  Progeny  Percent ectrodactyly Expected*  Observed  Arcsine Expected** Observed***  1. SWV  F1  0  1.2  2. F x  BC 1  6.3  6.3  3 . BC^  BC 2  21.4  22.1  27.56  4. BC 2  BC^  33.9  33.1  35.61  ,  50.6 .  5. CBA/J CBA/J  11.14 14.54  15.51 28.04 3^.84 44.96  * Theoretical percent ectrodactyly based on observed 50.6$ observed i n the CBA/J inbred s t r a i n . ** Theoretical arcsine from arcsine tables f o r large samples (n>50) by B l i s s (Snedecor and Cochran, 1967. PP. 569-571) corresponding to the t h e o r e t i c a l percent ectrodactyly. *** Observed arcsine i s generation mean of the mean weighted Freeman-Tukey arcsine f o r small samples ( n < 5 0 ) .  52 (see Snedecor and Cochran, 1967. pp. 5&9-571)  f o r  large  sample sizes corresponding to the expected mean percent ectrodactyly f o r that generation.  The. corresponding  observed  arcsine scores are the generation means of the mean weighted Freeman-Tukey a r c s i n e s . There was such close agreement of the observed generation means with the t h e o r e t i c a l means that s t a t i s t i c a l comparisons were not considered necessary.  Therefore, i t was assumed that  the three-locus model i s adequate to define the teratogenic s e n s i t i v i t y to acetazolamide  of the CBA/J s t r a i n when  compared with the r e s i s t a n t SWV s t r a i n . A discussion of the resorption response a f t e r acetazolamide  treatment has been l e f t to t h i s point because  i t i s best discussed i n the context of the backcross breeding program.  Kalter (1965) stated that " I t has often  been assumed i n t e r a t o l o g i c a l studies that f e t a l death i s the extreme a c t i o n of teratogens but not q u a l i t a t i v e l y a d i f f e r e n t expression of that action."  From his studies of response  differences i n mouse strains to g a l a c t o f l a v l n treatment, he found no c o r r e l a t i o n between the frequency of f e t a l death and the frequency of malformed fetuses and concluded  that  galactoflavin-induced f e t a l death and malformations were unrelated e f f e c t s .  Francis (1971) made an extensive  examination of the v a r i a t i o n s i n c l e f t palate induction and f e t a l death due to cortisone with four d i f f e r e n t inbred strains i n a d i a l l e l breeding design. conclude  Her data l e d her to  that f e t a l death was not an extreme form of the  53 t e r a t o g e n i c response but t h a t they are two separate responses to  cortisone. In  the i n i t i a l  of  CBA/J and SWV  at  10 A.M.  examination of the t e r a t o g e n i c  to a c e t a z o l a m l d e a d m i n i s t e r e d a t 750  and 4 P.M.  Chapter I I I ) ,  on day 10 of pregnancy  CBA/J e x h i b i t e d 27$  e c t r o d a c t y l y w h i l e SWV ectrodactyly.  for  (Table 2,  r e s o r p t i o n and  There was  47$  d e f i n i t e acetazolamide-induced SWV.  the more c r i t i c a l examination of the CBA/J  the b a c k c r o s s program (Table 6,  comparable  to the CBA/J response i n the  examination (Table 2 ) .  response  Chapter V ) , the mean  p e r c e n t r e s o r p t i o n f o r the 5 CBA/J s i r e groups was i t was  mg/kg  e x h i b i t e d 9$ r e s o r p t i o n w i t h no  f e t a l death i n CBA/J but none i n In  responses  24.7$ and  initial  However, when the i n d i v i d u a l  groups were examined, there was  sire  c o n s i d e r a b l e v a r i a t i o n i n the  r e s o r p t i o n responses which ranged from 2.3$  to 43.5$.  The  e c t r o d a c t y l y responses, on the o t h e r hand, f o r these CBA/J s i r e groups were t i g h t l y c l u s t e r e d around e c t r o d a c t y l y response.  T h i s was  the 50.6$ mean  one i n d i c a t i o n t h a t  fetal  death and e c t r o d a c t y l y a f t e r a c e t a z o l a m l d e treatment were not related. There appeared  to be a l o g i c a l p r o g r e s s i o n of the mean  r e s o r p t i o n f r e q u e n c i e s when t h i s parameter was p r o g e n i e s of the SWV Chapter V ) , B ^  (Table 7,  (Table 9,  Chapter VI) s i r e s .  I t was  (Table 8,  Chapter V ) ,  Chapter V) and B C 15.7$  examined i n the  2  f o r the SWV  (Table  12,  s i r e s and  i n c r e a s e d s l o w l y to 24.7$ f o r the CBA/J s i r e s d i s c u s s e d  54 above; however, the mean of the  s i r e s did not agree since  i t was 14.2$. An examination of the resorption frequencies of the progenies from the CBA/J, SWV, F^ and BC^ s i r e s was made using the Freeman-Tukey arcsine transformation. The means and variances from the transformed data were employed i n Wright's (1934) formula to see whether some meaningful estimate of the number of genetic factors involved i n t h i s resorption frequency difference could be made. The analysis was i d e n t i c a l to that used f o r the investigation of ectrodactyly.  An estimate of 0.29 l o c i resulted when the  variance of the F^ s i r e s was used to estimate the non-genetic variance.  No further manipulation of these data was made  since the variance of the resorption frequency found with the CBA/J s i r e s , that i s the CBA/J inbred s t r a i n , was so great r e l a t i v e to the other s i r e generations.  These data and  calculations were not Included i n this thesis since they were not germane to the i n v e s t i g a t i o n of the ectrodactyly problem; however, they are available from the author. In turn, the resorption frequency and ectrodactyly frequency f o r each s i r e group In each backcross generation were examined f o r a l o g i c a l a s s o c i a t i o n .  There was none.  In the BC 2 s i r e s (Table 12), the BG^ progenies from two BG 2 s i r e s (males # 91 and # 102) that behaved l i k e the CBA/J s t r a i n with respect to ectrodactyly response, had low resorption frequencies (14.0$ and 19.2$, respectively) r e l a t i v e to the other progeny groups.  Therefore, there  seemed to be no a s s o c i a t i o n of r e s o r p t i o n and e c t r o d a c t y l y responses to acetazolamlde treatment.  Since no a s s o c i a t i o n s  were found, i t was concluded t h a t they were two  independent  responses to a c e t a z o l a m l d e . Conclusion The p r e d i c t i o n from the t h r e e - l o c u s model of the i n d u c e d - e c t r o d a c t y l y frequency of t h i r d b a c k c r o s s g e n e r a t i o n was  tested.  The b r e e d i n g r e s u l t was i n c l o s e agreement w i t h  the p r e d i c t i o n .  I t was  concluded t h a t the t h r e e - l o c u s model,  w i t h s e n s i t i v i t y b e i n g a r e c e s s i v e t r a i t , would d e f i n e the sensitivity  of the CBA/J s t r a i n  to a c e t a z o l a m i d e - i n d u c e d  e c t r o d a c t y l y when compared w i t h the r e s i s t a n c e of the SWV strain. The a c e t a z o l a m i d e - i n d u c e d e c t r o d a c t y l y and f e t a l death appeared  to be independent  responses.  CHAPTER V I I EMBRYO TRANSFER EXPERIMENTS TO INVESTIGATE THE ACETAZOLAMIDE RESPONSE OF THE CBA/J AND SWV STRAINS There  i s c o m p e l l i n g evidence  acts d i r e c t l y  on the embryo.  t h a t suggests  Studies with  a p p l i c a t i o n of acetazolamide  i n rats  Intrauterine  ( S c o t t , 1970) and  hamsters (Layton, 1972, p e r s o n a l communication) t h a t more e c t r o d a c t y l y was produced  indicated  when the t e r a t o g e n was  d e p o s i t e d a t s i t e s c l o s e r t o the embryo.  The e x t e n s i v e  p h a r m a c o l o g i c a l s t u d i e s of Maren and E l l i s o n Ellison,  acetazolamide  (Maren and  1972a, 1972b, 1972c; E l l i s o n and Maren, 1972a,  1972b) w i t h r a t s d e f i n e d two p h y s i o l o g i c a l a c t i o n s t h a t were r e q u i r e d , a t l e a s t i n the dam, t o Induce e c t r o d a c t y l y i n the embryo:  c a r b o n i c anhydrase  imbalance.  i n h i b i t i o n and a potassium  They a l s o presented evidence  took p l a c e i n the embryo. to demonstrate  that these a c t i o n s  Suzuki and Takano (19&9) were a b l e  t h a t the c a r b o n i c anhydrase  enzyme was p r e s e n t  and a c t i v e i n the limb bud of the r a t embryo a t the a c e t a z o l a m l d e - s e n s l t i v e stage and t h a t i t c o u l d be i n h i b i t e d by acetazolamide a d m i n i s t e r e d t o the dam. In t h i s study, no malformed SWV o r SWV.CBA F^ embryos were found i n t r e a t e d SWV dams.  I f the s e n s i t i v e CBA/J  embryos, t r a n s f e r r e d t o SWV surrogate dams, responded e c t r o d a c t y l y t o acetazolamide  treatment,  56  t h i s would  with  57 demonstrate (1)  that:  the t e r a t o g e n i c environment  f o r the I n d u c t i o n of  e c t r o d a c t y l y does e x i s t i n SWV (2)  the r e s i s t a n c e of the SWV i s a r e s i s t a n c e of the SWV  dams and  s t r a i n to acetazolamide embryo p r o p e r .  M a t e r i a l s and Methods 1.  Embryo  transfer  procedure.  The embryo t r a n s f e r procedure was an  asynchronous  t r a n s f e r of day 3 embryos ( a t the "blastocyst stage) to day 2 pseudopregnant  r e c i p i e n t dams.  The techniques were  e s t a b l i s h e d i n t h i s l a b o r a t o r y by A l a n C. Peterson f o r use i n the p r o d u c t i o n of a r t i f i c i a l mosaic  or c h i m a e r l c mice  u s i n g the p r o t o c o l s d e s c r i b e d by Mullen and Whltten  (1972).  The embryo t r a n s f e r procedure f o r use i n e x p e r i m e n t a l t e r a t o l o g y was more f u l l y d e s c r i b e d by Takano, P e t e r s o n , B i d d l e and M i l l e r (1972, manuscript i n p r e p a r a t i o n ) . 2.  Developmental  s t a g i n g of t r a n s f e r r e d  Normal CBA/J and SWV  embryos.  pregnancies and t r a n s f e r  pregnancies of CBA/J embryos i n SWV  dams and SWV  embryos i n  CBA/J dams were terminated on day 10 between 12 noon and 3 P.M.  The developmental age of the embryos was  by somite  number.  determined  58  3.  Administration of acetazolamide to dams a f t e r embryo transfer* Acetazolamide was administered i n t r a p e r i t o n e a l l y to the  r e c i p i e n t dams a t 750 vug/kg per i n j e c t i o n a t 10 A.M. and 4 P.M. on day 10 of t h e i r pregnancy.  Day 0 i n this case was  the i n i t i a t i o n of t h e i r pseudopregnancy by mating with a vasectomized male i n preparation f o r embryo transfer on day 2 .  These pregnancies were terminated on day 18 and the  embryos were examined as described previously. Results and Discussion The asynchronous  transfer of day 3 blastocysts to day 2  pseudopregnant dams i n the transfer procedure necessitated the examination of embryonic development l a t e r i n the transfer pregnancy to determine the time at which to administer the acetazolamide.  I t was assumed that a morphological r a t i n g ,  based on the somite number, of the transferred embryos that was comparable with those of embryos from normal withins t r a l n matlngs would imply that, a t this chronological point i n pregnancy, the transferred embryos would have the same teratogenic s e n s i t i v i t y as those i n the normal w i t h i n - s t r a i n matings. The means and ranges of the somite number of CBA/J and SWV embryos from normal w l t h i n - s t r a i n matings are presented in Table 14. Their s i m i l a r i t y , a t l e a s t on the basis of this morphological c r i t e r i o n , was an important point to consider  59 TABLE  14  Development of Embryos i n Normal Matlngs and Embryo T r a n s f e r s Determined by Somite Number  Embryo  Somite number, day 10 of pregnancy  type  Mean + s.d.  1.  No. embryos  Range  Normal matings: a.  CBA/J  26.5 + 1.7  35  23  31  b.  SWV  27.4 + 3-3  84  21  34  Transferred  embryos:  a.  CBA/J i n SWV  25.2  5  17  31  b.  SWV  26.8  4  25  29  i n CBA/J  60  with respect to the response to acetazolamide of these two strains.  Their respective  s e n s i t i v i t y and resistance cannot  "by due to gross differences i n morphological development. The means and ranges of the somite number of CBA/J embryos transferred to SWV pseudopregnant dams and SWV embryos transferred to CBA/J pseudopregnant dams are a l s o l i s t e d i n Table 14. They were e s s e n t i a l l y i d e n t i c a l to those found i n the normal pregnancies a t t h i s chronological age. Therefore, the two types of transferred embryos, that were chronologically day 11 on day 10 of the r e c i p i e n t dams' pseudopregnancy, were morphologically s i m i l a r to day 10 embryos i n normal pregnancies.  Development had been retarded  by approximately one day between the time of asynchronous transfer of the day 3 blastocysts to day 2 pseudopregnant dams u n t i l t h e i r examination on day 10 of the r e c i p i e n t dams' pseudopregnancy.  I t was decided to administer the  acetazolamide to the r e c i p i e n t dams with transferred embryos at 10 A.M. and 4 P.M. on day 10 of t h e i r pseudopregnancy. Because of the l i m i t e d nature of t h i s study, the only classes of acetazolamide-treated and control transfers with the CBA/J and SWV s t r a i n s were those l i s t e d i n Table 1 5 . The SWV embryos transferred to CBA/J dams were.divided into two groups:  one treated with acetazolamide, the other without  treatment.  A l l CBA/J embryos transferred to SWV dams were  treated with acetazolamide.  In the two small treatment  samples, CBA/J embryos i n SWV dams responded with ectrodactyly while SWV embryos i n CBA/J dams were r e s i s t a n t  61 TABLE  15  Summary of Embryo T r a n s f e r s w i t h CBA/J and SWV and Treatment w i t h  lo  2  0  Pregnant p e r operated r e c i p i e n t dams.......... Implants p e r t r a n s f e r r e d embryos i n pregnant dams.....  3. A f f e c t e d l i t t e r s 4. R e s o r p t i o n s p e r implants  Acetazolamide  SWV embryos/ CBA/J dams  Observations  ....  5. E c t r o d a c t y l y per l i v e fetuses.  Strains  CBA/J embryos/ SWV dams  treated*  control**  treated  8/13  8/14  12/15  23/37  20/41  47/56 4/12  0/8  0/8  1/23  4/20  15/47  0/22  0/l6  6/32  * Treatment = 750 mg/kg per i n j e c t i o n a d m i n i s t e r e d l n t r a p e r l t o n e a l l y a t 10 A.M. and 4 P.M. on day 10 of r e c i p i e n t dams' pregnancy. ** C o n t r o l = no  treatment.  62 to the i n d u c t i o n of e c t r o d a c t y l y . The (1)  only v a l i d  the t e r a t o g e n i c  conclusions  p o s s i b l e from these d a t a a r e :  environment a f t e r acetazolamide  a d m i n i s t r a t i o n does e x i s t i n the SWV dams because the t r a n s f e r r e d CBA/J embryos responded w i t h e c t r o d a c t y l y , and (2)  the r e s i s t a n c e of the SWV s t r a i n to the a c e t a z o l a m i d e Induction  of e c t r o d a c t y l y i s a r e s i s t a n c e of the SWV  embryo because there were no a f f e c t e d SWV embryos i n t r e a t e d CBA/J dams. I t i s argued t h a t the o n l y comparisons t h a t should be made i n embryo t r a n s f e r experiments such as t h i s a r e w i t h those t h a t i n v o l v e embryo t r a n s f e r s .  The l i t t e r  size i n  t r a n s f e r pregnancies i s made s m a l l d e l i b e r a t e l y t o i n s u r e optimal  t r a n s f e r success a f t e r the e x t e n s i v e  McLaren and Mlchle  s t u d i e s of  (1956) w i t h t h i s t e c h n i q u e .  Therefore,  the proper c o n t r o l f o r the SWV embryos i n t r e a t e d CBA/J dams should  be CBA/J embryos t r a n s f e r r e d t o CBA/J dams and t h a t  f o r the CBA/J embryos t r a n s f e r r e d to SWV dams should embryos t r a n s f e r r e d to SWV dams.  be SWV  CHAPTER V I I I RESPONSE OF THE CBA/J AND SWV STRAINS TO DICHLORPHENAMIDE From a number of s t u d i e s , i t appears t h a t a l l potent inhibitors  of c a r b o n i c anhydrase cause the same t e r a t o g e n i c  l e s i o n as a c e t a z o l a m i d e .  Those t h a t have been t e s t e d and  proven t o be t e r a t o g e n i c a r e dichlorphenamide ( H a l l e s y and Layton, 1967), ethozzolamide (Wilson  e t a l . , 1968),  methazolamide (Maren, 1971), benzolamide (Maren and E l l i s o n ,  1972b) and 2 - a m i n o - l , 3 , 4 - t h l a d l a z o l e - 5 - s u l f o n a m i d e (CL 53^3) (Maren and E l l i s o n , 1972a). I t was d e c i d e d  to t e s t whether the r e s i s t a n c e of the  SWV s t r a i n t o a c e t a z o l a m l d e - i n d u c e d e c t r o d a c t y l y would remain with  other t e r a t o g e n i c c a r b o n i c anhydrase  inhibitors.  Dichlorphenamide was the o n l y one t e s t e d .  I f the SWV  maintained i t s r e s i s t a n c e w i t h  this  strain  ectrodactyly-inducing  agent, t h i s would i n d i c a t e t h a t SWV most l i k e l y possessed a g e n e r a l i z e d r e s i s t a n c e t o the t e r a t o g e n i c a c t i o n of t h i s c l a s s of drugs. SWV  s t r a i n with  Also, f u r t h e r biochemical  comparison of the  s e n s i t i v e s t r a i n s of mice would uncover the  n a t u r e of t h i s t e r a t o g e n i c r e s i s t a n c e and,' i n t u r n , out  point  the cause of the t e r a t o g e n i c s e n s i t i v i t y of other  strains.  63  Materials and Methods 1.  Preparation of dichlorphenamide solution f o r i n j e c t i o n . The following d i r e c t i o n s f o r solution of  dichlorphenamide were supplied by Dr. John E . Baer (Merck Sharp and Dohme).  To 300 mg of dichlorphenamide were added  1.0 ml of 1 N sodium hydroxide and the suspension was warmed u n t i l i t dissolved.  The f i n a l volume was adjusted to 6.0 ml  with d i s t i l l e d water r e s u l t i n g i n a f i n a l concentration of  5% dichlorphenamide. 2. Administration of dichlorphenamide and f e t a l examination. Dichlorphenamide was administered to pregnant dams a t 10 A.M. and k P.M. on day 10 by i n t r a p e r i t o n e a l i n j e c t i o n of 250 mg/kg per i n j e c t i o n .  With the 5$ s o l u t i o n , 0.005 ml/g  mouse was used. The pregnancies were terminated on day 18 and the uterine contents examined as described i n Chapter I I I . Results and Discussion Preliminary tests with dichlorphenamide, administered i n a single i n t r a p e r i t o n e a l i n j e c t i o n of 500 mg/kg a t 1 P.M. on day 10 f a i l e d .  This dose resulted i n death of both CBA/J  and SWV dams. When l t was reduced to 250 mg/kg, administered in the same way, dams of both strains were able to tolerate i t . The CBA/J and SWV dams were a l s o able to tolerate the 250 mg/kg dose of dichlorphenamide administered a t 10 A.M.  65 and 4 P.M.  on day 10, the same dosage schedule used i n most  of the acetazolamide s t u d i e s . are l i s t e d i n Table 16.  The r e s u l t s from this study  The CBA/J s t r a i n responded with  forelimb ectrodactyly while the SWV  s t r a i n did not.  The  resorption response was not elevated above the control (no treatment) l e v e l found i n Table 2 (Chapter I I I ) . Although the acetazolamide dosage regime of 250 mg/kg, administered at 10 A.M.  and 4 P.M.,  was not t r i e d with the  CBA/J s t r a i n , i t i s possible to roughly estimate what the response would be by extrapolation from the g r a p h i c a l l y displayed dose-response behaviour i n Figure 2 (Chapter I I I ) . Approximately 16-17$ ectrodactyly would be expected i n CBA/J treated with acetazolamide and t h i s i s i n the range of the response obtained with dichlorphenamide using t h i s dosage regime. I t i s impossible to say unequivocally that dichlorphenamide and acetazolamide have no teratogenic e f f e c t on the SWV  s t r a i n embryo. A l l attempts to use increasingly  higher doses to test f o r teratogenesis were stopped due to death of the SWV  dams; however, no obvious f e t a l e f f e c t s  were found i n those SWV  dams that survived.  The only  conclusion should be that, within the dosage l e v e l s used that were teratogenic to the CBA/J s t r a i n , the SWV  strain  was  r e s i s t a n t to any teratogenic or embryotoxlc e f f e c t s . Since two d i f f e r e n t potent carbonic anhydrase were examined and demonstrated  inhibitors  to be teratogenic In the CBA/J  s t r a i n , the resistance of the SWV  s t r a i n embryo i s most  probably a general resistance to t h i s class of teratogens.  66 TABLE 16 E f f e c t of Dichlorphenamide on CBA/J and SWV  Strain*  Affected litters  Resorptions/ implants  Ectrodactyly/ l i v e fetuses  No.  %  No.  11/72  CBA/J  3/8  10/72  14  SWV  0/6  9/85  11  0/76  18  0  * Treatment = 250 mg/kg per i n j e c t i o n administered i n t r a p e r i t o n e a l l y at 10 A.M. and 4 P.M. on day 10.  CHAPTER IX GENERAL DISCUSSION 1.  S t r a i n v a r i a t i o n s In t e r a t o g e n i c responses. Other s t r a i n v a r i a t i o n s i n the mouse t o d i f f e r e n t  t e r a t o g e n i c a c t i o n s a r e known and have been reviewed by Smlthberg  (1967).  The review i s s e v e r a l y e a r s o l d b u t the  s t a t e of the a r t has n o t advanced  much i n the i n t e r v e n i n g  years. In  examining  the s t u d i e s i n which the nature of these  s t r a i n v a r i a t i o n s was I n v e s t i g a t e d , a v e r y severe can be l a i d .  A l l , except Dagg et; a l . (1966) have based  t h e i r approaches genetics".  criticism  t o the g e n e t i c a n a l y s i s on "one-gene  When they, d i d n o t o b t a i n evidence from b r e e d i n g  t e s t s f o r the r e c o v e r y of the i n b r e d s t r a i n phenotype and low frequency responding phenotypes), investigations.  they stopped  (high their  V e r y heavy r e l i a n c e was p l a c e d on f i n d i n g  a s e g r e g a t i o n of d i s c r e t e response f r e q u e n c i e s . Dagg e_t a l . (1966) examined the h e r i t a b l e  differences  between two s t r a i n s of mice which d i f f e r e d In t h e i r responses t o induced c l e f t p a l a t e and Induced malformations by 5 - f l u o r o u r a c i l .  hindlimb  T h e i r backcross breeding  scheme and t h e i r employment o f Wright's  (1934) formula f o r  the e s t i m a t i o n o f the number of g e n e t i c f a c t o r s  involved  i n the t r a i t s were used as g u i d e l i n e s f o r the p r e s e n t  67  68 a n a l y s i s of the s t r a i n v a r i a t i o n i n a c e t a z o l a m i d e - i n d u c e d ectrodactyly.  Dagg and h i s co-workers estimated t h a t a  minimum of three and  four l o c i ,  r e s p e c t i v e l y , were i n v o l v e d  i n the i n d u c t i o n of c l e f t p a l a t e and malformed hlndlimbs 5-fluorouracil.  However, these were c o n s e r v a t i v e e s t i m a t e s  s i n c e t h e i r e s t i m a t e s of the n o n - h e r i t a b l e v a r i a n c e s of these t r a i t s were based of the two alone.  environmental  on averages  of v a r i a n c e s  i n b r e d s t r a i n and F^ s i r e s and not on the F^  The F^ s i r e v a r i a n c e s f o r the two  l a r g e compared w i t h those of the two the a v e r a g i n g procedure  reduced  e s t i m a t e s of the number of l o c i  t r a i t s were v e r y  Inbred s t r a i n s i r e s  Consequently,  i n v o l v e d i n the t r a i t s of  environmental v a r i a n c e s d e r i v e d from a l o n e , the e s t i m a t e s would be doubled respectively.  and  i f the  Induced c l e f t p a l a t e and malformed h i n d f e e t were based  eight l o c i ,  sires  the e s t i m a t e s of the  environmental v a r i a n c e s by h a l f .  and  by  on  the  the F^ s i r e v a r i a n c e s to approximately  six  There i s no j u s t i f i c a t i o n f o r  the use of one method of c a l c u l a t i o n over the other s i n c e the e f f e c t s of the i n d i v i d u a l genes Is not known and technique  i s based  on the assumption  i n a t r a i t has an e q u a l e f f e c t on i t . e s t i m a t e s of three and  four l o c i ,  the  t h a t each l o c u s Involved The  conservative  however,, tend to b i a s the  i n t e r p r e t a t i o n of these t r a i t s f o r those n o t i n i t i a t e d i n t h i s b l o m e t r i c a l approach to g e n e t i c a n a l y s i s . The  s t r a i n v a r i a t i o n i n the c o r t i s o n e i n d u c t i o n of  c l e f t p a l a t e i s a well-known t e r a t o l o g i c a l phenomenon i n mice and b e s t s e r v e s to i l l u s t r a t e most of the o t h e r  69 i n v e s t i g a t i o n s t h a t have been made of s t r a i n v a r i a t i o n s i n t e r a t o l o g i c a l responses.  F r a s e r and  demonstrated t h a t the A and low  F a l n s t a t (1951)  C57BL s t r a i n s e x h i b i t a high  frequency, r e s p e c t i v e l y , of c o r t i s o n e - i n d u c e d  palate.  palate  cleft  K a l t e r (1954) attempted a g e n e t i c a n a l y s i s of  variation.  He  demonstrated t h a t the  i n d u c t i o n was  dam  and  inbred  the genotype of the embryo played induction.  these t r a i t s was  s t r a i n ) of  the  a role in this  attempted s i n c e K a l t e r f e l t  the dam  trait  However, no meaningful a n a l y s i s of  i n s u f f i c i e n t progeny c o u l d be to c l a s s i f y  cleft  not a c y t o p l a s m l c a l l y - t r a n s m l t t e d  t h a t both the genotype ( t h a t i s the  c l e f t palate  this  s u s c e p t i b i l i t y to  and  and  that  c o l l e c t e d from any  f o r type of response.  c o r t i s o n e - t r e a t e d pregnancies to go  one  female  He a l l o w e d  to term and  scored  progeny a t b i r t h f o r the presence of c l e f t p a l a t e . have been r e l a t i v e l y easy to re-mate and  the the  I t would  t r e a t the dams  s e v e r a l times to c o l l e c t s u f f i c i e n t progeny. K a l t e r s b r e e d i n g d e s i g n was 1  sensitive A  strain sires.  b a c k c r o s s e d to the A  the  d a t a from r e c i p r o c a l F^ dams,  s i r e s , r u l e d out a maternal  cytoplasmlcally-transmltted performed one  The  a b a c k c r o s s to  sensitivity factor.  more b a c k c r o s s of BC^  He  females to the A  I f K a l t e r ' s data are examined i n the l i g h t of present  then  the  study of a c e t a z o l a m i d e - i n d u c e d e c t r o d a c t y l y ,  i n t e r e s t i n g observation  results.  sires.  an  I f the progeny mean  frequency response f o r induced c l e f t p a l a t e are examined i n a l o g i c a l progression  of mating types i n which the  cortisone  treatment of the dam was begun on day 11, the r e s u l t s i n 1 ? are obtained.  Table  It i s postulated trait and  of t e r a t o g e n i c s e n s i t i v i t y t o c o r t i s o n e i n the embryo  t h a t r e s i s t a n c e i s a dominant ( o r e s s e n t i a l l y dominant) Based on the 1 0 0 $ c l e f t p a l a t e response of the  trait. A  t h a t two independent l o c i c o n t r o l the  s t r a i n embryos, the expected BC^ ( c r o s s 2, Table  BCg  ( c r o s s 3 , Table  p a l a t e would be  1 7 ) progeny mean f r e q u e n c i e s  25.0$  and 56.3$. r e s p e c t i v e l y .  1 7 ) and  of c l e f t These  p r e d i c t e d mean f r e q u e n c i e s f o r the two b a c k c r o s s  generations  a r e too c l o s e t o the observed f r e q u e n c i e s from K a l t e r * s (1954) study  (Table 1 7 ) •  A r e i n v e s t i g a t i o n of t h i s c l e f t  palate  phenomenon should be made. In the case of c o r t i s o n e i n d u c t i o n of c l e f t p a l a t e , the genotype ( t h a t i s the i n b r e d s t r a i n ) of the dam i n f l u e n c e s the frequency  of induced  cleft  p a l a t e i n the progeny.  For  example, i n the case of the s e n s i t i v e A and the r e l a t i v e l y r e s i s t a n t C57BL s t r a i n s ,  the response of the F^ embryo i s  g r e a t e r i n s e n s i t i v e A s t r a i n dams than i t i s i n the more r e s i s t a n t C57BL s t r a i n dams. i n the dam and s e g r e g a t i o n  Both s e g r e g a t i o n  trait  of the s e n s i t i v i t y f a c t o r s i n the  embryo cannot be examined a t the same time. the t r a i t of c l e f t  of t h i s  Therefore, f o r  p a l a t e i n d u c t i o n i n the embryo, the  genotype of the dam must be kept constant and s e g r e g a t i o n of the t r a i t must be f o l l o w e d K a l t e r s breeding 1  through the s i r e .  Unfortunately,  program, by n e c e s s i t y f o r h i s main  study  of the dam e f f e c t , was the o p p o s i t e and s e g r e g a t i o n was  TABLE 17 Cortisone-Induced C l e f t Palate In Progeny of Backcross Matings with the A and C57BL S t r a i n s *  Dam  Sire  Progeny  C l e f t palate i n l i v e fetuses No.  1.  C57BL  A  2.  P  A  3. 4.  A  l  F  l  %  3/82  3.66  18/71  25.35 56.04  A  BC 2  51/91  A  A  36/36  100.  * Data are from Kalter (1954) f o r mating types i n which cortisone treatment of the pregnant dam was begun on day 11.  f o l l o w e d through the dam. observed  and  expected  Therefore,  the c o i n c i d e n c e of  g e n e r a t i o n mean f r e q u e n c i e s of Induced  c l e f t p a l a t e w i t h a two-locus model may The  important  the  be simply  fortuitous.  i n f o r m a t i o n t h a t i s m i s s i n g from K a l t e r ' s  (1954) d a t a i s some measure of the d i s t r i b u t i o n of a f f e c t e d and normal progeny per l i t t e r from which e s t i m a t e s g e n e t i c and non-genetic distributions, v a r i a t i o n was  v a r i a n c e s can be made.  From  these  I t would be p o s s i b l e to see what spread  of  p r e s e n t i n progeny g e n e r a t i o n s where the  t r a i t f o r s e n s i t i v i t y would be i n Table  of the  segregating  ( c r o s s e s 2 and  17).  There are f u r t h e r arguments f o r a r e i n v e s t i g a t i o n of s t r a i n v a r i a t i o n i n c o r t i s o n e - i n d u c e d c l e f t p a l a t e from work of Loevy (1963. s t r a i n and  two  w i t h the s e n s i t i v e Strong's  1968)  l e s s - s e n s i t i v e s t r a i n s , G3H  F o r some reason, unknown to me, Strong's A and C3H  utero with cortisone.  the A  and BALB/c.  Loevy (1963) used  of an F^ g e n e r a t i o n  the  treated in  From her d a t a , Loevy c o u l d only s t a t e  can o n l y s p e c u l a t e a t t h i s time as to the  g e n o t y p i c background needed f o r the e x p r e s s i o n of c l e f t palate.  I t c l e a r l y i n v o l v e s more than one  perhaps a t l e a s t t h r e e . "  There was  a b s o l u t e l y no  or a n a l y s i s made f o r the l a s t p a r t of her Unfortunately, review  the  s t r a i n s i n a b r e e d i n g program t h a t  extended to the examination  t h a t "One  3  t h i s d i d n o t prevent  of s t r a i n v a r i a b i l i t y  induced gene, and evidence  statement.  Smithberg (I967),  of t e r a t o g e n i c responses,  s t a t i n g t h a t "Loevy (I963) had  estimated  in his from  t h a t a minimum of  73  three p a i r s of genes were r e s p o n s i b l e f o r c l e f t  palate."  Loevy (1968) examined the c o r t i s o n e i n d u c t i o n of c l e f t p a l a t e i n backcross and  G3H  programs between (1) Strong's A  and (2) Strong's A s t r a i n and BALB/c.  strain  Her c r o s s e s  extended only to the BC^ progeny and no a n a l y s i s was performed other than a demonstration t h a t there a r e s t r a i n d i f f e r e n c e s i n the i n d u c t i o n of c l e f t and  p a l a t e i n the embryo  t h a t the genotype of the dam a l s o i n f l u e n c e s the  embryonic s e n s i t i v i t y . similar  t o those  Her c o n c l u s i o n s were  of K a l t e r (1954).  she demonstrated t h a t there  By u s i n g other  i s considerable  i n t e r a t o g e n i c response to c o r t i s o n e . r e a p p r a i s a l of some of her d a t a ,  essentially  strain  strains, variation  However, upon  there i s l i m i t e d  support f o r  e i t h e r a one- or two-locus mode o f i n h e r i t a n c e of the t r a i t of embryonic s e n s i t i v i t y based on the same assumptions used to r e - e v a l u a t e K a l t e r ' s d a t a . 2.  G e n e t i c model f o r acetazolamlde-lnduced The  present  ectrodactyly.  study has suggested a t h r e e - l o c u s model f o r  the i n h e r i t a n c e of s e n s i t i v i t y t o the i n d u c t i o n o f e c t r o d a c t y l y by acetazolamide compared w i t h SWV.  when the CBA/J s t r a i n i s  I t i s conceivable  t h a t other  genetic  models than t h a t of three l o c i may be p o s s i b l e to d e f i n e the i n d u c t i o n of e c t r o d a c t y l y by  acetazolamide.  With the dosage regime used i n the p r e s e n t program w i t h definite  the CBA/J and SWV s t r a i n s ,  breeding  there was a s m a l l b u t  response of the heterozygous CBA.SWV F  embryo but  74 no a f f e c t e d SWV.CBA F^ embryos were found, a l t h o u g h , i n t h i s l a t t e r case, the sample s i z e was s m a l l .  Does t h i s imply a  d i f f e r e n c e i n the t e r a t o g e n i c environment  of the CBA/J and  SWV dams w i t h the same dosage ( t h a t i s a dam e f f e c t ) o r i s i t simply a f u n c t i o n of sample s i z e ? suggested  The embryo t r a n s f e r study  t h a t the t e r a t o g e n i c environment  after  acetazolamide a d m i n i s t r a t i o n was p r e s e n t i n the SWV dam b u t i t was n o t designed  t o t e s t s u b t l e d i f f e r e n c e s between CBA/J  and SWV dams. The  t h r e e - l o c u s model I s based  on the genes f o r  t e r a t o g e n i c r e s i s t a n c e b e i n g dominant and t h e r e f o r e no a f f e c t e d F^ embryos a r e expected. acetazolamide was used  I f a lower dosage of  so t h a t no t r e a t e d CBA.SWV F^  embryos would e x h i b i t e c t r o d a c t y l y , would the t h r e e - l o c u s model s t i l l  f i t the c o r r e s p o n d i n g induced e c t r o d a c t y l y d a t a  from subsequent  b a c k c r o s s g e n e r a t i o n s t h a t would r e s u l t  from  t h i s lower dosage? These q u e s t i o n s must be t e s t e d t o f u r t h e r i n v e s t i g a t e the phenomenon of s t r a i n s e n s i t i v i t y and r e s i s t a n c e t o acetazolamlde. S t r a i n responses  t o the e c t r o d a c t y l y i n d u c t i o n of  a c e t a z o l a m i d e , d i f f e r e n t from t h a t of CBA/J, a r e known from a p r e l i m i n a r y study by Cathy C. A z a r and Dr. Margaret C. Green of the Jackson L a b o r a t o r y (Green, 1972, p e r s o n a l communication).  Using the acetazolamide dosage regime of  750 mg/kg a d m i n i s t e r e d i n t r a p e r i t o n e a l l y a t 10 A.M. and 4 P.M., they found t h a t C57BL/6J had approximately  the same  s e n s i t i v i t y as CBA/J "but C57BL/6J was more s e n s i t i v e on day 9 than on day 10.  They a l s o confirmed the f i n d i n g s of the  p r e s e n t study that CBA/J was more s e n s i t i v e on day 10 than on day 9.  E c t r o d a c t y l y was found i n the C3H/HeJ, SWR/J (not  to be confused w i t h SWV)  and DBA/2J s t r a i n s when t r e a t e d on  day 9 and i n the A / J s t r a i n when t r e a t e d on e i t h e r day 9 or day 10.  The order of d e c r e a s i n g s e n s i t i v i t y was C 5 7 B L / 6 J ,  C3H/HeJ and SWR/J but i n s u f f i c i e n t data were a v a i l a b l e f o r the DBA/2J and A / J s t r a i n s to e f f e c t i v e l y rank  them.  Dr. W. M. Layton, i n p e r s o n a l communication, found  that  a s t r a i n of mice w i t h the s i t u s i n v e r s u s mutant i s more s e n s i t i v e to acetazolamide on day 9 than on day 10.  Suzuki  and Takano (1969) a l s o found t h i s w i t h the ICR-JCL l i n e of mice. T h e r e f o r e , c o n s i d e r a b l e s t r a i n v a r i a b i l i t y e x i s t s both i n the frequency of response a c t i o n of a c e t a z o l a m i d e .  to acetazolamide and the time of  T h i s i m p l i e s t h a t more g e n e t i c  v a r i a b i l i t y e x i s t s than simply the CBA/J s e n s i t i v i t y and the SWV  r e s i s t a n c e and t h a t the i n v e s t i g a t i o n of the g e n e t i c  d i f f e r e n c e s between these two s t r a i n s , r e p o r t e d i n t h i s study, should o n l y be c o n s i d e r e d to be the b e g i n n i n g of t h i s analysis. Sewal Wright  (1934) l a i d a s e r i o u s charge a g a i n s t many  g e n e t i c i s t s , the i m p l i c a t i o n s of which have been ignored i n most i n v e s t i g a t i o n s of the g e n e t i c s of s t r a i n v a r i a t i o n s i n t e r a t o g e n i c responses.  He  stated:  There a r e many cases i n slow-breeding animals i n which a gene has been d e s i g n a t e d  76  on no more b a s i s than dominance i n of a c r o s s between t r u e b r e e d i n g s t r a i n s , a 3:1 r a t i o i n F? and a 1:1 r a t i o i n the b a c k c r o s s to the r e c e s s i v e s t r a i n . There i s , however, no c o n c l u s i v e evidence f o r or a g a i n s t i t s e x i s t e n c e u n t i l b r e e d i n g t e s t s have been made of the s e g r e g a t i n g generation. (p. 538) In  the p r e s e n t study, both approaches  t o the g e n e t i c  a n a l y s i s o f the b a c k c r o s s program suggested three independent  l o c i were r e s p o n s i b l e f o r the t r a i t of  teratogenic s e n s i t i v i t y .  P r e d i c t i o n s from the model were  confirmed by a f u r t h e r b a c k c r o s s g e n e r a t i o n .  In a d d i t i o n ,  two BCg s i r e s produced BC^ p r o g e n i e s from CBA/J dams w i t h e c t r o d a c t y l y response f r e q u e n c i e s comparable t o t h a t of the CBA/J s i r e s mated t o CBA/J dams and suggested genotype  had been r e c o v e r e d .  t h a t the CBA/J  However, f u r t h e r b r e e d i n g t e s t s  w i t h these two BCg s i r e s would be r e q u i r e d to demonstrate t h a t they do breed t r u e and the r e s u l t i n g  ectrodactyly  response of t h e i r progeny was t h a t of the CBA/J s t r a i n and no more. I t would be extremely i n t e r e s t i n g i f , by d e l i b e r a t e selection,  the frequency of a c e t a z o l a m i d e - i n d u c e d  e c t r o d a c t y l y i n a l i n e of mice d e r i v e d from these two BCg s i r e s c o u l d be r a i s e d Inbred s t r a i n .  s i g n i f i c a n t l y above t h a t of the CBA/J  C o n s i d e r a t i o n of the s t u d i e s of Waddington  (1953) w i t h D r o s o p h l l a melanogaster,  In which he d e s c r i b e d  the phenomenon of " g e n e t i c a s s i m i l a t i o n " suggests t h a t  this  might be p o s s i b l e w i t h the a c e t a z o l a m i d e - i n d u c e d ectrodactyly. Waddington (1953) was a b l e to induce a low frequency of  77 the  crossveinless  stock for  t r a i t "by heat shock of the pupae i n a w i l d  that d i d n o t e x h i b i t t h i s t r a i t n o r m a l l y .  and a g a i n s t  Selection  the appearance of the phenocopy r a p i d l y  Increased and decreased, r e s p e c t i v e l y , i t s frequency and, a f t e r lk g e n e r a t i o n s i n the upward s e l e c t e d s t r a i n ,  flies  were found that expressed the t r a i t even when they had n o t been exposed t o the heat shock. the heat shock, i n c r e a s e d  Further  s e l e c t i o n , without  and f i x e d the spontaneous frequency  of the c r o s s v e i n l e s s t r a i t a t almost complete penetrance. Therefore,  the genes which c o n t r o l l e d the e x p r e s s i o n  previously  induced t r a i t were a b l e  of a  to be s e l e c t e d or  a s s i m i l a t e d and, w i t h continued s e l e c t i o n , they allowed f o r the e x p r e s s i o n  of the t r a i t even i n the absence of the  abnormal environmental  stimulus.  There were no c r o s s v e i n l e s s f l i e s found i n the o r i g i n a l stock used by Waddington (1953)• (Waddington, 1957,  p. 177),  a d i f f e r e n t w i l d stock, s t a r t i n g population  However,  who repeated t h i s experiment w i t h  checked a l a r g e r number of the  and found a v e r y low percentage ( l e s s  than 1%) w i t h a n a t u r a l l y o c c u r r i n g phenotype.  Bateman  "low grade  From these few i n d i v i d u a l s , a  crossveinless"  crossveinless  stock was s e l e c t e d and when t e s t e d against, the a s s i m i l a t e d c r o s s v e i n l e s s stock derived  from subsequent heat shock of the  pupae and s e l e c t i o n of c r o s s v e i n l e s s a d u l t s , appeared to be g e n e t i c a l l y i d e n t i c a l . for  Therefore,  the c r o s s v e i n l e s s phenotype were a l r e a d y  o r i g i n a l populations  the two the genes  p r e s e n t i n the  but i n low frequency so t h a t only r a r e l y  did  i n d i v i d u a l s have s u f f i c i e n t of them to express the  crossveinless t r a i t . "by these procedures,  In most other phenocopy t r a i t s  there were no abnormal i n d i v i d u a l s i n (Waddington, 1957).  the s t a r t i n g p o p u l a t i o n s A s i m i l a r study w i t h  the acetazolamide  e c t r o d a c t y l y would p r o v i d e of t h i s induced  trait.  i n d u c t i o n of  f u r t h e r i n f o r m a t i o n on the  s e n s i t i v e Inbred  s t r a i n , a more r a t i o n a l d i s c u s s i o n that are  found i n d i f f e r e n t s e n s i t i v e i n b r e d s t r a i n s of mice. that d i f f e r e n t  to  t r a i t beyond t h a t found  can be made of the d i f f e r e n t response f r e q u e n c i e s  be  nature  I f i t i s p o s s i b l e , by s e l e c t i o n ,  change the t h r e s h o l d f o r t h i s induced i n any  studied  It  may  s t r a i n s have d i f f e r e n t g e n e t i c a l l y  determined t h r e s h o l d s f o r t h i s i n d u c t i o n of e c t r o d a c t y l y . I t should be p o i n t e d out t h a t g e n e t i c a s s i m i l a t i o n , s t a r t i n g w i t h i n b r e d l i n e s of f l i e s , proved (Waddington, 1957). t r a i t s i n Inbred (Roderick and  A l s o , s e l e c t i o n of v a r i o u s q u a n t i t a t i v e  s t r a i n s of mice has been u n s u c c e s s f u l  Schlager,  1966).  s e l e c t a h i g h frequency, mice would be  unsuccessful  T h e r e f o r e , any  attempts to  acetazolamide-responding  strain  of  s u c c e s s f u l o n l y w i t h an a r t i f i c i a l l y  heterogeneous s t o c k d e r i v e d from o u t c r o s s i n g s e v e r a l d i f f e r e n t inbred  strains.  With r e s p e c t to the p o s s i b i l i t y i n c r e a s e i n the frequency  of  of s e l e c t i n g f o r an  acetazolamide-induced  e c t r o d a c t y l y , the mutant p o s t a x i a l hemimelia (gene symbol should be d i s c u s s e d .  P o s t a x i a l hemimelia i s a  Mendelian r e c e s s i v e t r a i t and  px)  simple,  i s mapped i n l i n k a g e group XI  ( S e a r l e , 1964).  The homozygotes are s t e r i l e but have many  c h a r a c t e r i s t i c s i n common w i t h the e c t r o d a c t y l y syndrome.  acetazolamide-induced  The most common d e f e c t i n px/px  homozygotes i s a l o s s of d i g i t V on each f o r e l i m b .  More  severe cases show l o s s e s of d i g i t s IV and V and d i g i t s I I I , IV and V.  There  i s a r i g h t s i d e d predominance t o the  s e v e r i t y of malformations. humeral and  In a d d i t i o n ,  there are u l n a r ,  s c a p u l a r d e f e c t s and a few cases of h i n d l i m b  e c t r o d a c t y l y and  syndactyly.  The  c o i n c i d e n c e of the  c h a r a c t e r i s t i c s of p o s t a x l a l hemimelia  and  acetazolamide-  induced e c t r o d a c t y l y i s too obvious and warrants a c o n t i n u e d and more e x t e n s i v e i n v e s t i g a t i o n of the e c t r o d a c t y l y syndrome t h a t i s induced i n mice by acetazolamide and o t h e r s i m i l a r c a r b o n i c anhydrase 3.  inhibitors.  B i o c h e m i c a l b a s i s f o r s t r a i n d i f f e r e n c e s i n response  to  acetazolamide. The b a s i s f o r the r e s i s t a n c e of the SWV i n d u c t i o n of e c t r o d a c t y l y by acetazolamide  s t r a i n t o the  i s unknown.  It is  most probably a g e n e r a l r e s i s t a n c e to o t h e r potent c a r b o n i c anhydrase  i n h i b i t o r s s i n c e SWV  i s r e s i s t a n t t o the  df e c t r o d a c t y l y by dichlorphenamide. c a r b o n i c anhydrase  Since i n h i b i t i o n of  i s the o n l y known b i o c h e m i c a l f u n c t i o n of  t h i s group of compounds, a good working SWV  hypothesis i s that  has a r e s i s t a n t form of t h i s enzyme. The h y p o t h e s i s t h a t the s e n s i t i v e CBA/J and  SWV  induction  s t r a i n s may  resistant  d i f f e r w i t h r e s p e c t to a s e n s i t i v e  and  80 r e s i s t a n t c a r b o n i c anhydrase isoenzyme was  tested  by  l o o k i n g f o r e l e c t r o p h o r e t i c d i f f e r e n c e s i n t h i s enzyme i n a d u l t s of these two  strains.  There are known, g e n e t i c a l l y -  determined e l e c t r o p h o r e t i c v a r i a n t s of c a r b o n i c  anhydrase  i n the mouse which were f i r s t  erythrocytes  ( B i d d l e and  Petras,  both CBA/J and  SWV  1 67;  i s o l a t e d from the  B i d d l e and  Q  Krasny, 1970); however,  have the P r o - l b form of t h i s  anhydrase isoenzyme and  carbonic  t h i s isoenzyme from both s t r a i n s i s  i n h i b i t e d by acetazolamide on the s t a r c h g e l a f t e r electrophoresis.  A n o t h e r p o i n t i s t h a t the C57BL/6J  strain  which i s s e n s i t i v e to the t e r a t o g e n i c a c t i o n of acetazolamlde (Green, 1972,  personal  t h i s isoenzyme. l o c u s i s not  communication) has  Therefore,  involved with  the Pro-1  the P r o - l a form of  carbonic  anhydrase  this teratogenic resistance.  T h i s does not e l i m i n a t e  the r o l e of c a r b o n i c  anhydrase  i n t h i s t e r a t o g e n i c a c t i o n s i n c e , i n most mammalian  species  examined, s e v e r a l forms of the enzyme under separate  genetic  c o n t r o l have been i d e n t i f i e d  Shows,  1968).  One  (Tashian,  e r y t h r o c y t i c form of the enzyme has  d i o x i d e h y d r a t i o n a c t i v i t y and the other major form has a c t i v i t y and  low  preparations  low  high  esterase a c t i v i t y  carbon d i o x i d e  high esterase a c t i v i t y .  anhydrase enzymes i n other the same way;  S h r e f f l e r and  carbon while  hydration  The ,types of  carbonic  t i s s u e s have not been examined i n  however, examinations of f r a c t i o n a t e d l i v e r i n s e v e r a l s p e c i e s have r e v e a l e d  striking findings.  several  Maren, E l l i s o n , F e l l n e r and  Graham (1966)  found a supernatant f r a c t i o n i n r a t l i v e r , amounting to about  81 20% of the  total activity,  i n h i b i t i o n by v a r i o u s dog  t h a t was  almost r e f r a c t o r y to  sulfonamides such as acetazolamide  l i v e r d i d not have t h i s .  They r e p o r t e d  t h a t mouse  s t r a i n mentioned) and  r a b b i t l i v e r prepations  same as the r a t l i v e r  preparation.  I t i s possible that teratogenic embryo i s due  tested.  Difficulty  failed  to d e t e c t any  beyond the  SWV  in testing this by  (1968) on r a t embryos  enzyme a c t i v i t y u n t i l s e v e r a l days  sensitive period.  to r e f i n e the  the  anhydrase Isoenzyme.  h y p o t h e s i s w i l l a r i s e because the enzyme a s s a y s used W i l s o n , Maren, Takano and E l l i s o n  (no  r e s i s t a n c e of the  to r e s i s t a n c e of a c a r b o n i c  T h i s remains to be  behaved  but  techniques and  However, i t should be test this  hypothesis.  possible  CHAPTER X SUMMARY 1.  The SWV  Inbred s t r a i n of mice was found to be  r e s i s t a n t to the teratogenic action of acetazolamlde, a potent carbonic anhydrase i n h i b i t o r . 2.  The time of teratogenic action was examined i n the  sensitive CBA/J s t r a i n and mid-day 10 of pregnancy was  found  to be the most sensitive stage f o r the induction of ectrodactyly which i s the malformation produced by acetazolamide. 3.  The dose response to acetazolamide was examined i n  both the sensitive CBA/J and r e s i s t a n t SWV  strains.  Within  the dosage range examined, the frequency of ectrodactyly i n l i v e CBA/J fetuses Increased i n an apparent l i n e a r fashion with increased dose of acetazolamide while the resorption frequency increased i n a c u r v i l i n e a r fashion.  No  acetazolamide-induced ectrodactyly or f e t a l death was with the SWV  strain.  However, death of the SWV  found  dam occurred  at the highest dose examined. 4.  Reciprocal crosses with the r e s i s t a n t SWV  and  sensitive CBA/J strains indicated that resistance to the teratogenic action of acetazolamide i s dominant to sensitivity. 5.  Both a metrical analysis of the backcross program 82  83 u s i n g the s e n s i t i v e CBA/J dam and an examination of the g e n e r a t i o n mean frequency responses i n t h i s b a c k c r o s s program suggested t h a t three i n d e p e n d e n t l y s e g r e g a t i n g l o c i c o n t r o l l e d the t r a i t  o f embryonic s e n s i t i v i t y to the  t e r a t o g e n i c a c t i o n of a c e t a z o l a m i d e . 6. of  The p r e d i c t e d mean frequency of induced e c t r o d a c t y l y  the BC^ g e n e r a t i o n from t h i s t h r e e - l o c u s model was  demonstrated  by f u r t h e r b r e e d i n g t e s t s .  The s e n s i t i v e CBA/J  s t r a i n response frequency was r e c o v e r e d from two BCg s i r e s . 7.  No c o r r e l a t i o n was obvious between e c t r o d a c t y l y  i n d u c t i o n and f e t a l death a f t e r a c e t a z o l a m i d e The  administration.  two t r a i t s appear t o be separate responses to t h i s  teratogen. 8.  A c u r s o r y employment of the embryo t r a n s f e r  procedure w i t h the CBA/J and SWV s t r a i n s demonstrated the t e r a t o g e n i c environment  that  a f t e r acetazolamide  a d m i n i s t r a t i o n was p r e s e n t i n the r e s i s t a n t SWV s t r a i n dam and  t h a t r e s i s t a n c e of t h i s s t r a i n i s a p r o p e r t y of the  embryo. 9.  The t e r a t o g e n i c s e n s i t i v i t y and r e s i s t a n c e of the  CBA/J and SWV  s t r a i n s was demonstrated  to be the same f o r  dichlorphenamide, another potent c a r b o n i c anhydrase inhibitor. 10.  The r e s u l t s o f the above s t u d i e s were d i s c u s s e d i n  the c o n t e x t of other s t u d i e s of g e n e t i c v a r i a t i o n i n teratogenic responses.  F u r t h e r experiments were proposed t o  t e s t the g e n e t i c model presented i n t h i s study f o r  84 acetazolamide-induced e c t r o d a c t y l y . carbonic  anhydrase  perspective  V a r i a t i o n s i n the  enzyme were b r i e f l y d i s c u s s e d  of the v a r i a t i o n s i n the t e r a t o g e n i c  its Inhibitors.  t o g a i n some response t o  BIBLIOGRAPHY B i d d l e , F. G. and H. Krasny. 1970. The a s s o c i a t i o n of c a r b o n i c anhydrase a c t i v i t y with the non-hemoglobin e r y t h r o c y t i c p r o t e i n (Pro-1) of the house mouse. Can. J . Genet. G y t o l . 12: 374 ( a b s t r a c t ) . B i d d l e , F. G. and J . R. M i l l e r . 1972. G e n e t i c a n a l y s i s of the r e s i s t a n c e t o the t e r a t o g e n i c a c t i o n of acetazolamide i n mice. T e r a t o l o g y j?: 251 ( a b s t r a c t ) . B i d d l e , F. G. and M. L. P e t r a s . 1967. The i n h e r i t a n c e of a non-hemoglobin e r y t h r o c y t i c p r o t e i n i n Mus musoulus. G e n e t i c s j>7_: 943-949. Dagg, C. P., G. S c h l a g e r and A. Doerr. 1966. P o l y g e n i c c o n t r o l of the t e r a t o g e n i c i t y of 5 - f l u o r o u r a c i l i n mice. G e n e t i c s jQ: 1101-1117. E l l i s o n , A. G. and T. H. Maren. 1972a. The e f f e c t s of metabolic a l t e r a t i o n s on t e r a t o g e n e s i s . Johns Hopkins  Med.  J . 1J0: 87-94.  E l l i s o n , A. C. and T. H. Maren. 1972b. The e f f e c t of potassium metabolism on a c e t a z o l a m i d e - i n d u c e d teratogenesis. Johns Hopkins Med. J . 130: 105-115. F a l c o n e r , D. S. 196l. I n t r o d u c t i o n t o Q u a n t i t a t i v e Genetics. Ronald Press Co., New York. F r a n c i s , B. M. 1971• I n h e r i t a n c e of s e n s i t i v i t y t o the t e r a t o g e n i c and embryocidal e f f e c t s of c o r t i s o n e i n f o u r s t r a i n s of mice. Ph.D. T h e s i s , U n i v e r s i t y of M i c h i g a n , Ann A r b o r . F r a s e r , F. C. and T. D. F a l n s t a t . 1951. P r o d u c t i o n of c o n g e n i t a l d e f e c t s i n the o f f s p r i n g of pregnant mice t r e a t e d w i t h c o r t i s o n e . P e d i a t r i c s 8: 527-533. Green, M. C. 1972. The J a c k s o n L a b o r a t o r y , Bar Harbor, Maine. P e r s o n a l communication o f unpublished d a t a . H a l l e s y , D. W. and W. M. L a y t o n . 1967. Forelimb deformity of o f f s p r i n g of r a t s g i v e dichlorphenamide d u r i n g pregnancy. P r o c . Soc. Exp. B i o l . Med. 126: 6-8. K a l t e r , H. 195^. The i n h e r i t a n c e of s u s c e p t i b i l i t y t o the t e r a t o g e n i c a c t i o n of c o r t i s o n e i n mice. G e n e t i c s 39'  185-196.  85  86 K a l t e r , H. I965. I n t e r p l a y of I n t r i n s i c and e x t r i n s i c factors. Ghapt. 3. I n : T e r a t o l o g y ; P r i n c i p l e s and Techniques. J . G. W i l s o n and J . Warkany, ed. Univ. Chicago P r e s s , Chicago. Layton, W. M. 1971. golden hamsters.  T e r a t o g e n i c a c t i o n of acetazolamide i n T e r a t o l o g y 4: 95-102.  Layton, W. M. 1972. Dartmouth M e d i c a l S c h o o l , Hanover, New Hampshire. P e r s o n a l communication of unpublished d a t a . Layton, W. M. and D. W. H a l l e s y . 1965a. D e f o r m i t y of forelimb i n rats: a s s o c i a t i o n w i t h h i g h doses of acetazolamide. S c i e n c e 149: 306-308. Layton, W. M. and D. W. H a l l e s y . 1965b. Reply t o c r i t i c i s m of T. H. Maren (1965). S c i e n c e _15_0: 79. Loevy, H. 1963- G e n e t i c i n f l u e n c e s on Induced c l e f t p a l a t e i n d i f f e r e n t s t r a i n s of mice. Anat. Rec. 145: 117-122. Loevy, H. 1968. C o r t i s o n e - i n d u c e d t e r a t o g e n i c e f f e c t s i n mice. P r o c . Soc. Exp. B i o l . Med. 128: 841-844. Maren, T. H. 1965. F o r e l i m b d e f o r m i t y i n r a t s : with acetazolamide. S c i e n c e 150 79.  association  Maren, T. H. 1967. C a r b o n i c anhydrase: chemistry, p h y s i o l o g y and i n h i b i t i o n . P h y s i o l . Rev. 4£: 595-781. Maren, T. H. 1971. T e r a t o l o g y and c a r b o n i c anhydrase i n h i b i t i o n . A r c h . Ophthal. 8£: 1-2 ( e d i t o r i a l ) . Maren, T. H. and A. C. E l l i s o n . 1972a. The t e r a t o l o g i c a l e f f e c t of c e r t a i n t h i a d i a z o l e s r e l a t e d t o a c e t a z o l a m i d e , w i t h a note on s u l f a n i l a m i d e and t h i a z i d e d i u r e t i c s . Johns Hopkins Med. J . 130: 95-104. Maren, T. H. and A. C. E l l i s o n . 1972b. The t e r a t o l o g i c a l e f f e c t of benzolamide, a new carbonic anhydrase i n h i b i t o r . Johns Hopkins Med. J . 130: 116-123. Maren, T. H. and A. C. E l l i s o n . 1972c. The e f f e c t s of acetazolamide and amilorlde on tissue e l e c t r o l y t e s , with a note on the teratogenesis problem. J . Pharmacol. Exp. Ther. 181: 212-218. Maren, T. H., A. C. E l l i s o n , S. K. F e l l n e r and W. B. Graham. 1966. A study of hepatic carbonic anhydrase. Mol. Pharmacol. 2: 144-157.  87 Maren, T. H., E. Mayer and B. C. Wadworth. 1954. Carbonic anhydrase i n h i b i t i o n . I. The pharmacology of Diamox, 2-acetylamino-l,3»4-thiadiazole-5-sulfonamlde. Bull. Johns Hopkins Hosp. £5: 199-243. McLaren, A. and D. M i c h i e . 1956. S t u d i e s on the t r a n s f e r of f e r t i l i z e d mouse eggs to u t e r i n e f o s t e r - m o t h e r s . I. F a c t o r s a f f e c t i n g the i m p l a n t a t i o n and s u r v i v a l of n a t i v e and t r a n s f e r r e d eggs. J . Exp. B i o l . 33'- 394-416. M o s t e l l e r , F. and C. Youtz. 1961. T a b l e s of the FreemanTukey t r a n s f o r m a t i o n s f o r the b i n o m i a l and P o i s s o n d i s t r i b u t i o n s . B i o m e t r i k a 48: 433-440. Mullen, R. J . and W. K. Whitten. 1971. R e l a t i o n s h i p of genotype and degree of chlmerism i n coat c o l o r to sex r a t i o s and gametogenesis i n c h i m e r i c mice. J . Exp.  Z o o l . V[8:  165-176.  Roderick, T. H. and G. S c h l a g e r . 1966. Multiple factor i n h e r i t a n c e . Chapt. 9. In: B i o l o g y of the L a b o r a t o r y Mouse. E . L. Green, ed. McGraw-Hill Book Co., New York. S c o t t , W. J . 1970. E f f e c t s of i n t r a u t e r i n e a d m i n i s t r a t i o n of acetazolamide i n r a t s . T e r a t o l o g y 3_\ 261-268. S e a r l e , A. G. 1964. The g e n e t i c s and morphology of two ' l u x o i d mutants i n the house mouse. Genet. Res., Camb. 1  £: 171-197.  Smithberg, M. 1967. T e r a t o g e n e s i s i n i n b r e d s t r a i n s of mice. Advances i n T e r a t o l o g y 2: 257-288. Snedecor, G. W. and W. G. Cochran. I967. Statistical Methods. Iowa S t a t e Univ. P r e s s , Ames. S u z u k i , M. and K. Takano. I969. T e r a t o g e n i c e f f e c t of acetazolamide i n ICR mice. Cong. Anom. Suppl.: (abstract).  36  Takano, K., A. C. P e t e r s o n , F. G. B i d d l e and J . R. M i l l e r . 1972. The use of embryo t r a n s p l a n t a t i o n i n e x p e r i m e n t a l teratology: s t r a i n v a r i a t i o n i n triamcinolone-lnduced c l e f t p a l a t e . Manuscript i n p r e p a r a t i o n . Takano, K., S. Yokota and T. Nagata. 1971. Histochemical demonstration of c a r b o n i c anhydrase a c t i v i t y i n the r a t limbbud. Anat. Rec. I69: 484 ( a b s t r a c t ) . T a s h i a n , R. E., D. C. S h r e f f l e r and T. B. Shows. 1968. G e n e t i c and p h y l o g e n e t l c v a r i a t i o n i n the d i f f e r e n t m o l e c u l a r forms of mammalian e r y t h r o c y t i c c a r b o n i c anhydrases. Ann. N. Y. Acad. S c i . 151: 64-77.  88  V i c k e r s , T. H. 1972. Acetazolamlde J . Exp. Path. $2>. 5-21.  dysmelia  In r a t s .  Brit.  Waddington, C. H. 1 9 5 3 . G e n:e t i c a s s i m i l a t i o n of an a c q u i r e d c h a r a c t e r . E v o l u t i o n _7_ 118-126. Waddington, G. H. 1 9 5 7 . The S t r a t e g y of the Genes. A D i s c u s s i o n of Some A s p e c t s of T h e o r e t i c a l B i o l o g y . George A l l e n and Unwin L t d . , London. W i l s o n , J . G., T. H. Maren, K. Takano and A. E l l i s o n . 1968. T e r a t o g e n i c a c t i o n of c a r b o n i c anhydrase i n h i b i t o r s i n the r a t . T e r a t o l o g y _1: 51-60. Wright, S. 1934. The r e s u l t of c r o s s e s between i n b r e d s t r a i n s of guinea p i g s , d i f f e r i n g i n number of d i g i t s . G e n e t i c s 12: 5 3 7 - 5 5 1 . Wright, S. 1968. E v o l u t i o n and the G e n e t i c s of P o p u l a t i o n s . V o l o I . G e n e t i c and B i o m e t r i c F o u n d a t i o n s . Chapt. 1 5 . The G e n e t i c s of Q u a n t i t a t i v e V a r i a b i l i t y , pp. 3 7 3 - 4 2 0 . Univ. Chicago P r e s s , Chicago.  APPENDIX I D i s t r i b u t i o n of A c e t a z o l a m i d e - i n d u c e d D i g i t a l i n the CBA/J  Strain*  C l a s s of d i g i t a l anomaly 1. Normal 2. R - V abnormal  Male  Total  44  41  85  1  3. L - V abnormal  3  O ...  5. L - V absent 6.  Female  „  4. R - V absent  Anomalies  4 i  l  26  31 2  57 1  3  R - V absent L - V absent  0  3  3  7.  R - IV,V absent  4  1  5  8.  R - I I I , IV,V absent  2  0  2  7  0  7  1  0  1  9.  R - IV,V absent L - V absent 10. R - I I I abnormal*, IV,V absent L - V absent (* d i g i t I I I = b i f i d n a i l ) . . . . 11. R - III.IV.V absent L - IV,V absent... o . . . . o  1  0  1  12. R - V absent L - III,IV,V absent  1  0  1  13. R - IV,V absent L - III,IV,V absent  1  0  1  14. R - III,IV,V absent L - III,IV,V absent  1  0  1  96  Totals  76  172  * CBA/J f e t u s e s a r e from T a b l e 6; R = r i g h t f o r e l i m b , L = l e f t f o r e l i m b . Acetazolamide was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y to the dams a t 750 mg/kg a t 10 A.M. and 4 P.M. on day 10.  89  

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