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The fetal hydantoin syndrome : a mouse model Finnell, Richard H. 1978

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THE FETAL HYDANTOIN SYNDROME: A MOUSE MODEL by RICHARD H. FINNELL B . S c , U n i v e r s i t y of Oregon, 1975 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES i n the Genetics Program We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1978 © Richard H. F i n n e l l , 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M e d i c a l Genetics The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date A p r i l 1 1 , 1978 ABSTRACT The suspected t e r a t o g e n i c i t y o f Diphenylhydantoin (DPH) i n man i s important, e s p e c i a l l y to the 0.3 to 0.5% of pregnant women who are e p i l e p t i c and, t h e r e f o r e , candidates f o r a n t i c o n v u l s a n t drug therapy. To separate the t e r a t o g e n i c e f f e c t of e p i l e p s y from DPH treatment, an animal model c l o s e l y approximating the human c o n d i t i o n was developed to meet the f o l l o w i n g c r i t e r i a : (i) the t e s t animal must have spontaneous s e i z u r e s ( i i ) the s e i z u r e s must be c o n t r o l l e d or e l i m i n a t e d by:'DPH treatment ( i i i ) the drug must be administered o r a l l y (iv) serum DPH l e v e l s must f a l l w i t h i n the op t i m a l human t h e r a p e u t i c range between 5 and 20 micrograms per ml serum (v) treatment must begin p r i o r t o mating and continue throughout g e s t a t i o n (vi) the o f f s p r i n g of t r e a t e d animals must e x h i b i t the spectrum of malformations observed i n the o f f s p r i n g of e p i l e p t i c women The f i r s t c r i t e r i o n was met by mutant quaking (qk) mice. The s e i z u r e a c t i v i t y of these animals was reduced (from 2.1 to .34 s e i z u r e s per mouse day) by DPH treatment. To separate the e f f e c t of t h i s gene from t h a t of the DPH i n the e t i o l o g y o f the malformations, heterozygous (+/qk) and homozygous non-quaking (+/+) mice were a l s o s t u d i e d . M o n i t o r i n g o f DPH l e v e l s w i t h the SYVA Emit s p e c t r o p h o t o m e t r y assay.technique i n d i c a t e d serum c o n c e n t r a t i o n s w i t h i n the human t h e r a p e u t i c range a t 40 and 60 mg/kg body weight dosages. The i n c i d e n c e of f e t u s e s born wi t h s k e l e t a l or s o f t -t i s s u e a b n o r m a l i t i e s i n c r e a s e d w i t h i n c r e a s i n g DPH dosages. T h i s was observed i n a l l three genotypes. The a b i l i t y o f the unt r e a t e d quaking (qk/qk) dams to -produce normal o f f s p r i n g i m p l i c a t e s the drug r a t h e r than the mutant gene as the cause o f malformations. A p r e l i m i n a r y a p p l i c a t i o n o f t h i s animal model produced what can be c o n s i d e r e d the mouse e q u i v a l e n t of the f e t a l hydantion syndrome. The s i m i l a r i t i e s between the human and mouse syndromes i n c l u d e p r e n a t a l growth d e f i c i e n c y , n e u r a l , c a r d i a c , o r o f a c i a l , o c u l a r and g e n i t o u r i n a r y anomalies. F u r t h e r l a r g e - s c a l e a p p l i c a t i o n of the model should p r o v i d e i n s i g h t i n t o the r o l e of DPH i n the e t i o l o g y o f the malform-a t i o n s observed amongst the o f f s p r i n g of e p i l e p t i c women on hydantoin a n t i c o n v u l s a n t drug therapy. TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i I. INTRODUCTION 1 1. H i s t o r i c a l Perspective 1 2. Survey of Epidemiological Reports Up to 1975 6 3. Survey of Animal Experiments Up to 1977 15 4. The Fetal Hydantoin Syndrome: 1975-197 8 19 5. Purpose and Rationale of Present Study 24 I I . METHODOLOGY 28 1. Animals 2 8 2. DPH Treatment Levels 28 3. Calculation of Drug Doses 29 4. Diet Administration 30 5. Seizure Control Study 32 6. Determination of Serum DPH Levels 32 7. Experimental Design 34 a. Experimental Organism 34 b. Route of Adminstration 35 c. Matings 35 d. Fetal Examination 36 8. S t a t i s t i c a l Analysis 37 i v Page I I I . RESULTS 38 1. S e i z u r e C o n t r o l 38 2. Serum L e v e l s 38 3. Implantations and Resorptions 41 4. F e t a l Measurements 4 4 5. F e t a l Anomalies 4 4 IV. DISCUSSION 51 FIGURES 63 LITERATURE CITED 67 APPENDICES 7 3 APPENDIX A DPH Dose Conversion Table 7 3 APPENDIX B Water Intake Conversion Table 7 4 APPENDIX C DPH Dosage C a l c u l a t i o n Table 7 5 APPENDIX D A l i z a r i n . R e d S t a i n i n g Procedure f o r Skeletons 76 APPENDIX E ANOVA Tables 7 7 v LIST OF TABLES Table Page 1. R e t r o s p e c t i v e s t u d i e s on a n t i c o n v u l s a n t s a d m i n i s t e r -to e p i l e p t i c women and the frequency o f malformations as compared with the occurrence o f malformations i n untr e a t e d e p i l e p t i c s and i n n o n - e p i l e p t i c c o n t r o l s . 8 2. Frequency o f d i f f e r e n t malformations i n c h i l d r e n of mothers on a n t i c o n v u l s a n t s d u r i n g pregnancy. 13 3. E f f i c a c y o f DPH O r a l l y Administered to Quaking (qk/qk) Mice. 39 4. DPH Serum Co n c e n t r a t i o n s i n Non-Pregnant Females. 40 5. E f f e c t of Treatments on Water Intake and Serum DPH Co n c e n t r a t i o n s on Pregnant Females. 42 6. E f f e c t o f Treatments on Implantations and Re s o r p t i o n . 43 7. E f f e c t o f Treatments on L i v e B i r t h s , Sex, F e t a l Weights, and F e t a l A b n o r m a l i t i e s . 45 8. Types and Frequencies of S k e l e t a l Anomalies. 4 6 9. Types and Frequencies of S o f t T i s s u e Anomalies. 49 10. S i m i l a r i t i e s between the Human and Mouse F e t a l Hydantoin Syndrome. 53 v i LIST OF FIGURES F i g u r e s Page 1. O s s i f i c a t i o n anomalies of s u p r a o c c i p i t a l bone 63 and v e r t e b r a l centrae 2. S k e l e t a l anomalies i n c l u d i n g t r i a n g u l a r v e r t e b r a l c entrae 63 3. S k e l e t a l anomalies i n c l u d i n g absent sternabrae 63 4. Microphthalmia 64 5. Anophthalmia 64 6. ' P o l y d a c t y l y 64 7. Hydronephrosis 6 5 8. D i g i t a l h y p o p l a s i a and c o n t r o l 65 9. C l e f t p a l a t e and c o n t r o l 66 v i i ACKNOWLE DGEMENTS I wish t o express my thanks t o the members of my t h e s i s committee a t the U n i v e r s i t y o f B r i t i s h Columbia f o r t h e i r e f f o r t s d u r i n g the course o f t h i s p r o j e c t : Dr. C. 0. Person (Co-chairman) and Dr. A. J . F. G r i f f i t h s , Department of Botany and Dr. J . R. M i l l e r (Co-chairman) Department of Medical G e n e t i c s . In a d d i t i o n to the members of my committee, s p e c i a l thanks must be given t o Dr. C. W. Roberts, Department of P o u l t r y S c i e n c e , f o r the many e n l i g h t e n i n g c o n v e r s a t i o n s we have shared over the y e a r s . There are so many o t h e r s , too numerous t o name here, who pla y e d i n s t r u m e n t a l r o l e s i n c a r r y i n g out t h i s r e s e a r c h . I would e s p e c i a l l y wish t o thank Ms. R. Bunting and Ms. M. Hanson, Woodlands School; Mr. R. R o b i l l o , Department of Botany; Mr. B. Stewart, Department of Biom e d i c a l Communications; and Ms. M. P a r i s f o r her f a i t h f u l and accurate t y p i n g o f t h i s t h e s i s . To Dr. Ge r a l d (Buzz) Chernoff, my tea c h e r , my c o l l e a g u e and my very f i n e f r i e n d , my s i n c e r e thanks. Not o n l y f o r h i s t e c h n i c a l a s s i s t a n c e i n the c a r d i a c d i s s e c t i o n , but a l s o h i s p a t i e n t and c o n t i n u i n g i n t r o d u c t i o n i n t o the world o f experimental t e r a t o l o g y . My h e a r t f e l t thanks to Dr. C l a y t o n Person, without whose he l p t h i s t h e s i s would never have been completed. v i i i And of course, to my d e a r e s t of f r i e n d s , Susanna, and our c h i l d r e n , Yasmin and Alexander. I am f o r e v e r g r a t e f u l f o r t h e i r support and encouragement throughout the course of t h i s study. i x - 1 -CHAPTER I INTRODUCTION 1. HISTORICAL PERSPECTIVE E p i l e p s y , a d i s o r d e r c h a r a c t e r i z e d by paroxymal d i s c h a r g e s i n the b r a i n and by r e c u r r e n t s e i z u r e s , has plagued man s i n c e h i s b e g i n n i n g s . This can be evidenced by the l a r g e number of t r e p h i n e d p r e h i s t o r i c s k u l l s t h a t have been unearthed (Coatsworth and Penry, 1972). C e r t a i n l y a f r a c t i o n of the t r e p h i n i n g s r e p r e s e n t e a r l y man's f i r s t attempt to r i d h i m s e l f of the d i s e a s e , which the Greeks would l a t e r c a l l the "Sacred Disease" and which the C h r i s t i a n s would r e f e r to as the " F a l l i n g S i c k n e s s " (Tempkin, 1945; Lennox, 1960; u n l e s s otherwise i n d i c a t e d , the h i s t o r y t h a t f o l l o w s i s d e r i v e d from these sources.) Some of the e a r l i e s t treatments f o r e p i l e p s y were d e s c r i b e d by Hippocrates and h i s followers.? and much l a t e r by Galen. Both of these s c h o l a r s d i s m i s s e d the commonly h e l d n o t i o n t h a t e p i l e p s y was a form of demonic p o s s e s s i o n . There-f o r e , they p r e s c r i b e d s e c u l a r r a t h e r than s p i r i t u a l cures. T h e i r treatment f o r e p i l e p s y was d i e t a r y r e s t r a i n t based upon p a t h o l o g i c a l c o n s i d e r a t i o n . They f e l t t h a t i t was of v i t a l importance to keep d i g e s t i o n i n good o r d e r , o f t e n w i t h a c o n c o c t i o n of honey and v i n e g a r t h a t was drunk s e v e r a l times each day. I t i s noteworthy, t h a t from the 5th Century B.C.E. u n t i l the e a r l y t w e n t i e t h century, - 2 -every t r e a t i s e on e p i l e p s y s p e c i f i e d a r t i c l e s o f food t h a t should or should not be eaten. Hippocrates favoured a walk i n f r a g r a n t a i r and Galen sought e x o t i c d i e t a r y supplements. Galen's use of powdered human s k u l l s t a r t e d a p r a c t i s e t h a t was continued f o r a thousand years i n Europe. Another common p r e s c r i p t i o n was to d r i n k warm, human blo o d , which was u s u a l l y o b t a i n e d from a f a l l e n g l a d i a t o r i n Roman times. Blood from other sources continued to be p r e s c r i b e d i n Europe up to the Middle Ages. By the 4th Century A.D., the methods used to t r e a t e p i l e p s y i n c l u d e d d i e t e t i c , s u r g i c a l and pha r m a c o l o g i c a l techniques. Despite the te a c h i n g s of the H i p p o c r a t i c s c h o o l , which t h e o r i z e d the cause of s e i z u r e s to be due to 'excessive phlegm' o b s t r u c t i n g the passage of a i r to. the b r a i n , the demonic p o s s e s s i o n t h e o r i e s regained acceptance. T h i s r e b i r t h co-i n c i d e d w i t h the growing r o l e of the church i n the s o c i o -p o l i t i c a l framework of the Middle Ages. The gospel w r i t e r s recounted the t a l e of C h r i s t c u r i n g the e p i l e p t i c s by c a s t i n g out the demons w i t h h i s words. St. V a l e n t i n e became known as the patron s a i n t o f the e p i l e p t i c s , as were St. V i t u s and St. M i c h a e l , and those a f f l i c t e d with e p i l e p s y made pi l g r i m a g e s t o t h e i r p r i o r i e s , where h o s p i t a l s were b u i l t . The Church l e a d e r s even l i s t e d e p i l e p s y as one of the e i g h t c a r d i n a l contagious d i s e a s e s , which i n c l u d e d such heinous - 3 -d i s o r d e r s as bubonic plague, t u b e r c u l o s i s , s c a b i e s , anthrax and l e p r o s y . As P r o f e s s o r Siegmund A l b i c h , a 15th Century s c h o l a r proclaimed: "Therefore, n e i t h e r t a l k nor bathe w i t h them, s i n c e by t h e i r mere br e a t h they i n f e c t people." The m a j o r i t y of the p h y s i c i a n s d u r i n g the l a s t h a l f of the seventeenth century, and a g r e a t many i n the e a r l y p a r t of the e i g h t e e n t h century d i d not exclude demons or witches as the primary cause of e p i l e p s y . For those p h y s i c i a n s who d i d employ s e c u l a r c u r e s , the range of p h a r m a c o l o g i c a l remedies was o n l y as l i m i t e d as the.imagination of the p h y s i c i a n . The most o f t e n named herbs i n e p i l e p t i c cures i n c l u d e e l d e r , g a r l i c , m i s t l e t o e and peony. When peony began t o l o s e f a v o r , the v a l e r i a n r o o t was becoming i n c r e a s i n g l y important. The f a v o r i t e treatment of Thomas W i l l i s , a seventeenth century p r o f e s s o r of n a t u r a l p h i l o s o p h y a t Oxford, r e q u i r e d such e x o t i c s as powdered human s k u l l , dragon's blood, l i v e r of wolf and g a l l of boar w i t h d r i e d u r i n e . While these cures were, f o r the most part/ i n e f f e c t u a l , they were p r e f e r a b l e to the more r a d i c a l treatments of cautery and b r a i n surgery, which, more o f t e n than not, had secondary e f f e c t s t h a t were worse than the d i s e a s e i t s e l f . The n i n e t e e n t h century brought many advances f o r those s u f f e r i n g w i t h e p i l e p s y . For the f i r s t time e p i l e p t i c s were no longer c o n f i n e d to insane asylums. They were p r o v i d e d w i t h humane h o s p i t a l i z a t i o n t h a t p e r m i t t e d the. f i r s t s y s t e m a t i c , - 4 -c o n t r o l l e d s t u d i e s of the d i s o r d e r . From h i s experience i n French e p i l e p t i c h o s p i t a l s , E s q u i r o l d e s c r i b e d the importance of the p a t i e n t s ' confidence i n the d o c t o r ' s cure. T h i s p s y c h o l o g i c a l f a c t o r was more important than the a c t u a l treatment. The p r e v a i l i n g medical o p i n i o n i n the e a r l y n i n e t e e n t h century r e g a r d i n g e p i l e p s y was t h a t i t i n v o l v e d r e f l e x a c t i o n and c e r e b r a l angiospasm r e l a t e d to changes in the molecular s t a t e of the b r a i n . F u r t h e r , these changes were mediated by m a l n u t r i t i o n or p o i s o n i n g . Animal s t u d i e s by Kussmaul & Tenner d e s c r i b e d c o n v u l s i o n s due to 'sudden i n t e r r u p t i o n i n the n u t r i t i o n of the b r a i n ' . The f i r s t t r u l y e f f e c t i v e a n t i c o n v u l s a n t drug, sodium bromide, was i n t r o d u c e d by Charles Locock i n 1857. I t r e p l a c e d the i n e f f e c t u a l z i n c oxide treatment of t h a t time. While sodium bromide was an e f f e c t i v e treatment f o r e p i l e p s y , i t was not without s i d e e f f e c t s . P a t i e n t s c h r o n i c a l l y t r e a t e d w i t h t h i s drug complained of being sedated, had p s y c h i c d i s t u r b a n c e s , s k i n rashes and g a s t r i c di'S't'r-e'S's (Vida and Gerry, 197 3). Thus the search f o r a s a f e , e f f e c t i v e treatment f o r e p i l e p s y continued i n t o the t w e n t i e t h century, when the f i r s t p r a c t i c a l s y n t h e s i s of b a r b i t u r a t e s was achieved ( F i s h e r and Von Mering, 1903). I t was soon r e a l i z e d t h a t the h y p n o t i c p r o p e r t i e s possessed by b a r b i t a l were c h a r a c t e r i s t i c of a l l b a r b i t u r a t e s . A second b a r b i t u r a t e drug, p h e n o b a r b i t a l - 5 -(5-ethyl-5 p h e n y l b a r b i t u r i c acid) was i n t r o d u c e d i n t o medical p r a c t i c e by three independant r e s e a r c h e r s (Loewe, 1912; J u l i u s b u r g e r , 1912; Impens, 1912). In the same year A l f r e d Hauptmann r e c o g n i z e d the t h e r a p e u t i c v a l u e of p h e n o b a r b i t a l i n the treatment of e p i l e p s y (Hauptmann, 1912). I t i s s t i l l an important drug i n a n t i c o n v u l s a n t therapy, but i t s h y p n o t i c p r o p e r t y prevents p h y s i c i a n s from p r e s c r i b i n g h i g h enough doses to ensure f u l l p r o t e c t i o n a g a i n s t s e i z u r e s . Today the drug of c h o i c e i n the treatment of t o n i c -c l o n i c (grand mal) s e i z u r e s and p a r t i a l s e i z u r e s , whether f o c a l or not, i s d i p h e n y l h y d a n t o i n (DPH; D i l a n t i n ; r e g i s t e r e d trademark of Parke, D a v i s ) . The f i r s t s y n t h e s i s of 5,5-d i p h e n y l h y d a n t o i n was achieved by B l i t z (1908). I t s a n t i -c o n vulsant p r o p e r t i e s went unnoticed u n t i l M e r r i t t and Putnam (1938b)' demonstrated i t s e f f i c a c y a g a i n s t e l e c t r i c a l l y induced s e i z u r e s i n c a t s . L a t e r t h a t year they r e p o r t e d on the a n t i -c o n vulsant p r o p e r i t e s of DPH i n humans ( M e r r i t t and Putnam, 19 38a). T h i s drug i s p r e f e r r e d by many because i t i s non-s e d a t i v e and t h e r e f o r e tends to be l e s s t o x i c . The e f f e c t i v e n e s s of DPH i n c o n t r o l l i n g or e l i m i n a t i n g s e i z u r e s , shows t h a t medicine i n a s i n g l e century has come a long way by e l i m i n a t i n g the stigma attached to .the d i s e a s e and by r e p l a c i n g the c o n t r o v e r s i a l cures of more than a 1000 y e a r s . The p o s s i b i l i t y of more normal 1 : l i v e s f o r e p i l e p t i c s opened the prospect of marriage and c h i l d b e a r i n g to more - 6 -e p i l e p t i c woman than ever b e f o r e . T h i s i s p o s i n g , however, some new i s s u e s . The number of malformed c h i l d r e n born to e p i l e p t i c mothers has l e d many i n v e s t i g a t o r s to s p e c u l a t e t h a t a r e l a t i o n s h i p between e p i l e p s y , a n t i c o n v u l s a n t drugs and c o n g e n i t a l malformations e x i s t s . Although,there are r i s k f a c t o r s a s s o c i a t e d w i t h e p i l e p s y {anoxia, advanced maternal age, lower socio-economic s t a t u s and i n c r e a s e d i n c i d e n c e of hydramnios), which c o u l d be c o n t r i b u t i n g to the c o n g e n i t a l malformations ( F e d r i c k , 1973; Monson e t a l . , 1973; Janz, 1975), many suspect t h a t the malformations are i n s t e a d the r e s u l t of the t e r a t o g e n i c a c t i o n of the a n t i c o n v u l s a n t drugs. The next s e c t i o n w i l l examine the r e s u l t s of the s t u d i e s designed to r e v e a l these p o s s i b i l i t i e s . 2. SURVEY OF EPIDEMIOLOGICAL REPORTS UP TO 1975 Less than t h i r t y years a f t e r the c l i n i c a l i n t r o d u c t i o n of d i p h e n y l h y d a n t o i n , two German p h y s i c i a n s p u b l i s h e d a r e p o r t o u t l i n i n g the a s s o c i a t i o n between e p i l e p s y , a n t i c o n v u l s a n t drugs and c o n g e n i t a l malformations (Janz and Fuchs, 1964). T h i s survey of malformations amongst c h i l d r e n born to e p i l e p t i c mothers was completed s h o r t l y a f t e r the t h a l i d o m i d e (Kevadon) tragedy. A number of anecdotal case r e p o r t s f o l l o w e d , most not a b l y 'that of Meadow (1968) , who d e s c r i b e d s i x p a t i e n t s w i t h o r o f a c i a l c l e f t s whose mothers a l l r e c i e v e d DPH w h i l e pregnant. Of these s i x p a t i e n t s , four a l s o had c o n g e n i t a l h e a r t l e s i o n s . - 7 -U n t i l r e c e n t l y , a l l of these s t u d i e s on the outcome of e p i l e p t i c pregnancies have been r e t r o s p e c t i v e and i n c l u d e d d i f f e r e n t c o n t r o l groups, h i n d e r i n g comparisons between s t u d i e s . Table 1, drawn h e a v i l y from s i m i l a r t a b l e s i n Annegers e t a l . (1974) and Janz (1975), to which the reader i s r e f e r r e d f o r e x c e l l e n t review a r t i c l e s , i n c l u d e s the r e s u l t of e i g h t e e n surveys. The data have been t r a n s c r i b e d to f a c i l i t a t e p r e s e n t a t i o n i n t h i s format. In a number of s t u d i e s , the i n c i d e n c e of malformations was compared between e p i l e p t i c mothers r e c e i v i n g a n t i c o n v u l s a n t s and those who were not on drugs (Janz and Fuchs, 1964; Maroni and Markoff, 1969; S p e i d e l and Meadow, 1972; South, 1972; Lowe, 1973; F e d r i c k , 1973; StarresveId-Zimmerman e t a l . , 1973; Koppe e t a l . , 1973; Meyer, 1973; Monson e t a l . , 1973; Annegers e t a l . , 1974). The i n c i d e n c e of c o n g e n i t a l anomalies born to the t r e a t e d mothers i n these s t u d i e s ranges from a low of 2.2% (Janz and Fuchs, 1964) to a h i g h of 13.8% ( F e d r i c k , 1973), the average i n c i d e n c e being 7.2%. T h i s was n e a r l y twice the frequency (3.8%) recorded f o r e p i l e p t i c woman who were not on drugs d u r i n g t h e i r pregnancies. Two s t u d i e s show the'frequency of abnormal babies born to u n t r e a t e d mothers to be g r e a t e r than t h a t of t r e a t e d m o t h e r s , ( S t a r r e s v e l d -Zimmerman e t a l . , 1973; Meyer 1973). The s i g n i f i c a n c e of these o b s e r v a t i o n s i s u n c e r t a i n . In f i v e s t u d i e s t h a t had n o n - e p i l e p t i c p o p u l a t i o n s f o r c o n t r o l s there i s a t h r e e - f o l d i n c r e a s e i n the anomalies of c h i l d r e n born to t r e a t e d e p i l e p t i c s , over t h a t of the c o n t r o l p o p u l a t i o n (Elshove and - 8 -T a b l e 1. R e t r o s p e c t i v e s t u d i e s o n a n t i c o n v u l s a n t s a d m i n i s t e r e d t o e p i l e p t i c women a n d t h e f r e q u e n c y o f m a l f o r m a t i o n s a s c o m p a r e d w i t h t h e o c c u r r e n c e o f m a l f o r m a t i o n s i n u n t r e a t e d e p i l e p t i c s a n d i n n o n - e p i l e p t i c c o n t r o l s . L i v e b i r t h s t o t r e a t e d L i v e b i r t h s t o u n t r e a t e d m o t h e r s w i t h e p i l e p s y m o t h e r s w i t h e p i l e p s y R e f e r e n c e T o t a l M a l f n . % T o t a l M a l f n . J a n z & F u c h s (1964) 225 5 2.2 133 0 0 M a r o n i & M a r k o f f (1969) 21 1 4.8 14 0 0 E l s h o v e & Van E c k (1971) 65 10 15.4 - - -W a t s o n & S p e l l a c y (1971) 51 3 5.9 - - -S p e i d e l & Meadow (1972) 329 17 5.2 59 0 0 S o u t h (1972) 22 2 9.1 9 0 0 Lowe (1973) 134 9 6.7 111 3 2.7 F e d r i c k (1973) 217 30 13.8 19 2 10.5 K u e n s s b e r g & K n o x (1973) 48 3 6.2 _ _ _ M i l l a r & N e v i n s (1973) 110 7 6.4 _ _ S t a r r e s v e l d -Zimmerman e t a l . (19 7 3) 279 20 7.2 18 2 11.1 Koppe e t a l . (1973) 125 11 8.8 66 2 3.0 M e y e r (1973) 199 17 8.5 124 14 11 . 3 Monson e t a l . (1973) 205 11 5.5 101 3 3.0 B j e r k e d a l & B a h n a (1973) _ _ _ — _ N i s w a n d e r & W e r t e l e c k i (1973) _ _ _ _ A n n e g e r s e t a l . (1974) 141 10 7.1 56 1 1.8 B a i l e e t a l . (1975) 51 5 9.8 - - -T o t a l s 2222 161 7.2 711 27 3.8 - 9 -L i v e b i r t h s t o a l l e p i l e p t i c mothers T o t a l M a l f n . "5 L i v e b i r t h s to n o n - e p i l e p t i c c o n t r o l mothers T o t a l M a l f n . Q. "5 L i v e b i r t h s t o t a l p o p u l a t i o n T o t a l M a l f n . Q, *o 358 5 1.4 35 1 2.8 _ _ 65 0 0 12051 231 1.9 50 0 0 388 17 4.4 448 7 1.6 _ 31 2 6.4 _ — 7865 190 2.4 245 12 5.0 31877 877 2.8 236 32 13.6 649 36 5.5 — _ _ _ _ _ 14620 477 3.0 32227 1235 3.8 297 22 7.4 192 13 6.8 12300 426 3.5 323 31 9.6 — _ _ _ 306 14 4.6 50591 1240 2.5 _ _ 378 17 4.5 112328 2471 2.2 413 17 4.1 347097 9372 2.7 197 11 5.6 _ - - - - - - - - -3399 194 5.7 51083 1283 2.5 570365 15249 2.7 - 10 -Van Eck, 19 71; Watson and S p e l l a c y , 19 71; S p e i d e l and Meadow, 1972; F e d r i c k , 1973; Monson e t a l . , 1973) F e d r i c k (1973), i n her study,: was the o n l y major i n v e s t i g a t o r to u t i l i z e the n o n - e p i l e p t i c p o p u l a t i o n as case-c o n t r o l l e d s u b j e c t s . In most i n s t a n c e s the c o n t r o l mother was s e l e c t e d on the b a s i s o f s i m i l a r i t i e s w i t h the case p a t i e n t , i n terms of age, r a c e , p a r i t y , h o s p i t a l , year o f b i r t h and socio-economic s t a t u s ( F e d r i c k , 1973). When i t was not f e a s i b l e to employ the c a s e - c o n t r o l l e d techniques, other s t u d i e s used i n s t e a d the next h o s p i t a l b i r t h to a woman w i l l i n g to p a r t i c i p a t e i n . t h e s t u d i e s as c o n t r o l s (Watson and S p e l l a c y , 1971; S p e i d e l and Meadow, 1972) In o t h e r e p i d e m i o l o g i c a l s t u d i e s , i n v e s t i g a t o r s chose to use a l l h o s p i t a l b i r t h s d u r i n g a giv e n time i n t e r v a l (Elshove and Van Eck, 1971; South, 1972; Monson e t a l . , 1973), or a l l b i r t h s r e p o r t e d by g e n e r a l p r a c t i t i o n e r s (Kuenssberg and Knox, 1973). S t i l l o t h e rs used g e o g r a p h i c a l l y d e f i n e d r e g i o n s as c o n t r o l p o p u l a t i o n s (Lowe, 1973; M i l l a r and Nevins, 1973) . There are two l a r g e r e t r o s p e c t i v e s t u d i e s not mentioned so f a r , Niswander and W e r t e l e c k i (1973) and B j e r k e d a l and Bahna (197 3). The Niswander study r e p o r t e d a l l b i r t h s i n a m i l i t a r y h o s p i t a l i n .the years 1965 through 1971, and the l a t t e r study recorded a l l b i r t h s i n Norway - 11 -dur i n g 1967 and 1968. The i n c i d e n c e of malformations amongst c h i l d r e n born to women with diagnosed e p i l e p s y was 4.1% and 4.5%, r e s p e c t i v e l y . The comparable f i g u r e s i n the c o n t r o l p o p u l a t i o n s were 2.7% and 2.8%. U n f o r t u n a t e l y i n these s t u d i e s the e p i l e p t i c p a t i e n t s were not separated by t h e i r a n t i c o n v u l s a n t drug therapy s t a t u s . W i t h i n t h i s p o p u l a t i o n were women who, wh i l e diagnosed as being e p i l e p t i c , were not r e c e i v i n g a n t i c o n v u l s a n t drugs a t the time they were ascertained f o r t h i s survey.. T h i s may account f o r the lower i n c i d e n c e o f r e p o r t e d malformations than i n some of the o t h e r . s u r v e y s . The combined r e s u l t s of a l l e i g h t e e n surveys r e p o r t e d i n the l i t e r a t u r e by 1975 i n d i c a t e t h a t the r i s k o f producing c h i l d r e n w i t h malformations i s three times g r e a t e r f o r the e p i l e p t i c p o p u l a t i o n (7.2%) than i t i s f o r the ge n e r a l p o p u l a t i o n (2.7%). These f i g u r e s are based upon 2222 l i v e -b i r t h s t o t r e a t e d e p i l e p t i c mothers, and n e a r l y 600,0 00 l i v e b i r t h s r e p o r t e d i n . t h e g e n e r a l p o p u l a t i o n . The malform-a t i o n r a t e i n 711 l i v e b i r t h s to non-treated e p i l e p t i c women was 3.8%, a f i g u r e m a r g i n a l l y higher than f o r the ge n e r a l p o p u l a t i o n . T h i s has been a p o i n t o f c o n t e n t i o n i n many s t u d i e s , w i t h s e v e r a l authors c l a i m i n g t h a t e p i l e p t i c women are at an i n c r e a s e d r i s k r e g a r d l e s s of t h e i r drug therapy ( F e d r i c k , 1973; Monson e t a l . , 1973; Janz, 1975) and oth e r s m a i n t a i n i n g they are at no i n c r e a s e d r i s k ( S p e i d e l and Meadow, 1974), - 12 -While the r a t e o f malformations i n the group of t r e a t e d e p i l e p t i c s remains low, i t i s c l e a r l y h i g h e r than f o r the r e s t of the p o p u l a t i o n . The s p e c i f i c d e f e c t s t h a t are r e s p o n s i b l e f o r t h i s i n c r e a s e are o r o f a c i a l c l e f t s and c o n g e n i t a l h e a r t d e f e c t s (Table 2). The 1.8% i n c i d e n c e o f c l e f t l i p w i t h or without c l e f t p a l a t e , recorded f o r the c h i l d r e n of t r e a t e d e p i l e p t i c mothers, i s 7-13 times g r e a t e r than the f r e q u e n c i e s recorded f o r n o n - e p i l e p t i c c o n t r o l mothers i n the United King-dom (0.14%; S p e i d e l and Meadow, 1972), C a r d i f f (0.16%; Lowe, 1973), London (0.2%; South, 1972) and i n the Netherlands (0.27%; Elshove and Van Eck, 19 71). In e p i l e p t i c mothers not r e c e i v i n g a n t i c o n v u l s a n t s , t h e r e was not a s i n g l e case of c l e f t l i p , w i t h or without c l e f t p a l a t e , i n the 179 b i r t h s recorded i n the forementioned s t u d i e s ( S p e i d e l and Meadow, 197 2; Lowe, 1973; South, 1972). F u r t h e r , the average i n c i d e n c e of con-g e n i t a l h e a r t d e f e c t s among c h i l d r e n exposed p r e n a t a l l y to a n t i c o n v u l s a n t s i s f o u r f o l d g r e a t e r t h a t i t i s f o r a h e t e r -ogenous B r i t i s h p o p u l a t i o n ( S p e i d e l and Meadow, 1972) Not o n l y do the s t u d i e s examined p o i n t c o n c l u s i v e l y to an i n c r e a s e d r a t e o f malformation among o f f s p r i n g o f t r e a t e d e p i l e p t i c mothers (Table 1)', they a l s o r e v e a l , a c e r t a i n c o n s i s t e n c y i n the type o f malformation produced (Table 2). As a l l known teratogens i n man are a l s o t e r a t o -genic i n other s p e c i e s . ( S t a p l e s , 1972) i t i s l o g i c a l to i n i t i a l l y determine the t e r a t o g e n i c i t y of DPH i n lower animals. The f o l l o w i n g s e c t i o n e x p l o r e s the v a r i o u s animal - 13 -T a b l e 2. F r e q u e n c y o f d i f f e r e n t m a l f o r m a t i o n s i n c h i l d r e n o f m o t h e r s o n a n t i c o n v u l s a n t s d u r i n g p r e g n a n c y . MALFORMATIONS R e f e r e n c e No. o f P r e g n a n c i e s O r o f a c i a l c l e f t s C a r d i a c a n o m a l i e s A n e n -c e p h a l y J a n z & F u c h s (1964) 225 3 1 M a r o n i & M a r k o f f (1969) 21 E l s h o v e & Van E c k (1971) 65 5 2 1 W a t s o n & S p e l l a c y (1971) 51 1 S p e i d e l & Meadow (1972) 329 3 6 S o u t h (1972) 22 2 Lowe (1973) 134 1 1 1 F e d r i c k (1973) 217 1 2 K u e n s s b e r g & Knox (1973) 48 Monson e t a l . (1973) 205 2 3 1 M i l l a r & N e v i n (1973) 110 2 S t a r r e s v e l d -Zimmerman e t a l . (1973) 279 9 7 1 Koppe e t a l . (1973) 125 1 4 Me y e r (1973) 199 5 5 M i r k i n (1973) 7 2 H i l l e t a l . (1974) 28 1 2 A n n e g e r s e t a l . (1974) 141 3 8 B i a l e e t a l . (1975) 51 1 2 TOTAL 2257 41 44 4 PERCENTAGE 1.8 2.0 0.2 @ As c o m b i n a t i o n s o f m a l f o r m a t i o n s o c c u r , t h e t o t a l numbers o f c a s e s c a n be l o w e r t h a n t h e sum o f t h e d i f f e r e n t m a l f o r m a t i o n s s t a t e d . - 14 -M i c r o -c e p h a l y N e u r a l d e f e c t s H y d r o -c e p h a l y Hypo-s p a d i a s S k e l e t a l a n o m a l i e s G . I . d e f e c t s m i s c . @ # c a s e s m a l f n s . _ _ _ _ _ 1 _ 5 _ _ _ 1 _ _ _ 1 _ 1 _ _ 1 _ 1 ' 10 _ _ 1 _ _ _ 1 3 3 _ _ 2 1 2 2 17 - - - - - - - 2 - 1 - - 1 - 3 9 - 1 - 2 6 1 8 20 _ 1 1 _ 1 _ 3 1 - 2 - 3 - - 11 _ 1 _ — _ _ 4 7 _ 1 — 1 _ 1 20 - - - - - - 6 11 - 1 - - 7 - - 17 1 - - - - - - 3 - 1 - 1 - 1 1 7 - - - - - - 1 10 - - • - - 1 - 1 5 5 7 4 7 19 6 29 171 0.2 0.3 0.2 0.3 0.8 0.3 1.3 7.6 - 15 -s t u d i e s t h a t have been performed and the types and f r e q u e n c i e s o f the malformations t h a t have been produced. 3. SURVEY OF ANIMAL EXPERIMENTS UP TO 197.7. While the e p i d e m i o l o g i s t s and c l i n i c i a n s ammassed l a r g e r and l a r g e r numbers of p a t i e n t s f o r t h e i r surveys, a number of s t u d i e s were c a r r i e d out to determine the t e r a t o g e n -i c i t y of DPH i n other s p e c i e s . Massey (1966) i n j e c t e d A/J mice d a i l y w i t h DPH subcutaneously on g e s t a t i o n a l days 9-15 i n c l u s i v e . Using 1 2 . 5 , 25 and 50 mg/kg of body weight doses, she r e p o r t e d t h a t 42.8% of the o f f s p r i n g o f t r e a t e d mice h a d . o r o f a c i a l c l e f t s . T h i s was s i g n i f i c a n t l y h i g h e r than the 8-10% spontaneous r a t e ^ o f occurrence i n t h i s s t r a i n of mice. Gibson and Becker, .'(196 8) c a r r i e d out s i m i l a r i n v e s t -i g a t i o n s w i t h the s t r a i n s Swiss and A/J. They found o r o f a c i a l c l e f t s ( 1 5 . 2 % and 30.8%:, r e s p e c t i v e l y ) and reduced f e t a l s i z e and body weight when the dams were subcutaneously i n j e c t -ed with 50rmg/kg body weight dosages of DPH on days 11-13 of g e s t a t i o n . T h i s was not observed i n the mice t r e a t e d on days 9-11. F u r t h e r , when the dams r e c e i v e d DPH on days 1 1 - 1 3 , the A / J s t r a i n showed an i n c r e a s e i n r e s o r p t i o n to 35.4% compared wi t h 1 4 . 3 % i n the c o n t r o l s . Elshove (1969) found lower body weights and an i n c r e a s e d i n c i d e n c e ( 1 5 . 3 % ) o f c l e f t p a l a t e i n Swiss mice when t r e a t e d w i t h i n t r a p e r i t o n e a l - 16 -(ip) i n j e c t i o n s of 2.5, 1.9, or 1.75 mg of DPH. Interesting^" l y , when the mice were treated on days 10-13, there was 100% resorption, compared with a 15% incidence for mice that were injected on days 11-14. These studies indicate that the c r i t i c a l period for DPH teratogenicity, i s during the gestational days 10-14. Harbison and Becker (1969) administered various doses of DPH to mice at d i f f e r e n t gestational times. The dams were treated with either a single i p i n j e c t i o n of 150 mg/kg on days 8-15, or were given three subcutaneous injections i n lower doses on either days 8-10 or 12-14. Amongst the dams receiving single i n j e c t i o n s , those treated on days 10 or 14 had an 80% resorption rate, while those recieving DPH on other days had no increase i n the number of implants resorbed. A l l of the treated fetuses had low term weights and reduced crowMrump length, which was primarily the r e s u l t of shortening of the long bones. Harbison and Becker were the f i r s t to report defects other than c l e f t palate i n the offspring of dams treated with DPH. At the two higher dose lev e l s used i n t h e i r study (100 and 150 mg/kg), they reported c l e f t l i p , c l e f t palate, hydronephrosis, renal and intraperoneal hemorrhage and delayed o s s i f i c a t i o n and/or unfused sternabrae i n over 20% of progeny of treated dams. Other less commonly observed malformations included open eye/f ectrodactyly, i n t e r n a l hydrocephalus and - 17 -s p l i t c e r v i c a l c e n t r a . The mice t h a t were i n j e c t e d on g e s t a t i o n a l days 12-14 had e l e v a t e d f r e q u e n c i e s of o r o f a c i a l c l e f t s , while there were no malformations induced by t r e a t i n g the mothers on days 8-10. I n v e s t i g a t o r s at Guy's H o s p i t a l i n London s t u d i e d the t e r a t o g e n i c i t y of most of the commonly p r e s c r i b e d a n t i -c onvulsants e i t h e r by i n t u b a t i n g mice w i t h 40 or 120 mg/kg dose, or by mixing i n 250 mg/kg i n t o t h e i r feed on days 6-16. They found t h a t the i n c i d e n c e of c l e f t p a l a t e was e l e v a t e d when the mice r e c e i v e d the two h i g h e r doses ( S u l l i v a n and McElhatton, 1975). They a l s o noted a decrease i n f e t a l weights, but as the l i t t e r s i z e of the dams i n c r e a s e d , they were r e l u c t a n t t o a t t r i b u t e the decreased f e t a l weight to the a c t i o n of the drug. In a subsequent study ( S u l l i v a n and McElhatton, 19 77) these same i n v e s t i g a t o r s a dministered 15, 45 and 90 mg/kg DPH, i n t u b a t i n g dams on days 6-16. Though t h e r e was no i n c r e a s e i n the number of implants per l i t t e r o r i n .the r a t e of r e s o r p t i o n s i n the dams on DPH treatment, malformations were f r e q u e n t l y observed i n t h e i r o f f s p r i n g . Open eye d e f e c t s were seen i n f i v e of the seventeen l i t t e r s on the 4 5 mg/kg dose. At the h i g h e s t treatment l e v e l , reduced f e t a l weights, delayed o s s i f i c a t i o n of the hands (43%) and f e e t (62%), enl a r g e d c e r e b r a l v e n t r i c l e s and separated b a s i s p h e n o i d bones - 18 -w e r e t h e most;commonly o b s e r v e d d e f e c t s . When t h e s e r e s u l t s a r e c a l c u l a t e d o n a mean p e r c e n t a g e l i t t e r b a s i s , 42% o f t h e l i t t e r s h a d d e f e c t s a t t h e h i g h e r dose.:- Of t h e u n t r e a t e d c o n t r o l l i t t e r s , o n l y 0.62% h a d f e t u s e s w i t h m a l f o r m a t i o n s . An e x p l a n a t i o n f o r t h e a p p a r e n t l a c k o f i n c r e a s e i n t h e r e s o r p t i o n r a t e s i n t r e a t e d m o t h e r s was a d v a n c e d b y F r i t z a n d h i s c o - w o r k e r s ( 1 9 7 6 ) . They f e l t t h a t DPH c a u s e s e a r l y e m b r y o n i c l o s s , a n d t h a t t h e d r u g i s n ' t e m b r y o l e t h a l when a d m i n i s t e r e d a f t e r g e s t a t i o n a l d a y 14, I n t h e i r s t u d y t h e y p l a c e d t h e mouse u t e r i i n a 20% a q u e o u s s o l u t i o n o f ammonium s u l p h i d e t o v i s u a l i z e d e c i d u o m a t a t h a t w o u l d r e p r e s e n t e a r l y e m b r y o n i c r e s o r p t i o n . I n f o u r o f t h e dams t r e a t e d w i t h lOOmg/kg ( 1 3 % ) , ; a n d i n n i n e o f t h e dams on 170: mg/kg o f DPH (53%) o n l y d e c i d u o m a t a w e r e v i s i b l e . A f t e r t h e f o u r t e e n t h d a y o f g e s t a t i o n , e m b r y o n i c l o s s r e p r e s e n t e d o n l y 3% o f t h e t o t a l number o f i m p l a n t s i n t h e h i g h e s t DPH t r e a t e m e h t g r o u p . A l l t h e s e a n i m a l s t u d i e s c o n f i r m t h e s u s p e c t e d t e r a t o g e n i c p o t e n t i a l o f a n t i c o n v u l s a n t s o u t l i n e d i n t h e p r e v i o u s s e c t i o n . W h i l e o r o f a c i a l c l e f t s a r e t h e m o s t c o n s i s t e n t l y r e p o r t e d d e f e c t ( M a s s e y , 1966; G i b s o n and B e c k e r , 1968; H a r b i s i o n and B e c k e r , 1 9 6 9 , 1972; S u l l i v a n and M c E l h a t t o n , 1 9 7 5 , 1977; F r i t z e t a l . , 1 9 7 6 ) , s u c h a n o m a l i e s a s i n t e r n a l h y d r o c e p h a l u s , and o c u l a r d e f e c t s ( H a r b i s o n . . a n d B e c k e r , 19 69; S u l l i v a n a n d M c E l h a t t o n , 197 7) - 19 -have a l s o been observed. For the most p a r t , however, those malformations observed a t very h i g h doses a c t u a l l y r e p r e s e n t the. normal c o n d i t i o n s of a l e s s than eig h t e e n day mouse. 4. THE FETAL HYDANTOIN SYNDROME: 1975-1978 The e p i d e m i o l o g i c s t u d i e s d e s c r i b e d i n Secion 2 were designed p r i m a r i l y to i n v e s t i g a t e the a s s o c i a t i o n : , between maternal hydantoin consumption and s i n g l e d e f e c t s , such as c o n g e n i t a l h e a r t d e f e c t s or f a c i a l c l e f t s . However, th e r e were the o c c a s i o n a l r e p o r t s t t h a t i n d i c a t e d a broader range of malformations a s s o c i a t e d w i t h p r e n a t a l exposure to hydantoins (Meadow, 1970; S p e i d e l and Meadow, 1972; Loughnan e t a l . , 1973; H i l l e t al.. , 1974; Barr e t . a l . , 1974). In 1975, Hanson and Smith observed f i v e u n r e l a t e d c h i l d r e n at the Dysmorphology U n i t , U n i v e r s i t y of Washington, wi t h s i m i l a r dysmorphic f e a t u r e s , a l l of whom had been exposed i n u t e r o to hydantoin a n t i c o n v u l s a n t s . As t h i s p a t t e r n of malformations was d i s t i n c t from ot h e r malformation.-:: syndromes and had been observed o n l y i n the o f f s p r i n g o f women t a k i n g hydantoins, i t was r e f e r r e d to as the " f e t a l hydantoin syndrome''. These o r i g i n a l f i v e cases o f the f e t a l hydantoin syndrome showed a p a t t e r n of malformations t h a t i n c l u d e d c h a r a c t e r i s t i c ^ c r a n i o f a c i a l a b n o r m a l i t i e s such as a broad, low, n a s a l b r i d g e , e p i c a n t h i c f o l d s , s h o r t upturned nose, - 20 -o c u l a r h y p e r t e l o r i s m , p t o s i s or strabismus, prominent, low-set e a r s , wide mouth wit h t h i c k f l e s h y l i p s , t r i g o n -ocephaly w i t h metopic s u t u r a l : ; r i d g i n g and wide f o n t a n e l s . The s k e l e t a l d e f e c t s i n c l u d e d h y p o p l a s i a of the d i s t a l phalanges and n a i l s , a f i n g e r - l i k e thumb, and dermato-g l y p h i c p a t t e r n s c h a r a c t e r i z e d by low-arch r i d g e p a t t e r n s . Anomalies of the r i b s , sternum, and p o s i t i o n a l limb deform-i t i e s (calcaneovalgus d e f o r m i t y and pes c a v i s ) were a l s o observed. There i s a definite.^growth d e f i c i e n c y t h a t was p r e n a t a l i n i t s o r i g i n . P o s t n a t a l l i n e a r growth was a l s o s e v e r e l y r e t a r d e d ; 75% of normal. Developmental performance i s d e f i c i e n t , w i t h m i l d to moderate mental r e t a r d a t i o n u s u a l l y a s s o c i a t e d c w i t h microcephaly. Other f e a t u r e s , t h a t are commonly found i n these p a t i e n t s but are not c o n s i s t a n t as the f ofementioned f e a t u r e s , .".include: u m b i l i c a l and i n g u i n a l h e r n i a s , g e n i t a l anomalies i n c l u d i n g hypospadias, p i l o n d i a l sinus, o r o f a c i a l c l e f t s , r e n a l d e f e c t s and cardiac anomalies such as a t r i a l s e p t a l d e f e c t s , v e n t r i c u l a r s e p t a l d e f e c t s and pulmonary s t e n o s i s . R e t r o s p e c t i v e s t u d i e s p u b l i s h e d p r i o r to the d e s c r i p t i o n of the f e t a l hydantoin syndrome c o u l d o n l y c i t e r i s k f i g u r e s f o r t r e a t e d e p i l e p t i c mothers based upon the 7.2% frequency of malformations r e p o r t e d i n the o f f s p r i n g " , of e p i l e p t i c mothers on a n t i c o n v u l s a n t drug therapy. Since t h i s f i g u r e was d e r i v e d p r i m a r i l y from the i n c i d e n c e of f a c i a l c l e f t s and c o n g e n i t a l h e a r t l e s i o n s , and s i n c e these d e f e c t s - 21 -are but a s m a l l p o r t i o n of the anomalies comprising the f e t a l hydantoin syndrome, t h i s r i s k f i g u r e .would not be a c c u r a t e . Moreover, r e t r o s p e c t i v e s t u d i e s g e n e r a l l y r e l y upon a c l i n i c a l e v a l u a t i o n of an i n f a n t a t the time of or s h o r t l y a f t e r i t s b i r t h . C e r t a i n anomalies./ e s p e c i a l l y c a r d i a c d e f e c t s often, go undiagnosed u n t i l the c h i l d i s one year of age or o l d e r . Reporting s i n g l e malformations, as i s commonly done i n r e t r o s p e c t i v e s t u d i e s , has f a i l e d i n the p a s t to d e t e c t the major teratogens i n man (Hanson e t a l . , 1976). Most teratogens g i v e r i s e to a p a t t e r n of m u l t i p l e d e f e c t s , t h e r e f o r e , i t i s f a r b e t t e r to search f o r a p a t t e r n of malformations, r a t h e r than f o r a s i n g l e d e f e c t such as c l e f t p a l a t e . The most c o n s i s t e n t f e a t u r e s of t eratogens i n humans are p r e - and p o s t - n a t a l growth d e f i c i e n c i e s . These f e a t u r e s are o f t e n overlooked. Even i n the most thorough of the r e c e n t l y completed r e t r o s p e c t i v e s t u d i e s , the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t of the N a t i o n a l I n s t i t u t e of N e u r o l o g i c a l and Communicative D i s o r d e r s and Stroke, these more s u b t l e anomalies are not r e p o r t e d (Heinonen e t a l . , 1977). On the other hand, p r o s p e c t i v e s t u d i e s undertaken by i n v e s t i g a t o r s s p e c i f i c a l l y i n t e r e s t e d i n the t e r a t o g e n -i c i t y o f a n t i c o n v u l s a n t drugs have r e p o r t e d a frequency of malformations as high as 35.7% i n the o f f s p r i n g of t r e a t e d - 22 -e p i l e p t i c s ( M i r k i n , 1 9 7 1 ; H i l l . ; e t a l . , 1974; Hanson e t ; . a l . , 1 9 7 6 ) . T h i s i s a f o u r f o l d i n c r e a s e over the frequency d e r i v e d from the r e t r o s p e c t i v e s t u d i e s . Again these f i g u r e s may be questioned, as.:they might be e l e v a t e d s i n c e these p h y s i c i a n s are among the p r i n c i p a l proponents of the concept of the f e t a l hydantoin syndrome. The d i f f e r e n c e between p r o s p e c t i v e and r e t r o s p e c t i v e e p i d e m i o l o g i c a l techniques can l e a d to c o n f l i c t i n g c o n c l u s i o n s drawn from the same data source. For example, the study o f Shapiro and c o l l e a g u e s (1976) was based upon case" h i s t o r i e s c o l l e c t e d i n the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t . Shapiro e t a l . claimed t o have found ho evidence t h a t the major malformations amongst o f f s p r i n g of t r e a t e d e p i l e p t i c mothers were a s s o c i a t e d w i t h a n t i c o n v u l s a n t therapy. They concluded t h a t i t was p a r e n t a l e p i l e p s y , e i t h e r the mother or the f a t h e r , t h a t was r e s p o n s i b l e , :.ahd not the me d i c a t i o n . T h i s c o n c l u s i o n has been r e p u d i a t e d (Hanson e t a l . , 1976; Dansky, 1977) . Hanson e t a l . (1976) a l s o usedt'the r e s o u r c e s of the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t to o b t a i n 104 matched-c o n t r o l mothers i n t h e i r study to determine the frequency and v a r i a b i l i t y o f the f e t a l hydantoin syndrome. Though many of the dysmorphic f e a t u r e s c h a r a c t e r i s t i c of the f e t a l hydantoin syndrome were not e v a l u a t e d i n the Col-labor:-.:: a t i v e P r o j e c t , enough evidence was a v a i l a b l e f o r c o n f i d e n t diagnoses o f the syndrome i n 11 of the 104 c h i l d r e n (11%) born t o hydantoin t r e a t e d mothers. - 23 -Hanson e t a l . (1976) a l s o f o l l o w e d 23 S e a t t l e area e p i l e p t i c women on hydantoin drug therapy- through 35 pregnancies. They based t h e i r d i a g n o s i s of the f e t a l hydan-t o i n syndrome on the presence of a t l e a s t t hree of the fou r f e a t u r e s they c o n s i d e r to be d e f i n i t i v e of the syndrome. These are p r e n a t a l growth d e f i c i e n c y , p o s t n a t a l growth d e f i c i e n c y , microcephaly and mental r e t a r d a t i o n . On t h i s b a s i s , they found four c h i l d r e n (11%) wit h the f e t a l hydantoin syndrome, and an a d d i t i o n a l 11 c h i l d r e n (31%) wit h anomalies c o n s i s t e n t w i t h p r e n a t a l exposure t o hydantoins (Hanson e t a l . , 197 6). The d i a g n o s i s of the f e t a l hydantoin syndrome i n the o f f s p r i n g of women .receiving hydantoins d u r i n g pregnancy i n the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t was comparable with the r e s u l t s o f the S e a t t l e area study. The r i s k f i g u r e f o r the syndrome was 11%. To c l e a r l y separate the ^ t e r a t o g e n i c e f f e c t df the drug from t h a t of the d i s o r d e r , a l a r g e p r o s p e c t i v e study would be r e q u i r e d . T h i s study must i n c l u d e e p i l e p t i c women . not r e c e i v i n g a n t i c o n v u l s a n t ..drugs, but wit h the same s e v e r i t y o f the d i s o r d e r as those women r e c e i v i n g a n t i c o n -v u l s a n t therapy. An a d d i t i o n a l c o n t r o l group o f n o n - e p i l e p -t i c women i s needed, f o r the i n c r e a s e d r i s k a s s o c i a t e d . c a n on l y be determined by comparing the malformation r a t e s i n c h i l d r e n born to unt r e a t e d e p i l e p t i c women to those of the n o n - e p i l e p t i c p o p u l a t i o n . To make a group o f e p i l e p t i c women.not r e c e i v i n g a n t i c o n v u l s a n t s would imply withdrawing - 24 -the medication from them. This would r a i s e e t h i c a l c o n s i d e r a t i o n s . On the other hand, does not the p r e s c r i p t i o n of a suspective teratogen to a pregnant woman r a i s e i t s own s e r i e s of e t h i c a l questions? Therefore, an animal model tha t c l o s e l y ^ p a r a l l e l s the human s i t u a t i o n could circumvent t h i s dilemma and other such problems inherent i n research on human populations. 5. PURPOSE AND RATIONALE OF THE PRESENT STUDY E p i l e p t i c women make up approximately 0.3 to 0.5% of a l l pregnancies (Janz, 1975). Data from the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t suggests t h a t 2 per 1000 b i r t h s are i n f a n t s exposed to hydantoins (Hanson et a l . , 1976). Despite n e a r l y h a l f a century of experience w i t h the ant i c o n v u l s a n t DPH, i t s t e r a t o g e n i c p o t e n t i a l i n man i s s t i l l u n c e r t a i n . The human stu d i e s are confounded w i t h the heterogeneity of e p i l e p s y , the types and combinations of an t i c o n v u l s a n t drugs and t h e i r doses, and the methodology of p a t i e n t ascertainment. The animal stud i e s have been e q u a l l y unrewarding. In these s t u d i e s the drug was most o f t e n administered i n s i n g l e doses, or on s e l e c t e d days of g e s t a t i o n i n doses w e l l i n excess of maximal human the r a p e u t i c dose (Massey, 1966; Gibson and Becker, 1968; Harbison and Becker, 1969, 1972; S u l l i v a n and McElhatton, 19 75, 1977; F r i t z e t a l . , 1976). The route of a d m i n i s t r a t i o n : was most f r e q u e n t l y subcutaneous or i n t r a p e r i t o n e a l i n j e c t i o n or g a s t r i c i n t u b a t i o n . While such procedures produced - 25 -the a n t i c i p a t e d malformations, i t may traumatize the pregnant animal enough so as to confound the r e s u l t s . The'.inherent inadequacies of both the animal and human s t u d i e s and the complexity of the r e l a t i o n between the drug e f f e c t and the maternal metabolism j u s t i f i e s the development of an animal model. Should t h i s model produce the same spectrum of malformations d e f i n e d as the f e t a l hydantoin syndrome, i t would be i n v a l u a b l e i n answering q u e s t i o n s as fundamental as the a c t u a l e t i o l o g y of the d e f e c t s , as w e l l as the important c l i n i c a l problem of d e l i n e a t i n g those segments of the e p i l e p t i c p o p u l a t i o n who are at an i n c r e a s e d r i s k of producing a malformed c h i l d . A v a l i d animal model."of the f e t a l hydantoin syndrome should meet'".the f o l l o w i n g c r i t e r i a : 1. The animal must have spontaneous s e i z u r e s because of the p o s s i b l e b i o c h e m i c a l v a r i a n t s s p e c i f i c to the e p i l e p t i c c o n d i t i o n t h a t c o u l d i n f l u e n c e the route and r a t e of DPH metabolism. 2. The spontaneous s e i z u r e s must be c o n t r o l l e d or eliminatedr.by DPH treatment. 3. The drug must me administered o r a l l y . Human e p i l e p t i c s take D i l a n t i n more o f t e n as a p i l l or as a syrup than by i n j e c t i o n . 4. Serum l e v e l s must be monitored and a d j u s t e d to f a l l w i t h i n the o p t i m a l human t h e r a p e u t i c range between 5 and 20 - 26 -micrograms per ml serum (Miles et a l . , 1976; Jeavons, 1977). As there i s nol;constaht r a t i o between the amount of DPH admin-i s t e r e d and the serum c o n c e n t r a t i o n , i t i s important to deter-mine the blood serum l e v e l s of the drug.(Janz, 1975). 5. The DPH must be administered to the animal before mating and treatment must be continued throughout g e s t a t i o n . Studies on human e p i l e p t i c s have shown th a t when d a i l y t h e r a -p e u t i c doses are given o r a l l y , the drug accumulates and the serum concentration increases s l o w l y u n t i l a s t a b l e l e v e l i s reached when inta k e and e l i m i n a t i o n are equal,(Buchthal and Lennox-Buchthal, 1972). I t g e n e r a l l y takes 5 to 7 days at low doses, and somewhat longer at higher doses to.achieve the optimal t h e r a p e u t i c range (Pl a a , 1975; Harbson and Becker, 19 69). This time p e r i o d allows f o r the i n d u c t i o n of enzyme systems a s s o c i a t e d w i t h DPH metabolism t h a t cause the charact-e r i s t i c f a l l i n serum IgA and f o l i c a c i d l e v e l s reported i n chronic DPH use (Janz, 1975). 6. The model r e q u i r e s that the o f f s p r i n g of the tr e a t e d animals must e x h i b i t those malformations observed i n the o f f s p r i n g of e p i l e p t i c women on hydantoin therapy. Further, the frequency of the malformation must be s i g n i f i c a n t l y higher than t h e i r spontaneous occurrence. 7. A dose-responsive curve from 0 to 100% a f f e c t e d o f f s p r i n g , c h a r a c t e r i s t i c of t e r a t o g e n i c agents, should be observed„(Wilson, 1965). An animal model c l o s e l y approximating the human s i t u a t i o n - 27 -by meeting the aforementioned c r i t e r i a , should p r o v i d e i n s i g h t i n t o the t e r a t o g e n i c p o t e n t i a l of DPH. CHAPTER I I METHODOLOGY ANIMALS C 5 7 B 1 / 6 J , +/qk m i c e (Mus m u s c u l u s ) w e r e o b t a i n e d f r o m t h e J a c k s o n L a b o r a t o r i e s , B a r H a r b o r , M a i n e a n d h o u s e d w i t h t h e M e d i c a l G e n e t i c s b r e e d i n g c o l o n y i n t h e Z o o l o g y V i v a r i u m , U n i v e r s i t y o f B r i t i s h C o l u m b i a . From t h e s e a n i m a l s two b r e e d i n g l i n e s w e r e m a i n t a i n e d . One l i n e , C 5 7 B 1 / 6 J , +/+, t h e c o n t r o l m i c e w e r e i n t h e i r f o u r t h g e n e r a t i o n a t t h e commencement o f t h i s p r o j e c t . The o t h e r l i n e c o n s i s t e d o f C 5 7 B 1 / 6 J , +/qk and C 5 7 B 1 / 6 J , q k / q k m i c e , a l s o i n t h e i r f o u r t h g e n e r a t i o n . A l l a n i m a l s w e r e h o u s e d i n s t a n d a r d c l e a r p o l y c a r b o n a t e c a g e s , f e m a l e s i n p a i r s a n d m a l e s w i t h s i b s , a n d m a i n t a i n e d o n a 12 h o u r l i g h t c y c l e . T hey w e r e a l l o w e d a d l i b i t u m a c c e s s t o P u r i n a l a b o r a t o r y chow and t a p w a t e r u n l e s s o t h e r w i s e i n d i c a t e d . DPH TREATMENT LEVELS D r u g t r e a t m e n t w i t h 5 , 5 - D i p h e n y l h y d a n t o i n s o d i u m (DPH'; S i g m a C h e m i c a l s , S t . L o u i s , Mo.) was a d m i n i s t e r e d i n f o u r d o s e l e v e l s , 0, 2 0 , 40 and 60 mg/kg b o d y w e i g h t . W h i l e t h e s e l e v e l s may seem e x c e s s i v e i n c o m p a r i s o n t o t h e s t a n d a r d human t h e r a -p e u t i c d o s e o f 3.5 t o 10 mg/kg ( B u c h t h a l , and L e n n o x - B u c h t h a l , 1972) t h e m e t a b o l i c a c t i v i t y o f t h e mouse i n o x i d i z i n g d r u g s i s 20 t o 30 t i m e s h i g h e r ( B r o d i e e t a l . , 1 9 5 8 ) . When DPH i s g i v e n t o m i c e i n human d o s a g e s , i t i s e l i m i n a t e d s o r a p i d l y t h a t t h e s e r u m DPH l e v e l s f a l l b e l o w t h e s p e c i f i c i t y o f t h e SYVA E m i t spectrophotometric assay technique (SYVA, Palo A l t o , Cal.) used to measure serum DPH l e v e l s throughout the study. The lowest dose given to the dams tha t produced measurable serum DPH l e v e l s was 20 mg/kg. The highest dose l e v e l t h a t could be given to the dams without causing severe t o x i c i t y was 60 mg/kg. At t h i s dose the m a j o r i t y of females were f r e e of any c l i n i c a l manifesta-t i o n s of DPH i n t o x i c a t i o n , though some were l e t h a r g i c , i n d i c a t -i ng a m i l d t o moderate degree of t o x i c i t y . An intermediate dose of 40 mg/kg was included to comply w i t h the World Health Organization g u i d e l i n e s f o r teratogen t e s t i n g (Wilson, 1967). CALCULATION OF DRUG DOSES A stock s o l u t i o n of DPH c o n s i s t i n g of 1 mg DPH sodium to 1 ml tap water was made f r e s h d a i l y . The drug was measured out to one ten-thousandth of a gram i n a S a r t o r i o u s s i n g l e beam balance and was then t r a n s f e r r e d to Erlenmeyer F l a s k s . A f t e r the a d d i t i o n of an appropriate q u a n t i t y of water, the con tents of the f l a s k were repeatedly a s p i r a t e d by a pasteur p i p e t t to ensure t h a t the drug had completely d i s s o l v e d i n t o s o l u t i o n . The d e s i r e d q u a n t i t y of stock s o l u t i o n was measured out w i t h a disposable 10 ml s e r o l o g i c a l p i p e t t e , and was then d i l u t e d to a t o t a l of 50 ml. For those experiments i n v o l v i n g non-pregnant mice the d r i n k i n g s o l u t i o n was administered i n an in v e r t e d 50 ml B-D p l a s t i c syringe w i t h the needle end sealed by m e l t i n g , w i t h standard g l a s s d r i n k i n g tubes of 1.5 mm openings. For the l a t e r experiments w i t h pregnant dams, the d a i l y DPH water s o l u t i o n was p l a c e d i n F a l c o n 50 ml c o n i c a l graduated c e n t r i f u g e tubes w i t h a standard g l a s s d r i n k i n g tube. The amount o f stock s o l u t i o n r e q u i r e d f o r each cage was determined by c a l c u l a t i n g the animals' weight, average water consumption, and the treatment l e v e l i t was to r e c e i v e . Conversion t a b l e s were e s t a b l i s h e d t o f a c i l i t a t e r a p i d c a l c u l a -t i o n s . The f i r s t c o n v e r s i o n t a b l e (Appendix A) g i v e s the amount of DPH a mouse of a g i v e n weight must consume to be r e c e i v i n g the d e s i r e d dosage. The second c o n v e r s i o n t a b l e (Appendix B) c o r r e c t s f o r the a n i m a l s 1 f a i l u r e t o consume 50 ml of l i q u i d d a i l y . The product o f the valu e s obtained from these t a b l e s i s the amount o f stock s o l u t i o n r e q u i r e d f o r a g i v e n cage. A d e t a i l e d e x p l a n a t i o n of the usage of these t a b l e s can be found i n Appendix C. DIET ADMINISTRATION V i r g i n females 60 to 90 days o l d were used i n both the pha r m a c o l o g i c a l s t u d i e s on non-pregnant females and the t e r a t o l o g i c a l s t u d i e s . Although the ph a r m a c o l o g i c a l p r o p e r t i e s of DPH have been w e l l d e s c r i b e d i n both man and mouse (Gerber and A r n o l d , 1969; Kutt and Ve r e b e l y , 1970; K u t t , 1 9 7 1 ; Gerber and Lynn, 1972; Frey and Kampmann, 19 65; Glazko, 19 75) i t was necessary t o c h a r a c t e r i z e fundamental drug r e a c t i o n s i n s t r a i n C57B1/6J p r i o r to the s t a r t of the t e r a t o l o g i c a l experiments. For a l l s t u d i e s the mice were g r a d u a l l y i n t r o d u c e d to the drug to avo i d weight l o s s , s i c k n e s s and r e f u s a l t o d r i n k , which have been r e p o r t e d i n mice given l a r g e doses (0.3 mg/ml) of DPH (Frey and Kampmann, 1965). In order t o c o n s t r u c t a dose-response curve, the v a r i o u s doses of DPH were administered t o non-pregnant mice over a 28 day p e r i o d . The mice s t a r t e d out on treatment l e v e l 1 (0 mg/kg) f o r seven days, d u r i n g which t h e i r b a s e l i n e water consumption was determined. They were then e l e v a t e d i n a s t e p -wise f a s h i o n through the remaining three treatment l e v e l s . The mice remained at each l e v e l f o r seven days which allowed ample time f o r them t o become t o l e r a n t to the drug (Frey and Kampmann, 1965) and f o r t h e i r plasma DPH c o n c e n t r a t i o n t o reach a p l a t e a u (Plaa, 1975). The i n i t i a l accumulation f o l l o w e d by maintenance of a s t a b l e blood l e v e l i s an important c h a r a c t e r i s t i c of DPH and other c l i n i c a l l y proven a n t i c o n v u l s a n t s (Kutt, 1971). The dams used i n the t e r a t o l o g y study were randomly assigned to treatment l e v e l s 1, 3 or 4. As t h i s was p r i m a r i l y a s c r e e n i n g t e s t to see what p o s s i b l e malformations and t h e i r frequency may occur i n c h r o n i c a l l y t r e a t e d mice, o n l y the h i g h e s t doses were used. The mice were s t a r t e d out on l e v e l 1 (0 mg/kg) and every seventh day they proceeded t o the next hig h e r dose, u n t i l the pre-determined number of females were - 32 -maintained on t h e i r r e s p e c t i v e dosages. A l l dams were on t h e i r r e s p e c t i v e treatment l e v e l f o r a t l e a s t 14 days p r i o r t o the f i r s t attempt a t mating. 5. SEIZURE CONTROL STUDY A study of 12 C57 B1/6J, qk/qk female mice was conducted to determine the e f f e c t i v e n e s s o f o r a l l y a d m i n i s t e r e d DPH i n c o n t r o l l i n g t h e i r s e i z u r e s . The mice were housed i n p a i r s and allowed f r e e access t o water c o n t a i n i n g t h e i r dose of DPH. S t a r t i n g on l e v e l 1 (0 mg/kg), a background frequency of s e i z u r e s recorded i n terms of s e i z u r e s per mouse day was a s c e r t a i n e d f o r each mouse (Sidman e t a l . , 1965). During each seven day p e r i o d , the mice were observed f o r one hour on a t l e a s t 4 days. At 15 minute i n t e r v a l s d u r i n g the o b s e r v a t i o n p e r i o d , the animals were g e n t l y l i f t e d up by t h e i r t a i l s and slowly turned 180° ( G o l d s t e i n , 1973). Those animals t h a t e x h i b i t e d c l o n i c - t o n i c s e i z u r e s from the time the wire cage l i d had been removed u n t i l the time the l i d had been r e p l a c e d a f t e r t u r n i n g the mice, were recorded as having had a s e i z u r e . 6. DETERMINATION OF SERUM DPH LEVELS To determine dose-response curves f o r the four DPH t r e a t -ment l e v e l s , blood samples were drawn from the r e t r o - o r b i t a l s i n u s a f t e r each seven day p e r i o d on a giv e n treatment l e v e l . To maximize p o s s i b l e v a r i a b i l i t y i n blood DPH l e v e l s due to c i r c a d i a n f l u c t u a t i o n s the samples were c o l l e c t e d over a 14 - 33 -hour p e r i o d , 9 AM to 11 PM on the day of blood-sampling. Using a h e p a r i n i z e d Dade microhematocrit tube, approx-i m a t e l y 0.5 ml of blood was drawn a t each time. The b l o o d was c o l l e c t e d i n p l a s t i c micro c e n t r i f u g e tubes and spun down i n a S o r v a l l Superspeed RC2-B automatic r e f r i g e r a t e d c e n t r i f u g e a t 4°C f o r e i g h t minutes at 3,000 g. At l e a s t 50yul of serum was removed by s u c t i o n w i t h Corning 100yul g l a s s d i s p o s a b l e microsampling p i p e t t e s and t r a n s f e r r e d again to the p l a s t i c micro c e n t r i f u g e tubes. These were r e f r i g e r a t e d a t 4°C u n t i l the day of the assay. The SYVA Emit spectrophotometric assay "(SYVA, P a l o , A l t o , C a l . ) , which i s a homogeneous enzyme immunoassay technique, was used to determine serum DPH c o n c e n t r a t i o n s ( B a s t i a n i e t a l . , 1973). T h i s technique i n v o l v e s l a b e l i n g a drug, i n t h i s case DPH, w i t h the enzyme glucose-6-phosphate dehydrogenase !(G-6PDH) . An,'antibody to the drug binds to the drug-enzyme complex and b l o c k s the enzyme's a c t i v e s i t e . The f r e e drug i n the serum competes wi t h the enzyme-labeled drug f o r the antibody and the enzyme a c t i v i t y i s reduced. The r e s i d u a l enzyme a c t i v i t y i s d i r e c t l y r e l a t e d to the c o n c e n t r a -t i o n of drug (DPH)! present i n the sample. The a c t i v e enzyme converts NAD to NADH r e s u l t i n g i n an absorbance change t h a t i s measured s p e c t r o p h o t o m e t r i c a l l y (Anonymous, 1975; Schmidt e t a l . , 1977) . The assay procedure i n v o l v e s a 1:12 d i l u t i o n of the 50 u l sample wi t h 0.055 M T r i s h y d r o c h l o r i d e b u f f e r , pH 8.1 i n two s u c c e s s i v e steps w i t h a SYVA p i p e t t e r - d i l u t e r . Reagent A, c o n t a i n i n g 50yul of antibody p l u s 250yul of T r i s b u f f e r and reagent B, c o n t a i n i n g 50yal of the G-6PDH l a b e l e d DPH p l u s 250 u l of T r i s b u f f e r are mixed. The sample i s a s p i r a t e d i n t o a Beckman 2 4 spectrophotometer w i t h a s i p p e r system and thermal r e g u l a t e d flow c e l l s e t a t 30°C. Once the sample i s i n s i d e the spectrophotometer the Beckman 3115T p r i n t e r i s a u t o m a t i c a l l y a c t i v a t e d and the d i f f e r e n c e i n absorbance at 340 nm between 15 and 90 seconds a f t e r a s p i r a -t i o n i s recorded. As the sample i s d i l u t e d , up t o f i v e repeat measurements can be run f o r every sample. For the purposes of t h i s experiment d u p l i c a t e runs were performed on a l l samples. The average of the two v a l u e s was taken to be the serum concen-t r a t i o n o f DPH i n any sample. In the t e r a t o l o g i c a l study maternal blood samples were a l s o drawn, processed, and assayed i n t h i s manner j u s t p r i o r t o the death of the animal. EXPERIMENTAL DESIGN a. Experimental Organism The experimental p r o t o c o l was designed t o comply wi t h the c r i t e r i a e s t a b l i s h e d f o r an animal model of the f e t a l hydantoin syndrome. The f i r s t requirement c a l l e d f o r the use of ah - 35--o r g a n i s m w i t h a s p o n t a n e o u s s e i z u r e d i s o r d e r . F o r t h i s r e a s o n t h e mouse m u t a n t q u a k i n g ( q k ) , c h a r a c t e r i z e d by s p o n t a n e o u s l y r e c u r r i n g c o n v u l s i o n s i n homozygous ( q k / q k ) a n i m a l s , was s e l e c t e d ( S i d m a n e t a l . , 1 9 6 5 ) . F e m a l e m i c e o f t h r e e g e n o t y p e s w e r e r a n d o m l y a s s i g n e d t o one o f t h r e e t r e a t m e n t g r o u p s ( 0 , 40, and 60 mg/kg b o d y w e i g h t ) . To d e f i n e t h e r o l e o f t h e q u a k i n g gene (qk) i n t h e e t i o l o g y o f t h e m a l f o r m a t i o n s , dams t h a t w e r e h e t e r o z y g o u s (+/qk) f o r t h e m u t a n t gene and homozygous n o n - q u a k -i n g (+/+) dams w e r e a l s o i n c l u d e d i n t h e s t u d y . b. R o u t e o f D r u g A d m i n i s t r a t i o n The DPH was a d m i n i s t e r e d o r a l l y a s a s a l t s u s p e n s i o n i n t h e a n i m a l ' s d r i n k i n g w a t e r . The w a t e r i n t a k e o f e a c h c a g e was c h e c k e d d a i l y t o m a i n t a i n t h e p r o p e r d o s a g e . The d r u g t h e r a p y s t a r t e d two weeks p r i o r t o t h e f i r s t a t t e m p t a t m a t i n g and c o n t i n u e d t h r o u g h o u t g e s t a t i o n . Serum c o n c e n t r a t i o n s o f DPH w e r e d e t e r m i n e d b y means o f t h e SYVA E m i t s p e c t r o p h o t o m e t r y a s s a y t e c h n i q u e t o e n s u r e s e r u m l e v e l s t h a t w e r e w i t h i n t h e o p t i m a l human t h e r a p e u t i c r a n g e . c. M a t i n g s The m a t i n g s w e r e i n i t i a t e d b y p l a c i n g s i n g l e C57 B 1 / 6 J +/+ m a l e s i n t h e c a g e s o f p a i r e d f e m a l e s o f a l l t h r e e g e n o -t y p e s . The m i c e w e r e a l l o w e d two t o t h r e e h o u r s t o m a t e , a f t e r w h i c h t i m e t h e m a l e s w e r e r e t u r n e d t o t h e i r own c a g e s and t h e f e m a l e s w e r e e x a m i n e d . The p r e s e n c e o f a v a g i n a l p l u g was taken i n d i c a t i n g g e s t a t i o n a l day 1. The dams continued to be t r e a t e d w i t h DPH throughout g e s t a t i o n . On day 18, the mice were weighed, b l e d from the r e t r o -o r b i t a l s i n u s and 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 . d. F e t a l Examination Immediately a f t e r c e r v i c a l d i s l o c a t i o n , the maternal abdominal c a v i t y was opened and the u t e r i n e contents removed. The l o c a t i o n and p o s i t i o n of a l l l i v e born f e t u s e s and r e s o r p t i o n s i t e s were noted. The f e t u s e s were removed by s e v e r i n g the u m b i l i c a l cords and were checked f o r limb or mouth movements. Those f e t u s e s t h a t f a i l e d t o show any spontaneous or e l i c i t e d movements, or t h a t were p a l e i n c o l o u r , were regarded as dead and i n c l u d e d amongst the r e s o r p t i o n s f o r the s t a t i s t i c a l a n a l y s i s . The l i v e born f e t u s e s were weighed on the S a r t o r i o u s s i n g l e beam balance to one ten-thousanth of a gram. With the a i d of a W i l d d i s s e c t i n g microscope, the f e t u s e s were sexed and immediately examined f o r e x t e r n a l mal-formations of the head, p a l a t e , trunk, limbs and d i g i t s . Randomly, o n e - t h i r d of the f e t u s e s were p l a c e d i n 95% ETOH f o r a l i z a r i n red s t a i n i n g (Crary, 1962: Appendix D).' and the remaining tw o - t h i r d s p l a c e d i n Bouin'. s s o l u t i o n . T h i s l a t t e r group was examined f o r i n t e r n a l malformations u s i n g the f r e e -hand r a z o r blade techniques of Wilson (Wilson, 1965) and i t s m o d i f i c a t i o n (Barrow and T a y l o r , 1969). Those f e t u s e s t h a t had been s t a i n e d f o r s k e l e t a l examination were p l a c e d i n p e t r i d i s h e s f i l l e d w i t h g e l a t i n and examined under a d i s s e c t i n g microscope. The s o f t t i s s u e s e c t i o n s were a l s o examined w i t h a d i s s e c t i n g microscope, by p l a c i n g the 1-2 mm c r o s s s e c t i o n s l i c e s i n 70% ETOH i n white p o r c e l a i n spot p l a t e s . STATISTICAL ANALYSIS The 0.05 l e v e l o f s i g n i f i c a n c e was s e t f o r a l l s t a t i s t i c a l a n a l y s i s . The mean d i f f e r e n c e s between measurements were t e s t e d u s i n g a one-way a n a l y s i s of v a r i a n c e (Sokal & Rohlf, 1969). Where d i f f e r e n c e s were found to be s i g n i f i c a n t , a Duncan's new m u l t i p l e range t e s t was u t i l i z e d to d i s c e r n the source of the s i g n i f i c a n c e ( B l i s s , 1967). A l l c a l c u l a t i o n s i n c l u d i n g the d e s c r i p t i v e s t a t i s t i c s used i n the s e i z u r e c o n t r o l study and throughout the t e x t were performed on e i t h e r a Wang 3000 programable desk computer or a Texas Instruments Sr-50 hand c a l c u l a t o r . - 38 -CHAPTER I I I RESULTS 1. SEIZURE CONTROL The c r i t e r i a e s t a b l i s h e d f o r an animal model of the f e t a l hydantoin syndrome r e q u i r e d the experimental organism to e x h i b i t spontaneous s e i z u r e s t h a t are c o n t r o l l e d or e l i m i n a t e d by DPH treatment (see Page 27). On the average, homozygous (qk/qk) quaking mutant mice were s u b j e c t to more than two s e i z u r e s per mouse day and these were c o n t r o l l e d by DPH (Table 3). T h i s s a t i s f i e d the f i r s t two requirements of the mouse model. 2. SERUM LEVELS When the drug was a d m i n i s t e r e d o r a l l y f o r a seven day p e r i o d t o non-pregnant females of a l l t h r e e genotypes, the serum c o n c e n t r a t i o n s of DPH were found to i n c r e a s e w i t h i n c r e a s -i n g amounts of the drug a d m i n i s t e r e d (Table 4). When the 20 mg/kg body weight dose was administered, very low serum l e v e l s were recorded. These serum l e v e l s were f a r below those c o n s i d e r e d d e s i r a b l e f o r most human e p i l e p t i c s , and c o u l d be the r e s u l t of r a p i d drug o x i d a t i o n and e l i m i n a t i o n . The two h i g h e r doses r e s u l t e d i n serum l e v e l s t h a t f a l l w i t h i n the o p t i m a l human t h e r a p e u t i c range of 5 to 20 micrograms per ml of serum (Miles e t a l . , 1976; Jeavons, 1977). T h i s complied with the t h i r d and f o u r t h requirements of the animal model. - 39 -Table 3. E f f i c a c y o f DPH O r a l l y Administered to Quaking (qk/qk) Mice (n=12 females f o r each treatment) DPH Treatment S e i z u r e s * (mg/kg body weight) mean + SEM 2.06 + .74 20 1.96 + .98 40 0.77 + .72 60 0.34 + .41 *expressed as s e i z u r e s per mouse day Table 4. DPH Serum Co n c e n t r a t i o n s i n Non-pregnant Females. DPH Genotype Treatment Serum C o n c e n t r a t i o n * (mg/kg body weight) mean + SEM +/+ 0 0 20 2.06 + .15 (n=37 females f o r each 40 5.18 + .27** treatment) 60 12.06 + .56** +/qk 0 0 20 1.75 + .13 (n=21 females f o r each 40 4.59 + .39 treatment) 60 10.15 + .75** qk/qk 0 0 20 2.19 + .27 (n=17 females f o r each 40 6.29 + .62** treatment) 60 10.59 + .83** * expressed asyug per ml serum ** w i t h i n o p t i m a l human t h e r a p e u t i c range Table 5 shows the e f f e c t of v a r y i n g dose l e v e l s of DPH on water i n t a k e and serum DPH c o n c e n t r a t i o n s i n pregnant dams. The average water i n t a k e per mouse over a seven day p e r i o d was not s i g n i f i c a n t l y d i f f e r e n t amongst the genotypes (see Appendix E f o r ANOVA t a b l e s ) . The maternal serum l e v e l s of DPH (Table 5) appear to be e l e v a t e d over serum l e v e l s found a t a corresponding dose i n non-pregnant females (Table 4 ) , an o b s e r v a t i o n r e p o r t e d i n r a t s (Westmoreland and Bass, 1971). However, the data a v a i l a b l e were so l i m i t e d as to p r e c l u d e any v a l i d s t a t i s t i c a l a n a l y s i s . IMPLANTATIONS AND RESORPTIONS The e f f e c t of DPH treatment on i m p l a n t a t i o n and r e s o r p t i o n i s shown i n Table 6. The f i g u r e f o r implants i s based upon a l l l i v e and s t i l l b i r t h s p l u s any v i s i b l e r e s o r p t i o n s i t e s . As DPH dose l e v e l s were i n c r e a s e d , there were no s i g n i f i c a n t changes i n the number of implants. / S i m i l a r l y , t h e r e were no s i g n i f i c a n t changes i n the number of f e t u s e s resorbed as the l e v e l of administered DPH was i n c r e a s e d . In the homozygous quaking (qk/qk) mice, there was very l i t t l e d i f f e r e n c e between dams not on DPH and dams who r e c e i v e d 40 mg/kg body weight (24% and 22%, r e s p e c t i v e l y ) . Quaking mice (qk/qk) r e c e i v i n g the h i g h e s t dose o f DPH had no v i s i b l e r e s o r p t i o n s i t e s and these two dams d i d not c a r r y any o f f s p r i n g to term, though they d i d g a i n weight d u r i n g e a r l y pregnancy. T h i s extreme e f f e c t may be r e l a t e d t o maternal i n t o x i c a t i o n at t h i s h i g h dosage, Table 5. Effe c t of Treatments on Water Intake and Serum DPH Concentrations on Pregnant Females, Genotypes Number of Females DPH Treatment Daily Water Intake* Serum DPH** (mg/kg body wt.) mean + SEM mean + SEM +/+ 10.45 + .29 4 40 10.25 + .57 9.78 + .86 2 60 10.11 + .33 11.60 + .57 +/qk 1 0 10.39 + .68 0 1 40 11.21 + .59 11.20 + .0 1 60 10.51 + .63 20.00 + .0 qk/qk 2 0 9.49 + .61 0 3 40 10.75 + .61 7.75 + 1.97 2 60 11.27 + .53 *** *p:40.05 Level of significance **expressed asyug per ml serum ***blood samples hemolyzed T a b l e 6. E f f e c t o f DPH T r e a t m e n t o n I m p l a n t a t i o n a n d R e s o r p t i o n G e n o t y p e s Number o f F e m a l e s DPH T r e a t m e n t Number I m p l a n t s A v e r a g e I m p l a n t s * R e s o r p t i o n s Number* (mg/kg body wt.) •'mean + S E M : mean + S E M +/+ 4 0 32 o8.0 + .93 1.2 + .54 16 4 40 25 6.2 + .74 1.8 + .19 28 2 60 18 9.0 + .0 1.0 + .0 11 +/qk 1 0 5 5.0 + .0 1.0 + .0 20 1 40 8 8.0 + .0 2.0 + .0 25 1 60 9 9.0 + .0 3.0 + .0 33 q k / q k 2 0 17' 8.5 + . 35 2.0 + .0 24 3 40 32 10.75 + .19 2.3 + .98 22 2 60 (- — — — *p<£0.05 L e v e l o f s i g n i f i c a n c e **See t e x t f o r e x p l a n a t i o n - 44 -to which the mutant mice were p a r t i c u l a r l y susceptible. 4. FETAL MEASUREMENTS The results of measurements on l i v e b i r t h s , i . e . sex, average f e t a l weight and the percent abnormal are recorded i n Table 7. The sex r a t i o did not d i f f e r from the expected 1:1 ratio: .for a l l l i v e b i r t h s . The f e t a l weights were s i g n i f i c a n t l y decreased i n a l l three genotypes as the dose l e v e l of DPH was increased. There were, however, no s i g n i f i c a n t difference between genotypes i n the f e t a l weights recorded. 5. FETAL ANOMALIES The incidence of fetuses born with one or more skel -e t a l or soft tissue abnormalities increased with increasing DPH treatment l e v e l (Table 7). I t i s noteworthy that the homozygous quaking (qk/qk) dams who did not receive DPH were capable of producing normal of f s p r i n g . The only defect reported i n t h i s group was the delayed o s s i f i c a t i o n of the supraoccipital bone i n a single fetus, for an 8% background incidence of malformations amongst the progeny of untreated quaking (qk/qk) dams. This i s far below the 80% abnormal when the quaking (qk/qk) dams received 40 mg/kg DPH,(Table 7). Thus, the implication i s that i t i s the drug, and not the mat-ernal seizure disorder that i s responsible for the observed malformations. The most frequently observed s k e l e t a l malformations Table 7. E f f e c t of DPH Treatment on L i v e B i r t h s , Sex, F e t a l Weights and F e t a l A b n o r m a l i t i e s Number DPH L i v e Genotypes of Females Treatment B i r t h s Sex F e t a l Weight* Abnormal (mg/kg body wt.) F M mean + SEM Q, "O +/+ 4 0 27 13 14 .801 .02 11 4 40 18 9 9 .792 + .03 89 2 60 16 9 7 .689 + .04 100 +/qk 1 0 4 2 2 .858 + .02 0 1 40 6 3 3 . 830 + .04 83 1 60 6 4. 2 .639 + .02 100 qk/qk 2 0 13 7 6 1.021 + .04 8 3 40 25 15 10 ;797 + .05 80 2 60 0 0 0 0 — *p40.05 L e v e l of s i g n i f i c a n c e Table 8. Types and Frequencies of S k e l e t a l Anomalies V e r t e -DPH Fetuses Sterna- D i s t a l brae Genotype . Treatment Examined Occiput brae Phalanges Mandible M a x i l l a Nasal Centra Misc.* (mg/kg bd.wt.) +/+ 0 40 60 9 6 6 0 3 3 1 4 4 1 4 4 0 1 3 0 4 5 0 5 6 1 3 5 0 1 5 +/qk qk/qk 0 1 0 0 0 0 0 0 0 0 40 2 0 0 2 2 1 2 0 0 60 2 2 1 2 0 1 1 1 1 0 5 1 0 0 0 0 0 0 0 40 7 3 2 2 2 0 2 3 3 60 0 0 0 0 0 0 0 0 0 *Miscellaneous anomalies i n c l u d e : r i b a r t i c u l a t i o n d e f e c t s missing ischium and pubis bones missing i n t r a p a r i e t a l bone - 47 -(Table 8) included absent or delayed o s s i f i c a t i o n of the d i s t a l phalanges (40%), and: reduced nasal bones (43%). These anomal-ies were observed i n the progeny of the homozygous non-quaking (+/+) and heterozygotes (+/qk) dams at both treatment l e v e l s , and i n the l i t t e r s of quaking (qk/qk) .treated with 40 mg/kg of DPH. While a missing or reduced and misshapen, supraoccipital bone (Fig.l) and triangular vertebral centrae (Fig.2) were commonly observed amongst the offspring of the two homozygous genotypes (+/+ and qk/qk) at the 40 mg/kg dosage, these anomalies were not recorded among the limited number of fetuses from the heterozygous (+/qk) dams at t h i s treatment l e v e l . The maxillary defects found at both treatment lev e l s i n the homo-zygous non-quaking (+/+) and -the heterozygous (+/qk) dams' offspring were e n t i r e l y lacking amongst the fetuses of quaking (qk/qk) dams. Where more than"just a single sternabrae was found to be missing (Fig.3), i t was recorded as an anomaly. This was frequently observed at both dose lev e l s i n the homozygous non-quaking (+/+) dams' of f s p r i n g , i n the 40 mg/kg DPH treated quaking (qk/qk) dams' progeny, and only at the highest dose l e v e l i n the offspring of the heterozygous (+/qk) dams. Mandib-ular abnormalities were found at both drug l e v e l s amongst the homozygous non-quaking (+/+) dams' progeny, and i n the young of quaking (qk/qk) and heterozygous (+/qk) females treated with 40 mg/kg of DPH. The remaining defects of the sk e l e t a l system include an anomaly i n which the alignment of the vertebrae - 48 -i s such that t h e i r a r t i c u l a t i o n with the r i b s gives r i s e to the appearance of s p l i t vertebrael centrae. A number of fetuses were observed that had neither a pubis nor an ischium and one fetus had: no i n t r a p a r i e t a l bone. The r e s u l t s of soft tissue examinations are seen i n Table 9. The two most commonly observed categories of defects are ocular and renal abnormalities. The former include microphthalmia (Fig.4), anophthalmia (Fig.'5),. and lidgaps, while the renal defects were either hypoplastic kidneys or hydronephrosis (Fig. 7).„ While there were a few fetuses with clinodactyly and even one with Polydactyly (Fig. 6), the primary defect of the extremities was d i g i t a l hypoplasia (Fig. 8). The ocular and renal abnormalities were observed amongst o f f s p r i n g of homozygous non-quaking (+/+) and heterozygous (+/qk) dams at both treatment l e v e l s , as well as amongst the young df the quaking dams (qk/qk) given a 40: mg/kg dose of DPH. D i g i t a l hypoplasia . andi c l e f t palate (Fig. 9), were common at both dose leve l s for the homozygous non-quaking (+/+) , yet were only observed at the highest dose l e v e l amongst the young of the heterozygous (+/qk) dams, and at the 4 0 mg/kg for the quaking (qk/qk) dams. Dilated cerebral v e n t r i c l e s , often severe enough to be considered a mild form of in t e r n a l hydro-cephalus, were seen i n the progeny of the homozygous genotypes (+/+ and qk/qk), but were not recorded at the 60 mg/kg dosage amongst the young of the heterozygous (+/qk) dams. Another neural defect observed i n a single specimen .was the complete agenesis of the ri g h t cortex, and the secondary hyperplasia Table 9. Type and Frequencies of S o f t T i s s u e Anomalies D i l a t e d Hypo-DPH\ Fetuses Ven- p l a s t i c C l e f t Genotype Treatment Examined t r i c l e s C a r d i a c Ocular Renal G.I. D i g i t s P a l a t e Misc.* (mg/kg bd.wt.) +/+ 0 18 1 0 1 1 0 0 0 0 40 12 5 7 4 11 3 4 5 2 60 10 6 4 7 3 1 4 2 2 0 3 0 0 0 0 0 0 0 0 40 4 1 2 2 3 0 0 0 0 60 4 0 0 2 4 0 3 1 0 0 8 0 0 0 0 0 0 0 0 40 18 4 4 10 10 3 6 3 6 60 0 0 0 0 0 0 0 0 0 *Miscellaneous anomalies i n c l u d e : h y p o p l a s t i c lungs h y p o p l a s t i c adrenals hydroureters c l i n o d a c t y l y P o l y d a c t y l y hemangioma - 50 -of the l e f t c o r t e x . The f i n a l category of anomalies, t h a t o f the g a s t r o i n t e s t i n a l t r a c t , i n c l u d e d the d e f e c t s s i t u s i n v e r s u s , duodenal a t r e s i a , and i n one i n s t a n c e , tracheoeso-phogeal f i s t u l a . T h i s group of malformations was o n l y observed i n the homozygous genotypes (+/+ and qk/qk). A b n o r m a l i t i e s of the g r e a t v e s s e l s , i n a d d i t i o n to the d e f e c t s of the h e a r t proper, were determined by adopting a m o d i f i c a t i o n of Wilson's freehand r a z o r technique (Barrow and T a y l o r , 1969). D e f e c t i v e i n t r a v e n t r i c u l a r septa, the most common h e a r t anomaly observed i n human case r e p o r t s , was ; a l s o the most fre q u e n t c a r d i a c anomaly amongst the mice i n t h i s study. The other h e a r t d e f e c t s p r i m a r i l y i n v o l v e d the g r e a t v e s s e l s , such as pulmonary and a o r t i c a r c h w i t h p a t e n t ductus. O c c a s i o n a l l y there were m u l t i p l e h e a r t - d e f e c t s r a s s o c i a t e d with a s i n g l e f e t u s . One such specimen.had both pulmonary v a l v u l a r atresia;.and hemopericardium, the l a t t e r being the r e s u l t of the s u p e r i o r vena cava d e l i v e r i n g i t s bloodflow d i r e c t l y i n t o the p e r i c a r d i u m , r a t h e r than; i n t o the r i g h t atrium. In summary: maternal serum DPH c o n c e n t r a t i o n s and percent of abnormal o f f s p r i n g i n c r e a s e d w i t h i n c r e a s i n g doses of DPH. F u r t h e r , the average weight per f e t u s decreased as serum c o n c e n t r a t i o n s of DPH i n c r e a s e d . The s i g n i f i c a n c e of these r e s u l t s w i l l be d i s c u s s e d i n the f o l l o w i n g chapter. - 51 -CHAPTER IV DISCUSSION The human data c o l l e c t e d over the p a s t f o u r t e e n years suggest a p o s s i b l e r e l a t i o n between the maternal use of a n t i c o n v u l s a n t drugs and b i r t h d e f e c t s . Of the many a n t i c o n v u l s a n t s commercially a v a i l a b l e , D i l a n t i n (DPH) has been i m p l i c a t e d most o f t e n as the dysmorphogenic agent ( S p e i d e l and Meadow, 1974; Janz, 1975). Since 1975, the broad spectrum of malformations observed i n : the o f f s p r i n g of e p i l e p t i c women who r e c e i v e d hydantoin a n t i c o n v u l s a n t s throughout t h e i r pregnancies has become known as the f e t a l hydantoin syndrome (Hanson and Smith, 1975). The r e s u l t s r e p o r t e d i n the p r e v i o u s chapter f u r t h e r supports the t e r a t o -g enic p o t e n t i a l of DPH as w e l l as the n o t i o n of the f e t a l hydantoin syndrome. The purpose of t h i s study was to e s t a b l i s h whether or not the c r i t e r i a f o r an animal model of the f e t a l hydantoin syndrome c o u l d be achieved. The r e s u l t s s t r o n g l y suggest t h i s g o a l has been reached. But s i n c e the experimental d e s i g n p r e c l u d e d a d i r e c t d i a g n o s i s of p o s t n a t a l growth d e f i c i e n c y and developmental delay-both important f e a t u r e s of the human f e t a l hydantoin syndrome-it';may be argued t h a t the p a t t e r n of malformations observed d i f f e r s to a c e r t a i n e xtent from t h a t of the human s i t u a t i o n . However, the s t r i k i n g s i m i l a r i t y between the human and mouse f e t a l hydantoin syndrome, - 52-i n terms, o f s k e l e t a l a n d s o f t t i s s u e a n o m a l i e s ( T a b l e 10) , i s g o od e v i d e n c e t h a t t h i s c r i t e r i o n h a s s u f f i e n t l y b e e n f u l f i l l e d . F u r t h e r , t h e s e c r i t e r i a h a v e a l s o b e e n met: M i c e w i t h s p o n t a n e o u s s e i z u r e d i s o r d e r s h a v e h a d t h e i r s e i z u r e s c o n t r o l l e d b y o r a l l y a d m i n i s t e r e d DPH. The s e r u m l e v e l s o f t h e dams w e r e w i t h i n t h e o p t i m a l human t h e r a p e u t i c r a n g e , a n d when t h e dams w e r e m a t e d a f t e r c h r o n i c DPH t r e a t m e n t f o r two w e e k s , t h e y p r o d u c e d f e t u s e s w i t h m a l f o r m a t i o n s r e m i n -i s c e n t o f t h o s e o b s e r v e d i n t h e o f f s p r i n g o f human e p i l e p t i c women o n h y d a n t o i n t h e r a p y d u r i n g p r e g n a n c y . The d r u g e x p o s e d f e t u s e s w e i g h e d s i g n i f i c a n t l y l e s s t h a n t h e i r u n t r e a t e d c o n t r o l s . The l o w e s t w e i g h t r e c o r d e d f o r a v i a b l e o f f s p r i n g was 0.4917 gm. , s u g g e s t i n g a m i n i m a l w e i g h t f o r v i a b i l i t y o f t h i s s t r a i n t o be 0.5 gm. T h e r e ' w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h e f r e q u e n c y o f i m p l a n t s o f r e s o r p t i o n s a s t h e DPH d o s e l e v e l i n c r e a s e d . T h o s e s p e c i m e n s s t a i n e d f o r s k e l e t a l e x a m i n a t i o n p r e s e n t e d a p i c t u r e o f g e n e r a l i z e d g r o w t h d e l a y m a n i f e s t e d b y m i s s i n g o r r e d u c e d o s s i f i c a t i o n o f t h e s u p r a o c c i p i t a l b one ( F i g . 1 ) , h y p o p l a s i a o f t h e d i s t a l p h a l a n g e s , o f t e n a s s o c i a t e d w i t h r e d u c e d o s s i f -i c a t i o n o f t h e m e t a c a r p a l s and m e t a t a r s a l s , d o r s a l l y p o i n t e d t r i a n g u l a r s h a p e d v e r t e b r a l c e n t r a e , a n d t h e a b s e n c e o f more t h a n j u s t t h e f i f t h s t e r n e b r a e (Fig.T3). I n an e a r l i e r s t u d y a m i s s i n g f i f t h s t e r n a b r a e was c o n s i d e r e d a m a l f o r m a t i o n , h o w e v e r , t h e v e r y h i g h f r e q u e n c y o f t h i s f i n d i n g i n b o t h t h e t r e a t e d a n d u n t r e a t e d s p e c i m e n s was c o n s i d e r e d a p e c u l i a r i t y , o f t h e C 5 7 B 1 / 6 J s t r a i n , a n d n o t a p a t h o l o g i c a l s i t u a t i o n ( F r i t z Table 10. S i m i l a r i t i e s between the Human and Mouse F e t a l Hydantoin Syndrome Man Mouse 1. Growth D e f i c i e n c y X X 2. S k e l e t a l Anomalies X X 3. Neural Anomalies X X 4. C a r d i a c Anomalies X X 5. O r o f a c i a l C l e f t i n g X X 6. Ocular Anomalies X X 7. U r i n a r y Anomalies X X 8. G a s t r o i n t e s t i n a l Anomalies X X 9 . P e r i n a t a l Death X •? 10. Low Performance X •p e t a l . 1976.) Anomalies of the s o f t t i s s u e were observed f o r the most p a r t i n a l l t h r e e genotypes. The major e x c e p t i o n was c o n f i n e d t o the o f f s p r i n g o f the heterozygous (+/qk) dams t h a t had the s m a l l e s t sample s i z e o f the three genotypic groups. In t h i s group, no g a s t r o i n t e s t i n a l d e f e c t s were observed, c l e f t p a l a t e s and h y p o p l a s t i c d i g i t s were found o n l y a t the h i g h e s t dosage, w h i l e d i l a t e d v e n t r i c l e s and c a r d i a c anomalies were recorded o n l y a t the 40 mg/kg dose l e v e l . O r o f a c i a l c l e f t s , the predominant d e f e c t r e p o r t e d i n the e a r l y r e t r o s p e c t i v e e p i d e m i o l o g i c s t u d i e s on DPH t e r a t o g e n i c i t y , were observed i n l e s s than 20% of the t r e a t e d o f f s p r i n g o f a l l t h r e e genotypes. The p r e s e n t study was so designed as t o meet a s e l f -p r e s c r i b e d c r i t e r i o n t h a t an animal model of the f e t a l hydantoin syndrome should encompass. In so doing, the p r o t o c o l was d r a s t i c a l l y d i f f e r e n t from t h a t of a l l p r e v i o u s attempts t o study the t e r a t o g e n i c p o t e n t i a l o f DPH. T h e r e f o r e , i t i s not s u r p r i s i n g t h a t the r e s u l t s presented here are d i f f e r e n t i n c e r t a i n aspects from those p r e v i o u s l y r e p o r t e d (Massey, 1966; Gibson and Becker, 1968, Harbison and Becker, 1969/:1972 Elshove, 1969; M e r c i e r - P a r o t and Tuchmann-Duplessis, 1974; M i l l e r and Becker, 1975; S u l l i v a n and McElhatton, 1975, 1977; F r i t z e t a l . , 1976). - 55 -When t r y i n g to develop a human m o d e l i t i s an accepted p r a c t i c e i n experimental t e r a t o l o g y to a d m i n i s t e r the t e s t substance t o the organism by the same route o f a d m i n i s t r a t i o n t h a t w i l l be used c l i n i c a l l y i n humans,(Wilson, 1973). Most people take D i l a n t i n o r a l l y , e i t h e r by capsule or as a syrup. To date, a l l s t u d i e s have used e i t h e r subcutaneous (Massey, 1966; Gibson and Becker, 1968; M e r c i e r - P a r o t and Tuchmann-D u p l e s s i s , 1974; Harbison and Becker, 1969, 1972) or i n t r a -p e r i t o n e a l i n j e c t i o n (Elshove, 1969; Harbison and Becker, 1969, 1972; M i l l e r and Becker, 1975), g a s t r i c i n t u b a t i o n (Harbison and Becker, 1969; M i l l e r and Becker, 1975; S u l l i v a n and McEl-h a t t o n , 1975, 1977; F r i t z e t a l . , 1976) or as a mixture w i t h a powdered d i e t ( S u l l i v a n and McElhatton, 1975). The presen t study r e p r e s e n t s the f i r s t i n s t a n c e o f o r a l a d m i n i s t r a -t i o n v i a the animal's d r i n k i n g water. T h i s method has advantages of not s t r e s s i n g the pregnant dam by unnecessary h a n d l i n g arid e l i m i n a t e s i . t h e use o f v e h i c l e compounds such as carboxy-m e t h y l c e l l u l o s e , sodium hydroxide, sodium c h l o r i d e and propylene g l y c o l . The p r e s e n t study a l s o r e p r e s e n t s a departure from the m a j o r i t y o f s t u d i e s i n v e s t i g a t i n g t h e i . t e r a t o g e n i c i t y o f DPH, i n t h a t t h a t drug i s g i v e n p r i o r to and throughout g e s t -a t i o n , r a t h e r than .for three or fo u r days a t a time a t v a r i o u s stages throughout organogenesis (Massey, 19 66; Gibson and Becker, 1968; Harbison and Becker, 1969; M i l l e r and Becker, 1975). Although s h o r t term exposure i s c o n s i d e r e d t o be more p r e c i s e i n determining acute e m b r y o t o x i c i t y , i t does not - 56 -meet the needs of a l l experimental situations (Wilson, 1973)". In a model system designed to c l o s e l y p a r a l l e l the human sit u a t i o n , chronic administration i s e s s e n t i a l . Human e p i l e p t i c s are on drug: therapy i n most instances from the time the disorder i s diagnosed throughout the remainder of t h e i r l i v e s . Thus the drug metabolizing enzymes are f u l l y induced i n a woman long before any pregnancy i s contemplated. The c r i t i c i s m directed at long term .drug exposure i s that i t provides the organism enough time for adaptive reactions, such as the induction of l i v e r enzyme systems (Burns, 1970). This i s prec i s e l y what was intended by chronically administering the drug two weeks p r i o r to any attempted mating and then continuing treatment throughout gestation. With maternal enzyme systems f u l l y induced ther should be a minimum of f l u c t a t i o n i n maternal serum DPH concentrations and, therefore, the amount of drug available to the embryos should be constant. DPH has been shown to induce i t s own metabolism i n mice (Gerber and Arnold, 1969). When a drug i s capable of inducing, i t s e l f , serum concentrations of the drug can be expected to d i f f e r between an animal given a single i n j e c t i o n and the same animal repeatedly given the same dosage for a four day period (Conney and Burns, 1972). Thus, i n those studies that describe a highly teratogenic e f f e c t from a single treatment or even for repeated short periods of exposure (Massey, 1966; Gibson and Becker, 1968; Harbison and Becker, 1969,. 1972; M i l l e r and Becker, 1975), that e f f e c t - 57 -c o u l d p o s s i b l y b e r e d u c e d b y c h r o n i c DPH a d m i n i s t r a t i o n a l l o w i n g f o r t h e i n d u c t i o n o f t h e m e t a b o l i z i n g enzyme s y s t e m s . T h i s was d e m o n s t r a t e d i n r a t s b y K i n g a n d h i s c o l l e a g u e s (1965) u s i n g t h e a n t i h i s t a m i n e c h l o r c y c l i z i n e . When t h e d r u g was g i v e n o n d a y s 10 t o 1 5 , t h e r e was a h i g h i n c i d e n c e o f c l e f t p a l a t e . H o w e v e r , when t h i s d r u g was g i v e n i n t h e same 50 mg/kg d o s e on g e s t a t i o n a l d a y s 1 t o 1 5 , t h e i n c i d e n c e o f t h i s m a l f o r m a t i o n was g r e a t l y r e d u c e d . T h i s phenomenon may a c c o u n t f o r t h e l o w . [ . i n c i d e n c e o f c e r t a i n d e f e c t s , n a m e l y c l e f t p a l a t e . i n t h e p r e s e n t s t u d y a n d o t h e r s t u d i e s o f l o n g t e r m d r u g a d m i n a - s t r f a t i o n , ( S u l l i v a n a n d M c E l h a t t o n , 1 9 7 5 , 1977; F r i t z e t a l . 19 76) when c o m p a r e d w i t h t h o s e s t u d i e s o f a s h o r t t e r m d r u g t r e a t m e n t , ( M a s s e y , '.1966; G i b s o n a nd B e c k e r , 1968; H a r b i s o n and Becker!, 1969 , 1972; M e r c i e r - P a r o t a n d T u c h - . mann D u p l e s s i s , 1974; M i l l e r a n d B e c k e r , 1 9 7 5 ) . C e r t a i n a n o m a l i e s m e n t i o n e d i n o t h e r s t u d i e s w e r e n o t o b s e r v e d i n t h e p r e s e n t o n e . I n t h e i r s t u d i e s S u l l i v a n a n d M c E l h a t t o n ( 1 9 7 5 , 1977) o b s e r v e d a s e p a r a t e d b a s i s p h e n o i d b o n e ; no s u c h d e f e c t was o b s e r v e d i n t h e p r e s e n t s t u d y . The same a u t h o r s a l s o n o t e d f e t u s e s b o r n w i t h o p e n e y e s , a n o t h e r f e a t u r e n o t o b s e r v e d i n t h i s s t u d y , a l t h o u g h s e v e r a l m i c e w i t h l i d g a p s w e r e f o u n d . H a r b i s o n a n d B e c k e r (1969) r e p o r t e d t h e s k e l e t a l d e f e c t s e c t r o d a c t y l y , f u s e d v e r t e b r a e , s p l i t c e n t r a e a n d s h o r t e n i n g o f t h e l o n g b o n e s . L o n g bone l e n g t h was n o t m e a s u r e d i n t h e p r e s e n t s t u d y , a n d n e i t h e r c a s e s o f e c t r o d a c -t y l y n o r f u s e d v e r t e b r a e w e r e f o u n d . One' a n o m a l y , h o w e v e r , i n w h i c h t h e v e r t e b r a e w e r e n o t p r o p e r l y a l i g n e d d i d g i v e - 58 -the appearance of s p l i t c e n t r a . P e r i t o n e a l hemorrhage, a d e f e c t t h a t has a l s o been r e p o r t e d amongst the o f f s p r i n g of dams t r e a t e d w i t h DPH, was l a c k i n g from the p r e s e n t study (Harbison and Becker, 1969). Again, a p o s s i b l e e x p l a n a t i o n c o u l d be the d i f f e r e n t route of a d m i n i s t r a t i o n , as i n the study i n which t h i s d e f e c t was i n i t i a l l y r e p o r t e d an i n t r a -p e r i t o n e a l route of a d m i n i s t r a t i o n was used. The occurrence of reduced o s s i f i c a t i o n of s t e r n a b r a e , and the r e d u c t i o n of the mandible, m a x i l l a and n a s a l bones i n the p r e s e n t study r e p r e s e n t s the f i r s t time these malform-a t i o n s have been produced by o r a l a d m i n i s t r a t i o n of DPH. F u r t h e r , the v a r i o u s c a r d i a c , o c u l a r , g a s t r o i n t e n s t i n a l and d e f e c t s of the e x t r e m i t i e s , such as d i g i t a l h y p o p l a s i a , c l i n o d a c t y l y , and P o l y d a c t y l y observed amongst the t r e a t e d o f f s p r i n g , are the f i r s t time such anomalies have been observed i n rodent f e t u s e s exposed to DPH. T h i s wide spectrum of malformations c o u l d be the r e s u l t of the c h r o n i c a d m i n i s t r a t i o n , s i n c e repeated drug doses are known to produce a broader range of anomalies i n experimental animals than s h o r t term treatment (Wilson, 1973). I t has been demonstrated r e p e a t e d l y i n experimental t e r a t o l o g y t h a t c e r t a i n s p e c i e s are more s e n s i t i v e to a g i v e n t e r a t o g e n i c substance than are other s p e c i e s . The most .freq-u e n t l y c i t e d example i s t h a t of c l e f t p a l a t e i n d u c t i o n by c o r t i s o n e . Mice are h i g h l y s u s c e p t i b l e to the t e r a t o g e n i c - 59 -a c t i o n o f t h i s drug, while hamsters and guinea p i g s are l e s s s u s c e p t i b l e , and r a t s are seldom,, i f ever, a f f e c t e d (Wilson, 1973). T h i s s p e c i e s d i f f e r e n c e appears t o be t r u e f o r DPH as w e l l . In experiments w i t h rhesus monkeys, Wilson (1973) r e p o r t e d t h a t very h i g h doses of DPH d u r i n g c r i t i c a l p e r i o d s of primate.development d i d not induce c o n g e n i t a l malformations. When r a t s were used as the experimental organism, such mal-formations as anencephaly, hydrocephalus, c l e f t p a l a t e , hydro-ne p h r o s i s and s h o r t e n i n g of the long bones w e r e i r e p o r t e d o n l y when very h i g h doses (100 t o 200mg/kg) were used (Harbison and Becker, 1972; M e r c i e r - P a r o t and Tuchmann-Duplessis, 1974) and then malformations were never: i n excess of 30%. The m a j o r i t y o f the s t u d i e s t o date used mice, which appear to be the most s e n s i t i v e o f the three s p e c i e s , to the t e r a t o g e n i c a c t i o n o f DPH. Not onl y was the spectrum of d e f e c t s broader i n the mouse,-, but a l s o a h i g h e r percentage of abnormal f e t u s e s were observed at DPH doses lower than those r e s p o n s i b l e f o r producing malformations i n the r a t . Such s p e c i e s d i f f e r e n c e s must be taken i n t o account where the r e s u l t s of animal exper-iments are to be e x t r a p o l a t e d t o the human s i t u a t i o n . S t a p l e s , (1972) has suggested a p r o g r e s s i v e lowering of the t e s t doses to determine an embryolethal zone, a t e r a t o g e n i c zone, the zone of f e t a l growth r e t a r d a t i o n , the zone producing p o s t n a t a l f u n c t i o n a l a l t e r a t i o n , and t h e . t h r e s h o l d o f no e f f e c t . These must be obtained i n a v a r i e t y o f s p e c i e s b e f o r e adequate i n -s i g h t i s ob t a i n e d t o make p r e d i c t i o n s as t o what might occur i n humans. - 60 -The s i m i l a r i t i e s between the human and the mouse f e t a l hydantoin syndrome are l i s t e d i n Table 10. They i n c l u d e p r e n a t a l growth d e f i c i e n c y as evidenced by decreased f e t a l weights. Other s i m i l a r i t i e s i n c l u d e s k e l e t a l , n e u r a l , c a r d i a c , o r o f a c i a l , o c u l a r , "urinary, and g a s t r o i n t e s t i n a l d e f e c t s . While p e r i n a t a l death c o u l d not be observed d i r e c t l y , the high i n c i d e n c e o f c a r d i a c and u r i n a r y anomalies suggest t h a t t h i s too, may be a f e a t u r e o f the f e t a l hydantoin syndrome. F i n a l l y , low performance, which i s a major f e a t u r e of the human syndrome ( H i l l e t a l . , 1974; Hanson and Smith, 1975; Hanson e t a l . , 1976), c o u l d not be assessed due to the experimental d e s i g n . Any s i m i l a r i t y i n t h i s r e s p e c t must await i n v e s t i g a t i o n a t a l a t e r date. I n i t i a l l y , the experimental p r o t o c o l " c a l l e d f o r fou r l e v e l s of DPH treatment ( 0 , 2 0 , 40, and 60 mg/kg body weight) i n order to ..specif i c a l l y d e l i n e a t e any dose r e s p o n s i v e e f f e c t . Regretablyy t h i s format was amended to p r e l i m i n a r i l y t e s t o n l y the two h i g h e r treatment l e v e l s . T h i s was due to the l i m i t e d a v a i l a b l i t y . ~of animals o f the d e s i r e d age and genotype. The r e p r o d u c t i v e r a t e o f the homozygous quaking (qk/qk), dams i s u n f o r t u n a t e l y low. T h i s may be due to t h e i r s h o r t l i f e span o r the i n a b i l i t y of the males t o e l i c i t s a t i s f a c t o r y mounting postures from the females. In any case, these dams were always i n demand f o r experimentation, and l a r g e numbers c o u l d not be s e t a s i d e to produce heterozygous (+/qk) o f f s p r i n g . T h i s problem then, was r e s p o n s i b l e f o r the - 61 -very low sample s i z e of both the homozygous quaking (qk/qk) and the heterozygous (+/qk) females. Another reason behind the change i n p r o t o c o l centered upon u n c e r t a i n t y concerning c h r o n i c o r a l a d m i n i s t r a t i o n of DPH i n dosages t h a t produced serum l e v e l s w i t h i n the o p t i m a l human t h e r a p e u t i c range. I t was not y e t known whether these dosages would indeed produce any malformations at a l l . T h e r e f o r e , the lowest treatment l e v e l was e l i m i n a t e d . I t i s now c l e a r from the outcome of t h i s study t h a t these dosages w i l l not onl y produce malformations, but s p e c i f i c -a l l y those malformations observed i n the progeny o f e p i l e p t i c women on hydantoin a n t i c o n v u l s a n t s , a l b e i t not i n the same p r o p o r t i o n . , I t i s d i f f i c u l t t o assess an a c t u a l dose-responsive r e l a t i o n w i t h the a v a i l a b l e d a t a . C u r r e n t l y mice are r e c e i v i n g DPH treatment at the 20 mg/kg body weight dose l e v e l , and t h i s v a l u a b l e m i s s i n g b i t o f i n f o r m a t i o n w i l l be soon a v a i l a b l e f o r a n a l y s i s . At t h a t time, i t w i l l be p o s s i b l e to determine i f the seventh, and f i n a l , requirement o f the animal model w i l l have been met. At maternal, serum l e v e l s t h a t r e p r e s e n t what would be c o n s i d e r e d the lower l i m i t f o r c l i n i c a l e f f i c a c y i n humans, n e a r l y a l l of the malformations found i n the human f e t a l hydan-t o i n syndrome have been produced. While malformations have been r e p o r t e d i n the o f f s p r i n g o f women r e c e i v i n g a n t i c o n v u l s a n t s other than hydantoins (German e t a l . , 1970; Lowe, 1973), t h i s - 62 -s p e c i f i c p a t t e r n o f d e f e c t s was onl y observed i n i n f a n t s who r e c e i v e d i n ut e r o exposure t o hydantoin a n t i c o n v u l s a n t s (Hanson and Smith, 1975). I t i s of c l i n i c a l importance t o .. now determine the lowest dose, and s i m i l a r l y , the lowest serum c o n c e n t r a t i o n r e q u i r e d t o produce malformed c h i l d r e n . In c o n c l u s i o n , the i n t e n t i o n of t h i s study was t o design an animal model p a r a l l e l i n g as c l o s e l y as p o s s i b l e the human s i t u a t i o n . T h i s p r e l i m i n a r y t e s t of the -model has produced what can be co n s i d e r e d the mouse e q u i v a l e n t o f the f e t a l hydantoin syndrome. On the b a s i s o f the s i m i l a r i t i e s e x i s t i n g between the human and mouse f e t a l hydantoin syndromes, t h i s model s h a l l soon be reproduced on a much l a r g e r s c a l e . By i n c l u d i n g the 20 mg/kg body weight, and even lower.\.dosages, adequate data w i l l be a v a i l a b l e f o r a s s e s s i n g dose-responsive r e l a t i o n s . The d i f f e r e n c e between genotypes w i t h r e s p e c t to. serum DPH c o n c e n t r a t i o n s and the types and f r e q u e n c i e s of anomalies should a l s o be c l a r i f i e d . F i n a l l y , knowledge gained from t h i s animal model may someday p r o v i d e i n s i g h t i n t o the r o l e of DPH i n the e t i o l o g y o f the malformations observed i n the o f f s p r i n g of e p i l e p t i c women on hydantoin a n t i c o n v u l s a n t drug therapy. SKELETAL ANOMALIES x ! 2 magnification 63 F i g u r e 2 S k e l e t a l anomalies i n c l u d i n g t r i a n g u l a r v e r t e b r a l centrae 40 mg/kg DPH +/+ Dam F i g u r e 1 O s s i f i c a t i o n anomalies o s u p r a o c c i p i t a l bone and v e r t e b r a l c e n t r a e 40 mg/kg DPH qk/qk Dam F i g u r e 3 S k e l e t a l anomalies i n c l u d i n g absent s t e r n a b r a e 60 mg/kg DPH +/qk Dam SOFT TISSUE ANOMALIES - 64 x!2 m a g n i f i c a t i o n F i g u r e 4 Microphthalmia 60 mg/kg DPH +/+ Dam F i g u r e 5 Anophthalmia 60 mg/kg DPH +/+ Dam F i g u r e 6 P o l y d a c t y l y 40 mg/kg DPH qk/qk Dam ^ ^ ^ ^ • xl8 magnification - 65 -Figure 8 D i g i t a l hypoplasia and control 60 mg/kg DPH +/+ Dam xl8 magnification - 66 -Figure 9 C l e f t palate and control 40 mg/kg DPH +/+ Dams - 67 -LITERATURE CITED Annegars, J.F. , L.R. Elveback, W.A. Houser and L..T. Kurland, 1974 Do a n t i c o n v u l s a n t s have a t e r a t o g e n i c e f f e c t ? Arch. 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Meadow, S.R., 1970 Congenital abnormalities and anticonvulsant drugs. Proc. Royal Soc. Med. 63:48-49. Mercier-Parot, L. and H. Tuchmann-Duplessis, 1974 The dysmorpho-genic pot e n t i a l of phenytoin: Experimental observations. Drugs 8:340-353. Merr i t t , H.H. and T.J. Putnam, 19 38a Sodium diphenylhydantoinate i n .the treatment of convulsive disorders. J. Am. Med. Assoc. 111:1068-1073. Me r r i t t , H.H. and T.J. Putnam, 1938b A new series of anticonvul-sant drugs tested by experiments on animals. Arch. Neurol, psychiat. 39:1003-1015. Meyer, J.G., 1973 The:: t e r a t o l o g i c a l e f f e c t s of anticonvulsants and the effects on prenancy and b i r t h . Europ. Neurol. 10: 179-190. Miles, B.C., E.C. Attwood and R.M. Seddon, 1976 Serum-phenytoin. Lancet i:255. M i l l a r , J.H.D. and N.C. Nevin, 19 73 Congenital malformations and anticonvulsant drugs. Lancet i:328. M i l l e r , R.P. and B.A.Becker, 1975 Teratogenicity of o r a l diazepam and diphenylhydantoin i n mice. Toxicol. Appl. Pharmacol. 32:53-61. Mirkin, B.L., 1971 Diphenylhydantoin: Placental transport, f e t a l l o c a l i z a t i o n , neonatal metabolism, and possible teratogenic effects.- J:.. Paed. 78:329-337. Monson, R.R., L. Rosenberg, S.C. Hartz, S. Shapiro, O.P. Heinonen and D. Slone, 1973 Diphenylhydantoin and selected congenital malformations. N. Engl. J. Med. 289:1049-1052. - 71 -N i s w a n d e r , J.D. andW. W e r t e l e c k i , 1973 C o n g e n i t a l m a l f o r m a t i o n among o f f s p r i n g o f e p i l e p t i c women. L a n c e t i : 1 0 6 2 . P l a a , G.L., 1974 A c u t e t o x i c i t y o f a n t i e p i l e p t i c d r u g s . E p i l e p s i a , 1 6 : 1 8 3 - 1 9 1 . S c h m i d t , D., V. G o l d b e r g , P.J.M. G u e l e n , S. J o h a n n e s s e n , E. v a n d e r K l e i j n , J.W.A. M e i j e r , H. M e i n a r d i , A. R i c h e n s , H. S c h n e i d e r , Y. S t e i n - L a v i e and N. S y m a n n - L o u e t t e , , 1 9 7 7 E v a l u a t i o n o f new i m munoassay f o r d e t e r m i n a t i o n o f p h e n y t o i n a n d p h e n o b a r b i t a l : R e s u l t s , o f a E u r o p e a n C o l l a b o r a t i v e c o n t r o l s t u d y . E p i l e p s i a 1 8 : 3 6 7 - 3 7 3 . S h a p i r o , S., D. S l o n e , S.C. H a r t z , L. R o s e n b e r g , V. S i s k i n d , R.R. M o nson, A.A. M i t c h e l l , O.P. H e i n o n e n , J . I n d a n p a a n - K e i k k i l a , S. H a r o and L. S a x e n , 1976 A n t i c o n v u l s a n t s and p a r e n t a l e p i l e p s y i n t h e d e v e l o p m e n t o f b i r t h d e f e c t s . L a n c e t i : 2 7 2 - 2 7 5 . S i d m a n , R.L., M.C. G r e e n and S. H. A p p e l , 1965 " C a t a l o g o f t h e N e u r o l o g i c a l M u t a n t s o f t h e Mouse", H a r v a r d U n i v e r s i t y P r e s s , C a m b r i d g e . S o k a l , R.R. and F. J . R o h l f , 1969 " B i o m e t r y " , W.H. F r e e m a n , San F r a n c i s c o . S o u t h , J . , 1972 T e r a t o g e n i c e f f e c t o f a n t i c o n v u l s a n t s . L a n c e t i i : 1 1 5 4 S p e i d e l , B.D. and S.R. Meadow, 1972 M a t e r n a l e p i l e p s y and a b n o r m a l i t i e s o f t h e f e t u s and n e w b o r n . L a n c e t i i : 8 3 9 - 8 4 3 . S p e i d e l , B.D. and S.R. Meadow, 1974 E p i l e p s y , a n t i c o n v u l s a n t s and c o n g e n i t a l m a l f o r m a t i o n s . D r u g s 8:354-365. S t a p l e s , R.E., 1972 T e r a t o l o g y . I n : " A n t i e p i l e p t i c D r u g s " , D.M. Woodbury, J.K. P e n r y and R.P. S c h m i d t ( E d s . ) , R a v e n P r e s s , New Y o r k , pp. 5 5 - 6 2 . S t a r r e s v e l d - Z i m m e r m a n , A.A.E., W.J. v a n d e r K o l k , H. M e i n a r d i and J . E l s h o v e , 1973 A r e a n t i c o n v u l s a n t s t e r a t o g e n i c ? L a n c e t i i : 4 8 - 4 9 S u l l i v a n , F.M. and P.R. M c E l h a t t o n , 1975 T e r a t o g e n i c a c t i v i t y o f t h e a n t i e p i l e p t i c d r u g s p h e n o b a r b i t a l , p h e n y t o i n and p r i m i d o n e i n m i c e . T o x i c o l . A p p l . P h a r m a c o l . 3 4 : 2 7 1 - 2 8 2 . S u l l i v a n , F.M. and P.R. M c E l h a t t o n , 1977 A c o m p a r i s o n o f t h e t e r a t o g e n i c a c t i v i t y o f t h e a n t i e p i l e p t i c d r u g s c a r b a m a z e p i n e , c l o n a z e p a m , e t h o s u x i m i d e , p h e n o b a r b i t a l , p h e n y t o i n a n d p r i m i d o n e i n m i c e . T o x i c o l . A p p l . P h a r m a c o l . 4 0 : 3 6 5 - 3 7 8 . T e m p k i n , 0., 1945 "The F a l l i n g S i c k n e s s " , The J o h n s H o p k i n s P r e s s , B a l t i m o r e . - 72 -Vida, J.A. and E.H. Gerry, 1977 C y c l i c ureides. In: "Anti-convulsants", J.A. Vida (Ed.), Academic Press, New York, pp. 152-284. Watson, J.D. and W.N. Spellacy, 1971 Neonatal e f f e c t of maternal treatment with the anticonvulsant drug diphenylhydantoin. Obstet. Gynecol. 71:881-885. Westmoreland, B. and N.H. Bass, 1971 Diphenylhydantoin i n t o x i -cation during pregnancy. Arch. Neurol., 24:158-164. Wilson, J.G., 1965 Methods for administering agents and detect-ing malformations i n experimental animals. In: "Teratology: P r i n c i p l e s and Techniques", J. G. Wilson and J. Warkany (Eds.), University of Chicago Press, Chicago, pp. 262-277. Wilson, J.G. (Ed.), 1967 P r i n c i p l e s for the tes t i n g of drugs for teratogenicity. Wld. Hlth. Org. Teen. Rep. Ser. 364:5-18. Wilson, J.G., 1973 "Environment and B i r t h Defects", Acedemic Press, New York. • • - :.7-3 -APPENDIX A DPH DOSE 20 mg/kg 40 mg/kg 60 mg/kg Weight/gm Dose #2 Dose #3 Dose #4 60 1.20 2.40 3.60 59.5 1.19 2.38 3.57 59 1.18 2.36 3.54 58.5 1.17 2.34 3.51 58 1.16 2.32 3.48 57.5 1.15 2. 30 3.45 57 1.14 2.28 3.42 56.5 1.13 2.26 3.39 56 1.12 2.24 3.36 55.5 1.11 2.22 ' 3.33 55 1.10 2.20 3.30 54.5 1.09 2 .18 3.27 54 1.08 2.16 3.24 53.5 1.07 2.14 3.21 53 1.06 2.12 3.18 52.5 1.05 2.10 3.15 52 1.04 2.08 3.12 51.5 1.03 2.06 3.09 51 1.02 2.04 3.06 50.5 1.01 2.02 3.03 50 1.00 2.00 3.00 49.5 .99 1.98 2.97 49 .98 1.96 2.94 48.5 .97 1.94 2.91 48 .96 1.92 2 .88 47.5 .95 1.90 2.85 47 .94 1.88 2.82 46.5 .93 1.86 2.79 46 .92 1.84 2.76 45.5 .91 1.82 2.73 45 .90 1.80 2.70 44.5 .89 1.78 2.67 44 .88 1.76 2.64 43.5 .87 1.74 2.61 43 .86 1.72 2.58 42.5 .85 1.70 2.55 42 .84 1.68 2.52 41.5 .83 1.66 2.49 41 .82 1.64 2.46 40.5 .81 1.62 2.43 40 .80 1.60 2.40 39.5 .79 1.58 2.37 39 .78 1.56 2.34 38.5 .77 1.54 2.31 38 .76 1.52 2.28 37.5 .75 1.50 2.25 37 .74 1.48 2.22 36.5 .73 1.46 2.19 36 .72 1.44 2.16 35.5 .71 1.42 2.13 35 .70 1.40 2.10 ' 7;47 . -APPENDIX B Water Consumption per cage  5 ml 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17. 5 18 18.5 19 19.5 20 20.5 21 21.5 22 22.5 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 29 29.5 30 1 / F r a c t i o n of 50 ml 10 9.09 8.33 7.70 7.14 6 . 67 6.25 5.88 5.55 5.26 5.00 4.76 4.54 4.35 4.16 4.00 3. 85 3.70 3.57 3.45 3. 33 3.22 3.12 3.03 2.94 2.86 2.77 2.70 2.63 2.56 2.50 2.44 2.38 2.32 2.27 2.22 2.17 2.13 2.08 2.04 2.00 1.96 1.92 1.89 1.85 1.82 1.79 . 1.75 1.72 1.70 1.67 - (75 -APPENDIX C For example, a cage c o n t a i n i n g 50 grams of mice who were to r e c e i v e a 20 mg/kg dose averaged 10 ml of d r i n k i n g water d a i l y . From the f i r s t c o n v e r s i o n t a b l e (Appendix A) l o o k i n g across from 50 grams t o the 20 mg/kg dose, i s the value 1.0. On the second con-v e r s i o n t a b l e (Appendix B), across from 10 ml of water consumed, i s the value 5.0. The product of the two values o btained from the c o n v e r s i o n t a b l e s (1.0 and 5.0) i s the c o r r e c t amount of stock s o l u t i o n which w i l l then be d i l u t e d down to 50 ml t o t a l volume which w i l l be the a p p r o p r i a t e dose f o r t h i s cage. - (7:6 -APPENDIX D A l i z e r i n Red S t a i n i n g Procedure f o r Skeletons Fetuses are p l a c e d i n the f o l l o w i n g s o l u t i o n s : 1. 9 5% e t h a n o l f o r 24 hours 2. 1% KOH u n t i l animals are c l e a r and pink i n c o l o u r (24 hours) 3. 1% KOH and a l i z e r i n red s t a i n (1 or 2 drops) f o r 12-24 hours 4. g l y c e r i n and KOH (1:9) f o r 4 8 hours 5. g l y c e r i n and KOH (1:3) f o r 48 to 96 hours 6. g l y c e r i n and KOH (1:1) f o r 4 8 hours 7. s t o r e i n g l y c e r i n APPENDIX.E ANOVA Tables 1. WATER INTAKE source d.f . SS MS F Treatment 3 1.24.00 0 . 4133 0 .1270 E r r o r 116 • 377.5145 3 .2544 -T o t a l 119 378.7545 - -source d.f . SS MS F Genotype (+/+) 3 1.4011 0 .4670 0 .3797 E r r o r 36 44.2760 1 .2300 -T o t a l 39 45.6771 - -source d . f . SS MS F Genotypes (+/qk) 3 6.3968 2 .1322 0 .4904 E r r o r 36 156.5230 4 .3479 -T o t a l 39 162.9198 - -source d . f . SS MS F Genotype (qk/qk) 3 20.3071 6 .7690 1 .9111 E r r o r 36 127.5104 3 .5420 -T o t a l 39 147.8175 - -2. IMPLANTS source d.f . SS MS F Treatments 2 1.8211 0 .9105 0 .1421 E r r o r 14 89.7083 6 .4077 -T o t a l 16 91.5294 - 78 -source d . f . SS MS F Genotypes 2 17.729.4 8.8647 1.6817 E r r o r 14 73.8000 5.2714 . -T o t a l 16 91.5294 - -3. RESORPTIONS source d. f . SS MS F Treatments 2 0.8922 0.4461 0.3097 E r r o r 14 20.1666 1.4401 -T o t a l 16 21.0588 - -source d . f . SS MS F Genotypes 2 3.3588 1.6794 1.3283 E r r o r 14 17.7000 1.2643 -T o t a l 16 21.0588 - -4. FETAL WEIGHT source d.f. SS MS F Treatments 2 0.5518 0.2758 9.9928* E r r o r 108 2.9821 0.0276 -T o t a l 110 3.5339 - -(qk/qk) source d.f . SS MS F Treatment 1 0.4307 0.4307 10.5331* E r r o r 36 1.4720 0.0409 -T o t a l 37 1.9027 — — * - s i g n i f i c a n t (p<. 05) - 79 -(•+/qk) source d.f . SS. MS F Treatments 2 0, .1556 0, .0778 19, .2672* E r r o r 13 0, .0525 0, .0040 -J T o t a l 15 0. .2081 - -(+/+) source d.f. SS MS F Treatments 2. 0. . 1385 0, .0693 4, .0765* E r r o r 60 1. .0175 0. .0170 -T o t a l 62 1. .1560 — 

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