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The fetal alcohol syndrome in mice : an animal model Chernoff, Gerald F. 1977

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THE FETAL ALCOHOL SYNDROME IN MICE AN ANIMAL MODEL by GERALD F. CHERNOFF B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f M e d i c a l G e n e t i c s ) We a c c e p t t h i s t h e s i s as con f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Oc t o b e r , 1977 /-"N Gerald F. Chernoff, 1978 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives . It i s understood that copying or pub l i ca t i on of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Gerald P . Chernoff Department of Med ica l Genet ics The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Feb. 14, 1978 ABSTRACT Al t h o u g h the a d v e r s e e f f e c t s o f p r e n a t a l exposure t o a l c o h o l have been s u g g e s t e d s i n c e a n t i q u i t y , o n l y r e c e n t l y has a ' f e t a l a l c o h o l syndrome 1 been d e s c r i b e d i n human b e i n g s . S i n c e e t h i c a l c o n s i d e r a t i o n s l i m i t the types o f s t u d i e s p o s s i b l e w i t h humans, an animal model was d e v e l o p e d . CBA and C3H female mice, m a i n t a i n e d on a l i q u i d M e t r e c a l - e t h a n o l d i e t , r e c e i v e d from 0 t o 35 p e r c e n t e t h a n o l d e r i v e d c a l o r i e s (EDC) f o r a t l e a s t 30 days p r i o r t o and t h r o u g h o u t g e s t a t i o n . P r e n a t a l death and f e t a l a b n o r m a l i t i e s on day 18 o f g e s t a t i o n were r e l a t e d to maternal b l o o d a l c o h o l l e v e l s which i n c r e a s e d w i t h i n c r e a s i n g EDC. Reduced f e t a l w e i g h t s , s k e l e t a l , and n e u r a l anomalies were o b s e r v e d a t both low and h i g h maternal b l o o d a l c o h o l l e v e l s , w h i l e c a r d i a c and o c u l a r malform-a t i o n s , s i m i l a r t o t h o s e o b s e r v e d i n the human syndrome, e x h i b i t e d both a d o s e - r e s p o n s e e f f e c t and s t r a i n d i f f e r e n c e i n l i a b i l i t y , i n d i c a t i n g t h a t maternal c h r o n i c a l c o h o l i s m i s e m b r y o l e t h a l and t e r a t o g e n i c i n mice. In a second e x p e r i m e n t , the s t r a i n d i f f e r e n c e i n l i a b i l i t y was i n -v e s t i g a t e d u s i n g CBA, C3H, and C57 females m a i n t a i n e d on a 20 p e r c e n t EDC d i e t and mated i n a d i a l l e l e c r o s s . P r e n a t a l d e a t h , m a l f o r m a t i o n s , and f e t a l weights were d i r e c t l y r e l a t e d t o maternal b l o o d a l c o h o l l e v e l s , i i i i n d i c a t i n g a maternal e f f e c t . F e t a l a b n o r m a l i t i e s and maternal b l o o d a l c o h o l l e v e l s v a r i e d w i t h maternal s t r a i n (CBA > C3H > C57), and were i n v e r s e l y r e l a t e d to maternal a l c o h o l dehydrogenase a c t i v i t y . M i c r o -somal e t h a n o l o x i d i z i n g system i n d u c t i o n was d i r e c t l y a s s o c i a t e d w i t h i n -c r e a s e d f e t a l a b n o r m a l i t i e s , b e i n g g r e a t e s t i n CBA females and l o w e s t i n C57. The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t m a l f o r m a t i o n s o b s e r v e d i n the mouse and human syndrome a r e s i m i l a r , and t h a t l i a b i l i t y f o r t h e s e m a l f o r m a t i o n s i s dependent on maternal b l o o d a l c o h o l l e v e l s , which a r e det e r m i n e d by the r a t e o f maternal a l c o h o l m etabolism as w e l l as the amount o f maternal a l c o h o l consumption. ACKNOWLEDGEMENTS To P r o f e s s o r James R. M i l l e r , c h a i r p e r s o n o f my committee, t e a c h e r o f wisdom, and s o u r c e o f h e l p i n e v e r y phase o f t h i s s t u d y , I e x p r e s s my warmest a p p r e c i a t i o n . I t i s y o u r c o n s t a n t e n t h u s i a s m f o r e x p l o r i n g the gray a r e a r e l a t i n g e x p e r i m e n t a l r e s u l t s t o c l i n i c a l f i n d i n g s which p r o -v i d e d the f o u n d a t i o n upon which t h i s d i s s e r t a t i o n i s b u i l t . For t h e i r s u p p o r t , encouragement, and time i n v e s t e d i n t h i s p r o j e c t , I e x p r e s s g r a t i t u d e t o the members o f my committee: P r o f e s s o r C.W. "Bob" R o b e r t s , Dr. Derek A p p l e g a r t h , Dr. P a t r i c k McLeod, and Dr. Mi c h a e l C o r c o r a n . To E l i z a b e t h March, Jean McLeod, and the members o f the v i v a r i u m s t a f f , I o f f e r my a p p r e c i a t i o n f o r y o u r h e l p w i t h the d a i l y w e l l - b e i n g o f the mice, and y o u r w i l l i n g n e s s t o p r o v i d e both time and equipment f o r the c o m p l e t i o n o f t h i s s t u d y . I am g r a t e f u l t o my f r i e n d and c o l l e a g u e R i c h a r d F i n n e l l f o r the many l a t e e v e n i n g s s p e n t h e l p i n g w i t h the mouse ma t i n g s , as w e l l as the many hours o f p l e a s u r a b l e d i s c u s s i o n r e g a r d i n g v a r i o u s a s p e c t s o f t e r a t o g e n t e s t i n g . To the s t u d e n t s and s t a f f o f the M e d i c a l G e n e t i c s Department, I ex p r e s s thanks f o r the d i s c u s s i o n s you s h a r e d , and the warm memories you have s u p p l i e d . V A s p e c i a l note o f g r a t i t u d e i s due Dr. Kenneth Lyons Jones J r . , who p r o v i d e d the impetus f o r t h i s s t u d y w i t h h i s o r i g i n a l c l i n i c a l d e l i n e -a t i o n o f the syndrome, and who l a t e r as a f r i e n d and c r i t i c , f r e e l y gave o f h i s time i n d i s c u s s i n g the r e l a t i o n s h i p between the animal and human syndromes. To P r o f e s s o r D a v i d T. Suzuki and the members o f h i s l a b o r a t o r y from 1969 to 1970, I am p e r s o n a l l y i n d e b t e d f o r i n i t i a t i n g me t o the c r e a t i v e and p r a c t i c a l a s p e c t s o f e x p e r i m e n t a l g e n e t i c s . I e x t e n d a p p r e c i a t i o n t o the M e d i c a l Research C o u n c i l o f Canada and the D i s t i l l e d S p i r i t s C o u n c i l o f the U n i t e d S t a t e s , I n c . , f o r f i n a n c i a l l y s u p p o r t i n g t h i s p r o j e c t , both i n s u p p l i e s and s a l a r y , through g r a n t s t o P r o f e s s o r James R. M i l l e r . F i n a l l y , I would l i k e t o e x p r e s s my d e e p e s t thanks t o Win and Sacha f o r t h e i r unwavering f r i e n d s h i p , u n d e r s t a n d i n g , and s u p p o r t d u r i n g the e n t i r e t y o f t h i s p r o j e c t . vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ' i v LIST OF TABLES v i i i LIST OF FIGURES i x CHAPTER I. INTRODUCTION 1 H i s t o r i c a l P e r s p e c t i v e t o 1973 2 The F e t a l A l c o h o l Syndrome: 1973-1977. . . . 9 C l i n i c a l S t u d i e s 9 Animal S t u d i e s , ^ B e h a v i o r a l S t u d i e s 14 B i o c h e m i c a l S t u d i e s ^ E p i d e m i o l o g i c a l S t u d i e s . . . ^ I I . PURPOSE AND RATIONALE OF THE PRESENT STUDY 2 2 C r i t e r i a f o r an Animal Model 2 2 E t i o l o g i c C o n s i d e r a t i o n s 2 ° Experiments 2 9 I I I . METHODOLOGY 3 0 Experiment 1 30 Animals 30 D i e t s 3 0 D i e t A d m i n i s t r a t i o n 3 2 Matings and Pregnancy Management. . 3 3 F e t a l E x a m i n a t i o n , 3 3 D e t e r m i n a t i o n o f Blood A l c o h o l L e v e l s 3^ Data A n a l y s i s 3^ Experiment 2 3^ Animals . . . . . 3^ D i e t and A d m i n i s t r a t i o n 3 £ Matings and Pregnancy Management 3^ F e t a l E x a m i n a t i o n 3^ D e t e r m i n a t i o n o f B l o o d A l c o h o l L e v e l s 3' Enzyme P r e p a r a t i o n s 3^ A l c o h o l Dehydrogenase Assay . 3 8 Microsomal E t h a n o l O x i d i z i n g System Assay 3 8 v i i Page CHAPTER IV. RESULTS , , , 40 Experiment 1 40 Experiment 2. . 47 V. DISCUSSION 58 REFERENCES. 68 APPENDICES A. A l i z e r i n Red S t a i n i n g P r o c e d u r e f o r S k e l e t o n s . . . . 76 B. A n a l y s i s o f V a r i a n c e T a b l e s . 77 ANOVA f o r M a t e r n a l L i v e r Weight from TABLE 3 • . 78 ANOVA f o r M a t e r n a l B l o o d A l c o h o l L e v e l s by S t r a i n from T a b l e 3 , 79 ANOVA f o r Maternal B l o o d A l c o h o l L e v e l s Between S t r a i n s f rom TABLE 3 80 ANOVA f o r Implants from TABLE 4 81 ANOVA f o r R e s o r p t i o n s from TABLE 4 82 ANOVA f o r F e t a l Weights from TABLE 5 83 ANOVA f o r R e s o r p t i o n s from D i a l l e l e C r o s s , TABLES 8 and 9 84 ANOVA f o r F e t a l Weights from D i a l l e l e C r o s s , TABLES 10 and 11 85 ANOVA f o r F e t a l B l o o d A l c o h o l L e v e l s from TABLE 11 . . . . 86 ANOVA f o r F e t a l ADH A c t i v i t y by D i e t from TABLE 12 . . . . 87 ANOVA f o r F e t a l ADH A c t i v i t y by Mate r n a l Genotype from TABLE 12. . 88 ANOVA f o r Mat e r n a l Blood A l c o h o l L e v e l s from D i a l l e l e C r o s s , TABLE 13 89 ANOVA f o r Mate r n a l L i v e r Weight, ADH and MEOS A c t i v i t y from D i a l l e l e C r o s s , TABLE 13 90 r v i i i LIST OF TABLES Page T a b l e 1 C o m p o s i t i o n o f D i e t s , , . . 31 2 F e t a l Genotypes Generated With D i a l 1 e l e C r o s s 36 3 E f f e c t s o f D i e t s on C a l o r i c I n t a k e , L i v e r Weight and Blood A l c o h o l L e v e l s 41 4 E f f e c t o f D i e t s on I m p l a n t a t i o n and R e s o r p t i o n 42 5 E f f e c t o f D i e t s 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 i n CBA and C3H Females 45 6. Types and Frequency o f S k e l e t a l Anomalies 46 7 Types and Frequency o f S o f t T i s s u e Anomalies 48 8 I m p l a n t s , R e s o r p t i o n s and P e r c e n t R e s o r p t i o n s by F e t a l Genotype; D i e t 0 49 9 Implants, R e s o r p t i o n s and P e r c e n t R e s o r p t i o n s by F e t a l Genotype; D i e t 20 50 10 L i v e F e t u s e s , Weight, A b n o r m a l i t i e s and P e r c e n t Abnormal by F e t a l Genotype;:Dxet 0 51 11 L i v e F e t u s e s , Weight, A b n o r m a l i t i e s , P e r c e n t Abnormal and Blood A l c o h o l L e v e l s by F e t a l Genotype; D i e t 20 52 12. ADH A c t i v i t y by F e t a l Genotype and D i e t 54 13 Measurements o f M a t e r n a l Lifyer Weight, A l c o h o l Blood L e v e l s , ADH and MEOS A c t i v i t y i n Di a l l e l e C r o s s 55 14 S i g n i f i c a n t Sources o f V a r i a t i o n i n Measurements Determined by A n a l y s i s o f V a r i a n c e 57 15 S i m i l a r i t i e s Between the Human and Mouse F e t a l A l c o h o l Syndrome 60 16 T h e o r e t i c a l Rates o f T o t a l E t h a n o l O x i d a t i o n i n CBA, C3H and C57 Mice 66 tx LIST OF FIGURES Page F i g u r e 1 Dose Response Curve o f R e s o r p t i o n Rate 44 2 P e r c e n t Abnormal F e t u s e s f o r V a r y i n g Blood A l c o h o l L e v e l s . 62 CHAPTER I INTRODUCTION "Betsy; M a r t i n , widow, one c h i l d and one eye. Goes o u t c h a r i n g and washing by day; never had more than one eye, but knows her mother drank b o t t l e d s t o u t , and s h o u l d n ' t wonder i f t h a t caused i t " . C h a r l e s Dickens-1836 A l c o h o l , c l a i m e d by some t o be the e v i l o f mankind, and by o t h e r s t o be the s a l v a t i o n f o r a t r o u b l e d mind, has been a c o n t r o v e r s i a l sub-j e c t o f medical r e s e a r c h f o r a t l e a s t 200 y e a r s . R e c e n t l y , the d e s c r i p t i o n o f a f e t a l a l c o h o l syndrome i n humans has c o n t r i b u t e d f u r t h e r t o the c o n t r o v e r s y , and the q u e s t i o n posed by D i c k e n s ' B e t s y M a r t i n s t i l l a w a i t s an answer. The f e t a l a l c o h o l syndrome, which has been o b s e r v e d i n o f f s p r i n g o f c h r o n i c a l c o h o l i c women who c o n t i n u e heavy d r i n k i n g d u r i n g pregnancy, i s c h a r a c t e r i z e d by developmental and psychomotor d e l a y , p r e - and p o s t -n a t a l growth d e f i c i e n c y , i m p a i r e d i n t e l l e c t u a l performance, and c r a n i o -f a c i a l , c a r d i a c , and j o i n t a nomalies (Jones e t a l . , 1973). Because o f the magnitude o f a l c o h o l abuse i n North America, the e x i s t e n c e o f a f e t a l a l c o h o l syndrome has major s i g n i f i c a n c e f o r the p r e v e n t i o n o f con-g e n i t a l m a l f o r m a t i o n s and mental r e t a r d a t i o n . However, i t c a n , and has been, argued t h a t a l c o h o l i s m i s o n l y one v a r i a b l e common t o the syndrome, and t h a t o t h e r f a c t o r s such as maternal m a l n u t r i t i o n , d i s -r u p t i v e l i f e - s t y l e , and poor p r e n a t a l c a r e may c o n t r i b u t e t o the mal-f o r m a t i o n s o b s e r v e d . While the s e arguments may not be o f s i g n i f i c a n c e 2 t o the c l i n i c i a n d e a l i n g w i t h a f f e c t e d c h i l d r e n , they a r e o f r e l e v a n c e when d e l i n e a t i n g the e t i o l o g y and p o s s i b l e p r e v e n t i o n o f the syndrome, and t h e r e f o r e a re needed s u b j e c t s o f i n v e s t i g a t i o n . U n f o r t u n a t e l y , experiments t o e l u c i d a t e the p o s s i b l e e t i o l o g i c v a r i a b l e s o f the syndrome a r e not f e a s i b l e u s i n g human b e i n g s . C o n t r o l -l i n g c o n f o u n d i n g v a r i a b l e s would n e c e s s a r i l y impose r e s t r a i n t s on i n -d i v i d u a l freedom t h a t would be o f q u e s t i o n a b l e e t h i c s . Most i m p o r t a n t would be the q u e s t i o n o f a l l o w i n g an a l c o h o l i c woman t o c o n t i n u e a s u p e r v i s e d pregnancy knowing t h e r e i s an e l e v a t e d r i s k f o r an a f f e c t e d o f f s p r i n g , r e g a r d l e s s o f the e x a c t e t i o l o g y . F or t h e s e r e a s o n s , the o n l y p r a c t i c a l approach to s t u d y i n g t he p a t h o g e n e s i s o f t h i s syndrome i s by d e v e l o p i n g an a p p r o p r i a t e animal model. Once such a t o o l i s d e v e l o p e d , i t w i l l be p o s s i b l e to determine the e x a c t e t i o l o g y o f the syndrome as w e l l as i n v e s t i g a t e s e v e r a l o t h e r c l i n i c a l l y i m p o r t a n t i m p l i c a t i o n s o f the syndrome. H i s t o r i c a l P e r s p e c t i v e To 1973 The use o f drgus t o produce a l t e r e d s t a t e s o f c o n s c i o u s n e s s l i k e l y o r i g i n a t e d w i t h p r e h i s t o r i c man, and a l c o h o l , the p r o d u c t o f ferment-a t i o n , has p r o b a b l y been used by man as an i n t o x i c a t i n g agent s i n c e the i n v e n t i o n o f g l a z e d p o t t e r y (Green, 1974). With the use o f a l c o h o l by many c u l t u r e s o v e r l o n g p e r i o d s o f ti m e , v a r i o u s myths and s u s p i c i o n s have a r i s e n s u r r o u n d i n g i t s e f f e c t s . One such s u s p i c i o n , which e x i s t e d o v e r s e v e r a l c u l t u r e s , was t h a t a l c o h o l i n t o x i c a t i o n a t the time o f con-c e p t i o n o r d u r i n g pregnancy c o u l d r e s u l t i n abnormal o f f s p r i n g . Thus, Cart h a g e and S p a r t a i n a c t e d laws p r o h i b i t i n g newly wed c o u p l e s from d r i n k i n g s p i r i t s on t h e i r wedding n i g h t ; V u l c a n , the deformed b l a c k s m i t h 3 i n Greek mythology was the r e s u l t o f J u p i t e r ' s drunkeness; and the Holy B i b l e d e c l a r e s i n Judges 13:3-4, "Behold now, thou a r t b a r r e n , and b a r e s t n o t ; but thou s h a l l c o n c e i v e , and b ear a son. Now t h e r e f o r e be-ware, I pray t h e e , and d r i n k not wine o r s t r o n g d r i n k , and e a t not any u n c l e a n t h i n g " . The f i r s t documented r e p o r t s t o s u p p o r t t h o s e e a r l y s u s p i c i o n s ap-peared as a r e s u l t o f the E n g l i s h G i n Epidemic from 1720 t o 1751. D u r i n g t h i s p e r i o d , a law l i f t i n g t r a d i t i o n a l r e s t r i c t i o n s on d i s t i l l i n g was put i n t o e f f e c t p r o v i d i n g new g r a i n markets f o r the a r i s t o c r a t i c farm i n t e r e s t s . T h i s r e s u l t e d i n an abundant s u p p l y o f cheap g i n to the E n g l i s h p o pulace t h a t e v e n t u a l l y l e d t o a s o c i a l e p i d e m i c o f major p r o -p o r t i o n s . B i r t h r a t e s dropped, death r a t e s , e s p e c i a l l y f o r c h i l d r e n under f i v e , i n c r e a s e d , e v e n t u a l l y prompting the C o l l e g e o f P h y s i c i a n s t o p e t i t i o n P a r l i a m e n t f o r c o n t r o l on the d i s t i l l i n g t r a d e s , c i t i n g g i n as "A cause o f weak, f e e b l e , and d i s t e m p e r e d c h i l d r e n " (quoted i n George, 1965, p. 33). In such a s o c i a l c l i m a t e , i t i s not s u r p r i s i n g t h a t ab-s t i n e n c e groups formed and o f t e n used the n o t i o n o f p r e n a t a l i n s u l t as one o f the e v i l s o f a l c o h o l (see reviews by B a l l a n t y n e , 1902; Green, 1974; Warner and R o s e t t , 1975). A n e c d o t a l r e p o r t s , which seldom h e l d s c i e n -t i f i c v a l i d i t y , were quoted by a b s t i n e n c e p r e a c h e r s to b u i l d arguments such as t h a t s t a t e d i n a temperance manual from 1849 which c l a i m e d , " F a c t s a b u n d a n t l y show t h a t the c h i l d r e n o f mothers who d r i n k a l c o h o l a r e more l i k e l y than o t h e r s to become d r u n k a r d s , and i n v a r i o u s o t h e r ways to s u f f e r . O f t e n they a r e not so l a r g e and h e a l t h y as o t h e r c h i l d -r e n . They have l e s s s t r e n g t h o f e y e - s i g h t , l e s s f i r m n e s s o f q u i e t n e s s o f n e r v e s , l e s s c a p a c i t y o f g r e a t b o d i l y and mental achievement, and l e s s power to w i t h s t a n d the a t t a c k s o f d i s e a s e o r the v i c i s s i t u d e s o f c l i m a t e s 4 and season" (quoted i n Warner and R o s e t t , 1975, p. 1402). By the l a t e 1800s, animal experiments i n t e r a t o l o g y were b e i n g i n i t i a t e d i n F r a n c e , and the f i r s t e x p e r i m e n t a l s t u d i e s on the dangers o f p r e n a t a l exposure t o a l c o h o l began. T r e a t i n g c h i c k e n eggs by e i t h e r exposure to a l c o h o l vapor o r by d i r e c t i n j e c t i o n i n t o the egg w h i t e , Fere ( c i t e d by B a l l a n t y n e , 1902, p. 274) produced a v a r i e t y o f m a l f o r m a t i o n s i n c h i c k s . However, no m a l f o r m a t i o n s were o b s e r v e d by Mai r e t and Cambemale i n the o f f s p r i n g o f c o c k e r s p a n i e l dogs exposed t o a l c o h o l d u r i n g pregnancy ( c i t e d by Sandor and E l i a s , 1968, p. 53). Thus, the f i n d i n g s o f t h e s e e a r l i e s t experiments on the e f f e c t s o f p r e n a t a l ex-posure t o a l c o h o l were i n d i r e c t d i sagreement, s i g n a l l i n g t h e s t a r t o f many y e a r s o f c o n t r a d i c t o r y r e p o r t s . T h i s e a r l y s c i e n t i f i c c o n t r o v e r s y on the e f f e c t s o f a l c o h o l was b e s t e x e m p l i f i e d by B a l l a n t y n e i n h i s 1902 c l a s s i c monograph, Manual o f A n t e n a t a l P a t h o l o g y and Hygiene. In h i s review o f a l c o h o l i s m , B a l l a n t y n e (1902, p. 272-277) notes t h a t soon a f t e r the f i r s t animal e x p e r i m e n t s were r e p o r t e d , F o u r n i e r , n o t i n g a s i m i l a r i t y between a l c o h o l i s m and s y p h i l i s i n c a u s i n g s t r u c t u r a l d e f e c t s , r e p o r t e d o b s e r v i n g e c t r o d a c t y l y , d e f e c t s o f the o c c i p i t a l bone, h y d r o c e p h a l y , c r a n i a l asymmetry, p o r e n c e p h a l y , m i c r o c e p h a l y , and i n f a n t i l i s m i n c h i l d r e n o f a l c o h o l i c p a r e n t s . Support f o r t h i s f i n d i n g was c i t e d i n C a r r a r a ' s 1899 r e p o r t o f ne u r a l m a l f o r m a t i o n s i n the o f f s p r i n g o f guinea p i g s exposed t o a l c o h o l . However, i n a subsequent s t u d y i n r a b b i t s p r e n a t a l l y exposed t o a l c o h o l , B a l l a n t y n e notes t h a t no m a l f o r m a t i o n s were o b s e r v e d . At the t u r n o f the c e n t u r y , the d e m o n s t r a t i o n t h a t a l c o h o l c r o s s e d the p l a c e n t a i n r a b b i t s , g u i n e a p i g s , and human b e i n g s , s u p p o r t e d the n o t i o n t h a t a l c o h o l c o u l d have d e l e t e r i o u s e f f e c t s on the d e v e l o p i n g 5 embryo and f e t u s ( B a l l a n t y n e , 1902). Of p a r t i c u l a r i n t e r e s t were two papers by N i c l o u x ( c i t e d by B a l l a n t y n e , 1902, p. 273). The f i r s t s t u d y , u s i n g guinea p i g s , found b l o o d a l c o h o l l e v e l s a p p r o x i m a t e l y equal i n the mother and f e t u s a f t e r a one hour e q u i l i b r a t i o n p e r i o d . S i m i l a r l y , i n pregnant women g i v e n 60 cc o f rum a p p r o x i m a t e l y one hour p r i o r to de-l i v e r y , e x a m i n a t i o n o f f e t a l c o r d b l o o d as w e l l as p l a c e n t a r e v e a l e d e a s i l y d e t e c t a b l e amounts o f a l c o h o l , v e r i f y i n g t h a t i n humans and a n i m a l s , a l c o h o l c o u l d c r o s s the p l a c e n t a and e n t e r the f e t u s . D u r i n g t h i s same p e r i o d , W.C. S u l l i v a n (1899) used newly d e v e l o p e d e p i d e m i o l o g i c t e c h n i q u e s t o stu d y the e f f e c t s o f maternal a l c o h o l i s m i n a L i v e r p o o l p r i s o n p o p u l a t i o n . Of the 600 ob s e r v e d o f f s p r i n g born t o 120 female a l c o h o l i c s , 55 p e r c e n t were s t i l l b o r n o r d i e d under two y e a r s o f age; a s i g n i f i c a n t i n c r e a s e o v e r a c o n t r o l p o p u l a t i o n w i t h a s t i l l -b i r t h and i n f a n t m o r t a i l i t y r a t e o f 24 p e r c e n t . T h i s death r a t e o f o f f s p r i n g from a l c o h o l i c mothers i n c r e a s e d o v e r s u c c e e d i n g p r e g n a n c i e s , w i t h 34 p e r c e n t f o r f i r s t born and 72 p e r c e n t f o r s i x t h to t e n t h born. I f t h e mothers began to d r i n k a t l e a s t two y e a r s p r i o r t o t h e i r f i r s t p regnancy, the p e r i n a t a l m o r t a l i t y r a t e i n c r e a s e d t o 62 p e r c e n t f o r f i r s t b o r ns. Of p a r t i c u l a r i n t e r e s t was the o b s e r v a t i o n t h a t women who had adv e r s e p r e g n a n c i e s w h i l e a l c o h o l i c l a t e r bore h e a l t h y c h i l d r e n when f o r c e d t o a b s t a i n d u r i n g pregnancy because o f imprisonment. T h i s f i n d i n g w a r r a n t e d a pun from B a l l a n t y n e (1902, p. 275): "For the female h a b i t u a l d r u n k a r d , i t i s a p p a r e n t l y t h e b e s t t h i n g to be committed f o r a term o f imprisonment e a r l y i n her pregnancy; the p r i s o n baby may be the b e s t ! A sad f a c t , but a f a c t pregnant w i t h hope." F o l l o w i n g S u l l i v a n ' s i n i t i a l r e p r o d u c t i v e s t u d y , s e v e r a l i n v e s t i -g a t o r s , i n a v a r i e t y o f i n s t i t u t i o n a l s e t t i n g s , r e p o r t e d an a s s o c i a t i o n 6 between parenta l a l c o h o l i s m and o f f s p r i n g w i th e p i l e p s y , i d i o c y , and feeblemindedness (see review by Warner and R o s e t t , 1975). Then i n 1910, E lde r ton and Pearson repor ted a study of school c h i l d r e n from Edinburgh and Manchester t ha t f a i l e d to demonstrate a r e l a t i o n between maternal a l c o h o l i s m and abnormal phys ique , i n t e l l i g e n c e , o r d isease i n the o f f -s p r i n g . Ins t ead , they concluded tha t the low b i r t h w e i g h t s and observed a b n o r m a l i t i e s were the r e s u l t o f s o c i o l o g i c a l f a c t o r s which might be as-s o c i a t e d w i t h the tendency f o r abusive use of a l c o h o l . Th i s c o n c l u s i o n caused a fu ro r among abs t inence preachers , and a l s o po in ted out the n e c e s s i t y f o r f u r t h e r animal s tud ies i n a c o n t r o l l e d l a b o r a t o r y s e t t i n g . For the next severa l y e a r s , i n v e s t i g a t o r s us ing d i f f e r e n t animals and a d m i n i s t r a t i o n techniques reported c o n t r a d i c t o r y f i n d i n g s i n o f f -s p r i n g p r e n a t a l l y exposed to a l c o h o l . Most exhaus t ive was the work o f S tockard us ing f i s h embryos o f Fundulus h e t e r o c l i t u s , guinea p i g s , and c h i c k e n s . His e a r l i e s t work on f i s h blastomeres exposed to 2-5 percent d i l u t i o n s o f a l c o h o l repor ted o c u l a r malformations w i t h the extremes o f cyc lopy and microp tha lmia (S tocka rd , 1910). This was fo l l owed by a ten year study on guinea pigs i n which matings were made between normal females and a l c o h o l i z e d (exposed to a l coho l vapor) males , o r between a l c o h o l i z e d females and normal males. The l a t t e r c ross r e s u l t e d i n o f f -s p r i n g w i th e l eva ted p e r i n a t a l m o r t a l i t y ra tes and inc reased ra te o f malformat ions . These e f f e c t s p e r s i s t e d through seve ra l untreated gen-e r a t i o n s . Because o f the p o p u l a r i t y o f the then r e c e n t l y r e d i s c o v e r e d gene t ic laws o f Mendel , t h i s r e s u l t was i n t e r p r e t e d as being due to the t o x i c a c t i o n o f a l c o h o l on the germ c e l l s (S tocka rd , 1913; 1923). Re-peat ing F e r e ' s experiments w i th ch icken eggs exposed to a l c o h o l vapor , Stockard (1914) repor ted 100 percent malformed embryos a f t e r 14 to 20 7 hours o f t r e a t m e n t , and e m b r y o l e t h a l i t y w i t h t r e a t m e n t beyond 23 h o u r s . M a l f o r m a t i o n s r e s u l t e d from eye, neural and limb bud mai development, as w e l l as g e n e r a l r e t a r d a t i o n o f embryonic growth. In c o n t r a d i c t i o n t o S t o c k a r d ' s p o s i t i v e f i n d i n g s , P e a r l (1917) r e -p o r t e d t h a t t r e a t i n g hens f o r one hour w i t h a l c o h o l vapors r e s u l t e d i n fewer v i a b l e eggs, however, those t h a t d i d h a t c h were s u p e r i o r t o a con-t r o l s e r i e s i n terms o f m o r t a l i t y r a t e . A g a in u s i n g the p o p u l a r g e n e t i c n o t i o n s o f the day, t h e a u t h o r suggested t h a t a l c o h o l was u s e f u l as an i n t e r u t e r i n e s e l e c t i o n agent, s i n c e o n l y the most r e s i s t a n t embryos would reach h a t c h i n g . In 1923, McDowell r e p l i c a t e d S t o c k a r d ' s g u i n e a p i g e x p e r i -ments u s i n g mice and found an i n c r e a s e i n p r e n t a l m o r t a l i t y but no i n -c r e a s e i n the f r e q u e n c y o f m a l f o r m a t i o n s . S i m i l a r n e g a t i v e r e s u l t s were r e p o r t e d by N i c e (1912: 1917) i n a s e r i e s o f e x p e r i m e n t s u s i n g a l b i n o mice, and by Hanson and Cooper (1930) i n a s t u d y e x a m ining t e n gener-a t i o n s o f descendants from an o r i g i n a l group o f mice t r e a t e d w i t h a l c o h o l vapor. But most damaging t o S t o c k a r d ' s c r e d i b i l i t y was a s t u d y by Durhan and Woods (1932) t h a t f a i l e d t o f i n d an i n c r e a s e d m a l f o r m a t i o n r a t e i n the 6304 descendants o f 83 female guinea p i g s exposed to a l c o h o l vapor. The c o n f u s i o n r e s u l t i n g from the s e c o n t r a d i c t o r y r e p o r t s , as w e l l as the s o c i a l c l i m a t e s u r r o u n d i n g the end o f the American p r o h i b i t i o n , l e d to a g e n e r a l agreement t h a t p r e n a t a l exposure t o a l c o h o l was not d e l e t e r i o u s t o embryonic and f e t a l development. T h e r e f o r e , i n a p o p u l a r 1942 t e x t on a l c o h o l , Haggard and J e l l i n e k a t t r i b u t e d the f e t a l i n s u l t o b s e r v e d i n o f f s p r i n g o f a l c o h o l i c women t o poor n u t r i t i o n o r home environment r a t h e r than to a l c o h o l i t s e l f . T h i s same a t t i t u d e was r e -f l e c t e d by Roe (1944) who argued t h a t the h i g h m o r t a i l i t y r a t e , e p i l e p s y , 8 i d i o c y and p s y c h o s i s o b s e r v e d i n the o f f s p r i n g o f a l c o h o l i c women were the r e s u l t o f s o c i a l f a c t o r s r a t h e r than p h y s i c a l o r chemical damage due t o a l c o h o l . Even i n t h o s e animal s t u d i e s t h a t used the same r o u t e o f admin-i s t r a t i o n , i n c o n s i s t e n t r e s u l t s f a i l e d t o p r o v i d e i n s i g h t i n t o the a c t i o n o f a l c o h o l on the embryo o r f e t u s . Mirone (1952) was unable t o produce malformed o f f s p r i n g i n mice a d m i n i s t e r e d a l c o h o l o r a l l y , however the o f f s p r i n g o f female g u i n e a p i g s o r a l l y a d m i n i s t e r e d a l c o h o l t h r e e to f o u r times a week d i s p l a y e d s p e c i f i c m a l f o r m a t i o n s o f the c e n t r a l nervous system i n c l u d i n g abnormal f l a t t e n i n g o f t h e g y r i w i t h r e s u l t i n g s h a l l o w -ness o f f i s s u r e s , c e l l u l a r l e s i o n s i n the c o r t e x and b a s l g a n g l i a , edema, d i l a t i o n o f b l o o d v e s s e l s , hemorrhagic a r e a s , and g e n e r a l r e t a r d -a t i o n o f myel i n a t i o n a t b i r t h ( P a p a r a - N i c h o l s o n and T e l f o r d , 1957). L a s t l y , r a t s o r a l l y a d m i n i s t e r e d a l c o h o l p r i o r t o and d u r i n g pregnancy d e l i v e r e d o f f s p r i n g t h a t were normal a t b i r t h , but t h a t f a i l e d t o grow when nursed by a l c o h o l t r e a t e d mothers ( P i l s t r o m and K i e s s l i n g , 1967). U s i n g a m o d i f i c a t i o n o f F e r e ' s e a r l y e x p e r i m e n t s , Sandor and E l i as (1968) attempted t o examine the p a t h o g e n e s i s o f c h i c k embryos exposed t o a l c o h o l . Two p e r c e n t a l c o h o l i n j e c t e d i n t o the a i r space o f eggs a f t e r 30 hours i n c u b a t i o n r e s u l t e d i n a d i s t u r b a n c e o f c i r c u l a t i o n and growth r e t a r d a t i o n w i t h g e n e r a l i z e d m a l f o r m a t i o n o f the b r a i n v e s i c l e s and c audate n u c l e u s , e v e n t u a l l y r e s u l t i n g i n c h i c k m o r t a l i t y . By i n j e c t i n g eggs a t 23 hours i n c u b a t i o n and examining the embryos m i c r o s c o p i c a l l y a t 72 h o u r s , t h r e e o f t welve embryos were normal, w h i l e n i n e d i s p l a y e d de-formed s p i n a l c h o r d s , b r a i n v e s i c l e s and s o m i t e s , and developmental r e t a r d a t i o n (Sandor, 1968). These s t u d i e s , which s u g g e s t e d the mal-f o r m a t i o n s o c c u r i n e a r l y o r g a n o g e n e s i s , were extended t o r a t s . Females 9 were g i v e n 2 gm/kg a l c o h o l i n t r a v e n o u s l y on days s i x and seven o f g e s t -a t i o n , o r 1.5 gm/kg a l c o h o l i n t r a v e n o u s l y on g e s t a t i o n days s i x through e i g h t (Sandor and Amels, 1971). H a l f o f each l i t t e r was removed and examined on day 9.5 o f g e s t a t i o n , and the r e m a i n i n g h a l f on day 19.5. In th o s e embryos examined a t the e a r l i e r p e r i o d , t h e r e was an i n c r e a s e i n r e s o r p t i o n r a t e as w e l l as an i n c r e a s e i n t h e f r e q u e n c y o f dysmorphology i n c e n t r a l nervous system a n l a g e n , w h i l e i n those examined a t 19.5 days, t h e r e was o n l y an i n c r e a s e i n f e t a l r e s o r p t i o n and o c c u r r e n c e o f bone r e t a r d a t i o n . T h i s r e s u l t s u g g e s t s t h a t p r e n a t a l exposure t o a l c o h o l i n th e r a t most o f t e n r e s u l t s i n l e t h a l m a l f o r m a t i o n s , however i n thos e o f f -s p r i n g s u r v i v i n g , developmental r e t a r d a t i o n i s more common than mal-f o r m a t i o n s . T h i s second f i n d i n g i n the r a t was s u p p o r t e d by a human stu d y from t h e U n i v e r s i t y o f Washington t h a t a s s o c i a t e d maternal a l c o h o l i s m w i t h 41 p e r c e n t o f a group o f c h i l d r e n underweight f o r t h e i r g e s t a t i o n a l age. S u b s e q u e n t l y , 11 female a l c o h o l i c s were i d e n t i f i e d r e t r o s p e c t i v e l y , and te n o f t h e i r 12 c h i l d r e n were small f o r g e s t a t i o n a l age. F i v e o f the t e n i n f a n t s were r e t a r d e d i n performance on the G e s e l l o r Denver developmental s c a l e s , and e i g h t o f the c h i l d r e n f a i l e d t o t h r i v e g i v e n an adequate d i e t , w i t h weight and head c i r c u m f e r e n c e r e m a i n i n g below the t h i r d p e r c e n t i l e ( U l l e l a n d e t a l . , 1970). Three y e a r s l a t e r , t h e s e e i g h t c h i l d r e n were to become the f i r s t cases t o r e v i v e i n a d r a m a t i c way, an.awareness o f the e x i s t e n c e o f a f e t a l a l c o h o l syndrome. The F e t a l A l c o h o l Syndrome: 1973 t o 1977 C I i n i c a l S t u d i e s A f o l l o w - u p s t u d y on the e i g h t c h i l d r e n noted by U l l e l a n d t o have 10 r e t a r d e d growth was conducted by Jones and Smith a t the Dysmorphology U n i t , U n i v e r s i t y o f Washington, I t was found t h a t t h e s e c h i l d r e n d i s -p l a y e d a p a t t e r n o f m a l f o r m a t i o n s c h a r a c t e r i z e d by growth d e f i c i e n c y , which was more s e v e r e w i t h r e g a r d t o b i r t h l e n g t h than b i r t h w e i g h t ; s e v e r e p o s t n a t a l growth d e f i c i e n c y w i t h l i n e a r growth r a t e a v e r a g i n g 65 p e r c e n t o f normal, and average r a t e o f w e i g h t g a i n o n l y 30 p e r c e n t o f normal; mental r e t a r d a t i o n w i t h an average i n t e l l i g e n c e q u o t i e n t o f 63 i n t h o s e c h i l d r e n i n whom i t was measured; m i c r o c e p h a l y ; s h o r t p a l p e b r a l f i s s u r e s ; j o i n t a nomalies t h a t i n c l u d e d c o n g e n i t a l h i p d i s l o c a t i o n s , i n -a b i l i t y to extend the elbows c o m p l e t e l y , c a m p t o d a c t y l y o f toes and i n -a b i l i t y t o f l e x a t the m e t a c a r p a l - p h a l a n g e a l j o i n t s ; a l t e r a t i o n s o f palmar c r e a s e p a t t e r n s i n c l u d i n g r u d i m e n t a r y palmar c r e a s e s , a b e r r a n t a l i g n m e n t o f the palmar c r e a s e s , and/or s i n g l e upper palmar c r e a s e ; c a r d i a c a n o m a l i e s , t h e m a j o r i t y o f which were 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 f i n e motor d y s f u n c t i o n m a n i f e s t e d by a weak g r a s p , poor eye-hand c o o r d i n a t i o n , and t r e m u l o u s n e s s i n the newobrn p e r i o d (Jones e t a l . , 1973). The common p r e n a t a l f a c t o r s h a r e d by t h e s e c h i l d r e n was a s e v e r e c h r o n i c a l l y a l c o h o l i c mother who drank t h r o u g h o u t pregnancy; thus the term ' f e t a l a l c o h o l syndrome' was c o i n e d t o d e s c r i b e t h i s s p e c i f i c p a t t e r n o f m a l f o r m a t i o n s . Once t h i s i n i t i a l d e s c r i p t i o n was p u b l i s h e d , c a s e s t u d i e s from around the w o r l d began a p p e a r i n g i n the l i t e r a t u r e d e s c r i b i n g c h i l d r e n w i t h the syndrome ( B a r r y and O ' N u a l l a i n , 1975; B i e r i c h e t a l . , 1976; C h r i s t o f f e l l and S a l a f s k y , 1975; de Chateau, 1975; F e r r i e r e t a l . , 1973; Goetzman e t a l . , 1975; H a l l and O r e n s t e i n , 1974; I j a i y a e t a l . , 1976; Jones and Smith, 1973; L o i o d i c e e t a l . , 1975; L o s e r e t a l . , 1976; Manzke and G r o s s e , 1975; M u l v i h i l l and Yeager, 1976; M u l v i h i l l e t a l . , 11 1976; Noonan, 1976; Palmer e t a l , f 1974j R e i n h o l d e t al,, 1975; Root e t a l , , 1975; S a u l e , 1974; T e n b r i n k and B u c h i n , 1975). As e x p e r i e n c e w i t h t he syndrome i n c r e a s e d , anomalies o t h e r than t h o s e o r i g i n a l l y noted by Jones e t a l . were r e p o r t e d . U r o g e n i t a l anomalies i n c l u d i n g c r o s s e d f u s e d r e n a l e c t o p i a , r e n a l h y p o p l a s i a w i t h u r e t e r o ^ p e l v i c j u n c t i o n o b s t r u c t i o n , and p y e l o c a l y e c t a s i s were r e p o r t e d by T e n b r i n c k and Buchin (1975), and Goetzman e t a l , (1975). C a r d i o v a s c u l a r anomalies i n i t i a l l y r e p o r t e d i n 70 p e r c e n t o f the cases were shown t o o c c u r i n a p p r o x i m a t e l y 50 p e r c e n t o f the a f f e c t e d p a t i e n t s ( B a r r y and 0 K N u a l l a i n , 1975; L o s e r e t a l , , 1976; Noonan, 1976). A u t o p s i e s on 13 c h i l d r e n who d i e d near o r s h o r t l y a f t e r b i r t h r e v e a l e d e x t e n s i v e developmental anomalies o f the b r a i n i n c l u d i n g a b e r a t i o n o f neuronal m i g r a t i o n r e ^ s u i t i n g i n m u l t i p l e h e t e r o t o p i a s ; f u s i o n o f the a n t e r i o r s u p e r i o r g y r i through i n f i l t r a t i o n by l e p t o m e n i n g e a l hematomata o f g l i a l and neuronal c e l l s ; i n c o m p l e t e l y d e v e l o p e d c e r e b r a l c o r t e x as shown by r e l a t i v e a g y r i a and l a r g e l a t e r a l v e n t r i c l e s : ; ; and a g e n e s i s o f the corpus c a l l o s u m (Jones and Smith, 1973; J o n e s , 1975; C l a r r e n , 1977), Of major importance i n d e m o n s t r a t i n g t he p r e v a l a n c e o f the syndrome were t h r e e l a r g e s t u d i e s from F r a n c e , R u s s i a , and t h e U n i t e d S t a t e s , O r i g i n a l l y o v e r l o o k e d a t the time o f i t s p u b l i c a t i o n , the f i r s t s t u d y , by Lemoine and c o l l e a g u e s i n 1968, d e s c r i b e d 127 i n f a n t s born t o 69 French f a m i l i e s i n which t h e r e was c h r o n i c a l c o h o l i s m , Twenty p e r c e n t o f t h e s e c h i l d r e n had anomalies i n c l u d i n g c l e f t p a l a t e , m i c r o p t h a l m i a , limb m a l f o r m a t i o n s , c o n g e n i t a l h e a r t d i s e a s e , v i s e r a i a n o m a l i e s , pror. t r u d i n g f o r e h e a d , sunken na s a l b r i d g e , s h o r t upturned nose, r e t r a c t e d upper l i p , r e c e d i n g c h i n , and deformed e a r s , Many appeared h y p e r a c t i v e , had d e l a y e d psychomotor and language development, showed mental 12 r e t a r d a t i o n w i t h an average I.Q. o f 70, and, w i t h i n c r e a s i n g age had d i f f i c u l t y a t t e n d i n g t o t a s k s w i t h r e s u l t a n t b e h a v i o r a l problems a t s c h o o l . In the R u s s i a n s t u d y d e s c r i b i n g 98 p r e g n a n c i e s t o 18 c h r o n i c a l l y a l c o h o l i c women, 50 o f the p r e g n a n c i e s ended i n a b o r t i o n s , f i v e i n mis-c a r r i a g e s , and one i n a s t i l l b o r n , g i v i n g a t o t a l p e r i n a t a l m o r t a l i t y r a t e o f 57 p e r c e n t . Of the 42 r e s u l t i n g l i v e b i r t h s , 19 who were born b e f o r e t h e i r mother d e v e l o p e d a l c o h l i s m demonstrated v e g a t i v e , emotional and b e h a v i o r a l d i s o r d e r s which improved w i t h f a v o r a b l e m i c r o s o c i a l change. However, i n the 23 c h i l d r e n born a f t e r p r e n a t a l exposure t o a l c o h o l , 14 were m e n t a l l y r e t a r d e d and demonstrated o r g a n i c impairment o f t he c e n t r a l nervous system e a r l y i n i n f a n c y ( S h r u y g i n , 1974). The U n i v e r s i t y o f Washington s t u d y examined dysmorphogenic f e a t u r e s i n 41 c h i l d r e n born to c h r o n i c a l c o h o l i c women who c o n t i n u e d t o d r i n k d u r i n g pregnancy (Hanson e t a l . , 1976). In a d d i t i o n t o p r e v i o u s l y r e p o r t e d m a l f o r m a t i o n s o f the f e t a l a l c o h o l syndrome, two t o f i v e o f the c h i l d r e n had eye anomalies i n c l u d i n g m i c r o p t h a l m o s , i n t r a o c u l a r d e f e c t s , s t r a b i s m u s , and p t o s i s o f the e y e l i d s ; c l e f t p a l a t e ; m u s c u l a r s k e l e t a l a n omalies i n c l u d i n g p ectus excavatum; d i a p h r a g m a t i c a n o m a l i e s ; n a i l h y p o p l a s i a ; and cutaneous anomalies i n c l u d i n g pigmented neyfeandiih.i-rsiutttsm, Thus, t h e s e major s t u d i e s from s e p a r a t e p a r t s o f the w o r l d , a l o n g w i t h the many case r e p o r t s , v e r i f i e d the l o n g s u g g e s t e d a s s o c i a t i o n between maternal a l c o h o l i s m and a d v e r s e outcome o f pregnancy. Animal S t u d i e s S t i m u l a t e d by the human d a t a , s e v e r a l i n v e s t i g a t o r s attempted t o r e p l i c a t e the o b s e r v e d m a l f o r m a t i o n s i n a n i m a l s , In v i t r o t e s t s on day 9 and 10 r a t embryos demonstrated a d e c r e a s e i n somi t e s and r e t a r d e d b r a i n d i f f e r e n t i a t i o n a t a low a l c o h o l c o n c e n t r a t i o n (0.1 mg et h a n o l 13 per m l ) , w h i l e e m b r y o - l e t h a l i t y was o b s e r v e d a t h i g h e r c o n c e n t r a t i o n s ." (10 mg e t h a n o l per m l ) . E x t e n d i n g the st u d y t o i n u t e r o t e s t i n g , 5 ml o f 40 p e r c e n t e t h a n o l per kg was a d m i n i s t e r e d to 13 female r a t s on days 8-14 o f g e s t a t i o n . E x a m i n a t i o n o f f e t u s e s a t term r e v e a l e d an i n c r e a s e i n p o s t i m p l a n t a t i o n m o r t a l i t y and a s i g n i f i c a n t d e c r e a s e i n f e t a l w e i g h t ; however, no t e r a t o l o g i c a l e f f e c t s were o b s e r v e d ( S k o s y r e v a , 1973). When a l c o h o l was o r a l l y a d m i n i s t e r e d i n d r i n k i n g water f i v e weeks p r i o r to and d u r i n g g e s t a t i o n , l i t t e r s i z e and f e t a l w e i g h t was re d u c e d , but the o n l y anomalies r e p o r t e d i n the l i v e o f f s p r i n g were m i c r o c e p h a l y and gen-e r a l i z e d d e r m a t o l o g i c problems (Tze and Lee, 1975). Thus, attempts t o use the r a t as a model were u n s u c c e s s f u l . P o s s i b l e e x p l a n a t i o n s f o r t h i s f a i l u r e a r e t h a t t he maternal b l o o d a l c o h o l l e v e l s (average o f 61 mg pe r 100 ml b l o o d i n the Tze s t u d y ) were not s u f f i c i e n t t o cause f e t a l i n s u l t ; o f f s p r i n g were not examined f o r i n t e r n a l m a l f o r m a t i o n s ; and i n the Tze s t u d y , o f f s p r i n g may have been c a n n i b a l i z e d by the mothers a t b i r t h . In c o n t r a s t , mouse s t u d i e s have been s u c c e s s f u l i n p r o d u c i n g a l c o h o l i n d u c e d m a l f o r m a t i o n s , U s i n g t h e h y b r i d s t r a i n B6D2F^/J i n -j e c t e d i n t r a p e r i t o n e a l l y w i t h 0.030 ml p e r gram body wei g h t o f a 25 p e r -c e n t (v/v) s o l u t i o n o f 95 p e r c e n t e t h a n o l , K r o n i c k (1976) demonstrated t h a t f e t a l m a l f o r m a t i o n s were most p r e v a l e n t when maternal t r e a t m e n t oc-c u r r e d on days 8, 9, o r 10 o f g e s t a t i o n . M a l f o r m a t i o n s i n c l u d e d coloboma o f t he i r i s , which was p r e v a l e n t i n f e t u s e s exposed on days 8 o r 9; e c t r o d a c t y l y o f the forepaws, p r e v a l e n t on day 10; h y p o p l a s t i c a t r i a , which was i n f r e q u e n t l y o b s e r v e d from a l l t h r e e t r e a t m e n t days; hydron-e p h r o s i s , i n f r e q u e n t l y o b s e r v e d i n f e t u s e s t r e a t e d on days 9 o r 10; and exe n c e p h a l y o c c a s i o n a l l y o b s e r v e d i n f e t u s e s t r e a t e d on day 8 o r 9. F e t a l m o r t a l i t y i n c r e a s e d from 7 p e r c e n t w i t h t r e a t m e n t on g e s t a t i o n 14 day 7 t o a p p r o x i m a t e l y 45 p e r c e n t when females were t r e a t e d on day 10, 11, o r 12. T h i s s t u d y , which demonstrated c r i t i c a l time p e r i o d s f o r both the t e r a t o g e n i c i t y and e m b r y o l e t h a l i t y o f e t h a n o l a d m i n i s t e r e d i n t r a p e r i t o n e a l ^ , was s u p p o r t e d by a second s t u d y u s i n g C57B1/6J mice t h a t were o r a l l y a d m i n i s t e r e d a n u t r i t i o n a l l y adequate d i e t o f M e t r e c a l -e t h a n o l ( R a n d a l l , 1977). D i e t s c o n t a i n i n g 17, 25, o r 35 p e r c e n t e t h a n o l d e r i v e d c a l o r i e s (EDC) were a d m i n i s t e r e d to pregnant females on days 5 through TO o f g e s t a t i o n r e s u l t i n g i n b l o o d a l c o h o l l e v e l s r a n g i n g from l e s s than 40 mg per 100 ml b l o o d t o 300 mg per 100 ml b l o o d . F e t a l m o r t a l i t y , measured as r e s o r p t i o n s o r f e t u s e s dead a t b i r t h , i n c r e a s e d from 15 p e r c e n t a t the lo w e s t d i e t t o 25 p e r c e n t on the h i g h e s t d i e t w h i l e m a l f o r m a t i o n s , i n c l u d i n g s y n d a c t y l y , a d a c t y l y and e c t r o d a c t y l y o f the forepaws; m i c r o p t h a l m i a and a n o p t h a l m i a ; c a r d i o -v a s c u l a r anomalies i n v o l v i n g t h e major branches o f the a o r t a as w e l l as i n t e r c a r d i a c a b n o r m a l i t i e s ; h y d r o n e p h r o s i s and h y d r o u r e t e r ; and hyd r o c e p h a l u s and g a s t r o s c h i s i s , i n c r e a s e d from 8 p e r c e n t a t the low EDC d i e t s to 40 p e r c e n t a t the h i g h e s t d i e t s . A l t h o u g h both t h e s e mouse s t u d i e s r e p o r t e d m a l f o r m a t i o n s a s s o c i a t e d w i t h t he human syndrome, u n l i k e the human syndrome, no c l e a r p a t t e r n was o b s e r v e d . T h i s f a i l u r e t o r e p r o d u c e s y s t e m a t i c m a l f o r m a t i o n s c a s t s doubt on t h e a p p l i c a b i l i t y o f e q u a t i n g p r e n a t a l exposure to a l c o h o l d u r i n g o r g a n o g e n e s i s w i t h p r e -n a t a l exposure t h r o u g h o u t g e s t a t i o n . B e h a v i o r a l S t u d i e s The o b s e r v a t i o n o f e x t e n s i v e n e u r a l m a l f o r m a t i o n s i n a u t o p s i e d o f f -s p r i n g o f c h r o n i c a l c o h o l i c women (Jones and Smith, 1973; J o n e s , 1975; C l a r r e n , 1977) i s c o n s i s t e n t w i t h r e p o r t s o f i n t e l l e c t u a l and b e h a v i o r a l a b n o r m a l i t i e s i n c h i l d r e n p r e n a t a l l y exposed t o a l c o h o l . In a d d i t i o n 15 t o mental d e f i c i e n c y , I.Q. range o f 45 t o 105, 12 c h i l d r e n w i t h the syn -drome e x h i b i t e d b e h a v i o r a l a b e r a t i o n s i n c l u d i n g f i n e motor problems, weak and p r i m i t i v e g r a s p , poor f i g e r a r t i c u l a t i o n and d e l a y i n e s t a b -l i s h i n g hand dominance. As p r e s c h o o l e r s , they o f t e n had s e v e r e f e e d i n g problems, and tended t o be h y p e r a c t i v e w i t h poor a t t e n t i o n a l s k i l l s and problem s o l v i n g a b i l i t i e s ( S t r e i s s g u t h , 1976). In the 12 c h i l d r e n o f c h r o n i c a l c o h o l i c women who were measured f o r I.Q. a t 7 y e a r s o f age, s i x who l i v e d w i t h t h e i r mothers had a mean I.Q. o f 73, s i x who had sp e n t some time w i t h r e l a t i v e s had a mean I.Q. o f 84, and the c o n t r o l p o p u l a t i o n had a mean I.Q. o f 95. While t h i s i n d i c a t e s than an e n r i c h e d home environment might f a c i l i t a t e development i n m i l d l y a f f e c t e d c h i l d -ren w i t h o n l y b o r d e r l i n e r e t a r d a t i o n , c h i l d r e n w i t h the most s e v e r e s t i g m a t a o f t h e syndrome o f t e n were the most s e v e r e l y r e t a r d e d , and d i d not improve i n s p i t e o f e x c e l l e n t p o s t n a t a l c a r e (Jones e t a l . , 1974). In s t u d i e s on neonates p r e n a t a l l y exposed t o a l c o h o l , d e c r e a s e d r a p i d eye movement and q u i e t s l e e p , as w e l l as i n c r e a s e d f u s s i n e s s and c r y i n g have been r e p o r t e d (Landesman-Dwyer and K e l l e r , 1977). In ad-d i t i o n , d e c r e a s e d a c t i v i t y w i t h poor muscle tone and h a b i t u a t i o n as e v a l u a t e d by t h e B r a z e ! t o n Neonatal B e h a v i o r a l S c a l e has been noted ( S t r e i s s g u t h and B a r r , 1977) as has reduced l e a r n i n g i n an o p e r a n t t e s t i n g s i t u a t i o n ( M a r t i n , 1977). U n f o r t u n a t e l y the s i g n i f i c a n c e o f t h e s e f i n d i n g s i s u n c l e a r . Because o f h o s p i t a l d i s c h a r g e p r o c e d u r e s , the i n f a n t s were t e s t e d i n the f i r s t t h r e e days o f 1 i f e , " a n d t he r e s u l t s may have been confounded by a n e s t h e s i a o r o t h e r m e d i c a t i o n s g i v e n a t the time o f d e l i v e r y which were not f u l l y e l i m i n a t e d from t he i n f a n t a t t h e time o f t e s t i n g . F u r t h e r m o r e , s i n c e t h e r e i s no e v i d e n c e t h a t performance d u r i n g the neona t a l p e r i o d i s p r e d i c t i v e o f l a t e r l e a r n i n g 16 a b i l i t i e s , m e aningful r e s u l t s must a w a i t f u r t h e r s t u d i e s when the c h i l d -ren a re o l d e r . Animal s t u d i e s on b e h a v i o r and l e a r n i n g i n o f f s p r i n g p r e n a t a l l y ex-posed t o a l c o h o l , w h i l e g e n e r a l l y s u p p o r t i v e o f the human d a t a , f a i l t o g i v e u n e q u i v o c a l r e s u l t s . L e a r n i n g t o d i s c r i m i n a t e c o n t i n g e n c i e s i n a shock r e i n f o r c e m e n t punishment s i t u a t i o n was d e f i c i e n t when r a t s , p r e -n a t a l l y exposed t o a l c o h o l from day one o f g e s t a t i o n t o weaning were t e s t e d a t e i g h t months ( M a r t i n e t a l . , 1977). S i m i l a r l y , exposure t o a l c o h o l from day two o f g e s t a t i o n t o weaning i m p a i r e d T-maze and s h u t t l e -box l e a r n i n g when t e s t e d a t 21 to 33 days p o s t p a r t u r i t i o n ( S h a y w i t z , e t a l . , 1976). T h i s same d e f i c i e n c y i n s h u t t l e - b o x l e a r n i n g was ob-s e r v e d when r a t s , exposed to a l c o h o l t h r o u g h o u t g e s t a t i o n , were t e s t e d a t 1.5 months. However, when t h e s e a n i m a l s were t e s t e d a t a l a t e r age, 4.5 and s i x months, the d e f i c i e n c y was not o b s e r v e d , i n d i c a t i n g t h a t the n a t u r e o f the e a r l y d e f i c i t i n s h u t t l e - b o x l e a r n i n g was t r a n s i e n t (Auroux, 1973). In two out o f t h r e e s t u d i e s , a m b u l a t i o n i n an open-f i e l d s i t u a t i o n was i n c r e a s e d i n r a t s p r e n a t a l l y exposed to a l c o h o l ' , however w i t h age, the a c t i v i t y d e c r e a s e d ( M a r t i n , 1977; Bond and D i g i u s t o , 1976; Shaywitz e t a l . , 1976). While t h e s e s t u d i e s a re o f i n t e r e s t , t h e r e s u l t s s h o u l d be i n t e r p r e t e d c a r e f u l l y , because as men-t i o n e d e a r l i e r , p r e n a t a l exposure t o a l c o h o l has not been shown t o p r o -duce the c h a r a c t e r i s t i c m o r p h o l o g i c a b n o r m a l i t i e s o f the f e t a l a l c o h o l syndrome i n n e o n a t a l r a t s (Sandor and Amels, 1971; S k o s y r e v a , 1973; Tze and Lee, 1975). T h i s f a c t , a l o n g w i t h the c o n f o u n d i n g e f f e c t s o f c o n t i n u e d a l c o h o l a d m i n i s t r a t i o n through weaning, make c o n c l u s i o n s from t h e s e s t u d i e s t e n t a t i v e a t b e s t . In a s e r i e s o f s t u d i e s u s i n g mice, b e h a v i o r a l a b n o r m a l i t i e s i n 17 a n i m a l s p r e n a t a l l y exposed t o a l c o h o l were r e p o r t e d . O f f s p r i n g o f C57Bl/10Bg and DBA/l/Bg females a d m i n i s t e r e d a l c o h o l i n d r i n k i n g water p r i o r t o pregnancy and through day 14 p o s t - p a r t u r i t i o n d i s p l a y e d a r e d u c t i o n i n l a t e n c y t o a t t a c k and f i g h t i n g when measured as a d u l t s (Yanai e t a l . , 1976). When th e o f f s p r i n g were t e s t e d f o r a m b u l a t i o n , un-l i k e the r a t s t u d i e s , s c o r e s were reduced w i t h a noted i n c r e a s e d l a t e n c y t o l e a v e the i n i t i a l s quare ( G i n s b e r g e t a l . , 1976). Of p a r t i c u l a r i n t e r e s t was the f i n d i n g t h a t C57B1 mice p e r i n a t a l l y exposed t o a l c o h o l had an i n c r e a s e d s u s c e p t i b i l i t y t o a u d i o g e n i c s e i z u r e s . However, when c r o s s f o s t e r i n g t e c h n i q u e s were employed t o s e p a r a t e the p r e - and p o s t -n a t a l e f f e c t s o f a l c o h o l on t h i s i n c r e a s e d s u s c e p t i b i l i t y , i t was found t h a t the major d e t e r m i n a n t was the p o s t n a t a l exposure (Yanai e t a l . , 1975). T h e r e f o r e , w h i l e t h e s e s t u d i e s demonstrate t h a t e a r l y exposure to a l c o h o l can produce b e h a v i o r a l changes, the d a t a i n d i c a t e t h a t a t low; l e v e l s o f a l c o h o l exposure (maternal b l o o d a l c o h o l l e v e l s r a n g i n g from 20 t o 45 mg per 100 ml b l o o d ) , i t i s the e a r l y p o s t n a t a l exposure t h a t i s c r i t i c a l . T h i s p o s t n a t a l e f f e c t c o u l d be the r e s u l t o f f a u l t y n e u r a l m a t u r a t i o n i n e a r l y l i f e , e i t h e r as a d i r e c t i n s u l t from a l c o h o l , o r from a d e c r e a s e i n the q u a n t i t y o r q u a l i t y o f maternal m i l k p r o d u c t i o n . I t i s c l e a r t h a t b e h a v i o r a l s t u d i e s o f the f u t u r e , i f they a r e t o produce meaningful r e s u l t s , must employ c r o s s f o s t e r i n g t e c h n i q u e s . B i o c h e m i c a l S t u d i e s B i o c h e m i c a l s t u d i e s have been performed on r a t s p r e - and p o s t -n a t a l l y exposed t o a l c o h o l i n an attempt t o e x p l a i n t h e f u n c t i o n a l d e f i c i t s o b s e r v e d i n the syndrome. By f e e d i n g pregnant a n i m a l s a l c o h o l f o r two weeks p r i o r t o term and through l a c t a t i o n , p r o t e i n s y n t h e s i s measured by l e u c i n e i n c o r p o r a t i o n i n t o ribosomes was d e c r e a s e d i n both f e t a l and neonatal b r a i n , h e a r t and l i v e r (Rawat, 1975a; 1976; 1977). U s i n g the same t r e a t m e n t p r o t o c o l , a c e t y l c h o l i n e , the b r a i n e x c i t a t o r y n e u r o t r a n s m i t t e r , was d e c r e a s e d i n p r e n a t a l l y exposed f e t u s e s and neo-n a t e s , w h i l e the l e v e l s o f gamma-aminobutyric a c i d (GABA), an i n h i b i t o r y n e u r o t r a n s m i t t e r and i t s p r e c u r s o r , g l u t a m a t e , were i n c r e a s e d (Rawat, 1975b). R e c e n t l y , the d e m o n s t r a t i o n t h a t G A B A - t r a n s f e r a s e and glutamate d e c a r b o x y l a s e a c t i v i t i e s a r e d e c r e a s e d i n f e t a l and neonate b r a i n s o f a n i m a l s exposed t o a l c o h o l l e d Rawat (1977) t o s u g g e s t t h a t the metabolism o f GABA and glutamate i s r e d u c e d , r e s u l t i n g i n the h i g h e r o b s e r v e d l e v e l s . While i t i s tempting t o i n t e r p r e t t h e s e a l t e r a t i o n s i n n e u r o t r a n s m i t t e r l e v e l s and p r o t e i n s y n t h e s i s as b e i n g f a c t o r s i n the d e f i c i t s o b s e r v e d i n the human syndrome, i t s h o u l d f i r s t be remembered t h a t t h e r a t has not been shown t o be an a p p r o p r i a t e model f o r the syndrome, and s e c o n d l y , the e f f e c t s o f maternal a l c o h o l t r e a t m e n t d u r i n g l a c t a t i o n , which had s i g n i f i c a n t e f f e c t s i n the mouse b e h a v i o r a l s t u d y , have not been c a r e -f u l l y c o n t r o l l e d . T h e r e f o r e , w h i l e t h i s work has p r o m i s i n g p o s s i b i l i t i e s , the s i g n i f i c a n c e o f the r e s u l t s s h o u l d be viewed w i t h c a u t i o n . E p i d e m i o l o g i c a l S t u d i e s Of major importance from a c l i n i c a l p o i n t o f view i s a d e t e r m i n -a t i o n o f t h e f r e q u e n c y and v a r i a b i l i t y o f the f e t a l a l c o h o l syndrome i n the human p o p u l a t i o n . S e v e r a l e p i d e m i o l o g i c a l s t u d i e s , both r e t r o -s p e c t i v e and p r o s p e c t i v e have been i n i t i a t e d t o answer t h e s e q u e s t i o n s , but i n g e n e r a l the r e s u l t s have been i n c o n c l u s i v e . S t u d i e s from France (Kaminski e t a l . , 1976) and Germany (Mau and N e t t e r , 1974) have r e -p o r t e d t h a t mothers who consume a l c o h o l d u r i n g g e s t a t i o n have an i n -c r e a s e d i n c i d e n c e o f s h o r t p r e g n a n c i e s ( l e s s than 240 days) and s t i l l -b i r t h s ; but l i v e b i r t h s o f normal wei g h t and p h y s i c a l appearance. On 19 the o t h e r hand, an American s t u d y by R u s s e l l (1977) r e p o r t e d low b i r t h w e i g h t s i n o f f s p r i n g born t o women c l a s s i f i e d as h a v i n g an a l c o h o l r e -l a t e d p s y c h i a t r i c d i s o r d e r . In the German s t u d y , women who f r e q u e n t l y drank c o f f e e d u r i n g pregnancy had c h i l d r e n o f low b i r t h w e i g h t , s u g g e s t i n g t h a t i n the human p o p u l a t i o n , n u t r i t i o n a l and l i f e s t y l e d i f f e r e n c e s may confound t he e f f e c t o f a l c o h o l , making an a b s o l u t e d e t e r m i n a t i o n o f r i s k v e r y d i f f i c u l t . S i m i l a r c o n f o u n d i n g r e s u l t s have been noted i n two p r o s p e c t i v e s t u d i e s now b e i n g c a r r i e d out i n the U n i t e d S t a t e s . The l a r g e s t s t u d y , b e i n g c o n d u c t e d a t Loma L i n d a U n i v e r s i t y , has now r e p o r t e d t h a t i n the 1500 b i r t h s s t u d i e d t o d a t e , t he o n l y n o t a b l e e f f e c t o f a l c o h o l d r i n k i n g d u r i n g pregnancy i s low b i r t h w e i g h t . However, when c i g a r e t t e smoking i s t a k e n i n t o c o n s i d e r a t i o n , the low b i r t h w e i g h t s can be a t t r i b u t e d t o a c o m b i n a t i o n o f smoking and d r i n k i n g d u r i n g pregnancy, w i t h smoking h a v i n g t he major e f f e c t (Kuzma, 1977). In the Boston C i t y H o s p i t a l study ( R o s e t t e t a l . , 1976; O u e l l e t t e e t a l . , 1976; 1977), p r e l i m i n a r y r e p o r t s on 322 o f f s p r i n g f a i l e d t o show a c o r r e l a t i o n between the amount o f a l c o h o l consumed by the mother and the degree o f m a l f o r m a t i o n i n the o f f -s p r i n g . Hence, women c l a s s i f i e d as heavy d r i n k e r s (minimum o f one and o n e - h a l f d r i n k s p er day w i t h the o c c a s i o n a l i n t a k e o f a t l e a s t f i v e o r s i x d r i n k s ) bore c h i l d r e n w i t h t h e same degree o f f e t a l i n s u l t as women c l a s s i f i e d as moderate d r i n k e r s (more than one d r i n k p er month). Even more c o n f u s i n g was the f i n d i n g t h a t i n women who had been heavy d r i n k e r s i n t h e f i r s t t r i m e s t e r , but who reduced o r a b s t a i n e d i n the t h i r d t r i -m e ster, t he r a t e o f c o n g e n i t a l m a l f o r m a t i o n s i n t h e i r o f f s p r i n g was seven p e r c e n t as opposed to 41 p e r c e n t i n t h e o f f s p r i n g o f women who c o n t i n u e d heavy d r i n k i n g t h r o u g h o u t pregnancy. S i n c e t he m a j o r i t y o f c o n g e n i t a l m a l f o r m a t i o n s have t h e i r o r i g i n d u r i n g o r g a n o g e n e s i s i n the f i r s t t r i m e s t e r , such r e s u l t s a r e to be q u e s t i o n e d , The most r e a s o n a b l e e x p l a n a t i o n i s t h a t t h e s e women who d i d reduce t h e i r d r i n k i n g would have had c h i l d r e n w i t h a low c o n g e n i t a l m a l f o r m a t i o n r a t e even i f they had c o n t i n u e d t h e i r heavy d r i n k i n g p r a c t i c e t h r o u g h o u t g e s t a t i o n . Two s t u d i e s c o n d u c t e d a t the U n i v e r s i t y o f Washington have been most u s e f u l i n g i v i n g an i n d i c a t i o n o f the r i s k i n v o l v e d i n the syndrome. The f i r s t , u s i n g d a t a from t he 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 o f the N a t i o n a l I n s t i t u t e o f N e u r o l o g i c D i s e a s e and S t r o k e , e s t i m a t e d the f r e ^ quency o f a d v e r s e outcome o f pregnancy f o r c h r o n i c a l l y a l c o h o l i c women to be 43 p e r c e n t (Jones e t a l . , 1974), T h i s f i g u r e , which was a r r i v e d a t by n o t i n g t h a t o f the o f f s p r i n g o f 23 women who c h r o n i c a l l y drank a l c o h o l b e f o r e and d u r i n g pregnancy, f o u r d i e d p r i o r to one week o f age, and s i x had abnormal f e a t u r e s s u g g e s t i v e o f t h e f e t a l a l c o h o l syndrome, has been c r i t i c i z e d on the grounds o f sampling methodology ( R o s e t t , 1974; S t u r d e v a n t , 1974). S i n c e n a t i o n a l s u r v e y s have e s t i m a t e d t h a t f i v e p e r c e n t o f the female p o p u l a t i o n a r e heavy d r i n k e r s , among the 55,000 women t a k i n g p a r t i n the C o l l a b o r a t i v e Study, 2,700 would be e x p e c t e d t o f a l l i n t o t h e 'heavy' c l a s s i f i c a t i o n . S i n c e o n l y 23 cases o f maternal c h r o n i c a l c o h o l i s m were r e p o r t e d , the sample c o u l d r e p r e s e n t an extreme confounded by common n u t r i t i o n a l , s o c i o e c o n o m i c and d r i n k i n g v a r i a b l e s , o r an i n a c c u r a t e r e p o r t i n g o f maternal d r i n k i n g h i s t o r i e s . The p r e l i m i n a r y r e s u l t s from t he second s t u d y , p r o s p e c t i v e i n n a t u r e , s u g g e s t t h a t the e a r l y e s t i m a t e may a p p l y o n l y to c h r o n i c a l l y a l c o h o l i c women. In t h i s study,1.64 i n f a n t s , 74 born t o mothers consuming i n ex-ce s s o f one ounce o f a l c o h o l p er day, and 90 mothers consuming l e s s e r amounts, were examined. E l e v e n o f th e s e c h i l d r e n e x h i b i t e d c l i n i c a l 21 f e a t u r e s o f a l t e r e d growth and morphogenesis w i t h n i n e coming from the heavy d r i n k e r s and two from the low d r i n k i n g group, g i v i n g a t o t a l i n -c i d e n c e r a t e o f 12 p e r c e n t (Hanson and Smith, 1977). From t h e s e two s t u d i e s i t appears t h a t women who d r i n k i n exc e s s o f one ounce o f a l c o h o l d a i l y t h r o u g h o u t pregnancy have a r i s k o f 12 p e r c e n t f o r h a v i n g a c h i l d w i t h a l t e r e d growth and dysmorphogenesis, w h i l e women who a r e c h r o n i c a l c o h o l i c s and c o n t i n u e d r i n k i n g through pregnancy have an even g r e a t e r (43 p e r c e n t ) r i s k f o r an ad v e r s e outcome o f pregnancy. Whether t h e s e women have common n u t r i t i o n a l and s o c i o - e c o n o m i c f a c t o r s con-t r i b u t i n g t o the a b n o r m a l i t i e s o b s e r v e d can not be determined a t t h i s t i m e , nor can the e f f e c t s o f s m a l l e r amounts o f a l c o h o l . 22 CHAPTER II PURPOSE AND RATIONALE OF THE PRESENT STUDY Alt h o u g h many s t u d i e s have r e p o r t e d i n v e s t i g a t i n g the e p i d e m i o l o g i c , dysmorphic, b i o c h e m i c a l and b e h a v i o r a l e t i o l o g y o f the f e t a l a l c o h o l syndrome, l i t t l e i n s i g h t has been g a i n e d . The human s t u d i e s a r e con-founded w i t h unknown o r p o o r l y u n d e r s t o o d e n v i r o n m e n t a l v a r i a b l e s , and the animal s t u d i e s a r e based on the assumption t h a t f e t a l i n s u l t from p r e n a t a l exposure t o a l c o h o l i s e q u i v a l e n t i n a l l mammalian s p e c i e s ; an assumption not s u p p o r t e d by d a t a . C l e a r l y , an animal model o f the syndrome i s r e q u i r e d i f f u r t h e r s t u d i e s a r e g o i n g t o e l u c i d a t e the e t i o l o g y and p a t h o g e n e s i s o f the a b n o r m a l i t i e s o b s e r v e d i n the human popu-l a t i o n . I t i s the purpose o f t h i s s t u d y t o e s t a b l i s h such a model and to i n v e s t i g a t e some o f the f a c t o r s t h a t may i n f l u e n c e the degree o f i n s u l t found i n the p r e n a t a l l y exposed o f f s p r i n g . C r i t e r i a F o r An Animal Model When s e e k i n g an a p p r o p r i a t e model f o r the f e t a l a l c o h o l syndrome, one must q u e s t i o n t he a p p l i c a b i l i t y o f the t r a d i t i o n a l t e r a t o l o g i c a l method o f a d m i n i s t e r i n g t he s u s p e c t e d agent d u r i n g o r g a n o g e n e s i s o n l y . I t must be remembered t h a t the f e a t u r e s o f the human syndrom have been obs e r v e d o n l y i n o f f s p r i n g o f c h r o n i c a l c o h o l i c women, and t h e r e f o r e the s u s p e c t e d t e r a t o g e n i c agent i s not s i m p l y a l c o h o l , but the a c t i o n 23 o f c h r o n i c maternal a l c o h o l i s m . With t h i s i n mind, i t becomes r e a d i l y ap-p a r e n t t h a t t e s t i n g f o r a l c o h o l o n l y d u r i n g o r g a n o g e n e s i s i s not e q u i v a -l e n t t o t e s t i n g f o r the t e r a t o g e n i c i t y o f maternal c h r o n i c a l c o h o l i s m , and t h e r e f o r e a new s e t o f c r i t e r i a f o r t e s t i n g must be e s t a b l i s h e d . S i n c e c h r o n i c a l c o h o l i s m i s a d i s e a s e unique t o the human p o p u l a t i o n , the animal model, i f i t i s to be e f f e c t i v e i n d e l i n e a t i n g the e t i o l o g y , p r e v e n t i o n , and c u r e o f the human syndrome, must r e p l i c a t e where p o s s i b l e , the con-d i t i o n as o b s e r v e d i n the human p o p u l a t i o n ( F a l k e t a l . , 1972; M e l l o , 1973; L e s t e r and F r e e d , 1973). F u r t h e r m o r e , s i n c e i t i s the t e r a t o g e n i c i t y o f the maternal a c t i o n t h a t i s under q u e s t i o n , c r i t e r i a a p p l i c a b l e to t e r a t o l o g i c a l t e s t i n g must a l s o be f u l f i l l e d . By p a r a l l e l i n g c r i t e r i a f o r a d i a g n o s i s o f human a l c o h o l i s m ( C r i t e r i a Committee, 1972) and the p a t t e r n o f consumption i n mothers o f c h i l d r e n w i t h the f e t a l a l c o h o l syndrome ( U l l e l a n d , 1972), the f o l l o w i n g s e t o f c r i t e r i a was e s t a b l i s h e d . 1. O r a l Route o f A d m i n i s t r a t i o n : Most, i f not a l l c h r o n i c a l c o -h o l i c s i n g e s t r a t h e r than i n j e c t t h e i r a l c o h o l . T h i s i n t u r n i m p a i r s i n t e s t i n a l a b s o r p t i o n o f v i t a m i n s , which may r e s u l t i n a l t e r e d m e t a b o l i c p r o c e s s e s ( V i t a l e and C o f f e y , 1971). T h e r e f o r e , to s i m u l a t e the c o n d i t i o n o f c h r o n i c a l c o h o l i s m i n a n i m a l s , the a l c o h o l must be taken o r a l l y . 2. C x r c a d i a n D i s t r i b u t i o n o f A l c o h o l I n t a k e : In human c h r o n i c a l c o h o l i c s , d r i n k i n g t a k e s p l a c e t h r o u g h o u t the waking hours. Blood a l c o h o l l e v e l s , w h i l e f l u c t u a t i n g o v e r a 24 hour p e r i o d , seldom f a l l low enough to i n i t i a t e a l c o h o l w i thdrawal symptoms. T h e r e f o r e i n an animal model i t must be r e q u i r e d t h a t b l o o d a l c o h o l l e v e l s remain r e l a t i v e l y c o n s t a n t o v e r a 24 hour p e r i o d , t h e r e b y n e c e s s i t a t i n g an even i n t a k e o f a l c o h o l o v e r the n i g h t and day p e r i o d . 3. B l o o d A l c o h o l L e v e l s In t h e Human C h r o n i c A l c o h o l i c Range: 24 Human c h r o n i c a l c o h o l i c s m a i n t a i n b l o o d a l c o h o l l e v e l s r a n g i n g from 100 to 400 mg per 100 ml b l o o d . C o n s e q u e n t l y , the embryo/fetus o f a c h r o n i c a l c o h o l i c woman i s p o t e n t i a l l y exposed t o t h e s e same amounts. T h e r e f o r e , i n an animal model i t must be p o s s i b l e to a c h i e v e b l o o d a l c o h o l l e v e l s g r e a t e r than 100 mg per 100 ml b l o o d . 4. B e h a v i o r a l M a n i f e s t a t i o n o f I n t o x i f i c a t i o n : C h a r a c t e r i s t i c a l l y , the f i r s t s i g n s o f a l c o h o l i n t o x i c a t i o n a r e a t a x i a w i t h i n i t i a l hyper-k i n e s i s e v e n t u a l l y s u b s i d i n g t o l e t h a r g y . With time a t o l e r a n c e d e v e l o p s and the b e h a v i o r a l m a n i f e s t a t i o n s d e c r e a s e . The a c t u a l b a s i s f o r t h i s t o l e r a n c e i s unknown, however i t i s a common f e a t u r e o f a l c o h o l i s m and s h o u l d be o b s e r v e d i n the animal model. 5. Dependence: P h a r m a c o l o g i c a l dependence r e s u l t i n g from c h r o n i c a l c o h o l i s m i s m a n i f e s t e d by c h a r a c t e r i s t i c withdrawal symptoms when a l c o h o l i s removed from the a l c o h o l i c ' s d i e t . T h e r e f o r e , a s u r e s i g n t h a t an animal i s a l c o h o l i c i s the a b i l i t y t o demonstrate w i t h d r a w a l symptoms a f t e r removal o f a l c o h o l . 6. D i e t o f Adequate N u t r i t i o n a l S o urce: S i n c e one o f the c o n f o u n d i n g v a r i a b l e s i n human s t u d i e s has been the maternal d i e t a r y h a b i t s , i t i s e s s e n t i a l t h a t the animal model employ a d i e t t h a t meets the d a i l y n u t r i t i o n a l r e q u i r e m e n t s o f the animal used. T h i s i s not t o imply t h a t the animal i s not m a l n o u r i s h e d as a r e s u l t o f a l c o h o l d i s t u r b i n g ab-s o r p t i o n and m e t a b o l i c p r o c e s s e s , but r a t h e r t h a t the d i e t a v a i l a b l e t o the animal i s not d e f i c i e n t . 7. M a t e r n a l A l c o h o l i s m P r i o r to and D u r i n g Pregnancy: C h r o n i c a l c o h o l i s m has an a d v e r s e e f f e c t on almost e v e r y o r g a n o f the body. D i s e a s e o f the l i v e r , g a s t r o i n t e s t i n a l t r a c t , and the c a r d i o v a s c u l a r and h e m a t o p o i e t i c system a r e not uncommon ( S e i x a s , 1975). L i v e r 25 m e tabolism i s a l t e r e d and f a u l t y a b s o r p t i o n can l e a d to f o l a t e , t h i a m i n e , and magnesium d e f i c i e n c i e s ( L i e b e r , 1975). S i n c e a l l o f t h e s e maternal f a c t o r s may c o n t r i b u t e t o the f e t a l a l c o h o l syndrome, i t i s n e c e s s a r y to a d m i n i s t e r a l c o h o l b e f o r e pregnancy i n o r d e r t o a l l o w time f o r the v a r i o u s a l t e r a t i o n s caused by a l c o h o l i s m t o become m a n i f e s t . To f u r t h e r s i m u l a t e the human c o n d i t i o n , a l c o h o l t r e a t m e n t must then c o n t i n u e t h r o u g h o u t the pregnancy. 8. M a l f o r m a t i o n s i n O f f s p r i n g S i m i l a r t o Those i n the Human Syndrome: I f the model i s t o be u s e f u l i n f u r t h e r s t u d i e s o f t h i s syndrome, i t must p r o v i d e a s p e c i f i c p a t t e r n o f m a l f o r m a t i o n s t h a t a f f e c t the systems ob-s e r v e d i n the human syndrome. 9. Dose-Response E f f e c t : A c h a r a c t e r i s t i c o f t e r a t o g e n i c agents i s a dose r e l a t e d i n c r e a s e o f a f f e c t e d o f f s p r i n g , g o i n g from 0 t o 100 p e r c e n t ( W i l s o n , 1965). T h e r e f o r e , i f maternal c h r o n i c a l c o h o l i s m i s i n d e e d t e r a t o g e n i c , then such a dose-response e f f e c t s h o u l d be o b s e r v e d i n the model. I t i s p o s s i b l e t o meet the f i r s t s i x c r i t e r i a by i n d u c i n g a l c o h o l i s m w i t h s c h e u d l e i n d u c e d p o l y d i p s i a ( F a l k e t a l . , 1972), o r w i t h a M e t r e c a l -e t h a n o l l i q u i d d i e t ( F r e u n d , 1969). The p o l y d i p s i a t e c h n i q u e i s a t t r a c t i v e because i t a l l o w s the l e v e l o f a l c o h o l consumption i n a 24 hour p e r i o d t o be v a r i e d by the i n v e s t i g a t o r . U n f o r t u n a t e l y , f o r the method t o work, anim a l s must be k e pt a t a reduced weight i n s p e c i a l cages t h a t d e l i v e r the s p e c i f i e d amount o f food a t s p e c i f i c i n t e r v a l s . The l i q u i d d i e t u t i l i z e s the d i e t a r y s o u r c e o f c h o c o l a t e M e t r e c a l t o mask l a r g e q u a n t i t i e s o f a l c o h o l . With t h i s d i e t as the s o l e s o u r c e o f c a l o r i e s , a n i m a l s t e n d t o l o s e weight i n the e a r l y s t a g e s o f t r e a t m e n t , but soon r e t u r n t o t h e i r p r e t r e a t m e n t w e i g h t s . By u s i n g the l i q u i d d i e t t o f u l f i l l the f i r s t s i x 26 r e q u i r e m e n t s , i t i s p o s s i b l e t o t e s t t h e t e r a t o g e n i c i t y o f maternal c h r o n i c a l c o h o l i s m i n the mouse. E t i o l o g i c C o n s i d e r a t i o n s T e r a t o g e n i c agents o f e n v i r o n m e n t a l o r i g i n r e q u i r e a b i o l o g i c a l s y s -tem to a c t upon. T h i s i n t e r a c t i o n can b e s t be d e s c r i b e d i n terms o f a ' m u l t i f a c t o r i a l model' ( F r a s e r , 1976), where both e n v i r o n m e n t a l and b i o -l o g i c a l f a c t o r s d etermine how l i a b l e an embryo i s f o r t e r a t o g e n i c i n s u l t . In the f e t a l a l c o h o l syndrome, t he most l i k e l y c a n d i d a t e f o r the e n v i r o n -mental f a c t o r i s maternal c h r o n i c a l c o h o l i s m . B i o l o g i c a l f a c t o r s depend on the mechanism o f t e r a t o g e n e s i s , which can o c c u r e i t h e r by d i r e c t i n -s u l t on the d e v e l o p i n g o r ganism, o r i n d i r e c t i n s u l t v i a a l c o h o l induced a l t e r a t i o n i n maternal and/or f e t a l metabolism. D i r e c t i n s u l t , p o s s i b l e i n t h i s syndrome s i n c e a l c o h o l f r e e l y c r o s s e s the p l a c e n t a (Akesson, 1974), i m p l i e s t h a t l i a b i l i t y f o r m a l f o r m a t i o n i n -c r e a s e s as the amount o f t e r a t o g e n a v a i l a b l e f o r i n s u l t i n c r e a s e s . The amount o f a l c o h o l t h e embryo/fetus i s exposed t o w i l l t h e n be dependent on the amount o f maternal d r i n k i n g and the r a t e a t which a l c o h o l i s m e t a b o l i z e d , and thus e l i m i n a t e d from t he system. Acute doses o f a l c o h o l a r e m e t a b o l i z e d a l m o s t e x c l u s i v e l y i n the c y t o s o l f r a c t i o n o f l i v e r ( L u n d q u i s t , 1975). (1) CH 3CH 20H + NAD + ^ A D H * CH^CHO + NADH + H + In t h i s r e a c t i o n , g e n e r a l l y c o n s i d e r e d the r a t e l i m i t i n g s t e p i n a l c o h o l m e t a b o l i s m ( L u n d q u i s t , 1970), e t h a n o l i s o x i d i z e d t o a c e t a l d e h y d e by the n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e (NAD) - l i n k e d enzyme, a l c o h o l dehydrogenase (ADH). ADH isoenzymes, w i t h a l t e r e d a c t i v i t i e s , have been 27 d e s c r i b e d i n man (Ugarte e t a l . , 1970) and mouse (Sheppard e t a l . , 1968), and a f e t a l form d i f f e r e n t from t he a d u l t has been r e p o r t e d i n mice ( K r a s n e r e t a l . , 1974). With c h r o n i c a l c o h o l a d m i n i s t r a t i o n , a second pathway, the h e p a t i c microsomal e t h a n o l o x i d i z i n g system (MEOS), i s i n -duced ( L i e b e r and D e C a r l i , 1970). (2) CH 3CH 20H + NADPH + H + + 0 2 M E 0 S * CHgCHO + NADP + + H 20 In v i t r o s t u d i e s s u g g e s t t h a t a f t e r c h r o n i c a l c o h o l consumption, t h i s system may ac c o u n t f o r up t o 25 p e r c e n t o f a d u l t a l c o h o l m etabolism ( L i e b e r and D e C a r l i , 1970; 1972). Other s t u d i e s , f a i l i n g t o f i n d a c o r r e l a t i o n be-tween MEOS i n d u c t i o n and i n c r e a s e d a l c o h o l e l i m i n a t i o n , c a s t doubt on the s i g n i f i c a n c e o f t h i s pathway on i n v i v o m etabolism (Roach, 1973; Mezey, 1976). U n l i k e t h e ADH pathway, the MEOS i s not p r e s e n t i n t h e f e t a l l i v e r ( K a to, 1966), and thus i s e x c l u d e d from t h e f e t a l metabolism o f a l c o h o l . From the above, i t can be seen t h a t the embryo/fetus may be p r o t e c t e d a g a i n s t the d i r e c t a c t i o n o f a l c o h o l by both t he maternal and f e t a l ADH pathways, as w e l l as the maternal MEOS pathway. The r e l a t i v e importance o f t he maternal and f e t a l g e n e t i c c o n s t i t u t i o n i n the metabolism o f a l c o h o l , and thus t he amount a v a i l a b l e f o r i n s u l t , can be r e s o l v e d u s i n g a d i a l l e l e c r o s s . T h i s method, d e s c r i b e d by Schmidt (1919) as "the method o f complete i n t e r c r o s s i n g " , i n v o l v e s making a l l p o s s i b l e matings between two o r more s t r a i n s o f a n i m a l s . In the case o f t h r e e s t r a i n s (A,B,C), t h e r e a r e n i n e mating c o m b i n a t i o n s (AxA, AxB, AxC, BxA, BxB, BxC, CxA, CxB, CxC) r e s u l t i n g i n s i x f e t a l genotypes (AA, BB, CC, AB o r BA, AC o r CA, BC o r CB). By s c o r i n g t h e o f f s p r i n g f o r a phenotype such 28 as m a l f o r m a t i o n o r b l o o d a l c o h o l l e v e l , i t s h o u l d be p o s s i b l e t o d i s -t i n g u i s h between a maternal e f f e c t , where the f e t a l phenotypes a r e a s s o c i a t e d w i t h maternal genotype, and a f e t a l e f f e c t , where f e t a l pheno-typ e i s a s s o c i a t e d w i t h the f e t a l genotype. A l c o h o l i n d u c e d a l t e r a t i o n o f the maternal and f e t a l m e t a b o l i s m may o c c u r i n a number o f ways. M a l a b s o r p t i o n o f v i t a m i n s and m i n e r a l s can r e s u l t i n d e f i c i e n c i e s which t h e o r e t i c a l l y c o u l d have a d v e r s e consequences on a d e v e l o p i n g organism ( L i e b e r , 1975). A l c o h o l metabolism by ADH, e q u a t i o n ( 1 ) , r e s u l t s i n a r e d u c t i o n o f the NAD/NADH r a t i o , t h e r e b y i n -h i b i t i n g NAD-1inked r e a c t i o n s ( L i e b e r , 1975; L u n d q u i s t , 1975). The conse-quence o f t h i s i s not o n l y t he i n h i b i t i o n o f a l c o h o l m etabolism, but a l s o an i n h i b i t i o n o f l a c t a t e metabolism, r e s u l t i n g i n a c i d o s i s ( L i e b e r , 1975). T h i s a l t e r a t i o n i n the NAD/NADH r a t i o may be n o r m a l i z e d by i n -d u c t i o n o f the MEOS, e q u a t i o n ( 2 ) , which p r o v i d e s NADP + f o r t r a n s h y d r o x y -l a t i o n w i t h NADH, thus a c c e l e r a t i n g the ADH pathway by i n c r e a s i n g the amount o f a v a i l a b l e NAD (Veech e t a l . , 1969). I n d u c t i o n o f the MEOS a l s o r e s u l t s i n an appa r e n t i n d u c t i o n o f the microsomal drug d e t o x i f y i n g system, which not o n l y a c c e l e r a t e s drug metabolism i n the c h r o n i c a l c o h o l i c (Mezey, 1976), but a l s o m o d i f i e s hormone metabolism ( L i e b e r and D e C a r l i , 1973). Both mechanisms o f t e r a t o g e n e s i s may i n v o l v e the same b i o l o g i c a l f a c t o r s ; the r a t e o f a l c o h o l m etabolism by ADH and MEOS. In the case o f t e r a t o g e n e s i s by d i r e c t i n s u l t , an i n c r e a s e d r a t e o f met a b o l i s m would p r o -v i d e p r o t e c t i o n t o the d e v e l o p i n g o r g a n i s m by d e c r e a s i n g the amount o f t e r a t o g e n a v a i l a b l e f o r i n s u l t . In the second mechanism, t h i s same i n -c r e a s e d r a t e o f metabolism would prove d e l e t e r i o u s t o the d e v e l o p i n g organism s i n c e the e f f e c t s o f a l t e r e d metabolism, w i t h the d e s c r i b e d 29 harmful s i d e - e f f e c t s , would a l s o be i n c r e a s e d . An e x a m i n a t i o n o f the r o l e o f a l c o h o l m e t a b o l i s m as measured by ADH and MEOS a c t i v i t i e s would p r o v i d e i n s i g h t i n t o both t he mechanism and b i o l o g i c a l f a c t o r s r e s p o n s i b l e f o r the m a l f o r m a t i o n s o b s e r v e d i n t h i s syndrome. Experiments The goal o f the f i r s t e x p e r i ment i n t h i s s t u d y was t o e s t a b l i s h an animal model o f t h e f e t a l a l c o h o l syndrome. A t t h e same ti m e , by u s i n g two s t r a i n s o f mice to m a n i p u l a t e the g e n e t i c v a r i a b l e s a n d s e v e r a l doses o f c h r o n i c a l c o h o l consumption t o m a n i p u l a t e t he en v i r o n m e n t a l v a r i a b l e , the e n v i r o n m e n t a l and g e n e t i c f a c t o r s r e s p o n s i b l e f o r t h e m a l f o r m a t i o n s were i n v e s t i g a t e d . The second e x p e r i m e n t , which f o l l o w e d the f i r s t c h r o n o l o g i c a l l y , u t i l i z e d a d i a l l e l e c r o s s t o dete r m i n e the r e l a t i v e importance o f t h e maternal and f e t a l genotypes i n l i a b i l i t y f o r m a l f o r m a t i o n . F u r t h e r m o r e , i n an e f f o r t t o determine the mechanism o f t e r a t o g e n e s i s , and t h e r e b y g a i n i n s i g h t i n t o t h e c r i t i c a l b i o l o g i c a l f a c t o r s , m e t a b o l i c r a t e s as determined by ADH a c t i v i t i e s i n mothers and f e t u s e s , and MEOS a c t i v i t i e s from the mothers, were i n v e s t i g a t e d . 30 CHAPTER I I I METHODOLOGY Experiment 1 An i m a l s : Ml CBA/J and C3H/1g mice (Mus musculus) o b t a i n e d from 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 , U n i v e r s i t y o f B r i t i s h Columbia, were m a i n t a i n e d on a 12 hour l i g h t c y c l e i n the Zoolo 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 Columbia. Animals were housed 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 , females i n p a i r s and males s i n g l y , and a l l o w e d ad 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 tap water u n l e s s o t h e r w i s e n o t e d . D i e t s The l i q u i d d i e t s l i s t e d i n TABLE 1, d e f i n e d by the p e r c e n t o f e t h a n o l d e r i v e d c a l o r i e s (EDC) each c o n t a i n s (Freund, 1969; Freund and Walker, 1971), were f e d to females ad l i b i t u m . The l i q u i d p r e p a r a t i o n c o n s i s t e d o f c h o c o l a t e M e t r e c a l (Mead Johnson Co., E v a n s v i l l e , Ind.) c o n t a i n i n g 0.95 c a l p er ml and V i t a m i n D i e t F o r t i f i c a t i o n M i x t u r e ( N u t r i t i o n a l B i o -c h e m i c a l s , C l e v e l a n d , O h i o ) , 3 gm per l i t e r . D i e t 0 c o n t a i n e d t he b a s i c l i q u i d d i e t p l u s i s o c a l o r i c amounts o f s u c r o s e (87 p e r c e n t volume per volume and 3.5 c a l p er ml) f o r e t h a n o l . D i e t 15 c o n s i s t e d o f the l i q u i d d i e t p l u s 95 p e r c e n t volume per volume e t h y l a l c o h o l (5.25 c a l per m l ) , 31 TABLE 1 C o m p o s i t i o n o f D i e t s Ethanol (95% v/v) M e t r e c a l D i e t (% EDC) C a l o r i e s ml/100 C a l o r i e s ml/100 % % Lab Chow -0 - 100 100.0. 15 15 3.1 85 96.9 20 20 4.3 80 95.7 25 25 5.7 75 94.3 30 30 7.2 70 92.8 35 35 8.9 65 91.1 32 added such t h a t the f i n a l p r e p a r a t i o n c o n t a i n e d 15 p e r c e n t EDC. D i e t 20 c o n t a i n e d 20 p e r c e n t EDC, d i e t 25, 25 p e r c e n t EDC, and so on. The l i q u i d d i e t s were p r e p a r e d f r e s h d a i l y and were the o n l y s o u r c e o f c a l o r i e s . The p r e p a r a t i o n , c o m p o s i t i o n , and documentation o f n u t r i t i o n a l adequacy o f the l i q u i d d i e t s used were noted i n d e t a i l p r e v i o u s l y (Walker and Freund, 1971); each t r e a t m e n t d i e t c o n t a i n e d s e v e r a l times the minimum d a i l y r e q u i r e m e n t s o f a l l n u t r i e n t s , based on p r e v i o u s recommendations f o r mice (Walker and Z o r n e t z e r , 1974). Animals r e c e i v e d the l i q u i d s from i n v e r t e d 50-ml B-D p l a s t i c s y r i n g e s w i t h the ne e d l e end s e a l e d by m e l t i n g , through s t a n d a r d g l a s s d r i n k i n g tubes w i t h 1.5 mm openings e x t e n d i n g t o a p p r o x i m a t e l y 3 cm above the cage f l o o r . D a i l y f l u i d consumption and c a l o r i c i n t a k e was determined by measuring the amount o f f l u i d l e f t each day a t between 10 AM and 12 noon, o r 10 PM and 12 m i d n i g h t . C a l o r i c i n -take f o r c o n t r o l a n i m a l s on L a b o r a t o r y Chow was determined u s i n g m e t a b o l i c cages. D i e t A d m i n i s t r a t i o n To a v o i d weight l o s s and s i c k n e s s , which might have r e s u l t e d from an i n i t i a l i n t r o d u c t i o n to h i g h e t h a n o l d o s e s , a n i m a l s were i n t r o d u c e d t o the d i e t s i n s t a g e s . V i r g i n f e m a l e s , 60 t o 100 days o l d were taken o f f L a b o r a t o r y Chow and g i v e n d i e t 0. A f t e r t en days on t h i s d i e t , they were i n t r o d u c e d t o e t h a n o l by d i e t 15 o r 20, which was g i v e n f o r t e n days, f o l -lowed by the next h i g h e r e t h a n o l d i e t f o r t e n days, u n t i l the p r e d e t e r m i n e d number o f females w e r e i i n e a c h d i e t group. The females were then main-t a i n e d on t h e i r r e s p e c t i v e group d i e t s f o r a t l e a s t 30 days b e f o r e the i n i t i a l mating attempt. A t t h i s t i m e , a l l females had been on l i q u i d d i e t s 80 days, and on t h e i r r e s p e c t i v e group d i e t from 30 t o 80 days, depending on the d i e t group i . e . , d i e t 0, 80 days; d i e t 15, 70 days; 33 d i e t 35, 30 days. Matings and Pregnancy Management Matings between animals o f the same s t r a i n were i n i t i a t e d by i n t r o -d u c i n g an e s t r o u s female i n t o a males cage. To keep the males s o b e r and the females i n t o x i c a t e d , mating time was r e s t r i c t e d t o 1.5 hou r s , a t which time the mating p a i r was d e p r i v e d o f f o o d and water. T h i s was done to p r e v e n t c o n f o u n d i n g the r e s u l t s w i t h male a l c o h o l consumption, which has been r e p o r t e d to cause dominant l e t h a l m u t a t i o n s (Badr and Badr, 1975; K l a s s e n and Persaud, 1976). The pre s e n c e o f a c o p u l a t i o n p l u g was taken as i n d i c a t i n g day one o f g e s t a t i o n . Females were kept on t h e i r r e s p e c t i v e d i e t s t h r o u g h o u t g e s t a t i o n , and on day 18, were weighed and then 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 . F e t a l E x a m i n a t i o n A f t e r o p e n i n g the u t e r u s , r e s o r p t i o n s i t e s , l o c a t i o n and p o s i t i o n o f f e t u s e s were r e c o r d e d . F e t u s e s were removed by c u t t i n g the u m b i l i c a l c o r d s , and checked f o r l i f e by n o t i n g c o l o u r , and p r o b i n g u n t i l movement o f e i t h e r limbs o r mouth was o b s e r v e d . F e t u s e s t h a t f a i l e d t o show any movement and were p a l e i n c o l o u r were c o n s i d e r e d dead and counted w i t h the r e s o r p t i o n s . F e t u s e s were examined f o r e x t e r n a l m a l f o r m a t i o n s o f head, p a l a t e , l i m b s , d i g i t s , t r u n k and t a i l , and were sexed w i t h the a i d o f a W i l d d i s s e c t i n g m i c r o s c o p e . The l i v e f e t u s e s were then weighted on a M e t t l e r top l o a d i n g b a l a n c e and the wei g h t r e c o r d e d to one hundredth o f a gram. O n e - t h i r d o f the l i v e a n i m a l s were randomly a s s i g n e d f o r A l i z a r i n Red S S k e l e t a n s t a i n i n g ( C r a r y , 1962; Appendix A ) , and the remainder f i x e d i n Bouin's s o l u t i o n f o r subsequent e x a m i n a t i o n by f r e e -hand r a z o r s e c t i o n i n g ( W i l s o n , 1965). S k e l e t a l anomalies were determined 34 by o b s e r v i n g p r e p a r e d f e t u s e s i n g e l a t i n f i l l e d p e t r i d i s h e s under a d i s s e c t i n g m i c r o s c o p e . S o f t t i s s u e m a l f o r m a t i o n s were determined by ob-s e r v i n g 1-2 mm c r o s s s e c t i o n a l s l i c e s p l a c e d i n 70 p e r c e n t e t h a n o l i n white p o r c e l a i n s p o t p l a t e s under a d i s s e c t i n g m i c r o s c o p e . D e t e r m i n a t i o n o f Blood A l c o h o l L e v e l s Blood samples were taken by r e t r o - o r b i t a l b l e e d from females t h r e e days b e f o r e the f i r s t mating attempt, and by e i t h e r r e t r o - o r b i t a l b l e e d i n g o r c a r d i a c p u n c t u r e j u s t p r i o r t o k i l l i n g on day 18 o f g e s t a t i o n . To maximize any v a r i a b i l i t y i n b l o o d a l c o h o l l e v e l s due t o c i r c a d i a n f l u c t u -a t i o n s , random samples were c o l l e c t e d o v e r a d a i l y 16 hour p e r i o d , 10 AM to 2 AM. U s i n g 20 m i c r o l i t e r Drummond mi c r o c a p g l a s s b l o o d c o l l e c t i n g t u b e s , o r a 3 cc t u b e r c u l i n s y r i n g e armed w i t h a 21 x 1.5 n e e d l e f o r c a r d i a c p u n c t u r e , 1 t o 2 ml o f b l o o d was t r a n s f e r r e d i n t o a h e p a r i n r i n s e d 16 x 125 mm screw capped c u l t u r e tube and s t o r e d i n the r e f r i g e r a t o r 12 to 24 hours. At the time o f a s s a y , 0.1 ml o f serum o r plasma was d i l u t e d w i t h 4.9 ml o f a 0.9 p e r c e n t s o l u t i o n o f sodium c h l o r i d e , and 0.1 ml o f t h i s sample was used w i t h a C a l b i o c h e m ' A l c o h o l S t a t - P a k ' a s s a y k i t (Jones e t a l . , 1970). The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f 0.1 ml p r e -pared sample t o 2.6 ml o f prewarmed commercial r e a g e n t which c o n t a i n e d 50 mM TRIS b u f f e r (ph 8.8), .15 mM NAD, 13800 IU per l i t e r ADH, and a t r a p p i n g agent to remove a c e t a l d e h y d e from the r e a c t i o n m i x t u r e . A f t e r t e n minutes o f i n c u b a t i o n a t 30 degrees C, the molar e q u i v a l e n t s o f r e -duced NAD g e n e r a t e d i n the r e a c t i o n m i x t u r e were measured i n d u p l i c a t e , a g a i n s t water, i n q u a r t z c u v e t t e s w i t h 1 cm l i g h t p a t h , a t 340 nm on a Beckman DU s p e c t r o p h o t o m e t e r . The b l o o d a l c o h o l l e v e l s i n gm per 100 ml b l o o d were determined by t a k i n g the numerical d i f f e r e n c e between the sample and b l a n k (0.1 ml n i n e p e r c e n t w/v sodium c h l o r i d e i n 2.6 ml 35 commercial r e a g e n t ) a b s o r b a n c e s ; a method made p o s s i b l e by h a v i n g the volume o f r e a g e n t and sample f i x e d . Data A n a l y s i s Mean d i f f e r e n c e s o f measurements were t e s t e d a t the 0.05 l e v e l o f s i g n i f i c a n c e u s i n g a Type I A n a l y s i s o f V a r i a n c e (Sokal and R o h l f , 1969). Where s i g n i f i c a n t d i f f e r e n c e s were found, a Duncans M u l t i p l e Range T e s t was a p p l i e d t o determine the s i g n i f i c a n t d i f f e r e n c e s between means ( B l i s s , 1967). In the one measurement w i t h a known e x p e c t e d f r e q u e n c y , a Chi -square t e s t was u t i l i z e d a t the 0.05 l e v e l o f s i g n i f i c a n c e . Experiment 2 The methodology f o r Experiment 2 d e p a r t e d from t h a t d e s c r i b e d f o r Experiment 1 as f o l l o w s : Animals Ml In a d d i t i o n t o the CBA/J and C3H/1g mice p r e v i o u s l y mentioned, C57BL/6J mice o b t a i n e d from the J a c k s o n L a b o r a t o r i e s , Bar Harbor, Maine, were used. D i e t and A d m i n i s t r a t i on To c o n t r o l e n v i r o n m e n t a l v a r i a b l e s , a l l female mice were a d m i n i s t e r e d and m a i n t a i n e d o n a 20 p e r c e n t EDC d i e t as p r e v i o u s l y d e s c r i b e d f o r Experiment 1. Matings and Pregnancy Management Ml A l l p o s s i b l e matings between s t r a i n s CBA/J, C3H/lg , and C57BL/6J were made u s i n g a d i a l l e l e c r o s s . TABLE 2 i l l u s t r a t e s the f e t a l geno-types produced i n t h i s c r o s s . 36 TABLE 2 F e t a l Genotypes Generated w i t h a D i a l l e i C r o s s Males Females CBA C3H C57 CBA CBA/CBA CBA/C3H CBA/C57 C3H C3H/CBA C3H/C3H C3H/C57 C57 C57/CBA C57/C3H C57/C57 37 F e t a l E x a m i n a t i o n A f t e r e x t e r n a l e x a m i n a t i o n , the f e t u s e s were d e c a p i t a t e d , and one-h a l f o f the heads were p r e p a r e d f o r s k u l l s k e l e t a l s t a i n i n g as p r e v i o u s l y d e s c r i b e d , and the r e m a i n i n g h a l f were f i x e d i n Bouin's s o l u t i o n f o r b r a i n e x a m i n a t i o n by the f r e e h a n d r a z o r t e c h n i q u e . D e t e r m i n a t i o n o f Blood A l c o h o ! L e v e l s A f t e r d e c a p i t a t i o n , a m i x t u r e o f b l o o d and body f l u i d s was drawn from the j u g u l a r a r e a o f the f e t u s e s . L i t t e r s were p o o l e d t o o b t a i n the r e -q u i r e d volume o f b l o o d , and were then a n a l y z e d , a l o n g w i t h maternal samples, as d e s c r i b e d f o r Experiment 1. Enzyme P r e p a r a t i o n s 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 exposed and the l i v e r q u i c k l y removed. The l i v e r was washed i n a p p r o x i -mately 15 mis o f an i c e c o l d 1.15 p e r c e n t p o t a s s i u m c h l o r i d e (KC1) s o l u -t i o n , weighed, chopped, and homogenized i n f o u r volumes o f f r e s h i c e c o l d KC1 u s i n g an e l e c t r i c V i r t i s t i s s u e homogenizer w i t h a t e f l o n p e s t l e . T h i s p r o c e d u r e was m o d i f i e d f o r the f e t u s e s so t h a t l i v e r s were h e l d i n i c e c o l d 1.15 p e r c n e t KC1 u n t i l the e n t i r e l i t t e r had been d i s s e c t e d ( a p p r o x i m a t e l y 15 m i n u t e s ) , a f t e r which time the p o o l e d l i v e r s were handled as a s i n g l e sample. A f t e r a maximum o f one week f r e e z i n g , the homogenates were thawed and c e n t r i f u g e d i n a Beckman p r e p a r a t i v e c e n t r i -fuge a t 4 degrees C, f o r 30 minutes a t 10,000g. The s u p e r n a t a n t was then a d j u s t e d t o 50 mg per ml and used f o r the a s s a y o f a l c o h o l dehydrogenase (ADH) a c t i v i t y , and the p e l l e t was resuspended i n i c e c o l d 1.15 p e r c e n t KC1 to a c o n c e n t r a t i o n o f 400 mg l i v e r t i s s u e per ml. T h i s crude s o u r c e o f microsomes was then used t o determine the maternal microsomal enzyme 38 o x i d i z i n g system (MEOS) a c t i v i t y . A l c o h o l Dehydrogenase Assay The ADH a c t i v i t y was determined a c c o r d i n g t o the method o f Bonnichsen and B r i n k (1955), which u t i l i z e s the l i n e a r g e n e r a t i o n o f NADH i n c a l c u -l a t i n g the mean i n i t i a l r e a c t i o n v e l o c i t y . The r e a g e n t s , c o n s i s t i n g o f 10 mM g l y c i n e - s o d i u m h y d r o x i d e b u f f e r , pH 9.6, 72 mM e t h a n o l and 0.1 ml l i v e r s u p e r n a t a n t were prewarmed i n a 1 cm l i g h t path q u a r t z c u v e t t e to 30 degrees C, and the r e a c t i o n was then i n i t i a t e d by the a d d i t i o n o f 0.1 ml o f 1.5 mM NAD, g i v i n g a t o t a l r e a c t i o n volume o f 3.3 ml. The g e n e r a t i o n o f NADH was r e c o r d e d a t 0 and 3 min u t e s , i n d u p l i c a t e samples a t 340 nm i n a G i l f o r d s p e c t r o p h o t o m e t e r . V a l u e s f o r a b l a n k , p r e p a r e d e x a c t l y as the sample e x c e p t f o r the o m i s s i o n o f e t h a n o l , were s u b t r a c t e d from t h e sample v a l u e s . The ADH a c t i v i t i e s were then c a l c u l a t e d from the NADH m i l l i m o l a r a b s o r p t i v i t y ( t h e absorbance o f 1 micromole p er ml i n a 1 cm l i g h t path) o f 6.22 (Mezey e t a l . , 1968), and e x p r e s s e d as nmoles p er minute p er gm l i v e r f o r comparison w i t h a c t i v i t i e s r e p o r t e d i n the l i t e r -a t u r e . A c t i v i t y was a l s o e x p r e s s e d i n u n i t s c o r r e s p o n d i n g t o the change i n a b s o r p t i o n measured a t 340 nm per minute p er gm o f l i v e r p r o t e i n . T h i s u n i t c o r r e s p o n d s t o 161 nmoles o f a c e t a l d e h y d e formed per minute p er gm l i v e r p r o t e i n ( L i e b e r and D e C a r l i , 1970), and i s u s e f u l when d e t e r m i n i n g the t o t a l r a t e o f l i v e r e t h a n o l metabolism. P r o t e i n c o n t e n t was measured u s i n g the method o f Lowry e t a l . (1951). Microsomal Ethanol O x i d i z i n g System Assay The MEOS a c t i v i t y was determined by t h e method o f L i e b e r and D e C a r l i (1970) u s i n g the crude microsomal p r e p a r a t i o n . E t h a n o l o x i d i z i n g a c t i v i t y i n t h i s f r a c t i o n has been r e p o r t e d t o be comparable w i t h t h a t o f 39 the c o r r e s p o n d i n g i s o l a t e d microsomes ( L i e b e r and D e C a r l i , 1970), and has s u b s e q u e n t l y been used i n mouse MEOS s t u d i e s (Sze e t a l . , 1976). The i n c u b a t i o n m i x t u r e , c o n s i s t i n g o f 80 mM sodium phosphate b u f f e r , pH 7.4, 50 mM e t h a n o l , 0.3 mM NADPH, 5 mM magnesium c h l o r i d e , and 20 mM n i c o t i n a m i d e , was p l a c e d i n the main chamber o f a 15 ml Warburg r e a c t i o n v e s s e l and p r e i n c u b a t e d i n a c i r c u l a t i n g water bath a t 37 degrees C. At zer o t i m e , 0.1 ml o f the crude microsome p r e p a r a t i o n e q u i v a l e n t t o 40 mg l i v e r was added, and a f t e r f u r t h e r i n c u b a t i o n a t 37 degrees C f o r t en min u t e s , the r e a c t i o n was stoppe d by the a d d i t i o n o f 0.5 ml o f 70 p e r c e n t t r i c h l o r a c e t i c a c i d . The a c e t a l d e h y d e produced i n t h i s t e n minute p e r i o d was then a l l o w e d t o r e a c t w i t h 0.4 ml o f 1.5 mM s e m i c a r b a z i d e i n 17 mM po t a s s i u m phosphate b u f f e r , pH 7.0, which had p r e v i o u s l y been p l a c e d i n the c e n t e r w e l l o f the r e a c t i o n v e s s e l . A f t e r an o v e r n i g h t d i f f u s i o n p e r i o d a t room t e m p e r a t u r e , the c o n t e n t s o f the c e n t e r w e l l were c o l l e c t e d , and t he c o n c e n t r a t i o n o f a c e t a l d e h y d e bound to s e m i c a r b a z i d e was determined i n d u p l i c a t e a c c o r d i n g t o t h e method o f Gupta and Robinson (1966). In t h i s p r o c e d u r e , a 0.2 ml a l i q u o t from the c e n t e r w e l l i s d i l u t e d t o 1 ml and measured a t 224 nm a g a i n s t a blan k c o n t a i n i n g an e q u i v a l e n t amount o f s e m i c a r b a z i d e s o l u t i o n . Under t h e s e c o n d i t i o n s , 0.1 umole a c e t a l d e h y d e gave an absorbance o f 0.33, t h e r e b y a l l o w i n g e x p r e s s i o n o f the MEOS a c t i v i t y i n u n i t s c o r r e s p o n d i n g t o nanamoles o f a c e t a l d e h y d e produced per minute d u r i n g the i n i t i a l l i n e a r phase o f the r e a c t i o n . P r o t e i n was mea-su r e d by the method o f Lowry e t a l . (1951). 40 CHAPTER IV RESULTS Experiment 1_ The e f f e c t s o f v a r y i n g amounts o f EDC on maternal c a l o r i c i n t a k e , l i v e r weight' and b l o o d a l c o h o l l e v e l s a r e shown i n TABLE 3. D a i l y c a l o r i c i n t a k e averaged o v e r a t e n 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 w i t h i n s t r a i n s (see Appendix B f o r ANOVA t a b l e s on a l l a n a l y s e s ) . L i v e r w e i g h t s , taken a t day 18 o f g e s t a t i o n and e x p r e s s e d as grams per 100 grams body w e i g h t , were not s i g n i f i c a n t l y d i f f e r e n t w i t h i n s t r a i n s , nor were b l o o d a l c o h o l l e v e l s taken t h r e e days p r i o r t o mating and on day 18 o f g e s t a t i o n . M a t e r n a l b l o o d a l c o h o l l e v e l s on day 18 o f g e s t a t i o n were s i g n i f i c a n t l y d i f f e r e n t w i t h i n s t r a i n s on d i f f e r e n t d i e t s (CBA: 15 < 2 0 25.<30; C3H: <20 < 2 5 - c 3 0 - < 3 5 h i n c r e a s i n g the EDC s i g n i f i c a n t l y i n -c r e a s e d t he b l o o d a l c o h o l l e v e l . M a t e r n a l b l o o d a l c o h o l l e v e l s were a l s o s i g n i f i c a n t l y d i f f e r e n t between s t r a i n s o f tho s e d i e t s where comparisons were p o s s i b l e , w i t h the CBA h a v i n g a h i g h e r a l c o h o l l e v e l than the C3H ( D i e t s 20, 25, and 30, C3H <• CBA). TABLE 4 shows the e f f e c t s o f v a r i a b l e amounts o f EDC on i m p l a n t a t i o n s and r e s o r p t i o n s . On D i e t 30, CBA females had no v i s a b l e r e s o r p t i o n s i t e s and c a r r i e d no o f f s p r i n g to day 18 g e s t a t i o n even though they g a i n e d w eight e a r l y i n pregnancy. The same was t r u e f o r C3H females on D i e t 35. Because o f t h i s e m b r y o l e t h a l e f f e c t , no f e t a l data c o u l d be c o l l e c t e d f o r CBA on D i e t 30, o r C3H on D i e t 35. Average i m p l a n t a t i o n s per l i t t e r , TABLE 3 E f f e c t o f D i e t s on C a l o r i c I n t a k e , L i v e r Weight, and Blood A l c o h o l L e v e l s i n CBA and C3H Females S t r a i n D i e t D a i l y Cal Intake L i v e r ' Wt. Per 100 Gm Blood 1 A l c o h o l * 2 (X EDC) mean (SEM) mean (SEM) mear (SEM) mean (SEM) CBA Lab Chow 14.8 (0.47) 6.70 (0.16) 0 0 0 15.6 (0.48) 6.27 (0.24) 0 0 (n = 10 females 15 15.7 (0.60) 6.16 (0.31) 74 (2.4) 73 (2,0) f o r each d i e t ) 20 15.5 (0.52) 6.40 (0.24) 122 (5.0) 131 (2.3) 25 16.1 (0.52) 6.38 (0.28) 177 (2.3) 175 (3,0) 30 16.8 (0.53) 6.86 (0.24) 311 (10.9) 315 (6.7) C3H Lab Chow 15.4 (1.21) 5.64 (0.24) 0 0 0 16.0 (0.54) 5.68 (0.28) 0 0 (n = 10 females 20 16.1 (0.48) 5.72 (0.27) 83 (4.1) 87 (2,1) f o r each d i e t ) 25 16.4 (0.62) 5.55 (0.26) 142 (15.0) 146 (5.8) 30 16.3 (0.62) 5.72 (0.25) 279 (9.2) 278 (3,9) 35 16.1 (0.52) 5.67 (0.29) 372 (14.2) 373 (6,9) Measurement 1, taken t h r e e days p r i o r t o mating, and measurement two, taken a t day 18 g e s t a t i o n , a r e e x p r e s s e d as mg et h a n o l p er 100 ml b l o o d . 42 TABLE 4 E f f e c t o f D i e t s 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 n CBA and C3H Females S t r a i n D i e t Number Implants Average Implants R e s o r p t i o n s Number % (% EDC) mean (SEM) mean i (SEM) CBA Lab Chow 48 4.8 (0.51) 0,1 (0) 2 0 56 5.6 (0.50) 0 (0) 0 (n = 10 15 40 4.0 (0.44) 2.3 (0.47) 58 l i t t e r s f o r each d i e t ) 20 53 5.3 (0,56) 3.5 (0,67) 66 25 52 5.2 (0.49) 3.8 (0.62) 73 C3H Lab Chow 110 11.0 (0.91) 0,8 (0.32) 7 0 73 7.3 (0,63) 0 (0) 0 (n = 10 20 68 6.8 (0.71) 0 (0) 0 l i t t e r s f o r each d i e t ) 25 65 6.5 (0.37) 1.6 (0.37) 25 30 61 6.1 (0.67) 4,4 (0,52) 72 43 measured as l i v e and dead b i r t h s p l u s r e s o r p t i o n s i t e s , d i d not d i f f e r s i g n i f i c a n t l y between CBA f e m a l e s , however a s i g n i f i c a n t d i f f e r e n c e was found between C3H females on Lab Chow and thos e on l i q u i d d i e t s (Lab Cho > 0=20=25 > 3 0 ) . There were s i g n i f i c a n t l y more i m p l a n t s p er l i t t e r i n the D i e t 0, 20, and 25 females than i n D i e t 30 s u g g e s t i n g t h a t the l i q u i d d i e t as w e l l as hi g h b l o o d a l c o h o l c o n c e n t r a t i o n s e f f e c t i m p l a n t -a t i o n i n t h i s s t r a i n . In CBA f e m a l e s , t h e average number o f r e s o r p t i o n s per l i t t e r was s i g n i f i c a n t l y l e s s i n f e t u s e s from the Lab Chow and D i e t 0 compared t o thos e from t h e Ethanol c o n t a i n i n g d i e t s (Lab Chow=0 <£ 15=20= 25). S i m i l a r i l y , i n the C3H, the Lab Chow, D i e t 0, 20, and 25 females had s i g n i f i c a n t l y fewer r e s o r p t i o n s than females on D i e t 30 (Lab Chow=0=20= 25 -<30). These dat a d e m o n s t r a t i n g i n c r e a s e d p e r c e n t a g e o f r e s o r p t i o n w i t h i n c r e a s i n g EDC a r e i l l u s t r a t e d i n F i g u r e 1. F e t a l measurements o f l i v e b i r t h s , sex, average weight p er l i t t e r , a n d p e r c e n t w i t h one o r more a b n o r m a l i t i e s a r e shown i n TABLE 5. The sex r a t i o was not s i g n i f i c a n t l y d i f f e r e n t from the e x p e c t e d 1:1 r a t i o f o r a l l the o f f s p r i n g examined. Average CBA f e t a l w e i g h t s were s i g n i f i c a n t l y g r e a t e r i n the Lab Chow and D i e t 0 c o n t r o l s compared t o D i e t 15 f e t u s e s which were s i g n i f i c a n t l y h e a v i e r than f e t u s e s from D i e t s 20 and 25 (Lab Chow=0>15 >20=25). In the C3H, D i e t 0 f e t u s e s were s i g n i f i c a n t l y h e a v i e r than Lab Cow f e t u s e s , p o s s i b l y a r e f l e c t i o n o f the s m a l l e r l i t t e r s i z e i n the l a t t e r ( 0 > L a b Chow>20725=30). D i e t 20 f e t u s e s were l i g h t e r than t h o s e from females on Lab Chow, but h e a v i e r than those on D i e t s 25 and 30. The p e r c e n t o f f e t u s e s w i t h a t l e a s t one a b n o r m a l i t y i n c r e a s e d w i t h i n c r e a s i n g EDC i n both s t r a i n s . The most common s k e l e t a l a b n o r m a l i t y o b s e r v e d was an i n c o m p l e t e o r a p p a r e n t l y m i s s i n g o c c i p i t a l bone (TABLE 6 ) . 100r o o CO CD DC # 0 15 20 25 30 35 % Ethanol-derived calories Figure 1. Dose Response Curve of Resorption Rate 45 TABLE 5 E f f e c t o f D i e t s 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 i n CBA and C3H Females S t r a i n D i e t L i v e B i r t h s Sex F e t a l Wt, (gms) Abnormal {% EDC) F M mean 1 SEM) (*) CBA Lab Chow 47 24 23 0,97 ( 0,005) 2 0 56 25 31 0.95 ( 0.025) 0 (n = 10 : 15 17 7 10 0,64 ( 0.040) 59 l i t t e r s f o r each d i e t ) 20 18 10 8 0.51 ( 0.053) 100 25 14 7 7 0.51 ( 0.081) 100 C3H Lab Chow 102 61 41 1.14 ( 0.087) 2 0 73 35 38 1.27 < 0.018) 3 (n = 10 20 68 31 37 0.77 < ,0.040) 79 l i t t e r s f o r ,0.012) each d i e t ) 25 49 25 24 0.52 100 30 17 7 10 0,58 ,0.056) 100 I 46 TABLE 6 Types and Frequency o f S k e l e t a l Anomalies T o t a l F e t u s e s T o t a l S t r a i n D i e t Examined O c c i p u t Sternum Ribs Abnormal (% EDC) % CBA Lab Chow 15 1 0 0 7 0 18 0 0 0 0 15 6 6 3 0 100 20 6 6 6 4 100 25 5 5 5 3 100 C3H Lab Chow 35 2 0 0 6 0 24 1 0 0 4 20 22 18 6 5 82 25 16 16 7 9 100 30 6 6 4 6 100 47 T h i s was found i n both s t r a i n s a t t h e l o w e s t a l c o h o l c o n t a i n i n g d i e t . With the h i g h e r EDC, a p p a r e n t l y m i s s i n g s t e r n e b r a and r i b a n o m a l i e s , i n c l u d i n g f u s i o n and m i s a l i g n m e n t were produced. E x a m i n a t i o n o f s o f t t i s s u e s r e -v e a l e d a high p e r c e n t a g e o f b r a i n anomalies f o r both s t r a i n s a t the l o w e s t EDC d i e t s (TABLE 7 ) . These anomalies i n c l u d e d d i l a t e d o r immature c e r e -b r a l v e n t r i c l e s and absence o f t h e corpus c a l l o s u m . C a r d i a c anomalies i n c l u d i n g 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 hemopericardium, were o b s e r v e d a t t h e l o w e s t EDC d i e t , and the p e r c e n t a g e r o s e w i t h i n c r e a s i n g EDC. Open-l i d s a t b i r t h were found i n 100 p e r c e n t o f the CBA i n the h i g h e r two tre a t m e n t d i e t s , but c o u l d not be e v a l u a t e d i n the C3H s i n c e t h i s s t r a i n i s g e n e t i c a l l y o p e n - l i d d e d a t b i r t h . Exencephaly and g a s t r o s c h i s i s were ob s e r v e d i n both s t r a i n s a t h i g h e r EDC. To summarize the r e s u l t s o f Experiment 1, maternal b l o o d a l c o h o l l e v e l s , p e r c e n t r e s o p r t i o n , and p e r c e n t o f 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 EDC w h i l e average f e t a l w e i g h t d e c r e a s e d . F u r t h e r m o r e , a d i f f e r e n c e i n s t r a i n response was found i n the p e r c e n t r e s o r p t i o n and maternal b l o o d a l c o h o l l e v e l s measured on the same d i e t . Experiment 2^  TABLE 8 shows the number o f i m p l a n t s and r e s o r p t i o n s by f e t a l geno-ty p e s i n the d i a l l e l e c r o s s , D i e t 0. TABLE 9 shows the same i n f o r m a t i o n f o r D i e t 20. The average number o f r e s o r p t i o n s p e r l i t t e r f o r the t h r e e s t r a i n s used was not s i g n i f i c a n t l y d i f f e r e n t between f e t a l geneotypes on D i e t 0, but on D i e t 20, a s i g n i f i c a n t d i f f e r e n c e was found (CBA>C3H-C57). T h i s d i f f e r e n c e i s a t t r i b u t a b l e t o the c o n t r i b u t i o n o f the CBA maternal genotype. TABLE 10 shows the r e s u l t s o f D i e t 0 on the number o f l i v e f e t u s e s a v a i l a b l e f o r e x a m i n a t i o n , average f e t a l w e i g h t per l i t t e r , and number TABLE 7 Types and Frequency o f S o f t T i s s u e Anomalies S t r a i n D i e t T o t a l F e t u s e s Examined D i l a t e d B r a i n V e n t r i c l e s C a r d i a c Eyes Open G a s t r o -s c h i s i s Exence-p h a l y T o t a l Abnormal CBA (% EDC) Lab Chow 0 15 20 25 32 38 11 12 9 0 0 4 12 9 0 0 2 12 8 0 0 0 12 9 0 0 0 3 1 0 0 0 0 3 % 0 0 36 100 100 C3H Lab Chow 0 20 25 30 67 49 46 33 11 0 1 36 33 11 0 0 18 25 10 0 0 3 11 9 0 0 4 10 9 0 2 78 100 100 TABLE 8 Implants ( I ) , R e s o r p t i o n s (R) and P e r c e n t R e s o r p t i o n s (%R) by F e t a l Genotype; D i e t 0 Mai es Females CBA C3H C57 I 110 112 110 CBA R 4 4 3 %R 4 4 3 I 186 175 173 C3H R 6 5 5 %R 3 3 3 I 184 191 182 C57 R 5 8 8 %R 3 4 4 TABLE 9 Implants ( I ) , Resorpt ions (R) and Percent Resorp t ions (%R) by Fe ta l Genotype; D i e t 20 Males Females CBA C3H C57 I 101 108 108 CBA R 63 80 77 %R 62 74 71 I 172 157 179 C3H R 7 7 8 %R 4 4 4 I 179 186 180 C57 R 7 9 6 %R A 5 3 51 TABLE 10 Live Fetuses (LF), Weight (W), Abnormalities (A)* And Percent Abnormal (%A) by Fetal Genotype; Diet 0 Males Females CBA C3H C57 LF 106 108 107 CBA W** A 0.96 (0.02) 1 0.98 (0.02) 2 0.98 (0.02) 2 %A 1 2 2 LF 180 170 168 W 1.01 (0.02) 1.02 (0.03) 0,90 (0.02) C3H A 4 5 3 %A 2 3 2 LF 178 183 174 C57 W A 0.99 (0.02) 2 0.98 (0.02) 2 1.00 (0.01) 2 %A 1 1 1 * See text for explanation. ** Weight in grams n = 20 l i t t e r s . i s shown as mean (SEM) per fe ta l genotype group; 52 TABLE 11 Life Fetuses (LF), Weight (W), Abnormalities (A), Percent Abnormal (%A) and Blood Alcohol Levels (BAL) by Fetal Genotype; Diet 20 Males Females CBA C3H C57 LF 38 28 31 W* 0.42 (0.03) 0.44 (0.03) 0.47 (0.03) CBA A 38 28 31 %A 100 100 100 BAL** 140 (1.4) 139 (2.2) 138 (2.2) LF 165 150 171 W 0.76 (0.02) 0.75 (0.01) 0.75 (0.02) C3H A 126 124 138 %A 76 83 81 BAL 95 (2,3) 98 (2.4) 95 (4.6) LF 172 177 174 W 1.01 (0.01) 1.05 (0.03) 0.99 (0.01) C57 A 70 71 70 %A 41 40 40 BAL 45 (2.6) 43 (2.0) 43 (2.1) * Weight in l i t t e r s . grams is shown as mean (SEM) per fetal genotype group; n = 20 Blood alcohol level in mg per 100 ml blood is shown as mean (SEM) per fetal genotype group; n = 20 l i t t e r s . 53 and p e r c e n t o f f e t u s e s w i t h an a b n o r m a l i t y o f i n c o m p l e t e o r a p p a r e n t l y m i s s i n g o c c i p i t a l bone, o r d i l a t e d b r a i n v e n t r i c l e . TABLE 11 g i v e s the same data f o r d i e t 20, and a l s o i n c l u d e s the average f e t a l b l o o d a l c o h o l l e v e l f o r each f e t a l genotype. On D i e t 0, t h e r e was no s i g n i f i c a n t d i f -f e r e n c e i n the average f e t a l w e i g h t p e r l i t t e r between f e t a l g enotypes, and the p e r c e n t o f abnormal o f f s p r i n g remained below 3 p e r c e n t . On D i e t 20, t h e r e was a s i g n i f i c a n t d i f f e r e n c e i n average f e t a l w eight per l i t t e r , w i t h the major c o n t r i b u t i o n b e i n g due to a maternal e f f e c t (C57>C3H>CBA), The p e r c e n t o f abnormal o f f s p r i n g a l s o demonstrated t h i s maternal e f f e c t , as d i d the average f e t a l b l o o d a l c o h o l l e v e l s where the CBA was s i g n i f i -c a n t l y g r e a t e r than the C3H which was s i g n i f i c a n t l y g r e a t e r than the C57 ( CBA>C3H>C57). TABLE 12 shows the average f e t a l a l c o h o l dehydrogenase (ADH) a c t i v i t y f o r D i e t s 0 and 20. On both d i e t s t h e r e was a s i g n i f i c a n t d i f f e r e n c e be-tween f e t a l genotypes w i t h major c o n t r i b u t i o n s by both the maternal and p a t e r n a l genotype (C57>C3H >CBA). A s i g n i f i c a n t d i f f e r e n c e was found be-tween t r e a t m e n t s when maternal genotype and d i e t were used as the major s o u r c e o f v a r i a t i o n . In t h i s c a s e , the average a c t i v i t y on D i e t 20 was s i g n i f i c a n t l y g r e a t e r than t h a t o f D i e t 0. Measurements o f maternal l i v e r w e i g h t , a l c o h o l b l o o d l e v e l s , ADH a c t i v i t y , and microsomal e t h a n o l o x i d i z i n g system (MEOS) a c t i v i t y f o r the d i a l l e l e c r o s s a r e shown by s t r a i n f o r D i e t s 0 and 20 i n TABLE 13. There was no s i g n i f i c a n t d i f f e r e n c e i n l i v e r w e i g h t per 100 gm animal w i t h i n s t r a i n s on the d i f f e r e n t d i e t s , however t h e r e was a s i g n i f i c a n t d i f f e r e n c e between the s t r a i n s ( CBA>C3H>C57). Average maternal b l o o d a l c o h o l l e v e l s were s i g n i f i c a n t l y d i f f e r e n t between s t r a i n s (CBA> C3H> C57), and were i n the same d i r e c t i o n as o b s e r v e d i n the f e t u s e s (TABLE 1 1 ) . 54 TABLE 12 ADH A c t i v i t y (micromoles/min/gm l i v e r ) by F e t a l Genotype and D i e t Females D i e t CBA Males C3H C57 CBA C3H C57 0 20 0 20 0 20 mean (SEM) 0.86 (0.040) 0.94 (0.009) 1.09 (0.054) 1.13 (0.040) 1.54 (0.031) 1.74 (0.031) mean (SEM) 1.02 (0.031) 1.12 (0.040) 1.12 (0.054) 1.18 (0.042) 1 .52 (0.040) 1.93 (0.058) mean (SEM) 1.49 (0.058) 1.52 (0.027) 1.58 (0.045) 1.98 (0.045) 2.21 (0.045) 2.37 (0.139) 55 TABLE 13 Measurements o f Maternal L i v e r Weight , A l c o h o l Blood L e v e l s , ADH and MEOS A c t i v i t i e s i n D i a l l e l e Cross S t r a i n D i e t L i v e r W t J Blood A l c o h o l 2 ADH 3 MEOS4 (%EDC) mean (SEM) mean (SEM) mean (SEM) mean (SEM) 0 6.40 (0.66) — - 1.58 (0.04) 2.84 (0.22) CBA 20 6.40 (0.64) 135 (12,4) 1.54 (0.03) 10.64 (0.34) 0 5.55 (0.80) 1.86 (0,04) 9.54 (0.51) C3H 20 5.71 (0.66) 96 (12,6) 1.83 (0.04) 11.11 (0.41) 0 5.14 (0.53) 3.12 (0.05) 8.60 (0.49) C57 20 5.13 (0,73) 43 (6 .8) 3.17 (0.04) 9.81 (0.44) expressed as gm per 100 gm body weight (n - 60 per d i e t ) expressed as mg per 100 ml b lood (n = 60 per d i e t ) expressed as mmoles per min per gm l i v e r p r o t e i n (n = 10 per d i e t ) expressed as nmoles per min per mg l i v e r p r o t e i n (n = 10 per d i e t ) 56 ADH a c t i v i t i e s were a l s o s i g n i f i c a n t l y d i f f e r e n t between s t r a i n s (C57> C3H>CBA) and once a g a i n , f o l l o w e d the d i r e c t i o n o b s e r v e d i n the f e t u s e s (TABLE 1 2 ) . However, i n the a d u l t s , a s i g n i f i c a n t d i f f e r e n c e was not noted between t he d i e t groups. MEOS a c t i v i t y had a s i g n i f i c a n t i n t e r -a c t i o n between s t r a i n and d i e t . T h i s may be i n t e r p r e t e d as showing t h a t the MEOS a c t i v i t y i n CBA mice i s a f f e c t e d more by e t h a n o l i n the d i e t than i s the MEOS a c t i v i t y i n C3H and C57 mice. To summarize the r e s u l t s o f Experiment 2, b l o o d a l c o h o l l e v e l s , p e r c e n t r e s o r p t i o n s , and p e r c e n t abnormal f e t u s e s were g r e a t e s t i n f e t u s e s from CBA f e m a l e s , and l e a s t i n f e t u s e s from C57 f e m a l e s , r e g a r d l e s s o f p a t e r n a l genotype. M a t e r n a l and f e t a l ADH l e v e l s went i n the o p p o s i t e d i r e c t i o n w i t h t he C57 g r e a t e s t ; a d i f f e r e n c e between c o n t r o l and t r e a t m e n t a c t i v i t i e s was o n l y demonstrated i n the f e t a l a c t i v i t i e s . M a t e r n a l MEOS a c t i v i t i e s had an i n t e r a c t i o n e f f e c t between s t r a i n and t r e a t m e n t , w i t h the CBA showing t he g r e a t e s t i n c r e a s e w i t h e t h a n o l t r e a t m e n t . A summary o f the s i g n i f i c a n t s o u r c e s o f v a r i a t i o n i n measurements de t e r m i n e d by a n a l y s i s o f v a r i a n c e i n both experiments i s shown i n TABLE 14. 57 TABLE 14 S i g n i f i c a n t Sources o f V a r i a t i o n i n Measurements Determined by A n a l y s i s o f V a r i a n c e Measure D i e t Source o f V a r i a t i o n M a t e r n a l P a t e r n a l S t r a i n x D i e t M a t e r n a l Blood A l c o h o l Implants R e s o r p t i o n s L i v e r Wt. ADH MEOS X X X X X X X F e t a l Weight Blood A l c o h o l ADH X X X X X X 58 CHAPTER V DISCUSSION The p r i m a r y goal o f t h i s s t u d y was t o e s t a b l i s h a mouse model o f t h e f e t a l a l c o h o l syndrome, and t h e r e b y p r o v i d e a t o o l f o r f u r t h e r i n v e s t i -g a t i o n s . U s i n g the M e t r e c a l - e t h a n o l d i e t o f Freund (1969), female mice consumed i n t o x i c a t i n g l e v e l s o f a l c o h o l w i t h o u t a p p a r e n t i l l - e f f e c t s on c a l o r i c i n t a k e o r l i v e r w e i g h t (TABLE 3 ) , i n d i c a t i n g the absence o f d i e t a r y m a l n u t r i t i o n . With i n c r e a s i n g EDC d i e t s , maternal b l o o d a l c o h o l l e v e l s i n c r e a s e d from 0 to 373 mg per 100 ml b l o o d . The f a c t t h a t l e v e l s d i d not a p p r e c i a b l y v a r y o v e r a 16 hour t e s t i n g p e r i o d o r between t e s t i n g days a t l e a s t 21 days a p a r t i n d i c a t e s t h a t the d a i l y f l u c t u a t i o n was m i n i m a l , a f i n d i n g p r e v i o u s l y r e p o r t e d by Freund (1969). In CBA and C3H s t r a i n s o f mice, i n c r e a s i n g t h e EDC i n c r e a s e d both the r e s o r p t i o n and m a l f o r m a t i o n r a t e s (TABLES 4 and 5 ) . M a l f o r m a t i o n s , which i n c l u d e d both the s k e l e t a l system and s o f t t i s s u e s , e x h i b i t e d a s p e c i f i c d o s e - r e s p o n s e p a t t e r n i r r e s p e c t i v e o f f e t a l sex (TABLES 6 and 7 ) . Most common was a m i s s i n g o r reduced s u p r a o c c i p i t a l bone which o c c u r r e d a t a h i g h f r e q u e n c y from the l o w e s t EDC d i e t i n both s t r a i n s . B r a i n a n o m a l i e s , i n c l u d i n g d i l a t e d v e n t r i c l e s and a g e n e s i s o f the corpus c a l l o s u m were a l s o found from the l o w e s t EDC d i e t s , a t a s l i g h t l y lower f r e q u e n c y than t h e o c c i p i t a l abnorm-a l i t y . With i n c r e a s i n g EDC, c a r d i a c anomalies o f v e n t r i c u l a r s e p t a l de-f e c t s and hemopericardium, m i s s i n g s t e r n e b r a and v e r t e b r a l c e n t r a , and f u s e d and m a i a l i g n e d r i b s were noted a l o n g w i t h the b r a i n and s k u l l a n o m a l i e s . In a d d i t i o n , a t the h i g h e s t EDC d i e t s g a s t r o s c h i s i s and 59 e x e n c e p h a l y were f r e q u e n t l y o b s e r v e d . Open eye^-lids a t b i r t h , o b s e r v e d from D i e t s 20 and 25 i n the CBA, c o u l d not be e v a l u a t e d i n t h e C3H s i n c e t h i s s t r a i n i s g e n e t i c a l l y o p e n - l i d d e d a t b i r t h . S i m i l a r i t i e s between t h e mouse and human f e t a l a l c o h o l syndrome a r e shown i n TABLE 15. 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 e v i d e n c e d by low f e t a l w e i g h t and i n c o m p l e t e o s s i f i c a t i o n ; s k e l e t a l , n e u r a l , c a r d i a c and o c c u l a r a n o m a l i e s ; and p r e n a t a l wastage. U r o g e n i t a l d e f e c t s r e p o r t e d i n t he human syndrome ( T e n b r i n k and B u c h i n , 1975; Goetzman e t a l . , 1975) and i n the mouse s t u d i e s o f K r o n i c k (1976) and R a n d a l l (1977) were not ob-s e r v e d i n t h i s s t u d y , p o s s i b l y because o f a s t r a i n d i f f e r e n c e i n response t o i n d u c i n g such m a l f o r m a t i o n s , o r more l i k e l y because o f the e x a m i n a t i o n p r o c e d u r e used which e x c l u d e d d e t e c t i o n o f most u r o g e n i t a l anomalies w i t h the e x c e p t i o n o f o b v i o u s h y d r o n e p h r o s i s . P e r i n a t a l d e a t h , w h i l e not o b s e r v e d d i r e c t l y , was i n d i c a t e d by e x e n c e p h a l y and low f e t a l w e i g h t s . Because o f e x p e r i m e n t a l p r o t o c o l , performance c o u l d not be e v a l u a t e d . From the s i m i l a r i t i e s f ound, i t appears t h a t g i v e n c o n d i t i o n s c o n s i s t e n t w i t h human maternal a l c o h o l i s m , i t i s p o s s i b l e t o demonstrate a mouse model o f the f e t a l a l c o h o l syndrome. The f e a t u r e s o f the mouse syndrome e x h i b i t e d a d o s e - r e s p o n s e e f f e c t r a n g i n g from e m b r y o l e t h a l i t y a t h i g h EDC d i e t s t o n e u r a l m a l f o r m a t i o n s a t the l o w e s t dose t e s t e d . T h i s same e f f e c t was o b s e r v e d f o r r e s o r p t i o n and m a l f o r m a t i o n r a t e s (TABLES 4 and 5 ) , as w e l l as f e t a l w e i g h t s . T h i s f i n d i n g o f a d o s e - r e s p o n s e e f f e c t i n the absence o f d i e t a r y m a l n u t r i t i o n s u p p o r t s the n o t i o n t h a t an e n v i r o n m e n t a l f a c t o r , the amount o f maternal c h r o n i c a l c o h o l consumption p r i o r t o and d u r i n g g e s t a t i o n , s i g n i f i c a n t l y c o n t r i b u t e s t o the anomalies o b s e r v e d i n the syndrome. By h o l d i n g the EDC c o n s t a n t w h i l e v a r y i n g the maternal genotype, a 60 TABLE 15 S i m i l a r i t i e s Between the Human and Mouse F e t a l A l c o h o l 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 c c u l a r Anomalies X X 6. P r e n a t a l Wastage X X 7. P e r i n a t a l Death X X 8. Low Performance X ? 61 g e n e t i c f a c t o r was o b s e r v e d o p e r a t i n g i n the syndrome. Given t he same d i e t , CBA females had h i g h e r b l o o d a l c o h o l l e v e l s , lower f e t a l w e i g h t s , and i n c r e a s e d r a t e s o f m a l f o r m a t i o n and r e s o r p t i o n when compared t o the C3H females (TABLES 3, 4, and 5 ) . T h i s d i f f e r e n c e i n do s e - r e s p o n s e , i l l u -s t r a t e d f o r r e s o r p t i o n s i n F i g u r e 1, i s i n d i c a t i v e o f a g e n e t i c d i f f e r e n c e i n l i a b i l i t y f o r e m b r y o / f e t a l m a l f o r m a t i o n , w i t h t he CBA b e i n g the more l i a b l e . The a s s o c i a t i o n between EDC, maternal b l o o d a l c o h o l l e v e l s , and anomalous o f f s p r i n g s u g g e s t t h a t the c r i t i c a l f a c t o r i n f l u e n c i n g abnormal development i s the amount o f p r e n a t a l a l c o h o l e x p o s u r e , as measured by maternal b l o o d a l c o h o l l e v e l s . I t s h o u l d be noted t h a t the l o w e s t dose o f 43 mg per 100 ml b l o o d was w e l l below the 100 mg per 100 ml b l o o d used f o r a d i a g n o s i s o f human a l c o h o l i s m ( C r i t e r i a Committee, 1972). S i n c e t he f u l l e x p r e s s i o n o f m a l f o r m a t i o n s i n the f e t a l a l c o h o l syndrome has been r e p o r t e d o n l y i n the o f f s p r i n g o f c h r o n i c a l c o h o l i c women, the p o s s i b i l i t y e x i s t s t h a t a m i l d e r form o f the syndrome i n v o l v i n g o n l y n e u r a l malform-a t i o n s may be found i n the o f f s p r i n g o f women who have been moderate to heavy d r i n k e r s d u r i n g pregnancy. A d e t e r m i n a t i o n o f the lo w e s t dose r e -q u i r e d t o produce n e u r a l m a l f o r m a t i o n s would be e x t r e m e l y v a l u a b l e from a c l i n i c a l p o i n t o f view. The r e l a t i v e importance o f the maternal and f e t a l genotypes i n the syndrome was det e r m i n e d u s i n g a d i a l l e l e c r o s s . F e t a l i n s u l t was d i r e c t l y a s s o c i a t e d w i t h the maternal genotype, and was independent o f f e t a l geno-ty p e (TABLE 11). As b e f o r e , the maternal genotype determined maternal b l o o d a l c o h o l l e v e l s , and hence the degree o f f e t a l m a l f o r m a t i o n . By g r a p h i n g the p e r c e n t abnormal f e t u s e s v s . maternal b l o o d a l c o h o l l e v e l s ( F i g u r e 2 ) , i t i s r e a d i l y a p p a r e n t t h a t t he p e r c e n t o f abnormal young i s 100r £ o c X) CO c CD O i_ CD D_ 50 h s • CBA • C3H • C57 0 50 100 150 Maternal blood alcohol level (mg/100 ml blood) Figure 2. Percent Abnormal Fetuses for Varying Blood Alcohol Levels 63 s i m i l a r r e g a r d l e s s o f maternal s t r a i n , however, the d i e t o r amount o f d i e t a r y a l c o h o l r e q u i r e d t o reach a s p e c i f i c b l o o d a l c o h o l i s s t r a i n dependent. T h i s f i n d i n g i m p l i e s t h a t the q u a n t i t a t i v e b l o o d a l c o h o l l e v e l i s a more s e n s i t i v e i n d i c a t o r o f t h e maternal a l c o h o l i c s t a t e than the amount o f a l c o h o l i n the d i e t , s i n c e the l a t t e r can be i n f l u e n c e d by b i o -l o g i c a l f a c t o r s t h a t a r e u l t i m a t e l y under g e n e t i c c o n t r o l . The f i n d i n g o f a d i r e c t a s s o c i a t i o n between maternal genotype, b l o o d a l c o h o l l e v e l , and degree o f f e t a l i n s u l t sheds some l i g h t on the r e s u l t s o f the Boston C i t y H o s p i t a l s t u d y t h a t f a i l e d t o f i n d a c o r r e l a t i o n between the amount o f a l c o h o l consumed d u r i n g pregnancy and the degree o f f e t a l i n s u l t ( O u e l l e t t e , 1977). I t c o u l d w e l l be t h a t two females consuming an equal amount o f a l c o h o l would have v e r y d i f f e r e n t b l o o d a l c o h o l l e v e l s , and t h e r e f o r e o f f s p r i n g w i t h v a r y i n g degrees o f the syndrome. Measurement o f maternal b l o o d a l c o h o l l e v e l s a t d i f f e r e n t times t h r o u g h o u t g e s t a t i o n would be o f v a l u e i n t e s t i n g t h i s h y p o t h e s i s from the mouse model, and would be o f v a l u e f r o m . a c l i n i c a l p o i n t ' o f view, s i n c e i f t r u e , the amount o f maternal a l c o h o l consumption would be o n l y one o f two f a c t o r s d e t e r m i n i n g r i s k t o the o f f s p r i n g . E f f o r t s t o determine the f a c t o r s i n v o l v e d i n the maternal a l c o h o l c l e a r a n c e r a t e i n t h i s s t u d y were i n c o n c l u s i v e ; a r e s u l t commonly r e p o r t e d i n the l i t e r a t u r e (Mezey, 1976). H e p a t i c a l c o h o l dehydrogenase (ADH) a c t i v i t y d i f f e r e d i n t h e t h r e e s t r a i n s t e s t e d , w i t h female C57 h a v i n g the h i g h e s t a c t i v i t i e s and the CBA h a v i n g the l o w e s t (TABLE 13). T h i s f i n d i n g , which i s c o n s i s t e n t w i t h o t h e r s t u d i e s on s t r a i n d i f f e r e n c e s i n mouse ADH a c t i v i t y (Sze e t a l . , 1976; Belkap e t a l . , 1972; Sheppard e t a l . , 1968) s u g g e s t t h a t b l o o d a l c o h o l l e v e l s a f t e r c h r o n i c a l c o h o l i n t a k e a r e i n the o p p o s i t e d i r e c t i o n o f ADH a c t i v i t y . Whether t h e s e r e s u l t s a r e m eaningful 64 o r j u s t c o i n c i d e n t a l because o f the number o f s t r a i n s used i s unanswered by t h i s s t u d y , however, a f u r t h e r i n v e s t i g a t i o n would be o f v a l u e . F e t a l ADH a c i t i v i t i e s were i n f l u e n c e d by maternal and p a t e r n a l genotype, as w e l l ' a s by d i e t (TABLE 12). However, s i n c e f e t a l b l o o d a l c o h o l l e v e l s were dependent on maternal genotype r a t h e r than f e t a l ADH a c t i v i t y , i t can be assumed t h a t f e t a l m e tabolism o f a l c o h o l i s not o f s i g n i f i c a n c e i n t h i s syndrome. I n d u c t i o n o f the f e t a l ADH system r e p o r t e d p r e v i o u s l y by Sze e t a l . (1976), was a l s o o b s e r v e d i n t h i s s t u d y , however the b i o l o g i c a l s i g n i f i c a n c e o f t h i s i n d u c t i o n i s not known. An a l t e r n a t e pathway o f a l c o h o l metabolism, the microsomal e t h a n o l o x i d i z i n g system (MEOS), has been shown t o be i n d u c e d i n r a t s f o l l o w i n g c h r o n i c a l c o h o l a d m i n i s t r a t i o n ( L i e b e r and D e C a r l i , 1972). T h i s f i n d i n g , which has been c o n f i r m e d i n mice (Sze e t a l . , 1976; L i e b e r and D e C a r l i , 1974), was a l s o o b s e r v e d i n the p r e s e n t s t u d y . In the non-induced s t a t e , the t h r e e s t r a i n s had d i f f e r i n g a c t i v i t i e s w i t h the CBA females b e i n g the l o w e s t (TABLE 13). T h i s s u g g e s t s a g e n e t i c component o p e r a t i n g w i t h i n the system, an o b s e r v a t i o n a l s o found i n DBA and C57 mice (Sze e t a l . , 1976). When comparing i n d u c e d w i t h non-induced a c t i v i t i e s , an i n t e r a c t i o n between s t r a i n and d i e t o c c u r s , i n d i c a t i n g the degree o f i n d u c t i o n was much g r e a t e r i n CBA females than i n females o f the o t h e r two s t r a i n s . S i n c e the CBA females a l s o had the g r e a t e s t b l o o d a l c o h o l l e v e l s , t h i s f i n d i n g s u p p o r t s p r e v i o u s r e p o r t s t h a t the r o l e o f the MEOS i n v i v o may be minimal (Roach, 1973). However, the f a c t t h a t the a c t i v i t y i s g r e a t l y i n c r e a s e d i n the CBA s u g g e s t s t h a t perhaps the i n d u c t i o n may p l a y a n o t h e r r o l e i n the f e t a l a l c o h o l syndrome, namely, an a l t e r a t i o n o f maternal metabolism t h a t c o u l d i n f l u e n c e normal p r e n a t a l development. U n f o r t u n a t e l y , the c h a r a c t e r i s t i c s o f t h i s system have not been f u l l y e l u c i d a t e d , and an 65 i n v e s t i g a t i o n i n t o the e f f e c t s o f i n d u c t i o n on hormone met a b o l i s m , known t o be a f f e c t e d by i n d u c e d microsomal d e t o x i f y i n g systems ( L i e b e r and D e C a r l i , 1973), would be o f g r e a t v a l u e . To d etermine the t h e o r e t i c a l t o t a l o x i d a t i o n o f e t h a n o l per a n i m a l , the enzyme a c t i v i t i e s were c o n v e r t e d t o nanomoles o f a c e t a l d e h y d e produced per min per mg l i v e r p r o t e i n . By assuming the t o t a l l i v e r p r o t e i n was p r o p o r t i o n a l t o 1 i v e r weight i n a l l t h r e e s t r a i n s , i t can be seen from TABLE 16 t h a t the t h e o r e t i c a l r a t e o f e t h a n o l m etabolism ..could not a c c o u n t f o r t h e d i f f e r e n c e i n b l o o d a l c o h o l l e v e l s s i n c e t h e CBA females had the h i g h e s t m e t a b o l i c r a t e and the h i g h e s t b l o o d a l c o h o l l e v e l s . C l e a r l y , a n o t h e r f a c t o r o r s e t o f f a c t o r s must p l a y a major r o l e i n d e t e r m i n i n g e t h a n o l c l e a r a n c e r a t e s , which from t h i s s t u d y , appear t o be under a t l e a s t p a r t i a l g e n e t i c c o n t r o l . An u n d e r s t a n d i n g o f t h i s mechanism c o u l d l e a d t o a s i m p l e t e s t f o r e t h a n o l c l e a r a n c e r a t e , and t h e r e b y be o f v a l u e i n c o u n s e l l i n g f o r r i s k i n t h i s syndrome. The r e s u l t s from t h i s s t u d y s u g g e s t t h a t maternal c h r o n i c a l c o h o l i s m e x e r t s t e r a t o g e n i c i n s u l t by two mechanisms. The p o s i t i v e a s s o c i a t i o n be-tween f e t a l b l o o d a l c o h o l l e v e l s and r a t e o f m a l f o r m a t i o n s u p p o r t the mechanism o f d i r e c t a c t i o n on the embryo/fetus. As p r e n a t a l exposure to a l c o h o l i n c r e a s e s , anomalies o f t h e syndrome i n c r e a s e . At the same t i m e , i n d u c t i o n o f the MEOS i s a s s o c i a t e d w i t h i n c r e a s e d r a t e s o f f e t a l mal-f o r m a t i o n , s u g g e s t i n g m o d i f i c a t i o n o f maternal metabolism as a second mechanism o f t e r a t o g e n e s i s . A d e t a i l e d study o f t h e s e p o s s i b l e mechanisms would be o f h e l p i n e l u c i d a t i n g the p a t h o g e n e s i s o f t h i s syndrome. In c o n c l u s i o n , the r e s u l t s o f t h i s s t u d y , w h i l e answering s e v e r a l b a s i c q u e s t i o n s about the f e t a l a l c o h o l syndrome, have a l s o posed s e v e r a l q u e s t i o n s t h a t r e q u i r e f u r t h e r i n v e s t i g a t i o n . Having e s t a b l i s h e d a mouse TABLE 16 T h e o r e t i c a l Rate o f T o t a l Ethanol O x i d a t i o n i n CBA, C57 and C3H Mice T o t a l A c t i v i t y ' T o t a l A c t i v i t y S t r a i n ADH* MEOS* Times L i v e r Wt. p e r 100 gm Animal CBA 3.14 10.64 13.78 x 6.40 88.20 C57 6.45 9.81 16.26 x 5.14 83.58 C3H 3.76 11 .11 14.87 x 5.63 83.73 Ex p r e s s e d as nmoles a c e t a l d e h y d e produced p er min per mg l i v e r p r o t e i n . 67 model f o r t h e human syndrome, i t was p o s s i b l e t o demonstrate t h a t the degree o f f e t a l i n s u l t was dependent on the maternal b l o o d a l c o h o l l e v e l s . In t u r n , t h e s e l e v e l s a r e det e r m i n e d by the i n t e r a c t i o n o f an e n v i r o n -mental and b i o l o g i c a l component. A t l e v e l s e x c e e d i n g 131 mg a l c o h o l p er 100 ml b l o o d , a l l v i a b l e o f f s p r i n g e x h i b i t e d f e a t u r e s o f the syndrome. The l o w e s t b l o o d a l c o h o l l e v e l c o m p a t i b l e w i t h normal o f f s p r i n g has y e t t o be d e t e r m i n e d . The f a c t o r s i n v o l v e d i n maternal a l c o h o l e l i m i n a t i o n ap-pear t o be under g e n e t i c c o n t r o l , but what they a r e and how they a c t i s unknown a t t h i s t i m e . What i s c l e a r i s t h a t t h e MEOS a c t i v i t y i s g r e a t l y i n d u c e d i n t h e s t r a i n showing the g r e a t e s t l i a b i l i t y t o the syndrome, r a i s i n g t h e p o s s i b i l i t y t h a t a l t e r e d maternal metabolism may make secon-dary c o n t r i b u t i o n s t o the syndrome. Regarding the c l i n c i a l a s p e c t s o f the syndrome, a l l i n d i c a t i o n s from t h i s s t u d y s u g g e s t t h a t t h e r i s k t o the o f f s p r i n g i s not a f u n c t i o n o f amount o f maternal a l c o h o l consumption, but r a t h e r a f u n c t i o n o f the amount o f mat e r n a l consumption r e q u i r e d t o i n c r e a s e b l o o d a l c o h o l l e v e l s above a p a r t i c u l a r c r i t i c a l v a l u e . T h i s p r e s e n t s d i f f i c u l t i e s i n c o u n s e l l i n g f o r the syndrome, s i n c e the same amount o f a l c o h o l consumption by two women may r e s u l t i n d i f f e r i n g b l o o d a l c o h o l l e v e l s , and hence, d i f f e r e n t r i s k s to the o f f s p r i n g . U n t i l t h e time when a s i m p l e q u a n t i t a t i v e t e s t i s a v a i l a b l e , the b e s t means f o r r i s k d e t e r m i n a t i o n w i l l have t o r e l y on r e p e a t e d b l o o d a l c o h o l measure-ments t h r o u g h o u t pregnancy. While t h i s method may be used t o a l e r t t h e p h y s i c i a n o f a h i g h r i s k i n f a n t , i t i s l i m i t e d i n i t s a p p l i c a b i l i t y t o the ge n e r a l p o p u l a t i o n . 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APPENDIX A A l i z e r i n Red S t a i n i n g Procedure f o r S k e l e t o n s F e t u s e s a r e 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. 95% e t h a n o l f o r 24 hours 2. 1% KOH u n t i l a n i m a l s are c l e a r and pink i n c o l o r (24 hours) 3. 1% KOH and a l i z a r i n r e d s t a i n (1 o r 2 d r o p s ) f o r 12-24 hours 4. g l y c e r i n and KOH (1:9) f o r 48 hours 5. g l y c e r i n and KOH (1:3) f o r 48 t o 96 hours 6. g l y c e r i n and KOH (1:1) f o r 48 hours 7. s t o r e i n g l y c e r i n 77 APPENDIX B A n a l y s e s o f V a r i a n c e T a b l e s ANOVA f o r C a l o r i c Intake from TABLE 3 Sums o f Squares f o r S t a i n s Source o f V a r i a t i o n d . f , CBA C3H D i e t s 5 22,15 (NS)* 6.15 (NS) E r r o r 54 147.10 272.70 T o t a l 59 169.25 278,85 In t h i s and a l l o t h e r ANOVA T a b l e s , (NS) i n d i c a t e s not s i g n i f i c a n t , and (S) i n d i c a t e s s i g n i f i c a n t a t the 0.05 l e v e l . 78 ANOVA f o r Mat e r n a l L i v e r Weight from TABLE 3 Sums o f Squares f o r S t a i n s Source o f V a r i a t i o n d . f . CBA C3H D i e t s 5 3.51 (NS) 0.20 (NS) E r r o r 54 33.83 38.55 T o t a l 59 37.34 38.75 79 ANOVA f o r Maternal Blood A l c o h o l L e v e l s by S t r a i n from TABLE 3 Sums o f Squares f o r S t r a i n s Source o f V a r i a t i o n d . f , CBA C3H Subgroups 7 632606 (S) 1031733 (S) Day (A) 1 149 (NS) 263 (NS) D i e t (B) 3 632103 (S) 1031328 (S) A x B 3 354 (NS) 141 (NS) E r r o r 65 19826 "12170 T o t a l 79 652431 1043903 ANOVA f o r Maternal Blood A l c o h o l L e v e l s Between S t r a i n s from TABLE 3 Sums o f Squares f o r D i e t s Source o f V a r i a t i o n d . f . 20 25 30 S t r a i n 1 10341 (S) 4322 (S) 6808 (S) E r r o r 18 923 3837 5407 T o t a l 19 11264 8159 12215 SI ANOVA f o r Implants from TABLE 4 Sums o f Squares f o r S t r a i n s Source o f V a r i a t i o n d , f . CBA C3H D i e t E r r o r T o t a l 4 45 49 15.28 (NS) 113.70 128.98 157.32 (S) 231.10 388.42 ANOVA f o r R e s o r p t i o n s from TABLE 4 Sums o f Squares f o r S t a i n s Source o f V a r i a t i o n d . f . CBA C3H D i e t 4 131.72 (S) 133.12 (S) E r r o r 45 97.10 46.40 T o t a l 49 228.82 179.52 ANOVA f o r F e t a l Weights from TABLE 5 CBA Source o f V a r i a t i o n d , f . Sums o f Squares D i e t 4 5.724 (S) E r r o r 147 4.514 T o t a l 151 10.238 C3H Source o f V a r i a t i o n d . f . Sums o f Squares D i e t E r r o r T o t a l 4 304 308 24 r59 (S) 21.651 46.24 84 ANOVA f o r R e s o r p t i o n s from D i a l ! e l e C r o s s , TABLES 8 and 9 Source o f V a r i a t i o n d . f , Sums o f Squares f o r D i e t s 0 20 Subgroups 8 P a t e r n a l (A) 2 Ma t e r n a l (B) 2 A x B 4 E r r o r 163 T o t a l 179 1,211 (NS) 0.011 (NS) 1.011 (NS) 0.189 (NS) 40.450 41.661 444.10 (NS) 3.23 (NS) 435.60 (S) 5.27 (NS) 610.80 1054.90 85 ANOVA f o r F e t a l Weights from D i a l l e l e C r o s s , TABLES 10 and 11 Sums o f Squares f o r D i e t s Source o f V a r i a t i o n d . f . 0 20 Subgroup 8 0.049 (NS) 14.636 (S) P a t e r n a l (A) 2 0.007 (NS) 0.012 (NS) Mater n a l (B) 2 0.036 (NS) 14.594 (S) A x B 4 0.006 (NS) 0.030 (NS) E r r o r 163 1.220 3.704 T o t a l 179 1.269 18,340 86 ANOVA f o r Fe ta l Blood A l c o h o l Leve l s from TABLE 11 Source o f V a r i a t i o n d . f . Sums o f Squares Subgroups 8 68220.7 (S) Pa terna l (A) 2 7.6 (NS) Maternal (B) 2 68178.3 (S) A x B 4 34.8 (NS) E r r o r 28 1179.2 To ta l 44 69399.3 87 ANOVA f o r F e t a l ADH A c t i v i t y by D i e t from TABLE 12 Sums o f Squares f o r D i e t s Source o f V a r i a t i o n d . f . 0 20 Subgroup 8 6.701 (S) 9.442 (S) P a t e r n a l (A) 2 3.260 (S) 3.902 (S) Mater n a l (B) 2 3.328 (S) 5.302 (S) A X B 4 0.121 (NS) 0.238 (NS) E r r o r 28 0.387 0.640 T o t a l 44 7.088 10.482 ANOVA f o r F e t a l ADH A c t i v i t y by Maternal Genotype from TABLE 12 Source o f V a r i a t i o n d . f . Sums o f Squares Subgroups 5 9.9848 (S) Ma t e r n a l (A) 2 9.1331 (S) D i e t (B) 1 0.7971 (S) A x B 2 0.0546 (NS) E r r o r 79 0.7126 T o t a l 89 10.6974 ANOVA f o r M a t e r n a l Blood A l c o h o l L e v e l s from D i a l l e l e C r o s s , TABLE 13 Source o f V a r i a t i o n d . f . Sums o f Squares S t r a i n 2 256255 (S) E r r o r 177 21294 T o t a l 179 277550 ANOVA f o r Maternal L i v e r Weight, ADH and MEOS A c t i v i t y from D i a l l e l e C r o s s , TABLE 13 Sums o f Squares f o r Measurements Source o f V a r i a t i o n d . f . Weight ADH MEOS Subgroup 5 99.22 (S) 27.939 (S) 458.3 (S) S t r a i n (A) 2 97.46 (S) 27.932 (S) 134,4 (S) D i e t (B) 1 0.25 (NS) 0.002 (NS) 186.7 (S) A x B 2 0.51 (NS) 0,005 (NS) 137,2 (S) E r r o r 49 161.02 0.968 91,8 T o t a l 59 259,25 28.907 550,0 VO o 

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