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THE FETAL ALCOHOL SYNDROMEINMICE

AN ANIMAL MODEL

by

GERALDF.CHERNOFF

B . S c , U n i v e r s i t y o fB r i t i s h Columbia,1970

A THESIS SUBMITTEDINPARTIAL FULFILLMENTOF

THE REQUIREMENTSFOR THEDEGREEOF

DOCTOROFPHILOSOPHY

i n


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In pres enti ng th is t hes is in pa rt ia l f ul fi lm en t of the requirements for

an advanced degree at the Un iv er si ty of Br i t i s h Columbia, I agree that

the Li br ar y sh al l make it f re el y av ai la bl e fo r refe rence and study.

I fu rt he r agree tha t permi ssion fo r ext ens ive copying of thi s th es is

for s cho lar ly purposes may be granted by the Head of my Department or

by his re pr es en ta tiv es . It is understood that copying or pu bl ic at io n

of this thesis for financial gain sha l l not be al lowed without my

written permission.

Gerald P . Chernoff


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ABSTRACT

Although the adverse e f f e c t s of pre nat al exposure to al co ho l have

been suggested since a n t i q u i t y , onl y re ce nt ly has a ' f e t a l alcohol

syndrome 1 been described inhuman being s. Since e t h i c a l considerations

l i m i t the types of st ud ie s po ss ib le with humans, an animal model was

developed.

CBA and C3H female mice, maintained on a l i q u i d Metrecal-ethanol


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i ii

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 sandmater nal bl oo d

a l c o h o l l e v e l s v a r i e d with maternal s t r a i n (CBA > C3H > C57) , andwere

i n v e r s e l y r e l a t e d tomaternal a l c o h o l dehydrogenase a c t i v i t y . Micro

somal ethanol o x i d i z i n g system inductionwas d i r e c t l y ass oci ate d withi n

c r e a s e d f e t a l abn orm ali tie s, being grea testi n CBA females and lowesti n

C57.

The r e s u l t s o f t h i s study i n d i c a t e t ha t malfor mations observedi n

the mouse andhuman syndromeare s i m i l a r , and t h a t l i a b i l i t y f o rthe se

malformations i sdependent on maternal b loo d a l c o h o l l e v e l s , which are

determined by ther a t eo fmaternal a l c o h o l metabolism aswell as th e

amount o fmaternal a l c o h o l consumption.


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ACKNOWLEDGEMENTS

T o P r o f e s s o rJames R. M i l l e r , c h a i r p e r s o n o f my committee, te ac he r

o f wisdom, and s o u r c e o f h e l p i ne 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 syour con sta nt enthusia sm f o re x p l o r i n gth e

gray area 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 w h ic h p r o

v i d e d thef 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 t i m e i n v e s t e d i nt h i s p r o j e c t ,

I e x p re s s g r a t i t u d et o the members o f my c o mm i tt e e: P r o f e s s o rC.W. "Bob"


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V

A s p e c i a l note o f g r a t i t u d e i s d ue D r. Kenneth Lyons Jones J r . , whop 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 of 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 th e r e l a t i o n s h i p between the ani mal and human

syndromes.

To Professor David 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 in de bt ed f o r i n i t i a t i n g me to the creative

a nd 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 t h e M e d i c a l Research C o u n c i l o f Canada and

t he 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 t h e 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 , b o t h i n s u p p l i e s a nd s a l a r y , t h r o ug h 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 to ex pre ss my dee pes t thanks to Win and Sacha

f t h i i f i d h i d t d i d t d i t h


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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 eto 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 14B 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


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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 Procedure f o rS k e l e t o n s . . . . 76

B. A n a l y s i so fVariance Tables. 77

ANOVA f o rMaternal L i v e r Weight from TABLE 3 . 78ANOVA f o rMaternal Blood Alco ho l Le vel sby S t r a i n from

Table 3 , 79ANOVA f o rMaternal Blood Alco ho l Le vel s Between S t r a i n s

from TABLE 3 80ANOVA f o rImpla nts from TABLE 4 81

ANOVA f o r Resorptions from TABLE 4 82ANOVA f o r F e t a l Weights from TABLE 5 83


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vi ii

LIST OF TABLES

Page

Table

1 C om po si ti on o f D i e t s , , . . 31

2 F e t a l Genotypes Gener ated With D i a l1e l e Cr oss 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 Bl oo d

A l c o h o l L ev e ls 41

4 E f f e c t o f Di et s on Im pl ant at ion and Re so rp ti on 42

5 E f f e c t o f D i e t s o n L i v e B i r t h s , S ex , F e t a l Weights and Fetal

A b n o r m a l i t i e s i n CBA and C3H Females 45

6. Types and Frequency of Sk e l e t a l Anomalies 46

7 Types and Frequenc y of So f t Ti s su e Anomali es 48

8 Implants , Res orp tio ns and Perc ent Res orp tio ns by Fetal

Genotype; D i e t 0 49

9 Implants, Res orp tio ns and Per cen t Re sor pti ons by FetalG t D i e t 20 50


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tx

LIST OF FIGURESPage

F i g u r e

1 Dose Respon se Cur ve 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 rV a r y i n g B l o o d A l c o h o l L e v e l s . 62


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CHAPTER I

INTRODUCTION

"Betsy; Martin, widow,one c h i l d and oneeye. Goes out c h a r i n gand washingby day;never hadmore than one ey e, bu tknowshe r

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 ft 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 bysometo be th ee v i l o fmankind, and by others

t obe thes a l v a t i o n f o ra t r o u b l e d mind, hasbeen a c o n t r o v e r s ia l sub

j e c t o fm e d i c a l r e s e a r c h f o ra tl e a s t 200y 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 nhumans has c o n t r i b u t e d


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t o thec 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 , t he y ar e o f r e l e v a n c e

when d e l i n e a t i n g thee 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 thesyndrom e,

a n d t h e r e f o r e a reneeded 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 thep 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 thesyndrome ar e notf e a s i b l e u s in g human b e i n gs . C o n t r o l

l i n g confounding 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 ofq u e s t i on a b l e e t h i c s . Most important

would be theq u e s t i o n o fa l l o w i n g an a l c o h o l i cwoman 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 a n 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 thee x ac t e t i o l o g y . F orthese reas ons, theo n l y p r a c t i c a l approach to s t u d y i n g t h epathogenesis 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 li s

developed, i t w i l l be p o s s i b l e todetermine thee x a c t e t i o l o g y o f th e

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 important


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i n Greek mythology was t h e r e s u l t o f J u p i t e r ' s drunkeness; and th e Ho ly

B i b l e d e c l a r e s i n Judges 13:3-4, "Beh old now, thou a r t ba rr en , and

b a r e s t n o t ; bu t t ho u s h a l l c o n c e i v e , a n d bear a son. Now th e r e f o r e be

ware, I pra y th ee , and dr in k not wine or st ro ng dr i n k , and ea t not any

u n c l e a n t h i n g " .

Th e 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 a p

p ea r e d as a r e s u l t o f the E n g l i s h G i n Epidemic from 1720 to 1751. Du ri ng

t h i s p e r i o d , a l a w 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

pu t 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 t he a r i s t o c r a t i cfar m

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 gin to the

E n g l i s h populace 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, de at h 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 t h e 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 o n t h e d i s t i l l i n g

t r a d e s , c i t i n g gin as


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4

and season" (quoted i nWarner andRosett, 1975, p. 1402).

By the l a t e 1800s, animal experiments i nt e r a t o l o g ywere being

i n i t i a t e d i nFrance, and the f i r s t experimental studieson th e dangers

o f pren atal exposure toalcohol began. Tre ati ng chicken eggs by e i t h e r

exposure to alcohol vapor o r by d i r e c t i n j e c t i o n i n t o th e eggwh it e, Fere

( c i t e d by Ballantyne,1902, p. 274)produced a v a r i e t y o f malformations

i n c h i c k s . However, no malformations were observed byMai r e tand

Cambemale i n theo f f s p r i n g o fcocker spa nie l dogs exposed t oalc oho l

d u r i n g pregnancy ( c i t e d bySandor and E l i a s , 1968, p. 53 ). Thus,th e

f i n d i n g s o fthese e a r l i e s t experiments on th ee f f e c t s o fprenatalexposure t oalcoholwere i nd i r e c t disagreement, s i g n a l l i n g thes t a r t o f

many years o fcont rad icto ry reports.

T h i s e a r l y s c i e n t i f i c controversyon thee f f e c t s o falcohol was

b e s t e x e m p l i f i e d by

Ballantyne i n h i s 1902

c l a s s i c monograph,

Manual o f


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embryo andf 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 tweretwo

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 study,

u s i n g g u i n e a p i g s , f o u n d 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 e q u al i n the

mother andf e t u s a f t e ra onehour e q u i l i b r a t i o n p e ri o d. S i m i l a r l y , i n

pr e g n a n t women g i v e n 60 cc o f ruma p p r o x i m a t el y onehour p r i o rto de

l i v e r y , e x a m i n a t i o n o ff e t a l c o r d b l o o d asw e l l asp 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 fa l c o h o l , v e r i f y i n g t h a t i nhumansand

a n i m a l s , a l c o h o l c o u l d c r o s s thep l a c e n t a ande n t e r thef 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 developed

e p i d e m i o l o g i c t e c h n i q u e s t os t u d y thee f f e c t so fm a t er n a l 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 600o b s er v e d o f f s p r i n g b or n t o

1 2 0 f e m a l e 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 rd i e d under two years

o f age;a s i g n i f i c a n t i n c r e a se o v er 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 andi n f a n t m o r t a i l i t y r a te o f 24 p e r c e n t . T h i s d e a t h r a t eo f


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be tween p aren ta l a l c o h o l i s m and o f f s p r i n g wi th e p i l e p s y , i d i o c y , and

feeb lemi nded ness (see rev ie w by Warner and R o se t t , 197 5) . Then i n 191 0,

Elde r ton and Pearson repo rte d a s tudy 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 matern al

a l c o h o l i s m and abnormal phy s iqu e , i n t e l l i g e n c e , or d iseas e i n the o ff

s p r i n g . Ins tead , they conc luded that the low bi rt hw ei gh ts and observed

abnormali t ies were the resul t of 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 th the tendency fo r abus ive use of a l c o h o l . Thi s con c lu s i on

caused a fu ro r among ab st in en ce pr eac he rs , and al so po in te d out the

n e c e s s i t y f o r f u r t h e r a ni ma l s t u d i e s 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 s eve ra l yea r s , i nv es t ig a to r s us ing d i f f e r en t an ima ls

and ad mi n i s t r a t io n te chn iques r epor ted co n t rad ic to ry f in d i ng s in o f f

s p r i n g p r e n a t a l l y e x p o s e d t o a l c o h o l . Most exhaustive was the work of

Stocka rd u s i n g f i s h embryos of Fundulus h e t e r o c l i t u s , guinea p ig s , and


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hours o f treatment, a nd e m b r y o l e t h a l i t y w i t h treatment beyond 23 hours.

Malformations r e s u l t e d from ey e, ne ur al a nd li mb bud maidevelopment, as

w e l l as ge nera 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 ka 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 for 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 that did hatch 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 . Again usin g the popu lar gen eti c

no ti on s of the day, the author suggested th at al c oh ol was us ef ul as an

i n t e r u t e r i n e s e l e c t i o n agent, since only the most r e s i s t a n t embryos would

reac h ha tc hi ng . In 1923, McDowell r e p l i c a t e d S t oc k a rd ' s guinea 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 b u t no i n

cre ase i n the fre quen cy of malformations. S i m i l a r n e g a t i ve r e s u l t s were

reported by Nice (1912: 1917) i n a s e r i e s of experiments u s i n g a l b i n o

mice, and by Hanson and Cooper (1930) in a study examining te n gener


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i d i o c y and psychosis observed i n t h e o f f s p r i n g o f a l c o h o l i c women were

t he 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 those animal s t u d i e s t h a t used the same rou te 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 to prov ide in s i gh t in to the

ac ti on of al co hol on the embryo o r f e t u s . Mirone (1952) was unable to

produce malformed o f f s p r i n g i n mice a d m i ni s t e re d a l c o h o l o r a l l y , however

t h e o f f s p r i n g o f female guinea 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 t o

f o u r times a week d i s p l a y e d s p e c i f i c malformations of the central 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 he g y r i with r e s u l t i n g shallow

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 t he c o r t e x a nd b a s l g a n g l i a ,

edema, d i l a t i o n o f b l oo d v e s s e l s , hemorrhagic are as, and general re ta rd

a t i o n o fmyelin a t i o n a t b i r t h (Papara-Nicholson 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 an d d u r i n g pregnancy


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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 andseven 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 xthr ough

e i g h t (Sandor andAmels, 1971). H a l f o feach l i t t e r was removedand

examined on day 9.5 o f g e s t a t i o n , and th eremaining h a l f on day 19.5. In

those embryos examined a t th ee 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 thef r e q u e n c y o f dysmorphology

i n c e n t r a l nervous system anlagen, w h i l e i nthose examinedat 19.5 days,

t h e r e was o n l y an i n c r e a s e i nf 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 fbone

r e t a r d a t i o n . T h i s r e s u l t suggests t h a t p r e n a t a l exposure t oa l c o h o l i n

t h e r a tmost o f t e n r e s u l t s i nl e t h a l malformations, however i nthose 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 smore 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 s t u 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


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r e t a r d e d growth was co nd uc te d by Jon es and Smith a t the Dysmorphology

U n i t , U n i v e r s i t y o f Washington, It was found th at these 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 severe with regard to b i r t h length than b i r t h weight;

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 th l i n e a r growth r a t e a v e r a g i n g

65 pe rc en t o f nor mal , and ave rag e r a t e o f wei ght ga in on ly 30 pe rc en t of

normal; mental r et a rd a t io n wit h an average i n t e l l i g e n c e quotient of 63 in

t h o s e c h i l d r e n i nwhom 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 n o m a l i e s 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 t o ext end th e elbows completely, camptodactyly of toes and in

a b i l i t y t o f l e x a t t h e 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

ali gnm ent of the palmar cr ea se s, and/or s i n g l e upper palmar cr ea se ;

c a r d i a c an omali es, the ma jo ri ty of 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 ;


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11

1976; Noonan, 1976; Palmer e t a l , f1974j R e i n h o l d et 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 th e syndrome i n c r e a s e d , a n o m a l i e s o t h e r t h a n 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 a n o m a li e s 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 swere r e p o r t e d by T e n b r i n c k and

Buchin (1975), and Goetzman et a l , ( 1 97 5 ) . C a r d i o v a s c u l a r a n o m al i e s

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 c a s e s were shown to 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 rr y and 0 KN 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 n e a r 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 d e v e l o pm e n t a l

a n o m a l i e s 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 of n e u ro n a l m i g r a t i o n re^

s u i t i n g i nm 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 of the a n t e r i o r s u p e r i o r gyri

t h r o u g h 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 of g l i a l and neuronal


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12

r e t a r d a t i o n with anaverage I.Q. o f 70, an d,with 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 gtotask s with r e s u l t a n t beh avi ora l problemsa t

s c h o o l . In th eRussian study d e s c r i b i n g 98pregnanciest 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 thepre gna ncie s ended i na b o r t i o n s ,f i v e i nmis

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 57percent. Of t he 42 r e s u l t i n g l i v e b i r t h s , 19 whowere

born

b e f o r e t h e i r mother developed a l c o h l i s m demonstrated v eg at iv e, emotional

and be hav ior al di so rd er s which improved with fa vo ra bl e m i c r o s o c i a l

change. However, i n th e 23 c h i l d r e n born a f t e r pre nat al exposuret o

a l c o h o l , 14were mentally retardedanddemonstrated or ga ni c impairment

o f th ec e n t r a l nervous system e a r l y i nin fa nc y (Shru ygin , 1974). The

U n i v e r s i t y o fWashington stu dy examined dysmorphogenic fe at ur esi n 41

c h i l d r e n born toc h r o n i ca l c o h o l i cwomen who continuedt odrin k dur ing

pregnancy (Hanson e ta l . , 1976). Ina d d i t i o n t op r e v i o u s l y reported

l h l


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13

per m l ) ,whi le embryo-lethalitywas observed a t higher concentrations ."

(10 mg ethanol per ml) . Extending thestudy to inutero t e s t i n g , 5 ml of

40 percent ethanol 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 gest at io n. Examination o ff e t u s e sa tterm re vea ledan i n c r e a s ei 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 decrease i nf e t a l weight;

however, no t e r a t o l o g i c a l e f f e c t s were observed (Skosyreva, 1973). Whena l c o h o l was o r a l l y administeredi ndr in ki ng water f i v e weeks p r i o r to

and during ge st at io n,l i t t e r s i z e and f e t a l weight was reduced, bu t th e

o n l y anomalies reportedi n the l i v e o f f s p r i n g were microcephaly and gen

e r a l i z e d derm ato log ic problems (Tz e and Le e,

1975). Thus, atte mpts to

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 sf o r

t h i s f a i l u r e a ret h a tthematernal blo od a l c o h o l l e v e l s (average o f 61 mg

per 100 ml blood i n the Tzestudy) were not s u f f i c i e n t t ocause f e t a l

i n s u l t ; o f f s p r i n g were notexamined f o ri n t e r n a l malformations; and i n th e


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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 f e m a l e s were t r e a t e d on day 10,

11, or 12. T h i s s t u d y , which d e m o n s t r a t e d c r i t i c a l t i m e p e r i o d sf o r

both thet e r a t o g e n i c i t yand 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 s e c o n d 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 an 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 preg nant femal es

on days 5 t h r o u g h TO o fg e s t a t i o n r e s u l t i n gi nb l o o d a l c o h o l l e v e l s

r a n g i n g f r o m l e s s t h a n 40 mg per 100 ml b l o o d t o 300 mg pe r 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 deada tb i r t h , i n c r e a s e d f r o m 15 p e r c e n t a t thel o w e s t d i e t to 25 p e r c e n t on t he

h i g h e s t d i e t w h i l e m a l f o r m at 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 yand

e c t r o d a c t y l y o f thef o r e p aw s ; 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 a n o ma l i es i n v o l v i n g t h emajor

b r a n c h e s o f the a o r t a as well


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15

to mental d e f i c i e n c y , I.Q. range o f 45 to 105, 12 c h i l d r e n with 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 grasp, poor f i g e r a r t i c u l a t i o nand 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 hadsevere f e e d i n g

problems, andtended to be h y p e r a c t i v e with 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 womenwho were measured f o r I.Q. a t 7years o f age,

s i x who l i v e d with t h e i r mothershad a mean I.Q. of 73, s i x who had

spent some time with 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 nm i l d l y a f f e c t e d c h i l d

ren with 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 with themost severe

s t i g m a t a o f thesyndrome o f t e n were t h emost 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)


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16

a b i l i t i e s , meaningful r e s u l t s must await f u r t h e r s t u d i e s when the c h i l d

ren are 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 no f f s p r i n g p r e n a t a l l y ex

posed to 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 vo c a l r e s u l t s . L e a r n i n g to d i s c r i m i n a t e c o n t i n ge 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 nwas d e f i c i e n twhen r a t s , p r e

n a t a l l y exposed toa l c o h o l from day one o f g e s t a t i o n t oweaning were

t e s t e d a te 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 ,exposuret o

a l c o h o l from day two o fg e s t a t i o n toweaning i m p a i r e d T-maze and s h u t t l e -

bo x 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 yw i t z ,

e t a l . ,1976). T h i s same d e f i c i e n c y i ns 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 throughout 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 animals were t e s t e d a t a l a t e rage,

4 5 and s i x months the d e f i c i e n c y was not observed i n d i c a t i n g t h a t the


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17

animals 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 d ay 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 an d f i g h t i n gwhen measured as a du lt s

( Y an ai e t a l . ,1976). When t he o f f s p r i n g were t e s t e d f o r ambulation, un

l i k e t h e r a t s t u d i e s , s c o r e s were reduced with a noted i n c r e a s e d l a t e n c y

to leave the i n i t i a l square ( Gi n s be 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 t h e 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 to al co ho 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 ic 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 hn i q ue s were employed to se pa ra te the pre - and pos 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

that the major determinant was the po st na ta l exposure (Y an a i 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 alcohol

can produce b e h a v i o r a l changes the data i n d i c a t e th at at low; l e v e l s of


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f e t a l a n d n e o n a t a l b r a i n , h e a r t a nd l i v e r (Rawat, 1975a; 1976; 1977).

U s i n g t h e same tr e a tm e n t p r o t o c o l , a c e t y l c h o l i n e , t h e 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 e x p os e d f e t u s e s a nd neo

n a t e s , w h i l e t h e 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 an d i t s p r e c u r s o r , g l u t a m at e , were i n c r e a s e d (Rawat,

1975b). R e c e n t l y , t h e 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 a nd g l u t a m a t e

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 e x p o se d t o a l c o h o l l e d Rawat (1977) to sug ges t th at the metab olism

o f GABA a nd g l u t a m a t e i s r e d u c e d , r e s u l t i n g i n t h e h i g h e r o b s e r v e d l e v e l s .

W h i l e i t i s t e m p t i n g 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 a nd p r o t e i n s y n t h e s i s a s b e i n g f a c t o r s i n t h e d e f i c i t s o b s e r v e d

i n t h e human syndrome, i t s h o u l d f i r s t be remembered that the rat has

n o t been shown t o b e a n a p p r o p r i a t e model f o r t h e syndrome, a nd s e c o n d l y ,

t h e e f f e c t s o f m a t e r n a l 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


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19

t h e o t h e r hand, anAmerican 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 no f f s p r i n g bo rn 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 lr 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 th eGerman 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 hadc h i l d r e no f lowb i r t h weight, s u g g e s t i n g

t h a t i n thehuman po p u la t i on , n u t r i t i o n a land l i f e s t y l e d i f f e r e n c e smay

confound th ee f f e c to fa 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 ou n d i ng r e s u l t s have been n o t e d i n two p r o s p e c t i v e

s t u d i e s now b e in g c a r r i e d out i n theU 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 tLoma L in da 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 th e

1500 b i r t h s s t u d i e d t od a t e , th eo n l y n o t a b l e e f f e c to fa l c o h o l d r i n k i n g

d u r i n g pregnancy i s lowb i r t h weight. However,when c i g a r e t t e smoking

i s t a k en i n t o c o n s i d e r a t i o n , th e lowb i r t h w e i g h t s ca n 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


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c o n g e n i t a l malformations have t h e i r o r i g i n d u r i n g organogenesis in the

f i r s t t r i m e s t e r , such r e s u l t s a re to be q u e s t i o n e d , Themost r e a s o n a b l e

e x p l a n a t i o n i st 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 hadc h i l d r e n w i t h a lowc o n g e n i t a l malformation r a t e even i f they

h ad 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 throughout g e s t a t i o n .

Two studies conducted a t theU n i v e r s i t y o fWashington have been

most u s e f u l i ng i v i n g an i n d i c a t i o no f ther i s k i n v o l v e d i n thesyndr ome.

The f i r s t , using data from t heC o l l a b o r a t i v e P e r i n a t a l P r o j e c t of the

N a t i o n a l I n s t i t u t eo fN e u r o l o g i c D i s e a s e andS t r o k e , e s t i m a t e d thef r e ^

quency o fadverse outcomeo fpregnancy f o r c h r o n i c a l l y a l c o h o l i cwomen

t obe 43 p e r c e n t (Jones e ta 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 theo 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 andd u r i n g pregnancy, f o u r d i e d p r i o r to oneweeko f age ,

and six had abnormal f e a t u r e s s u g g e s t i v e o f the f e t a l a l c o h o l syndrome


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21

f e a t u r e s o fa l t e r e d growth andmorphogenes is wi th nine coming fromth e

heavy drinkersand twofrom th e lowdri nk in g group, g i v i n g a t o t a li n

cidence r a t eo f 12 percent (Hanson andSmith , 1977). From thesetwo

s t u d i e s i t appears th atwomen who d r i n ki nexcess o f oneounceo f

a l c o h o l d a i l y throughout pregnancy have a r i s k o f 12 percent for having

a c h i l d w i t h a l t e r e d growth anddysmorphogenesis, wh il ewomen who a r e

c h r o n i c a l c o h o l i c s andco nti nue d r i nk i n g through pregnancy have an even

g r e a t e r (43percent) r i s k f o ranadverse outcome o fpregnancy. Whether

these women have common n u t r i t i o n a l andsocio-economi c f a c t o r s con

t r i b u t i n g to th eabnorm alit ies observed can not bedetermined a t this

time, no r can th ee f f e c t s o fs m a l l e ramounts o f a l c o h o l .


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22

CHAPTER II

PURPOSE AND RATIONALE OF THE PRESENT STUDY

A l t h o u g h many s t u d i e s have r e p or t e d i n v e s t i g a t i n gthe 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 of thef e t a l a l c o h o l

syndrome, l i t t l e i n s i g h thasbeen g a i n e d . The human s t u d i e s a r econ

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

t h e a n i m a l s t u d i e s a r ebased on theassumption 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 se q u i v a l e n t i na l lmammalian s p e c i e s ; an

i t t d b d t C l l i l d l f th

23


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o f c h r o n i c m a t er n a l a l c o h o l i s m . With t h i s i nmind, i tbecomesr e a d i l yap

p a r e n t t h a t t e s t i n g f o ra 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 t h e t e r a t o g e n i c i t yo fm a t e rn a l 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 rt e s t i n gmust 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 t o be e f f e c t i v ei nd e l i n e a t i n g thee t i o l o g y , p r e v e n t i o n ,and cure o f th e human syndrome, must r e p l i c a t ewhere p o s s i b l e , thecon

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 ta l . ,1972; M e l l o ,

1 9 7 3 ; L e s t e r and F r e e d , 1973). Furthermore, s i n c e i t i s t h et e r a t o g e n i c i t y

o f them a t er n a l a c t i o n t h a t i sunder 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 eto

t e r a t o l o g i c a l t e s t i n gmust 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 fconsumption i nmothers o fc h i l d r e n wi t h thef e t a l a l c o h o l

syndrome ( U l l e l a n d , 1972), thef o l l o w i n g s e to fc r i t e r i a was e s t a b l i s h e d .

24


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Human c h r o n i c a l c o h o l i c s maintain blood a l c o h o l l e v e l s r an gi ng from 100

to 400 mg per 100 ml blo od . Con seq uen tly , the embr yo/f etus 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 to these same amounts. Therefore,

i n an animal model i tmust be po ss ib le to achi eve blood 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 blood.

4. Behavioral Man ife st ati on of 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 ,

th e f i r s t sig ns of a l c o h o l i n t o x i c a t i o n are a t a x i a with i n i t i a l hyper-

k i n e s i s eve ntu all y subsid ing to lethargy. With time a to le ra nc e develo ps

and the be ha vi or al ma ni fe st at io ns decrea se. The a c t u a l basis for this

t o l e r a n c e i sunknown, however i t i s acommon feature of alcoholism and

s h o u l d be observed in the animal model.

5. Dependence: Pharmacological dependence r e s u l t i n g from chronic

a l c o h o l i s m i s man ife ste 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 Th ere for e a sure s i g n that

25


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25

metabolism i s a l t e r e d a nd f a u l t y a b s o r p t i o n c an l e a d t o 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 t h e f e t a l a l c o h o l syndrome, i t i s neces sary

t o a d m i n i s t e r a l c o h o l b e f o r e pregnancy in or der to al lo 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 al co ho li sm to become m a n i f e s t . T o f u r t h e r

simulate the human c o n d i t i o n , a l c o h o l treatment must then continuethroughout the pregnancy.

8. Malformations i n O f f s p r i n g S i m i l a r t o Those in the Human Syndrome:

I f the model i s to be usef ul i n fu rt he r stu di es of t h i s syndrome, i tmust

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 malformations t h a t a f f e c t t h e systems ob

ser ved 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 in g from 0 to 100 pe rc en t

( W i l s o n , 1965). T h e r e f o r e , i fmaternal 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

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26

requirements, i ti sp o s s i b l e t ot e s t thet e r a t o g e n i c i t y o fmaternal

c h r o n i c a l c o h o l i s m i n themouse.

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 fenvironmental 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 tupon. T h i s i n t e r a c t i o ncanb e s t bed e s c r i b e d i nterms 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 environmental and b i o

l o g i c a l f a c t o r s determine how l i a b l e anembryo i sf o r t e r a t o g e n i c i n s u l t .

In thef e t a l a l c o h o l syndrome, themost 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 smaternal 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 themechanism o ft e r a t o g e n e s i s , which cano c c u r e i t h e r by d i r e c t i n

s u l t on thed e v e l o p i n g organism, 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 i n du c ed

a l t e r a t i o n i nmaternal 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 nt 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

t h l t (Ak 1974) i l i t h t l i b i l i t f lf ti i

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27

d e s c r i b e d i n man (Ugartee ta l . ,1970) andmouse (Sheppard e ta l . , 1968),

and a f e t a l form d i f f e r e n t from thea d u l t hasbeen r e p o r t e d i nmice

(Krasner e ta 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, theh e p a t i c microsomal ethanol 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 suggest 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 account forup t o 25percento fa d u l t a l c o h o l metabolism ( 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 to f i n d a c o r r e l a t i o nbe

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 ft h i s pathway on i nv i v o metabolism (Roach, 1973;Mezey,

1976). U n l i k e the ADH pathway, the MEOS i s notpresenti n the f e t a l

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28

as malformation o rblood a l c o h o l l e v e l , i t shouldbe p o s s i b l e t o d i s

t i n g u i s h between amaternal e f f e c t , where thef e t a l phenotypesar e

a s s o c i a t e d with maternal genotype, and a f e t a l e f f e c t , where f e t a l pheno-

type i sass oci ate d with thef e t a l genotype.

A l c o h o l induced a l t e r a t i o n o f thematernal and f e t a l metabolism may

occur i n anumbero fways. Malabsorptiono fvitaminsandmineralscan

r e s u l t i nd 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 could have adv ers e consequences

on a developing organism ( L i e b e r , 1975). Al co ho l metabolismby ADH,

equation ( 1 ) , r e s u l t s i n a r e d u c t i o no f the NAD/NADH r a t i o , thereby 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;Lun dqu ist , 1975). The conse

quenceo ft h i s i s noto n l ythei n h i b i t i o n o fa l c o h o l metabolism,bu t

a l s o an i n h i b i t i o n o fl a c t a t e metabolism, r e s u l t i n g i na c i d o s i s ( L i e b e r ,

1975). This a l t e r a t i o n i n the NAD/NADH r a t i o may be normalizedbyi n

d u c t i o n o f the MEOS equa tion ( 2) which prov ides NADP+

fo r transhydroxy

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29

harmful s i d e - e f f e c t s , would a l s o bei n c r e a s e d . Anexamination o f ther o l e

o f a l c o h o l metabolism asmeasured by ADH and MEOS a c t i v i t i e s would provide

i n s i g h t i n t o both themechanism andb i o l o g i c a l f a c t o r s res pon sib le fo r

the malformat ions observed i nt h i s syndrome.

Experiments

The goal o f thef i r s t experiment i nt h i s studywas toe s t a b l i s han

animal model o f thef e t a l a l c o h o l syndrome. At th esame time, byusing

two s t r a i n s o fmice tomanipulate theg e n e t ic v a r i a b l e s a n d s e v e ra l doses

o f c h r o n i c a l c o h o l consumption t omanipulate theenvironmental v a r i a b l e ,

the environmental andg 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 rt h emalforma tions

were i n v e s t i g a t e d .

The second experiment, which followedthef 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 st odetermine ther e l a t i v e importance o f th e

l d f l i l i b i l i t f lf i h

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CHAPTERI II

METHODOLOGY

Experiment 1

A n i m a l s :

Ml

CBA/J andC3H/1g mice (Musmusculus) o b t a i n e d f r o m t heMedic al

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 yo fB r i t i s h Columbia, were maintained

on a 12 hour l i g h t c y c l e i n the Z o o lo 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

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31

TABLE 1

Composition of D i e t s

Ethanol (95% v/v) Metrecal

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

% %

L b Ch

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added such t h a t the f i n a l prepa ratio n contained15percentEDC. D i e t 20

c o n t a i n e d 20percentEDC, d i e t 25, 25percent EDC, and so on . The l i q u i d

d i e t s were prepared f r e s h d a i l y andwere t heonl y source o f c a l o r i e s .

The pre pa rat ion , composi tion,anddocumentation o fn 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 nd e t a i l p r e v i o u s l y (Walkerand

Freund, 1971); each treatment d i e t contained s e v e r a l times theminimum

d a i l y requirements o fa l ln u t r i e n t s , based on previous recommendationsf o r

mice (Walker andZor net zer , 1974). Animals r e c e i v e d thel 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 syri nge s with theneedle ends e a l e d bym eltin g,

through standard g l a s s d r i n k i n g tubes with 1.5 mm openings extendingt oapproximately 3 cm above thecage 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 ewas determined bymeasuring theamount o f f l u i d l e f t each

day a tbetween10 AM and 12 noon, o r 10 PM and 12midnight. C a l o r i c i n

takef t l i l L b t Ch d t i d i t b li

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d i e t 35, 30 days.

Matings and Pregnancy Management

Matings between animals of thesame s t r a i n were i n i t i a t e d by i n t r o

ducing an estrous female i n t o a males c age . To keep th emales sob er and

th e 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 to 1.5 hours, a t

which time themating p a i r was deprivedo f food and water. Th is was done

to preven t confoundin g the r e s u l t s with male a l c o h o l consu mptio n, which

has been reportedto cause dominant l e t h a l mutations (Badr and Badr, 1975;

K l a s s e n and Persaud, 1976). The presence of a cop ula tio n plugwas taken

as i n d i c a t i n g day one of ge st at io n. Females were kept on t h e i rr e s p e c t i v e

d i e t s throughout gestation,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 Examination

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34

by observing prepared fetuses i n g e l a t i n f i l l e d p e t r i dishes under a

d i s s e c t i n g microscope. So ft t i s s u e malformations were deter min ed by ob

s e r v i n g 1-2 mm cross s e c t i o n a l s l i c e s pl ac ed i n 70 pe rc ent etha nol i n

white p o r c e l a i n spot p l a te s under a d i s s e c t i n g microscope.

Determination of Blood Alcohol Levels

Blood samples were taken by r e t r o - o r b i t a l bl ee d from fema les th re e

days before 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 bleeding

o r ca rd ia c puncture j u s t p r i o r to k i l l i n g on day 18 of g e s t a t i o n . To

maximize any v a r i a b i l i t y in blood alc oho l l e v e l s due to c i r c a d i a n f l u c t u

a t i o n s , random samples were c o l l e c t e d over a d a i l y 16 hour p e r i o d , 10 AM

to 2 AM. Using 20 m i c r o l i t e r Drummond microcap glass blood c o l l e c t i n g

tubes, or a 3 cc t u b er c ul i n sy ri ng earmed wit h a 21 x 1.5 ne ed le f o r

c a r d i a c pu nct ure , 1 to 2 ml of blood was t r a n s f e r r e di n t o a heparin rinsed

16 125 d l t t b d t d i th f i t 12

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commercial r eag ent ) abs orb anc es; a method made p o s s i b l e by having the

volume of reagent 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 at 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 of Var ian ce (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 es were found, a Duncans M u l t i p l e Range Test

was a p p l i e d to 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 ,

196 7). In the one measurement with a known exp ect ed fr eq ue nc y, a Chi -

square t e s t was u t i l i z e d at 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 Experi ment 2 de par ted from t h a t d e s c r i b e d for

Experiment 1 as f o l l o w s :

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TABLE 2

F e t a l Genotypes Generated with a D i a l l e i Cross

Males

Females CBA C3H C57

CBA CBA/CBA CBA/C3H CBA/C57

C3H C3H/CBA C3H/C3H C3H/C57

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F e t a l Examination

A f t e r e xte rna l examination, the fet use swere de ca pi ta te d, and one-

h a l f of the heads were prepared for s k u l l s k e l e t a l s t a i n i n g as pr ev iou sl y

d e s c r i b e d , and the remaining h a l f were f i x e d in Bouin's s o l u t i o n for

b r a i n examination by the freehand razor technique.

Determination of Blood Alcoho! Levels

A f t e r de ca pi ta ti on , a mixture of blood and body f l u i d s was drawn from

the ju gu la r area of the fet us es . L i t t e r s were pooled to obtain the re

q u i r e d volume of blood, and were then analyzed, along with maternal

samples, as de sc ri be d f o r Experiment 1.

Enzyme Preparations

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 yremoved. The l i v e r wash d

in approxi

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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 Ass ay

The ADH a c t i v i t y was determined ac cor din gto th emethod o f Bonnichsen

and Brink (1955), which u t i l i z e s thel i n e a r generationo f NADH i n c a l c u

l a t i n g themean i n i t i a l r e a c t i o nv e l o c i t y . Thereagents, c o n s i s t i n go f

10 mM glycine-sodium hydroxide buffer,pH 9.6, 72 mM ethanol and 0.1 ml

l i v e r supernatant were prewarmed i n a 1 cm l i g h t path quartz cuvetteto

30 degrees C, and th er e a c t i o nwas then i n i t i a t e d by th ea d d i t i o no 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 nvolumeo f 3.3 ml . Thegenera tion

o f NADH was recorded a t 0 and 3minutes, i ndu pl ic at e samples a t 340 nm

i n a G i l f o r d spectr ophotom eter. Values f o ra blank, prepared ex ac tl y

as thesample exc ept f o rtheomission o fe t h a n o l ,were subtracted from

the sample va lu es . The ADH a c t i v i t i e s were then ca lc ul at ed from the NADH

( f 1 l i 1

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the corresponding i s o l a t e d microsomes ( L i e b e randDe Ca rl i, 1970), and has

subsequently been used i nmouseMEOS studies (Sze e ta l . , 1976). The

i n c u b a t i o n mixture, c o n s i s t i n g o f 80 mM sodium phosphate bu 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 placed i n themain chamber o f a 15 mlWarburg reaction

v e s s e l andpreincubated i n a c i r c u l a t i n g water bath a t 37degrees C. At

z e r o time, 0.1 ml of thecrude microsome preparat ion equiva lentt o 40 mg

l i v e r was added, anda f t e r f u r t h e r i n c u b a t i o na t 37degrees C f o rte n

minutes, ther e a c t i o nwas stopped by th ea d d i t i o no f 0.5 ml o f 70 percent

t r i c h l o r a c e t i c a c i d . Theacet aldeh yde produced i nt h i s t e nminute pe ri od

was then allowed t ore ac t with 0.4 ml o f 1.5 mM semicarbazide i n 17 mM

potassium phosphate b u f f e r ,pH 7.0,which hadp r e v i o u s l ybeen placedi n

the center well o f ther e a c t i o n v e s s e l . A f t e ranovernight d i f f u s i o n

i d h f h ll l l d

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CHAPTER IV

RESULTS

Experiment 1_

The e f f e c t s o f v a r y i n g amounts of 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 re shown i n TABLE 3. D a i l y c a l o r i c

i n t a k e averaged over a ten 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 (seeAppendix B forANOVA t a b l e s on a l l a n a l y s e s ) . L i v e r

weights, taken at day 18 of 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 weight, 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 hi 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 tomating and on day 18 o f

TABLE 3


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E f f e c t o fD i e t s on C a l o r i c Intake, 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 yCalIntake

L i v e r 'Wt.Pe r

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 (0.2 6) 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 omating, an dmeasurement two,taken a t day 18 g e s t a t i o n ,ar eexpressed as mg e t h a n o l pe r 100 ml blood.

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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 an d C3H Females

S t r a in D i e t

Number

Implants

Average

Implants

R e s o r p t i o n s

Number %

(% EDC) mean (SEM) meani(SEM)

CBA Lab Chow 48 4.8 (0.51) 0,1 (0) 2

0 56 5.6 (0.50) 0 (0) 0

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measured as l i v e anddead b i r t h s p l us 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 wasfound between C3H females on Lab Chow and t h o s e on l i q u i d d i e t s(Lab

Cho > 0=20=25 >30). 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 e r l i t t e r

i n theD i e t 0, 20, an d 25 females than i nD i e t 30 s u g g e s t i n g t h a tth e

l i q u i d d i e t as

w e l l as

h i 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 nt h i s s t r a i n . In CBA f e m a l e s , t h eaverage number o f r e s o r p t i o n s

p er 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 nf e t u s e s from the Lab Chow and D i e t

0 compared t o t h o s e from t h eE t ha n ol c o n t a i n i n g d i e t s (Lab Chow=0


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100r

o

oCO

CDDC

#

0 15 20 25 30 35

% Ethanol-derived calories

Figure 1. Dose Response Curve of Resorption Rate

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TABLE 5

E f f e c t o fD 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 DietLive

B i r t h s SexF 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

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TABLE 6

Types andFrequencyo fS k e l e t a l Anomalies

TotalFetuses Total

S t r a i n D i e t Examined Oc c i p u t Sternum Ri bs Abnormal

(% EDC) %

CBA Lab Chow 15 1 0 0 7

0 18 0 0 0 0

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T h i s was found i nboth s t r a i n s at the lowest a l c o h o l c o n t a i n i n gd i e t . With

the higher EDC, a ppar entl y missing ster nebraand ri banomalies, i n c l u d i n g

f u s i o n and misalignment were produced. Examina tion o f s o f t t i s s u e s r e

v e a l e d a high percentage of b r a i n anomalies f o rboth s t r a i n s at the lowest

EDC d i e t s (TABLE 7) . Thes e anomalies inc lu de d d i l a t e d or immature cere

b r a l v e n t r i c l e s and absence o f thecorpus cal los um. Ca rd ia c anomaliesi 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 sand hemopericardium, were observed

a t the lowest EDC d i e t , and the percentage rose with 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 in 100 percentof the CBA in thehigher two

treatment d i e t s , but couldnot be evaluated i n th 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 open-lidded at b i r t h . Exencephaly and g a s t r o s c h i s i s were

observed i nboth s t r a i n s at higher EDC.

To summarize the r e s u l t s o f Experiment 1, maternal blo od 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 percent of abnormal o f f s p r i n g increased

TABLE 7


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TABLE 7

Types and Frequency o fS o f t T i s s u e Anomalies

S t r a i n Diet

TotalFetusesExamined

D i l a t e dBrain

V e n t r i c l e s CardiacEyesOpen

Gastro-s c h i s i s

Exence-ph al y

TotalAb no rm al

CBA

(% EDC)

Lab Chow

0

1520

25

32

38

1112

9

0

0

412

9

0

0

212

8

0

0

012

9

0

0

03

1

0

0

00

3

%

0

0

36100

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


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TABLE 8

Implants ( I ) ,R e s o r p t i o n s (R) and Percent

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

Fema le s CBA C3H C57

I 110 112 110

CBA R 4 4 3

%R 4 4 3


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TABLE 9

Implants ( I ) , Resorp tio ns (R) and Percent

Re so rp ti on s (%R) by Fe ta l Genot ype; D i e t 20

Males

Females CBA C3H C57

I 101 108 108

CBA R 63 80 77

%R 62 74 71

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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

CBAW**

A

0.96 (0.02)

1

0.98 (0.02)

2

0.98 (0.02)

2

%A 1 2 2

LF 180 170 168

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TABLE11

Life Fetuses (LF), Weight(W),Abnormalities (A),PercentAbnormal (%A)andBlood Alcohol Levels(BAL) byFetal Genotype;Diet20

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

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and per cen t of fe tu se s wit h an abn orm ali ty of incomp lete or ap pa re nt ly

m i s s i n g o c c i p i t a l bone, or d i l a t e d b r a i n v e n t r i c l e . TABLE 11 gives the

same data for d i e t 20, and a l s o in cl ud es the average f e t a l blood a l c o h o l

l e v e l f o r each f e t a l geno type . On D i e t 0, t h er 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 avera ge f e t a l weight per l i t t e r between f e t a l genotypes,

and the percent of abnormal o f f s p r i n g remain ed below 3 pe rc en t. On D i e t

20, th 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 weight per l i t t e r ,

with the major c o n t r i b u t i o n being due to a maternal e f f e c t (C57>C3H>CBA),

The p er ce nt 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 did the average f e t a l blood 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 tha n 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 Di e t s 0 and 20. On both d i e t s t he 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

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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 Diet

Females Diet CBA

Males

C3H C57

CBA0

20

0

mean (SEM)

0.86 (0.040)

0.94 (0.009)

1.09 (0.054)

mean (SEM)

1.02 (0.031)

1.12 (0.040)

1.12 (0.054)

mean (SEM)

1.49 (0.058)

1.52 (0.027)

1.58 (0.045)

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TABLE 13

Measurements of Maternal L i v e r Weight, Alcohol

Blood Levels, 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 Diet L i v e r W t J B lo o d Alco 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 )

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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)andonce aga in, fol lo wedthed i r e c t i o n observed i n thefet use s

(TABLE 12). However, i n thea 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 ewas not

noted between t h ed 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 andd i e t . Thismay 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 CBAmice i sa f f e c t e d more byethanol i n th e

d i e t than i s t he MEOS a c t i v i t y i n C3H an d C57 mice.

To summarize ther e s u l t s o fExperiment 2,blood alco hol l e v e l s ,

percent r e s o r p t i o n s ,andper cen t abnormal fe tu se swere greatesti nfetu ses

from CBAfemales, andl e a s t i nfe tu se s from C57females, reg ardl esso f

p a t e r n a l genotype. Mater nal and f e t a l ADH l e v e l s went i n theopp osit e

d i r e c t i o n with t h e C57g r e a t e s t ;ad i f f e r e n c ebetween c o n t r o land treatment

a c t i v i t i e s was only demonstrated i n thef e t a l a c t i v i t i e s . Maternal 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 andtreatmen t, withth e

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TABLE 14

S i g n i f i c a n t Sources of 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 Diet

Source of V a r i a t i o n

Maternal Paternal S t r a i n x Diet

Maternal

Blood A l c o h o l

Implants

X

X

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CHAPTER V

DISCUSSION

The primary goal of t h i s stu dy was to e s t a b l i s h a mouse model of the

f e t a l a l c o h o l syndrome, and ther eby pro vi de 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 . Using the Met reca l-e than ol d i e t of 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 without apparent i l l - e f f e c t s on

c a l o r i c int ake or l i v e r weight (TABLE 3), i n d i c a t i n g the absence of 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 blood 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 pe r 100 ml blood. The f a c t t h a t l e v e l s did not

a p p r e c i a b l y vary over a 16 hour t e s t i n g per iod or between t e s t i n g days

a t l e a s t 21 days apart i n d i c a t e s that the d a i l y f l u c t u a t i o n was mi ni ma l,

59


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exencephaly were fre que ntl y observed. Open eye^-lids at b i r t h , observed

from Di e ts 20 and 25 i n the CBA, could not be evaluated i n th 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 open -lid ded at b i r t h .

S i m i l a r i t i e s between the mouse and human f e t a l a l c o h o l syndrome are

shown i nTABLE 15. They inclu de prenatal growth d e f i c i e n c y as evidenced

by low f e t a l weight and incomplete o s s i f i c a t i o n ; s k e l e t a l , neur al, car dia c

and o cc u l a r anom alie s; and pre na ta lwastage. Urog eni tal defec ts repo rted

i n the human syndrome (Tenbrink and Buchin, 1975; Goetzman et a l . , 1975)

and in the mouse s tu di es of Kronick (1976) and Randall (1977)were not ob

s e r v e d i n t h i s study, p o s s i b l y because of a s t r a i n d i f f e r e n c e in response

t o ind uci ng such malfor mation s, or more l i k e l y because of the exa min ati on

procedure used which excluded d e t e c t i o n of most u ro ge ni ta l anomalies wi th

the ex cept ion of obvious hydrone phro sis. P e r i n a t a l dea th, wh il e not

observed d i r e c t l y , was i n d i c a t e d by exencephaly and low f e t a l weights.

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TABLE 15

S i m i l a r i t i e s Between theHuman 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

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g e n e t i c f a c t o r was observed oper atin gi n thesyndrome. Given thesame

d i e t , CBA females hadhigher blood a l c o h o l l e v e l s , lower f e t a l weights,

and in cr ea se d rat eso fmalformation andr e s o r p t i o nwhen compared to th e

C3H females (TABLES 3, 4, and5). Th is d i f f e r e n c e i ndose-response, i l l u -

s t r a t e d f o r r e s o r p t i o n si nFigure1, i si 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 embryo/fetal malform ation, with the CBA being themore

l i a b l e .

The a s s o c i a t i o n between EDC,maternal bloo 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 suggest thatthec 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 theamounto fprenatal a l c o h o l exposure, asmeasured by

maternal blood a l c o h o l l e v e l s . I tshould benoted th atthelo we st dose

o f 43 mg per 100 ml bloodwas well below the 100 mg per 100 ml blood used

f o r a d i a g n o s i so fhuman a l c o h o l i s m ( C r i t e r i a Committee, 1972). Si nc eth e

f u l l expressiono fmalformations i n thef e t a l a l c o h o l syndrome has been


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100r

ocX)CO

cCDOi_CD

D_

50h

s CBA

C3H

C57

0 50 100 150

Maternal blood alcohol level (mg/100 ml blood)

Figure2. Percent Abnormal Fetuses for VaryingBlood Alcohol Levels

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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 diet or 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 r e ac h 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 ndependent. T h i s f i n d i n g i m p l i e s t h a t t h e 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 t h e d i e t , s i n c e t h e l a t t e r c an 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, blood

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 results

of 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 co ul d we ll be th at 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 od a l c o h o l l e v e l s ,

an d 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 of 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 throughout g e s t a t i o n

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o r j u s t c o i n c i d e n t a l because of 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 of va lu e. Fe ta l

ADH a c i t i v i t i e s were i n f l u e n c e d b y maternal and paternal 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 metabolism o f a l c o h o l i s n o t o f s i g n i f i c a n c e i n

t h i s syndrome. Ind ucti on of 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

S ze e t a l . (1976), was a l s o observed i n t h i s s t u d y , however t h e 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 n o t known.

An a l t e r n a t e pathway o f a l c o h o l metabolism, the microsomal ethanol

o x i d i z i n g system (MEOS), has been shown to be induced in ra ts fo ll ow in 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 an d D e C a r l i , 1972). T h i s f i n d i n g ,

which has been confi rmed in mice (Sze et a l . , 1976; Li eb er and De Ca rl i,

1974), was a l s o observed i n the pr es en t stu dy. In the non-induced s t a t e ,

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i n v e s t i g a t i o n i n t o t h e e f f e c t s o f i n d u c t i o n on hormone metabolism, known

to be affected by induced microsomal d e t o x i f y i n g systems (Lieber and

D e C a r l i , 1973), would be of gr ea t va lu e.

To determine th e t h e o r e t i c a l t o t a l ox id at io n of ethanol per ani mal,

the enzyme a c t i v i t i e s were conv erte d to nanomoles o f acetaldehyde produced

per mi n 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 pr ot e in was

p r o p o r t i o n a l to 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 c an b e seen from

TABLE 1 6 t h a t t h e t h e o r e t i c a l r a t e o f e t h a n o l metabolism ..could not account

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 th e

h i g h e s t m e t a b o l i c r a t e a nd t h e 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 ,

another f a c t o r o r s e t o f f a c t o r s must play a major ro le i n dete rmin ing

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 to 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 could

le ad to a simp le t e s t f o r eth ano l cl ea ra nc e r a t e , and the reb y be of val ue


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TABLE 16

T h e o r e t i c a l Rate o fT 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 al 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

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model f o r thehuman syndrome, i twas p o s s i b l e t odemonstrate t h a tth e

degree o f f e t a l i n s u l twasdependent on th ematernal 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 sa r edetermined by th ei n t e r a c t i o no f an e n v i r o n

mental andb i o l o g i c a l component. At l e v e l s exceeding 131 mg a l c o h o lper

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 th esyndrom e.

The lowest blood alcohol level compatible w i t h normal o f f s p r i n g has y e t t o

be determined. Thef a c t o r s i n v o l v e d i nmaternal a l c o h o l e l i m i n a t i o nap

pear t o beunder g e n e ti c c o n t r o l , butwhat they a r e and how they a c t i s

unknown a tt h i s time. What i sc l e a r i st h a t the MEOS a c t i v i t yi sg r e a t l y

induced i n thes t r a i n showing theg r e a t e s t l i a b i l i t y to th esyndr ome,

r a i s i n g thep o s s i b i l i t y t h at a l t e r e d maternal metabolismmay make secon

dary c o n t r i b u t i o n s t o thesyndrome. Regarding thec 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 suggest t h a t t h er i s kto

t h e o f f s p r i n g i s not a f u n c t i o n o famounto fmaternal a l c o h o l consumption,

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