ocular hypertelorism, ptosis or strabismus, prominent, low-set ears, wide mouth ..... ment levels, blood samples were drawn from the retro-orbital sinus after each ...
THE F E T A L HYDANTOIN SYNDROME: A MOUSE MODEL by
B.Sc,
RICHARD H. FINNELL U n i v e r s i t y o f O r e g o n , 1975
THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES
in the G e n e t i c s Program
We a c c e p t to
this
thesis
the required
as
conforming
standard
THE UNIVERSITY OF BRITISH COLUMBIA May, 1978
©
R i c h a r d H. F i n n e l l ,
1978
In presenting this thesis in partial
fulfilment of the requirements for
an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this
thesis
for scholarly purposes may be granted by the Head of my Department or by his representatives.
It
is understood that copying or publication
of this thesis for financial gain shall not be allowed without my written permission.
Department of
Medical
Genetics
The University of B r i t i s h Columbia 2075 Wesbrook P l a c e
Vancouver, Canada V6T 1W5
Date
April
1 1 , 1978
ABSTRACT
The s u s p e c t e d t e r a t o g e n i c i t y o f D i p h e n y l h y d a n t o i n (DPH)
i n man i s i m p o r t a n t , e s p e c i a l l y t o t h e 0.3 t o 0.5%
of pregnant
women who a r e e p i l e p t i c
for anticonvulsant
drug
effect
f r o m DPH t r e a t m e n t ,
of epilepsy
approximating following (i) (ii)
therapy.
and, t h e r e f o r e ,
candidates
To s e p a r a t e t h e t e r a t o g e n i c an a n i m a l model c l o s e l y
t h e human c o n d i t i o n was d e v e l o p e d
t o meet t h e
criteria:
t h e t e s t a n i m a l must h a v e s p o n t a n e o u s the seizures
seizures
must be c o n t r o l l e d o r e l i m i n a t e d
by:'DPH
treatment (iii) (iv)
t h e d r u g must be a d m i n i s t e r e d serum DPH l e v e l s must therapeutic
range
fall
orally
within
t h e o p t i m a l human
between 5 and 20 m i c r o g r a m s p e r
ml serum (v)
t r e a t m e n t must b e g i n p r i o r t o m a t i n g throughout
(vi)
gestation
the offspring of treated spectrum
and c o n t i n u e
a n i m a l s must e x h i b i t t h e
of malformations
observed
i n the o f f s p r i n g
o f e p i l e p t i c women
The f i r s t The s e i z u r e .34 s e i z u r e s effect
criterion
activity
was met by m u t a n t q u a k i n g
o f t h e s e a n i m a l s was r e d u c e d
p e r mouse day) b y DPH t r e a t m e n t .
of this
gene f r o m
(qk) m i c e .
( f r o m 2.1 t o
To s e p a r a t e t h e
t h a t o f t h e DPH i n t h e e t i o l o g y o f
the malformations, heterozygous
(+/qk) and homozygous n o n -
quaking
(+/+) m i c e were a l s o s t u d i e d .
levels with indicated
t h e SYVA E m i t s p e c t r o p h o t o m e t r y
serum c o n c e n t r a t i o n s
within
r a n g e a t 40 and 60 mg/kg body w e i g h t
The tissue
Monitoring
incidence
abnormalities
of fetuses increased
assay.technique
t h e human
therapeutic
dosages.
born with
with
o f DPH
skeletal
or soft-
i n c r e a s i n g DPH d o s a g e s .
T h i s was o b s e r v e d
i n a l l three
untreated
(qk/qk) dams t o -produce n o r m a l o f f s p r i n g
quaking
implicates of
genotypes.
The a b i l i t y
o f the
t h e d r u g r a t h e r t h a n t h e mutant gene a s t h e c a u s e
malformations.
A preliminary
application of this
what c a n be c o n s i d e r e d hydantion
syndrome.
t h e mouse e q u i v a l e n t
The s i m i l a r i t i e s
mouse syndromes i n c l u d e p r e n a t a l cardiac,
orofacial,
a n i m a l model
ocular
produced
of the f e t a l
between t h e human and
growth d e f i c i e n c y , n e u r a l ,
and g e n i t o u r i n a r y
anomalies.
Further
l a r g e - s c a l e a p p l i c a t i o n o f t h e model s h o u l d
provide
insight
i n t o t h e r o l e o f DPH i n t h e e t i o l o g y o f t h e m a l f o r m -
a t i o n s o b s e r v e d amongst t h e o f f s p r i n g o f e p i l e p t i c women on hydantoin anticonvulsant
drug
therapy.
TABLE OF CONTENTS Page ABSTRACT
i i
TABLE OF CONTENTS
iv
LIST OF TABLES
vi
LIST OF FIGURES
vii
ACKNOWLEDGEMENTS
viii
I.
INTRODUCTION
1
1.
H i s t o r i c a l Perspective
1
2.
Survey o f E p i d e m i o l o g i c a l Reports Up to 1975
6
3.
Survey of Animal
Experiments
Up t o 1977
II.
15
4.
The F e t a l Hydantoin Syndrome:
1975-197 8
5.
Purpose and R a t i o n a l e o f Present Study
19 24
METHODOLOGY
28
1.
Animals
28
2.
DPH
28
3.
C a l c u l a t i o n o f Drug Doses
29
4.
Diet Administration
30
5.
S e i z u r e C o n t r o l Study
32
6.
Determination o f Serum DPH
7.
Experimental Design
8.
Treatment L e v e l s
Levels
32 34
a. Experimental Organism
34
b. Route of A d m i n s t r a t i o n
35
c. Matings
35
d. F e t a l Examination
36
S t a t i s t i c a l Analysis
iv
37
Page
III.
IV.
RESULTS
38
1.
Seizure Control
38
2.
Serum L e v e l s
38
3.
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
41
4.
F e t a l Measurements
44
5.
F e t a l Anomalies
44
DISCUSSION
51
FIGURES
63
LITERATURE
CITED
67
APPENDICES
73
APPENDIX A
DPH
APPENDIX B
Water I n t a k e C o n v e r s i o n
APPENDIX C
DPH
APPENDIX D
Alizarin.Red Staining for Skeletons
APPENDIX E
Dose C o n v e r s i o n
Table
Dosage C a l c u l a t i o n
ANOVA T a b l e s
v
Table Table
73 74 75
Procedure 76 77
L I S T OF TABLES Table 1.
2.
3.
Page R e t r o s p e c t i v e s t u d i e s on a n t i c o n v u l s a n t s a d m i n i s t e r t o e p i l e p t i c women and t h e f r e q u e n c y o f m a l f o r m a t i o n s as compared w i t h t h e o c c u r r e n c e o f m a l f o r m a t i o n s i n u n t r e a t e d e p i l e p t i c s and i n n o n - e p i l e p t i c c o n t r o l s . Frequency o f d i f f e r e n t malformations i n c h i l d r e n o f m o t h e r s on a n t i c o n v u l s a n t s d u r i n g p r e g n a n c y . Efficacy
o f DPH O r a l l y A d m i n i s t e r e d
8
13
t o Quaking
(qk/qk) M i c e .
39
4.
DPH Serum C o n c e n t r a t i o n s
5.
Effect
6.
DPH C o n c e n t r a t i o n s on P r e g n a n t F e m a l e s . E f f e c t o f T r e a t m e n t s on I m p l a n t a t i o n s and Resorption.
43
E f f e c t o f T r e a t m e n t s on L i v e B i r t h s , W e i g h t s , and F e t a l A b n o r m a l i t i e s .
45
7.
i n Non-Pregnant Females.
o f T r e a t m e n t s on Water I n t a k e
40
and Serum 42
Sex, F e t a l
8.
T y p e s and F r e q u e n c i e s
o f S k e l e t a l Anomalies.
46
9.
Types and F r e q u e n c i e s
of Soft
49
10.
Similarities Hydantoin
Tissue Anomalies.
between t h e Human and Mouse
Syndrome.
Fetal 53
vi
L I S T OF
FIGURES
Figures
Page
1.
O s s i f i c a t i o n anomalies o f s u p r a o c c i p i t a l and v e r t e b r a l c e n t r a e
2.
S k e l e t a l anomalies vertebral centrae
including
3.
Skeletal
including
4.
Microphthalmia
64
5.
Anophthalmia
64
6.
anomalies
63
triangular 63 absent
'Polydactyly
sternabrae
63
64
7.
Hydronephrosis
8.
Digital
9.
Cleft
65
hypoplasia
palate
bone
and c o n t r o l
and c o n t r o l
vii
65 66
ACKNOWLE DGEMENTS
I wish t o express thesis their
my t h a n k s t o t h e members o f my
committee a t the U n i v e r s i t y o f B r i t i s h efforts
Person
during
the course
Columbia f o r
of this project:
D r . C. 0.
( C o - c h a i r m a n ) a n d D r . A. J . F. G r i f f i t h s ,
Department
o f B o t a n y and D r . J . R. M i l l e r Medical
Genetics.
special
t h a n k s must be g i v e n
(Co-chairman) Department o f
I n a d d i t i o n t o t h e members o f my
of Poultry Science, we h a v e s h a r e d o v e r
t o D r . C. W. R o b e r t s ,
f o r t h e many e n l i g h t e n i n g
instrumental
conversations
t o o numerous t o name
roles i n carrying out this
I w o u l d e s p e c i a l l y w i s h t o t h a n k Ms. R. B u n t i n g Woodlands S c h o o l ;
Mr. R. R o b i l l o ,
Stewart, Department o f B i o m e d i c a l for her f a i t h f u l
To
and a c c u r a t e
Dr. G e r a l d fine
Department
the years.
T h e r e a r e s o many o t h e r s , who p l a y e d
committee,
research.
and Ms. M. Hanson,
D e p a r t m e n t o f B o t a n y ; Mr. B. Communications;
typing of this
(Buzz) C h e r n o f f ,
and Ms. M. P a r i s
thesis.
my t e a c h e r ,
and
my v e r y
his
technical assistance i n the cardiac dissection,
f r i e n d , my s i n c e r e t h a n k s .
h i s p a t i e n t and c o n t i n u i n g i n t r o d u c t i o n i n t o experimental Person,
teratology.
without
My h e a r t f e l t
whose h e l p
this
completed.
viii
here,
my
colleague
Not o n l y f o r but also
the world o f
thanks t o Dr. C l a y t o n
t h e s i s w o u l d n e v e r have b e e n
And and
o f c o u r s e , t o my
our c h i l d r e n ,
grateful
for their
the course of t h i s
dearest of friends,
Yasmin and A l e x a n d e r . support
I am
and e n c o u r a g e m e n t
study.
ix
Susanna,
forever throughout
-
1
-
CHAPTER I
INTRODUCTION 1.
HISTORICAL PERSPECTIVE Epilepsy, discharges
i n the
p l a g u e d man by
the
a disorder b r a i n and
have b e e n u n e a r t h e d of
call
the
the
would r e f e r
by
Galen.
notion
the
unless
Some o f by
that
the
Penry,
disease,
"Falling
from t h e s e
the
earliest
Hippocrates
Both of
and
they p r e s c r i b e d
Their
treatment
1972).
which the
which the
treatments his
Certainly first
Greeks would
Christians
(Tempkin,
1945;
history
that
for epilepsy
followers.? dismissed
and the
were
much
later
commonly
a form o f demonic p o s s e s s i o n .
secular
rather
o f honey and
than s p i r i t u a l
dietary restraint
consideration.
They f e l t
that
vinegar
t h a t was
I t i s noteworthy, that
5th
the
e a r l y twentieth
held There-
cures. based
i t was
i n good o r d e r ,
s e v e r a l t i m e s e a c h day. C e n t u r y B.C.E. u n t i l
that
sources.)
importance t o keep d i g e s t i o n
with a concoction
evidenced
skulls
i n d i c a t e d , the
f o r e p i l e p s y was
upon p a t h o l o g i c a l
be
has
e a r l y man's
Sickness"
these scholars
e p i l e p s y was
paroxymal
seizures, can
represent
otherwise
fore,
vital
of
by
prehistoric
( C o a t s w o r t h and
trephinings
i s derived
described
This
" S a c r e d D i s e a s e " and
t o as
Lennox, 1960; follows
recurrent
trephined
attempt to r i d h i m s e l f later
by
since his beginnings.
l a r g e number o f
a fraction
characterized
often
drunk from
century,
the
of
- 2 -
every
treatise
on e p i l e p s y s p e c i f i e d
articles
o f food
that
s h o u l d o r s h o u l d n o t be e a t e n .
Hippocrates Galen
sought
favoured
exotic dietary
a w a l k i n f r a g r a n t a i r and supplements.
powdered human s k u l l
started
for
a thousand
i n Europe.
was
t o d r i n k warm, human b l o o d , w h i c h was u s u a l l y
from
a fallen
sources Middle treat
years
gladiator
continued Ages.
a practise
Galen's t h a t was
A n o t h e r common
i n Roman t i m e s .
Blood
use o f continued
prescription obtained
from
other
t o be p r e s c r i b e d i n E u r o p e up t o t h e
By t h e 4 t h C e n t u r y
epilepsy included d i e t e t i c ,
A.D., t h e methods u s e d t o surgical
and p h a r m a c o l o g i c a l
techniques.
Despite the t e a c h i n g s o f the H i p p o c r a t i c s c h o o l , which theorized
t h e c a u s e o f s e i z u r e s t o be due t o ' e x c e s s i v e phlegm'
obstructing
t h e p a s s a g e o f a i r to. t h e b r a i n ,
possession theories regained
acceptance.
i n c i d e d w i t h t h e growing r o l e
framework o f t h e M i d d l e
recounted
the tale
casting
Ages.
saint
S t . M i c h a e l , and t h o s e
i n the socio-
The g o s p e l w r i t e r s
c u r i n g t h e e p i l e p t i c s by
o u t t h e demons w i t h h i s w o r d s .
known as t h e p a t r o n and
of Christ
This r e b i r t h co-
o f the church
political
t h e demonic
S t . V a l e n t i n e became
of the e p i l e p t i c s , afflicted
with
a s were S t . V i t u s
e p i l e p s y made
pilgrimages to their p r i o r i e s ,
where h o s p i t a l s were
The
e p i l e p s y a s one o f t h e e i g h t
Church l e a d e r s even l i s t e d
cardinal
contagious
d i s e a s e s , which i n c l u d e d such
built.
heinous
- 3 -
disorders and
as b u b o n i c
leprosy.
As
The of
tuberculosis,
scabies,
P r o f e s s o r Siegmund A l b i c h ,
scholar proclaimed: them, s i n c e by
plague,
a 15th
" T h e r e f o r e , n e i t h e r t a l k nor
t h e i r mere b r e a t h
they
infect
century,
and
who
as t h e p r i m a r y
physician.
o n l y as The
include elder, began t o l o s e
limited
garlic,
m i s t l e t o e and
f a v o r , the v a l e r i a n
peony.
becoming
seventeenth
century p r o f e s s o r of n a t u r a l philosophy
liver
of wolf
o f Thomas W i l l i s ,
e x o t i c s as powdered human s k u l l , and
gall
of boar
with dried
to
t h e more r a d i c a l
treatments
w h i c h , more o f t e n t h a n not, had worse than
The
the d i s e a s e
n i n e t e e n t h century brought For
longer confined to insane
humane h o s p i t a l i z a t i o n
at
Oxford,
While
blood, these
brain
effects
surgery,
t h a t were
itself.
suffering with epilepsy. no
a
t h e y were p r e f e r a b l e
o f c a u t e r y and secondary
increasingly
dragon's
urine.
c u r e s were, f o r t h e most p a r t / i n e f f e c t u a l ,
cures
When peony
The
such
treatment
the
important.
required
favorite
pharmacological
in epileptic
r o o t was
early
those p h y s i c i a n s
as t h e . i m a g i n a t i o n o f
most o f t e n named h e r b s
half
demons o r
For
d i d employ s e c u l a r c u r e s , t h e r a n g e o f
r e m e d i e s was
last
a g r e a t many i n t h e
cause of e p i l e p s y .
with
people."
p a r t of the e i g h t e e n t h century d i d not exclude witches
Century
bathe
m a j o r i t y of the p h y s i c i a n s d u r i n g the
the seventeenth
anthrax
the
first
asylums.
many a d v a n c e s f o r time
epileptics
those were
They were p r o v i d e d
t h a t p e r m i t t e d the. f i r s t
with
systematic,
- 4 -
controlled in
studies
French e p i l e p t i c
importance of cure. the
This
actual
century and
the
hospitals, Esquirol described
f a c t o r was
the
e p i l e p s y was
brain. or
nutrition
The
of
first
b r o m i d e , was the
with
Further,
that
practical
the
an
Mering,
properties
1903).
A
nineteenth
reflex action
c h a n g e s i n the
molecular
c h a n g e s were m e d i a t e d
to
by
Kussmaul
brain'.
e f f e c t i v e anticonvulsant by
Charles
Locock
treatment of
of
Patients being
and
i n 1857.
that
It
time.
sodium replaced
While
for epilepsy, i t
chronically treated
s e d a t e d , had
psychic and
e f f e c t i v e treatment
twentieth
of b a r b i t u r a t e s I t was
drug,
g a s t r i c di'S't'r-e'S's ( V i d a
for a safe,
i n t o the
p o s s e s s e d by
barbiturates.
i t involved
e f f e c t i v e treatment
search
synthesis
early
'sudden i n t e r r u p t i o n
skin rashes
continued
these
i n the
due
side effects.
Thus t h e
epilepsy
than
convulsions
drug complained
disturbances, 197 3 ) .
more i m p o r t a n t
studies
introduced
without
this
doctor's
Animal
i n e f f e c t u a l zinc oxide
not
the
poisoning.
truly
s o d i u m b r o m i d e was was
i n the
p r e v a i l i n g medical opinion
malnutrition
the
experience
treatment.
regarding
of
From h i s
p a t i e n t s ' confidence
& Tenner d e s c r i b e d
Von
disorder.
c e r e b r a l angiospasm r e l a t e d to
state
in
the
psychological
The
by
of
century,
was
when t h e
achieved
soon r e a l i z e d t h a t
the
(Fisher
Gerry, for first and
hypnotic
b a r b i t a l were c h a r a c t e r i s t i c o f a l l
second b a r b i t u r a t e
drug,
phenobarbital
-
5 -
( 5 - e t h y l - 5 p h e n y l b a r b i t u r i c a c i d ) was p r a c t i c e by
three
Juliusburger,
independant researchers
1912;
Impens, 1 9 1 2 ) .
Hauptmann r e c o g n i z e d in
the treatment
an
important
drug
drug
of c h o i c e i n the
s e i z u r e s and
was
seizures
in cats.
convulsant 19 3 8 a ) .
T h i s drug
The seizures,
by
years.
The
but
enough
e l i m i n a t i n g the
t o be
(1908).
they
registered 5,5-
Its
Merritt
anti-
and
r e p o r t e d on (Merritt
Putnam
less
stigma
Putnam,
has
t o .the
c u r e s o f more t h a n 1
non-
or e l i m i n a t i n g
century
attached
o f more n o r m a l
anti-
toxic.
in controlling
and
and
induced
the
many b e c a u s e i t i s
in a single
of marriage
tonic-
against e l e c t r i c a l l y
controversial
possibility
of
Dilantin;
until
i n humans
e f f e c t i v e n e s s o f DPH
prospect
still
i t s hypnotic
synthesis of
Blitz
i s p r e f e r r e d by
r e p l a c i n g the
opened t h e
by
Later that year
t h e r e f o r e tends
by
It is
s e i z u r e s , whether
(DPH;
first
i t s efficacy
shows t h a t m e d i c i n e
a l o n g way and
The
achieved
p r o p e r i t e s o f DPH
s e d a t i v e and
phenobarbital
treatment
partial
p r o p e r t i e s went u n n o t i c e d
(1938b)' d e m o n s t r a t e d
Alfred
protection against seizures.
trademark of Parke, D a v i s ) .
convulsant
same y e a r
(Hauptmann, 1 9 1 2 ) .
i s diphenylhydantoin
diphenylhydantoin
medical
1912;
p h y s i c i a n s from p r e s c r i b i n g h i g h
( g r a n d mal)
f o c a l or not,
In the
i n anticonvulsant therapy,
doses to ensure f u l l
Today t h e
(Loewe,
the t h e r a p e u t i c v a l u e of
of e p i l e p s y
property prevents
clonic
introduced into
: lives
for
come disease a
1000
epileptics
c h i l d b e a r i n g t o more
-
6
-
e p i l e p t i c woman t h a n e v e r b e f o r e . some new
issues.
The
e p i l e p t i c mothers has that and
however,
number o f m a l f o r m e d c h i l d r e n b o r n l e d many i n v e s t i g a t o r s t o
congenital malformations e x i s t s . factors associated with
maternal
age,
incidence
Janz,
of hydramnios), which could
1 9 7 5 ) , many s u s p e c t
result
of
the
drugs.
The
studies
designed
SURVEY OF
next
(Fedrick, t h a t the
teratogenic section w i l l
two
years
are
advanced increased
c o n t r i b u t i n g to
the
Monson e t a l . ,
1973;
instead
anticonvulsant
r e s u l t s of
the
possibilities.
TO
1975
a f t e r the
clinical
German p h y s i c i a n s
the
d r u g s and
congenital malformations
a s s o c i a t i o n between e p i l e p s y , (Janz
and
introduction
published
a
tragedy.
completed s h o r t l y a f t e r t h e
A number o f
n o t a b l y 'that o f Meadow orofacial
pregnant.
Of
lesions.
anecdotal
Fuchs,
thalidomide
case r e p o r t s
(1968) , who
described
1964).
s i x p a t i e n t s , four
epileptic
(Kevadon)
followed, six
c l e f t s whose m o t h e r s a l l r e c i e v e d these
report
anticonvulsant
s u r v e y o f m a l f o r m a t i o n s amongst c h i l d r e n b o r n t o
m o t h e r s was
heart
drugs
malformations are
examine t h e
outlining
with
1973;
EPIDEMIOLOGICAL REPORTS UP
of diphenylhydantoin,
be
and
a c t i o n of the
to reveal these
Less than t h i r t y
This
{anoxia,
status
to
speculate
Although,there
epilepsy
lower socio-economic
congenital malformations
2.
i s posing,
a r e l a t i o n s h i p between e p i l e p s y , a n t i c o n v u l s a n t
risk
the
This
a l s o had
most
patients DPH
while
congenital
-
Until of e p i l e p t i c different Table
1,
7
-
r e c e n t l y , a l l of
t h e s e s t u d i e s on
Janz
(1975), t o which the
excellent
review a r t i c l e s ,
surveys.
The
presentation
data
includes
format.
o f m a l f o r m a t i o n s was
and
(Janz
and
Fuchs,
Meadow, 1972;
The
1964;
South,
and
1972;
of c o n g e n i t a l
mothers i n t h e s e
studies
and
Fuchs,
to a high
the
average i n c i d e n c e
the
frequency
not
on
1964)
the'frequency of t o be
greater
these observations
increase
i n the
epileptics,
T h i s was
for epileptic
pregnancies.
1969;
Speidel
1973;
Two
treated (Janz
1973),
nearly
twice
woman who studies
were show
mothers
mothers,(Starresveld-
In
The five
s i g n i f i c a n c e of studies
f o r controls there
the
on
1973;
2.2%
born to untreated
treated
anomalies of
over t h a t of
of
(Fedrick,
Meyer 1 9 7 3 ) .
populations
were n o t
Annegers e t a l . , 1974).
7.2%.
i s uncertain.
the
epileptic
Koppe e t a l . ,
13.8%
abnormal b a b i e s
Zimmerman e t a l . , 1973;
non-epileptic
of
being
than t h a t of
studies,
Fedrick,
r a n g e s f r o m a low
their
facilitate
Markoff,
Lowe, 1973;
eighteen
anomalies born to the
(3.8%) r e c o r d e d
drugs d u r i n g
of
t h o s e who
M a r o n i and
Monson e t a l . , 1973;
incidence
result
compared between
S t a r r e s v e I d - Z i m m e r m a n e t a l . , 1973; Meyer, 1973;
is referred for
I n a number o f
mothers r e c e i v i n g a n t i c o n v u l s a n t s drugs
reader the
studies.
i n Annegers e t a l .
have b e e n t r a n s c r i b e d t o
in this
included
c o m p a r i s o n s between
drawn h e a v i l y f r o m s i m i l a r t a b l e s
incidence
outcome
p r e g n a n c i e s h a v e b e e n r e t r o s p e c t i v e and
c o n t r o l groups, h i n d e r i n g
(1974) and
the
c h i l d r e n born to
that
had
is a three-fold treated
control population
(Elshove
and
Table
1.
8 -
R e t r o s p e c t i v e s t u d i e s on a n t i c o n v u l s a n t s a d m i n i s t e r e d to e p i l e p t i c women a n d t h e f r e q u e n c y o f m a l f o r m a t i o n s a s compared w i t h t h e occurrence o f m a l f o r m a t i o n s i n u n t r e a t e d e p i l e p t i c s and i n n o n - e p i l e p t i c c o n t r o l s . Livebirths to treated mothers with e p i l e p s y
Reference
Livebirths to untreated mothers with e p i l e p s y
Total
Malfn.
%
Total
Malfn.
Janz & Fuchs
(1964)
225
5
2.2
133
0
0
Maroni & Markoff
(1969)
21
1
4.8
14
0
0
Elshove & Van Eck
(1971)
65
10
15.4
-
-
-
Watson & Spellacy
(1971)
51
3
5.9
-
-
-
Speidel & Meadow
(1972)
329
17
5.2
59
0
0
South
(1972)
22
2
9.1
9
0
0
Lowe
(1973)
134
9
6.7
111
3
2.7
Fedrick
(1973)
217
30
13.8
19
2
10.5
(1973)
48
3
6.2
_
_
_
(1973)
110
7
6.4
_
_
StarresveldZ i m m e r m a n e t a l . (19 7 3) 2 7 9
20
7.2
18
2
11.1
Kuenssberg Knox
&
Millar & Nevins
(1973)
125
11
8.8
66
2
3.0
(1973)
199
17
8.5
124
14
11. 3
(1973)
205
11
5.5
101
3
3.0
(1973)
_
_
_
—
Niswander & Wertelecki
(1973)
_
_
_
_
Annegers e ta l .
(1974)
141
10
7.1
56
1
Baile
(1975)
51
5
9.8
-
-
-
2222
161
7.2
711
27
3.8
Koppe
e ta l .
Meyer Monson
e ta l .
Bjerkedal Bahna
Totals
&
e ta l .
_
1.8
- 9 -
Total
Malfn.
358
5
1.4
35
1
2.8
"5
Total
0
50
0
0
7
1.6
4.4
448
31
2
6.4
_
245
12
5.0
236
32
13.6
22
7.4
192
13
6.8
323
31
9.6
306
14
4.6
378
17
4.5
413
17
4.1
197
11
5.6
3399
649
—
36
_
297
-
-
194
5.7
Q. "5
0
17
-
Malfn.
65
388
_
Livebirths population
Livebirths to non-epileptic c o n t r o l mothers
Livebirths to a l l e p i l e p t i c mothers
— 50591
Total
12051
total
Malfn.
Q,
*o
_
_
231
1.9
_ 7865
190
2.4
31877
877
2.8
5.5
—
_
_
_
14620
477
3.0
32227
1235
3.8
12300
426
3.5
_
_
1240
2.5
_ _
_
112328
2471
2.2
347097
9372
2.7 _
51083
1283
2.5
570365
15249
2.7
-
Van
E c k , 19 71;
1972;
Watson a n d S p e l l a c y ,
Fedrick,
controlled was
birth
to u t i l i z e
subjects.
I n most i n s t a n c e s
and s o c i o - e c o n o m i c
woman w i l l i n g
and
the case
1971; S p e i d e l
epidemiological
techniques,
studies
to a
as c o n t r o l s
and Meadow, 1972)
studies, i n v e s t i g a t o r s chose a given
time
interval
and Van E c k , 1971; S o u t h , 1972; Monson e t a l . , 1 9 7 3 ) ,
a l l b i r t h s reported Knox, 1 9 7 3 ) .
regions
When i t
the next h o s p i t a l b i r t h
to use a l l h o s p i t a l b i r t h s d u r i n g (Elshove
case-
t h e c o n t r o l mother
( F e d r i c k , 1973).
to participate in.the
In o t h e r
as
p a r i t y , h o s p i t a l , year o f
status
used i n s t e a d
(Watson and S p e l l a c y ,
major
the n o n - e p i l e p t i c population
i n terms o f age, r a c e ,
studies
and Meadow,
(1973), i n h e r study,: was t h e o n l y
n o t f e a s i b l e t o employ t h e c a s e - c o n t r o l l e d
other
or
Speidel
s e l e c t e d on t h e b a s i s o f s i m i l a r i t i e s w i t h
patient,
was
19 71;
1973; Monson e t a l . , 1973)
Fedrick investigator
10 -
by g e n e r a l
Still
p r a c t i t i o n e r s (Kuenssberg
others
used g e o g r a p h i c a l l y
defined
as c o n t r o l p o p u l a t i o n s
(Lowe, 1973; M i l l a r
and N e v i n s ,
1973) .
T h e r e a r e two l a r g e r e t r o s p e c t i v e s t u d i e s n o t mentioned
s o f a r , N i s w a n d e r and W e r t e l e c k i
Bjerkedal
and Bahna
all
(197 3 ) .
births i n a military
1971,
and t h e l a t t e r
(1973) and
The N i s w a n d e r s t u d y
h o s p i t a l i n .the y e a r s
study
recorded
reported
1965 t h r o u g h
a l l b i r t h s i n Norway
-
during
1967 and 1968.
11 -
The i n c i d e n c e
c h i l d r e n b o r n t o women w i t h 4.5%, r e s p e c t i v e l y . populations studies
were 2.7% a n d 2.8%.
anticonvulsant
were women who, w h i l e not
lower i n c i d e n c e
Unfortunately
status.
diagnosed
receiving anticonvulsant
ascertained f o r t h i s
f i g u r e s i n the c o n t r o l i n these
p a t i e n t s were n o t s e p a r a t e d
drug therapy
survey..
of reported
amongst
d i a g n o s e d e p i l e p s y was 4.1% and
The c o m p a r a b l e
the epileptic
of malformations
Within
as b e i n g
this
by t h e i r population
epileptic,
were
drugs a t t h e time they
were
T h i s may a c c o u n t
f o r the
m a l f o r m a t i o n s t h a n i n some
of the other.surveys.
The in
combined r e s u l t s
the l i t e r a t u r e
malformations
the
population
population births
(2.7%).
i s three
reported
T h e s e f i g u r e s a r e b a s e d upon 2222
in.the general
3.8%, a f i g u r e m a r g i n a l l y
s e v e r a l authors
a t an i n c r e a s e d
(Fedrick,
to non-treated higher
The m a l f o r m -
epileptic
risk
they
claiming
i n many
that e p i l e p t i c
regardless of their
a r e a t no i n c r e a s e d
women
than f o r the general
1973; Monson e t a l . , 1973; J a n z ,
maintaining 1974),
population.
live-
600,0 00
T h i s has been a p o i n t o f c o n t e n t i o n
studies, with are
o f producing
(7.2%) t h a n i t i s f o r t h e g e n e r a l
a t i o n r a t e i n 711 l i v e b i r t h s
population.
reported
times g r e a t e r f o r
t o t r e a t e d e p i l e p t i c mothers, and n e a r l y
livebirths
was
surveys
by 1975 i n d i c a t e t h a t t h e r i s k
children with epileptic
of a l l eighteen
risk
drug
women
therapy
1975) and o t h e r s ( S p e i d e l and Meadow,
-
12 -
While the r a t e o f malformations epileptics
r e m a i n s low, i t i s c l e a r l y
of the population. for
this
increase
defects
(Table
or without
The s p e c i f i c are o r o f a c i a l
2).
cleft
i n the group o f t r e a t e d
higher
defects clefts
The 1.8% i n c i d e n c e
palate, recorded
dom
1 9 7 3 ) , London Elshove
of cleft
the
genital
cleft
studies
1972).
heart
Further,
defects
population
an i n c r e a s e d
certain
consistency
genic
the average i n c i d e n c e
greater
o f con-
that
i t i s f o r a heter-
( S p e i d e l and Meadow, 1972)
(Table
i n the type
1)', t h e y
also reveal, a
of malformation
As a l l known t e r a t o g e n s
i n other
Lowe,
r a t e o f m a l f o r m a t i o n among o f f s p r i n g o f
e p i l e p t i c mothers
2).
179 b i r t h s r e c o r d e d i n
do t h e s t u d i e s e x a m i n e d p o i n t c o n c l u s i v e l y
treated
(Table
(0.27%;
among c h i l d r e n e x p o s e d p r e n a t a l l y t o
ogenous B r i t i s h
only
King-
(0.16%; Lowe,
( S p e i d e l and Meadow, 197 2;
i s fourfold
Not
i n the United
In e p i l e p t i c mothers n o t r e c e i v i n g
p a l a t e , i n the
anticonvulsants
to
than the frequencies
t h e r e was n o t a s i n g l e c a s e o f c l e f t l i p ,
forementioned South,
l i p with
f o r the c h i l d r e n of treated
and Meadow, 1 9 7 2 ) , C a r d i f f
and Van E c k , 19 7 1 ) .
or without
1973;
heart
(0.2%; S o u t h , 1972) and i n t h e N e t h e r l a n d s
anticonvulsants, with
that are responsible
f o r n o n - e p i l e p t i c c o n t r o l mothers
(0.14%; S p e i d e l
f o r the r e s t
and c o n g e n i t a l
e p i l e p t i c m o t h e r s , i s 7-13 t i m e s g r e a t e r recorded
than
i n man
produced
are also t e r a t o -
s p e c i e s . ( S t a p l e s , 1972) i t i s l o g i c a l t o
initially
d e t e r m i n e t h e t e r a t o g e n i c i t y o f DPH i n l o w e r
animals.
The f o l l o w i n g s e c t i o n e x p l o r e s
the various
animal
- 13 Table
2.
Frequency o f d i f f e r e n t malformations i n c h i l d r e n o f mothers on a n t i c o n v u l s a n t s d u r i n g pregnancy. MALFORMATIONS No. o f Pregnancies
Reference
Orofacial clefts
Cardiac anomalies
Anencephaly
Janz & Fuchs
(1964)
225
Maroni & Markoff
(1969)
21
Elshove & Van E c k
(1971)
65
Watson & Spellacy
(1971)
51
Speidel Meadow
(1972)
329
3
South
(1972)
22
2
Lowe
(1973)
134
1
1
Fedrick
(1973)
217
1
2
(1973)
48
(1973)
205
2
3
1
(1973)
110
2
StarresveldZ i m m e r m a n e t a l .( 1 9 7 3 )
279
9
7
1
Koppe e t a l .
(1973)
125
1
4
Meyer
(1973)
199
5
5
Mirkin
(1973)
7
2
Hill
(1974)
28
1
2
Annegers e t a l .
(1974)
141
3
8
Biale
(1975)
51
1
2
2257
41
44
&
Kuenssberg Knox
&
Monson
et a l .
Millar Nevin
&
et a l .
et a l .
TOTAL PERCENTAGE @
3
1
5
2
1
1
1.8
6
2.0
1
4 0.2
As c o m b i n a t i o n s o f m a l f o r m a t i o n s o c c u r , t h e t o t a l numbers o f c a s e s c a n b e l o w e r t h a n t h e sum o f t h e d i f f e r e n t m a l f o r m a t i o n s stated.
-
Neural Microcephaly defects
14
Hydrocephaly
Hypospadias
-
Skeletal G.I. anomalies defects
@ misc.
# cases malfns.
_
_
_
_
_
1
_
5
_
_
_
1
_
_
_
1
_
1
_
_
1
_
1
_
_
1
_
_
_
1
3
3
_
_
2
1
2
2
17
-
-
-
-
-
-
-
2
-
1
-
-
1
-
3
9
-
1
-
2
6
1
8
20
_
1
1
_
1
_
3
1
-
2
-
3
-
-
11
_
1
_
—
_
_
4
7
_
1
—
1
_
1
20
-
-
-
-
-
-
6
11
-
1
-
-
7
-
-
17
1
-
-
-
-
-
-
3
-
1
-
1
-
1
1
7
-
-
-
-
-
-
1
10
-
-
• -
-
1
-
1
5
5
7
4
7
19
6
29
171
0.2
0.3
0.2
0.3
0.8
0.3
1.3
'
10
7.6
studies of
3.
15 -
t h a t have b e e n p e r f o r m e d and t h e t y p e s and
t h e m a l f o r m a t i o n s t h a t have b e e n
SURVEY OF ANIMAL EXPERIMENTS UP TO While t h e e p i d e m i o l o g i s t s larger
frequencies
produced.
197.7. and c l i n i c i a n s ammassed
and l a r g e r numbers o f p a t i e n t s
fortheir
surveys,
number o f s t u d i e s were c a r r i e d o u t t o d e t e r m i n e t h e icity A/J
o f DPH i n o t h e r
species.
Massey
(1966)
inclusive.
Using
d o s e s , she r e p o r t e d mice h a d . o r o f a c i a l
12.5,
that
42.8%
clefts.
T h i s was s i g n i f i c a n t l y
weight
higher
i n this
strain
mice.
carried out s i m i l a r
i g a t i o n s w i t h t h e s t r a i n s S w i s s and A / J . clefts size
( 1 5 . 2 % and 30.8%:,
respectively)
with
They f o u n d o r o f a c i a l
and r e d u c e d
fetal
on
Further,
days 9-11.
11-13,
inject-
50rmg/kg body w e i g h t d o s a g e s o f DPH on d a y s 1 1 - 1 3 T h i s was n o t o b s e r v e d
35.4%
invest-
and body w e i g h t when t h e dams were s u b c u t a n e o u s l y
of g e s t a t i o n .
the A/J s t r a i n compared w i t h
i n t h e mice
when t h e dams r e c e i v e d
showed an i n c r e a s e
14.3%
cleft
palate
treated
DPH on d a y s
i n resorption to
i n the controls.
f o u n d l o w e r body w e i g h t s and an i n c r e a s e d of
days
of the o f f s p r i n g of treated
G i b s o n and B e c k e r , .'(196 8)
ed
injected
2 5 and 50 mg/kg o f body
than t h e 8-10% spontaneous r a t e ^ o f o c c u r r e n c e of
teratogen-
m i c e d a i l y w i t h DPH s u b c u t a n e o u s l y on g e s t a t i o n a l
9-15
a
Elshove incidence
(1969) (15.3%)
i n S w i s s m i c e when t r e a t e d w i t h i n t r a p e r i t o n e a l
- 16 (ip)
i n j e c t i o n s o f 2.5, 1.9, o r 1.75 mg o f DPH.
Interesting^"
l y , when the mice were t r e a t e d on days 10-13, there was 100%
r e s o r p t i o n , compared w i t h a 15% i n c i d e n c e
were i n j e c t e d on days 11-14.
f o r mice t h a t
These s t u d i e s i n d i c a t e t h a t the
c r i t i c a l p e r i o d f o r DPH t e r a t o g e n i c i t y , i s d u r i n g the g e s t a t i o n a l days 10-14.
Harbison and Becker
(1969) administered
various
doses o f DPH t o mice a t d i f f e r e n t g e s t a t i o n a l times.
The
dams were t r e a t e d w i t h e i t h e r a s i n g l e i p i n j e c t i o n o f 150
mg/kg on days 8-15, or were given three
subcutaneous
i n j e c t i o n s i n lower doses on e i t h e r days 8-10 o r 12-14. Amongst the dams r e c e i v i n g s i n g l e i n j e c t i o n s , those t r e a t e d on days 10 or 14 had an 80% r e s o r p t i o n r a t e , w h i l e those r e c i e v i n g DPH on other days had no i n c r e a s e i n t h e number of implants resorbed.
A l l o f the t r e a t e d f e t u s e s had low
term weights and reduced crowMrump l e n g t h , which was p r i m a r i l y the r e s u l t o f shortening
o f the long bones.
Harbison and Becker were the f i r s t other
defects
than c l e f t p a l a t e i n t h e o f f s p r i n g o f dams t r e a t e d
w i t h DPH. (100
to report
A t the two higher
dose l e v e l s used i n t h e i r
and 150 mg/kg), they r e p o r t e d
cleft
l i p , cleft
study
palate,
hydronephrosis, r e n a l and i n t r a p e r o n e a l hemorrhage and delayed o s s i f i c a t i o n and/or unfused sternabrae of t r e a t e d dams. i n c l u d e d open eye
i n over 20% o f progeny
Other l e s s commonly observed malformations /f
e c t r o d a c t y l y , i n t e r n a l hydrocephalus and
- 17 -
split
cervical
gestational clefts, the
centra.
days
12-14 had e l e v a t e d
frequencies of orofacial
while t h e r e were no m a l f o r m a t i o n s
m o t h e r s on days
teratogenicity
i n d u c e d by t r e a t i n g
8-10.
Investigators the
The m i c e t h a t were i n j e c t e d on
a t Guy's H o s p i t a l
i n London
studied
o f most o f t h e commonly p r e s c r i b e d
anti-
c o n v u l s a n t s e i t h e r b y i n t u b a t i n g m i c e w i t h 40 o r 120 mg/kg dose, 6-16.
o r by m i x i n g
i n 250 mg/kg i n t o
They f o u n d t h a t
and M c E l h a t t o n , 1975).
t h e two h i g h e r doses
(Sullivan
size
to attribute
o f t h e dams i n c r e a s e d ,
the decreased f e t a l
they
weight t o
a c t i o n o f the drug.
In these
a subsequent
same i n v e s t i g a t o r s
study
increase
6-16.
i n their
i n f i v e o f the seventeen
At the h i g h e s t treatment
delayed o s s i f i c a t i o n enlarged
Though t h e r e was no
i n t h e dams on DPH t r e a t m e n t ,
were f r e q u e n t l y o b s e r v e d
dose.
and M c E l h a t t o n , 19 77)
i n t h e number o f i m p l a n t s p e r l i t t e r
resorptions
were s e e n
(Sullivan
a d m i n i s t e r e d 15, 45 and 90 mg/kg
DPH, i n t u b a t i n g dams on d a y s
of
p a l a t e was
They a l s o n o t e d a d e c r e a s e i n f e t a l
w e i g h t s , b u t as t h e l i t t e r
the
f e e d on d a y s
the incidence of c l e f t
e l e v a t e d when t h e m i c e r e c e i v e d
were r e l u c t a n t
their
cerebral
ventricles
litters
rate
malformations
offspring.
level,
o f t h e hands
o r i n .the
Open e y e d e f e c t s
on t h e 4 5 mg/kg
reduced
fetal
(43%) and f e e t
weights,
(62%),
and s e p a r a t e d b a s i s p h e n o i d bones
- 18 w e r e t h e most;commonly are
defects.
c a l c u l a t e d o n a mean p e r c e n t a g e
litters
had defects
control
litters,
at the higher
only
An e x p l a n a t i o n the
observed
resorption rates
embryonic
loss,
early
(1976).
after
;
i n nine
DPH
(53%) o n l y
day
o f g e s t a t i o n , embryonic
total
number
isn't
causes
embryolethal In their
study
deciduomata that would
represent
I n f o u r o f t h e dams t r e a t e d
with
o f t h e d a m s o n 170: m g / k g o f
deciduomata were v i s i b l e .
of implants
t h a t DPH
i n a 20% aqueous s o l u t i o n o f
to visualize
(13%), and
They f e l t
g e s t a t i o n a l day 14,
embryonic r e s o r p t i o n .
lOOmg/kg
malformations.
lack of increase i n
and t h a t t h e drug
t h e mouse u t e r i
ammonium s u l p h i d e
Of t h e u n t r e a t e d
i n t r e a t e d m o t h e r s was a d v a n c e d b y
early
placed
dose.:-
f o r the apparent
and h i s co-workers
they
results
l i t t e r b a s i s , 42% o f t h e
0.62% h a d f e t u s e s w i t h
Fritz
when a d m i n i s t e r e d
When t h e s e
After the fourteenth
loss represented
i n the highest
only
3% o f t h e
DPH
treatemeht
the
suspected
group.
All
these
teratogenic previous
and
reported
McElhatton,
defect
Becker,
clefts
outlined i n the a r e t h e most
(Massey, 1966; Gibson and
and Becker,
1975, 1977; F r i t z
as i n t e r n a l
(Harbison..and
confirm
While o r o f a c i a l
1968; H a r b i s i o n
anomalies
studies
potential of anticonvulsants
section.
consistently Becker,
animal
1969, 1972; S u l l i v a n
e t a l . , 1976),
such
hydrocephalus,
and o c u l a r
19 6 9 ;
and McElhatton,
Sullivan
defects 197 7)
- 19 -
have a l s o b e e n o b s e r v e d .
F o r t h e most p a r t ,
those malformations observed represent day
4.
a t very
the. n o r m a l c o n d i t i o n s
high
however,
doses a c t u a l l y
o f a l e s s than
eighteen
mouse.
THE FETAL HYDANTOIN SYNDROME: The
epidemiologic
1975-1978
studies described
i n Secion
2
were d e s i g n e d p r i m a r i l y t o i n v e s t i g a t e t h e a s s o c i a t i o n : , between m a t e r n a l h y d a n t o i n s u c h as c o n g e n i t a l there
heart
c o n s u m p t i o n and s i n g l e d e f e c t s ,
defects
were t h e o c c a s i o n a l
or f a c i a l
(Meadow, 1970; S p e i d e l
Loughnan e t a l . , 1973; H i l l In
a t t h e Dysmorphology U n i t ,
prenatal
exposure
and Meadow, 1972;
e t al.. , 1974; B a r r
1975, Hanson and S m i t h o b s e r v e d
five
et.al.,
unrelated
a l l o f whom h a d b e e n
to hydantoin anticonvulsants.
As t h i s
pattern of
from o t h e r
and
i n t h e o f f s p r i n g o f women
malformation.-:: syndromes
h y d a n t o i n s , i t was r e f e r r e d t o as t h e " f e t a l
These o r i g i n a l
five
syndrome showed a p a t t e r n
low,
nasal bridge,
abnormalities
epicanthic
folds,
short
taking
hydantoin
cases o f the f e t a l
of malformations
characteristic^craniofacial
with
exposed
m a l f o r m a t i o n s was d i s t i n c t had been o b s e r v e d o n l y
1974).
children
U n i v e r s i t y o f Washington,
s i m i l a r dysmorphic f e a t u r e s , i n utero
However,
r e p o r t s t t h a t i n d i c a t e d a broader
range o f malformations a s s o c i a t e d w i t h to hydantoins
clefts.
that
syndrome''.
hydantoin included
s u c h as a b r o a d , upturned
nose,
ocular
hypertelorism,
low-set ears,
20
-
p t o s i s or
strabismus,
w i d e mouth w i t h t h i c k f l e s h y l i p s ,
o c e p h a l y w i t h m e t o p i c s u t u r a l : ; r i d g i n g and The
s k e l e t a l defects
p h a l a n g e s and
nails,
included
characterized
Anomalies of
ribs,
the
(calcaneovalgus
observed.
deformity
severely
retarded;
75%
of normal.
d e f i c i e n t , with mild
are
associatedcwith
commonly f o u n d
as
the
and
the
distal
dermato-
deform-
pes
also
cavis)
were
deficiency that
linear
g r o w t h was
Developmental
to moderate mental microcephaly.
Other but
pulmonary
are
features, not
and
cardiac anomalies
ventricular septal
defects
stenosis.
the
studies
published
p r i o r to
the
risk
figures for treated
epileptic
m o t h e r s b a s e d upon
7.2%
frequency of malformations reported
e p i l e p t i c m o t h e r s on
clefts
and
inguinal
pilondial
syndrome c o u l d
f i g u r e was
that
consistant
hydantoin
this
also
performance
fetal
of
of
renal defects
septal defects,
Retrospective description
was
retardation
g e n i t a l anomalies i n c l u d i n g hypospadias,
atrial
patterns.
f e a t u r e s , .".include: u m b i l i c a l and
sinus, o r o f a c i a l c l e f t s , s u c h as
fontanels.
p o s i t i o n a l limb
i n these p a t i e n t s
fo f e m e n t i o n e d
hernias,
and
Postnatal
of
trigon-
low-arch ridge
There i s a definite.^growth in i t s origin.
usually
by
wide
thumb, and
s t e r n u m , and
prenatal
is
hypoplasia
a finger-like
glyphic patterns
ities
prominent,
derived
congenital
anticonvulsant
drug therapy.
l e s i o n s , and
cite the
i n the o f f s p r i n g " ,
p r i m a r i l y from the
heart
only
incidence
since
these
Since of
facial
defects
-
are
but
a small
portion
21
of
h y d a n t o i n syndrome, t h i s
-
the
risk
anomalies comprising f i g u r e .would n o t
Moreover, r e t r o s p e c t i v e s t u d i e s clinical after
its birth.
defects of
age
evaluation
the
rise
the
a shortly
child
i s one
year
The
humans a r e
pre-
These f e a t u r e s thorough of
the
are
than
to
search
post-natal
Stroke,
detect Most
for a pattern
features
such
of
of as
teratogens
growth d e f i c i e n c i e s .
often overlooked.
r e c e n t l y completed
of Neurological
to
defects,
for a single defect
Collaborative Perinatal Project
Institute
past
of multiple
most c o n s i s t e n t and
i n the
i s commonly done
(Hanson e t a l . , 1 9 7 6 ) .
i t i s far better
palate.
failed
to a p a t t e r n
malformations, rather
and
upon
time of or
s i n g l e m a l f o r m a t i o n s , as
m a j o r t e r a t o g e n s i n man
therefore,
the
rely
accurate.
anomalies./ e s p e c i a l l y c a r d i a c
r e t r o s p e c t i v e s t u d i e s , has
cleft
be
fetal
older.
teratogens give
in
i n f a n t a t the
undiagnosed u n t i l
Reporting in
an
Certain
o f t e n , go or
of
generally
the
Even i n the
retrospective of
the
and
Communicative
t h e s e more s u b t l e
anomalies are
most studies,
National Disorders not
reported
(Heinonen e t a l . , 1977).
On by
the
other
hand, p r o s p e c t i v e
investigators specifically
icity
of
anticonvulsant
o f m a l f o r m a t i o n s as h i g h
studies
i n t e r e s t e d i n the
d r u g s have r e p o r t e d as
undertaken
35.7%
i n the
a
teratogen-
frequency
o f f s p r i n g of
treated
- 22 -
epileptics et;.al.,
1976).
frequency these
(Mirkin,
1971; Hill.;et
a l . , 1 9 7 4 ; Hanson
This i s a fourfold
d e r i v e d from
i n c r e a s e over the
the retrospective
studies.
f i g u r e s may be q u e s t i o n e d , as.:they m i g h t be e l e v a t e d
s i n c e t h e s e p h y s i c i a n s a r e among t h e p r i n c i p a l of the concept
of the f e t a l
The d i f f e r e n c e epidemiological drawn
from
collected
hydantoin
proponents
syndrome.
between p r o s p e c t i v e and r e t r o s p e c t i v e
techniques
can lead
t h e same d a t a s o u r c e .
S h a p i r o and c o l l e a g u e s
et
Again
to conflicting conclusions
F o r example, t h e study o f
( 1 9 7 6 ) was b a s e d
upon case" h i s t o r i e s
i n the C o l l a b o r a t i v e P e r i n a t a l P r o j e c t .
a l . c l a i m e d t o have f o u n d
malformations
ho e v i d e n c e
amongst o f f s p r i n g
that
of treated
Shapiro
t h e major
epileptic
m o t h e r s were a s s o c i a t e d w i t h a n t i c o n v u l s a n t t h e r a p y . concluded
that
or the father,
i t was p a r e n t a l e p i l e p s y , e i t h e r
Perinatal Project
c o n t r o l mothers i n t h e i r variability
(Hanson e t a l . ,
Hanson e t a l . ( 1 9 7 6 ) a l s o usedt'the
of the C o l l a b o r a t i v e
and
study
of the f e t a l
t o determine
hydantoin
hydantoin
ative Project, diagnoses born
1976;
resources
t o o b t a i n 104 matchedthe frequency
syndrome.
many o f t h e d y s m o r p h i c f e a t u r e s c h a r a c t e r i s t i c fetal
t h e mother
t h a t was r e s p o n s i b l e , :.ahd n o t t h e m e d i c a t i o n .
T h i s c o n c l u s i o n h a s been r e p u d i a t e d Dansky, 1 9 7 7 ) .
They
Though
of the
syndrome were n o t e v a l u a t e d i n t h e Col-labor:-.:: enough e v i d e n c e was a v a i l a b l e
f o rconfident
o f t h e syndrome i n 1 1 o f t h e 1 0 4 c h i l d r e n ( 1 1 % )
to hydantoin
t r e a t e d mothers.
- 23 Hanson e t a l . (1976) a l s o e p i l e p t i c women on h y d a n t o i n d r u g pregnancies. toin
They b a s e d
their
syndrome on t h e p r e s e n c e
features
f o l l o w e d 23 S e a t t l e t h e r a p y - t h r o u g h 35
diagnosis of the f e t a l of at least
t h e y c o n s i d e r t o be d e f i n i t i v e
These a r e p r e n a t a l growth d e f i c i e n c y , deficiency, microcephaly basis,
they found
and m e n t a l
four c h i l d r e n
syndrome, a n d an a d d i t i o n a l
The d i a g n o s i s o f t h e f e t a l
offspring in
the r e s u l t s o f t h e S e a t t l e for
from
clearly
with the f e t a l (31%)
with
hydantoin
anomalies
(Hanson e t a l . ,
h y d a n t o i n syndrome i n t h e during
pregnancy
P r o j e c t was c o m p a r a b l e
area study.
The r i s k
separate the ^ t e r a t o g e n i c
that of the disorder,
w o u l d be r e q u i r e d . not r e c e i v i n g severity vulsant tic
On t h i s
with
figure
t h e syndrome was 11%.
To drug
growth
t o hydantoins
o f women . r e c e i v i n g h y d a n t o i n s
the Collaborative Perinatal
o f t h e syndrome.
retardation.
11 c h i l d r e n
hydan-
three of the four
postnatal
(11%)
c o n s i s t e n t w i t h p r e n a t a l exposure 197 6 ) .
area
effect
df the
a large p r o s p e c t i v e study
T h i s s t u d y must i n c l u d e
e p i l e p t i c women .
a n t i c o n v u l s a n t ..drugs, b u t w i t h t h e same
of the disorder therapy.
a s t h o s e women r e c e i v i n g a n t i c o n -
An a d d i t i o n a l
control
group
of non-epilep-
women i s n e e d e d , f o r t h e i n c r e a s e d r i s k a s s o c i a t e d . c a n
o n l y be d e t e r m i n e d c h i l d r e n born
by comparing
the malformation
rates i n
t o u n t r e a t e d e p i l e p t i c women t o t h o s e o f t h e
non-epileptic population. women.not r e c e i v i n g
To make a g r o u p
a n t i c o n v u l s a n t s would
of epileptic imply
withdrawing
- 24 -
the m e d i c a t i o n from them. considerations.
T h i s would r a i s e e t h i c a l
On t h e o t h e r hand, does n o t t h e p r e s c r i p t i o n
of a suspective teratogen
t o a pregnant woman r a i s e i t s own
s e r i e s of e t h i c a l questions?
Therefore,
an a n i m a l model
t h a t c l o s e l y ^ p a r a l l e l s t h e human s i t u a t i o n c o u l d
circumvent
t h i s dilemma and o t h e r such problems i n h e r e n t i n r e s e a r c h on human p o p u l a t i o n s .
5.
PURPOSE AND RATIONALE OF THE PRESENT STUDY E p i l e p t i c women make up a p p r o x i m a t e l y 0.3 t o 0.5% of a l l p r e g n a n c i e s (Janz, 1975).
Data from t h e C o l l a b o r a t i v e
P e r i n a t a l P r o j e c t suggests t h a t 2 p e r 1000 b i r t h s a r e i n f a n t s exposed t o h y d a n t o i n s (Hanson e t a l . , 1976). h a l f a century
Despite
of experience with the anticonvulsant
t e r a t o g e n i c p o t e n t i a l i n man i s s t i l l u n c e r t a i n . s t u d i e s a r e confounded w i t h t h e h e t e r o g e n e i t y t y p e s and c o m b i n a t i o n s o f a n t i c o n v u l s a n t
was
most o f t e n a d m i n i s t e r e d
DPH, i t s
The human
of epilepsy, the
drugs and t h e i r d o s e s ,
and t h e methodology o f p a t i e n t a s c e r t a i n m e n t . have been e q u a l l y u n r e w a r d i n g .
nearly
The a n i m a l s t u d i e s
I n t h e s e s t u d i e s t h e drug
i n s i n g l e doses, o r on s e l e c t e d
days o f g e s t a t i o n i n doses w e l l i n excess o f maximal human t h e r a p e u t i c dose (Massey, 1966; G i b s o n and B e c k e r , 1968; H a r b i s o n and B e c k e r , 1969, 1972; S u l l i v a n and M c E l h a t t o n , 19 75, 1977;
F r i t z e t a l . , 1976).
The r o u t e o f a d m i n i s t r a t i o n : was
most f r e q u e n t l y subcutaneous o r i n t r a p e r i t o n e a l i n j e c t i o n or g a s t r i c i n t u b a t i o n .
W h i l e such p r o c e d u r e s produced
- 25
-
the a n t i c i p a t e d malformations,
i t may
a n i m a l enough so as t o c o n f o u n d
the
traumatize the results.
The'.inherent i n a d e q u a c i e s o f b o t h studies effect o f an
and and
the complexity of the r e l a t i o n the maternal metabolism
animal model.
spectrum
Should
of malformations
syndrome, i t w o u l d be fundamental
invaluable
clinical
of
population
the e p i l e p t i c
a malformed
A valid
of
as t h e f e t a l
The
who
a n i m a l model."of t h e
t h e same hydantoin questions
that
increased
well segments
risk
f e t a l hydantoin
a n i m a l must h a v e s p o n t a n e o u s
could
as
as
of
syndrome
criteria:
the p o s s i b l e b i o c h e m i c a l v a r i a n t s
condition
drug
development
of d e l i n e a t i n g those
a r e a t an
human
child.
s h o u l d meet'".the f o l l o w i n g 1.
j u s t i f i e s the
i n answering
problem
and
between t h e
as t h e a c t u a l e t i o l o g y o f t h e d e f e c t s ,
as t h e i m p o r t a n t
producing
the animal
t h i s model p r o d u c e
defined
pregnant
influence
specific
t h e r o u t e and
seizures
because
to the
epileptic
rate of
DPH
metabolism. 2.
The
eliminatedr.by 3. epileptics t h a n by
DPH
The
s e i z u r e s must be
c o n t r o l l e d or
treatment.
d r u g must me
take D i l a n t i n
administered o r a l l y .
more o f t e n
as a p i l l
Human
o r as a
syrup
injection. 4.
within
spontaneous
Serum l e v e l s
must be m o n i t o r e d
t h e o p t i m a l human t h e r a p e u t i c
range
and
adjusted to
between 5 and
fall 20
-
26 -
micrograms p e r ml serum ( M i l e s e t a l . , 1976; Jeavons,
1977).
As t h e r e i s nol;constaht r a t i o between t h e amount o f DPH admini s t e r e d and t h e serum c o n c e n t r a t i o n , i t i s i m p o r t a n t t o d e t e r mine t h e b l o o d serum l e v e l s o f t h e d r u g . ( J a n z , 1 9 7 5 ) . 5. The DPH must be a d m i n i s t e r e d t o t h e a n i m a l b e f o r e mating and t r e a t m e n t must be c o n t i n u e d throughout
gestation.
S t u d i e s on human e p i l e p t i c s have shown t h a t when d a i l y t h e r a p e u t i c doses a r e g i v e n o r a l l y , t h e drug accumulates
and t h e
serum c o n c e n t r a t i o n i n c r e a s e s s l o w l y u n t i l a s t a b l e l e v e l i s reached when i n t a k e and e l i m i n a t i o n a r e e q u a l , ( B u c h t h a l and Lennox-Buchthal,
1972).
I t g e n e r a l l y t a k e s 5 t o 7 days a t low
doses, and somewhat l o n g e r a t h i g h e r doses t o . a c h i e v e t h e o p t i m a l t h e r a p e u t i c range 19 69).
( P l a a , 1975; Harbson and B e c k e r ,
T h i s time p e r i o d a l l o w s f o r t h e i n d u c t i o n o f enzyme
systems a s s o c i a t e d w i t h DPH metabolism t h a t cause t h e c h a r a c t e r i s t i c f a l l i n serum IgA and f o l i c a c i d l e v e l s r e p o r t e d i n c h r o n i c DPH use ( J a n z , 1975). 6.
The model r e q u i r e s t h a t t h e o f f s p r i n g o f t h e
t r e a t e d a n i m a l s must e x h i b i t those m a l f o r m a t i o n s observed i n t h e o f f s p r i n g o f e p i l e p t i c women on h y d a n t o i n t h e r a p y .
Further,
t h e frequency o f t h e m a l f o r m a t i o n must be s i g n i f i c a n t l y h i g h e r than t h e i r spontaneous o c c u r r e n c e . 7.
A d o s e - r e s p o n s i v e curve from 0 t o 100% a f f e c t e d
o f f s p r i n g , c h a r a c t e r i s t i c o f t e r a t o g e n i c a g e n t s , s h o u l d be observed„(Wilson, 1965).
An a n i m a l model c l o s e l y a p p r o x i m a t i n g t h e human s i t u a t i o n
- 27 by m e e t i n g insight
the aforementioned
into
criteria,
the t e r a t o g e n i c p o t e n t i a l
should of
DPH.
provide
CHAPTER I I METHODOLOGY
ANIMALS C 5 7 B 1 / 6 J , +/qk m i c e
(Mus m u s c u l u s ) w e r e o b t a i n e d
from t h e
Jackson
L a b o r a t o r i e s , B a r Harbor, Maine and housed w i t h t h e
Medical
Genetics
University lines
of British
i n their
project.
were housed
females
i n pairs
light
Purina
i n t h e Zoology From t h e s e
line,
line
Vivarium,
animals
C 5 7 B 1 / 6 J , +/+,
fourth generation
C57B1/6J, qk/qk mice,
hour
One
The o t h e r
animals
colony
Columbia.
were m a i n t a i n e d .
mice were this
breeding
two
breeding
the control
a t t h e commencement o f
c o n s i s t e d o f C 5 7 B 1 / 6 J , +/qk
also i n their
and
fourth generation. A l l
i n standard
clear
polycarbonate
cages,
and males w i t h
sibs,
and m a i n t a i n e d
o n a 12
cycle.
They were a l l o w e d
ad l i b i t u m
l a b o r a t o r y chow and t a p w a t e r u n l e s s
access
otherwise
to indicated.
DPH TREATMENT L E V E L S Drug
treatment
Sigma Chemicals, levels, may
1972) 20
5,5-Diphenylhydantoin
S t . L o u i s , Mo.)
t o 30 t i m e s
to mice
activity
higher
levels
fall
( B u c h t h a l , and
(DPH';
i n four
dose
these
levels
human
thera-
Lennox-Buchthal,
o f t h e mouse i n o x i d i z i n g
(Brodie e t a l . , 1958).
i n human d o s a g e s ,
s e r u m DPH
While
i n comparison t o the standard
d o s e o f 3.5 t o 10 m g / k g the metabolic
sodium
was a d m i n i s t e r e d
0, 2 0 , 40 a n d 60 m g / k g b o d y w e i g h t .
seem e x c e s s i v e
peutic
with
When DPH
i t i s e l i m i n a t e d so r a p i d l y
below t h e s p e c i f i c i t y
drugs i s i s given that the
o f t h e SYVA
Emit
spectrophotometric
assay t e c h n i q u e
(SYVA, P a l o A l t o , C a l . )
used t o measure serum DPH l e v e l s t h r o u g h o u t t h e study.
The
lowest dose g i v e n t o t h e dams t h a t produced measurable serum DPH l e v e l s was 20 mg/kg.
The h i g h e s t dose l e v e l t h a t c o u l d be g i v e n t o t h e dams w i t h o u t c a u s i n g severe t o x i c i t y was 60 mg/kg.
A t t h i s dose
the m a j o r i t y o f females were f r e e o f any c l i n i c a l
manifesta-
t i o n s o f DPH i n t o x i c a t i o n , though some were l e t h a r g i c , i n g a m i l d t o moderate degree o f t o x i c i t y .
indicat-
An i n t e r m e d i a t e
dose o f 40 mg/kg was i n c l u d e d t o comply w i t h t h e World H e a l t h Organization guidelines f o r teratogen t e s t i n g
( W i l s o n , 1967).
CALCULATION OF DRUG DOSES A s t o c k s o l u t i o n o f DPH c o n s i s t i n g o f 1 mg DPH sodium t o 1 ml t a p water was made f r e s h d a i l y . out t o one t e n - t h o u s a n d t h
The d r u g was measured
o f a gram i n a S a r t o r i o u s s i n g l e
beam b a l a n c e and was then t r a n s f e r r e d t o Erlenmeyer F l a s k s . A f t e r t h e a d d i t i o n o f an a p p r o p r i a t e q u a n t i t y o f w a t e r , t h e con tents o f t h e f l a s k were r e p e a t e d l y a s p i r a t e d by a p a s t e u r p i p e t t t o ensure t h a t t h e drug had c o m p l e t e l y d i s s o l v e d i n t o
solution.
The d e s i r e d q u a n t i t y o f s t o c k s o l u t i o n was measured o u t w i t h a d i s p o s a b l e 10 ml s e r o l o g i c a l p i p e t t e , and was then t o a t o t a l o f 50 m l .
diluted
F o r those experiments i n v o l v i n g non-
pregnant mice t h e d r i n k i n g s o l u t i o n was a d m i n i s t e r e d i n an 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 w i t h t h e needle end s e a l e d
by m e l t i n g , w i t h s t a n d a r d g l a s s d r i n k i n g openings. daily
For the l a t e r
experiments
o f 1.5
with pregnant
centrifuge
The
tubes w i t h a standard g l a s s d r i n k i n g
by c a l c u l a t i n g
the animals' weight,
c o n s u m p t i o n , and t h e t r e a t m e n t Conversion tions.
level
t a b l e s were e s t a b l i s h e d
The f i r s t
the d e s i r e d dosage.
daily.
The s e c o n d
f o r the animals
1
(Appendix
conversion table
(Appendix
t o consume 50 ml o f these
DIET
e x p l a n a t i o n o f t h e usage o f t h e s e t a b l e s
i n Appendix
calcula-
receiving
t h e amount o f s t o c k s o l u t i o n r e q u i r e d f o r a g i v e n
found
B)
liquid tables cage.
c a n be
C.
ADMINISTRATION Virgin
females
60 t o 90 d a y s o l d were u s e d
p h a r m a c o l o g i c a l s t u d i e s on n o n - p r e g n a n t f e m a l e s teratological
studies.
o f DPH have b e e n w e l l
Although
d e s c r i b e d i n b o t h man
to characterize
and t h e
and mouse
fundamental
(Gerber
1 9 7 0 ; K u t t , 1 9 7 1 ; Gerber
L y n n , 1 9 7 2 ; F r e y and Kampmann, 19 6 5 ; G l a z k o ,
necessary
i n both the
the pharmacological p r o p e r t i e s
and A r n o l d , 1 9 6 9 ; K u t t and V e r e b e l y , and
rapid
A) g i v e s t h e amount
The p r o d u c t o f t h e v a l u e s o b t a i n e d f r o m
A detailed
water
i t was t o r e c e i v e .
must consume t o be
failure
tube.
c a g e was
average
to f a c i l i t a t e
conversion table
o f DPH a mouse o f a g i v e n w e i g h t
corrects
conical
amount o f s t o c k s o l u t i o n r e q u i r e d f o r e a c h
determined
mm
dams, t h e
DPH w a t e r s o l u t i o n was p l a c e d i n F a l c o n 50 ml
graduated
is
tubes
drug
19 75) i t was
reactions i n strain
C57B1/6J p r i o r
to the start
of the t e r a t o l o g i c a l
For a l l s t u d i e s
t h e m i c e were g r a d u a l l y
to avoid weight
loss,
have b e e n
reported
( F r e y and Kampmann,
introduced
s i c k n e s s and r e f u s a l
i n mice
given
to drink,
l a r g e doses
which
(0.3 mg/ml) o f DPH
a dose-response curve, the various
d o s e s o f DPH were a d m i n i s t e r e d t o n o n - p r e g n a n t The m i c e
started
was d e t e r m i n e d .
mice
over a
o u t on t r e a t m e n t l e v e l
(0 mg/kg) f o r s e v e n d a y s , d u r i n g w h i c h consumption
t o t h e drug
1965).
In o r d e r t o c o n s t r u c t
28 d a y p e r i o d .
experiments.
their
baseline
They were t h e n e l e v a t e d
1
water i n a step-
wise
fashion through the remaining three treatment l e v e l s .
mice
remained
a t each
level
f o r seven days which
t i m e f o r them t o become t o l e r a n t 1965) (Plaa,
and f o r t h e i r 1975).
plasma
The i n i t i a l
of a stable blood l e v e l and o t h e r c l i n i c a l l y
The assigned
dams u s e d
DPH c o n c e n t r a t i o n
test
a l l o w e d ample
( F r e y and Kampmann, t o reach a plateau
accumulation followed
by m a i n t e n a n c e
i s an i m p o r t a n t c h a r a c t e r i s t i c
proven anticonvulsants
i n the teratology
t o treatment l e v e l s
a screening
t o the drug
(Kutt,
s t u d y were
1, 3 o r 4.
The
o f DPH
1971).
randomly
As t h i s was p r i m a r i l y
t o s e e what p o s s i b l e m a l f o r m a t i o n s and t h e i r
f r e q u e n c y may o c c u r i n c h r o n i c a l l y h i g h e s t d o s e s were u s e d .
t r e a t e d mice, o n l y the
The m i c e were s t a r t e d
o u t on l e v e l 1
(0 mg/kg) a n d e v e r y s e v e n t h d a y t h e y p r o c e e d e d t o t h e n e x t h i g h e r dose, u n t i l
t h e p r e - d e t e r m i n e d number o f f e m a l e s were
- 32 m a i n t a i n e d on t h e i r respective first
5.
respective
treatment l e v e l
dosages.
A l l dams were on
f o r a t l e a s t 14 d a y s p r i o r t o t h e
attempt a t mating.
SEIZURE CONTROL
STUDY
A s t u d y o f 12 C57 B 1 / 6 J ,
qk/qk f e m a l e m i c e was c o n d u c t e d
to determine the e f f e c t i v e n e s s o f o r a l l y controlling allowed
their
on l e v e l
seizures
r e c o r d e d i n terms
4 days.
period, slowly
(Sidman
A t 15 m i n u t e
d o s e o f DPH.
frequency of
p e r mouse d a y was
e t a l . , 1965).
i n t e r v a l s during lifted 1973).
clonic-tonic seizures removed u n t i l
turning
their
i n p a i r s and
During each
t h e m i c e were o b s e r v e d f o r one h o u r on a t
turned 180° ( G o l d s t e i n ,
had been
after
of seizures
t h e a n i m a l s were g e n t l y
exhibited lid
containing
1 (0 mg/kg), a b a c k g r o u n d
f o r e a c h mouse
seven day p e r i o d ,
a d m i n i s t e r e d DPH i n
The m i c e were h o u s e d
f r e e access t o water
ascertained
6.
seizures.
Starting
least
their
the
up by t h e i r Those
observation tails
animals
that
from t h e time t h e w i r e
t h e t i m e t h e l i d h a d been
and
cage
replaced
t h e m i c e , were r e c o r d e d a s h a v i n g h a d a s e i z u r e .
DETERMINATION OF SERUM DPH LEVELS To d e t e r m i n e d o s e - r e s p o n s e c u r v e s f o r t h e f o u r ment l e v e l s , sinus
b l o o d samples
were drawn f r o m t h e r e t r o - o r b i t a l
a f t e r e a c h s e v e n day p e r i o d
To m a x i m i z e circadian
DPH t r e a t -
possible variability
on a g i v e n
treatment
level.
i n b l o o d DPH l e v e l s due t o
f l u c t u a t i o n s t h e samples
were c o l l e c t e d o v e r a 14
hour
period,
9 AM
Using imately
0.5
-
33
-
t o 11 PM
on
t h e day
ml
o f b l o o d was
i n p l a s t i c micro
a Sorvall
Superspeed
4°C
was
microsampling
Alto,
pipettes
of the
The
centrifuge
tubes
tubes.
SYVA E m i t
At
transferred
used
to determine
serum DPH
w i t h t h e enzyme
in this
c a s e DPH,
!(G-6PDH) .
drug-enzyme complex and
plastic
a t 4°C
t h e a n t i b o d y and
An,'antibody
immunoassay concentrations
involves
labeling
glucose-6-phosphate
t o the drug binds t o
b l o c k s t h e enzyme's a c t i v e
t h e enzyme a c t i v i t y
enzyme a c t i v i t y
c o n v e r t s NAD
site.
is directly
related
(DPH)! p r e s e n t i n t h e s a m p l e . t o NADH r e s u l t i n g
i n an
i s reduced. to the
The
absorbance
al.,
1977) .
the The
drug The
concentra-
active
enzyme
change
i s m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y (Anonymous, 1975; et
until
"(SYVA, P a l o ,
i n t h e serum competes w i t h t h e e n z y m e - l a b e l e d
of drug
serum
glass disposable
spectrophotometric assay
a drug,
residual
was
centrifuge
50yul of
T h e s e were r e f r i g e r a t e d
This technique
f r e e drug
blood
spun down i n
again to the
( B a s t i a n i e t a l . , 1973).
tion
least
C a l . ) , w h i c h i s a homogeneous enzyme
dehydrogenase
and
refrigerated
a t 3,000 g.
and
The
approx-
assay.
t e c h n i q u e , was
for
time.
s u c t i o n w i t h C o r n i n g 100yul
centrifuge
t h e day
drawn a t e a c h
RC2-B a u t o m a t i c
f o r e i g h t minutes
removed by
micro
blood-sampling.
a h e p a r i n i z e d Dade m i c r o h e m a t o c r i t t u b e ,
collected
at
of
that
Schmidt
The
assay procedure
50 u l sample w i t h in
i n v o l v e s a 1:12 d i l u t i o n
0.055 M T r i s
of the
hydrochloride buffer,
two s u c c e s s i v e s t e p s w i t h a SYVA
aspirated system
into
and t h e r m a l
printer
buffer
a r e mixed.
regulated flow c e l l
the spectrophotometer
i s automatically activated
absorbance
The
s e t a t 30°C.
experiment average
duplicate
Once t h e
t h e Beckman 3115T
and t h e d i f f e r e n c e i n
As t h e sample i s d i l u t e d ,
measurements c a n be r u n f o r e v e r y this
with a sipper
a t 340 nm b e t w e e n 15 and 90 s e c o n d s
i s recorded.
labeled
The sample i s
a Beckman 2 4 s p e c t r o p h o t o m e t e r
sample i s i n s i d e
tion
of Tris
and r e a g e n t B, c o n t a i n i n g 5 0 y a l o f t h e G-6PDH
DPH p l u s 250 u l o f T r i s
8.1
pipetter-diluter.
R e a g e n t A, c o n t a i n i n g 5 0 y u l o f a n t i b o d y p l u s 2 5 0 y u l buffer
pH
sample.
aspira-
up t o f i v e
repeat
For the purposes
r u n s were p e r f o r m e d
o f t h e two v a l u e s was t a k e n
after
of
on a l l samples.
t o be t h e serum
concen-
t r a t i o n o f DPH i n any s a m p l e .
In t h e t e r a t o l o g i c a l a l s o drawn, p r o c e s s e d ,
and a s s a y e d
to the death
of the animal.
EXPERIMENTAL
DESIGN
a.
Experimental The
criteria syndrome.
study maternal
blood
s a m p l e s were
i n t h i s manner j u s t
prior
Organism
e x p e r i m e n t a l p r o t o c o l was d e s i g n e d established The f i r s t
t o comply w i t h t h e
f o r an a n i m a l model o f t h e f e t a l requirement
called
hydantoin
f o r t h e u s e o f ah
- 35-organism
with
a spontaneous
t h e mouse m u t a n t q u a k i n g recurring selected
(Sidman
et al.,
(+/+) dams w e r e a l s o
b.
Route
o f Drug
started
two weeks p r i o r
of the quaking were
non-quak-
as a s a l t
The w a t e r
intake
throughout
gestation.
attempt
o f each The d r u g
c a g e was therapy
a t mating and
S e r u m c o n c e n t r a t i o n s o f DPH
b y means o f t h e SYVA E m i t
human t h e r a p e u t i c
suspension i n
serum l e v e l s
spectrophotometry
t h a t were w i t h i n t h e
range.
Matings
males
types.
matings
were i n i t i a t e d
i n t h e cages
The mice
a f t e r which and
(0, 40,
i n the study.
to the f i r s t
technique t o ensure
The +/+
included
t o m a i n t a i n t h e proper dosage.
were determined
c.
genotypes
o f t h e m a l f o r m a t i o n s , dams t h a t
water.
daily
optimal
the role
DPH w a s a d m i n i s t e r e d o r a l l y
checked
assay
of three
Administration
animal's drinking
continued
a n i m a l s , was
Female mice
To d e f i n e
reason
by s p o n t a n e o u s l y
(+/qk) f o r t h e m u t a n t g e n e a n d h o m o z y g o u s
ing
The
(qk/qk)
For this
a s s i g n e d t o one o f t h r e e t r e a t m e n t groups
(qk) i n t h e e t i o l o g y
heterozygous
the
1965).
60 m g / k g b o d y w e i g h t ) .
gene
disorder.
(qk), characterized
c o n v u l s i o n s i n homozygous
were randomly and
seizure
time
of paired
by p l a c i n g females
single
of a l l three
were a l l o w e d two t o t h r e e hours t h e males were r e t u r n e d t o t h e i r
t h e females were examined.
C57 B 1 / 6 J
The p r e s e n c e
of a
geno-
t o mate, own
cages
vaginal
p l u g was
t a k e n i n d i c a t i n g g e s t a t i o n a l day
continued On
day
18,
orbital
d.
t o be
treated with
sinus
and
killed
l o c a t i o n and
resorption severing
after cervical o p e n e d and
gestation.
from the
retro-
the
were r e g a r d e d
statistical the
a i d of
the
red
The
contents fetuses
were c h e c k e d that
failed
born
immediately
the
staining
to
show
limbs
and
1962:
i n 95%
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 u s i n g
been s t a i n e d
dishes
filled
of Wilson
Taylor,
for skeletal
with
g e l a t i n and
(Wilson,
1969).
of
the mal-
ETOH
A p p e n d i x D).' and
i n Bouin'. s s o l u t i o n .
for
digits.
g r o u p was
had
colour,
ten-thousanth
two-thirds
(Barrow and
in
were w e i g h e d
remaining
modification
any
examined f o r e x t e r n a l
trunk,
(Crary,
hand r a z o r b l a d e t e c h n i q u e s
or
resorptions
fetuses
f e t u s e s were p l a c e d
placed
by
d i s s e c t i n g microscope,
head, p a l a t e , of
and
f o r limb
amongst t h e
live
a Wild
removed.
t h a t were p a l e
s i n g l e beam b a l a n c e t o one
Randomly, o n e - t h i r d alizarin
born
maternal
f e t u s e s were removed
included
analysis.
f e t u s e s were s e x e d and of
uterine
movements, o r
dead and
With the
formations
and
Those f e t u s e s
Sartorious
a gram.
the
The
u m b i l i c a l cords
as
d i s l o c a t i o n , the
a l l live
s i t e s were n o t e d .
spontaneous or e l i c i t e d
for
dams
cervical dislocation.
p o s i t i o n of
mouth movements.
on
by
The
F e t a l Examination
a b d o m i n a l c a v i t y was
the
throughout
t h e m i c e were w e i g h e d , b l e d
Immediately
The
DPH
1.
This
the latter
the
1965)
and
Those f e t u s e s
e x a m i n a t i o n were p l a c e d
freeits
that in
petri
examined under a d i s s e c t i n g
microscope.
The
soft tissue sections
a d i s s e c t i n g microscope, slices
i n 70%
by p l a c i n g
were a l s o
t h e 1-2
ETOH i n w h i t e p o r c e l a i n
spot
mm
examined cross
with
section
plates.
S T A T I S T I C A L ANALYSIS The
0.05
analysis.
The
using
level
o f s i g n i f i c a n c e was
mean d i f f e r e n c e s
a one-way a n a l y s i s
multiple
range
significance descriptive throughout programable calculator.
t e s t was
(Bliss,
t o be
utilized
1967).
statistics
between measurements were
of variance
Where d i f f e r e n c e s were f o u n d
used
(Sokal
& Rohlf,
significant,
to discern
i n the s e i z u r e
tested
1969).
a Duncan's
the source of
A l l calculations including
t h e t e x t were p e r f o r m e d desk
set for a l l s t a t i s t i c a l
control
new the
the
study
and
on e i t h e r a Wang 3000
computer o r a T e x a s
Instruments
Sr-50
hand
-
38
-
CHAPTER I I I
RESULTS
1.
SEIZURE CONTROL The
criteria
hydantoin exhibit by
DPH
established
syndrome r e q u i r e d
spontaneous treatment
(qk/qk)
the e x p e r i m e n t a l organism
seizures
that
(see Page 27).
On
are c o n t r o l l e d the average,
q u a k i n g mutant m i c e were s u b j e c t
s e i z u r e s p e r mouse day (Table
f o r an a n i m a l m o d e l o f t h e
3).
This
and
the f i r s t
two
to
eliminated
homozygous
t o more t h a n
t h e s e were c o n t r o l l e d
satisfied
or
fetal
by
two
DPH
requirements of
the
mouse m o d e l .
2.
SERUM LEVELS When t h e d r u g was period
t o non-pregnant
administered o r a l l y
females of a l l t h r e e genotypes,
serum c o n c e n t r a t i o n s o f DPH ing
d o s e was
l e v e l s were r e c o r d e d . considered desirable the r e s u l t
of rapid
h i g h e r doses
These
serum
with
the t h i r d
and
and
i n serum l e v e l s
(Miles et a l . ,
range
1976;
increas-
serum
serum l e v e l s were f a r b e l o w
drug o x i d a t i o n
o p t i m a l human t h e r a p e u t i c
the
When t h e
a d m i n i s t e r e d , v e r y low
f o r most human e p i l e p t i c s ,
resulted
day
were f o u n d t o i n c r e a s e w i t h
amounts o f t h e d r u g a d m i n i s t e r e d ( T a b l e 4 ) .
20 mg/kg body w e i g h t
of
f o r a seven
and
elimination. that
fall
could
be
The
two
within
the
o f 5 t o 20 m i c r o g r a m s p e r
Jeavons,
1977).
This
f o u r t h requirements of the animal
those
ml
complied model.
-
Table
3.
39 -
E f f i c a c y o f DPH O r a l l y t o Q u a k i n g (qk/qk) (n=12
Administered Mice
females f o r each treatment)
DPH Treatment (mg/kg body w e i g h t )
Seizures* mean + SEM 2.06 + .74
20
1.96 + .98
40
0.77 + .72
60
0.34 + .41
*expressed
as s e i z u r e s p e r mouse d a y
Table
4.
DPH Serum C o n c e n t r a t i o n s
i n Non-pregnant
DPH Treatment
Genotype
Serum
(mg/kg body w e i g h t )
+/+ (n=37 f e m a l e s f o r each treatment)
0
(n=21 f e m a l e s f o r each treatment)
qk/qk
(n=17 f e m a l e s f o r each treatment)
* e x p r e s s e d a s y u g p e r ml
Concentration* mean + SEM
0
20
2.06 + .15
40
5.18 +
60 +/qk
Females.
.27**
12.06 + .56**
0
0
20
1.75 + .13
40
4.59 + .39
60
10.15 +
0
0
.75**
20
2.19 + .27
40
6.29 + .62**
60
10.59 + .83**
serum
** w i t h i n o p t i m a l human t h e r a p e u t i c
range
T a b l e 5 shows t h e e f f e c t on w a t e r The
intake
and
average water
not s i g n i f i c a n t l y
serum
DPH
o f v a r y i n g dose concentrations
levels
of
i n p r e g n a n t dams.
i n t a k e p e r mouse o v e r a s e v e n day p e r i o d different
E f o r ANOVA t a b l e s ) .
amongst t h e g e n o t y p e s
The m a t e r n a l serum
levels
of
DPH
levels
c o r r e s p o n d i n g dose i n non-pregnant
( T a b l e 4 ) , an
observation reported
i n rats
females
(Westmoreland
statistical
IMPLANTATIONS AND The is
and dose
changes
still
level
o f DPH
figure
f o r implants
b i r t h s p l u s any v i s i b l e
changes
resorption
DPH
was
increased.
and dams who
respectively).
t h e h i g h e s t d o s e o f DPH
very
sites.
As
t h e r e were
resorbed
no
as t h e
difference
between
40 mg/kg body w e i g h t
Quaking mice
had no v i s i b l e
any o f f s p r i n g
did
gain weight during
pregnancy.
may
be r e l a t e d
to maternal i n t o x i c a t i o n
(qk/qk)
resorption
t h e s e two dams d i d n o t c a r r y early
resorption
I n t h e homozygous
little
received
and
significant
/Similarly,
i n t h e number o f f e t u s e s
(qk/qk) m i c e , t h e r e was
22%,
any
i s b a s e d upon a l l
t h e r e were no
i n t h e number o f i m p l a n t s .
dams n o t on DPH (24% and
t r e a t m e n t on i m p l a n t a t i o n
The
of administered
quaking
as t o p r e c l u d e
RESORPTIONS
l e v e l s were i n c r e a s e d ,
significant
1971).
analysis.
shown i n T a b l e 6.
live DPH
effect
found a t a
and B a s s ,
However, t h e d a t a a v a i l a b l e were so l i m i t e d
was
(see A p p e n d i x
( T a b l e 5) a p p e a r t o be e l e v a t e d o v e r serum
valid
DPH
receiving sites
and
t o term, though they
This
extreme
at this
high
effect dosage,
Table 5.
Genotypes
E f f e c t o f Treatments on Water Intake and Serum DPH C o n c e n t r a t i o n s on Pregnant Females,
Number of Females
DPH Treatment (mg/kg body wt.)
qk/qk
mean + SEM
Serum DPH** mean + SEM
10.45 + .29
+/+
+/qk
Daily Water Intake*
4
40
10.25 + .57
9.78 + .86
2
60
10.11 + .33
11.60 + .57
1
0
10.39 + .68
1
40
11.21 + .59
11.20 + .0
1
60
10.51 + .63
20.00 + .0
2
0
9.49 + .61
3
40
10.75 + .61
7.75 + 1.97
2
60
11.27 + .53
***
*p:40.05 L e v e l o f s i g n i f i c a n c e **expressed asyug per ml serum ***blood samples hemolyzed
0
0
Table
6.
Genotypes
Effect
of
o f DPH T r e a t m e n t o n I m p l a n t a t i o n
Number Females
DPH Treatment
+/qk
qk/qk
Resorption
Number Implants
Average Implants* •'mean
(mg/kg b o d y w t . )
+/+
and
+
SEM:
Resorptions Number* mean
+
SEM
8.0
+ .93
1.2
+ .54
16
25
6.2
+ .74
1.8
+ .19
28
60
18
9.0
+ .0
1.0
+ .0
11
1
0
5
5.0
+ .0
1.0
+ .0
20
1
40
8
8.0
+ .0
2.0
+ .0
25
1
60
9
9.0
+ .0
3.0
+ .0
33
2
0
17'
8.5
+ . 35
2.0
+ .0
24
3
40
32
2.3
+ .98
22
2
60
(-
4
0
32
4
40
2
*p
