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Teratogenic Effects of Vigabatrin

i n T O Mo u s e F e t u s e sYOUSEF M. ABDULRAZZAQ,1 SALIM M.A. BASTAKI,2 AN D R. PADMANABHAN 3*1Departm ent of Pediatrics, Faculty of Medicine and H ealth S ciences, UAE University, Al A in, Un ited Arab E m irates2Departm ent of Pharm acology, Faculty of Medicine and Health S ciences, UAE University, Al Ain, U nited A rab Em irates3Departm ent of Anatomy, Faculty of Medicine and Health S ciences, UAE University, Al Ain , United Arab Em irates

ABSTRACT Vigabatrin (VGB) is a relativelyrecently introduced antiepileptic drug that enhances thebrain levels of gamma aminobutyric acid (GABA). Fewdata on its teratogenic effects appear to have beenreported. Our objective was to determine if VGB wasteratogenic in the TOmouse. Single doses of 300600

mg/ kg of VGB dissolved in saline were administeredintraperitoneally (IP) to groups of TO mice on one ofgestation days (GD) 712. The controls were salinetreated or untreated. No maternal toxic effects wereobserved in the 300 or 450 mg/ kg groups, and the600 mg/ kg dose was totally lethal to the mothers.Fetuses were collected on GD 18. Both 300 and 450mg/ kg doses induced a consistently significant intrauter-ine growth retardation irrespective of the developmen-tal stage at administration. VGB did not augment thespontaneous incidence of neural tubedefects character-istic of this strain, but accelerated destruction of thebrain in spontaneous exencephalic embryos. Mandibu-lar and maxillary hypoplasia, arched palate, cleft palate(two cases), limb defects (one case), and exomphaloswere observed in the malformed fetuses. The highincidence of exomphalos appears to be a unique resultof VGB treatment. Alizarin red-S/alcian blue-stainedskeletons revealed hypoplasia of mid facial bones,stage-dependent increase in the frequency of cervicaland lumbar ribs, rib fusion, and sternal and vertebralmalformations in the drug-treated fetuses. Middle anddistal phalanges of the forepaw and mid phalanges andtarsals of the hindpaw failed to ossify in a significantnumber of experimental fetuses. Homeotic shift interms of presacral vertebral number and a high inci-dence of lumbar and cervical ribs in the treated group

are suggestive of treatment-related alterations in geneexpression. In view of the paucity of human and animaldata on the reproductive toxicologic effects of VGB, theresults of the present study assume particular impor-tance and suggest that VGB should be used in preg-nancy with extreme caution. Teratology 55:165176,1997. r 1997 Wiley-Liss, Inc.

Epilepsy affects millions of people worldwide. It has

been estimat ed tha t a pproximat ely 0.5% of all pregnan -

cies occur in wom en with epileps y (Svigos, 84; Yerb y,

91). Epileptic women demonst ra te a h igher liability to

obstet ric complicat ions tha n n on-epilepticfemales (Mon-

touris et al., 79). Epilepsy in either paren t is reported

t o b e a s s oci a t ed w it h a n i n cr e a s ed r i sk of h a v i n g a

m a l for m e d i n fa n t (S h a p ir o e t a l ., 7 6; R ob er t a n d

Guibaud, 82; J an z, 82; Lindhout et al., 92; Naka ne

and Kaneko, 92). Congenital malforma tions are esti-ma ted t o be more comm on in infant s of epileptic women

treated with antiepileptic drugs (AEDs) than those of

n or m a l con t r o ls or n on -t r e a t e d e p il ep t ic m ot h e r s

(Meadow, 68; Speidel an d Mea dow, 72; Ann egers et a l.,

78; Kalter an d War ka ny, 83; Nak an e et a l., 80; Speidel

an d Meadow, 72; Kan eko et a l., 88). Both ma jor a nd m inor

malf or mations have been attr ibuted to AED t her apy

(Gailey et a l., 88; Koch et a l., 92). Almost all AEDs h ave

been found to induce some form of fetal malformations,

often described to constitute a syndrome named after

the drug in question. The calculated risk of malforma-

tions in fetuses exposed toAED is 710% (Lindhout and

Omtzigt, 94). Specific risk factors include h igh ma ter-

nal dosage of AED, low folate levels, drug polytherapy,and possible seizure occurrence during pregnancy. Epi-

demiological and experimental studies have corrobo-

ra ted t he t era tological effects of AEDs (Miyagawa et al.,

71; Hanson et al., 84; Dalessio, 85; Kelly, 84; Robert

an d Rosa , 83; Lam mer et a l., 87; Meadow, 91; Mar tin ez-

Fr ias, 90; Yerby, 91; Lindh out et al., 92; Na ka ne an d

Kaneko, 92; Finnel, 91; Padma nabh an and Ham eed,

94; Padma na bhan an d Ahmed, 96). One of the ques-

tions that remains yet unanswered is why only a small

per centage of the AED-exposed fetuses is affect ed,

whereas the majority are not malformed. It is possible

that within the population ther e a r e subpopulat i ons

with a genetic pr edisposit ion who, when exposed t o

AEDs at critical stages of development, become mal-for med (Khour y et al ., 92).One way of test ing t hi s

hypoth esis of dru g-sus ceptibility inter actionis to use

experimental animals with a known susceptibility to a

Contr act gran t s ponsor: UAE University.

*Correspondence t o: R. Padman abhan, Departm ent of Anatomy, Fa c-

ulty of Medicine and H ealth Sciences, UAE Universit y, P.O. Box 1766,

Al Ain, United Arab E mirat es.

Received 5 September 1996; accepted 3 March 1997

TERATOLOGY 55:165176 (1997)

r 1997 WILE Y-LISS, IN C.


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certain malformation(or malformations) and to deter-

mine if th e AED exposure would a ugment the liability.

The TO mouse is one such strain with a low frequency

of spontaneous neural tube defects (NTDs). We have

recently shown that valproic acid (VPA), a widely used

AE D , i n de ed i n t er a ct s w it h t h i s s u s ce p t ib il it y a n d

e n h a n ce s t h e N TD i n cid en ce (P a d m a n a bh a n a n dAhmed, 96). The s usceptibility is possibly related to th e

inability of the individual t o produce a dequat ely drug-

metabolizing enzymes that are genetically determined

(Strickler et al., 85). Buehler et al. (90) have ident ified

low levels of epoxide h ydrolase a ctivity in children with

fetal h ydantoin syndrome.

Vigabatrin (VGB)gamma-vinyl-GABAis a newly

introduced AED. It is an enzyme-activated, irreversible

inhibitor of GABA-transaminase which enhances brain

GABAlevels in experimenta l animals an d hum ans . The

increase in GABA levels following VGB treatment has

been shown to inhibit the luteinizing hor mone ( LH)

surge and prevent ovulation (Landa an d Donoso, 87)and to decr ease sexual behavior in th e r a t (Qur eshi et

al., 88). VGB a lso crosses th e h uma n placenta l barr ier

in isolat ed prepa ra tions (Challier et a l., 92). One case

each of diaphra gmatic hern ia (Kramer, 92) and hypo-

spadias (Lindhout a nd Omtzigt, 94) has been reported

following th e us e of VGB during pr egna ncy. Mater na lly

toxic doses of VGB h ave been reported t o induce cleft

palate in the rabbit (Merrel Dow file, cited by Grant and

Hee l, 91; Lindh out a nd O mt zigt, 94). To the bes t of our

knowledge, the teratogenic potential of VGB has not

been studied in sufficient detail. Our objectives in this

study were to find out if VGB is teratogenic in the TO

mouse an d to determ ine if VGB would intera ct with the

NTD liability as VPAdid.

MATERIALS AND METHODS

The TO mice used in t his stu dy were virgin females,

about 6 weeks of age an d a pproximat ely 30 g in weight.

T h ey w er e m a i n t a in e d on a com m e r ci a l l a bor a t or y

chow, tap water was provided ad libitum, an d th ey were

housed in light (12:12 hr light:da rk cycle)- an d temp era -

t u r e (2 2 6 1C)-controlled rooms. They were mat ed

with mal es of the same st ock in the eveni ng, and the

presence of a vaginal plug observed on the following

mor ni ng was taken t o indicate gestat ion day ( GD) 0.

Groups of mice were treated intraperitoneally (IP) with

a single dose of 300, 450, or 600 mg/kg of VGB (Marion

Merrell Dow, Paris, France) dissolved in physiological

saline on one of GD 712. This dose regimen was based

on our previous studies on VPA teratogenicity in this

str ain of mice (Padma nabhan and Ahmed, 96). The

contr ols wer e either untr eat ed or administer ed I P a

single dose of saline proportionate to th e h ighest toler-

ated dose of VGB, namely 450 mg/kg. The total fluid

injected corresponded to the body weight and did not

exceed 0.45 ml. The food and water consumption of all

the animals wer e r ecor ded thr oughout the st udy. Al l

animals were killed on GD 18 by cervical dislocation

and the fetuses were collected by hysterotomy. Total

implantation and r esor ption si tes wer e counted. The

fetuses were cleared of their membranes, blotted dry,

weighed and fixed in 95% ethanol, and subsequentl y

examined for malformations. The internal malforma-tions were observed by a modified meth od of Sterz an d

Lehmann (85) and then clear ed in 0.5% solut i on of

potassium hydroxide, processed for alizarin red-S and/or

alcian blue st aining of the skeleton (Inouye, 76), a nd

observed with a stereodissecting microscope. The con-

tr ol data r epor ted her e come f r om the untr eat ed ani -

mals. A total of 112 experimental and 11 control mice

w er e u s e d i n t h i s s t u d y. D a t a on fe t a l w e ig h t w er e

analyzed by Stu dents t- test and malfor mat i on fr e-

quency was analyzed by chi-squar e test with Yat es

corr ection (Elzey, 87). Significan ce was as sum ed wh en

P , 0.05.

RESULTS

M a t e r n a l e f f e c t s

Administration of single doses of 600 mg/kg of VGB

on one of GD 712 was complet ely leth al to th e mother s.

A total of 30 anima ls died within 23 days of trea tmen t.

Lower doses (300 or 450 mg/kg) caused a mild sedation

which lasted 1 or 2 hr. The food and water consumption

and body weight gain did not differ significantly from

those of the control. For example, the controls and the

higher dose (450 mg/kg VGB group treated on GD 8)

group registered on GD 18 increases of 80% and 76%,

respectively, over th eir initia l weight s.

F e t a l e f f e c t s

As noticed in our ear l ier exper iments, sa line t r eat -

ment did not pr oduce significant fetal al ter at i ons i n

compar ison t o the unt reat ed controls an d ther efore the

con t r ol d a t a r e p or t e d h e r e w er e ob t a in e d fr om u n -

tr eated animals. Ther e was a 6% incidence of embr y-

onic resorption in the control animals (Table 1). The

m od e st i n cr e a s e i n r e s or p t ion ob s er v ed i n t h e 3 00

mg/kg VGB trea tmen t group was not sta tistically signifi-

cant. An increase in the dose of VGB only produced a

significant augmentation in r esor ption i n t he GD 11

and 12 treatm ent groups. At least one or two embryos in

each treated litter appeared markedly growth retarded

(Fig. 1). Reduction in fetal body weight was hi ghly

significant (P , 0.05) in all experimenta l groups, irre-

spective of th e dose or sta ge of VGB trea tm ent (Table 1).

Exencephaly, maxillary-mandibular hypoplasia, arched

palate, cleft palate (two cases), and exomphalos oc-

curred in the VGB-treated litters. A low incidence of

sponta neous exen cephaly (3.03.5%) is cha ra cteristic of

the TO str ain used in this study. The dr ug tr eat ment

w a s n ot a s s ocia t e d w it h a n y a u gm e n ta t ion of t h e

background frequency of exencephaly. In VGB-treated

166 Y.M. ABDULRAZZAQ ET AL.


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exencephaly cases, open sku ll defects an d everted mes-

encephalon were associated with exophthalmia, cata-

r act , l ow set micr otia, and maxi llar y an d mandibular

hypoplasia. In some of them, the exposed brain tissue

h a d d e ge n e r a t ed a n d w a s r e p la ce d b y h e m or r h a g ic

capillaries. One of the two fetuses with cleft palate also

had severe mandibular retrognathia, left forelimb ectro-

dactyly of digits 13, and severe growth retardation. In

both fetuses with cleft pa late, the palata l shelves were

h y pop la s t ic (F i g. 2 ). T h e s h e lv es h a d r e a ch e d t h e

dorsum of the tongue but failed to oppose and fuse with

each other. Exompha los occurred in the GD 7, 10, and

F ig . 1 . Mouse fetus es of GD 18. A: Control. BF: Experimental group [(450 mg/kg of VGB, treated on GD

8 (B), 9 (C,D), and 11 (E,F)] shows growth retar dation (B,D), exencephaly a nd exophtha lmos (C),mandibular hypoplasia (C,D,E), and exomphalos (E,F).



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1 2 g r ou p s t r e a t e d w it h 4 50 m g /k g o f t h e d r u g; t h e

incidence was only significant in the GD 10 group. It

was char a ct er ized by t he pr esence of 210 loops of

intestine in the proximal segment of the umbilical cord

(Fig. 1). In one embr yo, similar loops were foun d t o have

her niated at the par aumbilical r egion of the vent r al

abdominal wall. Such anomalies were not observed in

an y of th e cont rol mice.

F ig . 2. A,B: Razor blade sections of mouse fetu ses of GD 18. A: Contr ol skeleton. Note the cleft palat e due

to p a la ta l sh e lf h y p op la sia (B) in th e tre a te d c a se (45 0 m g /k g o f VG B , tre a te d on G D 9 ). C: control

sk e le ton . O b serv e in th e a liza rin re d -S -sta in e d sk e le ton s of th e fore lim b s o f V GB -tre a te d fetu se s,

phalangeal hypoplasia (DF) and growth retardation.

VIGABATRIN TERATOGENICITY 169


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TA B LE 2 . A x i a l s k e l e t a l m a l f o r m a t i o n s i n d u c e d b y V GB i n m o u s e f e t u s e s 1

Skeletalelements

Treated

Unt reat edcon t r ol 300 m g/kg VGB 450

7 8 9 10 11 12 7 8 9

No. fet u ses 120 70 91 81 82 80 94 82 70 48

Hypoplaciafacialbon es 4 (3.3) 6 (8.6) 5 (5.5) 6 (7.4) 3 (3.2) 46 (56.1)* 33 (47.1)* 3 (6.3

Rib fu sion 1 (1.4) 3 (3.7) 18 (25.7)* 7/13/6 vert e-

bralcom p le m e n t 9 1 (7 6) 6 4 (9 3)* 7 7 (8 4. 6) 7 3 (9 0. 1)* 7 8 (9 5. 1)* 7 6 (9 5)* 8 9 (9 4. 7)* 7 4 (9 0. 2)* 6 7 (9 5. 7)* 4 7 (9 7.

Coccygealvert ebrae(5 6) 5 4 (4 5) 1 0 (1 4.3 )* 1 4 (1 5.4 )* 3 8 (46 .9) 2 2 (2 6.8 )* 3 0 (3 7.5 ) 35 (3 7.2 ) 3 9 (4 7.6 ) 38 (5 4.3) 3 0 (62 .

Coccygealvert ebrae(7 11 ) 6 1 (5 0.8 ) 5 8 (8 2.9 )* 7 1 (7 8)* 4 3 (5 3. 1) 5 1 (6 2.2 ) 4 3 (5 3.8 ) 50 (5 3.2 ) 3 8 (4 6.3 ) 2 1 (3 0)* 1 5 (3 1.

Lumbar ribs(r igh t ) 1 (0.8) 1 (1.4) 2 (2.2) 1 (1.2) 1 (1.2) 1 (1.3) 2 (2.1) 3 (3.7) 16 (22.9)* 3 (6.3

Lumbar ribs

(left ) 1 (0.8) 1 (1.4) 2 (2.2) 1 (1.2) 1 (1.2) 1 (1.3) 1 (1.1) 3 (3.7) 10 (14.3)* Cervical ribs(r igh t) 8 (6.7) 10 (14.3) 22 (24.2)* 11 (13.6) 3 (3.7) 2 (2.6) 2 (2.1) 44 (53.7)* 8 (11.4) 3 (6.3

Cervical ribs(left ) 8 (6.7) 10 (14.2) 14 (15.4) 8 (9.9) 3 (3.7) 2 (2.6) 2 (2.1) 30 (36.6)* 9 (12.9) 3 (6.3

S t e r n a ld efe ct s 3 (2 .5 ) 5 (7 .1 ) 2 0 (2 2)* 2 6 (4 .9 )* 1 0 (1 2.2 )* 3 (3 .8 ) 1 4 (1 4.9 )* 3 8 (4 6.3 )* 2 3 (3 2. 9)* 2 2 (4 5.

Forepawbones(,4 11 ) 2 1 (1 7.5 ) 8 (11 .4 ) 4 (4 .4 )* 1 6 (1 9. 8) 2 2 (2 6.8 ) 1 5 (1 8.8 ) 15 (1 6.0 ) 1 9 (2 3.2 ) 3 3 (4 7. 1)* 1 4 (2 9.

Forepawbones(54 1 2) 8 6 (8 1. 6) 6 2 (8 8. 6)* 8 7 (9 5. 6)* 6 5 (8 0. 2) 6 0 (7 3. 2) 6 5 (8 1. 3) 7 9 (8 4. 0) 6 3 (7 6. 8) 3 4 (4 8. 6)* 3 4 (7 0.

Hi ndpawbones (,1,5 , 1 2) 4 5 (3 7.5 ) 7 (1 0)* 3 8 (4 1.8 ) 4 7 (5 8)* 2 4 (2 9.3 ) 2 5 (3 1.3 ) 20 (2 1.3 )* 4 6 (5 6.1 )* 4 8 (6 8. 6)* 2 5 (5 2.

Hi ndpawbones (51,5, 12 or

m or e) 7 5 (6 2.5 ) 6 3 (90 )* 5 3 (5 8.2 ) 34 (4 2) 5 8 (70 .7 )* 5 5 (6 8.8 ) 74 (78 .7 )* 3 6 (4 3.9 ) 22 (3 1.4)* 2 3 (47 .Absence of

t a r sa ls 9 (12.9)* 19 (27.1)* 10 (20.MOD 2 (2.9) 33 (40.2)* 14 (8.5)* 5 (10.

1Num e r a ls in pa r e nthe se s a r e pe r c e nta ge s. F or e pa w bone s 5 4 m e ta c a r pa ls, 11 pha la nge s. Hindpa w bone s 5 1 ta r sa l, 5 m emiscellan eous ossificat ion defects (vertebr al a rch fusion, accessory vertebr al a rches, an d occipita lization of atlas).*P , 0.05, contr ol vs. experiment al.


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S k e l e t a l m a l f o r m a t i o n s

S k u l l . GD 7 a nd 8 were pa rticularly susceptible for

450 mg/kg of VGB-indu ced facial bone hypopla sia (Table

2); the nasal bone, premaxilla, maxilla, and mandible

w er e h y pop la s t i c i n t h e s e e m br y os . I n t h e g r ow t h -

r e t a r d ed fe t u s es of b ot h d os e g r ou p s , t h e p a r ie t a l,

i nter par ietal , and supr aoccipit al bones wer e poor lyos s ifi ed a n d t h e fon t a n e ll es w er e r a t h e r w id e . T h e

petrous pa rt of the t emporal bone a nd t he ossicles of the

inner ear were either hypoplastic or occasionally ab-

sent.

V ertebrae. The control embryos had 7 cervical, 13

thoracic, and 5 or 6 sacral vertebral arches and bodies

each; about 50% also had about 49 coccygeal vertebral

bodi es. About 24% had 25 pr esacr al (7 cer vical , 13

thoracic, an d 5 lumbar ) ver tebr ae and the r est had 26

presacral (7 cervical, 13 thoracic, and 6 lumbar) verte-

brae. All ha d at least 23 coccygeal arches ossified. In

t he GD 8 and 9 VGB- tr eated ( 450 mg/kg) embr yos,

t h e r e w a s a s ig n ifi ca n t i n ci de n ce of v er t e br a l a r ch

fusion (Table 2, F ig. 3); the fusion involved t he t hora cic

and lumbar regions. Occipitalization of the atlas verte-

bra occurred in a bout 7% of embryos of the 450 mg/kg

V G B g r o u p s t r e a t e d o n e i t h e r G D 7 o r 8 (P , 0.05).

Accessory vert ebra l arches wer e comm only found in t he

u p p er ce r vi ca l r e gi on , a l m os t a l wa y s u n i la t e r a l i n

p os it i on a n d oft e n fu s e d w it h t h e a d ja ce n t a r ch e s .

Hemivertebrae were occasionally observed in the lower

thoracic or upper lumbar regions. There was a signifi-

cant s hift from the 25 to 26 presacral vertebra l comple-

ment in al l but the 300 mg/ kg GD 8 tr eat ment gr oup.

Reduction in the number of ossified coccygeal vertebral

b od ie s w a s a ch a r a c t er i st i c f ea t u r e of i n t r a u t er i n e

gr owth r etar dati on (I UGR). I n t he 450 mg/kg VGB

gr oup t r eat ed on one of GD 812, ther e was a 4- f old

i ncr ease i n t he number of f et uses that had only 14

coccygeal vert ebra e ossified.

R i b s . T h ir t e en p a ir s of t h or a ci c r i b s (T R), a 1 %

incidence of lumba r r ibs an d a 7% frequ ency of cervical

ribs, were observed in the controls. The cervical ribs

att ached dorsally to the 7th cervical tr an sverse process;

a bar of car t i lage connected them ventr al ly t o t he 1st

TR. The l umbar r ibs, associated with t he fir st lumbar

ver t ebr a, wer e about 12 o r a l i t t l e m o r e t h a n 12 t h e

length of th e las t TR (Wilson, 73). Those r ibs less t ha n12 the 13th TR were not count ed as supernu mera ry ribs

and so wer e excluded. Dr ug-induced incr ease in the

i n ci de n ce of l u m ba r r i bs w a s h i gh l y s ig n ifi ca n t(P , 0.001) in the GD 8 450 mg/ kg VGB gr oup; in a

large number of these cases, there were 8 sternal ribs,

i n s t e a d o f t h e u s u a l 7 . I n c r e a s e i n t h e i n c i d e n c e o f

cervical ribs was most marked in the GD 7 450 mg/kg

VGB group (P , 0.001). Fu sion involved a ra nge of 24

adjacent ribs an d was often bilat era l in occurr ence. TR3

t o TR10 wer e commonly involved i n fusi on. Fusion

affected the shaft and neck of the ribs (Fig. 3). When

mor e pr oximal segments of the r ibs wer e f used, the

fusion extended t o include t he corresponding vertebra l

ar ches as well . Accessor y ossicles wer e somet imes

p r e se n t b et w ee n t h e v er t e br a e w h en r i b f u s ion oc-

curred.

S t e r n u m . The st ernu m of the contr ol embryos con-

sisted of 7 ster nebr ae; in about 3%, the 5t h ster nebr a

was relatively sma ller than t he rest. Absence or hypopla-

s ia of t h e 5 t h s t er n eb r a , fu s ion of 2 or m or e m idster nebr ae, an d displacement and malposi t ioning of

s t er n e br a e w er e com m on l y ob se r ve d in t h e d r u g-

tr eated embr yos and pr ef er entially in the 450 mg/ kg

group (P , 0.001). Agenesis a ffected invar iably th e 5th

stern ebra. In those fetuses with a lumba r rib, there was

a t e n d en cy for 8 s t e r n a l r i bs r a t h e r t h a n t h e u s u a l 7

(Fig. 4).

S k e l e t o n o f t h e l i m b s . Except for the bones of the

paws, th e rest of the limb sk eleton was n ot affected by

drug treatment. The control fetuses had 4 metacarpals

and 11 (18%) or 12 (82%) phalanges in the forepaw.

They a lso possessed 1 (or occasionally 2) ta rsal(s), 5

metatar sals, and at least 12 phalanges in t he hi ndpaw.

VGB tr eatment affected par ticular ly t he phal angealossification. Most remarkable in the 450 mg/kg group

was the fact th at t he pha langes of the middle and distal

p h a l a n g e s o f t h e f o r e p a w a n d m i d p h a l a n g e s o f t h e

hindpaw remained unossified (Figs. 2, 3). Between 13

and 27% of fetuses of this group did not have a single

ta rsa l bone ossified (Table 2).

D ISC U SSIO N

A thorough literature search showed few published

report s on fetotoxic effects of ma ter na l exposure t o VGB

in an imals and huma ns (Kr amer , 92; Li ndhout and

Omtzigt, 94; Marion Merrel Dow file, cited by Gra nt

and Heel, 91). Kra mer (92) observed a n infan t withdiaphr agmatic her nia following VGB usage dur i ng

pregnan cy. Lindhout and Omtzigt (94) reported 3 s ib-

l in g s, t h e fi r s t of w h i ch h a d h y pos p a d ia s w h en t h e

mother was treat ed with ph enytoin an d carbam azepine.

The second pr egnancy ended in miscar r iage. Dur i ng

her thir d pr egnancy she was on car bamazepi ne and a

male child was born with, in addition to hypospadias,

other featur es l ike br oad nasal br idge, high ar ched

palate, upwar d slant ing of the palpebral fissure, promi-

nent ear lobes, diast asis rectus abdominis, deep palmar

creases, bilater al clinodactyly of the 4th toes, and wide

internipple distance. That carbamazepine is known to

induce hypospadias is a fact (Lindhout et al ., 84;

Editorial, 91) that argues against VGB being the sole

causat ive factor in the r epor ted case. H owever, t hi s

report does not r ule out a possible role for VGB in t he

causation of the abnor malit ies seen. VGB was f ound

not to be ter atogenic in r ats at doses over a r ange of

150500 mg/kg/day. It produced a low incidence of cleft

p a la t e i n t h e r a b b it a t m a t e r n a l ly t ox ic d o sa g es of

150200 mg/kg/day; a d ose of 300 mg/kg/day wa s leth al

to the pr egnant rabbits (cited by Grant an d Heel, 91;

Lindh out a nd Om tzigt, 94).



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F

igure

3.



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The r esults of this study have shown that a si ngl e

dose of 600 mg/kg of VGB is leth al t o pregna nt TO mice.

Lower doses cause a modest increase in resorption, a

low frequency of craniofacial malformations, various

skeletal defects, and consistent growth retardation of

t h e fe t u s es . I n t h e a b s en ce of o b se r va b le m a t e r n a l

toxicity du e to VGB exposure, th e concordan ce of IUGR

in terms of a significant reduction in fetal weight and

skeletal gr owth inhibit ion should be inter pr et ed as a

dir ect action of th e dr ug on embr yonic gr owt h pr o-

F ig . 4 . Sterna of fetuses of GD 18, stained with alizarin red-S (A,DF ) and/or alizarin plus alcian blue

(B ,C). A: Control. The chest wall together with the sternum and costal elements are dissected out and

shown here. BF: Experimental group treat ed with 450 mg/kg of VGB on GD 8. Observe the 8 st ernal r ibs

(B), scrambling and fusion of stern ebrae (C,E,F), hypoplasia of 5th stern ebra (E,F), and th e unu sual sh ape

of the xyph oid process (C).

F i g . 3 . S k e le ton s of G D 1 8 fetu se s. A: Control. BH: F e tu se s of

experimental group treated with 450 mg/kg of VGB on GD 8 (BG) or

GD 11 (H). Observe occipitalizat ion of atlas vertebr a (open arr ow in B),

cervical ribs anteriorly attached to first rib (C; arrow) or free accessory

vertebral arch (D; arrow), typical lumbar ribs (LR in E), rib fusion

(arrow in E,F), hemivertebrae (open arrow in F), rudimenta ry lumbar

rib s (B ,F ), g row th re ta rd a tio n a n d a b se n ce o f c ra n ia l v a u lt in a n

e xe n ce p h a lic e m b ry o (G ), a n d a b se n ce of m e ta ta rsa ls, ta rsa ls, a n d

phalanges (H; stained with alizarin a nd alcian blue).



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cesses. One of our objectives of th is investiga tion was to

d e t er m i n e i f VG B w ou l d a u g m en t t h e s p on t a n e ou s

incidence ofexencepha ly char acteristic oft he TO mouse.

The r esults indicate that VGB did not i nfluence the

frequency of N TD and tha t VGB-liability gene(s)

interaction did not occur. However, it is important to

note here tha t t he open bra in of exencephalic fetuses ofthe drug-treated embryos had reached advanced stages

of hemorrhagic degeneration. It is not clear from this

study if the drug a ccelerated t he dest ruction of the open

neural tube. Our observation of thousands of embryos

of our colony of the TO str ain over a period of 5 years

indicates no spontaneous incidence of cleft palate, limb

malformat ions, an d exomphalos (Padma nabha n, un pub-

l ished da t a) . I n our study, 2 of the 93 f et uses tr eated

maternally (with 450 mg/kg of VGB) had cleft palate

but a large number of treated fetuses across the board

h a d a r ch e d p a la t e , s u g ge s t in g t h a t t h e d e ve lop in g

mouse palat e is par t icular l y vulner able t o t h e t er ato-

genic effect of VGB. The mid facial h ypoplasia , char ac-

terized by poor development of the nasal bone, premax-illa, ma xilla, and m an dible, is reminiscent of th e clinical

description of facial an omalies reported to r esult from

other anticonvulsant drugs in humans (Tein and Mac-

Gregor, 85) and in experimental a nimals (Padm ana b-

han an d Ha meed, 94; Pa dman abha n a nd Ahmed, 96).

There was a significant incidence of exomphalos in

the trea ted groups. This observation is of major impor-

t a n c e i n i n t er p r e t in g t h e fe t a l e ffe ct s of m a t e r n a l

exposur e to VGB, i n view of t he f act that abdominal

wall defects do not occur spont an eously i n the TO

strain. Abdominal wall defects are suggested to be of

vascular etiology (Van Allen, 81) an d d ue to disr upt ion

of a midline developmental field (Khoury et al., 89).

However, a careful exam ination of our mouse em bryoswith exomphal os r evealed no malfor mat i ons of t he

vitelline or other mesenter ic vessels. E xompha los of

VGB-treated embryos should therefore be considered a

distinctly different ma lforma tion, ar ising possibly from

a mistiming in the reduction of physiological umbilical

hern ia as opposed to the vas cular disruption sequence.

Skeletal malforma tions such a s rib fusion h ave been

report ed t o occur in VPA ter at ogenesis (Kao et a l., 81;

Ong et a l., 83; Pa dman abha n a nd H ameed, 94; Padma-

nabhan and Ahmed, 96). Appr oximately 25% of the

VGB gr oup of embr yos tr eated on GD 8 showed r ib

fusion in th is study. It is worth noting here th at Brown

and Colhoun (84) and our gr oup (Padma nabhan and

Ham eed, 94) have reported tha t VPA caus es defective

segment ati on of t he somites leading to ver t ebr al and

costal fusi on. Taken together, th ese dat a poi nt to th e

possibility that antiepileptic agents may share a com-

mon mechanism in embryonic dysmorphogenesis. The

implications of supernumerary ribs (SNR) in teratology

a r e d e b a t e d . M a t e r n a l s t r e s s d u r i n g p r e g n a n c y a n d

t r eatments with chemical s or dr ugs incl uding AED

(e .g ., VP A) h a v e b ee n s h ow n t o r e s u lt i n S N R i n

experimen ta l an imals (Wilson, 64, 73; Beyer an d Ch er-

noff, 86; Ong et al., 83; Pet rer e et al., 86; Pa dma na b-

han et al ., 94; Padman abhan and Ahmed, 96). VGB

administer ed on GD 7 accentuated the inci dence of

cer vical r ibs and injection on GD 8 augment ed t he

frequency of lumbar ribs and 8 sternal ribs. VGB also

significantly promoted the occurrence of 26 presacral

vertebral complement and sternal anomalies. Changesi n t h e n u m b e r of v e r t eb r a e , s t e r n eb r a e , a n d r i bs i n

response to teratogenic treatments commonly referred

t o a s a homeotic shift are considered a morphological

sign that developmental interference has occurred (Rus-

sel, 79). Ribs are a t hora cic cha ra cteristic. Persist ence

of th e costal elements in the lower cer vical r egion

(cer vical r ib) or upper lumbar r egion (lumbar r ib)

possibly results from misexpression of H ox 3.3 genes.

Such homeotic tr an sfor mations h ave been shown t o

occur in transgenic mice and after maternal treatment

with r etinoic acid, a key molecule in patt ern formation

(Kessel, 92; Kessel an d Gruss, 91; J egalian and De

Robertis, 92). Wherea s VPA indu ced a modera te red uc-

tion in t he frequency of 26 pr esacral vertebra l comple-ment, VGB enhanced the incidence significantly. The

mecha nism of th e differentia l response is not clear from

this study. That the phalangeal hypoplasia and failure

of ossification of the tarsal bones and of caudal verte-

brae were a s consistent as IUGR ma y be a reflection of

the overall growth-retarding effect of VGB treatment.

The phar ma cokinetics of most of t he anti epil epti c

agents are similar (Thomson and Brodie, 92). They are

well absorbed, bind to serum albumin (except VGB and

ethosuximide), and are mainly eliminated by metabo-

lism in th e liver. Their a bility to induce hepa tic monooxy-

genase or to act a s enzyme inhibitor s r esult s in dr ug-

drug interactions. Close relationships between serum

concentration and observed therapeutic and toxicologi-cal effects are well known for many of them, but such a

r elationship is n on- existent with VGB. VGB ha s an

elimination half-l ife of 57 hr and a r elat ivel y long

dura tion of action (2445 hr ) because it irr eversibly

b in d s t o t h e t a r ge t e n zy me . I t a p pe a r s t o h a ve a

negligible influence on hepatic metabolism and is ex-

creted in th e urine largely un changed (Gran t an d Heel,

91). I ts effects on the developing embr yos wil l be

deter mined by several factors such as th e dose, develop-

m e n t a l s t a g e a t a d m in i st r a t i on , a b il it y t o cr os s t h e

placenta l barr ier, ma terna l-fetal ph arm acokinetics, an d

genotype of the mother and the conceptus. The results

of th is stu dy in par ticular, and t hose of previous investi-

gators on AED teratogenesis in general, clearly showthat VGB is not as str ongly ter atogenic as VPA and

other AEDs and that VGB has a significantly deleteri-

ous effect on em bryonic growth.

ACKNOWLEDGMENTS

T h e a u t h or s a r e g r a t efu l t o P o n er y S . S a m a d , I ja z

Ahmed, and Mohamed Shafiullah for excellent techni-

cal support and Ashok Prasad for photographic assis-

tance. Ms. Janet C. Powell kindly read the manuscript



11/12

a n d m a d e m a n y u s e fu l s u gg es t ion s . T h is w or k w a s

g en e r ou s ly s u p p or t e d b y U A E U n iv er s it y a n d b y a

t r a v el g r a n t fr om H oe ch s t M a r ion R ou s s el of A bu

Dhabi.

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