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Prenatal Exposure to Aspartame and
Seizure Susceptibility
1'3Ellen F. Sperber, L2"3Solomon L. Mosh6, and 4Diana L. Dow-Edwards
Aspartame has been reported to be proconvulsant in rats and mice. To determine whether gestational exposure to aspartame alters susceptibility to
flurothyl-induced seizures in offspring, we administered either 500 or 750
mg/kg aspartame to pregnant guinea pigs throughout pregnancy. The offspring were exposed to flurothyl at age 30 days to determine the threshold for
clonic and tonic seizures. We also examined offspring from a gavaged control
group and a nontreated control group. There were no statistically significant
differences in either clonic or tonic seizure thresholds among all groups.
Therefore, aspartame administration throughout pregnancy in guinea pigs
had no epileptogenic potential in the flurothyl model. Key Words: Aspartame--Seizures--Development--Phenylalanine--Artificial sweetners-Guinea pigs.
Aspartame (ASP, N-I,-a-aspartyl-L-phenylalanine-l-methyl ester) is metabolized into three comp o n e n t s in the small intestine: p h e n y l a l a n i n e
(50%), aspartic acid (40%), and methanol (10% wt/
wt). Phenylalanine is an essential amino acid
which, w h e n consumed in excessive amounts, is
associated with various behavioral effects in humans, including seizures and mental retardation
Received April 25, 1994; accepted August 22, 1994.
From the Departments of 1 Neurology, ,~WPediatrics, and
3Neuroscience, Laboratory of Developmental Epilepsy,
Albert Einstein College of Medicine and Montefiore
Medical Center, Bronx, NY, and 4Department of Pharmacology & Anatomy/Cell Biology, Laboratory of Cerebral Metabolism, State University of New York, Health
Science Center, Brooklyn, NY, U.S.A.
Address correspondence and reprint requests to Dr.
E. F. Sperber at Albert Einstein College of Medicine,
Laboratory of Developmental Epilepsy, Rose F.
Kennedy Center, Room 316, 1410 Pelham Parkway S.,
Bronx, N.Y. 10461, U.S.A.
l- Epilepsy1995;8:51-56
© 1995 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
(1,2). In developing humans (3), hyperphenylalaninemia is associated with the mental retardation
and microcephaly of phenylketonuria (PKU). Animal models have d o c u m e n t e d neurochemical,
morphologic, and behavioral alterations induced
by increased phenylalanine levels during development (4-6). In animals, both acute and chronic administration of phenylalanine have been shown to
increase seizure susceptibility and impair learning
performance (7,8).
Aspartic acid does not cross the placenta to any
significant degree and thus is unlikely to induce
teratogenic effects. The effects of methanol on
brain development have not been adequately studied. A few studies reported a variety of neurologic
effects in adult humans after consumption of ASP
itself. High intake of ASP has been associated with
urticaria, mania, seizures, headaches, and migraines; other studies have refuted these findings
(9-11). A similar discrepancy exists in the animal
literature: Some studies report an effect of ASP on
behavior, and other studies negate these findings.
0896-6974/95/$10.00
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E. F. SPERBER ET AL.
Dailey et al. (12), Guiso et al. (13), and Tilson et al.
(14) reported that ASP had no effect on seizures
i n d u c e d by p e n t y l e n e t e t r a z o l , maximal electroshock (MES), quinolinic acid, or k i n d l i n g
whereas Kim et al. (15), Guiso et al. (13), and Pinto
and Maher (16) observed that ASP had a proconvulsant effect w h e n seizures were induced by
lidocaine, maximal electroshock seizures, pentylenetetrazol, or flurothyl.
ASP is widely consumed in a variety of food
products, including diet soda, Koolaid, and diet
desserts. However, federal regulations do not require that product labeling include the amount of
ASP in these items. According to the industry, diet
soda contains 208 mg ASP per 12-ounce can and
Koolaid contains 250 mg per 0.5-L serving. Pregnant women, in an effort to control their weight,
might be expected to consume relatively large
amounts of ASP since the product carries warning
labels only for phenylketonurics. Considerable
controversy exists concerning whether ASP consumption during pregnancy is safe for the fetus.
Previous work on developmental exposure to ASP
in guinea pigs identified impaired performance in
tests of aversion learning (17-19). The learning impairment was associated with maternal and fetal
phenylalanine levels that were within the range
that produce mental retardation in humans.
This research was designed to identify whether
intake of ASP that doubles maternal and fetal
plasma phenylalanine levels during pregnancy (5)
can alter seizure susceptibility in offspring. We selected the guinea pig as a model for two reasons:
(a) In the guinea pig, most brain development occurs in utero as it does in humans and the effects of
an altered maternal amino acid profile should be
studied on a fetus with an intact placenta; and (b)
the metabolism of ASP is more like that which occurs in h u m a n s rather than that which occurs in
other rodents, such as rats. The prenatally exposed
animals were examined for seizure susceptibility at
age 30 days because this age is well past weaning
(20 days) and correlates with the age at which
brain metabolism was examined (20).
Methods
Duncan Hartley guinea pigs weighing 525-575 g
(Camm Research, Wayne, NJ, U.S.A.) were gavaged once daily with vehicle (sesame oil, Sigma) or
500 or 750 mg/kg ASP (Sigma) from the first day of
pregnancy until parturition at - 6 5 days of gestation. The first day of pregnancy was defined as the
presence of open vaginal membranes coincident
52 J EPILEPSY, VOL. 8, NO. 1, 1995
with the presence of a male in the cage. A nontreated group was maintained in cases in which
pregnancy was initially undetected. All dams received standard housing conditiions with food and
water ad libitum, 12-h light/dark cycles, and lettuce twice a week. Females were housed in groups
of three and weighed daily; on gestation day 55
(G55), they were placed in individual cages until
the day of parturition. On the day of birth, all gavage treatments were stopped, pups were sexed,
measured, weighed, and given lettuce in addition
to regular chow each day thereafter. Pups were not
fostered. At age 23 days, guinea pigs were randomly selected from the four groups (no treatment, vehicle, ASP 500 mg/kg, and ASP 750 rag/
kg) and transferred to Albert Einstein College of
Medicine, where they were housed individually
under standard conditions. Food and water were
available ad libitum. At 30 days, all guinea pigs
were exposed to flurothyl.
Flurothyl (bis 2,2,2-tri flurothyl ether) is a volatile
convulsant agent established as an effective technique to measure the brain's threshold to generalized seizures in adult and immature animals (21).
The convulsions produced by flurothyl inhalation
are considered to represent a model of generalized
seizures. The guinea pigs were placed individually
in a sealed chamber measuring 9.2 L (19 x 22 x 10
cm). Flurothyl was infused at a constant rate of 20
~l/min by an infusion pump (Harvard Apparatus)
into the top of the chamber, where it dripped onto
a piece of filter paper and vaporized. As soon as a
generalized clonic or tonic seizure occurred, the
flurothyl infusion was discontinued. The following
variables were analyzed: latency to onset of forelimb clonus and latency to onset of tonic seizures
with loss of posture. From the latency data, the
flurothyl threshold to a clonic or tonic convulsion
was calculated by determining the amount of flurothyl necessary to elicit a clonic or a tonic convulsion, respectively (latency x rate of infusion). In
addition, the time between the two convulsive
endpoints was determined. Each animal was exposed to flurothyl once. Between trials, the flurothyl in the chamber was evacuated by a vacuum
pump and flushed with air and the filter paper was
replaced.
Results
Twenty guinea pigs (1 per litter) were included
in this study. Five received no treatment in utero;
the dams of the remaining 15 were exposed to either sesame oil vehicle (n = 3) or aspartame 500
ASPARTAME IN DEVELOPMENT AND SEIZURES
mg/kg (n = 6) or 750 mg/kg (n = 6) by daily gavage feedings. Effects of ASP on development were
described previously (5,17,20). No statistically significant effects on gestation length, litter size, or
birthweights were evident. Maternal weight gain
in the 750-mg/kg group tended to be lower than
that in the other three groups. There were no differences among guinea pigs in the various groups
in terms of growth parameters at age 30 days.
One-way analysis of variance showed no statistically significant differences among the groups
(Fig. 1), either in terms of clonic (F3,16 = 1.2, p >
0.05) or tonic seizures threshold (F3.16 = 1.7, p >
0.05). Furthermore, there was no statistically significant difference among the groups concerning
the interval separating forelimb clonic seizures
from generalized tonic seizures with loss of posture (F3,16 = 1 . 8 , p > 0.05).
Figure 1 suggests that nontreated (nongavaged)
animals had higher thresholds than animals exposed to gavage feedings in utero. Repetitive gavage may be associated with a potentially epileptogenic stress. Therefore, we analyzed the data by
grouping all gavaged animals (Fig. 2). Comparisons of thresholds to forelimb clonic seizures
showed that gavaged guinea pigs did not differ
from nongavaged guinea pigs (Fl,a8 = 2.4, p >
0.05). However, gavaged guinea pigs had significantly lower thresholds to generalized tonic seizures with loss of posture than did nontreated
pups (F1,18 = 4.95, p < 0.05). Accordingly, the
interval between the two convulsive points was
also significantly different (FIA8 = 5.5, p < 0.05).
Discussion
Our results indicate that ASP administration to
pregnant guinea pigs does not affect the suscepti-
Figure 1. Effects of two doses of aspartame (ASP) on flurothyl-induced
clonic and tonic seizures. ASP had no
significant effect on seizure susceptibility. Values are mean +- SE (in ~l).
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bility of the offspring to flurothyl-induced seizures. Although several reports have described the
effects of ASP on seizures in adult rats (12-15),
there has been only one study of rats exposed to
ASP during postnatal development (14); in that
study, rats were exposed to ASP (1,000 mg/kg)
during either postnatal days 3-13 or 21-35 and kindled at age 90 days. Consistent with our findings
with guinea pigs, the ASP-treated rats were not
significantly different from controls (14).
Several groups of investigators have examined
the effects of ASP on seizure susceptibility in adult
rats and mice, with conflicting results; e.g., Guiso
et al. (13) administered ASP 0.75-1 g/kg to fasted
rats and examined pentylenetetrazol-induced seizures. ASP 1 g/kg significantly increased the number of animals with tonic-clonic seizures, an effect
that could be mimicked by equivalent amounts of
phenylalanine. However, the increase in the number of animals showing seizures was not observed
in fed animals receiving ASP or in animals receiving ASP in three divided doses. On the other
hand, the same dose of ASP did not affect the CCs0
(mA) for tonic hindlimb extension induced by MES
or for EEG seizures induced by intrahippocampal
injection of quinolinic acid to nonfasted rats. This
study (13) and that of Torii et al. (22) emphasize
the ameliorating neurochemical and neurobehavioral effects of ASP w h e n administered with additional protein sources. Presumably, other large
neutral amino acids (LNAA), including tyrosine,
compete with phenylalanine for entry into brain by
the stereospecific saturable transport system and
block the dramatic increase in phenylalanine and
decrease in other LNNA, including tryptophan,
which occur in brain after increase in plasma phenylalanine alone (23). The flooding of brain with
phenylalanine alters neurotransmitter levels such
as norepinephrine and serotonin, which would al-
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No treatment
Vehicle
ASP 500mg/kg
ASP 750mg/kg
J EPILEPSY, VOL. 8, NO. 1, 1995 53
E. F. SPERBER ET AL.
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Figure 2. Effect of handling (during
gavage) during pregnancy on seizure
susceptibility of offspring. A: Effect on
flurothyl thresholds. B: Effect on interval between clonic and tonic seizures.
As compared with nontreated animals,
handling significantly decreased the
tonic seizure threshold by shortening
the interval between onset of a clonic
and a tonic seizure, respectively. Values are mean + SE in ~l (A) and in
seconds (B): *p K 0.05.
Gavage
No treatment
B
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ter seizure susceptibility (24,25). Therefore, examination of ASP effects in species such as rats, mice,
and guinea pigs that show both increased plasma
phenylalanine and tyrosine after ASP administration limits the relevance of the data to humans, in
which ASP increases plasma phenylalanine more
than tyrosine (10).
Pinto and Maher (16) attempted to minimize the
effect of increased plasma tyrosine after ASP administration by examining high doses of ASP,
which increase phenylalanine levels more than tyrosine levels in rodents. In their experiment, ASP
significantly increased the number of animals convulsing after pentylenetetrazol and decreased the
CDs0 for this drug. Again, the effect could be
blocked by administration of another LNAA (va54 J EPILEPSY, VOL. 8, NO. 1, 1995
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line) together with ASP. This study was repeated
using the same strain of mouse and experimental
conditions; Daily et al. (12) showed that ASP was
not a proconvulsant. One difference between the
studies was the weight of the mice and therefore
probably the age of the mice. Age-related changes
have been observed in seizure susceptibility of rodents to stimuli including environmental insults
during the second and third postnatal weeks (25).
Therefore, the slightly older age of animals examined in the latter study may have resulted in subtle
differences, such as a decrease in the excitability of
the central nervous system (CNS), thereby altering
the effect of ASP on seizure susceptibility.
One other factor that deserves consideration is
that of the effects of handling of the pregnant
ASPARTAME IN DEVELOPMENT A N D SEIZURES
guinea pigs d u r i n g gavage feedings. The results
indicate that the offspring of the three treated (gavaged) g r o u p s of d a m s h a d shorter latencies to onset of tonic-clonic seizures t h a n offspring of nontreated ( n o n h a n d l e d ) dams. Presumably, h a n d l i n g
increases stress in p r e g n a n t guinea pigs that m a y
result in h e i g h t e n e d CNS excitability in the offspring a n d alter seizure susceptibility. The observation that latency to onset of a clonic seizure was
not different b e t w e e n n o n t r e a t e d controls a n d gayaged offspring suggests that the t r e a t m e n t s altered
the m e c h a n i s m s responsible for expression of only
the tonic seizure. Clonic a n d tonic seizures a p p e a r
to h a v e different p a t h o p h y s i o l o g i c substrates. Gale
(26) p r o p o s e d that clonic seizures are p r e d o m i nantly of forebrain origin, w h e r e a s tonic seizures
originate in brainstem. Therefore, h a n d l i n g and
g a r a g i n g a p p e a r to cause functional changes in
b r a i n s t e m structures i n v o l v e d in tonic seizures.
The preferential i n v o l v e m e n t of b r a i n s t e m is the
direct result of the w a y in which the m a t u r a t i o n
process evolves d u r i n g gestation. Brainstem structures attain their maturity before forebrain structures a n d therefore are m o r e likely to be affected
(23,24).
In o u r study, two doses of ASP w e r e used: 500
and 750 mg/kg. These doses w e r e selected because
we previously d e m o n s t r a t e d that such doses double or triple maternal plasma p h e n y l a l a n i n e levels
(5). In h u m a n s , such high plasma p h e n y l a l a n i n e
levels t h r o u g h o u t p r e g n a n c y w o u l d be expected to
be associated with m e n t a l retardation. Doses of
ASP m u c h h i g h e r t h a n 750 mg/kg might have prod u c e d a p r o c o n v u l s a n t effect. The data of Pinto
a n d M a h e r (16) suggest, h o w e v e r , that a dosed e p e n d e n t r e s p o n s e m a y not exist, since a plateau
w a s r e a c h e d w i t h > l , 0 0 0 - m g / k g d o s e s in the
highly susceptible y o u n g mice.
O u r p r e v i o u s w o r k s h o w e d that 500 mg/kg ASP
t h r o u g h o u t p r e g n a n c y in g u i n e a pigs p r o d u c e s
offspring with impaired ability to p e r f o r m tests of
odor-associated learning at age 15 days. H o w e v e r ,
e x p o s u r e to ASP 500 and 750 mg/kg resulted in
offspring with n o alterations in basal rates of brain
glucose m e t a b o l i s m at age 30 days (20). Neither
dose of ASP c a u s e d changes in fetal g r o w t h parameters or brain weight. Therefore, o u r data indicate that chronic ASP c o n s u m p t i o n t h r o u g h o u t
p r e g n a n c y in guinea pigs has n o effect on g r o w t h
of offspring a n d causes only subtle n e u r o b e h a v i o r al changes e v i d e n t primarily w h e n the CNS is in
other t h a n baseline conditions. O u r results also
suggest that ASP effects m a y be potentiated b y
c o n c u r r e n t gestational stressors. Examination of
the effects of o t h e r s e i z u r e - i n d u c i n g t r e a t m e n t s
such as p e n t y l e n e t e t r a z o l or kindling w o u l d help
test w h e t h e r the CNS e x p o s e d to ASP t h r o u g h o u t
gestation is i n d e e d hyperexcitable.
Acknowledgment: This w o r k was s u p p o r t e d b y
N I H Grants No. NS-22766 a n d NS-30387-01A3 a n d
b y Grants No. NS-20253 from NINDS. We t h a n k
Drs. N. Ludvig and L. Scribani for assistance.
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