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I'~IUTT II1~,. E RW Q RT H ~ - . 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 SSDI 0896-6974(94)00009-0 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). ~ff~ ~ 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- 2oo ~ 11~ No treatment Vehicle ASP 500mg/kg ASP 750mg/kg J EPILEPSY, VOL. 8, NO. 1, 1995 53 E. F. SPERBER ET AL. A [] I C~on~ ~ I z ~ e Tonic ~ z u r e g 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 s ~ ~s s s ~ s s s ~ s s s s s s ~ s s ~ ~ ~s ~s~s~s~s~ ~s~s~s ~ ~ ~ ~ ~ ~ ~ ¢ ~ ¢ ¢ ~ ~ ~ ~ ¢ ~ ~ ~ ~ l ~ i~ ~ ~ ~ ~ ~ ~ No u'eatment 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 • ~~ s ~ s ~ s ~ s~~ s ~ s ~ s ~ s ~ s ~ s . s , ~ s s ~ s ~~ s s ~ s s ~ ~ s s s s s s s ~ s s ~ ~ ~ s ~ s ~ s ~ ~ ~ s s ~ s ~ s ~ s ~ ~ ~ s ~ s s ~ s s s s ~ ~ ~ ~ ~ ~ ~ ~ i ~ ~ Oav~e 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. References 1. Bauman ML, Kemper TL. 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