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Prevention of caffeine-induced cleft palate by l -glutamic acid
TOXICOLCX~Y
AND
APPLIED
PHARMACOLOGY
Prevention
35,123-128
(1976)
of Caffeine-Induced Cleft L-Glutamic Acid
Palate
by
EUGENE J. ZAWOISKI Department of Physiology, Jeferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received July IO, 1975; accepted September 5,1975
Prevention of Caffeine-Induced Cleft Palate by L-Glutamic Acid. Zawoiski, E. J. (1976). Toxicol. Appl. Pharmacol.35, 123-128. L-Glutamic acid produced a significant reduction in caffeine-induced congenital cleft palate when it wasused as a dietary supplement in gestating Swiss1CR:Ha albino mice. The mechanism of the protective action of L-glutamic acid is unknown but may be a reflection of the increased availability and utilization of amino acids, i.e., via transamination, or the result of the synthesis of intermediary metabolites, e.g., glutathione, that require L-glutamic acid. The antiteratogenic activity observed in this study wasconsistent with previous experiments which demonstrated that L-glutamic acid significantly reduced the incidence of cortisone-induced cleft palate and trypan blueindaced exencephaly and otocephaly. Cleft palate and harelip have been observed in animals following administration of therapeutic agents such as anti-inflammatory glucocorticoids (Fraser and Fainstat, 1951; Shah and Chaudhry, 1973; Walker, 1971), thalidomide (King and Kendrick, 1962), insulin (Brinsmade et al., 1956), carbutamide (Meyer and Isaac-Mathy, 1958), actinomycin D (Tuchmann-Duplessis and Mercier-Parot, 1959), and caffeine (Nishimura and,Nakai, 1960; Fujii and Nishimura, 1974). Although the amount of data on drug-induced cleft palate is voluminous, there is, however, a paucity of data concerning its prevention. Strean (1958) and Peer et al. (1958) observed that pyridoxine reduced the incidence of cleft palate in cortisone-treated mice, whereas Larsson (1962) reported that pyridoxine reduced the incidence of embryonic resorption but not that of cortisone-induced cleft palate. Larsson (1962) also showed that administration of vitamin Blz did not offer any protection against the adverse effects of cortisone in A/Jax mice. Zawoiski (1969) reported that animals fed either riboflavin- or pyridoxine-supplemented diets throughout gestation exhibited a significant reduction of cortisoneinduced cleft palate. Miller (1972) subsequently noted that the combined effects of cortisone administration and pyridoxine deprivation were mitigated when pyridoxine was given to mice during the time of palate shelf movement. Fujii and Nishimura (1974) demonstrated that a single intravenous injection of propranolol produced a significant decrease in caffeine-induced cleft palate, brachygnathia, and resorptions in ICR-SLC mice. Copyright 0 1976 by Academic Press, Inc. Au right.9 of reproduction in any form reserve. Printed in Oreat Britain
123
124
EUGENE
J. ZAWOISKI
Zawoiski (1974, 1975a, b) recently showed that t-glutamic acid not only exhibited protective activity against trypan blue-induced exencephaly and otocephaly but also cortisone-induced cleft palate in gestating Swiss ICR albino mice. Because of this protective action against different types of drug-induced congenital fetal malformations, it appeared of interest to examine the possibility that L-glutamic acid may be useful in preventing cleft palate induced by other chemotherapeutic agents. METHODS
Swiss 1CR:Ha albino mice weighing between 25 and 32 g were mated in breeding boxes using a 2 : 1 female : male ratio. The females were examined daily for the appearance of a vaginal mucous plug which was taken as evidence of copulation. The day the plug was found was designated as Day 1. All pregnant females were housed separately throughout the remainder of the gestation period and delivered by cesarean section on Day 16. The 30 females in the two test groups received on Day 13 a 200-mg/kg intraperitoneal injection of caffeine,’ prepared as a 1% solution in deionized water. The 22 females in the two control groups received an equivolumetric injection of the vehicle. The L-glutamic acid2 was administered in powdered diet (food-drug trituration) at a level of 83 mg/g of food from Day 2-16. The average daily oral intake of L-glutamic acid on Day 13 in the control group and caffeine-treated group was 0.48 and 0.47 g, respectively. The data obtained for the daily food intake, maternal and fetal body weights, implants,/ litter, and live fetuses/litter were evaluated by using Student’s t test. The values for the number of females that delivered a litter, the incidence of live fetuses, malformed fetuses, dead embryos, and resorptions were evaluated by using a chi-square test with Yates’ correction for continuity. The incidence of litters that had at least one fetus with a cleft palate was evaluated by using Fisher’s exact test (Sokal and Rohlf, 1973). RESULTS
L-Glutamic acid produced a significant degree of protection (p < 0.01) against caffeine-induced cleft palate (Table 1). The fetuses in the group that received caffeine and unsupplemented control diet exhibited an incidence of 18.3 % cleft palate compared to an incidence of only 4.1% (p < 0.01) in the group that received caffeine and L-glutamic acid. The incidence of cleft palate in the group that received caffeine and unsupplemented control diet was also significantly greater (p < 0.001) than the incidence (0.0%) in both of the control groups. There was no significant difference in the incidence of cleft palate in the females that received caffeine and L-glutamic acid and those in the control groups (Table 1). The incidence of litters that had at least one fetus with a cleft palate was significantly greater (p = 0.03) in the group that received caffeine and unsupplemented control diet (8/l 1) than in the group that received caffeine and L-glutamic acid (2/10). The incidence in the group that received caffeine and unsupplemented control diet was also significantly greater (p = 0.00004) than the incidence in the control groups (O/18). There was 1 Caffeine, quality control number 28, Matheson, Coleman and Bell, East Rutherford, New Jersey. 2 L-Glutamic acid (La-aminoglutaric acid; free acid), Lot 9X-2980, Sigma Chemical Company, St. Louis, Missouri.
OF L-GLUTAMIC
ACID
No. 12 9 86 84 0 2 0 9.6 9.3 0.42
% 90.0 95.3 0.0 4.7 0.0 2.3 0.0 -
97.7 0.0
75.0
%
L-Glutamic acid (8.3%)
Vehicle”
GROSS FETAL
a Deionized water, 20 ml/kg on Day 13. b Caffeine, 200 mg/kg on Day 13. c Percentage calculated in relation to the number of implants. d Percentage calculated in relation to the number of fetuses. ep < 0.001 compared to the group that received the vehicle and unsupplemented control diet. f p < 0.001 compared to the group that received the vehicle and L-glutamic acid. @p< 0.01 compared to the group that received caffeine and unsupplemented control diet. hp < 0.01 compared to the groupthat received the vehicle and unsupplemented control diet.
10 9 86 82 0 4 0 9.6 9.1 0.46
No.
TABLE 1 OF CAFFEINE-INDUCED
None
ON THE INCIDENCE
Females bred Litters Implants Live fetusesC Dead fetusesC Embryos and resorptionP Cleft palated Implants/litter Live fetuses/litter Average weight of the live fetuses (g)
Injection (ip) Dietary supplement
EFFECT
15 11 133 131 0 2 24 12.lh 11.9h 0.44
No.
None
MALFORMATIONS
MICE
-4.19
-
4 10.0 9.7 0.46 -
3.0
0.0
18.3’*’ -
0
0.0
66.7 97.0
3
10 100 97
73.3 98.5
-
%
1.5
15
No.
L-Glutamic acid (8.3 %)
-
%
ALBINO
Caffeineb
IN GESTATING
2
i
7
s B P
zi $
126
EUGENE
J. ZAWOISKI
no significant difference (p = 0.1) in the females that received caffeine and L-glutamic acid (2/10) and those in the control groups (O/18). The animals that received caffeine plus unsupplemented control diet also exhibited a significantly higher (p < 0.01) number of implants/litter and live fetuses/litter than the control group that received unsupplemented diet (Table 1). However, this is not relevant since the animals conceived and implanted 12 days before treatment. In addition, there was no significant increase or decrease in the incidence of dead fetuses, dead embryos, or resorptions in these two groups (Table 1). Both of the control groups exhibited larger increases in maternal body weight on Day 13 than the two caffeine-treated groups (Table 2). The average increase in the two TABLE 2 AVERAGE FOOD CONSUMPTION AND BODY WEIGHTS OF THE GESTATING FEMALES ON DAY 13
Injection” (ip)
Dietary* supplement
Vehicle Vehicle Caffeine Caffeine
None L-Glutamic acid None L-Glutamic acid
Increase in body weight
Food consumption
g 1.3 1.5 0.6 0.2
4.2 4.6 1.7 0.6
5.8’ 7.6 5.4d 5.6’
181 223 153* 166’
’ Vehicle, 20 ml/kg of deionized water on Day 13 ; caffeine, 200 mg/kg on Day 13. b L-Glutamic acid, 83 mg/g of powdered diet. =p < 0.05 compared to the group that received the vehicle and L-glutamic acid. *p < 0.01 compared to the group that received the vehicle and L-glutamic acid.
control groups was 4.2 and 4.6 %, respectively, whereas the average increase in the two caffeine-treated groups was 1.7 and 0.6 % (p < 0.05), respectively. A comparison of the average body weights of the live fetuses, using the litter as a sampling unit, did not reveal any significant differences (Table 1). The average food consumption of the animals in the control group that received glutamic acid was significantly higher (p < 0.05 and 0.01) than the average intake of the other three groups (Table 2). DISCUSSION The results demonstrate that gestating mice fed diets supplemented with L-glutamic acid exhibited a significant reduction in the incidence of caffeine-induced cleft palate. This was not entirely unexpected since it was previously reported that desiccated bovine serum albumin, a casein-derived hydrolysate, riboflavin, L-glutamic acid, and pyridoxine all had anti-teratogenic activity against drug-induced craniofacial malformations (Zawoiski, 1969, 1975a, b). It has also been well documented that both Lglutamic acid and pyridoxine play important roles in the protein metabolism of the developing fetus (Lehninger, 1970; Harper, 1973). Glutamic acid is important in several enzyme systems such as glutamate transaminase, aspartate-glutamate transaminase, and glutamate-pyruvate transaminase (Lehninger, 1970), while vitamin B-6 is essential
CAFFEINE-INDUCED
CLEIT
PALATE
127
as a coenzyme for amino acid metabolism and the transfer of amino acids into cells (Harper, 1973). The fact that L-glutamic acid exhibited protective activity against caffeine-induced cleft palate does not necessarily imply that glutamic acid was the active compound per se. Glutamic acid is directly or indirectly involved in the biotransformation and formation of a large number of compounds, including: a-ketoglutaric acid, glutathione, glutamine, y-aminobutyric acid, glutamic acid y-semialdehyde, aspartic acid, alanine, phenylacetylglutamine, arginine, proline, hydroxyproline, and pteroylyglutamic acid (Lehninger, 1970; Harper, 1973). It is not known whether any of these compounds mediate the antiteratogenic effect of L-glutamic acid. In light of the protective activity exhibited by r.-glutamic acid and pyridoxine against cortisone-induced cleft palate (Zawoiski, 1969, 1975b) and L-glutamic acid against caffeine-induced cleft palate, additional studies using L-glutamic acid either alone or in combination with vitamin B-6 as a source of pyridoxal phosphate may be of significance. ACKNOWLEDGMENT The author would like to express his gratitude to Dr. Gordon J. Eaton of the Institute for Cancer Research at Fox Chase, Philadelphia, Pennsylvania, for supplying the animals. REFERENCES BRINSMADE, A., BUCHNER, F. AND RUBSAAMEN, H. (1956). Missbildungen am Kaninchenembryo durch Insulininjektion beim Muttertier. Nuturwissenschaften 43, 259-265. FRASER, F. C. ANDFAINSTAT,T. D. (1951).Production of congenitaldefectsin the offspring of pregnantmicetreated with cortisone.Pediatrics 8, 527-533. FUJII, T. ANDNISHIMURA,H. (1974).Reduction in frequency of fetopathic effectsof caffeine in miceby pretreatmentwith propranolol. Teratology 10, 149-152. HARPER, H. A. (1973).Review ofPhysiological Chemistry, 14thed.LangeMedical Publications, Los Altos, Calif. KING, C. T. G. ANDKENDRICK, F. J. (1962).Teratogeniceffectsof thalidomidein the Sprague Dawley rat. Lancet 2, 1116. LARSSON, K. S. (1962).Studieson the closureof the secondarypalate.V. Attempts to study the teratogenicaction of cortisonein mice. Acta Odontol. &and. 20, 1-13. LEHNINGER, A. L. (1970).Biochemistry. The Molecular Basis of Cell Structure and Function. Worth, New York. MEYER,DER. ANDISAAC-MATHY,M. (1958).A propos de l’action teratogened’un sulfamide hypoglycemiant(N-sulfanityl, N-butyluree:Bz 55). Ann. Endocrinol. 19, 167-170. MILLER,T. J. (I 972).Cleft palate formation : A role for pyridoxine in the closure of the secondary palatein mice. Teratology 6, 351-356. NISHIMURA, H. ANDNAKAI, K. (1960).Congenitalmalformationsin offspring of micetreated by caffeine.Proc. Sot. Exp. Biol. Med. 104, 140-142. PEER,L. A., BRYAN,W., STREAN,L. P., WALKER,J. C., BERNHARD, W. G. ANDPECK,G. C. (1958).Induction of cleft palate in mice by cortisoneand its reduction by vitamins. .Z. Znt. CON. Surg. 30,249-254. SHAH, R. M. ANDCHAUDHRY, A. P. (1973).Hydrocortisone-inducedcleft palate in hamsters. Teratology 7, 191-194. SOKAL,R. R. ANDROHLF,F. J. (1973). Introduction to Biostatistics. W. H. Freeman, San Francisco. STREAN, L. P. (1958).The Birth of Normal Babies. Twayne Publishers,New York. T~~HMANN-D~LE~~I~,H. ANDMERCIER-PAROT, L. (1959).A propos de l’action teratogkne de l’actinomycine. C. R. Sot. Bioi. 153, 1697-1700.
128 WALKER,
EUGENE B.
J. ZAWOISKI
E. (1971). Induction of cleft palate in rats with antiinflammatory drugs. Teratology
4,39-42. E. J. (1969). Prevention of cortisone-induced cleft palate in albino mice. Proc. Pa. Acad. Sci. 43,200-204. ZAWOISKI, E. J. (1974). Prevention of trypan blue induced fetal malformations in gestating albino mice. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 33, 566 (abstr.). ZAWOISKI, E. J. (1975a). Prevention of trypan blue-induced exencephaly and otocephaly in gestating albino mice. Toxicol. Appl. Pharmacol. 31, 191-200. ZAWOISKI, E. J. (1975b). Prevention of cortisone-induced cleft palate and trypan blue-induced exencephaly by L-glutamic acid. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 34,774 (abstr.). ZAWOISKI,
AND
APPLIED
PHARMACOLOGY
Prevention
35,123-128
(1976)
of Caffeine-Induced Cleft L-Glutamic Acid
Palate
by
EUGENE J. ZAWOISKI Department of Physiology, Jeferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received July IO, 1975; accepted September 5,1975
Prevention of Caffeine-Induced Cleft Palate by L-Glutamic Acid. Zawoiski, E. J. (1976). Toxicol. Appl. Pharmacol.35, 123-128. L-Glutamic acid produced a significant reduction in caffeine-induced congenital cleft palate when it wasused as a dietary supplement in gestating Swiss1CR:Ha albino mice. The mechanism of the protective action of L-glutamic acid is unknown but may be a reflection of the increased availability and utilization of amino acids, i.e., via transamination, or the result of the synthesis of intermediary metabolites, e.g., glutathione, that require L-glutamic acid. The antiteratogenic activity observed in this study wasconsistent with previous experiments which demonstrated that L-glutamic acid significantly reduced the incidence of cortisone-induced cleft palate and trypan blueindaced exencephaly and otocephaly. Cleft palate and harelip have been observed in animals following administration of therapeutic agents such as anti-inflammatory glucocorticoids (Fraser and Fainstat, 1951; Shah and Chaudhry, 1973; Walker, 1971), thalidomide (King and Kendrick, 1962), insulin (Brinsmade et al., 1956), carbutamide (Meyer and Isaac-Mathy, 1958), actinomycin D (Tuchmann-Duplessis and Mercier-Parot, 1959), and caffeine (Nishimura and,Nakai, 1960; Fujii and Nishimura, 1974). Although the amount of data on drug-induced cleft palate is voluminous, there is, however, a paucity of data concerning its prevention. Strean (1958) and Peer et al. (1958) observed that pyridoxine reduced the incidence of cleft palate in cortisone-treated mice, whereas Larsson (1962) reported that pyridoxine reduced the incidence of embryonic resorption but not that of cortisone-induced cleft palate. Larsson (1962) also showed that administration of vitamin Blz did not offer any protection against the adverse effects of cortisone in A/Jax mice. Zawoiski (1969) reported that animals fed either riboflavin- or pyridoxine-supplemented diets throughout gestation exhibited a significant reduction of cortisoneinduced cleft palate. Miller (1972) subsequently noted that the combined effects of cortisone administration and pyridoxine deprivation were mitigated when pyridoxine was given to mice during the time of palate shelf movement. Fujii and Nishimura (1974) demonstrated that a single intravenous injection of propranolol produced a significant decrease in caffeine-induced cleft palate, brachygnathia, and resorptions in ICR-SLC mice. Copyright 0 1976 by Academic Press, Inc. Au right.9 of reproduction in any form reserve. Printed in Oreat Britain
123
124
EUGENE
J. ZAWOISKI
Zawoiski (1974, 1975a, b) recently showed that t-glutamic acid not only exhibited protective activity against trypan blue-induced exencephaly and otocephaly but also cortisone-induced cleft palate in gestating Swiss ICR albino mice. Because of this protective action against different types of drug-induced congenital fetal malformations, it appeared of interest to examine the possibility that L-glutamic acid may be useful in preventing cleft palate induced by other chemotherapeutic agents. METHODS
Swiss 1CR:Ha albino mice weighing between 25 and 32 g were mated in breeding boxes using a 2 : 1 female : male ratio. The females were examined daily for the appearance of a vaginal mucous plug which was taken as evidence of copulation. The day the plug was found was designated as Day 1. All pregnant females were housed separately throughout the remainder of the gestation period and delivered by cesarean section on Day 16. The 30 females in the two test groups received on Day 13 a 200-mg/kg intraperitoneal injection of caffeine,’ prepared as a 1% solution in deionized water. The 22 females in the two control groups received an equivolumetric injection of the vehicle. The L-glutamic acid2 was administered in powdered diet (food-drug trituration) at a level of 83 mg/g of food from Day 2-16. The average daily oral intake of L-glutamic acid on Day 13 in the control group and caffeine-treated group was 0.48 and 0.47 g, respectively. The data obtained for the daily food intake, maternal and fetal body weights, implants,/ litter, and live fetuses/litter were evaluated by using Student’s t test. The values for the number of females that delivered a litter, the incidence of live fetuses, malformed fetuses, dead embryos, and resorptions were evaluated by using a chi-square test with Yates’ correction for continuity. The incidence of litters that had at least one fetus with a cleft palate was evaluated by using Fisher’s exact test (Sokal and Rohlf, 1973). RESULTS
L-Glutamic acid produced a significant degree of protection (p < 0.01) against caffeine-induced cleft palate (Table 1). The fetuses in the group that received caffeine and unsupplemented control diet exhibited an incidence of 18.3 % cleft palate compared to an incidence of only 4.1% (p < 0.01) in the group that received caffeine and L-glutamic acid. The incidence of cleft palate in the group that received caffeine and unsupplemented control diet was also significantly greater (p < 0.001) than the incidence (0.0%) in both of the control groups. There was no significant difference in the incidence of cleft palate in the females that received caffeine and L-glutamic acid and those in the control groups (Table 1). The incidence of litters that had at least one fetus with a cleft palate was significantly greater (p = 0.03) in the group that received caffeine and unsupplemented control diet (8/l 1) than in the group that received caffeine and L-glutamic acid (2/10). The incidence in the group that received caffeine and unsupplemented control diet was also significantly greater (p = 0.00004) than the incidence in the control groups (O/18). There was 1 Caffeine, quality control number 28, Matheson, Coleman and Bell, East Rutherford, New Jersey. 2 L-Glutamic acid (La-aminoglutaric acid; free acid), Lot 9X-2980, Sigma Chemical Company, St. Louis, Missouri.
OF L-GLUTAMIC
ACID
No. 12 9 86 84 0 2 0 9.6 9.3 0.42
% 90.0 95.3 0.0 4.7 0.0 2.3 0.0 -
97.7 0.0
75.0
%
L-Glutamic acid (8.3%)
Vehicle”
GROSS FETAL
a Deionized water, 20 ml/kg on Day 13. b Caffeine, 200 mg/kg on Day 13. c Percentage calculated in relation to the number of implants. d Percentage calculated in relation to the number of fetuses. ep < 0.001 compared to the group that received the vehicle and unsupplemented control diet. f p < 0.001 compared to the group that received the vehicle and L-glutamic acid. @p< 0.01 compared to the group that received caffeine and unsupplemented control diet. hp < 0.01 compared to the groupthat received the vehicle and unsupplemented control diet.
10 9 86 82 0 4 0 9.6 9.1 0.46
No.
TABLE 1 OF CAFFEINE-INDUCED
None
ON THE INCIDENCE
Females bred Litters Implants Live fetusesC Dead fetusesC Embryos and resorptionP Cleft palated Implants/litter Live fetuses/litter Average weight of the live fetuses (g)
Injection (ip) Dietary supplement
EFFECT
15 11 133 131 0 2 24 12.lh 11.9h 0.44
No.
None
MALFORMATIONS
MICE
-4.19
-
4 10.0 9.7 0.46 -
3.0
0.0
18.3’*’ -
0
0.0
66.7 97.0
3
10 100 97
73.3 98.5
-
%
1.5
15
No.
L-Glutamic acid (8.3 %)
-
%
ALBINO
Caffeineb
IN GESTATING
2
i
7
s B P
zi $
126
EUGENE
J. ZAWOISKI
no significant difference (p = 0.1) in the females that received caffeine and L-glutamic acid (2/10) and those in the control groups (O/18). The animals that received caffeine plus unsupplemented control diet also exhibited a significantly higher (p < 0.01) number of implants/litter and live fetuses/litter than the control group that received unsupplemented diet (Table 1). However, this is not relevant since the animals conceived and implanted 12 days before treatment. In addition, there was no significant increase or decrease in the incidence of dead fetuses, dead embryos, or resorptions in these two groups (Table 1). Both of the control groups exhibited larger increases in maternal body weight on Day 13 than the two caffeine-treated groups (Table 2). The average increase in the two TABLE 2 AVERAGE FOOD CONSUMPTION AND BODY WEIGHTS OF THE GESTATING FEMALES ON DAY 13
Injection” (ip)
Dietary* supplement
Vehicle Vehicle Caffeine Caffeine
None L-Glutamic acid None L-Glutamic acid
Increase in body weight
Food consumption
g 1.3 1.5 0.6 0.2
4.2 4.6 1.7 0.6
5.8’ 7.6 5.4d 5.6’
181 223 153* 166’
’ Vehicle, 20 ml/kg of deionized water on Day 13 ; caffeine, 200 mg/kg on Day 13. b L-Glutamic acid, 83 mg/g of powdered diet. =p < 0.05 compared to the group that received the vehicle and L-glutamic acid. *p < 0.01 compared to the group that received the vehicle and L-glutamic acid.
control groups was 4.2 and 4.6 %, respectively, whereas the average increase in the two caffeine-treated groups was 1.7 and 0.6 % (p < 0.05), respectively. A comparison of the average body weights of the live fetuses, using the litter as a sampling unit, did not reveal any significant differences (Table 1). The average food consumption of the animals in the control group that received glutamic acid was significantly higher (p < 0.05 and 0.01) than the average intake of the other three groups (Table 2). DISCUSSION The results demonstrate that gestating mice fed diets supplemented with L-glutamic acid exhibited a significant reduction in the incidence of caffeine-induced cleft palate. This was not entirely unexpected since it was previously reported that desiccated bovine serum albumin, a casein-derived hydrolysate, riboflavin, L-glutamic acid, and pyridoxine all had anti-teratogenic activity against drug-induced craniofacial malformations (Zawoiski, 1969, 1975a, b). It has also been well documented that both Lglutamic acid and pyridoxine play important roles in the protein metabolism of the developing fetus (Lehninger, 1970; Harper, 1973). Glutamic acid is important in several enzyme systems such as glutamate transaminase, aspartate-glutamate transaminase, and glutamate-pyruvate transaminase (Lehninger, 1970), while vitamin B-6 is essential
CAFFEINE-INDUCED
CLEIT
PALATE
127
as a coenzyme for amino acid metabolism and the transfer of amino acids into cells (Harper, 1973). The fact that L-glutamic acid exhibited protective activity against caffeine-induced cleft palate does not necessarily imply that glutamic acid was the active compound per se. Glutamic acid is directly or indirectly involved in the biotransformation and formation of a large number of compounds, including: a-ketoglutaric acid, glutathione, glutamine, y-aminobutyric acid, glutamic acid y-semialdehyde, aspartic acid, alanine, phenylacetylglutamine, arginine, proline, hydroxyproline, and pteroylyglutamic acid (Lehninger, 1970; Harper, 1973). It is not known whether any of these compounds mediate the antiteratogenic effect of L-glutamic acid. In light of the protective activity exhibited by r.-glutamic acid and pyridoxine against cortisone-induced cleft palate (Zawoiski, 1969, 1975b) and L-glutamic acid against caffeine-induced cleft palate, additional studies using L-glutamic acid either alone or in combination with vitamin B-6 as a source of pyridoxal phosphate may be of significance. ACKNOWLEDGMENT The author would like to express his gratitude to Dr. Gordon J. Eaton of the Institute for Cancer Research at Fox Chase, Philadelphia, Pennsylvania, for supplying the animals. REFERENCES BRINSMADE, A., BUCHNER, F. AND RUBSAAMEN, H. (1956). Missbildungen am Kaninchenembryo durch Insulininjektion beim Muttertier. Nuturwissenschaften 43, 259-265. FRASER, F. C. ANDFAINSTAT,T. D. (1951).Production of congenitaldefectsin the offspring of pregnantmicetreated with cortisone.Pediatrics 8, 527-533. FUJII, T. ANDNISHIMURA,H. (1974).Reduction in frequency of fetopathic effectsof caffeine in miceby pretreatmentwith propranolol. Teratology 10, 149-152. HARPER, H. A. (1973).Review ofPhysiological Chemistry, 14thed.LangeMedical Publications, Los Altos, Calif. KING, C. T. G. ANDKENDRICK, F. J. (1962).Teratogeniceffectsof thalidomidein the Sprague Dawley rat. Lancet 2, 1116. LARSSON, K. S. (1962).Studieson the closureof the secondarypalate.V. Attempts to study the teratogenicaction of cortisonein mice. Acta Odontol. &and. 20, 1-13. LEHNINGER, A. L. (1970).Biochemistry. The Molecular Basis of Cell Structure and Function. Worth, New York. MEYER,DER. ANDISAAC-MATHY,M. (1958).A propos de l’action teratogened’un sulfamide hypoglycemiant(N-sulfanityl, N-butyluree:Bz 55). Ann. Endocrinol. 19, 167-170. MILLER,T. J. (I 972).Cleft palate formation : A role for pyridoxine in the closure of the secondary palatein mice. Teratology 6, 351-356. NISHIMURA, H. ANDNAKAI, K. (1960).Congenitalmalformationsin offspring of micetreated by caffeine.Proc. Sot. Exp. Biol. Med. 104, 140-142. PEER,L. A., BRYAN,W., STREAN,L. P., WALKER,J. C., BERNHARD, W. G. ANDPECK,G. C. (1958).Induction of cleft palate in mice by cortisoneand its reduction by vitamins. .Z. Znt. CON. Surg. 30,249-254. SHAH, R. M. ANDCHAUDHRY, A. P. (1973).Hydrocortisone-inducedcleft palate in hamsters. Teratology 7, 191-194. SOKAL,R. R. ANDROHLF,F. J. (1973). Introduction to Biostatistics. W. H. Freeman, San Francisco. STREAN, L. P. (1958).The Birth of Normal Babies. Twayne Publishers,New York. T~~HMANN-D~LE~~I~,H. ANDMERCIER-PAROT, L. (1959).A propos de l’action teratogkne de l’actinomycine. C. R. Sot. Bioi. 153, 1697-1700.
128 WALKER,
EUGENE B.
J. ZAWOISKI
E. (1971). Induction of cleft palate in rats with antiinflammatory drugs. Teratology
4,39-42. E. J. (1969). Prevention of cortisone-induced cleft palate in albino mice. Proc. Pa. Acad. Sci. 43,200-204. ZAWOISKI, E. J. (1974). Prevention of trypan blue induced fetal malformations in gestating albino mice. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 33, 566 (abstr.). ZAWOISKI, E. J. (1975a). Prevention of trypan blue-induced exencephaly and otocephaly in gestating albino mice. Toxicol. Appl. Pharmacol. 31, 191-200. ZAWOISKI, E. J. (1975b). Prevention of cortisone-induced cleft palate and trypan blue-induced exencephaly by L-glutamic acid. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 34,774 (abstr.). ZAWOISKI,