Diabetic embryopathy in C57BL/6J mice. Altered fetal sex ratio and impact of the splotch allele.

Maternal diabetes (types 1 and 2) induces a broad array of congenital malformations, including neural tube defects (NTDs), in humans. One of the difficulties associated with studying diabetic embryopathy is the rarity of individual malformations. In an attempt to develop a sensitive animal model for maternal diabetes-induced NTDs, the present study uses chemically induced diabetes in an inbred mouse model with or without the splotch (Sp) mutation, a putatively nonfunctional allele of Pax3. Pax3 deficiency has been associated with an increase in NTDs. Female C57BL/6J mice, either with or without the Sp allele, were injected intravenously with alloxan (100 mg/kg), and plasma glucose was measured 3 days later. A wide range of hyperglycemia was induced, and these diabetic mice were bred to C57BL/6J males, some carrying the Sp allele. Gestational-day-18 fetuses were examined for developmental malformations. Fetuses from matings in which either parent carried the Sp allele were genotyped by polymerase chain reaction. Maternal diabetes significantly decreased fetal weight and increased the number of resorptions and malformations, including NTDs. A significant correlation was found between the level of maternal hyperglycemia and the malformation rate. The sex ratio for live fetuses in diabetic litters was significantly skewed toward male fetuses. Matings involving the Sp allele yielded litters with significantly higher percentages of maternal diabetes-induced spina bifida aperta but not exencephaly, and this increase was shown to be associated with the presence of a single copy of the Sp allele in affected fetuses. Thus, Pax3 haploinsufficiency in this murine model of diabetic embryopathy is associated with caudal but not cranial NTDs.

Cocaine-induced embryonic cardiovascular disruption in mice.

The purpose of this study was to determine whether vascular disruption is a feature of cocaine-induced teratogenicity in early murine organogenesis. The embryotoxic effects of cocaine were assessed: (1) in vivo, (2) in embryos cultured in the presence of cocaine (in vitro), and (3) after cocaine was administered in vivo and the embryos subsequently cultured in the absence of cocaine (in vivo-in vitro). When cocaine (78 mg/kg) was administered in vivo on day 8 and embryos were assessed on day 10, significant vascular perturbations, in the form of vasodilation and hemorrhage, as well as neural defects, were observed. In the in vitro system, day 8 embryos were cultured for 48 hr in the presence of 0, 10, 20, 33, and 66 micrograms/ml cocaine. At 10 and 20 micrograms/ml, vascular perturbation was not seen, while at higher cocaine concentrations, development of the yolk sac vasculature was inhibited. Hemorrhage was not a feature of in vitro cocaine embryotoxicity. However, significantly increased incidences of neural defects were seen at concentrations of 20 micrograms/ml or greater. Finally, in the in vivo-in vitro system, 78 mg/kg cocaine was administered on day 8 in vivo and embryos were dissected after 15 min and cultured for 48 hr. Marked cardiovascular perturbation, as well as neural defects, were produced using this protocol. With cocaine treatment, only 26.6% of embryos had a functioning heartbeat and yolk sac circulation, compared to 85.6% of controls. This cardiovascular disruption was associated with pooling of blood in the embryo, with 59.9% of embryos exhibiting marked vasodilation and hemorrhage compared to 12.5% in controls. Additional manifestations of cardiovascular perturbation were edema and blisters observed in cocaine-treated embryos. Neural tube defects, including open neural tube (8.3%) and microcephaly/hypoplastic prosencephalon (30.0%), were also significantly increased in cocaine-treated embryos. The cardiovascular and neural effects produced by cocaine were dose-dependent (40, 20 mg/kg). Thus, administration of cocaine in the in vivo or in vivo-in vitro systems produced marked cardiovascular effects, while in vitro treatment did not. These results suggest that cocaine may elicit cardiovascular toxicity through a maternally mediated mechanism.

Role of oxygen free radicals in cocaine-induced vascular disruption in mice.

To test the hypothesis that cocaine-induced embryonic vascular disruption is mediated by oxygen free radicals, the antioxidants 2-oxothiazolidine-4-carboxylate (OTC) and alpha-phenyl-N-t-butyl nitrone (PBN) were employed. When cocaine (78 mg/kg) was administered on day 8 of gestation to ICR mice and embryos evaluated on day 10 (in vivo), 62.3% of cocaine-treated embryos showed increased vasodilation compared to 4.9% for controls, and 33.1% of the cocaine-exposed embryos showed marked hemorrhage compared to 3.3% for controls. In addition, cocaine increased the incidence of neural defects, in the form of open neural tube, hypoplastic prosencephalon, and microcephaly. Administration of OTC (0.25 and 0.5 mmol/kg) or PBN (300 mg/kg) prior to cocaine significantly reduced cocaine-induced vasodilation and hemorrhage, while not preventing neural defects. When cocaine (78 mg/kg) was administered in vivo on day 8 of gestation and embryos were dissected 15 min later and subsequently cultured for 48 hr in the absence of cocaine (in vivo-in vitro), marked vascular disruption was observed: normal yolk circulation/heartbeat was decreased to 26.6%, while edema/blisters and vasodilation/hemorrhage were increased to 45.6% and 59.6%, respectively. Administration of PBN (300 mg/kg) prior to cocaine completely prevented cocaine-induced vascular disruption. When cocaine was administered in vivo and PBN (300 micrograms/ml) was incubated with cultured embryos in vitro, the antioxidant only partially prevented cocaine-induced cardiovascular defects in this model. Neural defects produced by cocaine were not significantly affected by PBN, administered either in vivo or in vitro. Cocaine (78 mg/kg) administered in vivo stimulated lipid peroxidation maximally after 3 hr in both day 8 and day 9 embryos. When cocaine was incubated in vitro during embryo culture at 33 micrograms/ml, a concentration that produces nonspecific inhibition of growth and development, embryonic lipid peroxidation on day 9 was not affected. Finally, when PBN (300 mg/kg) was administered prior to cocaine (78 mg/kg) on day 8 of gestation, stimulation of lipid peroxidation by cocaine was prevented. These results suggest that cocaine-induced vascular disruption in early development is mediated by maternal production of oxygen free radicals.

Substance abuse in pregnancy: teratogenesis.

Substances of abuse include those that are legal (such as alcohol) and those that are illegal (street drugs). Many of these agents produce reproductive toxicity including intrauterine growth retardation. Teratogenesis is unproven with most of these agents. Alcohol is an exception, producing the fetal alcohol syndrome. Cocaine causes marked reproductive toxicity including decreased growth and morbidity. A number of birth defects have been associated with cocaine use including genitourinary, cardiac, and limb anomalies. The reproductive toxic and putative teratogenic effects of cocaine are probably associated with its well-known pharmacologic action causing vasoconstriction. From preliminary studies, it would appear that methamphetamine also produces reproductive toxic effects similar to those of cocaine.

Ethanol-induced limb defects in mice: effect of strain and Ro15-4513.

It is now thought that ethanol exerts many of its behavioral effects in the CNS by interaction with the gamma-aminobutyric acid (GABA) receptor, and it has been shown that the benzodiazepine reverse agonist Ro15-4513 reverses some of the CNS effects produced by ethanol. The hypothesis was tested that ethanol exerts its teratogenic effects through interaction with a putative embryonic GABA receptor by determining whether Ro15-4513 reverses ethanol-induced forelimb ectrodactyly in C57BL/6 mice. First, pregnant C57BL/6 dams were injected twice i.p. with ethanol (2.9 g/kg body weight, 4 hr apart) on day 10 of gestation: 49% of the fetuses were resorbed or dead and 46% of the survivors showed forelimb ectrodactyly. In contrast, when SWV mice were treated with ethanol, embryolethality was only 11.9% and no forelimb ectrodactyly was observed. In a second experiment, when ethanol (2.6 g/kg x 2) was administered to C57BL/6 mice, 34% resorptions and 31% forelimb ectrodactyly were observed. Ectrodactyly induced by ethanol was primarily of the forelimb and exclusively postaxial. Ethanol produced an unusual forelimb defect in a small number of instances where there was a postaxial autopod reduction defect coupled with a preaxial zeugopod reduction defect. Ro15-4513 administered alone (50 mg/kg x 2) was neither embryolethal nor teratogenic in C57BL/6 mice. To attempt to reverse the teratogenic effect of ethanol, dams that were injected 5 min before each ethanol administration with Ro15-4513 (0.5, 1, 2.5, 5, 10 mg/kg twice) showed no significant change in frequency of forelimb ectrodactyly compared to embryos treated with ethanol alone. However, resorptions increased significantly to 77% and 62% with the 5 and 10 mg/kg doses of Ro15-4513. Thus there appears to be an embryolethal interaction of Ro15-4513 with ethanol. Nevertheless, since Ro15-4513 did not reverse the teratogenic effect induced by ethanol, these results do not support the hypothesis that the teratogenic mechanism of ethanol is mediated through a putative embryonic GABA receptor.

Chloride transport in embryonic cells: effect of ethanol and GABA.

Ethanol and GABA (gamma-aminobutyric acid) and their interaction on 36Cl-influx were analyzed in cultured embryonic palate and limb mesenchymal cells in order to determine whether ethanol exerts its teratogenic action through a GABA receptor involved in embryogenesis. Cl- transport in secondary cultures of C57BL/6 palate mesenchymal cells was shown to consist of three systems including the electroneutral Cl-/HCO3- exchange (50%) and Na+/K+/Cl- cotransport (30%) pathways and the voltage-dependent Cl- channel (20%). Treatment with DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or SITS (4-acetamido-4'-isocyano-stilbene-2,2' disulfonic acid) in SWV palate cells inhibited the Cl-/HCO3- exchange pathway, while treatment with DIDS and bumetanide inhibited both the exchange and cation cotransport pathways, the residual Cl- influx inferred to be the electrogenic pathway. Inhibition of Cl- transport by anthracene-9-carboxylic acid confirmed the presence of the electrogenic Cl- pathway. It was observed that the rate of Cl- transport was significantly greater in palate cells of C57BL/6 mice than those of SWV mice. Also the rate of Cl- transport was significantly greater in secondary cultures of palate cells from C57BL/6 mice than from primary cultures of limb cells from the same strain. No evidence could be obtained that ethanol (10 to 100 mM) or GABA (3 X 10(-5) M) or their combination stimulated total Cl- influx in either C57BL/6 or SWV palate mesenchymal cells, putative voltage-dependent Cl- influx in C57BL/6 palate cells, or total Cl- influx in primary cultures of C57BL/6 limb mesenchymal cells.

Sites of serotonin uptake in epithelia of the developing mouse palate, oral cavity, and face: possible role in morphogenesis.

With the method of whole mouse embryo culture, together with immunocytochemistry with an antiserum to serotonin (5-HT), sites of 5-HT uptake were found to be transiently expressed in the epithelia of the developing palate, tongue, nasal septum, and maxillary and mandibular prominences during the period of active morphogenesis (embryonic days 12-14; or E12-14). These sites had the ability to take up 5-HT when added to the culture medium in the presence of the MAO inhibitor nialamide and an antioxiant, L-cysteine (NC), and could also be seen after exposure of embryos to the 5-HT precursor L-tryptophan (L-TRP) + NC. These sites were also visible after culturing embryos without any additives, which may have been due to the presence of L-TRP in one component of the culture medium (DMEM) or to 5-HT itself, which is present in relatively high amounts in fetal calf serum. At E12-13, the appearance of 5-HT immunoreactivity (IR) at these sites after treatment with 5-HT + NC was blocked by the 5-HT uptake inhibitor fluoxetine, providing further evidence that these are true sites of 5-HT uptake. However, fluoxetine did not completely block the appearance of these sites in E14 embryos after 5-HT + NC or L-TRP + NC although it was effective with NC alone. This finding could mean that at E14 5-HT uptake into these sites occurs by mechanisms not completely blocked by fluoxetine or that there is some limited capacity for 5-HT synthesis. Taken together with results from previous studies where 1) 5-HT has been reported to stimulate palatal shelf reorientation and palatal mesenchyme cell motility in vitro [Wee et al., J Embryol Exp Morphol 53:75-90, 1979; Zimmerman et al., J Craniofac Genet Dev Biol 3:371-385, 1983] and 2) long-term culturing of mouse embryos in the presence of 5-HT or fluoxetine has been shown to cause malformations of the craniofacial region (Lauder, Thomas, and Sadler, in preparation), the results of the present study suggest that 5-HT could act as a developmental signal in the palate, oral cavity, and face during the period of active morphogenesis.

Diazepam-induced cleft palate in the mouse and lack of correlation with the H-2 locus.

Cleft palate frequencies were studied in AJ and SW mice following either 1- or 2-day dosing schedules with the anxiolytic drug diazepam (DAZ). In all cases, mice were food and water deprived for 24 and 48 hours in the 1- and 2-day dosing schedules, respectively. High cleft palate frequencies in control mice of both strains resulting from 48-hour food and water deprivation (on days 13.5 and 14.5 of gestation) were reduced in mice deprived for 24 hours, indicating a stress related effect. Two-day dosing with DAZ (400 mg/kg) produced a net increase in cleft palate frequency in SW (33%) and AJ (18%) mice. Mice treated only on day 13.5 had reduced control and DAZ cleft palate frequencies, neither of which were significant. Clefting was significant but reduced following 1-day dosing on day 13/20 of gestation (13 days 20 hours) in SW mice (18%), whereas no clefting was seen in the AJ strain. This strain difference was shown not to be related to differences in developmental timing. Production of cleft palate seen in AJ mice after 2 days of dosing may be indicative of an interaction of DAZ with the stresses resulting from food and water deprivation. Genes of the major histocompatibility locus, H-2, have been shown to regulate cleft palate formation following glucocorticoid and phenytoin administration to mice. Despite pharmacological similarities between DAZ and phenytoin, comparison of cleft palate frequencies following administration of DAZ to various strains of mice of different H-2 haplotypes indicated that genes associated with the H-2 locus do not regulate DAZ-induced cleft palate in these strains.

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro.

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.

The effect of reducing ATP levels on reorientation of the secondary palate.

The force for directing palate shelf reorientation appears to be associated with elements of the presumptive hard palate (Brinkley & Vickerman, 1979; Bulleit & Zimmerman, 1985). The palatal elements that mediate this process do not require palate cells to be metabolically active for expression of the force. This contention was demonstrated using an in vitro system that allows substantial reorientation of the hard palate to occur. ATP levels were reduced by treatment with metabolic inhibitors and the degree of reorientation was measured 1 h following pretreatment with inhibitors. Treatment of cultured embryonic heads under anoxic conditions with 2,4-dinitrophenol or KCN had no effect on the degree of reorientation occurring in vitro. These agents reduced ATP levels by 71% and 62%, respectively. Treatment of cultured heads with 2-deoxy-D-glucose under anoxia also had no effect on reorientation. This treatment reduced ATP levels in embryonic heads by 92-94%. A similar reduction was observed if ATP levels were measured in palate tissue alone. The treatment of cultured heads with 2-deoxy-D-glucose and anoxia not only reduced levels of ATP but also reduced CTP, GTP and UTP. These results indicate that palate shelf reorientation is independent of cellular metabolic activity and supports the hypothesis that reorientation is dependent on a pre-existing infrastructure within the palate shelves.

Presence of gamma-aminobutyric acid in embryonic palates of AJ and SWV mouse strains.

The presence of gamma-aminobutyric acid (GABA) in the embryonic palate was sought as a criterion for its role in regulating palate development. GABA was measured by a gas chromatographic-mass spectrometric (GC-MS) method using the heptafluorobutyryl (HFB)-cyclohexyl-GABA derivative, which gave the necessary sensitivity and specificity to measure low levels of GABA in the presence of contaminating substances. GABA was measured in dissected embryonic palates at various times of development in the AJ mouse strain. GABA levels were lower in day 14 AJ palates (0.19 +/- 0.01 nmol/mg protein) than at days 13 (0.28 +/- 0.03) and 15 (0.30 +/- 0.04). Comparable levels were observed in fore- and hindlimbs at day 14, whereas levels were lower in embryonic tongue and higher, as was expected, in embryonic brain. To confirm the presence of GABA in the palate, it was analyzed in growing palate mesenchymal cells in primary and secondary cultures as well as in serum-free medium. In addition, GABA levels were compared in the SWV mouse strain; this strain exhibits a more efficient active uptake mechanism and diazepam produces a higher frequency of cleft palate in this strain than in AJ. SWV contained one and one-half to three times higher concentrations of GABA in excised palates and cultured palate cells than the AJ strain. Furthermore, when GABA levels in skin fibroblasts of the two strains were measured, SWV cells contained 2.7-fold greater GABA than AJ cells. The present results provide additional evidence for the role of GABA in palate development.

GABA uptake in embryonic palate mesenchymal cells of two mouse strains.

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate. Palate cells were capable of accumulating [3H]GABA by saturable uptake mechanisms characteristic of a high and low affinity active transport as indicated by temperature, Na+ ion and carrier dependence as well as Km and Vmax values that were comparable to other biological systems. The Vmax of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells. When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential responses to GABA.

The influence of the epithelium on palate shelf reorientation.

The intrinsic forces necessary for directing the reorientation of the secondary palate appear to reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could reorient regardless of whether it was intact or separated from the posterior third or presumptive soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing the epithelium, from either the presumptive oral or nasal surface followed by measurement of reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was shown to remove 41% of the oral epithelium leaving the majority of the basement membrane intact. The observed inhibition of reorientation did not appear to be a consequence of wound healing. Creation of wounds twice the area that was observed after treatment with EDTA and scraping inhibited reorientation minimally. These results suggest that the epithelium and particularly the anterior oral epithelium plays a major role in the reorientation of the murine secondary palate.

Role of neurotransmitters and teratogens on palate development.

Neurotransmitters regulate palate shelf reorientation. Acetylcholine and serotonin stimulate, whereas GABA inhibits reorientation. Serotonin stimulates cell movement in an in vitro chemotactic system. Diazepam may cause cleft palate by mimicking GABA. Diazepam sensitivity may be caused by genotypic differences in a GABA-ergic system in the embryo.

Role of neurotransmitters in palate development and teratologic implications.

It is hypothesized that neuropharmacologic agents are more teratogenic to humans. Since many neuropharmacologic agents function through neurotransmitter mechanisms, then neurotransmitters should function to regulate embryonic development. Evidence has been obtained that neurotransmitters do indeed function as biological signals in palate development. It has been shown that palate reorientation is modulated by neurotransmitters with a wide range of diversity, similar to the CNS. Thus serotonin and acetylcholine stimulate and GABA inhibits the reorientation process. Spatial diversity is also observed: serotonin functions at the anterior and acetylcholine at the posterior end, and GABA functions more efficiently at either end in different inbred strains. Many criteria for functioning neurotransmitters have been obtained. Both serotonin and GABA have been measured in the palate and developmental changes observed. Physiologic responses to serotonin have been monitored. Serotonin has been shown to stimulate palate cell motility as well as protein carboxyl methylation and cyclic GMP. The serotonin effects on protein carboxyl methylation and cyclic GMP could function to stimulate palate reorientation by modulating cell contractility and protein secretion. Further support for the hypothesis that neuropharmacologic agents could be teratogenic by perturbation of neurotransmitter mechanisms comes from studying GABA and diazepam. Evidence has been obtained that diazepam induces cleft palate by mimicking GABA in a functional GABAergic system in palate development. A significant finding is that genetic differences in both diazepam teratogenesis and in a GABAergic system have been observed. Comparing the SWV and AJ strains, the SWV mouse showed (1) a greater sensitivity to diazepam-induced cleft palate, (2) a greater sensitivity to GABA and diazepam inhibition of palate reorientation in embryo culture, (3) a greater concentration of palatal GABA and (4) a more efficient GABA uptake system. These results are suggestive that diazepam may be more teratogenic in individuals of both the mouse and human who possess a more developed GABAergic mechanism. If such a result would obtain in the human, these genotypic differences in GABA would probably not influence the individual to a significant degree in most situations, except for the pregnant woman being at high risk when treated with diazepam.

Histochemical localization of glycosaminoglycans during morphogenesis of the secondary palate in mice.

The hydration of hyaluronic acid (HA) accumulated in the secondary palatal processes is expected to exert an intrinsic tissue pressure that could, in part, provide the impetus for shelf reorientation. Glycosaminoglycans were histochemically localized in the A/J mouse palate during development (days 12 to 15) by specific enzymatic degradation followed by preferential staining with alcian blue under differential pH or MgCl2 concentration. The presence of HA and chondroitin sulphates A and C (CS) was demonstrated in proportions that differed regionally. At the time of reorientation (days 14 to 15) HA was the predominant staining component, being distributed according to the relative prominence of extracellular spaces (ECS). HA was present in higher concentration in the anterior than the posterior part of the palate, particularly in an area of low cell density adjoining the CS-rich mesenchyme of the maxillary process. This arrangement suggests that the maxillary process might provide a resilient incompressible structural base for the palate as its HA-rich ECS expands. Sulphated GAG, with CS being the predominant component, was localized for the most part on the oral-side mesenchyme both in the anterior and posterior palate. The most intense staining of sulphated proteoglycans occurred in association with the basal lamina along the presumptive oral-side. Mesenchymal cells along this region appeared condensed and may have been stabilized by these sulphated GAG providing structural constraints which might function in palate morphogenesis.

Rapid gas chromatographic--mass spectrometric quantitation of gamma-aminobutyric acid in biological specimens.

A mass fragmentographic method for gamma-aminobutyric acid (GABA) quantitation using the heptafluorobutyryl-cyclohexyl-GABA derivative is described. Both capillary and packed column gas chromatography were used. This procedure employs 2,2[2H2]GABA as an internal standard and allows the rapid, sensitive, and specific measurement of GABA with a minimum of sample clean-up. Application of the method is demonstrated in mouse embryonic brain, body, and palate and human platelets, plasma, cerebrospinal fluid, and urine.

The effects of dexamethasone on palate mesenchymal cell phospholipase activity.

Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with [3H]arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.

Neuropharmacologic teratogenesis and neurotransmitter regulation of palate development.

Neuropharmacologic agents may be more teratogenic to human beings by perturbing neurotransmitter mechanisms that regulate embryonic development. There is evidence that neurotransmitters function in mouse palate development: Serotonin and acetylcholine stimulate and GABA inhibits shelf reorientation. Both serotonin and GABA have been measured in the palate and uptake systems monitored. Serotonin stimulates cell motility, protein carboxyl methylation, and cyclic GMP. Diazepam (Valium) could cause cleft palate by mimicking GABA. Genetic differences in both diazepam teratogenesis and in a GABAergic system of the mouse have been observed. If human beings were to show genetic differences in a GABAergic system, some pregnant women could be at high risk when treated with diazepam.