Teratogenic effects of bis-diamine on the developing cardiac conduction system

BACKGROUND: Congenital heart defects, including conotruncal anomalies, are often associated with arrhythmias. Bis-diamine induces conotruncal anomalies in embryos when administered to pregnant female rats. To investigate the mechanism of arrhythmia in conotruncal anomalies, we histologically examined the development of the cardiac conduction system in this animal model. METHODS: A single dose of 200 mg of bis-diamine was administered to pregnant Wistar rats on ED 10.5 of pregnancy. The embryos were removed on each day from ED 11.5 to 15.5. Immunoexpression of HNK-1, connexin40, and connexin43 were examined in serial sections. The distribution pattern of TUNEL-positive cells around the conduction system was also examined. RESULTS: HNK-1 immunoreactivity was evident in interventricular septum, in both the control and the bis-diamine-treated embryos from ED 12.5. Although a chain of connexin40-immunoreactive cells from interventricular septum to trabeculae, corresponding to the His bundle and its branches, was demonstrated at ED 13.5 in the control embryos, this chain was first detected at ED 14.5 in the bis-diamine-treated embryos. Immunoexpression of connexin43 in the working myocardium was also less in the bis-diamine-treated embryos than in the control at ED 13.5. The number of TUNEL-positive cells in the interventricular septum was highest at ED 12.5 in the control and at ED 13.5 in the bis-diamine-treated embryos. Furthermore, these TUNEL-positive cells were HNK-1 negative, vimentin-positive, and alpha smooth muscle actin-positive. CONCLUSIONS: Bis-diamine disturbed the normal development of gap junctions and apoptosis of myofibroblasts around the HNK-1-positive conduction tissue through overall poor myocardial proliferation and growth.     

In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart

BACKGROUND: Maternal diabetes exposes embryos to periods of hyperglycemia. Glucose is important for normal cardiogenesis, and Glut-1 is the predominant glucose transporter in the embryo. METHODS: Pregnant mice were exposed to 6 or 12 hr hyperglycemia during organogenesis using intraperitoneal (IP) injections of D-glucose on gestational day (GD) 9.5 (plug = GD 0.5). Embryos were examined for morphology and total cardiac protein, and embryonic hearts were evaluated for Glut-1 protein and mRNA expression immediately after treatment (GD 9.75, GD 10.0), as well as on GD 10.5 and GD 12.5. RESULTS: IP glucose injections were effective in producing sustained maternal hyperglycemia. Maternal hyperglycemia for 6 or 12 hr on GD 9.5, followed by normoglycemia, produced a decrease in overall size and total cardiac protein in embryos evaluated on GD 10.5 but no difference on GD 12.5. Cardiac Glut-1 expression was immediately upregulated in embryos exposed to 6 or 12 hr maternal hyperglycemia. On GD 10.5, cardiac Glut-1 expression was not different in embryos exposed to maternal hyperglycemia for 6 hr but was downregulated in embryos exposed for 12 hr. On GD 12.5, cardiac Glut-1 expression in embryos exposed to maternal hyperglycemia on GD 9.5 for 6 or 12 hr, followed by normoglycemia, was not different from controls. The temporal pattern was the same for Glut-1 protein and mRNA expression. CONCLUSIONS: Hyperglycemia-induced alterations in Glut-1 expression likely interfere with balance of glucose available to the embryonic heart that may affect cardiac morphogenesis.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

The spatio-temporal expression pattern of cytoplasmic Cu/Zn superoxide dismutase (SOD1) mRNA during mouse embryogenesis

The cytoplasmic Cu/Zn-superoxide dismutase (SOD1) represents along with catalase and glutathione peroxidase at the first defense line against reactive oxygen species in all aerobic organisms, but little is known about its distribution in developing embryos. In this study, the expression patterns of SOD1 mRNA in mouse embryos were investigated using real-time RT-PCR and in situ hybridization analyses. Expression of SOD1 mRNA was detected in all embryos with embryonic days (EDs) 7.5–18.5. The signal showed the weakest level at ED 12.5, but the highest level at ED 15.5. SOD1 mRNA was expressed in chorion, allantois, amnion, and neural folds at ED 7.5 and in neural folds, notochord, neuromeres, gut, and primitive streak at ED 8.5. In central nervous system, SOD1 mRNA was expressed greatly in embryos of EDs 9.5–11.5, but weakly in embryos of ED 12.5. At EDs 9.5–12.5, the expression of SOD1 mRNA was high in sensory organs such as tongue, olfactory organ (nasal prominence) and eye (optic vesicle), while it was decreased in ear (otic vesicle) after ED 10.5. In developing limbs, SOD1 mRNA was greatly expressed in forelimbs at EDs 9.5–11.5 and in hindlimbs at EDs 10.5–11.5. The signal increased in liver, heart and genital tubercle after ED 11.5. In the sections of embryos after ED 13.5, SOD1 mRNA was expressed in various tissues and especially high in mucosa and metabolically active sites such as lung, kidney, stomach, and intestines and epithelial cells of skin, whisker follicles, and ear and nasal cavities. These results suggest that SOD1 may be related to organogenesis of embryos as an antioxidant enzyme.     

Improved development of rat embryos in culture during the period of craniofacial morphogenesis

To study the mammalian craniofacial development, the culture conditions of rat whole embryo during the period of major craniofacial morphogenesis were examined. The improved rotating apparatus which is gassed continuously was used. Rat embryos explanted at 11.5 days (plug day 0) developed in vitro for up to 72 hr, that is, throughout the period of major craniofacial morphogenesis, and cultured embryos showed normal facial formation. The medium was equilibrated with a gas mixture of 95% 02, 5% CO2. The 100% rat serum improved the protein content of embryos cultured for 48 hr compared with the medium consisting of 50% rat serum and 50% Tyrode solution, although somite number was not altered. Furthermore, 100% rat serum containing 2 mg/ml glucose was the best medium for supporting growth of embryos when it was measured by protein content. Thus, the best culture medium was pure rat serum containing 50 units/ml penicillin, 50 micrograms/ml streptomycin, and 2 mg/ml glucose. Protein content, body weight, craniofacial formation, and somite number of embryos cultured for 48 hr with continuous gassing were much better than those cultured with noncontinuous gassing.     

An in vivo/in vitro evaluation of the teratogenic action of excess vitamin A

Pregnant rats of CFHB strain were injected 81/2 days postcoitum with a 1% suspension of retinoic acid (RA) in arachis oil to give 20 mg RA per kg body weight. Control rats were injected with arachis oil only. After 26 hours, one uterine horn was removed from each rat and the embryos cultured in serum from untreated rats. The embryos in the other horn were allowed to continue development in vivo. After a further 48 hours the cultures were terminated and the second uterine horn removed from each rat. This provided four groups of embryos for comparison: (1) embryos from RA-treated rats, (2) cultured embryos from RA-treated rats, (3) embryos from control rats, and (4) cultured embryos from control rats. The results showed that the effects of the teratogen on the cultured embryos were similar to those on the embryos allowed to continue development for the same period in the mother. In both groups RA reduced protein synthesis, inhibited somite and limb bud formation, and caused various neural tube defects, particularly microcephaly and abnormalities in the closure of the anterior and posterior neuropores.     

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320–740 nm, 1600 lx) or decreased temperature (22 ± 1°C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37°C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37°C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos. Mol. Reprod. Dev. 50:30–34, 1998. © 1998 Wiley-Liss, Inc.     

Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia

By exposing chicken embryos to hypoxia (10%) acutely (2, 4, and 6 hr) during early development (2, 3, and 4 days) we tested the hypothesis that hypoxia has an impact on embryonic growth and impairs cardiac development at the time cardiac morphogenesis is taking place. After the hypoxic perturbation, the embryos were allowed to develop until day 9, when embryo mass, heart mass, and rate of oxygen consumption were recorded. Four-day-old embryos exposed to 6 hr of hypoxia showed an increased mortality (38.9% versus 18% for controls), indicating the immediate effect of hypoxia on survivability. While only 8% of the controls displayed morphological abnormalities, 3- and 4-day-old embryos exposed for 6 hr showed more frequent developmental abnormalities (25% and 30% respectively). No significant differences in embryo or heart mass were found except in 4-day-old embryos exposed for 2 hr. Mass-specific oxygen consumption was not different between controls and embryos exposed to hypoxia at 2 or 3 days of development, but it was increased in 4-day-old embryos exposed for 4 hr (P < 0.05). These results suggest that an acute hypoxic episode does not have an impact when occurring very early in development (days 2 or 3). However, when the hypoxic episode occurs on day 4, survivability is largely decreased. Considering the lack of permanent effects on the surviving embryos, we suggest that the early embryo resorts to a simple strategy of death or survival, and the individual capacity for survival must be based on interindividual differences rather than the existence of compensatory mechanisms. J. Exp. Zool. 286:450-456, 2000.     

Synchronous division of mouse two-cell embryos with nocodazole in vitro.

Mouse two-cell embryos were cultured in a medium supplemented with nocodazole or colcemid for 12.5-14.5 h in vitro, and development after elimination of these drugs was examined. All embryos cultured with nocodazole stopped at the metaphase of the second cell cycle. When nocodazole was removed, almost all embryos divided to the normal four-cell stage within 1 h and then developed into blastocysts (98%). The proportion of embryos that developed into young after transfer to recipients was not significantly different from the control (35 versus 36%), but the developmental ability of the embryos treated with colcemid was reduced, especially after transfer to recipients.     

N-CAM alterations in splotch neural tube defect mouse embryos.

The splotch (Sp) mouse is a model for both neurulation defects and defects in neural crest cell (NCC) derivatives. Since neurulation and NCC emigration from the neural tube occur at similar times in development, we suggest that these two events share a mechanism that, if disrupted, leads to malformations in both developmental pathways. Previous studies have shown that the underlying defect in these mutants may involve a mechanism that alters cellular organization and communication. Cell adhesion molecules (CAMs) have been linked with such interactions and because some, including N-CAM, are involved in neural development, we were interested in their pattern of expression in the splotch mutant. Immunolocalization studies showed similar temporospatial distributions of N-CAM antibody in embryonic day 9 mutants and controls. However, mutant embryos had a much higher intensity of anti-N-CAM fluorescence compared to controls. Further characterization using immunoblot analysis revealed that Sp mutants have an altered N-CAM polypeptide profile. Two N-CAM isoforms (Mr 140K and 180K, K = 10(3] are normally present at this time of development. However, extracts from Sp embryos display a heavier N-CAM species (Mr 200K), as well as an altered 140K isoform. Heterozygotes also exhibit a different N-CAM profile, displaying a band between 180K and 200K in addition to the normal 180K and 140K species. Microheterogeneity was also observed in mutant and heterozygous embryos carrying Spd, an allele of Sp. However, these differences were less dramatic than that of Sp. The Sp locus may be involved in post-translational modification of N-CAM. An aberration in N-CAM processing could be the primary target of the mutation that leads to the development abnormalities observed in this mouse mutant.     

Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat

The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity. and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1 microg/ml, estradiol-17beta; 0.5 microg/ml, FSH; 100 IU/ml, LH and 10% FCS on granulosa cell monolayer. After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PVA) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody. The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats.     

DNA hypomethylation of karyoplasts for bovine nuclear transplantation

The objective of this research was to evaluate if DNA hypomethylation in cells used as karyoplasts would improve development of bovine nuclear transplantation (NT) embryos. DNA from serum-fed (SF), serum-starved (SS), and 1, or 5 microM 5-azacytidine (5-aza-CR) treated cells was digested with a methylation sensitive enzyme, and evaluated for DNA methylation. A significant reduction in DNA methylation was observed in cells cultured for 48 or 72 hr in SS medium as well as in cells cultured for 48 hr in the presence of 5 microM 5-aza-CR when compared to cells cultured in SF medium. All other comparisons contained no significant differences when compared to controls. When donor cells were cultured in 5-aza-CR, SF, or SS treatment media for 48 hr, no significant difference was observed (P = 0.06) in blastocyst development rates after NT. One embryo produced by donor cells treated with 5-aza-CR established a pregnancy. Four pregnancies resulted from embryos produced by SS donor cell NT and 3 resulted from embryos produced by SF donor cell NT. Supplementation of the donor cell culture medium with 5-aza-CR was not beneficial for increasing blastocyst rate or establishing pregnancy after NT.     

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.     

A developmental study of the Desert hedgehog-null mouse testis.

Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in DROSOPHILA: A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5-12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.     

Cardiovascular anomalies in chick embryos produced by bis-diamine in dimethylsulfoxide.

N,N'-bis(dichloroacetyl)-1,8-octamethylenediamine(bis-diamin e) (100 micrograms) dissolved in dimethylsulfoxide (DMSO) was administered to early developing chick embryos (Hamburger-Hamilton stage 9-21) in order to clarify the teratogenic effects on the cardiovascular system and to determine whether bis-diamine interferes with the migration of neural crest cells. Of 346 cases, 154 (44.5%) survived. The incidence of cardiovascular anomalies was 149 out of 154 cases (96.8%). Infundibular ventricular septal defect, double outlet right ventricle, and persistent truncus arteriosus were the primary cardiac anomalies observed in this study. A high percentage of these anomalies were accompanied by hypoplasia of the right 6th aortic arch artery and persistent left 4th aortic arch artery. Particularly, administration of bis-diamine to chick embryos at stage 13 resulted in a high incidence of persistent truncus arteriosus (64.3%). Bis-diamine has been suspected to inhibiting the migration of neural crest cells. However, neural crest cells were observed in the tunica media of the great arteries and the truncal valves of persistent truncus arteriosus produced by bis-diamine in chimeric embryos at stage 13. Morphological changes such as cell death were not observed.     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

Maternal diabetes and cardiovascular malformations: predominance of double outlet right ventricle and truncus arteriosus

Most studies on the relationship of maternal diabetes to cardiovascular malformations (CVM) have been prospective investigations of pregnancy outcome and therefore could not identify associations with rare cardiac lesions. The results of a retrospective study shed new light on the risks of specific cardiac defects in diabetic pregnancies. The Baltimore-Washington Infant Study, a population-based case-control investigation of CVM, provides information on maternal diabetes reported in personal interviews. Among 2259 mothers of cases, 35 (1.5%) reported diabetes present before pregnancy (called "overt") and 95 (4.2%) reported diabetes only during pregnancy (called "gestational"). Among 2,801 mothers of controls, 14 (0.5%) had overt diabetes and 83 (3.0%) had gestational diabetes. Malformation-specific risks were expressed as odds ratios (OR) with 99.5% confidence intervals (CI). The strongest associations with overt maternal diabetes were found with double outlet right ventricle (OR 21.33; 99.5% CI 3.34, 136.26), and truncus arteriosus (OR 12.81; 99.5% CI 1.43, 114.64). No significant diagnosis-specific associations were found with gestational diabetes. Non-cardiac malformations were present in 23% of infants with CVM whose mothers had overt diabetes and in 26% of infants with CVM whose mother had gestational diabetes, in 32% of infants with CVM whose mothers did not have diabetes, and in 4% of controls. Double outlet right ventricle and truncus arteriosus are malformations dependent upon neural-crest-cell-derived ectomesenchymal tissues; these are precisely the conotruncal abnormalities that result from experimental ablation of the neural crest in chick embryos. The association with diabetes suggests a further etiologic link between these two lesions.     

Developmental defects of coronary vasculature in rat embryos administered bis-diamine

BACKGROUND: Conotruncal anomalies are often associated with abnormal coronary arteries. Although bis‐diamine is known to induce conotruncal defects, its pathological effects on coronary vascular development have not been demonstrated. This study sought to assess the teratogenic effects of bis‐diamine on coronary vascular development and the pathogenesis of this anomalous association. METHODS AND RESULTS: A single 200 mg dose of bis‐diamine was administered to pregnant Wistar rats at 10.5 days of gestation. Fifty‐two embryos from 10 mother rats underwent morphological analysis of the coronary arteries. Three embryos each were removed from four mothers on embryonic days (ED) 14.5, 15.5, 16.5, and 17.5 and used for immunohistochemical studies using the anti‐vascular cell adhesion molecule (VCAM)‐1 antibody. Conotruncal anomalies were detected in 48 of 52 embryos, and an aplastic or hypoplastic left coronary artery was found in all of them. In control embryos at ED 16.5, VCAM‐1‐positive epicardial cells were transformed into mesenchymal cells in vascular plexus, which appeared to differentiate into the endothelial cells of coronary vasculature. In the heart at ED 17.5, coronary vasculature was well developed and connected with coronary ostia near the aorta. However, poor epicardial–mesenchymal transformation and subsequent differentiation was revealed in bis‐diamine‐treated embryos at EDs 16.5 and 17.5, causing abnormal development of the coronary vasculature and incomplete connections with coronary ostia of the aorta. CONCLUSIONS: Anomalous coronary arteries in the bis‐diamine‐treated embryos are induced by the disruption of epicardial–mesenchymal transformation and subsequent poor development of coronary vasculature. Incomplete hatching of the coronary ostium is associated with abnormal truncal division. Birth Defects Res (Part B) 92:10–16, 2011. © 2010 Wiley‐Liss, Inc.     

Tolbutamide alters glucose transport and metabolism in the embryonic mouse heart

BACKGROUND: Tolbutamide is a sulfonylurea oral hypoglycemic agent widely used for the treatment of non insulin-dependent diabetes mellitus. Tolbutamide produces dysmorphogenesis in rodent embryos and becomes concentrated in the embryonic heart after maternal oral dosing. Tolbutamide increases glucose metabolism in extra-pancreatic adult tissues, but this has not previously been examined in embryonic heart. METHODS: CD-1 mouse embryos were exposed on GD 9.5 to tolbutamide (0, 100, 250, or 500 microg/ml) for 6, 12, or 24 hr in whole-embryo culture. Isolated hearts were evaluated for (3)H-2DG uptake and conversion of (14)C-glucose to (14)C-lactate. Glut-1, HKI, and GRP78 protein levels were determined by Western analysis, and Glut-1 mRNA was measured by RT-PCR. RESULTS: Cardiac (3)H-2DG uptake increased after exposure to 500 microg/ml tolbutamide for 6 hr, and 100, 250, or 500 microg/ml tolbutamide for 24 hr, compared to controls. Glycolysis increased after exposure to 500 microg/ml tolbutamide for 6 or 24 hr compared to controls. Glut-1 protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 12 or 24 hr, and Glut-1 mRNA increased in hearts exposed to 500 microg/ml tolbutamide for 24 hr compared to controls. HKI protein levels increased in hearts exposed to 500 microg/ml tolbutamide for 6 hr, but not 12 or 24 hr. There was no effect on GRP78 protein levels in hearts exposed to tolbutamide for 6, 12, or 24 hr. CONCLUSIONS: Tolbutamide stimulates glucose uptake and metabolism in the embryonic heart, as occurs in adult extra-pancreatic tissues. Glut-1 and HKI, but not GRP78, are likely involved in tolbutamide-induced cardiac dysmorphogenesis.