Uptake and blood pressure studies in the chick embryo following treatment with a teratogenic dose of dopamine

We report on the ventricular pressor response and regional (head, heart, and body) uptake of dopamine hydrochloride (DA) following topical application of a teratogenic dose to the stage 24 chick embryo. Embryos treated with DA exhibited a significant increase in mean ventricular blood pressure (MVBP) at 0.5 and 1 hour after treatment when compared to saline-treated control embryos. The time of the elevated MVBP correlated with the time of the peak uptake of DA in the embryonic heart. Even though the level of DA in the heart remained high for 9 hours, there was no measurable increase in blood pressure beyond 1.5 hours, suggesting unresponsiveness to DA or perhaps conversion of DA to its metabolites. These experiments have correlated a physiologic response of a known cardiovascular teratogen to its uptake in the embryo.     

Ventricular blood pressure and cardiac output changes in epinephrine- and metoprolol-treated chick embryos

The effects of a teratogenic dose (5 micrograms) of epinephrine on mean ventricular blood pressure (MVBP) and cardiac output (CO) at one and two hours after treating stage 24 chick embryos were investigated. Previous work demonstrated that a differential response in terms of cardiac rhythm during the first hour after epinephrine treatment was related to pathogenesis of two contrasting types of aortic arch malformations. Absence of one or more aortic arches occurred more frequently in embryos which developed a characteristic dysrhythmia, while persistence of the left fourth aortic arch (PL4AA) occurred more frequently in nondysrhythmic embryos. In this study, dysrhythmic epinephrine-treated embryos exhibited reductions in both MVBP and CO at one hour after treatment when compared to control values. Nondysrhythmic epinephrine-treated embryos exhibited elevated MVBP and no change in CO at one hour after treatment. MVBP and CO in recovered dysrhythmic and nondysrhythmic embryos were similar to control values at two hours following epinephrine treatment. MVBP and CO measurements were obtained from embryos which were pretreated with metoprolol and then subsequently treated with epinephrine. Metoprolol is a beta 1-adrenoreceptor antagonist which was previously shown to block the teratogenic effects of epinephrine and other catecholamines with beta 1-adrenoreceptor agonist properties. Pretreating embryos with metoprolol in this study reduced the dysrhythmogenic potential of epinephrine and also blocked the MVBP and CO changes observed in embryos treated with epinephrine alone. We conclude that pathogenesis of 1) abnormally absent aortic arches is related to dysrhythmogenesis, reduced MVBP, and reduced CO, and 2) an abnormally persistent left fourth aortic arch is related to elevated MVBP in the epinephrine model.     

The antidysrhythmic effect of metoprolol in the epinephrine treated chick embryo

It was confirmed through electrocardiography that within two hours after epinephrine treatment, four day chick embryos either maintained normal rhythm or developed a severe cardiac dysrhythmia (22/93, 24% dysrhythmic). The ECG dysrhythmia in epinephrine treated embryos were characterized by periods of bradycardia, asystole, and various supraventricular or ventricular dysrhythmias. Within four hours after treatment, dysrhythmic embryos either reestablished normal rhythmicity or died. Electrocardiographic data also demonstrated that metoprolol pretreatment will block epinephrine induced dysrhythmias (0/46, 0% dysrhythmic). We conclude that metoprolol possesses antidysrhythmic properties in the epinephrine treated chick embryo.     

Immunohistological localisation of monoclonal antibody R 24-recognized ganglioside Gilac2* in early chick embryos

The spatio-temporal cellular expression and biosynthesis of ganglioside Glac2 was investigated in early chick embryogenesis. For demonstration of embryonic Glac2-biosynthesis, chick embryos of stage 0 and of stages 4-5 were incubated in vitro in the presence of radioactive sugar precursors. It was found that chick embryos synthesize Glac2 as early as at the blastula stage as well as at the gastrula stage, both within the area pellucida and the area opaca. In contrast to the biosynthetical findings immunohistochemical staining of the chick embryos at various stages by aid of the mouse monoclonal antibody (mAb) R 24, specific for the immunoepitope NeuAc alpha, 8NeuAc alpha, 3Gal beta less than, as present on the ganglioside Glac2, revealed a spatio-temporal cellular pattern of expression of this ganglioside in early chick embryos. Immunohistochemical staining of the chick embryo at stage 0 shows that all cells of the embryo, the extraembryonic epiblast and the yolk endoderm included, are mAb R 24-positive. At the intermediate streak stage (stage 3), the cranial part of the deep layer, the so-called endophyll, is strongly mAb R 24-positive, whereas at the end of gastrulation (stage 5), mAb R 24-recognized epitopes appear to be restricted to a narrow band of deep-layer cells in the endophyllic crescent and to the yolk endoderm of the area opaca. At this stage, no labelling by the antibody is observed in cell layers of the future embryo. The beginning of neurulation (stage 7) is characterized by the expression of the mAb R 24-recognized epitope in the notochord, whilst the deep layer in the cranial part of the neural fold still expresses this epitope. No ecto- or mesodermal structures are stained by the antibody at this developmental stage. During further development (stage 12 and 13), mAb R 24-reactivity is restricted to the cranial part of the embryo with a preferential staining of cells of endodermal origin. At these stages, the notochord expresses mAb R 24 binding sites only in its cranial region. The spatial and temporal correlation between the presence of mAb R 24-recognized epitopes and the morphogenetic positioning of tissues may be indicative for a possible role of the ganglioside Glac2 in corresponding cellular interactions.     

Influence of L.5-hydroxytryptophan (L.5-HTP) on the development of chick embryo]

L.5-hydroxytryptophan (L.5-HTP) injections provoke, in the chick embryo, some malformations of the nervous system, when treated at 24 hours of incubation. The same treatement after 48 hours of incubation does not lead to malformations, but to a reduction in size which is as much obvious as the embryos are treated at a later stage. It seems that there could be some relation between the serotonin metabolism and the growth hormon secretion.     

Hemodynamic effects of acetylcholine in the chick embryo and differences from those in the rat embryo

It has been reported that acetylcholine induces cardiac anomalies in the chick embryo. Thus, we studied hemodynamic effects of this drug in the chick embryo and also compared them with those in the rat embryo since we found that the effect of caffeine was different between the chick and rat embryos. Acetylcholine was given at doses of 5, 0.5, and 0.05 micrograms into the vitelline vein in chick embryos at Hamburger-Hamilton stage 21 and at a dose of 0.5 micrograms into the placenta in rat embryos at gestational day 12. In the chick embryo, heart rate was reduced to 91, 88, and 87% of control at the end of injection of 0.05, 0.5, and 5 micrograms, respectively, then returned to the baseline level. Vitelline arterial blood pressure was 110% of control with 0.05 micrograms, 134% with 0.5 micrograms, and 142% with 5 micrograms at 1 min after injection. The dorsal aortic blood flow decreased with time after injection, but it was increased only by a 5 micrograms dose at the end of injection. The vascular resistance increased in a dose-dependent manner. In the rat embryo, the change of heart rate was qualitatively similar to that of the chick embryo. The blood pressure did not change significantly. The blood flow velocity at the outflow tract decreased at the end of injection, which indicated the decrease in cardiac output, along with slowing of heart rate, then returned to the control level thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

In adult systems, high homocysteine (HoCys) levels inhibit methylation reactions and can induce apoptosis in the central nervous system. In embryos, exogenous HoCys is teratogenic and is associated with neural tube defects. Because, methylation inhibitors and inducers of apoptosis can influence membrane composition, we have studied whether or not embryonic exposure to HoCys influenced membrane phospholipid levels, membrane fatty acid composition, and Caspase-3 activities in embryonic chick brains. Embryonic exposure to HoCys caused reduced brain phosphatidylcholine levels and increased levels of brain phosphatidylethanolamine. Exogenous HoCys also promoted decreased levels of long-chain, unsaturated membrane fatty acids and increased levels of saturated short-chain membrane fatty acids. These HoCys-induced brain membrane changes correlated with HoCys-induced increases in brain Caspase-3 activities, HoCys-induced reductions in brain mass, HoCys-induced reductions in embryo mass, and HoCys-induced reductions in the percentage of embryos that survived to 11 days of development (theoretical stage 37). Thus, HoCys-induced changes in brain membrane composition correlated with HoCys-induced apoptosis and reduced embryo viability.     

A study of the inductive capacity of post-nodal primitive-streak pieces after treatment with follicle-stimulating hormone

Summary Chick embryos at the primitive-streak stage were treatedin vitro with 0.002 and 0.008% follicle-stimulating hormone (FSH)(NIH-FSH-S1) for 24 hours. Post-nodal primitive-streak pieces 0.8 mm behind the node level were grafted into host chick embryos at the primitive-streak stage to assess the capacity of the grafted pieces to produce inductions.Control grafts from donors maintained under identical conditions were unable to cause induction; all of them were resorbed into the host embryo. The post-nodal pieces treated with FSH acquired capacity to induce neural tissue. The grafts seemed to induce foregut formation also. FSH appeared to have supported differentiation of the grafts into somites and mesenchyme.     

Pulmonary stenosis with ventricular septal defect, common aorticopulmonary trunk, and dextroposition of the aorta: morphologic and qualitative physiologic effects in caffeine-treated chick embryos

Effects of caffeine administration to Hamburger-Hamilton stage 19 chick embryos (3 days of incubation) were investigated. A morphologic study of the effect of caffeine on cardiogenesis showed that caffeine produced total cardiac malformations in the chick in a dose-related fashion. A maximum frequency of 70.6% was observed with 4.7 mg caffeine. Major malformations included common aorticopulmonary trunk and dextroposition of the aorta accompanied by ventricular septal defect with/without pulmonary stenosis. Qualitative analysis of cinegraphs following exposure of embryos to a single teratogenic dose of caffeine (3.5 mg/egg) produced marked alterations in cardiac function when compared with chick Ringer's controls. Within 3 minutes after exposure to caffeine, dilation of the common ventricle and weak ventricular contractility were observed and persisted for 1 hour. Dose-response data and microcinematographic observations suggest that caffeine induced cardiac anomalies by a direct toxic effect on the embryo rather than by altering cardiac cell function. Our data also suggest that pathophysiologic changes in cardiac function may play an important role in the pathogenesis of caffeine-induced cardiac anomalies in the chick embryo.     

MK801 antagonism of the prolonged depletion of striatal dopamine by amphetamine in iprindole-treated rats.

The administration of amphetamine to rats pretreated with iprindole to inhibit the metabolism of amphetamine results in a long-lasting depletion of striatal dopamine and its metabolites, DOPAC and HVA. Pretreatment with MK801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) subclass of excitatory amino acid receptors, antagonized the depletion of striatal dopamine, DOPAC and HVA 3 days after a single dose of amphetamine in iprindole-treated rats. MK801 pretreatment was effective up to 4 hours but not at 8 or 24 hours in preventing amphetamine effects on striatal dopamine, DOPAC and HVA.     

Strain differences in the teratogenicity induced by sodium valproate in cultured mouse embryos

Strain differences in the teratogenicity of valproic acid (VPA) have been reported in mice. Finnell and Chernoff (Proc. Grnwd. Genet. Ctr. 5:162-163, 1985) showed that 300 mg/kg of VPA twice a day on days 6-8 of gestation induced exencephaly in 82% of SWV embryos but in 0% of C57BL/6J embryos. In the present experiment, we have collected similar results and investigated this strain difference using whole embryo culture in an attempt to determine whether maternal or embryonic factors are responsible for the difference. Mouse embryos were explanted on day 8.5 (plug day 0), and embryos at the 6-8-somite stage were cultured for 48 hours in rat serum containing various doses of sodium valproate (NaVP). All the embryos died within 24 hours with 4.5-mM and higher doses of NaVP in C57BL/6NCr1BR (C57) and with 3.0-mM and higher doses in SWV. Unfused brain folds were recognized in embryos treated with 3.0-mM and higher doses in C57, and with 1.0-mM and higher doses in SWV. Irregular somite formation was observed in many embryos treated with 1.6-mM and higher doses in C57 and with 1.0-mM and higher doses in SWV. These results indicate that SWV embryos have 1.5-3 times the sensitivity of C57 embryos to the embryolethal and teratogenic effects of NaVP. Furthermore, the results suggest that the basis of the strain difference resides within the embryo rather than the mother.     

Lethal and teratogenic effects of long-term low-intensity radio frequency radiation at 428 MHz on developing chick embryo

Exposure of developing chick embryos to 428 MHz radio frequency (RF) radiation at a power density of 5.5 mW/cm2 for more than 20 days resulted in embryolethal and/or teratogenic effects and delayed hatching. These adverse biological effects were not due to any thermal effect of the RF radiation. We have demonstrated teratogenicity in the chick embryo as a result of protracted low-dose RF irradiation.     

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Comparative teratogenic effects of methyl salicylate applied orally or topically to hamsters

Methyl salicylate was administered topically to pregnant hamsters at 7d9h and the teratogenic results were compared with those obtained following oral treatment with the same compound. Both treatments produced the same defect in embryos recovered at day 9: failure of fusion of the neural tube, especially in the area of the developing brain. Analysis of serum salicylate levels following both treatments produced similar curves and indicated that teratogenic levels of salicylate can reach the maternal circulation after topical exposure.     

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Ham's F-10 or modified Dulbecco's phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Ham's F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .     

The effect of isoproterenol on cardiovascular function in the stage 24 chick embryo

The developing cardiovascular system of the chick embryo is susceptible to teratogenic effects of catecholamines. Yet the mechanism for the teratogenetic action is unclear. Since catecholamines affect cardiovascular physiology, we studied the acute effect of the beta-agonist isoproterenol on mean atrial pressure, heart rate, mean dorsal aortic blood flow, mean arterial pressure and vascular resistance in stage 24 chick embryos. Dorsal aortic blood velocity was measured with a 20-MHz pulsed-Doppler velocity meter and intravascular pressure was measured with a servo-null pressure system. Isoproterenol in doses of 2 X 10(-4) micrograms (2.5 micrograms/kg), 8 X 10(-4) micrograms (10 micrograms/kg), and 1.2 X 10(-3) micrograms (15 micrograms/kg) was injected intravenously in 5-microliters aliquots of chick Ringer's solution. Additional groups of embryos were treated with the beta-antagonist propranolol, and isoproterenol plus propranolol. Control embryos received 5 microliters chick Ringer's solution to assess the hemodynamic effects of a volume injection. We found that isoproterenol caused no change in mean atrial pressure, heart rate, or mean arterial pressure. However, isoproterenol caused a dose-related decrease in dorsal aortic blood flow and a 2.5-fold increase in vascular resistance. The effects of isoproterenol were blocked by propranolol, which suggested that the increase in vascular resistance was mediated by beta-receptor stimulation.     

Modulating effects of beta-naphthoflavone on the induction of SCEs by model compounds with emphasis on benzo[a]pyrene

The inducing capability of the synthetic flavonol beta-naphthoflavone (beta-NF) on cytochrome P-450 content was studied in primary chick embryo hepatocytes. In addition, the modulating effects of pretreatment with beta-NF on the induction of sister-chromatid exchanges (SCEs) in V79 cells by mutagens from different chemical classes were investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. Finally, the effects of pretreatment on benzo[a]pyrene (B(a)P) metabolism were studied in more detail. Pretreatment of cultured primary chick embryo hepatocytes with beta-NF resulted in a large increase in cytochrome P-450 content (a 2.8-fold increase after 31 h). Pretreatment with beta-NF had no effect on the level of SCEs induced by N-nitroso-dimethylamine (NDMA) and 2-aminoanthracene (2AA). Pretreatment with beta-NF resulted in a decrease in B(a)P-induced SCEs. This inhibitory potential was positively related to the beta-NF dose. However, there was an inverse relationship between the inhibitory action of beta-NF and the dose of B(a)P, at higher doses less inhibition was observed. When beta-NF was applied simultaneously with B(a)P the percentage of decrease was about the same as for pretreatment. Pretreatment with beta-NF followed by simultaneous application of beta-NF and B(a)P did not result in larger effects. In addition, subcellular fractions were prepared from chick embryos pretreated with beta-NF in ovo. The use of the S9 fraction resulted in a large decrease (80%) in the induction of SCEs in V79 cells by B(a)P whereas the use of the microsomal fraction resulted in a 70% increase in SCE induction compared with non-pretreated microsomes. Pretreatment with beta-NF in ovo gave rise to a large increase in aryl hydrocarbon hydroxylase (AHH) activity in the hepatic microsomal fraction. Increases were observed in the formation of all B(a)P metabolites. In particular the formation of the proximate carcinogenic and mutagenic metabolite B(a)P-7,8 dihydrodiol was increased 7-fold. The data strongly suggest that the inhibitory effects of pretreatment of cultured primary chick embryo hepatocytes with beta-NF cannot be ascribed to its inducing capabilities but instead seem to be due to the formation of an intracellular pool of beta-NF which acts as a competitive inhibitor for B(a)P metabolism.