Ethyl alcohol reduces cardiac output, stroke volume, and end diastolic volume in the embryonic chick.

It has been established that ethanol causes both human congenital cardiac malformations and structural intracardiac abnormalities in the embryonic chick. In view of a theory that reduced embryonic tissue hemodynamics are associated with the development of malformations, we attempted to determine whether or not a) ethanol altered cardiac blood flow and b) altered hemodynamics were a function of ethanol dose in the chick embryo. Cardiac function in Hamburger-Hamilton stage 19 chick embryos was recorded on videotape before and up to 10 hours after exposure to graded doses of ethanol. Parameters of cardiac function, including cardiac output, were determined from videotaped images by means of computer assistance. Cardiac output decreased in a linear fashion with dose for up to 3 hours after exposure to ethanol. The maximum relative percent decrease in cardiac output was directly related to the dose of ethanol administered. Furthermore, the time required after ethanol treatment for mean cardiac output to return to pretreatment and control values was also dose-dependent--lower doses of ethanol required less time for mean cardiac output to return to pretreatment and control values. Although relatively high doses of ethanol depress cardiac rate, we attribute the significant decrease in cardiac output primarily to parallel dose-dependent decreases in both stroke volume and end diastolic volume. Our data are consistent with the hypothesis that reduced embryonic cardiac blood flow during cardiogenesis is associated with the development of ethanol-induced intracardiac defects in chick embryos.

Cardioteratogenic dose of ethanol reduces both lactic dehydrogenase and succinic dehydrogenase activity in the bulbar ridges of the embryonic chick heart.

It has been established previously that the chick embryo is an appropriate model for studying the pathogenesis of ethanol-induced ventricular septal defect and that the growth and fusion of the proximal bulbar ridges are critical to ventricular septation within the embryonic heart. To determine whether enzyme activity was altered significantly in bulbar ridges in response to a cardioteratogenic dose of ethanol, we injected 0.32 ml of 50% ethanol in chick Ringer's saline (CRS) into the air cells of 3-day-old chick embryos (72-84 h incubation, Hamburger-Hamilton stages 18-19, Dekalb Delta strain). Embryos were harvested 48-60 h after treatment (stages 26-27). Hearts were removed, frozen and sectioned, and the tissues were processed for enzyme histochemical demonstrations of lactic and succinic dehydrogenase. Twelve ethanol-treated embryos and 12 CRS-treated controls were compared for lactic dehydrogenase (LDH) levels. Fourteen ethanol-treated embryos and 14 controls were compared for succinic dehydrogenase (SDH) levels. Formazan deposits were quantified by means of computer-assisted microdensitometry. Our results showed that ethanol decreased the mean LDH activity level by 36.3% (P = 0.005) and the mean SDH level by 40.8% (P < 0.02). Furthermore, ethanol significantly reduced LDH activity in 41.7% of the matched embryo pairs and significantly reduced SDH activity in 57.1% of the matched embryo pairs. Although some investigators have proposed that a disturbance in neural crest cell migration plays a major role in the pathogenesis of ethanol-induced cardiac malformations, this study provides evidence that the bulbar ridges of the embryonic heart are also potential target tissues for ethanol.

Cardioteratogenic dose of ethanol in the chick embryo results in albumin ethanol concentrations comparable to human blood alcohol levels.

Three-day-old chick embryos (Hamburger-Hamilton stages 18-19) were exposed to a dose of ethyl alcohol (0.32 ml of 50% ethanol) that causes cardiac malformations in 96.6% of the animals. Ethanol was administered into the air sac after 72-80 h of incubation. Samples of albumin at the opposite pole of the egg were drawn 0-50 h after treatment and quantitated for ethanol concentration with capillary gas-liquid chromatography. Ethanol concentrations in the albumin increased significantly (P < 0.05) at 2, 5 and 15 h after injection of ethanol, reached a maximum mean ethanol concentration at 20 h (217.3 +/- 23.5 mg dl-1), decreased significantly at 30 h to 175.4 +/- 27.5 mg dl-1, then increased again and stabilized at 40-50 h. Individual sample concentrations ranged from 0 mg dl-1 (at 0.5-2 h) to 286.5 mg dl-1 at 40 h. Ethanol concentrations in the albumin were comparable to human blood alcohol levels during intoxication (> 150 mg dl-1). Our results suggest that a potent cardioteratogenic dose of ethanol in the chick embryo is reasonable in terms of potential human embryo exposure.

Strain-dependent effect of ethanol on ventricular septal defect frequency in White Leghorn chick embryos.

We have tested the potential of a single dose of ethanol (0.20 ml 50% ethanol in chick Ringer's saline (CRS) administered into the air sac) to produce ventricular septal defect (VSD) in three distinct commercially available strains of White Leghorn chick embryo: stress-resistant Dekalb Delta strain, Hy-Vac SPF type V, and Hy-Vac SPF type L. Eggs were controlled for both size and developmental stage (Hamburger-Hamilton stage 18-19) at time of injection. The frequency of VSD in Dekalb Delta embryos was 4.0% (1/25), in Hy-Vac SPF type L embryos 9.1% (3/33), and in Hy-Vac SPF type V embryos 38.9% (14/36). Statistical analysis with the two-tailed Fisher Exact Test indicated that frequencies were not significantly different (P = 0.3215) when Dekalb Delta and Hy-Vac type L embryos were compared. However, the frequency of VSD for Hy-Vac type V embryos was significantly greater than that for either the Dekalb Delta or the Hy-Vac type L embryos (P < 0.005). All VSDs observed were located within the crista supraventricularis. When Hy-Vac SPF type V embryos were exposed to either 0.20 ml 50% ethanol in CRS or to 0.20 ml CRS (controls), ethanol-treated embryos showed a VSD incidence of 34.1% compared with a 3.6% incidence in the controls (P = 0.0017). These data suggest that ethanol is the cause of VSD in this strain. From the results of this study, we are led to conclude that different commercial strains of White Leghorn chick embryo show different susceptibilities to the induction of VSD by ethanol.

A computer-assisted image processing method for determining relative cardiac function in the chick embryo.

The general objective of this study was to develop a noninvasive method for efficiently and reproducibly determining relative cardiac function parameters in the chick embryo. The specific objectives of the study were 1) to develop several methods for computer-assisted image processing and quantitation of relative intraventricular blood volumes in the 3-day-old embryonic chick heart and 2) to compare methods for precision and with a previously established manual processing method. Images of the embryonic chick heart in ovo were recorded on videocassette tape, digitized, and enhanced by computer-aided histogram equalization. The area occupied by blood within the common ventricle was extracted by region-growing and spurious region removal algorithms and defined by the determination of edge-pixel coordinates. Edge-pixel coordinates of the longitudinal and transverse axes of the common ventricular blood region were located by three different methods, the lengths of the axes calculated, and volumes computed from the equation for determining volume of a prolate spheroid. Twenty-five images of the embryonic heart were randomly selected and processed. Volumes were calculated with each of the three methods on six different occasions. A coefficient of variation was calculated for each method. The intraobserver mean coefficient of variation for each method was 7.4%. When a 2-way ANOVA was conducted, mean coefficients of variation did not differ significantly for the three methods. However, computer processing (in addition to significantly reducing the time required to generate data) reduced the coefficient of variation observed in manual processing by 56.5%.

Cardioteratogenic dose of ethanol in the chick embryo results in egg white concentrations comparable to human blood alcohol levels.

Three-day-old chick embryos were exposed to a dose of ethyl alcohol (0.32 ml of 50% ethanol) that we previously demonstrated produces cardiac malformations in 96.6% of the animals. Ethanol was administered into the air sac at 72-80 h of incubation. Samples of egg white were drawn at 2, 6 and 24 h after treatment and analyzed by capillary gas-liquid chromatography. Ethanol concentrations were significantly higher at 6 and 24 h after exposure than at 2 h (P less than 0.01), but there were no differences in mean concentrations between 6 and 24 h (P greater than 0.2). Furthermore, concentrations (43-303 mg dl-1) were comparable to human blood alcohol levels during intoxication. These results suggest that the cardioteratogenic doses of ethanol administered to chick embryos in a previous study are not excessive in terms of potential human embryo exposure.

Protective effect of ouabain on adriamycin-induced cardiovascular anomalies in chick embryos.

Adriamycin (2.5-10.0 micrograms) was administered to 4 1/2 and 5 day embryonic chicks (Hamburger-Hamilton developmental stages 24-26) to investigate the effect of the drug on cardiovascular morphogenesis. The drug produced dose-related increases in both mortality rate and malformation frequency with a maximum incidence of 82% cardiovascular anomalies following a dose of 10.0 micrograms/egg (P less than .001 relative to saline controls). Frequencies of embryos with ventricular septal defect (P less than .005), dextroposition of the aorta (P less than .005), or aortic arch anomalies (P less than .05) were significantly higher than among controls. In a second study, embryos were pretreated with ouabain (12.2 micrograms), verapamil (0.5 micrograms), coenzyme Q10 (100 micrograms, 200 micrograms), or vitamin E (1.0 mg, 5.0 mg)--agents previously shown to protect against adriamycin-induced cardiotoxicity. Pretreatment of embryos with ouabain significantly reduced the incidence of cardiovascular malformations induced by adriamycin from 55 to 21% (P less than .05). A major protective effect was observed relative to the induction of ventricular septal defect, the frequency of which was reduced from 45 to 14% (P less than .05). However, administration of verapamil, coenzyme Q10, or vitamin E did not have an appreciable effect on adriamycin-induced frequencies of cardiovascular malformations. Negative inotropism is suggested as a mechanism for adriamycin-induced cardiac anomalies but warrants further study.

The effects of caffeine on the ultrastructure and mitochondrial function of the embryonic chick heart.

Results from this study indicate that caffeine (at an embryotoxic dose equal to the LD40 administered to 3-day chick embryos produced both ultrastructural and functional abnormalities in embryonic cardiac mitochondria. The principal effects of caffeine on the ultrastructure of embryonic myocardial cells were clearly suggestive of cellular injury and included: (1) a marked disruption of mitochondrial cristae with formation of intramitochondrial myelin-like figures and (2) intracellular edema. A biochemical analysis of mitochondrial function revealed that caffeine inhibited the capacity of mitochondria to oxidize succinate. However, when pyruvate and malate were employed as substrates for isolated mitochondria, caffeine did not significantly alter mitochondrial function. Interference with embryonic cardiac mitochondrial succinate oxidation and/or fragmentation of mitochondrial membranes are suggested as possible events in the pathogenesis of caffeine-induced cardiac cell injury which, in turn, may lead to the embryonic death of the chick.

Potentiating effects of verapamil on cardiovascular teratogenicity of phenobarbital in the chick embryos.

Teratogenic to subteratogenic doses of phenobarbital were administered to young chick embryos together with a calcium-blocking agent, verapamil, at doses where it alone induces no cardiovascular malformations. The cardiovascular teratogenicity of phenobarbital was significantly potentiated by verapamil (5 X 10(-11), 1 X 10(-10) mol). The alteration of calcium movement into the cell and/or depressed cardiac function following impaired calcium transport by verapamil are suggested as modus operandi in the potentiation of phenobarbital-induced cardiovascular malformations.

Cardiovascular malformations induced by bromodeoxyuridine in the chick embryo.

For the study of morphogenesis and early embryonic development, 5-bromodeoxyuridine (BUdR), a halogenated analogue of thymidine, is incorporated into replicating DNA and serves as a valuable tool. To study the teratogenicity of BUdR on the developing chick cardiovascular system, we topically administered graded doses of BUdR (32.6-325.6 nmol) in ovo during Hamburger-Hamilton stages 15 to 16. We also administered to a parallel group of embryos corresponding nanomole doses of thymidine during identical stages of development. In the thymidine-treated group, survival rates and cardiovascular anomaly rates did not differ statistically from those in the chick Ringer's control group. Both survival rates and cardiovascular anomaly rates in the BudR-treated group were dose-responsive. Among 78 embryos with cardiovascular anomalies induced by BUdR, vascular malformations were found in 96%. These anomalies included interruption of the right fourth aortic arch, absence or hypoplasia of the right and/or left sixth aortic arch, and persistence of the left fourth aortic arch. Interruption of the right fourth aortic arch was always associated with intracardiac anomalies. Intracardiac anomalies were found in 54% of the embryos; these included ventricular septal defect, double outlet right ventricle, and persistent truncus arteriosus. Subclavian artery malformations were noted in 95% of the embryos. Possible mechanisms for BUdR-induced malformations in the cardiovascular system of the chick are discussed.

The effects of cardioteratogenic doses of caffeine on cardiac function in the 3-day chick embryo.

The effects on embryonic cardiac function of caffeine administration (two non-cardioteratogenic and two cardioteratogenic doses) to Hamburger-Hamilton stage 19 (3-3 1/2 days of incubation) chick embryos were investigated. Using microcinephotoanalysis, we have determined that caffeine (1.0-4.7 mg/egg), within the initial three hours after treatment, produced a dose-dependent decrease in end diastolic volume, stroke volume, cardiac output, and ejection fraction. These effects were sustained for a longer period of time following dosing at a cardioteratogenic level (3.5-4.7 mg/egg). Caffeine (1.0-4.7 mg/egg) also increased cardiac rate with a maximum increase of 30% seen 60 minutes after treatment with doses of 2.7 mg and 3.5 mg. However, the increase in cardiac rate was not related to dose. At 20 hours after treatment, caffeine increased stroke volume, ejection fraction and cardiac output relative to the controls. End-diastolic volume and cardiac rate were not changed. These results are evidence for a biphasic effect of cardioteratogenic dosing with caffeine during the first 20 hours after treatment--initially a sustained decrease in cardiac output, suggesting decreased flow through the embryonic heart, followed by an increase in ejection fraction, suggesting increased cardiac workload.

Ethyl alcohol-induced cardiovascular malformations in the chick embryo.

Chick embryos incubated for 72-80 hours were exposed to various volumes (0.20-0.40 m1/egg) of 50% ethyl alcohol. Examination of embryos at day 14 of incubation showed that higher doses of ethanol decreased the survival rate of embryos compared with control embryos. Three major categories of cardiovascular malformations were observed in this study: intracardiac anomalies characterized primarily by isolated ventricular septal defect, ventricular septal defect with overriding aorta, double outlet right ventricle or common aorticopulmonary trunk; aortic arch anomalies; and subclavian artery anomalies. Frequencies of embryos with intracardiac anomalies were equal to or greater than 64.8% in the six groups exposed to ethanol. Administration of ethanol also induced high frequencies of embryos with subclavian artery anomalies (11.2-89.1%). Absence or hypoplasia of the right and/or left secondary subclavian artery was commonly associated with persistence of the corresponding primary subclavian artery. Bilateral absence and/or hypoplasia of the secondary subclavian arteries was more common than unilateral anomalies, whereas absence of the left secondary subclavian artery was more commonly observed than an absent right secondary subclavian artery. No embryos in the two control groups combined (n = 94) demonstrated aortic arch or subclavian artery anomalies.

The causes and underlying developmental mechanisms of congenital cardiovascular malformations: a critical review.

Cardiovascular malformations are the second most common type of birth defect, occurring in 5-8/1,000 livebirths with a still higher prevalence among stillborn infants and spontaneously aborted embryos and fetuses. Dealing effectively with the high frequency of heart defects means reducing the incidence of cardiac malformations in the world. In this paper we cite some of the genetic and environmental risk factors associated with congenital cardiovascular malformations, describe the putative biochemical nature of the genetic predisposition relative to environmentally induced teratogenesis, and either support or discourage the use of available methods as strategies in preventing cardiovascular anomalies.

The teratogenic effect of phenobarbital on the embryonic chick heart.

Phenobarbital was observed to produce cardiovascular malformations in embryonic chicks. Malformations included simple ventricular septal defect, ventricular septal defect associated with dextroposition of the aorta, double outlet right ventricle, and several types of aortic arch anomalies. Embryos were exposed to phenobarbital at doses of 1-25 mumol on day 4 of incubation (Hamburger-Hamilton developmental stage 24). Doses equal to and greater than 5 mumol phenobarbital (26 mg/kg egg) significantly increased the frequency of embryos with cardiovascular malformations compared with lesser doses and with saline. A significant reduction in heart rate and abnormal rhythm of the heart were observed in embryos treated with teratogenic doses of phenobarbital. No arrhythmia nor significant changes in heart rate were observed in embryos exposed to subteratogenic doses of phenobarbital or to saline.