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The adverse developmental effects of ethanol consumption have been documented in humans and in animal models. In animal models, the organ system affected by ethanol administration is dependent on the point in gestation at which the xenobiotic is administered. Previous studies have shown that an exposure of 24–48 hr beginning at the early somite stage in rodent conceptuses alters neural tube closure in vitro. However, the concentration and time dependency of this effect have not been fully defined. Whole embryo culture was therefore used to expose 3–6-somite mouse conceptuses (ICR strain) to ethanol at 300, 450, 600 and 800 mg/dl. The higher concentrations were selected to approximate the peak serum ethanol concentrations that have been shown to be teratogenic in vivo. A 24-hr exposure produced a concentration-dependent increase in neural tube defects (NTDs) and concomitant growth retardation. When shorter exposure periods were used (8, 10, 12 or 20 hr) the incidence of NTDs was dependent on the ethanol concentration and exposure period. At the 600 and 800 mg/dl concentrations an exposure of 8 hr or more produced NTDs, but shorter periods (4 and 6 hr) did not affect neural tube closure when evaluated at the end of a 24-hr culture period. At the 450 mg/dl concentration a 20-hr exposure induced NTDs, but a 12-hr exposure to this level did not. Exposure of conceptuses to ethanol for periods similar to their half-life in vivo did not induce NTDs and the highest concentration produced only a trend towards a reduction in protein content. When the incidence of NTDs was plotted against the area under the time and concentration curve (AUC) the correlation coefficient was 0.5779. An analysis of covariance indicated that the relationships between NTDs and AUC were similar at the 300 and 450 mg/dl concentrations and also at the 600 and 800 mg/dl concentrations. In contrast, the relationships between embryonic protein content and AUC did not differ at the 300, 450 and 600 mg/dl concentrations, but all differed from that at the 800 mg/dl level. These results indicate that ethanol-induced NTDs do not appear to be due solely to embryonic growth retardation. Additionally, ethanol-induced neural tube defects are a function of duration of exposure as well as of peak serum concentration.

The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 μ m or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 μ m . EME also induced defects at concentrations of 400 μ m or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 μ m or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 μ m , exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDs in vitro is higher than the teratogenic concentration in vivo . Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reported in vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.

The whole-embryo culture technique has been extensively used to evaluate embryonic growth and development using morphological criteria. However, the system also provides an opportunity to assess the status of embryonic metabolism to determine biochemical maturation and alterations induced by xenobiotics or changes in substrate availability. For example, using 14 C-labelled substrates the rates of product formation may be determined for a variety of foetal fuels such as glucose and the ketone body, β-hydroxybutyrate. During organogenesis glucose metabolism is characterized by high rates of glycolytic metabolism at the early-somite stage and an increased dependence on Krebs cycle concomitant with the establishment of the chorioallantoic placenta ( c . 30-somite stage) to initiate an increasingly ‘foetal’ type of pattern. The dependence of the neurulating embryo on glucose as a fuel is further demonstrated by the embryotoxic effects of insulin-induced hypoglycaemia. Although decreased glycolytic metabolism is the earliest effect of this treatment, both the Krebs cycle and oxidative pentose phosphate pathway are also inhibited. Therefore, the induction of malformations by hypoglycaemia is the result of a multifactorial biochemical alteration. In addition to glucose, mouse conceptuses have the capacity to metabolize alternative foetal fuels such as the ketone bodies d - and dl -BOHB at rates that are concentration dependent. However, the energy production from these substrates is limited by the low rates of Krebs-cycle metabolism at the early-somite stage.

Rates of 14 CO 2 production by mouse conceptuses in vitro from d - and dl -[3- 14 C]-β-hydroxybutyrate (βOHB) were determined during the period of organogenesis (days 9 through 12 of gestation) in the presence of 4 to 32 mmol/L dl -βOHB. During this time period the rates 14 CO 2 production from d -βOHB metabolism are concentration-dependent, increase on each day of gestation, and the site of metabolism appears to shift from the visceral yolk sac placenta to the embryo proper. In contrast to fetal and neonatal tissues, the rates of 14 CO 2 production from dl -βOHB oxidation is significantly greater than from d -βOHB suggesting that the utilization of the l -isomer may be equal to or greater than that of the d -form.

The effects of hypoglycemia on mammalian embryos undergoing neurulation (third to fourth week of human development) were investigated. Mouse embryos were maintained for 28 hours in whole embryo culture in serum collected from rats that had received 50 units of 100 United States Pharmacopeia insulin units per milliliter. Glucose concentrations used were 40, 60, 80; and 147 mg/dl (normal blood glucose in the pregnant mouse is 125 mg/dl). After the culture period embryos were evaluated for malformations and growth and compared with those maintained under eiuglycemic conditions. The results demonstrate that glucose concentrations approximately 50% of normal maternal levels were teratogenic but not growth inhibitory, whereas concentrations in the range of 30% to 40% of maternal levels were lethal to the embryo. Furthermore, a 14-hour exposure to reduced blood sugar in either the first or second half of the culture period produced malformations.