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The embryonic stem cell test (EST) is one of the more promising and extensively studied tests in the field of developmental toxicology. We evaluated the effect of a series of structurally related valproic acid analogues on cardiomyocyte differentiation in the EST. The goal of the present study was to determine the applicability of the EST by potency ranking within this chemical class. Cardiomyocyte differentiation was evaluated by morphological scoring as well as by gene expression analysis of cardiac markers using RT-PCR. All VPA analogues tested inhibited cardiomyocyte differentiation, with the exception of (±)-2-ethyl-4-methyl pentanoic acid, which correlates to their known in vivo developmental toxicity. Effects were also evident on gene expression of cardiomyocyte differentiation-regulated genes, such as MHC and Nkx2.5. Overall, the present results indicate that the assay can be a valuable screening tool in potency ranking of structurally related compounds.

Retinoids are essential in vertebrate reproduction and embryonic development. All-trans-retinoic acid (ATRA) is tightly regulated during these processes. CYP26A1 is mainly responsible for its degradation. To study the role of CYP26A1 during implantation, we applied R115866, a CYP26A1-specific antagonist, to rats during early gestation days (GD). On GD 6.5 and 12 samples were collected and the number of embryos was evaluated. ATRA concentration increased in uterus and serum, mRNA expression of CYP26A1 and CRABP2 increased in the liver, but not in the uterus. Uterine COX1 and 17βHSD mRNA expression was decreased. The number of embryos on GD 12 was not altered in this setting. It can be concluded that uterine expression of the analyzed retinoid-response genes during early gestation is not altered by this R115866 treatment and instead indirectly via ATRA. From our experiment we cannot confirm that ATRA obtains a major influencing role in the regulation of embryonic implantation.

The antiepileptic drug valproic acid (VPA) may produce NTDs because of interference with folate metabolism. Therefore, the possible interactions of VPA with the dihydrofolate reductase inhibitor trimethoprim (TM) was investigated. The combination of TM with sulfamethoxazol is used for treatment of urinary infections, the most common complications of pregnancy. TM (80 and 160 mg/kg) was given i.p. and orally, 0.5 and 1 h, respectively, prior to valproic acid (VPA, 300 and 400 mg/kg, s.c.) in day 8 pregnant NMRI mice. Fetuses were examined for exencephaly, resorptions, and fetal weight retardation on day 18 of gestation. TM (160 mg/kg, i.p.) produced no exencephaly or embryolethality, but increased fetal weight. Administration of TM (80 mg/kg, i.p.) increased VPA-induced exencephaly and fetal weight retardation but not embryolethality. Exencephaly rates induced by VPA (300 and 400 mg/kg) were 4% and 12.9% and were increased by coadministration of TM to 22.7% and 42.5%, respectively (P < 0.01). Oral TM also increased VPA-induced exencephaly and fetal weight retardation but with lower potency than i.p. injection. The observed effects were not due to altered VPA pharmacokinetics. These results support the view that VPA-induced neural tube defects may be mediated via an interaction with folate metabolism, and advise against TM-use in VPA-treated epileptics during pregnancy.

The diurnal variation of folate concentrations in mouse plasma and embryo between day 8.5 and day 9.5 of gestation (light cycle = 0900-2100) were determined by high-performance liquid chromatography. Folate concentrations in the embryo were high during the evening hours, decreased during the night, reached their lowest levels at 0500, and then increased again during the day. High levels of folates may be related to increased food intake by the pregnant mice. Small changes of the two major maternal plasma folate metabolites were observed. The relative amount of each folate metabolite in the embryo, as compared to the total folate concentration, remained in a narrow range. The main metabolites were tetrahydrofolic acid (THF) (32.4% +/- 2.1% of total folates), 5-CHO-THF (24.2% +/- 2.3%), and 10-CHO-THF (17.0% +/- 1.9%). A dramatic alteration of these ratios occurred only between 1100 and 1400. The relative content of THF increased (52.7% +/- 2.5%), whereas the relative concentration of 5-CHO-THF in the embryo decreased (6.5% +/- 1.9%). Before 1000 when the ratios of folate metabolites were stable, the rate of valproic acid-induced neural tube defects was reduced from 49% of living fetuses to 12% by coapplication of folinic acid via subcutaneously implanted minipumps. During the period in which dramatic changes in ratios between the folate metabolites in the embryo occurred, no protective effect of folinic acid on valproic acid-induced exencephaly could be observed. Our results indicate that the diurnal variation of folate metabolism in the embryo is important in regard to valproic acid teratogenesis and its protection by folate supplementation.

A direct teratogenic effect will depend on the concentration-time relationship of the drug or its active metabolite in the placental-embryonic compartment during the sensitive stages of gestation. Pharmacokinetic studies are therefore important for the interpretation and design of teratogenicity experiments, particularly for the problem of species differences, and for attempts to extrapolate experimental findings to the human (“risk assessment”). Pharmacokinetic studies are also crucial in elucidating whether the parent drug or a metabolite is the proximate or ultimate toxic entity. Finally, it is shown that structure-activity studies can greatly benefit from pharmacokinetic experiments to demonstrate whether the teratogenicity of a substance is related to its intrinsic activity or to the extent of placental transfer or both.

The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretin. The three aromatic retinoids were also tested for their direct inhibitory effect on chon-drogenesis in the limb bud mesenchymal cell “micromass” culture assay. After a standard dose of 100 nig/kg administered on day 11 of gestation in nmri mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all- trans -retinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all- trans -retinoic acid (ic50 of 12 ng/ml). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using hplc methods. Both etretinate and motretinide were metabolized in vivo to etretin, their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUC values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 × lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all- trans - and 13- cis -retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied.