Testing of pro-teratogens in the in the embryonic stem cell test: Simulation of metabolic conversion by treatment with defined mixtures of parent compounds with their respective metabolites

Lack of metabolic activity is one of the drawbacks of many in vitro assays. It still represents a major challenge to supplement existing in vitro methods with biotransformation systems. To estimate the amount of parent compound required of being converted into its major metabolite before affecting the outcome in the validated embryonic stem cell test (EST) we mimicked bioactivation by testing fix combinationsof selected chemicals with their respective teratogenic metabolite. Effects of parent compound/metabolite pairs were assessed separately and in combination at ratios of 80:20 and 50:50, thus mimicking incomplete bioactivation in the range of 20 and 50%, respectively. Valpromide (VPD), retinol (ROH) and albendazole(ABZ) were chosen as parent compounds together with their metabolites, i.e. valproic acid (VPA), all-trans retinoic acid (RA) and albendazole sulfoxide (ASO), respectively, and all were tested for their effects on mESC differentiation and cytotoxicity. The active metabolite VPA was 12-times more potent to inhibit differentiation into contracting cardiomyocytes compared to its parent, VPD, while no such significant differences were detected for half-maximal cytotoxic concentrations(IC50). Comparable inhibition of differentiation occurred in the 50:50 and 80:20 mixtures at calculated VPA concentrations of about 200-430 µM, thus indicating saturation of the developmental block in culture already at 20% conversion of the pretoxin. RA was 200- to 300-fold more potent in inhibiting cardiomyocyte differentiation and 700-fold more potent in reducing cell viability when compared to its mother compound, ROH. ABZ was more toxic (factor 40) in comparison to its metabolite ASO. Inhibition of differentiation as well as cytotoxicity could be associated with an ABZ concentration of about 0.2 - 0.4 µM. The EST, which it selflacks any metabolic competence, can be used as readout for assessing developmental toxicity of fixed pairs of compounds and its respective metabolites.