Teratogenic potency of valproate analogues evaluated by quantitative estimation of cellular morphology in vitro

To develop a simple prescreening system for teratogenicity testing, a novel in vitro assay was established using computer assisted microscopy allowing automatic delineation of contours of stained cells and thereby quantitative determination of cellular morphology. The effects of valproic acid (VPA) and analogues with high as well as low teratogenic activities-(as previously determined in vivo)-were used as probes for study of the discrimination power of the in vitro model. VPA, a teratogenic analogue (+/-)-4-en-VPA, and a non-teratogenic analogue (E)-2-en-VPA, as well as the purified (S)- and (R)-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively), were tested for their effects on cellular morphology of cloned mouse fibroblastoid L-cell lines, neuroblastoma N2a cells, and rat glioma BT4Cn cells, and were found to induce varying increases in cellular area: Furthermore, it was demonstrated that under the chosen conditions the increase in area correlated statistically significantly with the teratogenic potency of the employed compounds. Setting the cellular area of mouse L-cells to 100% under control conditions, the most pronounced effect was observed for (S)-4-yn-VPA (211%, P = < 0.001) followed by VPA (186%, P < 0.001), 4-en-VPA (169%, P < 0.001) and non-teratogenic 2-en-VPA (137%, P < 0.005) and (R)-4-yn-VPA (105%). This effect was independent of the choice of substrata, since it was observed on L-cells grown on plastic, fibronectin, laminin and Matrigel. However, when VPA-treated cells were exposed to an arginyl-glycyl-aspartate (RGD)-containing peptide to test whether VPA treatment was able to modulate RGD-dependent integrin interactions with components of the extracellular matrix, hardly any effect could be observed, whereas control cells readily detached from the substratum, indicating a changed substrate adhesion of the VPA-treated cells. The data thus indicate that measurement of cellular area may serve as a simple in vitro test in the early pre-screening evaluation of teratogenicity of novel therapeutic agents.