The overall goal of this project was to develop in vitro bioassays which would identify putative teratogens by their ability to impair the major developmental events of cell proliferation rate and differentiation, migration and cell-cell recognition. Five main approaches were adopted: 1) The synthesis of valproate analogues with varying in vivo teratogenic potential to establish structure-activity relationships and validate the in vitro systems under development; 2) Evaluation of in vitro systems which detect putative teratogens by virtue of their ability to reduce cell proliferation rate and induce differentiation using a C6 glioma cell line and rat embryo mesenchymal limb bud cells; 3) Development of cell lines engineered with human cell adhesion molecules (CAMs) to determine the influence of teratogens on cell-cell recognition events; 4) Establishment of an automated image analysis system to evaluate teratogen-induced alterations in cell morphology and motility; 5) Elucidation of potential molecular modes of teratogen action in the in vitro systems under development.
Over 50 substances structurally related to valproic acid (VPA) have been synthesised and their anticonvulsant and teratogenic potency evaluated in vivo. Based on these results six related analogues were selected and supplied to each partner in a coded manner for evaluation in the in vitro systems under development. These were VPA; the (+/-)-4-en-VPA teratogen and E-2-en-VPA non-teratogen; and the teratogenic (+/-)-4-yn-VPA racemate and its purified S(-)-4-yn-VPA and teratogenic R(+)-4-yn-VPA enantiomers which are teratogenic and non-teratogenic, respectively. VPA and related analogues tested exerted comparable G1 phase antiproliferative effects in C6 glioma and limb bud cells in a dose range of 0-3mM however, their relative potency did not correlate with in vivo teratogenicity. If inhibition of proliferation was coupled with an anti-differentiative action, the in vitro system predicted all teratogenic agents capable of inducing exencephalic rates of 5-44% in an expanded series of VPA structural analogues however, this did not correlate strictly to their relative in vivo potencies. The importance of this anti-differentiative action was confirmed using assays determining neuronal aggregation, migration and differentiation. VPA and all teratogenic analogues, at 3mM, inhibited neuronal cell aggregation and limb bud chondrocyte differentiation in a manner which exhibited a good correlation with their in vivo teratogenicity and was enantiomer-specific. Similar effects were obtained using the endpoints of cell morphology and migration. These were determined using a novel in vitro assay which employed computer assisted microscopy for the automated quantitation of change in cell contour and motility. VPA and related analogues induced significant decreases in random cell motility and shape in a manner which was related to their teratogenic potential and independent of the cell line employed. The most sensitive discrimination between teratogenic and non-teratogenic analogues was obtained by determining differential inhibition of primary neuronal neurite outgrowth stimulated by human cell adhesion molecules (L1 and N-cadherin). Half-maximal inhibition was observed at approximately 150uM for the teratogenic S(-)-4-yn-VPA enantiomer, but not the non-teratogenic R(+)-4-yn-VPA form. In the anti-differentiative assays, the effects of the non-teratogenic E-2-en-VPA analogue were indistinguishable from those of VPA, however, in vivo, this agent is a neurobehavioural teratogen, equipotent to VPA and with similar pharmacokinetics. Secondly, these assays failed to order the relative in vivo potency of the teratogenic analogues. With respect to mode of teratogen action, the results obtained are consistent with the existence of a defined structural pocket at which the primary action of VPA results in impaired signal transduction leading to downstream alterations in cytoskeletal assemblies with concomitant cell shape change and inhibition of neuritogenesis.
MAJOR SCIENTIFIC BREAKTHROUGHS:
The results obtained in this study raise a number of important issues with respect to the design of effective in vitro screens for teratogenic potential: 1) The most sensitive endpoints for teratogenic potential relate to perturbations of the differentiative process; 2) In vitro systems provide a qualitative evaluation of potential teratogenicity rather than an estimate of relative potency; 3) Validation will require expanding teratogen concept to include agents producing neurobehavioural deficits. Overall, the work has provided three novel screens for consideration of further validation and a molecular rationale for the mechanism of VPA-induced teratogenesis.
Funding SchemeCSC - Cost-sharing contracts
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