Prediction of embryotoxic potential using the ReProGlo stem cell-based Wnt reporter assay.

The ReProGlo assay was developed in 2009 to predict embryotoxic potential of drugs and chemicals by use of a stem cell-based in vitro system. It utilizes a luciferase reporter to detect drug-induced alterations in the canonical Wnt/ß-catenin signaling pathway, which is involved in regulation of early embryonic development. It allows the simultaneous determination of cell viability and luciferase reporter activity in a high throughput format. The present study was conducted within the framework of the EU ChemScreen-project. It (1) enlarges the original number of test-compounds from 17 to now 80, (2) introduces a new classification scheme and (3) anchors the results against a prediction scheme based on structural features of chemicals. The assay is applicable as stand-alone for priority setting or in a test battery.

Evaluation of an alternative in vitro test battery for detecting reproductive toxicants in a grouping context.

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.

ReProGlo: a new stem cell-based reporter assay aimed to predict embryotoxic potential of drugs and chemicals.

A stem cell-based reporter assay was developed to detect drug-induced alterations in the canonical Wnt/beta-catenin signaling pathway, which is involved in the regulation of early embryonic development. The so-called ReProGlo assay allows simultaneous determination of cell viability and luciferase reporter activity in a high throughput 96-well microtiter format. A clone of mouse embryonic stem (mES) cells stably expressing the SuperTopFlash reporter was established. This allows Wnt pathway activity determinations in undifferentiated mES cells and their differentiated descendants. Several test chemicals were analyzed in the new assay system. Known embryotoxicants like retinoic acid or lithium chloride induced concentration-dependent increases in reporter activity. The potency of valproic acid and a series of structural analogs to activate the Wnt pathway correlated well with their reported teratogenic activity in the mouse. Cyclophosphamide was also active but only after metabolic activation by hepatocytes. The new test may help to predict embryotoxic potential of chemicals.