Use of the fluorescence-activated cell sorter (FACS) for in vitro assays of developmental toxicity

Our objective is to predict embryotoxicity with reliable in vitro techniques. In several experimental systems, differentiation is accompanied by changes in the glycosylation pattern of cell-surface glycoconjugates. This is also the case with embryonal carcinoma cells. We have monitored the expression of receptors for wheat germ agglutinin (WGA). Murine embryonal carcinoma cells (P19 and F9) were exposed in vitro to xenobiotics for 1-3 days, then incubated successively with WGA-biotin (15 mug/ml) and streptavidin-phycoerythrin (SA-PE) (20 mug/ml), each for 30 min at room temperature. Cell-surface fluorescence was then analysed using a fluorescence-activated cell sorter (FACS). Exposure to 1 mum retinoic acid, a known inducer of differentiation, altered glycosylation as indicated by changes in WGA binding. Clear-cut effects were also observed after exposure to salts of arsenic (20 muM), or nickel (50 mum), and to methotrexate (1 mug/ml), fluorouracil (1.3 mug/ml) or actinomycin D (0.04 mug/ml). These compounds affected the percentage of positive cells the intensity of labelling, or both. Two non-teratogenic compounds (metronidazole and sulfonilamide) have also been tested and had no effect. Lectin histochemistry of embryonal carcinoma cells exposed to potentially toxic agents holds promise as a method for predicting embryotoxicity. FACS analysis allows rapid quantification.