Differentation of rat embryo cells in culture: Response following acute maternal exposure to teratogens and non‐teratogens

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Abstract
An in vivo–in vitro test system with high sensitivity to teratogens has been developed and validated. A single acute intra‐peritoneal injection of teratogens (18) and non‐teratogens (13) was administered to pregnant rats on the 12th day after fertilisation, and uteri were removed after 16 h by laparotomy. 34–36 Embryos somites were selected, and mid‐brain (CNS) and fore‐limb buds (LB) were dissected free and dispersed as single‐cell suspensions in Ham's F12 culture medium. The cells were cultured as micromass cell islands for 5 days, and discrete foci of neuronal cells differentiated in CNS cultures and chondrocytes in LB cultures. After 5 days, differentiation as determined by number of stainable foci of differentiated cells and 3H‐GABA incorporation in CNS or 35SO4 incorporation in LB and growth (as determined by total protein) were measured. Both differentiation and growth of CNS and LB cultures were markedly reduced following exposure of the dam to teratogens, whereas no significant effect was observed with non‐teratogens. One teratogen (amaranth) and one non‐teratogen (nitrilotriacetic acid) were classified as false negative and positive, respectively; the sensitivity of the test (proportion of teratogens correct) was therefore 92% and the specificity (proportion of non‐teratogens correct) was 94%. Inhibition of growth and differentiation in the rat embryo cell cultures following maternal exposure forms the basis of a short‐term in vitro test for teratogens.