The Comparative Testing of Eight Coded Chemicals in the Rat Limb Bud Micromass and Rat Embryo Culture Systems

Abstract

When cultured at high density, mesenchymal cells from rat limb buds proliferate and differentiate into chondrocytes. Inhibition of this in vitro chondrogenic process has been used for the preliminary evaluation of teratogenic potential. Alternatively, intact post-implantation rat embryos, maintained in short-term culture, provide a system for the in vitro study of abnormal development not limited to the skeletal system. Both systems isolate the test agent from maternal metabolism and pharmacokinetic restraints. In this study, drug-associated selective inhibition of alcian blue uptake by cartilage proteoglycans, in micromass cultures of limb bud cells prepared from 13-day-old rat embryos, was used to assess teratogenic potential in vitro following exposure for 48 hours to eight coded compounds (acetylsalicylic acid, isoniazid. penicillin G, saccharine, vincristine sulphate, 6-aminonicotinamide, retinoic acid, and amaranth). Following drug exposure, cultures were incubated for another 96 hours, and the cells were then fixed and stained with 0.5% alcian blue. Bound dye was then extracted and quantitated. In parallel cultures, cell viability was measured by neutral red uptake, and protein content was assayed by using the bicinchoninic acid method. Except for retinoic acid and vincristine sulphate, the maximum test concentration was 1000μg/ml. Inhibition of alcian blue uptake (> 50%) was noted at 0.001μg/ml vincristine sulphate, 0.5/μg/ml retinoic acid and 5μg/ml 6-aminonicotinamide, demonstrating that strong teratogens inhibit differentiation in micromass cultures at lower concentrations than those which affect limb cell viability. When the same eight compounds were tested in a 24-hour embryo culture model, dysmorphogenesis was evident at 0.005μg/ml vincristine sulphate, 0.1μg/ml retinoic acid and 0.3μg/ml 6-aminonicotinamide. For the other five chemicals, little or no toxicity was noted up to the maximum test concentration in either model. We conclude that the two test systems, both based on the developing rat embryo, are consistent with each other, and that either of them would be useful for the preliminary screening of potential teratogens.