De. Amacher et al., THE COMPARATIVE TESTING OF 8 CODED CHEMICALS IN THE RAT LIMB BUD MICROMASS AND RAT EMBRYO CULTURE SYSTEMS, ATLA. Alternatives to laboratory animals, 24(6), 1996, pp. 945-952
When cultured at high density, mesenchymal cells from rat limb buds pr
oliferate and differentiate into chondrocytes. Inhibition of this in v
itro 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 i
n vitro study of abnormal development not limited to the skeletal syst
em. Both systems isolate the test agent from maternal metabolism and p
harmacokinetic restraints. In this study, drug-associated selective in
hibition of alcian blue uptake by cartilage proteoglycans, in micromas
s cultures of limb bud cells prepared from 13-day-old rat embryos, was
used to assess teratogenic potential ill vitro following exposure for
48 hours to eight coded compounds (acetylsalicylic acid, isoniazid, p
enicillin G, saccharine, vincristine sulphate, B-aminonicotinamide, re
tinoic acid, and amaranth). Following drug exposure, cultures were inc
ubated 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 par allel cultures, cell viability was measured by neutral red upta
ke, and protein content was assayed by using the bicinchoninic acid me
thod. Except for retinoic acid and vincristine sulphate, the maximum t
est concentration was 1000 mu g/ml. Inhibition of alcian blue uptake (
greater than or equal to 50%) was noted at 0.001 mu g/ml vincristine s
ulphate, 0.5 mu g/ml retinoic acid and 5 mu g/ml B-aminonicotinamide,
demonstrating that strong teratogens inhibit differentiation in microm
ass cultures at lower concentrations than those which affect limb cell
viability. When the same eight compounds were tested in a 24-hour emb
ryo culture model, dysmorphogenesis was: evident at 0.005 mu g/ml vinc
ristine sulphate, 0.1 mu g/ml retinoic acid and 0.1 mu g/ml 6-aminonic
otinamide. For the ether five chemicals, little or no toxicity was not
ed up to the maximum test concentration in either model. We conclude t
hat the two test systems, both based on the developing rat embryo, are
consistent with each other, and that either of them would be useful f
or the preliminary screening of potential teratogens.