Elizabeth A. Horigan

Chondrogenesis: a model developmental system for measuring teratogenic potential of compounds.

A simple test for determining the teratogenic potential of compounds is described using embryonic limb bud cells in culture. These mesenchyme cells multiply and differentiate into chondrocytes during a 6-day culture period. The extent of chondrogenesis is assessed by staining for the cartilage specific proteoglycan with alcian blue. The amount of stain is then measured spectrophotometrically. Compounds which interfere with growth or differentiation reduce the amount of proteoglycan and as a consequence, reduce alcian blue staining. Compounds can be added directly to the media or be activated using several different metabolizing systems. The dose of a compound needed to reduce alcian blue staining by 50% is designated the teratogenic potential (TP50) of that compound. TP50s of proven teratogens compare favorably with in vivo teratogenic doses of the teratogens.

Detection of teratogenic compounds using differentiating embryonic cells in culture

SummaryMethods are described for screening for teratogenic compounds using differentiating neural crest and prechondrogenic limb bud mesenchyme cells in culture. Substances to be tested are either added directly to the culture medium or are combined in a dialysis bag with the postmitochondrial fraction from rat liver and certain cofactors. In the latter case, the compound and its metabolites are gradually released into the medium from the dialysis bag. The results obtained with 14 compounds demonstrate a positive relationship between teratogenicity in vivo and alterations in the growth or the differentiation of the cultured cells.

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.