Effect of some environmental contaminants on differentiation in micromass culture of rat embryo limb bud cells.

Minta M, Wlodarczyk B.

Department of Pharmacology and Toxicology, National Veterinary Research Institute, Pulawy, Poland.

Out of three in vitro tests validated so far for embryotoxicity prescreening tests, micromass culture of rat limb bud mesenchymal cells (LBC) is one candidate. The prediction model (PM) is based on the value of ID50 (50% inhibition concentration for differentiation) and allows to classify test chemical to one of three class of embryotoxicity: strong (III), weak (II) and non-embryotoxic (I). Confluent monolayer cultures were prepared from rat embryo limb buds, incubated for 5 days, fixed and stained for cell viability (Neutral red) and proteoglycans (Alcian blue). Proliferation and differentiation were determined spectrophotometrically at 540 and 620 nm, respectively. The method was checked in our laboratory with use of reference compounds, i.e. 5-Fluorouracyl (7 concentrations ranged from 15.63 to 1000 ng/ml) and Penicillin G (at 500 ug/ml). Cadmium chloride (CAS No. 10108-64-2), lead acetate (CAS No. 301-04-2) and sodium fluoride (CAS No.7681-49-4) were evaluated in this study. Concentrations used ranged from 0.17-125 ug/ml (Cd), 1.95-125 ug/ml (Pb), and 3.91-250 ug/ml (F). Mean ID50 values (the endpoint value derived from two separate experiments conducted on different days) were as follow: 2.95 ug/ml (Cd), 27.7 ug/ml (Pb), 24.9 ug/ml (F). Cadmium chloride was classified to group III, lead acetate and sodium fluoride -- to group II. Studies are in progress to evaluate more compounds of different teratogenic potential in vivo.

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