Uptake and binding of tritium from [chloroethyl3H] cyclophosphamide by rat embryos in vitro

Abstract

Day 10 rat embryos were exposed in vitro to [chloroethyl ³H] cyclophosphamide (³H‐CP) at 4 μg/ml over a 24‐hour period and the uptake and binding of labeled drug were monitored autoradiographically and biochemically. Autoradiographic analysis revealed that embryos exposed to ³H‐CP and a complete activating system exhibited radioactivity distributed throughout the embryo. Subsequent analysis indicated that the distribution of autoradiographic grains on a per cell basis ranged from 7.7 in surface ectoderm to 13.4 in the neuroepithelium. No correlation was found between the sensitivity of various embryonic tissues to the cytotoxic effects of CP and the number of grains per cell. Direct radiochemical analysis of the amount of tritium taken up and bound by embryos under bioactivating conditions (³H‐CP + S‐9 + cofactors) confirmed the autoradiographic analysis. Autoradiographic and radiochemical analyses demonstrate that embryos exposed under bioactivating conditions take up and bind approximately three times more tritium than embryos exposed under nonactivating conditions (³H‐CP + S‐9 without cofactors). Additional studies have demonstrated that uptake and binding of tritium from bioactivated ³H‐CP only are linear over the first 10 hours of incubation with no detectable increases thereafter.