During the past several years we have exploited the advantages of in vitro culture of mammalian (rat) embryos to gain new insights into molecular mechanisms (aspects) of teratogenesis. We have focused on one teratogen, cyclophosphamide (CP), a well-studied antineoplastic agent. Our results have, for the first time, delineated the in vitro dose-response curve for this teratogen, extablished the importance of metzbolism in teratogenicity, and, using a monofunctional derivative of CP, gained new insights into the molecular aspects of CP teratogenesis. Furthermore, using radioloabeled CP we have made the significant discovery that although CP is incorporated by all cells of the embryo, heart cells are completely resistant to the cytotoxic and teratogenic effects of CP while other tissues are extreemely sensitive. These findings provide us with a unique opportunity to investigate on of the intriguing and challenging problems of teratology, i.e., what are the molecular mechanisms by which teratogens kill certain cells but spare others during the teratogenic process, which eventually lead to birth defects? In an effort to understand the basis for this differential sensitivity, we will investigate several potential mechanisms. We will compare the incorporation of CP by resistant and sensitive cells to determine if availability of intracellular CP has a role in this differential sensitivity. Using the guanidine thiocyanate: cesium chloride procedure, we will compare the extent of CP binding to proteins and nucleic acids in sensitive and resistant tissues to determine which class of macromolecules serves as the primary target in terms of CP cytotoxicity. In the two tissue types we will characterize 1) the specific lesions produced in DNA using the alkaline elution procedure 2) the functional consequences of the alkylation of RNA using in vitro translation and 3) the spectrum of proteins alkylated using two-dimensional gel electrophoresis. Finally, we will examine the roles of: cellular proliferation, detoxification of CP, and, DNA repair in cell-specific cytotoxicity. These analyses will involve 3H-Thymidine autoradiography, high-pressure liquid chromatography and an autoradiographic analysis of DNA repair (unscheduled synthesis).
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