Embryotoxicity of the intercalating agents m‐AMSA and o‐AMSA and the epipodophyllotoxin VP‐16 in postimplantation rat embryos in vitro

Abstract

The intercalating agent, m‐AMSA, and the epipodophyllotoxin, VP‐16, both topoisomerase II—reactive anticancer agents, are also embryotoxic agents in rat embryos cultured in vitro. Quantifying the embryotoxic effects of these drugs revealed that the no observed adverse effect level (NOAEL) for m‐AMSA is 10 nM, the embryotoxic concentration range is 50–500 nM, and complete lethality is observed at 1 μM. In contrast, the NOAEL for o‐AMSA, an inactive isomer of m‐AMSA, is 1.0 μM, the embryotoxic concentration range is 10–100 μM, and complete lethality occurs at 200 μM. Based upon the concentrations of drugs required to produce 50% embryotoxicity or 50% malformed embryos, m‐AMSA exhibits a 200–500‐fold‐higher embryotoxicity compared to o‐AMSA. VP‐16 exhibits a NOAEL of 1.0 μM, an embryotoxic concentration range of 2–5 μM, and complete lethality at 10 μM. Compared to m‐AMSA, VP‐16 is approximately 10‐fold less embryotoxic. At appropriate concentrations, all three drugs were dysmorphogenic resulting in embryos that were characterized by hypoplasia of the prosencephalon with associated microopthalmia and dilation of the rhombencephalon. As a prelude to future studies focusing on the mechanism of drug‐induced embryotoxicity, we have used established biochemical and immunologic methods to identify and quantify topoisomerase II in rat embryos. In addition, we have demonstrated that the embryo topoisomerase II can be inhibited by both m‐AMSA and VP‐16. Finally, we have used a human cDNA probe to detect topoisomerase II mRNA in the rat embryo. Thus, the combination of the in vitro whole embryo culture and these biochemical/molecular assays should allow us to explore the role of a specific nuclear target, i.e., topoisomerase II, in the teratogenic effects of some commonly employed chemotherapeutic agents.