Monitoring of organ formation in rat embryos after in vitro exposure to azathioprine, mercaptopurine, methotrexate or cyclosporin A.

Rat embryos in the organ formation phase (days 9.5-11.5 post coitum) were cultivated in pure rat serum in the presence of an Aroclor 1254 pretreated liver microsomal preparation (S9-mix). Various concentrations of the immunosuppressive drugs azathioprine (AZ), 6-mercaptopurine (MP), methotrexate (MTX) or cyclosporin A (CS-A) were added at the beginning of the culture period. Forty-eight hours later, malformations were observed in the AZ, MP and MTX treated embryos at concentrations as low as 1 microgram/ml, 1.8 micrograms/ml and 0.05 microgram/ml, respectively. This indicates that these drugs have a direct effect on embryonic development. They selectively affected the rhombencephalic and telencephalic brain regions. Other malformations were seen in the caudal trunk, the heart and forelimb regions, and in the vesicular structures. It is suggested that the similarity of the pharmacological action of these drugs, that is, the DNA de novo synthesis inhibition, was the cause of the comparable types of malformations observed. Higher AZ, MP and MTX concentrations caused concentration-dependent increases in the types and incidences of malformations, as well as inhibited overall growth and differentiation. CS-A, a new type of immunosuppressant agent, had no effect on the morphogenetic events at the concentrations tested. These results are generally in agreement with the literature data, indicating that AZ, MP and MTX induce malformations in whole-animal systems, whereas CY-A does not. When AZ and MTX were assayed in the rat species in vivo, on the other hand, embryolethalities and retardations, but few malformations, were observed. The possibility of controlled exposure in vitro may, therefore, offer the advantage that clearer distinctions between embryolethal and teratogenic effects can be made.