Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system: Relationship of biotransformation to teratogenicity in vitro

Abstract

2-Acetylaminofluorene (AAF) produced abnormal, open neural tubes in cultured whole rat embryos only in the presence of an added, NADPH-dependent monooxygenase system. Reactive intermediary metabolites, including N-hydroxy-AAF, N-hydroxy-2-aminofluorene, 2-nitrosofluorene and N-acetoxy-AAF, each elicited embryonic malformations under culture conditions, but a statistically significant increase in the incidence of abnormal neurulation was not observed. Using [ 14C]AAF and high pressure liquid chromatography (HPLC) separation techniques, the biotransformation of AAF was studied under conditions in which embryos and the monooxygenase system were coincubated. The major metabolites produced cochromatographed with 5-hydroxy-AAF, 7-hydroxy-AAF, 9-hydroxy-AAF and 3-hydroxy-AAF. Other metabolic products also were detected. The embryonic effects of these major AAF metabolites were tested singly and in combination in the embryo culture system. Addition of 7-hydroxy-AAF to the embryo culture system resulted in open neural tubes in the absence of an added monooxygenase system. Other individual ring-hydroxylated metabolites produced retarded growth, but neurulation appeared normal. Ring-hydroxylated metabolites, added to the embryo culture system in combination in the same proportions as were formed during biotransformation in culture, also produced a marked increase in incidence of neural tube defects in the absence of an exogenous (added) biotransforming system. In combination with 3-, 5- and 9-hydroxy-AAF, 7-hydroxy-AAF exposure (86 μM) resulted in a 47% incidence of abnormal, open neural tubes. When tested individually, higher concentrations of 7-hydroxy-AAF (104μM) produced a lower percentage of malformed embryos (13%). The results suggested that 7-hydroxy-AAF was principally responsible for the neural tube defects caused by AAF following monooxygenase-dependent bioactivation, but that other metabolites also appeared to contribute to the observed effect.