Formation of glutathione adducts and 2-aminofluorene from 2-nitrosofluorene in postimplantation rat conceptuses in vitro

The formation of glutathione (GSH) adducts and 2-aminofluorene (AF), GSH-derived metabolic products from 2-nitrosofluorene (NOF), was examined as a possible mechanism of GSH-mediated protection from NOF embryotoxicity in the gestational day 10 (GD 10) rat conceptus in vitro. When added to whole embryo culture medium, NOF produced dose-dependent decreases in growth parameters and increases in the incidence of axial rotation defects in embryos cultured for 26 h. Culture of GD 10 rat conceptuses in 50 mu M NOF for 24 h following 2 h pretreatment with an irreversible inhibitor of glutathione disulfide reductase, 1,3-bis(2-chloroethyl)1-nitrosourea (BCNU, 25 CIM) did not result in statistically significant differences in morphology or biochemical parameters compared to NOF alone; viability, however, was decreased relative to controls. Nearly equal amounts of GS-AF(I), a stable S-oxide conjugate of GSH with NOF, AF, a GSH-dependent reaction product of NOF, and the parent NOF were recovered following short-term incubation of conceptuses with NOF (100 mu M) in serum-free medium. Stimulation of GSH synthesis with the cysteine prodrug 2-oxothiazolidine-4-carboxylate (OTC, 5 mM) resulted in a significant increase in AF concentrations (205% of control) and a decrease in NOF (50% of control) after 30 min. Sixty-minute exposure to the GSH depletor, diethylmaleate (DEM, 500 mu M), resulted in apparent reductions in both GS-AF, and AF by 36% and 34%, respectively, though these reductions were not statistically significant. Treatment with 25 mu M BCNU for 2 h, followed by exposure to 100 mu M NOF in serum-free medium resulted in a significant decrease in AF to 76% of control concomitant with lower GSH levels relative to NOF treatment alone. Exposure of conceptuses to 50 mu M NOF in complete medium following pretreatment with BCNU resulted in a reduction of GSH levels in the visceral yolk sac after 3 h rind in embryos after 5 h relative to controls. These data demonstrate that the intracellular protective effects oil GSH toward NOF embryotoxicity may act through a nonenzymatic mechanism of direct formation of GSH-NOF adducts in the day 10 rat conceptus in vitro, followed by the GSH-mediated conversion to a less toxic metabolite, AF.