title: Formation of glutathione adducts and 2-aminofluorene from 2-nitrosofluorene in postimplantation rat conceptuses in vitro.
abstract: 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 microM 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 microM) 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 microM) 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 microM), resulted in apparent reductions in both GS-AF(I) and AF by 36% and 34%, respectively, though these reductions were not statistically significant. Treatment with 25 microM BCNU for 2 h, followed by exposure to 100 microM 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 microM NOF in complete medium following pretreatment with BCNU resulted in a reduction of GSH levels in the visceral yolk sac after 3 h and in embryos after 5 h relative to controls. These data demonstrate that the intracellular protective effects of 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.
authors.affiliation: Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109-2029, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Berberian
authors.forename: G E
authors.initials: GE
authors.lastname: Eurich
authors.forename: G A
authors.initials: GA
authors.lastname: Rios
authors.forename: C
authors.initials: C
authors.lastname: Harris
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000493:pharmacokinetics* / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005449:Fluorenes / Q000032:analysis*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D008658:Inactivation, Metabolic; D009603:Nitroso Compounds / Q000737:chemistry / Q000493:pharmacokinetics*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002273:Carcinogens; D005449:Fluorenes; D009603:Nitroso Compounds; C012458:2-nitrosofluorene; C012177:2-aminofluorene; D005978:Glutathione; D002330:Carmustine