Characterization of hemoglobin adducts from a 4, 4'-methylenedianiline metabolite evidently produced by peroxidative oxidation in vivo.

Abstract

4,4'-Methylenedianiline (MDA) is a widely used mutagenic and carcinogenic industrial chemical. It is also a metabolite of 4, 4'-methylenediphenyl diisocyanate (MDI), which is used in the manufacturing of polyurethane foams. Biomonitoring of MDA, like other aromatic amines, is mainly carried out by GC/MS measurement of cysteine adducts in Hb from the nitroso metabolite, released by alkaline hydrolysis. In the present study it was investigated whether the formation of Hb adducts from non-nitroso metabolites of MDA can be used for the dosimetry of MDA. The study was carried out by treatment of mice with MDA and tritiated MDA or deuterated MDA and by identification of their products of reaction with Hb, after enzymatic hydrolysis of the globin and enrichment of the adducts. The main adduct, about 50% of the total amount of MDA associated with Hb, was characterized by MS and was shown to be a reaction product of MDA and the amino group of N-terminal valine in Hb, the derived structure being 1-[(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]benzene-4-azo-2-isovaleric acid. It is likely that this quinonoid MDA imine adduct to valine was formed by an attack of a metabolite formed through peroxidative oxidation of MDA, in analogy with earlier observed oxidation of some other aromatic amines, e.g., benzidine. The reactive intermediate is suggested to be [(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]-4-aminobenzene. The formation of the adduct was confirmed by incubating MDA with valine methyl ester in vitro in the presence of H2O2 and lactoperoxidase. Further, the same adduct was detected in MDI-exposed and control rats, the level in the exposed animals being about 60 times higher than in the controls. This study indicates that, at least in the mouse, extrahepatic peroxidative metabolism is an important pathway for the bioactivation of MDA, possibly leading to a genotoxic reactive intermediate. This study also demonstrates the usefulness of Hb adduct analysis for the identification of reactive intermediates in vivo.