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Characterization of hemoglobin adducts from a 4, 4'-methylenedianiline metabolite evidently produced by peroxidative
A Kautiainen1, C A Wachtmeister, L Ehrenberg
1Department of Environmental Chemistry, Wallenberg Laboratory, and Department of Radiobiology, Stockholm University, S-106 91 Stockholm, Sweden. antti@mk.su.se
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.
Insights
This study identifies a new hemoglobin (Hb) adduct formed from 4,4'-Methylenedianiline (MDA), a carcinogen. This discovery offers a novel method for biomonitoring MDA exposure and understanding its toxicological pathways.
Area of Science:
- Toxicology
- Biochemistry
- Chemical Engineering
Background:
- 4,4'-Methylenedianiline (MDA) is a mutagenic and carcinogenic industrial chemical.
- MDA is a metabolite of 4,4'-methylenediphenyl diisocyanate (MDI), used in polyurethane manufacturing.
- Current biomonitoring of MDA relies on measuring Hb adducts from its nitroso metabolite.
Purpose of the Study:
- Investigate Hb adducts formed from non-nitroso metabolites of MDA for improved dosimetry.
- Identify and characterize novel MDA-Hb adducts in vivo.
- Explore the role of peroxidative metabolism in MDA bioactivation.
Main Methods:
- Treatment of mice with labeled MDA (tritiated or deuterated).
- Enzymatic hydrolysis of globin and enrichment of adducts for identification.
- Mass spectrometry (MS) for adduct characterization and in vitro confirmation.
Main Results:
- A major Hb adduct, 1-[(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]benzene-4-azo-2-isovaleric acid, was identified, comprising ~50% of MDA-Hb adducts.
- This adduct is likely formed via peroxidative oxidation of MDA to a reactive quinonoid imine intermediate.
- The adduct was detected in MDI-exposed rats at significantly higher levels than controls.
Conclusions:
- Extrahepatic peroxidative metabolism is a key pathway for MDA bioactivation in mice.
- This pathway may lead to genotoxic reactive intermediates.
- Hb adduct analysis is valuable for identifying reactive intermediates in vivo.