Reactive intermediates formed during the peroxidative oxidation of anisidine isomers.
The ortho isomer of anisidine (2-methoxyaniline) causes urinary bladder tumors in both mice and rats while the para isomer (4-methoxyaniline) is inactive. Since the urinary bladder contains substantial peroxidase activity, we investigated the peroxidative metabolism of both o- and p-anisidine using horseradish peroxidase as a model enzyme. Both isomers were excellent reducing cofactors for the oxidized state of horseradish peroxidase (HRP), resulting in one-electron oxidation to free radicals. Using high-pressure liquid chromatography, we observed that HRP oxidized p-anisidine to a diimine metabolite which subsequently hydrolyzed to form a quinone imine. Also observed was a dimeric metabolite with an azo bond. Both the diimine and quinone imine metabolites were reactive toward nucleophiles. The quinone imine formed a conjugate with glutathione and was also reduced by glutathione or ascorbic acid. Higher concentrations of substrate (greater than 1 mM) led to the formation of polymeric products (tetramer). Similar metabolites (diimine, quinone imine, azo dimer, polymers) were observed with o-anisidine. Using tritium-labeled anisidine, we observed substantial metabolism-dependent covalent binding of both isomers to protein and DNA. These results demonstrate that horseradish peroxidase dependent metabolism of anisidine isomers yields similar metabolites, although some differences in reactivity of the respective intermediates with nucleophiles were observed.
Thompson
D C
DC
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Eling
T E
TE
D000814:Aniline Compounds / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000494:pharmacology*; D010084:Oxidation-Reduction
D000814:Aniline Compounds; D005609:Free Radicals; D004247:DNA; D006735:Horseradish Peroxidase; D005978:Glutathione