Some environmental alkylanilines, e.g., 3,5-dimethylaniline (3,5-DMA) may play a role in the etiology of human bladder cancer. The genotoxicity data of 3,5-DMA and its metabolites in cells indicated: 3,5-dimethylaminophenol (3,5-DMAP) had the highest  cytotoxicity and induced continuous dose-dependent production of reactive oxygen species (ROS). 3,5-DMAP at neutral or basic pH was spontaneously oxidized to 3,5-dimethyl-p-quinone imine (3,5-DMQI) by oxygen in the air. Then 3,5-DMQI reacted with the e-amino group of lysine and the thiol group of cysteine to form protein adducts both in vitro and in vivo. These aminophenol-adducts were continuously consuming the antioxidants or releasing ROS in cells via redox reactions. We developed a method combining activated thiol-sepharose and mass spectrometry to identify proteins modified by 3,5-DMAP. The quinone imine protein-adducts resulting from oxidation of aminophenol-adducts reacted with the thiol group on the agarose gel to selectively enrich modified proteins for identification. This method is high yield and could be potentially used to identify tissue proteins in animal models to facilitate the discovery of biomarkers and better understand the biological significance of protein-aminophenol in alkylanilines related diseases.

Identification of proteins modified by 3,5-dimethylaminophenol in human melanoma cancer cells

244th National Meeting (2012)

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