Intracellular Generation of ROS by 3,5-Dimethylaminophenol: Persistence, Cellular Response, and Impact of Molecular Toxicity

Epidemiological studies have demonstrated extensive human exposure to the monocyclic aromatic amines, particularly to 3,5-dimethylaniline, and found an association between exposure to these compounds and risk for bladder cancer. Little is known about molecular mechanisms that might lead to the observed risk. We previously suggested that the hydroxylated 3,5-dimethylaniline metabolite, 3,5-dimethylaminophenol (3,5-DMAP), played a central role in effecting genetic change through the generation of reactive oxygen species (ROS) in a redox cycle with 3,5-dimethylquinoneimine. Experiments here characterize ROS generation by 3,5-DMAP exposure in nucleotide repair-proficient and -deficient Chinese hamster ovary cells as a function of time. Besides, various cellular responses discussed herein indicate that ROS production is the principal cause of cytotoxicity. Fluorescence microscopy of cells exposed to 3,5-DMAP confirmed that ROS production occurs in the nuclear compartment, as suggested by a previous study demonstrating covalent linkage between 3,5-DMAP and histones. 3,5-DMAP was also compared with 3,5-dimethylhydroquinone to determine whether substitution of one of the phenolic hydroxyl groups by an amino group had a significant effect on some of the investigated parameters. The comparatively much longer duration of observable ROS produced by 3,5-DMAP (7 vs. 1 day) provides further evidence that 3,5-DMAP becomes embedded in the cellular matrix in a form capable of continued redox cycling. 3,5-DMAP also induced dose-dependent increase of H2O2 and center dot OH, which were determined as the major free radicals contributing to the cytotoxicity and apoptosis mediated via caspase-3 activation. Overall, this study provides insight into the progression of alkylaniline-induced toxicity.