o-Toluidine and its metabolites induce oxidative and nitrative DNA damage in human urinary bladder epithelial cells

o-Toluidine has been used for multiple industrial purposes, but causes bladder carcinogenesis in exposed workers. However, its underlying mechanism is still unclear. In this study, we examined oxidative and nitrative DNA damage caused by o-toluidine and its metabolites, 2-amino-m-cresol, 4-amino-m-cresol (4AC) and N-(4-hydroxy-2-methylphenyl) acetamide, using T24 human bladder epithelial cells. MTT assay showed that 4AC induced the most potent cytotoxicity among o-toluidine and its metabolites. In flow cytometry, 4AC showed the strongest ability of generating reactive oxygen species (ROS) and nitric oxide (NO) in exposed cells. Fluorescence immunocytochemistry revealed that 4AC significantly increased the staining intensities of an oxidative DNA lesion, 8-oxo-2'-deoxyguanosine, and a nitrative DNA lesion, 8-nitroguanine, at 5 nM. Their staining intensities were stronger than the other compounds. The comet assay showed that o-toluidine and its metabolites significantly increased the olive tail moment after the treatment with formamidopyrimidine DNA glycosylase. DNA damage was significantly reduced by transfection with small interfering RNA and antibodies against high-mobility group box-1 (HMGB1), receptor for advanced glycation end-products (RAGE) and Toll-like receptor (TLR) 9. These findings indicate that o-toluidine and its metabolites induce the release of HMGB1, which forms a complex with CpG DNA to bind to RAGE on neighboring cells and then is recognized by TLR9 in lysosomes, leading to ROS and NO production and DNA damage. In o-toluidine-induced carcinogenesis, TLR9-mediated inflammatory response and DNA damage induced by its metabolites, especially 4AC, may play a substantial role.