Cytotoxic Homo- and Hetero-Dimers of o -toluidine, o -anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o -toluidine ( o -Tol), o -anisidine ( o -Ans), and aniline (Ani), have not been completely determined. We have recently reported that o -Tol and o -Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p -semidine-type dimers, namely 2-methyl- N 4 -(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy- N 4 -(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o -Tol and o -Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o -Tol, o -Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p -semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 μM. Homo- and hetero-dimers containing the o -Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o -Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o -Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o -Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.