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.