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-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(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.

[1]  K. Wakabayashi,et al.  o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis. , 2021, Chemical research in toxicology.

[2]  Young-Man Cho,et al.  Expression of stem cell markers as useful complementary factors in the early detection of urinary bladder carcinogens by immunohistochemistry for γ-H2AX , 2020, Archives of toxicology.

[3]  K. Wakabayashi,et al.  A novel o-toluidine metabolite in rat urine associated with urinary bladder carcinogenesis. , 2020, Chemical research in toxicology.

[4]  Kenji Watanabe,et al.  Elevated levels of proinflammatory volatile metabolites in feces of high fat diet fed KK-Ay mice , 2020, Scientific Reports.

[5]  N. Miyoshi,et al.  Distinct differences in the mechanisms of mucosal damage and γ-H2AX formation in the rat urinary bladder treated with o-toluidine and o-anisidine , 2019, Archives of Toxicology.

[6]  C. Weight,et al.  Targeted and Untargeted Detection of DNA Adducts of Aromatic Amine Carcinogens in Human Bladder by Ultra-Performance Liquid Chromatography-High-Resolution Mass Spectrometry. , 2018, Chemical research in toxicology.

[7]  S. Miura,et al.  Effects of Sanyaku and Its Constituent Diosgenin on the Fasted and Postprandial Hypertriacylglycerolemia in High-Fat-Diet-Fed KK- A y Mice. , 2018, Journal of agricultural and food chemistry.

[8]  G. Miller,et al.  CYP2E1 hydroxylation of aniline involves negative cooperativity. , 2014, Biochemical pharmacology.

[9]  Susumu Tomono,et al.  Occurrence of cytotoxic 9-oxononanoyl secosterol aldehydes in human low-density lipoprotein. , 2013, Free radical biology & medicine.

[10]  Philip Levy Ho,et al.  Normal and neoplastic urothelial stem cells: getting to the root of the problem , 2012, Nature Reviews Urology.

[11]  W. M. Seganish,et al.  Preparation and palladium-catalyzed cross-coupling of aryl triethylammonium bis(catechol) silicates with aryl triflates. , 2004, The Journal of organic chemistry.

[12]  V. Havlíček,et al.  Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA. , 2002, Mutation research.