Archives of Toxicology

, Volume 93, Issue 3, pp 753–762 | Cite as

Distinct differences in the mechanisms of mucosal damage and γ-H2AX formation in the rat urinary bladder treated with o-toluidine and o-anisidine

  • Takeshi ToyodaEmail author
  • Kohei Matsushita
  • Tomomi Morikawa
  • Takanori Yamada
  • Noriyuki Miyoshi
  • Kumiko Ogawa
Genotoxicity and Carcinogenicity


Although aromatic amines are widely used as raw materials for dyes, some of them have been concerned about carcinogenicity in the urinary bladder. We examined early changes in histopathology and the formation of γ-H2AX, a biomarker of DNA damage, in the urinary bladder of rats to investigate the mechanisms of mucosal damage induced by monocyclic aromatic amines. 6-week-old male F344 rats were administered 0.4% or 0.8% o-toluidine, 0.3% or 1.0% o-anisidine, 0.4% 2,4-xylidine, 0.2% p-toluidine, or 0.6% aniline in the diet for 4 weeks. Animals were sequentially killed from day 2 to after 2 weeks of recovery, and histopathological and immunohistochemical analyses were performed. In the 0.8% o-toluidine group, there was sequential progression of bladder lesions, characterized by edematous changes and intramucosal hemorrhage at day 2 and formation of granulation tissue with mononuclear cell infiltration at week 1, followed by diffuse hyperplasia at weeks 2 and 4. In the 1.0% o-anisidine group, simple hyperplasia only with slight inflammation was detected from week 1. Whereas γ-H2AX-positive bladder epithelial cells in the 1.0% o-anisidine group were significantly increased in a time-dependent manner, transient increases in γ-H2AX-positive cells were detected at day 2 and week 1 in the 0.8% o-toluidine group. No apparent bladder lesions or increases in γ-H2AX formation were observed in any other groups. These results revealed different mechanisms of bladder mucosal damage associated with o-toluidine and o-anisidine. Moreover, immunohistochemical analysis for γ-H2AX suggested that both compounds may induce DNA damage in epithelial cells, mainly basal cells, of the bladder mucosa.


Aromatic amine γ-H2AX o-Anisidine o-Toluidine Rat Urinary bladder 



This work was supported by a Health and Labour Sciences Research Grant for the Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare, Japan. The authors thank Ayako Saikawa and Yoshimi Komatsu for expert technical assistance in processing histological materials.

Compliance with ethical standards

Conflict of interest

Takanori Yamada is an employee of Sanwa Kagaku Kenkyusho, Nagoya, Japan. All authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Takeshi Toyoda
    • 1
    Email author
  • Kohei Matsushita
    • 1
  • Tomomi Morikawa
    • 1
  • Takanori Yamada
    • 1
    • 2
  • Noriyuki Miyoshi
    • 3
  • Kumiko Ogawa
    • 1
  1. 1.Division of PathologyNational Institute of Health SciencesKawasakiJapan
  2. 2.Laboratory of Veterinary PathologyTokyo University of Agriculture and TechnologyFuchuJapan
  3. 3.Laboratory of Biochemistry, Graduate Program of Food Nutritional SciencesUniversity of ShizuokaShizuokaJapan

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