Promotion effects of acetoaceto-o-toluidide on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats

  • Nao Yukimatsu
  • Min Gi
  • Takahiro Okuno
  • Masaki Fujioka
  • Shugo Suzuki
  • Anna Kakehashi
  • Yukie Yanagiba
  • Megumi Suda
  • Shigeki Koda
  • Tatsuya Nakatani
  • Hideki WanibuchiEmail author
Genotoxicity and Carcinogenicity


Recent epidemiological studies have indicated that occupational exposure to the aromatic amine acetoaceto-o-toluidide (AAOT) was associated with a marked increase in urinary bladder cancers in Japan. However, little is known about the carcinogenicity of AAOT. To evaluate the urinary bladder carcinogenicity of AAOT, male and female F344 rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks followed by dietary administration of 0, 0.167, 0.5, or 1.5% AAOT for 31 weeks. The incidences and multiplicities of bladder tumors were significantly increased in the 0.5 and 1.5% groups of male and female rats in a dose-response manner. AAOT and seven downstream metabolites were detected in the urine of the male and female rats administered AAOT with levels increasing in a dose-dependent manner. The most abundant urinary metabolite of AAOT was the human bladder carcinogen o-toluidine (OTD), which was at least one order of magnitude higher than AAOT and the other AAOT metabolites. In a second experiment, male F344 rats were administered 0, 0.167, or 1.5% AAOT for 4 weeks. Gene expression analyses revealed that the expression of JUN and its downstream target genes was increased in the urothelium of male rats treated with 1.5% AAOT. These results demonstrate that AAOT promotes BBN-induced urinary bladder carcinogenesis in rats and suggest that overexpressed of JUN and its downstream target genes may be involved the bladder carcinogenicity of AAOT. In conclusion, AAOT, like other carcinogenic aromatic amines, is likely to be a carcinogen to the urinary bladder, and OTD metabolized from AAOT is the ultimate carcinogen.


AAOT OTD Bladder carcinogenicity Rat Occupational urinary bladder cancer 



This work was supported by a grant from Ministry of Health, Labor and Welfare, Japan, and Grant-in-Aid for Scientific Research form Japan Society for the Promotion of Science (JSPS KAKENHI Grant number 18H06347 and 18K09146). We are grateful to Dr. Michiharu Matsumoto for his statistical consultant (Japan Bioassay Research Center, Japan Organization of Occupational Health and Safety). We gratefully acknowledge the technical assistance of Rie Onodera, Keiko Sakata, Yuko Hisabayashi, and Yukiko Iura (Department of Molecular Pathology, Osaka City University Graduate School of Medicine School), and Emi Donoue (Research Support Platform, Osaka City University Graduate School of Medicine).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The manuscript does not contain clinical studies or patient data.

Supplementary material

204_2019_2605_MOESM1_ESM.docx (22 kb)
Supplementary file1 (DOCX 21 kb)


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Copyright information

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

Authors and Affiliations

  • Nao Yukimatsu
    • 1
    • 3
  • Min Gi
    • 1
    • 2
  • Takahiro Okuno
    • 1
  • Masaki Fujioka
    • 1
  • Shugo Suzuki
    • 1
  • Anna Kakehashi
    • 1
  • Yukie Yanagiba
    • 4
  • Megumi Suda
    • 4
  • Shigeki Koda
    • 4
  • Tatsuya Nakatani
    • 3
  • Hideki Wanibuchi
    • 1
    Email author
  1. 1.Department of Molecular PathologyOsaka City University Graduate School of MedicineOsakaJapan
  2. 2.Department of Environmental Risk AssessmentOsaka City University Graduate School of MedicineOsakaJapan
  3. 3.Department of UrologyOsaka City University Graduate School of MedicineOsakaJapan
  4. 4.Industrial Toxicology and Health Effects Research GroupNational Institute of Occupational Safety and Health, Japan (JNIOSH)KawasakiJapan

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