Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay

Masaharu Fujita, Yusuke Yamamoto, Shinichi Watanabe, Tsunetsugu Sugawara, Koji Wakabayashi, Yu Tahara, Nobuyuki Horie, Keiichi Fujimoto, Kei Kusakari, Yoshihiko Kurokawa, Tsuyoshi Kawakami, Kohichi Kojima, Hajime Kojima, Atsushi Ono, Yasuhiro Katsuoka, Hideto Tanabe, Hiroshi Yokoyama, Toshihiko Kasahara

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA—N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)—was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu 2+ promoted NAC oxidation significantly. When 0.25 μm of EDTA was added in the presence of Cu 2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

Original languageEnglish
Pages (from-to)191-208
Number of pages18
JournalJournal of Applied Toxicology
Volume39
Issue number2
DOIs
Publication statusPublished - Feb 1 2019

Keywords

  • ADRA (amino acid derivative reactivity assay)
  • Cu
  • DPRA (direct peptide reactivity assay)
  • EDTA (ethylenediaminetetraacetic acid)
  • NAC
  • OECD test guideline
  • in chemico alternative to animal testing
  • oxidation
  • skin sensitization

ASJC Scopus subject areas

  • Toxicology

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  • Cite this

    Fujita, M., Yamamoto, Y., Watanabe, S., Sugawara, T., Wakabayashi, K., Tahara, Y., Horie, N., Fujimoto, K., Kusakari, K., Kurokawa, Y., Kawakami, T., Kojima, K., Kojima, H., Ono, A., Katsuoka, Y., Tanabe, H., Yokoyama, H., & Kasahara, T. (2019). Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay. Journal of Applied Toxicology, 39(2), 191-208. https://doi.org/10.1002/jat.3707