α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity

Yosuke Maeda; Masahiro Takeyoshi; Takehisa Chuma; Hiroyuki Iwata

doi:10.1016/j.tox.2020.152476

α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity

Toxicology. 2020 Jun:439:152476. doi: 10.1016/j.tox.2020.152476. Epub 2020 Apr 23.

Authors

Yosuke Maeda¹, Masahiro Takeyoshi², Takehisa Chuma³, Hiroyuki Iwata⁴

Affiliations

¹ Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Japan. Electronic address: maeda-yosuke@ceri.jp.
² Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan.
³ The United Graduate School of Veterinary Science, Yamaguchi University, Japan; Joint Faculty of Veterinary Medicine, Kagoshima University, Japan.
⁴ The United Graduate School of Veterinary Science, Yamaguchi University, Japan.

PMID: 32335162
DOI: 10.1016/j.tox.2020.152476

Abstract

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.

Keywords: Adverse outcome pathway; Antioxidant response element (ARE); In vitro; Keratinocyte activation; Skin sensitization.

MeSH terms

Animal Testing Alternatives
Animals
Antioxidant Response Elements / drug effects*
Cell Survival / drug effects
Dermatitis, Allergic Contact / pathology*
Drug Evaluation, Preclinical / methods*
Humans
Keratinocytes / drug effects
Local Lymph Node Assay
Luciferases / metabolism
NF-E2-Related Factor 2 / drug effects*
Renilla / enzymology
Sensitivity and Specificity
Skin Tests
Thymidine Kinase / metabolism
Transcription, Genetic / drug effects*

Substances

NF-E2-Related Factor 2
Nfe2l2 protein, mouse
Luciferases
Thymidine Kinase