Development and assessment of new risk assessment methods

Title of Research:
15_PT01-01

Development of new in vivo screening method to validate thelow-dose effects induced by estrogenic chemicals using estrogen
reporter micePrincipal Investigator:
Tsuyoshi Nakanishi (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Collaborators:
Hisamitsu Nagase (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)

Summary of Research:
Objective: The uterotrophic bioassay has been proposed as a short-term in vivo screening test todetect the estrogenic properties of potentially endocrine disrupting chemicals by the Organisation
for Economic Co-operation and Development (OECD). Although the test is already standardized
as a OECD test guideline (Test No.440), there is a possibility that the test might overlook a certain
estrogenic properties which has no effect to the uterus. On the other hand, we recently generated
a new transgenic mouse carrying “E-Rep” reporter gene which consisted of the luciferase (Luc)with c-Myc tag under the control of estrogen-responsive elements coupled to a minimal promoter.
E-Rep mouse is a potential powerful tool for assessing the kinetics of gene activation by
estrogenic properties in vivo. We here established a new in vivo screening assay using E-Rep
mice with in vivo bioluminescence imaging to detect the estrogenic effects of xenobiotics andvalidated the assay to compare with the uterotrophic bioassay.
Methods: The experimental protocols of the new assay using E-Rep mice are based on theuterotrophic bioassay with some modifications. Briefly, E-Rep female mice (8 weeks of age) wereovariectomized and then fed phytoestrogen-free purified diet during the experimental period. After
7 days of ovariectomy, the mice were orally gaveged with 17α-ethinylestradiol (EE) for 7
consecutive days. The Luc activity was monitored in living animals with IVIS imaging at Day 1, 2, 4,
7. After last detection of in vivo bioluminescence imaging, mice were dissected and then theestrogenic effects were evaluated by measurement of uterine weight or detection of Luc activity atDay 7.
Results: Luc activity in the uterus increased significantly when EE was administered at 3
µg/kg/day or greater at Day 7. In contrast, the uterine weight rose significantly only when estradiol
was administered at 10 µg/kg/day. In addition, in vivo bioluminescence imaging analysis could
detect significant increment of Luc activity at above 0.3 µg/kg/day of EE, suggesting that detectionof Luc activity in E-Rep mice by in vivo bioluminescence imaging provide a more sensitive assayof estrogenic activity than the uterotrophic bioassay.
Conclusion: E-Rep mice with in vivo bioluminescence imaging provide a potential new assay
system to measure estrogenic activity of endocrine disrupting chemicals in multiple organs withhigh sensitivity.
Timeline: April, 2016 – March, 2017
Topics:1) Our research paper “Fluorene-9-bisphenol is anti-oestrogenic and may cause adverse
pregnancy outcomes in mice” published in Nature Communications was highlighted innatureasia.com (http://www.natureasia.com/en/research/highlight/11678).
2) Our research paper “Fluorene-9-bisphenol is anti-oestrogenic and may cause adverse
pregnancy outcomes in mice” published in Nature Communications featured as a daily news

Development and assessment of new risk assessment methods
entitled “BPA-free water bottles may contain another harmful chemical” in New Scientist(https://www.newscientist.com/article/2123098-bpa-free-water-bottles-may-contain-another-harmful-chemical/).
Publications:1) Zhang Z, Hu Y, Guo J, Yu T, Sun L, Xiao X, Zhu D, Nakanishi T, Hiromori Y, Li J, Fan X, Wan Y,
Cheng S, Li J, Guo X, Hu J, Fluorene-9-bisphenol is anti-oestrogenic and may cause adverse
pregnancy outcomes in mice, Nature Communications 8:14585 (2017)