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:
Background: The uterotrophic bioassay has been proposed as a short-term in vivo screening testto detect the estrogenic properties of potentially endocrine disrupting chemicals by theOrganisation for Economic Co-operation and Development (OECD). Although the test is alreadystandardized as a OECD test guideline (Test No.440), there is a possibility that the test mightoverlook 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 theluciferase (Luc) 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 byestrogenic properties in vivo. In the 4th term, we have established a provisional protocol for thenovel in vivo screening test, which employs bioluminescence imaging in the whole body as theend point for estrogenic action of test chemicals.
Objective: In the current term, we verified the validity of the provisional protocol by confirming
whether the estrogen-induced bioluminescence imaging in the whole body reflects the estrogenic
action in each organ. In addition, using the bone tissue-specific selective estrogen receptor
modulators (SERM), which have little effect on the uterus, we confirmed whether our novel in vivo
screening test have an advantage over 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) or SERMs for
7 consecutive days. The Luc activity was monitored in living animals with IVIS imaging at Day 1, 2,4 and 7. After last detection of in vivo bioluminescence imaging, mice were dissected andmeasured uterine weight in accordance with the uterotrophic bioassay. For measurement of daily
Luc activity induced by EE in each organ, EE-gaveged mice were dissected and removed eachorgan at Day 1, 2, 4 and 7.
Results: According to measurement of daily Luc activity in each organ, it was confirmed that theEE-induced bioluminescence imaging in the whole body reflects its estrogenic action in eachorgan. Furthermore In the experiment using the bone tissue-specific SERMs, our novel in vivo
screening test could determine the SERMs as positive estrogens, whereas the uterotrophic
bioassay could not determine a SERM even at a dose which show sufficient estrogen activity inthe bone tissue.
Conclusion: Our novel in vivo screening test using E-Rep mice with in vivo bioluminescenceimaging is an excellent method that can detect SERMs that cannot be detected by the uterotrophic
bioassay.
Timeline: April, 2017 – March, 2018

Development and assessment of new risk assessment methods
Topics:1) Dr. Tsuyoshi Nakanishi was awarded the 4th JCIA-LRI award for the research theme"Comprehensive research on reproductive toxicity of nuclear receptor-activated hazards andestablishment of its evaluation system".
Publications:
1) Nishioka Y, Tamai K, Onda M, Hiromori Y, Kimura T, Hu J, Nagase H, Nakanishi T, Potential
interference of oil vehicles on genital tubercle development during the fetal period in ICR mice,Biol. Pharm. Bull., 41:266-271 (2018)