Development and assessment of new risk assessment methods
Title of Research:
20_PT03-05

Establishment of an adverse outcome pathway for the evaluation ofdevelopmental neurotoxicity in chemical-induced hypothyroidism
Principal Investigator:
Tsuyoshi Nakanishi (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)(e-mail) nakanishi@gifu-pu.ac.jp
Collaborators:
Daisuke Matsumaru (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)(e-mail) matsumaru-da@gifu-pu.ac.jp
Summary of Research:
Recently, the concern over the toxicity of environmental chemicals that induce hypothyroidism
has been increased, since hypothyroidism during pregnancy can have a significant impact on thedevelopment of the fetal brain in human. However, the results of thyroid function disruption
detected by toxicity testing have not been sufficiently utilized for risk assessment of chemical
substances because the correlation between the degree of hypothyroidism during pregnancy andadverse effects on the development of the fetal brain remains unclear. In order to solve theproblem, we focused on differentiation markers of mature neurons and produced reportertransgenic mice (Tg mice) carrying luciferase (Luc2) and LacZ genes downstream of a neuronaldifferentiation marker promoter. In the past year, we have confirmed the usefulness of Tg mice inassessing developmental neurotoxicity and found the possibility of evaluating the effects ofhypothyroidism from early pregnancy on the infant brain using the antithyroid drug propylthiouracil(PTU). In this fiscal year, we analyzed the correlation between the expression profile of in vivo
imaging in the infant brain and maternal thyroid-related parameters when hypothyroidism wasinduced by exposure to various doses of PTU from early pregnancy.
PTU induced a dose-dependent decrease in triiodothyronine (T3)/thyroxine (T4) and anincrease in thyroid-stimulating hormone (TSH) in maternal blood. In histological analysis of thethyroid gland, symptoms of hypothyroidism such as thickening of follicular cells were observed
even at low-dose PTU exposure prior to significant changes in serum T3/T4/TSH levels. Inaddition, the expression of reporter molecules in the brain of the infants was also increased in adose-dependent manner of PTU. Furthermore, similar to the results of thyroid histological analysis
in the mother, the effects were observed even at low-dose without significant changes in the serum
levels of thyroid-related hormones.
These results suggest that in chemical-induced hypothyroidism, thyroid histology is a moresensitive indicator for detecting their effects than serum thyroid-related hormones, and that someadverse effects on brain development in the child may occur prior to changes in maternal serum
thyroid-related hormones.
Timeline: April, 2022 – March, 2023
Topics:1) Oral presentation at JCIA LRI Annual Workshop 2020 “Establishment of an adverse outcome
pathway for the evaluation of developmental neurotoxicity in chemical-induced
hypothyroidism” (On-line, August 26th, 2022)
2) Report in the Yomiuri Shimbun Morning Edition “Environmental Chemicals and the Brain:A
New Approach Methodology Using "Glowing Mice" - Gifu Pharmaceutical University, etc.,
Verify Effects on Children” (February 7th, 2023)
Publications:

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Development and assessment of new risk assessment methods
Ishida K, Tatsumi K, Minamigawa Y, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,Nakanishi T, Neuronal differentiation reporter mice as a new methodology for detecting in vivo
developmental neurotoxicity. Biochem. Pharmacol., 206:115332 (2022)
Ishida K, Matsumaru D, Shimizu S, Hiromori Y, Hisamitsu Nagase H, Nakanishi T, Evaluationof the estrogenic action potential of royal jelly by genomic signaling pathway in vitro and invivo. Biol. Pharm. Bull., 45:1510–1517 (2022)
Ishida K, Furukawa M, Kunitani M, Yamagiwa R, Hiromori Y, Matsumaru D, Hu J, Nagase H,Nakanishi T, Novel, highly sensitive, in vivo screening method detects estrogenic activity at
low doses of bisphenol A. J. Hazard. Mater. 445:130461 (2023).

Conference Presentations:1) Ishida K, Minamigawa Y, Tatsumi K, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,Nakanishi T, Validation of brain neuronal differentiation reporter mice for improved
developmental neurotoxicity evaluation，2022 ICCA-LRI & NITE Workshop, Yokohama/Japan,
June 2022
2) Tatsumi K, Ishida K, Minamigawa Y, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,Nakanishi T, Validation of brain neuronal differentiation tracer mice for improvement
developmental neurotoxicity evaluation, The 49th Annual Meeting of the Japanese Society ofToxicology (Sapporo), 2022.
3) Mori K, Ishida K, Minamigawa Y, Tatsumi K, Matsumaru D, Murashima A, Nagase H, Kanda Y,Nakanishi T, Imaging analysis of the offspring brain in a model of maternal hypothyroidism,
The 49th Annual Meeting of the Japanese Society of Toxicology (Sapporo), 2022.
4) Ishida K, Minamigawa Y, Tatsumi K, Mori K, Matsumaru D, Nagase H, Kanda Y, Takuma K,Nakanishi T, Establishment of in vivo neuronal differentiation tracing method for improved
developmental neurotoxicity evaluation, The 62nd Annual Meeting of the Japanese
Teratology Society (Kanazawa), 2022.
5) Ishida K, Matsumaru D, Nakanishi T, Perinatal hypothyroidism and DOHaD: Aiming for therisk assessment of neurodevelopmental disorders in offspring, Forum 2022 Pharmaceutical
Health Sciences/Environmental Toxicology (Kumamoto), 2022.
6) Ishida K, Tatsumi K, Minamigawa Y, Mori K, Matsumaru D, Murashima A, Nagase H, Kanda Y,Takuma K, Nakanishi T, Neuronal differentiation reporter mice as a new methodology fordetecting in vivo developmental neurotoxicity, In vivo imaging forum 2022 (Tokyo), 2022.