Research on the safety of new chemical substances including nanomaterials

Title of Research:
15_PT02-01

Construction of novel in vitro evaluation systems based on
genotoxic mechanisms of nanomaterials
Principal Investigator:Yukari Totsuka, Ph.D., Natl. Cancer Centr. Res. Inst.

Collaborators:
Dai Nakae, M.D., Ph.D. (Professor, Laboratory of Food Safety Assessment Science,Department of Nutritional Science and Food Safety, Faculty of Applied Biosciences, Tokyo
University of Agriculture),
Masatoshi Watanabe, M.D., Ph.D. (Professor, Yokohama National University, Graduate
School of Engineering),
Koichiro Hayashi (Division on Materials Research, Institute of Materials and Systems forSustainability, Nagoya University)

Summary of Research: To establish new in vitro evaluation systems for lung/skin toxicity
of nanomaterials, we aimed to establish assay models as listed below.

①

A novel in vitro genotoxicity assay model to assess lung toxicity using a co-culturesystem.

②

A novel in vitro assay model to assess skin toxicity using 3D human skinreconstitution models.

③

A novel in vitro assay model using 3D culture techniques such as tissue-slice andspheroids.

For the lung toxicity test, we established a co-culture system of GDL1 and RAW264 cells.
We evaluated nanomaterials having differences in physicochemical character by using thisco-culture system. In the present study, we chose multi-walled carbon nanotubes(MWCNTs) with different sizes, and magnetite (MGT) with/without surface modification as
test nanomaterials. When gpt delta mice were intratracheally instilled with multiple dosesof different sizes of MWCNTs, mutation frequency (MF) observed in the lungs was higher
than that of the vehicle control. However, the influence of size differences against MF was
not observed in the lungs of mice exposed to these MWCNTs. Supporting this, a similar
result was observed in the co-culture assay system. We also evaluated MF induced by MGT
with/without surface modification using the co-culture system, and there was a tendency
that higher MF was observed in the cells exposed to MGT with surface modification thanthat of MGT without surface modification. Therefore, it is suggested that the co-culture

Research on the safety of new chemical substances including nanomaterials
assay model can be considered as a suitable evaluation system for nanomaterial toxicity
for the lungs.
As a 3D skin model, we selected the LabCyte EPI model. Using biochemical andhistopathological techniques, we assessed cytotoxicity and ability of gold and silver
nanoparticles to invade into the skin using a reconstituted 3D human skin model (LabCyteEPI model, Japan Tissue Engineering Co., Ltd.) and HepG2 cells. In the reconstituted 3D
human skin model, neither gold nor silver nanoparticles killed or invaded into theepidermis up to 1000 µg/mL, while neither nanoparticle killed HepG2 cells up to 100 µg/mL.
It is thus suggested that gold or silver nanoparticles do not possess cytotoxicity or ability
to invade into the skin, at least under the present experimental conditions. Furthermore,
the LabCyte EPI model may be useful as a novel in vitro system to assess percutaneous
toxicity of nanomaterials.
On the other hand, since the respiratory system is susceptible to damage resulting frominhalation of nanomaterials, the assay of A549 spheroids layered on histological sections
was used for MGT induced cytotoxicity. Results of spheroid disruption by cellular adhesion
damage and cell viability by Alamar Blue assay showed the possibility of the assay of A549
spheroids layered on histological sections as a toxicological safety test.
Timeline: March 1st, 2016 – February 29th, 2017

Topics: “Construction of novel in vitro evaluation systems based on genotoxic mechanisms ofnanomaterials “ Presented at the poster session of the Annual Conference of New JCIA-LRI

Publications:
Journals:

1.

KatoT, Toyooka T, Ibuki Y, Masuda S, Watanabe M, Totsuka Y. Effect of Physicochemical
Character Differences on the Genotoxic Potency of Kaolin. Genes Environ., in press.

2.

Koichiro Hayashi, Yoshitaka Sato, Wataru Sakamoto, Toshinobu Yogo, “Theranostic
Nanoparticles for MRI-Guided Thermochemotherapy: Tight Clustering of Magnetic
Nanoparticles Boosts Relaxivity and Heat-Generation Power” ACS Biomaterials Science& Engineering, 3, 95–105, 2017.

Meetings:
1. TotsukaY, Watanabe M, Hayashi K, Nakae D: Development of a novel in vitro mechanism-based
evaluation system of the genotoxicity of nanomaterials 45th EEMGS(Copenhagen、Aug, 2016）
2. Sato H, Sakamoto Y, Nakae D, Totsuka Y: Effect of Physicochemical Character Differenceson genotoxic potency of MWCNTs 45th JEMS（Tsukuba、Nov, 2016）