Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids

Masami Komiya; Rikako Ishigamori; Mie Naruse; Masako Ochiai; Noriyuki Miyoshi; Toshio Imai; Yukari Totsuka

doi:10.3389/fgene.2021.768781

Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids

Front Genet. 2021 Nov 18:12:768781. doi: 10.3389/fgene.2021.768781. eCollection 2021.

Authors

Masami Komiya¹, Rikako Ishigamori², Mie Naruse², Masako Ochiai³, Noriyuki Miyoshi⁴, Toshio Imai^{1

2}, Yukari Totsuka^{1

5}

Affiliations

¹ Department of Cancer Model Development, National Cancer Center Research Institute, Tokyo, Japan.
² Department of Animal Experimentation, National Cancer Center Research Institute, Tokyo, Japan.
³ Food Environment Research Center, Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan.
⁴ Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
⁵ Laboratory of Environmental Toxicology and Carcinogenesis, School of Pharmacy, Nihon University, Chiba, Japan.

Abstract

Short-/middle-term and simple prediction studies for carcinogenesis are needed for the safety assessment of chemical substances. To establish a novel genotoxicity assay with an in vivo mimicking system, we prepared murine colonic/pulmonary organoids from gpt delta mice according to the general procedure using collagenase/dispase and cultured them in a 3D environment. When the organoids were exposed to foodborne carcinogens-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and acrylamide (AA)-in the presence of metabolic activation systems, mutation frequencies (MFs) occurring in the gpt gene dose-dependently increased. Moreover, the mutation spectrum analysis indicated predominant G:C to T:A transversion with PhIP, and A:T to C:G and A:T to T:A transversion with AA. These data correspond to those of a previous study describing in vivo mutagenicity in gpt delta mice. However, organoids derived from the liver, a non-target tissue of PhIP-carcinogenesis, also demonstrated genotoxicity with a potency comparable to colonic organoids. Organoids and PhIP were directly incubated in the presence of metabolic activation systems; therefore, there was a lack of organ specificity, as observed in vivo. Additionally, PhIP-DNA adduct levels were comparable in hepatic and colonic organoids after PhIP exposure. Taken together, the organoids prepared in the present study may be helpful to predict chemical carcinogenesis.

Keywords: PhIP; acrylamide; base substitution; gpt delta mice; murine organoids.