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

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.

The application of organoids in toxicity test of environmental pollutants

“…Intestine [99] Arsenic Diluted in media 0-100 μmol/L 24, 48, and 72 h Induce cell toxicity, oxidative stress, and gut barrier dysfunction Mouse intestine [100] Marine toxins okadaic acid (OA) and conotoxin (CgTx)…”

“…Studies have shown that the metabolic characteristics of induced pluripotent stem cells (iPSCs), hepatic endoderm cells and mature hepatocytes are not the same [97] . Compared with 2D cells, 3D organoids have unique advantages in maintaining metabolic function [98][99][100] .…”

“…Due to the limitations of culture technology, organoids typically contain off-target cell populations with low reproducibility, lack of cell complexity [93] and structure, in particular lack of a functional perfusion vascular network, leading to loss of metabolic function [100,140] . The use of organoids in the study of the toxicity of environmental pollutants is limited by these problems [109] .…”

Assessment of the genotoxicity of acrylamide

“…AA induced a modest increase (2‐fold at the highest tested dose) in mutation frequency at the knock‐in lacZ gene present in Hupki mouse embryo fibroblasts (Hölzl‐Armstrong et al, 2020a ) or a marginal increase (1.5‐fold) in the metabolically competent FE lung cell line (Hölzl‐Armstrong et al, 2020b ). In the presence of S9, gpt gene mutations were induced in lung cells in pulmonary organoid structures from gpt delta C57BL/6J mice (Komiya et al, 2021 ). The mutational spectrum (increases in GC > AT transitions, AT > TA and AT > CG transversions and deletions/insertions) was similar to that previously reported in vivo in the same transgenic mice (Ishi et al, 2015 ).…”