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.
Highlights
Safety evaluation is required to develop chemical substances, such as food additives and pharmaceuticals (Yamada and Honma, 2018)
The in vitro genotoxicity tests have been widely used as simplified safety evaluation tests for chemical substances, but it is difficult to predict carcinogenicity from these tests alone
We demonstrated that PhIP and AA showed strong genotoxicity in a dose-dependent manner in their target organs for carcinogenesis (Tables 1, 4)
Summary
Safety evaluation is required to develop chemical substances, such as food additives and pharmaceuticals (Yamada and Honma, 2018). We selected organoids derived from guanine phosphoribosyl transferase (gpt) delta mice (background strain C57BL/6J) (Nohmi et al, 2000) In this mouse model, point mutations and deletions are analyzed by gpt and Spi-selections, respectively, and are widely used in the field of environmental mutagenicity, including 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) and acrylamide (AA) (Figure 1) (Masumura et al, 1999; Masumura et al, 2000; Ishii et al, 2015). Point mutations and deletions are analyzed by gpt and Spi-selections, respectively, and are widely used in the field of environmental mutagenicity, including 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) and acrylamide (AA) (Figure 1) (Masumura et al, 1999; Masumura et al, 2000; Ishii et al, 2015) Both chemicals are known as foodborne carcinogens and are responsible for the development of some human cancers (de Conti et al, 2019; Adani et al, 2020; Bellamri et al, 2021; Le Marchand, 2021). We investigated mutagenicity in the liver, a non-target tissue for PhIP-carcinogenesis
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