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Engineered nanomaterials ( ENM ) are now used in a wide variety of fields, and, thus, their safety should urgently be assessed and secured. It has been suggested that inflammatory responses via the phagocytosis of  ENM  by macrophages is a key mechanism for their genotoxicity. The present study was conducted to establish a mechanism‐based assay to evaluate the genotoxicity of  ENM  under conditions simulating an in vivo situation, featuring a co‐culture system of murine lung resident cells ( GDL 1) and immune cells ( RAW 264.7).  GDL 1 were cultured with or without  RAW 264.7, exposed to a multi‐walled carbon nanotube ( MWCNT ), and then analyzed for mutagenicity and underlying mechanisms. Mutation frequencies induced in  GDL 1 by the  MWCNT  were significantly greater with the co‐existence of  RAW 264.7 than in its absence. Mutation spectra observed in  GDL 1 co‐cultured with  RAW 264.7 were different from those seen in  GDL 1 cultured alone, but similar to those observed in the lungs of mice exposed to the  MWCNT  in vivo. Inflammatory cytokines, such as  IL ‐1β and  TNF ‐α, were produced from  RAW 264.7 cells treated with the  MWCNT . The generation of reactive oxygen species and the formation of 8‐oxodeoxyguanosine in  GDL 1 exposed to the  MWCNT  were greater in the co‐culture conditions than in the single culture conditions. Based on these findings, it is indicated that inflammatory responses are involved in the genotoxicity of  MWCNT , and that the presently established, novel in vitro assay featuring a co‐culture system of tissue resident cells with immune cells is suitable to evaluate the genotoxicity of  ENM .

Establishment of an in vivo simulating co‐culture assay platform for genotoxicity of multi‐walled carbon nanotubes