A novel genotoxicity assay of carbon nanotubes using functional macrophage receptor with collagenous structure (MARCO)-expressing chicken B lymphocytes.

Although carbon nanotubes (CNTs) are promising nanomaterials, their potential carcinogenicity is a major concern. We previously established a genetic method of analyzing genotoxicity of chemical compounds, where we evaluated their cytotoxic effect on the DT40 lymphoid cell line comparing DNA-repair-deficient isogenic clones with parental wild-type cells. However, application of our DT40 system for the cytotoxic and genotoxic evaluation of nanomaterials seemed to be difficult, because DT40 cells only poorly internalized nanoparticles. To solve this problem, we have constructed a chimeric gene encoding a trans-membrane receptor consisting of the 5' region of the transferrin receptor (TR) gene (to facilitate internalization of nanoparticles) and the 3' region of the macrophage receptor with collagenous structure (MARCO) gene (which is a receptor for environmental particles). We expressed the resulting MARCO-TR chimeric receptor on DNA-repair-proficient wild-type cells and mutants deficient in base excision repair (FEN1 (-/-)) and translesion DNA synthesis (REV3 (-/-)). We demonstrated that the chimera mediates uptake of particles such as fluorescence-tagged polystyrene particles and multi-walled carbon nanotubes (MWCNTs), with very poor uptake of those particles by DT40 cells not expressing the chimera. MWCNTs were cytotoxic and this effect was greater in FEN1 (-/-)and REV3 (-/-) cells than in wild-type cells. Furthermore, MWCNTs induced greater oxidative damage (measured as 8-OH-dG formation) and a larger number of mitotic chromosomal aberrations in repair-deficient cells compared to repair-proficient cells. Taken together, our novel assay system using the chimeric receptor-expressing DT40 cells provides a sensitive method to screen for genotoxicity of CNTs and possibly other nanomaterials.