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Abstract: This study evaluated the biological responses to multi-walled carbon nanotubes (MWCNTs). Human monoblastic leukemia cells (U937) were exposed to As-grown MWCNTs and MWCNTs that were thermally treated at 1800 °C (HTT1800) and 2800 °C (HTT2800). Cell proliferation was highly inhibited by As-grown but not HTT2800. However, both As-grown and HTT1800, which include some impurities, were cytotoxic. Proteomics analysis of MWCNT-exposed cells revealed 37 protein spots on 2-dimensional electrophoresis gels that significantly changed (p <0.05) after exposure to HTT1800 with a little iron and 20 spots that changed after exposure to HTT2800. Peptide mass fingerprinting identified 45 proteins that included heat shock protein β-1, neutral α-glucosidase AB, and DNA mismatch repair protein Msh2. These altered proteins play roles in metabolism, biosynthesis, response to stress, and cell differentiation. Although HTT2800 did not inhibit cell proliferation or cause cytotoxicity in vitro, some proteins related to the response to stress were changed. Moreover, DJ-1 protein, which is a biomarker of Parkinson''s disease and is related to cancer, was identified after exposure to both MWCNTs. These results show that the cytotoxicity of MWCNTs depends on their impurities, such as iron, while MWCNTs themselves cause some biological responses directly and/or indirectly in vitro. Our proteomics-based approach for detecting biological responses to nanomaterials is a promising new method for detailed safety evaluations. [Copyright &y& Elsevier]