The impact of two types of multi-walled carbon nanotubes (MWCNTs) (12-14 nm) with different content of metallic impurities (purified and unpurified nanotubes) on peroxidation processes, the status of immune cells in healthy volunteers and gene expression combined to pathway analysis was studied in vitro. From the study it was shown that the main mechanism of action for both types of MWCNTs is induction of oxidative stress, the intensity of which is directly related to the amount of metallic impurities. Unpurified MWCNTs produced twice as high levels of oxidation than the purified CNTs inducing thus more intense mitochondrial dysfunction. All the above were also verified by gene expression analysis of 2 different human cellular cultures (lung epithelium and keratinoma cells) and the respective pathway analysis; modulation of genes activating the NFkB pathway is associated to inflammatory responses. This may cause a perturbation in the IL-6 signaling pathway in order to regulate inflammatory processes and compensate for apoptotic changes. A plausible hypothesis for the immunological effects observed in vivo, are considered as the result of the synergistic effect of systemic (mediated by cells of the routes of exposure) and local inflammation (blood cells). (C) 2015 Elsevier Ltd. All rights reserved.