S (20?0 g ml-1) Oxidative DNA damage at the highest tested concentrations (20?0 g ml-1) Dose-related cytotoxicity only at 2 h exposure 30 Pre-incubation of cells with NAC attenuated the DNA damage Statistically significant concentration- 48 dependent increase in MN No DNA damageUboldi et al. Particle and Fibre Toxicology (2016) 13:Page 10 ofTable 4 Genotoxicity studies on cobalt particles (Continued)WST-1 (1?0 g ml-1, 24 h) Comet assay (1?0 g ml-1, 2?4 h) FPG-modified comet assay (1?0 g ml-1, 2?4 h) Commercially available Co3O4P, Human DM-3189 site hepatocarcinoma Sigma-Aldrich (264 nm by DLS; (HepG2) cells 22 nm by TEM) LDH and MTT (5?0 g ml-1, 24?8 h) GSH/LPO/SOD/catalase (5?0?5 g ml-1, 24?8 h) Caspase-3 (5?0?5 g ml-1, 24?8 h) Comet assay Statistically significant viability reduction only at 40 g ml-1 Concentration-related DNA damage only at 40 g ml-1 Oxidative DNA damage Concentration- and time-dependent cytotoxicity Concentration- and time-related depletion of GSH and induction of LPO, SOD, and catalase Concentration- and time-dependent increase of caspase-3 activity Concentration- and time-dependent DNA damagefreshly isolated human peripheral leukocytes, whereas Co2+ did not exert any effect, and to explain these controversial results the authors suggested that the lack of DNA strand break induction by CoCl2 might be due to the fact that Co2+ are internalized less efficiently than CoP [45]. In Balb/3 T3 mouse fibroblasts, soluble CoP was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 more severe in inducing MN formation compared with Co2+: 1??0 M CoP exerted statistically significant but not dose-related chromosomal aberrations, whereas under the same experimental conditions Co2+ did not exert micronucleus formation [22]. Additionally, Ponti and coauthors observed, by comet assay, a statistically significant induction of DNA damage following exposure to CoP and Co2+, with the increased DNA damage induced by CoP not dose dependent whereas a dose-dependent effect was observed for Co2+ [22]. More recently, poorly soluble Co3O4P were shown to induce, in HepG2 cells, concentration- and timedependent primary DNA damage [43]. In BEAS-2B cells, we have observed a similar effect following 2 h and 24 h exposure. Additionally, Cavallo et al. observed that, in BEAS-2B cells, the primary DNA damage induced by Co3O4P could be observed only at high concentrations (20?40 g mL-1), while oxidative DNA damage, analyzed by performing a FPG-modified comet assay, was particularly evident at low concentrations (5?0 g mL-1) [30]. These results are only partially confirmed by our observations, by which the incubation of BEAS-2B cells with poorly soluble Co3O4P enhanced primary DNA strand breaks already at 10 g mL-1 cobalt, whereas at 2 h and 24 h exposure oxidative DNA lesions were significantly induced at much low cobalt concentrations (2.5 g mL-1 cobalt using FPG and 1.25 g mL-1 cobalt using hOGG1).The genotoxic potential of several types of nanoparticles, including cobalt nanoparticles, has been linked to their ability to induce oxidative stress [49?1]. For example, soluble CoOP exerted significant induction of ROS in human lymphocytes and in mouse peripheral blood mononuclear cells [52], as well as in the leukemic Jurkat, K562 and KG1-a cells [53]. In HepG2, the depletion of GSH and the induction of membrane lipid peroxidation was higher in the presence of poorly soluble Co3O4P than following exposure to CoCl2 [43]. Similarly, Papis and coauthors reported that, at equal cobalt concentrations, insoluble Co3O4P w.