Cobalt-based nanoparticles strongly diminish CalceinAM fluorescence independently of their cytotoxic potential in human lung cell line

Abstract

ObjectiveCobalt nanoparticles (NPs) when released into the air during industrial processes, can enter the lungs through inhalation, leading to potential toxicity and respiratory issues. The mechanism of cobalt toxicity is not well understood and needs further investigation. MethodsWe, therefore, studied the effects of two kinds of cobalt-based nanoparticles (NPs) on lung cells. These NPs were Co NPs (i.e. Cobalt NPs) and Co-Fe NPs (i.e. CoFe2O4 NPs) that were similar in size and shape but slightly differed for the surface iron composition. The average size of Co NPs was 28 ± 18 nm, and that of Co-Fe was 26 ± 13 nm. We exposed the cells to the NPs for 24 h and measured their viability by MTT assay and CalceinAM fluorescence imaging. Potential induction of reactive oxygen species (ROS) was detected by probes DHE specific for superoxide (O2•–) and DCFH-DA for hydrogen peroxide (H2O2). Antioxidant glutathione (GSH) quantification was followed by measuring apoptotic/necrotic potential. ResultsUsing Co NPs and Co-Fe NPs, we measured the IC50 for cell growth in A549 cells after 24 h of exposure. The values were 79 μg/mL and 163 μg/mL, respectively. We also observed that both NPs induced ROS and reduced the fluorescence of CalceinAM in the cells, but Co NPs had a stronger effect than Co-Fe NPs. This was correlated with the production of O2– by the NPs, which was higher for Co NPs than for Co-Fe NPs. On the other hand, H2O2 production was higher for Co-Fe NPs than for Co NPs. Our study suggests that the main cause of differential O2– production is the differential release Co2+ ion due to surface modification resulting in differential quenching of green (CalceinAM) fluorescence by both NPs. Both NPs induced apoptosis mode of cell death. ConclusionOur data from the use of naïve and surface-modified cobalt-based NPs, suggest that NAC can protect cells from cobalt-based NP toxicity by not only restoring cellular GSH levels but also by chelating released Co2+ ions from the NPs surfaces. Additionally, this study for the first time to our knowledge indicates that CalceinAM alone is not sufficient to assess the toxicity of cobalt-based NPs. Other methods should be used to confirm the cytotoxicity data of cobalt-based NPs.