Table 1.
Particle size distributions and surface area measurements.
Fig 1.
Primary size and morphology of the particles.
Nano- and micron-sized nickel metal (Ni-n, Ni-m1, Ni-m2) and nickel oxide particles (NiO-n) recorded with Transmission Electron Microscopy (TEM).
Fig 2.
Released amount of Ni in solution (aqueous Ni) compared with the total amount of Ni in the particles (Ni-n, Ni-m1, Ni-m2 and NiO-n). Release was analyzed after 0, 4 and 24 h incubation of the particle suspensions in cell medium or ALF (analyzed with AAS). Each bar represents the mean value of three independent experiments (n = 3), and the error bars the standard deviation of the mean value (±SD). Results for “0 h” correspond to measurements made directly after sonication of the particle dispersions, and therefore represent the starting point of the cell exposures.
Fig 3.
(A) Acellular ROS production of Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particles (20 μg mL-1 of total Ni) studied with the acellular DCFH-DA assay in the presence (+HRP) and absence (-HRP) of a catalyst (Horse Radish Peroxidase). The oxidative reactivity is presented as the change in fluorescence intensity when compared with control (PBS with DCFH). (B) Intracellular ROS production in A549 cells exposed to Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particles (20 μg mL-1 of total Ni) using the cellular DCFH-DA assay. Nano-sized CuO and H2O2 were used as positive controls. The ROS increase was calculated as mean slope per min and normalized to the unexposed control. The asterisk (*) assigns statistically significant (p<0.05) values compared with the corresponding control (PBS with DCFH ±HRP or unexposed cells).
Fig 4.
(A) 24 h and (B) 48 h exposure to Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particle suspensions at total Ni concentrations of 0.1, 1, 5, 10, 20 and 40 μg cm-2. CuO-nanoparticle suspensions (40 μg cm-2) were used as positive controls. Each bar represents the mean value of three independent experiments (n = 3), and each error bar the standard deviation of the mean (±SD). The asterisk (*) assigns statistically significant (p<0.05) values.
Fig 5.
Colony forming efficiency (CFE) after (A) 4 h and (B) 24 h exposure (and 7 days post-incubation) to Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particle suspensions at total Ni concentrations of 0.1, 1, 5, 10, 20 and 40 μg cm-2. CuO-nanoparticle suspensions (40 μg cm-2) were used as positive controls. Each bar represents the mean value of three independent experiments (n = 3), and each error bar the standard error of the mean (±SEM). The asterisk (*) assigns statistically significant (p<0.05) values.
Fig 6.
DNA damage analyzed with the comet assay after (A) 4 h and (B) 24 h of exposure to Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particle suspensions (20 μg cm-2 of total Ni). Cells exposed to CuO-nanoparticle suspensions (20 μg cm-2) were used as a positive control. The asterisk (*) is assigned for statistically significant (p<0.05) values. Each bar represents the mean value of three independent experiments (n = 3), and the error bars the standard deviation of the mean value.
Fig 7.
Particle uptake and intracellular localization.
A549 cells exposed to nano- and micron-sized nickel metal (Ni-n, Ni-m1, Ni-m2) and nickel oxide particles (NiO-n) recorded with Transmission Electron Microscopy (TEM) (A) directly after or (B) 24 h after a 4 h exposure (20 μg cm-2 of total Ni).
Fig 8.
A549 cell-associated Ni-fraction.
The amount of Ni that was taken up by the cells or bound to the cell membrane was analyzed with AAS after 4 h of exposure to Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particle suspensions (20 μg cm-2 of total Ni). The cell-associated Ni-fraction is presented as the percentage of the total amount of added Ni in the exposure suspensions. Each bar represents the mean value of three independent experiments (n = 3), and the error bars the standard deviation of the mean value.
Table 2.
Compilation of the responses of Ni metal (Ni-n, Ni-m1 and Ni-m2) and Ni oxide (NiO-n) particles to different assays in this study.