Fig 1.
High throughput analysis of 53BP1 foci using the CellInsight CX5 automated imagining and analysis system.
Cells were exposed for 24 h to 80 μM of MMS, then fixed and immunostained for 53BP1 foci. Nuclei were counterstained using Hoechst 33342. Fluorescence image of cell nuclei (A), 53BP1 foci (B) are acquired. Then, segmentation is performed using the HCS studio software, which selects nuclei that will be analysed, those that must not be considered, and counts 53BP1 foci in the selected nuclei (represented in blue, yellow and red, respectively, in D and E).
Fig 2.
Viability of cells exposed to model genotoxic substances.
Cell viability was evaluated using the WST-1 assay after 24 h of exposure to aflatoxin B1 (AFB1), methyl methanesulfonate (MMS), etoposide, hydroquinone (Hydroqu.), taxol, azidothymidine (AZT), N-nitroso-N-ethylurea (ENU), di-2(ethyl hexylphtalate) (DEHP) or propyl gallate (PG). The concentrations of these compounds are expressed in M (x-axis). Depicted are the mean values ± standard deviation of three independent experiments with five replicates per experiment (n = 15).
Fig 3.
53BP1 assay for genotoxicity assessment of model genotoxicants.
HCT116 cells were exposed to (a) aflatoxin B1, (b) methyl methanesulfonate, (c) etoposide, (d) hydroquinone, (e) taxol, (f) azidothymidine, (g) N-nitroso-N-ethylurea, (h) Di(2ethyl hexyl)phthalate, (i) propyl gallate for 24 h and then fixed or to (j) a 1 Gy X-ray irradiation and then fixed 30 min, 1 h, 1 h 30, 2 h or 24 h after irradiation. Then, the 53BP1 assay results was compared to that of γ-H2AX assay, on cells exposed to 1 Gy X-ray irradiation, 30 min after irradiation (k). All these samples were immunostained for 53BP1 (or γ-H2AX) foci, and foci were counted in each cell nucleus using automated fluorescence microscopy. Depicted are the mean number of foci per cell nucleus ± standard deviation of 3 independent experiments with 5 replicates per experiment (n = 15). Statistical significance: *p<0.05, exposed versus control; #p<0.05, 400 μM vs. 80 μM (MMS) or 12.5 μM, 25 μM or 50 μM vs. 5 μM (taxol).
Fig 4.
Impact of NBMs on cell viability.
Cell viability was evaluated using the WST-1 assay in HCT116 cells exposed to NMs and NBMs. (a) Metal-based NMs and NBMs: silver nanoparticles (AgNP), silver nanoparticles coated with hydroxyethylcellulose (AgHEC), gold nanoparticles (AuNP), gold nanorods (AuNR). (b) Organic NMs and NBMs: carbon nanoparticles (CNP), multi-walled carbon nanotubes (MWCNT), solid lipid nanoparticles (SLN). (c) Metal oxide NMs and NBMs: titanium dioxide (TiO2) and magnetite (Fe3O4). (d) Mineral NMs and NBMs: hydroxyapatite (HA), hydroxyapatite scaffold (HAsc) and iron-doped hydroxyapatite (FeHA). The impact of (CoO)(NiO) on cell viabiliy is also reported, as it used as positive control in the 53BP1 assay. Concentrations of NBMs are expressed in μg/mL (x-axis). Amine-functionalized polystyrene NPs (PS-NH2, 50 nm, 100 μg/mL, 24 h) were used as positive control in each assay (PS-NH2). Depicted are the mean values ± standard deviation of 3 independent experiments with 5 replicates per experiment (n = 15). Statistical significance: *p<0.05, exposed versus control.
Fig 5.
Genotoxicity screening of NBMs using the 53BP1 assay.
Genotoxicity was assessed in HCT116 cells exposed to NBMs for 24 h. (a) Metal-based NMs and NBMs; (b) metal oxide NMs and NBMs; (c) organic NMs and NBMs; (d) mineral NMs and NBMs. Cobalt nickel oxide (CoO)(NiO) NPs (20 μg/mL, 24 h) and etoposide (50 μM, 24 h) were included as positive controls. Depicted are the mean values ± standard deviation of 3 independent experiments with 5 replicates per experiment (n = 15). Statistical significance: *p<0.05, exposed versus control.
Fig 6.
Genotoxicity screening of NBMs using the cytochalasin-blocked micronucleus assay.
The per-centage of micronucleated cells in the binucleated cell population after 24 h exposure to the se-lected NBMs is shown. (a) Metal-based NMs and NBMs; (b) metal oxide NMs and NBMs; (c) organic NMs and NBMs; (d) mineral NMs and NBMs. Mitomycin C (MMC500, 500 ng/mL, 24 h) was used as a positive control. Depicted are the mean values ± standard deviation of 3 independent experiments with 5 replicates per experiment (n = 15). Statistical significance: *p<0.05, exposed versus control.
Fig 7.
ROS levels in cells exposed to NBMs.
ROS levels were assessed using the DHR123 assay, in cells exposed for 24 h to the whole series of tested NMs and NBMs (a), or for 0.5–24 h to Fe3O4 (b), SLN2 (c) or FeHA NPs (d). Tert-butyl hydroperoxide (THBP, 250 μM) was used as positive control. Depicted are the mean values ± standard deviation of 3 independent experiments with 5 replicates per experiment (n = 15). Statistical significance: *p<0.05, exposed versus control.