Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay

Abstract

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

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.