Enhanced genotoxicity of silver nanoparticles in DNA repair deficient Mammalian cells

Abstract

Silver nanoparticles (Ag-np) have been used in medicine and commercially due to their anti-microbial properties. Therapeutic potentials of these nanoparticles are being explored extensively despite the lack of information on their mechanism of action at molecular and cellular level. Here, we have investigated the DNA damage response and repair following Ag-np treatment in mammalian cells. Studies have shown that Ag-np exerts genotoxicity through double-strand breaks (DSBs). DNA-PKcs, the catalytic subunit of DNA dependent protein kinase, is an important caretaker of the genome which is known to be the main player mediating Non-homologous End-Joining (NHEJ) repair pathway. We hypothesize that DNA-PKcs is responsible for the repair of Ag-np induced DNA damage. In vitro studies have been carried out to investigate both cytotoxicity and genotoxicity induced by Ag-np in normal human cells, DNA-PKcs proficient, and deficient mammalian cells. Chemical inhibition of DNA-PKcs activity with NU7026, an ATP-competitive inhibitor of DNA-PKcs, has been performed to further validate the role of DNA-PKcs in this model. Our results suggest that Ag-np induced more prominent dose-dependent decrease in cell viability in DNA-PKcs deficient or inhibited cells. The deficiency or inhibition of DNA-PKcs renders the cells with higher susceptibility to DNA damage and genome instability which in turn contributed to greater cell cycle arrest/cell death. These findings support the fact that DNA-PKcs is involved in the repair of Ag-np induced genotoxicity and NHEJ repair pathway and DNA-PKcs particularly is activated to safeguard the genome upon Ag-np exposure.

Keywords: DNA damage and repair; DNA-PKcs; genotoxicity; silver nanoparticles.

Figure 1

Ag-np characterization. (A) Typical transmission electron microscopy (TEM) image, (B) UV-visible spectrum, (C) size distribution, (D) zeta potential, and (E) elemental analysis of silver nanoparticles (Ag-np) reconstituted after lyophilization. TEM images were captured with JEOL JSM 1220. The size distribution and zeta potential of Ag-np was measured with Zetasizer Nano.

Figure 2

Cell viability in mammalian cells following Ag-np treatment. Cell viability of (A) IMR-90 cells, (B) MO59K and MO59J cells, and (C) CHO AA8 and CHO V33 cells after 48 h treatment with Ag-np at different concentrations is shown. The cell viability percentage was normalized against the untreated controls (0 μg/ml) for Ag-np treatment alone or DMSO control (0 μM) for each cell type pre-treated with NU7026 (10 μM). The values represent the mean ± standard error of three sets of independent experiments; *denotes p < 0.05; **denotes p < 0.01; and ***denotes p < 0.001 as obtained using Student’s t-test, where the statistical significance between controls and Ag-np treated samples was analyzed.

Figure 3

Induction of DNA damage in Ag-np treated cells. DNA damage as measured by the Comet assay in different cell types following 48 h treatment with Ag-np (100 μg/ml). (A) Representative of SYBR Green-stained comets prepared from control (i) and Ag-np treated cells (ii). Magnification = 20×. (B) Mean tail moment (μm) represents the damage distribution in the attached cells. Mean ± standard error of three sets of independent experiments are shown. *Denotes p < 0.05 and **denotes p < 0.01.

Figure 4

Induction of micronuclei in cells following Ag-np treatment. (A) Representative image of binucleated cells without (Ai) and with micronuclei (Aii). White arrow (Aii) indicates the micronucleus formed among the binucleated cells. Cells and nuclei were stained with acridine orange (30 μg/ml). Magnification = 60×. (B) Data from CBMN show chromosomal aberrations. The data represent 1000 binucleated cells. *Denotes p < 0.05 and **denotes p < 0.01.

Figure 5

Induction of DNA double strand breaks in response to Ag-np treatment. (A) Representative of immunofluorescence images of γ-H2AX foci without (Ai) and with (Aii) 100 μg/ml Ag-np treatment, detected with anti-γ-H2AX antibodies. Nuclei were stained with DAPI (blue) and γ-H2AX was stained green with FITC. Magnification = 60×. (B) Represents mean no. of γ-H2AX induced in various cell types. *Denotes p < 0.05. (C) Western blots analysis of γ-H2AX following Ag-np treatment. Whole cells were trypsinized and lyzed. Equal amounts of proteins were loaded and separated using 12% SDS-PAGE, transferred to nitrocellulose membrane and immunoreacted with antibodies against proteins of interest. Actin was used as loading control. R denotes 48 h recovery.

Figure 6

Ag-np upregulated and activated DNA-PKcs and ATM in mammalian cells. Western blot analysis of DNA-PKcs and ATM following 48 h Ag-np treatment (100 μg/ml) and NU7026 (10 μM) in (A) IMR-90 cells, (B) MO59K cells and MO59J cells, and (C) CHO AA8 cells and CHO V33 cells. (D–F) Fold change for protein levels were obtained by densitometry analysis of X-ray films using Kodak molecular imaging software and normalized against actin levels to represent accurate values for proteins which deviated from the control.

Figure 7

Cell cycle analysis by flow-cytometry. Cell cycle histograms for (A) IMR-90 cells, (B) MO59K cells and MO59J cells, and (C) CHO AA8 cells and CHO V33 cells after 48 h exposure to Ag-np and NU7026 (10 μM) as measured by propidium iodide staining. Markers were set at regions of interest (subG1, G1, S, and G2/M), and the percentage of cells (events) under each area was generated using Microsoft excel.