Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

Abstract

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed.

Figure 1.

Characteristic TEM images (×60 k mag, 80kV) of the AgNPs utilised in all experiments. (A = 10nm, B = 20nm, C = 50nm, D = 100nm).

Figure 2.

DNA repair capacity of UV-induced damage in the presence or absence of AgNPs in repair competent bacteria. (A) Salmonella typhimurium strain TA102 and (B) Echerichia coli strain WP2 pKM101 bacteria were exposed to UV light for 3, 5, 15 or 30 s. Samples were either plated immediately (no hold) or allowed to repair for 60min in the presence or absence of AgNPs.

Figure 3.

Scanning transmission electron microscopy images of mammalian cells with EDS spectra showing the uptake of AgNPs. The highlighted red box indicates the silver signal observed for each of the respective areas obtained with EDS analysis. (A) 10nm AgNPs incubated with Jurkat cells. (B) 10nm AgNPs incubated with THP-1 cells. (C) 20nm AgNPs incubated with Jurkat cells. (D) 20nm AgNPs incubated with THP-1 cells. (E) 50nm AgNPs incubated with Jurkat cells. (F) 50nm AgNPs incubated with THP-1 cells. (G) 100nm AgNPs incubated with Jurkat cells. (H) 100nm AgNPs incubated with THP-1 cells. (I) Negative control (Jurkat cells incubated with 2mM sodium citrate buffer). (J) Negative control (THP-1 cells incubated with 2mM sodium citrate buffer).

Figure 4.

Mitochondrial activity of Jurkat (A) and THP-1 (B) cells exposed to various concentrations of 10, 20, 50 and 100nm AgNPs and AgNO₃ for 24h.

Figure 5.

Micronucleus induction by exposure of Jurkat and THP-1 cells to various concentrations of 10 (A), 20 (B), 50 (C) and 100 (D) nm AgNPs and AgNO₃ (E) for 24h. Ten thousand nucleated cells were assessed for the presence of micronuclei per treatment. Data are presented as the percentage of nucleated cells with micronuclei. Mitomycin C exposure for 4h was used as a positive control. * indicates P < 0.05.

Figure 6.

Alkaline Comet assay for DNA damage induced by 24-h exposure to AgNPs or AGNO₃. Jurkat cells were exposed to various concentrations of 10 (A), 20 (B), 50 (C) and 100 (G) nm AgNPs and AgNO₃ (D) for 24h. THP-1 cells were exposed to various concentrations of 10 (E), 20 (F), 50 (C) and 100 (G) nm AgNPs and AgNO₃ (H). Hydrogen peroxide exposure for 1h was used as a positive control. * indicates P < 0.05.