Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells

Abstract

Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5±14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 μg Ag(+) ion/l) could be dissolved after 24h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5-48 μg/cm(2) corresponding to 2.5-240 μg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 μg/cm(2) (corresponding to 60.8 μg/ml) and doubling of the percentage of DNA in tail at 48 μg/cm(2). However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the particles or by causing extensive agglomeration of the nanoparticles, with a possible reduction of the cellular uptake.

Potential risks and benefits of nanotechnology: perceptions of risk in sunscreens

“…These results suggested that Ag-NPs have genotoxic effects in BEAS 2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects. However, treatment of BEAS 2B cells with the PVP-coated Ag-NPs did not increase structural chromosomal aberrations (tested up to 182 µg/mL in 24-h treatment and 20.4 µg/mL in 48h treatment) or micronuclei (tested up to 182 µg/mL, 48-h treatment) where cytochalasin B was added at 6 h after starting the Ag-NP treatment (Nymark et al 2013); Ag-NPs inhibited mitoses at doses higher than 20.4 µg/mL and reduced the cytokinesis block cell proliferation index in a dosedependent manner.…”

“…The apparent conflicting results on the cytotoxicity of Ag-NPs may partly reflect differences among the studies in the type of Ag-NPs tested (primary particle size, coating, surface charge, solubility), cell type (uptake, sensitivity), and the technique used for exposing the cells such as dispersion method (dispersing agent, sonication, agglomeration, etc.) and exposure duration, resulting in variable exposure to Ag ions and/or Ag-NPs as well as their characteristics that may be important for toxicity (Ellegaard-Jensen et al 2012;Nymark et al 2013). showed a dose-dependent inhibition of the development of contracting cardiomyocytes for three different sizes of nanosilver (20 nm, 80 nm, and 113 nm diameter) in an embryonic stem cell assay.…”

“…Three studies on Ag-NPs in human bronchial epithelial BEAS 2B cells showed an induction of DNA damage, as assessed by the comet assay after exposure for 2 h (Ag-NPs <100 nm, no coating indicated; 190-762 µg/mL without S9 mix (Supernatant fraction obtained from an organ (usually liver) homogenate by centrifuging at 9000 g for 20 minutes in a suitable medium; this fraction contains cytosol and microsomes -Duffus et al 2007), 293-1172 µg/mL with S9 mix) , 24 h (Ag-NPs 43-260 nm, coating not indicated; 0.01-10 µg/mL) (Kim et al 2011a), and 4 h and 24 h (Ag-NPs 42.5±14.5 nm; coated with 15 wt% PVP; 16-48 µg/cm 2 i.e., 61-182 µg/mL) (Nymark et al 2013). In the last study (Nymark et al 2013), DNA damage increased in a dose-dependent manner from 8 up to 24 µg/cm 2 (20.4-91.2 µg/mL) but was not further increased at higher doses; as the PVP-coated Ag-NPs showed very low solubility to the medium used, the DNA-damaging effect was probably not due to Ag + ions in the medium but associated with cellular uptake of Ag-NPs.…”

“…Various mechanisms may be responsible for the in vitro genotoxicity of Ag-NPs, such as leaching of ionic Ag + from the particles in the culture medium, induction of reactive oxygen species (ROS) due to particle penetration through the cell membrane, and positive surface charge of Ag-NPs allowing interaction with negatively charged DNA El Badawy et al 2011;Kim et al 2011a;Nymark et al 2013). As the solubility of Ag particles is expected to depend on the available surface area, the size and coating of the particles is expected to affect the efficiency of ion release and the overall toxicity of the particles (Ahamed et al 2008(Ahamed et al , 2010Johnston et al 2010;Kawata et al 2009).…”

“…As the solubility of Ag particles is expected to depend on the available surface area, the size and coating of the particles is expected to affect the efficiency of ion release and the overall toxicity of the particles (Ahamed et al 2008(Ahamed et al , 2010Johnston et al 2010;Kawata et al 2009). The release of ions from the NPs could be more efficiently performed inside the cells than in the exposure medium, either due to the coating being degraded by the cell (Schrand et al 2008), or due to the Ag-NPs being ionized in the cells by a Trojanhorse type mechanism Nymark et al 2013).…”