Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells
Introduction
The dramatic expansion of the nanotechnology industry has prompted the need to investigate potential toxic effects of nano-sized particles (NPs) on human health as well as the environment. Such knowledge is of great importance for nanotechnology to grow in a responsible and sustainable manner.
The most common definition of nanoparticles includes particles ranging in size from 1 to 100 nm in diameter in at least one dimension under the term (Oberdorster et al., 2005, SCENIHR, 2009). Due to their unique size, they tend to posses novel physical, chemical and biological properties that make these materials superior. Silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (TiO2-NPs), which are among the top five NPs in pharmaceutical products, building materials and other consumer products, are pivotal in the development of nanotechnology (Cho et al., 2009, Park et al., 2010, Shukla et al., 2011).
While there are various potential routes of exposure to NPs, inhalation and ingestion appear to be the main routes. Some studies have reported that NPs are able to cross the blood–testis and blood–brain barrier (De Jong et al., 2008, Lankveld et al., 2010). Of particular concern is the fact that AgNPs have been documented to cross the blood–testis and blood–brain barrier in mice (Lankveld et al., 2010) and rats (Gromadzka-Ostrowska et al., personal communication).
NPs are capable of binding to cells as well as macromolecules like proteins and DNA (AshaRani et al., 2009b). When NPs come in contact with cells they are taken up by a variety of mechanisms that can lead to activation of cellular signalling processes producing reactive oxygen species (ROS), inflammation and finally cell cycle arrest or cell death (Ahamed et al., 2008, Ahamed et al., 2010, AshaRani et al., 2009b). Reduced rate of proliferation, impaired mitochondria function and induction of apoptosis and/or necrosis are among changes which have been observed in AgNP-exposed cells (Schrand et al., 2008, AshaRani et al., 2009a). Toxicity of AgNPs in higher eukaryotes have also been reviewed (Kruszewski et al., 2011). Notably, AgNPs are capable of entering the nucleus, and as such directly or indirectly interacting with nuclear material (AshaRani et al., 2009b, Kruszewski et al., 2011), leading to alterations in DNA integrity or affecting its synthesis. These perturbations may – via the causation of DNA damage or inhibition of cellular processes – result in the formation of mutant or tumorigenic cells. When such processes concern germline cells, the result may be altered spermatogenesis and fertility, subsequently affecting the reproduction rate and health of the offspring (Ema et al., 2010).
Reproductive toxicants targeting the germ line have the potential to cause alterations that can be passed on by genetic or epigenetic mechanisms to the next generation. Although there is increasing concern for the potential effects of the use of NPs on reproductive health, only few studies have been performed on testicular cells. The present study is therefore aimed at evaluating the cytotoxicity and DNA-damaging potential of AgNPs in NT2 cells and primary mouse testicular cells, employing TiO2-NPs as a benchmark. We show that AgNPs are more cytostatic and cytotoxic in testicular cells compared to TiO2-NPs, and tend to be more genotoxic.
Section snippets
NPs and their characterization
AgNPs were purchased from Plasmachem GmbH, Germany. TiO2-NPs were obtained from the European Commission Joint Research Center. The nominal sizes of AgNPs obtained were 20 and 200 nm, whereas TiO2-NPs were 21 nm (referred here to as Ag20, Ag200 and TiO2-NP, respectively). NPs were dispersed in dH2O/10X BSA/10 PBS in 8:1:1 ratio of 2 mg ml−1 stocks, as previously described (Bihari et al., 2008). The hydrodynamic sizes of NPs after the dispersion procedure were determined by dynamic light scattering
Cytotoxic effects of NPs inNT2 and primary mouse testicular cells
Fig. 1 shows micrographs of NT2 cells exposed to 100 μg ml−1 NPs for 24 h, with clearly reduced number of cells for the nano and submicron particles (Ag20 and Ag200), compared to control (untreated). The cellular metabolic activity, reflecting somehow the rate of proliferation of testicular cells, as assessed by the MTT assay, was reduced with increased exposure time and concentrations of Ag20 and Ag200 (Fig. 2). The data indicate that Ag20 and Ag200 affected the mouse primary testicular cells,
Discussion
Several particle features, such as type, size, zeta potential, dispersion/agglomeration status, as well as potential interaction with biomolecules, influence NP toxicity and hence their effects in humans. Generally, size (and hence the surface/mass ratio) has been considered as the most important factor for the toxicity of NPs (Hubbs et al., 2011). In the present study, we attempted to minimize variations due to inherent physical/biological properties of the particles used. As stated in the
Conclusions
Taken together, AgNPs appear to be more cytotoxic as well as cytostatic compared to TiO2-NPs in the primary testicular cells and cell line examined. With regards to genotoxicity, although weak and cell-type-specific, both NP types showed a tendency to cause DNA damage. Considering that TiO2-NPs appeared to activate the rate of cell proliferation, both NP types may have possible implications on reproduction as well as human and environmental health in general. Additionally, this study adds to
Conflict of interest
None.
Acknowledgements
This study was funded by the Polish-Norwegian Research Fund, Contract no. PNRF-122-AI-1/07. The support of other PNRF-122 team members not listed herein is greatly appreciated. Thanks to Daniel Pharm for technical assistance with the Comet assay.
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