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Toxicol Sci. Aug 2010; 116(2): 577–589.
Published online May 20, 2010. doi:  10.1093/toxsci/kfq148
PMCID: PMC2905406

Silver Nanoparticles Disrupt GDNF/Fyn kinase Signaling in Spermatogonial Stem Cells

Abstract

Silver nanoparticles (Ag-NPs) are being utilized in an increasing number of fields and are components of antibacterial coatings, antistatic materials, superconductors, and biosensors. A number of reports have now described the toxic effects of silver nanoparticles on somatic cells; however, no study has examined their effects on the germ line at the molecular level. Spermatogenesis is a complex biological process that is particularly sensitive to environmental insults. Many chemicals, including ultrafine particles, have a negative effect on the germ line, either by directly affecting the germ cells or by indirectly acting on the somatic cells of the testis. In the present study, we have assessed the impact of different doses of Ag-NPs, as well as their size and biocompatible coating, on the proliferation of mouse spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult testis. At concentrations ≥ 10 μg/ml, Ag-NPs induced a significant decline in SSCs proliferation, which was also dependent on their size and coating. At the concentration of 10 μg/ml, reactive oxygen species production and/or apoptosis did not seem to play a major role; therefore, we explored other mechanisms to explain the decrease in cell proliferation. Because glial cell line–derived neurotrophic factor (GDNF) is vital for SSC self-renewal in vitro and in vivo, we evaluated the effects of Ag-NPs on GDNF-mediated signaling in these cells. Although the nanoparticles did not reduce GDNF binding or Ret receptor activity, our data revealed that already at a concentration of 10 μg/ml, silver nanoparticles specifically interact with Fyn kinase downstream of Ret and impair SSC proliferation in vitro. In addition, we demonstrated that the particle coating was degraded upon interaction with the intracellular microenvironment, reducing biocompatibility.

Keywords: nanoparticle-protein interactions, silver nanoparticles, cell signaling, spermatogonial stem cells, Fyn kinase

Among antimicrobial agents, silver is unique because of its broad spectrum of action against ~650 different types of disease-causing organisms (Hardman et al., 2004; Lansdown, 2006). Current antimicrobial uses include coating of wound dressings and catheters with silver, which effectively reduces bacterial infections (Davenport and Keeley, 2005; Heggers et al., 2005). Although this metal is nontoxic to humans and animals in its bulk chemical form (Lansdown, 2006), it is unclear what impact nano-sized silver has on biological systems. With the advancement of nanotechnology, silver nanoparticles (Ag-NPs) have been synthesized and shown to be effective antimicrobial agents because of their ability to bind to proteins and interfere with bacterial and viral processes (Kim et al., 2007; Sun et al., 2005). Therefore, nano-sized silver is used for its antimicrobial properties in bandages, in coatings on clothing and other surfaces, and in paints (Cioffi et al., 2005; Jain et al., 2009; Percival et al., 2007; Samuel and Guggenbichler, 2004; Vigneshwaran et al., 2007). Perhaps one of the most interesting potential applications is the ability of Ag-NPs to bind to HIV-1 and prevent the virus from infecting host cells (Elechiguerra et al., 2005). Because Ag-NPs readily bind to proteins and glycoproteins, the interactions of these nanoparticles with tissues and cells must be evaluated.

Spermatogenesis is a complex process that is highly sensitive to environmental toxicants (Pryor et al., 2000). Ultimately, these effects can affect male fertility through a decrease in the amount of sperm produced or have negative consequences for the development of the offspring because of epigenetic alterations (Anway and Skinner, 2008; Boisen et al., 2001). Among emerging toxicants of concern for reproductive health are nanoparticles and their nonmanufactured counterpart, the ultrafine particles. The number of reports suggesting that these particles are toxic to many organs, including the testis, is increasing (De Jong and Borm, 2008; Li et al., 2009; Yauk et al., 2008). Most effects arise from their high surface to volume ratio, which can make the particles very reactive. Following systemic administration, nanoparticles easily penetrate very small capillaries throughout the body, therefore offering the most effective distribution to certain tissues. More importantly, nanoparticles can pass through epithelia and biological membranes and thus can affect the physiology of any cell in an animal body (Kashiwada, 2006; Kim et al., 2006). In addition to passing through the blood-brain barrier, nanoparticles penetrate the blood-testis barrier and distribute in the gonads (De Jong, 2008; Kim et al., 2006; Takeda, 2008; Yoshida, 2009).

We have previously demonstrated that mammalian spermatogonial stem cells (SSCs), which are at the origin of the germ line in the adult, are particularly sensitive to Ag-NPs (Braydich-Stolle et al., 2005). These nanoparticles induced a significant decline in cell proliferation; however, the molecular mechanisms leading to this decrease are not known. The growth factor glial cell line–derived neurotrophic factor (GDNF) is essential for SSC self-renewal in vivo and in vitro (Kubota et al., 2004; Meng et al., 2000). Mice overexpressing GDNF develop germ cell tumors, whereas GDNF-null mice show depletion of SSCs (Meng et al., 2001; Naughton et al., 2006). In addition, GDNF promotes SSC proliferation in vitro via the Src family kinase (SFK)/phosphoinositide 3 (PI3)-kinase/Akt pathway that leads to the upregulation of N-myc (Braydich-Stolle et al., 2007; Lee et al., 2007; Oatley et al., 2007). Because nanoparticles can directly interact with proteins (You et al., 2005), we sought to examine whether Ag-NPs can interact with components of the GDNF signaling pathway in SSCs, thus elucidating the molecular basis of growth inhibition. Furthermore, several studies have shown that altering the surface chemistry of nanoparticles is effective in preventing toxicity from the core nanomaterial (Chen et al., 2005; Dumortier et al., 2006; Gupta and Gupta, 2005; Wilhelm et al., 2003;). Therefore, in this study, we also have evaluated the role of surface chemistry in the interactions of Ag-NPs with SSCs.

Our data show that nanoparticles interfere with SSC proliferation in a dose- and size-dependent manner and that small-sized nanoparticles (10–25 nm) are more likely to promote apoptosis or the production of reactive oxygen species (ROS) in these cells. In addition, we show that Ag-NPs are able to disrupt components of the GDNF signaling pathway and that these effects are independent of the surface chemistry. The latter is because of the fact that the protective coating is likely degraded by the cellular environment.

MATERIALS AND METHODS

Silver Nanoparticle Characterization

Hydrocarbon-coated silver (Ag-HC) nanoparticles of 15, 25, and 80 nm diameters were synthesized and generously received in powder form from Dr Karl Martin of NovaCentrix, Austin, TX. For these nanoparticles, the surface hydrocarbons are not a continuous coating but rather an artifact of the manufacturing process that uses hydrocarbon to prevent sintering of the Ag-NPs during plasma synthesis. Polysaccharide-coated silver (Ag-PS) nanoparticles of 10, 25–30, and 80 nm diameters were received dispersed in solution from Dr Dan Goia's Laboratory at Clarkson University, Potsdam, NY. For these nanoparticles, the coating was continuous and intentional. The size, morphology, and dispersion of the nanoparticles were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). X-ray photoelectron spectroscopy (XPS) was used to determine surface coating composition (see Supplementary fig. 1 and Supplementary table 1).

Exposure to Nanoparticles

Various investigators in the field have experimentally evaluated the toxicity of nanoparticles based on their number/vol, wt/vol, or surface area/vol (reviewed by Teeguarden et al., 2007). For the present study, dosing was based on wt/vol because the only reasonable means of testing samples with an equal number of particles/vol would be to work with an extremely small sample size (1000–10,000 nanoparticles/ml) in order to limit differences in the other parameters (surface area, mass, and delivery rate). However, at this time, no technique is available that would allow the counting and handling of individual nanoparticles and provide a direct measure of the cellular dose (see Supplementary table 2).

The C18-4 Cell Line

The C18-4 cell line was established by stably transfecting type A spematogonia with the large T antigen gene (Hofmann, Braydich-Stolle, Dettin, et al., 2005). The cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium/nutrient F-12 Ham (DMEM/Ham's F-12; Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA), 2mM glutamine, and 1% penicillin/streptomycin. For experimental conditions, in particular when GDNF was required, FBS was replaced with 10% synthetic Nu-Serum at least 24 h before the assay to ensure a controlled environment (BD Biosciences, San Jose, CA). Cells were plated in 6- or 96-well tissue culture plates (Falcon; Fisher Scientific, Pittsburgh, PA) and incubated at 34°C and 5% CO2 in a humidified incubator.

Microscopy Analysis

Scanning electron microscope analysis.

To demonstrate binding of the Ag nanoparticles to the cell surface, scanning electron microscope images of C18-4 cells treated for 24 h with 10 μg/ml of the different Ag nanoparticles were taken on a Hitachi S-4800 at 5 kV. Prior to imaging, the cells were fixed with 4% paraformaldehyde, dehydrated through analytical grade 30–100% ethanol, mounted to aluminum stubs with double-sided carbon adhesive tape, and sputter coated with gold. At least 30 cells were observed for each conditions and each type of nanoparticles.

TEM analysis.

The C18-4 cells were seeded in 100-mm tissue culture dishes, and at 85% confluency, they were treated with 10 μg/ml of the different Ag nanoparticles. Twenty-four hours later, the cells were fixed in 2% paraformaldehyde/2.5% gluteraldehyde in PBS for 2 h. Thereafter, the cells were postfixed with 1% osmium tetroxide for 1 h, and then the cells were scraped from the plate to be processed for TEM. The cells were dehydrated using increasing concentrations of ethanol with three changes of 100% ethanol. The samples were then placed in 100% resin and cured overnight at 60°C in BEEM capsules. The samples were then sectioned on a Leica ultramicrotome at a thickness of 50–100 nm, collected on a TEM grid, and imaged using a Hitachi H-7600 microscope at 75–80 kV. At least 30 cells were observed for each conditions and each type of nanoparticles.

MTS assay.

Because the 3-(4,5,dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt (MTS) assay is a colorimetric assay, we first assessed if the range of nanoparticle concentrations used interfered with absorbance readings. In Supplementary figure 3, we report that for concentrations ≤ 60 μg/ml, the nonspecific absorbance of the particles alone in culture media is generally equal or below background. Cells were seeded into 96-well plates at a concentration of 5000 cells/well in culture media containing 10% Nu-Serum. The cells were grown to 80% confluency, and nanoparticles of different concentrations/sizes/coating were added to the cultures. After a further incubation of 24 h, the cells were washed and mitochondrial activity—a measure of cell viability—was assessed using the CellTiter 96 AQueous One Solution (Promega Corp., Madison, WI). The cell viability (%) relative to control wells containing cell culture medium without nanoparticles or PBS as a vehicle was calculated by [A]test/[A] control × 100, where [A]test is the absorbance (490 nm) of the test sample and [A]control is the absorbance of control sample. Each experiment was done in triplicate for a total number of three experiments, and the data are represented as the mean ± SD. A Student's t test was performed, and p < 0.05 indicated significance. The concentration at which cell proliferation started to decrease was determined at 10 μg/ml. Subsequently, C18-4 cells were seeded in 96-well plates at a density of 3000 cells/well in culture media containing 10% Nu-Serum. The following day, the cells were treated with 10 μg/ml of Ag nanoparticles (Ag 15-HC and Ag 10-PS), or polysaccharide alone, and 100 ng/ml of GDNF. Fresh GDNF was added daily for 6 days and cell viability was assessed at days 1, 3, and 6 using the MTS solution (Promega Corp.). Negative controls were cells without nanoparticle treatments. All experiments were done in triplicates for a total number of three experiments. Data were reported as the averages ± SDs. A Student's t test was performed, and p < 0.05 indicated significance.

ROS Assay

Cells were cultured in 24-well plates until 60–70% confluency in DMEM with 10% Nu-Serum. Because no effect on cell proliferation was seen with shorter incubation times, the cells were exposed to nanoparticles (10 μg/ml) for 48 h. After incubation, the cells were washed with warm PBS, and the monolayers were incubated with 25μM Carboxy DCFA for ROS staining or 1μM Hoechst solution for nuclear staining (Image IT Live Green Reactive Oxygen Species Detection Kit; Molecular Probe/Invitrogen). As a positive control, cells were treated with 0.03% hydrogen peroxide for 10 min to induce ROS production. The cells were incubated at 33°C for 30 min, protected from light to avoid bleaching of the fluorophores. After rinsing with PBS, the cells were observed with 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein/fluorescein isothiocyanate (FITC) filters. At least 300 cells were counted per culture condition (triplicates), and the percentage of apoptotic and necrotic cells was calculated over the total number of counted cells. The experiment was repeated three times, and the data are represented as the mean ± SD. A Student's t test was performed, and p < 0.05 indicated significance.

Apoptosis/Necrosis Assay

Cells were cultured in 24-well plates (Falcon; BD Biosciences). A total of 8500 cells were seeded with 500 μl DMEM and 10% Nu-Serum into each well, and the cells were treated with 10 μg/ml of nanoparticles for 48 h. Following nanoparticle treatment, the cells were washed with cold PBS and the Vybrant Apoptosis Assay Kit was used to assess apoptosis versus necrosis (Molecular Probes/Invitrogen, Carlsbad, CA). The cells were observed with an inverted fluorescence microscope (Olympus IX70) equipped with a DAPI filter (for nuclear Hoechst staining), a FITC filter (for YoPro apoptosis staining), and a rhodamine/Texas red filter (for propidium iodide necrosis staining). Approximately 300 cells were counted for each culture condition (triplicates) using the 20× objective, and cells were classified into three categories: live (no fluorescence), necrotic (fluorescence for propidium iodide and YoPro), or apoptotic (YoPro fluorescence only). The percentage of apoptotic and necrotic cells was calculated over the total number of counted cells. The experiment was repeated three times, and the data are represented as the mean ± SD. A Student's t test was performed, and p < 0.05 indicated significance.

Confocal Imaging and Lysosomal Staining

The C18-4 cells were plated in a 96-well BD Falcon imaging plate. Once they reached 80% confluency, the cells were dosed with 10 or 50 μg/ml of fluorescently labeled 25-nm Ag-HC in cell culture media supplemented with 10% Nu-Serum. The Ag-HC 25-nm nanoparticles were labeled with a coumarin dye. The lysosomes were stained using a polyclonal rabbit anti-lysosomal-associated membrane protein (LAMP)-2 antibody from Santa Cruz at a dilution of 1:250, and a secondary antibody goat anti-rabbit Alexa Fluor 647 from Invitrogen and the nuclei were counterstained with SYTOX Green from Invitrogen. The nanoparticles were excited at 375-nm wavelength and pseudocolored green, whereas the Alexa Fluor 647 was excited at a wavelength of 647 giving the lysosomes a red color. Any colocalization would appear yellow. The nuclei were excited at a wavelength of 488 nm and pseudocolored blue. The images were obtained on the BD Pathway 435 confocal microscope and ten 250-nm sections were obtained using the Z stack option, and the images presented here are the collapsed image from the 10-section Z stack. Using this approach, the uptake and localization of the Ag 25-nm particles in the lysosomes of the C18-4 cells was observed. Because of the small size of the NPs, there is sometimes a haze from the unagglomerated NPs that cannot be removed from the images. However, the larger agglomerates are clearer.

GDNF Binding Assay

To assess nanoparticle-GDNF binding, the particles were dispersed in DMEM 10% Nu-Serum at a concentration of 10 μg/ml and incubated for 48 h at 34°C. GDNF was then added at a final concentration of 100 ng/ml. Following 4-h incubation with GDNF, the media was centrifuged for 15 min at 15,000 × g to remove the nanoparticles from the media. The supernatant was then analyzed in triplicate for the presence of GDNF by using a direct ELISA. Briefly, microtiter plates were coated with the supernatants overnight at 4°C. The next morning, the plates were washed three times using PBS + 0.1% Tween 20 and blocked using 4% bovine serum albumin for 2 h at room temperature. Then a goat anti-GDNF antibody (#AF-212-NA, recognizing recombinant human—mouse and rat—GDNF; R&D systems, Minneapolis, MN) was added to the wells at the concentration of 1:100 and incubated overnight at 4°C. After washes, a donkey anti-goat IgG-HRP secondary antibody (#SC-2033; Santa Cruz) was added at the concentration 1:1000. Finally, the substrate (#SK6604; Vector) was added and the signal was read at 460 nm. Signals were normalized over the control (only media and GDNF). This assay was repeated three times, and the data were reported as averages ± SDs. A Student's t test was performed, and p < 0.05 indicated significance.

Immunoprecipitation and Western Blotting

The cells were cultured in 100-mm culture dishes with media containing 10% Nu-Serum until 80% confluency. They were exposed to 10 μg/ml HC- and PS-coated nanoparticles for 24 h before 4-h stimulation with GDNF (100 ng/ml recombinant rat GDNF; R&D Systems). Cells that were not exposed to nanoparticles, with or without 100 ng/ml GDNF for 4 h, were used as controls. After washing in cold PBS, the cells were lysed in a nondenaturing lysis buffer that contained phosphatase and protease inhibitors (Halt Protease Inhibitor and Halt Phosphatase Inhibitor kits, #78410 and #78420; Pierce). The samples (500 μg protein) were immunoprecipitated with an anti-Fyn antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and 30 μg of the immunoprecipitates were subjected to Western blot analysis. The membranes were probed using primary antibodies recognizing Fyn and phospho-SFKs (Upstate Technologies/Millipore, Temecula, CA). For quantitative analysis, the band intensities were evaluated with the Image J Analysis software (National Institutes of Health). The protein band intensities were standardized over the intensity of phosphorylated Fyn in the control samples without nanoparticles or GDNF. Data were generated in triplicate from two different experiments and are represented as the mean ± SD. A Student's t test was run, and p < 0.05 indicated significance.

Kinase Activity Assays

Ret activity assay.

The C18-4 cells were seeded in a 6-well plate and cultured in media containing 10% Nu-Serum until 80% confluency. They were exposed to 10 μg/ml HC- and PS-coated nanoparticles for 24 h, washed, and then stimulated for 4 h with GDNF (100 ng/ml recombinant rat GDNF; R&D Systems). Ret kinase was immunoprecipitated using an anti-Ret antibody (R&D Systems), and Ret activity was assessed using a tyrosine kinase activity kit (Chemicon/Millipore, Temecula, CA). Each experiment was performed in triplicate, and the data are represented as the mean ± SD. A Student's t test was performed, and p < 0.05 indicated significance.

Fyn kinase assays.

Kinase activity was determined using a tyrosine kinase activity kit (Chemicon/Millipore), and 15 ng of purified active Fyn (Chemicon/Millipore). The active Fyn was incubated for 45 min with 10 μg/ml of Ag-NPs or polysaccharide, and 10μM of the Src family inhibitor SU6656 was used as a control. Each experiment was performed in triplicate, and the data are represented as the mean ± SD. A Student's t test was performed and p < 0.05 indicated significance. Next, C18-4 cells were grown in 6-well culture plates in complete cell culture media containing 10% Nu-Serum until the cells were 80% confluent. The cells were then treated with 10 μg/ml of Ag-NPs, polysaccharide, or 10μM of SU6656 for 24 h. Next, the cells were washed and stimulated with 100 ng/ml of GDNF for 4 h, and proteins were isolated in a nondenaturing lysis buffer using osmotic lysis. Fyn kinase was immunoprecipitated using an anti-Fyn antibody (Santa Cruz Biotechnology), and Fyn activity was assessed using a tyrosine kinase activity kit (Chemicon/Millipore). Each experiment was performed in triplicate, and the data are represented as the mean ± SD. A Student's t test was performed, and p < 0.05 indicated significance.

Akt activity assay.

C18-4 cells were seeded in 6-well plates and cultured in media containing 10% Nu-Serum until 80% confluency. They were treated with 10 μg/ml of Ag-NPs or polysaccharide for 24 h. Next, the cells were washed and then treated with 100 ng/ml of GDNF for 4 h. Akt kinase was immunoprecipitated using an anti-Akt antibody (BioVision, Mountain View, CA), and the KinaseSTAR Akt Activity Assay Kit (BioVision) was used to determine Akt activity. The immunoprecipitated Akt kinase was incubated with a GSK-3α/ATP mixture to facilitate the kinase reaction, and then the level of phosphorylated GSK-3α protein was detected using a rabbit anti-phospho-GSK-3α (Ser 21)-specific antisera (1:1000 dilution) and a Cy3-labeled goat anti-rabbit secondary antibody (BioVision). The Cy3 levels were detected using a Gemini Spectra Max plate reader (Molecular Devices, Sunnyvale, CA) with an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each experiment was performed in triplicate, and the data are represented as the mean ± SD based on a percent control. A Student's t test was performed, and p < 0.05 indicated significance.

N-myc Expression Analysis

C18-4 cells were seeded in 6-well plates in media containing 10% Nu-Serum. After reaching 80% confluency, they were treated with 10 μg/ml of Ag-NPs or polysaccharide for 24 h. The cells were then treated with 100 ng/ml of GDNF overnight. The following day, RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). Using 1 μg of RNA, N-myc and β-actin expression were then assessed using the SuperScript One-Step qRT-PCR with Platinum Taq Kit (Invitrogen). The sequence of the primers used to detect mouse N-myc were 5′-ACTTCTACTTCGGCGG-3′, Tm = 47°C (forward), and 5′-TCTCCGTAGCCCAAT-3′, Tm = 44°C (reverse complement). The sequence of the primers used to detect β-actin were 5′-GGACTC CTATGTGGGTGACGA-3′, Tm = 59°C (forward), and 5′-GCCTCGGTGAGCAGC-3′, Tm = 52°C (reverse complement). The primers were designed and evaluated using the NetPrimer program and ordered from Integrated DNA Technologies (San Diego, CA). The qPCR was run on a Stratagene Mx3005P machine (Stratagene, San Diego, CA). Each experiment was performed in triplicate, and the data are represented as the mean ± SD based on a percent control. A Student's t test was performed, and p < 0.05 indicated significance.

Statistical Analysis

The data `were first critically examined to eliminate biologically inconsistent data, and then the Dixon test was used to confirm the elimination (critical level 5%). They were analyzed with the statistical analysis software Prism (GraphPad Software, San Diego, CA). For each nanoparticle growth inhibition curve, the nonparametric Krustal and Wallis test was used. For nanoparticle treatments where the nonparametric test showed a significant difference, a one-way ANOVA and post hoc Dunnet test was performed, followed by a Student's unpaired t test (one tailed) between the last nonsignificant toxic dose and the first toxic dose. Wherever this difference was significant, the t test was repeated with the next toxic dose (the nonparametric test was done to confirm the validity of results). In order to compare the different types of nanoparticles, the post hoc tests of Bonferroni were added to the ANOVA. The critical level of significance was chosen at 5% for each test.

RESULTS

Nanoparticle Characterization

We used silver nanoparticles of different sizes and surface coatings, such as hydrocarbon-coated silver (Ag-HC) nanoparticles of 15, 25, and 80 nm diameters and polysaccharide-coated silver (Ag-PS) nanoparticles of 10, 25–30, and 80 nm diameter. Ag 130-nm particles were used as a control because only particules between 1 and 100 nm in size are considered nanoparticles. The polysaccharide coating was used to promote a better dispersion of the nanoparticles and to make them more biocompatible (Lemarchand et al., 2005). TEM analysis confirmed the primary nanoparticle size and morphology, although the particles tend to highly aggregate because of the nature of the TEM preparation. The average nanoparticle coating was verified using TEM for the polysaccharide-coated particles and was found to be between 1 and 3 nm. In addition, DLS (Murdock et al., 2008) demonstrated that all silver nanoparticles aggregated to some extent after being dispersed in deionized water. Additional analysis and results are provided in the Supplementary tables 1 and 2 and Supplementary figures 1 and 2.

Nanoparticle Surface Binding, Uptake, and Localization in C18-4 Cells

As shown in Figures 1A and 1B, control cells and cells incubated with polysaccharide alone showed prominent round nuclei and an overall smooth surface appearance. However, the presence of nanoparticles on the cell surface is readily apparent and distinguishable from any sample preparation artifacts and cellular membrane irregularities by the high atomic contrast of Ag and round morphology of the nanoparticles (Figs. 1B–I). The largest particles (Ag 130) were found in aggregates of substantial sizes (~34 μm) spanning several cells with many other smaller agglomerates attached randomly to the surface of individual cells (Fig. 1C, arrows). Similar large aggregates were found on cells incubated with the second largest nanoparticles (Ag 80-HC) (Fig. 1D, arrows), whereas fewer were found attached to the surface of cells incubated with Ag 80-PS nanoparticles (Fig. 1G). Interestingly, the Ag 80-HC nanoparticles appear to surround the perimeter of the nucleus closer to the membrane edges. When the nanoparticles were reduced in size, their scattering at the cell surface was more uniform in the case of Ag 25-HC and Ag 15-HC (Figs. 1E and 1F) (arrows), compared with Ag 25-PS and Ag 10-PS nanoparticles (Figs. 1H and 1I). Membrane irregularities with little contrast differences from the rest of the cell suggest internalization of the Ag-NPs beneath the plasma membrane, which was apparent in the Ag-PS samples (Figs. 1G–I) (arrowheads). This internalization may have been masked in the other samples by the presence of many surface-bound nanoparticles.

FIG. 1.
Surface binding of coated silver nanoparticles by SSCs. Scanning electron micrographs of C18-4 cells treated with silver nanoparticles and taken at ×1300 magnification. The largest particles were rather found in aggregates, whereas the smaller ...

Because it was unclear if the nanoparticles truly were internalized, further examination with TEM was performed. After 24-h incubation with 10 μg/ml of Ag-NPs, ultrathin sections of the cells revealed that Ag-NPs were localized in membrane-bound intracellular vacuoles that appear to be endosomes by their size and shape (Figs. 2A–F, black arrows). The electron microscope data established that the Ag-NPs localized on the outside as well as the inside of the cells. Additionally, confocal microscopy on C18-4 cells immunostained against the LAMP-2 was performed. In control samples without nanoparticles, the lysosomes appeared red because of the Alexa Fluor 647 staining (Fig. 3A, arrows). In Figure 3B, yellow colocalization verified that some lysosomes contained Ag-NPs (arrowheads). At a concentration of 10 μg/ml, not all nanoparticles were confined to the lysosomes and many could be found in the cytosol of the C18-4 cells (stained green, arrows), indicating the potential for interaction with cytosolic proteins or other organelles. In comparison, at the higher concentration of 50 μg/ml, the nanoparticles were mainly localized within the lysosomes (arrowheads, Fig. 3C).

FIG. 2.
Internalization and localization of coated silver nanoparticles in SSCs. Transmission electron micrographs of C18-4 cells treated with 10 μg/ml silver nanoparticles (scale bar = 1 μm) confirming that the particles are internalized and ...
FIG. 3.
Lysosomal and cytosolic localization of coated silver nanoparticles in C18-4 cells. The nanoparticles were excited at a wavelength of 375 nm and pseudocolored green, whereas the lysosomes were immunostained and excited at a wavelength of 647 nm giving ...

Biocompatibility of Silver Nanoparticles

We first assessed the influence of particle size, coating, and concentration on cell proliferation (Fig. 4). Potential effects of free Ag+ ions released by the nanoparticles were assessed and found negligible at the concentrations used (Supplementary figs. 5 and 6). A decrease in proliferation was not observed at concentrations < 10 μg/ml for any type of nanoparticle, and the cells did not appear to be stressed at these concentrations, because no ROS production or signs of apoptosis were detected (data not shown). For concentrations ≥ 10 μg/ml, a decrease in cell proliferation was observed after 24 h of exposure, which was also dependent on particle size and coating. In particular, smaller nanoparticles (10–25 nm diameter) induced a greater decrease in viability than bigger nanoparticles (80 nm diameter), and the HC coating was more toxic than the PS coating. We then assessed the effect of silver nanoparticles size, coating, and concentration on ROS production and cell apoptosis. Previous work in our laboratory has shown that silver nanoparticles induce a dose-dependent increase in ROS production and apoptosis at concentrations > 10 μg/ml (Braydich-Stolle et al., 2005). At a concentration of 10 μg/ml, only Ag 10-PS seemed toxic and induced ROS production in 30% of the cells (Fig. 5A). Ag 10-PS also induced a slight increase in the number of apoptotic cells at this concentration (Fig. 5B).

FIG. 4.
Viability of C18-4 cells treated with different concentrations of coated silver nanoparticles. C18-4 cells were incubated for 24 h with HC- and PS-coated nanoparticles of different sizes and concentrations, and cell viability was assessed by using the ...
FIG. 5.
Effect of coated silver nanoparticles on cellular stress responses. A: ROS production in the C18-4 cells. Following a 48-h incubation with 10 μg/ml HC- and PS-coated nanoparticles, ROS production was assessed by using the Image IT Live Green ROS ...

Silver Nanoparticles Interfere with GDNF Signaling

To evaluate if nanoparticles induce a decrease in cell proliferation through an additional mechanism, which might be different from ROS production or apoptosis, we assessed their effect on the GDNF pathway. GDNF is a molecule essential for SSC self-renewal in vivo and for SSC proliferation in vitro. It is also stimulating the proliferation of the C18-4 cells, a model for SSCs (Hofmann, Braydich-Stolle, Dettin, et al., 2005). Culturing the cells with a concentration of silver nanoparticles of 10 μg/ml contributed to a decline in proliferation over time (Fig. 6A), and GDNF was unable to promote the proliferation of these cells regardless of nanoparticle size or coating (Fig. 6B). GDNF is known to signal in part through SFKs (Braydich-Stolle et al., 2007; Oatley et al., 2007), and we therefore used SU6656, a pharmacological inhibitor of Src kinases, as a control. GDNF proliferation was inhibited in the presence of this chemical. Furthermore, we observed that although the PS-coated particles were initially less cytotoxic to the cells (Fig. 4), over time, there was no difference in growth inhibition between the HC- and PS-coated silver nanoparticles (Fig. 6A). When C18-4 cells were treated with nanoparticles for 6 days, there was a size-dependent effect on the cell viability. The Ag 10-PS and the Ag 15-HC showed the greatest impact on viability after 6 days when compared with the other sizes (Fig. 6B).

FIG. 6.
Proliferation of C18-4 cells treated with coated silver nanoparticles. A: growth curve of C18-4 cells continuously exposed to 10 μg Ag 15-HC and Ag 10-PS silver nanoparticles and treated daily with GDNF during 6 days. The Ag 10-PS nanoparticles ...

GDNF Availability and Ret Activity in C18-4 Cells Treated with Silver Nanoparticles

Figure 7A shows that the presence of silver nanoparticles did not alter the levels of free GDNF in the culture media. Therefore, the nanoparticles were not binding to the growth factor and the amount of GDNF that was available to the C18-4 cells did not change significantly in the presence of nanoparticles. Furthermore, there was no significant change in the activation of the Ret receptor in presence of the silver nanoparticles (Fig. 7B). Taken together, the nanoparticles did not interfere with the extracellular components of the GDNF signaling pathway.

FIG. 7.
Effect of coated silver nanoparticles on the extracellular components of the GDNF signaling pathway. A: GDNF (100 ng/ml) was incubated for 48 h in presence of 10 μg/ml silver nanoparticles in tissue culture media. Thereafter, the particles were ...

Fyn Phosphorylation and Activity Following Exposure to Silver Nanoparticles

Because Fyn is the predominant Src kinase found in the C18-4 cells, we next investigated if Fyn expression or phosphorylation were altered in the presence of nanoparticles. The expression of the Fyn protein was not altered by silver nanoparticles; however, there was a significant decrease of its GDNF-dependent phosphorylation (Fig. 8A). Furthermore, in vitro and in vivo kinase assays were performed to evaluate changes in the kinase activity of the Fyn protein. When the silver nanoparticles were incubated with a purified active Fyn protein, there was a size-dependent decrease in Fyn activity and this occurred regardless of nanoparticle coating (Fig. 8B). The SU6656 Src inhibitor was used a control and Fyn activity was abolished in the presence of this inhibitor. Additionally, there was a significant decline in the Fyn activity in the C18-4 cells treated with silver nanoparticles, but this did not occur in a size- or coating-dependent manner (Fig. 8C).

FIG. 8.
Fyn kinase phosphorylation and activation in the presence of coated silver nanoparticles. A: phosphorylation of Fyn kinase. The C18-4 cells were treated with 10 μg/ml silver nanoparticles for 24 h. After adding GDNF for 4 h (100 ng/ml), Fyn kinase ...

Evaluation of Downstream Components of Fyn Following Exposure to Silver Nanoparticles

When the C18-4 cells were treated with the silver nanoparticles, GDNF was unable to increase the activation of the Akt protein (Fig. 9A). Furthermore, N-myc expression declined in the presence of the silver nanoparticles and did not increase when stimulated with GDNF (Fig. 9B).

FIG. 9.
Effect of coated silver nanoparticles in C18-4 cells downstream of Fyn. A: Effect on Akt activity: C18-4 cells were treated with 10 μg/ml silver nanoparticles for 24 h and then with 100 ng/ml GDNF for 4 h. Akt kinase was immunoprecipitated and ...

DISCUSSION

Because of their minute size, nanoparticles are showing novel physical and chemical characteristics that are different from the properties exhibited by the corresponding bulk materials (Cui and Gao, 2003). Silver nanoparticles are among the most commercialized nanoparticles because of their antimicrobial potential and are therefore highly attractive for potential applications in the manufacture of medical devices. An increasing number of studies have now shown the potential toxic effect of these compounds for human health. However, many mechanisms of action remain unanswered.

A number of studies have reported the in vitro effects of silver nanoparticles on a variety of somatic cell lines, and in vivo investigations also demonstrated damages to lung, brain, and liver tissues (Cha et al., 2008; Sharma et al., 2010; Sung et al., 2008) In addition, silver nanoparticles might be a concern for reproductive health because nanoparticles also reach the testes after systemic administration or inhalation (De Jong and Borm, 2008; Kwon et al., 2008; Yauk et al., 2008), and toxicity to Leydig cells has been reported (Komatsu et al., 2008; Li et al., 2009). Toxicants that impair normal reproductive functions are an important public health issue. Male sperm quality has decreased throughout the years, and several scientific studies have pointed to various solvents, pesticides, gases, metals, and other air pollutants as contributing to infertility (Mohallem et al., 2005; Utell and Frampton, 2000; Warheit et al., 2008). Semen quality is also affected by smoking because of epigenetic alterations (Elshal et al., 2008; Selevan et al., 2000; Yauk et al., 2008). There are also rising concerns about the impact of prenatal exposure to reproductive toxicants. Birth defects like hypospadias, undescended testes, and subsequent testicular cancer, in addition to semen quality, are disorders included in the concept of Testicular Dysgenesis Syndrome (Boisen et al., 2001). Nanoparticles could contribute to these defects because Li et al. (2009) recently demonstrated that in utero exposure to nanoparticles contained in diesel exhaust affects testicular function by suppressing testicular production of testosterone after inhibition of StAR and 17β-HSD. Although this study indicates an effect of nanoparticles on the physiology of Leydig cells, we recently demonstrated that specific metal nanoparticles reduce SSCs proliferation in vitro (Braydich-Stolle et al., 2005). In the present study, we sought to understand the molecular mechanisms leading to this effect, using silver nanoparticles (Ag-NPs) as a model. We chose nanoparticle concentrations (10–50 μg/ml) that are lower than what many others have used for in vitro and in vivo studies (De Jong et al., 2008; Yoshida et al., 2009; also reviewed in Oberdörster, 2010). In a recent report, Sung et al. (2009) analyzed the effects of subchronic 90-day inhalation of silver nanoparticles (0.6–3.0 × 106 nanoparticles/cm3) in rat. Nanoparticles were found in blood (~0.85–6.86 ng/ml) and in all tissues examined including testes, indicating a systemic distribution of silver nanoparticles by circulating blood. The authors describe alterations in lung function, lung inflammation, and nanoparticle accumulation in the liver and in female kidneys. However, there was no detailed analysis of testis histology. Although this study represents one of the most pertinent models to date for chronic exposure to silver nanoparticles, the concentrations used, as well as the concentrations used in our study, are difficult to relate to human exposures. Indeed, there is little data on workplace air concentrations and exposure to silver nanoparticles because of inhalation. Similarly, concentrations of silver nanoparticles released from consumer products as well as tissue exposures because of oral or cutaneous intake are not known. Therefore, the concentration/accumulation of silver nanoparticles in the seminiferous epithelium of the human testis upon chronic exposure remains to be determined. Yet, the likelihood of human exposure warrants a mechanistic understanding of their toxicity for risk assessment estimates.

We demonstrate here that Ag-NPs reduce SSC viability and proliferation mainly in a size- and concentration-dependent manner and that particle coating has no influence over time. Because the interactions of nanoparticles with tissues are not well known, we evaluated changes in the physicochemical properties of the nanoparticles and characterized them prior to, during, and after exposure to cells (see Supplementary data). These changes suggested that postexposure characterization of the nanoparticles was required to determine the impact of internalization and exposure to the cellular environment on the nanoparticle coating. Our data lead to the conclusion that the coating was degraded, in particular after integration into lysosomes, and the particles appear to be losing size because of loss of coating and Ag+ ions potentially dissociating into solution. Because ionic Ag by itself is toxic, we assessed the effect of media supernatants pretreated with silver nanoparticles on the C18-4 cells and found their influence negligible (see Supplementary figs. 5 and 6). This loss of coating strongly suggests the need to characterize nanomaterials at each step in the experimental process and demonstrates that the cellular environment has a major effect on the nanoparticle stability and biocompability. Furthermore, our results confirm that silver nanoparticles are more reactive in a size-dependent fashion (Carlson et al., 2008). Another reason for the increased toxicity of smaller nanoparticles might be a higher ability to pass through the plasma membrane and increasing surface interactions.

Many studies have shown that exposure to nanoparticles resulted in a dose-dependent cytotoxicity in cultured cells that is associated with increased generation of ROS (Asharani, Hande, 2009; Foldbjerg, 2009). In some studies, ROS production led to increased transcription of proinflammatory mediators via intracellular pathways, including calcium signaling (Stone et al., 2007). Several studies have also shown that an increase of oxidative stress leads to an increase in apoptosis as evidenced by increase of caspase activity (Ahamed et al., 2010) and DNA damage (Ahamed et al., 2008; Asharani, Low Kah Mun, et al., 2009). In the present study, we show that at low exposures, a decrease in cell proliferation is not associated with ROS production and apoptosis, except when the cells are incubated with the smaller Ag 10-PS nanoparticles. As explained above, this selective reactivity might be because of increased surface interactions in addition to the loss of the polysaccharide coating. However, it is also important to note that silver nanoparticles may have multiple cellular targets that vary among cell types (Asharani, Hande, et al., 2009).

Because oxidative stress and apoptosis could not entirely explain the decrease in cell viability and rate of proliferation, we investigated whether silver nanoparticles interfered with GDNF signaling. GDNF is a growth factor that is crucial for the self-renewal of SSCs in vivo and in vitro (Hofmann, Braydich-Stolle, and Dym, 2005; Kubota et al., 2004; Meng et al., 2000; Naughton et al., 2006), and a block of any step within its signaling pathway will reduce or stop cell proliferation. Silver nanoparticles have been shown to interact with proteins and could potentially block signaling by binding to GDNF or its receptor Ret, therefore abolishing ligand-receptor interaction, or by interfering with intracellular signaling molecules. Figure 10A illustrates the major proteins involved in mediating GDNF signaling in C18-4 cells (Braydich-Stolle et al., 2007), and identifies potential sites for disruption in the GDNF pathway. Figure 10B identifies actual disruption sites when the silver nanoparticles are present. Our data verified that the Ag-NPs were not interacting with the growth factor itself. In addition, there was no inhibition of Ret receptor phosphorylation, indicating that neither the nanoparticles nor free Ag+ ions interfere with GDNF binding or modify the structure of the extracellular domain of the receptor complex. Therefore, based on these results, it was highly possible that the mechanism of disruption occurs intracellularly and downstream of Ret. In addition, we showed that silver nanoparticles are able to significantly decrease the activity of a purified commercially available Fyn kinase, a SFK that plays a pivotal role in the GDNF-induced signaling cascade in the C18-4 cells. As predicted, our data also show that silver nanoparticles significantly affected the phosphorylating ability of intracellular Fyn, and this inhibition occurred independent of nanoparticle size or surface coating. Although free Ag+ ions could be responsible for some of the inhibitory effects by interacting with protein sulfhydryl groups intracellularly, our data indicate that at the concentration used, they at least do not impair the activity of Fyn kinase (Supplementary fig. 6). Disruption of GDNF signaling by silver nanoparticles is further supported by a decline of Akt kinase activity and N-myc expression, both downstream targets of the Fyn kinase (Braydich-Stolle et al., 2007). A similar change in biocompatibility over time and interference with the action of a growth factor was observed in neuroblastoma cells exposed to the Ag 25-HC and Ag 25-PS nanoparticles and then treated with nerve growth factor (Schrand et al., 2008), indicating that this phenomenon is not unique to C18-4 cells. Also, interference with vascular endothelial growth factor signaling by blocking Src phosphorylation has recently been reported in retinal endothelial cells, but the type of kinase involved is not known nor which downstream targets are affected (Sheikpranbabu et al., 2009). Taken together, we conclude that GDNF signaling in SSCs is disrupted by silver nanoparticles, which directly interact with Fyn kinase to prevent further activation of downstream signaling proteins. This ultimately inhibits the expression of N-myc, a transcription factor that activates key components of the cell cycle machinery (Fig. 10B). We also have illustrated that not only do nanoparticles have an effect on the intracellular environment but the cellular environment impacts the physicochemical properties of the nanoparticles as well. This study demonstrates that when researchers coat nanomaterials to make them “biocompatible,” these materials must be fully characterized and evaluated within the cellular environment prior to claiming biocompatibility.

FIG. 10.
Illustration of GDNF signaling disruption by coated silver nanoparticles. A: potential targets for disruption of GDNF signaling in C18-4 cells that inhibit proliferation. (1) Nanoparticles bind to GDNF and prevent receptor binding, (2) nanoparticles bind ...

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

Air Force Office of Scientific Research Project (JON # 2312A214 to S.H. and J.S.); the National Institutes of Health (HD044543 and HD054607 to M.C.H.); Postdoctoral fellowship from the National Research Council to L.K.B.S.; Eli Lilly graduate fellowship to B.L.; Biosciences and Protection Division, Human Effectiveness Directorate, Air Force Research Laboratory under the Oak Ridge Institute for Science and Education to A.S. and R.C.M.

Supplementary Material

[Supplementary Data]

Acknowledgments

The authors would like to thank Dr Rex Hess, Department of Veterinary Biosciences, University of Illinois, for helpful comments and discussions and Dr Kathy Schaefer for critical reading of the manuscript. We also thank Dr Karl Martin and Dr Dan Goia for generously supplying us with the nanomaterials used in this study, Scott Streiker from the Nanoscale Engineering Science and Technology Laboratory at the University of Dayton for assistance with the electron microscopy studies, and Tom Wittberg from the University of Dayton Research Institute Surface Chemistry Analysis Facility for assistance with the XPS analysis.

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