Introduction

In recent years, the development of new nanomaterials has become an emerging area of great interest that has revolutionized medical treatments, electronic technologies, and a number of industrial processes (Seal and Karn 2014). Among the different types of nanomaterials, silver nanoparticles (AgNPs) are one of the most widely used as antimicrobial agents, drugs and gene delivery vehicles and diagnostic bio-sensing materials (Austin et al. 2014; Maillard and Hartemann 2012; Schluesener and Schluesener 2013; Tolaymat et al. 2010). The emerging number of daily consumer products containing AgNPs (including cosmetics, textiles, food packaging, water purifiers and other household products) has led to some concerns, as released nanoparticles (NPs) may mean environmental and human health risks (Blaser et al. 2008; Kumar and Dhawan 2013; Sharma et al. 2014; Zhang et al. 2014).

Oral, inhalation, subcutaneous or intravenous administration of AgNPs to different mammalian species has shown to cause neuronal degeneration, chronic alveolar inflammation and renal and hepatocellular toxicity (Knudsen et al. 2014; Korani et al. 2013; Kovvuru et al. 2014; Li et al. 2013; Sarhan and Hussein 2014; Song et al. 2013). Increases in radical oxygen species (ROS) generation, mitochondrial dysfunction, DNA damage, expression of inflammatory cytokines, and apoptosis induction by caspase proteins, are the main cellular responses found in in vivo and in vitro studies after AgNP exposure (AshaRani et al. 2009; Böhmert et al. 2012; Jiang et al. 2013; Liu et al. 2010; Piao et al. 2011; Zhang et al. 2014).

Among a number of potential uses of AgNPs, these nanoparticles have been proposed for cancer therapy. The design of AgNPs with drugs or nucleic acids encased in a shell, coated with a peptide that enables targeted drug delivery into tumors, promises to revolutionize genetic therapies and biomedical research (Braun et al. 2014). However, the effect of the silver nanocore in cancer cells, as well as its collateral toxic effects on normal cells, should be carefully examined prior to this proposed application. A number of authors have reported that AgNPs have a direct anti-cancer effect due to their intrinsic ability to be internalized and induce apoptotic cell death (Guo et al. 2013; Jeyaraj et al. 2013; Ortega et al. 2015; Satapathy et al. 2013; Sriram et al. 2010). Most of these authors support the use of AgNPs for cancer treatment, although effects of AgNPs have been shown only after short exposure times. Conflicting results of AgNPs on the expression of cancer-related proteins have been reported by several authors, as vascular endothelial growth factor or epidermal growth factor signaling pathways (Comfort et al. 2011, 2014; Kang et al. 2011; Sriram et al. 2010). The disparate effects of AgNPs on the p53 tumor suppressor protein clearly demonstrate this problem (Ahamed et al. 2008; AshaRani et al. 2012; Kim et al. 2014; Lee et al. 2011; Mytych et al. 2015; Satapathy et al. 2013, 2014). Due to the relevant role in cell cycle control and in the induction of apoptotic signaling cascades, p53 activation or down-regulation could tip the balance to cell death to preserve genomic integrity, or to genomic degeneration and chemotherapy drug resistance. The p53 protein is inactivated in more than 50 % of human cancers (Rivlin et al. 2011). A number of authors have reported that AgNP toxicity is mediated by the p53 protein, and its inhibition-or silencing-significantly reduces the observed toxic effects (Foldbjerg et al. 2011; Kim et al. 2014; Satapathy et al. 2013, 2014).

Based on the results of the above studies, the present investigation was aimed at providing a better understanding of the dose- and time-dependent influence of AgNPs on p53. To achieve this goal, the human lung adenocarcinoma epithelial cell line A549 was exposed to 0, 10, 25, 50, 100 and 200 µg/mL of AgNPs, followed by a time course of cell viability for 72 h. We also determined the time course of expression of total and active p53 protein and the dependent proteins p21 and murine double minute 2 (MDM2). The expression of the mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD), the protein marker of DNA damage, phosphorylated histone H2A.X (γ-H2A.X), and the apoptotic protein caspase-3 was also assessed as indicators of cellular damage. The expression of all these proteins was also determined after daily exposure of the cells to 10 µg/mL of AgNPs for 7 days or after a discontinuous exposure to 10 µg/mL of AgNPs by treating the cells every 3 days for 15 or 30 days. Moreover, epigenetic changes as acetylation of the histone H3 protein, or changes in global DNA methylation patterns, were determined after 72 h of exposure to a range of concentrations of AgNPs, as well as intracellular radical oxygen species (ROS) generation. The results of the present study may help to explain why AgNPs have shown disparate results regarding the p53 protein.

Materials and methods

Characterization of AgNPs

Polyvinyl pyrrolidone (PVP)-coated AgNPs (99.9 % Ag, 0.3 % PVP, 20 nm) were purchased from NanoAmor (Houston, USA). AgNPs were dispersed in pure water (1 mg/mL) and were sonicated for 15 min at 35–40 W for mixing and forming a homogeneous dispersion. After deposition of the NPs suspension onto carbon film-coated Cu grids, characterization (size and morphology) was performed using a JEOL JEM-1011 (JEOL, USA) transmission electron microscope (TEM), operating at an accelerating range of voltages of 100–800 kV. A series of images from different TEM grid patches were processed with a particle analysis tool to establish size distributions using ImageJ software (Version 1.48). Particle size distributions were then used to calculate the specific surface area.

Cell culture and treatment conditions

The A549 lung adenocarcinoma cell line was used in this study. A549 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were grown in Ham’s F-12K (Gibco BRL, Life Technologies, Paisley, UK) medium supplemented with 10 % (v/v) fetal bovine serum (Gibco BRL, Life Technologies, Paisley, UK), 10 units/mL penicillin and 100 mg/mL streptomycin (Sigma, St. Louis, MO, USA). Cells were maintained in 5 % CO2 at 37 °C with 95 % humidity. Fresh water dispersions of AgNPs were prepared as stock solutions before cell treatment. To assess cell viability, cells were seeded in 96-well plates and treated with a range of concentrations (0, 10, 25, 50, 100 and 200 µg/mL) of AgNPs for 24, 48 and 72 h. For western blotting, cells were seeded in 60-mm culture dishes and treated at the same conditions as in the cell viability assessment. To determine protein expression after continuous and discontinuous long-term treatments by western blot, cells were seeded in 75 cm2 cell culture flasks. The culture medium was then replaced by new medium supplemented with 0 or 10 µg/mL of AgNPs every day for 7 days, or every 3 days for 15 or 30 days. For the observation of DCF fluorescence and extraction of DNA, cells were seeded in 24-well plates or 60 mm dishes, respectively, being exposed to a range of concentrations (0–200 µg/mL) of AgNPs for 72 h. The 0 µg/mL concentration was considered as control.

Measurement of cell viability

Cell viability was colorimetrically quantified using the metabolic dye [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT, Sigma, St. Louis, MO, USA). The MTT assay, a measure of the energy-generating potential of the cell, assesses mitochondrial function by measuring electron transfer potential. MTT was added to the cells at a final concentration of 500 µg/mL and incubated for 3 h. During that time, MTT was reduced to produce a dark blue formazan product. The medium was then removed, being the cells dissolved in DMSO. Formazan production was measured by the change in absorbance at 595 nm using a microplate reader (BioTek Power Wave XS, USA). The viability results are expressed as percentages, and the absorbance measured in the control cells was taken to be 100 %.

Western blot analysis

Aliquots containing 30 μg of protein per sample of A549 cell lysates were analyzed by western blot analysis. Briefly, samples were placed in sample buffer (0.5 M Tris–HCl pH 6.8, 10 % glycerol, 2 % [wt/vol] SDS, 5 % [vol/vol] 2-β-mercaptoethanol, 0.05 % bromophenol blue) and denatured by boiling at 95–100 °C for 5 min. Samples were then separated by electrophoresis on 12 % acrylamide gels. Proteins were subsequently transferred to Immobilon-P PVDF sheets (Millipore Corp, Bedford, MA, USA) using a transblot apparatus (Bio-Rad, Madrid, Spain). The membranes were blocked for 1 h with 5 % nonfat milk dissolved in TBS-T buffer (50 mM Tris, 1.5 % NaCl, 0.05 % Tween 20, pH 7.5). The blots were then incubated overnight with primary monoclonal antibodies against p53 (AbCam, Cambridge, UK), p53 phosphorylated on Ser15 (AbCam, Cambridge, UK), p21 (Biolegend, San Diego, CA, USA), MDM2 (AbCam, Cambridge, UK), caspase-3 (Biolegend, San Diego, CA, USA), histone H3 (AbCam, Cambridge, UK), histone H3 (acetyl K9 + K14 + K18 + K23 + K27) (AbCam, Cambridge, UK), γ-H2A.X phosphorylated on Ser139 (Biolegend, San Diego, CA, USA), MnSOD (Cell Signalling Technology, New England Biolabs, Beverly, MA, USA) or β-actin (Sigma, St. Louis, MO, USA). The blots were thoroughly washed in TBS-T buffer and incubated for 1 h with a peroxidase-conjugated immunoglobulin G antibody. Immunoreactive proteins were visualized using an Immun-Star Chemiluminescence Kit (Bio-Rad, Madrid, Spain) according to the manufacturer’s instructions. Chemiluminescent detection is based on the interaction of a chemiluminescent substrate with an enzyme (horseradish peroxidase substrate), which has been conjugated to an antibody. The light that was generated when the enzyme conjugate binds and interacts with the substrate was captured with a Chemidoc (Bio-Rad, Madrid, Spain), which permits to obtain digital images and to make a semi-quantification of the band intensity. The protein load was periodically monitored via the immunodetection of β-actin.

RNA isolation and complementary DNA synthesis

Total RNA was isolated from A549 cancer cells using a Qiagen RNeasy Kit according to the manufacturer’s protocol. The concentration and purity of total RNA were detected by spectrophotometry using UV absorbance at 260 and 280 nm. The quality of the RNA was assessed by electrophoresis on 1 % denaturing agarose gel. The RNA was reversed transcribed from 1 µg of total RNA from each sample, using a QuantiTect Reverse Transcription Kit (Qiagen Inc, Hilden, Germany), according to the manufacturer’s protocol. An identical reaction, without reverse transcriptase, was performed to verify the absence of genomic DNA. The complementary DNA (cDNA) was subsequently amplified by PCR, using human-specific primers (Table S1), with a PyroStart Fast PCR Master mix (2 ×) kit (Fermentas, Burlington, Canada) according to the manufacturer’s protocol. The PCR products were separated on a 1 % agarose gel, and only specific bands were detected. The non-reactivity of the primers with contaminant genomic DNA was tested by the inclusion of controls that omitted reverse transcriptase from the cDNA synthesis reaction.

Real-time reverse-transcription-PCR

Quantitative PCR for MnSOD, p53, caspase 3, p21, MDM2 and GAPDH was completed using the QuantiTect SYBR Green PCR kit (Qiagen Inc) according to the manufacturer’s protocol, and a Rotor-Gene Q Real-Time PCR cycler (Qiagen Inc). The thermal cycling comprised an initial step at 50 °C for 2 min, followed by a polymerase activation step at 95 °C for 15 min, and a cycling step with the following conditions: 40 cycles of denaturing at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. As oligonucleotides of varying lengths produce dissociation peaks at different melting temperatures, at the end of the PCR cycles, the PCR products were analyzed using a heat dissociation protocol in order to confirm that one single PCR product was detected by SYBR Green dye. Fluorescence data were acquired at the 72 °C step. The threshold cycle (Ct) was calculated by Rotor-Gene Q 2.0 software to identify significant fluorescence signals above noise, during the early cycles of amplification. The software calculated copy numbers for the target samples from the Ct by interpolating from a standard curve. The relative levels of the expression of the target genes were measured using GAPDH mRNA as an internal control according to the 2−ΔΔCt method.

Cellular reactive oxygen species assay

Oxygen radical production was detected using the dye dichlorofluorescein diacetate (DCF). After exposure to AgNPs, cells were subsequently washed twice with sterile PBS and pre-incubated for 30 min in Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 5.6 mM d-glucose and 5 mM HEPES, pH 7.4) containing 10 µM of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA; Sigma, St. Louis, MO, USA). CM-H2DCF-DA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative 2,7-dichlorofluorescein (DCFH) and thereby trapped within the cells. In the presence of a proper oxidant, DCFH is oxidized to the highly fluorescent 2,7-dichlorofluorescein (DCF). ROS generation was detected as a result of the oxidation of DCFH (excitation, 488 nm; emission, 515–540 nm) with fluorescence microscopy performed using an Olympus BX61 microscope (Olympus America Inc., NY, USA). For all samples, the effect of DCF photooxidation was minimized by collecting the fluorescent images under identical parameters, such as contrast and brightness.

Determination of the type of cell death

Cells positive apoptosis and necrosis detection was performed with Annexin V-FITC/Dead Cell Apoptosis Kit for Flow Cytometry (Invitrogen, Paisley, UK). The externalization of phosphatidylserine as a marker of early-stage apoptosis was measured through the binding of Annexin V-FITC, according to the manufacturer’s protocol. Briefly, 2 × 105 A549 cells were plated in 6-well plates with 2 mL of complete medium following treatment with 0, 10, 25, 50 100 and 200 µg/mL of PVP-AgNPs for 72 h. The cells were collected and re-suspended in 100 µl of 1× Annexin binding buffer. A549 cells were incubated with 5 µl of Annexin V-FITC and 1 µl of 100 µg/mL of propidium iodate (PI) solution in 1× Annexin binding buffer at room temperature for 15 min in the dark. Approximately 2 × 104 cells were analyzed using a FACScan flow cytometer (Coulter Corporation, Hialeah, FL, USA). Annexin V-positive/PI-negative, Annexin V-positive/PI-positive and Annexin V-negative/PI-positive cells represent cells in early apoptosis, or late apoptosis/necrosis, respectively.

DNA extraction and global DNA methylation assay

DNA was extracted from the cell cultures and purified using the SPEEDTOOLS DNA Extraction kit (Biotools, Spain) according to the manufacturer’s protocol. Lysis of sample is achieved by incubation of sample in a solution containing chaotropic ions in the presence of proteinase K. Addition of ethanol to the lysate creates the appropriate conditions for binding of DNA to the silica membrane of the column. Then, washing steps efficiently remove contaminations, being pure genomic DNA finally eluted under low ionic strength conditions in a slightly alkaline elution buffer. Subsequently, the global DNA methylation levels of the A549 samples were assessed with an ELISA-based commercial kit (MDQ1, Imprint® Methylated DNA Quantification Kit, Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol. The MDQ1 kit is a high-throughput, which detects methylated DNA using the capture and detection antibodies, then colorimetrically quantified. The amount of methylated DNA present in the sample is proportional to the absorbance measured at 488 nm. Quantification of global DNA methylation was obtained by calculating the amount of methylated cytosines in the sample relative to a positive control, which had been previously completely methylated.

Determination of AgNP uptake

To study the cellular uptake of PVP-AgNPs, intracellular concentrations of Ag metal were quantified using inductively coupled plasma mass spectrometry (ICP-MS). After exposure, A549 cells were rinsed three times with cold PBS, trypsinized, counted and centrifuged at room temperature. The cell pellets in each tube were re-suspended in complete medium to obtain a final concentration of 1 × 106 cells/mL. Aliquots of 1 mL were introduced in a microsampling quartz insert, being 65 % nitric acid (Suprapur, E. Merck) added to digest the samples. The microsampling inserts were then introduced in Teflon vessels and put into a microwave oven Star D (Milestone, Sorisole, Italy) (Gomez et al. 2008). All materials were previously washed with 10 % nitric acid in order to avoid any potential contamination. Ag concentrations were determined by means of a computer-controlled sequential ICP-MS (PerkinElmer Elan 6000) according to DIN EN ISO 17294-2. The detection limit, which was calculated as three times the standard deviation of the blank signal, was 0.02 μg/mL for Ag.

Statistical analysis

Data were evaluated by one-way ANOVA followed by Bonferroni’s post hoc test to identify significant differences between controls and treatments. All data were analyzed with the SPSS 20.0 software (SPSS Sciences, Chicago, IL). The level of statistical significance for all tests was set at p < 0.05. The results are displayed as the mean ± SD of at least three independent experiments. The relationships between the examined endpoints were assessed by correlation analysis using the Pearson method.

Results

TEM analysis of AgNPs

TEM was used to characterize the size and morphology of the PVP-coated AgNPs. Figure 1a shows TEM images revealing that the AgNPs had a spherical shape, although some agglomerations could be observed. Information on the mean size and SD was calculated by measuring two hundred NPs in random fields of view, in addition to images that showed the general morphology of the NPs. The mean size ± SD of AgNPs was 21.74 ± 7.76 nm. The frequency of AgNP size distributions is depicted in Fig. 1b.

Fig. 1
figure 1

Characterization of nanoparticle size and time course of A549 cell viability after AgNP exposure. a TEM images of representative single AgNPs show nearly spherical NPs. The scale bars are 20 nm. b Histogram of over two hundred single AgNPs measured by TEM shows the average diameter of NPs at 21.7 ± 7.7 nm. c Evaluation of A549 cell viability after 24, 48 and 72 h of exposure to AgNPs at 0, 10, 25, 50, 100 and 200 µg/mL. Cell viability data are expressed as the mean ± SD of four independent experiments. Significant differences relative to the control (0 µg/mL) were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05 versus control

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Effect of AgNPs on cellular viability

The time course of cell viability was assessed after exposure of A549 cells to various concentrations of AgNPs (Fig. 1c). A significant decrease (p < 0.05) was observed relative to control cells at all concentrations, after 24, 48 and 72 h of exposure to AgNPs. It must be noted that the decrease in cellular viability after 48 h was not significant compared with the values obtained after 24 h. In contrast, A549 cell death was more pronounced after 72 h of exposure to higher AgNP concentrations (17 ± 3, 31 ± 2, 39 ± 4, 54 ± 3 and 67 ± 5 %, for 10, 25, 50, 100 and 200 µg/mL, respectively).

AgNPs decreased p53, p21 and MDM2 protein levels

As observed in the blots of Fig. 2a, when compared with the control, the levels of the phosphorylated form of p53 and total p53 increased when the cells were exposed to 25 and 50 µg/mL of AgNPs, after 48 and 72 h of exposure. However, the levels of total p53 and the phosphorylated form declined sharply at 100 and 200 µg/mL during the same time intervals. Normalized phosphorylation levels (Fig. 2b) showed an increasing trend at 25 and 50 µg/mL, which was significantly higher (43 ± 7 %, p = 0.014) when the cells were exposed to 25 µg/mL after 48 h. In turn, a decreasing trend at 100 and 200 µg/mL, after 48 and 72 h, compared with the control was noted. These results indicate that the phosphorylation of p53 was directly influenced by the levels of total p53 protein, while the decrease in total p53 levels led to the diminished phosphorylation of p53. This same relationship was observed in the blots shown in Fig. 5a, c, in which the normalized phospho-p53/total-p53 ratio showed a significant decrease (39 ± 12 %, p = 0.042) when the A549 cells were exposed to 10 µg/mL of AgNPs daily for 7 days. Furthermore, discontinuous treatment every 3 days at the same concentration showed an increasing trend, but did not cause any significant changes compared with the control.

Fig. 2
figure 2

High doses of AgNPs decreased total and active p53, p21, MDM2 and caspase-3 protein expression after 72 h of exposure. A549 cells were exposed to 0, 10, 25, 50, 100 and 200 µg/mL AgNPs for 24, 48 and 72 h, and the expression of total and phosphorylated p53, p21, MDM2, caspase-3 and β-actin protein was measured by western blot analysis. a A western blot from one representative experiment is shown in the upper panel. b–e The intensity of the bands was determined by densitometric analysis, and the data were normalized using β-actin as an internal control. Relative levels of the data from three independent experiments are expressed as the mean ± SD in the lower panel. Significant differences relative to the control (0 µg/mL) were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05, **p < 0.01 and versus control

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Similarly, the expression of the p21 and MND2 proteins was coincident with the variability observed in the p53 protein, as it can be observed in Figs. 2a and 5a. The expression of both proteins showed an increasing trend at 25 and 50 µg/mL after 48 and 72 h, with a significant difference (44 ± 12 %, p = 0.022) for MDM2 at 50 µg/mL after 48 h of exposure (Fig. 2d). As with p53, both proteins suffered a notable decline (Fig. 2c, d), which was significant for p21 at 200 µg/mL (45 ± 6 %, p = 0.007), and for MDM2 at 100 (44 ± 8 %, p = 0.035) and 200 µg/mL (65 ± 16 %, p = 0.008) after 72 h of exposure. The MDM2 protein showed a significant increase (34 ± 7 %, p < 0.001) when the cells were discontinuously treated for 30 days, whereas its expression was significantly decreased (67 ± 7 %, p < 0.001) when the cells were continuously treated for 7 days (Fig. 5e). A similar significant decrease (45 ± 11 %, p < 0.001) was observed in P21 expression after daily treatment with 10 µg/mL (Fig. 5d). These findings were consistent with the changes observed in the p53 protein expression, as the expression of both proteins was largely influenced by the active form of p53.

AgNPs increased γ-H2A.X and MnSOD levels

γ-H2A.X levels were increased in a dose- and time-dependent manner (Fig. 3c), showing statistical significance upon exposure at 200 µg/mL (153 ± 54 %, p = 0.002) after 24 h, at 50 (75 ± 12 %, p = 0.044), 100 (115 ± 41 %, p = 0.001) and 200 µg/mL (245 ± 40 %, p < 0.001) after 48 h, and at 10 (70 ± 18 %, p = 0.003), 25 (134 ± 26 %, p < 0.001), 50 (217 ± 29, p < 0.001), 100 (299 ± 36 %, p < 0.001) and 200 µg/mL (409 ± 41 %, p < 0.001) after 72 h. Similarly, MnSOD expression also increased in a dose- and time-dependent manner, being significant upon exposure at 200 µg/mL (34 ± 12 %, p = 0.001) after 24 h, at 100 (52 ± 11 %, p < 0.001) and 200 µg/mL (76 ± 7 %, p < 0.001) after 48 h, and at 50 (43 ± 8 %, p = 0.044), 100 (65 ± 13 %, p = 0.012) and 200 µg/mL (135 ± 54 %, p < 0.001) after 72 h (Fig. 3d). A positive correlation was observed between γ-H2A.X and MnSOD expression (R = 0.91, p < 0.001), and between cell death and the expression of both proteins (R = 0.89 and R = 0.86, p < 0.001, respectively). A dose-dependent increase of ROS levels and the cell death after 72 h of exposure to AgNPs (Fig. 3f) supported this hypothesis. On the other hand, the same relationship was also observed in the daily treatment of cells with 10 µg/mL of AgNPs. In this case, MnSOD showed a significant increase (268 ± 109 %, p = 0.002), but did not show significant changes after discontinuous treatment for 15 or 30 days (Fig. 5f). In contrast, slightly elevated γ-H2A.X levels were noted after 15 (309 ± 73 %) and 30 (584 ± 115 %) days of discontinuous treatment (Fig. 5b). However, in comparison, daily exposure to 10 µg/mL of AgNPs presented a significant greater activation (2161 ± 596 %, p < 0.001) when compared to control.

Fig. 3
figure 3

High levels of AgNPs decreased histone H3 acetylation and increase global DNA methylation after 72 h of exposure, which correlated with an increase in ROS generation and MnSOD and γ-H2A.X protein expression. A549 cells were exposed to 0, 10, 25, 50, 100 and 200 µg/mL AgNPs for 24, 48 and 72 h, and the expression of total and acetylated histone H3, γ-H2A.X, MnSOD and β-actin protein was measured by western blot analysis. a A western blot from one representative experiment is shown in the upper panel. b–d The intensity of the bands was determined by densitometric analysis, and the data were normalized using β-actin as an internal control. e Measurements from the global DNA methylation assay after 72 h of exposure to AgNPs. f Morphology of A549 cells and intracellular ROS generation after 72 h of exposure to AgNPs. Data on the relative levels of western blot and on global DNA methylation from three independent experiments are expressed as the mean ± SD in the lower panel. Significant differences relative to the control (0 µg/mL) were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 and versus control

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AgNPs deregulates the gene expression of p53, Caspase 3, MDM2, p21 and MnSOD

The mRNA expression of p53, Caspase 3, MDM2, p21 and MnSOD genes were evaluated by real-time qPCR. Human lung adenocarcinoma epithelial cell line A549 was exposed to 0, 10, 25, 50, 100 and 200 µg/mL of AgNPs for 72 h (Fig. 4b). A549 cells were also daily exposed to 10 µg/mL of AgNPs for 7 days, or after a discontinuous exposure to 10 µg/mL of AgNPs every 3 days, for 15 or 30 days (Fig. 6a–e). The expression of p53 gene relative to control cells was found significantly down-regulated following exposure to 50 (35 ± 9 %, p = 0.001), 100 (49 ± 8 %, p = 0.000) and 200 µg/mL (78 ± 3 %, p = 0.000) after 72 h, and after daily exposure of the cells, to 10 µg/mL of AgNPs for 7 days (57 ± 13, p = 0.000). Similarly, transcript levels of caspase 3, p21 and MDM2 were also down-regulated in the same degree than p53, being significant upon exposure to 100 and 200 µg/mL after 72 h, and after daily exposure for 7 days. On the other hand, the expression of MnSOD at 100 (86 ± 15 %, p = 0.043) and 200 µg/mL (160 ± 65 %, p = 0.000) after 72 h, and after daily exposure for 7 days (133 ± 21, p = 0.021), was significantly up-regulated. These results are in agreement with those obtained from the protein expression. They suggest that alterations in the molecular mechanism that regulates the gene expression could be induced after AgNPs exposure.

Fig. 4
figure 4

A549 cells exposed to increasing concentrations of AgNPs induced early apoptosis or late apoptosis/necrosis cell death, in a dose-dependent manner, and decreased the mRNA expression of p53, p21, MDM2 and caspase-3 genes. A549 cells were exposed to 0, 10, 25, 50, 100 and 200 µg/mL AgNPs for 72 h. a Dot plot representing the percentage of Annexin V positive and Annexin V/Propidium Iodide staining in A549 cells, analyzed by flow cytometry. b The relative expression levels of the mRNAs encoding MnSOD, p53, caspase 3, p21 and MDM2 were determined by quantitative real-time RT-PCR assay. Data were normalized using the gene GAPDH as an internal control. The fold changes were calculated relative to the control. Data are expressed as the mean ± SD of three independent experiments. Statistical differences relative to the control were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 and versus control

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AgNPs increased global levels of DNA methylation and induced deacetylation of histone H3

Representative blots of acetylated histone H3 showed that exposure at 100 and 200 µg/mL, after 48 and 72 h, produced a deacetylation of the tails of histone 3 (Fig. 3a). In contrast, elevated levels of total histone H3 were found at the same concentrations and intervals of time. Thus, the normalized acetyl-H3/total-H3 ratio (Fig. 3b) showed a decreased value (below 1), with statistical significance at 100 µg/mL after 72 h (p = 0.021), and at 200 µg/mL after 48 (p = 0.001) and 72 h (p < 0.001). Deacetylation of the tails of histone H3 after 72 h was correlated with global DNA methylation (R = −0.81, p = 0.019), which showed a significant increase (13 ± 4 %, p = 0.012) at 200 µg/mL compared with the control (Fig. 3e). Epigenetic changes as DNA hypermethylation and deacetylation of the tails of histone H3 are associated with repressed chromatin structure. These epigenetic changes could be also related with the morphologic changes observed after 72 h of exposure to AgNPs (Fig. 3f). The cells did not show relevant morphological changes up to 50 µg/mL. However, apoptotic forms could be discerned. In contrast, catastrophic cell swelling, formation of membrane blebbing and a polymorphous cytoplasm, could be observed in the surviving cells as an adaptation to the injury caused by the 100 and 200 µg/mL AgNP exposure after 72 h.

AgNPs decreased pro-caspase-3 levels

As observed with p53, the pro-caspase-3 protein demonstrated a progressive increase (Fig. 2e), being significant at 50 µg/mL after 48 h (37 ± 16 %, p = 0.021) and 72 h (34 ± 18 %, p = 0.042) of exposure to AgNPs. Likewise, pro-caspase-3 decreased at high concentrations of AgNPs after 48 and 72 h. The decrease in protein expression was significant at 200 µg/mL after 72 h (56 ± 17 %, p = 0.021), and after daily exposure to 10 µg/mL (25 ± 9 %, p = 0.027) for 7 days (Fig. 5g). The cleaved form of caspase-3 showed the same pattern than its pro-form, being decreased at the same doses and intervals of times (Fig. S1).

Fig. 5
figure 5

Daily exposure to a low dose of AgNPs decreased the expression of total and phosphorylated p53, p21, MDM2 and caspase-3 protein and increased γ-H2A.X and MnSOD. A549 cells were treated every day for 7 days or every 3 days for 15 or 30 days with 0 or 10 µg/mL of AgNPs, followed by measurement of the expression of total and phosphorylated p53, p21, MDM2, caspase-3, γ-H2A.X, MnSOD and β-actin proteins by western blot analysis. a A western blot from one representative experiment is shown in the upper panel. b–g The intensity of the bands was determined by densitometric analysis, and the data were normalized using β-actin as an internal control. The relative levels of western blot data from three independent experiments are expressed as the mean ± SD in the lower panel. Significant differences relative to the control (0 µg/mL) were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 and versus control

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AgNPs induce early apoptosis, late apoptosis cells or necrosis cells in a dose-dependent manner

Cytometry flow analysis reveals that exposition of A549 cells to increasing concentrations of AgNPs induced changes in the distribution of early apoptotic, late apoptotic cells, and necrotic cells after 72 h of exposure (Fig. 4a). Annexin V/PI fluorescence revealed the ability of AgNPs to induce exclusively early apoptotic cell death at 10 (14.5 %) and 25 µg/mL (21.2 %). During this period, most cells were negative for PI, implicating the early stage of apoptosis. With increased concentrations of AgNPs (50, 100 and 200 µg/mL), A549 cells turned positive for PI in addition to membrane staining with Annexin V, consistent with a more advanced stage of apoptosis (27.5, 54.7 and 62.4 %, respectively). Low fraction of necrotic cells (2.1, 4.6 and 4.3 %, respectively) was detected at these same concentrations. These results indicate that single exposure to low doses of AgNPs (down 50 µg/mL) promotes early apoptosis cell death in A549 cells, and high doses (up to 50 µg/mL), late apoptotic/necrosis cell death.

Cellular uptake of AgNPs

We next investigated whether the differences in protein and gene expression could be explained by differences in cellular uptake. A549 cells were continuously or discontinuously exposed to 10 µg/mL of AgNPs, being the intracellular amount of silver quantified by ICP-MS (Fig. 6f). The highest accumulation of silver was observed after 7 days of exposure (97.1 ± 33.6 pg/cell), which was about 2.6–4.2 fold higher than discontinued treatment following 15 (23.1 ± 4.1 pg/cell) and 30 days (37.4 ± 6.3 pg/cell) of exposure.

Fig. 6
figure 6

Daily exposure to a low dose of AgNPs decreased the mRNA expression of p53, p21, MDM2 and caspase-3 genes and induced a higher cellular uptake of AgNPs. A549 cells were treated every day, for 7 days, or every 3 days for 15 or 30 days, with 0 or 10 µg/mL of AgNPs. The relative expression levels of the mRNAs encoding a caspase 3, b MnSOD, c p53, d p21 and e MDM2 were determined by quantitative real-time RT-PCR assay. Data were normalized using the gene GAPDH as an internal control. The fold changes were calculated relative to the control. Data are expressed as the mean ± SD of three independent experiments. Statistical differences relative to the control were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 versus control. f Intracellular amount of silver during the AgNP uptake in a 10 % FBS-supplemented medium, determined by ICP-MS. Data are given as mean ± SD (n = 3)

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Discussion

In spite of the considerable current information concerning the toxic effects of AgNPs, the controversy regarding the magnitude of their toxicity and their potential use as an anticancer agent is still under discussion. The present study was aimed at providing a better understanding of how the toxic action of AgNPs develops over time, and how about the effects after continuous or discontinuous exposure to AgNPs.

A number of investigations have found that after cellular uptake, the formation of lysosomes containing AgNPs deposits occurs. Due to the acidic lysosomal pH, the surface oxidation of the AgNPs and the release of silver ions can directly induce the generation of free radicals, which may diffuse through the lysosomal membrane to the cytosol and interfere with normal cellular functions (De Matteis et al. 2015; Greulich et al. 2011; He et al. 2012; Singh and Ramarao 2012). One of the main organelles susceptible to AgNP damage is the mitochondria. Silver ions can impair mitochondrial electron transfer chain complexes, which lead to the inhibition of adenosine triphosphate (ATP) synthesis, as well as an increase in the generation of hydroxyl radicals (AshaRani et al. 2009; Costa et al. 2010; Foldbjerg et al. 2011; Tan et al. 2014). In the present study, MnSOD protein expression was evaluated as an indicator of mitochondrial ROS production. We found that MnSOD protein and mRNA levels increased in a dose- and time-dependent manner during the 72 h of exposure, being positively correlated with a decrease in cell viability. In a previous investigation, Piao et al. (2011) observed that AgNPs induced cell death via ROS generation, and mitochondria-dependent apoptotic pathway activation, upon release of cytochrome c to the cytoplasm and the activation of caspases-9 and 3. In contrast, we found that increasing levels of MnSOD positively correlated with the γ-H2A.X. Several studies have reported DNA damage in nuclei after AgNP exposure by ROS generation (Arora et al. 2008; Kim et al. 2014; Xu et al. 2014). On the other hand, γ-H2A.X protein becomes phosphorylated when DNA double-strand breaks (DSBs) are produced in mammalian cells, resulting in specific foci of DNA damage sites (Guo et al. 2010).

Ataxia-telangiectasia mutated (ATM) protein is the main protein responsible for the phosphorylation of the Ser139 residue of histone H2A.X. ATM protein kinase is activated by oxidative substances and responds to DNA DSBs, triggered by the mobilization of intracellular signaling pathways, which initiate the response to DNA damage (Guo et al. 2010). Recently, Lim et al. (2012) reported an increase in active ATM protein after 48 h of exposure to 100 µg/mL of AgNPs in different mammalian cells types. Furthermore, another important target protein of ATM is p53, which is activated by phosphorylation of the Ser15 residue. After DNA damage, p53 leads to cell cycle arrest via activation of p21, promoting DNA repair processes or the induction of apoptosis. In the current study, phosphorylation of p53 on the Ser15 residue increased at lower doses after 48 and 72 h of exposure, coinciding with a moderate increase in γ-H2A.X and MnSOD levels. Surprisingly, when the levels of both proteins were drastically increased, phosphorylated and total p53 protein were down-regulated. Moreover, the time course levels of p21 and MDM2 proteins, which are both dependent on p53 for expression, showed the same pattern, resulting also decreased at high doses after 48 and 72 h. Disparate effects of AgNPs with respect to p53 protein expression have been reported. Ahamed et al. (2008), Kim et al. (2014) and Satapathy et al. (2013, 2014) showed that exposure to AgNPs increased the levels of active p53 protein in mouse embryonic stem cells, as well as BEAS-2B and HCT116 human cells. In turn, Mytych et al. (2015) and AshaRani et al. (2012) reported that total p53 protein decreased in HeLa, ACHN, IMR90 and U251 human cells. It must be taken into account that when p53 was shown to be activated, AgNPs with a large diameter (50–142 nm), or a short time of cell exposure (24 h), were used in the experimental procedure. However, when total levels of p53 decreased, small nanoparticle diameters (5–25 nm) and longer times of cell exposure were employed. The nanoparticle size could be an important factor in the generation of ROS and the expression of p53. It has been reported that smaller particles induced greater cellular damage at the same concentrations (Sun et al. 2013). DCF fluorescence revealed elevated ROS levels localized in nuclei at 100 and 200 µg/mL after 72 h. A prolonged time of nuclei exposure to ROS could trigger fatal mutations in the DNA sequences and chromosomal aberrations. It could influence the gene expression of p53, and consequently, the other genes indispensable for maintaining integrity. To support this hypothesis, we also determined the mRNA expression of p53, observing that it was down-regulated at the same manner that protein expression. A number of studies have shown that mRNA expression of p53 was down-regulated in the liver of different fish species and in human fibroblast cells after AgNP exposure (AshaRani et al. 2012; Chae et al. 2009; Christen et al. 2013; Mytych et al. 2015; Pham et al. 2012). Moreover, Mytych et al. (2015) also reported that the decrease in total p53 protein expression in human HeLa and ACHN cells was sustained after 144 h of AgNP exposure.

On the other hand, caspase-3 protein and gene expression displayed the same pattern of behavior as p53. Increasing levels of caspase-3 were produced at low concentrations of AgNPs after 48 and 72 h. However, these levels decreased at high doses. Although almost all existing studies report that caspase-3 increased after AgNP exposition (Piao et al. 2011; Satapathy et al. 2013), Arora et al. (2008) found the same pattern of behavior than that observed in the present study. The genetic predisposition of cancer cells to down-regulate the expression of apoptotic genes could partially explain this decreased protein and gene expression. Devarajan et al. (2002) showed that 75 % of breast tumor samples, as well as morphologically normal peritumoral tissue samples, lacked the caspase-3 transcript and caspase-3 protein expression. The lack of p53 and caspase-3 could deprive the cells of the ability to induce apoptosis, establishing necrosis as an alternative form of cell death. As reinforcement of this hypothesis, the cell morphology and flow cytometric analysis after 72 h of exposure to AgNPs showed that the number of apoptotic cells gradually increased in a dose-dependent manner at low doses. Subsequently, exposure to high doses showed significant morphologic changes such as abnormal sizes, hypertrophy and increases of late apoptosis/necrosis cell death. These changes at higher doses are coincident with the increased DCF fluorescence and the down-regulation of p53, p21 and caspase-3 protein expression after 72 h. A similar conclusion was also reached by AshaRani et al. (2009), who noted cytoskeletal deformations in U251 cells treated with 200 µg/mL of AgNPs during 48 h of exposure.

Another key finding of the current investigation was the fact that the daily exposure of cells to a fresh suspension of 10 µg/mL of AgNPs for 7 days showed the same molecular changes that were produced following a single exposure to 200 µg/mL after 72 h. A cellular disposition to the internalization of AgNPs could play an important role in the related cytotoxicity. We found that A549 cells, daily exposed to 10 µg/mL of AgNPs, really uptake concentrations of AgNPs, which could explain at least in part the differences with discontinued exposure. Cells have more capacity to uptake NPs when they are in a suspension than when they are agglomerated or sedimented (Albanese and Chan 2011; Lankoff et al. 2012). In relation to this, daily exposure to AgNPs could induce a chain reaction by which subsequently internalized NPs experienced higher and easier oxidation due to the pre-existence of higher ROS levels resulting from the initial exposure and cellular uptake of NPs. In addition, when the treatment was applied every 3 days, cells might have sufficient time to deal with the AgNP insult and attempt to restore normal levels prior to the next exposure. This could be the reason because discontinuous treatment for 15 or 30 days showed the same molecular pattern of behavior as the lower single doses of AgNPs after 72 h of exposure.

Other important findings of the present investigation have been the decrease in global acetylation levels of the tails of histone 3 protein, as well as the increase in global DNA methylation observed at higher doses of AgNPs, after 72 h of exposure. These epigenetic changes could also influence the gene expression of the p53 and caspase-3 proteins by hypermethylation of their promoter regions and consequent transcriptional silencing. Although there is not available information on the epigenetic effects induced by AgNPs, some studies have reported hypermethylation of the CpG island of PARP-1 gene, or global deacetylation of histones, after exposure to other types of NPs (Choi et al. 2008; Gong et al. 2010). It may be due to the fact that inhibition of mitochondrial ATP synthesis by AgNPs can induce a hypoxia-like state (Eom et al. 2013). It has been also reported that during hypoxic stress, histone H3 is deacetylated, being global DNA hypermethylated (Islam and Mendelson 2006; Robinson et al. 2012). In contrast, the levels of total histone H3 increased at high levels of AgNPs, after 48 and 72 h of exposure. This accumulation could be either a consequence of an increase in cell cycle arrest, or it could be due to some interference in the protein degradation pathway. Recently, Narayan et al. (2015) observed that accumulation of histones H3 and H4 correlated with a compromised protein degradation pathway in Alzheimer’s disease. With respect to this, Mangini et al. (2014) noted that AgNPs have high affinity to bind the ubiquitin protein in the formation of protein corona. The abduction of the ubiquitin protein necessary for proteasomal degradation by AgNPs could trigger histone H3 accumulation. To clarify this issue, further studies on the epigenetic alterations and compromised proteasomal degradation are still clearly needed.

Conclusions

The results of the current study demonstrate that daily exposure to low doses of AgNPs, or a single exposure to high doses of these nanoparticles for 72 h, decreased the levels of gene and protein expression of p53, p21, MDM2 and caspase-3 in A549 cells, with a high increase in ROS generation and DNA damage. However, the discontinuous exposure to low doses of AgNPS, or the single exposure to low doses for 72 h, could increase the levels of the active forms of p53 and caspase-3, as well as those of p21 and MDM2 protein. In addition, exposure to high doses of AgNPs induced higher levels of global DNA methylation, global histone H3 deacetylation, and accumulation of total histone H3 in A549 cells. The current results provide a better understanding about the human health risks and warn on the possibility that AgNPs may deprive cells of molecular pathways, which would delay or stop the development of tumors.