Apoptosis-mediated neurotoxicity and altered gene expression induced by silver nanoparticles

Abstract

Although not fully recognized, the neurotoxic effects of silver nanoparticles (Ag-NPs) are thought to occur through induction of oxidative stress and apoptosis. To investigate the exact underlying molecular mechanism, we aimed to explore the apoptotic effects of intraperitoneal injection of Ag-NPs and investigated the possible attributed changes in the mRNA expression of Bcl-2 and Bax genes in the rat hippocampus. Two in vivo sets of experiments, one to demonstrate apoptosis and the other to assess gene expression, were conducted on male Wistar rats. In each set, the first group, acting as control, received saline and the other three groups received Ag-NP at doses of 100, 200, and 400 ppm for five successive days. Ten days after the last injection, hippocampal tissue of the first set of rats was assessed for apoptosis using terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling staining. In the second set of experiments, mRNA expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction. Ag-NP treatment was shown to induce apoptosis in a dose-dependent manner. Furthermore, Ag-NP reduced mRNA level of Bcl-2 in the rat hippocampal cells at all investigated doses compared to the control group (p < 0.001). The mRNA level of Bax, on the other hand, was increased in these cells. The increase was significant compared to the control group at the doses of 200 ppm (p < 0.05) and 400 ppm (p < 0.001). Our results show that Ag-NPs reduce Bcl-2 and increase Bax genes expression, resulting in increased Bax/Bcl-2 ratios in rat hippocampal cells. This altered gene expression induces cell apoptosis and contributes to the neurotoxicity of Ag-NPs.

Keywords

Silver nanoparticles Bcl-2 gene Bax gene real-time PCR TUNEL

Introduction

Nanoparticles (NPs), defined as particles with one or more dimensions from 1 to 100 nm, are becoming extremely popular due to their unique physical and chemical properties. Silver NPs (Ag-NPs) are one of the most widely used nanomaterials mainly because of their bacteriostatic effects. In addition to occupational exposure, humans are exposed to Ag-NPs through medical instruments, personal care products, food services, water purification, building material, and textiles (Ahamed et al., 2010a; Hadrup and Lam, 2014; Sharma and Sharma, 2012).

Several in vitro and in vivo studies have confirmed toxicity of Ag-NPs to mammalian cell lines and animal models (Ahamed et al., 2010a; Hadrup and Lam, 2014). In particular, it has been shown that Ag-NPs can cross the blood–brain barrier, accumulate in the central nervous system, and induce neurotoxicity by damaging neuronal, glial, axonal, and endothelial cells (Ahamed et al., 2010a; Hoet et al., 2004; Tang et al., 2009). Many of these effects depend on the dose and route of NP administration.

Alteration in gene expression, generation of free radical-induced oxidative stress, and apoptosis induction are among the proposed mechanisms for Ag-NP neurotoxicity (Ahamed et al., 2010b; Lee et al., 2010; Rahman et al., 2009; Sharma and Sharma, 2012; Yin et al., 2013). However, the mechanism of action of this NP-induced apoptosis is still unclear.

The members of the Bcl-2 protein family determine the commitment of cells to apoptosis through their interplay on the mitochondria. Antiapoptotic groups, such as Bcl-xL and Bcl-2 itself, inhibit apoptosis, while proapoptotic members, such as Bax and Bcl-xS, induce the apoptosis process (Ashkenazi and Dixit, 1998; Czabotar et al., 2014; Ghribi et al., 2001). In this study, real-time polymerase chain reaction (PCR), one of the most sensitive and reliable methods for gene expression analysis, was implemented to detect changes in the expression of Bcl-2 and Bax genes in the rat hippocampus after exposure to Ag-NP.

Methods

Silver nanoparticles

Commercially available Ag-NPs (Ag, 99.99%, coated with ∼0.2 wt% Polyvinylpyrrolidone) were purchased from US Research Nanomaterials, Inc. (Houston, Texas, USA). The size and morphology of Ag-NPs were characterized by transmission electron microscopy (TEM, LEO 906; Carl Zeiss, Germany) after deposition of NP suspension onto carbon film-coated copper TEM grids. The AMT Imaging system (Advanced Microscopy Techniques Corp., Danvers, MA, USA) for the digital TEM camera was calibrated for size measurement of the NPs. A total of 186 Ag-NPs were measured in TEM for size distribution. The particle size distribution of the Ag-NPs was also assessed by dynamic light scattering (DLS) technique using Malvern Zetasizer Nano ZS (Malvern, UK) at 25°C.

Animal experiments were performed with three different concentrations of 100, 200, and 400 ppm, prepared from nanosilver colloids by a serial dilution method (Hogstrand and Wood, 1996). To form a homogeneous dispersion, each sample was sonicated for 10 min at 35–40 W before each experiment.

Animal groups

Two different sets of experiments, one to demonstrate apoptosis and one to assess gene expression, were conducted on 56 male Wistar rats weighing 200–250 g, at 8 weeks of age procured from Shahid Beheshti University. The rats were housed at 23 ± 2°C with a 12-h light/12-h dark cycle in the animal house of Parand Azad University and fed rodent chow. After 2 weeks of adaptation to the new environment, they were randomly allocated into eight groups of seven rats each; a control and three silver-treated groups for each of the two sets. For each set, the control groups received saline and experimental groups were administered nanosilver at concentrations of 100, 200, and 400 ppm for five consecutive days. Injections were performed intraperitoneally with a final volume of 1 cc for each dose. Ten days after the last injection, the rats were deeply anesthetized with chloroform and rapidly decapitated. The brains were dissected and placed on an ice-cooled cutting board. After removal of the meninges, extracted hippocampi were either fixed in 10% neutral buffered formalin for terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) staining or snap frozen in liquid nitrogen and stored at –70°C until later real-time PCR tests. All experiments conformed to the guidelines of the Ethical Committee of Parand Azad University.

TEM analysis

To demonstrate intracranial penetration of the Ag-NPs through the blood–brain barrier, one additional rat with similar characteristics was treated intraperitoneally with nanosilver at 200 ppm using the same 5-day regimen. Ten days after the last injection, the extracted hippocampus was pre-fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) (pH 7.4) for 2 h and post-fixed with 1% osmium tetroxide in the same buffer for another 2 h. After dehydration in an ascending series of ethanol, specimens were placed in propylene oxide and embedded in Epon 812 (TAAB, Berkshire, UK). Ultrathin sections (60–80 nm) were contrasted with uranyl acetate and lead citrate before being examined by TEM (LEO 906; Carl Zeiss).

TUNEL staining

The in situ cell apoptosis was identified by the TUNEL technique, using a commercial apoptosis kit (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Germany). In brief, hippocampal sections were deparaffinized with xylene, rehydrated through descending concentrations of ethanol, and rinsed in PBS twice for 10 min at room temperature (Khodarahmi et al., 2015). Proteinase-K digestion was applied as a pre-treatment for 20 min after which the sections were rinsed in PBS. Then, the TUNEL reaction was carried out according to the supplier’s instructions. Finally, the sections were counterstained with propidium iodide. A minimum number of 700 cells (approximately 100 cells per mice) were visualized microscopically in each group.

RNA extraction and cDNA synthesis

Total RNA of hippocampus tissue was isolated using the RNX-TM plus (CinnaGen Inc., Tehran, Iran). The quantity and purity of extracted RNA were determined using a spectrophotometer (NanoDrop ND-2000, Wilmington, Delaware, USA), and only extracted RNA samples with an A260/A280 ratio ranging from 1.8 to 2.0 were used for cDNA synthesis. Real-time transcription was performed with 1 µg of RNA and a first strand cDNA synthesis kit (Fermentas, Thermo Scientific, Waltham, MA, USA) according to manufacturer’s instructions.

Real-time quantitative PCR using SYBR Green

Real-time PCR was used to evaluate the quantitative expression of mRNA for Bcl-2 and Bax with β-actin as an internal standard. The relative quantification was performed in real-time PCR by measuring increased fluorescence light as a result of SYBR Green bonding using an ABI-7500 real-time PCR system (Applied Biosystems, Foster City, California, USA). Amplification was performed in a final volume of 20 µl, which included 1 µl of cDNA, 10 µl of SYBR Green Master Mix (TaKaRa Bio Inc., Shiga, Japan), 5 pmol of each complimentary primer in a volume of 0.8 µl, and 8.2 µl of deionized water. Sequences of Bcl-2, Bax, and β-actin primers used for real-time PCR are shown in Table 1. The amplification conditions were optimized as follows: predenaturation (95°C for 1 min) followed by 45 cycles of denaturation (95°C for 10 s each), annealing (60°C for 10 s), and extension (72°C for 34 s). Quantitative gene expression was analyzed by comparative cycle threshold (ΔΔCT) method (Livak and Schmittgen, 2001), using β-actin as an internal standard.

Table 1.

Primer sequences used for real-time PCR.

Gene	Primers	Product size	Reference
β-Actin	Forward: 5′-TCA CCA ACT GGG ACG ATA TG-3′ Reverse: 5′-GTT GGC CTT AGG GTT CAG AG-3′	119 bp	Min et al., 2014
Bcl-2	Forward: 5′-TGC GAC CTC TGT TTG ATT TC-3′ Reverse: 5′-GGT ATG CAC CCA GAG TGA TG-3′	90 bp	Takekoshi et al., 2012
Bax	Forward: 5′-TTG CTA CAG GGT TTC ATC CA-3′ Reverse: 5′-GAC ACT CGC TCA GCT TCT TG-3′	112 bp	Chen et al., 2009

PCR: polymerase chain reaction.

Data analysis

The data collected from the experiment were recorded and analyzed using SPSS version 22.0 statistical software package. All nominal data are expressed as mean ± standard deviation. Statistical significance of differences throughout this study was assessed using one-way variance analysis (Tukey’s test). A p-value of less than 0.05 was considered statistically significant.

Results

NP characterization

TEM and DLS were implemented to characterize the Ag-NP solution used in this study. The morphology of the Ag-NPs observed by TEM (Figure 1(a)) confirmed asphericity of their shape. The size distribution of the particles based on TEM is represented in Figure 1(b) and demonstrates an average size of 14.43 ± 5.57 nm. DLS intensity measurements (Figure 1(c)) showed three peaks, of which the first peak at 17.94 ± 5.56 nm is by far the most dominant concentration. Note that DLS intensity is not only dependent on particle concentration but also strongly biased by the particle size (scattering intensity is proportional to the particle diameter to the 6th power). In consequence, the intensity peaks demonstrated in Figure 1(c) do not reflect the true concentrations, rather the % Number shown in the associated table more closely approximates the true concentration of each peak. During our experimental period, no obvious aggregation was found in the stock solution and it stayed homogeneous, ensuring the consistency of the experiments with different incubation times.

NP translocation into the hippocampus

Electron microscopy of the Ag-NP-treated sample revealed NPs both in separated and aggregated forms in the hippocampus. Representative transmission electron micrographs of the hippocampus depicted in Figure 2 show the presence of NPs within the nuclei and cytoplasm.

NP-induced apoptosis

Representative photomicrographs of immunohistochemical staining with TUNEL is shown in Figure 3(a). The ratios of apoptotic cells are 9.1 ± 1.7, 23.9 ± 1.3, 36.7 ± 2.6, and 76.1 ± 2.4 in control, 100 ppm, 200 ppm, and 400 ppm groups, respectively (Figure 3(b)). As shown, Ag-NP induces a statistically significant increase in apoptosis at all investigated doses in a dose-dependent manner.

Altered gene expression

Expression of Bcl-2 and Bax genes was compared with that of β-actin for each sample using real-time quantitative PCR. The negative controls without target cDNA showed no signal increase. Figure 4 shows the effect of Ag-NP at doses 100, 200, and 400 ppm on the expression of Bcl-2 and Bax genes in the rat hippocampus. As illustrated, Ag-NP causes a statistically significant decrease in Bcl-2 mRNA levels at all administrated doses compared to the control group (p < 0.001) in a dose-dependent manner. Compared to the control group, Ag-NP induces a significant increase in expression of Bax gene at doses 200 and 400 ppm.

Discussion

Neurotoxic effects of Ag-NPs have been investigated in multiple studies using particles of various sizes, shapes, and concentrations, with different routes of administration (Ahamed et al., 2010a; Sharma and Sharma, 2012). Despite all efforts, data on the underlying mechanism(s) remain scarce. The purpose of the current study was first to demonstrate the apoptotic effects of Ag-NP on rat hippocampus at certain doses and then evaluate its adverse effects through assessment of the expression of two genes involved in apoptosis. Our results showed that intraperitoneal administration of Ag-NPs at doses 100, 200, and 400 ppm increases hippocampal cell apoptosis, reduces the expression of antiapoptotic Bcl-2 gene, and accentuates the expression of proapoptotic Bax gene, all in a dose-dependent manner.

Nanoscale particles including Ag-NP can enter the brain by disruption of the blood–brain barrier or be taken up directly into the brain by transsynaptic transport (Hoet et al., 2004; Lansdown, 2007). Along with our TEM visualizations, this has been demonstrated for the intraperitoneal route of administration by others (Rahman et al., 2009; Sharma et al., 2009). Studies have shown that Ag-NP has neurotoxic effects on the developing rat hippocampus and rat cerebellar granule cells (Rungby et al., 1987; Yin et al., 2013). It is also regarded as a “sequestered choroid plexus toxicant” (Zheng, 2001). At the cellular level, Ag-NPs have been shown to change neuronal cell morphology, reduce myelin, induce gliosis, and reduce locomotor activity (Sharma and Sharma, 2007; Zhang et al., 2013).

Based on multiple studies on different cell types from various species, possible mechanisms of Ag-NP-related toxicity include stimulation of oxidative stress, genotoxicity, and apoptosis (Kim and Ryu, 2013). Oxidative stress-related neurotoxicity of the Ag-NPs has been shown to be mediated through enhancement of reactive oxygen species (ROS) generation, depletion of antioxidant glutathione levels, disturbances in the calcium homeostasis, and activation of caspase-3 (Yin et al., 2013). Elevated ROS production also induces neurotoxicity in Ag-NP-treated neural tissues by altering the expression of the oxidative stress-related genes (Rahman et al., 2009).

In vitro evaluation of the Ag-NP-treated human and rat neural stem cell cultures has shown attenuated mitochondrial viability and upregulation of Bax protein expression, findings indicative of apoptosis-mediated neurotoxicity of Ag-NPs (Liu et al., 2015). The mitochondrial pathway of apoptosis, also known as intrinsic or Bcl-2 regulated, induces programmed cell death by interactions between three distinct subgroups of the Bcl-2 protein family on the mitochondrial outer membrane: Bh3 (the Bcl-2 homology 3) only proteins (which convey signals to initiate apoptosis), the pro-survival cell guardians such as Bcl-2 itself, and the proapoptotic effector proteins Bax (Bcl-2-associated X protein) and Bak (Bcl-2 antagonist/killer) (Czabotar et al., 2014). When sufficiently stimulated in response to various cytotoxic stresses, Bh3 only proteins neutralize the pro-survival and stimulate the proapoptotic proteins. The proapoptotic proteins Bax and Bak then begin to form the oligomers that permeabilize the mitochondrial outer membrane, which in turn releases apoptogenic factors such as cytochrome c into the cytosol. The cytochrome c released from the mitochondria promotes activation of caspase 9 and eventually effector caspases (caspaces 3, 7, and 6), which are known to play a vital role in both initiation and execution of apoptosis (Czabotar et al., 2014; Li et al., 1997).

In our in vivo study, Ag-NP was demonstrated to decrease the expression of pro-survival Bcl-2 and increase the expression of proapoptotic Bax genes in rat hippocampus, confirming the contribution of the apoptosis cascade in the cytotoxicity associated with Ag-NPs. This is in alignment with prior studies which semi-quantitatively showed modulation of the same genes in cells derived from human liver, hamster kidney, and human colon adenocarcinoma upon treatment with Ag-NP (Gopinath et al., 2010; Piao et al., 2011). Other downstream mediators of the apoptosis pathway such as caspases 9 and 3 have also shown an increase after exposure to Ag-NPs (Ahamed et al., 2010b; Gopinath et al., 2010; Piao et al., 2011; Yin et al., 2013). Although not thoroughly understood, the Ag-NP-related stimulation of the Bcl-2 protein family itself is postulated to occur by upregulation of p53 gene (Gopinath et al., 2010).

Engineered nanomaterials might be contaminated by endotoxins, common contaminants of bacterial origin, and induces significant synergistic toxic and inflammatory reactions. While this synergistic effect has not been observed for Ag-NPs (Herzog et al., 2014), its lack of assessment is a limitation to our study. Furthermore, to ensure availability of adequate hippocampal tissue for optimal performance of the real-time PCR test, we performed the TUNEL staining and gene expression analyses on two different sets of animals. Although both sets experienced similar laboratory conditions, possible different immunity responses in different animals may pose a limitation in our results.

In conclusion, our study showed that intraperitoneal administration of Ag-NPs promoted apoptosis of neural cells through overexpression of proapoptotic Bax and under-expression of antiapoptotic Bcl-2 genes in a dose-dependent manner. Due to the elevation in the Bax/Bcl-2 expression ratio, it is likely that apoptosis induced by Ag-NPs in the rat hippocampus is dependent on the mitochondrial pathway.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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