In Vivo Toxicity of Oral Administrated Nano-SiO2: Can Food Additives Increase Apoptosis?

Nano-silicon dioxide (nano-SiO2) has a great deal of application in food packaging, as antibacterial food additives, and in drug delivery systems but this nanoparticle, despite its wide range of utilizations, can generate destructive effects on organs such as the liver, kidney, and lungs. This study is aimed at investigating the toxicological effects of nano-SiO2 through apoptotic factors. For this purpose, 40 female rats in 4 groups (n = 10) received 300, 600, and 900 mg/kg/day of nano-SiO2 at 20–30 nm size orally for 20 days. Relative expression of Caspase3, Bcl-2, and BAX genes in kidney and liver was evaluated in real time-PCR. The results indicated the overexpression of BAX and Caspase3 genes in the liver and kidney in groups receiving 300 and 900 mg/kg/day of nano-SiO2. Bcl-2 gene was up-regulated in the liver and kidney at 600 mg/kg/day compared to the control group. Overexpression of the Bcl-2 gene in the kidney in 300 and 900 mg/kg/day recipient groups was observed (P ≤ 0.05). Histopathological examination demonstrated 600 mg/kg/day hyperemia in the kidney and lungs. In addition, at 900 mg/kg/day were distinguished scattered necrosis and hyperemia in the liver. The rate of epithelialization in the lungs increased. The nano-SiO2 at 300 and 900 mg/kg/day can induce more cytotoxicity in the liver and lung after oral exposure. However, cytotoxicity of nano-SiO2 at 600 mg/kg/day in the kidney and lung was noticed. Hence, the using of nano-SiO2 as an additive and food packaging should be more considered due to their deleterious effects.


Introduction
Nanoparticles have unrivaled physical and chemical characteristics, which affect their toxicity [1]. The tiny nanoparticles allow them to pass through cell membranes and other biological barriers [1]. Due to this property, these particles enter organisms quickly and at appropriate doses, causing cellular dysfunctions [1,2]. Other characteristics of these particles include a high surface-to-volume ratio, increasing their reactivity and, consequently, their toxicity [3][4][5]. Exposure to these nanomaterials causes harmful effects such as carcinogenesis [6], pulmonary inflammation [7], and apoptosis in the liver [8][9][10]. Silicon has a prominent role in hormonal control, prevention of heart disease [11] and osteoporosis, skin, and hair and nail disorders, atherosclerosis, and the removal of heavy and toxic metals from the brain [12,13]. Its anti-cancer, anti-atherosclerotic, and antidiabetic effects have also been studied [14]. Forms of silicon (including silicic acid or orthosilicic acid) are found in foods rich in fiber and drinking water [15,16]. In the 1980s, the association between adequate dietary silicon intake and the expected growth of animals (chickens and mice) was reported [17]. The organs and tissues with the highest silicon concentrations are connective tissue, bone, liver, skin, heart, muscle, kidneys, and lungs [18,19]. Silicon is an essential agricultural element that enhances plant tolerance to abiotic stresses [20][21][22][23]. Due to the widespread application of nano-SiO 2 in industry and medicine, including their use as additives in drug delivery, cosmetics, varnishes, printer toners, and food [24], this nanoparticle is the third most widely used nanomaterial after carbon and silver [25]. During these years, the possible adverse effects of these nanoparticles on 1 3 human health and the environment have been considered owing to their broad utilization [26]. Currently, little information is available on the potential hazards of nanomaterials on human health and the apoptotic protection properties of nanomaterials and nano-SiO 2 . Some studies illustrated that exposure to nano-SiO 2 arrests the cell cycle in phases S and G2 and disrupts double-strand breaks (DSBs) in DNA [27]. The formation of reactive oxygen species (ROS) can be considered a feasible mechanism for nano-SiO 2 , considering that silica has been demonstrated to cause oxidative and inflammatory responses [28]. Some research reported induction of apoptosis programmed cell death at cell line in vitro [29,30]. Apoptosis is described via depolymerization of the cytoskeleton, cell shrinkage, chromatin condensation, nuclear fragmentation, and phosphatidylserine transport to the cell surface [27,31]. Several genes and proteins, including Bcl-2, Caspase3, and BAX, are involved in the apoptotic pathway [32].
In the present study, we investigated the potentially harmful effects of exposure to nano-SiO 2 by measuring apoptotic markers (BAX, Bcl-2, and Caspase3) in the liver and kidney in all rats given 20 days of dietary intake at doses of 300, 600, and 900 mg/kg/day. Also, we examined histopathological changes in the kidney, liver, and lungs of experimental groups to confirm our findings.

Characterization of Nanoparticles
Nano-SiO 2 was purchased from the Iranian Nanomaterial Pioneers Co.

Animals and Doses
Forty female rats (weight 230 ± 35 g) were caged in 4 groups and acclimated for 1 week for adaptation to a standard humidity, temperature, and light period (12 h light/12 h dark). The rats were given free access to water and a commercial rodent pellet (2400 kcal kg −1 metabolic energy and 10,325 kcal kg −1 digestible energy; crude protein, 19.5%; crude fiber, 10%; phosphor, 0.7%; and calcium, 0.75%) provided from Pars Feed Company, Tehran, Iran. The controls and three experimental groups received normal saline and 300, 600, and 900 mg/kg/day of nano-SiO 2 , respectively. Nano-SiO 2 dissolved in normal saline and administered orally by gavage for 20 days. This study was carried out on female rats according to EU Directive 2010/63/EU for animal experiments. The study also has been approved by the Biomedical Ethics Committee of Tabriz University (Ethics number: IR.TABRIZU.REC.1400.010).

Clinical Observations
Clinical observations including mortality rate, behavioral changes, toxicity signs, food intake, and daily weight gain were evaluated for 20 days.

Sample Preparation
After 20 days of the experiment, the rats were euthanized by chloroform, and 50 mg of the liver, kidneys, and lungs were removed and kept at RNA Later (Thermo Fisher Scientific, Germany) for molecular studies. Histopathological samples were placed in normal saline and in hanks balanced salt solution (HBSS) and washed. The samples were finally placed in a 10% formalin solution for further histopathological studies. In the lab after fixation for 2 weeks, tissue specimens were processed using Autotechnicon (Shando Corporation, USA) with ethanol (descending percentages) and then xylol and impregnated and embedded with paraffin, then tissue blocks were cut into 5-µm sections, and microscopic slides were made of them. Finally, the slides were stained with hematoxylin and eosin.

Real-Time PCR
Relative expression of BAX, BCL-2, and Caspase3 genes was evaluated by SYBR™ Green Real-Time PCR in the triplet. RNA extraction was performed using AccuZol™ solution (Bioneer, Korea) according to the manufacturer's protocol. cDNA was synthesized (YTA, Iran) and applied to amplify the BAX, BCL-2, and Caspase3 genes in SYBR™ Green Master Mix (Yekta Tajhiz Azma Co., Iran) with specific primers (Table 1). Quantitative RNA extraction was performed by UV assay using a spectrophotometer. Realtime PCR reactions for all the amplified genes were set as below programs: holding at 95 °C for 10 min, step 1 (at 95 °C for 15 min), step 2 (60 °C for 30 s), step 3 (72 °C for 30 s), and melting (65-95 °C, wait for 90 s). Steps 1, 2, and 3 (40 cycles) and the melt stage were 1 cycle.

MTT Assay
For the cytotoxic assay, BHK-21 cells were seeded into 96-well tissue culture plates and overnight to 70-80% confluency. For treatment, all nanoparticles were suspended in complete DMEM (10% diluted nanoparticles with 90% DMEM containing PBS) [33], then the bath sonicated for 5 min was done to well mix the final concentrations of required nano-SiO 2 . BHK-21 cells were exposed to different concentrations (300, 600, and 900 mg) of nano-SiO 2 for 24, 48, and 72 h. Then, the viability of the treated cells was tested by MTT assay. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) stock solution was prepared by adding 1 ml PBS to 5 mg MTT (Sigma, USA) and put in a dark place. Then, 20 μl of the prepared stock solution was added to each experimental well (control cells and nano-SiO 2 -treated cells), vibrated for 10 min, and incubated at 37 °C for 3 h. After 3 h, the medium of each well was evacuated, 150 μl dimethyl sulfoxide (DMSO) was added, and the plate was shaken at 100 rpm for 10 min in darkness to dissolve formazan crystals, and a purple color noticeable developed. After going through these steps, the optical density of each well was measured by an ELISA reader (Sunrise RC, Tecan, Switzerland) at 570 nm.

Statistical Analysis
All data were reported as mean ± standard error (mean ± SEM) and determined using Prism 5 software. Oneway ANOVA and post hoc Tukey test were used to compare the difference between groups.

Histopathological Examination of the Nano-SiO 2 on Liver, Kidney, and Lung Tissues
The histopathological study illustrated that the nano-SiO 2 has no observable pathologic effects in the liver, kidney, and lung in the control group (Fig. 2a) and 300 mg/kg/ day groups (Fig. 2b). Histopathological examination of the 600 mg/kg/day group showed hyperemia in kidney and lung sections (Fig. 2c). It was observed that the rate of epithelialization (the number of types II pneumocytes) in the lungs augmented (Fig. 2d). Although the respiratory toxicity of silica is thought to be caused by the inhalation of nano-SiO 2 , our study exhibited oral consumption of nano-SiO 2 can also cause respiratory toxicity. In the 900 mg/kg/day group, scattered hepatocyte necrosis and hyperemia in the liver, severe hyperemia, epithelialization, and proliferation of type 2 pneumocytes in the lung were observed according qualitative description versus control group. Furthermore, hyperemia also was observed in the kidney (Fig. 2d).

Clinical Observation
Rats had no clinical symptoms during the treatment period compared to the controls.

The Effect of Nano-SiO 2 on Cell Cytotoxicity and Cell Viability
The MTT-based colorimetric cytotoxicity test was applied  Fig. 3, the effects of nano-SiO 2 on BHK-21 cells were dependent on dose and time. At doses of 300, 600, and 900 mg of nano-SiO 2 after 24 h and 48 h, it was observed significantly (P ≤ 0.05) that a cell decreased compared to the control group, whereas, after 72 h, the cell viability was not significantly diminished compared to the control group (Fig. 3).

The Impact of Nano-SiO 2 at the Diverse Concentration on Gene Apoptotic Factors
Relative expression of Caspase3, Bax, and Bcl-2 genes was calculated by normalizing all of the samples with the housekeeping gene (GAPDH). BAX gene upregulated significantly in the liver and kidney in groups receiving 300 mg and 900 mg nano-SiO 2 in comparison to the control group (Fig. 4). Caspase3 gene expression in the liver showed the highest expression (P ≤ 0.001) in the 300 mg nano-SiO 2 treatment group and 900 mg nano-SiO 2 recipient group (Fig. 5). Also, in the kidney, it was observed that Caspase3 gene expression was upregulated remarkably (P ≤ 0.001).
The expression of the Bcl-2 gene in the liver showed the highest expression in the 600 mg nano-SiO 2 recipient group (Fig. 6). However, the high level of expression of this gene at 600 mg was not obtained in the kidney (Fig. 5). The results of BAX and Caspase3 gene expression showed that these two genes have low upregulation at 600 mg, the same as the control group. Figure 7 shows relative gene expression of the Bax/Bcl-2 in the liver and kidney of rats. Gene expression of these genes together confirmed the apoptosis process.

Discussion
Although the nanomaterials have numerous convenient applications, they also have the potential to cause adverse effects at the cellular and intracellular levels [1]. Detrimental effects of nanoparticles may be due to their small size, chemical composition, surface charge, surface structure, solubility, shape, and cumulative behavior, which eventually causes different physiological, pathological, and metabolic changes in animals and humans [1,34]. Multiple studies have reported adverse events of nanoparticles such as airway inflammation and allergies, angiogenesis, liver and kidney damage, changes in blood cell counts, DNA damage, decreased keratinocyte viability, prothrombotic effects, and Parkinson's disease [35,36]. Nano-SiO 2 can administer by ingestion, intravenous, and intranasal inhalation and is distributed to the liver, lungs, brain, spleen, and kidneys, causing adverse effects on these organs [37]. In addition, the nanoparticle may stimulate or exacerbate inflammatory reactions by affecting immune-related cell populations in the lung [38].
In this study, we evaluated the rate of apoptotic response based on the received nanoparticle dose by realtime PCR. The Bax gene had the highest expression in Fig. 3 The effect of nano-SiO 2 on cell cytotoxicity and cell viability. At doses of 300, 600, and 900 mg of nano-SiO 2 after 24 h and 48 h, it was observed significantly (P ≤ 0.05) that a cell decreased compared to the control group, whereas, after 72 h, the cell viability was not significantly diminished compared to the control group the liver at 900 dose and the kidney at 300 mg/kg. Bcl-2 gene had the highest expression at 600 mg/kg dose in the liver and 300 mg/kg in the kidney. Caspase3 gene had the highest expression in the liver and kidney at a dose of 900 mg/kg. The previous investigation indicated that the nano-SiO 2 caused cytotoxicity in size, dose, and time-dependent manners [29]. Thibodeau et al.'s research on SiO 2 -induced apoptosis in rat alveolar macrophages reported that the cell exposure to silica contributed to apoptosis by activating Caspase3 and 9 [39]. Also, another study revealed that exposure to the nano-SiO2 can boost DNA damage, cell cycle arrest, proapoptotic factors, and anti-apoptotic factors such as Bax upregulation, Bcl-2 downregulation, caspase 3,7, 9 activity increase, and resulting in apoptosis [40]. The size, surface area, and surface chemistry of specific nanoparticles are thought to be involved in the production of ROS, which increases cell ROS levels to induce apoptosis by inducing inflammatory signaling cascades [41,42]. Thus, it appears that programmed cell death is induced by receptor-ligand and enzyme-substrate interactions, the kinetics.
MTT assay illustrated that after 24 h and 48 h, it was observed significantly on decreasing cells compared to the control group at all tests dosed with nano-SiO2. Exposure to nano-SiO 2 has been shown to cause oxidative stress in mice, but the mechanism by which oxidative Fig. 4 Relative gene expression of the Bax in the liver and kidney of rats which were treated with 300 mg/kg/day, 600 mg/kg/day, and 900 mg/kg/ day nano-SiO 2 in comparison to the control group. The expression level of the Bax gene in both liver and kidney with 300 and 900 mg/kg/day nano-SiO 2 was significantly upregulated (P ≤ 0.001). The error bars show mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05

Fig. 5
Relative gene expression of the Caspase3 in the liver and kidney of rats which were treated with 300 mg/kg/day, 600 mg/kg/day, and 900 mg/kg/ day nano-SiO 2 in comparison to the control group. The expression level of the Caspase3 gene in both liver and kidney with 300 and 900 mg/kg/day nano-SiO 2 was significantly upregulated (P ≤ 0.001) and (P ≤ 0.01), respectively. The error bars show mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05 stress and pro-inflammatory responses are involved in SiO 2 toxicity has not been well studied [43], but nanoparticle SiO 2 is capable of producing intracellular and extracellular ROS [44]. The production of ROS in kidney cells was significantly increased after exposure to nano-SiO 2 , notably at 20 nm [29,[45][46][47][48]. Nanostructured artificial amorphous silica (SAS) is used as a food additive (E551) in the food industry, but its single intravenous dose was between 7 and 17 mg/kg body weight for lethal mice [49,50]. Swensson et al. (1956) observed that after injection of daily doses of 0.25 mg in rabbits, no side effects were observed in the animal, but repeated intravenous injection of 30 mg/animal/day into rabbits caused death within a few days [50]. Daily doses of 5 and 10 mg and weekly doses of 30 mg/animal/week survived in rabbits, but liver fibrosis and renal degeneration were observed, so their study showed that the toxicity of amorphous silica particles increased with increasing particle size (in the range of 100-10 nm) is reduced [50]. The study by Dekkers et al. (2013) shows that the toxicity of SAS is size-dependent and highly toxic after intravenous injection [51]. Some studies have shown a dose-dependent Fig. 6 Relative gene expression of the Bcl-2 in the liver and kidney of rats which were treated with 300 mg/kg/day, 600 mg/ kg/day, and 900 mg/kg/day nano-SiO 2 in comparison to the control group. The expression level of Bcl-2 gene in both liver and kidney with 600 mg/kg/ day nano-SiO 2 was significantly upregulated (P ≤ 0.001) and (P ≤ 0.01), respectively. The error bars show mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05

Fig. 7
Relative gene expression of the Bax/Bcl-2 in the liver and kidney of rats which were treated with 300 mg/kg/day, 600 mg/kg/day, and 900 mg/ kg/day nano-SiO 2 in comparison to the control group. The expression level of Bax/Bcl-2 gene in both liver and kidney with 600 mg/kg/day nano-SiO 2 was significantly upregulated (P ≤ 0.001) and (P ≤ 0.01), respectively. The error bars show mean ± SD. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05 approach to inducing Si-NP airway inflammation [52]. Oxidative stress may be a key route in inducing the cytotoxicity of nanoparticles [53]. In other study, we showed dose-and size-dependent toxicity of nano-sized Zn particle. Toxicity of lower sizes of zinc nanoparticles because of their sizes and higher sizes showed dose dependent toxicity [54]. Our results that align with the previous research confirm that nano-SiO2 cytotoxicity is dependent on the time and dose of the nanoparticle.

Conclusion
Taking all the above-mentioned arguments into consideration, we believe that long-term use of this nanoparticle (high or low dose) may cause very severe effects on the function of the body cells and cause organ failure. However, we evaluated the effects of short-term/high dose of the SiO2. Longterm studies are needed to investigate the effects of using longer time and different dose exposure to SiO 2 .
In addition, we indicated that the entry of this nanoparticle into the body (through the respiratory tract or gastrointestinal tract) has side effects on the body.
Eventually, our recommendation is to try to limit the use of this nanoparticle in the industry (such as in food packaging) and use other alternative materials to decrease toxicity hazards due to exposing with this nanomaterial. The potential of nano-SiO 2 to enter the human body and food chains makes its risk assessment inevitable.
Acknowledgements The authors thank the Vice Chancellor for Research of University of Tabriz for the financial support.

Data Availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Declarations
Ethics Approval This study was carried out on female rats according EU Directive 2010/63/EU for animal experiments. The study also has been approved by Biomedical Ethics Committee of Tabriz University (Ethics number: IR.TABRIZU.REC.1400.010).

Competing Interests
The authors declare no competing interests.