Nowadays nanoparticles (NPs) are widely used due to their distinctive physical properties like enhanced surface and chemical reactivity, more surface area to mass ratio, high cell permeability (Hochella et al. 2008; Oberdörster et al. 2005). SiO2NPs are used for a wide range of applications like in rubber, plastic, adhesives, ceramics industries (Barbe et al. 2004; Rothen-Rutishauser et al. 2006). Moreover, it is also used extensively for biomedical applications including drug delivery, biomedical imaging and gene therapy, because of its ideal properties of resistant to biodegradation and biocompatibility in cellular environments (Hirsch et al. 2003; Moghimi et al. 2005; Slowing et al. 2008). Thus, threat of accidental release of these and other NPs to environment and ultimately to food chain is inevitable (Oberdörster et al. 2005). Documented studies on different organisms and cells suggested that SiO2NPs can have lethal effects on their development and growth (Arnold et al. 2013; Rodea-Palomares et al. 2010). So, wide use of SiO2NPs needs a comprehensive understanding of their potential genotoxic effects on animal and human health.

Allium test is widely used to analyze genotoxicity and cytotoxicity of environmental samples and as well as for chemicals (Gupta et al. 2018; Liman et al. 2013). This test is extensively used due to its many properties like high sensitivity, economic and easy to proceed, less number of chromosomes (2n = 16), and reproducibility of results (Rahman et al. 2017; Gupta et al. 2018, Caritá and Marin-Morales 2008; Chaparro et al. 2010; Liman et al. 2015). It also has been successfully employed as biomarker for nano and micro materials (Ghosh et al. 2011; Kaygisiz and Ciğerci 2017; Liman 2013; Rajeshwari et al. 2016). Comet assay is used to evaluate DNA damage and has been used to find the genotoxicity of nanoparticles due to its reliability, simplicity, low cost, sensitivity and versatility (Ghosh et al. 2015; Cvjetko et al. 2017; Ciğerci et al. 2015; De et al. 2016; Demir et al. 2014; Mangalampalli et al. 2018). Although, studies have been reported on the genotoxicity and cytotoxicity of silicon nanoparticles in human and other mammalian cells, but limited studies have been observed on cytotoxic and genotoxic behavior of SiO2NPs in plant cells. So, current study was designed to explore the cytotoxicity and genotoxicity of SiO2NPs by Comet and Allium ana-telophase tests. Characterization of SiO2NPs was also performed.

Materials and Methods

Silicon dioxide (10–20 nm particle size, CAS Number: 7631-86-9) were purchased from sigma Aldirich. Scanning and Transmission electron microscopic images were taken from Phenom ProX (Phenom-World BV, Eindhoven, Netherlands) and JEM-2100 (JEOL, Tokyo, Japan) with voltage of about 15 KV (SEM) and 200 KV (TEM). Energy Dispersive Xray Spectroscopy (EDX) and Zetasizer (Malvern Nano ZS90) were used to determine the size distribution, elemental analysis and zeta potential of particles.

A. Cepa organic bulbs were obtained from local market. Bulbs of about 25–30 mm were used. Nominal stock concentration of NPs was prepared by 300 mL double distilled water and making it of 500 µg/mL. Inductively coupled plasma-mass spectrometry (Thermo Scientific ICAP RQ ICP-MS,USA) was used to determine SiO2NPs concentration in the stock suspension, which was 115 ± 10 µg/mL in the suspension. Different concentrations (12.5, 25, 50 and 100 µg/mL) of SiO2NPs were prepared and suspended in distilled water. This was followed by sonication for about 30 min on ultrasonic water bath (Bandelin Sonorex Digitec DT100, Germany, 320 W, 35 kHz). The roots on reaching 2–3 cm in length were exposed to arbitrarily selected concentrations of SiO2NPs, distilled water (negative control) and MMS (10 µg/mL, positive control) at room temperature (21 ± 4 °C) for 4 h in the dark. After the exposure time period, ethanol:glacial acetic acid (3:1, v/v) solution was used to fix the root tips (0.7–1 cm) at 4 °C for 24 h. This was followed by washing with distilled water and then stored in 70% alcohol at 4 °C. The root tips were washed with distilled water and hydrolyzed at 60 °C by using 1 N HCl for about 8–10 min. Following this, 20–25 min of staining was done at room temperature by using Feulgen dye. The frequency of CAs (stickiness, distributed anaphase–telophase, chromosome laggards and anaphase bridge) and MI were measured as demonstrated by (Saxena et al. 2005) with minor modifications. It was done by using Nikon Eclipse Ci-L light microscope (Japan) that was equipped with a CMOS camera (Argenit, Kameram, Turkey). As per treatment, five slides were randomly taken and counted for scores. By using the given equation, CAs and MI were calculated .

MI = (total number of dividing cells/total cell numbers) × 100.

CA = (total number of abnormal cells/100 anaphase–telophase cells)× 100.

Comet assay was performed as demonstrated by (Tice et al. 2000) with slight modifications. Same concentrations (12.5, 25, 50 and 100 µg/mL) of NPs were used to expose the mersistem cells of A. cepa root. Ice cold Tris–MgCl2 nuclei isolation buffer (4 mM MgCl2·6H2O; 0.5% w/v Triton X-100, 0.2 M Tris, pH 7.5) was used to isolate nuclei from ten seedlings. Following this, 50 µL of cells were taken and mixed with 50 µL of LMPA (1.5% prepared) and poured over the slides that were pre coated with NMPA (1% prepared). After that, lysis was done in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for about 20 min at 4 °C. After lysis, slides were placed in electrophoresis tank and gel was run at 300 mA and 25V for 20 min at 4 °C. Neutralization was carried out by the 0.4 M Tris (pH 7.5) and stained with 70 µL EtBr solution (20 µg/mL). Fifty comets per slide were scored as from 0-undamaged to 4-complete damage, using fluorescence microscope (BAB, TAM-F, Turkey) equipped with a CCD camera (BAB, TC-5, Turkey). The analysis of arbitrary unit (AU) values of each treatment were calculated according to (Ciğerci et al. 2015).

Results and Discussion

Scanning electron microscope (SEM) was used to analyze the adsorption of SiO2NPs by the root surface. Figure 1a shows the roots from the samples that were lacking nanoparticle organization while Fig. 1b shows the roots that has adsorbed SiO2NPs at the surface. Figure 1d reveals the results of electron dispersive X-ray (EDX) studies which made it clear that detected particles were having silicon. By using TEM, physiochemical properties of SiO2NPs were recorded and presented in a Table 1. About 100 particles were randomly measured to calculate the mean size of SiO2NPs particles using TEM. Figures 1 and 2 shows size measurements of SiO2NPs that was obtained as 16.12 ± 3.07 nm. The average diameter of SiO2NPs was obtained in the range of 404.66 ± 93.39 nm by using water suspension with ultrahigh purity. On the other hand, pdI value of SiO2NPs was also recorded that was 0.513. Table 1 also shows the electrophoretic mobility of SiO2NPs particles and zeta potential (ζ) in ultrahigh purity water. Henry’s equation was used to calculate electrophoretic mobility (Baalousha et al. 2012).

Fig. 1
figure 1

SEM images of surface adhesion of SiO2NP son Allium root; a and c control roots showing the absence of particle adhesion to the surface and EDX analysis of SiO2NPs, b and d showing SiO2NPs adhered to the root surface and EDX analysis of SiO2NPs

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Table 1 Characterization of silicon dioxide nanoparticles (SiO2NPs)
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Fig. 2
figure 2

Transmission electron microscopy (TEM) images of silicon dioxide nanoparticles (SiO2NPs)

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The effects of SiO2NPs on MI and mitotic phases in the root tips of A. cepa after 4 h are shown in Table 2. Concentration dependent increase in MI was observed by the all concentrations of SiO2NPs but these were lower than the negative control group. Negative control showed the highest MI value (71.96 ± 1.16) while least (43.25 ± 0.52) was observed by the positive control group. There was an increase in the mitotic phases after the exposure of SiO2NPs, compared to the negative control group, except for the prophase at all concentrations and telophase at 25 µg/mL. Significant decrease of MI at lower concentrations may be due to inhibition of DNA synthesis (Sudhakar et al. 2001) or blocking of G2 phase, preventing the cell from entering mitosis (EI-Ghamery et al. 2000) or mitotic inhibition of chemical(s) (Sharma and Vig 2012) or blockage of specific cell cycle proteins which further stop DNA polymerase and other enzymes leading to antimitotic effect (Hidalgo et al. 1989; Türkoğlu 2015). Dose dependent increase in MI by exposure of SiO2NPs demonstrated that higher concentrations of SiO2NPs are less phytotoxic. It could be due that SiO2NPs appears to block mitosis at lower doses by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules (Jordan et al. 1993). SiO2NPs also have been used to check the cytotoxic effects of these on different cancerous cell lines (Gong et al. 2012; Lin et al. 2006; Wagner et al. 2009). (Park et al. 2009) also demonstrated the toxic effects of silica NPs in the embryonic stem cell even below the cytotoxic doses. Previously, it has been reported that silica NPs can easily agglomerate and influences the uptake of NPs into the cell (Lin and Haynes 2010; Napierska et al. 2010). Demir et al. (2015) also demonstrated that at highest concentration (100 µg/mL) of SiO2NPs agglomerated in Drosophila melanogaster cells which further decreases the penetration of NPs in to the cells. So, less amount of NPs might be eliminated by the own defence mechanism of plant cells (Demir et al. 2015). In our current study, lowest phytotoxicity of SiO2NPs at highest concentration could be suggestive of this mechanism. However, further studies on the cellular defense mechanism at molecular level after SiO2NPs exposure should be carried out.

Table 2 The effects of SiO2NPs on mitotic index and mitotic phases in the root tips of A. cepa after 4 h
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CAs (chromosome laggards, disturbed anaphase–telophase, stickness and anaphase bridge) induced by SiO2NPs in A. cepa root meristematic cells are shown in Table 3 and Fig. 3. It was observed that SiO2NPs induced significant CAs in A. cepa root anaphase–telophase cells compared to the control group. There was a concentration dependent increase in the total CAs by the SiO2NPs (r = 0.967, p = 0.01). Highest total anomalies (18.80 ± 0.45) were observed at 100 µg/mL, whereas least (11.2 ± 0.84) were observed at the 12.5 µg/mL dose. No significant difference was found between the total anomalies, at the highest concentration of studied NPs and the positive control group (p > 0.05). The highest frequency of disturbed anaphase–telophase was observed at all concentrations of SiO2NPs (except at 12.5 µg/mL) and stickiness at 50 and 100 µg/mL compared to the positive control group. The A. cepa cytotoxic test has been used to monitor the genotoxicity of NPs (Kaygisiz and Ciğerci 2017; Kumari et al. 2011; Liman 2013). Previously, a cell proliferation assay showed nontoxic effects at low dosages, but cell viability decreases at high dosages by the composite silica NPs on normal fibroblast and tumor cells (Chang et al. 2007). No toxic effects have been observed by the SiO2NPs to human mesothelioma cells and mouse embryonic fibroblast cells (Brunner et al. 2006). Amorphous fumed nano-silica (14 nm) showed the cytotoxicity in the human colon epithelial cell-line, when exposed for up to 24 h (Gerloff et al. 2009). Various in vitro studies of Silica NPs had showed the cytotoxic effects and oxidative stress in various model systems (Lin et al. 2006; Mu et al. 2012; Napierska et al. 2010; Sayes et al. 2007). It was demonstrated in human peripheral blood lymphocytes and HEK293 cells that different sizes of silica NPs have potential to interact with DNA and that can lead to primary DNA damage in these cells (Demir et al. 2013; Kaewamatawong et al. 2006; Kim et al. 2010). Whereas, in another study, no significant chromosome breakage and chromosome loss were observed in A549 human lung carcinoma cells (Gonzalez et al. 2010).

Table 3 Chromosomal anomalies induced by SiO2NPs in A. cepa root meristematic cells
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Fig. 3
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Anaphase–telophase anomalies in root tips of A. cepa induced by (SiO2NPs). a Disturbed anaphase–telophase, b chromosome laggards, c stickiness, d anaphase bridge

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In the current study, different chromosomal anomalies were observed like disturbed anaphase–telophase (Fig. 3a) and chromosome laggards (Fig. 3b). These could be due to the disturbed microtubules or failure of the chromosome to move toward the poles or deformation of spindle structure (Rajeshwari et al. 2016; Singh and Roy 2017). Anaphase bridge (Fig. 3d) formation could be due to chromosomes fusion or due to dicentric chromosome or changing activation of replication enzymes (EI-Ghamery et al. 2000). Increased chromosome aggregation, extra chromosomal intertwining of the chromatin fibers or depolymerization of DNA can cause the stickiness (Fig. 3c) of chromosomes (El-Ghamery and Mousa 2017; Türkoğlu 2015).

The results of DNA damage shown by the exposure of SiO2NPs in nuclei of A. cepa root meristems are presented in Table 4. The significant DNA damage was induced by the all concentrations of SiO2NPs compared to control group. The increase DNA damage showed a direct dose-response relationship (r = 0.974, p = 0.01). The highest DNA damage was observed by the positive control (153.33 ± 1.53) followed by the 100 µg/mL (149.67 ± 1.15) dose of SiO2NPs. While the least was observed by the negative control (2.67 ± 0.58) followed by the 12.5 µg/mL (122 ± 1.73) concentration.

Table 4 Detection of DNA damage in nuclei of A. cepa root meristems exposure to SiO2NPs using the Comet assay.
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The literature states that SiO2NPs can induce oxidative stress and formation of 8-OH-dG in a dose dependent manner. Which consequently results in the oxidative stress induced DNA damage (Gong et al. 2012). It is suggested that NPs induced DNA damage can be caused by the reactive oxygen species (ROS). Another suggestive mechanism of NPs mediated DNA damage is the creation of oxidants/genotoxic compounds by stimulating the target cells (Nel et al. 2006). In present study, the genotoxic effects of SiO2NPs in the A. cape cells could be due the ROS production. Different in vitro studies demonstrated size- and dose dependent cytotoxicity, enhanced production of ROS and pro-inflammatory stimulation by the nano-silica particles (Chen and von Mikecz 2005; Eom and Choi 2009). Different sizes of Silica NPs also showed the oxidative DNA damage in Drosophila haemocytes by the comet assay and Drosophila wing somatic mutation and recombination test (Demir et al. 2015).

Abnormal clusters of topoisomerase I (topo I) in the nucleoplasm, fibrogenesis and pro-inflammatory stimulation of endothelial cells was observed by the nano-silica in the Wistar rats (Chen and von Mikecz 2005; Chen et al. 2004). However, in another investigation, no toxic effects were shown by silicon NPs on mouse (Xue et al. 2006). Similarly, SiO2NPs did not cause the DNA damage in Daphnia magna and Chironomus riparius but caused mortality of these both species (Lee et al. 2009). In cultured mammalian cells, nano-silica also indicated the primary genotoxic and cytotoxic effects but not mutagenicity (Choi et al. 2011). Amorphous silica NPs (20–240 nm) showed no genotoxic effects on A549 human lung epithelial carcinoma cells and 3T3-L1 fibroblasts by alkaline comet assay (Chen et al. 2004; Park et al. 2009). Therefore, further studies are still needed to evaluate the genotoxic effects and mechanisms of silica NPs in different organisms along with different test systems.

It was revealed by the current study that SiO2NPs cause the cytotoxic and genotoxic effects on the root meristem cells of A. cepa.