Genetic effects of chemically and biosynthesized titanium dioxide nanoparticles in vitro and in vivo of female rats and their fetuses

With the increase in nanoparticles (NPs) products on the market, the possibility of animal and human exposure to these materials will increase. The smaller size of NPs facilitates their entrance through placental barriers and allows them to accumulate in embryonic tissue, where they can then be a source of different developmental malformations. Several toxicity studies with chemically synthesized titanium dioxide NPs (CTiO₂ NPs) have been recently carried out; although there is insufficient data on exposure to biosynthesized titanium dioxide NPs (BTiO₂ NPs) during pregnancy, the study aimed to evaluate the ability of an eco-friendly biosynthesis technique using garlic extract against maternal and fetal genotoxicities, which could result from repeated exposure to TiO₂ NPs during gestation days (GD) 6–19. A total of fifty pregnant rats were divided into five groups (n = 10) and gavaged CTiO₂ NPs and BTiO₂ NPs at 100 and 300 mg/kg/day concentrations. Pregnant rats on GD 20 were anesthetized, uterine horns were removed, and then embryotoxicity was performed. The kidneys of the mothers and fetuses in each group were collected and then maintained in a frozen condition. Our results showed that garlic extract can be used as a reducing agent for the formation of TiO₂ NPs. Moreover, BTiO₂ NPs showed less toxic potential than CTiO₂ NPs in HepG₂ cells. Both chemically and biosynthesized TiO₂ NP-induced genetic variation in the 16S rRNA sequences of mother groups compared to the control group. In conclusion, the genetic effects of the 16S rRNA sequence induced by chemically synthesized TiO₂ NPs were greater than those of biosynthesized TiO₂ NPs. However, there were no differences between the control group and the embryo-treated groups with chemically and biologically synthesized TiO₂ NPs.

GRAPHICAL ABSTRACT

Graphical Abstract. Schematic carton of the study showed the experimental strategy. It was designed by BioRender.com.

Introduction

In the coming decades, industrial production is expected to change as a result of the rapidly expanding field of nanotechnology. The high surface area and smaller particle size of NPs, along with their flexible design, functionalization, biocompatibility, and bioactivity, played a key role in tuning NPs in many applications. The major application is breaking the barriers among fundamental fields such as biology, chemistry, and physics. The application field is also expanding, including medical products, imaging techniques, sporting equipment, food production and agriculture, cosmetics, clothing cleaning products, personal care items, and toys for kids (1, 2). However, most nanomaterials have been introduced into the market based on claimed advantages, and the ecotoxicological potential of nanomaterial products is unclear to the scientific community (2, 3). The nanomaterials' physicochemical properties are due to their chemical nature, small size, surface composition, and aggregation (4).

For NP synthesis, there are two main approaches: the top-down method and the bottom-up method (5). The top-down approach starts with size reduction from the bulk material to the nanosized with physical methods such as ball milling and laser ablation (6), while the bottom-up approach needs a reducing agent to decrease the size of the particle. Chemical (for example, sodium hydroxide and potassium hydroxide) and biological [biomolecules from bacteria (6), fungi (7), yeast (8), virus (9), and plant extracts (10)] reducing agents can work perfectly for controlling the size of metals and metal oxides. Biomolecules are preferred due to their safety and biocompatibility with DNA, proteins, enzymes, polyphenols, flavonoids, and sugars (11). The simplest and quickest way to create NPs, which are based on the proteins and carbohydrates in biomolecules and act as a reducing agent to encourage the synthesis of metallic nanoparticles, is through plant extracts (12). Biomolecules have many chemically active groups such as hydroxyl, amine, and thiol groups (13). These groups will interact with metal ions in the solution via electron transfer, leading to oxidization from a positive oxidization state to a zero oxidization state, which guides the nucleation process.

Comparing biosynthesized NPs with those made using chemical methods, the biocompatibility of these NPs allows for a wide range of biomedical applications (14).

Exposure to TiO₂ NPs may change the cell membrane due to oxidative stress or even via Van der Waal forces with the cell wall, leading to defragmentation of the cell membrane and molecular structure, which induces genetic variation (15, 16). The generated reactive oxygen species (ROS) also oxidize DNA, resulting in mutations in DNA (15, 17). In addition, reactive oxygen species may promote inflammation, and oxidative stress and inflammation result in cell apoptosis (16, 18).

Recently, several toxicity studies with chemically synthesized titanium dioxide nanoparticles (CTiO₂ NPs) have been performed. Organs such as the kidney, liver, and spleen can suffer damage due to exposure to TiO₂ NPs (19–21), such as inflammation of renal tissue due to ROS. In addition, TiO₂ NPs accumulate in the kidney tissues and cause changes in embryogenesis during the first trimester of pregnancy (22, 23). In addition, oxidative stress significantly increases with increasing TiO₂ NP concentrations (24).

A repeated oral administration of CTiO₂ NPs in other experimental animals showed disturbances in metabolism (25). The results of the studies performed on rats as animal models revealed that after absorption of CTiO₂ NPs can enter the systemic circulation and cause organ injuries and inflammation (26).

Several in vivo and in vitro studies were carried out to assess the toxicity of green TiO₂ NPs. The reported toxicological data proposed that green TiO₂ promotes a higher safety profile with improved anticancer, antibacterial, and antiviral activities compared with chemically synthesized TiO₂ NPs (27). However, there are limited toxicological data on exposure to biosynthesized TiO₂ NPs from garlic extract (BTiO₂ NPs) during pregnancy. This study is based on the new method of biosynthesis of TiO₂ NPs by using garlic extract as a reducing agent, performing in vivo investigations on pregnant rats and their fetuses, and comparing the toxicity of BTiO₂ NP synthesis with that of CTiO₂ NP synthesis in both in vivo and in vitro.

Materials and methods

Chemicals and reagents

The following chemicals and reagents were used without any purification: titanium isopropoxide (TTIP), Dulbecco's modified Eagle's medium (DMEM), and penicillin–streptomycin (P/S) were purchased from Sigma–Aldrich Co., USA. Fetal bovine serum (FBS) was purchased from Biochrom, and 0.05% Trypsin-EDTA was purchased from HiMedia, India. Phosphate-buffered saline (PBS) was purchased from Serox, Germany, and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium and DMSO were purchased from Lobalohemia, India. Qiagen DNA mini kit and 100 bp DNA Ladder were purchased from GeneDireX (Germany). Liver hepatocellular cells (HepG₂ cell lines) were purchased from VACSERA, Giza, Egypt.

Synthesis of chemical TiO₂ NPs (CTiO₂ NPs)

TiO₂ NPs were created chemically using the coprecipitation method (28). In brief, 5 ml of titanium isopropoxide (TTIP) in 15 ml of propanol was used as a precursor solution, while the solvent solution was a 50/1 (V/V) mixture of distilled water and propanol. The precursor solution was added dropwise to the solvent solution after it had been heated to 70–90°C under continuous stirring for 2 h. As soon as the TiO₂ precursor was reduced to form TiO₂ NPs, a white precipitate started to form. The precipitate was centrifuged and allowed to cool at room temperature for the entire next day. The final precipitate was, then, dried at 100°C for 12 h and calcined at 400°C for 3 h after being washed three times with distilled water and once with ethanol.

Biosynthesis of TiO₂ NPs using garlic extract (BTiO₂ NPs)

Garlic (Allium sativum) water extract was used as a reducing agent for the biosynthesis of TiO₂ NPs. Approximately 20 g of washed and dried garlic in 150 ml of distilled water were boiled for 1 h to prepare the solvent solution. The precursor solution was prepared by adding 10 ml of TTIP to 150 ml of distilled water and vigorously stirring. Dropwise addition of a solvent solution (60 ml) of fresh garlic plant extract was made, while the precursor solution was continuously stirred for 2 h. The color of the solution changed from white to dark yellow, indicating that TTIP was reduced and BTiO₂ NPs were created. The formed precipitate was centrifuged and collected after being allowed to cool at room temperature for the entire night. The final precipitate was, then, dried at 100°C for 12 h and calcined at 400°C for 3 h after being washed three times with distilled water and once with ethanol.

TiO₂ NP characterizations

The produced TiO₂ NPs were categorized by X-ray diffraction (XRD) using X' Pert PRO-PAN, Malvern Panalytical, UK, diffractometer with cooper radiation (wavelength 1.54056 Å) at 40 kV and 30mA, high-resolution transmission electron microscopy (HETM) (JEOL, JEM 2100, Japan), Raman spectrometer (Horiba Jobin Yvon HR 800UV, Japan), and FTIR spectrophotometer (Model 6100, Jasco-Japan), with a resolution of 4.00 cm⁻¹and covers the wave number range of 4000–400 cm⁻¹ was used to determine the functional groups in the prepared samples. The optical absorption spectra of the prepared samples were evaluated with a UV–visible spectrophotometer (SPECORD 200 PLUS, Analytik Jena, Germany).

Cell viability

Liver hepatocellular cells (HepG₂ cell lines) were obtained from Vacsera (Giza, Egypt). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma–Aldrich, #D5796), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biochrom, #S0615) and 1% penicillin–streptomycin (P/S, Sigma–Aldrich, #P4333). Cells were grown in 75 cm² flasks (VACSERA Center, Cairo) and sub-cultured at approximately 80% confluency.

MTT assay was used to assess the mitochondrial function by reducing the tetrazolium dye MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium to the insoluble magenta formazan where a yellow tetrazolium is reduced to purple formazan in living cells (29). HepG₂ cells were seeded in 96-well culture plates with a total density of 1 × 10⁴ cells per well and incubated at 37°C and 5% CO₂. After 24 h of incubation, the culture medium was removed, and cells were rinsed with 100 μl PBS. Afterward, cells were exposed to both samples of TiO₂ NPs with different concentrations (0, 0.5, 1, 2, 4, and 8 mM) and then incubated at 37°C and 5% CO₂. After 24 h, cells were rinsed with PBS, and 80 μl of media without serum and 20 μl of MTT solution were added to each well and then incubated at 37°C for 3 h. Finally, the MTT solution was removed, then 100 μl of MTT solvent (DMSO) was added to each well, and the plates were warped in foil and shaken on an orbital shaker for 15 min. The absorbance at OD = 590 nm was recorded for each well with a Tecan infinite F50 absorbance microplate reader, and the cell viability was calculated using the following equation (29).

$\text{Cell viability}\%\text{ =}\frac{{\text{Absorbance}}_{\text{control}}{-\text{Absorbance}}_{\text{sample}}}{{\text{Absorbance}}_{\text{control}}}$

Animal experiment

In total, 20–30-week-old healthy female Albino Sprague–Dawley rats, weighing 180–220 g, were obtained from the animal house of the National Research Center Institute (Cairo, Egypt). The rats were housed in cages made of plastic, using a bedding material made of a wooden dust-free litter, and permitted a 2-week period of acclimation before the beginning of the study under hygienic measures and standard conditions (25 ± 2°C room temperature, 50% ± 15% relative humidity, and light–dark cycle of 12 h). All rats were given a commercial pellet diet and water ad libitum.

Experimental design and treatment

The study aimed to assess the ameliorator abilities of an eco-friendly biosynthesizing method using garlic (Allium sativum) extract as a reducing agent against maternal and fetal genotoxicities that could develop from repeated oral administration of TiO₂ NPs in pregnant mothers between gestation days 6 and 19. In total, 50 adult female rats were selected for this study and mated at night using healthy males in a ratio of 1:3. The day that sperm was observed in the vaginal smear, the next morning was regarded as GD1. After pregnancy detection, pregnant females were weighed and assigned into five equal groups, each of which comprises 10 pregnant mothers, as follows:

Group 1: control group, administrated distilled water by gavage (1 ml/day).

Group 2 and Group 3: CTiO₂ NP-treated groups received 100 and 300 mg/kg body weight (bw)/day, (30, 31) respectively.

Group 4 and Group 5: BTiO₂ NP-treated groups received 100 and 300 mg/kg bw/day, respectively. These concentrations of TiO₂ NPs of 100 and 300 mg/kg bw were chosen according to the World Health Organization (WHO) in 1969 (32), which reported that the LD₅₀ of TiO₂ for rats is greater than 12,000 mg/kg bw. Pregnant mothers were exposed to 100 mg/kg bw CTiO₂ NPs or BTiO₂ NPs, which is equal to approximately 6–7 g of TiO₂ NPs per 60–70 kg bw for humans (33).

TiO₂ NPs were suspended in distilled water. Throughout the dosing process, the dosage was continuously shaken to produce a homogenized suspension, and throughout the dosing procedure, the dosage was continuously stirred with a magnetic stirrer. All treatments were obtained by oral gavage and were freshly prepared. The pregnant rats' treatment starts from GD 6 to GD 19 (31). The schematic diagram of the study showed the experimental strategy (see Graphical abstract).

At the end of the experimental period on GD 20, all dams were euthanized by ether, and cesarean sections were performed. The uterine horns were removed, and then, the embryotoxicity was performed. The kidneys of mothers and fetuses in each group were collected and then maintained in a frozen condition.

DNA extraction, PCR amplification, and sequencing

Qiagen DNA mini kit was used to extract the genomic DNA from the kidneys of mothers and fetuses in each group by following the manufacturer's guidelines. The quality of DNA was examined by agarose gel electrophoresis with a 100bp DNA Ladder under UV light. The previous report stated that primers were used for 16S rRNA amplification (34). The polymerase chain reaction was executed in a final reaction volume of 40 μl, adding 20 μl of 2X Master Mix, 1 μl of each forward and reverse primer, 17 μl of nuclease-free water, and 1 μl genomic DNA. The PCR conditions were as follows: denaturing at 95°C for 4 min, then 35 cycles of denaturing at 94°C for 60 s, annealing at 48°C for 60 s, extension at 72°C for 60 s, and finishing with an extension at 72°C for 7 min. Amplification was confirmed by means of agarose gel electrophoresis at 1.5% containing ethidium bromide. The DNA sequencing was executed by Macrogen (South Korea). Sequence alignment was implemented using Clustal W (35).

Biopsy of fetal kidney

Fetal kidney biopsies from all embryos were extracted and thoroughly dipped in formalin solution to be fixative, followed by serial dehydration in ethanol, and embedding in paraffin wax. Kidney sections of approximately 5 microns were stained with hematoxylin and eosin (H&E) for histological findings (36).

Results

X-ray diffraction

The biosynthesis of NPs is an emerging technique that produces NPs with unique properties. Green nanotechnology was applied to the synthesis of TiO₂ NPs, and the formed NPs were characterized by several techniques. Structural analysis using XRD, HRTEM, and Raman spectroscopy revealed anatase phase formation, which indicates the complete reduction of titanium isopropoxide using garlic extract. No additional peaks for impurities, such as NaCl and Na₂TiO₃, were observed, indicating the high purity of the prepared samples. The X-ray diffraction (XRD) pattern of TiO₂ NPs (see Figure 1) revealed the phase and structural purity of TiO₂ NPs. All the recorded peaks belong to the anatase phase TiO₂ NPs, which support the reported card (JCPDS No. 21-1272). Although the diffraction peak of brookite B (121) was found in CTiO₂ NPs, it disappeared in BTiO₂ NPs (37).

FIGURE 1

Figure 1. XRD patterns of CTiO₂ and BTiO₂ NPs.

Debye–Scherer's equation was used for the calculation of the average crystal size of TiO₂ NPs (38).

$\begin{array}{l}d=\frac{0.89\lambda }{FWHMCos\theta }\end{array}$

where d is the average crystal size of TiO₂ NPs, λ is the wavelength of X-rays, 0.89 is a constant, θ is the diffraction angle, and FWHM is the full width at half maximum of XRD peaks recorded at diffraction angle 2θ. The calculated average crystalline size of CTiO₂ NPs was 48.11 nm, while it was increased to 53.31 nm for BTiO₂ NPs.

UV–visible spectroscopy

Both CTiO₂ and BTiO₂ samples have absorption spectra in the region below 400 nm. The spectral image displays the absorption peaks of TiO₂ NPs at wavelengths of 261.87 and 314 nm for CTiO₂ and BTiO₂ NPs, respectively (see Figure 2).

FIGURE 2

Figure 2. UV spectra of BTiO₂ and CTiO₂ NPs.

The optical band gap Eg of TiO₂ NPs was calculated by Tauc's equation (39) as follows:

$\begin{array}{l}\propto (hv)={A(hv-E_g)}^{\frac{m}{2}}\end{array}$

where ∝ is the absorption coefficient of TiO₂ NPs, the energy of incident light of wavelength λ was hv = hc/λ, (A) was constant, and (m) depends on the nature of the transition.

The direct band gaps of CTiO₂ and BTiO₂ NPs were 3.544ev and 2.660ev, respectively (see Figure 3).

FIGURE 3

Figure 3. Optical band gap of CTiO₂ and BTiO₂ NPs.

Raman spectra analysis of tio₂ NPs

The structural characterization of chemically and biosynthesized TiO₂ NPs with garlic extract by Raman spectroscopy is presented in Figure 3. The six active modes that belong to the anatase phase of TiO₂ were found in the two samples, which confirm the XRD result of the formation of anatase TiO₂ NPs. The low frequency O-Ti-O bending oscillation was observed for all samples as E_g(1), E_g(2), and B_1g modes (40). All sample frequency positions are higher than the reported values for bulk TiO₂ (143, 197, and 399 cm⁻¹, respectively). Moreover, higher frequency Ti-O strain oscillation was observed for all samples in E_g(3) and A_1g modes. The frequency position of E_g(3) has a lower frequency position than that of the bulk TiO₂ (639 cm⁻¹), while the frequency position of A_1g has a higher frequency position than that of the bulk TiO₂ (514 cm⁻¹). This result is attributed to a reduction in the crystal size of TiO₂ NPs that are produced chemically and biologically (41).

Fourier-transform infrared spectra

FTIR spectrum for CTiO₂ NPs is presented in Figure 4. The broad band at 3426.2 cm⁻¹ is assigned to the vibration of the hydroxyl group (42). The vibration band at 1151cm⁻¹ is due to Ti-O-Ti vibrations. The band at 1628 cm⁻¹ is described as the bending vibration of adsorbed water and Ti-OH (43). A band at 1376 cm⁻¹ is attributed to the asymmetrical C–H vibrations (43, 44). The band at 457.6 cm⁻¹ corresponds to the Ti–O –Ti. Vibrational bands appearing around 723 cm⁻¹ correspond to TiO₂ modes in the anatase phase (45–47).

FIGURE 4

Figure 4. Raman spectra of CTiO₂ and BTiO₂ NPs.

FTIR spectrum of garlic extract was studied to identify the presence of garlic function groups. The band at 3415.5cm⁻¹ describes the vibration of the hydroxyl group (O-H) and the presence of carbohydrates and amino acids. The bands at 2932 cm⁻¹ and 2890.2 cm⁻¹ represent the C-H stretching for lipids (48). Band 1631 cm⁻¹ represents the Amid I: C=O stretching for proteins. Band 1412.5cm⁻¹ represents the CH₂ vibration for lipids. The band at 1378.8 cm⁻¹ represents the C=S stretching for sulfur compounds. The band at 1274.4 cm⁻¹ represents the C-N stretching for amino acids. The band at 1120.5 cm⁻¹ represents the C-N stretching for amino acids, symmetric C-H stretching, and the vibration presence of antioxidant enzymes. The band at 1025.6 cm⁻¹ represents SO₃ symmetric stretching, mainly sulfur compounds. Bands at 931.2 cm⁻¹ and 817.5 cm⁻¹ represent the N-H stretching of proteins (49).

FTIR spectroscopy indicated the successful formation of TiO₂ NPs and the generation of the functional groups in garlic extract that are responsible for the reduction of Ti precursors. The FTIR spectrum of TiO₂ NPs is characterized by three broad bands, the first of which, between ~3800 and 3000 cm⁻¹, belongs to the stretching vibration of the hydroxyl group (O-H). The second band is around 1626 and 1638 cm¹, which belongs to the stretching of titanium. The third band, located approximately between 800 and 450 cm⁻¹, was the fingerprint of Ti-O stretching vibration. These bands were observed in all samples, which confirms the formation of TiO₂ NPs (50).

The biosynthesis of TiO₂ NPs using garlic extract was confirmed by the occurrence of functional groups in both TiO₂ and garlic. The functional groups of garlic at 1631 cm⁻¹, 1378.8 cm⁻¹, and 1120.5 cm⁻¹ were shifted to the lower wave numbers such as 1623.6 cm⁻¹, 1377 cm⁻¹, and 1117.4 cm⁻¹, respectively. This reflects the presence of C=O, C=S, and C-N and symmetric C-H stretching vibrations of antioxidant enzymes in the formation of TiO₂-garlic NPs. The band at 417.7 cm⁻¹ corresponds to the Ti–O –Ti. A vibrational band appeared around 716.8 cm⁻¹ due to TiO₂ modes in the anatase phase (51). The bands assigned for C-H stretching, CH₂ vibration, and symmetric C-H stretching disappeared in biosynthesized TiO₂NPs. In addition, bands corresponding to C=O, C=S, and C-N appeared in biosynthetic TiO₂ NPs. Consequently, C=O, C=S, and C-N functional groups are the cause of the bioreduction of titanium isopropoxide to TiO₂NPs.

High-resolution transmission electron microscope

HRTEM images of CTiO₂ and BTiO₂ NPs reflect a sort of agglomeration with a spherical or irregular spherical shape (see Figure 5). Particle size distribution of CTiO₂ and BTiO₂ NPs (see Figure 5) is estimated from HRTEM images. HRTEM images of the prepared samples showed a sort of agglomeration with an increase in the particle size of BTiO₂ NPs compared to CTiO₂ NPs, which was attributed to the presence of the biomolecules as they attracted other molecules due to the electrostatic force on their surfaces. Similar results were also obtained in the previous report (52, 53).

FIGURE 5

Figure 5. FTIR spectra of garlic extract and BTiO₂ and CTiO₂ NPs.

The average size is 18.46 ± 3.03 and 35.64 ± 4.9 nm for CTiO₂ and BTiO₂ NPs, respectively. These values are smaller than the calculated values from XRD results (48.19 and 53.317) for CTiO₂ and BTiO₂ NPs, respectively. This variation in size may be due to the agglomeration of TiO₂ NPs (51). The crystallite size calculated from Scherer's equation is the apparent size, which does not equal to the particle size, especially in the case of polydisperse NPs with aggregation, such as TiO₂ NPs.

Cytotoxicity

An accumulation of both TiO₂ NPs on the surface of the HepG₂ cells was found in the microscopic images (see Figure 6). Particularly, in the case of CTiO₂ NPs, this accumulation increased as NP concentration increased. The normalized cell viability is presented in Figure 7, and there is no observed toxicity for CTiO₂ and BTiO₂ NPs at low concentrations, while a small percentage of toxicity was observed for CTiO₂ at high concentrations (8 mM), as shown in Figure 8.

FIGURE 6

Figure 6. HRTEM images and particle size distribution of BTiO₂ and CTiO₂ NPs.

FIGURE 7

Figure 7. Microscopic images of HepG₂ cells after 24 h of exposure to BTiO₂ and CTiO₂ NPs at two concentrations (0.5 and 8 mM).

FIGURE 8

Figure 8. Cell viability of HepG₂ cells after 24 h of exposure to BTiO₂ and CTiO₂ NPs at two concentrations (0.5 and 8 mM).

The cytotoxicity of biosynthesized NPs can be tuned by several parameters, such as particle size, shape, and surface chemistry (54). It is reported that the biosynthesis of NPs can modify their surface due to the interaction with biomolecules, which, in turn, enhances the biocompatibility of the formed NPs (55). Similar behavior was observed in the cytotoxicity of our samples, as BTiO₂ NPs showed less toxic potential than CTiO₂ NPs. The accumulation of TiO₂ NPs on the surface of HepG₂ cells reduces the internalization rate and the cytotoxic effects (56–58).

Sequence variation using 16s rRNA gene

Herein, we assess the potential genetic effects of oral exposure to chemically and biosynthesized TiO₂ NPs with two doses (100 and 300 mg/kg body weight/day) during pregnancy.

In mothers, 538–543 bp of nucleotide sequences were obtained. The obtained sequences were deposited into GenBank, and the accession numbers are MZ782915, MZ782917, MZ782918, MZ782919, and MZ782920. The percentages of nitrogen bases are shown in Table 1. In the embryo, 540–550 bp of nucleotide sequences were obtained. The obtained sequences of embryos were submitted to GenBank, and the accession numbers are MZ788644, MZ788646, MZ788647, MZ788648, and MZ788649. The percentages of nitrogen bases are shown in Table 2.

TABLE 1

Table 1. Accession numbers, nucleotide frequencies, and their averages for 16S rRNA gene in the five mother groups.

TABLE 2

Table 2. Accession numbers, nucleotide frequencies, and their averages for 16S rRNA gene in the embryo in five groups.

In mothers, the P-distance among the groups was 0.0000 to 0.0027%. The highest P-distance (0.0027) was found between the control group and C100–TiO₂ NPs groups (Table 3 and Figure 9). In the embryo, the P-distance among the groups was 0.00. Overall, the distance value among all groups was 0.000% (Table 4).

TABLE 3

Table 3. Pairwise distances among mother-five groups using 16S rRNA gene.

FIGURE 9

Figure 9. Alignment of 16S rRNA partial sequences in mother-five groups. Dots refer to identical nucleotides, and A, T, C, and G refer to different nucleotides.

TABLE 4

Table 4. Pairwise distances among embryonic five groups using 16S rRNA gene.

Histopathological examination of fetal kidney

Histological findings of kidney tissue stained by H&E exhibited intact renal architectures in the control embryos (Figure 10A). Contrariwise, C100–TiO₂ NPs mg/kg/day treated group showed necrosis of the renal tissues compensated by focal mononuclear infiltrate and separation of the epithelium-lining tubules (Figure 10B). Similarly, C300–TiO₂ NPs treated group displayed severe histological damage characterized by necrosed and desquamated epithelial cells with extensive inflammation (Figure 10C). Regarding B100–TiO₂ NPs, they showed apparently normal kidney parenchyma (Figure 10D). Moreover, treated group with B300–TiO₂ NPs revealed healthy renal tissues with mildly congested blood vessels (Figure 10E).

FIGURE 10

Figure 10. (A–E) Transverse sections stained with H&E from fetal kidney of control (untreated) and experimental (treated) groups: (A) Light section of control showing intact nephritic tubules (arrow) and glomerulus (star). (B) Light section of C100–TiO₂ NPs showing separation of epithelium-lining tubules from the basement membrane (arrow), besides mononuclear infiltrate (star). (C) Light section of C300–TiO₂ NPs showing necrosis of the renal tubules replaced by inflammatory cells (arrow) as well as desquamated epithelial cells (star). (D) Light section of B100–TiO₂ NPs showing apparently healthy renal parenchyma (arrow). (E) Light section of B300–TiO₂ NPs showing healthy glomeruli (arrow) and mild congestion of the renal blood vessels (star). Scale bar = 200 μm.

Discussion

Oral consumption of TiO₂ NPs is considered one of the most common exposure scenarios due to exposure to TiO₂ NPs found in food, liquid products, and medicines (20, 46). Due to the smaller size and physiochemical properties of TiO₂ NPs, different cytotoxic and genotoxic behaviors were detected. Previous studies have demonstrated that NPs might pass biological barriers. Following intravenous injection at GD 16–17, TiO₂ NPs (35 nm) and silica (70 nm) NPs passed the mature blood placenta barrier (59).

Furthermore, another study observed that orally administrated ZrO₂ might pass the intestinal and maternal blood placenta barriers, and nanoparticles would accumulate in the fetal brain after three repeated oral doses to late-pregnancy mice (GD 16, 17, and 18) (60).

In most multicellular organisms, the mitochondrial DNA is maternally inherited, meaning it is inherited from the mother (61). 16S rRNA gene was primarily used for the identification of an organism, and thereafter, 16S rRNA sequencing was able to reclassify the organism into completely new species or even genera (62).

When compared with GC, the entire 16S rRNA gene exhibits AT richness (63). This was in coordination with our results, where the region amplified by the 16S rRNA gene was AT-rich.

Based on the results of 16S rRNA sequences of mothers, TiO₂ NPs caused genetic variation, where the TiO₂ NPs-treated groups were genetically distant from the control group that attributed to the effect of TiO₂ NPs. Landsiedel et al. (64) describe various studies on the genotoxicity of nanomaterials that contain TiO₂ NPs. They claim that the development of micronuclei, a sign of chromosomal and DNA damage, is evidence of the genotoxicity of TiO₂ NPs.

DNA damage can result from NPs that enter the body through the skin, mouth, or respiratory system. The oxidative stress and inflammation response associated with exposure to TiO₂ NPs can interrupt DNA structure, indirectly causing genetic effects. Being small enough, it can directly interact with DNA, causing genetic changes or even damaging the genetic material. When the nuclear membrane vanishes during mitosis, the entry of TiO₂ NPs into the nucleus is possible. The penetration of TiO₂ NPs and silica NPs into the nucleus was confirmed by many researchers. They reported that these small particles interact with intracellular proteins, causing aggregation, which can inhibit the replication, transcription, and proliferation processes (65, 66).

Some NPs can enter cell nuclei and may directly interfere with the structure and function of genomic DNA (67). TiO₂ NPs have been studied for their potential to cause cancer using assays that monitor gene mutations, chromosomal damage, indicative of potential clastogenic activity of the particles, and DNA strand breaks (29, 68–73). In the same context, TiO₂ NPs cause clastogenicity, genetic variation, oxidative DNA damage, and inflammation in vivo in mice. These outcomes were seen after just 5 days of water-based therapy (74).

On the contrary, the results of 16S rRNA sequences in embryos did not display differences between the control group and the TiO₂ NP-treated ones, which reflected that the TiO₂ NPs did not affect the genetic structure of the embryos. This is in coordination with the previous report (30) which reported that at doses up to 1000 mg/kg/day, there was no evidence of toxicity in the maternal or developmental tissues.

On the other hand, the histopathological findings showed that the embryonic renal tissue of the CTiO₂ NP-treated group showed significant necrosis compensated by focal mononuclear infiltrate, in addition to the separation of epithelium-lining tubules (75). The results indicate that exposure to BTiO₂ NPs reduced damage in the kidney tissue of the embryo. These results provide evidence that the biosynthesis of NPs can modify their surface due to the interaction with biomolecules, which, in turn, enhances the biocompatibility of the formed NPs.

The results of this study will provide worthy information on the developmental genotoxicity of CTiO₂ NPs and BTiO₂ NPs via repeated oral exposure, which can help in the process of hazard estimation of widely used nanoparticles.

Conclusion

Titanium dioxide NPs were biosynthesized with garlic extract as a reducing agent and have a semispherical shape. The biosynthesized protein did not show any structural disorder except for the increase in particle size, which, in turn, caused a little decrease in its cytotoxicity against HepG₂ cells. Moreover, the results of 16S rRNA sequences of mother groups showed that chemically synthesized TiO₂ NPs caused genetic variation compared to the control. The results of the maternal study showed that the biosynthesized TiO₂ NPs were less toxic compared to chemically synthesized TiO₂ NPs. However, the embryo-treated groups with both chemically and biosynthesized TiO₂ NPs did not display any differences compared to the control group. Our study was mainly designed for the experimental use of TiO₂ NPs and not for food or cosmetics; we recommend that the genetic variation be investigated more carefully.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement

The animal study was reviewed and approved by the Ethics of Animal Experiments Committee of South Valley University, Faculty of Science (Permit Number: 002/9/22).

Author contributions

ZK and AS: conception of the idea of the manuscript. ZK, AS, AE, IR, FZ, ZF, and MA: conceptualization and methodology. ZK, AS, AE, IR, FZ, ZF, and MA: formal analysis, writing, reviewing, and editing. All authors have substantially contributed to each step of manuscript preparation, study procedure, contributed to the article, and approved the submitted version.

Funding

This study was supported by the Slovak project KEGA no. 009UVLF-4/2021: Innovation and implementation of new knowledge of scientific research and breeding practice to improve the teaching of foreign students on the subject of animal husbandry.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

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Abbreviations

NPs, Nanoparticles; ROS, Reactive Oxygen Species; TTIP, Titanium isopropoxide; BTiO₂ NPs, Biosynthesized titanium dioxide NPs; CTiO₂ NPs, Chemically synthesized titanium dioxide NPs; GD, Gestation day; HepG₂ cells, Liver hepatocellular cells; DMSO, Dimethylsulfoxide; XRD, X-ray diffraction; FWHM, Full width at half maximum; HRTEM, High-resolution transmission electron microscope; FTIR, Fourier transformation infrared; DMEM, Dulbecco's Modified Eagle's Medium; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium; Large subunit ribosomal RNA, 16S rRNA gene.

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