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.

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 di erent developmental malformations. Several toxicity studies with chemically synthesized titanium dioxide NPs (CTiO NPs) have been recently carried out; although there is insu cient data on exposure to biosynthesized titanium dioxide NPs (BTiO 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 TiO NPs during gestation days (GD) -. A total of fifty pregnant rats were divided into five groups (n = ) and gavaged CTiO NPs and BTiO NPs at and mg/kg/day concentrations. Pregnant rats on GD 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 TiO NPs. Moreover, BTiO NPs showed less toxic potential than CTiO NPs in HepG cells. Both chemically and biosynthesized TiO NP-induced genetic variation in the S rRNA sequences of mother groups compared to the control group. In conclusion, the genetic e ects of the S rRNA sequence induced by chemically synthesized TiO NPs were greater than those of biosynthesized TiO NPs. However, there were no di erences between the control group and the embryo-treated groups with chemically and biologically synthesized TiO NPs. KEYWORDS S rRNA, titanium dioxide, developmental toxicity, biosynthesized, particle characterization

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 topdown 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 TiO 2 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 (CTiO 2 NPs) have been performed. Organs such as the kidney, liver, and spleen can suffer damage due to exposure to TiO 2 NPs (19)(20)(21), such as inflammation of renal tissue due to ROS. In addition, TiO 2 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 TiO 2 NP concentrations (24).
A repeated oral administration of CTiO 2 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 CTiO 2 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 TiO 2 NPs. The reported toxicological data proposed that green TiO 2 promotes a higher safety profile with improved anticancer, antibacterial, and antiviral activities compared with chemically synthesized TiO 2 NPs (27). However, there are limited toxicological data on exposure to biosynthesized TiO 2 NPs from garlic extract (BTiO 2 NPs) during pregnancy. This study is based on the new method of biosynthesis of TiO 2 NPs by using garlic extract as a reducing agent, performing in vivo investigations on pregnant rats and their fetuses, and comparing the toxicity of BTiO 2 NP synthesis with that of CTiO 2 NP synthesis in both in vivo and in vitro.

Synthesis of chemical TiO NPs (CTiO NPs)
TiO 2 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 TiO 2 precursor was reduced to form TiO 2 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 TiO NPs using garlic extract (BTiO NPs)
Garlic (Allium sativum) water extract was used as a reducing agent for the biosynthesis of TiO 2 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 BTiO 2 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.

TiO NP characterizations
The produced TiO 2 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 −1 and 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).

#S0615
) and 1% penicillin-streptomycin (P/S, Sigma-Aldrich, #P4333). Cells were grown in 75 cm 2 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). HepG 2 cells were seeded in 96-well culture plates with a total density of 1× 10 4 cells per well and incubated at 37 • C and 5% CO 2 . 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 TiO 2 NPs with different concentrations (0, 0.5, 1, 2, 4, and 8 mM) and then incubated at 37 • C and 5% CO 2 . 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).

Cell viability% =
Absorbance control − Absorbance sample Absorbance 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 ecofriendly biosynthesizing method using garlic (Allium sativum) extract as a reducing agent against maternal and fetal genotoxicities that could develop from repeated oral administration of TiO 2 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: CTiO 2 NP-treated groups received 100 and 300 mg/kg body weight (bw)/day, (30,31) respectively. Group 4 and Group 5: BTiO 2 NP-treated groups received 100 and 300 mg/kg bw/day, respectively. These concentrations of TiO 2 NPs of 100 and 300 mg/kg bw were chosen according to the World Health Organization (WHO) in 1969 (32), which reported that the LD 50 of TiO 2 for rats is greater than 12,000 mg/kg bw. Pregnant mothers were exposed to 100 mg/kg bw CTiO 2 NPs or BTiO 2 NPs, which is equal to approximately 6-7 g of TiO 2 NPs per 60-70 kg bw for humans (33).
TiO 2 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).

X-ray di raction
The biosynthesis of NPs is an emerging technique that produces NPs with unique properties. Green nanotechnology was applied to the synthesis of TiO 2 NPs, and the formed NPs were characterized Frontiers in Veterinary Science frontiersin.org . /fvets. . 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 Na 2 TiO 3 , were observed, indicating the high purity of the prepared samples. The X-ray diffraction (XRD) pattern of TiO 2 NPs (see Figure 1) revealed the phase and structural purity of TiO 2 NPs. All the recorded peaks belong to the anatase phase TiO 2 NPs, which support the reported card (JCPDS No. 21-1272). Although the diffraction peak of brookite B (121) was found in CTiO 2 NPs, it disappeared in BTiO 2 NPs (37). Debye-Scherer's equation was used for the calculation of the average crystal size of TiO 2 NPs (38).
where d is the average crystal size of TiO 2 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 CTiO 2 NPs was 48.11 nm, while it was increased to 53.31 nm for BTiO 2 NPs.

UV-visible spectroscopy
Both CTiO 2 and BTiO 2 samples have absorption spectra in the region below 400 nm. The spectral image displays the absorption peaks of TiO 2 NPs at wavelengths of 261.87 and 314 nm for CTiO 2 and BTiO 2 NPs , respectively (see Figure 2). where ∝ is the absorption coefficient of TiO 2 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 CTiO 2 and BTiO 2 NPs were 3.544ev and 2.660ev, respectively (see Figure 3).

Raman spectra analysis of tio NPs
The structural characterization of chemically and biosynthesized TiO 2 NPs with garlic extract by Raman spectroscopy is presented in Figure 3. The six active modes that belong to the anatase phase of TiO 2 were found in the two samples, which confirm the XRD result of the formation of anatase TiO 2 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 TiO 2 (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 TiO 2 (639 cm −1 ), while the frequency position of A 1g has a higher frequency position than that of the bulk TiO 2 (514 cm −1 ). This result is attributed to a reduction in the crystal size of TiO 2 NPs that are produced chemically and biologically (41).

High-resolution transmission electron microscope
HRTEM images of CTiO 2 and BTiO 2 NPs reflect a sort of agglomeration with a spherical or irregular spherical shape (see Figure 5). Particle size distribution of CTiO 2 and BTiO 2 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 BTiO 2 NPs compared to CTiO 2 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).
The average size is 18.46 ± 3.03 and 35.64 ± 4.9 nm for CTiO 2 and BTiO 2 NPs, respectively. These values are smaller than the calculated values from XRD results (48.19 and 53.317) for CTiO 2 and BTiO 2 NPs, respectively. This variation in size may be due to the agglomeration of TiO 2 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 TiO 2 NPs.

Cytotoxicity
An accumulation of both TiO 2 NPs on the surface of the HepG 2 cells was found in the microscopic images (see Figure 6). Particularly, in the case of CTiO 2 NPs, this accumulation increased as NP concentration increased. The normalized cell viability is presented in Figure 7, and there is no observed toxicity for CTiO 2 and BTiO 2 NPs at low concentrations, while a small percentage of Frontiers in Veterinary Science frontiersin.org . /fvets. . toxicity was observed for CTiO 2 at high concentrations (8 mM), as shown in Figure 8. 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 BTiO 2 NPs showed less toxic potential than CTiO 2 NPs. The accumulation of TiO 2 NPs on the surface of HepG 2 cells reduces the internalization rate and the cytotoxic effects (56-58).

Sequence variation using s rRNA gene
Herein, we assess the potential genetic effects of oral exposure to chemically and biosynthesized TiO 2 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.
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-TiO 2 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).

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-TiO 2 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-TiO 2 NPs treated group displayed severe histological damage characterized by necrosed and desquamated epithelial cells with extensive inflammation ( Figure 10C). Regarding B100-TiO 2 NPs, they showed apparently normal kidney parenchyma ( Figure 10D). Moreover, treated group with B300-TiO 2 NPs revealed healthy renal tissues with mildly congested blood vessels ( Figure 10E).

Discussion
Oral consumption of TiO 2 NPs is considered one of the most common exposure scenarios due to exposure to TiO 2 NPs found in  Alignment of S rRNA partial sequences in mother-five groups. Dots refer to identical nucleotides, and A, T, C, and G refer to di erent nucleotides.  (59). Furthermore, another study observed that orally administrated ZrO 2 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, TiO 2 NPs caused genetic variation, where the TiO 2 NPs-treated groups were genetically distant from the control group that attributed to the effect of TiO 2 NPs. Landsiedel et al. (64) describe various studies on the genotoxicity of nanomaterials that contain TiO 2 NPs. They claim that the development of micronuclei, a sign of chromosomal and DNA damage, is evidence of the genotoxicity of TiO 2 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 TiO 2 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 TiO 2 NPs into the nucleus is possible. The penetration of TiO 2 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). TiO 2 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, TiO 2 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 TiO 2 NP-treated ones, which reflected that the TiO 2 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 CTiO 2 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 BTiO 2 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 CTiO 2 NPs and BTiO 2 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 . /fvets. . little decrease in its cytotoxicity against HepG 2 cells. Moreover, the results of 16S rRNA sequences of mother groups showed that chemically synthesized TiO 2 NPs caused genetic variation compared to the control. The results of the maternal study showed that the biosynthesized TiO 2 NPs were less toxic compared to chemically synthesized TiO 2 NPs. However, the embryo-treated groups with both chemically and biosynthesized TiO 2 NPs did not display any differences compared to the control group. Our study was mainly designed for the experimental use of TiO 2 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.