Elimination of oxidative stress and genotoxicity of biosynthesized titanium dioxide nanoparticles in rats via supplementation with whey protein-coated thyme essential oil

The green synthesis of metal nanoparticles is growing dramatically; however, the toxicity of these biosynthesized particles against living organisms is not fully explored. Therefore, this study was designed to synthesize and characterize TiO2-NPs, encapsulation and characterization thyme essential oil (ETEO), and determination of the bioactive constituents of ETEO using GC-MS and evaluate their protective role against TiO2-NPs-induced oxidative damage and genotoxicity in rats. Six groups of rats were treated orally for 30 days including the control group, TiO2-NPs (300 mg/kg b.w)-treated group, ETEO at low (50 mg/kg b.w) or high dose (100 mg/kg b.w)-treated groups, and TiO2-NPs plus ETEO at the two doses-treated groups. Blood and tissues were collected for different assays. The GC-MS results indicated the presence of 21 compounds belonging to phenols, terpene derivatives, and heterocyclic compounds. The synthesized TiO2-NPs were 45 nm tetragonal particles with a zeta potential of −27.34 mV; however, ETEO were 119 nm round particles with a zeta potential of −28.33 mV. TiO2-NPs administration disturbs the liver and kidney markers, lipid profile, cytokines, oxidative stress parameters, the apoptotic and antioxidant hepatic mRNA expression, and induced histological alterations in the liver and kidney tissues. ETEO could improve all these parameters in a dose-dependent manner. It could be concluded that ETEO is a promising candidate for the protection against TiO2-NPs and can be applied safely in food applications.


Introduction
Recently, nanotechnology has developed rapidly in different sectors to improve human life leading to the production of several nanomaterials developed and extensively used in various fields including industry, food, medicine (Li et al. 2018;Sycheva et al. 2011;Wang et al. 2016;Zahin et al. 2020), personal healthcare (Sanders et al. 2012;Khosravi et al. 2012), toothpastes (Li et al. 2012), food packaging (Jovanović and Palić 2012;Philbrook et al. 2011), materials science (Zahin et al. 2020), and antimicrobial agents (Martínez-Gutierrez et al. 2012). Titanium dioxide NPs (TiO 2 -NPs) are the most commonly manufactured worldwide (Jomini et al. 2015). Inhalation of TiO 2 -NPs in mice leads to accumulating these NPs in the hepatic and cardiac tissue and transfer to the circulation after 24 h (Husain et al. 2015). After oral administration, TiO 2 -NPs accumulate in different organs mainly the liver, heart, brain, and lung inducing damage and inflammation to these organs (Geraets et al. 2014;Kandeil et al. 2020). Additionally, the abdominal injection of mice with TiO 2 -NPs accumulates these particles in different organs; induces severe damage to the liver, heart, and kidneys; and affects the serum lipids and sugar (Liu et al. 2009). TiO 2 -NPs activate the inflammatory processes, complement cascade in the heart, and innate immune responses mediated by the complement factor 3 in the blood. However, in the liver, these particles alter the gene expression especially that related to the acute phase response (Husain et al. 2015).
Essential oils (EOs) are the secondary metabolites extracted from different aromatic plants and are categorized as generally recognized as safe (GRAS) by the FDA (Hyldgaard et al. 2012). Thyme (Thymus vulgaris L.) is a medicinal aromatic plant (Lamiaceae family) that has been widely used in food, medicine, and agriculture (Morales 2002;Tao et al. 2014). Thyme essential oil (TEO) showed a variety of beneficial biological activities which include antioxidant, antitumor, antimicrobial, and antiinflammatory properties (Nikoli et al. 2014). However, the application of EOs in the food sector is faced by some challenges such as the interaction with different food matrix including proteins, starch, and fats (Hyldgaard et al. 2012), their strong volatile characteristic (Khalili et al. 2015). Besides, EOs may alter the sensory characteristics of the foods when used in high concentration; their poor solubility in the aqueous phase (Tao et al. 2014; Barbosa-Cánovas et al. 2009); and their sensitivity to light, heat, and oxygen during food processing, storage, and utilization (Woranuch and Yoksan 2013). To overcome these problems, the encapsulation process of EOs may be suitable to solve the challenges facing the applications of EOs by enhancing solubility and bioavailability, protecting against thermal and chemical degradation, and controlling the delivery release at the desired site and time (Tao et al. 2014). Moreover, the application of nanoparticlebased drug delivery system enhances clinical outcomes by improving drug internalization, enabling targeted delivery, enhanced permeability, prolonged circulation, easy biodistribution, and enhanced permeability rate, thereby improving therapeutic effectiveness of several natural agents (Bhattacharya et al. 2021;Kabir et al. 2021). The current study was designed to synthesize and characterize TiO 2 -NPs by green chemistry using orange peel extract, encapsulate and characterize TEO, and evaluate the potential protective activity of the encapsulated TEO (ETEO) against the oxidative damage and genotoxicity of TiO 2 -NPs in rats.

Preparation of TiO 2 -NPs
Fifty grams of the peel of orange was extracted for 2 h using 150 ml of deionized water at 90°C. The extract was filtered and was used for the biosynthesis of TiO 2 -NPs (Balashanmugam et al. 2013). TTIP (1.5 N) was dissolved in distilled water (100 ml), and the extract was added dropwise with constant stirring, and pH was maintained to 7 by continuous washing. The mixture was stirred continuously for 6 h in the light at room temperature. TiO 2 -NPs were synthesized, separated using Whatman filter paper, and washed with distilled water several times to remove any by-products. The obtained wet nanoparticles were dried at 80°C overnight and were calcined at 600°C for 4 h (Rao et al. 2015).
respectively, and the retention indices (Kovats index) of the separated volatile components were calculated using hydrocarbons as references (C7-C20, Aldrich Co.) as shown by Adams (2007).

Preparation of encapsulated thyme essential oil (ETEO)
WPI was dissolved in distilled water with stirring for 1 h. The solution was kept overnight at room temperature before emulsification. Tween 80 was added to the polymer as an emulsifier. Then, the essential oil was progressively added to the polymer solution with homogenization at 20,000 rpm for 10 min to form an emulsion. The polymer concentration was 20%, and the amount of essential oil used was 10% of the mass of the polymer concentration (Jinapong et al. 2008). The emulsion solution was encapsulated by spray drying.

Characterization of TiO 2 -NPs and ETEO
Scanning electron micrographs (SEM) for TiO 2 NPs were recorded on JEOL JAX-840A and JEOL JEM-1230 electron micro-analyzers, respectively. However, for ETEO, the droplets were placed onto a carbon-coated copper grid to form a thin liquid film and were negatively stained by one drop of uranyl acetate. The excess staining was removed using filter paper, and then the film was air-dried before the observation (Pecarski et al. 2014). The image acquisition was done using Orius 1000 CCD camera (GATAN, Warrendale, PA, USA). For measuring zeta potential, the sample of TiO 2 -NPs or ETEO was sonicated for 30-60 min just before assessment. The average diameter was calculated using zpw 388 version 2.14 nicomp software. The size distribution and the zeta potential of TiO 2 -NPs and ETEO were measured using a particle size analyzer (Nano-ZS, Malvern Instruments Ltd., UK).

Animals and experimental design
Sixty sexually mature male Sprague-Dawley rats (3 months old, 150-160 g) were supplied by the Animal House Lab, National Research Center (NRC), Dokki, Cairo, Egypt. The rats were housed in stainless steel cages in an artificially illuminated (12 h dark/light cycle) and thermally controlled (25 ± 1°C) room free from any source of chemical contamination at the Animal House Lab, NRC, Dokki, Cairo, Egypt. All animals were fed normal chow (Meladco Feed Co., Cairo, Egypt) and housed in filter top polycarbonate cages. The animals received humane care in compliance with the guidelines of the Animal Care and Use Committee of NRC and the National Institute of Health (NIH publication 86-23 revised 1985). After an acclimatization period of 1 week, the rats were divided into 6 groups (10 rats/group) and were treated orally using stomach tube for 30 days as follows: group 1, normal control received distilled water; group 2, rats treated with TiO 2 -NPs (300 mg/kg b.w) in an aqueous solution; group 3, rats treated with low dose (LD) of ETEO (50 mg/kg b.w); group 4, rats treated with high dose (HD) of ETEO (100 mg/kg b.w); and groups 5 and 6, rats treated with LD or HD of ETEO plus TiO 2 -NPs. Body weight was recorded each other day, and at the end of the treatment period (i.e., day 31), all animals have fasted for 12 h and weighed, and then blood samples were collected via the retro-orbital venous plexus under isoflurane anesthesia. Sera were centrifuged using a cooling centrifuge and kept at −20°C until used for the determination of ALT, AST, lipid profile creatinine, urea, TP, albumin, AFP, TNF-α, and CEA according to the kit's instructions. Immediately after blood samples were collected, all animals were euthanized, and samples of hepatic and renal tissue from each animal were dissected. A sample of the liver and kidney was weighed and homogenized in phosphate buffer (pH 7.4) and centrifuged at 1700 rpm and 4°C for 10 min, and the supernatant was separated and used for the estimation of MDA, NO, CAT, GPx, and SOD (Lin et al. 1998). Other samples of these organs from each animal were used for the histological examination. The tissue samples were fixed in 10% neutral formalin and paraffin-embedded. Sections (5 μm thickness) were stained with hematoxylin and eosin (H & E) for the histological examination (Bancroft et al. 1996). However, another sample of liver of each animal was quickly frozen using liquid nitrogen and kept at −80°C for the determination of gene expression.

Gene expression analysis
RNA was isolated from the liver samples using TRIZOL reagent. The concentration and the quality of RNA were determined by NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, USA), and only samples of high quality, with A260/A280 ratios between 1.8 and 2.2, were used. Reverse transcription-cDNA synthesis was carried out using a PreMix cDNA Kit (iNtRON Biotechnology, Korea). The obtained cDNA was kept at −20°С for later use or directly used as a semi-quantitative PCR (Sq-PCR) template. The expression of the selected genes was quantified using quantitative real-time PCR (RT-qPCR) performed in a One-Step SYBR Select Master Mix Kit as previously described (Kim et al. 2009). The gene-specific primer sequences for GAPDH, GPx, SOD, CAT, Bax, Bcl-2, and TNF-α are shown in Table 1. RT-qPCR was carried out on Stratagene Mx3005P RT-PCR System (Agilent Technologies) in a 20-μL reaction volume using, 1 μLcDNA, 10 μM of forward and reverse primers, 10 μL TOP real™ qPCR 2× PreMIX (SYBR Green with low ROX) (Enzynomics), and DNAse-free water. All samples were amplified in triplicates, and the amplification was conducted with a 15-min denaturation at 95°C, then 40 cycles of 95°C for 12 s, 56-63°C for 15 s, and 72°C for 30 s.
The PCR cycle number (C T ) was used for the calculation of expression level where the increased fluorescence curve passes across a threshold value. However, the relative expression of the target genes was obtained using the comparative C T (ΔΔC T ) method. The ΔC T was calculated by subtracting β-actin C T from that of the target gene, whereas ΔΔC T was obtained by subtracting the ΔC T of the calibrator from that of the test sample. The relative expression was calculated from the 2 −ΔΔCT formula based on the method of Pfaffl (2001) and Abdel-Wahhab et al. (2021).

Statistical analysis
Statistical analyses were carried using SPSS 16. Data were expressed as mean ± SE. Variables were compared using one-way ANOVA; post hoc Duncan's test and the significance of differences among means were determined at p ≤ 0.05.
The present results showed that addition of the extract of orange peel to TTIP and stirring in light for 6 h at the room temperature and pH 7 resulted in the synthesizing of the tetragonal structure of TiO 2 -NPs ( Fig. 1A) with an average size of 45 nm (Fig. 1B) and a zeta potential of −27.34 mV (Fig.  1C). Moreover, the SEM analysis of ETEO showed a round shape ( Fig. 1D) with an average particle size of 110 nm (Fig.  1E) and a zeta potential of -28.33 mV (Fig. 1F).
The in vivo results revealed that TiO 2 -NPs significantly decreased the body weight of rats; however; ETEO did not affect the body weight either at the low or high dose (Fig. 2). The co-treatment with TiO 2 -NPs plus ETEO at both tested doses improved the body weight, and no significant difference was observed between the two tested doses. The biochemical indices (Table 3) showed that all the tested parameters were significantly elevated in the group treated with TiO 2 -NPs ALT, AST, and creatinine were decreased significantly in the animals that received ETEO (LD), but no significant change was observed in Alb, TP, D. BIL, urea, and uric acid. However, administration of ETEO (HD) showed a significant decrease in the liver enzymes (ALT, AST) and kidney (creatinine and uric acid) and did not affect the other parameters. Co-administration with TiO 2 -NPs plus ETEO (LD) or ETEO (HD) improved all the biochemical parameters, and ETEO (LD) could normalize Alb, TP, and T. BIL; meanwhile, ETEO (HD) could normalize all these parameters except ALT and uric acid which were still higher than the control level. The current data (Table 4) showed the effect of different treatments on lipid profile. Treatment with TiO 2 -NPs increased the lipid profile parameters except HDL which was decreased significantly. Administration of ETEO (LD) increased HDL without any effect on the other lipid parameters; however, ETEO (HD) increased HDL and LDL with no effect on cholesterol or TG. All the lipid parameters were improved significantly in the groups that received the combined treatment, and the low dose could normalize TG, HDL, and LDL; besides, the high dose could normalize HDL and LDL. Additionally, TiO 2 -NPs induced a significant increase in hepatic and renal NO and MDA (Table 5). ETEO (LD) or ETEO (HD) decreased both parameters in a dose-dependent fashion. Animals that received TiO 2 -NPs plus ETEO (LD) or ETEO (HD) showed a significant improvement in these markers in a dose-dependent manner. Additionally, TiO 2 -NPs decreased the activity of the antioxidant enzymes (CAT, SOD, and GPx) in the hepatic and renal tissue (Table 6). Meanwhile, no significant changes were noticed in these enzymes in rats that received ETEO alone at the two doses  although the renal GPx and hepatic SOD were significantly increased in these groups. A significant improvement was observed in the animals that received combined treatment of TiO 2 -NPs plus ETEO, especially the hepatic GPx w h i c h w a s i n t h e n o r m a l r a n g e o f t h e c o n t r o l . Furthermore, TiO 2 -NPs administration also increased the serum TNF-α, CEA, and AFP (Table 7). ETEO at the low or the high dose did not affect TNF-α and AFP; however, CEA was decreased significantly than the control group. TNF-α returned to normal in the rats treated with TiO 2 -NPs plus ETEO (LD) or (HD); meanwhile, AFP and CEA were improved significantly especially in the group that received ETEO (HD). A quantitative PCR was carried out to estimate the changes in hepatic mRNA gene expression of pro-apoptotic Bax (Fig.  3A), TNF-α (Fig. 3B), and the antiapoptotic Bcl-2 ( Fig. 3C) in different treatment groups. TiO 2 -NPs alone increased significantly the mRNA expression of Bax and TNF-α and decreased Bcl-2 mRNA expression. Moreover, GAPDH (housekeeping glyceraldehyde-3-phosphate dehydrogenase gene) indicated an insignificant difference between the untreated control group and those treated with ETEO at the two tested doses in Bax, TNF-α, and Bcl-2 expressions. However, administration of TiO 2 -NPs plus ETEO at both tested doses could induce remarkable improvement in the transcript levels of these genes.
The cytogenetic study of antioxidant gene expression showed that TiO 2 -NPs administration markedly reduced the mRNA expression of GPx (Fig. 4A), SOD (Fig. 4B), and CAT ( Fig. 4C) compared to the control group. Treatment with ETEO (LD) or (HD) increased significantly the mRNA of GPx gene expression; however, these treatments did not affect SOD or CAT gene expression. Administration of ETEO at the tested doses plus TiO 2 -NPs improved the mRNA expression of these antioxidant genes towards the control level although these values still differed significantly from the control group.
The examination of liver sections of the control animals showed the hepatocytes with normal cytoplasm with vesiculated nuclei and separated by blood sinusoids radiating from the central vein (Fig. 5A). The liver section of the animals treated with ETEO (LD) showed no observable changes in hepatocytes architecture except few fibrous tissues around the central vein and bile duct hypertrophy (Fig. 5B); however, those treated with ETEO (HD) showed nearly normal hepatocytes architecture (Fig. 5C). The examination of liver sections of animals treated with TiO 2 -NPs showed marked histological alterations in hepatocytes as disorganization, vacuolar, and fatty degeneration shrunken hepatocytes with darkly stained pyknotic nuclei, patches of necrotic cells around the dilated and congested portal tract, and numerous mononuclear cellular infiltrations localized in dilated hepatic sinusoids (Fig. 5D). The liver of rats administrated with TiO 2 -NPs plus ETEO  (LD) showed several histological changes in hepatocytes as vacuolar, fatty degeneration, pyknotic nuclei, and an increase in mononuclear inflammatory cells (Fig. 5E). Additionally, the liver of animals in the group that received TiO 2 -NPs plus ETEO (HD) showed more improvement in hepatic cells stricter, but few inflammatory cells were also seen (Fig. 5F).
The examination of kidney cortex sections of the control animals showed the proximal convoluted tubules with high cuboidal acidophilic cells, narrow lumen, and distal convoluted tubules with low cuboidal cells. The renal corpuscle with the parietal layer of Bowman's capsule, glomerulus, and the urinal renal space was preserved (Fig. 6A). The kidney cortex sections of the rats treated with ETEO (LD) showed normal tubules; glomeruli with epithelial cells except some renal tubules showed a slight hyaline droplet in their lumen and widening of the Bowman's space (Fig. 6B). The kidney cortex of the animals treated with ETEO (HD) showed dilatation of tubular lumen with slight cytoplasmic hyaline, but the majority of renal tubules, glomeruli, and Bowman's capsule were nearly normal (Fig. 6C). The examination of the renal cortex of animals treated with TiO 2 -NPs showed vacuolar degeneration in the cytoplasm of tubular epithelial cells with deeply stained pyknotic nuclei in some renal tubules. Some of the lining epithelial cells were exfoliated into the lumen with hyaline droplets. The renal capsules were shrunken with a reduction in glomeruli mesangial cells and mononuclear cell infiltrations between degenerated tubule and renal corpuscle (Fig. 6D). The kidney cortex of animals treated with TiO 2 -NPs plus ETEO (LD) showed few foci of vacuolation in the tubular epithelial cells or hyaline cytoplasmic droplet in their lumen, but the majority of renal tubules and glomeruli were nearly normal (Fig. 6E). However, the renal cortex of the animals treated with TiO 2 -NPs plus ETEO (HD) showed few foci of vacuolation in the tubular epithelial cells or cytoplasmic droplet in their lumen with interstitial tubular mononuclear cell infiltrations, but the majority of renal tubules and glomeruli were nearly normal (Fig. 6F).
In the current work, the results of GC-MS analysis of ETEO revealed the presence of 21 bioactive compounds which belongs to phenols, terpene, and terpene derivatives. The phenolic compounds (thymol and carvacrol) represent 62.58 mg/g; however, the terpene and its derivatives represented 65.03 mg/g. These results were in good harmony with those reported in the literature (Amiri 2012;He et al. 2020;Nieto 2020;Youdim et al. 2002). However, the number and concentration of the compounds were somewhat different which is probably due to the difference in variety and the growing condition of the plant (Diniz do Nascimento et al. The results also showed that the use of orange peel extract as a green approach succeeded to synthesize TiO 2 -NPs in crystal shape with an average particle size of 45 nm and a zeta potential of −27.34 mV. Similar to these results, previous reports indicated that crystalline TiO 2 -NPs were synthesized in different size using the orange peel extract, and the size of these synthesized particles was smaller than those synthesized using chemical methods (Rao et al. 2015; Mobeen Amanulla and Sundaram 2019; Thakur et al. 2019). These authors suggested that the synthesis of TiO 2 -NPs using the orange peel extract was due to its high content of insoluble polysaccharides, soluble sugars, and polyphenols which act as reducing agents in addition to the amino acids, citric acid, and the carboxylic groups which act as stabilizing agents (Torrado et al. 2011). In a previous work, Patra and Baek (2014) reported that the extract concentration, reaction temperature, and pH are the main factors that affect the particle size, and the irregular shape of the synthesized TiO 2 -NPs may be due to the  The synthesized ETEO reported in this current study showed a round shape with an average size of 110 nm and a zeta potential of −28.33 mV. These results suggested that WPI, which was used as a wall material in the encapsulation process, enhanced the coalescence of droplets (Abdel-Wahhab et al. 2018). Moreover, the spherical shape of the particles indicated the presence of WPI in the wall of capsules (Eratte et al. 2014;Xu et al. 2013), and the negative zeta potential is also attributed to the negatively charged WPI in neutral pH which is mostly due to the carboxylate groups as the only charged functionalities present in WPI molecule (Eratte et al. 2014). Generally, the surface properties and the particle size have a critical role in nanoparticles uptake by the mucus membranes, and the size of 50-300 nm is the preferred size for uptake (Roger et al. 2010;El-Sayed and Kamel 2020) and affects the pharmacokinetics, tissue distribution, and clearance of nanoparticles (Sadat et al. 2016). Furthermore, the negative charge of zeta potential can enhance the dispersion of the droplets and increase the stability of the emulsion (McClements and Rao 2011), and the zeta potential higher than 30 mV and lower than −30 mV stimulates the high stability and prohibits the aggregation of the particles (Mohanraj and Chen 2006). In the in vivo study, animals were treated with TiO 2 -NPs alone or in combination with ETEO at a low or high dose. The selected dose of TiO 2 -NPs was based on Orazizadeh et al. (2014); however, the selected dose of ETEO was based on our previous work (El-Nekeety et al. 2011). The toxicity of TiO 2 -NPs was manifested primarily by the effect of body weight. Animals treated with TiO 2 -NPs alone showed a significant decrease in their final body weight than the control group; meanwhile, no significant change was noticed in body weight of the animals treated with ETEO at both tested doses. It was reported that ingestion of NPs induces disturbances in the digestion and the absorption of food components leading to a shortage of micro and macro elements in the body which in turn affect body weight (McClements et al. 2016). A similar decrease in body weight was reported in mice treated with different doses of TiO 2 -NPs at different time intervals. The mice showed toxic symptoms including loss of appetite which leads to a reduction in body weight (Chen et al. 2009). Administration of TiO 2 -NPs also reduces the number of villi in the intestine and reduces the surface responsible for nutrients absorption leading to malnutrition and the reduction of body weight (Duan et al. 2010). Furthermore, oral ingestion of TiO 2 -NPs can penetrate the intestinal mucosa (Ammendolia et al. 2017) leading to the damage and chronic failure of the epithelium tissue in the intestinal wall (Brun et al. 2014).
TiO 2 -NPs administration disturbed the biochemistry of the body as manifested by the significant elevation of liver and kidney function indices, serum cytokines (AFP, CEA, and TNF-α), oxidative markers (NO and MDA), cholesterol, TG, and LDL, and the significant reduction of total protein, albumin, HDL, and antioxidant enzyme activity. Previous in vitro studies reported that TiO 2 -NPs cause toxicity, genotoxicity (Kohen and Nyska 2002), and inflammation (Tucci et al. 2013;Wang et al. 2014;Zhao et al. 2013). The increase in liver and kidney indices reported that TiO 2 -NPs disturb the functions of these organs. The elevation of serum AST and ALT indicated the death or injury of hepatocytes (Thapa and Walia 2007). These enzymes are located in the cytoplasm of hepatic cells more than extracellular fluid, and their levels elevate in the serum if the hepatocytes are damaged (Dambach et al. 2005;Mohammed and Safwat 2020). Additionally, the elevation of urea, uric acid, and creatinine levels indicates the toxicity and dysfunction of the kidney (Ahamed et al. 2010). Thus, the increase of these kidney indices reported in the TiO 2 -NPs-treated group indicated the injury of renal tissue (Abdelhalim and Jarrar 2011;Fartkhooni et al. 2016). Administration of TiO 2 -NPs also disturbs lipid profile markers. The increase in TG, cholesterol, and LDL indicates that these nanoparticles affect the lipid metabolism through the effect on lipoprotein lipase enzyme (Ani et al. 2008) and/or the removing and transferring of the lipid fractions (Duan et al. 2010). Additionally, the increase of TG and cholesterol is associated with cardiovascular disease and other metabolic syndromes (Antoni et al. 2018;Reiner 2017). In addition to the disturbance in cholesterol and TG, the increase of LDL level is critical because it has a close association with arteriosclerosis; hence, TiO 2 -NPs can be considered a causative factor for the incidence of cardiovascular disorders (Chen et al. 2020). Hong et al. (2017) and Chen et al. (2015) reported similar results in mice and rats.
Previous reports proposed that oxidative damage is one of the mechanisms of TiO 2 -NPs-induced toxicity. These particles provoke the formation of ROS (reactive oxygen species) in different cell lines (Foroozandeh and Aziz 2015;Wang et al. 2014). In mammals, ROS induces damage to macromolecules such as proteins, lipids, carbohydrates, and nucleic acids mainly DNA (Abdel-Wahhab et al. 2020;Kelly et al. 1998;Shukla et al. 2014;Saquib et al. 2012). Lipid peroxidation (LP) probably changes the cell membrane structure resulting in disturbances in the vital functions of cells (Rikans and Hornbrook 1997). The oxidative damage induced by TiO 2 -NPs is attributed mainly to the generation of hydroxyl radical (•OH) (Reeves et al. 2008). Oral exposure to TiO 2 -NPs leads to the generation of •OH, a status of oxidative stress that occurs leading to the disturbances in lipids and accumulation of malondialdehyde levels and reduction of antioxidant capability in the hepatic tissue (Rajapakse et al. 2012). Also, exposure to TiO 2 -NPs was reported to decrease glutathione in the liver (Federici et al. 2007). The elevation of NO and MDA and the reduction of CAT, GPx, and SOD in the hepatic and renal tissues suggested the manifestation of oxidative damage and the disturbances in redox balance in these organs (Chen et al. 2020). The increase of ROS production reflexes the damage of DNA and the up-regulation of 8-hydroxyl deoxyguanosine (8-OHdG) in the hepatic and renal tissue (Trouiller et al. 2009), and the increase of MDA and the decrease of antioxidant enzymes in these organs may contribute to the cell apoptosis. The increase of ROS was reported to increase Nrf2 which consider the master regulator of the expression of several antioxidant genes, and the lack of Nrf2 increases the damage of DNA and increases the risk of cancer (Shi et al. 2015). Moreover, hydrogen peroxide (H 2 O 2 ) in the liver and kidney is accumulated due to the decrease in SOD activity leading to the inhibition of CAT activity (Latchoumycandane and Mathur 2002). This enzyme converts H 2 O 2 , the harmful by-product of the normal metabolic process, to H 2 O and O 2 and thus prevents the damage of cells and tissues (Sharma et al. 2014).
The increased level of serum cytokines reported in the current study in the animals treated with TiO 2 -NPs revealed the inflammatory response of these nanoparticles. These results supported the generation of ROS in TiO 2 -NPs-treated rats which leads to the reduction of the cell viability and stimulates the cytotoxicity via an apoptotic process (Salman et al. 2021;Müller et al. 2010). Thus, the toxicity of TiO 2 -NPs probably correlated to the surface chemistry of the particles which affects the inflammatory responses and the release of TNF-α and neutrophil-attracting chemokines (Iavicoli et al. 2011;Müller et al. 2010;Rossi et al. 2010), and the rate of release is the size and time-dependent (Wu and Tang 2018).
Gene expression assay is widely used as a quick and early biomarker to predict for TiO 2 NPs-induced potential liver injury and explore the possible mechanisms of their toxicities (Li et al. 2017). The administration of TiO 2 -NPs induced significant disturbances in the expression of antioxidant and apoptosis genes in hepatic tissue. The down-regulation of CAT, GPx, and SOD mRNA reported herein was harmonized with the biochemical findings and supported the hypothesis that TiO 2 -NPs induce oxidative stress via the exhaustion of antioxidant enzymes and suppress their gene expression. Moreover, TiO 2 -NPs also decreased Bcl-2 mRNA expression and up-regulated Bax and TNF-α mRNA in the liver. Bcl-2 proteins are family responsible for anti-apoptosis and control the mitochondrial integrity surface, while Bax is a proapoptotic protein. The balance of antiapoptotic Bcl-2 and the pro-apoptotic Bax proteins controls the sensitivity of the cell to apoptotic stimuli (Ilani et al. 2018). Additionally, Bcl-2 is found on the surface of mitochondria and prevents the release of cytochrome c in the plasma; however, Bax encourages the leakage of cytochrome c through the punching of the holes of the mitochondrial membrane (Kroemer et al. 2007). The imbalance between Bax and Bcl-2 also activates the pathway of caspase-dependent apoptotic (Peng et al. 2016). Thus, the generation of ROS after TiO 2 -NPs exposure disturbs the mitochondrial membrane potential as a result of apoptosis (Abdel-Wahhab et al. 2020;Zhao et al. 2009). Furthermore, the up-regulation of TNF-α mRNA in the hepatic tissue indicated the inflammatory response of TiO 2 -NPs and supported the earlier findings of the previous studies Gui et al. 2011;Trouiller et al. 2009). In this concern, Samak et al. (2018) reported that nanoparticles such as carbon black nanoparticles induce overproduction of the free radicals, which contributes to inflammation and subsequent cellular apoptosis at the gene expression level.
The histological examination of the liver and kidney tissues revealed that TiO 2 -NPs administration induced severe pathological changes in both tissues and confirmed the biochemical and cytogenetic results. Similar observations were reported in previous studies (Attia et al. 2013;Morgan et al. 2018) who reported disorganization of the hepatic cords with hepatocellular necrosis, macro, and microvascular steatosis. Moreover, Valentini et al. (2019) reported that the alterations on the liver tissue were mainly correlated with oxidative stress which is localized around the central vein. A close correlation between oxidative damage and anoxia of the tissue was reported in different organs (Chen et al. 2005;Pialoux et al. 2009). Moreover, the hepatic Kupffer cells are well known to be the most impacted cells by oxidative stress which may be due to their localization around the portal area in the liver sinusoids (Olmedo et al. 2008). On the other hand, the histological changes in the kidney tissues are characterized by the accumulation of protein materials in the lumen of many distal tubules and the collecting ducts found in the medulla which is less oxygenated compared to the proximal tubules and may be more exposed to the oxidative stress generated by TiO 2 -NPs (Epstein 1997).
The prophylactic activity of thyme essential oils is well documented in the literature; however, some limitations were reported for its application in the food and pharmaceutical sectors. To cope with these limitations, encapsulation technology was proposed. This technique was able to enhance the oil solubility and bioavailability, protect their active components against thermal or chemical degradation, and control the release of these components (Tao et al. 2014). ETEO in WPI was applied for the protection against TiO 2 -NPs-induced oxidative damage and genotoxicity. The antioxidant properties and the protective role of ETEO are focused on the major phenolic components mainly thymol and carvacrol (Ruberto and Baratta 2000). Beside these two major components, the other components in the oil such as linalool, myrcene, and yterpinene enhance the antioxidant activity of the oil (Youdim et al. 2002). Animals treated with ETEO alone exhibited significant improvements in all biochemical parameters, especially the antioxidant enzyme activity and oxidative markers suggesting that ETEO enhances the antioxidant activity. Previous reports indicated that thyme oil reduces the oxidation rate through the elimination of ROS or the breakdown of the peroxides to stable substances and prevents the promotion of further oxidation (El-Newary et al. 2017). ETEO also improved the body weight, biochemical parameters, cytokines, antioxidant enzymes, and their gene expression in rats treated with TiO 2 -NPs. These improvements are maybe due to the elimination of ROS generation which is responsible for protein damage and lipid oxidation of cell membrane as well as the disturbances in calcium homeostasis and increases the fluidity of membrane and the death of cells (Molavian et al. 2016). Furthermore, the high content of phenolic compounds in the oil was found to decrease the triglycerides and cholesterol in the hepatic and renal tissues (Abdel-Daim et al. 2019;Ebenyi et al. 2012). These compounds also prevent the secretion of pro-inflammatory factors through the reduction of lipopolysaccharides (De Andrade et al. 2017); thus, they are potent ROS scavengers' natural products (Ebenyi et al. 2012) and increase the production of GSH, SOD, and CAT (El-Banna et al. 2013). Several phenolic compounds such as ellagic acid showed a potential protective effect against iron nanoparticles-induced renal damage through the sirtuin1 activator . It was also reported that thyme oil can suppress TNF-α in mouse cells and inhibited cytochrome C oxidase-2 expression (Mahran et al. 2019). Generally, the protective role of bioactive components in ETEO may include the inhibition of cytochrome P450 activity and accelerate the regeneration of parenchyma cells, stabilizing the cell membrane and improvement of the antioxidant activity (Al-Fartosi et al. 2011). Previous reports indicated that encapsulation of thyme oil (TO) using different materials improves its properties. In this concern, several studies reported that the antioxidant activity, the thermal stability, and the release of the oil were improved when the oil was encapsulated using zein (Bilenler et al. 2015), chitosan (Detsi et al. 2020;Ghaderi-Ghahfarokhi et al. 2016;Khalili et al. 2015), Arabic gum (Cai et al. 2019), gelatin-Arabic gum (Gonçalves et al. 2017), and chitosan-Arabic gum (Hassani and Hasani 2018).
In our study, WPI was used as a wall in the encapsulation process; thus, we can propose another mechanism of the protective role of ETEO. WPI is well known to possess antioxidant activity due to its high content of amino acids mainly cysteine, β-lactoglobulin, α-lactoglobulin, and bovine serum albumin (Morr and Ha 1993). The amino acid cysteine helps to replenish intracellular GSH, the endogenous antioxidant responsible for peroxide detoxification (Gould and Pazdro 2019). Hence it acts as another source of antioxidants besides its role in the protection of the oil active ingredients and enhances the activity of ETEO.

Conclusion
The current results showed that 21 compounds were identified in TEO representing 130.85 mg/g oil and belong to phenols, terpene, and terpene derivatives class. Thymol, para-cymene, linalool, carvacrol, camphor, eucalyptol, γ-terpinene, borneol, terpinene-4-ol, βpinene, β-Phellandrene, β-myrcene, and Humuline in concentrations were the major compounds. TiO 2 -NPs can be synthesized using orange peel extract, and the resulted particles were tetragonal with an average size of 45 nm and zeta potential of −27.34 mV. The results also revealed that the encapsulation of TEEO using WPI resulted in round particles with an average size of 110 nm and a zeta potential of −28.33 mV. The biological study revealed that TiO 2 -NPs induced severe toxicity to the hepatic and renal tissues as manifested by the decrease of body weight, disturbed liver and kidney function, lipid profile parameters, increased serum cytokines, NO, and MDA. TiO 2 -NPs also increased the mRNA of pro-apoptotic and decrease the mRNA expression of antiapoptotic and antioxidant enzymes along with the pathological alterations in the liver and kidney tissues. ETEO did not induce any significant changes in all the parameters tested or the histological picture of the liver and kidney. Co-treatment with TiO 2 -NPs plus ETEO at both tested doses enhanced the antioxidant activity and alleviated the toxicity of TiO 2 -NPs in a dose-dependent manner. Encapsulation of TEO in WPI enhanced its antioxidant activity and may be a promising candidate to protect against TiO 2 -NPs-induced oxidative stress and genotoxicity. ETEO can be applied to overcome the problems associated with EOs and used in food or pharmaceutical applications for the protection against oxidative damage. In fact, there are some limitations in the current study which are worthy to be carried out in the future; we strongly recommended the some experiments including the release of bioactive components from ETEO along with the TRIF analysis for TiO 2 -NPs to identify the biomolecules responsible for nanoparticles stabilizing in the solution Abbreviations ALT, Alanine aminotransferase; Alb, Albumin; AFP, Alpha feta protein; ANOVA, Analysis of variance; AST, Aspartate aminotransferase; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 Associated X-protein; BC, Bowman's capsule; CEA, Carcinoembryonic antigen; CAT, Catalase; CV, Central vein; Cho, Cholesterol; cDNA, Complementary DNA; DNA, Deoxyribonucleic acid; D. BIL, Direct bilirubin; DT, Distal tubules; DLS, Dynamic light scattering; ETEO, Encapsulated thyme essential oil; ETEO (LD), Encapsulated thyme essential oil (low dose); ETEO (HD), Encapsulated thyme essential oil high dose); EOs, Essential oils; FID, Flame ionization detector; FDA, Food and Drug Administration; GC-MS, Gas chromatography-mass spectrometry; GRAS, Generally recognized as safe; GPx, Glutathione peroxidase; H & E, Hematoxylin and eosin stains; HDL, High density lipoprotein; GAPDH, Housekeeping glyceraldehyde-3-phosphate dehydrogenase gen; H2O 2 , Hydrogen peroxide; 8-OHdG, 8-hydroxyl deoxyguanosine; •OH, Hydroxyl radical; LDL, Low density lipoprotein; MDA, Malondialdehyde; mRNA, Messenger ribonucleic acid nitric oxide; NO, Nitric Oxide; Nrf2, Nuclear factor erythroid 2-related factor 2; RT-qPCR, qPCR quantitative real-time PCR; ROS, Reactive oxygen species; RNA, Ribonucleic acid; SEM, Scanning electron micrographs; Sq-PCR, Semi-quantitative PCR; SPSS, Statistical Package for the Social Sciences; SOD, Superoxide dismutase; TEOs, Thyme essential oils; TO, Thyme oil; TiO 2 -NPs, Titanium dioxide nanoparticles; TTIP, Titanium tetra isopropoxide; T. BIL, Total bilirubin; TP, Total protein; TNF-α, Tumor necrosis factor-alpha; WPI, Whey protein isolate Availability of data and material The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability The codes used during the current study are available from the corresponding author on reasonable request.