TiO2-NPs are widely utilized in several applications such as sauces, cheeses, ice cream, skimmed milk, food colorants, nutritional supplements, and confectionery products including chewing gum, candy, coating sweets, chocolate, and toothpaste (Bachler et al. 2015; Baranowska-Wójcik et al. 2020; Dudefoi et al. 2017; Peters et al. 2014). Weir et al. (2012) reported that chewing gums, sweets and candies contain high amounts of TiO2-NPs (<100 nm). The absorption of TiO2-NPs via the gastrointestinal tract leads to several organ toxicities including the liver, kidney, serum, testes, and seminal vesicle (Bu et al. 2010; Wang et al. 2007).
In the current work, the results of GC-MS analysis of ETEO revealed the presence of 21 bioactive compounds 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 probably due to the difference in variety and the growing condition of the plant (Diniz do Nascimento et al. 2010; Mutlu-Ingok et al. 2020; El-Guendouz et al. 2019). The results also showed that the use of orange peel extract as a green approach succeeded to synthesize TiO2-NPs in crystal shape with an average particle size was 45 nm and a zeta potential of -27.34 mV. Similar to these results, previous reports indicated that crystalline TiO2-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 syntheses of TiO2-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 particles size and the irregular shape of the synthesized TiO2-NPs may be due to the adhesion of phytochemicals in orange peel extract to particles (Swathi et al. 2019).
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 particles 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) and affect 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 stimulate the high stability and prohibit the aggregation of the particles (Mohanraj and Chen 2006).
In the in vivo study, animals were treated with TiO2-NPs alone or in combination with ETEO at a low or high dose. The selected dose of TiO2-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 TiO2-NPs was manifested primarily by the effect of body weight. Animals treated with TiO2-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 TiO2-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 TiO2-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 TiO2-NPs can penetrate the intestinal mucosa (Ammendollia et al. 2017) leading to the damage and chronic failure of the epithelium tissue in the intestinal wall (Brun et al. 2014).
TiO2-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, TriG, and LDL and the significant reduction of total protein, albumin, HDL and antioxidant enzyme activity. Previous in vitro studies reported that TiO2-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 herein indicated that TiO2-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 damaged (Dambach et al. 2005; Mohammed and Safwat 2020). Additionally, the elevation of urea, uric acid, and creatinine levels indicate the toxicity and dysfunction of the kidney (Ahamed et al. 2010). Thus, the increase of these kidney indices reported in the TiO2-NPs-treated group indicated the injury of renal tissue (Abdelhalim and Jarrar 2011; Fartkhooni et al. 2016).
Administration of TiO2-NPs also disturbs lipid profile markers. The increase in TriG, cholesterol, and LDL indicate 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 TriG 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 TriG, the increase of LDL level is critical because it has also had a close association with arteriosclerosis; hence, TiO2-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 TiO2-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. 20220; 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 TiO2-NPs is attributed mainly to the generation of hydroxyl radical (•OH) (Reeves et al. 2008). Oral exposure to TiO2-NPs, lead 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 TiO2-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 tissue reported herein suggested the manifestation of oxidative damage and suggesting 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 increase the damage of DNA and increases the risk of cancer (Shi et al. 2015). Moreover, hydrogen peroxide (H2O2) 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 H2O2, the harmful byproduct of the normal metabolic process, to H2O and O2 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 TiO2-NPs revealed the inflammatory response of these nanoparticles. These results supported the generation of ROS in TiO2-NPs-treated rats which leads to the reduction of the cell viability and stimulates the cytotoxicity via an apoptotic process (Müller et al. 2010). Thus, the toxicity of TiO2-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 TiO2 NPs-induced potential liver injury and exploring the possible mechanisms of their toxicities (Li et al. 2017). The administration of TiO2-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 is harmonized with the biochemical findings and supported the hypothesis that TiO2-NPs induce oxidative stress via the exhaustion of antioxidant enzymes and suppress their gene expression. Moreover, TiO2-NPs also decreased Bcl-2 mRNA expression and up-regulation of 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 pro-apoptotic protein. The balance of anti-apoptotic Bcl-2 and the pro-apoptotic Bax proteins control 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 TiO2-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 TiO2-NPs and supported the earlier findings of the previous studies (Chen et al. 2015; Gui et al. 2011; Trouiller et al. 2009).
The histological examination of the liver and kidney tissues revealed that TiO2-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 Kuffer 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 TiO2-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 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). The encapsulated thyme essential oil (ETEO) in WPI was applied for the protection against TiO2-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 y-terpinene 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 prevent the promotion of further oxidation (El-Newary 2017). ETEO also improved the body weight, biochemical parameters, cytokines, antioxidant enzymes, and their gene expression in rats treated with TiO2-NPs. These improvements may be due to the elimination of ROS generation which responsible for protein damage and lipid oxidation of cell membrane as well as the disturbances in calcium homeostasis and increase 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 tissue (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). 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 maybe include the inhibition of cytochrome P450 activity, accelerate the regeneration of parenchyma cells, stabilizing the cell membrane, improvement of the antioxidant activity (Al-Fartosi et al. 2011). Previous reports indicated that encapsulation of thyme oil (TO) using different materials improve 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; 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 dexification (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.