TiO₂ is extensively used in everyday life, especially as white pigment in painting, food additives and cosmetic industries, thanks to its high refractive index (Vittoriadiamanti, 2013). And also used in dyes and varnishes, textiles, paper, plastics, adhesives, plastics and rubber, printing inks, coated fabrics and textiles, also ceramics, roofing materials, toiletry, perfumery, detergent, toothpaste, soap, water disinfectant agents, drugs, photocatalytic, and antimicrobial compound (Jotham and Xuelia, 2018).It is the active ingredient in sunscreen by blocking absorption of the sun’s ultraviolet light (Chen et al., 2009).

Titanium dioxide (TiO₂) is used widely in biomedical applications and  implanted  devices( dental implants, joint replacements, cardiovascular stents, and spinal fixation devices) because of its excellent is a biocompatible compound with bioactive and bacteriostatic properties, blood compatibility, corrosion resistance, and negative surface charge in physiological solution that  resulting in the nucleation and growth of CaP phase and accelerated osteointegration. However, these implants release titanium under mechanical stress or altered physiological conditions such as low pH in nanometer size range (Vamanu et al., 2008; Shtansky et al., 2015)

It’s widely used as a powder and increasingly in a Nano particulate form, which considered nontoxic at the normal employed concentrations (Robert, 2013), The nanotechnology exponential evolution in the areas of its use, makes it coveted for commercial and medical applications (Shukla et al., 2011).

Titanium dioxide nanoparticles (TiO₂NP) enters the human body through inhalation, skin and eye contacts to be finally deposited in the lung acidic fluid lining, where it dissolution, leading to transient increases in Ti ions that permeates pulmonary cells nuclei and may directly embarrass the structure and function of genomic DNA producing severe pulmonary toxicity by oxidative stress and DNA damage (Liang et al., 2011). These pulmonary toxic effects are dose- and size-dependent, the smaller nanoTiO₂ particles (20 nm) the greater pulmonary inflammatory response in rats and mice (Wang et al., 2009) and causes inflammation, cell death and DNA damage by oxidative stress are caused by reactive oxygen species (ROS) under the presence of sunlight or UV light in mammalian cells, and reactive nitrogen species (RNS) accumulation (Liu et al., 2012; Imran, 2012).   Lipid    per    oxidation,   and   genes   expression

modifications involved in the oxidative or detoxification processes after NPs exposure. On the other hand, Idebenone is a synthetic product similar to coenzyme Q10. It works as an antioxidant and protects cells from oxidative damage (Azim et al., 2015).

In addition, TiO₂NP absorbed through gut because of its small dimension, and then transported to the blood causing adverse biological interactions in several organs. Moreover, it has been classified by the International Agency for Research on Cancer as Group 2B carcinogen, a “possible carcinogen to humans”. Different toxic effects on the lung functions were reported with various routes of exposure, such as the intragastric, intraperitoneal and intratracheal; dermal (Liang et al., 2011).

The pulmonary toxic effect after oral exposure was rarely studied. We are in need to discover a newly protective agent to neutralize the toxic effect of these nanoparticles that daily exposed. Because of this and due to their potential hazards and its wide utilization so its use is a great matter among health and environmental researchers so, this study was conducted aimed to improve the overall (TiO₂NP) use and decrease its related health hazards through the following objective among albino rats: to  assess the TiO₂NPs oral administration pulmonary induced toxic effect and to study  the beneficial role of Idebenone as a novel agent protecting the human body against this toxic effect.

An experimental clinical trial conducted on 75 adult albino rats of both sex from Faculty of Medicine Animal house at Zagazig University who received human care in compliance with the animal guidelines and ethical regulations in accordance with the guide for the care and use of laboratory animals (Institute of Laboratory Animal Resources et al., 1996).

The experiment phases

Fourteen days prior to the experiment, all animals were left to acclimatize and placed in plastic cages free from any source of chemical contamination under controlled conditions with an ambient range of temperature (22±2ËšC), relative humidity 50±5% and a 12 h light cycle with free access to tap water and balanced food (ad libtium) given before and during drug administration.

Preparation of the chemicals

Titanium dioxide nanoparticles (TiO₂NPs)

White  odourless  fine   powder   manufactured   by  SigmaË—Aldrisch chemical company, Germany was purchased from sigma Egypt. Its particle size 35 to 65 m²/g surface area and purity ≥99.5% trace metals basis.

Gum acacia

It is presented in a powder form and prepared by dissolving 10 g in 100 ml boiled distilled water. It was obtained from El-Nasr Pharmaceutical Chemicals Company, Egypt.

Idebenone

Capsules (45mg) were purchased from Sigma-Aldrich Co (St. Louis, MO, and USA) dissolved in distilled water. 

Rats were divided into five groups as follow; Group I (negative control) (n=15): each rat received regular diet and distilled water for 30 days to measure the basic parameters; Group II (positive control) (n=15): each rat orally gavaged 1 ml of 5% gum acacia solution(solvent of titanium dioxide) once daily; Group III (Idebenone treated group) (n=15): each rat orally gavaged(200 mg/Kg) daily disolved in 1 ml distilled water (Gunnar et al., 2009). The low therapeutic dose in human is 5 mg/Kg (Schulza et al., 1984) and high therapeutic dose is 2 tablets three times per day (Oscar et al., 2017); Group IV (Titanium Dioxide nanoparticles treated group (TiO₂NPs): (n=15); rats were orally gavaged with 250 mg/kg body weight Titanium dioxide nanoparticles (1/10 LD50) dissolved in 1ml of 5% gum acacia solution once daily (Rizk et al., 2017). This dose tested by Rizk et al. (2017) to produce cytotoxicity. Daily exposure of TiO2 NPs in food reaches as high as 0.5 to 9 g/kg (Weir et al., 2012).

Group V (TiO₂NPs + Idebenone treated group: (n=15); rats were treated with (250 mg/kg/day) TiO2NPs along with idebenone (200 mg/kg/day) both dissolved as before, gavaged once daily for 30 days.

Biochemical analysis (At the end of the experiment)

Rats were anaesthetized with ether then venous blood samples were collected from the retro orbital plexuses by micro capillary glass tubes under light ether anaesthesia (Johnson, 2007). Serum samples were obtained from centrifuged blood and maintained at -20°C to be used for estimation of reduced glutathione (GSH) by the method of Moron et al. (1979) and nitric oxide (NO), according to the method of Montgomery and Dymock (1961).

Preparation of bronchio-alveolar fluids (BALF)

The rat’s lung and trachea were bared by dissection, and then the left lung was tentatively clamped. The right lung was lavaged with 6 ml of warm normal saline, then, the BALF were centrifuged at 400 g for 10 min. The concentrations of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in BALF were analyzed using biochemical analysis kits (Shangbo, Beijing, China). SOD was determined by Kakkar et al. (1984) method, LDH by modified IFCC method in which rate of oxidation of NADH to NAD was measured as a decrease in absorbance that was proportional to the LDH activity in the sample (Varley et al., 1980).The reactions were measured using a UV/Vis spectrometer (Schwarze el al., 2006).

Histopathological examination and comet assay

The    lungs    were   removed   and   macroscopically  inspected  to determine any obvious abnormalities, then divided into two parts, the first part was fixed in 10% formalin for histopathological examination by light microscope according to Bancroft and Gamble (2002) as the followings, thick paraffin sections were placed on gelatin-coated slides and dried. Graded alcohol was used to hydrate the sections that stained with Hematoxylin stain for 2 mins and Eosin stain for 30 secs. The second part was put in saline for Comet assay to investigate DNA damage according to the method of Singh et al. (1988) and Khan et al. (2015) in Animal Reproductive Research Institute (ARRI) of Agricultural Research Centre of Ministry of Agriculture and Land Reclamation (Elharam, Giza).

Comet assay

Preparation of base slides

Low melting point agarose (LMPA 0.5%) and normal melting agarose (NMA 1.0%) were prepared. While NMA is hot, clean dry slides were dipped up to one-third of frosted area, removed and laid to dry.

Lung cell isolation

A small piece of the lung was placed in 1 ml cold Hank's Balanced Salt Solution "HBSS" containing 20 mM EDTA/10% Dimethylsulfoxide "DMSO", minced into fine pieces, let settle, removed and 5 to 10 ul mixed with 75 ul LMPA, and processed accordingly.

Electrophoresis of micro gel slides

Slides were arranged on the horizontal gel box beside each other. Electrophoresis buffer covers the slides for 20 mins to allow expression of alkali-labile DNA damage. The power was turned on and the current was 300 mA to electrophorese the slides for 30 mins. Then the power was turned off and the slides were stained with 80 ul IX Ethidium bromide "EtBr” leaved for 5 min and then dipped in chilled distilled water to remove excess stain. The slides were scored immediately.

Evaluation of DNA damage

For ideation of DNA damage, observations are made of EtBr-stained DNA using a 40× objective on a fluorescent microscope. A Komet 5 image analysis software developed by Kinetic Imaging, Ltd. (Liverpool, UK) linked to a CCD camera that was used to assess the quantitative and qualitative extent of DNA damage in the cells by measuring the length of DNA migration (tail length) and the percentage of migrated DNA in the tail (tail DNA%). Finally, the program calculates tail moment (correlation between tail length and tail DNA %). Generally, images of 100 (50×2) randomly selected cells are analyzed per sample. The mean value (for 100 cells) was calculated.

Statistical analysis

All the collected data were coded and analysed using SPSS version 20.0. The quantitative data were summarized and expressed as mean±standard deviation (X±SD), and analysed using by one-way analysis of variance (ANOVA) test, followed by least significant difference (LSD).  P-value  was  set  at  <0.05 for significant results, <0.01 for high significant result and <0.001 for very high significant expressed.

The studied 75 rates show no significant difference as regards age and sex. There was no statistical significant difference as regards the biochemical (blood and broncheo-alveolar lavage) and Comet test parameters among negative control (GI) positive control GII and idebenone groups (GIII) (Table 1 and Figure 1). While there were statistical significant difference as regards the biochemical (blood and broncheo-alveolar lavage) and Comet test parameters among in negative control (GI), titanium dioxide nanoparticle (GIV) and titanium dioxide nanoparticles + idebenone (GIII) groups (Table 2 and Figure 2).

Single cell gel electrophoresis

No significant differences regarding mean values of comet tail length, percentage of tail DNA (tail DNA%) and tail moment among –ve,+ve controls and idebenone groups (p>0.05) by ANOVA test, so we used negative control group  (I) as a standard reference for comparison with other treated groups (Table 1).  Table 2 showed significant difference among –ve control, TiO₂NPs and TiO2NPs+ idebenone regarding comet tail length (μm), tail DNA% and unit tail moment (P<0.001) by ANOVA test. Least significant test revealed significant increase in unit tail moment in both TiO2NPs and TiO2NPs+ idebenone groups when compared with control group (p<0.001) while no significant difference in TiO2NPs+idebenone when compared with TiO2NPs was detected(P>0.05) . (Figure 3) showed normal lung nuclei and undamaged cells in control group (fig1a) while abnormal tailed nuclei and damaged cells in TiO2NPs group (Figure 1b and c) and TiO₂NPs+idebenone groups (Figure 1d) less number of abnormal tailed nuclei and damaged cells were detected.

Histopathological

The light microscopical examination of hematoxylin and eosin (H&E) stained sections from the lung of control and idebenone groups, showed normal spongy histological appearance with numerous alveoli connected together with alveolar pores and opens into alveolar sacs, thin inter alveolar septa, bronchioles and blood vessels (Figures 4 and 5). while diffuse alveolar damage with marked consolidation of lung tissue, collapsed alveoli, marked thickening of interalveolar septa and extra-vasations of RBCs in the alveolar lumen, hyperemic foci,  thickened     wall     pulmonary     blood    vessels,   heavy infiltration with chronic inflammatory cells mainly lymphocytes and partial shedding of mucosal lining of bronchioles were detected in TiO2ONPs intoxicated group (Figures 6 to 8). In TiO₂NPs+ idebenone treated group lung sections showed normal alveoli with some collapsed alveoli scattered in-between, mild thickening of interalveolar septa and inflammatory cellular infiltration (Figure 9)

Intentional and unintentional exposures to titanium dioxide nanoparticles are by oral, ingestion, inhalation, dermal routesand by intravenous injection. It is one of the most widely utilized nanoparticles as its particles are small in size makes nanotechnology so useful in medicine and industry (Pileni, 2001).

Rosa et al. (2010) reported that the more decrease in the dimensions of nanoparticles the more thawing rate with more toxic effects. The extensive use of nano-particles causes potential risks for human and environmental biological system. It has great hazards to human health (Attia et al., 2013). The nanosized TiO₂ is used in numerous applications as colour of foods, cosmetics and environmental decontaminant of air, soil and water (Matt et al., 2014). TiO₂NPs can accumulate in body tissues causing inflammation, cell death, apoptosis, finally organ injury. Additionally, TiO₂NPs induce reactive oxygen species (ROS) production leading to DNA damage (Gao et al., 2013).

In the present study the administration of TiO₂NP showed significant decrease in serum oxiditave marker (GSH). This observation was in line with the results of Shukla et al. (2011) who reported that in vitro studies on human colon carcinoma cell line exposed to TiO₂NPs resulted in decreased viability, increased hydrogen peroxide free radicles and decreased glutathione (GSH) levels. This GSH level decrease can be due to either direct action of TiO₂NPs on its synthesis or interference with its action in scavenging free radicals.

The results of present study showed highly significant increase in mean values of serum NO. This result was in accordance with Gillis et al. (2014). High amounts of NO are released from the inducible nitric oxide synthase (iNOS) enzyme isoform evoked by inflammatory stimuli from variety of cell types. Oxidative stress and the rising levels of free radicals have a strong relation with the increased levels of inducible nitric oxide synthase (iNOS) protein in the lung with production of NO as a compensative mechanism (Porter et al., 2006).

Increased serum nitric oxide levels in TiO₂NPs intoxicated rats suggest that these nanoparticles can induce oxidative stress and increase pro inflammatory mediators (Kunal et al., 2009). Idebenone, a short chain benzoquinone related to coenzyme Q10 in its structure, is a potent antioxidant, electrons carriers and free radicals scavenger protecting bio membranes from oxidative damage induced by pollutants (Al-Rasheed  et  al., 2013).

The present study showed non-significant difference in mean values of serum NO in TiO₂NPs + Idebenone groups  when compared  with vet control group, but there was highly significant decrease in mean values of serum NO in TiO₂NPs + idebenone group when compared with TiO₂NPs intoxicated group. These results coincide with Suno et al. (2015) who reported that Idebenone has a potential preventive effect against lung inflammation caused by TiO₂NPs. The inhibition of the phagocytes production of reactive oxygen species (ROS) and inhibition of cytokine induced neutrophil chemo attractant (CINC) genes were the mechanisms of Idebenone. Improvement  of   oxidative  stress  was  associated  with decreased levels of pro-inflammatory mediators such as NO.

In our study, we found that TiO₂ NPS induced oxidative damage and inflammation in BALF by significant increase in LDH, MDA, IL6 and TNF-α with SOD decrease. There are seventeen proteins structurally different regardless of the composition and shape of nano particles which released in BALF (Rai et al., 2002). Nanoparticles exposure, stimulate expression of cytokines in lung epithelial   cells   and   in   lung    tissue.   Higher   protein concentrations in the nanoparticles exposed BALF samples are likely a result of plasma extravasation (Rao et al., 2005).

Lactate dehydrogenase leakage is a measure of membrane integrity damage. Nanoparticles induced LDH leakage in BALF, which revealed the damaging  impact of nanoparticles on cell membrane integrity (Liu et al., 2013).

Furthermore, the decreases of SOD values in exposed groups suggested that the balance between oxidation and anti-oxidation was destroyed in rats. It probably suggested that the acute toxicity primarily originated from

the cellular internalization of nanoparticles rather than physical damage on the cellular membrane (Lin et al., 2010).

In our study the concentrations of TNF-α, and IL-6 increased in BALF of rats exposed to TiO2 NPs. These results are in accordance with Lin et al. (2012) who stated that pro-inflammatory cytokines (TNF-α, IL-6) play an importantrole in regulating immunity. As pro-inflammatory  factors,   the  level  of IL-6  induced  by  the nanoparticles in BALF was significantly higher than that of the control group in his study.

A significant increase in mean values of serum GSH and BALF SOD and a significant decrease in mean values of serum BALF LDH, MDA, IL6 and TNF-α in TiO₂NPs + Idebenone group when compared with TiO₂NPs intoxicated group was detected. These results can be supported by Al-Rasheed et al. (2013) results who reported  that oral administration of Idebenone along with titanium dioxide nano particles resulted in restoration of renal GSH levels in rats. Idebenone can restore the decreased levels of GSH induced by TiO₂NPs through suppression of oxidative stress and lipid per oxidation together with neutralization of the excess amounts of free radicals perserving GSH in the reduced state.

In addition, Ciftci et al. (2012) reported that supplementation of idebenone completely prevents protein damage, apoptosis and lung injury caused by several toxic materials. In the present study, light microscope examination of sections of the lung of titanium dioxide nanoparticles intoxicated group showed collapsed alveoli, destruction of inter-alveolar septa with alveolar dilatation, thick inter-alveolar septa, and heavy infiltration of inflammatory cells, inflammatory exudates and extravasations of RBCs in the interstitium.

The results of the present study coincided with those of Hext et al. (2005) who noted signs of inflammation upon histo-pathological observation of female rats exposed to 10 mg/m³ TiO₂ concentration for 13 weeks as the nanoparticles were phagocyte by macrophage.

However, we observed that macrophages infiltrated TiO₂ nanoparticles in alveoli of treated rats. These findings are in line with the results of a previous study done by Bermudez et al. (2004), which showed degeneration, thickness of lung epithelial cells and particle-laden macrophages of the alveolar region.

Also, Stearna et al. (2011) explained thickening of inter-alveolar septa induced by TiO₂NPs by the increased interstitial collagen fiber deposition and marked cellular infiltration with lymphocytes, neutrophils, eosinophils and macrophages. Moreover, vascular congestion and cellular infiltration of the lung tissue caused by disruption of the endothelial barrier integrity and increased capillary permeability.

Furthermore, Park et al. (2008) demonstrated that uptake of TiO₂NPs in acidic lining of lung cells accelerates dissolution of these particles leading to lysosomal damage, mitochondrial disturbance and production of ROS and cytokines. The light microscope examination  of   sections   of   the   lung    of   TiO₂NPs + Idebenone group revealed partial improvement in histo-pathological changes. These results are in agreement with Nagy (2015) who reported that rats treated with Idebenone along with TiO₂NPs show well preserved lung tissue with partial improvement in lung histopathology. Also, Idebenone administration with TiO₂NPs was found to decrease congestion and inflammatory infiltration in the tissues.

Moreover, Nagai et al. (2015) mentioned that Idebenone, is a radical-scavenging antioxidant, has the ability to inhibit the induction of pulmonary oxidative stress, injury and inflammation observed in the rat lung 1 day after intra-tracheal instillation of TiO₂NPs. The Comet assay is a widely used assay in recording genotoxicity and DNA damage or repair of different pharmaceuticals and environmental chemical contaminants (Karlsson et al., 2015).

In this study the effect of TiO₂NPs was demonstrated at molecular level by Comet assay to investigate the ability of TiO₂NPs to generate DNA damage in off springs. Our results showed that administration of TiO₂NPs causes DNA strand breaks and disrepair of damaged DNA strands recorded by increase in unit tail moment.

These results are in accordance with Tao et al. (2014) who reported that different concentrations of TiO₂NPs are cytotoxic and genotoxic to different organs and cell lines in different organisms. This genotoxic effect reported to be dosing dependent Musarrat et al. (2009) and even after acute and sub-chronic exposure Gerloff et al. (2009).

These results could be explained by Singh et al. (2009) and Yang et al. (2009) who stated that increased ROS induced by nanoparticles in lysosomes can cause DNA mutations or induce single or double strand breaks. Dueto their small size, nanoparticles accumulate around the nucleus and few of them may diffuse through nuclear pores from where protein transport take place and this augment DNA damage caused by ROS (Simko et al., 2011).

Also, Hausladen and Stamler (1999) and Murphy(1999) concluded that unregulated production of  NO can lead to damage of cellular proteins, DNA damage, cell injury and death induced by TiO₂NPs. In addition, the presence of free titanium ions may be the cause of ROS-driven cytotoxicity and genotoxicity (Song et al., 2010).

The results of this work showed no-significant difference in mean values of unit tail moment in TiO₂NPs + Idebenone group when compared with TiO₂NPs intoxicated group, but there was highly significant increase when compared to negative control group.

These results are in agreement with the findings of Park et al. (2008) who reported that several in vitro experiments with cell lines indicate that Idebenone in the presence of transition metal ions acts as a pro-oxidant and increases the amount of damage to genetic material in human lymphocytes.

These results suggest that idebenone has a minimal protective effect on TiO2NPs induced DNA damage and these results could be explained by low dose of idebenone, short duration of supplementation or the fact that increased DNA damage needs long time with sufficient doses of Idebenone for the tissue to restore normal genetic and chromosomal appearance (Nagaoka et al., 2016).

TiO₂NPs oral administration induces oxidative stress and DNA damage in the lung tissue. Idebenone use with TiO2NP is considered a protective agent leads to attenuation of oxidative stress, pulmonary toxicity and slight improvement of DNA damage proved by Comet assay.

Caution use of TiO₂NPs to gain the benefits of nanotechnology and avoid its possible hazards also it’s highly recommended to use Idebenone as protective agent against TiO₂. Control the TiO₂NPs occupational exposure through continuous monitoring of work environment level, keep it within the recommended exposure limits, and involve its periodical clinical and laboratory examinations as a routine check-up among exposed workers. In addition to planning a health education program to Increase the awareness of workers about the TiO₂NPS materials, its hazards, proper handling and protective measurements nanoparticles toxicity. Finally, further studies with longer duration, is highly recommended in this interesting topic

The authors gratefully acknowledge the support and  help provided by all the staff of animal house, Zagazig University Hospitals.

The author has not declared any conflict of interest.