Abstract
Background: Titanium dioxide nanoparticles are among the most commonly used metal oxide nanoparticles in industrial products, such as cosmetics, sunscreens, food products, paints and drugs. They have been reported to elicit various adverse cellular effects including oxidative stress and deoxyribonuclic acid (DNA) damage. Aim: To evaluate the beneficial effect of idebenone against the titanium dioxide nanoparticles pulmonary toxicity in adult albino rats. Material and methods: Fifty adult male albino rats were classified into five groups. Group I: Negative control received regular diet and water. Group II: Positive control administered 1ml of 5% gum acacia solution (solvent of titanium dioxide) once daily. Group III: Each rat gavaged orally with 200 mg/kg b.w. idebenone once daily Group IV: Each rat received 1200 mg/kg b.w. titanium dioxide nanoparticles in 1ml of 5% gum acacia once daily. Group V: Each rat gavaged orally with 200 mg/kg b.w. idebenone then 1200 mg/kg b.w. titanium dioxide nanoparticles once daily. After 4 weeks, the rats of all groups were subjected to blood sample for estimating nitric oxide, reduced glutathione levels. Then the lung was subjected to histological examination. Cell suspension from the lung was examined to determine the extent of DNA damage by the Comet assay. Results: The results revealed that titanium dioxide nanoparticles induced significant decrease and increase in mean values of serum reduced glutathion and nitric oxide respectively, histological changes in the lung in the form of collapsed alveoli, destruction of interalveolar septa with alveolar dilatation, thick interalveolar septa, heavy infiltration of inflammatory cells and inflammatory exudates in comparison to negative control group. In addition, comet assay showed that titanium dioxide nanoparticles exposure produced DNA damage in lung cells. Upon supplementation of idebenone with titanium dioxide, nanoparticles produced normalization of the oxidative markers and partial to complete protection of pulmonary histological changes with moderate protective effects against DNA damage. Conclusion: Titanium dioxide nanoparticles exposure causes toxic effects on the lung that may be attributable to oxidative stress and administration of idebenone offers protection against its damaging effects.
Keywords: Idebenone; lung; nanoparticles; Titanium dioxide; Toxicity.
Introduction
Titanium dioxide (TiO2) has natural occurrance oxide of titanium. Refine titanium dioxide chemical is a delicate, white powder that produces a coruscate, white pigment (Baan, 2007).
Titanium dioxide has been used indyes and varnishes, textiles, paper , plastics,paints, adhesives, paper, plastics and rubber, printing inks, coated fabrics and textiles, also ceramics, roofing materials, Toiletry, Perfumery, Detergent, toothpaste, soap, water disinfectant agents, drugs, food additives, sunscreen and catalysts (Jotham and Xuelia, 2018)
Many people are exposed to titanium dioxide as an active ingredient in sunscreen protect ing a person’s skin by blocking absorption of the sun’s ultraviolet light that can cause sunburn (Chen et al., 2009).
Titanium is extensively used for a wide scope of implanted medical devices, such as dental implants, joint replacements, cardiovascular stents, and spinal fixation devices. However, under mechanical stress or altered physiological conditions such as low pH, Titanium-based implants can release amounts of particle debris; both in the micrometer and nanometer size range (Vamanu et al., 2008). The use of nanotechnology has seen an exponential evolution in the areas of its use, which makes them coveted for commercial and medical applications (Shukla et al., 2011).
Nanoparticles use is a matter of a great regard among health and environmental researchers due to their potential human and environmental embargo and its wide utilization. Titanium dioxide nanoparticles (TiO2NP) can enter the human body through inhalation, skin contact, and eye contact and finally deposited in the lungs throughout all the previous rout. Also after gut absorption of these nanoparticles because of its small dimension, they are transported to the blood causing adverse biological interactions in several organs. The major site of interaction with TiO2NPs is lung because of its acidic environment of the lining fluid result in TiO2NPs dissolution, leading to transient increases in the condensation of titanium ions and local pulmonary toxicity. In addition, it can permeat pulmonary cells nuclei and hence may directly embarrass the structure and function of genomic DNA producing severe pulmonary toxicity by oxidative stress and DNA damage. Moreover, TiO2NPs 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). 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).
Oxidative stress are caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulation that lead to cell inflammation, DNA destruction and death (liu et al., 2012).
Lipid per oxidation, and genes expression modifications involved in ant oxidative or detoxification processes were aimed by NPs exposure. On the other hand, Idebenone is a synthetic product similar to coenzyme Q-10. It works as an antioxidant and protects cells from oxidative damage (Schaffer et al., 2015).
The pulmonary toxic effect was rarely studied by oral route of exposure.we are in needto discover anewly protective agent to neutralize the toxic effect of these nanoparticles that daily exposed to.
The aim of this study was to confirm that oral administration of TiO2NPs induced pulmonary toxic effect and the beneficial role of idebenone as a novel agent protecting the human body against this toxic effect.
I- Material:
1. Chemicals:
*Titanium dioxide nanoparticles (TiO2NPs): white odourless fine powder manufactured by Sigma˗Aldrisch chemical company, Germany was purchased from Sigma Egypt. Its particle size 35-65 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 gm 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.
2. Animals: fifty adult male albino rats weighing between 200-220 g for each. They were obtained from Animal house of the Faculty of Medicine, Zagazig University. The study was performed at Animal house of the Faculty of medicine, Zagazig University. All animals 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).
All animals were left to acclimatize 14 days prior to the experiment and were placed in plastic cages free from any source of chemical contamination under controlled conditions with an ambient range of temperature (22±2˚c), relative humidity 50±5% and a 12 h light cycle with free access to tap water and balanced food rich in all stuffs important to maintain their health before and during drug administration. Rats were divided into five groups as follow:
Group I (negative control) (n=10): each rat received regular diet and distilled water for 30 days to measure the basic parameters
Group II (positive control) (n=10): animals received 1 ml of 5% gum acacia solution (solvent of titanium dioxide) by oral gavage once daily.
Group III (Idebenone treated group): (n=10) rats received (200 mg/Kg) dailydisolved in 1 ml distilled water (Gunnar et al., 2009)
Group III (Titanium Dioxide nanoparticles treated group (TiO2NPs): (n=10):˗ rats were treated gavaged with 1200 mg/kg body weight titanium dioxide nanoparticles (1/10 LD50) dissolved in 1ml of 5% gum acacia solution once daily (Eman et al., 2015).
Group IV (TiO2NPs + Idebenone treated group: (n=10):˗ rats were treated with (1200mg/kg/day) TiO2NPs along with idebenone (200mg/kg/day) both dissolved as before, gavaged once daily for 30 days.
3. 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 means of micro capillary glass tubes under light ether anaesthesia (Johnson, 2007). Blood samples were centrifuged to separate the serum 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).
4. Histopathological examination and comet assay:
After blood samples collection rats were sacrificed and the lungs were removed and grossly inspected to assess any gross 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. Lung sections were hydrated in graded alcohol and brought to distilled water. These sections were stained with Hematoxylin stain for 2 minutes and Eosin stain for 30 seconds. Then the sections were dehydrated in a graded series of alcohol, cleared with xylene and mounted with cover slips, the second part was put in saline for Comet assay to investigate DNA damage according to the method of Singh et al. (1988) & 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:
A. 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.
B. 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 – 10 ul mixed with 75 ul LMPA, and processed accordingly.
C. Electrophoresis of micro gel slides
Slides were placed side by side on the horizontal gel box close together as possible. The buffer reservoirs were filled with freshly made Electrophoresis Buffer until completely covers the slides for 20 minutes to allow for unwinding of the DNA and the expression of alkali-labile damage. The power was turned on and the current was adjusted to 300 milliamps ere to electrophorese the slides for 30 minutes. Then the power was turned off and the slides were gently lift from the buffer and placed on a drain tray. Slides were stained with 80ul IX Ethidium
Bromide “EtBr” leaved for 5 min and then dipped in chilled distilled water to remove excess stain. The
Slides were covered by cover slip and scored immediately.
D. Evaluation of DNA Damage
For visualization of DNA damage, observations are made of EtBr-stained DNA using a 40x 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 X 2) randomly selected cells are analyzed per sample. The mean value (for 100 cells) was calculated.
3-Statistical analysis:
All results Data collected were analyzed using SPSS version 20.0. Differences between multiple means (quantitative variables) were compared 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 as mean±standard deviation (X±SD).
RESULTS
1. Biochemical results:
Non significant differences regarding mean values of serum GSH and NO among negative control (I) , positive control (II) and idebenone groups (III) respectively were detected (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).
There were highly significant difference regarding mean values of serum GSH and NO in the TiO2NPs treated group (III) in comparison to negative control (I) and (TiO2NPs+ Idebenone )group (table 2).
2. Single cell gel electrophoresis (Comet assay):
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 2). 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 (1) showed normal lung nuclei and undamaged cells in control group (fig1a) while abnormal tailed nuclei & damaged cells in TiO2NPs group(fig 1b,c) and TiO2NPs+idebenone groups (fig 1d)less number of abnormal tailed nuclei & damaged cells were detected.
2.Histopathological results: The light microscopically 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 (Fig. 2,3).while diffuse alveolar damage with marked consolidation of lung tissue, collapsed alveoli, marked thickening of interalveolar septa and extravasations 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 (Fig.4,5,6). 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 (Fig. 7)
Table (1): Statistical Comparison of mean values of biochemical parameters (serum reduced glutathione (mmol/dl), nitric oxide (μmol/l) and Comet test analysis (comet tail length (μm), Tail DNA percentage, Unit tail moment) in negative control (-ve), positive control (+ve) and idebenone groups.
Group
Parameter -ve control group
N=10 +ve control group
N=10 Idebenone
group
N=10 P
Mean±SD
Biochemical
Parameters GSH(mmol/dl) 11.88 ± 0.79 11.93 ± 0.72 11.01 ± 0.75 0.848
NO(μmol/l) 41.43 ± 5.54 42.75 ± 5.11 44.26 ± 6.75 0.790
Comet test Tail length(μm) 4.69 ± 0.26 4.65 ± 0.25 4.69 ± 0.27 0.938
Tail DNA% 1.57 ± 0.26 1.61 ± 0.26 1.61 ± 0.27 0.235
Unit tail moment 15.47 ± 1.5 15.12 ± 1.5 15.55 ± 1.6 0.802
N= number of rats SD: Standard Deviation GSH: reduced glutathione mmol/dl: mill moles per deciliter. NO: Nitric oxide μmol/l: micromole per liter μm=micrometers
P: Analysis of variance test (ANOVA)
Table (2): Statistical Comparison of mean values of biochemical parameters (serum reduced glutathione (mmol/dl), nitric oxide (μmol/l) and Comet test analysis (comet tail length (μm), Tail DNApercentage, Unit tail moment) in different studied groups.
Group
Parameter -ve control group
N=10 Titanium Dioxide NPs
(N= 10) Titanium dioxide NPs + Idebenone
(N= 10) P
Mean±SD
Biochemical
Parameters GSH(mmol/dl) 10.88 ± 0.69 1.46 ± 0.64a 8.2 ± 1.02b <0.001
NO(μmol/l) 42.43 ± 5.44 114.26 ± 12.44 41.02 ± 8.12 <0.001
Comet test Tail length(μm) 4.69 ± 0.26 7.07 ± 0.10 6.74 ± 0.07 <0.001
Tail DNA% 1.57 ± 0.26 4.88± 0.13 2.48 ± 0.04 <0.001
Unit tail moment 15.47 ± 0.12 19.41 ± 1.3 16.09 ± 1.4 <0.001
NPs: nanoparticles N= number of rats SD: Standard Deviation -ve: negative GSH: reduced glutathione mmol/dl: mill moles per deciliter. NO: Nitric oxide μmol/l: micromole per liter μm=micrometers.
DISCUSSION
Titanium dioxide nanoparticles are one of the most widely spread utilized nanoparticles. The human body may be intentionally or unintentionally exposed to nanoparticles through oral ingestion, inhalation, dermal exposure and intravenous injection. While the small size of particles 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 nanoparticles causes potential risk for human and environmental biological system. It has a great dangerous 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 exposure can enter the body through many routes and accumulate in body tissues causing inflammation, cell death and apoptosis, finally organ injury and defeat. In addition, it has been recorded that TiO2NPs induce reactive oxygen species (ROS) leading to DNA damage (Gao et al., 2013).
In the present study the administration of TiO2NP showed significant decrease in serum GSH as a marker of oxidative stress. These observations were 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 decrease in GSH levels can be due to either direct action of TiO2NPs on GSH synthesis or interference with its action in scavenging free radicals.
Idebenone, a short chain benzoquinone structurally related to coenzyme Q10, is a potent antioxidant and electrons carriers also free radicals scavenger protecting bio membranes from oxidative damage induced by pollution (Al-Rasheed et al., 2013).
A significant increase in mean values of serum GSH 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 keeping GSH in the reduced state.
The results of present study showed highly significant increase in mean values of serum NO. These results were in accordance with Gillis et al. (2014).
High amounts of NO are released from the inducible nitric oxide synthase (iNOS) enzyme isoform in response to inflammatory stimuli from variety of cell types. 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).
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 compensatory mechanism (Porter et al., 2006).
The results of the present study showed no-significant difference in mean values of serum NO in TiO2NPs + idebenone group when compared with –ve 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 preventive effect against lung inflammation caused by TiO2NPs. This was attributed to inhibition of the production of reactive oxygen species (ROS) caused by activation of phagocytes, such as neutrophils and macrophages and suppression of cytokine induced neutrophil chemo attractant (CINC) genes. Improvement of oxidative stress was associated with decreased levels of proinflammatory mediators such as NO.
In addition, ciftci et al. (2012) reported that administration of moderate doses of idebenone almost 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 interalveolar septa with alveolar dilatation, thick interalveolar 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) and
Bermudez et al. (2002) also noted signs of inflammation upon histopathological observation of 6-week-old female rats exposed to 10 mg/m3 TiO2 concentration for 13 weeks (6 hr/day, 5 days/week) as the nanoparticles were phagocyte by macrophage.
The actual delivered amounts of TiO2 nanoparticles that are exposed to rats can be calculated by multiplying the aerosol concentration of TiO2 nanoparticles by the amount of inhaled air by rats, which was previously reported by Alexander et al. (2008). The rats inhaled higher amounts of nanoparticle; the toxic responses were more severe.
In addition, we have to consider the size of nanoparticle. We measured that mean particle size of approximately 80 nm in actual exposure atmosphere during study done by Sager et al. (2008).
However, we observed that macrophages infiltrated TiO2 nanoparticles in alveoli of treated rats. These findings are in line with the results of a previous inhalation study by Bermudez et al. (2004), which showed degeneration and thickness of lung epithelial cells and particle-laden macrophages of the alveolar region.
Also, stearna et al. (2011) explained thickening of interalveolar 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 could be caused by changes of the vascular integrity of the lung vessels causing disruption of the endothelial barrier and increased capillary permeability.
Furthermore, Parks 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, production of ROS and cytokines.
The light microscope examination of sections of the lung of TiO2NPs+idebenone group revealed partial improvement in histopathological 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 intratracheal instillation of TiO2NPs.
The comet assay is a widely used assay in ultimate research for DNA damage and repair, genotoxicity testing of novel chemicals and pharmaceuticals and environmental bio monitoring. However, comet assay has been working for toxicity assessment of highly reactive nanoparticles; and several studies had used it to investigate the potential toxicity of manufactured nanoparticles by assessing DNA strand breaks or oxidative DNA lesions ( 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 and cause long term side effects in off springs. Our results showed that administration of TiO2NPs causes DNA strand breaks and disrepair of damaged DNA strands evidenced by increase in unit tail moment.
These results are in accordance with TaoChen 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 Muserrat et al. (2009) and even after short term exposure Gerloff et al. (2009).
These results could be explained by Singh et al. (2009) & Yang et al. (2009) who stated that increased ROS induced by nanoparticles in lysosomes can cause DNA point mutations or induce single or double strand breaks. Due to 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) & Murphy, (1999) concluded that unregulated production of NO can lead to damage of cellular proteins, DNA damage, cell injury and death and this may be a cause of DNA damage 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 with 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 prooxidant 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).
CONCLUSION
Oral administration of TiO2NPs induced oxidative stress as well as DNA damage in the lung tissue. Idebenone administration along with TiO2NPs led to attenuation of oxidative stress and pulmonary toxicity induced by TiO2NPs, but there was slightly improved DNA damage proved by Comet assay. Titanium dioxide nanoparticles should be used cautiously to gain the benefits of nanotechnology and avoid its possible draw backs. Control occupational exposure to TiO2NPs by continuous monitoring of work environment level and keep it within the recommended exposure limits. Increasing the awareness of workers about the proper handling of TiO2NPS materials with periodical clinical and laboratory examinations are needed.
Conflict of interest:
There is no conflict of interest.
Acknowledgment:
The authors gratefully acknowledge the support and help provided by all the staff of animal House, Zagazig University Hospitals.