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Nano Progress
Research Article
In Vivo Interactions of Nanosized Titania Anatase and Rutile Particles Following Oral Administration
Abstract: Titanium dioxide nanoparticles (TiO2 NPs) have received growing attention for several biomedical applications such as photodynamic therapy, drug delivery, cell imaging etc. Anatase and rutile are important, naturally occurring polymorphs of TiO2. The present in vivo study in mice was carried out to investigate the effects (hemocompatibility, oxidative stress, genotoxicity and histopathology) of interactions of anatase (5-10 nm) and rutile NPs (600-800 nm) on biological components such as blood, tissues and cells following oral administration. These NPs were found to induce significant reduction in some of the hematological and serum biochemical parameters compared to untreated (p<0.05), indicating potential liver damage. The increase in levels of glutathione (p<0.05) and lipid peroxidation (p<0.05) in liver and spleen of mice treated with anatase and rutile compared to untreated tissues suggest the potential role of TiO2 NPs in inducing oxidative stress. Olive tail moment and %tail DNA were higher in liver and spleen of anatase and rutile treated mice compared with untreated mice (p<0.05) and indicate greater DNA damage in the treated tissues. Histopathological studies in mice treated with anatase and rutile NPs showed (i) liver damage as evident from the ballooning of hepatocytes in liver (anatase); focal portal inflammation, spotty necrosis and interface hepatitis in liver (rutile); (ii) red pulp congestion in spleen; and (iii) mild vascular congestion in lung. In conclusion, distribution pattern and interactions of anatase and rutile NPs with the biological milieu were found to be diverse and the resultant implications were established in distinct ways.
Keywords: Titania nanoparticles; Photodynamic therapy; Hemocompatibility; Oxidative stress; Histopathology; Genotoxicity.
Publication details: Received: 09th April 2020; Revised: 10th May 2020; Accepted: 11th May 2020; Published: 15th May 2020
1. Introduction
In recent years, Nanotechnology has received greater attention in terms of design and development of nanomaterials-based products for various biomedical applications such as disease diagnosis, imaging and therapy.[1] Nanoparticles (NPs) are administered by various routes for the aforementioned applications, with oral route preferred as the first choice as it is the most convenient, is inexpensive and typically a safe method of administration. Following oral administration, the NPs either distribute to the local tissues or are potentially transported to systemic circulation and consequently to different tissues of the body, where they may interact with cells.[2] Therefore, it is necessary to understand the interaction of NPs with biological components such as the blood, tissues/ cells and the consequences of those interactions at molecular and genetic levels from toxicity aspects.
Among different types of nanomaterials, Titanium dioxide (TiO2) has received a greater interest for several biomedical applications such as disease therapy, drug delivery, cell imaging, genetic engineering etc. owing to its unique photocatalytic properties, outstanding biocompatibility, high chemical stability and low toxicity.[3] TiO2 is also approved as a food additive in Europe (EU No 1129/2011 171),[4] and food grade TiO2 NPs do not elicit a noticeable effect on human gut microbial flora when tested in vitro at low concentrations.[5] In its natural state TiO2 exists as different polymorphs such as anatase and rutile (the most familiar phases); these are the most common crystalline phases used for photochemical applications. Anatase polymorph is 10-times more active than the rutile phase. The absorption edge for anatase is 386 nm (lies in the near ultraviolet range) and is 416 nm (in the visible range) for rutile.[6] Although rutile phase is thermodynamically more stable and possess low band gap energy (Eg) of 3.02 eV compared to 3.2 eV of anatase, hitherto anatase is preferred for photocatalysis due to its larger surface area.[7] Both of these polymorphs not only differ in their photoactivity, but the different crystallographic orientations of the same material may also display diverse activities.[8] Interestingly, efficient photocatalytic activity of TiO2 can be achieved by the synergistic effect of a mixture of anatase and rutile particles.[9] Moreover, nanoform of TiO2 particles has been used as effective sunscreen agents due to their potential UVB (290-320 nm) and UVA (320-400 nm) filtering properties.[10]
TiO2 NPs are one of the most promising candidates used in photodynamic therapy (PDT) in cancers due to their efficient photocatalytic activity when illuminated with UV radiation. The basic mechanism behind the therapeutic potential of TiO2 NPs is the generation of reactive oxygen species (ROS) (singlet oxygen and superoxide radicals) by these NPs in aqueous solution in presence of light. ROS modify respiratory pathways in mitochondria and cause the release of electron transfer proteins that trigger apoptosis of cancer cells.[11] The antitumor activity of photoexcited TiO2 NPs modified chemically with polyethylene glycol on C6 rat glioma cells suggests a novel therapeutic approach for glioma.[12] Similarly, considerable reduction in the survival rate of human colon carcinoma cells observed after exposure to photoexcited TiO2 NPs implies their potential applications in the treatment of colon cancer.[13] The potential of TiO2 NPs in efficient killing of cancer cells in vitro by the generation of ROS under ultrasound irradiation signifies their utility in sonodynamic therapy.[14] Recent studies have shown that PEGylated TiO2 NPs dramatically enhanced the potency of photochemical therapeutic agents towards cervical cancer cells and thus could be employed as excellent candidates for PDT of cervical cancer.[15]
The relevance of TiO2 NPs for drug delivery and cell imaging purposes is evident from previous reports: TiO2 NPs functionalized with a phosphate containing fluorescent molecule i.e., flavin mononucleotide and loaded with anticancer drug Doxorubicin were employed in experiments involving human breast cancer cells BT-20 with promising findings.[16] Likewise conjugation of polyethylenimine modified TiO2 NPs with folic acid and subsequent encapsulation with paclitaxel, an anticancer agent, resulted in light-triggered drug release and targeted therapy.[17] The worth of TiO2-DNA nanoconjugates (TiO2 NPs bound to single stranded DNA oligonucleotides) as gene targeting devices and imaging agents for tumor detection are being established, where these nanoconjugates specifically cleave mutated genomic DNA in a sequence-specific and inducible fashion,[18] in tumor cells.
The importance of TiO2 scaffolds in tissue engineering is apparent from an earlier study wherein formation of hydroxyapatite (the main inorganic component of natural bone and all calcified tissues) on the surface of TiO2 scaffolds in simulated body fluids due to the transformation of amorphous calcium carbonate coated onto the inner surface of TiO2 scaffolds indicates the bioactivity of the scaffolds.[19] The efficacy of TiO2 NPs in tissue engineering applications is also evident from a previous report, wherein presence of TiO2 NPs in nanoforms of TiO2-poly(lactide-co-glycolide) (PLGA) composite scaffolds significantly improved the function of osteoblasts.[20]
On the other side, research also suggests that these NPs induce oxidative stress, cytotoxicity, genotoxicity, inflammation, apoptosis etc.[21] According to some reports, anatase NPs are more toxic than the rutile phase,[22] as anatase NPs appeared to be internalized to a greater extent than their respective bulk materials namely, rutile NPs.[23] In contrast, some studies showed that rutile NPs induced higher toxicity than anatase.[24] Furthermore, the rutile phase appears to be better absorbed orally than the anatase phase even though the absorption is usually low.[25] On the other hand, the potential of both anatase and rutile in inducing damage to human dermal fibroblasts has been demonstrated in vitro.[26] Also the role of anatase particles in erythrocyte and platelet aggregation has also been reported in vitro.[27] In vivo, the intragastric administration of anatase particles in mice resulted in the accumulation of titanium in the liver, with the resultant aggregation of these particles in hepatocyte nuclei leading to inflammation, apoptosis and subsequently the liver dysfunction.[28] Intraperitoneally (IP) administered anatase particles were shown to accumulate in the spleen and induced congestion, lymph nodule proliferation of spleen tissue and splenocyte apoptosis.[29] The disparity in these findings may be because experiments conducted in vitro environment cannot reproduce the complexity of in vivo system, which is ideal for the evaluation of biological fate of NPs with respect to biodistribution, toxicity mechanisms etc.
Although many studies have evaluated the toxicity of TiO2 NPs prepared using different methods, on different healthy or tumor cell types and also in vivo, studies reporting an in depth and systematic evaluation of NPs interaction with the key components relevant to systemic exposure in the body to assess their toxicity potential are limited. Hence, we have investigated the influence of interactions of similar shaped (nearly spherical), differently sized and crystalline structures of TiO2 NPs (anatase and rutile) with the biological milieu in vivo. We have determined the consequences of these interactions at systemic, tissue, cellular and sub cellular levels by measuring hemocompatibility (hematology and serum biochemistry), oxidative stress, genotoxicity and histopathology parameters. Previous studies have revealed that TiO2 NPs are mainly distributed to liver, spleen, lung and kidney tissues following oral administration,[30] hence we had selected these organs for studying oxidative stress, genotoxicity and histopathology. To understand the mechanism of cell death, we measured oxidative damage by estimation of glutathione (GSH) and lipid peroxidation (LPO). The in vivo genotoxic effects were analyzed by comet and micronuclei (MN) assays. These investigations are expected to provide important information useful for the interpretation of clinical data.
2. Experimental Section
2.1. Materials
Ethylene diaminetetraacetic acid (EDTA) disodium salt, sodium lauroyl sarcosinate (SLS) and Normal melting agarose (NMA) were obtained from Sigma Aldrich Corporation (St. Louis, MO 63103, USA). Ultra-low gelling agarose was procured from BDH Electran, BDH laboratory supplies (Poole, England; 44415 2G). 4',6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI) were purchased from Sigma Aldrich Chemicals Pvt Ltd (Bangalore, Karnataka, India). Boric acid was obtained from Glaxo Laboratories Ltd (Mumbai, Maharashtra, India). Phosphate buffered saline (Ca2+, Mg2+ free PBS), Trypan blue, 2-Thiobarbituric acid (TBA), Trichloroacetic acid (TCA), Ellman’s reagent (DTNB reagent-5,5’-Dithiobis(2-Nitro Benzoic Acid) were purchased from Himedia Pvt Ltd (Mumbai, Maharashtra, India). All others chemicals obtained were of analytical reagent grade.
2.2. Methods
2.2.1. Synthesis of TiO2 Anatase and Rutile NPs:
2.2.1.1. TiO2 Anatase
Titanium isopropoxide was used as the precursor for the synthesis of TiO2 anatase. Titanium isopropoxide was dissolved in ethanol, stirred for 10 min and then this solution was added to the ice cold distilled water in an ice bath. The mixture was stirred at room temperature for about 20 min and then ammonium hydroxide (NH4OH) was directly added to the solution, and the hydrolytic reaction took place resulting in the precipitation of TiO2, and was stirred vigorously for 1 hr. The crystallization of amorphous TiO2 was carried out at 100ËšC for 18 hrs under constant stirring at 400-500 rpm speed and was due to hydrothermal reaction. After crystallization, the precipitate was collected, washed and then dried in a vacuum oven at room temperature.
2.2.1.2. TiO2 Rutile
TiO2 rutile nanocrystals were prepared by wet chemical precipitation method using titanium (IV) chloride (TiCl4) as a precursor. Titanium chloride was added drop-wise to ice cold distilled water of pH ~1 pre-adjusted using nitric acid (HNO3) and then ammonia was added to it. Furthermore the mixture was refluxed under mild boiling condition for 18 hrs under constant stirring. Then the precipitate was collected and washed by centrifugation to remove impurities and dried in ambient conditions.
2.2.2. Preparation and Physicochemical Characterization of Aqueous Dispersions of TiO2 Anatase and Rutile NPs
Aqueous dispersions of TiO2 anatase and rutile were prepared by the addition of nano powders to milli-Q water followed by stirring to obtain a concentration of 100 µg/ml (stock). Prior to use for the experiments, the stock suspension was vortexed, sonicated using a probe sonicator at 34 W 40% amplitude for 10 min and vortexed again to obtain uniform suspension.
The morphology (size and shape) of anatase was characterized by Transmission Electron Microscopy (TEM, Technai G2 200 KV, FEI, Netherlands), whereas morphology of the rutile particles was studied using Field Emission Scanning Electron Microscopy (FESEM, S-4300-SE/N, Hitachi, Japan). The powdered anatase and rutile samples were subjected to X-ray Diffraction (XRD, Bruker AXS D8 Advanced XRD) studies to understand their crystal structure and were also tested for their optical absorption property (λmax) using UV-Visible spectrophotometer (PE Lambda 650, Perkin–Elmer UV–Visible spectrometer).
2.2.3. Animals and Treatment
All animal handling procedures were carried out as per the regulations of Institutional Animal Ethics Committee (IAEC), Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi with their prior approval for using the animals. Male Swiss Albino Mice (6-weeks old, 27±3 g) were procured from INMAS central animal facility and were accommodated in a 12 h day and light cycle environment with accessibility of diet and water at the controlled temperature of 23±2ËšC, humidity of 55±5%. Animals were adapted to this environment for 7 days prior to the experiment and were fasted overnight prior to treatment.
The animals were divided into 3 groups containing 3 mice each: Group 1- Vehicle Control (administered water); Group 2- TiO2 anatase (1.6 mg/kg body weight) and Group 3- TiO2 rutile (1.6 mg/kg body weight) administered orally. All animals were weighed prior to and at the end of each treatment. Selection of NP doses for the proposed study was based on the maximum concentration that resulted in a stable dispersion and the selected doses were also believed to be in the authentic human exposure range.[31,32] After treatment for 72 hrs, the animals were anaesthetized with inhaled methoxyflurane (Penthrane, Abbott Laboratories, UK) and blood was collected by cardiac puncture for hematology and serum biochemical assays and the organs were collected immediately for isolation of single cells and for histopathology studies.
2.2.4. Hematology and Serum Biochemical Assays
Whole blood collected from three groups of mice were mixed with disodium EDTA and hematological parameters were evaluated {Red Blood Cell (RBC) count, Hemoglobin (Hgb), Hematocrit (HCT), Mean corpuscular Volume (MCV), Mean Corpuscular Hgb (MCH), MCH Concentration (MCHC), Red Cell Distribution Width (RDW), White Blood Cell (WBC) count, Platelet count, Mean Platelet Volume (MPV), Plateletcrit (PCT)} using Automated hematology analyzer (CELLTAC α NIHON KOHDEN, MEK-6450K, Japan). Serum was obtained by centrifugation of whole blood at 2500 rpm for 15 min. Serum biochemical parameters such as Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), urea, creatinine and total bilirubin were analyzed by a fully Automated Biochemistry analyzer (Roche, Hitachi 902, Manheim, Germany). Standard controls were run prior to each assay.
2.2.5. Oxidative Stress
2.2.5.1. Total GSH Measurement
A 10% (W/V) homogenate of tissues (liver, spleen, kidney of untreated, and anatase and rutile treated mice) were prepared in 0.1 M ice cold PBS (pH 7.4) by homogenization using hand-held homogenizer (IKA T10, Germany). GSH levels in the tissue samples were estimated using Beutler (1963) method.[33] Tissue homogenates were centrifuged at 8,000xg (10,000 rpm) for 10 min at 4ËšC (Combi 514R, Hanil, South Korea). The supernatants obtained were treated with precipitating reagent (in 1:2 ratio) containing metaphosphoric acid, disodium EDTA and NaCl and centrifuged for 10 min at 2500xg (Combi 514R, Hanil, South Korea). The supernatant obtained was used for analysis. 200 µl of supernatant was mixed with 650 µl of disodium hydrogen phosphate (Na2HPO4) buffer (0.3M) and 150 µl of Ellman’s reagent and the absorbance of yellow color developed was measured at 412 nm. A blank without sample was prepared similarly and absorbance was recorded. Total protein content in the supernatants obtained by centrifugation of tissue homogenates was estimated by Bradford’s method. Total GSH content was expressed as µmoles of GSH/mg protein.
2.2.5.2. LPO Assay
LPO content in the tissue homogenates of liver, spleen, and kidney prepared as mentioned in section 2.2.5.1 was measured according to the method described by Laughton et al 1989.[34] Briefly, 600 µl of tissue homogenate was incubated with 100 µl of TCA (10% W/V) at 37ËšC for 1 hr and then centrifuged at 1000xg (Combi 514R, Hanil, S Korea) for 10 min at room temperature. To the supernatant, 100 µl of TBA (0.67% W/V in 0.025 M NaOH) was added and incubated at 80ËšC for 30 min. The absorbance of pink coloured TBA-Malondialdehyde (MDA) complex developed was measured at 535 nm using Gen5 software (Powerwave XS2, Biotek, USA). A blank without sample was prepared similarly and the absorbance was recorded. The extent of LPO was expressed as µmoles of MDA/mg of protein.
2.2.6. Genotoxicity
2.2.6.1. Single Cell Gel Electrophoresis Assay (Comet Assay)
Liver, spleen, and kidney tissues of untreated, and anatase and rutile treated mice were minced and a single cell suspension were prepared in 0.1 M chilled PBS. Cell viability was assessed as per the guidelines of Tice et al 2000.[35] The cell suspensions were diluted with PBS and viability was checked with 0.4% trypan blue dye. Viable cells with intact cell membrane excluded the dye and were counted using hemocytometer under inverted phase contrast microscope (Olympus CKX 31). Neutral Comet assay was performed as per the method of Khaitan et al 2006.[36] Briefly, cell suspension containing approximately 10,000 cells was mixed with 0.75% ultra-low gelling agarose was layered onto microscopic slides pre-coated with 0.1% NMA and incubated at 4ËšC for 10 min. Lysis was performed by placing the slides in neutral lysis buffer (2.5% Sodium dodecyl sulphate-SDS, 1% SLS, 25 mM EDTA; pH 9.5) for 15 min at 25-30ËšC. Slides were then washed in distilled water for 5 min and electrophoresis was performed at 2 V/cm (400 mA) for 5 min at 10ËšC in electrophoresis buffer (90 mM Tris base, 90 mM Boric acid, 2.5 mM EDTA; pH 8.4). Slides were rinsed again in distilled water for 5 min, air dried at 45ËšC on a hot plate and stored in a cool humid box until use. Following rehydration in distilled water, comets were stained with PI (50 µg/ml in PBS) and images were acquired in Olympus BX60 Fluorescence microscope with appropriate fluorescence filter (WG; Olympus) using FA87 monochrome CCD camera (Grunding, Germany) and Optimas Image Analysis Software (Optimas USA; version 5.2). Analysis of DNA distribution in the comets was performed by measuring Olive tail moment (OTM) and %tail DNA using Komet 5.5 software, Kinetic imaging, USA.
2.2.6.2. MN Assay
Air-dried slides containing methanol-acetic acid (3:1 V/V) fixed cells of liver, spleen, and kidney of untreated, and anatase and rutile treated mice were stained with a DNA specific fluorochrome DAPI (3 µg/ml) as described previously (Khaitan et al 2006).[36] Slides were then examined under fluorescence microscope using UV excitation filter and fluorescing nuclei were visualized under blue emission filter. Cells containing MN were counted from >1000 binucleated cells by employing the criteria of Countryman and Heddle (1976).[37] The fraction of cells containing MN (M-fraction %) was calculated as follows:
M-fraction %= Nm/Nt X 100, where Nm is the number of cells with MN and Nt is the total number of cells analyzed.
2.2.7. Histopathological Studies
The organs (liver, spleen, kidney and lung) isolated from untreated, and anatase and rutile treated mice were fixed in 10% formalin for 48 hrs at room temperature. The tissues were dehydrated using different concentrations of alcohol (70%, 80%, 95% and 100%) and xylene (clearing agent) using Automatic Tissue Processor (Spencers, Model No. 1040-STP-004) and were embedded in paraffin blocks in Tissue Embedding System (Spencers, Model No. 3080-STE-004), which were then sliced into 5 µm in thickness under microtome and placed onto clean glass slides. Following hematoxylin-eosin (H&E) staining, the slides were examined by light microscopy for histopathological changes.
2.2.8. Statistical Analysis
Data is presented in terms of mean and standard error of mean (mean±SEM) for biological replicates from each group of experimental mice. The data was analyzed using one way analysis of variance (ANOVA) with Dunnett’s test wherever applicable using Graph pad prism 6.05 (Graph pad software, Inc., La Jolla, CA, USA) to illustrate the significant difference between the untreated and treated groups. p<0.05 was considered statistically significant in all the cases.
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3. Results and Discussions
3.1. Results
3.1.1. Physicochemical Characterization of TiO2 Anatase and Rutile NPs
TEM studies indicated that the TiO2 anatase NPs were nearly spherical morphology of with a size range between 5-10 nm (Fig. 1(a)) whereas SEM studies of TiO2 rutile showed that the tips of TiO2 nanorods radially self-assembled to form submicron sized (600-800 nm) spheres (Fig. 1(b)). Highly intense peaks observed in the XRD pattern matched the typical pure anatase and rutile crystal structures of TiO2 (Fig. 2(a) and (b)). XRD patterns of both the anatase and rutile powders matched with the JCPDS data no # 361451 which represented the 2 Theta values (2θ) = 25.31Ëš, 35.00Ëš, 37.85Ëš, 48.00Ëš, 53.88Ëš and 76.20Ëš with corresponding planes (101), (103), (004), (200), (105), (301) for anatase; 2θ = 27.46Ëš, 36.00Ëš, 41.25Ëš, 56.62Ëš and 68.90Ëš with corresponding planes (110), (101), (111), (220), (301) for rutile. The λmax obtained for anatase and rutile was 260 nm and 270 nm (Fig. 3(a) and (b)).
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3.1.2. Body weight and Organ Weight
The three groups of mice did not show any significant difference in terms of body weight. Likewise, no obvious difference was observed in the weight of organs (liver, lung and kidney) of anatase and rutile particle treated mice compared to untreated mice. However, color change and significant increase in the weight of mice spleen were observed in mice treated with anatase and rutile particles.
Name of the parameter | Untreated | Anatase treated | Rutile treated |
WBC (x103/µl) | 4.50±0.34 | 7.10±0.46a,b | 4.1±0.40 |
RBC (x106/µl) | 8.85±0.57 | 8.36±0.30 | 7.22±0.34 |
Hgb (g/dl) | 13.6±0.57 | 12.4±0.69 | 11.1±0.57a |
HCT% | 42.5±4.04 | 39.7±1.73 | 37.6±1.73 |
MCV (fl) | 48.0±5.19 | 47.5±1.81 | 52.1±1.73 |
MCH (pg) | 15.4±1.15 | 14.8±0.69 | 15.4±1.15 |
MCHC (g/dl) | 35.3±1.76 | 31.2±1.15 | 29.5±1.73 |
PLT (x103/µl) | 742±34.6 | 594±28.8a,b | 812±34.6 |
LY% | 71.3±5.77 | 71.5±4.09 | 83.4±2.88 |
GR% | 28.7±2.3 | 28.5±2.3 | 16.6±1.15a,b |
RDW% | 13.6±1.73 | 12.8±0.69 | 17.6±1.3b |
PCT% | 0.26±0.03 | 0.21±0.017 | 0.25±0.02 |
MPV (fl) | 3.5±0.28 | 3.6±0.23 | 3.1±0.11 |
PDW% | 20.4±2.88 | 20.4±1.15 | 20.2±1.15 |
asignificant difference (p<0.05) compared to untreated. bSignificant difference (p<0.05) between TiO2 anatase and rutile particles. WBC-White blood cell; RBC-Red blood cell; Hgb-Hemoglobin; HCT-Hematocrit; MCV-Mean corpuscular volume; MCH-Mean corpuscular Hgb; MCHC-Mean corpuscular Hgb concentration; PLT-Platelets; LY-Lymphocytes; GR-Granulocytes; RDW-Red cell distribution width; PCT-Plateletcrit; MPV-Mean platelet volume; PDW-Platelet distribution width. |
3.1.3. Hematology and Serum Biochemical Assays
Hematological parameters assessed from the blood of anatase and rutile treated mice, determined by automated hematology analyzer are listed in Table 1(a).
There was a significant increase in WBC count in anatase treated mice compared to untreated and rutile treated mice. Reduction in granulocytes% was observed in the mice treated with rutile treated mice compared with untreated and anatase treated mice. Significant decrease in platelet count was observed in anatase treated mice compared to untreated and rutile treated mice. Reduced Hgb and increased RDW levels were observed in rutile treated mice compared to untreated and anatase treated mice.
Serum biochemical parameters, in the serum as detected by autoanalyzer, are listed in Table 1(b). Levels of serum enzymes such as SGOT and SGPT were significantly lower in anatase treated mice compared to untreated mice. SGOT levels were significantly lower in rutile treated mice compared to untreated and anatase treated mice. Significant decrease in the levels of creatinine and bilirubin were observed in rutile treated mice compared to untreated and anatase treated mice.
Name of the parameter | Untreated | Anatase treated | Rutile treated |
SGOT (IU/l) | 83.0±2.30 | 25±2.30a | 15±1.73a,b |
SGPT (IU/l) | 25±2.30 | 16±1.73a | 23±2.30 |
Urea (mg/dl) | 53±4.6 | 53±2.88 | 48±2.88 |
Creatinine (mg/dl) | 0.15±0.01 | 0.12±0.01 | 0.07±0.005a,b |
Total bilirubin (mg/dl) | 0.35±0.02 | 0.30±0.017 | 0.09±0.01a,b |
aSignificant difference (p<0.05) compared to untreated; bSignificant difference (p<0.05) between TiO2 anatase and rutile NPs. SGOT- Serum glutamate oxaloacetate transaminase; SGPT- Serum glutamate pyruvate transaminase |
3.1.4. Oxidative stress
The potential of TiO2 NPs in inducing oxidative stress was evident from the increased levels of GSH in liver, and spleen of anatase and rutile treated mice compared to untreated mice. Furthermore GSH levels in spleen of mice treated with anatase were higher compared to those treated with rutile particles (Fig. 4(a)). LPO levels were significantly higher in the liver and spleen of mice treated with anatase compared to untreated and rutile treated mice (Fig. 4(b)). No significant changes in the levels of GSH and LPO were observed in the kidney of mice treated with anatase and rutile compared to untreated mice.
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Name of the sample
|
Olive tail moment (OTM)
|
%Tail DNA
|
||||
|
Liver
|
Spleen
|
Kidney
|
Liver
|
Spleen
|
Kidney
|
Untreated
|
1.28 ±0.06
|
2.34 ±0.04
|
1.96±0.017
|
11.68 ±0.75
|
20.68 ±1.12
|
22.51 ±0.61
|
Anatase treated
|
2.14 ±0.05a
|
3.57 ±0.07a,b
|
4.03 ±0.06a
|
22.2 ±1.73a
|
28.9 ±1.15a,b
|
22.6 ±1.73
|
Rutile treated
|
3.63 ±0.04a,b
|
3.02 ±0.05a
|
4.15±0.08a
|
21.3 ±1.05a
|
23.2±1.03
|
24.3 ±1.15
|
aSignificant difference (p<0.05) compared to untreated; bSignificant difference (p<0.05) between anatase and rutile treatments
|
3.1.5. Genotoxicity
Oxidative DNA damage in liver, spleen and kidney cells of mice treated with anatase and rutile was evaluated by comet assay and MN assay. Cell viability was found to be greater than 90% for all the samples. DNA damage was observed in liver, spleen and kidney of mice treated with anatase and rutile particles as evident by increase in OTM and %tail DNA compared to untreated mice (Table 2(a)). DNA damage was also indicated from the slightly increased formation of MN in mice treated with anatase and rutile particles compared to untreated (Table 2(b)).
Name of the sample
|
Micronuclei (%)±SEM
|
||
|
Liver
|
Spleen
|
Kidney
|
Untreated
|
0.7±0.11
|
1.3±0.17
|
1.7±0.4
|
Anatase treated
|
1.50±0.2
|
2.0±0.28
|
1.9±0.11
|
Rutile treated
|
1.2±0.17
|
1.5±0.11
|
2.0±0.23
|
3.1.6. Histopathological studies
Histological changes in liver, spleen, lung and kidney tissues of untreated mice and mice treated with anatase and rutile particles were observed under light microscope following H&E staining. Liver from anatase treated mice showed ballooning of hepatocytes (BH) (Fig. 5(b)). Focal portal inflammation (FPI), interface hepatitis (IH) and spotty necrosis (SN) were observed in liver from rutile treated mice (Fig. 5(c)). Spleen of anatase and rutile treated mice showed moderate red pulp congestion (Fig. 6). Lungs of mice treated with anatase and rutile particles showed mild pulmonary vascular congestion i.e., mild enlargement in the lung blood vessels (image not shown). No histological changes were observed in kidneys from anatase and rutile treated mice.
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3.2. Discussion
The interactions of anatase and rutile particles were investigated in mice following 72 hrs oral administration and measurement of hematological and serum biochemical parameters, oxidative stress, DNA damage and histopathological changes in the selected organs. Hematological data showed an increase in WBC count in anatase treated mice, indicating “leukocytosis”, which could be due to inflammatory response induced by these NPs. Significant decrease in platelet count observed in anatase treated mice compared to untreated and rutile treated mice leading to a condition known as “thrombocytopenia”, support the potential role of these NPs in platelet aggregation. Undesired platelet aggregation may be coupled with fractional or whole blockage of blood vessels leading to consequences such as cerebrovascular accident. The slight reduction in the RBC count of rutile treated mice compared to untreated mice suggests potential induction of hemolysis by these NPs. Significantly decreased Hgb in rutile treated mice might be attributed to the adsorption of Hgb onto the surface of these particles due to the interaction between Hgb and rutile, thus altering the surface functionality of NPs. Significantly increased RDW observed in rutile treated mice compared to untreated and anatase treated mice could be attributed to the variation in RBC size due to fragmentation. SGPT (ALT)/SGOT (AST) ratio, a more sensitive indicator of hepatic injury was significantly higher in both anatase (ALT/AST=0.64) and rutile (ALT/AST=1.53) treated mice compared to untreated mice (ALT/AST=0.3). Low levels of creatinine and bilirubin are indicators of liver damage.
The potential role of TiO2 NPs in inducing excess ROS formation, as evident from the previous report by Khan et al 2015,[38] is responsible for induction of oxidative stress. The increase in GSH levels in the present study may be a response to the excess ROS formation and might thereby slightly diminish LPO levels in kidney of mice treated with rutile particles. DNA damage was evident from the significant increase in the comet parameters of liver, spleen in anatase treated mice and liver of rutile treated mice compared to untreated mice and the damage was significantly higher in the spleen of mice treated with anatase than rutile. In the present study, DNA damage induced by anatase and rutile particles could be potentially due to either the direct interaction of NPs with cellular DNA and/or the interaction of higher concentrations of LPO products resulting from treatment with NPs (as these also evident from the increased LPO products as discussed in section 3.1.4). Increased DNA damage observed in liver and spleen of mice treated with anatase and rutile particles might be due to the enhanced absorption and distribution of these NPs in these tissues.[39]
Liver of anatase treated mice showed ballooning of hepatocytes (BH), a cause of extreme hepatocyte injury. Balloned hepatocytes are the enlarged hepatocytes with a central nucleus, surrounded by fluffy white cytoplasm and undergo apoptosis. The process of ballooning is irreversible, which leads to lytic necrosis and resulting in liver injury. Balloning observed in the present study may possibly be due to the massive damage to cytoskeletal proteins due to oxidative stress and the progressive retention of damaged structural proteins resulting in hepatocyte endoplasmic reticulum (ER) stress. ER stress confounds efforts to clear the ubiquitinated keratins, causing clumps of ubiquitinated cytokeratins to form in the swollen (balloned) cells.[40] FPI (suggesting stimulation of inflammatory response), IH and SN observed in the liver of rutile treated mice are the integral parts of morphologic spectrum of chronic hepatitis. The finding indicates that exposure to rutile particles result in their accumulation in liver and aggregation of these particles in the nuclei of hepatocytes and led to inflammation. As a result, the inflammatory infiltrates are extended to the adjacent parenchyma (leads to a condition known as IH), thereby hepatocytes undergo necrosis and the necrotic cells spread throughout the tissue both singly and in small groups led to liver injury. Granulocyte migration to the site of inflammation and their accumulation in the vicinity of necrotic liver cells might be the reason for significant decrease granulocytes% in the blood of rutile treated mice. The spleen of anatase and rutile treated mice showed moderate red pulp congestion as evident from the colour change and increased spleen weight (as mentioned in section 3.1.2). This may possibly resulted due to the distribution of TiO2 NPs to the red pulp of spleen led to congestion in the red pulp, which may leads to splenomegaly (enlargement in the spleen). The enlarged spleen may accumulate the WBC and platelets, and thus reducing their count in the blood and resulting in the decreased granulocytes% in the rutile treated mice and decreased platelet count observed in the mice treated with anatase. Even though low levels of serum creatinine and higher DNA damage were observed in the kidney of mice treated with anatase and rutile, no histological alterations were noticed in both the treatments.
Lungs of mice treated with anatase and rutile showed mild pulmonary vascular congestion i.e., mild enlargement in the blood vessels of lungs. An inhalation study by Oberdorster et al 1994,[41] reported that TiO2 NPs agglomerates of approximately 700 nm dissociate into smaller aggregates after deposition in the lung.
Retention of these smaller aggregates in lung epithelia may lead to mild enlargement in the lung blood vessels, a condition known as mild pulmonary vascular congestion.
In the present study, it is evident that the toxic effects observed in mice treated with TiO2 anatase and rutile NPs with the biological components were size and crystallinity dependent. This could be due to the differences in distribution pattern and the interaction mode of these NPs with the biological system and thus establish the resultant implications in distinct ways.
4. Conclusions
In summary, the present study showed that the acute exposure of TiO2 anatase and rutile NPs for 3 days resulted in histological alterations in liver and spleen of mice. Through evaluation of toxicity mediated by these particles, we found significant alterations in liver and spleen at the (i) tissue (ballooning of hepatocytes, focal portal inflammation, spotty necrosis, interface hepatitis; splenomegaly) (ii) cellular (induction of oxidative stress) (iii) and sub cellular (increased genotoxicity) levels. Significant alterations in the hematological parameters were found with respect to decreased platelet and granulocytes count. Furthermore the data secured from the present study would allow devising an association between the exposure of NPs and the observed toxicity. Such preclinical studies may aid in understanding the biological effects, and toxicity mechanisms associated with the NPs and might help in the interpretation of clinical data and design and development of safer NPs for biomedical applications. Additional investigations on the cellular uptake, cytotoxicity, inflammatory response, modifications in gene expression profile etc. of TiO2 NPs in vivo may present more information on the biological safety of the NPs designed for biomedical applications.
Acknowledgements
Authors are grateful to The Director, Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi for providing necessary facilities for animal experiments.
Conflicts of Interest
The authors declare no conflict of interest.
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