Introduction
The nanomaterials (NMs) have got various applications in optical, electronics, electrical, mechanical, magnetic, industrial, information technology. Advancements in nanotechnology have led to the development of the nanomedicine, which involves nanodevices for diagnostic and therapeutic purposes (Sahoo et al., 2007). These NMs are also used in sunscreens, cosmetics, food products, surface coatings, anti-bacterial, anti-fungal, antibiotic products (Zhang et al., 2012). A key requirement for the successful use of the nanoparticles (NPs) in biomedical applications is their good dispensability, colloidal stability in biological media, internalization efficiency, and low toxicity (Zhang et al., 2008). Although NPs are widely used in the field of biomedicine, there is a lack of information concerning the impact of manufactured NMs on human health as well as on the environment (El-Ansary and Al-Daihan, 2009). It has been reported that these NPs in general have different physicochemical properties from those of corresponding microparticles (MPs) such as higher specific surface area, increased surface reactivity, cell permeability and increased quantum effects as a function of their size (Hochella et al., 2008; Ivask et al., 2015). The utilization of NMs has increased throughout the world exponentially and the concerns also over their safe use for mankind and environment. Further, NPs modulate a broad range of biological responses resulting in cellular toxicity and genotoxicity (Zolnik et al., 2010). Therefore, toxicological profiling is necessary to understand the mechanism of NPs and MPs. Compounds found to be inert in the bulk form may become toxic at the nano form, because of their greater affinity to biological systems and reactivity in comparison to MPs (Nel et al., 2006; Oberdörster et al., 2005). The fact that NMs have been consistent in eliciting more pronounced toxicity than their micro counterparts was demonstrated in several studies with different NPs (Chen et al., 2008; Wang et al., 2007; Singh et al., 2013a; Arnold, 2013). As particle size becomes smaller, the ratio of surface atoms to those in the interior increases, which is the specialized role of concern of that material (Trindade et al., 2001). Consequently, NPs are able to get entry into the body via inhalation, dermal and oral routes. Most of the health hazards occur through the contact with nano-structured surfaces, or by the consumption of foods with nano sized colloids (Roco and Bainbridge, 2005).Some studies have shown that, upon administration of oral nano formulations to mice, they got accumulated in the liver and caused toxicity (Wang et al., 2007). Previous investigations have revealed that the NPs get absorbed through GI tract; first pass metabolism takes place and enters the systemic circulation. The entry of these NPs into GI tract could be via accidental ingestion through varied sources such as at manufacturing industries along with the drinking water or eating food contaminated with NPs (Ahamed et al., 2011).
Metal oxide NPs are used in various consumer applications includes nanomedicine, nanosensors and nanorobotics (Oberdörster, 2010). Zinc Oxide, Iron oxide, manganese oxide, magnesium oxide (MgO) NPs have high potential as contrast enhancement agents in MRI procedures to render excellent anatomical images (Estelrich et al., 2015). The potential cytotoxicity of NPs may be due to oxidative stress induced by reactive oxygen species (ROS). Thus, oxidative stress is a touchstone for comparing the toxic effects of NPs. In addition, the adverse effects of ROS generation might lead to apoptosis, which induces significant cell structure damage, membrane lipids, proteins, and the nuclear membrane (Xia et al., 2006). Many studies have demonstrated the ability of NPs to generate ROS in a cell-free environment. The evaluation of the oxidative potential of NPs is thus an important part of assessing their toxicity (El-Ansary and Al-Daihan, 2009).
MgO NPs have attracted wide scientific interest due to ease of synthesis, chemical stability and unique properties. Magnesium (Mg) plays an important role in the body, which mediates cell- extracellular matrix interactions, bone apatite structure and density. However, the toxic effects on humanity should also be of concern with the increased application of nano MgO. It is necessary to evaluate both the beneficial and toxic effects of these NMs in biological systems. Nano MgO are used in various fields like nano cryosurgery (Di et al., 2012), electrochemical biosensors (Patel et al., 2014), and to enhance the adhesion and proliferation of bone cells (Moorsom et al., 2014). MgO is also used as an antacid, detoxifying agent, and for bone regeneration (Bertinetti et al., 2009; Martinez-Boubeta et al., 2010). The cytotoxicity effects of MgO NPs have been investigated on human intestinal cell line and human cervical cancer cell line (Patel et al., 2013); human astrocytoma U87 cells (Lai et al., 2008); Human umbilical vein endothelial cells (HUVECs) proliferation (Ge et al., 2011); human cardiac microvascular endothelial cells (Sun et al., 2011); human liver cancer cell line (Kumaran et al., 2015).
Three different doses were used in the current investigation which ranged from highly toxic to least or no toxicity. The highest dose of 1000 mg/kg bw, which showed symptoms of toxicity was chosen to see the effect when large quantities of NPs are released accidentally into the environment, and they reach the human body (Kumari et al., 2014); Further, the low dose of 100 mg/kg bw, was chosen to speculate probable human exposure as the workers unintentionally get exposed to the NPs through hands and to the mouth during manufacturing processes (Singh et al., 2013b).
We are aware that the acute oral toxicity of MgO NPs and MPs in albino Wistar rats has not been investigated till date. In vivo study of NMs is important because animal systems are extremely complicated and interactions of these NPs with biological systems could lead to novel immune response, metabolism patterns, biodistribution and clearance which provides useful information on likely health hazards assessment in mankind (Fischer and Chan, 2007). Moreover, as GI tract is a crucial portal of entry of NPs in humans and animals, acute oral toxicity study of MgO NPs have been conducted following Organization for Economic Cooperation and Development test guidelines 420 (OECD, 2001). The characterization of NPs is required before predicting the toxicity to biological components (Murdock et al., 2008). Hence, in current study, the NPs and MPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), X- ray diffraction (XRD) and Brunner–Emmett–Teller (BET) analysis. The genotoxicity studies are crucial part of safety assessment of NPs in order to understand the mutations and cancer inductions. The in vivo genotoxicity was studied using comet assay (Tice et al., 2000), micronucleus test (MNT) (Schmid, 1975) and chromosomal aberration (CA) assay (Preston and Dean, 1987) Biochemical parameters are used to explore the NPs induced oxidative stress via reactive oxidative species (ROS). Biochemical markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione content (GSH), lipid peroxidation (MDA), lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD), and biochemical tests in serum (albumin, calcium, glucose, chloride, cholesterol, HDL cholesterol, triglycerides and creatinine) were evaluated in the treated rats in comparison with controls. Histopathological examination is necessary to determine the morphological changes due to NPs exposure and also for assessing the toxicological effects (Reddy et al., 2015). Therefore histopathology of the liver, kidney, heart, spleen, and brain was carried out from treated and control rats by haematoxylin and eosin staining method (H & E). Metal content analysis of NPs is essential to understand the amount of metal that enter in the target tissue or site to know their anatomic fate, biological effects, uptake and clearance. The amount of metal content in rat’s whole blood, liver, kidney, spleen, lungs, heart, brain, urine and feces was analyzed using inductively coupled plasma optical emission spectrophometry (ICP-OES).
Materials and methods
Chemicals:
MgO NPs (MgO < 40nm, 99.9%, CAS No. 1309-48-4) were purchased from Sigma Chemical Co. Ltd (St Louis, MO, USA). MgO MPs (fine powder, 99.9%, GRM1031, CAS No. 1309-48-4) (according to the manufacture’s data sheet) and all other chemicals were purchased from Himedia, Mumbai, India.
Characterization
The particles were characterized using TEM, BET, XRD, DLS and LDV analysis to evaluate the material size and morphology, surface area, crystal structure, size distribution, zeta potential and state of dispersion in Milli-Q water. Characterization of MgO NPs was performed to assess the size and morphology using a TEM (JEM-2100, JEOL, Japan). For the determination of average particle size of NPs and MPs, 100 particles were measured from different fields of view and pictures showing the general shape and structure of the particles. The specific surface area (m2/g) of the particles was determined by N2 adsorption– desorption measured at 77 K according to the BET protocol using a Quadrusorb-SI V 5.06 analyzer (M/S Quanta chrome Instruments Corporation, USA). The XRD pattern of the particles was documented on a Bruker AXS D-8 Advance powder X-ray diffractometer (Shimadzu, Japan). The instrument was operated at a current of 30 mA, voltage of 40 kV and utilizing a CuKα radiation (λ = 1.5406 Å). The size of the NPs and agglomerates was measured through DLS and LDV using a Malvern Zetasizer Nano-ZS (Malvern Instruments, UK. At the concentration of 40 µg/ml freshly prepared MgO NPs and MPs suspension in Milli-Q water were ultra sonicated using a probe sonicator (UP100H, Hielscher Ultrasonics GmbH, Teltow, Germany) for 10 min at 90% amplitude. The polydispersity index (PdI) was used to measure the different size ranges exist in the solution. The values of PdI scale lies in the range of 0 to 1, where the PdI scale value near to 0 indicates monodisperse and 1 indicates polydisperse state of the particles.
Animals
The animal model used for the study was female albino Wistar rats. They were procured from the National Institute of Nutrition (NIN), Hyderabad, India. The animals were 6-8 weeks old and had a weighs ranging from 120-140 g. The animals were housed in polypropylene cages, five animals kept per cage. The animals were given standard laboratory pellet diet and water ad libitum. They were kept at a humidity of 55-65%, temperature of 22±3°C and automated light cycles of 12 h light/12 h dark. The study design and experimental protocol was submitted to Institutional Animal Ethics Committee and the approval had a No. IICT/BIO/TOX/PG/25/06/2014/08.
Acute oral toxicity study
The acute oral toxicity of MgO NPs and MPs were assessed in accordance with the guideline 420, known as the “acute oral toxicity-fixed dose method” (OECD, 2001). According to the sighting study, a single rat was treated with an initial 5 mg/kg body weight (bw) dose. After careful observation and confirming no adverse signs and mortality a second dose of 50 mg/kg bw was administered to another rat followed by 300 mg/kg bw dose and maximum dose of 2000 mg/kg bw consecutively. Rats were fasted overnight prior to dosing. There was no evidence of toxic sign and death of rats at all the dose level in sighting study. Therefore, the main study was carried out with four female rats using 2000 mg/kg bw dose for MgO NPs and MPs. After the dosing the treated rats were monitored for a period of 14 days. The feed intake and bw were monitored daily during the observation period. Recordings were made regularly for mortality, signs and symptoms if any. At the end of the test period the animals were weighed and sacrificed for necropsy studies. Liver, kidneys, spleen, Heart, and brain tissues were collected for relative organ weight profile and histopathology studies.
Histopathological evaluation
Histopathological studies were conducted in the all visceral organs i.e. liver, kidneys, spleen heart and brain of female albino Wistar rats after acute oral exposure to 5, 50, 300 and 2000 mg/kg bw of MgO NPs and MPs to assess the changes in the tissue anatomy. After sacrifice, the tissues were collected and washed with ice cold normal saline (0.9 %) to remove any extraneous material. The tissues were fixed in formalin solution (10%). After the fixation, tissues were processed (Leica TP 1020) and embedded in paraffin blocks (Leica EG 1160). The paraffin blocks were sliced into 3 µm thick ribbons (Microm H 360) and placed on clean microscope slide. H & E dye was used for staining the tissue sections on a slide and analyzed for histological alterations using Nikon Eclipse E 80 microscope at ×400 magnification. A minimum of three slides per block were assessed for histological changes of the tissues.
Experimental design: genotoxicity, biochemical and bio distribution studies
Prior to treatment of rats, MgO NPs and MPs were suspended in Milli-Q water, properly ultra sonicated (UP100H, Germany) and vortexed before every treatment of the rats. The rats were divided into three main groups (5 rats in each group), a positive control (for the genotoxicity studies), negative control and experimental group. The experimental groups was further divided into low (100 mg/kg bw), medium (500 mg/kg bw) and high dose (1000 mg/kg bw). The doses were administered once using a suitable incubation cannula with volume 0.01 ml/1g bw. Control group received only Milli-Q water. A known mutagen CP, was used as the positive control at a dose 40 mg/kg bw and the volume injected 0.01 ml/g bw was given intraperitonially 24 h before sacrifice. Dose selection is on the basis of an initial range finding study. The highest dose was selected based on induction of a toxic effect without severe sufferings and mortality, whereas, the lowest dose demonstrated slight adverse effects. All treated rats were sacrificed by cervical dislocation at sampling times of 18h and 24h.
Genotoxicity parameters:
Comet Assay
The alkaline comet assay was conducted with whole blood of the rats after acute oral exposure to 100, 500 and 1000 mg/kg bw of MgO NPs and MPs for the evaluation of DNA damage. The test was carried out in accordance with method by Tice et al. (2000) and guidelines 489 (OECD, 2014). Whole blood was withdrawn from retro orbital plexus collected in heparinized tubes at 18h and 24 h after dosing. Cell viability was determined by the trypan blue exclusion assay (Pool‐Zobel et al., 1994). In brief 10 µl of whole blood (10,000-30,000 lymphocytes) was suspended in 120 µl of 0.5% LMA and embedded in between the layers of NMA (0.75%) and LMA (0.5%) on microscopic slides and spread uniformly with cover slip. The slides were kept for drying at 4°C for 10 min. The cover slip was then removed and slides were immersed in chilled lysis buffer (2.5 M NaCl, 0.1 M Na2 EDTA, 0.2 M NaOH, 1% Triton X-100, 10% DMSO, pH 10.0) for 10 h at 4°C for facilitating alkaline unwinding of DNA, all slides were soaked for 20 min in alkaline buffer (10 M NaOH, 200 mM Na2 EDTA, pH > 13.0) and then electrophoresis was performed at 25 V (or 300 mA) for 25 min. The neutralization of slides was done twice in 0.4 M tris buffer, pH 7.5, for 5 min and followed by methanol fixation. The slides were analyzed by staining with ethidium bromide (20 μg/ml) using a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan) and scored for DNA damage blindly in order to avoid bias. 50 randomly selected cells per slide (150 cells per rat) were analyzed to estimate the amount of DNA damage and expressed as the percentage of DNA in the comet tail. Quantification of DNA breakage was carried out by using CASP software version 1.2.2 (Comet Assay Software, CaspLab) to calculate the amount of DNA damage and expressed as a percentage of DNA in the comet tail.
Micronucleus test (MNT)
The MNT was performed in the bone marrow cells, which were extracted from femur and tibia of the rats by aspiration after acute oral exposure with MgO NPs and MPs at sampling times of 18 and 24h as per protocol of Schmid (1975) and guideline 474 (OECD, 1997a) with slight modifications. The extractions were placed in hypotonic solution made of 1% sodium citrate and centrifuged to get a pellet. The pellet was resuspended in 1% sodium citrate and used to prepare smears on microscopic slides and dried overnight in humidified air. The MNT in PBL cells was performed according to the protocol described by Celik et al. (2005) and guideline 474 (OECD, 1997a). Whole blood was collected from the retro-orbital plexus of rat and smears were made which were allowed to dry. The air dried slides were fixed in methanol and stained with 0.5% Giemsa for 3 min. The stained slides were used for the assessment of the micronucleus occurrence. Three slides were made for each animal and microscopically examined at ×1000 magnification. 2000 polychromatic erythrocytes (PCEs) per animal were assessed from the three slides and the micronucleated PCEs frequency (MN-PCEs) was recorded. The ratio of PCEs to normochromatic erythrocytes (NCEs) in the bone marrow and peripheral blood, was determined by examining 1000 cells from each animal and expressed as percentage (PCEs ×100/PCEs + NCEs).
Chromosomal Aberration assay
The method described by Preston and Dean and guideline 475 (OECD, 1997b) was used as a test method to carry out CA analysis in bone marrow cells. For analysis of metaphase cells, cell division was arrested by a mitotic inhibitor, colchicine (0.02%), injected intraperitonially 2h prior to sacrifice. The bone marrow was collected from femur and tibia by rinsing in hypotonic solution with 0.56% potassium chloride and centrifuged. Cells were then fixed in ice-cold Carnoy’s solution (methanol: acetic acid, 3:1 v/v) until the pellets were clean. After 24h refrigeration, cells were centrifuged and resuspended in fresh fixative i.e. Carnoy’s solution, dropped onto slides, dried and stained with Giemsa. Three slides for each animal were made by the flame-dried technique. Five hundred well spread metaphases per dose (100/animal) were selected to detect the presence of CAs. The mitotic index (MI) was determined with 1000 cells.
Biochemical assays
The whole blood samples were collected by puncturing the retro-orbital plexus of rats. For hematology parameters, the blood was collected in EDTA-coated tubes. For biochemical estimations, the samples collected in the tubes without anticoagulant were kept for 45 min at room temperature. The serum obtained was then centrifuged at 2500 rpm for 15 min. The liver and kidney were quickly dissected, washed in ice-cold saline and then with buffer of pH 7.4 (0.15 M Tris-HCl), dried and weighed. Tissues were then homogenized in ice-cold sucrose (0.25 M) to make 10% homogenate (w/v) with a Miccra D-1 high speed tissue homogenizer. Serum and tissue homogenates (liver and kidney) were used to analyse the reduced GSH content, catalase, SOD, LDH and lipid peroxidation as well for estimation of AST, ALT and AKP levels. All the biochemical indices were estimated after acute oral treatment with 100, 500 and 1000 mg/kg bw doses of MgO NPs and MPs using a spectrophotometer (Spectramax Plus, Molecular Devices, USA).
Hematological estimation
The blood parameters such as red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT) and mean corpuscular volume (MCV) were determined by using hematology analyzer ABX Micros ES 60 (HORIBA Medical, France).
AST, ALT and AKP levels
AST, ALT and AKP enzyme levels were measured according to procedure of Yatzidis et al. (1960) in serum as well as in tissue homogenates (liver and kidney). AST and ALT enzyme levels were expressed as µmol/h/ml in serum and µmol/h/mg of tissue homogenates (liver and kidney). The levels of AKP was determined following p-nitrophenyl phosphate (PNPP) method as described in kit from M/s. Siemens Healthcare Diagnostics Ltd., Baroda, Gujarat, India.
Reduced glutathione content
The GSH content was analyzed in serum, liver and kidney in according to the protocol of Jollow et al. (1974). The quantity of GSH present was expressed as µg GSH/ml of serum and µg GSH/g wet weight of tissue for tissue homogenates (liver and kidney).
Catalase
Catalase activity in serum and liver and kidney homogenates was determined by standard protocol of Sinha (1972). Quantity of H2O2 consumed was measured by reading the absorbance at 570 nm. The enzyme activity was expressed as U/mg protein.
Lipid peroxidation
Malondialdehyde (MDA) which is the end product of lipid peroxidation was measured in serum and tissue (liver and kidney) homogenates according to the procedure of Ohkawa et al. (1979) with slight modifications. The result of reaction among serum, tissue homogenates with thiobarbituric acid (TBA) and 15% of trichloroacetic acid (TCA) reagent formed TBA–MDA complex. The absorbance of this end product (pink colored) was estimated at 532 nm. The quantity of MDA was determined using a molar extinction coefficient of 1.56×105 M/cm and expressed as nmoles of MDA formed/gm wt of tissue. Protein was determined as described by Lowry et al. (1951).
Magnesium content analysis in tissues
The biodistribution study of acute dose of MgO NPs and MPs was carried out in female albino Wistar rats. The animals were placed in metabolic cages to collect the urine and feces samples along with control groups at sampling times of 24 and 72 h after dosing. Rats were sacrificed through cervical dislocation and whole blood, liver, kidneys, heart, brain and spleen were collected. The samples were processed using the method of Gómez et al. (1997). Nitric acid digested (overnight) samples were heated at 80°C for 10h, and heated additionally heating at 130-150°C for 30 min. Subsequently, perchloric acid (0.5ml, 70%) was added, followed by heating at 130-150oC for evaporating to dryness. Nitric acid (2%) was used to digest the dried sample and filtered with Whatman filter paper. The filtrate was made to a final volume of 5 ml with 2% nitric acid solution for ICP-OES analysis. The standard solution of Mg was serially diluted to 100, 50, 10, 1 ppm and wavelength of 418.66 nm was found to get intensity of samples. The Mg content in the samples was determined using ICP-OES (JY Ultima, JobinVyon, France).
Statistical analysis
The results were analyzed for the statistical significant changes between treated and control groups by two-way ANOVA. All results were expressed as mean and standard deviation (mean±SD) of the mean. Multiple pair-wise comparisons were done using the Bonferroni posttest to verify the significance of positive response. Statistical analyses were performed using GraphPad Prism 5 Software package for windows (GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance for all tests was set at P<0.01.