Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration

Mutagenesis, Oct 2014

The applications of cerium oxide nanoparticles (CeO2 NPs; nanoceria) extend to polishing agents, diesel fuel additives and as a putative antioxidant in therapeutics. Therefore, understanding the long-term toxic effects of CeO2 NPs is of particular importance. This study investigated the 28 days of repeated toxicity of 30, 300 and 600mg/kg body weight (bw)/day of nanoceria and CeO2 microparticles (MPs) in Wistar rats after oral exposure. Genotoxicity was analysed using comet, micronucleus (MN) and chromosomal aberration (CA) assays. The results demonstrated a significant increase in DNA damage in peripheral blood leukocytes and liver, MN and CA in bone marrow as well as MN in peripheral blood after exposure to CeO2 NPs at 300 and 600mg/kg bw/day. Significant alterations were observed in alkaline phosphatase and lactate dehydrogenase activity in serum and reduced glutathione content in the liver, kidneys and brain at 300 and 600mg/kg bw/day in a dose-dependent manner. Conversely, CeO2 MPs did not induce any significant toxicological changes. A much higher absorptivity and significant tissue distribution of CeO2 NPs was perceived in comparison to CeO2 MPs in a dose-dependent manner. A substantial fraction of CeO2 NPs was cleared by urine and faeces. Histopathological analysis revealed that CeO2 NPs caused alterations in liver, spleen and brain. Further, distinct difference in the data among genders was not obvious. In general, the results suggested that prolonged oral exposure to nanoceria has the potential to cause genetic damage, biochemical alterations and histological changes after retention in vital organs of rats at high concentrations.

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Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration

Mutagenesis Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration Monika Kumari 0 1 Srinivas Indu Kumari 1 Paramjit Grover 1 0 Department of Genetics, Osmania University , Osmania University Main Road, Hyderabad 500007, Andhra Pradesh , India 1 Toxicology Unit, Biology Division, Indian Institute of Chemical Technology , Hyderabad 500007, Andhra Pradesh , India The applications of cerium oxide nanoparticles (CeO2 NPs; nanoceria) extend to polishing agents, diesel fuel additives and as a putative antioxidant in therapeutics. Therefore, understanding the long-term toxic effects of CeO2 NPs is of particular importance. This study investigated the 28 days of repeated toxicity of 30, 300 and 600 mg/kg body weight (bw)/day of nanoceria and CeO2 microparticles (MPs) in Wistar rats after oral exposure. Genotoxicity was analysed using comet, micronucleus (MN) and chromosomal aberration (CA) assays. The results demonstrated a significant increase in DNA damage in peripheral blood leukocytes and liver, MN and CA in bone marrow as well as MN in peripheral blood after exposure to CeO2 NPs at 300 and 600 mg/kg bw/day. Significant alterations were observed in alkaline phosphatase and lactate dehydrogenase activity in serum and reduced glutathione content in the liver, kidneys and brain at 300 and 600 mg/kg bw/day in a dose-dependent manner. Conversely, CeO2 MPs did not induce any significant toxicological changes. A much higher absorptivity and significant tissue distribution of CeO2 NPs was perceived in comparison to CeO2 MPs in a dose-dependent manner. A  substantial fraction of CeO2 NPs was cleared by urine and faeces. Histopathological analysis revealed that CeO2 NPs caused alterations in liver, spleen and brain. Further, distinct difference in the data among genders was not obvious. In general, the results suggested that prolonged oral exposure to nanoceria has the potential to cause genetic damage, biochemical alterations and histological changes after retention in vital organs of rats at high concentrations. Introduction Nanoparticles (NPs) differ from larger materials in the number of atoms at the surface due to their small size (<100 nm). Their physical properties comprising high surface area per unit mass, enhanced chemical and surface reactivity, magnified cell permeability, which are different from bulk materials, have led to increased use of NPs in various commercial applications ( 1,2 ). However, size-dependent toxicity of various nanomaterials (NMs) has also been reported (3). Therefore, toxicological profiling of NMs, which have wide consumer value, is warranted. The ability of cerium oxide (CeO2) NPs (nanoceria) to switch oxidation states between III and IV based on environmental conditions makes CeO2 NPs comparable to that of biological antioxidants ( 4 ). Nanoceria has been included in the list of 14 representative manufactured NMs for testing owing to their existing commercial use and high production volume ( 5 ). The potential uses of CeO2 NPs incorporate pharmaceutical industry ( 6 ) and in nanotherapeutics ( 7 ). Among the several uses of CeO2 NPs, some are used as a polishing agent for glass mirrors, plate glass, television tubes, ophthalmic lenses and precision optics ( 8 ). In cosmetic industry, CeO2 NPs are used as UV-absorbing compound in sunscreen ( 9 ) and as a UV-scattering agent in non-irritating lipsticks ( 10 ). Additionally, CeO2 NPs are used as a diesel fuel-borne catalyst to reduce particulate matter ejection in emission control system of automobiles ( 11 ). Thereupon, hazard of accidental exposure to environment and their entry into human body through the food chain is inevitable ( 2 ). Some recent publications have suggested that the exposure to CeO2 NPs in various organisms and cells can cause adverse effects on their growth and development ( 12,13 ). The potential for human health hazards and the environmental effects following exposure to CeO2 NPs has been examined by in vitro studies ( 14–16 ). Further, there are studies that have investigated CeO2 NPs induced toxicity through inhalation, intratracheal instillation and intravenous (iv) administration routes of exposure in rats ( 17–20 ). On the contrary, there is scarcity of oral exposure reports. Therefore, the objective of the current study was to evaluate the toxicity of CeO2 NPs through oral route. Characterisation is important for the assessment of the particle behaviour in the biological system because physical and chemical properties are likely to play an important role in determining the behaviour, reactivity and potential toxicity of NPs ( 21 ). Hence in the present study, measurement of size, hydrodynamic diameter and surface charge of CeO2 NPs was described and compared with CeO2 microparticles (MPs). Genotoxicity studies are fundamental to assess hazardous effects of NPs as despair of genetic diseases and cancers is well known. Therefore, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. The comet assay is useful for the detection of DNA damage in genotoxicity testing. This assay in animal models can give information on secondary effects such as inflammation due to reactive oxygen species (ROS) production ( 22 ). Maffei et al. ( 23 ) suggested micronucleus test (MNT) as a promising biomarker for the early detection of colorectal cancer and demonstrated the significant association of micronucleus (MN) frequency with the clastogenic activity in the plasma. During anaphase, MN are formed from chromosomal fragments or whole chromosomes that are left behind when the nucleus divides. The chromosomal aberration (CA) test can diagnose agents that cause structural chromosome or chromatid breaks, dicentrics and other abnormal chromosomes, translocations that are implicated in the various human genetic diseases and cancers ( 24 ). Injuries to the liver and oxidative stress may increase alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and diminish the reduced glutathione (GSH) content. Therefore, evaluation of biochemical indices needs to be addressed. Further, NPs can have different toxicokinetic profiles such as absorption, distribution, elimination and biopersistence along with tissue pathology compared with MPs due to stronger interaction of NPs with biological systems ( 2 ). Hence, to gain an insight into the uptake, retention, kinetics and affects on tissue architecture, biodistribution and histopathology studies are also mandatory. Therefore, in the present investigation, a 28-day repeated oral dose study of CeO2 NPs and CeO2 MPs was carried in albino Wistar male and female rats. The effect of the particles on the behavioural symptoms, body weight (bw) and feed intake was examined. Genotoxicity assays such as the comet assay in peripheral blood leukocytes (PBL) and liver MNT in bone marrow and peripheral blood (PB) and CA in bone marrow cells were performed. Target biochemical enzymes namely ALP for liver damage and LDH for membrane damage were assessed in serum. GSH content assay was carried out to study oxidative stress in various tissues such as the liver, kidneys and brain. To get insight of morphological alterations, histopathological examination of the liver, kidneys, spleen, heart and brain was carried out from treated and control rats. Furthermore, biodistribution of cerium (Ce) in rat’s whole blood, vital organs, urine and faeces was analysed using inductively coupled plasma optical emission spectrometer (ICP-OES). The doses used to evaluate the toxicity of CeO2 NPs were 30, 300 and 600 mg/kg bw/day. The lowest treatment dose was to signify the conceivable human exposure. In order to ascertain the amount of Ce in animal’s organs as well as toxicity through accidental exposure to large amounts of CeO2 NPs, two higher doses were selected. Materials and methods NPs and chemicals CeO2 NPs (CeO2 < 25 nm, 99.95%, CAS No. 1306-38-3) and CeO2 MPs (CeO2 < 5µm, 99.9%, CAS No. 1306-38-3) were purchased from Sigma Chemical Co. Ltd (St Louis, MO, USA). Phosphate buffered saline (Ca2+, Mg2+ free; PBS), cyclophosphamide (CP), normal melting agarose (NMA), low melting agarose (LMA), etc. were also purchased from Sigma Chemical Co. Ltd. Characterisation of CeO2 NPs and CeO2 MPs The particles were characterised using transmission electron microscopy (TEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) to evaluate the material size, size distribution, state of dispersion and zeta potential of the NMs in the Milli-Q water. Characterisation of CeO2 NPs and CeO2 MPs was performed to assess the size and morphology using a TEM (JEM-2100, JEOL, Japan). The images were obtained from TEM with an accelerating voltage of 120 kV. The TEM was equipped with a plunge freezer and cryo transfer holder to fix specimens in the frozen state and fitted with a Gatan 2Kx2K CCD camera for acquiring high-resolution images. Particles were suspended in Milli-Q water at a concentration of 0.01 mg/ml, and one drop of suspension was placed on a carbon-coated copper TEM grid and evaporated at room temperature. The software for advanced microscopy techniques was used for the digital TEM camera. This software was calibrated for nanoscale size measurements for the accurate examination of NPs. For the size measurement, 100 particles were calculated from random fields of view and images showing the general morphology of the particles. The size of the NPs and agglomerates was measured through DLS and LDV using a Malvern Zetasizer Nano-ZS (Malvern Instruments, UK). This device uses a 4 mW He–Ne 633 nm laser to analyse the samples and an electric field generator for the LDV measurements. At the concentration of 40 µg/ml freshly prepared CeO2 NPs and MPs, suspension in Milli-Q water was ultrasonicated using a probe sonicator (UP100H, Hielscher Ultrasonics GmbH, Teltow, Germany) for 10 min at 90% amplitude. The high concentration of the suspension was further diluted two times and adjusted to a lower concentration of 20 µg/ml to acquire enough counts per second. The prepared samples were transferred to a 1.5 ml square cuvette for DLS measurements and 1 ml of the suspension was transferred to a Malvern Clear Zeta Potential cell for LDV measurement. The mean NPs diameter was calculated using the same software program as utilised in the NPs distribution, and the polydispersity index (PdI) was used to measure the size ranges present in the solution. The PdI scale ranges from 0 to 1, where 0 indicates monodisperse and 1 indicates polydisperse state of particles. Animals Male and female albino Wistar rats, aged 6–8 weeks and weighing 80–120 g, were obtained from the National Institute of Nutrition, Hyderabad, India. The animals were acclimatised for 1 week in groups of five in polypropylene cages. The animals were fed with a standard laboratory pellet diet and reverse osmosis water was provided ad libitum and maintained under standard conditions of humidity (55–65%), temperature (22 ± 3°C) and light (12 h light/12 h dark cycles). The study was approved by the Institutional Animal Ethics Committee. Treatment of animals Twenty-eight days of repeated dose oral toxicity study was conducted with male and female rats with CeO2 NPs and CeO2 MPs following Organization for Economic Co-operation and Development (OECD) Guideline 407 ( 25 ). For the treatment, rats were weighed daily and concentrations were calculated for the doses 30, 300 and 600 mg/kg bw for CeO2 NPs and CeO2 MPs and administered orally. The rats were administered once daily for 28 days using a suitable intubation cannula with dosing volume 2 ml/100 g bw. Particles were suspended in Milli-Q water, properly ultrasonicated (UP100H, Germany) and vortexed before every treatment of the rats. Animals were divided into four groups (10 rats; 5 males and 5 females in each group): control, low dose (30 mg/kg bw/ day), medium dose (300 mg/kg bw/day) and high dose (600 mg/kg bw/day) of CeO2 NPs and CeO2 MPs. Control group received only Milli-Q water. 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. Feed consumption and bw were monitored weekly for 4 weeks. All treated rats were sacrificed by cervical dislocation after 24h of last administration of a dose. A known mutagen CP was used as the positive control for genotoxicity studies at a dose 40 mg/kg bw and the volume injected was 0.01 ml/g bw. This was given intraperitoneally (ip) 24 h before sacrifice. Comet assay The alkaline comet assay was used for the assessment of DNA damage in the rats after 28 days of repeated oral exposure to the CeO2 NPs and CeO2 MPs. It was performed according to the method described by Tice et al. ( 26 ) with slight modifications. Liver samples were collected after the sacrificing of rats. Whole blood withdrawal was done in EDTA-coated tubes from retro-orbital plexus. The comet assay in liver tissue was performed following the technique described by Miyamae et al. ( 27 ). The tissue was removed from the rats after sacrificing at various time intervals, minced and suspended at ~100mg/ml in chilled homogenising buffer (pH 7.5) and homogenised gently at a speed of 500–800 r.p.m. Cell viability was determined by the trypan blue exclusion assay ( 28 ). Three slides were prepared for each experimental condition. In brief, microscope slides were pre-coated with 120 µl of 0.75% NMA in PBS and allowed to solidify overnight at 37°C after covering with cover slip for uniform layer. For second layer preparation, 10 µl of whole blood (10 000– 30 000 lymphocytes) or liver homogenate was suspended in 120 µl of 0.37% LMA. The suspension of cells and LMA was pipetted onto pre-coated slides and spread uniformly covering with cover slip. The slides were dried at 4°C for 10 min. A third layer of plain 0.37% LMA (120 µl) was applied and a cover slip was quickly put to get an even layer and dried at 4°C. After removing the cover slip, the 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% dimethyl sulphoxide, pH 10.0) for 10 h at 4°C. The slides were pre-soaked for 20 min in alkaline buffer (10 M NaOH, 200 mM Na2 EDTA, pH > 13.0) and then electrophoresis was performed at 25V adjusted at 300 mA for 20 min. The slides were neutralised twice in 0.4 M Tris buffer, pH 7.5, for 5 min and once in absolute methanol for 5 min. Coded slides were scored after staining with ethidium bromide (20 μg/ ml) using a fluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan) with a blue (488 nm) excitation filter and yellow (515nm) emission (barrier) filter at ×400 magnification. A total of 150 randomly selected cells per rat (50 cells per slide) were used to measure the amount of DNA damage and expressed as percentage of DNA in the comet tail. Quantification of DNA breakage was realised by using a Comet Image Analysis System, version Komet 5.5 (Single cell Gel Electrophoresis analysis company, Andor Technology 2005, Kinetic Imaging Ltd, Nottingham, UK). Micronucleus test The MNT in the rat bone marrow cells was carried out following the method described by Schmid ( 29 ). After 28 days of repeated oral treatment, the bone marrow was removed from both femur and tibia by aspiration into hypotonic solution of 1% sodium citrate and centrifuged at ~1000 r.p.m. for 5 min. The cell pellet was resuspended in a drop of 1% sodium citrate and a smear was prepared on a microscope slide and allowed to dry in humidified air overnight. The MNT in PB cells was performed according to the protocol described by Celik et al. ( 30 ) with some modifications and according to the OECD Guideline 474 ( 31 ). Whole blood was collected from retro-orbital plexus of rat from each group and smears were made on clean microscope slides. The slides were air dried, fixed in methanol for 2min and stained with 0.5% Giemsa stain prepared in PBS for 3 min. The stained slides were used for the assessment of the MN occurrence. Three slides were made for each animal; the slides were microscopically analysed at ×1000 magnification. Randomly, 2000 polychromatic erythrocytes (PCEs) per animal were selected from three slides and the frequency of micronucleated PCEs (MN-PCEs) was determined. In order to determine the ratio of PCEs to normochromatic erythrocytes (NCEs) in the bone marrow and PB, ~1000 cells from each animal were examined and the ratio was expressed as percentages: (PCEs × 100/PCEs + NCEs). CA assay The method described by Adler ( 32 ) was used for CA analysis and performed in bone marrow cells. It is globally recommended to follow the OECD Guideline 475 ( 33 ) as a test method to identify CAs. For analysis of metaphase cells, cell division was arrested by a mitotic inhibitor, colchicine (0.020%), 0.01 ml/g bw ip 2 h prior to sacrifice after 28 days of repeated oral treatment. The bone marrow was collected from femur and tibia bones by rinsing in hypotonic solution with 0.9% sodium citrate centrifuged at 2000 r.p.m. for 20 min. Cells were then fixed through several changes of ice-cold Carnoy’s solution (methanol:acetic acid, 3:1 v/v) until the pellets were clean. After refrigeration for at least 24 h, 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 per animal) were selected to detect the presence of CAs. The mitotic index (MI) was determined with 1000 or more cells. ALP activity assay The blood samples were collected in a tube without having anticoagulant after 28  days of repeated oral treatment with CeO2 NPs and CeO2 MPs. Serum (30  µl) was pooled by centrifuging blood at 1500 r.p.m. for 10 min for ALP activity determination. This assay was based on kinetic reaction. The activity of ALP was determined following p-nitrophenyl phosphate method as described in kit from M/s. Siemens Healthcare Diagnostics Ltd, Baroda, Gujarat. This method utilises p-nitrophenyl phosphate that is hydrolyzed by ALP into a yellow coloured product p-nitrophenol having maximal absorbance at 405 nm. The rate of reaction is directly proportional to the enzyme activity. The absorbance of the test was read against blank at 405 nm at intervals of 30 s for 2 min by using spectrophotometer (Spectra Max Plus, Molecular Devices, Sunnyvale, CA, USA). The enzyme activity was expressed as U/L using molar extinction coefficient of 18.75/mM/cm. LDH activity The activity of LDH was estimated in serum according to the procedures described by McQueen ( 34 ). In a quartz cuvette, 1000  µl of Sorensen phosphate buffer, 100  µl reduced nicotinamide adenine dinucleotide and 20  µl of serum were added and mixed well. Next, 150 µl sodium pyruvate was added and again mixed well. The absorbance was measured spectrophotometrically at 340 nm at 10-s intervals for 2 min using spectrophotometer. The LDH activity was expressed as µmol/h/ml using molar extinction coefficient of 6.3/mM/cm. Reduced GSH content The reduced GSH content was measured using the method of Jollow et al. ( 35 ). After 28  days of repeated oral exposure with CeO2 NPs and CeO2 MPs, rats were sacrificed and liver, kidneys and brain were removed. One gram of tissue from liver, kidneys and brain of treated and control rats was rinsed in ice-cold physiological saline, perfused with cold potassium chloride buffer (1.15% KCl and 0.5 mM EDTA) and homogenised in potassium phosphate buffer (KPB, 0.1 M, pH 7.4) using Miccra D-1 high-speed tissue homogeniser. An aliquot of 0.5 ml of each tissue homogenate was incubated with 0.5 ml of sulfosalicylic acid (4% w/v) for 1 h in ice and centrifuged at 10 000 r.p.m. for 10 min. A 0.4 ml aliquot of the supernatant was mixed with 0.4 ml of Ellman’s reagent, 5,5'-dithiobis-(2-nitrobenzoic acid (4 mg/ml in 5% sodium citrate) and 2.2 ml KPB (0.1 M, pH 7.4). The yellow colour developed was read at 412 nm. The amount of GSH present was expressed as µg GSH/g wet weight of tissue. Histopathological evaluation Histopathology was conducted in the different organs, i.e. heart, liver, spleen, brain and kidneys of albino Wistar rats after 28  days of repeated oral dose of CeO2 NPs and CeO2 MPs. After sacrifice, tissues were washed with 1% ice-cold saline and fixed in 10% neutral buffered formalin. The formalin-fixed tissues were processed in a Leica TP 1020 tissue processor and then embedded in paraffin blocks using Leica EG 1160 paraffin embedder. The paraffin blocks were sliced into ribbons of 3-µm thick sections using a Microm HM 360 microtome and mounted on a glass microscope slide. After that the slides were stained in haematoxylin and eosin (H&E) using a Microm HMS-70 stainer and examined with Nikon Eclipse E 800 microscope at ×400 magnification. A minimum of three random sections per slide and at least three different fields were assessed for histopathological conditions. Cerium content analysis in tissues The biodistribution study of the CeO2 NPs and CeO2 MPs in the female Wistar rats was carried upon 28  days of repeated oral treatment. The animals were placed in metabolic cages after treatment to collect the urine and faeces samples. Rats were sacrificed through cervical dislocation and whole blood, liver, kidneys, heart, brain and spleen were collected after 28 days. The samples were processed using the method of Gómez et  al. ( 36 ). The samples were predigested in nitric acid overnight, and heated at 80°C for 10 h, followed by additional heating at 130–150°C for 30 min. Subsequently, a volume of 0.5 ml of 70% perchloric acid was added, and the samples were again heated for 4 h and evaporated nearly to dryness. Following digestion, the samples were filtered, and 2% nitric acid was added to a final volume of 5ml for analysis. The standard solution of Ce was serially diluted to 100, 50, 10 and 1 p.p.m., and wavelength of 418.66 nm was found to get intensity of samples. The Ce content in the samples was determined using ICP-OES (JY Ultima, Jobin Vyon, France). Statistical analysis The statistical significant changes between treated and control groups were analysed by one-way analysis of variance. All results were expressed as mean ± SD. Multiple pair-wise comparisons were done using the Dunnett’s multiple comparison post-test to verify the significance of positive response. Statistical analyses were performed using GraphPad Instat Prism 3 Software package for windows (GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance for all tests was set at P < 0.05. Results Characterisation of CeO2 NPs and CeO2 MPs The data of physico-chemical characteristics of CeO2 particles that were determined by TEM, DLS and LDV analysis are shown in Table I. Over 100 particles were measured in random field to calculate the mean size of CeO2 NPs and CeO2 MPs using TEM. The size obtained of CeO2 NPs and CeO2 MPs was 24.2 ± 1.63 nm (Figure  1A) and 3.14 ± 1.29  μm (Figure  1B), respectively. The morphology of particles was observed as polyhedron crystals. The hydrodynamic diameter and PdI of CeO2 NPs in Milli-Q water suspension obtained by DLS was LDV −17.0 ND Zeta potential ζ (mV) Electrophoretic mobility (μm·cm/s V) −1.15 ND CeO2 NPs and CeO2 MPs at the concentration of 40 µg/ml were dispersed in Milli-Q water and mixing was done via probe sonication for 10 min just before estimations. ND = not detectable. pH 7.0 7.0 191.2 ± 20.25 and 0.304, respectively. The larger hydrodynamic diameter than the TEM size indicated that CeO2 NPs formed larger agglomerates in Milli-Q water suspension than in the dry state. Zeta potential (ζ) and electrophoretic mobility of CeO2 NPs in Milli-Q were quantified by LDV and found to be −17.0 mV and −1.15  µm·cm/s V, respectively, at pH 7.0. In case of CeO2 MPs, DLS and LDV data were found to be out of the detection limit (Table I). Animal observation, food consumption, bw and organ weight Mortality was not observed in male and female Wistar rats after 28 days of repeated doses of 30, 300 and 600 mg/kg bw/day of CeO2 NPs and CeO2 MPs in rats. However, rats treated with high dose of CeO2 NPs showed dullness, irritation and moribund symptoms. Further, both CeO2 NPs- and CeO2 MPs-treated animals showed insignificant loss in feed intake (Figure 2A), bw (Figure 2B) and relative organ weight (Figure 2C). Moreover, average was calculated combining data obtained from feed intake, bw and relative organ weight of male and female rats due to lack of apparent difference in data. The % loss in feed intake was in the range of 13.0–20.0% in CeO2 NPs-treated group and 1.7–12.4% in CeO2 MPs-treated group. Likewise, % bw loss was in the range of 10.9–16.7% and 8.0–10.2% in CeO2 NPs- and CeO2 MPs-exposed rats, respectively. Comet assay After 28 days of repeated oral treatment, a significant (P < 0.01) increase in % tail DNA was observed in the PBL and liver of male and female Wistar rats at 300 and 600 mg/kg bw/day dose of CeO2 NPs (Figure  3A and B). However, significant DNA damage was not observed at 30 mg/kg bw/day of CeO2 NPs and all doses of CeO2 MPs in comparison to control. In all samples, the cell viability by the trypan blue exclusion technique was >90% (data not shown). The mean % tail DNA of positive control rats was significantly (P < 0.01) higher compared with the control. Moreover, gender-dependent variation in % tail DNA was not apparent. Micronucleus test The MNT in bone marrow and PB was conducted after 28  days of oral treatment with 30, 300 and 600 mg/kg bw/ day of CeO2 NPs and CeO2 MPs in male and female Wistar rats. The data indicated statistically significant increment in MN-PCEs frequency in bone marrow (Table II) and PBL (Table III) in the CeO2 NPs-treated groups of male and female rats at 600 mg/kg bw/day (P  <  0.01) and at 300 mg/kg bw/ day (P < 0.05) in gender-independent manner. However, CeO2 MPs-treated groups did not show any significant increase in the frequency of MN-PCEs. On the other hand, CP-treated group induced a substantially significant (P  <  0.01) effect on MN-PCEs frequency. The dose of 600 mg/kg bw/day of CeO2 NPs exhibited a significant decrease in % PCEs in bone marrow of male and female rats in comparison to the negative control rats (Table II). CA assay The results of the CA assay in bone marrow cells examined after the oral administration of CeO2 NPs and CeO2 MPs for 28  days at various doses (30, 300 and 600 mg/kg bw/day) to male and female Wistar rats are shown in Tables IV and V, respectively. The structural changes observed were gaps, breaks, minutes and acentric fragments. The CeO2 NPs induced significant (P < 0.01) total aberrations including gaps in bone marrow cells at 300 and 600 mg/kg bw/day dose, whereas total aberrations excluding gaps incurred significantly (P  <  0.01) only at 600 mg/kg bw/day in the both male and female rats. Moreover, dose-dependent increments in structural aberrations were observed in bone marrow cells of CeO2 NPs-treated rats. However, reciprocal translocations were observed only in positive control group treated with CP but not in treated groups. The MI was used to conclude the rate of cell division. There was no significant decrease in MI after repeated exposure with CeO2 NPs and CeO2 MPs at any dose level. Moreover, there was no revelation of alterations in chromosomes by CeO2 MPs after repeated oral study compared with the control group (Tables IV and V). ALP activity The activity of serum ALP was found to be increased significantly (P  <  0.01) at 300 and 600 mg/kg bw/day of CeO2 NPs in both male and female rats. The percent increase in ALP activity in male was 18.31 and 37.48%, respectively, for the exposure with 300 and 600 mg/kg bw/day CeO2 NPs. Likewise, in female rats, 19.18 and 38.00% increase in ALP activity was documented at 300 and 600 mg/kg bw/day dose of CeO2 NPs, respectively. However, exposure of CeO2 MPs at all dose level and CeO2 NPs at 30 mg/kg bw/day dose did not induced significant increase in ALP activity and percent increase was in the range of 1–6% when compared to control in both male and female rats (Figure 4A). LDH activity The activity of serum LDH significantly increased (P < 0.01) at 300 and 600 mg/kg bw/day dose of CeO2 NPs after 28 days of repeated oral exposure. In male rats, percent increase in LDH activity with 300 and 600 mg/kg bw/day dose of CeO2 NPs with respect to control was observed as 78.56 and 179.53%, respectively. Similarly in female rats, LDH activity was noted to be 79.06 and 180.85% higher than the control at 300 and 600 mg/kg bw/day of CeO2 NPs. Conversely, CeO2 MPs and 30 mg/kg bw/day of CeO2 NPs did not induce significant damage to the cellular systems (Figure 4B). Treatments Dose (mg/kg bw/day) Male Data represented as mean ± SD, significantly different from control *P < 0.05 and **P < 0.01, n = 5 animals per group. aMilli-Q water (negative control). bCP (positive control). Data represented as mean ± SD, significantly different from control *P < 0.05 and **P < 0.01, n = 5 animals per group. aMilli-Q water (negative control). bCP (positive control). Significantly different from control at *P < 0.01. One hundred metaphases were analysed per animal; n = 5 animals per group. Data represented as mean ± SD; TA, total aberrations = structural aberrations. aNegative control (Milli-Q water). bCP (40 mg/kg bw). Reduced GSH content The 28 days of repeated oral treatment with CeO2 NPs revealed that GSH content in liver, kidneys and brain was inhibited in a dose-dependent manner compared with controls in both sexes of rats in gender-independent manner. There was significant inhibition in the GSH content in liver, kidneys and brain at 300 and 600 mg/kg bw/day dose of the CeO2 NPs-treated rats Significantly different from control at *P < 0.05 and **P < 0.01. One hundred metaphases were analysed per animal; n = 5 animals per group. Data represented as mean ± SD; TA, total aberrations = structural aberrations. aNegative control (Milli-Q water). bCP (40 mg/kg bw). respectively, at 300 and 600 mg/kg bw/day of CeO2 NPs. On the contrary, CeO2 MPs at all doses and CeO2 NPs at 30 mg/kg bw did not induce any significant alterations in GSH content in liver, kidneys and brain and this was in the range of 2–17% altogether. Histopathological examinations The slides of organs namely brain, heart, liver, spleen and kidneys stained with H&E were studied. All the organs were evaluated for any toxic changes as well as presence of any extraneous material deposits. In liver, significant damage was observed as dilated portal tract with exposure to CeO2 NPs in high dose groups (Figure  6B). Splenic hyperplasia (Figure  6F) was found along with inflammation in brain tissue (Figure 6J) upon exposure with 600 mg/kg bw/day of CeO2 NPs. However, there were no abnormal pathological changes in the kidneys and heart of the nanoceria-treated groups (Figure 6D and H). The histomicrographs of control liver, kidneys, spleen, heart and brain has been shown in Figure  6A, C, E, G and I, respectively. Significant histopathological changes were not found in the liver, kidneys, spleen, heart and brain of 600 mg/kg bw/day CeO2 MPs-treated rats (data not shown). Further, there were no evident effects in the low and medium dose groups of nanoand micro-sized CeO2. Tissue biodistribution of cerium particles The exposure of male and female Wistar rats with CeO2 NPs and CeO2 MPs repeatedly for 28 days with 30, 300 and 600 mg/ kg bw/day doses resulted in a significant increment in Ce concentration, indicating absorption of Ce (Figure  7). There was significant accumulation of Ce in all the tissues, i.e. liver, kidneys, brain, spleen, heart and whole blood (Figure 7A–F) in the groups of animals treated with CeO2 NPs. The distribution of Ce was maximum in 600 mg/kg bw/day followed by 300 and 30 mg/kg bw/day in male and female rats treated with CeO2 NPs. The maximum amount of Ce was distributed in the liver, kidneys, spleen and blood. However, the intake of Ce in various tissues was very high in the NPs-treated groups when compared to MPs-treated groups at all the dose level. Significant accumulation of Ce was observed in rats treated with CeO2 MPs at 300 and 600 mg/kg bw/day in the liver and spleen, whereas in kidneys and blood only at 600 mg/kg bw/ day. Moreover, significant amount of Ce was excreted in urine at 600 mg/kg bw/day of CeO2 MPs-treated rats. In the CeO2 NPs-treated rats, a significant amount of Ce was removed via urine in dose-dependent manner (Figure  7G), whereas CeO2 MPs-treated rats showed more excretion in faeces, probably due to their larger size (Figure 7H). Discussion The present study was executed to provide the first testimony of the genotoxic, biochemical and biodistribution of the Ce in different tissues, urine and faeces with CeO2 NPs and CeO2 MPs in albino Wistar male and female rats after 28  days of repeated oral treatment. The results obtained have revealed that CeO2 NPs induced toxic effects at medium and high doses without any severe distress symptoms and mortality. In contrast, CeO2 MPs did not demonstrate toxicity. Thus, CeO2 NPs can be categorised as substances with toxic effects when exposed at higher dose for a longer period of time. Moreover, the physical and chemical properties can influence NPs behaviour and may have an impact on toxicity. The specific physico-chemical parameters of NMs may govern unexpected biological interactions ( 37 ). To interpret the likely fate and behaviour of NPs in the environment, it is essential to establish their interaction in aqueous medium. Accordingly, in this study, the mean size of CeO2 NPs and CeO2 MPs was calculated and found to be 24.2 nm and 3.14  µm, respectively, in dry state, whereas hydrodynamic diameter in Milli-Q water was noted to be 191.2 nm. The difference in size in two states can be explained by possible agglomeration in aqueous medium due to physicochemical interactions between NPs, which tend to increase the particle diameter. Zeta potential measurement delves into surface charge and it was perceived to have an incipient instability of CeO2 NPs in suspension. The agglomeration of metal oxides in aqueous medium depends on particle charge and has a significant effect on their bioavailability. Moreover, zeta potential is a surface electrical characteristic that has been used to quantify the influence of NPs in biological cells ( 38 ). Several studies have revealed that NPs caused more toxicity than micron-sized particles ( 12,39 ). In this study, significant increase in % tail DNA in PBL and liver cells at a high and medium dose compared to control was found due to repeated CeO2 NPs exposure through comet assay. However, CeO2 MPs did not reveal any significant increase. The results suggest that CeO2 NPs may induce DNA lesion at alkali-labile sites in leukocytes. There are several in vitro studies depicting DNA damage with comet assay in divergent cellular systems with CeO2 NPs ( 40,41 ), which support our findings. In contrast, there are no in vivo studies with comet assay after oral exposure of CeO2 NPs. However, an investigation with zinc oxide (ZnO) NPs is in line with our results. In this study, mice treated for 14 continuous days resulted in significant increase in DNA damage with the comet assay in the liver (42). There was significant increment in MN-PCEs frequency in both bone marrow and PB cells in CeO2 NPs-treated groups. It is possible that clastogenic events are involved in the formation of these MN with CeO2 NPs. There are studies that have demonstrated that CeO2 NPs exposure induced chromosome damage and DNA lesion in distinct cellular systems using MNT and comet assay ( 41,43 ). Moreover, decrease in % PCEs suggests that cell death had occurred in the treated groups. There were significant chromosomal damages in bone marrow cells in CeO2 NPs high dose treated groups but not with CeO2 MPs. A decrease in MI results compared with the control groups suggested a slower progression of cells from the S (DNA synthesis) phase to the M (mitosis) phase of the cell cycle. It is most likely that this impairment in cell cycle progression is associated with CeO2 NPs and CeO2 MPs toxicity. During mitosis, NPs could interact with chromosomes and might introduce breaks into chromosomes or disturb the process of mitosis, mechanically or by chemical binding (24). Although there is no literature with CeO2 NPs utilising CA assay, a study with manganese oxide (MnO2) NMs has recorded structural aberrations in bone marrow cells of rats after 28 days of repeated dosing, which is in accordance with our results ( 44 ). In the present study, CeO2 NPs significantly increased the activities of ALP and LDH enzyme in the serum of exposed rats in dose-dependent manner, suggesting probable injuries to the tissues. The ALP is the foremost enzyme that is found to increase in liver disease. Similarly, serum activity of the ALP has been reported to elevate after 14 days of repeated oral exposure with ZnO NPs in mice ( 42 ). Nanoceria (8 nm) was also found to induce membrane damage and resultant cytotoxicity using LDH assay in H4IIE rat hepatoma cells ( 45 ). In the current study, in the liver, kidneys and brain of rats treated with 300 and 600 mg/kg bw/day dose of nanoceria, the GSH content declined significantly. GSH plays a crucial role in antioxidant defence, nutrient metabolism and regulation of cellular events. Its insufficiency contributes to oxidative stress, and this stress is prime culprit in aging and many diseases ( 46 ). The reason for this is that GSH effectively scavenges free radicals and other ROS (e.g. hydroxyl radical, lipid peroxyl radical, peroxynitrite and H2O2) directly and indirectly through enzymatic reactions ( 47 ). Similarly, cell membrane breakage along with decrease of GSH levels in different cellular systems due to nanoceria exposure has been documented ( 15,16,43 ). Additionally, histopathological damage to rats treated with 600 mg/kg bw/day dose of the nanoceria for 28  days showed alterations in the liver, spleen and brain tissues, such as dilation of hepatic portal tract, splenic hyperplasia and inflammation in brain tissue, respectively. Similar alterations were documented with other metal NPs ( 42,44 ). The evaluation of consequences of NMs exposure at the whole body and organ levels is important in their toxicokinetic study. Hence, another important aspect of this study was the tissue biodistribution of CeO2 NPs. The significant distribution of CeO2 NPs at all doses and higher dose of CeO2 MPs broadly destined to liver, spleen, kidneys, heart and brain through their translocation into the blood. The distribution was in dosedependent order. On the other hand, no gender-dependent tissue distribution was witnessed. A small but significant amount of Ce in nanoceria-treated rats was excreted in urine, whereas only higher dose of CeO2 MPs was found to expel Ce in urine. Because of larger average diameter of glomerular filtration pore, nanoceria of comparatively small size could leak out. The decrease in particle size might have enhanced the penetration of nanoceria into the systemic circulation ( 48 ). Further, large amount of either of the Ce particles were rapidly removed from the gastrointestinal system into faeces. Similar pattern of retention in liver and spleen of rats treated with 30 nm CeO2 NPs with single iv infusion was revealed after 14 days ( 49 ). The suggested mechanism for the tissue distribution was that the CeO2 NPs may have been phagocytised and transported to the liver or spleen by macrophages to help reduce the filtered amount or bound to haem in blood cells ( 7 ). Furthermore, limited gastrointestinal absorption of nanoceria was reported and their timedependent excretion via bile was found in faeces after oral dose ( 48 ). The biochemical alterations such as increased activity of serum ALP and LDH and reduction in liver, kidneys and brain GSH content can be probably explained by the accumulation of nanoceria. Tissues like liver and spleen are rich in mononuclear phagocyte cells and hence sensitive to CeO2 NPs exposure ( 49 ). Brain is sensitive to oxidative stress due to high metabolic activity. The GSH plays an important role in countering stress. Subsequently, nanoceria was reported to incite little oxidative stress in brain components after 20 h of iv infusion in rats ( 50 ). The body distribution of particles significantly depends on surface characteristics and the size of the particles irrespective of the uptake route ( 51 ). In support of the present findings, repeated oral dose studies performed with diverse NMs revealed that ZnO NPs (300 mg/kg) for 14 days in male mice and silver NPs and MnO2 NPs (30, 300 and 1000 mg/kg) for 28 days in male and female rats were found to be distributed into liver, spleen, brain and kidneys leading to several hepatic alterations after getting translocated into the blood ( 42,44,52 ). Persistence and clearance of NPs in different organs are the major determinants of the toxicity of NPs. There are no reports addressing the long-term impact of oral exposure of CeO2 NPs, whereas a study in rats demonstrated that a single iv infusion of aqueous dispersion of 85 mg/kg 30 nm nanoceria induced hepatic apoptosis after 30 days of exposure ( 53 ). The orally treated CeO2 NPs led to liver damage as exhibited by the histopathological study that showed dilated portal tract. The DNA damage, increased ALP level and decreased GSH content in liver cells were in concurrence with the probable hepatotoxicity. Moreover, our results suggest bioaccumulation of CeO2 NPs for the significant genotoxic potential in rats. However, the mechanisms of NPs genotoxicity are still not well understood. The possible mechanisms of genotoxicity may be either by direct interaction of NPs with the genetic material or by indirect damage from NPs-induced ROS or by toxic ions released from soluble NPs ( 54,55 ). Moreover, intracellular ROS generated in the metabolising cells could attack DNA base guanine and form mutagenic 8-OHdG lesions after unbalancing the redox potential in the cellular environment (56). Likewise, H2AX-Nox1/Rac1 pathway was reported as the possible mechanism for the DNA damage and intracellular ROS signalling for the induction of cell death ( 57 ). The data obtained with the tested compounds were gender independent. Further, the NPs showed more bioaccumulation compared with the MPs at the same dose level in male and female rats after 28 days of repeated oral dose study. Hence, particles in the nano-size range can enter into animal system via intestines. Moreover, high doses were used to provide detectable quantities after distribution into the animal system and were not intended to reflect human exposure. Further, there is always a wide scope for the next level of study in order to get conclusive information regarding CeO2 NPs toxicity. Funding This work was supported by Asian Office of Aerospace Research and Development (AOARD), Japan, under the grant no. FA2386-11-1-4085. 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Monika Kumari, Srinivas Indu Kumari, Paramjit Grover. Genotoxicity analysis of cerium oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral administration, Mutagenesis, 2014, 467-479, DOI: 10.1093/mutage/geu038