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Safety assessment and gastrointestinal retention of orally administered cerium oxide nanoparticles in rats – Scientific Reports

Preparation and characterization of CeO2 NPs (NM-212) suspension

CeO2 NPs (NM-212) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) (Product No. 22390; CAS No. 1306-38-3). CeO2 NPs were suspended in distilled water (DW) (stock concentration of 100 mg/mL) and the particle morphology was observed using TEM (JEM-3000F, 200 kV, JEOL Ltd., Tokyo, Japan) (Fig. 1). Energy-dispersive X-ray spectroscopy (EDS) analysis was also performed (JEM-2100F, JEOL Ltd., Tokyo, Japan) (Fig. 2). CeO2 NPs dispersion in DW is stable more than 5 days (Supplementary Information 3).

Figure 1
figure 1

Characterization of CeO2 NPs.

Figure 2
figure 2

EDS analysis of CeO2 NPs.

Animal husbandry

Five-week-old male (n = 55) and female specific-pathogen-free outbred Sprague Dawley rats (n = 55) supplied by Orient Bio Inc. (Seongnam-si, Republic of Korea). The animals were acclimatized to their new surroundings for seven days. To reduce the body weigh variation blew 10% in each group, one hundred rats were selected and those animals randomly assigned to four groups (one control group and three treatment groups) using the Pristima system (Version 7.3 Xybion Medical System Co., USA). Ten males and 10 females/group were assigned to the four groups (main group), and 5 males and 5 females/group were assigned to the 0 mg/kg (control group) and 1000 mg/kg (high-dose). Those 2 groups (recovery group) were used to check the delayed toxicity or the recovered effects. All animals at terminal sacrifices were euthanized using isoflurane with necropsy. And, remained animals (5 males and 5 females) were euthanized using isoflurane. During the all-study periods, all animals were housed 2–3 per cage and there was attached cage card, described animal information, in each cage. The body weight ranges prior to the start of dosing were 204.6–234.5 g for the males and 158.1–194.1 g for the females. All rats were housed in polycarbonate cages with bedding (Laboratory animal Aspen bedding, Abedd Baltic Ltd., Jelgava, Latvia) throughout the study period and provided sterilized tap water and pelleted food (PMI Nutrition International, USA). The rats were maintained under the following conditions: 12/12 h light/dark cycle, temperature 23 ± 3 °C, relative humidity 50 ± 10%, air ventilation 10–20 times/h, and light intensity 150–300 lx. This study was performed in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. The all experiments were reviewed and approved by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Toxicology [approval no. 2006–0180] and all experiments were performed according to the ARRIVE guidelines, the guidelines published by the OECD and the GLP regulations for Nonclinical Laboratory Studies of Korea Food Drug Administration21. No linestatistical methods were used to predetermine sample sizes, but our sample sizes are based on to those reported in previous test study22. Animal experiments were designed to use all animals for the analysis, except for those that died or suffered from issues related to animal welfare throughout the experiment.

Animal treatment

One hundred rats were divided into four groups, including a control group (n = 15, each sex), and three groups of orally administered CeO2 NPs (low dose: 10 mg/kg bw/day (n = 10, each sex), middle dose: 100 mg/kg bw/day (n = 10, each sex), high dose: 1000 mg/kg bw/day (n = 15, each sex). Dose formulations were administered by oral gavage once daily for 13 consecutive weeks. A daily oral dose of CeO2 NPs (NM-212) was administered to SD rats for 13 weeks (91 days), followed by a four week (28 days) recovery period. The animals of main group were sacrificed at Day 92 and animals of recorvery group were sacrificed at Day 119. Animals were dosed at a volume of 10 mL/kg. The dose volume was calculated based on the most recently measured body weight. Saline (vehicle control) was administered to the rats in the control group. Rats were divided into four groups and subjected to treatment doses of 0, 10, 100, and 1000 mg/kg bw/day CeO2 NPs, respectively. The effects of the CeO2 NPs were analyzed by clinical observation, body weight measurements, and recording of the rats’ food consumption. Serum chemistry and hematology examinations were performed to assess the clinical pathology of CeO2 NPs. In addition, during necropsy, gross observation, organ weight, histopathology, and immunohistochemistry were performed, and meaningful results were obtained in the stomach, jejunum, and colon.

Mortality and body weight

All measurements and examinations were performed using the Pristima system. The general condition and behavior of the experimental animals were checked once daily throughout the acclimation period. However, during the treatment period and the day before necropsy was performed clinical signs were examined and recorded twice daily (before and after dosing). The animals were weighed on the day of arrival, prior to randomization, before the administration of the first dose, once weekly thereafter, and on the day of necropsy.

Food consumption and ophthalmic examination

Cage food consumption was recorded once during the acclimation period and once weekly during the treatment period. The individual food consumption was calculated and expressed as g/rat/day. External eye examinations were performed on all animals during the acclimation period. However, at week 13 before necropsy both external and fundus examinations of the animals in the control and high-dose (1000 mg/kg bw/day) groups were performed using a binocular indirect ophthalmoscope (Vantage Plus Digital, Keeler Ltd., England). Prior to examination via binocular indirect ophthalmoscopy, a mydriatic compound (Midrin-P; Santen Pharmaceutical Co., Ltd., Japan) was administered to each eye.

Hematology analysis

All rats were fasted overnight before necropsy and blood samples were collected. Blood samples for hematological and clinical chemistry analyses were collected from the vena cava during necropsy. Samples for hematological analysis were collected in tubes containing EDTA-2 K. The following parameters were analyzed using an ADVIA 2120i hematology analyzer (Siemens, USA): white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained cells), and reticulocyte count. In addition, blood samples treated with 3.2% sodium citrate were analyzed for prothrombin and activated partial thromboplastin times using an ACL Elite Pro coagulation analyzer (Instrumentation Laboratory, Italy).

Biochemical analysis

Blood samples, simultaneously collected into tubes without anticoagulant for hematological and clinical chemistry analyses, were placed at room temperature for at least 90 min, and centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. The levels of glucose, blood urea nitrogen, creatinine, total cholesterol, total protein, albumin, albumin/globulin ratio, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, total bilirubin, alkaline phosphatase, gamma-glutamyl transferase, creatine kinase, calcium, inorganic phosphorus, sodium, potassium, and chloride were measured using an automatic analyzer (TBA 120FR NEO; Toshiba Corp., Tokyo, Japan).

Gross findings and histopathological examination

After blood sampling, the animals were sacrificed by exsanguination through the vena cava and aorta under isoflurane anesthesia. Complete necropsy was performed on all experimental animals. The absolute weights of the brain, pituitary gland, liver, kidneys, spleen, heart, lungs, thymus, salivary gland, thyroid gland, testes, epididymis, prostate, seminal vesicle, uterus, and ovaries were determined, and relative organ weights (% of terminal body weight) were calculated.

Following detailed external and internal examinations, tissue samples (skin, mammary gland, testes, epididymis, prostate, seminal vesicle, urinary bladder, ovaries, uterus, vagina, spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, heart, lungs, adrenal gland, liver, salivary gland, mesenteric lymph node, mandibular lymph node, thyroid gland, aorta, thymus, trachea, tongue, esophagus, sciatic nerve, skeletal muscle, sternum/marrow, femur/joint/marrow, thoracic spinal cord, Harderian gland, brain, pituitary gland, and eyes/optic nerve) were collected from each animal and preserved in 10% neutral-buffered formalin. The eyes and optic nerves were fixed in Davidson’s fixative, whereas the testes were fixed in Bouin’s fixative for approximately 48 h and then transferred to 70% ethanol. Preserved tissues were obtained from animals in the control and high-dose groups.

Immunohistochemistry and histopathological examination

Paraffin-embedded stomach and colon tissues were dewaxed using xylene and a graded alcohol series (100, 95, 70, and 50%). After washing with phosphate-buffered saline (PBS), the tissues were placed in an antigen retrieval solution (ENZO, Seoul, Korea) and permeabilized with PBS containing Tween-20 (PBST, 1%). After blocking with 5% (v/v) bovine serum albumin in PBST (0.01%), the tissues were incubated overnight at 4 °C with rabbit polyclonal antibodies against superoxide dismutase (SOD)-1, SOD-2 (Santa Cruz Biotechnology, Dallas, TX, USA), and cytochrome C (Cell Signaling Technology, Danvers, MA, USA). The tissues were then incubated with affinity-purified Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) and mounted with 4′,6-diamidino-2-phenylindole mounting medium. Finally, images were captured using an inverted phase-contrast fluorescence microscope (IX51, Olympus, Tokyo, Japan). In addition, stomach and colon tissues were sectioned, stained with 0.5% periodic acid solution for 5 min, stained with Schiff’s reagent for 15 min, and counterstained with hematoxylin solution for 2 min. All steps were performed at room temperature and the tissue sections were examined under a microscope.

Statistical analysis

The data were statistically analyzed using multiple comparison methods. When the Bartlett’s test showed no significant deviations from variance homogeneity, analysis of variance (ANOVA) was used to identify differences among group means. A p value of p < 0.05 was considered statistically significant. The Dunnett’s test was used to determine differences between the control and treatment groups when the data were significant based on ANOVA. Furthermore, when significant deviations from variance homogeneity were observed based on the Bartlett’s test, a non-parametric comparison test, the Kruskal–Wallis (H) test, was performed to determine any group mean differences (p < 0.05). When a significant difference was observed in the Kruskal–Wallis (H) test, the Dunn’s rank sum test was performed to quantify the specific pairs of group data that significantly differed from the mean. The Fisher’s exact test was used to compare pairs of data (including prevalence and percentage). The probability level was set at 1% or 5%. Statistical analyses were performed by comparing the data for the different dose groups with those of the control group, using the Pristima system.