Characterization of ZrO2-NPs. ZrO2-NPs were purchased from Sigma-Aldrich Chemical Company (Saint Louis, MO, USA) at 100.0% purity. The detailed characterization of the physicochemical properties of the ZrO2-NPs was reported in our previous study [10].
In Vivo Studies
Animal and Intravenous Injections. Seventy adult male Wistar rats (w 200.0±20 g each) were purchased from the Laboratory Animal Center, Southern Medical University, Guangzhou, China. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Jinan University. The ZrO2-NPs were primarily sterilized by gamma-ray radiation. Thereafter, the injection suspensions were prepared through the dispersion of ZrO2-NPs in 0.5% normal saline (NS). A single tail-vein injection of ZrO2-NPs (20 mg/kg-bw) was slowly administered to the rats. The rats were randomly divided into seven groups (ten in each group), including one control group. A group of ten animals injected with 0.5% NS was used as the controls. The rats were then sacrificed at six time points after the intravenous injections.
Perfusion and Harvesting of Tissues. The rats were deeply anesthetized by intraperitoneal injection of 2.3 mL of 6 mg/mL Nembutal. The rats were then sacrificed at six time points, i.e., 1 d, 3 d, 7 d, 10 d, 14 d and 28 d, after ZrO2-NP injection. Approximately 2-3 mL of blood was collected through cardiac puncture, and the rats were perfused through the left cardiac ventricle with NS until the venous return was clear. The liver tissues were harvested. All instruments and samples were thoroughly washed with water during harvesting to minimize adherent ZrO2-NPs. The samples were placed in cleaned Eppendorf tubes. The tubes were snap-frozen in liquid nitrogen and stored at -80°C until analysis.
Quantification of ZrO2-NPs in the Liver. The liver samples were weighed and then subjected to microwave-assisted digestion and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Briefly, the collected tissues (200 mg per sample) were primarily pre-digested overnight in concentrated nitric acid and H2O2 in an oven chamber. After digestion, the mixed solutions were then heated at 160°C using a high-pressure reaction container until the samples were completely digested. The solutions were then heated at 120°C until the remaining nitric acid was nearly evaporated to dryness. The resulting solutions were finally diluted to 2 mL with 1% nitric acid + 0.1% Triton-100 and analyzed using ICP-MS. Calibration standards of six Zr concentrations (0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 mg/L) were analyzed with each batch of samples. Each sample was spiked with 0.2 mg/L Ge as an internal standard.
Measurement of Cytokines. TNF-α, IL-1α, IL-1β, IL-6 and IL-8 in the liver after treatment with ZrO2-NPs and physiological saline for different intervals were measured using an enzyme-linked immunosorbent assay (ELISA) kit specifically for rats (Jiancheng Bioengineering Institute, Nanjing, China). The assays were performed following the manufacturer's instructions.
Ultrastructure Observation. Liver samples were fixed in 3% glutaraldehyde + 2% paraformaldehyde for 2 h. Small pieces of tissue (approximately 1 mm3) collected from these samples were washed with 0.1 mol/dm3 cacodylate buffer (pH 7.2-7.4) and post fixed in 1% osmium tetraoxide for 1 h. The samples were then dehydrated using a series of ethanol concentrations and embedded in Epon resin. Finally, TEM (H-7500, Hitachi, Tokyo, Japan) was used to visualize the ultrathin sections.
Histopathology and Immunohistochemical Analysis. The histological specimens were prepared as follows. Formalin-fixed samples were dehydrated with gradient solutions of ethanol and then embedded in paraffin. The tissue specimens were sliced with an automatic microtome and then stained with hematoxylin and eosin (H&E) for conventional morphological evaluation. Representative photos were captured with a charge-coupled device (CCD) digital camera fixed to a light-microscopy system (IX73, Olympus, Tokyo, Japan).
Furthermore, immunohistochemistry analysis was carried out to identify the specific toxicity effects. Proliferating cell death in tissues was determined using a Ki-67 assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Tissue sections were primarily incubated with the Ki-67 monoclonal antibody. Proliferating cells were stained with the Ki-67 monoclonal antibody. The mean positive-staining density was analyzed in randomly selected areas in each section at ×400 magnification using image analysis software (Image Pro Plus 7.0; Media Cybernetics, Rockville, MD, USA). The ratios of positively stained cells were determined according to the positively stained cell numbers divided by the total cell numbers.
Apoptotic cell death in tissues was determined using the Tunel assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. After incubation in the Tunel solution, tissue sections were immersed in 0.3% hydrogen peroxide in PBS and then incubated with peroxidase solution for 30 min at room temperature. Tissue sections were stained with 0.1% DAB solution and counterstained with hematoxylin. The presence of apoptotic cells was calculated. Positive-staining (brown) photographs were captured in randomly selected fields with image analysis software (Image Pro Plus 7.0, Media Cybernetics, Rockville, MD, USA).
Liver function assay. The levels of alanineaminotransferase (ALT), aspartate-aminotransferase (AST) and (lactate dehydrogenase, LDH) in serum were determined using a chemistry-immuno analyzer (VITROS 5,1 FS, Ortho-Clinical Diagnostics, NY, USA).
RNA-Seq Quantification. RNA-Seq quantifications of the expressed genes of the live samples were conducted 7 d post injection. Three samples of livers from rats injected with a single dose of ZrO2-NPs were harvested 7 d post injection. The samples were individually matched to tissues from noninjected control animals of a similar weight. The total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturers’ instructions. RNA was stored at -80°C. The RNA was then submitted to the BGI Genomics Core Facility Lab. mRNA with a polyA tail was primarily selected to remove the DNA probe. After purification, the targeted RNA was fragmented and then reverse transcribed into double-stranded cDNA (dscDNA) using an N6 random primer (BGI Genomics, Shenzhen, China). The dscDNA was end-repaired with phosphate at the 5’ end and sticky ‘A’s at the 3’ end. The PCR product was denatured by heat. The single-stranded DNA was then cyclized by splint oligo and DNA ligase. The RNA quality was analyzed using an Agilent 2100 Bioanalyzer (Agilent, Beijing, China). Sequencing qualification was then performed with the Gene Sequence Detector System BGI-SEQ 500 (BGI Genomics, Shenzhen, China). Differentially expressed genes (DEGs) were screened through the NOISeq method [18], with the default criteria for DEGs as follows: fold change ³2 and divergence probability ³0.8. Gene ontology (GO) and pathway enrichment analyses of the DEGs were performed using the PANTHER classification system (http://www.pantherdb.org/data/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/).
RT-qPCR Analysis. Based on the RNA-Seq quantification results, precise quantification of the gene expressions in the liver tissue was then conducted. The liver samples were kept at -80°C until analysis. Frozen tissues were homogenized and extracted using TRIzol Reagent (Gibco, Waltham, MA, USA) according to the manufacturer’s instructions. The extracted RNA was further purified using the Qiagen TM RNeasy Mini Kit (Dusseldorf, Germany) to remove any genomic DNA contamination. The total RNA was measured at 260 and 280 nm using an M5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The purity of the RNA sample was measured according to the 260/280 nm ratio, with expected values between 1.8 and 2.0. Synthesized complementary DNA (cDNA) was prepared from RNA samples (1000 ng) using the Prime Script TM RT Reagent Kit (TaKaRa, Tokyo, Japan). RT-qPCR was carried out using a commercial kit (SYBR Premix Ex Taq II, TaKaRa, Tokyo, Japan) and analyzed on a Lightcycler 480 Sequence Detector System (Roche, Basel, Switzerland). The amplification protocol was performed as follows: after 30 s of incubation at 94°C to activate the Hot Start DNA polymerase, 40 cycles of amplification were accomplished over 5 s at 94°C for denaturation and over 30 s at 60°C for annealing (fluorescence detection). The expression level of each target gene was normalized to its β-actin mRNA content. The primers used for PT-PCR are shown in Tab. 1.
In Vitro Studies
Cell Culture Treatment with ZrO2-NPs. The Hepg2 cell line (HB-8065) was obtained from the American type culture collection (ATCC). Hepg2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine, and 1% non-essential amino acids in a humidified atmosphere of 5% CO2 at 37°C. The media was changed every 3 days. Cells were removed from the culture dishes by rinsing in PBS and incubating in trypsin-EDTA solution. Cells were seeded onto each test substrate at 1 × 105 cells/ml in the same media for all assays. The ZrO2-NPs were sterilized by gamma-ray radiation and suspended in the culture medium. The stock solutions were then serially diluted to yield doses of 250 μg/ml. These samples were sonicated before exposure to the cells. After the cells were attached for 24 h in the full medium, the freshly dispersed ZrO2-NP suspensions were immediately applied to the cells. Cells free of ZrO2-NPs were set as the control.
Lipid Analysis. The total cholesterol (TC) and triglyceride (TG) levels in the supernatants of the HepG2 cells were determined by ELISA (Jiancheng Bioengineering Institute, Nanjing, China).
Intracellular ROS Measurement and Oxidative Stress. The reactive oxygen species (ROS) levels of the untreated control and ZrO2-NP-treated cells were determined by chemiluminescence using the Reactive Oxygen Species Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China) after 6 h of exposure. The SOD and MDA levels were independently measured using the reagent kits (Jiancheng Bioengineering Institute, Nanjing, China) after 24 h of exposure.
Cell-Cycle and Apoptosis Analyses. After incubation for 24 h, the cells were harvested by trypsinization and fixed with 70% ice-cold ethanol for 10 min at 4°C. After washing, the cell pellets were resuspended in propidium iodide (PI) staining buffer and incubated for 15 min at 37°C. The cell-cycle distributions were analyzed by a fluorescence-activated cell sorter (FACS; Gallios, Beckman Coulter, Brea, CA, USA) and Multicycle Mod Fit software (Beckman Coulter, Brea, CA, USA). The untreated control and ZrO2-NP-exposed cells were treated with cisplatin (20 mg/mL) for 24 h and then washed and resuspended. Then, 5 µL of annexin V-FITC conjugate and 10 µL of PI solution were added to 500 µL of each cell suspension, followed by incubation at room temperature for 15 min in the dark. Flow cytometry analysis was performed with FACS and analyzed by FlowJo express software (FlowJo LLC, Ashland, Oregon, USA).
Western-Blot Analysis. The Hepg2 cells were lysed in ice-cold protein lysis buffer (P0013, Biyuntian, Shanghai, China). After centrifuging the lysates at 12,000 rpm at 4°C for 10 min, the supernatants were collected and stored at -80°C until use. The lysates were then transferred to PVDF membranes. The membranes were incubated with 5% milk in PBS for 1 h and then incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-P53 (2524S, CST, Danvers, MA, USA), anti-P21 (ABS135943a, Absin, Shanghai, China), anti-Gadd45g (ABS132755a, Absin, Shanghai, China), anti-Bcl6 (sc-7388, Santa Cruz, Santa Cruz, CA, USA), anti-Foxo1 (2880S, Cell Signaling, Danvers, MA, USA), anti-Bcl2 (SC7382, Santa Cruz, Santa Cruz, CA, USA), anti-Bax (sc- 20067, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Caspase 3 (sc-7272, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Akt (ab8805; Abcam, France), anti-Irs2 (sc-390761, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Igfbp2 (sc-25285, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Gapdh (AB8245, Abcam, Cambridge, MA, USA). Blots were incubated with peroxidase-coupled secondary antibodies (BA1054, Boster, Pleasanton, CA, USA) for 1 h, and the protein concentration was measured using a bicinchoninic acid protein assay kit (23227, Thermo, Rockford, IL, USA).
Statistical Analysis. The data were analyzed using one-way ANOVA with Turkey’s HSD multiple comparison post-hoc tests, with P < 0.05 or P < 0.01 considered significant (SPSS v. 22.0, SPSS, Chicago, IL, USA).