Toxicity of Zirconia oxide Nanoparticles: Liver Biodistribution and Liver Damages

Background: Zirconia nanoparticles (ZrO 2 -NPs) have been increasingly used in industrial, biomedical and dental materials. However, the scientific basis for the toxicological effects of ZrO 2 -NPs is poorly elucidated, and the understanding of the underlying mechanism is still limited. Results: The hepatic biodistribution and toxicological effects of ZrO 2 -NPs after intravenous administration (20mg/kg bw) in vivo and the toxicological mechanism toward hepatocytes in vitro were investigated. The liver showed continuous ZrO 2 -NP accumulations liver over a 28-d period. Moreover, ZrO 2 -NPs induced oxidative stress and increased inflammatory responses and functional injury in the liver. Hepasteatosis and cell death were observed in histopathological and immunohistochemical studies. RNA-seq identified the main pathways involved in the metabolism, cellular process, and human diseases. The RT-qPCR analysis results showed that ZrO 2 -NP exposure caused the upregulation of P53, Foxo1, Gadd45g, P21, Caspase3, and PPARα and the downregulation of Igfbp2 and Akt in the liver in response to the ZrO 2 -NP treatment. Meanwhile, the results of the in vitro studies demonstrated that ZrO 2 -NPs exposure resulted in cytotoxicity in Hepg2 cells in a dose- and time-dependent manner. ZrO 2 -NPs were proven to induce oxidative stress, lipid accumulation, cell cycle arrest and cell apoptosis to Hepg2 cells. Western-blot analysis further proved the depression of Igfbp2, activation of Akt-mediated signaling pathway and P53-mediated signaling pathway for Hepg2 cells exposure to ZrO 2 NPs. Conclusions: This study proves that ZrO 2 -NPs have negative impacts on the liver and exhibit potential risks for non-alcoholic fatty liver disease (NAFLD). There is potential immunohistochemical studies. RNA-seq showed that the main pathways identified were involved in metabolism, cellular process, and human diseases. Meanwhile, the results of the in vitro studies demonstrated that ZrO 2 -NPs exposure resulted in cytotoxicity in Hepg2 cells in a dose- and time-dependent manner. Then ZrO 2 -NPs were proven to induce oxidative stress, lipid accumulation, cell apoptosis, Igfbp2 downregulation, Akt-mediated signaling pathway activation and P53-mediated signaling pathway activation. Overall, the present study indicates the potential toxicity of ZrO 2 - NPs toward the liver and a possible risk for NAFLD after exposure to a certain dosage and/or over a certain time period. There is potential concern over ZrO 2 -NPs hepatoxicity in biomedical applications and occupational exposure through large-scale production. However, the present study is a primarily study involving the ZrO 2 -NPs-triggered hepatic toxicological mechanism. Further studies involving precious gene expression analysis at both the mRNA and protein levels with the inhibition or promotion of key genes are needed to verify the ZrO 2 -NPs-induced hepatic toxicological pathways.

concern over ZrO 2 -NPs' hepatoxicity in biomedical applications and occupational exposure through large-scale production. Background ZrO 2-nanoparticles (ZrO 2 -NPs) have resulted in outstanding developments in the fields of industrial, biomedical and dental applications in recent years. Industrial uses of ZrO 2 -NPs include applications in foundry sands, refractories, and ceramics. ZrO 2 -NPs are also applied in biomedicine and dentistry, e.g., in the coatings of hip joint endoprostheses, dental prostheses, bone defect materials, biosensors and drug-loaded carriers [1][2][3][4].
However, NPs are difficult to degrade and can easily accumulate in organs. Because of their huge surface areas, NPs impact enzymes and other proteins, disrupting a series of biological processes in the body [5,6]. Thus, the extensive use of ZrO 2 -NPs may lead to environmental and human exposure, raising concerns about their potential impact and toxicity, especially via occupational exposure during manufacturing.
Studies have proven the potential toxicity of ZrO 2 -NPs. Alzahrani et al. [7] reported the apoptotic and DNA-damaging effects of yttria-stabilized ZrO 2 -NPs on human skin epithelial cells. Ye et al. [8] showed that ZrO 2 -NPs could induced ROS productions and apoptosis effects, which may have an impact on osteogenesis in Osteoblast-Like 3T3-E1 Cells. Yang et al. [9] reported that hollowed ZrO 2 -NPs induced oxidative damage in mice after intravenous injection. However, the scientific basis for the toxicological effects of ZrO 2 -NPs is poorly elucidated, and the understanding of the underlying mechanism is still finite, posing far-reaching challenges to their practical application. Our previous study investigated the distribution of ZrO 2 -NPs in various organs (including the spleen, heart, brain, kidney and lung) and proved that ZrO 2 -NPs exposure could induce oxidative stress, cell death and gene expression changes in various organs [10].
The liver provides the critical function of catabolism and biliary excretion of blood-borne particles, as well as serves as an important site for the elimination of foreign substances and particles [11]. Thus, the liver is a primary target organ in NPs distribution [12,13].
NPs can reach the liver through blood circulation or translocation from the other organs [13][14][15]. Therefore, full research on the hepatotoxicity of ZrO 2 -NPs and, especially, the potential molecular mechanisms is needed. Meanwhile, the biodistribution and clearance of ZrO 2 -NPs in the liver has not been clearly illustrated. The general scheme of NPs uptake and clearance occurs through three main mechanisms: renal, hepatobiliary, and mononuclear phagocyte system (MPS) mechanisms [14,16]. NPs can traverse the hepatic sinusoid and are primarily taken up by MPS after administration into the body. Depending on their physicochemical properties, NPs may filter out into the space of Disse and be endocytosed by hepatocytes. NPs are eventually eliminated through hepatobiliary or renal clearance [17]. As inorganic NPs, ZrO 2 -NPs have more stable cores and thus are almost non-degradable. Therefore, ZrO 2 -NPs are more likely to be retained in the liver for a long term.
In the present study, the distributions of ZrO 2 -NPs in rat livers after intravenous administration and the consequent potential particle-induced injuries and function alterations were estimated. RNA-Seq analysis and precious gene expression analysis of the liver were also conducted. Based on the outcomes of the in vivo studies, in vitro studies were further conducted using ZrO 2 -NPs incubated with Hepg2 cells to explore the mechanisms of ZrO 2 -NPs-induced liver damage. Therefore, this study provides overall insight into the hepatic effects of ZrO 2 -NPs.

Materials And Methods
Characterization of ZrO 2 -NPs. ZrO 2 -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 ZrO 2 -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 ZrO 2 -NPs were primarily sterilized by gamma-ray radiation. Thereafter, the injection suspensions were prepared through the dispersion of ZrO 2 -NPs in 0.5% normal saline (NS). A single tail-vein injection of ZrO 2 -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 ZrO 2 -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 ZrO 2 -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 ZrO 2 -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 H 2 O 2 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. 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 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/). penicillin/streptomycin, 1% L-glutamine, and 1% non-essential amino acids in a humidified atmosphere of 5% CO 2 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 × 10 5 cells/ml in the same media for all assays.

RT-qPCR
The ZrO 2 -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 The ZrO 2 -NPs were spherical with an average diameter of approximately 38 nm [10]. More detailed physicochemical properties of the ZrO 2 -NPs were reported in our previous study [10].
Dose of ZrO 2 -NPs adopted in this study. To date, very few data about the dose of ZrO 2 -NPs administration is available. In a recent study, mice were intravenously injected with hollowed ZrO 2 -NPs at single doses (100-500 mg/kg-bw) [9]. In the liver, it was reported that a single dose of NPs showed lower single-dose toxicity compared to that from multiple doses of NPs administration [19]. A dose of 20 mg/kg-bw ZrO 2 -NPs was chosen, since this was the dose used in our previous study without eliciting abnormal behavior from the rats [10]. Thus, the single dose of 20 mg/kg-bw ZrO 2 -NPs adopted in this study is a relative low dose. A dose of 20 mg/kg-bw in rats is equivalent to a human dose of 0.32 mg/kg bw, corresponding to approximately 19 mg for a human of 60 kg, according to the guidelines for dose translation from animals to humans.
Biodistribution of ZrO 2 -NPs. Fig. 2A demonstrates the biodistributions of the ZrO 2 -NPs accumulated in the liver at six time points after a single injection of ZrO 2 -NPs. The liver captured high amounts of ZrO 2 -NPs during a 28-d period post administration. Reportedly, the liver acts as a biological filtration system that sequesters 30-99% of administered nanoparticles from the bloodstream [13]. As inorganic NPs, ZrO 2 -NPs have more stable cores and are thus almost non-degradable. Therefore, in the present study, ZrO 2 -NPs persisted in the liver for at least a 28-d retention. Reportedly, inorganic NPs, such as gold [20] and iron oxide-coated NPs [21] could remain in the liver for 2-6 months after administration. From 1d to 10 d after administration, the contents in the liver gradually increased, reached a peak at 10 d, and then decreased ( Fig. 2A). Meanwhile, our previous study proved that the ZrO 2 -NPs accumulation in the lung began to decrease from 10 d after exposure [10]. The lungs primarily maintained high levels of ZrO 2 -NPs due to the lipophilic property of lung-lining fluid [22]. After acute exposure, the NPs can be translocated out of the lungs through lymphatic vessels [23] and/or the bloodstream to other organs over time. This can explain why the levels of ZrO 2 -NPs in the liver gradually increased and reached a peak at 10 d. Additionally, the levels of ZrO 2 -NPs in the liver began to decrease after 10 d post injection, which could have resulted from the elimination of ZrO 2 -NPs from the body.
NPs (<6 nm) can be cleared from the kidneys by renal clearance [24]. The MPS has been proven to take up NPs of a broad size scale spanning several hundreds of nanometers [25,26]. NPs smaller than the diameter of liver sinusoidal fenestrations (up to 150-200 nm) can extravasate into the space of Disse and interact directly with hepatocytes [27]. Thus, in the present study ZrO 2 -NPs (with a diameter of 38 nm) are more likely to be taken up and retained by the MPS or hepatocytes. If NPs are degradable by the MPS, their substituents may escape sequestration and return to blood circulation for eventual hepatobiliary or renal clearance. In hepatobiliary clearance, hepatocytes breakdown NPs and excrete them into the bile via the biliary system, after which the NPs traverse the entire gastrointestinal tract and are eliminated in feces [28]. Our previous study proved that feces showed ZrO 2 -NPs accumulations during a 28-d period post administration.
However, no ZrO 2 -NPs accumulation was detected in the kidneys or urine [10]. Based on these results, we believe ZrO 2 -NPs were cleared by hepatobiliary route in the present study. activate immune cells to release inflammatory cytokines such as TNF-α and IL-1β, which would further stimulate the expression of IL-6 and IL-8 [29]. It was revealed that NPsinduced ROS and TNF-α were important activators in inducing DNA strand breaks [30].

Oxidative Damage and Inflammatory Responses in the Rat
Thus, the tissue damage induced by ZrO 2 -NPs might be attributed to continuous inflammatory cytokine expressions and oxidative stresses.

Liver Function Analysis.
To further estimate the liver function damage, the serum biochemical parameters were assayed. The activities of ALT, AST and LDH in ZrO 2 -NPs-exposed liver samples increased significantly compared to the controls, indicating that ZrO 2 -NPs could induce hepatic function damage (Fig. 2E).

Histopathological, Immunofluorescence Evaluation and Ultrastructure
Observation. Histopathological evaluation proved the existence of steatosis and cell death in the liver. Excessive lipid droplet (LD) accumulation in hepatocytes can cause a lipotoxic response involving lysosome-dependent cell death [31]. Hepatic cord disorganizations were observed in the livers of rats 3 d and 7 d post exposure to ZrO 2 -NPs ( Fig. 3 C-F). Fatty degeneration of the liver cells was observed in the livers of rats 7 d post exposure to ZrO 2 -NPs (Fig. 3 E, F). Liver samples taken 10 d post exposure to ZrO 2 -NPs showed more severe hepatic cord disorganizations than did those taken 3 d post injection (Fig. 3 G, H). Significantly larger numbers of Ki-67 positively stained cells were observed in the liver 3d and 7d post injection (Fig. 3). The Ki-67 immunochemical test results (Fig.  4) reveal the ZrO 2 -NPs promotion effect on cell proliferative, indicating the increased possibility of an inflammatory response or tumor cell growth. Fig. 5 shows that the numbers of cells with nuclei positively stained by Tunel were significantly higher among the live cells 7 d after ZrO 2 -NPs exposure (P<0.01) than among the control cells, which indicates increased cellular apoptosis 7 d after ZrO 2 -NP exposure. Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease that refers to a wide spectrum of liver diseases that develop progressively from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, and even cirrhosis [32,33]. It is generally acknowledged that oxidative stress, inflammation, steatosis and cell death play essential roles in the development of NAFLD, although the pathogenesis of NAFLD has not been fully discovered [34]. Thus, based on the histopathological and immunofluorescence observation results, as well as the oxidative stress and inflammatory response tests, ZrO 2 -NPs exposure is likely to cause NAFLD. The ultrastructural changes in the liver show the generation of autophagosomes, as presented in Fig. 6. Reportedly, autophagy activation could alleviate hepatosteatosis [35].

RNA-Seq and RT-qPCR Analysis.
According to the studies above, ZrO 2 -NPs induced hepatic stress, especially by 7 d post exposure. Therefore, microarray analysis of the genes expressed in the liver 7 d post injection was conducted. In total, 68 gene expressions in the ZrO 2 -NPs-treatment groups (32 upreglated and 36 downregulated) were significantly different from those of the control group. All DEGs identified in the liver samples after exposure to ZrO 2 -NPs were annotated for GO enrichment analysis. Following exposure to ZrO 2 -NPs, the DEGs were assigned to 44 GO terms in all three ontologies (biological processes, cellular components and molecular functions), and biological processes appeared to capture most of these terms (Fig. 7A).
To identify the related pathways that induce liver tissue damage after exposure to ZrO 2 -NPs, we characterized the pathways in which the DEGs were involved in the KEGG database. The top first-level KEGG pathways were identified as related to metabolism, followed by organismal systems and human diseases (Fig. 7B). Top third-level KEGG pathway terms are shown in Tab. 2. Heat maps were generated to show the expression values of differently expressed transcripts for each group by color based on the read count, with the color changing from red to blue (Fig. 7C) The most differentially expressed gene in the liver was Bcl6, which showed the highest fold change of 5.11.

In Vitro Study
Cytotoxicity and ROS production from ZrO 2 -NPs Exposure. Treatment with ZrO 2 -NPs could induce significant cell death (>50 %) at a dose of 750 μg/ml after 24 h coincubation (Fig. 8A). Treatment with ZrO 2 -NPs could induce significant cell death (>50 %) at a dose of 1000 μg/ml after 12 h coincubation, and it caused the same toxic effect at 500 ug/mL after 24 h exposure (Fig. 8A). Our results showed that ZrO 2 -NPs triggered cytotoxicity in a dose-and time-dependent manner. Our findings were consistent with those of previous studies reported recently [7,8,36] .
To further examine the effect of ZrO 2 -NPs on the cell membrane permeability, an LDH assay was conducted in Hepg2 cells incubated with a series of concentrations of ZrO 2 -NPs for 12 h and 24 h. The percentage of LDH released into the culture medium was measured as an index of cellular death, and the results are presented in Figure 8B. The toxic effect was consistent with that observed in the cell viability evaluation results, also demonstrating a dose-and time-dependent manner. Fig. 8C demonstrates that the ROS levels of the cells exposed to ZrO 2 -NPs at concentrations from 0 ug/mL to 1000 μg/ml after 6 h exposure, which increased with the elevation of the ZrO 2 -NPs concentration.

ZrO 2 -NPs induced lipid accumulation and apoptosis. Excessive lipid droplet (LD)
accumulation in hepatocytes can drive a lipotoxic response. As shown by the in vivo study, hepatosteatosis, extra ROS generation, inflammatory responses and lipid metabolism gene changes were observed. We assume that hepatosteatosis could be the main mechanism behind ZrO 2 -NPs-induced liver toxicity. To further explore this assumption, an in vitro study for the evaluation of lipid accumulations and lipid metabolism-related protein expression changes was therefore carried out. According to the TG and TC accumulation evaluations (Fig. 9A), Hepg2 cell showed increased lipid accumulations upon exposure to ZrO 2 -NPs starting from a concertation of 500 μg/mL. Fig. 9 B, C and D show the upregulation of Foxo1 and the downregulation of Igfbp2 and Akt. The overexpression of Igfbp2 has been shown to reverse diabetes and steatosis in obese mice [37].
Downregulation of Igfbp2 was reported to be the maker of NAFLD [38]. As a symptom of metabolic disorders, hepatic lipid accumulation is strongly associated with insulin resistance [39]. Insulin resistance, which is characteristic of IRs/Akt depression, has been recognized for many years to be an essential component of NAFLD pathogenesis [40]. The downregulation of Akt reduces the phosphorylation of Foxo1, which leads to Foxo1 activation and results in the promotion of NAFLD [41]. Thus, based on our study, ZrO 2 -NPs are likely to induce hepatosteatosis via activation of the Akt/Foxo1 pathway or downregulation of Igfbp2.
A recent study investigated the toxicol profiles of several metal oxide NPs (including some rare earth oxides and transition metal oxides) in hepatocytes and proved that they could induce hepatocyte death by apoptosis [36]. However, the exact mechanism of ZrO 2 -NPsinduced hepatocyte death is unknown. As shown by Cell viability evaluation (Fig. 8A), ZrO 2 -NPs require a relatively higher concentration (>250 μg/mL) to cause a reduced percentage of viability. Fig. 10 shows that ZrO 2 -NPs exposure (250 μg/mL) could induce apoptosis in Hepg2 cells at 24 h, which is consistent with the in vivo Tunel test results.
Expressions of proapoptotic proteins-including P53, Bax, Caspase3, Gadd45g, and Bcl6 increased in the Hepg2 cells (Fig. 10C). Meanwhile, the expression of antiapoptotic protein Bcl2 decreased in the Hepg2 cells (Fig. 10C). Additionally, cell-cycle analysis (Fig. 10B) demonstrated that Hepg2 cell exposure to 250 μg/mL ZrO 2 -NPs resulted in G1/G0 arrest of the Hepg2 cells, allowing cellular repair or resulting in the activation of apoptosis signaling when severe DNA damage occurred [42]. The overexpression of cell cycleregulating proteins (P21 and Gadd45g) was also detected (Fig. 10C). These results have shown that exposure of HepG2 cells to ZrO 2 -NPs may trigger cell apoptosis through the upregulation of P53 and subsequently activate Bcl6 expression, thereby increasing the Bax/Bcl2 ratio and finally activating Caspase3 expression.

Conclusion
We investigated the hepatic biodistribution and toxicological effects of Overall, the present study indicates the potential toxicity of ZrO 2 -NPs toward the liver and a possible risk for NAFLD after exposure to a certain dosage and/or over a certain time period. There is potential concern over ZrO 2 -NPs hepatoxicity in biomedical applications and occupational exposure through large-scale production. However, the present study is a primarily study involving the ZrO 2 -NPs-triggered hepatic toxicological mechanism. Further studies involving precious gene expression analysis at both the mRNA and protein levels with the inhibition or promotion of key genes are needed to verify the

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Not applicable.

Consent for publication
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Competing interests
The authors declare that they have no competing interests. According to the gene expression analysis software, differences were statistically significant when P >0.80.