The potential ameliorative impacts of cerium oxide nanoparticles against fipronil-induced hepatic steatosis

Fipronil (FIP) is a phenylpyrazole insecticide that is commonly used in agricultural and veterinary fields for controlling a wide range of insects, but it is a strong environmentally toxic substance. Exposure to FIP has been reported to increase the hepatic fat accumulation through altered lipid metabolism, which ultimately can contribute to nonalcoholic fatty liver disease (NAFLD) development. The present study aimed to examine the function of cerium oxide nanoparticles (CeNPs) in protecting against hepatotoxicity and lipogenesis induced by FIP. Twenty-eight male albino rats were classified into four groups: FIP (5 mg/kg/day per os), CTR, CeNPs (35 mg/kg/day p.o.), and FIP + CeNPs (5 (FIP) + 35 (CeNPs) mg/kg/day p.o.) for 28 consecutive days. Serum lipid profiles, hepatic antioxidant parameters and pathology, and mRNA expression of adipocytokines were assessed. The results revealed that FIP increased cholesterol, height-density lipoprotein, triacylglyceride, low-density lipoprotein (LDL-c), and very-low-density lipoprotein (VLDL-c) concentrations. It also increased nitric oxide (NO) and malondialdehyde (MDA) hepatic levels and reduced glutathione peroxidase (GPx) and superoxide dismutase (SOD) enzyme activities. Additionally, FIP up-regulated the fatty acid-binding protein (FABP), acetyl Co-A carboxylase (ACC1), and peroxisome proliferator-activated receptor-alpha (PPAR-α). Immunohistochemically, a strong proliferation of cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba-1), cyclooxygenase-2 (COX-2) reactions in the endothelial cells of the hepatic sinusoids, and increased expression of caspase3 were observed following FIP intoxication. FIP also caused histological changes in hepatic tissue. The CeNPs counteracted the hepatotoxic effect of FIP exposure. So, this study recorded an ameliorative effect of CeNPs against FIP-induced hepatotoxicity.

Experimental animals. Twenty-eight healthy adult male albino rats (average weight 180 ± 10 g) were obtained from the Animal Breeding Unit, Faculty of Agriculture, Alexandria University. The animals were housed under a pathogen-free environment with controlled humidity, temperature (22 °C) and a 12 h light/dark cycle. The animal experiments were performed according to the Laboratory Animals of the National Institutes of Health (NIH) Care and Use Guidelines, and the study protocol was approved by the local authorities of Damanhour University, Egypt (DMU-2019-0023). Two weeks before the experiment was conducted, the animals were allowed to acclimatize the testing facility condition. Afterward, the rats were caged equally into four experimental groups, each consisting of seven rats. Group 1-control group (CTR) rats were orally received saline. Group 2-animals have orally received FIP solution (5 mg/kg bwt, 1/20 of the LD 50 ) 19,20 . Group 3-the rats have orally received CeNPs solution (35 mg/kg body weight) 21 . Group 4-animals orally received FIP (5 mg/kg) and CeNPs (35 mg/kg) solutions using gastric tube daily. During the experimental period, rats were daily observed for any abnormal behavior and clinical signs.
Blood and tissue sampling. On the 28th day of the experiment, all rats were prohibited from feeding overnight, weighed individually, and euthanized using an anesthesia system containing xylazine and ketamine HCl. Blood was gathered from the aortic vein, kept in anticoagulant-free test tubes, and the serum was isolated and kept at − 20 °C until lipid profile determination. The liver was excised and rinsed using physiological saline (NaCl 0.9%), wiped using filter paper and split into two sections -the first section kept rapidly at − 80 °C for biochemical and gene expression levels and the second section was used for histopathological and immunohistochemical examination after fixing in four percent paraformaldehyde (PFA) diluted in phosphate buffer saline (PBS) solution.

Serum biochemical test.
For examination of the serum lipid profile of the treated rats; triacylglycerol, cholesterol, HDL-c, LDL-c, and VLDL-c commercial kits were used, which were purchased from Bio-Diagnostics Co., Cairo, Egypt. The experiments were conducted following the manufacturer's instruction guidelines.
Immunohistochemical examination. Working dilutions, sources, methods, and antibodies for antigen recovery were listed in Table 1. The immunohistochemical technique in liver sections was investigated based on the method identified by Noreldin, et al. 28 and Noreldin,et al. 29 . Briefly, the paraffin sections were prepared with a thickness of 4 µm, deparaffinized by xylene and re-moistened in graded alcohol and washed with distilled water. Afterward, endogenous peroxidase was deactivated by 3% H 2 O 2 in absolute methanol for 30 min at 4 °C and washed again using PBS, blocking the nonspecific reaction at room temperature with 10% normal blocking serum for 60 min. Then, the sections were incubated overnight at 4 °C with the primary antibodies, washed with PBS, incubated for 60 min with biotin-conjugated goat anti-rabbit IgG antiserum or rabbit anti-goat IgG antiserum (Histofine kit, Nichirei Corporation) according to the species' primary antibody hosted. Then the sections were incubated for 30 min with streptavidin-peroxidase conjugate (Histofine kit, Nichirei Corporation) after washing with PBS. The streptavidin-biotin complex was further incubated for 3 min with a solution of 3,3′-diaminobenzidine tetrahydrochloride (DAB)-H 2 O 2 , pH 7.0. Finally, the sections were rinsed with distilled water and used Mayer's hematoxylin counterstain. A digital camera (Leica EC3, Leica, Germany) connected to a microscope (Leica DM500, Leica, Germany) was used to capture micrographs of the prepared sections. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for immunostaining intensities' quantification 30 . The mean of the inverse density of 10 randomly selected fields from different parts of 8 rats in each group was calculated byVis et al. 31 .

Results
Signs of toxicity. No obvious clinical signs or symptoms noticed all over the experimental period on rats that were exposed to FIP or/and CeNPs.
Serum biochemical findings. The FPN-intoxicated group showed significant elevation (P < 0.05) in the serum levels of cholesterol (19.0%), TAG (25.2%), LDL-c (66.0%) and HDL-c (47.6%) when compared with the CTR one (Table 3). Meanwhile, rats treated with CeNPs and FIP + CeNPs exhibited remarkable reductions (P ≤ 0.05) in serum cholesterol levels by 8.5 and 9.3%, respectively. Rats treated with CeNPs and FIP + CeNPs had similar content of TAG, VLDL-c, LDL-c, and HDL-c to that of the CTR group. The VLDL-c levels were not different in the serum of all treated groups (Table 3). Table 4 revealed that the FIP-treated group showed substantial increase (P ≤ 0.05) in MDA (68%) concentrations and decrease in GPx (39%) and SOD (46%) enzymes activities in the liver tissue in relative to the CTR one. The concentrations of MDA in liver tissue (37%) in CeNPs-treated rats was reduced significantly (P ≤ 0.05), while GPx and SOD enzyme activities were similar to normal CTR values (Fig. 2). Concentrations of serum NO were nearly comparable in CTR, CeNPs, and FIP + CeNPs groups. The FIP toxic effects on hepatic GPx, MDA, SOD, and NO were substantially reduced (P ≤ 0.05) by CeNPs administration indicating the effect of CeNPs in alleviating oxidative damage caused by FIP.

Liver lipid peroxidation and antioxidative indices. The results presented in
Histopathological investigation of hepatic tissue. The histological findings revealed a polygonal hepatocyte producing substantial anastomosis plaques in the liver tissue in the CTR animals with acidophilic cytoplasm (Fig. 1A). Hepatic cells exhibited one or two large central round heavy hematoxylin-stained nuclei. Moreover, one or more nucleoli were detected in some hepatic cells (Fig. 1A). Hepatocyte plates and the hepatic capillaries barriers (sinusoids) laminated the Disse's space, while hepatic capillary walls were bordered by Kupffer cells (Fig. 1A) and distinguished primarily by elongated and heavily stained nuclei. The tissues of the CeNPs group didn't show any histopathological effects (Fig. 1B). In contrast, the FIP group revealed portal vein congestion surrounded with lymphocytic infiltration, lymphocytic aggregation in between massive fatty degeneration, congested central vein, necrotic foci, and nuclear condensation ( Fig. 1C-E). It was noticed that CeNPs protected hepatic cells against FIP adverse effects, where it restored the normal architecture of liver tissues (Fig. 1F). The www.nature.com/scientificreports/ www.nature.com/scientificreports/ FIP treatment significantly increased the fatty bodies' degeneration and lymph aggregation compared to CeNPs and the CTR group, while CeNPs significantly countered these effects (Fig. 1G). CeNPs groups had the greatest PAS distribution in all hepatocytes ( Fig. 2A,B). Also, FIP caused a weak and uneven distribution of PAS and decreased glycogen content (Fig. 2C) in relation to the CTR one. Additionally, the FIP group that was treated with CeNPs had a moderate PAS reaction (Fig. 2D). FIP showed a significant decrease www.nature.com/scientificreports/ in PAS distribution concerning CTR and the CeNPs-treated group. CeNPs showed a high even distribution of PAS reaction in all hepatic lobules when combined with the FIP (Fig. 2E).
Immunohistochemistry. The FIP treatment showed a significant increase in caspase3 reaction concerning CTR-and CeNPs-treated groups. On the other hand, the FIP-treated group was protected with cerium as shown by a very weak caspase3 reaction (Fig. 3). www.nature.com/scientificreports/ Also, FIP-treated group showed strong PCNA reaction in the nuclei of most hepatocytes compared to CTR and CeNPs groups that showed negative PCNA reaction in the nuclei of hepatocytes, while FIP + CeNPs-treated group showed nearly negative PCNA reaction in nuclei of the hepatocytes (Fig. 4). www.nature.com/scientificreports/ Immunostaining of rat liver tissues by massive ionized calcium-binding adapter molecule1 (Iba-1) in the case of the FIP-intoxicated group showed a strong inflammatory process via strong cyclooxygenase (COX-2) reaction and Iba-1 positive hepatic macrophage in the hepatic sinusoids in comparison to fipronil group that treated with cerium nanoparticles. The latter group showed low IBA1 positive hepatic macrophage in the hepatic sinusoids. CTR and CeNPs groups showed normal distribution of the Iba-1positive hepatic macrophage in the hepatic sinusoids (Fig. 5) with a negative COX-2 reaction (Fig. 6). CeNPs attenuated FIP impacts on the endothelial cells of the hepatic sinusoids and showed a moderate reaction (Fig. 6). mRNA expression of FABP, ACC, PPAR-α, Caspase3, and Bcl-2. The results of mRNA expressions of FABP, ACC, PPAR-α, caspase3 and Bcl-2 genes in liver tissues were presented in Table 5. The results revealed up-regulation (P ≤ 0.05) in the comparative mRNA expressions of FABP, ACC, PPAR-α, and caspase3, while exhibited down-regulation in the Bcl-2 gene in the liver tissue of rats that received FIP when compared with the CTR animals. Meanwhile, CeNPs treatment down-regulated the mRNA expression of ACC and caspase3 and up-regulated the FABP, PPAR-α, and Bcl-2 compared to CTR. In the case of the FIP group that was treated with CeNPs, significant up-regulation in FABP, PPAR-α and Bcl-2 were observed. On the other hand, ACC and caspase3 mRNA expression were significantly down-regulated (Table 5).

Discussion
Hepatoxicity changes induced by FIP might be due to the imbalance of the antioxidant system, which might lead to the formation of ROS that activates the apoptotic process and disturbances in the lipid profile of rats 32,33 . Current results showed that CeNPs was a chemoprotective agent against FIP-induced hepatotoxicity. The hepatic cells were documented to have a significant and critical function in lipogeneses and glucose metabolism processes 34 . The disturbance of lipid and glucose metabolism might be observed by the excess accumulation of FA in the hepatic cells 35 . Consequently, increasing hepatic FA uptake 36 leads to an increase in the hepatic triglyceride (TG) levels, which induces NAFLD development 37 . The changes in lipid profile might be caused by the accumulation of a high level of FA in circulation and hepatic lipogenesis, resulting in TG accumulation in the liver while dietary lipids represent a large percentage of intrahepatic lipids 38,39 .
The current study explained a decreased weight gain in CeNPs-treated rats relative to CTR ones. Also, the animals treated with FIP showed a remarkable elevation in serum lipid profile (TAG, HDL-c, LDL-c, and cholesterol) compared to CTR, CeNPs, and FIP + CeNPs groups. The significant modification in lipids might be due to the oxidative stress produced by FIP, which ultimately leads to liver inflammation and injury. These results indicated that FIP induced lipogenesis and lipid accumulation via increased expression of FABP and ACC1 in comparison to the CeNPs-treated group, which showed downregulation of ACC1 along with the up-regulation of FABP like the CTR groups. Activated AMPK (adenosine monophosphate-activated protein kinase) that phosphorylates the main proteins associated with controlling lipid and carbohydrate metabolism, followed by ATP inhibition that consumes anabolic pathways, such as cholesterol, isoprenoid, and FA synthesis, hepatic gluconeogenesis 40 and induces lipolysis 41,42 . Some studies revealed that FIP increases adipogenesis by downregulating AMPKα, which disrupts lipid metabolism, which may demonstrate the high serum cholesterol level 20,33 . Fipronil insecticide has been reported to raise the levels of intracellular Ca +2 by altering the plasma membrane permeabilization 43 , leading to a disruption of adipogenesis and lipid metabolism process by the pathway mediated by Ca MKKβ and AMPK 44 .
FIP substantially elevated fatty acid synthase (FAS) and acetyl Co-A carboxylase (ACC) expression (the two rate-limiting enzymes for lipogenesis) 45 when compared with CTR group. Likewise, FABP (a protein that transports FA in the cytoplasm for metabolic process or storage) expression 46 , was significantly enhanced by FIP therapy relative to CTR 45 . Regarding CeNPs, interestingly, Rocca et al. 47 identified CeNPs as a new anti-obesity pharmaceutical formula after being tested in vitro and in vivo. They stated that CeNPs interfere with the adipogenic mechanism by decreasing the mRNA transcription of genes included in adipogenesis through inhibiting the accumulation of triglycerides found in 3T3-L1 pre-adipocytes. Intraperitoneal injection of CeNPs at a dose of 0.5 mg/kg did not show toxic symptoms in rats, however, a significant decrease in body weight and insulin, leptin, glucose, and TG plasma levels are observed in comparison to CTR group 47 . Our results are consistent with other studies that demonstrate the ability of CeNPs to activate AMPK which indicates inhibition of adipogenesis with decreased PPARαC/EBPα expression as well as adipogenic markers including ACC and FAS 48,49 .
Lipogenesis inhibition due to ACC phosphorylation may be the major regulatory step in FA synthesis and oxidation 50 . ACC stimulates the malonyl-CoA synthesis, which is a FA synthesis substrate and is inhibited by AMPK-mediated ACC phosphorylation 51 . Metabolism enhancement is considered as one of the anti-inflammatory actions of CeNPs mechanisms since that results reported herein have demonstrated that CeNPs substantially decreased the weight gain of rats, which might reduce the visceral obesity of rats (A. Rocca, S. Moscato, F. Ronca, S. Nitti, V. Mattoli, M. Giorgi, G. Ciofani, Pilot in vivo investigation of cerium dioxide nanoparticles as a novel anti-obesity pharmaceutical formulation, Nanomed. Nanotechnol., Biol. Med. 11 (7) (2015) 1725-1734). The mass of visceral adipose tissue in CeNPs-treated rats was approximately twice less than CTR 52 . PPARα is the most prevalent isotype in hepatic cells and participates in several lipid metabolism aspects 53 , such as FA synthesis, degradation, storage, transport, lipoprotein metabolism and ketogenesis throughout fasting. PPARα coordinates various de novo lipid synthesis pathways in the fed state to provide FA for hepatic TG storage during starvation times 54 . PPARα's mRNA expression in rats was the highest in tissues with high FA oxidation levels, such as kidney, heart, liver, and brown adipose tissue 55,56 . High-fat diet administration is often related to PPARα target genes hepatic expression participates in FA oxidation in wild-type mice, and it has been proposed the adaptive or protective effect of PPARα 57,58 . Few reports have investigated the outcomes of PPARα for treatments for NAFLD 59 . The improvement of β-FAO by PPARα agonist 60 might protect the mice from induced liver injury 61 . Also, the recent study explained the high PPARα expression in FIP-treated rats that might be Scientific Reports | (2021) 11:1310 | https://doi.org/10.1038/s41598-020-79479-5 www.nature.com/scientificreports/ due to the increased level of FFAs and b-oxidation that usually caused by FFAs load in the mitochondria. This increases the load on the endoplasmic reticulum, which leads to ROS production causing oxidative stress and  www.nature.com/scientificreports/ inflammatory pathway activation 62 . Besides its role in metabolism regulation, PPARα is also believed to have anti-inflammatory activity 63 . There has been cumulative evidence that pesticide toxicity inhibits redox homeostasis as well as oxidative damage induction. Several studies suggested that redox homeostasis disturbance was triggered during FIP toxicity was because of high ROS production 33,64,65 . Hepatotoxicity induced by FIP in this study might be due to increased levels of MDA and NO (the indicator of LPO) because of the high reactive oxygen metabolites production especially the hydroxyl radicals 33,66 , LPO play role in the disruption of the integrity of cellular membranes and implicated in liver injuries 67 . ROS can target cell membranes and other cellular molecules, resulting in protein oxidation, lipid peroxidation, caspase3 activation, and DNA damage 33,68 , leading to cell dysfunction 69,70 . The protective mechanisms toward oxidative stress mainly through balances mediated by non-enzymatic and enzymatic antioxidants 71 . In current study, the decrease in SOD and GPx activities of rats subjected to FIP might be due to the excess production of O 2 that is rapidly transformed by SOD and GPx to H 2 O 2 and water, respectively as reported in the liver of pregnant rats and their offspring due to the inadequate ROS detoxification produced by FIP in hepatocyte 33,66 .
It was recognized that liver cells showed the most active absorption and retention of CeNPs stored in the liver for eight weeks at least 62,72,73 . Recently 62 , demonstrated that the antioxidant activity of CeNPs was through increasing the antioxidant enzymes (SOD and GPx) with decreasing the lipid peroxidation markers (MDA and NO). CeNPs have a high ability to eliminate free radicals as soon as they are produced during ROS imbalance as they can inversely transform between Ce 3+ and Ce 4+ found on the surface 74 . The lipid peroxidation reduction after CeNPs administration diminishes ROS harmful effects on the hepatic tissue. The mechanism of scavenging of ROS/RNS of CeNPs depends on the physicochemical properties, the ability of CeNPs to absorb and release oxygen 75 . CeNPs can act as a catalyst that mimics the antioxidant enzyme SOD 76 . So, there are two ways that CeNPs might act as an antioxidant against FIP toxicity. The first is linked to its antioxidant activity because the intensification of lipid peroxidation in the liver is among the causes of the inflammation, while the second is controlled by AMPK-PPAR-α-signaling mechanism 77 . The inflammatory processes involved in the pathogenesis of liver damage and obesity-related NAFLD 78 . Furthermore, fat destroys liver tissues and induces neutrophil infiltration by releasing ROS and aggravating inflammatory processes 12,13 . High accumulation of neutral lipid (mostly TG in hepatic cell lipid droplets) begins the early NAFLD pathological stages 79 . NAFLD's pathogenesis is not well known but is suggested as a "two-hit" mechanism 80 . The first "hit" results in lipid aggregation and its mechanisms will likely include dysregulated lipid homeostasis such as de novo lipogenesis, β-oxidation, lipid storage and trafficking, and VLDL-c secretion 81 . This hepatic steatosis characterizes the liver to a "second hit" that results in inflammation, a primary pathophysiological symptom of steatohepatitis and advanced hepatic disorders 80,82 . Oxidative stress has been suggested as the main mediator of this "second hit" 80,83 . So, increased FFAs levels accompanied by PPAR-α stimulation, which in turn leads to ROSproduction and cell injury 81 .
The efficiency of CeNPs on liver tissues was demonstrated by restoring the tissues architecture; necrosis, inflammation, and a decline of dystrophy in rats exposed to the FIP corroborated the histopathological lesions observed in the study 84 . FIP group revealing congested central vein, massive fatty degeneration in the periportal areas, lymphocytic aggregation in between massive fatty degeneration, with lymphocytic infiltration. Also, necrotic foci, congested liver sinusoids, and fatty degeneration might be due to that FIP increase lipid peroxidation. The FIP group that was protected with cerium nanoparticles showed normal liver architecture similar to the results of 72 . Studies showed an important connection between PCNA, Iba-1, and COX-2 expression and the inflammatory reaction as well as mitotic division 85 . Iba-1 participates in macrophage inflammatory pathways, including proliferation, migration, and signal transduction 86 . Proliferating cell nuclear antigen (PCNA) is believed to have an important role in controlling DNA synthesis as well as cell proliferation 87 . Also, COX isozymes (COX-1 and 2) are particularly important, as they are the main NSAIDs targets 88 , that parameters detected using the immunohistochemical.
In current study, Iba-1 cells were observed in the FIP-treated group with strong PCNA and COX-2 reaction concentrated in the nuclei of the most hepatocytes that may be due to FIP induced liver injury while in case of FIP group that protected by CeNPs show low Iba-1 positive hepatic macrophage and nearly negative PCNA and COX-2 reaction in nuclei of the hepatocytes, So, liver regeneration is the expected physiological response. Also, FIP induces apoptosis by strong caspase3 reaction in all nuclei of hepatocytes in opposite to FIP group previously protected with cerium showing very weak caspase3 reaction in the nuclei of hepatocytes. That might be due to the cytotoxic activity of fatty acid that influences cell survival. Long term accumulation of lipids may lead to hepatocyte necrosis or apoptosis 89 . MDA and NO interacted directly with the DNA and triggered DNA adducts and nuclear condensation that promoted apoptosis via cytochrome C and further caspase3 activations as detected by immunostaining, along with the induced mitochondrial dysfunction. Several reports revealed that FIP triggered cell death through apoptotic pathways 20 .

Conclusions
In this study, FIP induced hepatotoxicity through disturbance in the serum lipid profile (VLDL-c, HDL-c, TAG, LDL-c) and cholesterol level might be due to increasing the mRNA fatty acid-binding protein expression and ACC genes and FIP can cause serious tissue injury in the liver caused by oxidative stress through increasing MDA and NO with decreasing SOD and GPx and apoptosis formation by increment caspase3 and decreasing BCL-2. While CeNPs could be used to activate the protective mechanisms against oxidative damage caused by FIP in the liver. Moreover, CeNPs effectively relieve the inflammatory processes in the blood of rats that may reduce obesity defects in liver damage.