iTRAQ-Based Quantitative Proteomics Analysis of Sprague-Dawley Rats Liver Reveals Peruorooctanoic Acid-Induced Urea Metabolism Dysfunction

Peruorooctanoic acid (PFOA) is a typical C8 representative compound of peruoroalkyl and polyuoroalkyl substances (PFAS) widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. Despite emerging scientic and public interest, the precise mechanisms of PFOA toxicity remain unclear. In this study, male rats were exposed to 1.25, 5, and 20 mg PFOA/kg body weight/day for 14 days. Urine samples were also collected and monitored by raising rats in metabolic cages. In vivo results demonstrate that PFOA exposure induces signicant hepatocellular hypertrophy and reduced urea metabolism. iTRAQ-based quantitative proteomics analysis of Sprague-Dawley (SD) rats livers identied 3,327 non-redundant proteins of which 112 proteins were signicantly upregulated and 80 proteins were downregulated. Gene ontology analysis revealed proteins are primarily involved in cellular, metabolic and single − organism processes. Among them, eight proteins (ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1) were related to oxidation of fatty acids and two proteins (ASS1 and CPS1) were found to be associated with urea cycle disorder. The downregulation of urea synthesis proteins ASS1 and CPS1 after exposure to PFOA was then conrmed through qPCR and western blot analysis. Together, these data demonstrate that PFOA exposure directly inuences urea metabolism and identify CPS1 as a potential regulatory target.


Introduction
Per uoroalkyl and poly uoroalkyl substances (PFASs) are a class of synthetic chemicals that are increasingly recognized as a new type of persistent organic pollutants (POPs). POPs contain high-energy C-F covalent bonds in which all hydrogen atoms may be replaced by uorine atoms. The archetypal PFAS, per uorooctanoic acid (PFOA), is a per uorocarboxylic acid with 8 C atoms. The uorinecontaining special structure of PFOA is responsible for its hydrophobicity, oleophobicity and extremely low surface tension. As such, PFOA is widely used in various commercial and industrial settings including manufacture of textiles, packaging materials, surfactants, pharmaceuticals and re-extinguishing foam In recent years, an increasing number of studies have revealed the toxic effects of PFOA accumulation in organisms (Andersen et al. 2008;Kennedy et al. 2004;Lau et al. 2007;Lau et al. 2004). In general, PFOA accumulation interferes with cellular lipid metabolism, leading to carcinogenicity, liver toxicity, developmental toxicity, immunotoxicity, endocrine interference and neurotoxicity. Previous reports from our lab have demonstrated that PFDoA exposure can restrict amino acid metabolism in rats and thereby in uence the synthesis of urea (Liu et al. 2016). In the following study, iTRAQ-based quantitative proteomics was utilized to screen for global proteomic pro le alterations in rat livers after exposure to PFOA. We hereby demonstrate the differential expression of ASL1, ASS1 and CPS1 and identify their role as PFOA sensitive genes related to the urea cycle. These ndings clarify the potential mechanisms responsible for PFOA toxicity in vivo and provide reference targets for future intervention and treatment of PFOA accumulation in humans. company (Nanjing, China) at 6-8 weeks of age. The rats were maintained in a SPF grade facility on a 12h light/12-h dark cycle and were allowed ad libitum access to a standard diet and pure water. The ambient temperature in the animal room was 23 ± 1℃ and the relative humidity was 60 ± 5%. After one week of adaptation, the rats were randomly separated into four groups of 10. The treatment rats were given doses of 1.25, 5, and 20 mg PFOA/kg body weight/day by oral gavage for 14 consecutive days.

Materials And Methods
The control animals were also treated with Milli-Q water, accordingly. At the end of the experiment, 7 rats from each group were weighed and anesthetized with sodium pentobarbital (45 mg/kg). Afterwards, blood was drawn from the inferior caval vein, and liver tissues were rapidly collected, weighed, rinsed with PBS, divided into small aliquots, ash frozen in liquid nitrogen before being stored at -80℃ until further analysis. The remaining three rats from each group were used for Clinicopathologic analysis. All procedures were performed in accordance with the Ethics Committee of Bengbu Medical College, Anhui Province. Urine collection and analyses 10 rats were randomly divided into 2 groups: high dosage group and normal control. Rats were raised in metabolic cages to collect urine samples of 24 h volume for 14 consecutive days. Urea concentration was determined by using commercially available kits (Nanjing Jianchen Bioengineering Institue, China). Histopathological Examination Three livers from each group were xed in freshly prepared paraformaldehyde (3.7% in DPBS) and processed sequentially in ethanol, xylene and para n. Tissues were then embedded in para n, sectioned (5 µm), and stained with hematoxylin and eosin (HE stains). Serum Biochemistry Analysis and creatinine (CR) were measured using cobas® 8000 modular analyser series (F. Hoffmann-La Roche Ltd). Protein Preparation, iTRAQ Labeling 3 individual liquid nitrogen frozen livers from normal control rats and three PFOA treated livers from 20 mg PFOA/kg/d group were randomly selected for iTRAQ based mass spectrometry analysis. Proteins were extracted by dissolving each liver sample in 300 µL of ice-cold 0.1 M Na 2 CO 3 and 10 mM sodium orthovanadate (pH 11) supplemented with protease inhibitor (Roche Complete EDTA Free) and phosphatase inhibitor (Roche), sonicated for 3 × 10 seconds and stored on ice. The bicinchoninic acid assay (BCA assay) was used to measure 200 µg proteins and mixed with urea/thiourea denaturation buffer to a nal concentration at 6 M urea, 2 M thiourea. All protein samples were trypsinized (mass spec grade, Promega). The tryptic peptides in the three biological samples from the control and PFOA-treated groups were labeled with iTRAQ reagents (isobaric tags 115, 116, and 117 for the control; 118, 119 and 121 for the treated group) (iTRAQ Reagent-8 Plex Multiplex Kit, AB Sciex). The iTRAQ labeling was performed according to the manufacturer's protocol. LC -MS/MS analysis Mass spectroscopic (MS) analysis was performed using an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Scienti c, USA) and coupled with an EASY-nLC HPLC system (Thermo Scienti c, USA). The iTRAQ labeled peptieds were loaded onto a C18-reversed phase column (3 µm-C18 resin, 75 µm ×15 cm) and separated on an analytical column (5 µm-C18 resin, 150 µm × 2 cm; GmbH, Ammerbuch, Germany) using mobile phase Buffer A: 0.5% formic acid / H 2 O and Buffer B: 0.5% FA/ACN at a ow rate of 300 nL / min, using a 150 min gradient. Spectra were acquired in Data Dependent Acquisition (DDA) mode. Database search for peptide and protein identi cation The raw mass data were analyzed using Thermo Proteome Discoverer version 1.4 (ver. 1.4.0.288; Thermo Fisher Scienti c) and with a false discovery rate (FDR) < 1% and expected cutoff or ion score < 5% (with 95% con dence) for searching the Uniprot Rat Complete Proteome database. The following options were used to identify the proteins: Peptide mass tolerance = ± 10 ppm, MS/MS tolerance = 0.6 Da, enzyme = trypsin, missed cleavage = 2, xed modi cation: iTRAQ 8plex (K) and iTRAQ 8plex (N-term), variable modi cation: oxidation (M), database pattern = decoy.

GO annotation and KEGG pathway analysis
To analyze the differentially expressed proteins in PFOA treated group compared with normal control group, Gene Ontology (GO) annotation of the identi ed proteins was performed by searching the GO Web site (http://www.geneontology.org) to catalog the molecular functions, cellular components, and biological processes. Protein interactions and biological pathways were determined using the ResNet database (version 6.5, Ingenuity Systems, Inc.) (KEGG) to better understand these differentially expressed proteins in relation to the published literature. RNA Isolation and Quantitative real-time PCR Rat livers were used for RNA extraction and subsequent qPCR assays. Total RNA of the liver samples was isolated using a Trizol reagent (Ambion, Thermo Fisher Scienti c, USA) and the isolation process was performed according to the manufacturer's instructions. Quantitative real-time PCRs (qPCR) were performed on a QuantStudio 3 Real-Time PCR System (Thermo Scienti c, USA) using a SYBR Green Real Master Mix Rox (Tiangen, China). The housekeeping gene GAPDH was used as an internal control. The information of the primer pairs are listed in the supplementary table S1. The relative quanti cation of target genes was calculated based on the 2 −ΔΔCT method. Western blotting authentication Protein extracts from the control and PFOA exposure group liver tissues were used for western blot analysis. The western blot is brie y as follows: Total proteins from liver of each rats were extracted with RIPA (Thermo Scienti c, USA) containing 1 mM PMSF (Sigma-Aldrich, USA) and 1% phosphatase inhibitor (Sigma-Aldrich, USA). The protein concentration was determined by using a BCA kit (cwbiotech, China). Approximately 40 µg of total protein was loaded on 10% sodium dodecyl sulfate (SDS)polyacrylamide-gels and then transferred to polyvinylidene uoride membrane (PVDF) tansblot membranes (Amersham Biosciences, Piscataway, NJ, USA). The blotted membranes were blocked in blocking buffer (TBST) for 1 h, and then incubated with primary antibodies dissolved by blocking buffer on a shaker overnight at 4°C. (The information of the primary antibodies is listed in supplementary table S2.) After washing with TBST for 3 times, the membranes were then incubated with uorescentconjugated anti-rabbit IgG as the secondary antibody for 1 h at room temperature, respectively. The immunoreactive bands were photoed and analyzed by Gel Doc XR + Gel Documentation System (BIO-RAD, USA). Statistical analyses Data were analyzed using SPSS for Windows 17.0 Software (SPSS, Inc., Chicago, IL) and presented as means with standard errors (mean ± SE). Differences between the control and treatment groups were determined using one-way analysis of variance (ANOVA). A P value of < 0.05 was considered statistically signi cant. OriginPro 2018 software was used to develop graphs (Origin Lab Corporation, USA).

Results
PFOA cause liver damage and in uence urea synthesis After 14 days, we nd that PFOA exposure may cause body weight loss and signi cant liver swelling (Fig. 1A-B). Both absolute and relative liver weight were signi cantly increased by PFOA exposure. HEstained liver slices from PFOA exposed mice also show signi cant liver swelling (Fig. 1C). In order to quantify the cell size, we counted the number of cells per unit area. The results show that the number of nuclei per area is signi cantly reduced following PFOA exposure (Fig. 1D); furthermore, this phenomenon has a clear dose-effect relationship with PFOA concentration.
We also monitored the effects of PFOA on metabolism of rats. 14 days of PFOA exposure had no signi cant in uence on daily food intake ( Fig. 2A). However, PFOA exposure had signi cant effect on urea metabolism. Rats exposed with PFOA had signi cantly lower urea concentration in urine compared with normal control rats (Fig. 2B). PFOA has effects on sera biochemical parameters To investigate the effect of PFOA on urea metabolism, we assayed 19 biochemical indexes in rat serum using a cobas® 8000 modular analyser series automatic biochemical analyzer. The results show that 8 indexes have signi cant changes compared with normal control group (Table 1) While the urea content of urine was signi cantly decreased in PFOA treated rats compared to normal rats, the level of urea in the serum of the 20 mg/kg/d treatment group was signi cantly increased.

Differentially Expressed Protein Identi cation and Relative Quanti cation by iTRAQ Analysis
Three individual samples were included in the iTRAQ experiment from the control and 20 mg PFOA/kg/d group. The MS/MS analysis identi ed a total of 25, 506 unique spectra matched to special peptides. Proteome Discover version 2.1 identi ed a total of 8,369 unique peptides from 2,868 proteins. Bioinformatics Analysis for Differential Expressed Proteins Induced by PFOA Heatmapping, Volcano plot analysis and Venn diagram packaging were used to explore the differentially expressed proteins in PFOA treated group compared with normal control (Fig. 3A, 3B, 3C). Among the 3,327 non-redundant proteins, 112 proteins were signi cantly upregulated and 80 proteins were downregulated. Signi cantly changed proteins are shown in the Volcano plot, where the cut log 2 (Fold Change) was set at 1 and the cutoff P value was 0.05 (Fig. 3B). Among the differentially expressed proteins, upregulated proteins are listed in Table 2 and supplementary table S4; while the downregulated  proteins are listed in Table 3.
To further characterize these differentially expressed proteins, we performed GO function annotation analysis via The Gene Ontology (GO) knowledgebase (http://geneontology.org/). Results show that the upregulated and downregulated proteins are mainly involved in the following three biological processes; cellular processes, metabolic process and single − organism processes, and that these processes are localized primarily within the cellular component. When classifying differential proteins by molecular function we nd that they are primarily associated with binding and catalytic activity (Fig. 4A, B).
KEGG Pathway analysis (http://www.kegg.jp/kegg/pathway.html) was also used to determine the involvement of differentially expressed proteins in metabolic and cell signaling pathways. The upregulated proteins were primarily involved in peroxisome, PPAR signaling pathway, fatty acid degradation and fatty acid metabolism; while the downregulated proteins were involved in chemical carcinogenesis, biosynthesis of amino acids and drug metabolism (Fig. 5). Pathway analysis of differentially expressed proteins identi ed in the rat livers Utilizing Ingenuity Pathway Analysis software, eight proteins (ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1) were found to be related to oxidation of fatty acid. (Fig. 6A). Two proteins (ASS1 and CPS1) were found to be associated with urea cycle disorder (Fig. 6B).

Effects of PFOA on urea synthesis related genes
To investigate the toxic effects of PFOA on urea synthesis related genes, we surveyed the transcription levels of three genes (ASL, ASS1 and CPS1) of key enzymes related to urea cycle using qRT PCR. Compared with the control group, the transcriptional levels of ASL remained unchanged (Fig. 7A), however, the mRNA transcriptional levels of ASS1 and CPS1 were signi cantly downregulated in the PFOA exposed groups in a dose-dependent manner (Fig. 7B,C). These results are consistent with the proteomic results. ASS1 and CPS1 are signi cantly reduced by PFOA exposure Expression levels of ASS1 and CPS1 were further veri ed via western blot. Results show expression levels of ASS1 and CPS1 are signi cantly downregulated in the PFOA exposed groups in a dose-dependent manner (Fig. 8A, B). These results are also consistent with the proteomic results.

Discussion
In the present study, the physiological effects of PFOA exposure and its role in liver toxicity was investigated in rat models. We hereby demonstrate that PFOA exposure produces signi cant body weight loss and liver swelling after 14 days exposure. These results are consistent with previous studies of PFOA exposure experiments in rodents (Lau et al. 2007;Starkov and Wallace 2002). Furthermore, we nd that PFOA exposure has a signi cant effect on urea metabolism. Rats exposed to PFOA have reduced urea concentration in urine compared with normal control rats. On the other hand, PFOA exposed rats presented with high urea concentration in the sera. A high urea content in serum rather than in urine may suggest that PFOA exposure either decreases the ability of the liver to metabolize urea, or that urea may leak into blood stream due to the hepatocyte damage.
Investigation of sera biochemistry reveals that levels of ALT, ALP and UREA increased signi cantly after PFOA exposure. The increased level of ALT and ALP implies that PFOA exposure contributes to liver damage and metabolic disfunction in rats. Levels of TG and TC were signi cantly decreased in the serum of treatment group suggesting a reduction in metabolic processes. Additionally, the levels of HDL-C and LDL-C were also decreased. A search of KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway and MetaboAnalyst metabolic pathway found that these indicators are involved in bile acid metabolic pathways and steroid and steroid hormone synthesis pathways.
iTRAQ-based quantitative proteomics were utilized to de ne the proteomic changes in rat livers after 14 days of PFOA exposure. Totally, 2,868 proteins were identi ed by MS, among which, 112 proteins were signi cantly upregulated and 80 proteins were downregulated. Two enzymes identi ed through quantitative proteomics analysis, ASS1 and CPS1, were found to be closely related to urea metabolism. The differential expression of ASS1 and CPS1, was then con rmed in western blotting experiments.
Con rming the downregulation of the enzymes involved in urea synthesis as a result of PFOA exposure provides potential targets for future intervention and treatment of PFOA toxicity.
Additionally, quantitative proteomics experiments also identi ed 8 differentially expressed proteins (ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1) all related to oxidation of fatty acid. ACOX1, ACOX2 and ACOX3 are enzymes related to the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. ACSL1 is an enzyme responsible for the conversion of free long-chain fatty acids into fatty acyl-CoA esters, and thereby plays a key role in lipid biosynthesis and fatty acid degradation. EHHADH is a bifunctional enzyme that is one of the four enzymes of the peroxisomal beta-oxidation pathway. ACCAA1 is a protein involved in the beta-oxidation system of the peroxisomes. The upregulation of these proteins in the livers of PFOA exposed rats implies an increase in liver fatty acid oxidation. This result is consistent with our nding of low levels of TG present in rat liver and sera. Previous studies have also suggested that PFOA exposure may cause accelerated fatty acid oxidation (Chen et al. 2020;Kudo et al. 2006;Yu et al. 2016). In our study, urea-cycle enzymes ASS1 and CPS1 were also downregulated after PFOA exposure, implying that urea synthesis is decreased in liver. Therefore, PFOA exposure accelerated the β-oxidation of fatty acid in the liver of rats, and at the same time inhibited the synthesis of urea in the liver.

Conclusion
In summary, after 14 days of PFOA exposure, rat livers displayed signi cant liver swelling and aberrant levels of TG, TC, HDL-C, LDL-C and urea. iTRAQ-based quantitative proteomics revealed that deregulated proteins ACOX1, ACOX2, ACOX3, ACSL1, EHHADH, GOT2, MTOR and ACAA1 are all related to oxidation of fatty acid while ASS1 and CPS1 are associated with urea cycle disorder. Overall, this study provides insight into speci c mechanisms of hepatotoxicity as a result of PFOA exposure. Data availability All data generated or analyzed during this study were included in this published article, Supplementary table S1, Supplementary table S2, Supplementary table S3, Supplementary table S4 were  availble from Springer link. Compliance with ethical standards Con icts of interest The authors declare no con ict of interest.
Ethical approval All authors declared that they had no known competing nancial interests or personal relationships that seemed to affect the work reported in this article. All authors followed the ethical responsibilities of this journal. Consent to participate and publish All authors participated and approved the nal manuscript to be published. *p < 0.05, **p < 0.01 Table 2. Lists of upregulated Proteins (higher than 2 fold) Identi ed by iTRAQ in rat livers after 20 mg/kg/day PFOA Exposure for 14 Days  Figure 1 PFOA exposure can cause body weight to lose and signi cant liver Swelling. A. Body weight gain during PFOA exposure for 14 days in each group. B. Organ index of liver (relative liver weight) was signi cantly increased by PFOA exposure. C. HE-stained liver slices from PFOA exposed mice compared with normal control (100x and 400x original mag). D. Nuclei number per unit area. Data points represent individual replicates (C: control; L: 1.25 mg/kg/d; M: 5 mg/kg/d; H: 20 mg/kg/d). Mean ± SEM; n = 10; *p < 0.05; **p < 0.01 (control group vs. PFOA treated groups).      Quantitative RT-PCR analysis of rat liver mRNA transcription levels of control and PFOA treated groups at various concentrations. Mean ± SEM; n = 6 *p < 0.05; **p < 0.01 (control group vs. PFOA treated groups). Asl: Argininosuccinate Lyase; Ass1: Argininosuccinate Synthase 1; Cps1: Carbamoyl-Phosphate Synthase 1.

Figure 8
Western blot analysis of rat liver protein expression levels of control and PFOA treated groups at various concentrations. A. Protein levels of CPS1 and ASS1 in rat livers after PFOA treatment. Protein intensities were normalized to the corresponding internal reference protein GAPDH level. B. Results from densitometry analysis of the western blots in A. Mean ± SEM; n = 6 *p < 0.05; **p < 0.01 (control group vs. PFOA treated groups).