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Gene Expression in Liver Injury Caused by Long-term Exposure to Titanium Dioxide Nanoparticles in Mice

Gene Expression in Liver Injury Caused by Long-term Exposure to Titanium Dioxide Nanoparticles in... Abstract Although liver toxicity induced by titanium dioxide nanoparticles (TiO2 NPs) has been demonstrated, very little is known about the molecular mechanisms of multiple genes working together underlying this type of liver injury in mice. In this study, we used the whole-genome microarray analysis technique to determine the gene expression profile in the livers of mice exposed to 10 mg/kg body weight TiO2 NPs for 90 days. The findings showed that long-term exposure to TiO2 NPs resulted in obvious titanium accumulation in the liver and TiO2 NP aggregation in hepatocyte nuclei, an inflammatory response, hepatocyte apoptosis, and liver dysfunction. Furthermore, microarray data showed striking changes in the expression of 785 genes related to the immune/inflammatory response, apoptosis, oxidative stress, the metabolic process, response to stress, cell cycle, ion transport, signal transduction, cell proliferation, cytoskeleton, and cell differentiation in TiO2 NP–exposed livers. In particular, a significant reduction in complement factor D (Cfd) expression following long-term exposure to TiO2 NPs resulted in autoimmune and inflammatory disease states in mice. Therefore, Cfd may be a potential biomarker of liver toxicity caused by TiO2 NPs exposure. titanium dioxide nanoparticles, mice, liver, gene expression profiling, inflammation, apoptosis, titanium dioxide nanoparticles Nanotechnology offers many benefits in areas such as imaging, water decontamination, and information and communication technologies, as well as the production of stronger, lighter materials (Colvin, 2004). Even the food industry is starting to introduce various nanoparticles (NPs) as food additives, food packaging components, or dietary supplements. Of these NPs, titanium dioxide (TiO2) NPs are applied in environmental decontamination of air, soil, and water (Cho et al., 2004; Choi et al., 2006; Esterkin et al., 2005) and are used for whitening and brightening foods, but can also be found in daily-use hygiene products such as toothpaste or sunscreens, or orally administered drugs (Powell et al., 2010). However, little is known about the potential toxicities and the underlying mechanisms of these metal oxide nanoparticles. Recent in vitro and in vivo studies have reported on the toxicity of TiO2 NPs. An in vitro study showed that both rutile and anatase TiO2 NPs impaired cell function in human dermal fibroblasts and decreased cell area, cell proliferation, cell mobility, and the ability of cells to contract collagen, with the latter being more potent in inducing cell damage (Pan et al., 2009). Petković et al. (2011) found that TiO2 NPs transiently upregulated mRNA expression of p53 and its downstream-regulated DNA damage response genes (mdm2, gadd45a, p21). Numerous reports have also unequivocally shown that exposure to TiO2 NPs caused liver dysfunction and oxidative damage in vivo. For example, Wang et al. (2007) reported that 5 g/kg TiO2 NPs administered by oral gavage increased the ratio of alanine aminotransferase (ALT) to aspartate aminotransferase (AST), the activity of lactate dehydrogenase (LDH), and liver weight, and caused hepatocyte necrosis. Our previous studies also found that ip injection of TiO2 NPs in mice for 14 consecutive days or intragastric administration for 60 consecutive days induced liver dysfunction (Liu et al., 2009), inflammatory responses (Cui et al., 2011; Ma et al., 2009), oxidative stress (Liu et al., 2010), and hepatocyte apoptosis of liver (Cui et al., 2010; Ma et al., 2009), which were closely associated with alterations in proinflammatory cytokines (Cui et al., 2011; Ma et al., 2009) or stress-related cytokine expression levels (Cui et al., 2010). Although the studies mentioned above were conducted to evaluate the toxicological effects of TiO2 NPs, studies on the multiple molecular mechanisms of TiO2 NP hepatotoxicity in animals and global changes in gene expression have not been assessed in animal model systems. We speculate that liver 
damage in mice following exposure to TiO2 NPs may have special biomarkers of toxicity. Microarray technology provides a high-throughput functional genomics approach toward a greater understanding of the complex and reciprocal interactions within the genome at the molecular level (Stover, 2004). Large-scale gene expression analysis provides a logical approach to studying the detailed mechanisms of chemical-induced organ toxicity and to the identification of potential biomarkers of toxicity (Yin et al., 2006). Recently, microarray was used to predict the toxicity of chemicals in various tissues, including the heart, aorta, and liver (Dutta et al., 2003). In this investigation, we aimed to determine the gene expression profile following TiO2 NP–induced liver injury and histopathological changes, ultrastructure, hematological parameters, and liver function. The data on gene expression profiling showed significant changes in genes involved in immune/inflammation responses, hepatocyte apoptosis, oxidative stress, and the metabolic process. These results may serve as a reference for future mechanistic studies on the effects of TiO2 NPs or other NPs on the liver of animals or humans. Materials and Methods Chemicals: preparation and characterization. Nanoparticulate anatase TiO2 was prepared via controlled hydrolysis of titanium tetrabutoxide. Details of the synthesis and characterization of TiO2 NPs were described in our previous reports (Hu et al., 2011; Yang et al., 2002). The average particle sizes of powdered TiO2 NPs suspended in 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4M solvent after 12 and 24 h incubation ranged from 5 to 6 nm, and the surface area of the sample was 174.8 m2/g. The mean hydrodynamic diameter of TiO2 NPs in HPMC solvent ranged from 208 to 330 nm (mainly 294 nm), and the zeta potential after 12 and 24 h incubation was 7.57 and 9.28 mV, respectively (Hu et al., 2011). Animals and treatment. It was previously demonstrated by Wang et al. (2007) that sensitivity to TiO2 NPs exposure was higher in CD-1 (ICR) female mice than in CD-1 (ICR) male mice (Wang et al., 2007). Therefore, CD-1 (ICR) female mice were used in this study. About 150 CD-1 (ICR) female mice (23 ± 2 g) were purchased from the Animal Center of Soochow University (China). All mice were housed in stainless steel cages in a ventilated animal room. Room temperature of the housing facility was maintained at 24 ± 2°C, with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 days. All procedures used in the animal experiments conformed to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (Bayne, 1996). In our preliminary experiments, we used TiO2 NPs suspensions at different concentrations (2.5, 5, and 10 mg/kg of body weight [BW]), which were administered intragastrically for 90 consecutive days. Treatment with 10 mg/kg BW TiO2 NPs resulted in severe organ damage (Gui et al., 2011; Sang et al., 2012), and this dose was used as the highest dose in subsequent experiments. The mice were randomly divided into two groups (n = 20): a control group (treated with 0.5% w/v HPMC) and an experimental group (10 mg/kg BW TiO2 NPs). The mice were weighed, and the TiO2 NPs suspensions were administered to the mice intragastrically every day for 90 days. Symptoms and mortality were carefully observed and recorded each day during the 90 days. After the 90-day period, all mice were weighed and then sacrificed after being anesthetized using ether. Blood samples were collected from the eye vein by rapidly removing the eyeball. Serum was collected by centrifuging blood at 2500 rpm for 10 min. The livers were quickly removed and placed on ice and then dissected and frozen at −80°C. Titanium content analysis. Livers were thawed, and approximately 0.1–0.3 g of the liver was weighed, digested, and analyzed for titanium content. Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co.) was used to analyze the titanium concentration in the samples. An indium concentration of 20 ng/ml was used as an internal standard, and the detection limit of titanium was 0.074 ng/ml. The data were expressed as nanograms per gram fresh tissue. Histopathological evaluation of liver. For pathological studies, all histopathological examinations were performed using standard laboratory procedures. The livers were embedded in paraffin blocks, then sliced into 5-mm-thick sections and placed onto glass slides. After hematoxylin–eosin staining, the slides were observed and photographs were obtained using an optical microscope (Nikon U-III Multi-point Sensor System). The identity and analysis of the pathology slides were unknown to the pathologist. Observation of hepatocyte ultrastructure. Livers were fixed using 2.5% glutaraldehyde in 0.1 mol/dm3 cacodylate buffer for 2 h, and the samples were washed three times with 0.1 mol/dm cacodylate buffer (pH 7.2–7.4) and postfixed for 1 h in 1% osmium tetraoxide. The specimens were dehydrated by a graded series of ethanol and embedded in Epon 812. Ultrathin sections obtained were contrasted with uranyl acetate and lead citrate and visualized using a JEOL 1010 transmission electron microscope (Hitachi Co., Japan). Hepatocyte apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). Hematological parameter determination. Blood samples were collected in tubes containing EDTA as anticoagulant. White blood cells (WBC), lymphocytes (LYMPH), and neutrophilic granulocytes (NEUT) were measured using a hematology autoanalyzer (Cell-DYN 3700). Biochemical analysis of liver function. Liver function was evaluated by serum levels of ALT, AST, alkaline phosphatase (ALP), LDH, total cholesterol (TCHO), and triglycerides (TG) using commercial kits (Bühlmann Laboratories, Switzerland). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A, Hitachi, Japan). Microarray assay. Gene expression profiles in liver tissue isolated from five mice in the control and TiO2 NP–treated groups were compared by microarray analysis using Illumina BeadChip (Illumina, San Diego CA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no. 1755; Austin, TX) according to the manufacturer’s protocol and stored at −80°C. RNA amplification is the standard method for preparing RNA samples for array analysis ( Kacharmina et al., 1999). Total RNA was then submitted to the Biostar Genechip Inc. (Shanghai, China), where RNA quality was analyzed using a BioAnalyzer, and cRNA was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized to a single array according to standard Illumina RNA Amplification Kit protocols for a total of arrays. Microarray data analysis. Illumina BeadStudio Application was used to analyze data generated in this study. This program identifies differentially expressed genes and establishes their biological significance based on the Gene Ontology (GO) Consortium (http://www.geneontology.org/GO.doc.html). Differentially expressed genes were identified using ANOVA, and the level of statistical significance was set at p < 0.05. A threshold of 13.0 was used to limit the data set to genes upregulated or downregulated with a diffscore of 13 or greater. Quantitative real-time PCR. The mRNA expression levels of Apoa4, Apoa5, Asah31, Cfd, Cyp2c55, Cyp51, Cyp7a1, Cyp7b1, Dhcr7, Ehhadh, Grb2, Jun, Ngfrap1, Nnmt, Nsdh1, Pmvk, Pvr12, Sc4mol, and Spsb2 in mouse livers were determined using quantitative real-time PCR (qRT-PCR) (Ke et al., 2000; Liu and Saint, 2002; Livak and Schmittgen, 2001). Synthesized cDNA was used for RT-PCR by employing primers, which were designed using Primer Express Software according to the software guidelines. PCR primer sequences are available on request. Statistical analysis. Statistical analyses were conducted using SPSS 17 software. Data were expressed as means ± standard deviation. One-way ANOVA was carried out to compare the differences in means among multigroup data. Dunnett’s test was performed when each data set was compared with the solvent–control data. Statistical significance for all tests was judged at a probability level of 0.05 (p < 0.05). Results Titanium ContentThe amount of titanium accumulated in the mouse liver was observed to be 2440 ng/g tissue for 90-day exposure. This accumulation of titanium may have caused liver dysfunction and tissue damage in mice, which were confirmed by subsequent assays of biochemical parameters and liver histological and hepatocyte ultrastructure observations. Histopathological Evaluation of LiverThe histological photomicrographs of liver sections are shown in Figure 1. In the control group, the liver tissue had no abnormal pathological changes (Fig. 1A). However, in the group exposed to 10 mg/kg BW TiO2 NPs, focal inflammatory cell infiltration, nucleus vacancy, vein congestion, and edema were observed in liver tissue (Fig. 1B), suggesting induction of the inflammatory response and hepatocyte damage by TiO2 NPs. Fig. 1. Open in new tabDownload slide Histopathology of the liver tissue in ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. (A) Control group shows normal hepatic architecture and hepatocytes; (B) 10 mg/kg BW TiO2 NPs group indicates focal inflammatory cell infiltration (white arrows) and vain congestion (black arrows). Fig. 1. Open in new tabDownload slide Histopathology of the liver tissue in ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. (A) Control group shows normal hepatic architecture and hepatocytes; (B) 10 mg/kg BW TiO2 NPs group indicates focal inflammatory cell infiltration (white arrows) and vain congestion (black arrows). Hepatocyte Ultrastructure EvaluationThe changes in hepatocyte ultrastructure in mouse liver are presented in Figure 2. It was observed that untreated mouse hepatocytes (in the control group) contained round nuclei with homogeneous chromatin (Fig. 2A). Hepatocyte ultrastructure in the group treated with 10 mg/kg BW TiO2 NPs showed classical morphological characteristics of apoptosis, such as mitochondrial swelling, nucleus chromatin condensation, or vacuolization (Fig. 2B). In addition, as seen in Figure 2B, TiO2 NPs aggregated in the cytoplasm or were deposited on karyotheca or in the nucleus. Fig. 2. Open in new tabDownload slide The changes of hepatocyte ultrastructure in liver of ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. 
(A) Control: Chromatin is well distributed, mitochondria turn round or oval and complete; (B) 10 mg/kg BW TiO2 NPs: Arrows indicate a significant shrinkage of the nucleus, nuclear vacuolation, mitochondrial swelling, and chromatin marginalization; (C) Magnification of the white circle of Figure 2(B) shows that TiO2 NPs were absorbed in the nuclear membrane of or entered nucleus in hepatocyte; (D) Magnification of the white circle of Figure 2(B) indicates that TiO2 NPs aggregated in the cytoplasm of hepatocyte. Fig. 2. Open in new tabDownload slide The changes of hepatocyte ultrastructure in liver of ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. 
(A) Control: Chromatin is well distributed, mitochondria turn round or oval and complete; (B) 10 mg/kg BW TiO2 NPs: Arrows indicate a significant shrinkage of the nucleus, nuclear vacuolation, mitochondrial swelling, and chromatin marginalization; (C) Magnification of the white circle of Figure 2(B) shows that TiO2 NPs were absorbed in the nuclear membrane of or entered nucleus in hepatocyte; (D) Magnification of the white circle of Figure 2(B) indicates that TiO2 NPs aggregated in the cytoplasm of hepatocyte. Hematological ParametersResults of hematological evaluation indicated that WBC, LYMPH, and NEUT in TiO2 NP–treated mice were gradually reduced (Table 1) (p < 0.05 or 0.01), i.e., the levels of WBC, LYMPH, and NEUT were decreased by 59.15, 69.78, and 58.90%, respectively. TABLE 1 Hematological Parameters and Biochemical Parameters in Mice by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* Note. Ranks marked with an asterisk or double asterisks means it is significantly different from the control (unexposed mice) at the 5 or 1% confidence level, respectively. Values represent means ± SE, n = 10. Open in new tab TABLE 1 Hematological Parameters and Biochemical Parameters in Mice by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* Note. Ranks marked with an asterisk or double asterisks means it is significantly different from the control (unexposed mice) at the 5 or 1% confidence level, respectively. Values represent means ± SE, n = 10. Open in new tab Assay of Liver FunctionSerum biochemical parameters were assayed to further evaluate the toxicity of TiO2 NPs on mouse liver. Table 1 lists the changes in biochemical parameters in mouse serum following administration of TiO2 NPs by gavage for 90 consecutive days. It was noted that TiO2 NPs exposure significantly increased the activities of ALT, AST, ALP, and LDH, and elevated the levels of total cholesterol (Tchol) and TG, suggesting that long-term exposure to TiO2 NPs resulted in hepatic dysfunction. Change in the Gene Expression ProfileMessenger RNA from liver tissues of the vehicle control group and the group exposed to 10 mg/kg BW TiO2 NPs for 90 consecutive days was analyzed with Illumina BeadChip. The results showed that more than 2.54% (1142 genes) of the total genes (45,000 genes) were significantly changed following long-term exposure to 10 mg/kg TiO2 NPs (Table 2). Of these 1142 genes, 664 genes were upregulated and 478 were downregulated. We used the ontology-driven clustering approach of Protein Analysis Through Evolutionary Relationships (PANTHER), which indicated that 785 of the 1142 genes were associated with immune/inflammatory response, apoptosis, oxidative stress, the metabolic process, response to stress, cell cycle, ion transport, signal transduction, cell proliferation, cytoskeleton, and cell differentiation (Fig. 3); the functions of another 357 genes were unknown (Fig. 3). Fig. 3 . Functional categorization of 1142 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER. Fig. 3 . Functional categorization of 1142 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER. TABLE 2 Genes Which Related to Immune Response, Inflammatory Response, Apoptosis, Oxidative Stress and Metabolic Process Altered Significantly by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Note. We classified the function of these genes according to gene Ontology in NCBI (http://www.ncbi.nlm.nih.gov/). Open in new tab TABLE 2 Genes Which Related to Immune Response, Inflammatory Response, Apoptosis, Oxidative Stress and Metabolic Process Altered Significantly by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Note. We classified the function of these genes according to gene Ontology in NCBI (http://www.ncbi.nlm.nih.gov/). Open in new tab Real-time Polymerase Chain ReactionTo verify the accuracy of the microarray assays, a number of genes that demonstrated significantly different expression patterns were chosen due to their association with immune response, oxidative stress, apoptosis, response to stress, the metabolic process, and cell proliferation. Most of the qRT-PCR results were in reasonable agreement with the microarray results (i.e., either up- or downregulation). For simple validation, 14 genes were verified by RT-PCR, all of which displayed expression patterns comparable with the microarray data (Table 3), with the exception of Grb2, which showed discrepancy between the two methods, suggesting possible problems with RNA handling or pipetting issues. TABLE 3 RT-PCR Validation of Selected Genes from Microarray Data Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Open in new tab TABLE 3 RT-PCR Validation of Selected Genes from Microarray Data Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Open in new tab Discussion The results of this study indicate that long-term exposure to 10 mg/kg BW TiO2 NPs results in titanium accumulation in mouse liver tissue, TiO2 NP aggregation in hepatocyte nuclei (Fig. 2B), liver inflammation (Fig. 1), and hepatocyte apoptosis (Fig. 2B), coupled with decreases in the levels of WBC, LYMPH, and NEUT and increases in ALT, AST, ALP, and LDH activities and TChol and TG levels (1). TiO2 NP–induced hepatic toxicity is closely related to NP size (in general, smaller NPs induce greater toxicity) (Oberdörster et al., 1994; Wang et al., 2007); however, toxicity is mainly dependent on TiO2 NPs surface area (Warheit et al., 2006). The surface area of TiO2 NPs is negatively correlated with its size. In this study, the average particle size in TiO2 NP powder was about 5 nm and the surface area was 174.8 m2/g (Hu et al., 2011; Yang et al., 2002); thus, exposure to these TiO2 NPs caused severe damage to mouse liver. The significant reduction in hematological parameters and liver dysfunction caused by exposure to TiO2 NPs may be related to damage of immune and metabolism function in mice, which may be associated with alterations in gene expression in the liver. To identify the molecular mechanisms caused by exposure to TiO2 NPs, microarray assays of RNA from mouse livers were performed to establish a global gene expression profile. These assays indicated that the expression levels of 1142 genes were significantly changed, and 785 of these genes were involved in inflammatory/immune response, apoptosis, oxidative stress, and the metabolic process. The main results are discussed below. Genes Related to Immune and Inflammatory ResponseA previous study demonstrated that TiO2 NPs significantly increased the mRNA and protein expression of TLR2, TLR4, and several inflammatory cytokines, including IKK1, IKK2, NF-κB, NF-κBP52, NF-κBP65, TNF-α, and NIK, thus inducing an inflammatory response in the liver of mice (Cui et al., 2011). In this study, focal inflammatory cell infiltration in liver tissue was observed (Fig. 1), and 39 genes (3.41% of 1142 genes) involved in immune and inflammatory responses were significantly changed as shown by microarray data (Table 2). Of these genes, 16 were upregulated and 21 were downregulated. In line with our data, Fujita et al. (2009) found that 15 genes involved in the inflammatory response and 23 genes involved in the immune response were upregulated at both 3 day and 1 month postexposure to C60 fullerene. These results suggest that nanoparticles may change the expression levels of key genes, then trigger toxic signal pathways, and finally induce an inflammatory response in the liver. Retinoic acid early transcript beta (Raet1b) plays a role in the positive regulation of the immune response in tumor cells, and its expression induces primary tumor rejection and triggers in vitro natural killer (NK) cell cytotoxicity and interferon-γ secretion (Hayakawa et al., 2002). Our data show that Raet1b was increased by 83.03-fold in the TiO2 NP–exposed group (Table 2), suggesting that TiO2 NPs induced Raet1b expression and then triggered NK cell cytotoxicity, which resulted in a reduction in immune capacity and the generation of inflammation in mouse liver. Chemokine (C-X-C motif) ligand 1 (CXCL1) is important for the expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine, and activation of NF-κB (Cai et al., 2010). In this study, Cxcl1 was overexpressed (with a diffscore of 70.45) following exposure to TiO2 NPs (Table 2); therefore, it activated proinflammatory cytokines and then led to hepatic inflammation. However, complement factor D (Cfd) was dramatically downregulated with a diffscore of –110.03 (Table 2). Cfd is a serine protease essential for activation of the alternative pathway in the complement system. Its main role is to eliminate foreign antigens and pathogens as part of the normal host response (Holers, 2000). Abrera-Abeleda et al. (2007) reported that Cfd gene knockout mice were a novel model of spontaneous mesangial immune complex glomerulonephritis. The inhibition of Cfd expression due to exposure to TiO2 NPs reduced the activity of C3b factor B, which is also known as C3 convertase. A small quantity of C3 convertase (C3bBb) is insufficient to cleave additional C3 molecules and generate C3a and C3b; therefore, TiO2 NP exposure causes further amplification of the immune/inflammatory programs in mouse liver (Fig. 4). Fig. 4. Open in new tabDownload slide Cfd network pathway obtained from network analysis of differentially expressed genes. Ingenuity Pathway Analysis software was used to construct and visualize molecular interaction networks. Fig. 4. Open in new tabDownload slide Cfd network pathway obtained from network analysis of differentially expressed genes. Ingenuity Pathway Analysis software was used to construct and visualize molecular interaction networks. Genes Related to ApoptosisChen et al. (2006) found that intratracheal instillation of TiO2 NPs in mice induced the differential expression of hundreds of genes including those related to apoptosis and the cell cycle. In this study, we observed hepatocyte apoptosis caused by TiO2 NPs (Fig. 2). To further clarify the molecular mechanism of apoptosis, we analyzed the apoptotic genes and found that 58 genes (5.07% of 1142 genes) were significantly 
altered by exposure to TiO2 NPs (Table 2). Of these 58 genes, 30 were upregulated and 28 were downregulated. Expression of the apoptotic genes, including cysteine-serine-rich nuclear protein-1 (Csrnp1) and Ddit4, were significantly upregulated, with diffscores of 111.74 and 42.40, respectively (Table 2). Csrnp1 has been identified as an immediate early gene that is strongly induced in response to IL-2 in mouse T cells (Gingras et al., 2007). We suggest that high expression of Csrnp1 may induce proinflammatory cytokine expression and then hepatocyte apoptosis. As a novel transcriptional target of p53, Ddit4, which is also known as REDD1, induced DNA damage (Ellisen et al., 2002). REDD1 encodes a shared transcriptional target that implicates reactive oxygen species (ROS) in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation. The overexpression of Ddit4 caused by TiO2 NPs may induce the accumulation of ROS, which leads to DNA oxidative damage and finally causes apoptosis. However, Bcl3 expression was significantly reduced following exposure to TiO2 NPs in the study. Kreisel et al. (2011) demonstrated a critical role for Bcl3 in regulating emergency granulopoiesis and suggested that Bcl3 prevents an acute inflammatory response in mouse lung injury. Bauer et al. (2006) indicated that apoptosis is inhibited by Bcl3 involving a Bim-independent pathway. Therefore, the typical apoptosis caused by exposure to TiO2 NPs implied that the upregulated pro-apoptosis genes and downregulated expression of Bcl3 may have a collaborative effect on inducing hepatocyte apoptosis. Genes Related to Oxidative StressPrevious studies have shown that TiO2 NPs can induce the production of ROS (such as O2•− and H2O2) and lipid peroxidation in the mouse liver, indicating that the liver suffered from oxidative stress (Cui et al., 2010; Ma et al., 2009), and demonstrated that TiO2 NPs exposure decreased the stress- related gene expression levels of superoxide dismutase, catalase, glutathione peroxidase, metallothionein, heat shock protein 70, glutathione S transferase, P53, and transferrin, and significantly increased cytochrome p450 1A expression in mouse liver (Cui et al., 2010). In this study, microarray data suggested that approximately 101 genes (8.84% of 1142 genes) related to oxidative stress were significantly changed in the TiO2 NP–exposed liver (Table 2). Of these 101 genes, 66 were upregulated and 35 were downregulated (Table 2). In this study, connective tissue growth factor (Ctgf) was highly expressed following exposure to TiO2 NPs, with a diffscore of 68.56. Ctgf is a multifunctional protein that coordinates complex biological processes during tissue development and remodeling. It also contributes to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and oxidative damage to biopolymers. Overexpression of Ctgf was demonstrated to induce pulmonary vascular remodeling and pulmonary hypertension (Chen et al., 2011). Therefore, we speculate that Ctgf may be a sensitive gene for TiO2 NP exposure. Its abnormal expression may cause the disorder of several biological processes involved in ROS production. ROS accumulated by exposure to TiO2 NPs lead to oxidative stress in the liver, thus causing hepatocyte apoptosis. Enoyl-coenzyme A (hydratase/3-hydroxyacyl coenzyme A dehydrogenase [Ehhadh]), a small protein encoded by this gene that uses dioxygen (O2) to oxidize organic molecules, contains some enzymes, and it degrades hydrogen peroxide (H2O2). Defects in this gene are a cause of peroxisomal disorders such as Zellweger syndrome (Stephen and David, 2000). In our study, Ehhadh was downregulated by TiO2 NP exposure, with a diffscore of −19.26. Reduced Ehhadh expression by TiO2 NPs may cause hepatocyte peroxisomal disorders and decrease antioxidative capacity or detoxification. Genes Related to Metabolic ProcessSeveral reports have addressed the transcriptomic changes involved in the hepatic metabolic process induced by diverse chemical toxicants. Richards et al. (2004) reported that hydrazine induced hepatic steatosis and necrosis in mice, and altered the expression of genes involved in lipid peroxidation/fatty acid synthesis and transport. Lee et al. (2007) indicated that valproic acid induced hepatotoxicity including microvesicular steatosis and necrosis in the liver following oral administration of a single dose of 100 mg/kg (low dose) or 1000 mg/kg (high dose) in mice. The expression of genes associated with the metabolic process was investigated. Functional categorization of genes altered by exposure to TiO2 NPs revealed that 135 genes (11.8% of 1142 genes) were involved in the lipid, fatty acid, and glutathione metabolic processes and steroid metabolism (Table 2). Notably, TiO2 NPs induced changes in the expression of genes involved in the biosynthetic pathways of both cholesterol and lipid metabolism. For example, the proprotein convertase subtilisin type 9 (Pcsk9) is involved in the cholesterol metabolic process and plays a key role in cholesterol homeostasis by binding the low-density lipoprotein receptor and targeting it toward degradation (Essalmani et al., 2011). Lee et al. (2005) reported that Acetyl-CoA acetyltransferase 2 (Acat2) provides the core cholesteryl ester of newly secreted very low-density lipoprotein, whereas lecithin:cholesterol acyltransferase adds cholesteryl ester during low-density lipoprotein particle formation. In this study, Pcsk9 and Acat2 were highly expressed following exposure to TiO2 NPs, with diffscores of 67.06 and 63.61, respectively (Table 2). TiO2 NPs inducing Proprotein convertase subtilisin/kexin type 9 (Pcsk9) and Acat2 overexpression may promote the biosynthesis of cholesteryl ester, thus, resulting in high levels of TChol in serum and liver dysfunction (Table 1). Conclusion After a single intragastric administration of 10 mg/kg BW TiO2 NPs for 90 consecutive days, accumulation of TiO2 NPs in liver tissue and even in hepatocyte nuclei was observed, which impaired liver function and induced a severe inflammatory response and hepatocyte apoptosis in mouse liver. The liver damage caused by long-term exposure to TiO2 NPs may be closely associated with significant changes in the expression of genes involved in immune and inflammatory responses, apoptosis, oxidative stress, and the metabolism process, and in particular, a reduction in Cfd expression. The obvious reduction in the expression of Cfd following TiO2 NPs exposure may trigger signaling cascades to activate inflammatory programs. Therefore, the application of TiO2 NPs in pharmacology; in popular sunscreens, toothpastes, and cosmetics; and in the decontamination of water and air should be performed with caution. FUNDING National Important Project on Scientific Research of China (Grant No. 2011CB933404), National Natural Science Foundation of China (Grant No. 30671782, 30901218, 30972504, 81172697), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Abrera-Abeleda M. A. Xu, Y. Pickering M. C. Smith R. J. Sethi S. ( 2007 ). 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Gene Expression in Liver Injury Caused by Long-term Exposure to Titanium Dioxide Nanoparticles in Mice

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© The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
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Research Article
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1096-6080
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10.1093/toxsci/kfs153
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22539623
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Abstract

Abstract Although liver toxicity induced by titanium dioxide nanoparticles (TiO2 NPs) has been demonstrated, very little is known about the molecular mechanisms of multiple genes working together underlying this type of liver injury in mice. In this study, we used the whole-genome microarray analysis technique to determine the gene expression profile in the livers of mice exposed to 10 mg/kg body weight TiO2 NPs for 90 days. The findings showed that long-term exposure to TiO2 NPs resulted in obvious titanium accumulation in the liver and TiO2 NP aggregation in hepatocyte nuclei, an inflammatory response, hepatocyte apoptosis, and liver dysfunction. Furthermore, microarray data showed striking changes in the expression of 785 genes related to the immune/inflammatory response, apoptosis, oxidative stress, the metabolic process, response to stress, cell cycle, ion transport, signal transduction, cell proliferation, cytoskeleton, and cell differentiation in TiO2 NP–exposed livers. In particular, a significant reduction in complement factor D (Cfd) expression following long-term exposure to TiO2 NPs resulted in autoimmune and inflammatory disease states in mice. Therefore, Cfd may be a potential biomarker of liver toxicity caused by TiO2 NPs exposure. titanium dioxide nanoparticles, mice, liver, gene expression profiling, inflammation, apoptosis, titanium dioxide nanoparticles Nanotechnology offers many benefits in areas such as imaging, water decontamination, and information and communication technologies, as well as the production of stronger, lighter materials (Colvin, 2004). Even the food industry is starting to introduce various nanoparticles (NPs) as food additives, food packaging components, or dietary supplements. Of these NPs, titanium dioxide (TiO2) NPs are applied in environmental decontamination of air, soil, and water (Cho et al., 2004; Choi et al., 2006; Esterkin et al., 2005) and are used for whitening and brightening foods, but can also be found in daily-use hygiene products such as toothpaste or sunscreens, or orally administered drugs (Powell et al., 2010). However, little is known about the potential toxicities and the underlying mechanisms of these metal oxide nanoparticles. Recent in vitro and in vivo studies have reported on the toxicity of TiO2 NPs. An in vitro study showed that both rutile and anatase TiO2 NPs impaired cell function in human dermal fibroblasts and decreased cell area, cell proliferation, cell mobility, and the ability of cells to contract collagen, with the latter being more potent in inducing cell damage (Pan et al., 2009). Petković et al. (2011) found that TiO2 NPs transiently upregulated mRNA expression of p53 and its downstream-regulated DNA damage response genes (mdm2, gadd45a, p21). Numerous reports have also unequivocally shown that exposure to TiO2 NPs caused liver dysfunction and oxidative damage in vivo. For example, Wang et al. (2007) reported that 5 g/kg TiO2 NPs administered by oral gavage increased the ratio of alanine aminotransferase (ALT) to aspartate aminotransferase (AST), the activity of lactate dehydrogenase (LDH), and liver weight, and caused hepatocyte necrosis. Our previous studies also found that ip injection of TiO2 NPs in mice for 14 consecutive days or intragastric administration for 60 consecutive days induced liver dysfunction (Liu et al., 2009), inflammatory responses (Cui et al., 2011; Ma et al., 2009), oxidative stress (Liu et al., 2010), and hepatocyte apoptosis of liver (Cui et al., 2010; Ma et al., 2009), which were closely associated with alterations in proinflammatory cytokines (Cui et al., 2011; Ma et al., 2009) or stress-related cytokine expression levels (Cui et al., 2010). Although the studies mentioned above were conducted to evaluate the toxicological effects of TiO2 NPs, studies on the multiple molecular mechanisms of TiO2 NP hepatotoxicity in animals and global changes in gene expression have not been assessed in animal model systems. We speculate that liver 
damage in mice following exposure to TiO2 NPs may have special biomarkers of toxicity. Microarray technology provides a high-throughput functional genomics approach toward a greater understanding of the complex and reciprocal interactions within the genome at the molecular level (Stover, 2004). Large-scale gene expression analysis provides a logical approach to studying the detailed mechanisms of chemical-induced organ toxicity and to the identification of potential biomarkers of toxicity (Yin et al., 2006). Recently, microarray was used to predict the toxicity of chemicals in various tissues, including the heart, aorta, and liver (Dutta et al., 2003). In this investigation, we aimed to determine the gene expression profile following TiO2 NP–induced liver injury and histopathological changes, ultrastructure, hematological parameters, and liver function. The data on gene expression profiling showed significant changes in genes involved in immune/inflammation responses, hepatocyte apoptosis, oxidative stress, and the metabolic process. These results may serve as a reference for future mechanistic studies on the effects of TiO2 NPs or other NPs on the liver of animals or humans. Materials and Methods Chemicals: preparation and characterization. Nanoparticulate anatase TiO2 was prepared via controlled hydrolysis of titanium tetrabutoxide. Details of the synthesis and characterization of TiO2 NPs were described in our previous reports (Hu et al., 2011; Yang et al., 2002). The average particle sizes of powdered TiO2 NPs suspended in 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4M solvent after 12 and 24 h incubation ranged from 5 to 6 nm, and the surface area of the sample was 174.8 m2/g. The mean hydrodynamic diameter of TiO2 NPs in HPMC solvent ranged from 208 to 330 nm (mainly 294 nm), and the zeta potential after 12 and 24 h incubation was 7.57 and 9.28 mV, respectively (Hu et al., 2011). Animals and treatment. It was previously demonstrated by Wang et al. (2007) that sensitivity to TiO2 NPs exposure was higher in CD-1 (ICR) female mice than in CD-1 (ICR) male mice (Wang et al., 2007). Therefore, CD-1 (ICR) female mice were used in this study. About 150 CD-1 (ICR) female mice (23 ± 2 g) were purchased from the Animal Center of Soochow University (China). All mice were housed in stainless steel cages in a ventilated animal room. Room temperature of the housing facility was maintained at 24 ± 2°C, with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 days. All procedures used in the animal experiments conformed to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (Bayne, 1996). In our preliminary experiments, we used TiO2 NPs suspensions at different concentrations (2.5, 5, and 10 mg/kg of body weight [BW]), which were administered intragastrically for 90 consecutive days. Treatment with 10 mg/kg BW TiO2 NPs resulted in severe organ damage (Gui et al., 2011; Sang et al., 2012), and this dose was used as the highest dose in subsequent experiments. The mice were randomly divided into two groups (n = 20): a control group (treated with 0.5% w/v HPMC) and an experimental group (10 mg/kg BW TiO2 NPs). The mice were weighed, and the TiO2 NPs suspensions were administered to the mice intragastrically every day for 90 days. Symptoms and mortality were carefully observed and recorded each day during the 90 days. After the 90-day period, all mice were weighed and then sacrificed after being anesthetized using ether. Blood samples were collected from the eye vein by rapidly removing the eyeball. Serum was collected by centrifuging blood at 2500 rpm for 10 min. The livers were quickly removed and placed on ice and then dissected and frozen at −80°C. Titanium content analysis. Livers were thawed, and approximately 0.1–0.3 g of the liver was weighed, digested, and analyzed for titanium content. Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co.) was used to analyze the titanium concentration in the samples. An indium concentration of 20 ng/ml was used as an internal standard, and the detection limit of titanium was 0.074 ng/ml. The data were expressed as nanograms per gram fresh tissue. Histopathological evaluation of liver. For pathological studies, all histopathological examinations were performed using standard laboratory procedures. The livers were embedded in paraffin blocks, then sliced into 5-mm-thick sections and placed onto glass slides. After hematoxylin–eosin staining, the slides were observed and photographs were obtained using an optical microscope (Nikon U-III Multi-point Sensor System). The identity and analysis of the pathology slides were unknown to the pathologist. Observation of hepatocyte ultrastructure. Livers were fixed using 2.5% glutaraldehyde in 0.1 mol/dm3 cacodylate buffer for 2 h, and the samples were washed three times with 0.1 mol/dm cacodylate buffer (pH 7.2–7.4) and postfixed for 1 h in 1% osmium tetraoxide. The specimens were dehydrated by a graded series of ethanol and embedded in Epon 812. Ultrathin sections obtained were contrasted with uranyl acetate and lead citrate and visualized using a JEOL 1010 transmission electron microscope (Hitachi Co., Japan). Hepatocyte apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). Hematological parameter determination. Blood samples were collected in tubes containing EDTA as anticoagulant. White blood cells (WBC), lymphocytes (LYMPH), and neutrophilic granulocytes (NEUT) were measured using a hematology autoanalyzer (Cell-DYN 3700). Biochemical analysis of liver function. Liver function was evaluated by serum levels of ALT, AST, alkaline phosphatase (ALP), LDH, total cholesterol (TCHO), and triglycerides (TG) using commercial kits (Bühlmann Laboratories, Switzerland). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A, Hitachi, Japan). Microarray assay. Gene expression profiles in liver tissue isolated from five mice in the control and TiO2 NP–treated groups were compared by microarray analysis using Illumina BeadChip (Illumina, San Diego CA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no. 1755; Austin, TX) according to the manufacturer’s protocol and stored at −80°C. RNA amplification is the standard method for preparing RNA samples for array analysis ( Kacharmina et al., 1999). Total RNA was then submitted to the Biostar Genechip Inc. (Shanghai, China), where RNA quality was analyzed using a BioAnalyzer, and cRNA was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized to a single array according to standard Illumina RNA Amplification Kit protocols for a total of arrays. Microarray data analysis. Illumina BeadStudio Application was used to analyze data generated in this study. This program identifies differentially expressed genes and establishes their biological significance based on the Gene Ontology (GO) Consortium (http://www.geneontology.org/GO.doc.html). Differentially expressed genes were identified using ANOVA, and the level of statistical significance was set at p < 0.05. A threshold of 13.0 was used to limit the data set to genes upregulated or downregulated with a diffscore of 13 or greater. Quantitative real-time PCR. The mRNA expression levels of Apoa4, Apoa5, Asah31, Cfd, Cyp2c55, Cyp51, Cyp7a1, Cyp7b1, Dhcr7, Ehhadh, Grb2, Jun, Ngfrap1, Nnmt, Nsdh1, Pmvk, Pvr12, Sc4mol, and Spsb2 in mouse livers were determined using quantitative real-time PCR (qRT-PCR) (Ke et al., 2000; Liu and Saint, 2002; Livak and Schmittgen, 2001). Synthesized cDNA was used for RT-PCR by employing primers, which were designed using Primer Express Software according to the software guidelines. PCR primer sequences are available on request. Statistical analysis. Statistical analyses were conducted using SPSS 17 software. Data were expressed as means ± standard deviation. One-way ANOVA was carried out to compare the differences in means among multigroup data. Dunnett’s test was performed when each data set was compared with the solvent–control data. Statistical significance for all tests was judged at a probability level of 0.05 (p < 0.05). Results Titanium ContentThe amount of titanium accumulated in the mouse liver was observed to be 2440 ng/g tissue for 90-day exposure. This accumulation of titanium may have caused liver dysfunction and tissue damage in mice, which were confirmed by subsequent assays of biochemical parameters and liver histological and hepatocyte ultrastructure observations. Histopathological Evaluation of LiverThe histological photomicrographs of liver sections are shown in Figure 1. In the control group, the liver tissue had no abnormal pathological changes (Fig. 1A). However, in the group exposed to 10 mg/kg BW TiO2 NPs, focal inflammatory cell infiltration, nucleus vacancy, vein congestion, and edema were observed in liver tissue (Fig. 1B), suggesting induction of the inflammatory response and hepatocyte damage by TiO2 NPs. Fig. 1. Open in new tabDownload slide Histopathology of the liver tissue in ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. (A) Control group shows normal hepatic architecture and hepatocytes; (B) 10 mg/kg BW TiO2 NPs group indicates focal inflammatory cell infiltration (white arrows) and vain congestion (black arrows). Fig. 1. Open in new tabDownload slide Histopathology of the liver tissue in ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. (A) Control group shows normal hepatic architecture and hepatocytes; (B) 10 mg/kg BW TiO2 NPs group indicates focal inflammatory cell infiltration (white arrows) and vain congestion (black arrows). Hepatocyte Ultrastructure EvaluationThe changes in hepatocyte ultrastructure in mouse liver are presented in Figure 2. It was observed that untreated mouse hepatocytes (in the control group) contained round nuclei with homogeneous chromatin (Fig. 2A). Hepatocyte ultrastructure in the group treated with 10 mg/kg BW TiO2 NPs showed classical morphological characteristics of apoptosis, such as mitochondrial swelling, nucleus chromatin condensation, or vacuolization (Fig. 2B). In addition, as seen in Figure 2B, TiO2 NPs aggregated in the cytoplasm or were deposited on karyotheca or in the nucleus. Fig. 2. Open in new tabDownload slide The changes of hepatocyte ultrastructure in liver of ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. 
(A) Control: Chromatin is well distributed, mitochondria turn round or oval and complete; (B) 10 mg/kg BW TiO2 NPs: Arrows indicate a significant shrinkage of the nucleus, nuclear vacuolation, mitochondrial swelling, and chromatin marginalization; (C) Magnification of the white circle of Figure 2(B) shows that TiO2 NPs were absorbed in the nuclear membrane of or entered nucleus in hepatocyte; (D) Magnification of the white circle of Figure 2(B) indicates that TiO2 NPs aggregated in the cytoplasm of hepatocyte. Fig. 2. Open in new tabDownload slide The changes of hepatocyte ultrastructure in liver of ICR mice caused by an intragastric administration with TiO2 NPs for 90 consecutive days. 
(A) Control: Chromatin is well distributed, mitochondria turn round or oval and complete; (B) 10 mg/kg BW TiO2 NPs: Arrows indicate a significant shrinkage of the nucleus, nuclear vacuolation, mitochondrial swelling, and chromatin marginalization; (C) Magnification of the white circle of Figure 2(B) shows that TiO2 NPs were absorbed in the nuclear membrane of or entered nucleus in hepatocyte; (D) Magnification of the white circle of Figure 2(B) indicates that TiO2 NPs aggregated in the cytoplasm of hepatocyte. Hematological ParametersResults of hematological evaluation indicated that WBC, LYMPH, and NEUT in TiO2 NP–treated mice were gradually reduced (Table 1) (p < 0.05 or 0.01), i.e., the levels of WBC, LYMPH, and NEUT were decreased by 59.15, 69.78, and 58.90%, respectively. TABLE 1 Hematological Parameters and Biochemical Parameters in Mice by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* Note. Ranks marked with an asterisk or double asterisks means it is significantly different from the control (unexposed mice) at the 5 or 1% confidence level, respectively. Values represent means ± SE, n = 10. Open in new tab TABLE 1 Hematological Parameters and Biochemical Parameters in Mice by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* . TiO2NPs (mg/kg BW) . Index . 0 . 10 . WBC (109/l) 7.1 ± 0.355 2.9 ± 0.1455** LYMPH (109/l) 5.89 ± 0.295 1.78 ± 0.089** NEUT (109/l) 1.46 ± 0.073 0.6 ± 0.03** ALT (U/l) 21 ± 1.05 26 ± 1.30* AST (U/l) 79 ± 3.95 103 ± 5.15* ALP (Ul) 96 ± 4.80 121 ± 6.05* LDH (U/l) 671 ± 33.55 850 ± 42.50* TChol (mmol/l) 2.03 ± 0.10 2.54 ± 0.13* Note. Ranks marked with an asterisk or double asterisks means it is significantly different from the control (unexposed mice) at the 5 or 1% confidence level, respectively. Values represent means ± SE, n = 10. Open in new tab Assay of Liver FunctionSerum biochemical parameters were assayed to further evaluate the toxicity of TiO2 NPs on mouse liver. Table 1 lists the changes in biochemical parameters in mouse serum following administration of TiO2 NPs by gavage for 90 consecutive days. It was noted that TiO2 NPs exposure significantly increased the activities of ALT, AST, ALP, and LDH, and elevated the levels of total cholesterol (Tchol) and TG, suggesting that long-term exposure to TiO2 NPs resulted in hepatic dysfunction. Change in the Gene Expression ProfileMessenger RNA from liver tissues of the vehicle control group and the group exposed to 10 mg/kg BW TiO2 NPs for 90 consecutive days was analyzed with Illumina BeadChip. The results showed that more than 2.54% (1142 genes) of the total genes (45,000 genes) were significantly changed following long-term exposure to 10 mg/kg TiO2 NPs (Table 2). Of these 1142 genes, 664 genes were upregulated and 478 were downregulated. We used the ontology-driven clustering approach of Protein Analysis Through Evolutionary Relationships (PANTHER), which indicated that 785 of the 1142 genes were associated with immune/inflammatory response, apoptosis, oxidative stress, the metabolic process, response to stress, cell cycle, ion transport, signal transduction, cell proliferation, cytoskeleton, and cell differentiation (Fig. 3); the functions of another 357 genes were unknown (Fig. 3). Fig. 3 . Functional categorization of 1142 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER. Fig. 3 . Functional categorization of 1142 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER. TABLE 2 Genes Which Related to Immune Response, Inflammatory Response, Apoptosis, Oxidative Stress and Metabolic Process Altered Significantly by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Note. We classified the function of these genes according to gene Ontology in NCBI (http://www.ncbi.nlm.nih.gov/). Open in new tab TABLE 2 Genes Which Related to Immune Response, Inflammatory Response, Apoptosis, Oxidative Stress and Metabolic Process Altered Significantly by an Intragastric Administration With TiO2 NPs for 90 Consecutive Days Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Symbol Gene ID Ontology DiffScore p val Symbol Gene ID Ontology DiffScore p val Immune response Raet1b NM_009016 Positive regulation of immune response to tumor cell 83.03 0 Igh-6 AK131185 Immunoglobulin-mediated immune response –17.75 0 Trim56 NM_201373 Innate immune response 36.14 0 Igh-VJ558 U58290 Immunoglobulin-mediated immune response –17.75 0 Icam1 NM_010493 Positive regulation of NF-kappaB transcription factor activity 31.80 0 Cfp NM_008823 Innate immune response –18.05 0 Nlrc5 NM_001033207 Innate immune response 31.24 0 H2-Eb1 NM_010382 Immune response –19.06 0 C1rl NM_181344 Innate immune response 30.07 0 Pvrl2 NM_008990 Positive regulation of immunoglobulin-mediated immune response –19.27 2.03E-10 H2-Q6 NM_207648 Immune response 27.23 0 Cd160 NM_001163496 MHC class I receptor activity –19.37 0.07 Mx2 NM_013606 Innate immune response 21.65 0 Igh-6 Z95476 Immunoglobulin-mediated immune response –20.92 0 Tbk1 NM_019786 Activation of innate immune response 18.68 0 Igh-VJ558 BF180947 Immunoglobulin-mediated immune response –20.92 0 Nr1d1 NM_145434 Negative regulation of toll-like receptor 4 signaling pathway 18.47 0 C1qb NM_009777 Innate immune response –22.78 0 Nkap NM_025937 Positive regulation of alpha-beta T cell differentiation 17.70 0 S1pr2 NM_010333 Innate immune response –24.35 0 Mbl2 NM_010776 Innate immune response 16.09 0 Spon2 NM_133903 Innate immune response –27.43 0 Il13 NM_008355 Immune response 15.69 0 Apoa4 NM_007468 Innate immune response in mucosa –36.25 0 Tcfe3 NM_172472 Humoral immune response 15.35 0 Tlr5 NM_016928 Innate immune response –37.43 0 H60a NM_001177775 Positive regulation of immune response to tumor cell –14.19 0.08 Igk AK036494 –91.24 0 Orm2 NM_011016 Regulation of immune system process –17.60 0 Cfd NM_013459 Innate immune response –110.03 0 Inflammatory response Cxcl1 NM_008176 Inflammatory response 70.45 0 Foxf1a NM_010426 Negative regulation of inflammatory response –17.81 0.14 Nr1h3 NM_013839 Positive regulation of toll-like receptor 4 signaling pathway 49.79 0 Foxfla AK087349 Negative regulation of inflammatory response –17.81 0.14 Adrb2 NM_007420 Negative regulation of inflammatory response 14.19 0 Ly86 NM_010745 Inflammatory response –19.19 0 Cxcl9 NM_008599 Inflammatory response –13.16 0 Il2ra NM_008367 Negative regulation of inflammatory response –22.46 0.48 Cnr2 NM_009924 Negative regulation of inflammatory response –13.97 0 Apoptosis Csrnp1 NM_153287 Apoptosis 111.74 0 Sgms1 NM_001168525 Negative regulation of apoptosis 13.21 0 Bcl6 NM_009744 Negative regulation of apoptosis 44.01 0 Slc9a1 NM_016981 Positive regulation of anti-apoptosis 13.01 0 Ddit4 NM_029083 Apoptosis 42.40 0 Irak2 NM_172161 Regulation of apoptosis –13.88 0 Tsc22d3 NM_001077364 Anti-apoptosis 42.19 0 Cdk5 NM_007668 Positive regulation of neuron apoptosis –14.61 0 Rps3a NM_016959 Induction of apoptosis 41.49 0 Senp1 NM_144851 Induction of apoptosis by extracellular signals –15.24 0.01 Krit1 NM_030675 Negative regulation of endothelial cell apoptosis 36.60 0 Lep NM_008493 Negative regulation of apoptosis –15.52 0.04 Chac1 NM_026929 Apoptosis 35.66 0 Epha2 NM_010139 Apoptosis –15.57 0.14 Inhba NM_008380 Induction of apoptosis 26.79 0 Arnt2 NM_007488 Negative regulation of apoptosis –15.64 0.23 Cebpb NM_009883 Anti-apoptosis 25.98 0 Txnip NM_001009935 Positive regulation of apoptosis –16.30 0 Plekhf1 NM_024413 Apoptosis 24.796 0 Ngfrap1 NM_009750 Induction of apoptosis via death domain receptors –16.32 0 Casp6 NM_009811 Positive regulation of apoptosis 24.16 0 Ube2m NM_145578 Positive regulation of neuron apoptosis –16.34 0 Arhgef2 NM_001198911 Induction of apoptosis by extracellular signals 23.86 0 Igf1 NM_010512 Anti-apoptosis –16.48 0 Aatf NM_019816 Induction of apoptosis by extracellular signals 23.28 0 Id1 NM_010495 Apoptosis –18.51 0 Dedd2 NM_207677 Induction of apoptosis via death domain receptors 22.45 0 Pglyrp1 NM_009402 Apoptosis –19.16 0.22 Atp2a1 NM_007504 Apoptosis in response to endoplasmic reticulum stress 21.17 0 Hoxa5 NM_010453 Positive regulation of apoptosis –19.70 0 Ube2b NM_009458 Negative regulation of apoptosis 20.17 0 Egfr NM_207655 Negative regulation of apoptosis –19.76 0 Fgd2 NM_001159538 Induction of apoptosis by extracellular signals 19.49 0 Alb NM_009654 Negative regulation of apoptosis –21.78 0.05 Prkra NM_011871 Induction of apoptosis 19.13 0 Net1 NM_019671 Induction of apoptosis by extracellular signals –22.23 0.02 Hsp90b1 NM_011631 Anti-apoptosis 18.99 0 Ngfrap1 NM_009750 Induction of apoptosis by extracellular signals –26.29 0 Rybp NM_019743 Apoptosis 17.68 0 Bcl3 NM_033601 Regulation of apoptosis –28.57 0 Apbb3 NM_146085 Negative regulation of apoptosis 17.56 0 Egfr NM_207655 Negative regulation of apoptosis –30.67 0 Mcl1 NM_008562 Negative regulation of apoptosis 16.88 0 Lcn2 NM_008491 Regulation of apoptosis –32.03 0 Hint2 NM_026871 Apoptosis 14.92 0 Jun NM_010591 Negative regulation of apoptosis –32.04 0 Pim3 NM_145478 Negative regulation of apoptosis 14.85 0 Bik NM_007546 Induction of apoptosis –38.09 0 Cflar NM_207653 Negative regulation of apoptosis 14.52 0 Peg3 NM_008817 Apoptosis –39.07 0.16 Trp53inp1 NM_001199105 Induction of apoptosis 14.45 9.13E-06 Nuak2 NM_001195025 Negative regulation of apoptosis –42.56 0 Puf60 NM_028364 Apoptosis 14.40 0 Cdkn1a NM_001111099 Positive regulation of anti-apoptosis –43.92 6.05E-06 Pdia3 NM_007952 Positive regulation of apoptosis 14.39 0 Ctrb1 NM_025583 Positive regulation of apoptosis –86.17 0.02 Ppargc1a NM_008904 Negative regulation of neuron apoptosis 14.20 0 Oxidative stress Ctgf NM_010217 Reactive oxygen species metabolic process 68.53 0 Acadvl NM_017366 Oxidation-reduction process 14.81 0 Sc4mol NM_025436 Oxidation-reduction process 68.39 0 Spryd4 NM_025716 Mitochondrion 14.65 0 Fdps NM_134469 Peroxisome 66.76 0 Ociad1 NM_001159889 Mitochondrion 14.33 0.02 Pmvk NM_026784 Peroxisome 63.86 0 Grsf1 NM_178700 Mitochondrion 14.24 0 Cyp2g1 NM_013809 Oxidation-reduction process 61.19 0 Pdhb NM_024221 Oxidation-reduction process 14.14 0 Sqle NM_009270 Oxidation-reduction process 58.93 0 Aldh1b1 NM_028270 Oxidation-reduction process 14.09 0 8430408G22Rik NM_145980 Mitochondrion 54.33 0 Coq10b NM_001039710 Mitochondrion 14.06 0 Gpd1 NM_010271 Oxidation-reduction process 52.14 0 Prr5l NM_001083810 Mitochondrion 13.64 0.03 Adh4 NM_011996 Oxidation-reduction process 46.10 0 Scd1 NM_009127 Oxidation-reduction process 13.50 0 Cyp51 NM_020010 Oxidation-reduction process 43.10 0 2410003K15Rik NM_029353 Mitochondrion 13.35 0 Cyp7a1 NM_007824 Oxidation-reduction process 42.24 0 Pecr NM_023523 Oxidation-reduction process 13.30 0 Dhcr7 NM_007856 Oxidation-reduction process 37.61 0 Impdh2 NM_011830 Oxidation-reduction process 13.09 0 Trak1 NM_175114 Mitochondrion 34.03 0 Mrps18c NM_026826 Mitochondrion 13.06 0 Rdh11 NM_021557 Oxidation-reduction process 33.22 0 Fmo4 NM_144878 Oxidation-reduction process 13.04 0 Aldh1a1 NM_013467 Response to oxidative stress 32.70 0 Gstt1 NM_008185 Glutathione peroxidase activity 13.04 0 Nsdhl NM_010941 Oxidation-reduction process 31.16 0 Bco2 NM_133217 Oxidation-reduction process –13.41 0 Sardh NM_138665 Oxidation-reduction process 29.13 0 Dhrs1 NM_026819 Oxidation-reduction process –13.51 0 Kmo NM_133809 Oxidoreductase activity 27.95 0 Decr2 NM_011933 Oxidation-reduction process –13.55 0 Stard4 NM_133774 Mitochondrion 26.36 0 Dct NM_010024 Oxidoreductase activity –13.79 0 Cyp2c54 NM_206537 Oxidation-reduction process 25.91 0 Gmpr NM_025508 Oxidation-reduction process –14.00 0 Dhdpsl NM_026152 Mitochondrion 24.94 0 Acot3 NM_134246 Peroxisome –14.19 0 Cyp2j9 NM_028979 Oxidation-reduction process 24.37 0 Akr1b10 NM_172398 Mitochondrion –14.20 0.08 Mt2 NM_008630 Nitric oxide-mediated signal transduction 24.27 0 Ugdh NM_009466 Oxidation-reduction process –14.44 0.65 Kdm5a NM_145997 Oxidation-reduction process 24.21 0 Pex11c NM_026951 Peroxisome –14.70 0 Mrps27 NM_173757 Mitochondrion 24.15 0 Mical2 NM_177282 Oxidation-reduction process –14.88 0 5730469M10Rik NM_027464 Oxidoreductase activity 23.93 0 Ddo NM_027442 Oxidation-reduction process –15.14 0 Tstd1 NM_001164525 Mitochondrion 23.747 0 Gtpbp5 NM_181424 Mitochondrion –15.41 0.10 Mmab NM_029956 Mitochondrion 23.02 0 Pnpo NM_134021 Oxidation-reduction process –15.53 0 Hmgcr NM_008255 Oxidation-reduction process 21.60 0 Blvrb NM_144923 Oxidation-reduction process –16.15 0 Ptcd3 NM_027275 Mitochondrion 21.56 0 Hsd17b6 NM_013786 Oxidation-reduction process –16.37 0 Glud1 NM_008133 Oxidation-reduction process 21.07 0 Por NM_008898 Oxidation-reduction process –16.49 0 Nox4 NM_015760 Oxidation-reduction process 20.82 0 Slc25a30 NM_026232 Mitochondrion –16.70 0 Qdpr NM_024236 Oxidation-reduction process 19.66 0 Ido2 NM_145949 Oxidation-reduction process –16.73 0.02 Cyp2c50 NM_001167877 Oxidation-reduction process 19.60 0 Ehhadh NM_023737 Oxidation-reduction process –19.26 0 Mtif2 NM_133767 Mitochondrion 19.52 0 Hsd3b5 NM_008295 Oxidation-reduction process –19.54 0 Slc25a16 NM_175194 Mitochondrial inner membrane 19.32 0 Suox NM_173733 Oxidation-reduction process –19.78 0 Gfm1 NM_138591 Mitochondrion 18.79 0 Cyp7b1 NM_007825 Oxidation-reduction process –20.35 0 Pdia6 NM_027959 Protein disulfide oxidoreductase activity 18.52 0 Cyba NM_007806 Oxidation-reduction process –21.82 0 Mtf1 NM_008636 Response to oxidative stress 18.24 5.71E-07 Pxmp4 NM_021534 Peroxisome –23.49 0 Rtn4ip1 NM_130892 Oxidation-reduction process 18.23 0 Gfod2 NM_027469 Oxidation-reduction process –25.19 0 Ppox NM_008911 Oxidation-reduction process 17.94 0 Cox8b NM_007751 Cytochrome c oxidase activity –25.23 0.38 Cyp2c55 NM_028089 Oxidation-reduction process 17.56 0 Gpx7 NM_024198 Oxidation-reduction process –27.03 0 Oxnad1 BC019806 Oxidation-reduction process 17.29 0 Cyp2b10 NM_009999 Oxidation-reduction process –32.32 0.07 Pyroxd1 NM_183165 Oxidation-reduction process 16.88 0 Decr1 NM_026172 Oxidation-reduction process –33.50 0 Gmppb NM_177910 Mitochondrion 16.82 0 Akr1c18 NM_134066 Oxidation-reduction process –44.32 0.61 Iyd NM_027391 Oxidation-reduction process 16.33 0 Dio1 NM_007860 Oxidation-reduction process –45.74 0.08 Nosip NM_025533 Negative regulation of nitric-oxide synthase activity 16.12 0 Mrpl10 NM_026154 Mitochondrion –45.85 9.13E-06 Bckdhb NM_199195 Oxidation-reduction process 16.06 0 Slc25a33 NM_027460 Mitochondrion –46.91 0 Aldh4a1 NM_175438 Oxidation-reduction process 15.11 0 — Oxidation-reduction process –52.35 0.82 Fmo3 NM_008030 Oxidation-reduction process 14.97 9.99E-07 Akr1b7 NM_009731 Oxidation-reduction process –73.14 0 Cybasc3 NM_201351 Oxidation-reduction process 14.95 0 Metabolic process Alas1 NM_020559 Porphyrin metabolic process 74.45 0 Cbs NM_144855 Regulation of cGMP metabolic process 13.45 0 Pcsk9 NM_153565 Induction of apoptosis 67.07 0 Cwf19l1 NM_001081077 Catalytic activity 13.45 0 Acat2 NM_009338 Metabolic process 63.61 0 Gstt3 NM_133994 Glutathione metabolic process 13.07 0 Gbe1 NM_028803 Carbohydrate metabolic process 52.75 0 Car11 NM_009800 Carbonate dehydratase activity 13.03 0.02 Stbd1 NM_175096 Carbohydrate metabolic process 49.85 0 Ufsp1 NM_027356 Metabolic process –13.06 0 Mccc2 NM_030026 Metabolic process 42.63 0 Hyal2 NM_010489 Metabolic process –13.09 0 Pm20d1 NM_178079 Metabolic process 42.10 0 Prune NM_173347 Hydrolase activity –13.09 0 St3gal4 NM_009178 Metabolic process 39.29 0 Ahsp NM_133245 Hemoglobin metabolic process –13.11 0.03 Sat2 NM_026991 Metabolic process 36.56 0 Pla2g6 NM_001199023 Metabolic process –13.21 0 Xylb NM_001199568 D-xylose metabolic process 35.60 0 Polr2d NM_027101 Cellular metabolic process –13.42 0 Bhmt NM_016668 Methyltransferase activity 35.18 0 Ear6 NM_053111 Metabolic process –13.45 0.29 Gfpt1 NM_013528 Carbohydrate biosynthetic process 31.83 0 Acot4 NM_134247 Lipid metabolic process –13.76 0 Rpia NM_009075 Ribose phosphate metabolic process 30.62 0 Nat1 NM_008673 Metabolic process –13.82 0 Agxt2 NM_001031851 Glyoxylate metabolic process 29.16 0 Pcx NM_001162946 Oxaloacetate metabolic process –13.85 0.11 Srr NM_013761 L-serine metabolic process 28.70 0 Ece2 NM_177941 Metabolic process –13.87 0 Ugt2b35 NM_172881 Aromatic compound catabolic process 28.40 0 Lyz1 NM_013590 Metabolic process –13.91 0 Plk3 NM_013807 Polo kinase kinase activity 27.53 0 Treh NM_021481 Metabolic process –14.13 0.53 Nudt16l1 NM_025839 Hydrolase activity 27.35 0 Gk2 NM_010294 Carbohydrate metabolic process –14.43 0.15 Gsta3 NM_001077353 Metabolic process 25.88 0 Pfas AK157402 Glutamine metabolic process –14.44 0 Thnsl2 NM_178413 Metabolic process 25.82 0 Lipn NM_027340 Lipid metabolic process –14.54 0.84 Upp2 NM_029692 Nucleoside metabolic process 25.72 0 Lhpp NM_029609 Metabolic process –14.85 0 Nmral1 NM_026393 Metabolic process 25.61 0 Aip NM_016666 Xenobiotic metabolic process –15.51 0 Man2a2 NM_172903 Metabolic process 25.40 0 Rabep2 NM_030566 GTPase activator activity –15.78 0 Ccrn4l NM_009834 Rhythmic process 25.29 5.71E-07 Daglb NM_144915 Lipid metabolic process –15.95 0 Mogs NM_020619 Oligosaccharide metabolic process 24.60 0 Pcsk7 NM_008794 Hydrolase activity –16.248 0 Car1 NM_009799 One-carbon metabolic process 23.91 0 Mgat4a NM_173870 Carbohydrate metabolic process –16.32 0 Gstm4 NM_026764 Nitrobenzene metabolic process 23.84 0 Pkp2 NM_026163 Heart development –16.41 0 Mobkl2b NM_178061 Kinase activity 23.76 0 Gmds NM_146041 GDP-mannose metabolic process –16.55 0 Tatdn1 NM_175151 Hydrolase activity 22.80 0 Gde1 NM_019580 Metabolic process –16.10 0 Safb NM_001163300 Hormone metabolic process 22.17 0 Klk1 NM_010639 Catalytic activity –17.15 0.87 Neu1 NM_010893 Metabolic process 22.04 0 Agpat2 NM_026212 Metabolic process –17.77 0 Nme7 NM_178071 Nucleotide metabolic process 21.98 0 Elovl2 NM_019423 Very long-chain fatty acid metabolic process –18.202 0 Corin NM_016869 Catalytic activity 21.89 0 Prtn3 NM_011178 Hydrolase activity –18.39 0.47 Lpin1 NM_001130412 Lipid metabolic process 21.48 0.02 Tgm1 NM_001161715 Protein metabolic process –18.43 0 Gstt2 NM_010361 Glutathione metabolic process 21.325 0 Pgpep1 NM_023217 Hydrolase activity –19.61 0 Phka2 NM_172783 Glycogen metabolic process 20.52 0 Tmprss2 NM_015775 Hydrolase activity –20.35 0 Peci NM_011868 Metabolic process 19.47 0 G6pc NM_008061 Steroid metabolic process –20.77 0 Ces1e NM_133660 Metabolic process 19.22 0 Chst13 NM_027928 Chondroitin sulfate biosynthetic process –21.05 0 Slc19a2 NM_054087 Thiamine-containing compound metabolic process 18.93 0 Pdxp NM_020271 Metabolic process –21.35 0 Ces2e NM_172759 Hydrolase activity 18.90 0 Anxa3 NM_013470 Positive regulation of DNA metabolic process –21.59 0 Mtmr12 NM_172958 Phosphatase activity 18.51 0 Apoa5 NM_080434 Positive regulation of triglyceride catabolic process –23.25 0 Atg4d NM_153583 Hydrolase activity 18.12 0 Agpat9 NM_172715 Metabolic process –23.50 0 Vldlr NM_013703 Cholesterol metabolic process 17.93 0.01 Lyz2 NM_017372 Metabolic process –23.74 0 Amy1 NM_007446 Carbohydrate metabolic process 17.82 0 Dpp7 NM_031843 Hydrolase activity –25.99 0 Hgfac NM_019447 Hydrolase activity 17.80 0 Cda NM_028176 Metabolic process –27.46 0 Thrsp NM_009381 Regulation of lipid biosynthetic process 17.67 9.13E-06 Agpat2 NM_026212 Metabolic process –27.66 0 Cdk10 NM_194446 Cyclin-dependent protein kinase activity 17.63 0 Chst8 NM_175140 Carbohydrate metabolic process –28.09 0 Yod1 NM_178691 ER-associated protein catabolic process 17.53 0.02 Klk1b27 NM_020268 Hydrolase activity –32.33 0 Hexdc NM_001001333 Metabolic process 16.79 0 Acot1 NM_012006 Acyl-CoA metabolic process –34.97 0 Acat3 NM_153151 Metabolic process 16.64 0 Nat8 NM_023455 Metabolic process –35.19 0.03 N4bp1 BC004022 Negative regulation of proteasomal ubiquitin-dependent protein catabolic process 15.21 0 Pdk4 NM_013743 Carbohydrate metabolic process –36.48 0.04 Ctdsp1 NM_153088 Hydrolase activity 15.21 0 Cpe NM_013494 Hydrolase activity Hyi NM_026601 Isomerase activity 15.07 0 Ppp1r3c NM_016854 Glycogen metabolic process –45.65 0 Slc2a2 NM_031197 Glucose transmembrane transporter activity 15.06 0.01 Csad NM_144942 Carboxylic acid metabolic process –51.19 0 Ugp2 NM_139297 Glucose 1-phosphate metabolic process 14.90 0 Pnliprp2 NM_011128 Lipid catabolic process –58.85 0.50 Galk1 NM_016905 Galactose metabolic process 14.86 0 Amy2a5 NM_001160152 Metabolic process –72.38 0 Rnaseh1 NM_011275 Metabolic process 14.70 0 Ctrc NM_001033875 Hydrolase activity –83.86 0.42 Adamts7 NM_001003911 Hydrolase activity 14.66 0 Cpa1 NM_025350 Hydrolase activity –87.31 0.82 Fggy NM_029347 Carbohydrate metabolic process 14.49 0 Ctrl NM_023182 Metabolic process –88.86 0.06 Lpin2 NM_001164885 Lipid metabolic process 14.44 0.01 Pnliprp1 NM_018874 Lipid metabolic process –92.09 0.04 5033411D12Rik NM_138654 Metabolic process 14.31 0 Rnase1 NM_011271 Hydrolase activity –100.336 0.408 Echdc3 NM_024208 Metabolic process 14.28 0 Cel NM_009885 Cholesterol catabolic process –104.65 0.11 Polg2 NM_015810 Metabolic process 14.13 0 Try4 NM_011646 Metabolic process –115.52 0.90 Gsta2 NM_008182 Metabolic process 14.10 0 Clps NM_025469 Steroid metabolic process –115.73 0.35 Capns1 NM_009795 Metabolic process 13.96 0 Cela3b NM_026419 Metabolic process –118.36 0.59 Serpind1 NM_008223 Serine-type endopeptidase inhibitor activity 13.83 0 Try5 NM_001003405 Metabolic process –128.77 0.51 Acss2 NM_019811 Metabolic process 13.71 0 Pnlip NM_026925 Lipid metabolic process –130.98 0.89 Nktr NM_010918 Peptidyl-prolyl cis-trans isomerase activity 13.47 0 Cela2a NM_007919 Catalytic activity –138.47 Note. We classified the function of these genes according to gene Ontology in NCBI (http://www.ncbi.nlm.nih.gov/). Open in new tab Real-time Polymerase Chain ReactionTo verify the accuracy of the microarray assays, a number of genes that demonstrated significantly different expression patterns were chosen due to their association with immune response, oxidative stress, apoptosis, response to stress, the metabolic process, and cell proliferation. Most of the qRT-PCR results were in reasonable agreement with the microarray results (i.e., either up- or downregulation). For simple validation, 14 genes were verified by RT-PCR, all of which displayed expression patterns comparable with the microarray data (Table 3), with the exception of Grb2, which showed discrepancy between the two methods, suggesting possible problems with RNA handling or pipetting issues. TABLE 3 RT-PCR Validation of Selected Genes from Microarray Data Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Open in new tab TABLE 3 RT-PCR Validation of Selected Genes from Microarray Data Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Function . Gene . ∆∆Ct . Fold . Microarray . Immune response Apoa4 1.302649 0.405381175 0.4282288 Metabolic Apoa5 0.551284 0.68241251 0.6019174 Cell proliferation Asah3l −0.523024 1.436964081 1.585262 Immune response Cfd 3.621761 0.081234648 0.1096267 Oxidative stress Cyp2c55 −2.396719 5.266041898 −3.789775 Oxidative stress Cyp51 −1.514383 2.85676628 2.127146 Oxidative stress Cyp7a1 −1.669531 3.181111632 2.05641 Oxidative stress Cyp7b1 0.403728 0.755902466 0.6108548 Oxidative stress Dhcr7 −0.833158 1.781580904 2.255726 Oxidative stress Ehhadh 0.38894 0.763690509 0.5382943 Response to stress Grb2 −0.138709 1.100919514 0.1723893 Apoptosis Jun 0.886755 0.540829218 0.5033823 Apoptosis Ngfrap1 0.209656 0.864743398 0.4900441 Oxidative stress Nsdhl −1.528868 2.885593343 2.057153 Open in new tab Discussion The results of this study indicate that long-term exposure to 10 mg/kg BW TiO2 NPs results in titanium accumulation in mouse liver tissue, TiO2 NP aggregation in hepatocyte nuclei (Fig. 2B), liver inflammation (Fig. 1), and hepatocyte apoptosis (Fig. 2B), coupled with decreases in the levels of WBC, LYMPH, and NEUT and increases in ALT, AST, ALP, and LDH activities and TChol and TG levels (1). TiO2 NP–induced hepatic toxicity is closely related to NP size (in general, smaller NPs induce greater toxicity) (Oberdörster et al., 1994; Wang et al., 2007); however, toxicity is mainly dependent on TiO2 NPs surface area (Warheit et al., 2006). The surface area of TiO2 NPs is negatively correlated with its size. In this study, the average particle size in TiO2 NP powder was about 5 nm and the surface area was 174.8 m2/g (Hu et al., 2011; Yang et al., 2002); thus, exposure to these TiO2 NPs caused severe damage to mouse liver. The significant reduction in hematological parameters and liver dysfunction caused by exposure to TiO2 NPs may be related to damage of immune and metabolism function in mice, which may be associated with alterations in gene expression in the liver. To identify the molecular mechanisms caused by exposure to TiO2 NPs, microarray assays of RNA from mouse livers were performed to establish a global gene expression profile. These assays indicated that the expression levels of 1142 genes were significantly changed, and 785 of these genes were involved in inflammatory/immune response, apoptosis, oxidative stress, and the metabolic process. The main results are discussed below. Genes Related to Immune and Inflammatory ResponseA previous study demonstrated that TiO2 NPs significantly increased the mRNA and protein expression of TLR2, TLR4, and several inflammatory cytokines, including IKK1, IKK2, NF-κB, NF-κBP52, NF-κBP65, TNF-α, and NIK, thus inducing an inflammatory response in the liver of mice (Cui et al., 2011). In this study, focal inflammatory cell infiltration in liver tissue was observed (Fig. 1), and 39 genes (3.41% of 1142 genes) involved in immune and inflammatory responses were significantly changed as shown by microarray data (Table 2). Of these genes, 16 were upregulated and 21 were downregulated. In line with our data, Fujita et al. (2009) found that 15 genes involved in the inflammatory response and 23 genes involved in the immune response were upregulated at both 3 day and 1 month postexposure to C60 fullerene. These results suggest that nanoparticles may change the expression levels of key genes, then trigger toxic signal pathways, and finally induce an inflammatory response in the liver. Retinoic acid early transcript beta (Raet1b) plays a role in the positive regulation of the immune response in tumor cells, and its expression induces primary tumor rejection and triggers in vitro natural killer (NK) cell cytotoxicity and interferon-γ secretion (Hayakawa et al., 2002). Our data show that Raet1b was increased by 83.03-fold in the TiO2 NP–exposed group (Table 2), suggesting that TiO2 NPs induced Raet1b expression and then triggered NK cell cytotoxicity, which resulted in a reduction in immune capacity and the generation of inflammation in mouse liver. Chemokine (C-X-C motif) ligand 1 (CXCL1) is important for the expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine, and activation of NF-κB (Cai et al., 2010). In this study, Cxcl1 was overexpressed (with a diffscore of 70.45) following exposure to TiO2 NPs (Table 2); therefore, it activated proinflammatory cytokines and then led to hepatic inflammation. However, complement factor D (Cfd) was dramatically downregulated with a diffscore of –110.03 (Table 2). Cfd is a serine protease essential for activation of the alternative pathway in the complement system. Its main role is to eliminate foreign antigens and pathogens as part of the normal host response (Holers, 2000). Abrera-Abeleda et al. (2007) reported that Cfd gene knockout mice were a novel model of spontaneous mesangial immune complex glomerulonephritis. The inhibition of Cfd expression due to exposure to TiO2 NPs reduced the activity of C3b factor B, which is also known as C3 convertase. A small quantity of C3 convertase (C3bBb) is insufficient to cleave additional C3 molecules and generate C3a and C3b; therefore, TiO2 NP exposure causes further amplification of the immune/inflammatory programs in mouse liver (Fig. 4). Fig. 4. Open in new tabDownload slide Cfd network pathway obtained from network analysis of differentially expressed genes. Ingenuity Pathway Analysis software was used to construct and visualize molecular interaction networks. Fig. 4. Open in new tabDownload slide Cfd network pathway obtained from network analysis of differentially expressed genes. Ingenuity Pathway Analysis software was used to construct and visualize molecular interaction networks. Genes Related to ApoptosisChen et al. (2006) found that intratracheal instillation of TiO2 NPs in mice induced the differential expression of hundreds of genes including those related to apoptosis and the cell cycle. In this study, we observed hepatocyte apoptosis caused by TiO2 NPs (Fig. 2). To further clarify the molecular mechanism of apoptosis, we analyzed the apoptotic genes and found that 58 genes (5.07% of 1142 genes) were significantly 
altered by exposure to TiO2 NPs (Table 2). Of these 58 genes, 30 were upregulated and 28 were downregulated. Expression of the apoptotic genes, including cysteine-serine-rich nuclear protein-1 (Csrnp1) and Ddit4, were significantly upregulated, with diffscores of 111.74 and 42.40, respectively (Table 2). Csrnp1 has been identified as an immediate early gene that is strongly induced in response to IL-2 in mouse T cells (Gingras et al., 2007). We suggest that high expression of Csrnp1 may induce proinflammatory cytokine expression and then hepatocyte apoptosis. As a novel transcriptional target of p53, Ddit4, which is also known as REDD1, induced DNA damage (Ellisen et al., 2002). REDD1 encodes a shared transcriptional target that implicates reactive oxygen species (ROS) in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation. The overexpression of Ddit4 caused by TiO2 NPs may induce the accumulation of ROS, which leads to DNA oxidative damage and finally causes apoptosis. However, Bcl3 expression was significantly reduced following exposure to TiO2 NPs in the study. Kreisel et al. (2011) demonstrated a critical role for Bcl3 in regulating emergency granulopoiesis and suggested that Bcl3 prevents an acute inflammatory response in mouse lung injury. Bauer et al. (2006) indicated that apoptosis is inhibited by Bcl3 involving a Bim-independent pathway. Therefore, the typical apoptosis caused by exposure to TiO2 NPs implied that the upregulated pro-apoptosis genes and downregulated expression of Bcl3 may have a collaborative effect on inducing hepatocyte apoptosis. Genes Related to Oxidative StressPrevious studies have shown that TiO2 NPs can induce the production of ROS (such as O2•− and H2O2) and lipid peroxidation in the mouse liver, indicating that the liver suffered from oxidative stress (Cui et al., 2010; Ma et al., 2009), and demonstrated that TiO2 NPs exposure decreased the stress- related gene expression levels of superoxide dismutase, catalase, glutathione peroxidase, metallothionein, heat shock protein 70, glutathione S transferase, P53, and transferrin, and significantly increased cytochrome p450 1A expression in mouse liver (Cui et al., 2010). In this study, microarray data suggested that approximately 101 genes (8.84% of 1142 genes) related to oxidative stress were significantly changed in the TiO2 NP–exposed liver (Table 2). Of these 101 genes, 66 were upregulated and 35 were downregulated (Table 2). In this study, connective tissue growth factor (Ctgf) was highly expressed following exposure to TiO2 NPs, with a diffscore of 68.56. Ctgf is a multifunctional protein that coordinates complex biological processes during tissue development and remodeling. It also contributes to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and oxidative damage to biopolymers. Overexpression of Ctgf was demonstrated to induce pulmonary vascular remodeling and pulmonary hypertension (Chen et al., 2011). Therefore, we speculate that Ctgf may be a sensitive gene for TiO2 NP exposure. Its abnormal expression may cause the disorder of several biological processes involved in ROS production. ROS accumulated by exposure to TiO2 NPs lead to oxidative stress in the liver, thus causing hepatocyte apoptosis. Enoyl-coenzyme A (hydratase/3-hydroxyacyl coenzyme A dehydrogenase [Ehhadh]), a small protein encoded by this gene that uses dioxygen (O2) to oxidize organic molecules, contains some enzymes, and it degrades hydrogen peroxide (H2O2). Defects in this gene are a cause of peroxisomal disorders such as Zellweger syndrome (Stephen and David, 2000). In our study, Ehhadh was downregulated by TiO2 NP exposure, with a diffscore of −19.26. Reduced Ehhadh expression by TiO2 NPs may cause hepatocyte peroxisomal disorders and decrease antioxidative capacity or detoxification. Genes Related to Metabolic ProcessSeveral reports have addressed the transcriptomic changes involved in the hepatic metabolic process induced by diverse chemical toxicants. Richards et al. (2004) reported that hydrazine induced hepatic steatosis and necrosis in mice, and altered the expression of genes involved in lipid peroxidation/fatty acid synthesis and transport. Lee et al. (2007) indicated that valproic acid induced hepatotoxicity including microvesicular steatosis and necrosis in the liver following oral administration of a single dose of 100 mg/kg (low dose) or 1000 mg/kg (high dose) in mice. The expression of genes associated with the metabolic process was investigated. Functional categorization of genes altered by exposure to TiO2 NPs revealed that 135 genes (11.8% of 1142 genes) were involved in the lipid, fatty acid, and glutathione metabolic processes and steroid metabolism (Table 2). Notably, TiO2 NPs induced changes in the expression of genes involved in the biosynthetic pathways of both cholesterol and lipid metabolism. For example, the proprotein convertase subtilisin type 9 (Pcsk9) is involved in the cholesterol metabolic process and plays a key role in cholesterol homeostasis by binding the low-density lipoprotein receptor and targeting it toward degradation (Essalmani et al., 2011). Lee et al. (2005) reported that Acetyl-CoA acetyltransferase 2 (Acat2) provides the core cholesteryl ester of newly secreted very low-density lipoprotein, whereas lecithin:cholesterol acyltransferase adds cholesteryl ester during low-density lipoprotein particle formation. In this study, Pcsk9 and Acat2 were highly expressed following exposure to TiO2 NPs, with diffscores of 67.06 and 63.61, respectively (Table 2). TiO2 NPs inducing Proprotein convertase subtilisin/kexin type 9 (Pcsk9) and Acat2 overexpression may promote the biosynthesis of cholesteryl ester, thus, resulting in high levels of TChol in serum and liver dysfunction (Table 1). Conclusion After a single intragastric administration of 10 mg/kg BW TiO2 NPs for 90 consecutive days, accumulation of TiO2 NPs in liver tissue and even in hepatocyte nuclei was observed, which impaired liver function and induced a severe inflammatory response and hepatocyte apoptosis in mouse liver. 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Toxicological Sciences – Oxford University Press

Published: Jul 1, 2012

Keywords: Key Words titanium dioxide nanoparticles mice liver gene expression profiling inflammation apoptosis titanium dioxide nanoparticles

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