in vivo 32 : 677-684 (2018) doi:10.21873/invivo.11293

cDNA Microarray Analysis and Influx Transporter OATP1B1 in Liver Cells After Exposure to Gadoxetate Disodium, a Gadolinium-based Contrast Agent in MRI Liver Imaging CHI-CHENG LU 1* , WEN-KANG CHEN 2* , JO-HUA CHIANG 3, YUH-FENG TSAI 4, YU-NING JUAN 5, PING-CHIN LIN 6, YEU-SHENG TYAN 7,8,9 and JAI-SING YANG 5

1Department of Pharmacy, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, R.O.C.; 2Department of Applied Cosmetology, National Tainan Junior College of Nursing, Tainan, Taiwan, R.O.C.; 3Department of Nursing, Chung Jen Catholic Junior College, Chiayi, Taiwan, R.O.C.; 4Department of Diagnostic Radiology, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, R.O.C.; 5Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan, R.O.C.; 6Department of Medical Imaging, Chia-Yi Christian Hospital, Chiayi, Taiwan, R.O.C.; 7Department of Medical Imaging, Chung Shan Medical University Hospital, Taichung, Taiwan, R.O.C.; 8School of Medical Imaging and Radiological Sciences, 9School of Medicine, Chung Shan Medical University, Taichung, Taiwan, R.O.C.

Abstract. Background/Aim: Gadoxetate disodium (Primovist western blotting. Results: Gadoxetate disodium at 5 and 10 or Eovist) is extensively used as a hepatospecific contrast agent mM failed to induce any cell cytotoxicity and morphological during magnetic resonance imaging (MRI) examinations. changes in Chang Liver cells. Our data demonstrated that However, there is no information determining whether gadoxetate disodium significantly enhanced the expression of gadoxetate disodium has a cytotoxic impact and/or affects 29 and suppressed that of 27. The SLCO1C1 (solute relative expression on liver cells. In the current study, we carrier organic anion transporter family member 1C1) mRNA investigated the effects of gadoxetate disodium on cytotoxicity expression was also increased by 2.62-fold (p-value=0.0006) and the levels of gene expression in human normal Chang in gadoxetate disodium-treated cells. Furthermore, we also Liver cells. Materials and Methods: The cytotoxic effect was checked and found that gadoxetate disodium up-regulated detected via methyl thiazolyl tetrazolium (MTT) assay and 4’,6- organic anion transporter polypeptide 1B1 (OATP1B1) diamidino-2-phenylindole (DAPI) staining. mRNA expression level and increased OATP uptake transporter gene SLCO1C1 was monitored by cDNA microarray and quantitative PCR mRNA expression. Conclusion: Our results provide evidence (qPCR) analysis. The protein levels were determined by regarding that gadoxetate disodium might be no cytotoxic effects on liver cells.

Gadoxetate disodium (C 23 H28 GdN 3Na 2O11 ; synonymous with This article is freely accessible online. gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid, disodium salt, Gd-EOB-DTPA) is a liver-specific paramagnetic *These Authors contributed equally to this work. contrast agent for magnetic resonance imaging (MRI) (1-4) Correspondence to: Dr. Jai-Sing, Yang, Department of Medical (Figure 1). Gadoxetate disodium can detect the focal liver Research, China Medical University Hospital, China Medical lesions and provide structural and functional responses in University, No. 2, Yude Road, Taichung 40447, Taiwan, R.O.C. Tel: hepatobiliary system (4, 5). For example, furthermost +886 422052121 ext. 2758, e-mail: [email protected]; or Dr. hepatocellular carcinoma (HCC) expresses hypo-intensity Yeu-Sheng Tyan, Department of Medical Imaging, Chung Shan compared to background liver cells in hepatobiliary-phase Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Road, (delayed phases) images on enhanced MRI (6-8). Many studies Taichung 40201, Taiwan, R.O.C. Tel: +886 424739595 ext. 32136, e-mail: [email protected] have investigated the transporters of gadoxetate disodium in hepatocellular cells and showed that organic anion transporting Key Words: Gadoxetate disodium, Chang liver cells, cDNA polypeptide 1 (OATP1) (one of the hepatocyte transporters) is microarray, influx transporter OATP1B1. a major target protein (9-13). On the other hand, excretion of

677 in vivo 32 : 677-684 (2018) gadoxetate disodium from hepatocytes into bile canaliculi occurs via the human canalicular multispecific organic anion transporter (multidrug resistance-associated protein 2; MRP2/ cMOAT) protein (14-16). It has been reported in a phase I trial that doses of 10, 25, 50, and 100 μmol/kg of gadoxetate disodium were well tolerated and showed no severe side-effects (17). To date, neither the cytotoxic effects of gadoxetate disodium on liver cells, nor the mRNA expression levels underlying its activity have been fully investigated. The aim of this study was to clarify the molecular mechanisms and gene expression profile involved in the effects of gadoxetate disodium on Figure 1. The chemical structure of gadoxetate disodium human normal Chang Liver cells, including the effects on the (C 23 H28 N3O11 .Gd.2Na). levels of OATP1B1 protein and of the OATP uptake transporter gene SLCO1C1 mRNA.

Materials and Methods 4 h and 24 h. Cells were sequentially washed with PBS, fixed in 4% formaldehyde for 15 min, and permeabilized in 0.1% Triton X-100 Reagents and chemicals. Gadoxetate disodium (Primovist) was for 15 min. Cells were then stained with 200 μl DAPI solution (1 obtained from Bayer Healthcare (Berlin, Germany). Minimum μg/ml) for 30 min at 37˚C in the dark. The integrity of nuclei was Essential Medium (MEM), penicillin/streptomycin, L-glutamine and visualized under a fluorescent microscope (Nikon Inc., Tokyo, trypsin-EDTA were purchased from BioConcept (Allschwil/BL, Japan), as previously described (20). Switzerland). Fetal bovine serum (FBS) was obtained from HyClone Laboratories, GE Healthcare Life Sciences (South Logan, UT, Western blot analysis . Cells prior to gadoxetate disodium challenge USA). The primary antibodies against OATP1B1 and β- actin, as (5 and 10 mM) for 4 h were scraped on the Trident RIPA lysis well as the mouse IgG antibody (HRP) secondary antibody were buffer (GeneTex, Hsinchu, Taiwan) and centrifuged at 1200 × g for bought from GeneTex (Hsinchu, Taiwan). All chemicals and 5 min at 4˚C. An aliquot of pelleted cells was used for protein reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, quantification as previously described (21, 22), and equal amounts USA) unless otherwise specified. of (40 μg) were separated on 10% acrylamide gels by SDS- electrophoresis and then transferred to the Immobilon-P Transfer Cell culture. The Chang Liver cell line was obtained from the Membrane (Merck Millipore, Billerica, MA, USA). After blocking American Type Culture Collection (ATCC, Rockville, MD, USA) and unspecific binding sites with 5% dry milk in PBST, the membranes maintained in MEM supplemented with 10% FBS, 2 mM L-glutamine were incubated with primary antibodies, diluted 1:1,000 in PBST- and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) 3% BSA overnight at 4˚C, and this was followed by incubation for at 37˚C in 5% CO 2. The cells were detached by 0.25% trypsin/0.02% 2 h at room temperature with the appropriate HRP-secondary EDTA and split every 2 to 3 days to maintain cell growth. antibody diluted 1:10,000 in PBST-3% BSA. All bands were normalized against β- actin, and band intensities were quantified by Cell viability assay. Cell viability was examined by the MTT ImageJ 1.47 program for Windows from NIH. method, as described previously with some modifications (18, 19). In brief, cells (2×10 4 cells/well) were dispensed in 96-well plates RNA extraction. Cells, after 5 and 10 mM of gadoxetate disodium and treated with different concentrations (0, 5 and 10 mM) of exposure for 2 h, were scraped and collected by centrifugation, and gadoxetate disodium for 4 h and 24 h. Per well, methyl thiazolyl total RNA was subsequently isolated by an RNeasy Mini Kit tetrazolium (MTT) (10 μl, 5 mg/ml) was added for an additional 1.5 (QIAGEN, Valencia, CA, USA). RNA quantity and purity were h-incubation. The optical density was measured under 570 nm with assessed at 260 nm and 280 nm using a Nanodrop (ND-1000; a spectrophotometer after the violet formazan crystal produced from Labtech International, Sussex, UK). Each sample (100 ng) was MTT was solubilized by 200 μl dimethyl sulfoxide (DMSO). Cell amplified and labeled using the GeneChip WT PLUS Reagent Kit viability was further calculated, and a percentage of alive versus (Thermo Fisher Scientific, Carlsbad, CA, USA) for expression dead cells was obtained. analysis according to the manufacturer’s instructions.

Morphological determination. Cells (2×10 5 cells/well) were seeded cDNA microarray analysis. Hybridization was performed against into 12-well plates and exposed to 5 and 10 mM of gadoxetate the Affymetrix Human Clariom S Array (Thermo Fisher Scientific, disodium for 4 h and 24 h, respectively. The cells were Carlsbad, CA, USA). The arrays were hybridized for 17 h at 45˚C photographed under a phase-contrast microscope to verify apoptotic and 60 rpm. Arrays were subsequently washed (Affymetrix Fluidics features before collection, as described elsewhere (20). Station 450, Thermo Fisher Scientific, Carlsbad, CA, USA) and stained with streptavidin-phycoerythrin (GeneChip Hybridization, 4’,6-diamidino-2-phenylindole (DAPI) for nucleic acid staining. Wash, and Stain Kit, Thermo Fisher Scientific, Carlsbad, CA, USA). Cells (2×10 5 cells/well) in 12-well plates were individually The chip was scanned on an Affymetrix GeneChip Scanner 3000 incubated with or without 5 and 10 mM of gadoxetate disodium for (Thermo Fisher Scientific, Carlsbad, CA, USA) as previously

678 Lu et al : Gadoxetate Disodium Increased OATP1B1 Levels

Figure 2. Effects of gadoxetate disodium on cell viability of Chang Liver cells. Cells were incubated with or without 5 and 10 mM of gadoxetate disodium for 4 h and 24 h, respectively. The cell viability was determined by MTT assay. Data are presented as the mean±SD (n=3). The different letters show significant differences (p<0.05) by the Duncan’s test.

described (23). The resulting data were analyzed using Expression CACCTTT, and human GAPDH-Reverse (R)-TAGCCAAATTCG Console software (Affymetrix Fluidics Station 450, Thermo Fisher TTGTCATACC; Human SLCO1C1-F-TGATGTGGCAGGACT Scientific, Carlsbad, CA, USA) with default RMA parameters. The AAC, and human SLCO1C1-R-GACAACCAGCAAGACAAG. genes regulated by gadoxetate disodium were considered to have a QPCR was performed in triplicate in a Applied Biosystems 7300 significant difference with a 2.0-fold change. Real-Time PCR System as previously reported (24).

Gene ontology and ingenuity pathway analysis. For detection of Statistical analysis. The results were acquired from three significantly over-represented (GO) biological independent experiments, and the means±standard deviation (SD) processes, the DAVID functional annotation clustering tool were reported. Data significance was evaluated by one-way ANOVA (http://david.abcc.ncifcrf.gov) was used (DAVID Bioinformatics followed by the Duncan’s multiple range test if ANOVA was Resources 6.7, Frederick, MD, USA). The significant list containing significant ( p< 0.05). the gadoxetate disodium and untreated control, complete with Affymetrix transcript identifiers, was uploaded from a Microsoft Excel Results spreadsheet onto the Ingenuity Pathway Analysis (IPA) software (QIAGEN) (https://www.qiagenbioinformatics.com/products/ingenuity Effect of gadoxetate disodium on cell viability, morphologic -pathway-analysis/). IPA recognized the Affymetrix identifiers and changes and DNA condensation of human normal Chang mapped the gadoxetate disodium to the IPA data analysis suite, generating maps to describe common pathways or molecular Liver cells . The cytotoxic effect on Chang Liver cells connections between gadoxetate disodium and untreated control on the cultured in the presence of different concentrations of list. Graphical representations of the molecular relationships between gadoxetate disodium was recorded. Our data demonstrated genes were generated using the IPA pathway analysis based on that gadoxetate disodium at 5 and 10 mM at 4 h and 24 h- processes showing significant ( p< 0.05) association. To identify the duration did not reduce the cell viability of Chang Liver cells potential function of the hub gene, GSEA (http://software. (Figure 2). No morphological change was observed between broadinstitute.org/gsea/index.jsp) was conducted to detect whether a series of defined biological processes were enriched in the gene rank. cells treated with gadoxetate disodium for 4 and 24 h and untreated control (Figure 3). Furthermore, no DNA Quantitative polymerase chain reaction (qPCR). Cells, after condensation (an apoptotic characteristic) occurred in the gadoxetate disodium (5 and 10 mM) exposure for 2 h, were untreated control and gadoxetate disodium-treated cells collected, and total RNA was subsequently isolated as above (Figure 4). Therefore, gadoxetate disodium exerts a non- described. The cDNA synthesis was performed using a High cytotoxic effect on Chang Liver cells. Capacity cDNA Reverse Transcription Kits (Applied Biosystems/ Thermo Fisher Scientific, Carlsbad, CA, USA). QPCR control with Effect of gadoxetate disodium on influx transporter OATP1B1 a 2X SYBR Green PCR Master Mix (Applied Biosystems) were amplified for SLCO1C1 gene under the same PCR parameters to protein level and SLCO1C1 mRNA of Chang Liver cells. The normalize the GAPDH quantitative data. PCR primers were as levels of OATP1B1 protein and SLCO1C1 gene have been follows: Human GAPDH-Forward (F)-ACACCCACTCCTC found to be mainly expressed on the sinusoidal membrane of

679 in vivo 32 : 677-684 (2018)

Figure 3. Effects of gadoxetate disodium on morphology of Chang Liver cells. Cells treated with or without 5 and 10 mM of gadoxetate disodium for 4 h and 24 h were examined and photographed for changes in cell morphology using a phase-contrast microscope as described in the “Materials and Methods” section.

human hepatocytes (25-27). To address how gadoxetate disodium caused a 2.62-fold increase in the expression of disodium interacts with Chang Liver cells, we determined OATP uptake transporter gene SLCO1C1 (p-value=0.0006). experimentally the level of OATP1B1 by western blotting and Hence, we suggest that the OATP uptake transporter gene qPCR methods. Our data indicated that gadoxetate disodium SLCO1C1 might exert a protective effect and play a vital role increased OATP1B1 protein expression in a concentration- in normal liver cells prior to gadoxetate disodium exposure. dependent manner (Figure 5A). Furthermore, qPCR analysis showed that gadoxetate disodium significantly up-regulated Discussion the organic anion transporter polypeptide (OATP) uptake transporter gene SLCO1C1 mRNA expression in treated cells, Gadolinium-based contrast agents (GBCA) are widely used for and this effect was concentration-dependent (Figure 5B). We enhanced magnetic resonance imaging (MRI) examinations (1, suggest that gadoxetate disodium can be rapidly pumped-out 4). Gadoxetate disodium was developed in order to reduce side extra-cellularly through modulating OATP1B1 and up- effects, and it is associated with other liver-specific regulating SLCO1C1 mRNA in Chang Liver cells. paramagnetic contrast agents on MRIs (3, 4). However, there is no report addressing the effects of gadoxetate disodium on Effect of gadoxetate disodium on gene expression of Chang cytotoxicity and its associated gene expression profile in human Liver cells by cDNA microarray analysis. The microarray normal Chang Liver cells. This study is the first to demonstrate analysis demonstrated that gadoxetate disodium up-regulated that gadoxetate disodium does not induce any cytotoxic effects 29 genes and down-regulated 27 genes in Chang Liver cells (Figures 2 and 3), DNA condensation or fragmentation (an after a 2 h exposure (Table I). Most of the differentially apoptotic characteristic) (Figure 4). It did however alter the expressed genes were associated with transmembrane transport mRNA expression profile of the transmembrane transport- (SLCO1C1 and IFT46); receptors (GABRA2, GHR and associated genes on Chang Liver cells (Table I). OR2L2); the DNA catabolic process (DNASE1, ZNF556 and Based on the changes in the gene expression profile in DZIP1); kinase activity (DGKD and MRAS); G-protein gadoxetate disodium-treated Chang Liver cells as shown by signaling (ARHGEF35 and RRAS); the apoptotic process cDNA microarray, we found that the cellular responses to (THAP2) and transcription (RP11-93O14.2, FAM65A, gadoxetate disodium treatment are multi-faceted and likely MAML3, MYH9, CUL4A, SULF2, SNX5, PTK2, to be mediated through a variety of signaling pathways. ARHGAP10, TSC1, ACVRL1, FAHD2A, DDX5, HIST1H1C, Gadoxetate disodium regulated the mRNA expression of KIDINS220, NAP1L2 and SH2B3) in cells following important genes, including those involved in anion gadoxetate disodium treatment. Among them, gadoxetate transmembrane transport ( SLCO1C1 and IFT46 ); receptors

680 Lu et al : Gadoxetate Disodium Increased OATP1B1 Levels

Figure 4. Effects of gadoxetate disodium on DNA condensation in Chang Liver cells. Cells treated with or without 5 and 10 mM of gadoxetate disodium after 4 h and 24 h were examined for DNA condensation and fragmentation using DAPI staining. To examine and photograph DNA condensation, a fluorescent microscope was used, as described in the “Materials and Methods” section.

(GABRA2, GHR , and OR2L2 ); the DNA catabolic process (DNASE1, ZNF556 , and DZIP1 ); kinase activity ( DGKD and MRAS ); G-protein signaling ( ARHGEF35 and RRAS ); the apoptotic process ( THAP2 ) and transcription ( RP11-93O14.2, FAM65A, MAML3, MYH9, CUL4A, SULF2, SNX5, PTK2, ARHGAP10, TSC1, ACVRL1, FAHD2A, DDX5, HIST1H1C, KIDINS220, NAP1L2 , and SH2B3 ) (Table I). Thus, regulation of these crucial genes may be responsible for effect of gadoxetate disodium on Chang Liver cells. It has been reported that several organic anion transporting polypeptides (OATPs) are involved in the uptake of gadoxetate disodium (28, 29). The OATP family of proteins includes OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1 (30). OATP1 is the first member of the OATP gene family, which has been isolated from the rat liver (26, 31, 32). OATP2 has been cloned from rat brain and expressed in the liver (32, 33). Furthermore, OATP has been cloned from human liver and has lower transport capacities for bile acid and organic anions (34-36). It has been documented that the organic anion transporters mediate mainly the transport of small anionic drugs such as salicylate, and acetylsalicylate (37, 38). Figure 5. Effects of gadoxetate disodium on influx transporter OATP1B1 OATP1B1 is most highly expressed in the human liver and is protein level and SLCO1C1 mRNA expression in Chang Liver cells. Cells mainly involved in the uptake of gadoxetate disodium into the were exposed to 5 and 10 mM of gadoxetate disodium for 4 h. (A) hepatocytes in conjunction with OATP8 (synonymous with OATP1B1 protein expression was analyzed by western blot. β- Actin OATP1B3) (39). OATP1B3 expression is strongly associated served as an internal control to ensure equal loading. The blot is a with Wnt signaling and represents the transporter of gadoxetate representative of three independent experiments. (B) cDNA produced from mRNA reverse transcription, was employed to examine SLCO1C1 mRNA disodium in hepatocellular carcinoma KYN-2 cells (10, 12). expression by qPCR analysis. GADPH, a house-keeping gene, was used Our results demonstrated that gadoxetate disodium increased as a control. Data are presented as the mean±SD (n=3). The different the protein level of OATP1B1 in Chang Liver cells (Figure letters (a-c) show significant differences (p<0.05) by the Duncan’s test.

681 in vivo 32 : 677-684 (2018)

Table I. Genes exhibiting > 2-fold changes in mRNA levels in Chang Liver cells following a 2-h treatment with gadoxetate disodium as identified using cDNA microarray.

ID Fold p-Value Gene GO Biological Process Term change symbol

TC1600010172.hg.1 3.42 0.0004 RP11-93O14.2 ; VPS35 Novel transcript TC1200009927.hg.1 2.67 0.0089 TAS2R20 Taste receptor, type 2, member 20 TC0400007853.hg.1 2.65 0.0068 EREG Epiregulin TC1200007043.hg.1 2.62 0.0006 SLCO1C1 Solute carrier organic anion transporter family, member 1C1 TC1700007671.hg.1 2.59 0.0115 C17orf78 17 open reading frame 78 TC1600008165.hg.1 2.43 0.0014 FAM65A Memczak2013 ALT_ACCEPTOR, ALT_DONOR, coding, INTERNAL, intronic best transcript NM_001193522 TC0400011957.hg.1 2.42 0.0028 MAML3 Memczak2013 ALT_ACCEPTOR, ALT_DONOR, coding, INTERNAL, intronic best transcript NM_018717 TC2200008597.hg.1 2.42 0.001 MYH9 Zhang2013 ALT_ACCEPTOR, ALT_DONOR, coding, INTERNAL, intronic best transcript NM_002473 TC1300008114.hg.1 2.34 0.0065 CUL4A Memczak2013 ALT_ACCEPTOR, ALT_DONOR, coding, INTERNAL, intronic best transcript NM_001008895 TC2000007581.hg.1 2.34 0.0011 SULF2 Memczak2013 ANTISENSE, coding, INTERNAL, intronic best transcript NM_001161841 TC2000008516.hg.1 2.27 0.0086 SNX5 Zhang2013 ALT_ACCEPTOR, ALT_DONOR, coding, INTERNAL, intronic best transcript NM_152227 TC0700012906.hg.1 2.23 0.0077 ARHGEF35 Rho guanine nucleotide exchange factor 35 TSUnmapped00000513.hg.1 2.22 0.0026 DGKD Diacylglycerol kinase TC0600007423.hg.1 2.19 0.0026 GPX5 Glutathione peroxidase 5 TC0800012005.hg.1 2.17 0.0053 PTK2 Transcript Identified by AceView, Gene ID(s) 5747 TC0400011706.hg.1 2.16 0.0112 FABP2 Fatty acid binding protein 2, intestinal TC0400010598.hg.1 2.16 0.0084 GABRA2 Gamma-aminobutyric acid (GABA) A receptor, alpha 2 TC0500007258.hg.1 2.16 0.002 GHR Growth hormone receptor TC1700008350.hg.1 2.11 0.003 NOG Noggin TC0400011052.hg.1 2.11 0.022 CXCL9 Chemokine (C-X-C motif) ligand 9 TC1600009533.hg.1 2.1 0.0092 MYH11 Myosin, heavy chain 11, smooth muscle TC0X00008736.hg.1 2.09 0.0446 FATE1 Fetal and adult testis expressed 1 TC0400008956.hg.1 2.08 0.0025 ARHGAP10 Transcript Identified by AceView, Entrez Gene ID(s) 79658 TC0900011820.hg.1 2.07 0.0029 TSC1 Transcript Identified by AceView, Entrez Gene ID(s) 7248 TC1600011345.hg.1 2.07 0.0026 DNASE1 Deoxyribonuclease I TC0200009700.hg.1 2.06 0.0258 GALNT13 Polypeptide N-acetylgalactosaminyltransferase 13 TC1200010719.hg.1 2.05 0.0162 ACVRL1 Memczak2013 ANTISENSE, CDS, coding, INTERNAL, intronic best transcript NM_001077401 TSUnmapped00000296.hg.1 2.04 0.0128 HYOU1 Hypoxia up-regulated 1 TC0X00008688.hg.1 2.01 0.0198 MAGEA9B ; MAGEA9 MAGE family member A9B; MAGE family member A9 TC1900011153.hg.1 –2.02 0.0124 RRAS Related RAS viral (r-ras) oncogene homolog TC0200008469.hg.1 –2.03 0.005 FAHD2A Transcript Identified by AceView, Entrez Gene ID(s) 51011 TC2200009013.hg.1 –2.04 0.0027 CDPF1 Cysteine rich, DPF motif domain containing 1 TC1900006603.hg.1 –2.05 0.0025 ZNF556 Zinc finger protein 556 TC0100015862.hg.1 –2.06 0.0046 S100A7 S100 calcium binding protein A7 TC0100012343.hg.1 –2.07 0.0215 OR2L2 Olfactory receptor, family 2, subfamily L, member 2 TC1700008602.hg.1 –2.07 0.0041 DDX5 Memczak2013 ANTISENSE, CDS, coding, INTERNAL best transcript NM_004396 TC1100012488.hg.1 –2.07 0.0146 IFT46 Intraflagellar transport 46 TC1300009514.hg.1 –2.08 0.0072 DZIP1 DAZ interacting zinc finger protein 1 TC0600007266.hg.1 –2.09 0.0031 HIST1H1C Memczak2013 ANTISENSE, CDS, coding, INTERNAL best transcript NM_005319 TC2000008121.hg.1 –2.09 0.0137 C20orf96 Chromosome 20 open reading frame 96 TC0200011670.hg.1 –2.1 0.0027 KIDINS220 Transcript Identified by AceView, Entrez Gene ID(s) 57498; 100216337 TC1200012639.hg.1 –2.11 0.002 HOXC6; HOXC4; HOXC5 Homeobox C6; homeobox C4; homeobox C5 TC0300008945.hg.1 –2.18 0.019 MRAS Muscle RAS oncogene homolog TC1500009056.hg.1 –2.25 0.0015 RASGRP1 RAS guanyl releasing protein 1 TC0500008785.hg.1 –2.26 0.0279 EGR1 Early growth response 1 TC0600007265.hg.1 –2.26 0.0015 HIST1H3C Histone cluster 1, H3c Table I. Continued

682 Lu et al : Gadoxetate Disodium Increased OATP1B1 Levels

Table I. Continued

ID Fold p-Value Gene GO Biological Process Term change symbol

TC0X00007686.hg.1 –2.28 0.0029 PBDC1 Polysaccharide biosynthesis domain containing 1 TC1600007959.hg.1 –2.29 0.0332 MT1E Metallothionein 1E TC0X00010052.hg.1 –2.35 0.0012 NAP1L2 Transcript Identified by AceView, Entrez Gene ID(s) 4674 TC1500006920.hg.1 –2.35 0.0011 C15orf54 Chromosome 15 open reading frame 54 TC0500013224.hg.1 –2.38 0.0019 SLC27A6 Solute carrier family 27 (fatty acid transporter), member 6 TC0200011013.hg.1 –2.39 0.001 FBXO36 F-box protein 36 TC1200012665.hg.1 –2.43 0.0159 THAP2 THAP domain containing, apoptosis associated protein 2 TC0300007296.hg.1 –2.45 0.0114 SCAP Memczak2013 ANTISENSE, CDS, coding, INTERNAL best transcript NM_012235 TC1200008877.hg.1 –2.69 0.0007 SH2B3 Transcript Identified by AceView, Entrez Gene ID(s) 10019 TC1400007706.hg.1 –2.73 0.0386 FOS FBJ murine osteosarcoma viral oncogene homolog

5A). In cDNA microarray analysis, we also found that 4 Schwope RB, May LA, Reiter MJ, Lisanti CJ and Margolis DJ: gadoxetate disodium regulated the anion transmembrane Gadoxetic acid: Pearls and pitfalls. Abdom Imaging 40(6) : 2012- transport SLCO1C1 mRNA expression (Table I). We also 2029, 2015. 5 Reis MA and Baroni RH: Liver-specific magnetic resonance validated the microarray results by qPCR analysis of SLCO1C1 contrast medium in the evaluation of chronic liver disease. gene expression (Figure 5B). The SLCO1C1 gene encodes a Einstein (Sao Paulo) 13(2) : 326-329, 2015. member of the organic anion transporter family. The encoded 6 Motosugi U, Bannas P, Sano K and Reeder SB: Hepatobiliary proteins include OATP1, OATPF, OATP-F, OATP14, mr contrast agents in hypovascular hepatocellular carcinoma. J OATP1C1, OATPRP5, and LC21A14, which are Magn Reson Imaging 41(2) : 251-265, 2015. transmembrane receptors that mediate the sodium-independent 7 Miyamoto S, Oshita A, Daimaru Y, Sasaki M, Ohdan H and uptake of thyroid hormones (30, 40). Our study is the first to Nakamitsu A: Hepatic sclerosed hemangioma: A case report and review of the literature. BMC Surg 15 : 45, 2015. demonstrate that gadoxetate disodium up-regulated SLCO1C1 8 Kakite S, Dyvorne H, Besa C, Cooper N, Facciuto M, mRNA expression in human normal Chang Liver cells by Donnerhack C and Taouli B: Hepatocellular carcinoma: Short- microarray (Table I) and qPCR analysis (Figure 5B). term reproducibility of apparent diffusion coefficient and In conclusion, the present study showed that OATP1B1 is intravoxel incoherent motion parameters at 3.0t. J Magn Reson an important mediator for gadoxetate disodium uptake in Imaging 41(1) : 149-156, 2015. human normal Chang Liver cells. 9 Kitao A, Matsui O, Yoneda N, Kozaka K, Kobayashi S, Koda W, Minami T, Inoue D, Yoshida K, Yamashita T, Yamashita T, Acknowledgements Kaneko S, Takamura H, Ohta T, Ikeda H, Sato Y, Nakanuma Y, Harada K, Kita R and Gabata T: Gadoxetic acid-enhanced magnetic resonance imaging reflects co-activation of beta- This work was partly supported by China Medical University catenin and hepatocyte nuclear factor 4alpha in hepatocellular Hospital, Taichung, Taiwan (grant no. DMR-107-123) and by the carcinoma. Hepatol Res 60(5) : 1674-1685, 2017. Ministry of Science and Technology, Taiwan (MOST grant no. 105- 10 Yoneda N, Matsui O, Kitao A, Kozaka K, Kobayashi S, Sasaki 2320-B-039-033-). M, Yoshida K, Inoue D, Minami T and Gabata T: Benign hepatocellular nodules: Hepatobiliary phase of gadoxetic acid- References enhanced mr imaging based on molecular background. Radiographics 36(7) : 2010-2027, 2016. 1 Yoon JH, Lee JM, Kim E, Okuaki T and Han JK: Quantitative liver 11 Yoneda N, Matsui O, Ikeno H, Inoue D, Yoshida K, Kitao A, function analysis: Volumetric t1 mapping with fast multisection b1 Kozaka K, Kobayashi S, Gabata T, Ikeda H, Nakamura K and inhomogeneity correction in hepatocyte-specific contrast-enhanced Ohta T: Correlation between gd-eob-dtpa-enhanced mr imaging liver mr imaging. Radiology 282(2) : 408-417, 2017. findings and oatp1b3 expression in chemotherapy-associated 2 Scali EP, Walshe T, Tiwari HA, Harris AC and Chang SD: A sinusoidal obstruction syndrome. Abdom Imaging 40(8) : 3099- pictorial review of hepatobiliary magnetic resonance imaging 3103, 2015. with hepatocyte-specific contrast agents: Uses, findings, and 12 Ueno A, Masugi Y, Yamazaki K, Komuta M, Effendi K, Tanami pitfalls of gadoxetate disodium and gadobenate dimeglumine. Y, Tsujikawa H, Tanimoto A, Okuda S, Itano O, Kitagawa Y, Can Assoc Radiol J 68(3) : 293-307, 2017. Kuribayashi S and Sakamoto M: Oatp1b3 expression is strongly 3 Unal E, Akata D and Karcaaltincaba M: Liver function associated with wnt/beta-catenin signalling and represents the assessment by magnetic resonance imaging. Semin Ultrasound transporter of gadoxetic acid in hepatocellular carcinoma. J CT MR 37(6) : 549-560, 2016. Hepatol 61(5) : 1080-1087, 2014.

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