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 genes and suppressed that of 27. The SLCO1C1 (solute relative gene 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) protein 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 proteins (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.