Research ArticleClinical Studies

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, WEN-KANG CHEN, JO-HUA CHIANG, YUH-FENG TSAI, YU-NING JUAN, PING-CHIN LIN, YEU-SHENG TYAN and JAI-SING YANG
In Vivo May 2018, 32 (3) 677-684;

Abstract

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

Gadoxetate disodium (C23H28GdN3Na2O11; synonymous with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid, disodium salt, Gd-EOB-DTPA) is a liver-specific paramagnetic contrast agent for magnetic resonance imaging (MRI) (1-4) (Figure 1). Gadoxetate disodium can detect the focal liver lesions and provide structural and functional responses in hepatobiliary system (4, 5). For example, furthermost hepatocellular carcinoma (HCC) expresses hypo-intensity compared to background liver cells in hepatobiliary-phase (delayed phases) images on enhanced MRI (6-8). Many studies have investigated the transporters of gadoxetate disodium in hepatocellular cells and showed that organic anion transporting polypeptide 1 (OATP1) (one of the hepatocyte transporters) is a major target protein (9-13). On the other hand, excretion of 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 human normal Chang Liver cells, including the effects on the levels of OATP1B1 protein and of the OATP uptake transporter gene SLCO1C1 mRNA.

Materials and Methods

Reagents and chemicals. Gadoxetate disodium (Primovist) was obtained from Bayer Healthcare (Berlin, Germany). Minimum Essential Medium (MEM), penicillin/streptomycin, L-glutamine and trypsin-EDTA were purchased from BioConcept (Allschwil/BL, Switzerland). Fetal bovine serum (FBS) was obtained from HyClone Laboratories, GE Healthcare Life Sciences (South Logan, UT, USA). The primary antibodies against OATP1B1 and β-actin, as well as the mouse IgG antibody (HRP) secondary antibody were bought from GeneTex (Hsinchu, Taiwan). All chemicals and reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA) unless otherwise specified.

Cell culture. The Chang Liver cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in MEM supplemented with 10% FBS, 2 mM L-glutamine and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in 5% CO2. The cells were detached by 0.25% trypsin/0.02% EDTA and split every 2 to 3 days to maintain cell growth.

Cell viability assay. Cell viability was examined by the MTT method, as described previously with some modifications (18, 19). In brief, cells (2×104 cells/well) were dispensed in 96-well plates and treated with different concentrations (0, 5 and 10 mM) of gadoxetate disodium for 4 h and 24 h. Per well, methyl thiazolyl tetrazolium (MTT) (10 μl, 5 mg/ml) was added for an additional 1.5 h-incubation. The optical density was measured under 570 nm with a spectrophotometer after the violet formazan crystal produced from MTT was solubilized by 200 μl dimethyl sulfoxide (DMSO). Cell viability was further calculated, and a percentage of alive versus dead cells was obtained.

Morphological determination. Cells (2×105 cells/well) were seeded into 12-well plates and exposed to 5 and 10 mM of gadoxetate disodium for 4 h and 24 h, respectively. The cells were photographed under a phase-contrast microscope to verify apoptotic features before collection, as described elsewhere (20).

4’,6-diamidino-2-phenylindole (DAPI) for nucleic acid staining. Cells (2×105 cells/well) in 12-well plates were individually incubated with or without 5 and 10 mM of gadoxetate disodium for 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 for 15 min. Cells were then stained with 200 μl DAPI solution (1 μg/ml) for 30 min at 37°C in the dark. The integrity of nuclei was visualized under a fluorescent microscope (Nikon Inc., Tokyo, Japan), as previously described (20).

Figure 1.
Figure 1.

The chemical structure of gadoxetate disodium (C23H28N3O11.Gd.2Na).

Western blot analysis. Cells prior to gadoxetate disodium challenge (5 and 10 mM) for 4 h were scraped on the Trident RIPA lysis buffer (GeneTex, Hsinchu, Taiwan) and centrifuged at 1200 × g for 5 min at 4°C. An aliquot of pelleted cells was used for protein quantification as previously described (21, 22), and equal amounts of proteins (40 μg) were separated on 10% acrylamide gels by SDS-electrophoresis and then transferred to the Immobilon-P Transfer Membrane (Merck Millipore, Billerica, MA, USA). After blocking unspecific binding sites with 5% dry milk in PBST, the membranes were incubated with primary antibodies, diluted 1:1,000 in PBST-3% BSA overnight at 4°C, and this was followed by incubation for 2 h at room temperature with the appropriate HRP-secondary antibody diluted 1:10,000 in PBST-3% BSA. All bands were normalized against β-actin, and band intensities were quantified by ImageJ 1.47 program for Windows from NIH.

RNA extraction. Cells, after 5 and 10 mM of gadoxetate disodium exposure for 2 h, were scraped and collected by centrifugation, and total RNA was subsequently isolated by an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). RNA quantity and purity were assessed at 260 nm and 280 nm using a Nanodrop (ND-1000; Labtech International, Sussex, UK). Each sample (100 ng) was amplified and labeled using the GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) for expression analysis according to the manufacturer's instructions.

cDNA microarray analysis. Hybridization was performed against the Affymetrix Human Clariom S Array (Thermo Fisher Scientific, Carlsbad, CA, USA). The arrays were hybridized for 17 h at 45°C and 60 rpm. Arrays were subsequently washed (Affymetrix Fluidics Station 450, Thermo Fisher Scientific, Carlsbad, CA, USA) and stained with streptavidin-phycoerythrin (GeneChip Hybridization, Wash, and Stain Kit, Thermo Fisher Scientific, Carlsbad, CA, USA). The chip was scanned on an Affymetrix GeneChip Scanner 3000 (Thermo Fisher Scientific, Carlsbad, CA, USA) as previously described (23). The resulting data were analyzed using Expression Console software (Affymetrix Fluidics Station 450, Thermo Fisher Scientific, Carlsbad, CA, USA) with default RMA parameters. The genes regulated by gadoxetate disodium were considered to have a significant difference with a 2.0-fold change.

Figure 2.
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.

Gene ontology and ingenuity pathway analysis. For detection of significantly over-represented Gene Ontology (GO) biological processes, the DAVID functional annotation clustering tool (http://david.abcc.ncifcrf.gov) was used (DAVID Bioinformatics Resources 6.7, Frederick, MD, USA). The significant list containing the gadoxetate disodium and untreated control, complete with Affymetrix transcript identifiers, was uploaded from a Microsoft Excel spreadsheet onto the Ingenuity Pathway Analysis (IPA) software (QIAGEN) (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/). IPA recognized the Affymetrix identifiers and mapped the gadoxetate disodium to the IPA data analysis suite, generating maps to describe common pathways or molecular connections between gadoxetate disodium and untreated control on the list. Graphical representations of the molecular relationships between genes were generated using the IPA pathway analysis based on processes showing significant (p<0.05) association. To identify the potential function of the hub gene, GSEA (http://software.broadinstitute.org/gsea/index.jsp) was conducted to detect whether a series of defined biological processes were enriched in the gene rank.

Quantitative polymerase chain reaction (qPCR). Cells, after gadoxetate disodium (5 and 10 mM) exposure for 2 h, were collected, and total RNA was subsequently isolated as above described. The cDNA synthesis was performed using a High Capacity cDNA Reverse Transcription Kits (Applied Biosystems/Thermo Fisher Scientific, Carlsbad, CA, USA). QPCR control with a 2X SYBR Green PCR Master Mix (Applied Biosystems) were amplified for SLCO1C1 gene under the same PCR parameters to normalize the GAPDH quantitative data. PCR primers were as follows: Human GAPDH-Forward (F)-ACACCCACTCCTCCACCTTT, and human GAPDH-Reverse (R)-TAGCCAAATTCGTTGTCATACC; Human SLCO1C1-F-TGATGTGGCAGGACTAAC, and human SLCO1C1-R-GACAACCAGCAAGACAAG. QPCR was performed in triplicate in a Applied Biosystems 7300 Real-Time PCR System as previously reported (24).

Statistical analysis. The results were acquired from three independent experiments, and the means±standard deviation (SD) were reported. Data significance was evaluated by one-way ANOVA followed by the Duncan's multiple range test if ANOVA was significant (p<0.05).

Results

Effect of gadoxetate disodium on cell viability, morphologic changes and DNA condensation of human normal Chang Liver cells. The cytotoxic effect on Chang Liver cells cultured in the presence of different concentrations of gadoxetate disodium was recorded. Our data demonstrated that gadoxetate disodium at 5 and 10 mM at 4 h and 24 h-duration did not reduce the cell viability of Chang Liver cells (Figure 2). No morphological change was observed between cells treated with gadoxetate disodium for 4 and 24 h and untreated control (Figure 3). Furthermore, no DNA condensation (an apoptotic characteristic) occurred in the untreated control and gadoxetate disodium-treated cells (Figure 4). Therefore, gadoxetate disodium exerts a non-cytotoxic effect on Chang Liver cells.

Effect of gadoxetate disodium on influx transporter OATP1B1 protein level and SLCO1C1 mRNA of Chang Liver cells. The levels of OATP1B1 protein and SLCO1C1 gene have been found to be mainly expressed on the sinusoidal membrane of human hepatocytes (25-27). To address how gadoxetate disodium interacts with Chang Liver cells, we determined experimentally the level of OATP1B1 by western blotting and qPCR methods. Our data indicated that gadoxetate disodium increased OATP1B1 protein expression in a concentration-dependent manner (Figure 5A). Furthermore, qPCR analysis showed that gadoxetate disodium significantly up-regulated the organic anion transporter polypeptide (OATP) uptake transporter gene SLCO1C1 mRNA expression in treated cells, and this effect was concentration-dependent (Figure 5B). We suggest that gadoxetate disodium can be rapidly pumped-out extra-cellularly through modulating OATP1B1 and up-regulating SLCO1C1 mRNA in Chang Liver cells.

Figure 3.
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.

Effect of gadoxetate disodium on gene expression of Chang Liver cells by cDNA microarray analysis. The microarray analysis demonstrated that gadoxetate disodium up-regulated 29 genes and down-regulated 27 genes in Chang Liver cells after a 2 h exposure (Table I). Most of the differentially expressed genes were associated with transmembrane transport (SLCO1C1 and IFT46); receptors (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) in cells following gadoxetate disodium treatment. Among them, gadoxetate disodium caused a 2.62-fold increase in the expression of OATP uptake transporter gene SLCO1C1 (p-value=0.0006). Hence, we suggest that the OATP uptake transporter gene SLCO1C1 might exert a protective effect and play a vital role in normal liver cells prior to gadoxetate disodium exposure.

Discussion

Gadolinium-based contrast agents (GBCA) are widely used for enhanced magnetic resonance imaging (MRI) examinations (1, 4). Gadoxetate disodium was developed in order to reduce side effects, and it is associated with other liver-specific paramagnetic contrast agents on MRIs (3, 4). However, there is no report addressing the effects of gadoxetate disodium on cytotoxicity and its associated gene expression profile in human normal Chang Liver cells. This study is the first to demonstrate that gadoxetate disodium does not induce any cytotoxic effects (Figures 2 and 3), DNA condensation or fragmentation (an apoptotic characteristic) (Figure 4). It did however alter the mRNA expression profile of the transmembrane transport-associated genes on Chang Liver cells (Table I).

Based on the changes in the gene expression profile in gadoxetate disodium-treated Chang Liver cells as shown by cDNA microarray, we found that the cellular responses to gadoxetate disodium treatment are multi-faceted and likely to be mediated through a variety of signaling pathways. Gadoxetate disodium regulated the mRNA expression of important genes, including those involved in anion transmembrane transport (SLCO1C1 and IFT46); receptors (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.

Figure 4.
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.

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). OATP1B1 is most highly expressed in the human liver and is mainly involved in the uptake of gadoxetate disodium into the hepatocytes in conjunction with OATP8 (synonymous with OATP1B3) (39). OATP1B3 expression is strongly associated with Wnt signaling and represents the transporter of gadoxetate disodium in hepatocellular carcinoma KYN-2 cells (10, 12). Our results demonstrated that gadoxetate disodium increased the protein level of OATP1B1 in Chang Liver cells (Figure 5A). In cDNA microarray analysis, we also found that gadoxetate disodium regulated the anion transmembrane transport SLCO1C1 mRNA expression (Table I). We also validated the microarray results by qPCR analysis of SLCO1C1 gene expression (Figure 5B). The SLCO1C1 gene encodes a member of the organic anion transporter family. The encoded proteins include OATP1, OATPF, OATP-F, OATP14, OATP1C1, OATPRP5, and LC21A14, which are transmembrane receptors that mediate the sodium-independent uptake of thyroid hormones (30, 40). Our study is the first to demonstrate that gadoxetate disodium up-regulated SLCO1C1 mRNA expression in human normal Chang Liver cells by microarray (Table I) and qPCR analysis (Figure 5B).

Figure 5.
Figure 5.

Effects of gadoxetate disodium on influx transporter OATP1B1 protein level and SLCO1C1 mRNA expression in Chang Liver cells. Cells were exposed to 5 and 10 mM of gadoxetate disodium for 4 h. (A) OATP1B1 protein expression was analyzed by western blot. β-Actin served as an internal control to ensure equal loading. The blot is a representative of three independent experiments. (B) cDNA produced from mRNA reverse transcription, was employed to examine SLCO1C1 mRNA expression by qPCR analysis. GADPH, a house-keeping gene, was used as a control. Data are presented as the mean±SD (n=3). The different letters (a-c) show significant differences (p<0.05) by the Duncan's test.

View this table:
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.

In conclusion, the present study showed that OATP1B1 is an important mediator for gadoxetate disodium uptake in human normal Chang Liver cells.

Acknowledgements

This work was partly supported by China Medical University Hospital, Taichung, Taiwan (grant no. DMR-107-123) and by the Ministry of Science and Technology, Taiwan (MOST grant no. 105-2320-B-039-033-).

Footnotes

  • ↵* These Authors contributed equally to this work.

  • This article is freely accessible online.

  • Received February 5, 2018.
  • Revision received March 6, 2018.
  • Accepted March 7, 2018.

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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, WEN-KANG CHEN, JO-HUA CHIANG, YUH-FENG TSAI, YU-NING JUAN, PING-CHIN LIN, YEU-SHENG TYAN, JAI-SING YANG
In Vivo May 2018, 32 (3) 677-684;
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