Chemicals
Cyclosporin A, chlorpromazine, valproic acid, amiodarone, paraquat, and isoniazid were purchased from Sigma (Zwijndrecht, the Netherlands). Williams E medium supplemented with Glutamax, penicillin/ streptomycin (pen/strep), D-Glucose, PBS were obtained from Invitrogen (Bleiswijk, the Netherlands).
Preparation and culture of liver slices
23-weeks old male C57BL/6 mice were obtained from Harlan (Horst, The Netherlands). Animals were kept for 1 week at a housing temperature of 22°C and at a relative humidity of 30–70%. The lighting cycle was 12-h light and 12-h dark. At the age of 24 weeks animals were sacrificed by an overdose of isoflurane. The treatment protocol was approved by the Ethical Committee for Animal Experiments at Wageningen University.
Immediately after the animals were killed the liver was perfused with PBS and placed in ice-cold Krebs–Henseleit buffer (KHB) (pH 7.4, supplemented with 11 mM glucose). Liver tissue was transported to the laboratory within approximately 30 min and cylindrical liver cores were produced using a surgical biopsy punch with diameter of 5 mm (KAI, SynErgo Europe, Romania). Liver cores were placed in a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL) filled with ice-cold KHB aerated with carbogen and supplemented with 11 mM glucose. Slices with a diameter of 5 mm, a thickness of 0.2 mm and a weight of approximately 6 mg were prepared. Immediately after preparation, slices were transferred into culture plates filled with pre-warmed (37°C) Williams E medium (WEM) supplemented with pen/strep. Three liver slices were pre-cultured in one well of the 6-well plate filled with 4 ml of WEM for one hour with continuous shaking (70 rpm). Incubations were performed in an oxygen controlled incubator (Galaxy 48 R New Brunswick, Nijmegen, the Netherlands) at 80% of oxygen; 5% CO2 and the remaining gas volume was filled up to 95% with N2. After one hour of pre-incubation, media were removed and replaced with fresh media containing test compounds or appropriate solvents. After incubations, samples were snap-frozen in liquid nitrogen and stored in −80°C for further analysis. Samples dedicated to histology were stored in 4% formaldehyde at room temperature.
Cytotoxicity analysis (dose selection)
PCLS were exposed to different model compounds inducing cholestasis, steatosis, necrosis, and to compounds that are not hepatotoxic in vivo. These compounds were selected based on literature search. As cholestatic model compounds cyclosporin A (CsA), and chlorpromazine (CPZ) were selected. For steatosis valproic acid [15] and amiodarone [16] were selected. As pro-necrotic drugs paraquat [17] and isoniazid [18] were used. To select a non-toxic dose eventually to be used for the gene expression profiling experiments, the following concentrations ranges were tested: cyclosporin A 0–100 μM, chlorpromazine 0–80 μM, valproic acid 0–500 μM, amiodarone 0–100 μM, paraquat 0–10 μM,and isoniazid 0–1000 μM,. CyclosporinA, chlorpromazine, amiodarone, paraquat were dissolved in DMSO, valproic acid was dissolved in ethanol (EtOH), and isoniazid was dissolved in PBS. The compounds were added to the culture medium at a final concentration of 0.1% vol/vol in an appropriate solvent (DMSO, EtOH, or PBS). Slices incubated with the solvents at 0.1% vol/vol served as controls. Toxicity was assessed with standard biochemical assays indicative for slices viability: LDH, ATP, and protein assay. Dose selection experiments were performed in slices obtained from at least two mice (see also Additional file 1: Figure S1, Additional file 2: Figure S2 and Additional file 3: Figure S3).
ATP and protein measurements
For each ATP and protein measurement a total of three co-cultured slices were placed in 400 μL Cell Lytic MT buffer (Sigma, Zwijndrecht, the Netherlands). In order to compare ATP and protein content in liver slices from different experiments, both ATP and protein were normalized on mass of liver slices. In order to determine the weight of the liver slices, three liver slices were removed from the culture well. Thereafter, the three liver slices were gently held in tweezers against a paper tissue for 2 seconds to let the tissue absorb the remaining medium. Next, the three slices were weighed on a regular laboratory balance and the mass was registered for each individual exposure, which was 18 mg ± 25% for three slices. Slices were homogenized (6500 g, 8°C) two times for 15 sec using a tissue homogenizer (Precellys 24 Bertin Technologies, Labmakelaar Benelux B.V. Rotterdam, The Netherlands). To remove cellular debris, the homogenates were centrifuged for 5 min (14000 g, 8°C) and the remaining supernatant was divided into two portions of 200 μL. One portion was stored at −80°C for protein measurements and the second 200 μL portion was mixed with 100 μL of ATP lytic buffer (ATPlite, Perkin Elmer, Oosterhout, The Netherlands) for ATP measurements. ATP was measured according to the manufacturer’s description using a microplate reader (Synergy TM HT Multi Detection Microplate Reader, Biotek Instruments Inc, Abcoude, the Netherlands) with settings for luminescence: 590/635 nm, top measurement, and sensitivity 230. ATP determination was performed in technical duplicates and luminescence values were recalculated into μM ATP in total liver slices extracts. ATP concentration was normalized on mass of liver slices used for one ATP extraction.
The protein concentration was determined according to the Bradford method (Protein assay, BioRad, Veenendaal, The Netherlands). Protein samples of 2 μL were diluted 80 times in PBS and measured according to the manufacturer’s instructions. BSA was used as a standard and each measurement was performed in duplicate. The protein content was normalized on mass of liver slices used for protein extraction.
Lactate dehydrogenase (LDH) assay
Lactate dehydrogenase activity in total tissue homogenate and in the culture medium of each slice was performed according to the manufacturer’s protocol (Cytotoxicity Detection Kit (LDH), Roche Diagnostics, Almere, the Netherlands). For the total LDH activity in slices, three slices were homogenized (6500 g, 8°C) twice for 15 sec using a tissue homogenizer (Precellys 24). Thereafter, culture medium or the slice homogenate were diluted 5 fold with PBS and mixed with 100 μL reaction mixture from the kit. After 15 minutes of incubation at room temperature, samples were measured at 490 nm using a Synergy TM HT Multi Detection microplate reader.
Triglycerides (TG) measurements
Three liver slices cultured together were used to make a homogenate in 300 μL buffer containing 10 mM Tris, 1 mM EDTA (pH 7.4) and 250 mM sucrose. Aliquots of 10 μL of the liver slices homogenates were used in the assay and measurements were performed according to the manufacturer’s protocol (triglycerides liquicolor mono, Instruchemie, Delfzijl, the Netherlands).
Bile acids measurements
For bile acids measurements, three slices were pooled and placed in 400 μL Cell Lytic MT buffer (Sigma, Zwijndrecht, the Netherlands). Slices were homogenized for two times 15 sec (6500 g, 8°C) using a tissue homogenizer (Precellys 24). Thereafter, the homogenates were centrifuged for 5 min (14000 g, 8°C) to remove cellular debris, and the supernatant was used for measurements. Bile acids quantification was performed according to the description in manufacturer’s protocol (Total Bile Acid Assay Kit Bioquant, Gentaur, Brussels, Belgium).
Histology
Slices were exposed for 24 and 48 hours to 40 μM CsA or 20 uM CPZ. For the 48 hours cultures, after 24 hours the culture medium was replaced with fresh medium. Immediately after incubation, slices were fixed in 4% buffered formaldehyde. After embedding in paraffin, cross-sections were prepared, stained with haematoxylin and eosin (HE) according to Mayer using standard procedures, and examined under a microscope with a 100 x magnification [19]. Fouchet staining for bile acids pigments and Periodic acid-Schiff (PAS) for glycogen were performed according to standard protocols (http://www.ihcworld.com/histology.htm). For each condition slices obtained from 5 mice were analysed. All pictures were taken using 100-fold magnification.
PCLS exposure (gene expression profiling)
To obtain RNA for transcriptome analysis, PCLS were cultured at the same conditions as described above. Slices were exposed for 24 hours to one concentration of the tested compounds or controls. Concentrations used in the exposure experiments were pre-selected in the dose selection experiments (cytotoxicity analysis). The highest concentration that did not cause significant toxicity assessed by biochemical assays (LDH, ATP, and protein assay) was selected. The concentrations used in the exposure experiments were as follows; for the cholestatic exposures 40 μM CsA and 20 μM CPZ. For the steatogenic exposures: 200 μM valproic acid, and 50 μM amiodarone. For the necrotic compounds: 5 μM paraquat and 1000 μM isoniazid. Liver slices obtained from five mice were used in five separate experiments in which exposures to toxic compound or vehicle control were performed at the same time. Since dose selection experiments and exposure experiments were performed at different days, ATP, LDH, and protein assays were performed again for the exposure experiments to confirm that the selected doses did also not affect slice viability in these experiments (Figure 1).
DNA microarray hybridizations
Gene expression analysis in liver slices incubated for 24 hours with the cholestatic, steatogenic, and necrotic compounds as well as the controls was performed on the HT Mouse Genome 430 PM array plate(s) using the Affymetrix GeneTitan system. RNA was extracted from three slices cultured and exposed together using the RNeasy Tissue Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions. RNA concentration and purity were assessed spectrometrically using a Nano Drop ND-1000 spectrophotometer (Isogen IJsselstein, The Netherlands) by measuring absorption ratios at 260/280 and 260/230 nm. The integrity of the RNA samples was examined using the Shimadzu MultiNA Bioanalyzer (Tokyo, Japan). Biotin- labelled cRNA was generated from high-quality total RNA with the Affymetrix 3’IVT Express Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s specifications with an input of 100 ng total RNA. The Agilent Bioanalyzer (Amstelveen, the Netherlands) and Shimadzu MultiNA Bioanalyzer (Tokyo, Japan) were used to evaluate the quality of cRNA in order to confirm if the average fragment size was according to Affymetrix’ specifications. Per sample, 7.5ug cRNA of the obtained biotinylated cRNA samples was fragmented and hybridized in a final concentration of 0.0375 ug /ul on the Affymetrix HT Mouse genome 430 PM array (Affymetrix, Santa Clara, CA, USA. After an automated process of washing and staining by the GeneTitan machine (Affymetrix, Santa Clara, CA, USA) using the Affymetrix HWS kit for Gene Titan, absolute values of expression were calculated from the scanned array using the Affymetrix Command Console v 3.2 software. The data Quality Control was performed in the program Affymetix Expression Console v 1.1 software to determine if all parameters were within quality specifications. The Probe Logarithmic Intensity Error Estimation (PLIER) algorithm method was used for probe summarisation [20].
In order to monitor the sample independent control and the performance of each individual sample during hybridization, hybridizations controls were added to the hybridization mixture. The sample dependent controls such as internal control genes, background values, and average signals were used to determine the biological variation between samples. In conclusion, all the data were within data Quality Control thresholds, according to Affymetrix Expression Console specifications. Non-normalized data in a form of the Cell Intensity File (*.CEL) were re-annotated (EntrezGene htmg430pm_Mm_ENTREZG) and the data were RMA normalized [20, 21].
Gene Set Enrichment Analysis (GSEA)
In order to identify differentially expressed gene sets related to diverse biological functions Gene Set Enrichment Analysis (GSEA) was applied. GSEA was performed using an open access bioinformatics tool (http://www.broadinstitute.org/gsea/index.jsp). In short, this method identifies biologically and functionally related genes affected due to experimental conditions. GSEA applies predefined gene sets that are based on literature or other experiments. Gene sets specify a group of genes specific for a certain biological process, GO ontology, pathway, or user defined groups. GSEA ranks all the genes on their expression ratios between a treatment and the control group and determines whether a particular gene set is significantly enriched at the top or the bottom of the ranked list [22]. Gene sets used in this study were created in an open access bioinformatics tool ANNI http://www.biosemantics.org/index.php?page=ANNI-2-0[23]. ANNI retrieves all the information available on known gene-gene associations present in Medline and can be used, among others, to create gene sets associated with simple queries, for example “inflammation” or “cholestasis”. For the purpose of this study we used several queries related to liver specific and liver non-specific processes. Summary of all the queries used for the creation of “the ANNI gene sets” is given in Additional file 4: Table S1. Genes present in at least five publications indicating an association with the specified queries were included in the ANNI gene sets.
For GSEA, also Gene Expression Omnibus microarray data relevant for human cholestasis were used (GSE46960). These gene expression data were generated in GeneChip® Human Gene 1.0 ST array (Affymetrix, CA) hybridisation experiments using human liver biopsies obtained from 64 infants with biliary atresia, 14 age-matched infants with cholestasis of other origin than biliary atresia, and from 7 deceased-donor healthy children.
MetaCore pathway analysis
The GSEA report output file informs which gene sets are significantly affected in the analysed groups. Additionally, it informs, which genes significantly contribute to this enrichment (significant genes). These significant genes were used as an input for the MetaCore pathway analysis. The pathway analysis was performed using Functional Ontology Enrichment/ Pathways Maps default option in MetaCore for mouse or human depending whether mouse or human data were analysed, respectively. MetaCore identifies pathways using a default enrichment analysis. The identified pathways were considered as significantly affected by the treatment if p <0.005.
Biomarker identification
In order to identify biomarkers indicative for the development of cholestasis, significant genes identified in GSEA were subjected to Venn analysis. Genes overlapping between CsA and CPZ treatments with absolute FC ≥1.5 were selected as potential biomarkers. Venn analysis was performed using an open access tool http://bioinfogp.cnb.csic.es/tools/venny/. Subsequently, biomarkers’ expression values (derived from the DNA microarray data) were compared with expression values for the same genes in slices treated with non-cholestatic drugs. In order to do so, the gene expression values were log2 transformed and median centered followed by hierarchical clustering using default options in Genesis (http://genome.tugraz.at/genesisserver/genesisserver_description.shtml). Additionally, the candidate biomarkers were uploaded to another open access bioinformatics tool denoted with Search Tool for the Retrieval of Interacting Genes/Proteins 8.2 (STRING) in order to perform functional clustering. STRING identifies and visualizes functional networks based on known and predicted protein-protein interactions [24]. Functional clustering of genes was performed in STRING using functional clustering function.
q-PCR
In further studies, expression of ten candidate biomarkers in PCLS was analyzed by q-PCR using the Biorad CFX96 TM Real-Time Detection System (Bio-Rad, Veenendaal, The Netherlands) with the following cycling conditions: 15 min 95°C followed by 40 cycles of 15 s 95°C and 1 min 60°C. Reactions were performed in 10 μl and contained 20 ng cDNA, 1x TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA), and depending on the analysed gene, 250 nM probe and 900 nM primer (for actin b) or 1xTaqMan gene expression assay (for the remaining genes) (Applied Biosystems, foster City, CA). Specific primer set for actinβ (ACTB) was developed with Primer Express 1.5 (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands) and sequences were as follows, probe: TGT CCC TGT ATG CCT CTG GTC GTA CCA C, forward primer: AGC CAT GTA CGT AGC CAT CCA, and reverse primer: TCT CCG GAG TCC ATC ACA ATG. Primers for Kruppel-like factor 15 (Klf15), bile acid-CoenzymeA:amino acid N-acyltransferase (Baat), ATP-binding cassette, sub-family G (WHITE), member 8 (Abcg8), ATP-binding cassette, and sub-family G (WHITE), member 5 (Abcg5), were purchased from Applied Biosystems. The numbers of assays from Applied Biosystems are given in Additional file 5: Table S2. Data were analyzed with SDS 2.0 software (Bio-Rad). For each sample, the RT-PCR reaction was performed in triplicate and the averages of the obtained threshold cycle values (CT) were processed for further calculations. To check for contaminating DNA, samples without reverse transcriptase (−RT reaction) were analysed as well. For normalization ACTB was used. Relative expression was calculated with ∆ (∆ (CT))-method [25].
Statistical analysis
Mann–Whitney test was used to calculate differences between controls and slices cultured with the tested compounds for ATP, protein, LDH, TG, and bile acids. The cut off for statistical significance was set at a p-value < 0.05.
Mann–Whitney (MW) test was used to determine if there was a significant difference in gene expression assessed by q-PCR in slices exposed to different compounds. A p-value <0.05 was considered significant.