Animals and treatment

Male Wistar rats were obtained from and kept at the ‘‘Division for Decentralized Biomedical Facilities of the Medical University of Vienna” under standardized SPF-conditions. Phenobarbital (PB, 5-Ethyl-5-phenyl-barbituric acid-sodium salt; Fluka, 04712) [29,30], admixed to drinking water, was administered to three rats which were 8 to 10 weeks old at a daily dose of 50mg/kg bodyweight. Two times a week the PB concentrations in the drinking water were adjusted to the body weights and the amount of water consumed. Five controls and four animals for the in vitro experiments were treated with tap water only. Animals were sacrificed by exsanguination under CO₂-asphyxation during the liver perfusion. The experiments were approved by the ‘‘Committee of Animal Protection of the Austrian Ministry of Sciences” (permission number 66009/157 II10b/2009) and performed according to Austrian regulations in accordance with the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory Animals” by the National Academy of Sciences.

Separation of liver cells and primary cultures

The following procedure was used for the in vitro and the in vivo experiments. To isolate and cultivate hepatocytes (HCs) and non-parenchymal cells (NPCs) at a functional state as naïve as possible, livers of rats were perfused with collagenase (Worthington CLS-2) as described before [31,32]. In the resulting cell suspension HCs were isolated from NPCs by low speed centrifugation followed by discontinuous density gradient centrifugation using Percoll [33]. The purity was found to be an average of 95.4% for hepatocyte fractions and 99.8% for NPC fractions. Cell preparations were used for further experimentation when the viability exceeded 90%, as determined by the trypan blue exclusion assay. HCs and NPCs were subsequently seeded on collagen-coated 6-well plates. HCs were seeded at a density of 4x10⁵cells/well in Williams’ medium E (Invitrogen) supplemented with glutamax, HEPES, gentamycin, H2 mix, ascorbat and 10% FCS. NPCs were seeded at a density of 3-4x10⁶cells/well in RPMI 1640 medium (Gibco Ltd.) supplemented with gentamycin and 10% FCS at 37°C for 2 hours. To determine the purity of the isolated cell fractions, cells were counted using microscope pictures and the ImageJ software (National Institutes of Health). After an attachment period of 2 hours cells were switched to serum-free medium (Williams medium E and RPMI 1640 medium, both supplemented as above without the 10% FCS) and kept at 37°C for further 24 hours in order to collect cell supernatants.

In vitro treatment of primary HCs and NPCs

In addition to the procedure described above, the in vitro treatment of cultures commenced 2h after plating of cells deriving from four untreated rats. Two rats were used for the cytokine treatment, while the other two were used for the PB treatment. HCs were treated with 10ng/ml interleukin-1ß (R&D Systems) and 5ng/ml interleukin-6 (R&D Systems) [34] for 24 hours, which induce the acute phase plasma protein synthesis in HCs [35–38]. 10ng/milliliter lipopolysaccharide (LPS, Sigma-Aldrich) was applied to the medium of NPCs for 24 hours [39–43]. HCs and NPCs were treated with 1mM PB (Fluka) for 24 hours.

Cellular sub-fractionation and protein sample processing (see also 44)

The serum-free supernatants were sterile filtered using a 0.2µm cellulose acetate filter (Whatman). One part of this filtrate was precipitated by adding 5x volume of -20°C tempered p.a. ethanol (Merck) and subsequent storing at -20°C for at least overnight. The other part was directly stored at -80°C for subsequent analyses by ELISA. During all steps samples were kept on ice. For harvesting of cytoplasmic and nucleic protein fraction, cells were gathered in isotonic buffer (10mM HEPES/NaOH pH=7.4, 10mM NaCl, 3.5mM MgCl2, 1mM EGTA, 0.25M Sucrose and 0.5% Triton X-100) and protease inhibitor mix (1mM PMSF; aprotinin, leupeptin and pepstatin, [1µg/ml] each). The cells were disrupted by sheer stress caused by syringing the cell lysates through 23G needles. The cytoplasmic fraction, in the supernatant, was separated from nuclei and membrane proteins as well as debris by centrifugation at 2300xg and 4°C for 5min and was subsequently precipitated by ethanol tempered to -20°C. The nuclei protein fraction was extracted from the remaining residue via a 10min incubation with an extraction buffer (10mM Tris/HCl pH=7.4, 1mM EDTA, 500mM NaCl) which act through osmotic pressure followed by a 1:10 dilution with a NP40 buffer (10mM Tris/HCl pH=7.4, 1mM EDTA, 0.5% NP40), to reduce the final NaCl concentration, for 15min. Nucleic proteins were separated from debris by centrifugation at 2328xg and 4°C for 5min and precipitated by ethanol tempered to -20°C.

After precipitation all fractions were centrifuged at 4700xg and 4°C for 25min. The resulting protein pellets were dissolved in sample buffer (7.5M urea, 1.5M thiourea, 4% CHAPS, 0.05% SDS, 100mM DTT) in a volume according to the protein amount / pellet size.

Shotgun analysis - 1D-SDS PAGE, in-gel tryptic digestion & MS analysis ('bottom up')

For 1D-SDS PAGE we used a 4% stacking and a 12% resolving polyacrylamide gel. Protein samples were loaded onto the gel and the electrophoresis ran until complete separation of a pre-stained molecular marker (Dual Color, Biorad, Hercules, CA) was visible. Gels were fixated with 50% methanol/10% acetic acid and MS compatible silver stained as described by Mortz, E. et al [45]. For tryptic digestion samples were cut into lanes to group proteins with a similar molecular weight, which improved the following LC-MS/MS analyses. These lanes were cubed and the proteins were destained, reduced and alkylated before digestion with trypsin (sequencing grad, Roche) at 37°C over night as described before [46]. After elution the peptide solutions were analysed by LC - MS/MS.

For reversed-phase chromatography we used a nano-flow LC (1100 Series LC system, Agilent) combined with the HPLC chip technology (Agilent). The chips consist of a 40nl Zorbax 300SB-C18 trapping column and a Zorbax 300SB-C18 (75µm x 150mm) separation column. The flow rate was 400nl/min, using a gradient from 0.2% formic acid and 3% ACN to 0.2% formic acid and 50% ACN over 40min or 60min for supernatants or the other two fractions, respectively.

Peptide identification was performed by MS/MS fragmentation analysis using an ion-trap mass spectrometer (XCT-Ultra, Agilent) combined with already described HPLC chips, which are eventually an orthogonal nanospray ion source (Agilent). For peak list-generation and spectrum identification, of the MS/MS data we used Spectrum Mill MS Proteomics Workbench software (Version A.03.03, Agilent). We searched our MS/MS data against the UniProtKB/Swiss-Prot protein database (Version 10^th August 2010; 519,348 entries). The settings were as follows: max. of 2 missed cleavages, minimum scored peak intensity (%SPI) of 70%, precursor mass tolerance of +/-1.5Da, product mass tolerance +/- 0.7. Two types of modification were considered, both arising during sample preparation. Carbamidomethylation of cysteine (deliberately) was set as fixed modification and oxidized methionine (artefact) was set as post-translational modification.

Generating protein maps and their interpretation

The resulting peptide-protein assignment list was reviewed by hand considering the following parameters. Spectrum Mill peptide sequence score values (sequence matching probability) of >13 counted as valid sequence assignments. Peptide sequences were valid, when at least one peptide sequence scored as much and did not occur in other proteins of this cell type. An estimated error rate was calculated by searching the sequences against a non-sense reversed database. Furthermore we included peptides scoring between 9 and 13 only if precursor m/z value, retention time and MS2 pattern were found similarly in at least one of our previous experiments and the peptide was thereby scoring above 13. With respect to protein inference, we chose the smallest number of proteins required to explain all observed peptides as described for ProteinProphet [47]. As our protein identification algorithm includes manual selection, we cannot calculate an exact false discovery rate.

Proteome analysis using emPAI (exponentially modified protein abundance index) values[48], comparisons and PB specific alterations were recorded and interpreted using the GPDE (Griss Proteomics Database Engine), a database specifically engineered for the identification and characterization of marker proteins [49].

ELISA

Arginase-1 protein levels were determined by a kit (Uscn Life Science Inc.; Houston, TX 77036) according to the user manual.

Isolation and proteome profiling of primary rat hepatocytes and non-parenchymal cells

Primary rat cells were obtained from untreated rats and rats treated with PB in vivo. Cell isolation was performed by liver perfusion followed by separation into hepatocytes (HCs) and non-parenchymal cells (NPCs) (see Figure 1). Cells were cultured for 24 hours to allow accumulation of secreted proteins. Subsequently, cells were fractionated into cytoplasm and nuclear extract. The protein fractions were further separated by SDS-PAGE and forwarded to proteome profiling via LC-MS/MS as described.

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Figure 1. Microscope images of HCs and NPCs in culture.

These pictures depict representative areas of untreated, PB in vitro as well as in vivo treated HCs and NPCs in culture extracted from microscopic images of equal magnification.

https://doi.org/10.1371/journal.pone.0076137.g001

We restricted our analyses to proteins which were identified by two peptides or more in two or more independent experiments. By this approach, we identified 1148 proteins in HCs and 1213 proteins in NPCs with an overlap of 966 proteins in both cell compartments. The 182 proteins being apparently specific for HCs indeed comprise a large number of known liver-specific proteins including, apolipoproteins and other serum proteins, cytochrome P450 isoenzymes, sulfotransferases, UDP-glucuronosyltransferases and many others (table 1-5, table S1 & S2). Furthermore, the 247 proteins identified in NPCs included many proteins known to be expressed in stromal cells. Representative for leukocytes are the surface antigens CD37, CD47, CD96 and CD166 as well as the chemokines CXCL1, CXCL2, and CCL6. Amongst known marker proteins for endothelial cells we identified endothelial cell-specific molecule 1, endothelial nitric oxide synthase and septin-2 (vascular endothelial cell specific protein 11), while MMP-3 and various collagens are characteristic for the stellate cells.

In vitro treatment of isolated primary cells

HCs were treated with IL-6 and NPCs with LPS, respectively. Upon treatment for 24 hours, the induction of several pro-inflammatory proteins was observed which confirms the responsiveness of these cells to stress stimuli under our experimental conditions (Figure 2).

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Figure 2. Proteome alterations induced by in vitro treatment of primary cells.

Part A) shows schematic representations of a cell and her three sub-compartments, namely the supernatant, the cytoplasm and the nucleus. The intensity of red represents the degree of amount of the selected protein found in the respective compartment in contrast to the other experiments. The higher intensity of red corresponds to a higher occurrence. This allows an easy comparison of the expression levels of a protein in different experimental setups.

NPCs induce the secretion of IL-1beta and TNF-alpha upon inflammatory stimulation with LPS. In vitro treatment with PB induced coronin-7 and ADP-ribosyl cyclase 1, which both are also induced by in vivo treatment. The expression of Hsp90, a stress response related protein, was increased upon LPS and PB treatment. Prostaglandin, a protein involved in promotion of proliferation in normal and preneoplastic cells, was induced upon LPS and in vivo PB treatment. HCs respond hardly to the in vitro treatment with PB. Treatment with IL-6 specifically induced the acute phase protein T-kininogen-2. UDP-glucuronosyltransferase 2B37 and the chaperone peptidyl-prolyl cis-trans isomerase D were induced by both in vitro stimulation experiments as well as by the in vivo treatment with PB. Carbamoyl-phosphate synthase is part of the urea cycle and has to be found in all four categories.

Proteins in NPC: (1) O35828 Coronin-7, (2) P16599 Tumor necrosis factor, (3) P34058 Heat shock protein HSP 90-beta, (4) Q63264 Interleukin-1 beta, (5) Q63921 Prostaglandin G/H synthase 1, (6) Q64244 ADP-ribosyl cyclase 1.

Proteins in HC: (1) P07756 Carbamoyl-phosphate synthase [ammonia], (2) P08932 T-kininogen 2, (3) P19488 UDP-glucuronosyltransferase 2B37, (4) Q6DGG0 Peptidyl-prolyl cis-trans isomerase D.

Part B) demonstrates the distribution of distinct proteins within the three fractions, supernatant, cytoplasm and nuclear protein fractions, underneath the respective treatment of the cells, which gives an overview of the responsiveness of the cells.

Abbr.: SN –proteome of the supernatant, Cyt – proteome of the cytoplasm, NE – proteome of the nuclear extract.

https://doi.org/10.1371/journal.pone.0076137.g002

To investigate direct drug effects, primary untreated cells were treated with 1mM PB for 24 hours and analysed by proteome profiling. Remarkably, this treatment had hardly any measureable effect on the proteome composition of HC and NPCs. A single protein was apparently induced in HC and two proteins only in NPCs, respectively. Proteins indicating stress such as chaperones including the heat shock protein family, stress-induced phosphoprotein, heme oxygenase 1 and DNAJ homolog subfamily members were not or hardly induced.

In vivo treatment of animals with phenobarbital

13 proteins were found to be newly induced in the cytoplasmic fraction of HCs by PB, including phase I drug metabolizing enzymes such as amine oxidase, phase II enzymes such as UDP-glucuronosyltransferases and glutathione S-transferases, the chaperone rotamase D, glutathione synthetases and the proto-oncogen c-Raf. The induction of estradiol 17-beta-dehydrogenase 8 may indicate alterations in steroid hormone homeostasis. The induction of CYP2B, probably via CAR, is a known positive control for PB action.

Table 1, 2 and 3 depict the most significant proteins found up-regulated in the secretome, cytoplasm or nuclear extract of HCs and NPCs in response to in vivo PB exposure. Proteins found up-regulated in the secretome of HCs are involved in the acute-phase response, inflammation response and the action of drugs. In the cytoplasm of HCs we found 82 proteins up-regulated. These proteins preferentially act in the acute-phase response, cell growth, immune response, inflammatory response as well as in response to cell stress, oxidative stress and wounding. In the nuclear extract of HCs we found 182 proteins up-regulated. These proteins are involved in nucleosome assembly, nucleocytoplasmic transport, mRNA processing, translation, protein localization to the nucleus, RNA processing, removal of superoxide radicals, anti-gen processing and presenting, protein methylation and cell redox homeostasis as well as are major nucleolar proteins of growing eukaryotic cells and proteins of the nuclear matrix.

In NPCs, 12 proteins were found newly induced, comprising proteins related to inflammation such as PAI-1 and S100-A10, cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 as well as altered immune cell functional state such as acetyl-CoA carboxylase 1 and ADP-ribosyl cyclase 1. Proteins found up-regulated in the secretome of NPCs are involved in the acute-phase response, inflammation response, and action of drugs and oxidative species. In the cytoplasm of NPCs we found 108 proteins up-regulated. These proteins exert function in tissue, acute phase, cell redox homeostasis, cell cycle, response to drug, cell adhesion, vesicle-mediated protein transport, stress response, positive regulation of MAPKKK cascade, cellular response to cell-matrix adhesion, response to ROS. In the nuclear extract of NPCs we found 78 proteins up-regulated which act on transcription regulation, mRNA processing, mRNA and protein transport, response to DNA damage stimulus and DNA repair.

Table 4 and 5 depict selected proteins found only in untreated rats, which means a down-regulation of these proteins in HCs and NPCs in response to in vivo PB exposure. This rather stringent selection criterion for down-regulated proteins improves the reliability of these results. In HCs, 6 proteins were found only in untreated rats, including the E3 ubiquitin-protein ligase UBR4, a protein involved in in membrane morphogenesis and cytoskeletal organization as well as the glutamine synthetase, which catalyses the production of glutamine and 4-aminobutanoate (gamma-aminobutyric acid, GABA). In NPCs, 28 proteins were found only in untreated rats, comprising proteins involved in cell adhesion, cell proliferation, liver development and protein transport.

Table S1 and S2 present the summarised proteome profiling results obtained with HCs (S1) and NPCs (S2) derived from PB treated rats (in vivo).

Pathway analysis of the in vivo data via Reactome

The alterations in the proteome were further analysed by Reactome, a public peer-reviewed pathway database from CSHL, OICR and EBI, which is cross-referenced to bioinformatics databases and allows the assignment of a given protein to one or more molecular pathways. When searching for the positively PB-induced proteome alterations, in both HCs and NPCs, proteins assigned to the categories of “gene expression”, “metabolism of proteins” and “3`UTR-mediated translational regulation” were most prominent among the top listed events (see table 6 & 7). With respect to molecular pathways, in HCs “Peroxisomal lipid metabolism”, “Class I MHC mediated antigen processing & presentation” and “Asparagine N-linked glycosylation” were listed on top (see table 8). In contrast, in NPCs “Protein folding”, “Dissolution of Fibrin Clot” and “Platelet Adhesion to exposed collagen” were listed on top (see table 9).

ELISA verification of arginase-1 variations

To verify selected LC-MS/MS data in a quantitative fashion, we conducted an ELISA for rat arginase-1. Arginase-1 was chosen, because of its ability to diminish anti-tumour immunity by interfering with the activation of T-cells [50]. LC-MS/MS results indicated a PB induced decrease in protein secretion of arginase-1 by HCs in case of in vivo treated rats. The ELISA results confirmed our LC-MS/MS results as demonstrated in Figure S1. In the secretome arginase-1 concentration was found decreased by a factor of 2.3. In the cytoplasmic protein fraction, arginase-1 abundance was found induced by PB more than three-fold, which corresponds very well to the LC-MS/MS results.