Additional file 1: of Cyclosporin A induced toxicity in mouse liver slices is only slightly aggravated by Fxr-deficiency and co-occurs with upregulation of pro-inflammatory genes and downregulation of genes involved in mitochondrial functions

Validation of known and putative Fxr target genes by q-PCR. Information about primers and functions of genes used in q-PCR to test expression of known and novel putative FXR target genes in WT-PCLS and FXRKO-PCLS exposed to endogenous FXR ligand. Numbers of Taqman assays are given in column “Taqman assay”. Information about functions of the tested genes is according to Gene Cards http://www.genecards.org/ . (PPTX 103 kb

Functional clustering of genes involved in energy metabolism (valproic acid).

<p>Genes related to energy metabolism identified by GSEA as being significantly altered upon valproic acid (VA) treatment were subjected to functional clustering in STRING. Functional clusters such as lipid synthesis, lipid catabolism, β-oxidation, glucose metabolism, and bile acid metabolism have been identified. Information about fold change (FC = treatment vs. control) for the analysed genes in individual mice is presented as a heat map. For further explanation of the networks see Fig. 3.</p

Functional clustering of genes involved in energy metabolism (tetracycline).

<p>Genes related to energy metabolism identified by GSEA as being significantly altered upon tetracycline (TET) treatment were subjected to functional clustering in STRING. Functional clusters such as lipid synthesis, β-oxidation, PPARα signaling, inflammation/apoptosis, amino acids (aa)/glucose/lipid metabolism, and cholesterol/bile acid homeostasis were identified. Information about fold change (FC = treatment vs. control) for the analysed genes in individual mice is presented as a heat map. For explanation of the networks see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086795#pone-0086795-g003" target="_blank">Fig. 3</a>.</p

Effects of steatogenic drugs on gene expression in mouse PCLS.

<p><b>A.</b> PCLS obtained from 5 mice were treated with 50 µM amiodarone (AMI), 200 µM of valproic acid (VA), 40 µM of tetracycline (TET) or vehicle for 24 h and subjected to Affymetrix microarray analysis. The biological processes in the heat map correspond to gene sets significantly affected according to GSEA (p<0.05, FDR<0.05). Processes that were upregulated are represented by red colour, the downregulated processes are depicted in green, and unaffected processes in black. <b>B.</b> Gene Ontology (GO) analysis of the significant genes identified by GSEA (p<0.05, FDR<0.05) was performed in DAVID. GO terms were considered to be significant if p<0.005, FDR<0.005. The significant GO terms were grouped into GO annotation clusters and are depicted as a heat map. For explanation of the colours see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086795#pone-0086795-g002" target="_blank">Figure 2A</a>.</p

Model Steatogenic Compounds (Amiodarone, Valproic Acid, and Tetracycline) Alter Lipid Metabolism by Different Mechanisms in Mouse Liver Slices

<div><p>Although drug induced steatosis represents a mild type of hepatotoxicity it can progress into more severe non-alcoholic steatohepatitis. Current models used for safety assessment in drug development and chemical risk assessment do not accurately predict steatosis in humans. Therefore, new models need to be developed to screen compounds for steatogenic properties. We have studied the usefulness of mouse precision-cut liver slices (PCLS) as an alternative to animal testing to gain more insight into the mechanisms involved in the steatogenesis. To this end, PCLS were incubated 24 h with the model steatogenic compounds: amiodarone (AMI), valproic acid (VA), and tetracycline (TET). Transcriptome analysis using DNA microarrays was used to identify genes and processes affected by these compounds. AMI and VA upregulated lipid metabolism, whereas processes associated with extracellular matrix remodelling and inflammation were downregulated. TET downregulated mitochondrial functions, lipid metabolism, and fibrosis. Furthermore, on the basis of the transcriptomics data it was hypothesized that all three compounds affect peroxisome proliferator activated-receptor (PPAR) signaling. Application of PPAR reporter assays classified AMI and VA as PPARγ and triple PPARα/(β/δ)/γ agonist, respectively, whereas TET had no effect on any of the PPARs. Some of the differentially expressed genes were considered as potential candidate biomarkers to identify PPAR agonists (i.e. AMI and VA) or compounds impairing mitochondrial functions (i.e. TET). Finally, comparison of our findings with publicly available transcriptomics data showed that a number of processes altered in the mouse PCLS was also affected in mouse livers and human primary hepatocytes exposed to known PPAR agonists. Thus mouse PCLS are a valuable model to identify early mechanisms of action of compounds altering lipid metabolism.</p></div

Comparative data analysis: relevance for mouse in vivo and human primary hepatocytes.

<p>Publically available transcriptomics data (Gene Expression Omnibus) relevant for the actions of known PPAR agonists in mouse liver <i>in vivo</i> and human primary hepatocytes were used. The heat map represents significant gene sets (GSEA p<0.05, FDR<0.05), which were subjected to HCA. Gene sets were obtained using the ANNI text mining tool. Processes that were upregulated are represented by red colour, the downregulated processes are depicted in green, and unaffected processes are in black. Ale stands for aleglitazar (double PPARα/γ agonist), Pio/Feno-pioglitazone/fenofibrate (PPAR γ/PPARα agonists), Tesa-Tesaglitazar (double PPAR γ/α agonist), AMI-amiodarone (PPAR γ agonist), VA-valproic acid (triple PPARα/(β/δ)/γ agonist), TET-tetracycline, Wy-Wy14643, FO-fish oil, m-mouse, h-human, PCLS-precision cut liver slices, PH-primary hepatocytes, L- liver in vivo.</p

Effect of valproic acid and amiodarone on PPARα, PPAR β/δ, and PPARγ gene reporter assays.

<p>Luciferase activity of PPARα CALUX cells upon exposure to PPARα agonists: GW7647 (A) and valproic acid (B). Luciferase activity of PPAR β/δ CALUX cells upon exposure to PPAR β/δ agonists: L-165, 041 (C), and valproic acid (D). Luciferase activity of PPAR<b>γ</b> CALUX cells upon exposure to PPAR<b>γ</b> agonists<b>:</b> rosiglitazone (E), valproic acid (F), and amiodarone (G). Data are corrected for solvent control values and expressed as means±standard errors (n = 3). X axis represents concentration of the compounds [M] and y axis represents luciferase units. AMI stands for amiodarone, VA-valproic acid, and TET-tetracycline.</p

Functional clustering of genes involved in energy metabolism (amiodarone).

<p>Genes related to energy metabolism identified by GSEA as being significantly altered upon amiodarone (AMI) treatment were subjected to functional clustering in STRING. Functional clusters such as lipid synthesis, β-oxidation, mitochondria, peroxisomes, and PPARα -dependent lipid metabolism were identified. Information about fold change (FC = treatment vs. control) for the analysed genes in individual mice is presented as a heat map. Genes that did not form connected nodes were removed from the presented clusters. Thicker lines represent stronger associations between genes. Inter-cluster edges are represented by dashed-lines. The bigger spheres represent genes coding for proteins with known structure. Smaller spheres represent genes coding proteins for which no structural information is available.</p

Viability of mouse liver slices upon treatment with steatogenic drugs.

<p>Liver slices were incubated for 24(AMI) 25, 50, and 100 µM, valproic acid (VA) 50, 200, and 500 µM, and tetracycline (TET) 5, 40, and 100 µM. ATP content (nmol/mg of protein) in slices treated with different concentrations of hepatotoxicants was compared to control slices. Each point is the mean±SD of 5 independent experiments (liver slices were isolated from livers of 5 mice) and each measurement was made in duplicate. There were no significant differences between the tested conditions.</p

Identification of potential biomarkers for tetracycline-like acting compounds in mouse PCLS.

<p>PCLS obtained from 4 or 5 mice were exposed for 24(amiodarone (A), valproic acid (B), or tetracycline (C)), cholestasis (cyclosporin A (D), chlorpromazine (E), or ethinyl estradiol (F)), necrosis (acetaminophen (G), isoniazid (H), or paraquat(I)), or controls. GSEA led to the identification of 19 genes downregulated by tetracycline (TET) treatment, which were considered as candidate biomarkers for TET-like acting compounds. mRNA expression values for the selected biomarkers are derived from DNA-microarrays, and results are presented as heat maps of log2, median centered gene expression values subjected to HCA. For explanation of the colours and abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086795#pone-0086795-g007" target="_blank">Figure 7</a>.</p