Project description: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 24h 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. From the transcriptomics data it was hypothesized that all 3 compounds affect peroxisome proliferator activated-receptor (PPAR) signalling. 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 mechanisms of action of compounds altering lipid metabolism. Two sets of candidate biomarkers could be used to screen compounds interfering with lipid metabolism by different mechanisms. Steatogenic compounds exposures Mouse precision cut liver slices (PCLS) were prepared from livers obtained from 5 different C57BL/6 male mice (24 weeks old). PCLS were cultured for 24 hours at 80% of oxygen; 5% CO2 and the remaining gas volume was filled up to 95% with N2. PCLS were exposed to model steatogenic, cholestatic, and necrotic compounds selected based on published reports. As steatogenic model compounds amiodarone (AMI), valproic acid (VA), and tetracycline (TET) were selected. As model cholestatic compounds cyclosporin A (CsA), chlorpromazine (CPZ), and ethinyl estradiol (EE) were applied. As necrotic compounds acetaminophen (APAP), isoniazid (ISND), and paraquat (PQ) were applied. To select a non-toxic dose eventually to be used for exposure experiments and subsequent gene expression profiling experiments, the following concentrations ranges were tested: AMI 0-100 μM, VA 0-500 μM, TET 0-100 μM , CsA 0-100 μM, CPZ 0-80 μM, EE 0-100 μM, PQ 0-10 μM, APAP 0-3000 μM and ISND 0-1000 μM. CsA, CPZ, AMI, PQ, EE were dissolved in DMSO, VA and TET were dissolved in ethanol (EtOH), and ISND 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. The viability of the slices was assessed by measuring the ATP content normalized on protein. Dose selection for the three steatogenic, cholestatic, and necrotic exposures were performed in 5 independent experiments with slices obtained from 5 mice. Concentrations that did not cause a decrease of the ATP level normalized on protein, compared to controls, were selected. For transcriptome analysis, PCLS were cultured at the same conditions as described above. The selected concentrations for steatogenic, cholestatic, and necrotic drugs were tested again in liver slices obtained from 5 different mice to re-confirm that the selected concentrations for the exposure experiments were not toxic. The concentrations used in the exposure experiments were for the steatogenic compounds: 50 μM for AMI, 200 μM for VA, and 40 μM for TET. For the cholestatic exposures 40 μM CsA, 20 μM CPZ, and 10 μM EE. For the necrotic compounds: 1000 μM APAP, 1000 μM ISND, and 5 μM PQ. Thereafter, RNA was extracted from three slices per each condition and after processing, the samples were hybridized on the HT Mouse Genome 430 PM array plate(s) using the Affymetrix GeneTitan system (CA, USA).

Project description: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 24h 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. From the transcriptomics data it was hypothesized that all 3 compounds affect peroxisome proliferator activated-receptor (PPAR) signalling. 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 mechanisms of action of compounds altering lipid metabolism. Two sets of candidate biomarkers could be used to screen compounds interfering with lipid metabolism by different mechanisms. Cholestatic compounds exposures Mouse precision cut liver slices (PCLS) were prepared from livers obtained from 5 different C57BL/6 male mice (24 weeks old). PCLS were cultured for 24 hours at 80% of oxygen; 5% CO2 and the remaining gas volume was filled up to 95% with N2. PCLS were exposed to model steatogenic, cholestatic, and necrotic compounds selected based on published reports. As steatogenic model compounds amiodarone (AMI), valproic acid (VA), and tetracycline (TET) were selected. As model cholestatic compounds cyclosporin A (CsA), chlorpromazine (CPZ), and ethinyl estradiol (EE) were applied. As necrotic compounds acetaminophen (APAP), isoniazid (ISND), and paraquat (PQ) were applied. To select a non-toxic dose eventually to be used for exposure experiments and subsequent gene expression profiling experiments, the following concentrations ranges were tested: AMI 0-100 μM, VA 0-500 μM, TET 0-100 μM , CsA 0-100 μM, CPZ 0-80 μM, EE 0-100 μM, PQ 0-10 μM, APAP 0-3000 μM and ISND 0-1000 μM. CsA, CPZ, AMI, PQ, EE were dissolved in DMSO, VA and TET were dissolved in ethanol (EtOH), and ISND 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. The viability of the slices was assessed by measuring the ATP content normalized on protein. Dose selection for the three steatogenic, cholestatic, and necrotic exposures were performed in 5 independent experiments with slices obtained from 5 mice. Concentrations that did not cause a decrease of the ATP level normalized on protein, compared to controls, were selected. For transcriptome analysis, PCLS were cultured at the same conditions as described above. The selected concentrations for steatogenic, cholestatic, and necrotic drugs were tested again in liver slices obtained from 5 different mice to re-confirm that the selected concentrations for the exposure experiments were not toxic. The concentrations used in the exposure experiments were for the steatogenic compounds: 50 μM for AMI, 200 μM for VA, and 40 μM for TET. For the cholestatic exposures 40 μM CsA, 20 μM CPZ, and 10 μM EE. For the necrotic compounds: 1000 μM APAP, 1000 μM ISND, and 5 μM PQ. Thereafter, RNA was extracted from three slices per each condition and after processing, the samples were hybridized on the HT Mouse Genome 430 PM array plate(s) using the Affymetrix GeneTitan system (CA, USA).

Project description: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 24h 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. From the transcriptomics data it was hypothesized that all 3 compounds affect peroxisome proliferator activated-receptor (PPAR) signalling. 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 mechanisms of action of compounds altering lipid metabolism. Two sets of candidate biomarkers could be used to screen compounds interfering with lipid metabolism by different mechanisms. Necrotic compounds exposures Mouse precision cut liver slices (PCLS) were prepared from livers obtained from 5 different C57BL/6 male mice (24 weeks old). PCLS were cultured for 24 hours at 80% of oxygen; 5% CO2 and the remaining gas volume was filled up to 95% with N2. PCLS were exposed to model steatogenic, cholestatic, and necrotic compounds selected based on published reports. As steatogenic model compounds amiodarone (AMI), valproic acid (VA), and tetracycline (TET) were selected. As model cholestatic compounds cyclosporin A (CsA), chlorpromazine (CPZ), and ethinyl estradiol (EE) were applied. As necrotic compounds acetaminophen (APAP), isoniazid (ISND), and paraquat (PQ) were applied. To select a non-toxic dose eventually to be used for exposure experiments and subsequent gene expression profiling experiments, the following concentrations ranges were tested: AMI 0-100 μM, VA 0-500 μM, TET 0-100 μM , CsA 0-100 μM, CPZ 0-80 μM, EE 0-100 μM, PQ 0-10 μM, APAP 0-3000 μM and ISND 0-1000 μM. CsA, CPZ, AMI, PQ, EE were dissolved in DMSO, VA and TET were dissolved in ethanol (EtOH), and ISND 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. The viability of the slices was assessed by measuring the ATP content normalized on protein. Dose selection for the three steatogenic, cholestatic, and necrotic exposures were performed in 5 independent experiments with slices obtained from 5 mice. Concentrations that did not cause a decrease of the ATP level normalized on protein, compared to controls, were selected. For transcriptome analysis, PCLS were cultured at the same conditions as described above. The selected concentrations for steatogenic, cholestatic, and necrotic drugs were tested again in liver slices obtained from 5 different mice to re-confirm that the selected concentrations for the exposure experiments were not toxic. The concentrations used in the exposure experiments were for the steatogenic compounds: 50 μM for AMI, 200 μM for VA, and 40 μM for TET. For the cholestatic exposures 40 μM CsA, 20 μM CPZ, and 10 μM EE. For the necrotic compounds: 1000 μM APAP, 1000 μM ISND, and 5 μM PQ. Thereafter, RNA was extracted from three slices per each condition and after processing, the samples were hybridized on the HT Mouse Genome 430 PM array plate(s) using the Affymetrix GeneTitan system (CA, USA).

Project description:Background: Studies in mice have shown that PPARÎ± is an important regulator of lipid metabolism in liver and a key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARÎ± in human liver. Here we set out to study the function of PPARÎ± in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARÎ± agonist Wy14643. Results: Quantitative PCR indicated that PPARÎ± is well expressed in human liver and human liver slices and that the classical PPARÎ± targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARÎ± activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARÎ± activation (q value<0.05). Many genes induced by PPARÎ± activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARÎ± activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon Î³-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARÎ± is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARÎ± activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARÎ± were identified that were commonly induced by PPARÎ± activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. Conclusion: Our manuscript demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARÎ± in human liver. Our data underscore the major role of PPARÎ± in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARÎ± in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease. Precision-cut liver slices, prepared from liver biopsies obtained from obese subjects undergoing bariatric surgery, were incubated with the peroxisome proliferator-activated receptor alpha (PPARÎ±) agonist Wy14643 or vehicle for 24hrs, after which gene expression was profiled by array.

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Transcriptional changes of precision-cut liver slices after 3 days of cultures in the absence or presence of 2.5 mM valproic acid sodium salt.

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