Dataset features


Application: Gene expression microarray analysis
Number of samples: 30
Release date: Dec 23 2013
Last update date: Sep 3 2014
Access: Public
Diseases: Fatty Liver, Fatty Liver, Alcoholic
Chemicals: Amiodarone, Tetracycline, Valproic Acid
Dataset link Model steatogenic compounds (amiodarone, valproic acid, and tetracycline) alter lipid metabolism by different mechanisms in mouse liver slices [Cholestatic compounds exposures]

Experimental Protocol

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).










Ewa Szalowska

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