Abstract
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.
Highlights
Drug induced fatty liver belongs to one of the most common forms of liver injury [1]
In Gene Set Enrichment Analysis (GSEA), we tested in total 47 gene sets related to general biological processes, hepatic functions, and functions unrelated to liver (Table S1)
We applied a toxicogenomics approach in combination with gene reporter assays to examine the value of mouse precision-cut liver slices (PCLS) as an alternative to animal testing and relevant model for humans
Summary
Drug induced fatty liver (steatosis) belongs to one of the most common forms of liver injury [1]. The exact molecular triggers resulting in lipid accumulation in the liver are largely unknown, but may arise from: 1) increased uptake of lipids, 2) elevated de novo lipogenesis, 3) impaired lipoprotein synthesis and secretion, and/or 4) reduced catabolism of fatty acids (FA) by peroxisomal/mitochondrial b-oxidation [3]. One of the most common causes of drug-induced steatosis is impairment of mitochondrial functions. Mitochondrial b-oxidation is the major process that eliminates FA, which accumulate in a form of TG in liver cells if not-catabolised. Consistent with these notions, many steatogenic drugs interfere directly with enzymes involved in b-oxidation [4]. Alterations in the expression of PPARa target genes involved in lipid catabolism (e.g. carnitine palmityltransferase 1 (Cpt1), 3-ketoacyl-CoA thiolase (Hadhb), acetyl-Coenzyme A acyltransferase 2 (Acaa2)), have been linked to the development of drug-induced steatosis [6]
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