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Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes.

Szalowska E, Stoopen G, Groot MJ, Hendriksen PJ, Peijnenburg AA - BMC Med Genomics (2013)

Bottom Line: The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR.No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ.Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: RIKILT - Institute of Food Safety, Wageningen UR, P,O, Box 230, 6700 AE Wageningen, the Netherlands. ewa.szalowska@wur.nl.

ABSTRACT

Background: Unexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties.

Methods: We employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis.

Results: Whole genome gene expression analysis showed that CsA up-regulated pathways related to NF-κB, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced "ballooning" of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients.

Conclusion: The present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

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Identification of potential biomarkers for cholestasis in PCLS. PCLS were exposed for 24 hours to model toxicants for cholestasis (cyclosporin A (CsA)-A, chlorpromazine (CPZ)-B), steatosis (amiodarone (A)-C, valproic acid (VA)-D, necrosis, isoniazid (ISND-E), paraquat (PQ)-F, and controls (ctr)). GSEA led to the identification of 73 genes for which the mRNA expression was down-regulated by both CsA and CPZ. 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. Red and green indicate expression higher or lower respectively than the average expression of all samples within the same heat map. Please note Fxr is also depicted as Nr1h4.
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Figure 6: Identification of potential biomarkers for cholestasis in PCLS. PCLS were exposed for 24 hours to model toxicants for cholestasis (cyclosporin A (CsA)-A, chlorpromazine (CPZ)-B), steatosis (amiodarone (A)-C, valproic acid (VA)-D, necrosis, isoniazid (ISND-E), paraquat (PQ)-F, and controls (ctr)). GSEA led to the identification of 73 genes for which the mRNA expression was down-regulated by both CsA and CPZ. 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. Red and green indicate expression higher or lower respectively than the average expression of all samples within the same heat map. Please note Fxr is also depicted as Nr1h4.

Mentions: In order to identify biomarkers that could be used for identification of drugs with cholestatic properties, genes were selected that, according to GSEA, significantly contributed to the enrichment of the analysed gene sets (p < 0.05, FDR < 0.25) in both CsA and CPZ treatments. In total 305 common candidate biomarkers were identified and further selection using FC ≥1.5 as criterion resulted in 73 genes. These genes could be categorized in five functional clusters: β-oxidation, biotransformation, BA metabolism, BA conjugation, and lipid metabolism (Figure 5). Hierarchical clustering of these genes, led to a very good separation between control and slices treated with CsA (Figure 6A). CPZ treated samples separated very well except for one control and one treated sample, which were mis-classified (Figure 6B). Furthermore, we analysed the expression levels of these 73 genes in liver slices exposed to two other classes of hepatotoxicants, i.e. steatogenic drugs (valproic acid and amiodarone) and necrotic drugs (paraquat and isoniazid). In contrast to the slices exposed to CsA and CPZ, hierarchical clustering of array data (of the 73 selected genes) from slices exposed to steatogenic (Figure 6C-D) and necrotic compounds (Figure 6E-F) did not show a clear separation between treatment and control.


Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes.

Szalowska E, Stoopen G, Groot MJ, Hendriksen PJ, Peijnenburg AA - BMC Med Genomics (2013)

Identification of potential biomarkers for cholestasis in PCLS. PCLS were exposed for 24 hours to model toxicants for cholestasis (cyclosporin A (CsA)-A, chlorpromazine (CPZ)-B), steatosis (amiodarone (A)-C, valproic acid (VA)-D, necrosis, isoniazid (ISND-E), paraquat (PQ)-F, and controls (ctr)). GSEA led to the identification of 73 genes for which the mRNA expression was down-regulated by both CsA and CPZ. 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. Red and green indicate expression higher or lower respectively than the average expression of all samples within the same heat map. Please note Fxr is also depicted as Nr1h4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852711&req=5

Figure 6: Identification of potential biomarkers for cholestasis in PCLS. PCLS were exposed for 24 hours to model toxicants for cholestasis (cyclosporin A (CsA)-A, chlorpromazine (CPZ)-B), steatosis (amiodarone (A)-C, valproic acid (VA)-D, necrosis, isoniazid (ISND-E), paraquat (PQ)-F, and controls (ctr)). GSEA led to the identification of 73 genes for which the mRNA expression was down-regulated by both CsA and CPZ. 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. Red and green indicate expression higher or lower respectively than the average expression of all samples within the same heat map. Please note Fxr is also depicted as Nr1h4.
Mentions: In order to identify biomarkers that could be used for identification of drugs with cholestatic properties, genes were selected that, according to GSEA, significantly contributed to the enrichment of the analysed gene sets (p < 0.05, FDR < 0.25) in both CsA and CPZ treatments. In total 305 common candidate biomarkers were identified and further selection using FC ≥1.5 as criterion resulted in 73 genes. These genes could be categorized in five functional clusters: β-oxidation, biotransformation, BA metabolism, BA conjugation, and lipid metabolism (Figure 5). Hierarchical clustering of these genes, led to a very good separation between control and slices treated with CsA (Figure 6A). CPZ treated samples separated very well except for one control and one treated sample, which were mis-classified (Figure 6B). Furthermore, we analysed the expression levels of these 73 genes in liver slices exposed to two other classes of hepatotoxicants, i.e. steatogenic drugs (valproic acid and amiodarone) and necrotic drugs (paraquat and isoniazid). In contrast to the slices exposed to CsA and CPZ, hierarchical clustering of array data (of the 73 selected genes) from slices exposed to steatogenic (Figure 6C-D) and necrotic compounds (Figure 6E-F) did not show a clear separation between treatment and control.

Bottom Line: The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR.No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ.Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: RIKILT - Institute of Food Safety, Wageningen UR, P,O, Box 230, 6700 AE Wageningen, the Netherlands. ewa.szalowska@wur.nl.

ABSTRACT

Background: Unexpected cholestasis substantially contributes to drug failure in clinical trials. Current models used for safety assessment in drug development do not accurately predict cholestasis in humans. Therefore, it is of relevance to develop new screening models that allow identifying drugs with cholestatic properties.

Methods: We employed mouse precision cut liver slices (PCLS), which were incubated 24 h with two model cholestatic compounds: cyclosporin A (CsA) and chlorpromazine (CPZ). Subsequently, transcriptome analysis using DNA microarrays and q-PCR were performed to identify relevant biological processes and biomarkers. Additionally, histology was carried out and levels of triglycerides (TG) and bile acids (BA) were measured. To verify the ex vivo mouse data, these were compared with publically available human data relevant for cholestasis.

Results: Whole genome gene expression analysis showed that CsA up-regulated pathways related to NF-κB, ER stress and inflammation. Both CsA and CPZ down-regulated processes related to extracellular matrix (ECM) remodelling, BA homeostasis, Fxr signalling, and energy metabolism. The differential expression of a number of characteristic genes (e.g. Abcg5, Abcg8, Klf15, and Baat) could be confirmed by q-PCR. Histology revealed that CsA but not CPZ induced "ballooning" of hepatocytes. No effects on TG and BA levels were observed after incubation of PCLS with CsA and CPZ. A substantial number of processes altered in CsA- and CPZ-treated mouse PCLS ex vivo was also found to be affected in liver biopsies of cholestatic patients.

Conclusion: The present study demonstrated that mouse PCLS can be used as a tool to identify mechanisms of action of cholestatic model compounds. The induction of general stress responses and down-regulated Fxr signalling could play a role in the development of drug induced cholestasis. Importantly, comparative data analysis showed that the ex vivo mouse findings are also relevant for human pathology. Moreover, this work provides a set of genes that are potentially useful to assess drugs for cholestatic properties.

Show MeSH
Related in: MedlinePlus