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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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Effects of cholestatic drugs on gene expression in mouse PCLS. PCLS obtained from 5 mice were treated with 40 μM cyclosporin A (CsA), 20 μM chlorpromazine (CPZ) or vehicle (DMSO) for 24 hours and subjected to Affymetrix microarray analysis. The biological processes in the heat map correspond to gene sets significantly affected according to GSEA analysis (p < 0.05, FDR < 0.25). Gene sets were obtained using the ANNI text mining tool. Processes that were up-regulated are represented by red color, the down-regulated processes are depicted in green, processes that were unchanged are depicted in black.
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Figure 2: Effects of cholestatic drugs on gene expression in mouse PCLS. PCLS obtained from 5 mice were treated with 40 μM cyclosporin A (CsA), 20 μM chlorpromazine (CPZ) or vehicle (DMSO) for 24 hours and subjected to Affymetrix microarray analysis. The biological processes in the heat map correspond to gene sets significantly affected according to GSEA analysis (p < 0.05, FDR < 0.25). Gene sets were obtained using the ANNI text mining tool. Processes that were up-regulated are represented by red color, the down-regulated processes are depicted in green, processes that were unchanged are depicted in black.

Mentions: Upon treatment of the slices for 24 h with 40 μM CsA or 20 μM CPZ, RNA was isolated and hybridized to DNA microarrays. In order to identify and to compare significantly enriched processes, GSEA was performed using gene sets made in the text-mining tool ANNI. The gene sets (listed in Additional file 4: Table S1) were related to diverse hepatic (e.g. lipid and glucose metabolism, inflammation, bile acid metabolism) and non- hepatic biological processes (e.g. adipogenesis, morphogenesis, tight junctions). As indicated in Figure 2, CsA and CPZ affected several of the gene sets/processes (p < 0.05, FDR < 0.25). Both compounds down-regulated processes related to cholesterol, bile acids, lipid, and glucose metabolism. The gene set related to Kupffer cells was also down-regulated by both compounds. CsA, but not CPZ, up-regulated processes related to inflammation (such as T cells, immunotoxicity, inflammation, and natural killer cells) and molecular processes involved in cell death (apoptosis, necrosis) and stress response (biological adaptation to stress, and sumoylation). Moreover, CsA down-regulated processes related to cirrhosis and stellate cells (Figure 2).


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)

Effects of cholestatic drugs on gene expression in mouse PCLS. PCLS obtained from 5 mice were treated with 40 μM cyclosporin A (CsA), 20 μM chlorpromazine (CPZ) or vehicle (DMSO) for 24 hours and subjected to Affymetrix microarray analysis. The biological processes in the heat map correspond to gene sets significantly affected according to GSEA analysis (p < 0.05, FDR < 0.25). Gene sets were obtained using the ANNI text mining tool. Processes that were up-regulated are represented by red color, the down-regulated processes are depicted in green, processes that were unchanged are depicted in black.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effects of cholestatic drugs on gene expression in mouse PCLS. PCLS obtained from 5 mice were treated with 40 μM cyclosporin A (CsA), 20 μM chlorpromazine (CPZ) or vehicle (DMSO) for 24 hours and subjected to Affymetrix microarray analysis. The biological processes in the heat map correspond to gene sets significantly affected according to GSEA analysis (p < 0.05, FDR < 0.25). Gene sets were obtained using the ANNI text mining tool. Processes that were up-regulated are represented by red color, the down-regulated processes are depicted in green, processes that were unchanged are depicted in black.
Mentions: Upon treatment of the slices for 24 h with 40 μM CsA or 20 μM CPZ, RNA was isolated and hybridized to DNA microarrays. In order to identify and to compare significantly enriched processes, GSEA was performed using gene sets made in the text-mining tool ANNI. The gene sets (listed in Additional file 4: Table S1) were related to diverse hepatic (e.g. lipid and glucose metabolism, inflammation, bile acid metabolism) and non- hepatic biological processes (e.g. adipogenesis, morphogenesis, tight junctions). As indicated in Figure 2, CsA and CPZ affected several of the gene sets/processes (p < 0.05, FDR < 0.25). Both compounds down-regulated processes related to cholesterol, bile acids, lipid, and glucose metabolism. The gene set related to Kupffer cells was also down-regulated by both compounds. CsA, but not CPZ, up-regulated processes related to inflammation (such as T cells, immunotoxicity, inflammation, and natural killer cells) and molecular processes involved in cell death (apoptosis, necrosis) and stress response (biological adaptation to stress, and sumoylation). Moreover, CsA down-regulated processes related to cirrhosis and stellate cells (Figure 2).

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