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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 cyclosporin A and chlorpromazine on the expression of genes involved in Fxr and Rxr-regulated pathways related to metabolism and/or transport of bile acids and lipids. Cyclosporin A (CsA) and chlorpromazine (CPZ) significantly affected Fxr-regulated cholesterol and bile acids cellular transport pathway (A), Bile acids regulation of glucose and lipid metabolism via Fxr(B), and Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr pathway (C). Blue and red bars indicate down-and up-regulation respectively of significantly affected genes. 1 and 2 indicate liver slices treated with CsA and CPZ respectively. Each bar represents average fold change of gene expression (treatment vs. control) in liver slices from five mice. ECM: extracellular matrix. For an explanation of the MetaCore symbols is referred to http://pathwaymaps.com/pdf/MC_legend.pdf.
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Figure 3: Effects of cyclosporin A and chlorpromazine on the expression of genes involved in Fxr and Rxr-regulated pathways related to metabolism and/or transport of bile acids and lipids. Cyclosporin A (CsA) and chlorpromazine (CPZ) significantly affected Fxr-regulated cholesterol and bile acids cellular transport pathway (A), Bile acids regulation of glucose and lipid metabolism via Fxr(B), and Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr pathway (C). Blue and red bars indicate down-and up-regulation respectively of significantly affected genes. 1 and 2 indicate liver slices treated with CsA and CPZ respectively. Each bar represents average fold change of gene expression (treatment vs. control) in liver slices from five mice. ECM: extracellular matrix. For an explanation of the MetaCore symbols is referred to http://pathwaymaps.com/pdf/MC_legend.pdf.

Mentions: In order to get insight into the molecular mechanisms of action of CsA and CPZ, the significantly altered genes identified by GSEA (1192 and 811 genes for CsA and CPZ respectively) were uploaded into MetaCore to identify significantly altered pathways. In total, we identified 204 and 125 significantly altered pathways for CsA and CPZ, respectively (p < 0.005) (Additional file 6: Table S3). For a general overview of the processes affected by the drugs, the identified pathways were grouped into broader functional categories using a MetaCore pathway classification output (Table 1, Additional file 6: Table S3). The analysis based on the functional categories showed that there was a substantial overlap between pathways affected by the two drugs (Table 1). Both drugs affected pathways related to immune function (immune response, chemotaxis), energy metabolism (pathogenesis of obesity, regulation of metabolism), and other biological processes (development, apoptosis and survival, signal transduction, cell adhesion, G-protein signalling, protein folding and processing). CsA affected additional functional categories, such as cell cycle and DNA damage (Table 1). In a further analysis, we focused on those pathways that according to MetaCore pathway analysis, were found to be significantly altered either by both drugs or specifically by one drug (p < 0.005). Both CsA and CPZ altered Fxr-regulated cholesterol and bile acids cellular transport (p = 6.8E-07 and p = 7.5E-08 respectively), Figure 3A, Additional file 6: Table S3. In this pathway both CsA and CPZ significantly down-regulated Fxr as well as its target genes involved in BA transport such as solute carrier family 10 member 2 (Slc10a2) and multi-drug resistance 1/ ATP-binding cassette, sub-family B, member 1 (Mdr1/Abcb1), Figure 3A. ATP-binding cassette, sub-family C, member 2/ multidrug resistance-associated protein 2 (Abcc2/ Mrp2), were down-regulated only by CsA, while Bsep/ ATP-binding cassette, sub-family B, member 11(Abcb11) and solute carrier family 10 (sodium/bile acid cotransporter family), member 1/ Na + −taurocholate cotransporting polypeptide (Slc10a1/Ntcp) were down-regulated only by CPZ (Figure 3A). Moreover, both CsA and CPZ down-regulated genes coding for cholesterol transporters such as Abcg5/Abcg8 and Scavenger receptor class B member 1 (Sr-Bi) as well as genes encoding for phospholipids transporter- multidrug resistance 3/ ATP-binding cassette, sub-family B (Mdr/Tap), member 4 (Mdr3/ Abcb4/Mdr2), (Figure 3A). Another process significantly affected by both CsA and CPZ is Bile acids regulation of glucose and lipid metabolism via Fxr (p = 7.2E-14 and p = 1.1E-09 respectively), (Figure 3B). Both, CsA and CPZ led to down-regulation of several genes in this pathway, e.g. Fxr (Nr1h4), retinoid X receptor α (Rxrα), small heterodimer partner (Shp), hepatocyte nuclear factor 4 alpha (Hnf4α), apolipoprotein B (Apob), very low-density lipoprotein (VLDL/Apoc2), liver X receptor α (Lxr α). Furthermore, both CsA and CPZ significantly down-regulated another pathway involved in lipid homeostasis: Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr (p = 5.8E-05 and p = 2.9E-04 respectively), (Figure 3C, Additional file 6: Table S3).


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 cyclosporin A and chlorpromazine on the expression of genes involved in Fxr and Rxr-regulated pathways related to metabolism and/or transport of bile acids and lipids. Cyclosporin A (CsA) and chlorpromazine (CPZ) significantly affected Fxr-regulated cholesterol and bile acids cellular transport pathway (A), Bile acids regulation of glucose and lipid metabolism via Fxr(B), and Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr pathway (C). Blue and red bars indicate down-and up-regulation respectively of significantly affected genes. 1 and 2 indicate liver slices treated with CsA and CPZ respectively. Each bar represents average fold change of gene expression (treatment vs. control) in liver slices from five mice. ECM: extracellular matrix. For an explanation of the MetaCore symbols is referred to http://pathwaymaps.com/pdf/MC_legend.pdf.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Effects of cyclosporin A and chlorpromazine on the expression of genes involved in Fxr and Rxr-regulated pathways related to metabolism and/or transport of bile acids and lipids. Cyclosporin A (CsA) and chlorpromazine (CPZ) significantly affected Fxr-regulated cholesterol and bile acids cellular transport pathway (A), Bile acids regulation of glucose and lipid metabolism via Fxr(B), and Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr pathway (C). Blue and red bars indicate down-and up-regulation respectively of significantly affected genes. 1 and 2 indicate liver slices treated with CsA and CPZ respectively. Each bar represents average fold change of gene expression (treatment vs. control) in liver slices from five mice. ECM: extracellular matrix. For an explanation of the MetaCore symbols is referred to http://pathwaymaps.com/pdf/MC_legend.pdf.
Mentions: In order to get insight into the molecular mechanisms of action of CsA and CPZ, the significantly altered genes identified by GSEA (1192 and 811 genes for CsA and CPZ respectively) were uploaded into MetaCore to identify significantly altered pathways. In total, we identified 204 and 125 significantly altered pathways for CsA and CPZ, respectively (p < 0.005) (Additional file 6: Table S3). For a general overview of the processes affected by the drugs, the identified pathways were grouped into broader functional categories using a MetaCore pathway classification output (Table 1, Additional file 6: Table S3). The analysis based on the functional categories showed that there was a substantial overlap between pathways affected by the two drugs (Table 1). Both drugs affected pathways related to immune function (immune response, chemotaxis), energy metabolism (pathogenesis of obesity, regulation of metabolism), and other biological processes (development, apoptosis and survival, signal transduction, cell adhesion, G-protein signalling, protein folding and processing). CsA affected additional functional categories, such as cell cycle and DNA damage (Table 1). In a further analysis, we focused on those pathways that according to MetaCore pathway analysis, were found to be significantly altered either by both drugs or specifically by one drug (p < 0.005). Both CsA and CPZ altered Fxr-regulated cholesterol and bile acids cellular transport (p = 6.8E-07 and p = 7.5E-08 respectively), Figure 3A, Additional file 6: Table S3. In this pathway both CsA and CPZ significantly down-regulated Fxr as well as its target genes involved in BA transport such as solute carrier family 10 member 2 (Slc10a2) and multi-drug resistance 1/ ATP-binding cassette, sub-family B, member 1 (Mdr1/Abcb1), Figure 3A. ATP-binding cassette, sub-family C, member 2/ multidrug resistance-associated protein 2 (Abcc2/ Mrp2), were down-regulated only by CsA, while Bsep/ ATP-binding cassette, sub-family B, member 11(Abcb11) and solute carrier family 10 (sodium/bile acid cotransporter family), member 1/ Na + −taurocholate cotransporting polypeptide (Slc10a1/Ntcp) were down-regulated only by CPZ (Figure 3A). Moreover, both CsA and CPZ down-regulated genes coding for cholesterol transporters such as Abcg5/Abcg8 and Scavenger receptor class B member 1 (Sr-Bi) as well as genes encoding for phospholipids transporter- multidrug resistance 3/ ATP-binding cassette, sub-family B (Mdr/Tap), member 4 (Mdr3/ Abcb4/Mdr2), (Figure 3A). Another process significantly affected by both CsA and CPZ is Bile acids regulation of glucose and lipid metabolism via Fxr (p = 7.2E-14 and p = 1.1E-09 respectively), (Figure 3B). Both, CsA and CPZ led to down-regulation of several genes in this pathway, e.g. Fxr (Nr1h4), retinoid X receptor α (Rxrα), small heterodimer partner (Shp), hepatocyte nuclear factor 4 alpha (Hnf4α), apolipoprotein B (Apob), very low-density lipoprotein (VLDL/Apoc2), liver X receptor α (Lxr α). Furthermore, both CsA and CPZ significantly down-regulated another pathway involved in lipid homeostasis: Rxr-dependent regulation of lipid metabolism via Ppar, Rar and Vdr (p = 5.8E-05 and p = 2.9E-04 respectively), (Figure 3C, Additional file 6: Table S3).

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