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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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Histological analysis of liver slices treated with CsA and CPZ. PCLS were cultured for 24 and 48 hours in the presence of DMSO (control) (A and B), 40 μM CsA (C and D) or 20 μM CPZ (E and F). Histology of PCLS treated with CsA revealed ballooning of hepatocytes at the outer parts of slices after 24 and 48 hours (C and D respectively). Histology of CPZ treated slices cultured for 24 and 48 hours (E and F respectively) revealed a slight increase in number of cells containing pycnotic nuclei compared to control slices cultured equally long (A and B).
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Figure 7: Histological analysis of liver slices treated with CsA and CPZ. PCLS were cultured for 24 and 48 hours in the presence of DMSO (control) (A and B), 40 μM CsA (C and D) or 20 μM CPZ (E and F). Histology of PCLS treated with CsA revealed ballooning of hepatocytes at the outer parts of slices after 24 and 48 hours (C and D respectively). Histology of CPZ treated slices cultured for 24 and 48 hours (E and F respectively) revealed a slight increase in number of cells containing pycnotic nuclei compared to control slices cultured equally long (A and B).

Mentions: Slices cultured for 24 and 48 hours with the reference compounds and controls were subjected to histological analysis. Upon incubation for 24 hours, some hepatocytes localized in the outer part of slices treated with CsA had developed a characteristic ballooned phenotype, which was even more evident after 48 hours and was absent in control slices. These hepatocytes were enlarged compared to control and had their nucleus in the centre (Figure 7). Based on the histological examination as well as transcriptome data analysis, showing alternations in lipid and BA metabolism, it could be envisaged that the enlargement of cells treated with CsA is due to TG or BA accumulation. Therefore, biochemical assays to quantify TG and BA levels were employed. However, compared to control, no increase of BA or TG in CsA treated slices was observed (Figure 8A and B). Additionally, we performed Periodic acid-Schiff (PAS) staining to detect glycogen, which theoretically could also accumulate in hepatocytes and lead to their enlargement. As expected, control slices showed substantial number of cells containing glycogen (Figure 8C). However, in the enlarged cells of the CsA treated slices no glycogen could be detected (Figure 8D). Finally, Fouchet staining was performed to examine whether CsA treatment resulted in the accumulation of bile, a process that is often accompanying cholestasis. Although, we did not detect bile accumulation in slices, Fouchet staining unexpectedly showed the presence of several vacuoles, which were absent in control slices (Figure 8E and F).


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)

Histological analysis of liver slices treated with CsA and CPZ. PCLS were cultured for 24 and 48 hours in the presence of DMSO (control) (A and B), 40 μM CsA (C and D) or 20 μM CPZ (E and F). Histology of PCLS treated with CsA revealed ballooning of hepatocytes at the outer parts of slices after 24 and 48 hours (C and D respectively). Histology of CPZ treated slices cultured for 24 and 48 hours (E and F respectively) revealed a slight increase in number of cells containing pycnotic nuclei compared to control slices cultured equally long (A and B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Histological analysis of liver slices treated with CsA and CPZ. PCLS were cultured for 24 and 48 hours in the presence of DMSO (control) (A and B), 40 μM CsA (C and D) or 20 μM CPZ (E and F). Histology of PCLS treated with CsA revealed ballooning of hepatocytes at the outer parts of slices after 24 and 48 hours (C and D respectively). Histology of CPZ treated slices cultured for 24 and 48 hours (E and F respectively) revealed a slight increase in number of cells containing pycnotic nuclei compared to control slices cultured equally long (A and B).
Mentions: Slices cultured for 24 and 48 hours with the reference compounds and controls were subjected to histological analysis. Upon incubation for 24 hours, some hepatocytes localized in the outer part of slices treated with CsA had developed a characteristic ballooned phenotype, which was even more evident after 48 hours and was absent in control slices. These hepatocytes were enlarged compared to control and had their nucleus in the centre (Figure 7). Based on the histological examination as well as transcriptome data analysis, showing alternations in lipid and BA metabolism, it could be envisaged that the enlargement of cells treated with CsA is due to TG or BA accumulation. Therefore, biochemical assays to quantify TG and BA levels were employed. However, compared to control, no increase of BA or TG in CsA treated slices was observed (Figure 8A and B). Additionally, we performed Periodic acid-Schiff (PAS) staining to detect glycogen, which theoretically could also accumulate in hepatocytes and lead to their enlargement. As expected, control slices showed substantial number of cells containing glycogen (Figure 8C). However, in the enlarged cells of the CsA treated slices no glycogen could be detected (Figure 8D). Finally, Fouchet staining was performed to examine whether CsA treatment resulted in the accumulation of bile, a process that is often accompanying cholestasis. Although, we did not detect bile accumulation in slices, Fouchet staining unexpectedly showed the presence of several vacuoles, which were absent in control slices (Figure 8E and F).

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