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Whole genome transcript profiling of drug induced steatosis in rats reveals a gene signature predictive of outcome.

Sahini N, Selvaraj S, Borlak J - PLoS ONE (2014)

Bottom Line: Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS.Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events.Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21).

View Article: PubMed Central - PubMed

Affiliation: Centre for Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.

ABSTRACT
Drug induced steatosis (DIS) is characterised by excess triglyceride accumulation in the form of lipid droplets (LD) in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP) to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014). Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1). Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21). Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.

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Hierarchical gene clustering.The average-linkage hierarchical clustering with Pearson correlation distance was applied. The values represent fold change of normalised DEGs of the selected 200 master genes summarised in Table S2. The data were analysed for each of the steatotic compounds at the highest dose and the 24 h time point after single dose and the highest dose after repeated treatment for 28 days. The steatosis compounds marked with a yellow bar are clearly segregated from non-steatotic compounds marked in grey. The boxes coloured in maroon represent DIS signature genes (see panel C of Figure 3) of which 5 (hallmarked with a blue box) group together.
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pone-0114085-g011: Hierarchical gene clustering.The average-linkage hierarchical clustering with Pearson correlation distance was applied. The values represent fold change of normalised DEGs of the selected 200 master genes summarised in Table S2. The data were analysed for each of the steatotic compounds at the highest dose and the 24 h time point after single dose and the highest dose after repeated treatment for 28 days. The steatosis compounds marked with a yellow bar are clearly segregated from non-steatotic compounds marked in grey. The boxes coloured in maroon represent DIS signature genes (see panel C of Figure 3) of which 5 (hallmarked with a blue box) group together.

Mentions: Hierarchical gene cluster analysis was performed to identify genes regulated in common based on their similar expression using the aforementioned 17 steatotic drugs in addition to 7 compounds (negative controls) that do not cause steatosis. A heatmap for the predicted DEGs was constructed based on the average-linkage hierarchical clustering with Pearson correlation distance by using Multi Experimental Viewer (MeV) software (http://www.tm4.org/mev.html). Only high dose animals and the time points 24 hrs (single dose) and 28 days (repeated dose) for each drug/chemical was considered with a significant log fold change ≥0.5 and p-value ≤0.05 as the filtering criteria. Genes significantly regulated among the master list of 200 genes were selected. The results show a clear separation between steatosis (in yellow) and non steatosis (in grey) clustering groups, except for CCL4 at day 28 which was grouped with the non-steatotic compounds (Figure 11). The Dendogram represents the distinct gene expression patterns for the study phenotypes with respect to dose and time; note, DIS signature genes grouped together in a cluster (ANGPTL4, FADS1, PKLR, FABP7 and LDLR).


Whole genome transcript profiling of drug induced steatosis in rats reveals a gene signature predictive of outcome.

Sahini N, Selvaraj S, Borlak J - PLoS ONE (2014)

Hierarchical gene clustering.The average-linkage hierarchical clustering with Pearson correlation distance was applied. The values represent fold change of normalised DEGs of the selected 200 master genes summarised in Table S2. The data were analysed for each of the steatotic compounds at the highest dose and the 24 h time point after single dose and the highest dose after repeated treatment for 28 days. The steatosis compounds marked with a yellow bar are clearly segregated from non-steatotic compounds marked in grey. The boxes coloured in maroon represent DIS signature genes (see panel C of Figure 3) of which 5 (hallmarked with a blue box) group together.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4254931&req=5

pone-0114085-g011: Hierarchical gene clustering.The average-linkage hierarchical clustering with Pearson correlation distance was applied. The values represent fold change of normalised DEGs of the selected 200 master genes summarised in Table S2. The data were analysed for each of the steatotic compounds at the highest dose and the 24 h time point after single dose and the highest dose after repeated treatment for 28 days. The steatosis compounds marked with a yellow bar are clearly segregated from non-steatotic compounds marked in grey. The boxes coloured in maroon represent DIS signature genes (see panel C of Figure 3) of which 5 (hallmarked with a blue box) group together.
Mentions: Hierarchical gene cluster analysis was performed to identify genes regulated in common based on their similar expression using the aforementioned 17 steatotic drugs in addition to 7 compounds (negative controls) that do not cause steatosis. A heatmap for the predicted DEGs was constructed based on the average-linkage hierarchical clustering with Pearson correlation distance by using Multi Experimental Viewer (MeV) software (http://www.tm4.org/mev.html). Only high dose animals and the time points 24 hrs (single dose) and 28 days (repeated dose) for each drug/chemical was considered with a significant log fold change ≥0.5 and p-value ≤0.05 as the filtering criteria. Genes significantly regulated among the master list of 200 genes were selected. The results show a clear separation between steatosis (in yellow) and non steatosis (in grey) clustering groups, except for CCL4 at day 28 which was grouped with the non-steatotic compounds (Figure 11). The Dendogram represents the distinct gene expression patterns for the study phenotypes with respect to dose and time; note, DIS signature genes grouped together in a cluster (ANGPTL4, FADS1, PKLR, FABP7 and LDLR).

Bottom Line: Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS.Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events.Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21).

View Article: PubMed Central - PubMed

Affiliation: Centre for Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany.

ABSTRACT
Drug induced steatosis (DIS) is characterised by excess triglyceride accumulation in the form of lipid droplets (LD) in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP) to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014). Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1). Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21). Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.

Show MeSH
Related in: MedlinePlus