Venn diagram of DEGs associated with steatosis and phospholipidosis.

<p>Panel A: DEGs associated with phospholipidosis under all conditions (single dose at 3, 6, 9 and 24 h and repeated treatment (3, 7, 14 and 28 days) at the low, mid and high dose for 12 phospholipidosis drugs). Note, none of the DEGs were commonly regulated amongst phospholipidosis drugs. Panel B: DEGs common amongst phospholipidosis and steatosis drugs. By applying less stringent criteria (regulation in any of the conditions, either by dose or time) a total of 26 genes were commonly regulated. Panel C: PPI network among the 26 common genes. Of these 58% proteins interact with each other. The strength of association is depicted with the thickness of the blue line between the interacting proteins (STRING 9.1, confidence view).</p

Whole Genome Transcript Profiling of Drug Induced Steatosis in Rats Reveals a Gene Signature Predictive of Outcome

<div><p>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 <i>in vivo</i> 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.</p></div

Electron microscopic images of steatosis and phospholipidosis.

<p>Depicted are examples of hepatic steatosis with lipid droplets (LD, see panel A and B). Hepatic phospholipidosis with concentric membranous structures known as lamellar bodies (arrow head) are shown in panel C and D. Note, the Kupffer cells, erythrocytes and space of Disse in panel D. LDs are also present around endoplasmic reticulum (ER, panel A and D) while mitochondria (marked with asterix) and peroxisomes (marked as P in panel B and C) appear to be in apposition ( =  close contact) with the lipid droplets and/inclusion bodies (Panel A and D). The lamellar bodies in the cytosol of Kupffer cells shown in panel C suggest their phagocytosis.</p

Hierarchical gene clustering.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone.0114085.s002" target="_blank">Table S2</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g003" target="_blank">Figure 3</a>) of which 5 (hallmarked with a blue box) group together.</p

Venn diagram of genes regulated in DIS.

<p>Panel A depicts the distribution of 9 commonly regulated genes under all conditions (see also panel C of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g003" target="_blank">Figure 3</a>) and were compared with frequently regulated genes depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g009" target="_blank">Figure 9</a> as well as 26 DEGs common amongst drugs causing phospholipidosis and steatosis (see panel C, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g006" target="_blank">Figure 6</a>). Among the three conditions, 4 genes, i.e. FABP7, FADS1, GK and PKLR were regulated alike. Panel B represents single dose (up to 24 hours) rat hepatocyte data that was compared with rat <i>in vivo</i> data under similar conditions. Here, 10 genes were in common in both of the conditions (ANGPTL4, CIDEC, CPT1, FABP7, FGF21, FOXA2, IRS2, MGLL, RXRG and VIM).</p

Experimental evidence of DIS signature genes to be expressed at the protein level in hepatic steatosis.

<p>Experimental evidence of DIS signature genes to be expressed at the protein level in hepatic steatosis.</p

Venn diagram of DEGs based on dose and time considerations.

<p>Panel A and B represent Venn diagrams of drug induced gene expression changes after single and repeated treatment at different doses and time points, respectively. For this purpose genes regulated at different doses (low, middle and high) and/or at least at three different time points (3, 6, 9 and/or 24 h) after single treatment were considered. The same analysis was carried out for animals treated repeatedly at the low, medium and high dose for 3, 7, 14 and 28 days. Panel C combines all DEGs after single and repeated dose/time considerations. Eventually, 9 genes were found to be regulated in common.</p

Representative images of H and E stained rat liver sections using 8 steatotic drugs.

<p>Examples of micro- or macrovesicular steatosis induced by 8 compounds after repeated high dose treatments for 28 days. Diltiazem  =  DIL, disulfiram  =  DSF, colchicine  =  COL, ethionalamide  =  ETH, ethanol  =  ETN and puromycin aminonucleoside  =  PAN, amitriptyline  = AMT. The images were retrieved from TG-GATE database (<a href="http://toxico.nibio.go.jp" target="_blank">http://toxico.nibio.go.jp</a>).</p

Drug induced steatosis and altered metabolic pathways.

<p>Shown is the predicted pattern of gene regulation in drug induced steatosis with respect to the 9 DIS signature genes (see panel C of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g003" target="_blank">Figure 3</a>). Drug induced toxicity enhances fatty acid lipolysis in adipocytes to increase fatty acid influx. This compensates for glycogen depletion in drug induced stress conditions where fatty acids serve the energy needs in hepatocytes; however, lipophilic drugs stored in adipocytes will also become systemically available to increase the burden on drug detoxification. Besides, drug-lipoprotein-complexes enter the liver via LDLR. Insufficient drug detoxification induces ER, mitochondrial and cellular stress that leads to LD biogenesis and associated with it are altered glucose and lipid metabolism. LD may function as temporary storage organelles for drug-TAG complexes thereby reducing the burden on detoxification and ROS production. Mitochondrial dysfunction leads to ischemia and insufficient ROS detoxification propagates lipotoxicity and inflammation. To sustain detoxification amino acids (AAs) and other molecular building blocks are diverted for synthesis of induced enzymes. This in turn affects cellular homeostasis including VLDL secretion and augments lipid accumulation in drug induced steatosis.</p

Biological processes of high frequency gene signatures.

<p>Depicted are common regulated genes (36 genes) after single and repeated treatment with high frequency using the information obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114085#pone-0114085-g008" target="_blank">Figure 8</a>. In all, 9 genes were commonly regulated with regard to their altered metabolic function.</p