A simple transcriptomic signature able to predict drug-induced hepatic steatosis

Abstract

It is estimated that only a few marketed drugs are able to directly induce liver steatosis. However, many other drugs may exacerbate or precipitate fatty liver in the presence of other risk factors or in patients prone to non-alcoholic fatty liver disease. On the other hand, current in vitro tests for drug-induced steatosis in preclinical research are scarce and not very sensitive or reproducible. In the present study, we have investigated the effect of well-characterized steatotic drugs on the expression profile of 47 transcription factors (TFs) in human hepatoma HepG2 cells and found that these drugs are able to up- and down-regulate a substantial number of these factors. Multivariate data analysis revealed a common TF signature for steatotic drugs, which consistently and significantly repressed FOXA1, HEX and SREBP1C in cultured cells. This signature was also observed in the livers of rats and in cultured human hepatocytes. Therefore, we selected these three TFs as predictive biomarkers for iatrogenic steatosis. With these biomarkers, a logistic regression analysis yielded a predictive model, which was able to correctly classify 92 % of drugs. The developed algorithm also predicted that ibuprofen, nifedipine and irinotecan are potential steatotic drugs, whereas troglitazone is not. In summary, this is a sensitive, specific and simple RT-PCR test that can be easily implemented in preclinical drug development to predict drug-induced steatosis. Our results also indicate that steatotic drugs affect expression of both common and specific subsets of TF and lipid metabolism genes, thus generating complex transcriptomic responses that cause or contribute to steatosis in hepatocytes.

Repression of the Nuclear Receptor Small Heterodimer Partner by Steatotic Drugs and in Advanced Nonalcoholic Fatty Liver Disease

“…The concentrations selected were always subcytotoxic (#IC 10 ), thus avoiding acute necrotic or apoptotic side effects (Benet et al, 2014). Moreover, we have previously shown that in these conditions, the selected steatotic drugs trigger lipid accumulation in HepG2 cells (Benet et al, 2014). We observed that most steatotic drugs (valproate, doxycycline, tetracycline, cyclosporine A, and tianeptine) negatively affected SHP expression (Fig.…”

“…Human hepatoma HepG2 cells were incubated for 24 hours with different drugs previously characterized and classified as nonhepatotoxic, hepatotoxic, phospholipidosic, and steatotic. The concentrations selected were always subcytotoxic (#IC 10 ), thus avoiding acute necrotic or apoptotic side effects (Benet et al, 2014). Moreover, we have previously shown that in these conditions, the selected steatotic drugs trigger lipid accumulation in HepG2 cells (Benet et al, 2014).…”

“…Amitriptyline, cyclosporin A, doxycycline, fluoxetine, flutamide, ketotifen, simvastatin, tamoxifen, tetracycline, tianeptine, tilorone, valproate, U0126 [1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene], and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] were acquired from Sigma-Aldrich (Madrid, Spain). Steatotic drugs and nonsteatotic compounds were chosen on the basis of previous information on their steatotic, phospholipidosic, hepatotoxic, or nonhepatotoxic effect (Benet et al, 2014). Stock solutions were prepared in dimethylsulfoxide (DMSO) or water and were diluted in a culture medium.…”

“…Then, liver tissues were homogenized twice for 10 seconds at 6000 rpm at 4°C in a Precellys 24 dual system equipped with a Criolys cooler. Tubes were centrifuged at 3000g for 5 minutes at 4°C, and total RNA was extracted from supernatants and reverse transcribed as described (Benet et al, 2014). Diluted cDNA was amplified in a LightCycler 480 Instrument (Roche Applied Science, Barcelona, Spain) using LightCycler DNA Master SYBR Green I (Roche Applied Science).…”

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

“…To further mature the cells, we investigated the role of cAMP signalling, as studies using hepatic cell lines have shown that activation of this pathway induces hepatic gene expression, in part through the induction of the peroxisome proliferator-activated receptor γ co-activator 1α (PGC1A; PPARGC1A -Human Gene Nomenclature Database), a co-activator that functions together with HNF4A to regulate the expression of many genes involved in hepatocyte function (Bell and Michalopoulos, 2006;Arpiainen et al, 2008;Benet et al, 2010;Dankel et al, 2010). Treatment of the aggregates with 8-bromoadenosine-3Ј,5Ј-cyclic monophosphate (8-Br-cAMP), a cell-permeable analogue of cAMP, from days 32 to 44 of culture significantly enhanced the expression of PGC1A (15-fold), G6P (25-fold) and TAT (33-fold) but not that of HNF4A (Fig.…”