Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures

Abstract

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Keywords: Adverse outcome pathway.; BSEP; Bosentan; Cholestasis; HepaRG; Metabolomics; Proteomics; Transcriptomics.

Fig. 1

(A) Immunostaining of BSEP (green) and nuclear staining (blue) of differentiated HepaRG cells showing BSEP localization. (B) Normalized relative gene expression (microarray) of BSEP (coded by the ABCCB11) in HepaRG cells exposed to bosentan (* Student t-test p < 0.05). (C) Accumulation of fluorescent probe substrate for BSEP (green) in bile canicular structures within the hepatocyte-like cell cluster of untreated HepaRG cells and cells exposed to bosentan (24 h/IC₁₀ concentration). Phase contrast (PhC) images show hepatocyte-like clusters of HepaRG cell culture surrounded by biliary like cells. (D) Quantification of fluorescence intensity in bile pockets of the bile pocket area in untreated cells and cells exposed to bosentan (24 h/IC₁₀ concentration) (* Student t-test p < 0.05). (E) HPLC-MS/MS quantification of cholic acid (CA) and glycocholic acid (GCA) (pmol/cm²) in HepaRG cell pellets of control samples and samples from cells exposed to bosentan.

Fig. 2

(A) Principle component analysis (PCA) plot of microarray data sets of HepaRG cells exposed for 1 h, 24 h and 24 h+72 h to 3 concentrations of bosentan, namely IC₁₀, IC₁₀/4 and IC₁₀/10, and respective vehicle controls. (B) Venn diagram of modulated genes in HepaRG cells exposed to different concentrations of bosentan (IC₁₀, IC₁₀/4 and IC₁₀/10) for 1 h, 24 h and 24 h+72 h (gene selection was based on a fold change modulation > 2 when compared to respective controls and Fisher’s p < 0.05).

Fig. 3

Heat map of NR1H4, NR1I2 and NR1I2 genes representing the downregulation of FXR, PXR and CAR in HepaRG cells exposed to bosentan (24 h/IC₁₀ concentration) (A). Heat map of a selection of modulated genes in HepaRG cells exposed to bosentan (24 h/IC₁₀ concentration) that correctly represent the corresponding KE of the AOP for cholestatic liver injury (Vinken et al., 2013) (B). Heat map of tumour necrosis factor (TNF), interleukin 6 (IL6) and interleukin 8 (IL8) in HepaRG cells exposed to bosentan (24 h/IC₁₀ concentration) (C).

Fig. 4

Alterations in metabolite production detected by NMR spectroscopy of media from HepaRG cells exposed to bosentan (24 h/IC₁₀ concentration) compared to control samples (** Student t-test p < 0.01; *** Student t-test p < 0.001).

Fig. 5

In vitro specifications of KE (green) related to the deteriorative cellular response (yellow background) and adaptive cellular response (pink background) of the AOP of cholestasis (Vinken et al., 2013).