Shingo Oda

MicroRNAs (miRNA) have received considerable attention as potential biomarkers for drug‐induced liver injury. We recently reported that the plasma levels of miR‐143‐3p and miR‐218a‐5p increased in severe cholestasis in rats. This study aimed to investigate whether these miRNAs increase in a severity‐dependent manner and to elucidate their pathophysiological roles in cholestasis. Male Sprague–Dawley rats were orally administered different doses of α‐naphthylisothiocyanate or 4,4‐methylenedianiline to induce acute cholestasis. They were also orally administered acetaminophen or thioacetamide to induce hepatocellular injury. We found that plasma miR‐143‐3p and miR‐218a‐5p levels increased in a dose‐dependent manner in cholestatic rats but not in hepatocellular injury. Bioinformatic analysis provided putative target genes of hsa‐miR‐218‐5p, rno‐miR‐218a‐5p, and mmu‐miR‐218‐5p, among which GNAI2, PPP1CB, and PPP2R5A were experimentally validated as their direct target genes in human cholangiocyte line MMNK‐1. Proliferation of MMNK‐1 cells was significantly suppressed after overexpression of miR‐218‐5p and transduction of siRNAs for GNAI2, PPP1CB, and PPP2R5A. In the cholestatic livers of rats, Ppp1cb and Ppp2r5a expression levels decreased, whereas Gnai2 expression levels increased compared with those in vehicle‐treated rats, suggesting that Ppp1cb and Ppp2r5a may be under the control of miR‐218a‐5p in vivo. In conclusion, our data suggest that miR‐218(a)‐5p is involved in the suppression of cholangiocyte proliferation by inhibiting the expression of PPP1CB and PPP2R5A, thereby contributing to the pathogenesis of cholestasis; and miR‐218a‐5p leaks into the plasma probably from damaged cholangiocytes in a severity‐dependent manner in rats. Therefore, miR‐218a‐5p overexpression could be one of the underlying mechanisms of acute cholestatic liver injury in rats.

Preventing clinical drug-induced liver injury (DILI) remains a major challenge, because DILI develops via multifactorial mechanisms. Immune and inflammatory reactions are considered important mechanisms of DILI; however, biomarkers from in vitro systems using immune cells have not been comprehensively studied. The aims of this study were (1) to identify promising biomarker genes for predicting DILI in an in vitro coculture model of peripheral blood mononuclear cells (PBMCs) with a human liver cell line, and (2) to evaluate these genes as predictors of DILI using a panel of drugs with different clinical DILI risk. Transcriptome-wide analysis of PBMCs cocultured with HepG2 or differentiated HepaRG cells that were treated with several drugs revealed an appropriate separation of DILI-positive and DILI-negative drugs, from which 12 putative biomarker genes were selected. To evaluate the predictive performance of these genes, PBMCs cocultured with HepG2 cells were exposed to 77 different drugs, and gene expression levels in PBMCs were determined. The MET proto-oncogene receptor tyrosine kinase (MET) showed the highest area under the receiver-operating characteristic curve (AUC) value of 0.81 among the 12 genes with a high sensitivity/specificity (85/66%). However, a stepwise logistic regression model using the 12 identified genes showed the highest AUC value of 0.94 with a high sensitivity/specificity (93/86%). Taken together, we established a coculture system using PBMCs and HepG2 cells and selected biomarkers that can predict DILI risk. The established model would be useful in detecting the DILI potential of compounds, in particular those that involve an immune mechanism.

Gefitinib (GEF) is the first selective tyrosine kinase inhibitor of epidermal growth factor receptor. It is associated with the occurrence of clinical drug-induced liver injury. Although GEF is metabolized to chemically reactive metabolites by cytochrome P450 3A and 1A enzymes and then conjugated to glutathione (GSH), whether these reactive metabolites contribute to GEF-induced toxicity remains unknown. In this study, we investigated whether GSH depletion can sensitize mice to liver injury caused by GEF. Male C57BL/6J mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO) at 700 mg/kg to inhibit GSH synthesis and then orally administered GEF at 500 mg/kg every 24 hr for 4 consecutive days. The coadministration of BSO and GEF increased plasma alanine aminotransferase (ALT) levels to approximately 700 U/L and 1600 U/L at 72 and 96 hr after the first administration, respectively, whereas the increase in plasma ALT levels in mice receiving GEF at 500 mg/kg alone was limited, suggesting that GSH plays a protective role in GEF-induced liver injury. Histological examination showed nuclear karyorrhexis and sporadic single hepatocyte death in the livers of BSO+GEF coadministered mice. In these mice, the hepatic expression levels of heme oxygenase 1 (Hmox1) and metallothionein 2 (Mt2) mRNA, caspase 3/7 enzymatic activity, and the amounts of 2-thiobarbiuric acid reactive substances were significantly increased, suggesting the presence of oxidative stress, which may be associated with hepatocellular death. Together, these results show that oxidative stress as well as the reactive metabolites of GEF are involved in GEF-induced liver injury in GSH-depleted mice.

Drug‐induced liver injury is a major problem in drug development and clinical drug therapy. Troglitazone (TGZ), a thiazolidinedione antidiabetic drug for the treatment of type II diabetes mellitus, was found to induce rare idiosyncratic severe liver injury in patients, which led to its withdrawal in 2000. However, in normal experimental animals in vivo TGZ has never induced liver injury. To explore TGZ hepatotoxic mechanism, we established a novel mouse model of TGZ‐induced liver injury. Administration of BALB/c female mice with a single intraperitoneal TGZ dose (300 mg/kg) significantly elevated alanine aminotransferase and aspartate aminotransferase levels 6 hours after the treatment. The ratio of oxidative stress marker glutathione/disulfide glutathione was significantly decreased. The increased hepatic mRNA levels of inflammation‐ and oxidative stress‐related factors were observed in TGZ‐treated mice. Subsequently, hepatic transcriptome profiles of TGZ‐exposed liver were compared with those of non‐hepatotoxic rosiglitazone. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was activated in TGZ‐induced liver injury. The activation of the JAK/STAT pathway promoted phosphorylation of STAT3 in TGZ‐treated mice. Consequently, upregulation of STAT3 activation increased mRNA levels of its downstream genes. In conclusion, a single intraperitoneal dose of TGZ exposure could induce liver injury in BALB/c female mice and, by a hepatic transcriptomic analysis, we found that the activation of JAK/STAT pathway might be related to TGZ‐induced hepatotoxicity.