Plasma miR-218a-5p as a biomarker for acute cholestatic liver injury in rats and investigation of its pathophysiological roles.

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.

Recent progress in the use of microRNAs as biomarkers for drug-induced toxicities in contrast to traditional biomarkers: A comparative review.

microRNAs (miRNAs) are small non-coding RNAs with 18-25 nucleotides. They play key regulatory roles in versatile biological process including development and apoptosis, and in disease pathogenesis, for example carcinogenesis, by negatively regulating gene expression. miRNAs often exhibit characteristics suitable for biomarkers such as tissue-specific expression patterns, high stability in serum/plasma, and change in abundance in circulation immediately after toxic injury. Since the discovery of circulating miRNAs in extracellular biological fluids in 2008, there have been many reports on the use of miRNAs as biomarkers for various diseases including cancer and organ injury in humans and experimental animals. In this review article, we have summarized the utility and limitation of circulating miRNAs as safety/toxicology biomarkers for specific tissue injuries including liver, skeletal muscle, heart, retina, and pancreas, by comparing them with conventional protein biomarkers. We have also covered the discovery of miRNAs in serum/plasma and their stability, the knowledge of which is essential for understanding the kinetics of miRNA biomarkers. Since numerous studies have reported the use of these circulating miRNAs as safety biomarkers with high sensitivity and specificity, we believe that circulating miRNAs can promote pre-clinical drug development and improve the monitoring of tissue injuries in clinical pharmacotherapy.

Expression of the chrXq27.3 miRNA cluster in recurrent ovarian clear cell carcinoma and its impact on cisplatin resistance.

Ovarian clear cell carcinoma (OCCC) is a histological subtype of epithelial ovarian cancer and exhibits dismal prognosis due to chemoresistance. Moreover, only few effective therapeutic options exist for patients with recurrent OCCC, and an understanding of its molecular characteristics is essential for the development of novel therapeutic approaches. In the present study, we investigated unique MicroRNAs (miRNA) profiles in recurrent/metastatic OCCC and the role of miRNAs in cisplatin resistance. Comprehensive miRNA sequencing revealed that expression of several miRNAs, including miR-508-3p, miR-509-3p, miR-509-3-5p, and miR-514a-3p was remarkably less in recurrent cancer tissues when compared with that in paired primary cancer tissues. These miRNAs are located in the chrXq27.3 region on the genome. Moreover, its expression was negative in omental metastases in two patients with advanced OCCC. In vitro analyses revealed that overexpression of miR-509-3p and miR-509-3-5p reversed cisplatin resistance and yes-associated protein 1 (YAP1) was partially responsible for the resistance. Immunohistochemistry revealed that YAP1 expression was inversely correlated with the chrXq27.3 miRNA cluster expression. In conclusion, these findings suggest that alteration of the chrXq27.3 miRNA cluster could play a critical role in chemoresistance and miRNAs in the cluster and their target genes can be potential therapeutic targets.

Models of Idiosyncratic Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a leading cause of attrition during the early and late stages of drug development and after a drug is marketed. DILI is generally classified as either intrinsic or idiosyncratic. Intrinsic DILI is dose dependent and predictable (e.g., acetaminophen toxicity). However, predicting the occurrence of idiosyncratic DILI, which has a very low incidence and is associated with severe liver damage, is difficult because of its complex nature and the poor understanding of its mechanism. Considering drug metabolism and pharmacokinetics, we established experimental animal models of DILI for 14 clinical drugs that cause idiosyncratic DILI in humans, which is characterized by the formation of reactive metabolites and the involvement of both innate and adaptive immunity. On the basis of the biomarker data obtained from the animal models, we developed a cell-based assay system that predicts the potential risks of drugs for inducing DILI. These findings increase our understanding of the mechanisms of DILI and may help predict and prevent idiosyncratic DILI due to certain drugs.

An in vitro coculture system of human peripheral blood mononuclear cells with hepatocellular carcinoma-derived cells for predicting drug-induced liver injury.

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.

Pharmacological evidence for the involvement of ryanodine receptors in halothane-induced liver injury in mice.

Drug-induced liver injury (DILI) is a major safety concern in drug development. Halothane (HAL), an inhaled anesthetic, induces severe and idiosyncratic liver injury. Ryanodine receptors (RyR) are major intracellular calcium release channels found on the plasma membrane of the endoplasmic reticulum (ER). It has been reported that disordered hepatic calcium homeostasis is a feature of HAL-induced liver injury (HILI) in guinea pigs. However, there are no reports on whether RyR could mediate the pathogenesis of HILI. The aim of the present study was to investigate the effect of RyR on HILI. Ryanodine (RYA, RyR agonist, 50 µg/kg, i.p.) was administered to BALB/c female mice 1 h before HAL administration (15 mmol/kg, i.p.), which significantly elevated plasma transaminase levels and induced severe hepatic inflammation and necrosis. In contrast, dantrolene sodium (DAN, RyR antagonist) treatment significantly suppressed HILI in a dose- and time-dependent manner and alleviated liver damage. The number of infiltrated neutrophils in the liver were higher in the group treated with HAL + RYA than in the group treated with HAL alone, while DAN treatment decreased neutrophil infiltration in HILI. The hepatic mRNA levels of proinflammatory cytokines; chemokines; and factors related to danger signals, neutrophils, oxidative and ER stress, pro-apoptosis, and RyR were significantly increased with RYA pretreatment, whereas these levels were decreased with DAN treatment. These results suggest that RYA exacerbates HILI, and DAN exerts a protective effect against HILI. Hence, our study provides a novel insight regarding the effect of RyR in the mechanism underlying HILI.

Exacerbation of gefitinib-induced liver injury by glutathione reduction in mice.

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.

Macrophage-derived extracellular vesicles regulate concanavalin A-induced hepatitis by suppressing macrophage cytokine production.

Acute liver failure is a clinical syndrome of severe hepatic dysfunction. Immune cells play an important role in acute liver failure. In recent years, the immunoregulatory function of extracellular vesicles (EVs) has been reported; therefore, it is inferred that EVs play a role in immune-mediated hepatitis. In this study, we investigated the immunoregulatory function of EVs in concanavalin A (Con A)-induced hepatitis. The mouse model was prepared by a single intravenous administration of 15 mg/kg Con A, in which there was a significant increase in the serum EVs number. In an in vitro study, the number of secreted EVs was also significantly increased in Con A-treated RAW264.7 cells, a mouse macrophage cell line, but not in Hepa1-6 cells, a mouse hepatoma cell line. In an in vitro EVs treatment study, EVs from Con A-treated mouse serum and Con A-treated RAW264.7 cells suppressed inflammatory cytokine production in Con A-stimulated RAW264.7 cells. miRNA sequencing analysis showed that the expression of mmu-miR-122-5p and mmu-miR-148a-3p was commonly increased in these EVs and EVs-treated cells. The pathways enriched in the predicted miRNA target genes included inflammatory response pathways. The mRNA levels of the target genes in these pathways (mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt and Rho/Rho-associated coiled-coil containing protein kinase pathways) were decreased in the EVs-treated cells. In an in vivo RNA interference study, the knockdown of liver RAB27A, an EVs secretion regulator, significantly exacerbated Con A-induced hepatitis. These data suggest that macrophage-derived EVs play an important role in Con A-induced hepatitis through immunoregulation.

Exploration of small RNA biomarkers for testicular injury in the serum exosomes of rats.

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats.

Unique miRNA profiling of squamous cell carcinoma arising from ovarian mature teratoma: comprehensive miRNA sequence analysis of its molecular background.

Owing to its rarity, the carcinogenesis and molecular biological characteristics of squamous cell carcinoma arising from mature teratoma remain unclear. This study aims to elucidate the molecular background of malignant transformation from the aspects of microRNA (miRNA) profiling. We examined 7 patients with squamous cell carcinoma and 20 patients with mature teratoma and extracted their total RNA from formalin-fixed paraffin-embedded tissues. Then we prepared small RNA libraries and performed comprehensive miRNA sequencing. Heatmap and principal component analysis revealed markedly different miRNA profiling in cancer, normal ovarian and mature teratoma tissues. Then we narrowed down cancer-related miRNAs, comparing paired-cancer and normal ovaries. Comparisons of cancer and mature teratoma identified two markedly upregulated miRNAs (miR-151a-3p and miR-378a-3p) and two markedly downregulated miRNAs (miR-26a-5p and miR-99a-5p). In addition, these findings were validated in fresh cancer tissues of patient-derived xenograft (PDX) models. Moreover, several miRNAs, including miR-151a-3p and miR-378a-3p, were elevated in the murine plasma when tumor tissues were enlarged although miR-26a-5p and miR-99a-5p were not elucidated in the murine plasma. Finally, we performed target prediction and functional annotation analysis in silico and indicated that targets genes of these miRNAs markedly correlated with cancer-related pathways, including 'pathway in cancer' and 'cell cycle'. In conclusion, this is the first study on miRNA sequencing for squamous cell carcinoma arising from mature teratoma. The study identified four cancer-related miRNAs that were considered to be related to the feature of malignant transformation. Moreover, miRNAs circulating in the murine plasma of the PDX model could be novel diagnostic biomarkers.

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3.

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.

Establishment of a mouse model of troglitazone-induced liver injury and analysis of its hepatotoxic mechanism.

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.

Experimental Evidence of Liver Injury by BSEP-Inhibiting Drugs With a Bile Salt Supplementation in Rats.

The bile salt export pump (BSEP, ABCB11) mediates bile acid efflux from hepatocytes into bile. Although the inhibition of BSEP has been implicated as an important mechanism of drug-induced liver injury (DILI), liver injury caused by BSEP-inhibiting drugs is rarely reproduced in experimental animals, probably due to species differences in bile acid composition between humans and rodents. In this study, we tested whether supplementation with chenodeoxycholic acid (CDCA) sodium, a hydrophobic bile salt, could sensitize rats to liver injury caused by a BSEP-inhibiting drug. A potent BSEP inhibitor, ketoconazole (KTZ), which is associated with clinical DILI, was intragastrically administered simultaneously with CDCA at a nontoxic dose once a day for 3 days. Plasma transaminase levels significantly increased in rats receiving CDCA+KTZ, whereas neither treatment with CDCA alone, KTZ alone nor a combination of CDCA and miconazole, a safe analog to KTZ, induced liver injury. In CDCA+KTZ-treated rats, most bile acid species in the liver significantly increased compared with treatment with vehicle or CDCA alone, suggesting that KTZ administration inhibited bile acid excretion. Furthermore, hepatic mRNA expression levels of a bile acid synthesis enzyme, Cyp7a1, and a basolateral bile salt influx transporter, Ntcp, decreased, whereas a canalicular phosphatidylcholine flippase, Mdr2, increased in the CDCA+KTZ group to compensate for hepatic bile acid accumulation. In conclusion, we found that oral CDCA supplementation predisposed rats to KTZ-induced liver injury due to the hepatic accumulation of bile acids. This method may be useful for assessing the potential of BSEP-inhibiting drugs inducing liver injury in vivo.

Establishment and characterization of a mouse model of rhabdomyolysis by coadministration of statin and fibrate.

Rhabdomyolysis is characterized by elevation of plasma creatine phosphokinase (CPK) level, and multiple organ disorders, especially renal failure, as well as approximately 50% of acquired rhabdomyolysis are caused by pharmaceuticals. Statins are known to cause rhabdomyolysis, and its incidence is ≥10 times higher with coadministration of statin and fibrate. The purpose of this study is to establish a mouse model of drug-induced rhabdomyolysis by coadministration of statin and fibrate to clarify the mechanisms of its myotoxicity. We administered lovastatin (LV) and gemfibrozil (GF) with a glutathione synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), to C57BL/6 J female mice once daily for 3 days. The plasma levels of CPK and aspartate aminotransferase (AST) were prominently increased, and the increase in plasma miR-206-3p and miR-133-3p levels, not the increase of miR-122-5p and miR-208-3p levels, suggested skeletal muscle-specific toxicity. The caspase 3/7 activity and mRNA levels of oxidative stress-related factors were elevated in skeletal muscle. Pharmacokinetic parameters showed that blood levels of statin were significantly increased by coadministered GF. The possibility of kidney injury was examined as in clinical rhabdomyolysis. In histological examination, vacuoles were observed in renal proximal tubules, and the plasma renal injury marker, lipocalin 2/neutrophil gelatinase-associated lipocalin (Lcn2/Ngal), was markedly increased in the mice coadministered LV and GF, suggesting mild complications of acute kidney injury. A quantitative comparison of the myotoxic potential of various statins was successfully performed using the present method. In this study, a rhabdomyolysis mouse model was established by coadministration of the clinically using statin and fibrate. This mouse model may be useful to identify drugs that have high risk for rhabdomyolysis.

Acute kidney injury model established by systemic glutathione depletion in mice.

Glutathione (GSH) is one of the most extensively studied tripeptides. The roles for GSH in redox signaling, detoxification of xenobiotics and antioxidant defense have been investigated. A drug-induced rhabdomyolysis mouse model was recently established in L-buthionine-(S,R)-sulfoximine (BSO; a GSH synthesis inhibitor)-treated normal mice by co-administration of antibacterial drug and statin. In these models, mild kidney injury was observed in the BSO only-treated mice. Therefore, in this study, we studied kidney injury in the GSH-depleted mouse. BSO was intraperitoneally administered twice a day for 7 days to normal mice. The maximum level of plasma creatine phosphokinase (351 487 ± 53 815 U/L) was shown on day 8, and that of aspartate aminotransferase was shown on day 6. Increased levels of blood urea nitrogen, plasma creatinine, urinary kidney injury molecule-1 and urinary creatinine were observed. An increase of mRNA expression level of renal lipocalin 2/neutrophil gelatinase-associated lipocalin was observed. Degeneration and necrosis in the skeletal muscle and high concentrations of myoglobin (Mb) in blood (347-203 925 ng/mL) and urine (2.5-68 583 ng/mL) with large interindividual variability were shown from day 5 of BSO administration. Mb-stained regions in the renal tubule and renal cast were histologically observed. In this study, the GSH-depletion treatment established an acute kidney injury mouse model due to Mb release from the damaged skeletal muscle. This mouse model would be useful for predicting potential acute kidney injury risks in non-clinical drug development.

A scrutiny of circulating microRNA biomarkers for drug-induced tubular and glomerular injury in rats.

Drug-induced acute kidney injury (AKI) is a frequent cause of adverse drug reaction. Serum creatinine (CRE) and blood urea nitrogen (BUN) are widely used as standard biomarkers for kidney injury; however, the sensitivity and specificity are considered to be low. In recent years, circulating microRNA (miRNAs) have been attracting considerable attention as novel biomarkers for organ injury, but there are currently no established miRNA biomarkers for drug-induced AKI. The present study aimed to identify plasma miRNAs that may enable early and specific detection of drug-induced tubular and glomerular injury through next-generation sequencing analysis. Six-week old male Sprague-Dawley (SD) rats were intravenously administered cisplatin (CSP, 6 mg/kg) and gentamicin (GEN, 120 mg/kg) to induce tubular injury. To create glomerular injury models, puromycin (PUR, 120 mg/kg) and doxorubicin (DOX, 7.5 mg/kg) were intravenously administered, and these models were always accompanied by tubular damage. Small RNA-sequencing was performed to analyze time-dependent changes in the plasma miRNA profiles. The cluster analyses showed that there were distinct plasma miRNA profiles according to the types of injury, and the changes reflected the progress of renal damages. In the differential analysis, miR-3473 was specifically up-regulated in the glomerular injury models. miR-143-3p and miR-122-5p were commonly down-regulated in all models, and the changes were earlier than the traditional biomarkers, such as plasma CRE and BUN. These data indicated that changes in the specific miRNAs in plasma may enable the early and sensitive detection of tubular and glomerular injuries. The present study suggests the potential utility of plasma miRNAs in the early and type-specific detection of drug-induced AKI.

Strain and interindividual differences in lamotrigine-induced liver injury in mice.

Lamotrigine (LTG) has been widely prescribed as an antipsychotic drug, although it causes idiosyncratic drug-induced liver injury in humans. LTG is mainly metabolized by UDP-glucuronosyltransferase, while LTG undergoes bioactivation by cytochrome P450 to a reactive metabolite; it is subsequently conjugated with glutathione, suggesting that reactive metabolite would be one of the causes for LTG-induced liver injury. However, there is little information regarding the mechanism of LTG-induced liver injury in both humans and rodents. In this study, we established an LTG-induced liver injury mouse model through co-administration with LTG and a glutathione synthesis inhibitor, l-buthionine-(S,R)-sulfoximine. We found an increase in alanine aminotransferase (ALT) levels (>10 000 U/L) in C57BL/6J mice, with apparent interindividual differences. On the other hand, a drastic increase in ALT was not noted in BALB/c mice, suggesting that the initiation mechanism would be different between the two strains. To examine the cause of interindividual differences, C57BL/6J mice that were co-administered LTG and l-buthionine-(S,R)-sulfoximine were categorized into three groups based on ALT values: no-responder (ALT <100 U/L), low-responder (100 U/L < ALT < 1000 U/L) and high-responder (ALT >1000 U/L). In the high-responder group, induction of hepatic oxidative stress, inflammation and damage-associated molecular pattern molecules in mRNA was associated with vacuolation and karyorrhexis in hepatocytes. In conclusion, we demonstrated that LTG showed apparent strain and interindividual differences in liver injuries from the aspects of initiation and exacerbation mechanisms. These results would support interpretation of the mechanism of LTG-induced liver injury observed in humans.

Comparative hepatic transcriptome analyses revealed possible pathogenic mechanisms of fasiglifam (TAK-875)-induced acute liver injury in mice.

Fasiglifam (TAK-875), a G protein-coupled receptor 40 (GPR40) agonist, was a drug candidate for type 2 diabetes. However, its development was terminated in phase 3 trials due to liver safety concerns. Although TAK-875 was reported to inhibit hepatobiliary transporters and disturb bile acid disposition, pathogenic mechanisms of TAK-875-induced liver injury are not fully understood. In this study, we sought to identify the mechanisms with a hepatic genome-wide transcriptomic analysis in a murine model. We demonstrated that, among the three GPR40 agonists, TAK-875, AMG-837, and TUG-770, only TAK-875 induced acute liver injury in mice. Transcriptome profiles of TAK-875-exposed liver was compared with those of non-hepatotoxic analogues AMG-837 and TUG-770 as negative controls and those of classical hepatotoxicants concanavalin A and carbon tetrachloride as positive controls. The comparative hepatic transcriptome analyses revealed the enrichment of genes involved in inflammation, endoplasmic reticulum (ER) stress, apoptosis, and hepatic lipid accumulation, suggesting that these events play pathophysiologic roles in the development of TAK-875-induced liver injury. These results were validated by quantitative PCR with significant changes in chemokines, danger signals, ER stress mediators, proapoptotic factors, and hepatic steatosis markers only in TAK-875-exposed liver. Pretreatment of TAK-875-administered mice with an ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated the liver injury. Consistent with the in vivo study, pretreatment of HepG2 cells with 4-PBA significantly improved the decrease of cell viability induced by TAK-875. In conclusion, by a comprehensive transcriptomic analysis, we found multiple possible processes that contribute to TAK-875-induced acute liver injury in mice.

Identification of Specific MicroRNA Biomarkers in Early Stages of Hepatocellular Injury, Cholestasis, and Steatosis in Rats.

Recently, studies on circulating microRNAs (miRNAs) as potential biomarkers of drug-induced liver injury (DILI) have received increasing attention. It has been demonstrated that miR-122 and miR-192, which are liver enriched, could be potential biomarkers of DILI; however, these miRNAs cannot discern types of injuries. In the present study, we comprehensively analyzed time-dependent plasma miRNA profiles in rats with drug- or chemical-induced hepatocellular injury, cholestasis, and steatosis with high-throughput miRNA sequencing. To enable the comparison of miRNA expression levels between DILI models with different severity and peak time of injuries, the stages of injury were defined as early, middle, and late, according to cluster patterns of miRNA expression profiles. Through differential analysis, we characterized miRNAs that were specifically up- or down-regulated in each DILI model. Several miRNAs were dramatically changed earlier than traditional biomarkers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). For example, in an acetaminophen (APAP)-induced hepatocellular injury model, plasma let-7b-5p was up-regulated as early as 3 h after dosing, whereas a significant change in ALT level was observed at 12 h. We then focused on the DILI type-specific miRNAs in plasma that were up-regulated at the early stage of injury. RT-qPCR analysis validated that let-7b-5p and miR-1-3p for hepatocellular injury, miR-143-3p and miR-218a-5p for cholestasis, and miR-320-3p for steatosis models showed significant increases in the early stage of the injuries. The present study suggests the utility of miRNAs as specific biomarkers for the early detection of DILI.

Comprehensive analysis of serum microRNAs in hepatic sinusoidal obstruction syndrome (SOS) in rats: implication as early phase biomarkers for SOS.

Sinusoidal obstruction syndrome (SOS) is a liver injury caused by clinical chemotherapy, of which pathogenesis is associated with the damage in liver sinusoidal endothelial cells (LSEC). The unavailability of appropriate specific biomarkers for the early diagnosis of SOS may potentially overlook SOS patients. In this study, we sought to find serum microRNAs (miRNAs) as non-invasive biomarkers for investigating SOS in rats. Male Sprague-Dawley rats were orally administered monocrotaline, and then, their livers and sera were collected after 0.25, 0.5, 1, 2, 4, and 7 days. The rats showed a typical SOS phenotype including LSEC damage as early as day 0.25, followed by severe hepatocyte damage on day 2, and developed hepatic fibrosis from days 4 to 7. The miRNA microarray showed that 65 serum miRNAs were increased in their levels on day 0.25, when LSEC damage was observed, while hepatocyte damage was absent. Among the increased serum miRNAs on days 0.25-1, miR-511-3p was enriched in normal LSECs and miR-21-5p was in both LSECs and hepatocytes, suggesting that they were released into blood from the damaged LSECs. The miR-122-5p, miR-192-5p, and miR-101b-3p, which were enriched in hepatocytes, reached the highest levels in serum on day 2, suggesting their utility as indicators for hepatocyte damage. No miRNA showing an increasing trend from days 4 to 7 was found as a biomarker for fibrosis. In conclusion, we found that LSEC-derived miR-21-5p and especially miR-511-3p in serum would serve as early phase biomarkers for SOS in response to LSEC damage.