Proteomic profiling in incubation medium of mouse,rat and human precision‐cut liver slices for biomarker detection regarding acute drug‐induced liver injury

Drug‐induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision‐cut liver slices (PCLS) exposed to liver injury‐inducing drugs for biomarker identification, using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3‐acetamidophenol, diclofenac and lipopolysaccharide for 24–48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP‐induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron‐regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3‐acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system. Copyright © 2013 John Wiley & Sons, Ltd.     

Prediction of drug‐induced liver injury using keratinocytes

Drug‐induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI‐associated genes such as S100 calcium‐binding protein A and interleukin (IL)‐1β are involved in drug‐induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI‐associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL‐1β basal expression. They also showed a higher inducibility of IL‐1β when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI‐associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI‐associated genes might be involved in the onset and progression of drug‐induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.     

Diagnostic and predictive performance and standardized threshold of traditional biomarkers for drug‐induced liver injury in rats

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug‐induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver‐operating characteristic (ROC) curve, using a free‐access database where 142 hepatotoxic or non‐hepatotoxic compounds were administrated to male rats (n = 5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in‐house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI. Copyright © 2014 John Wiley & Sons, Ltd.     

Prediction of clinically relevant drug‐induced liver injury from structure using machine learning

Drug‐induced liver injury (DILI) is the most common cause of acute liver failure and often responsible for drug withdrawals from the market. Clinical manifestations vary, and toxicity may or may not appear dose‐dependent. We present several machine‐learning models (decision tree induction, k‐nearest neighbor, support vector machines, artificial neural networks) for the prediction of clinically relevant DILI based solely on drug structure, with data taken from published DILI cases. Our models achieved corrected classification rates of up to 89%. We also studied the association of a drug's interaction with carriers, enzymes and transporters, and the relationship of defined daily doses with hepatotoxicity. The results presented here are useful as a screening tool both in a clinical setting in the assessment of DILI as well as in the early stages of drug development to rule out potentially hepatotoxic candidates.     

A modified multiparametric assay using HepaRG cells for predicting the degree of drug‐induced liver injury risk

The approach for predicting the degree of drug‐induced liver injury (DILI) risk was investigated quantitatively in a modified multiparametric assay using HepaRG cells. Thirty‐eight drugs were classified by DILI risk into five categories based on drug labels approved by the Food and Drug Administration (FDA) as follows: withdrawn (WDN), boxed warning (BW), warnings and precautions (WP), adverse reactions (AR), and no match (NM). Also, WP was classified into two categories: high and low concern. Differentiated HepaRG cells were treated with drugs for 24 h. The maximum concentration was set at 100‐fold the therapeutic maximum plasma concentration (C_max). After treatment with drugs, the cell viability, glutathione content, caspase 3/7 activity, lactate dehydrogenase leakage and albumin secretion were measured. As modified cut‐off values of each parameter, the TC₅₀ (toxic concentration that decreased the response by 50%) and EC₂₀₀ (effective concentration giving a response equal to 200% of controls) were calculated. In addition, the toxicity score (total sum score of the cytotoxic level of each parameter) was calculated. This modified multiparametric assay showed an 87% sensitivity and 87% specificity for predicting the DILI risk. The toxicity score showed a good predictive performance for WDN, BW and WP (high concern) categories [cut‐off: score ≥ 1; area under a receiver operating characteristic curve (ROC‐AUC): 0.88], and for WDN and BW categories (cut‐off: score ≥ 3; ROC‐AUC: 0.88). This study newly indicated that the degree of DILI risk might be predictable quantitatively by assessing the toxicity score in the modified multiparametric assay using HepaRG cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Kupffer cell‐mediated exacerbation of methimazole‐induced acute liver injury in rats

Methimazole (MTZ), an anti‐thyroid drug, is known to cause liver injury in humans. It has been demonstrated that MTZ‐induced liver injury in Balb/c mice is accompanied by T helper (Th) 2 cytokine‐mediated immune responses; however, there is little evidence for immune responses associated with MTZ‐induced liver injury in rats. To investigate species differences in MTZ‐induced liver injury, we administered MTZ with a glutathione biosynthesis inhibitor, L‐buthionine‐S,R‐sulfoximine (BSO), to F344 rats and subsequently observed an increase in plasma alanine aminotransferase (ALT) and high‐mobility group box 1 (HMGB1), which are associated with hepatic lesions. The hepatic mRNA expression of innate immune‐related genes significantly increased in BSO‐ and MTZ‐treated rats, but the change in Th2‐related genes was not much greater than the change observed in the previous mouse study. Moreover, an increase in Kupffer cells and an induction of the phosphorylation of extracellular signal‐regulated kinase (ERK)/c‐Jun N‐terminal kinase (JNK) proteins were accompanied by an increase in Toll‐like receptor 4 (TLR4) expression, indicating that Kupffer cell activation occurs through HMGB1‐TLR4 signaling. To elucidate the mechanism of liver injury in rats, gadolinium chloride, which inactivates the function of Kupffer cells, was administered before BSO and MTZ administration. The gadolinium chloride treatment significantly suppressed the increased ALT, which was accompanied by decreased hepatic mRNA expression related to innate immune responses and ERK/JNK phosphorylation. In conclusion, Kupffer cell‐mediated immune responses are crucial factors for the exacerbation of MTZ‐induced liver injury in rats, indicating apparent species differences in the immune‐mediated exacerbation of liver injury between mice and rats. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Association between genetic polymorphisms of SLCO1B1 and susceptibility to methimazole‐induced liver injury

Methimazole (MMI) has been used in the therapy of Grave's disease (GD) since 1954, and drug‐induced liver injury (DILI) is one of the most deleterious side effects. Genetic polymorphisms of drug‐metabolizing enzymes and drug transporters have been associated with drug‐induced hepatotoxicity in many cases. The aim of this study was to investigate genetic susceptibility of the drug‐metabolizing enzymes and drug transporters to the MMI‐DILI. A total of 44 GD patients with MMI‐DILI and 118 GD patients without MMI‐DILI development were included in the study. Thirty‐three single nucleotide polymorphisms (SNPs) in twenty candidate genes were genotyped. We found that rs12422149 of SLCO2B1, rs2032582_AT of ABCB1, rs2306283 of SLCO1B1 and rs4148323 of UGT1A1 exhibited a significant association with MMI‐DILI; however, no significant difference existed after Bonferroni correction. Haplotype analysis showed that the frequency of SLCO1B1*1a (388A521T) was significantly higher in MMI‐DILI cases than that in the control group (OR = 2.21, 95% CI = 1.11‐4.39, P = 0.023), while the frequency of SLCO1B1*1b (388G521T) was significantly higher in the control group (OR = 0.52, 95% CI = 0.29‐0.93, P = 0.028). These results suggested that genetic polymorphisms of SLCO1B1 were associated with susceptibility to MMI‐DILI. The genetic polymorphism of SLCO1B1 may be important predisposing factors for MMI‐induced hepatotoxicity.     

Establishment of a mouse model for amiodarone‐induced liver injury and analyses of its hepatotoxic mechanism

Drug‐induced liver injury (DILI) is the most frequent cause of post‐marketing warnings and withdrawals. Amiodarone (AMD), an antiarrhythmic, presents a risk of liver injury in humans, and its metabolites, formed by cytochrome P450 3A4, are likely more toxic to hepatocytes than AMD is. However, it remains to be clarified whether the metabolic activation of AMD is involved in liver injury in vivo. In this study, to elucidate the underlying mechanisms of AMD‐induced liver injury, mice were administered AMD [1000 mg kg^–1, per os (p.o.)] after pretreatment with dexamethasone [DEX, 60 mg kg^–1, intraperitoneal (i.p.)], which induces P450 expression, once daily for 3 days. The plasma alanine aminotransferase (ALT) levels were significantly increased by AMD administration in the DEX‐pretreated mice, and the liver concentrations of desethylamiodarone (DEA), a major metabolite of AMD, were correlated with the changes in the plasma ALT levels. Cytochrome c release into the hepatic cytosol and triglyceride levels in the plasma were increased in DEX plus AMD‐administered mice. Furthermore, the ratio of reduced glutathione to oxidized glutathione disulfide in the liver significantly decreased in the DEX plus AMD‐administered mice. The increase of ALT levels was suppressed by treatment with gadolinium chloride (GdCl₃), which is an inhibitor of Kupffer cell function. From these results, it is suggested that AMD and/or DEA contribute to the pathogenesis of AMD‐induced liver injury by producing mitochondrial and oxidative stress and Kupffer cell activation. This study proposes the mechanisms of AMD‐induced liver injury using an in vivo mouse model. Copyright © 2015 John Wiley & Sons, Ltd.     

Toxicological role of an acyl glucuronide metabolite in diclofenac‐induced acute liver injury in mice

The acyl glucuronide (AG) metabolites of carboxylic acid‐containing drugs are potentially chemically reactive and are suggested to be implicated in toxicity, including hepatotoxicity, nephrotoxicity and drug hypersensitivity reactions. However, it remains unknown whether AG formation is related to toxicity in vivo . In this study, we sought to determine whether AG is involved in the pathogenesis of liver injury using a mouse model of diclofenac (DIC)‐induced liver injury. Mice that were administered DIC alone exhibited significantly increased plasma alanine aminotransferase levels, whereas mice that were pretreated with the UDP‐glucuronosyltransferase inhibitor (−)‐borneol (BOR) exhibited suppressed alanine aminotransferase levels at 3 and 6 h after DIC administration although not significant at 12 h. The plasma DIC‐AG concentrations were significantly lower in BOR‐ and DIC‐treated mice than in mice treated with DIC alone. The mRNA expression levels of chemokine (C‐X‐C motif) ligand 1 (CXCL1), CXCL2 and the neutrophil marker CD11b were reduced in the livers of mice that had been pretreated with BOR compared to those that had been administered DIC alone, whereas mRNA expression of the macrophage marker F4/80 was not altered. An immunohistochemical analysis at 12 h samples revealed that the numbers of myeloperoxidase‐ and lymphocyte antigen 6 complex‐positive cells that infiltrated the liver were significantly reduced in BOR‐ and DIC‐treated mice compared to mice that were treated with DIC alone. These results indicate that DIC‐AG is partly involved in the pathogenesis of DIC‐induced acute liver injury in mice by activating innate immunity and neutrophils. Copyright © 2016 John Wiley & Sons, Ltd.     

Tissue influx of neutrophils and monocytes is delayed during development of trovafloxacin‐induced tumor necrosis factor‐dependent liver injury in mice

Idiosyncratic drug‐induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)‐induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX‐induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX‐treated mice, but even more so in control mice treated with the non‐DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh‐ and LVX‐treated mice whereas the amounts in TVX‐treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh‐ and LVX‐treated mice was markedly reduced or even absent in TVX‐treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti‐inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.     

Usefulness of urinary kidney injury molecule‐1 (Kim‐1) as a biomarker for cisplatin‐induced sub‐chronic kidney injury

We explored biomarkers suitable for monitoring sub‐chronic kidney injury using the three rat models of cisplatin (CDDP)‐induced kidney injury, which were designed to extend the current knowledge beyond the sub‐acute exposure period. In the pilot study, a single intravenous administration of 1.5 mg kg^–1 CDDP to rats was confirmed to result in no histopathological changes. Subsequently, CDDP was intravenously administered to rats at a dose of 1.5 mg kg^–1 for 4 days at 24‐h intervals (Experimental model 1) and for up to 10 weeks at weekly intervals (Experimental models 2 and 3), and the changes in blood and urine components, such as recently recommended urinary biomarkers (Kim‐1, clusterin and so on) and traditional blood biomarkers (blood urea nitrogen and serum creatinine), were examined together with the histopathological changes in renal tissues during the development of the kidney injury in each model. In these experimental models, a significant increase in urinary Kim‐1 was observed prior to the histopathological changes in renal tissues, and these changes were retained after the adverse histopathological changes. Significant changes in all of the other urinary biomarkers examined occurred along with the histopathological changes. In addition, the increase in urinary Kim‐1 after weekly treatment with CDDP for 4 weeks was reduced in a time‐dependent manner after cessation of the drug. The present findings indicate that urinary Kim‐1 is the most useful biomarker for CDDP‐induced rat sub‐chronic kidney injury among the biomarkers examined. Copyright © 2014 John Wiley & Sons, Ltd.