T‐helper cell‐mediated factors in drug‐induced liver injury

Drug‐induced liver injury (DILI) leads to a large burden on the healthcare system due to its potential morbidity and mortality. The key for predicting and preventing DILI is to understand the underlying mechanisms. Hepatic inflammation is one of the most common features of DILI. The inflammation can be attributed to the innate immune response. The adaptive immune system is also affected by the innate immune response resulting in liver damage. T‐helper cells are important regulators of acquired immunity. T‐helper cell‐mediated immune responses play pivotal roles in the pathogenesis of a variety of liver disorders. This review summarizes recent advances in the T‐helper cell‐mediated factors in DILI and potential mechanisms, which may lead to a better understanding of DILI. Copyright © 2015 John Wiley & Sons, Ltd.     

Lipopolysaccharide exposure augments isoniazide‐induced liver injury

Isoniazide (INH) is a classic antituberculosis drug associated with clinical idiosyncratic drug‐induced liver injury. It has been hypothesized that the interaction between a drug and modest inflammation results in a decreased threshold for drug toxicity. In this study, we tested the hypothesis that INH causes liver injury in rats when coadministered with lipopolysaccharide (LPS). Neither INH nor LPS alone caused liver injury. The coadministration of INH and LPS was associated with increases in serum and histopathological markers of liver injury. Tumour necrosis factor‐α expression was significantly increased in the coadministered group. The downregulation of the bile acid transporter, bile salt export pump, and multidrug resistance protein 2 at both mRNA and protein levels was observed. Furthermore, the level of Farnesoid X receptor, which regulates the bile salt export pump and multidrug resistance protein 2, were clearly decreased. These results indicate that the coadministration of nontoxic doses of LPS and INH causes liver injury; the disruption of biliary excretion is considered the primary inflammation‐related characteristic of INH‐induced hepatotoxicity. Copyright © 2014 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.     

基因芯片技术比较不同致病因素急性肝损伤的基因表达

目的 用基因芯片技术比较大鼠两种急性肝损伤模型基因表达的差异.方法 分别以网氯化碳(CCL)和95%肝大部切除术制备急性肝损伤大鼠模型,分为手术对照(A)组、手术造模(B)组、CCl_4对照(C)组和CCl_4造模(D)组,24～48 h取肝组织.将4组肝组织的mRNA制备成cDNA探针,与含1176条大鼠基因的微阵列进行杂交及扫描,计算机数据处理分析后两组数据比较.结果 CCl_4组筛选出上调基因共201条,手术组共筛选出上调基因138条,其中两组共同上调的基因主要包括激素及激素受体基因、G蛋白结合受体基因、代谢相关基因、应激反应基因、细胞周期相关基因及生长因子基因等.结论 CCl_4和肝大部切除所致急性肝损伤的发病机制具有一定相似性.     

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 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.     

抗肿瘤药物肝损伤研究进展

本文对抗肿瘤药物肝损伤的危险因素、诊断标准和临床分型、特异性易感基因检测的研究现状进行归纳,同时结合自身研究成果,总结抗肿瘤药肝损伤的防治措施,提出建设抗肿瘤药物肝损伤分子检测与临床评价技术平台,将对预测药物肝损伤的发生、指导临床化疗药物使用和新型药物开发具有重要的意义。     

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.     

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.     

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.     

Valproic acid induced liver injury: An insight into molecular toxicological mechanism

Valproic acid (VPA) is an anti-seizure drug that causes idiosyncratic liver injury. 2-propyl-4-pentenoic acid (Δ⁴VPA), a metabolite of VPA, has been implicated in VPA-induced hepatotoxicity. This review summarizes the pathogenesis involved in VPA-induced liver injury. The VPA induce liver injury mainly by i) liberation of Δ⁴VPA metabolites; ii) decrease in glutathione stores and antioxidants, resulting in oxidative stress; iii) inhibition of fatty acid β-oxidation, inducing mitochondrial DNA depletion and hypermethylation; a decrease in proton leak; oxidative phosphorylation impairment and ATP synthesis decrease; iv) induction of fatty liver via inhibition of carnitine palmitoyltransferase I, enhancing nuclear receptor peroxisome proliferator-activated receptor-gamma and acyl-CoA thioesterase 1, and inducing long-chain fatty acid uptake and triglyceride synthesis. VPA administration aggravates liver injury in individuals with metabolic syndromes. Therapeutic drug monitoring, routine serum levels of transaminases, ammonia, and lipid parameters during VPA therapy may thus be beneficial in improving the safety profile or preventing the progression of DILI.     

Combined 5‐hydroxymethylcytosine content of human leucocyte antigen‐B and human leucocyte antigen‐DQB1 as novel biomarker for anti‐tuberculosis drug‐induced liver injury

This study investigated the diagnostic value of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) contents of human leucocyte antigen (HLA)‐B and HLA‐DQB1 in anti‐tuberculosis drug‐induced liver injury (ADLI). In total, 110 ADLI patients and 120 patients without ADLI controls were enrolled. Enzyme‐linked immunosorbent assay (ELISA) was used to detect the 5‐mC and 5‐hmC content in DNA from peripheral blood leucocytes. The univariate analysis showed that smoking, drinking, and 5‐mC and 5‐hmC content of HLA‐B and HLA‐DQB1 were significantly associated with ADLI. After adjusting for drinking and smoking, we found that 5‐mC content of HLA‐B and HLA‐DQB1 were associated with ADLI (odds ratio [OR] = 0.251 and 0.347, respectively) and 5‐hmC contents of HLA‐B and HLA‐DQB1 were also associated with ADLI (OR = 1.848 and 4.705, respectively). Receiver operating characteristic (ROC) analysis indicated that the 5‐hmC contents of both HLA‐B and HLA‐DQB1 were more clinically significant than the 5‐mC contents were. The combined 5‐hmC level of HLA‐B and HLA‐DQB1 was the best diagnostic biomarker for ADLI, with the highest areas under the curve (AUC) for 0.953, sensitivity for 0.900 and specificity for 0.875. Therefore, combined 5‐hmC levels of HLA‐B and HLA‐DQB1 could be significant evidence for diagnosis of ADLI.     

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.     

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.     

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.     

利奈唑胺与卡泊芬净药物合用致肝损害分析(1例)

通过对1例住院患者用药前后临床表现的分析,阐述其发生药源性损害的原因。患者肝功能快速恶化与用药有较肯定的相关性,从临床表现的综合分析看,利奈唑胺与卡泊芬净的使用是患者肝损害快速加重的关键原因,然而,其药物说明书却没有这方面的提示。由于该二药都是上市不久的药物,新药的临床试验大多是相对"健康"的患者,涉及糖尿病患者与重症患者的情况很少,所以其在不同患者中的安全性值得重视。建议卡泊芬净与利奈唑胺尽可能避免联合使用,卡泊芬净与利奈唑胺应禁止与二甲双胍同时用药,必要时采取极慎重的态度。