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.     

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by DILI risk into five categories based on drug labels approved by the Food and Drug Administration (FDA) as follows: withdrawn (WDN), boxed warning (BW), warnings and precautions (WP), adverse reactions (AR), and no match (NM).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.     

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.     

中药导致急性肝损伤临床研究

目的：探讨中药所致急性肝损伤的临床特点。方法制定标准化的药物性肝损伤病案调查表,采用病例登记和随访,以及 RUCAM评分系统,从急性药物性肝损伤患者中筛选出中药导致急性肝损伤相关病例,分析中药所致肝损伤的类型、临床表现、肝损伤严重程度等。结果在132例中药导致急性肝损伤相关病例中,男性59例,女性73例,平均年龄（51．6±17．5）岁。肝细胞型肝损伤107例,胆汁淤积型肝损伤20例,混合型肝损伤5例。轻度肝损伤10例（7．6％）,中度肝损伤104例（78．8％）,重度肝损伤16例（12．1％）,致命性肝损伤2例（1．5％）。导致急性肝损伤的中药种类繁多,大多数药物系含有多种中药成分（26．5％）,或具体药物成分不详的中药（56．8％）。结论中药导致肝损伤以肝细胞型为主,少数病例为重症或致死性,临床医生需要重视中药肝毒性。     

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.     

Hepatotoxicity and liver injury induced by hydroxyapatite nanoparticles

As hydroxyapatite nanoparticles (HA NPs) are increasingly used in biomedical and biotechnological fields, risk assessment of HA NPs has attracted extensive attention. Nevertheless, little is known about the potential adverse effects of HA NPs on normal hepatocytes and the liver. In the present study, we conducted an in vitro study in which 80‐nm HA NPs were incubated with normal Buffalo rat liver (BRL) cells. By analyzing the changes in cell viability, apoptosis/necrosis and the mitogen‐activated protein kinase (MAPK) signaling pathway, we investigated the cytotoxicity and potential mechanism of HA NPs in hepatocytes. Furthermore, we used the serum hematology and histopathology examinations to explore the in vivo effects of HA NPs on the structure and function of the liver. Our results showed that exposure to HA NPs at a concentration above 200 µg ml^?1 decreased cell viability, increased levels of lactate dehydrogenase (LDH) leakage, induced apoptosis and necrosis, and triggered the MAPK signaling pathway in BRL cells in a dose‐dependent manner. Moreover, our in vivo study indicated that HA NPs increased the white blood cell count (WBC) and the levels of tumor necrosis factor‐α (TNF‐α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum, caused inflammatory cell infiltration at the portal area in the liver, and induced hepatic oxidative stress with elevated levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). These data demonstrate that at certain concentrations, 80‐nm HA NPs cause hepatotoxicity and liver injury. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic activation and drug‐induced liver injury: in vitro approaches for the safety risk assessment of new drugs

Drug‐induced liver injury (DILI) is a significant leading cause of hepatic dysfunction, drug failure during clinical trials and post‐market withdrawal of approved drugs. Many cases of DILI are unexpected reactions of an idiosyncratic nature that occur in a small group of susceptible individuals. Intensive research efforts have been made to understand better the idiosyncratic DILI and to identify potential risk factors. Metabolic bioactivation of drugs to form reactive metabolites is considered an initiation mechanism for idiosyncratic DILI. Reactive species may interact irreversibly with cell macromolecules (covalent binding, oxidative damage), and alter their structure and activity. This review focuses on proposed in vitro screening strategies to predict and reduce idiosyncratic hepatotoxicity associated with drug bioactivation. Compound incubation with metabolically competent biological systems (liver‐derived cells, subcellular fractions), in combination with methods to reveal the formation of reactive intermediates (e.g., formation of adducts with liver proteins, metabolite trapping or enzyme inhibition assays), are approaches commonly used to screen the reactivity of new molecules in early drug development. Several cell‐based assays have also been proposed for the safety risk assessment of bioactivable compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

七氟醚静吸复合麻醉对肝叶切除术患者肝肾功能的影响

目的 探讨七氟醚静吸复合麻醉对肝叶切除术患者肝肾功能的影响。方法 85例择期行开腹肝叶切除术患者随机分为七氟醚组(n=44)和丙泊酚组(n=41),麻醉诱导:七氟醚组所有患者吸入8%七氟醚,等意识丧失后,用0.2 mg·kg-1顺苯磺酸阿曲库胺和0.4 μg·kg-1舒芬太尼静脉注射;丙泊酚组所有患者将1～2 mg·kg-1丙泊酚、0.2 mg·kg-1顺苯磺酸阿曲库胺和0.4 μg·kg-1舒芬太尼静脉注射。麻醉维持:七氟醚组患者持续吸入2%～3%七氟醚,丙泊酚组患者将0.5～0.8 mg·kg-1·h-1丙泊酚持续静脉输注,维持BIS值在40～60之间。记录两组患者手术一般情况,分别于麻醉诱导前(T₀)、术毕(T₁)、术后1 d(T₂)、3 d(T₃)和5 d(T₄)时,检测两组患者肝肾功能指标。结果 与T₀时相比,两组患者T1～4时ALT升高,T1～3时AST升高,T2～4时TBil和DBil升高,T₂时ALB降低,差异均有统计学意义(P<0.05);七氟醚组患者T2～4时ALT、TBil和DBil均低于丙泊酚组,T2～3时AST低于丙泊酚组,T₂时ALB高于丙泊酚组,差异均有统计学意义(P<0.05);与T₀时相比,两组患者T2～4时Cystatin C和24 h尿微量白蛋白均升高,差异均有统计学意义(P<0.05);七氟醚组患者T2～4时Cystatin C和24 h尿微量白蛋白均低于丙泊酚组,差异均有统计学意义(P<0.05)。结论 相比于丙泊酚复合麻醉,七氟醚静吸复合麻醉更有利于减轻肝叶切除术中缺血—再灌注损伤对患者肝肾功能的影响。     

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.     

The predictors of complications in patients with drug‐induced liver injury caused by antimicrobial agents

Aliment Pharmacol Ther 31 , 1200–1207

Summary

Background Antimicrobials are the leading cause of idiosyncratic drug‐induced liver injury in most series. Aim To determine the incidence and the predictors of complications in patients with drug‐induced liver injury caused by antimicrobial agents requiring hospitalization. Methods Medical records of patients with drug‐induced liver injury caused by antimicrobial agents were identified by ICD‐10, for the period between 2002 and 2006. Clinical information and blood tests during hospitalization were recorded. The causality assessment of drug‐induced liver injury was determined by the Roussel UCLAF causality assessment method (RUCAM) scale. Results Of 47 594 in‐patient admissions per year, the annual incidence of drug‐induced liver injury was 0.03%. Male: female ratio was 7:3 with a median age of 47 years. Eighty reactions of drug‐induced liver injury were caused by anti‐tuberculosis drugs (85%) and by antibiotics (15%). The median (IQR) of RUCAM scale was 6 (5–8). A total of 36% had HIV infection and 9% of patients had diabetes mellitus. Median (IQR) duration of hospitalization was 9 (5–15) days. Serious complications and death were found in 27.5% and 26%, respectively. By a multivariable logistic analysis, the presence of jaundice was found to be significantly associated with an unfavourable outcome. Conclusion Although rare, antimicrobial agents‐related drug‐induced liver injury requiring hospitalization has a high mortality rate. The presence of jaundice predicts poor outcome.     

Isatidis Folium alleviates acetaminophen‐induced liver injury in mice by enhancing the endogenous antioxidant system

Isatidis Folium (IF) has been clinically combined with acetaminophen (APAP), but the rationality of combinational therapy is still ambiguous. In the present study, the protective effect and related mechanism of IF on APAP‐induced hepatotoxicity were evaluated. Hepatic histopathology and blood biochemistry investigations clearly demonstrated that IF could restore APAP‐induced hepatotoxicity. Liver distribution study indicated that the hepatoprotective effect of IF on APAP is attributed to the reduction of N‐acetyl‐p‐benzoquinone imine (NAPQI) in liver, which is a known hepatotoxic metabolite of APAP. Further study suggested the reduction is not via decreasing the generation of NAPQI through inhibiting the enzyme activities of CYP 1A2, 2E1, and 3A4 but via accelerating the transformation of NAPQI to NAPQI‐GSH by promoting GSH and decreasing GSSG contents in liver. Furthermore, IF significantly enhanced the hepatic activities of GSH‐associated enzymes in APAP‐treated mice. In summary, IF could alleviate APAP‐induced hepatotoxicity by reducing the content of NAPQI via enhancing the level of GSH and the followed generation of NAPQI‐GSH which might be ascribed to the upregulation of GSH‐associated enzymes.     

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.