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Sphingolipids are exceptionally diverse, comprising hundreds of unique species. The bulk of circulating sphingolipids are synthesized in the liver, thereby plasma sphingolipid profiles represent reliable surrogates of hepatic sphingolipid metabolism and content. As changes in plasma sphingolipid content have been associated to exposure to drugs inducing hepatotoxicity both in vitro and in rodents, in the present study the translatability of the preclinical data was assessed by analyzing the plasma of patients with suspected drug-induced liver injury (DILI) and control subjects. DILI patients, whether intrinsic or idiosyncratic cases, had no alterations in total sphingoid base levels and profile composition compared to controls, whereby cardiovascular disease (CVD) was a confounding factor. Upon exclusion of CVD individuals, elevation of 1-deoxysphingosine (1-deoxySO) in the DILI group emerged. Notably, 1-deoxySO values did not correlate with ALT values. While 1-deoxySO was elevated in all DILI cases, only intrinsic DILI cases concomitantly displayed reduction of select shorter chain sphingoid bases. Significant perturbation of the sphingolipid metabolism observed in this small exploratory clinical study is discussed and put into context, in the consideration that sphingolipids might contribute to the onset and progression of DILI, and that circulating sphingoid bases may function as mechanistic markers to study DILI pathophysiology.

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Drug‐induced liver injury (DILI) should be considered in all patients with recent elevation of liver tests without obvious etiology and normal hepatobiliary imaging. There is currently no biomarker that is helpful in diagnosis which relies on clinical and laboratory findings. Diagnosis is dependent on temporal relationship with a recently started drug or herbal and dietary supplement and elevated liver tests with exclusion of competing etiologies. The implicated agent should be discontinued and the patient should be observed closely. This is particularly important in patients with jaundice who have approximately 10% risk of liver related mortality and/or need for liver transplantation. There is no specific therapy for DILI which is only symptomatic such as for itching. Patients with jaundice and coagulopathy usually require hospitalization.

Drug-induced liver injury (DILI) is an unpredictable and feared side effect of antituberculosis treatment (AT). The present study aimed to identify clinical and genetic variables associated with susceptibility to AT-associated hepatotoxicity in patients with pulmonary tuberculosis treated with a standard protocol. Of 233 patients enrolled, 90% prospectively, 103 developed liver injury: 37 with mild and 66 with severe phenotype (DILI). All patients with mild hepatitis had a RUCAM score ≥4 and all patients with DILI had a RUCAM score ≥ 6. Eight clinical variables and variants in six candidate genes were assessed. A logistic multivariate regression analysis identified four risk factors for AT-DILI: age ≥ 55 years (OR:3.67; 95% CI:1.82–7.41; p < 0.001), concomitant medication with other hepatotoxic drugs (OR:2.54; 95% CI:1.23–5.26; p = 0.012), NAT2 slow acetylator status (OR:2.46; 95% CI:1.25–4.84; p = 0.009), and carriers of p.Val444Ala variant for ABCB11 gene (OR:2.06; 95%CI:1.02–4.17; p = 0.044). The statistical model explains 24.9% of the susceptibility to AT-DILI, with an 8.9 times difference between patients in the highest and in the lowest quartiles of risk scores. This study sustains the complex architecture of AT-DILI. Prospective studies should evaluate the benefit of NAT2 and ABCB11 genotyping in AT personalization, particularly in patients over 55 years.

The Biopharmaceutics Drug Disposition Classification system (BDDCS) is a four-class approach based on water solubility and extent of metabolism/permeability rate. Based on the BDDCS class to which a drug is assigned, it is possible to predict the role of metabolic enzymes and transporters on the drug disposition of a new molecular entity (NME) prior to its administration to animals or humans. Here, we report a total of 1475 drugs and active metabolites to which the BDDCS is applied. Of these, 379 are new entries, and 1096 are revisions of former classification studies with the addition of references for the approved maximum dose strength, extent of the systemically available drug excreted unchanged in the urine, and lowest solubility over the pH range 1.0–6.8 when such information is available in the literature. We detail revised class assignments of previously misclassified drugs and the literature analyses to classify new drugs. We review the process of solubility assessment for NMEs prior to drug dosing in humans and approved dose classification, as well as the comparison of Biopharmaceutics Classification System (BCS) versus BDDCS assignment. We detail the uses of BDDCS in predicting, prior to dosing animals or humans, disposition characteristics, potential brain penetration, food effect, and drug-induced liver injury (DILI) potential. This work provides an update on the current status of the BDDCS and its uses in the drug development process. Graphical abstract

In vitro models of human liver disease often fail to mimic the complex 3D structures and cellular organizations found in vivo. Precision cut liver slices (PCLS) retain the complex physiological architecture of the native liver and therefore could be an exceptional in vitro liver model. However, the production of PCLS induces a spontaneous culture-induced fibrogenic reaction, limiting the application of PCLS to anti-fibrotic compounds. Our aim was to improve PCLS cultures to allow compound-induced fibrosis induction. Hepatotoxicity in PCLS cultures was analyzed by lactate dehydrogenase leakage and albumin secretion, while fibrogenesis was analyzed by qRT-PCR and western blot for hepatic stellate cell (HSC) activation markers and collagen 6 secretion by enzyme-linked immunosorbent assays (ELISA). We demonstrate that supplementation of 3 mm mouse PCLS cultures with valproate strongly reduces fibrosis and improves cell viability in our PCLS cultures for up to 5 days. Fibrogenesis can still be induced both directly and indirectly through exposure to TGFβ and the hepatotoxin acetaminophen, respectively. Finally, human PCLS cultures showed similar but less robust results. In conclusion, we optimized PCLS cultures to allow for drug-induced liver fibrosis modeling.

Contains fulltext : 248670.pdf (Publisher’s version ) (Open Access) An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (C(max)) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC(10) values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

Background and Aims: We aimed to study clinical outcomes and liver biopsy features of alcoholic hepatitis (AH) patients on complementary and alternative medicines (CAMs) and to analyze the retrieved drugs for chemical and toxic components linked to drug-induced liver injury. Methods: We retrospectively assessed clinical, biochemical and liver biopsy features of AH patients on CAM with drug-induced liver injury (AH-CAM, n = 27) and compared them to a control group (classical AH, n = 29) on standard of care. Patients without liver biopsy evaluation and other causes for liver disease were excluded. Samples of the CAMs (n = 42) from patients were retrieved and assessed for chemical and toxins. Results: All were males, and significantly worse clinical presentation, biochemical severity, and liver disease scores were notable in patients with AH-CAM. Traditional Ayurvedic-polyherbal formulations were the most commonly used CAM. On liver histology, varying grades of severe-necrosis, severe hepatocellular, canalicular, cholangiolar cholestasis with predominant lymphocytic-portal-inflammation and varying grades of interface-hepatitis were noted in AH-CAM. Analysis of CAMs revealed presence of heavy metals up to 100,000 times above detectable range and adulterants, such as antibiotics, chemotherapy agents, nonsteroidal anti-inflammatory drugs, alcohols, antidepressants, anxiolytics, and recreational drugs. On follow up, a significantly higher number of patients with AH on CAM died at end of 1, 3- and-6-months compared to controls (37% vs. 83%, 29% vs. 62%, 18% vs. 52% respectively; p < 0.001). Conclusions: Patients with AH and CAM-related drug-induced liver injury have extremely poor short-term survival in the absence of liver transplantation compared to those patients with AH on evidence-based management. Early transplant referral and educating on and curbing of CAM use in severe liver disease through strict monitoring of unregulated traditional health practices can help ease the burden of liver-related death.

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation., Graphical abstract p62 inhibits the late injury phase of acetaminophen-induced liver injury (AILI) by increasing autophagic selective removal of acetaminophen-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.Image 1

Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of mechanisms-revealing data, but interpretative analysis tools specific for the testing systems (e.g. hepatocytes) are lacking. In this study, we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/ ), an R-Shiny-based implementation of weighted gene co-expression network analysis (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. The 398 gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, were perturbed by specific stressors and showed preservation in rat systems (rat primary hepatocytes and rat in vivo liver), with the exception of DNA damage and oxidative stress responses. A subset of 87 well-annotated and preserved modules was used to evaluate mechanisms of toxicity of endoplasmic reticulum (ER) stress and oxidative stress inducers, including cyclosporine A, tunicamycin and acetaminophen. In addition, module responses can be calculated from external datasets obtained with different hepatocyte cells and platforms, including targeted RNA-seq data, therefore, imputing biological responses from a limited gene set. As another application, donors' sensitivity towards tunicamycin was investigated with the TXG-MAPr, identifying higher basal level of intrinsic immune response in donors with pre-existing liver pathology. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data.

Drug-induced liver injury (DILI) has a very variable clinical and biochemical phenotype and differs widely in severity, from mild injury to life threatening liver failure. However, chronic injury has also been reported with a variable frequency, with a range from 3.4% to 39% of patients after 6-12 months after stopping the implicated agent. This wide range is probably related to various definition of chronic liver injury and variable selection of the DILI patients. The long-term sequalae of this chronic injury in terms of morbidity and mortality are unclear. Although rare vanishing bile duct syndrome, is associated with unfavorable prognosis with increased risk of chronic liver failure and need for liver transplantation. Other forms of long-term sequalae associated with DILI are progressive fibrosis, autoimmune like hepatitis, secondary sclerosing cholangitis, sinusoidal obstruction syndrome and as a common final stage the development of cirrhosis, portal hypertension and its complications. A new type of cancer immunotherapy, check point inhibitors can apart from the acute autoimmune like phenotype also lead to sclerosing cholangitis with cytotoxic T CD8+ cell infiltration in biliary tracts. DILI has been shown to have significant impact on health-related quality of life but very little is known on its psychological consequences in the long-term. Further investigations on the psychological disability in patients with chronic DILI particularly in those with chronic outcome are clearly needed in studies with structured long-term follow-up and periodic quality of live surveys.

Due to the improved effectiveness and safety of combined antiretroviral therapy, human immunodeficiency virus (HIV) infection has become a manageable, chronic condition rather than a mortal disease. However, HIV patients are at increased risk of experiencing non-AIDS-defining illnesses, with liver-related injury standing out as one of the leading causes of death among these patients. In addition to more HIV-specific processes, such as antiretroviral drug-related toxicity and direct injury to the liver by the virus itself, its pathogenesis is related to conditions that are also common in the general population, such as alcoholic and non-alcoholic fatty liver disease, viral hepatitis, and ageing. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of combined anti-HIV treatment due to their unique antiviral activity, high specificity, and acceptable toxicity. While first-generation NNRTIs (nevirapine and efavirenz) have been related largely to liver toxicity, those belonging to the second generation (etravirine, rilpivirine and doravirine) seem to be generally safe for the liver. Indeed, there is preclinical evidence of rilpivirine being hepatoprotective in different models of liver injury, independently of the presence of HIV. The present study aims to review the mechanisms by which currently available anti-HIV drugs belonging to the NNRTI family may participate in the development of liver disease.

The use of complementary and alternative medicines (CAMs) for treatment of acute and chronic diseases is on the rise world over, especially in Asian countries, and mostly in China and India. Drug-induced liver injury (DILI) due to CAM is increasingly reported in the literature from multiple centers all around the world and with large-number patient series published from the West, mostly based on nation-wide DILI networks and multicenter collaboration. Comprehensive DILI networks are lacking among major Asian countries with high incidence of CAM practices. Chinese medical societies dealing with drug toxicity, CAM practice and hepatobiliary disease have adopted an integrated approach to establishing identification, diagnosis and treatment of CAM-related DILI, representing a systematic approach that could be iterated by other countries for improving patient outcomes. In this exhaustive review, we provide published data on CAM-related DILI in Asia, with detail on incidences along with analysis of patient population and their clinical outcomes. Concise and clear discussion on commonly implicated CAM agents in major Asian countries and associated chemical and toxicology analyses as well as descriptions of liver biopsy findings are discussed with future directions.

Many drugs are known to potentially cause liver injury; however, only a few reports investigate the association between levofloxacin and acute liver failure (ALF). The case describes a 65-year-old man who was admitted with primary diagnoses of cerebrovascular accident (CVA) and acute coronary syndrome (ACS) who developed an upper respiratory tract infection for which he was started on levofloxacin. Following its administration, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased more than 100-fold above the upper limit of normal. Over the next 24 hours, AST peaked at 9334 U/L, ALT at 4525 U/L, prothrombin time to 24.6 seconds, international normalized ratio (INR) to 2.22, and serum ammonia to 157 µmol/L. The patient developed signs and symptoms of decompensated liver disease, namely hepatic encephalopathy (HE). Levofloxacin was discontinued immediately, and evidence-based treatment per society guidelines from The American Association for the Study of Liver Diseases consisting of IV n-acetylcysteine as well as lactulose and rifaximin was initiated. Such medical management resulted in clinical resolution of his ALF, but he had a poor overall prognosis and eventually succumbed to critical illness.

Drug-induced liver injury (DILI) is among the most common causes of acute liver injury and acute liver failure in the United States. Kratom is an herbal supplement made from the leaves of a tropical evergreen tree (Mitragyna speciosa) that is native to Southeast Asia. Due to its psychotropic and opioid-like activity, there has been an increase in its use as a recreational drug. Despite this increase, little is known regarding the toxicities and adverse effects though it is known to cause DILI. We present two cases of DILI associated with Kratom use.

A major challenge in drug development is safety and toxicity concerns due to drug side effects. One such side effect, drug-induced liver injury (DILI), is considered a primary factor in regulatory clearance. The Critical Assessment of Massive Data Analysis (CAMDA) 2020 CMap Drug Safety Challenge goal was to develop prediction models based on gene perturbation of six preselected cell-lines (CMap L1000), extended structural information (MOLD2), toxicity data (TOX21), and FDA reporting of adverse events (FAERS). Four types of DILI classes were targeted, including two clinically relevant scores and two control classifications, designed by the CAMDA organizers. The L1000 gene expression data had variable drug coverage across cell lines with only 247 out of 617 drugs in the study measured in all six cell types. We addressed this coverage issue by using Kru-Bor ranked merging to generate a singular drug expression signature across all six cell lines. These merged signatures were then narrowed down to the top and bottom 100, 250, 500, or 1,000 genes most perturbed by drug treatment. These signatures were subject to feature selection using Fisher’s exact test to identify genes predictive of DILI status. Models based solely on expression signatures had varying results for clinical DILI subtypes with an accuracy ranging from 0.49 to 0.67 and Matthews Correlation Coefficient (MCC) values ranging from -0.03 to 0.1. Models built using FAERS, MOLD2, and TOX21 also had similar results in predicting clinical DILI scores with accuracy ranging from 0.56 to 0.67 with MCC scores ranging from 0.12 to 0.36. To incorporate these various data types with expression-based models, we utilized soft, hard, and weighted ensemble voting methods using the top three performing models for each DILI classification. These voting models achieved a balanced accuracy up to 0.54 and 0.60 for the clinically relevant DILI subtypes. Overall, from our experiment, traditional machine learning approaches may not be optimal as a classification method for the current data.

[Background & Aims] Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period., [Methods] Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected., [Results] A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974–0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994–0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy’s law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%)., [Conclusions] AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid physicians in DILI characterisation and management., [Lay summary] Clinical information on drug-induced liver injury (DILI) collected from enrolled patients in the Spanish DILI Registry can guide physicians in the decision-making process. We have found that older patients with hepatocellular type liver injury and patients with additional liver conditions are at a higher risk of mortality. The type of liver injury, patient sex and analytical values of aspartate aminotransferase and total bilirubin can also help predict clinical outcomes., The present study has been supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional – FEDER (contract numbers: PI19/00883, PI16/01748, PI18/00901, PI18/01804, PI-0285-2016, PI-0274-2016, PI-0310-2018, PT17/0017/0020) and Agencia Española del Medicamento. CIBERehd and Plataforma ISCIII Ensayos Clinicos are funded by Instituto de Salud Carlos III. MRD holds a Joan Rodes (JR16/00015)/Acción B clinicos investigadores (B-0002-2019) and JSC a Rio Hortega (CM17/00243) research contract from ISCIII and Consejería de Salud de Andalucía.

The identification of specific HLA risk alleles in drug-induced liver injury (DILI) points toward an important role of the adaptive immune system in DILI development. In this study, we aimed to corroborate the role of an adaptive immune response in DILI through immunophenotyping of leukocyte populations and immune checkpoint expressions. Blood samples were collected from adjudicated DILI (n = 12), acute viral hepatitis (VH; n = 13), acute autoimmune hepatitis (AIH; n = 9), and acute liver injury of unknown etiology (n = 15) at day 1 (recognition), day 7, and day >30. Blood samples from patients with nonalcoholic fatty liver disease (NAFLD; n = 20) and healthy liver controls (HLCs; n = 54) were extracted at one time point. Leukocyte populations and immune checkpoint expressions were determined based on cell surface receptors, except for CTLA-4 that was determined intracellularly, using flow cytometry. At recognition, DILI demonstrated significantly higher levels of activated helper T-cell (P < 0.0001), activated cytotoxic T-cells (P = 0.0003), Th1 (P = 0.0358), intracellular CTLA-4 level in helper T-cells (P = 0.0192), and PD-L1 presenting monocytes (P = 0.0452) than HLC. These levels approached those of HLC over time. No significant differences were found between DILI and VH. However, DILI presented higher level of activated helper T-cells and CTLA-4 than NAFLD and lower PD-L1 level than AIH. Our findings suggest that an adaptive immune response is involved in DILI in which activated CD4+ and CD8+ play an important role. Increased expression of negative immune checkpoints is likely the effect of peripheral tolerance regulation. The present study has been supported by grants of Instituto de Salud Carlos III cofounded by Fondo Europeo de Desarrollo Regional – FEDER (contract numbers: PI19/00883, PI16/01748, P18-RT-3364-2020, and PT20/000127). CIBERehd and Plataforma ISCiii Ensayos Clínicos are funded by Instituto de Salud Carlos III. Funding for open access charge: Universidad de Málaga/CBUA. The funding sources had no involvement in the study design; in the collection, analysis, and interpretation of data; in the writing of the report, or in the decision to submit the manuscript for publication.

Due to the improved effectiveness and safety of combined antiretroviral therapy, human immunodeficiency virus (HIV) infection has become a manageable, chronic condition rather than a mortal disease. However, HIV patients are at increased risk of experiencing non-AIDS-defining illnesses, with liver-related injury standing out as one of the leading causes of death among these patients. In addition to more HIV-specific processes, such as antiretroviral drug-related toxicity and direct injury to the liver by the virus itself, its pathogenesis is related to conditions that are also common in the general population, such as alcoholic and non-alcoholic fatty liver disease, viral hepatitis, and ageing. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of combined anti-HIV treatment due to their unique antiviral activity, high specificity, and acceptable toxicity. While first-generation NNRTIs (nevirapine and efavirenz) have been related largely to liver toxicity, those belonging to the second generation (etravirine, rilpivirine and doravirine) seem to be generally safe for the liver. Indeed, there is preclinical evidence of rilpivirine being hepatoprotective in different models of liver injury, independently of the presence of HIV. The present study aims to review the mechanisms by which currently available anti-HIV drugs belonging to the NNRTI family may participate in the development of liver disease.

Introduction: Idiosyncratic drug-induced liver injury (DILI) is an unpredictable event, and there are no specific biomarkers that can distinguish DILI from alternative explanations or predict its clinical outcomes. Areas covered: This systematic review summarizes the available evidence for all biomarkers proposed to have a role in the diagnosis or prognosis of DILI. Following a comprehensive search, we included all types of studies in humans. We included DILI cases based on any threshold criteria but excluded intrinsic DILI, commonly caused by paracetamol overdose. We classified studies into diagnostic and prognostic categories and assessed their methodological quality. After reviewing the literature, 14 studies were eligible. Expert Opinion: Diagnostic studies were heterogeneous with regard to the study population and outcomes measured. Prognostic models were developed by integrating novel biomarkers, risk scores, and traditional biomarkers, which increased their prognostic ability to predict death or transplantation by 6 months. This systematic review highlights the case of need for non-genetic biomarkers that distinguish DILI from acute liver injury related to alternative etiology. Biomarkers with the potential to identify serious adverse outcomes from acute DILI should be validated in independent prospective cohorts with a substantial number of cases. This paper was funded by the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 821283 (www.imi.europa.eu). This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA. Translational Safety Biomarker Pipeline (TransBioLine): Enabling develop- ment and implementation of novel safety biomarkers in clinical trials and diagnosis of disease’ — ‘TransBioLine’ (‘action’). Grant Number: 821283.

Drug‐induced (secondary) sclerosing cholangitis has recently been observed with a number of agents (1). Ketamine is a remarkably safe anesthetic, providing sedation and analgesia as adjunct to or substitute for more traditional sedative medications. Although prior reports of recreational ketamine abuse have been associated with findings of secondary sclerosing cholangitis (SSC; 2,3), we report here a novel presentation of the syndrome in association with prolonged ketamine use in the intensive care unit.

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

Adicionalmente, el 30% de los pacientes con hepatitis E presentó eosinofilia y el 25 % linfopenia. Las enfermedades hepáticas subyacentes (OR=23,4, p20 veces el límite superior normal (OR=10,9, p = 0,002) se asociaron con el diagnóstico de hepatitis E. La seroprevalencia (anti-VHE IgG+) general fue 35%, distribuido uniformemente entre pacientes con sospecha de DILI (34%) y controles (39%) Conclusiones: El porcentaje de pacientes con hepatitis E es considerable en los pacientes con sospecha de DILI. En la infección aguda por hepatitis E así como en DILI, los pacientes de edad avanzada pueden tener un cuadro clínico más severo, especialmente cuando tienen patología hepática subyacente. Los pacientes con hepatitis E, pueden presentar manifestaciones clínicas de inmunoalergia de forma similar a los pacientes DILI por lo que, en ausencia de un diagnóstico serológico o microbiológico de VHE, la detección de estos signos y/o síntomas no ayudan a descartar un diagnóstico de VHE. La seroprevalencia del VHE es relativamente alta en España. Por todo lo anterior, se recomienda realizar una búsqueda activa para detectar una infección por el VHE en pacientes evaluados por sospecha de DILI, particularmente en pacientes de edad avanzada, con enfermedades hepáticas subyacentes y niveles altos de transaminasas. Fecha de lectura de Tesis Doctoral: 2 julio 2020. Antecedentes y objetivos: el daño hepático inducido por fármacos también denominado DILI (Drug Induced Liver Injury), se presenta con un amplio espectro fenotípico que requiere un diagnóstico diferencial extenso. El virus de la hepatitis E (VHE) no se descarta sistemáticamente durante la evaluación de los casos de hepatitis aguda en España. Los objetivos del estudio fueron evaluar el papel de la infección por el VHE en los pacientes con sospecha de DILI y establecer características diferenciales entre pacientes con diagnóstico de DILI y los pacientes con hepatitis E Métodos: se realizó un análisis de 265 pacientes con sospecha de DILI que fueron enviados para su valoración al Spanish DILI Registry y 108 controles con perfiles hepáticos normales. Se analizó la presencia de anti-VHE IgG en suero de todos los sujetos. En aquellos con muestras de suero extraídas dentro de los 6 meses posteriores al inicio del daño hepático (n=144), se analizó por duplicado el antígeno y los anticuerpos anti-VHE IgM . Además, la RT-PCR (ARN-VHE) se realizó externamente en 8 pacientes. Se consideró infección aguda por VHE a los pacientes con transaminasas elevadas e IgM, Ag y/o ARN-VHE positivos. Se analizaron también características demográficas, clínicas y de laboratorio de los pacientes DILI y con infección aguda por el VHE Resultados: De 144 pacientes, 12 (8%) fueron positivos para anti-VHE IgM. Aunque la edad media fue mayor en los pacientes con hepatitis aguda E que en los pacientes DILI, no hubo diferencias estadísticamente significativas (61 vs 55 años, respectivamente). Dos de los 3 pacientes que presentaron cuadros más graves de hepatitis aguda E eran pacientes mayores, de 74 y 75 años, y con importantes comorbilidades. El tercero era una paciente de 26 años con antecedentes de dos trasplantes previos de hígado. Los pacientes con hepatitis E presentaron en términos generales una sintomatología clínica similar a los pacientes DILI.

Background: Drug induced liver injury (DILI) is an increasing cause of acute liver injury especially with increasing need for pharmacotherapy of widening comorbidities amongst our ever-aging population. Uncertainty however remains regarding both acceptable and widely agreeable diagnostic algorithms as well a clear understanding of mechanistic insights that most accurately underpins it. In this review, we have explored the potential role of emerging novel markers of DILI and how they could possibly be integrated into clinical care of patients. Methods: We explored PUBMED and all other relevant databases for scientific studies that explored potential utility of novel biomarkers of DILI, and subsequently carried out a narrative synthesis of this data. As this is a narrative review with no recourse to patient identifiable information, no ethics committee's approval was sought or required. Results: Novel biomarkers such as microRNA-122 (miR-122) profiles, high mobility group box-1 (HMGB1), glutamate dehydrogenase (GLDH), and cytokeratin-18 (K-18), amongst others do have the potential for reducing diagnostic uncertainties associated with DILI. Conclusion: With the increasing validation of some of the novel liver biomarkers such as K-18, mir-122, HMGB-1, and GLDH, there is the potential for improvement in the diagnostic uncertainty commonly associated with cases of DILI.

Binny Thomas,1-3 Mahmoud Mohamed,1,3,4 Moza Al Hail,1-3Fatma Alzahra Y Awwad,1 Ramy M Wahba,1 Sabir B Hassan,1 Khalid Omar,1 Wessam El Kassem,1 Palivalappila Abdul Rouf1 1Hamad Medical Corporation, Doha, Qatar; 2Robert Gordon University, Aberdeen, Scotland, UK; 3Qatar University, Doha, 4Weill Cornell Medical College, Ar-Rayyan, Qatar Abstract: We report a case of 22-year-old primigravida presented to Women’s Hospital – Hamad Medical Corporation emergency with severe epigastric pain, nausea, and vomiting. On admission, she was dehydrated with remarkably worsening symptoms. Laboratory findings revealed significantly elevated liver enzymes with unknown etiology. Her past medical history showed an admission for nausea and vomiting 3weeks previously and she was discharged on antiemetics, and esomeprazole for the first time. Due to the predominantly elevated liver enzymes, the clinical pharmacist discussed the possibility of esomeprazole-induced adverse effects and suggested to suspend esomeprazole based on the evidence from literature review. The liver enzymes showed a substantial improvement within days after the discontinuation of the drug; however, a rechallenge was not done since it could have adversely affected the mother or the fetus. Using the Naranjo Adverse Drug Reaction Probability scales, the adverse reaction due to esomeprazole was classified as “probably”. Keywords: hyperemesis, drug-induced liver injury, esomeprazole, adverse drug reaction, ADR, proton pump inhibitor

Drug-induced liver injury (DILI) is a rare and potentially lethal condition associated with the use of many commonly-used medications, including inhaled fluticasone-vilanterol. Therefore, a careful review of medications should always be obtained in the setting of acute onset hepatic dysfunction. We present the first reported case of idiosyncratic drug-induced liver injury associated with the use of this medication.

Objectives: Z-Drugs (ZDs) have been developed to limit benzodiazepines (BZDs) abuse for sleep disorders. Data on the liver toxicity of zolpidem (ZLM) are lacking or anecdotal. The authors evaluated the presence of drug-induced liver injury (DILI) among a cohort of high-dose ZLM abusers. Methods: Retrospective study analyzing clinical records of 1112 consecutive patients admitted for BZDs detoxification from 2003 to 2018. Inclusion criteria: age >18 y.o.; ZLM abuse/dependence; high-dose ZDs abuse. Exclusion criteria: missing lab data; lack of informed consent. Main outcome was the presence of DILI measured as elevation of ALT/AST levels >250 U/l. Results: A total of 107 patients met the eligibility criteria. Liver enzymes alterations were present in 9.3% (95% CI 4.6-16.5%); one patient (0.9%, 95% CI 0.0-2.8%) showed DILI criteria. BMI significantly influenced transaminases levels. No correlations between duration nor doses of ZLM abuse and transaminases levels were found. Conclusion: The present study shows a very low prevalence of DILI among high-dose ZLM abusers. The prevalence of hypertransaminasemia was in line with general population. On one hand ZLM has a substantially safe liver profile but on the other hand ZLM abuse and dependence, especially at very high doses, represents an emerging problem.

Causality assessment in liver injury induced by drugs and herbs remains a debated issue, requiring innovation and thorough understanding based on detailed information. Artificial intelligence (AI) principles recommend the use of algorithms for solving complex processes and are included in the diagnostic algorithm of Roussel Uclaf Causality Assessment Method (RUCAM) to help assess causality in suspected cases of idiosyncratic drug-induced liver injury (DILI) and herb-induced liver injury (HILI). From 1993 until the middle of 2020, a total of 95,865 DILI and HILI cases were assessed by RUCAM, outperforming by case numbers any other causality assessment method. The success of RUCAM can be traced back to its quantitative features with specific data elements that are individually scored leading to a final causality grading. RUCAM is objective, user friendly, transparent, and liver injury specific, with an updated version that should be used in future DILI and HILI cases. Support of RUCAM was also provided by scientists from China, not affiliated to any network, in the results of a scientometric evaluation of the global knowledge base of DILI. They highlighted the original RUCAM of 1993 and their authors as a publication quoted the greatest number of times and ranked first in the category of the top 10 references related to DILI. In conclusion, for stakeholders involved in DILI and HILI, RUCAM seems to be an effective diagnostic algorithm in line with AI principles.

Background and Aims: Drug-induced liver injury with autoimmune features (AI-DILI) mimics the clinical presentation, and laboratory and pathologic features of idiopathic autoimmune hepatitis (AIH). We aimed to identify histopathologic hallmarks to differentiate these entities. Methods: All liver biopsies archived for the past 10 years were reviewed retrospectively to identify cases of recently detected liver injury associated with predominantly lymphoplasmacytic interphase hepatitis, positive markers for liver autoimmunity, and negative tests for viral hepatitis. Twenty cases were divided into AIH (n = 12) or AI-DILI (n = 8) groups. Blind qualitative evaluation of necroinflammatory changes and liver fibrosis were performed according to the Scheuer scoring system. Cellular densities were determined using ImageJ (V1.51t, National Institutes of Health, Bethesda, MD, USA). Fibrosis was assessed on Masson trichrome-stained slides, and collagen deposition was estimated following a protocol of color deconvolution. Results: Necroinflammatory changes as well as densities (portal and lobular) of neutrophils and eosinophils, intracellular cholestasis, and regenerative changes did not differ between the two groups (P ≥ 0.05). Neutrophil densities but not eosinophils showed a positive correlation with the severity of hepatocellular damage (r = 0.6 and 0.58, vs. alanine aminotransferase, P < 0.05). Ceroid-laden macrophages but not histiocytic aggregates appeared to be more common in AI-DILI (P < 0.05). AIH patients presented more often with evidence of chronic damage, including higher scores of fibrosis and collagen deposition, in comparison to AI-DILI (P < 0.05). Conclusions: Although there is no histologic feature pathognomonic for AI-DILI or AIH, advanced stages of liver fibrosis can be used to support the diagnosis of AIH in some cases. Definitive diagnosis of AI-DILI requires follow-up and demonstration of complete remission after drug withdrawal with no need for immunosuppression.

Several side-effects related to prolonged benzodiazepines (BZD) use have been reported. Given the primary role of liver in BZD metabolism, toxicity related to prolonged high-dose BZD use could be conceivable. No data are available on the long-term impact of high-dose BZD use on liver.A total of 201 BZD mono-abusers admitted to an Addiction Unit for detoxification were evaluated. Liver enzymes were evaluated at admission, before starting any treatment. An elevation of more than five times the upper limit of normal range (ULN) in serum ALT or conjugated bilirubin, or a combined elevation of AST, alkaline phosphatase and total bilirubin, one of which exceeding2 the ULN, was considered diagnostic for drug-induced liver injury.None of the evaluated subjects showed significant alterations of liver enzymes. Those with the highest transaminase levels were showing high body mass index. Twenty patients (10%) showed elevated gamma-glutamyl-transferase. No alteration of alkaline phosphatase, nor bilirubin was found in any patient. The average dosage of BZD was 307 mg of diazepam-equivalents for 7 years.Present data suggest that prolonged use of high-dose BZD, although very dangerous for several reasons, does not seem to produce a significant drug-induced liver injury.

Idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver failure resulting in liver transplantation or death. Prediction and diagnosis of iDILI remain a great challenge, as current models provide unsatisfying results in terms of sensitivity, specificity, and prognostic value. The absence of appropriate tools for iDILI detection also impairs the development of reliable biomarkers. Here, we report on a new method for identification of drug-specific biomarkers. We combined the advantages of monocyte-derived hepatocyte-like (MH) cells, able to mimic individual characteristics, with those of a novel mass spectrometry-based proteomics technology to assess potential biomarkers for Diclofenac-induced DILI. We found over 2,700 proteins differentially regulated in MH cells derived from individual patients. Herefrom, we identified integrin beta 3 (ITGB3) to be specifically upregulated in Diclofenac-treated MH cells from Diclofenac-DILI patients compared to control groups. Finally, we validated ITGB3 by flow cytometry analysis of whole blood and histological staining of liver biopsies derived from patients diagnosed with Diclofenac-DILI. In summary, our results show that biomarker candidates can be identified by proteomics analysis of MH cells. Application of this method to a broader range of drugs in the future will exploit its full potential for the development of drug-specific biomarkers. Data are available via ProteomeXchange with identifier PXD008918.

Introduction: In almost 50% of patients with drug-induced liver injury (DILI), the bile flow from the liver to the duodenum is impaired, a condition known as cholestasis. However, this toxic response only appears in a small percentage of the treated patients (idiosyncrasy). Prediction of drug-induced cholestasis (DIC) is challenging and emerges as a safety issue that requires attention by professionals in clinical practice, regulatory authorities, pharmaceutical companies, and research institutions. Area covered: The current synopsis focuses on the state-of-the-art in preclinical models for cholestatic DILI prediction. These models differ in their goal, complexity, availability, and applicability, and can widely be classified in experimental animals and in vitro models. Expert opinion: Drugs are a growing cause of cholestasis, but the progress made in explaining mechanisms and differences in susceptibility is not growing at the same rate. We need reliable models able to recapitulate the features of DIC, particularly its idiosyncrasy. The homogeneity and the species-specific differences move animal models away from a fair predictability. However, in vitro human models are improving and getting closer to the real hepatocyte phenotype, and they will likely be the choice in the near future. Progress in this area will not only need reliable predictive models but also mechanistic insights.

Introduction: In almost 50% of patients with drug-induced liver injury (DILI), the bile flow from the liver to the duodenum is impaired, a condition known as cholestasis. However, this toxic response only appears in a small percentage of the treated patients (idiosyncrasy). Prediction of drug-induced cholestasis (DIC) is challenging and emerges as a safety issue that requires attention by professionals in clinical practice, regulatory authorities, pharmaceutical companies, and research institutions. Area covered: The current synopsis focuses on the state-of-the-art in preclinical models for cholestatic DILI prediction. These models differ in their goal, complexity, availability, and applicability, and can widely be classified in experimental animals and in vitro models. Expert opinion: Drugs are a growing cause of cholestasis, but the progress made in explaining mechanisms and differences in susceptibility is not growing at the same rate. We need reliable models able to recapitulate the features of DIC, particularly its idiosyncrasy. The homogeneity and the species-specific differences move animal models away from a fair predictability. However, in vitro human models are improving and getting closer to the real hepatocyte phenotype, and they will likely be the choice in the near future. Progress in this area will not only need reliable predictive models but also mechanistic insights.

Increase in the number of new chemicals synthesized in past decades has resulted in constant growth in the development and application of computational models for prediction of activity as well as safety profiles of the chemicals. Most of the time, such computational models and its application must deal with imbalanced chemical data. It is indeed a challenge to construct a classifier using imbalanced data set. In this study, we analyzed and validated the importance of different sampling methods over non-sampling method, to achieve a well-balanced sensitivity and specificity of a machine learning model trained on imbalanced chemical data. Additionally, this study has achieved an accuracy of 93.00%, an AUC of 0.94, F1 measure of 0.90, sensitivity of 96.00% and specificity of 91.00% using SMOTE sampling and Random Forest classifier for the prediction of Drug Induced Liver Injury (DILI). Our results suggest that, irrespective of data set used, sampling methods can have major influence on reducing the gap between sensitivity and specificity of a model. This study demonstrates the efficacy of different sampling methods for class imbalanced problem using binary chemical data sets.

In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.

Purpose Paracetamol is one of the world’s most commonly used drugs. In overdose, it is well established to be hepatotoxic. The aim of this review was to identify factors that have been, or actually are, associated with the development of liver injury after paracetamol exposure in humans. Method Google Scholar and PubMed were searched on various dates between December 2016 and March 2017. Papers identified had their references analysed for further studies that might be relevant. Results At the time of writing, there was little good quality clinical evidence—from studies of paracetamol overdose or therapeutic use—to suggest that any groups of people are relatively protected from, or are at greater risk of, liver injury. The factors that were historically used to indicate higher risk in the UK have no good quality clinical evidence to support their re-introduction into clinical practice. The safe (and still effective) oral dose of paracetamol in patients weighing less than 50 kg has not been established. Conclusion There is no patient group that is unequivocally at elevated risk of paracetamol-induced liver toxicity. We propose two clinical scenarios that warrant further research. Firstly, there is a need to establish whether the dose of paracetamol should be reduced in patients with low body weight. Secondly, if or when genomic information regarding individual patients becomes readily available to inform prescribing, we propose the contribution of the genome to paracetamol toxicity should be re-investigated with robustly designed studies. Such studies could enhance the safe use of one of the most frequently taken drugs. Electronic supplementary material The online version of this article (10.1007/s00228-017-2356-6) contains supplementary material, which is available to authorized users.

Drug-induced liver injury (DILI) is a known adverse effect of both anti-tuberculosis (anti-TB) and antiretroviral (ARV) drugs. Recent studies highlight the implications of genetic predispositions to DILI. We performed a case-control study to identify Human Leukocyte Antigen-B (HLA-B) variant alleles associated with anti-TB and ARV co-treatment induced liver toxicity in Ethiopian TB and HIV co-infected patients. A total of 495 newly diagnosed TB and HIV co-infected patients were enrolled and received rifampicin based anti-TB and efavirenz based ARV therapy. Change in liver enzyme level from baseline was monitored 1st, 2nd, 4th, 8th, 12th, and 24th weeks after treatment initiation to identify patients who developed DILI (cases) and those who did not (treatment tolerants). Genomic DNA from 46 cases and 46 sex and age matched treatment tolerants were genotyped for HLA-B variant alleles using Olerup SSP®HLA-B DNA Typing Kits. The proportion of HLA-B*57 allele carriers in DILI cases (37.0%), particularly in those who developed cholestatic type of DILI (44.8%) was significantly higher compared with those who tolerated the treatment (2.2%). The HLA-B*57 allele frequency was significantly higher in cases (25%) than treatment tolerants (1.1%). In a multivariate logistic analysis, the proportion of patients carrying HLA-B*57 (P = 0.002) and HLA-B*14 (P = 0.014) alleles were significantly higher in DILI cases compared with treatment tolerants. HLA-B*57 was significantly associated with cholestatic (P = 0.001) and mixed (P = 0.017) types of liver toxicity, and mild-to-moderate severity (P = 0.001). Of all HLA-B*57 alleles detected, HLA-B*57:03 accounted 58.3% and HLA-B*57:02 accounted 41.7%. HLA-B*57:01 was not detected. The variant allele frequencies of HLA-B*57:03 (15.2% versus 0%) and HLA-B*57:02 (9.8% versus 1.1%) were significantly higher in the DILI cases than treatment tolerants (P < 0.03). We conclude that HLA-B*57 alleles (B*57:03 and B*57:02) confer susceptibility to the development of anti-TB and ARV drugs co-treatment induced liver toxicity, which is mainly of cholestatic type. The possible association of HLA-B*14 with anti-TB and ARV drugs co-treatment induced liver toxicity requires further investigations.

Ziel dieser Arbeit war es, ein neues in-vitro-Testsystem zur besseren Charakterisierung der Substanz-induzierten Leberschädigung (DILI) zu etablieren. Nach einer initialen Schädigung der Hepatozyten kann die Aktivierung des Immunsystems den ursprünglichen Schaden vergrößern und so zu schwerem Leberversagen führen. Dieser wichtige Einfluss der Immunantwort sollte durch die Berücksichtigung in einem Kokulturmodell zu einer verbesserten Vorhersage von DILI sowie zur Aufklärung der zugrunde liegenden molekularen Mechanismen führen. Dafür wurde eine Leberzelllinie mit einer in einen Makrophagen-ähnlichen Phänotyp differenzierten Immunzelllinie in einem indirekten Kokultursystem kombiniert, das den zellulären Austausch von Signalmolekülen erlaubte. Dadurch konnten in vivo beschriebene molekulare Ereignisse abgebildet werden, die zur Entstehung von Leberschäden führen. Im ersten Teil der vorliegenden Dissertation wurde daher ein Kokulturmodell mit HepG2- Zellen als hepatische Zelllinie und differenzierten THP-1-Zellen als Makrophagen-ähnliche Immunzelllinie etabliert. Zur Überprüfung des Modells wurde die Metabolisierung des leberschädigenden Antimyotikums Ketoconazol mittels Flüssigchromatographie-Massen- spektrometrie (LC-MS/MS) untersucht. Sieben reaktive Zwischenprodukte konnten nachge- wiesen werden, darunter das stark toxische N-Deacetyl-Ketoconazol. Anschließend wurden Veränderungen in Signalwegen in proteomischen Analysen mittels LC-MS/MS bestimmt. Dabei konnten die Inhibition der Sterol-Synthese sowie die Aktivierung des CXCL8-Signalwegs und der Nrf2-vermittelten oxidativen Stressantwort in kokultivierten HepG2-Zellen in vitro nachgewiesen werden. Diese Veränderungen führten wiederum zur Aktivierung der Zytokinexpression und -sekretion, die in PCR- und ELISA-Analysen gezielt analysiert wurden. Die Hinweise auf die Aktivierung des CXCL8-Signalwegs wurden durch den Nachweis der Expression und Sekretion mehrerer proinflammatorischer Zytokine, darunter neben CXCL8 auch TNF-alpha und IL-1beta, bekräftigt. Zusätzlich konnte in der behandelten Kokultur eine konzentrationsabhängige Zunahme des erst seit kurzem im Zusammenhang mit steriler Entzündung gefundenen möglichen Biomarkers HMGB1 nachgewiesen werden. In dem neu entwickelten Kokulturmodell wurde im zweiten Teil der Arbeit das aromatische Amin o-Anisidin, das u. a. in Tattoofarben enthalten sein kann, hinsichtlich einer Leberschädigung sowie deren Wirkmechanismus untersucht. Als Kontrolle dienten das para-Isomer p-Anisidin sowie Koffein. Die Untersuchung des Proteoms zeigte nach o- Anisidin-Behandlung eine Aktivierung des eIF2-Signalwegs sowie der Nrf2-vermittelten oxidativen Stressantwort. Darüber hinaus konnte in anschließenden Analysen eine konzen- trationsabhängige Zunahme von reaktiver Sauerstoffspezies (ROS) nachgewiesen werden. Auffällig war die Depolarisation des mitochondrialen Membranpotentials durch o-Anisidin- Behandlung. Diese korrelierte mit der Abnahme der intrazellulären ATP- Konzentration. Da in den o-Anisidin-behandelten Kokulturen keine erhöhte Expression proinflammatorischer Zytokine gezeigt werden konnte, ist eine Leberschädigung über einen immunvermittelten Mechanismus unwahrscheinlich. Allerdings konnte eine erhöhte HMGB1-Freisetzung nach o-Anisidin-Behandlung nachgewiesen werden. Dies unterstreicht die Bedeutung von HMGB1 als einen frühen Indikator für eine hepatische Schädigung. Im Gegensatz dazu induzierten weder p-Anisidin noch Koffein die Bildung von ROS. Koffein führte ebenfalls nicht zu Änderungen der Expression proinflammatorischer Zytokine. Die Verwendung der Huh-7-Zelllinie als mögliche Alternative zu den HepG2-Zellen zeigte bereits in Einzelkultur eine hohe Freisetzung von Zytokinen. Diese entspricht nicht beschriebenen in-vivo-Befunden. Die Huh-7-Zelllinie schien daher für den hier verfolgten Ansatz ungeeignet. Diese Ergebnisse zeigen, dass mit diesem in-vitro-Kokultursystem die Vorhersage toxischer Substanzen verbessert werden kann, da die Wechselwirkungen mit dem Immunsystem berücksichtigt werden können. HMGB1 scheint dabei ein neuer, möglicher Biomarker für geschädigte Hepatozyten und damit ein früher Indikator für eine Leberschädigung zu sein. Das in dieser Dissertation etablierte Kokulturmodell kann zukünftig für eine frühe Vorhersage der Hepatotoxizität von Substanzen dienen und so gleichzeitig zur Reduktion von Tierversuchen führen., The aim of the presented PhD thesis was the development of a new in vitro test system which allows for a better characterization and prediction of drug- induced liver injuries (DILI). After the initial damage of hepatocytes, the activation of the immune system can aggravate this damage and lead to severe liver failure. This important interaction should be included in a co-culture system and thus lead to an improved prediction of DILI as well as reveal the underlying mechanisms. Therefore a liver cell line and an immune cell line with a macrophage-like phenotype were combined to develop an indirect co- culture system. In this system soluble signalling molecules can be exchanged and thus molecular events which have been described in vivo can be analyzed in detail. In the first part of this work, a co-culture model with HepG2 cells as hepatic cell line and differentiated THP-1 cells as immune cell line was established. For the validation of the model system, the metabolism of the antifungal drug ketoconazole was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Seven reactive metabolites were detected, including the highly toxic N-deacetyl ketoconazole. Afterwards changes in signalling pathways were investigated by proteome analyses via LC-MS/MS. They revealed the inhibition of the sterol synthesis and the activation of the CXCL8 pathway and the Nrf2-mediated oxidative stress response in HepG2 cells in vitro. These changes lead to an increase in cytokine expression and secretion which was validated in targeted PCR and ELISA analyses. The activation of the CXCL8 signalling pathway could be verified by the detection of several proinflammatory cytokines including CXCL8, TNF-alpha and IL-1beta. In addition, a concentration dependent increase of HMGB1 protein could be detected in treated co-cultures which is an only recently described possible new biomarker. In the newly established co-culture model, the aromatic amine o-anisidine was tested regarding its mode of action. It was compared with its para-isomer p-anisidine and caffeine. The proteome analysis showed the activation of the eIF2 signalling pathway and the Nrf2-mediated oxidative stress response. Targeted approaches revealed the concentration dependent generation of reactive oxygen species (ROS) in o-anisidine treated cell cultures. Strikingly the mitochondrial membrane became depolarized by o-anisidine treatment. This correlated with the observed ATP decrease. As no increased expression of proinflammatory cytokines could be shown in treated co-cultures, a liver injury through an immune-mediated mechanism seems unlikely. The increased release of HMGB1 points towards its role as an early indicator of liver injury. In contrast to that neither p-anisidine nor caffeine induced the ROS generation. Caffeine did not change the expression of proinflammatory cytokines, either. Experiments using the Huh-7 cell line as a possible alternative to HepG2 showed already in single cultures the release of cytokines at high concentrations. This does not correlate with the described in vivo effects. Therefore the Huh-7 cell line is not suitable for the approach taken here. These results show that the use of this in vitro co- culture systems can lead to an improved prediction of toxic substances. They take the interactions with the immune system into account. HMGB1 seems to be a new, possible biomarker for damaged hepatocytes and thus an early indicator of a liver injury. The co-culture model established in this PhD thesis can lead to an early prediction of a drug’s hepatotoxicity and thus to a reduction of animal experiments.

Kratom ( Mitragyna speciosa) is a prevalent medicinal plant used mainly for the stimulant and analgesic properties provided through multiple alkaloid compounds. Over the past decade, use of kratom has increased despite the limited knowledge of toxicities and adverse side effects. With the current opioid epidemic, both patients and providers are seeking alternative methods to treat both addiction and pain control, and kratom as an alternative means of treatment has increasingly entered the mainstream. In this article, we present the clinical course of a 47-year-old male who developed fatigue, pruritus, and abnormal liver tests (with a mixed hepatocellular/cholestatic pattern) approximately 21 days after beginning kratom. After extensive evaluation including a negligible alcohol history, negative hepatitis serologies, and inconclusive imaging, the patient was diagnosed with drug-induced liver injury (DILI) caused by kratom. Nine months after his liver tests returned to normal, he took kratom again, and after a latency of 2 days, he developed fatigue, pruritus, and loss of appetite along with abnormal liver tests (with the same biochemical profile as previously), consistent with a positive rechallenge. We believe, through the use of the Roussel-Uclaf Causality Assessment Method and expert opinion, that this is a highly likely or definite example of kratom-induced DILI. With the gaining popularity of this drug, it appears that DILI may be an important complication of kratom for providers to recognize.

Background: The use of anti-tuberculosis drugs (ATD) is not uncommon to find side effects that complicate treatment. One of the side effects caused by ATD treatment is Drug Induced Liver Injury (DILI). Risk factors that trigger the incidence of DILI during tuberculosis (TB) treatment involving variety of factors. This study was conducted to determine the risk factors of DILI in pulmonary tuberculosis patient. Method: Conducted retrospective case-control study. Study subjects were all patients who underwent first category ATD treatment at Dr. Wahidin Sudirohusodo hospital of Makassar, Indonesia. Risk factors examined were age, gender, nutritional status, alcohol consumption, smoking, history of liver disease, consumption of other drug, ATD preparation of Fixed Dose Combination (FDC) and abnormality of SGPT/ total bilirubin control value at minimum of 2 weeks after starting treatment. Result: from total 47 samples who took part in the study, 10 samples (21.3%) suffered DILI. Samples characteristics who suffered from DILI, were an average age of 52.7 years old (P < 0.05), and consumption of other drug 29.4% (P < 0.05). There was no significant relationship between gender, nutritional status, alcohol consumption, smoking, and history of liver disease in the incidence of DILI. Conclusion: Old age and history of consumption of other drug has significant relationship as a risk factors for the incidence of DILI in TB patients who consume ATD, while no significant relationship found between gender, malnutrition, alcohol consumption, smoking, and history of liver disease.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery. Electronic supplementary material The online version of this article (doi:10.1007/s00204-017-2002-1) contains supplementary material, which is available to authorized users.

Drug-induced liver injury (DILI) is a clinically important adverse drug reaction, which prevents the development of many otherwise safe and effective new drugs. Currently, there is a lack of sensitive and specific biomarkers that can be used to predict, assess and manage this toxicity. The aim of this work was to evaluate gadoxetate-enhanced MRI as a potential novel biomarker of hepatobiliary transporter inhibition in the rat. Initially, the volume fraction of extracellular space in the liver was determined using gadopentetate to enable an estimation of the gadoxetate concentration in hepatocytes. Using this information, a compartmental model was developed to characterise the pharmacokinetics of hepatic uptake and biliary excretion of gadoxetate. Subsequently, we explored the impact of an investigational hepatobiliary transporter inhibitor on the parameters of the model in vivo in rats. The investigational hepatobiliary transporter inhibitor reduced both the rate of uptake of gadoxetate into the hepatocyte, k1, and the Michaelis–Menten constant, Vmax, characterising its excretion into bile, whereas KM values for biliary efflux were increased. These effects were dose dependent and correlated with effects on plasma chemistry markers of liver dysfunction, in particular bilirubin and bile acids. These results indicate that gadoxetate-enhanced MRI provides a novel functional biomarker of inhibition of transporter-mediated hepatic uptake and clearance in the rat. Since gadoxetate is used clinically, the technology has the potential to provide a translatable biomarker of drug-induced perturbation of hepatic transporters that may also be useful in humans to explore deleterious functional alterations caused by transporter inhibition. Copyright © 2013 John Wiley & Sons, Ltd.

Low-dose methotrexate (MTX) is part of the mainstay of rheumatoid arthritis treatment. Hepatotoxicity is among the most feared side effects of low-dose MTX and is associated with increased morbidity. At present, histological evaluation of liver biopsies is the gold standard to retrospectively diagnose MTX-induced liver damage. Genetic markers present an interesting opportunity to pre-emptively identify patients at risk for MTX-induced hepatotoxicity. Here, we will review the literature on candidate genetic markers for the risk of MTX-induced hepatotoxicity. These candidate genetic markers include polymorphisms in the gene encoding the enzyme MTHFR.

The aim of this study was to investigate the causes of elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in children. We analyzed the medical records for children aged 3 months to 18 years who presented to the hospital with ALT >45 IU/L and/or AST >50 IU/L, between 2012 and 2014, for various reasons, including those not related to liver disease. In total, 281 children met the study criteria. This group comprised of 125 (44.5%) females and 156 (55.5%) males. At the presentation, the most common patient complaint was fatigue (53.4%), while 15.7% of the patients reported no symptoms. The most common findings on the physical examination were jaundice and hepatomegaly. In 15% of the cases, the findings were normal. According to the diagnosis, the most common cause of the elevated transaminases were infections (34%), with hepatitis A virus (HAV) infection as the leading cause (18.9%). Drug-induced liver injury (DILI) was the cause in 18.1% of the cases and non-alcoholic fatty liver disease (NAFLD) in 11.1%. The highest transaminase levels were associated with HAV infection, while DILI and NAFLD caused only slightly elevated transaminases. Overall, our results show that the elevated transaminases in children are most often caused by infections, DILI, and NAFLD. In a majority of cases, elevated ALT and AST indicate liver disease, however, they could also be associated with conditions other than liver damage. Additionally, the elevated enzymes can be detected in completely healthy individuals.

Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals. Electronic supplementary material The online version of this article (doi:10.1007/s00204-016-1781-0) contains supplementary material, which is available to authorized users.

Primary human hepatocytes (hHEP), human HepaRG and HepG2 cell lines are the most used human liver-based in vitro models for hepatotoxicity testing, including screening of drug-induced liver injury (DILI)-inducing compounds. hHEP are the reference hepatic in vitro system, but their availability is limited and the cells available for toxicology studies are often of poor quality. Hepatic cell lines on the other hand are highly proliferative and represent an inexhaustible hepatic cell source. However, these hepatoma-derived cells do not represent the population diversity and display reduced hepatic metabolism. Alternatively, stem cell-derived hepatic cells, which can be produced in high numbers and can differentiate into multiple cell lineages, are also being evaluated as a cell source for in vitro hepatotoxicity studies. Human skin-derived precursors (hSKP) are post-natal stem cells that, after conversion towards hepatic cells (hSKP-HPC), respond to hepatotoxic compounds in a comparable way as hHEP. In the current study, four different human hepatic cell systems (hSKP-HPC, hHEP, HepaRG and HepG2) are evaluated for their capacity to predict hepatic toxicity. Their hepatotoxic response to acetaminophen (APAP) exposure is compared to data obtained from patients suffering from APAP-induced acute liver failure (ALF). The results indicate that hHEP, HepaRG and hSKP-HPC identify comparable APAP-induced hepatotoxic functions and that HepG2 cells show the slightest hepatotoxic response. Pathway analyses further points out that HepaRG cells show the highest predicted activation of the functional genes related to 'damage of liver', followed by hSKP-HPC and hHEP cells that generated similar results. HepG2 did not show any activation of this function.

Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p

The host-dependent nature of idiosyncratic drug-induced liver injury (iDILI) suggests that rare genetic polymorphisms may contribute to the disease. Indeed, a few mutations in key genes have already been identified using conventional human genetics approaches. Over 50 commonly used drugs can precipitate iDILI, making this a substantial medical problem. Only recently have human induced pluripotent stem cells been used as a research tool to discover novel iDILI genes and to study the mechanisms of iDILI in vitro. Here we review the current state of stem cell use in the investigation of iDILI, with a special focus on genetics. In addition, the concerns and difficulties associated with genetics and animal model research are discussed. We then present the features of patient-specific pluripotent stem cells (which may be derived from iDILI patients themselves), and explain why these cells may be of great utility. A variety of recent approaches to produce hepatocyte-like cells from pluripotent cells and the associated advantages and limitations of such cells are discussed. Future directions for the use of stem cell science to investigate iDILI include novel ways to identify new iDILI genes, a consideration of epigenetic impacts on iDILI, and the development of new and improved strategies for the production of hepatocytes from human pluripotent cells.

International audience; The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun. Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.