Mechanisms Of Drug-induced Liver Injury Research Articles

Novel reduced heteropolyacid nanoparticles for effective treatment of drug-induced liver injury by manipulating reactive oxygen and nitrogen species and inflammatory signals

With the rapid advancements in biomedicine, the use of clinical drugs has surged sharply. However, potential hepatotoxicity limits drug exploitation and widespread usage, posing serious threats to patient health. Hepatotoxic drugs disrupt liver enzyme levels and cause refractory pathological damage, creating a challenge in the application of diverse first-line drugs. The activation and deterioration of reactive oxygen and nitrogen species (RONS) and inflammatory signals are key pathological mechanisms of drug-induced liver injury (DILI). Herein, a novel reduced heteropolyacid nanoparticle (RNP) has been developed, possessing high RONS-scavenging ability, strong anti-inflammatory activity, and excellent biosafety. These features enable it to swiftly restore the redox and immune balance of the liver. Intravenous administration of RNP effectively scavenged RONS storm, reversing liver oxidative stress and restoring normal mitochondrial membrane potential and function. Furthermore, by inhibiting c-Jun-N-terminal kinase phosphorylation, RNP facilitated the restoration of nuclear factor erythroid 2-related factor 2-mediated endogenous antioxidant signaling, ultimately rescuing the liver function and tissue morphology in acetaminophen-induced DILI mice. Crucially, the high biocompatible RNP exhibited superior efficacy in the DILI mouse model compared to the clinical antioxidant N-acetylcysteine. This targeted therapeutic approach, tailored to address the onset and progression of DILI, offers valuable new insights into controlling the condition and restoring liver structure and function.

A computational framework to in silico screen for drug-induced hepatocellular toxicity.

Drug-induced liver injury (DILI) is the most common trigger for acute liver failure and the leading cause of attrition in drug development. In this study, we developed an in silico framework to screen drug-induced hepatocellular toxicity (INSIGHT) by integrating the post-treatment transcriptomic data from both rodent models and primary human hepatocytes. We first built an early prediction model using logistic regression with elastic net regularization for 123 compounds and established the INSIGHT framework that can screen for drug-induced hepatotoxicity. The 235 signature genes identified by INSIGHT were involved in metabolism, bile acid synthesis, and stress response pathways. Applying the INSIGHT to an independent transcriptomic dataset treated by 185 compounds predicted that 27 compounds show a high DILI risk, including zoxazolamine and emetine. Further integration with cell image data revealed that predicted compounds with high DILI risk can induce abnormal morphological changes in the endoplasmic reticulum and mitochondrion. Clustering analysis of the treatment-induced transcriptomic changes delineated distinct DILI mechanisms induced by these compounds. Our study presents a computational framework for a mechanistic understanding of long-term liver injury and the prospective prediction of DILI risk.

Acetaminophen Hepatotoxicity: Paradigm for Understanding Mechanisms of Drug-Induced Liver Injury.

Acetaminophen (APAP) overdose is the clinically most relevant drug hepatotoxicity in western countries, and, because of translational relevance of animal models, APAP is mechanistically the most studied drug. This review covers intracellular signaling events starting with drug metabolism and the central role of mitochondrial dysfunction involving oxidant stress and peroxynitrite. Mitochondria-derived endonucleases trigger nuclear DNA fragmentation, the point of no return for cell death. In addition, adaptive mechanisms that limit cell death are discussed including autophagy, mitochondrial morphology changes, and biogenesis. Extensive evidence supports oncotic necrosis as the mode of cell death; however, a partial overlap with signaling events of apoptosis, ferroptosis, and pyroptosis is the basis for controversial discussions. Furthermore, an update on sterile inflammation in injury and repair with activation of Kupffer cells, monocyte-derived macrophages, and neutrophils is provided. Understanding these mechanisms of cell death led to discovery of N-acetylcysteine and recently fomepizole as effective antidotes against APAP toxicity.

A network-based transcriptomic landscape of HepG2 cells uncovering causal gene-cytotoxicity interactions underlying drug-induced liver injury.

Drug-induced liver injury (DILI) remains the main reason for drug development attritions largely due to poor mechanistic understanding. Toxicogenomic to interrogate the mechanism of DILI has been broadly performed. Gene coregulation network-based transcriptome analysis is a bioinformatics approach that potentially contributes to improve mechanistic interpretation of toxicogenomic data. Here we performed an extensive concentration time course response-toxicogenomic study in the HepG2 cell line exposed to 20 DILI compounds, 7 reference compounds for stress response pathways, and 10 agonists for cytokines and growth factor receptors. We performed whole transcriptome targeted RNA sequencing to more than 500 conditions and applied weighted gene coregulated network analysis to the transcriptomics data followed by the identification of gene coregulated networks (modules) that were strongly modulated upon the exposure of DILI compounds. Preservation analysis on the module responses of HepG2 and PHH demonstrated highly preserved adaptive stress response gene coregulated networks. We correlated gene coregulated networks with cell death onset and causal relationships of 67 critical target genes of these modules with the onset of cell death was evaluated using RNA interference screening. We identified GTPBP2, HSPA1B, IRF1, SIRT1, and TSC22D3 as essential modulators of DILI compound-induced cell death. These genes were also induced by DILI compounds in PHH. Altogether, we demonstrate the application of large transcriptome datasets combined with network-based analysis and biological validation to uncover the candidate determinants of DILI.

Drug-induced liver injury: diagnosis of exclusion

Drug-induced liver injury (DILI) is a relevant issue in clinical practice and is still a diagnosis of exclusion. Despite the low incidence in the general population, DILI is the cause of most cases of acute hepatic injury and has a mortality rate of up to 50%. Despite many reports in the medical literature about the DILI mechanisms, a clear causal relationship between them, drugs, and risk factors has not been established. Current clinical practice is based on a combination of a thorough study of a history of risk factors, the timing of drug and dietary supplements' administration, and the analysis of laboratory and instrumental tests. It aligns with the international criteria of the Rousell Uclaf Causality Assessment Method (RUCAM), which is considered one of the main diagnostic algorithms for DILI. The article addresses current DILI classification, risk factors, diagnostic algorithms, causalities, clinical evaluation, promising liver function biomarkers, and specific treatment.

Cyclopia intermedia E. Mey protects against ROS-induced liver injury in HepG2/C3A cells.

Drug-induced liver injury (DILI) represents a significant clinical problem for which no standard treatment currently exists. Most DILI mechanisms center on oxidative stress resulting from glutathione depletion, excessive ROS production, and subsequent cell death. Flavonoid-rich Cyclopia intermedia E. Mey (honeybush) is a commercially important African herbal tea and is traditionally promoted as a restorative treatment. Cytotoxicity evaluation of a fermented C. intermedia extract in C3A hepatocytes confirmed the absence of toxicity up to 200 µg/mL using quantitative fluorescence microscopy with Hoechst 33342-PI dual-labeling. DPPH, NO scavenging, and ORAC assays established a strong antioxidant potential. Antioxidant bioavailability, assessed using the CAPe assay, indicated significant uptake at 100 µg/mL (p < 0.005). C. intermedia extract decreased TBHP-induced oxidative stress in C3A cells and attenuated TBHP-induced changes in thiol group levels at 200 µg/mL (p < 0.005) as confirmed by CellROX® Orange and ThiolTracker™ Violet staining, respectively. Apoptotic cell death was significantly reduced by the extract from 50 µg/mL (p < 0.005) as determined by Annexin-V FITC staining. These results suggest that C. intermedia can function as a hepatoprotectant and has potential as a treatment against DILI.

Combined analysis of 16S rDNA sequencing and metabolomics to find biomarkers of drug-induced liver injury

Drug induced liver injury (DILI) is a kind of liver dysfunction which caused by drugs, and gut microbiota could affect liver injury. However, the relationship between gut microbiota and its metabolites in DILI patients is not clear. The total gut microbiota DNA was extracted from 28 DILI patient and 28 healthy control volunteers (HC) and 16S rDNA gene were amplified. Next, differentially metabolites were screened. Finally, the correlations between the diagnostic strains and differentially metabolites were studied.The richness and uniformity of the bacterial communities decreased in DILI patients, and the structure of gut microbiota changed obviously. Enterococcus and Veillonella which belong to Firmicutes increased in DILI, and Blautia and Ralstonia which belong to Firmicutes, Dialister which belongs to Proteobacteria increased in HC. In addition, these diagnostic OTUs of DILI were associated with the DILI damage mechanism. On the other hands, there were 66 differentially metabolites between DILI and HC samples, and these metabolites were mainly enriched in pyrimidine metabolism and steroid hormone biosynthesis pathways. Furthermore, the collinear network map of the key microbiota-metabolites were constructed and the results indicated that Cortodoxone, Prostaglandin I1, Bioyclo Prostaglandin E2 and Anacardic acid were positively correlated with Blautia and Ralstonia, and negatively correlated with Veillonella.This study analyzed the changes of DILI from the perspective of gut microbiota and metabolites. Key strains and differentially metabolites of DILI were screened and the correlations between them were studied. This study further illustrated the mechanism of DILI.

An Immunological Perspective on the Mechanism of Drug Induced Liver Injury: Focused on Drugs for Treatment of Hepatocellular Carcinoma and Liver Transplantation.

The liver is frequently exposed to potentially toxic materials, and it is the primary site of clearance of foreign agents, along with many innate and adaptive immune cells. Subsequently, drug induced liver injury (DILI), which is caused by medications, herbs, and dietary supplements, often occurs and has become an important issue in liver diseases. Reactive metabolites or drug-protein complexes induce DILI via the activation of various innate and adaptive immune cells. There has been a revolutionary development of treatment drugs for hepatocellular carcinoma (HCC) and liver transplantation (LT), including immune checkpoint inhibitors (ICIs), that show high efficacy in patients with advanced HCC. Along with the high efficacy of novel drugs, DILI has become a pivotal issue in the use of new drugs, including ICIs. This review demonstrates the immunological mechanism of DILI, including the innate and adaptive immune systems. Moreover, it aims to provide drug treatment targets, describe the mechanisms of DILI, and detail the management of DILI caused by drugs for HCC and LT.

Drug-induced liver injury: a comprehensive review.

Drug-induced liver injury (DILI) remains a challenge in clinical practice and is still a diagnosis of exclusion. Although it has a low incidence amongst the general population, DILI accounts for most cases of acute liver failure with a fatality rate of up to 50%. While multiple mechanisms of DILI have been postulated, there is no clear causal relationship between drugs, risk factors and mechanisms of DILI. Current best practice relies on a combination of high clinical suspicion, thorough clinical history of risk factors and timeline, and extensive hepatological investigations as supported by the international Roussel Uclaf Causality Assessment Method criteria, the latter considered a key diagnostic algorithm for DILI. This review focuses on DILI classification, risk factors, clinical evaluation, future biomarkers and management, with the aim of facilitating physicians to correctly identify DILI early in presentation.

Drug-Induced Liver Injury in Tuberculosis: Mechanisms of Development and Diagnostic Methods

The review article discusses modern aspects of drug-induced liver injury (DILI) in patients with tuberculosis who are receiving etiotropic therapy. The main mechanisms of DILI, including toxic and idiosyncratic types, are described, as well as their pathogenetic, biochemical, and epidemiological differences. DILI can manifest as various clinicomorphological forms of liver damage, such as steatosis and steatohepatitis, acute and chronic hepatitis, mitochondrial cytopathy, cholestasis, sclerosing cholangitis, vascular injury, and others. The main diagnostic method for DILI is the detection of liver enzymes - transaminases and alkaline phosphatase - based on the degree of elevation and their ratio, which identify two main types of liver injury - hepatocellular and cholestatic - as well as a mixed variant. The article provides a scoring assessment of liver damage in a patient receiving chemotherapy to classify it as drug-induced liver injury.

Genetics of drug-induced liver injury: Current knowledge and future prospects.

Idiosyncratic drug-induced liver injury (DILI) remains an important clinical problem, both during drug development and the prescription of a range of licensed drugs. Although rare, the consequences are serious. Ongoing studies on genetic risk factors for DILI, especially genomewide association studies, have resulted in the identification of a number of genetic risk factors, including particular HLA alleles and a few non-HLA genes, both immune-related and metabolic. Some non-HLA associations, such as N-acetyltransferase 2 in isoniazid DILI and interferon regulatory factor 6 in interferon-beta DILI are likely to be drug-specific due to the role of the associated gene, but there is also evidence for polygenic susceptibility involving pathways such as oxidative and endoplasmic reticulum stress and mitochondrial function for DILI induced by multiple drugs. Increased knowledge of genetic risk factors should assist in better understanding underlying DILI mechanisms and help improve methods for identifying hepatotoxic drugs early in development. HLA allele-specific T cell proliferation together with in silico prediction of drug binding to specific HLA proteins have confirmed genetic findings for certain common causes of DILI. However, studies in hepatocytes exposed to high drug concentrations suggest toxicity that is not dependent on genotype also occurs. It seems likely that susceptibility to DILI involves several genetic risk factors combining with other factors that affect drug levels. Despite recent progress in detecting genetic risk factors for DILI, low positive predictive values mean that general implementation of genotyping prior to prescription of potentially hepatotoxic drugs is not useful currently.

Thiol “click” chromene mediated cascade reaction forming coumarin for in-situ imaging of thiol flux in drug-induced liver injury

Drug-induced organ damage has long been considered as a serious public health concern. Drug-induced liver injury (DILI) is one of the common clinical side effects of drugs and the main cause of acute liver failure. Previous evidence showed that there is a potential relationship between thiols and DILI. Many studies have reported that oxidative stress is involved in many drug-induced liver injury mechanisms. APAP, as the most representative drug for clinical DILI, is considered to cause large amounts of intracellular reactive oxygen species. However, thiols as the effective antioxidant maintain the normal redox balance of organisms and play a vital role in the liver injury and other diseases. Therefore, a new NIR fluorescent probe DCI-Ac-HMPC was developed based on thiol-chromene “click” reaction, which triggered the intramolecular cascade reaction to in-situ generate NIR coumarin fluorophore. In addition, we found that thiols levels in the liver of mice with DILI were significantly reduced, revealing a negative correlation between thiols and DILI. Furthermore, the therapeutic effects of three kinds of liver-protecting drugs on hepatotoxicity induced by acetaminophen (APAP) in vivo based on thiols fluctuations. This work provides a new idea for the in-situ synthesis strategy of NIR coumarin dyes, which is helpful for the diagnosis of DILI and the further study of the role of thiols in related diseases.

Discovery of bakuchiol as an AIM2 inflammasome activator and cause of hepatotoxicity

Ethnopharmacological relevancePsoralea corylifolia (P. corylifolia Linn.) is a traditional Chinese medicinal plant that exhibits significant aphrodisiac, diuretic, and anti-rheumatic effects. However, it has been reported to cause hepatic injury, but the precise mechanisms remain unclear. Aim of the studyTo evaluate the safety and risk of P. corylifolia and to elucidate the underlying mechanisms of drug-induced liver injury. Materials and methodsWestern blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, quantitative polymerase chain reaction (Q-PCR), and flow cytometry were used to explore the effect of bakuchiol (Bak), one of the most abundant and biologically active components of P. corylifolia, on the AIM2 inflammasome activation and the underlying mechanism. Furthermore, we used the lipopolysaccharides (LPS)-induced drug-induced liver injury (DILI) susceptible mice model to study the Bak-mediated hepatotoxicity. ResultsBak induced the maturation of caspase-1 P20, and significantly increased the expression of IL-1β and TNF-α (P < 0.0001) compared with the control group. Moreover, compared to the Bak group, knockdown of AIM2 inhibited Bak-induced caspase-1 maturation and significantly decreased the production of IL-1β and TNF-α, but knockout of NLRP3 had no effect. Mechanistically, Bak-induced AIM2 inflammasome activation is involved in mitochondrial damage, mitochondrial DNA (mtDNA) release, and subsequent recognition of cytosolic mtDNA. Our in vivo data showed that co-exposure to LPS and non-hepatotoxic doses of Bak significantly increased the levels of ALT, AST, IL-1β, TNF-α, and IL-18, indicating that Bak can induce severe liver inflammation (P < 0.005). ConclusionsThe result shows that Bak activates the AIM2 inflammasome by inducing mitochondrial damage to release mtDNA, and subsequently binds to the AIM2 receptor, indicating that Bak may be a risk factor for P. corylifolia-induced hepatic injury.

H2O2-Activated NIR-II Fluorescent Probe with a Large Stokes Shift for High-Contrast Imaging in Drug-Induced Liver Injury Mice.

Drug-induced liver injury (DILI) is the most common clinical adverse drug reaction, which is closely associated with the oxidative stress caused by overproduced reactive oxygen species. Hepatic H2O2, as an important biomarker of DILI, plays a crucial role in the progression of DILI. However, there remains a challenge to develop H2O2-activatable second near-infrared (NIR-II, 1000-1700 nm) small molecular probes with both a large Stokes shift and a long emission wavelength beyond 950 nm. Herein, we developed an activatable NIR-II fluorescent probe (IR-990) with an acceptor-π-acceptor (A-π-A) skeleton for real-time detection of H2O2 in vivo. In the presence of H2O2, nonfluorescent probe IR-990 was successfully unlocked by generating a donor-π-acceptor (D-π-A) structure and switched on intense NIR-II fluorescence, exhibiting a peak emission wavelength at 990 nm and a large Stokes shift of 200 nm. Moreover, it was able to detect H2O2 with high sensitivity and selectivity in vitro (LOD = 0.59 μM) and monitor the behavior of endogenous H2O2 in the HepG2 cell model of DILI for the first time. Notably, probe IR-990 was successfully applied in real-time imaging of endogenous H2O2 generation in the DILI mouse model, showing a high signal-to-background ratio of 11.3/1. We envision that IR-990 holds great potential as a powerful diagnosis tool for real-time visualization of H2O2 in vivo and revealing the mechanism of DILI in the future.

Diclofenac-Induced Cytotoxicity in Direct and Indirect Co-Culture of HepG2 Cells with Differentiated THP-1 Cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) frequently induce drug-induced liver injury (DILI). It is unclear whether macrophages such as M1 and M2 participate in NSAID-associated DILI; elucidating this relationship could lead to a better understanding of the detailed mechanism of DILI. We co-cultured human hepatoma HepG2 cells with M1 or M2 derived from human monocytic leukemia THP-1 cells to examine the roles of M1 and M2 in DIC-induced cytotoxicity. DIC was added to the direct or indirect co-cultures of HepG2 cells with M1 or M2 (HepG2/M1 or HepG2/M2, respectively) at cell ratios of (1:0, 1:0.1, 1:0.4, and 1:1). In both direct and indirect HepG2/M2 co-cultures (1:0.4), there was lower lactate dehydrogenase release compared with HepG2/M1 co-cultures. Other NSAIDs as well as DIC showed similar protective effects of DIC-induced cytotoxicity. There were only slight differences in mRNA levels of apoptosis- and endoplasmic reticulum stress-associated factors between M1 and M2 after DIC treatment, suggesting that other factors determined the protective effects of M2 on DIC-induced cytotoxicity. Levels of high mobility group box 1 (HMGB1) in the medium and the mRNA expression levels of HMGB1 receptors were different between M1 and M2 after DIC treatment. Increased HMGB1 concentrations and expression of toll-like receptor 2 mRNA in M1 were observed compared with M2 after DIC treatment. In conclusion, these results suggested that the HMGB1/TLR2 signaling axis can be suppressed in M2 but not M1, leading to the different roles of M1 and M2 in NSAID-induced cytotoxicity.

A Comprehensive Review on the Use of Herbal Dietary Supplements in the USA, Reasons for Their Use, and Review of Potential Hepatotoxicity

Herbal and dietary supplement (HDS) use has grown exponentially in the United States. Unfortunately, the incidence of HDS-related liver injury has proportionally increased. Despite the potential for certain HDSs to cause clinically significant liver injury, they are not regulated by the Food and Drug Administration. Recent efforts have been made to regulate HDSs but are far removed from the scrutiny of prescription medications. Scant literature exists on HDSs and their risks of causing liver injury. In this comprehensive review, we examine trends of HDS use in the United States and the pathophysiologic mechanisms of drug-induced liver injury (DILI) of certain HDSs. Finally, we review usage rates; benefits, if any; purported pathophysiology of DILI; and propensity for progression to fulminant hepatic failure of nine HDSs linked to clinically significant DILI.

Identification of New Toxicity Mechanisms in Drug-Induced Liver Injury through Systems Pharmacology.

Among adverse drug reactions, drug-induced liver injury presents particular challenges because of its complexity, and the underlying mechanisms are still not completely characterized. Our knowledge of the topic is limited and based on the assumption that a drug acts on one molecular target. We have leveraged drug polypharmacology, i.e., the ability of a drug to bind multiple targets and thus perturb several biological processes, to develop a systems pharmacology platform that integrates all drug–target interactions. Our analysis sheds light on the molecular mechanisms of drugs involved in drug-induced liver injury and provides new hypotheses to study this phenomenon.

The Potential Role of Metabolomics in Drug-Induced Liver Injury (DILI) Assessment.

Drug-induced liver injury (DILI) is one of the most frequent adverse clinical reactions and a relevant cause of morbidity and mortality. Hepatotoxicity is among the major reasons for drug withdrawal during post-market and late development stages, representing a major concern to the pharmaceutical industry. The current biochemical parameters for the detection of DILI are based on enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP)) and bilirubin serum levels that are not specific of DILI and therefore there is an increasing interest on novel, specific, DILI biomarkers discovery. Metabolomics has emerged as a tool with a great potential for biomarker discovery, especially in disease diagnosis, and assessment of drug toxicity or efficacy. This review summarizes the multistep approaches in DILI biomarker research and discovery based on metabolomics and the principal outcomes from the research performed in this field. For that purpose, we have reviewed the recent scientific literature from PubMed, Web of Science, EMBASE, and PubTator using the terms “metabolomics”, “DILI”, and “humans”. Despite the undoubted contribution of metabolomics to our understanding of the underlying mechanisms of DILI and the identification of promising novel metabolite biomarkers, there are still some inconsistencies and limitations that hinder the translation of these research findings into general clinical practice, probably due to the variability of the methods used as well to the different mechanisms elicited by the DILI causing agent.

Deriving time-concordant event cascades from gene expression data: A case study for Drug-Induced Liver Injury (DILI).

Adverse event pathogenesis is often a complex process which compromises multiple events ranging from the molecular to the phenotypic level. In toxicology, Adverse Outcome Pathways (AOPs) aim to formalize this as temporal sequences of events, in which event relationships should be supported by causal evidence according to the tailored Bradford-Hill criteria. One of the criteria is whether events are consistently observed in a certain temporal order and, in this work, we study this time concordance using the concept of "first activation" as data-driven means to generate hypotheses on potentially causal mechanisms. As a case study, we analysed liver data from repeat-dose studies in rats from the TG-GATEs database which comprises measurements across eight timepoints, ranging from 3 hours to 4 weeks post-treatment. We identified time-concordant gene expression-derived events preceding adverse histopathology, which serves as surrogate readout for Drug-Induced Liver Injury (DILI). We find known mechanisms in DILI to be time-concordant, and show further that significance, frequency and log fold change (logFC) of differential expression are metrics which can additionally prioritize events although not necessary to be mechanistically relevant. Moreover, we used the temporal order of transcription factor (TF) expression and regulon activity to identify transcriptionally regulated TFs and subsequently combined this with prior knowledge on functional interactions to derive detailed gene-regulatory mechanisms, such as reduced Hnf4a activity leading to decreased expression and activity of Cebpa. At the same time, also potentially novel events are identified such as Sox13 which is highly significantly time-concordant and shows sustained activation over time. Overall, we demonstrate how time-resolved transcriptomics can derive and support mechanistic hypotheses by quantifying time concordance and how this can be combined with prior causal knowledge, with the aim of both understanding mechanisms of toxicity, as well as potential applications to the AOP framework. We make our results available in the form of a Shiny app (https://anikaliu.shinyapps.io/dili_cascades), which allows users to query events of interest in more detail.

Newly identified genetic variants associated with idiosyncratic drug-induced liver injury.

Datasets of well characterized drug or herbal and dietary supplement-associated liver injury has provided a rich resource to identify genetic variants associated with hepatic injury that further supports the role of immune activation in drug-induced liver injury (DILI). Using DNA microarrays, whole genome sequencing, HLA-restricted DNA sequencing with appropriate ethnically matched population controls have identified HLA-specific genetic variants for drugs or botanical compounds with the same HLA variant associated with different agents. In addition to HLAs, two genes involved with immune signaling were also identified: a functional PTPN22 variant associated with increased DILI risk to any agent or clinical presentation and a variant in ERAP2 hepatic gene expression that trims peptide in preparation for presentation in the HLA cleft increased the risk for DILI in amoxicillin-clavulanate DILI when present with known HLA risk alleles. Variants in HLA and other genes involved in immune regulations further supports immune system activation in DILI. In the future, identifying these variants before exposure may minimize the risk for DILI events, help with assessment of drug causality for causing DILI and with greater understanding of DILI mechanisms, has important implication for future drug development.