The Evolving Profile of Idiosyncratic Drug-Induced Liver Injury.

Idiosyncratic drug-induced liver injury (DILI) is an infrequent but important cause of liver disease. Newly identified causes of DILI include the COVID vaccines, turmeric, green tea extract, and immune checkpoint inhibitors. DILI is largely a clinical diagnosis of exclusion that requires evaluation for more common causes of liver injury and a compatible temporal association with the suspect drug. Recent progress in DILI causality assessment includes the development of the semi-automated revised electronic causality assessment method (RECAM) instrument. In addition, several drug-specific HLA associations have been identified that can help with the confirmation or exclusion of DILI in individual patients. Various prognostic models can help identify the 5%-10% of patients at highest risk of death. Following suspect drug cessation, 80% of patients with DILI fully recover, whereas 10%-15% have persistently abnormal laboratory studies at 6 months of follow-up. Hospitalized patients with DILI with an elevated international normalized ratio or mental status changes should be considered for N-acetylcysteine therapy and urgent liver transplant evaluation. Selected patients with moderate to severe drug reaction with eosinophilia and systemic symptoms or autoimmune features on liver biopsy may benefit from short-term corticosteroids. However, prospective studies are needed to determine the optimal patients and dose and duration of steroids to use. LiverTox is a comprehensive, freely accessible Web site with important information regarding the hepatotoxicity profile of more than 1000 approved medications and 60 herbal and dietary supplement products. It is hoped that ongoing "omics" studies will lead to additional insight into DILI pathogenesis, improved diagnostic and prognostic biomarkers, and mechanism-based treatments.

Metabolomics-based strategy to assess drug hepatotoxicity and uncover the mechanisms of hepatotoxicity involved.

Toxicity studies, among them hepatotoxicity, are key throughout preclinical stages of drug development to minimise undesired toxic effects that might eventually appear in the course of the clinical use of the new drug. Understanding the mechanism of injury of hepatotoxins is essential to efficiently anticipate their potential risk of toxicity in humans. The use of in vitro models and particularly cultured hepatocytes represents an easy and robust alternative to animal drug hepatotoxicity testing for predicting human risk. Here, we envisage an innovative strategy to identify potential hepatotoxic drugs, quantify the magnitude of the alterations caused, and uncover the mechanisms of toxicity. This strategy is based on the comparative analysis of metabolome changes induced by hepatotoxic and non-hepatotoxic compounds on HepG2 cells, assessed by untargeted mass spectrometry. As a training set, we used 25 hepatotoxic and 4 non-hepatotoxic compounds and incubated HepG2 cells for 24 h at a low and a high concentration (IC10 and IC50) to identify mechanism-related and cytotoxicity related metabolomic biomarkers and to elaborate prediction models accounting for global hepatotoxicity and mechanisms-related toxicity. Thereafter, a second set of 69 chemicals with known predominant mechanisms of toxicity and 18 non-hepatotoxic compounds were analysed at 1, 10, 100 and 1000 µM concentrations from which and based on the magnitude of the alterations caused as compared with non-toxic compounds, we defined a "toxicity index" for each compound. In addition, we extracted from the metabolome data the characteristic signatures for each mechanism of hepatotoxicity. The integration of all this information allowed us to identify specific metabolic patterns and, based on the occurrence of that specific metabolome changes, the models predicted the likeliness of a compound to behave as hepatotoxic and to act through a given toxicity mechanism (i.e., oxidative stress, mitochondrial disruption, apoptosis and steatosis) for each compound and concentration.

Combining K-72 Hepatic Failure with 15 Individual T-Codes to Identify Patients with Idiosyncratic Drug-Induced Liver Injury in the Electronic Medical Record.

BACKGROUND: The aim of this study was to determine the utility of combining three K72 codes (hepatic failure) with 15 individual T-Codes (drug toxicity/poisoning) to identify potential DILI cases. METHODS: The EMR was searched for encounters that had a K72 code combined with a T-code that also met minimal liver injury laboratory criteria between 10/1/15 and 9/30/18. After manual chart review, a DILIN expert opinion causality score (1-5) was assigned to each case. RESULTS: Among the 345 patient encounters identified, mean age was 57 years, 53% were male, and 89% Caucasian. Thirty-seven cases (10.7%) were adjudicated as probable DILI with antibiotics being the most frequently identified suspect drugs. Of the 308 non-DILI cases, liver injury was most commonly due to congestive hepatopathy (38%) and hepatic metastases (15%). The probable-DILI cases were significantly more likely to have hepatocellular liver injury (57% vs 32.5%, p = 0.01), higher total bilirubin levels (7.7 vs 4.6 mg/dl, p = 0.03), and more severe liver injury scores (p < 0.01). The K72.0 (acute/ subacute hepatic failure) yielded the most DILI cases (29) compared to K72.9 (13) and K72.1 (0). The positive predictive value of the searching algorithm was 10.7% and improved to 15% when using only the K72.0 codes. CONCLUSIONS: K72 codes combined with drug poisoning T-codes had a low positive predictive value in identifying patients with idiosyncratic DILI. These data support further refinement of ICD-10-based algorithms to detect DILI cases in the EMR.

Delayed administration of N-acetylcysteine blunts recovery after an acetaminophen overdose unlike 4-methylpyrazole.

N-acetylcysteine (NAC) is the only clinically approved antidote against acetaminophen (APAP) hepatotoxicity. Despite its efficacy in patients treated early after APAP overdose, NAC has been implicated in impairing liver recovery in mice. More recently, 4-methylpyrazole (4MP, Fomepizole) emerged as a potential antidote in the mouse APAP hepatotoxicity model. The objective of this manuscript was to verify the detrimental effect of NAC and its potential mechanism and assess whether 4MP has the same liability. C57BL/6J mice were treated with 300 mg/kg APAP; 9 h after APAP and every 12 h after that, the animals received either 100 mg/kg NAC or 184.5 mg/kg 4MP. At 24 or 48 h after APAP, parameters of liver injury, mitochondrial biogenesis and cell proliferation were evaluated. Delayed NAC treatment had no effect on APAP-induced liver injury at 24 h but reduced the decline of plasma ALT activities and prevented the shrinkage of the areas of necrosis at 48 h. This effect correlated with down-regulation of key activators of mitochondrial biogenesis (AMPK, PGC-1α, Nrf1/2, TFAM) and reduced expression of Tom 20 (mitochondrial mass) and PCNA (cell proliferation). In contrast, 4MP attenuated liver injury at 24 h and promoted recovery at 48 h, which correlated with enhanced mitochondrial biogenesis and hepatocyte proliferation. In human hepatocytes, 4MP demonstrated higher efficacy in preventing cell death compared to NAC when treated at 18 h after APAP. Thus, due to the wider treatment window and lack of detrimental effects on recovery, it appears that at least in preclinical models, 4MP is superior to NAC as an antidote against APAP overdose.

Computational Models Using Multiple Machine Learning Algorithms for Predicting Drug Hepatotoxicity with the DILIrank Dataset.

Drug-induced liver injury (DILI) remains one of the challenges in the safety profile of both authorized and candidate drugs, and predicting hepatotoxicity from the chemical structure of a substance remains a task worth pursuing. Such an approach is coherent with the current tendency for replacing non-clinical tests with in vitro or in silico alternatives. In 2016, a group of researchers from the FDA published an improved annotated list of drugs with respect to their DILI risk, constituting "the largest reference drug list ranked by the risk for developing drug-induced liver injury in humans" (DILIrank). This paper is one of the few attempting to predict liver toxicity using the DILIrank dataset. Molecular descriptors were computed with the Dragon 7.0 software, and a variety of feature selection and machine learning algorithms were implemented in the R computing environment. Nested (double) cross-validation was used to externally validate the models selected. A total of 78 models with reasonable performance were selected and stacked through several approaches, including the building of multiple meta-models. The performance of the stacked models was slightly superior to other models published. The models were applied in a virtual screening exercise on over 100,000 compounds from the ZINC database and about 20% of them were predicted to be non-hepatotoxic.

Emerging and established modes of cell death during acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.

[Research advances in hepatocyte-like cells from human induced pluripotent stem cells and their application].

Induced pluripotent stem cells (iPSCs) have the potential of proliferation and differentiation into a variety of somatic cells, including hepatocyte-like cells (HLCs). HLCs from human iPSCs (hiPSC-HLCs) have similar features and functions as primary hepatocytes and are used as an efficient in vitro model of hepatocytes, which brings hope to studies on liver diseases and drug hepatotoxicity evaluation. This article reviews the research advances in hiPSC-HLCs and their application in the fields of disease model, drug hepatotoxicity evaluation, and cell transplantation and discusses the future perspectives of the application of hiPSC-HLCs.

RUCAM in Drug and Herb Induced Liver Injury: The Update.

RUCAM (Roussel Uclaf Causality Assessment Method) or its previous synonym CIOMS (Council for International Organizations of Medical Sciences) is a well established tool in common use to quantitatively assess causality in cases of suspected drug induced liver injury (DILI) and herb induced liver injury (HILI). Historical background and the original work confirm the use of RUCAM as single term for future cases, dismissing now the term CIOMS for reasons of simplicity and clarity. RUCAM represents a structured, standardized, validated, and hepatotoxicity specific diagnostic approach that attributes scores to individual key items, providing final quantitative gradings of causality for each suspect drug/herb in a case report. Experts from Europe and the United States had previously established in consensus meetings the first criteria of RUCAM to meet the requirements of clinicians and practitioners in care for their patients with suspected DILI and HILI. RUCAM was completed by additional criteria and validated, assisting to establish the timely diagnosis with a high degree of certainty. In many countries and for more than two decades, physicians, regulatory agencies, case report authors, and pharmaceutical companies successfully applied RUCAM for suspected DILI and HILI. Their practical experience, emerging new data on DILI and HILI characteristics, and few ambiguous questions in domains such alcohol use and exclusions of non-drug causes led to the present update of RUCAM. The aim was to reduce interobserver and intraobserver variability, to provide accurately defined, objective core elements, and to simplify the handling of the items. We now present the update of the well accepted original RUCAM scale and recommend its use for clinical, regulatory, publication, and expert purposes to validly establish causality in cases of suspected DILI and HILI, facilitating a straightforward application and an internationally harmonized approach of causality assessment as a common basic tool.

The assessment of the potential hepatotoxicity of new drugs by in vitro metabolomics.

Drug hepatotoxicity assessment is a relevant issue both in the course of drug development as well as in the post marketing phase. The use of human relevant in vitro models in combination with powerful analytical methods (metabolomic analysis) is a promising approach to anticipate, as well as to understand and investigate the effects and mechanisms of drug hepatotoxicity in man. The metabolic profile analysis of biological liver models treated with hepatotoxins, as compared to that of those treated with non-hepatotoxic compounds, provides useful information for identifying disturbed cellular metabolic reactions, pathways, and networks. This can later be used to anticipate, as well to assess, the potential hepatotoxicity of new compounds. However, the applicability of the metabolomic analysis to assess the hepatotoxicity of drugs is complex and requires careful and systematic work, precise controls, wise data preprocessing and appropriate biological interpretation to make meaningful interpretations and/or predictions of drug hepatotoxicity. This review provides an updated look at recent in vitro studies which used principally mass spectrometry-based metabolomics to evaluate the hepatotoxicity of drugs. It also analyzes the principal drawbacks that still limit its general applicability in safety assessment screenings. We discuss the analytical workflow, essential factors that need to be considered and suggestions to overcome these drawbacks, as well as recent advancements made in this rapidly growing field of research.

Liver injury during durvalumab-based immunotherapy is associated with poorer patient survival: A retrospective analysis.

Background: Durvalumab is approved for the treatment of lung cancer, advanced biliary tract cancers, and is also being evaluated in many other solid organ tumors. The aim of our study is to define the incidence, etiology, and outcomes of liver injury in consecutive patients receiving durvalumab-based immunotherapy. Patients and methods: Durvalumab treated patients between 1/2016 - 7/2020 were identified from the electronic medical record. Liver injury was defined as serum AST or ALT ≥ 5x upper limit of normal (ULN), ALP ≥ 2x ULN, bilirubin ≥ 2.5 mg/dl, or INR ≥ 1.5. Potential drug induced liver injury (DILI) cases were adjudicated using expert opinion scoring and confirmed with Roussel Uclaf Causality Assessment Method (RUCAM). Results: Amongst 112 patients, 58 (52%) had non-small cell lung cancer, the median age was 65 years, and 60% were male. The 21 (19%) liver injury patients were significantly more likely to harbor hepatic metastases (52% vs 17%, p=<0.001), experience tumor progression (67% vs 32%, p=0.01) or die (48% vs 11%, p<0.001) during follow-up compared to the 91 without liver injury. Using multivariate regression analysis, the development of liver injury during treatment as well as baseline hepatic metastases were independently associated with mortality during follow-up. Six of the 21 (29%) liver injury cases were adjudicated as probable DILI with four attributed to durvalumab and two due to other drugs (paclitaxel, pembrolizumab). Durvalumab was permanently discontinued in two DILI patients, three received corticosteroids, and one was successfully rechallenged. Only one patient with DILI developed jaundice, and none required hospitalization. Liver biochemistries normalized in all 6 DILI cases, while they only normalized in 27% of the 15 non-DILI cases (p=0.002). The 6 DILI patients also had a trend towards improved survival compared to those with other causes of liver injury. Conclusion: Liver injury was observed in 19% of durvalumab-treated patients and is associated with a greater likelihood of tumor progression and death during follow-up. The four durvalumab DILI cases were mild and self-limited, highlighting the importance of causality assessment to determine the cause of liver injury in oncology patients receiving immunotherapy.

Spatial Reconstruction of the Early Hepatic Transcriptomic Landscape After an Acetaminophen Overdose Using Single-Cell RNA-Sequencing.

An acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. A hallmark characteristic of APAP hepatotoxicity is centrilobular necrosis. General, innate mechanisms such as lower amounts of GSH and higher cytochrome P450 2e1 expression in pericentral (PC) hepatocytes are known to contribute to the differences in susceptibility to cell injury between periportal (PP) hepatocytes and PC hepatocytes. Although a sequence of molecular events involving formation of the reactive metabolite N-acetyl-p-benzoquinone imine, GSH depletion, oxidative stress, and c-Jun N-terminal kinase activation define the early cell stress trajectory following APAP exposure, their activation in PC versus PP hepatocytes is not well characterized. By using single-cell RNA-sequencing, we provide the first reconstruction of the early transcriptomic APAP liver lobule after validation of our methodology using human liver single-cell RNA-sequencing data. Two hours after APAP treatment, we find that PP hepatocytes progress along the APAP stress axis to oxidative stress, before resolving injury due to innate and adaptive mechanisms. However, PC hepatocytes continue along this stress axis as indicated by activation of mitogen-activated protein kinase genes, which is absent in PP hepatocytes. We also identify a population of glutamine synthetase enriched PC hepatocytes in close proximity to the central vein, where a stepwise induction of a stress program culminated in cell death. Collectively, these findings elucidate a molecular sequence of events distinguishing the differential response to APAP exposure between PP and PC hepatocytes and identify a subset of uniquely susceptible PC hepatocytes.

Oxidant Stress and Acetaminophen Hepatotoxicity: Mechanism-Based Drug Development.

Significance: Acetaminophen (APAP) is one of the quantitively most consumed drugs worldwide. Although safe at therapeutic doses, intentional or unintentional overdosing occurs frequently causing severe liver injury and even liver failure. In the United States, 50% of all acute liver failure cases are caused by APAP overdose. However, only one antidote with a limited therapeutic window, N-acetylcysteine, is clinically approved. Thus, more effective therapeutic interventions are urgently needed. Recent Advances: Although APAP hepatotoxicity has been extensively studied for almost 50 years, particular progress has been made recently in two areas. First, there is now a detailed understanding of involvement of oxidative and nitrosative stress in the pathophysiology, with identification of the reactive species involved, their initial generation in mitochondria, amplification through the c-Jun N-terminal kinase pathway, and the mechanisms of cell death. Second, it was demonstrated in human hepatocytes and through biomarkers in vivo that the mechanisms of liver injury in animals accurately reflect the human pathophysiology, which allows the translation of therapeutic targets identified in animals to patients. Critical Issues: For progress, solid understanding of the pathophysiology of APAP hepatotoxicity and of a drug's targets is needed to identify promising new therapeutic intervention strategies and drugs, which may be applied to humans. Future Directions: In addition to further refine the mechanistic understanding of APAP hepatotoxicity and identify additional drugs with complementary mechanisms of action to prevent cell death, more insight into the mechanisms of regeneration and developing of drugs, which promote recovery, remains a future challenge. Antioxid. Redox Signal. 35, 718-733.

Potential Effects of Coronaviruses on the Liver: An Update.

The coronaviruses that cause notable diseases, namely, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS) and coronavirus disease 2019 (COVID-19), exhibit remarkable similarities in genomic components and pathogenetic mechanisms. Although coronaviruses have widely been studied as respiratory tract pathogens, their effects on the hepatobiliary system have seldom been reported. Overall, the manifestations of liver injury caused by coronaviruses typically involve decreased albumin and elevated aminotransferase and bilirubin levels. Several pathophysiological hypotheses have been proposed, including direct damage, immune-mediated injury, ischemia and hypoxia, thrombosis and drug hepatotoxicity. The interaction between pre-existing liver disease and coronavirus infection has been illustrated, whereby coronaviruses influence the occurrence, severity, prognosis and treatment of liver diseases. Drugs and vaccines used for treating and preventing coronavirus infection also have hepatotoxicity. Currently, the establishment of optimized therapy for coronavirus infection and liver disease comorbidity is of significance, warranting further safety tests, animal trials and clinical trials.

COVID 19 and liver: An A-Z literature review.

The coronavirus pandemic has changed the priorities of the whole medical society. During the clinical course of COVID-19, it has been observed that hepatic injury occurs in a significant proportion of patients, particularly in those with severe or critical illness. In this literature review, we summarize the most recent studies, which covered the pathophysiology of COVID-19 induced liver injury including; hepatic pathological findings, therapy related liver damage, and the effects of the viral infection on pre-existing liver diseasesin context of the most recent recommendations. Conclusions: This review sheds light on the impact of COVID-19 infection on the liver, as well as the prognostic effect of liver laboratory markers on disease outcome. Temporal variations in liver parameters during disease course as well as different patterns of derangement are depicted. More intensive surveillance and individualized therapeutic approaches should be tailored for immunocompromised patients with advanced liver disease, hepatocellular carcinoma, and liver transplant patients. Despite the limited studies on COVID-19 infected patients with preexisting liver disease, this comprehensive overview provides a perspective on the management of liver disease during COVID-19.

Acetaminophen-induced apoptosis: Facts versus fiction.

An overdose of the widely used analgesic acetaminophen (APAP) is the most common cause of acute liver failure in the western world and hence is a clinically significant problem. Thus, mechanisms of APAP-induced hepatotoxicity have been the focus of extensive investigation for decades and it was established that APAP induces hepatocyte cell death by necrosis. Although APAP-induced necrosis shares some features of apoptosis induced by the intrinsic pathway, apoptotic cell death in this context was ruled out due to the absence of caspase activation and lack of protection by caspase inhibitors and missing morphological characteristics of apoptotic cells. Deeper mechanistic understanding of the cell death process after APAP in recent years has now revealed that cells die by programmed necrosis and apoptosis is not a relevant mode of cell death in this context. Hence, it is alarming to note that an increasing number of studies are being published purporting to indicate that APAP induces apoptotic cell death. These papers broadly measure "apoptotic markers" with questionable specificity such as Bax, Bcl-2 and caspase-3 protein expression, or use the terminal deoxynucleotidyl transferase dUTP nick end labeling assay as basis for the conclusion that there is apoptosis after APAP overdose. The misguided use of these apoptosis parameters in correlative studies without context or scientific rationale confuses the field and threatens to undo decades of careful mechanistic investigation into this topic. This review examines this emerging problem in detail and recommends approaches to correct it. RELEVANCE FOR PATIENTS: Hepatotoxicity and acute liver failure caused by an acetaminophen overdose is a serious clinical problem in western countries. Understanding the mode of cell death and the signaling pathways involved is critical for developing new therapeutic approaches. Recent trends to claim that apoptosis is a relevant mode of cell death in acetaminophen hepatotoxicity are not justified by sound scientific data and will not lead to effective new drug development.

Treatments in Covid-19 patients with pre-existing metabolic dysfunction-associated fatty liver disease: A potential threat for drug-induced liver injury?

Obese patients who often present metabolic dysfunction-associated fatty liver disease (MAFLD) are at risk of severe presentation of coronavirus disease 2019 (COVID-19). These patients are more likely to be hospitalized and receive antiviral agents and other drugs required to treat acute respiratory distress syndrome and systemic inflammation, combat bacterial and fungal superinfections and reverse multi-organ failure. Among these pharmaceuticals, antiretrovirals such as lopinavir/ritonavir and remdesivir, antibiotics and antifungal agents can induce drug-induced liver injury (DILI), whose mechanisms are not always understood. In the present article, we hypothesize that obese COVID-19 patients with MAFLD might be at higher risk for DILI than non-infected healthy individuals or MAFLD patients. These patients present several concomitant factors, which individually can favour DILI: polypharmacy, systemic inflammation at risk of cytokine storm, fatty liver and sometimes nonalcoholic steatohepatitis (NASH) as well as insulin resistance and other diseases linked to obesity. Hence, in obese COVID-19 patients, some drugs might cause more severe (and/or more frequent) DILI, while others might trigger the transition of fatty liver to NASH, or worsen pre-existing steatosis, necroinflammation and fibrosis. We also present the main mechanisms whereby drugs can be more hepatotoxic in MAFLD including impaired activity of xenobiotic-metabolizing enzymes, mitochondrial dysfunction, altered lipid homeostasis and oxidative stress. Although comprehensive investigations are needed to confirm our hypothesis, we believe that the current epidemic of obesity and related metabolic diseases has extensively contributed to increase the number of cases of DILI in COVID-19 patients, which may have participated in presentation severity and death.

High-Throughput Screening to Evaluate Inhibition of Bile Acid Transporters Using Human Hepatocytes Isolated From Chimeric Mice.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.

Biofabrication of Hepatic Constructs by 3D Bioprinting of a Cell-Laden Thermogel: An Effective Tool to Assess Drug-Induced Hepatotoxic Response.

A thermoresponsive Pluronic/alginate semisynthetic hydrogel is used to bioprint 3D hepatic constructs, with the aim to investigate liver-specific metabolic activity of the 3D constructs compared to traditional 2D adherent cultures. The bioprinting method relies on a bioinert hydrogel and is characterized by high-shape fidelity, mild depositing conditions and easily controllable gelation mechanism. Furthermore, the dissolution of the sacrificial Pluronic templating agent significantly ameliorates the diffusive properties of the printed hydrogel. The present findings demonstrate high viability and liver-specific metabolic activity, as assessed by synthesis of urea, albumin, and expression levels of the detoxifying CYP1A2 enzyme of cells embedded in the 3D hydrogel system. A markedly increased sensitivity to a well-known hepatotoxic drug (acetaminophen) is observed for cells in 3D constructs compared to 2D cultures. Therefore, the 3D model developed herein may represent an in vitro alternative to animal models for investigating drug-induced hepatotoxicity.

Engineered Liver-on-a-Chip Platform to Mimic Liver Functions and Its Biomedical Applications: A Review.

Hepatology and drug development for liver diseases require in vitro liver models. Typical models include 2D planar primary hepatocytes, hepatocyte spheroids, hepatocyte organoids, and liver-on-a-chip. Liver-on-a-chip has emerged as the mainstream model for drug development because it recapitulates the liver microenvironment and has good assay robustness such as reproducibility. Liver-on-a-chip with human primary cells can potentially correlate clinical testing. Liver-on-a-chip can not only predict drug hepatotoxicity and drug metabolism, but also connect other artificial organs on the chip for a human-on-a-chip, which can reflect the overall effect of a drug. Engineering an effective liver-on-a-chip device requires knowledge of multiple disciplines including chemistry, fluidic mechanics, cell biology, electrics, and optics. This review first introduces the physiological microenvironments in the liver, especially the cell composition and its specialized roles, and then summarizes the strategies to build a liver-on-a-chip via microfluidic technologies and its biomedical applications. In addition, the latest advancements of liver-on-a-chip technologies are discussed, which serve as a basis for further liver-on-a-chip research.

Mechanisms of Chronic Fialuridine Hepatotoxicity as Revealed in Primary Human Hepatocyte Spheroids.

Drug hepatotoxicity is often delayed in onset. An exemplar case is the chronic nature of fialuridine hepatotoxicity, which resulted in the deaths of several patients in clinical trials as preclinical studies failed to identify this human-specific hepatotoxicity. Conventional preclinical in vitro models are mainly designed to evaluate the risk of acute drug toxicity. Here, we evaluated the utility of 3D spheroid cultures of primary human hepatocytes (PHHs) to assess chronic drug hepatotoxicity events using fialuridine as an example. Fialuridine toxicity was only detectable after 7 days of repeated exposure. Clinical manifestations, including reactive oxygen species formation, lipid accumulation, and induction of apoptosis, were readily identified. Silencing the expression or activity of the human equilibrative nucleoside transporter 1 (ENT1), implicated in the mitochondrial transport of fialuridine, modestly protected PHH spheroids from fialuridine toxicity. Interference with the phosphorylation of fialuridine into the active triphosphate metabolites by silencing of thymidine kinase 2 (TK2) provided substantial protection, whereas simultaneous silencing of ENT1 and TK2 provided near-complete protection. Fialuridine-induced mitochondrial dysfunction was suggested by a decrease in the expression of mtDNA-encoded genes, which correlated with the onset of toxicity and was prevented under the simultaneous silencing of ENT1 and TK2. Furthermore, interference with the expression or activity of ribonucleotide reductase (RNR), which is critical to deoxyribonucleoside triphosphate (dNTP) pool homeostasis, resulted in selective potentiation of fialuridine toxicity. Our findings demonstrate the translational applicability of the PHH 3D spheroid model for assessing drug hepatotoxicity events which manifest only under chronic exposure conditions.