Model Of Drug-induced Liver Injury Research Articles

Control Compounds for Preclinical Drug-Induced Liver Injury Assessment: Consensus-driven systematic review by the ProEuroDILI Network

Background & AimsIdiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. This systematic review analyzed the compounds used in hepatotoxicity assays and established a list of DILI positive and negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from COST Action ProEuroDILI Network (CA17112). MethodsFollowing 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified ‘Toxicological Data Reliability Assessment Tool’. Results51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on the drug concentrations. ConclusionsThe extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative drugs is proposed for validating in vitro models for improving preclinical drug safety testing regimes. IMPACT AND IMPLICATIONSPrediction of human toxicity early in the drug development process remains a major challenge. For this, human in vitro models are becoming increasingly important, however, the development of more physiologically relevant liver models and careful selection of control DILI+ and DILI- drugs are requisites to better predict DILI liability of new drug candidates. Thus, this systematic study holds critical implications for standardizing validation of new in vitro models for studying drug-induced liver injury (DILI). By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. The results are of paramount importance to all the actors involved in the drug development process, offering a standardized approach to assess hepatotoxic risks. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

Interpretable machine learning in predicting drug-induced liver injury among tuberculosis patients: model development and validation study.

The objective of this research was to create and validate an interpretable prediction model for drug-induced liver injury (DILI) during tuberculosis (TB) treatment. A dataset of TB patients from Ningbo City was used to develop models employing the eXtreme Gradient Boosting (XGBoost), random forest (RF), and the least absolute shrinkage and selection operator (LASSO) logistic algorithms. The model's performance was evaluated through various metrics, including the area under the receiver operating characteristic curve (AUROC) and the area under the precision recall curve (AUPR) alongside the decision curve. The Shapley Additive exPlanations (SHAP) method was used to interpret the variable contributions of the superior model. A total of 7,071TB patients were identified from the regional healthcare dataset. The study cohort consisted of individuals with a median age of 47years, 68.0% of whom were male, and 16.3% developed DILI. We utilized part of the high dimensional propensity score (HDPS) method to identify relevant variables and obtained a total of 424 variables. From these, 37 variables were selected for inclusion in a logistic model using LASSO. The dataset was then split into training and validation sets according to a 7:3 ratio. In the validation dataset, the XGBoost model displayed improved overall performance, with an AUROC of 0.89, an AUPR of 0.75, an F1 score of 0.57, and a Brier score of 0.07. Both SHAP analysis and XGBoost model highlighted the contribution of baseline liver-related ailments such as DILI, drug-induced hepatitis (DIH), and fatty liver disease (FLD). Age, alanine transaminase (ALT), and total bilirubin (Tbil) were also linked to DILI status. XGBoost demonstrates improved predictive performance compared to RF and LASSO logistic in this study. Moreover, the introduction of the SHAP method enhances the clinical understanding and potential application of the model. For further research, external validation and more detailed feature integration are necessary.

Susceptibility to Hepatotoxic Drug-Induced Liver Injury Increased After Traumatic Brain Injury in Mice.

The early stages of brain injury can induce acute liver injury, which can be recovered in the short term. Continued medication treatment during hospitalization for brain injury alleviates the prognosis and contributes to a high incidence of drug-induced liver injury (DILI). We hypothesize that there is an interaction between changes in the hepatic environment after brain injury and liver injury produced by intensive drug administration, leading to an upregulation of the organism's sensitivity to DILI. In this study, mice models of TBI were established by controlled cortical impact (CCI) and models of DILI were constructed by acetaminophen (APAP). All mice were divided into four groups: Sham, TBI, APAP, and TBI+APAP, and related liver injury indicators in liver and serum were detected by Western blot, Quantitative real-time PCR (qRT-PCR), and immunohistochemical staining. The results suggested that liver injury induced in the early stages of brain injury recovered in 3 days, but this state could still significantly aggravate DILI, represented by higher liver enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), oxidative stress (increase in malondialdehyde [MDA] concentration and deregulation of glutathione [GSH] and superoxide dismutase [SOD] activities), inflammatory response (activation of the HMGB1/TLR4/NF-κB signaling pathway, and increased messenger RNA [mRNA] and protein levels of pro-inflammatory cytokines including tumor necrosis factor alpha [TNF-α], interleukin [IL]-6, and IL-1β), and apoptosis (TUNEL assay, upregulation of Bax protein and deregulation of Bcl-2 protein). In summary, our results suggested that TBI is a potential susceptibility factor for DILI and exacerbates DILI.

Comparative analysis of gene expression between mice and humans in acetaminophen-induced liver injury by integrating bioinformatics analysis

ObjectiveMice are routinely utilized as animal models of drug-induced liver injury (DILI), however, there are significant differences in the pathogenesis between mice and humans. This study aimed to compare gene expression between humans and mice in acetaminophen (APAP)-induced liver injury (AILI), and investigate the similarities and differences in biological processes between the two species.MethodsA pair of public datasets (GSE218879 and GSE120652) obtained from GEO were analyzed using “Limma” package in R language, and differentially expressed genes (DEGs) were identified, including co-expressed DEGs (co-DEGs) and specific-expressed DEGS (specific-DEGs). Analysis of Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed analyses for specific-DEGs and co-DEGs. The co-DEGs were also used to construct transcription factor (TF)-gene network, gene-miRNA interactions network and protein-protein interaction (PPI) network for analyzing hub genes.ResultsMouse samples contained 1052 up-regulated genes and 1064 down-regulated genes, while human samples contained 1156 up-regulated genes and 1557 down-regulated genes. After taking the intersection between the DEGs, only 154 co-down-regulated and 89 co-up-regulated DEGs were identified, with a proportion of less than 10%. It was suggested that significant differences in gene expression between mice and humans in drug-induced liver injury. Mouse-specific-DEGs predominantly engaged in processes related to apoptosis and endoplasmic reticulum stress, while human-specific-DEGs were concentrated around catabolic process. Analysis of co-regulated genes reveals showed that they were mainly enriched in biosynthetic and metabolism-related processes. Then a PPI network which contains 189 nodes and 380 edges was constructed from the co-DEGs and two modules were obtained by Mcode. We screened out 10 hub genes by three algorithms of Degree, MCC and MNC, including CYP7A1, LSS, SREBF1, FASN, CD44, SPP1, ITGAV, ANXA5, LGALS3 and PDGFRA. Besides, TFs such as FOXC1, HINFP, NFKB1, miRNAs like mir-744-5p, mir-335-5p, mir-149-3p, mir-218-5p, mir-10a-5p may be the key regulatory factors of hub genes.ConclusionsThe DEGs of AILI mice models and those of patients were compared, and common biological processes were identified. The signaling pathways and hub genes in co-expression were identified between mice and humans through a series of bioinformatics analyses, which may be more valuable to reveal molecular mechanisms of AILI.

Screening drug-induced liver injury through two independent parameters of lipid droplets and peroxynitrite with a π-extended coumarin-based NIR fluorescent probe

Drug-induced liver injury (DILI) or hepatotoxicity is a significant concern for public health. However, relying on a single biomarker for early DILI diagnosis poses a high risk of false positives. In this study, we reported a near-infrared fluorescent probe (BCOU-S), which enables independent visualization of LDs status and ONOO fluctuations. BCOU-S was constructed by combining a π-extended coumarin core as the NIR fluorophore and a methyl thioether group as the ONOO recognition site. BCOU-S could emit 655 nm NIR fluorescence excited at 518 nm, with a low detection limit of 27 nM and a large Stokes shift of 137 nm. The response mechanism to ONOO was confirmed by molecular orbital density function theory (DFT) and time-dependent DFT (TD-DFT) calculations. BCOU-S monitors LDs status through co-localization, oleic acid (OA) incubation, and starvation induction experiments. It sensitively distinguishes subtle changes in ONOO induced by different drugs in DILI cell models. In the acetaminophen (APAP)-induced DILI model, BCOU-S reveals significant LDs accumulation and increase in ONOO levels, establishing a clear time-effect and dose-effect relationship. Simultaneously monitoring ONOO levels and LDs accumulation, BCOU-S proves to be a more sensitive and effective tool for early DILI prevention, effectively avoiding false positives caused by changes in a single parameter. BCOU-S is a useful tool for further monitoring early DILI development.

Study based on the acetaldehyde dehydrogenase 2 gene polymorphism and acetaminophen-induced liver injury

Objective: To explore the association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and abnormal liver function-induced by acetaminophen (APAP) drugs. Methods: An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology. The obtained heterozygous mice were mated with opposite sex of heterozygotes. Genomic DNA was extracted from the tail of the offspring mouse. The polymerase chain reaction (PCR) method was used to determine the ALDH2 genotype. APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice. Blood and liver tissues of mice were collected for liver function index, HE staining, F4/80 immunohistochemistry, and other detections. The intergroup mean was compared using a one-way ANOVA. The LSD- t test was used for pairwise comparison. Results: ALDH2 knockout mice were bred successfully. The genotyping of the offspring was segregated into the wild-type (ALDH2(+/+)), heterozygous mutant (ALDH2(+/-)), and homozygous mutant (ALDH2(-/-)), respectively. Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) was not significantly increased in the blank control group (P < 0.05), while the ALT, AST,ALP, and TBil were all elevated in the APAP experimental group. The levels of ALT (P = 0.004), AST (P = 0.002), and TBil (P = 0.012) were significantly elevated among the mutant group compared to those in the wild-type group, and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group (P = 0.003, 0 and 0.006). In addition, the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group (P = 0.085) and wild-type group mice, but the difference was only statistically significant compared to wild-type mice (P = 0.002). HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration, necrosis, and increased inflammatory cell infiltration, which was mostly evident in mutant mice. Simultaneously, the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice, and its expression levels were significantly enhanced compared to the blank control group. Conclusion: APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism. The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling, and the ALDH2 gene defect may alleviate, to some extent, APAP-induced liver function abnormalities.

A Rationally Designed Prodrug for the Fluorogenic Labeling of Albumin and Theranostic Effects on Drug-Induced Liver Injury.

The development of small-molecular fluorogenic tools for the chemo-selective labeling of proteins in live cells is important for the evaluation of intracellular redox homeostasis. Dynamic imaging of human serum albumin (HSA), an antioxidant protein under oxidative stress with concomitant release of antioxidant drugs to maintain redox homeostasis, affords potential opportunities for disease diagnosis and treatment. In this work, we developed a nonfluorogenic prodrug named TPA-NAC, by introducing N-acetyl-l-cysteine (NAC) into a conjugated acceptor skeleton. Through combined thiol and amino addition, coupling with HSA results in fluorescence turn-on and drug release. It was reasoned that the restricted intramolecular motion of the probe under an HSA microenvironment after covalent bonding inhibited the nonradiative transitions. Furthermore, the biocompatibility and photochemical properties of TPA-NAC enabled it to image exogenous and endogenous HSA in living cells in a wash-free manner. Additionally, the released drug evoked upregulation of superoxide dismutase (SOD), which synergistically eliminated reactive oxygen species in a drug-induced liver injury model. This study provides insights into the design of new theranostic fluorescent prodrugs for chemo-selective protein labeling and disease treatments.

Research progress on rat model of drug-induced liver injury established by nonsteroidal anti-inflammatory drug (celecoxib) and royal jelly ameliorative effect.

NSAIDs, like celecoxib, are widely used to treat pain, fever, and inflammation, with celecoxib being particularly effective in managing arthritis symptoms and acute or chronic pain especially with its favorable gastrointestinal tolerability. The study aimed at exploring the effect of chronic administration of celecoxib on hepatic tissues in male albino rats. It also examined the royal jelly celecoxib interplay. 50 male albino rats in 5 equal groups; Group 1: received no drug. Group 2: received celecoxib (50 mg/kg/day, orally), for 30 successive days. Group 3: received celecoxib plus royal jelly (300 mg/kg/day, orally) for 30 successive days. Group 4: received celecoxib, for 30 days, then were left untreated for another 30 days. Group 5: received celecoxib plus royal jelly for 30 days, then were left untreated for another 30 days. Chronic celecoxib administration caused hepatotoxicity in male albino rats, with ameliorative effect of royal jelly. Celecoxib discontinuation significantly diminished the celecoxib-induced toxicity, and normal liver enzymes and serum protein levels were regained in the case of dual medications (celecoxib+RJ) discontinuation. Long-term celecoxib administration caused hepatotoxicity, with ameliorative effects of royal jelly against celecoxib-induced oxidative and apoptotic stress. In addition, it could be concluded that royal jelly may prove a useful adjunct in patients being prescribed celecoxib.

Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury

Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury. We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver invivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67). Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, invitro and invivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG. NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery. Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

Human liver organoid models for assessment of drug toxicity at the preclinical stage.

The hepatotoxicity of drugs is one of the leading causes of drug withdrawal from the pharmaceutical market and high drug attrition rates. Currently, the commonly used hepatocyte models include conventional hepatic cell lines and animal models, which cannot mimic human drug-induced liver injury (DILI) due to poorly defined dose-response relationships and/or lack of human-specific mechanisms of toxicity. In comparison to 2D culture systems from different cell sources such as primary human hepatocytes and hepatomas,, 3D organoids derived from an inducible pluripotent stem cell (iPSC) or adult stem cells are promising accurate models to mimic organ behavior with a higher level of complexity and functionality owing to their ability to self-renewal. Meanwhile, the heterogeneous cell composition of the organoids enables metabolic and functional zonation of hepatic lobule important in drug detoxification and has the ability to mimic idiosyncratic DILI as well. Organoids having higher drug-metabolizing enzyme capacities can culture long-term and be combined with microfluidic-based technologies such as organ-on-chips for a more precise representation of human susceptibility to drug response in a high-throughput manner. However, there are numerous limitations to be considered about this technology, such as enough maturation, differences between protocols and high cost. Herein, we first reviewed the current preclinical DILI assessment tools and looked at the organoid technology with respect to in vitro detoxification capacities. Then we discussed the clinically applicable DILI assessment markers and the importance of liver zonation in the next generation organoid-based DILI models.

Ratiometric Fluorescent Sensors Illuminate Cellular Magnesium Imbalance in a Model of Acetaminophen-Induced Liver Injury.

Magnesium(II) plays catalytic, structural, regulatory, and signaling roles in living organisms. Abnormal levels of this metal have been associated with numerous pathologies, including cardiovascular disease, diabetes, metabolic syndrome, immunodeficiency, cancer, and, most recently, liver pathologies affecting humans. The role of Mg2+ in the pathophysiology of liver disease, however, has been occluded by concomitant changes in concentration of interfering divalent cations, such as Ca2+, which complicates the interpretation of experiments conducted with existing molecular Mg2+ indicators. Herein, we introduce a new quinoline-based fluorescent sensor, MagZet1, that displays a shift in its excitation and emission wavelengths, affording ratiometric detection of cellular Mg2+ by both fluorescence microscopy and flow cytometry. The new sensor binds the target metal with a submillimolar dissociation constant─well suited for detection of changes in free Mg2+ in cells─and displays a 10-fold selectivity against Ca2+. Furthermore, the fluorescence ratio is insensitive to changes in pH in the physiological range, providing an overall superior performance over existing indicators. We provide insights into the metal selectivity profile of the new sensor based on computational modeling, and we apply it to shed light on a decrease in cytosolic free Mg2+ and altered expression of metal transporters in cellular models of drug-induced liver injury caused by acetaminophen overdose.

Scopoletin a potential phytochemical therapy for antitubercular treatment drug induced liver injury (ATT-DILI) model in Wistar rats.

The hepatoprotective properties of scopoletin have been explored in carbon tetrachloride (CCl4) induced liver injury but not in drug-induced liver injury (DILI) scenarios. Only N-acetyl-cysteine (NAC) has proven efficacy in DILI treatment. Accordingly, we conducted a study toassess the hepatoprotective action of scopoletin in the anti-tubercular treatment (ATT)-DILI model in Wistar rats, ifany. A total of 36 rats were evaluated, with six in each group. A 36-day ATT at 100 mg/kg dose for isoniazid, 300 mg/kg for rifampicin and 700 mg/kg for pyrazinamide were fed to induce hepatotoxicity in rats. Group I and II-VI received normal saline and ATT, respectively. Oral scopoletin (1,5 and 10 mg/kg) and NAC 150 mg/kg were administered in groups III, IV, V and VI, respectively, once daily for the last15days of the experiment. LFT monitoring was performed at baseline, days 21, 28, and 36. Rats were sacrificed for the histopathology examination. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and bilirubin levels were significantly increased in group II (receiving ATT) compared to normal control on day 28 and day 36 (p<0.05). All three doses of scopoletin and NAC groups led to the resolution of AST, ALT, ALP, and bilirubin changes induced by ATT medications effect beginning by day 28 and persisting on day 36 (p<0.01). An insignificant effect was observed on albumin and total protein levels. The effect was confirmed with antioxidants and histopathology analysis. The study confirms the hepatoprotective efficacy of scopoletin in a more robust commonly encountered liver injury etiology.

Molecular magnetic resonance imaging of liver inflammation using an oxidatively activated probe

Many liver diseases are driven by inflammation, but imaging to non-invasively diagnose and quantify liver inflammation has been underdeveloped. The inflammatory liver microenvironment is aberrantly oxidising owing in part to reactive oxygen species generated by myeloid leucocytes. We hypothesised that magnetic resonance imaging using the oxidatively activated probe Fe-PyC3A will provide a non-invasive biomarker of liver inflammation. A mouse model of drug-induced liver injury was generated through intraperitoneal injection of a hepatoxic dose of acetaminophen. A mouse model of steatohepatitis was generated via a choline-deficient, l-amino acid defined high-fat diet (CDAHFD). Images were acquired dynamically before and after intravenous injection of Fe-PyC3A. The contrast agent gadoterate meglumine was used as a non-oxidatively activated negative control probe in mice fed CDAHFD. The (post-pre) Fe-PyC3A injection change in liver vs. muscle contrast-to-noise ratio (ΔCNR) recorded 2min post-injection was correlated with liver function test values, histologic scoring assigned using the NASH Clinical Research Network criteria, and intrahepatic myeloid leucocyte composition determined by flow cytometry. For mice receiving i.p. injections of acetaminophen, intrahepatic neutrophil composition correlated poorly with liver test values but positively and significantly with ΔCNR (r= 0.64, p <0.0001). For mice fed CDAHFD, ΔCNR generated by Fe-PyC3A in the left lobe was significantly greater in mice meeting histologic criteria strongly associated with a diagnosis NASH compared to mice where histology was consistent with likely non-NASH (p= 0.0001), whereas no differential effect was observed using gadoterate meglumine. In mice fed CDAHFD, ΔCNR did not correlate strongly with fractional composition of any specific myeloid cell subpopulation as determined by flow cytometry. Magnetic resonance imaging using Fe-PyC3A merits further evaluation as a non-invasive biomarker for liver inflammation. Non-invasive tests to diagnose and measure liver inflammation are underdeveloped. Inflammatory cells such as neutrophils release reactive oxygen species which creates an inflammatory liver microenvironment that can drive chemical oxidation. We recently invented a new class of magnetic resonance imaging probe that is made visible to the scanner only after chemical oxidation. Here, we demonstrate how this imaging technology could be applied as a non-invasive biomarker for liver inflammation.

Hepatocyte DDX3X protects against drug-induced acute liver injury via controlling stress granule formation and oxidative stress

Drug-induced liver injury (DILI) is the leading cause of acute liver failure (ALF). Continuous and prolonged hepatic cellular oxidative stress and liver inflammatory stimuli are key signatures of DILI. DEAD-box helicase 3, X-linked (DDX3X) is a central regulator in pro-survival stress granule (SG) assembly in response to stress signals. However, the role of DDX3X in DILI remains unknown. Herein, we characterized the hepatocyte-specific role of DDX3X in DILI. Human liver tissues of DILI patients and control subjects were used to evaluate DDX3X expression. APAP, CCl4 and TAA models of DILI were established and compared between hepatocyte-specific DDX3X knockout (DDX3XΔhep) and wild-type control (DDX3Xfl/fl) mice. Hepatic expression of DDX3X was significantly decreased in the pathogenesis of DILI compared with controls in human and mice. Compared to DDX3Xfl/fl mice, DDX3XΔhep mice developed significant liver injury in multiple DILI models. DDX3X deficiency aggravates APAP induced oxidative stress and hepatocyte death by affecting the pro-survival stress granule (SG) assembly. Moreover, DDX3X deficiency induces inflammatory responses and causes pronounced macrophage infiltration. The use of targeted DDX3X drug maybe promising for the treatment of DILI in human.

Zebrafish as a model for drug induced liver injury: state of the art and beyond

Zebrafish as a preclinical drug induced liver injury (DILI) model provides multiple advantages ranging from ease of breeding and maintenance, availability of different strains and transgenic fish amenable to study liver function, and highly conserved liver structure and function with the human liver. In this review, the authors have aimed to provide an account of the metabolic enzymes that take roles in drug detoxification in both human and zebrafish in a comparative manner and exemplify several recent models in studying liver functionality. Moreover, the authors emphasize the difficulties associated with studying idiosyncratic DILI in preclinical models and propose that zebrafish could be an important complement to mice in testing functions of genes that are associated with DILI with respect to different drugs in human genome-wide association studies (GWAS) Catalog. Finally, this review highlights the state-of-the-art in the development of novel transgenic reporter strains that can be used to study degree and molecular mechanisms of hepatotoxicity caused by drugs in zebrafish. All of these will help researchers to use effectively the available resources in the zebrafish DILI models, while advocating potential leads that can be taken to provide advancements in a better understanding and treatment of DILI.

Molecular Idiosyncratic Toxicology of Drugs in the Human Liver Compared with Animals: Basic Considerations

Drug induced liver injury (DILI) occurs in patients exposed to drugs at recommended doses that leads to idiosyncratic DILI and provides an excellent human model with well described clinical features, liver injury pattern, and diagnostic criteria, based on patients assessed for causality using RUCAM (Roussel Uclaf Causality Assessment Method) as original method of 1993 or its update of 2016. Overall, 81,856 RUCAM based DILI cases have been published until mid of 2020, allowing now for an analysis of mechanistic issues of the disease. From selected DILI cases with verified diagnosis by using RUCAM, direct evidence was provided for the involvement of the innate and adapted immune system as well as genetic HLA (Human Leucocyte Antigen) genotypes. Direct evidence for a role of hepatic immune systems was substantiated by (1) the detection of anti-CYP (Cytochrome P450) isoforms in the plasma of affected patients, in line with the observation that 65% of the drugs most implicated in DILI are metabolized by a range of CYP isoforms, (2) the DIAIH (drug induced autoimmune hepatitis), a subgroup of idiosyncratic DILI, which is characterized by high RUCAM causality gradings and the detection of plasma antibodies such as positive serum anti-nuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), rarely also anti-mitochondrial antibodies (AMA), (3) the effective treatment with glucocorticoids in part of an unselected RUCAM based DILI group, and (4) its rare association with the immune-triggered Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) caused by a small group of drugs. Direct evidence of a genetic basis of idiosyncratic DILI was shown by the association of several HLA genotypes for DILI caused by selected drugs. Finally, animal models of idiosyncratic DILI mimicking human immune and genetic features are not available and further search likely will be unsuccessful. In essence and based on cases of DILI with verified diagnosis using RUCAM for causality evaluation, there is now substantial direct evidence that immune mechanisms and genetics can account for idiosyncratic DILI by many but not all implicated drugs, which may help understand the mechanistic background of the disease and contribute to new approaches of therapy and prevention.

Xanthene-Based Nitric Oxide-Responsive Nanosensor for Photoacoustic Imaging in the SWIR Window.

Shortwave infrared (SWIR) dyes are characterized by their ability to absorb light from 900 to 1400 nm, which is ideal for deep tissue imaging owing to minimized light scattering and interference from endogenous pigments. An approach to access such molecules is to tune the photophysical properties of known near-infrared dyes. Herein, we report the development of a series of easily accessible (three steps) SWIR xanthene dyes based on a dibenzazepine donor conjugated to thiophene (SCR-1), thienothiophene (SCR-2), or bithiophene (SCR-3). We leverage the fact that SCR-1 undergoes a bathochromic shift when aggregated for in vivo studies by developing a ratiometric nanoparticle for NO (rNP-NO), which we employed to successfully visualize pathological levels of nitric oxide in a drug-induced liver injury model via deep tissue SWIR photoacoustic (PA) imaging. Our work demonstrates how easily this dye series can be utilized as a component in nanosensor designs for imaging studies.

De novo design of a novel AIE fluorescent probe tailored to autophagy visualization via pH manipulation

BackgroundMacroautophagy is an essential cellular self-protection mechanism, and defective autophagy has been considered to contribute to a variety of diseases. During the process, cytoplasmic components are transported via autophagosomes to acidic lysosomes for metabolism and recycling, which represents application niches for lysosome-targeted fluorescent probes. Additionally, in view of the complexity of the autophagy pathway, it entails more stringent requirements for probes suitable for monitoring autophagy. Meanwhile, aggregation-induced emission (AIE) fluorescent probes have been impressively demonstrated in the biomedical field, which bring fascinating possibilities to the autophagy visualization.MethodsWe reported a generalizable de novo design of a novel pH-sensitive AIE probe ASMP-AP tailored to lysosome targeting for the interpretation of autophagy. Firstly, the theoretical calculation was carried out followed by the investigation of optical properties. Then, the performance of ASMP-AP in visualizing autophagy was corroborated by starvation or drugs treatments. Furthermore, the capability of ASMP-AP to monitor autophagy was demonstrated in ex vivo liver tissue and zebrafish in vivo.ResultsASMP-AP displays a large stokes shift, great cell permeability and good biocompatibility. More importantly, ASMP-AP enables a good linear response to pH, which derives from the fact that its aggregation state can be manipulated by the acidity. It was successfully applied for imaging autophagy in living cells and was proved capable of monitoring mitophagy. Moreover, this novel molecular tool was validated by ex vivo visualization of activated autophagy in drug-induced liver injury model. Interestingly, it provided a meaningful pharmacological insight that the melanin inhibitor 1-phenyl-2-thiourea (PTU)-induced autophagy was clearly presented in wild-type zebrafish.ConclusionsASMP-AP offers a simple yet effective tool for studying lysosome and autophagy. This is the first instance to visualize autophagy in zebrafish using a small-molecule probe with AIE characters, accurate lysosome targeting and simultaneous pH sensitivity. Ultimately, this novel fluorescent system has great potential for in vivo translation to fuel autophagy research.Graphical

Semi-Targeted Profiling of Bile Acids by High-Resolution Mass Spectrometry in a Rat Model of Drug-Induced Liver Injury.

Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.

Investigating bile acid-mediated cholestatic drug-induced liver injury using a mechanistic model of multidrug resistance protein 3 (MDR3) inhibition.

Inhibition of the canalicular phospholipid floppase multidrug resistance protein 3 (MDR3) has been implicated in cholestatic drug-induced liver injury (DILI), which is clinically characterized by disrupted bile flow and damage to the biliary epithelium. Reduction in phospholipid excretion, as a consequence of MDR3 inhibition, decreases the formation of mixed micelles consisting of bile acids and phospholipids in the bile duct, resulting in a surplus of free bile acids that can damage the bile duct epithelial cells, i.e., cholangiocytes. Cholangiocytes may compensate for biliary increases in bile acid monomers via the cholehepatic shunt pathway or bicarbonate secretion, thereby influencing viability or progression to toxicity. To address the unmet need to predict drug-induced bile duct injury in humans, DILIsym, a quantitative systems toxicology model of DILI, was extended by representing key features of the bile duct, cholangiocyte functionality, bile acid and phospholipid disposition, and cholestatic hepatotoxicity. A virtual, healthy representative subject and population (n = 285) were calibrated and validated utilizing a variety of clinical data. Sensitivity analyses were performed for 1) the cholehepatic shunt pathway, 2) biliary bicarbonate concentrations and 3) modes of MDR3 inhibition. Simulations showed that an increase in shunting may decrease the biliary bile acid burden, but raise the hepatocellular concentrations of bile acids. Elevating the biliary concentration of bicarbonate may decrease bile acid shunting, but increase bile flow rate. In contrast to competitive inhibition, simulations demonstrated that non-competitive and mixed inhibition of MDR3 had a profound impact on phospholipid efflux, elevations in the biliary bile acid-to-phospholipid ratio, cholangiocyte toxicity, and adaptation pathways. The model with its extended bile acid homeostasis representation was furthermore able to predict DILI liability for compounds with previously studied interactions with bile acid transport. The cholestatic liver injury submodel in DILIsym accounts for several processes pertinent to bile duct viability and toxicity and hence, is useful for predictions of MDR3 inhibition-mediated cholestatic DILI in humans.