scholarly journals Identification of Translational microRNA Biomarker Candidates for Ketoconazole-Induced Liver Injury Using Next-Generation Sequencing

2020 ◽  
Author(s):  
Dongying Li ◽  
Bridgett Knox ◽  
Binsheng Gong ◽  
Si Chen ◽  
Lei Guo ◽  
...  
Keyword(s):  
Liver Injury ◽  
Early Detection ◽  
Mirna Expression ◽  
Culture Media ◽  
Sequence Similarity ◽  
Drug Induced ◽  
Bioinformatics Analyses ◽  
Mirna Sequencing

Abstract Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Reliable and translational biomarkers are needed for early detection of DILI. microRNAs (miRNAs) have received wide attention as a novel class of potential DILI biomarkers. However, it is unclear how DILI drugs other than acetaminophen may influence miRNA expression or which miRNAs could serve as useful biomarkers in humans. We selected ketoconazole (KCZ), a classic hepatotoxin, to study miRNA biomarkers for DILI as a proof of concept for a workflow that integrated in vivo, in vitro, and bioinformatics analyses. We examined hepatic miRNA expression in KCZ-treated rats at multiple doses and durations using miRNA-sequencing and correlated our results with conventional DILI biomarkers such as liver histology. Significant dysregulation of rno-miR-34a-5p, rno-miR-331-3p, rno-miR-15b-3p, and rno-miR-676 was associated with cytoplasmic vacuolization, a phenotype in rat livers with KCZ-induced injury, which preceded the elevation of serum liver transaminases (ALT and AST). Between rats and humans, miR-34a-5p, miR-331-3p, and miR-15b-3p were evolutionarily conserved with identical sequences, whereas miR-676 showed 73% sequence similarity. Using quantitative PCR, we found that the levels of hsa-miR-34a-5p, hsa-miR-331-3p, and hsa-miR-15b-3p were significantly elevated in the culture media of HepaRG cells treated with 100 µM KCZ (a concentration that induced cytotoxicity). Additionally, we computationally characterized the miRNA candidates for their gene targeting, target functions, and miRNA/target evolutionary conservation. In conclusion, we identified miR-34a-5p, miR-331-3p, and miR-15b-3p as translational biomarker candidates for early detection of KCZ-induced liver injury with a workflow applicable to computational toxicology studies.

Drug-induced liver injury classification model based on in vitro human transcriptomics and in vivo rat clinical chemistry data

2014 ◽  
Vol 2 (4) ◽  
pp. 63-70 ◽  
Author(s):  
Danyel Jennen ◽  
Jan Polman ◽  
Mark Bessem ◽  
Maarten Coonen ◽  
Joost van Delft ◽  
...  
Keyword(s):  
Liver Injury ◽  
Clinical Chemistry ◽  
Classification Model ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Chemistry Data ◽  
Model Based ◽  
Injury Classification

scholarly journals circ-CBFB upregulates p66Shc to perturb mitochondrial dynamics in APAP-induced liver injury

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Zhecheng Wang ◽  
Yan Zhao ◽  
Ruimin Sun ◽  
Yu Sun ◽  
Deshun Liu ◽  
...  
Keyword(s):  
Liver Injury ◽  
Mitochondrial Dynamics ◽  
Circular Rna ◽  
Protective Effects ◽  
Bioinformatics Analyses ◽  
Hepatocyte Injury ◽  
Rna Immunoprecipitation ◽  
Competitive Endogenous Rna

Abstract p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.

scholarly journals Current Perspective: 3D Spheroid Models Utilizing Human-Based Cells for Investigating Metabolism-Dependent Drug-Induced Liver Injury

2020 ◽  
Vol 2 ◽  
Author(s):  
Christopher R. Cox ◽  
Stephen Lynch ◽  
Christopher Goldring ◽  
Parveen Sharma
Keyword(s):  
Liver Injury ◽  
Pharmaceutical Companies ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Current Perspective ◽  
Dosing Regimens ◽  
Underlying Mechanisms ◽  
Approved Drugs

Drug-induced liver injury (DILI) remains a leading cause for the withdrawal of approved drugs. This has significant financial implications for pharmaceutical companies, places increasing strain on global health services, and causes harm to patients. For these reasons, it is essential that in-vitro liver models are capable of detecting DILI-positive compounds and their underlying mechanisms, prior to their approval and administration to patients or volunteers in clinical trials. Metabolism-dependent DILI is an important mechanism of drug-induced toxicity, which often involves the CYP450 family of enzymes, and is associated with the production of a chemically reactive metabolite and/or inefficient removal and accumulation of potentially toxic compounds. Unfortunately, many of the traditional in-vitro liver models fall short of their in-vivo counterparts, failing to recapitulate the mature hepatocyte phenotype, becoming metabolically incompetent, and lacking the longevity to investigate and detect metabolism-dependent DILI and those associated with chronic and repeat dosing regimens. Nevertheless, evidence is gathering to indicate that growing cells in 3D formats can increase the complexity of these models, promoting a more mature-hepatocyte phenotype and increasing their longevity, in vitro. This review will discuss the use of 3D in vitro models, namely spheroids, organoids, and perfusion-based systems to establish suitable liver models to investigate metabolism-dependent DILI.

scholarly journals TAMH: A Useful In Vitro Model for Assessing Hepatotoxic Mechanisms

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Madison Davis ◽  
Brendan D. Stamper
Keyword(s):  
Liver Injury ◽  
Transforming Growth Factor ◽  
Chemical Exposure ◽  
Health Concern ◽  
Hepatocellular Injury ◽  
Drug Induced ◽  
Endogenous Factors ◽  
Drug Induced Liver Injury

In vitro models for hepatotoxicity can be useful tools to predict in vivo responses. In this review, we discuss the use of the transforming growth factor-αtransgenic mouse hepatocyte (TAMH) cell line, which is an attractive model to study drug-induced liver injury due to its ability to retain a stable phenotype and express drug-metabolizing enzymes. Hepatotoxicity involves damage to the liver and is often associated with chemical exposure. Since the liver is a major site for drug metabolism, drug-induced liver injury is a serious health concern for certain agents. At the molecular level, various mechanisms may protect or harm the liver during drug-induced hepatocellular injury including signaling pathways and endogenous factors (e.g., Bcl-2, GSH, Nrf2, or MAPK). The interplay between these and other pathways in the hepatocyte can change upon drug or drug metabolite exposure leading to intracellular stress and eventually cell death and liver injury. This review focuses on mechanistic studies investigating drug-induced toxicity in the TAMH line and how alterations to hepatotoxic mechanisms in this model relate to the in vivo situation. The agents discussed herein include acetaminophen (APAP), tetrafluoroethylcysteine (TFEC), flutamide, PD0325901, lapatinib, and flupirtine.

scholarly journals miR-15a-3p Protects Against Isoniazid-Induced Liver Injury via Suppressing N-Acetyltransferase 2 Expression

2021 ◽  
Vol 8 ◽  
Author(s):  
Xinmei Li ◽  
Heng Zhang ◽  
Lin Xu ◽  
Yuan Jin ◽  
Jiao Luo ◽  
...  
Keyword(s):  
Liver Injury ◽  
Drug Efficacy ◽  
Mature Mirnas ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Expression Activity ◽  
Underlying Mechanisms ◽  
Line Drug

Isoniazid (INH), an effective first-line drug for tuberculosis treatment, has been reported to be associated with hepatotoxicity for decades, but the underlying mechanisms are poorly understood. N-acetyltransferase 2 (NAT2) is a Phase II enzyme that specifically catalyzes the acetylation of INH, and NAT2 expression/activity play pivotal roles in INH metabolism, drug efficacy, and toxicity. In this study, we systematically investigated the regulatory roles of microRNA (miRNA) in NAT2 expression and INH-induced liver injury via a series of in silico, in vitro, and in vivo analyses. Four mature miRNAs, including hsa-miR-15a-3p, hsa-miR-628-5p, hsa-miR-1262, and hsa-miR-3132, were predicted to target the NAT2 transcript, and a negative correlation was observed between hsa-miR-15a-3p and NAT2 transcripts in liver samples. Further experiments serially revealed that hsa-miR-15a-3p was able to interact with the 3′-untranslated region (UTR) of NAT2 directly, suppressed the endogenous NAT2 expression, and then inhibited INH-induced NAT2 overexpression as well as INH-induced liver injury, both in liver cells and mouse model. In summary, our results identified hsa-miR-15a-3p as a novel epigenetic factor modulating NAT2 expression and as a protective module against INH-induced liver injury, and provided new clues to elucidate the epigenetic regulatory mechanisms concerning drug-induced liver injury (DILI).

Acetaminophen Responsive miR-19b Modulates SIRT1/Nrf2 Signaling Pathway in Drug-Induced Hepatotoxicity

2019 ◽  
Vol 170 (2) ◽  
pp. 476-488 ◽  
Author(s):  
Xing Liu ◽  
Hongqian Zhao ◽  
Chunyan Luo ◽  
Debin Du ◽  
Jinlong Huang ◽  
...  
Keyword(s):  
Liver Injury ◽  
Antioxidant Defense ◽  
Compensatory Mechanism ◽  
Drug Induced ◽  
Antioxidant Genes ◽  
Sirt1 Expression ◽  
Nrf2 Signaling Pathway ◽  
Pathophysiological Role

AbstractPrevious studies suggest that activation of SIRT1 protects liver from acetaminophen (APAP)-induced injury; however, the detailed mechanism of SIRT1 modulation in this process is still incomplete. Therefore, this study was to investigate the pathophysiological role of SIRT1 in APAP-mediated hepatotoxicity. We found that SIRT1 mRNA and protein were markedly upregulated in human LO2 cells and mouse liver upon APAP exposure. In vitro, the specific knockdown of SIRT1 expression ultimately aggravated APAP-evoked cellular antioxidant defense in LO2 cells. Moreover, lentivirus-mediated knockdown of hepatic SIRT1 expression exacerbated APAP-induced oxidative stress and liver injury, especially reduction of Nrf2 and subsequent downregulation of several antioxidant genes. Intriguingly, 30 mg/kg SRT1720, the specific SIRT1 activator, which greatly enhanced Nrf2 expression and antioxidant defense, and then eventually reversed APAP-induced hepatic liver injury in mice. Furthermore, APAP responsive miR-19b played an important role in regulating SIRT1 expression, whereas overexpression miR-19b largely abolished the induction of SIRT1 by APAP in vitro and in vivo. Specific SIRT1 3′-UTR mutation, which disrupted the interaction of miRNA-3′UTR, and successfully abrogated the modulation by miR-19b. Notably, hepatic miR-19b overexpression worsened the APAP-induced hepatotoxicity. In general, our results support the notion that the strong elevation of SIRT1 by APAP responsive miR-19b may represent a compensatory mechanism to protect liver against the drug-induced damage, at least in part by enhancing Nrf2-mediated antioxidant capacity in the liver.

Benchmark Concentrations for Untargeted Metabolomics vs. Transcriptomics for Liver Injury Compounds in in Vitro Liver Models

2021 ◽  
Author(s):  
David M Crizer ◽  
Sreenivasa C Ramaiahgari ◽  
Stephen S Ferguson ◽  
Julie R Rice ◽  
Paul E Dunlap ◽  
...  
Keyword(s):  
Liver Injury ◽  
Human Liver ◽  
Culture Media ◽  
Model Systems ◽  
Resolving Power ◽  
Untargeted Metabolomics ◽  
Metabolomics Data ◽  
Biological Responses

Abstract Interpretation of untargeted metabolomics data from both in vivo and physiologically-relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry (LCMS) analysis. The analyte features observed (with limited metabolites identitfied) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (i.e., sucrose, potassium chloride (KCl)). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

Modulation of NRF2/ARE pathway- and cell death-related genes during drug-induced liver injury

2021 ◽  
pp. 096032712110279
Author(s):  
BY Ghanim ◽  
MI Ahmad ◽  
QM Abdallah ◽  
LA Qatouseh ◽  
NA Qinna
Keyword(s):  
Cell Death ◽  
Liver Injury ◽  
Antioxidant Response ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Severity Of Injury ◽  
Primary Mouse ◽  
Dose Dependent

Transcriptional factor NRF2 is an emerging tool in reviewing mechanistic behavior of drug-specific injury pathways. Drug-induced liver injury (DILI) represents a major clinical concern that often manifests oxidative stress and cell death. Despite the pivotal role of NRF2 pathway in liver pathologies, it is questioned whether NRF2 activation or regulatory efficiency could be hindered in by the severity of DILI and progression of cell death. In this study, we evaluate NRF2 as a biomarker to DILI in comparison to severity of injury as well as explore stress mediating factors affecting Nrf2 expression. In vivo DILI model was established in C57BL/6 mice by acetaminophen (APAP) at different toxic doses, confirmed by dose-dependent liver pathological changes and accompanied with in vitro time- and dose-dependent depletion of GSH and SOD in isolated primary mouse hepatocytes. Increase in liver NRF2 translocation and cytosolic content was observed in 70 mg/kg APAP-treated mice. At this subtoxic dose, liver Nrf2 transcription was increased in mice by 18.3-fold, a prominent downregulation was seen in ARE (antioxidant response element) genes; Hmox1, Nqo1 and Glcm, and apoptotic Bcl2 regulating genes. In addition, upregulation in necrosis inducer Parp2 was associated to downregulation in Hmgb1. Collectively, expression of genes related to cell survival were regulated at mild APAP hepatotoxicity. By increasing APAP dose, hemorrhagic necrosis and impaired genetic transcription in both Nrf2 and several other genes were evident. In conclusion, NRF2/ARE system and cell death modulation is halted by the increase of chemical stress and found directly associated with DILI severity.

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