Involvement of Reactive Metabolites of Diclofenac in Cytotoxicity in Sandwich-Cultured Rat Hepatocytes

2017 ◽  
Vol 36 (3) ◽  
pp. 260-267 ◽  
Author(s):  
Atsushi Kawase ◽  
Ryota Hashimoto ◽  
Mai Shibata ◽  
Hiroaki Shimada ◽  
Masahiro Iwaki
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Hepatocytes ◽  
Protein Adducts ◽  
Reactive Metabolites ◽  
Uridine Diphosphate ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Protein Adduct ◽  
Acute Cytotoxicity ◽  
Cultured Rat Hepatocytes

Background and Objectives: Diclofenac (DIC) is metabolized to reactive metabolites such as diclofenac acyl-β-d-glucuronide (DIC-AG). It is possible that such reactive metabolites could cause tissue damage by formation of covalent protein adducts and other modification of cellular proteins or by induction of immune responses against its covalent protein adducts. However, the detailed mechanisms of idiosyncratic drug-induced liver injury (DILI) have been unclear. The objective is to clarify the involvement of DIC-AG and 4′hydroxydiclofenac (4′OH-DIC) in acute DILI. Methods: We examined the effects of inhibiting DIC-AG and 4′OH-DIC production on covalent protein adduct formation and lactate dehydrogenase leakage using sandwich-cultured rat hepatocytes (SCRHs). Results: After pretreatment of SCRH with (−)-borneol (BOR, a uridine diphosphate (UDP)-glucuronosyltransferase inhibitor) or sulfaphenazole (SUL, a cytochrome P450 2C9 inhibitor) for 30 minutes, intracellular concentrations of DIC, DIC-AG, and 4′OH-DIC were determined after further treating cells with 300 μM DIC for 3 hours. The decreased levels of reactive metabolites caused by BOR or SUL pretreatment resulted in decreased lactate dehydrogenase leakage from SCRH, although the formation of covalent protein adducts was not affected. Conclusion: These results suggested that both DIC-AG and 4′OH-DIC may be involved in acute cytotoxicity by DIC.

scholarly journals Novel Mechanisms of Valproate Hepatotoxicity: Impaired Mrp2 Trafficking and Hepatocyte Depolarization

2019 ◽  
Vol 171 (2) ◽  
pp. 431-442 ◽  
Author(s):  
Dong Fu ◽  
Panli Cardona ◽  
Henry Ho ◽  
Paul B Watkins ◽  
Kim L R Brouwer
Keyword(s):  
Rat Hepatocytes ◽  
Detectable Effect ◽  
Cellular Mechanisms ◽  
Drug Induced ◽  
Glycoprotein P ◽  
Recycling Endosomes ◽  
Drug Induced Liver Injury ◽  
Zonula Occludens ◽  
Early Endosomes ◽  
Cultured Rat Hepatocytes

Abstract Drug-induced liver injury (DILI) remains a major challenge in drug development. Although numerous mechanisms for DILI have been identified, few studies have focused on loss of hepatocyte polarization as a DILI mechanism. The current study investigated the effects of valproate (VPA), an antiepileptic drug with DILI risk, on the cellular mechanisms responsible for loss of hepatocyte polarization. Fully polarized collagen sandwich-cultured rat hepatocytes were treated with VPA (1–20 mM) for specified times (3–24 h). Hepatocyte viability was significantly decreased by 10 and 20 mM VPA. Valproate depolarized hepatocytes, even at noncytotoxic concentrations (≤5 mM). Depolarization was associated with significantly decreased canalicular levels of multidrug resistance-associated protein 2 (Mrp2) resulting in reduced canalicular excretion of the Mrp2 substrate carboxydichlorofluorescein. The decreased canalicular Mrp2 was associated with intracellular accumulation of Mrp2 in Rab11-positive recycling endosomes and early endosomes. Mechanistic studies suggested that VPA inhibited canalicular trafficking of Mrp2. This effect of VPA on Mrp2 appeared to be selective in that VPA had less impact on canalicular levels of the bile salt export pump (Bsep) and no detectable effect on P-glycoprotein (P-gp) canalicular levels. Treatment with VPA for 24 h also significantly downregulated levels of tight junction (TJ)-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of TJ proteins claudin 1, claudin 2, occludin, ZO1, and ZO3. These findings reveal that two novel mechanisms may contribute to VPA hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.

Recent Progress in Prediction Systems for Drug-induced Liver Injury Using in vitro Cell Culture

2020 ◽  
Vol 14 ◽  
Author(s):  
Shogo Ozawa ◽  
Toshitaka Miura ◽  
Jun Terashima ◽  
Wataru Habano ◽  
Seiichi Ishida
Keyword(s):  
Cell Culture ◽  
Recent Progress ◽  
Inflammatory Cells ◽  
Protein Adducts ◽  
In Vitro Assays ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Culture Systems ◽  
Cell Culture Systems

Background: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. Objective: In this study, the recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as for the limitations of in vitro cell culture systems for DILI research. Methods: Information related to DILI was collected through a literature search of the PubMed database. Results: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4+ helper T lymphocytes and recognition by CD8+ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells, or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILIinducing drugs with approximately 50% sensitivity and 90% specificity. Conclusion: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.

scholarly journals The acylation of proteins by xenobiotic amphipathic carboxylic acids in cultured rat hepatocytes

1988 ◽  
Vol 254 (1) ◽  
pp. 39-44 ◽  
Author(s):  
R Hertz ◽  
J Bar−Tana
Keyword(s):  
Protein Synthesis ◽  
Carboxylic Acids ◽  
Mode Of Action ◽  
Biological Effects ◽  
Rat Hepatocytes ◽  
Protein Adducts ◽  
Cultured Rat Hepatocytes ◽  
Acylated Proteins ◽  
Dose Dependent ◽  
Protein Acylation

Three xenobiotic amphipathic carboxylates, namely MEDICA 16, nafenopin and bezafibrate, which differ remarkably in their hydrophobic backbones, were found to acylate membrane and cytosolic liver proteins in cultured rat hepatocytes. The acylation patterns observed were time- and dose-dependent, and the acylated residue consisted of the original xenobiotic. The acylation patterns generated by the three xenobiotic carboxylates included common proteins which were acylated by the three xenobiotics (e.g. proteins of 32, 52, 56 and 72 kDa) as well as unique proteins which were specifically acylated by the respective xenobiotics. The acylation of liver proteins by either MEDICA 16 or nafenopin remained unaffected under conditions where protein synthesis was completely inhibited by cycloheximide. Protein acylation thus offers a common mode of action of xenobiotic amphipathic carboxylates, which may, however, result in diverse xenobiotyl-protein adducts. The xenobiotyl-acylated proteins might be involved in triggering some of the biological effects exerted by xenobiotic amphipathic carboxylates employed as hypolipidaemic effectors, peroxisomal proliferators or preadipocyte convertors.

Development and Application of a Transcriptomic Signature of Bioactivation in an Advanced In Vitro Liver Model to Reduce Drug-induced Liver Injury Risk Early in the Pharmaceutical Pipeline

2020 ◽  
Vol 177 (1) ◽  
pp. 121-139 ◽  
Author(s):  
Wen Kang ◽  
Alexei A Podtelezhnikov ◽  
Keith Q Tanis ◽  
Stephen Pacchione ◽  
Ming Su ◽  
...  
Keyword(s):  
Liver Injury ◽  
Human Model ◽  
Reactive Metabolites ◽  
In Vitro Test ◽  
Drug Induced ◽  
Vitro Assay ◽  
Drug Induced Liver Injury ◽  
Drug Candidates ◽  
Transcriptomic Signature

Abstract Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection.

scholarly journals Effect of P450-Inducers on the Hepatotoxicity of 2-Hexenal on the Leakage of Lactate Dehydrogenase from Primary Cultured Rat Hepatocytes.

Eisei kagaku ◽  
1997 ◽  
Vol 43 (6) ◽  
pp. 331-335
Author(s):  
Teruhisa HIRAYAMA ◽  
Akihiro YOSHIKAWA ◽  
Terue KASAI ◽  
Tetsushi WATANABE ◽  
Shunjiro OGAWA
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Hepatocytes ◽  
Primary Cultured Rat Hepatocytes ◽  
Cultured Rat Hepatocytes

scholarly journals Characterizing protein modifications by reactive metabolites using magnetic bead bioreactors and LC-MS/MS

2015 ◽  
Vol 51 (22) ◽  
pp. 4701-4703 ◽  
Author(s):  
Dandan Li ◽  
You-Jun Fu ◽  
James F. Rusling
Keyword(s):  
Magnetic Beads ◽  
Metabolic Enzymes ◽  
Magnetic Bead ◽  
Protein Adducts ◽  
Reactive Metabolites ◽  
Label Free ◽  
Protein Adduct ◽  
Identification Method ◽  
Ms Analysis ◽  
Detection And Identification

A label-free metabolite–protein adduct detection and identification method was developed using magnetic beads coated with metabolic enzymes as bioreactors to generate metabolite–protein adducts for LC-MS/MS analysis.

scholarly journals Selenium and Glutathione-Depleted Rats as a Sensitive Animal Model to Predict Drug-Induced Liver Injury in Humans

2019 ◽  
Vol 20 (13) ◽  
pp. 3141 ◽  
Author(s):  
Keisuke Goda ◽  
Kyotaka Muta ◽  
Yuzo Yasui ◽  
Shin-ichi Oshida ◽  
Kanae Kitatani ◽  
...  
Keyword(s):  
Animal Model ◽  
Liver Injury ◽  
Reactive Metabolites ◽  
Drug Induced ◽  
Deficient Diet ◽  
Drug Induced Liver Injury ◽  
Glutathione Peroxidase 1 ◽  
Se Deficiency ◽  
Repeated Dosing ◽  
Intact Rats

Drug-induced liver injury (DILI) is one of the most serious and frequent drug-related adverse events in humans. Selenium (Se) and glutathione (GSH) have a crucial role for the hepatoprotective effect against reactive metabolites or oxidative damage leading to DILI. The hepatoprotective capacity related to Se and GSH in rodents is considered to be superior compared to the capacity in humans. Therefore, we hypothesize that Se/GSH-depleted rats could be a sensitive animal model to predict DILI in humans. In this study, Se-deficiency is induced by feeding a Se-deficient diet and GSH-deficiency is induced by l-buthionine-S,R-sulfoxinine treatment via drinking water. The usefulness of this animal model is validated using flutamide, which is known to cause DILI in humans but not in intact rats. In the Se/GSH-depleted rats from the present study, decreases in glutathione peroxidase-1 protein expression and GSH levels and an increase in malondialdehyde levels in the liver are observed without any increase in plasma liver function parameters. Five-day repeated dosing of flutamide at 150 mg/kg causes hepatotoxicity in the Se/GSH-depleted rats but not in normal rats. In conclusion, Se/GSH-depleted rats are the most sensitive for detecting flutamide-induced hepatotoxicity in all the reported animal models.

Protective Effects of Water Extract of Clam on Normal and CCl4-Induced Damage in Primary Cultured Rat Hepatocytes

2010 ◽  
Vol 38 (06) ◽  
pp. 1193-1205 ◽  
Author(s):  
H.M. Chi ◽  
S.T. Chou ◽  
S.C. Lin ◽  
Z.Y. Su ◽  
Lee-Yan Sheen
Keyword(s):  
Lactate Dehydrogenase ◽  
Carbon Tetrachloride ◽  
Water Extract ◽  
Rat Hepatocytes ◽  
Control Group ◽  
Protective Effects ◽  
Primary Cultured Rat Hepatocytes ◽  
Defense Systems ◽  
Intracellular Glutathione ◽  
Cultured Rat Hepatocytes

The objective of this study was to investigate the effects of various concentrations and incubation times of water extract of clam (WEC) on glutathione, its antioxidant and the detoxification defense systems in normal and CCl4 -induced oxidative damaged primary rat hepatocytes. This study showed that when the hepatocytes were treated with WEC(0.14 ~ 1.68 mg/ml), the intracellular glutathione (GSH) levels, GSH/GSSG ratio, and the activities of GSH-related enzymes (GPx, GRd, and GST) were higher than those in the control at 24 or 48 hour treatments. However, the lactate dehydrogenase (LDH) leakage and microscopic observations did not differ from those of the control. Yet, when the hepatocytes were pretreated with various concentrations of WEC for 24 hours and then exposed to 5 mM carbon tetrachloride (CCl4) for 1 hour, at concentrations of WEC between 0.42 ~ 1.68 mg/ml, the viabilities, intracellular GSH level, and activities of GST and GPx were significantly increased compared to those of the CCl4 -treated control group ( p < 0.05). In conclusion, WEC could improve the viability and the capabilities of detoxification and antioxidation in hepatocytes by increasing the GSH level and the activities of GSH-related enzymes.

Assessment of reactive metabolites in drug-induced liver injury

2011 ◽  
Vol 34 (11) ◽  
pp. 1879-1886 ◽  
Author(s):  
Kye Sook Lee ◽  
Soo Jin Oh ◽  
Hwan Mook Kim ◽  
Ki Ho Lee ◽  
Sang Kyum Kim
Keyword(s):  
Liver Injury ◽  
Reactive Metabolites ◽  
Drug Induced ◽  
Drug Induced Liver Injury
Sign in / Sign up

Export Citation Format

Share Document