ABSTRACT
An ethanol extract complex of Descurainia sophia seeds and Peucedanum praeruptorum roots, called BP10A, has antitumor potential against colorectal cancer. In the present study, we evaluated the 28-day oral toxicity and the genotoxicity of BP10A. The subacute toxicity test was done through oral administration to mice. ICR mice (n = 10) received daily oral BP10A doses of 0, 500, 1000 and 2000 mg/kg for 28 consecutive days. During administration, general clinical signs, food consumption, organ weights, and hematologic, biochemical and histopathological parameters in male and female mice were assessed. No significant adverse effects up to the highest dose (2000 mg/kg) were found. The genotoxicity was evaluated using a battery of tests, including an in vitro bacterial reverse mutation (Ames) test, an in vivo micronucleus test using bone marrow cells in ICR mice and a chromosomal aberration test using CHL/IU cells. BP10A did not show any genotoxic signs in the Ames (up to 5000 µg/plate), micronucleus (up to 5000 mg/kg) and the chromosomal aberration tests (550-1750 µg/mL). Therefore, BP10A was considered safe based on the subacute toxicity and genotoxicity results, indicating that it is a useful pharmaceutical material with no adverse toxicity.
Subject(s)
Antineoplastic Agents/toxicity , Apiaceae/chemistry , Brassicaceae/chemistry , Chromans/toxicity , Colorectal Neoplasms/drug therapy , DNA Damage/drug effects , Plant Extracts/toxicity , Administration, Oral , Animals , Dose-Response Relationship, Drug , Female , Male , Mice , Mice, Inbred ICR , Models, Animal , Plant Extracts/administration & dosage , Plant Roots/chemistry , Seeds/chemistry , Toxicity TestsABSTRACT
In this study, we developed a multi-signal mitochondria-targeted fluorescent probe (NIR-Cys) for simultaneous detection of Cys and its metabolite, SO2. In the design of the probe, the acrylate group and the C[double bond, length as m-dash]C of the coumarin ring were used as the recognizing moiety for Cys and SO2, respectively. The probe exhibited high sensitivity, excellent specificity, and fast response. NIR-Cys was found to precisely target and visualize Cys metabolism in mitochondria of living cells with a multi-fluorescence signal. This probe is expected to be a useful tool for understanding Cys metabolism.
Subject(s)
Acrylates/chemistry , Chromans/chemistry , Coumarins/chemistry , Cysteine/analysis , Fluorescent Dyes/chemistry , Mitochondria/metabolism , Sulfur Dioxide/analysis , Acrylates/chemical synthesis , Acrylates/metabolism , Acrylates/toxicity , Animals , Cell Line, Tumor , Chromans/chemical synthesis , Chromans/metabolism , Chromans/toxicity , Coumarins/chemical synthesis , Coumarins/metabolism , Coumarins/toxicity , Cysteine/metabolism , Fluorescence , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Fluorescent Dyes/toxicity , Humans , Limit of Detection , Liver/metabolism , Mice , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Sulfites/analysis , Sulfites/chemistry , Sulfur Dioxide/metabolism , ZebrafishABSTRACT
Troglitazone, a member of the thiazolidinedione class of antidiabetic drugs, was withdrawn from the market because it causes severe liver injury. One of the mechanisms for this adverse effect is thought to be mitochondrial toxicity. To investigate the characteristics of troglitazone-induced liver toxicity in more depth, the toxicological effects of troglitazone on hepatocytes and liver mitochondria were investigated using a rat model of type 2 diabetes mellitus (T2DM). Troglitazone was found to increase mitochondrial permeability transition (MPT) in the liver mitochondria of diabetic rats to a greater extent than in control rats, whereas mitochondrial membrane potential and oxidative phosphorylation were not affected. To identify the factors associated with this increase in susceptibility to MPT in diabetic rats, we assessed the oxidative status of the liver mitochondria and found a decrease in mitochondrial glutathione content and an increase in phospholipid peroxidation. Moreover, incorporation of oxidized cardiolipin, a mitochondrion-specific phospholipid, was involved in the troglitazone-induced alteration in susceptibility to MPT. In conclusion, liver mitochondria display disease-associated mitochondrial lipid peroxidation in T2DM, which facilitates the higher susceptibility to troglitazone-induced MPT. Thus, greater susceptibility of liver mitochondria may be a host factor leading to troglitazone-induced hepatotoxicity in T2DM.
Subject(s)
Chromans/toxicity , Diabetes Mellitus, Type 2/metabolism , Hypoglycemic Agents/toxicity , Lipid Peroxidation , Mitochondria, Liver/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Thiazolidinediones/toxicity , Animals , Cardiolipins/metabolism , Chromans/adverse effects , Disease Models, Animal , Glutathione/metabolism , Hepatocytes/drug effects , Hypoglycemic Agents/adverse effects , Male , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/metabolism , Mitochondrial Permeability Transition Pore , Oxidative Phosphorylation/drug effects , Oxygen Consumption/drug effects , Phospholipids/metabolism , Rats, Zucker , Thiazolidinediones/adverse effects , TroglitazoneABSTRACT
Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/antagonists & inhibitors , Bile Acids and Salts/blood , Chemical and Drug Induced Liver Injury/etiology , Risk Assessment , Animals , Chromans/toxicity , Female , Male , Rats , Rats, Sprague-Dawley , Thiazolidinediones/toxicity , TroglitazoneABSTRACT
On the basis of reported antimycobacterial property of chroman-4-one pharmacophore, a series of chemically modified bis-spirochromanones were synthesized starting from 2-hydroxyacetophenone and 1,4-dioxaspiro[4.5] decan-8-one using a Kabbe condensation approach. The synthesized bis-spirochromanones were established based on their spectral data and X-ray crystal structure of 6e. All synthesized compounds were evaluated against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain, finding that some products exhibited good antimycobacterial activity with minimum inhibitory concentration as low as [Formula: see text]. Docking studies were carried out to identify the binding interactions of compounds II, 6a and 6n with FtsZ. Compounds exhibiting good in vitro potency in the MTB MIC assay were further evaluated for toxicity using the HEK cell line.
Subject(s)
Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacology , Chromans/chemical synthesis , Chromans/pharmacology , Mycobacterium tuberculosis/drug effects , Spiro Compounds/chemistry , Antitubercular Agents/metabolism , Antitubercular Agents/toxicity , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chemistry Techniques, Synthetic , Chromans/metabolism , Chromans/toxicity , HEK293 Cells , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Mycobacterium tuberculosis/metabolism , Protein Conformation , Structure-Activity RelationshipABSTRACT
A novel series of donepezil-trolox hybrids were designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological assays showed that these derivatives possessed moderate to good inhibitory activities against acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B) as well as remarkable antioxidant effects. The optimal compound 6d exhibited balanced functions with good inhibition against hAChE (IC50 = 0.54 µM) and hMAO-B (IC50 = 4.3 µM), significant antioxidant activity (41.33 µM IC50 by DPPH method, 1.72 and 1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation, and Aß1-42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d has very low toxicity and is capable of combating oxidative toxin (H2O2, rotenone, and oligomycin-A) induced neurotoxicity. Most importantly, oral administration of 6d demonstrated notable improvements on cognition and spatial memory against scopolamine-induced acute memory deficit as well as d-galactose (d-gal) and AlCl3 induced chronic oxidative stress in a mouse model without acute toxicity and hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a valuable candidate for the development of a safe and effective anti-Alzheimer's drug.
Subject(s)
Alzheimer Disease/drug therapy , Antioxidants/therapeutic use , Central Nervous System Agents/therapeutic use , Cholinesterase Inhibitors/therapeutic use , Chromans/therapeutic use , Indans/therapeutic use , Monoamine Oxidase Inhibitors/therapeutic use , Piperidines/therapeutic use , Amyloid beta-Peptides/drug effects , Animals , Antioxidants/pharmacology , Antioxidants/toxicity , Blood-Brain Barrier , Cell Line , Central Nervous System Agents/pharmacology , Central Nervous System Agents/toxicity , Chelating Agents/pharmacology , Chelating Agents/therapeutic use , Chelating Agents/toxicity , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/toxicity , Chromans/pharmacology , Chromans/toxicity , Copper , Donepezil , Drug Design , Drug Evaluation, Preclinical , Humans , Indans/pharmacology , Indans/toxicity , Male , Mice, Inbred ICR , Microglia/drug effects , Molecular Docking Simulation , Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase Inhibitors/toxicity , Neurotoxins/toxicity , Oxidants/toxicity , PC12 Cells , Peptide Fragments/drug effects , Piperidines/pharmacology , Piperidines/toxicity , Protein Aggregation, Pathological/drug therapy , Rats , Structure-Activity RelationshipABSTRACT
Global gene expression profiling is useful for elucidating a drug's mechanism of action on the liver; however, such profiling in rats is not very sensitive for predicting human drug-induced liver injury, while dedifferentiated monolayers of primary human hepatocytes (PHHs) do not permit chronic drug treatment. In contrast, micropatterned cocultures (MPCCs) containing PHH colonies and 3T3-J2 fibroblasts maintain a stable liver phenotype for 4-6 weeks. Here, we used MPCCs to test the hypothesis that global gene expression patterns in stable PHHs can be used to distinguish clinical hepatotoxic drugs from their non-liver-toxic analogs and understand the mechanism of action prior to the onset of overt hepatotoxicity. We found that MPCCs treated with the clinical hepatotoxic/non-liver-toxic pair, troglitazone/rosiglitazone, at each drug's reported and non-toxic Cmax (maximum concentration in human plasma) for 1, 7, and 14 days displayed a total of 12, 269, and 628 differentially expressed genes, respectively, relative to the vehicle-treated control. Troglitazone modulated >75% of transcripts across pathways such as fatty acid and drug metabolism, oxidative stress, inflammatory response, and complement/coagulation cascades. Escalating rosiglitazone's dose to that of troglitazone's Cmax increased modulated transcripts relative to the lower dose; however, over half the identified transcripts were still exclusively modulated by troglitazone. Last, other hepatotoxins (nefazodone, ibufenac, and tolcapone) also induced a greater number of differentially expressed genes in MPCCs than their non-liver-toxic analogs (buspirone, ibuprofen, and entacapone) following 7 days of treatment. In conclusion, MPCCs allow evaluation of time- and dose-dependent gene expression patterns in PHHs treated chronically with analog drugs.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Fibroblasts/drug effects , Hepatocytes/drug effects , Liver/drug effects , Transcriptome/drug effects , 3T3 Cells , Animals , Cells, Cultured , Chemical and Drug Induced Liver Injury/genetics , Chromans/toxicity , Coculture Techniques , Dose-Response Relationship, Drug , Fibroblasts/cytology , Gene Expression Profiling , Hepatocytes/cytology , Humans , Mice , Oxidative Stress/drug effects , Oxidative Stress/genetics , Primary Cell Culture , Rosiglitazone , Thiazolidinediones/toxicity , Toxicogenetics , TroglitazoneABSTRACT
Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.
Subject(s)
Apoptosis/drug effects , Biological Assay , Chemical and Drug Induced Liver Injury/etiology , Hepatocytes/drug effects , Liver/drug effects , Mitochondria, Liver/drug effects , Toxicity Tests/methods , Acetaminophen/toxicity , Biomarkers/metabolism , Caspase 3/metabolism , Caspase 7/metabolism , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Chromans/toxicity , Diclofenac/toxicity , Dose-Response Relationship, Drug , Ethanol/toxicity , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Isoxazoles/toxicity , Leflunomide , Liver/metabolism , Liver/pathology , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Oxygen Consumption/drug effects , Primary Cell Culture , Ranitidine/toxicity , Risk Assessment , Thiazolidinediones/toxicity , Time Factors , TroglitazoneABSTRACT
Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Pharmaceutical Preparations/metabolism , Adenosine Triphosphate/metabolism , Benzbromarone/analogs & derivatives , Benzbromarone/metabolism , Benzbromarone/toxicity , Cell Line , Cell Survival/drug effects , Chromans/metabolism , Chromans/toxicity , Cytochrome P-450 CYP2C9/metabolism , Cytochrome P-450 CYP2D6/metabolism , Cytochrome P-450 CYP3A/metabolism , Humans , Thiazolidinediones/metabolism , Thiazolidinediones/toxicity , TroglitazoneABSTRACT
INTRODUCTION: Preclinical in vivo QT measurement as a proarrhythmia essay is expensive and not reliable enough. The aim of the present study was to develop a sensitive, cost-effective, Langendorff perfused guinea pig heart model for proarrhythmia safety screening. METHODS: Low concentrations of dofetilide and cisapride (inhibitors of the rapid delayed rectifier potassium current, IKr) were tested alone and co-perfused with HMR-1556 (inhibitor of the slow delayed rectifier potassium current, IKs) in Langendorff perfused guinea pig hearts. The electrocardiographic rate corrected QT (QTc) interval, the Tpeak-Tend interval and the beat-to-beat variability and instability (BVI) of the QT interval were determined in sinus rhythm. RESULTS: Dofetilide and HMR-1556 alone or co-perfused, prolonged the QTc interval by 20±2%, 10±1% and 55±10%, respectively. Similarly, cisapride and HMR-1556 alone or co-perfused, prolonged the QTc interval by 11±3%, 11±4% and 38±6%, respectively. Catecholamine-induced fast heart rate abolished the QTc prolonging effects of the IKr inhibitors, but augmented the QTc prolongation during IKs inhibition. None of the drug perfusions increased significantly the Tpeak-Tend interval and the sinus BVI of the QT interval. DISCUSSION: IKs inhibition increased the QTc prolonging effect of IKr inhibitors in a super-additive (synergistic) manner, and the QTc interval was superior to other proarrhythmia biomarkers measured in sinus rhythm in isolated guinea pig hearts. The effect of catecholamines on the QTc facilitated differentiation between IKr and IKs inhibitors. Thus, QTc measurement in Langendorff perfused guinea pig hearts with pharmacologically attenuated repolarization reserve and periodic catecholamine perfusion seems to be suitable for preclinical proarrhythmia screening.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Drug Evaluation, Preclinical/methods , Heart/drug effects , Long QT Syndrome/chemically induced , Potassium Channel Blockers/toxicity , Animals , Catecholamines/pharmacology , Chromans/toxicity , Cisapride/toxicity , Coronary Circulation/drug effects , Delayed Rectifier Potassium Channels/drug effects , Drug Interactions , Electrocardiography/drug effects , Female , Guinea Pigs , Heart Rate/drug effects , In Vitro Techniques , Phenethylamines/toxicity , Sulfonamides/toxicity , Torsades de Pointes/chemically inducedABSTRACT
Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. This cell line was utilized to evaluate cytotoxicity of two bioactive (troglitazone and acetaminophen) and two non-bioactive (citrate and galactosamine) compounds by MTT assay. Cell viability significantly decreased upon treatment with bioactive drugs. Moreover, cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.
Subject(s)
Biological Assay , Cell Survival , Cytochrome P-450 CYP3A/metabolism , Nerve Tissue Proteins/genetics , Acetaminophen/toxicity , Activation, Metabolic , Cell Survival/drug effects , Chromans/toxicity , Citric Acid/toxicity , Cytochrome P-450 CYP3A/genetics , DNA Transposable Elements , Galactosamine/toxicity , Hep G2 Cells , Humans , Thiazolidinediones/toxicity , TroglitazoneABSTRACT
Mitochondrial dysfunction has been implicated in various drug-induced toxicities and genetic disorders. Recently, the zebrafish has emerged as a versatile animal model for both chemical and genetic screenings. Taking advantage of its transparency, various in vivo fluorescent imaging methods have been developed to identify novel functions of chemicals and genes in zebrafish. However, there have not been fluorescent probes that can detect mitochondrial membrane potential in living zebrafish. In this study, we identified a novel cyanine dye called ZMJ214 that detects mitochondrial membrane potential in living zebrafish from 4 to 8 days post fertilization and is administered by simple immersion. The fluorescence intensity of ZMJ214 in zebrafish was increased and decreased by oligomycin and FCCP, respectively, suggesting a positive correlation between ZMJ214 fluorescence and mitochondrial membrane potential. In vivo imaging of zebrafish stained with ZMJ214 allowed for the detection of altered mitochondrial membrane potential induced by the antidiabetic drug troglitazone and the antiepileptic drug tolcapone, both of which have been withdrawn from the market due to mitochondrial toxicity. In contrast, pioglitazone and entacapone, which are similar to troglitazone and tolcapone, respectively, and have been used commercially, did not cause a change in mitochondrial membrane potential in zebrafish stained with ZMJ214. Live imaging of zebrafish stained with ZMJ214 also revealed that knock-down of slc25a12, a mitochondrial carrier protein associated with autism, dysregulated the mitochondrial membrane potential. These results suggest that ZMJ214 can be a useful tool to identify chemicals and genes that cause mitochondrial dysfunction in vivo.
Subject(s)
Carbocyanines/chemistry , Fluorescent Dyes/chemistry , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/pathology , Optical Imaging , Animals , Anti-Bacterial Agents/toxicity , Anticonvulsants/toxicity , Benzophenones/toxicity , Chromans/toxicity , Disease Models, Animal , Hypoglycemic Agents/toxicity , Nitrophenols/toxicity , Oligomycins/toxicity , Optical Imaging/methods , Pioglitazone , Thiazolidinediones/toxicity , Tolcapone , Toxicity Tests/methods , Troglitazone , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Coupling of two distinct pharmacophores, tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimer's disease. Several of them showed improved inhibitory properties toward acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of nontoxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine-trolox hybrids exhibited low in vivo toxicity after im administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.
Subject(s)
Antioxidants/chemical synthesis , Antioxidants/pharmacology , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/pharmacology , Chromans/chemistry , Chromans/pharmacology , Tacrine/chemistry , Tacrine/pharmacology , Acetylcholinesterase/chemistry , Animals , Antioxidants/toxicity , Blood-Brain Barrier , Catalysis , Cholinesterase Inhibitors/toxicity , Chromans/toxicity , Drug Design , Free Radical Scavengers/chemical synthesis , Free Radical Scavengers/pharmacology , Hepatocytes/drug effects , Humans , Injections, Intramuscular , Kinetics , Ligands , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Molecular , Rats , Rats, Wistar , Tacrine/toxicityABSTRACT
In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1ß) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1ß and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.
Subject(s)
Acetaminophen/toxicity , Chemical and Drug Induced Liver Injury/immunology , Chromans/toxicity , Cytokines/metabolism , Fluoroquinolones/toxicity , Immunity, Innate/immunology , Inflammation Mediators/metabolism , Kupffer Cells/immunology , Naphthyridines/toxicity , Thiazolidinediones/toxicity , Animals , Cells, Cultured , Chemokines/metabolism , Down-Regulation/drug effects , Interleukin-10/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Kupffer Cells/metabolism , Male , Nitric Oxide/metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Troglitazone , Up-Regulation/drug effectsABSTRACT
INTRODUCTION: In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. METHODS: We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). RESULTS: The mean (± SEM) urea production was significantly higher in sandwich cultures from freshly-isolated hepatocytes (27.88 (± 0.96) nmol/cm(2)), compared to cultures from cryopreserved hepatocytes (22.86 (± 1.91) nmol urea/cm(2)). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (± SEM) DICI values for CsA 10 µM and Tro 75 µM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 µM) and Tro (≥ 100 µM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. DISCUSSION: In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes.
Subject(s)
Cholestasis/chemically induced , Chromans/toxicity , Cryopreservation , Cyclosporine/toxicity , Hepatocytes/drug effects , Thiazolidinediones/toxicity , Animals , Bile Acids and Salts/pharmacology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cholestasis/pathology , Cryopreservation/methods , Hepatocytes/pathology , Male , Rats , Rats, Wistar , TroglitazoneABSTRACT
OBJECTIVE: To characterize the mechanism of action of thiazolidinedione (TZD)-induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells. METHODS: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50µM) for 48h. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM). mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrial respiratory chain (MRC) complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p<0.05. RESULTS: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. CONCLUSION: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk:benefit profiles.
Subject(s)
Liver/drug effects , Mitochondria/drug effects , Thiazolidinediones/toxicity , Cell Line, Tumor , Chromans/toxicity , DNA, Mitochondrial/genetics , Electron Transport/drug effects , Humans , Hypoglycemic Agents/toxicity , Liver/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , Oxidative Stress/drug effects , Pioglitazone , Reactive Oxygen Species/metabolism , Rosiglitazone , TroglitazoneABSTRACT
DILI is a major safety issue during drug development and one of the leading causes for market withdrawal. Despite many efforts made in the past, the prediction of DILI using in vitro models remains very unreliable. In the present study, the well-established hepatocyte Collagen I-Matrigel™ sandwich culture was used, mimicking chronic drug treatment after multiple incubations for 14 days. Ten drugs associated with different types of specific preclinical and clinical liver injury were evaluated at non-cytotoxic concentrations. Mrp2-mediated transport, intracellular accumulation of neutral lipids and phospholipids were selected as functional endpoints by using Cellomics™ Arrayscan® technology and assessed at five timepoints (day 1, 3, 7, 10, 14). Liver specific functional impairments after drug treatment were enhanced over time and could be monitored by HCI already after few days and before cytotoxicity. Phospholipidosis-inducing drugs Chlorpromazine and Amiodarone displayed the same response as in vivo. Cyclosporin A, Chlorpromazine, and Troglitazone inhibited Mrp2-mediated biliary transport, correlating with in vivo findings. Steatosis remained difficult to be reproduced under the current in vitro testing conditions, resulting into false negative and positive responses. The present results suggest that the repeated long-term treatment of rat hepatocytes in the Collagen I-Matrigel™ sandwich configuration might be a suitable tool for safety profiling of the potential to induce phospholipidosis and impair Mrp2-mediated transport processes, but not to predict steatosis.
Subject(s)
Hepatocytes/drug effects , Amiodarone/administration & dosage , Amiodarone/toxicity , Animals , Cells, Cultured , Chlorpromazine/administration & dosage , Chlorpromazine/toxicity , Chromans/administration & dosage , Chromans/toxicity , Culture Techniques , Cyclosporine/administration & dosage , Cyclosporine/toxicity , Dopamine Antagonists/administration & dosage , Dopamine Antagonists/toxicity , Gene Expression Regulation/drug effects , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/toxicity , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/toxicity , Male , Potassium Channel Blockers/administration & dosage , Potassium Channel Blockers/toxicity , Rats , Rats, Wistar , Thiazolidinediones/administration & dosage , Thiazolidinediones/toxicity , TroglitazoneABSTRACT
The toxicity of parenterally administered vitamin E isomers, delta-tocotrienol (DT3) and gamma-tocotrienol (GT3), was evaluated in male and female CD2F1 mice. In an acute toxicity study, a single dose of DT3 or GT3 was administered subcutaneously in a dose range of 200 to 800 mg/kg. A mild to moderately severe dermatitis was observed clinically and microscopically in animals at the injection site at doses above 200 mg/kg. The severity of the reaction was reduced when the drug concentration was lowered. Neither drug produced detectable toxic effects in any other tissue at the doses tested. Based on histopathological analysis for both DT3 and GT3, and macroscopic observations of inflammation at the injection site, a dose of 300 mg/kg was selected as the lowest toxic dose in a 30-day toxicity study performed in male mice. At this dose, a mild skin irritation occurred at the injection site that recovered completely by the end of the experimental period. At a dose of 300 mg/kg of DT3 or GT3, no adverse effects were observed in any tissues or organs.
Subject(s)
Chromans/toxicity , Dermatitis, Contact/etiology , Irritants/toxicity , Vitamin E/analogs & derivatives , Administration, Cutaneous , Animals , Dermatitis, Contact/pathology , Female , Male , Mice , Skin/drug effects , Skin/pathology , Toxicity Tests, Acute , Vitamin E/toxicityABSTRACT
Advances in systems biology have allowed the development of a highly characterized systems pharmacology model to study mechanisms of drug-induced hepatotoxicity. In this issue of CPT, Yang et al. describe a model, DILIsym, used to characterize mechanisms of hepatotoxicity of troglitazone. Their modeling approach has provided new insight into troglitazone-induced hepatotoxicity in humans but is not associated with hepatotoxicity in rats, consistent with preclinical data for this drug.
Subject(s)
Bile Acids and Salts/physiology , Chemical and Drug Induced Liver Injury/etiology , Chromans/toxicity , Hypoglycemic Agents/toxicity , Thiazolidinediones/toxicity , Animals , Humans , Male , TroglitazoneABSTRACT
SPD-304 was discovered as a promising tumor necrosis factor alpha (TNF) antagonist that promotes dissociation of TNF trimers and therefore blocks the interaction of TNF and its receptor. However, SPD-304 contains a potentially toxic 3-alkylindole moiety, which can be bioactivated to a reactive electrophilic intermediate. A series of SPD-304 analogs was synthesized with the aim to diminish its toxicophore groups while maintaining the binding affinity for TNF. Incorporation of electron-withdrawing substituents at the indole moiety, in conjunction with elimination of the 6'-methyl group of the 4-chromone moiety, led to a significantly less toxic and equally potent TNF inhibitor.