ABSTRACT
Ivabradine, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel inhibitor, has been reported to induce photosensitivity-related visual disturbances such as phosphene in humans. Ivabradine-induced visual disturbances are caused by inhibition of HCN channels in the retina, and the mechanisms have been verified using HCN channel knockout mice and electroretinography (ERG). However, in rats, classical ERG using single flash light stimulus with standard analyses of waveform amplitude and latency has not revealed abnormal retinal function after administration of ivabradine. To verify whether retinal dysfunction after ivabradine administration was detectable in rats, we performed ERG using multistep flash light stimulation at the time when plasma concentration of ivabradine was high. Furthermore, the mechanism of the change in the waveform that appeared after the b-wave was investigated. Ivabradine and cilobradine, a selective HCN channel inhibitor, were administered subcutaneously to rats at 4-40 mg/kg as a single dose, and flash or long-duration ERG recordings at each light stimulus luminance were conducted 1.5 h after administration. Plasma and retinal concentrations of both compounds were measured immediately after the ERG recordings. In the flash ERG, prolongation of a- and/or b-wave latencies were detected at each light stimulus, and dose-dependent waveform changes after the b-wave were recorded at the specific light stimulus luminance for both compounds. These ERG changes increased in response to increasing plasma and retinal concentrations for both ivabradine and cilobradine. In the long-duration light stimulus ERG, a change in the waveform of the b-wave trough and attenuation of the c-wave were recorded, suggesting that the feedback control in the photoreceptor cells may be inhibited. This study revealed that the retinal dysfunction by HCN channel inhibitors in rats can be detected by multistep light stimulus ERG. Additionally, we identified that the inhibition of feedback current and the sustained responses in the photoreceptor cells cause the retinal dysfunction of HCN channel inhibitors in rats.
Subject(s)
Electroretinography , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Mice , Humans , Rats , Animals , Ivabradine , Retina , Vision, Ocular , Vision Disorders , Mice, Knockout , Photic StimulationABSTRACT
Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.
Subject(s)
Chemical and Drug Induced Liver Injury , Cytochrome P-450 CYP1A1 , Receptors, Aryl Hydrocarbon , Animals , Basic Helix-Loop-Helix Transcription Factors , Chemical and Drug Induced Liver Injury/genetics , Cytochrome P-450 CYP1A1/metabolism , Genes, Reporter , Hepatocytes/metabolism , Humans , Male , Rats , Receptors, Aryl Hydrocarbon/metabolismABSTRACT
BACKGROUND: Although clozapine-induced granulocytopenia (CIG) is less severe than clozapine-induced agranulocytosis (CIA), and some patients with CIG may not go on to develop serious complications, clozapine is discontinued in cases of both CIA and CIG. Understanding the pathogenic mechanisms of CIA/CIG could provide better management of clozapine therapy. Recently, as a mechanistic insight into adaptive immune systems, European groups reported clozapine-specific proliferative responses and clozapine-specific T cells using blood taken from patients with CIA and/or CIG. AIMS: The aims of our study are to support this mechanistic evidence and to investigate the difference in the lymphocyte response to clozapine between patients with CIG and those with CIA. METHODS: Lymphocyte stimulation tests (LSTs) were conducted using CD25-positive cell-depleted peripheral blood-derived mononuclear cells (PBMCs) isolated from blood of four Japanese patients with CIA, four patients with CIG, and nine clozapine-tolerant subjects. RESULTS: Three of four patients with CIA and one of four patients with CIG showed proliferative responses to clozapine with a stimulation index of greater than 2. In contrast, none of the nine clozapine-tolerant subjects showed any response to clozapine. Olanzapine did not stimulate PBMCs of patients with CIA, patients with CIG, or clozapine-tolerant subjects. CONCLUSIONS: Clozapine- and CIA-specific lymphocyte reactions in a Japanese population provided supportive evidence that the pathogenesis of CIA is based on adaptive immune reactions. In addition, patients with CIG who show a positive response to an LST may at the very least not be chosen for clozapine-rechallenge and further prospective studies are desirable to verify this hypothesis.
Subject(s)
Agranulocytosis , Antipsychotic Agents , Clozapine , Agranulocytosis/chemically induced , Antipsychotic Agents/adverse effects , Clozapine/adverse effects , Humans , Japan , Olanzapine/adverse effects , Prospective StudiesABSTRACT
The HLA class I allele HLA-A*33:03 is a risk factor for ticlopidine-induced liver injury. Herein, we show HLA class I-restricted ticlopidine-specific CD8+ T-cell activation in healthy donors expressing HLA-A*33:03. Cloned CD8+ T-cells proliferated and secreted IFN-γ in the presence of ticlopidine and autologous antigen presenting cells. A reduction of the T-cell response after blocking with HLA-class I and HLA-A*33 antibodies indicates that the interaction between drugs and the HLA allele detected in genetic association studies may be important for T-cell activation.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , HLA-A Antigens/metabolism , Lymphocyte Activation/drug effects , Ticlopidine/toxicity , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Genotype , HLA-A Antigens/genetics , Humans , Interferon-gamma/metabolismABSTRACT
It is unclear whether priming of naïve T cells to drugs is detectable in healthy human donors expressing different human leukocyte antigen (HLA) alleles. Thus, we examined T cell priming with drugs associated with HLA risk alleles and control compounds in 14 HLA-typed donors. Nitroso sulfamethoxazole and piperacillin activated T cells from all donors, whereas responses to carbamazepine and oxypurinol were only seen in donors expressing HLA-B*15:02 and HLA-B*58:01, respectively. Weak flucloxacillin-specific T cell responses were detected in donors expressing HLA-B*57:01 and HLA-B*58:01. These data show that the priming of T cells with certain drugs is skewed toward donors expressing specific HLA alleles.
Subject(s)
Drug-Related Side Effects and Adverse Reactions/immunology , HLA Antigens/immunology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Anti-Infective Agents/adverse effects , Anti-Infective Agents/immunology , Anticonvulsants/adverse effects , Anticonvulsants/immunology , Carbamazepine/adverse effects , Carbamazepine/immunology , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/immunology , HLA-B Antigens/immunology , Humans , Nitroso Compounds/adverse effects , Nitroso Compounds/immunology , Oxypurinol/adverse effects , Oxypurinol/immunology , Piperacillin/adverse effects , Piperacillin/immunology , Sulfamethoxazole/adverse effects , Sulfamethoxazole/immunology , T-Lymphocytes/immunologyABSTRACT
Isoniazid, rifampicin, pyrazinamide, and ethambutol are commonly used for the treatment of tuberculosis. Drug exposure is occasionally associated with liver and/or skin injury. The aim of this study was to determine whether drug-specific T-cells are detectable in patients with adverse reactions and if so characterize the nature of the T-cell response. Peripheral blood mononuclear cells (PBMC) from 6 patients with anti-tuberculosis drug-related adverse reactions (4 liver, 2 skin) were used to detect drug-responsive T-lymphocytes. Positive lymphocyte transformation test and/or ELIspot results were observed with all 6 patients. Over 3400 T-cell clones were generated from isoniazid, rifampicin, pyrazinamide, or ethambutol-treated PBMC. CD4+ clones from all 3 patients were activated to proliferate and secrete cytotoxic mediators (granzyme B, perforin, FasL) and effector (IFN-γ, Il-13) and regulatory (Il-10) cytokines with isoniazid, but not rifampicin, pyrazinamide, or ethambutol. Il-17 was not detected, while only 1 clone secreted Il-22. Isoniazid-responsive clones were not activated with other anti-tuberculosis drugs or isonicotinic acid albumin adducts. Activation of the clones with isoniazid was MHC class II-restricted and dependent on antigen-presenting cells. Most clones were activated rapidly even in the presence of the enzyme inhibitor 1-aminobenzotriazole. However, a time-dependent pathway of activation involving auto-oxidation of isoniazid was also observed. The discovery of isoniazid-specific CD4+ T-cell clones in patients with liver and skin injury suggests that the adaptive immune system is involved in the pathogenesis of both forms of iatrogenic disease.
Subject(s)
Antitubercular Agents/adverse effects , Chemical and Drug Induced Liver Injury/immunology , Isoniazid/adverse effects , Skin Diseases/immunology , T-Lymphocytes/immunology , Adolescent , Aged , Antitubercular Agents/immunology , Antitubercular Agents/therapeutic use , Clone Cells/immunology , Female , Humans , Isoniazid/immunology , Isoniazid/therapeutic use , Latent Tuberculosis/drug therapy , Male , Middle Aged , Skin Diseases/chemically induced , Tuberculosis, Pulmonary/drug therapyABSTRACT
A clinical association between a specific human leukocyte antigen (HLA) allele and idiosyncratic adverse drug reactions (IADRs) is a strong indication that IADRs are mediated by the adaptive immune system. For example, it is well-established that HLA-B*15:02 and HLA-B*57:01 are associated with carbamazepine-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and abacavir-induced hypersensitivity/flucloxacillin-induced liver injury, respectively. Drug-specific T-cells whose response is restricted by specific HLA risk alleles have been detected from IADR patients, also suggesting an adaptive immune pathogenesis. T-cells from carbamazepine SJS/TEN patients are activated by direct pharmacological interaction between carbamazepine and HLA-B*15:02 expressed on antigen presenting cells (APCs). Abacavir-specific, HLA-B*57:01-restricted T-cells are activated by APCs presenting peptides which are only displayed by the HLA molecule when abacavir is bound during peptide loading. Finally, HLA-B*57:01-restricted activation of T-cells from patients with flucloxacillin-induced liver injury is dependent on processing of drug protein adducts. Based on these observations, it is now possible to utilize blood from healthy drug-naïve volunteers to study the priming of naïve T-cells to drugs. Future development of these methodologies may lead to the development of assays that predict intrinsic immunogenicity of drugs and chemicals at the preclinical stage of drug development.
Subject(s)
Drug-Related Side Effects and Adverse Reactions/immunology , HLA Antigens/immunology , Pharmaceutical Preparations , Alleles , Drug-Related Side Effects and Adverse Reactions/genetics , HLA Antigens/genetics , HumansABSTRACT
Antituberculosis (TB) drug exposure is associated with a mild elevation of liver enzymes that occasionally develops into severe liver injury. Herein, we identify ethambutol- and rifampicin-specific CD4+ and CD8+ T-cell clones in a patient with fatal anti TB-related liver injury. The clones were activated to proliferate and secrete IFN-γ, Il-13, and granzyme B following drug treatment. Drug-responsive T-cells may contribute to the pathogenesis of antituberculosis-related liver failure.
Subject(s)
Antitubercular Agents/adverse effects , Chemical and Drug Induced Liver Injury/etiology , T-Lymphocytes/drug effects , Tuberculosis, Pulmonary/drug therapy , Fatal Outcome , Humans , Male , Middle Aged , T-Lymphocytes/immunology , Tuberculosis, Pulmonary/immunologyABSTRACT
A number of serious adverse drug reactions are caused by T cells. An association with HLA alleles has been identified with certain reactions, which makes it difficult to develop standardized preclinical tests to predict chemical liability. We have recently developed a T cell priming assay using the drug metabolite nitroso sulfamethoxazole (SMX-NO). We now report on reproducibility of the assay, establishment of a biobank of PBMC from 1000 HLA-typed volunteers, and generation of antigen-specific responses to a panel of compounds. Forty T cell priming assays were performed with SMX-NO; 5 gave weak responses (1.5-1.9) and 34 showed good (SI 2.0-3.9) or strong responses (SI > 4.0) using readouts for proliferation and cytokine release. Thus, SMX-NO can be used as a model reagent for in vitro T cell activation. Good to strong responses were also generated to haptenic compounds (amoxicillin, piperacillin and Bandrowski's base) that are not associated with an HLA risk allele. Furthermore, responses were detected to carbamazepine (in HLA-B*15:02 donors), flucloxacillin (in 1 HLA-B*57:01 donor) and oxypurinol (in HLA-B*58:01 donors), which are associated with HLA-class I-restricted forms of hypersensitivity. In contrast, naïve T cell priming to ximelagatran, lumiracoxib, and lapatinib (HLA-class II-restricted forms of hypersensitivity) yielded negative results. Abacavir, which activates memory T cells in patients, did not activate naïve T cells from HLA-B*57:01 donors. This work shows that the priming assay can be used to assess primary T cell responses to drugs and to study mechanisms T cell priming for drugs that display HLA class I restriction. Additional studies are required to investigate HLA-class II-restricted reactions.
Subject(s)
Drug Hypersensitivity/immunology , HLA Antigens/immunology , Histocompatibility Testing , Leukocytes, Mononuclear/drug effects , Lymphocyte Activation/drug effects , Sulfamethoxazole/analogs & derivatives , T-Lymphocytes/drug effects , Toxicity Tests/methods , Adult , Biological Specimen Banks , Cell Proliferation/drug effects , Cells, Cultured , Coculture Techniques , Cytokines/metabolism , Drug Hypersensitivity/metabolism , Female , Gene Frequency , HLA Antigens/genetics , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Male , Risk Assessment , Sulfamethoxazole/toxicity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Young AdultABSTRACT
Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.
Subject(s)
Isoniazid/metabolism , Lysine/metabolism , Serum Albumin/metabolism , Adult , Aged, 80 and over , Female , Humans , Isoniazid/adverse effects , Isoniazid/blood , Male , Microsomes, Liver/metabolism , Middle Aged , NADP/metabolism , Oxidation-Reduction , Protein BindingABSTRACT
The double null mutation of glutathione transferase, GSTM1 and GSTT1, is reported to influence troglitazone-associated abnormal increases of alanine aminotransferase and aspartate aminotransferase. However, no nonclinical data with a bearing on the clinical outcomes and underlying mechanisms have hitherto been reported. To investigate whether deficiency in GSTM1 and/or GSTT1 is related to troglitazone hepatotoxicity in vitro, the covalent binding level (CBL) (an index of reactive metabolite formation) and cytotoxicity of troglitazone and rosiglitazone, another thiazolidinedione but with low hepatotoxicity, were examined using human liver samples phenotyped for cytochrome P450s and genotyped for GSTM1 and GSTT1. Despite addition of GSH, CBLs of troglitazone and rosiglitazone in human liver microsomes were correlated with CYP3A (or CYP2C8) and CYP2C8 activities, respectively. With addition of recombinant GSTM1, the microsomal CBLs of troglitazone and rosiglitazone decreased. However, the CBLs of troglitazone in GSTM1/GSTT1 wild-type hepatocytes were unexpectedly higher than those in null hepatocytes. Although this discrepancy has not been fully explained, the GSTM1 and GSTT1 null mutations increased the cytotoxicity of troglitazone, independent of CYP3A or CYP2C8 activities. Furthermore, a GSH adduct of troglitazone, M2, limited to GSTM1 wild-type hepatocytes was detected. Of clear interest, GSTM1 and/or GSTT1 null mutation-dependent cytotoxicity and higher exposure to the reactive metabolite trapped as M2 as for troglitazone were not observed for rosiglitazone. This result might at least partly explain the findings related to clinical hepatotoxicity, suggesting that measurement of GSH adducts or cytotoxicity using GSTM1- and GSTT1-genotyped hepatocytes might offer an important in vitro system to assist in better prediction of idiosyncratic hepatotoxicity.
Subject(s)
Chromans/adverse effects , Glutathione Transferase/genetics , Hypoglycemic Agents/adverse effects , Microsomes, Liver/drug effects , Thiazolidinediones/adverse effects , Cells, Cultured , Chromatography, High Pressure Liquid , Genotype , Hepatocytes/drug effects , Humans , In Vitro Techniques , Microsomes, Liver/pathology , Tandem Mass Spectrometry , TroglitazoneABSTRACT
Whether lesion volume on diffusion-weighted MRI imaging (DWI) can reliably predict functional outcome in acute ischemic stroke is controversial. The aim of our study was to assess whether the Alberta Stroke Program Early CT Score (ASPECTS) on DWI is useful for predicting functional outcome in patients with anterior circulation infarction with a broad range of severities. Three-hundred and fifty patients with first-ever ischemic stroke in the anterior circulation within 24 h of onset were enrolled. We compared background characteristics, vital signs, laboratory data, and MRI findings between favorable (F) and unfavorable (U) outcome groups at 3 months, according to the modified Rankin Scale (mRS). The F and U groups were defined as having a mRS of 0-2 and 3-6, respectively. DWI ASPECTS was scored by DWI obtained 3-24 h after onset. Two-hundred and eighteen patients (62.3%) were classified into the F group and 132 patients (37.7%) into the U group. On univariate analysis, the F group patients were younger, had lower score of the National Institutes of Health Stroke Scale (NIHSS) at entry (5.7 ± 3.3 vs. 14.2 ± 6.0), male predominance, longer time after onset, lower rate of prior antithrombotic therapy, higher hematocrit and lower fibrinogen than the U group patients. Stroke subtype was different between the two groups, and F group patients had higher DWI ASPECTS score, lower leukoaraiosis and medial temporal atrophy score, and lower rate of early neurological deterioration (END) than the U group patients. Multiple logistic regression analysis revealed that NIHSS (p < 0.001), prior antithrombotic therapy (p = 0.013), ASPECTS (p = 0.002), and END (p < 0.001) were independent predictors of functional outcome. DWI ASPECTS can be an independent predictor for functional outcome, along with other clinical variables.
Subject(s)
Diffusion Magnetic Resonance Imaging/methods , Stroke/diagnosis , Stroke/physiopathology , Aged , Atrophy/pathology , Brain Mapping , Female , Humans , Image Processing, Computer-Assisted , Magnetic Resonance Imaging/methods , Male , Middle Aged , Middle Cerebral Artery/pathology , Prognosis , Retrospective Studies , Severity of Illness Index , Weights and MeasuresABSTRACT
There are few studies regarding functional outcome and lesion extent on diffusion-weighted MRI (DWI) in patients with posterior circulation (PC) infarction. The aim of our study was to assess whether a newly proposed posterior circulation Alberta Stroke Program Early CT Score (pc-ASPECTS) on DWI is useful for predicting functional outcome in PC patients. One hundred thirty-two patients with first-ever ischemic stroke in the posterior circulation within 24 h of onset who were admitted to our hospital were enrolled in the study. We compared background characteristics, vital signs, laboratory data, and MRI findings between favorable (F) and unfavorable (U) outcome groups at 3 months, according to the modified Rankin Scale (mRS). The F and U groups were defined as having a mRS of 0-2 and 3-6, respectively. pc-ASPECTS was scored by DWI obtained 12-36 h after onset. Ninety-eight patients (74.2%) were classified into the F group and 34 patients (25.8%) into the U group. On univariate analysis, F group patients were younger, had lower National Institutes of Health Stroke Scale (NIHSS) score at entry, and a lower rate of early neurological deterioration (END) and cardioembolic stroke than U group patients. On MRI, F group patients had lower leukoaraiosis and medial temporal atrophy score and higher pc-ASPECTS score on DWI compared to U group patients. Multiple logistic regression analysis revealed NIHSS (p < 0.001), END (p = 0.0057), pc-ASPECTS (p < 0.001), and leukoaraiosis (p = 0.0091) as independent predictors of functional outcome. pc-ASPECTS appears to be a powerful marker for predicting functional outcome, along with clinical severity and END. Leukoaraiosis may also be an independent predictor of functional outcome.
Subject(s)
Brain Ischemia/diagnosis , Brain Ischemia/physiopathology , Cerebrovascular Circulation , Stroke/diagnosis , Stroke/physiopathology , Age Factors , Aged , Analysis of Variance , Atrophy , Brain/pathology , Brain/physiopathology , Brain Ischemia/pathology , Diffusion Magnetic Resonance Imaging , Female , Humans , Leukoaraiosis/pathology , Logistic Models , Male , Middle Aged , Prognosis , Severity of Illness Index , Stroke/pathology , Temporal Lobe/pathology , Temporal Lobe/physiopathology , Time FactorsABSTRACT
Prediction of idiosyncratic drug-induced liver injury (DILI) is difficult, and the underlying mechanisms are not fully understood. However, many drugs causing DILI are considered to form reactive metabolites and covalently bind to cellular macromolecules in the liver. The objective of this study was to clarify whether the risk of idiosyncratic DILI can be estimated by comparing in vitro covalent binding (CB) levels among 12 positive compounds (acetaminophen, alpidem, bromfenac, carbamazepine, diclofenac, flutamide, imipramine, nefazodone, tacrine, ticlopidine, tienilic acid, and troglitazone) for DILI and 12 negative compounds (acetylsalicylic acid, caffeine, dexamethasone, losartan, ibuprofen, paroxetine, pioglitazone, rosiglitazone, sertraline, theophylline, venlafaxine, and zolpidem). After incubation with human liver microsomes in the presence of NADPH, there was a large overlap in the distribution of CB amounts between the positive and negative groups. On addition of UDP-glucuronic acid (UDPGA) as a cofactor for glucuronidation, the CB levels of bromfenac and diclofenac were increased. With addition of nucleophilic glutathione (GSH), values for most compounds were decreased. However, separation of the two groups on the basis of CB could not be improved by UDPGA or GSH. Furthermore, CB with human hepatocytes also failed to discriminate positive from negative compounds. Therefore, the CB amount alone is not sufficient for risk assessment of DILI. In contrast, when the CB amount was multiplied by the maximum daily dose, which may reflect maximum hepatic exposure, the two groups did become discriminated. Taken together, our findings suggest that the combination of CB amount and daily dose can estimate the risk of idiosyncratic DILI.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Liver/drug effects , Protein Binding , Chemical and Drug Induced Liver Injury/metabolism , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Glucuronic Acid/metabolism , Glucuronosyltransferase/metabolism , Glutathione/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver/metabolism , Metabolomics , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , NADP/metabolism , Reproducibility of Results , Risk Assessment , Risk FactorsABSTRACT
During the course of the study of UGT1A1 induction by bilirubin, we could not detect the induction of the reporter gene (-3174/+14) of human UGT1A1 in HepG2 by bilirubin (Mol. Biol. Rep. 31: 151-158 (2004)). In this report, we show the finding of the induction of the reporter gene of UGT1A1 by cortisol at 1 microM, a major natural cortico-steroid, with human glucocorticoid receptor (GR). RU486 of a typical GR antagonist at 10 microM inhibited the induction by cortisol from 5.9- to 1.8-fold. This result indicates that the induction by cortisol-GR is dependence on ligand-binding. This induction is caused by the UGT reporter gene itself, from the results of noinduction with control vector pGL2 (equal to pGV-C) in the presence of cortisol-GR. We confirmed that the induction of the reporter gene by cortisol is dependent on the position of proximal element (-97/-53) of UGT1A1. From this result, we concluded that the increase of corticosteroid in neonates must induce the elevation of UGT1A1 after birth and prevent jaundice. With the study of induction by corisol, we studied the influence of co-expression of PXR (pregnenolone xenobiotic receptor) with the UGT1A1 reporter gene and we could not find the induction of UGT1A1 expression in the presence of dexamethasone, rifampicin, or pregnenolone 16alpha-carbonitrile of the PXR ligands. These results suggest that the induction of UGT1A1 expression by GR is not mediated by PXR, unlike the induction of CYP3A4 through PXR.
Subject(s)
Glucocorticoids/pharmacology , Glucuronosyltransferase/metabolism , Hydrocortisone/pharmacology , Receptors, Glucocorticoid/antagonists & inhibitors , Base Sequence , Cell Line , Drug Antagonism , Glucuronosyltransferase/genetics , Humans , Mifepristone/pharmacology , Molecular Sequence Data , Pregnane X Receptor , Promoter Regions, Genetic , Receptors, Glucocorticoid/genetics , Receptors, Steroid/genetics , TransfectionABSTRACT
Previous study showed noinduction of the reporter gene (-3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by beta-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (-75/-63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [(32)P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe-protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.
Subject(s)
Glucuronosyltransferase/biosynthesis , Receptors, Glucocorticoid/physiology , Base Sequence , Cell Line , DNA Primers , Dexamethasone/pharmacology , Electrophoretic Mobility Shift Assay , Enzyme Induction , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/genetics , Humans , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Autoimmune hepatitis (AIH) is a disease of unknown etiology, characterized by liver-related autoantibodies. Autoimmune hepatitis is subdivided into two major types: AIH type 1 is characterized by the detection of ANA, SMA, ANCA, anti-ASGP-R, and anti-SLA/LP. Autoimmune hepatitis type 2 is characterized to be mainly related with drug-metabolizing enzymes as autoantigens, such as anti-LKM (liver-kidney microsomal antigen)-1 against CYP2D6, anti-LKM-2 against CYP2C9-tienilic acid, anti-LKM-3 against UGT1A, and anti-LC1 (liver cytosol antigen)-1 and anti-APS (autoimmune polyglandular syndrome type-1) against CYP1A2, CYP2A6, and others. Anti-LKM-1 sera inhibited CYP2D6 activity in vitro but did not inhibit cellular drug metabolism in vivo. CYP2D6 is the major target autoantigen of LKM-1 and expressed on plasma membrane (PM) of hepatocytes, suggesting a pathogenic role for anti-LKM-1 in liver injury as a trigger. Anti-CYP1A2 was observed in dihydralazine-induced hepatitis, and radiolabeled CYP1A2 disappeared from the PM with a half-life of less than 30 min, whereas microsomal CYP1A2 was stably radiolabeled for several hours. Main antigenic epitopes on CYP2D6 are aa 193-212, aa 257-269, and aa 321-351; and D263 is essential. The third epitope is located on the surface of the protein CYP2D6 and displays a hydrophobic patch that is situated between an aromatic residue (W316) and histidine (H326). Some drugs such as anticonvulsants (phenobarbital, phenytoin, and carbamazepine) and halothane are suggested to induce hepatitis with anti-CYP3A and anti-CYP2E1, respectively. Autoantibodies against CYP11A1, CYP17, and/or CYP21 involved in the synthesis of steroid hormones are also detected in patients with adrenal failure, gonadal failure, and/or Addison disease.
Subject(s)
Autoantibodies/immunology , Cytochrome P-450 CYP2D6/immunology , Hepatitis, Autoimmune/immunology , Animals , Cytochrome P-450 Enzyme System/immunology , Hepatitis, Autoimmune/enzymology , Humans , Inactivation, Metabolic/immunologyABSTRACT
Human UDP-glucuronosyltransferase (UGT) 1A1 is only enzyme in the conjugation of bilirubin for prevention of hyperbilirubinemia and jaundice. Deletion or mutation of the UGT1A1 gene causes Crigler-Najjar syndrome or Gilbert's syndrome. We previously reported the functional promoter region for expression of UGT1A1 [Hepatology Research 9, 152-163 (1997)]. We investigated the influence of some drugs on the transient transfection assay of the luciferase reporter gene containing the 5'-promoter region -3174/+14 of UGT1A1 in HepG2 cells. Among drugs investigated, dexamethasone was the most effective at the range of concentration of 10-100 microM, whereas stimulation by beta-estradiol was not found. We also could not find stimulation by bilirubin of the endogenous main substrate for UGT1A1. Stimulation by dexamethasone was continued for 48 hr. The luciferase reporter gene containing the 5'-region of -97/+14 was induced by dexamethasone but the gene of the 5'-region -53/+14 was not. The region -97/-53 is essential for induction by dexamethasone. This region contains HNF1 element, therefore, we speculated that dexamethasone directly and/or indirectly stimulates UGT1A1 expression through this HNF1 region in the promoter region of UGT1A1. Thus, we clarified that UGT1A1 was induced by dexamethasone and the key position was the region (-97/-53) in UGT1A1 promoter.
Subject(s)
Dexamethasone/pharmacology , Glucuronosyltransferase/biosynthesis , Transcription, Genetic , Base Sequence , Bilirubin/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Estradiol/metabolism , Exons , Gene Deletion , Genes, Reporter , Glucocorticoids/pharmacology , Humans , Luciferases/metabolism , Models, Genetic , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Time Factors , TransfectionABSTRACT
One of the Ser-tRNAs, Ser-tRNA(Sec), is converted to Sec-tRNA(Sec) by Sec synthase. This Ser-tRNA(Sec) is also converted to phosphoser-tRNA(Sec) by tRNA kinase. In this study, we analyzed of the products of phosphorylation with tRNA kinase. [3H]Ser-tRNA(Sec) purified on Sephacryl S-200 was phosphorylated with [gamma-32P]ATP by tRNA kinase. The product [32P][3H]phosphoser-tRNA was purified on Sephacryl S-200 and hydrolyzed with ribonuclease T2. The chromatogram of this hydrolyzate on DEAE-cellulose in 7 M urea buffer showed four peaks. The first peak of the pass-through fraction was seryl-adenosine liberated from the 3'-terminal of the tRNA. The second peak, eluted before the third peak containing inorganic phosphate, was phosphoseryl-adenosine. The major compound in the fourth peak was pGp. As a control experiment, non-acylated tRNA(Sec) was used as a substrate of phosphorylation and the product was analyzed. The chromatogram of the digest with ribonuclease T2 showed no peak of phosphoseryl-adenosine, but a peak of pGp was seen with the peak of inorganic phosphate. Thus, the major product in the presence of tRNA kinase was pGp, and a small but significant proportion of the radioactivity was found as phosphoserine in the presence of seryl residue on the 3'-CCA terminal of tRNA(Sec). These results indicated that tRNA kinase phosphorylates not only Ser-tRNA to phosphoser-tRNA but also Gp of the 5'-termini of tRNA to pGp. This study gives a new role to mammalian tRNA kinase.
Subject(s)
Guanosine Diphosphate/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Transfer, Ser/metabolism , Animals , Cattle , Chromatography , Phosphorus Radioisotopes , Phosphorylation , RNA, Transfer, Amino Acid-Specific/metabolism , RNA, Transfer, Amino Acyl/metabolism , Radioactive Tracers , TritiumABSTRACT
We analysed the predictive factors for relatively good prognosis in the patients with total anterior circulation infarction (TACI) defined by the Oxfordshire Community Stroke Project. One hundred patients with first-ever ischemic stroke within 6 hours of onset were enrolled. Outcomes were assessed at 3 months using the modified Rankin Scale (mRS), and rated as good if the mRS was 0 to 3, and as bad if the mRS was 4 or 5, or the patient was dead. These two groups were compared in terms of their clinical background, risk factors, vital signs, laboratory data, and cranial CT at the time of hospitalization and repeated CT at 24 to 48 hours after onset. There were 36 patients with good prognosis and 64 patients with bad prognosis. In univariate analysis, the patients with good prognosis were younger, had lower scores in the National Institutes of Health Stroke Scale (NIHSS, both total score and the score of consciousness sub-item), had fewer rates of deterioration within 7 days than the patients with bad prognosis. In the first cranial CT within 6 hours, the patients with good prognosis had fewer early CT signs including hyperdense middle cerebral artery sign, and brain swelling, as well as infrequent hypodensity more than 33% of total MCA territory, than the patients with bad prognosis. In the second CT during 24 to 48 hours, the patients with good prognosis had smaller lesions than the patients with bad prognosis. In multivariate analysis with logistic regression revealed that age < 70 years, low NIHSS score < or = 15, no deterioration in the acute phase, and small lesion on the second CT were independent predictors of good prognosis.