RESUMO
Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are key transcription factors for enzyme induction and lipid metabolism, respectively, in the liver. Treatment of mice with the CAR activator phenobarbital, an antiepileptic drug, increased plasma triglyceride levels and decreased the hepatic expression of PPARα target genes related to lipid metabolism. The increase in PPARα target gene expression induced by fenofibrate, a PPARα ligand, was inhibited by cotreatment with phenobarbital. CAR suppressed PPARα-dependent gene transcription in HepG2 cells but not in COS-1 cells. The mRNA level of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a coactivator for both CAR and PPARα, in COS-1 cells was much lower than in HepG2 cells. In reporter assays with COS-1 cells overexpressing PGC1α, CAR suppressed PPARα-dependent gene transcription, depending on the coactivator-binding motif. In mammalian two-hybrid assays, CAR attenuated the interaction between PGC1α and PPARα Chemical inhibition of PGC1α prevented phenobarbital-dependent increases in plasma triglyceride levels and the inhibition of PPARα target gene expression. These results suggest that CAR inhibits the interaction between PPARα and PGC1α, attenuating PPARα-dependent lipid metabolism. This might explain the antiepileptic drug-induced elevation of blood triglyceride levels. SIGNIFICANCE STATEMENT: Constitutive active/androstane receptor activated by antiepileptic drugs inhibits the peroxisome proliferator-activated receptor α-dependent transcription of genes related to lipid metabolism and upregulates blood triglyceride levels. The molecular mechanism of this inhibition involves competition between these nuclear receptors for coactivator peroxisome proliferator-activated receptor γ coactivator-1α binding.
Assuntos
Anticonvulsivantes/farmacologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Triglicerídeos/sangue , Animais , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Indução Enzimática/efeitos dos fármacos , Fenofibrato/farmacologia , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/farmacologia , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
To assess modification of thioacetamide-induced hepatotoxicity in mice fed a high-fat diet, male C57BL/6J mice were fed a normal rodent diet or a high-fat diet for 8 weeks and then treated once intraperitoneally with thioacetamide at 50 mg/kg body weight. At 24 and 48 hours after administration, massive centrilobular hepatocellular necrosis was observed in mice fed the normal rodent diet, while the necrosis was less severe in mice fed the high-fat diet. In contrast, severe swelling of hepatocytes was observed in mice fed the high-fat diet. In addition, mice fed the high-fat diet displayed more than a 4-fold higher number of BrdU-positive hepatocytes compared with mice fed the normal rodent diet at 48 hours after thioacetamide treatment. To clarify the mechanisms by which the hepatic necrosis was attenuated, we investigated exposure to thioacetamide and one of its metabolites, the expression of CYP2E1, which converts thioacetamide to reactive metabolites, and the content of glutathione S-transferases in the liver. However, the reduced hepatocellular necrosis noted in mice fed the high-fat diet could not be explained by the differences in exposure to thioacetamide or thioacetamide sulfoxide or by differences in the expression of drug-metabolizing enzymes. On the other hand, at 8 hours after thioacetamide administration, hepatic total glutathione in mice fed the high-fat diet was significantly lower than that in mice fed the normal diet. Hence, decreased hepatic glutathione amount is a candidate for the mechanism of the attenuated necrosis. In conclusion, this study revealed that thioacetamide-induced hepatic necrosis was attenuated in mice fed the high-fat diet.
RESUMO
We investigated the impact of glutathione transferases Mu 1 (GSTM1)- and glutathione transferase Theta 1 (GSTT1)-null genotypes on hepatic GST activities in humans and compared the results with those of Gstm1- and Gstt1-null mice. In liver with GSTM1/Gstm1-null genotype, GST activity toward p-nitrobenzyl chloride (NBC) was significantly decreased in both humans and mice. In addition, in liver with GSTT1/Gstt1-null genotype, GST activity toward dichloromethane (DCM) was significantly decreased in both humans and mice. Therefore, null genotypes of GSTM1/Gstm1 and GSTT1/Gstt1 are considered to decrease hepatic GST activities toward NBC and DCM, respectively, in both humans and mice. This observation shows the functional similarity between humans and mice for GSTM1 and GSTT1 toward some substrates. In the case of NBC and DCM, Gst-null mice would be relevant models for humans with GST-null genotype. In addition, decreases in GST activities toward 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one, and 1-chloro-2,4,-dinitrobenzene were observed in Gstm1-null mice, and a decrease in GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was observed in Gstt1-null mice. However, an impact of GST-null genotypes on GST activities toward these substrates was not observed in humans. In the case of these mouse-specific substrates, Gst-null mice may be relevant models for humans regardless of GST genotype, because GST activities, which are higher in wild-type mice than in humans, were eliminated in Gst-null mice. This study shows that comparison of hepatic GST activities between humans and mice using genotype information would be valuable in using Gst-null mice as human models.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Fígado/enzimologia , Animais , Feminino , Genótipo , Humanos , Fígado/efeitos dos fármacos , Masculino , Cloreto de Metileno/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Nitrobenzenos/farmacologia , Polimorfismo Genético , Especificidade por SubstratoRESUMO
A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.
Assuntos
Glutationa Transferase/genética , Metemoglobinemia/induzido quimicamente , Nitrobenzenos/toxicidade , Animais , Sequência de Bases , Primers do DNA , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sulfotransferases/metabolismo , Animais , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Humanos , Camundongos , Fenobarbital/metabolismo , Fosforilação , Sulfotransferases/genéticaRESUMO
Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/- mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/- mice. Although we conducted a repeated dose toxicity study in Sod2+/- mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/- mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/- mice. These results suggest that Sod2+/- mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.
Assuntos
Acetaminofen/farmacologia , Cromanos/farmacologia , Fígado/efeitos dos fármacos , Fígado/lesões , Superóxido Dismutase/genética , Tiazolidinedionas/farmacologia , Trifosfato de Adenosina/metabolismo , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Técnicas de Inativação de Genes/métodos , Hepatócitos/citologia , Hepatócitos/metabolismo , Heterozigoto , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Modelos Animais , Necrose/patologia , Sensibilidade e Especificidade , Fatores de Tempo , TroglitazonaRESUMO
Indium nitride (InN) luminescence is substantially enhanced by the introduction of a multilayer graphene interlayer, mitigating the lattice mismatch between the InN epilayer and the Gallium nitride (GaN) template on a sapphire substrate via weak van der Waals interaction between graphene and nitride layers. The InN epilayers are deposited by radio-frequency plasma-assisted molecular beam epitaxy (MBE), and are characterized by spatially-resolved photoluminescence spectroscopy using confocal microscopy. A small blue shift of the emission band from the band gap evidences a low density of equilibrium carriers, and a high quality of InN on multilayer graphene. A deposition temperature of ~375 °C is determined as optimal. The granularity, which is observed for the InN epilayers deposited on multilayer graphene, is shown to be eliminated, and the emission intensity is further enhanced by the introduction of an aluminum nitride (AlN) buffer layer between graphene and InN.
RESUMO
A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.
Assuntos
Fígado/metabolismo , Testes para Micronúcleos , Mutagênicos/toxicidade , Animais , Linhagem Celular , Cricetulus , Sistema Enzimático do Citocromo P-450/metabolismo , Masculino , Fenobarbital/farmacologia , Ratos Sprague-Dawley , beta-Naftoflavona/farmacologiaRESUMO
We previously reported that thioacetamide (TA)-induced hepatocellular necrosis was attenuated in mice fed a high-fat diet (HFD mice) compared with mice fed a normal rodent diet (ND mice). In this study, we investigated whether p38 mitogen-activated protein kinase (p38 MAPK) was involved in this attenuation. Western blot analysis revealed that hepatic phosphorylated p38 MAPK protein decreased at 8 and 24 hours (hr) after TA dosing in the HFD mice, while it decreased only at 24 hr in the ND mice in comparison to the time- and diet-matched, vehicle-treated mice. p38 MAPK regulates various biological functions including inflammation, therefore, hepatic metabolomics analysis focusing on pro-inflammatory lipid mediators was performed. At 24 hr after TA dosing, only one pro-inflammatory mediator, 12-hydroxyeicosatetraenoic acid (HETE), was higher in the HFD mice. On the other hand, in addition to 12-HETE, 15-HETE and 12-hydroxyeicosapentaenoic acid (HEPE) were higher and omega-3/omega-6 polyunsaturated fatty acids ratios were lower in the ND mice at 24 hr. These results of metabolomics indicated that less pro-inflammatory state was seen in HFD mice than in ND mice at 24 hr. Finally, to confirm whether the observed decrease in phosphorylated p38 MAPK could attenuate TA-induced hepatocellular necrosis, we showed that SB203580 hydrochloride, an inhibitor of p38 MAPK, partially attenuated TA-induced hepatic necrosis in ND mice. Collectively, these results suggest that a prompt decrease in phosphorylation of p38 MAPK after TA administration is one of the factors that attenuate TA-induced hepatic necrosis in HFD mice.
Assuntos
Dieta Hiperlipídica , Fígado/enzimologia , Necrose Hepática Massiva/induzido quimicamente , Necrose Hepática Massiva/terapia , Obesidade/etiologia , Tioacetamida/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Necrose Hepática Massiva/metabolismo , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/enzimologia , FosforilaçãoRESUMO
Comparison of 11 human nuclear receptor amino acid sequences revealed a conserved phosphorylation motif within their DNA-binding domains as an intramolecular signal that regulates proteolytic degradation. Nuclear receptors use this signal to either degrade or proscribe degradation through either the proteasome or nonproteasome pathways. A phosphomimetic farnesoid X receptor (FXR) S154D mutant neither bound to nor trans-activated an FXR-response element-driven reporter gene and was rapidly degraded in COS-1 cells. Ectopically expressed FXR had increased Ser154 phosphorylation in COS-1 cells after ligand treatment, and knock-down of the nuclear vaccinia-related kinase 1 (VRK1) greatly reduced this phosphorylation. FXR was phosphorylated at Ser154 in the nucleus of centrilobular hepatocytes only in ligand-treated mice. Thus, FXR Ser154 phosphorylation is a rheostat for activation and subsequent degradation that controls receptor levels and activity.
Assuntos
Fosforilação/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Serina/metabolismo , Motivos de Aminoácidos/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Células COS , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter/genética , Hepatócitos/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Receptores Citoplasmáticos e Nucleares/genética , Serina/genéticaRESUMO
PURPOSE: An anti-HER2 antibody-drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. EXPERIMENTAL DESIGN: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. RESULTS: DS-8201a exhibited a HER2 expression-dependent cell growth-inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1-insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. CONCLUSIONS: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1-insensitive HER2-positive cancers and low HER2-expressing cancers. Clin Cancer Res; 22(20); 5097-108. ©2016 AACR.
Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Camptotecina/análogos & derivados , Imunoconjugados , Neoplasias Pancreáticas/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Inibidores da Topoisomerase I/farmacologia , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Camptotecina/efeitos adversos , Camptotecina/farmacocinética , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Feminino , Histonas/metabolismo , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Macaca fascicularis , Maitansina/análogos & derivados , Maitansina/farmacologia , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor ErbB-2/imunologia , Trastuzumab/farmacologiaRESUMO
L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, was administered to mice via drinking water for 14 days in order to establish an animal model with continuously depleted levels of GSH. No toxicity was observed at 20 mM BSO, even though a significant decrease in liver weight was observed at 30 mM BSO. GSH levels in the liver, kidney, brain, lung, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, plasma and blood cells from mice given 20 mM of BSO were all less than those from the control mice continuously throughout a 24-hr period. The ratios of the GSH levels to that of the control were 46.4% and 16.7% in the liver and kidney, respectively, suggesting a decrease in GSH conjugation activity in vivo by GSH depletion. Liver cytochrome P450 content and UDP-glucuronosyltransferase activity to p-nitrophenol were not influenced by the BSO dosing. To confirm the adequacy of this GSH-depletion model, 0.125 or 0.25% of acetaminophen (APAP) was administered via diet to this model for 14 days. Nine out of the ten mice given both 20 mM BSO and 0.25% APAP died on Day 2, and remarkable necrosis was observed in the hepatocytes and renal tubular epithelium. Moreover, focal necrosis of hepatocytes with proliferation of fibroblasts was observed on Day 15 in some mice coadministered 20 mM BSO and 0.125% APAP. However, no toxicity was observed in mice given APAP alone. Based on these results, a mouse given 20 mM of BSO via drinking water for 14 days was concluded to be an animal model with continuously depleted levels of GSH in various organs without toxicity. This model shows high susceptibility to toxicity induced by chemicals which are metabolized to electrophilic and reactive metabolite(s), such as APAP.
Assuntos
Butionina Sulfoximina/toxicidade , Glutationa/deficiência , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Administração Oral , Alanina Transaminase/sangue , Animais , Butionina Sulfoximina/administração & dosagem , Creatinina/sangue , Sinergismo Farmacológico , Glutationa/análise , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Animais , Tamanho do Órgão/efeitos dos fármacos , Testes de Toxicidade , Água/administração & dosagemRESUMO
Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.
Assuntos
Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Conexinas/fisiologia , Acetaminofen/farmacocinética , Animais , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Conexinas/genética , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Testes de Função Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteína beta-1 de Junções ComunicantesRESUMO
INTRODUCTION: Glutathione S-transferases (GSTs) are Phase II drug-metabolizing enzymes that catalyze the conjugation of electrophilic compounds to glutathione. This reaction generally detoxifies reactive metabolites of xenobiotics, such as drugs and environmental chemicals, and therefore, GSTs are considered toxicologically important enzymes. Human GST genes display genetic polymorphisms, and the impact of null genotypes of GST Mu 1 (GSTM1) and GST Theta 1 (GSTT1) have been extensively studied. Recently, corresponding Gstm1- and Gstt1-null mice have been developed. AREAS COVERED: This review covers GSTM1- and GSTT1-null genotypes in humans and utilization of Gstm1- and Gstt1-null mice as animal models for absorption, distribution, metabolism, excretion and toxicity (ADMET) studies. Furthermore, the possibility of Gstm1- and Gstt1-null mice as models of humans with GSTM1- and GSTT1-null genotypes is discussed. EXPERT OPINION: The experimental approach to assess the impact of GSTM1- and GSTT1- null genotypes on ADMET is very limited so far. Gstm1- and Gstt1-null mice have potential as human models, since null genotypes of GSTM1/Gstm1 and GSTT1/Gstt1 decreased hepatic GST activities toward p-nitrobenzyl chloride and dichloromethane, respectively, in both humans and mice. This suggests functional similarity of GSTM1 and GSTT1 toward some substrates between humans and mice.
Assuntos
Glutationa Transferase/genética , Preparações Farmacêuticas/metabolismo , Animais , Genótipo , Humanos , Camundongos , Camundongos Knockout , Modelos Animais , Polimorfismo Genético , Especificidade da Espécie , Toxicologia/métodos , Xenobióticos/efeitos adversos , Xenobióticos/metabolismoRESUMO
We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3ß and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.
Assuntos
Acetaminofen/administração & dosagem , Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutationa Transferase/metabolismo , Fígado/efeitos dos fármacos , Administração Oral , Alanina Transaminase/sangue , Animais , Benzoquinonas/toxicidade , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Feminino , Glutationa/análise , Glutationa/metabolismo , Glutationa Transferase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Iminas/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/enzimologia , Fígado/patologia , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Knockout , FosforilaçãoRESUMO
The present study was designed to fully uncover sex and circadian modulatory effects on rat liver. Hepatic transcriptome analyses were performed at 4 hr intervals of a day-night cycle using young adult male and female rats. Sexually dimorphic genes, which were identified by a cross-sex comparison of time series data, included representative sex-predominant genes such as male- or female-predominant cytochrome P450 subfamilies (Cyp2c11, Cyp2c12, Cyp2c13, and Cyp3a2), sulfotransferases, and glutathione S-transferase Yc2. The identified sexually dimorphic genes were over-represented in the metabolism of retinols, xenobiotics, linoleic acids, or androgen and estrogen, or bile acid biosynthesis. Furthermore, transcription factor targets modeling suggested that transcription factors SP1, hepatocyte nuclear factor 4-alpha (HNF4-alpha), and signal transducer and activator of transcription 5b (STAT5b) serve as core nodes in the regulatory networks. On the other hand, Fourier transform analyses extracted universal circadian-regulated genes in both sexes. The circadian-regulated genes included clock or clock-controlled genes such as aryl hydrocarbon receptor nuclear translocator-like (Arntl), period homolog 2 (Per2), and D site albumin promoter binding protein (Dbp). The extracted cyclic genes were over-represented in major tissue activities, e.g. the urea cycle and the metabolism of amino acids, fatty acids, or glucose, indicating that the major liver functions are under circadian control. The transcription factor targets modeling suggested that transcription factors SP1, HNF4-alpha, and c-Myc proto-oncogene protein (c-MYC) serve as major hubs in the circadian-regulatory gene networks. Interestingly, transcription factors SP1 and HNF4-alpha are likely to orchestrate not only sexually dimorphic, but also circadian-regulated genes even though each criterion was rather mutually exclusive. This suggests the cross-talk between those regulations. Sexual dimorphism is likely to interact with circadian rhythmicity via overlapping gene regulatory networks on rat liver.
Assuntos
Ritmo Circadiano/genética , Perfilação da Expressão Gênica/métodos , Fígado/fisiologia , Caracteres Sexuais , Fatores de Transcrição ARNTL , Animais , Ritmo Circadiano/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA , Feminino , Glutationa Transferase/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Proteínas Circadianas Period , Proto-Oncogene Mas , Ratos , Ratos Endogâmicos F344 , Sulfotransferases/metabolismo , Fatores de TranscriçãoRESUMO
The secretory profile of growth hormone (GH) is sexually dimorphic in rats. In male transgenic (TG) rats expressing human GH (hGH) that we generated, the circulating levels of both hGH and endogenous GH are flattened with no male-type pulsatility. To elucidate the regulatory role of episodic GH profile on the liver, the hepatic transcriptome of male TG rats at the middle of the light and dark phases was characterized by genome-wide analyses as compared with that of male wild-type (WT) rats. Transcripts commonly up- or down-regulated regardless of the lighting conditions in TG rats were mainly enriched in the metabolism of xenobiotics. In TG rats, the gene expression profile was functionally feminized, verifying that the sexually dimorphic profile of GH rather than genetic sexuality is a stronger sex-determining factor on the hepatic transcriptome. The common transcripts which fluctuated during the day in both TG and WT rats were enriched in circadian rhythm signaling, and physiological rhythmicity was considered to be finely interconnected with liver metabolism via sexually dimorphic GH secretion. In contrast, some genes were differentially regulated in TG rats at only one of two time points measured, and others were fluctuated daily in only one genotype. In particular, some genes involved in the GH signaling pathway were included, suggesting the signal transduction is circadian-modulated depending upon the GH profile. Our transcriptome analyses clarified the regulatory role of episodic GH profile on the liver and strengthen the functional link between sexually dimorphic GH secretion, liver metabolism, and its circadian regulation.
Assuntos
Ritmo Circadiano , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônio do Crescimento Humano/biossíntese , Fígado/metabolismo , Animais , Ritmo Circadiano/genética , Glutationa Transferase/genética , Humanos , Immunoblotting , Fígado/enzimologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In order to characterize the hepatic effects of phenobarbital (PB) and clofibrate (CPIB) in dogs, PB and CPIB were administered to male beagle dogs for 14 days, and biochemical and histopathological examinations and comprehensive genomic and proteomic analyses, including GeneChip analysis and proteomics analysis using the 2-dimension difference gel electrophoresis (2D-DIGE) technique, were performed. Both compounds caused centrilobular hepatocellular hypertrophy, which were related to smooth endoplasmic reticulum (SER) proliferation in PB-treated dogs and to mitochondrial proliferation in CPIB-treated dogs. In the PB-treated dogs, drug-metabolizing enzyme induction was observed by Western blot and genomic analyses. CYP proteins could not be detected by the 2D-DIGE analysis, but increases in several endoplasmic reticulum (ER)-related proteins were observed. In the CPIB-treated dogs, drug-metabolizing enzyme induction was not clearly observed by any of Western blot, genomic and proteomic analyses. Genomic and proteomic analyses revealed that mitochondrial genes and proteins, including carnitine palmytoiltransferase II, acyl-CoA deheydrogenase and hydroxyacyl-CoA dehydrogenase, pyruvate carboxylase and ATP synthase beta chain were induced. There is a relatively good correlation among the morphology and the genomic and proteomic data, but some differences exist between the genomic and proteomic data. Comprehensive evaluation using these techniques in addition to morphological evaluation may provide a useful tool for safety assessment of the liver.
Assuntos
Clofibrato , Hepatomegalia/induzido quimicamente , Análise de Sequência com Séries de Oligonucleotídeos , Fenobarbital , Proteômica , Acil-CoA Desidrogenase/metabolismo , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Cães , Eletroforese em Gel Bidimensional , Retículo Endoplasmático Liso , Fígado/citologia , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias Hepáticas/genéticaRESUMO
Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC(0-24hr) based on free drug concentrations (3445 versus 844 microg.hr/ml) was observed in NAR when both rats showed the same level of AUC(0-24hr) based on the total drug concentrations (4436 versus 4237microg.hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed.
Assuntos
Clofibrato/farmacocinética , Clofibrato/toxicidade , Albumina Sérica/deficiência , Animais , Sistema Enzimático do Citocromo P-450/análise , Ácidos Graxos/metabolismo , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Albumina Sérica/análiseRESUMO
Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve(5-60) min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.