RESUMEN
Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.
Asunto(s)
Encéfalo/efectos de los fármacos , Creatinina/análogos & derivados , Pruebas de Toxicidad Crónica/métodos , Administración Oral , Animales , Encéfalo/metabolismo , Encéfalo/patología , Creatina/análogos & derivados , Creatina/sangre , Creatina/toxicidad , Creatinina/administración & dosificación , Creatinina/sangre , Creatinina/toxicidad , Femenino , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Ratas , Ratas Sprague-Dawley , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Factores de TiempoRESUMEN
Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50â¯mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3â¯h (overall median Tmax) across all doses with a mean t1/2 over 168â¯h of 55.5â¯h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27â¯at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25â¯mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmaxâ¯=â¯82.5â¯ng/mL and AUClast = 2521 h*ng/mL, on Day 27.
Asunto(s)
Antineoplásicos , Pirimidinas , Pirroles , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Perros , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/fisiología , Femenino , Células HEK293 , Humanos , Masculino , Nivel sin Efectos Adversos Observados , Neoplasias Pancreáticas/tratamiento farmacológico , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Pirimidinas/toxicidad , Pirroles/administración & dosificación , Pirroles/farmacocinética , Pirroles/toxicidadRESUMEN
Fx-5A peptide complex (Fx-5A), a High Density Lipoproteins (HDL) mimetic, has been shown to reduce atherosclerosis. The safety and toxicokinetics of Fx-5A administered IV by 30â¯min infusion at 8, 25 or 75â¯mg/kg body weight or vehicle, once every other day for 27 days, were assessed in cynomolgus monkeys. The Fx-5A was well tolerated at all doses. At the highest dose, there were statistically significant effects on hematology and clinical chemistry parameters that were considered non-adverse. Dose-dependent recoverable non-adverse erythrocytes morphological changes (acanthocytes, echinocytes, spherocytes, microcytes, and/or schistocytes) were observed. Fx-5A was not hemolytic in in-vitro fresh NHP or human blood assay. There were no Fx-5A-related statistically significant changes for any cardiovascular function, ECG or respiratory parameters, when compared to control. In addition, there were no Fx-5A-related effects on organ weights, macroscopic or microscopic endpoints. Finally, Fx-5A exhibited sporadic non-appreciable detection of anti-Fx-5A antibodies and a dose-dependent linear toxicokinetics with T1/2 value ranges from 2.7 to 6.2â¯h. In conclusion, the No Observed Adverse Effect Level was considered to be 75â¯mg/kg/day with associated exposures average Cmax and AUC0-last of 453⯵g/mL and 2232â¯h⯵g/mL, respectively, on Day 27.
Asunto(s)
Péptidos/farmacocinética , Péptidos/toxicidad , Esfingomielinas/farmacocinética , Esfingomielinas/toxicidad , Administración Intravenosa , Animales , Femenino , Péptidos y Proteínas de Señalización Intercelular , Lipoproteínas HDL , Macaca fascicularis , Masculino , Nivel sin Efectos Adversos Observados , Péptidos/sangreRESUMEN
Mouse models of acetaminophen (APAP) hepatotoxicity are considered relevant for the human pathophysiology. The C57BL/6 strain is most popular because it is the background strain of gene knock-out mice. However, conflicting results in the literature may have been caused by sub-strain mismatches, e.g. C57BL/6J and C57BL/6N. This study was initiated to determine the mechanism behind the sub-strain susceptibility to APAP toxicity. C57BL/6N and C57BL/6J mice were dosed with 200 mg/kg APAP and sacrificed at different time points. C57BL/6N mice developed significantly more liver injury as measured by plasma ALT activities and histology. Although there was no difference in glutathione depletion or cytochrome P450 activity between groups, C57BL/6N had a higher glutathione disulfide-to-glutathione ratio and more APAP protein adducts. C57BL/6N showed more mitochondrial translocation of phospho-JNK and BAX, and more release of mitochondrial intermembrane proteins apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), which caused more DNA fragmentation. The increased mitochondrial dysfunction was confirmed in vitro as C57BL/6N hepatocytes had a more precipitous drop in JC-1 fluorescence after APAP exposure. CONCLUSION: C57BL/6N mice are more susceptible to APAP-induced hepatotoxicity, likely due to increased formation of APAP-protein adducts and a subsequent enhancement of mitochondrial dysfunction associated with aggravated nuclear DNA fragmentation.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Western Blotting , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Susceptibilidad a Enfermedades , Glutatión/metabolismo , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/metabolismoRESUMEN
The inbred mouse strain C57BL/6J is widely used in models of immunological and infectious diseases. Here we show that C57BL/6J mice have a defect in neutrophil recruitment to a range of inflammatory stimuli compared with the related C57BL/6N substrain. This immune perturbation is associated with a missense mutation in Nlrp12 in C57BL/6J mice. Both C57BL/6J and NLRP12-deficient mice have increased susceptibility to bacterial infection that correlates with defective neutrophil migration. C57BL/6J and NLRP12-deficient macrophages have impaired CXCL1 production and the neutrophil defect observed in C57BL/6J and NLRP12-deficient mice is rescued by restoration of macrophage NLRP12. These results demonstrate that C57BL/6J mice have a functional defect in NLRP12 and that macrophages require NLRP12 expression for effective recruitment of neutrophils to inflammatory sites.
Asunto(s)
Quimiocina CXCL1/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/patología , Mutación , Neutrófilos/patología , Tularemia/inmunología , Animales , Secuencia de Bases , Movimiento Celular , Quimiocina CXCL1/deficiencia , Quimiocina CXCL1/inmunología , Susceptibilidad a Enfermedades , Francisella tularensis/inmunología , Expresión Génica , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Análisis de Supervivencia , Tularemia/genética , Tularemia/microbiología , Tularemia/mortalidadRESUMEN
UNLABELLED: Clinical evidence suggests that many cases of serious idiosyncratic drug-induced liver injury are mediated by the adaptive immune system in response to hepatic drug-protein adducts, also referred to as "drug-induced allergic hepatitis"; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug-induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane-induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty-four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b(+) Gr-1(high) cells were observed in the liver. Further study revealed a subpopulation of myeloid-derived suppressor cells within the CD11b(+) Gr-1(high) cell fraction that inhibited the proliferation of both CD4(+) and CD8(+) T cells. When CD11b(+) Gr-1(high) cells were depleted from the liver with Gr-1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin-4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD4(+) T cells, all features of an allergic reaction. When hepatic CD4(+) T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct-specific serum immunoglobulin and hepatotoxicity were reduced. CONCLUSION: Our data provide a rational approach for developing animal models of drug-induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease.
Asunto(s)
Antígeno CD11b/inmunología , Linfocitos T CD8-positivos/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Halotano/toxicidad , Hepatitis/inmunología , Células Mieloides/metabolismo , Alanina Transaminasa/metabolismo , Análisis de Varianza , Animales , Antígeno CD11b/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Hepatitis/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Células Mieloides/efectos de los fármacos , Óxido Nítrico/metabolismo , Distribución AleatoriaRESUMEN
UNLABELLED: Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A-induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1ß and tumor necrosis factor alpha. CONCLUSION: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug.
Asunto(s)
Anestésicos por Inhalación/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Citocinas/metabolismo , Halotano/efectos adversos , Interleucina-4/metabolismo , Animales , Concanavalina A , Femenino , Hepatitis Animal/metabolismo , Hepatocitos/metabolismo , Humanos , Ratones Endogámicos BALB C , Linfopoyetina del Estroma TímicoRESUMEN
UNLABELLED: Drug-induced liver injury (DILI) is a major health issue, as it remains difficult to predict which new drugs will cause injury and who will be susceptible to this disease. This is due in part to the lack of animal models and knowledge of susceptibility factors that predispose individuals to DILI. In this regard, liver eosinophilia has often been associated with DILI, although its role remains unclear. We decided to investigate this problem in a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected by flow cytometry in the liver within 12 hours and increased thereafter proportionally to liver damage. Chemokines, eotaxin-1 (CCL11) and eotaxin-2 (CCL24), which are known to attract eosinophils, increased in response to halothane treatment. The severity of HILI was decreased significantly when the study was repeated in wildtype mice made deficient in eosinophils with a depleting antibody and in eosinophil lineage-ablated ΔdblGata(-/-) mice. Moreover, depletion of neutrophils by pretreating animals with Gr-1 antibody prior to halothane administration failed to reduce the severity of HILI at antibody concentrations that did not affect hepatic eosinophils. Immunohistochemical staining for the granule protein, major basic protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis. CONCLUSION: Our findings indicate that eosinophils have a pathologic role in HILI in mice and suggest that they may contribute similarly in many clinical cases of DILI.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Eosinófilos/fisiología , Halotano/efectos adversos , Animales , Movimiento Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Quimiocina CCL11/metabolismo , Quimiocina CCL24/metabolismo , Comorbilidad , Modelos Animales de Enfermedad , Eosinofilia/epidemiología , Eosinófilos/patología , Femenino , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , PrevalenciaRESUMEN
In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4(-/-)) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4(-/-) mice was not due to elevated levels of hepatic APAP-protein adducts but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4(-/-) mice postacetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4(-/-) mice appeared to contribute toward increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK) since levels of phosphorylated JNK remained significantly higher in the IL-4(-/-) mice up to 24 h after APAP treatment. Overall, these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans but also in pathologies of the liver caused by other drugs and etiologies.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Glutatión/metabolismo , Interleucina-4/metabolismo , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocromo P-450 CYP2E1/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Interleucina-4/deficiencia , Interleucina-4/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
Acetaminophen/paracetamol-induced liver failure--which is induced by the binding of reactive metabolites to mitochondrial proteins and their disruption--is exacerbated by fasting. As fasting promotes SIRT3-mediated mitochondrial-protein deacetylation and acetaminophen metabolites bind to lysine residues, we investigated whether deacetylation predisposes mice to toxic metabolite-mediated disruption of mitochondrial proteins. We show that mitochondrial deacetylase SIRT3(-/-) mice are protected from acetaminophen hepatotoxicity, that mitochondrial aldehyde dehydrogenase 2 is a direct SIRT3 substrate, and that its deacetylation increases acetaminophen toxic-metabolite binding and enzyme inactivation. Thus, protein deacetylation enhances xenobiotic liver injury by modulating the binding of a toxic metabolite to mitochondrial proteins.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismo , Acetilación , Alanina Transaminasa/sangre , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Animales , Benzoquinonas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Iminas/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Unión ProteicaRESUMEN
C57BL/6 mice are widely used in biomedical research for the background of genetically engineered mice (GEM) and wild-type controls with the belief that the genetic background of GEM and control mice differ significantly by only one or more altered genes. This principle, however, does have limitations due in part to the existence of multiple substrains of C57BL/6 mice that should not be used interchangeably as they can differ both genetically and phenotypically. We show here that these mispairings do occur frequently and can lead to inaccurate and conflicting findings.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Concanavalina A/toxicidad , Ratones Endogámicos C57BL/genética , Ratones Transgénicos/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Mitógenos/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Técnicas de Silenciamiento del Gen , Genotipo , Ratones , Ratones NoqueadosRESUMEN
UNLABELLED: We previously reported that acetaminophen (APAP)-induced liver injury (AILI) in mice is associated with a rise in serum levels of the glucocorticoid (GC), corticosterone. In the current study, we provide evidence that endogenous GC play a pathologic role in AILI. Specifically, pretreatment of mice with the GC receptor (GCR) inhibitor, RU486 (mifepristrone), protected normal but not adrenalectomized mice from AILI, while pretreatment with dexamethasone, a synthetic GC, exacerbated AILI. RU486 did not affect the depletion of whole liver reduced GSH or the formation of APAP-protein adducts. It also had no effects on the formation of reactive oxygen species or the depletion of mitochondrial GSH or ATP. While RU486 pretreatment also protected against halothane-induced liver injury, it exacerbated concanavalin A (ConA)- and carbon tetrachloride (CCl(4))-induced liver injury, demonstrating the complexity of GC effects in different types of liver injury. CONCLUSION: These results suggest that under certain conditions, elevated levels of GC might represent a previously unappreciated risk factor for liver injury caused by APAP and other drugs through the diverse biological processes regulated by GCR.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Glucocorticoides/metabolismo , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Corticosterona/metabolismo , Dexametasona/metabolismo , Femenino , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Mifepristona/farmacología , Receptores de Glucocorticoides/antagonistas & inhibidores , Estrés FisiológicoRESUMEN
Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia. Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding, and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response, which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with calreticulin and calnexin in protein glycosylation. In this study, we found hyperoxia downregulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 small interfering RNA significantly knocked down ERp57 expression and reduced hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8 to 9.9% in hyperoxia-exposed cells and from 37.8 to 5.0% in tunicamycin-treated cells. The activation of caspase-3 induced by hyperoxia or tunicamycin was diminished and immunoglobulin heavy chain-binding protein/glucose-regulated protein 78-kDa (BiP/GRP78) induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1 to 14.3% in hyperoxia-exposed cells and from 14.0 to 21.2% in tunicamycin-treated cells. Overexpression of ERp57 also augmented tunicamycin-induced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxia- or tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction.
Asunto(s)
Citoprotección/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/metabolismo , Hiperoxia/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Tunicamicina/farmacología , Animales , Animales Recién Nacidos , Western Blotting , Caspasa 3/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Hiperoxia/patología , RatasRESUMEN
Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2(-/-) mice were treated with 300mg APAP/kg, 90% of JNK2(-/-) mice died of ALF compared to 20% of WT mice within 48h. The high susceptibility of JNK2(-/-) mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/enzimología , Hígado/efectos de los fármacos , Proteína Quinasa 9 Activada por Mitógenos/fisiología , Animales , Ciclina D , Ciclinas/metabolismo , Hígado/enzimología , Hígado/patología , Fallo Hepático Agudo/genética , Regeneración Hepática/genética , Masculino , Ratones , Ratones Mutantes , Proteína Quinasa 9 Activada por Mitógenos/genéticaRESUMEN
Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-gamma, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Interleucina-13/sangre , Acetaminofén/metabolismo , Alanina Transaminasa/sangre , Analgésicos no Narcóticos/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Silenciador del Gen , Glutatión/metabolismo , Interleucina-13/deficiencia , Interleucina-13/inmunología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patologíaRESUMEN
Drug-induced hepatitis remains a challenging problem for drug development and safety because of the lack of animal models. In the current work, we discovered a unique interaction that makes mice deficient in both IL-10 and IL-4 (IL-10/4-/-) highly sensitive to the hepatotoxic effects of acetaminophen (APAP). Male C57Bl/6 wild type (WT) and mice deficient in one or more cytokines were treated with 120 mg/kg APAP. Within 24 h after WT, IL-10-/-, IL-4-/-, or IL-10/4-/- mice were administered APAP, 75% of the IL-10/4-/- mice died of massive hepatic injury while all other genotypes were resistant to liver toxicity at this dose of APAP. The unique susceptibility of IL-10/4-/- mice was associated with reduced levels of liver glutathione and remarkably high serum levels of IL-6 and several proinflammatory factors including TNF-alpha, IFN-gamma, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and osteopontin (OPN) as well as nitric oxide (NO). IL-6 appeared to have a causal role in controlling the unique susceptibility of IL-10/4-/- mice to APAP-induced liver disease (AILD) because IL-6 neutralizing antibody reversed the high sensitivity of these mice to AILD. Moreover, IL-10/4/6-/- mice were also resistant to the enhanced susceptibility to AILD and expressed relatively low levels of most proinflammatory factor genes that were elevated in the IL-10/4-/- mice. In conclusion, liver homeostasis following AILD appears to be highly dependent on the activities of both IL-10 and IL-4, which together help prevent overexpression of IL-6 and other potential hepatotoxic factors.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Modelos Animales de Enfermedad , Interleucina-10/deficiencia , Interleucina-4/deficiencia , Interleucina-6/fisiología , Acetaminofén/administración & dosificación , Animales , Anticuerpos/farmacología , Arginasa/antagonistas & inhibidores , Arginasa/biosíntesis , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Predisposición Genética a la Enfermedad , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Interferones/biosíntesis , Interleucina-10/genética , Interleucina-4/genética , Interleucina-6/antagonistas & inhibidores , Interleucina-6/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Óxido Nítrico/biosíntesis , Óxido Nítrico/sangreRESUMEN
Drug-induced liver disease (DILD) continues to cause significant morbidity and mortality and impair new drug development. Mounting evidence suggests that DILD is a complex, multifactorial disease in which no one factor is likely to be an absolute indicator of susceptibility. As an approach to better understand the multifactorial basis of DILD, we recently compared the hepatic proteomes of mice that were resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD) wherein we identified potential risk factors and mechanistic pathways responsible for DILD. In this study, we have uncovered additional potential risk factors by comparing hepatic mRNA expression profiles of the same two strains of mice with that of SJLxB6-F1 hybrid (F1) mice, which were found to be of intermediate susceptibility to AILD. Global hepatic gene expression profiling over a 24 h period following APAP treatment revealed elevated patterns in the mRNA expression of cytoprotective genes in resistant SJL mice as compared to susceptible B6 mice, while F1 mice had intermediate mRNA expression levels of these genes. One of these genes encoded for heat shock protein (HSP) 70 whose relative protein expression among the three strains of mice was found to parallel that of their mRNA levels, suggesting that this protein had a protective role against AILD. However, there was no difference in the susceptibility of HSP70 knockout (KO) mice to AILD as compared to wild-type (WT) mice. There were also protoxicant genes, such as osteopontin (OPN), with elevated mRNA expression levels in the B6 mice as compared to the SJL mice and with intermediate levels in the F1 mice, suggesting that they may play a role in exacerbating liver injury after APAP treatment. In support of this hypothesis, OPN KO mice were found to be more resistant to AILD than WT mice. Additionally, the results from both the proteomic and the genomic studies were compared. The two approaches were found to be complementary to each other and not simply overlapping. Our findings suggest that comparative gene expression analysis of susceptible and resistant mouse strains may lead to the identification of factors that could have a role in determining the susceptibility of individuals to DILD.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas , Predisposición Genética a la Enfermedad , Hepatopatías/genética , Acetaminofén/química , Animales , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/deficiencia , Inyecciones Intraperitoneales , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Osteopontina , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factores de Riesgo , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Factores de TiempoRESUMEN
Recent experimental data suggest that the idiosyncratic nature of drug-induced liver disease (DILD) may be due in part to a deficiency of one or more hepatoprotective factors. In this study we have investigated whether interleukin (IL)-6 may also be one of these factors. Following the induction of liver injury with acetaminophen (APAP), a time-dependent increase in liver mRNA expression of IL-6 and its family members IL-11, leukemia inhibitory factor, and oncostatin M was observed in wild type (WT) mice, suggesting a possible hepatoprotective role played by this cytokine family. Indeed, mice lacking IL-6 (IL-6-/-) were more susceptible than were WT mice to APAP-induced liver injury. The increased susceptibility of the IL-6-/- mice was associated with a deficiency in the expression of hepatic heat shock protein (HSP)25, 32, and 40 as well as inducible HSP70 following APAP treatment. These results suggest that IL-6 and possibly other family members may protect the liver from injury, at least in part, by up-regulating the hepatic expression of several cytoprotective HSPs.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Proteínas de Choque Térmico/metabolismo , Interleucina-6/fisiología , Hígado/metabolismo , Acetaminofén , Animales , Citoprotección , Expresión Génica , Mediadores de Inflamación/metabolismo , Interleucina-6/genética , Cinética , Hepatopatías/genética , Hepatopatías/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/biosíntesisRESUMEN
Hepatic injury induced by various toxic agents, including acetaminophen (APAP), has been attributed, in part, to the production of proinflammatory cytokines and other mediators by resident Kupffer cells within the liver. However, recent evidence from our laboratory has demonstrated that hepato-protective factors, such as interleukin (IL)-10 and cyclooxygenase-derived mediators, are also upregulated in response to hepatic damage to help protect against exacerbated injury, and Kupffer cells have been suggested to be a source of these modulatory factors. In other models, Kupffer cells also serve important regulatory functions in pathophysiological states of the liver. Therefore, we reevaluated the role of Kupffer cells in a murine model of APAP-induced liver injury using liposome-entrapped clodronate (liposome/clodronate) as an effective Kupffer cell-depleting agent. We show that in contrast to pretreatment of mice with a widely used macrophage inhibitor, gadolinium chloride, which did not deplete Kupffer cells but moderately protected against APAP-induced hepatotoxicity as reported previously, the intravenous injection of liposome/clodronate caused nearly complete elimination of Kupffer cells and significantly increased susceptibility to APAP-induced liver injury as compared with mice pretreated with empty liposomes. This increased susceptibility was apparently unrelated to the metabolism of APAP since liposome/clodronate pretreatment did not alter APAP-protein adduct levels. Instead, Kupffer cell depletion by liposome/clodronate led to significant decreases in the levels of hepatic mRNA expression of several hepato-regulatory cytokines and mediators, including IL-6, IL-10, IL-18 binding protein and complement 1q, suggesting that Kupffer cells are a significant source for production of these mediators in this model. Our findings indicate that, in addition to their protoxicant activities, Kupffer cells can also have an important protective function in the liver through the production of a variety of modulatory factors which may counteract inflammatory responses and/or stimulate liver regeneration.