RESUMEN
ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be tumor suppressive. In some hepatocellular carcinoma patients, ARID1A was highly expressed in primary tumors but not in metastatic lesions, suggesting that ARID1A can be lost after initiation. Mice with liver-specific homozygous or heterozygous Arid1a loss were resistant to tumor initiation while ARID1A overexpression accelerated initiation. In contrast, homozygous or heterozygous Arid1a loss in established tumors accelerated progression and metastasis. Mechanistically, gain of Arid1a function promoted initiation by increasing CYP450-mediated oxidative stress, while loss of Arid1a within tumors decreased chromatin accessibility and reduced transcription of genes associated with migration, invasion, and metastasis. In summary, ARID1A has context-dependent tumor-suppressive and oncogenic roles in cancer.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas de Unión al ADN/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Oncogenes/genética , Animales , Western Blotting , Carcinogénesis/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Interferencia de ARN , Factores de TranscripciónRESUMEN
MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3'UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3'UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.
Asunto(s)
Acetaminofén/toxicidad , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A/genética , Sobredosis de Droga/metabolismo , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Adolescente , Sitios de Unión , Línea Celular , Niño , Preescolar , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sobredosis de Droga/sangre , Sobredosis de Droga/etiología , Sobredosis de Droga/genética , Femenino , Hepatocitos , Humanos , Masculino , MicroARNs/sangre , Biosíntesis de Proteínas/genética , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: A rapid and reliable point-of-care assay to detect acetaminophen protein adducts in the serum of patients with acute liver injury could improve diagnosis and management. AcetaSTAT is a competitive immunoassay used to measure acetaminophen protein adducts formed by toxic metabolites in serum samples from patients. We compared the accuracy of AcetaSTAT vs high-pressure liquid chromatography with electrochemical detection (HPLC-EC; a sensitive and specific quantitative analytic assay) to detect acetaminophen protein adducts. METHODS: We collected serum samples from 19 healthy individuals (no liver injury, no recent acetaminophen use), 29 patients without acetaminophen-associated acute liver injury, and 33 patients with acetaminophen-associated acute liver injury participating in the Acute Liver Failure Study Group registry. Each serum sample was analyzed by AcetaSTAT (reported as test band amplitude) and HPLC-EC (the reference standard). We also collected data on patient age, sex, weight, level of alanine aminotransferase on test day and peak values, concentration of acetaminophen, diagnoses (by site investigator and causality review committee), and outcome after 21 days. Differences between groups were analyzed using the Fisher exact test for categoric variables and the Kruskal-Wallis test or rank-sum test for continuous variables. RESULTS: AcetaSTAT discriminated between patients with and without acetaminophen-associated acute liver injury; the median AcetaSTAT test band amplitude for patients with acetaminophen-associated acute liver injury was 584 (range, 222-1027) vs 3678 (range, 394-8289) for those without (P < .001). AcetaSTAT identified patients with acetaminophen-associated acute liver injury with 100% sensitivity, 86.2% specificity, a positive predictive value of 89.2%, and a negative predictive value of 100%. Results from AcetaSTAT were positive in 4 subjects who received a causality review committee diagnosis of non-acetaminophen-associated acute liver injury; HPLC-EC and biochemical profiles were consistent with acetaminophen-associated acute liver injury in 3 of these cases. CONCLUSIONS: The competitive immunoassay AcetaSTAT shows a high degree of concordance with HPLC-EC results in identifying patients with acetaminophen-associated acute liver injury. This rapid and simple assay could increase early detection of this disorder and aid clinical management.
Asunto(s)
Acetaminofén/análisis , Inmunoensayo/métodos , Fallo Hepático Agudo/diagnóstico , Hígado/fisiopatología , Proteínas/química , Suero/química , Adulto , Anciano , Cromatografía Líquida de Alta Presión/métodos , Técnicas Electroquímicas/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Atención de Punto , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Phospholipids are an important class of lipids that act as building blocks of biological cell membranes and participate in a variety of vital cellular functions including cell signaling. Previous studies have reported alterations in phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) metabolism in acetaminophen (APAP)-treated animals or cell cultures. However, little is known about phospholipid perturbations in humans with APAP toxicity. In the current study, targeted metabolomic analysis of 180 different metabolites including 14 lysoPCs and 73 PCs was performed in serum samples from children and adolescents hospitalized for APAP overdose. Metabolite profiles in the overdose group were compared to those of healthy controls and hospitalized children receiving low dose APAP for treatment of pain or fever (therapeutic group). PCs and lysoPCs with very long chain fatty acids (VLCFAs) were significantly decreased in the overdose group, while those with comparatively shorter chain lengths were increased in the overdose group compared to the therapeutic and control groups. All ether linked PCs were decreased in the overdose group compared to the controls. LysoPC-C26:1 was highly reduced in the overdose group and could discriminate between the overdose and control groups with 100% sensitivity and specificity. The PCs and lysoPCs with VLCFAs showed significant associations with changes in clinical indicators of drug metabolism (APAP protein adducts) and liver injury (alanine aminotransferase, or ALT). Thus, a structure-dependent reduction in PCs and lysoPCs was observed in the APAP-overdose group, which may suggest a structure-activity relationship in inhibition of enzymes involved in phospholipid metabolism in APAP toxicity.
RESUMEN
3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Hepatocitos/efectos de los fármacos , Animales , Cromatografía Líquida de Alta Presión , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , NAD/efectos de los fármacos , NAD/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of acetaminophen oxidation revealed a biphasic kinetic profile that was best described by negative cooperativity (Hill coefficient=0.72). The best-fit mechanism for this relationship involved two binding sites with differing affinities (Ks=830µM and Kss=32mM). Introduction of styrene inhibited that reaction less than predicted by simple competition and thus provided evidence for a cooperative mechanism within the mixture. Likewise, acetaminophen acted through a mixed-type inhibition mechanism to impact styrene epoxidation. In this case, acetaminophen competed with styrene for CYP2E1 (Ki=830µM and Ksi=180µM for catalytic and effector sites, respectively) and resulted in cooperative impacts on binding and catalysis. Based on modeling of in vivo clearance, cooperative interactions between acetaminophen and styrene resulted in profoundly increased styrene activation at low styrene exposure levels and therapeutic acetaminophen levels. Current Michaelis-Menten based toxicological models for mixtures such as styrene and acetaminophen would fail to detect this concentration-dependent relationship. Hence, future studies must assess the role of alternate CYP2E1 mechanisms in bioactivation of compounds to improve the accuracy of interpretations and predictions of toxicity.
Asunto(s)
Acetaminofén/metabolismo , Inhibidores del Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Contaminantes Ambientales/metabolismo , Microsomas Hepáticos/enzimología , Estireno/metabolismo , Acetaminofén/química , Acetaminofén/toxicidad , Sitios de Unión , Unión Competitiva , Biotransformación , Inhibidores del Citocromo P-450 CYP2E1/química , Inhibidores del Citocromo P-450 CYP2E1/toxicidad , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Humanos , Técnicas In Vitro , Cinética , Microsomas Hepáticos/efectos de los fármacos , Modelos Biológicos , Modelos Químicos , Oxidación-Reducción , Estireno/química , Estireno/toxicidad , Especificidad por SustratoRESUMEN
Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.
Asunto(s)
Acetaminofén/envenenamiento , Ácidos y Sales Biliares/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Sobredosis de Droga/sangre , Acetaminofén/metabolismo , Adolescente , Alanina Transaminasa/metabolismo , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Niño , Preescolar , Diagnóstico Diferencial , Sobredosis de Droga/diagnóstico , Femenino , Ácido Glicoquenodesoxicólico/sangre , Ácido Glicodesoxicólico/sangre , Homeostasis , Humanos , Masculino , Metabolómica/métodos , Unión Proteica , Sensibilidad y Especificidad , Ácido Taurodesoxicólico/sangreRESUMEN
Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (â¦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ⦠5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.
Asunto(s)
Acetaminofén/metabolismo , Acetaminofén/toxicidad , Sistema Enzimático del Citocromo P-450 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Analgésicos no Narcóticos/metabolismo , Analgésicos no Narcóticos/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B(-/-) mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver.
Asunto(s)
Acetaminofén/química , Hepatocitos/efectos de los fármacos , Insulina/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Animales , Línea Celular , Supervivencia Celular , Silenciador del Gen , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Glutatión Transferasa/metabolismo , Hepatocitos/citología , Homeostasis , Humanos , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Rosiglitazona , Transducción de Señal , Suramina/química , Tiazolidinedionas/químicaRESUMEN
AIM: Long-chain acylcarnitines have been postulated to be sensitive biomarkers of acetaminophen (APAP)-induced hepatotoxicity in mouse models. In the following study, the relationship of acylcarnitines with other known indicators of APAP toxicity was examined in children receiving low-dose (therapeutic) and high-dose ('overdose' or toxic ingestion) exposure to APAP. MATERIALS & METHODS: The study included three subject groups: group A (therapeutic dose, n = 187); group B (healthy controls, n = 23); and group C (overdose, n = 62). Demographic, clinical and laboratory data were collected for each subject. Serum samples were used for measurement of APAP protein adducts, a biomarker of the oxidative metabolism of APAP and for targeted metabolomics analysis of serum acylcarnitines using ultra performance liquid chromatography-triple-quadrupole mass spectrometry. RESULTS: Significant increases in oleoyl- and palmitoyl-carnitines were observed with APAP exposure (low dose and overdose) compared with controls. Significant increases in serum ALT, APAP protein adducts and acylcarnitines were observed in overdose children that received delayed treatment (time to treatment from overdose >24 h) with the antidote N-acetylcysteine. Time to peak APAP protein adducts in serum was shorter than that of the acylcarnitines and serum ALT. CONCLUSION: Perturbations in long-chain acylcarnitines in children with APAP toxicity suggest that mitochrondrial injury and associated impairment in the ß-oxidation of fatty acids are clinically relevant as biomarkers of APAP toxicity.
Asunto(s)
Acetaminofén/efectos adversos , Carnitina/análogos & derivados , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Acetilcisteína/uso terapéutico , Adolescente , Factores de Edad , Alanina Transaminasa/sangre , Biomarcadores/sangre , Carnitina/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Niño , Preescolar , Análisis Discriminante , Femenino , Humanos , Análisis de los Mínimos Cuadrados , Masculino , Metabolómica , Factores SexualesRESUMEN
PURPOSE: Acetaminophen (APAP) protein adducts are a biomarker of APAP metabolism, reflecting oxidation of APAP and generation of the reactive metabolite N-acetyl-p-benzoquinone imine. High levels of adducts correspond to liver toxicity in patients with APAP-related acute liver failure. Adduct formation following low-dose exposure to APAP has not been well studied. APAP protein adducts were measured in blood samples collected from fasted individuals who participated in a crossover study of APAP (80 mg/kg) comparing extended release (ER) and immediate release (IR) formulations. METHODS: Adducts were quantified in all postdose blood samples using a validated high-performance liquid chromatography electrochemical detection (HPLC-EC) assay. RESULTS: Comparison of pharmacokinetic parameters for adducts did not reveal significant differences between ER and IR formulations, with one exception. Formation rates for adducts were faster for IR than the ER formulation (0.420 ± 0.157 vs. 0.203 ± 0.080 1/h), respectively. Maximum plasma concentrations (Cmax) of adducts for IR and ER were 0.108 (±0.020) and 0.100 (±0.028) nmol/ml serum, respectively, and were two orders of magnitude lower than adduct levels previously reported in adults with acute liver failure secondary to APAP. CONCLUSIONS: APAP protein adducts are rapidly formed following nontoxic ingestion of APAP at levels significantly lower than those associated with acute liver failure.
Asunto(s)
Acetaminofén/análogos & derivados , Acetaminofén/sangre , Analgésicos no Narcóticos/sangre , Proteínas/metabolismo , Acetaminofén/administración & dosificación , Acetaminofén/química , Adolescente , Adulto , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/química , Cromatografía Líquida de Alta Presión , Estudios Cruzados , Cisteína/metabolismo , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Ayuno , Femenino , Humanos , Masculino , Unión Proteica , Adulto JovenRESUMEN
In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.
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Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo I/fisiología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Western Blotting , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cisteína/metabolismo , Citoplasma/enzimología , Citoplasma/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Superóxido Dismutasa/metabolismoRESUMEN
HIF-1α is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1α. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1α in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1α in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48h and no staining was observed in centrilobular hepatocytes, the sites of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10mg/kg) reduced HIF-1α induction in APAP treated mice at 1 and 4h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1α induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism.
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Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Hipoxia/inducido químicamente , Estrés Oxidativo/fisiología , Acetaminofén/antagonistas & inhibidores , Alanina Transaminasa/sangre , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Hipoxia/metabolismo , Inmunohistoquímica , Inmunosupresores/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Nitroimidazoles/farmacología , Estadísticas no ParamétricasRESUMEN
Standard assays to assess acetaminophen (APAP) toxicity in animal models include determination of ALT (alanine aminotransferase) levels and examination of histopathology of liver sections. However, these assays do not reflect the functional capacity of the injured liver. To examine a functional marker of liver injury, the pharmacokinetics of indocyanine green (ICG) were examined in mice treated with APAP, saline, or APAP followed by N-acetylcysteine (NAC) treatment.Male B6C3F1 mice were administered APAP (200 mg/kg IP) or saline. Two additional groups of mice received APAP followed by NAC at 1 or 4 h after APAP. At 24 h, mice were injected with ICG (10 mg/kg IV) and serial blood samples (0, 2, 10, 30, 50 and 75 min) were obtained for determination of serum ICG concentrations and ALT. Mouse livers were removed for measurement of APAP protein adducts and examination of histopathology. Toxicity (ALT values and histology) was significantly increased above saline treated mice in the APAP and APAP/NAC 4 h mice. Mice treated with APAP/NAC 1 h had complete protection from toxicity. APAP protein adducts were increased in all APAP treated groups and were highest in the APAP/NAC 1 h group. Pharmacokinetic analysis of ICG demonstrated that the total body clearance (Cl(T)) of ICG was significantly decreased and the mean residence time (MRT) was significantly increased in the APAP mice compared to the saline mice. Mice treated with NAC at 1 h had Cl(T) and MRT values similar to those of saline treated mice. Conversely, mice that received NAC at 4 h had a similar ICG pharmacokinetic profile to that of the APAP only mice. Prompt treatment with NAC prevented loss of functional activity while late treatment with NAC offered no improvement in ICG clearance at 24 h. ICG clearance in mice with APAP toxicity can be utilized in future studies testing the effects of novel treatments for APAP toxicity.
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Acetaminofén/toxicidad , Acetilcisteína/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Modelos Animales de Enfermedad , Verde de Indocianina , Hígado/efectos de los fármacos , Acetaminofén/administración & dosificación , Acetaminofén/farmacocinética , Acetilcisteína/farmacocinética , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Animales , Antídotos/farmacocinética , Antídotos/uso terapéutico , Colorantes/farmacocinética , Verde de Indocianina/farmacocinética , Inyecciones Intravenosas , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos , Factores de TiempoRESUMEN
Acetaminophen (APAP)-induced liver toxicity occurs with formation of APAP-protein adducts. These adducts are formed by hepatic metabolism of APAP to N-acetyl-p-benzoquinone imine, which covalently binds to hepatic proteins as 3-(cystein-S-yl)-APAP adducts. Adducts are released into blood during hepatocyte lysis. We previously showed that adducts could be quantified by high-performance liquid chromatography with electrochemical detection following proteolytic hydrolysis, and that the concentration of adducts in serum of overdose patients correlated with toxicity. The following study examined the pharmacokinetic profile and clinical associations of adducts in 53 adults with acute APAP overdose resulting in acute liver failure. A population pharmacokinetic analysis using nonlinear mixed effects (statistical regression type) models was conducted; individual empiric Bayesian estimates were determined for the elimination rate constant and elimination half-life. Correlations between clinical and laboratory data were examined relative to adduct concentrations using nonparametric statistical approaches. Peak concentrations of APAP-protein adducts correlated with peak aminotransferase concentrations (r = 0.779) in adults with APAP-related acute liver failure. Adducts did not correlate with bilirubin, creatinine, and APAP concentration at admission, international normalized ratio for prothrombin time, or reported APAP dose. After N-acetylcysteine therapy, adducts exhibited first-order disappearance. The mean elimination rate constant and elimination half-life were 0.42 +/- 0.09 days(-1) and 1.72 +/- 0.34 days, respectively, and estimates from the population model were in strong agreement with these data. Adducts were detected in some patient samples 12 days post-ingestion. The persistence and specificity of APAP-protein adducts as correlates of toxicity support their use as specific biomarkers of APAP toxicity in patients with acute liver injury.
Asunto(s)
Acetaminofén/análogos & derivados , Acetaminofén/farmacocinética , Acetaminofén/envenenamiento , Analgésicos no Narcóticos/farmacocinética , Analgésicos no Narcóticos/envenenamiento , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/metabolismo , Acetaminofén/administración & dosificación , Acetaminofén/sangre , Acetilcisteína/uso terapéutico , Adulto , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/sangre , Antídotos/uso terapéutico , Teorema de Bayes , Benzoquinonas/metabolismo , Biomarcadores/sangre , Biotransformación , Bases de Datos como Asunto , Sobredosis de Droga , Femenino , Semivida , Humanos , Iminas/metabolismo , Fallo Hepático Agudo/tratamiento farmacológico , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Modelos Biológicos , Modelos Estadísticos , Dinámicas no Lineales , Adulto JovenRESUMEN
A simple, rapid method of determining the ibuprofen concentration in small volumes of human plasma (50 microl) by HPLC was developed. The sample was prepared for injection using a solid-phase extraction method, with naproxen as the internal standard. A 96-well extraction plate was used, easing sample preparation and allowing the simultaneous extraction of multiple plasma samples directly into the HPLC injection vials. Samples were stable at room temperature for at least 48 h prior to injection. The HPLC method used an ultraviolet detector with a 5-min run time and measured concentrations across the range typically seen with the clinical use of this drug. The calibration curve was linear across the concentration range of 0.78-100 microg/ml with a limit of quantitation (LOQ) of 1.56 microg/ml. The coefficient of variation for intra-day and inter-day precision was 6% or less with accuracies within 2% of the nominal values for low (4.5 microg/ml), medium (40 microg/ml) and high (85 microg/ml) ibuprofen concentrations. For ibuprofen concentrations at the LOQ, the intra-day and inter-day precision and accuracy were within 10 and 15%, respectively. Recovery was 87% or greater for ibuprofen. This method was used to analyze plasma samples for unknown ibuprofen concentrations in bioequivalence and limited food effect studies of different formulations of ibuprofen. Thus, this method has been fully validated and used in the analysis of unknown plasma samples for ibuprofen.