RESUMEN
Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.
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Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Ratones , Animales , Acetaminofén/toxicidad , Mitocondrias Hepáticas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Mitocondrias/metabolismo , Hepatocitos/metabolismo , Necrosis/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Adverse reactions to food are a common dermatological condition in dogs, requiring nutritional intervention using a novel or hydrolysate protein-based food. OBJECTIVE: To evaluate a therapeutic food containing egg and phytonutrients in dogs with food allergies using an activity monitor and core outcome set for canine atopic dermatitis (COSCAD'18) guidelines and in a controlled double-masked, multicenter, prospective clinical trial. ANIMALS: Adult dogs with a history of adverse food reaction as diagnosed by a food elimination trial were recruited from general practices. METHODS: After a 21-day baseline period, dogs were randomized to test or positive control (hydrolyzed protein) food for 21 days. Owner (pruritus visual analog score [PVAS], coat quality, food acceptance, and satisfaction) and veterinarian (canine atopic dermatitis lesion index [CADLI], physical examination) assessments were completed on days 0, 21, and 42. Dogs wore a collar-mounted activity monitor to record scratching and shaking behavior throughout the study. Statistical analysis included within-group comparison to baseline and between-group comparison at study end using a significance threshold of alpha = 0.05. RESULTS: At the end of the treatment period, all results were similar between groups for CADLI, PVAS, owner satisfaction, activity, and questionnaire data. Scores for hair dullness, brittleness, amount of dandruff, feces quality, and food acceptance were positive and not statistically different between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: The therapeutic test food was well-accepted and efficacious in managing signs of adverse reactions to food compared to baseline as well as compared to the positive control food.
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Dermatitis Atópica , Enfermedades de los Perros , Alérgenos , Animales , Dermatitis Atópica/veterinaria , Perros , Estudios Prospectivos , Prurito/veterinaria , Estados UnidosRESUMEN
BACKGROUND: Adverse reactions to food are a common dermatological condition in dogs, requiring nutritional intervention using novel or hydrolysate protein-based foods. OBJECTIVE: To evaluate a therapeutic food containing egg and phytonutrients in dogs with food allergies using an activity monitor and core outcome set for canine atopic dermatitis (COSCAD'18) in a controlled double-masked, multicenter, prospective clinical trial. ANIMALS: Adult dogs with a history of adverse food reaction as diagnosed by a food elimination trial were recruited from general practices. METHODS: After a 21-day baseline period, dogs were randomized to test or positive control (hydrolyzed protein) food for 21 days. Owner (pruritus visual analog score [PVAS], coat quality, food acceptance, and satisfaction) and veterinarian (canine atopic dermatitis lesion index [CADLI], physical examination) assessments were completed on days 0, 21, and 42. Dogs wore a collar-mounted activity monitor to record sleep, scratching, and shaking behavior throughout the study. Statistical analysis included within-group comparison to baseline and between-group comparison at study end using a significance threshold of alpha = 0.05. RESULTS: At the end of the treatment period, all results were similar between groups for CADLI, PVAS, owner satisfaction, activity, and questionnaire data. Scores for hair dullness, brittleness, amount of dandruff, feces quality, and food acceptance were positive and were not statistically different between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: The therapeutic test food was well-accepted and efficacious in managing signs of adverse reactions to food compared to baseline as well as compared to the positive control food.
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Dermatitis Atópica , Enfermedades de los Perros , Animales , Dermatitis Atópica/veterinaria , Perros , Estudios Prospectivos , Prurito/veterinaria , Reino UnidoRESUMEN
Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.
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Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Acetaminofén/administración & dosificación , Analgésicos no Narcóticos/administración & dosificación , Animales , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leupeptinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas/metabolismoRESUMEN
This retrospective study used 226 dogs and 296 cats to evaluate whether protein absorption was influenced by species, and within species, what influence increasing the percentage of total dietary protein, as plant protein, had on protein absorption. Each food was evaluated by at least one study with a minimum of six dogs or cats assigned to each study. Dietary inclusion of animal and plant based protein was calculated by analysis of ingredients and dietary inclusion level. Both dogs and cats were able to digest dietary plant protein, with protein digestibility in dogs unchanged as plant protein increased, while in cats, eating dry food, an increase in plant protein, was associated with increased protein digestibility. When individual plant high-concentration protein sources (excluding the protein from whole grains) were evaluated (i.e., soybean meal, soybean protein isolate, corn gluten meal, and rice protein concentrate) there was no response to increasing protein from these sources in the dog. In the cat, there was a significant positive effect on protein digestibility associated with an increasing concentration of corn gluten meal. In summary, as the dietary protein shifted from striated muscle and other animal proteins to plant based proteins, there was no effect in the dog, while in cats, increasing dietary plant protein was associated with increasing protein digestibility (5.5% increase at 50% protein from plants in dry cat food). Protein digestibility of food in dogs and cats is similar, if not enhanced, when the plant protein sources are concentrated from soybeans (soybean isolate, soybean meal), corn (corn gluten meal), or rice (rice protein concentrate).
RESUMEN
We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.
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Acetaminofén/envenenamiento , Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Compuestos Organofosforados/farmacología , Piperidinas/farmacología , Acetilcisteína , Animales , Antioxidantes/farmacología , Caspasa 3/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Formation of acetaminophen (APAP) protein adducts are a critical feature of APAP hepatotoxicity, and circulating protein adducts have recently been utilized in bioassays for identification of APAP overdose in humans. Despite their clinical significance, mechanisms of adduct release into the circulation are not well understood. Extracellular vesicles (EVs) are discrete membrane bound vesicles, which package cellular cargo and function in extracellular transport. Clarification of their role in transport of APAP adducts is relevant since adduct packaging within these vesicles could shield them from detection by antibody based methods, resulting in under-estimating adduct levels. Hence, this study evaluated EV release after APAP overdose in primary mouse hepatocytes and human HepaRG cells in vitro, in mice and APAP overdose patients in vivo and examined their role in transport of APAP-protein adducts. EVs were characterized by size and protein composition and the levels of APAP-protein adducts were measured. Significant elevations in circulating EV numbers were observed 6 h after APAP overdose in vivo and by 4 h in primary mouse hepatocytes in culture. EVs were also elevated in media from HepaRG cells by 24 h after APAP exposure, an effect recapitulated in APAP overdose patients, where EV numbers were elevated compared to healthy controls. Although APAP-protein adducts were elevated in circulation and media parallel to the increased exosome release, no detectable adducts were observed within EVs. This suggests that although APAP overdose enhances EV release from hepatocytes in mice and humans, it is not a significant mechanism of release of APAP protein adducts into circulation.
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Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Vesículas Extracelulares/efectos de los fármacos , Proteínas/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sobredosis de Droga/sangre , Sobredosis de Droga/etiología , Vesículas Extracelulares/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.
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Acetaminofén , Antiinflamatorios/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Conexina 43/antagonistas & inhibidores , Conexinas/antagonistas & inhibidores , Hígado/efectos de los fármacos , Péptidos/farmacología , Adenosina Trifosfato/metabolismo , Alanina Transaminasa/sangre , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Conexina 43/metabolismo , Conexinas/metabolismo , Citocinas/sangre , Citoprotección , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína beta1 de Unión ComunicanteRESUMEN
BACKGROUND & AIMS: Hypoxic hepatitis is a clinical condition precipitated by prolonged periods of oxygen deprivation to the liver. It can have several underlying causes. Despite its prevalence in critically ill patients, which can reach upwards of 10%, very little is known about the mechanisms of injury. Thus, we set out to measure previously identified circulating biomarkers in an attempt to describe mechanisms of injury following hypoxic hepatitis. METHODS: Plasma from patients diagnosed with hypoxic hepatitis was collected for this study. Biomarkers of hepatocellular injury, mitochondrial damage and cell death were measured. These results were compared against results obtained from well-characterized acetaminophen overdose patients. RESULTS: At peak injury, ALT measured 4082±606 U/L and gradually decreased over 5 days, corresponding to the clinically observed pattern of hypoxic hepatitis. Levels of GDH showed a similar pattern, but neither ALT nor GDH were significantly higher in these patients than in acetaminophen patients. Plasma levels of DNA fragments mimicked hepatocellular injury as measured by ALT and miRNA-122. Interestingly, we found a significant increase in caspase-cleaved cytokeratin-18; however, the full-length form greatly exceeded the cleaved form at the time of maximum injury (45837±12085 vs 2528±1074 U/L). We also found an increase in acHMGB1 at later time points indicating a possible role of inflammation, but cytokine levels at these times were actually decreased relative to early time points. CONCLUSIONS: The mechanism of injury following hypoxic hepatitis involves mitochondrial damage and DNA fragmentation. Importantly, necrosis, rather than apoptosis, is the main mode of cell death.
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Hepatitis/sangre , Hipoxia/sangre , Isquemia/sangre , Hígado/fisiopatología , Acetaminofén/toxicidad , Adolescente , Adulto , Alanina Transaminasa/sangre , Apoptosis , Biomarcadores/sangre , Fragmentación del ADN , ADN Mitocondrial/sangre , Femenino , Proteína HMGB1/sangre , Humanos , Isquemia/etiología , Queratina-18/sangre , Modelos Lineales , Hígado/irrigación sanguínea , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Mitocondrias/patología , Necrosis/etiología , Estados Unidos , Adulto JovenRESUMEN
Pannexins constitute a relatively new family of transmembrane proteins that form channels linking the cytoplasmic compartment with the extracellular environment. The presence of pannexin1 in the liver has been documented previously, where it underlies inflammatory responses, such as those occurring upon ischemia-reperfusion injury. In the present study, we investigated whether pannexin1 plays a role in acute drug-induced liver toxicity. Hepatic expression of pannexin1 was characterized in a mouse model of acetaminophen-induced hepatotoxicity. Subsequently, mice were overdosed with acetaminophen followed by treatment with the pannexin1 channel inhibitor 10Panx1. Sampling was performed 1, 3, 6, 24 and 48 h after acetaminophen administration. Evaluation of the effects of pannexin1 channel inhibition was based on a number of clinically relevant readouts, including protein adduct formation, measurement of aminotransferase activity and histopathological examination of liver tissue as well as on a series of markers of inflammation, oxidative stress and regeneration. Although no significant differences were found in histopathological analysis, pannexin1 channel inhibition reduced serum levels of alanine and aspartate aminotransferase. This was paralleled by a reduced amount of neutrophils recruited to the liver. Furthermore, alterations in the oxidized status were noticed with upregulation of glutathione levels upon suppression of pannexin1 channel opening. Concomitant promotion of regenerative activity was detected as judged on increased proliferating cell nuclear antigen protein quantities in 10Panx1-treated mice. Pannexin1 channels are important actors in liver injury triggered by acetaminophen. Inhibition of pannexin1 channel opening could represent a novel approach for the treatment of drug-induced hepatotoxicity.
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Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Conexinas/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Conexinas/genética , Conexinas/metabolismo , Citocinas/sangre , Citocinas/metabolismo , Sobredosis de Droga/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neutrófilos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Autosomal recessive polycystic kidney disease (ARPKD) is a severe monogenic disorder that occurs due to mutations in the PKHD1 gene. Congenital hepatic fibrosis (CHF) associated with ARPKD is characterized by the presence of hepatic cysts derived from dilated bile ducts and a robust, pericystic fibrosis. Cyst growth, due to cyst wall epithelial cell hyperproliferation and fluid secretion, is thought to be the driving force behind disease progression. Liver fibrosis is a wound healing response in which collagen accumulates in the liver due to an imbalance between extracellular matrix synthesis and degradation. Whereas both hyperproliferation and pericystic fibrosis are hallmarks of CHF/ARPKD, whether or not these two processes influence one another remains unclear. Additionally, recent studies demonstrate that inflammation is a common feature of CHF/ARPKD. Therefore, we propose a "pathogenic triumvirate" consisting of hyperproliferation of cyst wall growth, pericystic fibrosis, and inflammation which drives CHF/ARPKD progression. This review will summarize what is known regarding the mechanisms of cyst growth, fibrosis, and inflammation in CHF/ARPKD. Further, we will discuss the potential advantage of identifying a core pathogenic feature in CHF/ARPKD to aid in the development of novel therapeutic approaches. If a core pathogenic feature does not exist, then developing multimodality therapeutic approaches to target each member of the "pathogenic triumvirate" individually may be a better strategy to manage this debilitating disease.
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Enfermedades Genéticas Congénitas/complicaciones , Enfermedades Genéticas Congénitas/patología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/patología , Riñón Poliquístico Autosómico Recesivo/complicaciones , Riñón Poliquístico Autosómico Recesivo/patología , Animales , Modelos Animales de Enfermedad , Enfermedades Genéticas Congénitas/terapia , Humanos , Inflamación/complicaciones , Inflamación/patología , Cirrosis Hepática/terapia , Riñón Poliquístico Autosómico Recesivo/genética , Riñón Poliquístico Autosómico Recesivo/terapiaRESUMEN
Overdose of acetaminophen (APAP) causes severe liver injury and even acute liver failure in both mice and human. A recent study by Kim et al. (2015, Metformin ameliorates acetaminophen hepatotoxicity via Gadd45ß-dependent regulation of JNK signaling in mice. J. Hepatol. 63, 75-82) showed that metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP hepatotoxicity in mice. However, its exact protective mechanism has not been well clarified. To investigate this, C57BL/6J mice were treated with 400 mg/kg APAP and 350 mg/kg metformin was given 0.5 h pre- or 2 h post-APAP. Our data showed that pretreatment with metformin protected against APAP hepatotoxicity, as indicated by the over 80% reduction in plasma alanine aminotransferase (ALT) activities and significant decrease in centrilobular necrosis. Metabolic activation of APAP, as indicated by glutathione depletion and APAP-protein adducts formation, was also slightly inhibited. However, 2 h post-treatment with metformin still reduced liver injury by 50%, without inhibition of adduct formation. Interestingly, neither pre- nor post-treatment of metformin inhibited c-jun N-terminal kinase (JNK) activation or its mitochondrial translocation. In contrast, APAP-induced mitochondrial oxidant stress and dysfunction were greatly attenuated in these mice. In addition, mice with 2 h post-treatment with metformin also showed significant inhibition of complex I activity, which may contribute to the decreased mitochondrial oxidant stress. Furthermore, the protection was reproduced in JNK activation-absent HepaRG cells treated with 20 mM APAP followed by 0.5 or 1 mM metformin 6 h later, confirming JNK-independent protection mechanisms. Thus, metformin protects against APAP hepatotoxicity by attenuating the mitochondrial oxidant stress and subsequent mitochondrial dysfunction, and may be a potential therapeutic option for APAP overdose patients.
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Acetaminofén , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Metformina/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citoprotección , Modelos Animales de Enfermedad , Activación Enzimática , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Necrosis , Factores de TiempoRESUMEN
Use of natural products is increasingly popular. In fact, many patients with liver diseases self-medicate with herbal supplements. Resveratrol (RSV), in particular, is a common natural product that can reduce injury in experimental models of liver disease. Xenobiotic hepatotoxicity is a particularly important area-of-need for therapeutics. Drug-induced liver injury, for example, is the most common cause of acute liver failure (ALF) and ALF-induced deaths in many countries. Importantly, RSV protects against hepatotoxicity in animal models in vivo caused by several drugs and chemicals and may be an effective intervention. Although many mechanisms have been proposed to explain the protection, not all are consistent with other data. Furthermore, RSV suffers from other issues, including limited bioavailability due to extensive hepatic metabolism. The purpose of this article is to summarize recent findings on the protective effects of RSV in xenobiotic-induced liver injury and other forms of liver injury and to provide a critical review of the underlying mechanisms. New mechanisms that are more consistent with data emerging from the toxicology field are suggested. Efforts to move RSV into clinical use are also considered. Overall, RSV is a promising candidate for therapeutic use, but additional studies are needed to better understand its effects.
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Antioxidantes/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Estilbenos/uso terapéutico , Xenobióticos/toxicidad , Humanos , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/prevención & control , ResveratrolRESUMEN
Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury.
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Apoptosis , Biomarcadores/sangre , Hepatopatías/sangre , Hígado/patología , Daño por Reperfusión/sangre , Animales , Caspasa 3/sangre , Proteína HMGB1/sangre , Queratina-18/sangre , Ratones Endogámicos C57BL , MicroARNs/sangre , NecrosisRESUMEN
There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation.
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The effects of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen on transport attributable to P-glycoprotein were studied using Caco-2 cell monolayers in a transwell system, with rhodamine-123 as an index substrate for inhibition studies. The three compounds did not demonstrate differential flux between basal-apical and apical-basal directions in Caco-2 monolayers. The mean IC50 values for inhibition of rhodamine-123 transport were: 29 microM for tamoxifen; 26 microM for N-desmethyltamoxifen; and 7.4 microM for 4-hydroxytamoxifen. The three compounds were also evaluated as potential inhibitors of human CYP3A based on an in vitro model using triazolam hydroxylation by human liver microsomes as an index reaction. Mean (+/-SE) IC50 values versus formation of alpha-hydroxy-triazolam and 4-hydroxy-triazolam in human liver microsomes were, respectively: 23.5 (+/-3.9) and 18.4 (+/-5.3) microM for tamoxifen; 10.2 (+/-1.7) and 9.2 (+/-1.5) microM for N-desmethyltamoxifen; and 2.6 (+/-0.5) and 2.7 (+/-0.3) microM for 4-hydroxytamoxifen. Thus, tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen, do not appear to be substrates for transport by P-glycoprotein. However, tamoxifen has the potential to inhibit transport mediated by P-glycoprotein as well as CYP3A-mediated metabolism. Inhibitory effects of the principal metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen, may exceed those of the parent drug. Tamoxifen, and possibly its metabolites, may have the potential to cause drug interactions by inhibiting both drug transport and metabolism. This possibility requires further evaluation in clinical studies.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450 , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transporte Biológico , Células CACO-2 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/fisiología , Interacciones Farmacológicas , HumanosRESUMEN
The extraction, isolation and characterization of 29 natural products contained in Ginkgo biloba have been described, which we have now tested for their in-vitro capacity to inhibit the five major human cytochrome P450 (CYP) isoforms in human liver microsomes. Weak or negligible inhibitory activity was found for the terpene trilactones (ginkgolides A, B, C and J, and bilobalide), and the flavonol glycosides. However 50% inhibitory activity (IC50) was found at concentrations less than 10 microg L(-1) for the flavonol aglycones (kaempferol, quercetin, apigenin, myricetin, tamarixetin) with CYP1A2 and CYP3A. Quercetin, the biflavone amentoflavone, sesamin, as well as (Z,Z)-4,4'-(1,4-pentadiene-1,5-diyl)diphenol and 3-nonadec-8-enyl-benzene-1,2-diol, were also inhibitors of CYP2C9. The IC50 of amentoflavone for CYP2C9 was 0.019 microg mL(-1) (0.035 microM). Thus, the principal components of Ginkgo biloba preparations in clinical use (terpene trilactones and flavonol glycosides) do not significantly inhibit these human CYPs in-vitro. However, flavonol aglycones, the biflavonol amentoflavone and several other non-glycosidic constituents are significant in-vitro inhibitors of CYP. The clinical importance of these potential inhibitors will depend on their amounts in ginkgo preparations sold to the public, and the extent to which their bioavailability allows them to reach the CYP enzymes in-situ.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Ginkgo biloba , Microsomas Hepáticos/efectos de los fármacos , Extractos Vegetales/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Microsomas Hepáticos/enzimología , Extractos Vegetales/aislamiento & purificaciónRESUMEN
BACKGROUND: Components of grapefruit juice may impair the activity of intestinal cytochrome P450 (CYP) 3A enzymes, sometimes resulting in clinically important drug interactions. The time course of recovery from CYP3A inhibition after a single exposure to grapefruit juice is not clearly established. METHODS: Healthy volunteer subjects (N = 25) received a single 6-mg oral dose of the CYP3A substrate midazolam in the control condition without exposure to grapefruit juice. Two days later, midazolam was administered 2 hours after 300 mL of regular-strength grapefruit juice. Subjects were then randomly assigned to 3 different groups, receiving a third midazolam challenge at 26, 50, or 74 hours after exposure to grapefruit juice. The capacity of 6'7'-dihydroxybergamottin and bergamottin to inhibit human CYP3A was studied in vitro using human liver microsomes. RESULTS: The area under the plasma concentration curve (AUC) for midazolam increased by a factor of 1.65 (ratio compared with control) when midazolam was given 2 hours after grapefruit juice. At 26, 50, and 74 hours after grapefruit juice, the AUC ratios (mean AUC value at the indicated time divided by the mean control AUC on day 1) were 1.29, 1.29, and 1.06, respectively. The relationship of time after grapefruit juice exposure versus AUC increase over control indicated a recovery half-life estimated at 23 hours. The midazolam elimination half-life did not change significantly from the control value at any time after grapefruit juice exposure. 6'7'-Dihydroxybergamottin inhibited midazolam alpha-hydroxylation in vitro, with a mean 50% inhibitory concentration of 4.7 micro mol/L; preincubation of microsomes with 6'7'-dihydroxybergamottin greatly reduced the 50% inhibitory concentration to 0.31 micro mol/L, consistent with mechanism-based inhibition. Bergamottin itself had much weaker inhibitory potency compared to 6'7'-dihydroxybergamottin. CONCLUSIONS: A usual single exposure to grapefruit juice appears to impair the enteric, but not the hepatic, component of presystemic extraction of oral midazolam. Recovery is largely complete within 3 days, consistent with enzyme regeneration after mechanism-based inhibition. 6'7'-Dihydroxybergamottin was verified as a potent mechanism-based inhibitor of midazolam alpha-hydroxylation by CYP3A in vitro.