RESUMEN
Acetaminophen (APAP) overdose stands as the primary cause of acute liver failure in the United States. APAP hepatotoxicity involves hepatic glutathione (GSH) depletion and mitochondrial damage. To counteract the toxicity of APAP, the nuclear factor erythroid 2 like 2 (Nrf2) activates the expression of genes responsible for drug detoxification and GSH synthesis. In this study, we present evidence that the elimination of hepatocyte small heterodimer partner, a critical transcriptional repressor for liver metabolism, results in Nrf2 activation and protects mice from APAP-induced acute liver injury. Initial investigations conducted on wildtype (WT) mice revealed a swift downregulation of Shp mRNA within the first 24 h after APAP administration. Subsequent treatment of hepatocyte-specific Shp knockout (ShpHep-/-) mice with 300 mg/kg APAP for 2 h exhibited comparable bioactivation of APAP with that observed in the WT controls. However, a significant reduction in liver injury was observed in ShpHep-/- after APAP treatment for 6 and 24 h. The decreased liver injury correlated with a faster recovery of GSH, attributable to heightened expression of Nrf2 target genes involved in APAP detoxification and GSH synthesis. Moreover, in vitro studies revealed that SHP protein interacted with NRF2 protein, inhibiting the transcription of Nrf2 target genes. These findings hold relevance for humans, as overexpression of SHP hindered APAP-induced NRF2 activation in primary human hepatocytes. In conclusion, our studies have unveiled a novel regulatory axis involving SHP and NRF2 in APAP-induced acute liver injury, emphasizing SHP as a promising therapeutic target in APAP overdose-induced hepatotoxicity.
Asunto(s)
Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Humanos , Animales , Ratones , Acetaminofén/toxicidad , Acetaminofén/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hepatocitos/metabolismo , Hígado/metabolismo , Glutatión/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the western world. We report a novel signaling interaction between hepatocyte nuclear factor 4 alpha (HNF4α) cMyc and nuclear factor erythroid 2-related factor 2 (Nrf2) during liver injury and regeneration after APAP overdose. APPROACH AND RESULTS: APAP-induced liver injury and regeneration were studied in male C57BL/6J (WT) mice, hepatocyte-specific HNF4α knockout mice (HNF4α-KO), and HNF4α-cMyc double knockout mice (DKO). C57BL/6J mice treated with 300 mg/kg maintained nuclear HNF4α expression and exhibited liver regeneration, resulting in recovery. However, treatment with 600-mg/kg APAP, where liver regeneration was inhibited and recovery was delayed, showed a rapid decline in HNF4α expression. HNF4α-KO mice developed significantly higher liver injury due to delayed glutathione recovery after APAP overdose. HNF4α-KO mice also exhibited significant induction of cMyc, and the deletion of cMyc in HNF4α-KO mice (DKO mice) reduced the APAP-induced liver injury. The DKO mice had significantly faster glutathione replenishment due to rapid induction in Gclc and Gclm genes. Coimmunoprecipitation and ChIP analyses revealed that HNF4α interacts with Nrf2 and affects its DNA binding. Furthermore, DKO mice showed significantly faster initiation of cell proliferation resulting in rapid liver regeneration and recovery. CONCLUSIONS: These data show that HNF4α interacts with Nrf2 and promotes glutathione replenishment aiding in recovery from APAP-induced liver injury, a process inhibited by cMyc. These studies indicate that maintaining the HNF4α function is critical for regeneration and recovery after APAP overdose.
Asunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Masculino , Animales , Ratones , Acetaminofén/toxicidad , Regeneración Hepática/genética , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Ratones Endogámicos C57BL , Hígado/metabolismo , Hepatocitos/metabolismo , Glutatión/metabolismo , Ratones Noqueados , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismoRESUMEN
Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single ß-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.
Asunto(s)
Acetaminofén/toxicidad , Acetilglucosamina/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Glutatión/biosíntesis , Hígado/efectos de los fármacos , N-Acetilglucosaminiltransferasas/metabolismo , Acetaminofén/metabolismo , Acilación , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Glutatión/genética , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , Unión ProteicaRESUMEN
Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1-/- mice were overdosed with acetaminophen for 1, 6, 24 or 48h or were fed a choline-deficient high-fat diet for 8weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1-/- diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1-/- mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1-/- mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease.
Asunto(s)
Conexinas/genética , Citoprotección/genética , Eliminación de Gen , Hepatopatías/genética , Hígado/metabolismo , Proteínas del Tejido Nervioso/genética , Enfermedad Aguda , Animales , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Hígado/patología , Hepatopatías/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
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.
Asunto(s)
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
Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Sobredosis de Droga/enzimología , Receptores ErbB/fisiología , Hepatocitos/efectos de los fármacos , Regeneración Hepática , Acetaminofén/metabolismo , Analgésicos no Narcóticos/metabolismo , Animales , Receptores ErbB/antagonistas & inhibidores , Glutatión/metabolismo , Hepatocitos/enzimología , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/enzimología , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Estrés Oxidativo , Unión ProteicaRESUMEN
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.
Asunto(s)
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
BACKGROUND AND AIMS: Being goalkeepers of liver homeostasis, gap junctions are also involved in hepatotoxicity. However, their role in this process is ambiguous, as gap junctions can act as both targets and effectors of liver toxicity. This particularly holds true for drug-induced liver insults. In the present study, the involvement of connexin26, connexin32 and connexin43, the building blocks of liver gap junctions, was investigated in acetaminophen-induced hepatotoxicity. METHODS: C57BL/6 mice were overdosed with 300mg/kg body weight acetaminophen followed by analysis of the expression and localization of connexins as well as monitoring of hepatic gap junction functionality. Furthermore, acetaminophen-induced liver injury was compared between mice genetically deficient in connexin43 and wild type littermates. Evaluation of the toxicological response was based on a set of clinically relevant parameters, including protein adduct formation, measurement of alanine aminotransferase activity, cytokines and glutathione. RESULTS: It was found that gap junction communication deteriorates upon acetaminophen intoxication in wild type mice, which is associated with a switch in mRNA and protein production from connexin32 and connexin26 to connexin43. The upregulation of connexin43 expression is due, at least in part, to de novo production by hepatocytes. Connexin43-deficient animals tended to show increased liver cell death, inflammation and oxidative stress in comparison with wild type counterparts. CONCLUSION: These results suggest that hepatic connexin43-based signaling may protect against acetaminophen-induced liver toxicity.
Asunto(s)
Acetaminofén/efectos adversos , Analgésicos no Narcóticos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Conexina 43/genética , Hígado/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Conexina 43/análisis , Conexina 43/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
Connexin32 is the building block of hepatocellular gap junctions, which control direct intercellular communication and thereby act as goalkeepers of liver homeostasis. This study was set up to investigate whether connexin32 is involved in hepatotoxicity induced by the analgesic and antipyretic drug acetaminophen. To this end, whole body connexin32 knock-out mice were overdosed with acetaminophen followed by sampling at different time points within a 24-h time frame. Evaluation was done based upon a series of clinically and mechanistically relevant read-outs, including protein adduct formation, histopathological examination, measurement of alanine aminotransferase activity, cytokine production, levels of reduced and oxidized glutathione and hepatic protein amounts of proliferating cell nuclear antigen. In essence, it was found that genetic ablation of connexin32 has no influence on several key events in acetaminophen-induced hepatotoxicity, including cell death, inflammation or oxidative stress, yet it does affect production of protein adducts as well as proliferating cell nuclear antigen steady-state protein levels. This outcome is not in line with previous studies, which are contradicting on their own, as both amplification and alleviation of this toxicological process by connexin32 have been described. This could question the suitability of the currently available models and tools to investigate the role of connexin32 in acetaminophen-triggered hepatotoxicity.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Conexinas/metabolismo , Hígado/efectos de los fármacos , Acetaminofén/administración & dosificación , Acetaminofén/metabolismo , Alanina Transaminasa/sangre , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Conexinas/genética , Citocinas/sangre , Disulfuro de Glutatión/metabolismo , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteína beta1 de Unión ComunicanteRESUMEN
Chronic hepatitis C virus (HCV) infection predisposes patients to develop liver failure after acetaminophen (APAP) overdose. Mechanisms involved in this were explored using transgenic mice expressing the HCV structural proteins core, E1 and E2. Treatment of C57BL/6J mice with 200 mg/kg body weight APAP resulted in significant liver injury at 6 h as indicated by elevated ALT levels, focal centrilobular necrosis and nuclear DNA fragmentation. HCV transgenic mice showed a variable response, with approximately half the animals showing exacerbation of all parameters of liver injury, while the other half was protected. HCV transgenic mice with higher liver injury had lower liver glutathione levels, elevated mitochondrial oxidative stress and enhanced release of apoptosis-inducing factor (AIF) from the mitochondria. This was accompanied by induction of a higher ER stress response and induction of autophagy. Transgenic animals showing protection against liver injury had a robust recovery of liver glutathione content at 6 h when compared to wild-type animals, accompanied by reduction in mitochondrial oxidative stress and AIF release. This was accompanied by an elevation in glutathione S-transferase mRNA levels and activity, which suggests that an efficient clearance of the reactive intermediate may contribute to the protection against APAP hepatotoxicity in these mice. These results demonstrate that while HCV infection could exacerbate APAP-induced liver injury due to induction and amplification of mitochondrial oxidant stress, it could also protect against injury by activation of APAP scavenging mechanisms.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatitis C/complicaciones , Proteínas del Envoltorio Viral/efectos adversos , Animales , Autofagia/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Sustancias Protectoras/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Sustancias Protectoras/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND & AIMS: Hyperhomocysteinemia is often associated with liver and metabolic diseases. We studied nuclear receptors that mediate oscillatory control of homocysteine homeostasis in mice. METHODS: We studied mice with disruptions in Nr0b2 (called small heterodimer partner [SHP]-null mice), betaine-homocysteine S-methyltransferase (Bhmt), or both genes (BHMT-null/SHP-null mice), along with mice with wild-type copies of these genes (controls). Hyperhomocysteinemia was induced by feeding mice alcohol (National Institute on Alcohol Abuse and Alcoholism binge model) or chow diets along with water containing 0.18% DL-homocysteine. Some mice were placed on diets containing cholic acid (1%) or cholestyramine (2%) or high-fat diets (60%). Serum and livers were collected during a 24-hour light-dark cycle and analyzed by RNA-seq, metabolomic, and quantitative polymerase chain reaction, immunoblot, and chromatin immunoprecipitation assays. RESULTS: SHP-null mice had altered timing in expression of genes that regulate homocysteine metabolism compared with control mice. Oscillatory production of S-adenosylmethionine, betaine, choline, phosphocholine, glyceophosphocholine, cystathionine, cysteine, hydrogen sulfide, glutathione disulfide, and glutathione, differed between SHP-null mice and control mice. SHP inhibited transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1. Expression of Bhmt and cystathionine γ-lyase was decreased when mice were fed cholic acid but increased when they were placed on diets containing cholestyramine or high-fat content. Diets containing ethanol or homocysteine induced hyperhomocysteinemia and glucose intolerance in control, but not SHP-null, mice. In BHMT-null and BHMT-null/SHP-null mice fed a control liquid, lipid vacuoles were observed in livers. Ethanol feeding induced accumulation of macrovesicular lipid vacuoles to the greatest extent in BHMT-null and BHMT-null/SHP-null mice. CONCLUSIONS: Disruption of Shp in mice alters timing of expression of genes that regulate homocysteine metabolism and the liver responses to ethanol and homocysteine. SHP inhibits the transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1.
Asunto(s)
Ritmo Circadiano , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Homocisteína/metabolismo , Hiperhomocisteinemia/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Betaína-Homocisteína S-Metiltransferasa/genética , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Glucemia/metabolismo , Resina de Colestiramina , Ácido Cólico , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Etanol , Regulación Enzimológica de la Expresión Génica , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/metabolismo , Homeostasis , Homocisteína/sangre , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/inducido químicamente , Hiperhomocisteinemia/genética , Hiperhomocisteinemia/prevención & control , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Factores de Tiempo , Activación TranscripcionalRESUMEN
Acetaminophen (APAP) overdose causes severe and occasionally fatal liver injury. Numerous drugs that attenuate APAP toxicity have been described. However these compounds frequently protect by cytochrome P450 inhibition, thereby preventing the initiating step of toxicity. We have previously shown that pretreatment with allopurinol can effectively protect against APAP toxicity, but the mechanism remains unclear. In the current study, C3HeB/FeJ mice were administered allopurinol 18h or 1h prior to an APAP overdose. Administration of allopurinol 18h prior to APAP overdose resulted in an 88% reduction in liver injury (serum ALT) 6h after APAP; however, 1h pretreatment offered no protection. APAP-cysteine adducts and glutathione depletion kinetics were similar with or without allopurinol pretreatment. The phosphorylation and mitochondrial translocation of c-jun-N-terminal-kinase (JNK) have been implicated in the progression of APAP toxicity. In our study we showed equivalent early JNK activation (2h) however late JNK activation (6h) was attenuated in allopurinol treated mice, which suggests that later JNK activation is more critical for the toxicity. Additional mice were administered oxypurinol (primary metabolite of allopurinol) 18h or 1h pre-APAP, but neither treatment protected. This finding implicated an aldehyde oxidase (AO)-mediated metabolism of allopurinol, so mice were treated with hydralazine to inhibit AO prior to allopurinol/APAP administration, which eliminated the protective effects of allopurinol. We evaluated potential targets of AO-mediated preconditioning and found increased hepatic metallothionein 18h post-allopurinol. These data show metabolism of allopurinol occurring independent of P450 isoenzymes preconditions the liver and renders the animal less susceptible to an APAP overdose.
Asunto(s)
Acetaminofén/análogos & derivados , Aldehído Oxidasa/metabolismo , Alopurinol/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Cisteína/análogos & derivados , Hígado/efectos de los fármacos , Acetaminofén/administración & dosificación , Acetaminofén/efectos adversos , Aldehído Oxidasa/antagonistas & inhibidores , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cisteína/administración & dosificación , Cisteína/efectos adversos , Sobredosis de Droga , Glutatión/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/metabolismo , Masculino , Metalotioneína/metabolismo , Ratones , Ratones Endogámicos C3H , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxipurinol/metabolismo , Fosforilación , Xantina Oxidasa/metabolismoRESUMEN
At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.
Asunto(s)
Acetaminofén/metabolismo , Analgésicos no Narcóticos/metabolismo , Hígado/efectos de los fármacos , Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión/metabolismoRESUMEN
Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the western world. Controversy exists regarding the hypothesis that the hepatocyte injury is amplified by a sterile inflammatory response, rather than being the result of intracellular mechanisms alone. A recent study suggested that the purinergic receptor antagonist A438079 protects against APAP-induced liver injury by preventing the activation of the Nalp3 inflammasome in Kupffer cells and thereby preventing inflammatory injury. To test the hypothesis that A438079 actually affects the intracellular signaling events in hepatocytes, C57Bl/6 mice were treated with APAP (300 mg/kg) and A438079 (80 mg/kg) or saline and GSH depletion, protein adduct formation, c-jun-N-terminal kinase (JNK) activation, oxidant stress, and liver cell necrosis were determined between 0 and 6 h after APAP administration. APAP caused rapid GSH depletion, extensive protein adduct formation in liver homogenates and in mitochondria, JNK phosphorylation and mitochondrial translocation of phospho-JNK within 2 h, oxidant stress, and extensive centrilobular necrosis at 6 h. A438079 significantly attenuated GSH depletion, which resulted in a 50% reduction of total liver and mitochondrial protein adducts and substantial reduction of JNK activation, mitochondrial P-JNK translocation, oxidant stress, and liver injury. The same results were obtained using primary mouse hepatocytes. A438079 did not directly affect JNK activation induced by tert-butyl hydroperoxide and GSH depletion. However, A438079 dose-dependently inhibited hepatic P450 enzyme activity. Thus, the protective effect of A438079 against APAP hepatotoxicity in vivo can be explained by its effect on metabolic activation and cell death pathways in hepatocytes without involvement of the Nalp3 inflammasome.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Inhibidores Enzimáticos del Citocromo P-450 , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Piridinas/uso terapéutico , Tetrazoles/uso terapéutico , Animales , Proteínas Portadoras/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/patología , Inflamasomas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Pruebas de Función Hepática , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Autophagy is an evolutionarily conserved biological process that degrades intracellular proteins and organelles including damaged mitochondria through the formation of autophagosome. We have previously demonstrated that pharmacological induction of autophagy by rapamycin protects against acetaminophen (APAP)-induced liver injury in mice. In contrast, in the present study, we found that mice with the liver-specific loss of Atg5, an essential autophagy gene, were resistant to APAP-induced liver injury. Hepatocyte-specific deletion of Atg5 resulted in mild liver injury characterized by increased apoptosis and compensatory hepatocyte proliferation. The lack of autophagy in the Atg5-deficient mouse livers was confirmed by increased p62 protein levels and the absence of LC3-lipidation as well as autophagosome formation. Analysis of histological and clinical chemistry parameters indicated that the Atg5 liver-specific knockout mice are resistant to APAP overdose (500 mg/kg). Further investigations revealed that the bioactivation of APAP is normal in Atg5 liver-specific knockout mice although they had lower CYP2E1 expression. There was an increased basal hepatic glutathione (GSH) content and a faster recovery of GSH after APAP treatment due to persistent activation of Nrf2, a transcriptional factor regulating drug detoxification and GSH synthesis gene expression. In addition, we found significantly higher hepatocyte proliferation in the livers of Atg5 liver-specific knockout mice. Taken together, our data suggest that persistent activation of Nrf2 and increased hepatocyte proliferation protect against APAP-induced liver injury in Atg5 liver-specific knockout mice.
Asunto(s)
Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hígado/metabolismo , Proteínas Asociadas a Microtúbulos/deficiencia , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Caspasa 3/metabolismo , Proliferación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citoprotección , Modelos Animales de Enfermedad , Activación Enzimática , Glutatión/metabolismo , Hepatomegalia/genética , Hepatomegalia/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Factores de TiempoRESUMEN
Acetaminophen (APAP) overdose causes liver injury in humans and mice. DNA fragmentation is a hallmark of APAP-induced cell death, and nuclear translocation of apoptosis-inducing factor (AIF) correlates with DNA fragmentation after APAP overdose. To test the hypothesis that AIF may be a critical mediator of APAP-induced cell death, fasted male AIF-deficient Harlequin (Hq) mice and respective wild-type (WT) animals were treated with 200 mg/kg APAP. At 6 h after APAP, WT animals developed severe liver injury as indicated by the increase in plasma alanine aminotransferase (ALT) activities (8600 ± 1870 U/l) and 61 ± 8% necrosis. This injury was accompanied by massive DNA strand breaks in centrilobular hepatocytes (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] assay) and release of DNA fragments into the cytosol (anti-histone ELISA). In addition, there was formation of reactive oxygen (increase in liver glutathione disulfide (GSSG) levels and mitochondrial protein carbonyls) and peroxynitrite (nitrotyrosine [NT] staining) together with mitochondrial translocation of activated c-jun-N-terminal kinase (P-JNK) and release of AIF from the mitochondria. In contrast, Hq mice had significantly less liver injury (ALT: 330 ± 130 U/l; necrosis: 4 ± 2%), minimal nuclear DNA damage, and drastically reduced oxidant stress (based on all parameters) at 6 h. WT and Hq mice had the same baseline levels of cyp2E1 and of glutathione. The initial depletion of glutathione (20 min after APAP) was the same in both groups suggesting that there was no relevant difference in metabolic activation of APAP. Thus, AIF has a critical function in APAP hepatotoxicity by facilitating generation of reactive oxygen in mitochondria and, after nuclear translocation, AIF can be involved in DNA fragmentation.
Asunto(s)
Acetaminofén/toxicidad , Factor Inductor de la Apoptosis/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Western Blotting , Fragmentación del ADN , Sobredosis de Droga , Disulfuro de Glutatión/metabolismo , Hepatocitos/patología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Mitocondrias Hepáticas/metabolismo , Necrosis/inducido químicamente , Necrosis/metabolismo , Ácido Peroxinitroso/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
UNLABELLED: Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870±180U/L) and centrilobular necrosis at 6h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. CONCLUSIONS: the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/genética , Acetaminofén/administración & dosificación , Alanina Transaminasa/metabolismo , Analgésicos no Narcóticos/administración & dosificación , Animales , Fragmentación del ADN/efectos de los fármacos , Sobredosis de Droga , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Etiquetado Corte-Fin in Situ , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Necrosis/inducido químicamente , Transducción de Señal/efectos de los fármacosRESUMEN
Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif(-/-)) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.