RESUMEN
Alcohol-mediated liver injury is associated with changes in the level of the major cellular antioxidant glutathione (GSH). It is interesting to investigate if the changes in intracellular GSH level through exogenous agents affect the intracellular cysteine content and the protein adduct formation indicative of oxidative insult in chronic alcohol treated liver cells. In VL-17A cells treated with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) plus 100 mM ethanol, an increase in cysteine concentration which was accompanied by decreases in hydroxynonenal (HNE) and glutathionylated protein adducts were observed. Pretreatment of 100 mM ethanol treated VL-17A cells with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) had opposite effects. Thus, altered GSH level through exogenous agents may either potentiate or ameliorate chronic alcohol-mediated protein adduct formation and change the cysteine level in chronic alcohol treated VL-17A cells. The gene expression of non-treated and ethanol-treated hepatocytes in 2 microarray datasets was also compared to locate differentially expressed genes involved in cysteine metabolism. The study demonstrates that increased protein adducts formation and changes in cysteine concentration occur under chronic alcohol condition in liver cells which may increase alcohol-mediated oxidative injury.
Asunto(s)
Cisteína/metabolismo , Etanol/toxicidad , Glutatión/metabolismo , Estrés Oxidativo/efectos de los fármacos , Alcohol Deshidrogenasa/genética , Aldehídos/metabolismo , Familia 2 del Citocromo P450/genética , Etanol/metabolismo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Estrés Oxidativo/genética , Transcriptoma/efectos de los fármacosRESUMEN
PURPOSE: The deficiency of glutathione (GSH) has been linked to several diseases. The study investigated the role of GSH as a protective factor against hyperglycemia-mediated injury in VL-17A cells treated with 50 mM glucose. METHODS: The cell viability and different oxidative stress parameters including glyoxalase I activity were measured. RESULTS: GSH supplementation with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) increased the viability, GSH level and the GSH-dependent glyoxalase I activity in 50 mM glucose-treated VL-17A cells. Further, pretreatment of 50 mM glucose-treated VL-17A cells with NAC or UDCA decreased oxidative stress (levels of reactive oxygen species and protein carbonylation), apoptosis (caspase 3 activity and annexin V-propidium iodide positive cells) and glutathionylated protein formation, a measure of oxidative stress. GSH depletion with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) potentiated the decrease in viability, glyoxalase I activity and increase in oxidative stress and apoptosis, with decreased GSH levels in 50 mM glucose-treated VL-17A cells. CONCLUSION: Thus, changes in GSH levels with exogenous agents such as NAC, UDCA, BSO or DEM modulate hyperglycemia-mediated injury in a cell model of VL-17A liver cells.
Asunto(s)
Apoptosis , Glutatión/metabolismo , Hepatocitos/metabolismo , Hiperglucemia/metabolismo , Estrés Oxidativo , Acetilcisteína/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Antimetabolitos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Butionina Sulfoximina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Clonales , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Glucosa/efectos adversos , Glutatión/antagonistas & inhibidores , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Lactoilglutatión Liasa/metabolismo , Maleatos/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Ácido Ursodesoxicólico/farmacologíaRESUMEN
Gluthathione (GSH) is a major cellular antioxidant. The present study utilizing VL-17A cells exposed to chronic alcohol plus high glucose investigated the changes in oxidative stress, toxicity, and glyoxalase 1 activity as a detoxification pathway due to changes in GSH level through GSH supplementation with N-acetyl cysteine (NAC) or ursodeoxycholic acid (UDCA) and its depletion through buthionine sulfoximine (BSO) or diethyl maleate (DEM). Glyoxalase 1 plays an important role in detoxification of methylglyoxal which is formed as a precursor of advanced glycated end products formed due to high glucose mediated oxidative stress. Significant changes in glyoxalase 1 activity utilizing methylglyoxal or glyoxal as substrates occurred with NAC or UDCA or BSO or DEM supplementation in chronic alcohol plus high glucose treated VL-17A cells. NAC or UDCA administration in chronic alcohol plus high glucose treated VL-17A cells increased viability and decreased ROS levels, lipid peroxidation and 3-nitrotyrosine adduct formation. Similarly, GSH depletion with BSO or DEM had an opposite effect on the parameters in chronic alcohol plus high glucose treated VL-17A cells. In conclusion, modulation of GSH with NAC or UDCA or BSO or DEM leads to significant changes in oxidative stress, glyoxalase 1 enzyme activity and toxicity in chronic alcohol plus high glucose treated VL-17A cells.
Asunto(s)
Células/enzimología , Etanol/efectos adversos , Glutatión/metabolismo , Lactoilglutatión Liasa/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células/efectos de los fármacos , Células/metabolismo , Glucosa/efectos adversos , Glucosa/análisis , Humanos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Several antioxidants and agents having similar antioxidant effects are known to exert beneficial effects in ameliorating the injurious effects of hyperglycemia on liver in different diabetic in vitro and in vivo models. The review deals with some of the agents which have been shown to exert protective effects on liver against hyperglycemic insult and the various mechanisms involved. The different classes of agents which protect the diabetic liver or decrease the severity of hyperglycemia mediated injury include flavonoids, catechins, and other polyphenolic compounds, curcumin and its derivatives, certain vitamins, hormones and drugs, trace elements, prototypical antioxidants and amino acids. Some of the pronounced changes mediated by the antioxidants in liver exposed to hyperglycemia include decreased oxidative stress, and alterations in carbohydrate and lipid metabolism. Other mechanisms through which the agents ameliorate hyperglycemia mediated liver injury include decrease in oxidative DNA and protein damage, restoration of mitochondrial structural and functional integrity, decrease in inflammation and improved insulin signaling. Thus, antioxidants may prove to be an important mode of defense in maintaining normal hepatic functions in diabetes.
Asunto(s)
Antioxidantes/administración & dosificación , Diabetes Mellitus/tratamiento farmacológico , Hiperglucemia/tratamiento farmacológico , Hígado/lesiones , Estrés Oxidativo/efectos de los fármacos , Animales , Diabetes Mellitus/metabolismo , Humanos , Hiperglucemia/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismoRESUMEN
BACKGROUND: In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure. METHODS: In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied. RESULTS: Inhibition of CYP2E1 with 10µM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation. CONCLUSIONS: Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1. GENERAL SIGNIFICANCE: The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Inhibidores del Citocromo P-450 CYP2E1 , Glucosa/farmacología , Productos Finales de Glicación Avanzada/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Compuestos Alílicos/farmacología , Línea Celular , Citocromo P-450 CYP2E1/metabolismo , Humanos , Estrés Oxidativo/efectos de los fármacos , Pirazoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sulfuros/farmacologíaRESUMEN
Research on Cytochrome P450 2E1 (CYP2E1), a key enzyme in alcohol metabolism has been very well documented in literature. Besides the involvement of CYP2E1 in alcohol metabolism as illustrated through the studies discussed in the chapter, recent studies have thrown light on several other aspects of CYP2E1 i.e. its extrahepatic expression, its involvement in several diseases and pathophysiological conditions; and CYP2E1 mediated carcinogenesis and modulation of drug efficacy. Studies involving these interesting facets of CYP2E1 have been discussed in the chapter focusing on the recent observations or ongoing studies illustrating the crucial role of CYP2E1 in disease development and drug metabolism.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Investigación Biomédica , Citocromo P-450 CYP2E1/metabolismo , Hepatopatías/etiología , Estrés Oxidativo , Animales , Humanos , Hepatopatías/metabolismoRESUMEN
AIM: Ethanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde-acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated. MAIN METHODS: Chronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor - diallyl sulfide or ADH inhibitor - pyrazole or ADH and CYP2E1 inhibitor - 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis. KEY FINDINGS: Inhibition of CYP2E1 with 10 µM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury. SIGNIFICANCE: CYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.
Asunto(s)
Acetaldehído/metabolismo , Inhibidores del Citocromo P-450 CYP2E1 , Etanol/toxicidad , Malondialdehído/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/biosíntesis , Compuestos Alílicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP2E1/biosíntesis , Esquema de Medicación , Inhibidores Enzimáticos/farmacología , Etanol/administración & dosificación , Fomepizol , Células Hep G2 , Humanos , Pirazoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Sulfuros/farmacologíaRESUMEN
CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis-specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Interferón gamma/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Interleucina-4/metabolismo , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo , Tuberculosis Pulmonar/metabolismo , Adulto JovenRESUMEN
High glucose mediated oxidative stress and cell death is a well documented phenomenon. Using VL-17A cells which are HepG2 cells over-expressing alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) and control HepG2 cells, the association of ADH and CYP2E1 with high glucose mediated oxidative stress and toxicity in liver cells was investigated. Cell viability was measured and apoptosis or necrosis was determined through caspase-3 activity, Annexin V-propidium iodide staining and detecting decreases in mitochondrial membrane potential. Reactive oxygen species, lipid peroxidation and the formation of advanced glycated-end products were assessed. The levels of several antioxidants which included glutathione, glutathione peroxidase, catalase and superoxide dismutase were altered in high glucose treated VL-17A cells. Greater toxicity was observed in VL-17A cells exposed to high glucose when compared to HepG2 cells. Oxidative stress parameters were greatly increased in high glucose exposed VL-17A cells and apoptotic cell death was observed. Inhibition of CYP2E1 or caspase 3 or addition of the antioxidant trolox led to significant decreases in high glucose mediated oxidative stress and toxicity. Thus, the over-expression of ADH and CYP2E1 in liver cells is associated with increased high glucose mediated oxidative stress and toxicity.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Sistema Enzimático del Citocromo P-450/metabolismo , Glucosa/farmacología , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Familia 2 del Citocromo P450 , Humanos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Hyperglycemia or alcoholism can lead to impaired liver functions. Cytochrome P450 2E1 (CYP2E1) is elevated in hyperglycemia or alcoholism and plays a critical role in generating oxidative stress in the cell. METHODS: In the present study, we have used VL-17A cells that overexpress the alcohol metabolizing enzymes [alcohol dehydrogenase (ADH) and CYP2E1] to investigate the toxicity due to ethanol (EtOH) plus high glucose. Toxicity was assessed through viability assay and amount of acetaldehyde adduct formation. Oxidative stress parameters included measuring reactive oxygen species (ROS) levels and malondialdehyde adduct formation. Apoptosis was determined through caspase-3 activity, Annexin V- Propidium iodide staining, and changes in mitochondrial membrane potential. The effects of antioxidants and specific inhibitors of ADH and CYP2E1 on cell viability and ROS levels were also studied. RESULTS: When present together, EtOH plus high glucose-treated VL-17A cells exhibited greater oxidative stress and toxicity than other groups. Apoptosis was observed in liver cells treated with the toxins, and the EtOH plus high glucose-treated VL-17A cells exhibited apoptosis to the largest extent. A distinct and graded increase in CYP2E1 level occurred in the different groups of VL-17A cells. Further, antioxidants or inhibitors of ADH and CYP2E1 were effective in decreasing the observed oxidative stress and toxicity. CONCLUSIONS: The combined oxidative insult due to alcohol plus high glucose leads to greater liver injury, which may prove to be a timely warning for the injurious effects of alcohol consumption in diabetics.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Depresores del Sistema Nervioso Central/toxicidad , Citocromo P-450 CYP2E1/metabolismo , Etanol/toxicidad , Glucosa/toxicidad , Hepatocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Edulcorantes/toxicidad , Acetaldehído/metabolismo , Alcohol Deshidrogenasa/efectos de los fármacos , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Citocromo P-450 CYP2E1/efectos de los fármacos , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.
Asunto(s)
Hepatopatías Alcohólicas/genética , Animales , Biología Computacional , Bases de Datos como Asunto , Regulación de la Expresión Génica , Humanos , Ratones , Ratas , Transducción de Señal/genéticaRESUMEN
Cytochrome P450 2E1 (CYP2E1), a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury. The review deals with the changes in various cellular pathways in liver linked with the changes in regulation of CYP2E1 under hyperglycemic conditions. Some of the hepatic abnormalities associated with hyperglycemia-mediated induction of CYP2E1 include increased oxidative stress, changes in mitochondrial structure and function, apoptosis, nitrosative stress, and increased ketone body accumulation. Thus, changes in regulation of CYP2E1 are associated with the injurious effects of hyperglycemia in liver.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Hiperglucemia/patología , Hiperglucemia/fisiopatología , Hígado/enzimología , Hígado/lesiones , Animales , Diabetes Mellitus/patología , Diabetes Mellitus/fisiopatología , Dieta , Modelos Animales de Enfermedad , Humanos , Hígado/ultraestructura , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Estrés OxidativoRESUMEN
Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Etanol/toxicidad , Glutatión/metabolismo , Estrés Oxidativo/efectos de los fármacos , Alcohol Deshidrogenasa/metabolismo , Consumo de Bebidas Alcohólicas/efectos adversos , Carcinoma Hepatocelular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citocromo P-450 CYP2E1/metabolismo , Etanol/administración & dosificación , Etanol/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Factores de TiempoRESUMEN
The increasing prevalence of diabetes continues to be a major health issue world wide. Liver injury is highly relevant in diabetic subjects and Type 2 diabetes mellitus is an acknowledged risk factor for non-alcoholic fatty liver disease (NAFLD). Chronic hyperglycemia is an important feature of diabetes and hyperglycemia induced mitochondrial dysfunction in liver holds importance in context to NAFLD. Some of the hyperglycemia induced changes in mitochondria include decreased oxidative phosphorylation, increased oxidative stress and ultra structural abnormalities. The following review identifies the mechanisms through which hyperglycemia causes mitochondrial dysfunction in liver.
Asunto(s)
Diabetes Mellitus/metabolismo , Hiperglucemia/metabolismo , Mitocondrias Hepáticas/patología , Animales , Humanos , Hígado/patologíaRESUMEN
Hyperglycemia which characterizes diabetes, leads to several abnormalities in the cellular pathways. We examined the toxicity of glucose in human hepatoma HepG2 cells. HepG2 cells when incubated with 50mM glucose for 72h showed altered morphology i.e. presence of detached and shrunken rounded cells. Glucose treated HepG2 cells also exhibited a significant decrease in viability. Caspase-3 activity and Annexin V staining were significantly increased in glucose treated HepG2 cells, suggesting an apoptotic mode of cell death. Glucose induced apoptosis in HepG2 cells was a consequence of increased oxidative stress as evidenced by the increased reactive oxygen species (ROS) level, lipid peroxidation, protein carbonyl and 3-nitrotyrosine adduct formation. The intracellular antioxidant glutathione was found to be increased in HepG2 cells treated with glucose, possibly to aid the cells to overcome the persistent oxidative stress elicited by glucose in HepG2 cells. N-Acetyl cysteine, a precursor of glutathione and an antioxidant was effective in reversing the morphological changes, increasing the viability, decreasing the ROS level and 4-hydroxynonenal and 3-nitrotyrosine adduct formation, thus validating the role of oxidative stress as a major mechanism for glucose induced apoptosis in HepG2 cells. These results suggest that glucose induces apoptosis in liver cells through increased oxidative stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Glucosa/toxicidad , Anexinas/metabolismo , Carcinoma Hepatocelular , Caspasa 3/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosa/administración & dosificación , Humanos , L-Lactato Deshidrogenasa , Neoplasias Hepáticas , Manitol/farmacología , Coloración y Etiquetado , Factores de TiempoRESUMEN
Diabetes, characterized by chronic hyperglycemia, has reached serious epidemic proportions. It is also not infrequent to find increased incidence of liver injury in diabetics and hyperglycemia plays an important role in promoting liver injury through several mechanisms. The following review identifies the pathways through which hyperglycemia causes changes in liver of various animal models and liver cell culture models, and elucidates the mechanisms and consequences of hyperglycemia induced liver injury in humans. Some of the pathways which are hyperglycemia driven include increased oxidative and nitrosative stress, activation of stress signaling pathways and increased cytokine levels, impairment of protective mechanisms such as the expression of molecular chaperones and proteosome activity, and dysregulation of glucose and lipid metabolism. Thus, hyperglycemia induced changes in the liver's cellular environment in in vitro and in vivo models have been documented extensively in the literature.
Asunto(s)
Hiperglucemia/metabolismo , Hígado/metabolismo , Adaptación Fisiológica , Animales , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hiperglucemia/complicaciones , Hepatopatías/etiología , Hepatopatías/metabolismoRESUMEN
UNLABELLED: Cytochrome P450 2E1 (CYP2E1) activates several hepatotoxins and contributes to alcoholic liver damage. Obesity is a growing health problem in the United States. The aim of the present study was to evaluate whether acetone- or pyrazole-mediated induction of CYP2E1 can potentiate liver injury in obesity. CYP2E1 protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induced distinct histological changes in liver and significantly higher aminotransferase enzymes in obese mice compared to obese controls or acetone- or pyrazole-treated lean mice. Higher caspase-3 activity and numerous apoptotic hepatocytes were observed in the acetone- or pyrazole-treated obese mice. Increased protein carbonyls, malondialdehyde, 4-hydroxynonenal-protein adducts, elevated levels of inducible nitric oxide synthase, and higher 3-nitrotyrosine protein adducts were found in livers of acetone- or pyrazole-treated obese animals, suggesting elevated oxidative and nitrosative stress. Liver tumor necrosis factor alpha levels were higher in pyrazole-treated animals. The CYP2E1 inhibitor chlormethiazole and iNOS inhibitor N-(3-(aminomethyl)-benzyl) acetamidine abrogated the toxicity and the oxidative/nitrosative stress elicited by the induction of CYP2E1. CONCLUSION: These results show that obesity contributes to oxidative stress and liver injury and that induction of CYP2E1 enhances these effects.
Asunto(s)
Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Hepatopatías/complicaciones , Hepatopatías/genética , Obesidad/complicaciones , Acetona , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3/metabolismo , Catálisis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas , Aductos de ADN/metabolismo , Fragmentación del ADN/efectos de los fármacos , Inhibidores Enzimáticos , Regulación Enzimológica de la Expresión Génica , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Obesidad/genética , Proteínas/metabolismo , Pirazoles , ARN Mensajero/metabolismo , Solventes , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.
Asunto(s)
Citocromo P-450 CYP2E1/toxicidad , S-Adenosilmetionina/farmacología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Fragmentación del ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Hepatopatías/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias Hepáticas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , PirazolesRESUMEN
Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.
Asunto(s)
Benzoquinonas/farmacología , Citocromo P-450 CYP2E1/fisiología , Citocromo P-450 CYP2E1/toxicidad , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Hígado/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Hígado/enzimologíaRESUMEN
Freshly isolated peripheral blood lymphocytes from control rats were found to catalyze the N-demethylation of erythromycin, known to be mediated by cytochrome P450 3A (CYP3A) isoenzymes in rat liver. Pretreatment of rats with dexamethasone (100 mg/kgx3 days, i.p.), a CYP3A inducer, resulted in 3-4-fold increase in the activity of erythromycin demethylase (EMD) in freshly isolated peripheral blood lymphocytes. This increase in the enzyme activity was found to be associated with an increase in the rate of the reaction and affinity of the substrate towards the enzyme. Significant inhibition of the EMD activity on in vitro addition of ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised against rat liver CYP3A have suggested that EMD activity in blood lymphocytes is catalyzed primarily by CYP3A isoenzymes. Further, immunoblot analysis with polyclonal antibody raised against rat liver CYP3A revealed significant immunoreactivity, co-migrating with the liver isoenzyme, indicating constitutive expression of CYP3A in blood lymphocytes. Pretreatment with dexamethasone was found to significantly increase the expression of CYP3A protein in freshly isolated rat blood lymphocytes, as observed with liver. Likewise, significant CYP3A mRNA detected in control rat blood lymphocytes has further demonstrated constitutive expression of CYP3A isoenzymes in blood lymphocytes. Furthermore, several fold increase in CYP3A mRNA expression following pretreatment with dexamethasone showed similarities in the regulation of CYP3A isoenzymes in rat blood lymphocytes with the liver enzyme. The data suggest that the blood lymphocytes can be used to monitor tissue expression of CYP3A isoenzymes and validate the suitability of lymphocytes as surrogates of CYP status in less accessible target tissues.