RESUMEN
Reduced glutathione (GSH) and ascorbic acid (AA) are the two most abundant low-molecular-weight antioxidants in mammalian tissues. GclmKO knockout mice lack the gene encoding the modifier subunit of the rate-limiting enzyme in GSH biosynthesis; GclmKO mice exhibit 10-40% of normal tissue GSH levels and show no overt phenotype. GuloKO knockout mice, lacking a functional Gulo gene encoding L-gulono-γ-lactone oxidase, cannot synthesize AA and depend on dietary ascorbic acid for survival. To elucidate functional crosstalk between GSH and AA in vivo, we generated the GclmKO/GuloKO double-knockout (DKO) mouse. DKO mice exhibited spontaneous epileptic seizures, proceeding to death between postnatal day (PND)14 and PND23. Histologically, DKO mice displayed neuronal loss and glial proliferation in the neocortex and hippocampus. Epileptic seizures and brain pathology in young DKO mice could be prevented with AA supplementation in drinking water (1 g/L). Remarkably, in AA-rescued adult DKO mice, the removal of AA supplementation for 2-3 weeks resulted in similar, but more severe, neocortex and hippocampal pathology and seizures, with death occurring between 12 and 21 days later. These results provide direct evidence for an indispensable, yet underappreciated, role for the interplay between GSH and AA in normal brain function and neuronal health. We speculate that the functional crosstalk between GSH and AA plays an important role in regulating glutamatergic neurotransmission and in protecting against excitotoxicity-induced brain damage.
RESUMEN
Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn(2+)/(HCO(3)(-))(2) symporter, moving both ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.
Asunto(s)
Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Intrones , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Neomicina/farmacología , Placenta , Embarazo , Saco Vitelino/metabolismoRESUMEN
BACKGROUND & AIMS: Certain liver diseases have been associated with depletion of glutathione (GSH), the major antioxidant in the liver. A recent report about Gclc(h/h) mice with a hepatocyte-specific ablation of Gclc (the gene encoding the catalytic subunit of the rate-limiting enzyme in GSH synthesis) has shown an essential role of GSH in hepatic function. Gclc(h/h) mice develop severe steatosis and die of liver failure within one month, due to ~95% depletion of hepatic GSH; mitochondria are the major affected organelles, displaying abnormal ultrastructure and impaired functioning. METHODS: Gclc(h/h) mice were fed with L-N-acetylcysteine (NAC; 10 g/L) in drinking water, starting at postnatal day 18. RESULTS: Gclc(h/h) mice were rescued by use of NAC supplementation, and survived until adulthood. NAC replenished the mitochondrial GSH pool and attenuated mitochondrial damage, with accompanying diminished hepatic steatosis; however, abnormal liver biochemical tests, hepatocyte death, and hepatic oxidative stress persisted in the rescued mice. At 50 days of age, the liver from rescued Gclc(h/h) mice started to display characteristics of fibrosis and at age 120 days, macronodular cirrhosis was observed. Immunohistostaining for liver-specific markers as well as the expression profile of hepatic cytokines indicated that the repopulation of hepatocytes in the cirrhotic nodules involved the expansion of oval cells. CONCLUSIONS: Replenishment of mitochondrial GSH and restoration of mitochondrial function by NAC prevents mortality caused by the loss of hepatocyte GSH de novo synthesis, allowing steatosis to progress to a chronic stage. Thus, with NAC supplementation, Gclc(h/h) mice provide a model for the development of liver fibrosis and cirrhosis.
Asunto(s)
Acetilcisteína/administración & dosificación , Glutamato-Cisteína Ligasa/deficiencia , Cirrosis Hepática/etiología , Administración Oral , Animales , Antioxidantes/metabolismo , Secuencia de Bases , Citocinas/genética , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(-/-) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(-/-) mice were exposed to either 0.3 ppm ozone or filtered air for 48h. Ozone-induced lung hyperpermeability, as measured by total protein concentration in bronchoalveolar lavage fluid, was surprisingly lower in Gclm(-/-) mice than in wild-type mice. Lung hyperpermeability did not correlate with the degree of neutrophilia or with inflammatory gene expression. Pulmonary antioxidant response to ozone, assessed by increased mRNA levels of metallothionein 1 and 2, alpha-tocopherol transporter protein, and solute carrier family 23 member 2 (sodium-dependent vitamin C transporter) was greater in Gclm(-/-) mice than in Gclm(+/+) mice. These results suggest that compensatory augmentation of antioxidant defenses in Gclm(-/-) mice may confer increased resistance to ozone-induced lung injury.
Asunto(s)
Glutatión/deficiencia , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/genética , Ozono/efectos adversos , Animales , Líquido del Lavado Bronquioalveolar/citología , Diferenciación Celular , Glutamato-Cisteína Ligasa/genética , Glutatión/genética , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismoRESUMEN
Elevated oxidative stress and alteration in antioxidant systems, including glutathione (GSH) decrease, are observed in schizophrenia. Genetic and functional data indicate that impaired GSH synthesis represents a susceptibility factor for the disorder. Here, we show that a genetically compromised GSH synthesis affects the morphological and functional integrity of hippocampal parvalbumin-immunoreactive (PV-IR) interneurons, known to be affected in schizophrenia. A GSH deficit causes a selective decrease of PV-IR interneurons in CA3 and dendate gyrus (DG) of the ventral but not dorsal hippocampus and a concomitant reduction of beta/gamma oscillations. Impairment of PV-IR interneurons emerges at the end of adolescence/early adulthood as oxidative stress increases or cumulates selectively in CA3 and DG of the ventral hippocampus. Such redox dysregulation alters stress and emotion-related behaviors but leaves spatial abilities intact, indicating functional disruption of the ventral but not dorsal hippocampus. Thus, a GSH deficit affects PV-IR interneuron's integrity and neuronal synchrony in a region- and time-specific manner, leading to behavioral phenotypes related to psychiatric disorders.
Asunto(s)
Conducta Animal/fisiología , Relojes Biológicos/fisiología , Hipocampo/citología , Interneuronas/metabolismo , Estrés Oxidativo/fisiología , Parvalbúminas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adaptación Ocular/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Relojes Biológicos/efectos de los fármacos , Calbindina 2 , Calbindinas , Condicionamiento Clásico , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Estimulación Eléctrica/métodos , Electroencefalografía/métodos , Agonistas de Aminoácidos Excitadores/farmacología , Conducta Exploratoria/fisiología , Miedo , Conducta Alimentaria/fisiología , Regulación de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/deficiencia , Glutatión/deficiencia , Ácido Kaínico/farmacología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vías Nerviosas/fisiología , Oxidación-Reducción , Reconocimiento Visual de Modelos/fisiología , Recompensa , Proteína G de Unión al Calcio S100/metabolismo , Conducta Espacial/fisiologíaRESUMEN
Cellular senescence is the irreversible entry of cells into growth arrest. Senescence of primary cells in culture has long been used as an in vitro model for aging. Glutamate-cysteine ligase (GCL) controls the synthetic rate of the important cellular antioxidant glutathione (GSH). The catalytic subunit of GCL, GCLC, is catalytically active and essential for life. By contrast the modifier subunit of GCL, GCLM, is dispensable in mice. Although it is recognized that GCLM increases the rate of GSH synthesis, its physiological role is unclear. Herein, we show that loss of Gclm leads to premature senescence of primary murine fibroblasts as characterized by: (a) diminished growth rate, (b) cell morphology consistent with senescence, (c) increases in senescence-associated beta-galactosidase activity, and (d) cell cycle arrest at the G(1)/S and G(2)/M boundaries. These changes are accompanied by increased intracellular ROS, accumulation of DNA damage, and induction of p53 and p21 proteins. We also found that N-acetylcysteine increases intracellular GSH and prevents premature senescence in Gclm(-/-) cells. These results suggest that the control of GCLM, which in turn controls aspects of the cellular redox environment via GSH, is important in determining the replicative capacity of the cell.
Asunto(s)
Fibroblastos/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Subunidades de Proteína/metabolismo , Acetilcisteína/farmacología , Animales , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Femenino , Feto , Fibroblastos/patología , Depuradores de Radicales Libres/farmacología , Glutamato-Cisteína Ligasa/genética , Glutatión/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Subunidades de Proteína/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes. In astrocytes, all substances increased GSH levels and the activity of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). In neurons, curcumin and to a lesser extent tBHQ increased GCL activity and GSH levels, while quercetin decreased GSH and led to cell death. In the two cell types, the gene that showed the greatest increase in its expression was the one coding for the modifier subunit of GCL (GCLM). The increase in mRNA levels of GCLM was 3 to 7-fold higher than that of the catalytic subunit. In astrocytes from GCLM-knock-out mice showing low GSH (-80%) and low GCL activity (-50%), none of the substances succeeded in increasing GSH synthesis. Our results indicate that GCLM is essential for the up-regulation of GCL activity induced by curcumin, quercetin and tBHQ.
Asunto(s)
Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Neuronas/efectos de los fármacos , Análisis de Varianza , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Curcumina/farmacología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Glutamato-Cisteína Ligasa/química , Hidroquinonas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Quercetina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the past, CYP1A1 protein was known to be located in the endoplasmic reticulum (ER; microsomes). More recently, CYP1A1 was shown also to be targeted to the inner mitochondrial membrane; mitochondrial import is dependent on NH(2)-terminal processing that exposes a cryptic targeting signal. It is interesting that microsomal and mitochondrial CYP1A1 enzymes exhibit different substrate specificities, electron donors, and inducer properties. To understand the physiological functions of microsomal versus mitochondrial CYP1A1, we have generated three knock-in lines by altering the CYP1A1 NH(2) terminus. Cyp1a1(mtt/mtt) mice encode an NH(2)-terminal 31-amino acid-truncated protein, deleting the ER-targeting signal and exposing the cryptic mitochondrial-targeting signal. Cyp1a1(mtp/mtp) mice encode a protein carrying L7N and L17N mutations; this mutant lacks the signal recognition particle (SRP)-binding site and subsequent ER-targeting, but requires proteolysis by a cytosolic peptidase for mitochondrial import. Cyp1a1(mc/mc) mice encode a microsomal protein having R34D and K39I mutations, which abolish the mitochondrial targeting signal. After dioxin or beta-naphthoflavone treatment of these mouse lines, the CYP1A1 protein was shown to be located in the mitochondria of the Cyp1a1(mtp/mtp) and Cyp1a1(mtt/mtt) lines and in microsomes of the Cyp1a1(mc/mc) line. To test for differences in function, we compared the response to dietary benzo[a]pyrene (BaP). After 18 days of daily oral BaP, wild-type and Cyp1a1(mc/mc) mice were completely protected, whereas Cyp1a1(-/-) and Cyp1a1(mtp/mtp) mice showed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. Our data support the likelihood that it is the microsomal rather than mitochondrial CYP1A1 enzyme that protects against oral BaP toxicity.
Asunto(s)
Benzo(a)pireno/administración & dosificación , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Técnicas de Sustitución del Gen , Microsomas/enzimología , Mitocondrias/enzimología , Administración Oral , Secuencia de Aminoácidos , Animales , Pollos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Datos de Secuencia MolecularRESUMEN
The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.
Asunto(s)
Bicarbonatos/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Membrana/metabolismo , Metales/metabolismo , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Cadmio/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular , Células Cultivadas , Biología Computacional , Perros , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Simportadores/química , Simportadores/genéticaRESUMEN
The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/análisis , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP1A1/análisis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/análisis , Citocromo P-450 CYP1A2/genética , Tracto Gastrointestinal/enzimología , Animales , Benzo(a)pireno/farmacología , Western Blotting , Citocromo P-450 CYP1B1 , Inducción Enzimática/efectos de los fármacos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Dibenzodioxinas Policloradas/farmacologíaRESUMEN
The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.08 and 1.0+/-0.08 pmol/oocyte/h, and K(m) values of 0.48+/-0.08 and 0.26+/-0.09 microM, respectively; [b] ZIP8-mediated Cd(2+) uptake is most inhibited by Zn(2+), second-best inhibited by Cu(2+), Pb(2+) and Hg(2+), and not inhibited by Mn(2+) or Fe(2+); and [c] electrogenicity studies demonstrate an influx of two HCO3- anions per one Cd(2+) (or one Zn(2+)) cation, i.e. electroneutral complexes. Using Madin-Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that-similar to ZIP4-the ZIP8 eight-transmembrane protein is largely internalized during Zn(2+) homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn(2+) depletion.
Asunto(s)
Cadmio/farmacocinética , Proteínas de Transporte de Catión/metabolismo , Activación del Canal Iónico/fisiología , Riñón/metabolismo , Transporte de Proteínas/fisiología , Zinc/farmacocinética , Animales , Línea Celular , PerrosRESUMEN
UNLABELLED: Multidrug resistance-associated proteins (Mrps) are adenosine triphosphate-dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin-glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2-related factor-2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte-specific glutamate-cysteine ligase catalytic subunit-null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild-type and Nrf2-null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2-dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert-butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [-185 base pairs (bp)], Mrp3 (-9919 bp), and Mrp4 (-3767 bp). CONCLUSION: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps.
Asunto(s)
Glutatión/metabolismo , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Región de Flanqueo 5' , Animales , Antioxidantes/farmacología , Hidroxianisol Butilado/farmacología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Hepatocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Factor 2 Relacionado con NF-E2/genética , Regiones Promotoras Genéticas , Pirazinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Tionas , TiofenosRESUMEN
Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.
Asunto(s)
Separación Celular/métodos , Citocromo P-450 CYP1A1/deficiencia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/deficiencia , Citocromo P-450 CYP1A2/metabolismo , Animales , Benzo(a)pireno/farmacología , Técnicas de Cultivo de Célula , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Regulación de la Expresión Génica , Genotipo , Humanos , Mucosa Intestinal/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/genéticaRESUMEN
Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition. To characterize the enzyme structure-function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.
Asunto(s)
Glutamato-Cisteína Ligasa/química , Catálisis , Dominio Catalítico , Dimerización , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/biosíntesis , Subunidades de Proteína , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Técnicas del Sistema de Dos HíbridosRESUMEN
UNLABELLED: Oxidative stress is considered to be a critical mediator in liver injury of various etiologies. Depletion of glutathione (GSH), the major antioxidant in liver, has been associated with numerous liver diseases. To explore the specific role of hepatic GSH in vivo, we targeted Gclc, a gene essential for GSH synthesis, so that it was flanked by loxP sites and used the albumin-cyclization recombination (Alb-Cre) transgene to disrupt the Gclc gene specifically in hepatocytes. Deletion within the Gclc gene neared completion by postnatal day (PND)14, and loss of GCLC protein was complete by PND21. Cellular GSH was progressively depleted between PND14 and PND28-although loss of mitochondrial GSH was less severe. Nevertheless, ultrastructural examination of liver revealed dramatic changes in mitochondrial morphology; these alterations were accompanied by striking decreases in mitochondrial function in vitro, cellular ATP, and a marked increase in lipid peroxidation. Plasma liver biochemistry tests from these mice were consistent with progressive severe parenchymal damage. Starting at PND21, livers from hepatocyte-specific Gclc knockout [Gclc(h/h)] mice showed histological features of hepatic steatosis; this included inflammation and hepatocyte death, which progressed in severity such that mice died at approximately 1 month of age due to complications from liver failure. CONCLUSION: GSH is essential for hepatic function and loss of hepatocyte GSH synthesis leads to steatosis with mitochondrial injury and hepatic failure.
Asunto(s)
Hígado Graso/etiología , Glutamato-Cisteína Ligasa/deficiencia , Hepatocitos/metabolismo , Fallo Hepático/genética , Animales , Dominio Catalítico/fisiología , Hígado Graso/genética , Hígado Graso/patología , Glutatión/deficiencia , Hígado/patología , Fallo Hepático/patología , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/fisiología , Estrés OxidativoRESUMEN
Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Ácidos Grasos Omega-3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Proteínas de Ciclo Celular/química , Línea Celular , Proteínas Cullin/química , Ácidos Grasos Omega-3/química , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Estructura Molecular , Oxidación-Reducción , Estrés Oxidativo/genética , Activación TranscripcionalRESUMEN
Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The approximately 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney.
Asunto(s)
Lesión Renal Aguda/genética , Cadmio/toxicidad , Proteínas de Transporte de Catión/genética , Dosificación de Gen , Túbulos Renales Proximales/efectos de los fármacos , Testículo/efectos de los fármacos , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/fisiología , Cromosomas Artificiales Bacterianos/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Túbulos Renales Proximales/patología , Pulmón/metabolismo , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Necrosis , Fenotipo , ARN Mensajero/biosíntesis , Testículo/irrigación sanguínea , Testículo/patologíaRESUMEN
Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/fisiología , Carbazoles/metabolismo , Carcinógenos/metabolismo , Familia de Multigenes , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/fisiología , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/fisiología , Femenino , Humanos , Pulmón/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/fisiologíaRESUMEN
Some cytochrome P450 (CYP) heme-thiolate enzymes participate in the detoxication and, paradoxically, the formation of reactive intermediates of thousands of chemicals that can damage DNA, as well as lipids and proteins. CYP expression can also affect the production of molecules derived from arachidonic acid, and alters various downstream signal-transduction pathways. Such changes can be precursors to malignancy. Recent studies in mice have changed our perceptions about the function of CYP1 enzymes. We suggest a two-tiered system to predict an overall inter-individual risk of tumorigenesis based on DNA variants in certain 'early defence' CYP genes, combined with polymorphisms in various downstream target genes.
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Carcinógenos Ambientales/toxicidad , Transformación Celular Neoplásica/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Neoplasias/enzimología , Transducción de Señal , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biotransformación , Carcinógenos Ambientales/farmacocinética , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Redes y Vías Metabólicas , Ratones , Modelos Biológicos , Neoplasias/inducido químicamente , Neoplasias/genética , Farmacogenética , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Medición de Riesgo , Factores de Riesgo , Factores de Transcripción/metabolismoRESUMEN
Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.