RESUMEN
The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs - including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2) - at the 'stress-responding' cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'-UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wild-type) or mutant - pGL4.38-Cyp2a5_StREMut and pGL4.38-Cyp2a5_XREMut - reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC50 and EC50 of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wild-type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Bioensayo/métodos , Familia 2 del Citocromo P450/genética , Genes Reporteros/genética , Regiones Promotoras Genéticas/genética , Regiones no Traducidas 3'/genética , Células A549 , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Humanos , Luciferasas/genética , Células MCF-7 , Mutagénesis Sitio-Dirigida , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Pruebas de Toxicidad/métodosRESUMEN
Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.
Asunto(s)
Citocromo P-450 CYP2A6/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Citocromo P-450 CYP2A6/genética , Daño del ADN , Ensayo de Cambio de Movilidad Electroforética , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Bilirrubina/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hígado/enzimología , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/enzimología , Estrés Oxidativo , Transporte Activo de Núcleo Celular , Adaptación Fisiológica , Animales , Antioxidantes/farmacología , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Familia 2 del Citocromo P450 , Inhibidores Enzimáticos/farmacología , Ferredoxina-NADP Reductasa/metabolismo , Glucuronosiltransferasa/metabolismo , Cinética , Hígado/efectos de los fármacos , Masculino , Ratones Endogámicos DBA , Microsomas Hepáticos/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Carbonilación ProteicaRESUMEN
Most drugs are metabolized in the liver by cytochromes P450 (CYPs). Stress can modify CYP-catalyzed drug metabolism and subsequently, the pharmacokinetic profile of a drug. Current evidence demonstrates a gene-, stress- and species-specific interference in stress-mediated regulation of genes encoding the major drug-metabolizing CYP isozymes. Stress-induced up-regulation of CYPs that metabolize the majority of prescribed drugs can result in their increased metabolism and consequently, in failure of pharmacotherapy. In contrast, stress-induced down-regulation of CYP isozymes, including CYP2E1 and CYP2B1/2, potentially reduces metabolism of several toxicants and specific drugs-substrates resulting in increased levels and altered toxicity. The primary stress effectors, the adrenergic receptor-linked pathways and glucocorticoids, play primary and distinct roles in stress-mediated regulation of CYPs. Evidence demonstrates that stress regulates major drug metabolizing CYP isozymes, suggesting that stress should be considered to ensure pharmacotherapy efficacy and minimize drug toxicity. A detailed understanding of the molecular events underlying the stress-dependent regulation of drug metabolizing CYPs is crucial both for the design of new drugs and for physiology-based pharmacokinetic and pharmacodynamic modeling.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Farmacocinética , Estrés Psicológico/enzimología , Animales , HumanosRESUMEN
The mouse hepatic cytochrome P450 (CYP) 2A5 and its human orthologue CYP2A6 catalyse the metabolism of a number of drugs and toxins, such as halothane and aflatoxin B1. The enzymes are named "Coumarin 7-hydroxylase" and "Nicotine Hydroxylase", respectively, by virtue of their high affinity and specific activity towards these compounds. Bilirubin, the breakdown product of haem, has been suggested to be the endogenous substrate for both enzymes. Uniquely, CYP2A5 and CYP2A6 are induced during pathological conditions associated with liver injury when the function of most other CYP enzymes is compromised, which suggests an exceptional mode of regulation of the corresponding genes. Regulation of these genes is indeed complex where the promoters interact with multiple stress-activated transcription factors. The Cyp2a5 promoter contains a "stress-responding" cluster of binding motifs, which interact with major mediators of toxic insults including nuclear factor-E2 p45-related factor 2 (Nrf2) and aryl hydrocarbon receptor (AhR). These interactions are crucial in the up-regulation of the genes under stress conditions. Additionally, elevated transcription is also achieved through mRNA stabilisation mediated by interaction of the stress activated heterogenous ribonucleoprotein A1 (hnRNP A1) with the 3'UTR of the CYP2A5/CYP2A6 mRNA. The up-regulation via enhanced transcription combined with mRNA stabilisation, as seen in some of the stress situations, leads to a particularly strong, fast and persistent response. This review brings together knowledge obtained from studies in our laboratories and others' on regulation of Cyp2a5/CYP2A6 genes in response to toxic insults and toxicological significance of their catalytic activities that may provide clues to a functional role of the enzymes in relation to liver toxicity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Hepatopatías/patología , Hígado/patología , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Bilirrubina/metabolismo , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Regulación de la Expresión Génica , Humanos , Hepatopatías/genética , Ratones , Preparaciones Farmacéuticas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
The reporter gene assay AREc32 is based on the induction of the Nrf2 mediated oxidative stress response pathway in the human breast cancer cell line MCF7, where eight copies of the antioxidant response element (ARE) are linked to a reporter gene encoding for luciferase. The Nrf2-ARE pathway is responsive to many chemicals that cause oxidative stress, among them a large number of pesticides and skin irritants. We adopted and validated the AREc32 bioassay for water quality testing. tert-Butylhydroquinone served as the positive control, phenol as the negative control and other reactive chemicals were assessed for their specificity. An environmentally relevant reference chemical, benzo(a)pyrene was the most potent inducer of all tested chemicals. The concentration causing an induction ratio (IR) of 1.5 (EC(IR1.5)) was chosen as the effect benchmark value. The assay was applied to 21 water samples ranging from sewage to drinking water, including secondary treatment and various tertiary treatment options (ozonation, biologically activated carbon filtration, membrane filtration, reverse osmosis, advanced oxidation, chlorination, chloramination). The samples were enriched by solid phase extraction. In most samples the oxidative stress response was far more sensitive than cytotoxicity. The primary and secondary treated effluent exceeded the effect threshold IR 1.5 at a relative enrichment factor (REF) of 1, i.e., the native samples were active. All tertiary treated samples were less potent and their EC(IR1.5) lay between REF 1 and 10. The Nrf2 pathway was induced at a REF of approximately 10 for surface waters and drinking water, and above this enrichment cytotoxicity took over in most samples and quenched the induction. The blank (ultrapure water run through the sample enrichment process) was cytotoxic at an REF of 100, which is the limit of concentrations range that can be evaluated. Treatment typically decreased both the cytotoxicity and oxidative stress response apart from drinking water treatment where chlorination caused an increase in oxidative stress response, presumably due to the formation of disinfection by-products. This study demonstrates the relevance and applicability of the oxidative stress response pathway for water quality monitoring.
Asunto(s)
Bioensayo/métodos , Monitoreo del Ambiente/métodos , Genes Reporteros , Contaminantes Químicos del Agua/análisis , Elementos de Respuesta Antioxidante/genética , Línea Celular Tumoral , Agua Potable/química , Humanos , Luciferasas/análisis , Luciferasas/metabolismo , Estrés Oxidativo/genética , Aguas del Alcantarillado/química , Contaminantes Químicos del Agua/toxicidad , Purificación del AguaRESUMEN
Various hormonal and monoaminergic systems play determinant roles in the regulation of several cytochromes P450 (P450s) in the liver. Growth hormone (GH), prolactin, and insulin are involved in P450 regulation, and their release is under dopaminergic control. This study focused on the role of D2-dopaminergic systems in the regulation of the major drug-metabolizing P450s, i.e., CYP3A, CYP2C, and CYP2D. Blockade of D2-dopaminergic receptors with either sulpiride (SULP) or 4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol (L-741,626) markedly down-regulated CYP3A1/2, CYP2C11, and CYP2D1 expression in rat liver. This suppressive effect appeared to be mediated by the insulin/phosphatidylinositol 3-kinase/Akt/FOXO1 signaling pathway. Furthermore, inactivation of the GH/STAT5b signaling pathway appeared to play a role in D2-dopaminergic receptor-mediated down-regulating effects on these P450s. SULP suppressed plasma GH levels, with subsequently reduced activation of STAT5b, which is the major GH pulse-activated transcription factor and has up-regulating effects on various P450s in hepatic tissue. Levels of prolactin, which exerts down-regulating control on P450s, were increased by SULP, which may contribute to SULP-mediated effects. Finally, it appears that SULP-induced inactivation of the cAMP/protein kinase A/cAMP-response element-binding protein signaling pathway, which is a critical regulator of pregnane X receptor and hepatocyte nuclear factor 1α, and inactivation of the c-Jun N-terminal kinase contribute to SULP-induced down-regulation of the aforementioned P450s. Taken together, the present data provide evidence that drugs acting as D2-dopaminergic receptor antagonists might interfere with several major signaling pathways involved in the regulation of CYP3A, CYP2C, and CYP2D, which are critical enzymes in drug metabolism, thus affecting the effectiveness of the majority of prescribed drugs and the toxicity and carcinogenic potency of a plethora of toxicants and carcinogens.
Asunto(s)
Antipsicóticos/farmacología , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Receptores de Dopamina D2/fisiología , Sulpirida/farmacología , Animales , Antipsicóticos/efectos adversos , Antagonistas de los Receptores de Dopamina D2 , Etanolaminas/metabolismo , Factores de Transcripción Forkhead/fisiología , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hormonas/sangre , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/fisiología , Ratas , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Sulpirida/efectos adversosAsunto(s)
Arsenitos/toxicidad , Bilirrubina/orina , Estrés Oxidativo/efectos de los fármacos , Animales , Glutatión/análisis , Glutatión/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos DBA , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic "Bilirubin Oxidase" (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14-22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K(i) of 2.23 µM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human "Bilirubin Oxidase" where bilirubin is potentially a substrate and a regulator of the enzyme.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Bilirrubina/metabolismo , Biliverdina/metabolismo , Microsomas/metabolismo , Cromatografía Líquida de Alta Presión , Cicloheximida/farmacología , Citocromo P-450 CYP2A6 , Semivida , Células Hep G2 , Humanos , Microsomas/enzimología , Modelos Moleculares , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/enzimología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Stress is a critical player in the regulation of the major cytochrome P-450s (CYPs) that metabolize the majority of the prescribed drugs. Early in life, maternal deprivation (MD) stress and repeated restraint stress (RS) modified CYP expression in a stress-specific manner. In particular, the expression of CYP3A1 and CYP2C11 was increased in the liver of MD rats, whereas RS had no significant effect. In contrast, hepatic CYP2D1/2 activity was increased by RS, whereas MD did not affect it. The primary effectors of the stress system, glucocorticoids and epinephrine, highly induced CYP3A1/2. Epinephrine also induced the expression of CYP2C11 and CYP2D1/2. Further investigation indicated that AR-agonists may modify CYP regulation. In vitro experiments using primary hepatocyte cultures treated with the AR-agonists phenylephrine, dexmedetomidine, and isoprenaline indicated an AR-induced upregulating effect on the above-mentioned CYPs mediated by the cAMP/protein kinase A and c-Jun NH2-terminal kinase signaling pathways. Interestingly though, in vivo pharmacological manipulations of ARs using the same AR-agonists led to a suppressed hepatic CYP expression profile, indicating that the effect of the complex network of central and peripheral AR-linked pathways overrides that of the hepatic ARs. The AR-mediated alterations in CYP3A1/2, CYP2C11, and CYP2D1/2 expressions are potentially connected with those observed in the activation of signal transducer and activator of transcription 5b. In conclusion, stress and AR-agonists may modify the expression of the major CYP genes involved in the metabolism of drugs used in a wide range of diseases, thus affecting drug efficacy and toxicity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Hepatocitos/metabolismo , Receptores Adrenérgicos/metabolismo , Esteroide 16-alfa-Hidroxilasa/metabolismo , Estrés Fisiológico , Estrés Psicológico/metabolismo , Agonistas Adrenérgicos/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Células Cultivadas , Corticosterona/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Familia 2 del Citocromo P450 , Inducción Enzimática/efectos de los fármacos , Epinefrina/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Privación Materna , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos/química , Restricción Física/efectos adversos , Transducción de Señal/efectos de los fármacos , Esteroide 16-alfa-Hidroxilasa/genética , Estrés Psicológico/patologíaRESUMEN
We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic "BR oxidase". A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible "BR oxidase" where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/fisiología , Animales , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Hidrocarburo de Aril Hidroxilasas/metabolismo , Bilirrubina/metabolismo , Bilirrubina/fisiología , Biliverdina/biosíntesis , Cicloheximida/farmacología , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Inducción Enzimática , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Microsomas/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The Flinders Sensitive Line (FSL) rat model of depression exhibits some behavioral, neurochemical, and pharmacological features that have been reported in depressed patients and has been very effective in screening antidepressants. Major factor that determines the effectiveness and toxicity of a drug is the drug metabolizing capacity of the liver. Therefore, in order to discriminate possible differentiation in the hepatic drug metabolism between FSL rats and Sprague-Dawley (SD) controls, their hepatic metabolic profile was investigated in this study. The data showed decreased glutathione (GSH) content and glutathione S-transferase (GST) activity and lower expression of certain major CYP enzymes, including the CYP2B1, CYP2C11 and CYP2D1 in FSL rats compared to SD controls. In contrast, p-nitrophenol hydroxylase (PNP), 7-ethoxyresorufin-O-dealkylase (EROD) and 16alpha-testosterone hydroxylase activities were higher in FSL rats. Interestingly, the wide spread environmental pollutant benzo(alpha)pyrene (B(alpha)P) induced CYP1A1, CYP1A2, CYP2B1/2 and ALDH3c at a lesser extend in FSL than in SD rats, whereas the antidepressant mirtazapine (MIRT) up-regulated CYP1A1/2, CYP2C11, CYP2D1, CYP2E1 and CYP3A1/2, mainly, in FSL rats. The drug also further increased ALDH3c whereas suppressed GSH content in B(alpha)P-exposed FSL rats. In conclusion, several key enzymes of the hepatic biotransformation machinery are differentially expressed in FSL than in SD rats, a condition that may influence the outcome of drug therapy. The MIRT-induced up-regulation of several drug-metabolizing enzymes indicates the critical role of antidepressant treatment that should be always taken into account in the designing of treatment and interpretation of insufficient pharmacotherapy or drug toxicity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Trastorno Depresivo/enzimología , Modelos Animales de Enfermedad , Hígado/enzimología , Análisis de Varianza , Animales , Antidepresivos Tricíclicos/farmacología , Western Blotting , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A1/metabolismo , Dopamina/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hígado/efectos de los fármacos , Masculino , Mianserina/análogos & derivados , Mianserina/farmacología , Mirtazapina , Norepinefrina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Choline is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions in both humans and rodents. Various pathophysiological states have been linked to choline deprivation (CD). The aim of the present study was to determine the effect of CD upon biochemical, histological and metabolic alterations induced by drugs that affect hepatic functional integrity and various drug metabolizing systems via distinct mechanisms. For this purpose, paracetamol (ACET) or phenobarbital (PB) were administered to male Wistar rats that were fed with standard rodent chow (normally fed, NF) or underwent dietary CD. The administration of ACET increased the serum aspartate aminotransferase levels in NF rats, while CD restricted this increase. On the other hand, ACET suppressed alkaline phosphatase levels only in CD rats. Moreover, CD prevented the PB-induced increase of the mitotic activity of hepatocytes. The administration of ACET down-regulated CYP1A2 and CYP2B1 expression in CD rats, while up-regulating them in NF rats. The administration of PB suppressed CYP1A2 apoprotein levels in CD rats, whereas the drug had no effect on NF rats. The PB-induced up-regulation of CYP2B, CYP2E1 and CYP1A1 isozymes was markedly higher in CD than in NF rats. In addition, PB increased glutathione-S-transferase activity only in CD rats. Hepatic glutathione content (GSH) was suppressed by ACET in NF rats, whereas the drug increased GSH in CD rats. Our data suggest that CD has a significant impact on the hepatic metabolic functions, and in particular on those related to drug metabolism. Thus, CD may modify drug effectiveness and toxicity, as well as drug-drug interactions, particularly those related to ACET and PB.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Deficiencia de Colina/metabolismo , Hígado/efectos de los fármacos , Fenobarbital/farmacología , Alanina Transaminasa/sangre , Fosfatasa Alcalina/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Peso Corporal/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hígado/enzimología , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Ratas WistarRESUMEN
Nitrosamines are carcinogens that require metabolic activation by CYP enzymes in order to exert their carcinogenic effect. Species differences exist in their esophageal carcinogenic potency, with the rat being the most sensitive and the Syrian hamster a resistant species. In the latter, the liver is the main target organ. This difference does not apply to directly acting N-nitroso compounds, suggesting that tissue-specific metabolic activation is involved in hamster esophageal resistance to nitrosamines. We have previously shown that Cytochrome P450 2A3 (CYP2A3) is responsible for N-nitrosodiethylamine activation in the rat esophagus. In order to find a mechanistic explanation for the resistance of hamster esophagus for nitrosamines, we have compared the metabolism of NDEA between esophagus and liver of the hamster. Hamster esophagus is capable of activating NDEA (K(m)=1.02+/-0.44microM and V(max)=1.96+/-0.26nmol acetaldehyde/min/mg microsomal protein). However, the hamster liver showed a 40-fold higher catalytic efficiency (V(max)/K(m)) towards NDEA metabolism compared with its esophagus. Hamster esophagus expresses CYP2A8, CYP2A9 and CYP2A16, but not CYP2E1. An antibody against human CYP2A6 was able to inhibit NDEA metabolism in hamster esophageal, but not liver microsomes. Our results suggest that in the hamster esophagus, but not in the liver, most of the NDEA is metabolized by CYP2A enzymes, but with a rather poor efficiency when compared to the liver. This is in accordance with previous results showing that for the hamster, the main target organ of NDEA is the liver.
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Carcinógenos/metabolismo , Dietilnitrosamina/metabolismo , Neoplasias Esofágicas/metabolismo , Esófago/metabolismo , Alquilantes/metabolismo , Alquilantes/toxicidad , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Western Blotting , Carcinógenos/toxicidad , Cricetinae , Citocromo P-450 CYP2A6 , Dietilnitrosamina/toxicidad , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/genética , Esófago/efectos de los fármacos , Esófago/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Mesocricetus , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Nitrosaminas/metabolismo , Nitrosaminas/toxicidad , Especificidad de Órganos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1alpha and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1alpha expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1alpha expression vector demonstrated that PGC-1alpha is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4alpha response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1alpha binds, together with HNF-4alpha, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1alpha mediates the expression of Cyp2a5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4alpha. This strongly suggests that PGC-1alpha is the major factor mediating the fasting response of CYP2A5.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Ayuno/metabolismo , Hepatocitos/enzimología , Transactivadores/metabolismo , Xenobióticos/metabolismo , Adenoviridae/genética , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Técnicas de Cultivo de Célula , Células Cultivadas , GMP Cíclico/metabolismo , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Inducción Enzimática , Vectores Genéticos , Factor Nuclear 4 del Hepatocito/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transducción de Señal , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción , Activación Transcripcional , Transfección , Regulación hacia ArribaRESUMEN
We describe a novel cis-element in the 5' coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP)C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides downstream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is observed in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qualitative changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addition, we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.
Asunto(s)
Citostáticos/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico/genética , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Codón/genética , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dactinomicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Humanos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Sistemas de Lectura Abierta , Especificidad de Órganos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Sondas ARN/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The potential involvement of catecholamines and in particular of alpha(2)-adrenoceptor-related signalling pathways, in the regulation of drug-metabolizing enzymes by stress was investigated in Wistar rats after exposure to the environmental pollutant benzo(alpha)pyrene. For this purpose, total cytochrome P450 content, the CYP1A2 mRNA levels, 7-methoxyresorufin-O-dealkylase (MROD), 7-pentoxyresorufin-O-dealkylase (PROD) and p-nitrophenol hydroxylase activity levels were determined in the livers of rats exposed to repeated restraint stress after treatment with benzo(alpha)pyrene coupled with pharmacological manipulations of peripheral and/or central catecholamines and alpha(2)-adrenoceptors. The data show that stress is a significant factor in the regulation of CYP1A2 induction and that catecholamines play a central role in the stress-mediated modulation of hepatic CYP1A2 inducibility by benzo(alpha)pyrene. The up-regulating effect of stress on benzo(alpha)pyrene-induced CYP1A2 gene expression was eliminated after a generalized catecholamine depletion with reserpine. Similarly, in a state where only peripheral catecholamines were depleted and central catecholamines remained intact after guanethidine administration, the up-regulating effect of stress was eliminated. It is apparent that stress up-regulates the induction of CYP1A2 by benzo(alpha)pyrene mainly via peripheral catecholamines, while central catecholamines hold a minor role in the regulation. Pharmacological manipulations of alpha(2)-adrenoceptors appear to interfere with the effect of stress on the regulation of CYP1A2 inducibility. Either blockade or stimulation of alpha(2)-adrenoceptors with atipamezole and dexmedetomidine respectively, eliminated the up-regulating effect of stress on CYP1A2 benzo(alpha)pyrene-induced expression, while it enhanced MROD activity. In contrast, stress and pharmacological manipulations of catecholamines and alpha(2)-adrenoceptors did not affect total P450 content, the CYP2B1/2-dependent PROD and the CYP2E1-dependent p-nitrophenol hydroxylase activities. In conclusion, stress is a significant factor in the regulation of the CYP1A2 inducibility by benzo(alpha)pyrene, which in turn is involved in the metabolism of a large spectrum of toxicants, drugs and carcinogenic agents. Although the mechanism underlying the stress effect on CYP1A2 induction has not been clearly elucidated, it appears that peripheral catecholamines hold a predominant role, while central catecholamines and in particular, central noradrenergic pathways hold a minor role.
Asunto(s)
Benzo(a)pireno/farmacología , Catecolaminas/metabolismo , Citocromo P-450 CYP1A2/biosíntesis , Contaminantes Ambientales/farmacología , Estrés Fisiológico/enzimología , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Animales , Citocromo P-450 CYP1A2/genética , Dexmedetomidina/farmacología , Quimioterapia Combinada , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Guanetidina/farmacología , Imidazoles/farmacología , Inyecciones Intraperitoneales , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa 2/metabolismo , Reserpina/farmacología , Restricción FísicaRESUMEN
This study investigated the role of catecholamine-related signaling pathways in the regulation of hepatic cytochrome P450 (CYP2E1). Central and peripheral catecholamine depletion with reserpine down-regulated CYP2E1. On the other hand, selective peripheral catecholamine depletion with guanethidine increased CYP2E1 apoprotein levels. Enrichment of peripheral catecholamines with adrenaline suppressed p-nitrophenol hydroxylase activity (PNP). PNP activity was also markedly suppressed by l-DOPA. Stimulation of D(2)-receptors with bromocriptine up-regulated CYP2E1, as assessed by enzyme activity and protein levels, whereas blockade of D(2)-dopaminergic receptors with sulpiride down-regulated this isozyme. These findings indicate that central and peripheral catecholamines have different effects on CYP2E1. Central catecholamines appear related to the up-regulation, whereas the role of peripheral catecholamines is clearly related to the type and location of adrenoceptors involved. D(2)-receptor-linked signaling pathways have an up-regulating effect on CYP2E1, while D(1)-receptor pathways may down-regulate this isozyme. It is worth noting that the widespread environmental pollutant benzo(alpha)pyrene (B(alpha)P) altered the modulating effect of catecholaminergic systems on CYP2E1 regulation. In particular, whereas stimulation or blockade of adrenoceptors had no effect on constitutive PNP activity, exposure to B(alpha)P modified the impact of central and peripheral catecholamines and alpha(2)-adrenoceptors on CYP2E1 expression. It appears that under the influence of B(alpha)P, alpha(2)-adrenergic receptor-linked signaling pathways increased CYP2E1 apoprotein levels. Given that a wide range of xenobiotics and clinically used drugs are activated by CYP2E1 to toxic metabolites, including the production of reactive oxygen species (ROS), it is possible that therapies challenging dopaminergic receptor- and/or alpha(2)-adrenoceptor-linked signaling pathways may alter the expression of CYP2E1, thus affecting the progress and development of several pathologies.
Asunto(s)
Catecolaminas/farmacología , Citocromo P-450 CYP2E1/biosíntesis , Hígado , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Animales , Benzo(a)pireno/farmacología , Western Blotting , Antagonistas de los Receptores de Dopamina D2 , Regulación hacia Abajo , Isoenzimas , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Receptores de Dopamina D2/agonistas , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Glucocorticoids down regulate the inducible nitric oxide synthase (iNOS) gene both transcriptionally and post-transcriptionally. The post-transcriptional events are suggested to involve destabilization of the iNOS transcript although the molecular mechanisms for this effect are not known. Recently, our laboratory demonstrated a lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-induction-dependent interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I and hnRNPL with a destabilizing element contained in the 3'untranslated region (UTR) of iNOS mRNA. The aim of this study was to investigate if dexamethasone, which down regulates iNOS, is able to modulate this protein-mRNA interaction. As expected, dexamethasone inhibited the induction of iNOS by LPS and IFNgamma in RAW 264.7 cells, and destabilized the iNOS mRNA. Dexamethasone also counteracted the LPS/IFNgamma-induced disappearance of a gel shifted iNOS mRNA-protein complex containing hnRNPI and hnRNPL. UV cross-linking and Western blot analyses revealed that the RNA-binding and levels of hnRNPI, which decreased by LPS/IFNgamma treatment, were restored by dexamethasone. The results support our hypothesis that hnRNPI is pivotal in the post-transcriptional regulation of iNOS and strongly suggest that hnRNPI is one of the trans-acting factors mediating the post-transcriptional effects of dexamethasone.