RESUMEN
Recent data suggest that apart from its well-known role in the regulation of xenobiotic metabolizing enzymes, AhR is also involved in inflammation. However, the influence of inflammation on AhR expression remains unknown. Here, we demonstrated that proinflammatory conditions induced by either PMA or IL-1 ß enhance AhR expression in Caco-2 cells. This was associated with an increase in AhR promoter activity. By means of directed mutagenesis experiments and the use of proteasome inhibitors, we demonstrated that inflammation-induced AhR expression involved the NF κ B pathway but not AP-1. Moreover, conditioned media from PMA-treated Caco-2 cells were also able to induce AhR expression, and this induction was repressed by anti-IL-1 ß blocking antibodies. Similar results were obtained with conditioned media from PMA-treated THP-1 cells. Taken together, these data suggest that AhR could be involved in vivo in an inflammatory loop. AhR was recently suspected to be implicated in inflammatory bowel disease. Our results support this hypothesis and suggest that AhR could be a new target for inflammatory bowel disease patient management.
RESUMEN
In this work we demonstrate that Caco-2 cell treatment with WY-14643 (a potent PPARalpha agonist) causes an increase in AhR expression. Luciferase assays and directed mutagenesis experiments showed that induction mainly occurred at transcriptional level and involved a PPRE site located within the AhR promoter. These results were further confirmed by the use of PPARalpha knockout mice in which AhR induction by WY14643 was abrogated. In addition to CYP1 regulation, AhR has been described as being involved in inflammation, so we also studied the effect of AhR regulation by PPARalpha on the expression of some inflammation target genes. 3-Methylcholanthrene (a potent AhR agonist) increased the expression (mRNA) of the major inflammatory targets IL-1beta and MMP9. WY-14643 co-treatment abrogated the 3-methylcholanthrene pro-inflammatory effect. Hence the anti-inflammatory effect of PPARalpha overrides the pro-inflammatory effect of AhR.
Asunto(s)
Mediadores de Inflamación/metabolismo , PPAR alfa/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células CACO-2 , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación TranscripcionalRESUMEN
BACKGROUND AND AIMS: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. METHODS: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. RESULTS: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. CONCLUSIONS: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.
Asunto(s)
Citocromo P-450 CYP1A1/genética , PPAR alfa/fisiología , Adenocarcinoma , Secuencia de Bases , Carcinoma Hepatocelular , Línea Celular Tumoral , Cloranfenicol O-Acetiltransferasa/metabolismo , Neoplasias del Colon , Cartilla de ADN , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas , PPAR alfa/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Activación TranscripcionalRESUMEN
CYP1A1 is largely implicated in carcinogenesis. To date, it is known that this gene is induced by xenobiotics such as polycyclic aromatic hydrocarbons. In this study, we evaluated the effect of serum in the regulation of CYP1A1 gene expression. CYP1A1 mRNA level is induced 1) in HepG2 and HT29-D4 cells by 3-methylcholanthrene 2) only in HepG2 after treatment by serum. The CYP1A1 mRNA induction in HepG2 is the consequence at least in part of a transcriptional activation as was demonstrated by evaluation of the hnRNA level. HepG2 cells were transfected by a plasmid containing the 7.5 Kb of the CYP1A1 promoter and the CAT reporter gene. No CAT stimulation was observed after serum treatment. These results demonstrated that CYP1A1 is induced at a transcriptional level by a physiological compound contained in serum independently of the Ah receptor and the 7.5 Kb promoter region.
Asunto(s)
Sangre , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A1/genética , Neoplasias Hepáticas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Albúmina Sérica Bovina/farmacología , Células Tumorales Cultivadas/metabolismo , Adenocarcinoma/metabolismo , Animales , Bovinos , Cloranfenicol O-Acetiltransferasa/genética , Neoplasias del Colon/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Genes Reporteros , Humanos , Metilcolantreno/farmacología , ARN Nuclear Heterogéneo/análisis , ARN Mensajero/metabolismo , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We tested the hypothesis that dietary cholesterol modulate human ethanol-inducible CYP2E1 expression in vivo in circulating mononuclear cells. Healthy volunteers (n= 10) were submitted to a low fat low cholesterol diet for 4 days (day 0-day 3, LFLC). Cholesterol (595 +/- 56 mg/day) was then reintroduced for 7 days (day 4-day 10, LFHC). In the same time, controls subjects (n=7) did not change their habitual daily diet. CYP2E1 mRNA levels, evaluated in mononuclear cells, decreased in experimental subjects during both LFLC and LFHC from 100% to 53 +/- 5%, (p<0.001) with a main decrease during LFLC period (100% to 71 +/- 16%, p=0.05). Immunoreactive CYP2E1 showed a similar pattern and decreased from 100 to 62 +/- 12% during the trial (p<0.05). No significant change occured in control subjects. Between day 0 and day 11, changes in CYP2E1 mRNA correlated positively with plasma cholesterol (r2=0.67, p<0.001) and HDL cholesterol concentrations (r2=0.61, p<0.001). In contrast, no correlation was found between plasma fatty acids concentrations and CYP2E1 expression. The present results suggest that lipid factors regulate CYP2E1 expression, in vivo, in human mononuclear cells. In particular, plasma cholesterol concentrations may play an important role in this regulation.
Asunto(s)
Citocromo P-450 CYP2E1/genética , Grasas de la Dieta/farmacología , Etanol/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Adulto , Secuencia de Bases , Glucemia/análisis , Citocromo P-450 CYP2E1/metabolismo , Cartilla de ADN , Femenino , Humanos , Leucocitos Mononucleares/enzimología , Lípidos/sangre , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
We investigated the expression of c-myc in HT29-D4, HBL100 and Caco-2 cells treated with microtubule stabilising (paclitaxel) or depolymerising agents (vinblastine, nocodazole). After induction by epidermal growth factor (EGF), c-myc expression decreased in HT29-D4 cells treated with all the antimicrotubule agents. In HBL100 and Caco-2, when microtubules were stabilised with paclitaxel, c-myc expression also decreased. In contrast, its expression increased after treatment with depolymerising agents. In both cell lines, we also observed that depolymerising agents alone induced c-myc expression whilst paclitaxel had no effect. This mRNA induction was confirmed at the protein level. In HT29-D4, no variation of c-myc expression was observed. Then, we showed that the increase of mRNA level was due to activation of gene transcription. These results indicate that modulation of c-myc expression varied depending on the cell lines used and the type of antimicrotubule agents. This work provides a potential link between the microtubular network and c-myc gene expression.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Genes myc/genética , Microtúbulos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Western Blotting , Células CACO-2/metabolismo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29/metabolismo , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CYP1A1 is implicated in the bioactivation of procarcinogens such as polycyclic aromatic hydrocarbons. To date, no physiological compounds have been described as inducers of this gene. In this study, we have examined the role of serum in the regulation of CYP1A1 gene expression. After treatment of CaCo-2 cells with fetal bovine serum, CYP1A1 mRNA level increased to the same extent as that observed after 3-methylcholanthrene induction. The same effect was obtained after treatment with adult bovine or human serum. Evaluation of hnRNA level performed on CaCo-2 cells indicates that CYP1A1 induction by serum acts at least in part through transcriptional activation. Promoter region containing the XRE (1.56 kb) was tested in the CAT assay. No stimulation of this reporter gene was detected after serum treatment. These results demonstrate for the first time that physiological compound(s) contained in serum induces CYP1A1 gene expression by transcriptional activation independent of the AhR pathway.
Asunto(s)
Citocromo P-450 CYP1A1/genética , Receptores de Hidrocarburo de Aril/metabolismo , Adulto , Animales , Secuencia de Bases , Células CACO-2 , Bovinos , Cloranfenicol O-Acetiltransferasa/genética , Medios de Cultivo , Citocromo P-450 CYP1A1/biosíntesis , Cartilla de ADN/genética , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Metilcolantreno/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Carbamazepine (CBZ) is widely used in the treatment of epilepsy. The drug is principally metabolized by CYPs to 10, 11-epoxy carbamazepine (CBZ-E) but this metabolite more toxic than the parent drug, does possess anticonvulsant properties. In humans, CYP3A4, CYP2C8 and CYP1A2 have been shown to be implicated in CBZ biotransformation. Our purpose was to establish an experimental model to determine the interaction of CBZ with other antiepileptic drugs. We first identified the CYP isoforms that metabolized CBZ in rabbit. We used liver microsomes from rabbit treated with various compounds known to induce principally some CYPs subfamilies. Having tested all the compounds we demonstrated that only the animals treated with CYP3A inducers were able to metabolize CBZ strongly. The CBZ biotransformation was inhibited by anti CYP3A antibodies. All the CYP3A subfamily substrates specifically decrease CBZ-E formation. In our experiment we did not observe any inhibition with CYP2C substrate. These data provide evidence that in rabbit the CYP3A subfamily is primarily involved in CBZ metabolism. Using this model we investigated the interaction of CBZ with phenobarbital, phenytoin, ethosuccimide, primidone, progabide, vigabatrin and lamotrigine.
Asunto(s)
Anticonvulsivantes/farmacología , Hidrocarburo de Aril Hidroxilasas , Carbamazepina/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Anticuerpos/farmacología , Anticonvulsivantes/metabolismo , Anticonvulsivantes/uso terapéutico , Biotransformación , Carbamazepina/uso terapéutico , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/inmunología , Dextrometorfano/metabolismo , Dextrometorfano/farmacología , Interacciones Farmacológicas , Quimioterapia Combinada , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Epilepsia/tratamiento farmacológico , Hipnóticos y Sedantes/metabolismo , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Nifedipino/metabolismo , Nifedipino/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , ConejosRESUMEN
A strong overlap exists between gp170 and CYP3A substrates and inducers. In order to investigate a putative coregulation of MDR and CYPA gene expression, we measured their transcripts in human liver and after dexamethasone treatment in HepG2 cells or in different mouse tissues. In human liver, we observed no correlation between MDR1 and CYP3A4 expression, whereas these genes were coinduced by dexamethasone in HepG2 cells. In mouse liver treated with dexamethasone, mdr1b and Cyp3a were induced (5- and 2-fold, respectively). In adrenals, the main expressing gp170 tissue, Cyp3a, was increased while mdr1b was repressed (-51%). The expression of mdr1b increased in heart, brain, and colon and decreased in lung and kidney but Cyp3a was not detectable. In conclusion, human hepatic CYP3A4 and MDR1 are not corregulated but are coinducible. In vivo murine mdr1b and Cyp3a are coregulated by dexamethasone in liver and inversely regulated in adrenals.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Dexametasona/farmacología , Genes MDR/efectos de los fármacos , Oxidorreductasas N-Desmetilantes/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/biosíntesis , Cartilla de ADN/genética , Inducción Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Oxidorreductasas N-Desmetilantes/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
Cigarette smoking is a worldwide health problem and is the greatest risk factor for lung cancer. By activating procarcinogens, hepatic and extrahepatic cytochromes P450 can participate in lung carcinogenesis. Tobacco smoke contains numerous cytochrome P450 inducers, substrates, and inhibitors. In the present study we investigated, in male NMRI mice, the effects of cigarette smoke on hepatic and pulmonary cytochrome P450 expression and their possible role in the induction of DNA lesions such as DNA single strand breaks (SSB). Hepatic and pulmonary mouse cytochrome P450 isozymes involved in carcinogenesis (Cyp1a, 2b, 2e, 3a) were differently induced by cigarette smoke. Cyp2e1 mRNA was dramatically enhanced (12.7-fold increase) while Cyp2b10 mRNA remained unchanged and Cyp1a1 was decreased or not detected. Cyp3a protein and mRNA were not detected in lung, suggesting that this isozyme is not expressed in mouse pulmonary tissue. The SSB of DNA increased in lung and liver treated mice. In contrast no modification was observed in lymphocytes that barely expressed cytochromes P450. Cimetidine and propylene glycol reduced SSB of DNA induced by smoking in liver and lung cells. The inhibition (-70%) observed in lung following treatment by propylene glycol, a CYP2E1 inhibitor, suggested that this isozyme is at least in part involved in pulmonary DNA damage induced by tobacco smoke. The high concentration of CYP2E1 function and regulation in mammals suggests that this protein could be involved in pulmonary carcinogenesis in human smokers.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Daño del ADN , ADN de Cadena Simple , Isoenzimas/genética , Hígado/enzimología , Pulmón/enzimología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Carboxihemoglobina/análisis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/biosíntesis , Citocromos b5/biosíntesis , Citocromos b5/genética , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica , Isoenzimas/biosíntesis , Neoplasias Pulmonares/etiología , Masculino , Ratones , Oxidorreductasas N-Desmetilantes/genética , ARN Mensajero/análisisRESUMEN
The effect of cigarette smoke on the expression of several cytochromes P450 (CYP) and UDP-glucuronosyl-transferases (UGT) was studied in mice. The animals were exposed to cigarette smoke for 4 to 30 days. Enzymatic activities supported by CYP1A1, 1A2, 2B, 2E1 and the glucuronidation activity toward phenols were measured in lung, liver and kidney microsomes. Cigarette smoke induced several CYPs, especially in lung. CYP2E1 was more induced than CYP1A1 in this organ. The expression of CYP2E1 was also increased in kidney (5.6 times after 30 days). The glucuronidation in kidney was non-sensitive to the treatment whatever substrate used. In contrast, this activity was enhanced in liver and particularly in lung, in which the glucuronidation of 1-naphthol and 2-hydroxybiphenyl was increased by 122 and 180%, respectively. Interestingly, the times of induction differed according to the substrate used, thus suggesting the presence of different UGTs active toward phenols that were differentially affected by cigarette smoke. The UGT activities toward phenols were low in lung, when compared with those measured in liver or kidney. In conclusion, cigarette smoke greatly affected both glucuronidation activity and the hydroxylation reactions supported by CYPs in mouse liver and lung.
Asunto(s)
Sistema Enzimático del Citocromo P-450/análisis , Glucuronosiltransferasa/análisis , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Microsomas/efectos de los fármacos , Contaminación por Humo de Tabaco/efectos adversos , Animales , Citocromos b5/análisis , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Masculino , Ratones , Microsomas/enzimologíaRESUMEN
The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.
Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Secuencia de Bases , Estudios de Evaluación como Asunto , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Respuesta SOS en GenéticaRESUMEN
Several studies have shown in humans an association between renal carcinoma and cigarette smoking. Cigarette smoke contains numerous cytochrome P450 inducers and substrates. In the present study we investigated the effect of cigarette smoke on the regulation of murine cytochrome P450 expression in kidney and its possible role in the induction of single strand breaks in DNA. Results demonstrated that CYP2E1 (activity, protein, and MRNA) was induced by tobacco smoke (2.1, 5.6 and 20.8, respectively). We did not detect any CYP1A, CYP2B, and CYP3A using Western blot and RT-PCR experiments. We have analyzed the renal single strand breaks of DNA in control and treated mice. The results indicated an increase of single strand breaks of DNA in kidney from treated mice which paralleled the high inducibility of the CYP2E1. No significant difference was observed between lymphocytes (which expressed very low or undetectable cytochrome P450 levels) of control and treated mice.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Daño del ADN , Regulación Enzimológica de la Expresión Génica , Riñón/enzimología , Oxidorreductasas N-Desmetilantes/biosíntesis , Contaminación por Humo de Tabaco , Animales , Secuencia de Bases , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN , ADN de Cadena Simple , Inducción Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos , Microsomas/enzimología , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Reacción en Cadena de la Polimerasa , Valores de ReferenciaRESUMEN
We have isolated a cDNA (named P450 A) which is a variant of CYP2E1 presenting some differences in the 3' non coding region. Two substitutions and only one polyadenylation signal were detected on P450 A compared to CYP2E1 where there are two. With the aim of studying the frequency of utilization of these polyadenylation signals, probe A and probe J specific for P450 A and CYP2E1, respectively, were hybridized on human hepatic and extra-hepatic cDNA samples. Results indicated that in all tissues tested only P450 A using the first polyadenylation signal was detected. The CYP2E1 cDNA isolated by Song et al could be representative of a transcript either rarely represented among the Caucasian population, or expressed in a particular individual or ethnic population, or rapidly degraded.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas N-Desmetilantes/genética , Corteza Suprarrenal/metabolismo , Secuencia de Bases , Citocromo P-450 CYP2E1 , ADN Complementario/genética , Expresión Génica , Humanos , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/química , Poli A/metabolismo , ARN Mensajero/genéticaRESUMEN
We have demonstrated the presence of CYP2E1 protein in a catalytically active form in lung tumors, differences being observed between the tumors and normal tissues from the same patients. Indeed, a higher microsomal CYP2E1 N-nitrosodimethylamine (NDMA) demethylase activity was present in normal tissues compared to tumors and was accompanied by corresponding change in CYP2E1 protein concentration, as shown by Western blot analysis. The catalytic activity among tumors differed from that among normal tissues with statistical significance of p < 0.01. CYP2E1 mRNA was present in lung tumors and less expressed compared to normal tissues from the same individuals. In order to understand the regulation of CYP2E1 gene expression, we studied the methylation status of the CYP2E1 gene in human lung tumors and normal tissues from the same patients and observed that a hypomethylation was associated with a hypoexpression of the CYP2E1 gene in lung tumors.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Sistema Enzimático del Citocromo P-450/biosíntesis , Expresión Génica , Neoplasias Pulmonares/enzimología , Pulmón/enzimología , Microsomas/enzimología , Oxidorreductasas N-Desmetilantes/biosíntesis , Secuencia de Bases , Western Blotting , Carcinoma de Células Escamosas/genética , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/análisis , ADN/química , ADN/aislamiento & purificación , Cartilla de ADN , ADN de Neoplasias/química , ADN de Neoplasias/aislamiento & purificación , Humanos , Neoplasias Pulmonares/genética , Masculino , Metilación , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/análisis , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , FumarRESUMEN
The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas N-Desmetilantes/genética , Secuencia de Bases , Citocromo P-450 CYP2E1 , Expresión Génica , Humanos , Hígado/enzimología , Pulmón/enzimología , Metilación , Datos de Secuencia Molecular , Neurilemoma/enzimología , Placenta/enzimología , ARN Mensajero/genética , Piel/enzimologíaRESUMEN
Pulmonary and liver microsomes of male NMRI mice were used to study the inductive effect of cigarette smoke on various cytochrome P450 isoforms implicated in precarcinogen and premutagen bioactivation. The enzymatic activities catalyzed by CYP1A1, CYP2B, CYP2C, CYP2D, CYP2E1 and CYP3A were induced in liver microsomes. Immunoquantification of lung and liver CYP1A1, 2E1 and 3A demonstrated that 1) CYP1A1 was induced in lung and liver, 2) CYP3A subfamily was induced in liver and not detected in lung, 3) CYP2E1 was slightly induced in liver whereas its pulmonary expression was more largely increased (6.8 fold) than CYP1A1 (2.0 fold). This latter data suggests that CYP2E1, which is known to be expressed in human lung, could actively participate in pulmonary carcinogenesis induced by cigarette smoke.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Isoenzimas/biosíntesis , Pulmón/enzimología , Microsomas Hepáticos/enzimología , Microsomas/enzimología , Oxidorreductasas N-Desmetilantes/biosíntesis , Humo/efectos adversos , Animales , Biotransformación , Western Blotting , Carcinógenos/farmacocinética , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Inducción Enzimática , Isoenzimas/metabolismo , Masculino , Ratones , Oxidorreductasas N-Desmetilantes/metabolismo , Plantas Tóxicas , NicotianaRESUMEN
Vinblastine biotransformation was investigated by using a human liver microsomes library. The drug was converted into one major metabolite (M) upon incubation with the microsomes. A large interindividual variation in vinblastine metabolism was observed among the samples tested, with a 4.4 ratio between the lowest and the highest metabolic rates. The biotransformation of vinblastine followed Michaelis-Menten kinetics (Km = 6.82 +/- 0.27 microM and Vmax = 0.64 +/- 0.06 nmol/min/mg protein). The involvement of the cytochrome P450 3A subfamily in vinblastine metabolism was demonstrated by the following body of evidence: (a) the competitive inhibition of vinblastine biotransformation by cytochrome P450 3A specific probes with Ki values of 0.17, 22.5, 14.8, and 35.3 microM for ketoconazole, erythromycin, troleandomycin, and vindesine, respectively; (b) the immunoinhibition of vinblastine metabolism by polyclonal anti-cytochrome P450 3A antibodies; (c) the highly significant correlation between the level of cytochrome P450 3A determined by Western blots and vinblastine metabolism (r = 0.759, P < 0.001); (d) the highly significant correlation between erythromycin N-demethylase activity (mediated by cytochrome P450 3A) and vinblastine metabolism (r = 0.83, P < 0.001); (e) the significant correlation between the CYP3A4 mRNA level and vinblastine metabolism (r = 0.60, P < 0.1). Although vincristine and navelbine (members of the Vinca alkaloid family) also inhibit the metabolism of vinblastine, suggesting the involvement of the cytochrome subfamily in their respective metabolisms, other anticancer drugs currently associated with vinblastine in chemotherapy (etoposide, Adriamycin, lomustine, and teniposide) also interfere with vinblastine biotransformation. These metabolic drug interactions may alter the antitumor activity and/or toxicity of the drug during anticancer chemotherapy.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/metabolismo , Vinblastina/metabolismo , Adolescente , Adulto , Anticuerpos/farmacología , Secuencia de Bases , Unión Competitiva , Biotransformación , Western Blotting , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Interacciones Farmacológicas , Eritromicina/farmacología , Femenino , Humanos , Cetoconazol/farmacología , Cinética , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Familia de Multigenes , Oligodesoxirribonucleótidos , ARN Mensajero/metabolismo , Troleandomicina/farmacología , Vindesina/farmacologíaRESUMEN
This report characterizes the cytochrome P450 isozyme involved in clonazepam metabolism. This study was undertaken using a library of liver microsomal fractions prepared from untreated rabbits or those treated with drugs known to specifically induce various cytochrome P450 isozymes (ie P450 2B4 by phenobarbital, P450 1A1 and P450 1A2 by 3-methylcholanthrene and beta-naphthoflavone, P450 2E1 by acetone and ethyl alcohol, and P450 3A6 by erythromycin). Only microsomes obtained from phenobarbital-treated rabbits exhibited a type II binding spectrum upon addition of clonazepam (Ks(app) = 31.4 +/- 3.8 microM) and significantly metabolized clonazepam to 7-aminoclonazepam. Benzphetamine, which is a known substrate for P450 2B1 was also extensively metabolized by microsomes prepared from phenobarbital treated rabbits. This indicates that the same isozyme (P450 2B subfamily) was involved in the biotransformation of both substrates. Experiments performed on 14 human liver microsomal preparations showed a wide interindividual variability (from 1-4) and a good correlation (r = 0.70) between benzphetamine and clonazepam metabolism. Since P450 3A4 (nf25) was involved in benzphetamine metabolism, clonazepam was probably nitroreduced by the same isozyme. An oligonucleotide specific for the P450 3A4 gene subfamily was synthetized and used for hybridization on total RNA from human liver samples. Two transcripts of 2.2 and 3.0 kb were detected and the level of the 2.2 kb mRNA expression was significantly correlated (r = 0.61) with the intensity of clonazepam nitroreduction by the corresponding microsome batches.