RESUMEN
The cytochrome P4501A (CYP1A) enzymes play important roles in the metabolic activation and detoxification of numerous environmental carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, we tested the hypothesis that hepatic CYP1A2 differentially regulates mouse hepatic and pulmonary CYP1A1 expression and suppresses transcriptional activation of human CYP1A1 (hCYP1A1) promoter in response to 3-methylcholanthrene (MC) in vivo. Administration of wild-type (WT) (C57BL/6J) or Cyp1a2-null mice with a single dose of MC (100 µmol/kg i.p.) caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein content, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not Cyp1a2-null, animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for up to 8 days in both genotypes, with Cyp1a2-null mice displaying a greater extent of CYP1A1 expression. It is noteworthy that MC caused significant augmentation of human CYP1A1 promoter activation in transgenic mice expressing the hCYP1A1 and the reporter luciferase gene on a Cyp1a2-null background, compared with transgenic mice on the WT background. In contrast, the mouse endogenous hepatic, but not pulmonary, persistent CYP1A1 expression was repressed by MC in the hCYP1A1-Cyp1a2-null mice. Liquid chromatography-mass spectrometry experiments showed that CYP1A2 catalyzed the formation of 1-hydroxy-3-MC and/or 2-hydroxy-3-MC, a metabolite that may contribute to the regulation of CYP1A1 expression. In conclusion, the results suggest that CYP1A2 plays a pivotal role in the regulation of hepatic and pulmonary CYP1A1 by PAHs, a phenomenon that potentially has important implications for PAH-mediated carcinogenesis.
Asunto(s)
Carcinógenos Ambientales/toxicidad , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Metilcolantreno/toxicidad , Activación Transcripcional/efectos de los fármacos , Animales , Western Blotting , Carcinógenos Ambientales/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Hígado/enzimología , Pulmón/enzimología , Metilcolantreno/farmacocinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytochrome P450 (P450)1A1 plays a critical role in the metabolic activation and detoxification of polycyclic aromatic hydrocarbons (PAHs), many of which are potent human carcinogens. In this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1A1 expression in human hepatoma cells (HepG2) and that this phenomenon is mediated by sustained transcriptional activation of the CYP1A1 promoter. Treatment of HepG2 cells with MC resulted in marked induction (8-20-fold) of ethoxyresorufin O-de-ethylase activities, CYP1A1 apoprotein contents, and mRNA levels, which persisted for up to 96 h. MC also caused sustained transcriptional activation of the human CYP1A1 promoter for up to 96 h, as inferred from transient transfection experiments. Experiments with deletion constructs indicated that Ah response elements located at -886, -974, and -1047, but not -491, nucleotides from the start site, contributed to the sustained transcriptional activation of the CYP1A1 promoter. Electrophoretic mobility-shift and chromatin immunoprecipitation assays suggested that prolonged CYP1A1 induction was mediated by Ah receptor (AHR)-independent mechanisms. Experiments with [3H]MC and liquid chromatography-tandem mass spectrometry demonstrated rapid elimination of MC and its metabolites from the cells by 12 to 24 h, suggesting that these compounds did not elicit sustained CYP1A1 induction via the classical AHR-mediated pathway. In conclusion, the results of this study support the hypothesis that MC causes persistent induction of CYP1A1 in human hepatoma cells by mechanisms entailing sustained transcriptional activation of the CYP1A1 promoter via AHR-independent mechanisms. These observations have important implications for human carcinogenesis mediated by PAHs.
Asunto(s)
Carcinógenos/toxicidad , Citocromo P-450 CYP1A1/biosíntesis , Metilcolantreno/toxicidad , Línea Celular Tumoral , Citocromo P-450 CYP1A1/genética , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Receptores de Hidrocarburo de Aril/fisiología , Activación TranscripcionalRESUMEN
Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 µM)+RA (1 µM) were significantly reduced compared to those treated with BP only (P=0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 µM) for 24h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 µM RA was added. The cells were harvested 24h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 ± 34) than those in the BP/DMSO group (544 ± 33), P=0.032. Analysis of cell apoptosis showed an increase in BP+RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.
Asunto(s)
Anticarcinógenos/farmacología , Benzo(a)pireno/metabolismo , Aductos de ADN/metabolismo , Tretinoina/farmacología , Benzo(a)pireno/toxicidad , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Reparación del ADN , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismoRESUMEN
There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens. Cytochrome P450 (CYP)1A enzymes play key roles in the metabolic activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by 3-methylcholanthrene (MC) in vivo in rodents. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 and 1A2 in vivo by mechanisms entailing sustained transcriptional activation of the corresponding promoters. Adult male wild type (WT) (Cd-1) mice, transgenic mice expressing the human CYP1A1 promoter or the mouse CYP1A2 promoter were treated with the vehicle corn oil (CO) or the carcinogenic PAH, 3-methylcholanthrene (MC), once daily for 4days, and luciferase reporter gene expression was determined at 1, 8, 15, and 22days after MC withdrawal by bioluminescent imaging. Pulmonary and hepatic endogenous expression of CYP1A1 and 1A2 was also determined at the enzymatic, protein, and mRNA levels. The major findings were that MC elicited marked enhancement in the luciferase expression in the CYP1A1-luc as well CYP1A2-luc transgenic mice that was sustained for up to 22days, the magnitude of induction being more pronounced in the CYP1A1-luc mice. MC also caused persistent induction of endogenous CYP1A1 and 1A2 expression in the WT, CYP1A1-luc, and 1A2-luc mice for up to 22days. In conclusion, our results support the hypothesis that MC elicits sustained CYP1A1 and 1A2 expression by sustained transcriptional activation of the corresponding promoters. Thus, these novel transgenic models should be very useful for further understanding of the molecular mechanisms of persistent CYP1A induction, in relation to PAH-mediated carcinogenesis.
Asunto(s)
Carcinógenos/toxicidad , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Metilcolantreno/toxicidad , Regiones Promotoras Genéticas/efectos de los fármacos , Activación Transcripcional , Animales , Humanos , Masculino , Ratones , Ratones TransgénicosRESUMEN
Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0-72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2-4 fold), and mRNA levels (30-40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4-72 h), showed marked (2-6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD.
Asunto(s)
Citocromo P-450 CYP1A1/genética , Regulación Enzimológica de la Expresión Génica , Hiperoxia/complicaciones , Receptores de Hidrocarburo de Aril/metabolismo , Apoproteínas/metabolismo , Línea Celular , Citocromo P-450 CYP1A1/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Humanos , Luciferasas/metabolismo , Pulmón , Lesión Pulmonar/etiología , Lesión Pulmonar/fisiopatología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 micro g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 degrees C for 2h, giving rise to 9 adducts, as determined by (32)P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16h, followed by MC (1 micro M) treatment for 24h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.