RESUMO
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.
Assuntos
Carcinógenos Ambientais/toxicidade , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Metilcolantreno/toxicidade , Ativação Transcricional/efeitos dos fármacos , Animais , Western Blotting , Carcinógenos Ambientais/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1A2/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/enzimologia , Pulmão/enzimologia , Metilcolantreno/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.
Assuntos
Carcinógenos/toxicidade , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Desintoxicação Metabólica Fase II/fisiologia , Metilcolantreno/toxicidade , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP1B1 , Indução Enzimática/efeitos dos fármacos , Epóxido Hidrolases/biossíntese , Epóxido Hidrolases/genética , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Injeções Intraperitoneais , Isoenzimas/biossíntese , Isoenzimas/genética , Fígado/enzimologia , Pulmão/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
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.
Assuntos
Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/metabolismo , Adutos de DNA/metabolismo , Expressão Gênica , Metilcolantreno/farmacologia , Regiões Promotoras Genéticas , Animais , Linhagem Celular Tumoral , Citocromos , Indução Enzimática , Feminino , Genes Reporter , Humanos , Neoplasias Hepáticas Experimentais , Masculino , Metilcolantreno/metabolismo , Camundongos , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , TransfecçãoRESUMO
There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are bioactivated by the cytochrome P450 (P450) 1A family of enzymes to metabolites that are capable of covalently binding to DNA, a critical step in the initiation of carcinogenesis. We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P4501A expression for up to 45 days. Here, we tested the hypothesis that MC elicits persistent induction of other genes that are regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 micromol/kg) ip once daily for 4 days, and gene expression patterns were investigated using total liver RNA isolated from animals at 1, 15, and 28 days after MC withdrawal. Gene expression was studied by cDNA microarray analyses using 4608 unique clones from liver-derived expressed sequence tag (EST) libraries fortified with clones of known liver genes representing approximately 4000 genes. Several phase I (P4501A1, -1A2) and phase II [e.g., glutathione-S-transferase (GST)-M1, UDP-glucuronosyl transferases (UGT)] genes were persistently induced (3-10-fold) by MC for 15-28 days. The persistent induction of P4501A1 gene expression was confirmed by real time reverse transcriptase polymerase chain reaction (RT-PCR) experiments. MC also elicited a 5-fold persistent augmentation of acute phase genes such as orosomucoid 1 and alpha-1-acid glycoprotein (AGP), and this was accompanied by sustained liver damage and inflammation in the MC-exposed rats. In conclusion, our results strongly suggest that sustained induction of P4501A1 by MC is accompanied by persistent expression of other genes belonging to the Ah gene battery, as well as certain other genes involved in toxic responses. Elucidating the mechanisms of persistent induction of P4501A1 and other genes by MC might lead to a better understanding of the mechanisms of toxicity mediated by PAHs.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Metilcolantreno/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fase Aguda/genética , Animais , Citocromo P-450 CYP1A1/genética , Glucuronosiltransferase/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
Administration of supplemental oxygen is frequently encountered in infants suffering from pulmonary insufficiency and in adults with acute respiratory distress syndrome. However, hyperoxia causes acute lung damage in experimental animals. In the present study, we investigated the roles of the Ah receptor (AHR) in the modulation of cytochrome P4501A (CYP1A) enzymes and in the development of lung injury by hyperoxia. Adult male wild-type [AHR (+/+)] mice and AHR-deficient animals [AHR (-/-)] were maintained in room air or exposed to hyperoxia (>95% oxygen) for 24 to 72 h, and pulmonary and hepatic expression of CYP1A and lung injury were studied. Hyperoxia caused significant increases in pulmonary and hepatic CYP1A1 activities (ethoxyresorufin O-deethylase) and mRNA levels in wild-type (C57BL/6J) AHR (+/+), but not AHR (-/-) mice, suggesting that AHR-dependent mechanisms contributed to CYP1A1 induction. On the other hand, hyperoxia augmented hepatic CYP1A2 expression in both wild-type and AHR (-/-) animals, suggesting that AHR-independent mechanisms contributed to the CYP1A2 regulation by hyperoxia. AHR (-/-) mice exposed to hyperoxia were more susceptible than wild-type mice to lung injury and inflammation, as indicated by significantly higher lung weight/body weight ratios, increased pulmonary edema, and enhanced neutrophil recruitment into the lungs. In conclusion, our results support the hypothesis that the hyperoxia induces CYP1A1, but not CYP1A2, expression in vivo by AHR-dependent mechanisms, a phenomenon that may mechanistically contribute to the beneficial effects of the AHR in hyperoxic lung injury.
Assuntos
Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A2/biossíntese , Predisposição Genética para Doença/genética , Pulmão/metabolismo , Oxigênio/toxicidade , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Síndrome do Desconforto Respiratório/metabolismo , Animais , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Fígado/enzimologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/fisiologia , Síndrome do Desconforto Respiratório/enzimologia , Síndrome do Desconforto Respiratório/genéticaRESUMO
Benzo[a]pyrene (BP), a polycylic aromatic hydrocarbon (PAH), is a potent atherogen and carcinogen in laboratory animals. Since genotoxic mechanisms may contribute to the development of atherosclerosis by PAHs, we have tested the hypotheses that: 1) BP induces DNA adducts in mouse aortic smooth muscle cells (SMCs); 2) 3-hydroxybenzo[a]pyrene (3-OH-BP) and benzo[a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites; and 3) cytochrome P4501B1 (CYP1B1) mediates the activation of BP and its metabolites to ultimate genotoxic intermediates. Cultured mouse aortic SMCs were treated with BP, 3-OH-BP, or BPQ for 24 h, and DNA adduct formation was analyzed by (32)P-postlabeling. In some experiments, cells were pretreated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to BP or its metabolites. BP, 3-OH-BP, and BPQ induced formation of several DNA adducts that were not observed in dimethylsulfoxide-treated cells. Re- and cochromatography experiments indicated that 3-OH-BP and BPQ were proximate genotoxic metabolites of BP. DNA adduct formation was strongly inhibited by EP, a specific inhibitor of CYP1B1. BP treatment of SMCs resulted in induction of aryl hydrocarbon hydroxylase (AHH) activity and CYP1B1, but not CYP1A1, apoprotein. EP also blocked AHH induction by BP. In conclusion, the results of this study support the hypothesis that in SMCs, which are target sites for the development of atherosclerosis, the major bioactivation pathway of BP entails CYP1B1-mediated formation of the 3-OH-BP and BPQ, which are proximate genotoxic metabolites that may in turn get transformed to ultimate DNA-binding metabolites, which may contribute to atherogenesis by PAHs.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzo(a)pireno/metabolismo , Benzopirenos/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Animais , Biotransformação , Células Cultivadas , Citocromo P-450 CYP1B1 , DNA/efeitos dos fármacos , DNA/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Radioisótopos de FósforoRESUMO
There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (+/+)] mice or mice lacking the gene for the AHR were treated with a single dose (100 micromol/kg) of BP or MC, and hepatic DNA adducts were analyzed by (32)P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHR-null animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHR- and CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Adutos de DNA/metabolismo , DNA/efeitos dos fármacos , Fígado/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1B1 , DNA/metabolismo , Ativação Enzimática , Indução Enzimática , Homozigoto , Marcação por Isótopo , Fígado/enzimologia , Masculino , Metilcolantreno/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/enzimologia , Isótopos de FósforoRESUMO
The cytochrome P4501A enzymes play important roles in carcinogen metabolism. We reported previously that 3-methylcholanthrene (MC) elicits a persistent induction of hepatic, pulmonary, and mammary microsomal cytochrome P450 (P450) 1A enzymes for several weeks after MC withdrawal. In this study, we tested the hypothesis that CYP1A2, a liver-specific P450 isozyme, plays an important role in the mechanisms governing persistent CYP1A1 induction by MC in liver but not in extra-hepatic tissues such as lung, which is devoid of endogenous CYP1A2. Administration of wild-type (WT) or CYP1A2-null mice with MC (100 micromol/kg i.p.) once daily for 4 days caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein contents, 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 15 days in both the genotypes. Since MC is almost completely eliminated by day 15, we hypothesize that CYP1A2 contributes to the up-regulation of CYP1A1 in liver, but not lung, by a novel mechanism, presumably involving a CYP1A2-dependent persistent metabolite. The studies demonstrate tissue-specific differences in the regulation of CYP1A by MC, a phenomenon that may have implications for human carcinogenesis caused by environmental chemicals.