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1.
Toxicol Appl Pharmacol ; 226(3): 213-24, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17919675

RESUMO

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 microM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17beta-estradiol (E(2)) metabolism, whereas BKF levels greater than 1 muM inhibited E(2) metabolism. Time course studies showed that induction of CYP1-catalyzed E(2) metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.


Assuntos
Benzo(a)pireno/toxicidade , Neoplasias da Mama/enzimologia , Citocromo P-450 CYP1A1/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Fluorenos/toxicidade , Hidrocarboneto de Aril Hidroxilases , Benzo(a)pireno/metabolismo , Biotransformação/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1B1 , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Indução Enzimática/efeitos dos fármacos , Feminino , Fluorenos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia
2.
J Interferon Cytokine Res ; 23(11): 621-30, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14651776

RESUMO

The aim of the present work was to evaluate the induction and localization of Stat1, interferon (IFN) regulatory factor-1 (IRF-1), and IRF-2 after IFN-gamma exposure of human breast cancer cell lines, SKBR3, MDA468, MCF7, and BT20. Results from growth assays, Western staining, electrophoretic mobility shift assay (EMSA), and immunohistochemical staining were collated to test our hypothesis that immunohistochemical analysis of Stat1, IRF-1, and IRF-2 would provide additional information about the functionality of the IFN-gamma signaling pathway in human tumor lines. EMSA results showed that in each of four cell lines, Stat1 expression was increased and demonstrated functional activity after IFN-gamma stimulation. Western and EMSA analysis showed upregulation of IRF-1 but not IRF-2 in each cell line. Confocal microscopy of cells stained for Stat1, IRF-1, and IRF-2 confirmed the results and also provided novel information about the intracellular localization of proteins and intercellular variations in responses. The proportion of cells with IRF-1 stimulation and translocation was positively correlated with the IFN-gamma growth suppression in vitro. In conclusion, using four independent assays, we have demonstrated that heterogeneity in IFN-gamma-mediated upregulation of signal transduction proteins can be detected in vitro and that these differences can explain distinct cellular growth effects.


Assuntos
Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Interferon gama/farmacologia , Fosfoproteínas/metabolismo , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição , Anticorpos Monoclonais , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Microscopia Confocal , Proteínas Recombinantes , Fator de Transcrição STAT1 , Transdução de Sinais
3.
J Interferon Cytokine Res ; 23(9): 501-11, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14565859

RESUMO

Interferon (IFN) regulatory factor-1 (IRF-1) and IRF-2 play opposing roles in the regulation of many IFN-gamma-inducible genes. To investigate the signal transduction pathway in response to IFN-gamma in light of differences in growth effects, we selected four human breast carcinoma cell lines based on a spectrum of growth inhibition by IFN-gamma. MDA468 growth was markedly inhibited by IFN-gamma, and it showed substantial induction of IRF-1 mRNA but little IRF-2 induction. SKBR3 showed little growth inhibition and little induction of IRF-1 mRNA but significant induction of IRF-2 mRNA. HS578T and MDA436 growth inhibition and IRF-1/IRF-2 induction were intermediate. All four cell lines showed intact receptor at the cell surface and Stat1 translocation to the nucleus by immunostaining. By EMSA, there were marked differences in the induced ratio of IRF-1 and IRF-2 binding activity between the cell lines that correlated with growth inhibition. Finally, antisense oligonucleotides specific for IRF-1 attenuated IFN-gamma growth inhibition in MDA436 and MDA468, confirming the direct role of IRF-1 in IFN-gamma growth inhibition. Induction of IRF-1 causes growth inhibition in human breast cancer cell lines, and induction of IRF-2 can oppose this. The relative induction of IRF-1 to IRF-2 is a critical control point in IFN-gamma response.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferon gama/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras , Fatores de Transcrição , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Fator Regulador 1 de Interferon , Fator Regulador 2 de Interferon , Interferon gama/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Fosfoproteínas/genética , Transporte Proteico , RNA Mensageiro/metabolismo , Receptores de Interferon/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1 , Transativadores/metabolismo , Células Tumorais Cultivadas , Receptor de Interferon gama
4.
Anal Biochem ; 312(2): 162-6, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12531201

RESUMO

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Ensaio de Amplificação de Sinal de DNA Ramificado/métodos , Citocromo P-450 CYP1A1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Policíclicos/farmacologia , RNA Mensageiro/análise , Citocromo P-450 CYP1B1 , Fluorescência , Humanos , RNA Mensageiro/genética , Células Tumorais Cultivadas
5.
Drug Metab Dispos ; 30(3): 262-9, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11854143

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO(2) in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2- and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2- and 4-hydroxylation pathways at 3 microM BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO(2) caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 microM BAP because cotreatment with 10 microM NaAsO(2) inhibited the rates of the 2- and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO(2). The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO(2); however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO(2). These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.


Assuntos
Arsenitos/toxicidade , Hidrocarboneto de Aril Hidroxilases , Benzo(a)pireno/toxicidade , Citocromo P-450 CYP1A1/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Poluentes Ambientais/toxicidade , Compostos de Sódio/toxicidade , Western Blotting , Neoplasias da Mama , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Estradiol/metabolismo , Receptor alfa de Estrogênio , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Humanos , Hidroxilação , Proteínas de Membrana , Oxigenases de Função Mista/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Estrogênio/metabolismo , Células Tumorais Cultivadas
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