RESUMO
Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas produced by feeding 2-acetylaminofluorene or N-nitrosomorpholine was studied using isozyme-selective substrates, antibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) activities toward 4-methylumbelliferone, 1-naphthol, and benzo[a]pyrene-3,6-quinol were reversibly increased by short term feeding of 2-acetylaminofluorene but were persistently increased in hepatocyte nodules and differentiated hepatocellular carcinomas. Immunoblot analysis revealed that short term feeding of 2-acetylaminofluorene increased a Mr 55,000 polypeptide corresponding to the previously characterized UDP-GTI or phenol UDP-GT. However, in some hepatocyte nodules and hepatocellular carcinomas either the Mr 55,000 or a new Mr 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte nodules, which may contribute to the toxin-resistance phenotype frequently observed at cancer prestages.
Assuntos
Glucuronosiltransferase/biossíntese , Neoplasias Hepáticas Experimentais/enzimologia , RNA Mensageiro/análise , 2-Acetilaminofluoreno/farmacologia , Animais , Anticorpos , Indução Enzimática/efeitos dos fármacos , Glucuronosiltransferase/genética , Glucuronosiltransferase/imunologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Metilcolantreno , Ratos , Ratos EndogâmicosRESUMO
To investigate constitutive and inducible expression of rat phenol UDP-glucuronosyltransferase (UGT1A1) in liver and extrahepatic tissues, a selective cDNA probe for its unique exon 1 was utilized. 6-Hydroxychrysene was used as a functional probe of UGT1A1 activity. Constitutive expression of UGT1A1 was low in liver, but high in kidney, testis, epididymis and ovary. After treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms/kg for 7 days) the UGT1A1 mRNA level was markedly increased in liver (ca. 10-fold), and only moderately enhanced (up to 2-fold) in extrahepatic tissues where constitutive enzyme expression was high. UGT activity toward 6-hydroxychrysene was strongly inducible in liver (ca. 9-fold) and only moderately inducible in extrahepatic tissues (up to 2-fold). The results suggest complex tissue-specific regulation of UGT1A1 including positive and negative transcriptional factors and marked inducibility by TCDD in liver.
Assuntos
Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Dibenzodioxinas Policloradas/farmacologia , Animais , Crisenos/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Fígado/efeitos dos fármacos , Masculino , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
UDP-glucuronosyltransferases (UGTs), in addition to their role in overall pharmacokinetics, play important roles in local protection of cells against toxins and in the control of endogenous receptor ligands. UGT1A6, which conjugates planar phenols, appears to be expressed in many organs, but information on cell-specific expression in these organs is controversial or absent. Therefore, a non-isotopic in situ hybridization method was developed and applied to localize UGT1A6 expression in rat testis and brain. It was found that UGT1A6 is expressed in Sertoli cells and spermatogonia of rat testis and in brain neurons, in particular in hippocampal pyramidal cells and Purkinje cells of the cerebellum.
Assuntos
Encéfalo/enzimologia , Glucuronosiltransferase/biossíntese , Testículo/enzimologia , Animais , Encéfalo/metabolismo , Hibridização In Situ/métodos , Técnicas In Vitro , Masculino , Ratos , Ratos Wistar , Testículo/metabolismoRESUMO
The protooncogene c-myc was investigated in N-nitrosomorpholine-induced rat liver nodules to elucidate the role of altered DNA methylation in chemical carcinogenesis. Furthermore, Micrococcus luteus DNA and chicken erythrocyte DNA were modified in vitro by reactive metabolites of N-nitrosomorpholine, generated by P450-dependent monooxygenases. The modified DNAs were less methylated in vitro than control DNAs by DNA-(cytosine-5)-methyltransferase (DNA methylase). The DNA methylase assay and 32P-postlabeling analysis revealed lowered levels of DNA methylation in nodular DNA. In nodular tissue, c-myc messenger RNA levels were found to be increased compared to normal liver. DNA methylation analysis using the restriction endonucleases HpaII/MspI indicated hypomethylation in the first intron of c-myc DNA in liver nodules. The results suggest that genotoxic lesions may cause stably inherited, aberrant DNA methylation patterns which may be responsible for site-specific hypomethylation of the c-myc protooncogene in liver nodules.
Assuntos
Metilases de Modificação do DNA/metabolismo , Neoplasias Hepáticas/induzido quimicamente , Nitrosaminas , Proto-Oncogenes , Animais , DNA Bacteriano/metabolismo , DNA de Neoplasias/isolamento & purificação , Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Masculino , Metilação , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , RNA Neoplásico/isolamento & purificação , Ratos , Ratos EndogâmicosRESUMO
Cytochrome P4501A1 (CYP1A1) and the UDP-glucuronosyltransferase isoform UGT1A6 were studied in pharyngeal mucosa and squamous cancer tissue obtained from 27 male subjects (10 healthy nonsmoking volunteers, 10 smokers, and 7 smokers with pharyngeal cancer). CYP1A activity (7-ethoxyresorufin O-deethylase) was significantly induced in smokers as compared to nonsmokers (2.3 +/- 1.1 and 0.8 +/- 0.4 pmol x min[-1] x mg protein[-1], respectively). Immunoblot analysis demonstrated enhanced CYP1A1 protein in smokers. UGT activity towards 4-methylumbelliferone and 1-naphthol was also detectable in oropharyngeal mucosa. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis indicated that UGT activity was at least in part due to the expression of UGT1A6. In cancer tissue, CYP1A activity was decreased in comparison with surrounding healthy mucosa (1.2 +/- 0.9 in tumor tissue vs. 2.2 +/- 0.7 pmol x min[-1] x mg protein[-1], respectively), whereas means and medians of UGT activity were unchanged. The results suggest that phase I and II drug-metabolizing enzymes are detectable in oropharyngeal mucosa and that CYP1A activity is inducible by constituents of cigarette smoke.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Glucuronosiltransferase/metabolismo , Neoplasias Orofaríngeas/enzimologia , Faringe/enzimologia , Adulto , Humanos , Masculino , Mucosa/enzimologia , Fumar/metabolismoRESUMO
Transcriptional regulation and function of rat and human PAH-inducible UDP-glucuronosyltransferase (UGT) isoforms have been studied. 1. At least two PAH-inducible UGT isoforms are expressed in a variety of tissues, the rat isoforms UGT1A6 and UGT1A7, and the human isoforms UGT1A6 and UGT1A9. 2. For the rat and human UGT1A6 isoforms two modes of tissue- and cell-specific regulation were found, PAH-inducible and constitutive expression. 3. Transient transfection studies, using human UGT1A6/CAT fusion constructs and colon carcinoma Caco-2 cells, revealed that PAH induction of human UGT1A6 is mediated by the Ah receptor. 4. Cell-expressed UGT isoforms were used to study their function in PAH metabolism. Rat UGT1A7 and human UGT1A9 appear to be more efficient than the corresponding UGT1A6 isoforms in catalyzing glucuronide formation of PAH phenols and diphenols. Several isoforms may act together in the formation of benzo(a)pyrene-3.6-diol diglucuronide, the major glucuronide found in rat bile. The results suggest complex modes of transcriptional regulation of PAH-inducible UGTs. They also suggest a major role of these UGT isoforms in detoxication of PAHs.
Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Glucuronosiltransferase/genética , Receptores de Hidrocarboneto Arílico/genética , Transcrição Gênica/genética , Animais , Genes Reporter/genética , Glucuronatos/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Estrutura Molecular , Dibenzodioxinas Policloradas/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , RNA Mensageiro/genética , Ratos , Células Tumorais CultivadasRESUMO
We have used autoradiography of [125I]iodocyanopindolol [( 125I]-ICYP) to define the distribution and localization of beta-adrenergic receptors in the rat kidney. [125I]ICYP, a radiolabeled beta-adrenergic antagonist, proved to be an excellent probe to identify these receptors in kidney slices in that specific [125I]ICYP binding was 1) saturable, 2) competed stereoselectively by the agonist isoproterenol and the antagonist propranolol, and 3) competed by agonists in a classical rank order of potency for beta 1-adrenergic receptors (isoproterenol greater than epinephrine congruent to norepinephrine). Autoradiographic studies demonstrated that specific [125I]ICYP sites were present exclusively in the cortex and outer medulla and were localized in glomeruli, ascending limb, and distal tubule-cortical collecting duct. Autoradiography of probes like [125I]ICYP appears to offer a simple and rapid method to assess distribution of adrenergic receptors in the kidney and presumably in other tissues as well.
Assuntos
Rim/análise , Pindolol/análogos & derivados , Receptores Adrenérgicos beta/análise , Animais , Autorradiografia , Estudos de Avaliação como Assunto , Radioisótopos do Iodo , Iodocianopindolol , Rim/metabolismo , Masculino , Pindolol/metabolismo , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
beta-Adrenergic receptor subtypes were localized and differentiated in rat kidney slices by in vitro autoradiography using the nonselective beta-antagonist [125I]iodocyanopindolol in the presence of the selective agents betaxolol (beta 1) and zinterol (beta 2). [125I]Iodocyanopindolol binding to kidney sections in the presence of these agents could be differentiated into high- and low-affinity components as predicted by the subtype selectivity of the compounds. Autoradiography revealed that: beta-adrenergic receptors were highly concentrated within the renal cortex, especially in glomeruli and juxtaglomerular granule cells, and to tubular sites in the cortex and medulla; [125I]iodocyanopindolol labeling of the juxtaglomeruluar granule cells was abolished at lower concentrations (10(-8) M) of betaxolol than was the labeling of glomeruli (10(-6)--10(-4) M); and zinterol had little effect on labeling of juxtaglomerular granule cells and glomeruli unless high concentrations were used, whereas the tubular labeling in medulla was much more sensitive to incubation with zinterol. These results indicate that beta 1- and beta 2-adrenergic receptor subtypes are differentially distributed within the kidney: beta 1, predominantly contained in juxtaglomerular granule cells and glomeruli, and beta 2, predominantly in medullary tubules. In vitro autoradiography provides a useful means to examine different receptor populations in discrete tissue areas.
Assuntos
Rim/análise , Receptores Adrenérgicos beta/análise , Animais , Autorradiografia , Betaxolol , Ligação Competitiva , Etanolaminas/metabolismo , Iodocianopindolol , Masculino , Pindolol/análogos & derivados , Pindolol/metabolismo , Propanolaminas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Transcriptional regulation of human UGT1A6, a UDP glucuronosyltransferase isoform conjugating a wide variety of planar phenols, has been studied using transfection experiments with plasmids containing its 3-kb 5' upstream region and chloramphenicol acetyltransferase as reporter gene. Previously, two modes of expression of the isoform have been described: in colon carcinoma Caco-2 cells UGT1A6 was found to be TCDD-inducible, whereas in lung carcinoma A549 cells it was constitutively expressed. Therefore functional analysis of UGT1A6 regulation was carried out using these two cell lines. In the upstream region of human UGT1A6 one xenobiotic-responsive element (XRE) was found between-1498 and -1502 bp. In Caco-2 cells the reporter gene activity of the entire plasmid and of deletion mutants containing the XRE were TCDD-inducible, in contrast to experiments with a deletion mutant which did not contain the XRE. TCDD induction was marginal in transfection studies with A549 cells. Gel mobility shift analysis indicated that the aryl hydrocarbon receptor and its partner Arnt bind to the XRE. Furthermore, primer extension studies suggest cell-specific use of multiple TATA boxes. Hence, regulation of human UGT1A6 appears to be cell-specific including both constitutive and aryl hydrocarbon receptor-controlled expression.
Assuntos
Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/biossíntese , Receptores de Hidrocarboneto Arílico/metabolismo , Sequências Reguladoras de Ácido Nucleico , Translocador Nuclear Receptor Aril Hidrocarboneto , Sequência de Bases , Sítios de Ligação , Carcinoma/enzimologia , Neoplasias do Colo/enzimologia , Genes Reporter , Glucuronosiltransferase/genética , Humanos , Neoplasias Pulmonares/enzimologia , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Análise de Sequência de DNA , Deleção de Sequência , Transdução de Sinais , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Tissue-specific expression of human UGT1A6, a UDP-glucuronosyltransferase isoform conjugating a wide variety of planar phenols, has been studied using a reverse transcriptase-polymerase chain reaction. Use of intron-overlapping forward and reverse primers from exon 1 and 2 and of a "hot start" modification led to selective amplification of a UGT1A6 mRNA fragment. In addition, homologous competitor mRNA was synthesized, reverse transcribed, and coamplified to allow quantitation of UGT1A6 mRNA. Using these methods UGT1A6 mRNA could be demonstrated in liver, kidney, duodenum, and lung. Cell-specific regulation of UGT1A6 by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) was studied in various cell systems. TCDD induction was found in the human colon carcinoma cell line Caco-2 and in hepatocyte primary cultures. In contrast, in lung carcinoma A549 cells this isoform was constitutively expressed and not induced by TCDD.
Assuntos
Glucuronosiltransferase/biossíntese , Fígado/enzimologia , Dibenzodioxinas Policloradas/farmacologia , Duodeno/enzimologia , Indução Enzimática , Humanos , Mucosa Intestinal/enzimologia , Íntrons , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Valores de Referência , Fumar , Transcrição GênicaRESUMO
Induction of PAI-2 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been studied in human primary hepatocytes, hepatoma HepG2 cells and monocytic U937 cells, extending recent findings in human keratinocytes. PAI-2 represents a serpine-type protease inhibitor with wide-ranging implications in fibrinolysis, extracellular matrix proteolysis, growth factor activation and carcinogenesis. PAI-2 was induced by >10(-9) M TCDD in hepatocytes and HepG2 cells and by >10(-10) M TCDD in U937 cells. In the latter cell line, PAI-2 induction by TCDD and by 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been compared. TCDD appeared to be less efficient than TPA as an inducer of PAI-2. In contrast to induction by TPA, PAI-2 induction by TCDD was found to be biphasic, with an early peak of mRNA at 1-3 h and a late peak at 12-24 h. A biphasic response was also seen at the protein level although production of PAI-2 protein lagged behind the corresponding mRNA. PAI-2 is known to contain AP-1 sites, i.e. Jun/Fos protein-binding sites, in its promotor region. Hence, PAI-2 induction by TCDD has originally been conceived to be due to an indirect response, secondary to the induction of Jun/Fos proteins. Therefore, expression of jun/fos genes and their AP-1 activity were studied at the early phase of PAI-2 induction by TCDD. TCDD did not increase mRNA of c-fos, c-jun, junB or junD (in contrast to TPA which markedly increased the expression of c-fos and junB), nor did TCDD increase AP-1 activity. In conclusion, the findings suggest that PAI-2 induction by TCDD is not restricted to human keratinocytes but includes liver cells and monocytic U937 cells. The induction mechanism is complex but the early phase does not appear to involve Jun/Fos proteins.
Assuntos
Proteínas de Ligação a DNA , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Inibidores de Serina Proteinase/metabolismo , Fatores de Transcrição/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto , Carcinoma Hepatocelular/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Dactinomicina/toxicidade , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/toxicidade , Fator de Transcrição AP-1/metabolismo , Células Tumorais CultivadasRESUMO
Functions and regulation of selected human UDP-glucuronosyltransferases (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15) are summarized. Evidence for at least two PAH-inducible UGTs (UGT1A6 and UGT1A9) is presented, which, however, are also constitutively expressed in a tissue- and cell-specific manner. These isoforms have recently been characterized to conjugate planar and bulky phenols, respectively. Using a selective RT-PCR method, UGT1A6 expression was detected in a variety of tissues (liver, kidney, lung, intestine, and pharyngeal mucosa). PAH-inducible UGTs may cooperate in the metabolism of phenolic metabolites of benzo(a)pyrene. Studies with stably expressed isoforms suggest that UGT1A9 is responsible for the formation of benzo(a)pyrene-3.6-diphenol diglucuronide, the major biliary metabolite of benzo(a)pyrene.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glucuronosiltransferase/genética , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Indução Enzimática , Glucuronosiltransferase/fisiologia , Humanos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We have explored the localization of renal alpha 2-adrenoceptors by radioligand binding techniques and studies of 22Na+ influx into isolated renal tubular cells. Initially, we validated the use of the alpha 2-adrenergic antagonist [3H]rauwolscine as a high-affinity (Kd = 2.5 +/- 0.65 nM) probe that binds to a single class (Hill slope = 0.94 +/- 0.06) of alpha 2-adrenoceptors in rat renal cortical membranes (Bmax = 340 +/- 50 fmol/mg). [3H]Rauwolscine and [3H]yohimbine identified an identical number of sites, but [3H]rauwolscine bound with a several-fold higher affinity. Treatment of rats with 6-hydroxydopamine depleted renal norepinephrine greater than 80% but failed to alter the number or affinity of renal cortical alpha 2-receptors, thus suggesting that these alpha 2-receptors are not located on presynaptic sites. Further evidence for the postsynaptic localization of these receptors was obtained by in vitro autoradiographic studies, which showed that [3H]rauwolscine sites were preferentially located on proximal tubular cells, and by functional assays with isolated rabbit proximal tubular cells. In the latter studies, Na+-H+ antiport activity was stimulated up to 2-fold by the selective alpha 2-adrenergic agonist guanabenz, and this stimulation was blocked by yohimbine but not by the alpha 1-blocker prazosin. Although amiloride was able to block this response to guanabenz, radioligand binding studies demonstrated that amiloride can compete for [3H]rauwolscine sites at the concentrations used to block Na+-H+ antiport activity. These data suggest that alpha 2-adrenoceptors are present on proximal tubular cells in rats and rabbits and that these receptors appear to stimulate Na+-H+ antiport activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Túbulos Renais Proximais/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Amilorida/farmacologia , Animais , Autorradiografia , Guanabenzo/farmacologia , Hidroxidopaminas/farmacologia , Técnicas In Vitro , Córtex Renal/metabolismo , Cinética , Masculino , Oxidopamina , Prazosina/farmacologia , Coelhos , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Ioimbina/metabolismoRESUMO
Human colon carcinoma Caco-2 cells were used to study the induction of UDP glucuronosyltransferase (UGT) isoforms UGT1A6, UGT1A9, and UGT2B7 by aryl hydrocarbon receptor agonists and by antioxidant-type inducers with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and t-butylhydroquinone (TBHQ), respectively. Early- (PF11) and late-passage clones (TC7) of Caco-2 cells, which show low and high constitutive UGT1A6 expression, respectively, were selected. The following results were obtained: 1) In Caco-2 cells UGT activity (4-methylumbelliferone as substrate) was significantly enhanced by 10 nM TCDD or 40 to 80 microM TBHQ and 2) duplex reverse-transcription-polymerase chain reaction analysis showed for the first time that the expression of human UGT1A6, UGT1A9, and UGT2B7 was enhanced by 40 to 80 microM TBHQ; both UGT1A6 and UGT1A9 were induced by 10 nM TCDD, whereas UGT2B7 was not induced by TCDD. The results suggest that at least two human UGTs (UGT1A6 and UGT1A9) are inducible by aryl hydrocarbon receptor agonists and even more isoforms (UGT1A6, UGT1A9, and UGT2B7) are inducible by antioxidant-type inducers in Caco-2 cells.