RESUMO
Glucocorticoids precociously induce fetal rat UGT1A6 and potentiate polycyclic aromatic hydrocarbon (PAH)-dependent induction of this enzyme in vivo and in isolated rat hepatocytes. To establish whether induction was due to glucocorticoid receptor (GR), luciferase reporter vectors were tested in transfection assays with HepG2 cells. Using a reporter construct containing approximately 2.26 kilobases of the 5'-flanking region of the UGT1A6-noncoding leader exon (A1*), dexamethasone increased basal activity 3- to 7-fold in cells cotransfected with an expression plasmid for GR. PAH increased gene expression 23-fold, but the presence of dexamethasone only induced PAH-dependent expression by 1.5-fold, suggesting interaction between GR and the aryl hydrocarbon (Ah) receptor. Furthermore, the GR antagonist RU 38486 [17beta-hydroxy-11beta-(4-dimethylamino-phenyl)-17alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] was a partial agonist that increased, rather than inhibited, basal activity 3-fold. 5'-deletion analysis defined the 5'-boundary for a functional glucocorticoid-responsive unit between base pairs -141 and -118 relative to the transcription start site. This region contains the Ah receptor response element (AhRE), and both PAH and glucocorticoid-dependent gene activation were lost when this area was deleted. Mutation of a single base pair located in the AhRE region simultaneously reduced induction by PAH and increased glucocorticoid induction. Thus, the sequences of both the AhRE and glucocorticoid response elements seem to overlap, suggesting that Ah receptor binding may decrease glucocorticoid-dependent induction due to interactions of these two cis-acting elements. Mutation of a putative GRE located between base pair -81 and -95 reduced, but did not completely eliminate, glucocorticoid-dependent induction of the reporter, suggesting that a nonclassic mechanism of induction is involved in this response.
Assuntos
Glucocorticoides/farmacologia , Glucuronosiltransferase/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de Glucocorticoides/genética , Elementos de Resposta , Animais , Benzo(a)pireno/farmacologia , Linhagem Celular Tumoral , Dexametasona/farmacologia , Expressão Gênica , Genes Reporter , Glucuronosiltransferase/metabolismo , Humanos , Luciferases/metabolismo , Mifepristona/farmacologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Glucocorticoides/metabolismo , TransfecçãoRESUMO
Patients with Crigler-Najjar syndrome (CN) type I inherit an autosomal recessive trait for hyperbilirubinemia, which is characterized by the total absence of bilirubin UDP-glucuronosyltransferase (transferase) activity. The recent identification of two bilirubin transferase isoforms with identical carboxyl termini (Ritter, J. K., J. M. Crawford, and I. S. Owens. 1991. J. Biol. Chem. 266:1043-1047) led to the discovery of a unique locus, UGT1, which encodes a family of UDP-glucuronosyltransferase isozymes, including the two bilirubin forms (Ritter, J. K., F. Chen, Y. Y. Sheen, H. M. Tran, S. Kimura, M. T. Yeatman, and I. S. Owens. 1992. J. Biol. Chem. 267:3257-3261). The UGT1 locus features a complex of six overlapping transcriptional units encoding transferases, each of which shares the four most 3' exons (2, 3, 4, and 5) specifying the 3' half of the transferase coding regions (condons 289-533) and the entire 3' untranslated region of each mRNA. This gene model predicts that a single critical mutation in any of these four "common" exons may inactivate the entire family of encoded transferases. In agreement with this prediction, we show here that in the first CN type I individual analyzed (patient F.B.), a 13-bp deletion has occurred in exon 2. Analysis of product generated by the polymerase chain reaction and genomic DNA demonstrated that F.B. is homozygous for the defective allele (UGT1*FB), and that the consanguineous parents are both heterozygotic at this locus. The mutation is predicted to result in the synthesis of severely truncated bilirubin transferase isozymes that are lacking a highly conserved sequence in the carboxyl-terminus and the characteristic membrane (endoplasmic reticulum)-anchoring segment of the protein molecule.
Assuntos
Mapeamento Cromossômico , Síndrome de Crigler-Najjar/genética , Glucuronosiltransferase , Hexosiltransferases/genética , Mutação , Sequência de Bases , Pré-Escolar , Deleção Cromossômica , Éxons , Feminino , Humanos , Masculino , Dados de Sequência MolecularRESUMO
Accumulating evidence indicates that mutations in the human UGT1 gene locus abolish hepatic bilirubin UDP-glucuronosyltransferase activity and cause the subsequent accumulation of bilirubin to toxic levels in patients with Crigler-Najjar type 1 (CN-I). Genetic and biochemical criteria are required to link CN-I with mutations in UGT1. Here we present analysis of mutations at the UGT1 locus in three individuals that were clinically diagnosed with CN-I (two related and one unrelated). Each patient carries a single base substitution that alters conserved residues in the transferase enzyme molecule, serine to phenylalanine at codon 376 and glycine to glutamic acid at codon 309. Each was homozygous for the defect as demonstrated by sequencing and RFLPs. Mutant cDNAs, constructed by site-directed mutagenesis, inserted into expression vectors, and transfected into COS-1 cells, supported the synthesis of the bilirubin transferase protein but only cells transfected with the wild-type cDNA expressed bilirubin UDP-glucuronosyltransferase activity. The data provide conclusive evidence that alterations at Gly 309 and Ser 376 are the genetic basis for CN-I in these families. These results suggest that the two codons, located in conserved regions of the molecule, are part of the active site of the bilirubin enzyme.
Assuntos
Síndrome de Crigler-Najjar/genética , Glucuronosiltransferase/genética , Fígado/enzimologia , Mutação Puntual , Adolescente , Sequência de Aminoácidos , Animais , Sequência de Bases , Criança , Síndrome de Crigler-Najjar/enzimologia , Éxons , Família , Feminino , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , TransfecçãoRESUMO
At least three types of congenital nonhemolytic unconjugated hyperbilirubinemias, including the rare Crigler-Najjar (CN) diseases (Types I or II) and Gilbert's syndrome (affecting 6% of the population) are associated with either absent or reduced hepatic UDP-glucuronosyltransferase (transferase) activity towards the potentially toxic endogenous acceptor, bilirubin. Here, we review the biochemical studies associated with these deficiencies. Accumulated evidence from studies with an animal model of CN Type I syndrome, the Gunn strain of hyperbilirubinemic rats, suggested that multiple isozymes are absent. These confounding observations have been clarified by a flurry of reports which have revealed the molecular basis for the complex disease phenotype in the Gunn rat and by the isolation and description of a novel human gene complex, UGT1, which encodes multiple and independently-regulated transferase isozymes that contain identical carboxyl terminal regions (246 amino acids). Finally, we discuss the implications of the gene organization and genetic defects determined for four different CN Type I individuals as a basis for a model which explains the inheritance pattern and genotypes of other familial unconjugated hyperbilirubinemias.
Assuntos
Glucuronosiltransferase/genética , Hiperbilirrubinemia Hereditária/enzimologia , Hiperbilirrubinemia Hereditária/genética , Animais , Clonagem Molecular , Síndrome de Crigler-Najjar/enzimologia , Síndrome de Crigler-Najjar/genética , DNA/genética , Feminino , Doença de Gilbert/enzimologia , Doença de Gilbert/genética , Humanos , Masculino , Família Multigênica , Linhagem , Fenótipo , Ratos , Ratos GunnRESUMO
One of the most important mechanisms involved in host defense against xenobiotic chemicals and endogenous toxins is the glucuronidation catalysed by UDP-glucuronosyltransferase enzymes (UGT). The role of genetic factors in determining variable rates of glucuronidation is not well understood, but phenotypic evidence in support of such variation has been reported. In the present study, six single nucleotide polymorphisms were discovered in the first exon of the UGT1A7 gene, which codes for the putative substrate-binding domain, revealing a high structural heterogeneity at the UGT1 gene locus. The new UGT1A7 proteins differ in their primary structure at amino acid positions 129, 131 and 208, creating four distinct UGT1A7 allelic variants in the human population: UGT1A7*1 (N129 R131 W208), *2 (K129 K131 W208), *3 (K129 K131 R208), and *4 (N129 R131 R208). In functional studies, HEK cells stably transfected to express the four allelic UGT1A7 variants exhibited significant differences in catalytic activity towards 3-, 7-, and 9-hydroxy-benzo(a)pyrene. UGT1A7*3 exhibited a 5.8-fold lower relative Vmax compared to wild-type *1, whereas *2 and *4 had a 2.6- and 2.8-fold lower relative Vmax than *1, respectively, suggesting that these mutations confer slow glucuronidation phenotype. Kinetic characterization suggested that these differences were primarily attributable to altered Vmax. Additionally, it suggested that each amino acid substitutions can independently affect the UGT1A7 catalytic activity, and that their effects are additive. The expression pattern of UGT1A7 studied herein and its catalytic activity profile suggest a possible role of UGT1A7 in the detoxification and elimination of carcinogenic products in lung. A population study demonstrated that a considerable proportion of the population (15.3%) was found homozygous for the low activity allele containing all three missense mutations, UGT1A7*3. These findings suggest that further studies are needed to investigate the impact of the low UGT1A7 conjugator genotype on individual susceptibility to chemical-induced diseases and responses to therapeutic drugs.
Assuntos
Glucuronosiltransferase/genética , Mutação de Sentido Incorreto , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , Feminino , Genótipo , Glucuronosiltransferase/metabolismo , Humanos , Cinética , Masculino , Linhagem , Reação em Cadeia da PolimeraseRESUMO
This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.
Assuntos
Evolução Molecular , Genes , Glucuronosiltransferase/genética , Família Multigênica , Terminologia como Assunto , Sequência de Aminoácidos , Animais , Glucuronosiltransferase/química , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Using extracts of AtT-20 cell nuclei, protein binding sites on the POMC gene 5'-flanking region were examined with an exonuclease protection approach. One such binding site, located from -119 to -106 bp upstream from the mouse POMC gene transcription initiation site, which exhibited a close homology to the activator protein-2 (AP-2) site [1]. A double-stranded oligonucleotide containing this site was subsequently used in gel shift assays to demonstrate AP-2 consensus sequence binding activity in extracts of AtT-20 cell nuclei. Gel shift competition experiments using both homologous and heterologous competitor DNA sequences revealed that the AP-2 like factor(s) exhibited specific binding to the mouse AP-2 consensus sequence. Furthermore, AP-2 factor binding was also modulated by a CTF/NF1-like factor. Pretreatment of AtT-20 cell nuclear extracts with alkaline phosphatase prior to inclusion in gel shift assays led to a reduction in the intensities of AP-2 factor-specific bands, indicating a potential involvement of protein phosphorylation in AP-2 factor binding in AtT-20 cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes , Pró-Opiomelanocortina/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Genes Reguladores , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Neoplasias Hipofisárias , Fator de Transcrição AP-2 , Transcrição GênicaRESUMO
A 4-fold induction of hepatic microsomal cytochrome P-450 following 3 days of treatment of rats with clotrimazole (75 mg/kg), a potent monooxygenase inhibitor, greatly exceeded that evident from similar phenobarbital and dexamethasone treatment. The clotrimazole-induced microsomes exhibited a pattern of monooxygenase activities similar to that seen in microsomes from both phenobarbital- and dexamethasone-treated animals. Precautions were necessary to determine both monooxygenase activities and the full amount of cytochrome P-450 present in microsomes because of interference by residual clotrimazole in the microsomes.
Assuntos
Biotransformação/efeitos dos fármacos , Clotrimazol/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Imidazóis/farmacologia , Microssomos Hepáticos/enzimologia , Animais , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Masculino , Fenobarbital/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The results reported herein strongly suggest that increased dopaminergic and not increased serotonergic activity is responsible for methamphetamine-induced increases in the nigral concentration of substance P-like immunoreactivity (SPLI). Thus, treatment of rats with the specific dopamine (DA) uptake blockers amfonelic acid (AFA) and nomifensine caused elevations in the SPLI levels within the substantia nigra similar to that of methamphetamine (METH). In contrast, the specific serotonin uptake blockers citalopram and chlorimipramine were without significant effects on this substance P (SP) system. Additional studies revealed that the mechanisms whereby the DA uptake blockers and METH influence the striatonigral SP pathway are likely different. Specifically, AFA, unlike METH, altered the SP system without causing changes in the monoaminergic synthesizing enzymes tyrosine and tryptophan hydroxylase; in addition, pretreatment with reserpine abolished the AFA effect on nigral SPLI but did not interfere with METH-mediated changes in the SP system.
Assuntos
Dopamina/fisiologia , Substância P/metabolismo , Substância Negra/metabolismo , Animais , Citalopram , Clomipramina/farmacologia , Corpo Estriado/metabolismo , Masculino , Metanfetamina/farmacologia , Ácido Nalidíxico/análogos & derivados , Naftiridinas/farmacologia , Nomifensina/farmacologia , Propilaminas/farmacologia , Radioimunoensaio , Ratos , Ratos Endogâmicos , Reserpina/farmacologia , Serotonina/fisiologiaRESUMO
Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these reactions, except for chrysene diphenol diglucronide formation (Bock et al., Mol Pharmacol 42: 613-618, 1992). Enzyme kinetic studies of the glucuronidation of 6-hydroxychrysene (used as a stable PAH phenol) indicated that UGT1A7 conjugated this compound with a lower apparent Km value (0.1 microM) than UGT1A6 (10 microM). The results suggest that the two PAH-inducible UGTs may cooperate in conjugating PAH metabolites, but that UGT1A7 is more efficient.
Assuntos
Benzo(a)pireno/metabolismo , Crisenos/metabolismo , Glucuronatos/metabolismo , Glucuronosiltransferase/metabolismo , Linhagem Celular , Glucuronatos/análise , Glucuronosiltransferase/biossíntese , Himecromona/metabolismo , Cinética , Fenóis/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
OBJECTIVE: We compared the efficacy of in vivo and ex vivo liposome transfection in rat lung transplantation. METHODS: (1) Chloramphenicol acetyltransferase group: Fischer rats underwent isogeneic transplantation (n = 4 per group). Recipients were put to death on postoperative day 2 for chloramphenicol acetyltransferase activity. Ex vivo setting: Grafts received cDNA complexed or not with liposomes and were transplanted after 1.5 or 10 hours at 10 degreesC. In vivo setting: Donors were intravenously injected with cDNA complexed or not with liposomes. Lungs were harvested after 1.5 or 10 hours, preserved at 10 degreesC, and transplanted. (2) Transforming growth factor-beta1 group: Brown-Norway rats served as donors and Fischer rats as recipients. All grafts were preserved for 3 hours at 10 degreesC. On postoperative day 5, arterial oxygenation and histologic rejection scores were assessed. Ex vivo setting: Grafts received transforming growth factor-beta1 sense (n = 8) or antisense (n = 7) complexed with liposomes or cDNA alone (n = 5). In vivo setting: Donors were intravenously injected with liposome:transforming growth factor-beta1 sense cDNA (n = 7). Exposure time was 3 hours. RESULTS: (1) Chloramphenicol acetyltransferase-transfection was superior in the ex vivo group but was not statistically different for longer exposure times. (2) Transforming growth factor-beta1-arterial oxygenation was superior in the ex vivo liposome:sense group. cDNA alone was inefficient. Rejection scores were not statistically different between ex vivo and in vivo liposome:sense groups but were better when the ex vivo liposome:sense group was compared with the cDNA alone or the antisense groups. CONCLUSIONS: (1) With current liposome technology, the ex vivo route is superior to the in vivo approach; (2) cDNA alone does not provide transgene expression at levels to produce a functional effect.
Assuntos
Genes Reporter , Transplante de Pulmão , Transfecção/métodos , Animais , Cloranfenicol O-Acetiltransferase/genética , DNA Complementar , Expressão Gênica , Genes , Lipossomos , Pulmão/enzimologia , Transplante de Pulmão/patologia , Transplante de Pulmão/fisiologia , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Fator de Crescimento Transformador beta/genética , Transgenes , Transplante IsogênicoRESUMO
Administration of methamphetamine (METH) has been shown to increase substance P (SP) concentrations in striatonigral structures. Data described herein demonstrate that SP pathways associated with the mesolimbic systems also are influenced by METH treatment. Specifically, multiple doses of METH significantly decreased SP concentrations in the ventral tegmental area and the medial habenular nucleus; whereas, a single METH injection increased the levels of this neuropeptide in the ventral tegmental area. The possible significance of these observations is discussed.
Assuntos
Sistema Límbico/fisiologia , Metanfetamina/farmacologia , Substância P/fisiologia , Animais , Masculino , Ratos , Ratos Endogâmicos , Substância P/metabolismoRESUMO
Glucuronide conjugates represent one of the major types of naturally occurring phase 2 metabolites of xenobiotics and endobiotics. The process underlying their formation, glucuronidation, is normally considered detoxifying, because glucuronides usually possess less intrinsic biological or chemical activity than their parent aglycones and they are rapid excreted. However, a number of glucuronide conjugates are known that are active and may contribute to pharmacological activities or toxicities associated with their parent compounds. These include two classes of glucuronides with electrophilic chemical reactivity (N-O-glucuronides of hydroxamic acids and acyl glucuronides of carboxylic acids) and several types of glucuronides that impart biological effects through non-covalent interactions (morphine 6-O-glucuronide, retinoid glucuronides, and D-ring glucuronides of estrogens). Glucuronides may thus contribute to clinically significant effects, including environmental arylamine-induced carcinogenesis, drug hypersensitivity and other toxicities associated with carboxylic acid drugs, morphine analgesia, and cholestasis from estrogens. This review summarizes the rat and human UDP-glucuronosyltransferases that may be involved in the formation of bioactive glucuronides, including their substrate- and tissue-specificity and genetic and environmental influences on their activity. This knowledge may be useful for enhancing the therapeutic efficacy and minimizing the risk of adverse effects associated with xenobiotics that undergo bioactivating glucuronidation reactions.
Assuntos
Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Xenobióticos/farmacocinética , Animais , Biotransformação , Estrogênios/metabolismo , Humanos , Isoenzimas/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Three antimycotic N-substituted imidazoles, clotrimazole, tioconazole and miconazole, were able to induce hepatic microsomal cytochrome P-450 and monooxygenase reactions in both male and female hamsters to an extent similar to that seen with phenobarbital treatment. Imidazole treatment did not alter the cytochrome P-450 concentration, and ketoconazole treatment decreased it. Cytosolic sulfo- and glutathione transferases were not significantly altered by any imidazole. Induction of microsomal morphine glucuronosyltransferase activity by each compound generally paralleled the effect on cytochrome P-450 in females but induction was not evident in males. Clotrimazole treatment, in contrast to phenobarbital treatment, also caused a large induction of l-naphthol glucuronosyltransferase in females. The potential for antimycotic imidazoles to alter the hepatotoxicity of compounds will require consideration of the inductive changes in both Phase I and Phase II drug metabolizing enzymes in addition to their known inhibitory effects on Phase I oxidations. The inductive effects differ for each imidazole, and in the hamster model, depend upon the sex of the animal.
Assuntos
Clotrimazol/toxicidade , Imidazóis/toxicidade , Miconazol/toxicidade , Microssomos Hepáticos/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Cricetinae , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Glucuronatos/metabolismo , Cetoconazol/toxicidade , Masculino , Mesocricetus , Microssomos Hepáticos/enzimologia , Tamanho do Órgão/efeitos dos fármacos , Oxirredução , Fenobarbital/toxicidade , Fatores SexuaisAssuntos
Regulação Enzimológica da Expressão Gênica , Variação Genética , Glucuronosiltransferase/genética , Hominidae/genética , Isoenzimas/genética , Sequência de Aminoácidos , Animais , Bilirrubina/metabolismo , Éxons , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Dados de Sequência Molecular , Fenóis/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Treatment of male rats for 3 days with the N-substituted imidazole, clotrimazole, produced up to a 4-fold induction of hepatic microsomal cytochrome P-450. The monooxygenase activities induced varied with the dose administered. At low doses (less than 25 mg/kg), p-nitroanisole demethylase and aniline hydroxylase activities were induced. Only at higher doses were other monooxygenase activities (erythromycin and ethylmorphine demethylases and cytochrome P-450 metabolic-intermediate complex formation from troleandomycin) induced. Microsomal UDP-glucuronosyltransferase activity toward morphine was induced at low doses in a manner similar to that of p-nitroanisole demethylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomes indicated that low doses of clotrimazole caused the intensification of a 48,000 molecular weight protein band, whereas at high doses, there was a marked intensification of an additional 50,500 molecular weight protein, the same molecular weight band as was intensified in phenobarbital- and dexamethasone-induced microsomes. The observations suggest a phenomenon of "dose-differentiated" isozyme induction for cytochrome P-450.
Assuntos
Clotrimazol/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Imidazóis/farmacologia , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/biossíntese , Anilina Hidroxilase/biossíntese , Animais , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Eritromicina/metabolismo , Glucuronosiltransferase/metabolismo , Masculino , Oxirredutases O-Desmetilantes/biossíntese , Ratos , Troleandomicina/metabolismoRESUMO
Three daily administrations of N-substituted imidazole antimycotics, clotrimazole (CloTZ, 75 mg/kg/day), miconazole (MCZ, 150 mg/kg/day), or tioconazole (TCZ, 150 mg/kg/day), but not the 4,5-disubstituted imidazole cimetidine (350 mg/kg/day) or imidazole (200 mg/kg/day for 4 days), induced rat hepatic cytochrome P-450 and other drug-metabolizing enzymes. These findings paralleled in vitro observations where CloTZ, MCZ, and TCZ were several orders of magnitude more potent as inhibitors of p-nitroanisole O-demethylase activity in control male rat liver microsomes than cimetidine or imidazole. Although no marked difference in inhibitory potency was evident among the N-substituted imidazoles, there were qualitative and quantitative differences in the profiles and extents of induction of various cytochrome P-450-dependent monooxygenases and Phase II conjugation enzymes. Cytochrome P-450 was elevated dramatically by CloTZ (3-4 times the control) and to a lesser extent by MCZ and TCZ (congruent to 1.5 times the control). For all agents, there was an increase in metyrapone binding approximately equivalent to the additional (i.e. above control) cytochrome P-450. Despite the large difference in cytochrome P-450 induction by CloTZ, MCZ, and TCZ, these agents elevated p-nitroanisole demethylase and aniline hydroxylase to similar extents (3-5 X and 1-2 X control, respectively). All agents induced erythromycin and ethylmorphine demethylation in proportion to cytochrome P-450. Ethoxyresorufin O-de-ethylation was not substantially affected by any agent. Large differences in the extent and specificity of induction of microsomal glucuronide conjugations were also evident.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Imidazóis/farmacologia , Fígado/enzimologia , Preparações Farmacêuticas/metabolismo , Animais , Cimetidina/farmacologia , Clotrimazol/farmacologia , Sistema Enzimático do Citocromo P-450/análise , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Feminino , Fígado/efeitos dos fármacos , Masculino , Miconazol/farmacologia , Peso Molecular , Oxirredutases O-Desmetilantes/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
We report here that rats possess a hitherto unrecognized xenobiotic-inducible hepatic 7,8-dihydro-7,8-diol-benzo[a]pyrene (BPD) UDP-glucuronosyltransferase (UGT) activity. BPD UGT activity is induced in female F344 rat liver by treatment with the selective Phase 2 conjugation enzyme inducer oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione at 75-450 mg/kg per day for 3 days] and also by a polycyclic aromatic hydrocarbon-type inducer, beta-naphthoflavone (80 mg/kg per day for 3 days). Incubations of oltipraz-treated rat liver microsomes with racemic trans BPD (100 microM) resulted in formation of two fluorescent glucuronides that were resolved by silica thin layer chromatography (Rf 0.5 and 0.6). Incubations with either the (-) or (+) trans BPD isomers resulted in selective formation of the Rf 0.5 [designated -DS, for (-) diol specific] or Rf 0.6 [designated +DS, for (+) diol specific] glucuronide, respectively. The -DS and +DS BPD glucuronides were fluorescent under long wave ultraviolet irradiation, dependent on the presence of UDP-glucuronic acid in the incubation, and were beta-glucuronidase-sensitive. The inducing effect of oltipraz on BPD UGT activity was dose-dependent. The mean BPD UGT activity of the vehicle-treated control group was 0.05 +/- 0.02 nmol/mg per min compared with 0.53 +/- 0.07 nmol/mg per min in the group treated with oltipraz (450 mg/kg per day for 3 days) (P < 0.001). The apparent Km of the induced BPD UGT for BPD was 20 microM, suggesting that the enzyme has the capacity to bind and turnover BPD under physiological conditions. Pretreatment with beta-naphthoflavone, but not phenobarbital, induced BPD UGT activity to approximately the same extent as oltipraz. Neither oltipraz nor beta-naphthoflavone exhibited induction of BPD UGT in livers of homozygous Gunn rats, which lack functional UGT1-encoded isozymes. We conclude that the oltipraz- and polycyclic hydrocarbonresponsive BPD UGT is a member of the UGT1 family. The role of this isoform as a modifier of susceptibility to carcinogenesis elicited by B[a]P remains to be determined.
Assuntos
Benzo(a)pireno/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Anticarcinógenos/farmacologia , Indução Enzimática , Feminino , Glucuronatos/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Pirazinas/farmacologia , Ratos , Ratos Endogâmicos F344 , Tionas , TiofenosRESUMO
UDP-glucuronosyltransferase UGT1A7 catalyzes the glucuronidation of benzo(a)pyrene metabolites and other bulky aromatic compounds. Both UGT1A7 mRNA and an associated enzyme activity (benzo(a)pyrene7, 8-dihydrodioltransferase activity) are markedly increased in livers of rats treated with beta-naphthoflavone or 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz). Nuclear runoff assays show that the effects of both inducers are primarily due to transcriptional activation. A 27-kilobase region that included the UGT1A7/UGT1A6 promoter regions was cloned. Primer extension and RNase protection studies indicated >/=30 transcription start sites in five clusters between bases -85 and -40 respective to the translation start codon. There was no recognizable TATA box, but the promoter region is TA-rich. Sequence analysis revealed potential binding sites for CCAAT enhancer-binding protein, activator protein 1, and hepatic nuclear factors 1, 3, and 4, but no xenobiotic response elements or antioxidant response elements, implicated in the regulation of other genes by beta-naphthoflavone or oltipraz, were found. A UGT1A7 gene reporter plasmid directed strong constitutive expression in transient transfection assays using primary rat hepatocytes. Treatment with 3-methylcholanthrene or oltipraz had no effect compared with similarly treated pGL3-Basic-transfected cells. These results suggest that the regulatory elements controlling xenobiotic inducibility of UGT1A7 transcription are located either 5' or 3' of bases -1600 to +54. One possibility is that the polycyclic aromatic-mediated regulation of UGT1A7 occurs via the xenobiotic response element flanking the UGT1A6 locus 7 kilobase pairs downstream.