RESUMO
TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53(-/-) carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53(-/-) carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial-mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.
Assuntos
Claudinas/metabolismo , Genes p53 , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Prolactina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Various studies in animal tumour models have revealed the potential of fever-range whole body hyperthermia (FR-WBH) to be used in cancer therapy. To determine the safety of FR-WBH treatment in the clinic, patients with advanced solid tumours were heated in the outpatient setting to 39-39.5 degrees C for 3 or 6h, or 39.5-40 degrees C for 6h using the Heckel-HT 2000 apparatus. These WBH treatments were well tolerated, with no significant adverse events related to cardiac, hepatic, renal or pulmonary systems. In the majority of patients, flow cytometric analysis of peripheral blood leukocyte populations indicated that there were transient decreases in the number of circulating T lymphocytes and a concomitant decrease in the number of L-selectin positive lymphocytes in the peripheral blood. These findings closely mimic the affects seen previously in pre-clinical murine studies in which this same fever-like treatment was shown to inhibit tumour growth. These studies have established the safety of this treatment and will allow for future clinical trials where application of FR-WBH treatment can be combined with other anti-cancer therapies, including immunotherapy and chemotherapy.
Assuntos
Hipertermia Induzida/métodos , Neoplasias/terapia , Adulto , Animais , Feminino , Humanos , Hipertermia Induzida/efeitos adversos , Linfopenia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neoplasias Experimentais/terapia , SegurançaRESUMO
By using a batch in vitro anaerobic fecal fermentation model, we have shown that the fecal microflora can rapidly deconjugate rutin, isoquercitrin, and a mixture of quercetin glucuronides. High levels of beta,D-glucosidase, alpha,L-rhamnosidase, and beta,D-glucuronidase were present. Rutin underwent deglycosylation, ring fission, and dehydroxylation. The main metabolite, 3,4-dihydroxyphenylacetic acid, appeared rapidly (2 h) and was dehydroxylated to 3-hydroxyphenylacetic acid within 8 h. The pattern of in vitro fermentation of rutin was not changed by changing the pH (6.0 or 6.9), fermentation scale (10 or 1000 mL), or donors of the inoculum. Hydroxyphenylacetic acids were not methylated by colon flora in vitro. The colonic microflora has enormous potential to transform flavonoids into lower molecular weight phenolics, and these might have protective biological activities in the colon. The site of absorption of flavonoids and the form in which they are absorbed are critical for determining their metabolic pathway and consequent biological activities in vivo.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Fezes/microbiologia , Quercetina/metabolismo , Anaerobiose , Cromatografia Líquida de Alta Pressão , Fermentação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Metilação , Rutina/metabolismoRESUMO
Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.
Assuntos
Flavonoides/metabolismo , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Fígado/enzimologia , Cromatografia Líquida de Alta Pressão , Humanos , Cinética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The nature of quercetin conjugates present in blood after consumption of quercetin glucosides is still unclear. In this study, we analyzed plasma of volunteers that had consumed 325 micromol of either quercetin-3-glucoside or quercetin-4'-glucoside as an oral solution. Quercetin metabolites were extracted with acetonitrile/phosphoric acid and these extracts were analyzed using a high performance liquid chromatography with Coularray detection that distinguishes between the glucuronidated and the glucosylated forms of quercetin. No intact quercetin glucosides and only trace amounts of aglycone were found in human plasma, irrespective of the glucoside ingested. This was confirmed by spiking the plasma with glucoside standards. The major components in plasma had the same retention time as quercetin glucuronide standards. These plasma components disappeared after treatment of the plasma with bovine liver beta-glucuronidase, under reformation of quercetin, and showed the same oxidation pattern as the glucuronides. These results suggest that after consumption of quercetin glucosides, quercetin glucuronides are major metabolites in plasma.
Assuntos
Quercetina/análogos & derivados , Quercetina/administração & dosagem , Acetonitrilas , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Glucosídeos/sangue , Glucosídeos/metabolismo , Glucuronídeos/sangue , Glucuronídeos/metabolismo , Absorção Intestinal , Cinética , Ácidos Fosfóricos , Quercetina/sangueRESUMO
Although alpha-tocopherol (alpha-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, gamma-tocopherol (gamma-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of gamma-TOC and a high alpha-TOC:gamma-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the gamma-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 microM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of gamma-TOC (319.8 +/- 66.2 ng/10(6) cells) into V79 cells compared to alpha-TOC (66.9 +/- 6.4 ng/10(6) cells). Gamma-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, alpha-TOC acetate was partially protective against both genotoxins, whereas alpha-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of gamma-TOC into V79 cells, alpha-TOC but not gamma-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.
Assuntos
Oxidantes/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacos , alfa-Tocoferol/farmacologia , gama-Tocoferol/farmacologia , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cricetinae , Índice MitóticoRESUMO
NADPH-cytochrome P450 oxidoreductase (CYPOR), a flavoprotein localized in the nuclear envelope and endoplasmic reticulum of most cell types, is responsible for transferring electrons from NADPH to the cytochromes P450 as well as heme oxygenase, squalene epoxidase, and cytochrome b(5). CYPOR is encoded by a single gene and, similar to many housekeeping genes, has a TATA-less, GC-rich promoter with multiple Sp1 consensus sites. The current work has delineated the importance of multiple cis-acting elements contained within the proximal promoter for basal expression of the CYPOR gene. Transcription factor binding sites within this region included two upstream Sp1 motifs, a SEC element containing overlapping Sp1/Egr-1/CACCC box motifs, and a novel site designated the OxidoReductase Upstream element (ORU). Mutational modification of the ORU element, leading to a loss of protein binding, resulted in an approximately 90% decrease in transcriptional activity in H4IIE cells. Similarly, inactivation of the Egr-1/CACCC segment of the SEC element dramatically reduced promoter activity to less than 10% of wild-type, while mutagenesis of the contiguous Sp1 site did not affect basal transcription. Although both Sp1 sites contained within the minimal promoter were required for optimal expression in H4IIE cells, loss of these sites was compensated for by those Sp1 motifs located upstream of position 206, suggesting that Sp1 was acting as a position-independent enhancer. Hence, the CYPOR promoter was distinguished from the majority of TATA-less promoters in that Sp1 was not a primary transcriptional regulator and by the fact that the Sp1 binding site closest to the transcription start site was nonfunctional. Furthermore, both the SEC and ORU elements were essential for basal expression; however, the ORU element exhibited cell-specific differences in regulatory activity. Thus, several mechanisms appear to be in place to selectively alter the expression of the CYPOR gene.
Assuntos
Regulação Enzimológica da Expressão Gênica , NADH NADPH Oxirredutases/genética , Animais , Sítios de Ligação , Linhagem Celular , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Mutação , NADPH-Ferri-Hemoproteína Redutase , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.
Assuntos
Proteínas de Ciclo Celular/genética , Nucléolo Celular/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Células COS , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , DNA de Helmintos/genética , Feminino , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase/métodos , RNA de Helmintos/genética , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma mansoni/química , Schistosoma mansoni/genética , Homologia de Sequência de AminoácidosRESUMO
The single cell gel electrophoresis assay (comet assay) is an inexpensive, rapid and highly sensitive method for the determination of DNA damage, crosslinks, and alkaline-labile lesions in individual cells. A limitation of the procedure is that the microelectrophoretic gels must be scored rapidly as the comet configuration deteriorates on storage due to dehydration of the agarose and diffusion of DNA. The objectives of this study were firstly to evaluate drying regimes as rapid and simple methods of preservation of the microgels as close to their original fresh state as possible, and secondly to examine the effects of storage of the slides. Human hepatoma (HepG2) cells challenged for 30 min with hydrogen peroxide (H(2)O(2)) were used in the study. Microgel slides were prepared and evaluated immediately, or after drying with or without a methanol fixation step. Microgels that were dried at a variety of temperatures (22-50 degrees C) and re-hydrated did not differ in the values obtained for H(2)O(2)-induced DNA damage when compared to fresh samples. Samples could also be continually dried and re-hydrated over a period of up to 3 months with no obvious loss of information. In conclusion, drying of microgels represents a simple and inexpensive method of preserving comet assay slides.
Assuntos
Ensaio Cometa/métodos , Preservação de Tecido/métodos , Núcleo Celular/metabolismo , DNA/análise , Dano ao DNA/genética , Eletroforese em Gel de Ágar , Humanos , Peróxido de Hidrogênio , Metanol , Manejo de Espécimes , Temperatura , Células Tumorais CultivadasRESUMO
The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , NADPH-Ferri-Hemoproteína Redutase/genética , Tri-Iodotironina/farmacologia , Animais , Células COS , Masculino , Mutação , Regiões Promotoras Genéticas , Ratos , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacosRESUMO
Multiple cis-acting DNA sequences regulating expression of the rat liver NADPH-cytochrome P-450 oxidoreductase gene have been identified in transient transfection assays using promoter deletion constructs linked to the chloramphenicol acetyl transferase gene. The TATA-less promoter possesses nine GC-boxes which contain the consensus sequence for the transcription factor Sp1. While loss of the seven distal GC-boxes had minimal effect on transcriptional activity, deletion of the next 35 bp, from -206 to -172, resulted in approximately 90% loss of promoter activity. Contained within this region is an Sp1 binding site indicating that either (1) this particular consensus sequence was essential for transcription, (2) the two proximal GC boxes act in concert, or (3) a yet unidentified regulatory element resides within this 35-bp stretch. In addition, transfection experiments demonstrated that two separate distal regions (-622 to -1167 and -1500 to -2300) contain negative regulatory elements which down-regulate gene transcription in a position-independent manner. Mobility-shift analyses and DNase footprinting identified sequences in the proximal region of the promoter that bound proteins present in nuclear extracts. Four protected segments were observed within the first 100 bp upstream of the transcription start site; these include (1) the region encompassing the transcription start site (-7 to +4), (2) the region normally occupied by a TATA-box (-38 to -18), (3) the bases from -78 to -60 which contain the regulatory element CACC, and (4) bases -105 to -92 which include an Sp1 binding site. Hence, regulation of the NADPH-cytochrome P-450 oxidoreductase gene is controlled by both positive and negative regulatory elements, and, of the nine Sp1 consensus sites, the two proximal sites are sufficient to support basal transcription.
Assuntos
NADPH-Ferri-Hemoproteína Redutase/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Sequência Consenso , DNA/genética , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Fígado/enzimologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição Sp1/metabolismo , TATA BoxRESUMO
The untranslated first exon and the 5'-flanking region for the rat liver NADPH-cytochrome P450 oxidoreductase gene has been isolated from a Wistar-Furth genomic library. The remainder of the gene is composed of 15 exons which code for the mature protein and a 3'-nontranslated segment (T. D. Porter et al. Biochemistry, 1990, 29, 9814-9818). The 56-bp first exon resides 30.5 kb upstream from exon two, making the total gene length approximately 50 kb. While the region surrounding the start site (TCAGAGAC) was found to be homologous to a eukaryotic cap signal, the 5' flanking region possesses neither a TATA nor a CCAAT box. Instead it contains five GC-rich hexanucleotide consensus sequences for the transcription factor Sp1. These features clearly distinguish it from genes encoding other members of the mixed-function oxidase system, the cytochromes P450. Primer extension analysis and S1 nuclease mapping identified multiple transcriptional start sites. In many respects, the TATA-less oxidoreductase promoter resembles the promoter regions of dihydrofolate reductase and other housekeeping genes. Northern blot analysis demonstrates that this promoter is modulated by phenobarbital and trans-stilbene oxide, known inducers of oxidoreductase.
Assuntos
Fígado/enzimologia , NADPH-Ferri-Hemoproteína Redutase/genética , Regiões Promotoras Genéticas , Ratos Endogâmicos WF/genética , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Biblioteca Genômica , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Ratos , Mapeamento por Restrição , Transcrição GênicaRESUMO
The effects of 3-methylcholanthrene, butylated hydroxyanisole, and phenobarbital on the expression of glutathione S-transferase (GST, EC 2.5.1.18) were examined in the human parasite Schistosoma mansoni. GST specific activity toward 1-chloro-2,4-dinitrobenzene increased by 170% in parasites recovered from mice injected with 3-methylcholanthrene and 230% in parasites recovered from mice maintained on a diet containing butylated hydroxyanisole. These increases in specific enzyme activity were paralleled by accumulation of mRNA hybridizing to pGT16.4, a cDNA clone that encodes the most abundant GST subunit, SmGST-3. Northern hybridization analysis showed a 5-fold increase in mRNA hybridizing to pGT16.4 72 h after exposure to 3-methylcholanthrene, a 10-fold increase after 12 days exposure to butylated hydroxyanisole, and a 6-fold increase 16 h after treatment with phenobarbital. In contrast, no accumulation of mRNA hybridizing to either of two other cDNA clones that encode the SmGST-4 and SmGST-6 subunits was detected. Hybrid select translation using pGT16.4 combined with reverse-phase high-pressure liquid chromatographic analysis demonstrated that in addition to SmGST-3 mRNA, the clone also hybridized to mRNA species encoding the SmGST-1 subunit, a member of the same isoenzyme family. High-pressure liquid chromatographic analysis of GST affinity purified from butylated hydroxyanisole-exposed parasites revealed a 2.5-fold increase in the concentration of SmGST-1 and SmGST-3 present compared with an equivalent amount of tissue from control organisms. There was no change, however, in the SmGST-1 to SmGST-3 ratio (1:6), indicating that both subunits were induced to the same extent by this agent. The results of these studies suggest that alterations in GST expression may influence the parasite's survival within the host environment.
Assuntos
Hidroxianisol Butilado/farmacologia , Glutationa Transferase/biossíntese , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Schistosoma mansoni/enzimologia , Animais , Indução Enzimática/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Camundongos , RNA Mensageiro/genética , Xenobióticos/farmacologiaRESUMO
A soluble glutathione S-transferase isoenzyme, designated SmGST-4 was purified to apparent homogeneity in a single step from the cytosol of adult Schistosoma mansoni by selective elution of the enzyme from a glutathione-agarose affinity column using glutathione disulfide. SmGST-4, which comprised about 5% of the bound glutathione S-transferase activity, could be distinguished from the previously characterized glutathione S-transferase isoenzyme family (SmGST-1/2/3), by its unique chromatographic behavior, lower subunit M(r) (26,000), differences in substrate specificity and inhibitor sensitivity, and a lack of reactivity with antiserum to SmGST-3. The purified isoenzyme catalyzed the conjugation of several model xenobiotics including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and trans-4-phenyl-3-buten-2-one. Like the SmGST-1/2/3 isoenzyme family, SmGST-4 failed to catalyze the conjugation of a model epoxide substrate, 1,2-epoxy-3-(p-nitrophenoxy)propane. Because glutathione S-transferases from other organisms play a role in protecting cells against the toxic products of lipid peroxidation, SmGST-4 and the members of the SmGST-1/2/3 isoenzyme family were tested for their capacity to reduce cumene hydroperoxide and to catalyze the conjugation of 4-hydroxyalk-2-enals. Although all four isoenzymes catalyzed both reactions, the specific activity of SmGST-1, SmGST-2, and SmGST-3 toward cumene hydroperoxide was at least 10-fold greater than that of SmGST-4. In contrast, the latter more effectively conjugated a homologous series of 4-hydroxyalk-2-enal isomers.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glutationa Transferase/isolamento & purificação , Schistosoma mansoni/enzimologia , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Immunoblotting , Isoenzimas/antagonistas & inibidores , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Especificidade por SubstratoRESUMO
Dialyzed cytosol of adult Schistosoma mansoni worm pairs catalyzed the glutathione-dependent O-demethylation of dichlorvos (2,2-dichlorovinyl dimethylphosphate), the active form of the antischistosomal drug metrifonate, to form a thioether conjugate, S-methylglutathione, and desmethyl dichlorvos. The reaction rate was dependent on both time and protein concentration, and no product was formed when either dichlorvos or glutathione was omitted from the reaction mixture. Female worm cytosols were about 2.5-fold more active per milligram of protein that those of males. Partial purification of glutathione S-transferases from male worms by affinity chromatography on glutathione-agarose showed that the reaction could be catalyzed by a preparation containing the three major isoenzymes, but that the unbound fraction, which contains at least one additional form of the enzyme that is particularly active with epoxide substrates, was 16-fold more active toward dichlorvos than the bound fraction. S-Methylglutathione also was formed by S. mansoni worm pairs incubated in the presence but not in the absence of dichlorvos. Because GSH S-transferase-catalyzed metabolism of dichlorvos results in the formation of desmethyldichlorvos, which unlike the parent compound is not an effective acetylcholinesterase inhibitor, the reaction represents a pathway of detoxication in schistosomes. It is the first example of a clinically used schistosomicide shown to be detoxicated by a conjugation pathway. These results raise the possibility that dichlorvos detoxication by S. mansoni may help explain why this species is normally refractory to metrifonate.
Assuntos
Diclorvós/metabolismo , Glutationa Transferase/metabolismo , Schistosoma mansoni/enzimologia , Animais , Citosol/enzimologia , Dinitroclorobenzeno/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Glutationa/análogos & derivados , Glutationa/metabolismo , Inativação Metabólica , Cinética , Masculino , Metilação , Nitrobenzenos/metabolismoRESUMO
Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glutationa Transferase/metabolismo , Glutationa/metabolismo , 2,4-Dinitrofenol , Animais , Sequência de Carboidratos , Dinitrofenóis , Glutationa/análogos & derivados , Fígado/enzimologia , Dados de Sequência Molecular , Ratos , Especificidade por SubstratoRESUMO
Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.
Assuntos
Glutationa Transferase/análise , Isoenzimas/análise , Schistosoma mansoni/enzimologia , Animais , Ceco/enzimologia , Citoplasma/enzimologia , Feminino , Imunofluorescência , Immunoblotting , Masculino , Músculos/enzimologia , Schistosoma mansoni/citologiaRESUMO
The cytosolic fraction of adult Schistosoma mansoni contains glutathione S-transferase (EC 2.5.1.18) activity, determined with the prototype substrate 1-chloro-2,4-dinitrobenzene, that is 5- to 50-fold greater than that found in other metazoan parasites. A survey of several model substrates revealed that enzymes in male and female schistosomes have distinct but overlapping substrate specificities. Four forms of glutathione S-transferase were detected, three of which, SmGST-1, SmGST-2, and SmGST-3, were purified to apparent homogeneity by glutathione affinity chromatography and HPLC chromatofocusing. The purified enzymes displayed very similar catalytic and physicochemical properties. They could be distinguished by differences in activity with ethacrynic acid and trans-4-phenyl-3-buten-2-one, but not with aryl halide substrates. The isoelectric points of SmGST-1, SmGST-2, and SmGST-3 were estimated to be 7.2, 7.1, 6.9, respectively. A polyclonal antiserum to SmGST-3 cross-reacted with the other two forms, but not with other soluble schistosome proteins. Each of the purified enzymes displayed an apparent subunit molecular weight of 28,500 by polyacrylamide gel electrophoresis under denaturing conditions. Gel filtration chromatography yielded a molecular weight of 30,800 for the catalytically active form of the enzyme. Unlike all known glutathione S-transferases, the three enzyme forms purified from S. mansoni appear to be catalytically active monomeric proteins.