RESUMEN
Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.
Asunto(s)
Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/genética , Glioma/complicaciones , Glioma/genética , Isocitrato Deshidrogenasa/genética , Mutación/genética , Trombosis/etiología , Adulto , Anciano , Anciano de 80 o más Años , Oxidorreductasas de Alcohol/farmacología , Animales , Antineoplásicos/uso terapéutico , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Estudios de Cohortes , Femenino , Glioma/tratamiento farmacológico , Humanos , Masculino , Ratones , Persona de Mediana Edad , Trombina/metabolismo , Trombina/farmacología , Tromboplastina/metabolismo , Trombosis/tratamiento farmacológico , Trombosis/patologíaRESUMEN
OBJECTIVES: The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. METHODS: High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. KEY FINDINGS: Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. CONCLUSIONS: In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11ß-HSD1 in the male rats and humans.
Asunto(s)
Hígado/metabolismo , Metirapona/farmacocinética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/farmacología , Administración Oral , Oxidorreductasas de Alcohol/farmacología , Animales , Cromatografía Líquida de Alta Presión , Citosol/metabolismo , Humanos , Masculino , Metirapona/análogos & derivados , Metirapona/sangre , Metirapona/metabolismo , Microsomas Hepáticos/metabolismo , Ratas WistarRESUMEN
Human carbonyl reductase 1 (CBR1) plays a crucial role in cell survival and protects against oxidative stress response. However, its anti-inflammatory effects are not yet clearly understood. In this study, we examined whether CBR1 protects against inflammatory responses in macrophages and mice using a Tat-CBR1 protein which is able to penetrate into cells. The results revealed that purified Tat-CBR1 protein efficiently transduced into Raw 264.7 cells and inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2), nitric oxide (NO) and prostaglandin E2 (PGE2) expression levels. In addition, Tat-CBR1 protein leads to decreased pro-inflammatory cytokine expression through suppression of nuclear transcription factor-kappaB (NF-κB) and mitogen activated protein kinase (MAPK) activation. Furthermore, Tat-CBR1 protein inhibited inflammatory responses in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation when applied topically. These findings indicate that Tat-CBR1 protein has anti-inflammatory properties in vitro and in vivo through inhibition of NF-κB and MAPK activation, suggesting that Tat-CBR1 protein may have potential as a therapeutic agent against inflammatory diseases.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Antiinflamatorios/farmacología , Edema/tratamiento farmacológico , Productos del Gen tat/farmacología , Macrófagos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Animales , Oído Externo/patología , Edema/inducido químicamente , Edema/patología , Activación Enzimática/efectos de los fármacos , Lipopolisacáridos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Fracciones Subcelulares/efectos de los fármacos , Acetato de TetradecanoilforbolRESUMEN
Imbibitional chilling injury during germination causes agricultural losses, but this can be overcome by osmopriming. It remains unknown how membranes reorganize during germination. Herein, we comparatively profiled changes of membrane lipids during imbibition under normal and chilling temperatures in chilling-tolerant and -sensitive soybean seeds. We found three patterns of dynamic lipid remodelling during the three phases of germination. Pattern 1 involved a gradual increase in plastidic lipids during phases I and II, with an abrupt increase during phase III. This abrupt increase was associated with initiation of photosynthesis. Pattern 3 involved phosphatidic acid (PA) first decreasing, then increasing, and finally decreasing to a low level. Patterns 1 and 3 were interrupted in chilling-sensitive seeds under low temperature, which lead a block in plastid biogenesis and accumulation of harmful PA, respectively. However, they were rescued and returned to their status under normal temperature after polyethylene glycol osmopriming. We specifically inhibited phospholipase D (PLD)-mediated PA formation in chilling-sensitive seeds of soybean, cucumber, and pea, and found their germination under low temperature was significantly improved. These results indicate that membranes undergo specific and functional reorganization of lipid composition during germination and demonstrate that PLD-mediated PA causes imibibitional chilling injury.
Asunto(s)
Cucumis sativus/fisiología , Glycine max/fisiología , Lípidos de la Membrana/metabolismo , Pisum sativum/fisiología , Plastidios/metabolismo , Semillas/fisiología , Oxidorreductasas de Alcohol/farmacología , Membrana Celular/metabolismo , Clorofila/metabolismo , Frío , Cucumis sativus/efectos de los fármacos , Etanolaminas/metabolismo , Germinación , Pisum sativum/efectos de los fármacos , Semillas/efectos de los fármacos , Glycine max/efectos de los fármacos , Agua/metabolismoRESUMEN
Recent research performed in our laboratory (using a butyrylcholinesterase+choline oxidase enzyme electrode) suggested the validity of the biosensor approach using enzyme inhibition OPEEs (i.e. enzyme electrodes working in organic phase) in the case of organophosphorus and carbamate pesticides, which are poorly soluble in aqueous solutions. Since these pesticides are generally much more soluble in chloroform than in water, the present research aimed at analysing this class of pesticides using a tyrosinase inhibition OPEE operating in water-saturated chloroform medium. The tyrosinase biosensor was assembled using an oxygen amperometric transducer coupled to the tyrosinase enzyme, immobilized in kappa-carrageenan gel. Lastly a detailed comparison between the inhibition monoenzymatic tyrosinase and inhibition bienzymatic (butyrylcholinesterase+choline oxidase) OPEEs was performed and discussed in this work.
Asunto(s)
Técnicas Biosensibles , Carbamatos/farmacología , Técnicas de Química Analítica/métodos , Monofenol Monooxigenasa/química , Compuestos Organofosforados/farmacología , Plaguicidas/farmacología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/farmacología , Butirilcolinesterasa/química , Butirilcolinesterasa/farmacología , Calibración , Carragenina/química , Electroquímica/métodos , Enzimas Inmovilizadas/química , Geles , Modelos Químicos , Monofenol Monooxigenasa/antagonistas & inhibidores , Plaguicidas/químicaAsunto(s)
Aldehído Reductasa/biosíntesis , Aldehído Reductasa/sangre , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , Perfilación de la Expresión Génica , Neoplasias Pulmonares/patología , Fumar/efectos adversos , Oxidorreductasas de Alcohol/farmacología , Aldehído Reductasa/farmacología , Aldo-Ceto Reductasas , Humanos , Pronóstico , Transducción de Señal , Tretinoina/farmacología , Regulación hacia ArribaRESUMEN
A method for the quantitation of dityrosine in wheat flour and dough by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) using an isotope dilution assay with the internal standard 3,3'-(13)C(2)-dityrosine in the single-reaction monitoring mode was developed. The method consisted of the release of protein-bound dityrosine by hydrolysis in 4 mol/L hydrochloric acid/8.9 mol/L propionic acid for 24 h at 110 degrees C after addition of the internal standard, cleanup by C(18) solid-phase extraction, and HPLC-MS/MS. The limit of detection of dityrosine was 80 ng/g of sample (0.22 nmol/g), and the limit of quantitation was 270 ng/g of sample (0.75 nmol/g). The method was sensitive enough to analyze wheat flour and dough and to study the effect of flour improvers on the dityrosine content. Furthermore, the effect of the mixing time was studied. The dityrosine concentration in the flour was 0.66 nmol/g. After we mixed a dough to peak consistency, the dityrosine concentration doubled and remained constant on further mixing. Overdoses of hydrogen peroxide and hexose oxidase (HOX, E.C. 1.1.3.5) resulted in a strongly increased dityrosine content, whereas no increase of the dityrosine concentration was found after the addition of ascorbic acid and potassium bromate. Calculation of the percentage of dimeric tyrosine showed that less than 0.1% of the tyrosine residues of wheat protein were cross-linked. Therefore, dityrosine residues seem to play only a very minor role in the structure of wheat gluten.
Asunto(s)
Pan/análisis , Cromatografía Líquida de Alta Presión/métodos , Harina/análisis , Espectrometría de Masas/métodos , Triticum/química , Tirosina/análogos & derivados , Tirosina/análisis , Oxidorreductasas de Alcohol/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Bromatos/farmacología , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacologíaRESUMEN
d-2-hydroxyglutaric aciduria is a neurometabolic disorder with both a mild and a severe phenotype and with unknown etiology. Recently, a novel enzyme, d-2-hydroxyglutarate dehydrogenase, which converts d-2-hydroxyglutarate into 2-ketoglutarate, and its gene were identified. In the genes of two unrelated patients affected with d-2-hydroxyglutaric aciduria, we identified disease-causing mutations. One patient was homozygous for a missense mutation (c.1331T-->C; p.Val444Ala). The other patient was compound heterozygous for a missense mutation (c.440T-->G; p.Ile147Ser) and a splice-site mutation (IVS1-23A-->G) that resulted in a null allele. Overexpression studies in HEK-293 cells of proteins containing the missense mutations showed a marked reduction of d-2-hydroxyglutarate dehydrogenase activity, proving that mutations in the d-2-hydroxyglutarate dehydrogenase gene cause d-2-hydroxyglutaric aciduria.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/farmacología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Glutaratos/orina , Preescolar , Femenino , Perfilación de la Expresión Génica , Glutaratos/metabolismo , Humanos , Masculino , Mutación Missense , FenotipoRESUMEN
This study was designed to elucidate strain- and sex-related differences of carbonyl reductase activity in rat kidney by using the oral antidiabetic drug acetohexamide as substrate. The frequency distribution of carbonyl reductase activities in kidney microsomes of male Fischer 344 (Fischer), Sprague-Dawley, Wistar and Wistar-Imamichi (Wistar-IM) rats exhibited a marked strain-related difference. Furthermore, the enzyme activities in kidney microsomes of Fischer, Sprague-Dawley and Wistar rats were male-specific, resulting insignificant sex-related differences in these strains. There was no sex-related difference of carbonyl reductase activity in kidney microsomes of the Wistar-IM strain, which lacked its activity in both sexes. On the other hand, although carbonyl reductase activities were fully detectable in kidney cytosols from all the strains of male and female rats, no strain- or sex-related difference was observed among the cytosolic enzyme activities. These results provide new information for understanding the influence of internal factors on the renal metabolism of ketone-containing xenobiotics.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Acetohexamida/farmacología , Oxidorreductasas de Alcohol/análisis , Animales , Citosol/enzimología , Femenino , Hipoglucemiantes , Masculino , Microsomas/enzimología , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Ratas Wistar , Reproducibilidad de los Resultados , Factores Sexuales , Xenobióticos/metabolismoRESUMEN
Fewer than 20% of habitual smokers develop lung cancer, which suggests that genetic, environmental and nutritional factors contribute to the risk for developing this disease. Recently, five enzymes were shown to initiate the detoxification of nicotine-derived nitrosamine ketone (NNK), the most potent carcinogen present in tobacco. Importantly, four of these enzymes are potently inhibited by glycyrrhetinic acid, the main constituent of licorice. These observations might open novel and hitherto unexplored avenues for the risk assessment and prevention of tobacco-associated lung cancer.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Carcinógenos , Inactivación Metabólica , Neoplasias Pulmonares/inducido químicamente , Nitrosaminas/antagonistas & inhibidores , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antiinflamatorios/farmacología , Ácido Glicirretínico/farmacología , Humanos , Neoplasias Pulmonares/prevención & control , Nitrosaminas/metabolismo , Nitrosaminas/toxicidadRESUMEN
The human aldo-keto reductase 1C (AKR1C) isozymes are implicated in the pre-receptor regulation of steroid receptors, nuclear orphan receptors and membrane-bound ligand-gated ion channels. Human AKR members that may regulate the local concentration of steroid hormones include: AKR1C1, AKR1C2, AKR1C3, AKR1C4 and AKR1D1. Since, these enzymes are pluripotent, the physiological role for the human AKR1C isozymes is determined by their tissue-specific expression patterns and their substrate availability in target tissues. AKRs work in concert with short-chain dehydrogenases/reductases as switches to control ligand access to nuclear receptors. Consequently, they are potential targets in treating prostate cancer, breast cancer, endometriosis and endometrial cancer.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Hormonas Esteroides Gonadales/farmacología , Oxidorreductasas de Alcohol/efectos de los fármacos , Oxidorreductasas de Alcohol/farmacología , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Hormonas Esteroides Gonadales/fisiología , Humanos , Isoenzimas/química , Isoenzimas/farmacología , Isoenzimas/fisiologíaRESUMEN
UNLABELLED: The effectiveness of (11)C-choline PET in detecting various cancers, including prostate cancer, is well established. This study was aimed at developing an (18)F-substituted choline analog, (18)F-fluoroethylcholine (FECh), as a tracer of cancer detection. METHODS: No-carrier-added (18)F-FECh was synthesized by 2-step reactions: First, tetrabutylammonium (TBA) (18)F-fluoride was reacted with 1,2-bis(tosyloxy)ethane to yield 2-(18)F-fluoroethyl tosylate; and second, 2-(18)F-fluoroethyl tosylate was reacted with N,N-dimethylethanolamine to yield (18)F-FECh, which was then purified by chromatography. An automated apparatus was constructed for preparation of the (18)F-FECh injection solution. In vitro experiments were performed to examine the uptake of (18)F-FECh in Ehrlich ascites tumor cells, and the metabolites were analyzed by solvent extraction followed by various kinds of chromatography. Clinical studies of (18)F-FECh PET were performed on patients with untreated primary prostate cancer as follows: A dynamic (18)F-FECh PET study was performed on 1 patient and static PET studies were performed on 16 patients, and the data were compared with those of (11)C-choline PET on the same patients. RESULTS: (18)F-FECh was prepared in high yield and purity. The performance of the automated apparatus was excellent. The in vitro experiment revealed that (18)F-FECh was incorporated into tumor cells by active transport, then phosphorylated (yielding phosphoryl-(18)F-FECh) in the cells, and finally integrated into phospholipids. The clinical PET studies showed marked uptake of (18)F-FECh in prostate cancer. A dynamic PET study on 1 patient revealed that the blood level of (18)F-FECh decreased rapidly (in 1 min), the prostate cancer level became almost maximal in a short period (1.5 min) and it remained constant for a long time (60 min), and the urinary radioactivity became prominent after a short time lag (5 min). Static PET studies conducted under bladder irrigation showed no difference between (18)F-FECh uptake and (11)C-choline uptake in prostate cancer. However, (18)F-FECh gave a slightly higher spatial resolution of the image, which was attributed to the shorter positron range of (18)F. CONCLUSION: The synthesis of (18)F-FECh was easy and reliable. (18)F-FECh PET was very effective in detecting prostate cancer in patients. The chemical trap, consisting of active transport of (18)F-FECh and formation of phosphoryl-(18)F-FECh, seemed to be involved in the uptake mechanism of (18)F-FECh in tumors.
Asunto(s)
Colina/síntesis química , Radioisótopos de Flúor , Neoplasias de la Próstata/diagnóstico por imagen , Radiofármacos/síntesis química , Tomografía Computarizada de Emisión , Adenosina Trifosfato/farmacología , Oxidorreductasas de Alcohol/farmacología , Animales , Radioisótopos de Carbono , Carcinoma de Ehrlich/diagnóstico por imagen , Carcinoma de Ehrlich/metabolismo , Colina/análogos & derivados , Colina/química , Colina Quinasa/farmacología , Cromatografía Líquida de Alta Presión , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Trasplante de Neoplasias , Fosforilación , Radiofármacos/químicaRESUMEN
The 11-cis retinol dehydrogenase (11-cis-RoDH) enzyme catalyzes the oxidation of cis-retinols to their respective retinals, a rate limiting step in the formation of retinoic acids. Earlier, we have shown that the enzyme also exhibits an oxidative 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity that can convert 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) into dihydrotestosterone (DHT), the most potent natural androgen. 11-cis-RoDH could thus control the formation of two active hormones, namely 9-cis retinoic acid and DHT. Therefore, depending upon the substrate availability in the various tissues, this enzyme could provide different metabolites for specific cell functions. To further investigate the role of 11-cis-RoDH in the formation of DHT from 3alpha-diol, we stably expressed the enzyme in the human embryonic kidney cell line 293 (HEK-293). The transformation of 3alpha-diol by these cells was evaluated by assays using both microsomal fractions and intact cultured cells stably expressing 11-cis-RoDH. The results show that in the intact cells 11-cis-RoDH only catalyzes the oxidation of 3alpha-diol into DHT whereas the microsomal fraction catalyzes both the oxidation and the reduction reactions depending upon whether NAD(+) or NADH is added. Furthermore, we examined the ability of 11-cis-RoDH, through the production from 3alpha-diol of the active androgen DHT, to activate the androgen-responsive promoter of the prostate-specific antigen (PSA) gene. The co-transfection of the pCMV expression vector containing 11-cis-RoDH (pCMV-11-cisRoDH), a luciferase reporter gene driven by a PSA promoter (pCMV-PSA-Luc) and an androgen receptor (pCMV-hAR) showed that, in the presence of 3alpha-diol, the expression of the PSA promoter is increased by five to six-fold. Moreover, this stimulatory effect is inhibited by hydroxyflutamide, a well-known antiandrogen. These results suggest that 11-cis-RoDH could be involved in a non-classical pathway of androgen formation and might play a role in the modulation of the androgenic response in some peripheral tissues.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Andrógenos/fisiología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Línea Celular , Clonación Molecular , Genes Reporteros , Humanos , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Learning and maintenance of memory in mice intraperitoneally (IP) injected with choline oxidase (ChO, 6 units/g), a hydrolytic enzyme for choline (Ch), were assessed by means of a step-through passive-avoidance task. The ChO treatment induced a hydrolysis of free Ch in plasma, which in turn, induced a decrease in cerebral acetylcholine (ACh) release. In the learning test, the ChO-treated mice showed significant inhibition to learn the avoidance from electric shock. In the retention test, the impairment of the memory once established was not produced by posttreated ChO. We concluded that the decreased cerebral cholinergic neurotransmission induced by ChO retarded acquisition of passive-avoidance learning more readily than the maintenance of memory.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Animales , Reacción de Prevención/efectos de los fármacos , Colina/sangre , Masculino , Ratones , Tiempo de Reacción/efectos de los fármacosRESUMEN
An enzyme catalyzing the reduction of metyrapone, a diagnostic drug with a ketone group, was partially purified from liver microsomes of male rats. The partially purified metyrapone reductase had no ability to reduce acetohexamide, an oral antidiabetic drug with a ketone group, even though both metyrapone and acetohexamide are reduced in liver microsomes of male rats. These results clearly indicate that the reduction of these two drugs can be catalyzed by different enzymes. The partially purified metyrapone reductase was found to reduce aldehydes, ketones and menadione. The substrate specificities were in fair agreement with those of carbonyl reductase. However, the partially purified enzyme was strongly inhibited by inhibitors of aldehyde reductase, such as barbital, phenobarbital and sodium valproate.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Microsomas Hepáticos/enzimología , Acetohexamida/metabolismo , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/aislamiento & purificación , Oxidorreductasas de Alcohol/metabolismo , Animales , Femenino , Masculino , Metirapona/metabolismo , Ratas , Ratas Wistar , Especificidad por SustratoRESUMEN
Choline (Ch)-free plasma rats were induced successfully by intravenous (IV) injection of choline oxidase (ChO) (16). However, brain acetylcholine (ACh) levels were not affected by ChO treatment, maintaining the same levels as those in controls, although brain Ch levels were significantly decreased. To clarify the reasons for this, in vivo microdialysis was carried out in the treated rats' striata. The ChO treatment induced not only a 70% decrease of extracellular Ch levels but also a 40% decrease of extracellular ACh levels, reflecting the amount of ACh released from cholinergic terminals. In addition, plasma-bound Ch levels and choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities in the brain were examined in the rats receiving ChO treatment. No significant differences from controls were observed in these levels. The results suggest that: approximately 70% of striatal extracellular Ch is physiologically supplied from circulating plasma-free Ch; the inhibition of ACh release is related to the maintenance of tissue (intraneuronal) ACh levels under the condition of halting of the supply of free Ch from blood to the brain; if there is a compensative supply of free Ch from de novo synthesis, autocannibalism, or plasma-bound Ch, this may be supplied within neuronal cells, because the level of extracellular-free Ch maintained its depressed level even 11-14 h after ChO treatment.
Asunto(s)
Acetilcolina/metabolismo , Colina/sangre , Colina/metabolismo , Espacio Extracelular/metabolismo , Neostriado/metabolismo , Acetilcolinesterasa/metabolismo , Oxidorreductasas de Alcohol/administración & dosificación , Oxidorreductasas de Alcohol/farmacología , Animales , Colina O-Acetiltransferasa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Inyecciones Intravenosas , Masculino , Microdiálisis , Neostriado/citología , Neostriado/enzimología , Ratas , Ratas WistarRESUMEN
Dihydrodiol dehydrogenase(s) (DD) have been implicated in the detoxication of proximate (trans-dihydrodiol) and ultimate carcinogenic (anti-diol-epoxide) metabolites of polycyclic aromatic hydrocarbons (PAHs). These activities are catalyzed by soluble hydroxysteroid dehydrogenases and/or by aldehyde reductases. Molecular cloning indicates tha these enzymes have a high degree of sequence identity with members of the aldo-keto reductase super family. Substrate specificity studies indicate that non-K-region trans-dihydrodiols are the preferred substrates and that anti-dio-epoxides are not oxidized by the enzyme. The products of the DD reaction are transient catechols which auto-oxidize to PAH-o-quinones. As a consequence of this auto-oxidation superoxide anion, hydrogen peroxide and semiquinone radicals are generated. Studies on the biotransformation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene indicate that in subcellular fractions from uninduced rat liver, DD plays a significant role in the metabolism of this proximate carcinogen. Thus, the formation of benzo[a]pyrene-7,8-dione is only superseded by the formation of tetraols which are derived from the anti-diol epoxide of benzo[a]pyrene [anti-BPDE;(+/-)-anti-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene]. PAH-o-quinones produced by DD can inactivate the enzyme. These PAH-o-quinones also vary in their reactivity towards cellular nucleophiles, their cytotoxicity and their genotoxicity. Non-bay region and methylated bay-region PAH-o-quinones generated by DD are the most reactive Michael acceptors, and are also the most cytotoxic in hepatoma cells. Cytotoxicity results from the 1e- redox-cycling of the PAH-o-quinone, concomittant production of superoxide anion and a subsequent alteration in redoxstate. PAH-o-quinones are also genotoxic thus [3H]-benzo[a]pyrene-7,8-dione readily forms deoxyguanosine-adducts with native calf-thymus DNA, i.e., to the same extent as anti-BPDE. The cytotoxic and genotoxic properties of PAH-o-quinones suggest that DD may initiate a hitherto unrecognized pathway of PAH activation.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas , Compuestos Policíclicos/metabolismo , Oxidorreductasas de Alcohol/farmacología , Secuencia de Aminoácidos , Animales , Carcinógenos , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Compuestos Policíclicos/farmacologíaRESUMEN
Choline-free plasma (CFP) was induced in rats by intravenous (IV) injection of 56.0 x 10(2) units kg-1 of choline oxidase (ChO) which completely metabolized the free Ch circulating in the plasma for at least 15.0 h and caused subsequent significant decrease in the concentration of free Ch in the three brain regions examined, the striatum, hippocampus, and cortex. However, the treatment did not affect concentrations of acetylcholine (ACh) in these regions. By contrast, intraperitoneal (IP) injection of 1.0 mmol kg-1 Ch chloride resulted in a maximum concentration of free Ch in plasma in 5 min, after which tissue Ch in all regions examined increased (p < 0.001). Concomitant increases were observed in cortical and hippocampal ACh (p < 0.05) 20 min after the injection. It is thus suggested that the brain may possess compensative mechanisms to prevent the supply of free Ch from circulating to the brain during synthesis of ACh in the brain. It is also suggested that the CFP rat would be a useful and readily available animal model for future study.
Asunto(s)
Acetilcolina/metabolismo , Oxidorreductasas de Alcohol/farmacología , Encéfalo/metabolismo , Colina/metabolismo , Oxidorreductasas de Alcohol/administración & dosificación , Oxidorreductasas de Alcohol/metabolismo , Animales , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/metabolismo , Colina/sangre , Cuerpo Estriado/irrigación sanguínea , Cuerpo Estriado/metabolismo , Hipocampo/irrigación sanguínea , Hipocampo/metabolismo , Inyecciones Intravenosas , Cinética , Masculino , Especificidad de Órganos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The present studies were undertaken to investigate the enzymology of a fatal toxic syndrome that resulted from the absorption and subsequent oxidation of polyethylene glycol (PEG). The presence of organic acids of PEG in the blood of poisoned patients and in an animal model suggested that the metabolism of PEG involved sequential oxidations by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. A key question concerned the ability of ADH to initiate this pathway for oxidation of PEG. In the present studies the oxidation of PEG homologues by ADH was characterized. The polymer homologues of ethylene glycol from n = 1 to n = 8 were used as substrates. ADH catalyzed the oxidation of each of these PEGs. The oxidation of PEG was inhibited by the ADH inhibitor 4-methylpyrazole. With the exception of diethylene glycol, the Km decreased as the homologue number increased, and the Vmax decreased progressively through the series. The concentrations of PEG in the blood of poisoned humans and animals were 0.06 to 0.8 Km of ADH for all the PEG homologues above the triethylene glycol. These investigations establish ADH as a candidate enzyme for mammalian metabolism of PEG and thus suggest that specific inhibitors of ADH may prove to be useful as tools to treat PEG poisoning.
Asunto(s)
Oxidorreductasas de Alcohol/farmacología , Polietilenglicoles/metabolismo , Animales , Humanos , Cinética , Oxidación-ReducciónRESUMEN
We have developed a simple, specific, and sensitive method for plasma choline measurement based on the HPLC procedure of Potter et al. (J Neurochem 1983; 41: 188) for the measurement of acetylcholine and choline in neuronal tissue. The effluent from a reverse-phase column is mixed with choline oxidase in a post-column reaction coil to produce hydrogen peroxide which is monitored electrochemically. Plasma samples are prepared by deproteinization with perchloric acid. Choline is recovered quantitatively from the plasma, but an internal standard (homocholine) is added to compensate for any variation in electrode response. Choline can be measured in plasma samples containing less than 1 mumole per litre of plasma; the method response is linear in the 1-20 mumol/L range. Catecholamines and ascorbic acid do not interfere. The chromatography, enzymatic reactions, and electrochemistry all contribute to the specificity of the method.