RESUMEN
Bovine serum albumin (BSA) is highly water soluble and binds drugs or inorganic substances noncovalently for their effective delivery to various affected areas of the body. Due to the well-defined structure of the protein, containing charged amino acids, albumin nanoparticles (NPs) may allow electrostatic adsorption of negatively or positively charged molecules, such that substantial amounts of drug can be incorporated within the particle, due to different albumin-binding sites. During the synthesis procedure, pH changes significantly. This variation modifies the net charge on the surface of the protein, varying the size and behavior of NPs as the drug delivery system. In this study, the synthesis of BSA NPs, by a desolvation process, was studied with salicylic acid (SA) as the active agent. SA and salicylates are components of various plants and have been used for medication with anti-inflammatory, antibacterial, and antifungal properties. However, when administered orally to adults (usual dose provided by the manufacturer), there is 50% decomposition of salicylates. Thus, there has been a search for some time to develop new systems to improve the bioavailability of SA and salicylates in the human body. Taking this into account, during synthesis, the pH was varied (5.4, 7.4, and 9) to evaluate its influence on the size and release of SA of the formed NPs. The samples were analyzed using field-emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared, zeta potential, and dynamic light scattering. Through fluorescence, it was possible to analyze the release of SA in vitro in phosphate-buffered saline solution. The results of chemical morphology characterization and in vitro release studies indicated the potential use of these NPs as drug carriers in biological systems requiring a fast release of SA.
RESUMEN
Hypobromous acid (HOBr) is a powerful oxidant produced by stimulated neutrophils and eosinophils. Taurine, a non-protein amino acid present in high amounts in the leukocytes, reacts instantaneously with HOBr leading to their haloamine derivative taurine dibromamine (Tau-NBr2). Lysozyme is a bactericidal enzyme also present in leukocytes and in secretory fluids. The inhibition of lysozyme is a pathway for bacterial proliferation in inflammatory sites. Here, we investigated the inhibition of the enzymatic activity of lysozyme when it was submitted to oxidation by Tau-NBr2. We found that the oxidation of lysozyme by Tau-NBr2 decreased its enzymatic activity in 80%, which was significant higher compared to the effect of its precursor HOBr (30%). The study and comparison of Tau-NBr2 and HOBr regarding the alterations provoked in the intrinsic fluorescence, synchronous fluorescence, resonance light scattering and near and far-UV circular dichroism spectra of lysozyme and oxidized lysozyme revealed that tryptophan residues in the active site of the protein were the main target for Tau-NBr2 and could explain its efficacy as inhibitor of lysozyme enzymatic activity. This property of Tau-NBr2 may have pathological significance, since it can be easily produced in the inflammatory sites.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Embrión de Pollo/enzimología , Muramidasa/química , Taurina/farmacología , Animales , Bromatos/química , Dicroismo Circular , Huevos , Eosinófilos/química , Eosinófilos/metabolismo , Micrococcus/efectos de los fármacos , Muramidasa/antagonistas & inhibidores , Neutrófilos/química , Neutrófilos/metabolismo , Oxidación-Reducción , Taurina/análogos & derivados , Taurina/química , Triptófano/químicaRESUMEN
The excessive activation of neutrophils generates reactive oxygen species (ROS) and the secretion of primary granular enzymes, such as myeloperoxidase (MPO), which is implicated in numerous inflammatory diseases. The aim of this study was to evaluate chalcones as inhibitors of the chlorinating activity of MPO using in vitro and ex vivo assays. In addition to cytotoxic properties, the inhibition of respiratory burst, the scavenger capacity, and the oxidation potential were measured. 4'-Aminochalcone (1), 4'-amino-4- fluorochalcone (2), and 4'-amino-4-methylchalcone (3) exhibited potent inhibition of the chlorinating activity of MPO, as evaluated in a neutrophil system and a free cell system, to the following degree: (1) IC50 = 0.265 � 0.036 µmol L-1; (2) IC50 = 0.250 � 0.081 µmol L-1; and (3) IC50 = 0.250 � 0.012 µmol L-1. These values were similar to those for 5-fluorotryptamine (IC50 = 0.192 � 0.012 µmol L-1), a compound considered to be a potent MPO inhibitor. These aminochalcones were not toxic to neutrophils at concentrations below 100 µmol L- 1, as determined by the trypan blue exclusion assay. Compounds 1-3 presented a high oxidation potential (Epa1 â 0.80 V), low scavenger capacity against DPPH⢠and HOCl, and low inhibition of respiratory burst. These data indicated that aminochalcones are potent inhibitors of MPO chlorinating activity, a new property for chalcone derivatives, given that they are neither antioxidant agents nor inhibitors of respiratory burst. In conclusion, the selected aminochalcones have potential as pharmacological agents for inflammatory diseases.
Asunto(s)
Chalconas/química , Inhibidores Enzimáticos/química , Peroxidasa/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Chalconas/síntesis química , Chalconas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Halogenación , Humanos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oxidación-Reducción , Peroxidasa/metabolismo , Unión Proteica , Superóxidos/metabolismoRESUMEN
This study presents the increased efficiency of NADPH oxidase inhibition produced by esterification of protocatechuic acid (P0). Alkyl esters bearing chain lengths of 4 (P4), 7 (P7) and 10 (P10) carbons were synthesized and their oxidation potential, hydrophobicity, antiradical activity, inhibition of superoxide anion (O2°(-)), and the abilities to affect hypochlorous acid (HOCl) production by leukocytes and inhibit myeloperoxidase (MPO) chlorinating activity were studied. The increased hydrophobicity (logP, 0.81-4.82) of the esters was not correlated with a significant alteration in their oxidation potential (0.222-0.298 V). However, except for P10, the esters were ~ 2-fold more effective than the acid precursor for the scavenging of DPPH and peroxyl radicals. The esters were strong inhibitors of O2°(-) released by activated neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs). A correlation was found between the carbon chain length and the relative inhibitory potency. P7, the most active ester, was ~ 10-fold more efficient as NADPH oxidase inhibitor than apocynin. The esters strongly inhibited the release of HOCl by PMNs, which was a consequence of the inhibition of NADPH oxidase activity in these cells. In conclusion, as effective inhibitors of NADPH oxidase, the esters of protocatechuic acid are promising drugs for treatment of chronic inflammatory diseases. Moreover, this is the first demonstration that, besides the redox active moiety, the hydrophobicity can also be a determinant factor for the design of NADPH oxidase inhibitors.
Asunto(s)
Hidroxibenzoatos/química , NADPH Oxidasas/antagonistas & inhibidores , Técnicas Electroquímicas , Ésteres , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Hidroxibenzoatos/farmacología , Ácido Hipocloroso/toxicidad , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Superóxidos/químicaRESUMEN
Apatone™, a combination of menadione (2-methyl-1,4-naphthoquinone, VK3) and ascorbic acid (vitamin C, VC) is a new strategy for cancer treatment. Part of its effect on tumor cells is related to the cellular pro-oxidative imbalance provoked by the generation of hydrogen peroxide (H2O2) through naphthoquinone redox cycling. In this study, we attempted to find new naphthoquinone derivatives that would increase the efficiency of H2O2 production, thereby potentially increasing its efficacy for cancer treatment. The presence of an electron-withdrawing group in the naphthoquinone moiety had a direct effect on the efficiency of H2O2 production. The compound 2-bromo-1,4-naphthoquinone (BrQ), in which the bromine atom substituted the methyl group in VK3, was approximately 10- and 19-fold more efficient than VK3 in terms of oxygen consumption and H2O2 production, respectively. The ratio [H2O2]produced / [naphthoquinone]consumed was 68 ± 11 and 5.8 ± 0.2 (µM/µM) for BrQ and VK3, respectively, indicating a higher efficacy of BrQ as a catalyst for the autoxidation of ascorbic acid. Both VK3 and BrQ reacted with glutathione (GSH), but BrQ was the more effective substrate. Part of GSH was incorporated into the naphthoquinone, producing a nucleophilic substitution product (Q-SG). The depletion of BrQ by GSH did not prevent its redox capacity since Q-SG was also able to catalyze the production of reactive oxygen species. VK3/VC has already been submitted to clinical trials for the treatment of prostate cancer and has demonstrated promising results. However, replacement of VK3 with BrQ will open new lines of investigation regarding this approach to cancer treatment.
Asunto(s)
Humanos , Antineoplásicos/farmacología , Ácido Ascórbico/farmacología , Peróxido de Hidrógeno/metabolismo , Naftoquinonas/farmacología , Especies Reactivas de Oxígeno , Antineoplásicos/química , Ácido Ascórbico/química , Combinación de Medicamentos , Sustitución de Medicamentos , Naftoquinonas/química , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacos , /química , /farmacologíaRESUMEN
Apatone™, a combination of menadione (2-methyl-1,4-naphthoquinone, VK3) and ascorbic acid (vitamin C, VC) is a new strategy for cancer treatment. Part of its effect on tumor cells is related to the cellular pro-oxidative imbalance provoked by the generation of hydrogen peroxide (H2O2) through naphthoquinone redox cycling. In this study, we attempted to find new naphthoquinone derivatives that would increase the efficiency of H2O2 production, thereby potentially increasing its efficacy for cancer treatment. The presence of an electron-withdrawing group in the naphthoquinone moiety had a direct effect on the efficiency of H2O2 production. The compound 2-bromo-1,4-naphthoquinone (BrQ), in which the bromine atom substituted the methyl group in VK3, was approximately 10- and 19-fold more efficient than VK3 in terms of oxygen consumption and H2O2 production, respectively. The ratio [H2O2]produced / [naphthoquinone]consumed was 68 ± 11 and 5.8 ± 0.2 (µM/µM) for BrQ and VK3, respectively, indicating a higher efficacy of BrQ as a catalyst for the autoxidation of ascorbic acid. Both VK3 and BrQ reacted with glutathione (GSH), but BrQ was the more effective substrate. Part of GSH was incorporated into the naphthoquinone, producing a nucleophilic substitution product (Q-SG). The depletion of BrQ by GSH did not prevent its redox capacity since Q-SG was also able to catalyze the production of reactive oxygen species. VK3/VC has already been submitted to clinical trials for the treatment of prostate cancer and has demonstrated promising results. However, replacement of VK3 with BrQ will open new lines of investigation regarding this approach to cancer treatment.
Asunto(s)
Antineoplásicos/farmacología , Ácido Ascórbico/farmacología , Peróxido de Hidrógeno/metabolismo , Naftoquinonas/farmacología , Especies Reactivas de Oxígeno , Antineoplásicos/química , Ácido Ascórbico/química , Combinación de Medicamentos , Sustitución de Medicamentos , Humanos , Naftoquinonas/química , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacos , Vitamina K 3/química , Vitamina K 3/farmacologíaRESUMEN
Portadores de diabetes tipo-1 são acometidos por episódios freqüentes de acidose causada pelo aumento no metabolismo de ácido graxos com conseqüente acúmulo de acetoacetato (AcAc) e beta-hidroxibutirato (beta -OB). Neste trabalho estudou-se o efeito de concentrações patológicas destes metabólitos na lipoperoxidação, viabilidade e liberação da quimiocina CXCL8 (IL-8) por neutrófilos em cultura. Neutrófilos de indivíduos saudáveis foram isolados por gradiente de densidade (Histopaque 1077/1119) e incubados com os corpos cetônicos. A lipoperoxidação foi determinada pela presença de substâncias reagentes ao ácido tiobarbitúrico (TBARS). A viabilidade celular foi avaliada pela liberação da enzima lactato desidrogenase. A liberação de CXCL8 para o meio extracelular foi medida após cultura de 24 horas de neutrófilos estimulados por zymosan opsonizado por ensaio imunoenzimático (ELISA). O AcAc causou um aumento na lipoperoxidação dos neutrófilos e este efeito foi dependente da sua concentração (p < 0.05; r = 0.99146); não se observou efeito do beta-HOB. No estudo do efeito citotóxico, houve aumento dose-dependente da liberação da LDH até 40 mM de AcAc (p < 0.05); não se observou efeito do beta-HOB. A liberação de CXCL8 foi suprimida de modo dose-dependente por AcAc e beta-HOB. Estes resultados sugerem que o acúmulo de corpos cetônicos pode contribuir para aumentar o tempo de remissão de doenças e mesmo estar relacionado com a gravidade destas em indivíduos diabéticos.
Type-1 diabetes patients suffer from frequent episodes of acidosis caused by an increased fatty acid metabolism and consequently increased plasma level of acetoacetate (AcAc) and beta-hydroxybutyrate (beta-HOB). This article describes a study of the effects of pathological concentrations of AcAc and beta-HOB on lipoperoxidation, cell viability and the release of the CXCL8 (IL-8) cytokine by activated neutrophils. Neutrophils from healthy donors were isolated by density gradient (Histopaque® 1077/1119) and incubated with the ketone bodies. Lipoperoxidation was determined as thiobarbituric acid reactive substances (TBARS). The cell viability was evaluated by the release of intracellular lactate dehydrogenase. The release of CXCL8 was measured by ELISA in a 24-h culture of opsonized zymosanstimulated neutrophils. AcAc, but not beta-HOB, provoked a dose-dependent increase in the neutrophil membrane lipoperoxidation (p<0.05; r =0.9915). In the cytotoxicity assay, a dose-dependent release of LDH was observed when the neutrophils were incubated with AcAc in concentrations up to 40 mM (p<0.05). beta-HOB was devoid of effect. The release of CXCL8 was inhibited by AcAc and beta-HOB in a dose-dependent manner. In conclusion, these results suggest that the accumulation of ketone bodies in diabetic patients could be involved in their usually increased susceptibility to infection.
Asunto(s)
Diabetes Mellitus , Cuerpos Cetónicos , Neutrófilos , Supervivencia CelularRESUMEN
Maytenus ilicifolia is an important plant with potential on cancer treatment and has been largely used in Brazil and other countries. We have evaluated the crude ethanolic extract of M. ilicifolia as a potential antioxidant source using an assay based on the bleaching of the radical monocation 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(*+)) and by HOCl scavenger capacity. Trolox and uric acid were used as positive controls. The results indicated M. ilicifolia root bark as a great source of antioxidants based on its potential as scavenger of radicals.
Asunto(s)
Antioxidantes/química , Depuradores de Radicales Libres/química , Maytenus/química , Benzotiazoles , Cromanos/farmacología , Etanol/química , Concentración 50 Inhibidora , Corteza de la Planta/química , Extractos Vegetales/química , Ácidos Sulfónicos/metabolismo , Ácido Úrico/farmacologíaRESUMEN
Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37 degrees C in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 microM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 +/- 0.03 and 5.0 +/- 1.0 microM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.
Asunto(s)
Ácido Hipocloroso/metabolismo , Indoles/farmacología , Leucocitos/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Leucocitos/fisiología , Oxidación-ReducciónRESUMEN
Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37°C in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 æM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72 percent range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 æM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.
Asunto(s)
Humanos , Ácido Hipocloroso/metabolismo , Indoles/farmacología , Leucocitos/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Leucocitos/fisiología , Oxidación-ReducciónRESUMEN
The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 microM), indomethacin (12 microM), naproxen (160 microM), piroxicam (13 microM), and tenoxicam (30 microM) were incubated at 37 masculineC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 +/- 2% diclofenac, 90 +/- 2% indomethacin, 33 +/- 3% piroxicam, and 45 +/- 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 +/- 5% and diclofenac showed amplification in the light emission of 181 +/- 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 +/- 10, 45 +/- 3%), indomethacin (97 +/- 2, 100 +/- 1%), naproxen (56 +/- 8, 76 +/- 3%), piroxicam (77 +/- 5, 99 +/- 1%), and tenoxicam (90 +/- 2, 100 +/- 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Neutrófilos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/toxicidad , Mediciones Luminiscentes , Activación Neutrófila , Neutrófilos/metabolismo , Peroxidasa/efectos de los fármacos , RatasRESUMEN
The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37°C in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2 percent diclofenac, 90 ± 2 percent indomethacin, 33 ± 3 percent piroxicam, and 45 ± 6 percent tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5 percent and diclofenac showed amplification in the light emission of 181 ± 60 percent (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3 percent), indomethacin (97 ± 2, 100 ± 1 percent), naproxen (56 ± 8, 76 ± 3 percent), piroxicam (77 ± 5, 99 ± 1 percent), and tenoxicam (90 ± 2, 100 ± 1 percent), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.
Asunto(s)
Animales , Ratas , Antiinflamatorios no Esteroideos/farmacología , Neutrófilos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/toxicidad , Mediciones Luminiscentes , Activación Neutrófila , Neutrófilos/metabolismo , Peroxidasa/efectos de los fármacosRESUMEN
A growing body of evidence suggests that the pineal hormone, melatonin, has immunomodulatory properties, although very little is known about its effect on leukocytes. Therefore, we aimed to investigate the effect of melatonin and its oxidation product N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) on cytokine production by neutrophils and peripheral blood mononuclear cells (PBMCs). AFMK (0.001-1 mM) inhibits the lipopolysaccharide (LPS)-mediated production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) more efficiently in neutrophils than PBMCs. Moreover, the inhibitory activity of AFMK is stronger than that of melatonin. Interestingly, monocytes efficiently oxidize melatonin to AFMK. We conclude that neutrophils are one of the main targets for melatonin and that at least part of the effects described for melatonin on immune cells may be due to its oxidation product, AFMK. We also consider that the oxidation of melatonin may be an important event in the cross-talking between neutrophils and monocytes.
Asunto(s)
Citocinas/metabolismo , Kinuramina/análogos & derivados , Kinuramina/farmacología , Melatonina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Análisis de Varianza , Células Cultivadas , Humanos , Kinuramina/metabolismo , Melatonina/metabolismo , Oxidación-Reducción/efectos de los fármacosRESUMEN
We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657-662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H(2)O(2) ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O(-*)(2) or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.
Asunto(s)
Peroxidasa de Rábano Silvestre/metabolismo , Melatonina/metabolismo , Triptófano/metabolismo , Cromatografía Líquida de Alta Presión , Peróxido de Hidrógeno/metabolismo , Kinuramina/análogos & derivados , Kinuramina/metabolismo , Oxidación-ReducciónRESUMEN
The indole moeity is present in many substances of biological occurrence. Its metabolism, in most cases, involves an oxidative pathway. This study reports the oxidation of a series of indole derivatives, including several of biological origin, catalyzed by horseradish peroxidase in the presence of H2O2. Chemiluminescence emission was observed in most cases and its intensity and spectral characteristics were correlated with structural features of the substrates. The structures of the main products were determined. The participation of molecular oxygen and superoxide ion in the reaction was demonstrated and a general mechanism for product formation proposed. Since the oxidation of 2-methylindole proved to be highly chemiluminescent, its potentiality as a developing system for peroxidase-based assays was tested and showed to be very effective.
Asunto(s)
Peroxidasa de Rábano Silvestre/química , Indoles/química , Catálisis , Peroxidasa de Rábano Silvestre/análisis , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Mediciones Luminiscentes , Modelos Químicos , Estructura Molecular , Oxidación-Reducción , Especies Reactivas de Oxígeno/fisiología , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Superóxidos/químicaRESUMEN
In the presence of hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the production of N(1)-acetyl-N(2)-formyl-5-methoxykynuramine from melatonin. This reaction consumes oxygen and exhibits chemiluminescence in the 440-540 nm region. The excited cleavage product derived from the thermolysis of an intermediate dioxetane is suggested to be the emitting species. Chemiluminescence and the indole ring cleavage product were also observed when HRP/H(2)O(2) was replaced by phorbol myristate acetate or opsonized zymosan-activated neutrophils. Azide, a myeloperoxidase inhibitor, strongly suppressed melatonin oxidation. Superoxide dismutase has a strong inhibitory effect on light emission but catalase and uric acid are without effect on the emission. The oxidation of melatonin by activated neutrophils may be relevant to the in vivo functions of myeloperoxidase and melatonin. The possible biological implication of melatonin oxidation by neutrophils, especially in inflammatory conditions, is discussed.