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1.
J Med Chem ; 67(14): 12012-12032, 2024 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-38991154

RESUMEN

This study presents a new approach for identifying myeloperoxidase (MPO) inhibitors with strong in vivo efficacy. By combining inhibitor-like rules and structure-based virtual screening, the pipeline achieved a 70% success rate in discovering diverse, nanomolar-potency reversible inhibitors and hypochlorous acid (HOCl) scavengers. Mechanistic analysis identified RL6 as a genuine MPO inhibitor and RL7 as a potent HOCl scavenger. Both compounds effectively suppressed HOCl production in cells and neutrophils, with RL6 showing a superior inhibition of neutrophil extracellular trap release (NETosis). In a gout arthritis mouse model, intraperitoneal RL6 administration reduced edema, peroxidase activity, and IL-1ß levels. RL6 also exhibited oral bioavailability, significantly reducing paw edema when administered orally. This study highlights the efficacy of integrating diverse screening methods to enhance virtual screening success, validating the anti-inflammatory potential of potent inhibitors, and advancing the MPO inhibitor research.


Asunto(s)
Artritis Gotosa , Peroxidasa , Animales , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Artritis Gotosa/tratamiento farmacológico , Ratones , Humanos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Masculino , Ácido Hipocloroso , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Relación Estructura-Actividad , Evaluación Preclínica de Medicamentos
2.
Redox Biol ; 71: 103102, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38430684

RESUMEN

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.


Asunto(s)
Bromatos , Proteínas de la Matriz Extracelular , Fibrosis Pulmonar , Humanos , Animales , Ratones , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Bromuros/efectos adversos , Bromuros/metabolismo , Laminina/genética , Laminina/metabolismo , Matriz Extracelular/metabolismo , Pulmón/metabolismo , Peroxidasina , Colágeno Tipo IV/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Tirosina/metabolismo
3.
J Biol Chem ; 297(3): 101041, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34358560

RESUMEN

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.


Asunto(s)
ADP-Ribosilación , COVID-19/virología , Interferones/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , SARS-CoV-2/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Humanos
4.
J Biol Chem ; 296: 100494, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33667550

RESUMEN

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.


Asunto(s)
Cisteína/química , Cisteína/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Peróxido de Hidrógeno/química , Mutación , Oxidantes/química , Oxidantes/metabolismo , Oxidación-Reducción , Relación Estructura-Actividad
5.
Free Radic Biol Med ; 158: 115-125, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32702382

RESUMEN

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 ± 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 ± 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.


Asunto(s)
Cisteína , Peroxirredoxinas , Dominio Catalítico , Cisteína/metabolismo , Peróxido de Hidrógeno , Cinética , Oxidación-Reducción , Peroxirredoxinas/metabolismo
6.
Free Radic Biol Med ; 135: 227-234, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30862546

RESUMEN

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.


Asunto(s)
Antioxidantes/metabolismo , Proteínas de Homeodominio/genética , Oxidación-Reducción/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Proteínas de Homeodominio/antagonistas & inhibidores , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/microbiología , Compuestos Onio/farmacología , Oxidantes/metabolismo , Transducción de Señal/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Acetato de Tetradecanoilforbol/toxicidad
7.
Free Radic Biol Med ; 134: 394-405, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30699366

RESUMEN

Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP + ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.


Asunto(s)
Citoprotección , Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/citología , Insulinoma/patología , Chaperonas Moleculares/metabolismo , Estrés Oxidativo/efectos de los fármacos , Neoplasias Pancreáticas/patología , Prolactina/farmacología , Animales , Apoptosis , Glutatión/metabolismo , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma/tratamiento farmacológico , Insulinoma/metabolismo , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Oxidación-Reducción , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Transporte de Proteínas , Proteolisis , Células Tumorales Cultivadas
8.
Free Radic Biol Med ; 126: 177-186, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30118829

RESUMEN

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.


Asunto(s)
Inflamación/sangre , Neutrófilos/metabolismo , Peróxidos/metabolismo , Especies Reactivas de Oxígeno/sangre , Ácido Úrico/análogos & derivados , Catálisis , Línea Celular Tumoral , Radicales Libres/química , Radicales Libres/metabolismo , Humanos , Inflamación/patología , Espectrometría de Masas , Neutrófilos/química , Oxidación-Reducción , Peroxidasa/genética , Peroxidasa/metabolismo , Peróxidos/química , Peróxidos/aislamiento & purificación , Especies Reactivas de Oxígeno/aislamiento & purificación , Superóxidos/química , Superóxidos/metabolismo , Ácido Úrico/química , Ácido Úrico/aislamiento & purificación , Ácido Úrico/metabolismo
9.
Redox Biol ; 16: 179-188, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29510342

RESUMEN

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.


Asunto(s)
Neutrófilos/metabolismo , Peróxidos/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Ácido Úrico/análogos & derivados , Ácido Úrico/metabolismo , Catálisis , Diferenciación Celular/genética , Radicales Libres/metabolismo , Glutatión/metabolismo , Células HL-60/metabolismo , Células HL-60/microbiología , Humanos , Peróxido de Hidrógeno/metabolismo , Ácido Hipocloroso/química , Neutrófilos/microbiología , Oxidantes/metabolismo , Oxidación-Reducción/efectos de los fármacos , Peróxidos/química , Pseudomonas aeruginosa/patogenicidad , Ácido Úrico/química
10.
J Biol Chem ; 292(21): 8705-8715, 2017 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-28348082

RESUMEN

Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m-1 s-1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s-1) than Prx1 (k3 = 14.9 s-1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.


Asunto(s)
Eritrocitos/enzimología , Peróxidos/química , Peroxirredoxinas/química , Ácido Úrico/análogos & derivados , Disulfuros/química , Disulfuros/metabolismo , Humanos , Cinética , Oxidación-Reducción , Peróxidos/metabolismo , Peroxirredoxinas/metabolismo , Ácido Úrico/química , Ácido Úrico/metabolismo
11.
Chem Res Toxicol ; 28(8): 1556-66, 2015 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-26207674

RESUMEN

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.


Asunto(s)
Peróxido de Hidrógeno/química , Luz , Peróxidos/química , Ácido Úrico/análogos & derivados , Ácido Úrico/química , Cromatografía Liquida , Glutatión/química , Cinética , Estructura Molecular , Oxidación-Reducción , Estrés Oxidativo , Espectrometría de Masa por Ionización de Electrospray , Ácido Úrico/metabolismo
12.
Chem Biol Interact ; 203(2): 440-7, 2013 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-23523557

RESUMEN

The aim of this study was to investigate the involvement of the transient receptor potential ankyrin 1 (TRPA1) in haemorrhagic cystitis, the main side effect of cyclophosphamide-based chemotherapy. Hannover female rats received intraperitoneal (i.p.) injection of cyclophosphamide (three doses of 100 mg/kg, every other day, in a total of five days). This treatment was followed by the treatment with TRPA1 antagonist HC 030031 (50 mg/kg, p.o.). The threshold for hindpaw withdrawal or abdominal retraction to von Frey Hair and the locomotor activity were measured. The treatment with the TRPA1 antagonist HC 030031 significantly decreased mechanical hyperalgesia induced by cyclophosphamide without interfere with locomotor activity. Urodynamic parameters were performed by cystometry 24 h after a single treatment with cyclophosphamide (200 mg/kg, i.p.) in control and HC 030031 treated rats. Analyses of the urodynamic parameters showed that a single dose of cyclophosphamide was enough to significantly increase the number and amplitude of non-voiding contractions and to decrease the voided volume and voiding efficiency, without significantly altering basal, threshold or maximum pressure. The treatment with HC 030031 either before (100 mg/kg, p.o.) or after (30 mg/kg, i.v.) cyclophosphamide inhibited the non-voiding contractions but failed to counteract the loss in voiding efficiency. Our data demonstrates that nociceptive symptoms and urinary bladder overactivity caused by cyclophosphamide, in part, are dependent upon the activation of TRPA1. In this context, the antagonism of the receptor may be an alternative to minimise the urotoxic symptoms caused by this chemotherapeutic agent.


Asunto(s)
Ciclofosfamida/efectos adversos , Cistitis/inducido químicamente , Cistitis/complicaciones , Hemorragia/complicaciones , Hiperalgesia/tratamiento farmacológico , Canales Catiónicos TRPC/antagonistas & inhibidores , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Acetanilidas/farmacología , Acetanilidas/uso terapéutico , Animales , Antineoplásicos Alquilantes/efectos adversos , Cistitis/metabolismo , Cistitis/fisiopatología , Femenino , Hiperalgesia/complicaciones , Purinas/farmacología , Purinas/uso terapéutico , Ratas , Canal Catiónico TRPA1 , Vejiga Urinaria Hiperactiva/complicaciones , Urodinámica/efectos de los fármacos
13.
Pain ; 152(8): 1872-1887, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21620566

RESUMEN

In this study, we report that α,ß-amyrin, a plant-derived pentacyclic triterpene, reduced persistent inflammatory and neuropathic hyperalgesia in mice by a direct activation of the CB(1) and CB(2) cannabinoid receptors (CB(1)R and CB(2)R). The oral treatment with α,ß-amyrin (30 mg/kg) significantly reduced mechanical and thermal hyperalgesia and inflammation induced by complete Freund's adjuvant (CFA) and by partial sciatic nerve ligation (PSNL). The pretreatment with either CB(1)R or CB(2)R antagonists and the knockdown gene of the receptors significantly reverted the antinociceptive effect of α,ß-amyrin. Of note, binding studies showed that α,ß-amyrin directly bound with very high affinity to CB(1)R (K(i)=0.133 nM) and with a lower affinity to CB(2)R (K(i)=1989 nM). Interestingly, α,ß-amyrin, ACEA (CB(1)R agonist), or JWH-133 (CB(2)R agonist), at doses that caused antinociception, failed to provoke any behavioral disturbance, as measured in the tetrad assay. In addition, α,ß-amyrin largely decreased interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), keratinocyte-derived chemokine (KC) and interleukin 6 (IL-6) levels, and myeloperoxidase activity. Likewise, α,ß-amyrin prevented the activation of the transcriptional factors: nuclear factor κB (NF-κB) and cyclic adenosine monophosphate response element binding (CREB) and the expression of cyclooxygenase 2 in mice footpads and spinal cords. The present results demonstrated that α,ß-amyrin exhibits long-lasting antinociceptive and anti-inflammatory properties in 2 models of persistent nociception via activation of cannabinoid receptors and by inhibiting the production of cytokines and expression of NF-κB, CREB and cyclooxygenase 2.


Asunto(s)
Antiinflamatorios/uso terapéutico , Inflamación/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Ácido Oleanólico/análogos & derivados , Triterpenos Pentacíclicos/uso terapéutico , Receptores de Cannabinoides/metabolismo , Análisis de Varianza , Animales , Antiinflamatorios/química , Área Bajo la Curva , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Ciclohexanoles/farmacocinética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Edema/diagnóstico , Edema/tratamiento farmacológico , Edema/etiología , Ensayo de Inmunoadsorción Enzimática , Reacción Cataléptica de Congelación/efectos de los fármacos , Reacción Cataléptica de Congelación/fisiología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/fisiopatología , Locomoción/efectos de los fármacos , Masculino , Ratones , Neuralgia/fisiopatología , Ácido Oleanólico/química , Ácido Oleanólico/uso terapéutico , Oligodesoxirribonucleótidos Antisentido/farmacología , Umbral del Dolor/efectos de los fármacos , Triterpenos Pentacíclicos/química , Peroxidasa/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Cannabinoides/química , Receptores de Cannabinoides/genética , Tritio/farmacocinética
14.
J Biol Chem ; 286(15): 12901-11, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21266577

RESUMEN

Urate and myeloperoxidase (MPO) are associated with adverse outcomes in cardiovascular disease. In this study, we assessed whether urate is a likely physiological substrate for MPO and if the products of their interaction have the potential to exacerbate inflammation. Urate was readily oxidized by MPO and hydrogen peroxide to 5-hydroxyisourate, which decayed to predominantly allantoin. The redox intermediates of MPO were reduced by urate with rate constants of 4.6 × 10(5) M(-1) s(-1) for compound I and 1.7 × 10(4) M(-1) s(-1) for compound II. Urate competed with chloride for oxidation by MPO and at hyperuricemic levels is expected to be a substantive substrate for the enzyme. Oxidation of urate promoted super-stoichiometric consumption of glutathione, which indicates that it is converted to a free radical intermediate. In combination with superoxide and hydrogen peroxide, MPO oxidized urate to a reactive hydroperoxide. This would form by addition of superoxide to the urate radical. Urate also enhanced MPO-dependent consumption of nitric oxide. In human plasma, stimulated neutrophils produced allantoin in a reaction dependent on the NADPH oxidase, MPO and superoxide. We propose that urate is a physiological substrate for MPO that is oxidized to the urate radical. The reactions of this radical with superoxide and nitric oxide provide a plausible link between urate and MPO in cardiovascular disease.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Hiperuricemia/enzimología , Neutrófilos/enzimología , Peroxidasa/metabolismo , Superóxidos/metabolismo , Alantoína/biosíntesis , Alantoína/química , Enfermedades Cardiovasculares/enzimología , Humanos , Peróxido de Hidrógeno/química , Inflamación , NADPH Oxidasas/química , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Peroxidasa/química , Especificidad por Sustrato , Superóxidos/química , Ácido Úrico
15.
Clin Exp Pharmacol Physiol ; 36(3): 272-7, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18986332

RESUMEN

1. The aim of the present study was to investigate the role of redox modulation during the peripheral nociceptive transmission in vivo. The nociceptive response was evaluated by the amount of time that mice spent licking the footpad injected with glutamate (20 micromol/paw). Thiol groups in footpad tissue were quantified using a colourimetric reaction with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). 2. When coadministered with glutamate, the thiol alkylating agent iodoacetate (200 nmol/paw) caused significant antinociception in footpad tissue, in parallel with a decrease in free thiol groups. Treatment with the reducing agent dithiothreitol (200 nmol/paw) 5 min before glutamate and iodoacetate prevented the antinociception and thiol loss caused by iodoacetate. Injection of 100 nmol/paw ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), an in vitro redox modulator of the N-methyl-d-aspartate (NMDA) receptor, also prevented iodoacetate-induced antinociception. However, ebselen did not prevent thiol loss in the footpad. Dithiothreitol and ebselen had a synergic nociceptive effect with glutamate. 3. Alone, ebselen (100 nmol/paw) exhibited a pronociceptive effect. The nociception induced by ebselen was blocked by glutathione depletion induced by buthionine-sulphoximine (BSO; 2.5 micromol/paw). In addition, ebselen-induced nociception was prevented by 75 +/- 2% following injection of 5 nmol/paw MK-801 (an NMDA receptor antagonist). The nitric oxide synthase inhibitor N(G)-nitro-l-arginine (250 nmol/paw) had no effect on the nociception produced by ebselen. 4. In conclusion, the present paper reports on the effect of redox modulation on the glutamatergic system during peripheral nociceptive transmission in vivo. Antinociception was directly correlated with the availability of thiol groups, whereas the pronociceptive response of the reducing agents likely occurs via positive modulation of the NMDA receptor.


Asunto(s)
Analgésicos/farmacología , Conducta Animal/efectos de los fármacos , Dolor/prevención & control , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Alquilantes/farmacología , Animales , Azoles/toxicidad , Butionina Sulfoximina/farmacología , Modelos Animales de Enfermedad , Ditiotreitol/toxicidad , Maleato de Dizocilpina/farmacología , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Glutamato-Cisteína Ligasa/metabolismo , Ácido Glutámico , Glutatión/metabolismo , Yodoacetatos/farmacología , Isoindoles , Ratones , Compuestos de Organoselenio/toxicidad , Oxidación-Reducción , Dolor/inducido químicamente , Dolor/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sustancias Reductoras/toxicidad
16.
Basic Clin Pharmacol Toxicol ; 103(1): 43-7, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18598298

RESUMEN

This study investigated the role of the glutamatergic system on the antinociception caused by Polygala sabulosa hydroalcoholic extract (HE). The systems mediated by substance P, capsaicin, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were also investigated. P. sabulosa HE given orally produced a significant inhibition of glutamate-induced paw licking [ID(50) = 530.3 (416.7-674.8) mg/kg and inhibition of 79 +/- 6% at 1000 mg/kg]. The plant derivatives alpha-spinasterol, scopoletin and styryl-2-pyrones (compound 1 and 3) (10 mg/kg, intraperitoneally) inhibited 80 +/- 7%, 46 +/- 11%, 45 +/- 11% and 35 +/- 13% the nociceptive response caused by glutamate, respectively. Furthermore, P. sabulosa HE (500 mg/kg, orally) caused marked inhibition of nociceptive response induced by intrathecal injection of glutamate, N-methyl-d-aspartic acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, TNF-alpha and IL-1beta, with inhibitions of 44 +/- 7%, 55 +/- 4%, 38 +/- 10%, 61 +/- 7%, 76 +/- 9% and 100%, respectively. In contrast, P. sabulosa HE (500 mg/kg, orally) did not affect the biting response induced by the metabotropic glutamatergic receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, substance P and capsaicin. The locomotor activity was altered only in mice treated with a very high dose (1000 mg/kg) of P. sabulosa HE. Our results showed that the antinociceptive effects of P. sabulosa HE are associated with an inhibition of glutamatergic transmission and an inhibition of pathways dependent on pro-inflammatory cytokines. The plant derivatives alpha-spinasterol, scopoletin and styryl-2-pyrones play an important role on the antinociceptive effects of P. sabulosa HE.


Asunto(s)
Analgésicos/farmacología , Citocinas/fisiología , Dolor/tratamiento farmacológico , Polygala/química , Receptores de Glutamato/fisiología , Administración Oral , Analgésicos/uso terapéutico , Animales , Capsaicina/farmacología , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Interleucina-1beta/farmacología , Masculino , Ratones , Dolor/fisiopatología , Dimensión del Dolor , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Receptores de Neuroquinina-1/agonistas , Sustancia P/farmacología , Factor de Necrosis Tumoral alfa/farmacología
17.
J Ethnopharmacol ; 114(3): 355-63, 2007 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-17900839

RESUMEN

Humirianthera ampla Miers is a member of the Icacinaceae family and presents great amounts of di and triterpenoids. These chemical constituents in roots of Humirianthera ampla sustain not only the ethnopharmacological use against snake venom, but also some anti-inflammatory and analgesic properties of the plant. In this study we investigated the antinociceptive action of the ethanolic extract (EE) from roots of the Humirianthera ampla in chemical and thermal models of pain in mice. The oral treatment with ethanolic extract dose-dependently inhibited glutamate-, capsaicin- and formalin-induced licking. However, it did not prevent the nociception caused by radiant heat on the tail-flick test. The ethanolic extract (30 mg/kg) caused marked inhibition of the nociceptive biting response induced by glutamate, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), N-methyl-d-aspartate (NMDA) and substance P. The antinociception caused by ethanolic extract was significantly attenuated by naloxone, l-arginine, WAY100635, ondansetron or ketanserin, but not by caffeine or naloxone methiodide. In conclusion, the ethanolic extract from roots of Humirianthera ampla produces antinociception against neurogenic and inflammatory models of nociception. The mechanisms of antinociception involve nitric oxide, opioid, serotonin and glutamate pathways. Therefore, our results support the ethnopharmacological use of the Humirianthera ampla against inflammatory and painful process caused by snake venom.


Asunto(s)
Analgésicos/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales , Animales , Reacción de Prevención/efectos de los fármacos , Brasil , Relación Dosis-Respuesta a Droga , Femenino , Ácido Glutámico/farmacología , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Óxido Nítrico/biosíntesis , Raíces de Plantas/química , Ratas , Ratas Wistar , Sustancia P/farmacología
18.
Neurochem Res ; 31(4): 563-9, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16758366

RESUMEN

During the early postnatal period the central nervous system (CNS) is extremely sensitive to external agents. The present study aims at the investigation of critical phases where methylmercury (MeHg) induces cerebellar toxicity during the suckling period in mice. Animals were treated with daily subcutaneous injections of MeHg (7 mg/kg of body weight) during four different periods (5 days each) at the early postnatal period: postnatal day (PND) 1-5, PND 6-10, PND 11-15, or PND 16-20. A control group was treated with daily subcutaneous injections of a 150 mM NaCl solution (10 ml/kg of body weight). Subjects exposed to MeHg at different postnatal periods were littermate. At PND 35, behavioral tests were performed to evaluate spontaneous locomotor activity in the open field and motor performance in the rotarod task. Biochemical parameters related to oxidative stress (levels of glutathione and thiobarbituric acid reactive substances, as well as glutathione peroxidase and glutathione reductase activity) were evaluated in cerebellum. Hyperlocomotor activity and high levels of cerebellar thiobarbituric acid reactive substances were observed in animals exposed to MeHg during the PND 11-15 or PND 16-20 periods. Cerebellar glutathione reductase activity decreased in MeHg-exposed animals. Cerebellar glutathione peroxidase activity was also decreased after MeHg exposure and the lowest enzymatic activity was found in animals exposed to MeHg during the later days of the suckling period. In addition, low levels of cerebellar glutathione were found in animals exposed to MeHg during the PND 16-20 period. The present results show that the postnatal exposure to MeHg during the second half of the suckling period causes hyperlocomotor activity in mice and point to this phase as a critical developmental stage where mouse cerebellum is a vulnerable target for the neurotoxic and pro-oxidative effects of MeHg.


Asunto(s)
Conducta/efectos de los fármacos , Cerebelo/efectos de los fármacos , Cerebelo/crecimiento & desarrollo , Compuestos de Metilmercurio/toxicidad , Actividad Motora/efectos de los fármacos , Estrés Oxidativo , Animales , Animales Lactantes , Cerebelo/metabolismo , Femenino , Glutatión/metabolismo , Masculino , Ratones , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo
19.
Environ Res ; 102(1): 22-8, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16564521

RESUMEN

This study examined the exclusive contribution of methylmercury (MeHg) exposure through maternal milk on biochemical parameters related to the thiol status (glutathione (GSH) levels, glutathione peroxidase (GPx) and glutathione reductase (GR) activities) in the cerebellums of suckling mice. The same biochemical parameters were also evaluated in the cerebellums of mothers, which were submitted to a direct oral exposure to MeHg (10 mg/L in drinking water). With regard to the relationship between cerebellar function and motor activity, the presence of signs of motor impairment was also evaluated in the offspring exposed to MeHg during lactation. After the treatment (at weaning period), the pups lactationally exposed to MeHg showed increased levels of mercury in the cerebellum compared to pups in the control group and a significant impairment in the motor performance in the rotarod apparatus. In addition, these pups showed decreased levels of GSH in the cerebellum compared to pups in the control group. In dams, MeHg significantly increased the levels of cerebellar GSH and the activities of cerebellar GR. However, this was not observed in pups. This study indicates that (1) the exposure of lactating mice to MeHg causes significant impairments in motor performance in the offspring which may be related to a decrease in the cerebellar thiol status and (2) the increased GSH levels and GR activity, observed only in the cerebellums of MeHg-exposed dams, could represent compensatory pathophysiologic responses to the oxidative effects of MeHg toward endogenous GSH.


Asunto(s)
Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Compuestos de Metilmercurio/toxicidad , Actividad Motora/efectos de los fármacos , Animales , Animales Lactantes , Cerebelo/enzimología , Femenino , Lactancia , Masculino , Intoxicación del Sistema Nervioso por Mercurio/metabolismo , Intoxicación del Sistema Nervioso por Mercurio/fisiopatología , Ratones , Actividad Motora/fisiología , Embarazo , Distribución Aleatoria
20.
Environ Toxicol Pharmacol ; 19(2): 239-48, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21783482

RESUMEN

It is known that lead is toxic to several species of animals, and growing data support the participation of oxidative in lead toxicity. Selenium compounds, like diphenyl diselenide and Ebselen have a thiol-peroxidase like and other antioxidant properties. In this work, we determine whether these non-thiol-containing compounds with antioxidant properties could reverse the toxicity produced by Pb(2+). Lead acetate injection followed by injection with Ebselen or diphenyl diselenide did not change the levels of non-protein thiol groups (NPSH), whereas simultaneous treatment with lead plus Ebselen reduced NPSH levels in liver. Lead and Ebselen caused a marked reduction in TBARS level in kidney, whereas lead or selenium compounds did not change TBARS levels in brain or liver. Lead acetate inhibited, δ-aminolevulinate dehydratase (ALA-D) activity in blood, liver, kidney and brain. Selenium compounds did not change enzyme activity nor the inhibitory effect of lead acetate in kidney and liver. Ebselen reversed brain ALA-D inhibition caused by Pb(2+). Reactivation index for ALA-D by DTT was higher in lead-treated groups than control groups in all tissues. Lead acetate or selenium compounds did not demonstrate alteration on [(3)H]-glutamate uptake by synaptosomes, whereas lead acetate plus Ebselen showed an increase on [(3)H]-glutamate uptake. The results of the present study indicate that ALA-D inhibition antecedes the overproduction of reactive oxygen species, which is becoming well documented in the literature.

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