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1.
J Pharmacol Exp Ther ; 388(2): 624-636, 2024 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-38182415

RESUMEN

The primary response of proliferating bovine pulmonary artery endothelial cells (BPAECs) after X-ray irradiation [≤10 gray (Gy)] is shown to be transient cell-cycle arrest. Accompanying oxidant-linked functional changes within the mitochondria are readily measured, but increased autophagy is not. Radiation-induced apoptosis is negligible in this line-important because cells undergoing apoptosis release oxygen-derived species that can overwhelm/mask the radiation-associated species and their effects that we wish to investigate. Cells irradiated and cultured at 3% oxygen exhibited delayed cell-cycle arrest (6-8 hours after 10 Gy irradiation) compared with those maintained at 20% oxygen (2-4 hours after 10 Gy irradiation). At 3% oxygen, either only during or only after irradiation, results intermediate between 20% and 3% oxygen throughout were obtained. No variability in cell-cycle distribution was observed for unirradiated cells cultured under different prevailing oxygen levels. Mitochondrially localized manganese superoxide dismutase delayed the X-ray-induced cell-cycle changes when over-expressed in BPAEC, indicating superoxide to be one of the key oxygen-derived cytotoxic species involved in the radiobiological response. Also, the peroxynitrite biomarker 3-nitrotyrosine was elevated, whereas hydrogen peroxide levels were not. Lastly, the utility of the BPAEC for screening potential countermeasures to ionizing radiation is demonstrated with some quinoline derivatives. Three of the five compounds appeared mitigative, and all were protective. It is suggested that the oxidation-reduction chemistry of these compounds probably offers a reasonable explanation for their observed ameliorative properties. Furthermore, the results suggest a promising new direction in the search for lead compounds as countermeasures to the effects of ionizing radiation. SIGNIFICANCE STATEMENT: The primary radiological response of proliferating bovine pulmonary artery endothelial cells is cell-cycle arrest, starting soon after X-ray irradiation (1-10 Gy) at 20% O2 but delayed by 4 hours at systemic (3%) O2. Oxygen/superoxide is found to be radio-sensitizing in at least two distinct time windows, during and after the irradiation, with both responses antagonized by various hydroxyquinoline derivatives. Similar responses in many other cell lines are likely to be masked by elevated oxidants associated with apoptosis.


Asunto(s)
Células Endoteliales , Oxidantes , Animales , Bovinos , Rayos X , Oxidantes/farmacología , Superóxidos , Apoptosis/efectos de la radiación , Oxígeno
2.
J Pharmacol Exp Ther ; 388(2): 596-604, 2024 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-38182416

RESUMEN

There do not appear to be any established therapeutics for treating azide poisoning at this time, and presently available antidotes to cyanide poisoning are far from ideal, being particularly impractical for use if multiple victims present. The cobalt (II/III) complex of the Schiff-base ligand trans-[14]-diene (5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene (CoN4[14]) is shown to act as an effective antidote to both azide and cyanide toxicity in mice. Groups of animals challenged with an LD40 dose of NaCN (100 µmol/kg i.p.) exhibited significantly faster recovery from knockdown and fewer (zero) deaths if given CoN4[14] (50 µmol/kg i.p.) 2 minutes after the toxicant. Groups of animals challenged with an essentially lethal dose of NaCN (1.5 x LD50 = 150 µmol/kg i.p.) all survived if given the CoN4[14] (75 µmol/kg i.p.) 5 minutes before the toxicant dose. These data represent improved antidotal capability over the Food and Drug Administration-approved cobalt-based cyanide antidote hydroxocobalamin. Recovery of animals challenged sublethally with NaN3 (415 µmol/kg i.p.) was assessed employing a modified pole-climbing test. Mice given the CoN4[14] antidote (70 µg/kg i.p.) 5 minutes after the toxicant dose recovered twice as fast as the controls given no antidote. The interactions of cyanide and azide with CoN4[14] in vitro (buffered aqueous solutions) have been further investigated by a combination of spectroscopic approaches. The Co(II) form of the complex is able to bind two CN- anions while only binding a single N3 -, providing a reasonable explanation for the difference between their therapeutic abilities. SIGNIFICANCE STATEMENT: The Schiff-base complex CoN4[14] is shown to be an effective antidote to cyanide in mice, with improved therapeutic capabilities compared to the Food and Drug Administration-approved cobalt-containing hydroxocobalamin. CoN4[14] is also antidotal in mice toward azide poisoning, for which there is seemingly no approved therapy currently available. The activity toward cyanide involves a "redox-switching" mechanism that could be a common, but largely unrecognized, feature of all cobalt-based cyanide antidotes in use and under development.


Asunto(s)
Antídotos , Hidroxocobalamina , Estados Unidos , Animales , Ratones , Antídotos/farmacología , Antídotos/uso terapéutico , Hidroxocobalamina/farmacología , Hidroxocobalamina/uso terapéutico , Azidas , Cobalto/química , Cianuros/química , Bases de Schiff/química
3.
J Immunol ; 207(6): 1627-1640, 2021 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-34433619

RESUMEN

Silicosis is a lethal pneumoconiosis for which no therapy is available. Silicosis is a global threat, and more than 2.2 million people per year are exposed to silica in the United States. The initial response to silica is mediated by innate immunity. Phagocytosis of silica particles by macrophages is followed by recruitment of mitochondria to phagosomes, generation of mitochondrial reactive oxygen species, and cytokine (IL-1ß, TNF-α, IFN-ß) release. In contrast with LPS, the metabolic remodeling of silica-exposed macrophages is unclear. This study contrasts mitochondrial and metabolic alterations induced by LPS and silica on macrophages and correlates them with macrophage viability and cytokine production, which are central to the pathogenesis of silicosis. Using high-resolution respirometer and liquid chromatography-high-resolution mass spectrometry, we determined the effects of silica and LPS on mitochondrial respiration and determined changes in central carbon metabolism of murine macrophage cell lines RAW 264.7 and IC-21. We show that silica induces metabolic reprogramming of macrophages. Silica, as well as LPS, enhances glucose uptake and increases aerobic glycolysis in macrophages. In contrast with LPS, silica affects mitochondria respiration, reducing complex I and enhancing complex II activity, to sustain cell viability. These mitochondrial alterations are associated in silica, but not in LPS-exposed macrophages, with reductions of tricarboxylic acid cycle intermediates, including succinate, itaconate, glutamate, and glutamine. Furthermore, in contrast with LPS, these silica-induced metabolic adaptations do not correlate with IL-1ß or TNF-α production, but with the suppressed release of IFN-ß. Our data highlight the importance of complex II activity and tricarboxylic acid cycle remodeling to macrophage survival and cytokine-mediated inflammation in silicosis.


Asunto(s)
Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Dióxido de Silicio/química , Dióxido de Silicio/farmacología , Silicosis/inmunología , Animales , Supervivencia Celular/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Cristalización , Citocinas/biosíntesis , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fagocitosis/efectos de los fármacos , Fagosomas/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Silicosis/metabolismo
4.
Front Microbiol ; 11: 2113, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32983071

RESUMEN

The ubiquitous involvement of key iron-containing metalloenzymes in metabolism is reflected in the dependence of virtually all bacteria on iron for growth and, thereby, potentially provides multiple biomolecular targets for antimicrobial killing. We hypothesized that nitrosative stress, which induces damage to iron metalloproteins, would sensitize bacteria to the ferric iron mimic gallium(III) (Ga3+), potentially providing a novel therapeutic combination. Using both laboratory and clinical isolates of Pseudomonas aeruginosa, we herein demonstrate that Ga3+ and sodium nitrite synergistically inhibit bacterial growth under both aerobic and anaerobic conditions. Nitric oxide also potentiated the antimicrobial effect of Ga3+. Because many chronic pulmonary infections are found as biofilms and biofilms have very high antibiotic tolerance, we then tested the combination against biofilms grown on plastic surfaces, as well as the apical surface of airway epithelial cells. Ga3+ and sodium nitrite had synergistic antimicrobial activity against both biofilms grown on plastic and on airway epithelial cell. Both Ga3+ and various NO donors are (independently) in clinical development as potential antimicrobials, however, we now propose the combination to have some particular advantages, while anticipating it should ultimately prove similarly safe for translation to treatment of human disease.

5.
Chem Res Toxicol ; 33(2): 594-603, 2020 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-31922405

RESUMEN

Three cobalt-containing macrocyclic compounds previously shown to antagonize cyanide toxicity have been comparatively evaluated for the amelioration of sublethal azide toxicity in juvenile (7-8 weeks) Swiss-Webster mice. The lowest effective doses were determined for hydroxocobalamin, a cobalt porphyrin, and a cobalt-Schiff base macrocycle by giving the antidotes 5 min prior to the toxicant, 27 mg (415 µmol) /kg sodium azide. Both male and female mice were evaluated for their response to the toxicant as well as the antidotes, and no significant differences were noted once weight differences were taken into account. Two of the three compounds significantly decreased the recovery time of azide-intoxicated mice at 10 min after the administration of sodium azide, as determined by a behavioral test (pole climbing). Additionally, azide was determined to cause a several degree drop (∼3 °C) in measured tail temperature, and warming the mice led to a more rapid recovery. The mice were also shown to recover more rapidly when given sodium nitrite, 24 mg (350 µmol)/kg, 5 min after the toxicant; this treatment also suppressed the azide-induced tail temperature decrease. Electron paramagnetic resonance (EPR) measurements of mouse blood treated with sodium azide demonstrated the presence of nitrosylhemoglobin at levels of 10-20 µM which persisted for ∼300 min. The presence of the methemoglobin azide adduct was also detected by EPR at a maximum level of ∼300 µM, but these signals disappeared around 200 min after the administration of azide. The treatment of mice with 15N sodium azide proved that the nitrosylhemoglobin was a product of the administered azide by the appearance of a two-line hyperfine (due to the 15N) in the EPR spectrum of mouse blood.


Asunto(s)
Antídotos/farmacología , Complejos de Coordinación/farmacología , Modelos Animales de Enfermedad , Azida Sódica/antagonistas & inhibidores , Azida Sódica/toxicidad , Animales , Antídotos/administración & dosificación , Cobalto/química , Cobalto/farmacología , Complejos de Coordinación/administración & dosificación , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Hemoglobinas/análisis , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Masculino , Ratones , Azida Sódica/administración & dosificación
6.
Chem Res Toxicol ; 33(2): 333-342, 2020 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-31599574

RESUMEN

There is presently no antidote available to treat azide poisoning. Here, the Schiff-base compound Co(II)-2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaenyl dibromide (Co(II)N4[11.3.1]) is investigated to determine if it has the capability to antagonize azide toxicity through a decorporation mechanism. The stopped-flow kinetics of azide binding to Co(II)N4[11.3.1] in the absence of oxygen exhibited three experimentally observable phases: I (fast); II (intermediate); and III (slow). The intermediate phase II accounted for ∼70% of the overall absorbance changes, representing the major process observed, with second-order rate constants of 29 (±4) M-1 s-1 at 25 °C and 70 (±10) M-1 s-1 at 37 °C. The data demonstrated pH independence of the reaction around neutrality, suggesting the unprotonated azide anion to be the attacking species. The binding of azide to Co(II)N4[11.3.1] appears to have a complicated mechanism leading to less than ideal antidotal capability; nonetheless, this cobalt complex does protect against azide intoxication. Administration of Co(II)N4[11.3.1] at 5 min post sodium azide injection (ip) to mice resulted in a substantial decrease of righting-recovery times, 12 (±4) min, compared to controls, 40 (±8) min. In addition, only two out of seven mice "knocked down" when the antidote was administered compared to the controls given toxicant only (100% knockdown).


Asunto(s)
Antídotos/farmacología , Antídotos/uso terapéutico , Cobalto/farmacología , Complejos de Coordinación/farmacología , Bases de Schiff/farmacología , Azida Sódica/antagonistas & inhibidores , Azida Sódica/envenenamiento , Animales , Antídotos/administración & dosificación , Antídotos/química , Cobalto/administración & dosificación , Cobalto/química , Cobalto/uso terapéutico , Complejos de Coordinación/administración & dosificación , Complejos de Coordinación/química , Complejos de Coordinación/uso terapéutico , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Inyecciones Intraperitoneales , Masculino , Ratones , Bases de Schiff/administración & dosificación , Bases de Schiff/química , Bases de Schiff/uso terapéutico , Azida Sódica/administración & dosificación , Tasa de Supervivencia
7.
Chem Res Toxicol ; 32(8): 1630-1637, 2019 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-31241903

RESUMEN

The Co(II/III)-containing macrocycle, cobalt 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1(17)2,11,13,15-pentaenyl cation, or CoN4[11.3.1], is a potential cyanide-scavenging agent. The rate of reduction of Co(III)N4[11.3.1] by ascorbate is reasonably facile under pseudo-first-order conditions; a second-order rate constant of 11.7(±0.4) M-1 s-1 was determined at 25 °C and pH 7.4, along with the activation parameters for the reaction (ΔH⧧ = 53.9(±0.8) kJ mol-1; ΔS -79(±3) J mol-1 K-1). It follows that any cyanide-decorporating capability of the cobalt complex should depend more on the cyanide-binding characteristics of Co(II)N4[11.3.1] than the oxidized form. The kinetics of the reaction of cyanide with Co(II)N4[11.3.1] under anaerobic pseudo-first-order conditions is rapid and resulted in a linear dependence on the cyanide concentration, kHCN = 8 × 104 M-1 s-1, with a nonlinear intercept of 420 s-1 at 10 °C, pH 7.6. The observed reaction rate increases significantly with increasing pH. A rate law is suggested, kobs = k'[X] + (kHCN + kCNKa/[H+])[HCN], where kCN is estimated to be ∼2 × 106 M-1 s-1. Activation parameters for the reaction with HCN (ΔH⧧ = 10.7(±0.4) kJ mol-1; ΔS⧧ = -153(±1) J mol-1 K-1) suggest an associative mechanism. In the presence of excess oxygen, i.e., at higher levels than free oxygen in vivo, the reaction rate was too fast to be measured, and the final product was the oxidized complex, Co(III)N4[11.3.1], where any cyanide ligands had been lost. This is much more rapid than the oxidation of the parent compound by oxygen, for which a second-order rate constant of 0.5(±0.02) M-1 s-1 at 25 °C was obtained. The study has gone some way toward enhancing our understanding of the reaction of Co(II)N4[11.3.1] with cyanide. The fast reaction rate implies a high efficacy of the cyanide-scavenging capability of the complex and further supports the suggestion stemming from our previous work that Co(II)N4[11.3.1] could prove to be a better and more cost-effective cyanide antidote than the FDA-approved hydroxocobalamin.


Asunto(s)
Cobalto/química , Complejos de Coordinación/química , Cianuros/química , Compuestos Macrocíclicos/química , Sitios de Unión , Cinética , Estructura Molecular , Oxidación-Reducción , Bases de Schiff/química , Termodinámica
8.
Chem Res Toxicol ; 32(6): 1310-1316, 2019 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-31070361

RESUMEN

Phosphine (PH3) poisoning continues to be a serious problem worldwide, for which there is no antidote currently available. An invertebrate model for examining potential toxicants and their putative antidotes has been used to determine if a strategy of using Au(I) complexes as phosphine-scavenging compounds may be antidotally beneficial. When Galleria mellonella larvae (or wax worms) were subjected to phosphine exposures of 4300 (±700) ppm·min over a 20 min time span, they became immobile (paralyzed) for ∼35 min. The administration of Au(I) complexes auro-sodium bisthiosulfate (AuTS), aurothioglucose (AuTG), and sodium aurothiomalate (AuTM) 5 min prior to phosphine exposure resulted in a drastic reduction in the recovery time (0-4 min). When the putative antidotes were given 10 min after the phosphine exposure, all the antidotes were therapeutic, resulting in mean recovery times of 14, 17, and 19 min for AuTS, AuTG, and AuTM, respectively. Since AuTS proved to be the best therapeutic agent in the G. mellonella model, it was subsequently tested in mice using a behavioral assessment (pole-climbing test). Mice given AuTS (50 mg/kg) 5 min prior to a 3200 (±500) ppm·min phosphine exposure exhibited behavior comparable to mice not exposed to phosphine. However, when mice were given a therapeutic dose of AuTS (50 mg/kg) 1 min after a similar phosphine exposure, only a very modest improvement in performance was observed.


Asunto(s)
Antídotos/farmacología , Mariposas Nocturnas/efectos de los fármacos , Compuestos Orgánicos de Oro/farmacología , Fosfinas/antagonistas & inhibidores , Animales , Larva/efectos de los fármacos , Masculino , Ratones , Mariposas Nocturnas/crecimiento & desarrollo , Fosfinas/toxicidad
9.
Artículo en Inglés | MEDLINE | ID: mdl-30802621

RESUMEN

Caterpillars of the greater wax moth, Galleria mellonella, are shown to be a useful invertebrate organism for examining mitochondrial toxicants (inhibitors of electron transport) and testing putative antidotes. Administration of sodium azide, sodium cyanide, or sodium (hydro)sulfide by intra-haemocoel injection (through a proleg) results in a dose-dependent paralysed state in the larvae lasting from <1 to ~40 min. The duration of paralysis is easily monitored, because if turned onto their backs, the larvae right themselves onto their prolegs once they are able to move again. The efficacy of putative antidotes to the three toxicants can routinely be assessed by observing shortened periods of paralysis with larvae given toxicant and antidote compared to larvae administered only the same dose of toxicant. The validity of the approach is demonstrated with agents previously shown to be antidotal towards cyanide intoxication in mice; namely, sodium nitrite and CoN4[11.3.1] (cobalt(II/III) 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]-heptadeca-1(7)2,11,13,15-pentaenyl cation). These same compounds are shown to be antidotal towards all three toxicants in the G. mellonella caterpillars; findings that may prove important in relation to azide and sulfide poisonings, for which there are currently no effective antidotes available. The observation that sodium nitrite ameliorates cyanide toxicity in the larvae is additionally interesting because it unambiguously demonstrates that the antidotal action of nitrites does not require the involvement of methemoglobin, contributing to the resolution of an ongoing controversy.


Asunto(s)
Azidas/toxicidad , Cianuros/toxicidad , Complejo IV de Transporte de Electrones/metabolismo , Mariposas Nocturnas/enzimología , Sulfuros/toxicidad , Animales , Complejo IV de Transporte de Electrones/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Larva/efectos de los fármacos , Larva/enzimología , Mariposas Nocturnas/efectos de los fármacos
10.
Chem Res Toxicol ; 31(4): 259-268, 2018 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-29512376

RESUMEN

Four cobalt-containing macrocyclic compounds previously shown to ameliorate cyanide toxicity have been comparatively evaluated with an acute sublethal toxicity model in conscious (unanesthetized) adult male Swiss-Webster mice. All of the compounds (the cobalt-corrins cobalamin and cobinamide, a cobalt-porphyrin, plus a cobalt-Schiff base macrocycle) given 5 min prior to the toxicant dose significantly decreased the righting-recovery time of cyanide-intoxicated mice, but the doses required for maximal antidotal effect varied. Additionally, all of the compounds tested significantly reduced the righting-recovery time when administered at either 1 or 2 min after cyanide intoxication, but none of the compounds tested significantly reduced the righting-recovery time when delivered 5 min after the toxicant dose. Using the lowest effective dose of each compound determined during the first (prophylactic) set of experiments, neuromuscular recovery following cyanide intoxication in the presence/absence of the cobalt-based antidotes was assessed by RotaRod testing. All the compounds tested accelerated recovery of neuromuscular coordination, and no persistent impairment in any group, including those animals that received toxicant and no antidote, was apparent up to 2 weeks postexposures. The relative effectiveness of the cobalt compounds as cyanide antidotes are discussed and rationalized on the basis of the cyanide-binding stoichiometries and stability constants of the Co(III) cyano adducts, together with consideration of the rate constants for axial ligand substitutions by cyanide in the Co(II) forms.


Asunto(s)
Antídotos/farmacología , Cobalto/farmacología , Cianuros/antagonistas & inhibidores , Depuradores de Radicales Libres/toxicidad , Compuestos Organometálicos/farmacología , Animales , Antídotos/química , Cobalto/química , Cianuros/toxicidad , Masculino , Ratones , Estructura Molecular , Compuestos Organometálicos/química
11.
Chem Res Toxicol ; 30(12): 2100-2109, 2017 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-29088535

RESUMEN

Bovine pulmonary artery endothelial cells (BPAEC) respond in a dose-dependent manner to millimolar (0-10) levels of sodium sulfide (NaHS). No measurable increase in caspase-3 activity and no change in the extent of autophagy (or mitophagy) were observed in BPAEC. However, lactate dehydrogenase levels increased in the BPAEC exposed NaHS, which indicated necrotic cell death. In the case of galactose-conditioned BPAEC, the toxicity of NaHS was increased by 30% compared to that observed in BPAEC maintained in the regular glucose-containing culture medium, which indicated a link between mitochondrial oxidative phosphorylation and the mechanism of toxicant action. This is consistent with the widely held view that cytochrome c oxidase (complex IV of the mitochondrial electron-transport system) is the principal molecular target involved in the acute toxicity of "sulfide" (H2S/HS-). In support of this view, elevated NO (which can reverse cytochrome c oxidase inhibition) ameliorated the toxicity of NaHS and, conversely, suppression of endogenous NO production exacerbated the observed toxicity. Respirometric measurements showed the BPAEC to possess a robust sulfide oxidizing system, which was able to out-compete cytochrome c oxidase for available H2S/HS- at micromolar concentrations. This detoxification system has previously been reported by other groups in several cell types, but notably, not neurons. The findings appear to provide some insight into the question of why human survivors of H2S inhalation frequently present at the clinic with respiratory insufficiency/pulmonary edema, while acutely poisoned laboratory animals tend to either succumb to cardiopulmonary paralysis or fully recover without any intervention.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Óxido Nítrico/metabolismo , Arteria Pulmonar/efectos de los fármacos , Sulfuros/toxicidad , Animales , Bovinos , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Infusiones Parenterales , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Relación Estructura-Actividad , Sulfuros/administración & dosificación
12.
Chem Res Toxicol ; 30(12): 2197-2208, 2017 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-29116760

RESUMEN

In aqueous media at neutral pH, the binding of two cyanide molecules per cobinamide can be described by two formation constants, Kf1 = 1.1 (±0.6) × 105 M-1 and Kf2 = 8.5 (±0.1) × 104 M-1, or an overall cyanide binding constant of ∼1 × 1010 M-2. In comparison, the cyanide binding constants for cobalamin and a fully oxidized form of cytochrome c oxidase, each binding a single cyanide anion, were found to be 7.9 (±0.5) × 104 M-1 and 1.6 (±0.2) × 107 M-1, respectively. An examination of the cyanide-binding properties of cobinamide at neutral pH by stopped-flow spectrophotometry revealed two kinetic phases, rapid and slow, with apparent second-order rate constants of 3.2 (±0.5) × 103 M-1 s-1 and 45 (±1) M-1 s-1, respectively. Under the same conditions, cobalamin exhibited a single slow cyanide-binding kinetic phase with a second-order rate constant of 35 (±1) M-1 s-1. All three of these processes are significantly slower than the rate at which cyanide is bound by complex IV during enzyme turnover (>106 M-1 s-1). Overall, it can be understood from these findings why cobinamide is a measurably better cyanide scavenger than cobalamin, but it is unclear how either cobalt corrin can be antidotal toward cyanide intoxication as neither compound, by itself, appears able to out-compete cytochrome c oxidase for available cyanide. Furthermore, it has also been possible to unequivocally show in head-to-head comparison assays that the enzyme does indeed have greater affinity for cyanide than both cobalamin and cobinamide. A plausible resolution of the paradox that both cobalamin and cobinamide clearly are antidotal toward cyanide intoxication, involving the endogenous auxiliary agent nitric oxide, is suggested. Additionally, the catalytic consumption of oxygen by the cobalt corrins is demonstrated and, in the case of cobinamide, the involvement of cytochrome c when present. Particularly in the case of cobinamide, these oxygen-dependent reactions could potentially lead to erroneous assessment of the ability of the cyanide scavenger to restore the activity of cyanide-inhibited cytochrome c oxidase.


Asunto(s)
Cobalto/metabolismo , Corrinoides/metabolismo , Cianuros/metabolismo , Cianuros/toxicidad , Complejo IV de Transporte de Electrones/metabolismo , Oxígeno/metabolismo , Cobalto/química , Corrinoides/química , Cianuros/química , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/química , Estructura Molecular , Oxígeno/química
14.
Chem Res Toxicol ; 29(6): 1011-9, 2016 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-27104767

RESUMEN

The complex of cobalt(II) with the ligand 2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaene (CoN4[11.3.1]) has been shown to bind two molecules of cyanide in a cooperative fashion with an association constant of 2.7 (±0.2) × 10(5). In vivo, irrespective of whether it is initially administered as the Co(II) or Co(III) cation, EPR spectroscopic measurements on blood samples show that at physiological levels of reductant (principally ascorbate) CoN4[11.3.1] becomes quantitatively reduced to the Co(II) form. However, following addition of sodium cyanide, a dicyano Co(III) species is formed, both in blood and in buffered aqueous solution at neutral pH. In keeping with other cobalt-containing cyanide-scavenging macrocycles like cobinamide and cobalt(III) meso-tetra(4-N-methylpyridyl)porphine, we found that CoN4[11.3.1] exhibits rapid oxygen turnover in the presence of the physiological reductant ascorbate. This behavior could potentially render CoN4[11.3.1] cytotoxic and/or interfere with evaluations of the antidotal capability of the complex toward cyanide through respirometric measurements, particularly since cyanide rapidly inhibits this process, adding further complexity. A sublethal mouse model was used to assess the effectiveness of CoN4[11.3.1] as a potential cyanide antidote. The administration of CoN4[11.3.1] prophylactically to sodium cyanide-intoxicated mice resulted in the time required for the surviving animals to recover from "knockdown" (unconsciousness) being significantly decreased (3 ± 2 min) compared to that of the controls (22 ± 5 min). All observations are consistent with the demonstrated antidotal activity of CoN4[11.3.1] operating through a cyanide-scavenging mechanism, which is associated with a Co(II) → Co(III) oxidation of the cation. To test for postintoxication neuromuscular sequelae, the ability of mice to remain in position on a rotating cylinder (RotaRod test) was assessed during and after recovery. While intoxicated animals given CoN4[11.3.1] did recover ∼30 min more quickly than controls given only toxicant, there were no indications of longer-term problems in either group, as determined by continuing the RotaRod testing up to 24 h after the intoxications and routine behavioral observations for a further week.


Asunto(s)
Antídotos/farmacología , Cobalto/farmacología , Corrinoides/economía , Corrinoides/farmacología , Cianuros/antagonistas & inhibidores , Compuestos Macrocíclicos/economía , Compuestos Macrocíclicos/farmacología , Animales , Antídotos/química , Antídotos/economía , Cobalto/química , Cobalto/economía , Corrinoides/química , Cianuros/química , Cianuros/toxicidad , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Masculino , Ratones , Bases de Schiff/síntesis química , Bases de Schiff/química , Bases de Schiff/economía , Bases de Schiff/farmacología
15.
Chem Res Toxicol ; 29(3): 270-8, 2016 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-26692323

RESUMEN

The Co(III)-containing water-soluble metalloporphyrin cobalt(III) meso-tetra(4-N-methylpyridyl)porphine pentaiodide (Co(III)TMPyP) is a potential cyanide-scavenging agent. The rate of reduction of Co(III)TMPyP by ascorbate is facile enough that conversion to the Co(II)-containing Co(II)TMPyP should occur within minutes at prevailing in vivo levels of the reductant. It follows that any cyanide-decorporating capability of the metalloporphyrin should depend more on the cyanide-binding characteristics of Co(II)TMPyP than those of the administered form, Co(III)TMPyP. Addition of cyanide to buffered aqueous solutions of Co(II)TMPyP (pH 7.4, 25-37 °C) results in quite rapid (k2 = ∼10(3) M(-1) s(-1)) binding/substitution of cyanide anion in the two available axial positions with high affinity (K'ß = 10(10) to 10(11)). Electron paramagnetic resonance spectroscopic measurements and cyclic voltammetry indicate that cyanide induces oxidation to the Co(III)-containing dicyano species. The constraints that these observations put on plausible mechanisms for the reaction of Co(II)TMPyP with cyanide are discussed. Experiments in which Co(III)TMPyP and cyanide were added to freshly drawn mouse blood showed the same sequence of reactions (metalloporphyrin reduction → cyanide binding/substitution → reoxidation) to occur. Therefore, in cyanide-scavenging applications with this metalloporphyrin, we should be taking advantage of both the improved rate of ligand substitution at Co(II) compared to that at Co(III) and the increased affinity of Co(III) for anionic ligands compared to that of Co(II). Finally, using an established sublethal mouse model for cyanide intoxication, Co(III)TMPyP, administered either 5 min before (prophylaxis) or 1 min after the toxicant, is shown to have very significant antidotal capability. Possible explanations for the results of a previous contradictory study, which failed to find any prophylactic effect of Co(III)TMPyP toward cyanide intoxication, are considered.


Asunto(s)
Ácido Ascórbico/química , Cobalto/química , Cianuros/química , Depuradores de Radicales Libres/química , Metaloporfirinas/química , Compuestos Organometálicos/química , Animales , Sitios de Unión , Cobalto/sangre , Técnicas Electroquímicas , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Masculino , Metaloporfirinas/sangre , Ratones , Compuestos Organometálicos/sangre , Oxidación-Reducción
16.
Chem Res Toxicol ; 28(7): 1398-408, 2015 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-25951111

RESUMEN

There are currently no FDA-approved antidotes for H2S/sulfide intoxication. Sodium nitrite, if given prophylactically to Swiss Webster mice, was shown to be highly protective against the acute toxic effects of sodium hydrosulfide (∼LD40 dose) with both agents administered by intraperitoneal injections. However, sodium nitrite administered after the toxicant dose did not detectably ameliorate sulfide toxicity in this fast-delivery, single-shot experimental paradigm. Nitrite anion was shown to rapidly produce NO in the bloodstream, as judged by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin, together amounting to less than 5% of the total hemoglobin present. Sulfide-intoxicated mice were neither helped by the supplemental administration of 100% oxygen nor were there any detrimental effects. Compared to cyanide-intoxicated mice, animals surviving sulfide intoxication exhibited very short knockdown times (if any) and full recovery was extremely fast (∼15 min) irrespective of whether sodium nitrite was administered. Behavioral experiments testing the ability of mice to maintain balance on a rotating cylinder showed no motor impairment up to 24 h post sulfide exposure. It is argued that antagonism of sulfide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity of nitrite rather than formation of methemoglobin.


Asunto(s)
Antídotos/farmacología , Metahemoglobinemia/patología , Nitrito de Sodio/farmacología , Sulfuros/toxicidad , Animales , Aniones/química , Bovinos , Línea Celular , Cianuros/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Inyecciones Intraperitoneales , Masculino , Metahemoglobina/química , Metahemoglobina/metabolismo , Metahemoglobinemia/metabolismo , Ratones , Actividad Motora/efectos de los fármacos , Músculo Esquelético/fisiología , Miocardio/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico/metabolismo
17.
Biochim Biophys Acta ; 1834(9): 1894-900, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23624264

RESUMEN

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/ß-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.


Asunto(s)
Hemoglobina Glucada/metabolismo , Hemo/metabolismo , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Oxihemoglobinas/metabolismo , 2,3-Difosfoglicerato/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hemoglobina Glucada/química , Humanos , Óxido Nítrico/química , Oxidación-Reducción , Ácido Fítico/metabolismo , Unión Proteica
18.
Chem Res Toxicol ; 26(5): 828-36, 2013 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-23536974

RESUMEN

Isoamyl nitrite has previously been considered acceptable as an inhaled cyanide antidote; therefore, the antidotal utility of this organic nitrite compared with sodium nitrite was investigated. To facilitate a quantitative comparison, doses of both sodium nitrite and isoamyl nitrite were given intraperitoneally in equimolar amounts to sublethally cyanide-challenged mice. Righting recovery from the knockdown state was clearly compromised in the isoamyl nitrite-treated animals, the effect being attributable to the toxicity of the isoamyl alchol produced during hydrolysis of the isoamyl nitrite to release nitrite anion. Subsequently, inhaled aqueous sodium nitrite aerosol was demonstrated to ameliorate sublethal cyanide toxicity, when provided to mice after the toxic dose, by the more rapid recovery of righting ability compared to that of the control animals given only the toxicant. Aerosolized sodium nitrite has thus been shown by these experiments to have promise as a better alternative to organic nitrites for development as an inhaled cyanide antidote. The inhaled sodium nitrite led to the production of NO in the bloodstream as determined by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. The aerosol delivery was performed in an unmetered inhalation chamber, and in this study, no attempt was made to optimize the procedure. It is argued that administration of an effective inhaled aqueous sodium nitrite dose in humans is possible, though just beyond the capability of current individual metered-dose inhaler designs, such as those used for asthma. Finally, working at slightly greater than LD50 NaCN doses, it was fortuitously discovered that (i) anesthesia leads to significantly prolonged survival compared to that of unanesthetized animals and that (ii) the antidotal activity of nitrite anion was completely abolished under anesthesia. Plausible explanations for these effects in mice and their practical consequences in relation to testing putative cyanide antidotes are discussed.


Asunto(s)
Nitrito de Amila/análogos & derivados , Anestésicos/farmacología , Antídotos/farmacología , Cianuros/antagonistas & inhibidores , Cianuros/envenenamiento , Nitrito de Sodio/farmacología , Nitrito de Amila/farmacología , Nitrito de Amila/uso terapéutico , Animales , Espectroscopía de Resonancia por Spin del Electrón , Masculino , Ratones , Nitrito de Sodio/administración & dosificación , Nitrito de Sodio/uso terapéutico
19.
Am J Physiol Lung Cell Mol Physiol ; 304(5): L350-60, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23275622

RESUMEN

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.


Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Metalotioneína/metabolismo , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Animales , Femenino , Hiperoxia , Inflamación/inmunología , Metalotioneína/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/análisis , Mucosa Respiratoria/metabolismo
20.
Chem Res Toxicol ; 25(12): 2678-86, 2012 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-23148604

RESUMEN

The formation of Co(III)TMPyP(CN)(2) at pH 7.4 has been shown to be completely cooperative (α(H) = 2) with an association constant of 2.1 (±0.2) × 10(11). The kinetics were investigated by stopped-flow spectrophotometry and revealed a complicated net reaction exhibiting 4 phases at pH 7.4 under conditions where cyanide was in excess. The data suggest molecular HCN (rather than CN(-)) to be the attacking nucleophile around neutrality. The two slower phases do not seem to be present when cyanide is not in excess, and the other two phases have rates comparable to that observed for cobalamin, a known effective cyanide scavenger. Addition of bovine serum albumin (BSA) did not affect the cooperativity of cyanide binding to Co(III)TMPyP, only lowered the equilibrium constant slightly to 1.2 (±0.2) × 10(11) and had an insignificant effect on the observed rate. A sublethal mouse model was used to assess the effectiveness of Co(III)TMPyP as a potential cyanide antidote. The administration of Co(III)TMPyP to sodium cyanide intoxicated mice resulted in the time required for the surviving mice to right themselves from a supine position being significantly decreased (9 ± 2 min) compared to that of the controls (33 ± 2 min). All observations were consistent with the demonstrated antidotal activity of Co(III)TMPyP operating through a cyanide-binding (i.e., scavenging) mechanism.


Asunto(s)
Antídotos/farmacología , Cianuros/toxicidad , Metaloporfirinas/farmacología , Animales , Antídotos/química , Cianuros/química , Masculino , Metaloporfirinas/química , Ratones
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