Neurotoxicity of nitroxyl: Insights into HNO and NO biochemical imbalance

doi:10.1016/j.freeradbiomed.2005.07.007

Free Radical Biology and Medicine

Volume 39, Issue 11, 1 December 2005, Pages 1478-1488

https://doi.org/10.1016/j.freeradbiomed.2005.07.007 Get rights and content

Abstract

Nitroxyl anion (NO⁻), and/or its conjugate acid, HNO, may be formed in the cellular milieu by several routes under both physiological and pathophysiological conditions. Since experimental evidence suggests that certain reactive nitrogen oxide species can contribute significantly to cerebral ischemic injury, we investigated the neurotoxic potential of HNO/NO⁻ using Angeli's salt (AS), a spontaneous HNO/NO⁻-generating compound. Exposure to AS resulted in a time- and concentration-dependent increase in neural cell death that progressed markedly following the initial exposure. Coadministration of the donor with Tempol (1 mM), a one-electron oxidant that converts NO⁻ to NO, prevented its toxic effect, as did the concomitant addition of Fe(III)TPPS. Media containing various chelators, catalase, Cu/Zn superoxide dismutase, or carboxy-PTIO did not ameliorate AS-mediated neurotoxicity, ruling out the involvement of transition metal complexes, H₂O₂, O₂⁻, and NO, respectively. A concentration-dependent increase in supernatant protein 3-nitrotyrosine immunoreactivity was observed when cultures were exposed to AS under aerobic conditions, an effect lost in the absence of oxygen. A bell-shaped curve for augmented AS-mediated nitration was observed with increasing Fe(III)TPPS concentration, which contrasted with its linear effect on abating cytotoxicity. Finally, addition of glutamate receptor antagonists, MK-801 (10 μM) and CNQX (30 μM) to the cultures abrogated toxicity when given during, but not following, AS exposure; as did pretreatment with the exocytosis inhibitor, tetanus toxin (300 ng/ml). Taken together, our data suggest that under aerobic conditions, AS toxicity is initiated via HNO/NO⁻ but progresses via secondary excitotoxicity.

Introduction

Reactive nitrogen species have been determined to contribute to the pathogenesis of numerous pathological conditions including the evolution of cerebral ischemic brain damage. Overactivation of the Ca²⁺-dependent, constitutive, neuronal isoform of nitric oxide synthase (nNOS or NOS-1) contributes, in part, to the acute phase of tissue injury [1], [2], while the inducible isoform of NOS (iNOS or NOS-2) is involved in postcerebral ischemic injury progression [3], [4], [5], [6]. Although the free radical species (NO) is the best-known product of NOS, reports have suggested that nitroxyl (HNO/NO⁻) may be the principle intermediate in the oxidation of arginine by NOS [7], [8] and it is then converted to NO by suitable biological electron acceptors [7], [8], [9]. Thus, the form of NO that predominates may vary depending on cellular conditions. To wit, evidence suggests that HNO/NO⁻ formation could be favored over NO production under ischemic conditions. For instance, formation of HNO/NO⁻ is favored under conditions of decoupled enzymatic activity [10], [11], [12], [13]. Studies suggest nitrosothiols may generate HNO/NO⁻ via reaction with thiols [14] and hetereolytic decomposition under acidic conditions [15] and intracellular acidosis ensues following cerebral ischemia [16]. In addition, the reduction of NO to NO⁻ can be carried out by biological reducing agents. Such a conversion can be supported by cytochrome c [17], the release of which is also increased following cerebral ischemia [18], [19]. Pertinently, HNO/NO⁻ derived from Angeli's salt (3 μmol/kg) was shown to increase myocardial reperfusion injury in rabbits, while administration of an NO-generating compound prior to reperfusion was cardioprotective [20]. Coadministration of AS with ferricyanide, a one-electron oxidant that converts NO⁻ to NO, completely blocked the injurious effects of AS and exerted significant cardioprotective effects [20]. In addition, cerebrovascular infusion of Angeli's salt (AS, 1.7 mmol/min) to rats causes a significant increase in blood brain barrier permeability [21]. Thus, it seems plausible that the beneficial effect of NOS inhibition on myocardial and, by inference, cerebral ischemic injury could be due to the prevention of HNO/NO⁻ formation. Given this, we investigated the neurotoxic potential of HNO/NO⁻ using AS as a synthetic source.

Section snippets

Materials

Angeli's salt was either purchased from Alexis (San Diego, CA) or was a kind gift from Prof. Jon Fukuto (UCLA). Tempol-H was synthesized at the NCI (Bethesda, MD). Superoxide dismutase (bovine erythrocytes), catalase (Aspergillus niger), FeTPPS [5,10,15,20-tetrakis (4-sulfonatophenyl) prophyrinato ferric chloride], tetanus toxin (Clostridium tetani), and desferroxiamine mesylate were obtained from Calbiochem (San Diego, CA). MK-801 (dizocilpine maleate) and CNQX

Results

When murine mixed cortical cell cultures were exposed to AS, both a time- (Fig. 1A) and concentration–dependent (Fig. 1B) increase in neuronal cell death was observed. Loss of LDH (early cell death) occurred as early as 4 min following addition of AS (5 mM) with a significant increase evident after 1 h (Fig. 1A, black bars), a time at which the donor has been expended. Cells were washed free of experimental solutions at the times indicated and placed back into the 37°C incubator and LDH

Discussion

Several articles address the cytotoxic potential of AS [20], [45], [46], [47], [48], the most commonly used synthetic donor for the experimental study of HNO/NO⁻ under biological conditions [21], [25], [47], [48], [49], [50]. Using an in vitro preparation of plasmid DNA as well as calf thymus DNA, Ohshima's group found that HNO/NO⁻ generated from AS (100–500 μM) under aerobic conditions resulted in DNA strand breakage and base oxidation [47], a process enhanced by the presence of H₂O₂ [46].

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NO is a potent biological mediator regulating vascular tone, platelet function, leukocyte adhesion, and extravasations of leukocytes, but at high levels it plays critical roles in modulating inflammatory and degenerative diseases (12–15). HNO is an unstable weak acid (pKa = 11.4) (16,17), which readily dimerizes to yield N2O (16), and like NO displays both prooxidative and antioxidative effects (18–25). Hence, the ability of HXs to serve as HNO-donors and/or NO-donors under oxidizing environments is involved in both the beneficial and injurious activities.
Hydroxamic acids (RC(O)NHOH, HXs) display interesting chemical and biological properties. The pharmacological effects of HXs have been partially attributed to their ability to serve as HNO- or NO-donors under oxidative stress. We have demonstrated that one-electron oxidation of HXs by radiolytically borne radicals yields the respective transient nitroxide radicals, which decompose bimolecularly forming HNO. HXs oxidation by the metmyoglobin and H₂O₂ reactions system involves relatively milder oxidants such as ferryl myoglobin, which oxidize not only HXs but also the nitroxide radical, HNO and NO. The distinction between the effects of NO and HNO in vivo is masked by the reversible redox exchange between these two congeners and due to the Janus-faced behavior of NO and HNO. Acetohydroxamic acid potentiates H₂O₂-induced killing of Escherichia coli, whereas suberoylanilide hydroxamic acid (SAHA) protects mammalian cells subjected to oxidative stress. These results are similar to those observed for NO implying that in these systems HXs serve as NO-donors. In contrast, the effect of acetohydroxamic acid on Bacillus subtilis subjected to oxidative stress is similar to that of HNO. SAHA, but not valproic acid lacking the hydroxamate moiety, enhances the radiosensitization of hypoxic tumor cells in vitro. Given that NO, but not HNO, is a hypoxic cell radiosensitizer, the enhancement of tumor radioresponse by SAHA might involve its ability to serve as a NO-donor. It is concluded that HXs form HNO under oxidative stress, but can be considered as NO-donors if HNO oxidation competes with N₂O formation and with its reaction with potential biological targets.
Nitroxyl (HNO) reacts with molecular oxygen and forms peroxynitrite at physiological ph: Biological implications
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Additional research is needed to increase our understanding of the chemical biology and reactivity of HNO (11, 12). Several studies examining HNO in vitro demonstrated that Angeli's salt (HNO donor) exerts marked cytotoxicity that is oxygen-dependent (13–16). The endogenous production of HNO in mammalian systems has not been unambiguously shown to occur; however, there is considerable in vitro evidence for the intermediacy of this species from HNO donors.
Nitroxyl (HNO), the protonated one-electron reduction product of NO, remains an enigmatic reactive nitrogen species. Its chemical reactivity and biological activity are still not completely understood. HNO donors show biological effects different from NO donors. Although HNO reactivity with molecular oxygen is described in the literature, the product of this reaction has not yet been unambiguously identified. Here we report that the decomposition of HNO donors under aerobic conditions in aqueous solutions at physiological pH leads to the formation of peroxynitrite (ONOO⁻) as a major intermediate. We have specifically detected and quantified ONOO⁻ with the aid of boronate probes, e.g. coumarin-7-boronic acid or 4-boronobenzyl derivative of fluorescein methyl ester. In addition to the major phenolic products, peroxynitrite-specific minor products of oxidation of boronate probes were detected under these conditions. Using the competition kinetics method and a set of HNO scavengers, the value of the second order rate constant of the HNO reaction with oxygen (k = 1.8 × 10⁴ m⁻¹ s⁻¹) was determined. The rate constant (k = 2 × 10⁴ m⁻¹ s⁻¹) was also determined using kinetic simulations. The kinetic parameters of the reactions of HNO with selected thiols, including cysteine, dithiothreitol, N-acetylcysteine, captopril, bovine and human serum albumins, and hydrogen sulfide, are reported. Biological and cardiovascular implications of nitroxyl reactions are discussed.
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Suberoylanilide hydroxamic acid radiosensitizes tumor hypoxic cells in vitro through the oxidation of nitroxyl to nitric oxide
2014, Free Radical Biology and Medicine
The pharmacological effects of hydroxamic acids are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Previously, it was concluded that oxidation of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) by the metmyoglobin/H₂O₂ reaction system releases NO, which was based on spin trapping of NO and accumulation of nitrite. Reinvestigation of this system demonstrates the accumulation of N₂O, which is a marker of HNO formation, at similar rates under normoxia and anoxia. In addition, the yields of nitrite that accumulated in the absence and the presence of O₂ did not differ, implying that the source of nitrite is other than autoxidation of NO. In this system metmyoglobin is instantaneously and continuously converted into compound II, leading to one-electron oxidation of SAHA to its respective transient nitroxide radical. Studies using pulse radiolysis show that one-electron oxidation of SAHA (pK_a=9.56±0.04) yields the respective nitroxide radical (pK_a=9.1±0.2), which under all experimental conditions decomposes bimolecularly to yield HNO. The proposed mechanism suggests that compound I oxidizes SAHA to the respective nitroxide radical, which decomposes bimolecularly in competition with its oxidation by compound II to form HNO. Compound II also oxidizes HNO to NO and NO to nitrite. Given that NO, but not HNO, is an efficient hypoxic cell radiosensitizer, we hypothesized that under an oxidizing environment SAHA might act as a NO donor and radiosensitize hypoxic cells. Preincubation of A549 and HT29 cells with 2.5 μM SAHA for 24 h resulted in a sensitizer enhancement ratio at 0.01 survival levels (SER_0.01) of 1.33 and 1.59, respectively. Preincubation of A549 cells with oxidized SAHA had hardly any effect and, with 2 mM valproic acid, which lacks the hydroxamate group, resulted in SER_0.01=1.17. Preincubation of HT29 cells with SAHA and Tempol, which readily oxidizes HNO to NO, enhanced the radiosensitizing effect of SAHA. Pretreatment with SAHA blocked A549 cells at the G1 stage of the cell cycle and upregulated γ-H2AX after irradiation. Overall, we conclude that SAHA enhances tumor radioresponse by multiple mechanisms that might also involve its ability to serve as a NO donor under oxidizing environments.

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Original ContributionNeurotoxicity of nitroxyl: Insights into HNO and NO biochemical imbalance

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Biochem. Pharmacol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Cell

Biochim. Biophys. Acta

Neuron

Free Radic. Biol. Med.

Exp. Neurol.

J. Biol. Chem.

J. Neurosci. Methods

J. Biol. Chem.

Arch. Biochem. Biophys.

Nitric Oxide

J. Biol. Chem.

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

Free Radic. Biol. Med.

J. Biol. Chem.

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

Neuron

Neuron

Neuroscience

Neuron

Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase

Science

Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage

J. Neurosci.

Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage

Am. J. Physiol.

Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene

J. Neurosci.

Antisense oligodeoxynucleotide to inducible nitric oxide synthase protects against transient focal cerebral ischemia-induced brain injury [In Process Citation]

J. Cereb. Blood Flow Metab.

Inducible nitric oxide synthase expression in human cerebral infarcts

Acta Neuropathol. (Berl.)

Formation of free nitric oxide from L-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase

Proc. Natl. Acad. Sci. USA

No.NO from NO synthase

Proc. Natl. Acad. Sci. USA

Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase

Proc. Natl. Acad. Sci. USA

Original Contribution
Neurotoxicity of nitroxyl: Insights into HNO and NO biochemical imbalance