Peroxynitrite-mediated Oxidations Research Articles

Payload drug vs. nanocarrier biodegradation by myeloperoxidase- and peroxynitrite-mediated oxidations: pharmacokinetic implications.

With the advancement of nanocarriers for drug delivery into biomedical practice, assessments of drug susceptibility to oxidative degradation by enzymatic mechanisms of inflammatory cells become important. Here, we investigate oxidative degradation of a carbon nanotube-based drug carrier loaded with Doxorubicin. We employed myeloperoxidase-catalysed and peroxynitrite-mediated oxidative conditions to mimic the respiratory burst of neutrophils and macrophages, respectively. In addition, we revealed that the cytostatic and cytotoxic effects of free Doxorubicin, but not nanotube-carried drug, on melanoma and lung carcinoma cell lines were abolished in the presence of tumor-activated myeloid regulatory cells that create unique myeloperoxidase- and peroxynitrite-induced oxidative conditions. Both ex vivo and in vitro studies demonstrate that the nanocarrier protects the drug against oxidative biodegradation.

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: Modeling the impact of superoxide dismutase and nitric oxide diffusion

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO −) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O 2 − ) and nitric oxide ( NO), are formed at ratios (O 2 − / NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of NO preclude accumulation of O 2 − and NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation ( J NO 2 − Y ) during exposure to simultaneous fluxes of superoxide ( J O 2 − ) and nitric oxide ( J NO ), varying the radical flux ratios ( J O 2 − / J NO ), in the presence of carbon dioxide. With a basic set of reactions, J NO 2 − Y as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J NO 2 − Y as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O 2 − or NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3−) and nitrogen dioxide (NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3− and NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 × 109 and 7.6 × 106 M−1 s−1, respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 × 106 M−1 s−1. However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3− and NO2 radicals.

Kinetic and Mechanistic Studies of the Peroxynitrite-Mediated Oxidation of Oxymyoglobin and Oxyhemoglobin

Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO(2)) and oxyhemoglobin (HbFeO(2)) showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps. For myoglobin, we found that the small amount of deoxymyoglobin (MbFe(II)) which is in equilibrium with MbFeO(2) is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFe(IV)=O). Then, in the second step, MbFe(IV)=O is reduced by peroxynitrous acid to metmyoglobin (metMb). The second-order rate constant values obtained at pH 7.3 and 20 degrees C for the two steps are (5.4 +/- 0.2) x 10(4) and (2.2 +/- 0.1) x 10(4) M(-)(1) s(-)(1), respectively. Analogous studies with hemoglobin suggest that its reaction with peroxynitrite follows the same mechanism. In this case, the second-order rate constant values measured at pH 7.0 and 20 degrees C for the two steps are (8.8 +/- 0.4) x 10(4) and (9.4 +/- 0.7) x 10(4) M(-)(1) s(-)(1), respectively. A possible mechanism in the absence as well as in the presence of CO(2) and the relevance of these reactions in vivo are discussed.

Glutathione Peroxidase Protects against Peroxynitrite-mediated Oxidations: A NEW FUNCTION FOR SELENOPROTEINS AS PEROXYNITRITE REDUCTASE

There is a requirement for cellular defense against excessive peroxynitrite generation to protect against DNA strand breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. Here, we demonstrate a role of selenium-containing enzymes catalyzing peroxynitrite reduction using glutathione peroxidase (GPx) as an example. GPx protected against the oxidation of dihydrorhodamine 123 by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 x 10(6) M-1 s-1. Carboxymethylation of selenocysteine in GPx by iodoacetate led to the loss of "classical" glutathione peroxidase activity but maintained protection against peroxynitrite-mediated oxidation. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. When peroxynitrite was infused to maintain a 0.2 microM steady-state concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause the loss of classical GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx or by selenomethionine. The results demonstrate a novel function of GPx and potentially of other selenoproteins containing selenocysteine or selenomethionine, in the GSH-dependent maintenance of a defense line against peroxynitrite-mediated oxidations, as a peroxynitrite reductase.

Inhibition of Peroxynitrite-Mediated Oxidation of Glutathione by Carbon Dioxide

Peroxynitrite reacts with CO 2to form an adduct containing a weak O—O bond that can undergo homolytic and/or heterolytic cleavage to give other reactive intermediates. Because the peroxynitrite/CO 2reaction is fast and physiological concentrations of CO 2are relatively high, peroxynitrite-mediated oxidations of biological species probably involve the peroxynitrite–CO 2adduct and its subsequent reactive intermediates. We have examined the reaction of glutathione with peroxynitrite in the presence and absence of added bicarbonate. In the presence of added bicarbonate, CO 2competes with glutathione for peroxynitrite, resulting in a markedly decreased consumption of glutathione compared with that observed in the absence of added bicarbonate. However, the consumption of glutathione still is much higher than predicted from the assumption that the glutathione–peroxynitrite reaction is the only reaction that can consume glutathione in this system. These results suggest that glutathione partially, but not completely, traps intermediate(s) derived from the peroxynitrite and CO 2reaction. Some rate constants for the trapping of the intermediates are estimated by simulating the reactions, and possible mechanisms for the reaction of peroxynitrite with glutathione in the presence of added bicarbonate are discussed.

Peroxynitrite Reaction with Carbon Dioxide/Bicarbonate: Kinetics and Influence on Peroxynitrite-Mediated Oxidations

Peroxynitrite is a strong oxidant producedin vivoas the reaction product of superoxide anion and nitric oxide (k∼ 5 × 109m−1s−1) and can be formed and mediate reactions in the extracellular environment. It has recently been reported that peroxynitrite and carbon dioxide react in a second-order process (S. V. Lymar and K. Hurst (1995)J. Am. Chem. Soc.117, 8867–8868). Since one of the most abundant constituents of the extracellular milieu is bicarbonate anion (25 mmin plasma) which is in equilibrium with carbon dioxide (1.3 mmin plasma) we have further studied the kinetics of the reaction between peroxynitrite and carbon dioxide/bicarbonate and the effect of bicarbonate on different peroxynitrite-mediated oxidations. The apparent second-order rate constant for the reaction is (2.3 ± 0.1) × 103m−1s−1at 37°C and pH 7.4 and a pH-independent second-order rate constant of (5.8 ± 0.2) × 104m−1s−1at 37°C was obtained considering peroxynitrite anion and carbon dioxide as the reacting species. The enthalpy and entropy of activation are ΔH#= +10.7 ± 0.8 kcal mol−1and ΔS#= −6.5 ± 0.5 cal mol−1K−1, respectively. The presence of bicarbonate had variable influence on peroxynitrite-mediated oxidations. While bicarbonate significantly enhanced peroxynitrite-mediated nitration of aromatics, it partially inhibited the oxidation of thiols, dimethylsulfoxide, oxyhemoglobin, and cytochromec+2and totally inhibited the hydroxylation of benzoate. Spontaneous chemiluminescence studies suggest the formation of bicarbonate radicals during the interactions of peroxynitrite with carbon dioxide/bicarbonate. Our results support that peroxynitrite anion rapidly reacts with carbon dioxide to yield an adduct (ONOOCO−2) which can participate in oxidation and nitration processes, thus redirecting the primary reactivity of peroxynitrite.

32] Detection of secondary radicals from peroxynitrite-mediated oxidations by electron spin resonance

This chapter describes the simple electron spin resonance (ESR) techniques to detect and identify secondary radicals formed from peroxynitrite and mediated oxidations by ESR. The possibility of free radical formation from biomolecules makes the use of ESR an important approach to the understanding of peroxynitrite toxicity. The ESR spin-trapping technique is a valuable tool for studying highly transient free radicals, particularly oxygen-centered radicals that are impossible to detect by direct ESR in aqueous solutions at room temperature because of their short lifetimes and broad linewidths. The use of standard ESR and ESR spin-trapping techniques has provided direct evidence for the formation of free radical intermediates during peroxynitrite-mediated oxidation of ascorbate, desferrioxamine, 5,5-dimethylpyrroline N-oxide (DMPO), ethanol, formate, DMSO, and low and high molecular weight thiols. The chapter reviews the research study on the peroxynitrite-mediated one-electron oxidation of biomolecules that is an important event in its cytotoxic mechanism. Stationary ESR precludes direct kinetic analysis of rapid processes such as peroxynitrite-mediated oxidations however requires minimum quantities of reagents and can provide mechanistic clues.

Competitive reactions of peroxynitrite with 2′-deoxyguanosine and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG): Relevance to the formation of 8-oxodG in DNA exposed to peroxynitrite

We have examined the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) in reactions of peroxynitrite with 2′-deoxyguanosine (dG) and calf-thymus DNA. Peroxynitrite reacts with dG at neutral pH, but this reaction does not result in the buildup of 8-oxodG. We also do not find any evidence for the formation of 8-oxodG in calf-thymus DNA upon exposure to peroxynitrite. When 8-oxodG is mixed with 1000-fold excess dG and then allowed to react with peroxynitrite, about 50% of the 8-oxodG is destroyed. The preferential reaction of 8-oxodG is also evident when dG in calf-thymus DNA is partially oxidized in an Udenfriend system and then allowed to react with peroxynitrite. We suggest that 8-oxodG is not produced in peroxynitrite-mediated oxidations of dG and DNA or that it is produced but then is rapidly consumed in further reactions with peroxynitrite. Oxidized DNA bases frequently can be more oxidation sensitive than their corresponding progenitors and, therefore, may be present at low steady-state concentrations and not represent stable markers of oxidative stress status. The importance of the 8-oxodG/peroxynitrite reaction is discussed in relation to the formation of more stable, secondary oxidation products that might be more useful markers of DNA damage.