Spin trapping of glutathiyl and protein radicals produced from nitric oxide-derived oxidants

Despite the importance of protein radicals in cell homeostasis and cell injury, their formation, localization, and propagation reactions remain obscure, mainly because of the difficulties in detecting and characterizing radicals, in general, and protein radicals, in particular. New approaches based on spin trapping coupled with other methodologies are under development/testing but so far they have been applied mainly to the study of protein-tyrosyl and protein-tryptophanyl radicals. Here, our aim is to emphasize the importance of developing new methodologies for the detection of glutathyil and protein-cysteinyl radicals under physiological conditions. To this end, we summarize current EPR evidence supporting the view that glutathione and protein-cysteines are among the preferential targets of nitric oxide-derived oxidants and that they are oxidized to the glutathiyl and protein-cysteinyl radicals, respectively. The possible intermediacy of these species in the biological formation of mediators of protein-cysteine redox signaling, such as S-nitrosothiols and sulfenic acids, is also discussed.     

The generation of free radicals by nitric oxide synthase

Nitric oxide synthase (NOS) is an example of a family of heme-containing monooxygenases that, under the restricted control of a specific substrate, can generate free radicals. While the generation of nitric oxide (NO*) depends solely on the binding of L-arginine, NOS produces superoxide (O(2)*(-)) and hydrogen peroxide (H(2)O(2)) when the concentration of the substrate is low. Not surprisingly, effort has been put forth to understand the pathway by which NOS generates NO*, O(2)*(-) and H(2)O(2), including the role of substrate binding in determining the pathways by which free radicals are generated. By binding within the distal heme pocket near the sixth coordination position of the NOS heme iron, L-arginine alters the coordination properties of the heme iron that promotes formation of the perferryl complex NOS-[Fe(5+)=O](3+). This reactive iron intermediate has been shown to abstract a hydrogen atom from a carbon alpha to a heteroatom and generate carbon-centered free radicals. The ability of NOS to produce free radicals during enzymic cycling demonstrates that NOS-[Fe(5+)=O](3+) behaves like an analogous iron-oxo complex of cytochrome P-450 during aliphatic hydroxylation. The present review discusses the mechanism(s) by which NOS generates secondary free radicals that may initiate pathological events, along with the cell signaling properties of NO*, O(2)*(-) and H(2)O(2).     

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis.

Prostaglandins and NO. are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO. biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO. and PGHS, but this is not universally observed. Clarification of the role of NO. in PGHS catalysis has been attempted by examining NO. interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO. has not yet emerged. Available studies suggest that NO. may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2.-+NO.) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2.- from RAW 267.4 cells that produce NO. and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.     

Formation of nitric oxide from nitrous acid in ischemic tissue and skin.

Nitric oxide (NO) can form from nitrous acid under conditions of low pH and formation of the gas N2O3 is the rate-determining step. Published data allow us to calculate the rate at which NO forms from nitrite in a closed system such as circulating blood plasma. Because of the bimolecular reactions involved, and the very low concentration of nitrite, the rate of formation of NO is very slow. It might take at least 12 days, when the pH of nitrite solution is lowered, for the concentration of NO to reach a level sufficiently high to activate guanylyl cyclase and so it seems unlikely that naturally circulating nitrite is involved in vasodilation in ischemic tissue through its conversion into NO. It is more realistic to consider that NO is produced at biologically significant concentrations from nitrite in perspiration on the skin.     

The roles played by crucial free radicals like lipid free radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)-induced acute liver injury of mice

Mice were administered a single dose of carbon tetrachloride (CCl(4)) to induce acute liver injury. We found that lactate dehydrogenase (LDH) and glutamic pyruvic transaminase (GPT) levels in serum, as well as the level of thiobarbituric acid reaction substances (TBARS) in liver homogenate increased significantly in a manner both dose dependent and time dependent after CCl(4) administration. Such results suggest that the liver is susceptible to CCl(4) treatment and that lipid peroxidation is associated with CCl(4)-induced liver injury. The spin-trapping electron paramagnetic resonance (EPR) method was used to detect nitric oxide (NO) level in liver. The chemiluminescence method was also employed to measure the NO(2)(-)/NO(3)(-) concentration in serum. The NO levels in liver tissues and NO(2)(-)/NO(3)(-) concentration in serum were found to decrease significantly both in a dose-dependent manner and in time course after CCl(4) treatment. The nitric oxide synthase (NOS) II activity in the liver, in contrast, was found to increase significantly. Our study suggests that not only should the expression of NOS be analyzed but NO organ and blood concentration must be measured in the study of diseases involving nitric oxide. L-arginine treatment had no significant effect on the liver function of CCl(4)-treated mice. It was found that NO donor sodium nitroprusside (SNP; 50 or 100 microg/kg) treatment resulted in decreases of LDH, GPT, and TBARS levels, leading to a protective effect on CCl(4)-treated mice. On the other hand, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 or 300 mg/kg) treatment caused more severe liver damage. Moreover, we have found in an in vitro EPR study that SNP could scavenge lipid peroxyl radical LOO&z.rad;. The above results together suggest that NO may protect CCl(4)-induced liver injury through scavenging lipid radical, inhibiting the lipid peroxidation chain reaction. On the basis of our analysis, we put forth two explanations for the stated discrepancy between NOS II and NO production: (i) NO was used up gradually in terminating lipid peroxidation and (ii) NADPH was depleted (on the basis of correlation evidence only).     

Production and interaction of oxygen and nitric oxide free radicals in PMA stimulated macrophages during the respiratory burst

The activity of nitric oxide synthase (NOS) during the respiratory burst in phorbol-1,2-myristate-1,3-acetate (PMA) stimulated macrophages has been the topic of much debate in the literature. To help clarify the role of NOS, we have examined the chemiluminescence arising from peroxynitrite production, nitrite/nitrate and nitric oxide production, and oxygen consumption during the respiratory burst in PMA-stimulated macrophages. The Griess reaction was used to measure nitrite/nitrate, spin trapping with N-methyl D-glucamine dithiocarbamate (MGD)2-Fe2+ was used to quantify nitric oxide, and the spin probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-ol (TEMPOL) was used to measure oxygen consumption. Oxygen free radical production (hydroxyl and superoxide free radicals) was also investigated using the spin trap 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The chemiluminescence emitted by the PMA-stimulated macrophages and nitrite/nitrate in the culture system were both found to increase. However, the rate of nitric oxide release remained constant, indicating that the activity of NOS is not enhanced during the respiratory burst in PMA stimulated macrophages.     

Caloric restriction increases free radicals and inducible nitric oxide synthase expression in mice infected with Salmonella Typhimurium

It is well known that CR (caloric restriction) reduces oxidative damage to proteins, lipids and DNA, although the underlying mechanism is unclear. However, information concerning the effect of CR on the host response to infection is sparse. In this study, 6-month-old mice that were fed AL (ad libitum) or with a CR diet were infected with Salmonella serovar Typhimurium. EPR (electron paramagnetic resonance; also known as ESR (electron spin resonance)) was used to identify FRs (free radicals). These results were subsequently correlated with SOD (superoxide dismutase) catalytic activity, iNOS [inducible NOS (nitric oxide synthase) or NOSII] expression and NO (nitric oxide) content. EPR analysis of liver samples demonstrated that there was a higher quantity of FRs and iron-nitrosyl complex in infected mice provided with a CR diet as compared with those on an AL diet, indicating that CR was beneficial by increasing the host response to Salmonella Typhimurium. Furthermore, in infected mice on the CR diet, NOSII expression was higher, NO content was greater and spleen colonization was lower, compared with mice on the AL diet. No changes in SOD activity were detected, indicating that the NO produced participated more in the formation of iron-nitrosyl complexes than peroxynitrite. These results suggest that CR exerts a protective effect against Salmonella Typhimurium infection by increasing NO production.     

Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor

The aqueous decay and concomitant release of nitric oxide (*NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of *NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and *NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of *NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of *NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.     

Free radicals,cytokines and nitric oxide in cardiac failure and myocardial infarction

Myocardial infarction is the most common cause of congestive cardiac failure. Free radicals, cytokines, nitric oxide (NO) and antioxidants play a major role both in atherosclerosis and myocardial damage and preservation. In the early stages of atherosclerosis, neutrophils and monocytes infiltrate the intima and generate free radicals which damage the endothelial cells. As a result, production of NO and prostacyclin by the endothelial cells declines, which have cardioprotective actions. This also has relevance to the beneficial action of aspirin since, it can modulate both prostanoid and l-arginine-NO systems and NF-kB translocation. In both acute myocardial infarction and chronic congestive cardiac failure, the plasma levels of various inflammatory mediators such as interleukins and tumour necrosis factor- (TNF) are elevated. TNF, produced by the inflammatory cells and the myocardium, can suppress myocardial contractility and induce the production of free radicals, which in turn can further damage the myocardium. Transforming growth factor (TGF), polyunsaturated fatty acids and the glucose-insulin-potassium regimen can antagonize the harmful actions of TNF and protect the myocardium. This explains why efforts made to reduce the levels of pro-inflammatory cytokines have beneficial action and preserve the myocardium.     

Formation of nitrosothiols from gaseous nitric oxide at pH 7.4

Nitric oxide (NO) is generated in biological systems and plays important roles as a regulatory molecule. Its ability to bind to haem iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of S-nitrosothiols (SNOs). It has been suggested that S-nitrosohaemoglobin (-SNO Hb) and low molecular weight SNOs may act as reservoirs of NO. SNOs are formed in vitro, at strongly acidic pH values; however, the mechanism of their formation at neutral pH values is still debated. In this paper we report the anaerobic formation of SNOs (both high- and low-molecular weight) from low concentrations of NO at pH 7.4, provided Hb is also present. We propose a reaction mechanism entailing the participation of Fehaem in the formation of NO(+) and the transfer of NO(+) either to Cysbeta(93) of Hb or to glutathione; we show that this reaction also occurs in human RBCs.     

A hypothesis for the local control of osteoclast function by Ca2+, nitric oxide and free radicals

Several important conclusions have recently emerged fromin vitro studies on the resorptive cell of bone, the osteoclast. First, it has been established that osteoclast function is modulated locally, by changes in the local concentration of Ca²⁺ caused by hydroxyapatite dissolution. It is thought that activation by Ca²⁺ of a surface membrane Ca²⁺ receptor mediates these effects, hence providing a feedback control. Second, a number of molecules produced locally by the endothelial cell, with which the osteoclast is in intimate contact, have been found to affect bone resorption profoundly. For instance, the autocoid nitric oxide strongly inhibits bone resorption. Finally, reactive oxygen species have been found to aid bone resorption and enhance osteoclastic activity directly. Here, we will attempt to integrate these control mechanisms into a unified hypothesis for the local control of bone resorption.     

Interaction of the pyridoindole stobadine with alkoxyl and stable free radicals

Stobadine, a pyridoindole derivative which has been synthesized in search of new antiarrhytmic drugs, was able to interact with alpha-diphenyl-beta-picrylhydrazyl (DPPH^.) a stable free radical. Its scavenging potential was in the same order of magnitude as that of well known strong antioxidants. The scavenging effect of antioxidants tested increased in the order: stobadine, ascorbic acid, trolox, cysteine. Stobadine exerted a scavenging effect also towards alkoxyl radicals in a nonpolar solvent in the canthaxantin bleaching test. In this test stobadine was more effective than trolox.     

Formation of free radicals at photoreduction of allophycocyanin B

A photoinduced ESR signal is detected in the presence of 10(-2) M of dithionine in phycobilisomes and in fractions enriched in allophycocyanin B. The signal is characterized by the line width of 12G and g-factor of 2.004. It is demonstrated that the appearance of the free radicals is related to photoreduction of allophycocyanin B.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

A role for free radicals and nitric oxide in delayed recovery in aged rats with chronic constriction nerve injury

Using a reversible chronic constriction injury (CCI) model of neuropathic pain, we previously demonstrated that changes in thermal hyperalgesia correlate with the changes in peripheral microvascular blood flow in the affected paw, and that recovery can be assessed by normalization of both behavioral and vascular responses. Using the same model, this study examined age-related changes in recovery after nerve injury and the involvement of free radicals and nitric oxide (NO) in these changes. Four loose, nonconstrictive ligatures were applied to the sciatic nerve in the right, mid-thigh region of young and old (3 and 24 months) Sprague Dawley rats. All rats were monitored weekly (for 8-10 weeks) for their thermal threshold using a 46 degrees C water bath and some groups were used to examine endothelial and smooth muscle-dependent microvascular responses to substance P (SP) and sodium nitroprusside (SNP), respectively. These substances were perfused over the base of blisters raised on the footpad innervated by the injured nerve. Free radical activity in the sciatic nerve was assessed by measuring the activity of xanthine oxidase (XO) and lipid hydroperoxides (LPO). Young rats showed signs of recovery (reduction in thermal hyperalgesia and improvement of peripheral microvascular blood flow) from the fifth week. No signs of recovery were observed in old rats for 8 weeks, with some reduction in thermal hyperalgesia observed by weeks 9 and 10. XO activity was significantly higher in young injured nerves compared to sham (400%) and was even significantly greater in old injured nerves (680%). Similarly, old injured nerves showed 300% increase in LPO levels compared to sham. The role of reactive oxygen species (ROS) in delayed recovery in old rats was examined using the antioxidant tirilazad mesylate. Tirilazad (20 mg/kg) was injected intramuscularly (im) in the mid-thigh region starting on day 1 post CCI, (early treatment) or day 7 (late treatment). Levels of LPO in the injured sciatic nerves were significantly reduced using either early or late treatment, however tirilazad had opposing effects on recovery, prolonging or alleviating thermal hyperalgesia, respectively. The role of neuronal nitric oxide (nNO) was then examined using the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (3Br-7NI) (10 mg/kg). 3Br-7NI resulted in a significant alleviation of thermal hyperalgesia with improvement in the vascular responses from weeks 5 and 6 onwards. A combination of 3Br-7NI and tirilazad treatment was also used but did not show an additive effect. The results suggest that ROS and nNO contribute to delayed recovery of injured nerves in old rats and to the maintenance of thermal hyperalgesia and the reduction in microvascular blood flow in the area innervated by the injured nerve. The results also raise the notion that possible interaction of free radicals with NO to form peroxynitrite might be responsible for such delayed recovery. Ironically, this study also reveals a positive role for free radicals in tissue repair and raises the notion that early intervention with antioxidants could exert a negative effect on repair of injured nerves.     

Release of nitric oxide together with carbon-centered radicals from N-nitrosamines by ultraviolet light irradiation

Solutions of N-nitrosamines, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomorpholine and N-nitrosopyrrolidine in phosphate buffer (pH 7.4) were irradiated by ultraviolet (UV) light at room temperature. The N-nitrosamines were extensively degraded due to irradiation for 120 min in a time-dependent fashion as monitored by UV-absorption or high performance liquid chromatographic analysis. Carbon-centered radicals were generated from four N-nitrosamines during the short time irradiation of 10–60 s as monitored by electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-α-phenylnitrone as spin traps. Nitric oxide (NO) was generated during the short time irradiation as monitored by ESR technique using cysteine-Fe(II) complex, N-methyl-d-glucamine dithiocarbamate and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Significant amounts of nitrite (4–16%) from four N-nitrosamines and also a significant amount of nitrate (4%) was produced from N-nitrosodimethylamine during the irradiation time of 120 min. Released NO from the N-nitrosamines must be converted into nitrite through intermediary reactive nitrogen oxide species including nitrogen dioxide and dinitrogen trioxide in contact with dissolved oxygen.