Christopher C. Felix

Melanin photoreactions in aerated media: electron spin resonance evidence for production of superoxide and hydrogen peroxide.

Abstract Electron spin resonance measurements on aerated melanin suspensions during photoirradiation show changes in the microwave saturation of melanin free radicals and formation of adducts in the presence of spin traps. These observations indicate that oxygen is reduced to superoxide and hydrogen peroxide.

PHOTOIONIZATION OF MELANIN PRECURSORS: AN ELECTRON SPIN RESONANCE INVESTIGATION USING THE SPIN TRAP 5,5‐DIMETHYL‐1‐PYRROLINE‐1‐OXIDE (DMPO)

Abstract The photoionization of 3,4‐dihydroxyphenylalanine (dopa) and catechol has been studied by electron spin resonance spectroscopy using the free radical scavenger 5,5‐dimethyI‐1‐pyrroline‐1 ‐oxide as a spin trap for hydrated electrons and hydrogen atoms. The photochemistry of these materials is shown to resemble tyrosine in that both photoionization and photohomolysis (to give H) occur, with photoionization predominating (by a factor of 2.6 for dopa). Ionization of one of the phenolic hydroxyl groups increases the yield of radicals by a factor of 2. Action spectra and quantum yields for radical production are reported.

Photoreactions of melanin: a new transient species and evidence for triplet state involvement.

Abstract The detection of a new transient species in photoirradiated natural and synthetic melanins is reported. This species decays rapidly at both ambient and cryogenic temperatures and has an electron spin resonance spectrum whose time-profile reveals spin polarization effects (chemically induced dynamic electron polarization) characteristic of triplet state involvement. A possible mechanism for light absorption and degradation by melanins is suggested.

FREE RADICALS FROM PHOTOREDUCTION OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

—The anaerobic photoreduction of hematoporphyrin by organic reducing agents in fluid aqueous solutions has been studied by electron spin resonance spectroscopy. Efficient photoreduction occurs with many hydrogen‐atom or electron donors, including catechols, pyrogallol. hydroquinone. ascorbate, p‐phenylenediamine, cysteine and glutathione. A combination of direct and spin‐trapping measurements (using 2‐methyl‐2‐nitrosopropane) has been used to confirm production of radicals from both the porphyrin and the reducing agent in each system.

PHOTOLYSIS OF MELANIN PRECURSORS: FORMATION OF SEMIQUINONE RADICALS AND THEIR COMPLEXATION WITH DIAMAGNETIC METAL IONS

Abstract— Semiquinone free radicals have been generated by the UV photolysis of aqueous solutions of melanin precursors (catechols and catecholamines) and characterized by electron spin resonance (ESR) spectroscopy. Hyperfine parameters of the semiquinones are sensitive to the nature of the substituents on the aromatic ring, reflecting (i) the ionization state of the substituent; (ii) restricted rotation of methylene protons; and (iii) the presence of a chiral carbon center. In the presence of complexing metal ions (Zn2+, Cd2+) the semiquinones form chelate complexes whose ESR spectra differ from those of the uncomplexed radicals. Satellite peaks demonstrating hyperfine coupling to metal isotopes (67Zn, 111Cd and 113Cd) present in natural abundance were detected in several instances. Concentrations of metal complexes are much higher than those of the uncomplexed radicals, suggesting that diamagnetic metal ions may be useful in identifying the presence of semiquinone radicals of this kind in various systems.

NOX 100, a nitric oxide scavenger, enhances cardiac allograft survival and promotes long-term graft acceptance.

BACKGROUND We examined the role of nitrosative stress in allograft destruction. METHODS Rats undergoing cardiac transplants received NOX-100, a water-soluble nitric oxide (NO) scavenger with antioxidant properties, with or without low-dose cyclosporine (CsA). Graft survival, NO production, and nuclear factor kappa B (NF-kappaB) activity were studied. RESULT Using NOX-100 daily until rejection prolonged graft survival (11.6+/-0.6 vs. 7.4+/-0.2 days; P<0.05). Daily low-dose CsA (2.5 mg/kg im) for 7 days or until rejection also prolonged survival (12.6+/-0.5 and 21.6+/-1.6 days, respectively; P<0.01 vs. Controls). Low-dose CsA for 7 days and NOX-100 for 30 days prolonged graft survival (45.0+/-4.7 days; P<0.01 vs. all groups.). NOX-100 had no effect on whole blood CsA levels. Combination therapy until Day 100 resulted in 1 graft loss at Day 116 and indefinite survival in 3 animals (>300 days), which accepted a second WF strain heart without further immunosuppressive therapy but promptly rejected a third party (ACI) cardiac allograft. NOX-100 and CsA reduced nitrate and nitrite, and combination therapy completely normalized NO through to Day 30. Electron paramagnetic resonance spectroscopic analysis demonstrated reduction of signals for nitrosylmyoglobin and nitrosyl-heme with NOX-100 and elimination of signals with CsA alone or combination therapy. Activity of myocardial NF-kappaB decreased with monotherapy vs. untreated allografts. Combination therapy resulted in further inhibition of NF-kappaB up to Day 30. The extent of graft survival correlated with the extent of NO scavenging and NF-kappaB inhibition. Short-term combination therapy had no effect on graft lymphocytic infiltrate on Days 15, 20, and 30. CONCLUSION These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

Vascular protective actions of a nitric oxide aspirin analog in both in vitro and in vivo models of diabetes mellitus.

BACKGROUND Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals. MATERIAL AND METHODS Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae. RESULTS Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue. CONCLUSIONS Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.

The effect of low molecular weight chelators on iron chelation and free radical generation as studied by ESR measurement

The purpose of this work was to improve our current knowledge of the non-enzymatic mechanisms involved in brown rot fungal decay of wood, but also to improve our understanding of chelator-mediated reactions that may occur in other biological systems where low pH conditions may prevail. Several key steps (iron chelation and reduction) during early non-enzymatic wood decay processes have been studied by using electron spin resonance (ESR). It has been proposed that low molecular weight chelators as well as Fenton reagents are involved in brown rot decay, at least in early non-enzymatic stages. In this work, the binding between a catecholate model chelator and ferric iron was studied by ESR spectroscopy. The effects of the model chelator, Fenton reagents, as well as the reaction conditions on free radical generation were also studied using ESR spin-trapping techniques. The results indicate: (1) The relative quantity of the chelator-iron complex can be determined by measurement of the intensities of the characteristic g = 4.3 ESR signal. (2) The effects of the chelator:iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system can be determined using ESR spin-trapping techniques. (3) Data support the hypothesis that superoxide radicals are involved in chelator-mediated Fenton processes.

Detection by ESR of DMPO hydroxyl adduct formation from islets of langerhans

Electron spin resonance (ESR) spectroscopy together with spin trapping techniques and the application of state-of-the-art loop gap resonators was used to provide a direct measure of spontaneous oxygen radical production by homogenates of freshly isolated and cultured rat pancreatic islets. Using the spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect production by islets of an ESR-sensitive radical signal consisting of a quartet with intensity ratio of 1:2:2:1 and hyperfine splitting of aN = aH = 14.9 Gauss, which is consistent with the DMPO-OH adduct. The amplitude of the signal was decreased by decreasing amount of islets and not detected in the absence of islets. Formation of the DMPO-OH adduct was diminished by the hydroxyl radical scavengers (e.g., ethanol, dimethylsulfoxide, and dimethylthiourea). Only partial attenuation of signal was produced by incubation with an iron chelator or using chelex-treated buffers. The ESR signal was insensitive to the xanthine oxidase inhibitor, oxypurinol, or to superoxide dismutase, but was eliminated in a concentration-dependent manner by either potassium cyanide or catalase (but not heat-inactivated catalase). These observations suggest that the origin of the DMPO-OH arose not from free hydroxyl radicals but primarily from endogenous hydrogen peroxide production perhaps of mitochondrial origin. The development of this technology has implications for the potential measure of oxygen radical production in islet homogenates under pathologic conditions as well as to the application of other cell culture systems.

Non-heme iron protein: A potential target of nitric oxide in acute cardiac allograft rejection

We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis → Lewis) and mismatched (allografts: Wistar–Furth → Lewis) rats. On postoperative days (POD) 4–6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl–iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to ≈50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl–iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl–iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N6-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.