Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

Detection and characterization of free oxygen radicals induced protein adduct formation in differentiating macrophages.

Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.

Detection and identification of ethanal-derived spin-trapped free radicals using headspace thermal desorption gas chromatography-mass spectrometry (TD-GC-MS).

In this study, we demonstrate a novel approach to the detection and identification of the products of spin-trapped free radicals. Hydroxyl free radicals were generated by Fenton-based chemistry in the presence of ethanal and the spin-trapping agent N-tert-butyl-α-phenylnitrone (PBN). The resulting volatile compounds present in the reaction vial headspace were collected using thermal desorption (TD) and analysed by gas chromatography-mass spectrometry (GC-MS). Eleven compounds were detected in the headspace, and their identification was aided by using either a fluorinated or deuterated analogue of PBN as an alternative spin trap and/or deuterated ethanal (CD3CHO) as the secondary source of free radicals. The electron-ionisation (EI) mass spectra clearly demonstrate the "capture" of methyl radicals; two of the compounds detected were identified as containing one methyl group derived from ethanal, and four were shown to contain two methyl groups. This study demonstrates that sampling the reaction headspace using TD-GC-MS is a viable method for analysing products of free radical trapping, and potentially may be applied to a wide range of free radical systems.

Trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and genotoxic damage: Recent advances using the immuno-spin trapping technology.

Immuno-spin trapping detection of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrrloine N-oxide (DMPO) has made important contributions towards the understanding of DNA radicalization and genotoxicity at sites of inflammation. At sites of inflammation, one-electron oxidants and chloramines decay induce oxidation of genomic DNA, genotoxicity and cell transformation. Radicalization of DNA can result in either single- or double-strand breaks, or end-oxidation products at the sugar or bases. If not repaired, these modifications can lead to mutations and cell transformation. If trapped with DMPO, DNA-centered radical decay and subsequent formation of end-oxidation products are blocked. Herein we discuss recent literature regarding the use of immuno-spin trapping with DMPO to study DNA-centered radicals and their involvement in genotoxicity. This technique has shown the critical role of DNA radicalization in 8-oxo-dG formation and DNA strand breaks in isolated DNA, cells and in whole animals. Combination of technologies, including immuno-spin trapping and powerful chromatographic and sequencing techniques are needed to move forward the field towards the detection of specific genes that are susceptible to oxidative damage in cells located at sites of inflammation. This is important in order to provide novel information about genotoxicity mechanisms, as well as therapeutic possibilities of DMPO or its derivatives for preventing DNA-centered radical-mediated carcinogenesis.

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin trapping confirmation by using 13C-substitution.

Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumour, capacity to induce epidermal hyperplasia, and aptitude to induce allergic and irritant skin contact dermatitis are well known. These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl, alkoxyl, and alkyl radicals characterised basically by electron paramagnetic resonance (EPR) and spin-trapping (ST) techniques. To be a skin sensitiser CumOOH needs to covalently bind to skin proteins in the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O-O bond allows formation of unstable CumO•/CumOO• radicals rearranging to longer half-life specific carbon-centred radicals R• proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear which R• is precisely formed in the epidermis and thus involved in the sensitisation process. The aim of this work was to elucidate in conditions closer to real-life sensitisation which specific R• are formed in a 3D reconstructed human epidermis (RHE) model by using 13C-substituted CumOOH at carbon positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably methyl radicals derived from ß-scission of CumO• radicals occur in RHE through a one-electron reductive pathway suggesting that these could be involved in the antigen formation inducing skin sensitisation. We also describe a coupling between nitroxide radicals and ß position 13C atoms that could be of an added value to the very few examples existing for the coupling of radicals with 13C atoms.

In Vivo and In Situ Detection of Macromolecular Free Radicals Using Immuno-Spin Trapping and Molecular Magnetic Resonance Imaging.

SIGNIFICANCE: In vivo free radical imaging in preclinical models of disease has become a reality. Free radicals have traditionally been characterized by electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy coupled with spin trapping. The disadvantage of the ESR/EPR approach is that spin adducts are short-lived due to biological reductive and/or oxidative processes. Immuno-spin trapping (IST) involves the use of an antibody that recognizes macromolecular 5,5-dimethyl-pyrroline-N-oxide (DMPO) spin adducts (anti-DMPO antibody), regardless of the oxidative/reductive state of trapped radical adducts. Recent Advances: The IST approach has been extended to an in vivo application that combines IST with molecular magnetic resonance imaging (mMRI). This combined IST-mMRI approach involves the use of a spin-trapping agent, DMPO, to trap free radicals in disease models, and administration of an mMRI probe, an anti-DMPO probe, which combines an antibody against DMPO-radical adducts and an MRI contrast agent, resulting in targeted free radical adduct detection. CRITICAL ISSUES: The combined IST-mMRI approach has been used in several rodent disease models, including diabetes, amyotrophic lateral sclerosis (ALS), gliomas, and septic encephalopathy. The advantage of this approach is that heterogeneous levels of trapped free radicals can be detected directly in vivo and in situ to pin point where free radicals are formed in different tissues. FUTURE DIRECTIONS: The approach can also be used to assess therapeutic agents that are either free radical scavengers or generate free radicals. Smaller probe constructs and radical identification approaches are being considered. The focus of this review is on the different applications that have been studied, advantages and limitations, and future directions. Antioxid. Redox Signal. 28, 1404-1415.

N-tert-butylmethanimine N-oxide is an efficient spin-trapping probe for EPR analysis of glutathione thiyl radical.

The electron spin resonance (EPR) spin-trapping technique allows detection of radical species with nanosecond half-lives. This technique is based on the high rates of addition of radicals to nitrones or nitroso compounds (spin traps; STs). The paramagnetic nitroxides (spin-adducts) formed as a result of reactions between STs and radical species are relatively stable compounds whose EPR spectra represent "structural fingerprints" of the parent radical species. Herein we report a novel protocol for the synthesis of N-tert-butylmethanimine N-oxide (EBN), which is the simplest nitrone containing an α-H and a tertiary α'-C atom. We present EPR spin-trapping proof that: (i) EBN is an efficient probe for the analysis of glutathione thiyl radical (GS•); (ii) ß-cyclodextrins increase the kinetic stability of the spin-adduct EBN/•SG; and (iii) in aqueous solutions, EBN does not react with superoxide anion radical (O2-•) to form EBN/•OOH to any significant extent. The data presented complement previous studies within the context of synthetic accessibility to EBN and efficient spin-trapping analysis of GS•.

EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.

Spin trapping: an essential tool for the study of diseases caused by oxidative stress.

Electron spin resonance (ESR), called also electron paramagnetic resonance (EPR) together with the spin trapping technique, has allowed us to study and understand how free radicals are involved in various pathologies. In this review, the importance of spin trapping technique in the study of diseases such as cancer, diabetes, hypertension and parasitic diseases is discussed. In addition, advances in the use of this technique as therapeutic agents and other interesting applications as the immuno-spin trapping technique are reviewed.

Tendencias en la mortalidad por cáncer en México de 1980 a 2011 / Cancer mortality trends in Mexico, 1980-2011

Objetivo. Evaluar las tendencias de mortalidad por cáncer en México entre 1980 y 2011. Material y métodos. Se calcularon las tasas de mortalidad ajustadas por edad y sexo para todos los cánceres y para las 15 localizaciones más frecuentes mediante el método directo y tomando como población estándar la población mundial de 2010. Las tendencias en las tasas de mortalidad y el cambio porcentual anual para cada tipo de cáncer se estimaron a través de un modelo de regresión joinpoint. Resultados. A partir de 2004 y como consecuencia de la reducción de la mortalidad por cáncer de pulmón (-3.2% en hombres y -1.8% en mujeres), estómago (-2.1% en hombres y -2.4% en mujeres) y cérvix (-4.7%), se observó una disminución significativa (~1% anual) en la mortalidad por cáncer en general tanto en el grupo de todas las edades como en el de 35 a 64 años para ambos sexos. La mortalidad por otros cánceres como el de mama y el de ovario, en las mujeres o el de próstata, en los hombres, mostró un aumento sostenido. Conclusiones. Algunas de las reducciones en la mortalidad por cáncer pueden ser parcialmente atribuidas a la efectividad de los programas de prevención establecidos. Sin embargo, se requiere implementar registros adecuados de cáncer con base poblacional para evaluar el impacto real de estos programas, así como diseñar y evaluar intervenciones innovadoras que permitan desarrollar políticas de prevención más costo-efectivas.

Objective. To evaluate trends in cancer mortality in Mexico between 1980-2011. Material and methods. Through direct method and using World Population 2010 as standard population, mortality rates for all cancers and the 15 most frequent locations, adjusted for age and sex were calculated. Trends in mortality rates and annual percentage change for each type of cancer were estimated by joinpoint regression model. Results. As a result of the reduction in mortality from lung cancer (-3.2% -1.8% in men and in women), stomach (-2.1% -2.4% in men and in women) and cervix (-4.7%); since 2004 a significant (~1% per year) decline was observed in cancer mortality in general, in all ages, and in the group of 35-64 years of both sexes. Other cancers such as breast and ovarian cancer in women; as well as for prostate cancer in men, showed a steady increase. Conclusions. Some of the reductions in cancer mortality may be partially attributed to the effectiveness of prevention programs. However, adequate records of population-based cancer are needed to assess the real impact of these programs; as well as designing and evaluating innovative interventions to develop more cost-effective prevention policies.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps.

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated ß-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals.

The short lifetime of superoxide and the low rates of formation expected in vivo make detection by standard continuous wave (CW) electron paramagnetic resonance (EPR) challenging. The new rapid-scan EPR method offers improved sensitivity for these types of samples. In rapid-scan EPR, the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times, and data are processed to obtain the absorption spectrum. To validate the application of rapid-scan EPR to spin trapping, superoxide was generated by the reaction of xanthine oxidase and hypoxanthine with rates of 0.1-6.0 µM/min and trapped with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO). Spin trapping with BMPO to form the BMPO-OOH adduct converts the very short-lived superoxide radical into a more stable spin adduct. There is good agreement between the hyperfine splitting parameters obtained for BMPO-OOH by CW and rapid-scan EPR. For the same signal acquisition time, the signal/noise ratio is >40 times higher for rapid-scan than for CW EPR. Rapid-scan EPR can detect superoxide produced by Enterococcus faecalis at rates that are too low for detection by CW EPR.

Investigation of spin-trapping artifacts formed by the Forrester-Hepburn mechanism.

Free radical detection with ESR spin trapping relies on the specific addition of the radical to nitrone/nitroso compounds. It also has been proposed that spin traps can react in biological systems to give false-positive results. For nitrone spin traps, the reaction with nucleophiles, first described by Forrester and Hepburn, has been discussed as the most critical source of artifacts. For artifact identification, the ESR preincubation method may be used, which employs isotopically marked spin traps. Here we investigated the influence of fast sulfite-hydroxylamine equilibrium chemistry on the validity of this assay. Using the (faster) aspiration technique, we found that the Forrester-Hepburn mechanism also contributes to DMPO/(•)SO3(-) adduct formation during ferricyanide-mediated sulfite oxidation, but no evidence for artifactual DMPO/(•)SO3(-) formation was found if the more potent horseradish peroxidase was used. This is ESR evidence that the Forrester-Hepburn mechanism can occur under mild conditions, depending on the experimental details. This technique can also be used to test for other artifact mechanisms. We investigated the known ene reaction of DBNBS and tryptophan in more detail. We found that a strong artifact signal is induced by light; however, with atypically long incubations, we found that the artifact is also formed thermally.

[Antioxidant properties of the pollen exine polymer matrix].

The antioxidant properties of exine polymer matrix which forms the outer layer of pollen grain wall were studied. The main component of this matrix is sporopollenin - a unique biopolymer resistant to mechanical and chemical damage. The samples of isolated exine, purified from soluble compounds, were studied with EPR using stable nitroxyl radical TEMPO and DMPO spin trap. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduces TEMPO radical and eliminates hydroxyl radical. Also, the fluorometric analysis demonstrated that the exine eliminated H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4M NaOH, 170 degrees C) of ethers bonds a large amount of hydroxycinnamic acids has been released, and exines have lost their antioxidant capacity almost completely. The obtained results point to the ability of extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.

EPR studies on hydroxyl radical-scavenging activities of pravastatin and fluvastatin.

Statins are known clinically by their cholesterol reduction properties through the inhibition of HMG-CoA reductase. There is mounting evidence suggesting a protective role of statins in certain types of cancer, cardiac, and vascular disease through a mechanism that extends beyond their lipid lowering ability. The root mechanism of damage likely involves the inflammatory cascade, specifically compounds known as reactive oxygen species such as the hydroxyl radical. However, direct evidence for the hydroxyl-scavenging capacity of pravastatin and fluvastatin, two forms of statins being widely used to lower LDL cholesterol, is still lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-spin-trapping technique was utilized to determine the abilities of pravastatin and fluvastatin in scavenging hydroxyl radical generated from Fe(II) with H(2)O(2) system. In addition, we examined the effects of pravastatin and fluvastatin on oxidative-induced φX-174 RF I plasmid DNA damage. We have demonstrated here for the first time that pravastatin and fluvastatin at physiologically relevant concentrations significantly decreased formation of DMPO-OH adduct indicating that both compounds could directly scavenge hydroxyl radicals. However, pravastatin and fluvastatin were not able to directly protect against oxidative DNA plasmid damage. The hydroxyl radical sequestering ability of pravastatin and fluvastatin reported in this study may contribute to their beneficial use in certain types of cancer and in cardiovascular disease.

An amide-containing metal-organic tetrahedron responding to a spin-trapping reaction in a fluorescent enhancement manner for biological imaging of NO in living cells.

Metal-organic polyhedra represent a unique class of functional molecular containers that display interesting molecular recognition properties and fascinating reactivity reminiscent of the natural enzymes. By incorporating a triphenylamine moiety as a bright blue emitter, a robust cerium-based tetrahedron was developed as a luminescent detector of nitronyl nitroxide (PTIO), a specific spin-labeling nitric oxide (NO) trapper. The tetrahedron encapsulates molecules of NO and PTIO within the cavity to prompt the spin-trapping reaction and transforms the normal EPR responses into a more sensitively luminescent signaling system with the limit of detection improved to 5 nM. Twelve-fold amide groups are also functionalized within the tetrahedron to modify the hydrophilic/lipophilic environment, ensuring the successful application of biological imaging in living cells.

Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway.

Many terpenes used as fragrance compounds autoxidize when exposed to air, forming allylic hydroperoxides that have the potential to be skin contact allergens. To trigger the immunotoxicity process that characterizes contact allergy, these hydroperoxides are supposed to bind covalently to proteins in the skin via radical pathways. We investigated the formation of reactive radical intermediates from 7-hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol and 2-hydroperoxylimonene, responsible for the sensitizing potential acquired by autoxidized linalool and limonene. Both compounds were synthesized through new short and reproducible synthetic pathways. The hydroperoxide decomposition catalyzed by Fe(II)/Fe(III) redox systems, playing a key role in degradating peroxides in vivo, was examined by spin-trapping-EPR spectroscopy. Alkoxyl and carbon-centered free radicals derived from the hydroperoxides were successfully trapped by the spin-trap 5,5-dimethyl-1-pyrroline N-oxide, whereas peroxyl radicals were characterized by spin-trapping studies with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. Using liquid chromatography combined with mass spectrometry, we demonstrated the formation of adducts, via radical mechanisms induced by Fe(II)/Fe(III), between the hydroperoxides and N-acetylhistidine methyl ester, a model amino acid that is prone to radical reactions. Free radicals derived from these hydroperoxides can thus induce amino acid chemical modifications via radical mechanisms. The study of these mechanisms will help to understand the sensitizing potential of hydroperoxides.

Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling.

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Electron spin resonance. Part one: a diagnostic method in the biomedical sciences.

A review is presented of some of the ways in which electron spin resonance (ESR) spectroscopy may be useful to investigate systems of relevance to the biomedical sciences. Specifically considered are: spin-trapping in biological media; the determination of antioxidant efficiencies; lipid-peroxidation; the use of nitroxides as probes of metabolic activity in cells and as structumral probes of cell-membranes; ESR coupled with materials for radiation-dosimetry; food- and drug-irradiation; studies of enzyme systems and ofcyclodextrins; diagnosis of cancer and rheumatoid arthritis; measurement of oxidative stress in synovial tissue in preparation for joint replacement; determination of oxidative species during kidney dialysis; measurement of biological oxygen concentrations (oximetry); trapping in living cells of the endothelium-derived relaxing factor nitric oxide (NO); measurement of hydrogen peroxide; determination of drugs of abuse (opiates); ESR measurements of whole blood and as a means to determine the age of bloodstains for forensic analysis are surveyed, and also a determination of the aqueous volume of human sperm cells is described, among other topics.

Site-specific radical formation in DNA induced by Cu(II)-H2O2 oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS.

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H2O2 oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.