Pulmonary Neutrophilic Inflammation and Noncommunicable Diseases: Pathophysiology, Redox Mechanisms, Biomarkers, and Therapeutics.

Significance: Pulmonary neurophilic inflammation (PNI) is the homing and activation of neutrophil with damage to the microvasculature. This process is involved in pulmonary damage in patients exposed to airborne pollutants (exogenous stressors) and also to systemic inflammation/oxidative stress (endogenous stressors) associated with noncommunicable diseases (NCDs). Recent Advances: PNI is an important trigger of the early onset and progression of NCD in susceptible patients exposed to airborne pollutants. Irritation of the lung microvasculature by exogenous and endogenous stressors causes PNI. Circulating endogenous stressors in NCD can cause PNI. Critical Issues: Air pollution-triggered PNI causes increased circulating endogenous stressors that can trigger NCD in susceptible patients. Systemic inflammation/oxidative stress associated with NCD can cause PNI. Inflammation/end-oxidation products of macromolecules are also potential biomarkers and therapeutic targets for NCD-triggered PNI- and PNI-triggered NCD. Future Directions: Understanding the molecular mechanism of PNI triggered by exogenous or endogenous stressors will help explain the early onset of NCD in susceptible patients exposed to air pollution. It can also help undercover biomarkers and mechanism-based therapeutic targets in air pollutant-triggered PNI, PNI-triggered NCD, and NCD-triggered PNI.

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.

Diet-Induced Pulmonary Inflammation and Incipient Fibrosis in Mice: a Possible Role of Neutrophilic Inflammation.

Chicken fat and fructose are added into food-processing to reduce costs and enhance acceptability; however, these additives turn food into unhealthy and hypercaloric meals. Herein we have hypothesized that chronic feeding with chicken fat and fructose, together or by separate, can cause pulmonary redox and inflammatory changes. These changes are particularly related to neutrophils and myeloperoxidase, with consequent changes in the organ histophysiology. To test this hypothesis, we fed mice for 16 weeks with either control food (low-fat diet, LFD) or control food supplemented with 22% chicken fat and with or without 10% fructose in the drinking water. At the end of the feeding regimen, we measured redox and inflammatory changes in the lung with particular emphasis on neutrophil accumulation/activation and molecular-histological markers of fibrosis. Our results suggest that a diet supplemented with chicken fat and fructose causes additive effects on pulmonary oxidative stress, inflammation, and a pro-fibrotic status. Neutrophilic inflammation may play a critical role in pulmonary pathology associated with metabolic syndrome.

A soybean-based diet modulates cadmium-induced vascular apoptosis.

Cadmium (Cd) exposure has been associated with an increased risk of cardiovascular diseases. The diet is a modifiable source of protecting or damaging factors that may affect this risk. Herein we tested the hypothesis that a soybean-based diet (SBD) protects the vascular wall of the aorta against Cd-induced pro-inflammatory and pro-apoptotic effects. To test this hypothesis, we fed male Wistar rats for 60 days with a casein-based diet (CBD) or an SBD. These animals were also exposed to tap-water without (CBD-Co/SBD-Co) or with 15(CBD-15Cd/SBD-15Cd) or 100 (CBD-100Cd/SBD-100Cd) ppm of Cd. Inflammatory parameters (mRNAs and/or proteins) were measured in thoracic aorta tissue. These included inducible and endothelial nitric oxide synthases, cyclooxygenase-2, intracellular-adhesion molecule-1, and vascular cell-adhesion molecule-1. As pro-apoptotic parameters, we measured Bax and Bcl-2 mRNA/protein, as well as TUNEL positive cells in the aorta tissue. Compared to CBD-Co, inflammatory and apoptosis markers increased in the aorta with the concentration of Cd in the drinking water. These effects were not observed in either SBD-15Cd or SBD-100Cd, which were similar to CBD-Co. Cd content in serum and in aortas from animals fed CBD-Co/SBD-15Cd or CBD-Co/SBD-100Cd were similar suggesting that, if any, the effect of SBD is not due to changes in Cd bioaccumulation, but due to secondary effects linked to the composition of the dietary soybean flour. Our findings are consistent with a protective effect of an SBD against Cd-induced inflammation and apoptosis in the thoracic aorta in a rat model.

The nitrone spin trap 5,5­dimethyl­1­pyrroline N­oxide binds to toll-like receptor-2-TIR-BB-loop domain and dampens downstream inflammatory signaling.

The nitrone spin trap 5,5­dimethyl­1­pyrroline N­oxide (DMPO) dampens endotoxin-induced and TLR4-driven priming of macrophages, but the mechanism remains unknown. The available information suggests a direct binding of DMPO to the TIR domain, which is shared between TLRs. However, TLR2-TIR domain is the only TLR that have been crystallized. Our in silico data show that DMPO binds to four specific residues in the BB-loop within the TLR2-TIR domain. Our functional analysis using hTLR2.6-expressing HEKs cells showed that DMPO can block zymosan-triggered-TLR2-mediated NF-κB activation. However, DMPO did not affect the overall TLR2-MyD88 protein-protein interaction. DMPO binds to the BB-loop in the TIR-domain and dampens downstream signaling without affecting the overall TIR-MyD88 interaction. These data encourage the use of DMPO-derivatives as potential mechanism-based inhibitors of TLR-triggered inflammation.

A Mouse Model of Diet-Induced Obesity Resembling Most Features of Human Metabolic Syndrome.

Increased chicken-derived fat and fructose consumption in the human diet is paralleled by an increasing prevalence of obesity and metabolic syndrome (MS). Herein, we aimed at developing and characterizing a mouse model of diet-induced obesity (DIO) resembling most of the key features of the human MS. To accomplish this, we fed male C57BL/6J mice for 4, 8, 12, and 16 weeks with either a low-fat diet (LFD) or a high-chicken-fat diet (HFD) and tap water with or without 10% fructose (F). This experimental design resulted in the following four experimental groups: LFD, LFD + F, HFD, and HFD + F. Over the feeding period, and on a weekly basis, the HFD + F group had more caloric intake and gained more weight than the other experimental groups. Compared to the other groups, and at the end of the feeding period, the HFD + F group had a higher adipogenic index, total cholesterol, low-density lipoprotein cholesterol, fasting basal glycemia, insulin resistance, hypertension, and atherogenic index and showed steatohepatitis and systemic oxidative stress/inflammation. A mouse model of DIO that will allow us to study the effect of MS in different organs and systems has been developed and characterized.

Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals.

BACKGROUND: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas.

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.

Reduced blood nrf-2 mRNA in local overweight boys at risk of metabolic complications: a study in San Luis City, San Luis, Argentina.

BACKGROUND: Childhood overweight (OW) is a matter of public health concern because of its long-term impact on adulthood health. NF-E2-related factor 2 (Nrf-2) regulates the antioxidant/lipogenic response to a sustained positive energy balance that prevails during weight gain. Here we aimed at studying a possible link between OW and Nrf-2-dependent antioxidant/lipogenic response in a local population of boys at risk of metabolic complications. METHODS: We measured clinical and biochemical parameters related to lipid metabolism, oxidative stress, and metabolic syndrome in a population of OW boys [body mass index (BMI) percentile ≥85(th) and <95(th), n=22] and normal weight boys (NW; BMI percentile<85(th), n=27) from San Luis City, San Luis, Argentina. RESULTS: Compared to NW, OW boys had lower insulin sensitivity, an altered plasma lipid profile, and increased markers of oxidative stress and inflammatory fatty acids. OW boys also had a higher atherogenic index and peripheral insulin resistance than NW boys. We also found that glutathione peroxidase activity and the reduced glutathione to oxidized glutathione ratio were lower in OW boys than NW boys, suggesting that OW boys may have an altered antioxidant response to oxidative stress. Finally, Nrf-2 expression negatively correlated with metabolic syndrome parameters in OW boys. CONCLUSIONS: Our data suggest that OW boys have a reduced antioxidant and lipogenic response to a positive energy balance, resulting in oxidative stress, insulin resistance, and risk of developing metabolic complications. Our data also provide a rationale for nutritional interventions aimed at restoring Nrf-2 expression to reduce the risk of metabolic complications in OW boys.

In vivo detection of free radicals using molecular MRI and immuno-spin trapping in a mouse model for amyotrophic lateral sclerosis.

Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin trapping and molecular magnetic resonance imaging, in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A)-transgenic (Tg) mice, a model for ALS. For this study, the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (ip) over 5 days before administration (iv) of an anti-DMPO probe (anti-DMPO antibody covalently bound to an albumin-gadolinium-diethylenetriamine pentaacetic acid-biotin MRI contrast agent) to trap free radicals. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p < 0.05) or anti-DMPO probe concentrations measured from T1 relaxations (p < 0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescence-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via colocalization fluorescence microscopy. DMPO adducts were also confirmed in diseased/nondiseased tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p < 0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, noninvasive, in vivo diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models.

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide affects stress response and fate of lipopolysaccharide-primed RAW 264.7 macrophage cells.

The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly used to study free radicals. Due to its free radical trapping properties, DMPO is thought to reduce free radial-mediated oxidative damage and other related cellular responses. The purpose of this study was to assess the effect of DMPO on lipopolysaccharide (LPS)-induced inflammation, endoplasmic reticulum (ER) stress, and apoptosis in RAW 264.7 cells. The results showed that DMPO at 50 mM inhibited inducible nitric oxide synthase expression when added shortly after LPS treatment (≤3 h). Interestingly, DMPO increased anti-inflammatory heme oxygenase-1 (HO-1) expression and reversed LPS-induced decrease in HO-1 expression. LPS could increase cellular ER stress as indicated by C/EBP homologous protein (CHOP) induction; DMPO reduced LPS effect on CHOP expression. Unexpectedly, DMPO had a synergistic effect with LPS on increased caspase-3 activity. Overall, DMPO harbors multiple modulating effects but may induce apoptosis in LPS-stressed cells when given at 50 mM, an effective dose for its anti-inflammatory activity in vitro. Our data provide clues for further understanding of the nitrone spin trap with therapeutic potential.

In vivo imaging of immuno-spin trapped radicals with molecular magnetic resonance imaging in a diabetic mouse model.

Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.

Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide.

The free-radical-operated mechanism of death of activated macrophages at sites of inflammation is unclear, but it is important to define it in order to find targets to prevent further tissue dysfunction. A well-defined model of macrophage activation at sites of inflammation is the treatment of RAW 264.7 cells with lipopolysaccharide (LPS), with the resulting production of reactive oxygen species (ROS). ROS and other free radicals can be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a cell-permeable probe with antioxidant properties, which thus interferes with free-radical-operated oxidation processes. Here we have used immuno-spin trapping to investigate the role of free-radical-operated protein oxidation in LPS-induced cytotoxicity in macrophages. Treatment of RAW 264.7 cells with LPS resulted in increased ROS production, oxidation of proteins, cell morphological changes and cytotoxicity. DMPO was found to trap protein radicals to form protein-DMPO nitrone adducts, to reduce protein carbonyls, and to block LPS-induced cell death. N-Acetylcysteine (a source of reduced glutathione), diphenyleneiodonium (an inhibitor of NADPH oxidase), and 2,2'-dipyridyl (a chelator of Fe(2+)) prevented LPS-induced oxidative stress and cell death and reduced DMPO-nitrone adduct formation, suggesting a critical role of ROS, metals, and protein-radical formation in LPS-induced cell cytotoxicity. We also determined the subcellular localization of protein-DMPO nitrone adducts and identified some candidate proteins for DMPO attachment by LC-MS/MS. The LC-MS/MS data are consistent with glyceraldehyde-3-phosphate dehydrogenase, one of the most abundant, sensitive, and ubiquitous proteins in the cell, becoming labeled with DMPO when the cell is primed with LPS. This information will help find strategies to treat inflammation-associated tissue dysfunction by focusing on preventing free radical-operated proteotoxic stress and death of macrophages.

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells.

AIM: Exposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells. MAIN METHODS: RAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined. KEY FINDINGS: After cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15-60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production. SIGNIFICANCE: DMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.

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.

Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite.

Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical ((.)SO(3)(-)). This free radical further reacts with oxygen to form peroxymonosulfate anion radical ((-)O(3)SOO(.)) and the very reactive sulfate anion radical (SO(4)()), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H(2)O(2) is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO(4)()), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H(2)O(2) in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H(2)O(2) induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders.

Myeloperoxidase-induced genomic DNA-centered radicals.

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.