Immuno-spin trapping of macromolecules free radicals in vitro and in vivo - One stop shopping for free radical detection.

The only general technique that allows the unambiguous detection of free radicals is electron spin resonance (ESR). However, ESR spin trapping has severe limitations especially in biological systems. The greatest limitation of ESR is poor sensitivity relative to the low steady-state concentration of free radical adducts, which in cells and in vivo is much lower than the best sensitivity of ESR. Limitations of ESR have led to an almost desperate search for alternatives to investigate free radicals in biological systems. Here we explore the use of the immuno-spin trapping technique, which combine the specificity of the spin trapping to the high sensitivity and universal use of immunological techniques. All of the immunological techniques based on antibody binding have become available for free radical detection in a wide variety of biological systems.

Nitric oxide reverses drug resistance by inhibiting ATPase activity of p-glycoprotein in human multi-drug resistant cancer cells.

BACKGROUND: Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of drug efflux proteins, including P-170 glycoprotein (P-gp), an ATP-dependent efflux protein, is one of the main mechanisms responsible for multi-drug resistance (MDR). Because our previous studies have shown that nitric oxide (˙NO) or its related species inhibit the ATPase activities of topoisomerase II, we hypothesized that ˙NO should also inhibit the ATPase activity of P-gp and increase drug accumulation in MDR cells, causing a reversal of drug resistance. RESULTS: Cytotoxicity and cellular accumulation studies showed that ˙NO significantly inhibited the ATPase activity of P-gp in isolated membranes and in NCI/ADR-RES tumor cells, causing an increase in drug accumulation and reversals of adriamycin and taxol resistance in the MDR cells. While ˙NO had no effects on topoisomerase II-induced, adriamycin-dependent DNA cleavage complex formation, it significantly inhibited adriamycin-induced DNA double-strand breaks. Electron spin resonance studies showed an increase in adriamycin-dependent hydroxyl radical formation in the presence of an NO-donor. CONCLUSIONS: The reversal of drug resistance is due to inhibition of the ATPase activity by ˙NO, resulting in enhancement of the drug accumulation in the MDR cells. Furthermore, DNA damage was not responsible for this reversal of adriamycin resistance. However, formation of adriamycin-dependent toxic free radical species and subsequent cellular damage may be responsible for the increased cytotoxicity of adriamycin by ˙NO in NCI/ADR-RES cells. GENERAL SIGNIFICANCE: Appropriately designed NO donors would be ideal for the treatment of P-gp-overexpressing tumors in the clinic.

Switch of Mitochondrial Superoxide Dismutase into a Prooxidant Peroxidase in Manganese-Deficient Cells and Mice.

Superoxide radical anion (O2⋅‒) and other reactive oxygen species are constantly produced during respiration. In mitochondria, the dismutation of O2⋅‒ is accelerated by the mitochondrial superoxide dismutase 2 (SOD2), an enzyme that has been traditionally associated with antioxidant protection. However, increases in SOD2 expression promote oxidative stress, indicating that there may be a prooxidant role for SOD2. Here we show that SOD2, which normally binds manganese, can incorporate iron and generate an alternative isoform with peroxidase activity. The switch from manganese to iron allows FeSOD2 to utilize H2O2 to promote oxidative stress. We found that FeSOD2 is formed in cultured cells and in vivo. FeSOD2 causes mitochondrial dysfunction and higher levels of oxidative stress in cultured cells and in vivo. We show that formation of FeSOD2 converts an antioxidant defense into a prooxidant peroxidase that leads to cellular changes seen in multiple human diseases.

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease.

Exposure to (bi)sulfite (HSO3-) and sulfite (SO32-) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3-), peroxymonosulfate (-O3SOO.), and especially the sulfate (SO4. -) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease.

Elevated plasma 8-iso-prostaglandin F2α levels in human smokers originate primarily from enzymatic instead of non-enzymatic lipid peroxidation.

It is widely accepted that free radicals in tobacco smoke lead to oxidative stress and generate the popular lipid peroxidation biomarker 8-iso-prostaglandin F2α (8-iso-PGF2α). However, 8-iso-PGF2α can simultaneously be produced in vivo by the prostaglandin-endoperoxide synthases (PGHS) induced by inflammation. This inflammation-dependent mechanism has never been considered as a source of elevated 8-iso-PGF2α in tobacco smokers. The goal of this study is to quantify the distribution of chemical- and PGHS-dependent 8-iso-PGF2α formation in the plasma of tobacco smokers and non-smokers. The influences of gender and hormonal contraceptive use were accounted for. The distribution was determined by measuring the 8-iso-PGF2α/prostaglandin F2α (PGF2α) ratio. When comparing smokers (n = 28) against non-smokers (n = 30), there was a statistically significant increase in the 8-iso-PGF2α concentration. The source of this increased 8-iso-PGF2α was primarily from PGHS. When stratifying for gender, the increase in 8-iso-PGF2α in male smokers (n = 9) was primarily from PGHS. Interestingly, female smokers on hormonal contraceptives had increased 8-iso-PGF2α in both pathways, whereas those not on hormonal contraceptives did not have increased 8-iso-PGF2α. In conclusion, increased plasma 8-iso-PGF2α in tobacco smokers has complex origins, with PGHS-dependent formation as the primary source. Accounting for both pathways provides a definitive measurement of both oxidative stress and inflammation.

Synergistic enhancement of topotecan-induced cell death by ascorbic acid in human breast MCF-7 tumor cells.

Topotecan, a derivative of camptothecin, is an important anticancer drug for the treatment of various human cancers in the clinic. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I, other mechanisms, e.g., oxidative stress induced by reactive free radicals, have also been proposed. However, very little is known about how topotecan induces free radical-dependent oxidative stress in tumor cells. In this report we describe the formation of a topotecan radical, catalyzed by a peroxidase-hydrogen peroxide system. While this topotecan radical did not undergo oxidation-reduction with molecular O2, it rapidly reacted with reduced glutathione and cysteine, regenerating topotecan and forming the corresponding glutathiyl and cysteinyl radicals. Ascorbic acid, which produces hydrogen peroxide in tumor cells, significantly increased topotecan cytotoxicity in MCF-7 tumor cells. The presence of ascorbic acid also increased both topoisomerase I-dependent topotecan-induced DNA cleavage complex formation and topotecan-induced DNA double-strand breaks, suggesting that ascorbic acid participated in enhancing DNA damage induced by topotecan and that the enhanced DNA damage is responsible for the synergistic interactions of topotecan and ascorbic acid. Cell death by topotecan and the combination of topotecan and ascorbic acid was predominantly due to necrosis of MCF-7 breast tumor cells.

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells.

BACKGROUND: Topoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide (•NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide. METHODS: Here, we have evaluated the roles of • NO/ • NO-derived species in the stability and activity of topo II (α and ß) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of • NO on the ATPase activity of topo II. RESULTS: Treatment of purified topo IIα and ß with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. • NO-induced inhibition of these topo II (α and ß) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells. CONCLUSION: PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells. GENERAL SIGNIFICANCE: As tumors express nitric oxide synthase and generate • NO, inhibition of topo II functions by • NO/ • NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Chronic Endurance Exercise Impairs Cardiac Structure and Function in Middle-Aged Mice with Impaired Nrf2 Signaling.

Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling maintains the redox homeostasis and its activation is shown to suppress cardiac maladaptation. Earlier we reported that acute endurance exercise (2 days) evoked antioxidant cytoprotection in young WT animals but not in aged WT animals. However, the effect of repeated endurance exercise during biologic aging (WT) characterized by an inherent deterioration in Nrf2 signaling and pathological aging (pronounced oxidative susceptibility-Nrf2 absence) in the myocardium remains elusive. Thus, the purpose of our study was to determine the effect of chronic endurance exercise-induced cardiac adaptation in aged mice with and without Nrf2. Age-matched WT and Nrf2-null mice (Nrf2-/-) (>22 months) were subjected to 6 weeks chronic endurance exercise (25 meter/min, 12% grade). The myocardial redox status was assessed by expression of antioxidant defense genes and proteins along with immunochemical detection of DMPO-radical adduct, GSH-NEM, and total ubiquitination. Cardiac functions were assessed by echocardiography and electrocardiogram. At sedentary state, loss of Nrf2 resulted in significant downregulation of antioxidant gene expression (Nqo1, Ho1, Gclm, Cat, and Gst-α) with decreased GSH-NEM immuno-fluorescence signals. While Nrf2-/- mice subjected to CEE showed an either similar or more pronounced reduction in the transcript levels of Gclc, Nqo1, Gsr, and Gst-α in relation to WT littermates. In addition, the hearts of Nrf2-/- on CEE showed a substantial reduction in specific antioxidant proteins, G6PD and CAT along with decreased GSH, a pronounced increase in DMPO-adduct and the total ubiquitination levels. Further, CEE resulted in a significant upregulation of hypertrophy genes (Anf, Bnf, and ß-Mhc) (p < 0.05) in the Nrf2-/- hearts in relation to WT mice. Moreover, the aged Nrf2-/- mice exhibited a higher degree of cardiac remodeling in association with a significant decrease in fractional shortening, pronounced ST segment, and J wave elevation upon CEE compared to age-matched WT littermates. In conclusion, our findings indicate that while the aged WT and Nrf2 knockout animals both exhibit hypertrophy after CEE, the older Nrf2 knockouts showed ventricular remodeling coupled with profound cardiac functional abnormalities and diastolic dysfunction.

Classifying oxidative stress by F2-isoprostane levels across human diseases: A meta-analysis.

The notion that oxidative stress plays a role in virtually every human disease and environmental exposure has become ingrained in everyday knowledge. However, mounting evidence regarding the lack of specificity of biomarkers traditionally used as indicators of oxidative stress in human disease and exposures now necessitates re-evaluation. To prioritize these re-evaluations, published literature was comprehensively analyzed in a meta-analysis to quantitatively classify the levels of systemic oxidative damage across human disease and in response to environmental exposures. In this meta-analysis, the F2-isoprostane, 8-iso-PGF2α, was specifically chosen as the representative marker of oxidative damage. To combine published values across measurement methods and specimens, the standardized mean differences (Hedges' g) in 8-iso-PGF2α levels between affected and control populations were calculated. The meta-analysis resulted in a classification of oxidative damage levels as measured by 8-iso-PGF2α across 50 human health outcomes and exposures from 242 distinct publications. Relatively small increases in 8-iso-PGF2α levels (g<0.8) were found in the following conditions: hypertension (g=0.4), metabolic syndrome (g=0.5), asthma (g=0.4), and tobacco smoking (g=0.7). In contrast, large increases in 8-iso-PGF2α levels were observed in pathologies of the kidney, e.g., chronic renal insufficiency (g=1.9), obstructive sleep apnoea (g=1.1), and pre-eclampsia (g=1.1), as well as respiratory tract disorders, e.g., cystic fibrosis (g=2.3). In conclusion, we have established a quantitative classification for the level of 8-iso-PGF2α generation in different human pathologies and exposures based on a comprehensive meta-analysis of published data. This analysis provides knowledge on the true involvement of oxidative damage across human health outcomes as well as utilizes past research to prioritize those conditions requiring further scrutiny on the mechanisms of biomarker generation.

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•-) and H2O2 in the presence of NADH. Generation of the free radical O2•- and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

Role of cytochrome c in α-synuclein radical formation: implications of α-synuclein in neuronal death in Maneb- and paraquat-induced model of Parkinson's disease.

BACKGROUND: The pathological features of Parkinson's disease (PD) include an abnormal accumulation of α-synuclein in the surviving dopaminergic neurons. Though PD is multifactorial, several epidemiological reports show an increased incidence of PD with co-exposure to pesticides such as Maneb and paraquat (MP). In pesticide-related PD, mitochondrial dysfunction and α-synuclein oligomers have been strongly implicated, but the link between the two has not yet been understood. Similarly, the biological effects of α-synuclein or its radical chemistry in PD is largely unknown. Mitochondrial dysfunction during PD pathogenesis leads to release of cytochrome c in the cytosol. Once in the cytosol, cytochrome c has one of two fates: It either binds to apaf1 and initiates apoptosis or can act as a peroxidase. We hypothesized that as a peroxidase, cytochrome c leaked out from mitochondria can form radicals on α-synuclein and initiate its oligomerization. METHOD: Samples from controls, and MP co-exposed wild-type and α-synuclein knockout mice were studied using immuno-spin trapping, confocal microscopy, immunohistochemistry, and microarray experiments. RESULTS: Experiments with MP co-exposed mice showed cytochrome c release in cytosol and its co-localization with α-synuclein. Subsequently, we used immuno-spin trapping method to detect the formation of α-synuclein radical in samples from an in vitro reaction mixture consisting of cytochrome c, α-synuclein, and hydrogen peroxide. These experiments indicated that cytochrome c plays a role in α-synuclein radical formation and oligomerization. Experiments with MP co-exposed α-synuclein knockout mice, in which cytochrome c-α synuclein co-localization and interaction cannot occur, mice showed diminished protein radical formation and neuronal death, compared to wild-type MP co-exposed mice. Microarray data from MP co-exposed wild-type and α-synuclein knockout mice further showed that the absence of α-synuclein per se or its co-localization with cytochrome c confers protection from MP co-exposure, as several important pathways were unaffected in α-synuclein knockout mice. CONCLUSIONS: Altogether, these results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein, through its co-localization with cytochrome c or on its own, affects several biological pathways which contribute to increased neuronal death in an MP-induced model of PD.

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells.

BACKGROUND: Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions. METHODS: Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin. RESULTS: Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines. CONCLUSION: Inhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells. GENERAL SIGNIFICANCE: As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Reinterpreting the best biomarker of oxidative stress: The 8-iso-prostaglandin F2α/prostaglandin F2α ratio shows complex origins of lipid peroxidation biomarkers in animal models.

Oxidative stress is elevated in numerous environmental exposures and diseases. Millions of dollars have been spent to try to ameliorate this damaging process using anti-oxidant therapies. Currently, the best accepted biomarker of oxidative stress is the lipid oxidation product 8-iso-prostaglandin F2α (8-iso-PGF2α), which has been measured in over a thousand human and animal studies. 8-iso-PGF2α generation has been exclusively attributed to nonenzymatic chemical lipid peroxidation (CLP). However, 8-iso-PGF2α can also be produced enzymatically by prostaglandin-endoperoxide synthases (PGHS) in vivo. When failing to account for PGHS-dependent generation, 8-iso-PGF2α cannot be interpreted as a selective biomarker of oxidative stress. We investigated the formation of 8-iso-PGF2α in rats exposed to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) using the 8-iso-PGF2α/PGF2α ratio to quantitatively determine the source(s) of 8-iso-PGF2α. Upon exposure to a 120mg/kg dose of CCl4, the contribution of CLP accounted for only 55.6±19.4% of measured 8-iso-PGF2α, whereas in the 1200mg/kg dose, CLP was the predominant source of 8-iso-PGF2α (86.6±8.0% of total). In contrast to CCl4, exposure to 0.5mg/kg LPS was characterized by a significant increase in both the contribution of PGHS (59.5±7.0) and CLP (40.5±14.0%). In conclusion, significant generation of 8-iso-PGF2α occurs through enzymatic as well as chemical lipid peroxidation. The distribution of the contribution is dependent on the exposure agent as well as the dose. The 8-iso-PGF2α/PGF2α ratio accurately determines the source of 8-iso-PGF2α and provides an absolute measure of oxidative stress in vivo.

Formation and Implications of Alpha-Synuclein Radical in Maneb- and Paraquat-Induced Models of Parkinson's Disease.

Parkinson's disease (PD) is a debilitating, progressive, neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and motor deficits. Alpha-synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including PD; however, the mechanism that initiates and promotes intraneuronal alpha-synuclein aggregation remains unknown. We hypothesized protein radical formation as an initiating mechanism for alpha-synuclein aggregation. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of alpha-synuclein aggregation as well as to investigate the source of protein radical formation in the midbrains of Maneb- and paraquat-coexposed mice. Coexposure to Maneb and paraquat for 6 weeks resulted in active microgliosis, NADPH oxidase activation, and inducible nitric oxide synthase (iNOS) induction, which culminated in protein radical formation in the midbrains of mice. Results obtained with immuno-spin trapping and immunoprecipitation experiments confirmed formation of alpha-synuclein radicals in dopaminergic neurons of exposed mice. Free radical formation requires NADPH oxidase and iNOS, as indicated by decreased protein radical formation in knockout mice (P47phox(-/-) and iNOS(-/-)) and in mice treated with inhibitors such as FeTPPS (a peroxynitrite decomposition catalyst), 1400 W (an iNOS inhibitor), or apocynin (a NADPH oxidase inhibitor). Concurrence of protein radical formation with dopaminergic neuronal death indicated a link between protein radicals and disease progression. Taken together, these results show for the first time the formation and detection of the alpha-synuclein radical and suggest that NADPH oxidase and iNOS play roles in peroxynitrite-mediated protein radical formation and subsequent neuronal death in the midbrains of Maneb- and paraquat-coexposed mice.

Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone and/or polymerization of monomers into dimers, multimers, and sometimes insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions that have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine, and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion-limited rate with hydroxyl radical and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral, whereas others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and the weaknesses of some of the techniques used to detect these oxidative interactions, and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants, and microbes.

Loss of NOX-Derived Superoxide Exacerbates Diabetogenic CD4 T-Cell Effector Responses in Type 1 Diabetes.

Reactive oxygen species (ROS) play prominent roles in numerous biological systems. While classically expressed by neutrophils and macrophages, CD4 T cells also express NADPH oxidase (NOX), the superoxide-generating multisubunit enzyme. Our laboratory recently demonstrated that superoxide-deficient nonobese diabetic (NOD.Ncf1(m1J)) mice exhibited a delay in type 1 diabetes (T1D) partially due to blunted IFN-γ synthesis by CD4 T cells. For further investigation of the roles of superoxide on CD4 T-cell diabetogenicity, the NOD.BDC-2.5.Ncf1(m1J) (BDC-2.5.Ncf1(m1J)) mouse strain was generated, possessing autoreactive CD4 T cells deficient in NOX-derived superoxide. Unlike NOD.Ncf1(m1J), stimulated BDC-2.5.Ncf1(m1J) CD4 T cells and splenocytes displayed elevated synthesis of Th1 cytokines and chemokines. Superoxide-deficient BDC-2.5 mice developed spontaneous T1D, and CD4 T cells were more diabetogenic upon adoptive transfer into NOD.Rag recipients due to a skewing toward impaired Treg suppression. Exogenous superoxide blunted exacerbated Th1 cytokines and proinflammatory chemokines to approximately wild-type levels, concomitant with reduced IL-12Rß2 signaling and P-STAT4 (Y693) activation. These results highlight the importance of NOX-derived superoxide in curbing autoreactivity due, in part, to control of Treg function and as a redox-dependent checkpoint of effector T-cell responses. Ultimately, our studies reveal the complexities of free radicals in CD4 T-cell responses.

OKN-007 decreases free radical levels in a preclinical F98 rat glioma model.

Free radicals are associated with glioma tumors. Here, we report on the ability of an anticancer nitrone compound, OKN-007 [Oklahoma Nitrone 007; a disulfonyl derivative of α-phenyl-tert-butyl nitrone (PBN)] to decrease free radical levels in F98 rat gliomas using combined molecular magnetic resonance imaging (mMRI) and immunospin-trapping (IST) methodologies. Free radicals are trapped with the spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), to form DMPO macromolecule radical adducts, and then further tagged by immunospin trapping by an antibody against DMPO adducts. In this study, we combined mMRI with a biotin-Gd-DTPA-albumin-based contrast agent for signal detection with the specificity of an antibody for DMPO nitrone adducts (anti-DMPO probe), to detect in vivo free radicals in OKN-007-treated rat F98 gliomas. OKN-007 was found to significantly decrease (P < 0.05) free radical levels detected with an anti-DMPO probe in treated animals compared to untreated rats. Immunoelectron microscopy was used with gold-labeled antibiotin to detect the anti-DMPO probe within the plasma membrane of F98 tumor cells from rats administered anti-DMPO in vivo. OKN-007 was also found to decrease nuclear factor erythroid 2-related factor 2, inducible nitric oxide synthase, 3-nitrotyrosine, and malondialdehyde in ex vivo F98 glioma tissues via immunohistochemistry, as well as decrease 3-nitrotyrosine and malondialdehyde adducts in vitro in F98 cells via ELISA. The results indicate that OKN-007 effectively decreases free radicals associated with glioma tumor growth. Furthermore, this method can potentially be applied toward other types of cancers for the in vivo detection of macromolecular free radicals and the assessment of antioxidants.

Iron incorporation into MnSOD A (bacterial Mn-dependent superoxide dismutase) leads to the formation of a peroxidase/catalase implicated in oxidative damage to bacteria.

BACKGROUND: Mn/Fe-superoxide dismutase (SOD) is a family of enzymes essential for organisms to be able to cope with oxygen. These enzymes bound to their classical metals catalyze the dismutation of the free radical superoxide anion (O2(-)) to H2O2 and molecular oxygen. E. coli has the manganese-dependent SOD A and the iron-dependent SOD B. METHODS: Strains of E. coli overexpressing SOD A or SOD B were grown in media with different metal compositions. SODs were purified and their metal content and SOD activity were determined. Those proteins were incubated with H2O2 and assayed for oxidation of Amplex red or o-phenylenediamine, consumption of H2O2, release of iron and protein radical formation. Cell survival was determined in bacteria with MnSOD A or FeSOD A after being challenged with H2O2. RESULTS: We show for the first time that the bacterial manganese-dependent SOD A when bound to iron (FeSOD A) has peroxidase activity. The in vivo formation of the peroxidase FeSOD A was increased when media had higher levels of iron because of a decreased manganese metal incorporation. In comparison to bacteria with MnSOD A, cells with FeSOD A had a higher loss of viability when exposed to H2O2. GENERAL SIGNIFICANCE: The biological occurrence of this fundamental antioxidant enzyme in an alternative iron-dependent state represents an important source of free radical formation.

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the ß-globin chain of the Hb:Hp complex, including decreased ßCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.