Baicalein restrains proliferation, migration, and invasion of human malignant melanoma cells by down-regulating colon cancer associated transcript-1.

Baicalein (BAI) is an acknowledged flavonoids compound, which is regarded as a useful therapeutic pharmaceutical for numerous cancers. However, its involvement in melanoma is largely unknown. This study aimed to examine the anti-melanoma function of BAI and unraveled the regulatory mechanism involved. A375 and SK-MEL-28 were treated with BAI for 24 h. Then, CCK-8 assay, flow cytometry, and transwell assay were carried out to investigate cell growth, migration, and invasion. RT-qPCR was applied to detect the expression of colon cancer associated transcript-1 (CCAT1) in melanoma tissues and cells. The functions of CCAT1 in melanoma cells were also evaluated. Western blot was utilized to appraise Wnt/ß-catenin or MEK/ERK pathways. BAI restrained cell proliferation and stimulated cell apoptotic capability of melanoma by suppressing cleaved-caspase-3 and cleaved-PARP. Cell migratory and invasive abilities were restrained by BAI via inhibiting MMP-2 and vimentin. CCAT1 was over-expressed in melanoma tissues and down-regulated by BAI in melanoma cells. Overexpressed CCAT1 reversed the BAI-induced anti-growth, anti-migratory, and anti-invasive effects. Furthermore, BAI inhibited Wnt/ß-catenin and MEK/ERK pathways-axis via regulating CCAT1. Our study indicated that BAI blocked Wnt/ß-catenin and MEK/ERK pathways via regulating CCAT1, thereby inhibiting melanoma cell proliferation, migration, and invasion.

Baicalein retards proliferation and collagen deposition by activating p38MAPK-JNK via microRNA-29.

Immoderate proliferation and deposition of collagen generally result in hypertrophic scars and even keloids. microRNA-29 (miR-29) has been proved as a crucial regulator in these pathological processes. Although mounting evidence have proved baicalein (BAI) impairs scar formation, it is still incompletely understood whether miR-29 participated in the underlying mechanism. In the present study, NIH-3T3 cells were stimulated with BAI, and then cell viability was analyzed by cell counting kit-8 (CCK-8) and Western blot. We further analyzed total soluble collagen, collagen 1, and alpha-smooth muscle actin (α-SMA) in NIH-3T3 cells, which were exposed to transforming growth factor beta 1 (TGF-ß1)/BAI, using a Sircol assay kit, quantitative reverse transcription-PCR (qRT-PCR) and Western blot, respectively. Besides, the miR-29 inhibitor was transduced and its transfection efficiency was verified by qRT-PCR. Finally, the phosphorylated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) were examined by Western blot. BAI effectively retarded NIH-3T3 proliferation in a dose-dependent manner. Besides, TGF-ß1-induced deposition of total soluble collagen and synthesis of collagen 1 and α-SMA were repressed by BAI at mRNA and protein levels. However, miR-29 inhibitor reversed the effects of BAI. Remarkably, BAI promoted phosphorylated expression of p38MAPK and JNK while miR-29 inhibitor reversed its effects on the phosphorylated expression of p38MAPK and JNK. BAI effectively weakened the cell viability and repressed TGF-ß1-induced total soluble collagen as well as collagen 1 and α-SMA by upregulating miR-29. Mechanically, BAI activates the p38MAPK/JNK pathway by promoting miR-29.

Baicalein restrains proliferation, migration, and invasion of human malignant melanoma cells by down-regulating colon cancer associated transcript-1

Baicalein (BAI) is an acknowledged flavonoids compound, which is regarded as a useful therapeutic pharmaceutical for numerous cancers. However, its involvement in melanoma is largely unknown. This study aimed to examine the anti-melanoma function of BAI and unraveled the regulatory mechanism involved. A375 and SK-MEL-28 were treated with BAI for 24 h. Then, CCK-8 assay, flow cytometry, and transwell assay were carried out to investigate cell growth, migration, and invasion. RT-qPCR was applied to detect the expression of colon cancer associated transcript-1 (CCAT1) in melanoma tissues and cells. The functions of CCAT1 in melanoma cells were also evaluated. Western blot was utilized to appraise Wnt/β-catenin or MEK/ERK pathways. BAI restrained cell proliferation and stimulated cell apoptotic capability of melanoma by suppressing cleaved-caspase-3 and cleaved-PARP. Cell migratory and invasive abilities were restrained by BAI via inhibiting MMP-2 and vimentin. CCAT1 was over-expressed in melanoma tissues and down-regulated by BAI in melanoma cells. Overexpressed CCAT1 reversed the BAI-induced anti-growth, anti-migratory, and anti-invasive effects. Furthermore, BAI inhibited Wnt/β-catenin and MEK/ERK pathways-axis via regulating CCAT1. Our study indicated that BAI blocked Wnt/β-catenin and MEK/ERK pathways via regulating CCAT1, thereby inhibiting melanoma cell proliferation, migration, and invasion.

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.

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.

Role of nitric oxide in the chemistry and anticancer activity of etoposide (VP-16,213).

Originally identified as an innate cytotoxin, nitric oxide ((·)NO) formation in tumors can influence chemotherapy and exacerbate cancer progression. Here, we examined the hypothesis that (·)NO generation contributes to cancer cell drug resistance toward the widely used anticancer drug Etoposide (VP-16). The UV-vis spectrum of VP-16 was not changed by exposure of VP-16 to (·)NO in aqueous buffer. In contrast, reddish-orange compound(s) characteristic of o-quinone- and nitroso-VP-16 were readily generated in a hydrophobic medium (chloroform) in an oxygen-dependent manner. Similar products were also formed when the VP-16 radical, generated from VP-16 and horseradish peroxidase/H2O2, was exposed directly to (·)NO in chloroform in the presence of oxygen. Separation and spectral analysis of VP-16 reaction extracts by electron spin resonance and UV-vis indicated the generation of the phenoxy radical and the o-quinone of VP-16, as well as putative nitroxide, iminoxyl, and other nitrogen oxide intermediates. Nitric oxide products of VP-16 displayed significantly diminished topoisomerase II-dependent cleavage of DNA and cytotoxicity to human HL-60 leukemia cells. LPS-mediated induction of nitric oxide synthase in murine macrophages resulted in VP-16 resistance compared to Raw cells. Furthermore, (·)NO products derived from iNOS rapidly reacted with VP-16 leading to decreased DNA damage and cytotoxicity. Together, these observations suggest that the formation of (·)NO in tumors (associated macrophages) can contribute to VP-16 resistance via the detoxification of VP-16.

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.

Detection and imaging of the free radical DNA in cells--site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy.

Oxidative stress-related damage to the DNA macromolecule produces lesions that are implicated in various diseases. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Measurement of DNA damage has been a matter of debate as most of the available methods measure the end product of a sequence of events and provide limited information on the initial free radical formation. We report a measurement of free radical damage in DNA induced by a Cu(II)-H(2)O(2) oxidizing system using immuno-spin trapping supplemented with electron paramagnetic resonance. In this investigation, the short-lived radical generated is trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) immediately upon formation. The DMPO adduct formed is initially electron paramagnetic resonance active, but is subsequently oxidized to the stable nitrone adduct, which can be detected and visualized by immuno-spin trapping and has the potential to be further characterized by other analytical techniques. The radical was found to be located on the 2'-deoxyadenosine (dAdo) moiety of DNA. The nitrone adduct was repaired on a time scale consistent with DNA repair. In vivo experiments for the purpose of detecting DMPO-DNA nitrone adducts should be conducted over a range of time in order to avoid missing adducts due to the repair processes.

Simplified synthesis of isotopically labeled 5,5-dimethyl-pyrroline N-oxide.

5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment.

Evaluation of the Forrester-Hepburn mechanism as an artifact source in ESR spin-trapping.

Nitrone spin traps such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are commonly used for free radical detection. Though proven examples are rare, artifact formation must be considered. For example, the Forrester-Hepburn mechanism yields the same radical adduct as that formed by genuine radical trapping. A hydroxylamine is formed by nucleophilic attack of the substrate on DMPO and subsequently oxidized to the respective nitroxide radical. One potential candidate for this artifact is the sulfur trioxide radical adduct (DMPO/(•)SO(3)(-)), as detected in spin-trapping experiments with horseradish peroxidase and sulfite. It has previously been shown by NMR experiments that the hydroxylamine intermediate does indeed form, but no direct proof for the ESR artifact has been provided. Here, we used isotopically labeled DMPO with horseradish peroxidase and ferricyanide to test for the Forrester-Hepburn artifact directly in a spin-trapping experiment. Besides sulfite, we investigated other nucleophiles such as cyanide, cysteine, and glutathione. Neither sulfite nor biological thiols produced detectable spin-trapping artifacts, but with cyanide the relatively weak signal originated entirely from the nucleophilic reaction. The hydroxylamine intermediate, which is more abundant with cyanide than with sulfite, was identified as cyano-hydroxylamine by means of 2D NMR experiments. Although our study found that spin trapping provided authentic free radical signals with most of the substrates, the occurrence of the Forrester-Hepburn mechanism artifact with cyanide emphasizes the importance of isotope measurements with nucleophile substrates.

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.

Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red.

The photosensitized reduction of resorufin (RSF), the fluorescent product of Amplex Red, was investigated using electron spin resonance (ESR), optical absorption/fluorescence, and oxygen consumption measurements. Anaerobic reaction of RSF in the presence of the electron donor reduced nicotinamide adenine dinucleotide (NADH) demonstrated that during visible light irradiation (λ > 300 nm), RSF underwent one-electron reduction to produce a semiquinoneimine-type anion radical (RSF(•) ‾) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and NADH demonstrated, under irradiation with visible light, the production of the superoxide dismutase (SOD)-sensitive DMPO/(•)OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the RSF(•) ‾ was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH2). Both RSF(•) ‾ and RSFH2, when formed in an aerobic system, were immediately oxidized by oxygen, which regenerated the dye and formed superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode showed that molecular oxygen was consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirmed the production of superoxide and hydrogen peroxide.

Investigating the mechanisms of aromatic amine-induced protein free radical formation by quantitative structure-activity relationships: implications for drug-induced agranulocytosis.

Aromatic amine drugs have been associated with agranulocytosis (neutrophil depletion) for which the mechanism is unknown. We have previously shown that the metabolism of two aromatic amine drugs by human myeloperoxidase (MPO) results in phenyl radical metabolite formation and also in protein free radical formation on MPO. Because the concentration of drug required to produce a maximum signal for MPO protein free radical (MPO*) detection was different for each drug, this prompted us to consider that other aromatic amines may also show varying degrees of ability to induce MPO* formation. Immunoassay experiments using the immuno-spin-trapping technique were performed, which evaluated the potency of different aromatic amines containing the aniline substructure to generate the MPO*. Each reaction contained equal amounts of H(2)O(2), 5,5-dimethyl-1-pyrroline-N-oxide, MPO, and variable concentrations of aniline derivatives. Several physicochemical parameters for aniline derivatives were used to derive quantitative structure-activity relationship equations, which showed that the Hammett constant (sigma) best correlated with the MPO* formation for all aniline derivatives. More statistically robust equations were derived if the anilines were separated into mono- and disubstituted groups. However, some aniline derivatives did not induce MPO* formation. Using electron spin resonance spectroscopy, we evaluated the ability of all aniline derivatives tested to produce phenyl radical metabolites, as previously shown by spin trapping for the aromatic amine drugs. Interestingly, we found that only those aniline derivatives that produced a phenyl radical also formed MPO*. We propose that the phenyl radical is the reactive free radical metabolite responsible for generating the MPO*.

Lipid-derived free radical production in superantigen-induced interstitial pneumonia.

We studied the free radical generation involved in the development of interstitial pneumonia (IP) in an animal model of autoimmune disease. We observed an electron spin resonance (ESR) spectrum of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts detected in the lipid extract of lungs in autoimmune-prone mice after intratracheal instillation of staphylococcal enterotoxin B. The POBN adducts detected by ESR were paralleled by infiltration of macrophages and neutrophils into the bronchoalveolar lavage fluid. To further investigate the mechanism of free radical generation, mice were pretreated with the macrophage toxicant gadolinium chloride, which significantly suppressed the radical generation. Free radical generation was also decreased by pretreatment with the xanthine oxidase (XO) inhibitor allopurinol, the iron chelator Desferal, and the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Histopathologically, these drugs significantly reduced both the cell infiltration into the alveolar septal walls and the synthesis of pulmonary collagen fibers. Experiments with NADPH oxidase knockout mice showed that NADPH oxidase did not contribute to lipid radical generation. These results suggest that lipid-derived carbon-centered free radical production is important in the manifestation of IP and that a macrophage toxicant, an XO inhibitor, an iron chelator, and an iNOS inhibitor protect against both radical generation and the manifestation of IP.

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes.

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.

Procainamide, but not N-acetylprocainamide, induces protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.

Procainamide (PA) is a drug that is used to treat tachycardia in postoperative patients or for long-term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here, we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with PA/H 2O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis.

Cadmium generates reactive oxygen- and carbon-centered radical species in rats: insights from in vivo spin-trapping studies.

Cadmium (Cd) is a known industrial and environmental pollutant. In the present work, an in vivo spin-trapping technique was used in conjunction with electron spin resonance (ESR) spectroscopy to investigate free radical generation in rats following administration of cadmium chloride (CdCl2, 40 micromol/kg) and the spin trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 1 g/kg). In Cd-treated rats, POBN radical adducts were formed in the liver, were excreted into the bile, and exhibited an ESR spectrum consistent with a carbon-centered radical species probably derived from endogenous lipids. Isotope substitution of dimethyl sulfoxide [(CH3)2SO] with 13C demonstrated methyl radical formation (POBN/*13CH3). This adduct indicated the production of hydroxyl radical, which reacted with [(13CH3)2SO] to form *13CH3, which then reacted with POBN to form POBN/*13CH3. Depletion of hepatic glutathione by diethyl maleate significantly increased free radical production, whereas inactivation of Kupffer cells by gadolinium chloride and chelation of iron by desferal inhibited it. Treatment with the xanthine oxidase inhibitor allopurinol, the catalase inhibitor aminobenzotriazole, or the cytochrome P450 inhibitor 3-amino-1,2,4-triazole had no effect. This is the first study to show Cd generation of reactive oxygen- and carbon-centered radical species by involvement of both iron mediation through iron-catalyzed reactions and activation of Kupffer cells, the resident liver macrophages.

Electron transfer between a tyrosyl radical and a cysteine residue in hemoproteins: spin trapping analysis.

We investigated electron transfer between a tyrosyl radical and cysteine residue in two systems, oxyhemoglobin (oxyHb)/peroxynitrite/5,5-dimethyl-1-pyrroline N-oxide (DMPO) and myoglobin (Mb)/hydrogen peroxide/DMPO, using a combination of techniques including ESR, immuno-spin trapping (IST), and ESI/MS. These techniques show that the nitrone spin trap DMPO covalently binds to one or more amino acid radicals in the protein. Treating oxyHb with peroxynitrite and Mb with H2O2 in the presence of a low DMPO concentration yielded secondary Cys-DMPO radical adduct exclusively, whereas in the presence of high DMPO, more of the primary Tyr-DMPO radical adduct was detected. In both systems studied, we found that, at high DMPO concentrations, mainly tyrosyl radicals (Hb-Tyr42/Tyr24 and Mb-Tyr103) are trapped and the secondary electron-transfer reaction does not compete, whereas in the presence of low concentrations of DMPO, the secondary reaction predominates over tyrosyl trapping, and a thiyl radical is formed and then trapped (Hb-Cys93 or Mb-Cys110). With increasing concentrations of DMPO in the reaction medium, primary radicals have an increasing probability of being trapped. MS/MS was used to identify the specific Tyr and Cys residues forming radicals in the myoglobin system. All data obtained from this combination of approaches support the conclusion that the initial site of radical formation is a Tyr, which then abstracts an electron from a cysteine residue to produce a cysteinyl radical. This complex phenomenon of electron transfer from one radical to another has been investigated in proteins by IST, ESR, and MS.

An electron paramagnetic resonance investigation of the oxygen dependence of the arterial-venous gradient of nitrosyl hemoglobin in blood circulation.

Whether there is a nitrosyl hemoglobin (HbNO) gradient between the venous and the arterial parts of the circulatory system is a very controversial issue in nitric oxide research. We have carefully evaluated the measurement of HbNO concentration in blood using EPR generated in vivo by the NO donor DEANO under various oxygen tensions. We found that the absolute concentrations of HbNO in venous and arterial blood were the same within experimental error, independent of hemoglobin saturation; only the ratios of 5-coordinate and 6-coordinate HbNO differed. The HbNO concentration increased when the oxygen concentration breathed by the rats decreased in a manner that was linear in hemoglobin saturation. These results do not support the existence of an arterial-venous gradient of HbNO under our experimental conditions.

Aminoglutethimide-induced protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.

Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.