dimethyl-1-pyrroline-N-oxide Research Articles

Spin-trapping analysis for thermal degradation of poly(vinyl alcohol)

Spin-trapping electron spin resonance (ESR) and gel permeation chromatography (GPC) were applied to analyze the thermal degradation mechanisms of poly(vinyl alcohol) (PVA). The lifetime of the short-lived radical intermediate produced by thermal degradation at up to 200 °C was extended via the spin-trapping method for the purpose of investigating its molecular structure. Under the addition of the spin-trapping reagents dimethyl-1-pyrroline-N-oxide (DMPO) or 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS), into the PVA, the production of some radical intermediates, including hydroxyl (•OH) and some main chain radicals (–CH=CH–•CH– and •CH(OH)–CH2–), was confirmed via simulation analysis of the observed ESR spectra. The GPC measurement revealed that the cross-linking reaction occurred more often at temperatures above 100 °C and that the main chain scission also occurs significantly at around 200 °C. The elimination of •OH is therefore a key reaction in PVA degradation, and •CH(OH)–CH2– is produced by the β-scission from the main chain radical –CH2–•C(OH)–CH2–. This last radical was not observed in the present study; however, it should be produced through a hydrogen abstraction by •OH.

Insights into Interface Charge Extraction in a Noble-Metal-Free Doped Z-Scheme NiO@BiOCl Heterojunction

It is of great significance to thoroughly explore the interface charge extraction and migration in heterojunction systems, which could guide us to synthesize higher-efficiency photocatalytic materials. A novel noble-metal-free doped Z-scheme NiO@BiOCl heterojunction was found in this work. The corresponding heterostructure, interface electron extraction, and electron migration were investigated via first-principles calculation. 5,5′-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trapping electron spin resonance (ESR) and time-resolved photoluminescence (TRPL) tests were implemented to confirm the calculation results, which showed that electrons and holes stayed in the NiO (100) facet and BiOCl (110) facet, respectively. Owing to the large chemical potential of 2.40 V (vs ENHE) for the BiOCl valence-band hole, it possessed super activity to oxidize water into hydroxyl radicals or molecular oxygen. We hope this promising multifunctional photocatalytic material, therefore, NiO@BiOCl can be applied in advanced treatment of organic wastewater and oxygen production from photolysis water.

Exogenous PP2A inhibitor exacerbates the progression of nonalcoholic fatty liver disease via NOX2-dependent activation of miR21.

Nonalcoholic fatty liver disease (NAFLD) is an emerging global pandemic. Though significant progress has been made in unraveling the pathophysiology of the disease, the role of protein phosphatase 2A (PP2A) and its subsequent inhibition by environmental and genetic factors in NAFLD pathophysiology remains unclear. The present report tests the hypothesis that an exogenous PP2A inhibitor leads to hepatic inflammation and fibrogenesis via an NADPH oxidase 2 (NOX2)-dependent pathway in NAFLD. Results showed that microcystin (MC) administration, a potent PP2A inhibitor found in environmental exposure, led to an exacerbation of NAFLD pathology with increased CD68 immunoreactivity, the release of proinflammatory cytokines, and stellate cell activation, a process that was attenuated in mice that lacked the p47phox gene and miR21 knockout mice. Mechanistically, leptin-primed immortalized Kupffer cells (a mimicked model for an NAFLD condition) treated with apocynin or nitrone spin trap 5,5 dimethyl-1- pyrroline N-oxide (DMPO) had significantly decreased CD68 and decreased miR21 and α-smooth muscle actin levels, suggesting the role of NOX2-dependent reactive oxygen species in miR21-induced Kupffer cell activation and stellate cell pathology. Furthermore, NOX2-dependent peroxynitrite generation was primarily responsible for cellular events observed following MC exposure since incubation with phenylboronic acid attenuated miR21 levels, Kupffer cell activation, and inflammatory cytokine release. Furthermore, blocking of the AKT pathway attenuated PP2A inhibitor-induced NOX2 activation and miR21 upregulation. Taken together, we show that PP2A may have protective roles, and its inhibition exacerbates NAFLD pathology via activating NOX2-dependent peroxynitrite generation, thus increasing miR21-induced pathology.NEW & NOTEWORTHY Protein phosphatase 2A inhibition causes nonalcoholic steatohepatitis (NASH) progression via NADPH oxidase 2. In addition to a novel emchanism of action, we describe a new tool to describe NASH histopathology.

670nm light exposure increases the number of exosomes in the vessel bath and the number of endosomes in endothelial cells

In previous studies we demonstrated endothelial dependent vasodilation at 670nm red/near infrared light (670nm light) in healthy vessels and db/db mice related to S‐nitrosothiol release. Furthermore, the bath solution from intact 670nm light stimulated vessels could dilate 670nm light naïve vessels in a NO dependent manner. Therefore, we propose that 670nm light treatment induces endosome formation in endothelial cells (BAEC) and exosome release.METHODSBAEC were cultured on 4 chamber slides and treated with 100mW of 670nm light for 40seconds. Cells were immediately fixed and processed. BAEC were labeled with CD63, caveolin‐1, and FM95‐5 as endosome markers. Dimethyl‐1‐Pyrroline‐N‐Oxide (DMPO) was used to trap free radicals to form a nitroxide radical adduct which decays to produce a stable nitrone, which is recognized by the anti DMPO antibody. This enables us to recognize free radicals.Exosomes in the vessel bath of murine isolated facialis arteries after 670nm light exposure were counted with a nanosight particle counter and characterized by immunofluorescence. Briefly, the vessel bath was ultra‐centrifuged, the supernatant discarded, and the pellet was either analyzed by nanosight or cytospun onto slides. To identify exosomes, we labeled the cytospin with the styryl peptide FM95‐5 for lipid content, DMPO antibody to identify nitrone radicals in exosomes and von Willebrand Factor (vWF) to identify the origin of the exosomes.RESULTSBAEC exhibited higher fluorescence levels of the endosomal marker caveolin‐1 and CD63 after 670nm light treatment (Caveolin‐1: control 95.23 ± 2.73 vs 670nm light 111.53 ± 3.27, CD63: control 21.09 ± 0.74 vs 670nm light 62.47 ± 5.73). Light treatment also significantly increased DMPO (63.67 ± 6.78 vs. 670nm light 127.81 ± 14.87), FM5‐95 (control 8.33 ± 1.18 vs 670nm light 21.76 ± 1.92) and caveolin‐1(control 95.27 ± 2.73 vs 670nm light 111.53 ± 3.27) fluorescence intensity The number of exosomes in the vessel bath was increased after the 670nm light as demonstrated by nanosight (control 3.8 × 107 ± 410,000 vs. 670nm light 6.1×107 ± 294,000). DMPO fluorescence intensity was increased after 670nm light treatment in exosomes (control 87.43 ± 15.24 vs. 670nm light 133.99 ± 11.3) and endosomes (control 63.67 ± 6.78 vs. 670nm light 127.816 ± 14), and about 30% of FM 95‐5 positive exosomes were of endothelial cell origin as indicated by vWF positive labeling.In CONCLUSION 670nm light treatment induces endosome formation and exosome release. The intracellular vesicles have endosome and lipid droplet characteristics as demonstrated by CD63 and caveolin‐1 labeling. The endosomes and exosomes contain radical complexes as seen by DMPO labeling.Support or Funding InformationThis work was supported by the United States Veteran Health Administration, 1IK2BX002426 (NL).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

UVA-induced carbon-centred radicals in lightly pigmented cells detected using ESR spectroscopy

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2′-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2′-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid– guanine – radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.

Suicidal Inactivation of Methemoglobin by Generation of Thiyl Radical: Insight into NAC Mediated Protection in RBC

N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection against oxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC, studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in an H2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constants are ki = 8.5μM, kinact = 0.706 min(-1) and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NAC as a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidation product of NAC has been identified as the 5, 5'- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfur centered radical (a(N) = 15.2 G and a(H)=16.78 G). Binding studies indicate that NACox interacts at the heme moiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated from MetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatment reduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in the inactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC in hemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, and MetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.

Site-Specific Tryptophan Oxidation Induced by Autocatalytic Reaction of Polysorbate 20 in Protein Formulation

Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction. Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively. PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation. A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.

Indications of the reactive species in a heterogeneous Fenton-like reaction using Fe-containing zeolites

Heterogeneous Fenton-like reactions have potential applications in wastewater treatment. Recently, the use of zeolites containing iron species as catalysts in Fenton-like processes has gained increasing interest. While it has been assumed in the literature that hydroxyl radicals (OH ) are the dominant reactive species formed from H 2O 2 in the presence of Fe-zeolites, no direct evidence for this hypothesis exists so far. This work aims at the elucidation of the type of reactive species involved in the heterogeneous Fenton reaction using the Fe-containing zeolites Fe-ZSM5 and Fe-Beta. Electron paramagnetic resonance (EPR) measurements using dimethyl pyrroline-N-oxide (DMPO) as spin trap were carried out; the formation of the complex DMPO-OH was observed, which is often ascribed to a reaction with OH . However, significant differences were found in the kinetic deuterium isotope effect (KDIE) for methanol oxidation between the heterogeneous Fenton-like reaction and genuine OH reactions initiated by the homogeneous Fenton reaction. The KDIE for methanol oxidation in the heterogeneous reaction increased from 1.33 to 1.41 with increasing concentration of the OH scavenger bicarbonate, whereas the KDIE in the classical Fenton reaction is 1.07 and unaffected by the presence of quenchers. The significantly different values are a clear indication for the participation of additional oxidants beside OH . In addition, Fe-zeolites catalyzed the epoxidation of cyclohexene by H 2O 2. This type of reaction suggests the participation of a high-valent oxoiron complex (ferryl) in the wet peroxide oxidation of organic compounds catalyzed by Fe-zeolites.

Radical Scavenging Activity of the Japanese Traditional Food, Amazake

The traditional Japanese beverage amazake was evaluated as a possible treatment for oxidative stress. For this purpose, the radical scavenging activities of four kinds of amazake were investigated by determining diphenylpicrylhydrazyl radical, galvinoxyl radical and superoxide anion radical quenching activities using a spectrophotometric method and an electron spin resonance (ESR) technique. ESR measurements were used to assess the superoxide anion radical scavenging activity by employing dimethylpyrroline N-oxide as a spin trapping reagent. The ferricyanide-reducing activity and chelating activity were evaluated spectroscopically. The results indicated that amazake made from brown rice had stronger radical scavenging, metal chelating and metal reducing activities than amazake produced from white rice and sake cake (by-product of sake).

Intensity‐dependent Direct Solar Radiation‐ and UVA‐induced Radical Damage to Human Skin and DNA, Lipids and Proteins

Skin can be exposed to high-intensity UV-radiation in hot countries and during sunbed use; however, the free-radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free-radical generation in ex vivo human skin/substitutes +/- the spin-trap 5,5 dimethyl-1-pyrroline N-oxide (DMPO) exposed to solar-irradiation equivalent to Mediterranean sunlight. Skin-substitutes, model DNA-photosensitizer systems, lipids and proteins were also irradiated with low-intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail-keratin, tendon-collagen, phospholipid and DNA+melanin or riboflavin. In addition to lipid-derived (tentatively tert-alkoxyl/acyl-) and protein radicals detected with DMPO at lower intensities, isotropic carbon-, additional oxygen- and hydrogen-adducts were detected in solar-irradiated skin/substitutes at higher intensities. Carbon-adducts were detected in UVA-irradiated human skin cells, DNA+melanin or riboflavin and soybean-phospholipid. Anisotropic protein-adducts, comparable to adducts in solar-irradiated tendon-collagen, were absent in UVA-irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen-adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon- and oxygen-radicals) and part extracellular (protein- and hydrogen/H(+)+e(-) ), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid-oxidation is direct, DNA and protein-oxidation require photosensitisation.

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.

Electron Spin Resonance Spin Trapping Technique: Application to the Cleavage Process of Photoinitiators

AbstractThe cleavage of different photoinitiators (PIs) of polymerization is investigated by the ESR spin trapping using two different spin traps (STs): 5,5′‐dimethyl‐1‐pyrrolineN‐oxide (DMPO) and phenylN‐tert‐butyl nitrone (PBN). The hyperfine splitting constants for the different adducts are measured and provide a new set of data. These results show a difference of selectivity between the two (STs) and outline their complementarities.magnified image

Intramolecular Electron Transfer between Tyrosyl Radical and Cysteine Residue Inhibits Tyrosine Nitration and Induces Thiyl Radical Formation in Model Peptides Treated with Myeloperoxidase, H2O2, and NO2-: EPR SPIN TRAPPING STUDIES

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.

Clotrimazole Inhibits Hemoperoxidase of Plasmodium falciparum and Induces Oxidative Stress: PROPOSED ANTIMALARIAL MECHANISM OF CLOTRIMAZOLE

The mechanism of antimalarial activity of clotrimazole was studied placing emphasis on its role in inhibiting hemoperoxidase for inducing oxidative stress in Plasmodium falciparum. Clotrimazole, in the presence of H2O2, causes irreversible inactivation of the enzyme, and the inactivation follows pseudo-first order kinetics, consistent with a mechanism-based (suicide) mode. The pseudo-first order kinetic constants are ki = 2.85 microM, k(inact) = 0.9 min(-1), and t(1/2) = 0.77 min. The one-electron oxidation product of clotrimazole has been identified by EPR spectroscopy as the 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adduct of the nitrogen-centered radical (aN = 15 G), and as DMPO protects against inactivation, this radical is involved in the inactivation process. Binding studies indicate that the clotrimazole oxidation product interacts at the heme moiety, and the heme-clotrimazole adduct has been dissociated from the inactivated enzyme and identified (m/z 1363) by mass analysis. We found that the inhibition of hemoperoxidase increases the accumulation of H2O2 in P. falciparum and causes oxidative stress. Furthermore, the inhibition of hemoperoxidase correlates well with the inhibition of parasite growth. The results described herein indicate that the antimalarial activity of clotrimazole might be due to the inhibition of hemoperoxidase and subsequent development of oxidative stress in P. falciparum.

New Insights into the Stoichiometric and Catalytic Reactivity of Unsaturated Pd3(dppm)3COn+ Clusters (n = 0, 1) Towards Halocarbons – First Evidence for Inorganic By‐Products

AbstractThe title clusters, Pd3(dppm)3(CO)+ and Pd3(dppm)3(CO)0 can be electrochemically generated from the 1‐ and 2‐electron reductions, respectively, of the Pd3(dppm)3(CO)2+ cluster [dppm = bis(diphenylphosphanyl)methane; Pd32+]. Pd3+ reacts in a stoichiometric ratio with methyl iodide, MeI, and benzyl bromide, BzBr, in THF to provide the corresponding Pd3(X)+ adducts (X = I, Br respectively) as inorganic products. Other products are Bz2 and PhMe for BzBr but, for MeI, no organic product was observed (since they are too volatile). In the presence of the same substrates, Pd30 also reacts in a stoichiometric ratio to form the same organics and the Pd3‐(X)+ adducts (X = I and Br). However for MeI, the major inorganic product is the A‐frame Pd2(dppm)2(Me)2I+ binuclear complex. For BzBr, the corresponding A‐frame complex Pd2(dppm)2(Bz)2Br+ could not be detected. The spin‐trap agents, 2,2,6,6‐tetramethylpiperidin‐1‐oxyl (TEMPO) and 5,5′‐dimethyl‐1‐pyrroline N‐oxide (DMPO), have been used to demonstrate the intermediacy of the radical Bz·. The catalytic generation of “Bz·” was performed using two methods, i.e. 1) using a copper anode as the working electrode [Pd3‐(Br)+ + Cu – e– → Pd32+ + CuBr (s)] and 2) using a carbon cathode as the working electrode [Pd3(Br)+ + 2e– → Pd30 + Br–]. The chemical yields for Bz2 vary between 50 and 56 % and the Faradic yield is of the order of 90 % for method 1 and between 52 and 59 % for method 2 [taking into account the quantity of electricity necessary to reduce the catalyst Pd3‐(Br)+]. The X‐ray structure of Pd3(dppm)3(CO)(Br)+ is presented and the following parameters were recorded: monoclinic space group P21/n, a = 10.6546(2), b = 37.1091(7), c = 21.2714(7) Å, β = 91.55(1)o, V = 8407.3(4) Å3, Z = 4, R1 = 0.0581 [I > 2σ(I)], wR2 = 0.1478 (all data). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Hydroxyl Radical is the Active Species in Photochemical DNA Strand Scission by Bis(peroxo)vanadium(V) Phenanthroline

Bis(peroxo)vanadium(V) complexes are widely investigated as anticancer agents. They exert their antitumor and cyctotoxic effects through inhibition of tyrosine phosphatases and DNA cleavage, respectively. The latter process remains poorly understood. The mechanism of DNA cleavage by NH(4)[(phen)V(O)(eta(2)-O(2))(2)] (phen = 1,10-phenanthroline) was investigated. Kinetic studies on DNA cleavage revealed that the complex is a single-strand nicking agent with no specificity. EPR experiments using 2,2,6,6-tetramethyl-4-piperidone (TMP) and 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-traps for singlet oxygen and hydroxyl radical, respectively, implicated hydroxyl radical production upon photodecomposition of bis(peroxo)vanadium(V). This was corroborated by benzoate inhibition of DNA strand scission and stoichiometric oxidation of 2-propanol to acetone upon irradiation of bis(peroxo)vanadium(V) phenanthroline. High-resolution polyacrylamide gel analysis of the vanadium cleavage reaction and [Fe(II)EDTA](2)(-)/H(2)O(2) resulted in comigration of "ladder" pattern bands, which superimposed when both reactions were run on the same lane. These findings identify hydroxyl radical produced from the photooxidation of the peroxo ligand on vanadium as the active species in DNA cleavage.

Photoinduced Electron Transfer in Ruthenium Bipyridyl Complexes: Evidence for the Existence of a Cage with Molecular Oxygen

Ruthenium complexes with three bipyridyl ligands, one of which is modified by attaching one or two hydroxamic acids groups (Ru-1 and Ru-2, respectively), were synthesized. Using EPR spectroscopy, we have found that photoexcitation leads to formation of nitroxyl radicals. The nitroxyl radical concentration in Ru-2 increased dramatically in the presence of spin traps DMPO (5,5‘-dimethyl-1-pyrroline-N-oxide) and PBN (N-tert-butyl-α-phenylnitrone) characterized by strong affinity to superoxide radicals. We have attributed this behavior to the formation of a cage complex between Ru-2 and the superoxide radical. This paper concerns the study of cages formed between ruthenium complexes and molecular oxygen and the effect of functional groups attached to modified bipyridyl ligands on cage formation. The complex between Ru-2 and O_2 was formed in the ground state, probably with participation of the hydroxamic acid groups. The equilibrium constant of this complex was determined by EPR as K_(eq) ∼ 3 M^(-1). The formation of the Ru-2−O_2 complex is supported by the temperature-dependent rate of appearance of the EPR signal in the presence of PBN. Additional evidence comes from observation of paramagnetic shifts of the peaks in the 1H NMR spectrum of specific aromatic protons in the substituted bipyridyl ring upon exposure to O_2. Similar shifts were observed in the spectrum of Os-2, with osmium replacing ruthenium. Model compounds with functional groups that replace the hydroxamic acid or compounds without the metal center, but with the two hydroxamic acids, were synthesized. No shifts in the ^1H NMR spectra of these derivatives were observed in the presence of O_2. These results lead to the conclusion that both metal ions, Ru(II) or Os(II), and hydroxamic acid groups are essential components for the formation of the oxygen cage.

Simultaneous Production of Superoxide Radical and Singlet Oxygen by Sulphonated Chloroaluminum Phthalocyanine Incorporated in Human Low‐density Lipoproteins: Implications for Photodynamic Therapy¶

ABSTRACTSulfonated chloroaluminum phthalocyanines have been studied for their use in the photodynamic therapy (PDT) of tumors. Plasma low‐density lipoproteins (LDL) are important carriers of phthalocyanines in the blood, but on exposure to visible light, phthalocyanine‐loaded LDL undergo an oxidation process that propagates to erythrocytes. We attempted to identify the reactive species involved in LDL and erythrocyte oxidation by means of electron paramagnetic resonance (EPR) spectroscopy in the presence of 2,2,6,6‐tetramethyl‐4‐piperidone (TEMP) and the spin trap 5,5′‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). Irradiation of phthalocyanine‐loaded LDL in the presence of DMPO resulted in the formation of a four‐line EPR spectrum with relative intensity of 1:2:2:1 (aN= aH= 14.8 G), characteristic of DMPO‐hydroxyl radical spin adduct. This signal was sensitive to superoxide dismutase and slightly sensitive to catalase, but a mixture of the two enzymatic activities was the most efficient in promoting a decrease in the intensity of the EPR signal. In the presence of erythrocytes, an increase in the quartet intensity for a hematocrit of 1% and 4% was observed, decreasing for higher erythrocyte concentrations. The irradiation of phthalocyanine‐loaded LDL in the presence of TEMP resulted in the formation of a nitroxide radical, 2,2,6,6‐tetramethyl‐4‐piperidone‐N‐oxyl radical, intensity of which was sensitive to histidine, a singlet oxygen (1O2) quencher. Under both incubation conditions, with DMPO and TEMP, the formation of the respective EPR signals required the sensitizer (phthalocyanine), light and oxygen. Overall, the results are compatible with the simultaneous formation of superoxide anion and 1O2, implying that Type‐I and Type‐II mechanisms of photochemistry are simultaneously operative in phthalocyanine‐loaded LDL. However, for a constant LDL/phthalocyanine ratio, the formation of oxygen free radicals shows a biphasic behavior with the concentration of LDL increasing and reaching a plateau, whereas the formation of 1O2 increases linearly with LDL concentration. Erythrocytes at high (physiological) concentrations induced a decrease in the intensity of both EPR signals. The physiological relevance of these findings in the framework of PDT is briefly discussed.

Simultaneous production of superoxide radical and singlet oxygen by sulphonated chloroaluminum phthalocyanine incorporated in human low-density lipoproteins: implications for photodynamic therapy.

Sulfonated chloroaluminum phthalocyanines have been studied for their use in the photodynamic therapy (PDT) of tumors. Plasma low-density lipoproteins (LDL) are important carriers of phthalocyanines in the blood, but on exposure to visible light, phthalocyanine-loaded LDL undergo an oxidation process that propagates to erythrocytes. We attempted to identify the reactive species involved in LDL and erythrocyte oxidation by means of electron paramagnetic resonance (EPR) spectroscopy in the presence of 2,2,6,6-tetramethyl-4-piperidone (TEMP) and the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Irradiation of phthalocyanine-loaded LDL in the presence of DMPO resulted in the formation of a four-line EPR spectrum with relative intensity of 1:2:2:1 (a(N) = a(H) = 14.8 G), characteristic of DMPO-hydroxyl radical spin adduct. This signal was sensitive to superoxide dismutase and slightly sensitive to catalase, but a mixture of the two enzymatic activities was the most efficient in promoting a decrease in the intensity of the EPR signal. In the presence of erythrocytes, an increase in the quartet intensity for a hematocrit of 1% and 4% was observed, decreasing for higher erythrocyte concentrations. The irradiation of phthalocyanine-loaded LDL in the presence of TEMP resulted in the formation of a nitroxide radical, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl radical, intensity of which was sensitive to histidine, a singlet oxygen ((1)O(2)) quencher. Under both incubation conditions, with DMPO and TEMP, the formation of the respective EPR signals required the sensitizer (phthalocyanine), light and oxygen. Overall, the results are compatible with the simultaneous formation of superoxide anion and (1)O(2), implying that Type-I and Type-II mechanisms of photochemistry are simultaneously operative in phthalocyanine-loaded LDL. However, for a constant LDL/phthalocyanine ratio, the formation of oxygen free radicals shows a biphasic behavior with the concentration of LDL increasing and reaching a plateau, whereas the formation of (1)O(2) increases linearly with LDL concentration. Erythrocytes at high (physiological) concentrations induced a decrease in the intensity of both EPR signals. The physiological relevance of these findings in the framework of PDT is briefly discussed.

Simultaneous Production of Superoxide Radical and Singlet Oxygen by Sulphonated Chloroaluminum Phthalocyanine Incorporated in Human Low-density Lipoproteins: Implications for Photodynamic Therapy¶

Sulfonated chloroaluminum phthalocyanines have been studied for their use in the photodynamic therapy (PDT) of tumors. Plasma low-density lipoproteins (LDL) are important carriers of phthalocyanines in the blood, but on exposure to visible light, phthalocyanine-loaded LDL undergo an oxidation process that propagates to erythrocytes. We attempted to identify the reactive species involved in LDL and erythrocyte oxidation by means of electron paramagnetic resonance (EPR) spectroscopy in the presence of 2,2,6,6-tetramethyl-4-piperidone (TEMP) and the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Irradiation of phthalocyanine-loaded LDL in the presence of DMPO resulted in the formation of a four-line EPR spectrum with relative intensity of 1:2:2:1 (a(N) = a(H) = 14.8 G), characteristic of DMPO-hydroxyl radical spin adduct. This signal was sensitive to superoxide dismutase and slightly sensitive to catalase, but a mixture of the two enzymatic activities was the most efficient in promoting a decrease in the intensity of the EPR signal. In the presence of erythrocytes, an increase in the quartet intensity for a hematocrit of 1% and 4% was observed, decreasing for higher erythrocyte concentrations. The irradiation of phthalocyanine-loaded LDL in the presence of TEMP resulted in the formation of a nitroxide radical, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl radical, intensity of which was sensitive to histidine, a singlet oxygen ((1)O(2)) quencher. Under both incubation conditions, with DMPO and TEMP, the formation of the respective EPR signals required the sensitizer (phthalocyanine), light and oxygen. Overall, the results are compatible with the simultaneous formation of superoxide anion and (1)O(2), implying that Type-I and Type-II mechanisms of photochemistry are simultaneously operative in phthalocyanine-loaded LDL. However, for a constant LDL/phthalocyanine ratio, the formation of oxygen free radicals shows a biphasic behavior with the concentration of LDL increasing and reaching a plateau, whereas the formation of (1)O(2) increases linearly with LDL concentration. Erythrocytes at high (physiological) concentrations induced a decrease in the intensity of both EPR signals. The physiological relevance of these findings in the framework of PDT is briefly discussed.