Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.     

Application of a trityl-based radical probe for measuring superoxide

The use of triarylmethyl (trityl) free radical, TAM OX063, for detection of superoxide in aqueous solutions by electron paramagnetic resonance (EPR) spectroscopy was investigated. TAM is paramagnetic (EPR active), highly soluble in water and exhibits a single sharp EPR peak in aqueous media. It is also highly stable in presence of many oxidoreductants such as ascorbate and glutathione that are present in the biological systems. TAM reacts with superoxide with an apparent second order rate constant of 3.1 × 10³ M⁻¹ s⁻¹. The specific reactivity of TAM with superoxide, which leads to loss of EPR signal, was utilized to detect the generation of superoxide in various chemical (light/riboflavin/electron/donor), enzymatic (xanthine/xanthine oxidase), and cellular (stimulated neutrophils) model systems. The changes in the EPR line-width, induced by molecular oxygen, were utilized in the simultaneous determination of consumption of oxygen in the model systems. The effects of flux of superoxide and concentration of TAM on the efficiency of detection of superoxide were studied. The use of TAM for detection of superoxide offers unique advantages namely, (i) the utilization of very low concentration of the probe, (ii) its stability to bioreduction, and (iii) its use in the simultaneous determination of concentrations of superoxide and oxygen.     

Electron paramagnetic resonance imaging of tumor pO₂

Electron paramagnetic resonance imaging (EPRI) can be used to noninvasively and quantitatively obtain three-dimensional maps of tumor pO?. The paramagnetic tracer triarylmethyl (TAM), a substituted trityl radical moiety, is not toxic to animals and provides narrow isotropic spectra, which is ideal for in vivo EPR imaging experiments. From the oxygen-induced spectral broadening of TAM, pO? maps can be derived using EPRI. The instrumentation consists of an EPRI spectrometer and 7T magnetic resonance imaging (MRI) system both operating at a common radiofrequency of 300 MHz. Anatomic images obtained by MRI can be overlaid with pO? maps obtained from EPRI. With imaging times of less than 3 min, it was possible to monitor the dynamics of oxygen changes in tumor and distinguish chronically hypoxic regions from acutely hypoxic regions. In this article, the principles of pO? imaging with EPR and some relevant examples of tumor imaging are reviewed.     

EPR Oximetry Sensor—Developing a TAM Derivative for In Vivo Studies

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1?mM PBS, pH?=?7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH?=?7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250?mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.     

Effect of tamoxifen on the Notch signaling pathway in ovarian follicles of mice

ABSTRACT

We investigated the effect of tamoxifen (TAM) treatment on the Notch signaling pathway in mouse ovary. Mice were randomly divided into four groups. Control group A animals were untreated. Control group B animals were treated with the vehicle only. Animals of the 0.5 TAM group received 0.5 mg/day TAM. Animals of the 1.5 TAM group received 1.5 mg/day of TAM. TAM was injected subcutaneously for 5 days. Body weights were measured at the start and end of the experiment. Sections were stained using Crossman’s modified trichrome to examine general ovarian structure. Other sections were immunostained to demonstrate Jagged 1, Ki 67 and Notch 2. The TUNEL method was used to detect apoptosis. No significant differences in body weight or ovarian weight were found among the experimental groups. The number of primordial follicles was greater in the treatment groups than in the control groups, while the number of antral follicles and corpora lutea were reduced in the treatment groups. Cell proliferation rates were decreased by TAM treatment and cystic follicles were formed in the ovarian stroma. Notch 2 expression in the granulosa cells was increased following TAM administration, but no change was found in Jagged 1 expression. TAM administration suppressed follicular development and exhibited a negative effect on ovarian morphology. Our findings suggest that the Notch pathway participates in the action of TAM. We suggest that it may be useful to use Notch pathway regulators to adjust the effects of TAM on the ovary.     

The Synthesis Of Epr Differentiable Spinlabels And Their Coupling To Uridine

For EPR measurements of RNA, DNA, or proteins, the occurrence of the paramagnetic species is necessary. The aim of this work is to improve the synthesis of two different EPR spinlabels 2,2,6,6-tetra methyl-3,4-dehydro-piperidin-N-oxyl-4-acetylene (TEMPA) 6 and ¹⁵N-labeled TEMPA 6* and their coupling to uridine. The yield of the synthesis of TEMPA could be increased to 40% and the second nitroxide 2,2,6,6-tetramethyl-3,4-dehydro-piperidin-¹⁵N-oxyl-4-acetylene 6* could be synthesized with a yield of 11%.     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

Intraphagosomal oxygen in stimulated macrophages

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO₂ in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d₁₇-1-oxyl iodide (d-Cat₁)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl (¹⁵N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley-Liss, Inc.     

The generation of superoxide radicals by complex III in heart mitochondria and the antioxidant effect of dinitrosyl iron complexes at different partial pressures of oxygen

EPR spin trapping and EPR oximetry were used to study the superoxide radical generation in heart mitochondria from Wistar rats at various oxygen concentrations. Lithium phthalocyanine and TEMPONE-¹⁵N-D₁₆ were chosen to determine the oxygen content in a gas-permeable capillary containing mitochondria. TIRON was used as a spin trap. Several oxygen concentrations in the incubation mixture were tested; heart mitochondria were found to generate superoxide in complex III at various partial pressures of oxygen, including deep hypoxia (<5% O₂). Dinitrosyl iron complexes with glutathione (the drug Oxacom) exerted an antioxidant effect regardless of the partial pressure of oxygen; the magnitude and kinetic characteristics of the effect depended on the drug concentration.     

Lipopolysaccharide-induced alterations in oxygen consumption and radical generation in endothelial cells

Oxygen consumption rate (OCR) and generation of superoxide and nitric oxide (NO) in mouse aortic endothelial cells (MAECs) treated with lipopolysaccharide (LPS) were studied. The OCR was determined in cell suspensions at 37 °C by electron paramagnetic resonance (EPR) spectroscopy. LPS significantly altered the OCR in a dose and time-dependent fashion. The OCR was significantly elevated immediately following the treatment of MAECs with LPS (5 and 10 μg/ml) and NADPH (100 μM) whereas the same was depressed 1 h after exposure to similar conditions of incubation. Under similar experimental conditions, superoxide generation was also determined by EPR spectroscopy and cytochrome c reduction assays. A marginal increase in the superoxide production was observed when the cells were treated with LPS and NADPH alone whereas the same was further enhanced significantly when the cells were treated with LPS and NADPH together. The increase in oxygen consumption and superoxide production caused by LPS was inhibited by diphenyleneiodonium (DPI), suggesting the involvement of NAD(P)H oxidase. A significant increase in the NO production by MAECs was noticed 1 h after treatment with LPS and was inhibited by L-NAME, further suggesting the involvement of nitric oxide synthase (NOS). Thus, on a temporal scale, LPS-induced alterations in oxygen consumption by MAECs may be under the control of dual regulation by NAD(P)H oxidase and NOS. (Mol Cell Biochem 278: 119–127, 2005)     

Spin trap evidence for production of superoxide radical anions by purified NADPH-cytochrome P-450 reductase

Previous evidence for superoxide radicals as initial reduction products of oxygen by NADPH cytochrome P-450 reductase has been indirect. In this paper a technique is described to spin trap radicals produced in incubations of oxygen and reductase. Reference spin trap adducts were synthesized by adding phenyl-$\text{t}$-butyl nitrone (PBN) to superoxide radicals (PBN-OOH) or to hydroxyl radicals (PBN-OH). Both PBN adducts are stable in water or ethyl acetate for hours. Electron Paramagnetic Resonance (EPR) spectra measured in N₂-saturated ethyl acetate allow clear resolution of the hyperfine extrema of PBN-OH and PBN-OOH (2.1 and 4.5 G splitting, respectively). Comparison of EPR spectra from reductase and oxygen incubations with those of synthetic PBN-OOH suggest that superoxide radicals are the major primary reduction product of oxygen.     

Hydroxyl radical oxidation of guanosine 5′-triphosphate (GTP): requirement for a GTP–Cu(II) complex

Abstract

Levels of oxidized guanosine base in DNA have become a hallmark biomarker in assessing oxidative stress implicated in a variety of disease and toxin-induced states. However, there is evidence that the guanosine in the nucleotide triphosphate pool (GTP) is more susceptible to oxidation than guanosine residues incorporated into nucleic acids and this causes a substantial amount of the oxidized product, 8-oxoguanosine 5′-triphosphate (oxo⁸GTP), to accumulate in cell-free and in cell-culture preparations. Electron paramagnetic resonance (EPR) spectroscopy and direct EPR analysis of free radical production by copper sulfate and L-ascorbic acid demonstrates that the hydroxyl radical (HO^?) is produced via oxidation of Cu⁺ to Cu²⁺ while in a complex with GTP. This HO^? production is dependent on the availability of oxygen and the presence of GTP in the reaction milieu. Verification of free radical-mediated production of oxo⁸GTP is presented using HPLC with electrochemical detection and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-LTOF-MS). The sum of these results is presented in a novel mechanism of GTP oxidation by Cu²⁺ and L-ascorbic acid. A better understanding of the chemistry involved in this oxidative modification of GTP facilitates a more comprehensive understanding of its potential physiological consequences.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic radical.