Do stable nitroxide radicals catalyze or inhibit the degradation of hyaluronic acid?

Reactive oxygen-derived species and particularly OH radicals can degrade hyaluronic acid (HA), resulting in a loss of viscosity and a subsequent decrease in its effectiveness as a joint-lubricating agent. The production of OH in the vicinity of HA can be catalyzed by bound redox-active metals, which participate in the Haber-Weiss reaction. Damage to HA can also occur as a result of hypochlorite formed by myeloperoxidase (MPO). The protective reagents commonly used to inhibit oxidative stress-induced degradation of HA include antioxidative enzymes, such as SOD and catalase, chelators that coordinate metal ions rendering them redox-inactive, and scavengers of radicals, such as OH, as well as nonradical reactive species. In recent years, stable cyclic nitroxides have also been widely used as effective antioxidants. In many cases, nitroxide antioxidants operate catalytically and mediate their protective effect through an exchange between their oxidized and reduced forms. It was anticipated, therefore, that nitroxides would protect HA from oxidative degradation as well. On the other hand, nitroxides serve as catalysts in many oxidation reactions of alcohols, sugars and polysaccharides, including hyalouronan. Such opposite effects of nitroxides on oxidative degradation are particularly intriguing and the aim of the present study was to examine their effect on HA when subjected to diverse forms of oxidative stress. The results indicate that nitroxides protect HA from OH radicals generated enzymatically or radiolytically. The protective effect is attributable neither to the scavenging of OH nor to the oxidation of reduced metal, but to the reaction of nitroxides with secondary carbohydrate radicals-most likely peroxyl radicals.     

Neuroprotection and regeneration by extracellular metallothionein via lipoprotein-receptor-related proteins

Metallothionein has a well-documented protective and proregenerative effect in the mammalian brain, particularly following physical trauma and ischemia or during the onset of neurodegenerative disease. A range of mechanisms have been established for this, including metallothionein’s metal binding properties and its ability to scavenge free radicals. In recent years it has become apparent that metallothionein is present in the extracellular compartment of the central nervous system and that it can interact with cell surface receptors of the lipoprotein-receptor-related protein family, including lipoprotein-receptor-related protein 1 (LRP1) and megalin. These interactions activate intracellular pathways which are consistent with many of the observed effects of metallothionein in the central nervous system, including its effects on neurons, glial cells, and cells of the immune system. The evidence describing the release, receptor interactions, and subsequent physiological consequences of metallothionein is discussed in this review.     

Cyto- and genotoxic effects of novel aromatic nitroxide radicals in vitro

Because of the increasing interest in the use of nitroxide radicals as antioxidants and probes for various applications in biological systems, the question of their toxicity is of paramount importance. Cytotoxicity and mutagenicity studies have been extensively performed with the commercially available aliphatic nitroxides, and the general outcome is that these compounds are nonmutagenic and relatively noncytotoxic. In this study, the cytotoxicity and genotoxicity of a new class of aromatic nitroxides that we have synthesized (i.e., indolinonic and quinolinic nitroxides), whose antioxidant activity has been established in both chemical and biological systems, were evaluated and compared with those of two commercial nitroxides and with that of butylated hydroxytoluene (BHT). The mutagenicity assay was performed using Salmonella typhimurium tester strains TA98, TA100, and TA102, chosen on the basis of their ability to detect various types of mutations and their sensitivity to oxidative damage. None of the compounds tested were found to be mutagenic. The colony-forming assay (CFA) using Chinese hamster ovary (CHO) AS52 cells was employed for determining the cytotoxicity of the test compounds. On comparing the effective dose that inhibits the CFA by 50% (IC(50)), most of the compounds tested on an equal molar concentration basis were less toxic than BHT. Therefore, the overall results obtained correlate well with the data reported in the literature on the toxicity of aliphatic nitroxides and lend support to the possible use of these compounds as therapeutic antioxidants.     

Modulation of oxidative damage by nitroxide free radicals

Piperidine nitroxides like 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) are persistent free radicals in non-acidic aqueous solutions and organic solvents that may have value as therapeutic agents in medicine. In biological environments, they undergo mostly reduction to stable hydroxylamines but can also undergo oxidation to reactive oxoammonium compounds. Reactions of the oxoammonium derivatives could have adverse consequences including chemical modification of vital macromolecules and deleterious effects on cell signaling. An examination of their reactivity in aqueous solution has shown that oxoammonium compounds can oxidize almost any organic as well as many inorganic molecules found in biological systems. Many of these reactions appear to be one-electron transfers that reduce the oxoammonium to the corresponding nitroxide species, in contrast to a prevalence of two-electron reductions of oxoammonium in organic solvents. Amino acids, alcohols, aldehydes, phospholipids, hydrogen peroxide, other nitroxides, hydroxylamines, phenols and certain transition metal ions and their complexes are among reductants of oxoammonium, causing conversion of this species to the paramagnetic nitroxide. On the other hand, thiols and oxoammonium yield products that cannot be detected by ESR even under conditions that would oxidize hydroxylamines to nitroxides. These products may include hindered secondary amines, sulfoxamides and sulfonamides. Thiol oxidation products other than disulfides cannot be restored to thiols by common enzymatic reduction pathways. Such products may also play a role in cell signaling events related to oxidative stress. Adverse consequences of the reactions of oxoammonium compounds may partially offset the putative beneficial effects of nitroxides in some therapeutic settings.     

Application of Spin Traps to Biological Systems

Since 1971. when nitroxides were first reported to be bioreduced, several cellular enzymes, in addition to ascorbic acid. have been found to catalyze the reduction of nitroxides to their corresponding hydroxylami-nes. Numerous studies have demonstrated that cellular bioreduction of nitroxides are both dependent upon the structure of the nitroxide and cell type. For example, pyrrolidinyloxyls are considerably more resistant to bioreduction than their corresponding piperidinyloxyls. In addition, cellular levels of reductases present in freshly isolated rat hepatocytes are considerably greater than concentrations found in freshly isolated rat enterocytes. Thus, through the proper selection of a cell type and an appropriate nitroxide. one can study cellular-mediated free radical processes.

With the discovery that α-hydrogen-containing nitroxides, including 2, Z-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH) decompose rapidly in the presence of superoxide and thiols, the ability to determine if hydroxyl radical is generated during stimulation of human neutrophils, is in doubt. To explore the limits of spin trapping in this context. we have studied the effect of varying the rates of superoxide production. in the presence and absence of thiols, on the decomposition of DMPO-OH. In parallel studies, we have found that t-butyl α-methyl-4-pyridinyl-N-oxide nitroxide (4-POBN-CH₃) will not degrade in the presence of superoxide and a thiol. From these studies. we have determined that if hydroxyl radicals were generated as an isolated event in the presence of a continual flow of superoxide. spin trapping might not be able to detect its formation. Otherwise. spin trapping should be able to measure hydroxyl radicals. if continually generated, during activation of human neutrophils.     

Use of a Low-Frequency Esr Spectrometer: Implications for Spin-Trapping Free Radicals, In Situ

We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in siru measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) and 2.2-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem. we have increased the sensitivity of the spin trapping method by synthesizing a family of ¹⁵N-and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthinelxanthinc oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H₂O₂ to O₂ (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H₂O₂ decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.     

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).     

Inhibition of lipid peroxidation by spin labels. Relationships between structure and function

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.     

Nitroxides inhibit peroxyl radical-mediated DNA scission and enzyme inactivation

Nitroxides are cell-permeable stable radicals that protect biomolecules from oxidative damage in several ways. The mechanisms of protection studied to date include removal of superoxide radicals as SOD-mimics, oxidation of transition metal ions to preempt the Fenton reaction, and scavenging carbon-centered radicals. However, there is no agreement regarding the reaction of piperidine nitroxides with peroxyl radicals. The question of whether they can protect by scavenging peroxyl radicals is important because these radicals are formed in the presence of oxygen abundant in biological tissues. To further our understanding of the antioxidative behavior of piperidine nitroxides, we studied their effect on biochemical systems exposed to the water soluble radical initiator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). AAPH thermally decomposes to yield tert-amidinopropane radicals (t-AP(*)) that readily react with oxygen to form peroxyl radicals (t-APOO(*)). It has recently been reported that piperidine nitroxides protect plasmid DNA from t-AP(*) though not from t-APOO(*). The present study was directed at the question of whether these nitroxides can protect biological systems from damage inflicted by peroxyl radicals. The reaction of nitroxides with AAPH-derived radicals was followed by cyclic voltammetry and electron paramagnetic resonance spectroscopy, whereas the accumulation of peroxide was iodometrically assayed. Assaying DNA damage in vitro, we demonstrate that piperidine nitroxides protect from both t-AP(*) and t-APOO(*). Similarly, nitroxides inhibit AAPH-induced enzyme inactivation. The results indicate that piperidine nitroxides protect the target molecule by reacting with and detoxifying peroxyl radicals.     

Advances in quantitative proteomics

Large-scale protein quantification has become a major proteomics application in many areas of biological and medical research. During the past years, different techniques have been developed, including gel-based such as differential in-gel electrophoresis (DIGE) and liquid chromatography-based such as isotope labeling and label-free quantification. These quantitative proteomics tools hold significant promise for biomarker discovery, diagnostic and therapeutic applications. They are also important for research in functional genomics and systems biology towards basic understanding of molecular networks and pathway interactions. In this review, we summarize current technologies in quantitative proteomics and discuss recent applications of the technologies.     

Pulse radiolysis study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

Principles of the Metabolism of Nitroxides and Their Implications for Spin Trapping

A review of the principal interactions of nitroxides with cells suggests that if these same phenomena occur with spin adducts the result could be considerable experimental confusion and error. In particular, these could lead to differential rates of loss of spin adducts, thereby potentially invalidating conclusions on the amounts or even the types of free radicals that are trapped. In addition. shuttling of electrons between nitroxides and hydroxylamines also very significantly could alter the amounts and types of spin adducts that are observed.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Nitrones as therapeutics

Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones, including alpha-phenyl-tert-butylnitrone (PBN), have been shown to have potent biological activity in many experimental animal models. Many diseases of aging, including stroke, cancer development, Parkinson disease, and Alzheimer disease, are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development for stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also, in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic-noise-induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes cannot readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our studies and others in which the use of selected nitrones as novel therapeutics has been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases.     

Spin Trapping Using 2,2-Dimethyl-2H-Imidazole-1-Oxides

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na₂SO₃ and NaHSO₃. From the data obtained, a successful application of these new spin traps in biological systems can be expected.     

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.