In vivo evaluation of different alterations of redox status by studying pharmacokinetics of nitroxides using magnetic resonance techniques.

Free radicals, particularly reactive oxygen species (ROS), are involved in various pathologies, injuries related to radiation, ischemia-reperfusion or ageing. Unfortunately, it is virtually impossible to directly detect free radicals in vivo, but the redox status of the whole organism or particular organ can be studied in vivo by using magnetic resonance techniques (EPR and MRI) and paramagnetic stable free radicals - nitroxides. Here we review results obtained in vivo following the pharmacokinetics of nitroxides on experimental animals (and a few in humans) under various conditions. The focus was on conditions where the redox status has been altered by induced diseases or harmful agents, clearly demonstrating that various EPR/MRI/nitroxide combinations can reliably detect metabolically induced changes in the redox status of organs. These findings can improve our understanding of oxidative stress and provide a basis for studying the effectiveness of interventions aimed to modulate oxidative stress. Also, we anticipate that the in vivo EPR/MRI approach in studying the redox status can play a vital role in the clinical management of various pathologies in the years to come providing the development of adequate equipment and probes.

Reactive oxygen species and vascular remodeling in cardiovascular diseases / Especies reactivas de oxígeno y remodelado vascular en enfermedades cardiovasculares

Reactive oxygen species (ROS) are reactive derivatives of O2 metabolism produced by all types of vascular cells. ROS play an important role in both physiological and pathological situations by acting as intracellular signaling molecules which regulate vascular function and structure. Accordingly, oxidative stress is implicated among other processes in inflammation, hypertrophy, migration, growth/apoptosis and extracellular matrix protein turnover which are important processes involved in vascular remodeling in cardiovascular diseases. In the cardiovascular system, the major source of ROS is the NADPH oxidase family of enzymes composed by seven members where NOX-1 and NOX-4 are the main isoforms in vascular smooth muscle cells. This review highlights the importance of NOX-derived ROS in vascular biology and focuses on the potential role of oxidative stress in vascular remodeling

Las especies reactivas de oxígeno son derivados reactivos del metabolismo del O2 producido por todos los tipos celulares a nivel vascular. Las especies reactivas de oxígeno juegan un papel importante en situaciones tanto fisiológicas como patológicas mediante su actuación como moléculas de señalización intracelular que regulan la función y estructura vascular. De esta manera, el estrés oxidativo está implicado, entre otros procesos, en la inflamación, hipertrofia, migración, proliferación/apoptosis y reciclaje de proteínas de matriz extracelular, los cuales son procesos importantes implicados en el remodelado vascular durante enfermedades cardiovasculares. En el sistema cardiovascular, la mayor fuente de especies reactivas de oxígeno es la familia de enzimas NADPH oxidase formadas por siete miembros donde NOX-1 y NOX-4 son las principales isoformas en células musculares lisas vasculares. Esta revisión destaca la importancia de las especies reactivas de oxígeno derivadas de NOX en la biología vascular y se centra en el papel potencial del estrés oxidativo en el remodelado vascular

Ameliorating effect of selenium on chromium (VI)-induced oxidative damage in the brain of adult rats

Chromium is known for its wide toxic manifestations. This experiment aims to evaluate the effect of selenium against oxidative stress induced by chromium in the cerebrum and cerebellum. Female Wistar rats were randomly divided into four groups of six each: group I served as controls which received the standard diet; group II received drinking water K2Cr2O7 alone (700 ppm); group III received both K2Cr2O7 and Se (0.5 mg Na2SeO3/kg of diet); and group IV received Se (0.5 mg/kg of diet) for 3 weeks. The exposure of rats to K2Cr2O7 promoted oxidative stress in the cerebrum and cerebellum with an increase in malondialdehyde and a decrease of nonenzymatic antioxidant levels such as glutathione, nonprotein thiol, and vitamin C. An increase of enzyme activities like catalase, glutathione peroxidase, and superoxide dismutase activities was also observed. Acetylcholinesterase activity was inhibited after treatment with K2Cr2O7. Co-administration of Se restored the parameters cited above. The histopathological findings confirmed the biochemical results (AU)

NAD(P)H oxidase inhibiting with apocynin improved vascular reactivity in tail-suspended hindlimb unweighting rat

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Recent studies suggested that reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is of functional importance in modulating vascular tone, and we have previously detected excessive superoxide production in tail-suspended hindlimb unweighting (HU) rat cerebral and carotid arteries. HU rat was a widely used model to simulate physiological effects on the vasculature. The present study tended to investigate whether NAD(P)H oxidase inhibition with apocynin influences vasoconstriction, endothelium-dependent relaxation, and nitrite/nitrate (NOx) content in HU rat cerebral and carotid arteries. Vascular contractile and dilate responses were assessed in a myograph organ bath. NOx content in cerebral and carotid arteries was measured. We found enhanced maximal contractile response and impaired endothelium-dependent relaxation in HU rat basilar (P < 0.01) and common carotid artery (P < 0.05); however, chronic treatment of apocynin (50 mg/kg/day) partially reversed abnormal vascular response. Furthermore, 21-day HU increased arterial NOx content (P < 0.01) in cerebral and carotid arteries compared with control rats; however, apocynin treatment restored it toward near-normal values. These data demonstrated that NAD(P)H oxidase-derived oxidative stress mediated abnormal vasoreactivity though nitric oxide mechanism in the settings of simulated microgravity (AU)

Effects of L-Canavanine and ozone on vascular reactivity in septicemic rats

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Septicemia leads to oxidative stress with overproduction of reactive-oxygen species (ROS) and consumption of endogenous antioxidant enzymes. We tested a twofold hypothesis: (1) does oxidative stress (OxS) induced by sepsis acting alone or in concert with augmented inflammatory processes contributes to sepsis-related vascular dysfunction, and, (2) whether ozone (O3) and L-canavanine (CAV) mitigate the negative impact of the aforementioned phenomena. We investigated the relative impact of treatment with CAV and/or O3 on vascular OxS associated vascular functional changes in septicemic rats. For this study, 60 male Sprague–Dawley rats were used and divided into six experimental groups (n = 10): control group (C), sham-operated (Sham), septicemic rats (S), S rats treated with CAV (100 mg/kg. i.p; S + CAV), S rats treated with (..) (AU)

Influence of chemical activation of a 35% hydrogen peroxide bleaching gel on its penetration and efficacy--in vitro study.

OBJECTIVES: The aim of this study was to evaluate the effects of chemical activation of hydrogen peroxide (HP) gel on colour changes and penetration through the tooth structure. METHODS: One hundred and four bovine incisors were used. One dentine (CD) disc and one enamel-dentine (ED) disc were prepared from each tooth. They were positioned over artificial pulpal chambers and the bleaching was performed with an experimental 35% HP gel. Two control and six experimental groups were prepared. In the positive control group (PC) no chemical activator was used. In the negative control group (NC) the specimens did not receive any bleaching. Each experimental group received a different chemical activator (manganese gluconate-MG; manganese chlorite-MC; ferrous sulphate-FS; ferrous chlorite-FC; and mulberries root extract-MRE). After the bleaching procedure a sample of solution was collected from the artificial pulpal chamber and the HP concentration was measured. The data were analysed using ANOVA, Tukey's, and Dunnett's tests. RESULTS: The groups MG and FS showed a significantly lower penetration of HP than the PC group. For the parameter Delta E, all the groups, with the exception of the group MRE, showed a significantly higher means in relation to the PC group in ED colour. For dentine colour, just the groups MG and FS had significant differences in relation to PC. CONCLUSIONS: The addition of MG and FS decreases the penetration of HP. The chemical activation using metal salts tested was effective in increasing the bleaching effect.

Methylglyoxal augments intracellular oxidative stress in human aortic endothelial cells.

Methylglyoxal (MGO) is a non-enzymatic metabolite in the glycolytic pathway and its concentration in blood and tissues is elevated in diabetes and renal failure. MGO induces tissue injuries via ROS; however, the mechanism remains to be clarified. The present study examined the harmful actions of MGO. Human aortic endothelial cells were assessed under real-time fluorescent microscopy with continuous superfusion. Increases in intracellular ROS were measured with fluorescent indicator, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (DCFH-DA). The addition of MGO rapidly increased the ROS in a dose-dependent manner. The increment of DCF was entirely abolished by pre-treatment with superoxide anion scavenger and membrane-permeable catalase, indicating that MGO induces superoxide production. The increment was completely inhibited by 2-thenoyltrifluoroacetone or carbonyl cyanide 3-chlorophenylhydrazone and partially inhibited by N-methyl-L-arginine. These data suggest that MGO stimulates superoxide production from mitochondria and partially stimulates nitric oxide synthase in human endothelial cells.

ADME optimization and toxicity assessment in early- and late-phase drug discovery.

Integrating physicochemical, drug metabolism, pharmacokinetics, ADME, and toxicity assays into drug discovery in order to reduce the attrition rates in clinical development is reviewed. The review is organized around three main decision points used in discovery including hit generation, lead optimization and final candidate selection stages. The preclinical strategies used at each decision point are discussed from a drug discovery perspective. Typically, preclinical data produced at these stages use lower throughput assays, smaller amounts of compounds and operate within a timeframe that is consistent with the iterative cycle of most drug discovery research projects. Understanding the false positive rates of these drug discovery preclinical assays is a must in reducing attrition rates in development.

Protein synthesis patterns reveal a complex regulatory response to singlet oxygen in Rhodobacter.

Singlet oxygen (1O2) is a stress factor and signal in the facultative phototrophic bacterium Rhodobacter sphaeroides. In vivo protein labeling with L-[35S]-methionine and analysis by two-dimensional gel electrophoresis revealed that the synthesis of 61 proteins was changed in response to 1O2. After 1O2 treatment, protein synthesis patterns were distinct from those after H2O2 treatment but similar to those after high light exposure. This indicates regulatory mechanisms selective for different reactive oxygen species (ROS) and a response to light partly mediated by 1O2. Analysis of mutant strains support that the response to 1O2 is regulated mainly by rpoE (sigma E), but also a modulation of the sigma E dependent response by other factors and the existence of sigma E independent responses. The involvement of the RNA chaperon Hfq in the 1O2 response implies a role of small regulatory RNAs.

Sustained stress response after oxidative stress in trabecular meshwork cells.

PURPOSE: To investigate the mechanisms by which chronic oxidative stress may lead to a sustained stress response similar to that previously observed in the trabecular meshwork (TM) of glaucoma donors. METHODS: Porcine TM cells were treated with 200 microM H2O2 twice a day for four days and were allowed to recover for three additional days. After the treatment, TM cells were analyzed for generation of intracellular reactive oxygen species (iROS), mitochondrial potential, activation of NF-kappaB, and the expression of inflammatory markers IL-1alpha, IL-6, IL-8, and ELAM-1. Potential sources of iROS were evaluated using inhibitors for nitric oxide, nitric oxide synthetase, cyclooxygenase, xanthine oxidase, NADPH oxidase, mitochondrial ROS, and PKC. The role of NF-kappaB activation in the induction of inflammatory markers was evaluated using the inhibitors Lactacystin and BAY11-7082. RESULTS: Chronic oxidative stress simulated by H2O2 exposure of porcine TM cells resulted in the sustained production of iROS by the mitochondria. Inhibition of mitochondrial iROS had a significant inhibitory effect on the activation of NF-kappaB and the induction of IL-1alpha, IL-6, IL-8, and ELAM-1 triggered by chronic oxidative stress. Inhibition of NF-kappaB partially prevented the induction of IL-1alpha, IL-8, and ELAM-1, but not IL-6. CONCLUSIONS: Chronic oxidative stress in TM cells induced iROS production in mitochondria. This increase in iROS may contribute to the pathogenesis of the TM in glaucoma by inducing the expression of inflammatory mediators previously observed in glaucoma donors as well as the levels of oxidative damage in the tissue.

Cell-permeable, mitochondrial-targeted, peptide antioxidants.

Cellular oxidative injury has been implicated in aging and a wide array of clinical disorders including ischemia-reperfusion injury; neurodegenerative diseases; diabetes; inflammatory diseases such as atherosclerosis, arthritis, and hepatitis; and drug-induced toxicity. However, available antioxidants have not proven to be particularly effective against many of these disorders. A possibility is that some of the antioxidants do not reach the relevant sites of free radical generation, especially if mitochondria are the primary source of reactive oxygen species (ROS). The SS (Szeto-Schiller) peptide antioxidants represent a novel approach with targeted delivery of antioxidants to the inner mitochondrial membrane. The structural motif of these SS peptides centers on alternating aromatic residues and basic amino acids (aromatic-cationic peptides). These SS peptides can scavenge hydrogen peroxide and peroxynitrite and inhibit lipid peroxidation. Their antioxidant action can be attributed to the tyrosine or dimethyltyrosine residue. By reducing mitochondrial ROS, these peptides inhibit mitochondrial permeability transition and cytochrome c release, thus preventing oxidant-induced cell death. Because these peptides concentrate >1000-fold in the inner mitochondrial membrane, they prevent oxidative cell death with EC50 in the nM range. Preclinical studies support their potential use for ischemia-reperfusion injury and neurodegenerative disorders. Although peptides have often been considered to be poor drug candidates, these small peptides have excellent "druggable" properties, making them promising agents for many diseases with unmet needs.

Pro-oxidant actions of alpha-lipoic acid and dihydrolipoic acid.

There is strong accumulating evidence that a alpha-lipoic acid (LA) supplement is good insurance, and would markedly improve human health. LA is readily absorbed from the diet, transported to cells and reduced to dihydrolipoic acid (DHLA). Of the two compounds, DHLA evidently has greater antioxidant activity. Much research has focused on the antioxidant properties of these compounds. Aside from its antioxidant role, in vitro and in vivo studies suggest that LA and its reduced form DHLA also act as a pro-oxidant properties. Limited number of studies concerning the pro-oxidant potential of LA and DHLA were performed only in recent years. The ability of LA and/or DHLA to function as either anti- or pro-oxidants, at least in part, is determined by the type of oxidant stress and the physiological circumstances. These pro-oxidant actions suggest that LA and DHLA act by multiple mechanisms, many of which are only now being explored. LA has been reported to have a number of potentially beneficial effects in both prevention and treatment of oxygen-related diseases. Selection of appropriate pharmacological doses of LA for use in oxygen-related diseases is critical. On the other hand, much of the discussion in clinical studies has been devoted to the pro-oxidant role of LA. This aspect remains to be elucidated. In further studies, careful evaluation will be necessary for the decision in the biological system whether LA administration is beneficial or harmful.

Inactivation of wild-type p53 protein function by reactive oxygen and nitrogen species in malignant glioma cells.

Malignant gliomas are the most common primary brain tumors in adults, and the most malignant form, glioblastoma multiforme (GBM), is usually rapidly fatal. Most GBMs do not have p53 mutations, although the p53 tumor suppressor pathway appears to be inactivated. GBMs grow in a hypoxic and inflammatory microenvironment, and increased levels of the free radicals nitric oxide (NO) and superoxide () occur in these malignancies in vivo. Peroxynitrite (ONOO(-)) is a highly reactive molecule produced by excess NO and that can posttranslationally modify and inactivate proteins, especially zinc finger transcription factors such as p53. We demonstrated previously that GBMs have evidence of tyrosine nitration, the "footprint" of peroxynitrite-mediated protein modification in vivo, and that peroxynitrite could inhibit the specific DNA binding ability of wild-type p53 protein in glioma cells in vitro. Here we show that both authentic peroxynitrite and SIN-1 (3-morpholinosydnonimine hydrochloride), a molecule that decomposes into NO and to form peroxynitrite, can inhibit wild-type p53 function in malignant glioma cells. Concentrations of peroxynitrite associated with a tumor inflammatory environment caused dysregulation of wild-type p53 transcriptional activity and downstream p21(WAF1) expression.

Pharmacokinetics and metabolism of the reactive oxygen scavenger alpha-phenyl-N-tert-butylnitrone in the male Sprague-Dawley rat.

The pharmacokinetics of the spin-trap alpha-phenyl-N-tert-butylnitrone (PBN) was investigated in male Sprague-Dawley rats. Plasma concentrations after i.v. administration (10 mg/kg) declined monoexponentially with a terminal half-life of 2.01 +/- 0.35 h and total plasma clearance (CL(p)) and volume of distribution at steady state (Vd(ss)) averaged 12.37 +/- 3.82 ml/min/kg and 1.74 +/- 0.5 l/kg, respectively. The observed CL(p) was in close agreement with the blood clearance (CL(b)) value (11.5 ml/min/kg) predicted from in vitro liver microsomal incubations suggesting that PBN CL(p) in rats is predominantly due to hepatic metabolism. Peak plasma concentration (C(max)) following p.o. (20 mg/kg) and s.c. (30 mg/kg) PBN administration was 7.35 +/- 1.92 and 3.56 +/- 0.66 microg/ml, whereas the area under the concentration-time curve from 0 to infinity was 23.89 +/- 5.84 and 15.96 +/- 3.10 microg-h/ml, respectively. The mean oral bioavailability of PBN was 85.63 +/- 20.93%. Biotransformation studies indicated the P450 2C11-catalyzed hydroxylation of PBN to M1. Potential sites of hydroxylation included the benzylic carbon resulting in phenyl-N-tert-butylhydroxamic acid or the phenyl ring that would afford alpha-hydroxyphenyl-N-tert-butylnitrone (HOPBN). The structure of M1 was established as alpha-4-Hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) on the basis of: 1) obvious LC R(t) differences between M1 and the authentic hydroxamate standard, 2) P450 catalyzed hydroxylation of [(2)H]PBN that contained a deuterium instead of a hydrogen atom on its benzylic position and which afforded [(2)H]M1, and 3) comparison of the liquid chromatography-tandem mass spectrometry properties with a synthetic 4-HOPBN standard. We speculate that 4-HOPBN is an "active" PBN metabolite that provides an additive effect to the pharmacological action of PBN in vivo.

Pro-oxidant and antioxidant mechanisms of etoposide in HL-60 cells: role of myeloperoxidase.

Etoposide is an effective anticancer agent whose antitumor activity is associated with its phenolic E-ring, which can participate in intracellular redox cycling reactions. Myeloperoxidase (MPO)-catalyzed one-electron oxidation of the etoposide phenolic ring and/or interaction of this phenolic moiety with reactive radicals yields its phenoxyl radical, whose reactivity may determine the pro- or antioxidant effects of this molecule in cells. Using MPO-rich HL-60 cells, we directly demonstrated that both anti- and pro-oxidant activities of etoposide are realized in cells. Etoposide acted as an effective radical scavenger and antioxidant protector of phosphatidylethanolamine, phosphatidylcholine, and other intracellular phospholipids against H2O2-induced oxidation in HL-60 cells with constitutively high MPO activity and in HL-60 cells depleted of MPO by an inhibitor of heme synthesis, succinyl acetone. MPO-catalyzed production of etoposide phenoxyl radicals observed directly in HL-60 cells by electron paramagnetic resonance (EPR) did not result in oxidation of these membrane phospholipids, suggesting that the radicals were not reactive enough to trigger lipid oxidation. MPO-dependent pro-oxidant activity of etoposide was directly demonstrated by (a) the ability of intracellular reduced glutathione (GSH) to eliminate EPR-detectable etoposide phenoxyl radicals, (b) the ability of etoposide phenoxyl radicals to oxidize GSH and protein thiols (after preliminary depletion of intracellular GSH with a maleimide reagent, ThioGlo-1), and (c) the disappearance of these effects after depletion of MPO by pretreatment of cells with succinyl acetone. In addition, titration of intracellular GSH (in intact cells) using the maleimide reagent ThioGlo-1 resulted in remarkably augmented EPR-detectable etoposide phenoxyl radicals and enhanced etoposide-induced topoisomerase II-DNA covalent complexes. In conclusion, the phenolic moiety of etoposide acts as an effective free radical scavenger, accounting for its antioxidant action. Whereas one-electron oxidation of etoposide by free radical scavenging and/or by MPO results in a phenoxyl radical with low reactivity toward lipids, its high reactivity toward thiols is a determinant of its pro-oxidant effects in HL-60 cells.

The formation of DNA cleaving species during the reduction of chromate by ascorbate.

A detailed study of the ability of chromate in combination with ascorbate to induce DNA single-strand breaks in the absence of iron(II) and copper(II) has been carried out. In solutions containing 1 mM ascorbate and chromate in the range 0.1-1 mM extensive DNA cleavage occurred. Chromate alone or the final product of the chromate/ascorbate reaction were not responsible for the cleavage observed. Evidence is presented that an intermediate generated during the reduction of chromate is the reactive species. No strand breaks occurred upon addition of catalase, pointing to a role for peroxidic species in the steps leading to the generation of the cleaving species. The exclusion of oxygen led to a substantial decrease in the number of strand breaks. Furthermore, the formation of strand breaks declined with decreasing concentrations of phosphate in the phosphate buffers used as the incubation medium. No DNA strand breaks were induced in medium containing HEPES. These observations rule out chromium(V) as the agent directly responsible for the DNA degradation, as chromium(V) is formed during the reduction of chromate by ascorbate in HEPES buffer. Our results lead us to suggest that the DNA-damaging ability of chromate upon reduction by ascorbate arises from the activation of oxygen exacerbated by phosphate and points to a peroxo or superoxo complex involving chromium(V) or chromium(IV) as a possible candidate.