Antioxidant effects of nitric oxide.
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Animals↗
Biomedical subjects
J B Mitchell
Publications and source records attributed to J B Mitchell.
Hemodynamic effect of the nitroxide superoxide dismutase mimics.
Reactive oxygen species play critical roles in a number of physiologic and pathologic processes. Nitroxides are stable free radical compounds that possess superoxide dismutase (SOD) mimetic activity and have been shown to protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol, a cell-permeable hydrophilic nitroxide, protects against oxidative stress and also is an in vitro and in vivo radioprotector. In the course of evaluating the pharmacology and toxicity of the nitroxides, Tempol and another nitroxide, 3-carbamoyl-PROXYL (3-CP), were administered intravenously in various concentrations to miniature swine. Tempol caused dose-related hypotension accompanied by reflex tachycardia and increased skin temperature. Invasive hemodynamic monitoring with Swan Ganz catheterization (SGC) confirmed the potent vasodilative effect of Tempol. However, 3-CP had no effect on porcine blood pressure. The hemodynamic effects of Tempol and 3-CP are discussed in the context of differential catalytic rate constants for superoxide disumation that may impact systemic nitric oxide (NO) levels and lead to vasodilation. These findings are consistent with a role for the superoxide ion in the modulation of blood pressure and have potential implications for the systemic use of nitroxides.
Animals↗
Clinical radiosensitization: why it does and does not work.
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Antineoplastic Agents↗
A multidrug-resistant breast cancer cell line induced by weekly exposure to doxorubicin.
Attempts to study drug resistance in vitro have focused on continuous exposure of cell lines to cytotoxic agents that results in marked resistance. In an effort to examine drug resistance in an environment that is more typical of a clinical setting, monolayered human breast cancer cells (MCF-7) were exposed weekly for one hour to doxorubicin at a concentration that is achieved in vivo (0.1 microg/ml). After 20 weeks, the treated cell line (C20) was found to be resistant to doxorubicin by a factor of 2.0-2.5 at the 10 and 1% cell survival level compared to parent MCF-7 cells. Additionally, cross resistance to other chemotherapeutic agents including etoposide, vincristine, cisplatin, and mitomycin C was observed. Similar to other models of in vitro drug resistance, no modification of radiosensitivity was observed in C20 cells. The p170 glycoprotein was not overexpressed on C20 cells as assessed by the anti-p170 glycoprotein monoclonal antibody C219 staining nor was mRNA for the mdr-1 or MRP gene overexpressed. In addition, the mdr-blocking agent verapamil had no effect on the level of resistance encountered in tissue culture. C20 cells did not differ from the parent cell line with respect to cell cycle distribution, doubling time, GSH, GSH peroxidase, GSH reductase or GSH transferase levels. After a one-hour exposure to doxorubicin, lower intracellular doxorubicin levels were found in C20 cells compared to the parent line which provides at least a partial explanation for resistance, although the mechanism for this is unclear. Although the magnitude of resistance observed in the C20 cell line is low compared to other in vitro models of drug resistance, the modest level of drug resistance is probably sufficient to explain drug resistance seen clinically. The model of non-mdr mediated drug resistance presented may be a more relevant model for the evaluation of drug resistance in vitro.
Antineoplastic Agents↗
Principles of chemoradiation: theoretical and practical considerations.
Chemotherapy agents known to enhance the effects of radiation in preclinical studies have been used concurrently with radiotherapy in numerous clinical trials with the prospect of further enhancing radiation-induced local tumor control. While some success in several tumor histologies has been achieved using this approach, a major concern has been enhancement in normal tissue toxicity. This brief review addresses both theoretical and practical issues with respect to chemoradiation clinical trials. Recommendations for clinical trials are provided that, if implemented, can increase our understanding of basic mechanisms (in patients) and provide a more rational approach for future trials.
Animals↗
In vivo radioprotection and effects on blood pressure of the stable free radical nitroxides.
PURPOSE: The purpose of this study was to screen several water soluble nitroxides for in vivo radioprotection, to evaluate their pharmacology, and to measure the effect of nitroxides on systemic blood pressure as a means of exploring the mechanism of in vivo radioprotection. METHODS AND MATERIALS: A number of water soluble nitroxides were screened for in vivo radioprotection in C3H mice at a single radiation dose. Selected nitroxides were administered by the intraperitoneal route 10 minutes prior to a whole body radiation dose of 9 Gy. Electron paramagnetic resonance spectroscopy (EPR) was used to measure whole blood levels of nitroxides. The nitroxides were evaluated for effects on systemic blood pressure in C3H mice. RESULTS: All of the nitroxides studied demonstrated radioprotection compared to saline-treated controls. The 6-membered piperidine ring nitroxides including Tempol were reduced to the inactive hydroxylamine rapidly over 10-20 minutes. The 5-membered ring nitroxides were reduced more slowly over time. The 5-membered ring 3-carbamoyl-PROXYL did not produce a substantial decrease in systemic blood pressure after intraperitoneal administration compared to the other nitroxides studied. 3-carbamoyl-PROXYL was further evaluated over a range of whole body radiation doses and was found to provide radioprotection. CONCLUSION: All of the nitroxides studied provided radioprotection. In vivo radioprotection for all of the compounds except 3-carbamoyl-PROXYL may be at least partially explained by the induction of hypotension and bone marrow hypoxia. 3-carbamoyl-PROXYL provided in vivo radioprotection similar in magnitude to Tempol and had little effect on blood pressure compared to the other nitroxides. Other mechanisms for radioprotection, including scavenging of free radicals are likely. 3-carbamoyl-PROXYL should be evaluated further as a systemic radioprotector.
Animals↗
Redox generation of nitric oxide to radiosensitize hypoxic cells.
PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation. In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents. METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay. Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions. Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol. NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode. RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively. Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity. The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode. CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization. NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation.
Animals↗
Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage.
The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.
Animals↗
Protein folds and functions.
BACKGROUND: The recent rapid increase in the number of available three-dimensional protein structures has further highlighted the necessity to understand the relationship between biological function and structure. Using structural classification schemes such as SCOP, CATH and DALI, it is now possible to explore global relationships between protein fold and function, something which was previously impractical. RESULTS: Using a relational database of CATH data we have generated fold distributions for arbitrary selections of proteins automatically. These distributions have been examined in the light of protein function and bound ligand. Different enzyme classes are not clearly reflected in distributions of protein class and architecture, whereas the type of bound ligand has a much more dramatic effect. CONCLUSIONS: The availability of structural classification data has enabled this novel overview analysis. We conclude that function at the top level of the EC number enzyme classification is not related to fold, as only a very few specific residues are actually responsible for enzyme activity. Conversely, the fold is much more closely related to ligand type.
Binding Sites↗
Nitroxides tempol and tempo induce divergent signal transduction pathways in MDA-MB 231 breast cancer cells.
Tempol and tempo are stable free radical nitroxides that possess antioxidant properties. In this study, we examined the effects of these compounds on components of the mitogen-activated protein kinase signal transduction cascade. Tempo treatment (15 min) of MDA-MB 231 human breast cancer cells resulted in significant levels of tyrosine phosphorylation of several as yet unidentified proteins compared with equimolar concentration of tempol (10 mM). Both compounds caused tyrosine phosphorylation and activation of Raf-1 protein kinase (30 min, 2-3-fold). Interestingly, however, only tempol caused increased extracellular signal-regulated kinase 1 activity (2 h, approximately 3-fold). On the other hand, tempo, but not tempol, potently activated stress-activated protein kinase (2 h, >3-fold). Consistent with these data, tempol was found to be noncytotoxic, whereas tempo induced apoptotic cell death (2 h, >50%). Tempo treatment also resulted in significant elevation of ceramide levels at 30 min (54% over control) and 1 h (71% over control) posttreatment, preceding stress-activated protein kinase activation and apoptosis. These data suggest that in the absence of an environmental oxidative stress, tempol and tempo elicit distinct cellular signaling pathways. The recognition of the molecular mechanisms of nitroxide action may have important implications for biological effectiveness of these compounds.
Antioxidants↗
Preparation and EPR studies of lithium phthalocyanine radical as an oxymetric probe.
The electron paramagnetic resonance (EPR) spectrum of the paramagnetic center in solid lithium phthalocyanine, LiPc, exhibits a pO2 (partial pressure of oxygen)-dependent line width. The compound is insoluble in water and is not easily biodegradable and, therefore, is a useful spin probe for quantitative in vivo oxymetry. Because EPR spectrometry is potentially a useful technique to quantitatively obtain in vivo tissue pO2, such probes can be used to obtain physiological information. In this paper, a simple experimental procedure for the preparation of LiPc using potentiostatic electrochemical methods is described. The setup was relatively inexpensive and easy to implement. A constant potential ranging from 0.05 to 0.75 V versus Ag+/AgCl(s) was used for obtaining LiPc. The EPR spectral studies were carried out using spectrometers operating at X-band and at radiofrequency (RF) at different pO2 values to characterize the spectral response of these crystals. The results indicate that, depending on the electrolysis conditions, the products contain mixtures of crystals exhibiting pO2-sensitive and pO2-insensitive line widths. Electrolysis conditions are reported whereby the pO2-sensitive LiPc crystals were the predominant product. The influence of the working surface of the electrode and the electrolysis time on the yield were also evaluated. The crystals of LiPc were also studied using a time-domain RF EPR spectrometer. In time-domain EPR, the signals that survive beyond the spectrometer dead time are mainly the narrow lines corresponding to the pO2-sensitive crystals, whereas the signals arising from the pO2-insensitive component of LiPc were found not to survive beyond the spectrometer dead time. This signal survival makes the time-domain EPR method more sensitive for pO2 measurements using LiPc because the line width becomes very narrow at very low pO2 and, concomitantly, the relaxation time T2 longer, with no modulation or power saturation artifacts that are encountered as in the continuous wave (cw) mode. Further, minimal contributions from object motion in the spectral data obtained using time-domain methods make it an advantage for in vivo applications.
Crystallization↗
Design, synthesis and structure of a zinc finger with an artificial beta-turn.
We have incorporated a bicyclic beta-turn mimetic (BTD; beta-turn dipeptide) into a zinc finger, creating a zinc finger with an artificial beta-turn. The designed peptide chelates zinc and has the same fold as the unmodified native zinc finger (finger 3 of the human YY1 protein). A combination of 1H NMR and structure calculations reveals that, in solution, this zinc finger has a fold similar to the known wild-type crystal structure and to other zinc fingers containing the consensus sequence X3-Cys-X4-Cys-X12-His-X3-His-X. The peptide was designed with BTD between the chelating cysteine residues, with BTD forming a type II' beta-turn linking the two strands of a distorted anti-parallel beta-sheet. The C-terminal portion of the peptide forms a helix with zinc co-ordinating histidine residues on successive turns of the helix. This work represents a step towards developing methods by which parts of a target protein may be replaced by peptide mimetics.
Amino Acid Sequence↗
In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model.
Nitroxides are redox-sensitive probes, which are useful in noninvasively delineating tissue heterogeneity especially with respect to metabolic activity and tissue oxygenation. Recent studies have shown that nitroxides are in vitro and in vivo radioprotectors and selectively protect normal tissue compared to tumor tissue. It has been postulated that the basis for selective radioprotection of normal tissues is greater bioreduction of nitroxides in tumor tissue compared to normal tissue. The aim of the present study was to investigate the distribution and lifetime of nitroxides in tumor and normal tissues. Mice were implanted with tumor cells (RIF-1) in the thigh, and the tumor was allowed to grow to about 10-15 mm in diameter. After i.v. infusion of nitroxides, in vivo electron paramagnetic resonance spectroscopy and imaging of the tumor were performed using a specially built bridged-loop surface resonator. The pharmacokinetic and spatial distribution of the nitroxides in tumor tissue were followed and compared with those in normal tissue. Three-dimensional spatial images showed significant heterogeneity in the nitroxide distribution as well as reduction rates. The nitroxide reduction rates were significantly higher in tumors than in the normal tissue. Measurements using spin label oximetry showed a substantial difference in the level of oxygenation between normal tissue (muscle) and tumor tissue. Average pO2 levels in tumor tissue were found to be 3-fold lower than in a corresponding volume of normal tissue. The lower pO2 levels in tumor compared to normal tissue may explain the more rapid reduction of nitroxides in these tissues. This study demonstrates that electron paramagnetic resonance imaging can perform noninvasive anatomical as well as functional imaging and provide in vivo physiological information regarding cellular metabolism in tumor and normal tissues.
Animals↗
The cytotoxicity of nitroxyl: possible implications for the pathophysiological role of NO.
In addition to the broad repertoire of regulatory functions nitric oxide (NO) serves in mammalian physiology, the L-arginine:NO pathway is also involved in numerous pathophysiological mechanisms. While NO itself may actually protect cells from the toxicity of reactive oxygen radicals in some cases, it has been suggested that reactive nitrogen oxide species formed from nitric oxide synthase (NOS) can be cytotoxic. In addition to NO, the one electron reduction product NO- has been proposed to be formed from NOS. We investigated the potential cytotoxic role of nitroxyl (NO-), using the nitroxyl donor Angelis's salt, (AS; sodium trioxodinitrate, Na2N2O3) as the source of NO-. As was found to be cytotoxic to Chinese hamster V79 lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-(CN6)3-), which converts NO- to NO, afforded dramatic protection against AS-mediated cytotoxicity. Treatment of V79 cells with L-buthionine sulfoximine to reduce intracellular glutathione markedly enhanced AS cytotoxicity, which suggests that GSH is critical for cellular protection against the toxicity of NO-. Further experiments showed that low molecular weight transition metal complexes associated with the formation of reactive oxygen species are not involved in AS-mediated cytotoxicity since metal chelators had no effect. However, under aerobic conditions, AS was more toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA double strand breaks in whole cells, and this effect was completely prevented by coincubation of cells with ferricyanide or Tempol. The data in this study suggest that nitroxyl may contribute to the cytotoxicity associated with an enhanced expression of the L-arginine:NO pathway under different biological conditions.
Animals↗
Impact of atrial fibrillation on mortality, stroke, and medical costs.
BACKGROUND: The impact of atrial fibrillation (AF) on mortality, stroke, and medical costs is unknown. METHODS: We conducted a prospective cohort study of hospitalized Medicare patients with AF and 1 other cardiovascular diagnosis (CVD) compared with a matched group without AF (n = 26,753), randomly selected in 6 age-sex strata from 1989 MedPAR files of more than 1 million patients diagnosed as having AF. Stroke rates were also determined in another cohort free of CVD (n = 14,267). Total medical costs after hospitalization were available from a 1991 cohort. Cumulative mortality, stroke rates, and costs following index admission were adjusted by multivariate and proportional hazard regression analyses. RESULTS: Mortality rates were high in individuals with CVD, ranging from 19.0% to 52.1% in 1 year. Adjusted relative mortality risk was approximately 20% higher in patients with AF in all age-sex strata during each of the 3 years studied (P < .05). Incidence of stroke was high in individuals with CVD, 6.2% to 15.4% in 1 year, with and without AF, and was at least 5-fold higher than in individuals without CVD. In those with CVD, stroke rates were approximately 25% higher in women with AF (P < .05) but only 10% higher in men. Adjusted total Medicare spending in 1 year was 8.6- to 22.6-fold greater in men, and 9.8- to 11.2-fold greater in women with AF (P < .05). Second- and third-year costs were increased as well. CONCLUSION: Prevention of AF and treatment of patients with AF and associated CVD may yield benefits in reduced mortality and stroke as well as reducing health care costs.
Age Distribution↗
Increased mortality, blunted production of nitric oxide, and increased production of TNF-alpha in endotoxemic TGF-beta1 transgenic mice.
The expression of the inducible isoform of nitric oxide synthase (NOS2, iNOS) is increased in patients undergoing sepsis as well as in animal models in which septic shock is induced by injection of bacterial lipopolysaccharide (LPS). Transforming growth factor-beta1 (TGF-beta1) potently suppresses NO production both in vitro and in vivo. After intraperitoneal injection of LPS, mice over-expressing a cDNA coding for active TGF-beta1 in the liver (Alb/ TGF-beta1) exhibited reduced serum levels of the NO reaction products NO2(-) + NO3(-) compared with controls. Paradoxically, while endotoxemic Alb/ TGF-beta1 mice expressed much less NOS2 protein in peritoneal exudate cells than did endotoxemic wild-type mice, Alb/TGF-beta1 mice expressed more NOS2 mRNA and protein in both liver and kidney. Alb/ TGF-beta1 mice treated with LPS had eightfold higher serum tumor necrosis factor alpha (TNF-alpha) levels and experienced increased mortality compared with wild-type mice, which was associated with renal insufficiency. These results suggest that renal dysfunction, decreased production of NO, and/or increased production of TNF-alpha are associated with increased mortality of endotoxemic Alb/TGF-beta1 mice.
Alanine Transaminase↗
The medial frontal cortex and temporal memory: tests using spontaneous exploratory behaviour in the rat.
Evidence from several sources indicates that the frontal cortex is involved in memory involving time, or temporal memory, and in behavioral sequencing. Rats were presented with two familiar objects and the amount of exploration directed at each was recorded. Subjects directed more exploration at the object encountered further ago in the past, even though the two objects differed by only 1 h. After bilateral medial frontal cortex lesion, animals did not differentiate between old and recent objects. Performance on an object recognition test (novel versus familiar objects) that also used exploration time did not change as a result of the medial frontal cortex lesions.
Animals↗
Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide.
There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the "chemical biology of NO" to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented.
Animals↗