C.Murali Krishna

Superoxide Reaction with Nitroxides

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoptmetric (Cyt CIII) and chemiluminescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemiluminescence clearly demonstrated that the six-membered piperidine-1-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 x 10(4) to 1.2 x 10(5) M-1 s-1. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides, they may ultimately be more useful as superoxide oxide dismutase mimetic agents.

Superoxide reaction with nitroxide spin-adducts

The reactions of superoxide radical with persistent nitroxide spin-adducts or with stable spin-labels were studied using ESR spectrometry. Superoxide radicals were produced enzymatically using xanthine - xanthine oxidase or chemically by dissolving potassium superoxide in DMSO. Hydroxyl and methyl spin-adducts of the spin-trap DMPO were performed by sonolysis and subsequently reacted with superoxide radical. Superoxide-induced depletion of DMPO--OH obeyed second order kinetics. Contrary to previously published mechanisms, the reaction requires neither transition metal ions nor thiols. The depleted spin-adducts could not be restored by reoxidation with ferricyanide or copper +H2O2; thus, the superoxide-mediated destruction does not result in a mere one-electron reduction product. Superoxide also depletes other DMPO spin-adducts including DMPO--CH3 and DMPO--H, but not PBN--CH3. In addition, some 5-membered ring stable nitroxides are depleted by superoxide in a pseudo-zero order reaction. In studying systems which generate O2- and OH, the superoxide-induced destruction of DMPO--OH may well lead to erroneous conclusions regarding the primary radicals produced. In particular this reaction might be operative under circumstances where elevated rates of superoxide production take place, such as during oxygen consumption burst in phagocytosis, degranulation, or paraquat intoxication.

Nitroxide Sod-Mimics: Modes of Action

Low molecular weight superoxide dismutase mimics have been shown to afford protection from oxidative damage. Such SOD-mimics can readily permeate cell membrane achieving sufficiently high levels both inside and outside the cell to effectively detoxify intracellular O2-.. Preliminary findings also indicated that metal-based and metal-free SOD-mimics can protect hypoxic cells from H2O2-induced damage. The present study explored the possibility that SOD-mimics such as desferrioxamine-Mn(III) chelate [DF-Mn] or cyclic nitroxide stable free radicals could protect from O2-.-independent damage. Killing of monolayered V79 Chinese hamster cells was induced by H2O2 or by t-butyl hydroperoxide (t-BHP) and assayed clonogenically. Neither catalase nor native SOD protected the cells from t-BHP. In contrast, both DF-Mn and cyclic nitroxides protected suggesting cytotoxic processes independent of O2-. or of O2-.-derived active species. The inhibition of the damage by both metal-free and metal-based SOD mimics is attributable to either SOD-mimic reacting with reduced transition metal to block the Fenton reaction and/or intercepting and detoxifying intracellular organic free radicals.

The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.

Sonochemistry of volatile and non-volatile solutes in aqueous solutions : e.p.r. and spin trapping studies

Recent spin trapping studies of the free radical intermediates generated by the sonolysis of aqueous solutions are reviewed. Studies of rare gas saturated solutions of volatile solutes (e.g., methanol and ethanol) and of non-volatile solutes (acetate, amino acids, sugars, pyrimidines, nucleotides and surfactants) are consistent with the theory of three reaction zones in aqueous sonochemistry. The very high temperatures and pressures induced by acoustic cavitation in collapsing gas bubbles in aqueous solutions lead to the thermal dissociation of water vapour into hydrogen atoms and hydroxyl radicals. Reactions take place in the gas phase (pyrolysis reactions), in the region of the gas-liquid interface, and in the bulk of the solution at ambient temperature (similar to radiation chemistry reactions). By use of the rare gases with different thermal conductivities, the contributions of individual reaction steps with widely different energies of activation can be evaluated.

Evaluation of Tempol Radioprotection in a Murine Tumor Model

Tempol, a stable nitroxide free radical compound, is an in vitro and in vivo radioprotector. Previous studies have shown that Tempol protects C3H mice against whole-body radiation-induced bone marrow failure. In this study, the radioprotection of tumor tissue was evaluated. RIF-1 tumor cells were implanted in female C3H mice 10 d prior to radiation. Groups of mice were injected intraperitoneally with Tempol (275 mg/kg) or PBS followed 10 min later by a single dose of radiation to the tumor bed. Tumor growth curves generated after 10 and 33.3 Gy doses of radiation showed no difference in growth between the Tempol- and PBS-treated animals. A full radiation dose-response experiment revealed a tumor control dose in 50% of the animals in 30 d (TCD(50/30)) value of 36.7 Gy for Tempol-treated mice and 41.8 Gy for saline-treated mice suggesting no protection of the RIF-1 tumor by Tempol. Tumor pharmacokinetics were done to determine why Tempol differentially protected bone marrow and not tumor cells. Differential reduction of Tempol in the RIF-1 tumor and bone marrow was evaluated with EPR spectroscopy 10, 20, and 30 min after injection. Bioreduction of Tempol to its corresponding hydroxylamine (which is not a radioprotector) occurred to a greater extent in RIF-1 tumor cells compared to bone marrow. We conclude that the differences in radioprotection may result from enhanced intratumor bioreduction of Tempol to its nonradioprotective hydroxylamine analogue. The nitroxides as a class of compounds may provide a means to exploit the redox differences between normal tissues and tumors.

Topical application of nitroxide protects radiation-induced alopecia in guinea pigs

We have recently found that treatment of Chinese hamster V79 cells with the stable nitroxide radical TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) afforded significant protection against superoxide, hydrogen peroxide, and X-ray mediated cytotoxicity. Radiation-induced alopecia is a common radiotherapeutic problem. Topical application of TEMPOL was evaluated for possible protective effects against radiation-induced alopecia using guinea pig skin as a model. For single acute X-ray doses up to 30 Gy, TEMPOL, when topically applied 15 min prior to irradiation provided a marked increase in the rate and extent of new hair recovery when compared to untreated skin. TEMPOL was detected in treated skin specimens with electron paramagnetic resonance (EPR) spectroscopy. Similar measurements of blood samples failed to show any signal resulting from topical application, nor could TEMPOL be detected in brain tissue after application on the scalp. TEMPOL represents a new class of compounds with potential for selective cutaneous radioprotection without systemic absorption.

The role of singlet oxygen in the photohemolysis of red blood cells sensitized by phthalocyanine sulfonates.

Abstract The post‐illumination photohemolysis of human red blood cells was used as a model system for studying membrane damage sensitized by various phthalocyanine sulfonates. With a constant fluence and photolysis time at 670 ± 10 nm and with a fixed optical density of the sensitizer, the percentage of hemolysis was measured as a function of time after illumination; and from the usual sigmoidal shaped curves, the times for 50% hemolysis were determined. The most effective central cation was Al, followed by Zn; the metal‐free compound and those containing the paramagnetic ions Cu, Fe and Co were inactive. The sensitizer bound to the red blood cells was found to be effective for photohemolysis. Studies of the Al compound in 98.0% deuterated media (as measured by NMR) showed a definite deuterium isotope effect, suggesting the participation of singlet oxygen. This result was supported by the large protective effects of 3.3 raM and 13.3 mM tryptophan present during photolysis. Tryptophan reacts rapidly with singlet oxygen and with OH radicals. The much smaller effects observed with the same concentrations of mannitol and glycerol, OH radical scavengers which do not react with singlet oxygen, are consistent with the predominant role of singlet oxygen in photohemolysis sensitized by Al phthalocyanine sulfonate.

In vivo radioprotection and effects on blood pressure of the stable free radical nitroxides.

PURPOSEnThe 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.nnnMETHODS AND MATERIALSnA 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.nnnRESULTSnAll 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.nnnCONCLUSIONnAll 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.

A new approach for EPR detection of hydroxyl radicals by reaction with sterically hindered cyclic amines and oxygen.

Sterically hindered cyclic amines react with hydroxyl radicals in the presence of oxygen to yield stable nitroxide radicals which can be detected by EPR. This reaction provides a nonconventional spin‐trapping tool for detection of hydroxyl radicals.