Free Radical Research Communications

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Macrophages and hepatocytes from normal and BCG-primed mice have been spin-labelled in their membranes with 5- and 16-doxyl stearic acid. Incubation of spin-labelled cells from BCG-primed animals with lipopolysaccharide from E. coli 0111.B4 produced a detectable and transient disturbance in the cell membranes as reflected by an increase in the order parameter measured from the electron spin resonance spectra of 5-doxyl-stearate. This membrane disturbance was maximal at 3-4 hours of incubation and was only detected with cells from mice primed with BCG. Spectra obtained from the 16-doxyl-stearate-labelled cells showed no change in order parameter on incubation with lipopolysaccharide.
Plasmid and chromosomal DNA of E. coli during exponential growth are cleaved after treatment with copper(II)-1, 10-phenanthroline complex (1:2) without providing any exogenous reductant. About 1500 copper molecules per cell are present as estimated by atomic absorption analysis. Within the cell the endogenous reducing substances may have participated in the sequential oxidative reactions, which lead to the damage of DNA. A portion of the resultant DNA fragments originates from plasmid DNA as demonstrated by hybridization tests.
Free Br. atoms have been found to be generated upon eaq- and .OH radical induced degradation of 1,2-dibromoethane in aqueous solution. The relevant process is beta-bromine cleavage from CH2BrCH2 and CH2BrCHBr radicals, respectively. An absolute rate constant of k = 2.8 x 10(6) s-1 has been determined for the reaction CH2BrCH2----Br. + CH2 = CH2, while an estimate of k approximately 10(6) s-1 can be given for CH2BrCHBr----Br. + CH2 = CHBr. The Br. atoms have been identified through their reaction with Br- to Br2-. (k = 7.7 x 10(9) mol-1dm3s-1) and their reaction with 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonate) (k = 6.8 x 10(9) mol-1dm3s-1). Ethylene and vinyl bromide have been identified via GC. The results substantiate earlier findings that free radical induced mechanisms can serve as informative probe for metabolic processes.
Substances of low oxidation potential, which can also make available protons and hydrogen atoms, e.g. phenothiazines, NADH, and ascorbic acid efficiently reduce 1,2-dioxetanes to their vic-diols by single-electron-transfer; a significant side reaction is catalytic decomposition of dioxetanes into the corresponding ketone fragments.
ESR spin labels specific for skeletal proteins or cell-surface sialic acid have been used to monitor the interaction of 9-amino-1,2,3,4-tetrahydroacridine (THA) and its structural analogs with human erythrocyte membranes. The results suggest that THA significantly increases skeletal protein-protein interactions and may secondarily alter the physical state of the opposite side of the membrane. The fully aromatic analog of THA, 9-aminoacridine, showed even more pronounced effects on skeletal proteins than did THA. These results are discussed in relation to possible interaction sites of THA in erythrocyte ghosts and to potential mechanisms by which THA reportedly increases mental function of victims of Alzheimer's disease.
Electrochemical studies (reduction potential and reversibility) were performed on 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+). MPP+ gave reduction potentials in the range of -1.09 to -1.11 V in organic solvents in a process which was reversible. The reduction potential of MPDP+ was -0.64 V (irreversible). Possible relationships involving the electrochemical properties, oxy radical formation, and biological activity of these and related iminium species are discussed.
Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca2+ concentration ([Ca2+]i) produced by 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca2+ sequestration (Moore, G.A., McConkey, D.J., Kass, G.E.N., O'Brien, P.J. and Orrenius, S. FEBS Lett., 224, 331-336), (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca2+]i which was similar in magnitude to the [Ca2+]i elevation induced by the Ca2+ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca2+ transient, tBuBHQ elevated [Ca2+]i to a new steady state that was maintained for up to 15-20 min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca2+]i, [Ca2+]i rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca2+]i was transient, and not sustained. The hydroquinone mobilized the same intracellular Ca2+ pool as inositol 1,4,5-trisphosphate, but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. tBuBHQ stimulated glucose release from perifused hepatocytes, mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca2+ into the perfusate and inhibition of subsequent vasopressin-induced Ca2+ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca2+ sequestration and (Ca2+-Mg2+)-ATPase activity in microsomes isolated from the isolated perfused liver.(ABSTRACT TRUNCATED AT 250 WORDS)
Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1,6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and of TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1,6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1,6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release of lactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1,6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed. On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1,6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.
Several lines of evidence indicating a close relationship among ischemia, concentration of high-energy metabolites and onset of the "oxygen paradox" in reperfused tissues have been published. In this framework, we have recently studied the effects of exogenous fructose-1,6-bisphosphate on energy metabolism and on oxygen free radical damages of isolated rat heart subjected to anoxia and reoxygenation. In comparison with control groups, hearts perfused in the presence of 5 mM fructose-1,6-bisphosphate throughout the different perfusion conditions showed higher concentrations of energy metabolites at the end of anoxia, most of which were normalized after reperfusion. Furthermore, in comparison with control hearts, a reduction of tissue malondialdehyde and of lactate dehydrogenase release in the perfusate was observed in fructose-1,6-bisphosphate-perfused hearts. In this article we review most of the available data concerning the ability of fructose-1,6-bisphosphate to protect from ischemia and reperfusion damage outlining those recent findings which contributed both to clarify the pharmacological profile of the drug and to give an insight in its probable mechanism of action.
Thioredoxin reductase (TR) is a widely distributed flavoenzyme that provides reduced thioredoxin, a dithiol hydrogen donor for protein disulfide reduction and for the reduction of ribonucleotides to deoxyribonucleotides, the first unique step of DNA synthesis. Antitumor quinones were found to exhibit time- and concentration-dependent inhibition of purified rat liver TR that requires the presence of NADPH. Diaziquone initially shows competitive inhibition of the enzyme with 5,5'-dithiobis 2-nitrobenzoic acid as substrate with a Ki of 7.5 microM, which becomes non-competitive after 1 hour incubation with NADPH with a Ki of 0.5 microM. Doxorubicin shows non-competitive inhibition both initially and after 1 hr incubation with NADPH, with Ki values of 10 microM and 0.5 microM, respectively. Electron spin resonance spectroscopy showed the formation of semiquinone free radicals by TR incubated under anaerobic conditions with doxorubicin or diaziquone and NADPH. Redox cycling and formation of oxygen radicals does not play a major role in the inhibition of TR by antitumor quinones as shown by the minor effect on inhibition of removing O2, and the lack of effect of superoxide dismutase and catalase. Diaziquone causes time- and concentration-dependent inhibition of TR activity in intact A204 human rhabdomyosarcoma cells that is associated with growth inhibition. The results suggest that inhibition of TR by antitumor quinones could contribute to their growth inhibitory properties.
Silybin has been complexed in 1:1 ratio with phosphatidyl choline to give IdB 1016 in order to increase its bioavailability. The antioxidant and free radical scavenger action of this new form of silybin has been evaluated. One hour after the intragastric administration to rats of IdB 1016 (1.5 g/kg b.wt.) the concentration of silybin in the liver microsomes was estimated to be around 2.5 micrograms/mg protein corresponding to a final concentration in the microsomal suspension used of about 10 microM. At these levels IdB decreased by about 40% the lipid peroxidation induced in microsomes by NADPH, CCl4 and cumene hydroperoxide, probably by acting on lipid derived radicals. Spin trapping experiments showed, in fact, that the complexed form of silybin was able to scavenge lipid dienyl radicals generated in the microsomal membranes. In addition, IdB 1016 was also found to interact with free radical intermediates produced during the metabolic activation of carbon tetrachloride and methylhydrazine. These effects indicate IdB 1016 as a potentially protective agent against free radical-mediated toxic damage.
The protective effect of a novel synthetic zinc-carnosine chelate compound, zinc N-(3-aminopropionyl)-L-histidine (Z-103), on the gastric mucosal injury induced by ischemia-reperfusion was studied in rats. Ischemia and reperfusion injury was produced on the rat stomach by applying a small clamp to the celiac artery for 30 min and by removal of the clamp for 30 min. The decrease in the gastric mucosal blood flow was not influenced by the treatment with Z-103. The increase in total area of the erosions on the stomach after ischemia-reperfusion and the increase in lipid peroxides in the gastric mucosa were significantly inhibited by the oral administration of Z-103. In addition, Z-103 inhibited lipid peroxidation of rat brain homogenate and liver microsome in vitro. These results suggest that the protective effect of Z-103 against the aggravation of gastric mucosal injury induced by ischemia-reperfusion may be due to its inhibitory effect on lipid peroxidation.
The active site in bovine copper, zinc superoxide dismutase (Cu2, Zn2SOD) has been studied by 111Cd time differential Perturbed Angular Correlation (PAC) on enzyme with Zn2+ replaced by excited 111Cd2+. The PAC spectra obtained for both the oxidized and the reduced form of Cu2Cd2SOD show no asymmetry between the two Zn-sites in the dimeric enzyme. The spectra further reveal that a significant change has taken place at the Zn-site in the reduced form compared to the oxidized form. Semi-empirical calculations based on the Angular Overlap Model (AOM) and coordinates from the crystal structure of the native enzyme are in agreement with the experimental PAC data of the oxidized enzyme. The results indicate that Cd2+ coordinates in the same manner as Zn2+ and that the crystal structure of SOD is valid for the enzyme in solution. The PAC spectrum of the reduced enzyme can be explained by extending the AOM calculations to the enzyme in the reduced form and assuming that the imidazol ring of His61 is no longer bridging the copper and cadmium ions in the reduced state.
The Japanese herbal Kampo medicines TJ-9 (A), TJ-15 (B), TJ-23 (C), TJ-114 (D) and TJ-96 (E) were effective (2-5x less than alpha-tocopherol) in inhibiting a copper-induced peroxidation of low density lipoprotein. Kampo medicines dissolved in n-butanol formed stable free radical(s), detected by EPR spectroscopy as a single asymmetric line with g-value g = 2.005. The radical concentration increased in the order: C less than D approximately A approximately E less than B. When the Kampo medicines were oxidized in n-butanol by excess of PbO2 their radical concentration increased 7-15 fold and was in the order C less than D less than A approximately E much less than B. A relationship between the potency of the medicines to inhibit peroxidation of LDL and their ability to form stable free radicals upon oxidation was observed. The medicine which formed more radicals was more efficient in inhibiting peroxidation of LDL. In order to study whether Kampo medicines can reduce alpha-tocopherol radical, the alpha-tocopherol radical was generated by the reaction of alpha-tocopherol with UV irradiated di-tert-butylperoxide and by autooxidation of alpha-tocopherol in n-butanol (25 microliters ml-1) in air. In both systems vitamin-C greater than Kampo B decreased the concentration of the alpha-tocopherol radical and the EPR spectrum of Kampo B stable radical(s) appeared. The effect of other Kampo medicines was not clearly seen since their EPR spectra were superimposed with the spectrum of the alpha-tocopherol radical. The results indicate that Kampo medicines possess electron donor properties and ability to form stable radical(s). The results may contribute to understanding beneficial effects of Kampo medicines in diseases in which free radical damage is suggested.
The proposal is advanced that many anticancer agents may function via redox reactions resulting in generation of toxic oxy radicals which destroy neoplastic cells. Cyclic voltammetry was performed with some of the main types: iminium ions (protonated mAMSA derivatives), quinone derivatives (rhodamine 123) and metal complexes (nickel(II) salicylaldoximate). In addition, relevant literature data are provided. A rationale is offered that relates electrochemical data to physiological activity. © 1987 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), is commonly used for the detection of short-lived free radicals produced by neutrophils during their respiratory burst. The spin adducts of both the superoxide ion (O2-) and the hydroxyl radical (.OH) are detectable during this process. Since myeloperoxidase (MPO), which is also active during the respiratory burst, produces hypochlorous acid (HOCl) (HOCl) in the presence of chloride ions (Cl-) and hydrogen peroxide (H2O2), this species has been investigated as a possible source of the DMPO-OH adduct. At concentrations of hypochlorous acid between 0.1 and 0.7 mumol/ml the DMPO-OH spin adduct is detected using electron spin resonance (ESR) techniques. Two possible mechanisms for the formation of this adduct are proposed. These findings suggest that the product of MPO, namely hypochlorous acid, is a possible source of the hydroxyl spin adduct detected during the respiratory burst.
OPC-14117 is a potent drug which has both brain function activating effect and protective effect against cerebral ischemia. Occurrences of these effects might be expected due to superoxide dismutase-like activity of OPC-14117. The present study has been conducted to evaluate the active oxygen scavenging activity of OPC-14117 and to explain the mechanisms of its pharmacological activities. The reaction of OPC-14117 and superoxide anion, generated in potassium superoxide, was examined by electron spin resonance technique at both liquid nitrogen (77 K) and room (22 degrees C) temperatures. OPC-14117 showed a higher superoxide scavenging activity than that of alpha-tocopherol in an aprotic solvent system. The active moiety of OPC-14117 to provide the scavenging effect was found due to the phenolic hydroxyl group of its indan skeleton.
Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.
The catechol metabolite of the antitumor agent VP-16-213 and the ortho-quinone of VP-16-213--a secondary metabolite formed from the catechol--easily undergo auto-oxidation into a free radical at pH greater than or equal to 7.4. By elevation of the pH from 7.4 to 10, an increase in the production of the free radical was observed, which was accompanied by the formation of products with higher hydrophylicity than the catechol and ortho-quinone, as found by HPLC-analysis. The hyperfine structure of the free radical indicates that it is the semi-quinone radical of VP-16-213. At pH 12.5 a secondary radical is formed from the catechol and the ortho-quinone of VP-16-213 besides the semi-quinone radical. One-electron oxidation of the catechol with horseradish peroxidase/hydrogen peroxide resulted in the formation of the same radical as observed under alkaline conditions and subsequent oxidation to the ortho-quinone. If the ortho-quinone was incubated with NADPH cytochrome P-450 reductase, a free radical was detected by spin-trapping with POBN, but not without spin-trapping. Studies on inactivation of phi X174 DNA by the system ortho-quinone of VP-16-213/NADPH cytochrome P-450 reductase suggest that the semi-quinone radical may play a role in the process of inactivation of DNA.
Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 M NaCl at 20 degrees C, was 257 +/- 4 s. The signal recorded on incubation with HRP/H2O2 or MPO/H2O2 did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.
Tyrosinase-dependent activation of hydroxybenzenes forms reactive compounds, including catechols and o-quinones, and some of which show antitumor activity against pigmented melanomas. Since VP-16 is a phenoxy-containing antitumor drug, forms free radicals and reactive o-quinones during peroxidative activation, we evaluated the cytotoxicity of VP-16 to both tyrosinase-containing and non-tyrosinase-containing tumor cells. Our results show that VP-16 is significantly more cytotoxic to B-16/F-10 melanoma cells than human MCF-7 breast tumor cells. Phenylthiocarbamide, an inhibitor of tyrosinase activity, selectively decreased VP-16 toxicity only in melanoma cells. Furthermore, VP-16 was readily activated to its phenoxy free radical intermediate by purified tyrosinase, indicating tyrosinase may play a role in VP-16 toxicity in pigmented melanomas.
The effects of 17 beta estradiol, testosterone, the estradiol benzoate, and probucol on the oxidation kinetics of low density lipoprotein (LDL) in vitro in absorption presence of 10 microM Cu (II) are examined. Changes in the absorption at 234 nm (A234) and fluorescence (Ex340/Em420) are monitored. The kinetics of the changes observed let us suggest a precursor-product relationship between dienes and fluorochromes in the oxidized LDL. The addition of 17 beta estradiol and probucol to LDL results in a prolongation of the lag phase characterized by only insignificant formation of dienes and fluorochromes. The addition of testosterone and estradiol benzoate used as control compounds has no effect on the lag phase and thus no LDL stabilizing effect. Conditioned LDL which was incubated in F-10 medium before exposure to cultured P388D.1 macrophages increases the formation of cytoplasmic lipid droplets and of cellular cholesteryl esters. The LDL stabilizing compounds beta estradiol and probucol (but not testosterone) causes a reduction of the cholesteryl ester content of the cultured macrophages. Protection of LDL particles against oxidative damage apparently results also in lowering of cytoplasmic cholesteryl ester in cultured P388D.1 cells. We conclude that the known antiatherosclerotic potency of 17 beta estradiol may in part result from its LDL stabilizing activity.
The rate constant for the reaction of NO with ·O2- was determined to be (6.7 ± 0.9) × 109 1 mol-1 s-1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O2- and the formation of the product -OONO. The decay rate of -OONO, in the presence of 0.1 moll-1 formate, ranges from 1.2s-1 at pH 5 to about 0.2s-1 in strong base, the latter value probably reflecting catalysis by formate. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
We have previously shown (C.L. Borders, Jr. et al., (1989) Archives of Biochemistry and Biophysics, 268, 74-80) that the iron-containing (FeSOD) and manganese-containing (MnSOD) superoxide dismutases from Escherichia coli are extensively (greater than 98%) inactivated by treatment with phenylglyoxal, an arginine-specific reagent. Examination of the published primary sequences of these two enzymes shows that Arg-189 is the only conserved arginine. This arginine is also conserved in the three additional FeSODs and seven of the eight additional MnSODs sequenced to date, with the only exception being the MnSOD from Saccharomyces cerevisiae, in which it is conservatively replaced by lysine. Treatment of S. cerevisiae MnSOD with phenylglyoxal under the same conditions used for the E. coli enzymes gives very little inactivation. However, treatment with low levels of 2,4,6-trinitrobenzenesulfonate (TNBS) and acetic anhydride, two lysine-selective reagents that cause a maximum of 65-80% inactivation of the E. coli SODs, gives complete inactivation of the yeast enzyme. Total inactivation of yeast MnSOD with TNBS correlates with the modification of approximately 5 lysines per subunit, whereas 6-7 lysines per subunit are acylated with acetic anhydride on complete inactivation. It appears that the positive charge contributed by residue 189, lysine in yeast MnSOD and arginine in all other SODs, may be critical for the catalytic activity of MnSODs and FeSODs.
Exposure of hepatoma 1c1c7 cells to 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca2+, DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca2+ chelators, Quin 2 or BAPTA, to buffer the increase in cytosolic Ca2+ concentration induced by the quinone. DMNQ caused marked NAD+ depletion which was prevented when cells were preincubated with 3-aminobenzamide, an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity, or with either of the two Ca2+ chelators. However, 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage, NAD+ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca2+.
2,2'-Azo-bis(2-amidinopropane) thermolysis induces luminol luminescence. The luminescence intensity is quenched by SOD, catalase, Trolox and human blood serum. However, the time course of the light intensity profile is different for the different additives. In particular, the quenching efficiency of Trolox and human blood serum decreases with time after addition. Double quenching experiments show that SOD and Trolox are not competitive quenchers, while a simple competition can be established between Trolox and human blood serum in trapping a common intermediate. From the kinetic analysis of the data it is concluded that, at least at low additive concentrations, Trolox scavenges a luminol derived radical. Higher concentrations of Trolox or human blood serum produce induction times that are proportional to the additives concentrations. The possibility of employing luminol luminescence in the evaluation of TRAP levels and the capacity of biological samples to scavenge free radicals is discussed.
Aminoxyl radicals of the type R1N(O.)R2 are formed in the photochemical reaction between nitric oxide (NO.) and carbon-centered radicals R1. and R2.. R1. was formed from azo compounds such as 2,2'-azobisisobutyronitrile (AIBN): R1. = NC-C(CH3)2, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN): R1. = CH3-CH(CH3)-CH2-C(CN)CH3, or 4,4'-azobis(4-cyanovaleric acid) (ACVA): R1. = HOOC-(CH2)2-C(CN) CH3. R2. was derived from aliphatic substances such as methanol, ethanol, or 2-propanol by homolytic abstraction of a hydrogen atom brought about by R1. from the azo compounds.
Agarose gel electrophoresis of supercoiled pBR 322 DNA treated with AAPH. A. Dose dependence. Supercoiled pBR 322 DNA (10 pg/ml) was incubated with 0 (lane I), 0.01 (lane 2). 0. I (lane 3), 1 (lane 4) and 10 mM (lane 5 ) of AAPH at pH 7.4 and 37°C for 3 hr. B. Time course. The supercoiled DNA (10 pg/ml) was incubated with I mM AAPH at pH 7.4 and 37°C for 0 (lane I), I (lane 2), 3 (lane 3) and 5 hr (lane 4). Positions of a supercoiled form (form I), a nicked open circular form (form 11) and a linear form (form Ill) are indicated. 
When supercoiled plasmid DNA was incubated with 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH) at pH 7.4 in the presence and absence of oxygen, the DNA single strands were effectively cleaved. The breaking in the presence of oxygen was not inhibited by superoxide dismutase and catalase, but inhibited by mannitol, ethanol, butyl hydroxyanisole, thiol compounds, tertiary amines and spin trapping agents N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The breaking in the absence of oxygen was inhibited by ethanol, a tertiary amine and PBN. By electron spin resonance spin-trapping with PBN, the carbon-centered radical was detected both in the presence and the absence of oxygen. Hydroxyl radical was detected by use of DMPO only in the presence of oxygen. The DNA breaking activity of AAPH was found to be due primarily to the aliphatic carbon-centered radical. While the reactivity of carbon-centered radicals have received little attention, the aliphatic carbon-centered radical generated from AAPH was found to be highly reactive to break the DNA strands.
The chemical and enzymatic pathways of vitamin K1 epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase, E.C. reduction of vitamin K to the hydroquinone was a significant process in intact microsomes, but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K1 epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K1 10(-5) as rapidly as it reduced dichlorophenylindophenol (DCPIP). Reduction of 10 microM vitamin K1 by 200 microM NADH was not inhibited by 10 microM dicoumarol, whereas DCPIP reduction was fully inhibited. In contrast to vitamin K3 (menadione), vitamin K1 (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions, suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K1 epoxide and quinone, and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin, lapachol, and sulphaquinoxaline.
Dimethylammonium 2,4-dichlorophenoxyacetate (2,4-D . DMA) induced strand breaks in PM2 DNA when incubated with CuCl2, whereas 2,4-D . DMA alone or CuCl2 alone did not show any or only a negligible effect. The formation of single strand breaks increased linearly with time and concentration of 2,4-D . DMA. Neocuproine, a specific Cu(I) chelator totally prevented strand break formation. So did catalase (up to 100 mM 2,4-D . DMA), but DMSO had only a small protective effect. 2,4-Dichlorophenol, CO2 and formaldehyde were detected as reaction products of 2,4-D and CuCl2. From these results a redox reaction of Cu(II) and 2,4-D is proposed, which could explain the DNA damaging properties of CuCl2/2,4-D . DMA.
The industrial pollutant 2,4,5-trichlorophenol (2,4,5-TCP) was metabolized with postmitochondrial liver fraction from Aroclor-1254 induced rats. The generated metabolites induced single strand breaks in PM2 DNA. Among the metabolites produced are the 3,4,6-trichlorocatechol (TCC) and the 2,5-dichlorohydro-quinone (DCH), whereby the induction of DNA scission by DCH was approximately one hundred times greater than that of TCC. In the 2,4,5-TCP metabolization mixture radicals were observed by ESR. They were identified as the semiquinones of TCC and DCH. ESR studies confirmed that both TCC and DCH autoxidize in aqueous solution to their semiquinone radicals. The involvement of reactive oxygen species in the DNA strand scission was demonstrated by using DMSO, SOD, and catalase as scavengers. Inhibition of strand breaks with the scavenger enzymes did not give homogeneous results for DCH and TCC. This indicated that the directly damaging species might be different for DCH and TCC.
alpha-2,6-Difluorophenyl-N-tert-butylnitrone (F2PBN) was synthesized and evaluated. A number of alkyl adducts of F2PBN were studied by ESR/ENDOR. An additional hyperfine splitting (a triplet of doublets of doublets) is reported. The existence of two (one large, one small) F-hfsc's from the ortho-fluorine atoms in the phenyl ring of most alkyl adducts was confirmed by ENDOR spectroscopy.
The sensitivity of three human fibroblast lines, trisomic with respect to chromosome 21, to an anthracycline antibiotic carminomycin was compared with that of a normal fibroblast line using a 51Cr release assay. It was found that for an intermediate antibiotic concentration (10 microM) the sensitivity of trisomic fibroblasts, of increased content of Cu,Zn-superoxide dismutase was lower. These results suggest a role for superoxide-mediated membrane damage in the cytotoxic action of anthracycline antibiotics.
effectiveness in supporting carminomycin radical generation by various membrane-bound redox chainst 
Nuclear membranes from many tumors contain an unusual redox chain discovered originally in the Hepatoma 22a nuclear membranes which catalyzes superoxide dismutase-sensitive adrenaline oxidation to adrenochrome in the presence of either NADPH or NADH as electron donor, the reaction being inhibited by cyanide and azide. This redox chain can reduce anthracycline antitumor antibiotics adriamycin and carminomycin to their free radical states under anaerobic conditions. Evidence has been obtained for a higher stability of the carminomycin radical as compared to that of adriamycin. Operation of the nuclear membrane-bound redox chain can be a source of oxygen radical-mediated single strand breaks in DNA. The role of the nuclear membrane-associated electron transfer chain in augmenting the anticancer action of the anthracycline antibiotics is discussed.
Extracellular-superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme which, in contrast to the intracellular CuZn SOD, has affinity to the endothelium and a long vascular half-life. In the present study, the effects of EC-SOD C and CuZn SOD on reperfusion-induced myocardial damage were determined in rats subjected to 10 min of left coronary artery ligation followed by 24 h of reperfusion. Recombinant human EC-SOD C (rh-EC-SOD C) or the corresponding volume of the vehicle was administered after completion of the coronary ligation. CuZn SOD was given in two equal doses, the first dose directly after ligation and the second one 6 h later. At the end of the reperfusion period the myocardial damage was quantified by measuring the creatine kinase concentration (CK) in the reperfused part of the left ventricular free wall (LVFW), and expressed as a percentage of the concentration in the non-ischemic septum. In the group given the vehicle, 47 +/- 10 (mean +/- SD) of the CK remained in the reperfused LVFW. In the rats receiving rh-EC-SOD C the corresponding values for each dose: 1.4, 4.2 and 12.6 mg/kg were 55 +/- 12 (ns), 55 +/- 12 (ns) and 65 +/- 12% (p less than 0.05, vs. vehicle, Dunnett's multiple comparison test), respectively. Administration of CuZn SOD (2 x 10 mg/kg) resulted in 58 +/- 16% (ns) CK remaining in the LVFW.(ABSTRACT TRUNCATED AT 250 WORDS)
Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6 mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/kg IP 1/2 before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600 mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.
Pulse radiolysis studies of anisyl-3,4-semiquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have shown this semiquinone to be unreactive towards oxygen (k less than or equal to 10(5) M-1 s-1), although the reverse reaction of O2- with anisyl-3,4-quinone is very rapid (k = 8.7 x 10(8) M-1 s-1). Since 1,4 benzoquinone is also unreactive towards anisyl-3,4-semiquinone (k less than or equal to 10(5) M-1 s-1), the one-electron reduction potential, E17 (anisyl-3,4-quinone/anisyl-3,4-semiquinone), is likely to be considerably more positive than 0.1 V. This suggests that the cytotoxicity mechanism does not involve the generation of O2- and possible subsequent production of H2O2 and/or OH., leading to lipid peroxidation, as previously proposed, but rather involves as yet unknown reactions of anisyl-3,4-quinone. This quinone is unstable in water and its absorption spectrum was measured immediately (less than 0.1 s) following disproportionation of anisyl-3,4-semiquinone, before significant decay of the quinone had occurred.
Recently, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) has been applied to detect biological free radicals. However, DBNBS has various non-specific reactions which lead to preplexing results. Thus, we investigated some basic reactions of DBNBS in combination of other nitroso spin traps to assign DBNBS spin adducts derived from human platelets which presumably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (ST, LT, SS, and LS) in the presence of DBNBS (40 mM). The broad triplet due to ST was also observed by bubbling NO gas into a DBNBS solution. To identify ST, nitrosobenzene (NB) in dry dioxane was mixed with NO-saturated dioxane. The NB-NO spin adduct was observed but decomposed into diphenyl aminoxyl by the addition of H2O indicating that the primary adduct formed by the reaction of NO and DBNBS is unstable and turns into a dimerization product. Although ST could be eliminated by the inhibitor of EDRF, ST was shown to be produced by non-specific reactions. Another triplet was assigned to an S-centered radical because thiyl radicals which were generated from either the decomposition of S-nitrosothiol, or glutathione oxidation exhibited almost identical triplet signals. The other two sextets were assigned to C-centered radical adducts. Thus, DBNBS detected NQ-related, S-centered, and two C-centered radicals derived from human platelets. Special cautions are necessary for the identification of DBNBS spin adducts in a biological system to exclude artifactual radicals.
Porcine stress syndrome (PSS) which is an example of malignant hyperthermia (MH) in swine has previously been attributed to oxidative stress primarily due to an inherited antioxidant abnormality in MH susceptible (MHS) animals. C-phenyl-N-tert-butyl nitrone (PBN), a free radical spin trap, was selected to investigate whether free radicals are involved in MH. If free radicals cause the MH stress attack, then PBN should alter the time required for the onset of the stress attack, or perhaps protect the animal from experiencing the stress attack. In vivo phosphorus-31 (31P) magnetic resonance spectroscopy (MRS) was used to monitor metabolism in three to four week old normal and MHS piglets administered halothane as the stress challenge. Malignant hyperthermia was not reproducibly induced by halothane anesthesia. For those animals which did develop MH a dramatic fall in the level of PCr and a rise in the level of Pi was detected by 31P MRS. Intravenous administration of PBN prior to halothane exposure had no effect on the number of animals experiencing the stress attack. PBN does not appear to prevent, delay or reverse the onset of halothane-induced MH in three to four week old MHS piglets. The primary events leading to the MH syndrome do not appear to be influenced by the intervention of the type of free radicals normally trapped by PBN.
Four tissue compartments, differing in proton and inorganic phosphate concentration, were resolved by 31P-NMR spectroscopy in samples from dog hearts after cardioplegic treatment with HTK solution. Inversion of the physiological cytoplasmic-mitochondrial pH gradient was observed. The considerable ensuing acidosis of the matrix is discussed with regard to a possible delocalization of ferrous ions.
A prominent lesion in DNA exposed to oxidative free radicals results from the degradation of thymine leaving a formamido remnant. A 32P-postlabeling assay has been developed for the detection of the formamido lesion. The assay is based on the circumstance that the lesion prevents hydrolysis by nuclease P1 of the phosphoester bond 3' to the damaged nucleoside. Thus, a nuclease P1 plus acid phosphatase digest of DNA generates mostly nucleosides whereas the formamido lesion is rendered as a modified dinucleoside monophosphate. Dinucleoside monophosphates, but not nucleosides, are apt substrates for 32P-postlabeling by polynucleotide kinase. The assay was applied to calf thymus DNA X-irradiated in oxygenated solution. The formamido lesion could be detected down to a dose of a few Gy.
There was a 5-fold increase in cytotoxicity for cumene hydroperoxide, 10-fold for tert-butyl hydroperoxide and 25-fold for hydrogen peroxide, under metabolizing conditions (37 degrees C) in comparison to nonmetabolizing conditions (0 degrees C), when human P31 cells were exposed for 60 min. The induction of DNA single-strand breaks correlated poorly with cytotoxicity. Hydrogen peroxide was by far the most effective agent inducing single-strand breaks irrespective of temperature. Cumene hydroperoxide produced fewer strand breaks than tert-butyl hydroperoxide despite its greater cytotoxicity at either 37 degrees C or at 0 degrees C. The pattern of single-strand break induction did not change with temperature. The number of breaks, however, increased when the cells were exposed at 37 degrees C. The pattern of rejoining was similar for hydrogen peroxide- and tert-butyl hydroperoxide-induced breaks at both temperatures whereas the rejoining of cumene hydroperoxide-induced breaks deviated somewhat from this pattern. The results indicate that there is no clear-cut relationship between induction of DNA single-strand breaks and cytotoxicity after hydroperoxide exposure.
The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl)-methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non-enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10(-2)-10(-5) M CH 402 dose-dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.
In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg-1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%), 142.3 units (23.8%), and 22.9 units (3.8%) mg-1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%), 2.46 units (23.0%), and 0.70 units (6.5%) mg-1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing .OH radical from all intracellular compartments by the destruction of H2O2 which together with O2- is a precursor of .OH.
Superoxide dismutase (SOD) has demonstrated therapeutic potential for treating a variety of conditions including radiation injury, oxygen toxicity, reperfusion injury, and inflammation, especially arthritis. However, the native enzyme's short half-life in plasma (6 minutes in mice, 25 minutes in man) limits the enzyme's effectiveness in many applications, or requires infusion of large doses. High doses of SOD derived from either natural or rDNA sources may increase the potential for immunologic sensitization. One effective use of native SOD is intra-articular administration for treatment of arthritis, where injection of SOD into joints retards elimination (15 hour terminal half-life), allowing the effective use of lower doses. To overcome the limitations resulting from rapid clearance, various researchers have increased the persistence of SOD by cross-linking SOD or by attaching polymeric substances, including dextrans, albumin, Ficoll, polyvinyl alcohol or polyethylene glycol (PEG). PEG is relatively safe; however, the amount of modification by PEG, is the MW range 1,900-5,000 daltons, which is necessary to optimally increase serum persistence and reduce immunogenicity, results in the loss of much of the enzymatic activity. In this report we describe the preparation of SOD adducts containing 1 to 4 strands of high MW PEG (41,000-72,000 daltons). The MW range of these adducts, measured by steric exclusion HPLC based on protein standards, is 200,000 to over 1,000,000 daltons. The number of PEG strands attached per SOD dimer (32,000 daltons) was measured by HPLC. Because of the low degree of protein modification required to produce very high MW products, these PEG-SODs retain 90%-100% of the SOD activity of the native enzyme. Additionally, these very large adducts demonstrate longer persistence and lower immunogenicity and antigenicity compared to the more highly modified PEG-SODs containing low MW PEG (i.e., 7-16 strands of 5,000 dalton methoxy-PEG).
Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metabolized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.
(5-Nitro-2-furfurylidene)amino compounds bearing triazol-4-yl, benzimidazol-1-yl, pyrazol-1-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-1-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-1-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-1-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-1-yl and substituted triazol-4-yl derivatives.
When NADPH-cytochrome P-450 reductase isolated from rat liver microsomes was aerobically incubated with bleomycin, FeCl3, NADPH and DNA parallel NADPH and oxygen were consumed and malondialdehyde was formed. A similar parallelism of NADPH- and oxygen-consumption and malondialdehyde formation was observed when cell nuclei isolated from rat liver were incubated under the same conditions. The formation of malondialdehyde which was identified by HPLC and which was most likely released from oxidative cleavage of deoxyribose of nuclear DNA required oxygen, bleomycin, FeCl3 and NADPH. This indicates that a nuclear NADPH-enzyme, presumably NADPH-cytochrome P-450 reductase, is able to redox cycle a bleomycin-iron-complex which in the reduced form can activate oxygen to a DNA-damaging reactive species. The data suggest that the activity of this enzyme in the cell nucleus could play an important role in the cytotoxicity of bleomycin in tumor cells.
When aqueous solutions of the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) are treated with hydrogen peroxide in the presence of either FeII or light, the hydroxyl radical adduct DMPO-OH is formed, with a characteristic 4 line ESR spectrum. When oxy- or metmyoglobin is added to such a system the initial yield and the halife of DMPO-OH are reduced, and at high myoglobin concentrations (about 0.1 mmol dm-3) DMPO-OH becomes undetectable. Using the stable nitroxide 2,2,6,6-tetramethyl-1-piperidinyloxy-N-oxyl (TMPO) for comparison it was found that neither hydrogen peroxide nor myoglobin alone caused a loss of signal, but together a marked loss of signal was induced. From the evidence of these and other experiments it was concluded that the DMPO-OH adduct reacts with hydrogen peroxide and myoglobin to give non-paramagnetic products, and hence that the use of the DMPO spin trap to detect hydroxyl or other active radicals in systems containing physiological concentrations of myoglobin may give misleading results.
Top-cited authors
Barry Halliwell
  • National University of Singapore
Robert Huie
  • National Institute of Standards and Technology
Martina Dieber-Rotheneder
  • Medical University of Graz
Patrick Alexander Baeuerle
Ralf Schreck
  • Hochschule Aalen