Nadph-Cytochrome-P450 Reductase Promotes Hydroxyl Radical Production by the Iron Complex of ADR-925, the Hydrolysis Product of ICRF-187 (Dexrazoxane)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectropho-tometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.     

The displacement of iron(III) from its complexes with the anticancer drugs piroxantrone and losoxantrone by the hydrolyzed form of the cardioprotective agent dexrazoxane

Piroxantrone and losoxantrone are new DNA topoisomerase II-targeting anthrapyrazole antitumor agents that display cardiotoxicity both clinically and in animal models. A study was undertaken to see whether dexrazoxane or its hydrolysis product ADR-925 could remove iron(III) from its complexes with piroxantrone or losoxantrone. Their cardiotoxicity may result from the formation of iron(III) complexes of losoxantrone and piroxantrone. Subsequent reductive activation of their iron(III) complexes likely results in oxygen-free radical-mediated cardiotoxicity. Dexrazoxane is in clinical use as a doxorubicin cardioprotective agent. Dexrazoxane presumably acts through its hydrolyzed metal ion binding form ADR-925 by removing iron(III) from its complex with doxorubicin, or by scavenging free iron(III), thus preventing oxygen-free radical-based oxidative damage to the heart tissue. ADR-925 was able to remove iron(III) from its complexes with piroxantrone and losoxantrone, though not as efficiently or as quickly as it could from its complexes with doxorubicin and other anthracyclines. This study provides a basis for utilizing dexrazoxane for the clinical prevention of anthrapyrazole cardiotoxicity.     

The iron chelating cardioprotective prodrug dexrazoxane does not affect the cell growth inhibitory effects of bleomycin

The clinical use of bleomycin is limited by a dose-dependent pulmonary toxicity. Bleomycin is thought to be growth inhibitory by virtue of its ability to oxidatively damage DNA through its complex with iron. Our previous preclinical studies showed that bleomycin-induced pulmonary toxicity can be reduced by pretreatment with the doxorubicin cardioprotective agent dexrazoxane. Dexrazoxane is thought to protect against iron-based oxygen radical damage through the iron chelating ability of its hydrolyzed metabolite ADR-925, an analog of ethylenediaminetetraacetic acid (EDTA). ADR-925 quickly and effectively displaced either ferrous or ferric iron from its complex with bleomycin. This result suggests that dexrazoxane may have the potential to antagonize the iron-dependent growth inhibitory effects of bleomycin. A study was undertaken to determine if dexrazoxane could antagonize bleomycin-mediated cytotoxicity using a CHO-derived cell line (DZR) that was highly resistant to dexrazoxane through a threonine-48 to isoleucine mutation in topoisomerase IIalpha. Dexrazoxane is also a cell growth inhibitor that acts through its ability to inhibit the catalytic activity of topoisomerase II. Thus, the DZR cell line allowed us to examine the cell growth inhibitory effects of bleomycin in the presence of dexrazoxane without the confounding effect of dexrazoxane inhibiting cell growth. The cell growth inhibitory effects of bleomycin were unaffected by pretreating DZR cells with dexrazoxane. These results suggest that dexrazoxane may be clinically used in combination with bleomycin as a pulmonary protective agent without adversely affecting the antitumor activity of bleomycin.     

Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy

Abstract

Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO^?), hydrogen radical (H^?), and carbon-center radical (R^?). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO^? was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO ^? might have a crucial role in the pathogenesis of temporomandibular disorders.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

Prooxidant action of carazolol in the Fenton‐like reaction

Carazolol [4‐(2‐hydroxy‐3‐isopropyl‐amino‐propoxy)‐carbazole], a β₃‐adrenoceptor agonist, is clinically used in the treatment of hypertension, cardiac arrhythmias and angina pectoris. Despite the beneficial effect of the drug, its high dose may contribute to cardiotoxicity. This study was conducted to examine whether carazolol can influence hydroxyl radical formation by a Fenton‐like reaction [Co(II) + H₂O₂ + HO^‐] in the presence of ethylenediaminetetraacetic acid. The oxygen free radicals and singlet oxygen (¹O₂) formation was traced by three different assay methods: chemiluminescence (CL), an electron spin resonance (ESR) spin trapping with 2,2,6,6‐tetramethyl‐4‐piperidine and 5,5‐dimethyl‐1‐pyrroline‐1‐oxide, and spectrophotometric determination of ¹O₂ based on bleaching of p‐nitrosodimethylaniline. The effect of hydroxyl radical inhibitors and ¹O₂ quenchers on peroxidation of carazolol was also examined. The results indicated that carazolol enhanced the HO radical and ¹O₂ formation in a Fenton‐like reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Generation of hydroxyl radical from linoleic acid hydroperoxide in the presence of epinephrine and iron.

When linoleic acid hydroperoxide was reacted with ferrous iron, the electron spin resonance signals characteristic of the spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide and the hydroxyl radical were detected. Although the signals were not detected with the hydroperoxide and ferric iron, they were actually found if epinephrine was added, indicating that the hydroxyl radical could be generated from the hydroperoxide upon reduction of the latter with ferrous iron formed by epinephrine. The possible generation of the hydroxyl radical from lipid peroxides in vivo was discussed from the viewpoint of pathogenesis of lipid peroxide-related diseases.     

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Oxidation of Tris to One-Carbon Compounds in a Radical-Producing Model System,in Microsomes,in Hepatocytes and in Rats

The buffer substance tris(hydroxymethyl)aminomethane (Tris) is converted to formaldehyde in an hydroxyl radical producing model system and in rat liver microsomes, and to CO₂ in rat hepatocytes and in the intact rat. In microsomes, formaldehyde formation from Tris is inhibited by catalase, by the antioxidant propylgallate and by the iron chelator deferoxamine, formaldehyde formation is stimulated by the addition of Fe (II) EDTA. In hepatocytes, the formation of [¹⁴C] CO₂ from [¹⁴C] Tris is inhibited by propylgallate and by the iron chelator o-phenanthroline and is stimulated by the presence of a xanthine oxidase system plus Fe (II) EDTA in the medium. In the intact rat, the administration of [¹⁴C] Tris results in the exhalation of [¹⁴C] CO². The results indicate that an oxidant formed via a Fenton-type reaction, possibly the hydroxyl radical, may be involved in the formation of one-carbon compounds from Tris.     

In vivo monitoring of norepinephrine and hydroxyl free radical generation by ferrous iron in the myocardium with a microdialysis technique

1. We examined in vivo monitoring of norepinephrine and hydroxyl radical generation in rat myocardium with a microdialysis technique. For this purpose, we designed the microdialysis probe holding system which includes loose fixation of the tube and synchronization of the movement of the heart and the probe.2. The hydroxyl free radical (OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA) which can be measured electrochemically in picomole quantity by high performance liquid chromatography (HPLC).3. After probe implantation, norepinephrine concentration of dialysate decreased over the first 150 min and then reached an almost steady level. A positive linear correlation between the ferrous iron and OH formation trapped as 2,3-DHBA (R² = 0.960) and 2,5-DHBA (R² = 0.982) was observed using the microdialysis technique.4. The present results indicate that non-enzymatic oxidation in the extracellular fluid may play a key role in hydroxyl radical generation by ferrous iron.     

Esr Spin Trapping Studies Into of Nature of the Oxidizing Species Formed in the Fenton Reaction: Pitfalls Associated With of Use Of 5,5-Dimethyl-1-Pyrroline-n-Oxide in the Detection of the Hydroxyl Radical

Several investigators have challenged the widely held view that the hydroxyl radical is the primary oxidant formed in the reaction between the ferrous ion and hydrogen peroxide. In recent studies, using the ESR spin trapping technique, Yamazaki and Piette found that the stoichiometry of oxidant formation in the reaction between Fe²⁺ and H₂O₂ often shows a marked deviation from the expected value of 1:1 (I. Yamazaki and L. H. Piette (1990) J. Am. Chem. Soc. 113, 7588-7593). In order to account for these observations, it was suggested that additional oxidizing species are formed, such as the ferryl ion (FeO²⁺), particularly when iron is present at high concentration and chelated to EDTA.

In this paper it is shown that secondary reactions, involving the redox cycling of iron and the oxidation of the hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by iron, operate under the reaction conditions employed by Yamazaki and Piette. Consequently, the stoichiometry of oxidant formation can be rationalized without the need to envisage the formation of oxidizing species other than the hydroxyl radical. It is also demonstrated that the iron(III) complex of DETAPAC can react directly with DMPO to form the DMPO hydroxyl radical adduct (DMPO/OH) in the absence of hydrogen peroxide. Therefore, to avoid the formation of (DMPO/OH) as an artefact, it is suggested that DETAPAC should not be used as a reagent to inactivate containating adventitious iron in experiments using DMPO.     

The effect of melanin on iron associated decomposition of hydrogen peroxide

The effects of melanin on the iron-catalyzed decomposition of hydrogen peroxide to hydroxyl radicals and hydroxyl ions have been studied using electron spin resonance, spin trapping and visible light spectrophotometry. Melanin altered these reactions by several different mechanisms and consequently, depending on conditions, can significantly increase or decrease the yield of reactive products, including hydroxyl radicals. For low concentrations of ferrous ions, melanin decreased the yield of hydroxyl radicals due to binding of ferrous ions by melanin; ferrous ions bound to melanin did not decompose H2O2 efficiently. Melanins increased the rate of hydroxyl radical production if the predominant form of iron was ferric, due to the ability of melanin to reduce ferric to ferrous iron. Hydroxyl radical production in the presence of a strong chelator (e.g. EDTA) and melanin was greater than in the presence of a weak chelator (e.g. ADP) and melanin. Melanin also increased the rate of destruction of the DMPO-OH adduct.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.     

Detection of nucleic acid-derived radicals formed by alloxan

Pyrimidine base-derived radical spin adducts were detected in reaction mixtures containing pyrimidine bases, glutathione, and alloxan by the ESR spin trapping technique with a spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN). Pyrimidine nucleoside- and nucleotide-, and ribose- and deoxyribose-derived radical spin adducts of PBN were also observed. However, purine base- and nucleoside-derived radical spin adducts of PBN were not detected. A cytosine-derived radical spin adduct of PBN was not generated under anaerobic conditions. Catalase and mannitol inhibited the formation of the cytosine-derived radical spin adduct of PBN but superoxide dismutase (SOD) did not. EDTA stimulated it and desferrioxamine suppressed it nearly completely. From these results it is presumed that the hydroxyl radical is involved in the formation of the cytosine-derived radical spin adduct of PBN generated by alloxan.     

Electron Spin Resonance Studies on the Degradation of Hydroperoxides by Rat Liver Cytosol

Incubation of ¹BuOOH (in the concentration range 200μM to 20mM) with rat liver post-microsomal supernatant in the presence of the spin trap DMPO gives three radical species, which can be observed by electron spin resonance spectroscopy. The first of these is the ascorbyl radical (which decreases in concentration with time), the other two are identified as spin adducts of alkoxyl and carbon-centred radicals; these latter species increase in concentration with time. Addition of NADH, but not NADPH, led to an increase in concentration of the alkoxyl and carbon-centred radical adducts and a decrease in the concentration of the ascorbyl radical. Results obtained in the presence of iron chelators and other ligands suggest that the generating system is an NADH-dependent enzyme that reduces ¹BuOOH by one-electron to give initially the ¹BUO radical. Results from experiments carried out on dialysed cytosol samples lend support to this conclusion.     

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H₂O₂, hydroxyl radical, or peroxynitrite (ONOO⁻). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.     

Investigations of different chemiluminescent peaks in H2O2–SCN−–Cu2+–OH−–luminol flow system

In the H₂O₂–SCN^?–Cu²⁺–OH^?–luminol oscillatory system of chemiluminescence, the effects of the ingredient concentrations, temperature, flow rate and complexing agent on the oscillatory dynamics were investigated in a continuous‐flow stirred tank reactor (CSTR). The dynamical structure of two peaks during a period was discussed in detail. By addition of EDTA to the oscillating system, the peak I height decreased sharply while the peak II height was little affected, and the period kept constant. This may be due to the fast reaction between Cu(II) and EDTA and the highly stable complex Cu(II)–EDTA. From the experimental study and mechanism analysis, the chemiluminescent peak I corresponds to Cu(II) → Cu(I) transformation and the peak II corresponds to the Cu(I) → Cu(II) transformation process. The key species involving in the two‐transformation process are inferred to be superoxide radical and hydroxyl radical. Copyright © 2010 John Wiley & Son, Ltd.     

Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Studies on the antioxidant activity of some thiazolidinedione,imidazolidinedione and rhodanine derivatives having a flavone core

A series of flavonyl‐2,4‐thiazolidinedione, imidazolidinedione and rhodanine derivatives were tested for their antioxidant activity as scavengers of oxygen free radicals. Free radical scavenging activities, including superoxide anion radical , hydroxyl radical (HO^?) and 2,2′‐diphenyl‐1‐picrylhydrazyl free radical have been evaluated using chemiluminescence, electron paramagnetic resonance and spin trapping with 5,5‐dimethyl‐1‐pyrroline‐1‐oxide as a spin trap. Potassium superoxide in dimethylsulfoxide and 18‐crown‐6 ether were used for the production of . Hydroxyl radical was generated using the Fenton reaction. Ten of the eleven examined compounds exhibited decrease in chemiluminescence, but there were large differences in the decrease, ranging from 16% to 89%; also, two of these compounds increased light emission by about 200%. On the contrary, all compounds tested exhibited 30–68% scavenging HO^? and 25–96% scavenging the DPPH^? radical respectively. Possible mechanisms are proposed to explain the results. Copyright © 2014 John Wiley & Sons, Ltd.