Dinitrosyl iron complexes with glutathione in rat myocardial tissue during regional ischemia and postischemic reperfusion

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes.     

Transformations of dinitrosyl iron complexes in an isolated rat heart after introduction of this substance into perfusion medium

The objective of the present research was to study transformations of various physiological NO forms in an isolated rat heart, perfused with the medium containing dinitrosyl iron complexes with glutathione ligand (DNIC-GH). We showed that such aerobic perfusion resulted in accumulation of mostly diamagnetic NO physiological forms (S-nitrosothiols) in myocardial tissue. They were transformed into protein-bound mononuclear dinitrosyl iron complexes during subsequent total ischemia. Meantime, DNIC-GH injection on the onset of ischemia resulted in changes in the state of mitochondrial respiratory chain, characterized by the increase in myocardial concentration of flavosemiquinones.     

Effects of the donor of nitric oxide,dinitrosyl iron complex with glutathione,on blood circulation in healthy animals

We have found that the hypotensive effect of the nitric oxide donor dinitrosyl iron complex with glutathione was caused by a decrease in general peripheral resistance in healthy rats. This effect did not impair microcirculation and was accompanied by an increase in the myocardial contractile activity. Under the hypotension condition induced by dinitrosyl iron complex with glutathione, we did not find any changes in the oxygen or carbon dioxide tensions in the blood as compared to the control or any change in the acidic-basic blood state. Thus, the possible inhibitory influence of this complex on some enzymes and proteins in the animal body was not accompanied by effects on the heart, vessels, or blood. The dinitrosyl iron complex with glutathione induced a decrease in the arterial pressure only. We hypothesize that a new type of drugs for the treatment of cardiovascular diseases can be developed on the basis of such complexes and complexes with other thiol-containing ligands.     

Cardioprotective and antioxidant effects of apomorphine

Apomorphine is a potent antioxidant that infiltrates through biological membranes. We studied the effect of apomorphine (2 microM) on myocardial ischemic-reperfusion injury in the isolated rat heart. Since iron and copper ions (mediators in formation of oxygen-derived free radicals) are released during myocardial reperfusion, apomorphine interaction with iron and copper and its ability to prevent copper-induced ascorbate oxidation were studied. Apomorphine perfused before ischemia or at the commencement of reperfusion demonstrated enhanced restoration of hemodynamic function (i.e. recovery of the work index (LVDP x HR) was 69.2 +/- 4.0% with apomorphine pre-ischemic regimen vs. 43.4 +/- 9.01% in control hearts, p < 0.01, and 76.3 +/- 8.0% with apomorphine reperfusion regimen vs. 30.4 +/- 11.1% in controls, p < 0.001). This was accompanied by decreased release of proteins in the effluent and improved coronary flow recovery in hearts treated with apomorphine after the ischemia. Apomorphine forms stable complexes with copper and with iron, and inhibits the copper-induced ascorbate oxidation. It is suggested that these iron and copper chelating properties and the redox-inactive chelates formed by transition metals and apomorphine play an essential role in post-ischemic cardioprotection.     

The antitumor activity of the S-nitrosoglutathione and dinitrosyl iron complex with glutathione: Comparative studies

The antitumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma was found earlier with intraperitoneal administration of the complexes. This activity was also observed when this preparation was injected subcutaneously. The complex inhibited the tumor growth by 43% upon subcutaneous injection at a daily dose of 100 µM/kg (as calculated per one iron atom in the binuclear dinitrosyl iron complex) for 10 or 15 days. The effect was observed during the first 2 weeks after tumor transplantation. After this, the tumors began to grow at a rate that was equal to or even higher than that for the control animals. The mean survival time for the treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes were also effective against Ca-755 adenocarcinoma with intraperitoneal administration. In this case, however, the mean survival time for the treated animals only increased by 7%. It was also shown that S-nitrosoglutathione inhibited the growth of Lewis lung carcinoma and Ca-755 adenocarcinoma by 70 and 90%, respectively. However, in contrast to binuclear dinitrosyl iron complexes, the antitumor effect of S-nitrosoglutathione decreased with an increase in the daily dose of the compound from 200 to 400 µM/kg. The initial antitumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO that is released from both compounds. The subsequent suppression of the effect is caused by the activation of antinitrosative and antioxidant defense systems in tumors.     

Study of membranotropic and antioxidant activity of flavonoids and their complexes with ferric iron

We studied the interaction with liposomes and the antioxidant activity of flavonoid (quercetin, catechin, taxifolin) complexes with iron(III). It was found that the lipophilicity of complexes depends on an iron:flavonoid ratio and grows at a ratio of 1 to 1, while complexes in a 2: 1 ratio were the most effective to slow down the lipid peroxidation and restore radical 2,2-diphenyl-1-picrylhydrazyl. Thus, the stoichiometry of complexes formed in aqueous solution, may differ from the stoichiometry of complexes that most effectively protect membranes from peroxidation.     

Inhibition of mugineic acid-ferric complex uptake in barley by copper, zinc and cobalt

The interfering effects of copper, zinc, and cobalt on the uptake of mugineic acid-ferric complex were studied in barley ( Hordeum vulgare , cv. Minorimugi) grown in nutrient solution. Short-term uptake experiments of 3 h were performed utilizing both ionic and mugineic acid-complex forms of each metal at two different concentrations. Copper was most effective in decreasing iron uptake when added in an ionic form at either concentration. The inhibition order at higher concentrations followed Cu(II) > Zn(II) ≥ Co(II), Co(III), which is consistent with the stability constants of these metal complexes with mugineic acid. The displacement of iron from its mugineic acid complex by these metals is suggested as a probable explanation for the decreased iron uptake. The inhibitory effect of metal complexes with mugineic acid on iron uptake was only found in cases with higher concentrations of Cu(II) and Zn(II) complexes. Deformation of the specific iron transport system in the plasma membrane due to their adsorption may be responsible for this effect.     

Stereospecificity of siderophore-mediated iron uptake in Rhodotorula pilimanae as probed by enantiorhodotorulic acid and isomers of chromic rhodotorulate

Rhodotorulic acid (RA), a dihydroxamate siderophore produced by Rhodotorula pilimanae, forms 3:2 complexes with ferric and chromic ions (M2RA3) at pH 7. Kinetically inert chromic complexes of RA have been separated into geometrical isomers and for the first time partially resolved into optical isomers. The three isomers delta-cis, delta-trans, and lambda-trans were characterized by their visible and circular dichroism spectra. Inhibition by both delta-isomers of radiolabeled ferric RA uptake in R. pilimanae was equally effective. However the lambda-cis isomer was significantly less effective as an inhibitor. Concentration-dependent uptake kinetics were performed with ferric RA and the ferric complex of synthetic enantio-RA, which form predominantly delta and lambda complexes, respectively. The lambda-enantio-Fe2RA3 was 50% less effective in supplying iron to R. pilimanae than was Fe2RA3. An additional synthetic analog of RA, which lacks a carbonyl group at the diketopiperazine ring, exhibited the same uptake rates as ferric RA. We conclude that stereoselective recognition of optical isomers takes place during iron uptake mediated by RA and that this recognition primarily involves the right-handed delta coordination "propellor" of the metal center and its adjacent functionalities.     

Cardioprotective and Antioxidant Effects of Apomorphine

Apomorphine is a potent antioxidant that infiltrates through biological membranes. We studied the effect of apomorphine (2?μM) on myocardial ischemic-reperfusion injury in the isolated rat heart. Since iron and copper ions (mediators in formation of oxygen-derived free radicals) are released during myocardial reperfusion, apomorphine interaction with iron and copper and its ability to prevent copper-induced ascorbate oxidation were studied. Apomorphine perfused before ischemia or at the commencement of reperfusion demonstrated enhanced restoration of hemodynamic function (i.e. recovery of the work index (LVDP?×?HR) was 69.2±4.0% with apomorphine pre-ischemic regimen vs. 43.4±9.01% in control hearts, p<0.01, and 76.3±8.0% with apomorphine reperfusion regimen vs. 30.4±11.1% in controls, p<0.001). This was accompanied by decreased release of proteins in the effluent and improved coronary flow recovery in hearts treated with apomorphine after the ischemia. Apomorphine forms stable complexes with copper and with iron, and inhibits the copper-induced ascorbate oxidation. It is suggested that these iron and copper chelating properties and the redox-inactive chelates formed by transition metals and apomorphine play an essential role in post-ischemic cardioprotection.     

Enzymatic and structural modifications of mitochondrial NADH-ubiquinone reductase with autolysis as experimental model

Complex I (nicotinamide adenine dinucleotide-ubiquinone reductase) is a complex enzyme system located in the inner mitochondrial membrane. It has the ability to catalyze several different enzymatic reactions in electron transport, and is known to be one of the respiratory chain components most sensitive to ischaemia. Mitochondria and two complexes I (complex IA and complex IB) were isolated from normal and ischaemic myocardial tissue. Enzymatic activities, polypeptide composition, as well as other components such as non-haem iron, acid-labile sulphur and ubiquinone, were determined. The results indicated that complex IB reflected the enzymatic changes in the mitochondria during myocardial ischaemia, but complex IA did not. The lesion that resulted from ischaemia was localised as altered enzymatic activities due to a different polypeptide composition, as well as loss of ubiquinone and non-haem iron from complex IB.     

Dinitrosyl iron complexes with thiol-containing ligands in plant tissues

The formation of dinitrosyl iron complexes with thiol-containing ligands in plant tissues (parsley and apple leaves) in the presence of nitric monoxide was demonstrated using electron paramagnetic resonance. In two types of tissues dinitrosyl iron complexes are predominantly represented by the binuclear diamagnetic form. This diamagnetic form can be transformed in EPR-detectable mononitrosyl iron complexes with diethyldithiocarbamate due to the ability of diethyldithiocarbamate to accept the iron-mononitrosyl groups from iron-dinitrosyl fragments of binuclear complexes. A similar transformation was observed under the effect of diethyldithiocarbamate on a mononuclear paramagnetic form of dinitrosyl iron complexes. The significant amount of binuclear dinitrosyl iron complexes found in plant tissues suggests that these complexes can be considered as a “working form” of nitric monoxide, which is recognized now as a universal regulator of metabolic processes in plants as well as in other organisms.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

Hydroxyl radical production by the iron complex of the hydrolysis product of the antioxidant cardioprotective agent ICRF-187 (dexrazoxane)

SUMMARY

Dexrazoxane (ICRF-187) is now in clinical use for the prevention of doxorubicin-induced cardiotoxicity. This cardiotoxicity is thought to be due to iron-mediated oxidative stress. Dexrazoxane may be acting through its strongly metal ion binding rings-opened hydrolysis product ADR-925 by complexing iron. Since iron-chelates are known to be able to produce hydroxyl radicals, an electron paramagnetic resonance spin trapping study was undertaken to compare the hydroxyl radical-producing ability of the ferrous-ADR-925 complex with that of the ferrous complexes of ethylenediaminetetraacetic acid (EDTA) and the tetraacid analog of ADR-925 (DAPTA). In spectrophotometric studies it was shown that the ferrous-ADR-925 complex underwent aerobic oxidation 87 and 44 times slower than the ferrous complexes of EDTA or 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (DAPTA), respectively. In spite of the much slower oxidation of the ferrous-ADR-925 complex, it was, nonetheless, equally effective in producing hydrogen peroxide-dependent spin adducts. These spin adducts were produced from the reaction of the spin trap with free hydroxyl radical (HO^.), and with a transient iron oxidant with HO^.-like reactivity. Thus, it is concluded that ADR-925 acts by either complexing free iron or iron bound to doxorubicin, and forming a soluble iron complex that is less effective at producing site-specific oxygen radical damage.     

In vitro analysis of iron chelating activity of flavonoids

Flavonoids have been demonstrated to possess miscellaneous health benefits which are, at least partly, associated with iron chelation. In this in vitro study, 26 flavonoids from different subclasses were analyzed for their iron chelating activity and stability of the formed complexes in four patho/physiologically relevant pH conditions (4.5, 5.5, 6.8, and 7.5) and compared with clinically used iron chelator deferoxamine. The study demonstrated that the most effective iron binding site of flavonoids represents 6,7-dihydroxy structure. This site is incorporated in baicalein structure which formed, similarly to deferoxamine, the complexes with iron in the stoichiometry 1:1 and was not inferior in all tested pH to deferoxamine. The 3-hydroxy-4-keto conformation together with 2,3-double bond and the catecholic B ring were associated with a substantial iron chelation although the latter did not play an essential role at more acidic conditions. In agreement, quercetin and myricetin possessing all three structural requirements were similarly active to baicalein or deferoxamine at the neutral conditions, but were clearly less active in lower pH. The 5-hydroxy-4-keto site was less efficient and the complexes of iron in this site were not stable at the acidic conditions. Isolated keto, hydroxyl, methoxyl groups or an ortho methoxy-hydroxy groups were not associated with iron chelation at all.     

The role of oxidative stress in the toxicity of pyridoxal isonicotinoyl hydrazone (PIH) analogues

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.     

Effect of exogenous donors of nitric oxide and inhibitors of its enzymatic synthesis on experimental ischemic thrombosis in conjunctive veins of the rabbit eyes

The beneficial action of dinitrosyl iron complex with glutathione on conjunctive veins of eyes in rabbits with experimental thrombosis of conjunctive veins has been demonstrated. Aqueous solutions of dinitrosyl iron complexes were added subconjunctively at doses of 5.4-8.1 micromole per eye. The average duration of thrombosis by the action of dinitrosyl iron complex decreased from 6.4 days in control animals to 2 days. The addition of dinitrosyl iron complex resulted in blood flow recovery in occlusive vessels and prevented ischemia and necrosis of tissues. The enhancement of hemorrhagic activity induced by dinitrosyl iron complexes was abrogated with combined addition of the nonselective NO synthase inhibitor N-nitro-L-arginine. In contrast, S-nitrosoglutathione affected adversely the veins: the duration of thrombosis in experimental thrombosis of conjunctive veins increased to 7 days. Intensive hemorhage developed in the conjunctive. The formation of protein-bound dinitrosyl iron complexes was observed by the EPR method in eye tissues after the subconjunctive or parabulbar addition of dinitrosyl iron complex with glutathione. This was not the case when the complex was injected intravenously. It was shown that dinitrosyl iron complex with glutathione induces the blockade of pellet aggregation or strengthens the fibrinolytic activity of plasma of patients with eye vessel pathology. The beneficial action of dinitrosyl iron complexes on conjunctive veins was proposed to be due to the capacity of dinitrosyl iron complexes to donate NO primarily to its biological targets. The release of free NO molecules in large amounts is not characteristic for dinitrosyl iron complexes. This process is characteristic of S-nitrosoglutathione, which sharply increases the probability of the accumulation of peroxynitrite, which produces a toxic effect on cells and tissues.     

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.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

It has been established that albumin-bound dinitrosyl iron complexes can be destroyed by superoxide radicals generated in a xanthine-xanthine oxidase system. It was shown that peroxynitrite also effectively destroyed albumin-bound dinitrosyl iron complexes. At the same time, hydrogen peroxide and tert-butyl hydroperoxide did not stimulate the destruction of albumin-bound dinitrosyl iron complexes up to concentrations one order higher than the content of NO. The data have been obtained indicating that dinitrosyl iron complexes possess the vasodilatory activity. It has been proposed that peroxynitrite and superoxide radical, by causing the destruction of albumin-bound dinitrosyl iron complexes, affect the physiological properties of nitric oxide.     

Formation of a new type of dinitrosyl iron complexes bound to cysteine modified with methylglyoxal

It has been shown that interaction of cysteine dinitrosyl iron complexes with methylglyoxal leads to the formation of a new type of dinitrosyl iron complexes, EPR spectrum of these complexes essentially differs from spectra of dinitrosyl iron complexes containing unmodified thiol. The products of the cysteine reaction with methylglyoxal are hemithioacetals, Schiff bases and thiazolidines, which most likely serve as ligands for the new type of dinitrosyl iron complexes. It has been shown that the new type of dinitrosyl iron complexes as cysteine dinitrosyl iron complexes, which are physiological donors of nitric oxide, exert a vasodilator effect. It has also been found that the oxidative destruction of the new type of dinitrosyl iron complexes occurs at normal oxygen partial pressure, but these dinitrosyl iron complexes remain rather stable under hypoxia modeling. An assumption that the destruction of the new type of dinitrosyl iron complexes is caused by the formation of a bound peroxynitrite-containing intermediate is made.     

Effect of physico-chemical factors and species specificity on the structure of dinitrosyl iron complexes

Formation in mouse, rat and man's blood of iron nitrosyl complexes with pair thiol groups of proteins (complexes 2.03) was shown by ESR method. This formation was initiated by the introduction in blood in vitro or in vivo of low molecular dinitrosyl complexes of iron with phosphate, thiosulphate, cysteine or reduced gluthatione. Three forms of these complexes were found. They were characterized by ESR signals with rhombic or axial symmetry of g-factor tensor. These forms pass into one another under the effect of a number of thiol-containing compounds or at blood freezing. The life time of the complexes 2.03 in the blood in vivo is several hours.