Nitric oxide-induced conversion of cellular chelatable iron into macromolecule-bound paramagnetic dinitrosyliron complexes

One of the most important biological reactions of nitric oxide (nitrogen monoxide, *NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g=2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous *NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, *NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from *NO, and SIH added during the *NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free *NO, because addition of oxyhemoglobin prevents formation from either *NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by *NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free *NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of *NO and its effects on cellular iron homeostasis.     

Long-lasting hypotensive action of stable preparations of dinitrosyl-iron complexes with thiol-containing ligands in conscious normotensive and hypertensive rats

Previously we established the hypotensive action of nitric oxide donors, dinitrosyl-iron complexes (DNIC) with thiol-containing ligands, stored in frozen solution at 77K. In the present study, we tested recently designed water soluble dry powder preparations of DNICs keeping their characteristics in dry air for a long time. The complexes dissolved in PBS were injected intravenously into normotensive Wistar and spontaneously hypertensive SHR rats. The average arterial pressure (AAP) was recorded through preliminary implanted catheter in a carotid artery. The initial hypotensive action of DNIC with cysteine (DNIC-cys) was comparable to action of nitroprusside (SNP) but, in contrast to the latter, lasted for 20-120min depending on a doze. The blood DNIC content as detected by electronic paramagnetic resonance steadily decreased at this time. The hypotensive action of S-nitrosocysteine was similar to SNP while binding of iron in DNIC by batophenantroline-disulphonate prevented its hypotensive effect. These data suggest that long-lasting hypotensive action of DNICs may be caused by stable protein-bound DNICs forming in the process of transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNICs to thiol protein ligands. The relative initial dose-dependent effect of DNIC-cys was similar in Wistar and SHR but secondary AAP reduction was more profound in SHR. A substitution of cysteine in DNIC by thiosulphate resulted in markedly less initial AAP reduction while long-lasting effect was similar and substitution by glutathione smoothed initial AAP decline and stabilized AAP level in the second phase. Prolonged AAP reduction induced by DNIC-cys was considerably shortened in narcotized rats. Thus, dry preparations of DNICs preserve prolonged hypotensive activity.     

Mechanism of inhibition of catalase by nitro and nitroso compounds

Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested--nitroarginine, nitroglycerol, nitrophenol, and furazolidone--gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.     

Crucial role of lysosomal iron in the formation of dinitrosyl iron complexes in vivo

Dinitrosyl non-heme–iron complexes (DNIC) are found in many nitric oxide producing tissues. A prerequisite of DNIC formation is the presence of nitric oxide, iron and thiol/imidazole groups. The aim of this study was to investigate the role of the cellular labile iron pool in the formation of DNIC in erythroid K562 cells. The cells were treated with a nitric oxide donor in the presence of a permeable (salicylaldehyde isonicotinoyl hydrazone) or a nonpermeable (desferrioxamine mesylate) iron chelator and DNIC formation was recorded using electron paramagnetic resonance. Both chelators inhibited DNIC formation up to 50% after 6 h of treatment. To further investigate the role of lysosomal iron in DNIC formation, we prevented lysosomal proteolysis by pretreatment of whole cells with NH₄Cl. Pretreatment with NH₄Cl inhibited the formation of DNIC in a time-dependent manner that points to the importance of the degradation of iron metalloproteins in DNIC formation in vivo. Fractionation of the cell content after treatment with the nitric oxide donor revealed that DNIC is formed predominantly in the endosomal/lysosomal fraction. Taken together, these data indicate that lysosomal iron plays a crucial role in DNIC formation in vivo. Degradation of iron-containing metalloproteins seems to be important for this process.     

Dinitrosyl iron complexes with thiol ligands promote skin wound healing in animals

Dimeric dinitrosyl iron complexes (DNIC) with cysteine or glutathione as NO donors accelerated the healing of experimental skin wound in rats, as demonstrated by histological and histochemical examination. After two injections of an aqueous DNIC solution into the wound (total 5 μmol) on days 1 and 2 after surgery, the granulocyte volume in wound tissue on day 4 was 3–4 times greater than in the control. Higher DNIC doses provoked inflammation in the wound. Similar experiments with another NO donor S-nitrosoglutathione in equivalent amounts (10 μmol) adversely affected the wound. Addition of 2.5 μmol glutathione DNIC for 40 min produced EPR-detectable protein-bound DNIC (2.5 nmol) in wound tissue. Under the same conditions, 5 μmol S-nitrosoglutathione produced less than 10 pmol of protein-bound DNIC; an EPR-active nitrosyl hemoglobin complex was mainly formed (1.5–2.0 nmol) in this case. The beneficial effect of DNIC on the wound was suggested to be due to the delivery of NO to its targets without pronounced formation of cytotoxic peroxynitrite in wound tissue. In contrast, peroxynitrite could form upon administration of rapidly decomposed S-nitrosoglutathione, thereby aggravating the wound condition.     

Dinitrosyl iron complexes with thiolate ligands: Physico-chemistry,biochemistry and physiology

Some present-day concepts on the origin and functional activities of dinitrosyl iron complexes (DNIC) with thiolate ligands are considered. Nitric oxide (NO) including to DNIC increases its stability and ensures effective targeting of NO to organs and tissues. DNIC have a square–planar structure; unpaired electron is localized on the d_z2 orbital of the d⁷ iron atom. The formula of DNIC appears as {(RS^?)₂Fe⁺(NO⁺)₂….(^?SR)₂}^?; electron spin is S = 1/2. Conversion of an originally diamagnetic group, Fe²⁺(NO)₂ with electron configuration d⁸, into a paramagnetic Fe⁺(NO⁺)₂ group is a result of disproportionation of NO ligands and substitution of newly generated NO^? for NO. The nitrosonium ions present in DNIC impart to them high nitrosylating activity, e.g., ability to induce S-nitrosylation of thiols. The ability of S-nitrosothiols to form DNIC in a direct reaction with bivalent iron is a prerequisite to effective mutual conversions of DNIC and S-nitrosothiols. In this work, I consider some mechanisms of destructive effects of low-molecular DNIC on active centers of iron–sulfur proteins, ability of DNIC to express certain genes, to activate guanylate cyclase, to exert hypotensive, vasodilator effects, to inhibit platelet aggregation, to accelerate wound healing and to produce potent erective action. Recently a stabilized powder-like polymeric composition based on dimeric glutathione DNIC the water-soluble polymer in which was used as a filling agent was designed. The advantages of this stable DNIC-glutathione preparation include their ability to retain their physico-chemical and functional activities within at least one year. At present, the preparation undergo testing as a base for the design of a wide variety of broad-spectrum drugs.     

Beneficial effect of dinitrosyl iron complexes with thiol ligands on the rat penile cavernous bodies

Dinitrosyl iron complexes (DNIC) with thiol ligands were found to beneficially affect the state of the penile cavernous tissue upon its experimental denervation in rats. Histological and histochemical analysis showed that intracavernous administration of DNIC (twice weekly over six months) almost completely abolished the proliferation of endothelial cells typical of denervated cavernous tissue. On the other hand, this treatment sustained the mitotic activity of smooth myocytes and prevented the appearance of collagenase, a marker of their fibrotic transformation. The DNIC treatment had a pronounced effect on penile erection in neurotomized as well as in intact animals. Introduction of low-molecular DNIC into cavernous tissue was found to cause formation of protein-bound complexes observed by EPR and probably acting as depots of nitric oxide, ensuring steady erection.     

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.     

Formation of dinitrosyl iron complexes in cardiac mitochondria

It has been established that, in the presence of S-nitrosothiols, cysteine, and mitochondria, dinitrosyl iron complexes (DNIC) coupled to low-molecular-weight ligands and proteins are formed. The concentration of DNIC depended on oxygen partial pressure. It was shown that, under the conditions of hypoxia, the kinetics of the formation of low-molecular DNIC was biphasic. After the replacement of anaerobic conditions of incubation with aerobic ones, the level of DNIC came down; in this case, protein dinitrosyl complexes became more stable. We proposed that iron-and sulfur-containing proteins and low-molecular-weight iron complexes are the sources of iron for DNIC formation in mitochondrial suspensions. It was shown that a combination of DNIC and S-nitrosothiols inhibited effectively the respiration of cardiomyocytes.     

Prospects of designing the medicines with diverse therapeutic activity on the basis of dinitrosyl iron complexes with thiol-containing ligands

A stable hypotensive preparation (Oxacom) based on dinitrosyl iron complexes (DNIC) with glutathione has been developed. The preparation has successfully passed through pharmacological trials. The tests on volunteers have shown a high hypotensive activity of the preparation: a single intravenous infusion of its aqueous solution at a dose of 0.2 microM per kg of body weight led to a 20-30% decrease in arterial pressure, which persisted for a period of 15-20 h. Similar experiments on the animals demonstrated that aqueous solutions of DNIC with cysteine or glutathione exert also the hypotensive action due to their vasodilatory activity. Besides, these complexes accelerate wound healing and produce a potent erective action. There is reason to suggest that DNIC with thiol-containing ligands as NO donors can produce the cytotoxic action on the pathogenic mycobacteria Mycobacterium tuberculosis and, after respective treatment, inhibit cancer cell proliferation. These complexes can be used as analgetics, for inhibiting the adhesion process, in the therapy of preexplampsia, spermatogenesis pathologies, and in cosmetology for the treatment of skin injury.     

Dinitrosyl iron complexes with glutathione largely relieve rats of experimental endometriosis

A study was made of the effect of binuclear dinitrosyl iron complexes (DNIC) with glutathione in rats with experimental endometriosis. The latter was induced in an autotransplantation model, where two fragments of endometrium with myometrium (2 × 2 mm) from the left uterine horn were grafted to the inner surface of the anterior abdominal wall. After 4 weeks, the test animals received i.p. injections of 0.5 mL DNIC-glutathione at a dose of 12.5 μmol/kg daily for 12 days. This treatment more than halved the total volume of endometrioid tumors. Remarkably, tumor growths from grafts in control rats were often attended by tumors spontaneously arising nearby or in other locations; no such secondary tumors were observed in DNIC-treated animals. The EPR signal with g _av = 2.03 characteristic of protein-bound DNIC with thiol ligands was recorded in liver and endometrioid implants of control as well as treated animals. Activation of ribonucleotide reductase, detected by a doublet EPR signal at g = 2.0 with 2.3-mT hyperfine splitting, was found in small tumors. The beneficial effect of DNIC-glutathione was suggested to be due to DNIC breakdown near the tumors, with release of a large amount of molecular nitric oxide and nitrosonium ions that resulted in selective local cytotoxicity.     

Prospects of designing medicines with diverse therapeutic activity on the basis of dinitrosyl iron complexes with thiol-containing ligands

A stable hypotensive preparation (Oxacom) based on dinitrosyl iron complexes (DNIC) with glutathione has been developed. The preparation has successfully passed pharmacological trials. Tests on volunteers have shown a high hypotensive activity of the preparation: a single intravenous infusion of its aqueous solution at a dose of 0.2 μmol active substance per kg body wt led to a 20–30% decrease in arterial pressure, which persisted for 15–20 h. Similar experiments on animals demonstrated that aqueous solutions of DNIC with cysteine or glutathione also exert a hypotensive action due to their vasodilatory activity. Besides, these complexes accelerate wound healing and produce a potent erectile effect. There is reason to suppose that DNIC with thiol ligands as NO donors may be cytotoxic for pathogenic mycobacteria Mycobacterium tuberculosis and, after appropriate treatment, inhibit cancer cell proliferation. These complexes can be used as analgesics, for inhibiting the adhesion process, in treating preeclampsia, spermatogenesis pathologies, and in cosmetology for treatment of skin injury.     

Introduction of dinitrosyl iron complexes with thiol-containing ligands into animal organism by inhalation method

The possibility of water-soluble dinitrosyl iron complexes (DNIC) with thiol-containing ligands introduction into lungs and other tissues of mice by free inhalation of little drops (2–3 microns diameter) of the solutions of these complexes was investigated. Little drops of 2–20 mM solutions of the complexes were obtained by using an inhalation apparatus (compressor nebulizer). A cloud of these little drops was then inhaled by animals in a closed chamber. A maximal amount of protein-bound DNICs formed in mouse lungs was 0.6 micromoles per kilogram of tissue weight. The amount of DNIC in lungs, liver and blood decreased to the undetected level within 2–3 hours after inhalation. No cytotoxic effect of DNIC formed in lungs on Mycobacterium tuberculosis was found in mice infected with these mycobacteria.     

Protein-bound dinitrosyl-iron complexes appearing in blood of rabbit added with a low-molecular dinitrosyl-iron complex: EPR studies.

The formation of protein-bound dinitrosyl-iron complexes (DNIC) in blood plasma and packed red cell fraction has been demonstrated by the EPR method in the experiments on rabbits which were i/v injected with the low-molecular DNIC with thiosulphate. This formation was ensured by transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNIC onto serum albumin or hemoglobin molecules. Protein-bound DNICs appeared immediately after low-molecular DNIC injection followed with gradually decreasing their amounts. The complexes could be detected by EPR technique during more than two days. The addition of water-soluble NO scavenger, the iron complex with N-methyl-d-glucamine dithiocarbamate (MGD) resulted in decomposition of a part of protein-bound DNICs and in effective excretion of secondary products (mainly mononitrosyl-iron complexes with MGD) from the blood flow.     

Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging.

The nitrogen monoxide radical (NO*) forms paramagnetic mono- and dinitrosyl-iron complexes in biologic tissues. To establish a noninvasive technique for in vivo NO* imaging, we evaluated the suitability of these complexes as magnetic resonance (MR) contrast agents, making use of the ability of the unpaired electrons of the complexes to enter into dynamic nuclear polarization with water protons and hence produce enhancement on images generated by the technique of proton-electron-double-resonance imaging (PEDRI). Phantom solutions of synthetic nitrosyl-iron complexes (NICs) altered the signal intensity of PEDRI images. The dinitrosyl-iron complex (DNIC) with serum albumin induced a significantly larger signal alteration than the mononitrosyl-iron complex (MNIC) with dithiocarbamate. Exposure of rat liver to sodium nitroprusside (SNP) by ex vivo and in situ perfusion induced a composite X-band electron spin resonance (ESR) spectrum of the isolated liver characteristic of a MNIC and DNIC. On storage of the tissue, the MNIC signal disappeared and the DNIC signal intensity increased. Correspondingly, in cross-sectional PEDRI images taken at room temperature, the SNP-exposed livers initially exhibited a weak signal that strongly increased with time. In conclusion, NICs can be detected using PEDRI and could be exploited for in vivo NO* imaging.     

Physicochemistry of dinitrosyl iron complexes with thiolate ligands underlying their beneficial effect in endometriosis

Exogenous dinitrosyl iron complexes (DNIC) with thiolate ligands as NO and NO⁺ donors are capable of exerting both regulatory and cytotoxic effects on diverse biological processes similarly to those characteristic of endogenous nitric oxide. Regulatory activity of DNIC (vasodilatory, hypotensive, suppressing thrombosis, increasing erythrocyte elasticity, accelerating skin wound healing, inducing penile erection, etc.) is determined by their capacity of NO and NO⁺ transfer to biological targets of the latter (heme- and thiol-containing proteins, respectively) due to higher affinity of the proteins for NO and NO⁺ than that of DNIC. Cytotoxic activity of DNIC is provided by rapid DNIC decomposition under action of iron-chelating compounds, resulting in appearance of NO and NO⁺ in cells and tissues in high amounts. The latter mechanism is suggested to cause the blocking effect of DNIC as cytotoxic effectors on the development of benign endometrial tumors in rats with experimental endometriosis. It is also proposed that a similar mechanism can operate to cause at least a delay of malignant tumor proliferation under action of DNIC.     

Interaction of peroxynitrite and hydrogen peroxide with dinitrosyl iron complexes containing thiol ligands in vitro

The interaction of peroxynitrite with thiolate dinitrosyl iron complexes (DNIC) has been examined and compared with the interaction with H2O2. Peroxynitrite oxidized DNIC containing various thiolate ligands--cysteine, glutathione, and bovine serum albumin. Analysis of the oxidation suggested a two-electron reaction and gave third-order rate constants of (9.3 +/- 0.5).109 M-2.sec-1 for DNIC with BSA, (4.0 +/- 0.3).108 M-2.sec-1 for DNIC with cysteine, and (1. 8 +/- 0.3).107 M-2.sec-1 for DNIC with glutathione at 20 degrees C and pH 7.6. Peroxynitrite was more reactive towards DNIC than towards sulfhydryls. Addition of sodium dithionite after the reaction led to significant restoration of the EPR signal of DNIC with cysteine. The reaction of glutathione DNIC with H2O2 was about 600 times slower than with ONOO- and not reversed by sodium dithionite. Thus peroxynitrite, in contrast to hydrogen peroxide, changes the pool of nitrosocompounds which can be responsible for interconversion, storage, and transportation of nitric oxide in vivo.     

Inhibition of platelet aggregation by dinitrosyl iron complexes with low molecular weight ligands

The dinitrosyl iron complexes (DNIC) with thiosulphate, cysteine or phosphate were shown to inhibit in vitro (in citrate plasma) the human platelet aggregation induced by ADP, collagen or adrenaline. This effect cannot be explained by the toxic action of DNIC on the platelet membrane, since DNIC-pretreated platelets are capable of aggregating under the action of 10(-8) M/ml of phorbol ester, which is known to cause direct activation of protein kinase C. The antiaggregatory activity of DNIC exceeds that of Na-nitroprusside and seems to be due to nitric oxide capable to activate guanylate cyclase of platelets. Using the EPR method, it was shown that addition of DNIC to platelet-enriched plasma results in a rapid transfer of Fe(NO)2 groups to the coupled RS(-)-groups proteins of plasma and, apparently, of platelet membrane proteins. These protein DNIC seem to be the source of NO which inhibits human platelet aggregation.     

The dinitrosyl-iron complexes with cysteine block the development of experimental endometriosis in rats

It has been shown that the administration of 0,5 ml of 5 mM aqueous solution of dinitrosyl-iron complexes (DNIC) with cysteine alleviated the development of experimental endometriosis in rats induced by surgical way: the size of endometriomes decreased 1.85 times when the DNIC was added every day during 10 days. The effect was suggested to be due to cytotoxic action of NO molecules and nitrosonium ions (NO+) released from rapidly decomposed DNIC in animal organism on endometriome tissues.     

Dinitrosyl iron complexes with cysteine suppress the development of experimental endometriosis in rats

Administration of dinitrosyl iron complexes (DNIC) with cysteine suppressed the development of experimental (surgically induced) endometriosis in rats: the mean size of endometrioma was 1.85 times smaller if 0.5 mL of a 5 mM aqueous solution of DNIC had been injected daily for 10 days. It is supposed that NO molecules and nitrosonium ions (NO⁺), released from DNIC rapidly decomposed in the organism, prove cytotoxic for endometrioid tissue.