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The proposed in our studies mechanism of dinitrosyl iron complex (DNIC) formation through the main step of disproportionation of two NO molecules in complex with Fe2+ ion leads to emergence of the resonance structure of dinitrosyl-iron fragment of DNIC, [Fe2+(NO)(NO

It is shown that the inhalation of gaseous nitric oxide (gNO) or sprayed aqueous solutions of binuclear dinitrosyl iron complexes with glutathione or N-acetyl-L-cysteine by animals or humans provokes no perceptible hypotensive effects. Potentially, these procedures may be useful in COVID-19 treatment. The NO level in complexes with hemoglobin in blood decreases as the gNO concentration in the gas flow produced by the Plazon system increases from 100 to 2100 ppm, so that at 2000 ppm more than one-half of the gas can be incorporated into dinitrosyl complexes formed in tissues of the lungs and respiratory tract. Thus, the effect of gNO inhalation may be similar to that observed after administration of solutions of dinitrosyl iron complexes, namely, to the presence of dinitrosyl iron complexes with thiol-containing ligands in lung and airway tissues. With regard to the hypothesis posited earlier that these complexes can suppress coronavirus replication as donors of nitrosonium cations (Biophysics 65, 818, 2020), it is not inconceivable that administration of gNO or chemically synthesized dinitrosyl iron complexes with thiol-containing ligands may help treat COVID-19. In tests on the authors of this paper as volunteers, the tolerance concentration of gNO inhaled within 15 min was approximately 2000 ppm. In tests on rats that inhaled sprayed aqueous solutions of dinitrosyl iron complexes, their tolerance dose was approximately 0.4 mmol/kg body weight.

The role of nitric oxide as one of the universal regulators of metabolic processes in living organisms is considered. The results of experimental studies of NO association with cancer are reviewed. Antitumor and cytotoxic effects of various nitrogen oxide donors are described and the mechanisms of their action are discussed.

Addition of a 20-μL drop of a mixture containing 1 mM Fe2+ + 0.5 M glutathione to the surface of 0.5 M S-nitrosoglutathione solution, which is expected to form dinitrosyl iron complexes with glutathione, generated short-lived (0.2 s) concentric ring-shaped structures in the S-nitrosoglutathione solution. The number of the ring-like structures exceeded the number of concentric circles spread across the water surface (mechanical waves) when droplets of water fall into a body of an aqueous solution. It appears likely that the ring-shaped structures observed in the S-nitrosoglutathione solution are associated with an autowave distribution of products generated during the formation of dinitrosyl iron complexes with glutathione.

The influence of the nature of the ligands of nitric oxide-generating compounds, such as binuclear dinitrosyl iron complexes containing glutathione or mercaptosuccinate, on their tumor growth-inhibiting and cytotoxic effects has been studied in vivo and in vitro. The antitumor effect of the complex with glutathione exceeds that exerted by the complex with mercaptosuccinate. These complexes inhibit tumor growth (Lewis lung carcinoma) by 90 and 65%, respectively, compared to the control. These complexes are weakly cytotoxic against the MCF-7 human cancer cell line. The half-maximum inhibitory concentration of the complex with mercaptosuccinate at which the survival of cells is reduced by 50% (IC50) is 0.8 μmol/mL (640 μg/mL), whereas in the case of the complex with glutathione it is 2 μmol/mL (1600 μg/mL). It is assumed that the antitumor activity of iron complexes is determined by their ability to enter immunocompetent cells, which provide a direct route to tumor tissues.

The pathogenesis of diseases of the gastrointestinal tract in horses is accompanied by inflammation and oxidative stress and may be caused by a lack of nitric oxide, which controls various signaling pathways in the body. The level of nitrites, which are metabolites of nitric oxide in horses with various intestinal diseases, has been evaluated in blood serum at 3.60 ± 3.02 μM and 8.3 ± 6.0 μM in horses at ages of 7–26 years and 1–5 years, respectively. A sharp reduction in the nitrite concentrations was observed in all horses with intestinal diseases (3.39 ± 2.85 μM), especially in horses with tympanitic caecum (0.6 ± 0.4 μM) and obstruction of the colon (0.81 ± 0.5 μM).

This paper presents new results that confirm our previous inference that the binuclear form of biologically active dinitrosyl iron complexes (B-DNICs) with thiol-containing ligands (glutathione or N-acetyl-L-cysteine) may act as a donor of nitrosonium cations, which are responsible for S-nitrosothiol formation during B-DNIC decomposition in acid solutions under both aerobic and anaerobic conditions. The presence of nitrosonium cations within B-DNICs is determined by the dispropoportionation reaction of free-radical nitric oxide (NO) molecules while binding to Fe2+ cations (two molecules per one ion) during B-DNIC synthesis. When thiolic ligands are oxidized in DNICs or inactivated by thiol-specific reagents, the nitrosonium cations released during decomposition of these DNICs at neutral pH values are hydrolyzed and transformed to nitrite anions. A similar transformation occurs when mononuclear DNICs (M-DNICs) with nonthiolic ligands are decomposed at neutral pH values. It has been found that S-nitrosothiol formation in the decomposition of B-DNICs with thiolic ligands at acidic pH values can be inhibited by the presence of a two to threefold excess of free thiol molecules (outside the B-DNIC) with regard to the B-DNIC level. This inhibition is due to the reduction of nitrosonium cations induced by free thiol molecules and catalyzed by iron ions. The NO molecules that result from the reduction are released from the DNICs. Thus, both forms of DNICs, M and B, that form in living systems can act not only as donors of NO, which is now recognized as one of the universal regulators of metabolic processes, but also as donors of nitrosonium cations, which initiate S-nitrosation of low- and high-molecular-weight (protein-bound) thiols.

—The antitumor activity of S-nitrosoglutathione, bi- and mononuclear dinitrosyl iron complexes with different thiol-containing ligands such as glutathione, mercaptosuccinate, and thiosulfate was studied in models of transplantable murine solid tumors (Lewis lung carcinoma, Acatol adenocarcinoma, and Ca-755 adenocarcinoma). The highest antitumor activity, that is, 90% inhibition of tumor cell growth, was shown by the preparations of dinitrosyl iron complexes with glutathione via intravenous injection (Lewis lung carcinoma) and S-nitrosoglutathione via intraperitoneal injection (Ca-755 adenocarcinoma) to animals. Considering these results together with previous data on the antitumor activity of this type of compound, as well as the results of other researchers in this field, we hypothesized that the antitumor effect is mainly caused by the ability of these compounds to act as donors of nitrosonium ions. Nitrosonium ion-induced S-nitrosation of thiol-containing proteins on the surface of tumor cells leads to an increase in the intracellular oxidative capacity, thus promoting apoptosis and death of tumor cells.

The antitumor activity of the binuclear form of dinitrosyl iron complexes with mercaptosuccinate administered intravenously was studied in murine models of solid tumors: Lewis lung carcinoma and Acatol adenocarcinoma. The maximum effect, suppression of tumor growth by 65% in comparison to the control for Lewis carcinoma, was observed for a course of six intravenous injections in a daily dose of 2.5 μM/kg administered every 2 or 3 days over the period of days 1–17 after tumor transplantation.

It is shown that the efficacy of the photosensitized oxidation of organic substrates in aqueous media (with the oxidation of tryptophan being used as an example) does not depend on adding biologically active dinitrosyl iron complexes (DNICs) with thiol-containing ligands to the reaction medium if it contains Pluronic F127, a poly(ethylene oxide)–poly(propylene oxide)– poly(ethylene oxide) triblock copolymer, at concentrations higher than the critical micelle concentration. Photosensitizer molecules are localized in Pluronic micelles and are shielded from the damaging impact of NO• radicals that form upon the photodegradation of DNIC molecules. Photodynamic therapy (PDT) sessions (the photoinduced necrosis and apoptosis of cells in pathologically altered tissues and the simultaneous initiation of regeneration and repair of the treated tissues due to the photodecomposition of DNIC molecules) thus become fundamentally possible.

The appropriateness of verification of the possible antiviral effect of dinitrosyl iron complexes with thiol-containing ligands as donors of nitrosonium cations (NO+) is argued. There is reason to hope that treatment of the human respiratory tract and lungs with sprayed solutions of dinitrosyl iron complexes with glutathione or N-acetylcysteine (NAC) ​​as NO+ donors during COVID-19 infection can initiate S-nitrosylation of cellular proteases and thereby suppress viral infection.

The possibility that binuclear dinitrosyl iron complexes with glutathione and cysteine (DNIC-GSН and B-DNIC-Cys) have a strong cytotoxic effect on the growth of endometrioid tumours (EMT) in rats with surgically induced experimental endometriosis established in our previous studies has been supported with experimental data. The increase in the DNIC-GSН or B-DNIC-Cys dose from 10 (in our previous studies) to 20 μmol/kg (after i/p administration to experimental rats) fully suppressed the growth of uterine tissues implanted onto the inner surface of the abdominal wall. At 2 μmol/kg DNIC-GSН, the median value of EMT volume increased from 0 to 15 mm3, while the mean size of EMT—from 55 to 77 mm3 (data from EMT measurements in 10 experimental rats). After treatment of animals with B-DNIC with N-acetyl-L-cysteine (10 μmol/kg) known for its ability to penetrate easily through the cell membrane, the inhibiting effect on EMT growth diminished as could be evidenced from the transformation of ~30% of the implants into large-size EMT. Possible reasons for this phenomenon are discussed.

We have studied the effect of interactions between dinitrosyl iron complexes with thiol-containing ligands (DNIC-TL) and diglucamine salt of chlorine e6 (photoditazine, PD) on the rate of photosensitized oxidation of a model organic substrate – tryptophan – in the presence and absence of an amphiphilic polymer, Pluronic F127, as well as on the DNIC-TL and PD photostability. Using EPR and UV spectroscopy, we determined the rate constants for photodegradation of mono- and dinuclear DNIC-TL and PD, respectively. The presence of the photosensitizer and Pluronic F127 has been shown to have a negligible effect on the rate of photodestruction of mono- and dinuclear DNIC-TL, taking into account the changing DNIC-TL and PD concentrations in the photoexcitation conditions. At the same time, in the DNIC-TL presence, the rate of PD photodestruction increases, however, addition of Pluronic F127 leads to a decrease in the rate constant of PD photodestruction. The latter circumstance creates an opportunity for a simultaneous application of DNIC-TL and photodynamic therapy in the wound treatment without losing the PDT efficiency. Indeed, photodynamic therapy in combination with DNIC-TL facilitated skin wound healing in laboratory rats. As shown by a morphological study, application of the DNIC-TL-PD-F127 complex with the subsequent photoactivation was beneficial in reducing inflammation and stimulating regenerative processes.

—The objective of this work was to study the biological effect of dinitrosyl iron complexes (DNICs) with the glutathione ligand (GSH−DNICs) as a stabilized form of nitric oxide in a rat model of nitric oxide hyperproduction induced by inflammation. Administration of GSH−DNICs in endotoxin shock did not increase the total nitric oxide levels in rat organs, but exerted a protective effect by suppressing nitric oxide hyperproduction in the liver and led to an accumulation of the complexes with protein ligands in the kidney.

Dinitrosyl iron complexes (DNICs) with thiol-containing ligands occur in animal tissues as paramagnetic (EPR-active) mononuclear and diamagnetic (EPR-silent) binuclear species with the presence of nitric oxide. They provide stabilization and storage (within protein-bound DNICs) of nitric oxide, as well as its transport (within low-molecular-weight DNICs) to biological targets to serve as donors of not only nitric oxide itself but also of the nitrosonium ion (NO+). The latter function determines the ability of DNICs to S-nitrosylate various thiol-containing proteins. In this way, the complexes participate in a wide range of physiological and biochemical processes. With respect to the high and diverse level of biological activity and dose dependence of DNICs, they mimic the endogenous nitric oxide system. Taken together with the broad occurrence of DNICs (predominantly in the diamagnetic form) in animal tissues, this fact points to their role as an “operating form” of nitric oxide. It is thought that drugs designed on the basis of DNICs can substantially improve the efficiency of modern medical practice.

Two approaches to the synthesis of dinitrosyl iron complexes (DNIC) with glutathione and l -cysteine in aqueous solutions based on the use of gaseous NO and appropriate S-nitrosothiols, viz., S-nitrosoglutathione (GS-NO) or S-nitrosocysteine (Cys-NO), respectively, are considered. A schematic representation of a vacuum unit for generation and accumulation of gaseous NO purified from the NO 2 admixture and its application for obtaining aqueous solutions of DNIC in a Thunberg apparatus is given. To achieve this, a solution of bivalent iron in distilled water is loaded into the upper chamber of the Thunberg apparatus, while the thiol solution in an appropriate buffer (рН 7.4) is loaded into its lower chamber. Further steps, which include degassing, addition of gaseous NO, shaking of both solutions and formation of the Fe 2+ -thiol mixture, culminate in the synthesis of DNIC. The second approach consists in a stepwise addition of Fe 2+ salts and nitrite to aqueous solutions of glutathione or cysteine. In the presence of Fe 2+ and after the increase in рН to the physiological level, GS-NO or Cys-NO generated at acid media (pH The pattern of spin density distribution in iron-dinitrosyl fragments of DNIC characterized by the d 7 electronic configuration of the iron atom and described by the formula Fe + (NO + ) 2 is unique in that it provides a plausible explanation for the ability of DNIC to generate NO and nitrosonium ions (NO + ) and the peculiar characteristics of the EPR signal of their mononuclear form (M-DNIC).

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.

A significant antitumor activity of aqueous solutions of binuclear dinitrosyl iron complexes with glutathione was found when they were injected intravenously in a model of a solid malignant tumor, that is, Lewis carcinoma, in mice. Dinitrosyl iron complexes completely inhibited the tumor growth (by 100%) at doses of 20, 10, and 2 μmol/kg in the first 11 days after the beginning of experiment followed by tumor proliferation at a rate that was lowest for the lowest of the used doses. At day 16, the inhibition of tumor growth was 90% when a solution of dinitrosyl iron complexes was injected at a dose of 2 μmol/kg five times with an interval of 2 to 3 days between injections; whereas the inhibition of tumor growth did not exceed 70 and 30% at doses of 10 and 20 μmol/kg, respectively. Acceleration, rather than inhibition of carcinoma growth was observed at a dose of 100 μmol/kg. The tumor weight increased 1.5–2.0 times compared to the control values, depending on the time.

It has been established that treatment of mice with sodium nitrite, S-nitrosoglutathione and the water-soluble nitroglycerine derivative isosorbide dinitrate (ISDN) as NO donors initiates in vivo synthesis of significant amounts of EPR-silent binuclear dinitrosyl iron complexes (B-DNIC) with thiol-containing ligands in the liver and other tissues of experimental mice. This effect is especially apparent if NO donors are administered to mice simultaneously with the Fe2+-citrate complex. Similar results were obtained in experiments on isolated liver and other mouse tissues treated with gaseous NО in vitro and during stimulation of endogenous NO synthesis in the presence of inducible NO synthase. B-DNIC appeared in mouse tissues after in vitro treatment of tissue samples with an aqueous solution of diethyldithiocarbamate (DETC), which resulted in the transfer of iron-mononitrosyl fragments from B-DNIC to the thiocarbonyl group of DETC and the formation of EPR-detectable mononitrosyl iron complexes (MNIC) with DETC. EPR-Active MNIC with N-methyl-d-glucamine dithiocarbamate (MGD) were synthesized in a similar way. MNIC-MGD were also formed in the reaction of water-soluble MGD-Fe2+ complexes with sodium nitrite, S-nitrosoglutathione and ISDN.

Using the electron paramagnetic resonance (EPR) method, it was established that under aerobic conditions thiol (RS−) groups of thiol-containing ligands within the composition of paramagnetic mononuclear dinitrosyl iron complexes (M-DNIC) are converted into persulfide (RSS−) ligands in the presence of Na2S in water-ethanolic solutions thereby initiating the formation of paramagnetic M-DNIC with persulfide ligands. Correspondingly, the EPR signal of M-DNIC with thiol-containing ligands at g ⊥ = 2.04, g ∥ = 2.014, and g aver. = 2.031 is substituted for the EPR signal characteristic of M-DNIC with persulfide ligands at g ⊥ = 2.04, g ∥ = 2.02, and g aver. = 2.033. With the increase in the registration temperature to ambient temperature, the anisotropic shape of the EPR signal of M-DNIC with persulfide ligands recorded at 77 K and determined by the anisotropy of the g-factor turns into the symmetric shape with a halfwidth of 0.3–0.4 mT. The lack of the five-component hyperfine structure (HFS) of this EPR signal caused by the interaction of the unpaired electron with the nitrogen nuclei of two nitrosyl ligands is attributed to the compensation of the differently charged contributions of isotropic HFS formed as a result of the spin polarization of appropriate molecular orbitals. It is suggested that similar to M-DNIC with thiol-containing ligands, M-DNIC with persulfide ligands are characterized by the d 7 electronic configuration of the iron atom and the localization of the unpaired electron on MO(dz2). The incorporation of 57Fe into M-DNIC with persulfide ligands is accompanied by doublet HFS splitting of the EPR signal of M-DNIC by virtue of the mononuclear nature of the latter. It is concluded that M-DNIC with persulfide ligands may exist in the form of both low-molecular and protein-bound complexes and have relevance to the recently established biological activity of hydrogen sulfide and its derivatives.

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.

Composites of a collagen matrix and dinitrosyl iron complexes with glutathione (DNIC-GS) (in a dose of 4.0 μmoles per item) in the form of spongy sheets (DNIC-Col) were prepared and then topically applied in rat excisional full-thickness skin wound model. The effects of DNIC-Col were studied in comparison with spontaneously healing wounds (SpWH) and wounds treated with collagen sponges (Col) without DNIC-GS. The composites induced statistically and clinically significant acceleration of complete wound closure (21±1 day versus 23±1 day and 26±1 day for DNIC-Col, Col and SpWH, respectively). Histological examination of wound tissues on days 4, 14, 18 and 21 after surgery demonstrated that this improvement was supported by enhanced growth, maturation and fibrous transformation of granulation tissue and earlier epithelization of the injured area in rats treated with DNIC-Col composites benchmarked against Col and SpWH. It is suggested that the positive effect of the new pharmaceutical material on wound healing is based on the release of NO from decomposing DNIC. This effect is believed to be potentiated by the synergy of DNIC and collagen.

It has been shown that dinitrosyl iron complexes with thiol-containing ligands, bound with modified bovine serum albumin with high amount of thiol groups, appeared in baker yeast or in animal tissues in the presence of exogenous or endogenous nitric oxide, respectively, are represented predominantly by EPR-silent binuclear form. This form can be transformed into EPR-active mononuclear form of dinitrosyl iron complexes with an increase in pH to basic values, into EPR-active form of mononuclear iron nitrosyl complexes in case of bielectronic recovery of the binuclear form of dinitrosyl iron complexes or under the action of dithiocarbamate derivatives. The latter induced the transformation of dinitrosyl iron complexes into EPR-active mononitrosyl iron complexes with dithiocarbamates. A significant amount of binuclear dinitrosyl iron complexes with thiol-containing ligands in living systems and identical biological activity of these complexes and endogenous nitric oxide systems allow of considering endogenous binuclear dinitrosyl iron complexes as a "working form" of endogenous nitric oxide recognized now as a universal regulator of biological processes.

The dynamics of the oxidative metabolism of the blood of healthy donors (n = 30) who were exposed in vitro to nitric oxide in the form of gas or dinitrosyl iron complexes has been investigated. The intensity of lipid peroxidation and the level of malonic dialdehyde in plasma and red blood cells were determined, as well as the plasma antioxidant capacity and the level of superoxide dismutase activity in the blood samples. This study is the first to have characterized the specific features of the response of proand antioxidant systems of the blood to free or bound (into dinitrosyl iron complexes) nitrogen monoxide in vitro; in particular, the gas flow from the Plazon device was shown to have a pronounced prooxidant effect, which was moderately alleviated by tenfold dilution of the NO-containing mixture. The use of an experimental nitric oxide generator that was constructed at the Russian Federal Nuclear Center resulted in minimal pro-oxidant activity, while an aqueous solution of dinitrosyl iron complexes had a moderate antioxidant effect that was manifested as the limitation of lipid peroxidation in blood plasma and an increase of superoxide dismutase activity in erythrocytes.

The effective accumulation of binuclear dinitrosyl iron complexes with glutathione was shown after subcutaneous perilymphatic injection of an aqueous solution of dinitrosyl iron complexes into animal lung tissue at a single dose of 2 µmol per kg twice a day with an interval of 2 h. At 2 hours after the repeated administration, the concentration of these complexes was 16 µmol per kg of the tissue, which decreased during the last 2 hours to 7 μmol per kg of the tissue. Upon a single injection of binuclear dinitrosyl iron complexes with glutathione, their concentration 2 and 4 hours later was two times lower than that in the previous experiments. Presumably, at the obtained concentrations of dinitrosyl iron complexes, a bactericidal effect in the lungs could be observed against Mycobacterium tuberculosis and rapidly proliferating lung tumors.

Inhibition of growth of a transplanted solid tumor in BDF1 mice bearing Lewis lung carcinoma cells on days 1–5 and 7–11 after tumor transplantation was observed at daily intraperitoneal administration of an aqueous solution of binuclear form of dinitrosyl iron complexes with glutathione at a dose of 200 μmol/kg (relative to one Fe(NO)2 group in the complexes). Inhibition of tumor growth during the administration of complexes (for 11 days) was 70 and 85% when an iron complex: free glutathione ratio in solution was equal to 1: 1 and 1: 10, respectively. Accelerated tumor growth that was faster than in the control began after cessation of dinitrosyl iron complex administration. The selective accumulation of dinitrosyl iron complexes in the tumor, as well as the accumulation of nitrosyl complexes of heme proteins was detected by the EPR method. The latter were observed in the tumor and control animals. It is hypothesized that delayed tumor development during administration of the binuclear form of dinitrosyl iron complexes to mice is caused by inactivation of heme-containing proteins under the action of NO released from these complexes, which provides antinitrosative defenses produced in malignant tumors.

It has been established that intraperitoneal bolus administration of S-nitrosoglutathione (GS-NO) (12.5 μmoles/kg; 10 injections in 10 days), beginning with day 4 after transplantation of two 2-mm autologous fragments of endometrial tissue onto the inner surface of the abdominal wall of rats with surgically induced (experimenta) endometriosis failed to prevent further growth of endometrioid (EMT) and additive tumors, while treatment of animals with dinitrosyl iron complexes (DNIC) with glutathione (12.5 μmoles/kg, 10 injections in 10 days) suppressed tumor growth virtually completely. The histological analysis of EMT samples of GS-NO-treated rats revealed pathological changes characteristic of control (non-treated with GS-NO or DNIC) rats with experimental endometriosis. EPR studies established the presence of the active form of ribonucleotide reductase, a specific marker for rapidly proliferating tumors, in EMT samples of both control and GS-NO-treated animals. Noteworthy, in small-size EMT and adjacent tissues of DNIC-treated rats the active form of ribonucleotide reductase and pathological changes were not found.

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.

Dinitrosyl iron complexes have earned attention due to their applications as nitric oxide donors as well as their interesting electronic structures. Typical Fe–NO electromerism issues are complicated in this case by the presence of a second nitrosyl ligand. Each of the formal oxidation states of iron in such complexes – from Fe(III) to Fe(0) – have been shown to be subject to electromerism. Here, broken-symmetry density functional theory data are shown, that reveal asymmetry within iron dithiolate dinitrosyl models in several formal oxidation states. CASSCF calculations on selected models confirm the multiconfigurational character of the Fe(NO) 2 moiety, with the largest contributor at only 44% of the total weight of the wave function. This asymmetry is most noticeable in the excited states; it is further enhanced by solvation and reveals itself even in the ground state in dynamics calculations as well as in reaction pathways connecting the dinitrosyl state to a putative hyponitrite adduct.

The antitumor dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on a solid tumor in the mouse, Lewis lung carcinoma, was detected. The complexes being injected at doses of 21, 42, 105 mg/kg daily for 10 days blocked completely the development of the tumor for the first week after tumor cell implantation into animals. After that, the part of tumor cells which remained in intact alive state began to grow at a rate equal to that for control animals. The effect was proposed to be caused via formation of an antinitrosative defense system in the cells as a response to NO attack on cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. Data were obtained which were in line with the hypothesis.

Some recent data on the presence of mononuclear dinitrosyl iron complexes (M-DNIC) with persulfide (R-S-S−) ligands with a characteristic electron paramagnetic resonance signal at g ⊥ = 2.35 and g || = 2.02 (g aver. = 2.03) in biological systems (e.g., Escherichia coli cells and isolated iron–sulfur proteins) are reviewed. The generation of M-DNIC is controlled by inorganic sulfur (sulfide, S2−) whose binding to thiols gives persulfides. It is suggested that enhanced production of inorganic sulfur is a result of destruction of active centers of iron–sulfur proteins in the presence of NO or NO-containing compounds. Dinitrosyl iron complexes with thiol-containing ligands are the most active participants in this process. Inorganic sulfur may appear in biological systems during the synthesis or resynthesis of active centers of iron–sulfur proteins in response to activation of cysteine desulfurase, the key enzyme in sulfide synthesis from cysteine.

Using the electron paramagnetic resonance (EPR) and optical spectrophotometric methods, it has been established that biologically active, water-soluble dinitrosyl iron complexes (DNIC) with glutathione are predominantly represented by the diamagnetic binuclear form (B-DNIC) even in the presence of a 10-fold excess of glutathione non-incorporated into DNIC at neutral pH. With the increase in рН to 10-11, B-DNIC are fully converted into the paramagnetic mononuclear form (М-DNIC) with a characteristic EPR signal at g⊥=2.04, g‖=2.014 and gaver.=2.03. After treatment with a strong reducing agent sodium dithionite, both М- and B-DNIC are converted into the paramagnetic form with a characteristic EPR signal at g⊥=2.01, g‖=1.97 and gaver.=2.0. Both forms display similar absorption spectra with absorption bands at 960 and 640nm and a bend at 450nm. After oxidation by atmospheric oxygen, this situation is reversed, which manifests itself in the disappearance of the EPR signal at gaver.=2.0 and complete regeneration of initial absorption spectra of М- or B-DNIC with characteristic absorption bands at 390 or 360 and 310nm, respectively. Treatment of bovine serum albumin (BSA) solutions with gaseous NO in the presence of Fe(2+) and cysteine yields BSA-bound М-DNIC (М-DNIC-BSA). After treatment with sodium dithionite, the latter undergo transformations similar to those established for low-molecular М-DNIC with glutathione. Based on the complete coincidence of the optical and the EPR characteristics of sodium dithionite-treated М- and B-DNIC and other findings, it is suggested that sodium dithionite-reduced B-DNIC are subject to reversible decomposition into М-DNIC. The reduction and subsequent oxidation of М- and B-DNIC are interpreted in the paradigm of the current concepts of the initial electronic configurations of М- and B-DNIC (d(7) ({Fe(NO)2}(7)) and d(7)-d(7) ({Fe(NO)2}(7)-{Fe(NO)2}(7)), respectively).

The diamagnetic binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione can be easily synthesized in the air at ambient temperature. The synthetic protocol includes consecutive addition to distilled water of glutathione, which decreases the pH of the test solution to 4.0, a bivalent iron salt (e.g., ferrous sulphate) and sodium nitrite at the molar ratio of 2:1:1, with a subsequent increase in pH to neutral values. Under these conditions, the amount of B-DNIC formed is limited by initial nitrite concentration. In the novel procedure, 20 mM glutathione, 10 mM ferrous sulfate and 10 mM sodium nitrite give 2.5 mM B-DNIC with glutathione, while 5 mM glutathione remains in the solution. Bivalent iron (5 mM) is precipitated in the form of hydroxide complexes, which can be removed from the solution by passage through a paper filter. After the increase in рН to 11 and addition of thiols at concentrations exceeding that of DNIC tenfold, B-DNIC are converted into a mononuclear EPR-active form of DNIC (M-DNIC) with glutathione. B-DNIC preparations synthesized by using new method contain negligible amount of nitrite or S-nitrosoglutathione as a contaminations. All the steps of DNIC synthesis were characterized by using optical, EPR and HPLC methods. A long-lasting hypotensive action of DNIC formed was demonstrated.

The aim of this work is complex estimation of the nitric oxide action on whole blood of healthy people. We tested the reaction of whole human blood (n = 14) to the processing of it with cold NO-containing plasma. We performed direct sparging of blood samples by gaseous flow with NO in a special plant. Cold NO-containing plasma was generated by apparatus “Plazon” (Russia). We tested lactate dehydrogenase activity in direct and reverse reactions, aldehyde dehydrogenase and superoxide dismutase activity, total protein and lactate level, acid-base balance and the partial pressure of gases in blood. For integral assessment of energy metabolism changes a number of derivative parameters (coefficients of energy reaction balance and substrate provision) were used. Our experiments showed that the processing of whole human blood with NO-containing gas flow (NO concentration — 800 ppm) results in significant changes of its physical and chemical parameters. This exposure leads to inhibition of erythrocytes energy metabolism, decreasing plasma antioxidant reserves, developing moderate ionic disorders and acid-base disbalance in blood samples in vitro.

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes, that hampered the progress of these studies, were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several microM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was-considered previously.

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 gav = 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.

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.

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.

It has been found that heating of solutions of the binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione in a degassed Thunberg apparatus (рН 1.0, 70°С, 6 h) results in their decomposition with a concomitant release of four gaseous NO molecules per one B-DNIC. Further injection of air into the Thunberg apparatus initiates fast oxidation of NO to NO₂ and formation of two GS-NO molecules per one B-DNIC. Under similar conditions, the decomposition of B-DNIC solutions in the Thunberg apparatus in the presence of air is complete within 30-40 min and is accompanied by formation of four GS-NO molecules per one B-DNIC. It is suggested that the latter events are determined by oxidation of B-DNIC iron and concominant release of four nitrosonium ions (NO⁺) from each complex. Binding of NO⁺ to thiol groups of glutathione provokes GS-NO synthesis. At neutral рН, decomposition of B-DNIC is initiated by strong iron chelators, viz., о-phenanthroline and N-methyl-d-glucamine dithiocarbamate (MGD). In the former case, the reaction occurs under anaerobic conditions (degassed Thunberg apparatus) and is accompanied by a release of four NO molecules from B-DNIC. Under identical conditions, MGD-induced decomposition of B-DNIC gives two EPR-active mononuclear mononitrosyl iron complexes with MGD (MNIC-MGD) able to incorporate two iron molecules and two NO molecules from each B-DNIC. The other two NO molecules released from B-DNIC (most probably, in the form of nitrosonium ions) bind to thiol groups of MGD to give corresponding S-nitrosothiols. Acidification of test solutions to рН 1.0 initiates hydrolysis of MGD and, as a consequence, decomposition of MNIC-MGD and the S-nitrosated form of MGD; the gaseous phase contains four NO molecules (as calculated per each B-DNIC). The data obtained testify to the ability of B-DNIC with glutathione (and, probably, of B-DNIC with other thiol-containing ligands) to release both NO molecules and nitrosonium ions upon their decomposition. As far as nitrosyl iron complexes with non-thiol-containing ligands predominantly represented by the mononuclear mononitrosyl iron form (MNIC) are concerned, their decomposition yields exclusively NO molecules.

Current notions and new experimental data of the authors on physico-chemical features of dinitrosyl iron complexes with natural thiol-containing ligands (glutathione or cysteine), underlying the ability of the complexes to act as NO molecule and nitrosonium ion donors, are considered. This ability determines various biological activities of dinitrosyl iron complexes--inducing long-lasting vasodilation and thereby long-lasting hypotension in human and animals, inhibiting pellet aggregation, increasing red blood cell elasticity, thereby stimulating microcirculation, and reducing necrotic zone in animals with myocardial infarction. Moreover, dinitrosyl iron complexes are capable of accelerating skin wound healing, improving the function of penile cavernous tissue, blocking apoptosis development in cell cultures. When decomposed dinitrosyl iron complexes can exert cytotoxic effect that can be used for curing infectious and carcinogenic pathologies.

Studies with the use of a highly specific enzymatic sensor demonstrated that, contrary to the common opinion, normally nitrate is in fact not present in the most important physiological fluids. NO metabolites in the amniotic fluid and semen are mainly presented by NO donor compounds. Therefore, the intensity of NO synthesis can be evaluated by the total content of all its metabolites, but not by the widely used summary nitrite+nitrate content.

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.

It was shown that retinal ischemia entailed apoptosis in the inner layers of the retina. Administration of an NO-synthase inhibitor suppressed the development of ischemic apoptosis. To ascertain whether nitric oxide could induce the retinal apoptosis by itself, a nontoxic NO donor—dinitrosyl iron complex (DNIC) with glutathione—was injected into the vitreous body. DNIC at low concentrations induced apoptosis in the same retinal layers as in ischemia. However, with increasing DNIC doses, the number of apoptotic nuclei decreased markedly. Simultaneous administration of excess glutathione prevented apoptosis at any DNIC dose. The obtained data demonstrate the neurotoxic properties of the excess of nitric oxide in the retina.

Low-molecular dinitrosyl iron complexes with thiol-containing ligands (cysteine or glutathione) have recently been shown to be capable of modulating apoptosis. Being in the intact state, the complexes prevent apoptosis, and when decomposed, they initiate it. The possibility is considered of delivering the complexes to tumor tissues by ferromagnetic nanoparticles driven by an external magnetic field.

Although intermittent hypoxia is often associated with hypertension, experimental and clinical studies have demonstrated definite antihypertensive effects of some intermittent hypoxia conditioning (IHC) regimens. Mechanisms of this antihypertensive response are unknown. Endothelial dysfunction related to disturbed synthesis and/or reduced availability of nitric oxide (NO) has been linked to hypertension. Thus, experiments were conducted to determine if IHC can improve endothelium-dependent relaxation and formation of releasable vascular NO stores of young (4–8-week-old) spontaneously hypertensive rats (SHR). Rats were subjected to either IHC (9.5–10% O2, 5–10 min, 5–8 times per day, 20 d) or to sham conditioning. Endothelium-dependent relaxation to acetylcholine was measured in norepinephrine-precontracted, isolated aortic rings, and the size of NO stores was evaluated by percent relaxation to N-acetylcysteine (NAC), which releases stored NO. The capacity of aortic rings for NO storage was evaluated by the relaxation to NAC after prior incubation with an NO donor. IHC significantly suppressed the development of hypertension in young SHR. Endothelial function decreased from 54.7 ± 4.6% to 28.1 ± 6.4% relaxation to acetylcholine after 20 d of sham IHC, whereas endothelial function was sustained (60.3 ± 6.0% relaxation) in IHC rats. IHC also induced formation of available NO stores and enhanced the capacity of aortic rings to store NO. Therefore, the antihypertensive effect of IHC in young SHR is associated with prevention of endothelial dysfunction and with increased accumulation of NO stores in vascular walls.

The effect of NO donors (sodium nitroprusside, S-nitrosoglutathione, dinitrosyl-iron complexes) on the functional and mechanical properties of human platelets and red blood cells has been investigated. It has been established by atomic force microscopy that NO donor-induced platelet disaggregation is accompanied by changes in the elastic properties of cells. It has been shown that, in the presence of NO donors, the detergent-induced hemolysis of red blood cells is delayed, and the elasticity modulus of these cells decreases. The results obtained indicate that NO donors regulate the structural and functional properties of platelets and red blood cells.

Magnetic nanoparticles in five plant species growing on the Apsheron peninsula have been detected by the EPR method. The EPR spectra of these nanoparticles proved to be similar to those of synthesized magnetic nanoparticles. The result demonstrated that plants are capable of absorbing magnetic nanoparticles from the soil. The accumulation of nanoparticles in plants is confirmed by the presence of a broad EPR signal whose maximum position of the low-field component changes from g = 2.38 and half-width of the signal of 32 mT at room temperature to g = 2.71 and 50–55 mT at 80 K. The intensity of the broad EPR signal for plants grown in radioactively contaminated areas (170‐220 μR/h) was substantially lower compared with plants grown on clean soil. The parameters of the broad EPR signal and its dependence on the temperature of recording were identical with those for synthetic magnetic nanoparticles. The photosynthetic activity and changes in the genome of irradiated plants by the analysis of PCR products were studied.

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.

No pro-apoptotic effect of dinitrosyl iron complexes (DNIC) with glutathione, cysteine or thiosulfate was established after incubation of HeLa cells in Eagle’s medium. However, DNIC with thiosulfate manifested pro-apoptotic activity during incubation of HeLa cells in Versene’s solution supplemented with ethylene diamine tetraacetate (EDTA) known to induce the decomposition of these DNIC. The water-soluble о -phenanthroline derivative bathophenanthroline disulfonate (BPDS) had a similar effect on DNIC with glutathione during incubation of HeLa cells in Eagle’s medium. It was assumed that EDTA- or BPDS-induced pro-apoptotic effect of DNIC with thiosulfate or glutathione is coupled with the ability of decomposing DNIC to initiate S -nitrosylation of proteins localized on the surface of HeLa cells. Presumably, the pro-apoptotic effect of S -nitrosoglutathione (GS-NO) on HeLa cells preincubated in Eagle’s medium is mediated by the same mechanism, although the pro-apoptotic effect based on the ability of GS-NO to initiate the release of significant amounts of NO and its oxidation to cytotoxic peroxynitrite in a reaction with superoxide should not be ruled out either. No apoptotic activity was found in the presence of bivalent iron and glutathione favoring the conversion of GS-NO into DNIC with glutathione. It is suggested that interaction of HeLa cells with intact DNIC with glutathione or thiosulfate results in the formation of DNIC bound to cell surface proteins.