The Mild Toxic Effect of Dinitrosyl Iron Complexes during Inhalation of Syrian Hamsters in a “Nose-Only” Chamber

The antioxidant effect of dinitrosyl iron complexes (DNICs) was studied in various model systems. DNICs with glutathione ligands effectively inhibited Cu2+-induced peroxidation of low density lipoproteins (LDL). The antioxidant effect of DNICs with phosphate ligands and free reduced glutathione (GSH) was less pronounced. In addition, DNICs with glutathione suppressed the formation of reactive oxygen species during co-oxidation of lecithin liposomes and glucose. Free radical oxidation in this system was induced with a lipophilic azo initiator and evaluated by luminol-dependent chemiluminescence. NO sharply stimulated chemiluminescence during co-oxidation of glucose and liposomes, thus suggesting the formation of potent oxidants under these conditions. Glutathione DNICs scavenge the superoxide radical anion generated in the xanthine-xanthine oxidase system. Superoxide production was assessed by lucigenin-dependent chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy. Chemiluminescence revealed the dose-dependent character of antiradical effect of glutathione DNICs; moreover, these complexes turned out to be more efficient than GSH. EPR spectra of the adducts of the DEPMPO spin trap with free radicals suggest that the interaction of glutathione DNICs and superoxide does not result in the formation of the thiyl radical of glutathione. Here we propose a mechanism of the antioxidant action of glutathione DNICs, suggesting that unstable intermediate complexes are formed upon their interaction with superoxide or lipid radicals. Further, as a result of intramolecular rearrangement, these intermediates decompose without the free radical as the by-products.

The results of the study of the effect of mononuclear dinitrosyl iron complexes (DNICs) with functional sulfur-containing ligands (NO donors) on the cell viability and metabolism of human lung fibroblasts are presented, and the efficiency of their action is evaluated. It was shown that cationic DNICs increased the cell viability of fibroblasts and demonstrated the cytoprotective properties. Fluorescent analysis revealed that the DNICs compounds decrease the mitochondrial membrane potential but do not have a significant effect on the level of glutathione and reactive oxygen species in fibroblasts. It is assumed that the DNICs have the therapeutic potential for treating cardiovascular diseases.

Nitric oxide (NO) is a gaseous molecule which plays a key role in wound healing. Previously, we identified the optimal conditions for wound healing strategies using NO donors and an air plasma generator. The aim of this study was to compare the wound healing effects of binuclear dinitrosyl iron complexes with glutathione (B-DNIC-GSH) and NO-containing gas flow (NO-CGF) at their optimal NO doses (0.04 mmol for B-DNIC-GSH and 1.0 mmol for NO-CGF per 1 cm2) in a rat full-thickness wound model over a 3-week period. Excised wound tissues were studied by light and transmission electron microscopy and immunohistochemical, morphometrical and statistical methods. Both treatments had an identical stimulating impact on wound healing, which indicated a higher dosage effectiveness of B-DNIC-GSH compared to the NO-CGF. B-DNIC-GSH spray application reduced inflammation and promoted fibroblast proliferation, angiogenesis and the growth of granulation tissue during the first 4 days after injury. However, prolonged NO spray effects were mild compared to NO-CGF. Future studies should determine the optimal B-DNIC-GSH solution course for a more effective wound healing stimulation.

Abstract—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.

We studied the effect of dinitrosyl iron complexes with glutathione ligands on platelet aggregation with HeLa tumor cells. It was shown that dinitrosyl iron complexes not only inhibited cell aggregation, but being added at early stages can also block this process. These findings dictate further studies of dinitrosyl iron complexes as a compound reducing metastasizing and thrombus-formation in tumor patients.

Nitric oxide (NO) is an important signaling molecule that plays a key role in maintaining vascular homeostasis. Dinitrosyl iron complexes (DNICs) generating NO are widely used to treat cardiovascular diseases. However, the involvement of DNICs in the metabolic processes of the cell, their protective properties in doxorubicin-induced toxicity remain to be clarified. Here, we found that novel class of mononuclear DNICs with functional sulfur-containing ligands enhanced the cell viability of human lung fibroblasts and rat cardiomyocytes. Moreover, DNICs demonstrated remarkable protection against doxorubicin-induced toxicity in fibroblasts and in rat cardiomyocytes (H9c2 cells). Data revealed that the DNICs compounds modulate the mitochondria function by decreasing the mitochondrial membrane potential (ΔΨm). Results of flow cytometry showed that DNICs were not affected the proliferation, growth of fibroblasts. In addition, this study showed that DNICs did not affect glutathione levels and the formation of reactive oxygen species in cells. Moreover, results indicated that DNICs maintained the ATP equilibrium in cells. Taken together, these findings show that DNICs have protective properties in vitro. It was further suggested that DNICs may be uncouplers of oxidative phosphorylation in mitochondria and protective mechanism is mainly provided by the leakage of excess charge through the mitochondrial membrane. It is assumed that the DNICs have the therapeutic potential for treating cardiovascular diseases and for decreasing of chemotherapy-induced cardiotoxicity in cancer survivors.

Dinitrosyl iron complexes with thiol-containing ligands and apoptosis: Studies with HeLa cell cultures

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Hypochlorous acid (HOCl), one of the major precursors of free radicals in body cells and tissues, is endowed with strong prooxidant activity. In living systems, dinitrosyl iron complexes (DNIC) with glutathione ligands play the role of nitric oxide donors and possess a broad range of biological activities. At micromolar concentrations, DNIC effectively inhibit HOCl-induced lysis of red blood cells (RBCs) and manifest an ability to scavenge alkoxyl and alkylperoxyl radicals generated in the reaction of HOCl with tert-butyl hydroperoxide. DNIC proved to be more effective cytoprotective agents and organic free radical scavengers in comparison with reduced glutathione (GSH). At the same time, the kinetics of HOCl-induced oxidation of glutathione ligands in DNIC is slower than in the case of GSH. HOCl-induced oxidative conversions of thiolate ligands cause modification of DNIC, which manifests itself in inclusion of other ligands. It is suggested that the strong inhibiting effect of DNIC with glutathione on HOCl-induced lysis of RBCs is determined by their antioxidant and regulatory properties.

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.

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+)]. The latter allowed suggesting capacity of these complexes to function as donor of both neutral NO molecules as well as nitrosonium cations (NO+), which has been demonstrated in experiments. Analysis of biological activity of DNICs with thiol-containing ligands presented in this review demonstrates that NO molecules and nitrosonium cations released from the complexes exert respectively positive (regulatory) and negative (cytotoxic) effects on living organisms. It has been suggested to use dithiocarbamate derivatives to enhance selective release of nitrosonium cations from DNIC in living organisms followed by simultaneous incorporation of the released NO molecules into the biologically non-active mononitrosyl iron complexes with dithiocarbamate derivatives.

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.

The dynamics in the oxidative and energy metabolism and enzyme systems of blood detoxification in animals with thermal trauma injected with dinitrosyl iron complexes was explored. The positive effect of dinitrosyl iron complexes on the state of blood pro- and antioxidant systems in animals with experimental thermal injury having profound oxidative stress is shown. This effect is observed as a considerable reduction of the intensity (normalization) of lipid peroxidation processes against significant elevation of antioxidant potential of blood plasma. This tendency was also fixed in erythrocyte membranes. It is also stated, that dinitrosyl iron complexes clearly normalized erythrocyte energy metabolism already by the 3rd day after trauma. In addition, infusions of dinitrosyl iron complexes caused marked stimulation of aldehyde dehydrogenase catalytic activity in burned rats via mechanism, associated with enzyme detoxification properties.

The antioxidant/prooxidant effects of dinitrosyl iron complexes (DNICs), physiological donors of nitric oxide (NO•), are studied in reaction systems modeling processes with cytochrome c occurring in mitochondria under oxidative stress and leading to apoptosis. Using luminol-dependent chemiluminescence, DNICs with glutathione and phosphate ligands were shown to decrease the level of prooxidants in a reaction system containing ferricytochrome c and cumene hydroperoxide. Electron paramagnetic resonance (EPR) spectroscopy revealed that glutathione DNICs (DNICs-GS) intercepted the free radicals formed during the interaction between cytochrome c and tert-butyl hydroperoxide. DNICs-GS were also shown to prevent the formation of oligomeric forms of cytochrome c, which were induced by organic hydroperoxides. Reduced glutathione was less effective as an antioxidant than DNICs-GS or could even occasionally exhibit the prooxidant properties. Ferricytochrome c also catalyzed the formation of DNICs-GS with nitroxyl anion (NO−) taking part.

The effect of dinitrosyl iron complexes with glutathione and S-nitrosoglutathione on the development of experimental endometriosis in rats: A comparative studies