L. N. Kubrina

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (capital EM, Cyrillic-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussins red salt esters and can be prepared by treatment of aqueous solutions of Fe(2+) and thiols (small er, Cyrilliccapital EN, Cyrillic 7.4) with gaseous nitric oxide (NO) at the thiol:Fe(2+) ratio 1:1. capital EM, Cyrillic-DNICs are synthesized under identical conditions at the thiol:Fe(2+) ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)(2)}(7) at g(aver.)=2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at small er, Cyrilliccapital EN, Cyrillic 9-10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe(2+) ratio 1:2 results in their conversion into capital EM, Cyrillic-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-capital EM, Cyrillic-DNIC solutions results in cysteine oxidation-controlled conversion of capital EM, Cyrillic-DNICs first into cys-B-DNICs and then into the EPR-silent compound capital HA, Cyrillic able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound capital HA, Cyrillic is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe(2+) chelator small o, Cyrillic-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound capital HA, Cyrillic represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussins black salt thioesters and cannot be prepared by simple substitution of capital EM, Cyrillic-DNIC cysteine for glutathione. Treatment of capital EM, Cyrillic-DNIC with sodium dithionite attenuates the EPR signal at g(aver.)=2.03 and stimulates the appearance of an EPR signal at g(aver.)=2.0 with a hypothetical electronic configuration {Fe(NO)(2)}(9). These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g(aver.)=2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents capital EM, Cyrillic-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.

Dinitrosyl iron complexes with glutathione as NO and NO+ donors

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.

Antitumor activity of dinitrosyl iron complexes with glutathione

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.

An antinitrosative system as a factor in malignant tumor resistance to the cytotoxic effect of nitrogen monoxide

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.

[Mono- and Binuclear Dinitrosyl Iron Complexes with Thiol-containing Ligands in Various Biosystems].

It has been shown that dinitrosyl iron complexes with thiol-containing ligands that are bound with modified bovine serum albumin with a large number of thiol groups that occurred in baker yeast or in animal tissues in the presence of exogenous or endogenous nitric oxide, respectively, consist predominantly of the EPR-silent binuclear form. This form can be transformed into the EPR-active mononuclear form of dinitrosyl iron complexes with an increase in the pH to basic values, into the EPR-active form of mononuclear iron nitrosyl complexes in the case of two-electron reduction of the binuclear form of dinitrosyl iron complexes or under the action of dithiocarbamate derivatives. The latter process induced the transformation of dinitrosyl iron complexes into EPR-active mononitrosyl iron complexes with dithiocarbamates. The significant number of binuclear dinitrosyl iron complexes with thiol-containing ligands in living systems and the similar biological effects of these complexes and the system of endogenous nitric oxide allow us to consider endogenous binuclear dinitrosyl iron complexes as a “working form” of endogenous nitric oxide, which is now recognized as a universal regulator of biological processes.

Detection of autowave distribution of the concentration of a dinitrosyl iron complex with glutathione formed in an aqueous solution of S-nitrosoglutathione after addition of a mixture of glutathione and ferrous iron

The formation of dark green concentric autowaves of the distribution of the concentration of dinitrosyl iron complex (DNIC) with glutathione in a thin (0.3 mm thick) layer of 0.5 M solution of S-nitrosoglutathione in 15 mM HEPES buffer (pH 7.7) after applying on its surface a drop of a solution of glutathione (0.5 mM) and ferrous iron (1 mM) in the same buffer of volume 10 μL was detected. At regular intervals, the picture of autowaves changed in time intervals of 0.4–0.6 s over a period of 3 s after the application of the drop onto the solution. Then the structured picture of the distribution of DNIC dissipated, followed by a uniform green coloring of the solution caused by a uniform distribution of DNIC in it. It is assumed that the formation of autowaves is a consequence of the autooscillatory mode of the existence of a chemical system formed in a mixture of NO, low-molecular-weight thiols, and ferrous iron ions. DNIC with thiolate ligands and S-nitrosothiols arising in this system have a capacity for interconversion, and it is this process that may underlie the autooscillatory, autowave mode of functioning of the system. It is not ruled out that the existence of this system in cells and tissues of living organisms may provide the spatial and temporal organization of the regulation of the biological action of NO and its different endogenous compounds and derivatives.

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.

Physicochemical features of dinitrosyl iron complexes with natural thiol-containing ligands underlying the biological activities of these complexes

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