{"paper_id":"bd05963e-8e4f-47de-bb60-b4a731b8b4e9","body_text":"Citation: Vanin AF, Burgova EN and Adamyan LV. Dinitrosyl Iron Complexes with Glutathione Suppress \nSurgically Induced Experimental Endometriosis in Rats. Austin J Reprod Med Infertil. 2015; 2(4): 1019.\nAustin J Reprod Med Infertil - Volume 2 Issue 4 - 2015\nISSN : 2471-0393 | www.austinpublishinggroup.com \nVanin et al. © All rights are reserved\nAustin Journal of Reproductive Medicine & \nInfertility\nOpen Access\nAbstract\nThe review describes the results of our most recent investigations into \nmiscellaneous effects of Dinitrosyl Iron Complexes (DNIC) with Glutathione \nand S-Nitrosoglutathione (GS-NO) as donors of Nitric Monoxide (NO) on \nthe development of surgically induced endometriosis in rats. Whereas \nDNIC induced selective suppression of the growth of endometrioid tumors \nin implantation niduses, GS-NO enhanced tumour growth and lowered the \nimmune responsiveness of experimental rats. It was suggested that the \nselective cytotoxic action of DNIC with glutathione on endometrioid tumors is a \nresult of DNIC decomposition by endogenous iron chelators generated by tumor \ncells in order to provide the latter with iron required for fast tumor growth. The \nnitric monoxide released from DNIC either inside tumor cells or in their vicinity \nis oxidized to cytotoxic peroxynitrite and thus contributes to the strictly selective \ncytotoxic effect of DNIC on tumor cells. Such selectivity is not specific to GS-NO \nwhose decomposition is spontaneous and can take place in different divisions \nof the abdominal cavity.\nKeywords: Dinitrosyl iron complexes; Nitric oxide; S-Nitrosothiols; \nEndometriosis\nDinitrosyl Iron Complexes with Glutathione \nRepresent a “Working Form” of Nitric \nMonoxide (NO), One of the Most Universal \nRegulators of Biological Processes\nIt has been established that Nitric Monoxide (NO), one of the \nsimplest chemical compounds synthesized from L-arginine by the \nenzymatic route in the presence of three isoforms of NO-Synthesis \n(NOS), functions as one of the most universal regulators of an \nimmense variety of biological processes occurring in human and \nanimal organisms [4]. This activity is usually manifested at micro \nmolar steady-state concentrations of NO synthesized by constitutive \nisoforms of NOS, viz., the endothelial (eNOS) and neuronal (n-NOS) \nis forms [1]. At steady-state concentrations of NO ≥100 µM generated \nby inducible NOS (iNOS), NO molecules, or, more specifically, the \nproduct of their interaction with the superoxide, viz., Peroxynitrite \n(ONOO\n-) exerts various cytotoxic effects on cells and tissues by acting \nas a potent effector of cell-mediated immunity [4-6]. At physiological \nрН, the protonation of peroxynitrite gives a hydroxyl radical and \nnitrogen dioxide; both products are responsible for the cytotoxic \neffect of peroxynitrite [4,5]. Being free-radical compounds, NO and \nsuperoxide anions easily interact with each other by the diffusion-\ncontrolled mechanism resulting in a fast decrease of the NO content \nin cells and tissues. To prevent the NO decrease, the Nature utilizes \nthe ability of NO to initiate the reversible formation in biological \nsystems of endogenous nitroso derivatives, viz., S-Nitrosothiols (RS-\nNO) and Dinitrosyl Iron Complexes (DNIC) with thiol-containing \nligands [7-9], which are responsible for stabilization, deposition, \nmigration and transfer of NO to its biological targets. DNIC \nwith thiol-containing ligands, which are easily synthesized by the \nchemical route, exist in both paramagnetic, EPR-active mononuclear \nAbbreviations\nB-M-DNIC: Binuclear or Mononuclear Dinitrosyl Iron \nComplexes; EMT: Endometrioid Tumors; EPR: Electron \nParamagnetic Resonance; GS-NO: S-nitrosoglutathione; MNIC-\nDETC: Mononitrosyl Iron Complexes with Diethyldithiocarbamate; \nRCRPC: Russian Cardiological Research-and-Production Complex; \nRS-NO: S-Nitosothiol\nIntroduction\nPrevious studies carried out by the members of our research team \nat the Semenov Institute of Chemical Physics of the Russian Academy \nof Sciences have established that exogenous water-soluble Dinitrosyl \nIron Complexes (DNIC) with glutathione as Nitric Monoxide donors \n(NO) can selectively suppress the development of experimental \nendometriosis in rats induced by surgical transplantation of two 2-mm \nfragments of uterine tissue onto the inner surface of the abdominal \nwall [1-3]. This effect is manifested in early and more advanced steps \nof tumor growth. Another NO donor, viz., S-nitrosoglutathione, \nexerts a non-selective suppressive effect (if any) on tumor growth, \nwhich is directed against both endometrioid tumours and other \ntissues. By weakening the immune responsiveness in experimental \nanimals, S-nitrosoglutathione drastically enhances tumor growth \nin some of them. This paper is an overview of the results of our \nstudies into miscellaneous effects of DNIC with glutathione on the \ndevelopment of surgically induced experimental endometriosis in \nrats. It was established that the selectivity of cytotoxic effects of DNIC \non endometrioid tumors is determined by their physico-chemical \ncharacteristics and the ability to undergo decomposition inside or in \nthe vicinity of endometrioid tumours with a release of considerable \namounts of NO. \nReview Article\nDinitrosyl Iron Complexes with Glutathione Suppress \nSurgically Induced Experimental Endometriosis in Rats\nVanin AF1*, Burgova EN1 and Adamyan LV2\n1Semenov Institute of Chemical Physics, Russian \nAcademy of Sciences, Russia\n2Reproductive Medicine and Surgery, Moscow University \nof Medicine and Dentistry, Russia\n*Corresponding author: Anatolii F Vanin, Semenov \nInstitute of Chemical Physics, Russian Academy of \nSciences, Moscow, Russia\nReceived: July 06, 2015; Accepted: August 05, 2015; \nPublished: August 08, 2015\n\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 02\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\n(М-DNIC) and diamagnetic, EPR-silent binuclear (B-DNIC) forms; \ntheir chemical formulas appear as {(RS -)2 Fe(NO)2} and {(RS -)2 \nFe2(NO)4}, respectively [10-13]. B-DNIC represent thioethers of thiol-\ncontaining compounds (e.g, glutathione or cysteine) and Roussins’s \nred salt (chemical formula {(S)\n2 Fe2(NO)4}) [10]. The concentration of \nМ- and B-DNIC with thiol-containing ligands is determined by the \nchemical equilibrium shown in (Scheme 1): \nIn the presence of thiol excess (or, more specifically, of thiols \nionized at their sulfur atoms), the М-form dominates the solution; \nin the case of its deficit, it is the B-form that is predominant. It was \nshown [14] that М-DNIC are especially abundant in cultured animal \ncells; in animal tissues, DNIC with thiol-containing ligands are \nlargely represented by the binuclear form [15]. The biological activity \nof DNIC with thiol-containing ligands is determined by the ability \nof their iron-dinitrosyl fragments to produce neutral molecules \nof NO and Nitrosonium Ions (NO\n+) in full conformity with the \nchemical equilibrium between these fragments and their constituent \ncomponents, viz, iron ions and nitrosyl ligands (Scheme 2) [10,16]:\nFe+ (NO+)2 ⇔ Fe2+ + NO + NO+\nThe distribution of the spin density in  Fe+(NO+)2 shown in \nScheme 2 is consistent with the mechanism of formation of М-DNIC \nwith thiol-containing ligands established in our previous studies \n[10,16-18] (Scheme 3):\nIt was conjectured that binding of two NO molecules to Fe2+ ions in \nthe initial steps of DNIC formation leads to their dysproportionation \n(reciprocal single-electron oxidation-reduction), whereupon one NO \nmolecule is converted into a nitrosonium ion (NO +), while the other \none yields a Nitroxyl Ion (NO -). This conversion is a distinguishing \nfeature of the NO molecule; it is manifested in the gaseous phase \nand at high pressures and is described by the stoichiometric reaction \ndepicted in (Scheme 4) [19]:\n3NO → NO 2 + N2O\n The protonation of the nitroxyl ion (Scheme 3) is accompanied \nby its transformation into a Nitroxyl molecule (HNO), which further \ndissociates from the complex; subsequent recombination of two HNO \nmolecules gives nitrous oxide (N2O) and water. The coordination site \nof М-DNIC is immediately occupied by another NO molecule giving \nrise to paramagnetic М-DNIC with the d 7 electronic configuration of \nthe iron atom (Fe+). The distribution of the spin density in the Fe(NO)2 \nfragments in the form of Fe +(NO+) is equivalent to the formula \nFe2+(NO+)(NO) ([Fe(NO)2]7 in the Enemark-Feltham classification) \n[20]. Subsequent dimerization of М-DNIC yields B-DNIC (Scheme \n1).\nJudging from the distribution of the spin density in Fe(NO)\n2 \nfragments, their nitrosyl ligands represent easily hydrolyzable \nnitrosonium ions which do not confer stability on both types of \nDNIC. However, in reality the situation is different, if we take into \nconsideration the formation, in DNIC, of molecular orbital’s, which \ninclude d-orbital’s of iron and π-orbital’s of thiol-containing and \nnitrosyl ligands. High π-donor activity of sulfur atoms in thiol-\ncontaining ligands enables effective transfer of shared electronic \ndensity (unshared electron pairs) from sulfur atoms to nitrosonium \nions with high -acceptor activity. As a result, the values of the positive \ncharge on these ions, which determines their interaction with \nhydroxyl ions and, as a consequence, the hydrolysis of nitrosonium \nions, may decrease. This, in turn, hinders the hydrolysis of nitrosyl \nligands in DNIC without any effect on the distribution of spin density \nin these DNIC [16].\nIf, for one reason or another, thiol-containing ligands are released \nfrom М-DNIC, e.g, after establishing of a chemical equilibrium \nbetween М-DNIC and their constituent components, the distribution \nof the electron density between Fe(NO)\n2 fragments takes a pattern \ncharacteristic of electron spin density. This reaction is accompanied \nby a release of NO molecules and nitrosonium ions (Scheme 2). The \ntransfer of shared electronic density from bridging sulfur atoms \nin B-DNIC to iron atoms and nitrosyl ligands may significantly \ndecrease the electron density on their sulfur atoms. It is particularly \nthis decrease that determines the remarkable ability of B-DNIC with \nthiol-containing ligands to retain stability in strongly acidic media. \nLow electron density on thiol sulfur atoms drastically reduces the risk \nof their protonation and thus strengthens the bonding between thiol-\ncontaining ligands and iron atoms [16,21]. \nThe biological activity of М- and B-DNIC with thiol-containing \nligands, which is determined by their ability to act as nitrosonium \nion and NO donors [16,21], simulates the biological activity of \nendogenously produced NO. Similarly to endogenous NO, DNIC \nexert both beneficial (regulatory) and detrimental (cytotoxic) effect on \nvarious body cells and tissues. The former is a result of NO-induced \nactivation of guanylate cyclase, one of the key regulatory enzymes in \ncell signaling system [22], which, in its turn, is due to the ability of \nNO molecules to bind to heme iron in guanylate cyclase. This binding \nis accompanied by the formation of nitrosyl complexes of heme \niron and can significantly change the conformation of the protein \nglobule and thus modulate the biological activity of heme-containing \nproteins. As regards nitrosonium ions released from DNIC, their \nbiological activity is due to their ability to initiate S-nitrosation of \nthiol-containing proteins and interfere with their biological activity \n[23, 24].\nAs above, DNIC with natural thiol-containing ligands, e.g, \nglutathione or L-cysteine, can be easily prepared by chemical \nsynthesis [10,25,26]. These DNIC are readily soluble in water, do \nnot exert cytotoxic effects on biological objects and can be used \nwith equal efficiency in animal studies and in experiments on \nisolated tissues and cell cultures. Our recent studies carried out in \ncollaboration with researchers from other laboratories demonstrated \na beneficial (regulatory) effect of exogenous water-soluble DNIC \nwith various ligands (for the most part, with glutathione) on a vast \nvariety of physiological processes occurring in human and animal \norganisms and even in plants (Table 1). \nThe design of a novel DNIC-\nbased hypotensive drug, which got the name Oxacom ®, is one of the \nmost impressive recent achievements in this  area. After completion \nof pharmacological and clinical trials, which demonstrated high \n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 03\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\ntherapeutic activity of the new drug [31], Oxacom was put into mass \nproduction. The successful outcome of the pharmacological trials \nprompted the idea to examine the hypotensive activity of the drug \non healthy volunteers. In a new series of our experiments, a 3-4 min \nsingle (bolus) intravenous infusion of Oxacom (3-4 ml) at a dose \ncorresponding to 0.1 µmoles of B-DNIC with glutathione per kg of \nbody mass caused a fast (within several minutes) drastic (by ~ 20%) \ndrop of both systolic and diastolic pressure (from 137±4 to 110±4 \nmm Hg and from 85±2 to 61±4 mm Hg, respectively). During the \nnext 6-9h, the arterial pressure remained at a sufficiently low level \nwith a return to normal values after patient’s wakening on the next \nday. As stated earlier in this chapter, clinical trials of Oxacom as an \neffective tool for relieving hypertensive crises in human patients have \nalready been launched. Studies in this area are currently under way \nat the Russian Cardiological Research-and-Production Complex \n(RCRPC) in Moscow, at the Tomsk Cardiology Research Center \nand at other clinical establishments of Russia. The encouraging \nresults of the pioneering studies [50] allowed us to recommend \nOxacom as a highly effective hypotensive remedy for routine clinical \npractice. High clinical efficiency of Oxacom can be illustrated in the \nfollowing example. A 60-year-old male patient with a diagnosed acute \nhypertensive crisis was admitted to the in-patient department of \nRCRPC. A single intravenous dose of Oxacom (0.3 µ moles of DNIC \nwith glutathione per kg of body mass) caused a fast (within 30 min) \ndrop of both systolic and diastolic pressure (from 240/140 mm Hg to \n120/80 mm Hg), which remained at this level up to the moment of the \npatient’s discharge from the hospital (Figure 1). The cytotoxic effect of \nDNIC with thiol-containing ligands is manifested under conditions \nof their fast decomposition with a simultaneous release of significant \namounts of free NO and nitrosonium ions. A natural question \narises: what mechanism is responsible for this phenomenon? The \ndecomposition of DNIC can take place in response to acidification of \nintracellular compartments of DNIC or after treatment of the latter \nwith iron chelators. As can be inferred from the aforecited data, such \ndecomposition can hardly be related to acidification of the intracellular \nmedium because of sufficiently high acid resistance of B-DNIC with \nthiol-containing ligands [21]. As regards the М-form of B-DNIC, \nacidification is accompanied by the conversion of B-DNIC into acid-\nresistant B-DNIC with a decrease in the number of thiol-containing \nligands ionized at the sulfur atom (Scheme 1). Iron chelators or, \nmore specifically, bivalent iron (Fe\n2+) chelators destroy both forms of \nDNIC with a concomitant release of nitrosyl ligands (predominantly \nin the form of neutral NO molecules). Supporting evidence in favor \nof this hypothesis was obtained in experiments where B-DNIC with \nglutathione underwent decomposition by о-phenanthroline, one of \nthe most potent chelators of bivalent iron [21]. These data suggest \nthat nitrosonium ions released from DNIC according to Scheme 2 \nwere further reduced to NO by bivalent iron within the complex with \nо-phenanthroline.\nStudies on cultured HeLa cells established that decomposing \nDNIC with glutathione or thiosulfate ligands (≤ 0.5 m M) cannot \nthemselves produce a cytotoxic (proapoptotic) effect on HeLa \ncells [51]. The cytotoxic activity of these DNIC was studied \ncytofluorimetrically by fluorescence quenching of ethidium bromide \nintercolated into HeLa cell DNA. The fluorescence intensity of \nDNIC-Glutathione-treated HeLa cells was maintained at a level \n1 Potent vasodilatory and hypotensive effects [27-31]\n2 Inhibition of platelet aggregation [32-34]\n3 Antihypoxic effect on the myocardium [35]\n4 Increase of red blood cell viscosoelasticity [36]\n5 Acceleration of skin wound healing [37,38]\n6 Beneficial effect on rats with hemorrhage [39]\n7 Potent penile erective activity [40]\n8 Reduction of the size of the necrotic zone in experimental \nmyocardial infarction [41]\n9 Antiapoptotic effect on cultured normal animal and human cells [42, 43]\n10 Activation/inhibition of expression of certain genes [44-48]\n11 Enhanced assimilation of iron by plants with yellow rust disease [49]\nTable 1: The regulatory effects of DNIC with thiol-containing ligands on various \nphysiological processes.\nFigure 1:  The dynamics of changes in the systolic (1) and diastolic (2) \npressure and the pulse rate (3) of a 60-year-old male patient with hypertensive \ncrisis (240/140 mm Hg before treatment) after a single intravenous infusion of \nOxacom (7.5 mg/kg or 0.3 µmoles DNIC with glutathione/kg) 90 min (А) and \n480 min (B) after treatment. (The data were kindly supplied by Academician \nE.I. Chazov).\nFigure 2:  Upper panel: The histograms illustrating the lack of the \nproapoptotic effect of DNIC with glutathione (0.1, 0.2 and 0.5 mM) on HeLa \ncells after 22h incubation in Eagle’s medium. Solid lines - control, dotted \nlines – experimental animals. Lower panel: The histograms illustrating the \nproapoptotic effects of 0.05, 0.1 and 0.2 mM DNIC with thiosulfate (curves \n2-4). The cells were incubated in 0.5 mM Versene’s solution (EDTA) for 22h; \nmiddle – incubation of 0.2 mM DNIC with glutathione in the presence of 0.05 \nmM Bathophenanthroline Disulfonate (BPDS) (curve 3); right – 0.5 М GS-NO \n(curve 2). The cells were incubated in Eagle’s medium. In all histograms, \ncurve 1 is control. The left and central histograms (curves 2) - incubation of \nHeLa cells in the presence of 0.05 m M DNIC with thiosulfate in Versene’s \nsolution or 0.2 m M DNIC with glutathione in Eagle’s medium. Ordinate: \nnumber of cells (in rel. units) [51].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 04\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\ncorresponding to the diploid (2с) structure of DNA (Figure 2), top-\n22-h incubation of HeLa cells in Eagle’s medium). After incubation in \nthe presence of DNIC with thiosulfate in 0.5 mM Versene’s solution \n(ethylenediamine tetra acetate, EDTA), which normally initiates \nDNIC-Thiosulfate decomposition, the population of apoptotic cells \nincreased significantly with the DNIC concentration (Figure 2), \nbottom, left graph). A similar effect was observed after incubation \nof HeLa cells with DNIC-Glutathione + Bathophenanthroline \nDisulfonate (BPDS) (Figure 2), bottom, central graph). Another NO \ndonor, GS-NO, initiated apoptosis in HeLa cells in the absence of iron \nchelators (Figure 2, bottom, right), most probably, as a result of fast \nspontaneous decomposition of GS-NO and a concomitant release \nof considerable amounts of NO, which manifested strong apoptotic \nactivity against HeLa cells. It may be inferred from these data [51] \nthat in the course of their decomposition by iron chelators DNIC \nexert a cytotoxic effect on HeLa cells. This finding led us to suppose \nthat the cytotoxic activity of DNIC is manifested in the presence of \nendogenous iron chelators generated by rapidly proliferating cells and \ntissues in order to supply the latter with iron essential for their normal \ngrowth. Obviously, after incubation of cells and tissues with DNIC the \nlatter can undergo decomposition by endogenous iron chelators; the \niron released thereupon is utilized for maintaining the vital activity \nof biological objects. This reaction is accompanied by a release, from \nthe decomposing DNIC, of large amounts of NO, which were further \nconverted into cytotoxic peroxynitrite. Such is the mechanism that \nmay be responsible for the selectivity of cytotoxic effects on DNIC on \nrapidly proliferating cells and tissues, e.g., on cells of non-malignant, \ne.g., endometrioid, tumors. These findings suggest that exogenous \nDNIC with thiol-containing ligands simulate both beneficial \n(regulatory) and cytotoxic effects of endogenously produced NO. As, \njudging from the most recent data [14,15], these DNIC are formed in \nanimal tissues and cell cultures as major products in the presence of \nendogenous NO, they have every right to be regarded as a “working \nform” of endogenous NO, which determines their functional activity. \nIt is this particular form of NO that is responsible for the suppression \nof growth of endometrioid tumors in animals and man. Conclusive \nevidence in favor of this conclusion is given below.\nА Model of Surgically Induced Experimental \nEndometriosis in Rats: Benefits and Pitfalls\nA model of surgically induced experimental endometriosis in rats, \nwhich included transplantation of two fragments of uterine tissue (2 \nх 2 mm) onto the inner surface of the abdominal wall, was used as \na model of choice in our studies. The experiments were carried out \non adult female Wistar rats weighing 160 to 180 g supplied by the \n“Stolbovaya” Affiliated Nursery of the Russian Academy of Medical \nSciences. Throughout the 45-day observation period, the animals were \nhoused at the vivarium of the N.M. Emanuel Institute of Biochemical \nPhysics of the Russian Academy of Sciences, in full compliance with \nthe Guidelines of the Geneva Convention “International Principles \nfor Biomedical Research Involving Animals” (Geneva, 1990).\nProtocol: Induction of experimental endometriosis\nExperimental endometriosis was simulated in rats using a \nmodified surgical procedure described by Vernon and Wilson [52]. \nAll the animals were at the proestrus stage of the estrous cycle. Surgical \nmanipulations were performed in the supine position on a standard \nrat surgery board under thiopental anesthesia (0.06 g/kg wt) with \nxylazine (3 mg/kg wt) premedication and lasted 40-45 min. Tumor \ngrowth was induced by surgical transplantation of two autologous \nfragments (2 x 2 mm) of uterine tissue (the endometrium together \nwith the myometrium) excised from the left uterine horn onto the \nanterior surface of the abdominal wall. After termination of invasive \ntreatment, the rats were kept for 4 days under standard vivarium \nconditions (controlled environment, constant temperature (23±2\noС, \n12-h light/dark cycles). Standard dietary intake including free access to \nwater was used to accelerate the engraftment. The use of rodents (e.g. \nrats) for simulating endometriosis in experimental animals has pros \nand cons of its own. To obvious merits, one can relate their low cost, \neasy maintenance (in comparison with, e.g. primates) and a relatively \nshort (4-5 days) and frequent (70-80 cycles per year) estrous cycle \n(cf. 12 cycles in primates). Moreover, rodents are distinguished for \nlong-lasting (2 years) reproductive activity enabling the monitoring \nof various impacts of simulated endometriosis on the reproductive \ncycle and to follow the progress of impregnation, gestation and \ndelivery [53], and last but not least, surgical manipulations (including \nsurgically induced endometriosis) are tolerated by rodents more \neasily than by primates. The main pitfall of simulating endometriosis \nin rodents is their remote position on the evolutional scale relative \nto primates whose physiology is the closest to that of human beings. \nRodents have no menses [54], so their endometrium is not subject to \nmenstrual exfoliation. However, the extracellular matrix of the rodent \nuterus is highly susceptible to proteolysis depending on the collagen \ncontent in their excrements [55].\nB-DNIC with Glutathione Suppress the \nDevelopment of Surgically Induced \nEndometriosis in Rats\nAs stated earlier in this paper, in this study experimental \nendometriosis was simulated in rats by transplantation of two small \nFigure 3: The representative photo images of abdominal tissue samples of \nexperimental (B and D) and control (A and C) rats (Groups 1 and 2). The \npositions of EMT on panels A, C and D are indicated by arrows 1 and 2; on \npanel B, EMT are absent (the endometrial implant niduses are indicated by \narrows 1 and 2). On panel C, additive small-size tumors are indicated by \narrows 3-5 [2].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 05\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\n(2-mm) fragments of uterine tissue onto the inner surface of the \nabdominal wall. Thirty to forty-five days after surgery, the implants \ndeveloped into large-size (≤ 1 cm) oval-shaped Endometrioid Tumors \n(EMT) (Figure 3А and 3С); their growth ceased gradually during 2 \nmonths after surgery.\nFour days after surgery, Group 1 rats were given an \nintraperitoneally dose of B-DNIC with glutathione (6.25 µmoles/\nkg or 12.5 µmoles as calculated per one iron atom in B-DNIC). The \ntreatment course included one daily injection of B-DNIC and lasted \n10 days with subsequent two-week keeping of animals on a standard \nvivarium diet. After completion of treatment, EMT failed to be \ndetected in the majority of experimental rats (Figure 3В) (Table 2), \nwhile in the control group large-size EMT (mean volume ≤ 110 mm\n3) \ncontinued to develop throughout the observation period (30 days) \n(Figure 3А) (Table 2).\nIn Group 2 rats, B-DNIC injections (12.5 µmoles/kg daily, for 10 \ndays) were begun on day 30 after surgery, when the growth of large-\nsize tumors was complete, and were performed daily, for 10 days. In \nthe subsequent period, the animals were kept on a standard vivarium \ndiet for another 4–5 days. In these rats, the growth of EMT estimated \non day 45 after surgery appeared to be suppressed; several animals \ndisplayed the presence of only one (instead of two) tumor (Figure \n3D). It is noteworthy that the mean size of EMT was essentially the \nsame as that in Group 1 rats (control) estimated one month after \nsurgery (Table 2, lines 1 and 4). Control rats of Group 2 displayed the \npresence of two large-sizes EMT; their mean volume estimated 1.5 \nmonths after surgery was 150 mm\n3. Several animals of this group had \nmultiple small-size additive tumors (Figure 3С). In B-DNIC-treated \nexperimental rats of Group 2, additive tumors were absent. The \nhistopathological analysis of large-size EMT established complete \nlack of uterine endometrial cells responsible for tumor growth [54]. \nJudging from the data obtained, DNIC can indeed suppress the growth \nof rapidly proliferating EMT in full conformity with the aforesaid \nhypothesis. In experimental rats treated with the Fe\n2+ + glutathione \nmixture instead of B-DNIC with glutathione, the cytotoxic effect on \nEMT growth failed to be established [2]. Apparently, the inhibiting \neffect of B-DNIC with glutathione on tumor growth must be \nattributed to their nitrosyl ligands rather than to glutathione and \nbivalent iron. In the framework of the hypothesis on the role of \nendogenous iron chelators in the decomposition of B-DNIC, the \nlatter can take place either in the vicinity or in the interior of cells \nproducing these chelators. It is this ability of rapidly proliferating cells \nthat might determine the selectivity of the cytotoxic effect of DNIC \non these cells. Indeed, in our study the cytotoxic activity of B-DNIC \nwith glutathione was manifested only against EMT without any effect \non neighboring organs and tissues, including the abdominal wall and \nthe intestine. The measurements of EPR spectra in EMT samples of \nexperimental and control rats (Group 2) revealed the following. In \nrats treated intraperitoneally with B-DNIC-Glu (12.5 µmoles/kg), \nthe characteristic EPR signal of М-DNIC (the 2.03 signal) (g\n⊥ = 2.04, \ng = 2.014, g aver. = 2.03) [10] was recorded 10 min, 1h and 24h after \ninjection, respectively (Figure 4A,4B, and 4E). The appearance of the \nparamagnetic form of DNIC in biological objects in response to the \ntransfer of Fe(NO)\n2 groups from diamagnetic EPR-silent B-DNIC \nto thiol groups of proteins culminated in the formation of protein-\nbound М-DNIC. Evidence for their protein origin can be derived \nfrom the preservation of the anisotropic shape of their EPR signals \nwith the increase in the registration temperature from 77К to ambient \ntemperature (for explanation see [10]).\nThe intensity of the 2.03 signal (Figure 4A) recorded 10 min \nafter treatment of rats with B-DNIC corresponded to 500 nmoles of \nМ-DNIC per two EMT. After 1h, the signal intensity diminished to 6 \nnmoles of М-DNIC per two EMT with a further drop to 1 nmole per \ntwo EMT on the next day (Figure 4B and 4E). \nThe 2.03 signal with \nthe mean intensity corresponding to 1 nmole per two EMT was also \nrecorded in EMT of control animals of both groups (Figure 4F). In \naddition, these EMT produced an EPR signal with doublet (2.3 mТ) \nsplitting at g = 2.01 characteristic of the active form of Rib Nucleotide \nReductase (RNR) [56]. The intensity of the EPR signal in EMT of \nexperimental rats of both groups was three times lower than in control \n(Figure 4G). Noteworthy, an intense EPR signal corresponding to the \nactive form of RNR was recorded in samples of abdominal wall tissue \non the side opposite to EMT (Figure 4H). A 2.03 signal was recorded \nin the EPR spectra of blood samples collected 1h after treatment of rats \nMedian (min-max) Mean±SEM\nGroup 1\nControl rats (n = 10) 36 (2–599) 113±179\nExperimental rats (n = 10 0 (0–73) 7±17 p<0.001\nGroup 2\nControl rats (n = 10) 30 (2–866) 150±230\nExperimental rats (n = 10) 7 (0–759) 106±23 p<0.008\nTable 2: The results of statistic evaluation of the mean volumes of EMT (in mm3) \n(overall data for all animals) [2].\nFigure 4: The representative EPR spectra recorded in EMT (a, b, e-h) and \nblood (c, d) samples of experimental and control rats. a, b, e - EMT samples \nof Group 2 rats assayed 10 min, 1 h and 24h and blood samples (c, d) \nassayed 1h and 24h after intraperitoneally injection of DNIC with glutathione. \nf, g – EMT samples of control or experimental rats of Group 2, respectively. \nh - EPR spectrum of the abdominal wall of Group 2 rats (control) on the side \nopposite to EMT. The EPR spectra of exogenous (a-c) or endogenous DNIC \n(e-g) (g = 2.04, 2.014) and the active form of rib nucleotide reductase (RNR) \n(g = 2.01, doublet splitting) were recorded at 77K. The relative amplification of \nthe radio spectrometer is indicated to the right of the graph [2].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 06\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nwith B-DNIC (Figure 4C); its intensity corresponded to the М-DNIC \nconcentration of 8 nmoles/ml of blood. However, no such signal was \ndetected on the next day after B-DNIC treatment (Figure 4D). These \nresults suggest that 1h after treatment of control rats with B-DNIC \nwith glutathione the concentration of protein-bound М-DNIC \nformed in their EMT was by two orders of magnitude less than that \ndetermined 10 min after B-DNIC treatment (500 vs. 6 nmoles per \ntwo EMT), which testifies to the fast decomposition of DNIC and the \nappearance of significant amounts of NO in EMT samples. Further \noxidation of the latter to cytotoxic peroxynitrite seems to be the most \nprobable reason for the suppression of EMT growth in experimental \nrats. Noteworthy, this effect is selective and valid for EMT only.\nThe detection of М-DNIC in EMT samples of control (non-\ntreated with exogenous B-DNIC) rats testifies to activation of the \nsystem responsible for the NOS-induced synthesis of endogenous \nNO in abdominal tissues of experimental rats. However, the steady-\nstate concentration of NO in these EMT samples appeared to be \nsignificantly lower in comparison with the initial concentration \nof exogenous NO measured within the first 10 min after treatment \nof animals with exogenous B-DNIC (1 vs. 500 nmoles per two \nEMT), as could be judged from the concentration of М-DNIC \nformed thereupon. In all probability, this difference determines the \nmagnitude of the cytotoxic effect of exogenous B-DNIC on EMT. \nThe time-dependent drastic decrease of the М-DNIC concentration \nin EMT may be attributed to the transfer of the bulk of exogenous \nDNIC from abdominal tissues to circulating blood. Indeed, one hour \nafter treatment of rats with B-DNIC, the М-DNIC were detected in \nanimal blood at the concentration of 8 nmoles/ml. Considering that \nthe volume of the circulating blood did not exceed 10–15 ml, the \nconcentration of М-DNIC in whole blood was equal to 100 nmoles, \nwhich was significantly less than that measured in EMT samples 10 \nmin after B-DNIC treatment (500 nmoles per two EMT or the total \ndose of B-DNIC (2.5 µmoles per rat) as calculated per one iron atom \nin B-DNIC). These data are strongly suggestive of the drastic time-\ndependent decrease of М-DNIC in EMT samples in response to the \ndecomposition of B-DNIC in the vicinity of EMT. The detection of \nan EPR signal (g = 2.01, doublet splitting) corresponding to the active \nform of Rib Nucleotide Reductase (RNR) [56] was another important \nfinding of this study. The appearance of a form responsible for DNA \nsynthesis suggested enhanced proliferation of EMT in control animals \n[56]. Judging from the intensity of its EPR signal, the concentration of \nthe active form of RNR in the group of experimental rats diminished, \nwhich is consistent with the inhibiting effect of B-DNIC on EMT. \nInterestingly, the EPR signal corresponding to the active form of \nRNR was recorded on the opposite (with respect to EMT) side of \nthe abdominal wall, where EMT were absent. This finding points to \na high proliferative activity of abdominal tissue at large and may be \nresponsible for the appearance of small-size additive EMT in control \nrats (Group 2). The effects of other NO donors (e.g., S-nitrosothiols) \non the development of endometriosis were found to be different from \nthose of DNIC. The same group of Mexican investigators established \n[57] that prolonged (for several months) treatment of mice with \nsurgically induced endometriosis with the S-nitrosothiol derivative \nS-nitrosopenicillamine (SNAP) used at a much lower (in comparison \nwith DNIC) doses significantly enhanced the EMT growth instead \nof suppressing it. The immune status of SNAP-treated rats estimated \nby interleukin and interpheron content appeared to be notably \ndecreased suggesting enhanced proliferation of EMT. Similar results \nwere obtained in experiments on GS-NO-treated rats [3]. In this case, \nthe effect of GS-NO on rats with surgically induced endometriosis \nwas studied using the same protocol as in experiments on DNIC-\nGlu-treated rats where GS-NO (12.5 µmoles) was injected beginning \nwith day 4 after surgery; the treatment course lasted 10 days and \nincluded 10 injections. In parallel studies, this protocol was used for \nthe treatment of Group 1 rats with DNIC with glutathione. As in our \nprevious studies [2], treatment of experimental rats with B-DNIC \n(12.5 µmoles/kg calculated per one iron atom; daily, for 10 days) with \nsubsequent keeping on a standard vivarium diet over a period of two \nweeks culminated in complete suppression of EMT growth (Figure 5) \n(Table 3). In 6 out of 9 animals, EMT were absent, while in the rest (n \n= 3) their mean volume did not exceed 5 mm\n3.\nIn addition to large-size tumors grown from uterine tissue \nAnimals Median (min-max MeanS.D p\nControl (n=15) 42.4 (0–1838) 210±371\nRats treated with DNIC (n=9) 0.0 (0–44) 5±7 0.0001\nRats treated with GS-NO (n=9) (without regard for oversize tumors) 12.6 (0–339) 51±76 >0.1\nRats treated with GS-NO (n=9) (with regard for oversize tumors) 28.8 (0–25282) 1392±4916 >0.6\nTable 3: Statistic estimation of the total mean volume of EMT (in mm3) [3].\nFigure 5: The representative photoimages of tissue samples of DNIC-treated \n(A), control (B) and GS-NO-treated rats (C-E). (D,E) GS-NO-treated rats with \noversize EMT. The positions of EMT on panels A-D are marked by arrows 1 \nand 2. Panel B- additive small-size tumors (arrows 3 and 4). Panel E - arrows \n1-3 indicate EMT [1], multiple adhesions in the intestine (2) and the area of \nmassive dissemination of the abdominal wall with germinal tumors (3) [3].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 07\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nimplants, all control rats (n = 15) had multiple small-size additive \ntumors (Figure 5В) (n = 38). In all GS-NO-treated animals of this \ngroup (n = 9), the total number of EMT (including those developed \nfrom tissue implants plus additive tumors) was 30. Oversize’s EMT \nwere detected in 3 rats and were localized in the implant niduses. \nTheir size exceeded that in the control group by one or two orders \nof magnitude. Without regard for oversize tumors, the mean size \nof EMT in GS-NO-treated rats was 51±76 mm 3, i.e., it exceeded the \ncontrol value fourfold (Table 3). With regard to oversize tumors, \nthe mean tumor size exceeded that in the control group sevenfold \n(1392±4916 mm\n3). Similar results were obtained when these data \nwere recalculated per one animal (Table 4).\nThe histopathological data suggest that despite the obvious \nretardation (in comparison with control) of the EMT growth in the \nmajority of GS-NO-treated rats, their EMT contained significant \namounts of endometrial cells responsible for tumor growth. Their \nhistological characteristics were similar to those in control animals \n(Figure 6).\nThese data altogether indicate that GS-NO do not exert a \nbeneficial effect on the development of experimental endometriosis \nin rats. The slow growth of endometrioid tumors in GS-NO-treated \nrats can be attributed to the presence of multiple adhesions in the \nabdominal cavity (Figure 5Е), which prevent vascularization of EMT \nand, as a consequence, suppress their proliferation. If for one reason \nor another this does not take place, the growth of EMT continues, \nculminating in the appearance of oversize tumors.\nInteresting results were obtained during an EPR analysis of tissue \nsamples of rats with EMT (Figure 7). The EPR spectra of EMT samples \nof control and GS-NO-treated rats recorded in the final steps of these \nexperiments displayed the presence of EPR signals corresponding to \nthe active form of RNR, namely, a doublet EPR signal with a peak at g = \n2.01 and a 2.03 signal (Figure 7A and 7B). The former is characteristic \nof rapidly proliferating tissues [56] and may be considered as a \nmarker of enhanced proliferation of EMT, while the latter testifies to \nthe enhanced production of NO. Quite probably, this phenomenon \nrepresents a specific response of the immune system, where the \nsuppression of tumor growth is manifested in enhanced production \nof cytotoxic NO by immunocompetent cells. The detection, in several \nEMT samples, of an intense EPR signal of nitrosyl hemoglobin \ncomplexes (Figures 7D and 7E) provides conclusive evidence for this \nhypothesis. It is noteworthy that none of the tissue samples obtained \nfrom DNIC-treated rats with small-size EMT were able to generate \nthese EPR signals, with the exception of a weak EPR signal produced \nAnimals Median (min-max) Mean±S.D p\nControl rats (n=15) 393 (26–2449) 534 ±620\nRats treated with DNIC (n=9) 0 (0–55) 10±19 0.0003\nRats treated with GS-NO (n=9) (without regard for oversize tumors) 130 (0–431) 161±150 0.0005\nRats treated with GS-NO (n=9) (with regard for oversize tumors) 207 (0–25348) 4641±8472 >0.002\nTable 4: The mean size of EMT calculated per one animal (in mm3) [3].\nFigure 6: A histopathological view of EMT tissues of control (A-C), DNIC-\ntreated (D,E) and GS-NO-treated rats (F-H). (A-C) The auto graft represents \nan enlarged cystic endometrial gland with multiple small-size glandules. The \nlumen is filled with neutrophilic granules. The endometrial stroma is active \nand is infiltrated with neutrophils. (D,E) The endometriotic niduses display \na complete lack of endometrial glands, which are replaced by fibrous tissue \nand contain collagen depots and granulation tissue of different degrees \nof maturity. (F,C) The tissue samples of small-size tumors contain large \nendometriotic cysts with debris along the polymorph nuclear elements \nsuggestive of inflammation. Congestions of foamy macrophages are visible \non the periphery of endometrial lesions. Such patterns are characteristic of \noversize tumors (H). The macrophagial response is absent [3].\nFigure 7: The representative EPR spectra of the endogenous paramagnetic \ncenters recorded in EMT samples of control (a,b) and GS-NO-treated (b,e) \nrats at 77K. The EPR signal (c) is assigned to the free-radical centers in \na tissue sample of a DNIC-treated rat, consisting of a small-size EMT and \nsurrounding abdominal tissues [3].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 08\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nby endogenous free radicals at g = 2.0 (Figure 7C).\nThe formation of endogenous NO in EMT samples of control \nand GS-NO-treated rats was also observed during incorporation \nof NO into the spin trap Fe 2+-Diethyldithiocarbamate (DETC) and \nwas manifested in the appearance of EPR signals of mononitrosyl \niron complexes with DETC (MNIC-DETC) [58]. The spin trap was \nadministered to animals one hour prior to sacrifice. Treatment of rats \nwith and without EMT with B-DNIC was carried out using a similar \nprotocol. Previously, it was found [58] that the characteristic EPR \nsignal of MNIC-DETC recorded at 77К (g\n⊥ = 2.045; g  = 2.02) had \na triplet Hyperfine Structure (HFS) with splitting at 1.2 mТ (Figure \n8E). In all cases studied, this signal overlapped with the characteristic \nEPR signal of DETC complexes with endogenous Cu 2+, which had \na four-component HFS (Figure 8, lines 1–4) [58]. The appearance \nof an EPR signal of MNIC-DETC against the background of the \nintense EPR signal of Cu 2+-DETC was detected by the third (high-\nfield) component of the triplet HFS in the EPR signal of MNIC-DETC \n(Figure 8A and 8B). This component is specific to NO synthesis and \nwas present in the EPR spectra of EMT samples of both control and \nGS-NO-treated rats (Figure 8A and 8B), but not in the EPR spectra \nof B-DNIC-treated rats and of rats without EMT (Figure 8C and 8d).\nJudging from the intensity of this component, about 1–2 nmoles \nof NO per two EMT were incorporated into MNIC-DETC of \ncontrol and GS-NO-treated rats, which is commensurate with the \nconcentration of М-DNIC in EMT tissues of experimental animals \n(Figure 7A and 7B). These data provide additional evidence that in \nEMT tissues DNIC with thiol-containing ligands are predominantly \nrepresented by the М-form. A similar ratio between the М- and \nB-forms of endogenous DNIC was established in experiments with \ncultured isolated animal cells [14,59].\nThe results of yet another series of our experiments where GS-\nNO was used as a NO donor are in perfect agreement with those \nobtained by the Mexican team [57], viz., in contrast to DNIC with \nthiol-containing ligands, S-nitrosothiols were not only incapable to \nsuppress the development of experimental endometriosis in rats, \nbut even stimulated further growth of EMT. Therefore, it would be \nreasonable to suppose that the cytotoxic effect of S-nitrosothiols \nafter their chronic administration to rats was provided by their fast \ndecomposition and was accompanied by a release of considerable \namounts of NO. These events are prerequisite to the appearance, in \nthe animal organism, of significant amounts of cytotoxic peroxynitrite \nformed in response to the NO interaction with superoxide anions.\nSimilar to the cytotoxic effect of SNAP reported by the Mexican \ninvestigators [57], high cytotoxic activity of GS-NO, which in our \nstudies manifested itself in the deterioration of the immune status of \nexperimental rats, might be due to the fact that the decomposition of \nboth S-nitrosothiols affected not only the tissues surrounding EMT, \nbut involved the whole mass of the abdominal tissue. Correspondingly, \nthe cytotoxic effect of S-nitrosothiols might be directed not only \nagainst EMT, but also against other tissues. As regards the cytotoxic \neffect of DNIC, it might result from their decomposition either inside \nor in the vicinity of EMT. In the framework of this hypothesis, which \nhas every chance to be plausible, such decomposition is initiated by \nthe release, from rapidly proliferating EMT, of iron chelators able to \ninitiate the decomposition of DNIC and the appearance of significant \namounts of NO and cytotoxic peroxynitrite responsible for the \nselective apoptosis of EMT.\nDNIC with Glutathione Attenuate Pain \nAttacks in Rats with Experimental \nEndometriosis\nIn women, the main manifestations of endometriosis include low \nimpregnation capacity and a variety of pain syndromes, e.g., severe \ndysmenorrhea (excessive menstrual pains), severe dyspareunia \n(pelvic pain during sexual intercourse), dyschesia (menses-related \npelvic pains upon defecation) and chronic pelvic pains. In some \nfemales, the pain syndrome is concomitant with manifestations of \nsevere chronic diseases and pain syndromes, such as irritable bowel \nsyndrome, interstitial cystitis, and recurrent attacks of kidney stones, \nvulvodynia, migraine and fibromyalgias [60–63]. Chronic pelvic \npains can initiate psychological disturbances, such as chronic fatigue \nsyndrome, depression and anxiety [64]. The relationship between \nendometriosis and pain is still poorly understood; however, recent \ninvestigations into the nature of various pain syndromes in female \npatients and in animals with simulated endometriosis shed additional \nlight upon this problem. Many aspects of the hitherto unaccountable \npersistence of pelvic pains after surgical removal of EMT were \nelucidated in a series of brilliant investigations carried out by Berkley \net al. [65–68].\nThe role of Nitric Monooxide (NO) derivatives in neuronal \nprocesses is difficult to overestimate. Nitric oxide plays a crucial role \nin an immense diversity of signaling and pain processing reactions, \nincluding nociception and antinociception [69,70]. The fact that both \nthe signaling function and the binding of NO to its specific receptors \nFigure 8:  The representative EPR spectra recorded in EMT samples of \ncontrol (a) and GS-NO-treated rats (b), in a tissue sample of a DNIC-treated \nrat consisting of small-size EMT and surrounding tissues (c) and in abdominal \ntissue samples of an intact rat without endometriosis (d). All the animals were \ntreated with Fe2+-DETC as a NO trap. (e) - The EPR signal of MNIC- DETC. \n(Lines 1-4) -The four components of the hyperfine structure (HFS) of the EPR \nsignal of Cu\n2+-DETC complexes. The high-field component of the triplet HFS \nof the EPR signal of MNIC-DETC is indicated by an arrow (а). All the EPR \nspectra were recorded at 77K [3].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 09\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nare impossible without the involvement of free sulfhydryl groups \nof amino acids of high- and low-molecular-weight peptides, is now \ntaken as evidence [71,72]. The regulatory role of sulfhydryl groups is \ndetermined by their ability to interact with NO or, more specifically, \nwith its ionized form (NO\n+) to give S-nitrosothiols that stabilize NO. \nThis effect of NO can also be achieved through its incorporation into \nDNIC (see above). However, the ability of DNIC to accumulate and \nrelease NO in various pain signaling processes still remains to be \nelucidated.\nPrevious studies established that treatment of rats with \nsurgically induced endometriosis with DNIC-glutathione strongly \nsuppressed the further progress of the disease. It seemed, therefore, \nvery tempting to examine to what extent these DNIC reduce pain \nmanifestations in experimental rats. Daily 2h observations over \ncontrol and experimental rats with pain syndromes were carried \nout simultaneously by four investigators as described in [52]. The \nanimals were kept in individual cages (one animal per cage) located \nat a reasonable distance from one another. Selection of animals was \nperformed on a random principle; each rat was in the proestrus \nphase. To minimize the impact of individual factors, the groups of \nanimals (control – experiment) changed every day. The duration of \nposture-related pain manifestations [73] was measured with the help \nof a stop-watch.\nFigures 9 and 10 illustrate the changes in the mean duration of \na single pain attack and the total duration of the pain attacks during \nthe observation period (2h) (mean data from 4 animals) [74]. From \nFigure 9 it follows that the mean duration of a single pain attack \nin DNIC-treated and control rats diminished with time. The faster \ndecrease in this parameter in the experimental group pointed to the \nantinociceptive activity of DNIC. Estimation of the total duration \nof pain attacks within a 2h observation period gave similar results \n(Figure 10).\nThese studies also demonstrated that the mean duration of a single \npain attack in control rats increased on day 36 after surgery on day 8 \nafter the onset of treatment), which can be assigned to the influence of \nexternal and internal factors. The former include drastic fluctuations \nin atmospheric pressure, which has strong impacts on the behavioral \nactivity of experimental animals, and, possibly, estrous variations not \nestablished by the experimenters. (On the other hand, fluctuations \nof atmospheric pressure are known to exert similar effects on both \nexperimental and control animals).\nIt is not excluded that similar changes in the density of nerve \nfibers in endometrial implant niduses and, consequently, the increase \nin the overall duration of the pain attacks on week 5 after surgery also \ntook place in control rats. In the experimental group, the monotonous \ndecrease of these parameters was suggestive of the depletion of \nendogenous NO generated by the constitutive forms of NO synthesis \nby virtue of their inability to produce the antinociceptive effect. It \nmay thus be concluded that administration of NO within a complex \nwith reduced thiols opens up fresh opportunities for relieving pain \nmanifestations in animals and man.\nB-DNIC with Glutathione Suppress the \nProliferation of Transplanted Lewis Lung \nCarcinoma Cells in Early Steps of Tumor \nGrowth in Mice\nThe discovery of selective cytotoxic effects of B-DNIC with \nglutathione on cells of non-malignant Endometrioid Tumors \n(EMT) prompted the idea to investigate their activity against rapidly \nproliferating malignant tumors. The very first studies in this area \nestablished that daily intraperitoneal infusions of B-DNIC with \nglutathione to mice bearing Lewis lung carcinoma for 10 days can \nindeed inhibit the growth of this subcutaneous tumor in a dose-\ndependent manner, however, only in the initial steps of tumor \ndevelopment. In the subsequent periods, tumor growth continued at \nthe same or even higher rates in comparison with control (Figure 11) \n[75].\nInterestingly, simultaneous treatment of rats with B-DNIC with \nglutathione and a 10-fold excess of free glutathione notably enhanced \nthe cytotoxic effect of B-DNIC on Lewis carcinomas (Figure 11), most \nprobably, due to the high concentration of low-molecular DNIC in \nanimal blood. In the absence of free glutathione, the greater part of \nDNIC was bound to proteins as a result of which the cytotoxic effect \nof DNIC on tumor growth was significantly attenuated.\nThese findings suggest that despite their high cytotoxic activity \nFigure 9: The changes in the mean duration of a single pain attack (in sec) \nin the control and experimental groups (the total duration of the observation \nperiod was 12 days). The zero point corresponds to the onset of treatment \n(day 28 after surgery) [74].\nFigure 10:  The changes in the total duration of the pain attacks (in sec) \nrecorded during the 2-hour observation period. The zero point designates the \nonset of treatment (day 28 after surgery) [74].\nInternal factors include progressive hyperalgesia resulting from additional \nfunctional innervation in endometrial implant niduses [68]. In the aforecited \nstudies, ectopic endometrial cysts acquired elementary sensory and \nsympathetic innervation during the first two weeks after transplantation. \nDuring the next 3–4 weeks, these abnormalities turned to functional and \nbecome involved in neurogenic inflammation. By the end of the 4\nth–5th week, \nhyperalgesia became especially apparent due to the appearance, in the cyst \ninterior, of a multitude of sensory and sympathetic nerve fibers.\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 010\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nagainst non-malignant EMT, DNIC with glutathione are incapable \nto suppress the growth of malignant tumors. One should not rule out \nthe possibility that malignantly transformed cells possess an ability to \ngenerate antinitrosative protection proteins attenuating the cytotoxic \neffect of NO or, more specifically, of peroxynitrite generated from \nit. In this respect, the specific response of malignantly transformed \ncells and tissues to the cytotoxic effect of NO is similar to that \nestablished for bacterial cells. Previous studies have demonstrated \nthat the appearance of NO in bacterial cells initiates the expression \nof antinitrosative protection genes and, as a consequence, of proteins \nresponsible for the oxidation and reduction of exogenous NO and \nthus decrease appreciably its concentration in bacterial cells [76]. \nThe similarity of the specific responses of malignantly transformed \nand bacterial cells to NO indicates that a comprehensive search for \ncompounds able to suppress the activity of antinitrosative protection \nproteins is a promising approach to suppressing the growth of \nmalignant tumors through their treatment with NO donors, such as \nDNIC with thiol-containing ligands causing selective inhibition of \nrapidly proliferating cells and tissues.\nStipulating that in bacterial cells antinitrosative protection \nproteins are largely represented by heme-containing proteins or, \nmore specifically, by heme groups responsible for the oxidation-\nreduction of NO, it may be conjectured that in the presence of NO \nexcess heme groups of proteins undergo irreversible oxidation and \nthus become fully disabled. It is also quite probable that precisely \nthe same events took place in our studies where fast tumor growth \nwas recorded on day 12, i.e., immediately after cessation of DNIC \ntreatment. If the expression of the antinitrosative protection proteins \ndid occur at the genomic level, its activation might be initiated within \none or two days after exposure of malignant cells to DNIC as a NO \ndonor. Subsequent expression of antinitrosative protection proteins \nmight initiate the rapid growth of Lewis carcinomas. The lack of this \neffect in DNIC-treated rats suggests that NO released from DNIC \nsuppressed both the activity of antinitrosative protection proteins \nand the intensity of the metabolic processes occurring in malignantly \ntransformed cells. In its turn, the resumption of protein synthesis \nimmediately after cessation of DNIC treatment points to the release \nof the remainder of NO from EMT and, as a consequence, their fast \nproliferation. However, the results of the next series of our studies \nare at variance with this hypothesis, as can be evidenced from the \nchanges in the EMT mass, which did not differ from those observed \nafter prolonged (20-day) treatment of rats with B-DNIC (in press).\nThe Therapeutic Efficiency of Oxacom in \nthe Treatment of Endometriosis in Human \nPatients\nThe hypotensive drug Oxacom designed in collaboration with \na research team at RСRPC represents a dry powder obtained by \nlyophilization (–45 oС) of 19 mM citrate-phosphate buffer (рН \n7.4) containing 2.5 m M B-DNIC with glutathione, 13 m M free \nglutathione, 0.6 mМ dextran (M r = 40 kDa) and 0.9% NaCl. After \nlong-term storage of the degassed preparations in hermetically sealed \nampoules, the drug fully retained its physico-chemical and biological \ncharacteristics for a sufficiently long period of time (1–1.5 and more \nyears). The weight fraction of DNIC in the dry preparation was ∼ 4% \n[31].\nAs stated earlier in this chapter, Oxacom had successfully \nundergone clinical trials [31]. Its lethal i/v dose (LD\n50) for mice and \nrats is 3500–3600 and 3200–3300 mg/kg (70–72 and 64–66 µmoles \nof B-DNIC with glutathione/kg), respectively. A single intravenous \nor chronic dose of Oxacom did not produce any appreciable effect \non different cell populations of rabbit blood, while administration of \nthe drug to mice over a period of 1–19 days was not accompanied \nby any manifestations of mutagenic activity either against murine \nbone marrow cells or on the brood and body mass of newborn rats. \nThese animal studies demonstrated complete safety of Oxacom \nused at doses from 200 to 300 mg/kg (4–6 µmoles B-DNIC with \nglutathione per kg). Noteworthy, at the 0.5–1 µmole/kg dose B-DNIC \nwith glutathione induced a notable (by 50%) drop of arterial pressure \n[31,77].\nA question arises: if B-DNIC with glutathione (Oxacom) have \na beneficial effect on rats with surgically induced endometriosis, \nwill Oxacom produce a similar effect on human patients with \nendometriosis, one of the most rapidly progressing diseases among \nthe female population of our planet? Our animal studies established \nthat endometrioid tumors are non-malignant; hence, endometriosis \nis not a cancerous disease. Moreover, endometrioid tumors are highly \nsensitive to cytotoxic effects of DNIC with thiol-containing ligands. \nIn our studies, low (≤ 6 moles/kg) doses of B-DNIC with glutathione \nstrongly suppressed tumor growth in rats even after 10-day treatment \n[1-3]. But how high must the B-DNIC  dose be in order to suppress \nEMT growth in female patients at more advanced steps of the disease? \nIf the human dose exceeds radically the animal dose, the use of DNIC \nin the treatment of endometriosis in human patients is of limited \nutility. The reason is that high doses of DNIC exert strong hypotensive \neffects, which must be taken into consideration when DNIC are \nadministered to human patients by intraperitoneal or intravaginal \nroute. Remission of endometriosis in DNA-treated females also \npresents a problem, since the etiology of simulated endometriosis \nin animals and “natural” endometriosis in human beings is \nfundamentally different. In animals, surgically induced endometriosis \nconsists in transplantation of uterine tissue grafts onto the inner \nsurface of the abdominal wall and their subsequent development into \nlarge-size EMT. In our study, 10-day intraperitoneal treatment of rats \nwith DNIC in the initial steps of tumor growth suppressed further \ndevelopment and complete resolution of EMT (the endometrial \nimplant niduses contained only surgical threads whereby the implants \nhad been fixed on the inner surface of the abdominal wall). Infusions \nFigure 11: The changes in the mass of Lewis carcinomas in control mice and \nin mice treated with B-DNIC-Glu or B-DNIC-Glu + free glutathione (0.2 m M \nand 2 mM/kg, respectively) recorded on the 4th post-implantation day [75].\n\nAustin J Reprod Med Infertil 2(4): id1019 (2015)  - Page - 011\nVanin AF Austin Publishing Group\nSubmit your Manuscript | www.austinpublishinggroup.com\nof D NIC one month after surgical transplantation fully suppressed \nthe further growth of EMT, apparently due to the complete lack of \nEMT cells responsible for the fast growth of endometrioid implants \n(data from the histopathological analysis).\nThe totality of experimental data strongly suggest that remission \nof endometriosis in experimental animals was hardly possible, because \ntissue grafts did not contain any rapidly proliferating endometrial \ncells responsible for EMT growth. In female patients, this factor is \nabsent and endometriosis can be invoked by transplantation of \nendometrial cells onto the surface of the abdominal wall. However, \nthe factor initiating the release of endometrial cells from uterine \ntissue still remains to be established, since for reasons unknown the \nrelease of endometrial cells after cessation of DNIC treatment may \ninitiate remission of endometriosis. The results obtained by a research \nteam led by the coauthor of this review, Academician L.V.Adamyan, \nPresident of the Russian Society for Endometriosis, provide \nconclusive evidence that remission of endometriosis recorded in 75% \nof patients within two years after surgical treatment, can be assigned \nto the failure to remove small-size EMT in the course of the surgical \nprocedure. Such EMT might otherwise be easily eliminated through \nadministration of DNIC. As far as remission is concerned, it may \nbe related to the extraordinarily high susceptibility of some female \npatients to endometriosis and this fact should not in any way be \nneglected.\nIt remains to be hoped that the problem of remission and related \nproblems will soon be overcome through a comprehensive analysis of \ndirect effects of DNIC on human patients.\nAcknowledgement\nThis work has been supported by the Russian Foundation for \nBasic Research (Grant No 15-04-00708a) and the Presidium of the \nRussian Academy of Sciences (Program “Fundamental Sciences to \nMedicine, 2014).\nReferences\n1. Burgova EN, Tkachev NA, Vanin AF. [The dinitrosyl-iron complexes with \ncysteine block the development of experimental endometriosis in rats].  \nBiofizika. 2012; 57: 105-109.\n2. Burgova EN, Tkachev NÐ, Adamyan LV, Mikoyan VD, Paklina OV, Stepanyan \nAA, et al. Dinitrosyl iron complexes with glutathione suppress experimental \nendometriosis in rats.  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Biofizika. 2015; 60: 152-157.\n76. Green J, Rolfe MD, Smith LJ. Transcriptional regulation of bacterial virulence \ngene expression by molecular oxygen and nitric oxide. Virulence. 2013; 5: \n1-33.\n77. Lakomkin VL, Vanin AF, Timoshin AA, Kapelko VI, Chazov EI. Long-lasting \nhypotensive action of stable preparations of dinitrosyl-iron complexes with \nthiol-containing ligands in conscious normotensive and hypertensive rats.  \nNitric Oxide. 2007; 16: 413-418.\nCitation: Vanin AF, Burgova EN and Adamyan LV. Dinitrosyl Iron Complexes with Glutathione Suppress \nSurgically Induced Experimental Endometriosis in Rats. Austin J Reprod Med Infertil. 2015; 2(4): 1019.\nAustin J Reprod Med Infertil - Volume 2 Issue 4 - 2015\nISSN : 2471-0393 | www.austinpublishinggroup.com \nVanin et al. © All rights are reserved","source_license":"CC0","license_restricted":false}