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

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

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.     

Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.     

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

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

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-alpha-induced apoptosis of rat mesangial cells

Tumor necrosis factor-alpha (TNF-alpha) induces reactive oxygen species (ROS) that serve as second messengers for intracellular signaling. Currently, precise roles of individual ROS in the actions of TNF-alpha remain to be elucidated. In this report, we investigated the roles of superoxide anion (O-(2)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) in TNF-alpha-triggered apoptosis of mesangial cells. Mesangial cells stimulated by TNF-alpha produced O-(2) and underwent apoptosis. The apoptosis was inhibited by transfection with manganese superoxide dismutase or treatment with a pharmacological scavenger of O-(2), Tiron. In contrast, although exogenous H(2)O(2) induced apoptosis, TNF-alpha-triggered apoptosis was not affected either by transfection with catalase cDNA or by treatment with catalase protein or glutathione ethyl ester. Similarly, although ONOO(-) precursor SIN-1 induced apoptosis, treatment with a scavenger of ONOO(-), uric acid, or an inhibitor of nitric oxide synthesis, N(G)-nitro-L-argininemethyl ester hydrochloride, did not affect the TNF-alpha-triggered apoptosis. Like TNF-alpha-induced apoptosis, treatment with a O-(2)-releasing agent, pyrogallol, induced typical apoptosis even in the concurrent presence of scavengers for H(2)O(2) and ONOO(-). These results suggested that, in mesangial cells, TNF-alpha induces apoptosis through selective ROS. O-(2), but not H(2)O(2) or ONOO(-), was identified as the crucial mediator for the TNF-alpha-initiated, apoptotic pathway.     

Effects of reactive oxygen and nitrogen species on cyclooxygenase-1 and -2 activities

The effects of reactive oxygen species (superoxide anion radical--O(2)*-, hydrogen peroxide--H(2)O(2) and hydroxyl radical--*OH; the reaction products of xanthine plus xanthine oxidase system) and reactive nitrogen species [nitric oxide--NO*; from 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene--NOC7 and peroxynitrite--ONOO(-)] on the activities of purified cyclooxygenase (COX)-1 and -2 were studied. Xanthine plus xanthine oxidase suppressed the COX-1 and -2 activities in a xanthine oxidase concentration-dependent fashion. This effect was reversed by addition of catalase to the reactive oxygen species-generating system but not by superoxide dismutase or mannitol, indicating that H(2)O(2) is the responsible metabolite. NOC7 activated the COX-1 activity but inhibited the COX-2 activity at concentrations ranging from 1 to 50 microM. Experiments utilizing a NO* antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide revealed that the observed effects of NOC7 are caused by NO*.ONOO(-), a product of NO* and O(2)*-, both activated and inhibited the COX-1 and -2 activities, depending on ONOO(-) concentration. At a low concentration of ONOO(-) (5 microM) there was enhancement of the COX-1 and -2 activities, but with higher concentrations there was suppression of these two enzyme activities (COX-1, at 200 microM; COX-2, >50 microM). These results suggest that H(2)O(2), NO* and ONOO(-) can have different modulatory effects on the COX-1 and -2 activities.     

Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid.

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H(2)O(2)), peroxynitrite anion (ONOO(-)) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO(-) with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO(-) and H(2)O(2) were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5'dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman's reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.     

Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.     

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

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

小麦叶片顺乌头酸酶对NO和H2 O2 的敏感性

外源一氧化氮（nitric oxide,NO)供体硝普钠（sodium nitroprusside,SNP)和过氧化氢（hydrogen peroxide,H2O2)处理抑制小麦（Triticu aestivum L.)叶片顺乌头酸酶活性,抑制呈明显的浓度及时间效应;同时外源NO衍生代谢物过氧亚硝酸阴离子（peroxynitrite,ONOO^-)的供体3－morpholinosydnonimine hydrochlloride(SIN-1)和水杨酸（salicylic acid,SA)对酶活性也具有抑制作用,而且小麦叶片线粒体顺乌头酸酶对H2O2和SIN－1更敏感。分别以SNP与过氧化氢酶（catalase,CAT)专一性抑制剂氨基三唑（3－amino-1,2,4-triazole,3-AT)处理离体小麦叶片,发现在其内源H2O2含量上升的同时,顺乌头酸酶活性均呈浓度与时间依赖性下降趋势。表明NO除直接抑制顺乌头酸酶活性外,还可能经H2O2介导间接对顺乌头酸酶产生抑制作用。     

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

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

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

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

Effects of reactive species of oxygen and nitrogen on iron ion release from ferritin and synthesis of dinitrosyl iron complexes

Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.     

Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs

The antioxidant nordihydroguaiaretic acid (NDGA) has recently become well known as a putative anticancer drug. In this paper, it was evaluated the in vitro peroxynitrite (ONOO(-)), singlet oxygen ((1)O(2)), hydroxyl radical (OH(v)), hydrogen peroxide (H(2)O(2)), superoxide anion and hypochlorous acid (HOCl) scavenging capacity of NDGA. It was found that NDGA scavenges: (a) ONOO(-) (IC(50) = 4 +/- 0.94 microM) as efficiently as uric acid; (b) (1)O(2) (IC(50) = 151 +/- 20 microM) more efficiently than dimethyl thiourea, lipoic acid, N-acetyl-cysteine and glutathione; (c) OH(v) (IC(50) = 0.15 +/- 0.02 microM) more efficiently than dimethyl thiourea, uric acid, trolox, dimethyl sulfoxide and mannitol, (d) (IC(50) = 15 +/- 1 microM) more efficiently than N-acetyl-cysteine, glutathione, tempol and deferoxamine and (e) HOCl (IC(50) = 622 +/- 42 microM) as efficiently as lipoic acid and N-acetyl-cysteine. NDGA was unable to scavenge H(2)O(2). In an in vivo study in rats, NDGA was able to prevent ozone-induced tyrosine nitration in lungs. It is concluded that NDGA is a potent in vitro scavenger of ONOO(-), (1)O(2), OH(v), and HOCl and is able to prevent lung tyrosine nitration in vivo.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Why does iron abrogate the cytotoxic effect of S-nitrosothiols on human and animal cultured cells?

It was found that dinitrosyl iron complexes (DNIC) with thiol-containing ligands (cysteine or glutathione) of concentrations up to 1 mM produce no cytotoxic effect on cultured cells from human milk gland carcinoma (MCF-7). The cytotoxic action on MCF-7 cells was produced by S-nitrosocysteine: at a concentration of 1 mM, it induced the death of 50% cells. A more stable S-nitrosothiol, S-nitrosoglutathione, did not produce any cytotoxic effect at the same concentration. It is assumed that the negative action of nitrosocysteine is due to its rapid degradation, which results in the accumulation of large amounts of free NO molecules followed by their oxidation by superoxide ions to peroxynitrite, an efficient inhibitor of metabolic processes. These processes seem to be not characteristic of the more stable S-nitrosoglutathione. The cytotoxic effect of nitrosocysteine was completlly abrogated by the addition of 0.2 mM ferrous citrate complex to the medium. When S-nitrosoglutathione NO (0.5 mM) or S-nitrosoglutathione (0.5 mM) + Fe(2+)-citrate (0.2 mM) were added to the medium, protein-bound dinitrosyl iron complexes formed with the involvement of endogenous or exogenous iron were detected in cells. The amount of the complexes in the presence of exogenous iron increased four times, reaching the value of 1.6 nmole/5 x 10(6) cells. Therefore, it was proposed that the blockade of the cytotoxic action of S-nitrosoglutathione by iron complexes is due to Cys-NO transformation of S-nitrosocysteine into dinitrosyl iron complexes. The high stability of these complexes ensures only a gradual accumulation of nitric oxide in cells.