Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen.

Peroxynitrite (ONOO(-)/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O(2)) and nitric oxide ((*)NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO(-)), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, (-)ON=NO(2)(-)), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO(-) in the presence of oxygen is mainly characterized by peroxynitrite and not by HO( small middle dot) radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of (*)NO and O(2), probably by an electron-transfer mechanism. This prediction is supported by the fact that H(2)O(2) is formed during the decay of NO(-) in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO(-) in addition to the "classical" ( small middle dot)NO + O(2)() pathway.     

The mode of decomposition of Angeli's salt (Na2N2O3) and the effects thereon of oxygen,nitrite, superoxide dismutase,and glutathione

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation that the rates of decomposition of Angeli's salt under 20% O(2) or 100% O(2) were equal. Moreover, NO(2)(-), which inhibits this decomposition by favoring the back reaction, was more effective in the absence of agents that scavenge NO(-)/HNO. It is thus clear that Angeli's salt is a useful source of NO(-)/HNO for use in defined aqueous systems. The measurements made in the course of this work allowed approximation of the rate constants for the reactions of NO(-)/HNO with NO(2)(-), O(2), glutathione, or Cu, Zn superoxide dismutase. The likelihood of the formation of NO(-)/HNO in vivo is also discussed.     

Copper,zinc superoxide dismutase as a univalent NO(-) oxidoreductase and as a dichlorofluorescin peroxidase.

Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).     

Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening.

Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H2O2 plus Cu(II) caused predominant DNA damage at the 5' site of 5'-GGG-3' in the telomere sequence. Furthermore, H2O2 plus Cu(II) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in telomere sequences more efficiently than that in non-telomere sequences. NO plus O2- efficiently caused base alteration at the 5' site of 5'-GGG-3' in the telomere sequence. It is concluded that the site-specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.     

Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom.

The mechanism of DNA damage by hydrazine in the presence of metal ions was investigated by DNA sequencing technique and ESR-spin trapping method. Hydrazine caused DNA damage in the presence of Mn(III), Mn(II), Cu(II), Co(II), and Fe(III). The order of inducing effect on hydrazine-dependent DNA damage (Mn(III) greater than Mn(II) approximately Cu(II) much greater than Co(II) approximately Fe(III)) was related to that of the accelerating effect on the O2 consumption rate of hydrazine autoxidation. DNA damage by hydrazine plus Mn(II) or Mn(III) was inhibited by hydroxyl radical scavengers and superoxide dismutase, but not by catalase. On the other hand, bathocuproine and catalase completely inhibited DNA damage by hydrazine plus Cu(II), whereas hydroxyl radical scavengers and superoxide dismutase did not. Hydrazine plus Mn(II) or Mn(III) caused cleavage at every nucleotide with a little weaker cleavage at adenine residues, whereas hydrazine plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the GTC sequence. ESR-spin trapping experiments showed that hydroxyl radical is generated during the Mn(III)-catalyzed autoxidation of hydrazine, whereas hydrogen atom adducts of spin trapping reagents are generated during Cu(II)-catalyzed autoxidation. The results suggest that hydrazine plus Mn(II) or Mn(III) generate hydroxyl free radical not via H2O2 and that this hydroxyl free radical causes DNA damage. A possibility that the hydrogen atom releasing compound participates in hydrazine plus Cu(II)-induced DNA damage is discussed.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.     

The photorelease of nitrogen monoxide (NO) from pentacyanonitrosyl coordination compounds of group 8 metals.

The photodetachment of NO from [M(II)(CN)5NO]2- with M = Fe, Ru, and Os, upon laser excitation at various wavelengths (355, 420, and 480 nm) was followed by various techniques. The three complexes showed a wavelength-dependent quantum yield of NO production Phi(NO), as measured with an NO-sensitive electrode, the highest values corresponding to the larger photon energies. For the same excitation wavelength the decrease of Phi(NO) at 20 degrees C in the order Fe > Ru > Os, is explained by the increasing M-N bond strength and inertness of the heavier metals. Transient absorption data at 420 nm indicate the formation of the [M(III)(CN)5H2O]2- species in less than ca. 1 micros for M = Fe and Ru. The enthalpy content of [Fe(III)(CN)5H2O]2- with respect to the parent [Fe(II)(CN)5NO]2- state is (190 +/- 20) kJ mol(-1), as measured by laser-induced optoacoustic spectroscopy (LIOAS) upon excitation at 480 nm. The production of [Fe(III)(CN)5H2O]2- is concomitant with an expansion of (8 +/- 3) ml mol(-1) consistent with an expansion of the water bound through hydrogen bonds to the CN ligands plus the difference between NO release into the bulk and water entrance into the first coordination sphere. The activated process, as indicated by the relatively strong temperature dependence of the Phi(NO) values and by the temperature dependence of the appearance of the [Fe(III)(CN)5H2O]2- species, as determined by LIOAS, is attributed to NO detachment in less than ca. 100 ns from the isonitrosyl (ON) ligand (MS1 state).     

Mechanism of metal-mediated DNA damage and apoptosis induced by 6-hydroxydopamine in neuroblastoma SH-SY5Y cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin to produce an animal model of Parkinson's disease. 6-OHDA increased the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), a biomarker of oxidatively damaged DNA, and induced apoptosis in human neuroblastoma SH-SY5Y cells. Iron or copper chelators inhibited 6-OHDA-induced 8-oxodG formation and apoptosis. Thus, iron and copper are involved in the intracellular oxidatively generated damage to DNA, a stimulus for initiating apoptosis. This study examined DNA damage caused by 6-OHDA plus metal ions using (32)P-5'-end-labelled DNA fragments. 6-OHDA increased levels of oxidatively damaged DNA in the presence of Fe(III)EDTA or Cu(II). Cu(II)-mediated DNA damage was stronger than Fe(III)-mediated DNA damage. The spectrophotometric detection of p-quinone and the scopoletin method showed that Cu(II) more effectively accelerated the 6-OHDA auto-oxidation and H(2)O(2) generation than Fe(III)EDTA. This study suggests that copper, as well as iron, may play an important role in 6-OHDA-induced neuronal cell death.     

Antioxidation and DNA-Binding Properties of Binuclear Lanthanide(III) Complexes with a Schiff Base Ligand Derived from 8-Hydroxyquinoline-7-carboxaldehyde and Benzoylhydrazine

8-Hydroxyquinoline-7-carboxaldehyde (8-HQ-7-CA), Schiff-base ligand 8-hydroxyquinoline-7-carboxaldehyde benzoylhydrazone, and binuclear complexes [LnL(NO(3) )(H(2) O)(2) ](2) were prepared from the ligand and equivalent molar amounts of Ln(NO(3) )?6 H(2) O (Ln=La(3+) , Nd(3+) , Sm(3+) , Eu(3+) , Gd(3+) , Dy(3+) , Ho(3+) , Er(3+) , Yb(3+) , resp.). Ligand acts as dibasic tetradentates, binding to Ln(III) through the phenolate O-atom, N-atom of quinolinato unit, and C?N and ?O?C?N? groups of the benzoylhydrazine side chain. Dimerization of this monomeric unit occurs through the phenolate O-atoms leading to a central four-membered (LnO)(2) ring. Ligand and all of the Ln(III) complexes can strongly bind to CT-DNA through intercalation with the binding constants at 10(5) -10(6) M(-1) . Moreover, ligand and all of the Ln(III) complexes have strong abilities of scavenging effects for hydroxyl (HO(.) ) radicals. Both the antioxidation and DNA-binding properties of Ln(III) complexes are much better than that of ligand.     

Nitric oxide decreases the stability of DMPO spin adducts.

The effect nitric oxide (NO*) on the stability of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO* adduct, generated by porcine pulmonary artery endothelial cells in the presence of H2O2 and DMPO, or by a Fenton system (Fe(II)+H2O2) is degraded in the presence of the NO*-donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO*. A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/*CH3 and DMPO/*CH(CH3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO* in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO*, rather than with NO2* (formed from NO* and O2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO* may substantially affect the quantitative determination of DMPO adducts. We also show that NO2* radicals, generated by a myeloperoxidase/H2O2/nitrite system, also degrade DMPO/HO*. Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO* or NO2* radicals.     

Reactions of sulfur-nitrosyl iron complexes of "g=2.03" family with hemoglobin (Hb): kinetics of Hb-NO formation in aqueous solutions.

NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (VIII) have been investigated. The method for research has been based on the formation of Hb-NO adduct upon the interaction of hemoglobin with NO generated by complexes I-VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I-VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes (I-VI) with Hb is limited by the stage of NO release in the solution.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

Neurotoxicity of nitroxyl: insights into HNO and NO biochemical imbalance

Nitroxyl anion (NO-), and/or its conjugate acid, HNO, may be formed in the cellular milieu by several routes under both physiological and pathophysiological conditions. Since experimental evidence suggests that certain reactive nitrogen oxide species can contribute significantly to cerebral ischemic injury, we investigated the neurotoxic potential of HNO/NO- using Angeli's salt (AS), a spontaneous HNO/NO(-)-generating compound. Exposure to AS resulted in a time- and concentration-dependent increase in neural cell death that progressed markedly following the initial exposure. Coadministration of the donor with Tempol (1 mM), a one-electron oxidant that converts NO- to NO, prevented its toxic effect, as did the concomitant addition of Fe(III)TPPS. Media containing various chelators, catalase, Cu/Zn superoxide dismutase, or carboxy-PTIO did not ameliorate AS-mediated neurotoxicity, ruling out the involvement of transition metal complexes, H2O2, O2-, and NO, respectively. A concentration-dependent increase in supernatant protein 3-nitrotyrosine immunoreactivity was observed when cultures were exposed to AS under aerobic conditions, an effect lost in the absence of oxygen. A bell-shaped curve for augmented AS-mediated nitration was observed with increasing Fe(III)TPPS concentration, which contrasted with its linear effect on abating cytotoxicity. Finally, addition of glutamate receptor antagonists, MK-801 (10 microM) and CNQX (30 microM) to the cultures abrogated toxicity when given during, but not following, AS exposure; as did pretreatment with the exocytosis inhibitor, tetanus toxin (300 ng/ml). Taken together, our data suggest that under aerobic conditions, AS toxicity is initiated via HNO/NO- but progresses via secondary excitotoxicity.     

Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium.

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2 + Fe2+----HO. + HO- + Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH+HO.----R. + H2O; R. + O2----RO2.----products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g.1-1), cellobiose oxidase, O2 and Fe(III) (30 microM). An addition of catalase after 20-30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.