Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Comparative toxicity of the horse eosinophil peroxidase-H2O2-halide system and granule basic proteins

Stimulated eosinophils release cytotoxic granule constituents, including eosinophil peroxidase (EPO) and a group of granule basic proteins (GBP). EPO reacts with H2O2 formed by the respiratory burst and a halide to form cytotoxic oxidants. The relative potency of the EPO-H2O2-halide system and the GBP is considered here. Horse eosinophils were induced to degranulate, the degranulation products were separated by chromatography on Sephadex G-50 and comparable volumes of the column fractions were tested for toxicity to Escherichia coli and the schistosomula of Schistosoma mansoni in the presence and absence of H2O2 and halides. Both the EPO system and GBP were toxic. However, the peak EPO fraction could be diluted 1000-fold at pH 7.0 and 5000-fold at pH 5.0, and with a 10-fold dilution at pH 7.0 incubation time could be reduced to 5 s, with retention of bactericidal activity in the presence of H2O2 and halides, whereas the peak GBP fractions diluted 10-fold had a small bactericidal effect at 1 h which increased with prolongation of incubation to 24 h. A less than 1 log fall in E. coli viable cell count was produced by the GBP fractions under all conditions as compared to total destruction (greater than 5 log fall) with the EPO system. A 1000-fold dilution of the peak EPO fraction was schistosomulocidal in the presence of H2O2 and halides, with toxicity observed at 2 h with a 10-fold dilution. In contrast, no schistosomulocidal activity was observed at 18 h with a 10-fold dilution of the GBP fractions. However, toxicity was observed with a 5- or 50-fold increase in GBP concentration with maximum toxicity observed with fractions between the two major protein peaks. Thus, under the conditions employed, the EPO-H2O2-halide system contributed to a considerably greater degree to the toxic activity of the granule components than did the GBP.     

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Leukotriene production and inactivation by normal, chronic granulomatous disease and myeloperoxidase-deficient neutrophils

Appropriately stimulated neutrophils release peroxidase and undergo a respiratory burst to form hydrogen peroxide (H2O2) and hydroxyl radicals (OH). We report here that both the myeloperoxidase-H2O2-halide system and OH released in this way can degrade the leukotrienes (LT) formed by neutrophils. More LTB4 and LTC4 were recovered from the supernatants of chronic granulomatous disease neutrophils (which are unable to respond to stimulation with a respiratory burst) than from normal or myeloperoxidase-deficient neutrophils when stimulated with the calcium ionophore A23187. When radiolabeled LTC4 was added, 72% of the LTC4 was recovered from the chronic granulomatous disease cells in contrast to 0% from the myeloperoxidase-deficient and normal cells. Inhibitor studies using catalase, superoxide dismutase, azide, mannitol, or ethanol suggested that LTC4 degradation was mediated primarily by the myeloperoxidase system in normal cells and by OH in myeloperoxidase-deficient cells. LTC4 degradation by the cell-free myeloperoxidase-H2O2-halide system and the OH -generating acetaldehyde-xanthine oxidase-Fe2+ system had inhibitor profiles comparable to normal and myeloperoxidase-deficient neutrophils, respectively. LTC4 degradation products formed by the stimulated neutrophils and model systems included the 5-(S), 12-(R)- and 5-(S), 12-(S)-6-trans-isomers of LTB4. Thus phagocytes may modulate LT activity in inflammatory sites by the inactivation of these potent biologic mediators by at least two oxidative mechanisms.     

Proton translocation and ATP formation coupled to electron transport from H2O to the primary acceptor of photosystem 2.

1. The rate of electron transport from H2O to silicomolybdate in the presence of 3-(3-4-dichlorophenyl)-1,1-dimethylurea (diuron) (which involves the oxygen-evolving enzyme, the photochemistry of photosystem 2 and the primary electron acceptor of photosystem 2) is controlled by internal pH. This is based on the shift of the pH profile of the rate of electron transport upon addition of uncouplers, or by using EDTA-treated chloroplasts. Both stimulation and inhibition of electron transport by addition of uncouplers (depending on external pH) could be observed. These effects are obtained in the diuron-insensitive photoreductions of either silicomolybdate or ferricyanide. These experiments provide strong evidence that a proton translocating site exists in the sequence of the electron transport H2O leads to Q (the primary acceptor of photosystem 2). 2. The photoreduction of silicomolybdate in the presence of diuron causes the formation of delta pH. The value of delta pH depends on the external pH and its maximal value was shown to be 2.4. The calculated internal pH at different external pH values was found to be rather constant, namely between 5.1 -- 5.2. 3. Electron transport from H2O to silicomolybdate (in the presence of diuron) does not support ATP formation. It is suggested that this is due to the fact that the delta pH formed is below the "threshold" delta pH required for the synthesis of ATP. By adding an additional source of energy in the form of a dark diffusion potential created in the presence of K+ and valinomycin, significant amounts of ATP are formed in this system.     

Mast cell-mediated toxicity to schistosomula of Schistosoma mansoni: potentiation by exogenous peroxidase

Mast cells, when incubated in vitro with hydrogen peroxide (H2O2) and iodide, are cytotoxic to schistosomula of Schistosoma mansoni, as determined morphologically by dye exclusion, motility, and refractility and by transmission and scanning electron microscopy. When intact mast cells were incubated with schistosomula, mast cell degranulation with extracellular release of mast cell granules (MCG) was only observed in the presence of added H2O2 (10(-4) M). The secreted MCG, which contain small amounts of endogenous peroxidase activity, adhered to the surface of schistosomula. By 15 to 30 min, the mast cell-H2O2 system in the presence of iodide (10(-4) M) produced marked disruption of the tegumental and internal structures of the schistosomula. No helminthic damage was noted if any component of the incubation mixture (mast cells, H2O2 or iodide) was omitted. MCG could substitute for intact mast cells in the H2O2 and iodide-dependent cytotoxic system; MCG-mediated killing of schistosomula was inhibited by the hemeprotein inhibitor azide, suggesting that the cytotoxic reaction required endogenous peroxidase. The cytotoxicity was increased by eosinophil peroxidase bound to the MCG surface. These findings suggest a mechanism by which mast cells may contribute to the host cytotoxic response to helminths. H2O2 formed by nearby inflammatory cells may induce mast cell secretion, and the released MCG, through their endogenous peroxidase content (or bound eosinophil or neutrophil peroxidase), may react with H2O2 and a halide to form a system toxic to the adjacent helminth.     

Myeloperoxidase reduces the opsonizing activity of immunoglobulin G and complement component C3b

The effect of myeloperoxidase, hydrogen peroxide (H2O2) and a halide (Cl) on the opsonizing molecules in immunoglobulin G (IgG) and complement factor C3b was assayed. At concentrations of the enzyme (1 microgram/ml) that can be found in the extracellular fluid during inflammation, the myeloperoxidase-H2O2-Cl system inhibited the opsonizing effect of IgG and C3b measured as phagocytic uptake and superoxide generation. The effect was related to the enzymatic peroxidative activity of the protein. The presence of albumin (10 mg/ml) reduced the effect of myeloperoxidase with 10-20%. Taurine, which in the presence of myeloperoxidase-H2O2-Cl forms hydrophilic chloramines, and D-penicillamine, which scavenges HOCl, neutralize the inhibitory effect of myeloperoxidase. This suggests that either hypochlorous acid or lipophilic chloramines may exert its effect by oxidizing free sulphydryl groups exposed on the opsonizing ligands. Since the myeloperoxidase-H2O2-halide system also affects chemotactic factors, leukotrienes, proteinases and membrane receptors, the system may in several ways affect the development of the inflammatory response.     

Myeloperoxidase-catalyzed taurine chlorination: initial versus equilibrium rate

Myeloperoxidase (MPO) catalyzes the two-electron oxidation of chloride, thereby producing hypochlorous acid (HOCl). Taurine (2-aminoethane-sulfonic acid, Tau) is thought to act as a trap of HOCl forming the long-lived oxidant monochlorotaurine [(N-Cl)-Tau], which participates in pathogen defense. Here, we amend and extend previous studies by following initial and equilibrium rate of formation of (N-Cl)-Tau mediated by MPO at pH 4.0-7.0, varying H(2)O(2) concentration. Initial rate studies show no saturation of the active site under assay conditions (i.e. [H(2)O(2)] > or = 2000 [MPO]). Deceleration of Tau chlorination under equilibrium is quantitatively described by the redox equilibrium established by H(2)O(2)-mediated reduction of compound I to compound II. At equilibrium regime the maximum chlorination rate is obtained at [H(2)O(2)] and pH values around 0.4mM and pH 5. The proposed mechanism includes known acid-base and binding equilibria taking place at the working conditions. Kinetic data ruled out the currently accepted mechanism in which a proton participates in the molecular step (MPO-I+Cl(-)) leading to the formation of the chlorinating agent. Results support the formation of a chlorinating compound I-Cl(-) complex (MPO-I-Cl) and/or of ClO(-), through the former or even independently of it. ClO(-) diffuses away and rapidly protonates to HOCl outside the heme pocket. Smaller substrates will be chlorinated inside the enzyme by MPO-I-Cl and outside by HOCl, whereas bulkier ones can only react with the latter.     

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.     

Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myeloperoxidase-induced formation of chlorohydrins and lysophospholipids from unsaturated phosphatidylcholines

The formation of lysophosphatidylcholines and chlorohydrins from unsaturated phosphatidylcholines upon the treatment with the myeloperoxidase-hydrogen peroxide-chloride system was evaluated by means of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Lyso-products were primarily found in phosphatidylcholine samples containing highly unsaturated fatty acid residues such as arachidonic or docosahexenoic acid. On the other hand, chlorohydrins dominate in mono- or bis-unsaturated phosphatidylcholines. No formation of these products was detected in the absence of one of the components of the MPO-H(2)O(2)-Cl(-) system or in the presence of MPO inhibitors (sodium azide) or scavengers of hypochlorous acid (taurine, methionine). Thus, hypochlorous acid formed by the MPO-H(2)O(2)-Cl(-) system is responsible for the observed modification in unsaturated phosphatidylcholines. In the presence of the complete MPO system, lyso-products and chlorohydrins were only formed at pH values lower than pH 6.0 with an optimum at pH 4.3. In contrast, the reagent hypochlorous acid caused the formation of these products even at neutral pH values, indicating a clear dependence of the yield of products on the presence of undissociated HOCl. We conclude that the formation of lysophospholipids and chlorohydrins from unsaturated phosphatidylcholines by myeloperoxidase can be relevant in vivo under acute inflammatory conditions.     

Kinetics of the reaction of superoxide anion with ferric horseradish peroxidase

Reaction of horseradish peroxidase A2 and C with superoxide anion (O2-) has been studied using pulse radiolysis technique. Peroxidase C formed Compound I and an oxy form of the enzyme due to reaction of ferric enzyme with hydrogen peroxide (H2O2) and O2-, respectively. At low concentrations of O2- (less than 1 mM), O2- reacted with ferric peroxidase C nearly quantitatively and formation of H2O2 was negligible. The rate constant for the reaction was found to be increased below pH 6 and this phenomenon can be explained by assuming that HO2 reacts with peroxidase C more rapidly than O2-. In contrast the formation of oxyperoxidase could not be detected in the case of peroxidase A2 after the pulse, and only Compound I of the enzyme was formed. Peroxidase A2, however, produced the oxy form upon aerobic addition of NADH, suggesting that O2- can also react with peroxidase A2 to form the oxy form. The results at present indicate that the rate constant for the reaction of O2- with peroxidase A2 is smaller than 103 M-1.s-1.     

Anthrax toxin receptor 2 determinants that dictate the pH threshold of toxin pore formation

The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH approximately 6.0 or below when it is bound to ANTXR1, but only at pH approximately 5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.     

The heme environment of ovoperoxidase as determined by optical spectroscopy

Native ovoperoxidase exhibited an optical absorption spectrum with certain similarities to lactoperoxidase, but not horseradish peroxidase, over the pH range 4.5-11.5. Ovoperoxidase had three distinct spectral forms dependent on pH, with transitions at apparent pKa values of 6.6 and 3.0. Complexes of ovoperoxidase with CN-, N3-, F-, or when reduced and ligated to carbon monoxide, CN-, or pyridine, were distinct from other peroxidases. Ovoperoxidase formed two specific and different spectral derivatives at pH 6.0 and 8.0, either in the native state, or when combined with CN-, when reduced, or when reduced and ligated to CN-. The position of the Soret band when mixed with near-stoichiometric amounts of H2O2. This cycling was inhibited by phenylhydrazine, 3-amino-1,2,4-triazole, or low pH (less than or equal to 6). Compound II was formed when ovoperoxidase was mixed with ethyl hydrogen peroxide in a 1:3 ratio, but not with H2O2. With a great excess of H2O2, Compound III was formed at pH 8.0; at pH 6.0 or below, the Soret band shifted slightly with excess of H2O2, but Compound III was never formed. Even when ovoperoxidase was bound to proteoliaisin (Weidman, P. J., and Shapiro, B. M. (1987) J. Cell Biol. 105, 561-567), ovoperoxidase exhibited spectral characteristics of the free enzyme.     

Hydroxyl free radical production by the reaction of N-methyl-N'-nitro-N-nitrosoguanidine with hydrogen peroxide without exposure to light.

We examined hydroxyl free radical (.OH) production in the mixture of H2O2 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without exposure to light using the electron spin resonance spin-trapping technique. When the mixtures were protected from exposure to light, .OH was formed at pH 6.5 and above; it was not formed at pH 5.0 and below, consistent with our previous report. The amount of .OH trapped depended on the concentrations of MNNG and H2O2 and the pH. Nitrite ion was also detected colorimetrically at pH 6.5 and above, but not detected at pH 5.0 and below in the mixtures without exposure to light. Moreover, its production depended on the concentrations of MNNG and H2O2 and the pH. The formation of N-methyl-N'-nitroguanidine in the mixture at pH 7.8 was confirmed by thin-layer chromatography and melting point. These results suggest that nucleophilic attack by H2O2 on the nitroso nitrogen of MNNG results in the formation of N-methyl-N'-nitroguanidine and peroxynitrous acid, which degrades homolytically to yield .OH and nitrogen dioxide, resulting in the production of nitrite ion, at pH 6.5 and above without exposure to light.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Formation and morphology of struvite and newberyite in aqueous solutions at 25 and 37 degrees C

The influence of the initial reactant concentrations (c(i)(Mg)tot = 5.0 x 10(6) to 5.0 x 10(-1) mol dm(-3), c(i)(P)tot = c(i)(NH4)tot = 1.0 x 10(-3) to 5.0 x 10(-1) mol dm(-3)) and temperature (25 and 37 degrees C) on the composition and morphology of the precipitates formed in the system MgCl2-NH4H2PO4-NaOH-H2O at initial pHi = 7.40 has been investigated. Precipitation diagrams are presented showing the concentration regions within which different morphologies of solid phase have been formed. The solid phases aged for 24 hours were characterized by means of optical microscopy, FT-IR spectrophotometry, X-ray diffractometry and thermogravimetry. It was found that struvite was a predominant phase formed within the concentration region examined and newberyite was obtained only in the region where pH(24h) < 6.5. The influence of the initial pH on the formation and transformation of these two compounds were studied in the region 5.0 < or = pHi < or = 9.0 and the results are discussed.     

Hydroxylation and deglycosylation of 2'-deoxyguanosine in the presence of magnesium and nickel

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2'-deoxyguanosine (dG) that has been caused by ascorbic acid and H2O2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H2O2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H2O2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2-7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2'-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.     

Influence of chloride on modification of unsaturated phosphatidylcholines by the myeloperoxidase/hydrogen peroxide/bromide system

The leukocyte enzyme myeloperoxidase (MPO) is capable of catalyzing the oxidation of chloride and bromide ions, at physiological concentrations of these substrates, by hydrogen peroxide, generating hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively. Our previous results showed that the hypohalous acids formed react with double bonds in phosphatidylcholines (PCs) to produce chloro- and bromohydrins. Lysophosphatidylcholine (lyso-PC) is additionally formed in PCs with two or more double bonds. This study was conducted to determine the effect physiological chloride concentration (140 mM) has on the formation of bromohydrins and lyso-PC from unsaturated PC upon treatment with the myeloperoxidase/hydrogen peroxide/bromide (MPO/H2O2/Br-) system using physiological bromide concentrations (20-100 microM). The composition of reaction products was analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). With monounsaturated PC, we demonstrated that the rate and extent of mono-bromohydrin formation were higher in the samples with 140 mM chloride compared to those with no added chloride. Moreover, mono-bromohydrin came to be the major product and no mono-chlorohydrin was observed already at 60 microM bromide. We attributed these effects to the involvement of HOBr arising from the reaction of MPO-derived HOCl with bromide rather than to the exchange of bromide with chlorine atoms of chlorohydrins or direct formation of HOBr by MPO. The presence of chloride shifted the pH optimum for mono-bromohydrin formation (pH 5.0) toward neutral values, and a significant yield of mono-bromohydrin was detected at physiological pH values (7.0-7.4). For polyunsaturated PC, chloride enhanced also lyso-PC production, the effect being pronounced at bromide concentrations below 40 microM. The results indicate that at physiological levels of chloride and bromide, chloride promotes MPO-mediated formation of bromohydrins and lyso-PC in unsaturated phospholipids.