James K. Hurst

Relative Chlorinating, Nitrating, and Oxidizing Capabilities of Neutrophils Determined with Phagocytosable Probes

The capabilities of stimulated neutrophils to initiate intraphagosomal and extracellular chlorination, nitration, and other oxidative reactions has been evaluated using a fluorescent particle and soluble phenolic compounds as target molecules. Neutrophils activated by the soluble stimulus, phorbol myristate acetate, both chlorinated fluorescein that was covalently attached to polyacrylamide microspheres and initiated tyrosine dimerization. When nitrite ion was present at millimolar concentration levels in the medium, nitration of the phenolic rings also occurred; the relative extent of nitration increased as the nitrite concentration was increased. Myeloperoxidase (MPO) also catalyzed nitration and chlorination of fluorescein and the fluorescein-conjugated particles in cell-free solutions; the relative nitration yields increased with increasing [NO2 −]/[Cl−] ratios. Nitration did not involve intermediary formation of nitrating agents derived from reaction between MPO-generated HOCl and NO2 − because this reaction also occurred in chloride-free media and direct addition of HOCl to solutions containing NO2 − and fluorescein gave only chlorinated products. In marked contrast to these extracellular reactions, intraphagosomal nitration of the fluorescein-conjugated particles could not be detected (even at [NO2 −] as high as 0.1m), whereas chlorination of the probe was extensive. These data indicate that intraphagosomal aromatic nitration in neutrophils is negligible, although extracellular nitration of phenolic compounds by secreted MPO could occur at physiological concentration levels of NO2 −.

What really happens in the neutrophil phagosome

Current viewpoints concerning the bactericidal mechanisms of neutrophils are reviewed from a perspective that emphasizes challenges presented by the inability to duplicate ex vivo the intracellular milieu. Among the challenges considered are the influences of confinement upon substrate availability and reaction dynamics, direct and indirect synergistic interactions between individual toxins, and bacterial responses to stressors. Approaches to gauging relative contributions of various oxidative and nonoxidative toxins within neutrophils using bacteria and bacterial mimics as intrinsic probes are also discussed.

Cyclic transmembrane charge transport by pyrylium ions in a vesicle-based photocatalytic system

Solar energy is being used for power generation, but also attracts increasing interest as a renewable energy source for the photocatalytic production of useful chemicals. Simple systems based on vesicles with transmembrane redox mediators have been used to transform photon energy into long-lived, membrane-separated photoredox products. However, these systems are not suitable for high-throughput applications because the transmembrane electron carriers are oxidized inside the vesicle into charged species that are no longer able to readily traverse the membrane bilayer. This leads to continuous trapping of these carriers during photolysis and, ultimately, to the termination of the redox reaction due to accumulation of the available carriers within the vesicle interior. Living cells circumvent this problem by using quinones to simultaneously transport electrons and protons, thus allowing the carrier to remain neutral in its reduced and oxidized states and so retain the ability to undergo transmembrane diffusion throughout the redox cycle. But the incorporation of quinones into artificial systems is not practical because of their susceptibility to oxidative degradation and slow transmembrane diffusion. Here we describe an alternative mechanism for rapid electroneutral charge transport across vesicle membranes: we use pyrylium cations as the electron carrier, which undergo reversible ring-opening hydrolysis to form neutral diketones after deposition of the electron inside the vesicle. As the pyrylium cations are also the primary acceptors for the photoproduced electrons, our approach greatly simplifies the design of vesicle-based photocatalytic devices.

Bactericidal properties of hydrogen peroxide and copper or iron-containing complex ions in relation to leukocyte function

Various combinations of hydrogen peroxide, reductant (ascorbic acid and superoxide ion), and copper or iron salts and their coordination complexes were examined to determine their cytotoxicity toward several bacteria with diverse metabolic capabilities and cell envelope structures. Four sets of bactericidal conditions were identified, comprising: (1) high concentration levels (5-100 mM) of H2O2 in the absence of exogenous metal ions and reductant; (2) ferrous or ferric coordination complexes plus enzymatically generated O2.- and H2O2 at relatively low steady-state concentration levels; (3) cupric ion plus low concentration levels of H2O2 (1 microM-1 mM) and ascorbate (10 microM-4 mM); (4) cuprous ion (or cupric ion plus ascorbate) in the absence of O2 and H2O2. Rates of losses in viabilities increased proportionately with increases in the concentration of H2O2 in metal-free environments and with each of the components in the Cu2+/ascorbate/H2O2 bactericidal assay system. Oxidant levels required for equivalent killing increased with increasing cell densities of the bacterial suspensions over the range investigated (2 x 10(7)-2 x 10(9) cfu/ml). Other experimental conditions or other combinations of reagents, most notably Fe3+/ascorbate/H2O2 systems, did not generate bactericidal environments. The patterns of response of the three organisms tested, Streptococcus lactis, Escherichia coli, and Pseudomonas aeruginosa, were similar, suggesting common bactericidal mechanisms. However, preliminary evidence suggests that the lethal lesions caused by the various bactericidal conditions are distinct: As discussed, each of the four bactericidal conditions could conceivably be attained within the phagosomes of leukocytes, although none has as yet been identified.

Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking.

Cyclic transmembrane charge transport mediated by low-potential pyrylium ions.

We have investigated the capacity of a series of N-dialkylaminophenyl-substituted pyrylium and thiopyrylium ions to act as photosensitizers and redox mediators between reactants separated by bilayer membranes. These studies were prompted by earlier results indicating that simple trimethy- and triphenyl-substituted analogues could promote efficient photosensitized transmembrane redox between vectorially organized reactants by an electroneutral e(-)/OH(-) antiport mechanism. Unlike the dyes used in the earlier studies, the ions investigated herein absorb strongly throughout the visible absorption region and are therefore potentially useful in solar photoconversion processes. We demonstrate that these ions can carry out cyclic electron transport between phase-separated electron donors and occluded Co(bpy)(3)(3+) in several transversely organized vesicles. The quantum yields obtained were relatively low, but were independent of the membrane microviscosity, suggesting that transmembrane diffusion was not rate-limiting. Triphenylpyrylium and triphenylthiopyrylium ions were shown to be capable of acting as combined photosensitizers/redox relays, apparently by direct oxidation of either solvent (water) or buffer (acetate) ions from their triplet-excited state. These reactions did not require addition of separate photosensitizers and electron donors; as such, they represent a minimal photochemical scheme for effecting transmembrane charge separation. The low-potential visible-absorbing pyrylium ions were unable to function in this dual capacity, consistent with thermodynamic limitations. However, redox titrations established that the pyranyl radicals of these dyes should be capable of reducing H(+) to H(2) in weakly acidic solutions. Consistent with their strongly reducing nature, these dyes were shown to be capable of forming methyl viologen radical in photoinitiated transmembrane redox reactions.

Mechanistic Insight into Peroxydisulfate Reactivity: Oxidation of the cis,cis-[Ru(bpy)₂(OH₂)]₂O⁴⁺ "Blue Dimer"

One-electron oxidation of the μ-oxo dimer (cis,cis-[Ru(III)(bpy)2(OH2)]2O(4+), {3,3}) to {3,4} by S2O8(2-) can be described by three concurrent reaction pathways corresponding to the three protic forms of {3,3}. Free energy correlations of the rate constants, transient species dynamics determined by pulse radiolysis, and medium and temperature dependencies of the alkaline pathway all suggest that the rate-determining step in these reactions is a strongly nonadiabatic dissociative electron transfer within a precursor ion pair leading to the {3,4}|SO4(2-)|SO4(•-) ion triple. As deduced from the SO4(•-) scavenging experiments with 2-propanol, the SO4(•-) radical then either oxidizes {3,4} to {4,4} within the ion triple, effecting a net two-electron oxidation of {3,3}, or escapes in solution with ∼25% probability to react with additional {3,3} and {3,4}, that is, effecting sequential one-electron oxidations. The reaction model presented also invokes rapid {3,3} + {4,4} → 2{3,4} comproportionation, for which kcom ∼5 × 10(7) M(-1) s(-1) was independently measured. The model provides an explanation for the observation that, despite favorable energetics, no oxidation beyond the {3,4} state was detected. The indiscriminate nature of oxidation by SO4(•-) indicates that its fate must be quantitatively determined when using S2O8(2-) as an oxidant.

Oxidative Release of Copper from Pharmacologic Copper Bis(thiosemicarbazonato) Compounds

Intracellular delivery of therapeutic or analytic copper from copper bis-thiosemicabazonato complexes is generally described in terms of mechanisms involving one-electron reduction to the Cu(I) analogue by endogenous reductants, thereby rendering the metal ion labile and less strongly coordinating to the bis-thiosemicarbazone (btsc) ligand. However, electrochemical and spectroscopic studies described herein indicate that one-electron oxidation of CuII(btsc) and ZnIIATSM (btsc = diacetyl-bis(4-methylthiosemicarbazonato)) complexes occurs within the range of physiological oxidants, leading to the likelihood that unrecognized oxidative pathways for copper release also exist. Oxidations of CuII(btsc) by H2O2 catalyzed by either myeloperoxidase or horseradish peroxidase, by HOCl and taurine chloramine (which are chlorinating agents generated primarily in activated neutrophils from MPO-catalyzed reactions), and by peroxynitrite species (ONOOH, ONOOCO2-) that can form under certain conditions of oxidative stress are demonstrated. Unlike reduction, the oxidative reactions proceed by irreversible ligand oxidation, culminating in release of Cu(II). 2-Pyridylazoresorcinol complexation was used to demonstrate that Cu(II) release by reaction with peroxynitrite species involved rate-limiting homolysis of the peroxy O-O bond to generate secondary oxidizing radicals (NO2•, •OH, and CO3•-). Because the potentials for CuII(btsc) oxidation and reduction are ligand-dependent, varying by as much as 200 mV, it is clearly advantageous in designing therapeutic methodologies for specific treatments to identify the operative Cu-release pathway.

Cation-regulated viologen-mediated transmembrane electron transfer

A chemically switchable microheterogeneous redox system exhibiting vectorial charge transfer has been developed by asymmetrically organizing viologens across dihexadecylphosphate vesicle bilayers.

1-Carboxyethyl-4-cyanopyridinium-mediated photoinduced electron-proton cotransport across phosphatidylcholine vesicle membranes

Abstract Asymmetrically organized egg lecithin small unilamellar vesicles containing an electron acceptor (Co(bpy)33+) in the internal aqueous phase with a photosensitizer (ZnTPPS4−) and electron donor (dithiothreitol) in the external medium were prepared. When 1-carboxyethyl-4-cyanopyridinium ions were also present in the medium, illumination with visible light gave rise to net transmembrane oxidation–reduction; however, no transmembrane redox reaction was observed when this ion was absent. Transient spectroscopy revealed that the pyridinium derivative oxidatively quenched the 3ZnTPPS4− photoexcited ion, forming the neutral pyridinium radical, which subsequently diffused across the bilayer to reduce the occluded Co(bpy)33+ ion. Quenching rate constants in 20 mM Tris, pH 3.3, at room temperature were 4.5×109 M−1 s−1 and 2.1×109 M−1 s−1 in the absence and presence of vesicles, respectively; the characteristic time for transmembrane diffusion of the radical was 30 ms. Under continuous illumination, at least seven internal Co(bpy)33+ ions could be reduced to Co(bpy)32+ for each 1-carboxyethyl-4-cyanopyridinium ion present, implying that the redox mediator had cycled on average seven times across the membrane. Transmembrane reduction rates increased when the vesicle interior was made alkaline, consistent with the formally neutral 1-carboxyethyl-4-cyanopyridinium zwitterion being the diffusible form of the oxidized carrier. The data support a transmembrane transport model involving stepwise photosensitized reduction of the carrier, transmembrane transport of the neutral radial, reoxidation accompanied by proton release from the pendant carboxyl group, and diffusion of the oxidized zwitterion to the external medium with subsequent proton uptake to close the cycle.