Michael T. Ashby

Myeloperoxidase-induced genomic DNA-centered radicals

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.

Catabolite Control Protein A Controls Hydrogen Peroxide Production and Cell Death in Streptococcus sanguinis

Streptococcus sanguinis is a commensal oral bacterium producing hydrogen peroxide (H₂O₂) that is dependent on pyruvate oxidase (Spx) activity. In addition to its well-known role in bacterial antagonism during interspecies competition, H₂O₂ causes cell death in about 10% of the S. sanguinis population. As a consequence of H₂O₂-induced cell death, largely intact chromosomal DNA is released into the environment. This extracellular DNA (eDNA) contributes to the self-aggregation phenotype under aerobic conditions. To further investigate the regulation of spx gene expression, we assessed the role of catabolite control protein A (CcpA) in spx expression control. We report here that CcpA represses spx expression. An isogenic ΔccpA mutant showed elevated spx expression, increased Spx abundance, and H₂O₂ production, whereas the wild type did not respond with altered spx expression in the presence of glucose and other carbohydrates. Since H₂O₂ is directly involved in the release of eDNA and bacterial cell death, the presented data suggest that CcpA is a central control element in this important developmental process in S. sanguinis.

Influence of a model human defensive peroxidase system on oral streptococcal antagonism

Streptococcus is a dominant genus in the human oral cavity, making up about 20 % of the more than 800 species of bacteria that have been identified, and about 80 % of the early biofilm colonizers. Oral streptococci include both health-compatible (e.g. Streptococcus gordonii and Streptococcus sanguinis) and pathogenic strains (e.g. the cariogenic Streptococcus mutans). Because the streptococci have similar metabolic requirements, they have developed defence strategies that lead to antagonism (also known as bacterial interference). S. mutans expresses bacteriocins that are cytotoxic toward S. gordonii and S. sanguinis, whereas S. gordonii and S. sanguinis differentially produce H(2)O(2) (under aerobic growth conditions), which is relatively toxic toward S. mutans. Superimposed on the inter-bacterial combat are the effects of the host defensive mechanisms. We report here on the multifarious effects of bovine lactoperoxidase (bLPO) on the antagonism between S. gordonii and S. sanguinis versus S. mutans. Some of the effects are apparently counterproductive with respect to maintaining a health-compatible population of streptococci. For example, the bLPO system (comprised of bLPO+SCN(-)+H(2)O(2)) destroys H(2)O(2), thereby abolishing the ability of S. gordonii and S. sanguinis to inhibit the growth of S. mutans. Furthermore, bLPO protein (with or without its substrate) inhibits bacterial growth in a biofilm assay, but sucrose negates the inhibitory effects of the bLPO protein, thereby facilitating adherence of S. mutans in lieu of S. gordonii and S. sanguinis. Our findings may be relevant to environmental pressures that select early supragingival colonizers.

Small molecular, macromolecular, and cellular chloramines react with thiocyanate to give the human defense factor hypothiocyanite.

Thiocyanate reacts noncatalytically with myeloperoxidase-derived HOCl to produce hypothiocyanite (OSCN(-)), thereby potentially limiting the propensity of HOCl to inflict host tissue damage that can lead to inflammatory diseases. However, the efficiency with which SCN(-) captures HOCl in vivo depends on the concentration of SCN(-) relative to other chemical targets. In blood plasma, where the concentration of SCN(-) is relatively low, proteins may be the principal initial targets of HOCl, and chloramines are a significant product. Chloramines eventually decompose to irreversibly damage proteins. In the present study, we demonstrate that SCN(-) reacts efficiently with chloramines in small molecules, in proteins, and in Escherichia coli cells to give OSCN(-) and the parent amine. Remarkably, OSCN(-) reacts faster than SCN(-) with chloramines. These reactions of SCN(-) and OSCN(-) with chloramines may repair some of the damage that is inflicted on protein amines by HOCl. Our observations are further evidence for the importance of secondary reactions during the redox cascades that are associated with oxidative stress by hypohalous acids.

Hypochlorous Acid Reacts with the N-Terminal Methionines of Proteins to Give Dehydromethionine, a Potential Biomarker for Neutrophil-Induced Oxidative Stress

Electrophilic halogenating agents, including hypohalous acids and haloamines, oxidize free methionine and the N-terminal methionines of peptides and proteins (e.g., Met-1 of anti-inflammatory peptide 1 and ubiquitin) to produce dehydromethionine (a five-membered isothiazolidinium heterocycle). Amide derivatives of methionine are oxidized to the corresponding sulfoxide derivatives under the same reaction conditions (e.g., Met-3 of anti-inflammatory peptide 1). Other biological oxidants, including hydrogen peroxide and peroxynitrite, also produce only the corresponding sulfoxides. Hypothiocyanite does not react with methionine residues. We suggest that dehydromethionine may be a useful biomarker for the myeloperoxidase-induced oxidative stress associated with many inflammatory diseases.

Chapter 8 – Hypothiocyanite

Thiocyanate (SCN−) is the usual substrate for the human defensive peroxidases (components of human innate defense that include lactoperoxidase, salivary peroxidase, myeloperoxidase, and eosinophil peroxidase). The initial product of the peroxidase-catalyzed oxidation of SCN− by hydrogen peroxide is the antimicrobial agent hypothiocyanite (OSCN−). Stoichiometric methods of preparing OSCN− are also known. This review addresses the synthesis and chemistry of OSCN− and its derivatives in general, with particular attention paid to reactions that may be biologically relevant and to the evidence for putative biological intermediates. As SCN− is considered to be a pseudohalide in several aspects of reactivity, the inorganic chemistry of OSCN− is, not surprisingly, related to the corresponding hypohalites, albeit complicated by reactions of the cyano moiety of OSCN−.

Molecular Structure and Dynamic Properties of a Sulfonamide Derivative of Glutathione That Is Produced Under Conditions of Oxidative Stress by Hypochlorous Acid

Reduced glutathione (GSH) is a cornerstone of the antioxidant stratagem for eukaryotes and some prokaryotes. Hypochlorous acid (HOCl), which is produced by neutrophilic myeloperoxidase, reacts rapidly with excess GSH to yield mainly oxidized glutathione (GSSG). GSSG can be further oxidized to give first N-chloro derivatives and, later, higher oxidation states at the S centers. Under certain conditions, another major species that is observed during the oxidation of GSH by HOCl (and a minor species for other oxidants) exhibits a molecular mass that is 30 mass units heavier than GSH. This GSH+2O-2H species, which has been employed as a biomarker for oxidative stress, has been previously proposed to be a sulfonamide. Employing NMR spectroscopy and mass spectrometry, we demonstrate that the GSH+2O-2H species is indeed a nine-membered cyclic sulfonamide. Alternative formulations, including six-membered 1,2,5-oxathiazine heterocycles, have been ruled out. Remarkably, the sulfonamide exists as a 2:1 equilibrium mixture of two diastereomers. Isotope tracer studies have demonstrated that it is the Glu C alpha center that has undergone racemization. It is proposed that the racemization takes place via an acyclic imine-sulfinic acid intermediate. The glutathione sulfonamides are stable products of GSH that have been detected in physiological systems. Elucidation of the structures of the glutathione sulfonamides provides further impetus to explore their potential as biomarkers of hypochlorous acid formation.

Mechanism of Decomposition of the Human Defense Factor Hypothiocyanite Near Physiological pH

Relatively little is known about the reaction chemistry of the human defense factor hypothiocyanite (OSCN(-)) and its conjugate acid hypothiocyanous acid (HOSCN), in part because of their instability in aqueous solutions. Herein we report that HOSCN/OSCN(-) can engage in a cascade of pH- and concentration-dependent comproportionation, disproportionation, and hydrolysis reactions that control its stability in water. On the basis of reaction kinetic, spectroscopic, and chromatographic methods, a detailed mechanism is proposed for the decomposition of HOSCN/OSCN(-) in the range of pH 4-7 to eventually give simple inorganic anions including CN(-), OCN(-), SCN(-), SO(3)(2-), and SO(4)(2-). Thiocyanogen ((SCN)(2)) is proposed to be a key intermediate in the hydrolysis; and the facile reaction of (SCN)(2) with OSCN(-) to give NCS(═O)SCN, a previously unknown reactive sulfur species, has been independently investigated. The mechanism of the aqueous decomposition of (SCN)(2) around pH 4 is also reported. The resulting mechanistic models for the decomposition of HOSCN and (SCN)(2) address previous empirical observations, including the facts that the presence of SCN(-) and/or (SCN)(2) decreases the stability of HOSCN/OSCN(-), that radioisotopic labeling provided evidence that under physiological conditions decomposing OSCN(-) is not in equilibrium with (SCN)(2) and SCN(-), and that the hydrolysis of (SCN)(2) near neutral pH does not produce OSCN(-). Accordingly, we demonstrate that, during the human peroxidase-catalyzed oxidation of SCN(-), (SCN)(2) cannot be the precursor of the OSCN(-) that is produced.

Reactive Sulfur Species: Kinetics and Mechanism of the Equilibrium between Cysteine Sulfenyl Thiocyanate and Cysteine Thiosulfinate Ester in Acidic Aqueous Solution

The kinetics and mechanism of the hydrolysis of cysteine sulfenyl thiocyanate (CySSCN) to give cysteine thiosulfinate ester (CyS(=O)SCy) have been investigated between pH 0 and 4. The reaction is reversible. The hydrolysis of CySSCN is second-order in [CySSCN] and inverse first-order in [H+] and [SCN-]. The following mechanism is proposed for the hydrolysis of CySSCN (where the charge depends upon the pH): CySSCN0/+ + H2O <==>CySOH0/+ + SCN- + H+, CySOH0/+ + CySSCN0/+ --> CyS(=O)SCy0/+/2+ + SCN- + H+; k1 = 3.36 +/- 0.01 x 10-3 s-1, K1k2 = 0.13 +/- 0.05 Ms-1 (which yields k2/k-1 = 39 M). The observed rate law rules out alternative mechanisms for 1 <or= pH <or= 4, including the condensation of two molecules of cysteine sulfenic acid (CySOH) to give CyS(=O)SCy. The equilibrium can be approached from the opposite direction. The reaction of CyS(=O)SCy with SCN- to give CySSCN is first-order in [CyS(=O)SCy], [SCN-], and [H+] (for [H+] > 0.4 M). The following mechanism is proposed: CyS(=O)SCy2+ + H+ <==> CyS(OH)=SCy3+, Ka; CyS(OH)SCy3+ + SCN- --> CySOH+ + CySSCN+, k-2 = 0.239 +/- 0.007 M-2s-1/Ka M-1. Since cysteine sulfenic acids are known to play an important function in many enzymes, and SCN- exists in abundance in physiologic fluids, we discuss the possible role of sulfenyl thiocyanates in vivo.

AqpZ-Mediated Water Permeability in Escherichia coli Measured by Stopped-Flow Spectroscopy

We report that the water permeability of wild-type Escherichia coli during exponential growth is comparable to that of an aqpZ disruption mutant. In contrast, an increase in permeability is observed for the wild type at the onset of the stationary stage with no significant corresponding change in the permeability of the mutant.