M. Margaret Jefferson

Preparation and Characterization of Chloramines

Publisher Summary This chapter discusses the preparation and characterization of chloramines. Chloramines represent an important class of leukocyte oxidants and contribute to oxidative microbicidal, cytotoxic and cytolytic activities, the chemical modification of regulatory substances, and the uptake and metabolism of nitrogen compounds. These are products of the reaction of hypochlorous acid (HOCl) or other chlorinating agents with primary and secondary amines. The production of HOCl by leukocytes is the result of peroxidase-catalyzed oxidation of chloride (Cl – ) by hydrogen peroxide (H 2 O 2 ). HOCl is in equilibrium with hypochlorite (OCl – ) and chlorine (Cl 2 ). The most abundant N-moieties available for reaction with HOCl in biological systems are primary amino groups, such as taurine, polyamines, amino sugars, lysine residues, and protein amino termini. Leukocyte peroxidases catalyze the oxidation of bromide (Br – ), iodide (I – ), thiocyanate (SCN – ), and Cl – . N–Cl derivatives are used within minutes of preparation to avoid decomposition or rearrangement. However, most are stable at 4° for hours or days at the appropriate pH.

Cytotoxicity of Chloramines

Publisher Summary This chapter describes the cytotoxicity of chloramines. The binding of ligands to the membrane-surface receptors of neutrophilic leukocytes stimulates the reduction of oxygen (O 2 ) to superoxide (O 2 – ) and the secretion of cytoplasmic granule components including myeloperoxidase into the intracellular phagolysosome compartment and the extracellular medium. When neutrophils are incubated with target cells, nitrogen–chlorine (N–Cl) derivatives react with both types of cells so that results are influenced by the neutrophil:target cell ratio and the reactivity of N–Cl derivatives toward neutrophil and target cell components. Methods for evaluating the cytotoxicity of N–Cl derivatives consist of measuring oxidation, chlorination, or other chemical modifications of cell components and measuring lysis or the failure of specific structural or functional components of cells. Stimulated neutrophils undergo a progressive myeloperoxidase-dependent inhibition of O 2 and glucose metabolism. This inhibition results from the reaction of hypochlorous acid (HOCl) with ammonium (NH 4 + ) produced by the cells. Monochloramine (NH 2 Cl) is produced as a result and inactivates neutrophil components.

SECRETION AND INACTIVATION OF MYELOPEROXIDASE BY ISOLATED NEUTROPHILS

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues. Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the medium. Cells stimulated with phorbol myristate acetate (PMA) or opsonized zymosan particles secreted MPO and released superoxide free radicals (·O2 ‐). Dismutation of ·O2 ‐ produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the toxic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dismutase (SOD) to increase the rate of conversion of ·O2 ‐ to H2O2 had pH‐dependent effects on HOCl production. From pH 6.0 to 7.4, SOD promoted HOCl production by up to 500% but SOD had no effect at pH 7.6 and inhibited by 40 ± 10% at pH 7.8. In further experiments at pH 7.0, MPO activity in the cells decreased by 25 ± 2 and 44 ± 4% during 1‐h incubations with PMA and zymosan. Only 1 ± 0 and 3 ± 1% of the total activity was found in the medium, indicating that most of the secreted MPO was inactivated. Loss of activity was not accompanied by proteolytic destruction of the MPO protein, which was measured with anti‐MPO antibodies. SOD raised the amount of active MPO in the medium two‐ to sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect. The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but ·O2 ‐ production by ionophore‐stimulated cells was less than 4% of that of PMA‐ or zymosan‐stimulated cells and most of the secreted MPO was found active in the medium. When PMA‐stimulated cells were incubated with purified MPO, the added MPO activity was lost from the medium. Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium. The visible absorption spectrum of MPO was lost, indicating destruction of the iron‐containing prosthetic group. Loss of activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiological pH range, inactivation of MPO in the medium suppressed HOCl production. Inactivation required ·O2 ‐ but not HOCl, H2O2, or free iron. Inactivation of secreted MPO may limit MPO‐mediated damage to host tissues by stimulated neutrophils. J. Leukoc. Biol. 61: 293–302; 1997.

Hypothiocyanite Ion: Detection of the Antimicrobial Agent in Human Saliva

The median concentration of hypothiocyanite (OSCN-) in freshly collected whole saliva was 10 μM. The OSCN- concentration increased to a median value of 36 μM during incubation for one h at 37° in vitro. This increase was partially inhibited by adding certain sugars (especially sucrose). The results suggest that OSCN- is a naturally occurring component of human saliva. Also, dietary carbohydrate may inhibit OSCN- accumulation and antimicrobial action in saliva.

Peroxidase Antimicrobial System of Human Saliva: Requirements for Accumulation of Hypothiocyanite

Human saliva was fractionated to determine the components required for production and accumulation of the antimicrobial oxidizing agent, hypothiocyanite ion (OSCN-). The required components were: 1) peroxidase activity and thiocyanate ion (SCN -), 2) the saliva sediment, which produced hydrogen peroxide (H 2O2) in the presence of oxygen and a divalent cation, and 3) heatstable factors of the saliva supernatant. The supernatant factors were separated into high- and low-mol wt fractions. The high-mol wt fraction contained both peptide and carbohydrate, and its activity was partially inhibited by proteolytic treatment. The low-mol wt fraction contained carbohydrate and could be replaced by a number of mono- and di-saccharides. Glucosamine and N-acetyl glucosamine were the most effective, whereas neutral sugars such as sucrose were less effective. Sucrose competed with glucosamine, so that lower levels of OSCN- were obtained with increasing amounts of sucrose. The sugars stimulated production of H202 by the saliva sediment. Production of H202 was greater in the presence of glucosamine than of neutral sugars. Also, the ratio of OSCN- accumulation to H202 production was greater in the presence of glucosamine. The results suggest that peroxidase-mediated antimicrobial activity is modulated by the carbohydrate composition of whole saliva and by certain protein and glycoprotein components.

Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils

Abstract To investigate neutrophil interactions with mediators released by mast cells at sites of inflammation, stimulated neutrophils were incubated with histamine. No accumulation of chlorinated histamine derivatives was detected in the medium. Instead, histamine inhibited the formation of chloramine derivatives of other amines. Incubation with radiolabeled histamine resulted in rapid uptake of label into the cells, and most of the label could be extracted and recovered as histamine. About 3% of the label taken up was incorporated into acid-precipitable forms. Uptake depended on myeloperoxidase (MPO)-catalyzed formation of chlorinating agents. Uptake was promoted by adding MPO and blocked by the MPO inhibitor dapsone, catalase, scavengers for hypochlorous acid and chloramines, or in a low-chloride medium, but not by histamine receptor antagonists. Incubation of histamine with MPO, hydrogen peroxide, and chloride resulted in formation of mono- and di-chloramine derivatives of the primary amino group. Above pH 7.0, the chloramines were primarily in uncharged, lipophilic forms as indicated by partitioning into organic solvents. Histamine is a cation at neutral pH, but chlorination eliminated the charge on the amino group and shifted the pKa of the imidazole ring, resulting in formation of neutral histamine-chloramines. Incubation of neutrophils or other blood cells with radiolabeled histamine-chloramines resulted in rapid uptake of label, indicating membrane permeation by the uncharged, lipid-soluble forms. Incubation with labeled histamine-dichloramine also resulted in acid-precipitable incorporation. The results indicate that MPO-catalyzed chlorination of histamine could modulate histamine activity, tissue distribution, and metabolism at sites of inflammation.

Biomarkers of metastatic potential in cultured adenocarcinoma clones

Two-dimensional isoelectric focusing and gel electrophoresis followed by mass spectrometry were used to detect, measure, and identify changes in protein expression correlated with differences in the metastatic potential of cultured rat mammary adenocarcinoma cells. MTC is a non-metastatic cell clone derived from a primary tumor. MTLn2 and MTLn3 are low and high metastatic potential cell clones derived from lung metastases of the primary tumor. A total of 1,500 proteins was detected. The patterns of protein expression of MTLn2 and MTLn3 cells were similar. Only five spots had a threefold or greater statistically significant difference in staining intensity between MTLn2 and MTLn3 cells, whereas 70 spots differed between MTC and MTLn3 cells. Twenty spots were selected for further study, ten that had a positive correlation of staining intensity with metastatic potential and ten that had a negative (inverse) correlation. Of the 17 unique proteins that were identified, five have often been cited as tumor biomarkers. These included the positive biomarkers nucleophosmin (NPM) and 14-3-3 protein sigma and the negative biomarkers raf kinase inhibitor protein (RKIP), peroxiredoxin-2, and galectin-1. The only identified protein that was markedly higher in MTLn3 cells than in the less-metastatic MTLn2 cells was 14-3-3 protein sigma. The results indicate that increased metastatic potential is associated with positive and negative changes in expression of particular proteins. Proteins that are positively correlated with metastatic potential may prove more useful as clinical biomarkers, but those with negative correlations may still provide useful information about underlying mechanisms of metastatic spread.