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Residual chlorine and biofilm coexistence is inevitable in drinking water transmission and distribution networks. Understanding the microbial response and its mediated effects on disinfection byproducts under different categories of residual chlorine stress is essential to ensure water safety. The aim of our study was to determine the response of pipe wall biofilms to residual chlorine pressure in chlorine and chloramine systems and to understand the microbially mediated effects on the formation and migration of haloacetonitriles (HANs), typical nitrogenous disinfection byproducts. According to the experimental results, the biofilm response changes under pressure, with significant differences noted in morphological characteristics, the extracellular polymeric substances (EPS) spatial structure, bacterial diversity, and functional abundance potential. Upon incubation with residual chlorine (1.0 ± 0.2 mg/L), the biofilm biomass per unit area, EPS, community abundance, and diversity increased in the chloramine group, and the percentage of viable bacteria increased, potentially indicating that the chloramine group provides a richer variety of organic matter precursors. Compared with the chloramine group, the chlorination group exhibited increased haloacetonitrile formation potential (HANFP), with Rhodococcus (43.2%) dominating the system, whereas the prediction abundance of metabolic functions was advantageous, especially with regard to amino acid metabolism, carbohydrate metabolism, and the biodegradation and metabolism of foreign chemicals. Under chlorine stress, pipe wall biofilms play a stronger role in mediating HAN production. It is inferred that chlorine may stimulates microbial interactions, and more metabolites (e.g., EPS) consume chlorine to protect microbial survival. EPS dominates in biofilms, in which proteins exhibit greater HANFP than polysaccharides., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Response mechanisms of pipe wall biofilms in water supply networks under different disinfection strategy pressures and the effect of mediating halogenated acetonitrile formation”., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We studied the properties of N6-chloroadenosine phosphates (ATP, ADP, and AMP chloramines) as compounds with potentially increased antiplatelet efficacy determined by their binding to the plasma membrane of platelets. Chloramine derivatives of ATP, ADP, and AMP do not differ in their optical absorption characteristics: their absorption spectra are in the range of 220-340 nm with a maximum at 264 nm. Chloramines of adenosine phosphates are characterized by high reactivity with respect to thiol compounds. In particular, the rate constants of the reaction of N6-chloroadenosine-5'-diphosphate with N-acetylcysteine, reduced glutathione, dithiothreitol, and cysteine reach 59,000, 250,000, 340,000, and 1,250,000 M -1 ×sec -1 , respectively, and only 1.10±0.02 M -1 ×sec -1 with methionine. It has been found that N6-chloradenosine-5'-triphosphate is a strong inhibitor of platelet functions: it effectively suppresses ADP-induced cell aggregation (IC 50 in the whole blood is 5 μM) and inhibits aggregation of preactivated platelets and induces dissociation of their aggregates., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Histones are critical for the packaging of nuclear DNA and chromatin assembly, which is facilitated by the high abundance of Lys and Arg residues within these proteins. These residues are also the site of a range of post-translational modifications, which influence the regulatory function of histones. Histones are also present in the extracellular environment, following release by various pathways, particularly neutrophil extracellular traps (NETs). NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This suggests that histones could be targets for HOCl under conditions where aberrant NET release is prevalent, such as chronic inflammation. In this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl modified the histones in a dose- and time-dependent manner, resulting in structural changes to the proteins and the formation of a range of post-translational modification products. N-Chloramines are major products following exposure of the histones to HOCl and decompose over 24 h forming Lys nitriles and carbonyls (aminoadipic semialdehydes). Chlorination and dichlorination of Tyr, but not Trp residues, is also observed. Met sulfoxide and Met sulfones are formed, though these oxidation products are also detected albeit at a lower extent, in the non-treated histones. Evidence for histone fragmentation and aggregation was also obtained. These results could have implications for the development of chronic inflammatory diseases, given the key role of Lys residues in regulating histone function., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In this paper, for the first time, we show in chloraminated systems, the chloramine decaying proteins (CDP) play an important role in bulk water and biomass (biofilm) in resisting disinfectant. Extracellular polymeric substances in biofilm/biomass are known to protect microbes from disinfectants and toxic materials, but the exact mechanism(s) is/are not known. Starting with the seed from a nitrifying chloraminated reactor, two 5 L reactors were fed intermittently with either chloramine or ammonia containing nutrient solution. The degree of nitrification increased with time in both reactors despite an increase in soluble CDP in the chloraminated reactor, while soluble CDP decreased in the ammoniated one. The suspended biomass collected after eight months of operation from chloraminated reactor contained CDP and responded to short-term chloramine stress (1.5 h with initial 1.5 mg-Cl 2 ·L -1 ) by the additional production of soluble CDP. The suspended biomass from ammoniated reactor neither contained CDP nor produced soluble CDP as a stress response. The production, release and accumulation of CDP in biomass (biofilm) could be one of several mechanisms microbes use to defend against disinfectants (stress). The new understanding will pave the way for better disinfection management and better design of experiments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hydrogen peroxide (H 2 O 2 ) is one of the reactive oxygen species (ROS), endogenously produced during metabolism, which acts as a second messenger. In skeletal muscles, hypoxia- or hyperthermia-induced increase in H 2 O 2 might affect synaptic transmission by targeting the most redox-sensitive presynaptic compartment (Giniatullin et al., 2006). However, the effects of H 2 O 2 as a signal molecule have not previously been studied in different patterns of the synaptic activity. Here, using optical and microelectrode recording of synaptic vesicle exocytosis, we studied the use-dependent action of low concentrations of H 2 O 2 and other oxidants in the mouse neuromuscular junction. We found that: (i) H 2 O 2 at low micromole concentrations inhibited both spontaneous and evoked transmitter releases from the motor nerve terminals in a use-dependent manner, (ii) the antioxidant N-acetylcysteine (NAC) eliminated these depressant effects, (iii) the influence of H 2 O 2 was not associated with lipid oxidation suggesting a pure signaling action, (iv) the intracellular oxidant Chloramine-T or (v) the glutathione depletion produced similar to H 2 O 2 depressant effects. Taken together, our data revealed the effective inhibition of neurotransmitter release by ROS, which was proportional to the intensity of synaptic activity at the neuromuscular junction. The combination of various oxidants suggested an intracellular location for redox-sensitive sites responsible for modulation of the synaptic transmission in the skeletal muscle., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Earlier, we reported on soluble microbial products-mediated chloramine decay in nitrifying waters. However, we neither separated the agent(s) nor identified the factors that enhanced the production of chloramine-decaying soluble microbial products (cSMPs). Experiments were conducted by feeding reactor sets (each consisting of five reactors connected in series) with treated water (3-8 mg-DOC.L -1 ) obtained from a water treatment plant. The reactors simulated various nitrifying conditions that are experienced in a chloraminated system. In unfiltered samples obtained from nitrified reactors, about 89-93% of the dosed chloramine decayed within 40 h. The cSMP-mediated decay accounted for 21-39% of all chloramine decay in the samples from 0 to 5 mg-C.L -1 fed reactors and 15% in the samples from 7 to 8 mg-C.L -1 fed reactors. Microbial processes (mediated by nitrifiers and/or heterotrophs) and biomass-associated microbial products (BMPs) in insoluble form accounted for 13-21% for the reactors fed with 0-5 mg-C.L -1 and 34% for those fed with 7-8 mg-C.L -1 . The cSMPs were separable with a 30 kDa cut-off membrane but not with 50 or 100 kDa membranes, i.e., they were above 30 kDa but below 50 kDa in size, and were confirmed to be a protein(s). The protein(s) accelerated chloramine decay by accelerating chloramine auto-decomposition and nitrite oxidation. As opposed to the traditional belief, unknown factors accounted for approximately 34-53% in commonly encountered re-chloraminated nitrifying waters (2-5 mg-DOC.L -1 ). Understanding the identity and role of these factors - such as cSMPs, BMPs, heterotrophs - will lead to a better control of chloramine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Advanced technologies for controlled cell adhesion and detachment in novel biointerface designs profit from stimuli-responsive systems that are able to react to their environment. Here, a multilayer system made of thiolated chitosan and thiolated chondroitin sulfate was constructed, with the potential of switchable inter- and intramolecular thiol/disulfide interactions representing a redox-sensitive nanoplatform. Owing to the formation and cleavage of inherent disulfide bonds by oxidation and reduction, surface properties of the multilayer can be controlled toward protein adsorption/desorption and cell adhesion in a reversible manner. Oxidation of thiols by chloramine-T promotes fibronectin (FN) adsorption and fibroblast cell adhesion, whereas the reduction by tris(2-carboxyethyl)phosphine reverses these effects, leading to low FN adsorption and little cell adhesion and spreading. These effects on the biological systems are related to significant changes of wetting properties, zeta potential, and mechanical properties of these multilayer films. The system presented may be useful for biomedical applications as responsive and obedient surfaces in medical implants and support tissue regeneration.

The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H 2 O 2 ) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H 2 O 2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H 2 O 2 . Myeloperoxidase in the extract converted all the H 2 O 2 to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H 2 O 2 , the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H 2 O 2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As one of the most powerful biocides, N-halamine based antimicrobial materials have attracted much interest due to their non-toxicity, rechargeability, and rapid inactivation against a broad range of microorganisms. In this study, novel titanium dioxide-ADMH core-shell nanoparticles [TiO 2 @poly (ADMH-co-MMA) NPs] were prepared via miniemulsion polymerization using 3-allyl-5,5-dimethylhydantoin (ADMH) and methyl methacrylate (MMA) with nano-TiO 2 . The produced nanoparticles were characterized by FT-IR, TEM, TGA, and XPS. The UV stability of N-halamine nanoparticles has been improved with the addition of titanium dioxide. After chlorination treatment by sodium hypochlorite, biocidal efficacies of the chlorinated nanoparticles against S. aureus (ATCC 6538) and E. coli O157:H7 (ATCC 43895) were determined. The nanoparticles showed excellent antimicrobial properties against bacteria within brief contact time. In addition, in vitro cell cytocompatibility tests showed that the antibacterial nanoparticles had good biocompatibility., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Ammonia degradation was investigated in three batch reactors with differing initial concentrations of bacteria present in the same filtered water source based on pre-treatment filtration techniques. The potential for the bacterial community to degrade the ammonia present was determined in the absence of monochloramine, simulating a distribution system where a loss of disinfectant residual has occurred. Nitrification was observed in only one of the three batch reactors, whereas rapid microbiologically induced chloramine decay was present in two reactors. Results suggest that the microbial decay factor is not a valid tool for indication of nitrification, but may be used as an indicator of the occurrence of rapid monochloramine decay. Intact bacterial cell numbers did not to correlate with changes in ammonia, nitrite or nitrate concentrations and hence did not correlate with the nitrification observed. Neither use of the microbial decay factor or monitoring of ammonia oxidising prokaryotes provided an early indication for the occurrence of nitrification. Hence, monitoring of ammonia and nitrite would still be the most suitable tool for indicating nitrification., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

Hypochlorous acid (HOCl), an oxidant produced by myeloperoxidase (MPO), induces protein and lipid oxidation, which is implicated in the pathogenesis of atherosclerosis. Individuals with mildly elevated bilirubin concentrations (i.e., Gilbert syndrome; GS) are protected from atherosclerosis, cardiovascular disease, and related mortality. We aimed to investigate whether exogenous/endogenous unconjugated bilirubin (UCB), at physiological concentrations, can protect proteins/lipids from oxidation induced by reagent and enzymatically generated HOCl. Serum/plasma samples supplemented with exogenous UCB (≤250µM) were assessed for their susceptibility to HOCl and MPO/H2O2/Cl(-) oxidation, by measuring chloramine, protein carbonyl, and malondialdehyde (MDA) formation. Serum/plasma samples from hyperbilirubinemic Gunn rats and humans with GS were also exposed to MPO/H2O2/Cl(-) to: (1) validate in vitro data and (2) determine the relevance of endogenously elevated UCB in preventing protein and lipid oxidation. Exogenous UCB dose-dependently (P<0.05) inhibited HOCl and MPO/H2O2/Cl(-)-induced chloramine formation. Albumin-bound UCB efficiently and specifically (3.9-125µM; P<0.05) scavenged taurine, glycine, and N-α-acetyllysine chloramines. These results were translated into Gunn rat and GS serum/plasma, which showed significantly (P<0.01) reduced chloramine formation after MPO-induced oxidation. Protein carbonyl and MDA formation was also reduced after MPO oxidation in plasma supplemented with UCB (P<0.05; 25 and 50µM, respectively). Significant inhibition of protein and lipid oxidation was demonstrated within the physiological range of UCB, providing a hypothetical link to protection from atherosclerosis in hyperbilirubinemic individuals. These data demonstrate a novel and physiologically relevant mechanism whereby UCB could inhibit protein and lipid modification by quenching chloramines induced by MPO-induced HOCl., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In drinking water, monochloramine may promote ammonia–oxidizing bacteria (AOB) growth because of concurrent ammonia presence. AOB use (i) ammonia monooxygenase for biological ammonia oxidation to hydroxylamine and (ii) hydroxylamine oxidoreductase for biological hydroxylamine oxidation to nitrite. In addition, monochloramine and hydroxylamine abiotically react, providing AOB a potential benefit by removing the disinfectant (monochloramine) and releasing growth substrate (ammonia). Alternatively and because biological hydroxylamine oxidation supplies the electrons (reductant) required for biological ammonia oxidation, the monochloramine/hydroxylamine abiotic reaction represents a possible inactivation mechanism by consuming hydroxylamine and inhibiting reductant generation. To investigate the abiotic monochloramine and hydroxylamine reaction's impact on AOB activity, the current study used batch experiments with Nitrosomonas europaea (AOB pure culture), ammonia, monochloramine, and hydroxylamine addition. To decipher whether hydroxylamine addition benefitted N. europaea activity by (i) removing monochloramine and releasing free ammonia or (ii) providing an additional effect (possibly the aforementioned reductant source), a previously developed cometabolism model was coupled with an abiotic monochloramine and hydroxylamine model for data interpretation. N. europaea maintained ammonia oxidizing activity when hydroxylamine was added before complete ammonia oxidation cessation. The impact could not be accounted for by monochloramine removal and free ammonia release alone and was concentration dependent for both monochloramine and hydroxylamine. In addition, a preferential negative impact occurred for ammonia versus hydroxylamine oxidation. These results suggest an additional benefit of exogenous hydroxylamine addition beyond monochloramine removal and free ammonia release, possibly providing reductant generation.

Rectifying the accelerated chloramine decay after the onset of nitrification is a major challenge for water utilities that employ chloramine as a disinfectant. Recently, the evidence of soluble microbial products (SMPs) accelerating chloramine decay beyond traditionally known means was reported. After the onset of nitrification, with an intention to inactivate nitrifying bacteria and thus maintaining disinfectant residuals, breakpoint chlorination followed by re-chloramination is usually practiced by water utilities. However, what actually breakpoint chlorination does beyond known effects is not known, especially in light of the new finding of SMPs. In this study, experiments were conducted using severely nitrified chloraminated water samples (chloramine residuals <0.5 mg Cl2 L−1, nitrite residuals >0.1 mg N L−1 and an order of magnitude higher chloramine decay rate compared to normal decay) obtained from two laboratory scale systems operated by feeding natural organic matter (NOM) containing and NOM free waters. Results showed that the accelerated decay of chloramine as a result of SMPs can be eliminated by spiking higher free chlorine residuals (about 0.92 ± 0.03 to 1.16 ± 0.12 mg Cl2 L−1) than the stoichiometric requirement for breakpoint chlorination and nitrite oxidation. Further, accelerated initial chlorine decay showed chlorine preferentially reacts with nitrite and ammonia before destroying SMPs. This study, clearly demonstrated there is an additional demand from SMPs that needs to be satisfied to effectively recover disinfection residuals in subsequent re-chloramination.

Myeloperoxidase (MPO) produces hypohalous acids as a key component of the innate immune response; however, release of these acids extracellularly results in inflammatory cell and tissue damage. The two-step, one-pot Davis-Beirut reaction was used to synthesize a library of 2H-indazoles and 1H-indazolones as putative inhibitors of MPO. A structure-activity relationship study was undertaken wherein compounds were evaluated utilizing taurine-chloramine and MPO-mediated H2O2 consumption assays. Docking studies as well as toxicophore and Lipinski analyses were performed. Fourteen compounds were found to be potent inhibitors with IC50 values <1μM, suggesting these compounds could be considered as potential modulators of pro-oxidative tissue injury pertubated by the inflammatory MPO/H2O2/HOCl/HOBr system., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively, which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman and Halliwell, Biochemical and Biophysical Research Communications, 258, 168-172 (1999)). This report evaluates the significance of 3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid. The results demonstrate that 3-nitrotyrosine is chlorinated by hypochlorous acid or chloramines to form 3-chloro-5-nitrotyrosine. As 3-nitrotyrosine from in vivo samples is usually found within proteins rather than as free amino acid, we also examined the reaction of 3-nitrotyrosine modification in the context of peptides. The chlorination of 3-nitrotyrosine in peptides was observed to occur up to 700-fold faster than control reactions using equivalent amino acid mixtures. These results further advance our understanding of tyrosine chlorination and the use of 3-nitrotyrosine formed in vivo as a biomarker of reactive nitrogen species.

The efficiency of monochloramine disinfection was dependent on the quantity and composition of extracellular polymeric substances (EPS) in biofilms, as monochloramine has a selective reactivity with proteins over polysaccharides. Biofilms with protein-based (Pseudomonas putida) and polysaccharide based EPS (Pseudomonas aeruginosa), as well as biofilms with varied amount of polysaccharide EPS (wild-type and mutant P. aeruginosa), were compared. The different reactivity of EPS components with monochloramine influenced disinfectant penetration, biofilm inactivation, as well as the viability of detached clusters. Monochloramine transport profiling measured by a chloramine-sensitive microelectrode revealed a broader diffusion boundary layer between bulk and biofilm surface in the P. putida biofilm compared to those of P. aeruginosa biofilms. The reaction with proteins in P. putida EPS multiplied both the time and the monochloramine mass required to achieve a full biofilm penetration. Cell viability in biofilms was also spatially influenced by monochloramine diffusion and reaction within biofilms, showing a lower survival in the surface section and a higher persistence in the middle section of the P. putida biofilm compared to the P. aeruginosa biofilms. While polysaccharide EPS promoted biofilm cell viability by obstructing monochloramine reactive sites on bacterial cells, protein EPS hindered monochloramine penetration by reacting with monochloramine and reduced its concentration within biofilms. Furthermore, the persistence of bacterial cells detached from biofilm (over 70% for P. putida and ∼40% for polysaccharide producing P. aeruginosa) suggested that currently recommended monochloramine residual levels may underestimate the risk of water quality deterioration caused by biofilm detachment.

The aim of this study was to assess a possible role of monochloramine (NH2 Cl), one of the reactive chlorine species, which induce oxidative stress, on the proliferation of colorectal cancer cell line Caco-2. At concentrations ranging from 10 to 200 μM, NH2 Cl (14-61% inhibition), but not hypochlorous acid, dose-dependently inhibited the cell viability of Caco-2 cells. Experiments utilizing methionine (a scavenger of NH2 Cl), taurine-chloramine and glutamine-chloramine revealed that only NH2 Cl affects the cancer cell proliferation among reactive chlorine species, with a relative specificity. Furthermore, flow-cytometry experiments showed that the anti-proliferative effect of NH2 Cl is partially attributable to both apoptosis and G2/M cell cycle arrest. These results suggest that NH2 Cl has the potential to suppress colorectal cancer cell proliferation., (Copyright © 2013 John Wiley & Sons, Ltd.)

Chloramine is widely used in United States drinking water systems as a secondary disinfectant, which may promote the growth of nitrifying bacteria because ammonia is present. At the onset of nitrification, both nitrifying bacteria and their products exert a monochloramine demand, decreasing the residual disinfectant concentration in water distribution systems. This work investigated another potentially significant mechanism for residual disinfectant loss: monochloramine cometabolism by ammonia-oxidizing bacteria (AOB). Monochloramine cometabolism was studied with the pure culture AOB Nitrosomonas europaea (ATCC 19718) in batch kinetic experiments under drinking water conditions. Three batch reactors were used in each experiment: a positive control to estimate the ammonia kinetic parameters, a negative control to account for abiotic reactions, and a cometabolism reactor to estimate the cometabolism kinetic constants. Kinetic parameters were estimated in AQUASIM with a simultaneous fit to all experimental data. The cometabolism reactors showed a more rapid monochloramine decay than in the negative controls, demonstrating that cometabolism occurs. Cometabolism kinetics were best described by a pseudo first order model with a reductant term to account for ammonia availability. Monochloramine cometabolism kinetics were similar to those of ammonia metabolism, and monochloramine cometabolism was a significant loss mechanism (30-60% of the observed monochloramine decay). These results suggest that monochloramine cometabolism should occur in practice and may be a significant contribution to monochloramine decay during nitrification episodes in drinking water distribution systems., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

When chloramine is used as a disinfectant, managing an acceptable "residual" throughout the water distribution systems particularly once nitrification has set in is challenging. Managing chloramine decay prior to the onset of nitrification through effective control strategies is important and to-date the strategies developed around nitrification has been ineffective. This study aimed at developing a more holistic knowledge on how decaying chloramine and nitrification metabolites impact microbial communities in chloraminated systems. Five lab-scale reactors (connected in series) were operated to simulate a full-scale chloraminated distribution system. Culture independent techniques (cloning and qPCR) were used to characterise and quantify the mixed microbial communities in reactors maintaining a residual of high to low (2.18-0.03 mg/L). The study for the first time associates chloramine residuals and nitrification metabolites to different microbial communities. Bacterial classes Solibacteres, Nitrospira, Sphingobacteria and Betaproteobacteria dominated at low chloramine residuals whereas Actinobacteria and Gammaproteobacteria dominated at higher chloramine residuals. Prior to the onset of nitrification bacterial genera Pseudomonas, Methylobacterium and Sphingomonas were found to be dominant and Sphingomonas in particular increased with the onset of nitrification. Nitrosomonas urea, oligotropha, and two other novel ammonia-oxidizing bacteria were detected once the chloramine residuals had dropped below 0.65 mg/L. Additionally nitrification alone failed to explain chloramine decay rates observed in these reactors. The finding of this study is expected to re-direct the focus from nitrifiers to heterotrophic bacteria, which the authors believe could hold the key towards developing a control strategy that would enable better management of chloramine residuals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The biostability concept has been successfully used to predict the onset of nitrification in drinking water distribution systems, but in certain cases deficiencies have been observed in the predictions, indicating that modifications to parameters were needed. At the biostable disinfectant residual concentration (BRC), the rate of ammonia-oxidising bacterial (AOB) growth due to the substrate (free ammonia) and the rate of inactivation due to the disinfectant are balanced. Growth and inactivation rates vary greatly with temperature, but temperature is yet to be considered in the biostability equation. In this paper, two separate novel models are proposed which take into account the temperature effects on the biostability equation. First, a novel model of specific growth rate variability with temperature was shown to be valid for different bacterial species. Then, the biostability model was modified and validated for ammonia-oxidising bacterial activity using data collected from laboratory and full-scale distribution systems. The proposed model has two important uses: while the specific growth rate model and biostability model can be widely adopted for many microbes, the biostability model for AOB also has the potential to aid water utilities in disinfectant residual management throughout yearly temperature variations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: The activity of oxidants, such as halogens and active halogen compounds, decreases generally in the presence of proteinaceous material. A quantification of consumption effects was performed to judge the suitability of different representatives as antiseptics and their compatibility with pharmaceutical additives., Methods: An iodometric approach served to assess the temporal loss of oxidation capacity in the presence of peptone and fetal calf serum (FCS). The tested agents comprised active halogen compounds, well-known and in particular novel chloramine-based agents indicated for the topical treatment of infections., Key Findings: The decrease in oxidation capacity was higher in the presence of FCS than of peptone and correlated with the reactivity of the oxidants in both cases. The highest consumption rates were for active bromine compounds followed by hypochlorous acid and heterocyclic chlorimides, such as dichloro-isocyanuric acid, while N-chlorotaurine and related amine-based analogues were least consumed. The pH dependence was only remarkable for chloramine T., Conclusions: The observed consumption effects are the result of the differing oxidizing (chlorinating) potencies. Since consumption and irritation are founded on the very same reactions, representatives with low reactivity (N-chloro amino acids) are regarded as more tolerable and retain more oxidative capacity, which provides a more sustained antimicrobial activity., (© 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.)

Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The discovery of a microbially derived soluble product that accelerates chloramine decay is described. Nitrifying bacteria are believed to be wholly responsible for rapid chloramine loss in drinking water systems. However, a recent investigation showed that an unidentified soluble agent significantly accelerated chloramine decay. The agent was suspected to be either natural organic matter (NOM) or soluble microbial products (SMPs). A laboratory scale reactor was fed chloraminated reverse osmosis (RO) treated water to eliminate the interference from NOM. Once nitrification had set in, experiments were conducted on the reactor and feed waters to determine the identity of the component. The study showed the presence of SMPs released by microbes in severely nitrified waters. Further experiments proved that the SMPs significantly accelerated chloramine decay, probably through catalytic reaction. Moreover, application of common protein denaturing techniques stopped the reaction implying that the compound responsible was likely to be a protein. This significant finding will pave the way for better control of chloramine in the distribution systems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aims: Reactive oxygen species released from neutrophils during vascular inflammation could contribute to endothelial dysfunction seen in diseases such as atherosclerosis. Activated neutrophils generate hydrogen peroxide (H(2)O(2)) and hypochlorous acid (HOCl), as well as chloramines that are formed when HOCl reacts with amino compounds. These oxidants preferentially target thiol groups and thiol-containing proteins. The peroxiredoxins (Prxs) are thiol proteins that have high reactivity with H(2)O(2) and may also be sensitive to HOCl and chloramines., Results: We have investigated human umbilical vein endothelial cells and shown that their cytoplasmic (Prx1 and Prx2) and mitochondrial (Prx3) Prxs are oxidized when they are exposed to H(2)O(2), HOCl, or cell-permeable chloramines. H(2)O(2) converted the Prxs to hyperoxidized, inactive forms, with little accumulation of disulfide-linked dimers. The oxidized Prxs were reduced over hours, presumably due to the action of endothelial sulfiredoxin. In contrast to the hyperoxidation seen with H(2)O(2), HOCl and the chloramine derivatives of glycine and ammonia converted the Prxs to disulfide-linked dimers and dimerization was reversed within 10-30 min of oxidant removal. HOCl treatment caused thioredoxin reductase (TrxR) inhibition with no reversal of dimerization. The cytotoxicity of ammonia chloramine was increased when cells were pretreated with H(2)O(2) to hyperoxidize the Prxs, or when the chloramine was added in the presence of the TrxR inhibitor, auranofin., Innovation: We describe the novel observation that exposure of nucleated cells to inflammatory oxidants results in the accumulation of Prxs in the dimeric form., Conclusions: Endothelial cell Prxs are sensitive targets for neutrophil-derived oxidants and may protect against their damaging effects.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel "Screen and Insert" (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (30-65%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathione-methylfluorescein (GS-MF) and glutathione-monochlorobimane (GS-MCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GS-MCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GS-MF and GS-MCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation., (© 2012 Wiley Periodicals, Inc.)

Taste and odor issues occurring in new buildings were attributed to rapid loss of chloramine residual, high levels of microbes in the potable water system, and high water age due to use of advanced water conservation devices. Laboratory experiments confirmed that chloramine could decay rapidly in the presence of new copper pipe, providing a possible explanation for the rapid disinfectant loss in the new buildings. Higher temperature and lower pH also accelerated the rate of chloramine decay in copper pipes. The reaction was slowed by the addition of phosphate inhibitor or aluminum, which presumably formed barriers between the pipe wall and the chloramine in the bulk water. Additional research is needed to better understand how to maintain high quality water in buildings while also conserving water., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neutrophil-dependent reactions catalysed by myeloperoxidase (MPO) are thought to play important roles in the pulmonary pathobiology of cystic fibrosis (CF). Aerosolized thiol antioxidants such as glutathione (GSH) and N-acetylcysteine (NAC) are currently being utilized as therapeutics to modify CF respiratory tract oxidative processes. This study hypothesized that MPO in CF airway lining fluids may be a target of such therapeutics. MPO activity in sputum from 21 adult CF patients was found to be inversely associated with lung function (FEV(1)). In contrast, systemic inflammation (assessed by plasma C-reactive protein) was not correlated with lung function. Ex vivo studies revealed that GSH and NAC effectively scavenged N-chloramines in sputum and inhibited sputum MPO activity with potency exquisitely dependent upon MPO activity levels. Detailed kinetic analyses revealed that NAC and GSH inhibit MPO by distinct mechanisms. Activation of the key pro-inflammatory transcription factor NF-κB in cultured HBE1 cells was inhibited by GSH. The findings reveal that MPO activity and its reactive products represent useful predictors of the doses of inhaled thiol antioxidants required to ameliorate airway oxidative stress and inflammation in CF patients and provide mechanistic insight into the antioxidative/anti-inflammatory mechanisms of action of GSH and NAC when administered into the CF lung.

Drinking water biofilm development is affected by the available nutrient levels and the presence of disinfectants. Phosphorus is recognized as another important limiting nutrient besides organic carbon. In this study, drinking water biofilms were developed in annular reactors to examine the effects of phosphorus on the biofilm disinfections with free chlorine and monochloramine. Phosphorus addition was found to increase the biofilm cell number but decrease the exopolysaccharides (EPS) production. The disinfection efficacies of both free chlorine and monochloramine were increased when phosphorus was added into the reactor systems. At the same disinfection dosages, monochloramine showed greater biofilm removal efficiency than free chlorine. Monochloramine could be a better choice than free chlorine in biofilm disinfection when phosphate-based corrosion inhibitors are applied.

Service reservoirs play an important role in maintaining water quality in distribution systems. Several factors affect the reservoir water quality, including bulk water reactions, stratification, sediment accumulation and wall reactions. It is generally thought that biofilm and sediments can harbour microorganisms, especially in chloraminated reservoirs, but their impact on disinfectant loss on disinfectant loss has not been quantified. Hence, debate exists as to the extent of the problem. To quantify the impact, the reservoir acceleration factor (F(Ra)) is defined. This factor represents the acceleration of chloramine decay arising from all causes, including changes in retention time, assuming that the reservoir is completely mixed. Such an approach quantifies the impact of factors, other than chemical reactions, in the bulk water. Data from three full-scale chloraminated service reservoirs in distribution systems of Sydney, Australia, were analysed to demonstrate the generality of the method. Results showed that in two large service reservoirs (404 x 10(3) m(3) and 82 x 10(3) m(3)) there was minimal impact from biofilm/sediment. However, in a small reservoir (3 x 10(3) m(3)), the biofilm/sediment had significant impact. In both small and large reservoirs, the effect of stratification was significant., ((c) 2010 Elsevier Ltd. All rights reserved.)

Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

We investigated the mechanisms by which chlorine (Cl(2)) and its reactive byproducts inhibit Na(+)-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na(+) channels (ENaC) by measuring AFC in mice exposed to Cl(2) (0-500 ppm for 30 min) and Na(+) and amiloride-sensitive currents (I(Na) and I(amil), respectively) across Xenopus oocytes expressing human alpha-, beta-, and gamma-ENaC incubated with HOCl (1-2000 microm). Both Cl(2) and HOCl-derived products decreased AFC in mice and whole cell and single channel I(Na) in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I(Na) in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I(Na) by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the gamma-ENaC subunit restored the ability of HOCl to inhibit I(Na). Finally, I(Na) of oocytes expressing wild type alpha- and gamma-ENaC and a mutant form of beta ENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.

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.

In healthy cells, glutathione disulfide (GSSG) is rapidly reduced back to glutathione (GSH) by glutathione reductase to maintain redox status. The ratio of GSH/GSSG has been used as an indicator of oxidative stress. However, hypochlorous acid (HOCl) generated by the myeloperoxidase-H(2)O(2)-Cl(-) system of neutrophils converts GSH to irreversible oxidation products. Although several such products have been identified, yields of these compounds are very low in biological systems, and they cannot account quantitatively for thiol loss. In the current studies, we use liquid chromatography-mass spectrometry (LC-MS) to demonstrate that HOCl and chloramines oxidize GSSG to two irreversible products in high yield. The products, termed M-45 and M-90, are, respectively, 45 or 90 atomic mass units lighter than GSSG. The reaction pathway involves chloramine and aldehyde intermediates, and converts the gamma-glutamyl residues of GSSG to 5-hydroxybutyrolactam. Importantly, M-45 and M-90 were resistant to reduction by glutathione reductase. Moreover, the monohydroxylbutyrolactam M-45 accounted for >90% of the endogenous GSH oxidation products generated by activated neutrophils. Because the reaction pathway involves chlorinating intermediates, hydroxylbutyrolactams are likely to be specific products of HOCl, which is generated only by myeloperoxidase. Therefore, our observations implicate M-45 as a potential biomarker for myeloperoxidase activity in vivo.

The binding of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes and other cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by MALDI-TOF mass spectrometry hypochlorous acid and the myeloperoxidase-hydrogen peroxide-chloride system convert 1,2-dipalmitoyl-sn-glycero-3-phosphoserine into 1,2-dipalmitoyl-sn-glycero-3-phosphoacetaldehyde and 1,2-dipalmitoyl-sn-glycero-3-phosphonitrile. A transient chlorimine derivative was detected using 4-chloro-alpha-cyanocinnamic acid as matrix in mass spectrometry only at short incubation times and supplying HOCl in two-fold excess. The decay of transient chlorinated products was followed by changes in absorbance spectra using O-phospho-l-serine to model the behavior of the serine head group in phosphatidylserine. N-Chlorimine and N-monochloramine derivatives decayed with half-life times of 1.5 and 57 min, respectively, at 22 degrees C and pH 7.4. N-Dichloramines decayed within few seconds under these conditions.

When neutrophils phagocytose bacteria, they generate the cytotoxic agent hypochlorous acid (HOCl). The specific role that HOCl plays in bacterial killing is unclear. In the phagosome, it should react with neutrophil proteins to form protein chloramines and dichloramines. We investigated the stability of model dichloramines that are likely to be formed on N-terminal amino acids and Lys residues of proteins contained within phagosomes. Dichloramines were much more unstable than their analogous monochloramines. The stability was affected by substituents on the alpha-carbon. Amino acid dichloramines were extremely unstable, indicating that an alpha-carboxyl group facilitated decomposition. In general, the absence of a substituent enhanced stability. The carboxyl group on N-terminal Glu residues favored break down, but this effect was not apparent with Asp residues. Unstable dichloramines that contained a substituent on their alpha-carbon were cytotoxic and killed 50% of 10(5) Staphylococcus aureus (LD50) at a dose of approximately 2.5 nmol. Their cytotoxicity declined with time. The dichloramines of N-alpha-acetyl Lys and taurine were not bactericidal up to 10 nmol per 10(5) S. aureus. None of the analogous monochloramines were cytotoxic at this dose. Dichloramines decomposed to yield chlorimines, aldehydes, and the inorganic gases ammonia monochloramine (NH2Cl) and ammonia dichloramine (NHCl2). The LD50 values were determined for NH2Cl (0.37 +/- 0.14 nmol), NHCl2 (0.08 +/- 0.02 nmol), and HOCl (0.14 +/- 0.04 nmol). Stable products formed during the breakdown of dichloramines were not bactericidal. We propose a potential antimicrobial mechanism that explains in part how HOCl can react mainly with neutrophil components but still promote killing of phagocytosed bacteria. HOCl produced in phagosomes will react with amine groups on neutrophil proteins to form unstable dichloramines that will liberate cytotoxic NH2Cl and NHCl2. These gases will contribute to killing of ingested bacteria.

Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) are frequent complications in critically ill patients and are responsible for significant morbidity and mortality. So far, experimental evidence supports the role of oxidants and oxidative injury in the pathogenesis of ALI/ARDS. In this study, the antioxidant effects of conventional N-acetylcysteine (NAC) and liposomally entrapped N-acetylcysteine (L-NAC) were evaluated in experimental animals challenged with lipopolysaccharide (LPS). Rats were pretreated with empty liposomes, NAC, or L-NAC (25mg/kg body weight, iv); 4h later were challenged with LPS (E. coli, LPS 0111:B4) and sacrificed 20h later. Challenge of saline (SAL)-pretreated animals with LPS resulted in lung injury as evidenced by increases in wet lung weight (edema), increases in lipid peroxidation (marker of oxidative stress), decreases of lung angiotensin-converting enzyme (ACE) (injury marker for pulmonary endothelial cells) and increases in the pro-inflammatory eicosanoids, thromboxane B(2) and leukotriene B(4). The LPS challenge also increased pulmonary myeloperoxidase activity and chloramine concentrations indicative of neutrophil infiltration and activation of the inflammatory response. Pretreatment of animals with L-NAC resulted in significant increases in the levels of non-protein thiols and NAC levels in lung homogenates (p<0.05) and bronchoalveolar lavage fluids (p<0.001), respectively. L-NAC was significantly (p<0.05) more effective than NAC or empty liposomes in attenuating the LPS-induced lung injuries as indicated by the aforementioned injury markers. Our results suggested that the delivery of NAC as a liposomal formulation improved its prophylactic effectiveness against LPS-induced lung injuries.

After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.

Aims: Myeloperoxidase (MPO), a cardiovascular risk factor in humans, is an in vivo catalyst for lipoprotein modification via intermediate formation of reactive chlorinating species. Among the different lipoprotein classes, anti-atherogenic high-density lipoprotein (HDL) represents a major target for modification by hypochlorous acid (HOCl), generated from H2O2 by MPO in the presence of physiological chloride concentrations. As MPO was identified as an HDL-associated protein that could facilitate selective oxidative modification of its physiological carrier, the aim of the present study was to investigate whether and to what extent modification of HDL by HOCl affects the binding affinity of MPO in vitro., Methods and Results: We show that binding affinity of 125I-labelled MPO to HDL markedly increases as a function of increasing extent of HOCl modification of HDL. In contrast to native HDL, HOCl-HDL potently inhibits MPO binding/uptake by endothelial cells and effectively attenuates metabolism of MPO by macrophages. Reduction of HDL-associated chloramines with methionine strongly impaired binding affinity of MPO towards HOCl-HDL. This indicates that N-chloramines generated by HOCl are regulators of the high-affinity interaction between HOCl-HDL and positively charged MPO. Most importantly, the presence of HOCl-HDL is almost without effect on the halogenating activity of MPO., Conclusion: We propose that MPO-dependent modification of HDL and concomitant increase in the binding affinity for MPO could generate a vicious cycle of MPO transport to and MPO-dependent modification at sites of chronic inflammation.

The aim of this study was to investigate the direct iodination of the recently discovered peptide obestatin by LC-UV/ESI ion trap MS analysis. The influence of selected reaction parameters on obestatin iodination by chloramine-T, Iodo-Gen((R)) and lactoperoxidase was investigated by experimental design. Different responses, i.e. species percentage and yield, peptide recovery and iodination yield were evaluated. Mono-up till tetra-iodinated species are possible depending on the reaction conditions with electrophilic substitutions occurring at Tyr(16) and His(19) as confirmed by LC/MS/MS. The two possible mono-iodinated obestatin isomers, i.e. [I(1)-Tyr(16)]-obestatin and [I(1)-His(19)]-obestatin, could be chromatographically separated. Several significant main and quadratic effects, and interaction of factors were observed from which optimum conditions for a specific response could be derived. The highest impact on the response surface diagrams was overall attributed to the amount of iodide added. Synthesis methods were compared relative to the different response factors: lactoperoxidase was found to be the overall most robust iodination technique, and also gave the highest mono-iodinated species yield. The applicability of our research was demonstrated by non-carrier-added (125)I-radioiodination. To our knowledge, this is the first time an LC separation of mono-iodinated peptide isomers has been reported.

Disifin has emerged as a unique and very effective agent used in disinfection of wounds, disinfection of surfaces, materials and water, and other substances contaminated with almost every type of pathogenic microorganism ranging from viruses, bacteria, fungi and yeast, and, very possibly, protozoan parasites, as well. The major active component of Disifin is tosylchloramide sodium (chloramine T). However, the mechanism by which Disifin suppresses the activities of pathogenic microbial agents remains enigmatic. The molecular mechanisms, and the receptors and the signal transducing pathways responsible for the biological effects of Disifin are largely unknown. Despite considerable advances, enormous investigative efforts and large resources invested in the research on infectious diseases, microbial infection still remains a public health problem in many parts of the world. The exact nature of the pathogenic agents responsible for many infectious diseases, and the nature of the receptors mediating the associated inflammatory events are incompletely understood. Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns (PAMPs) by a family of transmembrane pattern-recognizing and signal transducing receptor proteins called Toll-like receptors (TLRs). The TLR family plays an instructive role in innate immune responses against microbial pathogens, as well as the subsequent induction of adaptive immune responses. TLRs mediate recognition and inflammatory responses to a wide range of microbial products and are crucial for effective host defense by eradication of the invading pathogens. Now, recent updates demonstrated the ability of Disifin-derived products, Disifin-Animal and Disifin-Pressant to effectively suppress the progression and activities of Chikungunya fever and that of avian influenza A virus [A/cardialis/Germany/72, H7N1: the agent of a highly pathogenic avian influenza (HPAI)] infection, respectively. Overall, the above findings led me to suggest that Disifin and TLRs may mechanistically overlap in the processes of executing their functions against pathogenic microbial organisms. Thus, elucidating and better understanding of the molecular underpinnings responsible for the biochemical effects of Disifin-products, and the nature and mode of the interaction(s) of Disifin with TLRs in the process of exerting their biological effects may open a novel dimension in the research of infectious diseases, which may provide novel therapeutic targets for the prevention and treatment of a wide range of infectious diseases.

Objectives: This study aims to investigate the importance of ISO11405 recommended storage regime for extracted teeth in surface disinfectant chloramine T (chlT) prior to use in biofilm or in vitro caries studies involving microorganisms. ChlT may be absorbed into dentin and undergoes breakdown with organic material., Methods: Extracted roots were stored in chlT (2 days), rinsed and transferred to distilled deionised water. HPLC at regular intervals determined chlT elution. At 4 weeks roots were boiled in water and eluent assessed with HPLC. ChlT breakdown (+/-organic material) over time was monitored with HPLC. ChlT minimum inhibitory/bactericidal concentration (MIC/MBC) against Lactobacillus acidophilus was evaluated using L. acidophilus broth and chlT serial dilutions., Results: No significant increase in chlT elution was detected between 2h and 4 weeks (ANOVA, Tukeys, p>0.05), although levels tended to increase with time. ChlT detected in water was 0.005%, corresponding to 0.05% in dentin. After boiling (4 weeks) chlT breakdown products in water corresponded to 0.015% in dentin. MIC/MBC of chlT against L. acidophilus was 0.031%., Significance: ChlT breakdown is accelerated by organic material. L. acidophilus is highly sensitive to chlT. ChlT readily leaches from dentin but rinsing does not reduce chlT concentration below MIC/MBC. Low levels of chlT may remain but will probably be in a less active form. Teeth disinfected in chlT for use in research involving bacteria must be stored in distilled water for at least 2h to reduce chlT concentration below MBC, although longer will give greater elution and breakdown.

Alanine and taurine sharply potentiate antiaggregant effects of hypochlorite on platelets in platelet-rich plasma. This effect is determined by more pronounced action of chloramine derivatives, products of interaction of added amino acids with hypochlorite. Platelets are more sensitive to the inhibitory effects of amino acid chloramine derivatives (biogenic chloramines) compared to erythrocytes and neutrophils. The antiaggregant effects of biobenic amines, as covalent platelet inhibitors, in platelet-rich plasma are characterized by their increased reaction capacity with molecular targets in cells. Quantitative parameter of this initial selectivity (ratio of rate constant of inactivation of platelet receptors to rate constant of side reaction with plasma proteins) far surpasses 1. N,N-Dichlorotaurine is a perspective antiaggreant among the studied biogenic chloramines. This agent is stable and exhibits specific pharmacological activity in all test systems, including animal model of thrombosis.

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

Cyanobacteria produce toxins that may contaminate drinking water sources. Among others, the presence of the alkaloid toxins cylindrospermopsin (CYN) and anatoxin-a (ANTX) constitutes a considerable threat to human health due to the acute and chronic toxicity of these compounds. In the present study, not previously reported second-order rate constants for the reactions of CYN and ANTX with chlorine and monochloramine and of CYN with potassium permanganate were determined and the influence of pH and temperature was established for the most reactive cases. It was found that the reactivity of CYN with chlorine presents a maximum at pH 7 (rate constant of 1265 M(-1)s(-1)). However, the oxidation of CYN with chloramine and permanganate are rather slow processes, with rate constants <1 M(-1)s(-1). The first chlorination product of CYN was found to be 5-chloro-CYN (5-Cl-CYN), which reacts with chlorine 10-20 times slower than the parent compound. The reactivity of ANTX with chlorine and chloramines is also very low (k<1M(-1)s(-1)). The elimination of CYN and ANTX in surface water was also investigated. A chlorine dose of 1.5 mg l(-1) was enough to oxidize CYN almost completely. However, 3 mg l(-1) of chlorine was able to remove only 8% of ANTX, leading to a total formation of trihalomethanes (TTHM) at a concentration of 150 microg l(-1). Therefore, chlorination is a feasible option for CYN degradation during oxidation and disinfection processes but not for ANTX removal. The permanganate dose required for CYN oxidation is very high and not applicable in waterworks.

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-l-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG2-, GSSG3-, and GSSG4- (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG2- is unreactive, whereas GSSG4- is about twice as reactive as GSSG3-. Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK1 = -7.47; GSSG2- = GSSG3- + H+, pK2 = 8.5; GSSG3- = GSSG4- + H+, pK3 = 9.5; GSSG3- + HOCl --> NCG3- + H2O, k4 = 2.7(2) x 106 M-1 s-1; GSSG4- + HOCl --> NCG4- + H2O, k5 = 3.5(3) x 107 M-1 s-1; NCG3- --> NDG4- + H+, k6 = fast; and NCG4- + HOCl --> NDG4- + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. NDG decomposes at pH 7.4 in a first-order process with kdec = 4.22(1) x 10-4 s-1 (t1/2 = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.