A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.

Effects of disinfectant type and dosage on biofilm's activity, viability, microbiome and antibiotic resistome in bench-scale drinking water distribution systems.

Drinking water distribution systems (DWDSs) are important for supplying high-quality water to consumers and disinfectant is widely used to control microbial regrowth in DWDSs. However, the disinfectant's influences on microbial community and antibiotic resistome in DWDS biofilms and the underlying mechanisms driving their dynamics remain elusive. The study investigated the effects of chlorine and chloramine disinfection on the microbiome and antibiotic resistome of biofilms in bench-scale DWDSs using metagenomics assembly. Additionally, the biofilm activity and viability were monitored based on adenosine triphosphate (ATP) and flow cytometer (FCM) staining. The results showed that both chlorine and chloramine disinfectants decreased biofilm ATP, although chloramine at a lower dosage (1 mg/L) could increase it. Chloramine caused a greater decrease in living cells than chlorine. Furthermore, the disinfectants significantly lowered the microbial community diversity and altered microbial community structure. Certain bacterial taxa were enriched, such as Mycobacterium, Sphingomonas, Sphingopyxis, Azospira, and Dechloromonas. Pseudomonas aeruginosa exhibited high resistance towards disinfectants. The disinfectants also decreased the complexity of microbial community networks. Some functional taxa (e.g., Nitrospira, Nitrobacter, Nitrosomonas) were identified as keystones in chloramine-treated DWDS microbial ecological networks. Stochasticity drove biofilm microbial community assembly, and disinfectants increased the contributions of stochastic processes. Chlorine had greater promotion effects on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and ARG hosts than chloramine. The disinfectants also selected pathogens, such as Acinetobacter baumannii and Klebsiella pneumonia, and these pathogens also harbored ARGs and MGEs. Overall, this study provides new insights into the effects of disinfectants on biofilm microbiome and antibiotic resistome, highlighting the importance of monitoring and managing disinfection practices in DWDSs.

Antiplatelet Action of Chloramine Derivatives of Adenosine Phosphates and Their Chemical Activity in Relation to Sulfur-Containing Compounds.

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 (IC50 in the whole blood is 5 µM) and inhibits aggregation of preactivated platelets and induces dissociation of their aggregates.

Tandem synthesis, cytotoxicity, and in silico study of new 1,3,4-oxadiazoles as potential thymidylate synthase inhibitors.

A new series of pyrrole-linked mono- and bis(1,3,4-oxadiazole) hybrids, attached to various arene units, was prepared using a two-step tandem protocol. Therefore, a benzohydrazide derivative was condensed with the appropriate aldehydes in ethanol at 80°C for 60-150 min to give the corresponding N-(benzoylhydrazones). Without isolation, the previous intermediates underwent intramolecular oxidative cyclization in dimethyl sulfoxide at 180°C for 90-200 min in the presence of chloramine trihydrate to afford the target hybrids. The cytotoxicity of all hybrids was examined in vitro against the MCF-7, HEPG2, and Caco2 cell lines. Arene-linked hybrids 4i and 4j, attached to p-nitro and p-acetoxy units, were the most potent ones, with IC50 values ranging from 5.47 to 8.80 and 12.75 to 21.22 µM, respectively, when tested on the above cell lines. At the tested concentrations of 5 and 7.5 µM, hybrid 4i inhibited thymidylate synthase (TS) with the best inhibition percentages of 72.3 and 91.3, whereas hybrid 4j displayed comparable inhibitory activity to the reference pemetrexed. Hybrid 4j had inhibition percentages of 62.7 and 82.6, whereas pemetrexed had inhibition percentages of 59.2 and 80.2, respectively. The capability of hybrids 4i and 4j as potential TS inhibitors is supported by molecular docking studies, while SwissADME predicts their efficacy as drug-like scaffolds.

Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria.

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.

Gating of the capsaicin receptor TRPV1 by UVA-light and oxidants are mediated by distinct mechanisms.

Redox-sensitivity is a common property of several transient receptor potential (TRP) ion channels. Oxidants and UVA-light activate TRPV2 by oxidizing methionine pore residues which are conserved in the capsaicin-receptor TRPV1. However, the redox-sensitivity of TRPV1 is regarded to depend on intracellular cysteine residues. In this study we examined if TRPV1 is gated by UVA-light, and if the conserved methionine residues are relevant for redox-sensitivity of TRPV1. Patch clamp recordings were performed to explore wildtype (WT) and mutants of human TRPV1 (hTRPV1). UVA-light induced hTRPV1-mediated membrane currents and potentiated both proton- and heat-evoked currents. The reducing agent dithiothreitol (DTT) prevented and partially reversed UVA-light induced sensitization of hTRPV1. UVA-light induced sensitization was reduced in the mutant hTRPV1-C158A/C387S/C767S (hTRPV1-3C). The remaining sensitivity to UVA-light of hTRRPV1-3C was not further reduced upon exchange of the methionine residues M568 and M645. While UVA-induced sensitization was reduced in the protein kinase C-insensitive mutant hTRPV1-S502A/S801A, the PKC-inhibitors chelerythrine chloride, staurosporine and Gö6976 did not reduce UVA-induced effects on hTRPV1-WT. While hTRPV1-3C was insensitive to the cysteine-selective oxidant diamide, it displayed a residual sensitivity to H2O2 and chloramine-T. However, the exchange of M568 and M645 in hTRPV1-3C did not further reduce these effects. Our data demonstrate that oxidants and UVA-light gate hTRPV1 by cysteine-dependent as well as cysteine-independent mechanisms. In contrast to TRPV2, the methionine residues 568 and 645 seem to be of limited relevance for redox-sensitivity of hTRPV1. Finally, UVA-light induced gating of hTRPV1 does not seem to require activation of protein kinase C.

Induction of the reactive chlorine-responsive transcription factor RclR in Escherichia coli following ingestion by neutrophils.

Neutrophils generate hypochlorous acid (HOCl) and related reactive chlorine species as part of their defence against invading microorganisms. In isolation, bacteria respond to reactive chlorine species by upregulating responses that provide defence against oxidative challenge. Key questions are whether these responses are induced when bacteria are phagocytosed by neutrophils, and whether this provides them with a survival advantage. We investigated RclR, a transcriptional activator of the rclABC operon in Escherichia coli that has been shown to be specifically activated by reactive chlorine species. We first measured induction by individual reactive chlorine species, and showed that HOCl itself activates the response, as do chloramines (products of HOCl reacting with amines) provided they are cell permeable. Strong RclR activation was seen in E. coli following phagocytosis by neutrophils, beginning within 5 min and persisting for 40 min. RclR activation was suppressed by inhibitors of NOX2 and myeloperoxidase, providing strong evidence that it was due to HOCl production in the phagosome. RclR activation demonstrates that HOCl, or a derived chloramine, enters phagocytosed bacteria in sufficient amount to induce this response. Although RclR was induced in wild-type bacteria following phagocytosis, we detected no greater sensitivity to neutrophil killing of mutants lacking genes in the rclABC operon.

Safety and Effectiveness of Monochloramine Treatment for Disinfecting Hospital Water Networks.

The formation of potentially carcinogenic N-nitrosamines, associated with monochloramine, requires further research due to the growing interest in using this biocide for the secondary disinfection of water in public and private buildings. The aim of our study was to evaluate the possible formation of N-nitrosamines and other toxic disinfection by-products (DBPs) in hospital hot water networks treated with monochloramine. The effectiveness of this biocide in controlling Legionella spp. contamination was also verified. For this purpose, four different monochloramine-treated networks, in terms of the duration of treatment and method of biocide injection, were investigated. Untreated hot water, municipal cold water and, limited to N-nitrosamines analysis, hot water treated with chlorine dioxide were analyzed for comparison. Legionella spp. contamination was successfully controlled without any formation of N-nitrosamines. No nitrification or formation of the regulated DBPs, such as chlorites and trihalomethanes, occurred in monochloramine-treated water networks. However, a stable formulation of hypochlorite, its frequent replacement with a fresh product, and the routine monitoring of free ammonia are recommended to ensure a proper disinfection. Our study confirms that monochloramine may be proposed as an effective and safe strategy for the continuous disinfection of building plumbing systems, preventing vulnerable individuals from being exposed to legionellae and dangerous DBPs.

Inhibition of DNA methylation in proliferating human lymphoma cells by immune cell oxidants.

Excessive generation of oxidants by immune cells results in acute tissue damage. One mechanism by which oxidant exposure could have long-term effects is modulation of epigenetic pathways. We hypothesized that methylation of newly synthesized DNA in proliferating cells can be altered by oxidants that target DNA methyltransferase activity or deplete its substrate, the methyl donor SAM. To this end, we investigated the effect of two oxidants produced by neutrophils, H2O2 and glycine chloramine, on maintenance DNA methylation in Jurkat T lymphoma cells. Using cell synchronization and MS-based analysis, we measured heavy deoxycytidine isotope incorporation into newly synthesized DNA and observed that a sublethal bolus of glycine chloramine, but not H2O2, significantly inhibited DNA methylation. Both oxidants inhibited DNA methyltransferase 1 activity, but only chloramine depleted SAM, suggesting that removal of substrate was the most effective means of inhibiting DNA methylation. These results indicate that immune cell-derived oxidants generated during inflammation have the potential to affect the epigenome of neighboring cells.

Identification of a crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor-1 (FGFR1) and the coreceptor beta-Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1-KLB after iodination with Na125 I and chloramine T, whereas human FGF19 retained its affinity for FGFR1-KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine-102 and cysteine-121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1-KLB upon iodination. Site-directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1-KLB affinity. The results showed that mutation of a single tyrosine-207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1-KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1-KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine-207 is the crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21.

Ameliorative effect of taurine-chloramine in azathioprine-induced testicular damage; a deeper insight into the mechanism of protection.

BACKGROUND: The male reproductive system is a sensitive and intricate process that can be distressed following exposure to various toxicants. Therapeutic drugs, especially chemotherapeutics, can also adversely affect male fertility by instigating hormonal changes leading to testicular cells injury. Azathioprine (AZA) is an effective anticancer drug, but some cases of testicular toxicity have been reported. The aim of this work was to investigate the protective effects of taurine chloramine (TAU-Cl), a reported antioxidant and antiinflammtory peptide, against AZA-induced testicular dysfunction in male rats and ascertain the contributing mechanisms. METHODS: Forty male rats were allocated into four equal groups; (i) normal control rats, (ii) TAU-Cl group (100 mg/kg b.w/day for 10 weeks, (iii) AZA group (5 mg/day for 4 weeks); (iv) TAU-Cl/AZA group. RESULTS: AZA caused increased DNA damage in the testes, and alterations in sex hormones and sperm quality, including sperm count, viability, and motility. Moreover, testicular tissue from the AZA-treated group had increased levels of oxidative stress indicator, MDA, and decreased activity of the antioxidant enzymes as superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT) levels. These deleterious events were accompanied by upregulated levels of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and protein expression of iNOS and NFκB-p65, interleukin-1beta (IL-1ß), and proapoptotic marker; caspase-9, together with decreased Bcl-2, NrF2 and hemeoxygenase (HO-1) expression. In contrast, TAU-Cl pretreatment significantly abrogated these toxic effects which were confirmed histologically. CONCLUSION: Pretreatment with TAU-Cl exerts a protective effect against AZA-induced male reproductive testicular atrophy. This finding could open new avenues for the use of TAU-Cl as a complementary approach to chemotherapy supportive care.

Chloramine T induced oxidative stress and the response of antioxidant system in Phanerochaete chrysosporium.

In this study, the effect of chloramine T (Chl-T) on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione S-transferase (GST); the levels of reduced (GSH) and oxidised glutathione (GSSG) and their ratios; and also membrane lipid peroxidation (LPO) levels in Phanerochaete chrysosporium were investigated in a dose- (0.25-1 mmol/L) and time-dependent (1.5-9 h) manner. The highest SOD activity was observed in 0.5 mmol/L Chl-T at 6th hour as 1.48-fold of its control. The observed highest level in CAT activities was 4.6-fold of control in 0.5 and 0.75 mmol/L at the 6th hour. The GSH levels that were over the control showed decreasing tendency from the beginning of incubation, except 0.25 mmol/L. In contrast with GSH level variations, GSSG levels reached 10.0-fold of its control by showing increasing tendency with the increases in concentration and time. While the GSH/GSSG ratios were over the control at 0.25 mmol/L during all incubation, they fell under the control values at the earlier hours of incubation with the increasing concentrations of Chl-T. Glutathione-related enzymes GSH-Px, GR and GST were also induced with Chl-T treatment, and the highest activities were 3.29-, 7.5- and 6.56-fold of their controls, respectively. On the other hand, the increases in LPO levels with increasing concentration and time up to 5.27-fold of its control showed that the inductions observed in antioxidant system could not prevent the Chl-T-based oxidative stress.

Detection of viable but non-culturable legionella in hospital water network following monochloramine disinfection.

BACKGROUND: Prevention of legionellosis remains a critical issue in healthcare settings where monochloramine (MC) disinfection was recently introduced as an alternative to chlorine dioxide in controlling Legionella spp. contamination of the hospital water network. Continuous treatments with low MC doses in some instances have induced a viable but non-culturable state (VBNC) of Legionella spp. AIM: To investigate the occurrence of such dormant cells during a long period of continuous MC treatment. METHODS: Between November 2010 and April 2015, 162 water and biofilm samples were collected and Legionella spp. isolated in accordance with standard procedures. In sampling sites where MC was <1.5mg/L, VBNC cells were investigated by ethidium monoazide bromide (EMA)-real-time polymerase chain reaction (qPCR) and 'resuscitation' test into Acanthamoeba polyphaga CCAP 1501/18. According to the Health Protection Agency protocol, free-living protozoa were researched in 60 five-litre water samples. FINDINGS: In all, 136 out of 156 (87.2%) of the samples taken from sites previously positive for L. pneumophila ST269 were negative by culture, but only 47 (34.5%) negative by qPCR. Although no positive results were obtained by EMA-qPCR, four out of 22 samples associated with MC concentration of 1.3 ± 0.5mg/L showed VBNC legionella resuscitation. The presence of the amoeba A. polyphaga in the hospital water network was demonstrated. CONCLUSION: Our study is the first report evidencing the emergence of VNBC legionella during a long period of continuous MC treatment of a hospital water network, highlighting the importance of keeping an appropriate and uninterrupted MC dosage to ensure the control of legionella colonization in hospital water supplies.

Decontamination of Bacillus spores adhered to iron and cement-mortar drinking water infrastructure in a model system using disinfectants.

Decontamination of Bacillus spores adhered to common drinking water infrastructure surfaces was evaluated using a variety of disinfectants. Corroded iron and cement-mortar lined iron represented the infrastructure surfaces, and were conditioned in a 23 m long, 15 cm diameter (75 ft long, 6 in diameter) pilot-scale drinking water distribution pipe system. Decontamination was evaluated using increased water velocity (flushing) alone at 0.5 m s-1 (1.7 ft s-1), as well as free chlorine (5 and 25 mg L-1), monochloramine (25 mg L-1), chlorine dioxide (5 and 25 mg L-1), ozone (2.0 mg L-1), peracetic acid 25 mg L-1) and acidified nitrite (0.1 mol L-1 at pH 2 and 3), all followed by flushing at 0.3 m s-1 (1 ft s-1). Flushing alone reduced the adhered spores by 0.5 and 2.0 log10 from iron and cement-mortar, respectively. Log10 reduction on corroded iron pipe wall coupons ranged from 1.0 to 2.9 at respective chlorine dioxide concentrations of 5 and 25 mg L-1, although spores were undetectable on the iron surface during disinfection at 25 mg L-1. Acidified nitrite (pH 2, 0.1 mol L-1) yielded no detectable spores on the iron surface during the flushing phase after disinfection. Chlorine dioxide was the best performing disinfectant with >3.0 log10 removal from cement-mortar at 5 and 25 mg L-1. The data show that free chlorine, monochloramine, ozone and chlorine dioxide followed by flushing can reduce adhered spores by > 3.0 log10 on cement-mortar.

Antiseptics and microcosm biofilm formation on titanium surfaces.

Oral rehabilitation with osseointegrated implants is a way to restore esthetics and masticatory function in edentulous patients, but bacterial colonization around the implants may lead to mucositis or peri-implantitis and consequent implant loss. Peri-implantitis is the main complication of oral rehabilitation with dental implants and, therefore, it is necessary to take into account the potential effects of antiseptics such as chlorhexidine (CHX), chloramine T (CHT), triclosan (TRI), and essential oils (EO) on bacterial adhesion and on biofilm formation. To assess the action of these substances, we used the microcosm technique, in which the oral environment and periodontal conditions are simulated in vitro on titanium discs with different surface treatments (smooth surface - SS, acid-etched smooth surface - AESS, sand-blasted surface - SBS, and sand-blasted and acid-etched surface - SBAES). Roughness measurements yielded the following results: SS: 0.47 µm, AESS: 0.43 µm, SB: 0.79 µm, and SBAES: 0.72 µm. There was statistical difference only between SBS and AESS. There was no statistical difference among antiseptic treatments. However, EO and CHT showed lower bacterial counts compared with the saline solution treatment (control group). Thus, the current gold standard (CHX) did not outperform CHT and EO, which were efficient in reducing the biofilm biomass compared with saline solution.

Antiseptics and microcosm biofilm formation on titanium surfaces

Abstract Oral rehabilitation with osseointegrated implants is a way to restore esthetics and masticatory function in edentulous patients, but bacterial colonization around the implants may lead to mucositis or peri-implantitis and consequent implant loss. Peri-implantitis is the main complication of oral rehabilitation with dental implants and, therefore, it is necessary to take into account the potential effects of antiseptics such as chlorhexidine (CHX), chloramine T (CHT), triclosan (TRI), and essential oils (EO) on bacterial adhesion and on biofilm formation. To assess the action of these substances, we used the microcosm technique, in which the oral environment and periodontal conditions are simulated in vitro on titanium discs with different surface treatments (smooth surface - SS, acid-etched smooth surface - AESS, sand-blasted surface - SBS, and sand-blasted and acid-etched surface - SBAES). Roughness measurements yielded the following results: SS: 0.47 µm, AESS: 0.43 µm, SB: 0.79 µm, and SBAES: 0.72 µm. There was statistical difference only between SBS and AESS. There was no statistical difference among antiseptic treatments. However, EO and CHT showed lower bacterial counts compared with the saline solution treatment (control group). Thus, the current gold standard (CHX) did not outperform CHT and EO, which were efficient in reducing the biofilm biomass compared with saline solution.

Biocidal efficacy of monochloramine against planktonic and biofilm-associated Naegleria fowleri cells.

AIMS: Free-living amoebae (FLA) in aqueous systems are a problem for water network managers and health authorities because some are pathogenic, such as Naegleria fowleri, and they have also been reported to operate as reservoirs and vectors of several pathogenic bacteria. Therefore, to better control the occurrence of such amoebae, we evaluate the efficacy of monochloramine against planktonic forms (trophozoites and cysts) and also biofilm-associated cells of N. fowleri as FLA are often associated with biofilms. METHODS AND RESULTS: From a freshwater biofilm growing in a pilot reactor and inoculated with N. fowleri, we obtained Ct values ranging from 4 to 17 mg Cl2 min l(-1) at 25°C and pH 8·2 on both planktonic and biofilm associated cells. In addition, the inactivation pattern of biofilm associated was intermediate between those of trophozoïtes and cysts. CONCLUSIONS: The monochloramine efficiency varies with the life stage of N. fowleri (trophozoïte, cyst, and biofilm-associated). The sensitivity to disinfectant of amoeba, that is, trophozoïtes and cysts, in the biofilm life stage is as high as that of their planktonic cyst form. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives Ct values for cysts and biofilm-associated N. fowleri. This may impact on water treatment strategies against amoebae and should be considered when controlling N. fowleri in man-made water systems such as cooling towers or hot water systems.

Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine.

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin ß3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

Monochloramine suppresses the proliferation of colorectal cancer cell line Caco-2 by both apoptosis and G2/M cell cycle arrest.

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.

Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants.

Free-living amoebae are naturally present in water. These protozoa could be pathogenic and could also shelter pathogenic bacteria. Thus, they are described as a potential hazard for health. Also, free-living amoebae have been described to be resistant to biocides, especially under their cyst resistant form. There are several studies on amoeba treatments but none of them compare sensitivity of trophozoites and cysts from different genus to various water disinfectants. In our study, we tested chlorine, monochloramine and chlorine dioxide on both cysts and trophozoites from three strains, belonging to the three main genera of free-living amoebae. The results show that, comparing cysts to trophozoites inactivation, only the Acanthamoeba cysts were highly more resistant to treatment than trophozoites. Comparison of the disinfectant efficiency led to conclude that chlorine dioxide was the most efficient treatment in our conditions and was particularly efficient against cysts. In conclusion, our results would help to adapt water treatments in order to target free-living amoebae in water networks.