Possible nonimmunological toxicological mechanisms of vesnarinone-associated agranulocytosis in HL-60 cells: role of reduced glutathione as cytotoxic defense.

This study was conducted as part of an investigation into the cause of vesnarinone-associated agranulocytosis. When HL-60 cells were exposed to vesnarinone for 48 hr, little cytotoxicity was observed, although reduced glutathione (GSH) content decreased in a concentration-dependent manner. Significant cytotoxicity and reactive oxygen species (ROS) production were observed when intracellular GSH content was reduced by treatment with L-buthionine-(S, R)-sulphoximine. The involvement of myeloperoxidase (MPO) metabolism was suggested, as when HL-60 cells were exposed to a reaction mixture of vesnarinone-MPO/H2O2/Cl-, cytotoxicity was also observed. In contrast, the presence of GSH (1 mM) protected against these cytotoxic effects. Liquid chromatography-mass spectrometry analysis of the MPO/H2O2/Cl- reaction mixture revealed that vesnarinone was converted into two metabolites, (4-(3,4-dimethoxybenzoyl)piperazine [Metabolite 1: M1] and 1-chloro-4-(3,4-dimethoxybenzoyl)piperazine [Metabolite 2: M2]). M2 was identified as the N-chloramine form, a reactive metabolite of M1. Interestingly, M2 was converted to M1, which was accompanied by the conversion of GSH to oxidized GSH (GSSG). Furthermore, when HL-60 cells were exposed to synthetic M1 and M2 for 24 hr, M2 caused dose-dependent cytotoxicity, whereas M1 did not. Cells were protected from M2-derived cytotoxicity by the presence of GSH. In conclusion, we present the first demonstration of the cytotoxic effects and ROS production resulting from the MPO/H2O2/Cl- metabolic reaction of vesnarinone and newly identified the causative metabolite, M2, as the N-chloramine metabolite of M1, which induces cytotoxicity in HL-60 cells. Moreover, a protective role of GSH against the cytotoxicity was revealed. These findings suggest a possible nonimmunological cause of vesnarinone agranulocytosis.

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.

Composition of active bacterial communities and presence of opportunistic pathogens in disinfected and non-disinfected drinking water distribution systems in Finland.

Many factors, including microbiome structure and activity in the drinking water distribution system (DWDS), affect the colonization potential of opportunistic pathogens. The present study aims to describe the dynamics of active bacterial communities in DWDS and identify the factors that shape the community structures and activity in the selected DWDSs. Large-volume drinking water and hot water, biofilm, and water meter deposit samples were collected from five DWDSs. Total nucleic acids were extracted, and RNA was further purified and transcribed into its cDNA from a total of 181 water and biofilm samples originating from the DWDS of two surface water supplies (disinfected with UV and chlorine), two artificially recharged groundwater supplies (non-disinfected), and a groundwater supply (disinfected with UV and chlorine). In chlorinated DWDSs, concentrations of <0.02-0.97 mg/l free chlorine were measured. Bacterial communities in the RNA and DNA fractions were analysed using Illumina MiSeq sequencing with primer pair 341F-785R targeted to the 16S rRNA gene. The sequence libraries were analysed using QIIME pipeline, Program R, and MicrobiomeAnalyst. Not all bacterial cells were active based on their 16S rRNA content, and species richness was lower in the RNA fraction (Chao1 mean value 490) than in the DNA fraction (710). Species richness was higher in the two DWDSs distributing non-disinfected artificial groundwater (Chao1 mean values of 990 and 1 000) as compared to the two disinfected DWDSs using surface water (Chao1 mean values 190 and 460) and disinfected DWDS using ground water as source water (170). The difference in community structures between non-disinfected and disinfected water was clear in the beta-diversity analysis. Distance from the waterworks also affected the beta diversity of community structures, especially in disinfected distribution systems. The two most abundant bacteria in the active part of the community (RNA) and total bacterial community (DNA) belonged to the classes Alphaproteobacteria (RNA 28 %, DNA 44 %) and Gammaproteobacteria (RNA 32 %, DNA 30 %). The third most abundant and active bacteria class was Vampirovibrionia (RNA 15 %), whereas in the total community it was Paceibacteria (DNA 11 %). Class Nitrospiria was more abundant and active in both cold and hot water in DWDS that used chloramine disinfection compared to non-chlorinated or chlorine-using DWDSs. Thirty-eight operational taxonomic units (OTU) of Legionella, 30 of Mycobacterium, and 10 of Pseudomonas were detected among the sequences. The (RT)-qPCR confirmed the presence of opportunistic pathogens in the DWDSs studied as Legionella spp. was detected in 85 % (mean value 4.5 × 104 gene copies/100 ml), Mycobacterium spp. in 95 % (mean value 8.3 × 106 gene copies/100 ml), and Pseudomonas spp. in 78 % (mean value 1.6 × 105 gene copies/100 ml) of the water and biofilm samples. Sampling point inside the system (distance from the waterworks and cold/hot system) affected the active bacterial community composition. Chloramine as a chlorination method resulted in a recognizable community composition, with high abundance of bacteria that benefit from the excess presence of nitrogen. The results presented here confirm that each DWDS is unique and that opportunistic pathogens are present even in conditions when water quality is considered excellent.

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.

Lysine and Arginine Reactivity and Transformation Products during Peptide Chlorination.

Chlorine reactions with peptide-bound amino acids form disinfection byproducts and contribute to pathogen inactivation by degrading protein structure and function. Peptide-bound lysine and arginine are two of the seven chlorine-reactive amino acids, but their reactions with chlorine are poorly characterized. Using N-acetylated lysine and arginine as models for peptide-bound amino acids and authentic small peptides, this study demonstrated conversion of the lysine side chain to mono- and dichloramines and the arginine side chain to mono-, di-, and trichloramines in ≤0.5 h. The lysine chloramines formed lysine nitrile and lysine aldehyde at ∼6% yield over ∼1 week. The arginine chloramines formed ornithine nitrile at ∼3% yield over ∼1 week but not the corresponding aldehyde. While researchers hypothesized that the protein aggregation observed during chlorination arises from covalent Schiff base cross-links between lysine aldehyde and lysine on different proteins, no evidence for Schiff base formation was observed. The rapid formation of chloramines and their slow decay indicate that they are more relevant than the aldehydes and nitriles to byproduct formation and pathogen inactivation over timescales relevant to drinking water distribution. Previous research has indicated that lysine chloramines are cytotoxic and genotoxic to human cells. The conversion of lysine and arginine cationic side chains to neutral chloramines should alter protein structure and function and enhance protein aggregation by hydrophobic interactions, contributing to pathogen inactivation.

Overlooked Iodo-Disinfection Byproduct Formation When Cooking Pasta with Iodized Table Salt.

Iodized table salt provides iodide that is essential for health. However, during cooking, we found that chloramine residuals in tap water can react with iodide in table salt and organic matter in pasta to form iodinated disinfection byproducts (I-DBPs). While naturally occurring iodide in source waters is known to react with chloramine and dissolved organic carbon (e.g., humic acid) during the treatment of drinking water, this is the first study to investigate I-DBP formation from cooking real food with iodized table salt and chloraminated tap water. Matrix effects from the pasta posed an analytical challenge, necessitating the development of a new method for sensitive and reproducible measurements. The optimized method utilized sample cleanup with Captiva EMR-Lipid sorbent, extraction with ethyl acetate, standard addition calibration, and analysis using gas chromatography (GC)-mass spectrometry (MS)/MS. Using this method, seven I-DBPs, including six iodo-trihalomethanes (I-THMs) and iodoacetonitrile, were detected when iodized table salt was used to cook pasta, while no I-DBPs were formed with Kosher or Himalayan salts. Total I-THM levels of 11.1 ng/g in pasta combined with cooking water were measured, with triiodomethane and chlorodiiodomethane dominant, at 6.7 and 1.3 ng/g, respectively. Calculated cytotoxicity and genotoxicity of I-THMs for the pasta with cooking water were 126- and 18-fold, respectively, compared to the corresponding chloraminated tap water. However, when the cooked pasta was separated (strained) from the pasta water, chlorodiiodomethane was the dominant I-THM, and lower levels of total I-THMs (retaining 30% of the I-THMs) and calculated toxicity were observed. This study highlights an overlooked source of exposure to toxic I-DBPs. At the same time, the formation of I-DBPs can be avoided by boiling the pasta without a lid and adding iodized salt after cooking.

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.

Effects of different types of nitrogen sources in water on the formation potentials of nitrogenous disinfection by-products in chloramine disinfection process based on isotope labeling.

Nitrogenous disinfection by-products (N-DBPs) raise increasing concerns because of their high genotoxicity, cytotoxicity, and carcinogenicity compared to carbonaceous disinfection by-products (C-DBPs). Nitrogen-containing disinfectants, dissolved organic nitrogen (DON), and inorganic nitrogen may all promote the formation of N-DBPs. Therefore, it is urgent to explore the dominant nitrogen source of N-DBPs under the coexistence of multiple nitrogen sources. In this study, the effects of amino acids, nitrate, ammonia, and chloramine as different types of nitrogen sources on the formation of five N-DBPs were investigated systematically, including chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), bromochloroacetonitrile (BCAN), dibromoacetonitrile (DBAN) and dichloroacetamide (DCAcAm). L-Aspartic acid (L-Asp) as the organic nitrogen source showed a high potential on the formation of N-DBPs by forming acetonitrile intermediates. Ammonia as the inorganic nitrogen source consumed oxidants and changed the existing form of chloramine, thus inhibiting the formation of N-DBPs. Instead of providing nitrogen to N-DBPs, nitrate as a salt promoted the volatilization of N-DBPs, thereby reducing the detected N-DBPs. Furthermore, an isotope labeling method was applied to clearly trace the nitrogen sources of N-DBPs via GC-MS with electron ionization. 15N-chloramine, 15N-amino acid, 15N-nitrate and 15N-ammonia were selected as the corresponding isotopic nitrogen sources. The results indicated that chloramine was the major nitrogen contributor to five N-DBPs during the chloramination of L-Asp under the coexistence of multiple nitrogen sources, ranging from 61 % to 79 %. The influence of environmental factors (reaction time, pH, and bromide) on the formation of N-DBPs during chloramination was also investigated. There was competition between brominated N-DBPs and chlorinated N-DBPs in chloramination. With the increase of reaction time or bromine, the formation potentials of chlorinated N-DBPs gradually decreased, while brominated N-DBPs gradually increased. Moreover, higher pH inhibited the generation of N-DBPs.

Chloramine-T-Enabled Mass Spectrometric Analysis of C═C Isomers of Unsaturated Fatty Acids and Phosphatidylcholines in Human Thyroids.

Specific locations of carbon-carbon double bonds (C═C) in lipids often play an essential role in biological processes, and there has been a booming development in C═C composition analysis by mass spectrometry. However, a universal derivatization and fragmentation pattern for the annotation of C═C positions in lipids is still challenging and attractive. To expand this field in lipidomics, a flexible and convenient N-tosylaziridination method was developed, with high derivatization efficiency, sensitivity, and specificity. The derivatization was very fast (15 s), and C═C numbers as well as locations could be pinpointed specifically in tandem mass spectra. By qualitative and quantitative studies of paratumor and tumor thyroid tissues of human beings, the total content of unsaturated fatty acids was suggested to be increased in tumor tissues, and good correlations in and between lysophosphatidylcholines and phosphatidylcholines were revealed by Spearman analysis. Further studies of C═C isomers showed that n-6/n-3 ratios were closely associated with human thyroid tumorigenesis, and high ratios of n-6/n-3 isomers seemed to suffer a high risk of carcinogenesis. Other isomers were not very representative; however, C═C in n-9/n-7 could also be significant for oncology research. Generally, it is supposed that both total amounts and C═C isomer ratios were related to cancer, and N-tosylaziridine derivatization could provide an alternative strategy for the C═C isomer study of disease models.

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.

Radiosynthesis and in silico bioevaluation of 131 I-Sulfasalazine as a highly selective radiotracer for imaging of ulcerative colitis.

This study demonstrated the tracking of ulcerative colitis, which is considered a stressful immune disease. Although there are many ways to test for this disease including dependence on gases, dyes, and painful anal endoscopy, these treatment modalities have many disadvantages. Hence, it is the utmost need of time to discover new methods to detect this chronic immune disease and to avoid the defects of traditional methodologies. Sulfasalazine (SSD) was labeled with iodine-131 (half-life: 8 days, Energy: 971 keV) under optimum reaction conditions including the amount of reducing agent, pH factor, chloramine-T (Ch-T) amount, and incubation period. Characterization was performed using 1 H/ 13 C-NMR, ESI-MS, and HPLC (UV/ Radio) techniques. The biodistribution study was performed in normal and ulcerative mice models, and in silico molecular docking study was performed to evaluate the possible mechanism of action to target peroxisome proliferator-activated receptor gamma (PPARγ). The high radiolabeling yield of [131 I]-sulfasalazine ([131 I]-SSD) was achieved ≥90% through the direct labeling method with radioactive iodine-131 in the presence of chloramine-T (100 µg). The radiotracer [131 I]-SSD was observed to be stable in normal saline and freshly eluted serum up to 12 hr at ambient temperature (37℃ ± 2℃). The radiotracer [131 I]-SSD showed the highest uptake in the targeted organ (i.e., ulcerative colon) which was observed to be ≥75% injected dose per gram (% ID/g) organ for 24 hr postinjection (p.i). Furthermore, in silico data collected from molecular modeling analysis of SSD and [131 I]-SSD with antimicrobial protein (PDB code: 3KEG) and peroxisome proliferator-activated receptor gamma (PPARγ) (PDB code: 4XTA) showed azoreductase activity and high binding potential for PPAR-γ site, respectively. The results of biological studies obtained in this study enlighten the usefulness of radiotracer [131 I]-SSD as a potential imaging agent for ulcerative colitis.

Avaliação de ph externo utilizando diferentes soluções irrigantes e medicações intracanais / External ph evaluation using different irrigation solutions and intracanal medications

O objetivo do presente estudo foi mensurar o pH externo radicular de dentes bovinos que foram desinfetados em solução de cloramina por 7 dias. Neste estudo foram utilizadas soluções irrigadoras, hipoclorito de sódio 1%, clorexidina 0,12% e hipoclorito de sódio 2,5% associadas ao edta e as medicações intracanais, hidróxido de cálcio, hidróxido de cálcio com PMCC e clorexidina gel 2%. O preparo químico mecânico foi realizado com as limas easy logic e as soluções foram agitadas com ultrassom durante 3 minutos e após os dentes foram imersos em água deionizada em eppendorfs estéreis e mantidos em estufa a 37°C. O pH externo foi analisado utilizando as fitas medidoras de pH no período de 24 horas, 48 horas, 7 dias, 10 dias e 15 dias. A normalidade dos valores obtidos de cada ensaio foi testada através do teste Kolmogorof- Smirnov, e o teste estatístico foi ANOVA de uma via e comparações múltiplas de Tukey. Os resultados mostraram que houve diferença estatística nos valores de pH nos grupos avaliados dentro dos tempos (p< 0,05). Concluiu-se que mesmo com as variações de pH nos períodos avaliados, as medicações e as soluções irrigantes podem ser utilizadas de forma associadas na endodontia, com o intuito de eliminar o maior número de microrganismos dos canais radiculares(AU)

The objective of the present study was to measure the external root pH of bovine teeth that were disinfected in chloramine solution for 7 days. Irrigating solutions, 1% sodium hypochlorite, 0.12% chlorhexidine and 2.5% sodium hypochlorite associated with edta and intracanal medications, calcium hydroxide, calcium hydroxide with PMCC and chlorhexidine gel 2%. The mechanical chemical preparation was performed with the easy logic files and the solutions were shaken with ultrasound for 3 minutes and after the teeth were immersed in deionized water in eppendorfs and kept in an oven at 37 ° C. The external pH was analyzed using the pH measuring tapes in the period of 24 hours, 48 hours, 7 days, 10 days and 15 days. The normal values obtained from each test were tested using the Kolmogorof-Smirnov test, and the statistical test was one-way ANOVA and Tukey's multiple comparisons. The results showed that there was a statistical difference in pH values in the groups evaluated within the times (p< 0.05). It was concluded that even with pH variations in the evaluated periods, medications and irrigating solutions can be used in an associated way in endodontics, in order to eliminate the largest number of microorganisms in the root canals(AU)

Pleiotropic Properties of Valsartan: Do They Result from the Antiglycooxidant Activity? Literature Review and In Vitro Study.

Valsartan belongs to angiotensin II type 1 (AT1) receptor blockers (ARB) used in cardiovascular diseases like heart failure and hypertension. Except for its AT1-antagonism, another mechanism of drug action has been suggested in recent research. One of the supposed actions refers to the positive impact on redox balance and reducing protein glycation. Our study is aimed at assessing the antiglycooxidant properties of valsartan in an in vitro model of oxidized bovine serum albumin (BSA). Glucose, fructose, ribose, glyoxal (GO), methylglyoxal (MGO), and chloramine T were used as glycation or oxidation agents. Protein oxidation products (total thiols, protein carbonyls (PC), and advanced oxidation protein products (AOPP)), glycooxidation products (tryptophan, kynurenine, N-formylkynurenine, and dityrosine), glycation products (amyloid-ß structure, fructosamine, and advanced glycation end products (AGE)), and albumin antioxidant activity (total antioxidant capacity (TAC), DPPH assay, and ferric reducing antioxidant power (FRAP)) were measured in each sample. In the presence of valsartan, concentrations of protein oxidation and glycation products were significantly lower comparing to control. Moreover, albumin antioxidant activity was significantly higher in those samples. The drug's action was comparable to renowned antiglycation agents and antioxidants, e.g., aminoguanidine, metformin, Trolox, N-acetylcysteine, or alpha-lipoic acid. The conducted experiment proves that valsartan can ameliorate protein glycation and oxidation in vitro in various conditions. Available animal and clinical studies uphold this statement, but further research is needed to confirm it, as reduction of protein oxidation and glycation may prevent cardiovascular disease development.

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.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

The competitive effect of different chlorination disinfection methods and additional inorganic nitrogen on nitrosamine formation from aromatic and heterocyclic amine-containing pharmaceuticals.

Amine-containing pharmaceuticals formed nitrosamines that are nitrogenous disinfection byproducts of public concerns due to their carcinogenicity. The objective of this study was to investigate the co-effect of additional inorganic nitrogen in different forms (ammonium, nitrite, and nitrate) and different disinfection approaches (chlorination, monochloramination, dichloramination, and two-step chlorination) on eight nitrosamine formation from four widely used pharmaceuticals. N-nitrosodimethylamine (NDMA) was the main species formed. The presence of N-nitrosomethylethylamine (NMEA), nitrosomorpholine (NMor), and N-nitrosopiperidine (NPip) was found in certain experiments. For one-step chlorination, the influential factors, in decreasing order of importance, were the molecular structural characteristics of the pharmaceutical, oxidation method, and presence and form of additional nitrogen. In four pharmaceuticals with comparative structures, the availability of amine intermediates during degradation was the key to higher nitrosamine yields. Monochloramine significantly enhanced nitrosamine formation from four pharmaceuticals. NDMA formation by adding hypochlorous acid and ammonium separately were lower than those during monochloramination. During two-step chlorination, NDMA formation was enhanced at certain pre-chlorine doses (e.g., a Cl/N molar ratio of 20 or 4). The pre-chlorine dose changed the Cl/N ratio. As the ratio was increased, the combined chlorine residual was formed and decreased. When the ratio was high, breakpoint chlorination possibly occurred enhancing NDMA formation. While NDMA formation was successfully inhibited by two-step chlorination, ammonium brought the NDMA yields of these pharmaceuticals back to the range observed in chloramination, suggesting the importance of ammonium control for limiting NDMA formation from pharmaceuticals during two-step chlorination.

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.

[Effect of disinfectant with benzethon chloramine and isopropanol as main active ingredients on the accuracy of dental impression].

OBJECTIVE: To assess the effect of disinfectant (Cavicide) with benzethon chloramine and isopropanol as main active ingredients disinfectant on dental impression accuracy. METHODS: The effect of Cavicide on three impression materials (alginate, polyether and vinylpolysiloxane) were assessed using a standard model. The standard model was digitized by an extraoral scanner (IScan D103i, Imetric). For each kind of impression materials, thirty impressions were taken following the manufactures' instruction in the same conditions. Subsequently, the impressions were randomly divided into three groups, with ten impressions in each group. After the impression taking was completed, the three groups underwent pure water rinse for 1 min (blank control, BC), 2% glutaraldehyde solution immersion disinfection for 30 min (glutaraldehyde, GD), and Cavicide solution spray disinfection for 5 min (Cavicide, CC), respectively. All the impressions were digitized by the extraoral scanner (IScan D103i, Imetric) after disinfection and exported to a dedicated three-dimensional analysis software (Geomagic Qualify 2014, Geomagic, USA). In the software, the digital models of the impressions were trimmed to teeth and then superimposed with the digitized standard model via best-fit alignment. Root mean square (RMS) was used to evaluate the deviations between the impression and the standard model. The deviation in the anterior and posterior regions was evaluated respectively. One-way ANOVA test and the LSD post-hoc test were used to compare the deviations between the three groups (P < 0.05). The color map of each superimposition was saved for visual analysis. RESULTS: For the polyether and vinylpolysiloxane materials, the difference between the three groups was not statistically significant (P=0.933, P=0.827). For the alginate material, the difference in posterior region between group GD and group BC, as well as group GD and group CC were statistically significant (GD vs. BC, P=0.001; GD vs. CC, P=0.002), while the difference between group BC and group CC was not statistically significant (P=0.854). The visual analysis showed an obvious deviation in the buccal-lingual direction in group GD. CONCLUSION: Disinfectant (Cavicide) with benzethon chloramine and isopropanol as main active ingredients using spray disinfection has no effect on the accuracy of the alginate, polyether and vinylpolysiloxane impressions.

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.

UV/sulfite chemistry to reduce N-nitrosodimethylamine formation in chlor(am)inated water.

The disinfection by-product N-nitrosodimethylamine (NDMA) is a major concern in water quality management due to its carcinogenicity. Thus, a proper pretreatment is necessary to mitigate NDMA formation upon periodic chloramination by removing precursors, such as ranitidine (RNT). This study investigated the effect of UV/sulfite pretreatment on NDMA formation from an RNT-spiked tap and chloraminated synthetic swimming pool (SSP) water. At UVC intensity of 2.1 mW cm-2 and 0.5 mM of sulfite, UV/sulfite chemistry showed complete degradation of 20 µM RNT within 30 min. It was found that SO4•- primarily reduced the NDMA formation potential (FP) of RNT, while hydrated electrons effectively mitigated the pre-formed NDMA in the SSP water. The UV/sulfite pretreatment alleviated NDMA formation during post-chloramination (24 h) by up to 82%, outperforming the commonly employed advanced oxidation processes such as UV/H2O2. However, in the presence of bromide ions, the effectiveness of UV/sulfite pretreatment was seriously deteriorated, although the bromide ion itself was found to inhibit the NDMA formation from RNT especially at pH < 8 during chloramination. Mass spectrometric analysis indicated that the NDMA-FP of RNT could be removed by UV/sulfite principally via N-methylation, dealkylation, and oxygen transfer pathways. Consequently, UV/sulfite could be used as an alternative unit process for water treatment with reduced NDMA formation.