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Comparative studies of Iodo-bead and chloramine-T methods for the radioiodination of human alpha-fetoprotein.

Human alpha-fetoprotein (h-AFP) of iodination grade was radiolabeled with Na125I by chloramine-T (conventional method) and by Iodo-bead (solid-phase method) in our laboratory under standard and varied conditions, such as quantity of h-AFP and volume of the reaction mixture. We have slightly modified the chloramine-T method and developed a protocol for the Iodo-bead method of iodination. Our results show that while the chloramine-T method yields radiolabeled h-AFP of apparently 40% higher specific radioactivity (SR) than those of the Iodo-bead method, the latter appears to be a simpler, more controllable and milder method, giving rise to a more stable population of [125I]h-AFP. This is supported by the results of radioactive counting of [125I]h-AFP collected from a Sephadex G-50 column after radioiodination, storage stability and immunoreactivity with anti-h-AFP.

Chloramines↗

Quinidine blocks cardiac sodium current after removal of the fast inactivation process with chloramine-T.

To determine if the fast sodium current inactivation process is necessary for sodium current (INa) blockade by quinidine, we studied the effects of quinidine on INa in guinea-pig ventricular myocytes treated with chloramine-T, which removes the fast inactivation process of INa. Following exposure to chloramine-T (2 mM), INa amplitude was reduced at all voltages and INa decay was irreversibly prevented. Quinidine (10 microM) produced resting block of INa of 36 +/- 2% (n = 5) at the peak potential of -30 mV in chloramine-T treated myocytes. Quinidine decreased INa in a dose-dependent manner. The half-blocking concentration (KD) was 1.9 +/- 0.2 x 10(-5) M (n = 4). The steady-state inactivation curve (hx) was shifted in the negative potential direction (-5.2 +/- 0.4 mV, n = 4). Even after removal of the fast inactivation process of INa, use-dependent block was observed in the presence of quinidine when various depolarizing pulse durations (5 ms approximately 200 ms) were applied repetitively at intervals of 300 ms approximately 2 s. Longer depolarizing pulses and higher frequency pulse trains produced greater use-dependent block. Use-dependent block was also enhanced at more positive holding potentials. These results suggest that quinidine produces both resting block and use-dependent block of sodium channels in the absence of the fast INa inactivation process.

Animals↗

Marked improvement of a substance P radioimmunoassay by reduction of 125I-labelled [Tyr8]-substance P prepared by the chloramine-T method with mercaptoethanol and subsequent purification by reversed-phase liquid chromatography.

[Tyr8]-substance P was radiolabelled with 125I by the application of the chloramine-T method. Due to the high oxidative potential of the 125I-chloramine-T system the purified reaction product was converted into a derivative, which presumably had been oxidized to the corresponding sulphoxide at Met11. This conversion was shown by reversed-phase high-performance liquid chromatography after consecutive reduction-oxidation experiments of the freshly prepared radiopeptide. The oxidized derivative exhibited only negligible binding to substance P receptors isolated from rat brain homogenates. However, in contrast, it showed marked cross-reaction to the antibody raised in rabbits against synthetic SP(1-11). The variance in the quantification of identical samples was marked, and the measurement of concentrations in the lower pg/ml range was not sensitive enough to determine levels of substance P-like immunoreactivity in human cerebrospinal fluid. Assay sensitivity could be substantially improved and variance significantly decreased by the use of a radiopeptide, which had been labelled by the chloramine-T method and which had been subsequently reduced with mercaptoethanol and purified by liquid chromatography.

Chloramines↗

Exposure to chloramines in a green salad processing plant.

Acute eye and upper respiratory irritation was reported at an industrial facility that processes green salads in water containing hypochlorite. The irritant agents were chloramines resulting from the reaction of hypochlorite and nitrogen compounds coming from the sap proteins released when the vegetables were cut. Specific sampling and analytical methods were developed in order to assess the workers' exposure. Monochloramine, dichloramine and nitrogen trichloride were found in the atmosphere, which can explain the irritation phenomena of which the workers complained. In the washing room (where salads are processed), the total chloramine concentration ranged from 0.4 to 16 mg.m-3, depending on the day and the location of the area sampling points. The exposure of workers determined by personal sampling ranged from 0.2 to 5 mg.m-3. In a previous study, with swimming pool instructors exposed to the same pollutants, irritation phenomena generally appeared at chloramine values of around 0.5 mg.m-3.

Chloramines↗

Morphometric assessment of epidermal and mucous-biofilm changes caused by exposure of trout to chloramine-T or formalin treatment.

Juvenile rainbow trout were exposed to therapeutic concentrations of formalin or chloramine-T to assess the effects of these chemicals on the morphology of the piscine epidermis and its mucous coat. Repeated treatment, once weekly for 4 weeks, with either chemical did not affect the mucous coat of the epithelium or the degree of folding of the basal lamina. However, treated fish had increased numbers of highly dense vesicles within the apical portions of epithelial cells. The epidermal mucous cells of chloramine-T-treated fish were significantly smaller than in controls. This effect was not noted in formalin-treated fish. Treatment with either chemical resulted in a significantly thinned epidermis. It is concluded that although chloramine-T and formalin may continue to be useful in the aquaculture industry they cause potentially harmful alterations to fish skin.

Animals↗

Determination of N-chloramines in As-samra chlorinated wastewater and their effect on the disinfection process.

Chlorine is the most widely used disinfectant of wastewater due to its capacity to inactivate most pathogenic microorganisms quickly. However, chlorine reacts with natural organic compounds present in wastewater to produce some undesirable organic byproducts. One such class of compounds is the nitrogenous compounds. The reaction between chlorine and compounds containing a nitrogen atom with one or more hydrogen atoms attached to it will form chloramines which have lower disinfection efficiency. Eighty percent of the wastewater generated in Jordan is treated at the Khirbet As-Samra wastewater treatment plant for eventual use in agriculture. In this study efficiency of chlorination was studied by collecting samples from the effluent of the treatment plant. The yield concentration of N-chloramines upon chlorination was determined. Efficiency of disinfection process as a function of contact time, concentration of chlorine dosage, concentration of nitrogenous compound and pH were studied. In this study, it has been found that at a chlorine dosage of 15 mg/L and contact time of 15 min, the percentage total coliform kill in As-samra wastewater sample was 100%. After addition of histidine, glycine and phenylalanine (15 mg/L in each case) to the wastewater sample, the percentage of total coliform kill dropped to 58, 78 and 79% respectively. When chlorine dosage was increased to 24 mg/L the percentage total coliform kill reached 96, 99 and 100% in wastewater samples treated with 5 mg/L histidine, glycine and phenylalanine, respectively. The percentage total coliform kill dropped to zero in wastewater samples treated with histidine, glycine and phenylalanine at a concentration of 30 mg/L each. This work supports the theory that amino acids and ammonia preferentially react with chlorine to form N-chloramine which significantly reduces the disinfection efficiency of the chlorination process.

Ammonium Chloride↗

Comparison of bacterial regrowth in distribution systems using free chlorine and chloramine: a statistical study of causative factors.

Bacterial regrowth was investigated over a 15-month period in distribution systems (DSs) of Durham and Raleigh in North Carolina. These two water utilities were chosen because they are adjacent to one another, have similar service area characteristics, and treat surface waters of similar characteristics with conventional processes (coagulation-sedimentation and dual-media filtration). The finished waters have similar chemical quality and regrowth potential as measured by assimilable organic carbon (AOC). The major difference in treatment is the choice of final disinfectants (chlorine in Durham and chloramine in Raleigh). Ten sampling sites (monthly sampling) were chosen in each system to give wide geographic coverage and correspondingly, a wide range of water residence times. Significant losses were observed in both chlorine and chloramine residual in the DSs that produced bacterial regrowth as measured by heterotrophic plate count (HPC). The frequency distributions for log HPC (133 observations from Durham and 135 observations from Raleigh) were statistically the same in the chlorinated and chloraminated DSs. A correlation analysis indicated that disinfectant residual is the most important factor determining HPC level. However, the resulting R2 value for a non-linear regression model that also included AOC, temperature, and pH as independent variables was less than 0.7. Bacterial regrowth as measured by HPC, is dependent upon a complex interaction of chemical, physical, and operational parameters that may not be captured by such a simple statistical relationship.

Bacteria↗

Influence of the order of reagent addition on NDMA formation during chloramination.

The formation of the potent carcinogen, N-nitrosodimethylamine (NDMA), during chlorine disinfection has caused significant concern among drinking water and wastewater recycling utilities practicing intentional or unintentional chloramination. Previous research modeled NDMA formation as arising from a reaction between monochloramine and organic nitrogen precursors, such as dimethylamine, via an unsymmetrical dimethylhydrazine (UDMH) intermediate. Contrary to the importance of monochloramine indicated by previous studies, hypochlorite formed an order of magnitude more NDMA than monochloramine when applied to a secondary municipal wastewater effluent containing excess ammonia. Experiments involving variation of the order that each reagent (i.e., hypochlorite, ammonium chloride, and dimethylamine) was added to solution suggest two factors that may be more important for NDMA formation than the presence of monochloramine: (i) the chlorination state of organic nitrogen precursors and (ii) the partial formation of dichloramine. Although dichloramine formation was most influenced by the pH conditions under which inorganic chloramine formation was performed, mixing effects related to the order of reagent addition may be important at full-scale plants. Chloramination strategies are suggested that may reduce NDMA formation by nearly an order of magnitude.

Ammonium Chloride↗

Triclosan reactivity in chloraminated waters.

Triclosan, widely employed as an antimicrobial additive in many household personal care products, has recently been detected in wastewater treatment plant effluents and in source waters used for drinking water supplies. Chloramines used either as alternative disinfectants in drinking water treatment or formed during chlorination of nonnitrified wastewater effluents have the potential to react with triclosan. This study examined triclosan reactivity in chloraminated waters over the pH range of 6.5-10.5. Experimental and modeling results show that monochloramine directly reacts with the phenolate form of triclosan; however, the reaction is relatively slow as evinced by the second-order rate constant k(ArO)-NH2Cl = 0.025 M(-1) s(-1). Kinetic modeling indicates that for pH values less than 9.5, reactions between triclosan and two monochloramine autodecomposition intermediates, hypochlorous acid (k(ArO)-HOC = 5.4 x 10(3) M(-1) s(-1)) and dichloramine (k(ArO)-NHCl2 = 60 M(-1) s(-1)), are responsible for a significant percentage of the observed triclosan decay. The products of these reactions include three chlorinated triclosan byproducts as well as 2,4-dichlorophenol and 2,4,6-trichlorophenol. Low levels of chloroform were detected after 1 week at pH values of 6.5 and 7.5. The slow reactivity of triclosan in the presence of chloramines explains the recalcitrance of this species in nonnitrified wastewater effluents.

Ammonia↗

Nitrosamine formation pathway revisited: the importance of chloramine speciation and dissolved oxygen.

Nitrosamine formation during chloramination previously has been linked to a reaction between monochloramine and organic nitrogen precursors via unsymmetrical dialkylhydrazine intermediates. Our results demonstrate the critical importance of dichloramine and dissolved oxygen. We propose a new nitrosamine formation pathway in which dichloramine reacts with secondary amine precursors to form chlorinated unsymmetrical dialkylhydrazine intermediates. Oxidation of these intermediates by dissolved oxygen to form nitrosamines competes with their oxidation by chloramines. Even when preformed monochloramine was applied, our model explained nearly all N-nitrosodimethylamine formation from the traces of dichloramine formed via monochloramine disproportionation. We suggest that, in contrast to unsymmetrical dialkylhydrazines, the weak, nonpolar nature of the N-Cl linkage in chlorinated unsymmetrical dialkylhydrazine intermediates enables incorporation of dissolved oxygen to form nitrosamines. With the improved understanding of the nitrosamine formation pathway, strategies are suggested that could significantly reduce nitrosamine formation during chloramination.

Chloramines↗

Electronic structures of some antimicrobial N-chloramines. Possible existence of intramolecular hydrogen bonding and its effect on germicidal efficiency.

The photoelectron spectra of eight N-chloramines and N,N-dichloramines derived from either alpha-aminoisobutyric acid or 2-amino-2-methylpropanol have been measured. The lone-pair ionization potentials obtained from the photoelectron spectra have been interpreted to indicate that a substantial intramolecular interaction exists between tne N-H function and the various oxygen lone pairs of the N-chloramines. Such an intramolecular interaction for the N-chloramines can explain at least in part why these molecules are less potent as antimicrobial agents than are the N,N-cichloramine analogues for which a similar intramolecular interaction is impossible.

Anti-Infective Agents, Local↗

Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin.

The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences.

Animals↗

Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis.

Proteins labeled with either (3)H by reductive methylation or (125)I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. However, (125)I-labeled proteins were apparently degraded at rates far exceeding their (3)H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3-5 times more (125)I-labeled bovine serum albumin and X. laevis serum proteins lacking albumin were degraded than the corresponding (3)H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of (125)I-labeled vitellogenin was 15 times greater than that of [(3)H]vitellogenin labeled in vivo (60% compared to 4%). Moreover, examination of the relative amounts of (3)H- compared to (125)I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable (3)H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the (3)H-labeled ligands. Passage of dissolved oocytes previously exposed to (125)I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount ( approximately 90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine. The evolution of either iodotyrosine or free iodine would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). We conclude, therefore, that apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.

Animals↗

Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis.

Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.

Adenosine Triphosphate↗

Shock chloramination: potential treatment for Chironomidae (Diptera) larvae nuisance abatement in water supply systems.

In the early 1990s, infestations of midge larvae (Chironomidae, Chironomus sp.) were discovered in the potable water system of Tel Aviv, Israel. Control measures, such as draining and cleaning tanks, spraying water into the tank's air space, and electrocution traps of midge adults, were either inadequate or ineffective. In this system, monochloramine concentrations of up to 0.75 mg/liter are used routinely as a secondary disinfectant. This chemical was tested in the laboratory as a toxicant of midge larvae. The mortality of 4th instar midge larvae after short exposure to high chloramine concentrations (LC50 values of 32 mg/liter for 75 min) suggested the efficacy of instituting a Shock Chloramination treatment program. Tanks were partially drained until they contained only 20 cm of water and were then temporarily disconnected. Chloramine was added to this water to produce a concentration of approximately 70 mg/liter for 1-2 h. Subsequently, all dead chironomids were flushed out, and the tank was refilled to attain the operational volume of water. A 2nd identical treatment of water in the tank was suggested 7 d later to kill midges from reproductive adults and egg-masses that survived the 1st treatment. This treatment program was tested in commercial covered tanks and gave complete control of these pests for 6-10 wk. These results suggest that this treatment program may effectively prevent midge outbreaks in Israel's drinking water supply system during the height of the summer.

Animals↗

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T.

Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.

Bacillus subtilis↗

Oxidation of methionine residues in bovine growth hormone by chloramine-T.

The methionine residues in bovine growth hormone were chemically modified wih chloramine-T at pH 7.4 using different chloramine-T methionine molar ratios. Methionine 4 was shown to be the most reactive followed in decreasing order by methionines 148, 123 and 178. With a 50-fold molar excess of chloramine-T over methionine almost full oxidation of all the methionine residues was achieved. The oxidation of the four residues of methionine does not change the growth promoting activity of the hormone.

Amino Acid Sequence↗

Effect of pH, application technique, and chlorine-to-nitrogen ratio on disinfectant activity of inorganic chloramines with pure culture bacteria.

The influence of pH, application technique, and chlorine-to-nitrogen weight ratio on the bactericidal activity of inorganic chloramine compounds was determined with stock and environmental strains of Escherichia coli, Salmonella spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae. The rate of inactivation increased from 1.5 to 2 times as the chlorine-to-nitrogen weight ratio was adjusted from 2:1 to 5:1, 5 to 6 times as the pH was decreased from 8 to 6, and 5 to 6 times as the concentration was increased from 1 to 5 mg/liter. Separate additions of free chlorine and ammonia (concurrent addition and preammoniation) into seeded water at or below pH 7.5 resulted in killing comparable to that observed with free chlorine (99% inactivation in less than 20 s). At pH 8, inactivation by separate additions was considerably slower and was comparable to that by prereacted chloramine compounds (99% inactivation in 25 to 26 min). Determination of the effectiveness of inorganic chloramine compounds as primary disinfectants for drinking water must consider the method of application, pH and concentrations of chlorine and ammonia.

Chloramines↗