Novel evidence on iodoacetic acid-induced immune protein functional and conformational changes: Focusing on cellular and molecular aspects.

Elevated levels of iodide occur in raw water in certain regions, where iodination disinfection byproducts are formed during chloramine-assisted disinfection of naturally iodide-containing water. Iodoacetic acid (IAA) is one of the typical harmful products. The mechanisms underlying IAA-induced immunotoxicity and its direct effects on biomolecules remained unclear in the past. Cellular, biochemical, and molecular methods were used to investigate the mechanism of IAA-induced immunotoxicity and its binding to lysozyme. In the presence of IAA, the cell viability of coelomocytes was significantly reduced to 70.8 %, as was the intracellular lysozyme activity. Upon binding to IAA, lysozyme underwent structural and conformational changes, causing elongation and unfolding of the protein due to loosening of the backbone and polypeptide chains. IAA effectively quenched the fluorescence of lysozyme and induced a reduction in particle sizes. Molecular docking revealed that the catalytic residue, Glu 35, which is crucial for lysozyme activity, resided within the docking range, suggesting the preferential binding of IAA to the active site of lysozyme. Moreover, electrostatic interaction emerged as the primary driving force behind the interaction between IAA and lysozyme. In conclusion, the structural and conformational changes induced by IAA in lysozyme resulted in impaired immune protein function in coelomocytes, leading to cellular dysfunction.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

R-hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis-A new entry to the carrier protein family Lipocalines.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.

Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group.

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.

[A novel method for the selective enrichment of persulfidated peptides based on iodoacetic acid functionalized dendrimer].

Protein persulfidation is an important oxidative translational modification which plays vital roles in many important processes including cellular senescence, endoplasmic reticulum stress, vasorelaxation, and apoptosis. The proteome-wide analysis of persulfidation is of great importance; therefore, this study combines filter-aided sample preparation with an iodoacetic acid functionalized polyamidoamine dendrimer to enrich persulfidated peptides (denoted as filter-aided dendrimer enrichment strategy, FADE). To evaluate the performance of this strategy, the synthetic persulfidated standard peptide was spiked into bovine serum albumin (BSA) digests at a mass ratio of 1:100, and was successfully identified by FADE. Moreover, in combination with stable isotope labelling by amino acids in cell culture technology, the FADE strategy was applied to enrich persulfidated peptides from NaHS-stimulated SHSY5Y cells over a concentration gradient, resulting in the identification of 163 persulfidated peptides. Bioinformatic analysis indicated that persulfidation might play important roles in the central nervous system.

Superoxide dismutase response and the underlying molecular mechanism induced by iodoacetic acid.

Given the ubiquity of iodinated disinfection by-products (I-DBPs) in drinking water and their prominent toxicity, it is of vital significance to evaluate I-DBPs toxicity and explore the underlying mechanism. The toxicity of iodoacetic acid (IAA), a typical type of I-DBPs, might be linked with oxidative stress. However, it remains unknown for the response of antioxidant enzyme superoxide dismutase (SOD) in the mouse primary hepatocytes when exposed to IAA and the underlying mechanism. This study explored SOD response to IAA and the underlying mechanisms at the molecular and cellular levels. Under IAA exposure, the observed increase of SOD activity in the hepatocytes was caused by the increase of SOD production via ROS stimulation and the increase of SOD molecular activity. Molecular experiments showed that IAA binds to SOD molecule mainly via electrostatic forces with one binding site around the active site and six binding sites in the surface of protein. The binding interaction leads to the conformational changes of SOD and the disruption of protein aggregates. This work could offer basic data for the comprehensive understanding of the adverse effects of IAA and references for assessing the harmful effects of DBPs.

Differential roles of ice crystal, endogenous proteolytic activities and oxidation in softening of obscure pufferfish (Takifugu obscurus) fillets during frozen storage.

Obscure pufferfish (Takifugu obscurus) softening during frozen storage remains to be solved. This study was therefore aimed to provide explanations by differentiate the roles of three potential factors in fish softening. The influences of ice crystal, endogenous proteolytic activities, and oxidization were distinguished by treatment of fish fillets with liquid nitrogen, iodoacetic acid, and tea polyphenol with ascorbic acid, respectively. This distinguishing method was verified to be effective by investigation in ice crystal microstructure, endogenous proteolytic activities and lipid and protein oxidation. In comparison of three factors, it showed that the shear force of fish fillets with smaller ice crystals was about 15.5% and 13.7% higher than those with the inhibition of endogenous proteolytic activities and oxidation respectively, indicating the dominant role of ice crystal in frozen fish softening. Besides, quality decline of frozen fish was initially fast and then slowed down during the storage.

Mix-and-Match Proteomics: Using Advanced Iodoacetyl Tandem Mass Tag Multiplexing To Investigate Cysteine Oxidation Changes with Respect to Protein Expression.

Cysteine redox state has been identified as one of the key biological influences behind protein structure and/or function. Altered protein redox state has been shown to cause significant physiological changes and can leave proteins with changed sensitivity to oxidative stress. Protein redox-state changes are recognized as an important mediator of disease, cellular abnormalities, and environmental changes, and therefore their characterization is of interest. Isotopic or isobaric labeling followed by sample multiplexing and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) allows relative comparison of protein expression levels or of protein redox states between several samples. Combining analysis of protein expression level and redox state into one analysis would add an extra dimension and permit the normalization of protein redox changes with protein abundance. To achieve this, we have developed a quantitation workflow that uses commercially available cysteine-reactive tandem mass tags (iodoTMT) to differentially label cysteine residues, and we have applied it to two Leishmania mexicana cell lines that have previously shown different responses to oxidative stress. The individually labeled samples have been pooled in different combinations to create multiple sixplex samples in order to study the relationship between cysteine oxidation and overall protein expression, as well as providing information about protein oxidation levels in each cell line. The results highlight 11 proteins that are differentially expressed between the two cell lines and/or have significant redox changes. This advanced multiplexing method effectively demonstrates the flexibility of tandem mass tags and how they can be used to maximize the amount of information that can be acquired.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

Rapid oxidation of iodide and hypoiodous acid with ferrate and no formation of iodoform and monoiodoacetic acid in the ferrate/I-/HA system.

Toxic and odorous iodinated disinfection byproducts (I-DBPs) could form in the chemical oxidation of iodine-containing water. A critical step for controlling the hazardous I-DBPs is to convert the iodine species into stable and harmless iodate (IO3-) while inhibiting the accumulation of highly reactive hypoiodous acid (HOI). Herein, the oxidation of I- and HOI with ferrate was investigated, and the formation profile of HOI was determined based on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) coloring method through a stopped-flow spectrophotometer. The second-order rate constants (kapp) of ferrate with HOI decreased from 1.6 × 105 M-1s-1 to 8.3 × 102 M-1s-1 as the solution pH varied from 5.3 to 10.3, which were 7.5, 7.2 and 13.8 times higher than that of ferrate with I- at pH 6.0, 7.0 and 8.0, respectively. Compared with other oxidants such as ozone, hypochlorous acid, chloramine and potassium permanganate, ferrate would swiftly oxidize HOI formed in the I- oxidation process. For the ferrate oxidation of I-containing water, HOI was swiftly oxidized to IO3- from pH 5.0 to 9.0. Phosphate buffer promoted the oxidation of I- while inhibited the oxidation of HOI with ferrate. When 5 mgC/L of humic acids (HA) existed in the solution, no formation of iodoform and monoiodoacetic acid (MIAA) was observed in the oxidation of iodide (20 µM) with ferrate (from 10 µM to 80 µM). These results suggested that ferrate oxidation could be an effective method for the control of I-DBPs in iodine-containing water treatment.

Drinking water disinfection byproduct iodoacetic acid interacts with catalase and induces cytotoxicity in mouse primary hepatocytes.

Disinfection byproducts (DBPs) are produced during the disinfection of drinking water and pose a hazard to human health. As a typical type of DBPs, iodoacetic acid (IAA) exhibits prominent cytotoxicity in mammalian cell systems which links with oxidative stress. However, little is known about the relationship of catalase (CAT) with the cytotoxicity of IAA and the adverse effects of IAA to CAT. This study investigated the effects of IAA on the cell viability and CAT activity in the mouse primary hepatocytes. It was shown that IAA exposure induced the loss of cell viability and the increase of intracellular CAT activity. Intracellular CAT activity significantly increased due to the stimulation of CAT production under IAA exposure. The molecular CAT activity was inhibited due to the direct interaction of IAA with HIS 74 and TYR 357 around the active sites of CAT. IAA binds to CAT with (4.05 ±â€¯1.98) sites via van der Waals and hydrogen bonding interactions, resulting in the loosening of protein skeletons and the change of protein size.

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA.

Modification of Cysteine.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is described. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples. © 2017 by John Wiley & Sons, Inc.

[Effect of Three Typical Disinfection Byproducts on Bacterial Antibiotic Resistance].

The effect of typical disinfection byproducts (DBPs) on bacterial antibiotic resistance was investigated in this study. chlorodibromomethane (CDBM), iodoacetic acid (IAA) and chloral hydrate (CH) were selected, which belong to trihalomethanes (THMs), haloacetic acids (HAAs) and aldehydes, respectively. After exposure to the selected DBPs, the resistance change of the tested strains to antibiotics was determined. As a result, all of the three DBPs induced Pseudomonas aeruginosa PAO1 to gain increased resistance to the five antibiotics tested, and the DBPs ranked as IAA > CH > CDBM according to their enhancement effects. Multidrug resistance could also be enhanced by treatment with IAA. The same result was observed in Escherichia coli K12, suggesting that the effect of DBPs on antibiotic resistance was a common phenomenon. The mechanism was probably that DBPs stimulated oxidative stress, which induced mutagenesis. And the antibiotic resistance mutation frequency could be increased along with mutagenesis. This study revealed that the acquisition of bacterial antibiotic resistance might be related to DBPs in drinking water systems. Besides the genotoxicological risks, the epidemiological risks of DBPs should not be overlooked.

Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

Rotating Ring-Disk Electrode and Quantum-Chemical Study of the Electrochemical Reduction of Monoiodoacetic Acid and Iodoform.

This study examined the electrochemical (EC) reduction of monoiodoacetic acid (MIAA) and iodoform (CHI3), which are typical iodine-containing disinfection byproducts (I-DBPs). Experiments carried out using the method of a rotating ring-disk electrode (RRDE) with a gold working electrode showed that the reduction of CHI3 and MIAA is diffusion-controlled. The MIAA diffusion coefficient was determined to be (1.86 ± 0.24)·10(-5) cm(2) s(-1). The yield of the iodide ion formed as a result of MIAA or CHI3 reduction was affected by the presence of dissolved organic matter (DOM) and resorcinol. Increasing concentrations of DOM or resorcinol did not affect the EC reduction of the examined I-DBPs, but the formation of iodide was suppressed. This indicated that free iodine, ·I, was formed as a result of the first step in the EC reduction of MIAA and CHI3. This also indicated that the pathway of the EC reduction of MIAA and CHI3 was different from that typical for the reduction of Br- and Cl-containing DBPs, in which case Br(-) or Cl(-) tend to be formed as a result of the electron transfer. Quantum-chemical (QC) calculations confirmed the thermodynamic likelihood of and possible preference to the formation of free iodine species as a result of the EC reduction of MIAA, CHI3, and other I-DBPs.

In Vitro Cytotoxicity and Adaptive Stress Responses to Selected Haloacetic Acid and Halobenzoquinone Water Disinfection Byproducts.

The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts (DBPs). Some of these are mutagenic, genotoxic, teratogenic, and cytotoxic, as well as potentially carcinogenic both in vivo and in vitro. We investigated the in vitro biological activity of five DBPs: three monohaloacetic acids (monoHAAs) [chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA)] and two novel halobenzoquinones (HBQs) [2,6-dichloro-p-benzoquinone (DCBQ) and 2,6-dibromo-p-benzoquinone]. We focused particularly on cytotoxicity and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoxic to the Caco-2 cell line after a 4 h exposure, and all DBPs induced both of the adaptive stress response pathways, Nrf2/ARE and p53, in the micromolar range, as measured by two ß-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA ∼ BAA > DCBQ ∼ DBBQ > CAA. Induction of oxidative stress was previously proposed to be the molecular initiating event (MIE) for both classes of DBPs. However, comparing the levels of activation of the two pathways uncovered that the Nrf2/ARE pathway was the more sensitive endpoint for HAAs, whereas the p53 pathway was more sensitive in the case of HBQs. Therefore, the DNA damage-responsive p53 pathway may be an important piece of information to fill in a gap in the adverse outcome pathway framework for the assessment of HBQs. Finally, we cautiously compared the potential risk of the two novel HBQs using a benchmarking approach to that of the well-studied CAA, which suggested that their relative risk may be lower than that of BAA and IAA.

Identification of reduction-susceptible disulfide bonds in transferrin by differential alkylation using O(16)/O(18) labeled iodoacetic acid.

Stabilization of native three-dimensional structure has been considered for decades to be the main function of disulfide bonds in proteins. More recently, it was becoming increasingly clear that in addition to this static role, disulfide bonds are also important for many other aspects of protein behavior, such as regulating protein function in a redox-sensitive fashion. Dynamic disulfide bonds can be taken advantage of as candidate anchor sites for site-specific modification (such as PEGylation of conjugation to a drug molecule), but are also frequently implicated in protein aggregation (through disulfide bond scrambling leading to formation of intermolecular covalent linkages). A common feature of all these labile disulfide bonds is their high susceptibility to reduction, as they need to be selectively regulated by either specific local redox conditions in vivo or well-controlled experimental conditions in vitro. The ability to identify labile disulfide bonds in a cysteine-rich protein can be extremely beneficial for a variety of tasks ranging from understanding the mechanistic aspects of protein function to identification of troublesome "hot spots" in biopharmaceutical products. Herein, we describe a mass spectrometry (MS)-based method for reliable identification of labile disulfide bonds, which consists of limited reduction, differential alkylation with an O(18)-labeled reagent, and LC-MS/MS analysis. Application of this method to a cysteine-rich protein transferrin allows the majority of its native disulfide bonds to be measured for their reduction susceptibility, which appears to reflect both solvent accessibility and bond strain energy.

Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution.

A multiple-label stable isotope dilution assay for quantifying glutathione (GSH), glutathione disulfide (GSSG), and glutathione sulfonic acid in erythrocytes was developed. As the internal standards, [(13)C3,(15)N]glutathione, [(13)C4,(15)N2]glutathione disulfide, and [(13)C3,(15)N]glutathione sulfonic acid were used. Analytes and internal standards were detected by LC-MS/MS after derivatization of GSH with iodoacetic acid and dansylation of all compounds under study. The calibration functions for all analytes relative to their respective isotopologic standards revealed slopes close to 1.0 and negligible intercepts. As various labelings of the standards for GSH and GSSG were used, their simultaneous quantitation was possible, although GSH was partly oxidized to its disulfide during analysis. The degree of this artifact formation of GSSG was calculated from the abundance of the mixed disulfide formed from unlabeled GSH and its respective standard. Thus, the detected GSSG amount could be corrected for the artifact amount. In this way, the amount of GSSG in erythrocytes was found to be less than 0.5% of the GSH concentration. Similar to GSSG, the detected amount of glutathione sulfonic acid was found to be formed at least in part during the analytical process, but the degree could not be quantified.

Genotoxic and clastogenic effects of monohaloacetic acid drinking water disinfection by-products in primary human lymphocytes.

The haloacetic acids (HAAs) are the second-most prevalent class of drinking water disinfection by-products formed by chemical disinfectants. Previous studies have determined DNA damage and repair of HAA-induced lesions in mammalian and human cell lines; however, little is known of the genomic DNA and chromosome damage induced by these compounds in primary human cells. The aim of this study was to evaluate the genotoxic and clastogenic effects of the monoHAA disinfection by-products in primary human lymphocytes. All monoHAAs were genotoxic in primary human lymphocytes, the rank order of genotoxicity and cytotoxicity was IAA > BAA >> CAA. After 6 h of repair time, only 50% of the DNA damage (maximum decrease in DNA damage) was repaired compared to the control. This demonstrates that primary human lymphocytes are less efficient in repairing the induced damage by monoHAAs than previous studies with mammalian cell lines. In addition, the monoHAAs induced an increase in the chromosome aberration frequency as a measurement of the clastogenic effect of these compounds. These results coupled with genomic technologies in primary human cells and other mammalian non-cancerous cell lines may lead to the identification of biomarkers that may be employed in feedback loops to aid water chemists and engineers in the overall goal of producing safer drinking water.