Pyruvate remediation of cell stress and genotoxicity induced by haloacetic acid drinking water disinfection by‐products

Monohaloacetic acids (monoHAAs) are a major class of drinking water disinfection by‐products (DBPs) and are cytotoxic, genotoxic, mutagenic, and teratogenic. We propose a model of toxic action based on monoHAA‐mediated inhibition of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as a target cytosolic enzyme. This model predicts that GAPDH inhibition by the monoHAAs will lead to a severe reduction of cellular ATP levels and repress the generation of pyruvate. A loss of pyruvate will lead to mitochondrial stress and genomic DNA damage. We found a concentration‐dependent reduction of ATP in Chinese hamster ovary cells after monoHAA treatment. ATP reduction per pmol monoHAA followed the pattern of iodoacetic acid (IAA) > bromoacetic acid (BAA) >> chloroacetic acid (CAA), which is the pattern of potency observed with many toxicological endpoints. Exogenous supplementation with pyruvate enhanced ATP levels and attenuated monoHAA‐induced genomic DNA damage as measured with single cell gel electrophoresis. These data were highly correlated with the S_N2 alkylating potentials of the monoHAAs and with the induction of toxicity. The results from this study strongly support the hypothesis that GAPDH inhibition and the possible subsequent generation of reactive oxygen species is linked with the cytotoxicity, genotoxicity, teratogenicity, and neurotoxicity of these DBPs. Environ. Mol. Mutagen. 54:629–637, 2013. © 2013 Wiley Periodicals, Inc.     

Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products

Cytotoxicity and genotoxicity assays were used to analyze drinking water disinfection by-products (DBPs) in Chinese hamster ovary (CHO) AS52 cells. The DBPs were chosen because they are common in drinking water, resulting from conventional disinfection using chlorination and chloramination. Data were also available to compare these results with cytotoxicity and mutagenicity studies in Salmonella typhimurium. The rank order in decreasing chronic cytotoxicity measured in a microplate-based assay was bromoacetic acid (BA) > 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > KBrO(3) > tribromoacetic acid (TBA) > EMS (ethylmethanesulfonate, positive control) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The induction of DNA strand breaks by these agents was measured by alkaline single-cell gel electrophoresis (SCGE, comet assay) and the rank order in decreasing genotoxicity was BA > MX > CA > DBA > TBA > EMS > KBrO(3), while DCA and TCA were refractory. BA was more cytotoxic (31x) and genotoxic (14x) than MX in CHO cells. BA was over 400x more genotoxic than potassium bromate. The brominated haloacetic acids (HAAs) were more cytotoxic and genotoxic than their chlorinated analogs. The HAAs expressed a statistically significant inverse relationship in CHO cell cytotoxicity and genotoxicity as a function of increased numbers of halogen atoms per molecule. A quantitative comparison was conducted with results from a previous study with cytotoxicity and mutagenicity in S. typhimurium. There was no correlation between chronic CHO cell and bacterial cell cytotoxicity. DBP-induced CHO cell cytotoxicity was not related to mutagenic potency in S. typhimurium. Cytotoxicity in CHO cells was statistically significant and highly correlated to CHO cell genotoxicity. Finally, we determined that the DBP genotoxic potency in CHO cells and the mutagenic potency in S. typhimurium were not related. This suggests that toxicity data in S. typhimurium did not quantitatively predict the toxic effects of DBPs in mammalian cell systems. The microplate CHO cell cytotoxicity and genotoxicity assays were well suited for the analysis of DBPs, especially when the quantity of test material is limited.     

DNA damage and oxidative stress in human liver cell L‐02 caused by surface water extracts during drinking water treatment in a waterworks in China

Because of the daily and life‐long exposure to disinfection by‐products formed during drinking water treatment, potential adverse human health risk of drinking water disinfection is of great concern. Toxicological studies have shown that drinking water treatment increases the genotoxicity of surface water. Drinking water treatment is comprised of different potabilization steps, which greatly influence the levels of genotoxic products in the surface water and thus may alter the toxicity and genotoxicity of surface water. The aim of the present study was to understand the influence of specific steps on toxicity and genotoxicity during the treatment of surface water in a water treatment plant using liquid chlorine as the disinfectant in China. An integrated approach of the comet and oxidative stress assays was used in the study, and the results showed that both the prechlorination and postchlorination steps increased DNA damage and oxidative stress caused by water extracts in human derived L‐02 cells while the tube settling and filtration steps had the opposite effect. This research also highlighted the usefulness of an integrated approach of the comet and oxidative stress assays in evaluating the genotoxicity of surface water during drinking water treatment. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

A review on the 40th anniversary of the first regulation of drinking water disinfection by-products

Water disinfection, primarily by chlorination, is one of the greatest achievements of public health. However, more than half a century after its introduction, studies in the 1970s reported that (a) chlorine interacted with organic matter in the water to form disinfection by-products (DBPs); (b) two DBPs, chloroform and bromoform, both trihalomethanes (THMs), were rodent carcinogens; (c) three brominated THMs were mutagenic; in six studies chlorinated drinking waters in the United States and Canada were mutagenic; and (d) in one epidemiological study there was an association between bladder cancer mortality and THM exposure. This led the U.S. Environmental Protection Agency to issue its first DBP regulation in 1979. Forty years later, >600 DBPs have been characterized, 20/22 have been shown to be rodent carcinogens, >100 have been shown to be genotoxic, and 1000s of water samples have been found to be mutagenic. Data support a hypothesis that long-term dermal/inhalation exposure to certain levels of the three brominated THMs, as well as oral exposure to the haloacetic acids, combined with a specific genotype may increase the risk for bladder cancer for a small but significant population group. Improved water-treatment methods and stricter regulations have likely reduced such risks over the years, and further reductions in potential risk are anticipated with the application of advanced water-treatment methods and wider application of drinking water regulations. This 40-year research effort is a remarkable example of sustained cooperation between academic and government scientists, along with public/private water companies, to find answers to a pressing public health question.     

2010年广州市越秀区中小学校直饮水机直饮水卫生状况调查

目的了解2010年广州市越秀区中小学校直饮水机卫生状况。方法随机抽取42所学校作为调查对象。其中中学20所、小学22所,共设监测点54个。每个监测点分别采集管网分梢近端水、入机自来水、直饮水各1份水样。每份水样均检测浑浊度、臭和味、色度、肉眼可见物、pH值、挥发酚类、总大肠菌群、耐热大肠菌群、耗氧量、锰、铅、三氯甲烷、砷、四氯化碳、亚硝酸盐等共15个项目。结果检测管网分梢近端水、入机自来水、直饮水共162份水样,臭和味、色度、肉眼可见物、挥发酚类、总大肠菌群、耐热大肠菌群、锰、铅、三氯甲烷、砷、四氯化碳等共11项全部合格,耗氧量、亚硝酸盐、pH值、浑浊度等4项均存在不合格的情况。结论越秀区学校直饮水机总体情况良好,直饮水的主要不合格项目为耗氧量、亚硝酸盐、pH值和浑浊度,总合格率需进一步提高。     

In vitro toxicity and genotoxicity assessment of disinfection by‐products,organic N‐chloramines

Disinfection by‐products (DBPs) are of concern to both water industries and health authorities. Although several classes of DBPs have been studied, and there are regulated safe levels in disinfected water for some, a large portion of DBPs are not characterized, and need further investigation. Organic N‐chloramines are a group of DBPs, which can be formed during common disinfection processes such as chlorination and chloramination, but little is known in terms of their toxicological significance if consumed in drinking water. Only a few in vitro studies using bacterial assays have reported some genotoxic potential of organic N‐chloramines, largely in the context of inflammatory processes in the body rather than exposure through drinking water. In this study, we investigated 16 organic N‐chloramines produced by chlorination of model amino acids and amines. It was found that within the drinking water‐relevant micromolar concentration range, four compounds were both cytotoxic and genotoxic to mammalian cells. A small reduction of cellular GSH was also observed in the treatment with these four compounds, but not of a magnitude to account for the cytotoxicity and genotoxicity. The results presented in this study demonstrate that some organic N‐chloramines, at low concentrations that might be present in disinfected water, can be harmful to mammalian cells. © Environ. Mol. Mutagen. 2012. Published 2011 Wiley‐Liss, Inc.     

Immunotoxicity of dibromoacetic acid administered via drinking water to female B6C3F1 mice

Dibromoacetic acid (DBA) is a disinfection by-product commonly found in drinking water as a result of chlorination/?ozonation processes. The Environmental Protection Agency estimates that more than 200 million people consume disinfected water in the United States. This study was conducted to evaluate the potential immunotoxicological effects of DBA exposure when administered for 28 days via drinking water to B₆C₃F₁ mice, at concentrations of 125, 500, and 1000?mg/L. Multiple endpoints were evaluated to assess innate, humoral, and cell-mediated immune components, as well as host resistance. Standard toxicological parameters were unaffected, with the exception of a dose–responsive increase in liver weight and a decrease in thymus weight at the two highest exposure levels. Splenocyte differentials were affected, although the effects were not dose–responsive. Exposure to DBA did not significantly affect humoral immunity (immunoglobulin M [IgM] plaque assay and serum IgM anti-sheep erythrocyte titers) or cell-mediated immunity (mixed-leukocyte response). No effects were observed on innate immune function in either interferon-γ-induced in vitro macrophage cytotoxic activity or basal natural killer (NK)-cell activity. Augmented NK-cell activity (following exposure to polyinosinic-polycytidylic acid) was decreased at the low dose, however the effect was not dose–responsive. Finally, DBA exposure had no effect on resistance to infection with either Streptococcus pneumoniae or Plasmodium yoelii, or challenge with B16F10 melanoma cells. With the exception of changes in thymus weight, these results indicate that DBA exposure resulted in no immunotoxic effects at concentrations much larger than those considered acceptable in human drinking water.     

Calling time on responsible drinking: A qualitative study of perceptions of information on alcohol product labels

Objectives

This study aimed to explore (a) how people interpret responsible drinking messages on alcohol product labels, and (b) the acceptability of including health information on labels.

Design

Qualitative interviews.

Methods

Face-to-face semi-structured interviews were conducted with 20 people aged 21–63; 18 were classified risky drinkers using AUDIT-C. They were shown three sets of alcohol product labels: one including three responsible drinking messages (drink responsibly), one with three positively worded health messages (drinking less reduces risks) and one with three negatively worded health messages (drinking more increases risks). Health messages included information about cancer, liver and heart disease.

Results

Thematic analysis identified three themes: ambiguity about alcohol labelling; identifying oneself as responsible; and acceptability of enhanced product labelling. Participants were critical of responsible drinking messages and wary of conflicting health information in the media. They positioned themselves as responsible, knowledgeable drinkers and distanced themselves from problem drinkers. They did not appear to support the inclusion of health information on labels; however, novel information was considered more impactful.

Conclusions

Responsible drinking messages were seen by our sample as an alcohol industry ploy. Although health messages about cancer were seen as potentially impactful, the ability of consumers to position themselves as unproblematic drinkers means that they may not see the information on the label as relevant to themselves. Understanding factors that increase the personal relevance of messages is needed, alongside an exploration of a wider range of methods for alcohol health communication.     

Catalase has a key role in protecting cells from the genotoxic effects of monomethylarsonous acid: A highly active metabolite of arsenic

Although it is widely known that arsenic‐contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H₂O₂). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat^+/+ and Cat^?/? primary splenic lymphocytes to monomethylarsonous acid (MMA^III) to identify the potential role of H₂O₂ in mediating cellular effects of this metalloid. The results showed that the Cat^?/? lymphocytes are more susceptible to MMA^III than the Cat^+/+ lymphocytes by a small (1.5‐fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat^+/+, Cat^+/?, and Cat^?/? hepatocytes to determine if the Cat^?/? cells were more susceptible to MMA^III than lymphocytes. The results showed that the Cat^?/? hepatocytes exhibit higher levels of DNA strand breakage than the Cat^+/+ (approximately fivefold) and Cat^+/? (approximately twofold) hepatocytes exposed to MMA^III. Electron spin resonance using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide as the spin‐trap agent detected the generation of ·OH via MMA^III when H₂O₂ was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMA^III. Environ. Mol. Mutagen. 54:317–326, 2013. © 2013 Wiley Periodicals, Inc.     

Mutation spectra in salmonella of chlorinated,chloraminated, or ozonated drinking water extracts: Comparison to MX

Drinking water samples were prepared in a pilot-scale treatment plant by chlorination (Cl₂), chlorami-nation (NH₂Cl), ozonation (O₃), or O₃ followed by Cl₂ or NH₂Cl; and the nonvolatile acidic organics of the raw and treated waters were extracted by XAD/ethyl acetate and evaluated for mutagenicity in Salmonella (-S9). The extracts were 2–8 times more mutagenic in TA100 than in TA98, and the mutagenic potencies of the water extracts ranked similarly in both strains: Cl₂ > O₃ + Cl₂ > NH₂Cl > O₃ + NH₂Cl > O₃ > raw. 3-Chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone (MX), which was estimated to account for ～-20% of the mutagenic activity of the extracts, was shown to be the most potent compound tested thus far in a prophage-in-duction assay in Escherichia coli and a forward-mutation assay in Salmonella TM677. The mutations in ～2,000 revertants of TA98 and TA 100 induced by MX and the water extracts were analyzed by colony probe hybridization and polymerase chain reaction/DNA sequence analysis. The water extracts and MX produced similar mutation spectra, which consisted in TA100 of predominantly of GC → TA transversions in the second position of the CCC (or GGG) target of the hisG46 allele. This spectrum resembles that produced by large aromatic compounds and is distinct from that produced by alkylating agents and the semivolatile drinking water mutagen dichloroacetic acid. In TA98, MX and those water extracts resulting from the introduction of the chlorine atom produced 50–70% hotspot 2-base deletions and 30–50% complex frameshifts (frameshifts with an adjacent base substitution—mostly GC → TA transversions as found in TA100). No other compound or mixture is known to induce such high frequencies of complex frameshifts. These results suggest that MX and “MX-like” compounds (possibly halogenated aromatics, such as halogenated polycyclic aromatic hydrocarbons) account for much of the mutagenic activity and specificity of the nonvolatile organics in drinking water and that these halogenated organics are especially capable of promoting misincorporation by the DNA replication complex. This study provides further evidence that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, i s in the public domain in the United States of America.

     

Advanced glycation end product Nε‐carboxymethyllysine induces endothelial cell injury: the involvement of SHP‐1‐regulated VEGFR‐2 dephosphorylation

N^ε‐carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes‐induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML‐related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)‐mediated SH2 domain‐containing tyrosine phosphatase‐1 (SHP‐1) activation by CML inhibits the VEGF receptor‐2 (VEGFR‐2, KDR/Flk‐1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR‐2 activation in parallel with the increased SHP‐1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP‐1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP‐1 with VEGFR‐2. Consistently, SHP‐1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [³H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML‐increased SHP‐1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP‐1 and CML was increased, but phospho‐VEGFR‐2 staining was decreased in the aortic endothelium of streptozotocin‐induced and high‐fat diet‐induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP‐1‐regulated VEGFR‐2 dephosphorylation through NADPH oxidase‐derived ROS is involved in the CML‐triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Multilayer Thin Films by Layer‐by‐Layer Assembly of Hole‐ and Electron‐Transport Polyelectrolytes: Optical and Electrochemical Properties

Summary: In this paper, we present the synthesis of a series of p‐type and n‐type semiconducting polyelectrolytes with triarylamine, oxadiazole, thiadiazole and triazine moieties. The synthesized polymeric hole and electron transport materials were examined optically and electrochemically using UV/Vis spectroscopy, PL spectroscopy and CV. Based on the optical and electrochemical data, each of the energy levels were calculated and all values suggested that they were promising hole‐ (p‐type) or electron‐transport (n‐type) materials for devices. Moreover, the synthesized ionic polymers were suitable for LBL thin film deposition from dilute polymer solutions and the multilayers were fully characterized by UV/Vis, PL spectroscopy and CV.

     

Human T‐cell receptor V gene segment of alpha and beta families: A revised primer design strategy

The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A semi‐qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies.     

The who's who of T‐cell differentiation: Human memory T‐cell subsets

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single‐cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T‐cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T‐cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.     

Genetic toxicology and toxicogenomic analysis of three cigarette smoke condensates in vitro reveals few differences among full‐flavor,blonde, and light products

Cigarette smoking leads to various detrimental health outcomes. Tobacco companies produce different brands of cigarettes that are marketed as reduced harm tobacco products. Early examples included “light” cigarettes, which differ from regular cigarettes due to filter ventilation and/or differences in chemical constituents. In order to establish baseline similarities and differences among different tobacco brands available in Canada, the present study examined the cytotoxicity, mutagenicity, clastogenicity, and gene expression profiles of cigarette smoke condensate (CSC) from three tobacco products, encompassing a full‐flavor, blonde, and “light” variety. Using the Salmonella mutagenicity assay, we confirmed that the three CSCs are mutagenic, and that the potency is related to the presence of aromatic amines. Using the Muta?Mouse FE1 cell line we determined that the CSCs were clastogenic and cytotoxic, but nonmutagenic, and the results showed few differences in potencies among the three brands. There were no clear brand‐specific changes in gene expression; each brand yielded highly similar expression profiles within a time point and concentration. The molecular pathways and biological functions affected by exposure included xenobiotic metabolism, oxidative stress, DNA damage response, cell cycle arrest and apoptosis, as well as inflammation. Thus, there was no appreciable difference in toxicity or gene expression profiles between regular brands and products marketed as “light,” and hence no evidence of reduced harm. The work establishes baseline CSC cytotoxicity, mutagenicity, and expression profiles that can be used as a point of reference for comparison with data generated for products marketed as reduced harm and/or modified risk tobacco products. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

The human lung cell line A549 does not develop adaptive protection against the DNA‐damaging action of formaldehyde

The alkaline comet assay was used to further characterize the induction of DNA‐protein crosslinks (DPX) by formaldehyde (FA) and their removal in the human lung cell line A549. DPX were indirectly measured as the reduction of gamma ray‐induced DNA migration. Repeated treatments of A549 cells with low FA concentrations (up to 100 μM) did not lead to significant differences in the induction of DPX in comparison with a single treatment. Pretreatment with higher FA‐concentrations (200 μM and above) enhanced the crosslinking effect. There was no indication for an adaptive protection against the induction of DPX by FA. These findings are in agreement with RT‐PCR measurements of the expression of genes that encode the main enzymes involved in FA detoxification. A549 cells exposed to FA (50–300 μM) for 1, 4, or 24 hr did not reveal altered expression of the GSH‐dependent formaldehyde dehydrogenase (FDH, which is identical to alcohol dehydrogenase 3; ADH3), the cytosolic aldehyde dehydrogenase 1 (ALDH1A1) and the mitochondrial ALDH2. Pretreatment of A549 cells with a low FA concentration (50 μM) also did not enhance the removal of DPX induced by higher FA concentrations. Taken together, these results suggest that A549 cells do not develop adaptive protection against the genotoxic action of FA. Neither metabolic inactivation of FA nor the repair of FA‐induced DPX seems to be enhanced in cells pretreated with FA. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.