Faulty gap-filling in nucleotide excision repair leads to double strand break formation in senescent cells.

The change of repair efficiency of UV-induced pyrimidine dimers due to aging was examined in replicatively senesced fibroblasts. The fibroblasts with repeated passages showed the characteristics of cellular senescence including irreversible cell cycle arrest, elevated ß-galactosidase activity and senescence-associated secretory phenotype. The incision efficiency of oligonucleotide containing UV lesions was similar regardless of cell doubling levels, but the gap filling process was impaired in replicatively senescent cells. The releases of XPG, PCNA and RPA from damaged sites were delayed, which might have disturbed the DNA polymerase progression. The persistent single stranded DNA (ssDNA) was likely converted to double strand breaks (DSBs), leading to ATM phosphorylation and 53BP1 foci formation. γ-H2AX induction mainly occurred in G1 phase in senescent cells, not in S phase like in normal cells, indicating replication stress-independent DSBs might be formed. Mre11 having nuclease activity accumulated to damaged sites at early time point after UV irradiation but not released in senescent cells. The pharmacological studies using specific inhibitors for the nuclease activity suggested that Mre11 contributed to the enlargement of ssDNA gap, facilitating the DSB formation.

Glucose starvation impairs NER and γ-H2AX after UVB irradiation.

Glucose is the major source for energy production. As tumor cells have higher glucose requirement, combination of glucose restriction and radio- and chemo-therapy has been explored. In this study, impairment of UVB-induced DNA damage repair response (DDR) by glucose starvation was revealed. Human keratinocytes and skin carcinoma cells were cultured in medium containing 0, 2.5, 5.5 and 25 mM glucose. Glucose restriction suppressed cell proliferation and histone acetylation. UVB exposure formed similar levels of pyrimidine dimers in all glucose conditions, whereas the repair tended to be delayed in low glucose medium. The repair molecules, TFIIH and XPG, were accumulated to DNA damaged sites regardless of glucose supply levels, but the release was delayed in glucose-starved cells. The remaining pyrimidine dimers would induce the collapse of replication forks during S phase, resulting in phosphorylation of histone H2AX (γ-H2AX), but the γ-H2AX in cells cultured in glucose-deleted medium was unexpectedly decreased. This might be due to the suppression of DNA replication by glucose deletion. This was further confirmed by the decrease in the formation of DNA double strand breaks in glucose-starved cells. These results suggested that condition of energy supply might affect UV-induced DDR.

Butyrate Enhances γ-H2AX Induced by Benzo[a]pyrene.

Benzo[a]pyrene (BaP) is known to form DNA adduct following metabolic activation, which causes phosphorylation of histone H2AX (γ-H2AX). Recent studies have shown that histone deacetylase (HDAC) inhibitors enhanced BaP-induced CYP1A1 gene expression. In this study, we examined the relationship between the HDAC inhibitor-augmented metabolic activation and BaP-induced γ-H2AX. Sodium butyrate (SB), a typical HDAC inhibitor, enhanced BaP-induced γ-H2AX. The enhanced DNA damage was further confirmed by biased sinusoidal field gel electrophoresis, which detects DNA double-strand breaks. SB remarkably augmented BaP-induced CYP1A1 gene expression, and CYP1A1-overexpressing cells showed elevated generation of γ-H2AX. Furthermore, SB enhanced intracellular oxidation after treatment with BaP. These results suggested that SB-induced CYP1A1 upregulation facilitated BaP metabolism, which might result in excess DNA adducts or oxidative DNA damages, leading to augmentation of γ-H2AX.

Protective role of electrophile-reactive glutathione for DNA damage repair inhibitory effect of dibromoacetonitrile.

Dibromoacetonitrile (DBAN) is a disinfection byproduct (DBP) and linked with cancer in rodents, but the mechanism of its carcinogenicity has not been fully elucidated. We recently reported that DBAN induced inhibition of nucleotide excision repair (NER). In this study, we investigated if glutathione (GSH) is involved in the DBAN-induced inhibition of NER. Human keratinocytes HaCaT were pretreated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete intracellular GSH. BSO treatment markedly potentiated the DBAN-induced NER inhibition as well as intracellular oxidation. The recruitment of NER proteins (transcription factor IIH, and xeroderma pigmentosum complementation group G) to DNA damage sites was inhibited by DBAN, which was further exacerbated by BSO treatment. Our results suggest that intracellular GSH protects cells from DBAN-induced genotoxicity including inhibition of DNA damage repair.

Inhibition of nucleotide excision repair and damage response signaling by dibromoacetonitrile: A novel genotoxicity mechanism of a water disinfection byproduct.

Dibromoacetonitrile (DBAN) is a carcinogenic disinfection byproduct (DBP) but how it precipitates cancer is unknown. Nucleotide excision repair (NER) is a versatile repair mechanism for removing bulky DNA lesions to maintain genome stability, and impairment of this process is associated with cancer development. In this study, we found that DBAN inhibited NER and investigated its mechanism with other DNA damage responses. Human keratinocytes HaCaT were treated with DBAN followed by ultraviolet (UV) as a model inducer of DNA damage, pyrimidine dimers, which require NER for the removal. DBAN pretreatment exacerbated UV-cytotoxicity, and inhibited the repair of pyrimidine dimers. DBAN treatment delayed the recruitment of NER proteins, transcription factor IIH (TFIIH) and xeroderma pigmentosum complementation group G (XPG), to DNA damaged sites, and subsequent gap filling process. Moreover, DBAN suppressed the UV-induced double strand breaks (DSBs) formation, as well as phosphorylated histone H2AX (γ-H2AX), a widely used DNA damage marker. Altogether, DBAN could negatively impact the NER process and phosphorylation pathway responding to DNA damage. This study was the first to identify the inhibition of NER and damage response signaling as a genotoxicity mechanism of a class of DBPs and it may serve as a foundation for DBP carcinogenesis.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-Induced Histone Acetylation via α7nAChR-Mediated PI3K/Akt Activation and Its Impact on γ-H2AX Generation.

A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

Low extracellular pH inhibits nucleotide excision repair.

Nucleotide excision repair (NER) is the main pathway to repair bulky DNA damages including pyrimidine dimers, and the genetic dysregulation of NER associated proteins is well known to cause diseases such as cancer and neurological disorder. Other than the genetic defects, 'external factors' such as oxidative stress and environmental chemicals also affect NER. In this study, we examined the impact of extracellular pH on NER. We prepared the culture media, whose pH values are 8.4 (normal condition), 7.6, 6.6 and 6.2 under atmospheric CO2 conditions. Human keratinocytes, HaCaT, slightly died after 48 h incubation in DMEM at pH 8.4, 7.6 and 6.6, while in pH 6.2 condition, marked cell death was induced. UV-induced pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), were effectively repaired at 60 min and 24 h, respectively, which were remarkably inhibited at pH 6.6 and 6.2. The associated repair molecule, TFIIH, was accumulated to the damaged sites 5 min after UVC irradiation in all pH conditions, but the release was delayed as the pH got lower. Furthermore, accumulation of XPG at 5 min was delayed at pH 6.2 and 6.6, and the release at 60 min was completely suppressed. At the low pH, the DNA synthesis at the gaps created by incision of oligonucleotides containing pyrimidine dimers was significantly delayed. In this study, we found that the low extracellular pH inhibited NER pathway. This might partially contribute to carcinogenesis in inflamed tissues, which exhibit acidic pH.

Long-wavelength UVA enhances UVB-induced cell death in cultured keratinocytes: DSB formation and suppressed survival pathway.

Solar UV radiation consists of both UVA and UVB. The wavelength-specific molecular responses to UV radiation have been studied, but the interaction between UVA and UVB has not been well understood. In this study, we found that long-wavelength UVA, UVA1, augmented UVB-induced cell death, and examined the underlying mechanisms. Human keratinocytes HaCaT were exposed to UVA1, followed by UVB irradiation. Irradiation by UVA1 alone showed no effect on cell survival, whereas the UVA1 pre-irradiation remarkably enhanced UVB-induced cell death. UVA1 delayed the repair of pyrimidine dimers formed by UVB and the accumulation of nucleotide excision repair (NER) proteins to damaged sites. Gap synthesis during NER was also decreased, suggesting that UVA1 delayed NER, and unrepaired pyrimidine dimers and single-strand breaks generated in the process of NER were left behind. Accumulation of this unrepaired DNA damage might have led to the formation of DNA double-strand breaks (DSBs), as was detected using gel electrophoresis analysis and phosphorylated histone H2AX assay. Combined exposure enhanced the ATM-Chk2 signaling pathway, but not the ATR-Chk1 pathway, confirming the enhanced formation of DSBs. Moreover, UVA1 suppressed the UVB-induced phosphorylation of Akt, a survival signal pathway. These results indicated that UVA1 influenced the repair of UVB-induced DNA damage, which resulted in the formation of DSBs and enhanced cell death, suggesting the risk of simultaneous exposure to high doses of UVA1 and UVB.

Evaluation of Histidine Reactivity and Byproduct Formation during Peptide Chlorination.

The covalent modifications resulting from chlorine reactions with peptide-bound amino acids contribute to pathogen inactivation and disinfection byproduct (DBP) formation. Previous research suggested that histidine is the third most reactive of the seven chlorine-reactive amino acids, leading to the formation of 2-chlorohistidine, 2-oxohistidine, or low-molecular-weight byproducts such as trihalomethanes. This study demonstrates that histidine is less reactive toward formation of chlorine transformation products (transformation time scale of hours to days) than five of the seven chlorine-reactive amino acids, including tyrosine (transformation time scale of minutes). Chlorine targeted tyrosine in preference to histidine within peptides, indicating that chlorine reactions with tyrosine and other more reactive amino acids could contribute more to the structural modifications to proteins over the short time scales relevant to pathogen inactivation. Over the longer time scales relevant to disinfection byproduct formation in treatment plants or distribution systems, this study identified ß-cyanoalanine as the dominant transformation product of chlorine reactions with peptide-bound histidine, with molar yields of ∼50% after 1 day. While a chlorinated histidine intermediate was observed at lower yields (maximum ∼5%), the cumulative concentration of the conventional low-molecular-weight DBPs (e.g., trihalomethanes) was ≤7%. These findings support the need to identify the high-yield initial transformation products of chlorine reactions with important precursor structures to facilitate the identification of unknown DBPs.

Cell line-dependent difference in glutathione levels affects the cigarette sidestream smoke-induced inhibition of nucleotide excision repair.

We recently reported that cigarette sidestream smoke (CSS) induced inhibition of nucleotide excision repair (NER) and the cause was NER molecule degradation by aldehydes contained in CSS [Carcinogenesis39, 56-65, 2018; Mutat. Res.834, 42-50, 2018]. In this study, we examined the relationship between intracellular glutathione (GSH) levels and CSS-induced NER inhibition. CSS treatment decreased the intracellular GSH level in human keratinocytes HaCaT, in which the repair of pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) after UVB irradiation was suppressed. We used l-buthionine-(S,R)-sulfoximine (BSO) to artificially deplete intracellular GSH level. BSO treatment remarkably accelerated the CSS-induced NER inhibition. The NER inhibition by CSS was attributed to the delay of accumulation of NER molecules (TFIIH and XPG) to DNA damaged sites, which was further enhanced by BSO treatment. CSS degraded TFIIH, and BSO promoted it as expected. Formaldehyde (FA), a major constituent of CSS, showed similar intracellular GSH reduction and NER inhibition, and BSO promoted its inhibitory effect. Five cultured cell lines showed considerable variability in intrinsic GSH levels, and CSS-induced NER inhibitory effect was significantly correlated with the GSH levels. Chemicals like aldehydes are known to react not only with proteins but also with DNA, causing DNA lesions targeted by NER. Our results suggest that the tissues and cells with low intrinsic GSH levels are susceptible to treatment with CSS and electrophilic compounds like aldehydes through NER inhibition, thus leading to higher genotoxicity and carcinogenicity.

Formaldehyde inhibits UV-induced phosphorylation of histone H2AX.

Formaldehyde (FA) is widely known to cause DNA damage. Recently, our study showed that FA can also inhibit a repair process of DNA damage, nucleotide excision repair (NER). DNA damage response (DDR) involving activation of phosphorylation pathways is important for the accuracy of the repair process, and the inhibition of the accurate repair would raise mutation rate, leading to cancer. We herein investigated whether FA influences phosphorylation of histone H2AX (γ-H2AX), an intermediate player of DDR signaling pathways. Human keratinocytes HaCaT were treated with FA and then exposed to UV known to generate clear γ-H2AX signal. UV-induced γ-H2AX was inhibited by FA in a dose-dependent manner. The repair of pyrimidine dimers was inhibited by FA, while the recruitments of γ-H2AX-related proteins, Mre11 and 53BP1, to damaged sites were also delayed. Mre11, Nbs-1, H2AX and ATM were not degraded after treatment with FA as opposed to NER-related protein, TFIIH. On the other hand, FA inhibited phosphorylation of ATM which acts upstream of γ-H2AX. These results suggest that FA can affect the repair of DNA damage via inhibition of the phosphorylation pathways of H2AX.

Serum electrolytes can promote hydroxyl radical-initiated biomolecular damage from inflammation.

Chronic inflammatory disorders are associated with biomolecular damage attributed partly to reactions with Reactive Oxygen Species (ROS), particularly hydroxyl radicals (•OH). However, the impacts of serum electrolytes on ROS-associated damage has received little attention. We demonstrate that the conversion of •OH to carbonate and halogen radicals via reactions with serum-relevant carbonate and halide concentrations fundamentally alters the targeting of amino acids and loss of enzymatic activity in catalase, albumin and carbonic anhydrase, three important blood proteins. Chemical kinetic modeling indicated that carbonate and halogen radical concentrations should exceed •OH concentrations by 6 and 2 orders of magnitude, respectively. Steady-state γ-radiolysis experiments demonstrated that serum-level carbonates and halides increased tyrosine, tryptophan and enzymatic activity losses in catalase up to 6-fold. These outcomes were specific to carbonates and halides, not general ionic strength effects. Serum carbonates and halides increased the degradation of tyrosines and methionines in albumin, and increased the degradation of histidines while decreasing enzymatic activity loss in carbonic anhydrase. Serum electrolytes increased the degradation of tyrosines, tryptophans and enzymatic activity in the model enzyme, ketosteroid isomerase, predominantly due to carbonate radical reactions. Treatment of a mutant ketosteroid isomerase indicated that preferential targeting of the active site tyrosine accounted for half of the total tyrosine loss. The results suggest that carbonate and halogen radicals may be more significant than •OH as drivers for protein degradation in serum. Accounting for the selective targeting of biomolecules by these daughter radicals is important for developing a mechanistic understanding of the consequences of oxidative stress.

Aldehyde-mediated protein degradation is responsible for the inhibition of nucleotide excision repair by cigarette sidestream smoke.

We recently reported that cigarette sidestream smoke (CSS) can delay nucleotide excision repair (NER), which was due to the inhibition of repair protein accumulation to DNA damage sites. However, the mechanisms how the protein recruitment was inhibited remains unclear. We hypothesized that aldehydes in CSS could be a candidate taking a role for the inhibition, and tested our hypothesis by removing aldehydes from CSS using cigarette-filters. The NER inhibition potency of CSS or filtered CSS (F-CSS) was compared using human keratinocyte cell line, HaCaT. Cigarette-filters were able to reduce total aldehydes in CSS by half. Pretreating cells with CSS and F-CSS enhanced UVB-induced cell death, with the effect of CSS weakened by filtration. CSS strongly inhibited the repair of UVB-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts (6-4PPs), where the recruitments of repair molecules, TFIIH and XPG, were slowed down. F-CSS showed similar inhibition of NER and accumulation of related proteins, but the effect was weaker than CSS. Semicarbazide (SEM), an aldehyde-trapping agent, alleviated the NER delay induced by both CSS and F-CSS, further confirming that aldehydes in CSS were the main cause for the inhibition of NER and that the different amounts of aldehydes in CSS and F-CSS were responsible for the different inhibition efficiency. Furthermore, TFIIH level was decreased by treatment with CSS and restored in the presence of proteasome inhibitor, indicating that the degradation of NER proteins might be the cause of the inhibition of NER-protein recruitment. These results supported our hypothesis that aldehydes in CSS are the main contributor for the NER inhibition via protein degradation, and reconfirmed that exposure to CSS without filtration could be a severe threat to human health.

Chlorotyrosines versus Volatile Byproducts from Chlorine Disinfection during Washing of Spinach and Lettuce.

Following the Food Safety Modernization Act of 2011 in the U.S., guidelines for disinfection washes in food packaging facilities are under consideration to control pathogen risks. However, disinfectant exposures may need optimization because the high concentrations of chlorine disinfectant promote the formation of high levels of disinfection byproducts (DBPs). When chlorine doses up through the 200 mg/L as Cl2 range relevant to the current practice were applied to spinach and lettuce, significant DBP formation was observed, even within 5 min at 7 °C. Concentrations of volatile chlorinated DBPs in washwater were far higher than typically observed in disinfected drinking water (e.g., 350 µg/L 1,1-dichloropropanone). However, these DBPs partitioned to the aqueous phase and so represent a greater concern for the disposal or reuse of washwater than for consumer exposure via food. The volatile DBPs represent the low-yield, final products of chlorination reactions with multiple biomolecular precursors. The initial, high-yield transformation products of such reactions may represent a greater concern for consumer exposure because they remain bound within the biopolymers in food and would be liberated during digestion. Using protein-bound tyrosine as an example precursor, the concentrations of the initial 3-chlorotyrosine and 3,5-dichlorotyrosine transformation products from this one precursor in the leaf phase were comparable to, and, in the case of some lettuces, exceeded, the aggregate aqueous concentration of volatile DBPs formed from multiple precursors. Chlorotyrosine formation increased when spinach was shredded due to the greater accessibility of chlorine to proteins in the leaf interiors. The cytotoxicity of chlorotyrosines to Chinese hamster ovary cells was higher than any of the trihalomethanes regulated in drinking water.

Investigation of nuclear enzyme topoisomerase as a putative molecular target of monohaloacetonitrile disinfection by-products.

Disinfection by-products occur widely as the unintended effect of water disinfection and are associated with toxicity and adverse human health effects. Yet the molecular mechanisms of their toxicity are not well understood. To investigate the molecular basis of hyperploidy induction by monohaloacetonitriles, the interaction of monohaloacetonitriles with topoisomerase II in Chinese hamster ovary cells was examined. We showed a concentration-dependent inhibition of DNA decatenation activity of topoisomerase under acellular conditions while in vitro monohaloacetonitrile treatment expressed mixed results. The working hypothesis, that topoisomerase II is a molecular target of monohaloacetonitriles, was only partially supported. Nevertheless, this research serves as a starting point toward molecular mechanisms of toxic action of monohaloacetonitriles.

Acetonitrile and N-Chloroacetamide Formation from the Reaction of Acetaldehyde and Monochloramine.

Nitriles and amides are two classes of nitrogenous disinfection byproducts (DBPs) associated with chloramination that are more cytotoxic and genotoxic than regulated DBPs. Monochloramine reacts with acetaldehyde, a common ozone and free chlorine disinfection byproduct, to form 1-(chloroamino)ethanol. Equilibrium (K1) and forward and reverse rate (k1,k-1) constants for the reaction between initial reactants and 1-(chloroamino)ethanol were determined between 2 and 30 °C. Activation energies for k1 and k-1 were 3.04 and 45.2 kJ·mol(-1), respectively, and enthalpy change for K1 was -42.1 kJ·mol(-1). In parallel reactions, 1-(chloroamino)ethanol (1) slowly dehydrated (k2) to (chloroimino)ethane that further decomposed to acetonitrile and (2) was oxidized (k3) by monochloramine to produce N-chloroacetamide. Both reactions were acid/base catalyzed, and rate constants were characterized at 10, 18, and 25 °C. Modeling for drinking water distribution system conditions showed that N-chloroacetamide and acetonitrile concentrations were 5-9 times higher at pH 9.0 compared to 7.8. Furthermore, acetonitrile concentration was found to form 7-10 times higher than N-chloroacetamide under typical monochloramine and acetaldehyde concentrations. N-chloroacetamide cytotoxicity (LC50 = 1.78 × 10(-3) M) was comparable to dichloroacetamide and trichloroacetamide, but less potent than N,2-dichloroacetamide and chloroacetamide. While N-chloroacetamide was not found to be genotoxic, N,2-dichloroacetamide genotoxic potency (5.19 × 10(-3) M) was on the same order of magnitude as chloroacetamide and trichloroacetamide.

Toxic impact of bromide and iodide on drinking water disinfected with chlorine or chloramines.

Disinfectants inactivate pathogens in source water; however, they also react with organic matter and bromide/iodide to form disinfection byproducts (DBPs). Although only a few DBP classes have been systematically analyzed for toxicity, iodinated and brominated DBPs tend to be the most toxic. The objectives of this research were (1) to determine if monochloramine (NH2Cl) disinfection generated drinking water with less toxicity than water disinfected with free chlorine (HOCl) and (2) to determine the impact of added bromide and iodide in conjunction with HOCl or NH2Cl disinfection on mammalian cell cytotoxicity and genomic DNA damage induction. Water disinfected with chlorine was less cytotoxic but more genotoxic than water disinfected with chloramine. For both disinfectants, the addition of Br(-) and I(-) increased cytotoxicity and genotoxicity with a greater response observed with NH2Cl disinfection. Both cytotoxicity and genotoxicity were highly correlated with TOBr and TOI. However, toxicity was weakly and inversely correlated with TOCl. Thus, the forcing agents for cytotoxicity and genotoxicity were the generation of brominated and iodinated DBPs rather than the formation of chlorinated DBPs. Disinfection practices need careful consideration especially when using source waters containing elevated bromide and iodide.

Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 µM IAN, 12 µM BAN and 900 µM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of chromosomes may contribute to cancer induction and perhaps be involved in the induction of adverse pregnancy outcomes associated with long-term consumption of disinfected water. Here we present the first observation of the induction of hyperploidy by a class of DBPs.

Chloroacetonitrile and n,2-dichloroacetamide formation from the reaction of chloroacetaldehyde and monochloramine in water.

Combined chlorine is increasingly being used as an alternative disinfectant to free chlorine to maintain a residual in drinking water distribution systems mainly because it would reduce the formation of regulated disinfection byproducts (DBPs) trihalomethanes and haloacetic acids. However, the use of combined chlorine could promote the formation of currently unregulated nitrogenous DBPs (N-DBPs) such as haloacetonitriles and haloacetamides that are found to be more cyto- and genotoxic than regulated DBPs. Monochloramine quickly reacts with chloroacetaldehyde, a DBP formed during primary disinfection with free chlorine, forming and reaching pseudoequilibrium (equilibrium constant K1 = 1.87 × 10(3) M(-1)) with the carbinolamine 2-chloro-1-(chloroamino)ethanol. 2-Chloro-1-(chloroamino)ethanol undergoes slow dehydration to form the imine 1-chloro-2-(chloroimino)ethane that decomposes at a faster rate to chloroacetonitrile. 2-Chloro-1-(chloroamino)ethanol is also oxidized by monochloramine to produce the previously unreported DBP N,2-dichloroacetamide. The carbinolamine dehydration step was found to be acid/base catalyzed (k2(0) = 3.30 × 10(-6) s(-1), k2(H) = 2.43 M(-1) s(-1), k2(OH) = 3.90 M(-1) s(-1)). In contrast, N,2-dichloroacetamide formation was observed to be only base catalyzed (k3(OH) = 3.03 × 10(4) M(-2) s(-1)). N,2-dichloroacetamide cytotoxicity (LC50 = 2.56 × 10(-4) M) was found to be slightly lower compared to that reported for chloroacetamide but higher than those of di- and trichloroacetamide.

Development and performance characterization of a polyamide nanofiltration membrane modified with covalently bonded aramide dendrimers.

A first generation of amine terminated aramide dendrimers (G1-NH2) was covalently attached to the polyamide (PA) active layer of a commercially available nanofiltration (NF) membrane. Amide bonds between G1-NH2 and PA free carboxylic groups were formed by activation of the carboxylic groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or 2-chloro-1-methylpyridinium iodide (CMPI), followed by aminolysis. Dendrimer attachment was assessed by indirectly measuring the concentration of carboxylic groups and amine groups before and after membrane modification with RBS using yttrium and tungstate ions (Y(3+) and WO4(2-)) as ion probes. RBS analyses showed a decrease in the concentration of carboxylic groups and an increase in amine groups on the membrane active layer, consistent with dendrimers attaching covalently to the active layer. Permeation experiments with Rhodamine WT (R-WT) revealed that the water and solutes permeability decreased after modification with dendrimer G1-NH2. Water permeability of G1-NH2 modified membrane decreased by 16-19% using EDC combined with sulfo-N-hydroxysuccinimide (s-NHS), and by 17-33% using CMPI. The permeability of the electrolyte BaCl2 decreased by 54% after G1-NH2 modification using EDC/s-NHS and only by 20% using CMPI, the latter consistent with a weaker Donnan exclusion effect. The permeability of the larger solute R-WT decreased by 82% in modified G1-NH2 membranes when using EDC/s-NHS, and 64% for cross-linking reagent CMPI. Thus, the use of EDC/s-NHS was more favorable because it resulted in higher gains in solute rejection with lower losses in water permeability.