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

Butyrate Enhances γ‑H2AX Induced by Benzo[<i>a</i>]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

Chloroacetonitrile and <i>N</i>,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 <i>K</i>₁ = 1.87 × 10³ 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 <i>N</i>,2-dichloroacetamide. The carbinolamine dehydration step was found to be acid/base catalyzed (<i>k</i>₂⁰ = 3.30 × 10^–6 s^–1, <i>k</i>₂^H = 2.43 M^–1 s^–1, <i>k</i>₂^OH = 3.90 M^–1 s^–1). In contrast, <i>N</i>,2-dichloroacetamide formation was observed to be only base catalyzed (<i>k</i>₃^OH = 3.03 × 10⁴ M^–2 s^–1). <i>N</i>,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

Acetonitrile and <i>N</i>‑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 (<i>K</i>₁) and forward and reverse rate (<i>k</i>₁,<i>k</i>_–1) constants for the reaction between initial reactants and 1-(chloroamino)­ethanol were determined between 2 and 30 °C. Activation energies for <i>k</i>₁ and <i>k</i>_–1 were 3.04 and 45.2 kJ·mol^–1, respectively, and enthalpy change for <i>K</i>₁ was −42.1 kJ·mol^–1. In parallel reactions, 1-(chloroamino)­ethanol (1) slowly dehydrated (<i>k</i>₂) to (chloroimino)­ethane that further decomposed to acetonitrile and (2) was oxidized (<i>k</i>₃) by monochloramine to produce <i>N</i>-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 <i>N</i>-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 <i>N</i>-chloroacetamide under typical monochloramine and acetaldehyde concentrations. <i>N</i>-chloroacetamide cytotoxicity (LC₅₀ = 1.78 × 10^–3 M) was comparable to dichloroacetamide and trichloroacetamide, but less potent than <i>N</i>,2-dichloroacetamide and chloroacetamide. While <i>N</i>-chloroacetamide was not found to be genotoxic, <i>N</i>,2-dichloroacetamide genotoxic potency (5.19 × 10^–3 M) was on the same order of magnitude as chloroacetamide and trichloroacetamide

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

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

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 (Y³⁺ 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-<i>N</i>-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