Halobenzoquinones Research Articles

Magnetic multi-enzyme cascade combined with liquid chromatography tandem mass spectrometry for fast DNA digestion and quantitative analysis of 5-hydroxymethylcytosine in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA.

Occurrence and transformation of newly discovered 2-bromo-6-chloro-1,4-benzoquinone in chlorinated drinking water

Halobenzoquinones (HBQs) have been reported as an emerging category of disinfection byproducts (DBPs) in drinking water with relatively high toxicity, and the previously reported HBQs include 2,6-dichloro-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2,6-diiodo-1,4-benzoquinone, 2-chloro-6-iodo-1,4-benzoquinone, and 2-bromo-6-iodo-1,4-benzoquinone. In this study, another HBQ species, 2-bromo-6-chloro-1,4-benzoquinone (2,6-BCBQ), was newly detected and identified in drinking water. The occurrence frequency and levels of 2,6-BCBQ were investigated, and its cytotoxicity was evaluated. Since the formed 2,6-BCBQ was found to be not stable in chlorination, its transformation kinetics and mechanisms in chlorination were further studied. The results reveal that 2,6-BCBQ was generated from Suwannee River humic acid with concentrations in the range of 4.4–47.9 ng/L during chlorination within 120 h, and it was present in all the tap water samples with concentrations ranging from 1.5 to 15.7 ng/L. Among all the tested bromochloro-DBPs, 2,6-BCBQ showed the highest cytotoxicity on the human hepatoma cells. The transformation of 2,6-BCBQ in chlorination followed a pseudo-first-order decay, which was significantly affected by the chlorine dose, pH, and temperature. Seven polar chlorinated and brominated intermediates (including HBQs, halohydroxybenzoquinones, and halohydroxycyclopentenediones) were detected in chlorinated 2,6-BCBQ samples, according to which the transformation pathways of 2,6-BCBQ in chlorination were proposed. Besides, four trihalomethanes and four haloacetic acids were also generated during chlorination of 2,6-BCBQ with molar transformation percentages of 1.6–13.7%.

Quantitative identification of halo-methyl-benzoquinones as disinfection byproducts in drinking water using a pseudo-targeted LC-MS/MS method

Halobenzoquinones (HBQs) as disinfection byproducts (DBPs) in drinking water is prioritized for research due to their prevalent occurrence and high toxicity. However, only fifteen HBQs can be identified among a high diversity using targeted LC-MS/MS analysis in previous studies due to the lack of chemical standards. In this study, we developed a pseudo-targeted LC-MS/MS method for detecting and quantifying diverse HBQs. Distinct fragment characteristics of HBQs was observed according to the halogen substituent effects, and extended to the development of a multiple-reaction-monitoring (MRM) method for the quantification of the 46 HBQs that were observed in simulated drinking water using non-targeted analysis. The fragmentation mechanism was supported by the changes of Gibbs free energy (ΔG), and a linear relationship between the ΔG and the ionization efficiency of analytes was developed accordingly for quantification of these 46 HBQs, 30 of which were lack of chemical standards. It is noted that 29 of the 30 newly-identified HBQs were halo-methyl-benzoquinones (HMBQs), which were predicted to be carcinogens related with drinking-water bladder cancer risk and be more toxic than non-methyl HBQs. Using the new method, twelve HMBQs were detected in actual drinking water samples with concentrations up to 100.4 ng/L, 3 times higher than that reported previously. The cytotoxicity in CHO cells of HMBQs was over 1-fold higher than that of non-methyl-HBQs. Therefore, HMBQs are an essential, highly toxic group of HBQs in drinking water, which deserve particular monitoring and control.

Neurotoxicity and transcriptome changes in embryonic zebrafish induced by halobenzoquinone exposure

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) with a widespread presence in drinking water that exhibit much higher cytotoxicity than regulated DBPs. However, the developmental neurotoxicity of HBQs has not been studied in vivo. In this work, we studied the neurotoxicity of HBQs on zebrafish embryos, after exposure to varying concentrations (0-8 µmol/L) of three HBQs, 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ), 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ) for 4 to 120 hr post fertilization (hpf). HBQ exposure significantly decreased the locomotor activity of larvae, accompanied by significant reduction of neurotransmitters (dopamine and γ-aminobutyric acid) and acetylcholinesterase activity. Furthermore, the expression of genes involved in neuronal morphogenesis (gfap, α1-tubulin, mbp, and syn-2α) were downregulated by 4.4-, 5.2-, 3.0-, and 4.5-fold in the 5 µmol/L 2,5-DCBQ group and 2.0-, 1.6-, 2.1-, and 2.3-fold in the 5 µmol/L 2,5-DBBQ group, respectively. Transcriptomic analysis revealed that HBQ exposure affected the signaling pathways of neural development. This study demonstrates the significant neurotoxicity of HBQs in embryonic zebrafish and provides molecular evidence for understanding the potential mechanisms of HBQ neurotoxicity.

Fast coupling and detoxification of aqueous halobenzoquinones by extracellular nucleophiles: The relationship among structures, pathways and toxicity

Halobenzoquinones (HBQs), a metabolism of pentachlorophenol, arouse increasing concern for their high toxicity. This paper systematically studied the influences of 31 extracellular nucleophiles on the removal of dichlorobenzoquinones (DCBQs). The results showed that at pH 6.0–8.0, the transformation of DCBQs followed a pattern of pseudo first-order reaction. With the presence of different thiol compound (ratio 1:1), the apparent rate constant of 25DCBQ and 26DCBQ was between 0.028 and 0.204 min−1 and 0.061–0.156 min−1, respectively, which was 6.6–48.3 times faster than direct hydrolysis and 1.9–23.4 times of that with histidine, a typical amino acid. This was attributed to the different transformation pathways of DCBQs with thiol or amino groups. According to the MS spectrum and transition state theory, thiol compounds was stably incorporated into DCBQs via Michael addition or substitution, which also decreased the oxidative stress of DCBQs by 35.5–86.4% and cytotoxicity of hydrolyzed DCBQ by 44.9–100%, respectively. Conversely, amino acids promoted the hydrolysis of DCBQs by forming unstable AA-DCBQ intermediates that can be further hydrolyzed to OH-DCBQs, leading to an increased selectivity of OH-DCBQs, and increased the oxidative stress of 25DCBQs by 0.2–31.2% and cytotoxicity of hydrolyzed 25DCBQ by 42.0–52.7%. Such difference was inherently related to the nucleophilicity of different atoms. Moreover, it was revealed that the maximum condensed local nucleophilicity index of nucleophiles (Nk,max) can joint describe the relationship among nucleophilic structures, transformation pathways and toxicity of DCBQs. This paper provided a comprehensive perspective on the fast removal and detoxification of HBQs in extracellular environment.

Formation of chlorinated halobenzoquinones during chlorination of free aromatic amino acids

Chlorinated halobenzoquinones (HBQs) widely exist in drinking water as emerging disinfection byproducts (DBPs), which have attracted significant attention due to their wide occurrence and high toxicity. In this study, the formation of chlorinated HBQs from the three free aromatic amino acids, tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe), during chlorination was investigated, the formation pathways of chlorinated HBQs were explained based on the detected intermediates and influence factors. The results revealed that four chlorinated HBQs, including 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,3,5-trichloro-1,4-benzoquinone, 2,3,5,6-tetrachloro-1,4-benzoquinone and 2,6-dichloro-3-methyl-1,4-benzonquinone, were formed in chlorination of the three free aromatic amino acids, and 2,6-DCBQ was the dominant species among the formed chlorinated HBQs. Of the three free aromatic amino acids, Trp and Tyr presented relatively high yields of chlorinated HBQs than Phe. Moreover, ten intermediates were successfully detected (e.g., N,2-dichloroaniline from Trp, 2,4,6-trichlorophenol from Tyr) according to the isotope and fragment information obtained using high resolution mass spectrometry. The formation pathways of chlorinated HBQs from Trp and Tyr were proposed to include electrophilic addition, electrophilic substitution, oxidation, deacidification and dehydration reaction, and further validated using theoretical calculation. The yields of chlorinated HBQs during chlorination of the free aromatic amino acids were significantly affected by free chlorine dosage, pH and temperature.

Molecular composition of halobenzoquinone precursors in natural organic matter in source water

Halobenzoquinones (HBQs) are emerging disinfection byproducts generated during the reaction of chlorine disinfectant with natural organic matter (NOM) in source water. In this study, the correlations between molecular weight and HBQs generation of river NOM was evaluated. The compositional and functional characteristics of primary HBQs precursors were revealed by using Orbitrap mass spectrometry combined with molecular tagging. The NOM fraction larger than 50 kDa resulted in approximately 9 times more HBQs (50.9 ± 2.7 ng/mgC) than low molecular weight fractions. Significant correlations were found between the yields of HBQs and lignin-like and highly oxygen compounds in NOM, suggesting their critical roles in HBQs formation. Derivatizating the aldehydes, ketones, hydroxyl and carboxyl groups in NOM could reduce HBQs yields by 90.7%–100%. Unraveling the molecular characteristics of HBQs precursors in NOM would greatly benefit the prediction of HBQs yields of different source water, and develop more efficient disinfection byproduct control strategies.

Transcriptomic analysis of adult zebrafish heart and brain in response to 2, 6-dichloro-1, 4-benzoquinone exposure

Halobenzoquinones (HBQs) are emerging and widespread disinfection byproducts (DBPs), but their toxicological mechanisms to aquatic organisms remain elusive. Herein, we evaluated oxidative stress, cardiac toxicity, and cerebral toxicity after 2, 6-dichloro-1, 4-benzoquinone (2,6-DCBQ) exposure in zebrafish. Adult zebrafish were respectively exposed to 0.25, 0.5, and 1 μM 2,6-DCBQ for 96 h. The mortality rate of 2,6-DCBQ (1 μM) was 10%, while the LC50 value was 1.532 μM. Besides, 2,6-DCBQ exposure caused irregularity and elimination of myocardial fiber in the heart, and the pyknosis of nuclears and the agglutination of chromatin in the brain. We measured the 2,6-DCBQ-induced oxidative stresses in the heart and brain. Additionally, the glutathione (GSH) content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and total antioxidant capacity (T-AOC) were significantly inhibited. To better understand the potential toxicity of 2,6-DCBQ, transcriptomic analysis was performed in the control and 1 μM group after 96 h exposure. As a result, 545 and 1228 differentially expressed genes (DEGs) were detected in the heart and brain, respectively. GO analysis revealed that these DEGs were primarily enriched in blood vessel development, vasculature development, and oxidoreductase activity in the heart; response to stimulus, nervous system development, and oxidoreductase activity in the brain. KEGG enrichment analysis indicated that the DEGs were mainly enriched in VEGF signaling pathway and vascular smooth muscle contraction pathway in the heart; neuroactive ligand-receptor interaction, and NOD-like receptor signaling pathway in the brain. These findings exposed the underlying toxicity mechanism of 2,6-DCBQ exposure on zebrafish cardiovascular and brain systems.

Hydrolysis and Chlorination of 2,6-Dichloro-1,4-benzoquinone under conditions typical of drinking water distribution systems

Halobenzoquinones (HBQs) are emerging disinfection by-products (DBPs) that are postulated drivers of bladder carcinogenicity. Prior assessments of 2,6-dichloro-1,4-benzoquinone (DCBQ) occurrence in drinking water distribution systems have revealed a gradual decline with increasing distance from points of entry. While this signals a degradation pathway, there is limited quantitative data on rate of that degradation. A systematic evaluation of DCBQ hydrolysis was performed, resulting in a rate law that is first order in both hydroxide [OH−] and [DCBQ]. The impact of temperature on that rate was characterized according to the Arrhenius relationship. Under the conditions tested (pH~7.2, T = 20°C) chloramine did not significantly impact DCBQ concentrations. However, DCBQ was rapidly degraded in solutions containing free available chlorine (FAC). Kinetic analysis showed non-integer order with respect to FAC. Further investigation led to a model that invoked reaction with dichlorine monoxide (Cl2O) as well as FAC.

Transformation of emerging disinfection byproducts Halobenzoquinones to haloacetic acids during chlorination of drinking water

Halobenzoquinones (HBQs) are emerging disinfection byproducts of high toxicity and frequent occurrence in drinking water. HBQs are unstable and can be transformed to hydroxylated HBQs (OH-HBQs) and haloacetic acids (HAAs) in the presence of multiple reactive species, which are generated during photolysis of chlorine. In this study, we investigated the occurrence of HBQs in the effluent of 10 water treatment systems and household tap water across China, and demonstrated the kinetic and mechanism of 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) degradation to HAAs by the solar/chlorine processes. HBQs were detected in all samples at concentrations from 1.2 to 42.7 ng/L. The degradation of 2,6-DCBQ during solar/chlorine treatment followed the pseudo-first order kinetics. HOCl/OCl- and ClO. contributed to 47.9% and 50.2% of 2,6-DCBQ degradation, whereas the contributions of Cl. and .OH were <1.5%. The degradation rates of 2,6-DCBQ were stable in the presence of chloride, but decreased by 4.3% in the presence of 0.1 mgC/L natural organic matter (NOM). ClO. and Cl. directly attacked the aromatic ring of 2,6-DCBQ to generate HAAs, whereas .OH reacted with OH-HBQs, the intermediate products of 2,6-DCBQ to generate HAAs. Unraveling the mechanism of free radical mediated 2,6-DCBQ transformation would greatly benefit the development of effective HBQ elimination technologies at water treatment plants in the future.

Occurrence and toxicity of halobenzoquinones as drinking water disinfection byproducts

Halobenzoquinones (HBQs) are emerging unregulated drinking water disinfection byproducts (DBPs) that are more toxic than regulated DBPs. This study aimed to determine the distribution and formation of HBQs in drinking water from water treatment plants in China, compare their chronic cytotoxicity and their induction of chromosomal damage in Chinese hamster ovary cells, and analyze the correlation of HBQ toxicity with their physicochemical parameters. Two HBQs, 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) and 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), were detected in finished water and tap water in China. The concentrations were in the ranges of <2.6–19.70 ng/L for 2,6-DCBQ and <0.38–1.8 ng/L for 2,6-DBBQ. Chemical oxygen demand and residual chlorine were positively correlated with HBQ formation. The HBQ concentration was lower in a drinking water treatment plant using chlorine dioxide. High Ca2+ in tap water decreased the HBQ level. The rank order of HBQ by cytotoxicity was 2-chloro-1,4-benzoquinone > 2,3-diiodo-1,4-benzoquinone > 2,6-diiodo-1,4-benzoquinone > 2,6-dibromo-1,4-benzoquinone > 2,5-dibromo-1,4-benzoquinone > 2,5-dichloro-1,4-benzoquinone > 2,6-dichloro-1,4-benzoquinone > tetrachloro-1,4-benzoquinone > 2,3,6-trichloro-1,4-benzoquinone, and for their genotoxicity, 2,5-dichloro-1,4-benzoquinone > 2,6-dichloro-1,4-benzoquinone > 2,3-diiodo-1,4-benzoquinone > 2,6-diiodo-1,4-benzoquinone > tetrachloro-1,4-benzoquinone > 2,5-dibromo-1,4-benzoquinone > 2,6-dibromo-1,4-benzoquinone > 2,3,6-trichloro-1,4-benzoquinone. The cytotoxicity of six dihalo-HBQs was negatively correlated with the octanol-water partition coefficient (r = −0.971, P < 0.05), molar refractivity (r = −0.956, P < 0.05), energy of the highest occupied molecular orbital (EHOMO) (r = −0.943, P < 0.05), and polar surface area (r = −0.829, P < 0.05). The genotoxicity of these three pairs of dihalo-HBQ isomers followed the same order as their EHOMO values. This study reveals the occurrence and formation of HBQs in drinking water in China and systematically evaluates the chromosomal damage caused by nine HBQs in mammalian cells.

Hydrolysis characteristics and risk assessment of a widely detected emerging drinking water disinfection-by-product—2,6-dichloro-1,4-benzoquinone—in the water environment of Tianjin (China)

Halobenzoquinones (HBQs) are an emerging class of drinking water disinfection byproducts (DBPs) that have been frequently detected in drinking water and are highly relevant to bladder cancer. Among the studied HBQs, 2,6-dichloro-1,4-benzoquinone (DCBQ) had the highest detection frequency and concentrations in drinking water. However, compared to other countries, the studies on HBQs that are being conducted in China, especially those on HBQs in drinking water, are not sufficient. Therefore, the concentrations of DCBQ in the Tianjin drinking water supply system were investigated in two seasons (winter and summer), and the risk that is posed by DCBQ in drinking water was evaluated for the first time. In addition, since HBQs are prone to hydrolysis in neutral and alkaline environments, identification of the hydrolytic characteristics of DCBQ at various pH values and in the real water environment is essential for better describing the environmental behavior of DCBQ; hence, the hydrolysis characteristics of DCBQ in phosphate buffers with various pH values and in four water samples were also examined in our study. The results demonstrated that DCBQ was widely detected in the drinking water treatment process and distribution systems, and the average concentration in our study (12.0 ng/L) was at a moderately high level compared with the reported concentration of DCBQ in the drinking water distribution networks. The risk quotient (RQ) of DCBQ is equivalent to that of trihalomethanes (THMs); thus, the relatively low concentrations of DCBQ should also be considered. Furthermore, the results demonstrated that the hydrolysis of DCBQ follows first-order reaction kinetics, the reaction rate accelerates as the pH of the phosphate buffer system increases, and the rate of hydrolysis of DCBQ in drinking water is affected not only by the pH but also by other environmental factors, such as the organic matter concentration. Therefore, further investigation is necessary to identify the main factor of DCBQ hydrolysis in real water environments.

Cytotoxicity and genotoxicity assays of halobenzoquinones disinfection byproducts using different human cell lines.

Recently, halobenzoquinones (HBQs) disinfection byproducts, including 2,6-dichloro-1, 4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1, 4-benzoquinone (DCMBQ), 2,3,6-trichloro-1, 4-benzoquinone (TCBQ), and 2,6-dibromobenzoquinone (DBBQ), have been of increasing concern due to their reported ability to induce oxidative damage, and thus genotoxicity. However, data on the risk of genotoxicity due to chromosomal damage by HBQs are still scarce. Here, the cytotoxicity and genotoxicity of the four HBQs were assessed using human cell lines (bladder cancer 5637 cells, colon carcinoma Caco-2 cells, and gastric MGC-803 cells). The four HBQs exhibited significant concentration-response relationships in all the three cell lines. Cytotoxicity of DCBQ, DCMBQ, TCBQ, and DBBQ, represented by the 50% concentration of inhibition (IC50 ) values, were 80.8-99.5, 41.0-57.6, 122.1-146.6, and 86.9-93.8 μM, respectively. The lowest effective concentrations for cellular micronuclei induction in the cell lines by DCBQ, DCMBQ, TCBQ, and DBBQ were 50-75, 20-41.5, 87.4-100, and 50 μM, respectively. 5637 and Caco-2 cells were more sensitive to the cytotoxic and genotoxic effects of HBQs than MGC-803 cells. These results show that HBQs can induce chromosomal damage; DCMBQ induced the highest cytotoxicity and genotoxicity in all the cell lines, and TCBQ caused the least toxicity.

A novel automated method for the quantification of ten halobenzoquinones in drinking water using online solid-phase extraction coupled with liquid chromatography tandem mass spectrometry

When assessing occurrence and exposure to toxic chemicals in drinking water, developing methods that are sensitive and efficient is paramount. A new method was developed for the quantification of ten halobenzoquinones (HBQs), a class of disinfection by-products (DBPs) in drinking water, which have been shown to be more toxic than most regulated DBPs. This method uses a small sample volume with online solid phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Previous methods used offline SPE coupled with standard addition, and a pre-concentration step. This requires larger volumes of sample, solvent, and standards. The current method has comparable limits of quantification (0.2–166 ng/L), requires minimal sample preparation, and analysis is almost entirely automated. It also includes 2,6-dibromo-3‑chloro-5-methyl-1,4-benzoquinone which was not included in previous methods. A stability test was conducted over a one-week period with different preservatives, including ascorbic acid, sodium thiosulfate, and formic acid. Method optimization included source temperature, SPE size, sample volume, and SPE loading time. This method was validated using drinking water collected from four different drinking water plants; spike recoveries of HBQs were between 70–111%, relative standard deviations of <20%, and linearity of >0.98. Further, using this method, we report the highest concentration of 2,6-dibromo-1,4-benzoquinone found in drinking water (254 ng/L).

Occurrence, Formation, and Oxidative Stress of Emerging Disinfection Byproducts, Halobenzoquinones, in Tea.

Halobenzoquinones (HBQs) are frequently detected disinfection byproducts (DBPs) in drinking water with high toxicity and relevance to public health. In this study, we characterized the occurrence, formation, and oxidative stress of the HBQs in tea. 2,6-DCBQ and TetraC-1,2-BQ were identified in all prepared teas at total concentrations of 1.3-2.0 ng/L. 2,6-DCBQ originated from drinking water DBPs, while TetraC-1,2-BQ originated from tea leaves or were generated during tea polyphenol chlorination. HBQs in tea induced the formation of reactive oxygen species and semiquinone radicals, and the oxidative stress could be depleted by tea polyphenols, e.g., (-)-epigallocatechin gallate (EGCG). High-resolution mass spectrometry analysis indicated that the HBQs combined with EGCG and formed adducts at a ratio of 1:1 or 2:1 with the binding sites on the A ring and B ring of EGCG. The viability of HepG2 cells exposed to 50 μM 2,6-DCBQ was increased from 20.0% to 65.2% when 50 μM of EGCG was added. These results demonstrated that various HBQs can occur in tea due to the HBQ DBPs in drinking water, the leachate from tea leaves, and the chlorination of tea polyphenols; furthermore, the oxidative stress and cellular toxicity induced by HBQs in tea could be decreased by tea polyphenols. This is the first study to report HBQs in tea, elucidate the sources of HBQs, and assess relevant health risks.

Occurrence and formation of halobenzoquinones in indoor and outdoor swimming pool waters of Nanning City, Southwest China.

Disinfection byproducts (DBPs) in swimming pool have elicited increasing worldwide concern due to their potential health risks. However, only trihalomethanes (THMs) are regulated by several local governing bodies. Studies indicated that specific unregulated DBP classes would drive disinfected water toxicity in addition to THMs. Halobenzoquinones (HBQs), a type of emerging unregulated DBPs, have been shown to be possible bladder cancer carcinogens. This study aimed to determine the distribution and formation of HBQs in indoor and outdoor swimming pool waters of Nanning City, Southwest China. Seven HBQs in water from seven public indoor and outdoor swimming pools were examined using an effective ultra-performance liquid chromatography-tandem mass spectrometry method. Results suggest the presence of 2,6-dichloro-1,4-benzoquinone in all the swimming pool waters in the range of 4.56-45.30ng/L. Furthermore, 2,6-dibromo-1,4-benzoquinone and 3,4,5,6-tetrachloro-1,2-benzoquinone (TetraC-1,2-BQ) were detected in two pools at concentrations of < 0.38-14.20 and < 0.54-2.60ng/L, respectively. The swimming pool water featured higher HBQs than input tap water, and TetraC-1,2-BQ was only detected in pool water. Higher HBQ levels were observed in the indoor pools than in the outdoor pools. These findings demonstrate that low NH3-N, high chloride, humic acid, chemical oxygen demand, and UV254 in the indoor pools increased the HBQ formation. This study is the first to reveal the occurrence and formation of HBQs in water from Chinese indoor and outdoor swimming pools. The findings should be useful in the management of these governing factors and HBQ controls in swimming pools.

Assessment of the in vitro toxicity of the disinfection byproduct 2,6-dichloro-1,4-benzoquinone and its transformed derivatives

An emerging class of unregulated disinfection byproducts, halobenzoquinones (HBQs), has gained recent interest following suggestions of enhanced toxicity compared to regulated disinfection byproducts. While the kinetics of HBQ hydrolysis in water have been well characterized, the stability of HBQs in cell culture media, a critical parameter when evaluating toxicity in vitro, has been overlooked. The objective of this study was: (1) to contrast the stability of a prevalent HBQ, 2,6-dichloro-1,4-benzoquinone (DCBQ), in cell culture media and water, and (2) to evaluate the cytotoxicity of parent and transformed DCBQ compounds as well as the ability of these compounds to generate intracellular reactive oxygen species (ROS) in normal human colon cells (CCD 841 CoN) and human liver cancer cells (HepG2). The half-life of DCBQ in cell media was found to be less than 40 min, compared to 7.2 h in water at pH 7. DCBQ induced a concentration-dependent decrease in cell viability and increase in ROS production in both cell lines. The parent DCBQ compound was found to induce significantly greater cytotoxicity compared to transformed DCBQ products. We demonstrate that the study design used by most published studies (i.e., extended exposure periods) has led to a potential underestimation of the cytotoxicity of HBQs by evaluating the toxicological profile primarily of transformed HBQs, rather than corresponding parent compounds. Future in vitro toxicological studies should account for HBQ stability in media to evaluate the acute cytotoxicity of parent HBQs.

Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways.

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce reactive oxygen species and oxidative damage in vitro. However, the impacts of HBQs on oxidative-stress-related gene expression have not been investigated. In this study, we examined alterations in the expression of 44 genes related to oxidative-stress-induced signaling pathways in human uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show the structure-dependent effects of HBQs on the studied gene expression. After 2 h of exposure, the expression levels of 9 to 28 genes were altered, while after 8 h of exposure, the expression levels of 29 to 31 genes were altered. Four genes ( HMOX1, NQO1, PTGS2, and TXNRD1) were significantly upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation, and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter the gene expression of oxidative-stress-related signaling pathways and contributes to the understanding of HBQ-DBP-associated toxicity.

Halobenzoquinone-mediated assembly of amino acid modified Mn-doped ZnS quantum dots for halobenzoquinones detection in drinking water

Halobenzoquinones (HBQs) were reported as disinfection byproducts (DBPs) which had potential risk of bladder cancer. In this paper, a highly selective analytical method for HBQs was developed by HBQs-mediated assembly of amino acid modified Mn-doped ZnS/Quantum Dots (Mn: ZnS QDs). In the presence HBQs, a charge-transfer complex (CTC) was formed between aromatic rings of HBQs and the primary amino groups on the surface of the QDs. The formation of CTC led to the aggregation of QDs, as a result fluorescence decreasing occurred. The decrease was correlated with the concentration of HBQs. Then a fluorescence sensor array for discrimination of three kinds of HBQs including 2,6-Dichloro-1,4-benzoquinone (DCBQ), 2,6-Dibromo-1,4-benzoquinone (DBBQ) and 2,3,6-trichloro-1,4-benzoquinone (TCBQ) was developed. Four kinds of amino acids including cysteine, threonine, tyrosine and tryptophan were embellished on the Mn: ZnS QDs. The different extents of aggregation led to different fluorescence decreasing effect, thus distinct fluorescence patterns were created. It showed that three kinds of HBQs could be discriminated successfully by fluorescence sensor array at a range of concentrations through principal component analysis (PCA). The unknown samples were predicted by with a stepwise linear discriminant analysis (SLDA) using Mahalanobis distance as a selection criterion with accuracy of 100%. Remarkably, the practicability of the proposed sensor array was further validated by identification of three kinds of HBQs at different concentrations in real drinking water samples. Compared to LC/MS/MS, this fluorescent sensor array-based method was proved to be more convenient since the nanoparticles can be prepared flexibly according to the property of the target.

Characterization of Mechanisms of Glutathione Conjugation with Halobenzoquinones in Solution and HepG2 Cells.

Halobenzoquinones (HBQs) are a class of emerging disinfection byproducts. Chronic exposure to chlorinated drinking water is potentially associated with an increased risk of human bladder cancer. HBQ-induced cytotoxicity involves depletion of cellular glutathione (GSH), but the underlying mechanism remains unclear. Here we used ultrahigh performance liquid chromatography-high resolution mass spectrometry and electron paramagnetic resonance spectroscopy to study interactions between HBQs and GSH and found that HBQs can directly react with GSH, forming various glutathionyl conjugates (HBQ-SG) in both aqueous solution and HepG2 cells. We found that the formation of HBQ-SG varies with the initial molar ratio of GSH to HBQ in reaction mixtures. Higher molar ratios of GSH to HBQ facilitate the conjugation of more GSH molecules to an HBQ molecule. We deduced the reaction mechanism between GSH and HBQs, which involves redox cycling-induced formation of halosemiquinone (HSQ) free radicals and glutathione disulfide, Michael addition, as well as nucleophilic substitution. The proposed reaction rates are in the following order: formation of HSQ radicals > substitution of bromine by GSH > Michael addition of GSH on the benzoquinone ring > substitution of chlorine by GSH > substitution of the methyl group by GSH. The conjugates identified in HBQ-treated HepG2 cells were the same as those found in aqueous solution containing a 5:1 ratio of GSH:HBQs.