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 (<i>HMOX1</i>, <i>NQO1</i>, <i>PTGS2</i>, and <i>TXNRD1</i>) 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

Emerging Disinfection Byproducts, Halobenzoquinones: Effects of Isomeric Structure and Halogen Substitution on Cytotoxicity, Formation of Reactive Oxygen Species, and Genotoxicity

Halobenzoquinones (HBQs) are a structurally diverse class of water disinfection byproducts. Here, we report a systematic study on the effects of isomeric structure and the type and number of halogen substitutions of HBQs on their cytotoxicity, formation of reactive oxygen species (ROS), and genotoxicity. Dynamic responses and IC₅₀ histograms were obtained using real-time cell analysis, clearly ranking the cytotoxicity of the HBQs in Chinese hamster ovary (CHO-K1) cells. Strong isomeric structure effects were shown with 2,5-HBQ isomers inducing greater cytotoxicity than their corresponding 2,6-HBQ isomers (<i>P</i> < 0.05). HBQ-halogen substitution groups also influence cytotoxicity, as cytotoxicity increases across the dihalogenated HBQs: iodo- > bromo- > chloro-HBQs (<i>P</i> < 0.05). Determination of HBQ-induced ROS further supports isomeric structure and halogen substitution effects. HBQ-induced genotoxicity was shown as increased levels of 8-hydroxy-2′-deoxyguanosine and p53 protein. Pearson correlation analysis of the HBQ toxicity measurements with their physicochemical parameters demonstrates that dipole moment and the lowest unoccupied molecular orbital energy are two major structural influences on toxicity (<i>r</i> = −0.721 or −0.766, <i>P</i> < 0.05). Dipole moment also correlates with isomer toxicity. This study suggests that formation and occurrence of highly toxic iodo-HBQs and 2,5-HBQs warrant further investigation to fully assess the impact of HBQs in drinking water

Multidrug Resistance Protein 4 (MRP4/ABCC4) Protects Cells from the Toxic Effects of Halobenzoquinones

Halobenzoquinones (HBQs) are frequently detected disinfection byproducts (DBPs) in treated water. Recent studies have demonstrated that HBQs are highly cytotoxic and capable of inducing the generation of reactive oxygen species (ROS) and depleting cellular glutathione (GSH). Multidrug resistance proteins (MRPs/ABCCs) are known to play a critical role in the elimination of numerous drugs, carcinogens, toxicants, and their conjugated metabolites. In general, little is known about the roles of transporters in DBP toxicity. Here, we hypothesize that MRPs may play roles in the detoxication of HBQs. To test this hypothesis, we used human embryonic kidney 293 (HEK293) cells stably expressing MRPs (MRP1, 3, 4, and 5) and HEK293 cells with empty vector (HEK-V) to examine the comparative cytotoxicity of four HBQs: 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), and 2,3,6-trichloro-1,4-benzoquinone (TriCBQ). The cytotoxicity (IC₅₀) of the four HBQs in HEK-MRP1, -MRP3, -MRP4, and -MRP5 cells and the control HEK-V cells clearly showed that MRP4 had the most significant effect on reducing the toxicity of the four HBQs. To further support MRP4-mediated detoxication of HBQs, we examined the HBQ-induced ROS levels in HEK-MRP4 and HEK-V cells. ROS levels were significantly reduced in HEK-MRP4 cells compared with HEK-V cells after HBQ treatment. Furthermore, it was found that MRP4-mediated detoxication of the HBQs was GSH dependent, as the cytotoxicity of the HBQs was increased in GSH-depleted HEK-MRP4 cells in comparison to HEK-MRP4 cells. The GSH-dependent protection of cells from HBQs supports the possibility of HBQ–GSH conjugate efflux by MRP4. This study demonstrates a role for MRP4 in cellular protection against HBQ DBP-induced toxicity and oxidative stress

Real-Time Cell-Electronic Sensing of Coal Fly Ash Particulate Matter for Toxicity-Based Air Quality Monitoring

The development of a unique bioassay for cytotoxicity analysis of coal fly ash (CFA) particulate matter (PM) and its potential application for air quality monitoring is described. Using human cell lines, A549 and SK-MES-1, as live probes on microelectrode-embedded 96-well sensors, impedance changes over time are measured as cells are treated with varying concentrations (1 μg/mL–20 mg/mL) of CFA samples. A dose-dependent impedance change is determined for each CFA sample, from which an IC₅₀ histogram is obtained. The assay was successfully applied to examine CFA samples collected from three coal-fired power plants (CFPs) in China. The samples were separated into three size fractions: PM2.5 (<2.5 μm), PM10-2.5 (2.5 μm < <i>x</i> < 10 μm), and PM10 (>10 μm). Dynamic cell-response profiles and temporal IC₅₀ histograms of all samples show that CFA cytotoxicity depends on concentration, exposure time (0–60 h), and cell-type (SK-MES-1 > A549). The IC₅₀ values differentiate the cytotoxicity of CFA samples based on size fraction (PM2.5 ≈ PM10-2.5 ≫ PM10) and the sampling location (CFP2 > CFP1 ≈ CFP3). Differential cytotoxicity measurements of particulates in human cell lines using cell-electronic sensing provide a useful tool for toxicity-based air quality monitoring and risk assessment

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 μM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC₅₀ values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 μM, respectively, more toxic than Roxarsone, whose 24 h IC₅₀ values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 μM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone

Analytical and Toxicity Characterization of Halo-hydroxyl-benzoquinones as Stable Halobenzoquinone Disinfection Byproducts in Treated Water

Exposure to chlorination disinfection byproducts (DBPs) is potentially associated with an increased risk of bladder cancer. Four halobenzoquinones (HBQs) have been detected in treated drinking water and have shown potency in producing reactive oxygen species and inducing damage to cellular DNA and proteins. These HBQs are unstable in drinking water. The fate and behavior of these HBQs in drinking water distribution systems is unclear. Here we report the high-resolution mass spectrometry identification of the transformation products of HBQs as halo-hydroxyl-benzoquinones (OH-HBQs) in water under realistic conditions. To further examine the kinetics of transformation, we developed a solid-phase extraction with ultrahigh-performance liquid chromatography tandem mass spectrometry (SPE–UHPLC–MS/MS) method to determine both the HBQs and OH-HBQs. The method provides reproducible retention times (SD < 0.05 min), limits of detection (LODs) at subnanogram per liter levels, and recoveries of 68%–96%. Using this method, we confirmed that decrease of HBQs correlated with increase of OH-HBQs in both the laboratory experiments and several distribution systems, supporting that OH-HBQs were more stable forms of HBQ DBPs. To understand the toxicological relevance of the OH-HBQs, we studied the in vitro toxicity with CHO-K1 cells and determined the IC₅₀ of HBQs and OH-HBQs ranging from 15.9 to 72.9 μM. While HBQs are 2-fold more toxic than OH-HBQs, both HBQs and OH-HBQs are substantially more toxic than the regulated DBPs

Post-Horse River Wildfire Surface Water Quality Monitoring Using the Water Cytotoxicity Test

Version 1.0 - External Review Draft. Supporting Dataset can be found at: Kinniburgh, David; Huang, Dorothy; Moe, Birget; Dey, Indranil; Luong, Jennie; Xie, Li; Tesfazgy, Milly; Demofsky, Paige; Parmentier, Spencer; Gabos, Stephan; Zhang, Weiping; Reichert, Megan; Wang, Nina Ching Yi; Ellehoj, Erik; Hatfield Consultants, 2023, "Dataset for: Post-Horse River Wildfire Surface Water Quality Monitoring Using the Water Cytotoxicity Test", https://doi.org/10.5683/SP3/ICGLUE, Borealis, V1.The 2016 Horse River wildfire had a significant environmental impact on the Regional Municipality of Wood Buffalo (RMWB) in Northern Alberta, with a burn area exceeding 580,000 hectares. To understand the impact of this unprecedented event on water quality in the RMWB, water samples were collected from surface waters, drinking water treatment plants, wastewater treatment plants, and taps over the three-year period immediately proceeding the wildfire, beginning in May 2017. Samples were collected from sites directly impacted by the Horse River wildfire (Fort McMurray), as well as sites upstream (Athabasca) and downstream (Fort McKay, Fort Chipewyan) from the impacted area. Each water sample was tested using the cell-based water cytotoxicity assay, an in-house developed bioassay with quality control criteria and previous application to environmental testing. The underlying technology of the assay allows for non-invasive and continuous monitoring of human HepG2 cells, providing more human health relevant toxicity information than traditional assays with non-mammalian targets. Comparative toxicity values incorporating both concentration and temporal cellular response data were determined for each sample, allowing for the identification of trends across geographic location, source (surface, treatment plant, tap), and time. Complementary chemical analysis, including routine water chemistry and trace element analysis, was also performed to evaluate chemical components that may have influenced the measured cellular response and to observe trends in contaminant concentrations across time and sampling location. The surface water samples with the highest measured cytotoxicity were collected in 2017 from Fort McMurray, which was directly impacted by the Horse River wildfire. Sites located further downstream in Fort McKay and Fort Chipewyan were less cytotoxic, indicating dilution may have impacted the distribution of the wildfire contaminants. Trace chemical analysis revealed elevated concentrations of sixteen trace elements in these samples, which were highest in samples from the impacted area and lower in sites downstream. A second spike in toxicity was observed in many surface waters and drinking water source locations in 2019, which may reflect other wildfire events that occurred in the province that year. Among the wastewater treatment plants evaluated, all four facilities demonstrated effective treatment across their treatment streams, but the effluent from Fort McMurray, the only facility to utilize clarifiers, was the least cytotoxic. Exceedances of Canadian drinking water guideline values were evaluated in all sample types. Linear regression analysis found positive correlations between 1) concentrations of routine testing parameters and cytotoxicity in wastewater influent and 2) concentrations of trace elements and cytotoxicity in surface and source waters. Many samples containing trace amounts of targeted chemicals still presented with high cytotoxicity, indicating that untargeted substances or mixture effects impacted the cellular response and bioassay testing can complement traditional chemical analysis approaches for environmental monitoring. The water cytotoxicity assay provides numerous advantages, including limited sample preparation, small sample volume requirements (< 10 mL), and simple testing procedures. The testing method is also data rich, providing quantitative information that can be used to compare samples exhibiting low or high cytotoxicity. The results of this three-year investigation indicate that the water cytotoxicity assay has strong potential for application to routine environmental monitoring (to complement chemical-based monitoring programs), and to identify high toxicity samples that require further assessment/remediation as part of the investigation of emergency situations (e.g. an industrial spill).Othe