Halobenzoquinones (HBQs), a new class of disinfection byproducts (DBPs), occur widely in treated drinking water and recreational water. The main concern regarding human exposure to DBPs stems from epidemiological studies that have consistently linked the consumption of chlorinated drinking water with an increased risk of developing bladder cancer. The U.S. Environmental Protection Agency and Health Canada have set regulations on the amount of DBPs in drinking water to minimize the risk. However, these regulated DBPs do not account for the increased risk of bladder cancer because they have different target organs or lower magnitudes of risk based on animal carcinogenesis studies. Because of the pervasive exposure to DBPs, identification of DBPs relevant to human health has become one of the important research targets to address DBP-associated health concerns. Quantitative structure-toxicity relationship (QSTR) analysis has predicted HBQs to be potential bladder carcinogens. Therefore, this perspective focuses on the chemical and toxicological characterization of HBQs. In vitro cytotoxicity experiments have shown that HBQs induce greater cytotoxicity and/or greater developmental toxicity than most of the regulated DBPs. Cellular mechanistic studies indicate that HBQs are capable of producing reactive oxygen species (ROS) either within cells or in solution, depleting cellular glutathione levels, and influencing cellular antioxidant enzymes, which further induces oxidative stress and oxidative damage to cellular proteins and DNA. Oxidative damage to DNA was demonstrated in the form of significant increases in cellular levels of 8-hydroxydeoxyguanosine (8-OHdG), DNA strand breaks, and apurinic/apyrimidinic (AP) sites. HBQs can also form DNA adducts, affect genome-wide DNA methylation, and inhibit DNA repair enzymes. These findings demonstrate that HBQs are highly cytotoxic and potentially genotoxic and carcinogenic, although in vivo data corroborating this is not available. To fully understand the potential adverse health effects and cancer risk due to HBQ exposure, multidisciplinary research is required regarding human exposure, health risk assessment, and toxicological mechanisms of HBQs.

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.

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 (P < 0.05). HBQ-halogen substitution groups also influence cytotoxicity, as cytotoxicity increases across the dihalogenated HBQs: iodo- > bromo- > chloro-HBQs (P < 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 (r = -0.721 or -0.766, P < 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.

Four halobenzoquinones (HBQs), 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 recently confirmed as disinfection byproducts (DBPs) in drinking water; however, their toxicological information is scarce. Here, we report that HBQs are cytotoxic to T24 bladder cancer cells and that the IC50 values are 95 μM for DCBQ, 110 μM for DCMBQ, 151 μM for TCBQ, and 142 μM for DBBQ, after a 24-h exposure. The antioxidant N-acetyl-l-cysteine (NAC) significantly reduces the cytotoxicity induced by the four HBQs, supporting the hypothesis that oxidative stress contributes to the cytotoxicity of HBQs. To further explore the oxidative mechanisms of cytotoxicity, we examined HBQ-induced production of reactive oxygen species (ROS) in T24 cells, and measured 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyls, and malondialdehyde (MDA) adducts of proteins, markers of oxidative damage to DNA, proteins, and lipids, respectively. All four HBQs generated intracellular ROS in T24 cells in a concentration-dependent manner. HBQs also produced 8-OHdG in genomic DNA of T24 cells, with the highest levels of 8-OHdG induced by DCMBQ. Protein carbonylation was significantly increased in T24 cells that were incubated with each of the four HBQs for 24 h. However, MDA adduct formation, a marker of lipid peroxidation, was not affected by any of the four HBQs tested. These results suggest that the ROS-induced oxidative damage to DNA and protein carbonylation are involved in the observed toxicity of HBQs in T24 cells.

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The developmental toxicity of water disinfection byproducts remains unclear. Here we report the study of halobenzoquinone (HBQ)-induced in vivo developmental toxicity and oxidative stress using zebrafish embryos as a model. Embryos were exposed to 0.5-10 μM of individual HBQs and 0.5-5 mM haloacetic acids for up to 120 h postfertilization (hpf). LC values of the HBQs at 24 hpf were 4.6-9.8 μM, while those of three haloacetic acids were up to 200 times higher at 1900-2600 μM. HBQ exposure resulted in significant developmental malformations in larvae, including failed inflation of the gas bladder, heart malformations, and curved spines. An increase in reactive oxygen species was observed, together with a decrease in superoxide dismutase activity and glutathione content. Additionally, the antioxidant N-acetyl-l-cysteine significantly mitigated all HBQ-induced effects, supporting that oxidative stress contributes to HBQ toxicity. Further experiments examined HBQ-induced effects on DNA and genes. HBQ exposure increased 8-hydroxydeoxyguanosine levels, DNA fragmentation, and apoptosis in larvae, with apoptosis induction related to changes in the gene expression of p53 and mdm2. These results suggest that HBQs are acutely toxic, causing oxidative damage and developmental toxicity to zebrafish larvae.

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