In Vitro Cytotoxicity and Adaptive Stress Responses to Selected

Article pubs.acs.org/crt

In Vitro Cytotoxicity and Adaptive Stress Responses to Selected Haloacetic Acid and Halobenzoquinone Water Disinfection Byproducts Erik Procházka,*,† Beate I. Escher,†,‡,§ Michael J. Plewa,∥,⊥ and Frederic D. L. Leusch† †

Smart Water Research Centre, Australian Rivers Institute, School of Environment, Griffith University, Gold Coast, Queensland 4222, Australia ‡ Cell Toxicology, Helmholtz Centre for Environmental Research−UFZ, 04318 Leipzig, Germany § Environmental Toxicology, Center for Applied Geosciences, Eberhard Karls University Tübingen, 72074 Tübingen, Germany ∥ Department of Crop Sciences, ⊥Safe Global Water Institute and the Science and Technology, Center of Advanced Materials for the Purification of Water with Systems, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *

ABSTRACT: The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts (DBPs). Some of these are mutagenic, genotoxic, teratogenic, and cytotoxic, as well as potentially carcinogenic both in vivo and in vitro. We investigated the in vitro biological activity of five DBPs: three monohaloacetic acids (monoHAAs) [chloroacetic acid (CAA), bromoacetic acid (BAA), and iodoacetic acid (IAA)] and two novel halobenzoquinones (HBQs) [2,6dichloro-p-benzoquinone (DCBQ) and 2,6-dibromo-p-benzoquinone]. We focused particularly on cytotoxicity and induction of two adaptive stress response pathways: the oxidative stress responsive Nrf2/ARE and DNA-damage responsive p53 pathways. All five DBPs were cytotoxic to the Caco-2 cell line after a 4 h exposure, and all DBPs induced both of the adaptive stress response pathways, Nrf2/ARE and p53, in the micromolar range, as measured by two β-lactamase-based reporter gene assays. The decreasing order of potency for all three endpoints for the five DBPs was IAA ∼ BAA > DCBQ ∼ DBBQ > CAA. Induction of oxidative stress was previously proposed to be the molecular initiating event (MIE) for both classes of DBPs. However, comparing the levels of activation of the two pathways uncovered that the Nrf2/ARE pathway was the more sensitive endpoint for HAAs, whereas the p53 pathway was more sensitive in the case of HBQs. Therefore, the DNA damage-responsive p53 pathway may be an important piece of information to fill in a gap in the adverse outcome pathway framework for the assessment of HBQs. Finally, we cautiously compared the potential risk of the two novel HBQs using a benchmarking approach to that of the well-studied CAA, which suggested that their relative risk may be lower than that of BAA and IAA.

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INTRODUCTION The disinfection of drinking water is one of the major public health successes of the modern era.1 The use of highly reactive disinfectants such as chlorine (Cl2), chloramine (NH2Cl), chlorine dioxide (ClO2), and ozone (O3) has drastically reduced water-borne infections in the developed world; however, an unintended consequence is the creation of many disinfection byproducts (DBPs). DBPs are generated following the reaction of the disinfectant(s) with natural organic matter, anthropogenic contaminants, and bromide/iodide present in the source water.2,3 To date, over 600 DBPs have been identified in drinking waters,4 but this quantity represents merely a fraction of the total organic halogen (TOX) generated. A small number of DBPs has undergone systematic, quantitative, and comparative toxicological analyses,4−6 and current chemical analysis can explain only a small portion of genotoxicity and cytotoxicity observed in in vitro bioassays.7,8 © 2015 American Chemical Society

Research in test animals, in vitro studies, epidemiological evidence, and toxicological modeling demonstrate that many DBPs are cytotoxic, genotoxic, mutagenic, teratogenic, and potentially carcinogenic and may induce adverse pregnancy outcomes.4,5,9−18 DBPs belong to a wide variety of chemical classes.4,14 In this study, we focus on the ubiquitous haloacetic acids (HAAs) and novel halobenzoquinones (HBQs). HAAs are pervasive DBPs with relatively simple structures, low Henry’s law constants, strong acidity (low pKa), low hydrophobicity (low Kow of the neutral form), and high water solubility of the anionic form.19 They represent the second most common class of DBPs after trihalomethanes (THMs) and can occur in drinking water at concentrations up to a hundred micrograms per liter for Received: July 1, 2015 Published: September 1, 2015 2059

DOI: 10.1021/acs.chemrestox.5b00283 Chem. Res. Toxicol. 2015, 28, 2059−2068

Article

Chemical Research in Toxicology chloroacetic acid (CAA)20 and in low micrograms per liter for bromoacetic (BAA)21 and iodoacetic acids (IAA)22 (Table 1).

oxo-2′-deoxyguanosine (8-OHdG), protein carbonyls, and malondialdehyde (MDA), indicating that oxidative stress may play a role in the observed cytotoxicity.17 Reduced cellular toxicity following the coincubation with the detoxification enzyme glutathione (GSH) in vitro also points to the role of oxidative stress in HBQ cytotoxicity.39 Wang and Li40 measured in vitro cytotoxicity of HBQs as well as their corresponding hydroxylated transformation products, OH-HBQs, in the cricetine CHO-K1 cell line. The 24 h IC50 values for HBQs were 27.3 μM for DCBQ, 11.4 μM for DCMBQ, 45.5 μM for TriCBQ, and 19.8 μM for DBBQ. Hydroxylation decreased the cytotoxicity of the parent compound by a factor of 2, with 24 h IC50 values for OHHBQs reported at 61.0 μM for OH-DCBQ, 20.4 μM for OHDCMBQ, 64.4 μM for OH-TriCBQ, and 42.8 μM for OHDBBQ.40 More recently, Li et al.41 reviewed available chemical and toxicological information on HBQs and included a further realtime impedance-based cytotoxicity (RTCA)42 endpoint, which confirmed the cytotoxicity of HBQs to be in the micromolar range. The authors concluded that the in vitro evidence of oxidative stress-induced DNA damage may indicate that HBQs are likely to be genotoxic and carcinogenic.41 Current data on HBQs, therefore, suggest that they have the potential to contribute to the overall carcinogenicity associated with exposure to disinfected drinking water. Several signaling pathways are involved in the cellular response to oxidative, genotoxic, and proteotoxic stresses. From these pathways, those primarily involved in adaptive stress response are NFE2L2 (Nrf2), NFE2L1 (Nrf1), p53, heat shock factor (HSF), and unfolded protein response (UPR).43 In this study, we focused primarily on the Nrf2/ARE and p53 pathways because the genes associated with these two pathways were shown to be induced following exposure to both individual DBPs as well as disinfected drinking waters.15,23,44−47 Nrf2/ARE and p53 signaling pathways are two important adaptive stress response signaling pathways that provide protection against toxic insults.43,48 The Nrf2/ARE pathway, also known as the Keap1-Nrf2 pathway, serves as a cellular protective mechanism against the cumulative damaging effects of reactive oxygen species (ROS) and toxic electrophiles that accumulate as a result of either natural biological processes, such as respiration, or exposure to toxic chemicals.49 The p53 tumor suppressor gene acts to preserve the stability of the genome and to limit oncogenic potential by leading adaptive responses to genotoxic stress by inducing cell cycle arrest, senescence, and apoptosis.50 The p53 pathway, therefore, plays a pivotal role in cellular differentiation and tumor suppression. The pathway is activated in response to a variety of events, including DNA damage, genotoxic insult, or oxidative stress.43 The objective of the current study was to quantify the in vitro cytotoxicity of two novel HBQs (DCBQ and DBBQ) and three well-characterized monoHAAs (CAA, BAA, and IAA) in human epithelial colorectal adenocarcinoma (Caco-2) cells as well as to investigate their level of activation of two adaptive cellular stress pathways, p53 and Nrf2/ARE, related to DNA damage and oxidative stress response, respectively. The Caco-2 colorectal adenocarcinoma cell line was chosen for cytotoxicity analysis because it has been reported to positively correlate with in vivo rodent toxicity.51 Additionally, the Caco-2 cell line may be relevant to the digestive tract, which would be impacted by contaminants in drinking water, such as DBPs, even though direct inference of effects would be difficult due to both the

Table 1. CAA Bioanalytical Equivalent Concentrations (BEQs) Relative to Occurrence Data for HAAs and HBQsa

CAA BAA IAA DCBQ DBBQ a

max occurrence conc. (μg/L)

CAABEQ(cytotox) (μg/L)

CAABEQ(ARE‑bla) (μg/L)

CAABEQ(p53RE‑bla) (μg/L)

24420 3.7521 1.7822 0.2831 0.0431

240 110 62 2.1 0.30

240 38 32 0.40 0.052

240 38 29 1.1 0.10

Note that all BEQs are rounded to two significant figures.

HAAs have been identified as being genotoxic and cytotoxic and able to induce cellular DNA damage and oxidative stress response via several pathways, including ATM, MAPK, p53, BRCA1, BRCA2, ATR, and Nrf2/ARE.8,23−25 In addition, it was recently shown that monoHAAs cause their toxic effects via direct irreversible inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and subsequent induction of oxidative stress.24,26 A number of in vivo rodent studies provide data on toxicity of monoHAAs while offering some evidence for genotoxicity and tumorigenicity.27−29 HBQs constitute a relatively recently identified group of halogenated DBPs. The first reported, as well as the most abundant, HBQ in drinking water was 2,6-dichloro-1,4benzoquinone (DCBQ),30 detected in drinking water treatment plants across the U.S. and Canada at concentrations of 4.5− 274.5 ng/L.31 Other members of this group were subsequently identified and detected in drinking water,31,32 including 2,6dibromo-1,4-benzoquinone (DBBQ) at 0.05); however, CAA was significantly less toxic than both BAA and IAA (one-way ANOVA, Tukey’s posthoc test, p ≤ 0.05). Both bromo- and iodo-DBPs have gained increasing attention due to their relatively high toxicity when compared to that of their chlorinated counterparts.45,69−71 Iodoacetic acid was previously found to be powerfully cytotoxic (IC50 = 2.77 μM) and genotoxic in vitro in mouse embryonic fibroblast NIH3T3 cells, causing DNA damage, as measured by γ-H2AX, and increased tail moment in single-cell gel electrophoresis.71 The IAA-transformed NIH3T3 foci formed aggressive fibrosarcomas after they were inoculated into Balb/c nude mice, demonstrating that IAA has biological activity consistent with that of a human carcinogen.71 The two HBQs were approximately equitoxic in the Caco-2 cells (one-way ANOVA, Tukey’s posthoc test, p > 0.05; Table 2). This is consistent with previously published data on the cytotoxicity of HBQs in the T24 bladder cancer cell line, where DCBQ was only marginally more toxic than DBBQ.17 In the Caco-2 cell viability assay, the IC50 values of the two HBQs fell between those of CAA and BAA. The descending rank order of cytotoxicity was as follows: IAA ∼ BAA > DCBQ ∼ DBBQ > CAA (statistical significance determined by oneway ANOVA, Tukey’s posthoc test, p ≤ 0.05; Table 2). Nrf2/ARE Pathway Activation. All three HAAs and two HBQs were found to activate the Nrf2/ARE pathway. The descending rank order of potency was the same as that for the effect on cell viability/cytotoxicity: IAA ∼ BAA > DCBQ ∼ DBBQ > CAA (significant difference determined by one-way ANOVA, Tukey’s posthoc test, p ≤ 0.05; Table 3). Table 3 provides the ECIR1.5 values calculated from the linear portions of the concentration−effect curves. For comparison, the positive control for the assay, tert-butylhydroquinone (tBHQ), had an average ECIR1.5 of 3.8 μM, which was approximately as potent as IAA. A comparison of the HAAs demonstrated BAA was approximately 10× more potent than CAA and that IAA was about 18× more potent than CAA. The HBQs were only slightly more potent than CAA: DCBQ, 1.5× and DBBQ, 1.3× (Table 3). The results confirm previous observations on the mode of action of HAAs, which strongly indicated that they cause toxicity by inducing oxidative stress.24,26,44−46,58 As for the benzoquinone DBPs, Du et al.17 demonstrated that HBQs cause protein carbonylation and ROS-induced oxidative damage to DNA.17 This was confirmed by Li et al.39 who demonstrated that their cytotoxicity to the T24 cell line can be attenuated by coincubation with glutathione (GSH).39 Cellular depletion of GSH has been observed previously following cellular exposure to various quinone-containing compounds either by conjugation between the quinone and GSH or oxidation of GSH to glutathione disulfide (GSSG).41 Lower endogenic levels of GSH may, therefore, result in increased sensitivity to the cytotoxic effects of HBQs and other quinones.72 Likewise, dysfunction or alteration of the Nrf2mediated enzymatic antioxidant system, resulting in lower activity of glutathione reductase, responsible for reduction of

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RESULTS AND DISCUSSION HBQ Reactivity. Both investigated HBQs are highly reactive40 and were observed to undergo transformation upon addition to the test media measurable by a change in color detectable at a wavelength of approximately 515 nm up to 24 h post application to the assay media. The change did not occur in the carrier solvent (methanol) (data not shown). A similar effect was observed previously by Lente et al.,67 who reported reduction of DCBQ to 2,6-dichlorohydroquinone, OH-DCBQ, and molecular oxygen by aqueous photoreaction;67 however, the observation was not mentioned in any of the toxicological literature on HBQs published to date. The observed transformation could alter the cellular response to the tested agents, as it indicates that the HBQs were directly undergoing a reaction with the components of the assay media. However, the transformation we observed during the Caco-2 assay did not appear to significantly affect the cytotoxicity of the HBQs in vitro. We performed comparative assays immediately after addition and after 1 h of preincubation in the media, and in both cases, the responses (IC50) were comparable (Figure S1). Interestingly, there was a marked difference in cytotoxicity to the CHO cells when they were dosed immediately and when they were dosed following a short incubation of the HBQs in the assay media (F12) (Figure S2). Caco-2 Cell Viability. The three monoHAAs followed a descending order of cytotoxicity: IAA ∼ BAA > CAA (Table 2; Table 2. Acute in Vitro Impact of Selected DBPs on Caco-2 Cell Line Viability after 4 h Exposure compound/endpoint CAA BAA IAA DCBQ DBBQ

log IC50/Mb ± SE −2.93 −4.37 −4.47 −3.79 −3.82

± ± ± ± ±

0.04 0.06 0.06 0.03 0.02

IC50b (μM)

REPa

1200 42 34 160 150

1.0 29 35 7.5 8.0

a

Relative effect potency values, REP = IC50(CAA)/IC50(i), where i is a specific DBP. bMean value.

see Figure S3 for concentration−effect curves). This is consistent with findings of others.23,25,68,69 Plewa et al.69 hypothesized that, similar to the reactivity of methyl halides, which are potent alkylating agents reacting via a SN2 mechanism, the reactivity of monoHAAs also depends on the C−X bond dissociation energy, which, in turn, is related to the bond length. There is a good correlation between the atomic 2062


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Chemical Research in Toxicology Table 3. Activity of Selected DBPs in Two in Vitro Cell-Based Reporter Gene Assaysa ARE-blab log (ECIR1.5/M) CAA BAA IAA DCBQ DBBQ

−4.14 −5.15 −5.40 −4.31 −4.26

ARE-bla ECIR1.5 (μM) ± SE

REP(ARE)

p53RE-blab log (ECIR1.5/M)

± ± ± ± ±

1.0 10 18 1.5 1.3

−4.00 −4.99 −5.20 −4.58 −4.50

71 7.1 3.9 49 55

25 1.7 0.9 7 12

p53RE-bla ECIR1.5 (μM) ± SE

REP(p53)

± ± ± ± ±

1.0 10 16 3.7 3.1

100 10.2 6.4 27 32

20 2.7 2.7 4 2

Reporter genes p53RE-bla and ARE-bla were used, and activity is expressed as log ECIR1.5/M and μM (including relative effect potency values, REP = ECIR1.5(CAA)/ECIR1.5(i), where i is a specific DBP). bMean value. a

for the present study (IC50 values: DCBQ = 11.3 μM and DBBQ = 14.4 μM; for concentration−effect curves, see Figure S5), but it provided similar results to those obtained in a 24 h assay using T24 cells as reported by Du et al.17 (Figure 1).

GSSG back to GSH,73 may result in increased cellular sensitivity to HBQs. Taken together, our present study confirmed the previous findings that indicated that HBQs are cytotoxic via induction of oxidative stress. In addition, our in vitro investigation demonstrated HBQs’ high level of activity in triggering the oxidative stress response mediated by the Nrf2/ARE signaling pathway. Both DCBQ and DBBQ exhibited higher potency than the ubiquitous CAA, but they did not reach the potency of either BAA or IAA. p53 Pathway Activation. The DBPs in this study activated the p53 pathway. The descending rank order of potency was again the same as that for the effect on cell viability/cytotoxicity and for Nrf2/ARE pathway activation: IAA ∼ BAA > DCBQ ∼ DBBQ > CAA (significant difference determined by one-way ANOVA, Tukey’s posthoc test, p ≤ 0.05; Table 3). Table 3 provides the ECIR1.5 values calculated from the linear portions of the dose−response curves observed. For comparison, BAA was approximately 10× more potent than CAA, and IAA was 16× more potent than CAA. CAA was tested previously with the same assay, and the ECIR1.5 of 100 μM agreed well with the literature value of 86 μM.8 As for the HBQs, DCBQ and DBBQ were approximately 4× and 3× as potent as CAA, respectively, and DBBQ was practically equipotent with DCBQ (Table 3). The results indicate that p53 pathway activation plays an important role in the in vitro toxicity mechanism of the analyzed DBPs. Attene-Ramos et al.23 previously showed a clear induction of genes related to several pathways, including p53, ATM, ATR, MAPK, BRCA1, and BRCA2, using toxicogenomic analysis of the three monoHAAs in nontransformed, normal human cell line, FHs 74 Int.23 These pathways are involved in cellular processes and mechanisms related to DNA damage repair, cell cycle progression, and apoptosis,23 which suggests that both HAAs and, based on our current results, HBQs interfere with the cell cycle and potentially cause DNA damage via the induction of oxidative stress. Other adverse effects on, or modifications of, DNA, such as mutagenicity, DNA methylation, and inhibition of DNA replication and repair enzymes, have also been identified as being potentially significant for eliciting HBQ toxicity and require further study.41 Assay Sensitivity vs Responsiveness. Different in vitro cell systems provide various levels of sensitivity. This can be seen in the differing EC/IC values obtained among the different systems and may depend on various factors, such as inherent sensitivity of a particular cell line to toxic insult, the length of exposure, and other factors (e.g., growth media serum content, metabolic activity). The 4 h Caco-2 cell (MTS) cytotoxicity assay used in this study was up to 10-fold less sensitive for the tested DBPs than the 24 and 72 h CHO and CHO-K1 cytotoxicity assays previously reported25,40,68 as well as the CHO assay performed

Figure 1. Comparison of the in vitro cytotoxicity of different cell lines depicting differences in their sensitivity. Studies referenced are as follows: CHO-K1 (24 h), HBQs;40 T24 (24 h), HBQs;17 CHO (72 h), HAAs;25 CHO-K1 (24 h), HAAs;68 Caco-2 (24 h), HAAs and HBQs; and CHO (72 h), HBQs (present study). Data are presented as log (IC50/M).

Comparable results were obtained in the Caco-2 (MTS) cytotoxicity assay at 4 and 24 h of exposure (Figure 1), suggesting that most of the detected cytotoxicity in this cell line occurred within the first few hours following exposure to the selected DBPs. HBQs induced higher cytotoxicity in CHO cells than they did toward Caco-2 (colon) and T24 (kidney) cells. The data indicate that CHO cells exhibit higher sensitivity to the cytotoxic effects of the studied DBPs (HBQs in particular). It is important to note that the Caco-2 cell line carries a mutation in the p53 gene, which renders it p53 deficient74 and possibly affects the cell line’s sensitivity to genotoxic agents. However, the difference in sensitivity in vitro may be a result of inherent heterogeneity in the Nrf2-mediated cytoprotection systems between species and/or tissues from which the cells originate. We speculate that these results may be due to lower levels of antioxidant enzymes (e.g., GSH) and ultimately higher sensitivity to oxidative and electrophilic stress. In the case of HAAs, the ARE-bla assay was responsive at lower concentrations than those used in the p53RE-bla assay (Figure 2). The ARE-bla assay provides an indication of the level of Nrf2/ARE pathway activation due to the formation of reactive oxygen species (ROS) and the resulting cellular oxidative stress. The cellular oxidative stress response is, hence, the most sensitive of the three investigated endpoints for the three HAAs. This observation is also supported by the results of studies done previously.24,26 2063


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The lack of guideline values for BAA and IAA as well as the novel HBQs create a problem in determining the potential risk associated with concentrations found in drinking water. To overcome this issue, we expressed the biological activity of the different compounds as relative effect potency (REPs) (Table 1), which, when multiplied by the occurrence concentrations for individual compounds, provides a bioanalytical equivalent concentration (BEQ) that can be cautiously compared to the existing guideline value for CAA. The maximum CAA concentration found in drinking water reported in the literature exceeds both the WHO and Australian guideline values. It is important to stress that this is a maximum detected value and may not represent the overall occurrence of CAA in drinking water; however, it can be used as a worst case scenario. The other monoHAAs (BAA, IAA), when expressed as CAA-BEQ concentrations, would exceed the WHO guideline value of 20 μg/L for each of the three endpoints; however, they would not exceed the Australian guideline value. Interestingly, neither of the HBQs expressed as CAA-BEQ exceeded the WHO guidelines for CAA, suggesting that, when considering their combined biological activity and occurrence, they may not pose as significant a risk as that of HAAs. Although quantitative in vitro assays provide high resolving power, we need to be cautious in using the BEQ values for hazard assessment, as in vitro activity does not necessarily translate directly to in vivo toxicity. Additionally, we have considered only individual compounds; therefore, HBQs may still contribute to overall mixture effects of disinfected drinking water. Future Directions. The quest for identification and characterization of toxic DBPs still continues. An adverse outcome pathway (AOP) framework52−54 to investigate the biological action of xenobiotics characterized by the presentation of a logical sequence of events or processes within a biological system can be used to understand adverse effects of chemical compounds and to refine their risk assessment.54 The application of the three in vitro assays in the present study identified oxidative stress as a dominant cause of cytotoxicity for the three HAAs and p53 pathway activation, for the two HBQs; these results are strongly supported by the findings of other studies.17,24,39,58 The adaptive stress responses described here occur later in the cellular toxicity pathway than MIEs investigated previously and are, therefore, more integrative because they take into account the cellular defense and repair mechanisms. Arguably, they are more suited to the benchmarking exercise performed in our study. Nevertheless, there may still be molecular targets, transcription factors, and pathways other than Nrf2/ARE and p53 that could play a role in the overall toxic response. Future research should focus on the application of genomic methods to investigate the role of other molecular pathways in the toxicity of both individual DBPs as well as, and perhaps more importantly, mixtures of DBPs. 80 The genomic studies may also lead to the identification of biomarkers that can be used to identify susceptible populations more sensitive to adverse biological effects of exposure to DBPs. The affected cellular pathways identified this way can provide insight into the etiology of disease associated with DBP exposure.76 Finally, the DBPs, both individually and in mixtures, need to be studied in vivo by applying the knowledge gained from the in vitro assessments to understand their effects at higher levels of biological organization.

Figure 2. Assay responsiveness: Caco-2 acute cytotoxicity (presented as log IC50/M) versus ARE-bla and p53RE-bla activation (presented as log ECIR1.5/M).

In the case of HBQs, we detected higher activity in the p53 pathway in comparison to that of the Nrf2/ARE pathway (Figure 2), which suggests that DNA damage is induced by mechanism other than ROS-mediated oxidative and redox stress. In addition to ROS-mediated DNA damage, other benzoquinone compounds, such as metabolites of polycyclic aromatic hydrocarbons or estrogens, are known to produce DNA adducts, which have been implicated as being potential contributors to their observed carcinogenicity.75 Even though both classes of DBPs activate the Nrf2/ARE pathway, the mechanism of ROS formation is indeed different for each of the DBP classes and is not a mechanism per se but a resulting insult that causes cellular dysfunction.76 In monoHAAs, the primary cellular target is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), whose inhibition leads to pyruvate starvation and mitochondrial stress that leads to the generation of ROS.24,26,58 Meanwhile, HBQs are directly electrophilic compounds capable of damaging cellular macromolecules such as proteins, lipids, and DNA.17 The ability of DBPs to activate the Nrf2/ARE pathway is particularly important, as the ROS from exposure to DBPs can cause cellular oxidative stress and initiate genotoxic damage and the process of chemical carcinogenesis as well as participate in tumor promotion via modulation of cellular redox potential.77 The cytoprotective adaptive response via the activation of the Nrf2/ARE pathway is important not only to attenuate the ROS-mediated damage to cellular macromolecules and to prevent cellular dysfunction leading to cell death but also to inhibit potential chemical carcinogenesis in its early stages.48 Comparative Risk. HAAs, with the exception of CAA, lack a specific drinking water guideline in most jurisdictions worldwide. The guideline for drinking water quality set by the World Health Organization (WHO) for CAA is 20 μg/L,37 whereas the Australian drinking water guideline (ADWG) value is significantly higher at 150 μg/L.78 The U.S. regulates HAAs as a sum of the five most commonly found HAAs in U.S. drinking waters, CAA, dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), BAA, and dibromoacetic acid (DBAA), collectively known as HAA5.79 This makes direct comparison with levels of individual compounds difficult. Furthermore, given the large differences in potency of the different HAAs, a sum parameter that does not differentiate between the individual constituents is inadvisable. 2064


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ASSOCIATED CONTENT

LOAEL, lowest observable adverse effect level; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MeOH, methanol; MIE, molecular initiating event; Mit-C, mitomycin-C; MTS, methyltetrazolium salt; MX, mutagen X, 3chloro-4-(dichloromethyl)-5-hydroxy-5H-furan-2-one; N-DBP, nitrogenous disinfection byproduct; NDMA, N-nitrosodimethylamine; NFE2L1, nuclear factor, erythroid 2-like 1; NFE2L2, nuclear factor, erythroid 2-like 2; NH2Cl, monochloramine; NIH3T3, Mus musculus embryonic fibroblast cell line; NOM, natural organic matter; NRU, neutral red uptake; Nrf1, nuclear factor erythroid 2 (NF-E2)-related factor 1; Nrf2, nuclear factor erythroid 2 (NF-E2)-related factor 2; O3, ozone; OH-DBBQ, 3hydroxyl-2,6-dibromo-p-benzoquinone; OH-DCBQ, 3-hydroxyl-2,6-dichloro-p-benzoquinone; OH-DCMBQ, 3-hydroxyl-2,6dichloro-3-methyl-p-benzoquinone; OH-HBQ, hydroxyl-halobenzoquinone; OH-TriCBQ, 5-hydroxyl-2,3,6-trichloro-p-benzoquinone; p53, cellular tumor antigen p53; p53RE, cellular tumor antigen p53 response element; p53RE-bla, Invitrogen GeneBLAzer p53RE-bla reporter gene assay; QA/QC, quality assurance/quality control; QSTR, quantitative structure− toxicity relationship; REP, relative effect potency; ROS, reactive oxygen species; RTCA, real-time cell analyzer assay; SN2, bimolecular nucleophilic substitution; tBHQ, tert-butylhydroquinone; TCAA, trichloroacetic acid; THM, trihalomethane; TOX, total organic halogen; TriCBQ, 2,3,6-trichloro-pbenzoquinone; UPR, unfolded protein response

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00283. Detailed methods for the cytotoxicity, p53RE-bla, and ARE-bla assays; cytotoxicity dose−response curves for each investigated compound; graphical representation of the p53RE-bla and ARE-bla endpoints; and dose− response curves for the chronic CHO cytotoxicity data for the two halobenzoquinones (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: e.prochazka@griffith.edu.au. Phone: +61 7 5552 7814. Funding

E.P. is a recipient of an Australian Postgraduate Award (APA) Scholarship administered by Griffith University, and the project was funded by the Griffith University’s funding scheme for graduate research students. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ben Matthews for technical assistance and Dr. Peta Neale for her assistance and thoughtful advice on various elements of the data analysis and manuscript preparation.

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ABBREVIATIONS 8-OHdG, 8-hydroxy-2′-deoxyguanosine; γ-H2AX, histone H2A family member X, phosphorylated on serine 139; ADWG, Australian Drinking Water Guidelines; AOP, adverse outcome pathway; ARE, antioxidant response element; ARE-bla, Invitrogen GeneBLAzer ARE-bla reporter gene assay; ATM, ataxia telangiectasia mutated gene; ATR, ataxia telangiectasia and Rad3 related gene; BAA, monobromoacetic acid; Balb/c, albino laboratory strain of mouse Mus musculus; BEQ, bioanalytical equivalent concentration; BRCA1, breast cancer 1, early onset gene; BRCA2, breast cancer 2, early onset gene; C-DBP, carbonaceous disinfection byproduct; C−X, carbon− halogen bond; CAA, monochloroacetic acid; Caco-2, human colorectal adenocarcinoma cell line; CHO, Chinese hamster ovary cell line; CHO-K1, Chinese hamster ovary cell line, subclone K1; Cl2, molecular chlorine; ClO2, chlorine dioxide; CTI, cytotoxicity index; DBP, disinfection byproduct; DBAA, dibromoacetic acid; DBBQ, 2,6-dibromo-p-benzoquinone; DCAA, dichloroacetic acid; DCBQ, 2,6-dichloro-p-benzoquinone; DCMBQ, 2,6-dichloro-3-methyl-p-benzoquinone; DMEM, Dulbecco’s modified Eagle’s medium; DMEM/F12, Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture; DWG, Drinking Water Guideline; ECIR1.5, concentration producing an induction ratio of 1.5; F12, Ham’s F12 nutrient mixture; FBS, fetal bovine serum; FRET, fluorescence resonance energy transfer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GlutaMAX, L-glutamine supplement; HAA, haloacetic acid; HAA5, sum of mass concentrations of five haloacetic acids; HAN, haloacetonitrile; HBQ, halobenzoquinone; Hep G2, human hepatocellular carcinoma cell line; HSF, heat shock factor; IAA, monoiodoacetic acid; IC50, median inhibition concentration; Keap1, Kelch-like ECHassociated protein 1; Kow, octanol−water partition coefficient; 2065


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