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. 2014 Oct 2;141(2):335–343. doi: 10.1093/toxsci/kfu088

Glutathione-Mediated Detoxification of Halobenzoquinone Drinking Water Disinfection Byproducts in T24 Cells

Jinhua Li 1, Wei Wang 1, Hongquan Zhang 1, X Chris Le 1, Xing-Fang Li 1,1
PMCID: PMC4833019  PMID: 24812012

Abstract

Halobenzoquinones (HBQs) are a new class of drinking water disinfection byproducts (DBPs) and are capable of producing reactive oxygen species and causing oxidative damage to proteins and DNA in T24 human bladder carcinoma cells. However, the exact mechanism of the cytotoxicity of HBQs is unknown. Here, we investigated the role of glutathione (GSH) and GSH-related enzymes including glutathione S-transferase (GST) and glutathione peroxidase (GPx) in defense against HBQ-induced cytotoxicity in T24 cells. The HBQs are 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,6-dibromobenzoquinone (DBBQ). We found that depletion of cellular GSH could sensitize cells to HBQs and extracellular GSH supplementation could attenuate HBQ-induced cytotoxicity. HBQs caused significant cellular GSH depletion and increased cellular GST activities in a concentration-dependent manner. Our mass spectrometry study confirms that HBQs can conjugate with GSH, explaining in part the mechanism of GSH depletion by HBQs. The effects of HBQs on GPx activity are compound dependent; DCMBQ and DBBQ decrease cellular GPx activities, whereas DCBQ and TriCBQ have no significant effects. Pearson correlation analysis shows that the cellular GSH level is inversely correlated with ROS production and cellular GST activity in HBQ-treated cells. These results support a GSH and GSH-related enzyme-mediated detoxification mechanism of HBQs in T24 cells.

Keywords: halobenzoquinones, disinfection byproducts, glutathione, glutathione S-transferase, glutathione peroxidase

Disinfection of drinking water is a great public health success and has been demonstrated for more than a century to substantially reduce and control waterborne diseases, such as cholera and typhoid (Cutler and Miller, 2005). Disinfection can unavoidably produce disinfection byproducts (DBPs) resulting from reactions of the disinfectants (such as chlorine, chloramine, ozone, and chlorine dioxide) with natural organic matter in the source water (Krasner et al., 2006; Richardson et al., 2007). The main concern of exposure to DBPs is based on the epidemiological observation of potentially increased risk of human cancers (e.g., bladder cancer (Villanueva et al., 2007), colorectal cancer (Rahman et al., 2010), and skin cancer (Karagas et al., 2008)) and developmental toxicity (Costet et al., 2012; Savitz et al., 2005; Tardiff et al., 2006; Waller et al., 1998). Of more than 600 identified DBPs in the disinfected drinking water, only a small number (<90) of DBPs have toxicological results reported (Richardson et al., 2007). In addition, the regulated DBPs cannot account for the increased risk of bladder cancer on the basis of animal carcinogenic studies (Bull et al., 2011). Therefore, it is important to study the toxicological mechanisms of newly identified DBPs to understand their relevance to cancer risk.

We have recently identified four halobenzoquinones (HBQs) in disinfected drinking water as a new class of DBPs: 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,6-dibromobenzoquinone (DBBQ) (Zhao et al., 2010). A quantitative structure toxicity relationship (QSTR) model has predicted HBQs as plausible bladder carcinogens and thus they have been proposed to be priority DBPs (Bull et al., 2006). We have previously reported that these four HBQs were cytotoxic to T24 human bladder carcinoma cells. The four HBQs can induce cellular oxidative stress via generation of reactive oxygen species (ROS), leading to oxidative damage to DNA and proteins (Du et al., 2013). Additionally, HBQs have been observed to interact with oligonucleotides in solutions (Anichina et al., 2010), and induce DNA methylation (Zhao et al., 2013). However, the exact mechanism of the cytotoxicity of HBQs is unknown. Because oxidative stress is partly responsible for their toxic effects, HBQs may influence the endogenous antioxidant defense system, which has not been studied to date.

Glutathione (GSH) and glutathione-related enzymes constitute the primary defense mechanism against cytotoxicity of xenobiotics, especially oxidative stress. GSH is a tri-peptide, comprised of glutamate, cysteine, and glycine. It is an abundant nonprotein thiol in cells and is found at millimolar concentration levels (0.1–10 mM), depending on cell types (Ehrlich et al., 2007; Hayes and McLellan, 1999). GSH serves as a detoxificant via either a conjugation reaction catalyzed by glutathione S-transferase (GST) or a reduction of hydrogen peroxide by glutathione peroxidase (GPx) (Hayes and McLellan, 1999). Cellular GSH synthesis is associated with two enzymes, glutamate cysteine synthetase (GCS), and glutathione synthetase (GS). We hypothesize that the GSH defense system plays an important role in protecting cells from cytotoxicity induced by HBQs. This is supported by the literature of toxicity of quinones, because quinones can react readily with sulfur nucleophiles (covalent binding), such as GSH or cysteine residues on proteins, leading to depletion of cellular GSH levels (Bolton et al., 2000). In this study, we will use buthionine sulfoximine (BSO), an irreversible inhibitor of GCS as BSO can bind to GCS tightly (Griffith, 1999), to explore whether the GSH depletion would affect the cytotoxicity of HBQs. We will also include a parallel assay to examine whether extracellular GSH supplementation would affect the cytotoxicity of HBQs. The objectives of this study are to explore the role of the GSH system in detoxification of HBQs in T24 cells, and to examine the effects of HBQs on cellular GSH levels and glutathione-related enzyme activities including GPx and GST. The results are important to the understanding of mechanisms of toxicity of HBQ DBPs.

MATERIALS AND METHODS

Reagents

Standards of DCBQ, DCMBQ, TriCBQ, and DBBQ were purchased from Sigma-Aldrich, Shanghai Acana Pharmtech, INDOFINE Chemical Company, and Fluka, respectively. HBQs were dissolved in methanol (HPLC grade, Fisher Scientific) and stored at −20°C in sterile amber glass vials. BSO (Sigma) was dissolved in complete McCoy's 5A medium at a concentration at 20mM as stock solution and stored at 4°C. GSH was dissolved in complete McCoy's 5A medium at a concentration at 100 mM as stock solution and stored at −20°C. Protease inhibitor cocktail was purchased from Sigma-Aldrich and stored at −20°C. Triton X-100 was purchased from Fisher Scientific.

Cell culture

The human bladder epithelial carcinoma cell line, T24, was obtained from ATCC (Manassas, VA) and cultured in McCoy's 5A modified medium (ATCC) plus 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin/streptomycin (100 U/100 μg/ml) (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2.

Effects of glutathione depletion on cell viability

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure cell viability. T24 cells were cultured in the 96-microwell plates. The viability of the cells was measured at 490 nm using a Microplate Spectrometer (Bio-Rad, Benchmark Plus). T24 cells were pretreated with or without BSO for 12 h. When the cells reached 70–80% confluence, the media were removed and replaced with media containing varying concentrations of HBQs. After treatment with HBQs for 24 h, the viability of the cells was measured. Untreated cells were included as negative control. All experiments were repeated twice in triplicate.

Effects of exogenous glutathione on cell viability

To study the effects of exogenous GSH on cell viability and HBQ-cytotoxicity, the MTS assay was used as described above. T24 cells were seeded in the 96-microwell culture plate in the presence of BSO. After 12 h, the medium in half of the microwells was replaced with 10 mM of GSH/well in medium, and in the other half of the microwells with the culture medium without addition of GSH. After another 12 h, the old medium was aspirated, and the T24 cells were washed by phosphate buffered saline (PBS) twice, followed by treatment with varying concentrations of the four HBQs. After the cells were treated with HBQs for 24 h, the cell viability was measured. Untreated cells were also measured as the negative control. All experiments were repeated twice in triplicate.

Measurement of cellular glutathione levels

T24 cells were seeded in a 60 mm dish. After the cells reached 70–80% confluence, they were treated with varying concentrations (3/6, 4/6, 5/6, and 1 24 h-IC50) of HBQs. The negative control and solvent control (0.4% methanol, vol/vol) were also included. After 24 h of HBQ exposure, the cells were trypsinized, collected, and centrifuged at 1700 rpm, for 3 min. Cell pellets were transferred to 1.5 ml-microcentrifuge tubes, washed twice with ice-cold PBS and resuspended in ice-cold metaphosphoric acid (MPA). After homogenization, the solution was centrifuged at 10,000 × g at 4°C for 10 min and then the supernatant was used for the measurement of GSH levels according to the manufacturer's instructions (Bioxytech-GSH 400, OxisResearch, Portland, OR). The assay was performed in microcentrifuge tubes and transferred to flat-bottom 96-well plates for absorbance measurement at 400 nm. The pellet from the centrifugation was dissolved in 100 μl of 0.1 M NaOH and the protein concentration was determined using the Bio-Rad microprotein assay in a 96-well plate using bovine serum albumin (BSA) as the standard. The GSH level was expressed as nmol GSH/mg cellular protein or nmol GSH/106 cells, and then as the percentage of the control.

Measurement of cellular glutathione S-transferase (GST) activity

T24 cells were seeded and exposed to HBQs as described for the GSH assay. After 24 h of HBQ exposure, the cells were collected using a rubber policeman and washed with ice-cold PBS and then lysed in GST sample buffer provided in a GST Colorimetric Activity Assay Kit (BioVision, Mountain View, CA). A freeze-thaw process was used to break cell membranes: freezing in liquid nitrogen for 15 min and thawing on ice. The cell lysate was centrifuged at 10,000 × g for 15 min at 4 °C and the supernatant was collected and stored at −80 °C until analysis.

The GST colorimetric activity assay is based on the GST-catalyzed reaction between GSH and a GST substrate, CDNB (1-chloro-2,4-dinitrobenzene). Formation of CDNB-GSH can be measured with a spectrophotometer at 340 nm. Measurements were performed every minute over a 5-min period. One unit of GST activity is defined as the amount of enzyme producing 1 mmol of CDNB-GSH/min under the conditions of the assay. Protein concentration was determined by the Bio-Rad microprotein assay in a 96-well plate using BSA as the standard.

Measurement of cellular glutathione peroxidase (GPx) activity

To obtain a cell lysate for the GPx activity assay, T24 cells were exposed to HBQs as described in the GSH assay. The cells were collected by a rubber policeman in ice-cold PBS, and resuspended in an ice-cold 1× assay buffer (HT Glutathione Peroxidase Assay Kit, Trevigen, Gaithersburg, MD) containing 1% protease inhibitor cocktail and 1% Triton X-100. The cell suspensions were incubated on ice with periodic vortexing for 30 min. The cell lysate was centrifuged at 10,000 × g for 15 min at 4°C, and the supernatant was collected and stored at −80°C until assayed. The protein concentration of the cleared cell lysate was determined with the Bio-Rad microprotein assay in a 96-well plate using BSA as the standard.

GPx activity was measured using a HT Glutathione Peroxidase Assay Kit. This assay measures the GPx activity indirectly by a coupled reaction with glutathione reductase (GR). Because GPx catalyzes the reduction of hydrogen peroxide using GSH as a substrate and forms glutathione disulfide (GSSG) and water, GR is used to reduce GSSG to GSH and decrease the NADPH level. The decreased level of NADPH measured at 340 nm is directly proportional to GPx activity in the sample. Measurements were taken every minute for 10 time points. One unit of GPx activity is defined as the amount of enzyme that will cause the oxidation of 1 nmol of NADPH to NADP+ per minute at 25 °C. The protein concentration was determined by the Bio-Rad microprotein assay in a 96-well plate using BSA as the standard.

Data analysis

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA) and OriginPro 8.5 (OriginLab Corporation, Northampton, MA). Experimental results were expressed as mean ± standard deviation (SD). Differences between the treated groups with and without BSO were determined by Student's t-test. One-way analysis of variance (ANOVA, followed by Dunnett's post hoc Multiple Comparisons) was used for multiple comparisons among treatment and control groups. Pearson correlation analysis was used to study the relationships between GSH level and enzyme activity or ROS production. Differences were considered statistically significant at p < 0.05.

RESULTS

GSH-Mediated Detoxification of HBQs in T24 Cells

We first hypothesize that GSH plays one of the key roles in detoxification of HBQs. To confirm this hypothesis, we have examined the cytotoxicity of HBQs to T24 cells when intracellular GSH is depleted and when GSH is supplemented in culture media. First, we identified the optimal concentration of BSO to deplete GSH levels with minimal toxicity to T24 cells. After T24 cells were pretreated with BSO ≤ 50 μM, the cell viability was maintained at >90%. Therefore, BSO (50 μM) was used for the following experiments (Supplementary table 1).

With or without the pretreatment with BSO, the concentration dependent toxic effects of HBQs were obtained as shown in viability curves (Fig. 1). The IC50 values for HBQs are presented in Supplementary table 2. Without the BSO pretreatment, the IC50 values are DCBQ (95 μM), DCMBQ (110 μM), TriCBQ (151 μM), and DBBQ (142 μM). With the BSO pretreatment, the IC50 values are DCBQ (63 μM), DCMBQ (22 μM), TriCBQ (94 μM), and DBBQ (70 μM). Compared with no pretreatment of BSO, the IC50 of HBQs significantly decreased by 1.5–4.9 fold (p < 0.0001). The toxicity of HBQ compounds can be ranked as DCBQ > DCMBQ > DBBQ > TriCBQ in the absence of BSO and as DCMBQ > DCBQ ≥ DBBQ > TriCBQ in the presence of BSO. The results clearly demonstrate that T24 cells are more sensitive to DCMBQ with pretreatment with BSO than the other three HBQs, suggesting that predepletion of GSH dramatically increases the susceptibility of T24 cells to DCMBQ-cytotoxicity.

FIG. 1.

FIG. 1.

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Effects of BSO and HBQs on viability of T24 cells. T24 cells were exposed separately to four HBQs for 24 h in the absence and presence of pretreatment with 50 μM BSO in the culture media. Quantitative determination of viable cells was performed with the MTS assay. Error bars represent SD of triplicate data and two separate experiments (n = 6).

After confirming the role of intracellular GSH in detoxification of HBQs, we examined whether exogenous GSH can also assist with detoxification. Figure 2 shows the viability of T24 cells treated with HBQs with and without pretreatment with GSH. The pretreatment of GSH significantly reduces the cytotoxic effects of HBQs.

FIG. 2.

FIG. 2.

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Effects of exogenous GSH on HBQ-cytotoxicity in T24 cells. T24 cells were exposed to 4 HBQs for 24 h after the cells were with or without the pretreatment of 10mM of exogenous GSH. Quantitative determination of viable cells was performed by MTS assay. Error bars represent SD of triplicate data and two separate experiments (n = 6).

Taken together, GSH depletion enhanced the cytotoxicity of HBQs and GSH supplementation attenuated the HBQ-induced cytotoxicity in T24 cells, supporting the hypothesis that GSH plays one of the key roles in detoxification of HBQs.

Effects of HBQs on the Intracellular Levels of Free Reduced GSH

We further hypothesize that HBQ cytotoxicity is associated with the depletion of intracellular GSH induced by HBQs. To confirm this hypothesis, we studied the effects of HBQs on free GSH levels in T24 cells. Solvent control experiments show that the amount of methanol used in HBQ solutions does not induce statistically significant change in GSH levels (Supplementary fig. 1).

Figure 3 presents the cellular GSH levels after HBQ treatment at the same concentration of equivalent biological response (3/6, 4/6, 5/6, and 1 IC50). Because DCMBQ demonstrated significantly reduced IC50 when cellular GSH was depleted, two lower concentrations (1/20 and 1/4 IC50) were used in this study. The reduction in cellular GSH levels is clearly dependent on the concentrations of HBQs (p < 0.05) (one-way ANOVA analysis of variance, followed by Dunnett's post hoc test). At half the IC50 value, the cellular GSH is 57% (DCBQ), 16% (DCMBQ), 62% (TriCBQ), and 92% (DBBQ) of the control; at the IC50 value, the cellular GSH is 23% (DCBQ), 7% (DCMBQ), 25% (TriCBQ), and 35% (DBBQ) of the control. These results indicate that at the same concentration of equivalent biological response, DCMBQ displays a higher ability to deplete cellular GSH compared with the other three HBQs.

FIG. 3.

FIG. 3.

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Effect of HBQs on the cellular GSH levels in T24 cells. GSH levels were determined after 24 h of exposure to HBQs and compared to the levels of the negative control. Error bar represents SD of triplicate data and two separate experiments (n = 6). *p < 0.05, HBQ treatment groups compared with negative control.

Effects of HBQs on Cellular Glutathione S-Transferase (GST) Activity

To further explain the role of GSH in the cellular response to HBQ treatment, we also evaluated the changes of GSH-associated enzymes in HBQ-treated T24 cells: GST, which indicates the conjugation of GSH, and GPx, which is involved in the reduction reaction.

To investigate the impact of HBQs on GST activity in T24 cells, we used a colorimetric assay to measure the CDNB-GSH conjugate that is catalyzed by GST. Figure 4 shows alteration of the cellular GST activity when T24 cells were treated with varying concentrations of individual HBQs at the same concentration used in GSH assay. GST activity increases with the increase of HBQ concentrations used to treat T24 cells. At the same concentration of equivalent biological response (1/2 24 h-IC50 to 24 h-IC50), the GST activity over the control increased significantly (*p < 0.05): 1.2–2.5 fold (DCBQ), 2.5–3.5 fold (DCMBQ), 1.5–4.5 fold (TriCBQ), and 1.4–2.0 fold (DBBQ), when the concentrations of HBQs used were 75 and 95 mM for DCBQ, 55–110 μM for DCBMQ, 100–150 μM for TriCBQ, and 118–142 μM for DBBQ. In parallel, the solvent control displayed no change in GST activity compared with the negative control (Supplementary fig. 2).

FIG. 4.

FIG. 4.

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Effect of HBQ compounds on cellular glutathione S-transferase (GST) activity. T24 cells were exposed to HBQs for 24 h. GST activity was measured using a colorimetric assay, and the 340 nm absorbance obtained was the measure of the formation of the CDNB-GSH conjugate which was compared to the amount of cellular total protein. Bar graphs show the differences in the means between HBQ-exposed cells and control cells (*p < 0.05). Error bars indicate SD of six determinations from two separate experiments.

Effects of HBQs on Cellular Glutathione Peroxidase (GPx) Activity

Figure 5 shows the cellular GPx activity after treatment with varying concentrations of HBQs. No change in GPx activity was observed in the cells exposed to 0.4% methanol compared with the negative control (Supplementary fig. 3). The effects of HBQs on GPx activity are compound dependent. When the cells were treated with each HBQ at the concentrations of 1/2 IC50–IC50, DCBQ and TriCBQ did not change GPx activity, whereas DCMBQ (1/4 IC50–IC50) and DBBQ (1/2 IC50–IC50) significantly reduced GPx activity, compared to the negative control (*p < 0.05). At the same concentration of equivalent biological response (1/2 24 h-IC50 to 24 h-IC50), GPx activity was deceased to 75–83% for DCMBQ and 65–72% for DBBQ compared with the negative control.

FIG. 5.

FIG. 5.

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Effect of four HBQs on cellular glutathione peroxidase (GPx) activity. T24 cells were exposed to HBQs for 24 h. The GPx activity was investigated indirectly with GR in a couple-enzyme reaction. The bar graphs show the differences in the means between HBQ-exposed cells and control cells (*p < 0.05). Error bars indicate SD of six determinations from two separate experiments.

Correlation between Cellular GSH Level and GST Activity, GPx Activity, or ROS Production

Finally, we conducted a Pearson correlation analysis between cellular GSH level and GST activity, GPx activity, and ROS production (Table 1). The cellular GSH level is inversely correlated with ROS production and cellular GST activity in the cells treated with the four HBQs; the correlation was highly significant for DCBQ and DCMBQ, but moderate for TriCBQ and DBBQ. The cellular GSH level was significantly correlated with cellular GPx activity in DCMBQ-treated cells, but not in the cells treated with the other three HBQs. To sum up, HBQs can deplete the cellular GSH level and induce production of ROS and GST activity.

TABLE 1. Pearson Correlation Analysis of Cellular GSH Level with Cellular GST Activity, GPx Activity and ROS Production After 24 h in HBQ-treated T24 Cells.
HBQ group GST activity (log value)a GPx activity (log value)a ROS production (log value)b
r p value r p value r p value
GSH level (log value)a DCBQ −1.00** 0.00 0.81 0.19 −0.96* 0.04
DCMBQ −0.96** 0.00 0.91* 0.01 −0.96** 0.01
TriCBQ −0.62 0.38 −0.78 0.22 −0.89 0.11
DBBQ −0.92 0.08 0.03 0.97 −0.95 0.05
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aData presented in this study. Data is from six determinations from two separate experiments.

bData summarized from our previous study (Du et al., 2013).

*Correlation is significant at the level of 0.05.

**Correlation is significant at the level of 0.01.

DISCUSSION

We first examined the role of GSH in the defense against HBQ-induced cytotoxicity in the bladder cancer cell line T24, because our previous studies have shown that T24 cells were more sensitive to some DBPs, including nitrosamines and phenazine, and it is relevant to the potential risk of bladder cancer due to exposure to DBPs (Boyd et al., 2008; Villanueva et al., 2007; Zhou et al., 2012). The involvement of GSH in detoxifying the HBQ-induced cytotoxicity was first supported by two sets of evidence: GSH depletion enhanced the cytotoxicity induced by HBQs and GSH supplementation reduced HBQ-induced cytotoxicity. A decrease of intracellular GSH was observed in T24 cells exposed to HBQs. These observations indicate that HBQ cytotoxicity is associated with depletion of intracellular GSH. We then examined whether the GSH depletion was due to conjugation or reduction reaction through analysis of the effects of HBQs on GST and GPx enzyme activities. Our results indicate that GST plays a more important role compared with GPx, which led us to propose a hypothesis that GSH depletion is mainly due to conjugation. To confirm this hypothesis, we examined HBQ-GSH conjugation using mass spectrometry analysis (Supplementary data). After incubating GSH with individual HBQs for 1 h, we identified the peaks corresponding to the conjugation products (HBQ-GSH). Supplementary figure 4 shows the measured (black) and theoretical (red) accurate mass and isotope pattern of the conjugation products of GSH with DCBQ, DCMBQ, TriCBQ, and DBBQ, respectively. The accurate mass measurements are in agreement with the theoretical mass and isotopic patterns (red) of the conjugates consisting of one molecule of HBQ bound with one molecule of GSH. The mass accuracies are all better than 10 ppm. Our results are consistent with the determination of GSH conjugation with quinones (Nakamura et al., 2003; Rossi et al., 1986).

The pretreatment of 50 μM BSO increased the cytotoxicity of DCMBQ up to five-fold and that of other three HBQs two-fold. This result is consistent with studies reporting that BSO sensitizes T24 cells to increase cytotoxicity of other compounds (Byun et al., 2005; Jiang et al., 2014), and increases ML-1 cells’ susceptibility to toxic effects of hydroquinone (Trush et al., 1996). GSH supplementation reduces cytotoxic effects of HBQs, suggesting that T24 cells can uptake GSH. Other cells, kidney cells, and the small intestinal cells can also uptake GSH (Deneke and Fanburg, 1989; vanKlaveren et al., 1997). The protection of extracellular GSH against HBQ-cytotoxicity is consistent with our previous result that N-acetylcysteine (NAC) treatment prevents HBQ-induced cytotoxic effects (Du et al., 2013), and also consistent with other findings that exogenous GSH can protect cells and mice from toxicity (Hagen et al., 1986; Jin et al., 2010; Lash et al., 1986).

HBQs depleted cellular GSH in a concentration-dependent manner. Several studies have shown that GSH depletion is one of the key mechanisms for hydroquinone (HQ) and benzoquinone (BQ) toxicity in some human cells (Luo et al., 2008; Rubio et al., 2011; Smith, 1999; Trush et al., 1996). One previous study reported that cellular GSH was decreased to 38% of the control, after HepG2 cells were incubated with HQ (50μM) for 1 h (Luo et al., 2008). In another study, relative GSH content over the control was decreased to 80% for HQ and 60% for BQ, after Beas-2B cells were treated with 20 μM of HQ or BQ for 2 h (Rubio et al., 2011). Compared with these results of HQ and BQ, HBQs displayed a much higher ability to deplete cellular GSH. At the highest concentration (IC50) value we used, the cellular GSH is depleted to 7–35% of the control. Among the four HBQs, DCMBQ has the highest ability to deplete cellular GSH levels. We have previously observed that DCMBQ can generate higher levels of 8-OHdG adducts in T24 compared with the other three HBQs (Du et al., 2013). It is likely that higher depletion of GSH by DCMBQ may make T24 cells more sensitive to DNA damage and lead to enhanced formation of DNA adducts. Quinones are Michael acceptors and react readily with sulfur nucleophiles like GSH, leading to depletion of cellular GSH levels (Bolton et al., 2000). Moreover, conjugation of HQ and BQ to GSH resulted in the formation of less toxic metabolites with increased solubility and excretion (Snyder et al., 1988). Our mass spectrometry results of interactions of HBQs with GSH support the HBQ-GSH conjugation. Therefore, the conjugation between HBQs and GSH is proposed to be one of the major pathways of HBQ-induced depletion of cellular GSH levels.

The findings of the effects of HBQs on the activities of GST and GPx are supported by the study of BQ-treated MCF7 cells that no significant change occurs in the gene expression of GPx whereas GST gene expression was significantly enhanced after 24 h treatment (Baigi et al., 2008). The induction of intracellular antioxidants, such as detoxifying enzymes (e.g., GST), is critical for detoxification of quinones and benzene (Nakamura et al., 2003; Rubio et al., 2011; Zhou et al., 2009). The induction of GST was also observed in inorganic arsenic, and similar pathway is also at work for many heavy metal ions, e.g., Cd2+ (Prins et al., 2014; Schuliga et al., 2002). The induction of GST and other GSH-related enzymes are mostly regulated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is the principal regulator of the antioxidant response element (ARE)-driven cellular defense system (Biswas and Rahman, 2009). The lower cellular GPx activity in HBQ-treated T24 cells suggests that GPx was consumed to defend against cellular ROS. As a cofactor, GSH is indispensable in the process by which GPx catalyzed hydrogen peroxide or hydroperoxide into water (Brigelius-Flohe and Kipp, 2009; Li et al., 2000). Because HBQs significantly depleted GSH levels, little cellular GSH left for GPx to use. Also, catalase (CAT) is able to convert hydrogen peroxide into water, without the need for GSH as a cofactor (Li et al., 2000). Therefore, the effects of HBQs on cellular GPx activity were not as significant as on GST activity.

Based on these results, we propose the possible pathways of GSH-mediated detoxification of HBQs (Fig. 6). Low concentrations of HBQs can cause cellular ROS production, GSH depletion, and oxidative stress, which lead to the activation of related genes (such as Nrf2/ARE) and to the induction of cellular antioxidants (such as GST) to detoxify HBQs. The data presented here support a possible pathway for the toxic mechanisms of HBQs, a class of drinking water DBPs.

FIG. 6.

FIG. 6.

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Possible protection pathway involving GSH against HBQ cytotoxicity. Exposure to HBQs causes cellular GSH depletion and ROS production, generating oxidative stress, which leads to activation of related genes (such as Nrf2/ARE) and induction of cellular antioxidants (such as GST) to detoxify HBQs in T24 cells.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjornals.org/.

FUNDING

Natural Sciences and Engineering Research Council (NSERC) of Canada; Alberta Innovates-Energy and Environmental Solutions; Alberta Health; China Scholarship Council (to J.L.).

Supplementary Material

Supplementary Data
supp_141_2_335__index.html (947B, html)

Acknowledgments

The authors would like to thank Dr Steve E. Hrudey and Dr Elaine Leslie for their advice on our study and Ms Katerina Carastathis for her edition of the manuscript.

REFERENCES

  1. Anichina J., Zhao Y. L., Hrudey S. E., Le X. C., Li X. F. Electrospray ionization mass spectrometry characterization of interactions of newly identified water disinfection byproducts halobenzoquinones with oligodeoxynucleotides. Environ. Sci. Technol. 2010;44:9557–9563. doi: 10.1021/es1024492. [DOI] [PubMed] [Google Scholar]
  2. Baigi M. G., Brault L., Neguesque A., Beley M., El Hilali R., Gauzere F., Bagrel D. Apoptosis/necrosis switch in two different cancer cell lines: Influence of benzoquinone- and hydrogen peroxide-induced oxidative stress intensity, and glutathione. Toxicol. In Vitro. 2008;22:1547–1554. doi: 10.1016/j.tiv.2008.06.008. [DOI] [PubMed] [Google Scholar]
  3. Biswas S. K., Rahman I. Environmental toxicity, redox signaling and lung inflammation: The role of glutathione. Mol. Aspects Med. 2009;30:60–76. doi: 10.1016/j.mam.2008.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolton J. L., Trush M. A., Penning T. M., Dryhurst G., Monks T. J. Role of quinones in toxicology. Chem. Res. Toxicol. 2000;13:135–160. doi: 10.1021/tx9902082. [DOI] [PubMed] [Google Scholar]
  5. Boyd J. M., Huang L., Xie L., Moe B., Gabos S., Li X. F. A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals. Anal. Chim. Acta. 2008;615:80–87. doi: 10.1016/j.aca.2008.03.047. [DOI] [PubMed] [Google Scholar]
  6. Brigelius-Flohe R., Kipp A. Glutathione peroxidases in different stages of carcinogenesis. Biochim. Biophys. Acta. 2009;1790:1555–1568. doi: 10.1016/j.bbagen.2009.03.006. [DOI] [PubMed] [Google Scholar]
  7. Bull R. J., Reckhow D. A., Li X. F., Humpage A. R., Joll C., Hrudey S. E. Potential carcinogenic hazards of non-regulated disinfection by-products: Haloquinones, halo-cyclopentene and cyclohexene derivatives, N-halamines, halonitriles, and heterocyclic amines. Toxicology. 2011;286:1–19. doi: 10.1016/j.tox.2011.05.004. [DOI] [PubMed] [Google Scholar]
  8. Bull R. J., Reckhow D.A., Rotello V., Bull O. M., Kim J. Use of Toxicological and Chemical Models to Prioritize DBP Research. Denver, CO: Awwa Research Foundation; 2006. [Google Scholar]
  9. Byun S. S., Kim S. W., Choi H., Lee C., Lee E. Augmentation of cisplatin sensitivity in cisplatin-resistant human bladder cancer cells by modulating glutathione concentrations and glutathione-related enzyme activities. BJU Int. 2005;95:1086–1090. doi: 10.1111/j.1464-410X.2005.05472.x. [DOI] [PubMed] [Google Scholar]
  10. Costet N., Garlantezec R., Monfort C., Rouget F., Gagniere B., Chevrier C., Cordier S. Environmental and urinary markers of prenatal exposure to drinking water disinfection by-products, fetal growth, and duration of gestation in the PELAGIE Birth Cohort (Brittany, France, 2002–2006) Am. J. Epidemiol. 2012;175:263–275. doi: 10.1093/aje/kwr419. [DOI] [PubMed] [Google Scholar]
  11. Cutler D., Miller G. The role of public health improvements in health advances: The twentieth-century United States. Demography. 2005;42:1–22. doi: 10.1353/dem.2005.0002. [DOI] [PubMed] [Google Scholar]
  12. Deneke S. M., Fanburg B. L. Regulation of cellular glutathione. Am. J. Physiol. 1989;257:L163–L173. doi: 10.1152/ajplung.1989.257.4.L163. [DOI] [PubMed] [Google Scholar]
  13. Du H. Y., Li J. H., Moe B., McGuigan C. F., Shen S. W., Li X. F. Cytotoxicity and oxidative damage induced by halobenzoquinones to T24 bladder cancer cells. Environ. Sci. Technol. 2013;47:2823–2830. doi: 10.1021/es303762p. [DOI] [PubMed] [Google Scholar]
  14. Ehrlich K., Viirlaid S., Mahlapuu R., Saar K., Kullisaar T., Zilmer M., Langel U., Soomets U. Design, synthesis and properties of novel powerful antioxidants, glutathione analogues. Free Radic. Res. 2007;41:779–787. doi: 10.1080/10715760701348611. [DOI] [PubMed] [Google Scholar]
  15. Griffith O. W. Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic. Biol.Med. 1999;27:922–935. doi: 10.1016/s0891-5849(99)00176-8. [DOI] [PubMed] [Google Scholar]
  16. Hagen T. M., Brown L. A., Jones D. P. Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type-II cells. Biochem. Pharmacol. 1986;35:4537–4542. doi: 10.1016/0006-2952(86)90776-8. [DOI] [PubMed] [Google Scholar]
  17. Hayes J. D., McLellan L. I. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic. Res. 1999;31:273–300. doi: 10.1080/10715769900300851. [DOI] [PubMed] [Google Scholar]
  18. Jiang J., Yuan X., Zhao H., Yan X., Sun X., Zheng Q. Licochalcone A inhibiting proliferation of bladder cancer T24 cells by inducing reactive oxygen species production. Biomed. Mater. Eng. 2014;24:1019–1025. doi: 10.3233/BME-130899. [DOI] [PubMed] [Google Scholar]
  19. Jin Y. P., Zhao F. H., Zhong Y. A., Yu X. Y., Sun D., Liao Y. J., Lv X. Q., Li G. X., Sun G. F. Effects of exogenous GSH and methionine on methylation of inorganic arsenic in mice exposed to arsenite through drinking water. Environ. Toxicol. 2010;25:361–366. doi: 10.1002/tox.20509. [DOI] [PubMed] [Google Scholar]
  20. Karagas M. R., Villanueva C. M., Nieuwenhuijsen M., Weisel C. P., Cantor K. P., Kogevinas M. Disinfection byproducts in drinking water and skin cancer? A hypothesis. Cancer Cause Control. 2008;19:547–548. doi: 10.1007/s10552-008-9116-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krasner S. W., Weinberg H. S., Richardson S. D., Pastor S. J., Chinn R., Sclimenti M. J., Onstad G. D., Thruston A. D. Occurrence of a new generation of disinfection byproducts. Environ. Sci. Technol. 2006;40:7175–7185. doi: 10.1021/es060353j. [DOI] [PubMed] [Google Scholar]
  22. Lash L. H., Hagen T. M., Jones D. P. Exogenous glutathione protects intestinal epithelial-cells from oxidative injury. Proc. Natl. Acad. Sci. U.S.A. 1986;83:4641–4645. doi: 10.1073/pnas.83.13.4641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li S. J., Yan T., Yang J. Q., Oberley T. D., Oberley L. W. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res. 2000;60:3927–3939. [PubMed] [Google Scholar]
  24. Luo L. H., Jiang L. P., Geng C. Y., Cao J., Zhong L. F. Hydroquinone-induced genotoxicity and oxidative DNA damage in HepG2 cells. Chem.-Biol. Interact. 2008;173:1–8. doi: 10.1016/j.cbi.2008.02.002. [DOI] [PubMed] [Google Scholar]
  25. Nakamura Y., Kumagai T., Yoshida C., Naito Y., Miyamoto M., Ohigashi H., Osawa T., Uchida K. Pivotal role of electrophilicity in glutathione S-transferase induction by tert-butylhydroquinone. Biochemistry-US. 2003;42:4300–4309. doi: 10.1021/bi0340090. [DOI] [PubMed] [Google Scholar]
  26. Prins J. M., Fu L., Guo L., Wang Y. Cd(2+)-induced alteration of the global proteome of human skin fibroblast cells. J. Proteome Res. 2014;13:1677–1687. doi: 10.1021/pr401159f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rahman M. B., Driscoll T., Cowie C., Armstrong B. K. Disinfection by-products in drinking water and colorectal cancer: A meta-analysis. Int. J. Epidemiol. 2010;39:733–745. doi: 10.1093/ije/dyp371. [DOI] [PubMed] [Google Scholar]
  28. Richardson S. D., Plewa M. J., Wagner E. D., Schoeny R., DeMarini D. M. Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: A review and roadmap for research. Mutat. Res.-Rev. Mutat. 2007;636:178–242. doi: 10.1016/j.mrrev.2007.09.001. [DOI] [PubMed] [Google Scholar]
  29. Rossi L., Moore G. A., Orrenius S., Obrien P. J. Quinone toxicity in hepatocytes without oxidative stress. Arch. Biochem. Biophys. 1986;251:25–35. doi: 10.1016/0003-9861(86)90047-0. [DOI] [PubMed] [Google Scholar]
  30. Rubio V., Zhang J., Valverde M., Rojas E., Shi Z. Z. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. In Vitro. 2011;25:521–529. doi: 10.1016/j.tiv.2010.10.021. [DOI] [PubMed] [Google Scholar]
  31. Savitz D. A., Microbial/Disinfection By-Products Research Council (U.S.), AWWA Research Foundation., and United States. Environmental Protection Agency . Drinking Water Disinfection By-products and Pregnancy Outcome. Denver, CO: AWWA Research Foundation; 2005. [Google Scholar]
  32. Schuliga M., Chouchane S., Snow E. T. Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethal concentrations of inorganic arsenic. Toxicol. Sci. 2002;70:183–192. doi: 10.1093/toxsci/70.2.183. [DOI] [PubMed] [Google Scholar]
  33. Smith M. T. Benzene, NQO1, and genetic susceptibility to cancer. Proc. Natl. Acad. Sci. U.S.A. 1999;96:7624–7626. doi: 10.1073/pnas.96.14.7624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Snyder C. A., Sellakumar A. R., James D. J., Albert R. E. The carcinogenicity of discontinuous inhaled benzene exposures in Cd-1 and C57b1/6 Mice. Arch. Toxicol. 1988;62:331–335. doi: 10.1007/BF00293618. [DOI] [PubMed] [Google Scholar]
  35. Tardiff R. G., Carson M. L., Ginevan M. E. Updated weight of evidence for an association between adverse reproductive and developmental effects and exposure to disinfection by-products. Regul. Toxicol. Pharm. 2006;45:185–205. doi: 10.1016/j.yrtph.2006.03.001. [DOI] [PubMed] [Google Scholar]
  36. Trush M. A., Twerdok L. E., Rembish S. J., Zhu H., Li Y. B. Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities. Environ. Health Perspect. 1996;104:1227–1234. doi: 10.1289/ehp.961041227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. vanKlaveren R. J., Demedts M., Nemery B. Cellular glutathione turnover in vitro, with emphasis on type II pneumocytes. Eur. Respir. J. 1997;10:1392–1400. doi: 10.1183/09031936.97.10061392. [DOI] [PubMed] [Google Scholar]
  38. Villanueva C. M., Cantor K. P., Grimalt J. O., Malats N., Silverman D., Tardon A., Garcia-Closas R., Serra C., Carrato A., Castano-Vinyals G., et al. Bladder cancer and exposure to water disinfection by-products through ingestion, bathing, showering, and swimming in pools. Am. J. Epidemiol. 2007;165:148–156. doi: 10.1093/aje/kwj364. [DOI] [PubMed] [Google Scholar]
  39. Waller K., Swan S. H., DeLorenze G., Hopkins B. Trihalomethanes in drinking water and spontaneous abortion. Epidemiology. 1998;9:134–140. [PubMed] [Google Scholar]
  40. Zhao B., Yang Y., Wang X., Chong Z., Yin R., Song S. H., Zhao C., Li C., Huang H., Sun B. F., et al. Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism. Nucleic Acids Res. 2013;42:1593–1605. doi: 10.1093/nar/gkt1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhao Y. L., Qin F., Boyd J. M., Anichina J., Li X. F. Characterization and determination of chloro- and bromo-benzoquinones as new chlorination disinfection byproducts in drinking water. Anal. Chem. 2010;82:4599–4605. doi: 10.1021/ac100708u. [DOI] [PubMed] [Google Scholar]
  42. Zhou H. F., Kepa J. K., Siegel D., Miura S., Hiraki Y., Ross D. Benzene metabolite hydroquinone up-regulates chondromodulin-I and inhibits tube formation in human bone marrow endothelial cells. Mol. Pharmacol. 2009;76:579–587. doi: 10.1124/mol.109.057323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhou W. J., Lou L. J., Zhu L. F., Li Z. M., Zhu L. Z. Formation and cytotoxicity of a new disinfection by-product (DBP) phenazine by chloramination of water containing diphenylamine. J. Environ. Sci.-China. 2012;24:1217–1224. doi: 10.1016/s1001-0742(11)60926-1. [DOI] [PubMed] [Google Scholar]

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