Characterization of Mechanisms of Glutathione Conjugation with

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Characterization of Mechanisms of Glutathione Conjugation with Halobenzoquinones in Solution and HepG2 Cells Wei Wang, Yichao Qian, Jinhua Li, Naif Aljuhani, Arno G. Siraki, X. Chris Le, and Xing-Fang Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05945 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Environmental Science & Technology

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Characterization of Mechanisms of Glutathione Conjugation with

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Halobenzoquinones in Solution and HepG2 Cells

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Wei Wanga,b*, Yichao Qianb, Jinhua Lib, Naif Aljuhanic, Arno G. Sirakic, X. Chris Leb,d

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and Xing-Fang Lib*

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a

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310058, China

Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang

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b

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Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta,

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Edmonton, Alberta, T6G 2G3, Canada

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c

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Alberta, T6G 2H7, Canada

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d

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T6G 2G2, Canada

Division of Analytical and Environmental Toxicology, Department of Laboratory

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton,

Department of Chemistry, Faculty of Science, University of Alberta, Edmonton, Alberta,

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*

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Email: [email protected].

Corresponding authors: Xing-Fang Li, Email: [email protected]. Wei Wang,

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1 ACS Paragon Plus Environment


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Abstract

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Halobenzoquinones (HBQs) are a class of emerging disinfection byproducts (DBPs).

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Chronic exposure to chlorinated drinking water is potentially associated with an

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increased risk of human bladder cancer. HBQ-induced cytotoxicity involves depletion of

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cellular glutathione (GSH), but the underlying mechanism remains unclear. Here we used

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high performance liquid chromatography-high resolution mass spectrometry and electron

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paramagnetic resonance spectroscopy to study interactions between HBQs and GSH, and

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found that HBQs can directly react with GSH, forming various glutathionyl conjugates

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(HBQ-SG) in both aqueous solution and HepG2 cells. We found that the formation of

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HBQ-SG varies with the initial molar ratio of GSH to HBQ in reaction mixtures. Higher

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molar ratios of GSH to HBQ facilitate the conjugation of more GSH molecules to an

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HBQ molecule. We deduced the reaction mechanism between GSH and HBQs, which

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involves redox cycling-induced formation of halosemiquinone free radicals and

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glutathione disulfide, Michael addition, as well as nucleophilic substitution. The

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proposed reaction rates are in the following order: formation of HSQ radical >

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substitution of bromine by GSH > Michael addition of GSH on benzoquinone (BQ)

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ring > substitution of chlorine by GSH > substitution of methyl group by GSH. The

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conjugates identified in HBQ-treated HepG2 cells were the same as those found in

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aqueous solution containing a 5:1 of GSH:HBQs.

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Disinfection of drinking water inactivates pathogenic microorganisms; thus, it is

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an essential water treatment step for the prevention of waterborne diseases. However, this

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process inevitably generates disinfection byproducts (DBPs) from the reactions between

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disinfectants (e.g., chlorine and chloramines) and natural organic matter in raw water.1

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Epidemiological studies have repeatedly observed associations between long-term

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consumption of disinfected water and an increased risk of bladder cancer,2–4 or adverse

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reproductive effects.5–7 Currently, several commonly-detectable DBPs, such as

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trihalomethanes (THMs) and haloacetic acids (HAAs), are regulated.8–10 The available

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toxicity evidence suggests that the carcinogenic potencies of these regulated DBPs are

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insufficient to account for the observed adverse health effects.11–16

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We have frequently detected four halobenzoquinones (HBQs) as DBPs in

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drinking water and recreational waters in North America: 2,6-dichloro-(1,4)-

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benzoquinone (2,6-DCBQ), 3,5-dichloro-2-methyl-(1,4)-benzoquinone (DCMBQ), 2,3,6-

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trichloro-(1,4)-benzoquinone (TriCBQ), and 2,6-dibromo-(1,4)-benzoquinone (2,6-

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DBBQ).17–22 HBQs are predicted to be probable DBPs relevant to the observed bladder

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cancer risk on the basis of their structure.23,24 In vitro cytotoxicity experiments confirmed

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that HBQs are highly cytotoxic and potentially genotoxic.25,26 HBQs readily produce

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intracellular reactive oxygen species (ROS), causing dysfunction in cellular antioxidant

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systems and damaging protein and DNA.25–28

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Glutathione (GSH), composed of cysteine (Cys), glutamic acid (Glu), and glycine

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(Gly), is the most abundant tripeptide thiol in cells, and serves as the major endogenous

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antioxidant protecting cells from HBQ toxicity.27,29,30 In a lab-controlled study, a

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concentration-dependent depletion of cellular GSH levels was correlated with increased



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HBQ cytotoxicity in T24 bladder cancer cells when concentrations of HBQs were in the

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range of 0–142 µM.27 Although cellular GSH depletion has been associated with HBQ

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cytotoxicity, the underlying mechanism remains unknown. A study of HBQ and GSH

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conjugation in solution revealed that HBQs readily conjugate to GSH, with one molecule

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of GSH bound to each HBQ.27 Thus, it is likely that GSH-HBQ conjugation plays a key

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role in GSH depletion in vitro as well. To examine GSH-HBQ conjugation in vitro, we

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have selected a human liver carcinoma cell line, HepG2, as these cells contain a high

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concentration of intracellular GSH,29,30 and GSH-conjugation is known to occur primarily

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in the liver.31,32 Therefore, HepG2 cells will serve as a good model to characterize the

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interactions between GSH and HBQs in mammalian cells.

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In this study, we examined the interactions between GSH and HBQs using high

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performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS)

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and electron paramagnetic resonance (EPR) spectroscopy, and found that intracellular

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GSH depletion by HBQs is attributable to both the direct conjugation of GSH to HBQs

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and the oxidation of GSH to glutathione disulfide (GSSG). We further elucidated the

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interaction mechanisms, which were found to involve Michael addition, nucleophilic

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substitution, free radical formation and desulfurization.

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Experimental Section

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Chemicals and Solvents. DCMBQ (≥98%) and TriCBQ (≥98%) were synthesized by

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Shanghai Acana Pharmtech (Shanghai, China); 2,6-DBBQ (≥98%) was purchased from

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Indofine Chemical Company (Hillsborough, NJ); 2,6-DCBQ (≥98%), L-glutathione

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reduced (HPLC grade, ≥98%) and L-glutathione oxidized (HPLC grade, ≥98%) were


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purchased from Sigma-Aldrich (St. Louis, MO). OptimaTM LC/MS grade water and

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methanol were purchased from Fisher Scientific (Nepean, ON, Canada). The purity was

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confirmed by UHPLC-UV and HPLC-MS analysis. Formic acid (HPLC grade, 50% in

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water) was purchased from Fluka (via Sigma-Aldrich). Superoxide dismutase (SOD) was

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purchased from MP Biomedicals (via Fisher Scientific). Ethyl alcohol (EtOH) and 5,5-

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Dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Dojindo Molecular

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Technologies (via Cedarlane Laboratories, Burlington, ON, Canada), and dimethyl

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sulfoxide (DMSO) was purchased from Sigma-Aldrich.

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Liquid Chromatography ─ Mass Spectrometry Analysis. The separation of conjugates

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was achieved by an ultra-high performance liquid chromatography (UHPLC) system

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(Agilent 1290 Infinity Quaternary LC series) coupled with a Luna C18(2) column (100 ×

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2.0 mm i.d., 3 µm; Phenomenex, Torrance, CA) at room temperature (25 oC). The mobile

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phase consisted of solvent A (0.1% FA in water) and solvent B (0.1% FA in methanol)

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with a flow rate of 0.17 mL/min. The gradient program was optimized as: B was linearly

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increased from 2% to 50% in 30 min; B was rapidly increased from 50% to 90% in 0.01 s

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and kept for 35 min; and finally, B was changed to 2% in 0.01 s and kept for 40 min for

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column equilibration. The sample injection volume was 20 µL.

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A quadrupole time-of-flight mass spectrometer (AB SCIEX TripleTOF® 5600

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MS, AB SCIEX, Concord, ON, Canada) was coupled with UHPLC to obtain the isotope

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pattern and fragment information of the conjugation products. The TripleTOF instrument

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(mass resolution) was tuned every three hours using an AB SCIEX calibration solution

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for the AB SCIEX TripleTOF 5600 system (Concord, ON, Canada). To obtain the

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information of all possible unknown conjugation products and to reduce the background



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interference, we developed an information dependent acquisition (IDA) method. In the

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IDA method, we set two simultaneous experiments: (1) negative ToF MS survey scan and

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(2) negative product ion scan. For the ToF survey scan, the specific conditions were: ion

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source voltage, -4500 V; gas I, 60 arbitrary units; gas II, 60 arbitrary units; curtain gas, 25

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arbitrary units; source temperature, 450 ºC; declustering potential (DP), -90 V;

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accumulation time, 0.25 s; and scan range, m/z 100–3000. For the negative product ion

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scan, a maximum of four parent ions in each survey scan were selected for MS/MS

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analysis. The criteria to initiate the MS/MS scan included: (a) the m/z of the parent ion

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was greater than 100 and smaller than 1250 (the maximum m/z that the instrument can

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measure); (b) the intensity of the parent ion was higher than 50 cps; (c) the charge state of

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the parent ion was between 1 and 4; and (d) the isotope within 4 Da was excluded in the

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same cycle. The background was subtracted dynamically. The related parameters were set

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as follows: collision energy (CE), -40 V; collision energy spread (CES), 10 V;

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accumulation time, 0.2 s; and scan range, m/z 30–3000. The accurate masses of HBQs

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were set in the inclusion list to track the peaks of HBQs at all times. PeakViewTM

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software (AB SCIEX) was used for data analysis.

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The stock solution of GSH (100 mM in water) was prepared daily prior to the

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experiments. Reaction mixtures were prepared by vortex-mixing 10 µmol solid standards

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of HBQs with a 10 mL aqueous GSH solution. The concentrations of GSH in solution

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were selected on the basis of intracellular GSH levels reported in cells (0.1-11 mM)29,30

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and to produce sufficient concentrations of conjugates for identification. Thus, the

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reaction mixtures contained 1 mM HBQ with varying concentrations of GSH (0.1, 0.3,

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0.5, 1, 3, 5, 10, and 100 mM). All reactions took place in amber bottles with Teflon caps


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to avoid light irradiation. The reaction mixtures were diluted 10 times with Optima water

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prior to UHPLC-QToF MS analysis.

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Electron Paramagnetic Resonance Analysis. Electron paramagnetic resonance (EPR)

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spectroscopy analysis was performed at room temperature using a Bruker Elexys E-500

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spectrometer. The 200-µL reaction solution was transferred to a flat cell for immediate

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scan. In all analyses, the Q value was at 1900 ± 100, and the frequency was 9.8143 ±

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0.0001 GHz. The scan range was from 3440 G to 3540 G, the modulation amplitude was

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1.0 G, and the sweep time was 60 s.

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Stock solutions (100 mM) of 2,6-DBBQ and 2,6-DCBQ were separately prepared

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by dissolving solid standard (purity ≥ 98%) into methanol (Optima LC-MS grade), while

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a fresh stock solution (100 mM) of GSH was prepared in water (Optima LC-MS grade)

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prior to each experiment. DMPO was added to water and then mixed with the stock

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solutions of GSH and 2,6-DBBQ or 2,6-DCBQ for EPR spin trapping. To account for

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solvent effects, we analyzed a series of controls: 1) pure water (Optima LC-MS grade); 2)

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1% methanol (Optima LC-MS grade) in water; 3) 100 mM DMPO in water; and 4) 50

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mM GSH with 100 mM DMPO in water.

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Collection of Treated Cells and Culture Medium. The human hepatocellular carcinoma

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cell line, HepG2, was obtained from the American Type Culture Collection (ATCC,

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Manassas, VA). The cells were incubated in 60 mm dishes and maintained at 37 oC and

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5% CO2 in a humidified incubator. The culture medium was Eagle’s Minimum Essential

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Medium (ATCC; #30-2003) supplemented with 10% fetal bovine serum (Sigma; #F1051)

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and 1% of 1000 U penicillin/1000 µg streptomycin solution (Gibco; #15140-122). To

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select proper doses of HBQs, a pre-experiment was conducted to determine the IC50



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values of HBQs following the method described in our previous study.25 When the

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confluency of cells reached 70-80%, HBQs were dosed at half concentrations of the 24 h-

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IC50 values: 36 µM for 2,6-DCBQ, 85 µM for DCMBQ, 97.5 µM for TriCBQ, and 85

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µM for 2,6-DBBQ. The cells were collected by trypsinization and washed with

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Dulbecco’s phosphate-buffered saline (DPBS) three times. The cell pellets were

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resuspended in 100 µL of ice-cold formic acid (5%), homogenized for 1 min, and

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centrifuged at 10,000 x g at 4 oC for 10 min. The extracts were analyzed using the same

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LC-MS method used for the analysis of the mixed solutions of GSH and HBQs. We

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prepared control samples that 1) only contain culture media and HBQs, but no HepG2

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cells; and 2) only contain culture media and HepG2 cells, but no HBQs. No conjugates

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were detected in the control samples.

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Results and Discussion

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Identification of GSH and 2,6-DCBQ Conjugates by UHPLC-MS/MS. Our results

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show that GSH and 2,6-DCBQ can form conjugates readily, reacting completely within 5

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min. Figure 1 shows typical chromatograms of the UHPLC-QToF MS analysis of a 10

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times diluted solution of the reaction mixture containing 1 mM 2,6-DCBQ with GSH

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concentrations of A) 0.1 mM, B) 1 mM, and C) 5 mM. When the chromatograms of the

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reaction mixtures were compared with those of the blank and of pure solutions of GSSG,

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GSH, and 2,6-DCBQ (Figure S1), several new peaks were detected in the reaction

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mixture of 2,6-DCBQ with GSH.

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To elucidate the structures of these conjugation products, we developed an

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information dependent analysis (IDA) method using ultra-high performance liquid

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chromatography – quadrupole time-of-flight mass spectrometry (UHPLC-QToF MS).


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The IDA method acquired the accurate mass measurements by ToF scan and the MS/MS

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spectra of candidate precursors by product ion scan in the same run. Figure 2 shows the

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scan spectra of the parent ions of four conjugates (1-1, 2-1, 3-1, and 4-1) and their

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MS/MS spectra (1-2, 2-2, 3-2, and 4-2). For example, Peak 7 at retention time 14.5 min

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in Figure 1 has a molecular ion of m/z 753.1287 with the isotopic pattern shown in Figure

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2 (2-1). The measured accurate masses correspond to the chemical formula

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[C26H34N6O14S2Cl]- with a mass accuracy of 4.3 ppm. The MS/MS spectrum of Peak 7

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was obtained by the IDA product ion scan, as shown in Figure 2 (2-2). Several

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characteristic fragments of GSH-conjugates were identified in the MS/MS spectrum: the

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fragment ion of m/z 306.0772 corresponding to GSH; m/z 272.0893 resulting from the

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elimination of H2S from GSH; m/z 254.0786 from the elimination of both H2S and H2O

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from GSH; m/z 143.0463 and m/z 128.0375 attributed to the cleavage of the γGlu-Cys

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amide bond of 272.0893. These fragments confirmed that this peak corresponds to a GSH

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conjugate. The fragments m/z 172.9472 and 206.9346 correspond to sulfur-monochloro-

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hydroquinone (S-MCHQ) and sulfur-thiol-monochloro-hydroquinone (S-SH-MCHQ)

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radicals, supporting that the formation of the sulfur-quinone bond is a result of the

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conjugation of GSH with 2,6-DCBQ. Fragments m/z 444.0549 and m/z 480.0332

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correspond to sulfur-glutathionyl-hydroquinone (S-SG-HQ) and sulfur-glutathionyl-

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monochloro-benzoquinone (S-SG-MCBQ), respectively. Fragments m/z 624.0875 and

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m/z 717.1554 are formed from the elimination of Glu or H2O from 2-monochloro-3,6-

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diglutathionyl-hydroquinone (2-MC-3,6-DiSG-HQ). All peaks in the dependent product

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ion scan correspond to fragments of 2-MC-3,6-DiSG-HQ, with mass accuracy better than

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3.2 ppm.



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Figure 2 presents the MS and MS/MS spectra of mono-, di-, tri- and tetra-SG

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conjugates. The ToF scan spectra (1-1, 2-1, 3-1, and 4-1) of the parent ions (black line)

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match their theoretical isotope patterns (red line) of [M-H]- (for Compounds 1–3) and

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[M-2H]2- (for Compound 4). Their MS/MS spectra (Figures 1-2, 2-2, 3-2, and 4-2) match

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with the fragments of the proposed chemical structures. Similarly, we used the accurate

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masses of parent ions and their MS/MS spectra to identify other products.

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In total, we identified 11 conjugates of 2,6-DCBQ with GSH, including mono-

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SG-BQ, di-SG-BQ, tri-SG-BQ, and tetra-SG-BQ conjugates, as well as their isomers.

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Table 1 summarizes the chemical formulas, putative structures, and formation pathways

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(presented with simplified reaction components) of the 11 conjugates identified in the

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reaction mixture of 2,6-DCBQ and GSH. It should be noted that one mass (formula) may

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represent several isomers of a conjugate. The chemical structures of isomers are proposed

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on the basis of the dipole moment (calculated using Chem3D UltraTM). The isomer with a

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high dipole moment is of high polarity, thus its retention time on the C18 column is

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shorter. For example, Peak 5 (retention time of 10.7 min) and Peak 7 (retention time of

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14.5 min) have the same MS and MS/MS spectra corresponding to monochloro-

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diglutathionyl-(1,4)-benzoquinone (MC-DiSG-BQ). We propose that Peak 5 is 2-chloro-

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5,6-diglutathionyl-(1,4)-benzoquinone (2-MC-5,6-DiSG-BQ) with a higher dipole

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moment of 7.204 Debye, and Peak 7 is 2-chloro-3,5-diglutathionyl-(1,4)-benzoquinone

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(2-MC-3,6-DiSG-BQ) with a lower dipole moment of 2.570 Debye. In addition, HBQs

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coexist with halo-semiquinones (HSQs) and halohydroquinones (XHQs) through

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reversible redox reactions.33,34 Neither LC separation nor MS spectra can distinguish the


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co-existing [M-H]- ion of XHQ, [M]- ion of HSQ, and [M+H]- ion of HBQ. Therefore,

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peaks 4–14 may represent a mixture of three chemical forms.

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The Reaction Pathways between GSH and 2,6-DCBQ. The identification of various

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GSH conjugates led to further investigation of the binding stoichiometry of 2,6-DCBQ

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with GSH. When the ratio of GSH:2,6-DCBQ was 0.1, mono-SG and di-SG substituted

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2,6-DCBQ formed: 2,6-dichloro-3,5-diglutathionyl-(1,4)-benzoquinone/hydroquinone

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(2,6-DC-3,5-DiSG-BQ or 2,6-DC-3,5-DiSG-HQ, Peak 9) and 2,6-dichloro-3-

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glutathionyl-(1,4)-benzoquinone/hydroquinone (2,6-DC-3-SG-BQ or 2,6-DC-3-SG-HQ,

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Peak 10). Michael addition of GSH on 2,6-DCBQ forms 2,6-DC-3-SG-HQ that can be

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oxidized by oxygen or 2,6-DCBQ to form 2,6-DC-3-SG-BQ. A second GSH attacks 2,6-

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DC-3-SG-BQ to form 2,6-DC-3,5-DiSG-HQ and 2,6-DC-3,5-DiSG-BQ. The proposed

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reaction pathways are as follows:

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2,6-DCBQ + GSH ↔ 2,6-DC-3-SG-HQ (Figure 1, Peak 10)

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2,6-DC-3-SG-HQ (Figure 1, Peak 10) + O2 ↔ 2,6-DC-3-SG-BQ (Figure 1, Peak

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10) + H2O2 2,6-DC-3-SG-HQ (Figue 1, Peak 10) + 2,6-DCBQ ↔ 2,6-DC-3-SG-BQ (Figure 1, Peak 10) + 2,6-DCHQ 2,6-DC-3-SG-BQ (Figure 1, Peak 10) + GSH ↔ 2,6-DC-3,5-DiSG-HQ (Figure 1, Peak 9) 2,6-DC-3,5-DiSG-HQ (Figure 1, Peak 9) + O2 ↔ 2,6-DC-3,5-DiSG-BQ (Figure 1, Peak 9) + H2O2 2,6-DC-3,5-DiSG-HQ (Figure 1, Peak 9) + 2,6-DCBQ ↔ 2,6-DC-3,5-DiSG-BQ (Figure 1, Peak 9) + 2,6-DCHQ



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When the GSH:2,6-DCBQ ratio was increased to 1, additional dechlorinated SG-

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conjugates were formed: 2-chloro-5,6-diglutathionyl-(1,4)-benzoquinone/hydroquinone

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(2-MC-5,6-DiSG-BQ or 2-MC-5,6-DiSG-HQ, Peak 5), 2-chloro-3,6-diglutathionyl-(1,4)-

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benzoquinone/hydroquinone (2-MC-3,6-DiSG-BQ or 2-MC-3,6-DiSG-HQ, Peak 7), 2,6-

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diglutathionyl-(1,4)-benzoquinone/hydroquinone (2,6-DiSG-BQ or 2,6-DiSG-HQ, Peak

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12), 2-chloro-3,5-diglutathionyl-(1,4)-benzoquinone/hydroquinone (2-MC-3,5-DiSG-BQ

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or 2-MC-3,5-DiSG-HQ, Peak 13), and 2-chloro-6-glutathionyl-(1,4)-

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benzoquinone/hydroquinone (2-MC-6-SG-BQ or 2-MC-6-SG-HQ, Peak 14). The loss of

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chlorine from 2,6-DCBQ to form these conjugates indicated that GSH substitutes the

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chlorine on the benzoquinone (BQ) ring through a nucleophilic substitution reaction.

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XHQs are not believed to react with GSH because of the higher electron density of

259

XHQs, and chlorine would be unfavorable as a leaving group.35

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2,6-DCBQ + GSH ↔ 2-MC-6-SG-BQ (Figure 1, Peak 14) + HCl

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2-MC-6-SG-BQ + GSH ↔ 2,6-DiSG-BQ (Figure 1, Peak 12) + HCl

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These dechlorinated conjugation products can further undergo Michael addition to form

263

more glutathionylated products.

264 265 266

2-MC-6-SG-BQ + GSH ↔ 2-MC-5,6 (or 3,6; or 3,5)-DiSG-HQ (Figure 1, Peak 5, 7 or 14) When GSH:2,6-DCBQ was increased to 5, tri-SG- and tetra-SG-conjugates

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emerged: triglutathionyl-(1,4)-benzoquinone/hydroquinone, (TriSG-BQ or TriSG-HQ,

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Peak 4), 2-monochloro-3,5,6-triglutathionyl-(1,4)-benzoquinone/hydroquinone (2-MC-

269

3,5,6-TriSG-BQ or 2-MC-3,5,6-TriSG-HQ, Peak 6), and tetraglutathionyl-(1,4)-

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benzoquinone/hydroquinone (TetraSG-BQ or TetraSG-HQ, Peak 3). Some mono- and di-


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SG conjugates were still present (Figure 1C). These conjugates coexisted and achieved

272

chemical equilibrium.

273 274

2-MC-5,6 (or 3,6; or 3,5)-DiSG-BQ (Figure 1, Peak 5, 7 or 14) + GSH ↔ 2-MC3, 5, 6-TriSG-HQ (Figure 1, Peak 6)

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2-MC-5,6 (or 3,6; or 3,5)-DiSG-BQ + GSH ↔ TriSG-BQ (Figure 1, Peak 4) + HCl

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2,6-DiSG-BQ + GSH ↔ TriSG-HQ (Figure 1, Peak 4)

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TriSG-BQ + GSH ↔ TetraSG-HQ (Figure 1, Peak 3)

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2-MC-3,5,6-TriSG-BQ + GSH ↔ TetraSG-BQ (Figure 1, Peak 3) + HCl

280

The redox reaction between HQ to BQ derivatives can be a two-electron reduction

281

or two sequential one-electron reduction steps through the formation of semiquinone

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radicals (SQ).33 To examine the possible production of SQ in the reaction process, we

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analyzed the reaction mixture of 2,6-DCBQ and GSH at varying molar ratios using EPR

284

spectroscopy. Figure 3 shows that 2,6-dichloro-(1,4)-semiquinone radical (2,6-DCSQ•–)

285

was present in solution, and the intensity of the radical decreased as a function of

286

increased GSH level. When GSH was increased to 100 µM, 2,6-DCSQ•– was

287

undetectable. 2,6-DCBQ can undergo a one-electron transfer reaction, forming 2,6-

288

DCSQ•–:

289

2,6-DCBQ + e – ↔ 2,6-DCSQ•– (Figure S2-A and S3-A)

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A sequential one-electron transfer reaction forms 2,6-dichloro-(1,4)-hydroquinone (2,6-

291

DCHQ):

292

2,6-DCSQ•–+ e – + 2H + ↔ 2,6-DCHQ



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In an attempt to detect other transient free radicals, we analyzed the mixture using

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EPR spin trapping with 100 mM DMPO. Figure S2 shows the signals detected when

295

DMPO was added to the reaction mixture of 2,6-DCBQ with GSH. In addition to 2,6-

296

DCSQ•–, we detected the apparent signal of the DMPO/•OH spin adduct. However, the

297

signal of the DMPO/•OH spin adduct was not depleted when DMSO or SOD was added

298

(Figure S3). Thus, the DMPO/•OH signal is not an indication of the formation of

299

superoxide or hydroxyl radicals in the reaction system. It may be a background signal or

300

it may arise from the oxidation of DMPO by photoexcited 2,6-DCBQ.36,37 The

301

mechanism we propose is analogous to that proposed previously by Monroe and Eaton

302

(1996) for menadione:38 Light

(photoexcited)

303

2,6-DCBQ ሱۛሮ 2,6-DCBQ*

304

2,6-DCBQ* + DMPO → 2,6-DCSQ•– + DMPO•+

305

DMPO+• + H2O → DMPO/•OH + H+

306

With the increase of GSH, both 2,6-DCSQ•– and DMPO/•OH decreased. When

307

GSH was increased to 5 mM, all free radical species were undetectable. Because the

308

conjugation of GSH to 2,6-DCBQ will increase the electron intensity, the conjugation

309

products are more easily oxidized than 2,6-DCBQ.

310

2,6-DCHQ + O2 ↔ 2,6-DCBQ + H2O2

311

H2O2 + 2,6-DCSQ•– ↔ •OH + OH– + 2,6-DCBQ39

312

2,6-DCSQ•– radical or hydroxyl radical oxidized GSH to form GSSG40,41

313

2,6-DCSQ•– + e– + 2GSH ↔ 2,6-DCHQ + GSSG (Figure 1, Peak 2)

314

2•OH + 2GSH ↔ GSSG (Figure 1, Peak 2) + H2O


Page 15 of 36


315

The amount of •OH is very limited, if it forms at all; thus, the major reactant is the 2,6-

316

DCSQ•– radical.

317

In summary, three typical reactions between GSH and 2,6-DCBQ were involved:

318

nucleophilic substitution of chlorine on the BQ ring to form glutathionyl BQs; Michael

319

addition of GSH to the BQ ring to form glutathionyl HQs; and reversible redox reactions

320

between HBQ, HSQ•– or XHQ along with the oxidation of GSH to GSSG (Figure 4A).

321

With increasing GSH:2,6-DCBQ ratios, the conjugation ratio of GSH to 2,6-DCBQ is

322

also increased, meaning mono- and di-SG substituted BQs are further glutathionylated to

323

tri- and tetra-SG BQs. Figure 4B illustrates the proposed pathways of GSH conjugation to

324

2,6-DCBQ. We identified seven conjugates of TriCBQ and GSH, and found that the

325

reaction mechanisms between TriCBQ and GSH are similar to those between 2,6-DCBQ

326

and GSH.

327

The Reaction Pathways between GSH and DCMBQ. When DCMBQ was incubated

328

with GSH, five conjugation products and GSSG were identified in the mixture using

329

UHPLC-MS/MS. The names, formulas, and simplified formation mechanisms of these

330

conjugates are summarized in Table S1. In addition to the three typical reactions, we

331

observed the substitution of a methyl group by a glutathionyl group. The proposed

332

reaction process is shown in Figure S4. GSH attacks the methyl-connected carbon on the

333

BQ ring of triglutathionyl-methyl-benzoquinone (TriSG-MBQ, Compound 9-1) to form

334

Compound 9-2, with a subsequent elimination of a hydrogen from a methyl group and an

335

SG group to form Compound 9-3. Addition of GSH or H2O (H2O can serve as a hydrogen

336

donor) to the double bond can form HQ Compound 9-4 and BQ Compound 9-5. A similar



Page 16 of 36

337

reaction process forms TetraSG-HQ (Compound 9-7) and TetraSG-BQ (Compound 9-8),

338

sequentially.

339

The Reaction Pathways between GSH and 2,6-DBBQ. We analyzed 2,6-DBBQ

340

solution using EPR, and found the 2,6-dibromo-(1,4)-benzosemiquinone radical (2,6-

341

DBSQ•−) in aqueous solution. When 1 mM 2,6-DBBQ was incubated with different

342

concentrations of GSH, only the 2,6-DBSQ•− radical was found, and the intensity of the

343

radical decreased with an increasing GSH:2,6-DBBQ ratio (Figure S5). The 2,6-DBSQ•−

344

radical signal was undetectable when the GSH concentration was increased to 300 µM

345

([GSH]:[2,6-DBBQ] = 0.3). In addition to the 2,6-DBSQ•− radical, we also detected the

346

formation of the DMPO/•OH adduct using DMPO spin trapping (Figure S6). Similarly

347

with 2,6-DCBQ, the intensity of 2,6-DBSQ•− and DMPO/•OH decreased as GSH

348

increased.

349

Nine glutathionyl conjugates were identified in the mixture of 2,6-DBBQ and

350

GSH using UHPLC-MS/MS: TetraSG-BQ or TetraSG-HQ (Peak 3), TriSG-BQ or TriSG-

351

HQ (Peak 4), 2-bromo-3,5-diglutathionyl-(1,4)-benzoquinone/hydroquinone (2-MB-3,5-

352

DiSG-BQ or 2-MB-3,5-DiSG-HQ, Peak 6), 2-bromo-3,5,6-triglutathionyl-(1,4)-

353

benzoquinone/hydroquinone (2-MB-TriSG-BQ or 2-MB-TriSG-HQ, Peak 7), 2-bromo-

354

5,6-diglutathionyl-(1,4)-benzoquinone/hydroquinone (2-MB-5,6-DiSG-BQ or 2-MB-5,6-

355

DiSG-HQ, Peak 9), 2-bromo-3-glutathionyl-(1,4)-benzoquinone/hydroquinone (2-MB-3-

356

SG-BQ or 2-MB-3-SG-HQ, Peak 10), 2,6-dibromo-3,5-diglutathionyl-(1,4)-

357

benzoquinone/hydroquinone (2,6-DB-3,5-DiSG-BQ or 2,6-DB-3,5-DiSG-HQ, Peak 12),

358

and 2-bromo-6-glutathionyl-(1,4)-benzoquinone/hydroquinone (2-MB-6-SG-BQ or 2-

359

MB-6-SG-HQ, Peak 14), and 2,6-DiSG-BQ or 2,6-DiSG-HQ (Peak 16) (Figure S7 and


Page 17 of 36


360

Table S2). These products are formed from free radical reactions, Michael addition, and

361

nucleophilic substitution, similar to the reactions between 2,6-DCBQ and GSH. An

362

important difference is that the substitution of bromine by GSH is favored over Michael

363

addition. Several debrominated compounds were identified at low GSH:2,6-DBBQ ratios,

364

including 2-MB-3-SG-HQ (Peak 10), 2,6-DB-3,5-DiSG-HQ (Peak 12), 2-MB-6-SG-HQ

365

(Peak 14), and 2,6-DiSG-HQ (Peak 16) (Figure S7A). Thus, the reactions follow the

366

order: substitution of bromine by GSH > Michael addition of GSH on the BQ ring >

367

substitution of chlorine by GSH.

368

In addition, some minor products were identified in the mixtures of 2,6-DBBQ

369

with GSH: bromo-glutathionyl-desulfurized glutathione-benzoquinone/hydroquinone

370

(MB-SG-G-BQ or MB-SG-G-HQ, Peak 5, 8 and 11) and bromo-glutathionyl-vulcanized

371

glutathione-benzoquinone/hydroquinone (MB-SSG-BQ or MB-SSG-HQ, Peak 13). G is

372

the desulfurized glutathione, and SSG is disulfide glutathione. We propose the formation

373

pathway of these G or SSG conjugates as follows (Figure S8): β-elimination of cysteine

374

or GSH has been reported to form dehydropeptide 13-2, releasing hydrogen sulfide

375

(H2S).42–44 The 2,6-DBSQ•– radical reacts with Compound 13-2 to form a carbon-

376

centered radical, 13-3. GSH donates a hydrogen to the carbon-centered radical, acting as

377

a free radical scavenger. This reaction happens very rapidly, thus we did not capture the

378

intermediate radical.45 The conjugation product 13-4 changes to the more stable HQ-form,

379

2,6-DB-3-G-HQ anion (Compound 13-5), which is further oxidized to 2,6-DB-3-G-BQ

380

(Compound 13-6). GS substitutes bromine on 2,6-DB-3-G-BQ to form 2-MB-5-G-6-SG-

381

HQ and 2-MB-5-SG-6-G-HQ (Compound 13-7), corresponding to Peaks 5 and 8 in



382

Figure S7. Meanwhile, GSH reacts with H2S forming GSSH,46 which further react with

383

2,6-DCBQ to produce a GSS conjugate (Compound 13-8).

Page 18 of 36

384

GSH+H2S → GSSH+H2

385

2,6-DBBQ+GSSH → MB-GSS-BQ (Figure S7, Peak 13) + HBr

386

Identification of Conjugation Products in HBQ Treated HepG2 Cells. After

387

confirming that 2,6-DCBQ can conjugate with GSH in an aqueous solution, we aimed to

388

study how HBQs react with GSH inside cells. When HepG2 cells were exposed to 2,6-

389

DCBQ, GSSG and eight conjugation products of 2,6-DCBQ and GSH were identified in

390

the cell extracts. Based on their retention times, accurate masses, and MS/MS spectra, the

391

eight GSH conjugates were the same as those identified in the reaction mixture of GSH

392

and 2,6-DCBQ at a molar ratio of 5:1. The three conjugates identified at the molar ratio

393

of GSH to 2,6-DCBQ of 0.1:1 were not identified in cells. This is reasonable, as cellular

394

levels of GSH are between 1 and 11 mM,29,30 which is more than 100 times higher than

395

the dose of 2,6-DCBQ (36 µm).

396

We collected cells after 10 min, 20 min, 30 min, 2 h, and 4 h exposure to 2,6-

397

DCBQ. The intensities of each conjugate as a function of exposure time are shown in

398

Figure 5. Only mono- and di-SG conjugates were identified after the 10-min exposure.

399

Eight conjugates were all identified in the cell extracts after 20-min of treatment. The

400

intensity of mono- and di-glutathionylated conjugates was reduced compared to the

401

increase in intensity of tri- and tetra-glutathionylated conjugates at 4 h. This result

402

supports the sequential conjugation of 2,6-DCBQ by GSH. Similarly, the conjugation

403

products identified in HepG2 cells treated with DCMBQ, TriCBQ, or 2,6-DBBQ were

404

also found to be the same as those detected in the mixtures of GSH:DCMBQ,


Page 19 of 36


405

GSH:TriCBQ, or GSH:2,6-DBBQ at 5:1 ratios, respectively. Only TetraSG-BQ was not

406

identified in DCMBQ treated cells, indicating that the substitution of the methyl group by

407

GSH may be unfavourable in cells (Figure S9). The sequential conjugation trends were

408

also observed in HepG2 cells treated with DCMBQ, TriCBQ, and 2,6-DBBQ. Although

409

the conjugates identified in solution and in vitro are similar, the evidence is insufficient to

410

conclude that the intracellular transformation of HBQs are mostly non-enzymatic. The

411

glutathione transferases (GST) are reported to catalyze the nucleophilic attack of GSH on

412

electrophilic substrates,47 and our previous study found that HBQs increased cellular GST

413

activities in a concentration-dependent manner in T24 cells.27 Future studies to compare

414

conjugation with and without GST inhibitors are necessary to distinguish enzymatic and

415

non-enzymatic reactions.

416

These results provide insight into the interactions of HBQs with GSH involving

417

three reaction pathways: the redox cycling reactions between HBQs and XHQs to form

418

HSQ free radicals and GSSG, Michael addition of GSH to the BQ ring, and nucleophilic

419

substitution of halo groups by GSH. The reactions follow the order: formation of HSQ

420

radical > substitution of bromine by GSH > Michael addition of GSH on the BQ ring >

421

substitution of chlorine by GSH > substitution of methyl groups by GSH. The unique

422

differences detected in GSH conjugation with 2,6-DBBQ and DCMBQ from chloro-BQs

423

suggest that halogens affect GSH-HBQ interactions. The conjugates identified in HepG2

424

cells were all identified in aqueous solutions of GSH and HBQs with a molar ratio of 5:1,

425

demonstrating intracellular GSH and HBQ interactions.

426 427

ASSOCIATED CONTENT



428

Supporting Information: The Supporting Information is available free of charge on the

429

ACS publication website at DOI:

430

Two tables and nine figures showing additional results to support the findings.

Page 20 of 36

431 432

AUTHOR INFORMATION

433

*Corresponding Author

434

E-mail: [email protected]

435

ORCID: Xing-Fang Li: 0000-0003-1844-7700

436

Corresponding Author

437

Email: [email protected]

438

ORCID: Wei Wang: 0000-0001-7066-6076

439 440

ACKNOWLEDGEMENTS

441

The study was supported by grants from the Natural Sciences and Engineering Research

442

Council of Canada, Alberta Health, and the Fundamental Research Funds for the Central

443

Universities. W. Wang acknowledges the Izaak Walton Killam Memorial Scholarship. We

444

appreciate the assistance of Dr. Derrick Clive from the Department of Chemistry,

445

University of Alberta, and of Dr. Benzhan Zhu at the Research Center for Eco-

446

Environmental Sciences, Chinese Academy of Sciences, to explain the reaction

447

mechanisms.

448 449 450

References


Page 21 of 36

451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495


(1). 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. (2). Villanueva, C. M.; Cantor K. P.; Cordier, S.; Jaakkola, J. J. K.; King, W. D.; Lynch, C. F.; Porru, S.; Kogevinas, M. Disinfection byproducts and bladder cancer - A pooled analysis Epidemiology. 2004, 15, 357–367. (3). 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.; Marcos, R.; Rothman, N.; Real, F. X.; Dosemeci, M.; Kogevinas, M. Bladder cancer and exposure to water disinfection by-products through ingestion, bathing, showering, and swimming in pools. Am. J. Epidemiol. 2007, 165, 148–156. (4). Hrudey, S. E.; Backer, L. C.; Humpage, A. R.; Krasner, S. W.; Michaud, D. S.; Moore, L. E.; Singer, P. C.; Stanford, B. D. Evaluating evidence for association of human bladder cancer with drinking-water chlorination disinfection by-products. J. Toxicol. Environ. Health B. Crit. Rev. 2015, 22, 666–684. (5). Rice, G.; Teuschler, L. K.; Speth, T. F.; Richardson, S. D.; Miltner, R. J.; Schenck, K. M.; Gennings, C.; Hunter, E. S. III.; Narotsky, M. G.; Simmons, J. E. Integrated disinfection by-products research: Assessing reproductive and developmental risks posed by complex disinfection by-product mixtures. J. Toxicol. Environ. Health A 2008, 71, 1222–1234. (6). Colman, J.; Rice, G. E.; Wright, J. M.; Hunter, E. S.; Teuschler, L. K.; Lipscomb, J. C.; Hertzberg, R. C.; Simmons J. E.; Fransen, M. Osier, M. Narotsky, M. G. Identification of developmentally toxic drinking water disinfection byproducts and evaluation of data relevant to mode of action. Toxicol. Appl. Pharmacol. 2011, 254, 100– 126. (7). Nieuwenhuijsen, M. J.; Martinez, D.; Grellier, J.; Bennett, J.; Best, N.; Iszatt, N.; Vrijheid, M.; Toledano, M. B. Chlorination disinfection by-products in drinking water and congenital anomalies: review and meta-analyses Environ. Health. Perspect. 2009, 117, 1486–1493. (8). World Health Organization (WHO). Guidelines for drinking-water quality,.4th ed.; 2011. http://www.who.int/water_sanitation_health/publications/2011/dwq_guidelines/en/. Accessed: Jan 5, 2016. (9). Health Canada. (2012) Guideline for Canadian drinking water quality, Ontario, Canada, sixth ed., http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/2012-sum_guideres_recom/index-eng.php. (10). United States Environmental Protection Agency (US EPA). National Primary Drinking Water Regulations: Stage 2 Disinfectants and Disinfection Byproducts Rule; Final Rule. 2006, 71, 388–493. (11). Richardson, S. D. Disinfection by-products and other emerging contaminants in drinking water. Trends. Anal. Chem.2003, 22, 666–684. (12). Richardson, S. D.; Kimura, S. Y. Water analysis: emerging contaminants and current issues. Anal. Chem. 2016, 88, 546–582. (13). Itoh, S.; Gordon, B. A.; Callan. P.; Bartram. J. Regulations and perspectives on disinfection by-products: importance of estimating overall toxicity. J. Water Supply Res. T. 2011, 60, 261–274.



496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541

Page 22 of 36

(14). Wagner, E. D.; Plewa, M. J., CHO cell cytotoxicity and genotoxicity analyses of disinfection by-products: an updated review. J. Environ. Sci. 2017, 58, 64-76. (15). Plewa, M. J.; Wagner, E. D.; Richardson, S. D., TIC-Tox: A preliminary discussion on identifying the forcing agents of DBP-mediated toxicity of disinfected water. J. Environ. Sci. 2017, 58, 208-216. (16). Plewa, M. J.; Wagner, E. D., Charting a new path to resolve the adverse health effects of DBPs. In Occurrence, Formation, Health Effects, and Control of Disinfection By-Products, Karanfil, T.; Mitch, W.; Westerhoff, P.; Xie, Y., Eds. Am. Chem. Soc.: Washington, D.C., 2015; Vol. 1190, pp 3-23. (17). Qin, F.; Zhao, Y. Y.; Zhao, Y.; Boyd, J. M.; Zhou, W.; Li, X-F. A toxic disinfection by-product, 2,6-dichloro-1,4-benzoquinone, identified in drinking water. Angew. Chem. Int. Ed. 2010, 49,790–792. (18). Zhao, Y.; 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. (19). Zhao, Y.; Anichina, J.; Lu, X.; Bull, R. J.; Krasner, S. W.; Hrudey, S. E.; Li, X-F. Occurrence and formation of chloro- and bromo-benzoquinones during drinking water disinfection. Water Res. 2012, 46, 4351–4360. (20). Wang, W.; Qian, Y.; Boyd, J. M.; Wu, M.; Hrudey, S. E.; Li, X-F. Halobenzoquinones in swimming pool waters and their formation from personal care products. Environ. Sci. Technol. 2013, 47, 3275–3282. (21). Wang, W.; Qian, Y.; Li, J.; Moe, B.; Huang, R.; Zhang, H.; Hrudey, S. E.; Li, X.-F. Analytical and toxicity characterization of halo-hydroxyl-benzoquinones as stable halobenzoquinone disinfection byproducts in treated water. Anal Chem. 2014, 86, 4982– 4988. (22). Wang, W.; Birget Moe, B.; Li, J.; Qian, Y.; Zheng, Q.; Li, X.-F. Analytical characterization, occurrence, transformation, and removal of the emerging disinfection byproducts halobenzoquinones in water. Trends Anal. Chem. 2016. 85, 97-110. (23). Bull, R. J.; Reckhow, D. A.; Rotello, V.; Bull, O. M.; Kim, J. Use of Toxicological and Chemical Models to Prioritize DBP Research; American Water Works Association Research Foundation and American Water Works Association: Denver, CO, 2006. (24). 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. (25). 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. (26). Zhao, B.; Yang, Y.; Wang, X.; Chong, Z.; Yin, R.; Song, S. H.; Zhao, C.; Li, C.; Huang, H.; Sun, B. F.; Wu, D.; Jin, K. X.; Song, M.; Zhu, B. Z.; Jiang, G.; Danielsen, J. M. R.; Xu, G. L.; Yang, Y. G.; Wang, H. Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism. Nucleic. Acids. Res. 2014, 42, 1593−1605. (27). Li, J. H.; Wang, W.; Zhang, H. Q.; Le, X. C.; Li, X.-F. Glutathione-mediated detoxification of halobenzoquinone drinking water disinfection byproducts in T24 Cells. Toxicol. Sci. 2014, 141, 335–343. 22 ACS Paragon Plus Environment

Page 23 of 36

542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586


(28). Li, J. H.; Wang, W.; Moe, B.; Wang, H. L.; Li, X.-F. Chemical and toxicological characterization of halobenzoquinones, an emerging class of disinfection byproducts. Chem. Res. Toxicol. 2015, 28, 306–318. (29). Hayes, J.D.; MeLellan, L.I. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic. Res. 1999, 31, 273-300. (30). 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, 2823-2830. (31). Simic, T.; Savic-Radojevic, A.; Pljesa-Ercegovac, M.; Matic, M.; Mimic-Oka, J. Glutathione S-transferases in kidney and urinary bladder tumors. Nat. Rev. Urol. 2009, 6, 281-289. (32). Mulder, T.P.; Court, D.A.; Peters, W.H. Variability of glutathione S-transferase alpha in human liver and plasma. Clin. Chem. 1999, 45(3), 355-359. (33). El-Najjar, N.; Gali-Muhtasib, H.; Ketola, R. A.; Vuorela, P.; Urtti, A.; Vuorela, H. The chemical and biological activities of quinones: overview and implications in analytical detection. Phytochem Rev. 2011, 10, 353–370. (34). Ma, W.; Long, Y. T.; Quinone/hydroquinone-functionalized biointerfaces for biological applications from the macro- to nano-scale. Chem. Soc. Rev. 2014, 43, 30–41. (35). Song, Y.; Wagner, B. A.; Witmer, J. R.; Lehmler, H. J.; Buettner G. R. Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones. Proc. Natl. Acad. Sci. USA 2009, 106, 9725–9730 (36). McCormick, M. L.; Denning, G. M.; Reszka, K. L.; Bilski, P.; Buettner, G.R.; Rasmussen, G.T.; Railsback, M.A.; Britigan, B.E. Biological effects of menadione photochemistry: Effects of mendione on biological systems may not involve classical oxidant production. Biochemical J. 2000, 350, 797–804. (37). Alegria, A. E.; Ferrer, A.; Santiago, G.; Sepulveda, E.; Flores, W. Photochemistry of water-soluble quinones. Production of the hydroxyl radical, singlet oxygen and the superoxide ion. J. Photochem. Photobio. A 1999, 127, 57–65. (38). Monroe, S.; Eaton, S. S. Photo-enhanced production of the spin adduct 5,5dimethyl-1-pyrroline-N-oxide/center dot OH in aqueous menadione solutions. Arch. Biochem. Biophys. 1996, 329, 221–227. (39). Zhu, B. Z.; Mao, L.; Huang, C. H.; Qin, H.; Fan, R. M.; Kalyanaraman, B.; Zhu, J. G. Unprecedented hydroxyl radical-dependent two-step chemiluminescence production by polyhalogenated quinoid carcinogens and H2O2. Proc. Natl. Acad. Sci. USA. 2012, 109, 16046–16051. (40). Madrasi, K.; Joshi, M. S.; Gadkari, T.; Kavallieratos, K.; Tsoukias, N. M. Glutathiyl radical as an intermediate in glutathione nitrosation. Free Radic. Bio. Med. 2012, 53, 1968–1976. (41). Wardman, P. Evaluation of the "radical sink" hypothesis from a chemical-kinetic viewpoint. J Radioanal. Nucl. Chem. 1998, 232, 23–27. (42). Zhao, Y.; Wang, H.; Xian, M. Cysteine-activated hydrogen sulfide (H2S) donors. J. Am. Chem. Soc. 2011, 133, 15–17. (43). Zhao, Y.; Biggs, T. D.; Xian, M. Hydrogen sulfide (H2S) releasing agents: chemistry and biological applications. Chem. Commun. 2014, 50, 11788–11805.



587 588 589 590 591 592 593 594 595 596 597 598 599 600

Page 24 of 36

(44). Fonvielle, M.; Mellal, D.; Patin, D.; Lecerf, M.; Blanot, D.; Bouhss, A.; Santarem, M.; Mengin-Lecreulx, D.; Sollogoub, M.; Arthur, M.; Etheve-Quelquejeu, M. EtheveQuelquejeu, M. Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemXWv Aminoacyl Transferase. Chem-Eur J. 2013, 19, 1357–1363. (45). Willson, R. L. Free radical repair mechanisms and the interactions of glutathione and vitamins C and E. Radioprotectors and Anticarcinogens, eds Nygaard OF, Simic MG(Academic Press, London), 1982, pp. 1–23. (46). Chung, H. S.; Wang, S. B.; Venkatraman, V.; Murray, C. I.; Van, Eyk J. E. Cysteine oxidative posttranslational modifications emerging regulation in the cardiovascular system. Circ. Res. 2013, 112, 382–392. (47). Eaton, D. L.; and Bammler, T. K. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol. Sci. 1999, 49, 156−164.


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601 602 603

Figure Legends

604

mechanism of each new peak identified in the mixture of 2,6-DCBQ with GSH.

605

Figure 1. The UHPLC-ToF chromatograms of the reaction mixtures of GSH and 2,6-

606

DCBQ at different ratios. A) GSH:2,6-DCBQ = 0.1, B) GSH:2,6-DCBQ = 1, and C)

607

GSH:2,6-DCBQ = 5.

608

Figure 2. The MS and MS/MS spectra of mono-, di-, tri-, and tetra-glutathionyl-

609

benzoquinones: 1) 2,6-DC-SG-HQ, 2) 2-MC-3,6-DiSG-HQ, 3) TriSG-HQ, and 4)

610

TetraSG-HQ. 1-1, 2-1, 3-1 and 4-1 are the ToF MS spectra of the parent ions (blue line),

611

in accordance with the theoretical isotope pattern of proposed [M-H]- or [M-2H]2- anions

612

(red line); 1-2, 2-2, 3-2, and 4-2 are the dependent MS/MS spectra of the parent isotope

613

with highest intensity.

614

Figure 3. Semiquinone radical detected in the reaction between GSH and 2,6-DCBQ.

615

Reactions were carried out in ddH2O and [2,6-DCBQ] = 1 mM. (A) [GSH] = 0,

616

[GSH]:[2,6-DCBQ] = 0, pH = 6.8; (B) [GSH] = 10 µM, [GSH]:[2,6-DCBQ] = 0.01, pH

617

= 6.6; (C) [GSH] = 30 µM, [GSH]:[2,6-DCBQ] = 0.03, pH = 6.5; (D) [GSH] = 50 µM,

618

[GSH]:[2,6-DCBQ] = 0.05, pH = 6.4; (E) [GSH] = 100 µM, [GSH]:[2,6-DCBQ] = 0.1,

619

pH = 6.2; (F) [GSH] = 300 µM, [GSH]:[2,6-DCBQ] = 0.3, pH = 6.2. The center peak was

620

g = 2.00538.

621

Figure. 4. Summary of the proposed reaction mechanism of GSH and HBQs. (A) Overall

622

reactions involved in the conjugation of GSH on chlorinated HBQs. X is the substitution

623

group, Cl, Br, or CH3; a is the number of the substituted groups, equal to 1, 2 or 3. (B)

624

Proposed reaction pathways of HBQs with GSH, using 2,6-DCBQ as an example. [O] is

625

the oxidant, which could be oxygen gas or a less glutathionylated quinone. The molecules

626

in (B) identified by LC-MS/MS are described in Table 1.

627

Figure. 5. The ToF ion intensity of 2,6-DCBQ-GSH conjugates in 2,6-DCBQ treated cells

628

as a function of exposure time. The error bars represent standard deviations of triplicate

629

samples.

Table 1. The retention time, chemical formula, possible structures, and formation



Page 26 of 36

Table 1. The retention time, chemical formula, possible structures, and formation mechanism of each new peak identified in the mixture of 2,6-DCBQ with GSH. No. Retention Name time (min) 1 3.2 glutathione

Formula

Structure

C10H17N3O6S

Formation Reactant

(GSH) 2

3

4

5

4.9

7.6

8.8

10.7

glutathione

C20H32N6O12S2

2GS

disulfide

(GSSG)

2,3,5,6-tetraglutathionyl-

C46H66N12O26S4

4GS+2,6-DCBQ-2Cl-

(1,4)hydroquinone/

(TetraSG-HQ)

2H

benzoquinone

(TetraSG-BQ)

2,3,5-triglutathionyl-

C36H51N9O20S3

(1,4)hydroquinone/

(TriSG-HQ)

benzoquinone

(TriSG-BQ)

2-monochloro-5,6-

C26H35N6O14S2Cl

diglutathionyl-(1,4)

(2-MC-5,6-DiSG-HQ)

hydroquinone/benzoquinone

(2-MC-5,6-DiSG-BQ)

3GS+2,6-DCBQ-2Cl-H

2GS+2,6-DCBQ-Cl-H


Page 27 of 36


No. Retention Name time (min) 6 11.5 2-monochloro-3,5,6-

7

8

9

14.5

15.0

17.9

Formula

Structure

C36H50N9O20S3Cl

triglutathionyl-(1,4)

(2-MC-3,5,6-TriSG-HQ)


(2-MC-3,5,6-TriSG-BQ)

2-monochloro-3,6-

C26H35N6O14S2Cl


(2-MC-3,6-DiSG-HQ)


(2-MC-3,6-DiSG-BQ)

2-monochloro-6-

C16H20N3O8SCl

glutathionyl-(1,4)

(2-MC-6-SG-HQ)


(2-MC-6-SG-BQ)

2,6-dichloro-2,5-

C26H34N6O14S2Cl2


(2,6-DC-2,5-DiSG-HQ)


(2,6-DC-2,5-DiSG-BQ)

2,6-dichloro-3-

C16H19O8N3SCl2

glutathionyl-(1,4)

(2,6-DC-3-SG-HQ)


(2,6-DC-3-SG-BQ)

2,6-dichloro-(1,4)

C6H2Cl2O2


(2,6-DCBQ)

3GS+2,6-DCBQ-Cl-2H

2GS+2,6-DCBQ-Cl-H

GS+2,6-DCBQ-Cl-H

OH Cl

Cl

GS

10

11

19.3

22.3

Formation

2GS+2,6-DCBQ-2H

SG OH

GS+2,6-DCBQ-H

Reactant

(2,6-DCHQ)



No. Retention Name time (min) 12 13.2 2,6-diglutathionyl-

C26H34N6O14S2

(1,4)hydroquinone/

(2,6-DiSG-HQ)

benzoquinone

(2,6-DiSG-BQ)

2-monochloro-3,5-

C26H33ClN6O14S2


(2-MC-3,5-DiSG-HQ)


(2-MC-3,5-DiSG-BQ)

2-monochloro-6-

C16H18ClN3O8S

glutathionyl-(1,4)

(2-MC-6-SG-HQ)


(2-MC-6-SG-BQ)

13

14

16.2

17.3

Formula

Structure

Page 28 of 36

Formation 2GS+2,6-DCBQ-2Cl

2GS+2,6-DCBQ-Cl-2H

GS+2,6-DCBQCl-H


Page 29 of 36


Figure 1. The UHPLC-ToF chromatograms of the reaction mixtures of GSH and 2,6-DCBQ at different ratios. A) GSH:2,6-DCBQ = 0.1, B) GSH:2,6-DCBQ = 1, and C) GSH:2,6-DCBQ = 5.



Page 30 of 36


Page 31 of 36


Figure 2. The MS and MS/MS spectra of mono-, di-, tri-, and tetra-glutathionyl-benzoquinones: 1) 2,6-DC-SG-HQ, 2) 2-MC-3,6DiSG-HQ, 3) TriSG-HQ, and 4) TetraSG-HQ. 1-1, 2-1, 3-1 and 4-1 are the ToF MS spectra of the parent ions (blue line), in accordance with the theoretical isotope pattern of proposed [M-H]- or [M-2H]2- anions (red line); 1-2, 2-2, 3-2, and 4-2 are the dependent MS/MS spectra of the parent isotope with highest intensity.



Page 32 of 36

Figure 3. Semiquinone radical detected in the reaction between GSH and 2,6-DCBQ. Reactions were carried out in ddH2O and [2,6-DCBQ] = 1 mM. (A) [GSH] = 0, [GSH]:[2,6-DCBQ] = 0, pH = 6.8; (B) [GSH] = 10 µM, [GSH]:[2,6-DCBQ] = 0.01, pH = 6.6; (C) [GSH] = 30 µM, [GSH]:[2,6-DCBQ] = 0.03, pH = 6.5; (D) [GSH] = 50 µM, [GSH]:[2,6-DCBQ] = 0.05, pH = 6.4; (E) [GSH] = 100 µM, [GSH]:[2,6-DCBQ] = 0.1, pH = 6.2; (F) [GSH] = 300 µM, [GSH]:[2,6-DCBQ] = 0.3, pH = 6.2. The center peak was g = 2.00538.


Page 33 of 36


A

B

OCl

OH Cl

2GSH + e-

GSSG

Cl

Cl

O

OH e2e- + 2H+

O Cl

Cl

GSH Michael Addition O

OH

Cl

Cl H O [O] Cl 2

GS

GS O

GS

Cl SG Substitution of Cl

GSH Michael Addition OH

O OH GS

OH

Cl

O

SG

GS

GS

Cl

GS

Cl

GS

OH

O

OH

SG

Or

Cl [O] H2O Cl

GS

O

O Cl

GSH Michael Addition

Cl

SG Substitution of Cl

SG OH [O]

SG

[O]

H2O

O SG Substitution of Cl

H2O

O

GSH Michael Addition OH

O

O GS

Cl

O

OH

GS

Cl

GS GS

SG SG

GS GS

SG O


O

H2O SG Substitution of Cl

OH SG

H2O [O]

OH [O]

O

O

SG Substitution of Cl GS

SG

Cl

GS

SG


O GS

SG

GS O

SG

GS O

SG

GS OH



Page 34 of 36

Figure 4. Summary of the proposed reaction mechanism of GSH and HBQs. (A) Overall reactions involved in the conjugation of GSH on chlorinated HBQs. X is the substitution group, Cl, Br or CH3; a is the number of the substituted group, equal to 1, 2 or 3. (B) Proposed reaction pathways of HBQs with GSH, using 2,6-DCBQ as an example. [O] is the oxidant, which could be oxygen gas or a less glutathionylated quinone. The molecules in (B) identified by LC-MS/MS are described in Table 1.


Page 35 of 36


Figure 5. The ToF ion intensity of 2,6-DCBQ-GSH conjugates in 2,6-DCBQ treated cells as a function of exposure time. The error bars represent standard deviations of triplicate samples.



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TOC


Characterization of Mechanisms of Glutathione Conjugation with

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