Halobenzoquinone-Induced Alteration of Gene Expression Associated

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Ecotoxicology and Human Environmental Health

Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative Stress Signaling Pathways Jinhua Li, Birget Moe, Yanming Liu, and Xing-Fang Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06428 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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Environmental Science & Technology

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Halobenzoquinone-Induced Alteration of Gene Expression Associated with Oxidative

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Stress Signaling Pathways

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Jinhua Li,1,2* Birget Moe,2,3 Yanming Liu,2 and Xing-Fang Li2*

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1. Department of Health Toxicology, School of Public Health, Jilin University, Changchun, Jilin, China 130021 2. Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G3 3. Alberta Centre for Toxicology, Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1

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* Corresponding authors: Xing-Fang Li: [email protected], 1-780-492-5094; Jinhua Li:

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[email protected]

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ACS Paragon Plus Environment

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Abstract

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Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) that effectively induce

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reactive oxygen species and oxidative damage in vitro. However, impacts of HBQs on oxidative

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stress related gene expression have not been investigated. In this study, we examined alteration

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in the expression of 44 genes, related to oxidative stress-induced signaling pathways, in human

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uroepithelial cells (SV-HUC-1) upon exposure to six HBQs. The results show structure-

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dependent effects of HBQs on the studied gene expression. After 2 h exposure, the expression

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levels of 9 to 28 genes were altered, while after 8 h exposure, the expression levels of 29 to 31

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genes were altered. Four genes, HMOX1, NQO1, PTGS2, and TXNRD1, were significantly

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upregulated by all six HBQs at both exposure time points. Ingenuity pathway analysis revealed

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that the Nrf2 pathway was significantly responsive to HBQ exposure. Other canonical pathways

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responsive to HBQ exposure included GSH redox reductions, superoxide radical degradation,

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and xenobiotic metabolism signaling. This study has demonstrated that HBQs significantly alter

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gene expression of oxidative stress related signaling pathways and contributes to the

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understanding of HBQ-DBP-associated toxicity.

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TOC graphic

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Introduction Disinfection of drinking water is necessary to inactivate pathogenic microorganisms;

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however, it leads to the inadvertent formation of drinking water disinfection byproducts (DBPs)

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from the reaction(s) of disinfectants (e.g. chlorine, chloramine, chlorine dioxide, or ozone) with

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natural organic matter in source water.1 Human exposure to DBPs in drinking water is a concern

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driven by epidemiological findings that suggest a potential association between consumption of

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chlorinated drinking water and an increased risk of developing bladder cancer.2 Although eleven

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DBPs, including the trihalomethanes (THMs) and haloacetic acids (HAAs), are under regulation

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by the United States Environmental Protection Agency (US EPA), these regulated DBPs do not

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appear to account for the observed increased bladder cancer risk based on animal carcinogenicity

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studies.3

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Halobenzoquinones (HBQs) are an emerging class of DBPs that have been found to

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occur widely in treated tap water and recreational waters at levels up to 300 ng/L.4-6 HBQs have

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also been predicted as potential bladder carcinogens by quantitative structure–toxicity

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relationship (QSTR) analysis,7 though limited experimental data is available concerning the

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toxicological effects of HBQs. HBQs have shown greater cytotoxicity than regulated DBPs in

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Chinese hamster ovary (CHO) cells,8, 9 while also inducing cytotoxic effects in T24 human

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bladder carcinoma cells with half-inhibitory concentrations (IC50) at micromolar levels.

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Oxidative stress has been identified as one of the main mechanisms of HBQ-induced cytotoxicity

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due to production of intracellular reactive oxygen species (ROS), depletion of the cellular

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antioxidant glutathione (GSH), inhibition of cellular antioxidant enzymes, and oxidative damage

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to DNA and proteins.10-12 A comparison of HBQ structure and its biological effects identified

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isomeric structure and the number and identity of halogen substitutions as significant structural

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features of HBQs resulting in the observed differences in cytotoxicity, ROS generation, and

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genotoxicity.13 The results of these in vitro studies suggest that HBQs are likely to be genotoxic

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through oxidative stress-induced DNA damage, the extent of which can be influenced by HBQ

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structure; however, the molecular basis for this is unclear.

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Oxidative stress is one of the major contributors to the development of cancer.14-15 In

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response to increased levels of ROS and oxidative stress, several cell signaling pathways are

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involved to induce adaptive stress responses through modulation of stress–response genes.16 Of

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these, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway is one of the most


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important pathways to protect cells against oxidative stress.33-34 As a transcription factor, Nrf2

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regulates the expression of a series of antioxidant genes through its interaction with the

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antioxidant response element (ARE), which is located on the regulatory region of these genes.17

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Animal studies have shown that susceptibility to chemical-induced urinary bladder cancer

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significantly increased in Nrf2-deficient mice in comparison to wild-type mice.18 It has also been

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reported that the Nrf2 pathway was activated in human cells and in rats by other DBPs, such as

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HAAs.19-20 However, the effects of HBQ exposure on the expression of Nrf2 pathway-related

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genes have not yet been studied. The participation of the Nrf2 signaling pathway and the

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response of Nrf2-regulated genes to HBQ exposure remain unclear.

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Microarrays are a useful tool to identify multiple pathways or monitor whole gene

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expression simultaneously after exposure to environmental toxicants.21 In addition, TaqMan

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probe-based real-time PCR arrays are useful to provide quantitative data on gene expression.22

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The TaqMan PCR array method has been successfully demonstrated to study the potential

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toxicity pathways of some DBPs, such as HAAs,19, 23-26 bromate,27-28 and 3-chloro-4-

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(dichloromethyl)-5-hydroxy-5H-furan-2-one (MX).29 Therefore, we propose to use this method

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to examine the impact of HBQs on gene expression.

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The objective of this study was to demonstrate the impact of HBQs on gene expression,

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particularly on the genes associated with oxidative stress signaling pathways. To achieve this, we

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used a customized TaqMan Array to investigate the changes in expression of genes related to

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oxidative-stress induced signaling pathways in human uroepithelial cells exposed to HBQs. A

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panel of 44 functional genes were selected because they are associated with identified HBQ-

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induced cellular effects. These genes cover nine functional gene groups, including transcription

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factors, the GSH production/utilization pathway, the thioredoxin system pathway, stress

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responsive genes, antioxidant genes, membrane transporters, cell cycle regulation, and oxidative

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DNA damage repair. We selected an immortalized, non-tumorigenic human urinary tract

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epithelial cell line, SV-HUC-1, for analysis, as it is a commonly used bladder tissue model.30 The

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use of this cell line will provide more relevant information on gene expression comparison to

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data obtained from human cancer cell lines. We will also compare the difference in gene

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response between HBQ isomers to identify the molecular effects that underlie the different

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biological responses observed in vitro.13 Thus, this study will provide insights into the molecular

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basis of antioxidant defense against HBQ-induced oxidative stress in human uroepithelial cells.


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Materials and Methods

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Reagents. Standards of 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,5-dichloro-1,4-

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benzoquinone (2,5-DCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ) were purchased

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from Sigma-Aldrich (St. Louis, MO, USA). 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ)

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and 2,3,6-trichloro-1,4-benzoquinone (TriCBQ) were purchased from Shanghai Acana

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Pharmtech (Shanghai, China), while 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ) was purchased

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from Indofine Chemical Company (Hillsborough, NJ, USA). The chemical structure, molecular

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weight, and purity of these HBQs are included in Table S1 of the Supporting Information. HBQ

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stock solutions were prepared by dissolving each HBQ in 100% methanol (HPLC grade; Fisher

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Scientific, Ottawa, ON, Canada) and were stored at -20°C in sterile amber glass vials.

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Cell culture. The human uroepithelial cell line, SV-HUC-1, was obtained from American Type

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Culture Collection (ATCC® CRL9520™; Manassas, VA, USA). The cells were maintained in

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Kaighn's modification of Ham's F-12 (F-12K) medium (ATCC) containing 10% fetal bovine

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serum (FBS; Sigma-Aldrich) and 1% penicillin/streptomycin (100 U/100 µg/ml; Invitrogen,

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Burlington, ON, Canada) at 37°C in a 5% CO2 incubator.

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HBQ-induced cytotoxicity and detection of reactive oxygen species (ROS). The cytotoxicity

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of each HBQ was examined using real-time cell analysis (RTCA; ACEA Biosciences, San

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Diego, CA, USA). ROS formation was determined using 2’, 7’-dichlorofluorescein diacetate

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(DCFH-DA) (Sigma-Aldrich), a commonly used fluorogenic dye for the quantification of

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intracellular ROS generation.31 The detailed procedures for these experiments are described in

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the Methods section of the Supporting Information.

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Real-time reverse transcription (RT)-PCR for time-course gene expression analysis. SV-

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HUC-1 cells were seeded into 6-well plates (Corning Costar, Fisher Scientific) at a density of 1 ×

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106 cells per well. After 24 h growth, cells were treated with each HBQ (2,6-DCBQ, 2,5-DCBQ,

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2,6-DBBQ, 2,5-DBBQ, TriCBQ, DCMBQ) at concentrations of equivalent biological response

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(IC20). At each indicated exposure time point (0.5, 1, 2, 4, 6, 8, and 24 h), cells were harvested

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for RNA extraction. Total RNA was extracted using TRIzol (Invitrogen) according to the

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manufacturer’s instructions. The resultant DNA-free RNA samples were quantified with a

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NanoVue Plus™ spectrophotometer (Biochrom, Cambridge, UK) and stored at -80°C. RNA

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integrity number (RIN) values were determined using an Agilent 2100 Bioanalyzer (Santa Clara,


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CA, USA), and RNA purity was determined by the A260/A280 ratio. Only RNA with a ratio within

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the range of 1.8-2.0 was reverse transcribed into cDNA using SuperScript™ III Reverse

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Transcriptase (Invitrogen) with random primers (Invitrogen) according to the manufacturer’s

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instructions. The collected cDNA samples were stored in sterile microcentrifuge tubes at -20°C.

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The cDNA was used as a template for real-time quantitative PCR using the Fast SYBR®

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Green PCR kit (Applied Biosystems, Carlsbad, CA, USA). The primers were synthesized by

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Integrated DNA Technologies (Coralville, IA, USA), and the sequences are presented in Table

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S2. Real-time fluorescence detection was performed using a StepOnePlus™ Real-Time PCR

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system (Applied Biosystems). The detailed protocols for these experiments are described in the

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Methods section of the Supporting Information.

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Two representative genes, NFE2L2, which encodes the transcription factor Nrf2, and

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NADPH:quinone oxidoreductase 1 (NQO1), a target gene of Nrf2, were used to select the most

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informative exposure time points for the array experiments. Gene expression was determined

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using cycle threshold (Ct) values. A comparative Ct method was used for relative quantification.

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The Ct values were first normalized to the housekeeping gene glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH) present in the same samples (∆Ct) and were then expressed as the

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fold-change over concurrent negative control groups (no HBQ treatment; culture medium)

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prepared at each treatment time point (2-∆∆Ct). Duplicate samples were used in individual

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experiments, and three independent experiments were performed (n = 3).

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TaqMan Array for pathway gene expression analysis. After 2 h and 8 h HBQ exposure at

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concentrations equal to their respective IC20 value (2,6-DCBQ, 2,5-DCBQ, 2,6-DBBQ, 2,5-

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DBBQ, DCMBQ, TriCBQ), total RNA was isolated and reverse transcribed to cDNA as

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described previously. A customized TaqMan Array in a 96-well fast plate was synthesized by the

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manufacturer (Thermo Scientific, Waltham, MA) using the plate format shown in Figure S1.

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Table S3 describes the 44 genes selected for the array, categorized into nine functional gene

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groups, including transcription factors, the GSH production/utilization pathway, the thioredoxin

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system pathway, stress responsive genes, antioxidant genes, membrane transporters, cell cycle

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regulation, and oxidative DNA damage repair. Four reference control genes [18S ribosomal

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RNA (18S rRNA), GAPDH, hypoxanthine phosphoribosyltransferase 1 (HPRT1), β-

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glucuronidase (GUSB)] and 44 target genes were included in duplicate on each 96-well plate.

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Real-time PCR analysis was conducted using a StepOnePlus™ Real-Time PCR system.


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Additional details regarding the protocol for these experiments are described in the Methods

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section of the Supporting Information. The comparative Ct method was used for relative

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quantification. The Ct values were first normalized to the four reference control genes in the

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same samples (∆Ct) and expressed as the fold-change over the concurrent negative control

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groups (no HBQ treatment; culture medium) prepared at each treatment time point (2-∆∆Ct)..

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Duplicate samples were analyzed in each replicate experiment. Two independent experiments of

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the gene expression array were completed (n = 2).

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Ingenuity pathway analysis (IPA). Briefly, the genes with significantly altered expression were

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uploaded into the IPA software (www.ingenuity.com) for canonical pathway analysis, which was

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based on the IPA library of canonical pathways. The significance of the association between our

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gene list and a canonical pathway was measured by Fisher's exact test.

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Data analysis. Data are expressed as the mean ± SEM or the mean ± SD. CI values were

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normalized by the RTCA software (ACEA Biosciences) and were used to fit dose–response

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curves to obtain the half inhibitory concentrations, IC50, and 20% inhibitory concentrations, IC20,

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for each HBQ at specific time points. Student’s t-test was performed to determine the statistical

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significance of the gene expression between negative control groups and HBQ treatment groups.

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Fitting of dose-response curves and statistical analysis were conducted using GraphPad Prism

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5.0 software (San Diego, CA). Associations were considered statistically significant at P < 0.05.

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Results and Discussion

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Optimization of experimental conditions for the TaqMan Array. To obtain reliable gene

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expression profiles, we first optimized the conditions for several key experimental parameters,

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including the treatment concentrations of the HBQs and the exposure time. An optimized

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treatment concentration was identified as one that exhibits low cytotoxicity, but generates

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sufficient amounts of intracellular ROS to elicit a measurable biological response, specifically

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the induction of oxidative stress and the subsequent activation of the Nrf2 pathway. To select

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low cytotoxic concentrations of each HBQ, we used RTCA to continuously monitor the response

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of SV-HUC-1 cells to different concentrations of HBQs for 80 h. Figure S2 shows the

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cytotoxicity response profiles for SV-HUC-1 cells exposed to each HBQ. The response profiles

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clearly show that HBQ-induced cytotoxicity in SV-HUC-1 cells is concentration-dependent.

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From the concentration–response data, we determined IC20 values, which are concentrations


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eliciting a 20% decrease in normalized cell index values in comparison to the untreated control

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cells (no HBQ treatment; culture medium). The IC20 values, therefore, exhibit low cytotoxicity

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and allow for a comparison between HBQ-induced effects based on equivalent concentrations of

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biological response. The 24-h IC20 values (mean ± SD) were determined to be 13 ± 2.3 µM (2,6-

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DCBQ), 17 ± 1.1 µM (2,5-DCBQ), 10 ± 1.4 µM (2,6-DBBQ), 12 ± 0.9 µM (2,5-DBBQ), 17 ±

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2.9 µM (DCMBQ), and 24 ± 3.3 µM (TriCBQ). When SV-HUC-1 cells were pre-treated with 1

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mM N-acetyl-L-cysteine (NAC) prior to HBQ treatment, higher concentrations of HBQs were

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needed to induce a full concentration–response (Figure S3) and resulted in 24, 48, and 72 h IC50

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values that were significantly higher than the IC50 values of HBQs in cells without NAC

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pretreatment (Figure S4). Because NAC can function as either a source of thiol in cells or as an

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antioxidant to scavenge ROS directly,32 these results demonstrate HBQ-induced oxidative stress

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in SV-HUC-1 cells, which is consistent with results from T24 human bladder carcinoma cells.10

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To confirm that a treatment concentration equal to the IC20 value for each HBQ can

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generate sufficient ROS to elicit a measurable biological response in the TaqMan Array, we

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measured intracellular ROS levels after HBQ treatment from 0.5 h to 72 h. Figure S5

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summarizes the time-course of cellular ROS generation after each HBQ treatment at their

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respective IC20 values. All six HBQs share a similar temporal trend of ROS production: ROS

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production increases from 0.5h, peaks at 8h, and then slowly decreases over the remainder of the

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exposure period. For all time points examined, ROS generation by each HBQ was statistically

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greater than the concurrent negative control group (one-way ANOVA, P < 0.05). This is

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consistent with a study of the quinone compound menadione, where ROS production increased

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over time until peaking at 8 h after treatment in HepG2 cells.33 Based on our RTCA and ROS

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results, the IC20 values are an optimal testing concentration for the TaqMan Array.

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To select the optimal exposure time points to represent both early and late responses

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within the Nrf2 pathway, we examined the time-dependent effects of HBQ treatment on the

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expression of two representative genes. The two genes examined were NFE2L2, representing the

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transcription factor Nrf2, and NQO1, representing a target gene of Nrf2. RNA integrity was

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assessed to ensure its high quality prior to RT-PCR. Therefore, only RNA with high RIN values

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were used (Figure S6; RIN > 9, Figure S7). Exposure to 24-h IC20 concentrations of each HBQ

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resulted in an initial increase in NFE2L2 expression during the first 2 h of exposure, with a

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subsequent decrease in expression after 4 h exposure (Figure S8A). Because 2 h exposure to each


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HBQ significantly increased NFE2L2 expression (P < 0.05), it was chosen as the early response

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time point for our array experiments. Each HBQ also increased the Nrf2 downstream target gene,

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NQO1, in a time-dependent manner (Figure S8B). The expression of NQO1 increased gradually

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from 2 h, peaked at 8 h, and subsequently decreased at 24 h, guiding our selection of 8 h as the

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late response time for our array experiments. Thus, in the following experiments, we analyzed

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the gene expression changes in SV-HUC-1 cells under the optimized conditions of 2 h and 8 h

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HBQ treatment at concentrations equal to their respective 24-h IC20 values.

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Modulation of oxidative-stress induced pathways by HBQs. The 44 genes selected for our

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customized TaqMan Array focus on the Nrf2 signaling pathway due to its significant impact on

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the ability of cells to protect against oxidative stress, the main mechanism of HBQ-induced

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cytotoxicity. Additional genes were also selected that underlie known HBQ-induced effects,

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including oxidative DNA damage10, 12 and p53 upregulation13, 34. Figure 1 summarizes the

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relationships between the selected genes and known HBQ-induced effects alongside key

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oxidative stress-induced pathways. Hence, the modulation of these genes after HBQ exposure

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will reveal the molecular basis of HBQ-induced responses observed in vitro. Under the

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optimized experimental conditions, each HBQ altered the transcription of multiple genes at the

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early and late exposure time points, including significant alterations in genes from all nine

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functional gene groups. Table S4 lists the genes that exhibited significant changes in expression

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(P < 0.05) after HBQ exposure. After 2 h exposure, each HBQ altered expression levels in 9 to

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28 genes, while after 8 h exposure, each HBQ altered expression levels in 29 to 31 genes. As

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predicted, all six HBQs upregulated NFE2L2, the gene which encodes Nrf2, indicating that

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HBQs are able to activate the Nrf2 signaling pathway. This has also been shown previously in an

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ARE-dependent β-lactamase-based reporter gene assay, which indicated that both 2,6-DCBQ and

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2,6-DBBQ activated Nrf2 with a similar level of potency.34 Interestingly, in this study, we

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observed that the modulation of NFE2L2 was time-dependent. While 2,5-DCBQ and 2,5-DBBQ

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upregulated NFE2L2 at the late exposure time point, the remaining four HBQs exhibited

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upregulation at the early exposure time point. The ability of compounds to activate the Nrf2

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pathway has been shown to correlate with their reactivity toward thiol groups of Kelch-like

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ECH-associated protein-1 (Keap1) cysteine residues, which inhibit the ubiquitination of Nrf2.

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This indicates that the structure of HBQs may influence their ability to activate Nrf2, which is

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supported by evidence from tert-butylhydroquinone (tBHQ) and 1,2-naphthoquinone.35


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Furthermore, because HBQs readily bind to thiols in GSH with differing affinity,36 it is likely

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they could exhibit differing reactivity toward the thiol groups of Keap1, affecting their ability to

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activate Nrf2.

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The main targeted genes of the Nrf2 signaling pathway are antioxidant genes and stress

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responsive genes. While previous studies have indicated activation of Nrf2 by HBQs, no

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evidence has been presented that demonstrates downstream activation of genes regulated by Nrf2.

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The selected genes in these functional groups may indicate how SV-HUC-1 cells detoxify HBQs

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and/or its induced ROS, or mount an inflammatory response. NQO1 is a detoxification enzyme

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specific to quinones, catalyzing a two-electron reduction of quinones to their less toxic

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hydroquinone form, a major detoxification pathway of quinones.37 As expected, NQO1 was

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upregulated in SV-HUC-1 cells exposed to any of the six HBQs. Alternatively, N-

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acetyltransferase 1 (NAT1) and NAT2 encode N-acetyl-transferases that promote xenobiotic

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metabolism via transfer of an acetyl group.38 No consistent pattern in expression of NAT1 or

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NAT2 was observed across the six HBQs, but overall, both genes were downregulated, indicating

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that this pathway is not likely a defense against HBQs as is the activation of NQO1.

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HBQs are potent inducers of intracellular ROS.9 Superoxide dismutase (SOD) catalyzes

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the conversion of superoxide to hydrogen peroxide (H2O2) or O2. At 8 h exposure, SOD1 and

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SOD2 were significantly upregulated by 2,5-DCBQ and 2,5-DBBQ, but downregulated by 2,6-

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DBBQ and TriCBQ. Thus, it is possible that the 2,5-HBQs induce higher amounts of superoxide

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than the other HBQs. Also, the downregulation of SOD could possibly be in response to the

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upregulation of other superoxide antioxidants in 2,6-DBBQ- or TriCBQ-treated cells, rendering

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SOD obsolete. The clearest evidence for the production of a specific type of ROS by all six

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HBQs relates to the expression of antioxidant genes related to the detoxification of H2O2. The

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genes regulating catalase (CAT), cytoglobin (CYGB), glutathione peroxidase (GPX2), and

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prostaglandin-endoperoxide synthase 2 (PTGS2) were all significantly upregulated by each HBQ

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at 8 h exposure, suggesting these four genes may coordinate to eliminate H2O2. The genes

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associated with the heme peroxidase superfamily, lactoperoxidase (LPO) and myeloperoxidase

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(MPO), were not modulated by HBQ exposure.

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The oxidant/antioxidant balance within cells is tightly controlled, with oxidants playing a

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key role in cellular processes such as metabolism and cell signaling. The induction of oxidative

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stress can affect the modulation of oxidants in addition to the more commonly assayed


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antioxidants. NADPH oxidase (NOX) proteins are the main non-mitochondrial source of ROS

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within cells, generating superoxide.39 Both NOX5, encoding NOX5, and cytochrome b-245 beta

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chain (CYBB), encoding NOX2, were modulated by HBQ exposure. Hence, it is possible that

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superoxide radicals produced by HBQ-induced upregulation of CYBB could result in the

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concomitant downregulation of NOX5, balancing the production and removal of superoxide

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radicals after exposure to low doses of HBQs. It is clear, however, that other genes are involved

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to maintain this balance, as the four HBQs most strongly upregulating CYBB, 2,5-DCBQ (17-

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fold increase), 2,6-DBBQ (14-fold increase), 2,5-DBBQ (20-fold increase), and TriCBQ (14-fold

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increase), also exhibited differing modulation of the SOD genes (SOD1, SOD2). A more

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comprehensive screen of genes associated with the oxidative balance of superoxide is necessary

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to understand these findings.

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Inflammation is a complex cellular response that is closely linked to xenobiotic-induced

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oxidative stress.40 Hemeoxygenase-1 (HMOX1) encodes the hemeoxygenase-1 enzyme (HO-1),

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which is able to cleave heme to produce carbon monoxide (CO), biliverdin, and Fe(II).41-42

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Among the 44 genes screened in our panel, HMOX1 exhibited the highest upregulation, with

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expression levels 7- to 50-fold greater after 8 h HBQ exposure and 5- to 10-fold higher after 2 h

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HBQ exposure. The significant increase of HMOX1 expression suggests that iron regulation may

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play a role in the detoxication of HBQs by controlling intracellular levels of superoxide with

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SOD. In addition, the protective role of HMOX1 against oxidative stress is believed to be

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through its product CO, which is an important signaling molecule in anti-inflammatory

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pathways.43-44 The PTGS2 gene is also involved in the anti-inflammatory process, although it is

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not modulated by Nrf2. PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyzes the

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conversion of arachidonic acid to prostaglandin H2 (PGH2) and further to prostaglandins (PGs),

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a primary component of the anti-inflammatory process.45 The upregulation of both HMOX1 and

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PTGS2 suggests a possible pro-inflammatory response in SV-HUC-1 cells after HBQ exposure.

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This is significant, as inflammation has been linked to the development of every stage of cancer40

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and to the development of different types of cancer, including bladder cancer.46 Furthermore, the

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induction of COX-2 itself has been linked to the development of bladder cancer,47-48 which is the

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target organ of DBP exposure identified in several epidemiological studies.

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The most prominent thiol-dependent antioxidant systems present in cells are the GSH and

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thioredoxin systems,49 both of which contain targeted genes in the Nrf2 signaling pathway. After


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8 h exposure, all six HBQs induced a two-fold upregulation of glutamate–cysteine ligase

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modifier subunit (GCLM), glutamate–cysteine ligase catalytic subunit (GCLC), and glutathione

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reductase (GSR). GCLM and GCLC are composed of glutamate cysteine ligase (GCL), which is

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the rate-limiting enzyme for GSH synthesis.50-51 GSR is responsible for the reduction of oxidized

328

GSH to reduced GSH.51 Thus, it is clear from the upregulation of these genes that Nrf2 increased

329

the synthesis of GSH in response to HBQ-induced GSH depletion, as the ability of HBQs to

330

deplete and/or conjugate GSH has been demonstrated in HBQ-treated cells.11, 36 The conjugation

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of GSH to xenobiotics is catalyzed by glutathione transferase (GST). In our study, GSTT1,

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GSTZ1, and GSTP1 were mostly downregulated, although GSTP1 was slightly upregulated in

333

four HBQs after 2 h exposure. Because HBQ-GSH conjugation occurs rapidly in vitro, the

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overall downregulation of GST isoforms implies that HBQ-GSH conjugation in cells may be

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mostly non-enzymatic.

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Although HBQ-GSH interactions have been well-characterized, the ability of HBQs to

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react with other thiol groups in cells is unclear. The thioredoxin system is composed of

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thioredoxin (TXNRD), peroxiredoxin (PRDX), and sulfiredoxin (SRXN). After 2 h HBQ

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exposure, the gene expression levels of thioredoxin reductatse 1 (TXNRD1) increased 2- to 3-fold,

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and increased 2- to 5-fold after 8 h exposure to each HBQ. TXNRDs are a class of enzymes that

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reduce oxidized thioredoxin to reduced thioredox.52 Because the altered transcription of TXNRD1

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is affected by all six HBQs at both exposure time points, thioredoxin and TXNRD1 may be

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sensitive targets for HBQs in uroepithelial cells, providing the first evidence that demonstrates

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that HBQs can affect the thioredoxin system. SRXN1 was also upregulated at 2 h (2,6-DBBQ,

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TriCBQ and DCMBQ) and 8 h exposure (2,6-DCBQ, 2,5-DCBQ, 2,5-DBBQ, and DCMBQ).

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Benzoquinone-induced induction of SRXN1 was also observed in rodent and human cells

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exposed to tBHQ.53, 54 Because SRXN proteins re-activate PRDX proteins,55 an upregulation in

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PRDX genes would be expected. However, we found that most of the PRDX genes were

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unaffected or downregulated after HBQ exposure.

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The multi-drug resistance proteins (MRPs) have been shown to protect cells from HBQ-

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induced cytotoxicity and oxidative stress in cell models overexpressing MRP.56 The genes that

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encode the MRPs, the ABCC genes, are target genes of Nrf2. After 8 h HBQ exposure, ABCC2

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was significantly upregulated by five of the HBQs (not DCMBQ), indicating that Nrf2 may

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control the induction of ABCC2 (MRP2) to increase efflux of HBQs or HBQ-GSH conjugates.


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However, from our results, it is clear that the expression of ABCC genes varied widely amongst

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the different HBQs, indicating that individual HBQs may affect MRP expression differently.

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This could have important toxicodynamic implications for HBQs, as the MRPs are known to

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affect xenobiotic efflux and distribution.57

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Two genes included in our 44-gene panel that are not regulated by Nrf2, but are

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associated with HBQ-induced effects, are 8-oxoguanine glycosylase (OGG1) and cyclin-

361

dependent kinase inhibitor 1 (CDKN1A). In response to DNA damage, cells can directly repair

362

the damaged DNA or stop proliferation through cell cycle arrest. Interestingly, the DNA damage

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repair gene, OGG1, was downregulated by all six tested HBQs at 8 h exposure. HBQs have been

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shown to increase levels of 8-hydroxydeoxyguanosine (8-OHdG),10, 13 a key biomarker of

365

oxidative DNA damage,58 which is primarily repaired through the DNA base excision repair

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(BER) pathway by 8-oxoguanine DNA glycosylase, encoded by OGG1.59 Therefore, HBQs

367

could induce oxidative DNA damage (8-OHdG), while also decreasing DNA repair capacity.

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This phenomena has also been observed with estrogen, with estrogen-mediated oxidative stress

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suggested to play an important role in estrogen-induced breast carcinogenesis.60 Because the

370

decreased expression of OGG1 has been shown to be associated with tumor development,61 these

371

findings suggest the importance of examining the carcinogenic potential of HBQs.

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CDKN1A, regulated by p53, encodes the cyclin-dependent kinase inhibitor 1 (p21)

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protein, which regulates progression of the cell cycle at G1 and S phases.62 CDKN1A was

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significantly upregulated by all six HBQs at 2 h exposure, and by most of the HBQs at 8 h

375

exposure, indicating that SV-HUC-1 cells may activate the p53 pathway to promote p21-

376

regulated cell cycle arrest to protect against HBQ-induced DNA damage. This is consistent with

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several recent reports. A significant increase in p53 protein expression was detected in CHO

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cells treated with HBQs (2,5-DBBQ, 2,6-DCBQ, and 2,5-DCBQ) after 24 h exposure,13 while

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activation of the p53 pathway after 2,6-DCBQ and 2,6-DBBQ exposure in Caco-2 cells was also

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detected using a β-lactamase reporter system.34 Furthermore, cell cycle arrest was induced by

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2,6-DCBQ and 2,6-DBBQ exposure in human neural stem cells.63 It is also possible that p21 can

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activate the Nrf2 signaling pathway to protect cells from oxidative stress by releasing Nrf2 from

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its inhibitor Keap1.64-65 Taken together, p53 may be activated by HBQs to induce p21 to regulate

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cell cycle arrest, while p21 may also simultaneously activate the Nrf2 pathway to protect against

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HBQ-induced oxidative stress.


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Differences of transcriptome changes between HBQ isomers. 2,5-HBQs have been shown to

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induce higher cytotoxicity and greater ROS production than their corresponding 2,6-HBQ

388

isomers.13 Although this difference has been associated with HBQ structure, as dipole moment is

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correlated with HBQ isomer reactivity and resulting toxicity, determining the underlying

390

differences in biological activity between isomer pairs may help to further elucidate the

391

differences in cellular response that have been observed. Figure 2 shows the difference in gene

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expression between the HBQ isomer pairs in SV-HUC-1 cells. Interestingly, 2,6-DCBQ and 2,6-

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DBBQ both upregulate NFE2L2, while 2,5-DCBQ and 2,5-DBBQ both downregulate Keap1 at

394

the 2 h exposure time point. This indicates that at the earlier exposure time, both the 2,6- and 2,5-

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HBQ isomers are able to activate the Nrf2 signaling pathway; however, it appears that this

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activation is achieved in different ways. Tetrachlorobenzoquinone has also been observed to

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activate NFE2L2, but not Keap1, in HepG2 cells.66 Another interesting finding among the HBQ

398

isomers is that the 2,6-HBQs significantly increased the expression levels of GPX2 and CAT at 2

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h, while the 2,5-HBQs did not. Because GPX2 and CAT encode proteins responsible for reducing

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H2O2, the greater expression of these two genes could decrease the cellular ROS induced by 2,6-

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HBQs. This could explain the greater toxicity induced by 2,5-HBQs in comparison to their 2,6-

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HBQ isomers. The third and most important finding was that 2,5-DCBQ and 2,5-DBBQ induced

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significantly higher expression levels of nine genes (2 h: HMOX1; 8 h: GCLC, GSR, TXNRD1,

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CYBB, CYGB, OGG1, HMOX1, SOD1, SOD2) than their corresponding 2,6-HBQ isomers (P

Halobenzoquinone-Induced Alteration of Gene Expression Associated

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