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TIV 2508 12 November 2010 1 No. of Pages 9, Model 5G Toxicology in Vitro xxx (2010) xxxxxx Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit 2 Essential role of Nrf2 in protection against hydroquinone3 and benzoquinone-induced cytotoxicity 4 Valentina Rubio a,b, Jiawei Zhang b,1, Mahara Valverde a, Emilio Rojas a,, Zheng-Zheng Shi b 5 a Departamento de Medicina Genmica y Toxicologa Ambiental, Instituto de Investigaciones Biomdicas, Universidad Nacional Autnoma de Mxico, Ciudad Universitaria, 6 04510 Mxico D.F., Mexico 7 b The Methodist Hospital Research Institute, Department of Radiology, The Methodist Hospital, Houston, TX 77030, United States 8 article info 21 30 11 Article history: 12 Received 9 August 2010 13 Accepted 29 October 2010 14 Available online xxxx 15 Keywords: 16 Hydroquinone 17 Benzoquinone 18 Nrf2 19 ARE-driven 20 Apoptosis 21 Glutathione 22 abstract Benzene is a well-established human carcinogen. Benzene metabolites hydroquinone (HQ) and benzoquinone (BQ) are highly reactive molecules capable of producing reactive oxygen species and causing oxidative stress. In this study, we investigated the role of the Nrf2, a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes, in defense against HQ- and BQ-induced cytotoxicity in cultured human lung epithelial cells (Beas-2B). When the cells were exposed to HQ or BQ the activity of an ARE reporter was induced in a dose-dependent manner, meanwhile Nrf2 protein levels were elevated and accumulated in the nucleus. Increased expression of well-known Nrf2-dependent proteins including NQO1, GCLM, GSS and HMOX was also observed in the HQ/BQ-treated cells. Moreover, transient overexpression of Nrf2 conferred protection against HQ- and BQ-induced cell death, whereas knockdown of Nrf2 by small interfering RNA resulted in increased apoptosis. We also found that the increased susceptibility of Nrf2-knockdown cells to HQ and BQ was associated with reduced glutathione levels and loss of inducibility of ARE-driven genes, suggesting that deficiency of Nrf2 impairs cellular redox capacity to counteract oxidative damage. Altogether, these results suggest that Nrf2-ARE pathway is essential for protection against HQ- and BQ-induced toxicity. 2010 Published by Elsevier Ltd. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 1. Introduction gaseous chemicals consisting primarily of nitrogen oxides (NOX), 46 carbon monoxide (CO), sulfur dioxide (SO2), ozone (O3), and vola- 47 43 Air pollution is a worldwide problem and has become a major tile organic compounds (VOCs) (Ferm et al., 2006; Han and Naeher, 48 44 environmental health issue (Chen et al., 2007; WHO, 2005). Air 2006). In recent years, due to increasing emissions from outdoor 49 45 pollution is defined as a mixture of particulate matter (PM) and (Riediker et al., 2003; Rodolfo Sosa et al., 2009) and indoor sources 50 (Carrer et al., 2000), and individual activities (e.g. smoking) (Ser- 51 rano-Trespalacios et al., 2004), populations in large urban areas 52 Abbreviations: ARE, antioxidant response element; BQ, benzoquinone; CO, carbon monoxide; ED50, effective dose 50; GCLM, glutamate cysteine ligase are exposed to high levels of VOCs (Tovalin et al., 2006), predominantly, monocyclic aromatic hydrocarbons, in particular benzene, 53 54 modifier subunit; GSH, glutathione; GSS, glutathione synthetase; GSTs, glutathione-S-transferases; HMOX1, heme oxygenase 1; H2O2, hydrogen peroxide; HQ, hydroquinone; OH, hydroxyl radical; Keap1, Kelch-like ECH-associated protein 1; Maf, musculoaponeurotic fibrosarcoma oncogene; MTS, [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; toluene, ethylbenzene, and isomers of xylene (m-, o-, p-xylene) (Tovalin-Ahumada and Whitehead, 2007; Tovalin et al., 2006). Exposure to benzene has been associated with aplastic anemia, leukemia and lymphoma (Snyder, 2002; Yin et al., 1996). Besides 55 56 57 58 NQO1, NAD(P)H dehydrogenase quinone 1; NOX, nitrogen oxides; Nrf2, nuclear factor (erythroid-derived 2)-like 2; O3, ozone; ROS, reactive oxygen species; siRNA, short interference RNA; SO2, sulfur dioxide; O2 , superoxide; UGTs, UDP-glucuronosyltransferases; VOCs, volatile organic compounds. Corresponding author. Address: Departamento de Medicina Genmica y Toxicologa Ambiental, Instituto de Investigaciones Biomdicas, Universidad Nac- its oncogenic effect on hematopoietic tissue, recent studies have correlated benzene exposure with tumor formation in human (Yin et al., 1996) and animal (Maltoni et al., 1989; Snyder et al., 1988) lungs, indicating that the lung is also a target of benzene-induced toxicity. Benzene toxicity is attributed to its metabolism, 59 60 61 62 63 ional Autnoma de Mxico, Coyoacn, Apdo. Postal 70228, Cdigo Postal 04510 Mxico D.F., Mexico. Tel.: +52 55 56 22 9177; fax: +52 55 55 50 0048. E-mail address: emilior@servdor.unam.mx (E. Rojas). 1 Present address: Cancer Institute (National Ministry of Education Key Laboratory of Cancer Prevention and Intervention) the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China. mainly in the liver (Koop et al., 1989; Nedelcheva et al., 1999; Ross, 2000; Snyder et al., 1989) and probably in the lungs (Powley and Carlson, 2000, 2001, 2002; Sheets et al., 2004), which leads to the formation of reactive metabolites hydroquinone (1,4-benzenediol or 1,4-hydroquinone; HQ) and its oxidized form benzoquinone 64 65 66 67 68 0887-2333/$ - see front matter 2010 Published by Elsevier Ltd. doi:10.1016/j.tiv.2010.10.021 Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 No. of Pages 9, Model 5G 2 V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 69 (1,4-benzoquinone or p-benzoquinone; BQ). HQ and BQ are highly streptomycin/ml (Invitrogen Corporation, Carlsbad CA) at 37 C in 131 70 reactive molecules and, through redox cycling, they produce reac- a 5% CO2 incubator. 71 tive oxygen species (ROS) (Bolton et al., 2000), including superox- 132 72 ide O2 , hydrogen peroxide (H2O2), nitric oxide (NO) and 2.2. Plasmids and transient transfections 73 ultimately hydroxyl radical OH, resulting in oxidative stress 133 74 (Luo et al., 2008; Snyder and Hedli, 1996) and oxidative damage The plasmids pcDNA-Nrf2-V5 (referred as Nrf2-V5) (Jain et al., 134 75 to DNA (Abernethy et al., 2004; Luo et al., 2008), proteins, and lip- 2005) and pGL2B-NQO1-ARE-LUC (referred as ARE-LUC) (Dhaksh- 135 76 ids (Gut et al., 1996; Winn, 2003). Moreover, addition of antioxi- inamoorthy and Jaiswal 2000) were a kind gift from Dr. Anil Jaiswal 136 77 dant enzymes (e.g. catalase) and N-acetyl cysteine, a glutathione (University of Maryland). Transient transfection of Beas-2B cells 137 78 precursor (GSH), has been shown to block oxidative damage in- were carried out using Lipofectamine 2000 following the manufac- 138 79 duced by these metabolites (Barreto et al., 2009; Ruiz-Ramos turer's instructions (Invitrogen Corporation, Carlsbad CA). Briefly, 139 80 et al., 2005) confirming the role of ROS production and oxidative the cells were seeded in 6-well plates at a density of 140 81 stress in HQ and BQ cytotoxicity. 3 105 cells/well (>90% confluence). Twenty-four hours after plat- 141 82 To counteract damage induced by oxidative stress, cells have ing, cells were transfected with 4 lg of either the above plasmids 142 83 developed an adaptive defense mechanism that leads to rapid or the pcDNA 3.1 empty vector diluted in Opti-MEM media (Invit- 143 84 and efficient induction of detoxifying enzymes (phase II enzymes) rogen Corporation, Carlsbad CA). 144 85 and antioxidants (Kang et al., 2005). Induction of these molecules 86 is through a cis-acting element in the promoter region known as 2.3. siRNA transfection 145 87 the antioxidant response element (ARE) (Lee et al., 2005). The nu- 88 clear factor (erythroid-derived 2)-like 2 (Nrf2), a basic leucine zip- Control siRNA (50-UAACGACGCGACGACGUAATT-30 and 50-UUA 146 89 per member of the cap `n' collar family of transcription factors CGUCGUCGCGUCGUUATT-30) and siRNA targeting human Nrf2 siR- 147 90 (Shen et al., 2004), is the principal regulator of the ARE-driven cel- NA (Lee et al., 2008) (50-GCUUUUGGCGCAGACAUUCTT-30 and 50- 148 91 lular defense system. Under homeostatic conditions, Nrf2 is pres- GAAUGUCUGCGCCAAAAGCTG-30) were obtained from Ambion 149 92 ent in the cytoplasm attaching to an actin-binding protein named Inc. (Austin, TX). Beas-2B cells were transiently transfected with 150 93 Kelch-like ECH-associated protein 1 (Keap1) (Lee et al., 2007). 100 nM of control siRNA or Nrf2 siRNA mixed with DharmaFect1 151 94 Keap1 functions as a suppressor of Nrf2 by retaining it in the cyto- Transfection Reagent (Dharmacon, Lafayette, CO) according to the 152 95 sol and enhancing its proteasomal degradation (Lo and Hannink, manufacturer's protocol. 153 96 2006). Exposure to electrophiles and ROS uncouples the Nrf2 97 Keap1 complex, leading to the release of Nrf2 and its nuclear trans- 2.4. Luciferase assay 154 98 location where it dimerizes with other transcription factors such as 99 members of the small Maf (musculoaponeurotic fibrosarcoma Twenty-four hours after transfection with ARE-LUC, the cells 155 100 oncogene) family (Motohashi et al., 2004). Binding of these hetero- were seeded in 96-well plates at a density of 1 104 cells/well 156 101 dimers to ARE enables transcriptional activation of many target and treated with conditions as elsewhere indicated. After the treat- 157 102 genes including those encoding antioxidants (e.g. GSH), drug- ment, luciferase activity was determined using the Bright-Glo 158 103 metabolizing enzymes (Phase I and Phase II), drug-efflux pumps Luciferase Assay System (Promega Corporation, Madison WI) 159 104 (Phase III), 26S proteasome subunits, heat shock proteins, growth according to the manufacturer's instructions. Luminescence was 160 105 factors, and transcription factors (Hayes and McMahon, 2009; Itoh recorded using a FLUOstar Optima plate reader (BMG Labtech 161 106 et al., 1997; Owuor and Kong 2002). The up-regulation of these Inc., Cary, NC). 162 107 genes promotes cell survival and protection against oxidative dam- 108 age (Lee et al., 2004; Li et al., 2005). 2.5. Nuclear extraction 163 109 It has been demonstrated that the Nrf2-dependent adaptive re- 110 sponse provides a pivotal defense mechanism against environmen- Nuclear extracts were prepared using the NE-PER system 164 111 tal hazards, including various air pollutants (reviewed in (Osburn (Pierce Chemical Co., Rockford IL) following the manufacturer's 165 112 and Kensler, 2008) and (Rubio et al., 2010)). In this study, we have recommendations. Briefly, 3 105 control and treated cells were 166 113 explored the role of Nrf2 in protection against benzene metabolites harvested and suspended in 100 ll of the cytoplasmic extraction 167 114 HQ and BQ in human lung cells. Our results demonstrate that these reagent I (CER I) and incubated on ice for 10 min. The cytoplasmic 168 115 metabolites are able to induce ARE-driven gene expression through extraction reagent II (CER II, 5 lL) was then added, vortexed for 5 s, 169 116 the activation of Nrf2. However, knockdown of Nrf2 greatly en- incubated on ice for 1 min and centrifuged (16,000g) at 4 C for 170 117 hances HQ- and BQ-induced cytotoxicity and cell death, and the in- 5 min. The pellet was suspended in 50 ll of nuclear extraction re- 171 118 creased susceptibility of the Nrf2-knockdown cells is associated agent (NER) and incubated on ice for 40 min, vortexing for 15 s 172 119 with reduced levels of GSH and loss of induction of ARE-driven every 10 min. After incubation, the sample was centrifuged at 173 120 genes, suggesting that Nrf2 is essential for the survival of lung cells 4 C (16,000g) for 5 min and the supernatant was collected and 174 121 against the toxic effects of these benzene metabolites. frozen at 80 C until further use. 175 2.6. Cell viability 176 122 2. Materials and methods Cell viability was assessed by the [3-(4,5-dimethylthiazol-2-yl)- 177 123 2.1. Chemicals and cell culture 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 178 inner salt (MTS)-based assay following the manufacturer's instruc- 179 124 All chemicals used in this study were purchased from Sigma tions (Promega Corporation, Madison, WI). 180 125 Aldrich (St. Louis, MO). Human bronchial epithelial cells (Beas- 126 2B) were obtained from American Tissue Culture Collection (ATCC, 2.7. Flow cytometry 181 127 Rockville MD). Beas-2B cells were grown in Dulbecco's Modified 128 Eagle's Medium (DMEM, South Logan UT) supplemented with Analysis of different stages of apoptosis was performed using 182 129 10% fetal bovine serum (FBS, Invitrogen Corporation, Carlsbad Annexin-V/PI and Annexin-V/7-AAD staining kits (BD Pharmigen, 183 130 CA) and antibiotics (100 U penicillin/ml and 100 lg San Jose, CA) following manufacturer's protocol, and analyzed by 184 Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 No. of Pages 9, Model 5G V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 3 185 flow cytometry (BD LSR II cytometer). Briefly, 2 105 HQ or BQ- method according to the manufacturer's guidelines (GE Healthcare, 211 186 treated cells were harvested, washed with PBS, stained with either Pittsburgh, PA). Antibodies used were purchased from the follow- 212 187 Annexin-V/PI or Annexin-V/7-AAD for 15 min at room temperature ing suppliers: Nrf2, HMOX1, GSS, NQO1, GCLM and lamin A from 213 188 and analyzed by flow cytometry. Santa Cruz Biotechnology (Santa Cruz, Biotechnology Inc., Santa 214 Cruz, CA); b-actin and GAPDH from SigmaAldrich (SigmaAldrich, 215 189 2.8. GSH measurement St. Louis, MO); and anti-rabbit, anti-goat and anti-mouse IgG peroxidase linked whole antibody from GE Healthcare (GE Healthcare, 216 217 190 GSH was assessed using the intracellular thiol probe ThiolTrac- Waukesha, WI). 218 191 ker Violet (Invitrogen Corporation, Carlsbad CA) following the 192 manufacturer's instructions. Briefly, Beas-2B cells were treated 3. Results 193 with various concentrations of either HQ or BQ in serum-free med- 219 194 ia for 2 h, washed twice with PBS, and stained with ThiolTracker 3.1. Induction of ARE-driven gene expression and Nrf2 activation by 195 Violet for 30 min at 37 C. Then, the dye was replaced with pre- HQ and BQ 196 warmed PBS and fluorescence intensity (410 nm excitation/ 220 221 197 520 nm emission) was measured using a FLUOstar Optima plate To examine the capability of HQ and BQ in the induction of ARE- 222 198 reader (BMG Labtech Inc., Cary, NC). driven gene expression, human bronchial epithelial cells, Beas-2B, 223 were transiently transfected with a luciferase reporter plasmid dri- 224 199 2.9. Western blot ven by the NQO1 gene ARE (referred as ARE-LUC) (Dhakshinamoor- 225 thy and Jaiswal, 2000). The cells were exposed to various 226 200 Cell lysates were prepared in lysis buffer (Cell Signaling Tech- concentrations (520 lM) of HQ or BQ for 16 h and assayed for 227 201 nology, Danvers, MA) supplemented with protease inhibitor cock- luciferase activity. These doses were found to produce mild to 228 202 tail (Thermo Fisher Scientific Inc., Rockford, IL) followed by moderate stress to the cells without causing appreciable cytotoxic- 229 203 centrifugation at 16,000g for 10 min at 4 C. Total protein was ity that may interfere with the reporter gene assay (Crisman et al., 230 204 quantified using a bicinchoninic acid kit (Thermo Fisher Scientific 2007; Fan and Wood, 2007). Both HQ and BQ treatments resulted 231 205 Inc., Rockford, IL). Equal amounts of protein were loaded in a in a dose-dependent induction of ARE-driven luciferase activity 232 206 420% gradient polyacrylamide gel (Invitrogen Corporation, Carls- (Fig. 1A). When used at a lower concentration (5 lM), HQ seemed 233 207 bad, CA) and transferred to a PVDF membrane. The membrane was to be a stronger ARE inducer than BQ. However, when a higher con- 234 208 blocked with 5% non-fat milk solution and sequentially incubated centration (20 lM) was used, they induced luciferase activities at a 235 209 with primary antibody and enzyme-conjugated secondary anti- comparable rate ($2.5-fold). We then evaluated if the observed 236 210 body. The bands were visualized using the chemiluminescence ARE induction could correlate with the activation of the Nrf2 237 Fig. 1. Induction of ARE-driven gene expression and activation of Nrf2 by HQ and BQ. (A) Beas-2B cells were transfected with the ARE-LUC reporter vector. Forty-eight hours after transfection, the cells were treated with various concentrations of HQ or BQ for $16 h and luciferase activity was measured. Data are expressed as fold of induction of luciferase activity compared to vehicle-control (dimethyl sulfoxide) (mean SD; n = 3). (B) Exposure to either HQ or BQ increases Nrf2 protein expression and causes Nrf2 nuclear accumulation. Beas-2B cells were treated with 20 lM of HQ or 10 lM of BQ for 4 h. Nuclear extracts and whole-cell lysates were prepared as described in Section 2. b- actin and lamin A antibodies were used as loading controls for whole-cell lysates and nuclear extracts respectively. (C) Up-regulation of Nrf2-dependent enzymes. Western blot analysis of Beas-2B cells treated with up to 20 lM of HQ or 10 lM of BQ for 16 h. b-actin antibody was used as loading control. Quantification of band intensity was performed by ImageJ version 1.42q software (developed by the National Institute of Health) (right panel) and expressed as percentage of induction compare to vehicle-treated cells (control). Data are representative of three independent experiments with similar results. GCLM, glutamate cysteine ligase modifier subunit; GSS, glutathione synthetase; HMOX1, heme oxygenase 1; NQO1, NAD(P)H dehydrogenase quinone 1. Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 No. of Pages 9, Model 5G 4 V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 238 pathway. A rapid elevation of Nrf2 protein was detected in whole- translocation and protein accumulation leading to the transcrip- 260 239 cell lysates prepared after a short term treatment (4 h) with either tional up-regulation of ARE-driven genes in Beas-2B cells. 261 240 HQ or BQ (Fig. 1B). Since Nrf2 nuclear translocation is a key event 241 in the activation of this pathway, we examined Nrf2 nuclear accu- 3.2. Protection against HQ- and BQ-induced cytotoxicity and cell death 262 242 mulation in either HQ or BQ treated Beas-2B cells. Western blot by Nrf2 243 analysis showed that HQ and BQ led to Nrf2 accumulation in nucle- 263 244 ar extracts after 4 h treatment (Fig. 1B). These data indicate that To evaluate the role of Nrf2 in HQ- and BQ-induced cytotoxicity, 264 245 Nrf2 is rapidly stabilized and mobilized to the nucleus in response we explored whether Nrf2 overexpression could confer cytoprotec- 265 246 to HQ and BQ treatments. Next, to determine the effect of Nrf2 acti- tion against these xenobiotics in lung epithelial cells. Beas-2B cells 266 247 vation on gene expression, levels of several well-known Nrf2 target were transfected with Nrf2-V5 plasmid to establish the transient 267 248 proteins were analyzed. These included GSH synthesis enzymes overexpression of wild-type Nrf2 (Fig. 2A). Transfected cells were 268 249 glutamate cysteine ligase modifier subunit (GCLM) and glutathione treated with different concentrations of either HQ or BQ for 5 h 269 250 synthetase (GSS), the drug-metabolizing enzyme NAD(P)H dehy- and cell viability was assessed by MTS reduction assay. As shown 270 251 drogenase quinone 1 (NQO1), and the antioxidant enzyme heme in Fig. 2B, cells overexpressing Nrf2 exhibited significantly attenu- 271 252 oxygenase 1 (HMOX1). As shown in Fig. 1C, induction of these pro- ated loss of viability caused by HQ or BQ treatments compared to 272 253 teins was confirmed in cells exposed to either HQ or BQ for 16 h. control cells, as reflected by either raised viability curves 273 254 Levels of HMOX and GCLM were markedly increased by both com- (Fig. 2B) or increased effective dose 50 (ED50, yielding 50% cell via- 274 255 pounds, whereas expression of GSS and NQO1 only became detect- bility) (Fig. 2C). To further stress the importance of Nrf2 in protec- 275 256 able after treatment with either HQ or BQ. Noteworthy, HQ tion against HQ and BQ cytotoxicity, Nrf2 in Beas-2B cells was 276 257 treatment caused a greater accumulation of HMOX1 (7-fold induc- silenced by siRNA (short silencing RNA) and tested for their sus- 277 258 tion) as compared to the BQ treatment (2-fold). These results dem- ceptibility to these metabolites. Nrf2 siRNA transfection led to a 278 259 onstrate that HQ and BQ activate Nrf2 by triggering its nuclear knockdown of Nrf2 protein to an undetectable level as determined 279 Fig. 2. The role of Nrf2 in protection against HQ- and BQ-induced cytotoxicity. Overexpression experiments (AC). (A) Western blot analysis of Nrf2 expression in Beas-2B cells transiently transfected with pcDNA 3.1 or Nrf2-V5 plasmid. b-actin was used as loading control. (B) Beas-2B cells overexpressing Nrf2 were exposed to various concentrations of either HQ or BQ for 5 h and cytotoxicity was determined by MTS reduction assay. The curves represent the percentage of viability of treated cells relative to untreated-control cells (mean SD; n > 3). Note: data are representative of at least three independent experiments with similar results. (C) Based on the cytotoxicity assays, effective dose 50 (ED50) was calculated for HQ and BQ in control and Nrf2-overexpressing cells (mean SD; n > 4). Knockdown experiment (DF). (D) Western blot analysis of Nrf2 in Beas-2B cells transfected with control siRNA or siRNA targeting Nrf2. b-actin was used as loading control. (E) Forty-eight hours after siRNA transfection, Beas-2B cells were exposed to various concentrations of either HQ or BQ for 5 h and cytotoxicity was evaluated by MTS reduction assay. The curves represent the percentage of viability of treated cells relative to untreated-control cells (mean SD; n > 3). Note: data are representative of at least three independent experiments with similar results. (F) Based on the cytotoxicity **p < 0.01. assays, effective dose 50 (ED50) was calculated for HQ and BQ in control and Nrf2-knockdown Beas-2B cells (mean SD; n > 4). Student's t-test *p < 0.05, Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 No. of Pages 9, Model 5G V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 5 280 by Western blot analysis (Fig. 2D). The Nrf2-knockdown cells Nf2 deficiency increases susceptibility of Beas-2B cells to these 311 281 exhibited increased susceptibility to either HQ- or BQ-induced compounds. 312 282 cytotoxicity as reflected by lowered viability curves (Fig. 2E) and 283 decreased ED50s for HQ and BQ as compared to control-siRNA 3.3. Reduced GSH levels and loss of inducibility of ARE-driven genes in 313 284 transfected cells (Fig. 2F). Nrf2-knockdown cells 314 285 Since the above cell viability assay (MTS) was based on measur- 286 ing mitochondrial function and unable to differentiate the pro- GSH is the most abundant cellular non-protein thiol, it plays a 315 287 cesses of cell death in detail, we further assessed cytotoxicity of central role in maintaining cellular redox status and protecting 316 288 HQ and BQ in Nrf2 overexpressing and knockdown cells using cell against oxidative damage (Zhang et al., 2009). GSH depletion ap- 317 289 death-based assays. Apoptosis was determined by annexin V/PI pears to be one of the key mechanisms underlying HQ and BQ tox- 318 290 (overexpression experiments) and annexin V/7-AAD (knockdown icity in other human cells (Luo et al., 2008; Smith, 1999; Trush 319 291 experiments) staining followed by flow cytometry analysis. et al., 1996). We measured GSH levels in Beas-2B cells treated with 320 292 Although pronounced induction of cell death was observed in both various concentrations of either HQ or BQ for a short period (2 h). 321 293 controls and Nrf2 overexpressing Beas-2B cells after the treatment As shown in Fig. 5A, cellular GSH decreased in a doseresponse 322 294 of either HQ or BQ (4 h, 50 lM) (Fig. 3), Nrf2 overexpressing cells manner in both treatments, with relatively greater depletion of 323 295 showed a relatively lower rate of cell death compared to control GSH in BQ treatment. Since Nrf2 is a master regulator of antioxi- 324 296 cells (empty vector transfected cells) (Fig. 3). Noteworthy, early dant transcriptional response, we hypothesized that the initial 325 297 apoptotic cells (annexinV+/PI-) were, in fact, slightly increased in depletion of GSH by HQ and BQ could trigger an Nrf2-dependent 326 298 the Nrf2-overexpressing cells, but the overall apoptotic process up-regulation of detoxifying enzymes and antioxidants, which 327 299 was remarkably reduced in these cells. In addition, we also tested could help detoxify these compounds (Bolton et al., 2000; Moran 328 300 if lack of Nrf2 could enhance apoptotic cell death induced by either et al., 1999; Smith, 1999). In order to delineate the role of Nrf2 329 301 HQ or BQ (Fig. 4). After HQ treatment (4 h, 50 lM) $40% of Nrf2- in this detoxification mechanism, we studied the effect of Nrf2- 330 302 knockdown cells were dead or in later stages of apoptosis whereas knockdown in the same cellular model. As expected, Nrf2-knock- 331 303 only $20% of control cells were dead or apoptotic (Fig. 4A and C). down cells had lower basal levels of GSH compared to the con- 332 304 Moreover, the percentage of viable cells was significantly lower trol-transfected cells (80%) (Fig. 5B). When the siRNA transfected 333 305 in Nrf2-knockdown cells than that of control cells. Similar results cells were treated with either 20 lM of HQ or 10 lM of BQ for 334 306 were obtained from the BQ treatment, in which $60% of Nrf2- 16 h, protein levels of HMOX and GSS were markedly increased 335 307 knockdown cells were dead or in later stages of apoptosis but only in control-siRNA transfected cells, but this induction was totally 336 308 $40% of control cells were the case (Fig. 4B and D). Taken together, absent in Nrf2-knockdown cells (Fig. 5C). These results suggest 337 309 these results demonstrate that Nrf2 overexpression attenuates that the previously observed enhanced HQ/BQ cytotoxicity in 338 310 cytotoxicity and cell death induced by both HQ and BQ whereas Nrf2-knockdown cells is due to decreased production of GSH and 339 Fig. 3. Nrf2 overexpression renders protection against HQ- and BQ-induced apoptotic cell death induction. Forty-eight hours after transfection with control or Nrf2-V5 plasmid Beas 2-B cells were exposed to 50 lM of either HQ or BQ for $4 h. Apoptosis and cell death induction was evaluated by Annexin-V/PI staining followed by flow cytometry analysis. Data from 1 104 cells were acquired. Representative flow cytometry profiles are presented for HQ (A) and BQ (C) treated cells. The percentage of cell population in each quadrant is indicated. (B) and (D) Data shown as mean SD of three independent experiments. Q1, dead cells (annexinV-/PI+); Q2, late apoptotic cells (annexinV+/PI+); Q3, viable cells (annexinV-/PI-); Q4, early apoptotic cells (annexinV+/PI-). Student's t-test *p < 0.05, **p < 0.01. Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 6 V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx No. of Pages 9, Model 5G Fig. 4. Nrf2-knockdown enhances HQ- and BQ-induced apoptotic cell death. Forty-eight hours after transfection with control or Nrf2 siRNA, Beas 2-B cells were exposed to 50 lM of either HQ or BQ for $4 h. Apoptosis and cell death induction was evaluated by Annexin-V/7-AAD staining followed by flow cytometry analysis. Data from 1 104 cells were acquired. Representative flow cytometry profiles are presented for HQ (A) and BQ (C) treated cells. The percentage of cell population in each quadrant is indicated. (B) and (D) Data shown as mean SD of three independent experiments. Q1, dead cells (annexinV-/7AAD+); Q2, late apoptotic cells (annexinV+/7AAD+); Q3, viable cells (annexinV-/7AAD-); Q4, early apoptotic cells (annexinV+/7AAD-). Student's t-test *p < 0.05, **p < 0.01. Fig. 5. Reduced GSH and loss of inducibility of HOMX1 and GSS in Nrf2-knockdown cells. (A) Beas-2B were treated at the indicated concentrations for 2 h followed by measurement of GSH intracellular content (mean SD; n = 4). (B) GSH content was measured 48 h after transfection with either control siRNA or Nrf2 siRNA (mean SD; n = 3) as described in Section 2. (C) Forty-eight hours after transfection, control and Nrf2-knockdown cells were treated with the indicated concentrations of HQ or BQ for 16 h. After treatment, protein levels of Nrf2, HMOX1 and GSS were determined by Western blot analysis. GAPDH was used as loading control. Quantification of bands intensity was performed by ImageJ version 1.42q software (developed by the National Institute of Health) (right panel) and expressed as percentage of induction compared to vehicle-treated cells (control). Data are representative of three independent experiments with similar results. Student's t-test *p < 0.05. Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 340 loss of inducibility of Nrf2-dependent antioxidant enzymes includ341 ing HMOX and enzymes responsible for GSH metabolism (e.g. GSS). 342 Therefore, the deficiency of Nrf2 reduces cellular redox capacity to 343 counteract oxidative damage induced by HQ and BQ. No. of Pages 9, Model 5G 7 344 4. Discussion 345 In this study, we used human bronchial epithelial cells (Beas- 346 2B) as a cellular model to explore the role of the Nrf2 pathway in 347 the defense against HQ- and BQ-induced cytotoxicity in lung. We 348 have shown that both HQ and BQ are able to induce ARE-driven 349 gene expression in the lung cells, as reflected by an increase in 350 ARE-dependant luciferase reporter activity (Fig. 1A). HQ and BQ 351 exposures also lead to increased Nrf2 total protein levels, as well 352 as Nrf2 nuclear accumulation (Fig. 1B). It is well established that Fig. 6. Model detoxification pathway involved in the activation of Nrf2. Exposure to 353 Nrf2 nuclear accumulation and protein stabilization are indicators benzene metabolites HQ and BQ causes GSH depletion and ROS generation, 354 of Nrf2 activation (Li et al., 2005; Niture et al., 2009). Hence, these resulting in changes in the redox status of the cell, which triggers nuclear translocation of Nrf2 and the activation of the Nrf2/ARE pathway, in turn leading to 355 results reveal that HQ and BQ are Nrf2 activators, capable of induc- up-regulation of Phase II detoxification enzymes and enzymes responsible for the 356 ing ARE-driven gene expression. Furthermore, we confirmed that synthesis of endogenous antioxidants such as GSH. This auto-regulation mechanism 357 HQ and BQ can induce endogenous ARE-driven genes, including is suggested as a therapeutic target to prevent or reduce benzene toxicity using 358 those encoding GSH synthesis enzymes GCLM and GSS, the drug- pharmaceutical or natural dietary agents. 359 metabolizing enzyme NQO1, and the antioxidant enzyme HMOX1. 360 The induction of detoxifying enzymes (phase II enzymes) and anti- 361 oxidants is critical for detoxification of benzene metabolites. NQO1 To further elucidate the protective role of Nrf2 against HQ- and 403 362 and GSH have been found to be key intrinsic molecules that defend BQ-induced cytotoxicity and cell death, two strategies were used 404 363 against benzene-induced toxicity in hematopoietic cells (reviewed to manipulate Nrf2 levels: gene overexpression and knockdown. 405 364 in (Bolton et al., 2000) and (Ross and Zhou, 2009)). NQO1 is a fla- While Nrf2 overexpression significantly reduced the cytotoxicity 406 365 voprotein that catalyzes the beneficial two-electron reduction of and apoptotic cell death induced by either HQ or BQ (Figs. 2 and 407 366 benzoquinone to hydroquinone, preventing unwanted one-elec- 3), knockdown of Nrf2 by siRNA led to the opposite effects (Figs. 408 367 tron reduction which results in formation of ROS, thus avoiding re- 4 and 5). These results are in agreement with several previous re- 409 368 dox cycling of semiquinone radicals and the subsequent covalent ports in which neurons and kidney cells with Nrf2 overexpression 410 369 modification of cellular components (Vasiliou et al., 2006). It has showed enhanced resistance against oxidant-induced cell death 411 370 been clearly demonstrated that NQO1 induction protects against (Chen and Shaikh, 2009; Shih et al., 2003), however, Nrf2-knock- 412 371 cell death induced by HQ (Smith 1999) and BQ (Flescher and Sny- down bone marrow stromal cells exhibited increased sensitivity 413 372 der, 1995). Moreover, conjugation of HQ and BQ to GSH and other to HQ and BQ (Zhu et al., 2006). Finally, we demonstrated that 414 373 endogenous substrates (e.g. glucuronic acid) enables the formation (1) reduced cellular GSH (Fig. 5B) and (2) impaired induction of 415 374 of less reactive and toxic metabolites, with increased solubility and ARE-driven genes including GSS and HMOX (Fig. 5C), both due to 416 375 excretion (Snyder et al., 1993). Induction of the GSH synthesis en- the loss of Nrf2, are the mechanistic explanations for the observed 417 376 zymes and enzymes that catalyze the conjugation reactions (such enhanced susceptibility of Nrf2-knowckdown cells to HQ and BQ. 418 377 as glutathione-S-transferases (GSTs) and UDP-glucuronosyl- In other words, once the ``auto-regulation'' mechanism (Fig. 6) is 419 378 transferases (UGTs)) is mostly regulated by Nrf2 (Biswas and Rah- disrupted, e.g., by inactivation of Nrf2, cells are more susceptible 420 379 man, 2009; Cho et al., 2002). Nrf2-dependent gene expression not to these compounds. It is noteworthy that BQ appears to be rela- 421 380 only counteracts oxidative stress produced by HQ and BQ expo- tively more potent than HQ in terms of depleting GSH and causing 422 381 sures, but also participates in the biotransformation and excretion cytotoxicity (Figs. 1, 4 and 5). Compared to HQ, BQ is considered a 423 382 of these metabolites. Our study demonstrated that exposure to less stable, but more reactive electrophile (Gaskell et al., 2005) 424 383 either HQ or BQ causes a rapid GSH depletion (Fig. 5A) likely due capable of reacting directly with cellular components (e.g. DNA 425 384 to production of ROS or direct conjugation with the compounds and proteins) and GSH (Snyder and Hedli, 1996). Nevertheless, 426 385 (Luo et al., 2008; Smith, 1999; Trush et al., 1996), leading to Nrf2 manipulation of Nrf2 significantly affects cytotoxicity induced by 427 386 activation and subsequent induction of GCLM and GSS (Fig. 1C), either HQ or BQ (Figs. 24). 428 387 two major enzymes involved GSH synthesis (Biswas and Rahman, Our study demonstrated that the Nrf2/ARE pathway is essential 429 388 2009). On the other hand, HQ and BQ are Michael reaction accep- in the intrinsic defense against inhaled benzene and its metabolites 430 389 tors (Bolton et al., 2000), compounds that have been shown to re- HQ and BQ. This knowledge is critical for the development of strat- 431 390 act with SH groups of Keap1 leading to Nrf2 nuclear translocation egies to help susceptible populations exposed to these air pollu- 432 391 and the subsequent activation of the pathway (Dinkova-Kostova tants. One of the most prominent developing strategies to 433 392 et al., 2001; Wang et al., 2010). It is more likely that both of these prevent or reduce the benzene-induced damage is to enhance the 434 393 two mechanisms of Nrf2 induction contribute to the observed Nrf2 Nrf2-dependent antioxidant response. It has been suggested that 435 394 activation. These results allow us to assemble a series of conse- failure to induce functional NQO1 contributes to increased risk of 436 395 quential events: exposure to HQ and BQ ? ROS produc- benzene hematotoxicity (Moran et al., 1999) and induction of this 437 396 tion ? depletion of GSH and generation of oxidative enzyme by a well-known Nrf2 inducer, (Kwak et al., 2001) was 438 397 stress ? Nrf2 activation ? induction of ARE-driven gene expres- proven to increase protein levels preventing HQ-induced apoptosis 439 398 sion ? detoxification of HQ and BQ and repair of oxidative damage in hematopoietic cells. Furthermore, NQO1 can facilitate the for- 440 399 (Fig. 6). This suggests that there exists an auto-regulation mecha- mation of less toxic and soluble metabolites that are easier to be 441 400 nism (Rubio et al., 2010) for the detoxification of HQ/BQ com- eliminated (Schrenk et al., 1996). Recently, several dietary chemo- 442 401 pounds and it is largely dependent on the activation of the Nrf2/ preventive compounds (e.g. curcuminoids, resveratrol, and isothio- 443 402 ARE pathway. cyanates) have been reported to increase Nrf2 levels, disassociate 444 Please cite this article in press as: Rubio, V., et al. Essential role of Nrf2 in protection against hydroquinone- and benzoquinone-induced cytotoxicity. Toxicol. in Vitro (2010), doi:10.1016/j.tiv.2010.10.021 TIV 2508 12 November 2010 No. of Pages 9, Model 5G 8 V. Rubio et al. / Toxicology in Vitro xxx (2010) xxxxxx 445 Nrf2 of its cytosolic inhibitor Keap1, or/and enhance Nrf2 protein 446 stabilization, resulting in the activation of Nrf2-dependent gene 447 expression (reviewed in (Kwak et al., 2004) and (Jeong et al., 448 2006)). Use of these common and abundant compounds could 449 prove to be a simple and safe strategy to provide protection against 450 benzene- and benzene metabolite-induced toxicity. With tens of 451 millions of people living and working in densely populated cities 452 and industrial areas, such a preventive strategy if successful 453 could have a significant impact on lessening the public health bur454 den from exposure to oxidant pollutants. 455 Acknowledgment 456 This work is supported by the Methodist Hospital Research 457 Institute Faculty Seed Fund to Z.-Z.S. and partially by DGAPA458 UNAM grant number IN212107. V.R. is a recipient of a CONACYT 459 fellowship. 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