Benzene’s metabolites alter c-MYB activity via reactive oxygen species in HD3 cells

Toxicology and Applied Pharmacology 222 (2007) 180 – 189 www.elsevier.com/locate/ytaap

Benzene’s metabolites alter c-MYB activity via reactive oxygen species in HD3 cells☆ Joanne Wan a , Louise M. Winn a,b,⁎ a

Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada b School of Environmental Studies, Queen’s University, Kingston, Ontario, Canada Received 9 March 2007; revised 20 April 2007; accepted 24 April 2007 Available online 21 May 2007

Abstract Benzene is a known leukemogen that is metabolized to form reactive intermediates and reactive oxygen species (ROS). The c-Myb oncoprotein is a transcription factor that has a critical role in hematopoiesis. c-Myb transcript and protein have been overexpressed in a number of leukemias and cancers. Given c-Myb’s role in hematopoiesis and leukemias, it is hypothesized that benzene interferes with the c-Myb signaling pathway and that this involves ROS. To investigate our hypothesis, we evaluated whether benzene, 1,4-benzoquinone, hydroquinone, phenol, and catechol generated ROS in chicken erythroblast HD3 cells, as measured by 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) and dihydrorhodamine-123 (DHR-123), and whether the addition of 100 U/ml of the antioxidating enzyme superoxide dismutase (SOD) could prevent ROS generation. Reduced to oxidized glutathione ratios (GSH:GSSG) were also assessed as well as hydroquinone and benzoquinone’s effects on cMyb protein levels and activation of a transiently transfected reporter construct. Finally we attempted to abrogate benzene metabolite mediated increases in c-Myb activity with the use of SOD. We found that benzoquinone, hydroquinone, and catechol increased DCFDA fluorescence, increased DHR-123 fluorescence, decreased GSH:GSSG ratios, and increased reporter construct expression after 24 h of exposure. SOD was able to prevent DCFDA fluorescence and c-Myb activity caused by benzoquinone and hydroquinone only. These results are consistent with other studies, which suggest metabolite differences in benzene-mediated toxicity. More importantly, this study supports the hypothesis that benzene may mediate its toxicity through ROS-mediated alterations in the c-Myb signaling pathway. © 2007 Elsevier Inc. All rights reserved. Keywords: Benzene; c-Myb; Benzoquinone; Hydroquinone; Phenol; Catechol

Introduction Benzene is a ubiquitous environmental contaminant that is found in cigarette smoke, automobile exhaust, and industrial Abbreviations: ROS, reactive oxygen species; DHR-123, dihydrorhodamine-123; DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; PEG-SOD, polyethylene glycol conjugated superoxide dismutase. ☆ Preliminary reports of this research were presented at the 43rd annual meeting of the Society of Toxicology (U.S.A.), March 21–24th 2004; the 44th annual meeting of the Society of Toxicology (U.S.A.), March 6–10th 2005; the 39th Annual Symposium of the Society of Toxicology of Canada, December 4– 5th 2006; and at the International Symposium on Recent Advances in Benzene Toxicity, Munich, Germany, October 9–12th 2004. ⁎ Corresponding author. Department of Pharmacology and Toxicology and School of Environmental Studies, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Fax: +1 613 533 6412. E-mail address: [email protected] (L.M. Winn). 0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2007.04.016

processes (reviewed in Golding and Watson, 1999). Human exposure to benzene has been linked to hematopoietic disorders such as acute myeloid leukemia, myelodysplasic syndromes, and aplastic anemia (Descatha et al., 2005; Irons et al., 2005; Lan et al., 2004). It is generally believed that benzene must be metabolized by cytochrome P450 2E1 (CYP2E1) to mediate its toxicity as mice lacking CYP2E1 activity display less benzenemediated toxicity and produce less hydroxylated metabolites than normal mice (Valentine et al., 1996). The main metabolites of benzene include 1,4-benzoquinone, hydroquinone, catechol, and phenol (Snyder and Hedli, 1996). Some of these metabolites are formed in the liver and others through subsequent metabolism in the bone marrow by myeloperoxidases (Schattenberg et al., 1994; Thomas et al., 1990). These metabolites are then thought to be reactive or can produce reactive oxygen species (ROS).

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ROS are key signaling molecules that are produced through normal metabolic processes and through the metabolism of exogenous compounds. Some species of ROS include the superoxide anion, hydrogen peroxide, and the hydroxyl radical (reviewed in Hancock et al., 2001). The production of ROS is tightly regulated and the cell possesses detoxifying mechanisms such as catalase, glutathione and superoxide dismutase (SOD) to maintain appropriate cellular ROS levels. However should an excess of ROS be produced, oxidative stress can occur and lead to altered cellular signaling (reviewed in Adler et al., 1999). Evidence for ROS formation after benzene exposure has been shown through in vitro models as benzene’s metabolites increase cellular ROS and oxidative DNA damage in HL60 cells (Kolachana et al., 1993; Zhang et al., 1993). Benzene or benzene’s metabolites’ ability to induce DNA damage has been shown in vitro, in mice, and in humans as measured through the comet assay (Hiraku and Kawanishi, 1996; Pan et al., 2003; Sul et al., 2005). Support for the role of ROS in benzene induced DNA damage has been shown through the ability of the antioxidative enzyme catalase to reduce benzene-metaboliteinduced aberrant DNA recombination (Winn, 2003). In addition to effects on DNA, benzene-mediated oxidative stress may alter the behavior of proteins. For example, exposure to the benzene metabolite trans,trans-muconaldehyde increased activator protein-1 (AP-1) binding and protein levels in HL60 cells and mouse bone marrow (Ho and Witz, 1997). Another potential ROS sensitive protein is the c-Myb oncoprotein (Myrset et al., 1993). The c-Myb transcription factor is the cellular variant of the protein produced by the avian myeloblastosis virus (v-Myb), which was initially discovered for its role in causing acute monoblastic leukemia in chickens (reviewed in Oh and Reddy, 1999). As an example, c-Myb’s sensitivity to oxidative conditions is thought to be in due in part to the oxidation of a key cysteine residue in the R2 region of its DNA binding domain, which affects DNA binding affinity (Myrset et al., 1993). c-Myb has a critical role in hematopoiesis as knockout mice die in utero due to an inability to proceed with mature hematopoiesis (Mucenski et al., 1991). Furthermore, levels of c-myb transcript are higher in immature cells and decrease as the cell differentiates (Ramsay et al., 1986). c-myb RNA has been found to be more stable in some human cases of acute myeloid leukemia (Baer et al., 1992) and overexpressed in various leukemias and colon cancers (Biroccio et al., 2001; Gopal et al., 1992; Hulette et al., 1992). Elevated c-Myb protein levels have been measured in T-cell lymphoma cell lines (Siegert et al., 1990) and experimental overexpression of full length cMyb has been found to block terminal differentiation of various hematopoietic cell types (Fu and Lipsick, 1997; Kaspar et al., 2005). This suggests that the activation of c-Myb is an oncogenic event. Given c-Myb’s role in hematopoiesis and the evidence supporting a role for c-Myb in leukemogenesis, we hypothesize that c-Myb is a target for benzene-mediated leukemogenesis, which involves ROS. We have previously shown that the benzene metabolite catechol, but not phenol or benzene itself was able to increase c-Myb activity and c-Myb phosphorylation in the HD3 chicken erythroblast cell line (Wan and Winn, 2004).

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Furthermore, we published preliminary data suggesting that the benzene metabolites hydroquinone and benzoquinone alter cMyb activity via phosphorylation and can generate ROS (Wan et al., 2005). To expand on these preliminary results and to confirm the role of ROS in this mechanism, the current study evaluated the protective effects of the antioxidant enzyme SOD against benzene-metabolite-induced alterations in c-Myb signaling and ROS production. We also measured reduced to oxidized glutathione ratios (GSH:GSSG) to determine whether benzene’s metabolites induced oxidative stress in these cells. Our results support a role for ROS production and deregulation of c-Myb activity after exposure to some of benzene’s metabolites. Materials and methods Chemicals. All tissue culture media reagents were obtained from Invitrogen Corp. (Burlington, ON). Nonproprietary dual luciferase assay reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) with the exception of bovine serum albumin (Fisher Scientific Ltd., Ottawa, ON) and coelenterazine (Calbiochem-Novabiochem Corp., La Jolla, CA). Benzene was purchased from Fisher Scientific Ltd. Phenol, catechol, 1,4-benzoquinone, and hydroquinone were obtained from Sigma-Aldrich Chemical Co. and were at least 99.9% pure. Cell culture. Chicken erythroblast HD3 cells were kindly obtained from Scott A. Ness (Department of Molecular Genetics and Microbiology, University of New Mexico, USA). Cells were maintained at 37 °C/5% CO2 in DMEM supplemented with 4% fetal bovine serum, 4% fetal calf serum, 2% chicken serum, 1.0 mM HEPES, 0.1 mM sodium pyruvate, 0.01 mM MEM nonessential amino acids, 10 units/ml penicillin–streptomycin, and 2 mM Lglutamine. Antioxidant and metabolite exposure. 100 U/ml polyethylene-glycol conjugated superoxide dismutase (PEG-SOD) (Sigma-Aldrich Chemical Co.) was administered 1 h prior to the addition of benzene or its metabolites when required. The polyethylene glycol conjugated form of SOD was used because of enhanced cellular uptake and increased protein half-life (Beckman et al., 1988). Benzene and its metabolites were dissolved at 10 mM in full media and stored in aliquots at − 80 °C before use. The concentration of hydroquinone, benzoquinone, and catechol used for fluorescent dye and antioxidant studies was based on levels required to obtain a statistically significant increase in cMyb activity after 24 h of exposure. The benzene and phenol concentrations used were similar to concentrations used in previous studies in our laboratory (Wan and Winn, 2004). The concentration of most benzene metabolites used was not cytotoxic after 24 h of exposure according to the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. 50 μM benzoquinone caused a 35 ± 10% decrease in cell viability after 24 h of exposure. ROS detection. HD3 chicken erythroblasts were plated in 24-well plates at a density of 1.5 × 106 cells per well, allowed to incubate overnight, and were exposed to 50 μM benzoquinone, 50 μM hydroquinone, 300 μM benzene, 300 μM phenol, and 100 μM catechol for 1, 12, or 24 h prior to harvest. Treated cells were also exposed to 10 μM of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) (Invitrogen Corp.) for 2 h prior to harvest or to 10 μM dihydrorhodamine-123 (DHR-123) (Invitrogen Corp.) for 30 min prior to harvest. Dyes were initially dissolved in DMSO before final dilutions were performed. Cells were then trypsinized and resuspended in 5 mg/ml of propidium iodide (PI) (Sigma-Aldrich Chemical Co.) in phosphate buffered saline and analyzed using flow cytometry (excitation of 505 nm, 505 nm, and 536 nm; emission of 535 nm, 534 nm, and 617 nm for DCFDA, DHR-123, and PI respectively). PI was used to exclude apoptotic and necrotic cells from analysis. DCFDA results are reported as percentage of live cells that positively stained for DCFDA. DHR-123 results are reported as normalized mean fluorescence values (median fluorescence of sample / median fluorescence of unexposed control).

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Reduced to oxidized glutathione determination. Cells were seeded in 6-well dishes at a density of 4.0 × 106 cells/well in 3 ml of full media. Forty eight hours later, the cell media were replaced and cells were exposed to 300 μM benzene, 300 μM phenol, 50 μM hydroquinone, 50 μM benzoquinone, or 100 μM catechol for varying time points in 3 ml of media prior to harvest by trypsinization. Samples were then homogenized with 100 μl of 5-sulfosalicyclic acid (5% w/v) and centrifuged at 12,000×g. Supernatant was collected and stored at − 80 °C until analysis. Total glutathione (GSHt) was measured according to the enzymatic recycling method (Brehe and Burch, 1976) further modified for microtiter analysis (Baker et al., 1990). A 50 μl aliquot of each sample was incubated with 1.5 μl of 2-vinylpyridine and 10 μl of 10% triethanolamine for 1 h to block GSH and allow for GSSG readings. Samples were read in a 96-well plate in triplicate. Kinetic readings at 405 nm for 2 min were taken with a Bio-Tek Synergy HT multidetection microplate reader with KC4 v3.3 software (Bio-Tek Instruments Inc., Winooski, VT). A standard curve using 0–150 pmol of GSSG per well was run in conjunction with samples. GSH content was determined by multiplying values by 2 to obtain GSHt and then subtracting 2GSSG from GSHt. All reagents were obtained from Sigma-Aldrich Chemical Co. Transfections. A plasmid containing the chicken mim-1 promoter linked to firefly luciferase was obtained from Scott A. Ness (Department of Molecular Genetics and Microbiology, University of New Mexico, USA). HD3 cells were seeded at a density of 0.8 × 106 cells/well in 500 μl of full media in 24-well dishes and transfected as previously described (Leverson et al., 1998). In total, 0.6 μg of DNA, 2 μL Lipofectamine™, and 200 μl of Opti-MEM™ (Invitrogen Technologies) were used per well. DNA content included 0.25 μg of mim-1 promoter linked firefly luciferase reporter construct (Ness et al., 1989) and an appropriate amount of empty vector (pGEM; Promega Corp., Madison, WI.) to attain equal amounts of DNA. In addition, 50 ng of thymidine kinase promoter linked to renilla luciferase vector (pRL-TK; Promega Corp.) was co-transfected into each well to control for transfection efficiency. The mim-1 promoter contains three c-Myb recognition sequences and is fairly specific for c-Myb activation in HD3 cells (Ness et al., 1989). After metabolite exposure, cells were washed with 1 × 1 ml of cold phosphate buffered saline (PBS) and lysed with 0.1 ml of 1× Passive Lysis Buffer (Promega Corp.). Dual luciferase activities of 20 μl of cell lysate were measured using a nonproprietary dual luciferase assay (Dyer et al., 2000) or through a proprietary kit (Promega Corp.) and a Lumat LB 9507 Variable Injector Luminometer (Berthold Technologies GmbH and Co., Germany). Results are reported as a mean of at least 3 independent experiments performed in triplicate. To control for day to day differences in transfection efficiencies, relative luciferase values were further normalized by dual luciferase readings from unexposed cells. Immunoblotting. Cells were seeded in 6-well dishes at a density of 4.0 × 106 cells/well in 3 ml of full media. Forty eight hours later, the cell media were replaced and cells were exposed to a final concentration of 50 μM benzoquinone or hydroquinone for 6 or 24 h in 3 ml of media prior to harvest. Cells were then washed with 2 ml of cold PBS and collected through trypsinization. Cells from each well were resuspended in 100 μl of buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2% (w/v) NP-40, and 1.0 mM EDTA, and protease inhibitors. Eighty micrograms of prepared sample was then separated by SDS– PAGE in a 10% polyacrylamide gel and transferred to a polyvinylidene diflouride carrier membrane (Millipore Co., Bedford, MA). Protein concentration of each supernatant was measured using the Bio-Rad Laboratories’ Bradford Protein Assay (Mississauga, ON) and an Ultrospec 3100 Pro scanning spectrophotometer (Biochrom Ltd., UK). After the transfer, the membrane was incubated with rabbit polyclonal IgG antibody with an epitope mapping the carboxy terminus of murine c-Myb (Santa Cruz Biotechnology Inc., Santa Cruz, CA) in 5% milk Tris-buffered saline containing Tween 20 (TBS-T) (25 mM Tris–HCl, 140 mM NaCl, 2 mM KCl, 0.05% (v/v) Tween 20) blocking solution. The membrane was washed with TBS-T and incubated in horseradish peroxidase conjugated anti-rabbit IgG secondary antibody (GE Healthcare Bio-Sciences Inc., Baie d’Urfé, QC) in 5% milk TBS-T. The membrane was then washed with TBS-T and developed using the ECL chemiluminescence detection system (GE Healthcare Bio-Sciences Inc.) and Kodak X-OMAT scientific imaging film.

To control for differences in protein loading, the membrane was then washed with TBS-T and incubated in stripping solution (0.7% (v/v) β-mercaptoethanol, 2% (v/v) SDS, 62.5 mM Tris–HCl, pH 6.7) for 30 min at 55 °C. This was followed by blocking nonspecific binding sites in 10% milk TBS-T overnight followed by incubation in mouse monoclonal anti-α-tubulin IgG antibody (Sigma-Aldrich Canada Co.) followed by washing with TBS-T and incubation in horseradish peroxidase linked sheep anti-mouse IgG secondary antibody (GE Healthcare Bio-Sciences Inc.) in 5% blocking solution. To measure phosphorylated c-Myb protein levels in hydroquinone or benzoquinone treated cells, HD-3 cells were seeded and treated as above in 6-well plates. Cells were exposed to 50 μM of hydroquinone or benzoquinone for 20 h before harvest and purification with the Qiagen PhosphoProtein Purification Kit (Qiagen Inc., Mississauga, ON). Twenty-five percent of collected phosphorylated protein lysate was run in duplicate under aforementioned SDS–PAGE conditions and similarly probed with c-Myb antibody. Cells were stripped and reprobed with anti-α-tubulin antibody to serve as a loading control (SigmaAldrich Canada Co.). Densitometry. Immunoblot bands were quantified by scanning blots and measuring their relative optical density (ROD) using ImageJ (Rasband, 1997). ROD measurements of c-Myb were normalized to α-tubulin content for each well. α-Tubulin protein expression levels did not significantly change upon benzoquinone or hydroquinone exposure (data not shown). Day to day differences in film exposure were accounted for by expressing treated sample values as a percentage of untreated cell values on each blot. Statistical analysis. Results were analyzed using a computerized statistical program (GraphPad Prism 4.0). Transfection and immunoblotting studies involving multiple time points and metabolite concentrations were first compared using a two-way analysis of variance (ANOVA) followed by a one-way ANOVA for each time point and a Dunnett’s post-test to compare treated groups against unexposed controls. Other experimental results were subjected to one-way ANOVA followed by a Newman–Keuls post-test to perform multiple comparisons or a Dunnett’s post-test to compare against non-exposed controls. Data for the phosphorylated protein immunoblotting were subjected to a oneway ANOVA followed by a Student’s t-test to compare values from exposed cells to unexposed cells. The p value for all analyses was set to b0.05. Values are presented as mean ± standard deviation (SD).

Results ROS detection ROS were detected in HD3 cells by measuring DCFDA and DHR-123 fluorescence. These lipophillic dyes readily enter the cell and are oxidized to fluorescent moieties (Crow, 1997). Exposure to 50 μM benzoquinone [F(3,16) = 3.88, p = 0.029], 50 μM hydroquinone [F(3,24) = 18.28, p b 0.001], and 100 μM catechol [F(3,20) = 14.47, p b 0.001] significantly increased the number of cells staining for DCFDA after 24 h of exposure (Fig. 1). However, benzene or phenol exposure did not increase DCFDA fluorescence in HD3 cells. Pretreatment with 100 U/ml PEG-SOD was able to prevent DCFDA fluorescence in cells exposed to 50 μM hydroquinone or 50 μM benzoquinone, but not to 100 μM catechol for 24 h (Fig. 2). 46 ± 10% of cells were DCFDA fluorescent after 24 h of benzoquinone exposure, this fell to 22 ± 14% in the PEG-SOD with benzoquinone group. Baseline DCFDA staining for benzoquinone was 19 ± 13%. For the hydroquinone exposed cells 76 ± 16% of live cells were DCFDA positive compared to the 44 ± 18% in the hydroquinone and PEG-SOD exposed group, and 16 ± 20% in unexposed controls. PEG-SOD pretreatment was able to maintain DCFDA fluorescence levels at baseline for benzoquinone but not for

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ratios were significantly lower as early as 1 h after benzene metabolite exposure, as determined by a Dunnett’s post-test after a one-way ANOVA for each metabolite. As an example, GSH:GSSG ratios in 50 μM benzoquinone exposed cells dropped from 163 ± 7.6 to 107 ± 24.9 at 1 h, to 80.5 ± 19.9 after 24 h of exposure. Exposure to benzene or phenol did not decrease the GSH:GSSG ratio in HD3 cells. Hydroquinone decreased GSH:GSSG from baseline levels of 170 ± 15 to 53 ± 40 at 24 h. 100 μM catechol lowered GSH:GSSG from baseline levels of 180 ± 16, to 111 ± 42 at 1 h, to 74 ± 25 at 24 h of exposure. Decreases in GSH:GSSG ratios were metabolite dependent as hydroquinone, catechol, and benzoquinone were able to decrease the GSH:GSSG ratio, but benzene and phenol were not. c-Myb activity after hydroquinone and benzoquinone exposure To determine whether c-Myb activity would alter after exposure to benzoquinone or hydroquinone, we examined c-Myb Fig. 1. ROS production in HD3 cells as measured by percentage of live cells staining positive for DCFDA fluorescence after exposure to hydroquinone (HQ), benzoquinone (BQ), catechol (CAT), phenol (PH), or benzene (BZ) for 1, 12, or 24 h. Number of replicates for each metabolite are as follows: PH (n = 4), BZ (n = 3), HQ (n = 6), BQ (n = 5), CAT (n = 6). Data points represent a mean of percentage live cells staining positive for DCFDA fluorescence ± SD. Apoptotic and necrotic cells were excluded from analysis by resuspending cells in propidium iodide prior to analysis by flow cytometry. ⁎p b 0.05 compared to unexposed controls for each metabolite. In the presence of the positive control, 44 mM of hydrogen peroxide for 1 h (n = 5, not shown), 84% ± 19 of cells stained positive for DCFDA.

hydroquinone exposed cells. However, DCFDA fluorescence for PEG-SOD and hydroquinone treated cells was lower than for hydroquinone alone. This shows that superoxide is a key ROS produced in HD3 cells after hydroquinone and benzoquinone exposure. ROS detection with dihydrorhodamine-123 Exposure to benzoquinone, hydroquinone, and catechol increased DHR-123 fluorescence after 24 h to 1.6 ± 0.3, 1.5 ± 0.2, and 1.4 + 0.2 fold of control fluorescence levels respectively (Fig. 3). An increase in DHR-123 fluorescence was also observed with phenol as early as 1 h (2.0 ± 0.8 fold of control); however an increase at 24 h (1.63 ± 0.28 fold of control) was no longer statistically significant from the unexposed group based on a Dunnett’s post-test performed after a one-way ANOVA. Increases in DHR-123 mean fluorescence were not observed with benzene exposure. Preincubation with 100 U/ml PEGSOD for 1 hour prior to metabolite exposure did not decrease DHR-123 fluorescence for any of the metabolites that were used (data not shown). Glutathione status Exposure to 50 μM benzoquinone, 50 μM hydroquinone, and 100 μM catechol decreased the reduced to oxidized glutathione ratio (GSH:GSSG) (Fig. 4). In some cases, GSH:GSSG

Fig. 2. 100 U/ml PEG-SOD pretreatment for 1 h prevents (A) 50 μM benzoquinone, (B) 50 μM hydroquinone, but not (C) 100 μM catechol mediated ROS production after 24 h or exposure as measured by DCFDA fluorescence. Data are expressed as the mean percent of live cells staining positive for DCFDA fluorescence ± SD from three independent determinations. ⁎ indicates statistically significant difference from unexposed controls (p b 0.05). α indicates statistically significant difference from PEG-SOD pretreated, metabolite exposed group (p b 0.05). Apoptotic and necrotic cells were excluded from analysis by resuspending cells in propidium iodide prior to flow cytometric analysis.

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luciferase reporter construct. PEG-SOD did not prevent catechol mediated increases in c-Myb activity. Similar to our DCFDA results, this suggests that the ROS produced in HD3 cells differ for each of benzene’s metabolites and that superoxide is a product of hydroquinone and benzoquinone exposure. Discussion

Fig. 3. ROS production in HD3 cells as measured by DHR-123 fluorescence levels after exposure to 300 μM benzene, 300 μM phenol, 50 μM benzoquinone, 50 μM hydroquinone, or 100 μM catechol for 1, 12, or 24 h. Data are expressed as mean fluorescence ratios ± SD (median fluorescence of exposed cells / median fluorescence of unexposed controls) for at least 3 independent experiments. ⁎ indicates statistical significance from unexposed control (p b 0.05). Apoptotic and necrotic cells were excluded from analysis by resuspending cells in propidium iodide prior to flow cytometric analysis.

activity through a dual-luciferase assay at 6 and 24 h after exposure to varying concentrations of these metabolites. Benzoquinone [F(5,18) =6.87, p = 0.001] and hydroquinone [F(5,18) = 14.81, p b 0.001] had a concentration dependent effect on reporter construct activation in HD3 cells (Fig. 5). Significant increases in c-Myb activity were observed after 24 h of exposure to 50 μM hydroquinone (188 ± 37% of control RLU) or 50 μM benzoquinone exposure (175 ± 31% of control RLU). Immunoblotting Exposure to 5 or 50 μM hydroquinone or benzoquinone for 6 or 24 h did not cause an increase in c-Myb protein levels (Fig. 6). However, exposure to 50 μM of hydroquinone or benzoquinone increased phosphorylated c-Myb protein levels in HD3 cells [F(2,14) = 4.377, p = 0.04] (Fig. 7). Relative c-Myb/α-tubulin protein levels after exposure to hydroquinone or benzoquinone were 1.061 ± 0.34 and 0.92 ± 0.28 respectively. These c-Myb/αtubulin levels were significantly higher than those measured in unexposed cells (0.58 ± 0.09) (Fig. 7). Due to day to day blot exposure consistency, values for phosphorylated c-Myb protein levels are expressed as a ratio of c-Myb/α-tubulin without normalization for day to day variations in exposure. Antioxidants and c-Myb activity Exposure to 50 μM benzoquinone, 50 μM hydroquinone, and 100 μM catechol for 24 h increased c-Myb activity to 187 ± 44, 258 ± 38, and 227 ± 34% of control levels (Fig. 8). Preincubation with 100 U/ml of PEG-SOD for 1 h prevented increases in benzoquinone mediated activity and partly abrogated hydroquinone mediated increases in activation of the mim-1/

These studies show that the benzene metabolites benzoquinone, hydroquinone, and catechol produce ROS, cause oxidative stress, and alter c-Myb signaling in HD3 cells. These results support our previously published study focusing on catechol (Wan and Winn, 2004) and expand upon our preliminary results suggesting that hydroquinone and benzoquinone can also interfere with normal c-Myb signaling (Wan et al., 2005). In the present study we measured glutathione status and DHR-123 fluorescence and evaluated the effects of antioxidants. ROS produced by these metabolites of benzene have an effect on the regulation of a key hematopoietic protein, c-Myb. Pretreatment with PEG-SOD before benzoquinone and hydroquinone exposure prevented increases in DCFDA fluorescence, but not DHR-123 fluorescence, suggesting that superoxide is a product of these two metabolites. DHR-123 and DCFDA recognize similar ROS moieties such as peroxynitrite (Crow, 1997) and hydrogen peroxide (Royall and Ischiropoulos, 1993). However, DHR-123 is oxidized to form the fluorescent and cationic rhodamine-123, which then sequesters in mitochondria (Chen, 1989). The selectivity of rhodamine-123 for mitochondria suggests that it measured mitochondrial ROS more selectively than other sources of intracellular ROS. Interestingly, phenol was able to increase DHR-123 fluorescence but not DCFDA fluorescence. This increase was temporally inconsistent with what was observed for hydroquinone, benzoquinone, and catechol in our cells and was only observed with one of the dyes. To rule out a direct phenol/DHR-123 interaction, we incubated phenol and DHR-123 in the presence and absence of cell media for up to 30 min and did not see any differences in fluorescence compared to the dye alone (data not shown). Furthermore, phenol did not fluoresce in this cell free system. Phenol mediated DHR-123 fluorescence may have occurred due to mitochondrial specific cytochrome P450s. However, if this was the case, we would have expected to see a similar DHR-123 fluorescence increase with benzene exposure as both phenol and benzene have been shown to be substrates of this enzyme in rat livers (Karaszkiewicz and Kalf, 1990). As we did not see such an increase in DHR-123 fluorescence with benzene, and because this phenol mediated DHR-123 increase in fluorescence occurred as early as 5 min of incubation (data not shown), we hypothesize that mitochondrial specific P450s were not involved. Another explanation for the increase in DHR-123 fluorescence could be phenol’s ability to inhibit mitochondrial respiration (Izushi et al., 1988). If phenol was able to generate mitochondrial ROS in this manner, we would expect it to be detoxified by enzymes such as metallothionein, which can be induced by mitochondrial inhibitors (Futakawa et al., 2006). Catechol mediated increases in ROS levels and c-Myb activity were not diminished by PEG-SOD, and hydroquinone’s

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Fig. 4. Oxidative stress in HD3 as measured by reduced to oxidized glutathione ratios (GSH:GSSG). Cells were exposed to (A) 300 μM benzene, (B) 300 μM phenol, (C) 50 μM benzoquinone, (D) 50 μM hydroquinone, or (E) 100 μM catechol for up to 24 h. Data are expressed as means from 4 (hydroquinone and phenol) or 5 (benzoquinone, benzene, or catechol) independent experiments ± SD. ⁎ indicates statistical significance from unexposed control (p b 0.05).

effects were only partly attenuated by PEG-SOD. It is possible that hydrogen peroxide and other ROS were generated after exposure to these metabolites as PEG-SOD was unable to fully abrogate their effects. Furthermore, partial effectiveness of PEG-SOD on hydroquinone’s effects could be due to the removal of superoxide radicals that autooxidize hydroquinone into benzoquinone (Greenlee et al., 1981). Catechol can be converted to 1,2-benzoquinone by myeloperoxidases, and the reverse reaction is mediated by NAD(P)H-quinone oxidoreductase. Hydroquinone and 1,4-benzoquinone can also undergo this interconversion with the same enzymes (reviewed in Kim et al., 2006). Both quinones can undergo NADH-dependent redox cycling, via a semiquinone radical, to produce ROS (Bolton et al., 2000). Catechol and hydroquinone have been shown to have different redox properties, with catechol displaying an initially

slower ability to generate superoxide via the oxidation of Cu2+ and subsequent reduction of cytochrome-c in vitro (Hirakawa et al., 2002). This difference in superoxide generating ability could be a reason why SOD was effective at attenuating responses caused by benzoquinone and hydroquinone, but not catechol. It should be noted that serum proteins were present in the cell media during metabolite and fluorescent dye exposure. The components of fetal calf serum have been shown to increase ROS-mediated benzoquinone genotoxicity in human peripheral blood mononuclear cells within 2 h of exposure (Fabiani et al., 2005). Furthermore, the genotoxicity of serum and benzoquinone was attenuated by SOD and catalase in that study. While the presence of serum could have accounted for the effects observed with benzoquinone, catalase was not effective at attenuating ROS production in our system and significant increases

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Fig. 5. c-Myb activity levels as determined by activation of a c-Myb responsive luciferase reporter construct in HD3 cells. Cells were co-transfected with a cMyb responsive construct containing the chicken mim-1 promoter and the firefly luciferase gene, and a renilla luciferase expressing construct to act as an internal control. Cells were exposed to 1, 5, 20, 50, or 100 μM of (A) benzoquinone or (B) hydroquinone for 6 or 24 h. Data are expressed as a percentage of relative luciferase units of unexposed controls + SD from 4 independent determinations performed in triplicate. ⁎ indicates statistically significant difference from unexposed controls (p b 0.05).

in dye fluorescence were not observed until 24 h of exposure. Therefore the mechanism behind ROS production and increased c-Myb activity in our study is qualitatively different than the serum mediated mechanism observed by Fabiani et al. (2005). In addition, catechol and hydroquinone have been shown to have radical scavenger activity and may interfere with the oxidation of fluorescent dyes such as DHR-123 (Kim et al., 2005; Ordoudi et al., 2006). Performing another assay to assess ROS production such as the ferrous ion oxidation in the presence of xylenol orange (FOX) assay, which is selective for hydrogen peroxide formation (Jiang et al., 1991), would clarify our results. Hydrogen peroxide formation has been previously shown to occur after benzene metabolite exposure (Rao and Snyder, 1995). To address whether peroxide was formed in HD3 cells after benzene metabolite exposure, we tried to attenuate observed ROS and c-Myb activity responses by incubating cells with 200 U/ml of PEG-catalase for 1 h prior to benzene meta-

Fig. 6. c-Myb protein levels as measured through SDS–PAGE followed by western blotting. HD3 cells were exposed to 5 or 50 μM of (A) benzoquinone or (B) hydroquinone for 6 or 24 h. Data are expressed as normalized c-Myb/αtubulin ratios + SD from 7 (benzoquinone) or 8 (hydroquinone) independent experiments. c-Myb protein levels did not differ between treatment groups. Representative blots are shown above each graph.

Fig. 7. Phosphorylated c-Myb protein levels after exposure to 50 μM of benzoquinone (BQ) or 50 μM of hydroquinone (HQ) for 20 h. Whole cell lysates were processed by a commercially available phospho-protein purification kit followed by SDS–PAGE and western blotting. Bars represent the mean c-Myb/ α-tubulin ratios + SD from 5 independent experiments. Statistically significant differences between exposed cells and control cells have been indicated with an asterisk (p b 0.05). Representative blots have been displayed above the graph.

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Fig. 8. c-Myb activity after HD3 cell pretreatment with PEG-SOD (P.SOD) followed by exposure to (A) 50 μM of benzoquinone, (B) 50 μM of hydroquinone, or (C) 100 μM catechol exposure for 24 h. Data are expressed as a percentage of relative luciferase units of unexposed controls + SD from 3 independent determinations. ⁎ indicates statistically significant difference from unexposed controls p b 0.05). α indicates statistically significant difference from PEG-SOD pretreated controls (p b 0.05). γ indicates statistically significant difference from the cells exposed to both PEG-SOD and benzene metabolite (p b 0.05).

bolite exposure (data not shown). However, we were not able to modify responses for any of the metabolites (data not shown). Surprisingly, catalase levels in the HD3 cell line decreased after exposure to 200 U/ml of PEG-catalase for 1 h or 24 h as measured through western blotting (data not shown). It is unclear why this particular phenomenon was observed as catalase has been successfully used in HL60 cells and CHO cells to prevent benzene-mediated damage (Hiraku and Kawanishi, 1996; Winn, 2003). The decreases in GSH:GSSG ratios indicate that benzene’s metabolites caused oxidative stress in HD3 cells and this was observed as early as 1 h after exposure with benzoquinone. The measurement of GSH:GSSG through enzymatic recycling is an accepted method of measuring oxidative stress (Pastore et al., 2003). However, as we did not measure protein content in our acidified samples, we cannot accurately assess total glutathione levels with this assay. Therefore, the decrease in GSH:GSSG ratios could be due to conjugation of benzene’s metabolites to

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glutathione (Ludewig et al., 1989) or through the detoxification of hydrogen peroxide via glutathione peroxidase (Colinas et al., 1996). Increases in c-Myb signaling occurred at the same time as increased ROS and oxidative stress were measured. Furthermore, the use of the antioxidant enzyme SOD was able to completely or partially abrogate increases in reporter gene activation and ROS production in our cells after benzoquinone and hydroquinone, but not catechol exposure. This suggests that ROS, in particular superoxide, has a role in altering c-Myb activity. The increases in reporter construct activation after hydroquinone, benzoquinone, and catechol exposure were not due to increased c-Myb protein expression. Rather, phosphorylation of c-Myb appears to have been responsible for increased activity. Similar to our previously reported findings for catechol, we observed significant increases in phosphorylated c-Myb protein levels after benzoquinone and hydroquinone exposure (Wan and Winn, 2004). Oxidative stress has been shown to alter the phosphorylation status of various protein signaling pathways (reviewed in Lopez-Neblina and Toledo-Pereyra, 2006). Phosphorylation of c-Myb at a casein kinase II (CK2) site located at serines 11 and 12 was found to increase DNA binding (Ramsay et al., 1995), but hyperphosphorylation of c-Myb increased its proteolytic breakdown and decreased its DNA binding affinity (Bies et al., 2000). In our hands HD3 cell transfection with the Pim-1 kinase, a known phosphorylator of c-Myb (Winn et al., 2003), increased c-Myb reporter gene activation (Wan and Winn, 2004). In our cell system, it is possible that phosphorylation of c-Myb is a reason for the increased reporter gene activation that was observed; however we are currently evaluating whether other upstream co-activators of cMyb such as the p300 acetyltransferase (Tomita et al., 2000) and CREB-binding protein (CBP) (Dai et al., 1996) are involved in increasing c-Myb activity after benzene metabolite exposure. Other co-activators of c-Myb include the CAAT-enhancer binding protein family (C/EBP) (Tahirov et al., 2002) and p100 (Leverson et al., 1998). c-Myb was also shown to be redox sensitive (Myrset et al., 1993) and oxidative stress could have altered c-Myb protein conformation and thus affect reporter construct activation. Other studies have shown that benzene’s metabolites, through ROS, can alter signaling pathways. As an example, the ability of ROS from benzene metabolite exposure to alter protein activity has been observed for the MAP/ERK pathway (Ruiz-Ramos et al., 2005). Furthermore, activator protein-1 (AP-1) and NF-κB DNA binding was found to increase after exposure to the benzene metabolite trans,trans-muconaldehyde in HL60 cells and after benzene exposure in mice (Ho and Witz, 1997). Benzene exposure has also been shown to increase protein levels of the cyclin-dependent kinase inhibitor p21, via a p53 dependent mechanism, in C57BL/6 mouse myeloid progenitor cells (Yoon et al., 2001). Our studies have shown that some of benzene’s metabolites can produce ROS in HD3 cells and that this ROS can lead to increases in c-Myb signaling. Furthermore, different benzene metabolites produced different species of ROS. We showed that hydroquinone and benzoquinone, but not catechol lead to the production of superoxide in our cells.

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The concentrations of benzene, phenol, hydroquinone, catechol, and benzoquinone were higher than what humans would be exposed to on a daily basis. Similar concentrations have been previously used to investigate rat embryo toxicity (Chapman et al., 1994), clastogenicity in human lymphocytes (Anderson et al., 1995), and sister chromatid exchange in Syrian hamster embryo cells (Tsutsui et al., 1997). With the exception of benzoquinone, the concentrations of metabolites used were not cytotoxic. We hypothesize that the effects observed with benzoquinone were not due to cytotoxicity as similar ROS and cMyb signaling phenomena were observed with non-cytotoxic concentrations of hydroquinone. Furthermore, western blotting analysis in our laboratory found that HD3 cells had less than 0.05-fold myeloperoxidase content than HL60 cells and mouse bone marrow, and low amounts of CYP2E1 using rat liver microsomes as a loading control (data not shown). The lower levels of metabolizing enzymes could account for less cytotoxicity observed with this cell line and the amount of time required for effects to occur. In conclusion, our results support the hypothesis that benzene-mediated leukemogenesis may occur through the cMyb signaling pathway. This mechanism is metabolite and ROS dependent and involves activation of c-Myb through phosphorylation rather than increases in c-Myb protein expression. Given that exposure to environmental toxicants, including benzene, have been proposed to be a primary cause for the increase in certain leukemias in developed countries, further elucidation of how benzene mediates leukemogenesis is certainly warranted. Acknowledgments This work was supported by a research grant from the Canadian Institutes of Health Research (CIHR). L.M.W. is the recipient of a HRF/CIHR Rx&D Career Researcher Award. J.W. is a recipient of a CIHR Doctoral Research Award. References Adler, V., Yin, Z., Tew, K.D., Ronai, Z., 1999. Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18, 6104–6111. Anderson, D., Yu, T.W., Schmezer, P., 1995. An investigation of the DNAdamaging ability of benzene and its metabolites in human lymphocytes, using the comet assay. Environ. Mol. Mutagen. 26, 305–314. Baer, M.R., Augustinos, P., Kinniburgh, A.J., 1992. Defective c-myc and c-myb RNA turnover in acute myeloid leukemia cells. Blood 79, 1319–1326. Baker, M.A., Cerniglia, G.J., Zaman, A., 1990. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal. Biochem. 190, 360–365. Beckman, J.S., Minor Jr., R.L., White, C.W., Repine, J.E., Rosen, G.M., Freeman, B.A., 1988. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance. J. Biol. Chem. 263, 6884–6892. Bies, J., Feikova, S., Bottaro, D.P., Wolff, L., 2000. Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/ Thr protein phosphatases. Oncogene 19, 2846–2854. Biroccio, A., Benassi, B., D’Agnano, I., D’Angelo, C., Buglioni, S., Mottolese, M., Ricciotti, A., Citro, G., Cosimelli, M., Ramsay, R.G., Calabretta, B., Zupi, G., 2001. c-Myb and Bcl-x overexpression predicts poor prognosis in colorectal cancer: clinical and experimental findings. Am. J. Pathol. 158, 1289–1299.

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