NF-E2 Related Factor 2 Activation and Heme Oxygenase-1 Induction

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Chem. Res. Toxicol. 2007, 20, 1242–1251

Articles NF-E2 Related Factor 2 Activation and Heme Oxygenase-1 Induction by tert-Butylhydroquinone Protect against Deltamethrin-Mediated Oxidative Stress in PC12 Cells Huang Yuan Li,† Yu Fang Zhong,‡ Si Ying Wu,§ and Nian Shi*,| Department of Occupational and EnVironmental Health, Major Subject of EnVironment and Health of Fujian Key UniVersities, School of Public Health, Fujian Medical UniVersity, Fuzhou 350004 China, Institute of EnVironmental Pollution and Health, School of EnVironmental and Chemical Engineering, Shanghai UniVersity, Shanghai 200072 China, Department of Epidemiology and Health Statistics, Major Subject of EnVironment and Health of Fujian Key UniVersities, School of Public Health, Fujian Medical UniVersity, Fuzhou 350004 China, and Department of Health Toxicology, MOE Key lab of EnVironment and Health, School of Public Health, Tongji Medical College, Huazhong UniVersity of Science and Technology, Wuhan 430030 China ReceiVed March 10, 2007

Recent findings suggest that oxidative stress caused by pyrethroid pesticides could be closely involved in the neurotoxicity. tert-Butylhydroquinone (tBHQ) is a known inducer of Nrf2-mediated transcription, and treatment of cells with tBHQ can confer protection against H2O2 and 6-hydroxydopamine (6-OHDA). In this study, we investigated the neuroprotective effect of tBHQ against deltamethrin (DM)-induced oxidative stress using rat PC12 adrenal pheochromocytoma cells. The pretreatment of PC12 cells with tBHQ significantly reduced DM-induced generation of reactive oxygen species (ROS) and increased intracellular ionized calcium ([Ca2+]i). We also observed that DM or tBHQ induced the expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), a Nrf2-regulated gene. In addition, the Nrf2 antioxidant responsive element (ARE) pathways activated by tBHQ caused a partial inhibition of the DM-induced Nrf2 and HO-1 expression. Altogether, our data clearly indicate that an activation of Nrf2/ARE pathways in PC12 cells by tBHQ treatment protects cells from DM-induced oxidative stress and regulates DM- mediated adaptive responses in PC12 cells via translocation of Nrf2. Introduction Pyrethroid pesticides, the major class of insecticides, are commonly used in agriculture and urban settings due to their high potency and selectivity as nerve poisons and low persistent residues compared with other classes of insecticides (1). Human exposure to pyrethroids is widespread. Deltamethrin ((S)a-cyano3-phenoxybenzyl-(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate, DM), one of the most potent pyrethroid insecticides with a cyano substituant, produces the prototypical type II neurological syndrome (also named “choreoathetosis with salivation” or “CS” syndrome) of pyrethroids, which is characterized by salivation without lacrimation followed by jerking leg movements and progressive writhing convulsions (choreoathetosis) (1, 2). We have previously shown that deltamethrin inhibits dopamine biosynthesis in dopamine content by the reduction of tyrosine * To whom correspondence should be addressed. Mailing address: Department of Health Toxicology, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road No.13, Wuhan 430030, China. E-mail: [email protected]. Tel.: +86 27 83692720. † Department of Occupational and Environmental Health, Fujian Medical University. ‡ Institute of Environmental Pollution and Health, Shanghai University. § Department of Epidemiology and Health Statistics, Fujian Medical University. | Huazhong University of Science and Technology.

hydroxylase mRNA and protein levels in PC12 cells (3, 4) and causes increased apoptosis in brain that is accompanied by increased expression of p53 and Bax, which are pro-apoptotic, and decreased expression of Bcl-2, which is antiapoptotic (5–7). Recently, Bloomquist and co-workers showed that deltamethrin selectively increases dopamine release and uptake in the dopaminergic nerve terminals of the striatum in mice (8). These data provide further evidence supporting the hypothesis that environmental/occupational exposure to pyrethroid pesticides may produce specific damage to dopaminergic neuronal cells. Although numerous studies have been conducted, the molecular mechanism underlying pyrethroid neurotoxicity is still not well understood. It has been reported that the induction of oxidative stress and alteration of antioxidant system by pyrethroids occur in rats (9–12). Accumulating evidence indicates that oxidative stress might be responsible for the progression of dopaminergic neurodegeneration (13). Overproduction of reactive oxygen species (ROS) injured cell components such as lipids and DNA and resulted in the impairment of mitochondrial respiration followed by energy failure (14, 15). The induction of genes that encode these antioxidant enzymes has been reported to be mediated via activation of the antioxidant responsive element (ARE) located in the 5′-flanking region of these genes (16–18). The ability of Nrf2 to up-regulate the expression of antioxidant genes via ARE suggests that increasing Nrf2 activity may

10.1021/tx700076q CCC: $37.00  2007 American Chemical Society Published on Web 08/04/2007

Nrf2 ActiVation by tBHQ and Deltamethrin

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Table 1. Primer Sequences Used in RT-PCR Analysis gene name

accession no.

primer sequence for PCR

size of expected product (bp)

HO-1

NM 012580

441

GAPDH

XM 216696

5′-ACAGAAGAGGCTAAGACCG-3′ 3′-TTACCTTCCTCTACGGAC-5′ 5′-AAATGGGTGATGCTGGTG-3′ 3′-TGAGCGAGTTCTAACAGTCG-5′

provide a useful system to combat oxidative insults. Overexpression of Nrf2 in cell culture protects against oxidative damage elicited by nitric oxide, H2O2, and glutamate (19, 20). Because the Keap1–Nrf2 pathway is an inducible system, various chemical agents have been identified that can stimulate the pathway, leading to up-regulation of cytoprotective genes. tertButylhydroquinone (tBHQ) is a known inducer of Nrf2-mediated transcription and treatment of cells with tBHQ can confer protection against H2O2 and 6-hydroxydopamine (6-OHDA) (21–23). These in vitro studies provide support for up-regulating Nrf2 activity to neutralize oxidative insults. The chemical induction of antioxidant and detoxification genes has been shown to be protective against a number of different oxidative insults, including H2O2, 6-OHDA, and 3-morpholinosydnonimine (SIN1) but not against 1-methyl-4-phenylpyridinium (MPP+) toxicity (21–24). To further expand on the types of oxidative damage that may be attenuated by up-regulating Nrf2 activity, we studied the effects of DM-mediated oxidative insult. In the present study, we investigated whether induction of the Nrf2 with tBHQ would protect against DM-mediated oxidative stress using PC12 cells as an in vitro model. The results presented show that the pretreatment of PC12 cells with tBHQ significantly reduced DM-induced generation of reactive oxygen species (ROS) and the increase of intracellular ionized calcium ([Ca2+]i). We also observed that DM or tBHQ induced the expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), a Nrf2-regulated gene. In addition, the Nrf2-antioxidant responsive element (ARE) pathways activated by tBHQ caused a partial inhibition of the DM-induced Nrf2 and HO-1 expression. The present study found that Nrf2 activation and subsequent antioxidant protection may be a mechanism by which tBHQ protect neuronal cell against DM neurotoxicity.

Materials and Methods Reagents and Instruments. Deltamethrin (98.5%) was obtained from Roussel-Uclaf Corp (Romainville Cedex, France). 6-OHDA (6-OHDA hydrobromide with ascorbic acid, Sigma-Aldrich, St. Louis, MO) was kind gift from Dr. Yang Peng (HUST). Fura-2/ AM (Calbiochem, San Diego, CA) TRIZOL reagent was obtained from Invitrogen Life Technologies (California). rTaq (recombinant Taq) DNA polymerase, ReverTra Ace (Moloney murine leukemia virus reverse transcriptase RNaseH-), RNase inhibitor were obtained from Toyobo Co., Ltd. (Osaka, Japan). All primers were synthesized by Bioasia Biologic Technology Co., Ltd. (Shanghai, China). Random primers were obtained from Promega (Shanghai, China); dNTPs were obtained from Shengong Co., Ltd. (Shanghai, China). Anti-Nrf2 antibodies (C-20, sc-722, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were kind gift from Professor Ma Qiang (National Institute for Occupational Safety and Health, Atlanta, GA). Anti-HO-1 antibodies (Santa Cruz Biotechnology, Inc.), DMEM (HyClone, Utah), and rabbit streptavidin/peroxidase-9000 Kit were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG antibodies were purchased from Proteintech Group Inc. (Chicago, IL). tert-Butylhydroquinone (tBHQ), dimethyl sulfoxide (DMSO), 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA), RNase A, and propidium iodide (PI) were purchased from

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Sigma Chemical Co. (St. Louis, MO). NC membranes were from Millipore Company (Bedford, MA). Enhanced chemiluminescence reagent were from HyClone-Pierce Company. X-ray film was from KODAK (Xiamen, China). T-Gradient thermoblock PCR (Biometra CO., Germany), HVE50 gel imaging system, electrophoresis chamber (Bio-Rad CO.), RF5300 fluorescent spectrophotometer (Hitachi, Japan), ultraviolet spectrophotometer (Shimadzu Co., Japan), Olympus FV500 laser scan confocal microscopy (LSCM) (Olympus Co., Japan), and CO2 constant temperature incubator (Sheldon) were used. PC12 Cells Culture and Treatment. PC12 cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere of 10% CO2/90% air. The medium was changed every other day, and cells were plated at an appropriate density according to each experimental scale. After 24 h incubation, cells were switched to medium-free heat-inactivated fetal bovine serum for treatment. Cells were incubated with or without 40 µM tBHQ for 16 h, followed by exposure to 10 µM DM for 1 h. DM and tBHQ were dissolved in DMSO. The final concentration of DMSO was 0.1% (v/v) and had no effect on the parameters measured. Assay of the Cellular Contents of Reactive Oxygen Species. The intracellular generation of reactive oxygen species was measured using 2′,7′-dichlorofluorescin, which is oxidized to the fluorescent form 2′,7′-dichlorofluorescein by H2O2 and other reactive oxygen species (25). ROS was measured as described previously (25) with modifications. Briefly, after treatment with DM or vehicle, the treated cells were washed three times with icecold phosphate-buffered saline (PBS) and then incubated with 10 µM 2′,7′-dichlorofluorescin diacetate (2′,7′-dichlorodihydrofluorescein diacetate; 100 mM in dimethyl sulfoxide) for 30 min at 37 °C. The cellular free radical content was assayed by measuring 2′,7′dichlorofluorescein fluorescence by using a fluorescent spectrophotometer (excitation at 485 nm/emission at 535 nm, bandwidth 5 nm). Intracellular Ca2+ Measurements. The intracellular free calcium levels ([Ca2+]i) in PC12 cells were quantified by fluorescence of the Ca2+ indicator dye fura-2 as described previously (26). Preparation of Cytosolic and Nuclear Extracts. Cells were treated with various chemicals as detailed in the respective figure legends. Nuclear and cytosolic extracts were isolated as described previously (27) with modifications. Briefly, cells were washed with cold PBS and resuspended in cold buffer A (lysis buffer 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.5 mg/mL benzamidine, 0.1 mg/mL leupeptin, and 1.2 mg/mL aprotinin). The cells were allowed to swell on ice for 15 min; then 7.5 µL of 10% (v/v) NP-40 was added and vortex mixed vigorously for 10 s. The homogenate was centrifuged for 50 s at 16 000g, and the supernatant was used as cytosolic extract. The nuclear pellet was resuspended in cold buffer B (extraction buffer 20 mM HEPES, pH 8.0, 1 mM EDTA, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.5 mg/mL benzamidine, 0.1 mg/mL leupeptin,1.2 mg/mL aprotinin, and 20% glycerol). All the protein fractions were stored at -70 °C until use, and the protein concentrations were determined by Bradford method with bovine serum albumin as a standard. Immunoblot Analysis. Briefly, protein samples were resolved on 10% (w/v) SDS–polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were blocked for 1 h in Tris-buffered saline (TBS), 0.1% (v/v) Tween-20, and 5% (w/v) nonfat dry milk,

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Figure 1. DCF fluorescence intensity increased by DM was attenuated by tBHQ. PC12 cells were pretreated with 40 µM tBHQ or vehicle for 16 h. After the medium was removed, PC12 cells were exposed to 10 µM DM for 1 h. The treated cells were loaded with H2DCFDA (10 µM), and DCF fluroscence intensity (Arbitrary units of DCF fluroscence) was measured using a fluorescent spectrophotometer. Values are mean ( standard deviation of three determinations. * indicates P < 0.001 as compared with control group; § indicates P < 0.05 as compared with control group. ( indicates P < 0.001 as compared with DM group.

followed by an overnight incubation with primary antibody diluted in the same buffer (Nrf2 1:500, GAPDH 1:8000). After washing with 0.1% (v/v) Tween 20 in TBS, the membrane was incubated with peroxidase-conjugated secondary antibody for 1 h and then washed and developed using the ECL chemiluminescent detection system followed by visualization with X-ray film. Quantitation of the results was performed by Bandscan 4.3 software. Densitometric analyses were normalized against the signal obtained by coincubating the membranes with GAPDH. Immunocytochemical Detection of Nrf2 and HO-1. Nrf2 was immunocytochemically detected on the cells that first attached to the coverslips coated with poly(L-lysine) according to the method of Deptala et al. (28) with minor modifications. Briefly, at the end of treatment, medium was aspirated. Then the cells were washed with PBS and fixed in ice-cold 95% ethanol for 15 min at -20 °C twice. After washing in PBS, cells were permeabilized in 0.2% (v/v) Triton X-100 in PBS for 15 min, washed, and incubated with normal goat serum for 30 min at room temperature. The cells were first treated with anti-Nrf2 polyclonal rabbit antibody (C-20) (1: 200) or anti-HO-1 antibody (1:50) for 18 h at 4 °C and subsequently incubated with FITC-labeled goat antirabbit IgG (H+L) for 30 min at 37 °C. Cellular DNA was then counterstained by addition of 1 mL of a solution containing 5 µg/mL of propidium iodide (PI) and 100 µg/mL RNase A for 35 min at room temperature. After washing the cells with PBS, coverslips were mounted on microscope slides with 50% (v/v) glycerin buffer solution. The coverslips were put on the stage of the laser scanning confocal microscopy with oil objective lens of UPLAPO 100×, and experiment parameters were optimized to ensure optimal signal detection. The experiment parameters were maintained when all cases of the same item were determined. Data of images were recorded by Fluoview version 4.3.53. Images were analyzed by image-Pro Plus version 5 (Media Cyberbetics, Inc.) and Adobe Photoshop 8.0 (Adobe, Inc.). Nrf2 was detected immunocytochemically with FITC-labeled antibody, and its presence in the nucleus as compared with the cytoplasm was monitored by measuring the green (FITC) fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), over the cytoplasm, and over the whole cell, respectively. The image analysis was made by an investigator who was unaware of the cell identity. The steps of immunocytochemical detection of HO-1 were similar to those of Nrf2, except the step of PI counterstain. The expression

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Figure 2. Effect of tBHQ on the increase in [Ca2+]i induced by DM. PC12 cells were exposed to 40 µM tBHQ for 16 h following treatment with DM. Values are mean ( standard deviation of three determinations. * indicates P < 0.05 as compared with control group. # indicates P < 0.05 as compared with DM group.

of HO-1 in PC12 cells was monitored by measuring the green fluorescence over the whole cell. Semiquantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis. Total RNA was isolated from PC12 cells using TRIzol reagent according to manufacturer’s instructions. The purity and quantity of the RNA was determined by A260/A280 ratios and A260, respectively. For RT-PCR, first-strand cDNA was synthesized using random primers. Briefly, reverse transcription reactions (RT) were carried out in the reaction system of 20 µL reaction volume containing total RNA 2.0 µL (ca 3.0 µg), random primer 0.5 µL, 5× RT buffer 5.0 µL, 10 mmol/L dNTPs 2.0 µL, ReverTra ACE 1.0 µL (100 U), Rnase inhibitor 0.5 µL (20 U), diethylpyrocarbonate-treated (DEPC) H2O 14.0 µL. The temperature cycling conditions were as follows: incubation at 30 °C for 10 min, at 42 °C for 45 min, and then at 90 °C for 5 min. PCR was carried out sequentially. The volume of PCR reaction system is 50.0 µL,containing cDNA 1.0 µL, 10 pmol/µL of sense primer 2.0 µL, 10 pmol/µL of antisense primer 2.0 µL, rTaq enzyme 0.5 µL (2.5 U), 10× Taq enzyme buffer (added with Mg2+) 5.0 µL, 10 mmol/L dNTPs 1.0 µL, ddH2O 38.5 µL. The primers were designed by the software primer 5 and confirmed with the sequences in the NCBI database. The primer sequences selected from detected gene for cDNA amplification were shown in Table 1 (sense and antisense primer, respectively). The temperature cycling conditions were as follows: predenaturation at 94 °C for 3 min, 32 cycles of denaturation at 94 °C for 30 s, annealing 55 °C for 40 s, and extension at 72 °C for 40 s, and a final extension at 72 °C for 5 min. As an internal control, a 188-bp DNA fragment of a rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also amplified. In this experiment, 2 µg of total RNA was reverse transcribed in a 50 µL reaction volume. A 5 µL aliquot of each PCR amplified product were resolved by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light. For quantification, photographs showing PCR products were scanned by HVE50 and analyzed by using Biocapt MW Image software (Vilber Lourmat, France). For respective samples, the PCR product values were normalized to the GAPDH PCR product values. Statistical Analysis. Each data bar represents the mean values ( SD (standard deviations) of at least three independent experiments in all cases. Results were analyzed using SPSS (Statistical Package for the Social Science) for Windows (version 12.0). Differences between groups were analyzed by one-way analysis of variance (ANOVA). If the F values were significant, the least significant



Figure 3. Effect of tBHQ on DM-induced expression of Nrf2 protein in PC12 cells. (A) Cells were incubated with or without 40 µM tBHQ for 16 h, followed by exposure to 10 µM DM for 1 h. Protein expression was examined by immunoblot analysis. Equal amounts of protein (45 or 55 µg) extract were loaded in each lane of 8.0% SDS–PAGE gels, and the blot was probed with the polyclonal anti-Nrf2 antibody (1:500) overnight. The protein was visualized by chemiluminescence. A representative immunoblot of Nrf2 is shown. An immunoblot for GAPDH, a cytoplasmic marker, was used as internal control and indicated that the nuclear fraction was not contaminated by cytoplasmic fractions. (B) Quantification of Nrf2 densitometric analysis. Ratio ) relative level of Nrf2 protein expression in experimental group/relative level of Nrf2 expression in control group. Values are mean ( standard deviation of three determinations. * indicates P < 0.001 as compared with control group; dotted cross symbol indicates P < 0.05 as compared with control group. # indicates P < 0.001 as compared with DM group; f indicates P < 0.01 as compared with DM group. ∆ indicates P < 0.001 as compared with tBHQ group.

difference (LSD) posthoc test was used to compare multiple groups. A P value of e0.05 was considered statistically significant in all cases.

Results Pretreatment with tBHQ Reduced DM-Induced ROS Generation and the Increase of Intracellular Ionized Calcium ([Ca2+]i). DM-induced neuronal cell death seems to be caused by ROS generation. First, we observed that DM markedly induced ROS production in PC12 cells (Figure 1). To address the mechanism by which tBHQ reduces DM-induced cell oxidative stress, we examined whether pretreatment of cells with tBHQ suppresses DM-induced ROS generation. As shown in Figure 1, the pretreatment of cells with 40 µM tBHQ for 16 h significantly reduced DM-induced ROS generation, whereas tBHQ treatment alone showed no effect on ROS generation. In addition, it has been demonstrated that increase of intracellular ionized calcium ([Ca2+]i) plays a key role in the cell death process caused by DM. We examined the effects of tBHQ pretreatment on DM-induced increase of [Ca2+]i. The exposure of PC12 cells to 10 µM DM for 1 h induced the increase of [Ca2+]i (Figure 2). The pretreatment of cells with 40 µM tBHQ for 16 h reduced DM-induced increase of [Ca2+]i, whereas tBHQ treatment alone increased the amount of [Ca2+]i (Figure 2). Taken together, these data indicate that DM stimulates both oxidative stress and intracellular calcium signaling events in

PC12 cells, whereas pretreatment with tBHQ suppresses these cell responses to DM. Effect of DM or 6-OHDA on Nrf2 Activation and HO-1 Expression. PC12 cells were extracted and fractionated into cytosolic and nuclear fractions to determine the relative distribution of Nrf2. In both the nuclear and cytosolic fractions, there was an increase in Nrf2 protein levels in the PC12 cells treated with DM compared with cells treated with vehicle (Figures 3 and 4). The fractionation between nucleus and cytosol was specific, as indicated by the presence of the cytosolic marker GAPDH only in the cytosolic fraction (Figure 3). Densitometric analyses revealed a 1.3-fold and 2.4-fold increase in Nrf2 levels in the nuclear and cytoplasmic fractions, respectively. We also noticed that the Nrf2 signal in the cytoplasmic fraction was a single band, whereas Nrf2 signal appeared as a triplication in the nuclear fraction. This difference could be due to the antibody recognizing a nonspecific band not present in the cytoplasm or could indicate the presence of a modified isoform of Nrf2 not present in the cytoplasm. In addition, Nrf2 migrated with an apparent molecular weight of about 100 kDa, which is significantly larger than the predicted size of 66 kDa. This anomalous migration has been previously described in other systems (29, 30). According to the methods of Shukla et al. (31), the proteins from nuclear fraction resolved on parallel gels were either stained for protein with Coomassie brilliant blue or analyzed for target protein by immunoblot. Except for an obvious elevation in the 100 kDa molecular weight fraction,


Li et al.

Figure 4. Effect of tBHQ on DM-increased Nrf2 immunofluorescent intensity in PC12 cells. PC12 cells were grown on coverslips and treated with or without tBHQ, followed by DM exposure for 1 h. Nrf2 subcellular localization was determined as described in Materials and Methods. Coverslips were mounted on glass slides with fluorescent mounting medium and visualized using a LSCM with a 100× objective. (A) Shown are images reprentative of a typical experiment. Scale bar ) 20 µm. (B) Quantification of Nrf2 immunofluorescent intensity. Ratio ) Nrf2 immunofluorescent intensity of experiment group/Nrf2 immunofluorescent intensity of control group. Values are mean ( standard deviation of three determinations. * indicates P < 0.05, dotted cross symbol indicates P < 0.001, and f indicates P < 0.001 as compared with control group. # indicates P < 0.05 and g indicates P < 0.001 as compared with DM group. ∆ indicates P < 0.05 as compared with tBHQ group.

the protein pattern in PC12 cells treated with DM or tBHQ is very similar to the pattern observed in cells treated with vehicle, suggesting that the nuclear protein loaded in the gel lane was strictly equal (data not shown) and the 100 kDa band represented specific binding of the Nrf2 antibody. The increased Nrf2 expression induced by DM was accompanied by Nrf2 nuclear translocation as determined by immunofluorescence (Figure 4). We used anti-Nrf2 antibodies and LSCM to visualize the subcellular distribution of Nrf2 and to semiquantitatively determine Nrf2 expression. In PC12 cells treated with vehicle, Nrf2 fluorescence was found to distribute throughout the cells including cytoplasm and nucleus (Figure 4). The changes in fluorescence of PC12 cells treated with DM are shown in Figure 4. Administration of DM for 1 h led to the obvious increase in the green (FITC) fluorescence intensity

measured over cell (Fcell), as well as in the integrated green (FITC) fluorescence intensity measured over nuclear (Fn) and cytoplasmic area (Fc) compared with control group. The Fn/Fc ratio was markedly increased in DM-treated PC12 cells (Figure 4). The enhanced Nrf2 fluorescence intensity of cell and increased Fn/Fc ratio in PC12 cells treated with DM suggests that DM induces Nrf2 expression and activates Nrf2, respectively. In fact, fluorescence microscopy of Nrf2 confirmed the increased localization of Nrf2 into the nucleus. The enhanced expression of Nrf2 and its presence in the nucleus suggest that the message levels of antioxidant and phase II detoxification genes would be increased due to inducible activity of Nrf2 on ARE-containing genes. To address this issue, we looked at the message level of HO-1, a known Nrf2regulated antioxidant gene, by RT-PCR analysis. Within 1 h



Figure 5. Effect of tBHQ on DM-induced expression of HO-1 mRNA in PC12 cells. Cells were incubated with or without 40 µM tBHQ for 16 h, followed by exposure to 10 µM DM for 1 h. mRNA expression was examined by semiquantitative RT-PCR. Values are mean ( standard deviation of three determinations. Ratio ) relative level of HO-1 mRNA expression in experimental group/relative level of HO-1 mRNA expression in control group: * indicates P < 0.001 as compared with control group; # indicates P < 0.001 as compared with DM group; ∆ indicates P < 0.001 as compared with tBHQ group.

after treatment of PC12 cells with DM, HO-1 mRNA was increased by 1.7-fold and remained elevated for at least 17 h (Figure 5). Increased levels of HO-1 mRNA paralleled a greater increase in HO-1 protein (Figure 6), which was approximately 6.4-fold (vs control) 1 h after treatment. Some studies indicated that DM may produce specific damage to dopaminergic neuronal cells (3, 4, 8). 6-OHDA is a potent neurotoxin widely used to model dopaminergic degeneration in both in vitro and in vivo models of Parkinson’s disease (PD). To confirm that the neurotoxic action by DM is similar to the action of 6-OHDA, 6-OHDA treatment is used as a positive control for Nrf2 activation and HO-1 induction. HO-1 expression is known be regulated in part by the ARE and has been previously shown to respond to 6-OHDA in vivo and in vitro (32–34). Nrf2 activation and increased HO-1 expression is seen in 6-OHDA-treated cultures (Figure 7). These data suggested that the PC12 cell response for DM is similar to the response for 6-OHDA in Nrf2 activation and increased HO-1 expression. tBHQ Induces the Expression of HO-1 via the Activation of Nrf2. The level of Nrf2 protein is kept relatively low under basal conditions due to proteasomal degradation of Nrf2. Activation of the Keap1–Nrf2 pathway leads to stabilization of Nrf2 protein by freeing it from Keap1 repression (35). To determine whether the Keap1–Nrf2 pathway is active in PC12 cells, we examined the stability of Nrf2 protein following treatment with the chemical inducer tBHQ. Nrf2 protein is barely detected in untreated PC12 cells but is dramatically up-regulated

after treatment with 40 µM tBHQ for 16 h post-treatment (Figure 3). tBHQ also stimulated an increase in Nrf2 nuclear translocation in the PC12 cells as determined by immunoblot and immunocytochemistry (Figures 3 and 4). Treatment with both tBHQ and DM does significantly decrease Nrf2 induction over tBHQ alone (Figure 3). This suggests that DM suppress the Nrf2 induction by tBHQ pretreatment. In addition to stimulating Nrf2 translocation, treatment of PC12 cells with tBHQ also induced HO-1 mRNA and protein expression (Figures 5 and 6), indicating that the antioxidant response pathway was induced at 40 µM. Furthermore, the addition of antioxidant tBHQ caused a partial inhibition of the DM-induced HO-1 expression (Figures 5 and 6).

Discussion In this study, we demonstrated that the pretreatment of PC12 cells with tBHQ reduced DM-induced oxidative stress. The pretreatment with tBHQ suppressed DM-induced ROS generation and the increase of intracellular ionized calcium. The generation of excess reactive oxygen species (ROS) and uncontrolled increases in intracellular ionized calcium ([Ca2+]i) are two events common to many forms of neurodegeneration (36–39). The studies from our laboratory show that exposure to high dose of deltamethrin would interfere with intracellular free Ca2 + concentration and apoptotic rate in rat neural cells, and increased [Ca2+]i is thought to play a multiple role in the complex pathways leading to neuronal apoptosis (40, 41).


Li et al.

Figure 6. Effect of tBHQ on DM-increased HO-1 immunofluorescent intensity in PC12 cells. PC12 cells were grown on coverslips and treated with or without 40 µM tBHQ, followed by DM exposure for 1 h. HO-1 protein level was determined as described in Materials and Methods. Coverslips were mounted on glass slides with fluorescent mounting medium and visualized using a LSCM with a 100× objective. (A) Shown are images reprentative of a typical experiment. (B) Quantification of HO-1 immunofluorescent intensity. Ratio ) HO-1 immunofluorescent intensity of experiment group/HO-1 immunofluorescent intensity of control group. Values are mean ( standard deviation of three determinations: * indicates P < 0.001 as compared with control group; # indicates P < 0.001 as compared with DM group; ∆ indicates P < 0.001 as compared with tBHQ group.

DM may contribute to cell death by increasing intracellular ionized calcium ([Ca2+]i), suggesting that tBHQ may in part confer protection by altering DM-induced [Ca2+]i signals. To examine this possibility, we measured [Ca2+]i of fura-2-loaded cultures of tBHQ- and vehicle-pretreated cells during DM treatment. DM exposure induced an increase in [Ca2+]i that was significantly higher in tBHQ-pretreated cells. Thus, antioxidant upregulation may contribute to protection during oxidative stress by stabilizing [Ca2+]i. However, this inhibition of DM-enhanced intercellular calcium level by tBHQ was not absolute, suggesting that the effect may not be solely due to oxidative stress. In addition, since oxidative stress may induce cytotoxicity by multiple pathways, [Ca2+]i stabilization may not be the only mechanism responsible for the protective effect of tBHQ. DM caused increased ROS production, increased [Ca2+]i, and eventually neuronal cell death via apoptosis (12, 40, 41). Therefore, the cells’ antioxidant potential and calcium-buffering capacity are very important in protecting neurons from the harmful effects of this neurotoxin. Nrf2 plays an important role in the maintenance of calcium homeostasis as well as antioxidant potential. Lee et al. reported that Nrf2-/- neurons were more susceptible to increased [Ca2+]i induced by ionomycin or 2,5di-(t-butyl)-1,4-hydroquinone, and microarray analysis identified several calcium-binding proteins and calcium sensors that play an important role in neuroprotection against increased [Ca2+]i. (42). To determine the mechanism by which the pretreatment with tBHQ reduces DM-induced ROS generation and [Ca2+]i elevation, we examined the effect of tBHQ on the expression of HO-1 in PC12 cells. Treatment of cells with 40 µM tBHQ for 16 h induced HO-1 mRNA and protein expression (Figures 5 and 6). The induction of HO-1 by the treatment with tBHQ should increase the intracellular antioxidative potential, leading to an

enhancement of resistance to cell injuries associated with oxidative stress. It has been reported that the induction of HO-1 expression by tBHQ is mediated via the activation of ARE in human neuroblastoma IMR32 cells (43). Using the immunoblot and fluorescence microscopy, therefore, we examined whether tBHQ causes the activation of Nrf2 in PC12 cells. In the present study, Nrf2 was found to be markedly induced in PC12 cells exposed to a nontoxic concentration of tBHQ. The translocation of Nrf2 into the nucleus following tBHQ treatment was associated with a marked increase in HO-1 expression. The direct implication of Nrf2 in the regulation of the HO-1 gene via the ARE has been recently demonstrated by utilizing Nrf2 dominant negative mutants (Nrf2M). It has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury (44, 45). This agrees with previous work that HO enzyme activity and HO-1 expression of both protein and mRNA were increased by DM treatment in vivo (46). The induction of HO-1 contributes to the protection against DM-induced oxidative stress. Treating cells with tBHQ, a strong inducer of phase II detoxification enzymes via activation of Nrf2, can protect cells from oxidative stress (47). The protective effect conferred by tBHQ may not simply be due to an increase in one gene but the coordinate up-regulation of many genes. Recent reports demonstrated that the apoptotic process and development of cell injury in human neuroblastoma cells or primary neural cultures resulting from H2O2-induced oxidative stress was attenuated through tBHQ activation of ARE (48, 49). In addition, primary neuronal or astrocytic cultures from Nrf2-/- mice showed increased sensitivity to H2O2-, rotenone-, or MPP+-induced



Figure 7. Effect of 6-OHDA on Nrf2 activation and HO-1 expression in PC12 cells. (A) Cells were incubated with 100 µM 6-OHDA for 17 h. Protein expression was examined by immunoblot analysis. Ratio ) relative level of Nrf2 protein expression in experimental group/relative level of Nrf2 protein expression in control group. (B) mRNA expression was examined by semiquantitative RT-PCR. Ratio ) relative level of HO-1 mRNA expression in experimental group/relative level of HO-1 mRNA expression in control group. (C) HO-1 protein level was determined as described in Materials and Methods. Coverslips were mounted on glass slides with fluorescent mounting medium and visualized using a LSCM with a 100× objective. Shown are images representative of a typical experiment. Ratio ) HO-1 immunofluorescent intensity of experiment group/HO-1 immunofluorescent intensity of control group. Values are mean ( standard deviation of three determinations. * indicates P < 0.001 and dotted cross symbol indicates P < 0.05 as compared with control group.


cytotoxicity. Microarray analysis dissected a cluster of detoxification enzymes and antioxidant genes, which may be involved in the protective effect observed by treatments with tBHQ (20, 24, 25). All of these findings suggest that Nrf2-dependent ARE activation by tBHQ is responsible for up-regulation of many key genes, such as HO-1, NQO1, GCLR, ferritin, thioredoxin reductase (TR), glutathione reductase (GR), copper/ zinc superoxide dismutase (SOD1), and multiple heat shock proteins, that together construct a potent antioxidant network. Pretreatment with tBHQ reduced but did not abolish both Nrf2 and HO-1 activation by DM. Moreover, induction of the Nrf2/ARE–HO-1 pathway with tBHQ was able to significantly reduce cell oxidative stress due to DM in vitro. These results suggest that tBHQ reduces cell oxidative stress due to DM in vitro through this pathway of Nrf2/ARE. 6-Hydroxydopamine (6-OHDA), a hydroxylated analog of dopamine (DA), is potent neurotoxin widely used to model dopaminergic degeneration in both in vitro and in vivo models of PD (49). 6-OHDA undergoes autoxidation to generate reactive quinones, H2O2 and free radicals (49). Treatment with this agent causes the generation of ROS and subsequent apoptotic cell death in cultured neural cells and neuroblastoma cell lines. Here we show that 6- OHDA or DM similarly activates the Nrf2 and HO-1 in cultured PC12 cells, suggesting that the PC12 cells response for DM is similar to the response for 6-OHDA in Nrf2 activation and increased HO-1 expression. Recently it has been demonstrated that up-regulation of Nrf2 activity by a variety of measures provided significant protection against 6-OHDA toxicity in SH-SY5Y cells (24). The relative levels and types of free radicals generated from these treatments may be same and could be quenched by the antioxidant genes regulated by Nrf2. Although the Nrf2 protein is induced by 6-OHDA or DM, it is clear that this cell host response is insufficient to quell toxicity. However, further induction of the Nrf2 may protect against cell oxidative stress. Preliminary in vitro data shown herein imply that preactivation with tBHQ can protect against DM-induced cell oxidative stress. Calkins et al. have shown that Nrf2-mediated protection is efficacious in the malonate model of Huntington’s disease (50). Based on these results, Nrf2-mediated protection could be efficacious in neurotoxicity induced by DM in vivo. In conclusion, the treatment with tBHQ conferred resistance against DM-induced oxidative stress in PC12 cells. The protective effect of tBHQ seems to be due to an increase of antioxidative potential, such as induction of HO-1, via Nrf2 activation. In this study, we assessed cell oxidative stress using assays that measure ROS production and increase of intracellular ionized calcium, which may represent the type of cell oxidative stress initiated by the oxidative insults. Future studies incorporating cell death assays will be needed to fully understand the relationship between Nrf2 activation and DM-mediated cell toxicity. The results presented indicate that increasing Nrf2 transcription of ARE-containing genes can be a potentially useful mechanism to protect cells against oxidative stress. Therefore Nrf2 activation and stabilization by pharmacological modulation (tBHQ) might be viable strategies to prevent a wide spectrum of oxidative stress-related neuronal cell injuries. Acknowledgment. This work was supported by National Natural Science Foundation in China (Grant 30371225). AntiNrf2 antibodies were a kind gift from Prof. Ma Qiang (NIOSH). The technical help for LSCM and image analysis was supplied by Prof. Zhang Min-Hai of Laboratory of LSCM, Tongji Medical College, Huazhong University of Science and Technol-

Li et al.

ogy. We gratefully acknowledge the contribution of these professors to our work.

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