Induction of Reactive Oxygen Species by Bisphenol A and Abrogation of Bisphenol A-Induced Cell Injury by DJ-1

Hiromasa Ooe*,{dagger}, Takahiro Taira*,{dagger}, Sanae M. M. Iguchi-Ariga{dagger},{ddagger} and Hiroyoshi Ariga*,{dagger},1

* Graduate School of Pharmaceutical Sciences, {ddagger} Graduate School of Agriculture, Hokkaido University, Sapporo 060-0812, Japan, and {dagger} CREST, Japan Science, Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

1 To whom correspondence should be addressed at Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, 060-0812, Japan. Fax: 81–11–706–4988; E-mail: hiro{at}pharm.hokudai.ac.jp.

Received June 3, 2005; accepted August 8, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DJ-1 was first identified as an activated ras-dependent oncogene. DJ-1 is related to male fertility, and its expression in sperm decreases in response to exposure to a number of reproductive toxicants. DJ-1 has been associated with the onset of familial Parkinson's disease (PD) in humans, and has been found to have activity against oxidative damage by eliminating reactive oxygen species (ROS). In this study, we investigated the role of DJ-1 in oxidative stresses by administration of bisphenol A (BPA), which has been reported to induce oxidative stress in rodents, to male mice and cultured cells. In male mice, we found that BPA significantly increased the expression level of DJ-1 in the sperm and brain. In cultured Neuro2a and GC1 cells, we found that BPA induced ROS production and significantly compromised mitochondrial function concomitant with elevated expression and oxidization of DJ-1. DJ-1 was found to maintain the complex I activity against BPA-induced oxidative stress after the localization in mitochondria. The results showed that DJ-1 plays a role in the prevention of mitochondrial injury-induced cell death.

Key Words: bisphenol A; reactive oxygen species; DJ-1; cell death; oxidative stress; mitochondria.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DJ-1 was first identified by our group as a novel candidate of the oncogene product that transformed mouse NIH3T3 cells in cooperation with activated ras (Nagakubo et al., 1997Go). DJ-1 is 20-kDa protein comprising 189 amino acid residues that is expressed in various tissues with particularly high levels of expression in the testis, sperm, and brain (Bandopadhyay et al., 2004Go; Wagenfeld et al., 2000Go). DJ-1 was later found to be a positive regulator of the androgen receptor (Niki et al., 2003Go; Taira et al., 2004aGo; Takahashi et al., 2001Go) and p53 (Shinbo et al., 2005aGo) and to be a negative regulator of PTEN tumor suppressor (Kim et al., 2005Go). DJ-1 has also been reported to be related to infertility of rats and mice and to participate in fertilization for sperm to penetrate into the zonae pellucida of eggs (Klinefelter et al., 2002Go; Okada et al., 2002Go; Wagenfeld et al., 1998Go, 2000Go; Welch et al., 1998Go; Yoshida et al., 2003Go). Recently, DJ-1 has been shown to be responsible for onset of familial Parkinson's disease (PD), PARK7 (Bonifati et al., 2003Go), and 11 mutations in familial and sporadic forms of PD have been reported (Abou-Sleiman et al., 2003Go; Hague et al., 2003Go).

Reactive oxygen species (ROS), including superoxide hydrogen peroxide (H2O2), and hydroxyl radical (HO), damage various cell components such as unsaturated lipids, proteins, and nucleic acids. Oxidative stress is caused by ROS in cells in which large amounts of ROS are produced by alternative activities of scavenger proteins or by dysfunction of the mitochondrial respiratory chain pathway with reduction of complex I activity. Complex I (NADH:ubiquinone oxidoreductase) catalyses the first step in the mitochondrial electron transport chain, by which electrons from the oxidation of NADH are used to convert oxygen to water, the energy liberated being trapped in ATP formation and ultimately used as the body's energy source. Oxidation of nucleic acid, lipid, and protein is thought to result in the onset of various diseases, including cancer, infertility, and neurodegenerative diseases such as PD (Agarwal et al., 2003Go; Benhar et al., 2002Go; Golden et al., 2002Go; Rego and Oliveira, 2003Go). Expression of DJ-1 and oxidation of DJ-1 have been shown to be induced in cells that had been administered ROS-inducing chemicals (Kinumi et al., 2004Go; Mitsumoto et al., 2001Go; Mitsumoto and Nakagawa, 2001Go), and abnormal oxidative forms of DJ-1 have been found in some patients with sporadic forms of PD (Bandopadhyay et al., 2004Go). We and other groups have shown that some DJ-1 is located in mitochondria in addition to the cytoplasm and nucleus (Shendelman et al., 2004Go; Shinbo et al., 2005aGo; Zhang et al., 2005Go) and that translocation of DJ-1 to mitochondria was stimulation by oxidative stress (Blackinton et al., 2005Go; Jin et al., 2005Go; Li et al., in pressGo). Precise functions of DJ-1 in mitochondria are, however, not clear.

In addition to its transcriptional activity as a coactivator, we previously reported that DJ-1 plays a role in the anti-oxidative stress reaction, in which reactive oxygen species were eliminated in vitro and in vivo by oxidizing DJ-1 itself, and that mutations of DJ-1, including various mutations found in PD patients, lead to oxidative stress-induced cell death (Taira et al., 2004bGo; Yokota et al., 2003Go). Other groups also reported anti-oxidative activity of DJ-1 (Canet-Aviles et al., 2004Go; Martinat et al., 2004Go; Shendelman et al., 2004Go). It has been shown that, of three cysteines at amino acid numbers 46, 53, and 106 in DJ-1, C106 was first oxidized by addition of SO3H or SO2H, followed by oxidation of C46 and C53 with a dose of H2O2 added to cultured cells (Kinumi et al., 2004Go), and that C106 was required for DJ-1 to exert activity against oxidative stress (Canet-Aviles et al., 2004Go; Martinat et al., 2004Go; Shendelman et al., 2004Go; Taira et al., 2004b; Takahashi-Niki et al., 2004Go). In addition to oxidation of DJ-1, DJ-1 has been reported to be conjugated with SUMO-1 on a lysine residue at amino-acid number 130 (K130) (Shinbo et al., in pressGo). Sumoylation of DJ-1 at K130 has been found to essential for DJ-1 to exert its full activities, including activities for ras-dependent transformation, cell growth stimulation, and anti-oxidative stress (Shinbo et al., in pressGo). Loss of these functions of DJ-1 is therefore thought to lead to the onset of PD.

Some endocrine disruptors (EDs) such as lindane, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and bisphenol A (BPA) have been shown to induce oxidative stress in the brain, liver, kidney, testis, and epididymal sperm in rodents (Bindhumol et al., 2003Go; Chitra et al., 2003Go; Junqueira et al., 1988Go; Kabuto et al., 2003Go, 2004Go; Latchoumycandane et al., 2002Go; Stohs et al., 1991Go). Furthermore, BPA and some bisphenols have been reported to reduce mitochondrial function (Nakagawa and Toyama, 2000Go), but the precise mechanisms underlying these phenomena have not been elucidated.

In this study, we analyzed the effect of DJ-1 on BPA-induced cell death. The results showed that BPA induced oxidative stress in mitochondria and that DJ-1 plays a role as a scavenger of ROS to prevent cell death.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment.
C57BL/6 male mice at 5 weeks of age were purchased from Sankyo Laboratory, Sapporo, Japan. The mice were acclimated to the laboratory for 1 week prior to the experiments and then divided into the following three groups: one group of mice at 6 weeks of age orally administered daily bisphenol A (BPA, Wako) dissolved in corn oil at 10 or 100 µg/kg body weight for 1 or 2 weeks, one group of mice given corn oil (5 ml/kg) alone for the same period, and one group of mice not treated. At 24 h after last administration, the mice were killed, and their brain and cauda epididymides were removed. To purify sperm, sperm was extracted from the cauda epididymes of male mice with a syringe, transferred to a tube containing 5% Ficoll in phosphate-buffered saline (PBS), and centrifuged at 14,000 rpm for 5 min. The pellet fraction containing purified sperm and brain were stored at –80°C until use. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Committee for Animal Research at Hokkaido University.

Cells and treatment.
Mouse Neuro2a and GC1 cells were purchased from American Tissue Culture Collection. All the cells used in this study were cultured in Dulbecco's modified medium supplemented with 10% calf serum. Cells were then administered various concentrations of BPA dissolved in dimethyl sulfoxide (DMSO) for 24 or 48 h. As a vehicle control, 0.1% DMSO was added to the medium.

Western blotting and isoelectric focusing.
Proteins were prepared from whole tissues or cultured cells for Western blot analysis as follows. The brain and sperm were homogenized in 20 mM phosphate buffer (pH 7.5) containing 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, and protease inhibitors. Cultured cells were homogenized in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 1 mM PMSF, and protease inhibitors. Extracts were then centrifuged at 12,000 rpm for 5 min, and their supernatants were used. To analyze proteins in mitochondria by Western blotting, cells were homogenized in a buffer containing 0.25 M sucrose, 10 mM Tris–HCl (pH 7.3), and 1 mM EDTA and then centrifuged at 1000 x g for 10 min. Their supernatant fractions were then centrifuged at 12,000 x g for 15 min, and the pellet fractions (mitochondrial-enriched fractions) were suspended in a buffer containing 0.25 M sucrose, 10 mM Tris–HCl (pH 7.3), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.1 mM PMSF. Concentration of mitochondria protein was measured by using a BCA protein assay kit (PERBIO), and 20 µg of mitochondria protein was used for Western blotting. For isoelectric focusing, cells were homogenized in PBS containing 2% NP-40 and centrifuged at 12,000 rpm for 5 min, and their supernatants were used. Proteins in these extracts were then separated in a 12.5% polyacrylamide gel containing SDS or in an isoelectric focusing gel of pH 5–8, transferred onto nitrocellulose membranes, and reacted with an anti-DJ-1 antibody, which was an affinity-purified rabbit anti-DJ-1 polyclonal antibody described previously (Nagakubo et al., 1997Go), an anti-complex I monoclonal antibody cocktail (Mito Science), or an anti-actin antibody (Chemicon). The proteins on the membranes were then reacted with IRDye800- or Alexa Fluor680-conjugated secondary antibodies followed by visualization using an infrared imaging system (Odyssey, LI-COR).

Flow cytometric analysis.
Nero2a and GC1 cells were mixed with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes) or 2-[6-( 4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) (Daiichi Kagaku) in Hank's solution containing 10 mM HEPES-KOH (pH 7.4) for 30 min in the dark, and their fluorescences were measured using a FACScan flow cytometer (Becton-Dickinson) with excitation at 488 and emission at 530 nm. The amounts of fluorescences were also quantified using a "CELL QUEST" program as a value of "median".

Indirect immunofluorescence.
Cells were mixed with 500 nM MitoTracker-Green (Molecular Probes) for 40 min and then reacted with 5 µM MitoSOX Red (Molecular Probes) for 10 min. The cells were then fixed with a solution containing 4% paraformaldehyde and reacted with an affinity-purified rabbit anti-DJ-1 polyclonal antibody. The cells were then reacted with a Cy-5-conjugated anti-rabbit IgG and observed under a confocal laser fluorescent microscope.

siRNA.
The nucleotide sequences for siRNAs targeting DJ-1 were 5'-CCUUGCUAGUAGAAUAAACdTdT-3' and 3'-dTdTGGAACGAUCAUCUUAUUUG-5' for upper and lower strands, respectively. siRNA-targeting luciferase was purchased from Greiner (Japan). Twenty nM siRNA was transfected into cells using Lipofectamine 2000 (Invitrogen) according to the supplier's manual.

Complex I activity.
A mitochondria-enriched fraction was prepared as described above. Seventy µg of mitochondria protein and 0.05 mM ubiquinone 1 were added to 35 mM phosphate buffer (pH 7.4) containing 2.65 mM sodium cyanide, 5 mM MgCl2, 1 mM EDTA, 1 mg/ml bovine serum albumin, and 2 µg/ml antimycin in a final volume of 0.48 ml. After incubation of the mixture at 37°C for 2 min, 0.02 ml of 5 mM NADH solution was added to the reaction mixture, and decrease in absorbance was measured by a spectrophotometer at 340 nm for 4 min.

Cell viability assay.
Cell viability was measured by a methyl thiazolyl tetrazolium (MTT) assay using a cell counting kit 8 (DOJINDO).

Statistical analyses.
Data are expressed as means ± SD. Statistical analyses were performed using analysis of variance (one-way ANOVA) followed by unpaired Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Change in DJ-1 Expression in Male Mice and Cultured Cells Caused by BPA Administration
Since it has been reported that concentrations of extracted BPA from food such as anchovy, herring, and sardine and from saliva are 10–50 µg/gliq and 90–931 µg/h, respectively, (Biles et al., 1999Go; Olea et al., 1996Go), we chose 10 and 100 µg/kg body weight/day of BPA as the low and high concentrations, respectively. Although these concentrations of BPA to mice are relatively high, these concentrations have been used in several studies to examine the molecular mechanism of BPA action (see recent references, Al-Hiyasat et al., 2004Go; Nikaido et al., 2004Go; Toyama et al., 2004Go). These two concentrations of BPA dissolved in corn oil were administered orally to C57BL/6N male mice for 1 or 2 weeks, and corn oil alone was administered to mice as a vehicle control. Another control group consisted of untreated mice. Expressions of DJ-1 in the brain and sperm after administration of BPA were then examined by Western blotting (Figs. 1A and 1B, upper panels), and intensities of bands were measured using an infrared imaging system (Figs. 1A and 1B, lower panels). The results showed that expression levels of DJ-1 in the brain and sperm were significantly increased over a period of 2 weeks by 1.5- to 2-fold compared to those in the vehicle control in the case of administration of high-dose BPA. In the case of administration of low-dose BPA, however, there was little change in DJ-1 expression. These results indicate that BPA stimulates DJ-1 expression in mice.



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FIG. 1. Expression of DJ-1 in male mice after administration of BPA. Proteins extracted from the brain and sperm were analyzed by Western blotting with anti-DJ-1 and anti-actin antibodies. Intensities of bands were quantified as described in Materials and Methods. Means for each treatment group are represented in the bar graphs for the expression of DJ-1 relative to actin (error bars indicate standard deviation). (A) brain (n = 4). (B) sperm (n = 4). Asterisks indicate significant difference from the vehicle control; *p < 0.05; **p < 0.01.

 
To investigate the effect of BPA on DJ-1 expression in vitro, BPA dissolved in DMSO was administered to cultured mouse Neuro2a and GC1 cells, cells that originate from neuronal cells and spermatogonia immortalized with p53, respectively. Viabilities of Neuro2a and GC1 cells in the presence of BPA were first determined by an MTT assay (Fig. 2A). At 48 h after BPA administration, viabilities of Neuro2a and GC1 cells had significantly decreased at concentrations of more than 50 µM and 100 µM BPA, respectively, suggesting that Neuro2a cells are more sensitive than GC1 cells to BPA. We therefore used BPA at concentrations less than 50 and 100 µM for administration to Neuro2a and GC1 cells, respectively, in the following experiments. Expression levels of DJ-1 after administration of BPA for 24 and 48 h in cells were then determined by Western blotting (Figs. 2B and 2C). Results showed that expressions of DJ-1 in Neuro2a and GC1 cells increased with BPA administration in time- and dose-dependent manners, and the expression levels were 2.5-fold higher than in vehicle control cells.



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FIG. 2. Expression of DJ-1 in cultured cells after administration of BPA. Nueo2a and GC1 cells were administered various concentrations of BPA. (A) 48 h after administration, viability of cells was measured by an MTT assay. At 24 and 48 h after BPA administration, proteins extracted from cells were analyzed by Western blotting with anti-DJ-1 and anti-actin antibodies. Intensities of bands were quantified, as described in Materials and Methods, to measure relative expressions (DJ-1/actin) in Neuro2a (B) and GC1 cells (C). (B) Neuro2a (n = 5). (C) GC1 (n = 5). Asterisks indicate significant difference from the vehicle control; *p < 0.05; **p < 0.01.

 
Production of ROS in Neuro2a and GC1 Cells Induced by BPA Administration
ROS levels in Neuro2a and GC1 cells after administration of BPA were measured by using flow cytometry (Fig. 3). At various times after administration of 50 or 100 µM BPA, cells were reacted with DCFH-DA, which detects various ROS (Kobzik et al., 1990Go). BPA was found to induce the production of ROS in Neuro2a cells in a time-dependent manner (Fig. 3A). In GC1 cells, on the other hand, ROS production peaked at 12 h after BPA administration and decreased with time (Fig. 3B). Furthermore, to measure highly reactive oxygen species (hROS) such as hydoxyradical (·OH) and peroxynitrite (ONOO), cells that had been administered BPA were reacted with HPF, which only detects hROS (Setsukinai et al., 2003Go). H2O2 added to both Neuro2a and GC1 cells at a concentration of 100 µM for 1 h was not detected by HPF (Figs. 3C and 3D). BPA, on the other hand, was found to generate hROS production in both Neuro2a and GCI cells at 48 h after administration (Figs. 3C and 3D). The results indicate that BPA induces the production of various ROS, including hROS, in Neuro2A and GC1 cells.



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FIG. 3. Production of ROS in cultured cell induced by BPA administration. Neuro2a (A) and GC1 cells (B) were administered 50 and 100 µM BPA, respectively. At various times after administration, cells were treated with 10 mM DCFH-DA for 30 min, and levels of ROS were analyzed by flow cytometry. To detect hROS produced in cells, Neuro2a (C) and GC1 cells (D) were similarly administered BPA. At 48 h after administration, cells were treated with 10 mM HPF for 30 min, and levels of hROS were analyzed by flow cytometry. Cells were added with 100 mM H2O2 for 1 h, and their hROS levels were also analyzed. Amounts of fluorescences were quantified and were shown in an insert or beside of each figure. Experiments were carried out more than five times.

 
pI Shift of DJ-1 in Neuro2a and GC1 Cells Induced by BPA Administration
As described in the introductory section, two types of modification, sumoylation and oxidation, occur on DJ-1. Various oxidized forms of DJ-1 have been found in in vitro cultured cells after oxidative stress (Kinumi et al., 2004Go; Mitsumoto et al., 2001Go; Mitsumoto and Nakagawa, 2001Go), and DJ-1 has been found to eliminate ROS by oxidizing DJ-1 itself in vitro and in vivo (Taira et al., 2004bGo). We have further found that of three cysteines in DJ-1, C106 was first oxidized, and other two cysteines, C46 and C53, were then oxidized by using LC-MS and LC-MS/MS analyses (Kinumi et al., 2004Go). pI shift of DJ-1, which indicates oxidation of DJ-1, is thought to be important for DJ-1 to exert its functions.

Proteins were extracted from Neuro2a and GC1 cells at various times after administration of BPA, separated on isoelectric focusing gels, and analyzed by blotting with an anti-DJ-1 antibody (Figs. 4A and 4B, upper panels). The same aliquots of proteins on filters were also reacted with an anti-actin antibody to show loading controls, and intensities of bands were measured by an infrared imaging system (Figs. 4A and 4B in lower panels, 4C and 4D). The results showed that the amounts of oxidized DJ-1 in both Neuro2a and Gc1 cells increased with BPA administration in time- and dose-dependent manners (Figs. 4A and 4B). The rate of oxidized DJ-1/unoxidized DJ-1 was significantly increased at 48 h after administration of 50 µM BPA in Neuro2a cells and at 24 h after administration of 100 µM BPA in GC1 cells (Figs. 4C and 4D), indicating that BPA stimulates oxidization of DJ-1 in Neuro2a and GC1 cells.



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FIG. 4. pI shift of DJ-1 in cultured cells induced by BPA administration. Neuro2a (A) and GC1 cells (B) were administered BPA as described in Figures 3A and 3B. At 48 h after administration, proteins extracted from cells were separated on isoelectric focusing gels and analyzed by blotting with an anti-DJ-1 antibody. Aliquots of proteins were also analyzed by Western blotting with an anti-actin antibody. Intensities of bands were quantified as described in Materials and Methods, and relative expressions (DJ-1/actin) are shown (A, Neuro2a; B, GC1). Relative rates of the oxidized forms of DJ-1/unoxidized forms of DJ-1 to that in untreated cells (untreated) at 0 h are also shown (C, Neuro2a; D, GC1). Arrowheads indicate oxidized DJ-1. (A) Neuro2a (n = 5). (B) GC1 (n = 5). Asterisks indicate significant difference from the vehicle control; *p < 0.05; **p < 0.01.

 
Change in Localization of DJ-1 in Neuro2a and GC1 Cells Following BPA Administration
DJ-1 has been shown to be localized both in the cytoplasm and nucleus and to be translocated from the cytoplasm to nucleus upon mitogen stimulation (Nagakubo et al., 1997Go) and UV irradiation (Shinbo et al., 2005aGo). Recent studies have also shown that some DJ-1 is also localized in mitochondria (Blackinton et al., 2005Go; Canet-Aviles et al., 2004Go; Jin et al., 2005Go; Li et al., 2005Go; Shinbo et al., in pressGo). We therefore examined the localization of DJ-1 after administration of BPA to cells. Neuro2a and GC1 cells that had been administered BPA were stained with MitoTrakker-Green, which stains mitochondria, and with MitoSOX-Red, which is a mitochondrial indicator. MitoSOX-Red is permealized into cells and selectively targeted to mitochondria, in which MitoSOX-Red is oxidized by but not by other ROS (see manufacturer's homepage, http://probes.invitrogen.com/lit/bioprobes47/bp47_5.pdf). Cells were also reacted with a rabbit anti-DJ-1 polyclonal antibody and then with a Cy5-conjugated anti-rabbit IgG and were visualized under a confocal laser microscope. These reactions give green, red, and blue colors, respectively, and these images were merged (Figs. 5A and 5B). DJ-1 was found to be localized in the cytoplasm and nucleus without BPA or with vehicle administration in Neuro2a and GC1 cells as reported in other cell types (Nagakubo et al., 1997Go; Shinbo et al., 2005aGo). After BPA administration, on the other hand, expression levels of DJ-1 were increased in both Neuro2a and GC1 cells concomitant with an increase in signals of -oxidized mitochondria (Figs. 5A and 5B). Intensities of the red fluorescence of MitoSOX-Red corresponding to levels of in mitochondria were further measured by flow cytometry, and stimulation of production by BPA administration in both Neuro2a and GC1 cells was confirmed (Figs. 5C and 5D).



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FIG. 5. Localization of DJ-1 after administration of BPA in cultured cells. Neuro2a (A) and GC1 cells (B) were administered 50 and 100 µM BPA. At 48 h after administration, cells were reacted with 500 nM MitoTracker-Green for 40 min, 5 mM MitoSOX-Red for 10 min, and an anti-DJ-1 antibody followed by reaction with an Cy5-conjugated anti-rabbit IgG, and then visualized under a confocal laser microscope. The images were merged (merge). Levels of superoxide () in mitochondria of Neuro2a (C) and GC1 cells (D) were also measured by flow cytometry, and the amounts of fluorescences quantified were shown under each figure. These experiments were carried out more than five times. Proteins extracted from Neuro2a (E) and GC1 cells (F) at 48 h after administration were analyzed by Western blotting with an anti-20-kDa subunit of a complex I monoclonal antibody and anti-actin antibody. (E) Neuro2a (n = 5). (F) GC1 (n = 5). Asterisks indicate significant difference from the vehicle control; *p < 0.05; **p < 0.01.

 
It is interesting that the intensity of green fluorescence of MitoTracker-Green decreased in Neuro2a and GC1 cells that had been administered 50 µM BPA and 100 µM BPA for 48 h, suggesting that ROS compromised the mitochondria. To explore this possibility, expressions of a 20-kDa subunit of mitochondrial complex I in cells were examined by Western blotting with an anti-20-kDa subunit antibody, and intensities of bands were quantified. The results showed that levels of the 20-kDa subunit of mitochondrial complex 1 in both Neuro2a and GC1 cells decreased with BPA administration in a dose-dependent manner (Figs. 5E and 5F). Moreover, the increased expression level of DJ-1 was found to be localized on the damaged mitochondria (see the merged figures, in which the color changed to white yellow). These results suggest that increased produced by BPA compromises mitochondria and induces DJ-1 expression and that some of DJ-1 is localized in the injured spots.

Dysfunction of Mitochondrial Complex 1 in Cells Induced by BPA Administration
Dysfunction of mitochondria is thought to be responsible for the onset of neurodegenerative disorders, including PD, and DJ-1 is a causative gene of familial PD. Dysfunction of mitochondrial complex 1 has been found in PD patients, and rats or mice that had been administered drugs that injure mitochondrial complex 1 have been reported to show PD-like phenotypes, including dopaminergic neuronal cell death (see recent reviews, Corti et al., 2005Go; Shen and Cookson, 2004Go; Tretter et al., 2004Go). Moreover, since we have found that BPA induced production of ROS in the mitochondria of Neuro2a and GC1 cells and injured the mitochondria, activity and expression levels of subunits of mitochondorial complex 1 were examined. Neuro2a and GC1 cells were administered various concentrations of BPA. At 48 h after administration, mitochondria-rich fractions were prepared, and their activities and expression levels of subunits of complex 1, including subunits of 39, 30, and 20 kDa, were analyzed by Western blotting with anti-complex 1 subunit antibodies (Fig. 6). The expression of DJ-1 in mitochondrial-rich fractions was also examined. While the expression levels of all of the subunits of mitochondrial complex 1 decreased with BPA administration in a dose-dependent manner, complex I activity level was found to increase in Neuro2a cells or to hardly change in GC1 cells at low concentrations of BPA and then to significantly decrease in a dose-dependent manner at high concentrations of BPA (decrease at more than 50 and 100 µM BPA in Neuro2a and GC1 cells, respectively) (Figs. 2, 6A–6D). It is notable that patterns of complex 1 activities, initial increase and then decrease, paralleled those of DJ-1 expression in cells (Figs. 6E and 6F). To assess the relationship between DJ-1 expression and complex 1 activity, siRNA targeting the DJ-1 or luciferase gene, which is a nonspecific control, was transfected into Neuro2a and GC1 cells to knock down expression of the respective gene, and activities of complex 1 were measured at 3 days after transfection. Introduction of these siRNAs into cells was confirmed not to affect the expressions of subunits of complex 1 (Figs. 6C and 6D). Although siRNA against luciferase affected neither the expression of DJ-1 nor the activity of complex 1, siRNA against DJ-1 reduced both the expression level of DJ-1 and activity level of complex 1 to 10% and 70% of those without siRNA in Neuro2a and GC1 cells, respectively (Figs. 6E and 6F, lanes 7–9). These results suggest that BPA abrogates complex I activity by disrupting complex 1 and that DJ-1-knockdown decreases complex I activity without decrease in complex I expression. The results also suggest that the initial increase in the level of DJ-1 expression upon BPA administration that leads to ROS production plays a role in maintenance of complex 1 activity.



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FIG. 6. Dysfunction of mitochondria in cultured cell induced by BPA administration. Neuro2a (A) and GC1 cells (B) were administered various concentrations of BPA. At 48 h after administration, mitochondria-rich fractions were prepared from cells, and complex 1 activities were measured using 70 µg proteins as described in Materials and Methods. Twenty µg of proteins were analyzed by Western blotting to detect 39-, 30-, and 20-kDa subunits of complex 1. Intensities of bands were quantified as described in Materials and Methods, and relative expression of each subunit to that of untreated cells (untreated) at 0 h is also shown (C, Neuro2a; D, GC1). DJ-1 expression in Neuro2a (E) and GC1 cells (F) that had been administered BPA was analyzed by Western blotting with an anti-DJ-1 antibody. Twenty nM siRNAs against luciferase or DJ-1 were transfected into cells. At 72 h after transfection, complex 1 activity and expressions of subunits of complex 1 and DJ-1 were analyzed as described above. (A, C, and E) Neuro2a (n = 6). (B, D, and F) GC1 (n = 7). Asterisks indicate significant difference from the control and transfection of siRNA targeting the luciferase gene; *p < 0.05; **p < 0.01.

 
Abrogation of BPA-Induced Cell Death by DJ-1 in Neuro2a and GC1 Cells
To investigate the role of DJ-1 in BPA-induced cell injury, siRNA against DJ-1 was transfected into Neuro2a and GC1 cells using Lipofectamine 2000 to reduce the expression levels of DJ-1 in the cells. siRNA targeting the luciferase gene was used as a negative control, and transfection using Lipofectamine 2000 alone without siRNA was used as a vehicle control. BPA was then administered into cells at 24 h after transfection of siRNA. At 48 h after administration of BPA, cell viabilities were examined by an MTT assay (Fig. 7). The concentrations of BPA used are those that induce cell death as shown in Figure 2A, 50 and 100 µM into Neuro2a cells and 100 and 200 µM of BPA. We first confirmed that the expression levels of DJ-1 in transfected cells were reduced to 10% of those in nontransfected cells as described in Figures 6E and 6F, and that cell death did not occur after transfection of siRNA into cells without BPA administration (data not shown). Neuro2a and GC1 cells transfected with siRNA against DJ-1 were found to become significantly more susceptible to cell death than those transfected with siRNA against luciferase or with a buffer alone.



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FIG. 7. Sensitization of BPA-induced cell death in DJ-1-knockdown cells. Neuro2a (A) and GC1 cells (B) were transfected with siRNA against DJ-1 or luciferase by lipofectamine plus (Invirogen). At 24 h after transfection, cells were administered various concentrations of BPA. At 48 h after administration, viabilities of cells were measured by an MTT assay. (A) Neuro2a (n = 5). (B) GC1 (n = 5). Asterisks indicate significant difference from transfection of siRNA targeting the luciferase gene; *p < 0.05; **p < 0.01. Symbols of "untreated, vehicle, siLuc and siDJ-1" in figures indicate cells not transfected, transfected with lipofectamine plus alone, transfected with siRNA targeting the luciferase gene, and transfected with siRNA targeting the DJ-1 gene, respectively.

 
To further investigate the role of DJ-1 in BPA-induced cells death, Nero2a and GC1 cells were transfected with an expression vector for DJ-1, pcDNA3-F-DJ-1, using Lipofectamine 2000, and BPA was administered to cells 24 h after transfection. At 48 h after administration of BPA, cell viabilities were examined by an MTT assay (Fig. 7). Transfection with vector alone, pcDNA3-F, and that without plasmid DNA were used as negative (vector) and vehicle controls. Expression of transfected FLAG-tagged DJ-1 in transfected Neuro2a and GC1 cells was confirmed by Western blotting with an anti-DJ-1 antibody (Figs. 8A and 8B). In these blottings, relatively equal expression levels of endogenous DJ-1 in Neuro2a and GC1 cells were observed. In contrast to the case of transfection with siRNA into cells, Neuro2a and GC1 cells transfected with DJ-1 were found to become significantly more resistant to cell death than those transfected with a vector or with a buffer alone (Figs. 8C and 8D). These results suggest that DJ-1 is one of the proteins that prevent BPA-induced cell death in Neuro2a and GC1 cells.



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FIG. 8. Abrogation of BPA-induced cell death by DJ-1 in Neuro2a and GC1 cells. (A, B) Neuro2a and GC1 cells in 6-well plates were transfected with 650 and 400 ng of pcDNA3-F-DJ-1 or pcDNA3-F, respectively, using Lipofectamine 2000 (Invitrogen) according to the supplier's manual. At 72 h after transfection, proteins extracted from Neuro2a (A) and GC1 (B) cells were analyzed by Western blotting with anti-DJ-1 (ab4150, Abcam) and anti-actin (Chemicon) antibodies. Proteins were then reacted with an IRDye800-conjugated second antibody and visualized by an infrared imaging system (Odyssey, LI-COR). (C, D) Neuro2a and GC1 cells in 96-well plates were transfected with 16 and 10 ng of pcDNA3-F-DJ-1 or pcDNA3-F, respectively, using Lipofectamine 2000. At 24 h after transfection, Neuro2a (C) and GC1 (D) cells were administered various concentrations of BPA. At 48 h after administration, viabilities of cells were measured by an MTT assay. (C) Neuro2a (n = 5). (D) GC1 (n = 5). Asterisks indicate significant difference from transfection of pcDNA3, *p < 0.05; **p < 0.01. Symbols of "untreated, vehicle, vector and DJ-1" in figures indicate cells not transfected, transfected with lipofectamine plus alone, transfected with pcDNA3-F, and transfected with pcDNA3-F-DJ-1, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stresses caused by excess ROS production in mitochondria and microsomes are known to damage nucleic acid, lipid, and protein, resulting in the onset of various diseases, including cancer, infertility, and neurodegenerative diseases such as PD. Chemicals with structures similar to that of BPA have been reported to accumulate in adipose tissues and to be partitioned preferentially into membranes containing interior hydrophobic protein rather than the polar hydrophilic part (Law et al., 1986Go; Nunez et al., 2001Go). Since the mitochondria membrane is composed of this type of protein, BPA is thought to accumulate in the mitochondrial membrane, resulting in an uncoupling of the oxidative phosphorylation, thereby inhibiting complex I activity. The molecular mechanism of ROS production by BPA, however, remains unclear.

In this study, we examined the expression and oxidative levels of DJ-1 after administration of BPA to mice and cultured cells, and we found that expression of DJ-1 was induced by BPA-induced ROS production concomitant with oxidation of DJ-1. We also found that BPA compromised mitochondria, resulting in reduction of the activity of mitochondrial complex 1 and that upon injury of mitochondria by BPA, an elevated expression level DJ-1 was observed on the injured mitochondria to restore the activity of complex 1. Since DJ-1 was located in the cytoplasm, nucleus, and mitochondria in various types of cells (Bandopadhyay et al., 2004Go; Nagakubo et al., 1997Go; Shendelman et al., 2004Go; Shinbo et al., 2005aGo, in pressGo; Wagenfeld et al., 2000Go; Zhang et al., 2005Go), and some DJ-1 was translocated to mitochondria after oxidative stress (Blackinton et al., 2005Go; Jin et al., 2005Go; Li et al., 2005Go), the localization of DJ-1 in mitochondria in the cells that had been administered BPA was thought to affect BPA-induced ROS production.

The activity of DJ-1 to sustain the activity of complex 1 was, however, observed at 50 and 100 µM BPA in Neuro2a and GC1 cells, respectively, and this activity was lost over these concentrations. Since, at high concentrations of BPA, cells began to die and the amount of DJ-1 also decreased, it is thought that the amount of DJ-1 is not sufficient to sustain the activity of complex 1. Alternatively, although DJ-1 contains three cysteines, and oxidation of a cysteine at amino acid number 106 (C106) is essential for DJ-1 to exert its anti-oxidative stress activity (Taira et al., 2004bGo; Takahashi-Niki et al., 2004Go), oxidation of all three cysteines by excess ROS, which is produced by high doses of BPA, for instance, may result in loss of its activity (Kinumi et al., 2004Go; Taira et al., 2004bGo).

Although DJ-1 is an abundant protein and approximately 5 x 105 molecules/cell of DJ-1 is present in cells, it is likely that, in addition to DJ-1, other ROS-scavenging proteins such as superoxide dismutase, glutathione peroxidase and catalase, and proteins belonging to other redox systems participate in protecting cells from BPA-induced cell injury. We have found that drastic changes in expressions of genes, including genes related to stress, apoptosis, oxidative stress, and neurotoxicity, occurred in DJ-1 knockdown and that expressions of some genes were regulated by DJ-1 (Nishinaga et al., in pressGo), suggesting that in addition to anti-oxidative activity of DJ-1, transcriptional function of DJ-1 toward genes related to stress functions is important.

In this study, levels of ROS production peaked at 12 h after BPA administration in GC1 cells and then decreased at 24 and 48 h. ROS production in Neuro2A cells, however, continued to occur during these periods. In the kidney, liver, and testis, an isoform of UDP-glucuronosyltransferase (UGTs) has been shown to metabolize BPA to BPA-glucuronide (Reinhechel et al., 1995Go). Although the presence of UGTs in GC1 cells has not been examined, BPA produced at 12 h after BPA administration might be metabolized in GC1 cells.

The present study showed that BPA induced production of various ROS, including highly reactive hydroxy radicals, and ROS have been shown to attack mitochondrial complex I (Yokota et al., 1999Go). Nakagawa and Toyama reported reduction of complex 1 activity after incubation of a mitochondria-rich fraction with BPA in vitro, but degradation of mitochondria and production of ROS were not examined (Nakagawa and Toyama, 2000Go). Since the present study showed that BPA induces ROS production and inhibits complex I activity by disrupting complex I, it would be interesting to examine whether degradation of complex 1 by BPA also occurs in vitro using an electron microscopy and whether this degradation is restored by DJ-1.

Environmental factors are thought to trigger the onset of PD. Several neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTT), rotenone, 6-hydroxydopamine (6-OHDA), and a metabolite of dopamine itself, have been reported to injure mitochondria, resulting in dopaminergic neuron death (Jenner, 2003Go). Recently, BPA has been reported to affect movement and to induce dopaminergic neuron death when BPA was administered to embryonal mice and into the substantia nigra of adult rats, respectively, though ROS levels were not examined (Kabuto et al., 2004Go; Mizuo et al., 2004Go; Suzuki et al., 2003Go). BPA has been detected in several tissues and the blood serum of humans (Ikezuki et al., 2002Go; Schonfelder et al., 2002Go) and accumulates in adipose tissue and membranes, creating the potential for long-term exposure of humans to a low dose of BPA. Human exposure to PBA, and our resent results, suggest that BPA could be a risk factor for the onset of familial and sporadic PD. Furthermore, there are many factors in the environment that cause oxidative stress to humans, including smoking, UV light, alcohol, and environmental contaminants. These factors, including BPA, might act by themselves or in combination as risk factors for diseases such as cancer, infertility, and neurodegenerative diseases.

Since the activity of mitochondrial complex I was significantly diminished by siRNA targeting DJ-1 in the cells of this study, it is possible that DJ-1 has a novel function to maintain complex I activity. In DJ-1-knockdown cells, complex 1 was still intact. Although the precise molecular basis of this reaction is not clear at present, it is important to clarify its mechanism.


    ACKNOWLEDGMENTS
 
We thank Yoko Misawa and Kiyomi Takaya for their technical assistance. This work was supported by grants-in-aid from the Ministry of Education, Science, Culture, Sports and Technology of Japan and the Ministry of Health, Labor and Welfare of Japan. Conflict of interest: none declared.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abou-Sleiman, P. M., Healy, D. G., Quinn, N., Lees, A. J., and Wood, N. W. (2003). The role of pathogenic DJ-1 mutations in Parkinson's disease. Ann. Neurol. 54, 283–286.[CrossRef][ISI][Medline]

Agarwal, A., Saleh, R. A., and Bedaiwy, M. A. (2003). Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil. Steril. 79, 829–843.[CrossRef][ISI][Medline]

Al-Hiyasat, A. S., Darmani, H., and Elbetieha, A. M. (2004). Leached components from dental composites and their effects on fertility of female mice. Eur. J. Oral. Sci. 112, 267–272.[CrossRef][ISI][Medline]

Bandopadhyay, R., Kingsbury, A. E., Cookson, M. R., Reid, A. R., Evans, I. M., Hope, A. D., Pittman, A. M., Lashley, T., Canet-Aviles, R., Miller, D. W., et al. (2004). The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson's disease. Brain 127, 420–430.[Abstract/Free Full Text]

Benhar, M., Engelberg, D., and Levitzki, A. (2002). ROS, stress-activated kinases and stress signaling in cancer. EMBO Rep. 3, 420–425.[Abstract/Free Full Text]

Biles, J. E., White, K. D., McNeal, T. P., and Begley, T. H. (1999). Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods. J. Agric. Food Chem. 47, 1965–1969.[CrossRef][ISI][Medline]

Bindhumol, V., Chitra, K. C., and Mathur, P. P. (2003). Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology 188, 117–124.[ISI][Medline]

Blackinton, J., Ahmad, R., Miller, D. W., van der Brug, M. P., Canet-Aviles, R. M., Hague, S. M., Kaleem, M., and Cookson, M. R. (2005). Effects of DJ-1 mutations and polymorphisms on protein stability and subcellular localization. Mol. Brain Res. 134, 76–83.[ISI][Medline]

Bonifati, V., Rizzu, P., van Baren, M. J., Schaap, O., Breedveld, G. J., Krieger, E., Dekker, M. C., Squitieri, F., Ibanez, P., Joosse, M., et al. (2003). Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299, 256–259.[Abstract/Free Full Text]

Canet-Aviles, R. M., Wilson, M. A., Miller, D. W., Ahmad, R., McLendon, C., Bandyopadhyay, S., Baptista, M. J., Ringe, D., Petsko, G. A., and Cookson, M. R. (2004). The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl. Acad. Sci. U.S.A. 101, 9103–9108.[Abstract/Free Full Text]

Chitra, K. C., Latchoumycandane, C., and Mathur, P. P. (2003). Induction of oxidative stress by bisphenol A in the epididymal sperm of rats. Toxicology 14, 119–127.

Corti, O., Hampe, C., Darios, F., Ibanez, P., Ruberg, M., and Brice, A. (2005). Parkinson's disease: From causes to mechanisms. C. R. Biol. 328, 131–142.[ISI][Medline]

Golden, T. R., Hinerfeld, D. A., and Melov, S. (2002). Oxidative stress and aging: Beyond correlation. Aging Cell 1, 117–123.[CrossRef][ISI][Medline]

Hague, S., Rogaeva, E., Hernandez, D., Gulick, C., Singleton, A., Hanson, M., Johnson, J., Weiser, R., Gallardo, M., Ravina, B., et al. (2003). Early-onset Parkinson's disease caused by a compound heterozygous DJ-1 mutation. Ann. Neurol. 54, 271–274.[CrossRef][ISI][Medline]

Ikezuki, Y., Tsutsumi, O., Takai, Y., Kamei, Y., Taketani, Y. (2002). Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum. Reprod. 17, 2839–2841.[Abstract/Free Full Text]

Jenner, P. (2003). Oxidative stress in Parkinson's disease. Ann. Neurol. 53(Suppl. 3), S26–S36.[CrossRef][ISI][Medline]

Jin, J., Meredith, G. E., Chen, L., Zhou, Y., Xu, J., Shie, F. S., Lockhart, P., and Zhang, J. (2005). Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson's disease. Mol. Brain Res. 134, 119–138.[ISI][Medline]

Junqueira, V. B., Simizu, K., Van Halsema, L., Koch, O. R., Barro, S. B., and Videla, L. A. (1988). Lindane-induced oxidative stress. I. Time course of changes in hepatic microsomal parameters, antioxidant enzymes, lipid peroxidative indices and morphological characteristics. Xenobiotica 18, 1297–1304.[ISI][Medline]

Kabuto, H., Amakawa, M., and Shishibori, T. (2004). Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice. Life Sci. 74, 2931–2940.[CrossRef][ISI][Medline]

Kabuto, H., Hasuike, S., Minagawa, N., and Shishibori, T. (2003). Effects of bisphenol A on the metabolisms of active oxygen species in mouse tissues. Environ. Res. 93, 31–35.[CrossRef][ISI][Medline]

Kim, R. H., Peters, M., Jang, Y., Shi, W., Pintilie, M., Fletcher, G. C., DeLuca, C., Liepa, J., Zhou, L., Snow, B., et al. (2005). DJ-1, a novel regulator of the tumor suppressor PTEN. Cancer Cell 7, 263–273.[CrossRef][ISI][Medline]

Kinumi, T., Kimata, J., Taira, T., Ariga, H., and Niki, E. (2004). Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide mediated oxidation in vivo in human umbilical vein endothelial cells. Biochem. Biophys. Res. Commun. 317, 722–728.[CrossRef][ISI][Medline]

Klinefelter, G. R., Welch, J. E., Perreault, S. D., Moore, H. D., Zucker, R. M., Suarez, J. D., Roberts, N. L., Bobseine, K., and Jeffay, S. J. (2002). Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro. Andrology 23, 48–63.

Kobzik, L., Godleski, J. J., and Brain, J. D. (1990). Oxidative metabolism in the alveolar macrophage: Analysis by flow cytometry. J. Leukoc. Biol. 47, 295–303.[Abstract/Free Full Text]

Latchoumycandane, C., Chitra, K. C., and Mathur, P. P. (2002). The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the antioxidant system in mitochondrial and microsomal fractions of rat testis. Toxicology 171, 127–135.[CrossRef][ISI][Medline]

Law, P., Campbell, S. D., Lepock, J. R., and Kruuv, J. (1986). Effects of butylated hydroxytoluene on membrane lipid fluidity and freezethaw survival in mammalian cell. Cryobiology 23, 317–322.[CrossRef][ISI][Medline]

Li, H. M., Niki, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2005). Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress. Free Radic. Res. (in press).

Martinat, C., Shendelman, S., Jonason, A., Leete, T., Beal, M. F., Yang, L., Floss, T., and Abeliovich, A. (2004). Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: An ES- derived cell model of primary parkinsonism. PLoS Biol. 2, e327.[CrossRef][Medline]

Mitsumoto, A., and Nakagawa, Y. (2001). DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin. Free Radic. Res. 35, 885–893.[ISI][Medline]

Mitsumoto, A., Nakagawa, Y., Takeuchi, A., Okawa, K., Iwamatsu, A., and Takanezawa, Y. (2001). Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat. Free Radic. Res. 35, 301–310.[ISI][Medline]

Mizuo, K., Narita, M., Miyagawa, K., Narita, M., Okuno, E., and Suzuki, T. (2004). Prenatal and neonatal exposure to bisphenol-A affects the morphine-induced rewarding effect and hyperlocomotion in mice. Neurosci. Lett. 356, 95–98.[CrossRef][ISI][Medline]

Nagakubo, D., Taira, T., Kitaura, H., Ikeda, M., Tamai, K., Iguchi-Ariga, S. M. M., and Ariga, H. (1997). DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras. Biochem. Biophys. Res. Commun. 31, 509–513.

Nakagawa, Y., and Toyama, S. (2000). Metabolism and cytotoxicity of bisphenol A and other bisphenols in isolated rat hepatocytes. Arch. Toxicol. 74, 99–105.[CrossRef][ISI][Medline]

Nikaido, Y., Yoshizawa, K., Danbara, N., Tsujita-Kyutoku, M., Yuri, T., Uehara, N., and Tsubura, A. (2004). Effects of maternal xenoestrogen exposure on development of the reproductive tract and mammary gland in female CD-1 mouse offspring. Reprod. Toxicol. 18, 803–811.[CrossRef][ISI][Medline]

Niki, T., Takahashi-Niki, K., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2003). DJBP: A novel DJ-1-binding protein, negatively regulates the androgen receptor by recruiting histone deacetylase complex, and DJ-1 antagonizes this inhibition by abrogation of this complex. Mol. Cancer Res. 1, 247–261.[Abstract/Free Full Text]

Nishinaga, H., Takahashi-Niki, K., Taira, T., Andreadis, A., Iguchi-Ariga, S. M. M., and Ariga, H. (2005). Expression profiles of genes in DJ-1-knockdown and L166P DJ-1 mutant cells. Neurosci. Lett. (in press).

Nunez, A. A., Kannan, K., Giesy, J. P., Fang, J., and Clemens, L. G. (2001). Effects of bisphenol A on energy balance and accumulation in brown adipose tissue in rats. Chemosphere 42, 917–922.[CrossRef][ISI][Medline]

Okada, M., Matsumoto, K., Niki, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2002). DJ-1, a target protein for an endocrine disrupter, participates in the fertilization in mice. Biol. Pharm. Bull. 25, 853–856.[CrossRef][ISI][Medline]

Olea, N., Pulgar, R., Perez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, A. M., and Sonnenschein, C. (1996). Estrogenicity of resin-based composites and sealants used in dentistry. Environ. Health Perspect. 104, 298–305.[ISI][Medline]

Rego, A. C., and Oliveira, C. R. (2003). Mitochondrial dysfunction and reactive oxygen species in excitotoxicity and apoptosis: Implications for the pathogenesis of neurodegenerative diseases. Neurochem. Res. 28, 1563–1574.[CrossRef][ISI][Medline]

Reinhechel, T., Wiswedel, I., Noack, H., and Augustin, W. (1995). Electrophoretic evidence for the impairment of complexes of the respiratory chain during iron/ascorbate induced peroxidation in isolated rat liver mitochondria. Biochim. Biophys. Acta 1239, 45–50.[ISI][Medline]

Schonfelder, G., Wittfoht, W., Hopp, H., Talsness, C. E., Paulk, M., and Chahoud, I. (2002). Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ. Health Perspect. 110, A703–A707.[ISI][Medline]

Setsukinai, K., Urano, Y., Kakinuma, K., Majima, H. J., and Nagano, T. (2003). Development of Novel Fluorescence Probes That Can Reliably Detect Reactive Oxygen Species and Distinguish Specific Species. J. Biol. Chem. 278, 3170–3175.[Abstract/Free Full Text]

Shen, J., and Cookson, M. (2004). Mitochondria and dopamine. New insights into recessive parkinsonism. Neuron 43, 301–304.[CrossRef][ISI][Medline]

Shendelman, S., Jonason, A., Martinat, C., Leete, T., and Abeliovich, A. (2004). DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation. PLoS Biol. 2, e362.[Medline]

Shinbo, Y., Taira, T., Niki, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2005a). DJ-1 restores p53 transcription activity inhibited by Topors/p53BP3. Int. J. Oncol. 26, 641–648.[ISI][Medline]

Shinbo, Y., Niki, T., Taira, T., Ooe, H., Takahashi-Niki, K., Maita, C., Seino, C., Iguchi-Ariga, S. M. M., and Ariga, H. (2005b). Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities. Cell Death Diff. (in press).

Stohs, S. J., Alsharif, N. Z., Shara, M. A., al-Bayati, Z. A., and Wahba, Z. Z. (1991). Evidence for the induction of an oxidative stress in rat hepatic mitochondria by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Adv. Exp. Med. Biol. 283, 827–831.[Medline]

Suzuki, T., Mizuo, K., Nakazawa, H., Funae, Y., Fushiki, S., Fukushima, S., Shirai, T., and Narita, M. (2003). Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: Enhancement of the methamphetamine-induced abuse state. Neuroscience 117, 639–644.[CrossRef][ISI][Medline]

Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2004a). Co-localization with DJ-1 is essential for the androgen receptor to exert its transcription activity that has been impaired by androgen-antagonists. Biol. Pharm. Bull. 27, 574–577.[CrossRef][ISI][Medline]

Taira, T., Saito, Y., Niki, T., Iguchi-Ariga, S. M. M., Takahashi, K., and Ariga, H. (2004b). DJ-1 has a role in antioxidative stress to prevent cell death. EMBO Rep. 5, 213–218.[Abstract/Free Full Text]

Takahashi, K., Taira, T., Niki, T., Seino, C., Iguchi-Ariga, S. M. M., and Ariga, H. (2001). DJ-1 positively regulates the androgen receptor by impairing the binding of PIASx alpha to the receptor. J. Biol. Chem. 276, 37556–37563.[Abstract/Free Full Text]

Takahashi-Niki, K., Niki, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2004). Reduced anti-oxidative stress activities of DJ-1 mutants found in Parkinson's disease patients. Biochem. Biophys. Res Commun. 320, 389–397.[CrossRef][ISI][Medline]

Toyama, Y., Suzuki-Toyota, F., Maekawa, M., Ito, C., and Toshimori, K. (2004). Adverse effects of bisphenol A to spermiogenesis in mice and rats. Arch. Histol. Cytol. 67, 373–381.[CrossRef][ISI][Medline]

Tretter, L., Sipos, I., and Adam-Vizi, V. (2004). Initiation of neuronal damage by complex I deficiency and oxidative stress in Parkinson's disease. Neurochem. Res. 29, 569–577.[CrossRef][ISI][Medline]

Wagenfeld, A., Yeung, C. H., Shivaji, S., Sundareswaran, V. R., Ariga, H., and Cooper, T. G. (2000). Expression and cellular localization of contraception-associated protein. J. Androl. 21, 954–963.[Abstract/Free Full Text]

Wagenfeld, A., Yeung, C. H., Strupat, K., and Cooper, T. G. (1998). Shedding of a rat epididymal sperm protein associated with infertility induced by ornidazole and alpha-chlorohydrin. Biol. Reprod. 8, 1257–1265.

Welch, J. E., Barbee, R. R., Roberts, N. L., Suarez, J. D., and Klinefelter, G. R. (1998). SP22: A novel fertility protein from a highly conserved gene family. J. Androl. 19, 385–393.[Abstract/Free Full Text]

Yokota, H., Iwano, H., Endo, M., Kobayashi, T., Inoue, H., Ikushiro, S., and Yuasa, A. (1999). Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in rat liver. Biochem. J. 340, 405–409.[CrossRef][ISI][Medline]

Yokota,T., Sugawara, K., Ito, K., Takahashi, R., Ariga, H., and Mizusawa, H. (2003). Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition. Biochem. Biophys. Res. Commun. 312, 1342–1348.[CrossRef][ISI][Medline]

Yoshida, K., Sato, Y., Yoshiike, M., Nozawa S., Ariga, H., and Iwamoto, T. (2003). Immunocytochemical localization of DJ-1 in human male reproductive tissue. Mol. Reprod. Dev. 66, 391–397.[CrossRef][ISI][Medline]

Zhang, L., Shimoji, M., Thomas, B., Moore, D. I., Yu, S.-W., Marupudi, N. I., Torp, R., Torgner, I. A., Ottersen, O. P., Dawson, T. M., et al. (2005). Mitochondrial localization of the Parkinson's disease related protein DJ-1: Implications for pathogenesis. Hum. Mol. Genet. 14, 2063–2073.[Abstract/Free Full Text]





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