In Vitro Neurotoxicity of PBDE-99: Immediate and Concentration

Dec 3, 2009 - Concentration-Dependent Effects on Protein Expression in Cerebral. Cortex Cells. Henrik Alm,*,† Birger Scholz,† Kim Kultima,† Anna...
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In Vitro Neurotoxicity of PBDE-99: Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells Henrik Alm,*,† Birger Scholz,† Kim Kultima,† Anna Nilsson,‡ Per E. Andre´n,‡ Mikhail M. Savitski,§ Åke Bergman,| Michael Stigson,† Åsa Fex-Svenningsen,⊥ and Lennart Dencker† Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Sweden, Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Sweden, Department of Cellular and Molecular Biology, Uppsala University, Sweden, Department of Environmental Chemistry, Stockholm University, Sweden, and Institute of Medical Biology, Anatomy and Neurobiology, University of Southern Denmark, Denmark Received August 13, 2009

Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentrationdependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 µM). Changes on a post-translational level were studied using a 1 h exposure to 30 µM PBDE-99. The effects of 24 h exposure to 3 and 30 µM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 µM PDBE99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 µM, but not 10 or 30 µM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes. Keywords: 2D-DIGE • Neurotoxicity • ESI-LTQ • Development • PBDE-99

Introduction Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in many consumer products used on a daily basis, such as textiles, carpets, polyurethane foams, electronic cables, television sets and computers.1 These lipid soluble, persistent compounds bioaccumulate in the adipose tissue of humans and animals. High levels of PBDEs have been found in human breast milk, which together with house dust constitute the major exposure routes for human infants.2 Despite the long-term use of PBDEs, relatively little is known about their potentially toxic effects, especially the mechanisms of these effects. Despite the banned use and production of penta- and * To whom correspondence should be addressed. Henrik Alm, Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, Box 594, SE-751 24 Uppsala, Sweden. E-mail: Henrik.Alm@ farmbio.uu.se. Tel: +46 18 471 42 65. Fax: +46 18 471 42 53. † Department of Pharmaceutical Biosciences, Uppsala University. ‡ Laboratory for Biological and Medical Mass Spectrometry, Uppsala University. § Department of Cellular and Molecular Biology, Uppsala University. | Stockholm University. ⊥ University of Southern Denmark.

1226 Journal of Proteome Research 2010, 9, 1226–1235 Published on Web 12/03/2009

octa-brominated diphenylethers (BDEs) in the EU and several U.S. states,3 the global use, environmental accumulation and incomplete degradation of these substances ensure that these compounds will be the focus of attention in decades to come. Furthermore, the PBDE levels in populations of Asian countries such as China and India4,5 are likely to continue to rise, with the possibility of future consequences on the environment as well as human health to emerge. It is now well-established that pre- and postnatal exposure to congeners of PBDEs in rodents disrupts normal brain development.6–11 As a consequence, more research has focused on the mechanisms behind PBDE neurotoxicity, and in vitro systems with neuronal and astroglial cell preparations have been used for these studies.12–16 The end-points are often based on targets and pathways known to be affected by polychlorinated biphenyls (PCBs)2 or other halogenated environmental toxicants, reflecting that PCBs and PBDEs share aspects of toxicity, or the use of a conventional approach for new sets of compounds. Previous in vitro studies of PBDE-99 in human astrocytoma cells demonstrated that high PBDE-99 concentrations (50-100 10.1021/pr900723c

 2010 American Chemical Society

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µM) impair cell viability and induce apoptosis. At the highest concentration tested (100 µM), low levels of protein kinase C (PKC) translocation were observed. Another study in primary cerebellar rat granule (CGN) cells reported PBDE-99-induced PKC activation with levels as low as 10 µM PBDE.17 The same concentration of PBDE-99 was required to induce calcium uptake in isolated microsomes and mitochondria from rat frontal cortex, hippocampus and hypothalamus.18 PBDE-99 (at 10 µM) has also demonstrated moderate potency in endocrine disruption in in vitro screening systems based on human hepatoma, osteoblast and breast cancer cells.19 A recent interaction study using human neuroblastoma cells showed that simultaneous coexposure to PBDE-47 and PBDE-99 can induce synergistic neurotoxic effects, assessed as reduction in cell viability and induction of oxidative stress, in particular at low concentrations of PBDE-47.20 To our knowledge, no large-scale in vitro genomic or proteomic approach has been used thus far to investigate PBDE-induced neurotoxicity. In the present study, twodimensional difference gel electrophoresis (2D-DIGE) was used to analyze differences in protein expression in cultured cortical cells isolated from prenatal rat fetuses after exposure to various concentrations of PBDE-99 (3, 10, and 30 µM). On the basis of our previous in vivo studies of the effects of PBDE-99 in the hippocampus, striatum21 and cortex,22 we used 24 h as the exposure time to analyze the early effects of PBDE-99.

Experimental Procedures Animals and Treatments. This study was approved by the regional ethics committee for research on animals (permit no. 2006/48 of May 5, 2006, Uppsala, Sweden) and performed in accordance with the policies of the Society for Neuroscience. Preparation, Culture, And Exposure of Primary Cells from Fetal Rat Cortex. Female Sprague-Dawley rats were purchased already mated (B&K, Sollentuna, Sweden). The day of the vaginal plug was designated gestation day (GD) 0. The animals underwent 1 week of acclimatization, and were sacrificed on GD21 using an overdose of CO2. The fetuses were collected from the dams under sterile conditions and placed in a 100-mm Petri dish with cold L15 medium (Leibovitz, Gibco). The brains were dissected, and the cortex was isolated and transferred to a new dish containing fresh ice-cold L15. The cortices from 9-13 fetuses were pooled in each preparation. Meninges were removed and the cortices were mechanically dissociated using a Pasteur pipet. Dissociated cells were transferred to a 10-mL tube containing L15 medium, and collected by centrifugation at 1100 rpm for 5 min at room temperature. The pellet was resuspended in fresh L15 medium and recentrifuged at 1100 rpm for 5 min. The pellet was resuspended in culture medium (Neurobasal medium, 600 µM glutamine, 1% penicillin-streptomycin, 2% B-27 supplement) and filtered (cell strainer 40 µm, Falcon) to avoid cell clumps. Cells were plated at 8 × 105 cells/mL on poly lysine-coated 60mm cell culture dishes (Sarstedt), and grown at 37 °C in 5% ¨ rn et al.,23 CO2. PBDE-99, which was synthesized according to O was dissolved in dimethylsulfoxide (DMSO) and added to the cell culture medium 24 h after seeding. Six groups were exposed to varying concentrations of treatments for 24 h: 0.3, 3, 10, and 30 µM PBDE-99, DMSO, and control. Two additional groups were treated for only 1 h: 30 µM PBDE-99 and control. 2D-DIGE. The protein lysates from a total of 28 PBDE-99 treated samples were analyzed: samples treated with 3 µM for 24 h (n ) 4), 10 µM for 24 h (n ) 4), 30 µM for 1 h (n ) 4) and

24 h (n ) 6), and controls (n ) 10). The high concentration samples (1 h, 30 µM) were analyzed along with the set of samples exposed for 24 h (24 h 3 µM, 10 µM, and 30 µM). For 2D-DIGE, control, treated, and pooled protein samples (50 µg each) were labeled with cyanine dye Cy5, Cy3 and Cy2, following the manufacturer’s instructions for CyDye DIGE Fluor minimal dyes (GE Healthcare), except that only half of the recommended amount of fluorescent dye was used, as earlier described.24 The pooled sample was a mixture of equal amounts of protein from all samples in the experiment. We used a dye-swap loop design for individual samples. Before the first-dimension isoelectric focusing (IEF), a 50 µg aliquot from each of the three labeling mixes (see above) was combined with DeStreak rehydration buffer and 0.5% (v/v) Pharmalytes (GE Healthcare) that covered the pH interval (pH 3-11 NL) of the IPG strips to give a final volume of 450 µL. Gel rehydration of the 24 cm IPG strips (GE Healthcare) with the 450 µL of rehydration buffer (including the protein sample) was performed at room temperature in the dark for 12 h according to the manufacturer’s instructions. IEF was run on an IPGPhor (GE Healthcare) at 500 V for 1 h, 1 kV for 1 h, and 8 kV until a total of 64 kVh was reached. After IEF, the strips were equilibrated by gentle shaking (2 × 15 min) in a buffer containing 50 mM Tris-HCl (pH 6.8), 6 M urea, and 2% sodium dodecyl sulfate (SDS), supplemented with 2% dithiothreitol (DTT) in the first equilibration step and 2.5% iodoacetamide in the second. For the second dimension SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the equilibrated strips were placed on top of large format 12.5% polyacrylamide gels and run using an Ettan DALTsix large-format vertical system (GE Healthcare). All gels were run at 5 W for 45 min and then increased to 11 W; gels were run until the bromophenol blue dye front reached the bottom of the gel. The temperature was kept constant at 27 °C. Scanning and Image Analysis. The gels were scanned using a fluorescence scanner (Typhoon 9400, GE Healthcare, Uppsala, Sweden) at 100-µm resolution. The fluorescent images of the 2D-DIGE gels were analyzed using the DeCyder software suite, version 5.02 (GE Healthcare) with default settings; the software was set to find 3000 spots per image. The Cy2, Cy3, and Cy5 images for each gel were merged in the Differential In-gel Analysis DeCyder module, and spot boundaries were detected automatically. Spots reflecting dust particles and other artifacts were excluded from further analysis. We matched spots from the different gels using the automatic matching function in the Biological Variance Analysis DeCyder module after prior landmark settings, and confirmed the spots manually by comparing each spot against the master gel. Volume data and coordinate data for each spot were exported using the DeCyder XML toolbox. Three gels from the 1 h exposure group were subsequently stained with Pro-Q Diamond phosphor stain according to the manufacturer’s instructions. Spot Picking and MS Analysis. 2D-DIGE gels that use 3 × 50 µg CyDye labeled protein aliquots do not provide large quantities of protein, making MS-based identification difficult. We previously demonstrated that the intensities of labeled proteins (3 × 50 µg CyDye) in a 2D-DIGE gel are unaffected by the addition of unlabeled protein;21 therefore, we added 150 µg of unlabeled proteins from a randomly selected sample onto a DIGE gel designated for spot picking. Selected spots were then digested using an Ettan spot picker/digester per the manufacturer’s instructions (GE Healthcare, Uppsala, Sweden). The tryptic digest of each spot was dissolved in 10 µL of 0.25% Journal of Proteome Research • Vol. 9, No. 3, 2010 1227

research articles (v/v) acetic acid, and 5 µL was desalted on a Nano-Precolumn (LC Packings, Amsterdam, The Netherlands) using Ettan MDLC (GE Healthcare). The digest was then separated by a 20-min gradient from 3% to 80% acetonitrile in 0.25% acetic acid on a 15-cm, 75 µM inner diameter, C18 capillary column (LC Packings, Amsterdam, The Netherlands). The peptides were electro-sprayed into a linear ion trap mass spectrometer (LTQ, Thermo Electron, San Jose, CA) at a flow rate of approximately 150 nL/min. The spray voltage was 1.8 kV, the ES source capillary temperature was 160 °C, and 35 units of collision energy were used to obtain peptide fragmentation. One zoom scan spectrum and one tandem mass spectrometry (MS/MS) scan spectrum were collected in a data-dependent acquisition manner following each full-scan mass spectrum. The dynamic exclusion feature enabled sequence information for as many codetected peptides as possible. The tandem mass spectra generated in the nano-LC MS/MS run were converted into dta files using Xcalibur (Version 2.0 SR2) and the dta files were then combined into mgf files by using an in-house developed script. The information from the electro-spray ionization MS and MS/ MS spectra was used to search against protein sequence data in the Swiss-Prot rodent database (release 51.3, 19 267 entries) using MASCOT (Matrix Science Ltd.).25 The settings were partial oxidation of methionine (+16 Da), cysteine alkylation (+57 Da), peptide mass tolerance of 1.5 Da, and fragment ion mass tolerance of 0.8 Da. Trypsin was specified as the digesting enzyme with a maximum of one missed cleavage. The criteria for positive identification of a protein were two or more peptides, with each having a MASCOT score of 33 or higher from the same protein. If more than one protein was matched by the peptides, we selected and reported the protein with the best score, most matched peptides and the most likely identity considering the identities surrounding the spot on the gel. Supplemental file S6 lists all proteins the data supports equally well. 2D-DIGE Data Analysis. For the 24 h samples, the data was ‘SC-2D’ normalized to account for spatial and intensity dye effects.26 After normalization, the three comparisons (or effects) of interest were estimated by the least-squares method using the lmFit() Limma function.27 The gels were treated as blocks in the linear model so that effects were estimated within gels where possible. The dyes were treated as a fixed factor to account for differences between the Cy5, Cy3 and Cy2 channels. The pool channel samples, which were originally created by mixing equal amounts of proteins from each sample, were consequently assumed to contain 6/20 Ctl, 4/20 3 µM, 4/20 10 µM and 6/20 30 µM. The correlation between the spots of the three dyes within each gel was first estimated using the dupcor() function28 in Limma. To test for differences in expression levels between groups, moderated F and t-statistics were calculated for the effects 3vC, 10vC and 30vC using the eBayes() function in Limma, (see refs 27 and 29). We included spots for which we had at least 50% nonmissing values in all of the four treatment groups (control, 3, 10, 30 µM), resulting in a primary data set (concentration-dependent) with a total of 816 spots (in which, of 292 identified). As the additional 30 µM 1 h study only contained two sample groups (control vs treatment), this data was normalized using the method ‘2D loess’, with the pool channel (Cy2) excluded in the statistical analysis, as recommended.26 To find spots that differed between the two 1 h sample groups, we used a linear model that accounts for dye-specific differences for the Cy3 and Cy5 channels, and calculated moderated t-statistics27 using 1228

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Alm et al. the eBayes() function in Limma. Gels from the 24 h samples were included to get a better estimate of dye-specific differences. We included spots with a maximum of one missing value that were successfully matched to the 24 h samples, resulting in a secondary smaller data set (time-concentration) with a total of 345 spots (in which, of 156 identified). In both data sets, spots with p < 0.05 and log2 |0.2| were considered significantly different. Multivariate Data Analysis. An unsupervised multivariate analysis by principal component analysis (PCA) using the SIMCA-P software (version 11.5, Umetrics AB, Umeå, Sweden) resulted in PCA models for both data sets (larger, 816 spots; smaller, 345 spots). Expression Profile Analysis. Short-Time Series Expression Miner (STEM)30 was used to plot the expression profiles of all the proteins in the larger concentration-response data set (Supplemental Data S1). STEM compares sequential groups, such as concentration-response or time-dependent data series. To correlate the STEM expression profiles with gene ontology analysis (GO) for the 292 identified proteins,31 we created a special GO-annotation to accommodate the isoform-rich data from the 2D-DIGE study (Supplemental Data S2). Assuming only minor differences in the general functionality between isoforms of a given protein within a given GO-annotation, a GO set incorporating each isoform (designated Id1a, _Id1b.. .Id1n for the identified protein Id1) was created for all 292 identified proteins. Multiple testing in STEM model profiling was performed using Bonferroni correction with a significance level of 0.05. The significance of STEM profiles was calculated by comparing the number of proteins assigned to the number of expected proteins by a permutation test (50 permutations) and the profiles’ p-value. Default STEM parameters were used throughout, except that we used no normalization, and a minimum absolute expression change of 0.20. In addition, for the cluster profile, we used a minimum correlation of 0.75. For the STEM GO analysis, all electronically inferred annotations (IEA) were excluded, the minimum hierarchical GO level was set to 2, and the minimum number of proteins used for GO analysis was set to 5. Multiple hypothesis correction for actual size based GO enrichment analysis was performed using 10000 randomized samples. The maximum number of STEM profiles was set to 98. The bioinformatics database DAVID32 was used to functionally annotate the 292 identified and differently expressed proteins for additional STEM-independent GO analysis. Western Blot. Gap43 has previously been shown to be responsive to PBDE-99 exposure in rodents in vivo; this was the reason we wanted to analyze the expression of Gap43 and the active form p-Gap43 in cultured cerebral cortex cells after PBDE-99 exposure. Western blot analysis was performed to assess Gap43 expression (1:1500, sc-10786, Santa Cruz Biotechnology, CA) and p-Gap43 levels (1:750, sc-17109, Santa Cruz Biotechnology, CA). After incubation with horseradish peroxidase-conjugated secondary antibody (1:5000, sc-2004, Santa Cruz Biotechnology, CA), the protein images were developed on film (Cronex 5 light-sensitive film, Agfa Gevert, MA) using the ECL Advance Western blotting detection system (GE Healthcare, Uppsala Sweden) according to the manufacturer’s description. In the Western blot experiment, we also included a 24 h 0.3 µM exposure group to assess the effects on Gap43 expression after low concentration PBDE-99 exposure. Gene Expression. Cortical cells were cultured and treated with DMSO, 3 or 30 µM PBDE-99 as described above. The cells

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Figure 1. Characterization of the larger 2D-DIGE concentration-response data set. (A) Representative 2D-DIGE gel image. Magnified regions show phosphostained protein spots and corresponding DIGE spots. (B) Cell death of cortical cells after exposure to different concentrations of 24 h PBDE-99 and a positive DMSO control (*p < 0.05, **p < 0.01). (C) Three-dimensional principal component analysis (PCA) scatter plot using the first three principal components and explaining ∼58% of the variation in the data. Ctl stands for controls (red, six samples), U3 equals 3 µM (green, four samples), U10 equals 10 µM (blue, four samples) and U30 equals 30 µM (black, six samples). (D) The proportion of total number of matched 2D-DIGE spots (816 spots) that are differentially regulated after 24 h PBDE-99 for the different concentrations compared to the controls (fold change cutoff log2 0.2, orange p < 0.01, yellow p < 0.05).

were lysed and harvested using the Qiagen Mini-prep kit (Qiagen, CA) according to the manufacturer’s instructions. The gene expression study was performed using the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) set, which consisted of 38 784 70-mer probes printed on two slides. The microarrays were obtained from Systems Toxicology Group, MRC Toxicology Unit, Leicester, U.K. (http://www.systems-toxicology.com). Six microarrays were used for each group in a dye-swap design, in which control and 3 µM samples were compared in one set, and control and 30 µM samples in a second set. Labeling and Hybridization. Briefly, 10 µg of total RNA was reverse transcribed using 1.0 µL of the anchored oligo dT23N2 (8 µg/µL) and 1 µL of pentadecamers (10 nmol/µL). After heating to 95 °C for 5 min and 70 °C for 10 min, the tubes were snap cooled on ice for 1 min. After addition of dNTPs and superscript, the solution was mixed and incubated at 50 °C for 3 h. After RNA hydrolysis and cDNA purification, Cy3 and Cy5 dyes were coupled to the cDNA samples. The labeled cDNA was cleaned using quick PCR columns (Qiagen, CA), and the labeling was quantified using nanodrop (ND-1000, SaveenWerner, Sweden). For hybridization, 1 µL of tRNA and 380 µL of water were added, and the solution was placed on Microcon 30 columns and centrifuged for 7 min at 12 000 rpm. The probe was dried using a speed vac for 15 min in room temperature until completely dry. The probe was resuspended in 50 µL of genisphere buffer and an equal amount of water, and then denatured at 100 °C for 5 min and incubated at 42 °C for 1 h. Fifty microliters of probe was placed on each array slide, followed by a coverslip. Five milliliters of water was placed in

the bottom of the Genetix hybridization chamber, and the array slides were then inserted in the chamber and left to incubate overnight at 42 °C; slides were then washed three times in wash solution (0.03% SDS in SSC). Microarray Data Analysis. The mouse oligonucleotide clones were used to search against the rat genome using cross-species megaBLAST (NCBI) using default parameters. Only clones with at least 95% sequence identity were used in the study. The microarrays were scanned using a GenePix 4000B scanner (Axon, Foster City, CA) at 10 µM resolution. The photomultiplier tube voltage settings were varied to obtain maximum signal intensities while saturating less than 0.1% of the spots. Images were analyzed using GenePix Pro 6.0.1.22 (Axon) software, utilizing the option to find irregular features. Spots with visible artifacts or containing fewer than 40 pixels, 20% or more saturated pixels or a median intensity less than double background intensity were flagged as “bad”. The background was estimated using “morphological closing” followed by “opening” in the GenePix Pro software. To remove systematic sources of variation, within-print-group loess normalization33 was performed, in which the relative weight of 0.1 was given to spots flagged as missing or bad, using a span setting of 0.4. The background subtraction was estimated using “normexp” method with an offset of 50.34 The base 2 logarithm (log2) ratio of the median spot intensity was used to quantify the fold change in relative gene expression levels. A minimum of 50% of nonmissing values was used as filtering criteria to include a spot in the statistical analysis. A total of 19 766 clones were used for the 30 µM study and 16 840 for the 3 µM study after filtering. Journal of Proteome Research • Vol. 9, No. 3, 2010 1229

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Figure 2. Expression profiles of differentially expressed proteins in concentration-dependent 2D-DIGE set. (A) Number of significant up (+) and down (-) regulated proteins between the different concentrations and controls (fold change threshold log2 0.2 and p < 0.05). (B) The upper Venn diagram shows all shared differentially expressed proteins in the different concentrations compared to controls. The lower Venn diagram displays all shared differentially expressed and identified proteins in the different concentrations compared to controls (fold change threshold log2 0.2 and p < 0.05). The green spots show the number of proteins that exhibit the same type of differential regulation (up vs up regulation, down vs down regulation). The red spots show the number of proteins that exhibit the different types of differential regulation (down vs up regulation, up vs down regulation). (C) Overview of significantly enriched STEM expression profiles (colored profiles, p < 0.0001). The significant profiles are represented by 283 out of 816 spots. Same color profiles are similar to each other (orange profiles, 141 spots; pink profiles, 102 spots; blue profile, 29 spots; brown profile, 11 spots). (D) Top STEM expression profiles for 4 selected significant GO groups. All groups include profiles where the 3 µM induce an expression change which is in the opposite direction to the expression change after exposure to 10 and 30 µM. Profiles with an asterisk are significantly enriched in GO-group proteins (*p < 0.05, **p < 0.01).

A linear model was employed to account for dye-specific differences and to assess differential expression moderated t-statistics, and lod scores were calculated.27 A p-value of 0.005 (p < 0.005)24 was used as the threshold for differential expression. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). One microgram of total RNA from each sample was reverse transcribed into cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems) with random hexamer primers according to the manufacturer’s instructions. Reactions excluding MultiScribe Reverse Transcriptase (Applied Biosystems) were performed as negative controls. PCR primers were designed using the Primer3 software (http://frodo.wi.mit.edu/) with the rat genome as template. cDNA targets at a 20-fold final dilution were amplified in replicate wells (three biological replicates and three technical replicates for each biological replicate) using optimized primer concentrations in 1× qPCR Mastermix (12.5 µL of SYBR Green I Mastermix with 10 nM fluorescein, 5 µL of forward and reverse primer (250 nM), and 2.5 µL of nuclease-free water) in a Bio-Rad Icycler IQ System (Bio-Rad, CA) with the following thermal profile: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C in 25 µL. PCR products were checked by 1230

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monitoring melting curves. For each concentration, three biological replicates, each analyzed in triplicate, were used. For each gene, the mean normalized gene expression (MNE) was calculated based on the ratio between the target gene and three reference genes according to Muller35 and as previously described.36,37

Results Electrospray ionization linear trap quadrupole (ESI-LTQ) mass spectrometry (MS) was used to identify a total of 292 proteins differentially expressed in a concentration-dependent manner in the large data set; 156 of these proteins were also identified in the smaller time-concentration data set (Supplemental Data S3). The 24 h PBDE-99 Exposure. Cultured cortical cells displayed significant levels of cell death upon treatment with 10 and 30 µM PBDE-99, indicating the lowest dose (3 µM) to be subtoxic. The highest concentration (30 µM) resulted in >30% cell death (Figure 1B). A principal component analysis (PCA) of the 816 spots in the larger data set generated a model using three components to explain 60.8% of the variation (PC1

In Vitro Neurotoxicity of PBDE-99 explained 39.3% of the variation, PC2 explained 11.8% and PC3 explained 9.7%). While the PCA plot showed relatively small differences between controls and lower doses, the high 24 h PBDE-99 concentration was clearly separate from the rest of the data (Figure 1C). A pairwise comparison approach using a cutoff at log2 0.2 (and p < 0.05) revealed 62, 46, and 443 differentially expressed spots compared to controls in the 3, 10, and 30 µM exposure groups, respectively, after 24 h (Supplemental Data S3). Of these, 48, 43, and 238 proteins, respectively, were successfully identified. Less than 10% of all matched spots were differentially regulated at the subtoxic concentration of 3 µM and the slightly toxic 10 µM, whereas 50-60% were affected at the highest (toxic) concentration (Figure 1D). STEM analysis of the larger concentration-response data set showed a number of expression profiles to be overrepresented (Figure 2C). Among the profiles, 23% of the larger data set (190 spots) was generally downregulated in response to 30 µM treatment, and 140 spots (or 17%) were upregulated with 30 µM treatment. Several gene ontology (GO) terms were significantly enriched for both individual and clusters of STEM profiles. Among them were Cytoskeleton, Actin cytoskeleton, Microtubule cytoskeleton and Axon guidance (Figure 2D). Overall, we detected somewhat fewer upregulated than downregulated spots (p < 0.05, log2 |0.2|) in response to treatment with various concentrations of PBDE-99 compared to controls (Figure 2A). Upon closer inspection, there were almost no differentially expressed proteins/spots in common between the lower doses (p < 0.05, log2 |0.2|), whereas we detected a larger overlap between the low doses and the high dose (Figure 2B). Interestingly, the proteins/spots in common between the lowest (3 µM) and highest dose (30 µM) (3 vs 30) displayed different fold change properties than those in common between 10 and 30 µM (10 vs 30). Equal numbers of proteins/spots were up- and downregulated from 3 vs 30 (41 spots, 28 identified), whereas proteins/spots from 10 vs 30 (28 spots, 26 identified) generally displayed the same expression patterns. Only nine proteins were significantly affected in all exposure groups compared to controls: two isoforms of stathmin (STMN1), eukaryotic translation initiation factor 1A (EF1A), Psmd8 protein (Q3B8P5), transaldolase (TALDO), 26S proteasome non-ATPase regulatory subunit 7 (PSD7), tubulin beta-5 chain (TBB5), serine/threonine-protein phosphatase 2A (2AAA) and pyruvate kinase isozymes M1/M2 (KPYM). Our previous study on the effects of 24 h of PBDE-99 treatment on the neonatal mouse cortex demonstrated a large expression effect on cytoskeleton-related proteins. A GO-based annotation (using the DAVID database32) of the proteins identified in this study found 38% (18 proteins) of the proteins differentially expressed at 3 µM to be cytoskeleton related (annotation cluster 9 in the functional annotation using “cytoskeleton organization and biogenesis”, “cytoskeletal protein binding” and “cytoskeleton”), compared to 9% (4 proteins) and 19% (45 proteins) differentially expressed in response to 10 and 30 µM treatment, respectively. The 1 h PBDE-99 Exposure. A PCA model (two components where PC1 explains 36.6% of the variation and PC2 explains 12.8% of the variation) using the smaller time-concentration data set clearly separated the 24 h 30 µM concentration samples from the other samples (Figure 3A). Hierarchical clustering of the data from the smaller data set also showed that the 24 h 30 µM group does not cluster well with the other groups. The 10 µM group clustered better with controls than with the 3 µM

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Figure 3. Principal component analysis (PCA) and hierarchical clustering based on data from the smaller DIGE data set. (A) All 28 DIGE samples visualized in a two-dimensional scatter plot that uses the two first components (DC1-DC2). The model uses expression intensities from 345 spots (156 identified) and explains 50% of the variation in the data. The circle corresponds to a Hotelling’s T2 tolerance at 0.95. (B) Unsupervised hierarchical clustering. Two-way clustering of the group median expression values for all 345 proteins was used to visualize differences between the exposure groups.

group (Figure 3B). Using pairwise comparisons, a total of 69 spots were found to be differentially regulated (p < 0.05, log2 |0.2|) after 1 h exposure to 30 µM PBDE-99; of the 69 spots, 31 were identified (Supplemental Data S4) and included proteins such as several isoforms of the cytoskeleton-associated stathmin. To assess differences in phosphorylation of the proteins, three gels were stained with the Pro-Q Diamond phosphoprotein gel stain (a representative gel image is presented in Figure 1A). A large number of proteins were phosphorylated after 1 h exposure to 30 µM PBDE-99. Proteomic Similarities between in Vivo and in Vitro PBDE-99 Exposure. We compared the protein identities in our data sets with results from our previous 2D-DIGE study in postnatal day (PND) 11 mouse cortex after a single oral exposure to 12 mg (21 µM) of PBDE-99/kg body weight on PND10.22 While pointing out that there are developmental differences between a PND11 mouse and GD 21 rats from which the cells used in these experiments were taken, a total of 33 nonredundant protein identities (154 when including all in vivo and in vitro isoforms) were identical between the in vivo analysis and our present data (Figure 4). All 33 proteins are over 90% identical on the amino acid sequence level between mouse and rat (data not shown), and 26 were significantly (p < 0.05) differentially expressed in vivo. Among the most significantly affected proteins in vivo (FABP7, ACTB, GRP78, PP1R7, QCR2, FABP5), alterations were seen for many of them at low PBDE-99 concentrations in vitro (Figure 4). One out of eight in vitro isoforms of dihydropyrimidinase-related Journal of Proteome Research • Vol. 9, No. 3, 2010 1231

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Figure 4. Comparison between PBDE-99 neurotoxicity in vivo and in vitro. A total of 33 protein identities were identical between previous in vivo experiments (cortex from 24 h PBDE-99 exposure on PND10) in neonatal mice22 and the present in vitro study. The in vivo significance levels are seen to the left of the identity column, in vitro data to the right. Also included are microarray gene expression significance levels when possible.

protein 3 in this study is especially interesting since Dpyl3 is significantly affected in vivo and this isoform shows aberrant expression after both 1 and 24 h PBDE-99 exposure in vitro. The protein identities were cross-referenced against mouse MEEBO microarrays results from 24 h 3 µM and 30 µM exposures to determine whether there was a correlation between PBDE-99-induced changes in mRNA and protein expression. To minimize cross species effects on probe hybridization, we only used expression data from probes >95% identical in sequence between rat and mouse (after sequence comparison and filtering, 16 840 probes for the 3 µM study and 19 766 for the 30 µM study). We only detected a weak correlation between mRNA and protein changes in response to 3 µM treatment, whereas the more toxic high dose resulted in mRNA and protein changes that showed a greater correlation. Realtime qPCR analysis of selected genes (FABP7, FABP5, DPYL3 and DDX3X) encoding proteins listed in Figure 4 did not demonstrate significant differences in mRNA expression (p > 0.05), with the exception of DPYL3 mRNA, which was significantly downregulated after 24 h of 30 µM PBDE-99 (p < 0.05, Supplemental Data S5). A GO and GSEA analysis of the microarray data from the two comparisons (3 µM vs controls, and 30 µM vs controls) did not reveal any clear patterns (data not shown). 1232

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Gap43. In addition to our mainly proteomic analysis of PBDE-99-exposed primary cortical cells, we also analyzed mRNA and protein levels of growth-associated protein-43 (Gap43), a protein not detected in our present 2D-DIGE study but one we previously found to be sensitive to 24 h PBDE-99 exposure in both in vivo neonatal mouse striatum21 and cerebral cortex.22 Interestingly, qPCR analysis showed that Gap43 mRNA level significantly increased after 24 h of 0.3 µM exposure, and was significantly downregulated after 24 h 30 µM (Figure 5), suggesting different effects of low and high concentrations of PBDE-99 on mRNA expression. Western blot analysis did not reveal any correlation between Gap43 protein levels and mRNA expression, except after exposure to the highest concentration where cytotoxicity likely influences the downregulation of both mRNA and protein. However, we found a significant increase in levels of the Ser-41 phosphorylated form of Gap43 at the lowest (nontoxic) doses (0.3 and 3 µM).

Discussion Despite the well-known general toxicity of various PBDEs, the actual mechanisms underlying their neurotoxicity remain poorly understood. Our earlier proteomic studies on PBDE99-induced neurotoxicity 24 h after exposure in neonatal

In Vitro Neurotoxicity of PBDE-99

Figure 5. Effects on Gap43 mRNA and protein expression. (A) Effects on Gap43 mRNA after 24 h exposure to PBDE-99. A mean normalized gene expression (MNE) was calculated based on the ratio between target and three reference genes according to Muller35 and as previously described.36,37 Three biological replicates were used for each dose group, and three technical replicates for each biological replicate. Dose of 0.3 µM PBDE-99 significantly upregulated the mRNA expression (p < 0,01) and 30 µM significantly downregulated the mRNA expression (p < 0,05). Error bars display standard error of the mean (SEM). (B) Effects on GAP43 protein expression after 24 h exposure to PBDE99. No apparent effects except a downregulation at the 30 µM concentration. (C) Effects on phosphorylated Gap43. The 0.3 and 3 µM doses show upregulated protein expression. (D) Tubulin β-III was used as reference protein. The staining intensity was similar for DMSO and all PBDE concentrations used in the experiment. Representative images are shown.

cortex22 indicated altered neuronal (or glial) maturation and effects on neurite sprouting processes. The aim of this study was to examine changes in the primary cortical cell proteome in response to different concentrations of PBDE-99, ranging from noncytotoxic and in the range of the doses used in vivo (3 µM), to clearly cytotoxic doses (30 µM). Our findings demonstrate that the biological effects of low concentrations of PBDE-99 exposure are fundamentally different than effects of high concentrations of exposure. Although 24 h 10 µM exposure induced cell death, there was no general increase in significantly affected proteins compared to 3 µM treatment (Figure 1B,D). Upon examination of proteins/spots differentially expressed at both 10 µM and the clearly cytotoxic 30 µM dose (compared to control), most proteins/spots show the same regulation effects. In contrast, very few proteins/spots were differently expressed in response to both the lowest dose (3 µM) and either the 10 or 30 µM exposure. The near complete lack of differentially expressed proteins shared between the low doses (3 and 10 µM) while we detected a larger overlap between each of the lower doses with the highest dose (30 µM) is peculiar. The larger overlap between 10 and 30 µM exposures makes sense in the light of cytotoxicity induction; both 10 and 30 µM induce significant reductions in cell viability (Figure 1B). Indeed, several in vitro studies using PBDE-99 or DE-71 in different cell lines have shown cytotoxicity induction at or below the 10 µM concentration level.20,38–40 A possible explanation for the larger overlap between 3 and 30 µM compared to 3 and 10 µM PBDE-99 is that while the proteins are sensitive to both low and high concentrations of

research articles the substance, they for the most part have different regulation effects. A shared protein that is significantly upregulated at 3 µM is most often significantly down regulated at 30 µM PBDE99. That we observed significant cell death at levels as low as 10 µM PBDE-99 in GD21 primary cortical neurons makes these cells much more sensitive to PBDE-99 cytotoxicity than the human astrocytoma cell line reported by Madia et al.,15 in which no effects were seen even at 100 µM, as assessed by trypan blue assay. The reduced cell viability at 10 µM indicates that the GD21 cells used in this study are more sensitive than the previously reported GD17 cells, where reduced cell viability was seen at 30 µM.41 This difference in sensitivity may reflect that the GD17 cultures consist almost exclusively of neurons, while the GD21 cultures also contain astrocytes.42 Our previous study on the effects of PBDE-99 in mouse neonatal cortex reported aberrant expression of a large number of cytoskeleton-related proteins.22 In this study, the expression of cytoskeletal proteins was also affected upon treatment with various doses of PBDE-99, although most prominently in response to 3 µM (18 proteins) and 30 µM (45 proteins) treatment, with only 4 proteins affected with 10 µM. Effects on cytoskeleton-associated proteins after exposure to the highest concentration of PBDE-99 are not surprising, considering the increased cell death detected with 30 µM treatment. However, the effects observed after exposure to the noncytotoxic 3 µM concentration suggest that other regulatory aspects of cytoskeletal functions may be affected. The STEM GO analysis suggests different effects of high and low concentrations of PBDE-99 on axon guidance (Figure 2D). This possibility is supported by data demonstrating that as low as 0.3 µM PBDE-99 affects post-translational modification of Gap43 to a more active neurite-stimulatory form (Ser-41 phosphorylated).43 Dihydropyrimidinase-related protein 3 (Dpyl3), involved in neuronal plasticity and neurite outgrowth and extension,44 also functions in the regulation of cytoskeletal organization.45,46 In this study, several Dpyl3 isoforms displayed significantly altered expressions after treatment with both low and high concentrations of PBDE-99. Dpyl3 expression is also altered in mouse cerebral cortex after neonatal PBDE-99 exposure.22 Interestingly, no cell death-independent changes were observed in the mRNA levels of Dpyl3 in this study. Instead, we found that specific Dpyl3 protein isoforms were affected within just 1 h of PBDE-99 exposure. As 1 h may not be long enough for alterations in mRNA levels to result in detectable changes in protein synthesis, and no changes in gene expression were observed, it seems reasonable to assume that changes in Dpyl3 isoforms reflect alterations in post-translational modifications. We found that all of the resolved Dpyl3 isoforms were stained in the Pro-Q Diamond phosphostain, suggesting that changes in the number of phosphor-groups on the Dpyl3 isoforms results in their separation, or that other unidentified PTMs are involved. To conclude, the present investigation shows that PBDE-99 alters the levels of cytoskeletal proteins already at low micromolar nontoxic concentrations in cultured cerebral cortex cells, an effect that is consistent with previous in vivo data.41 Our data define a group of proteins known to be affected both in vivo and in vitro, some of them within a very short time span. Since PBDE-99-mediated effects are evident as soon as 1 h of exposure, we hypothesize that the effects are, at least in part, mediated by changes in post-translational modifications of Journal of Proteome Research • Vol. 9, No. 3, 2010 1233

research articles these proteins. Proteins such as dihydropyrimidinase-related protein 3 and Gap43 are particularly interesting candidates for future studies of PBDE-99 neurotoxicity. Since the molar concentration in the brain after neonatal in vivo exposure is in the same order of magnitude used in this study (based on ref 47), we speculate that PBDE-99, and possibly other PBDEs, may disrupt cortical development in rodents by directly affecting cytoskeletal organization in a noncytotoxic, possibly even stimulatory manner. The importance of a functional and dynamic cytoskeletal organization for neurodevelopmental events can hardly be overstated. The proper interaction of the actin cytoskeleton and microtubules steers neuronal development and in so doing mediates axonal branching as well as dendritic spine and synapse formation. Any disruption of this molecular interplay will likely have dramatic effects on both brain architecture and function. Thus, if the effects of PBDE99 on cytoskeletal proteins holds true also after detailed, scrutinizing in vivo studies, the effects on behavior seen after developmental PBDE-99 exposure can be related to a miswiring of the adult rodent brain.

Acknowledgment. This work was supported by the The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (2008-805). The authors wish to thank Dr. Timothy Gant at the University of Leicester for kindly providing the MEEBO arrays. Supporting Information Available: Normalized data used for STEM expression analysis, isoform adapted Gene Ontology (GO) annotations used in STEM analysis, identified proteins in all exposure groups, differentially regulated proteins after 1 h exposure to 30 µM PBDE-99 and mean MNE values for the 5 genes which were analyzed with qRT-PCR including primer sequences and mean efficiency values. all protein identities for each spot in the experiment. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) de Wit, C. A. An overview of brominated flame retardants in the environment. Chemosphere 2002, 46 (5), 583–624. (2) Costa, L. G.; Giordano, G. Developmental neurotoxicity of polybrominated diphenyl ether (PBDE) flame retardants. Neurotoxicology 2007, 28 (6), 1047–67. (3) Vonderheide, A. P.; Mueller, K. E.; Meija, J.; Welsh, G. L. Polybrominated diphenyl ethers: Causes for concern and knowledge gaps regarding environmental distribution, fate and toxicity. Sci. Total Environ. 2008, 400 (1-3), 425–36. (4) Jin, J.; Liu, W.; Wang, Y.; Yan Tang, X. Levels and distribution of polybrominated diphenyl ethers in plant, shellfish and sediment samples from Laizhou Bay in China. Chemosphere 2008, 71 (6), 1043–50. (5) Binelli, A.; Sarkar, S. K.; Chatterjee, M.; Riva, C.; Parolini, M.; Bhattacharya, B. D.; Bhattacharya, A. K.; Satpathy, K. K. Concentration of polybrominated diphenyl ethers (PBDEs) in sediment cores of Sundarban mangrove wetland, northeastern part of Bay of Bengal (India). Mar. Pollut. Bull. 2007, 54 (8), 1220–9. (6) Branchi, I.; Alleva, E.; Costa, L. G. Effects of perinatal exposure to a polybrominated diphenyl ether (PBDE 99) on mouse neurobehavioural development. Neurotoxicology 2002, 23 (3), 375–84. (7) Branchi, I.; Capone, F.; Alleva, E.; Costa, L. G. Polybrominated diphenyl ethers: neurobehavioral effects following developmental exposure. Neurotoxicology 2003, 24 (3), 449–62. (8) Eriksson, P. Brominated flame retardants: a novel class of developmental toxicants in our environment. Environ. Health Perspect. 2001, 109 (9), 903–8. (9) Eriksson, P.; Viberg, H.; Jakobsson, E.; Orn, U.; Fredriksson, A. A brominated flame retardant, 2,2′,4,4′,5-pentabromodiphenyl ether: uptake, retention, and induction of neurobehavioral alterations in mice during a critical phase of neonatal brain development. Toxicol. Sci. 2002, 67 (1), 98–103.

1234

Journal of Proteome Research • Vol. 9, No. 3, 2010

Alm et al. (10) Viberg, H.; Fredriksson, A.; Eriksson, P. Neonatal exposure to the brominated flame retardant 2,2′,4,4′,5-pentabromodiphenyl ether causes altered susceptibility in the cholinergic transmitter system in the adult mouse. Toxicol. Sci. 2002, 67 (1), 104–7. (11) Viberg, H.; Fredriksson, A.; Eriksson, P. Neonatal exposure to polybrominated diphenyl ether (PBDE 153) disrupts spontaneous behaviour, impairs learning and memory, and decreases hippocampal cholinergic receptors in adult mice. Toxicol. Appl. Pharmacol. 2003, 192 (2), 95–106. (12) Kodavanti, P. R.; Ward, T. R. Differential effects of commercial polybrominated diphenyl ether and polychlorinated biphenyl mixtures on intracellular signaling in rat brain in vitro. Toxicol. Sci. 2005, 85 (2), 952–62. (13) Mariussen, E.; Fonnum, F. The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles. Neurochem. Int. 2003, 43 (4-5), 533–42. (14) Kodavanti, P. R.; Derr-Yellin, E. C. Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons. Toxicol. Sci. 2002, 68 (2), 451–7. (15) Madia, F.; Giordano, G.; Fattori, V.; Vitalone, A.; Branchi, I.; Capone, F.; Costa, L. G. Differential in vitro neurotoxicity of the flame retardant PBDE-99 and of the PCB Aroclor 1254 in human astrocytoma cells. Toxicol. Lett. 2004, 154 (1-2), 11–21. (16) Zhang, M.; He, W. H.; He, P.; Xia, T.; Chen, X. M.; Wang, A. G. [Effects of PBDE-47 on oxidative stress and apoptosis in SH-SY5Y cell]. Zhonghua Laodong Weisheng Zhiyebing Zazhi 2007, 25 (3), 145–7. (17) Kodavanti, P. R.; Ward, T. R.; Ludewig, G.; Robertson, L. W.; Birnbaum, L. S. Polybrominated diphenyl ether (PBDE) effects in rat neuronal cultures: 14C-PBDE accumulation, biological effects, and structure-activity relationships. Toxicol. Sci. 2005, 88 (1), 181– 92. (18) Coburn, C. G.; Curras-Collazo, M. C.; Kodavanti, P. R. In vitro effects of environmentally relevant polybrominated diphenyl ether (PBDE) congeners on calcium buffering mechanisms in rat brain. Neurochem. Res. 2008, 33 (2), 355–64. (19) Hamers, T.; Kamstra, J. H.; Sonneveld, E.; Murk, A. J.; Kester, M. H.; Andersson, P. L.; Legler, J.; Brouwer, A. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants. Toxicol. Sci. 2006, 92 (1), 157–73. (20) Tagliaferri, S.; Caglieri, A.; Goldoni, M.; Pinelli, S.; Alinovi, R.; Poli, D.; Pellacani, C.; Giordano, G.; Mutti, A.; Costa, L. G. Low concentrations of the brominated flame retardants BDE-47 and BDE-99 induce synergistic oxidative stress-mediated neurotoxicity in human neuroblastoma cells Toxicol In Vitro DOI: 10.1016/ j.tiv.2009.08.020. Published online: Aug 29, 2009. (21) Alm, H.; Scholz, B.; Fischer, C.; Kultima, K.; Viberg, H.; Eriksson, P.; Dencker, L.; Stigson, M. Proteomic evaluation of neonatal exposure to 2,2 4,4 5-pentabromodiphenyl ether. Environ. Health Perspect. 2006, 114 (2), 254–9. (22) Alm, H.; Kultima, K.; Scholz, B.; Nilsson, A.; Andren, P. E.; FexSvenningsen, A.; Dencker, L.; Stigson, M. Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex. Neurotoxicology 2008, 29 (4), 628–37. (23) o¨rn, U.; Eriksson, L.; Jakobsson, E.; Bergman, Å. Synthesis and characterization of polybrominated diphenyl ethers - Unlabeled and radiolabeled tetra-, penta-, and hexabromodiphenyl ethers. Acta Chem. Scand. 1996, 50, 802–807. (24) Sonck, K.; De Keersmaecke, S.; Vanderleyden, J. DIGE protein labeling; how low can you go? Mol. Cell. Proteomics 2005, 4 8 (Suppl. 1), 290–8. (25) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551–67. (26) Kultima, K.; Scholz, B.; Alm, H.; Skold, K.; Svensson, M.; Crossman, A. R.; Bezard, E.; Andren, P. E.; Lonnstedt, I. Normalization and expression changes in predefined sets of proteins using 2D gel electrophoresis: a proteomic study of L-DOPA induced dyskinesia in an animal model of Parkinson’s disease using DIGE. BMC Bioinf. 2006, 7, 475. (27) Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 2004, 3, Article3. (28) Smyth, G. K.; Michaud, J.; Scott, H. S. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 2005, 21 (9), 2067–75. (29) Lo¨nnstedt, I, S. T. Replicated microarray data. Stat. Sin. 2002, 12, 31–46.

research articles

In Vitro Neurotoxicity of PBDE-99 (30) Ernst, J.; Nau, G. J.; Bar-Joseph, Z. Clustering short time series gene expression data. Bioinformatics 2005, 21, i159–68. (31) Ernst, J.; Bar-Joseph, Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinf. 2006, 7, 191. (32) Dennis, G., Jr.; Sherman, B. T.; Hosack, D. A.; Yang, J.; Gao, W.; Lane, H. C.; Lempicki, R. A. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 2003, 4 (5), P3. (33) Yang, Y. H.; Dudoit, S.; Luu, P.; Lin, D. M.; Peng, V.; Ngai, J.; Speed, T. P. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002, 30 (4), e15. (34) Ritchie, M. E.; Silver, J.; Oshlack, A.; Holmes, M.; Diyagama, D.; Holloway, A.; Smyth, G. K. A comparison of background correction methods for two-colour microarrays. Bioinformatics 2007, 23 (20), 2700–7. (35) Muller, P. Y.; Janovjak, H.; Miserez, A. R.; Dobbie, Z. Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques 2002, 32 (6), 1372-41376, 1378-9. (36) Kultima, K.; Fernandez, E. L.; Scholz, B.; Gustafson, A. L.; Dencker, L.; Stigson, M. Cadmium-induced gene expression changes in the mouse embryo, and the influence of pretreatment with zinc. Reprod. Toxicol. 2006, 22 (4), 636–46. (37) Jergil, M.; Kultima, K.; Gustafson, A. L.; Dencker, L.; Stigson, M. Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects. Toxicol. Sci. 2009, 108 (1), 132– 48. (38) Giordano, G.; Kavanagh, T. J.; Costa, L. G. Neurotoxicity of a polybrominated diphenyl ether mixture (DE-71) in mouse neurons and astrocytes is modulated by intracellular glutathione levels. Toxicol. Appl. Pharmacol. 2008, 232 (2), 161–8. (39) Reistad, T.; Fonnum, F.; Mariussen, E. Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabro-

(40)

(41)

(42) (43) (44) (45)

(46) (47)

mocyclododecane (HBCD) in rat cerebellar granule cells in vitro. Arch. Toxicol. 2006, 80 (11), 785–96. Yu, K.; He, Y.; Yeung, L. W. Y.; Lam, P. K. S.; Wu, R. S. S.; Zhou, B. DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells in vitro. Toxicol. Sci. 2008, 104 (2), 341–51. Alm, H.; Kultima, K.; Scholz, B.; Nilsson, A.; Andren, P. E.; FexSvenningsen, A.; Dencker, L.; Stigson, M. Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex. Neurotoxicology 2008, 294, 628–37. Sauvageot, C. M.; Stiles, C. D. Molecular mechanisms controlling cortical gliogenesis. Curr. Opin. Neurobiol. 2002, 12 (3), 244–9. Dent, E. W.; Meiri, K. F. Distribution of phosphorylated GAP-43 (neuromodulin) in growth cones directly reflects growth cone behavior. J. Neurobiol. 1998, 35 (3), 287–99. Quinn, C. C.; Gray, G. E.; Hockfield, S. A family of proteins implicated in axon guidance and outgrowth. J. Neurobiol. 1999, 41 (1), 158–64. Franken, S.; Junghans, U.; Rosslenbroich, V.; Baader, S. L.; Hoffmann, R.; Gieselmann, V.; Viebahn, C.; Kappler, J. Collapsin response mediator proteins of neonatal rat brain interact with chondroitin sulfate. J. Biol. Chem. 2003, 278 (5), 3241–50. Rosslenbroich, V.; Dai, L.; Baader, S. L.; Noegel, A. A.; Gieselmann, V.; Kappler, J. Collapsin response mediator protein-4 regulates F-actin bundling. Exp. Cell Res. 2005, 310 (2), 434–44. Vijverberg, H. P.; van den Berg, M. Re: Viberg, H. et al. Neurobehavioral derangements in adult mice receiving decabrominated diphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicol. Sci. 2003, 76 (1), 112–20. Toxicol. Sci. 2004, 79, (1), 205-6; author reply 207-8.

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