Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry; Rochester, New York 14642
Received August 13, 2004; accepted November 3, 2004
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ABSTRACT |
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Key Words: neurogenesis; bHLH/PAS; Arnt; CYP 1A1/1B1; dioxin.
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INTRODUCTION |
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Most, if not all TCDD-induced pathology is mediated via binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily (Gu et al., 2000). The AhR resides in the cytosol bound to 90 kD heat shock protein (hsp90) and several other chaperone proteins (Gu et al., 2000
). Upon ligand binding, hsp90 dissociates from AhR and the receptor translocates to the nucleus. After dimerizing with the aryl hydrocarbon receptor translocator protein (Arnt), also a member of the bHLH/PAS family, the complex binds dioxin-response elements (DREs), located in the 5' region upstream of responsive target genes (Gu et al., 2000
), most notably a group of proteins termed the "AhR gene battery" (Nebert et al., 2000
). The mechanisms involved in AhR-mediated transcription have been elucidated primarily by the investigation of cytochrome P450 (CYP) gene regulation (Whitlock 1999
). Exposure to TCDD induces CYP1A1 and 1B1 gene transcription coordinately with dose-dependent and time-dependent increases in protein levels (Filbrandt et al., 2004
; Hakkola et al., 1997
; Whitlock, 1999
). TCDD, through interaction with the AhR, is also known to regulate the expression of a wide array of additional drug-metabolizing enzymes, genes that participate in cell cycle regulation, and inflammatory mediators (Lai et al., 1996
; Nebert et al., 2000
; Puga and Elferink, 2002
). Despite the understanding of the molecular mechanisms by which TCDD modulates gene regulation, specific roles for AhR during brain development and in neurotoxicity are poorly understood.
Evidence at the cellular and molecular levels supports the contention that inappropriate AhR activation by xenobiotics during development could lead to neurotoxicity in the cerebellum. For example, prenatal exposure to 7,12-dimethylbenz[a]anthracene, an Ah-R ligand and a known carcinogen, was shown to profoundly disrupt cerebellar cytoarchitecture (Kellen et al., 1976). At the molecular level, a recent study revealed that the cerebellum contained the highest levels of DRE binding in the adult rat brain and that indigo, a putative endogenous AhR ligand, stimulated DRE binding in cerebellar granule neurons (Kuramoto et al., 2003
). Furthermore, gestational TCDD exposure was shown to modulate the developmental expression profile of Sp1, a transcription factor involved in growth and differentiation, in rat cerebellum and cerebral cortices (Nayyar et al., 2002
), but the biological significance requires clarification. Although the developing cerebellum is potentially vulnerable to AhR-mediated neurotoxicity, the precise cellular localization and transcriptional activity of AhR in this brain region requires additional study.
Considering that AhR is present during critical phases of histogenesis in several organs (Abbott et al., 1995; Abbott and Probst, 1995
) and is widely expressed throughout the adult central nervous system (CNS) (Petersen et al., 2000
), it is conceivable that AhR might normally regulate neurogenesis and differentiation during brain development. It is hypothesized that perinatal exposure to TCDD disrupts endogenous AhR signaling events during brain development. This study determined that AhR is expressed and transcriptionally active in cerebellar granule neuroblasts throughout a critical period of postnatal neurogenesis. Several processes, which include proliferation, migration, differentiation, synaptogenesis, and programmed cell death (apoptosis), occur during this time (Altman and Bayer, 1997
; Goldowitz and Hamre, 1998
; White and Barone, 2001
). Furthermore, TCDD was shown to reduce DNA synthesis and cell survival in a granule precursor cell model system that maintains the ability to proliferate in vitro. These observations suggest potential roles for AhR in cerebellar granule neuron maturation and raise the possibility that TCDD might interfere with these actions by displacing an endogenous ligand from its normal developmental functions.
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MATERIALS AND METHODS |
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Experimental animals. All animals were maintained on a 12-h light/dark cycle with food and water provided ad libitum and kept in accordance with the guidelines set by the University of Rochester University Committee on Animal Resources and the American Association for Laboratory Animal Science. Five- to six-day-old C57Bl/6 J mice were purchased from Jackson Laboratories (Bar Harbor, ME). AhR / mice modified at exon 2 (Schmidt et al., 1996) were also obtained from Jackson Laboratories and backcrossed onto the wild-type C57Bl/6 J background for at least 10 generations. C57Bl/6 J mice were purchased from Jackson Laboratory, and DRE-lacZ animals were generated as previously described (Willey et al., 1998
). Animals were generated using the p21lac plasmid construct, which consists of 2 DRE-Ds, a TATA box, lacZ reporter, and the SV40 intron and polyadenylation signal. Mice were maintained as a heterozygous colony backcrossed onto wild-type C57Bl/6 J background for greater than 10 generations. Polymerase chain reaction (PCR) analysis of tail DNA was performed to determine transgene status of animals.
In vivo TCDD treatment. DRE-lacZ mice were injected with 30µg TCDD/kg dissolved in olive oil or vehicle alone when AhR protein is at peak levels on postnatal day 10 in the developing cerebellum. This dose has been previously shown to produce maximal induction of aryl hydrocarbon hydroxylase activity in C57Bl/6 J mice (Poland and Glover, 1975). Animals were perfused 1824 h later with saline, followed by 4% paraformaldehyde. This time period after TCDD exposure was previously reported to be sufficient for detection of ß-galactosidase activity in tissue sections obtained from transgenic animals (Nazarenko et al., 2001
).
ß-galactosidase staining. In situ localization of ß-galactosidase (ß-gal) activity was determined on a histochemical substrate, 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-gal). After perfusion, whole brains were fixed in 4% paraformaldehyde overnight. Brains were transferred to 30% sucrose for 24 h. Cerebella were cut into 30-µm sagittal sections with a freezing sliding microtome. Sections were incubated with X-gal for 16 h at 37°C in a humidified chamber and then rinsed with DPBS before mounting. Wild-type animals were injected with olive oil to evaluate endogenous ß-galactosidase activity.
Cell culture. Primary granule cell cultures were prepared as previously described (Gao et al., 1991; Opanashuk and Hauser, 1998
; Opanashuk et al., 2001
). Briefly, cerebella were quickly dissected from postnatal day 56 C57/Bl6 mice and the meninges were removed prior to the addition of trypsin and DNAse. Dissociated cells were then passed through a 30-µm nylon mesh (Spectrum Laboratories) and centrifuged through a 35%:60% Percoll gradient (Amersham Biosciences, Piscataway, NJ). Cells were then rinsed and preplated for 90 min in poly-D-lysine (0.1 mg/ml; Sigma, St. Louis, MO) coated flasks. Cells were then resuspended in MEM containing 10% horse serum, 5% fetal bovine serum, glucose (9 mg/ml), glutamine (292 mg/ml), and penicillin (0.1%). Cells were plated in 96-well plates at a density of 3 x 105 cells/well and maintained in a humidified atmosphere of 5% CO2/95% air at 37°C to facilitate reaggregate formation. After 24 h, reaggregates were then transferred to serum-free MEM (SFM) containing B27 and N2 supplements, glucose (9 mg/ml), glutamine (292 mg/ml), and penicillin (0.1%), and plated on high molecular weight poly-D-lysinecoated plates (0.1 mg/ml). This time point is designated day in vitro 1 (DIV 1).The characterization of these cultures, as previously described (Gao et al., 1991
) by morphological and immunocytochemical criteria, indicated that >98% of these cells were of the granule neuron lineage.
Western blot analysis. Cerebellar tissue or cells were homogenized in phosphate buffered saline (PBS) containing 0.1% Triton X-100 and antiprotease cocktail (Roche Molecular Biochemical, Manheim, Germany). Total protein concentrations were determined by the microBCA assay (Pierce, Rockford, IL). Proteins (2075 µg) were fractionated on 7% acrylamide gels and transferred to Immun-Blot PVDF membranes (BioRad, Hercules, CA). Membranes were blocked with 5% powdered milk containing 0.2% Tween-20 and probed with antibodies specific for AhR (1:5000; Biomol, Plymouth Meeting, PA), ARNT (1:2000; Novus, Littleton, CO), CYP1A1 (1:2000; Xenotech LLC, Kansas City, KS), or CYP1B1 (1:5,000; a generous gift from Dr. Colin Jefcoate, University of Wisconsin, Madison, WI), overnight at 4°C. Membranes were then probed with the appropriate horseradish peroxidase conjugated secondary antibodies (1:5000; Jackson Immunoresearch, Westgrove, PA) for 2 h at room temperature. Proteins were visualized by chemiluminescence (ECL Amersham Biosciences, Piscataway, NJ). All immunoblots were stripped and reprobed with anti-ß-actin (1:5000; Sigma, St Louis, MO) to confirm equal protein loading. Because the expression of actin and several other housekeeping proteins are developmentally regulated in the brain, the membranes were stained with Ponceau S to verify even transfer of proteins in individual samples during postnatal cerebellar development. AhR, Arnt, or CYP proteins were not detected on blots that were incubated with either secondary antibodies alone or with nonspecific IgG.
Real-time PCR. RNA was isolated using TRIZOL reagent following instructions provided by the manufacturer (Invitrogen). RNA was quantified by spectroscopy then concentrated with 5 M ammonium acetate and ethanol precipitation. Samples were resuspended in DEPC (0.1% diethylpyrocarbonate)-treated water. First strand cDNA was synthesized from 4 µg RNA using the SuperScript II First Strand cDNA Synthesis Kit (Invitrogen). Prior to real-time PCR analysis, samples were phenol/chloroform extracted and ethanol precipitated. Real-time quantitative PCR was accomplished with Taqman PCR primers and Universal MasterMix (PE Applied Biosystems, Foster City, CA) according to the TaqMan EZ RT-PCR (reverse-transcriptase-polymerase chain reaction) User Bulletin #2 Protocol (PerkinElmer). Intron spanning primers with the following sequences were designed using Primer Express Software to reduce genomic amplification. AhR: (F) CGGCTTCTTGCAAAACACAGT (R) GTAA ATGCTCTCGTCCTTCTTCATC (Probe) FAM-AGTCCAATGCACGCTT; CYP1A1: (F) GACCTTCCGGCATTCATCCT (R) TCAGACTTGTATCTCTTGTGGTGCT (Probe) FAM-CGTCCCCTTCACCATCCCCCA-BHQ-1; CYP1B1: (F) TGGCTGCTC ATCCTCTTCACC (R) CCCACAACCTGGTCCAACTC (Probe) FAM-TTCAGGCCCG CGTG-BHQ-1; ß-Actin: (F) GCTTCTTTGCAGCTCCTTCGT (R) CCAGCGCAGCGA TATCG (Probe) HEX-CGCCACCAGTTCGCCATGGA-BHQ-1. Amplified products were fluorometrically detected by the BioRad iCycler analyzer. Plasmid DNA containing CYP1A1, CYP1B1, or ß-actin was used to generate standard curves. All standards and samples were run in duplicate. CYP1A1 and CYP1B1 levels were normalized to relative ß-actin levels. Data are expressed as fold induction compared to vehicle control (DMSO), which was arbitrarily set at one.
Immunocytochemistry. After a 24-h reaggregation period, granule neurons were plated into 8-well culture chamber slides (Lab-Tek, VWR, Bridgeport, NJ) coated with 0.1 mg/ml poly-D-lysine. After 24 h, cells were treated with DMSO or 10 nM TCDD for 1560 min. After the incubation period, the medium was removed and cells were rinsed with DPBS then fixed with 4% paraformaldehyde for 30 min at room temperature. After rinsing, neurons were incubated in PBS containing 10% BSA and 0.3% Triton X-100 (PBST) for 30 min at room temperature. Reaggregates were then incubated overnight at 4°C in PBST containing AhR antibody (1:800; Biomol, Plymouth Meeting, PA). After rinsing, cells were incubated in PBST containing Alexa Fluor 546 goat anti-rabbit secondary antibody (1:1000; Molecular Probes, Eugene, OR) for 90 min at room temperature. Cellular nuclei were subsequently stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI). Slides were cover-slipped with anti-fade (Molecular Probes, Eugene, OR) as mounting media. Fluorescence was visualized using Nikon Eclipse TS100 inverted microscope. Images were taken at 40x magnification with SPOT Advanced software. AhR was not detected in granule neurons isolated from AhR knockout animals or in cultures treated with secondary antibody alone.
Thymidine incorporation. Thymidine incorporation into DNA was measured by procedures similar to those previously described (Gao et al., 1991; Opanashuk and Hauser, 1998
; Opanashuk et al., 2001
). After reaggregation for 24 h, EGL cells were transferred to SFM. TCDD was added to cultures for 36 h and cells were labeled with 2 µCi/ml 3H-thymidine for the last 12 h of the treatment period. Reaggregates were subsequently harvested onto filter paper with TCA washes and repeated water elution with a Skatron cell harvester, as previously described (Gao et al., 1991
; Opanashuk and Hauser, 1998
; Opanashuk et al., 2001
). After the filters dried, thymidine labeling was measured by liquid scintillation counting. At least four independent determinations from separate cell preparations were evaluated for each treatment.
Cell survival assay. Neuron viability was assessed with a commercially available kit (Live/Dead Cytotoxicity Kit, Molecular Probes, Eugene, OR), as previously described (Opanashuk and Hauser, 1998; Opanashuk et al., 2001
). EGL reaggregates were transferred to SFM and plated onto poly-L-lysinecoated glass coverslips at the same densities used for the thymidine incorporation. After 24 h in SFM, EGL cells were treated with TCDD for 24 h. Cultures were then rinsed three times with DPBS (Gibco BRL) and incubated for 40 min at 34°35°C in DPBS containing ethidium homodimer and calcein-AM. Ethidium binds to DNA in dead neurons and emits red fluorescence, while calcein-AM is converted by esterases within living cells and emits a green fluorescence. The proportion of surviving neurons was determined as a percentage of the number of live + dead neurons. About 800 neurons were counted in each culture using a Nikon Eclipse T100 fluorescent microscope (40x) with the experimenter blinded to the conditions. At least four cultures were evaluated for each experimental treatment and each culture consisted of cells pooled from separate litters.
Statistical analyses. Data were expressed as mean ± standard error of the mean (SEM). All experiments were completed at least four times. Data were analyzed by analysis of variance (ANOVA) with Statview (version 5.0). The Fisher's post hoc test was used for individual comparisons
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RESULTS |
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DISCUSSION |
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For AhR to disrupt the molecular events that regulate brain development after TCDD exposure, the receptor must be transcriptionally functional. The data from this study strongly indicate that cerebellar granule neuroblasts are responsive to dioxin and that AhR is transcriptionally active. The transgenic DRE-LacZ reporter mouse (Willey et al., 1998) served as an ideal system with which to explore the spatial distribution of transcriptionally active AhR in the developing cerebellum. A maximally inducing dose of TCDD (Poland and Glover, 1975
) administered on PND 10 revealed activation of the DRE-LacZ reporter gene in cerebellar granule neuroblasts residing in the EGL, molecular layer, and IGL, suggesting that an AhR-mediated signaling pathway was stimulated. The absence of detectable DRE-LacZ reporter gene activation in tissues from mice not treated with TCDD does not exclude the possibility that AhR normally participates in granule neuron development. Only a single time point was examined in the present investigation, and the sensitivity of the exogenous LacZ gene may be lower than particular endogenous DRE-containing genes. Nevertheless, the DRE-LacZ reporter mouse will be useful to elucidate endogenous roles for AhR by examining the spatiotemporal expression of transcriptional activity throughout brain development. Furthermore, this transgenic model will facilitate identifying additional cellular and anatomical targets of AhR-mediated developmental TCDD neurotoxicity.
At the cellular level, ligand interaction stimulates AhR to transit from the cytosol into the nucleus, where the receptor dimerizes with Arnt prior to activating gene transcription by binding to DREs (Gu et al., 2000). TCDD stimulated nuclear translocation of AhR in cultured granule neuroblasts rapidly following treatment. Interestingly, AhR was intermittently expressed in the nuclei of vehicle-treated and untreated control granule cells. This phenomenon has been previously been observed in other tissue types (Chang and Puga, 1998
; Singh et al., 1996
). It is not clear whether nuclear expression can be attributed to the presence of an endogenous ligand or a ligand-independent process. After entering the nucleus, AhR regulates the expression of several genes, termed the "Ah target gene battery" (Nebert et al., 2000
), of which the best characterized are certain CYP metabolic enzymes. The CYP family members are heme-containing enzymes that are responsible for the metabolic oxidation of a wide variety of exogenous and endogenous substrates (Whitlock, 1999
). Although TCDD was recently shown to induce CYP1A1 mRNA in adult rat cerebellar granule neurons (Huang et al., 2000
), the developmental regulation of AhR target gene expression has not been examined at the cellular level. In the current study, TCDD stimulated concentration-dependent and time-dependent increases in CYP1A1 and 1B1 mRNA in cerebellar granule neuroblast cultures. Elevated CYP1A1 and 1B1 protein levels accompanied alterations in gene expression. These findings indicate that TCDD modulates CYP expression, most likely via interaction with the AhR, in developing cerebellar granule neuroblasts.
Although TCDD served as a means to examine the transcriptional activity of AhR by monitoring CYP 1A1 and 1B1 expression, the altered profiles of these metabolic enzymes produced after environmental exposures could impair normal brain function. The CYP family members are heterogeneously distributed at modest levels in several brain regions (Hedlund et al., 2001), which suggests that the CNS participates in bioactivation or detoxification of environmental toxicants and the regulation of endogenous substances. Previous studies have indicated that the CYP isozymes normally participate in the metabolism of neurotransmitters, endogenous steroids, and neurosteroids in the brain (Miksys and Tyndale, 2002
). Often inert exogenous or endogenous compounds are bioactivated to reactive metabolites by CYP1A1, which can lead to oxidative stress production and cell death (Nebert et al., 2000
), potentially mechanisms by which TCDD could mediate neurotoxicity. The relationships between AhR-mediated CYP induction, neuronal development, and TCDD neurotoxicity obviously require further study.
The most compelling issues that emerge from this study are related to the impact of reduced granule neuroblast DNA synthesis and survival on cerebellar development and ultimately function. Cerebellar granule neuron precursors (GNP) originate in the rostral aspect of the rhombic lip, and then migrate laterally to form the external granule cell layer (EGL) on the dorsal surface of the cerebellum during the embryonic period in rodents (Altman and Bayer, 1997; Goldowitz and Hamre, 1998
). During the postnatal period in rodents, GNPs proliferate in the outer EGL, leave the cell cycle, and move into the inner EGL before migrating through the molecular and Purkinje cell layers and settling in the IGL, where terminal differentiation and synaptogenesis occur. A tightly regulated spatiotemporal program of gene expression orchestrates these molecular events during granule neuron maturation. Considering that TCDD treatment decreased DNA synthesis and cell survival in granule neuroblast cultures, environmental exposure could influence final granule neuron numbers in the cerebellum by interfering with normal proliferative and apoptotic programs. Such disruption of granule neuron production could adversely affect cell interactions and neural circuitry formation, thereby leading to functional abnormalities.
Although it is premature to speculate about functional consequences of TCDD exposure, our observations are the first to demonstrate that AhR and ARNT proteins are expressed and transcriptionally active in cerebellar granule cells during a critical period of neuronal maturation. Most importantly, the reduction in DNA synthesis and cell survival in cultured granule neuroblasts after TCDD treatment leads to the speculation that TCDD may impede granule neuron production by diverting AhR from its endogenous function. Findings from ongoing and future studies will provide information regarding the mechanisms by which perinatal TCDD exposure affects neurogenesis, perhaps by dysregulating the expression of candidate genes involved in the normal proliferation, differentiation, and apoptosis programs during granule neuron development. Inappropriate activation of AhR after exposure to an environmental toxicant such as TCDD could interfere with the requisite gene profiles for neuronal maturation and disrupt the proper formation of brain circuitry, which might ultimately engender adverse neurobehavioral or neurological outcomes.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Box EHSC, 575 Elmwood Avenue, Rochester, NY 14642. Fax: 5852732591. E-mail: lisa_opanashuk{at}urmc.rochester.edu
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