A single prenatal exposure to the endocrine ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2010 | 115 | 897–909

doi: 10.1111/j.1471-4159.2010.06974.x

BioPharmaNet–DIMORFIPA, University of Bologna, Ozzano Emilia, Italy

Abstract Polychlorinated dibenzo-dioxins, furans and dioxin-like polychlorinated biphenyls are ubiquitous in foodstuffs of animal origin and accumulate in the fatty tissues of animals and humans. The most toxic congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a lipophilic endocrine-disrupting molecule that accumulates in adipose tissue, placenta and milk. polychlorinated biphenyls and TCDD are known to interfere with thyroid hormone metabolism and signaling in the developing brain. As thyroid hormone is critical in the myelination process during development, we investigated the effect of a single dose of TCDD prenatal exposure (gestational day 18) on the myelination process. A semi-quantitative analysis of oligodendrocyte markers at different stages of maturation was performed in the offspring’s medulla oblongata, cerebellum, diencephalon and telenchephalon at different postnatal

days (2/3, 14, 30 and 135). The most significant alterations observed were: (i) cerebellum and medulla oblongata: altered expression of oligodendroglial lineage and platelet-derived growth factor alpha receptor, myelin basic protein (MBP) mRNAs (P2/3, P135) and MBP protein (P135); (ii) diencephalon: increase in platelet- derived growth factor alpha receptor mRNA level (P2/3); (iii) telenchephalon: decrease in MBP mRNA expression. The oligodendroglial generation capability of adult neural stem/precursor cells obtained ex vivo from TCDD and vehicle-treated dams was then explored. TCDD impairs neurosphere proliferation and retards CNPase-positive cell generation from adult neurospheres. Keywords: myelin basic protein, neural stem cells, oligodendrocyte precursor cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Neurochem. (2010) 115, 897–909.

Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system by interfering with hormone signaling. Many EDCs derives by industrial processes and are persistent organic pollutants remaining available for uptake and bioaccumulation over a long period of time (Thundiyil et al. 2007). Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans are widespread, persistent and highly toxic environmental pollutants from industrial and domestic wastes burning processes or production of herbicides (Boas et al. 2009). Dioxins are ubiquitous, persisting in the environment and accumulating in the food chain of animal origin as well as in fatty tissues of animals and humans (Domingo et al. 2002; Vehlow et al. 2006). The most toxic congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which accumulates in adipose tissue, placenta and milk (Suzuki et al. 2005). Dioxins contained in maternal blood are all transferred to the fetal circulation via the placenta (Hirako et al. 2005). Moreover, numerous studies worldwide have also demonstrated that when human milk is

contaminated with PCDDs, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls (PCBs), the breastfed infant is consequently further exposed during lactation (Seo et al. 1995). Thus, foetus and infant are highrisk subjects for the possible adverse effects of TCDD contamination. Received June 11, 2010; revised manuscript received July 22, 2010; accepted August 6, 2010. Address correspondence and reprint requests to Mercedes Fernańdez, PhD, BioPharmaNet-DIMORFIPA, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (Bologna), Italy. E-mail [email protected] Abbreviations used: AhR, aryl hydrocarbon receptor; bFGF, basic fibroblast growth factor; CNPase, 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase; DIV, days in vitro; EDCs, endocrine-disrupting chemicals; EGF, epidermal growth factor; GFAP, glial fibrillary acid protein; MBP, myelin basic protein; Olig-1, oligodendroglial lineage; PCBs, polychlorinated biphenyls; PCDDs, polychlorinated dibenzo-p-dioxins; PDGFa-R, platelet derived growth factor alpha receptor; OPCs, oligodendrocyte precursor cells; SVZ, subventricular zone; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxin; TH, thyroid hormone.

2010 The Authors Journal of Neurochemistry 2010 International Society for Neurochemistry, J. Neurochem. (2010) 115, 897–909

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Increasing evidence indicates that dioxin and dioxin-like substances severely influence the development and maturation of living organisms by acting at different levels. TCDD binds the dioxin/aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor and member of the bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim) family of chemosensors and developmental regulators (HaarmannStemmann and Abel 2006). However, the TCDD action mechanism is still far from being fully understood. Other mechanisms by which TCDD may exert its effects involve cross-talk with estrogen (Ohtake et al. 2003; Matthews et al. 2005), progestin (Kuil et al. 1998), androgen (Jana et al. 1999; Morrow et al. 2004) and thyroid hormone (TH) (Yamada-Okabe et al. 2004) receptor pathways. Finally, TCDD might be responsible also for indirect effects on gonadal and thyroid dysfunction. Although many studies have so far been published on the consequences of acute exposure to different EDCs, much less attention has been devoted to the chronic, long-lasting consequences of a single exposure, in particular during the prenatal period. In this study, we focus our attention on the possible effects of TCDD prenatal exposure on myelination, a developmental process strictly dependent on a correct hormonal balance and in particular involving the thyroid axis. TCDD was administered in single doses to dams, and myelination was investigated in pups at 2/3, 14, 30 and 135 postnatal days by quantitative analysis of gene products (mRNAs and protein) expressed at different stages of oligodendrocyte maturation, that is, undifferentiated neural precursors committed to oligodendroglial lineage (Olig-1), oligodendrocyte precursor cells (NG2); proliferating, nonmyelinating oligodendrocytes [platelet derived growth factor alpha receptor (PDGFa-R ) – and myelinating oligodendrocytes [2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase (CNPase ) – and myelin basic protein (MBP)]. As adult neural stem and precursor cells of the subventricular zone (SVZ) participate in remyelination during adult life, we also investigated the capability to generate mature oligodendocytes in vitro of neural stem and precursor cells obtained from the SVZ of adult rats prenatally exposed to TCDD as compared with control rats. Oligodendoglial lineage potential was investigated in vitro by neurosphere and differentiation assay.

Materials and methods Materials The description of materials and the purchasing companies has been included in Appendix S1. Animals TCDD or vehicle alone (corn oil) was administered to dams (DarkAgouti strain) orally (os) as a single dose, 0.7 lg/kg, on gestational day 18. The offspring from these two experimental groups was used to perform all the experiments described herein. For mRNA and

protein studies, animals were killed on days 2/3 (between the second and the third day), 14, 30 and 135 postnatal; the brain was removed and the following areas dissected: telencephalon, diencephalon, medulla oblongata and cerebellum. Dissected areas to be used for RNA and protein extract preparations were deep-frozen. For immunohistochemistry, half of the cerebellum was fixed by immersion. Three male animals for each experimental group (vehicle- and TCDD-treated mothers offspring) and for each timepoint (postnatal day) studied were used. For the in vitro studies (neurosphere assay), 2-month-old female offspring were used. Three independent neurosphere cultures were performed using from three to four animals per group. Both, in vivo and in vitro experiments were performed blind or at least in duplicate. Animal care and treatments were in accordance with European Community Council Directives of November 24, 1986 (86/609/ EEC), approved by the intramural committee of Bologna University and the Italian Health Ministry, in compliance with the guidelines published in the National Institutes of Health, Guide for the Care and Use of Laboratory Animals (2003). Protein/total RNA isolation TRI reagent was used for simultaneous isolation of proteins and total RNA from the different areas mentioned above by following manufacturer’s instructions (see Appendix S1). Western blotting This procedure was performed for the quantitative analysis of myelin protein expression. Equal amounts of protein from the different samples were separated in 15% sodium dodecyl sulfate– polyacrylamide gels and electroblotted to nitrocellulose membranes. After incubation with primary and secondary antibodies, densitometric analysis of band proteins was performed (see Appendix S1). RNA reverse transcription and relative quantitative real-time PCR Total RNAs were retrotranscribed and cDNAs were processed for real-time PCR. Reactions were performed in the Mx3005PTM realtime PCR system (Stratagene, La Jolla, CA, USA) using SYBRgreen I dye. See Appendix S1 for all technical details. For the analysis of the data obtained from real-time PCR experiments, it was decided to consider vehicle-treated rat offspring at the adult stage time-point, postnatal day P135, as the reference group for comparing gene expression of Olig-1, PDGFaR and MBP in the areas in which they were studied. Immunohistochemistry Morphological analysis of myelination was focused on the cerebellum. The detailed procedures for tissue fixation and immunohistochemistry are included in Appendix S1. Images of processed sections were taken using an Olympus Provis IX70 motorized microscope equipped with an F-View II camera and Cell* Imaging Software (Mu¨nster, Germany) and the images shown (Figs 5 and 7) were generated using Adobe Photoshop 6.0 and Adobe Illustrator 9.2 software. Neurosphere culture assay and Immunocytochemistry The subventricular zone was carefully dissected and tissue processed as previously described for the preparation of neurospheres in serumfree conditions (Fernańdez et al. 2004, 2009). Epidermal growth


Dioxin and myelination

factor (EGF) (20 ng/mL) and basic fibroblast growth factor (bFGF) (10 ng/mL) were added to the medium every 48 h. Procedures followed for cell proliferation, differentiation and immunocytochemistry have been included in Appendix S1.

in vitro studies, we used Student’s t-test. Results were considered significant when p < 0.05. PrismGraph software (GraphPad Software, San Diego, CA, USA) was used to perform the statistical analyses and for the preparation of the graphs.

Statistical analysis We used Student’s t-test for the study of TCDD incidence on the number of born animals, sex and body weight gain (Table S1). For the in vivo myelination timing studies, we used two-way analysis of variance (ANOVA) and the Bonferroni post-test for the study of two independent variables (development- and treatment-effect) in the analysis of mRNA expression and protein tissue levels. For the

Results Animals, myelination markers and data presentation TCDD (0.7 lg/kg, os) at day 18 of gestation is mildly toxic for gestation, as indicated by the reduced number of offspring. Within the offspring, TCDD provoked a shift of

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Fig. 1 Effect of TCDD prenatal exposure on PDGFalphaR and MBP mRNA developmental expression profile. PDGFalphaR mRNA levels decrease in the course of development in the telencephalon (a), diencephalon (b), and medulla oblongata (c). TCDD treatment of pregnant rats causes significant differences in the PDGFalphaR mRNA expression in offspring at P2/3 and P14 in the telencephalon, diencephalon and medulla oblongata. Results are presented as mean values ± SEM. The ANOVA results of TCDD treatment and significant results obtained after the Bonferroni post-test are reported in the graph. (a) Two-way ANOVA, development effect p < 0.0001, F(3,38) = 23.99. (b) Two-way ANOVA, development effect ***p < 0.0001, F(3,40) = 94.67; treatment effect ***p < 0.0001, F(1,40) = 24.85; interaction ***p < 0.0001, F(3,40) =

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13.48. Bonferroni post-test, development times: P2/3 ***p < 0.0001; P135 *p < 0.05. (c) Two-way ANOVA, development effect ***p < 0.0001, F(3,34) = 55.19. The expression profile of MBP during development was similar in all the areas (d, e, f), increasing from P2/3 until P30, then decreasing at P135; a TCDD significant effect was observed in the medulla oblongata (f). (d) Two-way ANOVA, development time ***p < 0.0001, F(3,33) = 203.8. (e) Two-way ANOVA, development time ***p < 0.0001, F(3,36) = 222.4. (f) Two-way ANOVA, development time ***p < 0.0001, F(3,27) = 48.27; treatment *p = 0.0434,F(1,27) = 4.493. PDGFalphaR, platelet-derived growth factor; MBP, myelin basic protein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.



sex ratio toward females, with a lower number being obtained from TCDD-exposed rats. We also observed a lower weight gain in male offspring from TCDD-exposed rats at 50 days postnatal (Table S1). Offspring were then killed at 2/3, 14, 30 and 135 days after birth, using only males in the sample collection. Included in the study were regions having a different white/ grey matter ratio, that is, cerebral cortex (telencephalon), cerebellum, diencephalon, medulla oblongata. The gene expression level studies thus consisted of oligodendrocytes at different maturation stages, for example, Olig-1, which is

expressed during oligodendroglial commitment of neural stem cells (Ligon et al. 2006), PDGF-aR as marker for oligodendrocyte precursor cells (OPCs) (Polito and Reynolds 2005), MBP as marker for myelinating oligodendrocytes (Boggs et al. 2006). Offspring from vehicle-treated rats were thus considered at the P135 (adult stage) time-point as the reference group for comparing gene expression of both vehicle and TCDD offspring. Results of mRNA analysis are expressed as fold of increase with respect to adult, vehicle-exposed animals. In Figs 1–4, data are expressed as mean ± SEM. Results of

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Fig. 2 Effect of TCDD prenatal exposure on developmental expression of MBP 21.5 and 18.5 kDa isoforms protein. At P2/3, the expression was very low or even undetectable but increased in the course of development and reached its highest value at the longest time studied. Results are presented as values ± SEM. The ANOVA result for TCDD treatment and the significant results obtained after Bonferroni post-test are reported in the graph (*p < 0.05). (a) Twoway ANOVA: development time *p = 0.0471, F(3,8) = 4.174; treatment *p = 0.0121, F(1,8) = 10.43. Bonferroni post-test, development time:

P135 *p < 0.05. (b) Two-way ANOVA, development time *p = 0.0177, F(3,8) = 6.173; treatment *p = 0.0343, F(1,8) = 6.493; interaction *p = 0.0477, F(3,8) = 4.152. Bonferroni post-test, development time: P135 *p < 0.05. (c) Two-way ANOVA, development time ***p = 0.0009, F(3,8) = 16.3; (d) Two-way ANOVA, development time **p = 0.0022, F(3,8) = 12.47. (e) Two-way ANOVA: development time **p = 0.0032, F(3,8) = 11.13; treatment *p = 0.025, F(1,8) = 7.57. (f) Two-way ANOVA: development time **p = 0.0057, F(3,8) = 9.17; treatment **p = 0.0058, F(1,8) = 13.87.



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Fig. 3 Effect of TCDD prenatal exposure on Olig-1, PDGFalphaR and MBP mRNA developmental expression profile in the cerebellum. The expression profile of Olig-1 and PDGFalphaR was similar, decreasing as development progressed, whereas MBP mRNA presented a mirror expression profile increasing from P2/3 until P30, then decreasing at slightly decreasing in the adult stage. TCDD treatment induced significant changes in the expression profile of Olig-1, PDGFalphaR and MBP mRNA. Results are presented as mean values ± SEM. The ANOVA result for TCDD treatment and significant results obtained

after Bonferroni post-test are reported in the graph (**p < 0.001). (a) Two-way ANOVA, development time ***p < 0.0001, F(3,36) = 157.6; treatment *p = 0.0191, F(1,36) = 6.021; interaction *p = 0.0149, F(3,36) = 3.99. Bonferroni post-test, development time: P135 **p < 0.01. (b) Two-way ANOVA, development time ***p < 0.0001, F(3,37) = 135.2. (c) Two-way ANOVA, development time ***p < 0.0001, F(3,35) = 98.18; interaction *p = 0.406, F(3,33) = 3.084. (d) Representative agarose gel of Olig-1, PDGFalphaR and MBP PCR products. Olig-1, oligodendrocyte transcription factor 1.

two-way ANOVAs are detailed in the legend to the figures, whereas the overall treatment effect is also reported in the graphs below the TCDD columns, when significant. Moreover, the results of Bonferroni’s post hoc test are also indicated by asterisks (p < 0.05).

analysis indicates that developmental profile of PDGF-aR mRNA in TCDD-offspring differs from vehicle in the telencephalon, where expression of the OPCs markers is higher in TCDD (Fig. 1). On the contrary, the expression profile of the mature oligo marker MBP mRNA is lower in the medulla oblongata, a highly myelinated area (Fig. 1). In the same area, also MBP level is lower in TCDD offspring (Fig. 2). The MBP protein isoforms are differentially regulated during development: the 21.5 and 18.5 kDa, which are considered the mature isoforms, progressively increase over the investigated times in all the areas studied, being very low expressed at P2/3 and P14 and highly expressed at P30 and P135 (Figs 2, 4a and b); in contrast, the 17 kDa isoform, decreases in the course of development. This smaller isoform was found to be highly expressed at P14, about two times more than at P30 and P135 (Fig. 4c). Figure 4d shows a representative western blot experiment of the MBP isoforms in the cerebellum at all the developmental time-points studied in vehicle (lanes 1–4) and TCDD- prenatally exposed animals (lanes 5–8). The expression of the 21.5 and 18.5 kDa isoforms increases over time while the expression of the 17 kDa MBP isoform decreases after 2 weeks.

Timing of myelination in offspring of vehicle- and TCDDtreated animals A temporally regulated expression of mRNAs encoding for marker proteins of immature (PDGFa-R) and mature (MBP) oligodendrocytes was observed in all the investigated areas (Fig. 1: telencephalon, diencephalon, medulla oblongata; Fig. 3: cerebellum). In all investigated areas, PDGFa-R expression, the marker for OPCs, is high at P2/3 and P14, then dramatically decreases at P30 and P135. On the contrary, MBP mRNA expression, the marker for mature oligos, has a mirror profile, increasing up to P30, then with expression again reduced at P135. In the cerebellum, Olig-1 gene is highly expressed at P2/3, remaining high at P14 and P30, then decreasing in adulthood. Prenatal TCDD-treatment alters the developmental profile of some of the investigated parameters. The overall ANOVA



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Fig. 4 Effect of TCDD prenatal exposure on developmental expression of MBP protein in the cerebellum. The 17-kDa isoform showed a different expression pattern compared with 21.5- and 18.5-kDa isoforms, reaching its highest expression at P14 and then dropping as development proceeded. Results are presented as mean values ± SEM. Statistics: two-way ANOVA and Bonferroni post-test. The ANOVA results for TCDD treatment are reported in the graph. (a) Twoway ANOVA development time ***p = 0.0007, F(3,14) = 10.54; interac-

tion *p = 0.0408, F(3,14) = 3.6. (b) Developmental time ***p < 0.0001, F(3,14) = 20.12. (c) Developmental time ***p < 0.0001, F(3,8) = 20.30. (d) Representative immunoblot of cerebellum from TCDD-prenatally exposed rats (lanes 5–8) and control animals (lanes 1–4) is reported. A single band of 36 kDa in the upper western-blots corresponds to the GAPDH protein. The size of the standard protein marker has been indicated on the left.

Quite interesting, the expression profile of Olig-1 in the cerebellum remains higher compared to vehicle in TCDD offspring (Fig. 3), whereas MBP protein level is not different in vehicle and TCDD offspring (Fig. 4). Specificity of PCR products for all pair of primers used has been checked (Fig. 3d). Immunohistochemical staining was performed on protein markers, which are expressed at different maturation stages of oligodendrocytes. In particular, NG2 is expressed by OPCs (Polito and Reynolds 2005); Rip was recently identified as CNPase, a non-compact myelin protein expressed by early stage oligodendrocytes; MBP is an oligodendrocyte-specific protein that is essential for oligodendrocyte morphogenesis at the later stages of cell differentiation (Rogister et al. 1999; Watanabe et al. 2006). Representative micrographs including the simple lobule of cerebellar cortex are reported in Fig. 5, on the left side of the panel. Images show decreased expression of NG2 and increased expression of Rip, CNPase and MBP during postnatal development. At P2, a large number of NG2positive OPCs is observed (Fig. 5a), and staining intensity decreases over time (Fig. 5b and c). Conversely, immuno-

staining for mature oligodendrocytes is absent at P2 (Rip, Fig. 5g; CNPase, Fig. 5m; MBP, Fig. 5s), appears at P14 (Fig. 5h, n and t) and takes on an adult pattern at P30 (Fig. 5i, o and u) where a strong staining of the subcortical white matter is observed. In TCDD exposed animals, NG2 at P2and CNPase at P30 are stronger than in vehicle exposed, age-matched animals. Effect of prenatal TCDD treatment on oligodendroglial lineage from SVZ-derived neurospheres in adult rats To investigate whether the mature CNS retains the effects in oligodendroglial generation related to prenatal TCDD exposure, the neural stem/progenitor cells from adult rats born from TCDD- and vehicle-treated dams were studied. Neurospheres were derived from the SVZ of adult animals and maintained in two different culture conditions to enable them, respectively, first to proliferate and then to differentiate. For proliferation studies, cells were cultured free-floating in the presence of mitogens (EGF + bFGF). Formed neurospheres were counted after 2, 4 and 5 days in vitro (DIV), photographed for counting and diameter analysis (Fig. 6). Data from three independent experiments



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Fig. 5 Developmental myelination in vehicle and prenatally TCDDexposed rats, as illustrated by immunohistochemistry for OPCs and mature oligodendrocyte markers. Markers for OPCs (NG2, a–f), early stage (Rip, g–l, and CNPase, m–r) and late stage (MBP, s–y) oligodendrocytes are illustrated in the cerebellum of vehicle (P2/3, a, c; P14, g–i; P30, m–o; P135, s–u) and TCDD (P2/3; P14; P30; P135)

prenatally treated rats. Images show decreased developmental expression of NG2 and increased developmental expression of Rip, CNPase and MBP expression. In TCDD-exposed animals, NG2 at P2/ 3 and CNPase at P30 are stronger than in vehicle-exposed, agematched animals. Panels a, g, m, s, d, j, p, v, scale bar 200 lm. Remaining panels, scale bar 100 lm.

were averaged in the results. Although the number of primary neurospheres generated from the two experimental groups does not differ, neurospheres generated from TCDD exposed animals are significantly smaller, thus suggesting a lower proliferative capability. Cells obtained by neurosphere splitting were then seeded onto poly-L-lysine coated surfaces and grown as a monolayer without mitogens; antigen expression analysis was performed at 3 and 9 DIV. Morphology of immunoreactive generated cells is shown in Fig. 7a–h, where the cell count from three independent experiments is also reported (Fig. 7i). Although the percentage of undifferentiated precursors (nestin-positive) and

neuroblasts (DCX-positive) does not differ between vehicleand TCDD-derived spheres, the glial lineage and maturation of adult neurospheres is affected by prenatal exposure to TCDD. In particular, although the percentage of astrocytes increases from 3 to 9 DIV in vehicle-derived cells, there is a significantly lower percentage of glial fibrillary acid protein (GFAP)-positive cells at 9 DIV in TCDD compared with vehicle-derived cells (#p < 0.05). Moreover, oligodendroglial maturation is also affected. Although a drastic reduction was observed in the percentage of NG2-positive OPCs between 3 and 9 DIV in both groups, there is a significantly lower percentage of CNPase-positive cells at



suggesting a dissociation in MBP synthesis. Indeed, when it was attempted to generate in vitro mature oligodendrocytes from neural progenitors and stem/precursor cells derived from the SVZ of adult rats born from TCDD-exposed mothers, a significant defect was found in generating mature oligodendrocytes and astrocytes, possibly reflecting a delay of the in vitro maturation process. It was therefore concluded that a single prenatal exposure to TCDD at gestational day 18 alters the timing of developmental oligodendrocyte generation/maturation and also produces long-lasting effects that could affect myelin repair potential during adulthood. (a)

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Fig. 6 Effect of TCDD prenatal exposure on proliferation of neurospheres obtained from the SVZ of adult rats. Images illustrate typical neurospheres obtained from vehicle and TCDD-exposed dams, cultured in suspension in the presence of mitogens for 2, 4 and 5 days in vitro (DIV). TCDD exposure, although not significantly, modifies the number of generated neurospheres (a) and reduces the proliferation rate as indicated by the smaller size of generated neurospheres (b). Data are expressed as mean ± SEM. Statistics: Student’s t-test, # p < 0.05. Scale bar 100 lm.

9 DIV in TCDD than vehicle-derived cells (#p < 0.05). It should be noted that many cells at 9 DIV still co-express undifferentiated precursor markers (nestin) and lineagespecific markers (DCX for neural lineage; GFAP for astroglial lineage; NG2, CNPase and MBP for oligodendroglial lineage) (data not shown); the percentage of cells in Fig. 7i thus exceeds 100%.

Discussion The present study reports that a single prenatal exposure to TCDD at mildly toxic dose for the mother modifies myelination timing in neonatal rat. Some molecular and cellular alterations persist until adulthood in some of the brain areas studied (e.g. Olig-1 and PDGFaR in the diencephalon, Olig-1 and MBP mRNA, MBP protein in the cerebellum). Figure 8 summarizes the results obtained in the cerebellum in vehicle (gray) and TCDD (red) offspring in time-–course manner, and asterisks points the significant differences between the two groups. The overall representation illustrates that undifferentiated markers for oligo lineage persists in TCDD-adult offspring, although MBP mRNA expression level is lower. A dysfunction in proper myelination related to TCDD exposure is also suggested by the higher MBP protein content,

Effect of 700 ng/kg TCDD oral gestational administration on dams and offspring TCDD 700 ng/kg was classified as an intermediate to high dose in the Covance GLP study after a single administration during gestation (Bell et al. 2007a, 2010). As indicated by Bell et al. (2007a, 2010) and other studies (Ishihara et al. 2007), prenatal TCDD affects the number of pups born, body weight gain and sex ratio towards females of offspring, as has been also described in this study. When administered at the dose and administration route used in this study, TCDD accumulates in the foetus and pups for 21 days, thus lasting until 2 weeks postnatal (Emond et al. 2004; Li et al. 2006; Bell et al. 2007b). In particular, when 800 ng/kg were administered at gestational day 15, high levels of TCDD were found in the brain at postnatal day 5 and detectable levels were still present at PND 120 (Kakeyama et al. 2003). The fact that more than 10% of administered TCDD accumulates in the encephalon (Hurst et al. 2000) guarantees brain exposure to TCDD over the critical period of gliogenesis and myelination, corresponding to the end of gestation and the early postnatal period (Rice and Barone 2000). Although it does not induce permanent thyroid diseases either in the mother or in the offspring, it transiently alters thyroid function (Seo et al. 1995; Crofton and Zoeller 2005). A decrease in total and free T4 and an increase in thyroid stimulating hormone plasma levels has been described after gestational and lactational exposure to TCDD which is followed by hyperplasia of thyroid follicular cells (Nishimura et al. 2003, 2005). In view of the aim of this study, this is quite significant because myelination is a THdependent process and TH exerts its main role during the perinatal period (Rodrı´guez-Penã 1999; Bernal and Guadanõ-Ferraz 2002; Zoeller and Rovet 2004; Bernal 2007; Ahmed et al. 2008; Darras 2008; Ceballos et al. 2009). Molecular, cellular and morphogenic processes during oligodendrocyte development and myelination require a spatially, temporally and quantitatively orchestrated sequence of genetically and epigenetically driven events in gestational, perinatal and postnatal periods, which also includes different cell types and a correct exposure to hormones and vitamins (Miller and Mi 2007). Considering the long half-life of TCDD, the administration of TCDD at gestation day 18



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Fig. 7 Effect of TCDD prenatal exposure on spontaneous differentiation neural precursor and stem cells obtained from the SVZ of adult rats. (a–h) Immunostaining of cells derived from SVZ neurospheres after mitogen withdrawal at 9 DIVs. (a, b) NG2-positive cells; (c, d) CNPase-positive cells; (e, f) MBP-positive cells; (g, h) GFAP-positive cells. (a, c, e, g) Cells from prenatal vehicle exposure; (b, d, f, h) cells from TCDD prenatal exposure. Graph (i) shows quantitative results, expressed as a percentage, of undifferentiated precursors (nestin),

neuroblasts (doublecortin, DCX), astrocytes (GFAP, glial fibrillary acid protein), and oligodendrocytes at different differentiation stages (NG2, oligodendrocyte precursor cells; CNPase, early differentiated oligodendocytes; MBP, late differentiated oligodendocytes). A smaller number of CNPase-positive oligodendrocytes and astrocytes is generated from TCDD prenatally exposed rats compared with vehicle. Data are mean ± SEM. Statistics: Student’s t-test, #p < 0.05. Scale bar 100 lm.

guarantees the exposure to the endocrine disruptor in the most critical phase of myelination (Zoeller and Rovet 2004; Bernal 2005; Ceballos et al. 2009). The present study included the analysis of molecular markers, which are expressed by oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes throughout their maturation process. Using the markers indicated, in vehicle-exposed rats a developmental profile was observed that is characterized by the progressive decline of Olig-1 and PDGFaR and a mirrorimage increase in MBP in all investigated areas. Moreover, a wave of MBP mRNA expression at P30 precedes the MBP 18.5 and 21.5 kDa protein increase in adult rats. The content of MBP 17 kDa, the developmental isoform of MBP (Boggs 2006), is higher at P14, then declining until adulthood. The overall profile of oligodendroglial lineage and maturation correspond to the myelination process described (Bradl and Lassmann 2010) and provide a reliable framework in which quantitatively investigate the effect of prenatal exposure to TCDD on the timing of molecular events critically involved in myelination.

The major alteration observed in TCDD-prenatally exposed rats is the higher expression of undifferentiated markers, for example, Olig-1 and PDGFaR, which lasts until adulthood, that is, in the diencephalon and cerebellum, and the altered MBP content, which is lower in mature cerebellum, diencephalon and medulla oblongata, and higher in the telencephalon. These could be the results of a maturation defect and/or delay in OPCs turning into myelinating oligodendrocytes. This also correlated with the lower MBP mRNA expression described in the cerebellum and medulla oblongata of rats exposed to PCBs during gestation (Zoeller et al. 2000). This hypothesis is confirmed by the ex vivo analysis of oligodendrogenesis in adult TCDD- and vehicle-exposed rats, as analyzed by neurosphere assay. It was found that neurospheres generated from TCDD-exposed offspring brains are smaller than those generated from vehicle offspring, which points to a reduced proliferative capability. Moreover, fewer astrocytes and CNPase-postive cells are formed at 9DIV after mitogen withdrawal. This suggests that a long-lasting gliogenesis



Fig. 8 Effect of TCDD prenatal exposure on cerebellum oligodendrocyte marker expression. Overview of oligodendrocyte markers expression profile in the cerebellum at postnatal days studied. Asterisks indicate significant values. The experimental schedule within the scheme of the embryonic and postnatal rat period has been included at the bottom.

defect in the mature brain could occur after prenatal TCDD exposure. Interestingly, prenatal exposure to PCB significantly reduces cell density in the corpus callosum, without affecting the proportion of myelin associated glicoprotein- (oligodendrocyte) or GFAP-positive (astrocyte) cells (Sharlin et al. 2006). A quite similar result was obtained after TCDD exposure using the neurosphere assay. However, fetus exposure to dioxin also alters myelin the transcription factor 1-like gene, one of the transcription factors favouring oligodendrocyte maturation (Nicolay et al. 2007). Different molecular mechanisms are possibly involved in altered oligodendrocyte maturation. One possibility is related to a direct effect of TCDD on OPCs via AhR receptor during the critical period for OPCs proliferation, migration and maturation. AhR was recently indicated as an important player in epithelial cell (Hall et al. 2010), T lymphocyte (Marshall and Kerkvliet 2010) and hematopoietic stem cell (Singh et al. 2009) differentiation, possibly representing a cross-road of different pathways critical to cell cycle regulation, mitogen-activated protein kinase cascades, differentiation and apoptosis (Ma et al. 2009). However, the complex effect described in this study could also be related to indirect effects of TCDD on endocrine function, as oligodendrocyte maturation and white matter track development are under hormonal control. One possibility is that the maturation defect and/or delay of OPCs is related to the alteration of the thyroid function induced by TCDD and, consequently, to the altered TH drive for oligodendrocyte generation and maturation and myelin protein expression.

TH is a key regulator of oligodendrocyte lineage and maturation acting at different levels and different times (Rodriguez-Penã 1999; Bernal and Guadanõ-Ferraz 2002; Bernal 2007; Zoeller and Rovet 2004; Darras 2008; Calza et al. 2010), as indicated by studies in genetically modified animals (Baas et al. 1997, 2002; O’Shea and Williams 2002), by the analysis of myelination in hypo- and hyperthyroid animals (Jagannathan et al. 1998; Schoonover et al. 2004; Obregon et al. 2007) and by in vitro studies on OPCs (Durand and Raff 2000). Thus, as perinatal exposure to TCDD alters maternal, neonatal and infant thyroid function, and also TH receptor-mediated gene expression and pathways, transient dysthyroidism could be part of the described effects (Beischlag et al. 2004; Yamada-Okabe et al. 2004; Casey 2005; Wang et al. 2005; Giacomini et al. 2006; Nagayama et al. 2007). However, astrocytes are the cells responsible for the local synthesis of the active form of TH in the brain (Guadanõ-Ferraz et al. 1997; Pallud et al. 1997). The action of PCBs and dioxins on TH nuclear receptors is actually regarded as a possible mechanism to explain neurodevelopmental problems described also in humans after accidental prenatal exposure to these pollutants (Fritsche et al. 2005; Zoeller and Crofton 2005; Wilhelm et al. 2008). Another mechanism by which TCDD may exert its effects is cross-talk with estrogen (Ohtake et al. 2003; Matthews et al. 2005), progestin (Kuil et al. 1998) and androgen receptor (Jana et al. 1999; Morrow et al. 2004) (Beischlag et al. 2004) downstream pathways, as already mentioned. Notably, also prenatal exposure to the PCB mixture Aroclor 1254 alters the development of cells



responsible for myelination (Sharlin et al. 2006), also inducing a transient increase in MBP mRNA expression, and these effects are not believed to be simply a function of PCB-induced dysthyroidism (Zoeller et al. 2000).

Conclusions In recent years, growing attention has been devoted to the intrauterine life period as a possible risk factor for postnatal functional development, maturation, aging and for disease development. Results presented in this study suggest that there are long-term consequences due to TCDD intra-utero exposure on myelin and gliogenic potential of mature CNS. In particular, the altered gliogenic capacity of adult neural stem and precursor cells needs to be further investigated as regards the potential implications for CNS vulnerability and repair capability, also in the context of demyelinating diseases.

Acknowledgements This work was supported by MIUR PRIN 2007, project NUMBER 2007 4 SPYCM (LC). Assistance of Giovanni Vitale for TCDD oral administration is gratefully acknowledged. (G. Vitale, Dept Biomedical Sciences, University of Modena and Reggio Emilia, Italy).

Supporting information Additional Supporting information may be found in the online version of this article: Appendix S1. Supplementary Materials and methods. Table S1. TCDD prenatal exposure effects on offspring. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

References Ahmed O. M., El-Gareib A. W., El-Bakry A. M., Abd El-Tawab S. M. and Ahmed R. G. (2008) Thyroid hormones states and brain development interactions. Int. J. Dev. Neurosci. 26, 147–209. Baas D., Bourbeau D., Sarlie`ve L. L., Ittel M. E., Dussault J. H. and Puymirat J. (1997) Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia 19, 324–32. Baas D., Legrand C., Samarut J. and Flamant F. (2002) Persistence of oligodendrocyte precursor cells and altered myelination in optic nerve associated to retina degeneration in mice devoid of all thyroid hormone receptors. Proc. Natl Acad. Sci. USA 99, 2907– 2911. Beischlag T. V., Taylor R. T., Rose D. W., Yoon D., Chen Y., Lee W. H., Rosenfeld M. G. and Hankinson O. (2004) Recruitment of thyroid hormone receptor/retinoblastoma-interacting protein 230 by the

| 907

aryl hydrocarbon receptor nuclear translocator is required for the transcriptional response to both dioxin and hypoxia. J. Biol. Chem. 279, 54620–54628. Bell D. R., Clode S., Fan M. Q. et al. (2007a) Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. I: No decrease in epididymal sperm count after a single acute dose. Toxicol. Sci. 99, 214–231. Bell D. R., Clode S., Fan M. Q. et al. (2007b) Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin in the developing male Wistar(Han) rat. II: Chronic dosing causes developmental delay. Toxicol. Sci. 99, 224–233. Bell D. R., Clode S., Fan M. Q. et al. (2010) Interpretation of studies on the developmental reproductive toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring. Food Chem. Toxicol. 48, 1439–1447. Bernal J. (2005) Thyroid hormones and brain development. Vitam. Horm. 71, 95–122. Bernal J. (2007) Thyroid hormone receptors in brain development and function. Nat. Clin. Pract. Endocrinol. Metab. 3, 249– 259. Bernal J. and Guadanõ-Ferraz A. (2002) Analysis of thyroid hormonedependent genes in the brain by in situ hybridization. Methods Mol. Biol. 202, 71–90. Boas M., Main K. M. and Feldt-Rasmussen U. (2009) Environmental chemicals and thyroid function: an update. Curr. Opin. Endocrinol. Diabetes Obes. 16, 385–391. Boggs J. M. (2006) Myelin basic protein: a multifunctional protein. Cell. Mol. Life Sci. 63, 1945–1961. Bradl M. and Lassmann H. (2010) Oligodendrocytes: biology and pathology. Acta Neuropathol. 119, 37–53. Calza` L., Fernańdez M. and Giardino L. (2010) Cellular approaches for stimulating CNS remyelination: Thyroid hormone to promote myelin repair via endogenous stem and precursor cells. J. Mol. Endo. 44, 13–23. Casey B. (2005) Environmental contaminants and maternal thyroid function. Am. J. Obstet. Gynecol. 193, 1889–1890. Ceballos A., Belinchon M. M., Sanchez-Mendoza E., Grijota-Martinez C., Dumitrescu A. M., Refetoff S., Morte B. and Bernal J. (2009) Importance of monocarboxylate transporter 8 for the blood–brain barrier-dependent availability of 3,5,3¢-triiodo-L-thyronine. Endocrinology 150, 2491–2496. Crofton K. M. and Zoeller R. T. (2005) Mode of action: neurotoxicity induced by thyroid hormone disruption during development – hearing loss resulting from exposure to PHAHs. Crit. Rev. Toxicol. 35, 757–769. Darras V. M. (2008) Endocrine disrupting polyhalogenated organic pollutants interfere with thyroid hormone signalling in the developing brain. Cerebellum 7, 26–37. Domingo J. L., Bocio A., Nadal M., Schuhmacher M. and Llobet J. M. (2002) Monitoring dioxins and furans in the vicinity of an old municipal waste incinerator after pronounced reductions of the atmospheric emissions. J. Environ. Monit. 4, 395–399. Durand B. and Raff M. (2000) A cell-intrinsic timer that operates during oligodendrocyte development. Bioessays 22, 64–71. Emond C., Birnbaum L. S. and DeVito M. J. (2004) Physiologically based pharmacokinetic model for developmental exposures to TCDD in the rat. Toxicol. Sci. 80, 115–133. Fernańdez M., Pirondi S., Manservigi M., Giardino L. and Calza` L. (2004) Thyroid hormone participates in the regulation of neural stem cells and oligodendrocyte precursor cells in the central nervous system of adult rat. Eur. J. Neurosci. 20, 2059–2070. Fernańdez M., Paradisi M., Giardino L. and Calza` L. (2006) To know neural stem properties from diseased brains: a critical step for brain



repair, in Neural Stem Cell Research (Greer E. V., ed.), pp. 77–97. Nova Science Publishers Inc, New York. Fernańdez M., Paradisi M., Del Vecchio G., Giardino L. and Calza` L. (2009) Thyroid hormone induces glial lineage of primary neurospheres derived from non-pathological and pathological rat brain: implications for remyelination-enhancing therapies. Int. J. Dev. Neurosci. 27, 769–778. Fritsche E., Cline J. E., Nguyen N. H., Scanlan T. S. and Abel J. (2005) Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: clue for involvement of thyroid hormone receptors. Environ. Health Perspect. 113, 871–876. Giacomini S. M., Hou L., Bertazzi P. A. and Baccarelli A. (2006) Dioxin effects on neonatal and infant thyroid function: routes of perinatal exposure, mechanisms of action and evidence from epidemiology studies. Int Arch Occup Environ Health 79, 396–404. Guadanõ-Ferraz A., Obregoń M. J., St Germain D. L. and Bernal J. (1997) The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc. Natl Acad. Sci. USA 94, 10391–10396. Haarmann-Stemmann T. and Abel J. (2006) The arylhydrocarbon receptor repressor (AhRR): structure, expression, and function. Biol. Chem. 9, 1195–1199. Hall J. M., Barhoover M. A., Kazmin D., McDonnell D. P., Greenlee W. F. and Thomas R. S. (2010) Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol. Endocrinol. 24, 359–369. Hirako M., Aoki M., Kimura K., Hanafusa Y., Ishizaki H. and Kariya Y. (2005) Comparison of the concentrations of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in maternal and fetal blood, amniotic and allantoic fluids in cattle. Reprod. Toxicol. 20, 247–254. Hurst C. H., DeVito M. J., Setzer R. W. and Birnbaum L. S. (2000) Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in pregnant Long Evans rats: association of measured tissue concentrations with developmental effects. Toxicol. Sci. 53, 411–420. Ishihara K., Warita K., Tanida T., Sugawara T., Kitagawa H. and Hoshi N. (2007) Does paternal exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) affect the sex ratio of offspring? J. Vet. Med. Sci. 69, 347–352. Jagannathan N. R., Tandon N., Raghunathan P. and Kochupillai N. (1998) Reversal of abnormalities of myelination by thyroxine therapy in congenital hypothyroidism: localized in vivo proton magnetic resonance spectroscopy (MRS) study. Brain Res. Dev. Brain Res. 109, 179–86. Jana N. R., Sarkar S., Ishizuka M., Yonemoto J., Tohyama C. and Sone H. (1999) Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells. Biochem. Biophys. Res. Commun. 256, 462–468. Kakeyama M., Sone H., Miyabara Y. and Tohyama C. (2003) Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters activitydependent expression of BDNF mRNA in the neocortex and male rat sexual behavior in adulthood. Neurotoxicology 24, 207–217. Kuil C. W., Brouwer A., van der Saag P. T. and van der Burg B. (1998) Interference between progesterone and dioxin signal transduction pathways. Different mechanisms are involved in repression by the progesterone receptor A and B isoforms. J. Biol. Chem. 273, 8829– 8834. Li B., Liu H. Y., Dai L. J., Lu J. C., Yang Z. M. and Huang L. (2006) The early embryo loss caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin may be related to the accumulation of this compound in the uterus. Reprod. Toxicol. 21, 301–306. Ligon K. L., Kesari S., Kitada M., Sun T., Arnett H. A., Alberta J. A., Anderson D. J., Stiles C. D. and Rowitch D. H. (2006) Develop-

ment of NG2 neural progenitor cells requires Olig gene function. Proc. Natl Acad. Sci. USA 103, 7853–7858. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Ma C., Marlowe J. L. and Puga A. (2009) The aryl hydrocarbon receptor at the crossroads of multiple signaling pathways. EXS. 99, 231–257. Marshall N. B. and Kerkvliet N. I. (2010) Dioxin and immune regulation: emerging role of aryl hydrocarbon receptor in the generation of regulatory T cells. Ann. N Y Acad. Sci. 1183, 25–37. Matthews J., Wihleń B., Thomsen J. and Gustafsson J. A. (2005) Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. Mol. Cell. Biol. 25, 5317–5328. Miller R. H. and Mi S. (2007) Dissecting demyelination. Nat. Neurosci. 10, 1351–1354. Morrow D., Qin C., Smith R., III and Safe S. (2004) Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation. J. Steroid Biochem. Mol. Biol. 88, 27–36. Nagayama J., Kohno H., Kunisue T., Kataoka K., Shimomura H., Tanabe S. and Konishi S. (2007) Concentrations of organochlorine pollutants in mothers who gave birth to neonates with congenital hypothyroidism. Chemosphere 68, 972–976. Nicolay D. J., Doucette J. R. and Nazarali A. (2007) Transcriptional control of oligodendrogenesis. Glia 55, 1287–1299. Nishimura N., Yonemoto J., Miyabara Y., Sato M. and Tohyama C. (2003) Rat thyroid hyperplasia induced by gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology 144, 2075–2083. Nishimura N., Yonemoto J., Nishimura H., Ikushiro S. and Tohyama C. (2005) Disruption of thyroid hormone homeostasis at weaning of Holtzman rats by lactational but not in utero exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 85, 607–614. Obregon M. J., Calvo R. M., Del Rey F. E. and de Escobar G. M. (2007) Ontogenesis of thyroid function and interactions with maternal function. Endocr. Dev. 10, 86–98. Ohtake F., Takeyama K., Matsumoto T. et al. (2003) Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423, 545–550. O’Shea P. J. and Williams G. R. (2002) Insight into the physiological actions of thyroid hormone receptors from genetically modified mice. J. Endocrinol. 175, 553–570. Pallud S., Lennon A. -M., Ramauge M., Gavaret J.-M., Croteau W., Pierre M., Courtin F. and St. Germain D. L. (1997) Expression of the Type II Iodothyronine Deiodinase in Cultured Rat Astrocytes Is Selenium-dependent. J. Biol. Chem 272, 18104–18110. Polito A. and Reynolds R. (2005) NG2-expressing cells as oligodendrocyte progenitors in the normal and demyelinated adult central nervous system. J. Anat. 207, 707–716. Rice D. and Barone S., Jr (2000) Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108, 511–533. Rodrı´guez-Penã A. (1999) Oligodendrocyte development and thyroid hormone. J. Neurobiol. 40, 497–512. Rogister B., Ben-Hur T. and Dubois-Dalcq M. (1999) From neural stem cells to myelinating oligodendrocytes. Mol. Cell. Neurosci. 14, 287–300. Schoonover C. M., Seibel M. M., Jolson D. M., Stack M. J., Rahman R. J., Jones S. A., Mariash C. N. and Anderson G. W. (2004) Thyroid hormone regulates oligodendrocyte accumulation in developing rat brain white matter tracts. Endocrinology 145, 5013–5020.



Seo B. W., Li M. H., Hansen L. G., Moore R. W., Peterson R. E. and Schantz S. L. (1995) Effects of gestational and lactational exposure to coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid hormone concentrations in weanling rats. Toxicol. Lett. 78, 253– 262. Sharlin D. S., Bansal R. and Zoeller R. T. (2006) Polychlorinated biphenyls exert selective effects on cellular composition of white matter in a manner inconsistent with thyroid hormone insufficiency. Endocrinology 147, 846–858. Singh K. P., Casado F. L., Opanashuk L. A. and Gasiewicz T. A. (2009) The aryl hydrocarbon receptor has a normal function in the regulation of hematopoietic and other stem/progenitor cell populations. Biochem. Pharmacol. 77, 577–587. Suzuki G., Nakano M. and Nakano S. (2005) Distribution of PCDDs/ PCDFs and Co-PCBs in human maternal blood, cord blood, placenta, milk, and adipose tissue: dioxins showing high toxic equivalency factor accumulate in the placenta. Biosci. Biotechnol. Biochem. 69, 1836–1847. Thundiyil J. G., Solomon G. M. and Miller M. D. (2007) Transgenerational exposures: persistent chemical pollutants in the environment and breast milk. Pediatr. Clin. North Am. 54, 81–101. Vehlow J., Bergfeldt B. and Hunsinger H. (2006) PCDD/F and related compounds in solid residues from municipal solid waste incineration – a literature review. Waste Manag. Res. 24, 404–420. Wang S. L., Su P. H., Jong S. B., Guo Y. L., Chou W. L. and Pa¨pke O. (2005) In utero exposure to dioxins and polychlorinated biphenyls

| 909

and its relations to thyroid function and growth hormone in newborns. Environ. Health Perspect. 113, 1645–1650. Watanabe M., Sakurai Y., Ichinose T., Kotani M. and Itoh K. (2006) Monoclonal antibody Rip specifically recognizes 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterasi in oligodendrocytes. Neurosci. Res. 84, 525–533. Wilhelm M., Wittsiepe J., Lemm F. et al. (2008) The Duisburg birth cohort study: influence of the prenatal exposure to PCDD/Fs and dioxin-like PCBs on thyroid hormone status in newborns and neurodevelopment of infants until the age of 24 months. Mutat. Res. 659, 83–92. Yamada-Okabe T., Aono T., Sakai H., Kashima Y. and Yamada-Okabe H. (2004) 2,3,7,8-Tetrachlorodibenzo-p-dioxin augments the modulation of gene expression mediated by the thyroid hormone receptor. Toxicol. Appl. Pharmacol. 194, 201–210. Zoeller R. T. and Crofton K. M. (2005) Mode of action: developmental thyroid hormone insufficiency – neurological abnormalities resulting from exposure to propylthiouracil. Crit. Rev. Toxicol. 35, 771–781. Zoeller R. T. and Rovet J. (2004) Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J. Neuroendocrinol. 16, 809–818. Zoeller R. T., Dowling A. L. and Vas A. A. (2000) Developmental exposure to polychlorinated biphenyls exerts thyroid hormone-like effects on the expression of RC3/neurogranin and myelin basic protein messenger ribonucleic acids in the developing rat brain. Endocrinology 141, 181–189.
