Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat

Toxicology and Applied Pharmacology 237 (2009) 127–136

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring Daniela Cocchi a, Giovanni Tulipano a, Alessandra Colciago d, Valeria Sibilia b, Francesca Pagani b, Daniela Viganò c, Tiziana Rubino c, Daniela Parolaro c, Patrizia Bonfanti e, Anita Colombo e, Fabio Celotti d,⁎ a

Division of Pharmacology and Toxicology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Milan, Italy Department of Structural and Functional Biology, University of Insubria, Varese, Italy d INBB Research Unit, Department of Endocrinology, Pathophysiology and Applied Biology University of Milan, Italy e Department of Environmental Sciences, University of Milan-Bicocca, Milan, Italy b c

a r t i c l e

i n f o

Article history: Received 31 October 2008 Revised 4 March 2009 Accepted 9 March 2009 Available online 24 March 2009 Keywords: Polychlorinated biphenyls Growth hormone Somatostatin Bone Rat

a b s t r a c t Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following longterm PCB exposure. Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic–pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females. © 2009 Elsevier Inc. All rights reserved.

Introduction Polychlorinated biphenyls (PCBs) are widespread environmental pollutants (Winneke et al., 2002). Due to their lipophilicity and structural stability, PCBs accumulate in higher levels of food chains (Pinto et al., 2008). Despite that their production and use have been banned for decades in many industrialized countries, these compounds are still present in the environment and in biological samples from different areas in the world. Remarkably, recent studies have been performed on people living in an industrialized town in North Italy where for about 50 years a PCB factory caused substantial ⁎ Corresponding author. E-mail address: [email protected] (F. Celotti). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.03.008

pollution of superficial waters used for irrigating farmland. A strong association between high levels of PCBs in humans and consumption of food produced in the most polluted area was found (Donato et al., 2006). Since PCBs accumulate in adipose tissue and milk (Safe, 1990) and can easily cross the placenta, mammalian offspring is likely to be exposed to higher concentrations of these pollutants during prenatal development and breast-feeding than at any other time (Kaya et al., 2002; Colciago et al., 2006). PCBs still represent a major toxicological issue. PCBs can induce neurobehavioral toxicity in animals and humans by interfering with neurotransmitter systems and signal transduction pathways after affecting gene expression profile (Basha et al., 2006; Royland et al., 2008). Moreover, PCBs form a subgroup of endocrine-disruptors in that they are believed to alter thyroid hormones (Toni et al., 2005) and

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sex-steroid secretion and activity (Schrader and Cooke, 2003; Janosek et al., 2006). In turn, these hormones are important regulators of neuronal development (Chung et al., 2001; Kaya et al., 2002; Steinberg et al., 2007). Elevated serum PCBs have been also associated with diabetes in humans (Codru et al., 2007; Everett et al., 2007). Thyroid hormones, estrogens and androgens play an important role in somatic growth and influence differentiation and function of several target tissues during development and in the adulthood. In detail, thyroid hormones and gonadal steroids are known to influence the development and the activity of growth hormone (GH)–Insulinlike growth factor-1 (IGF-1) axis (Muller et al., 1999). The physiological pattern of growth hormone (GH) secretion is regulated through a complex neuroendocrine system which includes two main hypothalamic peptides, GH-releasing hormone (GHRH) and somatostatin (SS). These peptides exert stimulatory and inhibitory activity on somatotroph cells, respectively and are the final mediators of metabolic, endocrine and neural influences modulating GH secretion. The different pattern of GH secretion between male and female animals, particularly evident in rodents, strictly depends on gonadal steroids (Muller et al., 1999). Among the various effects exerted by sex steroids and thyroid hormones on different tissues, a key role in the control of bone homeostasis has been documented. In particular, sex steroids play an important role in bone growth and the attainment of peak bone mass. They are responsible for the sexual dimorphism of the skeleton which emerges during adolescence. The effects of androgens and estrogens on bone growth and metabolism involve a direct interaction with their own receptor in chondrocytes, osteoblasts and osteocytes (Van der Eerden et al., 2003; Vanderschueren et al., 2004). This paper represents the first part of a multitasked study in which PCB liver and brain accumulation, along with the measurements of many morpho-functional parameters have been evaluated from birth to adulthood in animals exposed to PCB during fetal development and breast-feeding. The experiments described in the present paper were aimed at evaluating whether PCB exposure in the rat can induce longterm alterations of GH-axis activity and bone mass in the adult offspring. At this purpose, we measured somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we described sex-related alterations of hypothalamic somatostatin expression, pituitary GH expression and bone structure in adulthood, following long-term PCB exposure. The second part of the study focuses on PCB effects on the hypothalamic expression levels of sex-steroid receptors and the main enzymes involved in the formation of sex-steroid metabolites, the development of the reproductive system and sexual- and non-reproductive behaviours (see Colciago et al., submitted for publication). A number of studies have faced the effects of commercial mixtures of PCBs (Aroclor) in animals. Actually, the distinct PCBs (congeners) have different chemical characteristics which influence their accumulation, uptake and metabolism in the environment and in organisms, giving rise to marked differences in PCB congener composition between the commercial mixtures and the pool of compounds accumulated in fat and breast milk (Ramos et al., 1997; Bachour et al., 1998; Lanting et al., 1998). We focused our attention on a previously unstudied reconstituted mixture (RM) composed by selecting PCB 138, PCB 153 and PCB 180, as indicators of PCBs abundance in biological matrix as brain, liver, muscle, lung (Bachour et al., 1998), and a dioxin-like congener, PCB126, generally detected in trace because of its very high toxicity. The selected PCBs are highly chlorinated and this property causes a slowdown in the metabolism and makes easy their accumulation in tissues. Based on in vitro studies, the di-ortho, poly-chloro substituted biphenyls, PCB-138, -153 and -180 can compete with the binding of the natural ligand to estrogen receptors. PCB 138 has significant dose-dependent antiandrogenic activity, also (Bonefeld-Jorgensen et al., 2001). The pentachloro substituted biphenyl PCB 126 is a non-ortho coplanar PCB and

bind to the aryl hydrocarbon receptor (AHR) (Maier et al., 2007) and is known to affect the female reproductive system (Sakurada et al., 2007). Materials and methods Animal care and PCB administration. During the study, the animals were housed in the Department of Endocrinology Animal Facility, University of Milan and maintained in accordance with the guidelines of the Italian Ministry of Health for the care and use of laboratory animals (Decreto legge 116/92). Time pregnant Sprague–Dawley rats (CD SPF/VAF, Charles Rivers, Calco, Italy) were individually housed in animal quarters in normal condition (light schedule: 14 h light–10 h dark; environmental temperature: 21–23 °C). They were fed a standard pellet diet and water was provided ad libitum. Sperm positive vaginal smears were considered as day 0 of gestation. Five control and 5 treated dams were euthanized at gestation day 20 (GD20) and fetuses, delivered by laparatomy, were sexed by screening for the presence of the SRY gene in DNA on a brain fragment (Poletti et al., 1998). All the other dams delivered spontaneously and the day of birth was considered as postnatal day 1 (PN1). On PN1, litter sizes and total weight were determined and the pups were examined for malformations. Five dams/group and their litters were euthanized at PN12 and PN21, respectively. After PN21, the remaining litters were reared for sex. Female and male rats were housed separately, four rats per cage. Animals have been weighed two times/week from the beginning of the treatment to the end of the experiments. Fetal toxicity was evaluated at GD20 as number of fetuses/dam, male/female ratio, number of underdeveloped foetuses, fetal weight, and the presence of macroscopic malformations. After delivery, the number of dead pups/litter, the male/female ratio, and the survival rate were also evaluated. To avoid variations of the parameters under investigation related to the estrous cycle, all the assays were performed in samples obtained from diestrous animals. The reconstituted PCB mixture (RM) was composed by two hexachlorobiphenyls (PCB138 and PCB153), one heptachlorobiphenyl (PCB180) and one coplanar pentachlorobiphenyl (PCB 126). All the compounds (guaranteed purity 100%) were supplied by Chemical Research (Roma, Italy). PCB 138, 153 and 180 were present in the mixture at the same concentration (each representing one third of the total); since PCB 126 is normally present in human milk at very low concentrations (Ramos et al., 1997), the compound was added to the total mixture in a 1/10,000 ratio. Dams were treated subcutaneously from GD15 to GD19 with 10 mg/kg/day of the mixture, dissolved in 0.1 ml peanut oil and then left to deliver spontaneously. During breastfeeding, dams were treated with the same dose given twice a week till weaning (PN21). It was calculated that this treatment schedule provided an average RM daily intake of 4 mg/kg. The control rats were injected with the corresponding volume of vehicle. Analysis of PCB concentrations in brain and liver. At each experimental endpoint, fragments of brain and liver were collected, snap frozen in liquid nitrogen and stored at − 80 °C until determination of PCB content by gas-chromatography/mass spectrometry as previously described (Pravettoni et al., 2005). Briefly, samples of approximately 0.1 g were homogenized with sodium sulphate anhydrous and spiked with 25 ng of PCB 40 and PCB 128 (Dr. Esternstorfer, Augsburg, Germany), to check recovery efficiencies of methodology. A pre-extraction (24 h) was performed with n-hexane (PESTANAL, Riedel-De Haen, Seelze, Germany) to clean the Soxhlet extraction apparatus and thimbles. All samples were Soxhlet extracted for 24 h in n-hexane. The extracts were reduced in a rotary evaporator under vacuum to 5 ml, transferred to a vial and reduced under a gentle nitrogen flow to 2 ml, and then added an equal volume of sulphuric acid and mixed with vortex for 5 min. When separation of the two layers was achieved, the hexane layer was pipetted off and charged on

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Ultra-clean SPE Florisil (500 mg/4 ml, Alltech). The column was eluted with 8 ml of ethylacetate:hexane 1:1 and 1 ml of hexane. The sample was then reduced under a gentle stream of nitrogen to 0.5 μl and transferred to 2 ml GC vials. All extracts were further concentrated under a gentle stream of nitrogen to facilitate solvent exchange to 25 μl of dodecane containing two internal standards (PCB 30 and PCB 141 kindly given as gifts by Dr. Esternstorfer, Augsburg, Germany) for subsequent PCB quantification analysis. To identify and quantify PCB residues in rat tissues, the extracts were analysed by Gas Chromatography (CG) coupled with Mass Spectrometry (MS). Further GC–MS details are given in Pravettoni et al. (2005). Lipid content was determined by extracting the samples in an ultrasonic apparatus for 2 h using a mixture of n-hexane:acetone (1:1). The percent lipids by dry weight were calculated by weighting the extracts evaporated. Mean recovery of each analyte ranged between 70 and 110% and the reproducibility was calculated by means of a triplicate analysis giving an overall 10% error. Series of procedural blanks were analyzed periodically. Reported values are blank corrected. Method detection limit was determined as the instrument detection limit of the lowest concentration standard of each analyte, which was 0.5 pg/μl for each congener. Analytical (instrument) variation is typically b 10% as measured by repeated injections of samples. Dissections and hormone determinations. At the moment of sacrificing the animals, trunk blood was collected in EDTA containing tubes and the plasma was separated by centrifugation and stored at −20 °C until hormone determinations by radioimmunoassays using commercial kits (rat IGF-1 RIA, Mediagnostic, Germany; free T3- and free T4-RIA, MP Biomedicals, Asse-Relegem, Belgium; testosterone luminescence immunoassay, IBL-Hamburg, Germany). The whole brain and the anterior pituitary were dissected, frozen on dry ice immediately and stored at −80 °C until mRNA analysis. The right tibia was dissected free from soft tissue, fixed in 10% formaldehyde solution for epiphyseal disk width measurement (Tibia test). Left tibiae and femurs were excised and fixed in 10% formaldehyde solution for dual energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Determination of somatostatin expression in the hypothalamus. The probe consisted of a 45-mer oligonucleotide directed against bases of the rat somatostatin mRNA sequence (5′-CCA GAA GAA GTT CTT GCA GCC AGC TTT GCG TTC CCG GGG TGC CAT-3′, MWG oligo synthesis, Italy). The sequence has been kindly suggested by Michele Zoli (Modena, Italy). Oligonucleotides were labelled by tailing the 3′ end with deoxyribonucleotidyl terminal transferase (Roche Diagnostic, Italy) and α[35S]dATP (PerkinElmer, Italy) and incubated at 37 °C for 30 min. The reaction was stopped by addition of 0.2 M EDTA and the labelled probe was purified using mini spin columns (Quick Spin, Roche Diagnostics, Italy). The extent of probe labelling was assessed using β-scintillation counting. On PN60, male and female rats were killed by decapitation and their brain were rapidly removed, frozen on dry ice and stored at −80 °C until processing. 12 μm coronal sections were cut on a cryostat and thaw mounted on gelatin-coated slides. Sections were briefly dried and stored at −80 °C until they were processed for in situ hybridization as described by Parolaro et al. (1993) with slight modifications. Prior to the hybridization reaction, tissue sections were fixed with 4% paraformaldehyde in phosphate-buffered saline solution, acetylated with 0.25% acetic anhydrate in 0.1 M triethanolamine/ 0.9% NaCl for 10 min, dehydrated with a graded alcohol series, and then defatted in chloroform for 10 min. The sections were then hybridized with 35S-labeled oligonucleotides (5 × 105 dpm/μl) in hybridization buffer at 42 °C for 18 h. The hybridization buffer contained 50% deionized formamide, 4 × standard saline citrate (SSC), 10% dextran sulphate, 1 × Denhardt's solution (0.02% Ficoll, 0.02%

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polyvinylpyrrolidone, 0.02% bovine serum albumin), 10 mM dithiothreitol, 500 μg/ml of salmon sperm DNA, 250 μg/ml of tRNA. Following the hybridization reaction, sections were washed (5 × 15 min) at 63 °C in 1 × SSC. Dried sections were exposed to autoradiographic film (BioMax MR Kodak) for 7 days. Film autoradiograms were subjected to regional optical density determinations using a computerized analysis system provided by Immagini & Computer (Milan, Italy) consisting of a dual scanner Artixscan 1800F connected to a personal computer (PC). Image were analysed using Image-ProPlus 5.0 (MediaCybernetics, Silver Sprimng, USA). Each area of both side of the brain was traced with the mouse cursor using the atlas of Paxinos and Watson (2005) as reference, and light transmittance was determined as the grey level (optical density, O.D.). The optical density measurements were made within the linear range as determined using 35S standards prepared in the laboratory. Determination of growth hormone expression in the anterior pituitary. Total RNA was isolated from each pituitary by the single-step acid guanidinium thiocyanate–phenol–chloroform extraction method (Trireagent, Sigma). Total RNA sample (15 μg/sample) from the pituitary was run on a 1.2% formaldehyde/agarose gel and transferred to nylon membranes (Hybond N, Amersham International). Filters were hybridized with a rat GH cDNA sequence kindly provided by Dr. F. De Noto (University of California, San Francisco, CA, USA). The probe was labeled using the Megaprime DNA labeling system (Amersham) with [-32P]dCTP to a specific activity of 1 × 109 dpm/μg DNA. The size of the GH mRNA transcript detected by Northern hybridization, 0.8 kb, corresponds to that reported previously (Cocchi et al., 1999). Control of the amount of the RNA loaded was performed by reprobing the filters with [-32P]dCTP-labeled GAPDH cDNA. After hybridization, autoradiography was carried out at −70 °C for 12 h with intensifying screens using Hyperfilm-MP (Amersham). Determination of cartilage epiphyseal disk width. Tibiae were split in the midsagittal plane and washed extensively in water. After a 30 min long incubation in absolute acetone, bones were washed in water and then placed in freshly prepared 2% AgNO3. Two minutes later, they were rinsed in deionized water and exposed to bright light until the calcified portions appeared dark brown. After a 1 min long incubation in 10% sodium thiosulfate, the specimens were washed with water extensively and stored in 80% ethanol. The width of the uncalcified epiphyseal cartilage was measured at stereoscope (dissecting microscope, Zeiss, Germany) using a calibrated micrometer eyepiece. For each tibia, the width value was obtained as a mean of 8–10 consecutive measures along the cartilage line. Bone structure analysis of tibiae and femurs. DEXA measurements of tibiae and femurs were carried out with Hologic QDR 4000 densitometer (Hologic Inc., Waltham, MA, USA) in the ultra high resolution mode with a longitudinal line spacing of 0.254 mm, implemented with a collimatir 1.0 mm in diameter and with the high resolution software (version 4.47) adapted for small animals. Bones were placed on a plexiglass platform on their caudal surface and whole bone planar area (cm2), mineral content (BMC, mg) and bone mineral density (BMD, mg/cm2) were measured. PQCT measurements of tibia were performed using a Stratec Research SA+pQCT scanner (Stratec Medizintechnick GmbH, Pforzheim, Germany) with a voxel size of 100 μm. In order to orient the long axes of the bones parallel to the image planes, the excised bone specimens were placed in a plastic tube filled with saline and centered in the gantry of the machine. The correct longitudinal positioning was determined by means of an initial scout scan. Proximal tibial metaphysis were scanned in the horizontal plane using 3 consecutive cross-sectional images (0.5 mm distance from each other) at 10% of total length of the bone. Tibial diaphysis was scanned at the midpoint (50%) of the total bone length.

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The scans were analyzed with PQCT software 6.00 B. At the methaphysis, we used the measuring mode for dividing total bone into cortical bone or cancellous bone in peel mode 20, that can detect the inner threshold automatically. In each transverse image, we measured Area (mm2), BMC (mg/mm) and BMD (mg/cm3) of total bone and respectively of cancellous and cortical + subcortical regions. At the mid-diaphysis, the cortical bone was detected by separation mode 1 with a threshold value of 710 mg/cm3. In this region, we measured the parameters of Total and Cortical bone Area, BMC and BMD. We also measured cortical thickness (mm), endosteal and periosteal perimeters (MM). The X–Y and polar-axis strength strain indices (xSSI, ySSI, pSSI) were also calculated by software. Statistics. The statistical significance (P b 0.05) of the differences in the mean values between the distinct experimental groups was evaluated by either a parametric or a non-parametric test when appropriate. The number of animals used for each determination (corresponding to the number of samples) and the statistics used are detailed for the results in the legends of figures and tables and in the running text.

Results PCB accumulation in brain and liver of exposed dams and pups To ascertain the effective exposure risk during development, the concentrations of each PCB congener were measured by gaschromatography/mass spectrometry in the brain and liver of the offspring at birth (GD20), during infancy (PN12), at weaning (PN21) and in adulthood (PN60). The results, including cumulative PCB concentrations and the relative amount of each congener, are shown in Fig. 1. In the same figure, PCB levels in the brains and livers of dams are also reported, for comparison. Small amounts of PCB 126 were detected only in maternal and offspring livers at PN12 and PN60 (Fig. 1B). At the other two time points in liver and in all brain samples, PCB 126 was not analytically detectable. No significant differences were observed in bioaccumulation between male and female animals, both in brain and liver. PCB bioaccumulation in dam brains is already detectable at GD20 and progressively increases during the lactation period reaching levels statistically higher at PN21 than at GD20 and PN12. These amounts decrease slightly in dam brains at PN60. On the

Fig. 1. Brain (A) and hepatic (B) concentrations of each PCB congener as measured by gas-chromatography/mass spectrometry. Results are reported as mean ± SEM. Statistical differences were evaluated with Duncan's multiple comparison after ANOVA. Panel A: a p b 0.01 vs GD20 and PN12 dams; b p b 0.01 vs GD20, PN21 and PN60 offspring of the same sex. Panel B: a p b 0.05 vs GD20, PN12 and PN21 dams; b p b 0.05 vs PN12 and PN21 dams; c p b 0.01 vs GD20, PN21 and PN60 offspring of the same sex. d p b 0.01 vs GD20 and PN60 offspring of the same sex. ⁎⁎p b 0.01 vs offspring of both sexes at the same time point.

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contrary, PCBs accumulate in offspring brains during gestation and the first period of lactation (till PN12), and then decrease in the second part of lactation. The levels detected at PN12 are statistically different from those evidenced in all the other time points considered. A further decrease is evident after weaning, even though detectable amounts of the pollutants are still present in the adult animals. At GD20 and PN12, PCB concentration in offspring brains, expressed as μg/g wet weight, is higher, but not statistically different, than that observed in the corresponding mother brains, whereas starting from weaning PCB levels in mother brains become statistically higher than that measured in the offspring (Fig. 1A). The relative amount of each congener compared to the total quantity of congeners in the mixture is statistically unchanged even if there is a trend of accumulation of PCB 153 both in mother and offspring brains in the three postnatal time intervals considered (Fig. 1A). The considerable levels of PCB congeners detected in maternal liver on GD20, decrease progressively during the lactation period, reaching levels statistically lower than those measured at the end of gestation. The tested congeners start to accumulate again at PN60 in dam liver, rising to amounts significantly higher than those measured at the end of gestation and lactation. In the offspring, the high levels of PCB congeners, detected in the first period of lactation (PN12), are statistically different from the other three time points considered, whereas no difference was evidenced between the end of gestation and adulthood (PN60) (Fig. 1B). Considering the levels of maternal and offspring PCB based on wet weight, the amounts of the compounds at GD20 are significantly lower in fetal than in maternal liver, while during lactation period it is observed as a statistically significant increase of PCB concentration in offspring liver compared to the mothers. If the relative amount of each congener compared to the total quantity of congeners in the mixture is considered, the ratio remains unchanged and no selective accumulation trend is evidenced both in mother and offspring livers. General toxicity indices and developmental parameters Body weight of dams from GD15 to weaning was not influenced by PCB treatment; neither have we observed signs of gross toxicity in offspring at birth nor differences in the number of pups per litter and in the percentage of males per litter between control and exposed animals. Moreover, PCB exposure did not increase postnatal mortality, as shown by no difference in the survival rate (data not shown). The effect of PCB exposure on offspring growth is shown in Fig. 2: body weight of pups did not differ between control and exposed

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Table 1 Body weight, relative organ weight and Testosterone (T) plasma levels in adult (PN60) female and male animals after developmental exposure to PCB.

a

Body weight (g)

b Relative ovarian weight T plasma levels in diestrous animals (ng/ml)

a

Body weight

b

Relative testes weight Relative prostate weight T plasma level (ng/ml) b

Control

PCB-exposed

Females 226.8 ± 23.6 (4.6 ± 3.2/4 litters) 58 ± 6 0.142 ± 0.028

204.9 ± 14.8⁎⁎ (5.8 ± 2.2/5 litters) 57 ± 7 0.141 ± 0.032

Males 343.6 ± 24.3 (6.6 ± 3.3/5 litters) 885 ± 63 104 ± 13 4.9 ± 1.9

316.8 ± 17.9⁎⁎ (8 ± 2.4/5 litters) 908 ± 77 95 ± 16 7 ± 3.5

a Data are expressed as mean ± SD. The mean ± SD of the number of pups/litter and the number of litters are reported in parentheses. b Organ weights are given as mean ± SD of mg/100 g body weight. ⁎⁎ p b 0.01 vs the corresponding controls (Student's t test).

animals until weaning. Afterward, there was a constant and significant reduction of the growth curves of exposed pups both in male (F = 66.70; P b 0.001) and in female (F = 27.79; P b 0.001) animals, that was still significant in adults of both sexes (Table 1). No significant alterations in ovary-, prostate- and testis weights relative to body weight, were observed at the moment of sacrificing the rats (Table 1). The effects on puberty onset, estrous cyclicity and sex behaviour are described in the second part of the study (see Colciago, submitted for publication). Plasma hormone concentrations and cartilage epiphyseal disk width The PCBs used in this study had no discernible effects on plasma concentrations of free-T4 and free-T3, as measured during prepubertal development (PN21) and in the adulthood (PN60) (Table 2). Based on two-way ANOVA, no significant differences in mean plasma IGF-1 concentrations between male and female rats were observed at weaning (PN21). Conversely, gender-related differences were observed in adult rats, with 0.5 times higher plasma IGF-1 levels in males vs females (F = 8.197, P b 0.01). As to the effects of treatment, PCB-exposed rats showed higher plasma IGF-1 levels as compared to the respective controls at PN21 (F = 4.929, P b 0.05). This effect was not sex-related (Table 3). The differences between the means did not attain statistical significance in the adulthood at PN60 (F = 0.914, NS), anymore. The analysis at stereoscope following staining with AgNO3 revealed a significant increase in the width of the tibial epiphyseal cartilage, an index of GH-axis activity on peripheral tissues, for both male- and female PCB-treated animals compared to the respective controls at PN 60 (Fig. 3A). Testosterone levels in adults of both sexes were unaffected by perinatal PCB exposure (Table 1).

Table 2 Effects of exposure to PCBs during prenatal life and breast-feeding on plasma thyroid hormone (pmol/l) concentrations at the moment of euthanizing the animals. Age PN21

Fig. 2. Effects of exposure to PCBs during fetal development and breast-feeding on somatic growth from PN 21 to PN 60. The data from PN21 to PN60 (mean ± SEM, N = 16) were analyzed by non-linear regression analysis, calculating the best fit of the variables in the model (R2 from 0.95 to 0.98). Differences between control and exposed animals were assessed by comparison of the best fits obtained (P b 0.001 both for males and females).

PN 60

Hormone F T4 F T3 T4/T3 F T4 F T3 T4/T3

Males

Females

Control

PCB-exposed

Control

PCB-exposed

13 ± 2 2±1 6±1 32 ± 3 5 ± 0.6 6.4 ± 1

11 ± 3 2±1 5±2 29 ± 4 4.7 ± 0.7 6.2 ± 1.5

16 ± 3 4±2 6±2 28 ± 3 5.8 ± 0.5 4.8 ± 0.9

14 ± 3 3±1 5±2 25 ± 2 5 ± 0.4 5 ± 1.5

Data represent the mean ± SD of 10 to 12 animals for each group. Based on unpaired t test analysis, the differences between PCB-exposed rats and the respective controls did not attain statistical significance.

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Table 3 Effects of exposure to PCBs during prenatal life and breast-feeding on plasma IGF-1 concentrations (ng/ml) at the moment of euthanizing the animals. Age

Control

PCB-exposed

PN 21 PN 60

Females 152 ± 38 950 ± 130

254 ± 30⁎ 1150 ± 280

PN 21 PN 60

Males 155 ± 27 1520 ± 150⁎⁎

236 ± 61⁎ 1630 ± 140

Data represent the mean ± SD, N = 10 to 14 for each group. ⁎ P b 0.05 vs age and sex matched controls. ⁎⁎ P b 0.01 vs PN 60 females.

Determination of pituitary GH expression and hypothalamic somatostatin expression At weaning (PN21), the pituitary GH mRNA content did not differ significantly between PCB-treated- and vehicle-treated rats (data not shown). Conversely, at PN60, northern blot analysis revealed a significant decrease of pituitary GH mRNA content in the anterior pituitary of male rats from mothers treated with PCBs, as compared

Fig. 3. (A) Effects of exposure to PCBs during fetal development and breast-feeding on the width of the tibial epiphyseal cartilage in the adult rats (PN 60), as measured at stereoscope using a calibrated micrometer eyepiece. Results are the means ± SEM of ten to fourteen animals (⁎P b 0.05 versus the respective controls, unpaired t test). (B) Effects of exposure to PCBs during fetal development and breast-feeding on pituitary GH mRNA content evaluated by Northern blot analysis in the adult rats on PN 60. The density of the autoradiographic bands was quantified by Analytical Imaging Station FLA 2000 (Fujifilm) and the amount of GH mRNA was expressed as a percentage of GAPDH mRNA. Results are the means± SEM of six to eight animals for each group (⁎P b 0.05 vs the respective controls; unpaired t test and non-parametric Mann–Whitney's test gave similar results). (C) A portion of the autoradiographic sheet containing male rat mRNAs, is shown.

with the respective controls. No significant changes were observed in female littermates (Figs. 3B and C). In situ hybridization analysis revealed a clear-cut increase of SS mRNA expression in the hypothalamic periventricular nucleus of male and female rats (PN 60) from mothers treated with PCBs versus the respective controls. SS mRNA levels were unchanged in the central arcuate nucleus whereas increased expression of the neuropeptide was observed in the lateral arcuate nucleus of male PCB-exposed rats (Fig. 4). Bone structure analysis of tibiae and femurs The exposure to PCBs resulted in decreased whole bone planar area, as assessed by DEXA measurements of total tibiae in both male and female adult offspring. DEXA revealed a significant decrease of bone mineral content (BMC), restricted to male rats. Bone mineral density (BMD) appeared unchanged versus controls. This finding could be consequent to the concomitant decrease of both area and BMC (Fig. 5).

Fig. 4. Effects of exposure to PCBs during fetal development and breast-feeding on somatostatin mRNA levels in three different hypothalamic areas, as measured by in situ hybridization in rats euthanized at PN 60. Results are the means ± SEM of six animals (⁎⁎P b 0.01; ⁎P b 0.05 versus the respective controls; unpaired t test and non-parametric Mann–Whitney's test gave similar results). Representative bright-field microphotographs of emulsion autoradiograms showing somatostatin mRNA-related signal in the periventricular nucleus (A–D) and in the central and lateral arcuate nuclei (E–F), are included. Panel A, control female rats. Panel B, PCB-treated female rats. Panel C and E, control male rats. Panel D and F, PCB-treated male rats.

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Table 4 Effects of exposure to PCBs during fetal development and breast-feeding on the bone structure at the proximal metaphysis (panel A) and the mid-diaphysis (panel B) of tibiae in the adult rats, as measured by PQTC. Sample

Parameter

Female rats Control

A. Proximal metaphysis Total bone Area BMC BMD Cancellous Area bone BMC BMD Cortical + Area subcort. bone BMC BMD B. Mid-diaphysis Total bone Area BMC BMD Cancellous Area bone BMC BMD Periosteal perimeter Endosteal perimeter Bone thickness

Male rats PCB-exposed Control

PCB-exposed

15.97 ± 0.32 15.63 ± 0.28 23.84 ± 0.37 8.00 ± 0.21 8.17 ± 0.19 9.22 ± 0.23 501.0 ± 10.3 525 ± 8.91 387.7 ± 6.1 7.19 ± 0.14 7.04 ± 0.13 10.74 ± 0.16

22.92 ± 0.37 9.10 ± 0.19 398.0 ± 7.8 10.34 ± 0.17

1.93 ± 0.11 262.4 ± 14.5 8.77 ± 0.17

2.08 ± 0.09 198.7 ± 10.8 12.58 ± 0.20

1.99 ± 0.09 2.01 ± 0.1 283 ± 12.1 184 ± 6.5 8.66 ± 0.16 13.09 ± 0.20

6.10 ± 0.12 698 ± 9.6

5.79 ± 0.22 704 ± 8.31

7.20 ± 0.15 554 ± 7.65

7.01 ± 0.10 562 ± 7.90

5.48 ± 0.14 4.66 ± 0.11 851 ± 13.9 3.29 ± 0.08

5.25 ± 0.10 4.47 ± 0.09 848 ± 5.73 3.15 ± 0.05

6.65 ± 0.12 5.57 ± 0.06 837 ± 14.4 4.00 ± 0.05

6.16 ± 0.11⁎⁎ 5.26 ± 0.07⁎⁎ 855.5 ± 8.88 3.82 ± 0.05⁎

4.14 ± 0.11 3.97 ± 0.08 4.97 ± 0.09 1260 ± 4.2 1259 ± 2.8 1243 ± 5.8 8.96 ± 0.09 8.78 ± 0.08 10.03 ± 0.09

4.72 ± 0.06⁎⁎ 1234 ± 4.9 9.54 ± 0.07⁎⁎

5.09 ± 0.34

5.05 ± 0.29

6.33 ± 0.08

6.02 ± 0.05⁎⁎

0.562 ± 0.01 0.522 ± 0.02 0.612 ± 0.005 0.593 ± 0.01⁎

Results are the means ± SEM of 10–12 animals. ⁎P b 0.05; ⁎⁎P b 0.01 versus the respective controls (unpaired t test). Units: Area, mm2 — BMC, mg/mm — BMD, mg/mm3 — perimeter and thickness, mm.

Fig. 5. DEXA measurements of tibiae in the adult offspring (PN 60) from mothers treated with PCB during pregnancy and lactation versus the respective controls. Results are the means ± SEM of 10–12 rats for each group. ⁎P b 0.05; ⁎⁎P b 0.01 vs the respective controls (unpaired t test).

The results of PQTC analysis of tibiae are shown in Table 4. The exposure to PCBs did not induce any significant changes in all bone parameters measured in trabecular bone at the proximal metaphysis (Table 4, panel A). Conversely, cortical area as well as cortical BMC resulted in a significant decrease in male PCBs-treated rats, compared with controls, at the mid-diaphysis (Table 4, panel B). In females, there was no difference between PCB-treated and control animals in all bone parameters. The analysis of bone cortical substructure revealed a significant reduction of bone thickness, periosteal and endosteal perimeters in PCB-treated rats compared to controls (Table 4, panel B). The impairment of bone thickness was accompanied by a reduction in bone strain strength, as assessed by calculation of X-, Y- and polarstrain strength indices (Fig. 6).

range much higher than that previously found in subjects naturally exposed to environmental pollutants (Bachour et al., 1998; Lanting et al., 1998). The quantitative time-course evaluation of PCB bioaccumulation in dam and offspring brains presents significant differences: the bioaccumulation rises exponentially in dams till PN21, conversely in the offspring it increases from birth to the first half of lactation and then tends to slightly decrease, being still detectable at PN60. This trend, which has been observed for PCB 153 and PCB 126 also in nursing mice (Lee et al., 2002) does not seem to be related to an increase of the developing pup adipose mass, since the same authors did not find significant differences in PCB accumulation in pup carcasses at the same time frames (Lee et al., 2002). A possible explanation is an increased PCB brain mobilization, coupled to their degradation by the hepatic detoxification mechanisms. In line with

Discussion This study confirmed and extended information on long-lasting consequences of developmental exposure of rats to a reconstituted PCB mixture. We decided to focus on the four PCB compounds most abundant in biological extracts (Ramos et al., 1997; Bachour et al., 1998, Lanting et al., 1998) and their far-reaching effects in the adult offspring, as far as GH-axis activity and bone mass homeostasis are concerned. As expected, the administration of the RM to dams from the last part of gestation to the end of lactation results in a significant accumulation of PCBs in the brain of both dams and offspring in a

Fig. 6. Effects of exposure to PCBs during fetal development and breast-feeding on bone strain strength, as measured by PQTC at the mid-diaphysis of tibiae in the adult rats (PN 60). Results are the means ± SEM of 10–12 rats for each group. ⁎⁎P b 0.01 vs the respective controls (unpaired t test).

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this hypothesis, preliminary observations by the group of Santagostino (Colombo et al., 2006) detected a progressive induction of CYP1A1/2 and CYP2B1/2 in the liver of offspring exposed to the same RM used in the present experiments. A further decrease of PCB brain content is evident after weaning, possibly linked to a progressive mobilization during post-weaning growth. Nonetheless, detectable amounts of the pollutants are still present in the PN60 brains. This explanation is supported by the PCB pharmacokinetics in dam and offspring liver where the lactating dams have the lowest PCB concentration, whereas offspring show the highest concentrations in early lactation period (PN12). Small levels of PCB 126 were analytically detectable only in maternal and offspring liver at PN12 and PN60, while this congener was below the detection limit in the other time points and in all brain samples. The absence of analytically measurable PCB126 levels in brain could be explained with the concept of the liver as a “depot organ” for different halogenated compounds (Chen et al., 2001); TCDD and dioxin-like congeners, such as PCB126, induce besides CYP1A1, CYP1A2, an isoform able to bind and sequester these compounds in liver. As already indicated, a strong induction of CYP1A1 and CYP1A2 in maternal and offspring livers was evidenced by Colombo et al. (2006). On the whole these results confirm early studies that placenta does not represent an efficient barrier in limiting the transfer of PCBs to foetuses and that lactation is the major source of exposure in the pups (Lee et al., 2002). The treatment schedule adopted in the present experiments neither produces general and reproductive toxicity (as demonstrated by the same litter size and survival rate between control and exposed animals), nor does it interfere with the achievement of the main endpoints of sexual function, since the relative gonadal and prostate weights, plasma testosterone levels and ovarian cyclicity are unaffected. However, small changes in puberty onset and male sexual behaviour have been observed (see Colciago et al., submitted for publication). The finding that testosterone levels in adult males are unaffected by PCB exposure contrasts with many studies (Ulbrich and Stahlmann, 2004) indicating that in utero exposure to PCB mixtures or to single congener damages testicular steroidogenesis and thus testosterone production. One possible explanation of this difference is that in our experiments PCB treatments started in the last part of gestation (e.g. GD15), when fetal Leydig cells are already differentiated and functional (Weisz and Ward, 1980). A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. This trend is similar to what was observed in rats after treatments with technical (Goldey et al., 1995; Hany et al., 1999) or reconstituted (Kaya et al., 2002) PCB mixtures at comparable exposure levels, as well as in human children at background environmental levels (Patandin et al., 1999). Thus, it appears that the influence on body weight gain is a common developmental effect of PCBs. It is well known that somatic growth is under the influence of GH/ IGF-1, thyroid hormones and sex steroids. A few studies have described a hypothyroid effect by PCBs and dioxin-like compounds (Craft et al., 2002; Chevrier et al., 2007). Craft et al. have reported that PCB126 and PCB153 decrease serum T4 in rats after subchronic treatment, due to induced hepatic microsomal T4 glucuronidation activity. In the present study, the decrease in plasma T4 and T3 concentrations after exposure to a PCB mixture containing PCB153 and PCB126, did not attain the statistical significance at weaning as well as in the adult rats. Based on the dose–response curves shown by Craft et al., the difference in the average daily intake of these specific congeners between the two studies can fully account for the different results. A long-lasting consequence on hypothalamic–pituitary GH axis was indeed observed for the PCB-exposed offspring in adulthood, in that there was a significant increase of SS expression in periventricular nucleus (PeVN) of both male and female rats. It is worth remembering that SS-positive neurons in PeVN play a key role in generating

pulsatile GH release from pituitary. SS-positive neurons are also localized in the arcuate nucleus (ARC), in close proximity of GHRHreleasing neurons and are likely involved in a direct intra-hypothalamic control of GHRH release (Muller et al., 1999). The developmental exposure to PCBs increased significantly SS mRNA levels in the lateral ARC and decreased GH mRNA levels in the pituitary of adult male rats. The increase of SS expression in the ARC might account for the decrease of pituitary GH expression by enhancement of the inhibitory control on GHRH release. To date, there have been few experimental studies designed to assess the possible adverse effects of PCBs on bone metabolism. It has been reported that administration of PCB126 to adult rats impairs bone mineralization and reduces bone strain strength (Lind et al., 1999, 2000). The exposure of rats to commercial PCB mixture (Aroclor 1254) induces a decrease in femur length and increases bone fragility. Furthermore, it has been reported that long-term Aroclor 1254 administration increases bone resorption either in normal male rats or in ovariectomized mature rats (Ramajayam et al., 2007). The present study has demonstrated that exposure to PCBs during preand perinatal life is associated to long-lasting effects on bone structure in adulthood. In particular, we have shown that PCBs caused a significant reduction of BMC and cortical bone thickness restricted to male rats. The mechanisms responsible for the gender difference in the adverse effects of PCBs on bone mass detected in the present study remain to be clarified. However, it can be speculated that the higher sensitivity of male rats compared to females to the negative bone effects of PCBs could depend on an influence of PCBs in the function of the somatotropic axis. This assumption is supported by the above mentioned data showing that PCB-exposed male rats show a reduction in pituitary GH mRNA content. Furthermore, we have shown that tibial diaphysis was more severely impaired than tibial metaphysis following PCBs administration. This is in keeping with reduced promoting activity of GH on longitudinal bone growth and periosteal bone formation (Ohlsson et al., 1998) and suggest that the increased width of the tibial cartilage, as observed at microscope after staining of mineralized tissue, may be actually the outcome of the retardation of bone formation in PCB-exposed animals. The effects of GH on bone may be direct or mediated by GHinduced production of IGF-1 (Rosen and Donahue, 1998). IGFs are among the most important growth factors secreted by skeletal cells and are considered autocrine regulators of osteoblastic functions (Canalis et al., 1989). In addition to regulating osteoblast activity, IGF1 can enhance osteoclast recruitment and differentiation either directly or indirectly via receptor activator of nuclear factor-kB ligand (RANKL) expression (Rubin et al., 2002). It is well known that critical for the bone resorptive process is the balance between osteoprotegerin (OPG) and RANKL. The receptor for RANKL (RANK) is present on osteoclasts and their precursors and when bound activates them to resorb bone. OPG, blocks the effects of RANKL by neutralizing and preventing its binding on RANK. In the present study, plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning but the difference did not attain the statistical significance at PN60. The increase of circulating IGF-1 after exposure to PCB, was not related to increased pituitary GH expression, suggesting direct effects of PCBs on IGF-1 release from the liver. We did not measure plasma GH concentrations in blood samples collected at the endpoint, since they would be scarcely representative due to the pulsatile release of GH from pituitary. The temporary alteration of plasma IGF-1 levels could account for the increased width of the tibial epiphyseal cartilage disk in PCB-exposed rats at the endpoint, even in the absence of GH-axis activation. Moreover, it is possible that high levels of IGF-I could have activated bone resorption resulting in decreased BMC. In line with this hypothesis are in vitro and clinical studies showing that IGF-1 increased RANKL and decreased OPG expression in mouse stromal cells and that IGF-1 treatment to postmenopausal women decreased

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serum OPG (Rubin et al., 2002). Considering that estrogens have stimulatory effects on OPG protein and gene expression, the effects of estrogens may mask the effect of IGF-1 on circulating OPG and explain the unchanged bone parameters detected in PCBs-exposed female rats. Remarkably, PCBs caused a reduction of bone mass, bone mineral content and cortical bone thickness, without affecting bone mineral density. It is well known that estrogens and androgens are responsible for the skeletal sexual dimorphism during childhood and thereafter they maintain bone integrity in both genders (Duan et al., 2001). Androgens stimulate chondrocyte maturation, methaphysial ossification and the growth of the long bones (Vanderschueren et al., 2004), whereas estrogens exert a dual effect. Low estrogen levels promote the pubertal growth spurt whereas growth plate fusion is mediated by high levels of estrogens (Van der Eerden et al., 2003). The larger bone size in men with both a larger diameter and greater cortical thickness in the long bones confer biomechanical advantages and could be responsible for the lower incidence of fragility fractures compared with women (Compston, 2001). In adulthood gonadal sex steroids are key hormones for the maintenance of bone mass since conditions of estrogen or androgen deficiency induce bone loss by increasing the rate of bone remodelling with bone resorption exceeding bone formation (Manolagas et al., 2002). As to the present study, the mechanisms responsible for the adverse effects of the reconstituted PCB mixture on bone mass in male rats, remain to be clarified. The lack of any effect on the main endpoints of sexual development does not support the hypothesis of long-lasting antiandrogenic effects produced by developmental exposure to PCBs. Remarkably, it has been previously shown that exposure to a reconstituted PCB mixture reduced serum concentrations of 1,25dihydroxyvitamin D3 in dams, even at the lowest exposure levels used, and in offspring at the highest exposure levels (Lilienthal et al., 2000). Moreover, a clear relationship between circulating 1,25-dihydroxyvitamin D3, calcium, phosphate and hepatic PCB load has been observed in the grey seals living in the Baltic sea. High levels of PCBs have been associated with increased bone resorption and bone lesions in these animals (Routti et al., 2008). PCB-induced effects on vitamin D3 metabolites might account for the reduced bone mineral content of tibiae and the retardation of bone mineralization at the epiphyseal growth plate in the male adult rats. However, this hypothesis can not explain the sex-related differences, with unchanged bone parameters detected in PCB-exposed female rats. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring. We observed significant changes in hypothalamic neurons, in pituitary GH-secreting cells, in cartilage and bone tissues. Actually, the direct and pleiotropic effects of PCBs on distinct tissues make it difficult to distinguish between primitive effects of PCBs and the following systemic consequences, during long-lasting exposure to these pollutants. Noteworthy, PCB effects are often sex-specific, the males appearing more sensitive than females. Acknowledgments This study was supported by a grant from the Italian MIUR (PRIN 2004). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. References Bachour, G., Failing, K., Georgii, S., Elmadfa, I., Brunn, H., 1998. Species and organ dependence of PCB contamination in fish, foxes, roe deer, and humans. Arch. Environ. Contam. Toxicol. 35, 666–673. Basha, M.R., Braddy, N.S., Zawia, N.H., Kodavanti, P.R., 2006. Ontogenetic alterations in prototypical transcription factors in the rat cerebellum and hippocampus following perinatal exposure to a commercial PCB mixture. Neurotoxicology 27, 118–124.

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