ToxSci Advance Access originally published online on April 13, 2007
Toxicological Sciences 2007 98(1):178-186; doi:10.1093/toxsci/kfm086
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Polybrominated Diphenyl Ethers and ortho-Substituted Polychlorinated Biphenyls as Neuroendocrine Disruptors of Vasopressin Release: Effects during Physiological Activation In Vitro and Structure–Activity Relationships
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* Environmental Toxicology Graduate Program, University of California, Riverside, California 92521
Cellular and Molecular Toxicology Branch, Neurotoxicology Division, NHEERL, ORD, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Department of Cell Biology and Neuroscience, University of California, Riverside, California 92521
1 To whom correspondence should be addressed at Cellular and Molecular Toxicology Branch, Neurotoxicology Division, B 105-06, NHEERL/ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-0717. E-mail: kodavanti.prasada{at}epa.gov.
Received January 17, 2007; accepted April 7, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The neuropeptide, vasopressin (VP) is synthesized in magnocellular neuroendocrine cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the mammalian hypothalamus. VP has multiple functions including maintenance of body fluid homeostasis, cardiovascular control, learning and memory, and nervous system development. Polybrominated diphenyl ethers (PBDEs), used as additive flame retardants, have been shown to interfere with hormone metabolism and function. Previously, we demonstrated that the technical polychlorinated biphenyl (PCB) mixture, Aroclor 1254, inhibits somatodendritic VP release from the SON of osmotically stimulated rats. The objectives of the current study were to test whether PBDEs affect central VP release in a similar manner and to determine the potency of several PCB and PBDE congeners in order to identify a common mode of action for these persistent chemicals. The current work shows that the commercial PBDE mixture (DE-71) significantly decreased somatodendritic VP release from rat SON punches in a strain-independent manner. In addition, the specific congeners PBDE 47 and PCB 47 (15 and 5µM) were also neuroactive in this system. To explore structure/activity relationships, we compared the effects of PBDE 77 with PCB 77. PBDE 77, but not PCB 77 significantly reduced VP release. These results show that like PCBs, PBDEs perturb signaling mechanisms responsible for hormone release, and that environmentally relevant PBDE congeners are more neuroactive than the commercial mixtures with noncoplanarity of these compounds playing a role in promoting neuroactivity.
Key Words: polychlorinated biphenyls; polybrominated diphenyl ethers; neurotoxicity; supraoptic nucleus; neuroendocrine disruption; hypothalamus; intracellular signaling; organohalogen compounds; osmoregulation; structure–activity relationships.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Persistent organic chemicals that are used in industrial applications are of major concern for human and ecosystem health. Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are two classes of such persistent organohalogen compounds that are endocrine disrupting agents and share similar structural features (Fig. 1). Owing to their persistence, the manufacture of PCBs in the USA was discontinued in 1977; however, PCBs are still found in significant quantities in the environment (Lang, 1992
). In contrast to PCBs that were used in the early 1970s, PBDEs are currently being produced for use as flame-retarding compounds in a variety of consumer goods including electrical appliances, building materials, foams, and upholstery furnishings (Hale et al., 2006). Over time PBDEs leach into the environment when household wastes decompose in landfills or are incompletely incinerated (Wilford et al., 2005). Due to their high production, lipophilic characteristics, and persistence, PBDEs are now found in air and water as well as in fish, birds, marine mammals, and humans, and in many cases their concentrations are increasing over time (Hites, 2004; Petreas et al., 2003).
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Although humans and wildlife suffer adverse health consequences resulting from exposure to environmental endocrine disrupting chemicals (Crews and McLachlan, 2006
; Vos et al., 2000) to date, the neuroendocrine effects of environmental chemicals have focused primarily on disruption of the hypothalamic-pituitary-thyroid axis (Cooper et al., 2000; Kodavanti, 2005; Toni et al., 2005) and the hypothalamic-pituitary-gonadal axis (Gore et al., 2002; Legler and Brouwer, 2003). In the current study, we investigated the effects of in vitro exposure to two classes of organohalogen compounds on neuroendocrine mechanisms associated with the hypothalamo-neurohypophysial system (HNS), known to govern osmoregulation through the neuropeptide, vasopressin (VP) which regulates blood volume and plasma osmolality. In response to dehydration, detected as a rise in plasma osmolality, VP is released into the bloodstream from the neurohypophysis. VP performs its antidiuretic function in the periphery by acting upon the kidney to increase water retention. Vasopressinergic magnocellular neurosecretory cells (MNCs) that release VP are primarily located in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. In response to dehydration, VP is also released centrally within the hypothalamic parenchyma, from the soma and dendrites of MNCs (Ludwig, 1998; Neumann et al., 1993). Central VP release modulates the characteristic firing patterns of MNCs that are important for maintaining efficient hormonal output when the system is under physiological demand (Cazalis et al., 1985; Gouzenes et al., 1998). An additional central vasopressinergic system originating in the suprachiasmatic nucleus, the bed nucleus of the stria terminalis, and the medial amygdaloid nucleus regulates cognitive functions including learning and memory and social behaviors (Bielsky and Young, 2004; Ring, 2005).
Recent studies suggest that postnatal exposure to PBDEs is much greater than in utero exposure. It is currently believed that inadvertent ingestion of house dust is the largest contributor to exposure for humans at all life stages except for infancy during which lactational transfer from breast milk is the primary route of exposure to PBDE (Fischer et al., 2006; Jones-Otazo et al., 2005). Breast milk from women in the United States has been reported to have considerably higher concentrations than those found in their Swedish counterparts (Schecter et al., 2003). Because high levels of PBDEs are present in breast milk they can be transferred to the nursing infant (Mazdai et al., 2003; Noren and Meironyte, 2000). As there are particular concerns about infant health due to the sensitivity of the developing brain to the effects of organohalogen compounds (Eriksson et al., 2002; Stein et al., 2002), elevated PBDE body burdens in women of childbearing age warrant further investigation in regard to the endocrine disrupting potential of these compounds.
A prominent diphenyl ether (DE) mixture used in the manufacture of textiles and polyurethane foams is DE-71 (Hale et al., 2001). DE-71 is composed of the congeners 2,2',4,4',5-pentabromodiphenyl ether (PBDE 99; pentaBDE) and 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47; tetraBDE) as the major constituents (LaA Guardia et al., 2006). A number of studies suggest that DE-71 has the potential to disrupt endocrine homeostasis and reproductive development (Stoker et al., 2004, 2005) and this mixture has been shown to significantly decrease circulating total serum thyroxin (T4) in perinatally exposed rats (Ellis-Hutchings et al., 2006).
Our previous work has shown that at environmentally relevant doses, the commercial PCB mixture (Aroclor 1254) administered in vivo reduces somatodendritic VP release and exaggerates systemic release into the circulation in response to dehydration (Coburn et al., 2005). Furthermore, Aroclor 1254 (20µM) added directly to SON tissue punches from dehydrated rats suppresses local VP release, suggesting that direct actions on MNCs are sufficient to explain the inhibition of VP release without the need to invoke effects on other forebrain osmosensitive regions (Coburn et al., 2005). It has been demonstrated that somatodendritic VP release is dependent on an evoked rise in both intracellular Ca2+ (Sabatier et al., 2004) and nitric oxide (Gillard et al., 2007). Because Aroclor 1254 and DE-71 have been shown to disrupt calcium buffering in microsomes and mitochondria isolated from adult rat brain (Kodavanti and Ward, 2005) and NOS is inhibited by specific PCB congeners (Sharma and Kodavanti, 2002), it is likely that PBDEs could disrupt signaling mechanisms required for VP release in response to an osmotic challenge. Therefore, the objectives of the present study were to (1) test if DE-71 inhibits VP release in a manner similar to Aroclor 1254; (2) test if the neuroendocrine effects of Aroclor 1254 and DE-71 are rat strain specific; (3) compare the potency and efficacy of specific environmentally relevant PBDE and PCB congeners; and (4) compare the structure–activity relationship (SAR) between two identically substituted congeners, 3,3',4,4'-tetrabromodiphenyl ether (PBDE 77, noncoplanar congener) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77, coplanar congener).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Long–Evans hooded and Sprague–Dawley male rats (250–350 g; 70–80 days old) were obtained from Charles River Laboratory (Raleigh, NC) and housed in pairs in an AAALAC approved animal facility. Food and water were provided ad libitum. Temperature was maintained at 21 ± 2°C and relative humidity at 50 ± 10% with a 12-h light/12-h dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of the National Health and Environmental Research Laboratory at U.S. Environmental Protection Agency (EPA), in compliance with National Institutes of Health guidelines.
Chemicals.
The technical PBDE mixture DE-71 (Lot # 1550OI18A) and the PBDE congener, 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) were gifts from Great Lakes Chemical Corporation (West Lafayette, IN). PBDE 77 (3,3',4,4'-tetrabromodiphenyl ether) was purchased from the Biochemical Institute for Environmental Carcinogens, Lurup 4, D-22927 Grosshansdorf, Germany. PCB 47 (2,2',4,4'-tetrachlorobiphenyl) and the PCB technical mixture Aroclor 1254 (Lot # 124-191) were purchased from AccuStandard, Inc., New Haven, CT. All PBDE and PCB congeners were 98–100% pure and dissolved in dimethyl sulfoxide (DMSO).
In vitro tissue preparation.
SON tissue punches were prepared as described (Coburn et al., 2005). Briefly, after decapitation, brains were quickly removed, and placed in cold oxygenated artificial cerebrospinal fluid (aCSF). The base Locke's solution (290 mOsm, pH 7.4, 37°C) was adapted from that described previously (Hussy et al., 2001), and is a glucose-containing, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–buffered Locke's solution consisting of (in mM): NaCl (132), KCl (5), CaCl2 (2), MgCl2 (2), KH2PO4 (1.2), HEPES (10), and glucose (10). To achieve final osmolalities of 295 and 350 mOsm, NaCl was added to the base solution yielding a hypertonic, hyperosmotic solution. The SON was dissected bilaterally from 1-mm coronal sections placed on an ice-cold slide. SON tissue punches were transferred to an individual static well of a multiwell plate containing aCSF. Each well contained the combined bilateral SON from one rat brain in a static volume of 500 µl of incubation solution (analysate). Acutely dissected SON punches were maintained in the wells with continuous oxygenation (95/5% O2/CO2). All punches incubated in normosmotic aCSF (295 mOsm; pH 7.4, 37°C) during a 30-min equilibration period after which bathing solution containing basal VP release was discarded. SON punches then received either fresh normosmotic aCSF or hyperosmotic aCSF (350 mOsm, pH 7.4, 37°C). We have recently reported that plasma osmolality values for rats subjected to hyperosmotic stimulation in vivo are similarly elevated with most values ranging between 335 and 345 mOsm (Gillard et al., 2007). A total of 225 rats were used for these experiments and each data point represents the combined bilateral nuclei for one rat.
In vitro toxicant exposure.
SON tissue punches were exposed to two different concentrations of mixtures. Tissue was exposed to a higher concentration of 17.6 µg/ml (PBDE-71) and 10 µg/ml (Aroclor 1254) and a lower concentration of 5.8 µg/ml (DE-71) and 3.3 µg/ml (Aroclor 1254). Although the dose values differ on a weight basis the concentrations are identical in terms of molarity (approximately 30µM for the higher dose and 10µM for the lower dose). Two concentrations were also chosen for the congeners tested in this work. Tissue was exposed to either a 15 or 5µM concentration of PBDE 47 or PCB 47. Because no significant effect was observed when tissue was exposed to 15µM PCB 77 studies were not conducted using a lower concentration. Control tissue was exposed to DMSO vehicle only. In previous studies, we have shown that the final concentration of DMSO in the assay buffer (< 0.5% vol/vol) does not significantly affect VP release from the SON tissue punch preparation used in this work (Coburn et al., 2005).
Tissue was exposed to the toxicants or DMSO vehicle during the 30-min equilibration period at 37°C in 295 mOsm aCSF. Following the toxicant exposure, 295 mOsm aCSF was replaced with 500 µl of fresh 350 mOsm aCSF and incubated for an additional 10-min experimental period, after which aliquots of analysates were removed and frozen for subsequent VP analysis. With the exception of the normosmotic control group all tissues were incubated in 350 mOsm aCSF for the 10-min experimental period to stimulate VP release. Finally, SON punches were collected in cold buffer containing protease inhibitor cocktail (Protease inhibitor cocktail III from Calbiochem, San Diego, CA), homogenized, and frozen for later total protein determination using the bicinchoninic acid method (Pierce, Rockford, IL).
Quantification of VP by enzyme immunoassay.
As described previously (Gillard et al., 2007), VP content in individual aliquots of incubation solution (25 µl) was measured without extraction, using a highly specific, commercially available competitive enzyme immunoassay kit (arg8-vasopressin Correlate-Enzyme-Immunoassay kit, Assay Designs, Ann Arbor, MI) with a manufacturer-specified sensitivity of 3.52 pg/ml and intra- and interassay coefficients of variation of 7.9% and 8.5%, respectively. The samples were assayed in duplicate. In separate experiments using DE-71 or Aroclor 1254 (30 µg/ml) incubated without brain tissue, this assay showed no significant cross-reactivity. VP concentrations (pg/ml) in the sample aliquots were calculated using a four-parameter curve-fitting computer program (STATLIA; Brendan). VP values for each punch were then normalized to the protein measured in the tissue punch and expressed as pg/µg protein.
Statistical analysis.
Findings presented in this manuscript represent pooled data from 10 separate experiments (each experiment consisted of 20–25 animals). For VP, each rat contributed one measurement consisting of analysates of combined bilateral SON nuclei. Values for all groups are presented as the mean ± SEM. Statistical differences were tested at p < 0.05 using a Student's t-test for two-group comparisons. When data met normal distribution and equal variance assumptions, multiple group comparisons were analyzed using a general linear one-way analysis of variance (ANOVA). When ANOVA yielded statistically significant effects, Dunnett's multiple comparison test (
< 0.05) was used to detect specific post hoc differences (Prism3.0, GraphPad Software, Inc.)
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Commercial PBDE and PCB Mixtures on VP Release from SON Tissue Punches
Central release of neurohypophysial hormones by MNCs is an important autoregulatory mechanism influencing their systemic output in response to physiological stimuli (Ludwig and Leng, 1998
). To address the potential impact of PBDEs on hormone release mechanisms, SON tissue punches were subjected to hyperosmotic stimulation in vitro. Previous work has shown that increasing the osmolality of the perfusate of SON punches obtained from normosmotic rats can more than double somatodendritic VP release in vitro (Gillard et al., 2007). Because SON MNCs project axons to the neurohypophysis and because their axons have few, if any, collaterals located within the SON, VP measured from SON punches in vitro reflects secretion primarily from the soma and/or dendrites (Ludwig and Leng, 2006; Swanson and Sawchenko, 1983).
In agreement with previous studies (Gillard et al., 2007; Ludwig, 1998), Figure 2 shows a significant (T2.98, 19; p = 0.008) compensatory increase in VP secreted within the SON of osmotically stimulated tissue relative to normosmotic controls (11.7 ± 1.1 vs. 7.3 ± 1.1 pg/µg protein (n = 20–24)). This neuroendocrine response to hyperosmotic stimulation is significantly reduced after exposure to DE-71 or Aroclor 1254 (F4,60 = 4.093; p < 0.01). At equimolar concentrations (17.6 µg/ml DE-71 and 10.0 µg/ml Aroclor 1254), the two mixtures significantly reduced osmotically stimulated SON VP release. VP values for hyperosmotic, hyperosmotic plus DE-71 and hyperosmotic plus Aroclor 1254 treated groups were 11.7 ± 1.1, 5.6 ± 1.1, and 6.2 ± 1.5 pg/ug protein (n = 10-24). However, when the in vitro concentrations were reduced to one-third that of the higher dose values (5.8 µg/ml DE-71 and 3.3 µg/ml Aroclor 1254) no significant effect was observed.
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Effects of PBDE and PCB Mixtures on VP Release in Two Strains of Rats
To determine if toxicant effects on VP release are rat strain specific, we tested the effects of DE-71 (17.6 µg/ml) and Aroclor 1254 (10.0 µg/ml) in another common strain of laboratory rodent, the Long–Evans hooded rat. As seen before for Sprague–Dawley rats (Fig. 2), Figure 3 shows a significant (T4.26,27; p = 0.0002) compensatory increase in VP secreted within the SON of osmotically stimulated tissue relative to normosmotic controls obtained from Long–Evans rats (12.0 ± 1.1 vs. 5.5 ± 1.0 pg/µg protein (n = 13–16)). Consistent with our findings in Sprague–Dawley rats, osmotically induced VP release was suppressed (F2,40 = 3.927; p < 0.05) by DE-71 and Aroclor 1254. VP values for hyperosmotic, hyperosmotic plus DE-71, and hyperosmotic plus Aroclor 1254 groups were 12.0 ± 1.1, 7.4 ± 1.6, and 7.2 ± 1.6 pg/µg protein (n = 13–16). These findings show that inhibition of VP release was similar in the two rat strains suggesting that the effect of these two groups of toxicants is not genotype specific. Since the inhibition of VP release by these two compounds is similar, PBDEs and PCBs may be exerting their effects via a common mode of action.
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SARs for the Effects of Individual PBDE and PCB Congeners on VP Release
To investigate the neuroactive potential of specific PBDE and PCB congeners, we compared the effects of PBDEs 47 and 77 with PCBs 47 and 77. PBDE 47 is the predominant congener found in most environmental and human samples (Hites, 2004
) and is a significant component of the commercial mixture, DE-71 (LaA Guardia et al., 2006). PCB 47 was selected for comparison with PBDE 47. ANOVA indicated a significant decrease in congener-induced inhibition of osmotically stimulated VP release (F6,64 = 2.971; p < 0.05). For these studies, two doses of the toxicants, 15 and 5µM, were used. As shown in Figure 4, exposure to 15µM PBDE 47 and PCB 47 significantly (p < 0.05) reduced osmotically stimulated peptide release to normal control levels indicating that osmotically stimulated release was obliterated by toxicant treatment. VP values for hyperosmotic, hyperosmotic plus PBDE 47, and hyperosmotic plus PCB 47 were 11.6 ± 1.6, 6.6 ± 1.5, and 6.1 ± 1.4 (n = 9–16) pg/µg protein. Even at low concentrations (5µM), exposure to these congeners obliterated stimulated hormone release in this system, a finding in contrast to that seen with commercial mixtures at lower doses. Exposure to 5µM of PBDE 47 and PCB 47 significantly (p < 0.05) decreased osmotically stimulated SON VP release to normal control levels. VP values for hyperosmotic, hyperosmotic plus PBDE 47, and hyperosmotic plus PCB 47 were 11.6 ± 1.6, 5.9 ± 1.4, and 6.0 ± 1.1 pg/µg protein (n = 7–16). These results indicate that 2,2',4,4'-tetrabromodiphenyl ether (PBDE 47) and 2,2',4,4'-tetrachlorobiphenyl (PCB 47, noncoplanar congener) are more neuroactive than the parent commercial mixtures they compose.
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To explore the SAR in this system, two additional congeners were chosen for study. For this work, the non–ortho-substituted 3,3',4,4'-tetrachlorobiphenyl (coplanar; PCB 77) and the less-coplanar 3,3',4,4'-tetrabromodiphenyl ether (PBDE 77) (Fig. 1) were selected based on previous SAR studies indicating that coplanar congeners are less active in neuronal preparations (Kodavanti and Tilson, 1997
). These two compounds were selected because they have a similar halogen substitution pattern, but they differ in their coplanarity. ANOVA yielded a significant effect of treatment with these congeners (F2,31 = 3.453; p < 0.05) and a post hoc analysis showed that the two congeners differed in their activity. As shown in Figure 5, PBDE 77 significantly (p < 0.05) inhibited stimulated SON VP release. In contrast, PCB 77 (15µM) failed to demonstrate a significant effect (p > 0.1). VP values for hyperosmotic, hyperosmotic plus PBDE 77 and hyperosmotic plus PCB 77 were 11.6 ± 1.6, 6.2 ± 1.1, and 9.2 ± 0.9 pg/µg protein (n = 9–16).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The widespread use of PBDEs and increased contamination in the environment has led to the rising concern about the potential adverse health effects to wildlife and humans. PBDEs are currently found in human blood, adipose tissue, and breast milk and environmental levels of these toxicants are increasing (Gill et al., 2004
; Ikonomou et al., 2002; Rayne et al., 2003). The concentrations of toxicants used in the current work (5–30µM) are consistent with other investigators measuring neurotoxicity in a variety of in vitro preparations. For example, it has been demonstrated that in a cell-free preparation Aroclor 1254 and DE-71 inhibit microsomal and mitochondrial 45Ca2+ -uptake with IC50 values of 4–24µM (Kodavanti and Ward, 2005) and Mariussen and Fonnum (2001) have reported inhibition of dopamine and serotonin uptake in rat brain synaptic vesicles with Aroclor mixtures at EC50 values of 4–33µM. Furthermore, effects reported in the current work are observed at concentrations and exposure times where cytotoxicity was not observed. Kodavanti and Ward (2005) using % lactate dehydrogenase leakage as an index reported an absence of effect in neuronal cultures exposed to 50 µg/ml (
100µM) DE-71 up to 4 h of exposure. Relative to environmental concentrations, the doses used in this study are within a degree or two of magnitude. Alarmingly, a study comparing PBDE levels in a California family found the highest concentrations in the youngest member, an 18-month-old toddler, who expressed serum levels of PBDE 47 at 200 ng/g lipid weight (0.4µM) (Fischer et al., 2006).
In an earlier report, we found that basal intranuclear VP release was nearly identical in control and PCB-treated rats, suggesting that the effect of PCBs is selective, with the function of the system being noticeably impaired only during conditions of physiological stimulation. In addition, we showed that in vitro exposure to Aroclor 1254 (20µM) completely abolished dehydration-induced central VP release from SON tissue punches. In the current study, we demonstrate that, like Aroclor 1254 (10 µg/ml), exposure to 17.6 µg/ml DE-71 significantly reduced the VP release response to hyperosmotic stimulation in SON tissue punches. At the doses selected, PCB and PBDE mixtures are equally potent. It should be noted that although the dose values differ on a weight basis (10.0 µg/ml Aroclor 1254 vs. 17.6 µg/ml DE-71), the concentrations are identical in terms of molarity (
30µM). It is noteworthy that when the mixtures' dose was decreased to one-third the higher concentration (5.8 µg/ml DE-71 vs. 3.3 µg/ml Aroclor 1254), the reduction in VP release failed to meet statistical significance even though the response to dehydration appeared to be dampened. One interpretation of this finding is that some of the congeners composing the mixture are neuroactive in this system while others, such as the coplanar congeners, may not be. Therefore, by reducing the concentration of the neuroactive congeners in the incubation media their interactions with specific cellular and intracellular targets are diminished which, in turn, attenuates the inhibition of the measured effect, in this case, VP release.
Our data show that the neuroendocrine effect of PBDEs in the MNC system is similar for both Sprague–Dawley and Long–Evans rat strains suggesting that these chemicals may work through a similar mode of action. In SON MNC's local hyperosmolality elicits calcium influx via stretch-inactivated cation channels (Zhang and Bourque, 2006), which leads to both depolarization and increased firing activity. Depolarization-induced Ca2+ influx through Ca2+ channels and/or through N-methyl-D-aspartate-type glutamate receptors triggers exocytosis of VP-containing vesicles and liberation of peptide into the extracellular space. In turn, VP binds to VP autoreceptors on SON MNCs (Gillard et al., 2007; Hurbin et al., 2002) that ultimately couple to adenylate cyclase as well as phospholipase C, leading to an influx of Ca2+ through voltage gated ion channels and liberation from internal stores (Dayanithi et al., 1996; Gouzenes et al., 1999; Sabatier et al., 2004). It has been shown that Aroclor 1254 and DE-71 inhibit calcium buffering in mitochondria and microsomes isolated from adult male rat brain (Kodavanti and Ward, 2005). PKC (protein kinase C) translocation from the cytosol, a calcium-dependent signaling event, has also been shown to be increased in a dose-dependent manner by both Aroclor 1254 and DE-71 (Kodavanti and Ward, 2005). Moreover, PKC can phosphorylate NOS, facilitating NO production required for dehydration-induced VP release (Gillard et al., 2007). Related to this Sharma and Kodavanti (2002) have shown decreased hypothalamic NOS activity after exposure to PCB congeners. While not yet fully understood, these effects seen in a cell-free system may underlie the neuroendocrine disrupting effects demonstrated in the current work and may have physiological consequences for systemic output of VP hormone and body water homeostasis.
Our data address the SARs of PCB and PBDE neuroendocrine effects. Because it has been shown that PBDE 47 significantly increases phorbol ester binding at concentrations as low as 10µM (Kodavanti et al., 2005) and ortho-substituted hydroxylated PCB congeners perturb calcium buffering mechanisms at concentrations as low as 3µM (Kodavanti et al., 2003), we chose to test the effect of these two congeners at 15 and 5µM. Results from these experiments showed that both the PBDE and PCB congener reduced dehydration-induced hormone release to levels that were comparable to normal controls, which in vivo likely deranges the response of this system to an osmotic challenge. It is important to compare the potency of the mixtures shown in Figure 2 with the congeners shown in Figure 4. For the commercial mixtures DE-71 and Aroclor 1254, the lower concentration tested (10µM) failed to inhibit peptide release. In contrast, the specific congeners significantly inhibited VP release at concentrations as low as 5µM indicating that in MNCs, these congeners are more neuroactive than the technical mixtures they compose. PBDE 47 has been shown to disrupt endocrine homeostasis and reproductive development by inhibiting the binding of androgens to the androgen receptor in vivo (Stoker et al., 2005) and by decreasing circulating thyroid hormone levels in mice (Hallgren et al., 2001). In addition, neonatal exposure to PBDE 47 results in permanent aberrations in spontaneous behavior and habituation capability that appears to worsen with age (Eriksson et al., 2001). PBDE 47 is found in major quantities in biological and environmental samples (Morland et al., 2005; Schecter et al., 2004) and is the predominant congener found in breast milk taken from nursing mothers with a mean concentration of 40.8 (ng/g/lipid) (Schecter et al., 2003).
PCBs share similar molecular features with PBDEs, but the two families of compounds are not structurally identical. In contrast to PCBs, PBDE congeners are more noncoplanar in nature due to the oxygen bridge linking the biphenyl rings (Fig. 1). In an effort to deduce the mode of action of toxicants evaluated in this study, we compared the activity of PCB 77, a non–ortho-substituted (coplanar) congener with PBDE 77 that shares an identical halogen substitution pattern but is less coplanar. As shown in Figure 5, PBDE 77 significantly reduced hormone release from stimulated SON MNCs, but PCB 77 failed to do so. These findings are in agreement with those of others who have reported a lack of neuroactivity for coplanar PCB congeners for such endpoints as Ca2+ homeostasis (Kodavanti and Tilson, 1997), neuronal apoptosis induction (Howard et al., 2003), and altered membrane structure in cerebellar granule cells (Tan et al., 2004). The results obtained in the current study, in conjunction with those reported by others, underscore the concept that substitution patterns (PCBs) or molecular structure (PBDEs) favoring noncoplanarity promotes their activity in vitro.
Neuropeptides that are released from dendrites act as autocrine or paracrine signals at their site of origin, but can also act at distant brain targets to evoke long-lasting changes in behavior (Ludwig and Leng, 2006). Central VP is involved in learning, memory, and social recognition in a number of species (Faiman et al., 1992; Ferguson et al., 2002; Ring, 2005), and decreased central VP function contributes to deficits in social engagement and social bond formation characteristic of autistic spectrum disorders (Hammock and Young, 2006). In addition, Jentsch et al. (2003) have shown that VP plays a critical role in regulating visual attention and cognition. In addition, recent work by Lein et al. (2007) has demonstrated that developmental exposure to Aroclor 1254 significantly decreases neurite outgrowth in rats, which is noteworthy because VP has been shown to facilitate neuronal process outgrowth in cortical neurons in vitro (Chen et al., 2000). Therefore, our findings raise the consideration that while genetic factors certainly play a role in developmental disabilities, environmental chemicals may also be causative factors for various disturbances of the brain, such as autism and attention deficit disorders (Kodavanti, 2005).
In conclusion the findings presented in this study extend and confirm our earlier observations demonstrating an inhibitory effect of a commercial PCB mixture on dehydration-induced central VP release in vitro. Because central VP release is responsible not only for endocrine regulation of body fluid and cardiovascular function but also plays a role in cognitive functions such as learning and memory, our findings could apply broadly to other brain regions and higher brain function.
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NOTES |
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Preliminary findings were presented at the 26th Symposium on Halogenated Environmental Organic Pollutants and POPs (Dioxin2006) meeting in Oslo, Norway (August 19–26, 2006).
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
M.C.C.C. and P.R.S.K. share equal last authorship on this paper.
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ACKNOWLEDGMENTS |
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The authors thank Dr Elizabeth Gillard for her technical assistance and Drs Ram Ramabhadran and Tammy Stoker for their comments on an earlier version of this paper. Work described in this paper was partially funded through EPA contract EP06D000030 awarded to C.G.C. and an National Science Foundation award to M.C.C.C.
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