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Inhibition of Vasopressin Release in the Rat Supraoptic Nucleus by Exposure to the Pbde Mixture (De-71) in Vitro.
Citation:
COBURN, C. G., M. C. CURRAS-COLLAZO, AND PRASADA RAO S. KODAVANTI. Inhibition of Vasopressin Release in the Rat Supraoptic Nucleus by Exposure to the Pbde Mixture (De-71) in Vitro. Presented at 26th International Symposium on Halogenated Environmental Organic Pollutants and POPs (Dioxin 2006), Oslo, NORWAY, August 21 - 25, 2006.
Description:
Introduction
Persistent organic pollutants (POPs) are long-lived toxic organic compounds and are of major concern for human
and ecosystem health1,2 . Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are
examples of such chemicals. PBDEs and PCBs belong to a group of pollutants called polyhalogenated aromatic
hydrocarbons and therefore, are structurally related. Owing to their persistence, the manufacture of PCBs was
discontinued in 1977, however, PCBs are still found in significant quantities in the environment. PBDEs are
currently being manufactured and used in large quantities as flame-retardant additives in polymers, especially in the
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manufacture of a variety of electrical appliances, building materials, foams and upholstery furnishings. PBDEs in
these materials are not chemically bound to plastic and foam, and therefore, easily leach out when items containing
PBDEs decompose in landfills or are incompletely incinerated. Over time, these compounds become ubiquitous
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contaminants in the environment because of their high production, lipophilic characteristics, and persistence.
PBDEs are now found in air, water, fish, birds, marine mammals, and humans, and in many cases their
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concentrations are increasing over time. In addition, PBDE levels found in several biological and environmental
samples from the United States were several-fold higher than the levels found in European countries4,5 .
Our recent work showed that at environmentally relevant dosages, the commercial PCB mixture (Aroclor 1254)
administered in vivo suppresses dehydration-induced dendritic release of vasopressin (VP) and exaggerates systemic
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release into the circulation. In addition, when Aroclor 1254 (20 µM) was administered directly to tissue punches
from supraoptic nucleus (SON) of dehydrated rats in vitro, dendritic VP release was abolished, suggesting that
direct actions on magnocellular neuroendocrine cells (MNCs) are sufficient to inhibit VP release without the need
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for effects on other osmosensitive regions. The objectives of the present study were to determine if in vitro
exposure to PBDEs, (which are structurally similar to PCBs) induce comparable inhibition of dendritic release of
VP.
Methods and Materials
Animals: Long-Evans hooded and Sprague Dawley male rats (300-400g) were obtained from Charles River
Laboratory (Raleigh, NC) and housed in pairs in AAALAC approved animal facilities. Food and water were
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provided ad libitum. Temperature was maintained at 21± 2C and relative humidity at 50 ± 10% with a 12-h
light/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. EPA in compliance with NIH guidelines.
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SON Punch Preparation: SON tissue punches were prepared as described. Briefly, after decapitation, brains were
quickly removed, and placed in cold oxygenated artificial cerebrospinal fluid (aCSF). The SON was dissected
bilaterally from 1 mm coronal sections placed on an ice-cold slide. Each sample was transferred to an individual
static well containing aCSF (pH 7.4, 37oC). Each well contained the bilateral SON from one rat brain in a total
volume of 500 µl of incubation solution (analysate). Acutely-dissected samples were maintained in the wells with
continuous oxygenation (95/5% O2/CO2). Control samples were incubated in normal aCSF (290 mOsm/L). SON
punches were osmotically stimulated by incubation in hyperosmotic aCSF (350 mOsm/L).
In Vitro Toxicant Exposure: Tissue punches were exposed to either toxicant or DMSO vehicle for a 30-minute
period at 37oC in 290 mOsm aCSF. Following the toxicant exposure, 290 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 and nitric oxide (NO) analysis. With the exception of the
normosmotic control group, all tissues were incubated in 350 mOsm aCSF for the 10-minute experimental period to
stimulate NO and VP release. Finally, SON punches were collected in cold buffer containing protease inhibitor
cocktail, homogenized, and frozen for later protein determination using the bicinchoninic acid method (Pierce).
Quantification of VP: VP content in perfusate samples was measured using enzyme-immunoassay (arg8-vasopressin
correlate EIA kit, Assay Designs) with a sensitivity of 3.39 pg/ml. VP values for each sample were normalized to
the total protein present in each SON sample to control for variations in the size of the SON punches of origin and
expressed as pg/ml/µg protein.
Quantification of NO: Aliquots of the analysate of each SON tissue punch were assayed for NO by the Griess
Method (Oxford Biomedical, enzymatic kit) for detection of the oxygenation product of NO, nitrite. The reaction
detects as little as 0.5 µM NO (using an 85 µl sample). The final product of the Griess reaction was then read at 540
nm on a Molecular Devices Spectra Max 190 plate reader. For the assay, 85 µl of incubation solution was analyzed
to determine the concentration of NO (µM). The total NO produced by each punch (pmols) in the original 500 µl
incubation volume was then normalized by dividing by total protein in the punch. Final NO values were expressed
as pmol/µg protein.
Statistical Analysis: Data collected for this study were analyzed for main effects of toxicant or hydration state by
separate one-way ANOVAs using Sigma Stat software. General linear, or repeated measures ANOVA was used
where data met normal distribution/equal variance assumptions. Where overall significance of (p<0.05) was
obtained, post hoc multiple comparisons were used to detect specific differences.
Results and Discussion
The degree to which PBDEs constitute a human health hazard is not yet clear and the potential ability of these
substances to compromise neuroendocrine systems has not been fully investigated. To begin to address the
potential deleterious effects of PBDEs on neuroendocrine activity, SON punches were removed from adult male rats
and their VP release was measured. In agreement with previous studies6,7 , Figure 1 shows a significant
compensatory increase in VP secreted within the SON of stimulated tissue relative to normosmotic controls
(30.8±3.45 n=14 vs. 20.92±2.99 n=16 pg/ml/µg; p<0.05). Interestingly, the osmotically-induced rise in SON VP is
significantly (p<0.001) reduced by exposure to either Aroclor 1254 or DE-71. As shown in Figure 1 panel A,
exposure to 8.8 µg/0.5 ml and 5.0 µg/0.5ml of DE-71 and Aroclor 1254, respectively, inhibited osmotically-induced
VP release by 64 and 60%, respectively. VP values for hyperosmotic, hyperosmotic plus DE-71 and hyperosmotic
plus Aroclor 1254 groups are 30.8 ± 3.45, (n=14),11.22 ± 2.17 (n=10) and 12.31 ± 2.99 (n=10), respectively.
Preliminary results in panel B show the effect of lower concentration of these mixtures (2.9 µg/ml and 1.65 µg/ml)
of DE-71 and Aroclor 1254, respectively on VP release. Early data indicate that at these lower concentrations, VP
release may be attenuated, but the values fail to meet statistical significance due to the limited numbers of animals
used in the experiment.
The in vitro effects of PBDEs on SON vasopressin release is noteworthy because this punch preparation is
functionally isolated from possible actions of toxicants on osmoreceptors such as the subfornical organ or organum
vasculosum of the lamina terminalis, which act pre-synaptically upon MNCs to influence plasma VP levels in the
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intact animal. These data suggest that the toxicants within the mixtures are acting directly upon the MNCs.