RP150 (PED-1117-861197) Evaluation of Brominated Flame Retardants in Relationship to Bottlenose Dolphin Immunity.
Start time: 8:00 AM
Peden-Adams, M^{1, 2}, Romano, T^{1, 2}, Rice, C^{1, 3}, Lee, A¹, EuDaly, J¹, Mitchum, G⁵, Bossart, G^{1, 4}, Fair, P^{1, 5}, ¹ Medical University of South Carolina, Charleston, SC, USA² Mystic Aquarium and Institute for Exploration, Mystic, CT, USA³ Clemson University, Clemson, SC, USA⁵ NOAA/NOS/CCEHBR, Charleston, SC, USA⁴ Harbor Branch Oceanographic Institute, Ft. Pierce, FL, USA
Many consumer products contain brominated flame retardants (BFR) leading to increased levels contaminating the environment. Polybrominated diphenyl ethers (PBDEs) are used in large quantities as additives in plastics, textiles, and synthetic building materials. In rodents few studies have assessed immunotoxicity in relation to BFRs, but those that have observed suppression of IgM production and decreased T-cell proliferation. As part of a dolphin health assessment study, biomarkers of immune function were correlated with PBDE blubber levels. Whole blood and plasma samples were collected from 51 male bottlenose dolphins in the Indian River Lagoon (IRL) of Florida and the Charleston (CHS) Harbor of South Carolina during the summer of 2003. Ages ranged from 3.5 to 28 years. PBDE concentrations (6 congeners) and immune parameters (lymphocyte proliferation, NK cell activity, lymphocyte immunophenotypes, plasma lysozyme, phagocytosis, total serum IgG, and C-reactive protein) were measured. Correlations were assessed for all animals with-in and among sites. With all animals combined the strongest correlation observed was lysozyme activity and total PBDEs (Rs= -0.5438). When separated by location NK cell activity in IRL males was positively correlated to PBDE 47 (Rs= 0.5043) and total MHCII+ cells (Rs= -0.4947) and CD4+ T-cells (Rs= -0.4754) in IRL males were correlated with total PBDEs. Total IgG in CHS males was positively correlated with PBDE 47 and total PBDEs (Rs= 0.5586 and 0.4097, respectively). These data suggest that immune parameters are altered in marine mammals with exposure to PBDEs. Further efforts identifying risk to disease is required following PBDE exposure.

RP151 (PED-1117-861950) Do PCBs Modulate Immunity in Atlantic Bottlenose Dolphin?
Start time: 8:00 AM
Peden-Adams, M^{1, 2}, Romano, T^{1, 2}, Rice, C^{1, 3}, Lee, A¹, EuDaly, J¹, Mitchum, G⁵, Bossart, G^{1, 4}, Fair, P^{1, 5}, ¹ Medical University of South Carolina, Charleston, SC, USA² Mystic Aquarium and Institute for Exploration, Mystic, CT, USA³ Clemson University, Clemson, SC, USA⁵ NOAA/NOS/CCEHBR, Charleston, SC, USA⁴ Harbor Branch Oceanographic Institute, Ft. Pierce, FL, USA
It has been suggested that PCBs suppress immunity in bottlenose dolphin. Previous studies, however, were limited by correlations using small sample sizes. As part of a dolphin health assessment study, biomarkers of immune function were correlated with PCB levels in blubber. Whole blood and plasma samples were collected from 51 male bottlenose dolphins in the Indian River Lagoon (IRL) of Florida and the Charleston (CHS) Harbor of South Carolina during the summer of 2003. Ages ranged from 3.5 to 28 years. PCB concentrations (72 congeners) and immune parameters (lymphocyte proliferation, NK cell activity, lymphocyte immunophenotypes, plasma lysozyme, phagocytosis, total serum IgG, and C-reactive protein) were measured. Correlations were assessed for all animals with-in and among sites. With all animals combined the strongest correlation observed was CD21+ B-cells and octachlorinated PCBs (Rs= -0.5806). When separated by location NK cell activity in IRL males was positively correlated to dichloroinated PCBs (Rs= 0.5852) and total IgG and CD+21 cells in CHS males were correlated with dichlorinated (Rs=0.5479) and octachlorinated (-0.5061) PCBs, respectively. Lymphocyte proliferation did not correlate with PCB levels, contrasting with previous reports. These data indicate that PCBs may alter immunity in marine mammals. Further efforts should be made to clarify health hazards to PCBs in these protected species.

RP152 (MAR-1117-867658) Characterization of fluorotelomer alcohols as estrogen-like compounds by a combination of in vitro bio-assays.
Start time: 8:00 AM
Maras, M.¹, Vanparys, C.¹, Van de Vijver, K.¹, Berger, U.², Barber, J.³, Blust, R.¹, De Coen, W.¹, ¹ Laboratory for Ecophysiology, Biochemistry and Toxicology, Antwerp, Belgium² Norwegian Institute for Air Research, The Polar Environmental Centre, Tromso, Norway³ Environmental Science Department, Lancaster University, Lancaster, United Kingdom
During past years, major research efforts in environmental health sciences have been devoted to the development of easy to perform and reliable in vitro bio-assays. In vitro cell based assays have already shown their usefulness for studying, for instance, endocrine disrupting chemicals. This project started with the well known E-screen assay, in which chemicals are tested for their capacity to re-induce the proliferation of growth arrested breast cancer cells. Besides well known xeno-estrogen 4-nonylphenol, diverse perfluorinated compounds, such as perfluorosulfonate (PFOS), perfluorononanoic acid (PFNA), perfluoroctanoic acid (PFOA), and the fluorotelomer alcohols 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH) were analysed. Both fluorotelomer alcohols were able to re-induce MCF-7 cell proliferation. In order to confirm estrogen-like properties, two additional assays were performed. Cell cycle analysis by flow cytometry reveals whether exposures to chemicals during 24 hours leads to increased percentages of cells in the S(ynthesis) phase of the cell cycle. While an increased percentage is regarded as a measure of estrogenicity, this assay again confirmed the estrogen-like properties of the tested fluorotelomer alcohols. Finally, we analyzed gene expression of well known estradiol-responsive genes in the MCF-7 breast cancer cells. While the expression levels of genes such as TFF1 (Trefoil factor 1) or PGR (Progesterone receptor) were unchanged upon exposures to PFOS, PFNA or PFNA, significant inductions were observed by fluorotelomer alcohols. We also tested all perfluorinated compounds in combination with faslodex, which is a well known anti-estrogenic compound that interferes with binding to the estrogen receptor. Latter assay showed that faslodex was able to undo the estrogenic effects of 6:2 FTOH and 8:2 FTOH, from which can be concluded that the estrogen receptor is involved. In conclusion, fluorotelomer alcohols were characterized as estrogen-like chemicals in vitro, by a combination of the above described in vitro bio-assays.

RP153 (FLY-1117-816686) Comparative sensitivity of androgenic effects in medaka (Oryzias latipes) following exposure to 17-trenbolone.
Start time: 8:00 AM
Flynn, K¹, Lothenbach, D¹, Hammermeister, D¹, Haselman, J¹, Sheedy, B¹, Whiteman, F¹, Johnson, R¹, ¹ US EPA, Mid-Continent Ecology Division, Duluth, MN, USA
In response to the Food Quality Protection Act, EPA is developing protocols to screen chemicals for their potential to disrupt endocrine activity in animals. Similar efforts are ongoing with the member countries of the Organization for Economic Development and Cooperation (OECD). Vitellogenin (Vtg) induction has been shown in fish to be a reliable biomarker for exposure to estrogenic chemicals. Similar biomarkers for exposure to androgens are not as well developed. Furthermore, androgen exposure to developing fish can induce hermaphrodism or even sex reversal. Detection of sex reversal can be difficult without knowledge of the genotypic sex of individual animals. Identifying the sex determining gene, DMY, in individual medaka simplifies this analysis. To obtain information about the relative sensitivities of several phenotypic response in medaka following exposure to the androgen 17-trenbolone, and to compare these responses to the genotypic sex of individuals, medaka were exposed in a flow-through exposure systems to the waterborne chemical at one of 5 concentrations in ng/L; 4, 14, 45, 150, 500, and controls. Fish were exposed for 21-days in each treatment, which consisted of 6 replicates and 10 fish per replicate. After exposure the fish were sacrificed to assess the following endpoints; weight and length, anal fin papillary processes, Vtg protein, gonadal sex, genotypic sex (DMY), Vtg mRNA, and choriogenin (Chg) mRNA. The observed LOEC was 150 ng/L for increased weight, and lowered liver Vtg in females. The LOEC for papillary processes on the anal fin of females was 45 ng/l.

RP154 (RIC-1117-821084) The effects of acute exposure to Indirubin-3-monoxime on lymphoid CYP1A expression and select immune functions in channel catfish.
Start time: 8:00 AM
Babcock, A¹, Rice, C¹, ¹ CIET/Biological Sciences, Clemson University, Clemson, SC, USA
Indirubin (IR) is an indole metabolite of tryptophan and is the active anti-neoplastic ingredient in extracts from the Chinese herb Polygonum tinctgorium. At least in mammalian systems, indirubin binds to the cytosolic aryl hydrocarbon receptor (AhR) with high affinity and activates the translocation of the ligand-receptor complex to the nucleus, leading to gene activation. Several studies suggest that tryptophan and its metabolites may be the endogenous ligand for the AhR. Moreover, it is now known that tryptophan levels have dramatic effects on antigen-specific immunity in vertebrates. However, immunotoxicologists have established that virtually any xenobiotic that binds to the AhR with high affinity (e.g., TCDD and co-planar PCBs) modulate immune functions in a variety of animal models. We exposed channel catfish to 3 mM/kg IR, PCB-126, or carrier control via a single i.p. injection and three days later examined lymphoid CYP1A induction and quantified circulating lysozyme, total IgM, C-reactive protein levels. Compared to fish receiving carrier control, only PCB-126 treatment induced lymphoid CYP1A, while both IR and PCB-126 suppressed circulating lysozyme and IgM. C-reactive protein was not altered by either treatment. This study suggests that indirubin may be a potent immunotoxic compound and that channel catfish are useful models for further studying its effects on immune functions in vertebrates. However, based on lymphoid CYP1A as in indicator of AhR binding and activation, it appears that IR may not be as potent as PCB-126 in fish. A critical step in further characterizing the AhR-binding and activation potential of IR in fish will be to examine hepatic CYP1A and to further evaluate dose-response relationships over longer periods of time.

RP155 (TAL-1117-831121) Reptile macrophages as indicators of the consequences of estradiol exposure on innate immune responses.
Start time: 8:00 AM
Melson, D.¹, Burnham, D.¹, Jarrell, V.¹, Talent, L.¹, ¹ Oklahoma State University, Stillwater, OK, USA
This project was part of an ongoing investigation to establish the western fence lizard (Sceloporus occidentalis) as a laboratory model for immunological studies, specifically related to macrophage function. Macrophages play crucial roles in both innate and humoral immune responses and have been studied extensively in mammalian systems. However, no complete model exists for reptile studies. Ethinylestradiol is a synthetic estrogenic hormone used in birth control pills and treatment of menopause symptoms. Estrogenic compounds are commonly found in the environment particularly downstream of water treatment facilities due to their use in birth control, menopausal treatment, as well as various industrial procedures. Protocols were developed to obtain baseline information on lizard macrophage function, followed by examining estradiol treated lizards to assay the effects of exposure to this compound. Subjects were sexually mature adult descendants of lizards collected from the San Joaquin Valley in California. Experimental groups included untreated control lizards, with low, medium and high doses of ethinylestradiol based on subject weight. Subjects were kept for approximately 2 weeks post-treatment before eliciting peritoneal macrophages for in vitro testing. Samples from within each treatment group were utilized for this study. Initial results indicate a decrease in harvested macrophage density at low levels of ethinylestradiol exposure with an increase in phagocytic activity under the same conditions.

RP156 (POL-1117-834433) Stress response and contaminants in male Lesser Scaup from the northern boreal forest.
Start time: 8:00 AM
Pollock, B¹, Machin, K², ¹ Department of Toxicology, University of Saskatchewan, Saskatoon, SK, Canada² Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada
Field work was conducted in the boreal forest along the Ingraham Trail (62N, 114W) approximately 25 km east of Yellowknife, Northwest Territories, Canada. From June 4 until June 21, 2004, 14 female and 36 male Lesser Scaup (Aythya affinis) were captured and 1.5ml of blood was collected from the jugular or brachial vein. Each duck received a unique combination of nylon nasal markers of varying colour and shape, then were released and observed to identify pair status (paired or unpaired). Of the 36 male Lesser Scaup captured, 13 were recaptured and euthanized for contaminant analysis. Blood samples were assayed for corticosterone and liver was used for selenium analysis. Selenium was measured using Inductively Coupled Plasma Spectroscopy using a hydride generator (ICP-HG). Paired individuals had higher circulating levels of corticosterone in response to handling stress than unpaired males, while paired males had lower selenium (0.74 +/- 0.16 ppm) compared to unpaired males (1.01 +/- 0.34 ppm). Some studies have shown a negative relationship between the residual corticosterone concentration and selenium. Paired males may spend less time on staging grounds on the lower Great Lakes which is thought to be the likely source of selenium exposure for lesser scaup, thus accumulating less selenium. Reduced corticosterone synthesis in the adrenals may indicate elevated selenium levels and could have an impact on the ability of ducks to respond to stressors.

RP157 (RIC-1117-820569) A monoclonal antibody for quantifying lysozyme as a biomarker of innate immunity in all fish.
Start time: 8:00 AM
Beckham, M¹, Billings, S¹, Rice, C¹, ¹ CIET/Biological Sciences, Clemson University, Clemson, SC, USA
Lysozyme destroys the cell wall of gram positive and some gram negative bacteria, and is associated with innate immunity in all vertebrates. Circulating lysozyme activity is fairly high in fish undergoing infection and is often modulated in fish following exposure to chemical contaminants. Lysozyme in fish is routinely quantified as enzymatic activity in serum or plasma (40 uL minimum) at a low pH (e.g., 5.9). However, the significance of lysozyme activity in circulation is questionable because the pH of body fluids is normally above 7, far above the optimal pH for lysozyme activity, thus rendering the enzyme inactive in circulation. More than likely, circulating lysozyme is a spill-over from phagocyte activity in the tissues and lymphoid organs, thereby serving as an indicator of heightened innate immunity. Moreover, circulating lysozyme at physiological pH may have immunological functions other than bacterial destruction. Furthermore, the amount of serum or plasma required for standard lysozyme activity (20 − 40 L) is more than routinely acquired from very small fish. We developed a monoclonal antibody (mAb M24-2) for quantifying lysozyme in mummichog, Fundulus heteroclitus, then examined its cross-reactivity with different species of teleosts. A single protein of 14-15 Kd mass was identified by the mAb in all samples using as little as 2 L material. ELISAs were used to quantify circulating lysozyme protein in mummichogs collected from the field at a US EPA superfund site in VA on the Elizabeth River and compared with reference fish. Circulating lysozyme protein correlated with enzyme activity when comparing populations of mummichogs, however protein levels were less variable than enzymatic activity. Using mAb M24-2, researchers can quickly and accurately quantify circulating lysozyme protein in small fish, which are commonly used in laboratory aquatic toxicity studies, as well as in fish collected from the field in a variety of applications.

RP158 (VIL-1117-661346) Quantitative RT-PCR assays for fathead minnow gonadotropin (FSH and LH subunits) mRNA expression: cDNA cloning and assay development.
Start time: 8:00 AM
Villeneuve, D.¹, Miracle, A.², Degitz, S.¹, Korte, J.¹, Kahl, M.¹, Jensen, K.¹, Ankley, G.¹, ¹ U.S. EPA Mid-Continent Ecology Division, Duluth, MN, USA² Pacific Northwest National Laboratory, Richland, WA, USA
Pituitary gonadotropins play an important role in the regulation of reproduction in teleost fish. In an effort to further enhance the utility of the fathead minnow (Pimephales promelas) as a model organism for endocrine disruptor research we set out to develop methods for measuring the expression of mRNAs coding for fathead minnow lutenizing hormone-like (LH) and follicle-stimulating hormone-like (FSH) -subunits. Complementary DNA (cDNA) was prepared from total RNA extracted from a pool of fathead minnow pituitary tissue. Partial cDNA sequences were isolated using a combination of degenerate and non-degenerate primers. The fathead minnow sequences obtained shared considerable nucleotide sequence identity (79-92%) with LH and FSH cDNA sequences available for closely related cyprinids (e.g. Mylopharyngodon piceus, Cyprinus carpio, Carassius auratus, and Danio rerio). Gene specific probes and primers were designed and fathead minnow LH and FSH mRNA standards were prepared from gel purified cDNA using high yield in vitro transcription. Quantitative RT-PCR protocols were developed and used to measure LH and FSH mRNA expression in pituitary samples collected from control fish as well as fish exposed to the methoxytriazine herbicide, prometon, (50 and 1250 g/L) for 21 d. LH mRNA expression ranged from 1.2 x 10⁵ to 5.3 x 10⁶ copies/ng total RNA. FSH mRNA expression ranged from 7.8 x 10⁴ to 2.3 x 10⁷ copies/ng total RNA. Exposure to prometon did not significantly affect mean FSH (p=0.205) or LH (p=0.825) mRNA expression in fathead minnow pituitary tissue (Power > 0.95). The QPCR methods developed were statistically robust and appear suitable for application in additional studies aimed at characterizing the expression of LH and FSH mRNAs over the course of fathead minnow gonad development and reproductive cycles, and following exposure to other potential endocrine disrupting compounds. The contents of this abstract do not necessarily reflect U.S. EPA policy.

RP159 (DUF-1117-760471) Are we underestimating PCB-induced immunotoxicity in contaminated aquatic sites by only using CYP1A induction to assess toxicity?
Start time: 8:00 AM
Zelikoff, J¹, Duffy, J¹, ¹ New York University School of Medicine, Tuxedo, NY, USA
Binding to, and activation of, the aryl hydrocarbon receptor (AhR) is well-defined and believed to be a pre-requisite for polychlorinated biphenyl (PCB)-induced toxicity, including immunotoxicity. In recent years studies have emerged in which noncoplanar PCB congeners have been shown to alter both innate and cell-mediated immune parameters. The objective of this study was to determine the impact of a noncoplanar (PCB 153) and coplanar (PCB 126) PCB congener on the immune response and to investigate an AhR-independent mechanism of PCB-induced immunotoxicity using a feral fish species. Bluegill sunfish, injected intraperitoneally (i.p.) with equitoxic doses of either PCB 126 (0.01 or 1.0 g /g BW) or PCB 153 (5.0 or 50.0 g /g BW), were sacrificed after 1, 3, 7, 14 or 21 d and hepatic CYP1A induction, phagocyte-mediated superoxide (O₂^.-) production and T- and B-lymphocyte proliferation were measured. Results demonstrated that PCB-induced hepatic CYP1A induction was not correlated with immune alterations. The coplanar congener PCB 126 significantly inhibited B-lymphocyte proliferation at 1 d post-injection, while hepatic CYP1A induction was observed only at 3, 7, 14 and 21 d post-injection. Moreover, in the absence of CYP1A induction, treatment of fish with 50.0 g PCB 153/g BW suppressed T- and B-lymphoproliferation for up to 7 d and increased phagocyte-mediated extracellular (O₂^.-) production at 3 d post-injection. Studies have been initiated to examine the role of the neuroimmune axis, specifically the serotonergic component, as a potential mechanism of PCB-induced immunotoxicity. Preliminary studies using parachlorophenylalanine (a specific inhibitor of the rate-limiting enzyme in serotonin [5-HT] synthesis, tryptophan hydroxylase) have demonstrated that similar to that observed for other fish species, 5-HT is important for regulating the immune response of bluegill. The lack of correlation in this study between hepatic CYP1A induction and immune dysfunction in PCB-exposed fish, questions whether field studies which utilize only CYP1A induction to assess exposure are actually underestimating both PCB exposure and potential risk. Hudson River Foundation Graduate Fellowship and USACEHR No. DAMD 17-99-9011.

RP160 (MCM-1117-809053) Reproductive assessments of fish populations collected in Canadian Areas of Concern.
Start time: 8:00 AM
McMaster, M¹, Tetreault, G¹, Boyko, C¹, Brown, S¹, Sherry, J, ¹ Environment Canada, Burlington, Ontario, Canada
Environment Canada has undertaken studies in Canadian Areas of Concern (AOCs) to determine the current state of fish and wildlife health. Phase One (2001-2005) is focusing on conditions in AOCs of the lower Great Lakes. The studies presented here will focus on AOCs in Lake Ontario including Toronto Harbour and the Bay of Quinte as well as the Niagara River and Cornwall on the St. Lawrence River. Two different fish species were collected from exposed and reference sites and examined for reproductive health. As part of this study our laboratory measures circulating levels of reproductive sex steroids, the production of these steroid using an in vitro gonadal incubation assay as well as gonadal histology for differences in gonadal development due to exposure. These reproductive endpoints are compared to other indicators of overall fish health as well as other endocrine endpoints including thyroid function and circulating vitellogenin levels. Reproductive alterations have been demonstrated at other Canadian AOCs previously looked at. This study is ongoing and its overall goal is to reassess all Canadian AOCs for evidence of endocrine disruption.

RP161 (WEB-1117-736098) Effects of polychlorinated biphenyls on UDP-GT activity and thyroid function in Japanese quail.
Start time: 8:00 AM
Webb, C.¹, McNabb, F. M. A. ¹, ¹ Virginia Tech, Blacksburg, VA, USA
For several decades, chemical contaminants, including polychlorinated biphenyls (PCBs), have been associated with detrimental effects on wildlife in several regions of the Great Lakes. Included in these effects are alteration of thyroid gland function, which may lead to hypothyroidism. Growth and differentiation of many tissues are regulated by thyroid hormones. One mechanism by which PCBs cause hypothyroidism in mammals is by induction of a liver enzyme, uridine diphosphate-glucuronosyltransferase (UDP-GT), that glucuronidates thyroxine (T4). Glucuronidation of T4 increases hormone solubility in water, thereby increasing its excretion in bile. Induction of UDP-GT by PCBs in mammals has been studied extensively, but there is little research in this area on birds. We had two objectives: (1) to develop and validate an assay using labeled T4 as substrate for measuring UDP-GT activity in birds; and (2) to determine the effects of Aroclor 1254, an industrial PCB mixture, on UDP-GT activity and thyroid function in weanling mice and Japanese quail chicks. Using the T4-specific assay that we developed, we found that UDP-GT activity was linearly and significantly induced by PCBs and that plasma T4 was significantly decreased by PCBs in both quail and mice. However, plasma triiodothyronine (T3) was significantly decreased by PCBs in quail but was significantly increased in mice. In control animals, quail had lower enzyme activity than mice. Quail had less enzyme induction by PCBs than mice, and the decrease in plasma T4 also was less in quail than mice. Supported by a Virginia Tech Graduate Research Development Project grant.

RP162 (FOR-1117-639903) Capillary Electrophoresis Method for Characterization of Steroid Hormone Profiles.
Start time: 8:00 AM
Foran, C¹, Holland, L¹, ¹ West Virginia University, Morgantown, WV, USA
Endocrine disrupting chemicals with different mechanisms of action have consequences for the production, activity and excretion of steroid hormones. Several xenoestrogens are known to alter the concentration of single circulating hormones, as measured by enzyme immunosorbant assay. However, mixtures of contaminants and long-term exposures will result in complex biological interactions and many potential consequences for steroid metabolism. Therefore, we have developed a method for separation, identification, and quantification of several steroid hormones from a single 100 l sample. This method is compatible with samples of volume limited, such as plasma. Currently, we are using this protocol for matrix verification to identify estrogens, androgens and progestins in extracts of pooled serum. The first raw capillary electrophoresis method is capable of separating eight steroids in under 4 minutes. The steroids targeted for chemical profiling are estriol, 17-estradiol, estrone, testosterone, 11-ketotestosterone, androstenedione, 17,20-dihydroxyprogesterone, and progesterone. Several figures of merit for the raw capillary electrophoresis method have been determined: migration time reproducibility (0.34 % RSD n=5), linear range (conc: 25 M - 100M mass: 7.5 pg - 30 pg, R² = 0.992), and extraction efficiency of 10 l plasma sample (72%). In addition, nano-liquid chromatography and mass spectrometry have been used to profile estrogen, androgen, and progesterone derivative. This provides a means for identification of steroids that are structurally different from the 8-component sex steroid library in the capillary electrophoresis screen. Ultimately this new analytical tool will provide rapid information about changes in steroid metabolism induced in fish upon exposure to mixtures of endocrine disrupters.

RP163 (CHE-1117-716787) Effects of thyroid disruption on the expression of RC3 gene in developing Japanese quail brain.
Start time: 8:00 AM
Chen, Y.¹, McNabb, F. M. A.¹, ¹ Virginia Tech, Blacksburg, VA, USA
Thyroid hormones (THs) have critical roles in vertebrate brain development by regulating the expression of TH-responsive genes during specific developmental time windows. Hypothyroidism during these time windows results in irreversible brain defects. Our hypothesis is that hypothyroidism, from exposure to endocrine disrupting chemicals, will alter the expression of TH-responsive genes in the brain of developing birds. To test this hypothesis, we are studying the expression of TH-responsive genes in the brain of developing Japanese quail exposed to thyroid disrupting chemicals (perchlorate and PCBs). We selected the RC3 gene, which codes for a neuron-specific, calmodulin-binding protein involved in regulating neuronal Ca2+ dynamics, as the first gene to study. A sharp increase in RC3 gene expression coincides with the onset of synaptogenesis during embryonic development in mammals. Hypothyroidism decreases RC3 mRNA level in both developing and adult mammals and leads to central nervous system disorders. We have isolated and sequenced RC3 cDNA from total RNA of embryonic and adult Japanese quail brain. The RC3 mRNA levels in both normal and hypothyroid brains at different developmental stages are being analyzed by Northern blot. Plasma THs and thyroid gland TH content will be measured in the same embryos and chicks and correlated with the RC3 mRNA levels. Supported by grants from Sigma Xi and the Grad. Res. Developm. Project.

RP164 (DEW-1117-637041) The developmental immunotoxicity of dibutyltin dichloride in Sprague-Dawley rats.
Start time: 8:00 AM
DeWitt, J¹, Copeland, C², Luebke, R², ¹ Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC, USA² Immunotoxicology Branch, Experimental Toxicology Division, NHEERL, US EPA, Research Triangle Park, NC, USA
Methyl- and butyltin compounds used as stabilizers in polyvinyl chloride (PVC) pipe production are of concern as they leach from supply pipes into drinking water and have been associated with multisystem toxicity. This study assessed immune function in Sprague-Dawley (CD) rats developmentally exposed to the stabilizer dibutyltin dichloride (DBTC). Individually housed pregnant CD dams were given drinking water containing 0, 10 or 25 mg DBTC/L in 0.5% Alkamuls from gestational day six to post-natal day 21 (PND21, weaning). Offspring were exposed via gestation and lactation (indirect group) or via gestation and lactation plus oral gavage with 0, 1.0, or 2.5 mg DBTC/kg body weight/dose for a total of ten doses given three times/week beginning at PND3 (direct group). Water consumption and body weights (BW) were recorded bi-weekly in dams and three times weekly in offspring. Water consumption was statistically decreased in dams consuming 25 mg DBTC/L, although BW did not vary by dose. Delayed-type hypersensitivity (DTH), primary and secondary antibody responses to sheep red blood cells, and natural killer (NK) cell activity were evaluated in separate groups of immunologically mature treated and control offspring. Direct DBTC exposure decreased BW gain in both sexes from the 25 mg/L group. DTH response and NK cell activity did not vary by dose for any group. IgM was suppressed in females from the direct group and IgG was elevated in males from the indirect group. However, as these effects occurred only within the high dose of 25 mg DBTC/L, which is a concentration a million times higher than levels of DBTC reported in drinking water, our data suggest that DBTC is unlikely to be associated with developmental immunotoxicity at concentrations found in drinking water supplies. This abstract does not reflect EPA policy and was supported by UNC/EPA Cooperative Training Agreement CT829472.