M4 AM Endocrine Disruption in Fish|
Monday, 14 November 2005: 8:00 AM - 11:40 AM in Ballroom 4
28 (ARU-1117-722946) Modulation of the StAR protein and P450scc in the brain of juvenile Atlantic salmon (Salmo salar) is a novel aspect of nonylphenol toxicity.
Start time: 8:00 AM
Arukwe, A.1, 1 Department of Biology, Norwegian Univ. of Sci. & Technol. (NTNU), Trondheim, NORWAY
Gene expression patterns for key brain steroidogenic (StAR, P450scc, CYP11), xenobiotoc- and steroid-metabolizing enzymes (CYP1A1 and CYP3A) have been investigated in waterborne nonylphenol (NP; 5, 15 and 50 g/L) treated juvenile Atlantic salmon (Salmo salar), in addition to control and vehicle (ethanol control) exposed fish and sampled at different time intervals (0, 3 and 7 days) after exposure. Gene expression patterns were studied using real-time PCR and qualitatively using Northern hybridization. Treatment of juvenile salmon with noylphenol caused significant induction of StAR protein mRNA at day 7 post-exposure in the group receiving 15g NP/L. P450scc was first induced in the group treated with 5g NP/L at day 7, thereafter an apparent nonylphenol-concentration dependent decreases in P450scc mRNA were observed. Exposure to vehicle, ethanol, caused a significant induction of P450scc mRNA at day 3 post-exposure. CYP11 mRNA was significantly induced at day 3 after exposure to 5g NP/L, thereafter CYP11 mRNA levels were inhibited below control levels in the 15 and 50g NP/L groups at day 3. At day 7, significant induction of CYP11 mRNA was observed only in the group exposed to 15g NP/L. For CYP1A1 mRNA, an apparent nonylphenol concentration-dependent decreases were observed at day 7 post-exposure. CYP3A mRNA was significantly induced by all exposure concentrations at day 7. When exposed groups were compared, CYP3A transcript was significantly induced between 5 and 15g NP/L, and decreased between 15 and 50g NP/L. The ethanol control showed a significant reduction of CYP3A mRNA at day 3 post-exposure. The present study has demonstrated variations in three key steroidogenic proteins, xenobiotic and steroid metabolizing cytochrome P450 isozyme levels in the brain of nonylphenol exposed juvenile salmon. Thus representing a novel aspect of neuroendocrine effects of nonylphenol in fish that need to be studied in more detail.
29 (MAC-1117-981604) Devlopment of partial and full lifecycle reproductive bioassays in the estuarine mummichog, Fundulus heteroclitus .
Start time: 8:20 AM
MacLatchy, D1, Dube, M2, Peters, R1, Shaughnessy, K1, 1 University of New Brunswick, Saint John, NB, Canada2 University of Saskatchewan, Saskatoon, SK, Canada
We have developed, in a northern, western Atlantic saltwater minnow, the mummichog (Fundulus heteroclitus), a short-term (7-day) exposure bioassay for reproductive endocrine responses and a lifecycle (adult, embryo, larval, juvenile, adult) exposure to assess population-level effects. The bioassays have been validated by exposures to 17-ethynyl estradiol (EE2), an environmentally-relevant estrogenic EDS (endocrine disrupting substance) in fish. We have optimized whole organism (plasma steroid and vitellogenin) and tissue (gonadal steroid production) assays for this species and have developed spawning and grow-out procedures for the full lifecycle protocol. In the short-term bioassay, at high (>250 ng/L) EE2 exposures, the effect on males was depression of androgen steroidogenesis and plasma steroid levels. In females, high EE2 depressed gonadal production and circulating E2 levels; however, EE2 concentrations <100 ng/L caused increased gonadal production and plasma E2. Male and female plasma vitellogenin responded in a concentration-dependent fashion to EE2. Endocrine perturbations resulted in changes in reproductive status and progeny development using rhe full lifecycle bioassay. EE2 causes delays in time to hatch and cumulative hatch in progeny from exposed adults and larvae. At 100ng/L, survival of juvenile fish was higher, some gonadal male fish displayed female secondary sex characteristics, and sex ratios were skewed toward females. In addition, post-hatch males had decreased gonad sizes and females showed increased hepatic vitellogenin. The protocols used are specifically designed to provide mechanistic information linked back to endocrine-mediated effects. Cost-effective short-term bioassays occur in a timeframe suitable to identifying key endocrine responses, while longer-term bioassays link impairments in endocrine homeostasis to effects on reproduction and development. Research in the lab is currently focusing on further standardization of the bioassay as well as approaches to compare effects of EDSs on freshwater and marine/estuarine fish.
30 (SCO-1116-518416) Evidence for estrogenic endocrine disruption in the open sea.
Start time: 8:40 AM
Scott, A1, Hylland, K2, Katsiadaki, I1, Witthames, P3, Thain, J4, 1 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth, Dorset, UK2 NIVA, Oslo, Norway3 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Lowestoft, Suffolk, UK4 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Burnham-on-Crouch, Essex, UK
In 2002, CEFAS started to look for evidence of estrogenic endocrine disruption in the Atlantic cod (Gadus morhua), a species that lives its entire life cycle in the open sea. Collections of cod have now been made from several areas on the continental shelf. In at least three of these areas (off Iceland, the Shetland Box and the southern North Sea) male cod have been found with elevated levels of VTG. Furthermore, elevated VTG levels in males show a strong positive correlation with the size of the fish. These findings raise several questions, the main ones being: are elevated VTG levels a sign of endocrine disruption or of a natural aging process in males? is endocrine disruption a problem for the cod (perhaps being linked to the sharp decline of this species in the North Sea)? is it a problem for the consumer? The synthesis of VTG by the liver requires estrogen stimulation. Thus, if VTG in males were a natural process, we would expect to find elevated estradiol concentrations in the plasma of those males that also have elevated VTG. This we have not found (and nor have we found any of these males to be intersex). It thus seems probable that the causative agent is of external origin. We hypothesise that large cod pick up estrogenic compounds via the food chain. This hypothesis is based upon the fact that the size of fish at which VTG levels rise sharply (ca. 5 kg) is also the size at which cod change their diet from mainly nektonic (free-swimming) to benthic (bottom-living) organisms. We acknowledge the financial support of CEFAS and DEFRA.
31 (VAN-1117-858168) Toxicogenomic evaluation of salmon exposed to municipal waste water effluents discharged into the Pacific Ocean.
Start time: 9:00 AM
van Aggelen, G1, Osachoff, H1, Skirrow, R1, Bruno, J1, 1 Environment Canada, North Vancouver, BC, Canada
As a project funded under the Georgia Basin Action Plan, the Toxicology Section of Environment Canada,s Pacific Environmental Science Centre (PESC) conducted toxicological studies on a select number of pharmaceuticals and personal care products (PPCPs) and effluents from municipal waste water treatment plants that discharge into the Georgia Basin (Pacific Ocean). The purpose of these studies was to determine the potential for the effluents and PPCPs, at receiving water concentrations, to cause endocrine disruptor effects in salmonids. The PESC Chemistry section profiled sewage effluents by analyzing for: sterols, metals, PCBs, acidic drugs, musks, and phthalates. PESC used both conventional toxicological endpoints and gene array technology (genomics) to determine potential molecular level toxicity resulting from exposure to endocrine disrupting substances contained in the effluent. A rainbow trout (RBT) cDNA microarray has been developed in-house that can be used with Pacific salmonid species, such as chinook (Oncorhynchus tshawytscha) and coho (O. kisutch). The RBT microarray comprises 207 cDNA gene transcripts representing broad gene classes such as growth, immune response, metabolism, oncogenesis, transcription and reproduction. Toxicogenomic testing involved hatchery-raised underyearling Chinook salmon (O. tshawytscha) acclimated to seawater (26 %o salinity) and then exposed to whole Municipal Waste Water Effluent (MWWE; raw sewage) at receiving water concentrations. Discussion will draw together results of molecular endpoints (microarray and vitellogenin protein data) with physiological changes and chemistry data.
Start time: 9:20 AM
32 (SCH-1117-731514) In vivo Bioassay Guided Fractionation of Marine Sediment Extracts from the Southern California Bight for Estrogenic Activity.
Start time: 10:00 AM
Schlenk, D1, Sapozhnikova, Y.1, Irwin, M.1, Reddy, S.2, Brownawell, B2, Kolodziej, E.3, Sedlak, D.3, Montagne, D.4, Armstrong, J.5, Snyder, S.6, 1 University of California, Riverside, Riverside, CA, USA2 Stonybrook University, Stonybrook, NY, USA3 University of California, Berkeley, Berkeley, CA, USA4 Los Angeles County Sanitation District, Whittier, CA, USA5 Orange County Sanitation District, Fountain Valley, CA, USA6 Southern Nevada Water Authority, Las Vegas, NV, USA
The exposure and uptake of environmental estrogenic compounds have been reported in previous studies of demersal flatfish species in the central Southern California Bight (SCB). The objective of this study was to evaluate the feminizing activity of marine sediments from the SCB by using in vivo vitellogenin (Vtg) assays in male or juvenile fish. In 2003, sediments were collected near wastewater outfalls serving the counties of Los Angeles (LACSD) and Orange (OCSD), and the city of San Diego (SD). Cultured male California Halibut (CH) (Paralichthys californicus) were either directly exposed to sediments for 7 days or treated with two intraperitoneal injections of sediment extract over seven days. 17B-Estradiol (E2) equivalent values ranged from 1-90 ug/kg with LACSD > SD > OCSD. Measurable concentrations of E2 were observed in all sediment extracts and ranged from 0.16 to 0.45 ng/g. Estrone (E1) was only observed in sediments near the LACSD outfall (0.6 ng/g). Alkylphenols and alkylphenol ethoxylates were observed in all sediment samples but were highest near the OCSD outfall where concentrations of nonylphenol were 3200 ng/g. Fractionation studies of the LACSD sediment extract failed to demonstrate relationships between Vtg expression and 62 analytes including E2, which was observed in whole extract (2.9 ng/g). Oxybenzone (1.6 ng/g) was identified in bioactive fractions as well as unknown compounds of relatively high polarity. These results indicate estrogenic compounds other than classic natural and xenoestrogens may contribute to estrogenic activity of sediments from the SCB.
33 (MEN-1117-738508) Endocrine disruption in winter flounder from the New York/New Jersey Harbor Estuary.
Start time: 10:20 AM
Mena, L1, Pesa, M1, Dove, A1, McElroy, A1, 1 Stony Brook University, Stony Brook, NY, USA
The New/New Jersey Harbor Estuary receives sewage input from more than 10 million people, along with run-off from combined sewer overflows, industrial discharges and coastal landfills. These inputs have resulted in some of the most contaminated sediments in the nation. We have been studying winter flounder, Pleuronectes americanus, as a model benthic organism to assess potential endocrine disruption in this urban estuary. Non spawning adult founder from an area known to contain high levels of nonylphenol and estradiol in sediments in Jamaica Bay show subtle sex specific alterations in plasma sex steroid levels and vitellogenin (VTG) as compared to reference fish. There is also evidence of altered expression and activity of CYP1A forms in these fish. A wider survey of young-of-the-year (YOY) flounder from multiple sites within Jamaica Bay shows widespread evidence of elevated VTG, indicating increased sensitivity of younger fish. Histopathological evaluation of the gonads showed skewed sex ratios indicating that the numbers of male fish are very limited in Jamaica Bay both in adult and YOY populations as compared to other sites evaluated. Ongoing work is being done to assess the status of flounder from other areas in the harbor, and effects of contaminated sediment exposure on developing embryos. This additional information should provide us with a more complete picture of endocrine disruption on resident flatfish in a major urban estuary.
34 (VIN-1117-827482) Endocrine disruption in an estuarine fish, Gillichthys mirabilis: Linking reproductive parameters with sediment chemistry.
Start time: 10:40 AM
Vines, C1, Anderson, S1, Bennett, W1, Teh, S1, Denison, M1, Baston, D1, Hwang, H1, Brooks, A2, Cherr, G, 1 University of California, Davis, Davis, CA, USA2 University of California, Santa Barbara, Santa Barbara, CA, USA
The Pacific Estuarine Ecosystem Indicator Research Consortium (EPA, EaGLes program) has been developing a suite of biomarker responses in common, resident species of California estuaries as an integrative tool for determining overall wetland condition. As part of this project, we analyzed reproductive biomarker responses in the longjaw mudsucker, Gillichthys mirabilis, collected from or outplanted at marshes in Northern and Southern California. Gillichthys is a ubiquitous, indigenous species that utilizes burrows for reproduction, and as a result is exposed to contaminants through direct contact with sediment as well as absorption from water and food. While Gillichthys displays some vertical integration from channels to marsh plain, their limited home range provides valuable information on contaminant exposure at specific locations within marshes. Reproductive parameters measured were presence of choriogenins (egg shell proteins) in male/immature fish, gross and histological examination of gonads for abnormalities (ovotestis, tumors), and apoptosis in ovaries. A subset of fish were analyzed for tissue levels of organic contaminants, including PCBs, known endocrine disrupting compounds. Sediments collected from stations where fish were trapped or outplanted were analyzed for a variety of chemicals (PCBs, PAHs, metals, pesticides), and extracts of these sediments were utilized in a reporter gene mammalian cell bioassay specific for estrogenic activity. Fish collected from a known contaminated marsh in San Francisco Bay (Stege Marsh) exhibited increased numbers of male/immature fish with choriogenins, particularly at stations where the corresponding sediments contained high levels of PCBs and increased estrogenic potential. Fish outplanted at selected stations at Stege showed a similar trend, and also showed elevated levels of PCBs in tissues. There was also a higher incidence of ovotestes, gonadal tumors, and apoptosis at the contaminated marsh (Stege Marsh). Reproductive impairment in Gillichthys, including endocrine disruption, appears to be a useful indicator for toxicant stress on a native fish.
35 (JOH-1117-837300) Xenoestrogen exposure and altered reproductive timing in Puget Sound English sole.
Start time: 11:00 AM
Johnson, L1, Lomax, D1, Myers, M1, Swanson, P1, Felli, L1, West, J2, O'Neill, S2, 1 NOAA Fisheries - Northwest Fisheries Science Center, Seattle, WA, United States2 Washington State Department of Fish and Wildlife, Olympia, WA, United States
Vitellogenin, a yolk protein produced in the liver of oviparous animals in response to estrogens, normally occurs only in sexually mature females with developing eggs. However, males can synthesize vitellogenin when exposed to environmental estrogens, making the abnormal production of vitellogenin in male animals a useful biomarker for xenoestrogen exposure. We surveyed English sole from a number of sites in Puget Sound, Washington for evidence of xenoestrogen exposure, using vitellogenin production in males as an indicator. Significant levels of vitellogenin were found in male fish from several urban sites, with especially high numbers of fish affected in Elliott Bay in Seattle. At the Elliott Bay sites where abnormal vitellogenin production was observed in male sole, female English sole were entering vitellogenesis earlier in the season than sole from other sites, and final egg maturation and spawning appeared to be delayed. Additionally, female sole were maturing at a smaller size and younger age at Elliott Bay sites that at other reference sites in Puget Sound. In male sole, spawning was delayed at the Elliott Bay sites, but other aspects of reproductive development were unchanged. We are currently investigating the impact of environmental estrogens on the reproductive physiology of English sole by measuring steroid hormone levels and expression of StAR protein, which is involved in steroid biosynthesis.
36 (VID-1117-830276) Sexual Abnormalities in Southern Coastal California Coastal Flatfish Near Municipal Wastewater Outfalls.
Start time: 11:20 AM
Vidal , D. E.1, Bay , S. M.1, Gully , J.2, Hagstrom , R.1, 3, Irwin , M.4, Kelley, K. M.3, Montagne , D. 2, Reyes , J. 3, Schlenk , D.4, 1 Southern California Coastal Water Research Project, Westminster, CA, USA2 Los Angeles County Sanitation District, Whittier, CA, USA3 California State University at Long Beach, Long Beach, CA, USA4 University of California at Riverside, Riverside, CA, USA
Intersex is a condition that is now frequently encountered in fish living in areas near wastewater outfalls. The occurrence of intersex has been linked with exposures to endocrine-disrupting compounds present in the environment, which mimic the actions of the female sex hormone 17--oestradiol. When affected by estrogenic endocrine disruptor compounds male fish develop oocytes in the testicular tissue. The purpose of this study was to evaluate histological gonadal sections of English sole (Parophrys vetulus) and hornyhead turbot (Pleuronichthys verticalis) to determine the presence of intersex as part of an endocrine disruption survey in southern Califonia (USA) costal flatfish. Gonadal tissue was also used to determine the stage of gonad maturity and other abnormalities such as female atresia, which may be the result of endocrine disruptors in fish. Although the number of fish examined per site was not very large, testis-ova formations were found in some male English sole and hornyhead turbot near outfalls. Fish affected by intersex also showed evidence of elevated production of vitellogenin (an egg yolk precursor) and estradiol. An increase in oocyte atresia was also found in female fish of both species collected in suspected contaminated areas. These partial results warranted the planning of a more complete and rigorous study to further investigate and assess endocrine disruption in flatfish near Southern California municipal wastewaters.