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VITELLOGENIN GENE EXPRESSION IN FATHEAD MINNOWS EXPOSED TO EE2 IN A WHOLE LAKE DOSING EXPERIMENT
Citation:
Lazorchak, J M., R Flick, D L. Lattier, K. Kidd, V. Palace, M E. Smith, B E. Wiechman, T. Hodge, AND G P. Toth. VITELLOGENIN GENE EXPRESSION IN FATHEAD MINNOWS EXPOSED TO EE2 IN A WHOLE LAKE DOSING EXPERIMENT. Presented at Society of Environmental Toxicology and Chemistry, Austin, TX, November 8-13, 2003.
Impact/Purpose:
The indeterminate condition of exposure indicator research stands to change markedly with the ability to connect molecular biological technologies with cellular or tissue effects and outcomes. Three focal areas of ecological research aim to develop a sequence of approaches where "the earliest recognizable signatures of exposure" (i.e., unique patterns of up- and down-regulated genes and proteins) are identified for numerous stressors, demonstrable in case studies and incorporated into Agency, State and Regional studies supported by EMAP and other programs.
Area 1, Computational Toxicology Research: Exposure assessment has historically been based on use of chemical analysis data to generate exposure models. While biological activity of chemicals has been recognized to be important for exposure risk assessments, measurement of such activity has been limited to whole organism toxicity tests. Use of molecular approaches will:
improve extrapolation between components of source-to-outcome continuum (source , exposure , dose , effect , outcome)
Using a systems modeling approach, gene and protein expression data, in small fish models (fathead minnow and zebrafish), will be integrated with metabolomic and histopathological data. This will assist in prediction of environmental transformation and chemical effects based on structural characteristics, and enhance quantitative risk assessments, including areas of uncertainty such as a basis for extrapolation of effects of endocrine disrupting chemicals, interspecies extrapolation, complex chemical mixtures and dose-response assessment.
Area 2, Ecological Research-Environmental Diagnostics: Development of molecular diagnostic indicators contributes to several of the GPRA Diagnostic Research Goals. Methods will employ DNA microarray technology and expression proteomics, focusing on species of relevance to aquatic ecosystem risk assessment. Significantly, these diagnostic indicators will open the door to understanding subcellular interactions resulting from exposure to complex chemical mixtures.
define relationship between genetic disposition of populations and degree/specificity of stressor-specific gene transcriptional response in aquatic organisms (fish and invertebrates)
identify of chemical mixture induced transcriptional "patterns" using microarrays and hyperspectral scanning - via collaboration with DOE Sandia National Labs
apply molecular indicators to watershed level stressor study, including pilot studies with targeted pesticides and toxins indicators
develop molecular indicators of exposure for invertebrates (Daphnia, Lumbriculus, Chironomus)
Area 3, Exposure Research in Endocrine Disruptors:
Subobjective 1: Develop exposure methods, measurement protocols, and models for assessment of risk management practices of endocrine disrupting compounds. As risk management approaches are identified and developed, there will be a need to identify, adapt and develop bioassay screening tools and other analytical methods to assess their efficacy. Measurements research will be performed to define management needs. This effort will entail cross-lab participation from NRMRL, NERL and NHEERL.
Subobjective 2: Determine extent of environmental and human exposures to EDCs, characterize sources and factors influencing these exposures, develop and evaluate risk management strategies to reduce exposures. In order to develop effective risk management strategies, it is important to understand the extent of exposures to endocrine disrupting compounds and factors influencing source-to-exposure-to-dose relationships.
apply molecular indicators of exposure to estrogenic compounds in selected wastewater treatment plants located in ten USEPA Regions
identify differential gene expression following exposure of fathead minnows to environmental androgens and androgen-like compounds
apply molecular indicators of exposu
Description:
A whole-lake endocrine disruption experiment was conducted by Fisheries and Oceans Canada at the Experimental Lakes Area (ELA), northwestern Ontario in 2001 and 2002. This experiment examined population, organism, biochemical and cellular-level effects in lake trout, white sucker, fathead minnow, and pearl dace exposed to environmentally-relevant (4-6 ng/L) concentrations of the synthetic estrogen, 17a ethynylestradiol. The USEPA collaborated in this study by evaluating vitellogenin gene expression (Vtg) in male fathead minnow livers: 1) from indigenous fish collected from the dosed and control lakes four times, once before dosing and up to three times after dosing; 2) in 2001 from indigenous minnows collected from the control lake and deployed for 1, 3, 7 and 13 days in the dosed and control lakes; and 3) in 2001 and 2002 from Cincinnati cultured minnows exposed to water collected from both ELA lakes and shipped to Cincinnati. RT-PCR methods were used for measuring Vtg expression. In addition to water exposures, embryo-larval fish were exposed to control and dosed lake sediment elutriates for 5 days. Gene expression in indigenous male fatheads collected at all time intervals from the dosed lake showed a constant level of elevation. Gene expression in the 2001 deployment study was detected within 24 hours after deployment of control fish into the treated lake and stayed elevated for the entire 13-day study. Highly variable gene expression was found in fry exposed to dosed lake sediments but no significant gene expression was found in fry exposed to control lake sediments. Results indicate that RT-PCR analyses of total RNA can be used to provide a rapid and timely estimate of exposure to estrogenic substances.