Citation:

Lazorchak, J M., J. M. Hemming, J. Allen, D. B. Huggett, B. W. Brooks, D L. Lattier, M E. Smith, B E. Wiechman, T V. Reddy, AND D A. Gordon. EVALUATION OF A WASTEWATER DISCHARGE USING VITELLOGENIN GENE EXPRESSION AND PLASMA PROTEIN LEVELS IN MALE FATHEAD MINNOWS. Presented at Society of Environmental Toxicology and Chemistry, Baltimore, MD, November 11-15, 2001.

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:

Liver vitellogenin gene expression and plasma vitellogenin protein presence, indicators of exposure of fish to estrogens, were measured in male fathead minnows (Pimephales promelas) caged at two locations in a constructed wetland below a sewage treatment plant effluent outfall in Pecan Creek, TX. Control fish were held in activated charcoal-dechlorinated tap water in the laboratory at similar densities. Adult fish from the USEPA, National Exposure Research Laboratory's Aquatic Research Facility were shipped to the University of North Texas and held for a 2-day acclimation period before deployment. Ten fish were collected at 0, 1, 2, 4, 7, 14 and 21 days. At these intervals male secondary sex characteristics were scored; hepatic and gonadal somatic indices (HSI, GSI) were calculated; livers analyzed for vitellogenin gene expression; blood analyzed for vitellogenin protein; and hematocrit and fish condition factor were determined. Equilenin, estradiol, estrone and ethynylestradiol were analyzed in water collected at the two exposure locations on days 1, 7 and 21. Plasma vitellogenin protein was detected in fish at site 1 within 2 days of exposure and peaked within 7 days. Liver vitellogenin gene expression followed a similar trend. HSI at site 1 was significantly elevated by the end of the exposure period, but GSI was not simultaneously decreased. Results indicate that liver vitellogenin gene expression and plasma protein were higher in male fish caged closest to the wastewater discharge than in laboratory controls. Additionally, at site 1, the secondary sex characteristics fatpad thickness and number of breeding tubercles decreased with exposure time and were significantly decreased toward the end of the exposure period.

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