Citation:

Lazorchak, J M., V. Palace, R. E. Evans, K. Wautier, R Flick, B E. Wiechman, K. Kidd, AND D L. Lattier. MOLECULAR, PROTEIN AND HISTOLOGICAL INDICATORS IN FATHEAD MINNOWS EXPOSEDF TO EE2 IN A WHOLE-LAKE DOSING EXPERIMENT. Presented at Society of Environmental Toxicology & Chemistry, Portland, OR, November 14-18, 2004.

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 from 2001 to 2003. This experiment examined population, organism, biochemical and cellular-level effects in lake trout, white sucker, fathead minnow and pearl dace exposed to environmentally-relevant concentrations of the synthetic estrogen, 17a-ethynylestradiol. The USEPA collaborated in this study by evaluating vitellogenin (Vtg) gene expression in male fathead minnow and male pearl dace livers from indigenous fish collected from the dosed and reference lakes. In addition, for the same collections of fathead minnows and pearl dace, concentrations of vitellogenin protein were determined in whole body homogenates using an indirect competitive ELISA assay and histological analyses were done on liver and gonads. Gene expression results indicate that male fathead minnows and pearl dace show induced vitellogenin gene expression throughout the dosing season and females of both fish show levels of gene expression beyond the normal breeding season. Vitellogenin protein levels were significantly elevated over background levels and over those in fish collected from control lakes. Histological sections of fathead minnow testes from Lake 260 revealed widespread fibrosis and inhibited development. In addition, EE2-exposed fish had kidney anomalies including edema within and between the kidney tubules, and hyaline deposits in the tubule cells. Liver tissues from fish from Lake 260 also had reduced glycogen stores and increased liver cell size when compared to baseline data from the same lake and reference lake fish.

Record Details: