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GENE EXPRESSION AND GENETIC DAMAGE INDICATORS IN FISH EXPOSED TO VARYING STREAM CONDITIONS IN A MIDWESTERN WATERSHED
Citation:
Chang, L W., J R. Meier, F B. Daniel, J M. Lazorchak, D L. Lattier, AND G P. Toth. GENE EXPRESSION AND GENETIC DAMAGE INDICATORS IN FISH EXPOSED TO VARYING STREAM CONDITIONS IN A MIDWESTERN WATERSHED. 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:
Newly developed molecular diagnostic exposure indicators were evaluated as part of a riparian zone assessment for environmental stressors. Water and fish samples collected from 12 headwater tributaries in the Little Miami River Basin were evaluated for the presence of estrogenic compounds and polycyclic aromatic hydrocarbons, measured by vitellogenin (Vg) and cytochrome P4501A1 (P450) gene expression, respectively; and genotoxicants, measured by single cell gel electrophoresis (SCG) and micronucleus (MN) assays. Larval fathead minnows (Pimephales promelas) were exposed to stream water and a laboratory control water for 24 hours. Total RNA was isolated and Vg and P450 gene expression were measured using designed synthetic oligonucleotide primers in quantitative RT-PCR. Fish blood samples obtained from bluegill sunfish (Lepomis macrochirus) and white suckers (Catostomus commersoni) were used for the SCG and MN analyses. For the SCG assay, the tail moment parameter was analyzed by computerized image analysis. Micronucleus frequencies were analyzed in polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE), and the PCE:NCE ratio determined. Results indicated an increase in P450 gene expressio in fathead minnow larvae exposed to water samples from all 12 streams as compared to control laboratory water, the highest being observed in Turtle Creek. A marginal increase in Vg gene expression was observed only in three streams. Neither the SCG or MN parameters from fish blood cells showed evidence of significant exposure to genotoxic contaminants in any of the streams. Relational databases for a range of field-measured stream condition paramerters--land cover metrics, nutrient levels, and in-stream habitat measures--will be discussed.