You are here:
ECOTOXICOGENOMICS: EXPOSURE INDICATORS USING ESTS AND SUBTRACTIVE LIBRARIES FOR MULTI-LIFE STAGES OF PIMEPHALES
Citation:
Lattier, D L., R Flick, J M. Lazorchak, G P. Toth, M E. Smith, D. E. Williams, AND B E. Wiechman. ECOTOXICOGENOMICS: EXPOSURE INDICATORS USING ESTS AND SUBTRACTIVE LIBRARIES FOR MULTI-LIFE STAGES OF PIMEPHALES. 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:
Ecotoxicogenomics is research that identifies patterns of gene expression in wildlife and predicts effects of environmental stressors. We are developing a multiple stressor, multiple life stage exposure model using the fathead minnow (Pimephales promelas), initially studying four chemicals across three early life stages. We report studies of fathead minnows exposed to 17-a-ethinyl estradiol (0, 2, 5, 10 and 20 ng/L) for 24 h at the following life stages: 7 days old, 14 days and 60 days. These stages correspond to distinct developmental windows prior to notable reproductive organogenesis. Early development was targeted because of the genomic plasticity and the legions of transcriptional programs occurring during ontogeny. Following exposure, total RNA was isolated from organisms pooled by age and across doses. This RNA was used to construct forward and reverse cDNA subtraction libraries for each life stage, or to identify expressed sequence tags (ESTs) using differential display. Several differentially expressed products show identity with structurally characterized teleost sequences in GenBank. Subtractive cDNA clones will be used to construct an exposure-specific Pimephales microarray. The identical approach will be applied with a pesticide, common environmental metal, and a potent inducer of the P4501A1 gene. Following generation of these libraries, a multi-exposure microarray will be assembled. This will yield the foundation of expressed gene patterns that correlate with single or complex toxicant exposures, and the relative bioavailable concentrations of environmental stressors. This approach will also identify genes critical in early development, the disruption of which could influence reproductive outcomes, and detect differential expression patterns influenced by chronic chemical exposure, in comparison to acute onset of exposure.