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DEVELOPMENT OF DNA MICROARRAYS FOR ECOLOGICAL EXPOSURE ASSESSMENT
Citation:
Miracle, A L. AND D L. Lattier. DEVELOPMENT OF DNA MICROARRAYS FOR ECOLOGICAL EXPOSURE ASSESSMENT. Presented at Science Forum 2003, Washington, DC, May 5-7, 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:
EPA/ORD is moving forward with a computational toxicology initiative in FY 04 which aims to integrate genomics and computational methods to provide a mechanistic basis for prediction of exposure and effects of chemical stressors in the environment.
The goal of the present research is to develop a genomic screening tool (DNA microarrays) for identification of unique patterns of genes that are "turned on" or "turned off" by specific environmental stressors. DNA microarrays are made with hundreds to ten thousands of gene sequences arrayed in a manner conducive for rapid screening of a large number of genes at one time. Until recently, microarray production was limited to organisms in which sequence information was known, such as human or mouse. Through a variety of modern molecular biological techniques, hundreds of genes from a standard aquatic organism used extensively in toxicity testing (e.g., the fathead minnow, Pimephales promelas) are now known. The ongoing effort to build a gene sequence database for the fathead minnow will soon result in the ability to assess changes in gene expression as a result of an aquatic environmental exposure. More importantly, the examination of patterns of gene expression changes resulting from environmental mixtures (multiple stressors) can be rapidly assessed using this type of molecular platform, and compared to single stressor patterns. Signatures of gene expression for specific stressors will then be used to develop specific, diagnostic exposure indicators, which is a vital component in the development of non-invasive computational models for toxicological risk assessment. In partnership with other ORD labs, a better understanding of the pathways from exposure to outcome can be achieved for building computational toxicology. This technology will ultimately reduce the uncertainty in assessing risk of stressors in the environment.