Citation:

FLICK, R. W. AND J. M. LAZORCHAK. DEVELOPMENT OF MARKERS OF EXPOSURE TO COPPER, NONYLPHENOL AND ATRAZINE USING SUTRACTIVE HYBRIDIZATION. Presented at SETAC North America, Milwaukee, WI, November 11 - 15, 2007.

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:

Daphnia have been among the most commonly used test organisms for aquatic toxicity studies due to their sensitivity to contaminants. This sensitivity makes them excellent candidates for development as sentinels to monitor the occurrence of chemicals in the environment. The sequencing of the Daphnia pulex genome provides an opportunity to develop gene expression assays as indicators of exposure to sublethal concentrations of environmental stressors. In order to gauge the utility of such assays, we have produced subtraction libraries from Daphnia exposed to three chemicals that are commonly found in the environment: copper, nonylphenol and atrazine. For each of these three subtractions at least 480 clones from the forward subtraction and 480 clones from the reverse subtraction were selected for differential screening by reverse northern analyses. Based on the results of these analyses, 50 to 85 clones from each subtraction were identified as differentially expressed. Clones that exhibited the greatest differences in spot intensity were chosen for sequencing. The resulting sequences were compared to the Daphnia pulex genome database using the BLAST algorithm. A small number of candidate genes were identified for further analysis. We are in the process of generating quantitative real time PCR primers from these sequences, which we will use to verify differential expression in an independent set of exposed and control Daphnia. The development of a suite of these gene expression markers will enable us to use deployed Daphnia to diagnose the presence of contaminants in aquatic environments.

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