Citation:

Miracle, A L., G P. Toth, AND D L. Lattier. THE PATH FROM MOLECULAR INDICATORS OF EXPOSURE TO DESCRIBING DYNAMIC BIOLOGICAL SYSTEMS IN AN AQUATIC ORGANISM: MICROARRAYS AND THE FATHEAD MINNOW. Ecotoxicology 12(6):457-462, (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:

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the discovery of genes indicative of environmental exposure in the USEPAs long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Novel cDNAs and coding sequences will be used in DNA microarray analyses for pattern identification of stressor-specific, differentially up- and down-regulated genes. The methods currently used to discover genes in this organism, for which few annotated nucleic acid sequences exist, are cDNA subtraction libraries, differential display, exploiting PCR primers for known genes of other members of the family Cyprinidae and use of degenerate PCR primers designed from regions of moderate protein homology. Single or multiple genes noted as being differentially expressed in microarray analyses will then used in separate studies to measure bioavailable stressors in the laboratory and field. These analyses will be accomplished by quantitative RT-PCR (QRT-PCR). Moving from analysis of single gene exposures to the global state of the transcriptome offers possibilities that those genes identified by DNA microarray analyses might be critical components of dynamic biological systems and networks, wherein chemical stressors exert toxic effects through various modes of action. Additionally, the ability to discriminate bioavailability of stressors in complex environmental mixtures, and correlation with adverse effects downstream from these early molecular events, presents challenging new ground to be broken in the area of risk assessment.

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