Citation:

Meier, J R., L W. Chang, S E. Franson, F B. Daniel, G P. Toth, J M. Lazorchak, AND P A. Wernsing. ASSESSMENT OF GENETIC DAMAGE INDICATORS IN FISH IN LABORATORY, MESOCOSM AND WATERSHED STUDIES. Presented at Society of Environmental Toxicology and Chemistry, Salt Lake City, UT, November 18-22, 2002.

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 micronucleus (MN) and single cell gel electrophoresis (SCG) ("Comet") techniques for measuring DNA damage are being evaluated for their potential use as indicators of exposure of fish populations. Laboratory studies employed acute exposures of bluegill sunfish to five model genotoxic agents. Field mesocosm studies used caged common carp exposed to alachlor, atrazine, or benzo(a)pyrene for durations up to 60 days. The same genetic damage indicators were included in an integrated evaluation of biological, chemical, and physical stream parameters in two midwestern watersheds, one predominantly agricultural and the other urban dominated. For the watershed assessments, the target fish species selected were sunfish sp., white suckers, central stonerollers, and creek chubs. The tail length, tail moment and % tail DNA parameters were included in the SCG analysis; the percentage polychromatic erythrocytes (PCE) and the frequency of MN were used in the MN analysis. The laboratory studies indicated an optimal sampling time of 6-8 days for MN and 1 day for SCG. The mesocosm exposures to pesticides indicated significant SCG responses by day 1 and peak responses by day 4. The watershed studies showed significant variation among sites for both the comet and % PCE parameters. Multivariate statistical analyses are aimed at relating these parameters to the range of conditions (water chemistry, land cover, biotic assemblages, pesticide concentrations) observed in these streams. MN frequencies were extremely low in all species of the stream-collected fish, suggesting this may not be a sufficiently sensitive indicator for field assessments. These results demonstrate the utility of conducting studies at different scales to help in the development and interpretation of genetic damage indicators for assessing ecological exposure.

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