Citation:

Reddy, T V., J M. Lazorchak, M E. Smith, P E. Grimmett, D F. Bender, B E. Wiechman, AND D L. Lattier. THE INSTABILITY OF ESTROGENIC CHEMICALS DURING LABORATORY STATIC EXPOSURE CONDITIONS WITH MALE FATHEAD MINNOWS. Presented at Society of Environmental Toxicology & Chemistry, Portland, OR, November 14-18, 2004.

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:

Endocrine disrupting chemicals (EDCs) such as Para-nonylphenol (NP), estradiol (E2), estrone (E1), estriol (E3) and ethynylestradiol (EE2) are shown to be ubiquitous in surface waters, sediments and sludge. These EDCs are known to induce vitellogenin gene (Vg) expression in male fathead minnows. We have observed that the aforementioned EDCs are not stable during static laboratory conditions under high biomass to volume exposures at 25oC, with aeration at the rate of 100 bubbles minute-1 and a loading rate of 3 g fish Liter-1 instead of the standard rate of 0.5 g fish Liter-1. Male fat head minnows were exposed to NP at nominal concentrations for 24 hours and no detectable levels of vitellogenin gene (Vg) transcripts were seen. We checked the analytical values of NP in exposure water and found that measured values were only 2% of the nominal values. Additional experiments revealed that within 4 hours of exposure 98% of the NP was lost, of which 30% of nominal dose was bound to external body mucous of the fish and the remaining 68% was unaccounted. Subsequent analysis of E1, E2, E3 and EE2 under similar test conditions revealed that E1 and E3 showed losses similar to NP while, E2 and EE2 exhibited less concentration decline (20-30%). In order to maintain analytical concentrations close to the nominal concentrations, NP was supplemented continuously at appropriate concentrations by means of a peristaltic pump. Under this protocol, the final measured concentrations were close to 80% of the nominal dose and, in addition, Vg expression was observed in a dose dependant manner. These results show the importance of the exposure regimen in determining/interpreting the effects of EDCs in fish.

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