Citation:

Knoebl, I, D Villeneuve, M. Kahl, K M. Jensen, D Kuehl, AND G Ankley. AROMATASE-B (CYP 19B) EXPRESSION IN FATHEAD MINNOWS (PIMEPHALES PROMELAS) EXPOSED TO PERFLUOROOCTANE (PFOS) AND THE AROMATASE INHIBITOR FADROZOLE. 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:

Perfluorooctane sulfonate (PFOS) is a fluorinated organic contaminant that is globally distributed in both humans and wildlife. PFOS belongs to a family of perfluorinated sulfonates that are highly persistent in the environment and have been commercially produced for over 40 years. These compounds are used in paint, polishes, food packaging, fire-fighting foams and fabric treatment. Although PFOS has been measured in a variety of wildlife, information as to its toxic mode of action (MOA) is scarce. PFOS may be an inhibitor of CYP19 aromatase, the P450 enzyme that converts testosterone to estradiol in vertebrates. To evaluate this possible MOA of PFOS in fish, we have developed a quantitative real-time PCR (TaqMan) assay to measure induction or inhibition of the aromatase gene in the brains and ovaries of fathead minnows in response to exposure to test chemicals. An aromatase cDNA sequence was isolated and cloned from the brains of fathead minnows and was used to construct a standard curve and to design sequence-specific primers and a probe. The assay was optimized and used to quantitate CYP19 gene expression in fish exposed to PFOS and to a known aromatase inhibitor, the pharmaceutical fadrozole. Fish were exposed to fadrozole (50, 17.7, 5.55, 1.85 ug/L) for 7 days, and to PFOS (.03, 0.3, 0.1, 1.0 mg/L) for 21 days. We present data comparing aromatase gene expression in brains and ovaries of the exposed fish, and make comparisons to aromatase enzyme activity in those tissues.

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