Citation:

KNOEBL, I., R. WANG, G. P. TOTH, E. F. ORLANDO, M. S. SEPULVEDA, D. H. MILLER, E. J. DURHAM, K. M. JENSEN, E. A. MAKYNEN, AND D. L. VILLENEUVE. ALTERATIONS IN THE TRANSCRIPTOME AND PROTEOME OF ZEBRAFISH (DANIO RERIO) EXPOSED TO FADROZOLE, A MODEL AROMATASE INHIBITOR. Presented at SETAC, Baltimore, MD, November 13 - 17, 2005.

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:

Fadrozole is a reversible, competitive inhibitor of aromatase activity and therefore an endocrine-disrupting compound (EDC) that disrupts steroidogenesis by inhibiting the conversion of testosterone to 172-estradiol. While fadrozole is a therapeutic drug with generally no environmental relevance, it does serve as a relatively specific aromatase inhibitor and, as such, is useful for the discovery of potential diagnostic markers. In this study, we examined the effects of fadrozole on gene and protein expression in tissues comprising the hypothalamic-pituitary-gonadal (HPG) axis in reproductively mature zebrafish (Danio rerio). Fish were exposed to two concentrations of fadrozole (25 and 100 �g/L) or control water. Transcriptome and proteome changes in brain, liver, and gonads were determined using 22,000 gene microarrays and two-dimensional gel electrophoresis followed by peptide analysis via mass spectroscopy, respectively. Brain and gonad aromatase activities were also determined. Fathead minnows (Pimephales promelas) exposed to fadrozole exhibit altered reproductive biology (e.g., decreased plasma estradiol and vitellogenin and decreased fecundity). Integrating these data and the alterations in gene and protein expression in the zebrafish documented here will help identify key genes and proteins of the HPG axis affected by fadrozole exposure, thereby providing a more definitive description of its modes/mechanisms of action (MOA). The combination of genomic and proteomic approaches along with whole organism endpoints and future metabonomic work and systems-biology modeling will identify new molecular biomarkers of exposure to fadrozole and other compounds with similar MOA and link these biomarkers to effects that are relevant for both diagnostic and predictive risk assessments.

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