Citation:

Bencic, D C., D. C. Volz, P. Chen, D. E. Hinton, AND S. W. Kullman. XENOBIOTIC INDUCED ORGAN-SPECIFIC GENE EXPRESSION AND MACRO/MICROARRAY DEVELOPMENT IN MEDAKA (ORYZIAS LATIPES). 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:

As part of an ongoing effort to understand and address the short and long-term consequences of increasing levels of environmental contaminants, we used suppressive subtractive hydridization (SSH) to develop gene expression profiles from Japanese medaka (Oryzias latipes) exposed to various prototypical xenobiotics. SSH analysis provided an unbiased, open-ended tool for screening gene expression change sin male medaka tissues following 48 h exposure to the endocrine disrupting compound 17B-estradiol (E2), the hypolipidemic pharmaceutical ciprofibrate (CF) or the persistent organic pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The actions of these xenobiotics are typically mediated via very specific and different nuclear receptors: the estrogen receptor for E2, the peroxisome proliferator-activated receptor a for CF, and the aryl hydrocarbon receptor for TCDD. Furthermore, while each is known to strongly alter the expression of specific genes (E2, CF and TCDD strongly upregulate vitellogenin, peroxisome membrane protein 70, and cytochrome P450 1A, respectively), much less is known about organ-specific gene expression responses. We sequenced 1800 clones and subsequently identified approximately 600 differentially expressed, non-redundant genes from male medaka brain, liver, and testis following E2, CF or TCDD exposure. Of these total identified genes, less than 5% were shared among all three organs or xenobiotics, suggesting xenobiotic- and organ-specific responses at the level of gene expression. Many of the highly responsive genes and their corresponding proteins are being investigated as potentially novel markers of toxicity using approaches such as real-time RT-PCR, in situ hybridization, and immunohistochemistry. Furthermore, the results of these xenobiotic-iduced gene expression studies were used to develop a DNA macro-microarray to assess aquatic exposure and effect, with an emphasis on the linkage between gene expression and altered phenotype. The array technology will help us link molecular events and biochemical alterations following exposure and provide a mechanism to evaluate exposure and effects at both the individual and population levels.

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