Citation:

Knoebl, I, J. L. Blum, AND N Denslow. THE USE OF GENE ARRAYS TO DETERMINE TEMPORAL GENE INDUCTION IN SHEEPSHEAD MINNOWS EXPOSED TO E2. 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:

Gene arrays provide a means to study differential gene expression in fish exposed to environmental estrogens by providing a "snapshot" of the genes expressed at a given time. Such array data may also uncover previously unknown biochemical pathways affected by estrogenic compounds. To study the temporal induction of certain estrogen responsive genes, we have expanded an existing cDNA gene array {Larkin, 2002 #1342; Larkin, 2003 #1340} developed for the sheepshead minnow (SHM) (Cyprinodon variegates) to include over 200 additional genes that may be differentially regulated by EDCs. These genes were obtained from SHM liver cDNA libraries, suppressive subtractive hybridization libraries of liver mRNA from SHM exposed to known EDCs (methoxychlor, nonylphenol) or from differential display analyses. The gene arrays were used to examine temporal gene induction in livers of adult male SHM exposed to 100 ng/L and 500 ng/L 17 -estradiol for 6, 12, 24 or 48 hr. The estrogen receptor alpha gene was induced beginning at 6 hr, but the level of induction did not increase with longer exposure times. The estrogen responsive genes (vitellogenins [VTG 1, VTG2] and zona radiata [ZRP2, ZRP3]) were upregulated beginning 12 hr after exposure with the induction level increasing as exposure time increased. VTG1 was expressed at a 10-fold higher level than VTG2 after 48 hr. Other genes that appear to be upregulated over 48 hr include ATP synthase 6, serum amyloid protein, tryptophan 2,3 dioxygenase, a secretory phospholipase precursor and several unidentified genes. Several genes appear to be downregulated including fibrinogen, cytochrome b and cytochrome c oxidase. A number of genes increase after 6 and 12 hr of exposure, but are down-regulated to levels below those of unexposed fish after 48 hr. Those genes include chitinase, a scavenger receptor, C type lectin S and several unidentified genes. Quantitative real-time PCR (TaqMan) was used to validate the array results for VTG1, VTG2, ZRP2 and ZRP3. Through the use of gene arrays, temporal patterns of altered gene expression can be determined for key genes involved in metabolic pathways, and these changes can be quantified by real-time PCR.

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