Citation:

Biales, A, C. Leutenegger, D. Riordan, V. deVlaming, J M. Lazorchak, AND D L. Lattier. COMPARISON OF QPCR METHODS FOR THE DETECTION OF VITELLOGENIN EXPRESSION IN 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:

Male fathead minnows (FHM) normally express little if any of the egg yolk precursor protein vitellogenin (Vg). However, when exposed to estrogenic compounds such as 17a-ethynylestradiol (EE2), transcriptional levels of Vg rise dramatically and result in decreased fecundity and increased mortality rates. For this reason, measuring the expression of Vg in male FHM is a biologically relevant and useful tool for assessing exposure to estrogenic compounds. Recent advances in gene expressiion analysis have led to the establishment of two PCR-based methods, Sybrgreen and Taqman, which allow real-time monitoring of gene expression levels. These real-time platforms are both rapid and sensitive; however, their relative efficacy and applicability in the context of monitoring environmental quality has not been adequately evaluated. To this end, we compare the sensitivity, reproducibility and practical application of these two methods in gene expression monitoring. The Vg gene was cloned and used as a standard curve to determine the limit of sensitivity of Vg between the two experimental platforms. We also determined the reproducibility of quantification across a range of concentrations through comparison of standard deviations of replicate reactions. The ability to monitor Vg expression in multiple fish species with little manipulation of established protocols is desirable. Therefore, we determined the applicability of each QPCR protocol (Taqman or Sybrgreen) to monitor Vg expression in additional cyprinid fish species. Finally we have compared the limit of detection in male FHMs exposed to 2.5 ng/L, 5 ng/L or 10 ng/L EE2 using two different established protocols for both the Sybrgreen and Taqman methods to illustrate the importance of method optimization and to assess cross lab variability. Our results will have important implications in the establishment of gene expression based assays for monitoring water quality.

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