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EXPOSURE METHOD CONSIDERATIONS FOR MEASURING VITELLOGENIN EXPRESSION IN LARVAL AND MALE FATHEAD MINNOWS (PIMEPHALES PROMELAS)
Citation:
Reddy, T V., J M. Lazorchak, D. E. Williams, B E. Wiechman, D L. Lattier, L W. Chang, AND G P. Toth. EXPOSURE METHOD CONSIDERATIONS FOR MEASURING VITELLOGENIN EXPRESSION IN LARVAL AND MALE FATHEAD MINNOWS (PIMEPHALES PROMELAS). Presented at Society of Environmental Toxicology and Chemistry, Baltimore, MD, November 11-15, 2001.
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:
Our laboratory has developed methods for measuring the expression of the vitellogenin (Vg) gene in larval and adult male fathead minnows. During this development we found several conditions that affect background Vg levels and we observed preconditions for the expression of this gene. The first finding was that there was some basal level of Vg expression in early life stages of larval fathead minnows (4-24 h old). We hypothesize that this was due to maternal transfer. The second finding was that the males showed some level of Vg expression when housed together with females or separated from females for some period of time. We hypothesize that this was a population control mechanism due to male-female feedback interactions. To evaluate maternal transfer of Vg message in larval fathead minnows we measured Vg expression at 4, 24, 48 and 72 h post hatch. To evaluate male-female association effects we measured Vg expression in males mixed with females for up to 6 months, then separated from females for up to 4 weeks. Results of the larval tests indicated that there was detectable Vg expression at 4 and 24 h, but not in larvae that were older than 24 h post hatch. In addition, when larvae that were <24 h old and 24-48 h old were exposed to ethynylestradiol, greater Vg expression was found in the <24 hr old exposed larvae compared to 24-48 h old larvae.