You are here:
Sensitivity of the vitellogenin assay: ability to discriminate among exposure concentrations in adult fathead minnow
Citation:
FLICK, R. W. AND M. A. CRANE. Sensitivity of the vitellogenin assay: ability to discriminate among exposure concentrations in adult fathead minnow. Presented at SETAC 2011, Boston, MA, November 13 - 17, 2011.
Impact/Purpose:
Ecological investigation in the present Task have been partitioned into three focal research areas: (1) Core Computational Toxicology research, (2) Ecological research and (3) Molecular Diagnostics and endocrine disrupting compounds. Three areas of core and applied research will be consequential on development of molecular indicators diagnostic for exposure to specific xenobiotic, natural stressors, and complex mixtures thereof, in freshwater fish and invertebrates. Research is focused on Agency’s long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Although numerous molecular biological approaches are exploited, foremost methods leading to development of molecular indicators are assembly and manufacture of DNA microarrays containing transcriptionally relevant gene sequences of fathead minnow, and the detection of novel or differentially expressed proteins by means of 2-D polyacrylamide gel electrophoresis followed by mass spectrometry. An extensive effort in 'gene discovery' research with the fathead minnow has been the primary emphasis of respective research areas, since high-throughput genome sequencing efforts-such as those in human and mouse-have not been directed toward organisms used in aquatic toxicity testing such as fathead minnows. Gene discovery research is fundamental to identified research areas, and is expected to be augmented by high-throughput cDNA sequencing data arising through a collaborative effort with the Dept. of Energy, Joint Genome Institute. Also, proteins induced to differential expression by chemical exposure will be used in ‘reverse genetics’ approach wherein knowledge of protein sequence will make possible the investigation of gene function and associated mechanistic biology. Following development and validation, fathead minnow microarrays, along with critical indicators identified by expression proteomics, will enable extensive molecular profiling studies, the hypotheses of which are that unique patterns of gene expression will be detected in targeted tissues of fathead minnows exposed to individual chemical stressors. Identification of unique, differentially expressed genes will then provide the basis for stressor-specific, quantitative molecular indicators, theory and methods that can be readily transferred to investigators within USEPA Regions, Tribes and states.
Description:
Vitellogenin is often used to infer exposure of an organism to estrogenic substances. Vitellogenin gene induction and protein levels increase, up to a point, with concentration of estrogen and duration of exposure. A biomarker such as vitellogenin should exhibit sufficient sensitivity that it can discriminate among exposure concentrations to an extent greater than simple presence/absence of a contaminant. The goals of this study were to investigate the sensitivity of the vitellogenin assay and to determine if longer exposures increased sensitivity under controlled laboratory exposures. Fathead minnows were exposed to concentrations of ethinyl estradiol ranging from 0.05 to 100 ng/L for 48 hr, 96 hr and seven days in a flow-through diluter system. Vitellogenin gene expression was quantified in liver tissue and vitellogenin protein was quantified in plasma. Gene expression patterns were similar at 48 and 96 hr, but at 7 days, expression was highly variable over many concentrations and the ability of the assay to discriminate among concentrations less than at the other two time points. Shorter exposures may provide greater sensitivity than longer exposures and are likely more tractable and cheaper to perform.