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Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture--Presentation
Citation:
BIALES, A. D., D. C. BENCIC, K. A. BLOCKSOM, D. L. LATTIER, R. W. FLICK, AND J. M. LAZORCHAK. Proteomic analysis of a model fish species exposed to individual pesticides and a binary mixture--Presentation. Presented at Society of Toxicology Meeting, Washington, DC, March 05 - 11, 2011.
Impact/Purpose:
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. Ultimately, proposed research will provide environmental science and risk assessment communities with advanced molecular biological indicator methods, complete with validation studies and guidance for their application.
Description:
Pesticides are nearly ubiquitous in surface waters of the United States, where they often are found as mixtures. The molecular mechanisms underlying the toxic effects of sub-lethal exposure to pesticides as both individual and mixtures are unclear. The current work aims to identify and compare differentially expressed proteins in brains of male fathead minnows (Pimephales promelas) exposed for 72h to permethrin, terbufos, and a binary mixture of both. Twenty-four proteins were found to be differentially expressed among all treatments relative to the control using an ANOVA followed by a Dunnett's post-hoc test. One protein was found to be differentially expressed among all treatment groups and one protein was in common between the terbufos and the mixture group. Fifteen spots were successfully sequenced using LC-MS/MS sequencing. Proteins associated with the ubiquitin-proteasome system, glycolysis, the cytoskeleton and hypoxia were enriched. As a second objective, we attempted to establish protein expression signatures (PES)for individual permethrin and terbufos exposures. We were unable to generate a useable PES for terbufos; however, the permethrin PES was able to distinguish between control and permethrin-exposed individuals in an independent experiment with an accuracy of 87.5%. This PES also accurately classified permethrin exposed individuals when the exposure occurred as part of a mixture. The identification of proteins differentially expressed as a result of pesticide exposure represent a step forward in the understanding of mechanisms of toxicity of permethrin and terbufos. They also allow a comparison of molecular responses of the binary mixture to single exposures. The permethrin PES is the first step in establishing a method to determine exposures in real-world scenarios.