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DEVELOPMENT OF PROTEIN PROFILE TECHNOLOGY TO EVALUATE ECOLOGICAL EFFECTS OF ENVIRONMENTAL CHEMICALS USING A SMALL FISH MODEL
Impact/Purpose:
The research objectives are to: 1) Develop protein profiles in aquatic species which are indicative of the adverse effects of specific classes of chemicals; 2) Apply such biomarkers to the development of a model screening method for detection of EDC effects in wildlife and other animals; and 3) Using these biomarkers, investigate and develop screening methods to extrapolate sub-lethal effects from surrogate species to other species including endangered species.
Description:
The rationale for this research is: i) Protein expression changes with life stage, disease, tissue type and environmental stressors; ii) Technology allows rapid analysis of large numbers of proteins to provide protein expression profiles; iii) Protein profiles are used as specific biomarkers of effects of bioactive compounds; and iv) This technology should provide biomarkers of the effects of a variety of pesticides and toxic chemicals in the environment and provide an understanding of their mode of action. This research is designed to identify endocrine-mediated effects using rapid high through-put protein fingerprinting techniques to provide EPA with predictive tools for chemical screening and prioritization and enhanced interpretation of exposure, hazard identification and dose-response information. Current endocrine testing methods are animal intensive and lack the rapid throughput necessary to screen the large number (>80,000) of chemicals required under FQPA and SDWAA. As a part of a tiered approach new molecular techniques are being developed to assist EPA in evaluating and prioritizing chemicals for testing. Ultimately a tiered testing approach will be used to screen and assay these thousands of compounds. In-silico QSAR models will be employed to identify compounds with the greatest likelihood of disrupting endocrine systems through known, modeled endocrine pathways. The small number of compounds identified by the computer models as potential EDCs may then be subjected to in vivo screening tests, such as presented in this task, to assist in prioritization of chemicals before more costly Tier 2 definitive testing is initiated. Recently, a cost effective method has been developed to simultaneously analyze large numbers of proteins in biological samples to provide rapid tissue-specific protein expression profiles. We are currently exploring the utility of fish plasma protein profiling as a rapid and cost effective means to screen large chemical inventories for pathway specific toxicity in vertebrate species. As proof of concept, GED scientists are using a ProteinChip� Biomarker System which employs a Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometer (SELDI-TOF MS) to examine protein patterns associated with exposure to EDCs, specifically, estrogenic compounds. Plasma samples from control and treated fish are applied to ProteinChip arrays producing spectral fingerprints characteristic for a specific estrogenic pathway as determined using pattern recognition software. After establishing a definitive baseline with the native ligand, 17�-estradiol, other compounds representative of specific modes of estrogenic toxicity including receptor-mediated and non-receptor mediated pathways will be evaluated. Specific protein profiles generated for known estrogenic pathways will then be incorporated into a predictive estrogen-responsive pattern recognition model. Chemicals with unknown properties, or tissues supplied from field collected organisms can be tested and evaluated for estrogenic activity based on comparison to the model. Although the patterns generated will be specific to the species tested, the protocol should be readily transferrable to any species or chemical toxicity mechanism of interest.