Proteomic analysis (the comprehensive qualitative and quantitative characterization of protein expression/abundance in and organism) holds great promise for the study of mechanistic ecotoxicology, but implementation of this powerful approach in studies of aquatic toxicology have been limited to date. Although two-dimensional electrophoresis remains one of the most common techniques for proteome characterization, significant limitations are associated with this gel-based method. In collaboration with the Duke Proteomics Core Facility within the Institute for Genome Science and Policy, we are developing a gel- and label-free high throughput technique for proteome characterization in small fish based on nanoUHPLC with Q-TOF, data-independent MS/MS analysis.
Our initial work in this project has demonstrated the use of this method for the evaluation of differential hepatic proteomic profiles of fathead minnows (Pimephales promelus) exposed to the potent anti-estrogen fadrozole. Using our gel-free method, we have identified ~1,000 proteins within the liver of fathead minnows exposed to fadrozole, with > 60 proteins differentially regulated.
We are applying the same method to evaluate the biological effects of contaminant mixtures on fathead minnows exposed to Kiawah Island, SC stormwater. We expect that the complex mixture of endocrine disruptors, wastewater contaminants, pesticides, and turf-management chemicals in these stormwaters will perturb numerous biological pathways within the fish. The proteomic data being generated is used in biological pathway analysis to elucidate mechanisms of response following exposure.
Transcriptomic, proteomic, and metabolomic methods are beginning to be applied in the study of mechanistic ecotoxicology; however, seldom have these techniques been employed to examine the biological response of an organism to environmental stressors in a comprehensive manner, simultaneously. As a complement to the detailed proteomic studies described above, we are developing and implementing new methods suitable for mulitple 'omic' analyses on the same tissue in support of systems biology studies linking multiple levels of biological organization and identifying molecular initiating events that result in adverse outcome pathways. We are testing this approach by assessing endocrine disrupting effects in fathead minnows exposed to single EDC stressors (e.g. fadrozole) or complex mixtures of contaminants (e.g. Kiawah Island stormwater ponds) using transcriptomic, proteomic, and metabolomic analyses on the same hepatic tissue. This systems biology approach not only provides an integrated understanding of toxicity but also will provide a pivotal platform for predictive toxicology.