Proteomic Analysis of a Model Fish Species Exposed to Individual Pesticides and a Binary Mixture

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 72 h to permethrin (7.5 μg/L), terbufos (57.5 μg/L) and a binary mixture of both. Twenty-four proteins were found to be differentially expressed among all three treatments relative to the control using an ANOVA followed by a Dunnett's post hoc test (p ≤0.05). 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.