Concentration–response concept in ecotoxicoproteomics: Effects of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome

Concentration–response experiments, based on the testing of less replicates in favour of more exposure concentrations, represent the typical design of choice applied in toxicological and ecotoxicological effect assessment studies using traditional endpoints such as lethality. However, to our knowledge this concept has not found implementation in the increasingly applied OMICS techniques studying thousands of molecular endpoints at the same time. The present study is among the first applying the concentration–response concept for an ecotoxicoproteomics study. The effects of six different concentrations in the low effect range (<LC₂₀) of the PAH phenanthrene to the proteome of the ecotoxicological vertebrate model zebrafish (Danio rerio) embryo were investigated (two replicates per concentration) after 5 days exposure. Proteomics analyses were performed on organism extracts using 2-DE DIGE. Protein abundance profiles of around 713 protein spots were studied. About one-third of the protein signals could be detected to show robust reactions correlating with stressor concentration. Within this group, 65 protein signals showed significant changes compared to controls already at 1% lethal concentration (LC₀₁). Interestingly, 28 proteins significantly reacted at very low concentrations (<LC₀₁) and showed an exposure concentration dependent regulation status. Characteristic protein spots were identified by mass spectrometry. With the results of the present study the utility and several benefits using a concentration–response approach in proteomics studies could be shown. These included (i) knowledge about and the ability to model concentration dependent dynamics of molecular endpoints, (ii) to gain information about sensitivity of the molecular response in comparison to traditional endpoints and (iii) to help selecting the most promising protein spots for further investigations such as protein identification and biomarker studies. Using this experimental design based on testing of several exposure concentrations and less replicates might provide a step forward in getting increased output from toxicoproteomics studies.