Shotgun Ecotoxicoproteomics of Daphnia pulex: Biochemical Effects of the Anticancer Drug Tamoxifen

Abstract

Among pollutants released into the environment by human activities, residues of pharmaceuticals are an increasing matter of concern because of their potential impact on ecosystems. The aim of this study was to analyze differences of protein expression resulting from acute (2 days) and middle-term (7 days) exposure of aquatic microcrustacean Daphnia pulex to the anticancer drug tamoxifen. Using a liquid chromatography-mass spectrometry shotgun approach, about 4000 proteins could be identified, providing the largest proteomics data set of D. pulex published up to now. Considering both time points and tested concentrations, 189 proteins showed a significant fold change. The identity of regulated proteins suggested a decrease in translation, an increase in protein degradation and changes in carbohydrate and lipid metabolism as the major effects of the drug. Besides these impacted processes, which reflect a general stress response of the organism, some other regulated proteins play a role in Daphnia reproduction. These latter results are in accordance with our previous observations of the impact of tamoxifen on D. pulex reproduction and illustrate the potential of ecotoxicoproteomics to unravel links between xenobiotic effects at the biochemical and organismal levels. Data are available via ProteomeXchange with identifier PXD001257.

Quantitative Proteomics Reveals Remodeling of Protein Repertoire Across Life Phases of Daphnia pulex

“…Despite previous reports that quantitative proteomics in Daphnia presents significant challenges, we were able to obtain a deeper coverage of the proteins expressed across very different states of life cycle and conditions, using our standard proteomic sample preparation protocols and analysis procedures and identifying over 5000 proteins. The only previous study that was similarly successful in identifying individual D. pulex proteins utilized the label‐free MS protocol. There is a well‐established tradeoff between the depth of coverage and accuracy of quantitation when comparing label‐free and labeled MS proteomics analysis with that label free protocol allowing deeper coverage given the same running time per sample, but has a significantly higher variance among samples .…”

“…Here, we report proteome differences between parthenogenetically produced embryos, juveniles, adults, and sexually produced resting eggs that can be used as a base‐line reference for protein expression analysis across the life cycle of an organism. Previous advances in Daphnia proteomics identified difficulties in obtaining a large number of intact and identifiable proteins due to high protease activity in tissue extracts, often resulting in a relatively small number of proteins reported (often 500–1500 out of over 25 000 predicted protein coding genes; the highest number reported close to 4000).…”

“…Despite these difficulties, a variety of proteomic responses have been reported, including responses to various ecological stimuli, such as temperature, food availability, hypoxia, microgravity, and presence of predators or toxicants . Other studies focused on characterization of specific groups of proteins, such as neuropeptides and hormones .…”

“…Despite these difficulties, a variety of proteomic responses have been reported, including responses to various ecological stimuli, such as temperature, [5,6] food availability, [6] hypoxia, [7] microgravity, [8] and presence of predators [9][10][11] or toxicants. [4,[12][13][14] Other studies focused on characterization of specific groups of proteins, such as neuropeptides and hormones. [15] Yet, Daphnia proteome remains largely uncharacterized, [3] including the lack of proteomics data across different life cycle phases.…”

Proteomic Profile of Daphnia pulex using Data‐Independent Acquisition Mass Spectrometry and Ion Mobility Separation

“…Cultures of D. pulex were allowed to acclimatize to these conditions for a minimum of four generations prior to harvesting. Approximately 50 adult females were placed in algae free media for 12-14 h, filtered using ultra-soft [3] and Colbourne et al [1] C) Venn diagrams comparing proteins identified from proteomic analysis employing in-gel digestion (Gel), in-solution digestion (Soln), data-independent acquisition (MS E ), and/or MS E with ion mobility separation (HDMS E ). nylon mesh, frozen immediately in liquid nitrogen, and stored at −80°C.…”