Department Environmental Toxicology

mTOR pathway and its role in mediation of chemical effects on growth in fish

 

To date, thousands of fish are being used annually to perform toxicity tests needed to inform environmental risk assessment of chemicals. To reduce the use of animals, alternative (non-animal) toxicity testing methods need to be developed.

Our approach involves working with permanent cell lines of fish origin, where chemically induced molecular changes known to affect cell growth and proliferation can be monitored and used to predict chemical effects in the whole animal. Currently, we focus on the mechanistic target of rapamycin (mTOR) pathway, because (i) it is a major cellular signalling pathway involved in the regulation of cell growth and proliferation in eukaryotes, and (ii) there are indications that the mTOR pathway could be involved in the mediation of chemical effects on growth, but these interactions have not yet been systematically explored, especially in aquatic organisms such as fish.

In the project called “Functionality of the mTOR pathway in cultured fish cells and its role in molecular mechanisms underlying chemical effecs on cell population growth”, we use in vitro cultured zebrafish (Danio rerio) PAC2 cells as a model to investigate the architecture and functionality of the mTOR pathway, as well as its susceptibility to chemicals and the concomitant effects on growth and proliferation of fish cells. Since the activity of the mTOR pathway is chiefly regulated by protein phosphorylation, a major hurdle precluding a comprehensive investigation of mTOR pathway activity in fish so far has been the lack of fish-specific antibodies for (de)phosphorylated proteins of interest. Here, we will address this need by developing a mass spectrometry-based workflow for targeted analysis of phosphorylation dynamics for selected proteins within the mTOR pathway. The pipeline will include protein extraction and digestion, followed by enrichment of phosphopeptides and mass spectrometric assays to quantify phosphorylated and dephosphorylated protein forms using heavy-labeled peptide counterparts.

The established workflow will provide a valuable tool to study phosphorylation-based molecular signaling within the mTOR pathway in fish cells and, by inference, in fish in general. This could represent a significant methodological advancement since the respective antibodies for non-mammalian proteins are not always available.

Phosphoprotein dynamics will be measured upon intentional modulation of mTOR pathway (i.e., using specific pharmacological inhibitors or activators of this pathway), as well as in response to chemical exposures, and correlated to growth-related outcomes and other relevant physiological parameters in fish cells. A better understanding of the mTOR-mediated signalling and its role in growth regulation in fish cells, as well as its potential disruption by chemicals, could provide an avenue to develop a new non-animal toxicity test for predicting chemical effects on fish growth without using the actual fish

Publications

Groh, K. J.; Suter, M. F. -J. (2020) Mass spectrometry in ecotoxicology, In: Sidona, G.; Banoub, J. H.; Di Gioia, M. L. (Eds.), Toxic chemical and biological agents. Detection, diagnosis and health concerns, 93-108, doi:10.1007/978-94-024-2041-8_6, Institutional Repository
Tierbach, A.; Groh, K. J.; Schoenenberger, R.; Schirmer, K.; Suter, M. J. -F. (2020) Characterization of the mercapturic acid pathway, an important phase II biotransformation route, in a zebrafish embryo cell line, Chemical Research in Toxicology, 33(11), 2863-2871, doi:10.1021/acs.chemrestox.0c00315, Institutional Repository
Tierbach, A.; Groh, K. J.; Schönenberger, R.; Schirmer, K.; Suter, M. J. -F. (2018) Glutathione S-transferase protein expression in different life stages of zebrafish (Danio rerio), Toxicological Sciences, 162(2), 702-712, doi:10.1093/toxsci/kfx293, Institutional Repository
Groh, K. J.; Carvalho, R. N.; Chipman, J. K.; Denslow, N. D.; Halder, M.; Murphy, C. A.; Roelofs, D.; Rolaki, A.; Schirmer, K.; Watanabe, K. H. (2015) Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: II. a focus on growth impairment in fish, Chemosphere, 120, 778-792, doi:10.1016/j.chemosphere.2014.10.006, Institutional Repository
Groh, K. J.; Suter, M. J. -F. (2015) Stressor-induced proteome alterations in zebrafish: a meta-analysis of response patterns, Aquatic Toxicology, 159, 1-12, doi:10.1016/j.aquatox.2014.11.013, Institutional Repository

Contact

Dr. Nikolai Huwa Postdoctoral Scientist Tel. +41 58 765 5653 Send Mail

Team Members

Dr. Ksenia Groh Group Leader Tel. +41 58 765 5182 Send Mail
René Schönenberger Lab Technician Tel. +41 58 765 5105 Send Mail

Funding

Eawag Discretionary Funds