Department Environmental Toxicology

Proteomics for ecotoxicology

Photo: Réne Schönenberger
Desalting

Proteins play crucial roles in maintaining and directing cellular structure, metabolism and functions, through serving as structural components, enzymes, signaling molecules, transporters, ion pumps or transcription factors, etc. Extracellularly displayed or excreted proteins are also involved in cell-cell communication and development of tissue and organ architecture. It is therefore not surprising that molecular-level actions of many chemicals involve effects on the abundance, posttranslational modifications, structure or function of certain proteins. These changes can in turn affect cellular phenotypes and propagate further to the level of tissues and organs, finally manifesting at the whole organism level.

Proteomics focuses on studying proteins and their alterations as part of natural biological variation or in response to stimuli or stressors. Compared to transcriptomics, i.e. the analysis of mRNA transcripts’ abundance, proteomics have so far been less widely employed in toxicological research. This may have been due to technological challenges, higher cost and (perceived) lower amount of information obtained in a typical proteomics experiment compared to a transcriptomics study. However, driven by the developments in the biomedical arena, proteomics technologies have been constantly improving in the recent years, thus opening up exciting avenues for a broader application in the ecotoxicological research studies as well. Therefore, the bioanalytics team at the Department of Environmental Toxicology works on adapting mass spectrometry-based bottom-up proteomics analysis pipelines for various applications in ecotoxicology.

Depending on the research question, both global and targeted proteomics experiments can be designed.

Discovery-mode global proteomics can provide a broad overview of multiple processes and functions governed by proteins, allowing identification of specific components or pathways affected by exposure to a particular stressor. In terms of mass spectrometry analysis, both data-dependent acquisition (DDA) and data-independent acquisition (DIA) can be employed, with the latter offering an improvement in the sensitivity and reproducibility of label-free quantitation.

Specific proteins of interest identified through a global proteomics analysis or based on a particular hypothesis, can be studied by targeted proteomics methods, typically based on selected reaction monitoring (SRM). For example, we are employing targeted proteomics to glutathione S-transferases (GST), an important phase II biotransformation enzyme family involved in detoxification of electrophiles, in fish early life stages and cellular models.

Both global and targeted proteomics can also be employed to study post-translational modifications of proteins. For example, phosphorylation/dephosphorylation of proteins is known to be involved in signaling cascades playing important roles in basic cell physiology and cellular response to stressors. We are currently developing a pipeline for mass spectrometry-based targeted analysis of phosphorylation dynamics within the mechanistic target of rapamycin (mTOR) pathway, suspected to be involved in the mediation of chemical effects on growth in fish. The developed workflow could then be applied to study other protein networks. Mass spectrometry-based targeted proteomics assays could thus provide a valuable tool to study phosphorylation-based molecular signaling in fish, where this mechanistic aspect has been so far largely neglected due to challenges with obtaining species-specific antibodies for (de)phosphorylated proteins of interest.

Publications

Bakker, E.; Bleiner, D.; Groh, K. (2021) Perspectives and future directions of the division of analytical sciences of the Swiss Chemical Society, Chimia, 75(5), 455-456, doi:10.2533/chimia.2021.455, Institutional Repository

Characterization of the mercapturic acid pathway, an important phase II biotransformation route, in a zebrafish embryo cell line

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high-throughput screening in toxicology and chemical hazard assessment.
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

Mass spectrometry in ecotoxicology

Risk assessment of chemical effects in the environment requires the understanding of the fate and behavior of anthropogenic chemicals in natural and technical systems, which is the focus of environmental chemistry. The exposure data obtained by environmental chemists are in turn used to evaluate the significance of toxicological effects in organisms, as studied by environmental toxicologists. Mass spectrometry-based techniques are frequently applied to monitor the exposure or investigate the effects of chemicals, particularly their mechanism of action. These techniques include, for example, targeted and non-targeted chemical analytics as well as diverse –omics methods. This chapter illustrates the application of mass spectrometry in environmental chemistry and toxicology using research projects carried out at our institute, with a particular focus on the aquatic environment.
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

Glutathione S-transferase protein expression in different life stages of zebrafish (Danio rerio)

Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations.
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

Tralopyril bioconcentration and effects on the gill proteome of the Mediterranean mussel Mytilus galloprovincialis

Antifouling (AF) systems are used worldwide as one of the most cost-effective ways of protecting submerged structures against heavy biofouling. The emergence of environmentally friendly AF biocides requires knowledge on their environmental fate and toxicity. In this study we measured the bioconcentration of the emerging AF biocide tralopyril (TP) in the Mediterranean mussel Mytilus galloprovincialis and investigated the effects of TP on the mussel gill proteome following acute (2 days) and chronic (30 days) exposure, as well as after a 10-day depuration period. The experiments were carried out with 1 μg/L TP; blank and solvent (5 × 10−5% DMSO) controls were also included. Proteomics analysis was performed by mass spectrometry-based multidimensional protein identification technology (MudPIT). Differentially expressed proteins were identified using a label-free approach based on spectral counts and G-test. Our results show that TP is rapidly accumulated by mussels at concentrations up to 362 ng/g dw (whole tissues), reaching steady-state condition within 13 days. Ten days of depuration resulted in 80% elimination of accumulated TP from the organism, suggesting that a complete elimination could be reached with longer depuration times. In total, 46 proteins were found to be regulated in the different exposure scenarios. Interestingly, not only TP but also DMSO alone significantly modulated the protein expression in mussel gills following acute and chronic exposure. Both compounds regulated proteins involved in bioenergetics, immune system, active efflux and oxidative stress, often in the opposite way. Alterations of several proteins, notably several cytoskeletal ones, were still observed after the depuration period. These may reflect either the continuing chemical effect due to incomplete elimination or an onset of recovery processes in the mussel gills. Our study shows that exposure of adult mussels to sublethal TP concentration results in the bioconcentration of this biocide in the tissues and modulates the expression of several proteins that may intervene in important metabolic pathways.
Oliveira, I. B.; Groh, K. J.; Stadnicka-Michalak, J.; Schönenberger, R.; Beiras, R.; Barroso, C. M.; Langford, K. H.; Thomas, K. V.; Suter, M. J. -F. (2016) Tralopyril bioconcentration and effects on the gill proteome of the Mediterranean mussel Mytilus galloprovincialis, Aquatic Toxicology, 177, 198-210, doi:10.1016/j.aquatox.2016.05.026, Institutional Repository

Mass spectrometric target analysis and proteomics in environmental toxicology

Mass spectrometric techniques are widely used in environmental toxicology. One major application is the quantitative determination of chemical pollutants in environmental compartments. This is increasingly linked with biological effects assessment in an approach called effect-directed analysis, which, as the term says, allows focusing on samples that cause an effect in in vitro or in vivo test systems. Identification of the chemical(s) causing an effect is done by submitting the active sample to a classical target analysis using established methods. If the causative agent is not part of the list of target analytes, scan-dependent MS/MS analyses have to be performed and active samples compared to controls. This then allows to narrow-down the elemental composition of compounds primarily found in active samples, find functional groups and substructures, and potentially identify the unknowns.
Equally important for a refined risk assessment is the determination of actual internal concentrations in organisms, which reduces uncertainties in predicting toxicity thresholds across chemicals and species.
An entirely new level in environmental toxicology has been reached with the application of novel techniques such as proteomics and metabolomics. They allow investigating the molecular response of a model organism to environmental challenge. Ideally this leads to the identification of robust biomarkers of exposure and the identification of conserved stress response pathways which can be used to extrapolate to other species and predict adverse effects of novel chemical stressors or even their mixtures.
This chapter gives an introduction into effect-directed analysis and environmental proteomics.
Groh, K. J.; Suter, M. J. -F. (2014) Mass spectrometric target analysis and proteomics in environmental toxicology, In: Banoub, J. (Eds.), Detection of chemical, biological, radiological and nuclear agents for the prevention of terrorism, 149-167, doi:10.1007/978-94-017-9238-7_10, Institutional Repository

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.
Groh, K. J.; Schönenberger, R.; Eggen, R. I. L.; Segner, H.; Suter, M. J. -F. (2013) Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics, General and Comparative Endocrinology, 193, 210-220, doi:10.1016/j.ygcen.2013.07.020, Institutional Repository

Linking proteome responses with physiological and biochemical effects in herbicide-exposed Chlamydomonas reinhardtii

Exposure to a toxicant causes proteome alterations in an organism. In ecotoxicology, analysis of these changes may allow linking them to physiological and biochemical endpoints, providing insights into subcellular exposure effects and responses and, ultimately mechanisms of action. Based on this, useful protein markers of exposure can be identified. We investigated the proteome changes induced by the herbicides paraquat, diuron, and norflurazon in the green alga Chlamydomonas reinhardtii. Shotgun proteome profiling and spectral counting quantification in combination with G-test statistics revealed significant changes in protein abundance. Functional enrichment analysis identified protein groups that responded to the exposures. Significant changes were observed for 149–254 proteins involved in a variety of metabolic pathways. While some proteins and functional protein groups responded to several tested exposure conditions, others were affected only in specific cases. Expected as well as novel candidate markers of herbicide exposure were identified, the latter including the photosystem II subunit PsbR or the VIPP1 protein. We demonstrate that the proteome response to toxicants is generally more sensitive than the physiological and biochemical endpoints, and that it can be linked to effects on these levels. Thus, proteome profiling may serve as a useful tool for ecotoxicological investigations in green algae.
Nestler, H.; Groh, K. J.; Schönenberger, R.; Eggen, R. I. L.; Suter, M. J. -F. (2012) Linking proteome responses with physiological and biochemical effects in herbicide-exposed Chlamydomonas reinhardtii, Journal of Proteomics, 75(17), 5370-5385, doi:10.1016/j.jprot.2012.06.017, Institutional Repository

Global proteomics analysis of testis and ovary in adult zebrafish (Danio rerio)

The molecular mechanisms controlling sex determination and differentiation in zebrafish (Danio rerio) are largely unknown. A genome-wide analysis may provide comprehensive insights into the processes involved. The mRNA expression in zebrafish gonads has been fairly well studied, but much less data on the corresponding protein expression are available, although the proteins are considered to be more relevant markers of gene function. Because mRNA and protein abundances rarely correlate well, mRNA profiles need to be complemented with the information on protein expression. The work presented here analyzed the proteomes of adult zebrafish gonads by a multidimensional protein identification technology, generating the to-date most populated lists of proteins expressed in mature zebrafish gonads. The acquired proteomics data partially confirmed existing transcriptomics information for several genes, including several novel transcripts. However, disagreements between mRNA and protein abundances were often observed, further stressing the necessity to assess the expression on different levels before drawing conclusions on a certain gene's expression and function. Several gene groups expressed in a sexually dimorphic way in zebrafish gonads were identified. Their potential importance for gonad development and function is discussed. The data gained in the current study provide a basis for further work on elucidating processes occurring during zebrafish development with use of high-throughput proteomics.
Groh, K. J.; Nesatyy, V. J.; Segner, H.; Eggen, R. I. L.; Suter, M. J. -F. (2011) Global proteomics analysis of testis and ovary in adult zebrafish (Danio rerio), Fish Physiology and Biochemistry, 37(3), 619-647, doi:10.1007/s10695-010-9464-x, Institutional Repository

Proteomics for the analysis of environmental stress responses in prokaryotes

Prokaryotic microorganisms constitute a large proportion of the inhabitants of aquatic and terrestrial ecosystems, greatly contributing to the maintenance of the natural nutrient cycles and the overall balance in their habitat. Having to adjust to ever-changing environmental conditions, bacteria and archaea have evolved sophisticated systems allowing them to respond and adjust to various stresses encountered, be it physical, chemical, or starvation-related. Some prokaryotes are able to tolerate extreme environments inaccessible for other organisms, such as those displaying temperature extremes, very high salinity, acidity, heavy metal contamination or other pollution, or nutrient limitation. This makes them interesting and useful research models for both investigating the mechanisms of stress response and searching for novel biomarkers of toxicity. In addition, microbes are well known for their outstanding catabolic versatility. They can, for instance, use many different electron donors and acceptors, and can drive dehalogenation, denitrification, sulfate reduction, and many other chemical reactions. Consequently, some species play an important role in bioremediation processes including detoxification and mineralization of contaminants [see Chapters 43–46, Vol. II], while others are used in a wide array of biotechnological applications (see also Chapters in Sections 6 and 7, Vol. II). All these features make the microorganisms important, interesting, and useful research models (see Sections 6 and 7, Vol. II).
Groh, K. J.; Nesatyy, V. J.; Suter, M. J. -F. (2011) Proteomics for the analysis of environmental stress responses in prokaryotes, In: de Bruijn, F. J. (Eds.), Handbook of molecular microbial ecology. Volume I: Metagenomics and complementary approaches, 605-625, doi:10.1002/9781118010518.ch66, Institutional Repository

Contact

Dr. Ksenia Groh Group Leader Tel. +41 58 765 5182 Send Mail

Team members

René Schönenberger Lab Technician Tel. +41 58 765 5105 Send Mail

Funding

Eawag