The use of non-targeted proteomics and in vitro bioassays as a non-destructive approach towards the development of biomarkers of contaminant exposure in threatened marine wildlife Author Chaousis, Steph Published 2019-10-31 Thesis Type Thesis (PhD Doctorate) School School of Environment and Sc DOI https://doi.org/10.25904/1912/1259 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/389085 Griffith Research Online https://research-repository.griffith.edu.au The use of non-targeted proteomics and in vitro bioassays as a non-destructive approach towards the development of biomarkers of contaminant exposure in threatened marine wildlife Stephanie Chaousis, BSc, Honours Supervision by Dr. Jason P van de Merwe & Prof. Frederic DL Leusch School of Environment and Science Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy on the 24th of April 2019 Abstract Biomarkers of chemical exposure and effect are an important tool for monitoring the health of threatened species that are vulnerable to the adverse effects of prolonged contaminant exposure. However, there are many challenges that have limited the discovery of new biomarkers of chemical exposure in protected species, particularly the constraint on the use of destructive methods such as in vivo experimentation. This thesis first examines the current methods of biomarker discovery as reported in the literature to identify what methods future research in this field should focus on. A systematic quantitative review of methods of non-destructive biomarker discovery in wildlife highlighted the hinderance of these limitations on current research as well as the paucity of studies harnessing in vitro techniques (Chapter 2). The use of in vitro bioassays is not uncommon in ecotoxicology, however, these assays are largely targeted at one or several known markers. Non-targeted proteomics analyses allow for the discovery of new biomarkers, which is important considering wildlife may continually be exposed to new compounds and mixtures. Therefore the application of in vitro and in vivo non- targeted proteomic analysis as a tool for biomarker discovery was examined. To do so, sea turtles were used as a model species due to their priority conservation status and their susceptibility to the adverse effects of contaminant exposure. Cell lines derived from sea turtles exposed in vitro to environmentally relevant contaminants were used as a model for the in vitro analyses. Firstly, experimental sources of variation on protein expression (time, concentration and contaminant type) were explored (Chapter 3) revealing a strong effect of exposure time on observed proteomic changes and little effect from concentration. Furthermore, potential candidate biomarkers were discovered and the relevance of this method to in vivo molecular responses was demonstrated with the observation of known in vivo markers of exposure dysregulated in response to chemical exposure. Then, the influence of biological sources of variation (tissue type) i were examined with cells derived from several tissue types exposed in vitro to contaminants (Chapter 4). Different tissue types showed a different response to contaminant exposure and known in vivo biomarkers of exposure were again observed. Furthermore, potential new biomarker candidates were identified that would be beneficial for future research in this field to explore. Finally, non-targeted proteome profiling was applied to biological samples of wild-caught animals to examine its potential in biomarker discovery (Chapter 5). The proteome of the blood plasma of three Southeast Queensland sea turtle populations exposed to different chemical profiles were compared, and distinct differences in population protein expression were observed. These differences indicated altered immune states between populations, which could be caused by contaminant and/or pathogen exposure. In conclusion, this novel method of non-targeted proteomic analysis of both in vitro and in vivo samples provides a wealth of information about how contaminants affect sea turtles at the cellular and whole organism level and is a promising avenue to enhance wildlife toxicology. However, it was discovered that it is challenging to draw correlations between in vivo and in vitro global protein expression, and furthermore that sources of experimental variation can greatly influence the outcomes. Therefore, it is vital that future studies on this area focus on reproducibility of these sensitive methods before fully utilising them for directing wildlife toxicology research. ii Statement of originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. Signature __________________________________ Date: 24/04/2019 Stephanie Chaousis iii Statement of ethical clearance This work was conducted in accordance with animal ethics requirements approved by the Griffith University Animal Ethics Committee (ENV/13/15/AEC). Signature __________________________________ Date: 24/04/2019 Stephanie Chaousis iv Table of contents Abstract………………………………………………………………...………………...i Statement of originality………………………………………………………….……...iii Statement of ethical clearance…………………………………………………...……...iv List of tables…………………………………………………………………...…...…..vii List of figures…………………………………………………………………….....…..ix Acknowledgements……………….………………………………………...………….xii Acknowledgement of funding………………………………………………...…….…xiv Contribution of others to this thesis…………………………………....…………….…xv Publications during candidature………………………………….………..…….…......xv Conference presentations based on thesis………………………………..……...…....xvii Chapter 1: Introduction…………………………………………………………...……..1 1.1 Thesis objectives………………………………………….……….……..….……....7 1.2 Thesis structure…………………………………………………..…………….…....8 1.3 References……………………………………………………...……………..……10 Chapter 2: Charting a path towards non-destructive biomarkers in threatened wildlife: A systematic quantitative literature review…………………………………...……..……19 2.1 Abstract………………………………………………………………………….….20 2.2 Keywords……………………………………………………………….……….….20 2.3 Introduction……………………………………………………….…...………..….20 2.4 Methods……………………………………………………………….……………25 2.4.1 Search tools and parameters………………………………………………….…..25 2.4.2 Categorisations and exclusions…………………………………………...………26 2.4.2.1 Broad experimental categories………………………………………...……….26 2.4.2.2 Detailed experimental categories…………………………………………….…27 2.4.2.3 Classification of methods and quality of experimental design…………….…...28 2.5 Results………………………………………………………………..………...…..29 2.5.1 Search results and experimental categories…………………………………...….29 2.5.2 Classification of methods and quality of experimental design……………......….30 2.6 Discussion…………………………………………………………..…………...….32 2.6.1 Experimental categories……………………………………………..……….......32 2.6.2 Methodological categories…………………………………..…………..…….....36 2.6.2.1 Method 1: Correlation of biomarkers in destructive and non-destructive samples……………………………………………………………………………..…..36 2.6.2.2 Method 2: Correlation of multiple biomarkers in non-destructive samples only……………………………………………………………………………….….....39 2.6.2.3 Method 3: Correlation between site/treatment and detection of biomarkers in non-destructive samples……………………………………………………………......44 2.6.2.4 Method 4: Validation of methods for detection of potential non-destructive biomarkers with no contaminant correlations made…………………………..………..45 2.6.2.5 Method 5: In vitro methods using non-destructive samples……………………46 2.6.3 Future directions for non-destructive biomarker discovery in threatened wildlife………………………………………………………………………………….50 2.7 References……………………………………………………………………….…54 v Supporting information for Chapter 2...………………………..………………………66 Chapter 3: Influence of concentration and exposure duration on global protein expression of green sea turtle primary skin cells exposed to polychlorinated biphenyl (PCB) 153 and perfluorononanoic acid (PFNA)…………………………………..…...77 3.1 Abstract……………………………………………………………………………..79 3.2 Key words…………………………………………………………………………..80 3.3 Introduction……………………………………………………………………..….80 3.4 Methods………………………………………………………………………...…..84 3.4.1 Chemicals…………………………………………………………………….…..84 3.4.2 Primary cell culture………………………………………………………………85 3.4.3 Validation that exposure concentrations are not cytotoxic……………….……...85 3.4.4 24 and 48 h exposure bioassays for proteomic analysis………………………….86 3.4.5 Protein extraction…………………………………………………………………86 3.4.6 Protein quantification and digestion…………………………………………..….87 3.4.7 LC-MS/MS…………………………………………………………………….…88 3.4.8 Data analysis………………………………………………………………..….…88 3.5 Results and discussion……………………………………………..………….……91 3.5.1 Cytotoxicity results and cell morphology…………………….…………….….…91 3.5.2 Effect of concentration, exposure time and contaminant on total number of dysregulated proteins……………………………………………………………..….....93 3.5.3 Practical aspects of the methodology used for cellular bioassay proteomic analysis..………………………………………………………………………………..97 3.5.4 Effect of treatments on up and down regulation and compound-specific protein dysregulation………………………………………………………………….……..…98 3.5.5 In vitro expression of known in vivo biomarkers expressed by cells exposed to PCB153 for 24 h………………………………………………………………………101 3.6 Conclusion……………………………………………………………….....……..104 3.7 References……………………………………………………………...……...….105