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

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 nontargeted 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) 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.