https://hdl.handle.net/10388/16267

Per- and poly-fluoroalkyl substances (PFASs) are a diverse group of over 12,000 synthetic, amphiphilic compounds known for a resistance to degradation that has led to some compounds having a widespread, global presence. Despite not being considered acutely potent compared to other environmental pollutants, PFASs can undergo extensive biomagnification and bioaccumulation in aquatic environments. This results in both humans and wildlife being chronically exposed to PFASs and resulting in some PFASs also being ubiquitous in blood and tissues, with associated toxic effects. While specific substances of concern have been phased-out or banned, other PFASs that are emerging as alternative substances are still produced and are being released into the environment. These “replacement” PFASs are emerging as potential contaminants of concern but remain under-studied and un-tested with relatively unknown toxicity profiles. Therefore, this dissertation aimed to (1) provide an assessment of two such emerging replacement PFASs, the cyclic perfluoroethylcyclohexane sulphonate (PFECHS) and the short-chain perfluorobutane sulphamide (FBSA), (2) investigate the interaction potential of PFECHS and FBSA with the legacy PFAS perfluoroctane sulphonate (PFOS), (3) compare the genetic expression of the emerging and legacy PFASs using next-generation sequencing techniques, and (4) investigate the feasibility of connecting high-throughput results of genetic expression (transcript abundance) obtained from new approach methodologies to individual and population level adverse outcomes. To complete these objectives, this dissertation employed both in vitro and in vivo techniques. To characterize the toxic potential of PFECHS and identify the molecular mechanisms of PFAS exposure, zebrafish embryos were exposed to a range of concentrations in either an acute (up to 96 hours post fertilization) or chronic (up to 21 days post-hatch) scenario before higher organizational level responses were evaluated (abnormalities, morphology, mortality, growth, and development). Targeted (RT-qPCR) and un-targeted (RNA-sequencing) transcriptomic expression was also evaluated. Transcriptomic datasets obtained from RNA-seq were used to model dose-response for genes/transcripts and calculate benchmark concentrations of toxicant-responsive genes/pathways. These benchmark concentrations were applied to obtain transcriptomic points of departure (tPoDs). Molecular responses determined from the entire transcriptome for each PFAS were also applied to adverse-outcome pathway network-based analysis to quantitatively link gene expression alterations to individual and population-level responses. Results demonstrated that while PFECHS and FBSA were less potent than PFOS under both in vitro and in vivo exposures, PFECHS demonstrated similar apical mechanisms of toxicity as PFOS and all the tested PFASs led to similar molecular dysregulation, regardless of the reduced predicted accumulation potential of the replacements. These results support work elsewhere that reductions in accumulation potential do not necessarily correlate with reductions in toxic potential. Mixtures of the replacement and legacy PFASs interacted mainly synergistically, albeit in a concentration-, ratio-, and endpoint-dependent manner. Cytotoxic synergism was observed at higher concentrations for PFOS-PFECHS and PFOS-FBSA combinations and for all ratio and concentration levels for PFECHS-FBSA combinations. Combinations investigating phospholipidosis were strictly additive. These results highlighted the need to consider both ratio and exposure concentration when predicting the effects of PFAS mixtures. Estimated tPoDs for each PFAS were protective of apical points of departure observed in this study. PFOS had the lowest tPoD identified to date within zebrafish with a value of 2.5 μg/L. These results suggest that while tPoD determination is a viable method for ranking and quantifying the toxic potentials of PFASs, they can also reveal unexpected toxic potencies for individual substances. Although the application of network-based analyses did not reveal any quantitative links to individual and population level outcomes based on the adverse outcome pathway framework, qualitative linkage of key events was in support of previously published adverse outcomes of PFAS exposure. Overall, this dissertation demonstrated that although the replacement PFASs, PFECHS and FBSA, may appear less toxically potent when using metrics from traditional high-level toxicity tests, the underlying mechanisms and effects indicate they are more similar to legacy substances like PFOS than originally predicted. Furthermore, novel methods such as RNA-sequencing can unveil unexpected toxicity of well-studied legacy PFASs like PFOS. Finally, this dissertation demonstrates the feasibility of novel, network-based analyses that can be applied to the high-throughput analysis of PFASs, which has been sorely lacking in research of complex chemical classes. These results call into question not only the current perceived safety of replacement PFASs, but also the current human and environmental health safety thresholds, which remain mainly based on single compound exposures and traditional toxicity endpoints.

https://hdl.handle.net/10388/16267