ABSTRACT
Per- and polyfluoroalkyl substances (PFAS) have emerged as high priority contaminants due to their ubiquity and pervasiveness in the environment. Numerous PFAS co-occur across sources of drinking water, including areas of North Carolina (NC) with some detected concentrations above the Environmental Protection Agency's health advisory levels. While evidence demonstrates PFAS exposure induces harmful effects in the liver, the involvement of extracellular vesicles (EVs) as potential mediators of these effects has yet to be evaluated. This study set out to evaluate the hypothesis that PFAS mixtures induce dose-dependent release of EVs from liver cells, with exposures causing differential loading of microRNAs (miRNAs) and PFAS chemical signatures. To test this hypothesis, a defined PFAS mixture was prioritized utilizing data collected by the NC PFAS Testing Network. This mixture contained three substances, PFOS, PFOA, and PFHxA, selected based upon co-occurrence patterns and the inclusion of both short-chain (PFHxA) and long-chain (PFOA and PFOS) substances. HepG2 liver cells were exposed to equimolar PFAS, and secreted EVs were isolated from conditioned media and characterized for count and molecular content. Exposures induced a dose-dependent release of EVs carrying miRNAs that were differentially loaded upon exposure. These altered miRNA signatures were predicted to target mRNA pathways involved in hepatic fibrosis and cancer. Chemical concentrations of PFOS, PFOA, and PFHxA were also detected in both parent HepG2 cells and their released EVs, specifically within a 15-fold range after normalizing for protein content. This study therefore established EVs as novel biological responders and measurable endpoints for evaluating PFAS-induced toxicity.
ABSTRACT
Extracellular vesicles (EVs) represent small, membrane-enclosed particles that are derived from parent cells and are secreted into the extracellular space. Once secreted, EVs can then travel and communicate with nearby or distant cells. Due to their inherent stability and biocompatibility, these particles can effectively transfer RNAs, proteins, and chemicals/metabolites from parent cells to target cells, impacting cellular and pathological processes. EVs have been shown to respond to disease-causing agents and impact target cells. Given that disease-causing agents span environmental contaminants, pathogens, social stressors, drugs, and other agents, the translation of EV methods into public health is now a critical research gap. This paper reviews approaches to translate EVs into exposure science, toxicology, and public health applications, highlighting blood as an example due to its common use within clinical, epidemiological, and toxicological studies. Approaches are reviewed surrounding the isolation and characterization of EVs and molecular markers that can be used to inform EV cell-of-origin. Molecular cargo contained within EVs are then discussed, including an original analysis of blood EV data from Vesiclepedia. Methods to evaluate functional consequences and target tissues of EVs are also reviewed. Lastly, the expanded integration of these approaches into future public health applications is discussed, including the use of EVs as promising biomarkers of exposure, effect, and disease. IMPACT STATEMENT: Extracellular vesicles (EVs) represent small, cell-derived structures consisting of molecules that can serve as biomarkers of exposure, effect, and disease. This review lays a novel foundation for integrating EVs, a rapidly advancing molecular biological tool, into the field of public health research including epidemiological, toxicological, and clinical investigations. This article represents an important advancement in public health and exposure science as it is among the first to translate EVs into this field.
