PILOT: Center for Appalachian Research in Environmental Sciences: Transcriptional Effects of Per and Poly Fluorinated Alkyl Substances

Per and poly fluorinated alkyl substances (PFAS)1 are man made surfactant chemicals of concern to human health due to their detectable levels in most individuals, their persistent nature and ubiquitous presence in our environment. The long-term consequences of human exposure to PFAS are difficult to assess due in large part to their diverse chemical structures, regional differences in human exposures and poorly understood mechanisms of action. One possible deleterious consequence of exposure to PFAS is suggested by studies in humans and laboratory animals that associate elevated circulating levels of PFAS, in particular PFOA (perfluorooctanoic acid) and PFOS (perfluorooctanesulfonic acid), with increased circulating cholesterol levels which is an established risk factor for coronary artery disease. Understanding the mechanisms linking PFAS exposures to alterations in circulating cholesterol levels would improve our ability to determine how PFAS exposures impact human health and might inform strategies for improved clinical management of PFAS exposed individuals. Despite the importance of the question, attempts to identify the mechanisms by which PFOA and PFOS alter cholesterol homeostasis have yielded contradictory and inconclusive results. In humans, for example, serum levels of PFOA have been found to be inversely correlated with the expression of genes involved in reverse cholesterol transport (i.e., CYP7A1). However, preclinical studies with animal models indicate that PFOA primarily upregulates cholesterol biosynthesis via upregulation of nuclear hormone receptors such as PPARá. PFOA has also been found to increase liver size and expression of genes involved in proliferation [3-5]. Use of cultured hepatocytes and either metabolomics or transcriptomic approaches have either failed to generate consistent results or report results obtained using high levels of either PFOA or PFOS that are not relevant to human health. To resolve this issue, we propose to use a liver microtissue model (hLiMT, 3D human liver microtissue)[6] that retains much of the normal physiology and function of the liver to examine the impact of PFOA and PFOS on transcriptional pathways linked to cholesterol homeostasis. The objective of this project is to identify the molecular mechanism(s) by which PFOA and PFOS increase cholesterol levels. We hypothesize that exposure to PFOA and PFOS inappropriately activates key nuclear hormone receptors in the liver which subsequently results in increased synthesis of cholesterol. It is important to note that elevated cholesterol may arise from either 1) increased intake of dietary intake, 2) increased de novo cholesterol synthesis or 3) cholesterol absorption. By focusing on liver-specific events, we will begin to discriminate between these possibilities. Two aims are proposed to test this hypothesis. In aim #1, we will verify the impact of PFOS and PFOA on hepatic proliferation and cholesterol/lipid accumulation in a 3D human liver microtissue model. In aim #2, we will identify differentially expressed profiles of genes relative to cholesterol homeostasis and induced by PFOA treatment in 3D human liver microtissues. Successful completion of these aims will lead to the identification of biomarkers and vulnerable populations,assessment of the risk of exposure to PFAS and will inform therapeutic interventions.