The Effects of Perfluorooctanesulfonic acid (PFOS) on Human Umbilical Vein Endothelial Cells (HUVECs) Proliferation and Gene Expression and its Implications on Fetal Development

Abstract

Polyfluoro-alkyl substances (PFAS) are widely distributed environmental contaminants linked to human toxicity and developmental delays, especially low birthweight (LBW). In this study, Human Umbilical Vein Endothelial Cells (HUVECs) were exposed to the PFAS perfluorooctanesulfonic acid (PFOS). After 48-hours, their proliferation, and differential gene expression were assessed. A small, yet significant, reduction in proliferation was seen at 50 μg/mL and 75 μg/mL. RNA sequencing showed that estrogen response and notch signaling pathways were significantly altered. This study increases our understanding of how PFAS may interfere with endothelial cell (HUVECs) functions which may have larger effects on fetal growth, development, and birthweight.

Figure 1. Effect of HUVEC Exposure to PFOS on Cell Proliferation and Gene Expression

(A) HUVECs were treated with PFOS treatments (Complete media, 0.6% DMSO, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL PFOS) along with the EdU thymine analog for 48 hours. Post treatment the Click-it EdU protocol was followed to measure the amount of fluorescence from the incorporated EdU thymine into the cell’s DNA. The Click-it Edu experiment was conducted three independent times with five wells for each treatment group. In an ANOVA (F _{5, 84} = 3.92, p = 0.003) followed by a post-hoc Tukey test (α = 0.05), PFOS had significant reduction in proliferation in the 50 μg/mL and 75 μg/mL compared to the control (complete media and DMSO). NS = not significant, * = p < 0.05, n=3. (B) Heatmap of the top 20 differentially expressed genes by adjusted p value between media control and 50 μg/mL PFOS after 48 hours of exposure. (C) Average log ₂ fold change in the top 20 differentially expressed genes (by adjusted p value) in PFOS exposed HUVECs compared to control HUVECs. Differential expression was compared using limma-voom, and lowly expressed genes were removed. Contrasts measured in limma-voom were Control versus PFOS. Contrast testing was set to a minimum log ₂ fold change of 0.58 and a p -value adjusted threshold of 0.01, significance was tested relative to a fold-change threshold. (D) GOseq pathway analysis displaying the top ten over-represented categories of differentially expressed genes in biological processes in HUVECs after 50 μg/mL of PFOS for 48 hours