Exposure of Ldlr-/- Mice to a PFAS Mixture and Outcomes Related to Circulating Lipids, Bile Acid Excretion, and the Intestinal Transporter ASBT.

Background: Previous epidemiological studies have repeatedly found per- and polyfluoroalkyl substances (PFAS) exposure associated with higher circulating cholesterol, one of the greatest risk factors for development of coronary artery disease. The main route of cholesterol catabolism is through its conversion to bile acids, which circulate between the liver and ileum via enterohepatic circulation. Patients with coronary artery disease have decreased bile acid excretion, indicating that PFAS-induced impacts on enterohepatic circulation may play a critical role in cardiovascular risk.

Objectives: Using a mouse model with high levels of low-density and very low-density lipoprotein (LDL and VLDL, respectively) cholesterol and aortic lesion development similar to humans, the present study investigated mechanisms linking exposure to a PFAS mixture with increased cholesterol.

Methods: Male and female $Ldl{r}^{-/-}$ mice were fed an atherogenic diet (Clinton/Cybulsky low fat, 0.15% cholesterol) and exposed to a mixture of 5 PFAS representing legacy, replacement, and emerging subtypes (i.e., PFOA, PFOS, PFHxS, PFNA, GenX), each at a concentration of $2\mathrm{mg}/L$, for 7 wk. Blood was collected longitudinally for cholesterol measurements, and mass spectrometry was used to measure circulating and fecal bile acids. Transcriptomic analysis of ileal samples was performed via RNA sequencing.

Results: After 7 wk of PFAS exposure, average circulating PFAS levels were measured at 21.6, 20.1, 31.2, 23.5, and $1.5\mu g/\mathrm{mL}$ in PFAS-exposed females and 12.9, 9.7, 23, 14.3, and $1.7\mu g/\mathrm{mL}$ in PFAS-exposed males for PFOA, PFOS, PFHxS, PFNA, and GenX, respectively. Total circulating cholesterol levels were higher in PFAS-exposed mice after 7 wk ($352\mathrm{mg}/\text{dL}$ vs. $415\mathrm{mg}/\text{dL}$ in female mice and $392\mathrm{mg}/\text{dL}$ vs. $488\mathrm{mg}/\text{dL}$ in male mice exposed to vehicle or PFAS, respectively). Total circulating bile acid levels were higher in PFAS-exposed mice ($\mathrm{2,978}\text{\hspace{0.17em}pg}/\mu L$ vs. $\mathrm{8,496}\text{\hspace{0.17em}pg}/\mu L$ in female mice and $\mathrm{1,960}\text{\hspace{0.17em}pg}/\mu L$ vs. $\mathrm{4,452}\text{\hspace{0.17em}pg}/\mu L$ in male mice exposed to vehicle or PFAS, respectively). In addition, total fecal bile acid levels were lower in PFAS-exposed mice ($\mathrm{1,797}\text{\hspace{0.17em}ng}/\mathrm{mg}$ vs. $682\text{\hspace{0.17em}ng}/\mathrm{mg}$ in females and $\mathrm{1,622}\text{\hspace{0.17em}ng}/\mathrm{mg}$ vs. $670\text{\hspace{0.17em}ng}/\mathrm{mg}$ in males exposed to vehicle or PFAS, respectively). In the ileum, expression levels of the apical sodium-dependent bile acid transporter (ASBT) were higher in PFAS-exposed mice.

Discussion: Mice exposed to a PFAS mixture displayed higher circulating cholesterol and bile acids perhaps due to impacts on enterohepatic circulation. This study implicates PFAS-mediated effects at the site of the ileum as a possible critical mediator of increased cardiovascular risk following PFAS exposure. https://doi.org/10.1289/EHP14339.