Exposure to a mixture of legacy, alternative, and replacement per- and polyfluoroalkyl substances (PFAS) results in sex-dependent modulation of cholesterol metabolism and liver injury

Abstract

Background: Epidemiological studies have shown Per- and polyfluoroalkyl substances (PFAS) to be associated with diseases of dysregulated lipid and sterol homeostasis such as steatosis and cardiometabolic disorders. However, the majority of mechanistic studies rely on single chemical exposures instead of identifying mechanisms related to the toxicity of PFAS mixtures.

Objectives: The goal of the current study is to investigate mechanisms linking exposure to a PFAS mixture with alterations in lipid metabolism, including increased circulating cholesterol and bile acids.

Methods: Male and female wild-type C57BL/6J mice were fed an atherogenic diet used in previous studies of pollutant-accelerated atherosclerosis and exposed to water containing a mixture of 5 PFAS representing legacy, replacement, and alternative subtypes (i.e., PFOA, PFOS, PFNA, PFHxS, and GenX), each at a concentration of 2 mg/L, for 12 weeks. Changes at the transcriptome and metabolome level were determined by RNA-seq and high-resolution mass spectrometry, respectively.

Results: We observed increased circulating cholesterol, sterol metabolites, and bile acids due to PFAS exposure, with some sexual dimorphic effects. PFAS exposure increased hepatic injury, demonstrated by increased liver weight, hepatic inflammation, and plasma alanine aminotransferase levels. Females displayed increased lobular and portal inflammation compared to the male PFAS-exposed mice. Hepatic transcriptomics analysis revealed PFAS exposure modulated multiple metabolic pathways, including those related to sterols, bile acids, and acyl carnitines, with multiple sex-specific differences observed. Finally, we show that hepatic and circulating levels of PFOA were increased in exposed females compared to males, but this sexual dimorphism was not the same for other PFAS examined.

Discussion: Exposure of mice to a mixture of PFAS results in PFAS-mediated modulation of cholesterol levels, possibly through disruption of enterohepatic circulation.

Keywords: Bile acids; Cardiometabolic disease; Cholesterol; Hyperlipidemia; Liver injury; PFAS toxicity; Per- and polyfluoroalkyl substances.

Figure 1.. PFAS exposure induces sex-dependent liver injury

Male and female C57BL/6J mice were exposed to vehicle water or the PFAS mixture for 12 weeks: female+vehicle (n=10), female+PFAS (n=9), male+vehicle (n=10), and male+PFAS (n=10). (A) Male and female liver sections were stained for H&E. Male PFAS mouse: The hepatocytes are swollen with eosinophilic cytoplasm, indicating hepatocyte injury. Scattered acidophil bodies (hepatocyte necrosis) are also present (arrows). Female PFAS mouse: the portal triad demonstrates mild chronic inflammation with lymphocytes. The hepatocytes are swollen with eosinophilic cytoplasm, consistent with injury. In this mouse, hepatocellular and canalicular cholestasis (yellow pigment) is also present. (B) Liver weight given as a percentage of total body weight. (C) Liver sections were analyzed and quantified for number of inflammatory foci or eosinophilic cells per field of view. (D) Percentage of hepatocytes demonstrating eosinophilic cytoplasm (E) Effects of PFAS exposure on hepatic gene expression of liver injury- and toxicant-associated fatty liver disease-related markers. (F) Effects of PFAS exposure on hepatic gene expression of oxidative stress-related markers. (G) Effects of PFAS on 5-Hete metabolite levels. Bars represent mean ± S.E.M. of ten mice (RNA sequencing) or seven mice (Metabolomics Scaled Counts) in each group. Statistical significance for all (p<0.05) was determined by two-way ANOVA analysis and post-hoc comparisons by Holm-sidak method.

Figure 2.. PFAS exposure modulates genes related to metabolism of bile acids and fatty acids

Hepatic mRNA was isolated and analyzed via RNA sequencing. Mice in each treatment group: female+vehicle (n=10), female+PFAS (n=9), male+vehicle (n=10), and male+PFAS (n=10). (A) Heatmap of PFAS-induced effects and sex-related effects on hepatic gene expression pattern for bile acid metabolism-related genes. (B) Heatmap of PFAS and sex-related effects on hepatic gene expression pattern for fatty acid metabolism-related genes. Red = indicates scaled raw counts. Yellow/Blue = indicates fold-change (FC) of the interaction between PFAS exposure and sex. #Statistically significant interaction (p<0.05) between PFAS exposure and sex was determined by Two-Way ANOVA. *Statistically significant effect from PFAS exposure.

Figure 3.. Effects of PFAS exposure and sex on the enterohepatic circulation

This schematic depicts pathways related to cholesterol and bile acid metabolism and transport. Perturbations in these pathways due to PFAS exposure, or the interaction of PFAS exposure and gender, are indicated by color. Red: Significant PFAS induction. Blue: Significant PFAS reduction. Bolded Black*: Significant PFAS x Sex interaction. Black: No significant effect. ¹Not measured. Changes in gene expression patterns analyzed by RNA sequencing are shown in italics. Significance is determined at p-values adjusted for multiple comparisons (q<0.05). Changes in metabolic signature are shown outlined in black. Genes and metabolites shown in the Gut portion of the schematic are inferred from hepatic results.