Perfluoroalkyl substances and severity of nonalcoholic fatty liver in Children: An untargeted metabolomics approach

Abstract

Background: Toxicant-associated steatohepatitis has been described in adults but less is known regarding the role of toxicants in liver disease of children. Perfluoroalkyl substances (PFAS) cause hepatic steatosis in rodents, but few previous studies have examined PFAS effects on severity of liver injury in children.

Objectives: We aimed to examine the relationship of PFAS to histologic severity of nonalcoholic fatty liver disease (NAFLD) in children.

Methods: Seventy-four children with physician-diagnosed NAFLD were recruited from Children's Healthcare of Atlanta between 2007 and 2015. Biopsy-based liver histological features were scored for steatosis, lobular and portal inflammation, ballooning, and fibrosis. Plasma concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonic acid (PFHxS), and untargeted plasma metabolomic profiling, were determined using liquid chromatography with high-resolution mass spectrometry. A metabolome-wide association study coupled with pathway enrichment analysis was performed to evaluate metabolic dysregulation associated with PFAS. A structural integrated analysis was applied to identify latent clusters of children with more severe form of NAFLD based on their PFAS levels and metabolite pattern.

Results: Patients were 7-19 years old, mostly boys (71%), Hispanic (51%), and obese (85%). The odds of having nonalcoholic steatohepatitis (NASH), compared to children with steatosis alone, was significantly increased with each interquartile range (IQR) increase of PFOS (OR: 3.32, 95% CI: 1.40-7.87) and PFHxS (OR: 4.18, 95% CI: 1.64-10.7). Each IQR increase of PFHxS was associated with increased odds for liver fibrosis (OR: 4.44, 95% CI: 1.34-14.8), lobular inflammation (OR: 2.87, 95% CI: 1.12-7.31), and higher NAFLD activity score (β coefficient 0.46; 95% CI: 0.03, 0.89). A novel integrative analysis identified a cluster of children with NASH, characterized by increased PFAS levels and altered metabolite patterns including higher plasma levels of phosphoethanolamine, tyrosine, phenylalanine, aspartate and creatine, and decreased plasma levels of betaine.

Conclusions: Ηigher PFAS exposure was associated with more severe disease in children with NAFLD. PFAS may be an important toxicant contributing to NAFLD progression; however larger, longitudinal studies are warranted to confirm these findings.

Figure 1.. Dysregulated metabolic pathways associated with plasma PFAS (including PFOA, PFOS and PFHxS) in children with NAFLD.

The vertical axis represents the pathways (blue circles) with radius representing the numbers of associated metabolite features. The horizontal axis represents the negative log₁₀ (P-value) of each pathway. Only pathways with >5 hits of significant metabolites (overlapping size) are shown.

Figure 2.. Structural integrated analysis of PFAS composite variable and plasma metabolites that identify a cluster of children with increased odds of having NASH.

Blue lines represent positive effects and grey lines indicate negative effects, with the width proportional to the effect size. The thick blue line connecting PFAS to “Cluster 2” indicates an association with higher levels of PFAS as compared to lower levels with “Cluster 1” (indicated by a thin blue line). The blue lines connecting clusters to metabolites indicate positive associations, while the grey lines connecting clusters to metabolites indicate negative associations. The red line indicates an increased risk for NASH associated with “Cluster 2” (OR=4.24), compared to those in “Cluster 1” (OR=1, reference).