Integrated Spheroid-to-Population Framework for Evaluating PFHpA-Associated Metabolic Dysfunction and Steatotic Liver Disease

The rising prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), particularly among pediatric populations, requires identification of modifiable risk factors to control disease progression. Per- and polyfluoroalkyl substances (PFAS) have emerged as potential contributors to liver damage; however, their role in the etiology of MASLD remains underexplored. This study aimed to bridge the gap between human epidemiological data and in vitro experimental findings to elucidate the effect of perfluoroheptanoic acid (PFHpA), a short chain, unregulated PFAS congener on MASLD development. Our analysis of the Teen-LABS cohort, a national multi-site study on obese adolescents undergoing bariatric surgery, revealed that doubling of PFHpA plasma levels was associated with an 80% increase in MASLD risk (OR, 1.8; 95% CI: 1.3–2.5) based on liver biospies. To further investigate the underlying mechanisms, we used 3D human liver spheroids and single-cell transcriptomics to assess the effect of PFHpA on hepatic metabolism. Integrative analysis identified dysregulation of common pathways in both human and spheroid models, particularly those involved in innate immunity, inflammation, and lipid metabolism. We applied the latent unknown clustering with integrated data (LUCID) model to assess associations between PFHpA exposure, multiomic signatures, and MASLD risk. Our results identified a proteome profile with significantly higher odds of MASLD (OR = 7.1), whereas a distinct metabolome profile was associated with lower odds (OR = 0.51), highlighting the critical role of protein dysregulation in disease pathogenesis. A translational framework was applied to uncover the molecular mechanisms of PFAS-induced MASLD in a cohort of obese adolescents. Identifying key molecular mechanisms for PFAS-induced MASLD can guide the development of targeted prevention and treatment.