Abstract

Background:
Per- and polyfluoroalkyl substances (PFASs) are long-lasting environmental pollutants that accumulate in apex predators. The mechanistic consequences of PFAS exposure in endangered large felids remain poorly characterized.

Aim:
This study aimed to characterize molecular and pathological features consistent with PFAS-associated multiorgan injury in the endangered Persian leopard (Panthera pardus tulliana) using integrative molecular and pathological approaches.

Methods:
A complete necropsy was performed on a deceased wild adult male Persian leopard (P. pardus tulliana) with clinical signs consistent with multi-organ dysfunction. Tissues from the adrenal cortex, myocardium, renal cortex, liver, and hypothalamus were analyzed using single-cell RNA sequencing, spatial transcriptomics (10x Genomics Visium), transmission electron microscopy, targeted PFAS quantification (LC-MS/MS), high-resolution mitochondrial respirometry, targeted steroidomics, and immunohistochemistry.

Results:
Elevated perfluorooctane sulfonate (PFAS) concentrations were detected across all examined tissues, with PFOS as the predominant congener. Ultrastructural analysis revealed severe mitochondrial damage characterized by cristolysis, membrane rupture, and intramitochondrial vacuolation, accompanied by a 57% reduction in oxidative phosphorylation capacity. Spatial transcriptomics demonstrated coordinated downregulation of steroidogenic and oxidative phosphorylation gene networks, spatially juxtaposed with IL18, TNF, and CCL20-expressing inflammatory microdomains. Single-cell profiling identified 31 transcriptional clusters. Steroidogenic cell populations exhibited reduced mitochondrial gene expression, whereas T-cell subsets showed increased expression of inhibitory checkpoint receptors (PDCD1, LAG3, TIGIT, and CTLA4), suggesting immune exhaustion. Targeted steroidomics revealed marked suppression of cortisol, corticosterone, aldosterone, and pregnenolone levels. Immunohistochemistry confirmed reduced mitochondrial mass, impaired mitophagic flux, and elevated oxidative DNA damage.

Conclusion:
To the best of our knowledge, this study provides the first detailed molecular and pathological characterization of PFAS-associated multi-organ injury in an endangered large felid. Convergent disruption of mitochondrial, endocrine, and immunometabolic pathways is identified as a key feature associated with PFAS accumulation, with implications for conservation toxicology and wildlife health assessment.

Key words: PFAS; Persian leopard; Panthera pardus tulliana; Mitochondrial dysfunction; Spatial transcriptomics; Immunometabolic disruption.