PFOA effects on osteoblast differentiation: Involvement of oxidative stress and endocannabinoid receptors

• PFOA exposure leads to catalase upregulation maintenance under oxidative stress. • High-dose PFOA (50–100 µM) reduces CB1 and CB2 protein levels. • PFOA slightly enhances calcium deposition at 50 µM. Perfluorooctanoic acid (PFOA), a persistent perfluoroalkyl substance (PFAS), has been implicated in bone mineral density loss and defective osteogenesis. In this study, by employing human fetal osteoblast (hFOB1.19), we investigated whether PFOA interferes with osteoblast differentiation by altering the transcription of genes involved in osteogenesis, and protein levels of oxidative stress defense and cannabinoid receptors (CBs). The hFOB 1.19 were exposed to increasing PFOA concentrations (1–100 µM) for seven days, representing supra-environmental concentration commonly used in mechanistic in vitro studies. Osteogenic markers were then evaluated at transcriptional level together with matrix deposition, and the results were compared to an untreated control group. Exposure to PFOA at 10 µM increased the expression of osteocalcin ( BGLAP ) encoding for a protein involved in calcium deposition. Catalase (CAT) protein levels were upregulated at 1 µM PFOA, while superoxide dismutase (SOD1) did not change, suggesting a selective antioxidant response to oxidative perturbation. Notably, this increase in CAT correlated with a trend toward RUNX2 upregulation, possibly representing a compensatory mechanism to preserve differentiation under oxidative stress. In addition, the highest concentration of PFOA modulated the endocannabinoid system (ECS), reducing CB1 and CB2 protein levels. Despite these molecular changes, Alizarin Red staining revealed a borderline and not statistically significant enhanced calcium deposition only at 50 µM PFOA, suggesting potentially aberrant mineralization. Overall, our findings suggest that PFOA perturbs osteoblast differentiation through oxidative stress-linked mechanisms and CBs modulation, with catalase emerging as a key protective mediator of osteogenic competence under environmental contaminants. Furthermore, the observed dysregulation of CB1 and CB2 receptors indicates that the ECS itself may represent a direct target of PFOA action in osteoblasts.