Perfluorooctanoic acid and its alternatives disrupt the osteogenesis and osteoclastogenesis balance: Evidence from the effects on cell differentiation process.

In the present study, we investigated the effects of a representative of the per- and polyfluoroalkyl substance (PFAS) chemical group, namely perfluorooctanoic acid (PFOA), and its alternatives (perfluorobutanoic acid [PFBA] and the hexafluoropropylene oxide dimer acid [GenX]) on bone homeostasis, a process that mainly depends on osteoblast (OB) and osteoclast (OC) activities at the cellular level. C3H10T1/2 cells and bone marrow macrophages (BMMs) were respectively induced into OBs and OCs, and treated with PFOA, PFBA, and GenX at doses of 0.25, 2.5, and 25 μM. Real-time PCR, ALP activity analysis, ARS staining, and TRAP staining were performed to assess cell differentiation. To explore the indirect effects on OC differentiation, conditioned media were collected from 7-day PFAS-treated C3H10T1/2 cells during differentiation. RANKL/OPG expression and secretion levels in C3H10T1/2 cells were determined, and BMMs induced into OCs were incubated in conditioned media for two days. PFOA suppressed osteogenesis characterized by the decreased mRNA expression of OB-specific molecules, ALP activity, and calcium deposition, and promoted osteoclastogenesis as evidenced by upregulated osteoclastic genes and increased number of TRAP-positive cells. Although the PFOA alternatives did not affect early osteogenesis, calcium deposits were significantly reduced and osteoclastogenesis was facilitated. The results suggested that PFOA and its alternatives could directly disturb the balance between osteogenesis and osteoclastogenesis. In addition, PFOA and its alternatives enhanced the RANKL/OPG ratio during early OB differentiation, and more BMMs stimulated with the conditioned medium differentiated into OCs, indicating the indirect stimulation on OC differentiation by PFOA and its alternatives. These findings highlight the potential skeletal hazards of PFASs, particularly emerging alternatives that might be ignored, offering a toxicological basis for further exploration.