Naphthalene exposure inhibits osteogenic differentiation via the reactive oxygen species–ubiquitin pathway

While acute and chronic toxicities of naphthalene have been well documented, its effects on osteogenesis remain unexplored. In this study, we investigated the toxicity of naphthalene on osteoblast function using osteoblast-like MG-63 cells. Naphthalene at concentrations of 5-50 μM, which were low enough to not affect cell viability, effectively suppressed alkaline phosphatase (ALP) activity and matrix mineralization in MG-63 cells. Moreover, naphthalene downregulated the expression of osteogenic markers, including runt-related transcription factor 2, Osterix, ALP, collagen type 1 alpha 1, and osteopontin, and phosphate-regulating endopeptidase homolog X-linked, at both the mRNA and protein levels. The expression of β-catenin and its associated proteins was also suppressed following naphthalene treatment, leading to a reduction in nuclear accumulation of β-catenin. Naphthalene enhanced the intracellular reactive oxygen species level and suppressed nuclear expression of β-catenin signaling and its target molecules, including glycogen synthase kinase 3β, cyclin D1, and c-Myc, and these proteins were restored by treatment with the antioxidant N-acetylcysteine. Treatment with lithium chloride and MG132 confirmed that naphthalene-induced reactive oxygen species generation increased GSK3β activity, thereby promoting the proteasomal degradation of β-catenin and suppressing osteogenic activity in MG-63 cells. Interestingly, MG-63 cells cultured as spheroids exhibited enhanced osteogenic potential, providing a more physiologically relevant model. In conclusion, this study is the first to demonstrate the inhibitory effects of naphthalene on osteogenic differentiation in MG-63 cells, as validated using a 3D spheroid model. Further research on the molecular mechanisms underlying naphthalene's effects, particularly its role in the Wnt/β-catenin/GSK3β pathway, is needed.