5-Hydroxymethylfurfural induces skeletal toxicity associated with ROS accumulation by disrupting cartilage and bone remodeling in zebrafish larvae.

5-Hydroxymethylfurfural (5-HMF) is a heat-induced Maillard reaction product in sugar-rich foods and some pharmaceutical products. Although 5-HMF has been associated with cytotoxicity, genotoxicity and developmental toxicity, its effects on skeletal development and bone-resorbing cells remain incompletely understood. Here, we investigated whether 5-HMF disrupts skeletal development in zebrafish larvae and whether reactive oxygen species (ROS) are involved. Wild-type zebrafish and Tg(sox9b:GFP) and Tg(sp7:EGFP) reporter lines were exposed to 10, 25 or 50 μg/mL 5-HMF from 8 h post-fertilization. Cartilage, mineralized bone, osteoblasts, osteoclast-like cells, ROS production and skeletal gene expression were assessed using Alcian blue staining, alizarin red staining, fluorescence imaging, TRAP staining and RT-qPCR. 5-HMF reduced craniofacial cartilage development, caused abnormal chondrocyte morphology, decreased craniofacial mineralization and osteoblast reporter signals, and increased TRAP-positive osteoclast-like activity. These phenotypes were accompanied by increased ROS accumulation, decreased sod1, sod2 and cat expression, suppressed chondrogenic and osteogenic marker expression, and elevated markers associated with matrix degradation and osteoclast differentiation. Co-treatment with the ROS scavenger N-acetyl-l-cysteine partially restored cartilage morphology, bone mineralization and osteoblast reporter signals, while reducing TRAP staining. These findings indicate that high concentrations of 5-HMF induce ROS-associated skeletal toxicity in zebrafish larvae by disrupting the balance between bone formation and resorption. These results provide mechanistic evidence of skeletal toxicity under high-exposure conditions and support further evaluation at exposure-relevant levels.