Linking di-2-ethylhexyl phthalate (DEHP) exposure to osteoporosis through population analysis, network toxicology, and experimental evidence

Osteoporosis is a major public health problem, and environmental toxicants such as di(2-ethylhexyl) phthalate (DEHP) have been implicated as potential risk factors. However, the molecular mechanisms linking DEHP exposure with osteogenic impairment remain unclear. The association between DEHP exposure and osteoporosis risk using NHANES data, and employed network toxicology and molecular docking to identify key regulatory targets. To further verify these predictions, wet-lab experiments including Western blotting and quantitative PCR (qPCR) were conducted in osteoblast-related cells under control (CON), osteoporosis (OP), DEHP, and combined OP+DEHP conditions. Epidemiological analysis revealed that higher DEHP exposure was associated with increased osteoporosis risk. In the fully adjusted model, participants in the highest quartile of ln∑DEHP had higher odds of osteoporosis compared with those in the lowest quartile (OR = 1.32, 95% CI = 0.76–1.66, P for trend <0.001). Network toxicology analysis identified HDAC1 as a central hub gene, while RUNX2 emerged as a key osteogenic regulator. Western blotting and qPCR further demonstrated significant upregulation of HDAC1 and downregulation of RUNX2, with the most pronounced changes observed in the OP+DEHP group. These results suggest that DEHP exposure may contribute to osteogenic impairment and increased osteoporosis risk, potentially through epigenetic dysregulation involving HDAC1 and suppression of RUNX2. • Higher urinary DEHP exposure was associated with increased odds of osteoporosis in adults aged 50 years and older. • An integrative framework combining NHANES data, network toxicology, and molecular docking was used to identify mechanistic targets of DEHP-induced bone toxicity. • HDAC1 was identified as a central hub gene, while RUNX2 was highlighted as a key regulator of osteogenic differentiation affected by DEHP exposure. • Wet-lab validation confirmed DEHP-induced upregulation of HDAC1 and suppression of RUNX2 in osteoblast-related cells. • These findings suggest that environmental phthalates may contribute to osteogenic impairment through epigenetic dysregulation.