Individual and binary exposure of short- and long-chain phthalate esters decreases viability and inhibits cellular respiration in human lung cells

Phthalate esters (PAEs) such as dimethyl phthalate (DMP), diethyl phthalate (DEP), bis-2(ethylhexyl) phthalate (DEHP), and di-n-octyl Phthalate (DOP) are synthetic chemicals used as solvent stabilisers and plasticizers in commercial and industrial products. Emerging literature suggests that semi-volatile PAEs (e.g., DEHP) could be toxic to human lung cells, but atmospheric concentration regulations on PAEs remain limited. This work evaluated the individual and combined toxicity of prolonged exposure to environmentally relevant concentrations of DMP, DEP, DEHP, and DOP (10 μM - 1 mM) on Calu-3 human sub-bronchial gland cells. Individual exposure to the tested PAEs yielded the following maximal inhibitory concentration (IC₅₀) at 24 h (4.46 mM DMP, 2.05 mM DEP), 72 h (3.07 mM DMP, 927 μM DEP), and 168 h (418 μM DMP, 106 μM DEP, 343 μM DEHP, 400 μM DOP). Concentration addition and independent action toxicity models predicted synergism and additive effects, respectively, at 72 h exposure with the DMP-DEP combination. Cellular oxygen (O₂) consumption was measured via high-resolution respirometry. Exposure of live cells to individual or binary mixtures of PAEs inhibited cellular O₂ consumption in a concentration specific manner, indicating direct inhibition of mitochondrial respiratory flux. The results of this work raise a concern on potential long-term adverse effects of atmospheric levels of DMP, DEP, and DOP on the human respiratory system. To the best of our knowledge, this is the first report on the cytotoxic effects of prolonged exposure to DMP, DEP, and DOP and the first to assess the toxicity of binary mixtures of DMP-DEP and DEHP-DOP in a human lung cell line. The results also raise a concern about whether current workroom air quality regulations set to a maximum of 5 mg/m³ on DMP, DEP, and DEHP are safe for human health.