Determination of phthalate esters in milk and evaluation of their effect on beta-TC3 cells and potential modulation of vitamin D3

The increasing presence of phthalates in dairy products as endocrine-disrupting chemicals (EDCs) poses a significant risk to metabolic health. However, the relationship between milk fat content, phthalate concentration, and pancreatic beta-cell dysfunction remains poorly understood. This study aimed to quantify phthalate migration in pasteurized milk and evaluate their impact on beta-TC3 cell functionality, while assessing the modulatory potential of Vitamin D3. Phthalate concentrations (DEHP, DMP, DEP, DBP, DOP, and BBP) in low-fat and full-fat pasteurized milk were quantified using HPLC-DAD. Standardized mixtures based on these detected concentrations were applied to beta-TC3 cells. Oxidative stress markers (MDA, GSH, FRAP), MTT-based viability, and insulin secretion dynamics were evaluated over 72 h, with and without Vitamin D3 treatment. The mean concentrations of phthalates ((DEHP), (DMP), (DEP), (DBP), (DOP), (BBP)) in low-fat milk were 49.8 ± 4.6, 90.6 ± 7.82, 57 ± 4.06, 131.2 ± 8.22, 42.6 ± 6.58, 31 ± 3.87) µg/L (, and in full-fat milk were 92.2 ± 6.76, 149.6 ± 5.77, 102.2 ± 5.58, 199.6 ± 8.08, 100.8 ± 4.96, and 70.8 ± 6.45) µg/L (, respectively. Exposure to phthalate mixtures resulted in a significant, time-dependent increase in oxidative stress and a decline in both cell viability and functional capacity. According to the MTT assays, cell viability at 72 h dropped significantly to 83.1% in the low-fat milk group and reached a minimum of 73.4% in the full-fat milk group. This cytotoxicity was accompanied by a marked rise in lipid peroxidation (MDA levels) and a depletion of antioxidant defenses, characterized by reduced glutathione (GSH) and ferric reducing antioxidant power (FRAP), with the most severe oxidative damage observed in the full-fat milk model. Functionally, phthalates caused a profound suppression of insulin secretion. In the low-fat milk group, insulin levels decreased to 289.1 pg/mL under low glucose and 618.2 pg/mL under high glucose conditions at 72 h. In the full-fat milk group, insulin secretion under high glucose was limited to 550 pg/mL. Notably, while the addition of Vitamin D3 showed a measurable capacity to reduce oxidative markers and partially improve viability in the low-fat model, it was unable to fully restore the insulin secretory function in the presence of the higher phthalate concentrations found in the full-fat matrix.