Comparison of embryotoxicity of di(2-ethylhexyl) phthalate using mouse and human embryonic stem cell test models in vitro

Abstract

To establish a mouse embryonic stem cell test (mEST) model and human embryonic stem cell test (hEST) model, to evaluate the embryotoxicity of di(2-ethylhexyl) phthalate (DEHP). We developed mEST and hEST models according to the European Centre for the Validation of Alternative METHODS (ECVAM). We used penicillin G (PN-G) as the standard negative reference and 5-fluorouracil (5-FU) as the standard positive reference, respectively, to verify validity of the models. Based on model validity, mouse embryonic stem cells D3 (mESC-D3), mouse Balb/c-3T3 (3T3), and human embryonic stem cells H9 (hESC-H9) were administered different concentrations of DEHP (15.6, 31.2, 62.5, 125.0, 250.0, 500.0, and 1 000.0 μg/ml) for 7 days. A cell counting Kit-8 was used to detect the 50% inhibitory proliferation concentration (IC50) of mESC-D3 cells, 3T3 cells, and hESC-H9 with DEHP. mESC-D3 and hESC-H9 were treated with DEHP (15.6, 31.2, 62.5, 125.0, 250.0 μg/ml, and 500.0 μg/ml) for 10 days based on the cytotoxicity results. At day 10, the expression of cardiomyocyte differentiation gene alpha-myosin heavy chain (α-MHC) was detected by real-time PCR and the 50% inhibition of cardiomyocycte differentiation (ID50) determined. Based on the values of IC50 and ID50, functions Ⅰ, Ⅱ and Ⅱ could be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions. Nontrophoblast lineage both ES cells were cultured under optimal conditions and highly expressed hESC markers OCT4 , SSEA4, and TRA-1-60. The embryoid bodies formed were uniform in size and shape, and these results were highly repeatable. The PN-G and 5-FU results coincided with the prediction by ECVAM. Validation of our EST models was satisfactory. RESULTS of the three endpoints of DEHP in mEST were 197.3 μg/ml (IC50 3T3), 210.0 μg/ml (IC50 D3) and 246.8 μg/ml (ID50 D3). DEHP was evaluated to be a nonembryotoxic compound based on values of function Ⅰ (7.78), function Ⅱ (7.58) and function Ⅲ (-7.79). The three endpoints of DEHP in hEST were 195.4 μg/ml (IC50 3T3), 184.8 µg/ml (IC50 D3), and 84.3 µg/ml (ID50). By comparing the values of function Ⅰ (3.21), function Ⅱ (5.77), and function Ⅲ (-6.46), DEHP was evaluated to be weakly embryotoxic. DEHP was determined to be a nonembryotoxic compound by mEST and weakly embryotoxic by hEST. Therefore, hEST is a more sensible model for the evaluation of DEHP embryotoxicity.