Effects of di-(2-ethylexyl) phthalate on apoptosis of cytotrophoblasts in early pregnancy

Abstract

To investigate the influence of di-(2-ethylexyl) phthalate (DEHP) on cell apoptosis and expression of Bcl-2 and bax in cultured human first trimester cytotrophoblasts. Human first trimester cytotrophoblasts were cultured with DEHP at concentration of 0, 25, 50, 100 µmol/L for 24 hours. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and flow cytometer method. The expression of apoptosis-associated genes, including Bcl-2 and bax, were detected by reverse transcription (RT)-PCR in cultured cytotrophoblast cells. The protein expression of Bcl-2 and bax in cytotrophoblast cells was measured by western blot. (1) The expression of Bcl-2: when incubated with DEHP at concentration of 0, 25, 50 and 100 µmol/L, the expression of Bcl-2 were 1.00 ± 0.05, 1.03 ± 0.04, 1.04 ± 0.03, 1.04 ± 0.04, which did not show statistical difference (P > 0.05). The expression of Bcl-2 protein were 0.11 ± 0.02, 0.11 ± 0.04, 0.12 ± 0.02, 0.12 ± 0.03, which also didn't reach statistical difference (P > 0.05). (2) The expression of bax: when incubated with DEHP at concentration of 50 and 100 µmol/L, the expression of bax protein were 0.63 ± 0.04 and 0.81 ± 0.04, which were significantly higher than 0.23 ± 0.05 with DEHP at 0 µmol/L (P < 0.05). The expression of bax mRNA were 0.96 ± 0.04 and 1.02 ± 0.04, which was significantly higher than 0.81 ± 0.05 with DEHP at 0 µmol/L (P < 0.05). (3) Apoptosis: when incubated with DEHP at concentration of 50 and 100 µmol/L for 24 hours, the apoptotic cell ratio were (18.8 ± 2.6) % and (20.3 ± 2.0) % by annexin V-FITC/PI staining, which were significantly higher than (10.6 ± 1.4) % at 0 µmol/L and (18.1 ± 4.6) % and (19.5 ± 1.2) % by TUNEL staining, which were significantly higher than (11.2 ± 3.1) % at 0 µmol/L of DEHP (P < 0.05). DEHP could induce apoptosis of cytotrophoblast cells by increasing bax gene expression, but had no effect on Bcl-2 expression.