Placental P-glycoprotein inhibition enhances susceptibility to Di-(2-ethylhexyl)-phthalate induced cardiac malformations in mice: A possibly promising target for congenital heart defects prevention

Abstract

Backgrounds: Reducing toxicants transplacental rates could contribute to the prevention of congenital heart defects (CHDs). Placental P-glycoprotein (P-gp) plays a vital role in fetal toxicants exposure and subsequently affects the risk of toxicants-induced birth defects. However, data on the role of placental P-gp in decreasing toxicants-induced cardiac anomalies is extremely limited. This study aimed to explore the protective role of placental P-gp in reducing the risk of Di-(2-ethylhexyl)-phthalate (DEHP) induced cardiac anomalies in mice.

Methods: The C57BL mice were randomly divided into four groups: the vehicle group (corn oil, n = 10), 500mg/Kg DEHP group (n = 15), 3mg/Kg verapamil group (n = 10) and 500mg/Kg DEHP & 3mg/Kg verapamil group (n = 20). Pregnant dams in different group received respective intervention by gavage once daily from E6.5-14.5. Maternal weights were monitored every day and samples were collected at E15.5. HE staining was used to examine fetal cardiac malformations. Real-time quantitative PCR (RT-qPCR) and Western-Blot were applied to detect Nkx2.5/Gata4/Tbx5/Mef2c/Chf1 mRNA and protein expression, respectively. The mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) was also determined using RT-qPCR.

Results: Co-administration of verapamil and DEHP significantly elevated fetal cardiac malformation rates, in comparison with the DEHP group, the verapamil group and the vehicle group. Different phenotypes of cardiac anomalies, including septal defects and ventricular myocardium noncompaction, were noted both in the DEHP group and the DEHP & verapamil group. The ventricular myocardium noncompaction appeared to be more severe in the DEHP & verapamil group. Fetal cardiac PPARγ mRNA expression was notably increased and Gata4/Mef2c/Chf1 expression was markedly decreased in the DEHP & verapamil group.

Conclusion: Placental P-gp inhibition enhances susceptibility to DEHP induced cardiac malformations in mice.

Fig 1

Effect of maternal Di-(2-ethylhexyl)-phthalate (DEHP) and verapamil exposure from E6.5 to E14.5 on maternal bodyweights (A), fetal weights (B) and placental weights (C). Differences among different groups were determined by ANOVA followed by a Student–Newman–Keuls multiple comparisons. N = 10, 15, 20 and 10 for vehicle, 500mg/Kg DEHP, 500mg/Kg DEHP & 3mg/Kg verapamil and 3mg/Kg verapamil group, respectively. Data were expressed as Means±SEM. *P<0.05, **P<0.01, ***P<0.001 in comparison with the verapamil group and the vehicle group.

Fig 2. Fetal cardiac malformations induced by maternal Di-(2-ethylhexyl)-phthalate (DEHP) and verapamil exposure from E6.5 to E14.5.

The phenotypes of fetal cardiac malformations included septal defects (B, D, E, G). The magnification used was 4×and 10×.

Fig 3. Fetal cardiac malformations induced by maternal Di-(2-ethylhexyl)-phthalate (DEHP) and verapamil exposure from E6.5 to E14.5.

The phenotype of fetal cardiac malformations included ventricular myocardium noncompaction (G, H, M, N). The magnification used was 4×and 10×.

Fig 4. Effect of maternal Di-(2-ethylhexyl)-phthalate (DEHP) and verapamil exposure from E6.5 to E14.5 on fetal heart PPARγ/Nkx2.5/Gata4/Tbx5/Mef2c/Chf1 mRNA expression at E15.5.

Differences among different groups were determined by ANOVA followed by a Student–Newman–Keuls multiple comparisons. N = 10, 15, 20 and 10 for vehicle, 500mg/Kg DEHP, 500mg/Kg DEHP & 3mg/Kg verapamil and 3mg/Kg verapamil group, respectively. Data were expressed as Means±SEM. *P<0.05, **P<0.01, ***P<0.001 in comparison with the verapamil group and the vehicle group.

Fig 5. Effect of maternal Di-(2-ethylhexyl)-phthalate (DEHP) and verapamil exposure from E6.5 to E14.5 on fetal heart Nkx2.5/Gata4/Tbx5/Mef2c/Chf1 protein expression at E15.5.

Differences among different groups were determined by ANOVA followed by a Student–Newman–Keuls multiple comparisons. N = 10, 15, 20 and 10 for vehicle, 500mg/Kg DEHP, 500mg/Kg DEHP & 3mg/Kg verapamil and 3mg/Kg verapamil group, respectively. Data were expressed as Means±SEM. *P<0.05, **P<0.01, ***P<0.001 in comparison with the verapamil group and the vehicle group.