Research Article: 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

Date Published: May 14, 2019

Publisher: Public Library of Science

Author(s): Changqing Tang, Chunyan Luo, Yimin Hua, Kaiyu Zhou, Hongyu Duan, Fan Ma, Yi Zhang, Yifei Li, Dajian Qiu, Chuan Wang, Cheryl S. Rosenfeld.


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.

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.

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.

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

Partial Text

Worldwide congenital heart defect (CHD) is one of the most common birth defects, occurring in 7 to 8 per 1000 live births in China. The origins of CHDs are closely related to fetal toxicants exposure [1–4]. Previously, our epidemiological survey have illustrated that maternal exposure to phthalates, the most commonly used plasticizer, could increase the risk of CHD [5]. Thereafter, our animal study further proved that maternal exposure to di-(2-ethylhexyl)-phthalate (DEHP), which accounts for 80% of phthalate production in China, could result in various types of fetal cardiac anomalies in mice [6]. Except for merely exploring the mechanism regarding adverse effect of phthalates/DEHP on fetal cardiac development, seeking specific targets for decreasing transplacental transfer rates of phthalates/DEHP could be a promising alternative for CHDs prevention.

As shown in Fig 1, the vehicle group and the verapamil group demonstrated similar maternal weight gain without significant difference. The dams both in the DEHP group and the DEHP & verapamil group showed a significantly declining trend of maternal bodyweight at each time point from E7.5 onwards compared with the verapamil group and the control group. By the time of sample collection at E15.5, maternal bodyweight in the DEHP & verapamil group was lower than that of the DEHP group. However, when the weight gain of the dam was adjusted with the number of the fetuses delivered by cesarean section, there were no significant differences in the weight gain between the DEHP group and the DEHP & verapamil group, indicating that the decreased body weight of pregnant dams was most likely caused by both a loss of the fetuses (the sums of dead fetuses, abortion fetuses, resorption fetuses and even the pre‐implantation loss), but not a toxic effect of DEHP on the dam itself. In addition, the fetus both in the DEHP group and the DEHP & verapamil group had general growth retardation; the bodyweights of live fetuses both in the two groups were significantly decreased at E15.5 in comparison with the vehicle group and the verapamil group. A significantly decreased placental weight was also observed in the DEHP group and in the DEHP & verapamil group.

Highly expressed early in fetus and placenta, P-gp appears to be one of the most characterized and abundant transporter in placenta [7–11, 13]. Several studies had displayed that placental P-gp had a protective role in fetal development by restricting the penetration of xenobiotics from maternal circulation into fetal compartment [9, 13, 29, 30]. As far back as 1998, an animal study performed by Lankas GR et al.[17]had found that fetuses deficient in Abcb1 (-/-) were 100% susceptible to cleft palate resulting from exposure to avermectin, a known teratogenic substrate of P-gp, whereas the heterozygotes (-/+) littermates were less sensitive and the homozygous (+/+) fetuses with abundant P-gp were totally protected from the effects of teratogens. Afterwards, another study [15] revealed the similar results and they noticed that the mice strains with knockout Abcb1 genes showed susceptibility to cleft palate following the administration of phenytoin to the pregnant dams. Among 48 fetuses with cleft palate, 36 had the heterozygous (Abcb1a+/-) genotype and 12 were homozygous for the knockout gene (Abcb1a-/-). Additionally, el-Ashmawy, I. M. et al., [28] observed that verapamil could dramatically enhance the susceptibility of fetal developmental disorders induced by ivermectin via P-gp inhibition. With regard to clinical studies, two researches [31, 32] have reported that 3435C>T polymorphism of ABCB1 gene could affect the risk of toxicants-induced birth defects, which might be explained by the alteration of placental P-gp expression and efflux activity. Moreover, a recent epidemiological case-control survey [33] documented that several drug classes that are substrates for P-gp were shown to have a higher user rate in mothers of cases with specific anomalies. The use of this subset of drugs in combination with other P-gp substrates increased the risk for specific anomalies (OR 4.17, 95% CI 1.75–9.91), and the addition of inhibitors further increased the risk (OR 13.03, 95% CI 3.37–50.42). These findings discussed above provided strong evidences that placental P-gp play a vital role in fetal toxicants exposure and subsequently affect the risk of toxicants-induced birth defects. However, up to date, data on the role of placental P-gp in toxicants-induced cardiac developmental malformations is extremely limited.




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