The placenta constitutes a boundary between the fetal and maternal environments, being involved in the exchange of nutrients and waste products between the mother and the fetus. A compromised uterine blood flow and placental formation and development — placentation — causes reduced availability of nutrients and oxygen for the fetus, as observed in pathologies such as fetal growth restriction and preeclampsia.
Pregnant women are frequently exposed to several xenobiotics due to lifestyle factors such as diet, smoking, drug abuse, alcohol consumption, or therapeutic drugs. For instance, pregnant women often take medication for the treatment of conditions such as epilepsy, hypertension, depression, and type II and gestational diabetes. This work focused on two xenobiotics, ethanol (EtOH) and metformin (METF).
EtOH is one of the most frequently-used drugs worldwide, and although most women avoid alcohol during pregnancy, many of them begin to reduce consumption only upon pregnancy recognition. In the US, in 2017, about 10% of pregnant women reported alcohol use. Alcohol consumption during pregnancy can lead to brain, craniofacial, cardiovascular, and limb defects collectively termed Fetal Alcohol Spectrum Disorders. EtOH was chosen for this study because previous experiments from our group showed a detrimental effect of this compound on term trophoblasts (Keating et al. 2009).
METF is a biguanide agent that reduces hyperglycemia. METF is widely used as first-line treatment in type 2 diabetes and polycystic ovary syndrome (PCOS) and is becoming increasingly accepted as an alternative to insulin during pregnancy for the management of type 2 and gestational diabetes and PCOS. Considering that 1 to 14% of all pregnancies are affected by gestational diabetes (with prevalence varying with population-specific factors such as genetics, environment, and screening and diagnostic methods), METF exposure during pregnancy is gaining an expressive importance. We decided to investigate the effect of METF because its effect on the placenta and its long-term effect on fetal physiology is not clearly elucidated, and because it was recently found to possess an anticancer effect, thus interfering with cell characteristics such as proliferation and apoptosis.
In this work, we investigated the influence of two xenobiotics, namely EtOH — and its metabolite acetaldehyde (ACA) — and METF, on placentation-related processes (cell viability and proliferation, culture growth, migratory capacity and apoptosis index) of a human first-trimester extravillous trophoblasts (EVTs) cell line (HTR-8/SVneo cell line). EVTs are fully-specialized trophoblasts displaying an invasive and proliferative phenotype, and they are the key cell type involved in the placentation process. Moreover, the effect of and EtOH and METF on nutrient (glucose (GLU) and folic acid (FA)) uptake by EVTs cells was also investigated because an adequate nutrition of EVT itself is critical for the normal process of placentation.
We verified that EtOH, ACA, and METF (24h) significantly reduced cell proliferation rates, culture growth, viability, and migratory capacity of HTR-8/SVneo cells. We also verified that both EtOH (100 μM) and METF (1 mM) increased the apoptosis index and inhibited 3H-deoxy-D-glucose (3H-DG) and 3H-folic acid (3H-FA) uptake.
Moreover, we verified that PI3K, mTOR, JNK, and intracellular signaling pathways were involved in the effect of EtOH upon 3H-FA uptake and in the effect of METF upon cell viability, and mTOR and JNK in the effect of EtOH upon cell viability and 3H-DG uptake. These intracellular signaling pathways were investigated because they are known to be involved in regulation of placentation-related processes. More specifically, JNK pathways regulate migration in different cell types using multiple molecular mechanisms, and PI3K and mTOR are major signaling components that play a crucial role in growth factor-mediated first-trimester EVT cell migration. Indeed, several growth factors are known to increase trophoblast motility by inducing activation of PI3K and consequently by downstream activating Akt and mTOR. Of note, mTOR signaling is influenced by a large number of upstream regulators such as amino acids, growth factors, and free fatty acids.
In conclusion, we have shown that EtOH and METF have a detrimental effect on placentation-related processes of HTR-8/SVneo cells. Moreover, mTOR, JNK, and PI3K appear to mediate some of these negative effects.
These findings are described in the article entitled Involvement of mTOR, JNK and PI3K in the negative effect of ethanol and metformin on the human first-trimester extravillous trophoblast HTR-8/SVneo cell line, recently published in the European Journal of Pharmacology. This work was conducted by Ana Correia-Branco, Elisa Keating, and Fátima Martel from the University of Porto.