Influence Of Pregnant Mice To Triclosan On Placental Development And Underlying Molecular Mechanisms

Triclosan(TCS),a broad-spectrum antimicrobial agent,is used in clinical settings and in various personal care and consumer products.TCS was detected in 100%and 51%of urine and cord blood samples,respectively,obtained from 181 expectant mothers in New York.This compound has also been identified in the mother's milk.We reported high urinary TCS levels in spontaneous abortion patients.Animal experiments have shown that the exposure of pregnant mice to TCS(100 mg/kg)causes decreases in fetal body weight and viability.Thyroid hormones,which consist of triiodothyonine(T3)and thyroxine(T4),play a critical role in fetal growth and development.The treatment with TCS decreased total serum T3 and T4 in pregnant rats and mice.Published data consistently document a relationship between maternal thyroid hormones and feto-placental development.Maternal thyroid deficiency is associated with intrauterine growth restriction,fetal distress and low birth weight.Treatment of pregnant rats with TCS(10 mg/kg)decreases the levels of total serum T4 and T3 and causes decreases in pup body weights.In mammals,the major determinant of intrauterine growth is the delivery of nutrients to the fetus via the placenta.Placenta nutrient transport is dependent on placental size,morphology,nutrient transporter capacity/availability.Several lines of evidence suggest that activation of thyroid hormone receptors can trigger the phosphatidylinositol 3-kinase(PI3K)/Akt-mTOR and ERK signaling pathways to enhance the placental development.Furthermore,the thyroid hormones can modulate the expression and function of syncytiotrophoblast amino acid transporter transports(SNAT1?SNAT2?SNAT4?TAUT)and glucose transporters(GLUT1?GLUT3).Therefore,it is proposed that TCS-induced reductions in thyroid hormone levels during pregnancy affect placental development and function.ObjectiveTo determine influence of pregnant mice to triclosan on placental development and underlying molecular mechanismsMaterials and Methods1.Female mice received the oral administration of TCS(1,4 and 8 mg/kg/day,termed 1-TCS,4-TCS and 8-TCS mice)from gestational day(GD)6-18.2.Placentas were fixed in 4%paraformaldehyde and embedded in paraffin wax.Sections(5 ?m)were stained with hematoxylin and eosin(HE).Placental volumes densities were measured.3.Immunohistochemistry of BrdU and PCNA in placentas was performed.The number of BrdU-positive(BrdU+)cells and PCNA-positive(PCNA+)cells were count.4.Placental amino acid and glucose transporter activity were measured by radioimmunoassay.5.Enzyme linked immunosorbent assay(ELISA)was used to examine levels of serum T3,T4,Thyroid stimulating hormone(TSH),estrogen(E2),progesterone(P4),IL-6,IL1? and TNF?.6.Levels of Akt,mTOR,p70S6K and ERK1/2 protein and phosphorylation were examined by Western blotting analysis.7.Levels of PCNA,Cyclin D3,SNAT1,SNAT2,SNAT4,TAUT,GLUT1and GLUT3 mRNA were measured by RT-PCR.Results1.In comparison with control mice,the numbers of live TCS-fetuses on GD19 were slightly decreased in 8-TCS mice,but not in 1-TCS mice or 4-TCS mice.The ratio of fetal loss in 8-TCS mice was higher than that in control mice.2.The placental sizes and the placental weights of GD19 8-TCS mice were less than those of control mice.Additionally,the stereohistological analysis revealed that overall placental volumes and labyrinth zone volumes were significantly reduced in 8-TCS mice compared to control mice.We did not observe the placental thrombi or hemorrhaging,or tissue necrosis in TCS mice.3.The numbers of BrdU+ cells and PCNA+ cells in the labyrinth zone of 8-TCS mice was less than that in control mice.The levels of placental PCNA and Cyclin D3 in 8-TCS mice were lower those in control mice,but in 1-TCS mice and 4-TCS mice did not differ from control mice.4.The activity of SNAT per gram of placenta was significantly reduced in 8-TCS mice compared to control mice,but its activity in 4-TCS mice and 1-TCS mice was not significantly different from its activity in control mice.In addition,the activity of glucose transporter per gram of placenta was lower in 8-TCS mice than in control mice.At all time points,[14C]-MeAIB and[14C]-methyl-D-glucose accumulation per gram of fetus was the same in 8-TCS mice as the control mice,indicating that the fetuses were receiving the appropriate amounts of radioactive label for their size.5.The levels of placental either SNAT1 and SNAT4 mRNA or GLUT1 mRNA were reduced in 8-TCS mice compared to control mice,while the levels of SNAT2 and GLUT1 mRNA were not different between control mice and 8-TCS mice.6.In comparison with controls,8-TCS mice exhibited reduced total T3 and T4 levels and slightly elevated TSH levels.There were no significant differences in the levels of serum E2 and P4 between control mice and TCS mice.7.The levels of placental Akt,mTOR,p70S6K phosphorylation on GD17 were reduced in 8-TCS mice,which could be rescued by the administration of L-thyroxinein(T4)from GD15 to GD17.By contrast,no changes in the levels of ERK1/2 phosphorylation were observed in TCS mice compared to control mice.8.8-TCS mice treated with the T4 replacement from GD10-13 corrected the reductions in the numbers of BrdU+ cells and the levels of PCNA mRNA.The T4 replacement from GD15-17 rescued the activity of amino acid transporter and glucose transporter in 8-TCS mice.These effects were blocked by rapamycin.The levels of GLUT1 mRNA in 8-TCS mice were increased by the T4 replacement,but this change was not affected by rapamycin.The treatment of 8-TCS with T4 from GD10-17 could rescue the decrease in fetal body weight,which was sensitive to rapamycin.ConclusionThe present study has provided the morphological and functional evidence that exposure to 8 mg/kg TCS during gestation affects placental development and placental nutrient transport,leading to decreases in fetal body weight.