Effects Of Environmental Endocrine Disruptor Perfluorooctane Sulfonic Acid On Thyroid Function And Fetal Intrauterine Growth Restriction

Objective:As a kind of persistent organic pollutant,perfluoroalkyl and polyfluoroalkyl substances（PFASs）are often used in surfactants,detergents,fire-extinguishing foam,leather preparation,food packaging and so on because of their properties of chemical stability,thermal stability and tensile.PFASs can be directly discharged into the environment during the process of their production and usage,where it enters the body mainly through diet.Current studies have confirmed that PFASs has reproductive toxicity,embryo toxicity,thyroid toxicity and other toxic effects.At the early stages of life,it is susceptible to PFASs,so in recent years there has been increasing concern about the effects of PFASs on human health.Among all PFASs,perfluorooctane sulfonate（PFOS）and perfluorooctanoic acid（PFOA）attract the most attention.Previous birth cohort studies have shown an inverse correlation between PFOS and PFOA and fetal birth outcomes（such as birth weight,birth length and/or head circumference）,but epidemiological findings remain controversial.A meta-analysis showed a negative correlation between PFASs and total T4,and their toxic effects may vary depending on the concentration of PFASs.Animal experiments have shown that PFASs can disrupt the hypothalamic-pituitary-thyroid（HPT）axis and affect the secretion and function of thyroid hormones.Even mild thyroid hormone fluctuations during pregnancy can have adverse effects on pregnancy outcomes and offspring cognition,increasing the risk of complications associated with adverse pregnancy outcomes,such as premature delivery,abortion,and low birth weight.Whether PFASs cause adverse birth outcomes by interfering with thyroid hormones and their related placental mechanisms remains unclear.As an important organ in the development of the fetus,the placenta provides glucose,amino acids and other nutrients for developing the fetus during pregnancy.Both placental dysplasia and fetal undernutrition can cause fetal intrauterine growth restriction.Thyroid hormones are crucial to the development of the fetus and placenta,and the fetus’s thyroid hormones depend on the mother’s supply.The placenta can express thyroid hormone receptors,and activation of the thyroid hormone receptors can activate the phosphorylation of AKT-mTOR and ERK signaling pathways,thereby regulating placental development and the function of nutrient transporters.Studies have confirmed that PFOS can have adverse effects on fetal growth and development through the placenta,but the underlying placental mechanism is still unknown.The main objective of this study was to clarify the relationship between PFOS exposure concentration,thyroid function,and offspring birth weight by detecting the PFOS in the urine of pregnant women in combination with the results of case-control studies in the population.A model was established by exposing pregnant mice with different concentrations of PFOS to determine the effects of exposure to PFOS during pregnancy on maternal-fetal thyroid function and intrauterine growth of offspring,and to explore the effects of PFOS exposure on thyroid hormone receptor signaling and the potential placental mechanism affecting intrauterine growth in young mice.Based on the correlation between PFOS,thyroid function and fetal weight and the possible biological pathway,we explored the specific mechanism by which thyroid hormone may mediate the relationship between PFOS and intrauterine growth restriction.Research methods:1.To clarify the relationship between PFOS exposure concentration,thyroid function,and offspring birth weight,we collected pregnant women in cities such as Shenyang and Dalian of Liaoning province（areas with an appropriate amount of iodine）who had lived locally for more than 5 years and who were in the first trimester of singleton pregnancy at the appropriate age（19-40 years old）.Fasting blood was collected in the morning,serum TSH,FT₄,TPOAb and TgAb were detected,and urine samples were collected in the morning.Except for other obvious thyroid diseases and complications during pregnancy,the offspring were divided into the group with small for gestational age（SGA）and the group with appropriate for gestational age（AGA）according to the follow-up.The relationship between PFOS,thyroid function,and offspring birth weight was clarified by detecting the concentration of PFOS in urine.2.To clarify the effects of PFOS exposure during pregnancy on maternal-fetal thyroid function during pregnancy and intrauterine growth and development of offspring mice,the pregnant mice were randomly divided into 6 groups,including solvent blank control groups（PFOS 0 mg/kg/day,PFOS-0）,PFOS exposure group:PFOS 1 mg/kg/day（PFOS-1）,PFOS 5 mg/kg/day（PFOS-5）,PFOS 10 mg/kg/day（PFOS-10）,PFOS 15mg/kg/day（PFOS-15）and PFOS 20 mg/kg/day（PFOS-20）.During the whole pregnancy（GD0-GD17）,PFOS was administered by gavage to establish a mouse poisoning model.All the mice were sacrificed at GD13 and GD18.The number of pups,abortions,stillbirths,and birth weights of the offspring mice were recorded.Collect serum from female and fetal mice,and the serum thyroid hormone levels were detected by ELISA.In order to further clarify the potential placental mechanism of PFOS exposure during pregnancy on offspring’s intrauterine growth restriction,female placenta was collected and partly used for total RNA extraction and then the mRNA expression levels detection of thyroid hormone-related receptors（TR,TSHR）,the nutrient thyroid hormone transporters,deiodinase and placenta-related growth factors,part of the female placenta was used for Western blotting to detect the protein expression levels of thyroid hormone receptors and mTOR,and part of the placenta was used for histopathology detection,The area of each part of the placenta in each group was compared by H&E staining.3.In order to further explore the possible biological pathways of PFOS mediating intrauterine growth restriction in offspring through THs,the pregnant mice were randomly divided into 4 groups,including solvent blank control groups（PFOS 0mg/kg/day,PFOS-0）,PFOS 20 mg/kg/day high concentration exposure group（PFOS-20）,PFOS 20 mg/kg/day high concentration exposure combined with thyroid hormone intervention group（P-20+LT₄）,PFOS 20 mg/kg/day high concentration exposure combined with thyroid hormone and rapamycin intervention group（P-20+LT₄+Ra）.The changes of the birth weight of offspring mice and the thyroid function of female mice were observed.Western blotting was used to detect the total protein and phosphorylation levels of AKT-mTOR-pS6K1 and ERK.The mRNA levels and related protein expression levels of amino acid transporters（SNAT1,SNAT2,and SNAT4）of placenta-related nutrient transporters and glucose nutrient transporters（GLUT1,GLUT3）were detected.Results:1.A total of 313 pregnant women were included,including 133 cases in the SGA group and 180 cases in the normal control AGA group.Comparing the general information of pregnant women in the two groups,the birth weight of offspring in the SGA group was significantly lower than that of the AGA group（p<0.05）,and the other baseline indicators had no statistical difference（p>0.05）.There was no difference in the levels of thyroid function.The urine PFOS level in pregnant women in the SGA group was higher than that in the AGA group,in pregnant women with TPOAb/TgAb positive,the PFOS concentration in pregnant women in the SGA group was significantly higher than that in the AGA group（p<0.05）.In the SGA group,when TSH≥4 mIU/L,the PFOS concentration was significantly higher than TSH<4mIU/L（p<0.05）.When TPOAb is positive or TgAb is positive,PFOS in urine is positively correlated with TSH;when TgAb is positive,PFOS is negatively correlated with FT₄.2.Exposure to PFOS at different concentrations during pregnancy did not affect the implantation rate and abortion rate of the offspring,but the exposure to PFOS caused a significant decrease in the birth weight of the offspring,and the incidence of intrauterine growth restriction（IUGR）in the PFOS exposure group increased significantly with the increase in the concentration of PFOS,showing an obvious dose-response relationship.Exposure to PFOS during pregnancy affected the changes of thyroid hormones in the serum of females and offspring,leading to a decrease in female TT₄ and TT₃ levels and an increase in TSH levels at GD18,which then resulted in a hypothyroidism effect.In the offspring,the concentration of TT₃ significantly decreased,and TT₄ showed an increasing trend.At GD13,only TSH increased,while TT₄ and TT₃ did not decrease significantly.After PFOS exposure,with the increase of PFOS concentration,the proportion of labyrinth area of the placenta decreased,while the proportion of junction area increased.With the increase of PFOS exposure during pregnancy,the gene expression of placental thyroid hormone receptor TR-αwas significantly down-regulated,while TR-βwas significantly down-regulated only in the high-concentration PFOS-20 group,and there was no significant difference in TSHR expression.The gene expressions of placenta T₃and T₄ transporters MCT8 and OATP1c1 were significantly decreased with the increase of PFOS concentration.Compared with the PFOS-0 group,the deiodinase 2（Dio2）gene expression was significantly down-regulated,while the deiodinase 3（Dio3）gene expression was significantly increased in the PFOS-20 group.The expressions of VEGF,GLUT1 and SNAT1 genes in PFOS-15 group and PFOS-20 group were significantly down-regulated.PFOS exposure can significantly inhibit the protein level of TR-αand the phosphorylation level of mTOR,but has no significant effect on TSHR.3.After the LT₄ intervention,the birth weight of the offspring in the P-20+LT4group was significantly higher than that of the P-20 group,but still significantly lower than that of the control group.PFOS-20 can significantly inhibit the phosphorylation levels of AKT-mTOR-pS6K1 and ERK signaling pathways.LT₄ can partially restore the phosphorylation levels of AKT-mTOR-PS6K1,but cannot restore the phosphorylation levels of ERK.When m TOR pathway was inhibited by rapamycin,the phosphorylation level of ERK was restored to a certain extent.After the administration of LT₄,the gene expression of placental nutrient transporters GLUT1,SNAT1,and SNAT2 was up-regulated,but not significantly（p>0.05）,and the protein level of GLUT1 recovered significantly.Conclusion:Exposure to PFOS during pregnancy can cause hypothyroidism and intrauterine growth restriction in offspring.PFOS interferes with thyroid hormones to a certain extent,through inhibit phosphorylation of AKT-mTOR-PS6K1 and ERK,causing nutrient transport disorders and resulting in intrauterine growth restriction of offspring.