Effects Of Prenatal DEHP Exposure To Hypothalamus-pituitary-thyroid Axis Of Adult Offspring Rats

Objective:The major objective is to investigate the potential interference effects of prenatal DEHP exposure through detecting the expression of related gene in hypothalamus,pituitary gland and HPT axis and changes of thyroid morphology and function of adult offspring rats.Methods:(1)32 pregnant rats were randomly assigned into four groups(n = 8 per group)and treated with corn oil(vehicle control),2 mg/kg,10 mg/kg or 50 mg/kg DEHP by gavage from G14 to G19.The dosing volume was 1 ml/100 g.Postnatal day 0(PND 0)was determined by the birth of pup.From P21,the pups were weaned and were housed four same-sex littermates per cage.(2)On PND70,one male and one female rat per litter(n = 8 per sex per group)were randomly chosen.The rats were perfused by 4% paraformaldehyde(PFA)through the transcardial pathway.Thyroids were fixed in paraformaldehyde,processed and trimmed,embedded in paraffin,sectioned to a thickness of 6 ?m and stained with hematoxylin and eosin(H&E)for microscopic examination.(3)Randomly select one male and one female rat in the same way as above.After decapitation,the trunk blood was collected for serum hormone assays.Full coagulation was allowed at room temperature.Serum was separated after centrifugation at 700 g for 10 minutes and then stored at-80?.Serum total TSH,T4 and T3 concentrations were detected by Luminex xMAP technique.(4)Rat brain were collected and prepared into 1mm thick slices,according to rat brain stereotaxic atlas,determine the hypothalamic arcuate nucleus and paraventricular nucleus position and use a with punch needle with a diameter of 1.25 mm for punching.The relative expression levels of mRNA of Trh,Trh receptor,Tsh,Npyand Npy-Y1 receptor were determined by real-time fluorescent quantitative polymerase chain reaction(RT-PCR).Results:(1)The thyroid of DEHP treated animals exhibited signs for hyperactivity,as indicated by an increase in the number of thyroid follicular epithelial cells and the cytoplasm that appeared foamy and vacuolated was observed in the enlarged follicular.Follicular cavity was smaller.(2)There was no significant difference in serum TSH level of female rats between the treatment groups.The concentration of TSH in serum were differences between each group of male rats.Compared with the control group,the serum TSH level in 10mg/kg and 50mg/kg DEHP treatment group decreased(P<0.05);There was no significant difference in serum T4 level of female and male rats;The concentration of T3 in the serum were difference between each group of female rats.Compared with the control group,the serum T3 level in the 2mg/kg DEHP treatment group was lower(P<0.05).The concentration of T3 in serum were no difference between each group of male rats.(3)There was no significant difference in the expression of Trh gene between each group of female and male rats;There was no difference in the expression of Trh receptor gene between each treatment group of female rat.The expression of Trh receptor gene in the pituitary gland of male rats was significantly different between the treatment groups.Compared with the control group,the expression level of Trh receptor gene in 2mg/kg and 10mg/kg DEHP group were higher(P<0.05).(4)There was no significant difference in the expression of Tsh gene in female and male rats.(5)The expression of Npy gene in the female rats were no difference between each group.The expression of Npy gene in male rats was significantly different between each treatment group.Compared with the control group,the expression level of Npy gene in 50 mg/kg DEHP treatment group decreased(P<0.05);There was no significant difference in the expression of Npy-Y1 receptor gene in female and male rats between each treatment group.Conclusions:(1)Prenatal DEHP exposure could interferes with the structure and function of thyroid tissue of adult offspring rat.(2)The mechanism of prenatal DEHP exposure on the thyroid gland may be related to the interference with the expression of Trh receptor mRNA and the Npy mRNA in the hypothalamic ARC nuclear area which regulating the synthesis of TRH of the HPT axis.(3)The mechanism of prenatal DEHP exposure on the thyroid gland may have sex difference.(4)The effect of prenatal DEHP exposure may have low-dose effects on adult offspring rats.