| Objective:Thyroid cancer is a common endocrine malignancy.According to the data of annual cancer report in China,breast cancer is the first malignant tumor in urban women,followed by lung cancer,colorectal cancer,thyroid cancer and gastric cancer.With the improvement of examination methods and the health awareness of the population,the incidence of thyroid cancer shows an increasing trend year by year,which is the highest in coastal areas,lower in inland areas,and higher in urban areas than in rural areas.The incidence of thyroid cancer was significantly different between the sexes,with a ratio of about 1:3 to 1:4 between men and women.In addition,the incidence of thyroid cancer in women of reproductive age was higher,and the incidence of postmenopausal showed a gradual decline.As the incidence of thyroid cancer increases year by year,more and more studies have been focus on its pathogenesis,but the mechanism is still unclear.Radiation,excess iodine,endocrine disorders and genetic factors are known to be related to the occurrence of the thyroid disease.Changes in thyroid physiological function and abnormal expression of related genes also affect thyroid hormone synthesis,secretion,transport and metabolism.At the same time,external stimuli such as thyroid disruptors also affect the occurrence and development of thyroid tumors and related diseases.In particular,the environmental endocrine disruptors widely exposed in human life in recent years will interfere with the hypothalamic-pituitary-thyroid axis,disturb thyroid hormone homeostasis,and affect the normal physiological function of thyroid.Among them,as the main raw materials and plasticizers of plastic products,di(2-ethylhexyl)phthalate(DEHP)and bisphenol A(BPA)are widely used in daily production and our life,which can be detected in the environment and human body,and have become relatively serious chemical pollutants.In consideration of the existence of multiple mixtures of environmental pollutants in our life,the diversification of human exposure,and the mixed mode may present different toxic effects from the individual substances,so the study on the toxic effects of a single pollutant can no longer meet the actual needs.As one of the target organs of DEHP and BPA,it is still unclear whether exposure to alone or in combination will affect the development of thyroid cancer and the mechanism of action.At first,the study used a 3×3 factorial design to confirm the effects of adolescent exposure to DEHP and BPA on thyroid function and hormone homeostasis,and found that the interaction between the two interfered with thyroid function.Suggesting a long-term alone and joint exposure of EDCs can increase the susceptibility of thyroid cancer,through the construction of environmental endocrine disruptors exposed animal model clarify phthalate(2-ethyl)with bisphenol A alone and joint exposure via TSHR activate the downstream signal transduction pathways,inducing HDAC6 expression,and regulating tumor suppressor gene PTEN,promoting thyroid cancer.The present study will provide reasonable safety evaluation of environmental endocrine disruptors,and novel theoretical basis of the preventive for thyroid related diseases.Methods:Firstly,according to the 3×3 factorial design,63 healthy 4-week-old female SD rats were used.DEHP(0,150 mg/kg,750 mg/kg)and BPA(0,20 mg/kg,100 mg/kg)were gavaged for 42 days.Observed growth situation,recorded the rats weight and organ coefficient.Tissues of rats in formalin fixed,paraffin embedding tissue section,microscopic observation rat thyroid pathological changes after HE staining.Then detect the levels of TT3,TT4,FT3,FT4 and TSH in the blood of rats by enzyme linked immunosorbent(ELISA).STITCH database was used to identify potential targets for DEHP and BPA,and Cytoscape software was used to perform a DEHP-BPA-target interaction network.KEGG pathway enrichment analysis was performed on potential targets of DEHP and BPA using DAVID database.The m RNA and protein expressions of ESR1 and its downstream transcription factor CREB were detected by RT-PCR and immunohistochemical staining(IHC).Secondly,160 healthy female SD rats of 4 weeks were used to perform the rat thyroid tumor model,which was randomly divided into the carcinogen DMD(diethylnitrosamine DEN,n-methyl nitrosamine MNU and diisopropanol nitrosamine DHPN)(DA groups)and sham administration group(DN groups).The DA group was divided into four groups:control,DEHP150 mg/kg,BPA20mg/kg and DEHP150 mg/kg+BPA20 mg/kg groups,with 20 rats in each group,and the DN group was also divided into the above four groups.Namely DA group animals according to 100 mg/kg intraperitoneal injection of DEN on the first day,and on the 5,8,11,14 days according to 20 mg/kg intraperitoneal injection of MNU,the first week and the third week according to the 0.1%DHPN by water,animals in DN group given saline injections and daily drinking water.Then animals were given DEHP and BPA alone and combination for 30 weeks,and the growth status,body weight of rats in each group were observed and recorded during the experiments.At the end of experiments,all tissues of the rats were retained,the changes of organ coefficients of the rats were analyzed.After HE staining,the thyroid tissues were scanned under the microscope,and the films were read by the pathologists.The incidence and types of thyroid tumors of the rats in each group were calculated.The expression of tumor markers PCNA and Tg in thyroid tissues of rats in each group was determined by IHC,and the results were analyzed by IHC score.ESR1,TSHR,HDAC6,PTEN and c-MYC protein expressions and phosphorylation of CREB and AKT in the thyroid tissues of rats in each group were detected by westernblot,further revealing the promotion of DEHP and BPA alone and combination on thyroid neoplasia and the possible mechanism.Finally,human thyroid cancer cells BCPAP and normal thyroid epithelial cells Nthy-ori3-1 were treated with MEHP and BPA alone or in combination for 24 h and 48 h.CCK8 was used to detect the proliferation of MEHP and BPA on thyroid cancer cells.Immunofluorescence and transwell assay were used to detect the expression of Ki67 and PCNA and cell migration ability.The protein expressions of HDAC6,PTEN and c-MYC,the phosphorylation of CREB,Akt and ERK were detected by westernblot,and the m RNA changes of HDAC6 and PTEN were detected by RT-PCR.HDAC6 knockout were performed by small interfering RNA.Meanwhile,Tub A,a selective inhibitor of HDAC6,was used to treat the cells.The changes of related factors in the downstream signaling pathway were detected by westernblot to verify that MEHP and BPA inhibit PTEN through HDAC6 and activate downstream AKT phosphorylation.Ch IP experiment testing histone H3K9ac enrichment of PTEN gene promoter region in thyroid cancer cells.Then westernblot and PCR detection H3K9ac levels and PTEN gene expression after inhibit HDAC6 respectively,further confirmed that the inhibitor of HDAC6 can inhibit the effects of BPA and MEHP,such as the inhibition of PTEN and activation of AKT signaling pathways,mainly through the promoter region histone H3K9acetylation modification PTEN expression.Results:1.In the 42 days study,the body weight of the rats in each group increased steadily,with no significant difference between the groups.Compared with the control group,the thyroid organ coefficient of the DEHP750+BPA100 treatment group was significantly lower,and even than that of the DEHP750 treatment group alone(P<0.05).ELISA showed that the serum TT3 levels of the DEHP150 and BPA exposed groups were significantly lower than that of the DEHP150 exposed group alone(P<0.05),and the TT3level of DEHP750+BPA20 group was significantly lower than that of the DEHP750exposed group alone(P<0.05).Compared with the control group,the serum TT4 levels of rats exposed to DEHP750,BPA and the combination of them were significantly lower(P<0.05),while the combined exposure of DEHP and BPA further reduced the TT4 levels of rats exposed to DEHP alone(P<0.05).That is,combined exposure of DEHP and BPA interfered with thyroid hormone homeostasis,while serum FT3,FT4 and TSH levels of rats in each treatment group were not significantly changed compared with the control group.Pathological results showed that the number of follicular epithelial cells in thyroid tissues of rats exposed to DEHP750 and BPA100 increased,and DEHP750 was significantly higher than that of the control group(P<0.05).2.STITCH,Swiss Target databases were used to identify potential targets for DEHP and BPA,and Cytoscape software was used to visualize the DEHP-BPA-target network.Among them,ESR is the common target of the two compounds,suggesting that ESR may act as an intermediary between DEHP and BPA.KEGG pathway enrichment analysis focus on the estrogen receptor signaling pathways in the top 10.Then we tested the ESR and downstream related transcription factors CREB phosphorylation of thyroids in rats,the results showed DEHP750 was significantly increased the expression of ESR1(P<0.05)and phosphorylation levels of CREB(P<0.05).However,the expression of ESR1 in DEHP750+BPA100 group was significantly down-regulated compared with DEHP750(P<0.05),which preliminarily evaluated the effect of EDCs mixed exposure on thyroid hormone homeostasis.3.During the long-term exposure of DEHP and BPA alone and in combination,the growth of the rats in DN group(without DMD pretreatment)was stable,the food intake and body weight increased gradually,the reaction was rapid,the body hair was thick and smooth,the vulva was dry,and the stool was granular.The rats in group DA(pretreated with DMD)grow slowly,showed low spirits,and wet hair around the vulva,with more loose stool.The death time of the rats in the DMD group was earlier,but there was no significant difference between the survival curve of the control group.However,after the pretreatment with DMD,the rats in the treatment group in DA group all grow slowly.Compared with the control group,the weight of the rats in the treatment group in DA group showed a significant decrease(P<0.05).In the DN group,there was no significant change in the thyroid viscera coefficient of the rats in each treatment group,the liver coefficient of the rats in the DEHP treatment group was significantly higher than that in the control group(P<0.05),while no significant change was observed in other organ coefficients.In DA group,each rat thyroid organ coefficient of treatment group were significantly lower than control(P<0.05),significantly increased the organ coefficients of liver and kidney(P<0.05),including BPA and DEHP combined treatment group rats renal organ coefficient was also significantly higher than the two individual treatment group(P<0.05).4.In the DN group,thyroid neoplasms were not observed in either treatment group.The number of follicular epithelial cells in the thyroid tissues of rats treated with DEHP and BPA alone increased significantly,and the diameter of thyroid follicles decreased.Moreover,significant pathological changes such as follicular fragmentation were observed in the thyroid tissues of rats treated with DEHP and BPA alone.In group DA,a variety of pathologic changes were observed:papillary carcinoma,myeloid carcinoma,precancerous lesion(hyperplasia or adenoma),and partial inflammatory response.In the DA group,the incidence of thyroid tumors in BPA and DEHP+BPA treated group was significantly higher than that in the control group(P<0.05).The incidence of precancerous lesions in each treatment group was statistically different from that in the control group(P<0.05).The positive rate of PCNA and Tg in thyroid tissues of rats in DN group was not significantly different from that in the control group.DA group,BPA and DEHP+BPA treatment group had a significantly higher positive rate of PCNA thyroid tissue in rat control group(P<0.05),at the same time,BPA and DEHP separate and combined treatment group rat thyroid Tg strong positive rate was significantly higher than control group(P<0.05),indicating successful,thyroid tumor model in rats was established and proved DEHP and BPA exposure of the carcinoma in tumorigenesis.5.Further analysis was made on the oncogenic mechanism of DEHP and BPA exposure alone and in combination on thyroid neoplasms in rats.In DN group and DA group,no significant difference was observed in the expression of thyroid ESR1 protein in rats treated with DEHP and BPA alone and in combination with control group.In the DN group,BPA and DEHP+BPA treatment group rat thyroid TSHR protein expression was significantly higher than that of control group(P<0.01),and thyroid both single and combined treatment group rats after CREB phosphorylation level was also significantly higher than that of control group(P<0.05),BPA and DEHP thyroid HDAC6 express individual treatment group rats increases,the joint treatment group is significantly higher than control(P<0.05),the treatment group rats thyroid PTEN protein expression and AKT phosphorylation levels not seen significant change.In DA group,the treatment group rats thyroid TSHR expression showed a trend of increase,CREB phosphorylation level of BPA and DEHP alone and combined treatment group were significantly higher than control(P<0.05),the HDAC6 protein expression are on the rise,DEHP and DEHP+BPA treatment group are significantly higher than the control group(P<0.05),the of PTEN expression of each groups were significantly lower,BPA and DEHP+BPA treatment groups were significantly lower than the control group(P<0.05),The levels of thyroid AKT phosphorylation in all treatment groups were significantly higher than those in the control group(P<0.05).6.Both MEHP and BPA could significantly induce the proliferation of BCPAP cells in the concentration range of 10-9?10-6mol/L.After treatment with 10-7mol/L alone or in combination for 24 h,the expression of Ki67 in the nucleus and the migration ability of BCPAP cells were significantly increased(P<0.05).BPA alone and combined with MEHP could significantly induce CREB phosphorylation and accumulation into the nucleus,and then up-regulate HDAC6 protein and m RNA expression after 24 h and 48 h treatment(P<0.05).BPA combined with MEHP could significantly inhibit PTEN protein and the m RNA levels in BCPAP cells after 48 h treatment(P<0.05),and had significant inhibitory effect on cytoplasmic membrane PTEN(P<0.05)and activate downstream Akt phosphorylation.BPA alone and combined with MEHP also induced significantly up-regulation of c-MYC protein expression after 48 h treatment(P<0.05).At the same time,the total amount of?-catenin protein in BCPAP cells treated by BPA alone or combined with MEHP for 24 h and 48 h did not change significantly,but the immunofluorescence detection showed that the nucleation of?-catenin in BCPAP cells treated with BPA alone or combined with MEHP for 24 h were increased significantly.7.HDAC6 was inhibited by small interfering RNA and HDAC6 selective inhibitor Tub A,respectively.The expression of PTEN protein was not significantly changed,but the phosphorylation of Akt was significantly down-regulated(P<0.05).On this basis,the cells were treated with MEHP and BPA alone or in combination,and it was found that the inhibition of PTEN disappeared,and the activation of Akt phosphorylation of BCPAP cells by MEHP and BPA was also blocked,and the c-MYC induction of BCPAP cells by MEHP and BPA alone or in combination was inhibited.The enrichment of H3K9Ac in the PTEN promoter region of BCPAP cells was confirmed.After inhibiting HDAC6,MEHP and BPA were given separately or in combination,the protein expression of H3K9Ac in BCPAP cells was significantly up-regulated(P<0.05),and the m RNA level of PTEN was significantly up-regulated compared with the control group(P<0.05).8.In addition,MEHP and BPA could significantly induce the proliferation of Nthy-ori3-1 cells in normal thyroid epithelium at10-6 mol/L,and the expression of PCNA in the nucleus was significantly increased after treated with MEHP and BPA alone or in combination after 24h treatment.Nthy-Ori3-1cells treated with BPA alone or combined with MEHP for 24 h and 48 h could significantly induce CREB protein phosphorylation and up-regulate HDAC6 protein and m RNA expression(P<0.05),but the protein and m RNA levels of PTEN were not significantly changed,and the plasma membrane PTEN was not significantly changed,and the phosphorylation level of Akt serine(Ser473)was significantly upregulated after 48 h treatment with BPA alone or combined with MEHP(P<0.05).After 24 h and 48 h treatments,BPA combined with MEHP could significantly induce ERK phosphorylation in Nthy-ori3-1 cells(P<0.05),the level of ERK phosphorylation was significantly decreased after HDAC6 knockdown.On this basis,the cells treated with MEHP and BPA alone or combined with MEHP partially recovered the activation of Akt phosphorylation in Nthy-or3-1 cells,while the level of ERK phosphorylation in MEHP alone group was still significantly lower than that in the control group.Conclusion:1.High-dose exposure to DEHP and BPA leads to proliferation of thyroid follicular epithelial cells in rats.In addition,DEHP alone can up-regulate the expression of ESR1 in adolescent rat thyroid tissues,while combined exposure with BPA may reduced the up-regulation caused by DEHP alone,and the combined effect of them may interfere with thyroid hormone homeostasis through ESR1-related signaling pathways.2.Long-term exposure to BPA alone or in combination with DEHP can promote thyroid tissue hyperplasia in female rats,up-regulate HDAC6,and increase the expression of oncogene c-myc.3.Long-term combined exposure of DEHP and BPA has no carcinogenicity on the thyroid,but can produce cancer-promoting effects,leading to an increase in the incidence of thyroid tumors in rats.In addition,BPA can enhance the effect of DEHP alone exposure,up-regulate the expression of HDAC6 protein,inhibit the tumor suppressor gene PTEN,and activate the phosphorylation of downstream AKT.It also induces the expression of the oncogene c-MYC and increases the susceptibility to thyroid tumors.4.BPA alone and in combination with MEHP,the metabolite of DEHP,can significantly induce the proliferation and migration of human thyroid cancer cells BCPAP,which is dependent on HDAC6.It can modify H3K9ac in the promoter region of tumor suppressor gene PTEN to inhibit PTEN,activate downstream AKT signal pathway,and simultaneously up-regulate the expression of oncogene c-MYC.5.BPA alone and in combination with MEHP can significantly induce the proliferation of human thyroid normal epithelial cells Nthy-ori3-1,which may be partially dependent on HDAC6,and then activate downstream ERK signaling pathway. |
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