Studies On The Endocrine Disrupting Effects Of Phthalates And The Molecular Mechamisms Of Their Effects On Thyroid Hormone Disrupting

Phthalate is a class of chemical compounds widely used in industrial activities. They are mainly used as plasticizers for polyvinyl chloride (PVC) resins, adhesives, and cellulose film coating. Phthalates can be easily released into the environment through volatilization and leaching from plastics during the process of production of plastics and plastic materials or their disposal. Organisms can bioaccumulate phthalate compounds and the subsequent adverse effects on the environment and organisms can not be ignored.PartⅠAssessing effects of phthalates on endocrine disrupting by reporter gene assaysObjectiveThe estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) mediate luciferase reporter gene assays were used to detect (anti)estrogen, (anti) androgen and (anti) thyroid hormone activities of di-2-ethylhexyl phthalate (DEHP), di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP), respectively. And the hormone activities of the test chemicals were compared to each other.Method1. ER mediate luciferase reporter gene assay: The CV-1 cell line was co-transfected with the rat ER expression vector rERα/pCI and the luciferase reporter plasmid pERE-aug-Luc. ER agonist and antagonist activity were generally performed by quantifying the induction of the reporter gene Luc product by the test chemicals and measure the ability of test compounds to inhibit the induction of Luc caused by E2, respectively.2. AR mediate luciferase reporter gene assay: The MDA-kb2 cell line, stably transformed with murine mammalian tumor virus (MMTV)-luciferase was treated with compounds alone and with flutamide, the potent antagonist of AR to detect the AR agonist activity of the test chemicals. And the MDA-kb2 cell line was exposed to chemicals alone with DHT to investigate the AR antagonist activity of the compounds.3. TR mediate luciferase reporter gene assay: The CV-1 cells were transfected with pGal4-L-TRβwhich was an expression vector coding for the ligand binding domain (LBD) of TRβfused to the DNA binding domain of Gal4, and Gal4 responsive luciferase reporter pUAS-tk-luc. TR agonist and antagonist activity were generally performed by quantifying the induction of the reporter gene Luc product by the tested chemicals and measure the ability of tested compounds to inhibit the induction of Luc caused by T3, respectively.Result1. ER mediate luciferase reporter gene assay: For DEHP and MBP, the induction of luciferase was not significantly higher than vehicle control, while DBP increased the expression of Luc when it reached the maximal induction of 2.6-fold at high concentration. None of the compounds could inhibit the Luc expression induced by E2.2. AR mediate luciferase reporter gene assay: DBP, MBP and DEHP induced the expression of luciferase with EC50 of 6.17×10?6, 1.13×10?5 M and exceeding 1×10?4, respectively. Co-treatment of chemicals with 1.0×10?5M flutamide significantly reduced expression of the luciferase gene. MBP possessed stronger agonist activity than DBP. When the test chemicals were co-administered with 1.0×10?9 M DHT, each suppressed the luciferase expression significantly. The IC50 value of DBP, MBP and DEHP was 1.05×10?6, 1.22×10?7 M and exceeding 1×10?4 M, respectively. The strength of inhibition of the tested phthalates was DEHP< DBP <MBP.3. TR mediate luciferase reporter gene assay: DBP, MBP and DEHP suppressed the expression of Luc induced by 5×10-9 M T3 with IC50 of 1.31×10?5, 2.77×10?6M and exceeding 1.0×10?4 M, respectively. DBP and MBP exhibited the stronger antagonist activity than DEHP, especially the metabolite. None of them could induce the expression of the luciferase. Conclusion1. Neither DEHP nor MBP had estrogenic activity, while DBP displayed weak estrogenic activity. None of them possessed anti-estrogenic activity.2. The test chemicals displayed androgenic activity. The strength of androgenicity of the test phthalates was DEHP< DBP <MBP, and the metabolite MBP possessed stronger agonist activity than DBP. The test chemicals displayed anti-androgenic activity. The strength of antiandrogenicity of the test phthalates was DEHP< DBP <MBP. MBP possessed stronger antagonist activity than DBP.3. The test chemicals displayed anti-thyroid hormone activity. The strength of anti-thyroid hormone activity of the test phthalates was DEHP< DBP <MBP. MBP possessed stronger anti-thyroid hormone activity than DBP. None of them possessed TR agonist activity.4. The three test phthalates simultaneously act as agonists and/or antagonists via two or more hormonal receptors to interfere endocrine system.PartⅡAssessing the effects of phthalates on thyroid hormone system using Xenopus laevis and exploring the potential molecular mechanismsObjectiveAssessing the effects of phthalates on thyroid hormone system using Xenopus laevis and exploring the molecular mechanisms of the effects.Methods1. Groups of twenty tadpoles reached stage 51were placed in 5L of deionized water containing nominal concentrations of 2, 10, 15 mg/l DBP and MBP, 1nM T3 and 0.005% DMSO as solvent control for 21 days. On day 22, developmental stage, whole body length, interocular distance and body-to-tail length ratio were determined in all tanks.2. Head and tail tissues of three randomly selected individuals tadpoles from each group were used for RNA extraction. Expression of TRβ, RXRγ, TSHαand TSHβmRNA in head and TRβ, RXRγmRNA in tail were analyzed by quantitative real-time polymerase chain reaction (qPCR). Considering the difference stages of tadpoles may be a factor that would interfere the expression of the target genes, we also analyzed the expression of these genes of the tadpoles reached stage 57 in different groups.3. Using mammalian two-hybrid assay in vitro, we investigated whether DBP or MBP can affect the interaction between co-repressor Silence Mediator of Retinoic acid and Thyroid hormone (SMRT) and thyroid hormone receptor.4. Genomic DNA was isolated from brain and tail pools of different groups of X. laevis. Three samples of head or tail were mix together as a pool and each treatment group contain two pools of head and tail. Bisulfite sequencing was used to characterize the DNA methylation patterns and changes at the promoter region of TRβgene in X. laevis.Results1. The median stage of tadpoles immerged in 15 mg/l DBP and MBP was stage 54 and 53, while tadpoles kept in the solvent control and positive control reached stage 57 and 58 respectively. The test chemicals did not affect the body-to-tail length ratio of the tadpoles, but significantly reduced the interocular distance at the highest concentration. And it should be noticed that the whole body length of tadpoles treated with 10 and 15 mg/l MBP was significantly less than solvent control while only 15 mg/l DBP was.2. When determined in head tissue of tadpole exposed day 21, TRβand RXRγmRNA expression was significantly increased in tadpoles exposed to T3, while no significant differences were detectable in TSHαand TSHβmRNA expression between solvent control and T3 treatment groups. The expression of TRβand RXRγmRNA was decreased by DBP and MBP in a concentration-dependent manner. Nominal TSHαand TSHβinduction compared to solvent control was noted in the higher concentration treated groups of DBP. However, mRNA expression of both TSH subunits were dramatically increased in low concentration 2 mg/l DBP and MBP. Interestingly, the tails had different patterns of TRβand RXRγmRNA expression. In contrast with the down-regulation of TRβmRNA caused by DBP and MBP in head tissues, the expression of TRβwas up-regulated in tail tissues, especially in the treatment groups of 2 mg/l DBP and MBP. The mRNA expression of RXRγin T3 treatment group was decreased, whereas the levels of RXRγmRNA were dose-dependently increased by DBP and MBP, which was completely opposite of the RXRγexpression pattern in the head. In the head tissue, different groups of tadpoles reached stage 57 had the same tendency of TRβ, RXRγand TSH subunits expression with the tadpoles at different stages except for down-regulation of RXRγand TSHβboth caused by T3. But in tails the expression of TRβand RXRγwere totally different from the tadpoles at different stages. The expression of TRβwas significantly decreased in all concentrations of DBP and MBP treatment groups, especially in MBP. However, almost no significant differences to the solvent control group were detected in mRNA expression levels of RXRγin response to T3, DBP or MBP in tail tissue.3. DBP and MBP enhanced the interactions between SMRT and TR in a dose-dependent manner even though the addition of 1nM T3 released the interactions.4. A total of 240 and 242 clones were studied for TRβgene in head and tail tissue of X. laevis respectively. We here show that the promoter region of TRβwas almost fully methylated (96.8%, with CpG 11 unmethylated in one clone) in head of X. laevis, whereas only 62% of the clones were completed methylated in tail tissue. Complete promoter methylation in the TRβgene was identified in head and tail tissues in all groups. In the head tissue, the frequency of clone with complete methylation in all treated groups is lower than in solvent control groups, especially the groups treated with 2 and 10 mg/l MBP (P < 0.05). However, no significant difference in tail was found except for the group of T3. Notably, CpG 2 was highly hypomethylated in tail of T3 group compared with solvent controls (P < 0.05). Conclusion1. DBP and MBP could lead to retardation of metamorphic development of X. laevis, and the MBP seemed to possess stronger activity.2. DBP and MBP can affect the metamorphic development of X. laevis throuth several molecular mechanism, including affecting the gene expression, enhanceing the interactions between co-repressor SMRT and TR, the methylation status of promoter region of TRβgene. The current findings highlighted the danger of DBP and MBP as environmental thyroid disruptors, and provided the information in vivo for the risk assessments of the two compounds for human health.