An In Vitro Investigation Of Endocrine Disrupting Effects Of Steroid Hormone

Endocrine disrupting compounds?EDCs?have been attracted great concerns because of their harmful impacts.With the further research,the steroids draw people's attention.They mainly exist in the incomplete treated drainage of feedlot and wastewater treatment plant.Therefore,it makes great significance to establish and study the disrupting effects of steroid hormone.Firstly,we developed a method to analyze 4 commonly used steroid hormone intermediates by SPE/OSE-HPLC analysis,and then applied to analyze their concentrations in sewage treatment plants of one pharmaceutical plant in Zhejiang Province.Secondly,a human glucocorticoid receptor??hGR??mediated dual-luciferase reporter gene system to qualitatively and quantitatively evaluate the endocrine disrupting effects was adopted.Finally,the study was followed-up by investigations of steroldogenesis gene expression in human adrenoeortical carcilnoma cell?H295R?using qRT-PCR.The research contents and results are as followings:?1?To develop a pre-treatment method of SPE,the influential factors such as the elution solvent and volume,washing solvent were investigated.At 10 mg/L,20 mg/L and 40 mg/L spiking levels,the recoveries were bigger than 79.88%,and relative standard deviations?RSD,n=3?were less than 11.58%,Presenting certain stability.Additionally,to develop a pre-treatment method of OLE,the influential factors such as the extration solvent and volume,extration time were investigated.At 50 mg/L,100mg/L and 150 mg/L spiking levels,the recoveries were bigger than79.17%,and relative standard deviations?RSD,n=3?were less than11.66%,presenting certain stability.With methanol as mobile phase,the four compounds can realize complete separation within 20 minutes by HPLC.Using external standard method for quantitative analysis,the correlation coefficients of standard curve are good.Under the optimized conditions,the limits of detection are from15²0?g/L,the limits of quantification are from 50¹20?g/L?In the actual samples,the content of AD?androstenedione?is 36.35 mg/g,HEP?11?-hydroxy-16?,17-epoxyprogesterone?is1.42mg/L,21-DOF?21-deoxycortisol?is 37.99 mg/L,and 21-DOE?21-deoxycortisone?was measured in the both sampls,the content is 194.35mg/L and 78.82 mg/g,respectively.?2?Results from the dual-luciferase reporter gene assay demonstrated that AD,HEP,21-DOE,21-DOF all exhibited the hGR?antagonistic activity.The significant inhibition of 5×10^-88 HC was observed at 10^-77 M for AD,HEP and at 10^-66 M for 21-DOE,21-DOF.In addition,AD,21-DOF,21-DOE and HEP inhibited the glucocorticoidactivity via hGR?with RIC₂₀values at 2.69×10^-88 M,2.63×10^-88 M,4.47×10^-77 M and 1.02×10^-77 M,respectively.It was our first attempt to adopt the human glucocorticoid receptor??hGR??mediated dual-luciferase reporter gene system to qualitatively and quantitatively evaluate the endocrine disrupting effects of four steroid intermediats.?3?After a 24 h exposure 10^-88 M,10^-77 M and 10^-66 M tested chemicals to H295R cell,qRT-PCR was used to detect mRNA levels of steroidogenesis genes.Results demostrated that all the tested chemicals could surpress the expression of HMGR,StAR and stimulate the expression of CYP11B1,CYP11B2;mRNA level of other related genes were altered by inhibition or stimulation.This further shows that AD,HEP,21-DOE and 21-DOF have endocrine disrupting effects at molecular gene level for the first time.