Study On Receptor Reporter Gene Assays For Screening Environmental Endocrine Disruptors

The receptor reporter gene assay, also called receptor transcriptional activation assay, is an in vitro methods to screen the environmental endocrine disruptors (EEDs) proposed by the US EPA. On the basic of receptor-mediated theory, the receptor reporter gene assays are designed to identify substances that might interfere with normal hormone activity by acting as an agonist or antagonist through ligand-receptor interaction. The gene containing hormone responsive elements (HRE) was cloned into a reporter gene plasmid using gene reconstructing technique, and then the reporter gene was regulated by the HRE. The constructed reporter plasmid and the expression plasmid containing the receptor gene were cotransfected into a host cell to create an artificial gene expressionsystem. The receptor reporter gene assays provide a relatively simple way to indirectly reveal whether a substance can activate or inhibit the transcriptional activation of receptor-regulated genes by the measurement of the reporter gene product, typically an enzyme or a protein. The assays have an advantage over the receptor binding assays because they measure the biological response to receptor binding (i.e., RNA transcription) and thus, unlike receptor binding assays, can distinguish an agonist from an antagonist. In this study, the African green monkey kidney cell CV-1 was cotransfected with two different types of plasmids including expression plasmid containing the cDNA of the receptor and reporter plasmid containing the reporter gene regulated by HER to create the AR and ER reporter gene assays, respectively.Part â…  Study on the androgen receptor reporter gene assayObjectiveIn order to provide a model for screening the (anti)androgen effects of chemicals, the AR reporter gene assay was developed using the chloramphenicol acetyl transferase (CAT) as reporter gene. The assay was applied to evaluate the (anti)androgen effects of some pesticides including organochlorine pesticide p'p-dichlorodiphenyldichloroethylene (p'p-DDE), pyrethroid pesticide fenvalerate (Fen), organophosphorus pesticide phoxim (Pho) and alkylphenol chemicals including octylphenol (OP), nonylphenol (NP), bisphenol A (BPA). MethodsThe oligonucleotides of the mouse mammary tumor virus longterminal repeat (MMTV-LTR) containing four androgen responsive element (ARE) sequences and TATA promoter were inserted into the kpn I and Bgl II sites of pCAT3-basic to construct the new plasmid pMMTV-CAT. The CV-1 was cotransfected with three plasmids including the AR expression plasmid AR/pcDNA3.1 containing the cDNA of AR, constructed reporter plasmid pMMTV-C AT containing the CAT regulated by ARE and the control plasmid pSV-P-Gal to create the AR reporter gene assay. The reference androgen 5oc-dihydrotestosterone (5oc-DHT) was used to verify the performance of the assay. AR agonism was generally performed by quantifying the induction of the reporter gene CAT product in response to activation of the AR by the test chemicals. AR antagonism was performed by measure the ability of a test substance to inhibit the induction of the reporter gene CAT product by 5oc-DHT. Result(1) The sensitivity of the assay was 2.06xl0^-10M 5a-DHT. The intro coefficient of variation (CV) of the induction of 5oc-DHT was 9.3%, and the inter CV was 10.9%. The reference androgen 5a-DHT showed no significant effect in inducing CAT activity, when the CV-1 cell was not transfected with AR expression plasmid AR/pcDNA3.1. While the CV-1 cell was cotransfected with the three plasmids, the CAT activity was induced obviously at 10-9M and the maximal magnitude of the fold induction of > 70 fold of that of the negative control was achieved at 10-8M. The maximal effect contained at a plateau level at the ranges of 10-8M-10-6M. The CAT activity could not be induced by the glucocorticoid receptor (GR) agonist dexamethasone at the tested dosages. At the concentration ranges of 10-7M -10-5M, nilutamide significantlyHinhibited the CAT activity induced by 0.5nM 5a-DHT.( 2 ) p'p-DDE, Fen and Pho did not show androgeni...