ABSTRACT
cDNAs encoding alligator (caiman), Caiman crocodilus and whiptail lizard, Cnemidophorus uniparens estrogen receptors (ERs) were cloned and sequenced. This is the first report of full-length cDNA sequences for reptilian ERs, to our knowledge. The full-length alligator (caiman) ER cDNA (1764 bp, 587 amino acid residues) shows 68% amino acid homology with the full-length whiptail lizard ER cDNA (1746 bp, 581 amino acid residues). The respective ligand binding E domains have 87 and 83% amino acid homology with human ER while the DNA binding C domains show 100% amino acid homology with the human, rat and chicken forms. When the cDNAs were inserted into the pRc/RSV vector and transfected into HeLa cells with a reporter plasmid, the encoding proteins were confirmed to be functional through the interaction of the receptor-ligand complex with the estrogen responsive element (ERE).
Subject(s)
Alligators and Crocodiles/genetics , Cloning, Molecular , Lizards/genetics , Receptors, Estrogen/genetics , Reptilian Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , Genes, Reporter , HeLa Cells , Humans , Male , Molecular Sequence Data , Receptors, Estrogen/chemistry , Reptilian Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
A metabolic activation system with an S9 fraction or liver microsomes was applied to a reporter gene assay in vitro for the screening of estrogenicity of chemicals. The endpoint (luciferase) was luciferase induction in cells transfected with a reporter plasmid containing an estrogen-responsive element linked to the luciferase gene. Compounds were applied to the reporter gene assay system after pretreatment or simultaneous treatment with an S9 fraction or liver microsomes. Both trans-stilbene and methoxychlor themselves showed no or little estrogenicity, but when they were treated with an S9 fraction or liver microsomes, they demonstrated strong effects, indicating their metabolites to be estrogenic. When four pyrethroid insecticides were subjected to this assay system, however, they showed no estrogenicity even with liver microsome or S9 mix treatment.
Subject(s)
Estradiol/pharmacology , Genes, Reporter , Luciferases/genetics , Methoxychlor/pharmacology , Microsomes, Liver/metabolism , Stilbenes/pharmacology , Animals , Biotransformation , Breast Neoplasms , Carcinogens/pharmacokinetics , Carcinogens/pharmacology , Drug Combinations , Drugs, Chinese Herbal/pharmacology , Female , Glycyrrhiza , HeLa Cells , Humans , Insecticides/pharmacokinetics , Insecticides/toxicity , Methoxychlor/pharmacokinetics , Paeonia , Plasmids , Pyrethrins/pharmacokinetics , Pyrethrins/toxicity , Rats , Stilbenes/pharmacokinetics , Transfection/methods , Tumor Cells, CulturedABSTRACT
The progesterone receptor (PR) is associated with physiological events such as implantation and the maintenance of pregnancy. Recently, it has become a social concern that chemicals may exert agonistic or antagonistic effects on hormone receptors. Therefore, we examined the effects of various chemicals on the human PR, with a focus on pyrethroid insecticides, using three in vitro methods. Eight pyrethroid insecticides (fenvalerate, d-allethrin, d-phenothrin, prallethrin, empenthrin, permethrin, cypermethrin and imiprothrin), examples of environmental pollutants and positive control chemicals were subjected to a reporter gene assay (luciferase assay) using human breast cancer T-47D cells, a two-hybrid assay and a binding assay using the same whole cells or receptors (cell-free). In none of these did the eight pyrethroid insecticides show any binding to the PR, agonistic or antagonistic effects.
