ABSTRACT
In the present study, potential interaction between natural estrogens i.e., estrone (E(1)), estradiol (E(2)) and estriol (E(3)) with human estrogen receptor (hER) was seen by in silico study. Molecular docking studies were carried out using Glide and ligand docking program. The binding affinity, assessed by Glide score, indicates stronger interaction of E(3) with hER followed by E(2) and E(1). Real-time PCR analysis of vga and vgb expressions, in the liver of different groups of Channa punctatus injected with the three natural estrogens, supported the docking analysis and indicated E(3) to be the most potent estrogen in inducing vga and vgb expressions followed by E(2) and E(1). This study lays the groundwork for studying interactions of various estrogenic substances with different estrogen receptors and to assess estrogenicity of various chemicals which are being released into the environment by employing molecular docking technique.
Subject(s)
Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Models, Molecular , Perciformes/metabolism , Vitellogenins/metabolism , Amino Acid Sequence , Analysis of Variance , Animals , Computer Simulation , DNA Primers/genetics , Estrogen Receptor alpha/genetics , Gene Expression Profiling , Liver/metabolism , Male , Molecular Sequence Data , Molecular Structure , Protein Binding , Real-Time Polymerase Chain Reaction/veterinary , SoftwareABSTRACT
A novel incomplete vitellogenin (VgC) was purified from the plasma of estradiol-treated male murrel, Channa punctatus, by gel filtration chromatography. The native mass of VgC protein was 180 kDa, and it resolved as a single peptide of 100 kDa on SDS-PAGE. The peptide on subjecting to matrix-assisted laser desorption/ionization-time of flight produced a peptide mass fingerprint. On tandem mass spectrometry, some of these peptides showed mass to charge (m/z) ratio and amino acid sequence similarity with VgC peptides of other teleosts. Phylogenetic analysis revealed a similarity of murrel VgC with fish species of the order Perciformes. Semi-quantitative RT-PCR assay was developed to study expression of vgc gene at variable levels of estradiol exposure. Presence of VgC in males indicates that fish has been exposed to estrogens; hence, it can be used as a biomarker for estrogenic exposure.
Subject(s)
Biomarkers/blood , Perciformes/genetics , Phylogeny , Vitellogenins/genetics , Amino Acid Sequence , Animals , Chromatography, Gel/veterinary , Electrophoresis, Polyacrylamide Gel/veterinary , Estradiol/pharmacology , Likelihood Functions , Male , Models, Genetic , Molecular Sequence Data , Real-Time Polymerase Chain Reaction , Sequence Homology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , Vitellogenins/blood , Vitellogenins/isolation & purificationABSTRACT
The present study was undertaken to characterize different vitellogenins in Channa punctatus. Protein purification by gel chromatography followed by fast protein liquid chromatography (FPLC) revealed existence of two different Vg forms. Liquid chromatography tandem mass spectrophotometry (LC-MS/MS) suggested the existence of Vga and Vgb. Cloning of partial sequences of vga and vgb mRNA and phylogenetic analysis substantiated the existence of two vitellogenins. Real time PCR for vga and vgb genes from liver of estradiol-17ß (E2) treated fish reveals difference in expression levels of transcripts of these two genes. vgb is expressed at lower dose of estradiol suggesting a higher sensitivity to estradiol. The present study thus proposes different regulatory control for the expression of these two genes and vgb as a superior biomarker than vga to assess exposure of C. punctatus to environmental estrogens.
