ABSTRACT
Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.
Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Diethylhexyl Phthalate/toxicity , Environmental Pollutants/toxicity , Estrogen Receptor alpha/drug effects , Estrogen Receptor alpha/physiology , Estrogens , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lactoferrin/biosynthesis , Lactoferrin/genetics , Lactoferrin/metabolism , MCF-7 Cells/drug effects , MCF-7 Cells/metabolism , Mass Spectrometry/instrumentation , Microchemistry/instrumentation , Neoplasm Proteins/drug effects , Neoplasm Proteins/physiology , Neoplasm Proteins/metabolism , Neoplasms, Hormone-Dependent/pathology , Proteomics , Tamoxifen/antagonists & inhibitors , Tamoxifen/pharmacologyABSTRACT
A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.