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Cellosaurus publication CLPUB00334

Publication number CLPUB00334
Authors Brennan J.C.
Title Development, optimization, and application of nuclear receptor dependent cell-based bioassays for endocrine disruptors.
Citation Thesis PhD (2015), University of California Davis, United States
Web pages https://search.proquest.com/openview/063ba5d9526a3cb41897df9a6c774d81/1?pq-origsite=gscholar&cbl=18750&diss=y
Abstract Endocrine disrupting chemicals (EDCs) include a wide variety of structurally diverse compounds that affect normal signaling pathways in most all animal species by multiple mechanisms. Given the global distribution of EDCs in environmental, biological and food samples and commercial products, detection, relative quantitation, and assessment of their biological/toxicological effects is of paramount importance. While instrumental methods allow detection and quantitation of specific (known) EDCs in sample extracts, these methods do not allow identification and characterization of new EDCs. Accordingly, mechanism-based bioanalytical methods have been developed to assist in the identification and characterization of matrices and extracts containing EDCs. The so-called chemically-activated luciferase expression (CALUX) bioassays and related CALUX-type bioassays represent a collection of nuclear receptor-based recombinant cell bioassays for detection of EDCs, including estrogenic-, androgenic-and dioxin-like chemicals. Although CALUX assays are much cheaper and quicker than instrumental analysis and whole animal assays, there is a critical need to make these assays as biologically/toxicologically relevant and consistent with our current understanding of endocrine disruption in order to provide optimal data for selection of chemicals (or mixtures) which warrant in vivo assessment. This includes improved assay characteristics (i.e., minimal detection limits (MDLs)) and response to EDCs, inclusion of more receptors (i.e. multiple subtypes of a given nuclear receptor) and consideration of receptor cross-talk and related mechanisms. In this dissertation, I describe two significantly improved CALUX bioassay systems for EDCs. The first is a set of species-specific Ah receptor (AhR)-responsive CALUX cell lines that exhibit dramatically increased sensitivity (10-100-fold lower MDL (to sub pM concentrations)) and responsiveness to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like compounds (DLCs), compared to current dioxin-responsive CALUX cell lines. I demonstrate the utility of these novel AhR-responsive CALUX bioassays in analysis of sediment extracts. The second CALUX cell line (estrogen receptor (ER)-CALUX) is a modification of our existing estrogen-responsive human cell line (BG1Luc4E2). Although this cell line has received official regulatory approval from the USEPA and OECD (to the point of being included in the USEPA Endocrine Disruptor Screening Program) and is currently being utilized by the Tox21 program for chemical screening purposes, this cell line only expresses one of the two known human ER subtypes (ERalpha). Since both ER subtypes (ERalpha and ERbeta) can differ in their ligand-specificity, cross-talk with other signaling and transcription factors and gene induction profiles, optimal cell bioassay assays should be able to detect the full spectrum of ER-active chemicals (i.e., activators/repressors of ERalpha and/or ERbeta). Accordingly, we stably transfected the ERalpha-containing BG1LucE2 CALUX cell line with a human ERbeta expression vector and screening of the resulting BG1LucERbeta clones with the ERbeta-selective ligands genistein and Br-ERbeta-041 allowed identification and isolation of a clonal cell line (BG1LucERbetac9) expressing ERbeta and highly responsive to ERbeta- selective ligands. Comparison of the response of BG1Luc4E2 and BG1LucERbetac9 cell lines to a chemical library of 176 compounds revealed that the ERalpha/ERbeta-containing BG1LucERbetac9 cell line can identify a greater number of potential estrogenic chemicals than the ERalpha- containing BG1Luc4E2 cell line. Since cross-talk between the AhR and ER has been identified as a potential mechanism for endocrine disruption in animals exposed to DLCs (which act to repress ER gene signaling), in the final chapter I explored cross-talk between AhR and ER in a human breast carcinoma cell line (SKBR3). I specifically focused on the effect of ERalpha/ERbeta on AhR gene signaling in transiently transfected cells. In SKBR3 cells (which lack functional ER), we observed an ERalpha subtype- specific enhancement of AhR-dependent reporter gene expression (from the transfected AhR-responsive plasmid pGudLuc6.1) in the absence of added E2 and a repression of the ER-dependent enhancement of AhR signaling by stimulating transfected ERalpha with E2, PPT, or fulvestrant (no repression was observed with ERbeta). The repressive effect of E2 could be abolished by co-incubation of E2-activated ERalpha-transfected SKBR3 cells with the ER antagonist 4-hydroxytamoxifen, demonstrating the requirement for functionally active ERalpha. Our ER-dependent repressive results in SKBR3 cells indicate that AhR has selective interactions with ERa versus ERbeta. The results presented in this dissertation demonstrate that the CALUX reporter vectors and stable cell lines not only have application for highly sensitive high throughput screening for EDCs systems, but for in-depth mechanistic studies examining crosstalk between receptor signaling pathways which can play a critical role in endocrine disruption.
Cell lines CVCL_DD13; BG1LucERbc9