Development Of In Vitro Systems To Study Endocrine Disruptors

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Environmental estrogenic endocrine disruptors (EDs) are a health concern as the general population is exposed to a variety of EDs at low doses. Rapid screening and in depth understanding of how the body responds to EDs and their mixtures are crucial for toxicological evaluation and health protection. In this work, in vitro systems, including stable cells lines with estrogen induced green fluorescence protein (GFP) expression and micro cell culture analog (microCCA) devices, were developed to study EDs. A dual cell-line GFP expression system was constructed to study EDs with activities as estrogen receptor agonists or antagonists. The two stable cell lines, Ishikawa-GFP (uterine origin) and MCF7-GFP (breast origin), displayed increased intracellular GFP intensity with estrogens. This system also responded in a tissue specific manner to selective estrogen receptor modulators, raloxifene and tamoxifen. Our results also suggested no low dose synergistic effects between the tested estrogenic compounds. Physiologically realistic microCCA devices were developed to help predict in vivo responses to EDs. The silicon microCCA device contained a co-culture of three cell lines: MCF7-GFP, Ishikawa-GFP and HepG2/C3A (liver origin). Recirculation of medium mimicked the time dependent changes in drug concentrations and the microfluidic shear conditions inside a body. An in-line bubble trap was incorporated into the system to overcome air bubble related device failure. Interactions between hydrophobic surfaces and the fluid were investigated. Significant surface adsorption of estrogen to polypropylene based tubing (BPT) was observed. Contaminants leaching out of the BPT tubing were also found to be estrogen receptor agonist. A pneumatic micropump with a pumping rate ~3 ul per min was build with PTFE tubing replacing BPT tubing. Detection of estrogens was demonstrated on a microCCA device integrated with the micropump. Further, a photopolymerizable hydrogel based method was developed for direct patterning of multiple types of cells into their individual cell culture chambers in a PDMS microCCA. The use of this device as a biosensor was demonstrated by monitoring PEG encapsulated cells in microCCA exposed to a model toxicant, Triton X-100. The results indicate the potential use of PDMS microCCA devices to study EDs.

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