Bioassay Core/ Judy L. Bolton

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The new Bioassay Core (Core C) will provide bioassay support for all Projects with an array of validated, robust, and efficient bioassays, which have been previously established in Project 2. The following specific aims are proposed: 1. Evaluate botanical extracts for their chemopreventive properties. We will analyze the antioxidative activity, the NAD(P)H:quinone oxidoreductase 1 (NQ01) inducing properties, the antiinflammatory activity, and aromatase enzyme inhibiting properties of the extracts/compounds. When extracts/compounds will be active in the NQ01 assay, we will analyze these samples in the antioxidative response element luciferase assay and in the glutathione-S-transferase induction assay. The induction of several Phase I metabolism enzymes is regulated by the xenobiotic response element (XRE). Inducer of Phase I enzymes interfere with the metabolism of other drugs and endogenous estrogen; therefore, the extracts will be tested in the XRE-luciferase assay. 2. Evaluate botanical extracts/compounds for hormonal activity (efficacy). The alkaline phosphatase assay in Ishikawa cells will be employed as the major assay to test the botanicals for estrogenic and antiestrogenic activities. Active fractions will be analyzed for estrogen receptor (ER)a and ERB selectivity in an ERa and ERB-ERE-luciferase assay in breast cancer cells. If promising samples have been found, we will test for ER ligands in the ERa and ERB competitive radioactive binding assay. As progestogenic activity is important for endometrium protection, the progesterone response element luciferase and progesterone receptor competitive binding assay will be conducted. Finally, the selective serotonin reuptake inhibition assay (SSRI) will be employed, since SSRIs have been described to alleviate menopausal symptoms. 3. Evaluate botanicals for their distribution profile, safety, and efficacy in vivo. When active extracts/compounds have been determined, the ovariectomized rat model to analyze the distribution and metabolism profile (Project 3), the safety, and efficacy will be performed. With these assays. Core C directly supports the whole Center efforts by establishing toxic, interfering, and active compounds, thus providing bioactive marker compounds for the standardization of new botanicals.