Notes: CYP450 activity was measured using the P450-Glo™ Assays for CYP3A4, CYP2B6 and CYP1A2, and viability was measured using two separate luminescent assays, one of which being CellTiter-Glo® Cell Viability Assay. (4877)

Notes: Humans are exposed to a cocktail of low-dose chemicals in the environment, through diet, and through medication. However, most studies looking at compound toxicity, look at these compounds in isolation, not as they might be encountered in the environment—in mixtures. The pregnane X receptor (PXR) is a xenoreceptor that has been identified by the US Environmental Protection Agency as a major target of environmental and dietary chemicals, and many studies have highlighted the role of nuclear receptors like PXR in transducing the deleterious effects of endocrine disrupting compounds in the environment. The authors of this study used compound screening and functional analysis to demonstrate that the combined use of an environmentally persistent organochlorine pesticide and the active component of contraceptive pills (17α-ethinylestradiol) produces synergistic effects on PXR and expression of its target gene, the cytochrome P450 gene, CYP34A. Gene expression of CYP3A4 was measured using a luciferase reporter created in a pGL3-basic backbone. Activity of the CYP3A4 protein was measured in primary human hepatocytes using the P450-Glo™ CYP3A4 Assay with Luciferin-IPA, and cell number was normalized using the CellTiter-Glo® Luminescent Cell Viability Assay. (4579)

Notes: Here the authors describe development of an HTS cell viability assay protocol for use with cultured cyropreserved human primary hepatocytes. Cryopreserved hepatocytes for culturing were prepared as suspensions and dispensed at 2,000 or 4,000 cells/5µl/well in collagen I-coated 1,536-well plates. Cells were allowed to attach and then 23nl of each test compound was added in a dilution series from 2.8nM to 92µM, and cells incubated for 24 or 40 hours. Five microliters of CellTiter-Glo^® Reagent was added and cells were incubated 30 minutes before reading the luminescent output. IC₅₀ values for 12 compounds were determined; a summary of the protocol is provided in Table 1 of the paper. Cultured cryopreserved hepatocytes were assayed for function using the P450-Glo^® CYP3A4 assay with the Luciferin-IPA substrate. (4182)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)