Oxidative phosphorylation assay and

In addition to the foregoing, three further examples in this list (numbers 5-7) deserve consideration. These are (5) interaction of endocrine disrupters with the estrogen receptor, (6) the action of uncouplers of oxidative phosphorylation, and (7) mechanisms of oxidative stress. Until now only the first is well represented by biomarker assays that have been employed in ecotoxicology. 

Different types of organisms such as daphnia, mussels, algae, and fish have been extensively incorporated in toxicity tests for water assessment systems [65], Most of these assays are developed as test systems with few as laboratory-based sensor systems. Membranes with their active enzyme system have also been implemented in the development of toxicity kits and sensors. An example is the MitoScan Kit (Harvard BioScience, Inc., Holliston, MA), which uses fragmented inner mitochondrial membrane vesicles isolated from beef heart (EPA, 2005 [9]). The submito-chondrial particles contain complexes of enzymes responsible for electron transport and oxidative phosphorylation. When specific toxins are in the sample, the enzyme reactions are slowed or inhibited, and these are monitored spectophotometrically at 340 mn. This is still in a bioassay test kit format but may be developed to optical sensor system. 

Based on the structure of dioxapyrrolomycin and related compounds, it was postulated that the insecticidal activity of these compounds was due to the uncoupling of oxidative phosphorylation. This was subsequently confirmed through mouse-liver mitochondrial assays [6]. 

In this chapter, we describe a protocol for the systematic perturbation of patient-derived cell lines using small-molecule probes, which is both scalable to high-throughput workflow and generalizable to a variety of assays. In the protocol presented here, multiple patient-derived lymphoblastoid cell lines either mutant or wild type at a defined genetic locus (e.g., HNF4a) are perturbed by an annotated chemical library. After sufficient incubation, cells are subjected to a phenotypic assay, in this case a luminescence-based readout of cellular ATP content, that aims to quantify the effect of compounds on oxidative phosphorylation, viability, or other relevant traits. The effect of each compound is expressed as a metric that reflects the difference in compound-induced phenotypes between mutant and wild-type cells. These ratios are then... 

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