Mitochondrial Bioenergetics

The purpose of this contract is to understand basal levels of mitochondrial bioenergetics in different brain regions and other tissues from Brown Norway rats at different life-stages (1 month, 4 month, 12 month and 24 month old rats). This project will serve to establish constitutive (basal) levels of mitochondrial bioenergetics in different brain regions and other tissues under communities research area (SHC: Community Public Health Work; Task# 2.2.1.4) to improve our understanding of chemically-induced life-stage susceptibility and alterations in mitochondrial bioenergetics as a mode of action. More specifically, the contractor shall analyze several parameters related to mitochondrial respiration including basal complex I driven respiration, Complex Ill ATP synthesis rate, Complex IV oligomycin rate, maximum respiratory capacity and the respiratory control ratio (RCR =State Ill/State V) as a measure of mitochondrial integrity using the state-of-the art Flux Analyzer from Seahorse Bioscience (XF analyzer: 24-well format). In addition enzymes related to mitochondrial bioenergetics (Complex I, Complex II, Complex IV, and pyruvate dehydrogenase complex activities) will be assayed in a 96-well plate format. Please refer to the Research Protocol Methods below and QA Narrative statement (attached) for quality assurance requirements and assay procedures in accomplishing the required services. Samples to be analyzed, includes 5 biological replicates per age group (1, 4, 12 & 24 months of age) for each of 8 tissues (frontal cortex, cerebellum, striatum, hippocampus, brain stem, liver, heart, and lung) for a total of 160 samples (5 x 4 x 8 = 160). Brown Norway rats for the study will be obtained by EPA and shipped to University of Kentucky, Lexington, KY immediately after the contract is awarded. In addition, EPA will purchase and supply three kits each for enzyme assays of mitochondrial bioenergetics (Complexes I, II, and IV enzyme activities). Mitochondrial respiration assays using the Seahorse instrument and enzyme assays related to mitochondrial bioenergetics shall be carried out by the Spinal Cord & Brain Injury Research Center, University of Kentucky. The information obtained will assist in further elucidating chemically-induced life-stage susceptibility and possible mode(s) of action. 3