P450s (CYPs) and a number of other proteins are bimodally targeted to mitochondria (mt-CYP) in addition to their well- established ER or cytoplasmic destination. Work in the PI's laboratory has led to the discovery of a new family of "chimeric" signals, which direct the bimodal targeting of CYPs and a number of nuclear hormone receptors to more than one subcellular compartment. Two major mechanisms have been described for the activation of cryptic mitochondria targeting signals: 1) N-terminal processing by a cytosolic endoprotease, and 2) signal activation by PKA or PKC-mediated phosphorylation. Work during the current grant period has resulted in the characterization of cytoplasmic endoprotease which activates mt-targeting signals of a large number of proteins. Results also show that mt-CYP2D6 may have activity for the metabolism of neurotoxin MPTP and other drugs. Additionally mt-CYP1B1 and CYP2C6 are induced in cardiomyocytes by hypoxia and myocardial ischemia. The objective of this proposal is to initiate studies on pathophysiological roles of mt- CYPs and HO-1 in drug metabolism, ROS production, and cellular toxicity: 1) Define the role of xenobiotic-induced cytosolic endoprotease in modulating mt- targeting of proteins such as GR, RxR1, ER2, and other proteins on mt-biogenesis, and mt- function. Role of these stress induced proteins on mt transcription, mtDNA packaging, DNA damage and ROS production will be studied. Mechanism of induction of p90 (polyserase 1) gene by BNF, Dex and hypoxia will be studied. 2) The role of mt-CYP2D6 in the metabolism of drugs such as bufuralol, tamoxifen, chlorpromazine and toxicants such as MPTP will be investigated. Stable cell lines predominantly expressing either the mt-CYP2D6 or mc-CYP2D6 in combination with selective silencing of CYP reductase, adrenodoxin (Adx), and MAO knock out and CYPR knock out mouse models will be used to investigate the role of mt-CYP2D6 in drug metabolism, drug-induced mitochondrial dysfunction. 3) The role of mt-targeted CYP1B1, CYP2C6, and other CYPs in H9C2 cardiomyocytes subjected to hypoxia and mouse hearts subjected to ischemia will be investigated and the role of these CYPs in ROS production, and mt-toxicity will be studied. Novel mitochondria targeted EPR probe Mito-DEPMPO (DEPMPO conjugated to triphenylphosphonium ion) and Mito-DHE will be used for measuring mitochondrially produced ROS and mt-targeted Adx peptides will be generated for selective inhibition of mt- CYPs in whole cells and tissues. PUBLIC HEALTH RELEVANCE: The proposal is focused to elucidate mechanisms by which xenobiotic agents induce cellular toxicity and oxidative or chemical damage. The proposal is aimed to elucidate the role of mitochondria targeted CYP2D6, 2C6 and 1B1 and also a number of xenobiotic inducible hormone receptors in inducing mitochondrial dysfunction, mtDNA damage and ROS production using state of the art assay systems. Finally, the role of mitochondria targeted CYPs in ischemia reperfusion injury will be studied using cell and animal models.