Cytochromes P450 (CYPs), including CYPs 1A1 and lA2, are of particular importance in the metabolism and bioactivation of toxic xenobiotics, including environmental pollutants such as PCBs and TCDDs. The toxicity of such planar halogenated aromatic hydrocarbons (pHAHs) is in part mediated by the CYP-dependent production of reactive oxygen species (ROS). The quantitative dose-related linkages of pHAH -induced uncoupling of CYP1As to produce reactive oxygen have not been investigated in a coherent manner with regard to species and pHAH.
The specific aims are to determine quantitative pHAH-mediated linkages between uncoupling of CYP1As and ROS production in vitro and in cell lines for a consistent set of human and model organism CYP1 As, including both mice and zebrafish. TCDD has been shown to also increase the mitochondrial production of ROS. The relative balance of TCDD-dependent ROS production between mitochondria and CYP1As located in the endoplasmic reticulum is critical for evaluating the contributions of these two mechanisms to the overall production of ROS within a cell. Determination of the relative importance of CYPIA-mediated ROS stress versus mitochondrial production of ROS will be accomplished by localization of ROS production using confocal microscopy of fluorescent reactive oxygen probes. The mechanisms of CYP1A inactivation as a result of uncoupling will be investigating by determining the modification of the heme moiety and active site amino acid residues using HPLC and MALDI-ToF. Recently identified AhR-agonistic persistent pollutants, such as polybrominated diphenyl ethers will also be investigated.
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