ABSTRACT: PROJECT 4 Many Superfund (SF) chemicals (e.g., PAHs, PCBs, Chlorinated solvents) have been reported to induce reactive oxygen species (ROS) in several cell types. Preliminary studies suggest that most of the ROS induced by SF chemicals originates from the mitochondria and that CCl4-induced cardiomyocyte cell death is partly caused by impaired proteasome activity. The hypothesis that chronic exposure to xenobiotics leads to cellular dysfunction and cell death mainly via mitochondrial oxidative stress (MOS) will be tested. MOS results in excessive production of ROS, increased levels of oxidized mitochondrial proteins, lipids, and DNA, decreased ATP levels, and depolarized mitochondrial membrane potential. Project 4 will further explore whether MOS- related increases in ROS levels affects several cellular pathways, including cellular proteostasis, apoptosis, endoplasmic reticulum (ER) function, and fibrosis. Moreover, preliminary data generated by the research team suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) produce cardiotoxicity by MOS and impaired proteostasis. Since NSAIDs, such as diclofenac, are designed to be bioactive and bioaccumulate in animals, such as fish, it is very likely that these compounds would pose a larger problem than many current SF chemicals in the near future. Among the project aims is to investigate the effects of drug (diclofenac)-SF chemical mixtures. These hypotheses will be tested in cell cultures and chronic animal models. The research team will develop cell-based assays to evaluate the mechanistic basis of xenobiotics effects on MOS and proteostasis (with Core A, Core B and Project 5). This project will evaluate the effects of chronic exposure to xenobiotics on increased MOS, apoptosis, fibrosis, heart damage and associated alterations of plasma oxidized protein levels (with Core A, and B), and develop methods to monitor MOS and impaired proteostasis from chronic exposure to chemicals (with Project 1, 2 and 3 as well as Core A and B). The long term goal of this work is to develop a high-content and medium throughput bioassay to test the potentials of SF chemicals to cause MOS and proteostasis, and obtain a biomarker of MOS associated biological effects for bio-fluid analysis. Overall, this project will investigate a novel hypothesis that current SF chemicals, as well as potential future SF chemicals, cause cellular dysfunction mainly via MOS.