1. Apoptosis, controlled cell death, is a natural process of development. Recently it has been found that mitochondria play an important role in apoptosis through a signaling cascade. Reactive oxygen species (ROS) are byproducts of oxidative phosphorylation and indicative of cellular stress. Excess ROS are damaging to the cell and can lead to cytochrome c release from mitochondria and subsequent activation (cleavage) of caspase-3, an event that commits the cell to apoptosis. Many aging problems, including progressive disorders such as Parkinson’s disease, stem from mitochondrial dysfunction. Recent studies found that many of the effects of aging can be replicated in cells through treatment with a mitochondrial toxin, rotenone. Rotenone is natural substance used in many pesticide products that inhibits complex I of the electron transport chain. Mouse fibroblast cells in this study were infected with rotenone and subsequently given an antioxidant as a means of recovery. A recent therapeutic option for treatment of aging complications focuses on the use of a synthetic antioxidant targeting the mitochondria. PMX- 500F, a lipoylcarnitine derivative, is a synthetic antioxidant that has been found to reduce ROS in mice. Rotenone treated culture mouse fibroblasts in this study were co- treated with PMX-500F as a potential therapy for ROS and rotenone-induced apoptosis. Using a cell culture system, rotenone induced damage through inhibition of mitochondrial complex I and generation of ROS will be counteracted by the antioxidant, PMX-500F. Co-treatment with PMX-500F will result in reductions in: 1. ROS (as measured by DCF-DA fluorescence). 2. Apoptosis (as measured by nuclear morphology). 3. Activation of caspase-3 (as measured by western blotting). Using a cell culture system, rotenone induced damage through inhibition of mitochondrial complex I and generation of ROS will be counteracted by the antioxidant, PMX-500F. Co-treatment with PMX-500F will result in reductions in: 1. ROS (as measured by DCF-DA fluorescence). 2. Apoptosis (as measured by nuclear morphology). 3. Activation of caspase-3 (as measured by western blotting). NIH/3T3 cells were grown at 37°C and 5.0% CO 2 in complete medium. Photomicrographs were taken when viewed with an Olympus CK2 microscope equipped with digital camera. Hoechst 33342 (Sigma-Aldrich®) staining was used to view cells under a Nikon Eclipse E800M microscope under a DAPI filter. ROS measurements were performed using dichlorofluorescein diacetate (DCF-DA) fluorescence over a 45 minute time period at 488 nm excitation and 525 nm emission in a Tecan Infinite® M200 machine using Magellan software (Männedorf, Switzerland). Western blot procedures were performed by running extracts from cells on 12% acrylamide gels and transfer to PVDF membranes. Membranes were incubated overnight in primary antibodies, washed, and incubated with secondary (HRP-conjugated) antibody. Pierce® ECL western blotting substrates was used for detection using X- ray film. Merial Veterinary Scholars Program, MitoCure Foundation, Auburn University, Kodeeswaran Parameshwaran, Michael C. Kohn, Auburn University Small Animal Teaching Hospital Department of Radiology Recent research has focused on lessening the effects of aging by targeting mitochondria and their role in apoptosis. The mitochondrial toxin, rotenone, was previously used in vivo as model of aging and in the evaluation of a possible therapeutic antioxidant, PMX- 500F. The purpose of this study was to further determine the effects of rotenone, a common pesticide and complex I inhibitor, and the antagonism of PMX-500F on NIH/3T3 cells. Specifically, the primary goals were to determine the mechanism of action of PMX-500F and to define a set of in vitro parameters for future studies on the antioxidant. Cells were treated with 2 µM rotenone and 2 µM PMX-500F for 24 and 48 hours. The results illustrate that PMX-500F failed in rescuing NIH/3T3 cells from the effects of rotenone at the chosen concentration and time points. Further studies need to be performed to definitively characterize antioxidant effects on mitochondrial function. PMX-500F did not reduce proapoptotic factors of treated NIH/3T3 cells to control levels. It is concluded that at a concentration of 2 µM PMX-500F and 2 µM rotenone for 24 and 48 hour treatment periods, PMX-500F did not reduce ROS levels, cleaved caspase-3 levels, or percent apoptosis in NIH/3T3 fibroblast cells. An incidental finding was that PMX-500F alone lowered ROS (as compared to rotenone), apoptotic factors, and cell overgrowth. After 24 hour treatment, a 21.1% increase in ROS values were seen in cells with rotenone, 9.9% in cells with PMX- 500F, and 15.2% in cells with both rotenone and PMX-500F. After 48 hour treatment, ROS values increased 40.7% with rotenone, 23.4% with PMX-500F, and 63.5% with both. Apoptosis in 24 hour treatments indicated an increase of 23% with rotenone, 38% with both, and a decrease of 0.5% with PMX-500F. After 48 hours, apoptosis was 36% with rotenone, 28% with both, and -3% with PMX-500F. Values represent a comparison of untreated cells. Western blotting showed the highest increase in cleaved caspase-3 in both rotenone and rotenone plus PMX-500F cells for 24 hour treatment. 48 hour treatment showed the highest increase in cleaved caspase-3 in rotenone plus PMX-500F cells. Hannah P. Findlay, Michael H. Irwin, and Carl A. Pinkert Department of Pathobiology, College of Veterinary Medicine, Auburn University ABSTRACT INTRODUCTION RESULTS METHODS HYPOTHESIS CONCLUSION ACKNOWLEDGEMENTS Total caspase-3* Cleaved caspase-3 24 hour treatment A- No treatment B- 2 µM rotenone C- 2 µM PMX-500F D- 2 µM rotenone + 2 µM PMX-500F A B C D 48 hour treatment 2µM rotenone 2µM rotenone + 2µM PMX-500F 2µM PMX-500F No treatmentNormal cells Apoptotic cells A B C D *Detects both cleaved and uncleaved caspase-3.