EFFECTS OF GLUTATHIONE PEROXIDASE 1, GLUTATHIONE PEROXIDASE 4 AND COPPER, ZINC-SUPEROXIDE DISMUTASE GENE KNOCKOUTS ON ENZYME ACTIVITIES, BODY SELENIUM AND RESISTANCE TO OXIDATIVE AND NITROSATIVE STRESSES

This thesis addresses two studies, 1) characterization of a glutathione peroxidase 4 (GPx4) haploid insufficient (+/-) mouse and 2) effects of different levels of glutathione peroxidase 1 (GPx1) and Cu, Zn superoxide dismutase 1 (SOD1) gene dosage on enzyme expression and responses to paraquat (PQ), diquat (DQ) and/or acetaminophen (AP) toxicity. These studies are united in a general way by addressing response to oxidative stress with altered antioxidant enzyme expression and in a more specific way by involvement of two selenium (Se) containing glutathione peroxidases (GPx1 and GPx4) and SOD1. All three of these enzymes are believed to be involved in in vivo metabolism of reactive oxygen species (ROS). GPx4 is structurally and functionally unique among selenoperoxidases since it functions as a monomer and is able to metabolize phospholipid hydroperoxides. GPx4 is more resistant to Se depletion than other selenoperoxidases and deletion of both GPx4 alleles is embryonic lethal, indicating an important role that is not yet fully understood. Experiment 1 examined the effects of deletion of one gpx4 allele at baseline and with a ROS challenge. GPx4+/and wild-type (WT or GPx4+/+) mice were injected with 24 mg/kg body weight of the ROS generator PQ or phosphate buffered saline (PBS) control and sacrificed 4 h later. GPx4+/mice had decreased GPx4 activity in lung, liver, kidney and testis, from 24 to 39% (P 72 h). SOD1 knockout was also associated with 28 to 34% decreases in GPx1 activity, (P < 0.05; +/+ vs. +/and P = 0.078 +/+ vs. -/-), depending on SOD1 copy number). Plasma ALT peaked between 20-40 h (11740 ± 1074) with AP treatment and <20 h with DQ treatment (2129 ± 537) but was unchanged in all surviving mice. In summary, there are a variety of responses to antioxidant enzyme knockouts and not all are detrimental to defense against oxidative stress. In Experiment 1, gpx4 deletion reduced tissue GPx4 activity and testis Se without increased susceptibility to PQ toxicity. In Experiment 2, SOD1 knockout was associated with high resistance to AP toxicity, high susceptibility to DQ toxicity and decreased GPx1 activity while GPx1 knockout was associated with increased AP and DQ mortality. Overall, the effect of SOD1 knockout on resistance to AP lethality was greater than the effect of GPx1 knockout on AP lethality. BIOGRAPHICAL SKETCH Michael Salvatore Scimeca was born on September 11, 1975 in Kingston, NY and grew up in New York State’s Mid-Hudson Valley. He graduated from Poughkeepsie Day School in 1993 and attended Cornell University. In 1997, he graduated with a BS degree in Animal Science. That same year he moved to Boston to take a job as a research technician at Harvard Medical School / Massachusetts Eye and Ear Infirmary studying hereditary retinal diseases. In 1999 he married his college sweetheart, Clay Chiment and in the following year, took a job in the Division of Comparative Medicine at the Massachusetts Institute of Technology studying Helicobacter and gastrointestinal cancer. In fall of 2001, he returned to the Cornell University Department of Animal Science to pursue his graduate studies. His daughter, Laia, was born on April 5, 2005.