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 < 0.05) lower activity than WT. GPx4+/- mice had a 34% (P < 0.05) decrease in testis Se concentration. GPx4+/- had no effect on Se concentration, protein carbonyl formation (measure of oxidized protein) or GPx1 activity in other tissues or GPx3 and alanine aminotransferase (ALT) activity in plasma. In summary, deletion of one gpx4 allele demonstrated a range of effects on GPx4 activities and Se concentrations, but did not affect susceptibilities to pro-oxidant-induced protein oxidation in various tissues of mice. SOD1 and GPx1 are often considered to detoxify ROS in the cytosol. SOD1 produces hydrogen peroxide from superoxide which GPx1 reduces to water. A dramatic increase in ROS lethality has been found in cells and mice lacking GPx1 or SOD1 (GPx1-/- or SOD1-/-) whereas GPx1-/- hepatocytes are protected against reactive nitrogen species (RNS) induced cell death. Experiment 2 compared responses of mice with various gene dosages of both SOD1 and GPx1 in a 72 h survival trial to the ROS generator DQ (25 mg/kg body weight) or the putative in vivo RNS generator AP (600 mg/kg body weight). Although this experiment was limited by sample size and group death rates, some general trends emerged. The SOD1 knockout allele decreased AP-induced mortality and increased DQ-induced mortality (P < 0.05). The GPx1 knockout allele increased mortality from both AP and DQ (P < 0.05). In combination, mice with only one functional copy in total of SOD1 and GPx1 (GPx1+/-|SOD1-/- or GPx1-/-|SOD1+/-) had significantly decreased DQ survival time but unchanged AP survival time (P < 0.05). AP-treated GPx1+/-|SOD1+/+ mice died significantly earlier than control (<40 h) but GPx1+/-|SOD1+/- survival time was not significantly different from control (>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.
superoxide dismutase; glutathione peroxidase; knockout; mouse; reactive oxygen species; reactive nitrogen species; copper; selenium
dissertation or thesis