Perturbation of cellular redox homeostasis to a more oxidized state has been linked to adverse human health effects such as diabetes and cancer. However, the impact of altering the regulation of redox homeostasis during development is not fully understood. Specifically, this project investigates the role of the Nrf2 antioxidant response pathway and its effect on glutathione (GSH; cellular redox buffer) at critical windows of development. To explore this, we used zebrafish embryos (Danio rerio) as a model due to the function of GSH and the Nrf2 being conserved among vertebrates. We exposed zebrafish embryos to three Nrf2 activators: two antioxidant enhancing molecules: sulforaphane (SFN; 40 µM) and Dimethyl fumarate (DFM; 7µM) as well as the pro-oxidant tert-Butylhydroquinone (tBHQ; 1µM) for 6 hours at critical windows development: 24, 48, 72 hours post fertilization (hpf). Following exposure, we visualized Nrf2 protein levels and glutathionylation rates using immunohistochemistry and confocal imaging. We found that changes in Nrf2 expression were dependent on the tissue type with there being significant changes in Nrf2 when looking at the pancreatic beta cells. Also in the beta cells, exposure to SFN, tBHQ, and DMF were found to increase Nrf2 translocation into the nucleus. Most notably, all three Nrf2 activators significantly altered glutathionylation levels depending on the time-point the zebrafish were exposed. SFN and tBHQ were also found to significantly increase glutathionylation at 48 and 72hpf, but led to a significant decrease at 96hpf while DMF increased glutathionylation at all three time-points. Interestingly, there was little correlation between Nrf2 protein levels and glutathionylation, but zebrafish with a mutated Nrf2 did have significantly different glutathionylation rates than the wild type fish. This suggests that oxidative stress is not the sole regulator of glutathionylation and instead Nrf2 may also be regulating glutathionylation through GSH storage. My data indicate that the effects of Nrf2 activation on Nrf2 levels and glutathionylation depend on the timing of exposure to the perturbing chemicals and the tissue type. Finding these windows of development where redox homeostasis is most sensitive in humans can allow for possible preventative and/or protective measures to oxidative stress during development.

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Isoflavones have antioxidant activities in vivo, however, their antioxidative potential against oxidative stress initiated by exercise was not explored. The first study investigated the effect of high-genistin isoflavone (HGI) supplementation on erythrocyte antioxidant enzymes and tissues' thiobarbituric reactive substances (TBARS) in acutely exercised one-year old rats. All tissue genistein concentrations increased after exercise. Ingestion of HGI seemingly enhanced running time to exhaustion, and maintained glutathione peroxidase (GPx) and catalase (CAT) activities decreased due to exercise. The second study investigated the dose effect of HGI supplementation. Genistein concentrations were significantly higher (P<0.05) in tissues of rats fed the 1045 PPM HGI diet than in rats fed 522 or 209 PPM HGI diets and increased the glutathione (GSH)/total glutathione (TGSH) ratio (P<0.03). Reductions of the in vivo MDA concentrations (P<0.05) were observed only in the plasma of rats fed 522 and 1045 PPM HGI diets compared to those fed 0 PPM (-1.08, -0.82, and 0.03 mM, respectively). Therefore, isoflavones at 522-1045 PPM HGI diet have antioxidative effects in rats. The last two studies investigated the effect of isoflavone supplementation on the modulation of erythrocyte antioxidant enzyme activities, glutathione homeostasis, and other oxidative biomolecules in healthy young men undergoing 80%VO2pk exercise. In Study 3 exercise at 80%VO2pk increased oxidative stress which was best demonstrated by increased superoxide dismutase (SOD) activity (16.5%), GSH/TGSH ratio, in vivo MDA (12.6%), plasma uric acid (4.9%) and ferric reducing/antioxidant ability (FRAP) ( 7.8%). Therefore, 30 minutes 80% VO2pk exercise induced oxidative stress in moderately active college men. In study 4, four-week HGI supplementation produced plasma genistein and daidzein concentrations of 499 and 415 ng/ml, which were significantly increased to 633 and 539 ng/ml by exercise (P=0.04 and P=0.05). Isoflavones significantly decreased in vivo pre-exercise plasma MDA (P<0.05), increased pre-exercise blood TGSH (P=0.01) and pre-exercise erythrocyte SOD activity (P=0.0006), and maintained the decreased activities of GPx due to exercise at pre-exercise levels. Results demonstrated that isoflavones had antioxidant activity in vivo under normal physiological conditions in healthy young men. They also maintaining GPx activity which was decreased due to exercise, however, isoflavones may not overcome all oxidative stress initiated by intense exercise. / Ph. D.

Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

In previous work, mouse lines were selected for resistance (R) or for susceptibility (S) to fescue toxicosis based upon reductions in post-weaning growth rate caused by an endophyte-infected diet. The first objective of the current experiment was to determine whether long term reproduction of S mice was more severely depressed than that of R mice by the toxic diet. The second objective was to quantify glutathione-S-epoxytransferase (GST) and uridine diphosphate glucuronosyl-transferase (UDPGT) activities in R and S dams form the experiment and to determine whether reproduction during continuous cohabitation and liver detoxification enzyme activities were correlated within line x diet groups. Effects of the toxic diet were detectable within the first litters produced. Reproduction was more seriously influenced by the toxic diet within the S line than within the R line when these measures were compared within four equal time phases. The effects of the toxic diet on reproduction were greatest early in the experiment; by the fourth time phase differences among line x diet groups, with the exception of litter weight, were not significant. Percentage differences in total reproduction were greater between S mice fed the non-toxic diet and S mice fed the toxic diet than those between the R mice fed the non-toxic and toxic diets. Averaged across diets, GST activities were higher in R mice, but UDPGT activities were not significant. Within R line mice, GST was correlated with three reproductive measures, but UDPGT activity was not correlated with reproduction within any line x diet group. / Master of Science