The central thesis of this project is that damage caused by reactive nitrogen species, e.g. 3-nitrotyrosine (Tyr-NO2), constitutes a marker of disease progression/severity. A new sensitive electrochemiluminescence ELISA was optimised and validated for Tyr-NO2 measurement, giving a lower limit of quantification of 0.04 nM BSA-NO2, intra- and inter-assay CVs of 6.5% and 11.3%, an average recovery of 106 ± 3% and average linearity 0.998 ± 0.001. Nitrative stress, carbonyl stress and C-reactive protein (CRP) concentrations were measured before and after major elective surgery. CRP measurements confirmed the induction of an inflammatory response. Median serum Tyr-NO2 levels increased post-surgery to a median (inter-quartile range) value of 0.97 (0 – 1.7) fmol nitrated BSA (BSA-NO2) equivalents/mg protein compared with a pre-surgery level of 0.59 (0 – 1.3) fmol BSA-NO2 equivalents/mg protein (p<0.05). Oxidative damage was confirmed by serum protein carbonyl levels (p<0.05). In a second pre-/post- surgery study, patients who developed sepsis postoperatively had significantly higher serum Tyr-NO2 levels one day prior to diagnosis (median (IQR) 4.5 (1.65 – 8.21) fmol BSA-NO2 equivalents/mg protein) compared to patients without sepsis (1.2 (0.74 – 5.97) fmol BSA-NO2 equivalents/mg protein; p<0.05). Tyr-NO2 levels have not previously been measured before clinical diagnosis. However, Tyr-NO2 did not improve upon CRP as a diagnostic marker (area under the curve: Tyr-NO2 0.69 versus CRP 0.88). Nitrate (NO3¯) supplementation in healthy smokers was also studied. Plasma Tyr-NO2 levels were unaltered by supplementation or smoking status. Salivary nitration was unaffected by smoking and decreased with NO3¯ supplementation: the median (IQR) pre-supplementation was 0.67 (0.31-1.14) and post-supplementation was 0.43 (0.12-0.61) pmol BSA-NO2 equivalents/mg protein. Ozone-based chemiluminescence was utilised for nitrite (NO2¯) and NO3¯ measurement as indicators of ˙NO production. Plasma and salivary NO2¯ and NO3¯ concentrations increased significantly with NO3¯ supplementation (p<0.05). In contrast to published studies, brain frontal lobe Tyr-NO2 levels were not higher in dementia: the median (IQR) levels in dementia were 0.29 (0.19-0.57) and in non-dementia controls were 0.3 (0.22-0.55) pmol BSA-NO2 equivalents/mg protein. However, the median brain tissue NO2¯ concentration was significantly higher in the Alzheimer’s disease group (p<0.05). Western blotting revealed that nitration was predominantly in a few select proteins, with TOF-MS/MS analysis suggesting haemoglobin is one of these proteins. Measurement of nitrative stress using ozone-based chemiluminescence and an electrochemiluminescence-based-ELISA overcomes earlier methodological flaws, such as low sensitivity. Detection of total Tyr-NO2 in different inflammatory states indicates that its measurement could have potential as a marker of disease, but measurement of nitration in specific proteins may be more informative than total Tyr-NO2.

Nuclear transfer (NT), or cloning, which is the transfer of a donor nucleus to a recipient enucleated oocyte, has been successfully achieved to produce viable offspring in many species. The process is very inefficient, as reprogramming of the donor nucleus is required, and losses are high throughout development. Placentation abnormalities are a common feature amongst cloned animals. Incomplete nuclear reprogramming and erroneous epigenetic imprinting may contribute to aberrant protein transcription and DNA mutations, affecting mitochondrial metabolism and inducing cellular stress. In vitro produced embryos under high oxygen culture conditions may also suffer oxidative stress, with the resulting reactive oxygen species causing mitochondrial DNA mutations and cellular stress similar to clones. In this study, expression of oxidative stress protein markers (Hsp60, SOD2, Hsp70) in NT cotyledons were compared to artificial insemination (AI) at different time points of gestation (days 50, 100, and 150). As a continuum of the oxidative stress investigation in cloned cotyledons, in vitro produced embryos were cultured under 20% oxygen compared to the control 7% oxygen laboratory standard culture, with oxidative stress protein markers examined between the groups at blastocyst stage (day 7) and day 15. Embryo morphology was also observed to determine apparent physiological differences between the treatment and control embryos. No previous studies to date have investigated the developmental effects of oxidative stress in day 15 bovine embryos. The significant differences in oxidative stress proteins observed at several time points in the NT and AI groups were not repeatable, possibly due to sample freeze/thaw degradation. Morphological differences observed between embryos cultured in 20% oxygen and control groups were visually apparent, although not quantified. At day 15 manganese superoxide dismutase expression was significantly lower in the 20% group compared to control. The 20% oxygen group did not show higher heat shock protein 60 expression than control, however the same results have been observed in another study at blastocyst stage. The results of this study suggest that the effect of oxidative stress on embryonic development is evident yet inconclusive in bovine NT cotyledons, however does not appear apparent in day 15 embryos following culture in 20% oxygen.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Pro-inflammatory cytokines released from immune cells infiltrating the endocrine pancreas in Type 1 Diabetes (T1D) induce the generation of reactive oxygen and nitrogen species (ROS/RNS). Cytokines are in part cytotoxic to ƓÒ-cells via the production of peroxynitrite (ONOO-). ƓÒ-cell are weakly protected against the toxicity of ROS/RNS because of limited expression of antioxidant enzymes. The purpose of this study was to evaluate the expression and regulation of Peroxiredoxins (Prdxs/PRDXs), a new family of antioxidant enzymes in islet ƓÒ-cell. Peroxiredoxin 5 (Prdx5) is ubiquitously expressed in mammals and it exhibits a range of cellular roles including cytoprotective antioxidant defence. Human PRDX5 possesses a peroxynitrite reductase activity but its role in ƓÒ-cell defence was not investigated yet. In a first set of experiments, the localization of the Prdx family was analyzed in rodent pancreas. Prdx1 was preferentially found in the non-b-cells of the islet and in exocrine tissue. Prdx2, Prdx3 and Prdx5 were present in b and non-b-cells, while Prdx4 and Prdx6 were poorly expressed. Then, we investigated the modulation of Prdx mRNA and protein expression levels by cytokines in adult rat isolated islets. Prdx1, Prdx2 and Prdx3 expression was not modified while Prdx5 mRNA was upregulated. However, Prdx5 protein was downregulated, which could involve ubiquitination and proteasomal degradation. Little is known about the PRDX antioxidant enzyme expression in human islets. In a second set of experiments, we investigated the expression and regulation of the 6 PRDXs in human islet preparations facing the context of T1D pathogenesis. PRDX 2, 3, 5, 6 were observed in the exocrine part of the pancreas. PRDX2 and PRDX6 were preferentially expressed in islet ƓÑ cells rather than in ƓÒ cells. PRDX3 and PRDX5 were localized in ƓÑ cells as well as in ƓÒ cells. PRDX4 was detected neither in exocrine nor in endocrine tissue. Islets exposed to a mixture of cytokines showed a downregulation of PRDX2, 3, 5, 6 mRNA expression, as was also the case for PRDX5 protein. This study demonstrated that a clear difference between human and rodent species does exist in terms of tissue localization, expression and regulation of Prdxs by cytokines. Finally, we performed Prdx5 overexpression or silencing in insulin secreting cell line INS-1E. Overexpression of Prdx5 was effective against a stress induced by SIN-1 but not against the cytokines mixture. On the opposite, silencing Prdx5 expression decreased the cell viability. Then, the hypothesis that the vulnerability of islets to cytokines mixture was due to the Prdx5 downregulation was not demonstrated. However, the modification of Prdx5 expression would in part be responsible for the high sensitivity of ƓÒ-cell to peroxynitrite. In conclusion, this study featured the presence of some Prdxs/PRDXs in islet cells, and the regulation of their expression by cytokines. They intervene in protection against ONOO- toxicity but their implication against cytokine agression remain to be more precisely evaluated.

Drug induced idiosyncratic agranulocytosis has been attributed to oxidation by hypochlorite formed by bone marrow myeloperoxidase (MPO). Idiosyncratic liver toxicity could also involve drug oxidative activation by cytochrome P450 (in hepatocytes) or MPO (in Kupffer cells or infiltrating neutrophil/macrophages). Such drug reactive metabolites could cause cytotoxicity or release “danger signals” that attract immune cells which release H2O2 resulting from nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation. In vivo animal studies have shown that low level tissue inflammation markedly increased drug-induced tissue toxicity which was prevented by immune cell inhibitors and increased by cell activators. It is suggested that idiosyncratic drugs are much more toxic, taken during symptomless inflammation periods. Furthermore, it is hypothesized that hepatocytes are much more susceptible to some idiosyncratic drugs if they are exposed to hydrogen peroxide (H2O2)/myeloperoxidase or cytokines released by inflammatory cells. A hepatocyte inflammation model, in which hepatocytes were exposed to a non-toxic H2O2 generating system and peroxidase, was found to be much more vulnerable to four idiosyncratic drugs e.g., troglitazone, isoniazid, hydralazine and amodiaquine. The molecular cytotoxic mechanisms for this marked increase in cytotoxicity were investigated as follows: 1) A P450/H2O2-catalyzed pathway not involving oxidative stress e.g., hydralazine and isoniazid; 2) A P450/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g., hydrazine (an isoniazid metabolite) and hydralazine; and 3) A peroxidase/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g,, hydralazine, amodiaquine and troglitazone. Before cytotoxicity ensued, GSH oxidation, protein carbonyl formation and often lipid peroxidation occurred followed by a decrease in mitochondrial membrane potential indicating that oxidative stress was the molecular mechanism of cytotoxicity. In summary, a H2O2-enhanced hepatocyte system in the presence and absence of peroxidase may prove useful for a more robust screening of drugs for assessing the enhanced drug toxicity risk associated with taking drugs during periods of inflammation.

Drug induced idiosyncratic agranulocytosis has been attributed to oxidation by hypochlorite formed by bone marrow myeloperoxidase (MPO). Idiosyncratic liver toxicity could also involve drug oxidative activation by cytochrome P450 (in hepatocytes) or MPO (in Kupffer cells or infiltrating neutrophil/macrophages). Such drug reactive metabolites could cause cytotoxicity or release “danger signals” that attract immune cells which release H2O2 resulting from nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation. In vivo animal studies have shown that low level tissue inflammation markedly increased drug-induced tissue toxicity which was prevented by immune cell inhibitors and increased by cell activators. It is suggested that idiosyncratic drugs are much more toxic, taken during symptomless inflammation periods. Furthermore, it is hypothesized that hepatocytes are much more susceptible to some idiosyncratic drugs if they are exposed to hydrogen peroxide (H2O2)/myeloperoxidase or cytokines released by inflammatory cells. A hepatocyte inflammation model, in which hepatocytes were exposed to a non-toxic H2O2 generating system and peroxidase, was found to be much more vulnerable to four idiosyncratic drugs e.g., troglitazone, isoniazid, hydralazine and amodiaquine. The molecular cytotoxic mechanisms for this marked increase in cytotoxicity were investigated as follows: 1) A P450/H2O2-catalyzed pathway not involving oxidative stress e.g., hydralazine and isoniazid; 2) A P450/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g., hydrazine (an isoniazid metabolite) and hydralazine; and 3) A peroxidase/H2O2-catalyzed oxidative stress-mediated cytotoxic pathway e.g,, hydralazine, amodiaquine and troglitazone. Before cytotoxicity ensued, GSH oxidation, protein carbonyl formation and often lipid peroxidation occurred followed by a decrease in mitochondrial membrane potential indicating that oxidative stress was the molecular mechanism of cytotoxicity. In summary, a H2O2-enhanced hepatocyte system in the presence and absence of peroxidase may prove useful for a more robust screening of drugs for assessing the enhanced drug toxicity risk associated with taking drugs during periods of inflammation.

No description available.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 77-86).

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Introduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K). Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide. Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway. Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.