Iron homeostasis is regulated by iron regulatory proteins, IRP1 and IRP2, which bind to iron responsive elements (IRE) in the mRNA of proteins of iron metabolism such as ferritin (iron storage). IRP2 undergoes iron-mediated degradation, and this pathway shares notable similarities with that of hypoxia-inducible factor 1 (HIF-1). It has been reported that oxidative stress marked by increased reactive oxygen species (ROS) signal HIF-1 stabilization in hypoxia. The role of ROS in IRP2 regulation is not well-established. We show that the degree of hypoxia induces differential effects on iron-mediated degradation of IRP2, such that IRP2 levels are 3-fold higher when exposed to 0.1% O 2 compared to 3% O2 after 4 hours of iron treatment. Hydrogen peroxide (H2O2) affects IRP2 by inducing IRE-binding activity after 12 hours, which is accompanied by decreased ferritin levels. Furthermore, the ability of H2O2 to protect IRP2 against iron-dependent degradation is similar to that of hypoxia. Finally, both intracellular and extracellular sources of oxidative stress protect IRP2 from ascorbate-mediated degradation. Taken together, these results support a role of ROS in protecting IRP2 against iron-mediated degradation and indicate that oxidative stress modulates downstream effects of IRP2.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

Exposure to the anticonvulsant valproic acid (VPA) is associated with a 7.5% rate of major malformations and a 1-2% incidence of neural tube defects (NTDs). Although the teratogenic outcomes resulting from VPA use during pregnancy were first identified in the 1980s, the mechanisms by which VPA induces birth defects are not fully elucidated. Based on evidence in the literature, the studies in this thesis examined the role of in utero VPA exposure on oxidative stress and epigenetic alterations in the developing embryo to provide further mechanistic insight into VPA’s teratogenic pathway. The first study investigated the role of oxidative stress in VPA-induced teratogenesis. Using CD-1 mice, catalase was shown to protect against VPA-induced effects on developmental and morphological parameters in both whole embryo culture and in vivo models. Studies in whole embryo culture demonstrated that markers of oxidative damage were not altered by VPA; however, VPA increased apoptosis in the neuroepithelium, which was attenuated by the addition of catalase. The second objective addressed epigenetic modifications induced by VPA in an in vivo mouse model. Maternal administration of VPA resulted in increased acetylation of histones H3 and H4, increased methylation of histone H3K4, and decreased methylation of histone H3K9. Furthermore, these changes were localized to VPA target tissues including the neuroepithelium, heart, and somites. Global DNA methylation in the embryo was not altered by VPA. The final objective was to determine VPA’s effect on a marker of DNA damage, markers of cell cycle proteins, and a marker of apotosis in vivo. Maternal administration of VPA resulted in a rapid increase of γH2A.X, a marker of DNA double strand breaks (DSBs). Increased expression of p27KIP1, a cyclin-dependent kinase inhibitor, and activated caspase-3, a marker of apoptosis, were observed and these changes were localized to the neuroepithelium of developing embryos. In conclusion, this thesis supports the hypothesis that VPA-induced increases in ROS production and HDAC inhibition may lead to altered gene expression patterns and consequently teratogenic effects, namely NTDs. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2011-05-24 13:12:33.778

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Folic acid can effectively reduce blood homocysteine levels. Recent studies have shown that hyperhomocysteinemia is also associated with liver disorders. However, the underlying mechanisms remain unclear. The general objective of my study was to investigate the biochemical and molecular mechanisms of homocysteine-induced liver injury and abnormal lipid metabolism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 weeks. An elevation of serum aminotransferases activities (indicator for liver injury) and an increase in hepatic lipid peroxidation were observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production led to oxidative stress in the liver. Reduction of oxidative stress by inhibiting superoxide anion production ameliorated hyperhomocysteinemia-induced liver injury. A significant elevation of hepatic and serum cholesterol concentrations in hyperhomocysteinemic rats was observed, exclusively due to increased expression of HMG-CoA reductase in hepatocytes. The molecular mechanisms of homocysteine-induced adverse effects were further investigated in isolated rat hepatocytes and in human hepatoma cells (HepG2). Hcy stimulated HMG-CoA reductase expression in hepatocytes via activation of transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element binding protein (CREB) and nuclear factor Y (NF-Y). Activation of these 3 transcription factors was detected in hyperhomocysteinemic rat liver and in homocysteine-treated hepatocytes. Pretreatment of hepatocytes with inhibitors for individual transcription factors effectively attenuated Hcy-induced HMG-CoA reductase mRNA expression. Supplementation of folic acid in diet significantly reduced serum homocysteine level and effectively inhibited hyperhomocysteinemia-induced superoxide anion production, resulting in amelioration of oxidative stress-mediated liver injury in hyperhomocysteinemic rats. These results reflected a protective role of folic acid in hyperhomocysteinemia-induced liver injury. In conclusion, the present study demonstrates that (1) hyperhomocysteinemia can cause oxidative stress and liver injury; (2) homocysteine stimulates cholesterol biosynthesis in hepatocytes via transcriptional regulation of HMG-CoA reductase expression; (3) supplementation of folic acid offers a hepatoprotective effect during hyperhomocysteinemia. Oxidative stress and accumulation of cholesterol in the liver contribute to liver injury associated with hyperhomocysteinemia. The role of folic acid in maintaining good health may extend beyond the cardiovascular system to encompass hyperhomocysteinemia-associated liver disorders.

Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Understanding the molecular basis of doxorubicin (Dox)-induced oxidative stress leading to cardiomyopathy is crucial to finding cardioprotective strategies to manage this important clinical problem. Improving the antioxidant defenses of cardiac cells could be one strategy for cardioprotection. The role of oxidative stress in Dox-induced cardiotoxicity as well as testing the efficacy of antioxidant Vitamin C (Vit C) in offering protection to cardiomyocytes was investigated. As stem cells have been suggested to play a role in this cardiotoxicity, Dox-mediated oxidative stress effects, with and without Vit C, on the stem cell antigen-1 (Sca) positive cells from heart as well as bone marrow were also examined. Our time-course studies of the effects of Dox on the isolated cardiomyocytes showed that the phosphorylation of mitogen-activated protein kinases and p53 followed the rise in reactive oxygen species (ROS) production. Dox also downregulated the Sodium-dependent Vit C Transporter-2 (SVCT-2) and this may have enhanced Dox-induced increase in oxidative stress. Pro-apoptotic markers Bax/Bcl-xL ratio and caspase 3 cleavage were higher after the activation of stress-induced pathways and viability of cells was decreased. Dox-induced increase in apoptosis and decrease in cell viability depended in part on the activation of p38/JNK and p53 proteins, but not on the ERK protein. Exposure to Dox, increased membrane leakage, autophagy and lipid peroxidation. On the other hand, Dox decreased overall antioxidant capacity as well as expression of the endogenous antioxidant enzymes glutathione peroxidase, Cu/Zn superoxide dismutase and catalase. Dox affected Sca-1 positive cells in a prominent manner which was marked by a dose-dependent increase in cell loss, cell leakage and ROS levels as well as decrease in cellular ATP levels. Vit C pre-treatment prior to the addition of Dox delayed and reduced Dox-induced injury to cardiomyocytes, preserving viability. Vit C was able to blunt the decrease in SVCT-2 as well as Dox-induced oxidative stress. Vit C also offered protection to Sca-1 positive cells by partially preventing Dox-induced changes to these cells. The data presented in this thesis improves our knowledge of the molecular mechanisms leading to Dox-induced cardiotoxicity as well as suggest cardioprotection by Vit C.

Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

Šiame darbe tirta ŽPV infekcijos įtaką nėščiosios organizmo antioksidacinės sistemos būklei bei vertintas vietinio imuniteto atsakas į šią infekciją. Nustatytas tiriamųjų infekuotumas ŽPV; identifikuoti atskirų tipų ŽPV; palygintas tiriamųjų infekuotumas ŽPV tarp pirmojo ir trečiojo nėštumo trimestrų; ištirta antioksidacinės sistemos būklė bei vietinį imunitetą rodančių interleukinų (IL-10 ir IL-12) koncentracijų pokyčiai nėštumo metu priklausomai nuo ŽPV infekcijos. Atsižvelgiant į tai, kad Lietuvoje yra didelis sergamumas gimdos kaklelio vėžiu bei mirtingumas nuo jo, mūsų nustatytas nėščiųjų infekuotumas ŽPV bei identifikuoti ŽPV tipai leido įvertinti infekuotumo ŽPV pokyčius nėštumo metu didelės gimdos kaklelio vėžio rizikos populiacijoje ir sudarė galimybę palyginti gautus duomenis su kitų šalių autorių gautais duomenimis. Įrodyta, kad nėštumo metu moterims vystosi oksidacinis stresas, kurio lygis nepriklauso nuo infekuotumo ŽPV. Nustatyta, kad IL-10 ir IL-12 koncentracijos pokyčiai gimdos kaklelio nuoplovose (vietinis imunitetas) susiję su Th1 ir Th2 limfocitų funkcija ir priklauso nuo infekuotumo ŽPV. / This study analyses the impact of HPV infection for antioxidative state of pregnant woman, and the response of local immunity to this infection. In order to achieve this aim the following goals were: to determine HPV infection in pregnant women; to determine it’s types; to compare infection between first and third trimesters of pregnancy; to study the antioxidative system state during pregnancy due to HPV infection; to study interleukins (IL-10 and IL-12), which show local immunity, concentration changes during pregnancy due to HPV infection. Considering a high morbidity and mortality from cervical cancer in Lithuania, our established HPV infection in pregnant women and identified types of this virus enabled us to determine changed of HPV infection during pregnancy in high risk cervical cancer population. Also there is possibility to compare obtained data with that from authors in other countries. It is established, that women develop oxidative stress during pregnancy, and that its level did not depend on HPV infection. Changes of IL-10 and IL-12 concentrations in cervicovaginal washing fluid (local immunity) are related to Th1 and Th2 lymphocytes function, and depend on HPV infection.

Nanotechnology as a science is emerging rapidly. As materials are synthesized and utilized at the nanometer size scale, concerns of potential health and safety effects are arising. In an effort to elucidate the physicochemical characteristics of nanoparticles influential in toxicological studies, surface properties of metal oxide and carbonaceous nanoparticles were measured. These properties include zeta potential, dissolution and surface-bound chemical components. Subsequently, the role of these properties in oxidative stress was examined in vitro. This work identifies the influence that pH has on the zeta potential of nanoparticles. The zeta potential has the ability to alter colloidal stability, as the largest nanoparticle agglomerate is seen at or near the isoelectric point for each of the particles tested. Furthermore, it was observed that metal oxide nanoparticles which exhibit a charged surface at physiological pH, lead to decreased in vitro cellular viability as compared to those that were neutral. Thus, nanoparticle zeta potential may be an important factor to consider when attempting to predict nanoparticle toxicity. Real world exposure to nanoparticles is a mixture of various particulates and organics. Therefore, to simulate this particle mixture, iron oxide (Fe2O3) and engineered carbon black (ECB) were utilized in combination to identify potential synergistic reactions. Following in vitro exposure, both nanoparticle types are internalized into endosomes, where liberated Fe3+ reacts with hydroquinone moieties on the ECB surface yielding Fe2+. This bioavailable iron may then generate oxidative stress through intracellular pathways including the Fenton reaction. As oxidative stress is common in particulate toxicology, a comparison between the antioxidant defenses of epithelial (A549) and mesothelial (MeT-5A) cell lines was made. The A549 cell line exhibits alterations in the NRF2-KEAP1 transcription factor system and therefore retains high basal levels of phase II antioxidants. Both cell types were exposed to 33 nm silica where intracellular oxidant generation coupled with markers of oxidative stress were observed. While the MeT-5A cells exhibited a decrease in cell viability, the A549 cell line did not. Therefore, proper characterization of both material and biological systems prior to toxicity testing will help to further define the risks associated with the use of nanotechnology.