Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Elevated circulating free fatty acids (FFA) cause hepatic insulin resistance; however, the mechanisms for this process are incompletely understood. The objective of the studies in the thesis was to examine whether protein kinase C (PKC)-delta (d), oxidative stress, and the serine kinase IkBa kinase (IKK) B are causally involved in FFA-induced hepatic insulin resistance. To test this, we infused rats with lipid with or without inhibitors of the aforementioned factors for 7h, during the last 2h of which a hyperinsulinemic-euglycemic clamp was performed. In Study 1, inhibition of hepatic PKC-d using antisense oligonucleotide prevented FFA-induced membrane translocation of PKC-d, which is a marker of its activation, in parallel with prevention of lipid-induced hepatic insulin resistance, without affecting lipid-induced peripheral insulin resistance. These results implicate PKC-d as a causal mediator of FFA-induced hepatic insulin resistance. In Study 2, the antioxidant N-acetyl-L-cysteine (NAC) prevented lipid-induced hepatic insulin resistance in conjunction with reversal of lipid-induced increase in markers of IKKB and c-Jun NH2-terminal kinase 1 (JNK1) activation, and of impairment of insulin signaling, without affecting PKC-d membrane translocation and increase in phosphorylated p38 mitogen-activated protein kinase (MAPK) induced by lipid infusion. These findings suggested that oxidative stress is a causal mediator of lipid-induced hepatic insulin resistance upstream of IKKB and JNK1, and potentially downstream of PKC-d and p38 MAPK. In Study 3, sodium salicylate, an IKKB inhibitor, prevented FFA-induced hepatic insulin resistance via restoration of hepatic insulin signaling, thus implicating IKKB as a causal factor in the process. Together, the results from these studies demonstrate that PKC-d, oxidative stress, and IKKB are causally involved in FFA-induced hepatic insulin resistance and suggest that the sequence for the process is: FFA -> PKC-d -> oxidative stress -> IKKB -> impaired hepatic insulin signaling.

An important mechanism involved in the pathogenesis of type 2 diabetes is elevation of plasma free fatty acids which induce insulin resistance and may impair both β-cell function and mass (β-cell lipotoxicity). The objective of my thesis was to investigate the role of oxidative stress in β-cell lipotoxicity, using in vivo, ex vivo, and in vitro models. I used in vivo models of 48h i.v. oleate or olive oil infusion in Wistar rats followed by hyperglycemic clamps, or islet secretion studies ex vivo, and in vitro models of 48h exposure to oleate in isolated islets. My first study showed that 48h oleate infusion decreased the insulin response to a hyperglycemic clamp, an effect prevented by coinfusion of the antioxidants N-acetylcysteine and taurine. Similar to the findings in vivo, 48h infusion of oleate decreased glucose stimulated insulin secretion (GSIS) ex vivo, and induced oxidative stress in isolated islets, effects prevented by coinfusion of the antioxidants N-acetylcysteine, taurine, or tempol. Islets exposed to oleate or palmitate showed a decreased insulin response to high glucose and increased levels of oxidative stress, effects prevented by taurine. Therefore, my data are the first demonstration that oxidative stress plays a role in the decrease in β-cell secretory function induced by prolonged exposure to FFA, in vitro and in vivo. My second study addressed downstream effects of oxidative stress involving inflammation. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the IKKβ inhibitor salicylate. GSIS in isolated islets was impaired by olive oil or oleate and restored by salicylate. These results suggest a potential role for both oxidative stress and inflammation in lipid-induced β-cell dysfunction. My third study addressed downstream effects of oxidative stress involving β-cell insulin signalling. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the tyrosine phosphatase inhibitor bisperoxovanadate. GSIS in isolated islets was impaired by olive oil or oleate and restored by bisperoxovanadate, suggesting a role of FFA in decreasing β-cell function by induction of β-cell insulin resistance.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Estrés oxidativo e inflamación parecen jugar un papel importante en el proceso de envejecimiento relacionado con la disfunción del sistema cardiovascular humano. Hábitos de vida tales como fumar, consumo de alcohol y ejercicio físico, pueden alterar el estado redox de los tejidos y afectar la relación de los marcadores de estrés oxidativo con la edad. Se requiere Información sobre el estado oxidativo de las poblaciones humanas para verificar la función de daño oxidativo en el envejecimiento y los cambios relacionados con el sistema cardiovascular, con el fin de identificar cuáles son los factores de riesgo cardiovascular más influyentes en el daño oxidativo asociado al envejecimiento y el papel de la dieta y el estilo de vida en esta relación. Este estudio se realizó en una población de 160 personas sanas o con patología leve tratada y compensada, distribuidos uniformemente en un rango de edad de 16 a 84 años. Lo datos recogidos de cada paciente fueron; antropométricos, bioquímica general, personales, dietéticos y se determinó la concentración de variables relacionadas con el estrés oxidativo, inflamación y/o riesgo cardiovascular: Mieloperoxidasa (MPO), Homocisteina, Proteína C Reactiva (PCR) ultrasensible y Malondialdehido (MDA). Resultados. La edad se correlaciona con numerosos marcadores de riesgo cardiovascular aunque no lo hace con los de estrés oxidativo. La PCR, la MPO y el MDA se correlacionan positivamente con los triglicéridos. El MDA se correlaciona con el colesterol; la PCR con el Índice Aterogénico; el IMC y la circunferencia de cintura resultaron las variables que más influyen sobre la concentración de triglicéridos e Índice aterogénico. El MDA se correlaciona positivamente con la edad en individuos menores de 65 años. El consumo de frutas y verduras se asociaría negativamente al MDA en una regresión múltiple incluyendo solo individuos de 40 a 65 años. Los resultados de este estudio indican que las variables lipídicas son las que mejor se correlacionan con el estrés oxidativo. El IMC y la circunferencia de cintura se han comportado como las variables más influyentes sobre los valores de lipídicos. Los hábitos dietéticos relacionados con el consumo de frutas y verduras ayudan a disminuir la concentración de MDA que tiende a incrementarse con la edad en individuos de 40 a 65 años en nuestra población. / Oxidative stress and inflammation appear to play an important role in the process of ageing and in the associated dysfunction of the human cardiovascular system. Lifestyle habits such as smoking, alcohol consummation, physical exercises, might alter the redox state of body tissues and affect the relationship of oxidative stress markers with age. Information about the oxidative status of human populations is required to verify the role of oxidative damage in the ageing related changes of the cardiovascular system, and to identify what are the most influential cardiovascular risk factors on the oxidative damage associated to ageing and the influence of lifestyle in this relation. This study was conducted in a population of 160 healthy individuals or with mild disease treated and compensated, in an evenly distributed range of age of age 16 to 84 years. General biochemical, anthropometric data, personal, dietary supplements and determining the concentration of variables related to oxidative stress and / or cardiovascular risk: Myeloperoxidase (MPO), Homocysteine, ultrasensitive C-reactive protein (CRP) and Malondialdehyde (MDA) was collected of each patient. Information about individual lifestyle patterns were also collected. Interactions between variables were assessed by correlation (Pearson or Spearman) and multiple regression analyses. Results. There were not gender differences in the variables of oxidative stress and inflammation assessed in this study. hsCRP was significantly associated with age, Framingham index, waist circumference, body mass index, blood glucose and plasma triglycerides. MDA plasma concentration was related to the values of cholesterol and triglycerides blood levels. MPO was associated with the plasma concentration of triglycerides and the atherogenic index. BMI and waist circumference are variables with more influence in the concentration of triglycerides and atherogenic index. The MDA was positively correlated with age in individuals younger than 65 years. The consumption of fruits and vegetables were negatively associated with MDA in a multiple regression including only individuals from 40 to 65 years. These results indicate that plasma concentrations of triglycerides and total cholesterol positively associate with oxidative stress and inflammation in a general human population. In addition, due to the body mass index appears to be the most influential factor on the lipid plasma levels, it may be a good marker of this association. Eating habits associated with the consumption of fruits and vegetables help reduce the concentration of MDA, which tends to increase with age in individuals 40 to 65 years in our population.

Las especies reactivas del oxígeno (ROS), superóxido (O2o-), peróxido de hidrógeno (H2O2), y radical hidroxilo (OHo), se generan a partir de la reducción parcial del oxígeno molecular durante procesos metabólicos como la respiración o tras la exposición a ciertos agentes ambientales como las radiaciones UV. Estas ROS pueden reaccionar con biomoléculas como lípidos, proteínas y DNA e inactivar su función, por lo que las células han desarrollado actividades enzimáticas que se encargan de mantener niveles no-tóxicos de estos oxidantes. Se llama estrés oxidativo a la situación en la cual se produce un incremento en la concentración intracelular de ROS como consecuencia de un aumento en la generación o una disminución en la degradación de las mismas. En respuesta a estrés oxidativo, la célula activa rutas de señalización y factores de transcripción específicos que activan la expresión de proteínas antioxidantes encargadas de reestablecer los niveles redox intracelulares y de reparar los desperfectos causados por estos oxidantes. La levadura Schizosaccharomyces pombe es un organismo modelo ideal para el estudio de las respuestas a estrés oxidativo en las células eucariotas ya que posee sensores específicos a estrés oxidativo como el factor de transcripción Pap1 (pombe AP-1-like) y rutas de respuesta global a estrés, como las descritas en las células de mamífero, que son activadas por diferentes tipos de estrés. En el centro de esta ruta de respuesta global a estrés se encuentra la MAPK (Mitogen-activated protein kinase) Sty1. El factor de transcripción Pap1, de localización citoplasmática basal, se acumula en el núcleo en respuesta a estrés oxidativo. Este cambio de localización subcelular es debido a la inhibición del exporte nuclear dependiente de Crm1, aunque se desconocía el mecanismo molecular utilizado por este factor de transcripción para sensar y responder a oxidantes como H2O2 y dietilmaleto (DEM). Los resultados obtenidos indican que H2O2 oxida de forma reversible dos residuos de cisteína de Pap1 induciendo, seguramente, la formación de un puente disulfuro intramolecular, mientras que, DEM actúa como un agente alquilante que modifica de forma irreversible los residuos de cisteína del dominio C-terminal de Pap1. El gen que codifica para el factor de transcripción Pap1 fue aislado inicialmente como un gen que, en elevado número de copias, confería a las células un fenotipo de resistencia a ciertas drogas como estaurosporina. Esto es debido a que, tras acumularse en el núcleo en respuesta a estrés oxidativo, Pap1 activa la transcripción de genes implicados tanto en la respuesta antioxidante como en la resistencia a multidrogas. Todos aquellos genes que, al igual que pap1 fueron identificados por su implicación en la resistencia a multidrogas, codifican para proteínas que regulan la actividad del factor de transcripción Pap1. hba1 fue el único gen relacionado con resistencia a multidrogas, cuyo producto génico, una proteína con un dominio de unión a Ran (Ran-binding domain), Hba1, no había sido relacionado con la actividad de Pap1. Uno de los objetivos de mi trabajo experimental era el de determinar si Hba1 tenía un papel en la regulación de la actividad de Pap1. Nuestros resultados indican que la proteína Hba1, localizada en el nucleoplasma de la célula, participa en el exporte nuclear mediado por Crm1 de ciertas proteínas como el factor de transcripción Pap1 y la MAPK Sty1, aunque no de otras como la proteína PKI. Por ello, la pérdida de función de Hba1, por sobreexpresión o deleción del gen hba1, induce la localización nuclear constitutiva de Pap1 y Sty1 en ausencia de estrés. Esta localización nuclear de Pap1 es suficiente para la activación transcripcional de sus genes diana. Por lo tanto, el fenotipo de resistencia aumentada a multidrogas de las cepas en las que se ha perdido la actividad de la proteína Hba1, es debido a la acumulación de Pap1 en el núcleo en condiciones de no-estrés.

The main objective of this proposal was to investigate the effect of vitamin B6 on colon carcinogenesis in vivo. Two in vivo studies were conducted to determine the role of vitamin B6 as a biological modifier of colon carcinogenesis. It is hypothesized that vitamin B6 may serve as an antioxidant in vivo and will modulate colon carcinogenesis. In the first study, a 2X3 factorial experimental design was used to determine if three different levels of vitamin B6, classified as low, normal and high in conjunction with two different levels of protein intake, classified as normal or high, will affect post-initiation stages of colon carcinogenesis, in Sprague-Dawley rats. Male Sprague-Dawley male were injected with azoxymethane for two weeks (15mg/kg/week) and then one week later they were allocated to different dietary treatment groups. After eight weeks, the effects of dietary treatment on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of preneoplastic lesions, aberrant crypt foci (ACF), were evaluated. The lowest level of vitamin B6 intake with a high protein diet reduced the growth and development of ACF. Vitamin B6 had no significant effect on the oxidative stress markers. The level of protein was an important variable in modulating the levels of 3-nitrotyrosine and 8-OH-DG which were lower in high protein groups than normal protein counterparts. The objective of the second study was to investigate if a supraphysiological (5 fold higher than normal level) dosage of vitamin B6 could have an antioxidant effect in a metabolically compromised state like obesity and thereby lower the risk of colon cancer. Female Zucker obese (Zk-OB) rats received normal (Zk-OBN, 7 mg/kg) or high (Zk-OBH, 35 mg/kg) vitamin B6 (Pyridoxine-HCl) diets two weeks prior to, during and six weeks following injection with colon carcinogen AOM. The effects of supplemental vitamin B6 on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of ACF were carried out. High intake of vitamin B6 significantly lowered liver weights and plasma cholesterol compared to the normal intake (p≤0.05). Zk-OBH rats had significantly reduced number of ACF compared to Zk-OBN (p≤0.05). Hepatic GSH increased in the ZK-OBH group with a concomitant decrease in GPx activity. The findings demonstrate that in Zucker Obese rats, a high B6 intake augmented the antioxidant potential and decreased sensitivity to colon carcinogenesis. These findings suggest that high vitamin B6 plays an important therapeutic role in the compromised state of obesity.

There is an increasing concern over the safety of engineered nanoparticles (ENPs) to humans and the environment. It is important that the environmental risks of these particles to be tested under research and regulatory schemes, e.g. Nano Risks to the environment & Human Health (NanoReTox) under Seventh Framework Programme (FP7) in EU. Due to their unique properties and the fact that their detection and characterization in complex matrices is challenging, classic analytical methods and test approaches for assessing environmental risk may not be appropriate for ENPs. In this paper I present the challenges associated with ENPs exposure to the estuarine environment and the testing of a chosen ENPs to generate data on ecotoxicity in the test estuarine organisms for further consideration of risk assessment of marine environment. Careful consideration was given to the selection of the test materials (benthic organisms Nereis diversicolor and Scrobicularia plana), the test system and the test exposure conditions (CdS ENPs and aqueous CdS added to filtered natural seawater with a concentration of 10 µg L-1). Evaluation of the exposure effects was carried out by behavioral tests (burrowing kinetics and feeding rate) and biochemical responses (quantification of biomarkers). Multispecies Freshwater Biomonitor®  (MFB) tests and GST analysis results show significant differences in between control group and CdS NPs exposure one, indicating that CdS NPs are potential to cause sublethal effects in test organisms. Our knowledge in environmental risk assessment of ENPs is still limited. Coordinated research is required to gain a better understanding of the factors and processes affecting ENP fate and effects in the environment as well as to develop more usable, robust and sensitive methods for characterization and detection of ENPs in environmental systems. / NanoReTox, FP7 NMP Work Programme