Cleva Villanueva

Subcellular and cellular locations of nitric-oxide synthase isoforms as determinants of health and disease

The effects of nitric oxide in biological systems depend on its steady-state concentration and where it is being produced. The organ where nitric oxide is produced is relevant, and within the organ, which types of cells are actually contributing to this production seem to play a major determinant of its effect. Subcellular compartmentalization of specific nitric oxide synthase enzymes has been shown to play a major role in health and disease. Pathophysiological conditions affect the cellular expression and localization of nitric oxide synthases, which in turn alter organ cross talk. In this study, we describe the compartmentalization of nitric oxide in organs, cells, and subcellular organelles and how its localization relates to several relevant clinical conditions. Understanding the complexity of the compartmentalization of nitric oxide production and the implications of this compartmentalization in terms of cellular targets and downstream effects will eventually contribute toward the development of better strategies for treating or preventing pathological events associated with the increase, inhibition, or mislocalization of nitric oxide production.

Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs.

The antioxidant nordihydroguaiaretic acid (NDGA) has recently become well known as a putative anticancer drug. In this paper, it was evaluated the in vitro peroxynitrite (ONOO− ), singlet oxygen (1O2), hydroxyl radical (OH√), hydrogen peroxide (H2O2), superoxide anion and hypochlorous acid (HOCl) scavenging capacity of NDGA. It was found that NDGA scavenges: (a) ONOO− (IC50 = 4 ± 0.94 μM) as efficiently as uric acid; (b) 1O2 (IC50 = 151 ± 20 μM) more efficiently than dimethyl thiourea, lipoic acid, N-acetyl-cysteine and glutathione; (c) OH√ (IC50 = 0.15 ± 0.02 μM) more efficiently than dimethyl thiourea, uric acid, trolox, dimethyl sulfoxide and mannitol, (d) (IC50 = 15 ± 1 μM) more efficiently than N-acetyl-cysteine, glutathione, tempol and deferoxamine and (e) HOCl (IC50 = 622 ± 42 μM) as efficiently as lipoic acid and N-acetyl-cysteine. NDGA was unable to scavenge H2O2. In an in vivo study in rats, NDGA was able to prevent ozone-induced tyrosine nitration in lungs. It is concluded that NDGA is a potent in vitro scavenger of ONOO− , 1O2, OH√, and HOCl and is able to prevent lung tyrosine nitration in vivo.

Antioxidant-Induced Stress

Antioxidants are among the most popular health-protecting products, sold worldwide without prescription. Indeed, there are many reports showing the benefits of antioxidants but only a few questioning the possible harmful effects of these “drugs”. The normal balance between antioxidants and free radicals in the body is offset when either of these forces prevails. The available evidence on the harmful effects of antioxidants is analyzed in this review. In summary, a hypothesis is presented that “antioxidant-induced stress” results when antioxidants overwhelm the body’s free radicals.

The detection of brain ischaemia in rats by inductive phase shift spectroscopy.

Ischaemia in the brain is an important clinical problem that is often monitored and studied with expensive devices such as MRI and PET, which are not readily available in low economical resource parts of the world. We have developed a new less expensive tool for non-invasive monitoring of ischaemia in the brain. This is a first feasibility study describing the concept. The system is based on the hypothesis that electromagnetic properties of the tissue change during ischaemia and that measuring the electromagnetic properties of the bulk of the brain with non-contact means can detect these changes. The apparatus we have built and whose design we describe here consists of two electromagnetic coils placed around the head. The system measures the bulk change in time of the phase difference between the electromagnetic signal on the two coils in a range of frequencies. A mathematical model simulating the device and the measurement is also introduced. Ischaemia was induced in the brain of rats by occlusion of the right cerebral and carotid arteries. Experimental subjects were monitored for 24 h. Inductive phase shift measurements were made at five frequencies in the range of 0.1-50 MHz eight times during the observation period. An ex vivo estimation of the percentage of necrosis in the ischemic subjects at t = 24 h was done. The mathematical model was also applied to the experimental tested situation. The results of both experiments and theory show significant phase shifts increase as a function of frequency and ischaemia time. The theoretical and experimental results suggest that the tested technique has the potential to detect the processes and level of ischaemia in the brain by non-invasive, continuous, bulk volumetric monitoring with a simple and inexpensive apparatus.

Inductive Phase Shift Spectroscopy for Volumetric Brain Edema Detection: An Experimental Simulation

This study evaluates experimentally an induction based non-invasive technique for detection of changes of fluid volume through phase shift measurements as a possible method for volumetric brain edema monitoring. An induction coil - spherical head model was build and tested. The model involves two different diameter coils coaxially centered on a two- compartment glass sphere head model centrally placed with respect to the coils. Three different fluid volumes of physiological saline in 20 ml increments were used to simulate different edema levels. Phase shift of the impedance coils as a function of relative fluid volume was measured at five frequencies (40, 50, 100, 200 and 300 MHz) by a commercial vector network analyzer. The results show significant phase shift increase as a function of frequency and fluid volume. The experiments with the coil- spherical head system suggest that the tested technique has the potential to become a practical configuration for non-invasive volumetric brain edema monitoring.

Effects of Exercise on Oxidative Stress in Rats Induced by Ozone

Oxidative stress (OS) induced by acute exercise is reduced by chronic exercise. Ozone (O3) exposure produces OS. The aim of this study was to determine if aerobic exercise (AE) reduced OS produced by O3. A pilot experiment was performed with male Wistar rats submitted to AE (trained to swim 90 min/day). Adaptation to exercise was demonstrated three weeks after training by means of changes in reduced nitrates (NOx) in plasma. Therefore, two-week training was chosen for the following experiments. Six of twelve trained rats were exposed to O3 (0.5 ppm, 4 h/day, one hour before exercise). Two groups of sedentary animals (n = 6 each) were used as controls, one of which was exposed to O3. At the end of the experiments NOx, 8-isoprostane (8-IP), malondialdehyde (MDA), superoxide dismutase (SOD) activity, and carbonyls (CBs) were measured in plasma. CBs did not change in any group. O3-induced OS was manifested by reduced NOx and SOD activity, as well as increased 8-IP and MDA. Exercise significantly blocked O3 effects although SOD was also decreased by exercise (a greater drop occurring in the O3 group). It is concluded that AE protects against OS produced by O3 and the effect is independent of SOD.

Oxychlorine species suppress postsurgical adhesions in rats

BACKGROUND Surgically induced adhesions complicate up to 100% of abdominal surgeries. Food and Drug Administration-approved treatments are generally not only less effective than desired but they also have major contraindications. Oxychlorine species, including chlorine dioxide (ClO2), suppress scar formation in infected wounds without affecting keratinocytes while reducing fibroblast proliferation. The aim of the present study was to evaluate the effect of oxychlorine solutions containing ClO2 on adhesion formation. METHODS Male Wistar rats were subjected to Buckenmaier model of surgical adhesions and treated with either oxychlorine solutions containing ClO2 (40-150 ppm) or isotonic saline solution. To increase the severity of adhesions, peritonitis was produced by intraperitoneal administration of a diluted nonlethal dose of feces (50 mg/kg). Wound strength of the healed wound was measured to evaluate the effects of oxychlorine solutions. In addition, an oxychlorine solution of lesser efficacy (at 100 ppm) was compared with three available anti-adhesion materials. RESULTS Reproducibility of the model was validated in 26 rats. Oxychlorine solutions containing ClO2 (40-110 ppm) significantly reduced postsurgical adhesion formation without affecting the strength of the healed wound. Higher concentrations (120 and 150 ppm) had no effect. Fecal peritonitis significantly increased, and solutions with ClO2 at 110 ppm significantly reduced adhesion formation. The effect of the oxychlorine solution was significantly greater than that of Interceed, Guardix, Seprafilm, and isotonic saline solution. CONCLUSIONS ClO2-containing oxychlorine solutions could be an innovative strategy for the suppression of surgical adhesion formation, with the additional advantage of contributing antiseptic properties.

Free Radicals and Neuronal Recovery from an Ischaemic Penumbra: A Review

Stroke remains the second leading cause of death worldwide. The major problem is that the therapeutic window is short and no accepted treatment is completely efficient. Even though there is evidence of free radical participation in the pathophysiology of stroke, no beneficial effects of antioxidants have been demonstrated in clinical assays. Moreover, some reports paradoxically indicate that antioxidants could be harmful and that oxidative stress preconditioning could reduce the long-term effects of stroke. There are two major areas within the ischaemic zone: (1) the core, where neuronal necrosis develops in minutes, and (2) the penumbra surrounding the core, where some neurons could eventually be recovered over an extended time. The present review specifically focuses on the role of free radicals in the life or death of brain cells (mainly neurons) within the ischaemic penumbra. It also analyses the effects of oxidative stress on bloodbrain barrier disruption. In conclusion, we postulate a cascade of events that follow cerebral ischaemia and what type of therapeutic approach could eventually change the effect of free radicals on neuronal recovery from an ischaemic penumbra.

Impedance spectroscopy assisted by magnetic nanoparticles as a potential biosensor principle for breast cancer cells in suspension

Breast cancer (BC) is the leading cause of cancer death in women worldwide, with a higher mortality reported in undeveloped countries. Ideal adjuvant therapeutic strategies require the continuous monitoring of patients by regular blood tests to detect circulating cancer cells, in order to determine whether additional treatment is necessary to prevent cancer dissemination. This circumstance requires a non-complex design of tumor cell biosensor in whole blood with feasibility for use in poor regions. In this work we have evaluated an inexpensive and simple technique of relative bioimpedance measurement, assisted by magnetic nanoparticles, as a potential biosensor of BC cells in suspension. Measurements represent the relative impedance changes caused by the magnetic holding of an interphase of tumor cells versus a homogenous condition in the frequency range of 10-100 kHz. The results indicate that use of a magnet to separate tumor cells in suspension, coupled to magnetic nanoparticles, is a feasible technique to fix an interphase of tumor cells in close proximity to gold electrodes. Relative impedance changes were shown to have potential value as a biosensor method for BC cells in whole blood, at frequencies around 20 kHz. Additional studies are warranted with respect to electrode design and sensitivity at micro-scale levels, according to the proposed technique.

Modelo Biofísico Bidimensional que Simula el Efecto Ablativo Focalizado de Tejido Renal Cancerígeno a través de Radiofrecuencia y Nanopartículas Ferromagnéticas

A bi-dimensional biophysical model to simulate a selective ablative effect in renal cancer was designed and tested. The model involves symmetrical regions of tumor/healthy renal tissue, a single circular coil to generate an alternating magnetic field in the microwave range and the presence of magnetic nanoparticles in the tumor region. The bi-dimensional biophysical model was solved by a finite element technique on the basis of electromagnetic and heat transfer considerations. The simulation produced induced current densities and surfaces of temperature gradients in tumor/healthy renal tissues with and without nanoparticles presence. The induced current density in the tumoral region increased as a function of the nanoparticles presence and was manifested by an increase in the temperature gradient. The simulation results showed that the radio-frequency exposition of renal tumor tissue influenced by magnetic nanoparticles has the potential to being a selective thermal ablation technique for renal cancer therapy