Inflammation, DNA-centered radicals, and oxidative genotoxicity: The role of HOCl produced by myeloperoxidase in carcinogenesis

Myeloid cells (macrophages and neutrophils) infiltrate and synthesize myeloperoxidase (MPO) in sites of inflammation, producing gentotoxicity. In RAW 264.7 macrophages, bacterial lipopolysaccharide (LPS) induces superoxide radical anion, nuclear deformation (nuclear protuberances), MPO synthesis, biomolecule oxidation and cell death. “Freezing” LPS-triggered macrophage activation with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) prevented cell activation and death. Oxidation of proteins and genomic DNA was also blocked, with formation of protein- and DNA-DMPO nitrone adducts, as analyzed by immuno-spin trapping with a polyclonal anti-DMPO serum. Interestingly, confocal microscopy analysis of these cells showed that MPO, genomic DNA, and DNA-DMPO nitrone adducts co-localized in the nuclear protuberances. These observations, and the fact that DNA is negatively charged and MPO is a cationic protein, suggest a role for uptaken or newly synthesized MPO in oxidative genotoxicity induced by myeloid cells in sites of inflammation. 
In order to understand MPO-induced formation of DNA-centered radicals, we studied DNA-DMPO nitrone adducts in calf thymus DNA treated with micromolar concentrations of hypochlorous acid (HOCl) added as a bolus or generated in situ by the MPO/H2O2/Cl- system in the presence of DMPO. We also investigated DNA-DMPO nitrone adducts inside living cells containing MPO. The cell models we used were: i) human leukemia (HL)-60 cells, which overexpress MPO, ii) RAW 264.7 macrophages activated with LPS (1 ng/ml for 24 h), to induce MPO, and iii) A549 human airway epithelial cells pre-loaded with human MPO. When these cells were activated with the phorbol ester PMA, the number of 6-thioguanine-resistant cells with the hypoxanthine-guanine phosphoribosyl transferase (HRPT) mutation increased. This mutation was prevented by each of the following: the NADPH oxidase inhibitor apocynin; the MPO inhibitors salicylhydroxamic acid and 4-aminobenzoic acid hydrazide; the cell-permeable HOCl scavenger resveratrol; and DMPO, which traps DNA-centered radicals and prevents further oxidation. 
Genomic DNA-centered radicals and further mutagenesis induced by activated myeloid cells in sites of inflammation can be prevented by blocking MPO activity, preventing formation of and/or scavenging HOCl, or trapping DNA-centered radicals. Our findings provide new therapeutic avenues for preventing carcinogenesis induced by infiltration and activation of myeloid cells in sites of inflammation, for example, in the lung exposed to particulate matter. SUPPORTED BY NIEHS 5R00ES015415-03
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Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.Facultad de Ciencias Médica

Neutrophilic Inflammation Induces Genotoxicity in a Mouse Model of Acute-distress Respiratory Syndrome

The transit of neutrophils through pulmonary microvasculture is controlled by the expression of adhesion molecules whose expression is under the control of the nuclear factor-kB (NF-kB) and by the expression of integrins on the surface of the neutrophils. It is known that the activation of leukocytes in circulation is manifested by activation of NADPH oxidase-2 (NOX-2) and release of enzymes and proteases from their granules.Fil: Ramirez, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Claveles Casas, Florencia Nahir. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: López, Cristofer Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Barrera, Florencia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Di Sciullo, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Gomez Mejiba, Sandra E.. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Bioquímica y Ciencias Biológicas; ArgentinaFil: Hinojosa Vera, Kathleen Fabiola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Chacon, Inalen del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaSfRBM's 26th Annual MeetingLas VegasEstados UnidosSociety for Redox Biology and Medicin

A Mouse Model of Diet-Induced Obesity Resembling Most Features of Human Metabolic Syndrome

Increased chicken-derived fat and fructose consumption in the human diet is paralleled by an increasing prevalence of obesity and metabolic syndrome (MS). Herein, we aimed at developing and characterizing a mouse model of diet-induced obesity (DIO) resembling most of the key features of the human MS. To accomplish this, we fed male C57BL/6J mice for 4, 8, 12, and 16 weeks with either a low-fat diet (LFD) or a high-chicken-fat diet (HFD) and tap water with or without 10% fructose (F). This experimental design resulted in the following four experimental groups: LFD, LFD + F, HFD, and HFD + F. Over the feeding period, and on a weekly basis, the HFD + F group had more caloric intake and gained more weight than the other experimental groups. Compared to the other groups, and at the end of the feeding period, the HFD + F group had a higher adipogenic index, total cholesterol, low-density lipoprotein cholesterol, fasting basal glycemia, insulin resistance, hypertension, and atherogenic index and showed steatohepatitis and systemic oxidative stress/inflammation. A mouse model of DIO that will allow us to study the effect of MS in different organs and systems has been developed and characterized

Immuno-spin trapping of protein and DNA radicals: “tagging” free radicals to locate and understand the redox process

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied <em>in situ</em> and in real time in order to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively-generated modification of biomolecules, <em>in situ</em> and in real time, by detecting protein- and DNA-centered radicals. The purpose of this method article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline <em>N</em>-oxide (DMPO)