Elucidation Of Cellular Damage During Exposure To Oxidat

Heart, Lung, And Blood Institute

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Abstract

Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of research: (a) Regulation of apoptosis. Previous studies demonstrated that high concentrations of manganese ions [Mn(II)] induces apoptosis in cultured HeLa and NIH3T3 cells by a caspase 12-mediated cytochochrome c independent pathway. The Mn(II)-induced apoptosis is associated with increases in the cellular levels of reactive oxygen species (ROS), activation of procaspase 12, and up-regulation of manganese superoxide dismutase [Mn(II)SOD]. The promoter region and the 5'-untranslated region of the mouse caspase 12 gene were isolated and sequenced, and the functional analysis of the caspase gene is now under investigation. (b) Role of apoptosis in aging. Results of previous studies demonstrated that treatment of cultured leukemia NB4 cells with arsenite leads to an increase in the levels of oxidized proteins and to apoptosis, and that inhibition of apoptosis leads to an even greater increase in the accumulation of oxidized proteins. The possibility that the observed age-related increase in levels of oxidized proteins is due in part to a loss in ability to eliminate oxidatively damaged cells by apoptosis and replace them with good cells is supported by our studies showing that treatment of cultured leukemia NB4 cells with arsenite leads to an increase in levels of oxidized proteins and to apoptosis, and that inhibition of apoptosis leads to an even greater accumulation of oxidized proteins. Further studies are in progress to determine if, in fact, aging is associated with a decrease in the ability to induce apoptosis. (c) Examination of apoptosis in unicellular organisms. The possibility that apoptosis may occur in unicellular organisms is supported by results of studies showing that yeast contain a gene (YCA1) with high structural homology to the mammalian caspases, and that this gene is able to mediate programmed cell death in yeast. We have initiated studies to determine the effects of acute oxidative stress on the chronological aging of a wild type strain of Saccharomyces cerevisiae containing normal levels of YCA1 and of a mutant homozygous strain that lacks YCA1, as well as a heterozygous strain, and on the susceptibility of these strains to oxidative stress. (d) Effect of ribonucleic acid (RNA) oxidation on translational efficiency. Oxidative modification of RNA is associated with several neurological disorders. To study the effect of ribonucleic acid oxidation on its translational efficiency, RNA encoding the luciferase gene was subjected to oxidation by hydrogen peroxide and its ability to produce luciferase when incubated in reticulocyte lysate was examined. Activity of the luciferase protein translated from oxidized RNA is considerably lower than that of normal luciferase preparations. Further studies have shown that the products formed in the oxidation of RNA vary, depending upon the composition of the system used to generate reactive oxygen species. In addition to 8-oxo-guanine, other low molecular weight compounds are formed. The identity of these products is under investigation. (e) Effect of bicarbonate buffer on the iron-catalyzed oxidation of low density lipoprotein (LDL). It is well established that oxidation of LDL is implicated in arteriosclerosis. Previous in vitro attempts to elucidate mechanisms of LDL oxidation by various reactive oxygen generation systems have been carried out using non-physiological phosphate buffer systems. Based on results of our earlier studies showing that metal-catalyzed oxidation of proteins and amino acids is greatly enhanced when reactions are carried out in buffers composed of physiological concentrations of bicarbonate and carbon dioxide, we initiated studies to examine the effects of bicarbonate buffer on the oxidation of LDL. We found that substitution of bicarbonate for phosphate buffers led to substantial increases in the rates of LDL oxidation as measured by the generation of malondialdehyde, protein carbonyl, and methionine sulfoxide derivatives. Collectively, results of these studies suggest that the iron + peroxide-dependent oxidation of LDL is stimulated by interactions between ferrous iron, bicarbonate, and various iron chelators to form complexes having redox potentials that favor Fenton chemistry.