Spin trapping endogenous radicals in MC-1010 cells: evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts.

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO2) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO2 oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (AN = 15.9 G; AH = 22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO2 treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe2+ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO2 oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of AH4 = 1.8 G.

Nucleotide chloramines and neutrophil-mediated cytotoxicity.

Hypochlorite is a reactive oxidant formed as an end product of the respiratory burst in activated neutrophils. It is responsible for killing bacteria and has been implicated in neutrophil-mediated tissue injury associated with the inflammatory process. Although hypochlorite is a potent cytotoxic agent, the primary mechanism by which it exerts its effect is unclear. This review examines evidence that the primary event in hypochlorite cytotoxicity is the loss of adenine nucleotides from the target cell. This loss appears to be mediated by the formation of adenine nucleotide chloramines which are reactive intermediates with a free radical character and are capable of forming stable ligands with proteins and nucleic acids.

Electron spin resonance studies of the reaction of hypochlorite with 5,5-dimethyl-1-pyrroline-N-oxide.

The reaction of hypochlorous acid with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was found to yield 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX). In addition to DMPOX, 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) and an unidentified chlorine-containing radical species were also observed under neutral and near-neutral conditions. Through the use of [17O]HOCl and the hydroxyl radical scavengers ethanol and formate, it was established that DMPO-OH was derived from hydration of DMPO rather than the spin-trapping of hydroxyl radical. Furthermore, kinetic studies and the incorporation of 17O showed that DMPO-OH was readily oxidized to DMPOX and that this reaction was acid and base catalyzed. Under strongly alkaline conditions, DMPOX reversibly formed another species, presumably the enolate, that had a four-line ESR signal identical to that of DMPO-OH. Eventually, carbon-centered adducts appeared whose ESR signals were consistent with the formation of DMPO condensation products.

Hypochlorite-modified adenine nucleotides: structure, spin-trapping, and formation by activated guinea pig polymorphonuclear leukocytes.

Adenosine and its nucleotides react with hypochlorite to form unstable products that have been identified as the N6 chloramine derivatives. These chloramines spontaneously oligomerize, form stable adducts with proteins and nucleic acids, and are converted with loss of chlorine to the original nucleoside or nucleotide by reducing agents. The chloramines are associated with a free radical, and the spin-trapping of adenosine chloramine with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) yielded a mixture of unstable nitroxyl adducts that corresponded to nitrogen-centered radicals from the parent nucleoside. When activated guinea pig polymorphonuclear leukocytes were stimulated with phorbol myristate acetate to produce hypochlorite, they actively incorporated [14C]adenosine into acid-insoluble products by a process that was dependent on oxygen and inhibited by azide and thiols. These findings suggest that adenine nucleotide chloramines are generated by activated phagocytic cells and form ligands with proteins and nucleic acids as observed in model systems. The results imply that nucleotide chloramines are among the cytotoxic and possibly mutagenic factors that are associated with the inflammatory process.

Nucleotide modification, a radical mechanism of oxidative toxicity.

Reduced nicotinamide adenine dinucleotide (NADH) reacts rapidly with hypochlorite to form five major products separable by reversed-phase high-pressure liquid chromatography (HPLC). The involvement of a free radical mechanism is indicated by an electron spin resonance (ESR) signal as well as unusual pH changes and the uptake of oxygen. The present work suggests that hypochlorite may contribute to the cytotoxic activity of phagocytic cells through its ability to modify important cellular components by means of radicals generated by its reaction with reduced pyridine nucleotides.

ADP-ribosylation of isolated rat islets of Langerhans.

Incubation of isolated rat islets of Langerhans with [adenine-2,8-3H]NAD+ results in rapid incorporation of 3H into acid-insoluble products. The major site of incorporation appears to be the cell membrane. The reaction is inhibited by nicotinamide, an ADP-ribosylation inhibitor, and stimulated by arginine, an ADP-ribose acceptor. The results demonstrate that islet membrane proteins can be ADP-ribosylated in the absence of exogenous ADP-ribosylating agents and suggest that ADP-ribosylation plays a role in pancreatic islet cell function.

Reduction of cytochrome c by hydrogen peroxide and its inhibition by superoxide dismutase.

Ferricytochrome c is slowly converted by hydrogen peroxide to an equilibrium mixture of ferricytochrome c and ferrocytochrome c, and in the process, the hydrogen peroxide is decomposed. The reductant appears to be superoxide anion, produced from the reaction of hydrogen peroxide with oxygen. Because the reduction of ferricytochrome c by hydrogen peroxide is inhibited by superoxide dismutase, we propose that the enzyme acts by converting superoxide anion to a dimerized product that is less active as a reductant.

Formation of reduced nicotinamide adenine dinucleotide peroxide.

Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.

Mitochondrial chemiluminescence elicited by acetaldehyde.

Acetaldehyde-dependent chemiluminescence has been found to be a sensitive technique for the study of superoxide and hydrogen peroxide formation in beef heart mitochondria. The system responds to ATP and antimycin A with increased emission intensities and to ADP and rotenone with decreased intensities, indicating that the chemiluminescence reflects the energy status of the mitochondrion. These effects are based on the ability of acetaldehyde to react with superoxide and hydrogen peroxide to form metastable intermediates which decay spontaneously with the emission of light. Additionally, these intermediates can react with cyanide to give alternative products which can also decay with the emission of light, the cyanide-evokable chemiluminescence. The interaction of acetaldehyde with mitochondria is complex because acetaldehyde can serve as a hydrogen source for NADH and as an inhibitor (at high concentration) of electron transport, and appears to be a reducing agent for a heat-stable site that autoxidatively generates HOOH from O2-.. Inasmuch as acetaldehyde is a metabolite of ethanol, this broad spectrum of reactivity may play a role in the hepatic and cardiac toxicity that is associated with alcoholism. The heat-stable site that generates HOOH from O2-. has been studied further and appears to contain vicinal dithiol which is primarily responsible for the cyanide-evokable chemiluminescence.

Dowex-1-induced reactions of reduced nicotinamide adenine dinucleotide (NADH).

Dowex 1-formate has been found to cause both anomerization and oxidation of NADH, and when NADH is chromatographed on a column of this resin, the major products observed are NAD+ and alpha-NAD+. Competing with the oxidation reaction is the conversion of NADH and alpha-NADH to unstable acid-modification products that subsequently break down during chromatography to give ADP-ribose and a variety of neutral and cationic degradation products. The effects of Dowex 1-formate on NADH differ from those of acid as oxidation is minimal when NADH is incubated in acid solution, although anomerization, acid-modification, and degradation to ADP-ribose and other products readily occur. The neural and cationic acid-degradation products that are formed from acid-modified NADH have been resolved by chromatography into 12 compounds, 6 of which react with 3-methyl-2-benzothiazolinone hydrazone and thus are identified as carbonyls. These substances gradually disappear from acid solution over a period of days and are replaced by polymeric pigments.

Nonenzymic hydrogen transfer between reduced and oxidized pyridine nucleotides.

Mixtures of NADH and NADP+ or NADPH and NAD+ were incubated and periodically assayed for hydrogen transfer by measuring the formation of NADPH and NADH with glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) and lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27), respectively. Each mixture showed a steady nonenzymic transfer of hydrogen from the reduced to the oxidized pyridine nucleotide to yield a product that was completely enzymically active. The results demonstrate the specific nonenzymic transfer of hydrogen from NADH and NADPH to the pyridine C-4 position of NADP+ and NAD+, respectively.

Physiology aspects of pyridine nucleotide regulation in mammals.

Tissue levels of NAD+ appear to be regulated primarily by the concentration of extracellular nicotinamide, which in turn is controlled by the liver in a hormone-sensitive manner. Hepatic regulation involves the conversion of excess serum nicotinamide to 'Storage NAD+' and inactive excretory products, and the replenishment of serum nicotinamide by the hydrolysis of 'Storage NAD+.' Tryptophan and nicotinic acid contribute to 'Storage NAD+,' and thus are additional sources of nicotinamide. In response to administered nicotinamide, there is a preferential utilization of ATP and PRPP (5-phosphorylribose-1-pyrophosphate) for the biosynthesis of NAD+. This biosynthetic priority, whose purpose appears to be the conservation of intracellular nicotinamide, may explain why nicotinamide inhibits RNA and DNA synthesis in regenerating tissues and why elevated nicotinamide levels are toxic to growing animals and to mammalian cells in culture.

Mitochondrial glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent KM's for NADP+ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.