At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

This review focuses on the role of oxidative processes in atherosclerosis and its resultant cardiovascular events. There is now a consensus that atherosclerosis represents a state of heightened oxidative stress characterized by lipid and protein oxidation in the vascular wall. The oxidative modification hypothesis of atherosclerosis predicts that low-density lipoprotein (LDL) oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis. In support of this hypothesis, oxidized LDL can support foam cell formation in vitro, the lipid in human lesions is substantially oxidized, there is evidence for the presence of oxidized LDL in vivo, oxidized LDL has a number of potentially proatherogenic activities, and several structurally unrelated antioxidants inhibit atherosclerosis in animals. An emerging consensus also underscores the importance in vascular disease of oxidative events in addition to LDL oxidation. These include the production of reactive oxygen and nitrogen species by vascular cells, as well as oxidative modifications contributing to important clinical manifestations of coronary artery disease such as endothelial dysfunction and plaque disruption. Despite these abundant data however, fundamental problems remain with implicating oxidative modification as a (requisite) pathophysiologically important cause for atherosclerosis. These include the poor performance of antioxidant strategies in limiting either atherosclerosis or cardiovascular events from atherosclerosis, and observations in animals that suggest dissociation between atherosclerosis and lipoprotein oxidation. Indeed, it remains to be established that oxidative events are a cause rather than an injurious response to atherogenesis. In this context, inflammation needs to be considered as a primary process of atherosclerosis, and oxidative stress as a secondary event. To address this issue, we have proposed an "oxidative response to inflammation" model as a means of reconciling the response-to-injury and oxidative modification hypotheses of atherosclerosis.

Role of Oxidative Modifications in Atherosclerosis

Few nonphagocytic cells are known to generate reactive oxygen intermediates. Based on horseradish peroxidase-dependent, catalase-inhibitable oxidation of fluorescent scopoletin, seven human tumor cell lines constitutively elaborated H2O2 at rates (up to 0.5 nmol/10(4) cells/h) large enough that cumulative amounts at 4 h were comparable to the amount of H2O2 produced by phorbol ester-triggered neutrophils. Superoxide dismutase-inhibitable ferricytochrome c reduction was detectable at much lower rates. H2O2 production was inhibited by diphenyleneiodonium, a flavoprotein binder (concentration producing 50% inhibition, 0.3 microM), and diethyldithiocarbamate, a divalent cation chelator (concentration producing 50% inhibition, 3 microM), but not by cyanide or azide, inhibitors of electron transport, or by agents that inhibit xanthine oxidase, polyamine oxidase, or cytochrome P450. Cytochrome b559, present in human phagocytes and lymphocytes, was undetectable in these tumor cells by a sensitive spectrophotometric method. Mouse fibroblasts transfected with human tyrosinase complementary DNA made melanin, but not H2O2. Constitutive generation of large amounts of reactive oxygen intermediates, if it occurs in vivo, might contribute to the ability of some tumors to mutate, inhibit antiproteases, injure local tissues, and therefore promote tumor heterogeneity, invasion, and metastasis.

https://cancerres.aacrjournals.org/content/canres/51/3/794.full.pdf

Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells

A metabolic pathway of activated macrophages (M phi) involving oxidation of the guanido nitrogens of L-arginine is required for inhibition of growth and respiration of some target cells. The goal of this study was to identify the M phi metabolite(s) that induce these injuries. The stable products of the L-arginine pathway, NO2- and NO3-, were incapable of causing cytostasis under coculture conditions. However, NO2- became cytostatic upon mild acidification, which favors its transformation into nitrogen oxides of greater reactivity. This suggested that NO. (and/or NO2), recently identified as an M phi metabolite of L-arginine, could be a mediator. Authentic NO. caused cytostasis and respiratory inhibition in L1210 cells in a dose-dependent manner. The mitochondrial lesions caused by NO. were confined to complex 1 and 2, a pattern of injury identical to that seen after coculture with activated M phi. Inclusion of NO. scavenger systems prevented cytostasis from developing in M phi-L1210 cocultures. Thus, M phi-generated NO. can account for L-arginine-dependent cytostasis and respiratory inhibition.

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Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host-pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

https://www.pnas.org/content/pnas/97/16/8841.full.pdf

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

Cells in culture are exposed to marked oxidative stress, H2O2 being one of the predominant agents. Pyruvate and other alpha-ketoacids reacted rapidly, stoichiometrically, and nonenzymatically with H2O2, and they protected cells from its cytolytic effects. All five human and murine cell types studied, both malignant and nonmalignant, released pyruvate at an initial rate of 35-60 microM/h/2.5 X 10(6) cells when placed in 1 ml pyruvate-free medium. After 6-12 h a plateau of 60-150 microM pyruvate was attained, corresponding to concentrations reported for normal human serum and plasma. The rate of pyruvate accumulation was almost doubled in the presence of exogenous catalase, suggesting that released pyruvate functions as an antioxidant. The rate of pyruvate accumulation was dependent on cell number. Succinate, fumarate, citrate, oxaloacetate, alpha-ketoglutarate, and malate were not secreted in significant amounts from P815 cells; export was specific for pyruvate and lactate among the metabolites tested. Extracellular pyruvate was in equilibrium with intracellular stores. Thus, cells conditioned the extracellular medium with pyruvate at the expense of intracellular pyruvate, until homeostatic levels were attained in both compartments. We propose that cells plated at low density in the absence of exogenous pyruvate fail to thrive for two reasons: prolonged depletion of intracellular pyruvate and prolonged vulnerability to oxidant stress.

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Secretion of pyruvate. An antioxidant defense of mammalian cells.

Nine human cell types, six of them malignant, displayed a marked resistance to lysis by hydrogen peroxide (LD50, 2-20 mM). Of the reactive oxygen intermediates generated extracellularly, only H2O2 lysed all the cell types. OH was lytic to one of four, OI- to one of one, and O-2 to none of four cell types tested. Resistance to oxidative lysis did not correlate with specific activity of catalase, glutathione (GSH) peroxidase, other peroxidases, or glutathione disulfide reductase, or with specific content of GSH. Resistance to H2O2 seemed to occur via mechanisms distinct from those responsible for cellular consumption of H2O2. Consumption was inhibitable by azide and was probably due to catalase in each cell type. In contrast, resistance to oxidative lysis occurred via distinct routes in different cells. One cell type used the GSH redox cycle as the primary defense against H2O2, like murine tumors previously studied. Other cells seemed to utilize catalase as the major defense against H2O2. Nonetheless, with both catalase and the GSH redox cycle inhibited, all the human cells tested exhibited an inherent resistance to oxidative lysis, that is, resistance independent of detectable degradation of H2O2.

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C J DeBoer

Papers

Secretion of pyruvate. An antioxidant defense of mammalian cells.

Resistance of human tumor cells in vitro to oxidative cytolysis.