Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation "atheronals" for this previously unrecognized class of steroids.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H2O2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H 2O3) and ozone (O3) from singlet oxygen ( 1 O 2 * ) and water. Given that neutrophils have the potential both to produce 1 O 2 * and to bind antibodies, we considered that these cells could be a biological source of O 3. We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O 3. This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated 1 O 2 * . Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O 3 to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation. N eutrophils (PMNs) are the most abundant leukocytes in the bloodstream. Their function is the killing of bacteria and fungi, in part by the triggering of an oxidative burst that is composed of a set of enzymatic and chemical reactions ultimately leading to the formation of hypohalous acid, 1 O 2 * , and hydroxyl radical (HO • ) (1, 2). The first step in this cascade, the reduction of dioxygen, is initiated by the enzyme NAD(P)H oxidase. This oxidase is a complex enzyme composed of five components: gp91 phox (with phox being phagocyte oxidase), a heavily glycosylated 56-kDa protein that contains the electroncarrying components of the oxidase; p67 phox , p47 phox , and p22 phox , which are proteins named according to their approximate molecular weights; and rac2, a low molecular weight GTPase. In the resting cell, p47 phox and p67 phox form a complex in the cytosol (which also contains p40 phox , a protein whose effect on oxidase activity is unclear), whereas gp91 phox and p22 phox are in the membrane. When the PMN is activated by antibody-coated bacteria, p47 phox is phosphorylated on particular serines and moves to the membrane to assemble the active oxidase, carrying with it its cargo of p67 phox and the enigmatic p40 phox . Rac 2, also necessary for oxidase activity, picks up a GTP and moves into the oxidase assembly. The NAD(P)H oxidase then produces superoxide anion (O 2•Ϫ ) (Eq.

Ischemia and reperfusion both contribute to tissue damage after myocardial infarction. Although many drugs have been shown to reduce infarct size when administered before ischemia, few have been shown to be effective when administered at reperfusion. Moreover, although it is generally accepted that a burst of reactive oxygen species (ROS) occurs at the onset of reperfusion and contributes to tissue damage, the source of ROS and the mechanism of injury is unclear. We now report the finding that chloramphenicol administered at reperfusion reduced infarct size by 60% in a Langendorff isolated perfused rat heart model, and that ROS production was also substantially reduced. Chloramphenicol is an inhibitor of mitochondrial protein synthesis and is also an inhibitor of a subset of cytochrome P450 monooxygenases (CYPs). We could not detect any effect on mitochondrial encoded proteins or mitochondrial respiration in chloramphenicol-perfused hearts, and hypothesized that the effect was caused by inhibition of CYPs. We tested additional CYP inhibitors and found that cimetidine and sulfaphenazole, two CYP inhibitors that have no effect on mitochondrial protein synthesis, were also able to reduce creatine kinase release and infarct size in the Langendorff model. We also showed that chloramphenicol reduced infarct size in an open chest rabbit model of regional ischemia. Taken together, these findings implicate CYPs in myocardial ischemia͞reperfusion injury.C urrent treatment of myocardial infarction is directed at the restoration of blood flow to the ischemic region and reduction of myocardial oxygen demand. However, during reperfusion, the heart undergoes further damage due, in large part, to the generation of reactive oxygen species (ROS) (1). It is clear that permanent ischemia results in necrotic cell death. However, it is unclear whether reperfusion itself induces apoptosis or merely permits the manifestation of cell death processes that were initiated and irreversibly committed to during ischemia. Moreover, the relative contributions to tissue injury by the ischemic phase and by reperfusion have been difficult to evaluate. Resolving this question carries important therapeutic implications, as efforts directed toward treating reperfusion injury would have limited value if most cell death were predestined during ischemia.Although we initially hypothesized that the protective effect of chloramphenicol was caused by inhibition of mitochondrial protein synthesis, we did not see down-regulation of mitochondrial-encoded proteins after chloramphenicol infusion. The energy-sparing effects of inhibition of cytosolic protein synthesis have been described (2), and our previously reported observation that mitochondrial elongation factor Tu is phosphorylated during ischemia suggests a similar process may take place in the mitochondria (3). However, because chloramphenicol also inhibits some cytochrome P450 monooxygenases (CYPs), it was important to determine whether that inhibitory effect was relevant to cardioprotection. In this re...

The proatherogenic properties of the cholesterol 5,6-secosterols (atheronal-A and atheronal-B), recently discovered in atherosclerotic arteries, have been investigated in terms of their effects on monocyte/macrophage function. A fluorescent analogue of atheronal-B (1) (50 microM), when cultured in either aqueous buffer (PBS) or in media containing fetal calf serum (10%), is rapidly taken-up into cultured macrophage (J774.1 or RAW 264.7) cells and accumulates at perinuclear sites (within 1 h). Co-incubation of macrophage cells (J774.1) with atheronal-A (25 microM) and atheronal-B (25 microM) when complexed with low-density lipoprotein (LDL) (100 microg/mL) leads to a significant upregulation of scavenger receptor class A (approximately 3-fold increase relative to LDL alone, p < 0.05) but not CD36, showing that cultured macrophages respond to LDL-complexed atheronals in a manner highly analogous to acetylated LDL rather than oxidized LDL. Both atheronal-A and atheronal-B in solution exhibit a dose-dependent (0-25 microM) induction of chemotaxis of cultured macrophages (p < 0.001). When complexed with LDL (100 microg/mL), atheronal-A (but not atheronal-B) induces a dose-dependent (0-25 microM, p < 0.05) upregulation of the cell-surface adhesion molecule endothelial (E)-selectin on vascular endothelial cells (HUVECs). LDL (100 microg/mL) complexed atheronal-B (25 microM) but not atheronal-A induces cultured human monocytes (THP-1) to differentiate into macrophage cell lineage. When these in vitro data are taken together with the already known effects of cholesterol 5,6-secosterols on foam cell formation and macrophage cytotoxicity, the atheronals possess biological effects that if translated to an in vivo setting could lead to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, with the major leukocyte player in inflammatory artery disease. As such, the atheronal molecules may be a new association, in the already complex inter-relationship, between inflammation, cholesterol oxidation, the tissue macrophage, and atherosclerosis.