Cell-derived reactive oxygen species
With the exception of unusual circumstances such as exposure to UV light, ionizing radiation and other high energy sources, ROS are generally produced in cells by electron transfer reactions, which can be enzymatic or non-enzymatic. Epithelial cells, resident macrophages, endothelial cells and recruited inflammatory cells, such as neutrophils, eosinophils, monocytes and lymphocytes, generate ROS in response to increased levels of secretagogue stimuli. Activation of macrophages, neutrophils and eosinophils results first in the formation of O2•−, which is rapidly converted to H2O2 by SOD, and •OH is formed non-enzymatically in the presence of Fe2+ as a secondary reaction. ROS produced by phagocytes at sites of inflammation are a major cause of cell and tissue damage associated with various chronic inflammatory lung diseases.
Reactive oxygen species are generated intracellularly from several sources, including mitochondrial respiration, cytochrome P-450, the NADPH oxidase system, xanthine/xanthine oxidase and metabolism of arachidonic acid.13–19 The major ROS generating enzyme is NADPH oxidase, a membrane-bound multi-component enzyme complex that is present in phagocytes as well as non-phagocytic cells.13,15 In addition to NADPH oxidase, phagocytes contain other ROS generating enzymes, the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase. Activation of these peroxidases results in the formation of the potent oxidants HOCl and hypobromous acid (HOBr) from H2O2 in the presence of chloride (Cl-) and bromide (Br-) ions, respectively.
The oxidant burden produced by eosinophils is substantial, since these cells possess several times greater capacity for generating O2•− and H2O2 than neutrophils, and the content of eosinophil peroxidase in eosinophils is several times higher than that of MPO in neutrophils. Therefore eosinophils, which are present in increased numbers in the airways of asthmatic patients, have a major role in ROS generation in airway inflammation.
Detailed studies have shown that metals, including iron (Fe), copper (Cu), cadmium (Cd), chromium (Cr), mercury (Hg), nickel (Ni), vanadium (V) and several other metals, possess the ability to produce reactive radicals.20 While Fe, Cu, Cr, V and cobalt (Co) undergo redox-cycling reactions, the primary toxicity of a second group of metals, including Hg, Cd and Ni, is due to depletion of glutathione (GSH) and bonding to the sulphydryl groups of proteins. Arsenic (As) is thought to bind directly to critical thiols or to be involved in the formation of H2O2 under physiological conditions. The unifying factor in the toxicity of all these metals is the generation of ROS. The redox state of the cell is largely linked to an Fe (and Cu) redox couple and is maintained within strict physiological limits. It has been suggested that Fe regulation ensures that there is no free intracellular Fe. However, under conditions of stress, an excess of superoxide radical releases ‘free Fe’ from Fe-containing molecules. The released Fe2+ ion can participate in the generation of the highly reactive hydroxyl radical, catalysed by the Fenton reaction (Eqn 1).19,21–23
The superoxide anion participates in the Haber–Weiss reaction (Eqn 3), which is a combination of a Fenton reaction and the reduction of Fe3+ by O2•−, yielding Fe2+ and O2 (Eqn 2).19
Cigarette smoke and inhaled oxidants
Inhalation of volatile substances in cigarette smoke, as well as fine particulate matter, may increase ROS levels in the lungs.3–5 Inhalation of cigarette smoke and airborne pollutants, either oxidant gases such as O3 and sulphur dioxide (SO2), or particulate air pollution, results in direct lung damage as well as the activation of inflammatory responses in the lungs. Cigarette smoke is a complex mixture of over 4700 chemical compounds, including high concentrations of oxidants (1014 oxidant radicals/puff).24 The cellular mechanisms resulting in oxidative stress induced by smoking are complex and poorly understood. However, there is striking evidence for oxidative stress and an imbalance between oxidants and antioxidants in smokers.25 The gas phase of cigarette smoke contains largely short-lived oxidants such as O2•− and nitric oxide (NO), which react to form the highly reactive peroxynitrite (ONOO-) molecule.26 The tar phase of cigarette smoke contains long-lived radicals such as the semiquinone radicals, which can react with O2•− to form •OH and H2O2.27,28 The tar phase of cigarette smoke also generates H2O2 continuously for long periods in aqueous media.27,28 Cigarette smoking is also correlated with increased concentrations of MPO.29
According to the USA Environmental Protection Agency, the burden of particulate matter in ambient air due to chemical (31%) and non-chemical (35%) industrial processes, as well as transportation (27%) and fuel combustion (35%), is increasing worldwide.30 Many types of inhaled particles have the ability to generate free radicals in biological systems and to activate oxidative stress-response signalling pathways in cells.31 Ambient particulate matter may also induce oxidative DNA damage in lung epithelial cells.32 In addition, a recent study revealed that inhalation of exogenous H2O2 increased lung vascular permeability in an animal model.33
The hydroxyl radical can eliminate a hydrogen atom from PUFA resulting in the formation of lipid radicals, which can interact further with oxygen to generate the lipid peroxyl radical. If the resulting lipid peroxyl radical is not reduced by antioxidants, lipid peroxidation occurs. In contrast to free radicals, products of lipid peroxidation are generally stable, can diffuse within, or even escape from the cell, and attack targets far from the site of the original free radical event. In addition to their cytotoxic properties, lipid peroxides are increasingly recognized as being important in signal transduction for a number of inflammatory responses.34