1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

Virtually all inflammatory mediators investigated to date seem to be dysregulated in the inflamed intestinal mucosa of patients with inflammatory bowel disease. However, which of these are actually involved in the initiation and perpetuation of intestinal tissue damage is still not fully understood. Amongst these mediators are the reactive oxygen metabolites, produced in large amounts by the massively infiltrating leucocytes. These reactive oxygen metabolites are believed to constitute a major tissue-destructive force and may contribute significantly to the pathogenesis of inflammatory bowel disease.

This paper provides a concise overview of reactive oxygen metabolite biochemistry, the types of cell and tissue damage potentially inflicted by them, and the endogenous antioxidants which should prevent these harmful effects. An up-to-date summary of the available human experimental data suggests that reactive oxygen metabolite-mediated injury is important in both the primary and downstream secondary pathophysiological mechanisms underlying intestinal inflammation. Nonetheless, how the individual components of the mucosal antioxidant enzymatic cascade respond to inflammatory conditions is a neglected area of research. This particular aspect of intestinal mucosal oxidative stress therefore merits further study, in order to provide a sound, scientific basis for the design of antioxidant-directed treatment strategies for inflammatory bowel disease patients.


  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

One of the major fundamental tissue-destructive mechanisms is oxidative stress through an excessive release of reactive oxygen metabolites (ROM).1–3 Numerous in vitro and in vivo studies have shown convincingly that ROM, a term used for all metabolites of molecular oxygen, including oxygen free radicals, are capable of causing, directly, reversible and irreversible damage to any oxidizable biomolecule. Consequently, they have been implicated in cell or tissue damage of practically every disease.4–12

In this review, we discuss why ROM are notoriously suspect in inflammatory diseases and, in particular, evaluate the evidence implicating oxidative stress in the pathogenesis of intestinal inflammation.

Superoxide anion (O2•). The primary ROM is superoxide anion (O2•). O2• is formed from the single-electron reduction of molecular oxygen, and is an oxygen free radical because it contains an unpaired electron. O2• is generated through a variety of sources in both physiological and pathophysiological conditions. Probably most pertinent to the pathogenesis of intestinal inflammation, however, is its generation by neutrophils and macrophages.13–16 These cells, on interaction with pro-inflammatory agents, such as cytokines, immune complexes or bacterial products, undergo a so-called respiratory burst. This process involves a sudden stimulus-induced activation of the membrane-bound enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which in turn evokes the release of large amounts of ROM. Although it is beyond doubt that O2• generation by phagocytes (and to a lesser extent by eosinophils, lymphocytes and fibroblasts) is essential for an effective host defence against bacterial infection, its continuous overproduction during inflammatory processes may also cause extensive tissue destruction.17

In addition to certain exogenous O2• sources, such as cigarette smoke and ionizing radiation,1 that are particularly relevant in lung diseases8, 11 and radiation pathologies,18 O2• is generated intracellularly. In the mitochondrion, electrons continuously leak from their carriers within the respiratory chain, passing directly on to molecular oxygen. As much as 1–5% of the total oxygen consumption by normal tissues might thus be transformed into O2•,19, 20 which makes the mitochondrion the major endogenous intracellular O2• production site under non-inflammatory conditions.21 Significant amounts of O2• can also be generated by a variety of endogenous enzyme systems, such as the peroxisomal enzyme xanthine oxidase, which is activated on re-introduction of oxygen after periods of hypoxia (reviewed in Kooij22).

Despite the enormous potential for its production, O2• itself is not considered as a particularly reactive intermediate.2, 23–25 It does not rapidly cross lipid membrane bilayers and it dismutates spontaneously at physiological pH [reaction (1)]:

  • image(1)

Paradoxically, however, the danger in O2• lies in its neutralization. Reaction (1), accelerated by the superoxide dismutases (SOD; discussed below), is the first of a cascade of other oxidant reactions (illustrated in Figure 1), yielding much more powerful ROM, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and the hydroxyl radical (OH•).


Figure 1. Simplified schematic representation of reactive oxygen metabolite (ROM) reactions in intestinal inflammation. Letters indicate the sources of ROM (A, neutrophil NADPH oxidase; B, xanthine oxidase; C, mitochondrial NADPH cytochrome p450 reductase). Numbers correspond to the (anti)oxidant reactions in the text. The large grey arrows indicate possible ROM targets (membrane lipids, proteins, DNA, matrix blood vessels, bacteria). Fe, ferrous iron; H 2O2, hydrogen peroxide; HOCl, hypochlorous acid; MT, metallothionein; NO, nitric oxide; O2•, superoxide anion; OH•, hydroxyl radical; ONOO, peroxynitrite.

Download figure to PowerPoint

Hydrogen peroxide (H2O2). Any system producing O2• will also produce H2O2 and, consequently, inflammatory phagocytes also generate and release remarkable amounts of H2O2.13–16 Although H2O2 has been shown to directly exert non-specific irreversible damage to epithelial cells,26 it is generally considered as a relatively weak ROM. Its high in vivo reactivity, therefore, is not only attributed to its stability and diffusibility, but particularly to its ability to react with partially reduced metal ions, such as Fe2+ or Cu+, to form OH• in the so-called Fenton reaction [reaction (2)]:27

  • image(2)

The formation of OH• from H2O2 can be bypassed through the two-electron reduction of H2O2 to water, catalysed by catalase (CAT) [reaction (3)] or glutathione peroxidase (GPO) [reaction (4)]:

  • image(3)
  • image(4)

Hypochlorous acid (HOCl). Instead of being neutralized to water, H2O2 can also be metabolized by the enzyme myeloperoxidase to form the potent chlorinating as well as oxidizing agent HOCl (commonly known as bleach) [reaction (5)]:

  • image(5)

This reaction is specifically considered to be relevant in inflammatory processes, as the haemoprotein myeloperoxidase is one of the most abundant proteins in phagocytes. It is estimated to represent 5% of neutrophil protein and 1% of monocyte protein, and myeloperoxidase is believed to be present in human macrophages as well.28, 29 When activated, neutrophils can secrete myeloperoxidase extracellularly.30

HOCl is estimated to be 100–1000 times more toxic than O2• or H2O2, and seems to have distinct biochemical targets.31 For example, it is capable of inactivating essential enzymes,31 of oxidizing plasma membrane thiol (SH) groups,32 of disrupting certain protein and plasma membrane functions33 and of decreasing the adhesive properties of some extracellular matrix components.34 In addition, exposure to HOCl seems to increase endothelial permeability through the mobilization of cellular Zn2+ molecules.35 No specific HOCl neutralizing enzyme has been found as yet, but HOCl can be removed by reaction with albumin and ascorbic acid.36

Hydroxyl radical (OH•). OH• is considered to be the most reactive ROM.24, 37 In contrast with H2O2, for instance, OH• inactivates the pivotal mitochondrial enzyme pyruvate dehydrogenase,38 depolymerizes gastrointestinal mucin and directly inflicts DNA damage.39, 40 OH• is formed from H2O2 through the Fenton reaction [reaction (2)] or from O2• through another transition metal-dependent reaction, called the iron-catalysed Haber–Weiss reaction [reaction (6)]:41

  • image(6)

Recently, OH• has been shown to be produced via certain alternative, but inflammation-relevant, pathways. They include the generation of OH• during the inactivation of Cu/Zn-SOD by H2O2,42 and through the interactions between O2• and HOCl [reaction (7)],43 HOCl and reduced iron ions [reaction (8)]44 and H2O2 and nitric oxide (NO) [reaction (9)]:45

  • image(7)
  • image(8)
  • image(9)

Again, no known enzyme exists to facilitate the detoxification of OH•. OH•-induced tissue damage may be prevented, however, by the binding (‘sequestration’) of transition metal ions by, for instance, albumin, caeruloplasmin, ferritin, transferrin and metallothionein.1

Reactive nitrogen metabolites (RNM). In analogy with ROM, reactive nitrogen metabolites (RNM) are derived from nitrogen, and include NO and peroxynitrite. In recent years, considerable interest has been shown in the role of RNM in cellular redox reactions, and an overwhelming amount of papers have been published to describe their role in inflammation. Although NO is a highly lipid-soluble molecule with a considerably long half-life, capable of diffusing several cell diameters from its site of synthesis, there is still much debate as to whether NO production is actually harmful to inflamed tissues (reviewed in Grisham et al.46 and Kubes and McCafferty47). It is also unclear whether the NO-producing enzyme, nitric oxide synthase, is expressed by inflammatory cells.

NO itself probably is not particularly noxious and may even have some beneficial, inflammation-reducing, effects (reviewed in Lefer and Lefer48). For example, NO has been shown to protect epithelial cells against H2O2-mediated toxicity,49, 50 to protect macrophages from cytokine-induced cytotoxicity51 and to diminish leucocyte binding to endothelial cells.52 In addition, through its very rapid interaction with O2•[reaction (10)], NO may form a sink for O2• and its toxic downstream ROM derivatives.53–55

  • image(10)

However, the product of reaction (10), the peroxynitrite anion, is generally believed to be considerably more reactive and damaging than its precursors.56 Moreover, it has a relatively long lifetime and passes easily through lipid bilayers.57 Peroxynitrite oxidizes mitochondrial membrane lipids,58 is believed to damage sodium channels in the colon59 and increases microvascular permeability and oedema formation.60 Peroxynitrite formation, which depends on the existence of sufficient levels of available O2• and NO, may be self-limiting through the capability of NO to inactivate NADPH oxidase, the enzyme responsible for phagocytic O2• production.61 On the other hand, nitric oxide synthase seems to be capable of producing O2• itself.62 In the absence of a specific peroxynitrite detoxifying mechanism, proteins such as haemoglobin and neutrophilic myeloperoxidase, which are abundantly present during inflammation, are thought to play a major defensive role against peroxynitrite.63

Oxidative damage

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

Excessive generation of ROM and RNM may, in principle, result in the attack and damage of all cellular and extracellular components (see Figure 1). It is important to realize, however, that the overall high reactivity and short half-life of ROM implies that the tissue damage they inflict is generally close to the site of ROM generation. In addition, as stated by McCord,3 reactivity and toxicity are not synonymous. A particular ROM may not be particularly bioreactive, but may still be extremely toxic when it strikes a crucial target effectively.

Membrane lipids. The polyunsaturated fatty acids located within the cell membrane lipid bilayer are major targets for ROM attack.1, 64 They are particularly effectively attacked by OH•, thereby initiating the process of lipid peroxidation. Once initiated, lipid peroxidation continues as a chain reaction to generate lipid hydroperoxides and aldehydes, and a single oxidative event can thus affect many lipid molecules. The accumulation of hydroperoxides in the cell membrane has a profound effect on its fluidity and, as such, on the activity of transmembrane enzymes, transporters, receptors and other membrane proteins.65, 66 As a result, lipid peroxidation causes changes in membrane permeability and selectivity, and ultimately leads to alterations in cell volume homeostasis and cellular metabolism.67 Moreover, hydroperoxides and aldehydes are directly toxic to cells and organelles,68 have neutrophil chemotactic properties69 and may regulate cytokine production.70

Lipid peroxidation accelerates only when cellular detoxification systems have failed to remove the precursors of OH•, in particular H2O2, effectively. Once initiated, lipid peroxidation is most successfully combated by lipid-soluble antioxidants such as α-tocopherol (vitamin E),2 although it has also been shown that NO can act as a chain-breaking antioxidant against lipid peroxidation.71

Proteins. Proteins are the most abundant cell constituents, which make them important ROM targets.72 Moreover, a relatively minor structural (oxidative) modification of a single protein can lead to a marked change (in most cases lowering) in its biological activity. Similar to lipid peroxidation, OH• seems to be most effective in inducing oxidative protein damage.72 The process of protein oxidation frequently introduces new functional groups, such as hydroxyls and carbonyls, which contribute to altered function, turnover and degradation.73 Secondary effects include protein fragmentation, cross-linking and unfolding.73

Another permanent modification that can adversely affect protein function is the nitration of protein-bound tyrosines by peroxynitrite.74, 75 Tyrosine is an important amino acid, involved in (de)phosphorylation reactions and signal transduction pathways,72 and its nitration may not only compromise protein function, but may also have serious consequences in cellular regulation. It is of particular interest that the enzymes (SOD) which catalyse reaction (1) have been identified as specific protein targets of nitration. Nitration of cytoplasmic Cu/Zn-SOD has been reported to occur in vitro without loss of enzymatic activity,76 whereas tyrosine nitration of mitochondrial Mn-SOD is associated with the loss of enzyme function, and has been detected in rejected human kidney allografts.77 It should be mentioned, however, that protein nitration and inactivation might also occur through a peroxynitrite-independent, myeloperoxidase-catalysed, pathway.78, 79

DNA. Both nuclear and mitochondrial DNA are known targets of ROM attack,80, 81 which can result in many types of DNA modification. The most common are base hydroxylations and strand cleavage, leading to adenosine triphosphate depletion and gene mutations, which can in turn result in malignant transformation or cell death. O2• is relatively unreactive with DNA,82 and other ROM implicated in DNA damage are peroxynitrite83, 84 and NO, which may directly damage chromatin.85 The most substantial portion of DNA modifications, however, is thought to involve in situ produced OH• as the attacking species.39, 40 Once H2O2 escapes from its cytosolic neutralizing enzymes and reaches the nucleus, it will react with chromatin-bound iron (or copper released from oxidatively damaged Cu/Zn-SOD),42, 86 producing OH•in situ, which in turn will attack nearby DNA residues.87 Substantial protection against OH•-mediated DNA damage may come from the thiol-rich protein metallothionein (see below). Metallothionein is not only a powerful OH• scavenger,88 it also accumulates in the cell nucleus during certain phases of the cell cycle.89

Apoptotic cell death. Cell death can follow two distinct pathways: necrosis or apoptosis.90 Apoptosis (or controlled cell death) differs from necrosis (chaotic cell death due to overt injury) by distinct morphological and biochemical features, such as chromatin condensation, membrane surface blebbing, DNA fragmentation and, finally, the breakdown and autodigestion of the cell into a series of smaller units (apoptotic bodies). Although the early biochemical events that dictate the mode of cell death are still unclear, several lines of evidence implicate ROM as modulators of apoptosis.91In vitro exposure to low doses of ROM, or a depletion of cellular antioxidants, has been shown to result in apoptosis,92–97 and, conversely, apoptosis can be blocked by the addition of antioxidant compounds.98–101

Reactive oxygen metabolites as immunomodulators

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

The traditional view of ROM as directly causing non-specific injury to cells through a series of local chemical reactions has, in recent years, been complemented by numerous in vitro reports that show more subtle, immunomodulatory effects of ROM. O2•, for example, has been shown to mediate the infiltration and accumulation of neutrophils at sites of inflammation,102, 103 and to be involved in the mobilization of arachidonic acid.104 Furthermore, H2O2 appears to act as a neutrophil chemoattractant,105 to elicit leucocyte rolling,106 to activate T lymphocytes107 and to induce angiogenesis.107 H2O2, like O2•, is also capable of mobilizing arachidonic acid.104

Most of these oxidant effects are thought to be mediated by shared, redox-sensitive, regulatory pathways, which are discussed in detail elsewhere.108 Apparently, ROM are involved in the (in)activation of a variety of kinases and transcription factors, their up- or down-regulation depending on the magnitude of the redox change. In particular, the transcription factors nuclear factor-κB and activator protein-1 have received much attention in this respect. They are activated during intestinal inflammation in epithelial and inflammatory cells,109 where they lead to the up-regulation of a number of inflammatory genes, including those encoding tumour necrosis factor-α, interleukin-1, -6 and -8, inducible nitric oxide synthase, major histocompatibility complex class I antigens and the adhesion molecules E-selectin and vascular cell adhesion molecule-1.108, 110 Redox-mediated, nuclear factor-κB/ activator protein-1-induced gene expression may be of particular relevance in the context of chronic inflammatory processes. Some of the cytokines whose genes are switched on by nuclear factor-κB, such as tumour necrosis factor-α and interleukin-1, are themselves activators of nuclear factor-κB.111 Moreover, interleukin-1 and tumour necrosis factor-α are also known to induce cellular ROM production,112–114 and ROM, in particular H2O2, have been shown to activate nuclear factor-κB.111, 115, 116 Evidence exists that ROM and pro-inflammatory cytokines work synergistically to further intensify transcription factor activation.111, 117 In this context, it is also of interest to note that another major tissue-destructive force, an extensive group of proteases called the matrix metalloproteinases,118, 119 also appears to be under ROM control, probably through the same regulatory pathways. In fact, the genes that code for matrix metalloproteinases and their inhibitors are modulated by activator protein-1,120, 121 and ROM at concentrations achieved at sites of inflammation can activate multiple matrix metalloproteinase family members122, 123 and at the same time inactivate their inhibitors,124 thereby allowing uncontrolled protease and collagenase activity to damage tissues. Such ROM-/cytokine-/transcription factor-regulated self-sustaining regulatory loops may contribute to the perpetuation and exacerbation of chronic inflammation and tissue damage, particularly when the local immune response is ‘hyper-responsive’ and fails to successfully down-regulate the immune reaction. The inflammatory bowel diseases show signs suggestive of such mechanisms.125

Inflammatory bowel disease

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

The expression of the inflammatory bowel diseases, Crohn's disease and ulcerative colitis, is generally thought to depend on the interplay of environmental, genetic and immunological factors (reviewed in Fiocchi125). Yet, an integrated concept explaining the initiating event(s) and/or fundamental abnormalities in inflammatory bowel disease in relation to the pathophysiological changes has not yet emerged. It does seem certain that inflammatory bowel disease is amplified and propagated by an uncontrolled and sustained host immune response (reviewed in Brandtzaeg et al.126), as the disease is paralleled by an extensive inflammatory infiltrate in the lamina propria, consisting of polymorphonuclear neutrophils, eosinophils and plasma cells. However, the final steps leading from such an excessive and enduring mucosal immune activation to tissue injury are still not fully understood. Ultimately, only a limited number of effector mechanisms, including ROM, might be responsible for the excessive cellular/tissue damage, chronic inflammation and destruction of normal tissue that is observed in inflammatory bowel disease.

Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

The chronic presence of numerous, activated, myeloperoxidase-containing phagocytes in the inflamed intestine of inflammatory bowel disease patients implies a prolonged and intense mucosal exposure to an arsenal of toxic and damaging agents, including ROM and RNM. Yet, attempts to directly quantify ROM/RNM levels in the inflammatory bowel disease intestinal mucosa have been limited. The main reason for this lies undoubtedly in the technical difficulty of measuring ROM and RNM directly in cells and tissues, due to their short biological half-lives.127 Over the years, several easy-to-use techniques have been developed, including histochemistry128–132 and colorimetric assays,133–136 but they all lack sufficient sensitivity and specificity. More reliable techniques, such as electron spin resonance spectroscopy136, 137 and chemiluminescence,138 involve highly specialized and expensive laboratory equipment. Moreover, most of such methods are conducted ex vivo, and their extrapolation to the in vivo situation has been questioned.139

Nevertheless, several laboratories have assessed ROM levels directly in inflammatory bowel disease by applying chemiluminescence techniques in colonic biopsy specimens of ulcerative colitis and Crohn's disease patients.140–143 Their results were highly consistent: when compared with normal control mucosa, ROM production was considerably increased in the inflammatory bowel disease samples, was positively correlated with inflammatory bowel disease activity and appeared to be neutrophil-derived. In analogy, direct NO measurements in ulcerative colitis and Crohn's disease patients revealed greatly increased colonic concentrations.144–146 Again, these could be correlated with clinical and endoscopic indices of disease activity. Additional evidence of increased NO production in inflammatory bowel disease has been obtained indirectly, through the analysis of the mucosal levels of the NO-producing enzyme nitric oxide synthase. In numerous studies, nitric oxide synthase activity and protein levels were reported to be up-regulated in the inflamed inflammatory bowel disease mucosa.144, 147–157 Thus, both direct and indirect measurements of ROM/RNM levels in inflammatory bowel disease strongly suggest an increased mucosal production of and exposure to O2•, NO or their downstream metabolites.

Evidence of oxidative damage in inflammatory bowel disease

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

Excessive inflammatory ROM production in the gut has been held responsible for the enhanced electrolyte and water secretion, culminating in diarrhoea, in inflammatory bowel disease patients.158 However, experimental evidence of in vivo oxidant injury in inflammatory bowel disease is scarce, non-circumstantial and generally lacks causative importance. Most has been obtained from drug-induced intervention studies aimed at reducing the generation or effects of ROM in various animal models of intestinal inflammation and in inflammatory bowel disease patients (reviewed in Kruidenier and Verspaget,159 and see below). In fact, direct evidence has been provided in one study only, in which McKenzie et al. showed that the loss of glyceraldehyde-3-phosphate dehydrogenase enzyme activity in colon epithelial crypt cells, harvested directly from inflamed lesions of Crohn's disease and ulcerative colitis patients, resulted from oxidation by HOCl.160

Instead, measurement of oxidative injury in vivo, and hence in human inflammatory bowel disease, has commonly relied on the assessment of oxidatively modified marker molecules.40, 127, 132, 161, 162 The increased levels of malondialdehyde and 4-hydroxynonenal, found in colonic biopsies from inflammatory bowel disease patients,163, 164 for instance, provide evidence for excess lipid peroxidation reactions. This is also true of the increased breath ethane and pentane excretion in these patients,165–167 which are non-invasive markers of lipid peroxidation, and have been correlated with disease activity in most cases. With regard to protein damage, the carbonyl content of cells/tissues has been widely used as a convenient marker of oxidative protein damage,168 whereas the nitration of tyrosine residues in proteins to 3-nitrotyrosine is an indication of the presence of peroxynitrite-modified proteins.56, 74 In colonic biopsies from Crohn's disease and ulcerative colitis patients, the protein carbonyl content has been reported to be increased,143 as has the immunohistochemical expression of 3-nitrotyrosine in mainly the lamina propria mononuclear cells of both Crohn's disease and ulcerative colitis mucosa.149, 152, 154 Obvious correlations with disease activity, however, have never been established. Mucosal DNA oxidation in human inflammatory bowel disease has been evaluated in one study only, in which the DNA oxidation product, 8-hydroxy-2′-deoxyguanosine, was found to be increased in Crohn's disease biopsies.143 More information is available regarding the role of apoptosis in inflammatory bowel disease.169 Markers of this mode of cell death include Fas and Fas-ligand, and DNA strand breaks detected by electrophoresis or immunohistochemistry. From a number of studies, the pattern emerges that, in the inflammatory bowel disease mucosa, apoptosis is increased in the epithelium,170–172 but decreased in T lymphocytes and neutrophils.173–176 These disturbances may have important pathogenetic implications, because they may lead to an increase of epithelial turnover and the accumulation of inflammatory cells,169 thereby hampering immune down-regulation. To date, however, no evidence exists to indicate that the defective apoptosis in inflammatory bowel disease is directly mediated through excessive ROM production.

Antioxidant defences

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

All of these findings strengthen the concept that an uncontrolled, excessive ROM/RNM production and/or disturbances of the redox status in inflammatory diseases, such as inflammatory bowel disease, may have a considerable impact on the course of the disease. On the one hand, ROM and RNM pose a serious threat of deleterious effects by oxidizing and damaging important cellular structures and macromolecules, and, on the other, they can have a profound effect on the expression of a variety of immune and inflammatory molecules, thereby influencing the recruitment and activation of inflammatory cells and tissue-destructive mechanisms. Obviously, it is an absolute necessity for cells and tissues to carefully regulate ROM/RNM levels, even more so under inflammatory conditions. To do so, cells are equipped with an elaborate antioxidant defence system.2 Roughly, the components of this system can be categorized into non-enzymatic and enzymatic antioxidants.

Non-enzymatic antioxidants

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

This group includes several dietary compounds with antioxidant properties, which normally originate from natural sources, such as fruits, vegetables and plant extracts (reviewed in Aruoma177). In particular, certain minerals (e.g. zinc), vitamins (C and E) and the flavonoids found in these extracts are considered to be of prime interest in this context, and have also been exploited commercially.2, 177

In addition, the human gut naturally contains and/or produces a variety of non-enzymatic antioxidant defences.2 These include water-soluble agents, such as glutathione, metallothionein, ascorbic acid (vitamin C), uric acid and some plasma proteins, as well as lipid-soluble defences, such as α-tocopherol (vitamin E), bilirubin and ubiquinol (reduced coenzyme Q10).

Glutathione. Reduced glutathione, a tripeptide with a reactive sulphydryl group, can act on multiple levels of antioxidant defence. Apart from being a substrate for the antioxidant enzyme GPO (see below), glutathione serves as a scavenger of several ROM, including O2•, OH•, peroxynitrite and lipid hydroperoxides.2, 178 In addition, it has been shown to be involved in the direct repair of oxidative DNA lesions, and in the protection against ROM- or cytokine-induced apoptosis.94, 179 During its antioxidant function, reduced glutathione is converted to its oxidized state, upon which it is reduced back again by glutathione reductase.

Metallothionein. The ‘sequestration’ of free metal ions is an important antioxidant defence mechanism, preventing OH• formation via the Fenton and Haber–Weiss reactions [reactions (2) and (6)]. Metallothionein is a small thiol-rich protein that effectively binds potentially harmful metals, such as copper and zinc (reviewed in Davis and Cousins180). As a result of its high thiol content, however, an additional asset of metallothionein function is its capability to directly scavenge OH•.181 These characteristics make metallothionein an effective inhibitor of O2•-, H2O2- and peroxynitrite-dependent lipid peroxidation,182, 183 and its nuclear localization enables metallothionein to protect against oxidative DNA damage and apoptosis.88, 183–185

Localization studies of metallothionein in the normal gut revealed it to be expressed in the cytoplasm and nucleus of luminal and crypt enterocytes.186–188 Metallothionein synthesis is related to cellular zinc homeostasis,180 but also seems to be under inflammatory cytokine control. The metallothionein gene contains activator protein-1 binding sequences, and inflammatory cytokines, such as interleukin-1, interleukin-6, tumour necrosis factor-α and interferon-γ, have been shown to induce metallothionein.180, 189 Metallothionein is consumed during OH• scavenging,181 and can be damaged on exposure to O2• and HOCl through the mobilization of zinc.190

Superoxide dismutases. Soon after the discovery of SOD activity by McCord and Fridovich in 1969,191 it became clear that this enzyme is absolutely necessary to maintain life in aerobic organisms.19 As mentioned before, SOD detoxify O2• by converting it to H2O2[reaction (1)] in what appears to be the fastest enzyme-catalysed reaction known.192 In consequence, loss of SOD function might induce cellular oxidative toxicity through an increase in O2• levels or, more importantly, in its downstream metabolites, such as OH• or peroxynitrite. In humans, three forms of SOD have been identified, each with distinct distribution and metal components.19

Cu/Zn-SOD is a cyanide-sensitive homodimer of approximately 32 kDa, which is diffusely located throughout the cytoplasm and, to a lesser extent, in the nucleus, but is absent in mitochondria.193, 194 It is by far the most abundant SOD isoform, constituting approximately 70% of the total SOD activity,195 and can be found in the epithelium and all types of phagocytes in most organs.196 Apart from O2•, Cu/Zn-SOD can also accept H2O2 as a substrate to interact with reduced copper (Cu+). This reaction reduces H2O2, forming OH• adducts of nearby target molecules (such as Cu/Zn-SOD itself).42 In addition, Cu/Zn-SOD can accept peroxynitrite as a substrate,76 forming nitronium ions that can subsequently transfer nitrate groups to tyrosines on various proteins. The dual role of SOD as an O2• scavenger and an H2O2 producer is increasingly being recognized.197 In humans, overexpression of Cu/Zn-SOD is thought to be associated with Down's syndrome pathogenesis,198 and the Cu/Zn-SOD gene has been linked to the motor neuron degenerative disorder amyotrophic lateral sclerosis.199

The second SOD isoform Mn-SOD is a homotetramer of 96 kDa containing one manganese atom per subunit;200 it is exclusively located in the mitochondria.194 Mn-SOD constitutes approximately 15% of the total SOD activity in most tissues,195 where it has been detected in epithelial cells as well as in phagocytes.200 The importance of this particular SOD isozyme and the potential toxicity of mitochondrially produced O2• have been effectively illustrated in Mn-SOD knockout mice, which die within several days after birth.201 Cu/Zn-SOD knockouts and extracellular-SOD knockouts, on the contrary, survive quite well until they are stressed.202, 203

Extracellular-SOD is the third, and most recently described, SOD family member.204 It is the dominant SOD isoform in the plasma and the interstitium,194 and is the only known extracellular enzyme to scavenge O2•. It is a secretory, tetrameric, copper- and zinc-containing glycoprotein of 135 kDa with a high affinity for glycosaminoglycans, such as heparin.204 Due to this heparin affinity, it may exist at very high concentrations in unique extracellular compartments, whereas it is estimated to make up only 0.5–17% of the total SOD activity in tissues.195 In the human lung, extracellular-SOD has a specific distribution in the connective tissue matrix and smooth muscle cells around larger vessels and airways.205 Based on these findings, it has been speculated to contribute to the protection of collagen matrix elements against ROM, or to be involved in the modulation of vascular tone by the prevention of NO conversion to peroxynitrite.206

Catalase and glutathione peroxidase.  Although CAT and GPO share their substrate H2O2[reactions (3) and (4)], both enzymes have certain distinct features. CAT is one of the most efficient enzymes known: it cannot be saturated by H2O2 at any concentration. In mammalian cells, CAT is largely contained in peroxisomes207 and some of it seems to be secreted. GPO performs the role of CAT in the extracellular environment. It is a largely selenium-dependent enzyme that is predominantly found in the cytoplasm, but also in mitochondria and peroxisomes.207 At least five GPO isozymes are present in mammals, some of which have been localized to mature intestinal absorptive epithelial cells.208

Functionally, GPO has a much higher affinity for H2O2 than CAT,209 and only GPO can react effectively with lipid hydroperoxides210 and prevent peroxynitrite-mediated oxidations.211 In human inflammatory cells, GPO and its substrate glutathione are preferably found in monocytes compared to neutrophils, whereas CAT is found in much higher levels in neutrophils than in monocytes.196 All of these observations concur with various in vitro data,212, 213 which indicate that the glutathione/GPO redox cycle acts as the primary defence against a low, continuous exposure to H2O2, whereas CAT becomes more significant in conditions of acute, severe oxidative stress.

Expression regulation. Only a balanced, co-ordinate action of these three antioxidant enzymes secures a low steady state concentration of ROM in the cell, and hence their activities need to be very precisely regulated. The regulation of SOD isozyme expression has been investigated in numerous in vitro and in vivo studies. All indicate that Cu/Zn-SOD is expressed constitutively, whereas Mn-SOD is highly inducible. Cu/Zn-SOD gene transcription seems to be under the regulation of Sp1-related factors,214 and is marginally affected by exposure to cytokines,188, 215–219 oxidants220–222 or other forms of stress, such as ultraviolet irradiation.218 In macrophages, Cu/Zn-SOD activity can be induced by thyroid hormones and insulin.223 The Mn-SOD gene contains consensus sites for nuclear factor-κB and activator protein-1 transcription factor binding,224–226 which makes it easily inducible under inflammatory conditions. Profound up-regulation of Mn-SOD expression has been demonstrated following exposure to various oxidants,220–222, 227, 228 tumour necrosis factor-α,188, 215–219, 226, 229, 230 interleukin-1,215–217, 226 interleukin-6,218 interferon-γ,216, 231 irradiation218 or endotoxins.215, 226, 232 Insulin and dexamethasone have been shown to reduce Mn-SOD activity in both macrophages and intestinal epithelial cells.223, 233 The expression of extracellular-SOD, like that of Cu/Zn-SOD, is not influenced by its substrate or other ROM.221 It can, however, be elevated by interferon-γ and is depressed by interleukin-1, tumour necrosis factor-α and particularly transforming growth factor-β.216

Much less information is available concerning the expression regulation of the H2O2-metabolizing enzymes CAT and GPO. Their expression levels were induced after exposure to H2O2,220, 222 but treatment with O2• or tumour necrosis factor-α had no effect.188, 219, 220 Insulin increased the activities of macrophage CAT and GPO, whereas thyroid and glucocorticoid hormones reduced GPO activity.223

Autocatalytic inactivation. Any antioxidant enzyme introduced to a site of inflammation will be susceptible to attack by the ROM and RNM present. Indeed, in vitro experiments have shown that practically all ROM are capable of inactivating one or several antioxidant enzymes. Exposure to O2•, for instance, led to a rapid inhibition of CAT,234, 235 but not of GPO activity,235 and H2O2 depressed the activities of Cu/Zn-SOD42, 236 and GPO.235 Low concentrations of neutrophil-derived HOCl very quickly inactivated GPO, whereas CAT was inactivated at higher HOCl levels and Cu/Zn-SOD was not easily affected by HOCl at all.237 Similarly, OH• has been reported to inhibit CAT and GPO activities, but not Cu/Zn-SOD.237 As far as RNM are concerned, NO has been shown to inactivate CAT and GPO.238 On the other hand, it was capable of rescuing Cu/Zn-SOD from H2O2-mediated inactivation.236 Exposure to peroxynitrite effectively inactivated both Mn-SOD239 and GPO.240

Antioxidant enzyme levels in inflammatory bowel disease

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

The differences in the regulation of expression between SOD, CAT and GPO may not only reflect their different roles in normal physiology, but may also endanger the efficient removal of ROM under inflammatory conditions such as inflammatory bowel disease. A decrease in antioxidant enzyme activity, or an unbalanced overexpression of one of these enzymes, may increase the vulnerability of cells to ROM.241 For instance, an increase in SOD would deplete the cell of O2•, but would increase H2O2 production, which might be deleterious unless sufficient CAT or GPO was available. Likewise, excess GPO could unnecessarily deplete glutathione and/or NADPH reserves, even though CAT was present. This concept of an imbalanced antioxidant enzyme response has been appreciated in several (inflammatory) diseases.242–244

In inflammatory bowel disease, the endogenous intestinal expression of SOD isozymes has been investigated in a limited manner (see Table 1). Focusing on Cu/Zn-SOD only, decreased SOD protein and activity levels have been reported in resected mucosa from inflammatory bowel disease patients with active disease,251 and in inflamed mucosal biopsies from Crohn's disease and ulcerative colitis patients.143, 254 No information is available regarding the other SOD isoforms, Mn-SOD and extracellular-SOD. Intestinal CAT and GPO activity levels in inflammatory bowel disease have been evaluated in only a handful of studies. These enzymes do not seem to be affected by the inflammatory process,255–258 despite the fact that some smaller studies have reported an increased GPO activity in inflammatory bowel disease mucosa.245, 252

Table 1.  Reported changes in the levels of endogenous tissue antioxidants in human inflammatory bowel disease
  1. CAT, catalase; CD, Crohn's disease; EC, extracellular; GPO, glutathione peroxidase; ROM, reactive oxygen intermediate(s); SOD, superoxide dismutase; UC, ulcerative colitis.

Reduced glutathioneGPO substrate; scavenges ROM[DOWNWARDS ARROW]CD ileum (n=12)245
=CD colon (n=7);  tendency to [DOWNWARDS ARROW] in UC (n=8)246
[DOWNWARDS ARROW]UC (n=26);  tendency to [DOWNWARDS ARROW] in CD colon (n=14)247
=UC (n=28)248
[DOWNWARDS ARROW]CD ileum (n=26);  not related to steroid intake249
MetallothioneinChelates metals; scavenges OH•[DOWNWARDS ARROW]Immunohistochemistry, CD ileum  (n=6); related to steroid intake187
[DOWNWARDS ARROW]CD (n=29) and UC (n=12);  not related to medication251
[UPWARDS ARROW]Immunohistochemistry,  CD ileum/colon (n=22) and UC (n=48);  not related to medication253
Total SOD activityDetoxifies O2[DOWNWARDS ARROW]UC (n=27);  negatively correlated with disease activity254
=UC (n=25)255
Cu/Zn-SODDetoxifies O2[DOWNWARDS ARROW]Protein content,  CD (n=29) and UC (n=12);  not related to medication251
[DOWNWARDS ARROW]=Enzyme activity,  [DOWNWARDS ARROW] in CD colon (n=38),=in UC (n=29)143
Mn-SODDetoxifies O2Unknown  
EC-SODDetoxifies O2Unknown  
CATDetoxifies H2O2=CD colon (n=19) and UC (n=37)256
=CD colon (n=6)257
=UC (n=31)258
=UC (n=25)255
GPODetoxifies H2O2; inhibits  lipid peroxidation[UPWARDS ARROW]CD ileum (n=12)245
=UC (n=31)258
=UC (n=25)255

Antioxidant therapy: the radical solution?

  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References

In summary, the hypothesis that oxygen radicals are pathogenic factors in inflammatory bowel disease is certainly not ridiculous. All the available experimental evidence suggests that ROM/RNM-mediated events are important in both the primary and downstream secondary pathophysiological mechanisms underlying intestinal inflammation. If so, do antioxidant treatment regimens deserve a place in the inflammatory bowel disease clinic? In fact, attenuating oxidative stress in inflammatory bowel disease patients has already been a therapeutic strategy for 50 years. Commonly used drugs, in particular sulfasalazine and its active moiety 5-aminosalicylic acid, are potent ROM scavengers (reviewed in Miles and Grisham259). In addition, there have been attempts to specifically prevent or attenuate intestinal oxidative stress through either the inhibition of ROM-producing enzymes or the direct scavenging of ROM. Most investigations have been carried out in animal models of colitis and have been extensively reviewed elsewhere.159 Specific antioxidant trials in inflammatory bowel disease patient groups are rare, and all are uncontrolled. Some of them have been withdrawn.260, 261 In fact, only two studies remain. Published in the mid-1980s, high positive response rates (> 80% remission) were observed when patients with severe Crohn's disease (n=30) or ulcerative colitis (n=4) were treated with free or liposomal-encapsulated bovine Cu/Zn-SOD.262, 263 Since then, no further SOD-based clinical trials in inflammatory bowel disease patients have been reported. Obviously, the therapeutic applicability of natural SOD has its limitations in terms of its limited cell permeability, short circulating half-life, immunogenicity and cost of production. Several innovative antioxidant agents have recently been developed that have overcome these limitations, and these await further clinical evaluation.264

Before we start introducing any drug with antioxidant activity into the gut, however, it is imperative that we learn more about the status of the endogenous antioxidant defences in the normal and inflamed intestinal mucosa. To date, data on the mucosal concentration, activity and localization of the most important (anti) oxidant enzymes in Crohn's disease and ulcerative colitis are scarce or, at most, fragmentary. We also need to understand their association with parameters of oxidative damage. In combination with animal experiments designed to evaluate the functional relationship between the (transgenic) expression of antioxidant enzymes and the development and course of intestinal inflammation, these studies might give a fresh impulse to the application and development of antioxidant therapy for inflammatory bowel disease.


  1. Top of page
  2. Summary
  4. Reactive oxygen metabolites
  5. Biochemistry and characteristics
  6. Oxidative damage
  7. Cellular targets of oxidative attack
  8. Reactive oxygen metabolites as immunomodulators
  9. Inflammatory bowel disease
  10. Evidence of increased reactive oxygen metabolite levels in inflammatory bowel disease
  11. Evidence of oxidative damage in inflammatory bowel disease
  12. Antioxidant defences
  13. Non-enzymatic antioxidants
  14. Antioxidant enzymes
  15. Regulation of antioxidant enzyme expression
  16. Antioxidant enzyme levels in inflammatory bowel disease
  17. Antioxidant therapy: the radical solution?
  18. References
  • 1
    Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine, 2nd edn. Oxford: Clarendon Press, 1989.
  • 2
    Halliwell B. Antioxidants in human health and disease. Ann Rev Nutr 1996; 16: 3350.
  • 3
    McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000; 108: 6529.
  • 4
    Vlessis AA, Goldman RK, Trunkey DD. New concepts in the pathophysiology of oxygen metabolism during sepsis. Br J Surg 1995; 82: 8706.
  • 5
    Chapple ILC. Role of free radicals and antioxidants in the pathogenesis of the inflammatory periodontal diseases. J Clin Pathol: Mol Pathol 1996; 49: M24755.
  • 6
    Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996; 32A: 308.
  • 7
    Delanty N, Dichter MA. Oxidative injury in the nervous system. Acta Neurol Scand 1998; 98: 14553.
  • 8
    Saugstad OD. Chronic lung disease: the role of oxidative stress. Biol Neonate 1998; 74S: 218.
  • 9
    Reid GM, Tervit H. Sudden infant death syndrome: oxidative stress. Med Hypotheses 1999; 52: 57780.
  • 10
    Lefer DJ, Granger DN. Oxidative stress and cardiac disease. Am J Med 2000; 109: 31523.
  • 11
    MacNee W. Oxidants/antioxidants and COPD. Chest 2000; 117: 303S317S.
  • 12
    Robberecht W. Oxidative stress in amyotrophic lateral sclerosis. J Neurol 2000; 247S: I/1I/6.
  • 13
    Rosen GM, Pou S, Ramos CL, et al. Free radicals and phagocytic cells. FASEB J 1995; 9: 2009.
  • 14
    Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 1998; 92: 300717.
  • 15
    Babior BM. Phagocytes and oxidative stress. Am J Med 2000; 109: 3344.
  • 16
    Nathan CF. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 1987; 80: 155060.
  • 17
    Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320: 36576.
  • 18
    Panes J, Granger N. Neutrophils generate oxygen free radicals in rat mesenteric microcirculation after abdominal irradiation. Gastroenterology 1996; 111: 9819.
  • 19
    Fridovich I. Superoxide radical and superoxide dismutases. Ann Rev Biochem 1995; 64: 97112.
  • 20
    Turrens JF. Superoxide production by the mitochondrial respiratory chain. Biosci Rep 1997; 17: 38.
  • 21
    Richter C, Schweizer M. Oxidative stress in mitochondria. In: Scandalios JG, ed. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1997: 169200.
  • 22
    Kooij A. A re-evaluation of the tissue distribution and physiology of xanthine oxidoreductase. Histochem J 1994; 26: 889915.
  • 23
    Wakefield A, Snakey E, Dhillon A, et al. Pathogenesis of Crohn's disease: multifocal gastrointestinal infarction. Lancet 1989; 2: 105762.
  • 24
    Ma TY, Hollander D, Freeman D, et al. Oxygen free radical injury of IEC-18 small intestinal epithelial cell monolayers. Gastroenterology 1991; 100: 153343.
  • 25
    Baker SS, Campbell CL. Rat enterocyte injury by oxygen-dependent processes. Gastroenterology 1991; 101: 71620.
  • 26
    Mulier B, Rahman I, Watchorn T, et al. Hydrogen peroxide-induced epithelial injury: the protective role of intracellular nonprotein thiols (NPSH). Eur Respir J 1998; 11: 38491.
  • 27
    Wardman P, Candeias LP. Fenton chemistry: an introduction. Radiat Res 1996; 145: 52331.
  • 28
    Tsuruta T, Tani K, Hoshika A, et al. Myeloperoxidase gene expression and regulation by myeloid growth factors in normal and leukemic cells. Leuk Lymphoma 1999; 32: 25767.
  • 29
    Cohen AB, Chenoweth DE, Hugli TE. The release of elastase, myeloperoxidase, and lysozyme from human alveolar macrophages. Am Rev Respir Dis 1982; 126: 2417.
  • 30
    King CC, Jefferson MM, Thomas EL. Secretion and inactivation of myeloperoxidase by isolated neutrophils. J Leuk Biol 1997; 61: 293302.
  • 31
    Schraufstätter IU, Browne K, Harris A, et al. Mechanisms of hypochlorite injury of target cells. J Clin Invest 1990; 85: 55462.
  • 32
    Carr AC, Winterbourn CC. Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid. Biochem J 1997; 327: 27581.
  • 33
    Zavodnik IB, Lapshina EA, Zavodnik LB, et al. Hypochlorous acid damages erythrocyte membrane proteins and alters lipid bilayer structure and fluidity. Free Rad Biol Med 2001; 30: 3639.
  • 34
    Vissers MCM, Thomas C. Hypochlorous acid disrupts the adhesive properties of subendothelial matrix. Free Rad Biol Med 1997; 23: 40111.
  • 35
    Tatsumi T, Fliss H. Hypochlorous acid and chloramines increase endothelial permeability: possible involvement of cellular zinc. Am J Physiol 1994; 267: H15971607.
  • 36
    Yan LJ, Traber MG, Kobuchi H, et al. Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch Biochem Biophys 1996; 327: 3304.
  • 37
    Lubec G. The hydroxyl radical: from chemistry to human disease. J Invest Med 1996; 44: 32446.
  • 38
    Tabatabaie T, Potts JD, Floyd RA. Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase. Arch Biochem Biophys 1996; 336: 2906.
  • 39
    Takeuchi T, Nakajima M, Morimoto K. Relationship between the intracellular reactive oxygen species and the induction of oxidative DNA damage in human neutrophil-like cells. Carcinogenesis 1996; 17: 15438.
  • 40
    Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition. Mutat Res 1999; 443: 3752.
  • 41
    Kehrer JP. The Haber–Weiss reaction and mechanisms of toxicity. Toxicology 2000; 149: 4350.
  • 42
    Yim MB, Chock PB, Stadtman ER. Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. Proc Natl Acad Sci USA 1990; 87: 500610.
  • 43
    Candeias LP, Patel KB, Stratford MRL, et al. Free hydroxyl radicals are formed on reaction between neutrophil-derived species superoxide anion and hypochlorous acid. FEBS Lett 1993; 333: 1513.
  • 44
    Candeias LP, Strafford MR, Wardman P. Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron (II) complex. Free Rad Res 1994; 20: 2419.
  • 45
    Nappi AJ, Vass E. Hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide. Biochim Biophys Acta 1998; 1380: 5563.
  • 46
    Grisham MB, Jourd'heuil D, Wink DA. Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation. Am J Physiol 1999; 276: G31521.
  • 47
    Kubes P, McCafferty D-M. Nitric oxide and intestinal inflammation. Am J Med 2000; 109: 1508.
  • 48
    Lefer AM, Lefer DJ. Nitric oxide. II. Nitric oxide protects in intestinal inflammation. Am J Physiol 1999; 276: G57275.
  • 49
    Wink DA, Cook JA, Pacelli R, et al. Nitric oxide (NO) protects against cellular damage by reactive oxygen species. Toxicol Lett 1995; 82/83: 2216.
  • 50
    Kim H, Kim K. Effect of nitric oxide on hydrogen peroxide-induced damage in isolated rabbit gastric glands. Pharmacology 1998; 57: 32330.
  • 51
    Scivittaro V, Boggs S, Mohr S, et al. Peroxynitrite protects raw 264.7 macrophage from lipopolysaccharide/interferon-γ-induced cell death. Biochem Biophys Res Commun 1997; 241: 3742.
  • 52
    Binion DG, Rafiee P, Ramanujam KS, et al. Deficient iNOS in inflammatory bowel disease intestinal microvascular endothelial cells results in increased leukocyte adhesion. Free Rad Biol Med 2000; 29: 8818.
  • 53
    Packer MA, Porteous CM, Murphy MP. Superoxide production by mitochondria in the presence of nitric oxide forms peroxynitrite. Biochem Mol Biol Int 1996; 40: 52734.
  • 54
    Xia Y, Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 1997; 94: 69548.
  • 55
    Reiter CD, Teng R-J, Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000; 275: 32 460–6.
  • 56
    Murphy MP, Packer MA, Scarlett JL, et al. Peroxynitrite: a biologically significant oxidant. Gen Pharmacol 1998; 31: 17986.
  • 57
    Marla SS, Lee J, Groves JT. Peroxynitrite rapidly permeates phospholipid membranes. Proc Natl Acad Sci USA 1997; 94: 14 243–8.
  • 58
    Gadelha FR, Thomson L, Fagian MM, et al. Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation. Arch Biochem Biophys 1997; 345: 24350.
  • 59
    Bauer ML, Beckman JS, Bridges RJ, et al. Peroxynitrite inhibits sodium uptake in rat colonic membrane vesicles. Biochim Biophys Acta 1992; 1104: 8794.
  • 60
    Greenacre S, Ridger V, Wilsoncroft P, et al. Peroxynitrite: a mediator of increased microvascular permeability? Clin Exp Pharmacol Physiol 1997; 24: 8802.
  • 61
    Ródenas J, Mitjavila MT, Carbonell T. Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes. Am J Physiol 1998; 274: C82730.
  • 62
    Xia Y, Dawson VL, Dawson TM, et al. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci USA 1996; 93: 67704.
  • 63
    Arteel GE, Briviba K, Sies H. Protection against peroxynitrite. FEBS Lett 1999; 445: 22630.
  • 64
    Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41: 181928.
  • 65
    Ohyashiki T, Ohtsuka T, Mohri T. A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives. Biochim Biophys Acta 1986; 861: 3118.
  • 66
    Jourd'Heuil D, Vaananen P, Meddings JB. Lipid peroxidation of the brush-border membrane: membrane physical properties and glucose transport. Am J Physiol 1993; 264: G100915.
  • 67
    Chen JJ, Bertrand H, Yu BP. Inhibition of adenine nucleotide translocator by lipid peroxidation products. Free Rad Biol Med 1995; 19: 58390.
  • 68
    Aw TY. Determinants of intestinal detoxification of lipid hydroperoxides. Free Rad Res 1998; 28: 63746.
  • 69
    Curzio M, Esterbauer H, Poli G, et al. Possible role of aldehydic lipid peroxidation products as chemoattractants. Int J Tissue React 1987; 9: 295306.
  • 70
    Jayatilleke A, Shaw S. Stimulation of monocyte interleukin-8 by lipid peroxidation products: a mechanism for alcohol-induced liver injury. Alcohol 1998; 16: 11923.
  • 71
    Hogg N, Kalyanaraman B. Nitric oxide and lipid peroxidation. Biochim Biophys Acta 1999; 1411: 37884.
  • 72
    Stadtman ER, Berlett BS. Reactive oxygen-mediated protein oxidation in aging and disease. Chem Res Toxicol 1997; 10: 48594.
  • 73
    Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997; 324: 118.
  • 74
    Ischiropoulos H, Al-Mehdi AB. Peroxynitrite-mediated oxidative protein modifications. FEBS Lett 1995; 364: 27982.
  • 75
    Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998; 356: 111.
  • 76
    Ischiropoulos H, Zhu L, Chen J, et al. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 1992; 298: 4317.
  • 77
    MacMillan-Crow LA, Crow JP, Kerby JD, et al. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci USA 1996; 93: 11 853–8.
  • 78
    Eiserich JP, Hristova M, Cross CE, et al. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998; 391: 3937.
  • 79
    Sampson JB, Ye Y, Rosen H, et al. Myeloperoxidase and horseradish peroxidase catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide. Arch Biochem Biophys 1998; 356: 20713.
  • 80
    Henle ES, Linn S. Formation, prevention, and repair of DNA damage by iron/hydrogen peroxide. J Biol Chem 1997; 272: 19 095–8.
  • 81
    Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci USA 1997; 94: 5149.
  • 82
    Bielski BHJ, Cabelli DE, Arudi RL. Reactivity of HO2/O2 radicals in aqueous solution. J Phys Chem Ref Data 1985; 14: 1041100.
  • 83
    Cuzzocrea S, Caputi AP, Zingarelli B. Peroxynitrite-mediated DNA strand breakage activates poly(ADP-ribose) synthethase and causes cellular energy depletion in carrageenan-induced pleurisy. Immunology 1998; 93: 96101.
  • 84
    Kennedy M, Denenberg AG, Szabó C, et al. Poly(ADP-ribose) synthethase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells. Gastroenterology 1998; 114: 5108.
  • 85
    Nguyen T, Brunson D, Crespi CL, et al. DNA damage and mutation in human cells exposed to nitric oxide in vivo. Proc Natl Acad Sci USA 1992; 89: 30304.
  • 86
    Park J-W, Floyd RA. Glutathione/Fe3+/O2-mediated DNA strand breaks and 8-hydroxydeoxyguanosine formation. Enhancement by copper, zinc superoxide dismutase. Biochim Biophys Acta 1997; 1336: 2638.
  • 87
    Meneghini R. Iron homeostasis, oxidative stress, and DNA damage. Free Rad Biol Med 1997; 23: 78392.
  • 88
    Chubatsu LS, Meneghini R. Metallothionein protects DNA from oxidative damage. Biochem J 1993; 291: 1938.
  • 89
    Tsujikawa K, Imai T, Kakutani M, et al. Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes. FEBS Lett 1991; 283: 23942.
  • 90
    McConkey DJ. Biochemical determinants of apoptosis and necrosis. Toxicol Lett 1998; 99: 15768.
  • 91
    Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Rad Biol Med 2000; 29: 32333.
  • 92
    Gardner AM, Xu F-H, Fady C, et al. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Rad Biol Med 1997; 22: 7383.
  • 93
    Sandoval M, Liu X, Mannick EE, et al. Peroxynitrite-induced apoptosis in human intestinal epithelial cells is attenuated by mesalamine. Gastroenterology 1997; 113: 14808.
  • 94
    Zucker B, Hanusch J, Bauer G. Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous oxygen species. Cell Death Diff 1997; 4: 38895.
  • 95
    Clément M-V, Ponton A, Pervaiz S. Apoptosis induced by hydrogen peroxide is mediated by decreased superoxide anion concentration and reduction of intracellular milieu. FEBS Lett 1998; 440: 138.
  • 96
    Bosca L, Hortelano S. Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators. Cell Signal 1999; 11: 23944.
  • 97
    Warren MC, Bump EA, Medeiros D, et al. Oxidative stress-induced apoptosis of endothelial cells. Free Rad Biol Med 2000; 29: 53747.
  • 98
    Baker AF, Briehl MM, Dorr R, et al. Decreased antioxidant defence and increased oxidant stress during dexamethasone-induced apoptosis: bcl-2 prevents the loss of antioxidant enzyme activity. Cell Death Diff 1996; 3: 20713.
  • 99
    Akahoshi T, Namai R, Sekiyama N, et al. Rapid induction of neutrophil apoptosis by sulfasalazine: implications of reactive oxygen species in the apoptotic process. J Leuk Biol 1997; 62: 81726.
  • 100
    Moreno-Manzano V, Ishikawa Y, Lucio-Cazana J, et al. Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-α-induced apoptosis of rat mesangial cells. J Biol Chem 2000; 275: 12 684–91.
  • 101
    Ottonello L, Frumento G, Arduino N, et al. Immune complex stimulation of neutrophil apoptosis: investigating the involvement of oxidative and nonoxidative pathways. Free Rad Biol Med 2001; 30: 1619.
  • 102
    Shibuya H, Ohkohchi N, Tsukamoto S, et al. Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver. Hepatology 1997; 26: 11320.
  • 103
    Nishikawa M, Kakemizu N, Ito T, et al. Superoxide mediates cigarette smoke-induced infiltration of neutrophils into the airways through nuclear factor-κB activation and IL-8 mRNA expression in guinea pigs in vivo. Am J Respir Cell Mol Biol 1999; 20: 18998.
  • 104
    Martínez J, Moreno JJ. Influence of superoxide radical and hydrogen peroxide on arachidonic acid mobilization. Arch Biochem Biophys 1996; 336: 1918.
  • 105
    Klyubin IV, Kirpichnikova KM, Gamaley IA. Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils. Eur J Cell Biol 1996; 70: 34751.
  • 106
    Johnston B, Kanwar S, Kubes P. Hydrogen peroxide induces leukocyte rolling: modulation by endogenous antioxidant mechanisms including NO. Am J Physiol 1996; 271: H61421.
  • 107
    Monte M, Davel LE, Sacerdote de Lustig E. Hydrogen peroxide is involved in lymphocyte activation mechanisms to induce angiogenesis. Eur J Cancer 1997; 33: 67682.
  • 108
    Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal 1999; 11: 114.
  • 109
    Rogler G, Brand K, Vogl D, et al. Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastroenterology 1998; 115: 35769.
  • 110
    Barnes PJ, Karin M. Nuclear factor-κB — A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336: 106671.
  • 111
    Janssen-Heininger YMW, Macara I, Mossman BT. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-κB. Requirement of Ras/mitogen-activated protein kinases in the activation of NF-κB by oxidants. Am J Respir Cell Mol Biol 1999; 20: 94252.
  • 112
    Meier B, Radeke HH, Selle S, et al. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-α. Biochem J 1989; 263: 53945.
  • 113
    Adamson GM, Billings RE. Tumor necrosis factor induced oxidative stress in isolated mouse hepatocytes. Arch Biochem Biophys 1992; 294: 2239.
  • 114
    Lo YY, Conquer JA, Grinstein S, et al. Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species. J Cell Biochem 1998; 69: 1929.
  • 115
    Meyer M, Pahl HL, Baeuerle PA. Regulation of the transcription factors NF-κB and AP-1 by redox changes. Chem Biol Interact 1994; 91: 91100.
  • 116
    Janssen-Heininger YMW, Poynter ME, Baeuerle PA. Recent advances towards understanding redox mechanisms in the activation of nuclear factor κB. Free Rad Biol Med 2000; 28: 131727.
  • 117
    Schoonbroodt S, Legrand-Poels S, Best-Belpomme M, et al. Activation of the NF-κB transcription factor in a T-lymphocytic cell line by hypochlorous acid. Biochem J 1997; 321: 77785.
  • 118
    Pender SL, Tickle SP, Docherty AJP, et al. A major role for matrix metalloproteinases in T cell injury in the gut. J Immunol 1997; 158: 158290.
  • 119
    Baugh MD, Perry MJ, Hollander AP, et al. Matrix metalloproteinase levels are elevated in inflammatory bowel disease. Gastroenterology 1999; 117: 81422.
  • 120
    Borden P, Heller RA. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Expr 1997; 7: 15978.
  • 121
    Wenk J, Brenneisen P, Wlaschek M, et al. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated induction of matrix-degrading metalloprotease-1. J Biol Chem 1999; 274: 25 869–76.
  • 122
    Rajagopalan S, Meng XP, Ramasamy S, et al. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest 1996; 98: 25729.
  • 123
    Brenneisen P, Briviba K, Wlaschek M, et al. Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free Rad Biol Med 1997; 22: 51524.
  • 124
    Frears ER, Zhang Z, Blake DR, et al. Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite. FEBS Lett 1996; 381: 214.
  • 125
    Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998; 115: 182205.
  • 126
    Brandtzaeg P, Haraldsen G, Rugtveit J. Immunopathology of human inflammatory bowel disease. Springer Semin Immunopathol 1997; 18: 55589.
  • 127
    Rumley AG, Paterson JR. Analytical aspects of antioxidants and free radical activity in clinical biochemistry. Ann Clin Biochem 1998; 35: 181200.
  • 128
    Briggs RT, Robinson JM, Karnovsky ML, et al. Superoxide production by polymorphonuclear leukocytes. A cytochemical approach. Histochemistry 1986; 84: 3718.
  • 129
    Oshitani N, Kitano A, Okabe H, et al. Location of superoxide anion generation in human colonic mucosa obtained by biopsy. Gut 1993; 34: 9368.
  • 130
    Dannenberg AM, Schofield BH, Rao JB, et al. Histochemical demonstration of hydrogen peroxide production by leukocytes in fixed-frozen tissue sections of inflammatory lesions. J Leuk Biol 1994; 56: 43643.
  • 131
    Karnovsky MJ. Cytochemistry and reactive oxygen species: a retrospective. Histochemistry 1994; 102: 1527.
  • 132
    Frank J, Pompella A, Biesalski HK. Histochemical visualization of oxidant stress. Free Rad Biol Med 2000; 29: 1096105.
  • 133
    Pick E, Keisari Y. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods 1980; 38: 16170.
  • 134
    Halliwell B, Grootveld M, Gutteridge JMC. Methods for the measurement of hydroxyl radicals in biochemical systems: deoxyribose degradation and aromatic hydroxylation. Methods Biochem Res 1988; 33: 5990.
  • 135
    Nims RW, Cook JC, Krishna MC, et al. Colorimetric assays for nitric oxide and nitrogen oxide species formed from nitric oxide stock solutions and donor compounds. Methods Enzymol 1996; 268: 93105.
  • 136
    Mueller S. Sensitive and non-enzymatic measurement of hydrogen peroxide in biological systems. Free Rad Biol Med 2000; 29: 4105.
  • 137
    Togashi H, Shinzawa H, Matsuo T, et al. Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging. Free Rad Biol Med 2000; 28: 84653.
  • 138
    Faulkner K, Fridovich I. Luminol and lucigenin as detectors for O2. Free Rad Biol Med 1993; 15: 44751.
  • 139
    Fridovich I. Superoxide anion radical (O2), superoxide dismutases, and related matters. J Biol Chem 1997; 272: 18 515–7.
  • 140
    Keshavarzian A, Sedghi S, Kanofsky J, et al. Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe. Gastroenterology 1992; 103: 17785.
  • 141
    Simmonds NJ, Allen RE, Stevens TRJ, et al. Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease. Gastroenterology 1992; 103: 18696.
  • 142
    Sedghi S, Fields JZ, Klamut M, et al. Increased production of luminol enhanced chemiluminescence by the inflamed colonic mucosa in patients with ulcerative colitis. Gut 1993; 34: 11917.
  • 143
    Lih-Brody L, Powell SR, Collier KP, et al. Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease. Dig Dis Sci 1996; 41: 207886.
  • 144
    Rachmilewitz D, Stamler JS, Bachwich D, et al. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Gut 1995; 36: 71823.
  • 145
    Herulf M, Ljung T, Hellström PM, et al. Increased luminal nitric oxide in inflammatory bowel disease as shown with a novel minimally invasive method. Scand J Gastroenterol 1998; 33: 1649.
  • 146
    Rachmilewitz D, Eliakim R, Ackerman Z, et al. Direct determination of colonic nitric oxide level — A sensitive marker of disease activity in ulcerative colitis. Am J Gastroenterol 1998; 93: 40912.
  • 147
    Boughton-Smith NK, Evans SM, Hawkey CJ, et al. Nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Lancet 1993; 342: 33840.
  • 148
    Godkin AJ, De Belder AJ, Villa L, et al. Expression of nitric oxide synthase in ulcerative colitis. Eur J Clin Invest 1996; 26: 86772.
  • 149
    Singer II, Kawka DW, Scott S, et al. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 1996; 111: 87185.
  • 150
    Ikeda I, Kasajima T, Ishiyama S, et al. Distribution of inducible nitric oxide synthase in ulcerative colitis. Am J Gastroenterol 1997; 92: 133941.
  • 151
    McLaughlan JM, Seth R, Vautier G, et al. Interleukin-8 and inducible nitric oxide synthase mRNA levels in inflammatory bowel disease at first presentation. J Pathol 1997; 181: 8792.
  • 152
    Dijkstra G, Moshage H, Van Dullemen HM, et al. Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease. J Pathol 1998; 186: 41621.
  • 153
    Gupta SK, Fitzgerald JF, Chong SKF, et al. Expression of inducible nitric oxide synthase (iNOS) mRNA in inflamed esophageal and colonic mucosa in a pediatric population. Am J Gastroenterol 1998; 93: 7958.
    Direct Link:
  • 154
    Kimura H, Hokari R, Miura S, et al. Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis. Gut 1998; 42: 1807.
  • 155
    Kolios G, Rooney N, Murphy CT, et al. Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines. Gut 1998; 43: 5663.
  • 156
    Leonard N, Bishop AE, Polak JM, et al. Expression of nitric oxide synthase in inflammatory bowel disease is not affected by corticosteroid treatment. J Clin Pathol 1998; 51: 7503.
  • 157
    Zhang X-J, Thompson JH, Mannick EE, et al. Localization of inducible nitric oxide synthase mRNA in inflamed gastrointestinal mucosa by in situ reverse transcriptase-polymerase chain reaction. Nitric Oxide 1998; 2: 18792.
  • 158
    Gaginella TS, Kachur JF, Tamai H, et al. Reactive oxygen and nitrogen metabolites as mediators of secretory diarrhea. Gastroenterology 1995; 109: 201928.
  • 159
    Kruidenier L, Verspaget HW. Antioxidants and mucosa protectives: realistic therapeutic options in inflammatory bowel disease? Med Inflamm 1998; 7: 15762.
  • 160
    McKenzie SJ, Baker MS, Buffinton GD, et al. Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest 1996; 98: 13641.
  • 161
    De Zwart LL, Meerman JHN, Commandeur JNM, et al. Biomarkers of free radical damage. Applications in experimental animals and in humans. Free Rad Biol Med 1999; 26: 20226.
  • 162
    Winterbourn CC, Kettle AJ. Biomarkers of myeloperoxidase-derived hypochlorous acid. Free Rad Biol Med 2000; 29: 4039.
  • 163
    Chiarpotto E, Scavazza A, Leonarduzzi G, et al. Oxidative damage and transforming growth factor beta 1 in pretumoral and tumoral lesions of human intestine. Free Rad Biol Med 1997; 22: 88994.
  • 164
    Selley ML. Determination of the lipid peroxidation product 4-hydroxy-2-nonenal in clinical samples by gas chromatography—negative-ion chemical ionisation mass spectrometry of the O-pentafluorobenzyl oxime. J Chromatogr B: Biomed Sci Appl 1997; 691: 2638.
  • 165
    Sedghi S, Keshavarzian A, Klamut M, et al. Elevated breath ethane levels in active ulcerative colitis: evidence for excessive lipid peroxidation. Am J Gastroenterol 1994; 89: 221721.
  • 166
    Pelli MA, Trovarelli G, Capodicasa E, et al. Breath alkanes determination in ulcerative colitis and Crohn's disease. Dis Colon Rectum 1999; 42: 716.
  • 167
    Aghdassi E, Allard JP. Breath alkanes as a marker of oxidative stress in different clinical conditions. Free Rad Biol Med 2000; 28: 8806.
  • 168
    Chevion M, Berenshtein E, Stadtman ER. Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Rad Res 2000; 33: S99108.
  • 169
    Levine AD. Apoptosis: implications for inflammatory bowel disease. Inflamm Bowel Dis 2000; 6: 191205.
  • 170
    Lee FD. Importance of apoptosis in the histopathology of drug related lesions in the large intestine. J Clin Pathol 1993; 46: 11822.
  • 171
    Iwamoto M, Koji T, Makiyama K, et al. Apoptosis of crypt epithelial cells in ulcerative colitis. J Pathol 1996; 180: 1529.
  • 172
    Sträter J, Wellisch I, Riedl S, et al. CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis. Gastroenterology 1997; 113: 1607.
  • 173
    Boirivant M, Marini M, Di Felice G, et al. Lamina propria T cells in Crohn's disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis. Gastroenterology 1999; 116: 55765.
  • 174
    Ina K, Itoh J, Fukushima K, et al. Resistance of Crohn's disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance. J Immunol 1999; 163: 108190.
  • 175
    Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat Med 2000; 6: 5838.
  • 176
    Brannigan AE, O'Connell PR, Hurley H, et al. Neutrophil apoptosis is delayed in patients with inflammatory bowel disease. Shock 2000; 13: 3616.
  • 177
    Aruoma OI. Free radicals: dietary advantages and disadvantages. In: Scandalios JG, ed. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1997: 84160.
  • 178
    Harding JJ, Blakytny R, Ganea E. Glutathione in disease. Biochem Soc Trans 1996; 24: 8813.
  • 179
    Xu Y, Jones BE, Neufeld DS, et al. Glutathione modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor α toxicity. Gastroenterology 1998; 115: 122937.
  • 180
    Davis SR, Cousins RJ. Metallothionein expression in animals: a physiological perspective on function. J Nutr 2000; 130: 10858.
  • 181
    Thornalley PJ, Vašák M. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta 1985; 827: 3644.
  • 182
    Thomas JP, Bachowski GJ, Girotti AW. Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins. Biochim Biophys Acta 1986; 884: 44861.
  • 183
    Cai L, Klein JB, Kang YJ. Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage. J Biol Chem 2000; 275: 38 957–60.
  • 184
    Rossman TG, Goncharova EI. Spontaneous mutagenesis in mammalian cells is caused mainly by oxidative events and can be blocked by antioxidants and metallothionein. Mutat Res 1998; 402: 10310.
  • 185
    Deng DX, Cai L, Chakrabarti S, et al. Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein. Toxicology 1999; 134: 3949.
  • 186
    Clarkson JP, Elmes ME, Jasani B, et al. Histological demonstration of immunoreactive zinc metallothionein in liver and ileum of rat and man. Histochem J 1985; 17: 34352.
  • 187
    Elmes ME, Clarkson JP, Jasani B. Histological demonstration of immunoreactive metallothionein in rat and human tissues. Experientia 1987; 52: 5337.
  • 188
    Janssen AML, Van Duijn W, Oostendorp-Van de Ruit MM, et al. Metallothionein in human gastrointestinal cancer. J Pathol 2000; 192: 293300.
  • 189
    Sato M, Sasaki M, Hojo H. Antioxidative roles of metallothionein and manganese superoxide dismutase induced by tumor necrosis factor-α and interleukin-6. Arch Biochem Biophys 1995; 316: 73844.
  • 190
    Fliss H, Ménard M. Oxidant-induced mobilization of zinc from metallothionein. Arch Biochem Biophys 1992; 293: 1959.
  • 191
    McCord JM, Fridovich I. Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244: 604955.
  • 192
    Klug D, Rabani J, Fridovich I. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J Biol Chem 1972; 247: 483942.
  • 193
    Crapo JD, Oury T, Rabouille C, et al. Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci USA 1992; 89: 10 405–9.
  • 194
    Oury TD, Chang LY, Day BJ, et al. Compartmentalization of radical reactions. In: DaviesKJA, UrsiniFS, eds. The Oxygen Paradox. Padova: CLEUP University Press, 1995: 195207.
  • 195
    Marklund SL. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J 1984; 222: 64955.
  • 196
    Pietarinen-Runtti P, Lakari E, Raivio KO, et al. Expression of antioxidant enzymes in human inflammatory cells. Am J Physiol 2000; 278: C11825.
  • 197
    Offer T, Russo A, Samuni A. The pro-oxidative activity of SOD and nitroxide SOD mimics. FASEB J 2000; 14: 121523.
  • 198
    Kedziora J, Bartosz G. Down's syndrome: a pathology involving the lack of balance of reactive oxygen species. Free Rad Biol Med 1988; 4: 31730.
  • 199
    Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993; 362: 5962.
  • 200
    Whittaker JW. Manganese superoxide dismutase. Metal Ions in Biological Systems 2000; 37: 587611.
  • 201
    Li Y, Huang TT, Carlson EJ, et al. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet 1995; 11: 37681.
  • 202
    Reaume AG, Elliott JT, Hoffman EK, et al. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 1996; 13: 437.
  • 203
    Carlsson LM, Jonsson J, Edlund T, et al. Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia. Proc Natl Acad Sci USA 1995; 92: 62648.
  • 204
    Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci USA 1982; 79: 76348.
  • 205
    Oury TD, Chang L-Y, Marklund SL, et al. Immunocytochemical localization of extracellular superoxide dismutase in human lung. Lab Invest 1994; 70: 88998.
  • 206
    Oury TD, Day BJ, Crapo JD. Extracellular superoxide dismutase: a regulator of nitric oxide bioavailability. Lab Invest 1996; 75: 61736.
  • 207
    Singh AK, Dhaunsi GS, Gupta MP, et al. Demonstration of glutathione peroxidase in rat liver peroxisomes and its intraorganellar distribution. Arch Biochem Biophys 1994; 315: 3318.
  • 208
    Tham DM, Whitin JC, Kim KK, et al. Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract. Am J Physiol 1998; 275: G146371.
  • 209
    Jones DP, Eklow L, Thor H, et al. Metabolism of hydrogen peroxide in isolated hepatocytes: relative contribution of catalase and glutathione peroxidase in decomposition of endogenously generated H2O2. Arch Biochem Biophys 1981; 210: 50516.
  • 210
    McCay P, Gibson D, Fong K, et al. Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Biochim Biophys Acta 1976; 431: 45968.
  • 211
    Sies H, Sharov VS, Klotz L-O, et al. Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase. J Biol Chem 1997; 272: 27 812–7.
  • 212
    Hata Y, Kawabe T, Hiraishi H, et al. Antioxidant defenses of cultured colonic epithelial cells against reactive oxygen metabolites. Eur J Pharmacol 1997; 321: 1139.
  • 213
    Masaki H, Okano Y, Sakurai H. Differential role of catalase and glutathione peroxidase in cultured human fibroblasts under exposure of H2O2 or ultraviolet B light. Arch Dermatol Res 1998; 290: 1138.
  • 214
    Seo SJ, Kim HT, Cho G, et al. Sp1 and C/EBP-related factor regulate the transcription of human Cu/Zn SOD gene. Gene 1996; 178: 17785.
  • 215
    Visner GA, Dougall WC, Wilson JM, et al. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. Role in the acute inflammatory response. J Biol Chem 1990; 265: 285664.
  • 216
    Marklund SL. Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts. J Biol Chem 1992; 267: 6696701.
  • 217
    Valentine JF, Nick HS. Acute-phase induction of manganese superoxide dismutase in intestinal epithelial cell lines. Gastroenterology 1992; 103: 90512.
  • 218
    Isoherranen K, Peltola V, Laurikainen L, et al. Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines. J Photochem Photobiol B: Biol 1997; 40: 28893.
  • 219
    Siemankowski LM, Morreale J, Briehl MM. Antioxidant defenses in TNF-treated MCF-7 cells: selective increase in MnSOD. Free Rad Biol Med 1999; 26: 91924.
  • 220
    Shull S, Heintz NH, Periasamy M, et al. Differential regulation of antioxidant enzymes in response to oxidants. J Biol Chem 1991; 266: 24 398–403.
  • 221
    Strålin P, Marklund SL. Effects of oxidative stress on expression of extracellular superoxide dismutase, CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts. Biochem J 1994; 298: 34752.
  • 222
    Röhrdanz E, Kahl R. Alterations of antioxidant enzyme expression in response to hydrogen peroxide. Free Rad Biol Med 1998; 24: 2738.
  • 223
    Pereira B, Costa Rosa LFBP, Safi DA, et al. Hormonal regulation of superoxide dismutase, catalase, and glutathione peroxidase activities in rat macrophages. Biochem Pharmacol 1995; 50: 20938.
  • 224
    Ho YS, Howard AJ, Crapo JD. Molecular structure of a functional rat gene for manganese-containing superoxide dismutase. Am J Respir Cell Mol Biol 1991; 4: 27886.
  • 225
    Zhang N. Characterization of the 5′ flanking region of the human MnSOD gene. Biochem Biophys Res Commun 1996; 220: 17180.
  • 226
    Kiningham KK, Xu Y, Daosukho C, et al. Nuclear factor κB-dependent mechanisms coordinate the synergistic effect of PMA and cytokines on the induction of superoxide dismutase 2. Biochem J 2001; 353: 14756.
  • 227
    Warner BB, Stuart L, Gebb S, et al. Redox regulation of manganese superoxide dismutase. Am J Physiol 1996; 271: L1508.
  • 228
    Jackson RM, Parish G, Helton ES. Peroxynitrite modulates MnSOD gene expression in lung epithelial cells. Free Rad Biol Med 1998; 25: 46372.
  • 229
    Warner BB, Burhans MS, Clark JC, et al. Tumor necrosis factor-α increases Mn-SOD expression: protection against oxidant injury. Am J Physiol 1991; 260: L296301.
  • 230
    Kuratko CN, Constante BJ. Linoleic acid and tumor necrosis factor-α increase manganese superoxide dismutase activity in intestinal cells. Cancer Lett 1998; 130: 1916.
  • 231
    Harris CA, Derbin KS, Hunte-McDonough B, et al. Manganese superoxide dismutase is induced by IFN-γ in multiple cell types. Synergistic induction by IFN-γ and tumor necrosis factor or IL-1. J Immunol 1991; 147: 14954.
  • 232
    Tian L, White JE, Lin H-Y, et al. Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin. Am J Physiol 1998; 275: C7407.
  • 233
    Valentine JF, Nick HS. Glucocorticoids repress basal and stimulated manganese superoxide dismutase levels in rat intestinal epithelial cells. Gastroenterology 1994; 107: 166270.
  • 234
    Kono Y, Fridovich I. Superoxide radical inhibits catalase. J Biol Chem 1982; 257: 57514.
  • 235
    Pigeolet E, Corbisier P, Houbion A, et al. Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Mech Ageing Dev 1990; 51: 28397.
  • 236
    Kim YS, Han S. Nitric oxide protects Cu, Zn-superoxide dismutase from hydrogen peroxide-induced inactivation. FEBS Lett 2000; 479: 258.
  • 237
    Aruoma OI, Halliwell B. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. Biochem J 1987; 248: 9736.
  • 238
    Asahi M, Fujii J, Suzuki K, et al. Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity. J Biol Chem 1995; 270: 21 035–9.
  • 239
    Yamakura F, Taka H, Fujimura T, et al. Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine. J Biol Chem 1998; 273: 14 085–9.
  • 240
    Padmaja S, Squadrito GL, Pryor WA. Inactivation of glutathione peroxidase by peroxynitrite. Arch Biochem Biophys 1998; 349: 16.
  • 241
    Michiels C, Raes M, Toussaint O, et al. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Rad Biol Med 1994; 17: 23548.
  • 242
    Moysan A, Marquis I, Gaboriau F, et al. Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts. J Invest Dermatol 1993; 100: 6928.
  • 243
    Picardo M, Grammatico P, Rocella F, et al. Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma. J Invest Dermatol 1996; 107: 3226.
  • 244
    Gaetani P, Pasqualin A, Rodriguez y Baena R, et al. Oxidative stress in the human brain after subarachnoid hemorrhage. J Neurosurg 1998; 89: 74854.
  • 245
    Iantomasi T, Marraccini P, Favilla F, et al. Glutathione metabolism in Crohn's disease. Biochem Med Metab Biol 1994; 53: 8791.
  • 246
    Buffinton GD, Doe WF. Depleted mucosal antioxidant defences in inflammatory bowel disease. Free Rad Biol Med 1995; 19: 9118.
  • 247
    Ruan EA, Rao S, Burdick JS, et al. Glutathione levels in chronic inflammatory disorders of the human colon. Nutr Res 1997; 17: 46373.
  • 248
    Holmes EW, Yong SL, Eiznhamer D, et al. Glutathione content of colonic mucosa. Evidence for oxidative damage in active ulcerative colitis. Dig Dis Sci 1998; 43: 108895.
  • 249
    Sido B, Hack V, Hochlehnert A, et al. Impairment of intestinal glutathione synthesis in patients with inflammatory bowel disease. Gut 1998; 42: 48592.
  • 250
    Miralles-Barrachina O, Savoye G, Belmonte-Zalar L, et al. Low levels of glutathione in endoscopic biopsies of patients with Crohn's colitis: the role of malnutrition. Clin Nutr 1999; 18: 3137.
  • 251
    Mulder TPJ, Verspaget HW, Janssens AR, et al. Decrease in two intestinal copper/zinc containing proteins with antioxidant function in inflammatory bowel disease. Gut 1991; 32: 114650.
  • 252
    Sturniolo GC, Mestriner C, Lecis PE, et al. Altered plasma and mucosal concentrations of trace elements and antioxidants in active ulcerative colitis. Scand J Gastroenterol 1998; 33: 6449.
  • 253
    Brüwer M, Schmid KW, Metz KA, et al. Increased expression of metallothionein in inflammatory bowel disease. Inflamm Res 2001; 50: 28993.
  • 254
    Dagli Ü, Balk M, Yücel D, et al. The role of reactive oxygen metabolites in ulcerative colitis. Inflamm Bowel Dis 1997; 3: 2604.
  • 255
    Durak I, Yasa MH, Bektas A, et al. Mucosal antioxidant defense is not impaired in ulcerative colitis. Hepato-Gastroenterology 2000; 47: 10157.
  • 256
    O'Morain C, Smethurst P, Levi AJ, et al. Organelle pathology in ulcerative and Crohn's colitis with special reference to the lysosomal alterations. Gut 1984; 25: 4559.
  • 257
    Aimone-Gastin I, Cable S, Keller JM, et al. Studies on peroxisomes of colonic mucosa in Crohn's disease. Dig Dis Sci 1994; 39: 217785.
  • 258
    Bhaskar L, Ramakrishna BS, Balasubramanian KA. Colonic mucosal antioxidant enzymes and lipid peroxide levels in normal subjects and patients with ulcerative colitis. J Gastroenterol Hepatol 1995; 10: 1403.
  • 259
    Miles AM, Grisham MB. Antioxidant properties of aminosalicylates. Methods Enzymol 1994; 234: 55572.
  • 260
    Salim AS. Role of oxygen-derived free radical scavengers in the management of recurrent attacks of ulcerative colitis: a new approach. J Lab Clin Med 1992; 119: 7107.
  • 261
    Hammerschmidt DE, Gross AG. Allegations of impropriety in manuscripts by Aws S. Salim: examination and withdrawal of journal aegis. The Executive Editorial Committee of the Journal of Laboratory and Clinical Medicine. J Lab Clin Med 1994; 123: 7959.
  • 262
    Niwa Y, Somiya K, Michelson AM, et al. Effect of liposomal-encapsulated superoxide dismutase on active oxygen-related human disorders. A preliminary study. Free Rad Res Commun 1985; 1: 13753.
  • 263
    Emerit J, Pelletier S, Tosoni-Verlignue D, et al. Phase II trial of copper zinc superoxide dismutase (CuZnSOD) in treatment of Crohn's disease. Free Rad Biol Med 1989; 7: 1459.
  • 264
    Salvemini D, Riley DP, Cuzzocrea S. SOD mimetics are coming of age. Nat Rev Drug Disc 2002; 1: 36774.