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A wide variety of oxygen free radicals and other reactive oxygen species can be formed in the human body and in food systems. Transition metal ions accelerate free‐radical damage. Antioxidant defenses, both enzymic and nonenzymic, protect the body against oxidative damage, but they are not 100% efficient, and so free‐radical damage must be constantly repaired. Nonenzymatic antioxidants are frequently added to foods to prevent lipid peroxidation. Several lipid antioxidants can exert prooxidant effects toward other molecules under certain circumstances, and so antioxidants for food and therapeutic use must be characterized carefully. Methods of measuring oxidative damage and trapping free radicals in vivo are briefly discussed. Such methods are essential in checking proposals that increased intake of food‐derived antioxidants (such as antioxidant vitamins) would be beneficial to humans.
Damage to DNA by oxygen radicals and other reactive oxygen/nitrogen/chlorine species occurs in vivo despite the presence of multiple antioxidant defence and repair systems. Such damage is thought to make a significant contribution to the age-related development of cancer. Modulation of oxidative DNA damage by diet thus constitutes a "biomarker" putatively predictive of the effect of diet on cancer incidence, provided that DNA damage can be accurately quantitated by validated methods. Current issues addressed in this article include the problems of artifactual DNA oxidation during isolation and analysis, the relative merits of different analytical methods, the advantages and disadvantages of relying on measurement of 8-hydroxy-deoxyguanosine (8OHdG, 8-oxodG) as an index of oxidative DNA damage, and the limited data that are so far available on how diet can affect "steady-state" levels of oxidative DNA damage in humans. It appears that such damage can be modulated by vegetable intake, although the effects of vegetables may be mediated by components different from the "classical" antioxidants vitamin C, α-tocopherol and β-carotene.
1. Hydroxyl radicals (OH) are thought to be generated at sites of inflammation and to contribute to tissue damage. All anti-inflammatory drugs tested were able to scavenge ˙ OH generated in free solution at almost diffusion-controlled rates (rate constants about 1010 M−1 s−1).2. Much ˙ OH generation in vivo occurs at specific sites, where bound metal ions (such as Fe2+) react with H2O2 to produce ˙ OH that immediately attacks the site. Only ˙ OH scavengers that have sufficient metal-binding ability to withdraw metal ions from this site can protect against site-specific damage.3. All anti-inflammatory drugs tested were able to protect against site-specific damage by ˙ OH in a simple model system in vitro. Penicillamine, diclofenac sodium, piroxicam, azathioprine, primaquine, chloroquine and hydroxychloroquine were especially effective.4. The ability of an anti-inflammatory drug to protect against ˙ OH formation in vivo depends not only on its rate constant for reaction with ˙ OH, but also on its metal-binding ability and on the geometry and redox potential of any metal complex formed.
Recent studies have shown that low concentrations of H2O2 are produced endogenously by nonphagocytes after wounding. We observed that H2O2 at such concentrations can stimulate proliferation as well as migration of keratinocytes in a scratch-wound assay. Both wounding and H2O2 can induce phosphorylation of ERK1/2 via EGFR, but the activation of ERK1/2 by H2O2 is more sustained and can last more than 8h. Sustained ERK1/2 activation is required for the increased proliferation and migration induced by H2O2. The p38 MAPK was also found to be phosphorylated upon treatment with H2O2 but it was not required for H2O2-induced migration or proliferation. Furthermore, it was observed that there is a cross talk between the ERK1/2 and the p38 pathways whereby inhibition of either pathway can lead to activation of the other. As a result, the motogenic effects of H2O2 were further enhanced when p38 was inhibited. Our data are consistent with the view that H2O2 may play an important signaling role in wound healing.
Review Article| January 01 1996 Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer Helen WISEMAN; Helen WISEMAN *Department of Nutrition and Dietetics, King's College London, Campden Hill Road, London W8 7AH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Barry HALLIWELL Barry HALLIWELL †Pharmacology Group, King's College London, Manresa Road, London SW3 6LX, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1996) 313 (1): 17–29. https://doi.org/10.1042/bj3130017 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation Helen WISEMAN, Barry HALLIWELL; Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1 January 1996; 313 (1): 17–29. doi: https://doi.org/10.1042/bj3130017 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. The Biochemical Society, London © 19961996 Article PDF first page preview Close Modal You do not currently have access to this content.
A bstract : Peroxynitrite is implicated in numerous human diseases. Hence, there is considerable interest in potential therapeutic peroxynitrite scavengers. It has been claimed that uric acid is a powerful peroxynitrite scavenger. We previously observed that uric acid is a powerful inhibitor of tyrosine nitration induced by peroxynitrite, but fails to prevent α 1 ‐antiproteinase (α 1 ‐AP) inactivation induced by peroxynitrite. However, the reactivity of peroxynitrite is significantly modified by bicarbonate and this has not been considered in evaluating the scavenging activity of uric acid and other endogenous antioxidant compounds. In the presence of bicarbonate (25 mM), the ability of uric acid, ascorbate, Trolox, and GSH to inhibit peroxynitrite‐mediated tyrosine and guanine nitration is decreased. Protection against peroxynitrite‐mediated α 1 ‐AP inactivation is also decreased by ascorbate, Trolox, and GSH, but it is enhanced by uric acid. Bicarbonate also inhibits the ability of these compounds to prevent peroxynitrite‐mediated ABTS radical cation formation. However, the abilities of these antioxidants to prevent peroxynitrite‐mediated bleaching of pyrogallol red are enhanced by bicarbonate. These results show that physiologic concentrations of bicarbonate substantially modify the ability of uric acid to prevent peroxynitrite‐mediated reactions. This study highlights the need to use several different assays in the presence of physiologically relevant concentrations of bicarbonate when assessing compounds for peroxynitrite scavenging, in order to avoid misleading results.
Free radicals and other “reactive oxygen species” (ROS) are formed continuously in the human body, both deliberately and by “accidents of chemistry.” Our endogenous antioxidant defenses are inadequate to completely prevent damage by ROS. Hence, diet-derived antioxidants may be of special importance in delaying or preventing the onset of diseases in which ROS are involved, such as cardiovascular diseases, chronic inflammatory diseases, neurodegenerative diseases, and some forms of cancer. Determining the optimal intake of antioxidant nutrients is one of the greatest challenges in the nutrition/free radicals field today, but the possibility of side effects from supraoptimal doses must not be ignored.
Diets rich in fruits and vegetables are associated with decreased risk of cardiovascular disease and cancer. Biomarkers of oxidative DNA damage and lipid peroxidation can be used to establish the role of antioxidants in this protection and the optimal intake of those antioxidants. This concept is based on the presumptions that oxidative DNA damage is a significant contributor to the age-related development of some cancers and that lipid peroxidation plays a key role in the development of cardiovascular disease. Mass spectrometric measurements of various families of isoprostanes (F2-, F3-, and F4-isoprostanes) and of multiple DNA base oxidation products are probably the most promising biomarkers for use in human nutritional intervention studies. Biomarker studies should precede, as well as accompany, major intervention trials that measure disease incidence. The use of biomarkers provides a logical scientific basis for major intervention trials of antioxidants; such trials will, in turn, eventually validate or disprove the biomarker concept.
SUMMARY Recent research suggests that H 2 O 2 is a normal metabolite in both plants and animals and is not particularly cytotoxic. The impact of H 2 O 2 on cell metabolism is discussed with particular reference to photorespiration. Catalase may function by preventing the formation of excessive concentrations of H 2 O 2 and by using H 2 O 2 in the peroxidatic oxidation of compounds such as methanol and formic acid. Radicals (such as OH and O 2 ‐ ) and ‘excited’ oxygen are far more damaging to living organisms. The formation of these radicals in biological systems is described. Superoxide dismutase plays a key role in protection against such radicals, but glutathione peroxidase is also involved.
Incubation of aqueous solutions of 2-nitropropane in air causes a slow oxidation reaction that generates H(2)O(2). Purified horseradish peroxidase catalyses the oxidation of such preincubated 2-nitropropane solutions according to the equation: [Formula: see text] The pH optimum is 4.5 and K(m) for 2-nitropropane is 16mm. Other nitroalkanes or nitro-aromatics tested are not oxidized at significant rates by peroxidase. H(2)O(2) or 2,4-dichlorophenol increases the rate of 2-nitropropane oxidation by peroxidase. Catalase inhibits the reaction completely. Superoxide dismutase or mannitol, a scavenger of the hydroxyl radical, OH(.), each inhibits partially. Aniline and guaiacol are also powerful inhibitors of 2-nitropropane oxidation. It is suggested that peroxidase uses the traces of H(2)O(2) generated during preincubation of 2-nitropropane to catalyse oxidation of this substrate into a radical species that can reduce O(2) to the superoxide ion, O(2) (-.).O(2) (-.), or OH(.) derived from it, then appears to react with more nitropropane, generating further radicals and H(2)O(2) to continue the oxidation. Inhibition by aniline and guaiacol seems to be due to a competition for H(2)O(2).
Peroxynitrite, formed by reaction of superoxide and nitric oxide, appears to be an important tissue-damaging species generated at sites of inflammation. In this paper, we compare the abilities of several biological antioxidants to protect against peroxynitrite-dependent inactivation of α1-antiproteinase, and to inhibit tyrosine nitration upon addition of peroxynitrite. GSH and ascorbate protected efficiently in both systems. Uric acid inhibited tyrosine nitration but not α1-antiproteinase inactivation. The possibility that ascorbic acid is an important scavenger of reactive nitrogen species in vivo is discussed.Key Words: Peroxynitriteascorbatetyrosine nitrationα1-antiproteinasereactive nitrogen speciesrheumatoid arthritismethionineGSH
Dichlorofluorescin diacetate (DCFDA) is a popular fluorescence-based probe for reactive oxygen species (ROS) detection in vitro and in vivo, and has been used for this purpose in C. elegans. DCFDA is first deacetylated by endogenous esterases to dichlorofluorescein (DCFH), which can react with several ROS to form the fluorophore DCF (reviewed by Halliwell and Gutteridge, 2007). The DCFDA assay in C. elegans is sometimes performed using lysed worms, following high intensity sonication. This process disrupts the outer cuticle and internal membranes, causing intracellular, as well as intraorganelle, contents to be released. Worm lysis will cause the release of transition metal ions such as iron. Free iron may participate in redox cycling to generate ROS, for example, by Fenton chemistry (Halliwell and Gutteridge, 2007). Therefore, apparent ROS levels detected in lysed worms may be un-physiological. However, DCFDA readily diffuses into cells where it undergoes deacetylation, and this allows ROS measurement in whole worms. If whole animals are utilized, cells are not disrupted and iron remains sequestered. Using DCFDA and a synchronized worm cohort, we measured the amount of ROS in equivalent numbers of lysed and whole worms. The gradient of the linear regression curve (expressed as ΔRFU/Δmin) (Figure 1) is the rate of fluorescent DCF production. This gradient can be, very approximately, converted to the rate of H2O2-equivalent ROS production (nmol/min) by spiking known amounts of H2O2. It should, however, be noted that H2O2 itself does not oxidize DCFH, and hence, the free radical reactions resulting in increased DCF signal following H2O2 spiking are likely complex. Therefore, the calibration ratio can only be considered to provide an approximate estimation of ROS produced. Nevertheless, we found that the rate of H2O2-equivalent production was more than an order of magnitude faster in lysed (4.1x10nmol/min/worm) compared to whole animals (3.6x10 nmol/min/ worm). Using the O2 consumption rate measured in the same worm cohort (0.018 nmol/min/worm), the % of O2 consumed leading to ROS generation can be estimated by dividing the rate of H2O2-equivalent production by the rate of O2 consumption. Using this approach, we found apparent ROS production to be around 23% for lysed worms, but only 2% for whole worms. Current estimates of in vivo mitochondrial ROS production range from 0.2 to 2% (Balaban et al., 2005 PMID: 15734681). Assuming all ROS measured using the DCFDA assay to be of mitochondrial origin, ROS production is clearly overestimated by a factor of at least 10-fold, and may be as much as 100-fold, in lysed worms compared to whole worms. Therefore, when using the DCFDA assay, whole worms should be utilized instead.
Freshly-voided human urine contains significant concentrations of hydrogen peroxide (H2O2). This H2O2 appears to arise in whole or in part by superoxide-dependent autoxidation of urinary biomolecules. Since instant coffee also contains high levels of H2O2, we examined the effect of coffee drinking on urinary levels of H2O2. Studies on healthy human volunteers showed that coffee drinking is rapidly and reproducibly followed by increased levels of H2O2 detectable in the urine for up to 2 h after drinking the coffee. The levels of H2O2 detected in urine suggest that exposure of human tissues to H2O2 may be greater than is commonly supposed. It is possible that H2O2 in urine could act as an antibacterial agent, and that H2O2 is involved in the regulation of glomerular function.
