Publications

Reactive oxygen species and reactive nitrogen species are formed in the human body. Endogenous antioxidant defences are inadequate to scavenge them completely, so that ongoing oxidative damage to DNA, lipids, proteins and other molecules can be demonstrated and may contribute to the development of cancer, cardiovascular disease and possibly neurodegenerative disease. Hence diet-derived antioxidants may be particularly important in protecting against these diseases. Some antioxidants (e.g. ascorbate, certain flavonoids) can exert pro-oxidant actions in vitro, often by interaction with transition metal ions. The physiological relevance of these effects is uncertain, as is the optimal intake of most diet-derived antioxidants. In principle, these questions could be addressed by examining the effects of dietary composition and/or antioxidant supplementation upon parameters of oxidative damage in vivo. The methods available for measuring steady-state damage (i.e. the balance between damage and repair or replacement of damaged molecules) and the actual rate of damage to DNA, proteins and lipids are reviewed, highlighting areas in which further methodological development is urgently required.

Abstract The first organisms to evolve on the Earth were anaerobes: there was initially little or no O2 intheatmosphere. As the O2 content rose, some organisms adapted by evolving antioxidant defence systems to protect themselves against the toxic effects of O2 (see Chapter 1). The term ‘ antioxidant’ is widely used in newspapers, magazines, and the scientific literature, but it is rarely defined. Often it is used to refer only to antioxidants than can scavenge radicals, such as superoxide dismutase, vitamin E, and ascorbic acid (vitamin C). Let us explore this further.

Phenolic compounds are not completely absorbed in the small intestine and so enter the colon, where they might exert physiological effects. To identify phenolics that are present in normal human colon, fecal water was prepared from 5 free-living volunteers with no dietary restrictions and analyzed by gas chromatography-mass spectrometry. Daily measurements were also performed on a single individual to examine the variation more closely. Levels of polyphenols were variable between individuals. Naringenin and quercetin had mean concentrations of 1.20 and 0.63 μM. All other flavonoids examined were present ≤0.17 μM. Simple phenolic and other aromatic acids were present at much higher concentrations. The major components were phenylacetic acid, 479 μM; 3-phenylpropionic acid, 166 μM; 3-(4-hydroxy)-phenylpropionic acid, 68 μM; 3,4-dihydroxycinnamic acid, 52 μM; benzoic acid, 51 μM; 3-hydroxyphenylacetic acid, 46 μM; and 4–hydroxyphenylacetic acid, 19 μM. Other phenolic acids ranged from 0.04 to 7 μM. Decreased dietary phenolic intake caused a decrease in polyphenol and monophenolic acid concentration in fecal water 24 h later. This study is the first to measure the range of aromatic compounds in human fecal water and demonstrates that phenolic acid concentrations are high. The biological effects of phenolics may play an important role in colon function.

Abstract— When oxidizing NADH or dihydroxyfumarate, horseradish peroxidase exists mainly as compound III, which is formed by a reaction between O ‐ 2 and the enzyme. Certain phenols stimulate peroxidase‐catalysed oxidation of NADH or dihydroxyfumarate, apparently because they bring about the breakdown of compound III to active enzyme. Superoxide dismutase and catalase, added at any time during the reaction, inhibit dihydroxyfumarate oxidation by peroxidase. This is attributed to generation of the dihydroxyfumaryl radical during H 2 O 2 ‐dependent oxidation of dihydroxyfumarate by peroxidase. This radical reduces O 2 to O ‐ 2 , which can then react with more dihydroxyfumarate. Superoxide dismutase added during the course of the reaction does not inhibit NADH oxidation by peroxidase, since O ‐ 2 , although generated in the reaction mixture, reacts only slowly with NADH. Lactate dehydrogenase or Mn 2+ stimulate NADH oxidation by peroxidase because they catalyse a reaction between NADH and O ‐ 2 . Oxidation of NADH by peroxidase in the presence of certain phenols and Mn 2+ is very rapid, and requires both H 2 O 2 and O ‐ 2 . O ‐ 2 also seems to be necessary to initiate NADH oxidation by peroxidase. Aromatic compounds are hydroxylated by a mixture of peroxidase and dihydroxyfumarate. Hydroxylation is prevented by superoxide dismutase or by scavengers of the hydroxy radical (.OH). O ‐ 2 is insufficiently reactive to hydroxylate aromatic rings directly and so may give rise to .OH, which is the true hydroxylating species. Hydroxyl radical probably does not arise by the Haber‐Weiss reaction. H 2 O 2 , but not O ‐ 2 , is involved in the oxidation of thiol compounds by peroxidase.

Activated phagocytes produce ‘reactive oxygen, halogen and nitrogen species’ that help to kill some types of microorganism. How these species destroy microorganisms remains, however, an enigma: both direct oxidative damage and indirect damage (whereby reactive species promote the actions of other antibacterial agents) are involved, and no single mechanism is likely to account for the killing of all microorganisms. Phagocyte-derived reactive species are known to injure human tissues and to contribute to inflammation. Recently, however, we have learned that they can also be anti-inflammatory by modulating the immune response. These data have implications for the proposed use of antioxidants to treat inflammation.

Green tea and red wine are claimed to have health benefits because of their high content of polyphenolic compounds, but they have also been reported as mutagenic in some test systems. In this paper, we show that a commonly used cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM), catalyses oxidation of green tea and red wines to generate H2O2. The level of H2O2 produced from green tea accounted for all of the cytotoxic effects of this beverage on PCl2 cells. By contrast, H2O2 was only responsible for part of the cytotoxicity of the red wines examined. Our data illustrate the danger of extrapolating from cell culture studies to predict the effects of complex beverages in vivo.

Abstract This chapter gives examples of how oxygen radicals and other reactive species (RS) are useful. It describes how enzymes such as ribonucleotide reductase (tyrosyl, glycyl, or deoxyadenosyl radicals, the last from S-adenosylmethionine [SAM] or adenosylcobalamin), other 'radical SAM' enzymes, pyruvate formate lyase (glycyl radicals), galactose and glyoxal oxidases, and indoleamine (tyrosyl radical) or tryptophan dioxygenases use RS to bring about reactions. The roles of RS in inflammasome formation in response to damage-associated and pathogen-associated molecular patterns is described, and how RS are employed by neutrophils, macrophages, monocytes, and eosinophils to kill micro-organisms. The contributions of superoxide (generated by NADPH oxidases), hypochlorous acid, singlet O2, and peroxynitrite to killing, and the mechanisms evolved by some bacteria to resist them, are presented. Contributions of these RS to acute and chronic inflammation are detailed. The roles of NADPH oxidases and dual oxidases in other cell types (e.g. respiratory tract and gastrointestinal epithelium, lymphocytes, platelets, osteoblasts, osteoclasts, blood vessels) are described, e.g. in regulation of blood pressure and bone turnover. Involvement of RS in thyroid hormone biosynthesis, degradation of lignin, defence mechanisms of beetles, fertilization of eggs, and the defence mechanisms used by plants (e.g. the RS-generating respiratory burst oxidase homologues) against pathogens are presented. Cyclooxygenases and lipoxygenases are reviewed, in relation to the production of prostaglandins and leukotrienes in animals and its regulation by RS, as well as the roles of lipoxygenases in plants. The mechanisms of light production by green fluorescent protein and other systems (e.g. pholasin, aequorin) are described.

Hydrogen peroxide (H 2 O 2 ) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H 2 O 2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H 2 O 2 may be greater than is commonly supposed: substantial amounts of H 2 O 2 can be present in beverages commonly drunk (especially instant coffee), in freshly voided human urine, and in exhaled air. Levels of H 2 O 2 in the human body may be controlled not only by catabolism but also by excretion, and H 2 O 2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Urinary H 2 O 2 levels are influenced by diet, but under certain conditions might be a valuable biomarker of ‘oxidative stress’.

This article reviews what is presently known about the biological roles of the diet-derived compound ergothioneine (ET). ET seems important to humans because it is rapidly taken up from the diet by a transporter largely or completely specific for ET, and once taken up it is retained within the body for weeks or months. The various possible functions of ET in vivo are explored. Much emphasis has been placed on the antioxidant properties of ET, but although these are well established in vitro, the evidence that antioxidant activity is the principal function of ET in vivo is weak. ET is not unique in this: The evidence for the antioxidant roles of vitamin C and polyphenols such as the flavonoids in vivo is also weak. By contrast, α-tocopherol has demonstrated in vivo antioxidant effects in humans.