The wanderings of a free radical

doi:10.1016/j.freeradbiomed.2008.11.008

Free Radical Biology and Medicine

Volume 46, Issue 5, 1 March 2009, Pages 531-542

https://doi.org/10.1016/j.freeradbiomed.2008.11.008 Get rights and content

Abstract

In my career I have moved from chemistry to biochemistry to plant science to clinical chemistry and back again (in a partial way) to plants. This review presents a brief history of my research achievements (ascorbate–glutathione cycle, role of iron in oxidative damage and human disease, biomarkers of free radical damage, and studies on atherosclerosis and neurodegeneration) and how they relate to my research activities today. The field of free radicals/other reactive species/antioxidants underpins all of modern Biology. These agents helped to drive human evolution and the basic principles of the field are repeatedly found to be relevant in other research areas. It was an exciting field when I started some 40 years ago, and it still is today, but some major challenges must be faced.

Introduction

I am honored and delighted to receive the 2008 Lifetime Achievement Award from the Society for Free Radical Biology and Medicine (SFRBM), and when asked to write a paper for Free Radical Biology and Medicine, I thought that it was an opportunity (reviewers permitting!) to think back on my career in this field and see what I have learned. My bachelors degree was in Biochemistry but contained a great deal of Chemistry, for which I have always been grateful [1]. A firm grasp of chemical basics is very useful in the free radical/antioxidant field, since many of the wilder claims, such as “the high reactivity of O₂^- in aqueous solution” or “the enormous antioxidant activity of melatonin” can easily be seen to be unlikely on the basis of a modicum of chemical knowledge. Superoxide is selective in its reactions, although the targets that it does attack are crucial to cell metabolism [2], [3], whereas melatonin is a mediocre antioxidant, unlikely at physiological levels to exert direct antioxidant effects in vivo [4], [5], [6]. That does not, of course, mean that melatonin cannot have beneficial effects [7]. Even today, when I look at the structure of molecules such as edaravone, I wonder to what extent direct antioxidant activity really contributes to their apparent biological actions [8], [9].

Section snippets

Early days: Plants

My Ph.D. was on plant biochemistry, investigating a process called “photorespiration” [10], [11]. Plants fix CO₂ in the light using the enzyme ribulose bisphosphate carboxylase, forming two molecules of phosphoglycerate. If CO₂ levels are low and O₂ high, this enzyme can react with O₂ instead of CO₂, giving phosphoglycerate and phosphoglycolate. After conversion of the latter to glycolate, it is oxidized by glycolate oxidase in peroxisomes, forming H₂O₂ and glyoxylate [10]. We were able to show

Plants in context

Working on plants was a good start to a research career, since plants are of enormous importance in the free radical/antioxidant field. First, they supply us with the essential biradical, O₂. As they do, plants expose themselves to high levels of O₂ and so are rich in antioxidant defences and repair systems against oxidative damage to help them deal with O₂ toxicity [10], [28], [29], [30]. Second, plants supply a range of antioxidants to humans—some known to be important in vivo (ascorbate,

Antioxidants and pro-oxidants

Human diets contain not only antioxidants but also many pro-oxidants (Table 2), including phytoprostanes [49], F₂-isoprostanes [50], other lipid peroxides [51], aldehydes [45], [52], [53], and H₂O₂ [54]. Hence the human stomach is an interesting place to observe complex free radical chemistry [43], [44], [45], [55], [56]. This abundance of pro-oxidants was brought home to me when we investigated the mechanism behind early reports that tea [57] and coffee [58] are mutagenic in bacterial test

The “fad” for total antioxidant capacity (TAC) determinations

A wide range of assays is available to measure “total antioxidant capacity” of body fluids and plant extracts (reviewed in [28], [63], [64], [65]). These assays are easy to do and publish the data, so people do them and fill up the literature with descriptions of changes in plasma/serum TAC in disease X or the excellent in vitro antioxidant capacity of fruit Y or vegetable Z. Interpreting the physiological significance of the results needs a clear grasp of the chemistry involved [28], [63], [64]

Free radicals, human disease, and Darwinian medicine

Since my first academic position was in a medical school [1], I soon gave up plants to focus on fundamental free radical work of relevance to human disease. Worldwide interest in free radicals was awakening at that time, and simple assays such as the TBA test (“that bloody assay [75]”) were being widely used to demonstrate increased “lipid peroxidation” or “free radical damage” in almost every human disease examined. The assumption often was that free radicals cause diseases, and antioxidants

Irony then and now

Another feature of my work with John Gutteridge was our investigation and emphasis on the role of iron and other transition metals in catalyzing free radical reactions, and the introduction of the concept that the safe sequestration of such metals in non-redox-active forms can be regarded as a component of the antioxidant defence network, this component being particularly important in the extracellular milieu [110], [111], [112], [113]. Iron ions catalyse the conversion of less reactive species

Iron and atherosclerosis revisited

In 1982, we found that advanced human atherosclerotic lesions contain iron “catalytic” for free radical reactions [167]. However, this could be due to the cell necrosis (Fig. 3A) that is known to occur in advanced lesions, and it does not tell us whether or not such iron is important in the early development of atherosclerosis. Hence recently, we have used an animal model to examine the role of iron in the early stages of atherosclerosis. In rabbits fed a cholesterol-rich diet, atherosclerosis

The antioxidant paradox

When I was young, the field of free radicals and antioxidants was simple: free radicals are bad, antioxidants must be good. Indeed, despite all the recent discoveries about signaling and other metabolic roles for H₂O₂ and some other reactive oxygen species [28], [79], [80], [81], [82], [83], I still believe that overall they are bad over the long human life span, contributing to ageing and certain age-related diseases, especially cancer and neurodegeneration (as reviewed earlier). But why then

An old Chinese proverb—The biomarker concept

Mention of biomarkers brings to mind an old Chinese saying “It is difficult to find a black cat in a dark room….” The challenge for us is to measure low levels of oxidative damage products in the presence of a vast excess of unmolested biomolecules in human tissues and body fluids [28], [150], and the even bigger challenge is to elucidate what these oxidized biomolecule levels mean (Table 4). My research group has long been involved in the development of methodology for measuring oxidative

Cell culture and other artifacts

A vast amount of knowledge about metabolism, signal transduction, and other life events has come from studies of cells in culture. Yet cell culture is an abnormal state—the in vivo extracellular environment is missing and cell culture media are contaminated with transition metal ions [224]. Some media, such as DMEM (Dulbecco's modified Eagle's medium) contain added inorganic iron, usually as Fe(NO₃)₃, giving them even greater pro-oxidant properties [224]. In addition, there is gross hyperoxia.

Conclusion

When I began work in this “new field” of free radicals and antioxidants some 40 years ago, I wondered how long it would last. In my past 40 years of research, I have sometimes felt that the field is “in a rut” or has gone “stale”. Usually just at that time something new explodes to reorientate and revitalise our thinking. Journals and conferences devoted to the field have proliferated and all are doing well. Perhaps I can summarise by saying that free radicals/other reactive

Acknowledgments

I am delighted to acknowledge support of my work by the distinguished Tan Chin Tuan family through its Centennial Professorship funds, plus the following funding agencies who have kindly supported my research work in Singapore: Biomedical Research Council, National Medical Research Council, Office of Life Sciences, National Research Foundation, Singapore Totalisator Board and the Academic Research Fund of the National University of Singapore, and the Ministry of Education.

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Review ArticleThe wanderings of a free radical

Abstract

Introduction

Section snippets

Early days: Plants

Plants in context

Antioxidants and pro-oxidants

The “fad” for total antioxidant capacity (TAC) determinations

Free radicals, human disease, and Darwinian medicine

Irony then and now

Iron and atherosclerosis revisited

The antioxidant paradox

An old Chinese proverb—The biomarker concept

Cell culture and other artifacts

Conclusion

Acknowledgments

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Toxicol. In Vitro

Chem. Phys. Lipids

Biochim. Biophys. Acta

Lancet

Arch. Biochem. Biophys.

Am. J Clin. Nutr.

Arch. Biochem. Biophys.

Curr. Opin. Plant Biol.

Free Radic. Biol. Med.

Methods Enzymol.

Mutat. Res.

Adv. Clin. Chem.

J. Nutr.

Biochem. Biophys. Res. Commun.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

FEBS Lett.

Free Radic. Biol. Med

Curr. Opin. Cell Biol.

Free Radic. Biol. Med.

Trends Biochem. Sci.

Free Radic. Biol. Med.

Cancer Detect. Prev.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

How I become a biochemist

IUBMB Life

The role of iron–sulfur clusters in in vivo hydroxyl radical production

Free Radic. Res.

Cellular defenses against superoxide and hydrogen peroxide

Annu. Rev. Biochem.

Medical implications of melatonin: receptor-mediated and receptor-independent actions

Adv. Med. Sci.

Edaravone inhibits rheumatoid synovial cell proliferation and migration

Free Radic. Res.

Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia-reperfusion injury of the liver by limiting free radical-mediated tissue damage

Free Radic. Res.

Chloroplast metabolism: The structure and function of chloroplasts in green leaf cells

Oxidative decarboxylation of glycollate and glyoxylate by leaf peroxisomes

Biochem. J.

Oxidation of formate by peroxisomes and mitochondria from spinach leaves

Biochem. J.

Non-enzymic catalysis of oxalate decarboxylation by light and flavins

Biochem. J.

Flavin-mononucleotide-sensitized photo-oxidation of glyoxylate in Good's buffers

Biochem. J.

Superoxide dismutase: a contaminant of bovine catalase

Biochem. J.

Univalent reduction of molecular oxygen by spinach chloroplasts on illumination

J Biol. Chem.

Hydroxylation of p-coumaric acid by illuminated chloroplasts. The role of superoxide

Eur. J. Biochem.

Evidence for sodium azide as an artifact mediating the modulation of inducible nitric oxide synthase by C-reactive protein

J. Cardiovasc. Pharmacol.

Failure of endotoxin-free SOD to reduce some paw edemas and adjuvant arthritis in rats

Review Article
The wanderings of a free radical