Free radicals and other reactive species (RS) are thought to play an important role in many human diseases. Establishing their precise role requires the ability to measure them and the oxidative damage that they cause.


This article first reviews what is meant by the terms free radical, RS, antioxidant, oxidative damage and oxidative stress.


It then critically examines methods used to trap RS, including spin trapping and aromatic hydroxylation, with a particular emphasis on those methods applicable to human studies.


Methods used to measure oxidative damage to DNA, lipids and proteins and methods used to detect RS in cell culture, especially the various fluorescent ‘probes' of RS, are also critically reviewed.


The emphasis throughout is on the caution that is needed in applying these methods in view of possible errors and artifacts in interpreting the results.





Keywords: Cell culture, free radical, reactive species, antioxidant, oxidative stress, oxidative damage, fluorescent probe, lipid peroxidation, superoxide


Introduction
Free radicals and other ‘reactive oxygen (ROS)/nitrogen/chlorine species' (for an explanation of these terms see Table 1) are widely believed to contribute to the development of several age-related diseases, and perhaps, even to the aging process itself (Halliwell & Gutteridge, 1999; Sohal et al., 2002) by causing ‘oxidative stress' and ‘oxidative damage' (terms explained in Table 2). For example, many studies have shown increased oxidative damage to all the major classes of biomolecules in the brains of Alzheimer's patients (Halliwell, 2001; Butterfield, 2002; Liu et al., 2003). Other diseases in which oxidative damage has been implicated include cancer, atherosclerosis, other neurodegenerative diseases and diabetes (Hagen et al., 1994; Chowienczyk et al., 2000; Halliwell, 2000a, 2001, 2002a, 2002b; Parthasarathy et al., 2000). If oxidative damage contributes significantly to disease pathology (Table 3 lists the criteria needed to establish this), then actions that decrease it should be therapeutically beneficial (Halliwell, 2001; Lee et al., 2002a; Liu et al., 2003). If the oxidative damage is involved in the origin of a disease, then successful antioxidant treatment should delay or prevent the onset of that disease (Halliwell, 1991, 2002a, 2002b; Galli et al., 2002; Steinberg & Witztum, 2002). To establish the role of oxidative damage (Table 3), it is therefore essential to be able to measure it accurately. For example, the failure of interventions with antioxidants such as vitamin E, β-carotene or ascorbate to decrease disease incidence in several human intervention trials may have simply been due to the failure of these compounds to decrease oxidative damage in the subjects tested (Halliwell, 1999a, 2000c; Levine et al., 2001; Meagher et al., 2001). In this review, we will examine the methods available to measure reactive species (RS) and oxidative damage, with a particular emphasis on those applicable to human studies.



Table 1

Nomenclature of reactive species





Table 2

Some key definitions





Table 3

Criteria for implicating RS as a significant mechanism of tissue injury in human disease




Measuring RS in vivo: basic principles

Some fascinating techniques such as L-band electron spin resonance (ESR) with nitroxyl probes and magnetic resonance imaging spin trapping are under development to measure RS directly in whole animals (e.g. Berliner et al., 2001; Han et al., 2001; Utsumi & Yamada, 2003), but no probes are currently suitable for human use. Most RS persist for only a short time in vivo and cannot be measured directly. There are a few exceptions: examples include H2O2 (discussed below), and perhaps, NO•, in the sense that serum levels of NO2− have been claimed to measure vascular endothelial NO• synthesis (Kelm et al., 1999), despite the fact that NO2− is quickly oxidized to NO3− in vivo (Kelm et al., 1999; Oldreive & Rice-Evans, 2001). Essentially, there are two approaches to detecting transient RS: 


attempting to trap these species and measure the levels of the trapped molecules and


measuring the levels of the damage done by RS, that is, the amount of oxidative damage.




Sometimes other approaches are used. They include measurements of erythrocyte antioxidant defences and of total antioxidant activity of body fluids; falls in these parameters are often taken as evidence of oxidative stress. Erythrocytes cannot synthesize proteins, however, and their antioxidant enzyme levels may drop as they ‘age' in the circulation (Denton et al., 1975). Thus changes in their levels are more likely to reflect changes in the rates of red blood cell turnover: if this slows down, the circulating erythrocytes will be older on average and so levels of antioxidant enzymes in them will appear to fall. Vice versa, if an intervention accelerates red cell removal or increases erythropoiesis, levels of antioxidants in red cells will seem to rise. Hence, such data should be interpreted with caution.

Depending on the method that is used to measure it, the plasma or serum ‘total antioxidant capacity' (TAC) usually involves major contributions from urate, ascorbate and sometimes albumin −SH groups (Benzie & Strain, 1996; Halliwell & Gutteridge, 1999; Prior & Cao, 1999; Rice-Evans, 2000; Bartosz, 2003), although different methods measure different things (Schlesier et al., 2002; Bartosz, 2003). Thus, for example, if plasma albumin levels fall, TAC will fall. If urate levels rise, TAC will rise. The multiple changes in blood chemistry that occur in sick people mean that TAC changes should be interpreted with caution. TAC is also influenced by diet, often because consumption of certain foods may produce changes in plasma ascorbate and/or urate levels (Halliwell, 2003b).

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Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?

A major development of carcinogenesis research in the past 20 years has been the discovery of significant levels of DNA damage arising from endogenous cellular sources. Dramatic improvements in analytical chemistry have provided sensitive and specific methodology for identification and quantitation of DNA adducts. Application of these techniques to the analysis of nuclear DNA from human tissues has debunked the notion that the human genome is pristine in the absence of exposure to environmental carcinogens. Much endogenous DNA damage arises from intermediates of oxygen reduction that either attack the bases or the deoxyribosyl backbone of DNA. Alternatively, oxygen radicals can attack other cellular components such as lipids to generate reactive intermediates that couple to DNA bases. Endogenous DNA lesions are genotoxic and induce mutations that are commonly observed in mutated oncogenes and tumor suppressor genes. Their mutagenicity is mitigated by repair via base excision and nucleotide excision pathways. The levels of oxidative DNA damage reported in many human tissues or in animal models of carcinogenesis exceed the levels of lesions induced by exposure to exogenous carcinogenic compounds. Thus, it seems likely that oxidative DNA damage is important in the etiology of many human cancers. This review highlights some of the major accomplishments in the study of oxidative DNA damage and its role in carcinogenesis. It also identifies controversies that need to be resolved. Unraveling the contributions to tumorigenesis of DNA damage from endogenous and exogenous sources represents a major challenge for the future.

/pdf/oxyradicals-and-dna-damage-1ctpd5dr95.pdf

Oxyradicals and DNA damage

Increasing interest in the health benefits of tea has led to the inclusion of tea extracts in dietary supplements and functional foods. However, epidemiologic evidence regarding the effects of tea consumption on cancer and cardiovascular disease risk is conflicting. While tea contains a number of bioactive chemicals, it is particularly rich in catechins, of which epigallocatechin gallate (EGCG) is the most abundant. Catechins and their derivatives are thought to contribute to the beneficial effects ascribed to tea. Tea catechins and polyphenols are effective scavengers of reactive oxygen species in vitro and may also function indirectly as antioxidants through their effects on transcription factors and enzyme activities. The fact that catechins are rapidly and extensively metabolized emphasizes the importance of demonstrating their antioxidant activity in vivo. In humans, modest transient increases in plasma antioxidant capacity have been demonstrated following the consumption of tea and green tea catechins. The effects of tea and green tea catechins on biomarkers of oxidative stress, especially oxidative DNA damage, appear very promising in animal models, but data on biomarkers of in vivo oxidative stress in humans are limited. Larger human studies examining the effects of tea and tea catechin intake on biomarkers of oxidative damage to lipids, proteins, and DNA are needed.

Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions.

Free radicals are ubiquitous in our body and are generated by normal physiological processes, including aerobic metabolism and inflammatory responses, to eliminate invading pathogenic microorganisms. Because free radicals can also inflict cellular damage, several defences have evolved both to protect our cells from radicals--such as antioxidant scavengers and enzymes--and to repair DNA damage. Understanding the association between chronic inflammation and cancer provides insights into the molecular mechanisms involved. In particular, we highlight the interaction between nitric oxide and p53 as a crucial pathway in inflammatory-mediated carcinogenesis.

Radical causes of cancer

Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies

Exposure to their precursors (e.g., amines, nitrate/nitrite, NOx) can lead to formation in the human body of N-nitroso compounds (NOC), a class of potent animal carcinogens, which are also suspected of being carcinogenic in man. A non-invasive method, the ‘N-nitrosoproline (NPRO) test’, for estimating endogenous nitrosation in man was developed in our laboratory. This test, which monitors 24-hr-excretion of urinary N-nitrosamino acids. is now applied in clinical and field studies, with the aim of measuring nitrosamine exposure and of identifying dietary, lifestyle, and host factors, or disease states, that affect nitrosation in man. Results from such studies are used to identify populations/individuals at high risk for cancers of the stomach, oesophagus, and oral cavity possibly caused by endogenous nitrosamines, and to indicate preventive measures by which the body burden of endogenous nitroso carcinogens can be lowered efficiently.

Jagadeesan Nair

Papers

Urinary N-nitrosamino acids as indices of endogenous formation of N-nitroso compounds.