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Home , Lipid peroxidation, Oxidative stress, Radical (chemistry)

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What Is Oxidative Stress?

JMAJ 45(7): 271–276, 2002

Toshikazu YOSHIKAWA* and Yuji NAITO**

Professor* and Associate Professor**, First Department of Medicine, Kyoto Prefectural University of Medicine

Abstract:Oxidative stress is well known to be involved in the pathogenesis of lifestyle-related diseases, including , hypertension, mellitus, ischemic diseases, and malignancies. Oxidative stress has been defined as harm- ful because free radicals attack biological such as , pro- teins, and DNA. However, oxidative stress also has a useful role in physiologic adaptation and in the regulation of intracellular . Therefore, a more useful definition of oxidative stress may be “a state where oxidative forces exceed the systems due to loss of the balance between them.” The biomarkers that can be used to assess oxidative stress in vivo have been attracting interest because the accurate measurement of such stress is necessary for inves- tigation of its role in lifestyle diseases as well as to evaluate the efficacy of treat- ment. Many markers of oxidative stress have been proposed, including hydro- peroxides, 4-hydroxynonenal, isoprostan, 8-hydroxyguanine, and ubiquinol-10. To prevent the development of lifestyle diseases, advice on how to a healthy life should be given to individuals based on the levels of oxidant and antioxidant activity assessed by pertinent biomarkers. Individual genetic information should also be taken into consideration. Key words:Oxidative stress; Free radicals; Active oxygen; Biomarkers

Introduction and oxidative DNA damage, but also physi- ologic adaptation phenomena and regulation The close association between oxidative of intracellular signal transduction. From a stress and lifestyle-related diseases has become clinical standpoint, if biomarkers that reflect well known. Oxidative stress is defined as a the extent of oxidative stress were available, “state in which oxidation exceeds the antioxi- such markers would be useful for physicians to dant systems in the body secondary to a loss of gain an insight into the pathological features of the balance between them.” It not only causes various diseases and assess the efficacy of hazardous events such as drugs.

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 124, No. 11, 2000, pages 1549–1553).

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Table 1 Major Active Oxygen Species true that the high reactivity of these oxygen metabolites is utilized to control various bio- מO ⅐ 2 logical phenomena. H2O2 HO⅐ From a biological viewpoint, various oxygen- 1 O2 derived free radicals have been attracting HOO⅐ radical attention for the following reasons: Various LOOH Alkylhydroperoxide ⅐ active oxygen species are generated in the LOO Alkylperoxyl radical body during the process of utilizing of oxygen. LO⅐ Alkoxyl radical Hypochlorite Because the body is furnished with elaborate מCIO O Ferryl ion mechanisms to remove active oxygen speciesםFe4 O Periferryl ion and free radicals, these by-products of oxygenםFe5 NO⅐ are not necessarily a threat to the body under physiological conditions. However, if active oxygen species or free radicals are generated excessively or at abnormal sites, the Free Radicals, Active Oxygen balance between formation and removal is lost, Species, and Oxidative Stress resulting in oxidative stress. Consequently, active oxygen species and free radicals can Usually, an is composed of a central attack molecules in biological membranes and nucleus with pairs of electrons orbiting around tissues, thus inducing various diseases. In other it. However, some and molecules have words, oxidative stress is defined as a “state unpaired electrons and these are called free harmful to the body, which arises when oxida- radicals. Free radicals are usually unstable and tive reactions exceed antioxidant reactions highly reactive because the unpaired electrons because the balance between them has been tend to form pairs with other electrons. An lost.” oxygen (O2) undergoes four-electron However, oxidative stress is actually useful reduction when it is metabolized in vivo. Dur- in some instances. For example, oxidative stress ing this process, reactive oxygen metabolites induces to prepare the birth canal for are generated by the excitation of electrons delivery. Also, biological defense mechanisms secondary to addition of energy or interaction are strengthened by oxidative stress during with transition elements. The reactive oxygen appropriate physical exercise and . metabolites thus produced are more highly Therefore, a more useful definition of oxidative reactive than the original oxygen molecule and stress may be a “state where oxidation exceeds are called active oxygen species. Superoxide, the antioxidant systems because the balance hydrogen peroxide, hydroxyl radicals, and between them has been lost.” singlet oxygen are active oxygen species in the narrow sense. Active oxygen species in a broad Biomarkers of Oxidative Stress sense are listed in Table 1. Only active oxygen species having an , indicated The biomarkers that can be used to assess with a dot above and to the right of the chemi- oxidative stress have been attracting interest cal formula in the table, are free radicals. because the accurate assessment of such stress For aerobic organisms, a mechanism to is necessary for investigation of various patho- remove these highly reactive active oxygen logical conditions, as well as to evaluate the species is essential to sustain life. Therefore, efficacy of drugs. Assessment of the extent of various antioxidant defense mechanisms have oxidative stress using biomarkers is interesting developed in the process of evolution. It is also from a clinical standpoint. The markers found

272 JMAJ, July 2002—Vol. 45, No. 7 OXIDATIVE STRESS

O2 á oxygen radical LOO been the most frequently used marker of oxi- á á IH dative stress partly because lipid peroxidation LH L LOO stable products (Fig. 1) is a very important mechanism of cell membrane destruction. Lipid peroxidation is a chain reaction by which unsaturated fatty acids LOOH LH (cell membrane components) are oxidized in various pathological conditions. membrane injury When a hydrogen atom is removed from a cellular injury molecule for some reason, the free tissue injury radical chain reaction proceeds as shown in Fig. 1 The chain reaction causing lipid peroxidation Fig. 1. Thus, radicals that can be involved in the extraction of hydrogen atoms from lipids include the hydroxyl radical (HO⅐), the hydro- peroxyl radical (HOO⅐), the lipid peroxyl radi- in blood, urine, and other biological fluids may cal (LOO⅐), and the alkoxyl radical (LO⅐). provide information of diagnostic value, but it Metal-oxygen complexes, particularly - would be ideal if organs and tissues suffering oxygen complexes, are also important in vivo. from oxidative stress could be imaged in a man- The peroxidation chain reaction propagates ner similar to CT scanning and MR imaging. In itself once it has started. The process by which recent years, attempts have been made to use lipid radicals (L⅐) are generated from lipids electron techniques for this pur- (LH) is called the chain initiation reaction. pose, but it will take time before such methods Lipid radicals (L⅐) thus generated react imme- can be applied to . diately with oxygen, resulting in the formation Because the body is not necessarily fully of LOO⅐, which attacks another lipid and protected against oxidative damage, some of removes a hydrogen atom from it, resulting in its constituents may be injured by free radicals, the formation of lipid (lipid per- and the resultant oxidative products have oxide; LOOH) and another L⅐. This new L⅐ also usually been used as markers. Many markers reacts with oxygen and forms LOO⅐, which have been proposed, including lipid peroxides, attacks another lipid to generate lipid peroxide, , and 4-hydroxynonenal as so lipid peroxide accumulates as the chain reac- markers for oxidative damage to lipids; iso- tion proceeds. prostan as a product of the free radical oxi- Gastric mucosal injury occurs in patients dation of ; 8-oxoguanine with extensive burns. Before the development (8-hydroxyguanine) and thymineglycol as indi- of mucosal lesions, the blood level of - cators of oxidative damage to DNA; and vari- derived substances that react with thiobarbi- ous products of the oxidation of and turic acid shows an increase. Then these sub- amino acids including carbonyl protein, stances also increase in the gastric mucosa, hydroxyleucine, hydrovaline, and nitrotyro- leading to the development of mucosal lesions. sine. Lipid peroxide was assessed in clinical The free radical peroxidation of lipids is an samples even in relatively early studies, and important factor in local injury to cell mem- the analytical methods for this substance have branes and impairment of the activity of improved. and receptors bound to the mem- The famous method of Yagi, which measures brane, and the lipid peroxide thus produced substances that react with thiobarbituric acid, can affect even remote organs. has been widely used in both clinical and Among the agents that protect the body experimental studies. Such substances have from lipid peroxidation, is consid-

JMAJ, July 2002—Vol. 45, No. 7 273 T. YOSHIKAWA and Y. NAITO

0.8 40 quently, vitamin E levels seem unlikely [םwithout Cu2] CoQH2-10 to be a useful biomarker of oxidative stress. In 0.6 30

M) addition, vitamin E is lipid soluble, so its blood

Ȑ VE 0.4 20 level varies depending on the lipid content. M) Ȑ When plasma is incubated at 37°C VC 0.2 10 in air, the concentrations of and PC-OOH CE-OOH lipid peroxides change as shown in Fig. 2. Of 0.0 0 the three antioxidants, decreases 0.8 40 first, followed by reduced coenzyme Q-10 ם2 CoQH2-10 [with 5ȐM Cu ] 0.6 30 (ubiquinol-10). This suggests that vitamin C and ubiquinol-10 are the antioxidants that are VE Vitamin C and vitamin E ( 0.4 20 most sensitive to oxidative stress. Vitamin E CE-OOH Ubiquinol-10 and lipid hydroperoxide ( may be protected by vitamin C and ubiquinol- 0.2 VC 10 10 because it is an important antioxidant. Vita- PC-OOH 0.0 0 min C and ubiquinol-10 levels were measured 01020304050 to assess oxidative stress in patients with vari- Time (hr) ous diseases. In patients with chronic

CoQH2-10; coenzymeQ10 hepatitis, liver chirrhosis, and liver , the VC; vitamin C, VE; vitamin E PC-OOH; phosphatidylcholine hydroperoxide vitamin C and ubiquinone-10 (oxidized coen- CE-OOH; cholesterylester hydroperoxide zyme Q-10) levels were significantly decreased Fig. 2 Changes of antioxidants and generation of lipid and increased, respectively, when compared peroxides during incubation of human plasma with those in the control group, with a signifi- at 37¡C in air Source: Yamamoto, Y. et al.: Oxidative Damage and cant percent increase of oxidized coenzyme Repair. ed. Davies, K.J.A., Pergamon Press, 1991; Q-10. In contrast, there was no significant dif- pp.287Ð291. ference of the vitamin E level.

Oxidative Stress as a Biological ered to be the most important. This vitamin has Modulator and as a Signal (Fig. 3) attracted attention as an antioxidant because it can scavenge lipid peroxyl radicals and hence Oxidative stress not only has a cytotoxic stop the propagation of the free radical chain effect, but also plays an important role in the reaction. The lipid peroxyl radical removes a modulation of messengers that regulate essen- hydrogen atom from the of vita- tial cell membrane functions, which are vital for min E and the molecule that has accepted the survival. It affects the intracellular status, hydrogen atom is stabilized. In turn, vitamin E leading to the activation of protein kinases, is converted into a radical, which is also stable including a series of receptor and non-receptor and less reactive. Consequently, this vitamin E- kinases, protein kinase C, and the derived radical is unlikely to attack lipids and MAP kinase cascade, and hence induces vari- perpetuate the chain reaction. Instead, it is ous cellular responses. These protein kinases thought to react with another peroxyl radical play an important role in cellular responses and thus become stable. This antioxidant reac- such as activation, proliferation, and differen- tion protects biological membranes from injury tiation, as well as various other functions. caused by free radicals and lipid peroxides. Accordingly, the protein kinases have attracted However, lipid peroxides are still generated the most attention in the investigation of in the plasma despite the presence of an the association between oxidative stress and adequate concentration of vitamin E. Conse- disease.

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Activation of protein kinases

Tyrosine kinase Src family Oxidative stress Syk/ZAP-70 family Active oxygen EGF receptors Cellular responses species Protein kinase C Activation Ischemia Proliferation Inflammation system MAP kinase cascade system Inflammatory MEK-ERK pathway reaction light SEK1-JNK pathway Stress Anticancer drugs MKK3/6-p38 pathway protection Heavy metals Activation of transcription factors AP-1 NF-␬B Nrf2

Fig. 3 Oxidative stress and cellular responses

Oxidative stress can influence many biologi- plays in the activation of NF-␬B, many new cal processes such as apoptosis, viral prolifer- findings have been obtained recently. Stimu- ation, and inflammatory reactions. In these lation with tumor factor (TNF)-␣, processes, gene transcription factors such as phorbol myristate (PMA), interleukin nuclear factor-␬B (NF-␬B) and activator (IL)-1, lipopolysaccharide, viral , and protein-1 (AP-1) act as oxidative stress sensors ultraviolet light to the generation of active through their own oxidation and reduction oxygen species, which function as a second cycling. This type of chemical modification of messenger in the activation of NF-␬B. The by oxidation and reduction is called mitochondrial respiratory chain is considered reduction-oxidation (redox) regulation. to be the major source of active oxygen species. The transcription factor NF-␬B undergoes In cells lacking mitochondria, damage caused translocation from the cytoplasm to the nucleus by TNF-␣ and NF-␬B dependent IL-6 produc- in response to an extracellular signal. This tion is suppressed. It has also been shown that translocation induces its ability to bind to DNA, antimycin A, an inhibitor of mitochondrial elec- leading to transcriptional up-regulation of the tron transport, increases the intracellular gen- expression of many genes related to inflamma- eration of active oxygen species and enhances tion and immunity. Thus, NF-␬B seems to be the activation of NF-␬B. In resting cells, NF-␬B involved in development and aggravation of is bound to I␬B and remains in the cytoplasm. many diseases. Recently, it was also suggested An extracellular signal causes the that this factor may be involved in the process of these two molecules and I␬B decomposes, of because it is located upstream whereupon NF-␬B migrates to the nucleus and to a series of transcription regulation factors activates transcription. and because it possesses the ability to suppress The phosphorylation cascade that produces apoptosis. the NF-␬B/I␬B complex has been shown to With respect to the role that oxidative stress depend on the interaction between proteins

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derived from activation of IL-1 and TNF recep- store massive amounts of genetic information tors. The activation of NB-␬B requires a signal on DNA microchips and has provided various derived from active oxygen species. The possible efficient computer programs for analysis, thus involvement of active oxygen species in the promising rapid progress in this field. release of NF-␬B is partly suggested because Many daily habits are closely associated with I␬B undergoes phosphorylation via a group of oxidative stress, which is augmented by smok- kinases involved in a phosphorylation cascade. ing, drinking, and an irregular diet. In Japan, Induction of the expression of thioredoxin by dietary habits have undergone a marked change active oxygen species is also involved in the over the years. When the energy intake related activation of NF-␬B, since thioredoxin gives to major nutrients is calculated, lipids provide NF-␬B the ability to bind to DNA in a process over 25%, reflecting this change. Many envir- that is regulated by redox reactions. onmental factors can generate active oxygen NF-␬B seems to be the key transcription species and DNA damage caused by such oxy- factor for elucidating the relationship of oxida- gen radicals is extremely serious because it may tive stress to lifestyle diseases and identifica- be related to carcinogenesis. To prevent the tion of the precise mechanisms involved may development of lifestyle diseases, instructions lead to the development of new therapies for on how to lead a healthy life should be given such diseases. individually depending on the level of antioxi- dant activity assessed by pertinent biomarkers. Conclusion Individual genetic information should also be taken into consideration when giving such The causes of lifestyle diseases can be divided instructions. Such health issues may become into three major categories, which are genetic, central to medical care in the 21st century. habitual, and environmental. Many of the genes that are associated with biological oxidative stress have been identified, with the genes for REFERENCES NO synthetase (NOS) and oxygenase 1) Yoshikawa, T.: A Guide to Free Radicals. Part (HO) being considered as candidates for such 2. Sentan Igaku Sha, Tokyo, 1998. diseases. However, lifestyle diseases are often 2) Yoshikawa, T.: Medicine of Free Radicals. Shin- multifactorial, so it is difficult to identify the dan to Chiryo Sha, Tokyo, 1997. causative factors. Recent progress in the field 3) Yoshikawa, T.: Science of Free Radicals. Kou- of molecular biology has made it possible to dan Sha Saientifikku, Tokyo, 1997.

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