0307-4412(94)00097-2

DIETARY ANTIOXIDANTS PROTECTION AGAINST OXIDATIVE STRESS

SHAHAB UDDIN and SARFRAZ AHMAD

D e p a r t m e n t s of Med ic ine and Phys io logy L o y o l a Unive r s i ty o f Chicago M a y w o o d , Ill inois 60153, U S A

Introduction Several lines of evidence indicate that the active oxygen-induced and free radical- mediated oxidation of biological molecules, membranes and tissues are closely related to a variety of pathological events. 1 Diverse biological processes such as inflammation, carcinogenesis, aging, stroke and photobiological effects, appears to involve reactive oxygen species. Oxidative metabolism, which is a normal biological process associated with a variety of metabolic activities in aerobes, is also capable of generating highly reactive oxygen flee radicals. 2 These active oxygen species include the superoxide anion radical, hydrogen peroxide, the hydroxyl radical, and singlet oxygen. Oxidative damage inflicted by these active free radicals is referred to as oxidative stress. Some of the major molecular targets of these agents are DNA, 3 proteins, 4 carbohydrates and lipids. 5

Transition metal ions are important in the production of radical species. The ability of these ions to move electrons is the basis for the formation and propagation of many of the most toxic radical reactions. For example, superoxide anion is relatively nonreactive in aqueous solution, but in the presence of hydrogen peroxide and a transition metal such as iron, the extremely reactive hydroxyl radical may be generated. This pathway, known as the iron-catalyzed Haber-Weiss reaction as represented by following reactions, is a Fenton chemistry and has been extensively studied. Although its role in pathology is not well established, the extensive measures taken by cells to minimize the presence of free metal ions such as iron and copper (ie the presence of iron- and copper-binding proteins) indirectly indicate that such reactions are detrimental to biological systems.

Fe 3+ 0 2 - - . . . . . . . . . > Fe 2+ + 02 (1)

Fe 2+ + H 2 0 2 . . . . . . . . . . > OH- + O H - (2)

Combining 1 and 2:

Fe 3+ + 02- + H 2 0 2 . . . . . . . . . . > 0 2 + OH" + O H - (3)

Most of the compounds which are antioxidant, also behave as pro-oxidant. The mechanisms of their pro- and antioxidant properties are not very well understood but it is conceivable that their molecular structures allow them to undergo autoxidation in the presence of oxygen and transition metal ions. This autoxidation would generate reactive oxygen species, which might be responsible for their pro-oxidation behavior. Their molecular structures would also allow them to react with oxygen free radicals, produced by other compounds, providing them with radical-trapping properties. This mechanism would account for their antioxidant behavior. Thus, these molecules have the potential of acting as both pro- and antioxidants, depending on the redox state of their biological environment. In the cellular environment, these two opposing effects may be competitive and each of these compounds may have dual role in mutagenesis and carcinogenesis. Such a dual role of antioxidants in the modification of chemical carcinogenesis has been reported. 6

As organisms evolved to use oxygen in energy metabolism, mechanisms also developed to minimized the generation of random free radical oxidation. First among these was the compartmentation of oxidative metabolism in mitochondria. Molecular oxygen and reactive free radical species are also tightly bound to specific enzyme classes such as the cytochrome system during oxidative phosphorylation. In order to limit free radical formation, transition metals such as copper and iron, which can catalyze free radical formation, are tightly bound to transport and storage proteins.

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Several enzyme systems exist within the cell for the neutralization and thus control of free radical formation. Superoxide dismutase catalyzes the transformation of superoxide. radicals. Catalase and glutathione transferase neutralize hydrogen peroxide and fatty acid radicals. Such deleterious reactions are also controlled by non-enzymatic antioxidants which eliminate pro-oxidants and scavenge free radicals, and the term 'antioxidant' is often used in a restricted sense to denote this activity. A broader definition of an antioxidant is any substance that when present at low concentration compared to those of its oxidizable substrate, significantly delays or inhibits the oxidation of that substrate. 7 Antioxidants have received much attention recently and the function and synthesis of the naturally occurring as well as the synthetic antioxidant drugs have been the subject of extensive studies. The present article reviews some aspects of antioxidants of biological significance.

Dietary carotenoids as cellular antioxidants

[3-Carotene is the essential precursor of retinol or vitamin A (Fig 1). Carotenoids are widely distributed in nature where they play important role(s) in the protection of cells and organisms from oxidative damage. Due to their highly conjugated double-bond system, carotenoids are extremely efficient quenchers of singlet oxygen. It has been proposed that dietary B-carotene in combination with other antioxidants protects against lipoprotein oxidation and thus may play a potentially important role in retarding the progression of atherosclerosis. Oxidized low density lipoproteins have been associated with atherosclerosis, and antioxidants that reduce the rates of degradation of low density lipoproteins also prevent the progression of atherosclerosis in rabbits. 8 Dietary [3-carotene is reported to afford protection against the progression of cortical, nuclear and mixed cataract of the lens. 9 As the ocular lens is physiologically damaged by oxyradicals, the role of carotene is thought to exert its protective effect by countering the effects of these active oxygen species thereby preserving membrane integrity. Thus, [3-carotene may be useful for prophylaxis or therapy against cataract formation.

The best studied association of carotenoids and disease has been that of carotenoids and cancer. Dietary [3-carotene is associated with the decreased risk of several neoplasms, including cervical cancer. However, the most consistent association is that of decreased dietary [3-carotene with lung cancer, particularly squamous cell cancer. Several studies indicate that carotenoids protect against neoplasia in animals. [3-Carotene protects against UV- and chemically-induced carcinogenesis and against tumor implants in a variety of regimens in mice, rats and hamsters. ~0 In addition, carotenes inhibited the proliferation of a human neuroblastoma cell line and suppressed the level of N-myc messenger RNA.10

Vitamin E or tocopherols In recent years, a general role of tocopherol (Fig 1) as an antioxidant in human plasma has been recognized. ~t The vitamin E content of membranes often controls the susceptibility of microsomal membranes, low density lipoproteins, hepatocytes, or whole organs to damage by peroxidizing agents such as hydroxyl radicals, aloxyl radicals, peroxyl radicals, singlet oxygen and perhaps a number of oxygen metal complex. These agents not only damage the lipids but produce as secondary intermediates, lipid hydroperoxides, which

CH3

(~H 3 .CH3 C['I 3 ~.

#-CAROTENE

R CHCH2OH

H H 3 HO CH3

TOCOPHEROL ASCORBATE

Figure 1 Structures of [3-carotene, tocopherol and ascorbate

BIOCHEMICAL EDUCATION 23(1) 1995

can decompose into aloxyl and organic peroxyl radicals, and thus initiate the chain reaction of lipid peroxidation. Tocopherois protect lipids by scavenging peroxyl radicals without reacting in further chain-propagation steps. Vitamin E has been suggested to act as an antioxidant defense agent for the eye lens. It was shown that lens lipid peroxidation in vitro, the result of photochemical insult, was decreased by vitamin E. Oxidative stress has also been suggested to be a mechanism of cigarette smoke toxicity, an association proposed from the results of several studies of vitamins E and C, and pathological effects of cigarette smoking. Patch et a112 found little vitamin E in bronchoalveolar lavage from cigarette smokers compared to non-smokers, and concluded that the lung lining fluid was deficient in this compound in smokers. It has been reported that chronic cigarette smoking lowered the serum vitamin C content in smokers.13

Ascorbic acid or vitamin C

Ascorbic acid or vitamin C (Fig 1) is essential for the protection of humans against scurvy. The ascorbic acid activity of vitamin C lies in its role as an essential cofactor in hydroxylation reactions that are involved in the biosynthesis of stable cross linked collagen. 14 This and other metabolic functions of ascorbic acid are derived from its strong reducing potential. The same reducing property makes vitamin C an excellent antioxidant, capable of scavenging a wide variety of different oxidants. For example, ascorbic acid has been shown to scavenge superoxide, hydrogen peroxide, hydroxyl radical, aqueous peroxyl radicals and singlet oxygen. ~5 Ascorbate also protects against endogenous oxidative DNA damage in human serum. Epidemiological studies show a correlation between low dietary levels of vitamin C (blood concentration) and ischemic heart disease and cancer. 16 Much of the evidence for a possible role of vitamin C as a protective agent against cardiovascular disease and cancer suggests that the incidence of these conditions is lower in populations that have an abundant intake of leafy green vegetable or fruit. 17 Ascorbic acid in the diet is reported to act as an anticarcinogenic agent in rodents treated with either UV radiation, benzo[a]pyrene or nitrite.18 Ascorbate is considered to be the most important antioxidant in extracellular fluids and many cellular activities of antioxidants are associated with the actions of this important vitamin.

Antioxidant thiols Thiol groups act as intracellular antioxidant by scavenging free radicals and through enzymatic reactions. Glutathione (Fig 2) is often the first line of defence against tissue injury from the administered or metabolically generated electrophiles. The critical role of the tripeptide is to protect tissue from the reactive xenobiotic metabolites, has stimulated considerable interest in underlying the biochemical mechanisms. Alkylation of electro-

H2N~HCH2CH2CON'HC~HCONHCH2COOH

COOH CH2SH

GLUTATHIONE

Figure 2 Glutathione is c-h-glutamyl-L-cysteinyl-L-glycine

philic metabolites or reduction of reactive oxygen species are considered to be the major protective mechanisms of glutathione.19 Glutathione is the most important cellular thiol which acts as a substrate for several transferases, peroxides and other enzymes that prevent and/or mitigate the deleterious effects of oxygen free radicals. The protection is an interesting aspect of its function since this water-soluble thiol prevents damage in a lipid environment. This protection is enzymatically mediated and also dependent on vitamin E which has been attributed to oxidant neutralizing, lipid peroxidase and/or tocopherol- radical generating activities. Depending on the experimental conditions, dihydrolipoate and other thiols can also protect membranes non-enzymatically by preventing lipid peroxidation and by sparing tocopherol. This suggests that the thiols associated with the membrane proteins may also have an antioxidant role, and that the protection of protein thiols by glutathione would be considered along with the prevention of peroxidation and sparing the tocopherols. 15 The glutathione concentration may be influenced by the dietary sulfur amino acids. Glutathione levels are changed more efficiently by L-2-oxothiazol- idine-4-carboxylate, which is an effective antagonist of acetaminophen-caused liver damage. Acetaminophen is thought to be toxic through radical and quinone oxidizing metabolites. Dietary glutathione may be an effective anticarcinogen against aflatoxin. TM

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Antioxidant elements

Flavonoids

Synthetic anticarcinogen

The role of the antioxidant minerals in the etiology of human cancer has been reviewed. 21 Evidence exists for the involvement of low levels of dietary intake of manganese, copper or zinc as risk factor in this regard. However, selenium is emerging as a dietary factor that might prove to be of major significance as prophylactic agent against cancer. Selenium is also present in the active site of glutathione peroxidase (also known as selenite) significantly inhibits the induction of mammary tumor by viruses. Selenium also inhibits the transformation of mouse mammary cells. 22 It has also been proposed that selenium involves in the liver mixed function oxidase system that is responsible for the metabolism of chemical carcinogens. The contention that the protective effect of selenium is mediated through glutathione peroxidase are achieved at the levels of selenium considerably lower than those needed to prevent cancer, and the proposal that the high levels of selenium are necessary because the element is toxic to rapidly proliferate the tumor cells. 23

Flavonoids are a group fo naturally occurring benzopyrene derivatives with low molecular weights (Fig 3) and are ubiquitous in photosynthesizing cells. They are widely distributed in plants kingdom, of course, including herbs used to folk medicines in China for centuries. Flavonoids have been reported by many authors to have anti-infammatory, antiallergic, antivira124 properties, inhibiting DNA synthesis in tumor cells and inhibiting tumor promotion. 25 Many flavonoids are also found to be the strong free radical scavengers and antioxidants. 26 It is known tht excessive free radical production and lipid peroxidation in vivo may cause many kinds of disease. The superoxide anion scavenging activity and antioxidation of several flavonoids have been recently reported. Morin and hispidulin have very strong inhibition of lipid peroxidation while their scavenging activity is very weak. On the other hand, naringin, as it has strong scavenging activity, does not inhibit lipid peroxidation at all. Thus, the scavenging of superoxide anions is not the only mechanism for the inhibition of lipid peroxidation. Some flavonoids, such as quercetin, are known as hydroxyl scavengers 26 and metal ion chelators. These properties of flavonoids may also contribute to their inhibition of lipid peroxidation.

The synthetic antioxidant drugs are the subject of extensive studies. There are several such type of chemicals which exert scavenging effect on reactive species carcinogenesis. Most extensive work in this direction has been done with the phenolic antioxidants. Protection against chemical carcinogenesis is probably the most challenging experimental finding obtained with phenolic antioxidants. Protection has most often been localized in the initiation phase. Post-initiation protection does, however, also occur as evident by a number of genuine post-treatment experiments. 27 Extensive work has been done on the

OH (a) (b) ~ O H

HO.~ A .0,. A ~ ~ R1 H O a X Y..oX X v . , ,

T 11 T -o. ..R 3 0 OH 0

(c) OH

OH 0

Figure 3 Flavonoid structure. (a) shows the ring system: (b) quercetin, (c) naringenin

B I O C H E M I C A L E D U C A T I O N 23(1) 1995

6

synthetic phenolic antioxidant, butylated hydroxyanisol (BHA) and butylated hydroxy- toluene (BHT) (Figure 4). The BHA and BHT are added in food as preservatives which prevent oxygen-induced lipid peroxidation. In addition to lipid hydroperoxyl radicals, other radical species including the hydroxyl radical, can be trapped by phenolic antioxidant; a number of them interact with the superoxide anion radical) It has been demonstrated that BHT is capable of scavenging the radical formed from N-Methyl-N- nitroso guanidine (MNNG) and hydrogen peroxide. 28 The BHT also protect against MNNG-induced gastric cancer. 28 Experiments with BHT in rats have shown inhibitory effects of allopurinol-induced nephrotoxicity. 29

Ebselen (Fig 4), a synthetic 5 selenium-containing heterocycle has been found to have antioxidant activity in ADP-Fe-induced rat liver microsomes and isolated hepatocytes. 3° A protective action of ebselen against acute gastric mucosal injury induced by toxic agents, stress and ischemic reperfusion has also been reported. 3 Recent study has shown that synthetic carotenoid, polyketones and capsorubin isomers act efficient quenchers of singlet molecular oxygen. The broad spectrum of biological activity of radical-scavenging phenolic antioxidants indicates that regulation of biological process via endogenous oxidant is important. However, many of the targets of such endogenous oxidants are still to be identified.

OH

( C H 3 ) 3 C ~ / C ( C H 3 ) 3

CH 3

Figure 4 Structures o f (a) B H T and (b) ebselen

Conclusions and perspectives

References

Antioxidants are of primary value biologically by restricting the damage that reactive free radicals can do to the cell and the cellular components. Antioxidants may afford varying degrees of protection against the cell damage caused by these reactive oxygen free radicals. The antioxidant(s) is(are) much more likely to be effective against chemical carcinogens that are metabolically activated to the reactive free radical intermediate that are self propagating than in situations where the metabolic activation results in non-radical reactive intermediates such as epoxides or carbonium ions. Free radical disturbances may be of primary and major significance in some important human diseases and intoxication, and it is a general proposition that antioxidant administration and/or intake may turn out to be of substantial value in the treatment of such cases. Although, there are a number of dietary antioxidants which have been identified, and some are still in the process of developing and testing, that would help us to understand as to how the antioxidants function. This understanding should prove to be valuable more effectively in treating diseases that have as their basis of some oxidants or free radical involvements.

x Sies, H (1991) Oxidative Stress: Oxidants and Antioxidants, Academic Press, London 2Flohe, L (1982) In: Pryor, W A (editor) Free Radicals in Biology, Academic Press, New York 5, 223-254 3Uddin, S (1994) Biochem Mol Biol lnt 32, 341-347 4Wolf, S P and Dean, R T (1986) Biochem J 234, 339-403 5Mascio, P D, Murphy, M E and Sies, H (1991) Am J Clin Nutr 53, 1945-2005 6Kahl, R (1986) J Environ Sci Health C4(1), 47-92

7Halliwell, B and Gutteridge, J M C (1989) Free Radicals in Biology and Medicine, second edition, Clarendon Press, Oxford

BLue, G and Fruchart, J (1991) Am J Clin Nutr 53, 206S-209S 9Leske, M C, Chylack, L T, Jr and Wu, S U (1991) Arch Opthalmol 109, 244-251

~°Murakoshi, M, Takayasu, J, Kimura, O, Kohmura, E, Nishino, H, Iwashima, A, Okuzumi, J, Sakai, T, Sugimoto, T, Imanishi, J and Iwasaki, R (1989) J Natl Cancer lnst 81, 1649-1652

J l Packer, L (1992) Proc Soc Exp Biol Med 200, 271-276

t2Patch, E R, Daseki, H, Mohammed, J R, Cornwell, D G and Davis, W B (1989) J Clin Invest 77, 789-796 ~3Smith, J L and Hodges, R E (1987) Ann N YAcad Sci 498, 144-152

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14jaffe, G M (1986) In: Machin, L (editor), Handbook of Vitamins: Nutritional, Biochemical and Clinical Aspects, pp 199-206, Marcel Decker, New York

tSFrie, B, England, L and Ames, B N (1989) Proc Natl Acad Sci USA 86, 6377-6381

t6Gey, K F, Brubacker, G B and Sthelin, H B (1987) Am J Clin Nutr 45, 1368-1377

17Acheson, R M and Williams, D D R (1983) Lancet 1, 1191-1193

18Terraa, J and Matsushiba, S (1988) Free Radical Biol Med 4, 205-208

mSies, H (1988) ISI Atlas of Science: Biochemistry 1, 109-204 2°Novi, A M (1981) Science 212, 541-542

21Diplock, A T (1990) Med Oncol Tumor Pharmacother 7, 193-198

22Chatterjee, M and Banerjee, M R (1982) Cancer Lett 17, 187-195 23Diplock, A T (1991) Am J Clin Nutr 53, 189S-193S

24Harsteen, B (1983) Biochem Pharmacol 32, 1141-1148

25Nakamura, Y, Colburn, H N and Ginhart, T D (1985) Carcinogenesis 6, 229-235 26Husain, R, Cillard, J and Cillard, P (1987) Phytochemistry 26, 2489-2491

27Mizimoto, K, Ito, N, Kitazawa, S, Tsutsumi, M, Denda, A and Konishi, Y (1989) Carcinogenesis 10, 1491- 1494

28Mikuni, T, Tatsuta, M and Kamachi, M (1987) J Natl Cancer lnst 79, 281-283

29Ansari, N D and Rajaram, S (1992) Res Commun Chem Pathol Pharmacol 75,221-229

3°Narayanaswami, V and Sies, H (1990) Free Radical Res Commun 10, 237-244

3~ Ueda, S, Yoshikawa, T, Takahashi, S, Naito, Y, Oyamada, H, Takemura, T, Morita, Y, Tanigawa, T, Sugino, S and Kondo, M (1990) In: Emerit, I (editor), Antioxidants in Therapy and Preventive Medicine, pp 187-191, Plenum, New York

Multiple-choice Type Questions

1 Antioxidant is: (a) a substance which catalyzes the formation of free

radicals (b) a substance which neutralizes the formation of free

radicals (c) an oxygen free radical (d) none of the above

2 The antioxidant properties of a substance depends on: (a) The molecular structure of the substance (b) The redox state of the surrounding environment (c) Presence of oxygen and transition metal ions (d) All of the above

3 The free radical causes damage to: (a) Lipids and carbohydrates (b) Proteins (c) DNA (d) All of the above

4 Dietary carotenoids are efficient quenchers of singlet oxygen:

(a) Due to their wide distribution in nature (b) Due to their highly conjugated double-bond system (c) Due to the presence of methyl group in their

structure , (d) None of the above

5 Vitamin E protect lipids by: (a) Scavenging peroxyl radical (b) Chelating metal ions (c) Reducing molecular oxygen (d) All of the above

Selenium is present: (a) In circulating blood (b) At the active site of glutathione peroxidase (c) In mitochondria (d) In the intestine

The glutathione level depends on: (a) Dietary sulphur (b) Blood oxygen level (c) Transition metal ions (d) Dietary carotenoids

8 Which of the following oxidant?

(a) Its oxidizing potential (b) Its reducing potential (c) (a) & (b) (c) None of the above

makes vitamin C an anti-

The flavonoid quercetin is: (a) Widely distributed in plant kingdom (b) A hydroxyl radical scavenger (c) A metal ion chelator (d) All of the above

10 Ebselen and BHT are: (a) Synthetic anticarcinogens (b) Naturally occurring compounds (c) Only found in plants (d) None of the above

Answers: 1 (b) 2 (d) 3 (d) 4 (b) 5 (a) 6 (b) 7 (a) 8 (b) 9 (d) 10 (a)

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