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Mechanisms of Pro- and Antioxidation1

November 27, 2004

EXPANDED ABSTRACT

KEY WORDS: * antioxidants * prooxidants * dietary fat

Prooxidants often are considered synonymous with reactive species, toxic substances that can cause oxidative damage to major constituents of biological systems. In contradistinction, antioxidants are defined as any substance that, when present at low concentrations compared with that of the oxidizable substrate, significantly prevents the prooxidant-initiated oxidation of that substrate. In this context, and based on the potential of free radical reactions contributing to the degradation of biological systems, it was suggested that it might be possible to reduce the extent of damage caused by free radicals through 3 dietary changes: i) reducing energy intake (i.e., lowering the level of free radical reactions arising in the course of normal metabolism), ii) minimizing dietary components that increase the level of free radical reactions (e.g., polyunsaturated fats), and iii) supplementing the diet with one or more free radical-reaction inhibitors (antioxidants) (1).

Step i, lowering energy intake, was intended to diminish excess levels of harmful radicals resulting from electron loss at specific dehydrogenase reactions and the electron transport chain. The rationale rests on the assumption that the more metabolic fuels are stoked, the greater the electron loss, resulting in greater free radical formation. Support for this rationale is found in Tannenbaum’s 1940 studies, which concluded, “Persons of average weight, or less, were less likely to develop cancer as those who are overweight” (2). These observations were reaffirmed some 63 y later in a large prospective study: “Increased body weight was associated with increased death rates for all cancers combined and for cancers at multiple sites” (3).

With regard to Steps ii and iii, lipid peroxidation exemplifies the type of chain reaction initiated by free radicals. Antioxidants can terminate such reactions. Both the phenolic antioxidant BHT and the carotenoid β-carotene affect UV carcinogenesis. However, there is a lack of correlation between physicochemical and pathophysiological responses in both instances. The bimolecular rate constant for the reaction of BHT with model peroxyl radicals is low compared with that of β-carotene. Moreover, both can efficiently inhibit lipid peroxidation in biological membranes. Indeed, the influence on UV carcinogenesis of both antioxidants is reported to diminish as the level of dietary fat decreases, pointing to the involvement of lipid peroxidative reactions. Nevertheless, although increased dietary levels of (n-6) fatty acids exacerbate UV carcinogenesis, reflected in a shortened tumor latent period and increased tumor multiplicity, (n-3) fatty acids significantly inhibit UV carcinogenesis (4,5). Both (n-6) and (n-3) fatty acids are polyunsaturated, compelling one to explore alternative mechanisms to lipid peroxidation as an explanation for both dietary lipid-modulated UV carcinogenesis and its inhibition by BHT.

Aside from membrane lipid, other in vivo targets include strand scission of membrane proteins and cross-linking. Of particular importance is disulfide cross-linking resulting from oxidation of sulfhydryl groups. When stratum corneum from BHT-treated and control animals was isolated and tested for spectral transmission, the transmission of wavelengths from 280 to 320 nm (wavelengths most effective in carcinogenic induction) was ~65% greater in samples from the control group compared with the group fed BHT (6). The protective effect exhibited by the stratum corneum in animals fed BHT could not be attributed to BHT-induced alteration of physical dimensions, because neither the number of layers nor the thickness of the stratum corneum differed from controls. It was proposed that BHT elicited its protective response by virtue of its antioxidant properties. During the normal maturation (oxidation) of the stratum corneum, which is composed principally of keratin, sulfhydryl moeties are protected from oxidation by BHT, resulting in fewer disulfide cross-links. Certainly, the degree of cross-linking has long been known to alter the X-ray diffraction patterns of keratin, and it is expected that optical properties are altered as well. Thus, the mode of action of BHT in inhibiting UV carcinogenesis appears to be related to UV dose diminution resulting from an increased spectral absorbance of the stratum corneum. Hence, we have an antioxidant producing chemical changes in a nonliving tissue with potentially important effects on the incidence of skin cancer.

β-Carotene, conversely, has no such effect on epidermal transmission, and, although its initially reported anticarcinogenic potential was based on the carotenoid’s specific capacity to quench singlet oxygen, scavenge oxy-radicals, and terminate free radical reactions, it actually exacerbates UV carcinogenesis under certain dietary conditions (7,8). β-Carotene also may act as a prooxidant at high oxygen pressures and under oxidative stress conditions. Many strongly oxidizing species, especially peroxyl radicals, convert the carotenoid to the 1-electron oxidized form, i.e., the β-carotene radical cation (9). This radical cation exhibits a reduction potential of about 1000 mV and itself represents a strong oxidizing agent (10). Furthermore, depending on the microenvironment, it has a rather long lifetime, suggesting that if left unrepaired, the radical cation could inflict considerable damage on biological membranes. Formation of the radical cation could help explain the procarcinogenic action of supplemental β- carotene, with respect to both UV carcinogenesis in mice and increased occurrence of lung cancer among smokers (11). Based on the relative electron transfer rate constants for interaction between β-carotene, α-tocopherol (vitamin E) and ascorbic acid (vitamin C), a mechanism was proposed by which β-carotene could react with both vitamins E and C to quench radical reactions (12) (Fig. 1).

However, either removing or increasing dietary vitamin C in β-carotene-supplemented semisynthetic diets did not affect the carotenoid’s exacerbation of UV carcinogenesis (13). Reducing the level of vitamin E in β-carotene-supplemented semisynthetic diets further exacerbatated UV carcinogenesis, suggesting a vitamin E and β-carotene interaction. Because β-carotene has no exacerbative effect when it is added to closed-formula rat diets, the modulation of carcinogenesis must be dependent on the interaction of other dietary factors that are either absent or present in ineffectual concentrations in the semidefined diet. Those factors could be other carotenoids, their isomers, or some yet unidentified phytochemical(s). Although our data do not support the proposed mechanism involving vitamin C repair of the carotenoid radical cation, such a mechanism, to the point of repair, could explain the procarcinogenic characteristic of β-carotene. Indeed, the mechanism might be applicable in humans who have a nutritional requirement for vitamin C; for example, vitamin C levels are notoriously low in smokers.

FIGURE 1 Proposed schema for the interactions of vitamins E, vitamin C, and β-carotene in quenching radical reactions, based on relative electron rate constants. α-Tocopherol first intercepts an oxyradical and forms the tocopherol radical cation, which, in turn, is repaired by β-carotene to form the β- carotene radical cation. This radical is repaired by ascorbic acid. Low plasma vitamin C, in the case of smokers, might limit normal repair of the carotenoid radical cation (upper arrow). In the case of mice, which have no dietary requirement for vitamin C, with excess levels under conditions of oxidative stress (e.g., UV), oxidized (dehydro or ascorbate radical) vitamin C could act as a prooxidant and stoichiometrically inhibit the repair mechanism (lower arrow).

In summary, the difficulties associated with antioxidant supplementation are exemplified by the different anti- or prooxidant reactions and anti- or procarcinogenic responses observed under physicochemical and physiological conditions, respectively. This illustrates some of the complexities that must be considered when supplementing the diet with one or more antioxidants and presents a paradox reflected in the complex relation between chemical mechanisms and biological modes of action of antioxidants. Recent clinical and experimental data suggest that antioxidant supplementation of the complex and intricately balanced natural antioxidant defense system as a cancer prevention strategy will demand extreme caution.

1 Presented as part of the conference “Free Radicals: The Pros and Cons of Antioxidants,” held June 26-27 in Bethesda, MD. This conference was sponsored by the Division of Cancer Prevention (DCP) and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Department of Health and Human Services (DHHS); the National Center for Complementary and Alternative Medicine (NCCAM), NIH, DHHS; the Office of Dietary Supplements (ODS), NIH, DHHS; the American Society for Nutritional Science; and the American Institute for Cancer Research and supported by the DCP, NCCAM, and ODS. Guest editors for the supplement pu\blication were Harold E. Seifried, National Cancer Institute, NIH; Barbara Sorkin, NCCAM, NIH; and Rebecca Costello, ODS, NIH.

LITERATURE CITED

1. Harman, D. (1962) Role of free radicals in mutation, cancer, ageing and maintenance of life. Radiation Res. 16: 753-763.

2. Tannenbaum, A. (1940) Relationship of body weight to cancer incidence. Arch. Pathol. 30: 509-517.

3. Calle, E. E., Rodriguez, C., Walker-Thurmond, K. & Thun, M. J. (2003) Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N. Engl. J. Med. 348: 1625-1638.

4. Orengo, I. F., Black, H. S., Kettler, A. H. & Wolf, J. E., Jr. (1989) Influence of dietary menhaden oil upon carcinogenesis and various cutaneous responses to ultraviolet radiation. Photochem. Photobiol. 49: 71-77.

5. Black, H. S., Thornby, J. L, Gerguis, J. & Lenger, W. (1992) Influence of dietary omega-6, -3 fatty acid sources on the initiation and promotion stages of photocarcinogenesis. Photochem. Photobiol. 56: 195-199.

6. Koone, M. D. & Black, H. S. (1986) A mode of action for butylated hydroxytoluene-mediated photoprotection. J. Invest. Dermatol. 87: 343-347.

7. Black, H. S. (1998) Radical interception by carotenoids and effects on UV carcinogenesis. Nutr. Cancer 31: 212-217.

8. Black, H. S., Okotie-Eboh, G. & Gerguis, J. (2000) Diet potentiates the UV-carcinogenic response to beta-carotene. Nutr. Cancer 37: 173-178.

9. Tinkler, J. H., Tavender, S. M., Parker, A. W., McGarvey, D. J., Mulroy, L. & Truscott, T. G. (1996) Investigation of carotenoid radical cations and triplet states by laser flash photolysis and time-resolved resonance Raman spectroscopy: observation of competitive energy and electron transfer. J. Am. Chem. Soc. 118: 1756-1761.

10. Edge, R. & Truscott, T. G. (2000) The carotenoids-free radical interactions. Spectrum 3: 12-20.

11. The α-Tocopherol, β-Carotene Cancer Prevention Study Group (1996) The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330: 1029-1035.

12. Edge, R., Land, E. J., McGarvey, D., Mulroy, L. & Truscott, T. G. (1998) Relative one electron reduction potentials of carotenoid radical cations and the interactions of carotenoids with the vitamin E radical cation. J. Am. Chem. Soc. 120: 4087-4090.

13. Black, H. S. & Gerguis, J. (2003) Modulation of dietary vitamins E and C fails to ameliorate β-carotene exacerbation of UV carcinogenesis in mice. J. Nutr. Cancer 45: 36-45.

Homer S. Black2

Department of Dermatology, Baylor College of Medicine, Houston, TX 77030

2 To whom correspondence should be addressed. E-mail: hblack@bcm.tmc.edu.

Copyright American Institute of Nutrition Nov 2004




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