Archives of Biochemistry and Biophysics Vol. 385, No. 1, January 1, pp. 20 27, 2001 doi:10.1006/abbi.2000.2149, available online at http://www.idealibrary.

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MINIREVIEW Antioxidant and Prooxidant Properties of Carotenoids

Andrew J. Young* ,1 and Gordon M. Lowe
*School of Biological and Earth Sciences and School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK

Received August 7, 2000, and in revised form September 26, 2000; published online December 7, 2000

The ability of dietary carotenoids such as -carotene and lycopene to act as antioxidants in biological systems is dependent upon a number of factors. While the structure of carotenoids, especially the conjugated double bond system, gives rise to many of the fundamental properties of these molecules, it also affects how these molecules are incorporated into biological membranes. This, in turn, alters the way these molecules interact with reactive oxygen species, so that the in vivo behavior may be quite different from that seen in solution. The effectiveness of carotenoids as antioxidants is also dependent upon their interaction with other coantioxidants, especially vitamins E and C. Carotenoids may, however, lose their effectiveness as antioxidants at high concentrations or at high partial pressures of oxygen. It is unlikely that carotenoids actually act as prooxidants in biological systems; rather they exhibit a tendency to lose their effectiveness as antioxidants. 2001 Academic Press Key Words: antioxidant; carotenoid; prooxidant; membrane; peroxyl-radical.

An antioxidant may be broadly dened as any substance that when present at low concentrations compared to those of an oxidisable substrate, signicantly delays or prevents oxidation (1). Carotenoids are regarded as effective antioxidants, but with the exception of the well documented role of carotenoids such as -carotene as quenchers of singlet oxygen ( 1O 2, see below), little evidence has been forthcoming to support the view that carotenoids actually function as antioxidants in vivo.
1 To whom correspondence should be addressed at School of Biological and Earth Sciences, Liverpool John Moores University, Byrom St., Liverpool, L3 3AF, UK. Fax: 44-151-207 3224. E-mail: a.j.young@livjm.ac.uk.

The majority of epidemiological studies consistently and clearly show that increased consumption of foods rich in -carotene is associated with a reduced risk of lung and some other cancers (2). A similar relationship has been found for levels of -carotene in the plasma (2, 3). In contrast, results from recent intervention studies (the Beta-Carotene and Retinol Efcacy Trial CARET (4) and the Alpha-Tocopherol, Beta-Carotene Cancer Prevention StudyATBC (5)) indicate that exposure of individuals taking supplemental -carotene to high-intensity (6) cigarette smoke (or those individuals suffering from asbestosis, 7) increases lung cancer incidence. A number of authors have put forward theories as to why -carotene may exhibit such procarcinogenic activity under these conditions. In particular, Mayne and colleagues (7, 8) and, more recently, Wang and Russell (9) have highlighted a number of factors that may explain the apparent prooxidant behavior of -carotene in these studies. These include the generation of relatively high amounts of deleterious oxidation products (typically epoxides) of -carotene brought about by exposure to the reactive oxygen species (ROS) 2 found in tobacco smoke (or produced as a consequence of asbestosis, 7), or by metabolic processes via increased retinoic acid catabolism or interference with retinoid signal transduction (9). In this article, we consider a number of other factors, some of which have been previously highlighted by Britton (10), which may inuence the antioxidant (or prooxidant) activities of carotenoids in biological systems, namely: (i) the structure (i.e., size, shape, and the nature, position, and number of substituent groups) and physical form (aggregated or monomeric, cis or trans conguration, etc.) of the carotenoid molecule;
2

Abbreviation used: ROS, reactive oxygen species.

0003-9861/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.

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(ii) the location or site of action of the carotenoid molecule within the cell; (iii) the potential for interaction with other carotenoids or antioxidants (especially vitamins C and E). (iv) the concentration of carotenoid; and (v) the partial pressure of oxygen. In particular, we will ask the following question: Could the antioxidant (or even apparent prooxidant) behavior of carotenoids result from factors other than by direct interaction with ROS? In answering this question, it is worthwhile reviewing the ways in which our views concerning the function of carotenoids in biological systems have altered in recent years. Until quite recently, the function of carotenoids in photosynthetic tissues, for example, was thought to be restricted to the well documented roles of energy transfer (light-harvesting) and quenching of 1 O 2. However, it is now clear that these carotenoids can actually behave in a quite different manner as well. The structure of the carotenoid molecule effectively dictates how these molecules are incorporated into and, subsequently, affect or control their local environment. Different carotenoids have been shown to exert profound effects on the organization and hence function of the pigmentprotein complexes associated with light-harvesting (1113). Observations such as these have challenged the traditional views of carotenoid function in plants, highlighting the fact that these molecules may act differently in vivo compared to free in solution.
1. CAROTENOID STRUCTURE

The properties, and therefore functions, of a carotenoid molecule are primarily dependent upon its structure and hence its chemistry. In particular, the conjugated CAC double bond system is considered to be the single most important factor in energy transfer reactions, such as those found in photosynthesis (14). It is this feature of the molecule that also permits the quenching of 1O 2. The clinical use of -carotene as an effective treatment for erythropoietic protoporphyria, in which 1O 2 is produced via sensitization of free porphyrins accumulated in the skin, is well established (15, 16). Interactions with other ROS produced in the body, including radicals such as superoxide (O 2 ) and nonradicals such as hydrogen peroxide (H 2O 2), will, however, be dependent upon other mechanisms (see below). In human plasma and tissues, a wide range of carotenoids have been identied including cyclic (e.g., -carotene, -carotene) and acyclic carotenes (e.g., lycopene, phytoene) together with a number of xanthophylls (e.g., zeaxanthin, lutein, and -cryptoxanthin), all of which can be directly derived from dietary sources (17). In addition, a number of carotenoid me-

tabolites and potential oxidation products of dietary carotenoids such as lycopene, lutein, and zeaxanthin, have been found in plasma, generally only at trace levels (17). For example, Khachik and colleagues (18) have reported the presence of 2,6-cyclolyopene-1,5-diols in plasma, and acyclo-retinol has been detected in chylomicrons (19). Although a number of these oxidation products may be produced by interaction of carotenoids with ROS under controlled conditions (20, 21), their functional signicance (if any) is not yet fully understood (see 22). The antioxidant behavior of a carotenoid molecule (whether mediated by direct or indirect means) is dependent on its structure but also the nature of the oxidizing species itself. Quenching constants for 1O 2 have been determined for a range of carotenoids in solution and reveal a clear relationship between structure of the carotenoid and the ability to quench this particular ROS by energy transfer (2325). This ability is directly related to the position of the excited state energy levels which are primarily governed by the length of the conjugated CAC chain, but may be inuenced by other structural features. Chemical quenching (as opposed to energy quenching) of 1O 2 by carotenoids can also occur, leading to the destruction (bleaching) of the carotenoid molecule (26). Both reactions require a very close interaction between the carotenoid and the 1O 2 molecules. The nature and position of substituent groups on the carotenoid molecule may also govern its antioxidant ability. The reactivity of a number of carotenoids with ROS has been examined (27, see also 10) and differences in reactivity between molecules shown to be related to differences in their electron density proles. For example, the effect of the keto-groups at C(4) and C(4) in astaxanthin and canthaxanthin (which signicantly affects their electron density proles) or hydroxy groups at C(4) and C(4) in isozeaxanthin is to reduce the reactivity of these molecules by preventing hydrogen abstraction from these positions (10, 27, 28). The preferred sites for epoxidation of astaxanthin and canthaxanthin are different from that seen with -carotene or zeaxanthin (10). Carotenoids may interact with free radicals in three main ways, namely electron transfer (Eq. [1]), hydrogen abstraction (Eq. [2]) and addition of a radical species (Eq. [3]) (10, 29). ROO ROO ROO CAR 3 ROO CAR 3 ROOH CAR 3 ROO CAR CAR CAR

[1] [2] [3]

In the human body, a range of ROS are produced, including 1O 2, OH, O 2 , and H 2O 2 (1). The mechanisms

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and rate of scavenging of free radicals by carotenoids in solution is strongly dependent upon the nature of the ROS itself (30). Carotenoids such as -carotene are very reactive to peroxyl radicals but less so to OH and O 2 (31). To date, the majority of the fundamental information regarding the antioxidant potential of carotenoids with these various ROS (especially radicals) has been gathered in vitro by challenging individual carotenoids (often simply in organic solvents, simple micelles or liposomes) with individual ROS. While such experiments serve to provide fundamental information on the interactions between carotenoid and ROS they do not represent a complex in vivo situation. In vivo, the carotenoid content and composition of the vast majority (if not all) tissues is heterogeneous. Carotenoids are also present in lower concentrations than typically used in vitro (10), and rather being free in solution they are generally associated with some form of lipoprotein complex (see below). Although carotenoid interactions have readily been demonstrated in organic solutions, it is much more difcult to demonstrate such interactions in vivo. Other properties of the carotenoid molecule are affected by its structure. The solubility of carotenoids in aqueous solutions is extremely poor, effectively restricting them to the hydrophobic regions of biological systems. However, this is greatly inuenced by carotenoid structures so that different compounds (e.g., carotenes and xanthophylls) will be incorporated quite differently into membranes (see below). Solubility is affected by the presence and nature of polar substituent groups and cis/trans isomerization (10).
2. CAROTENOIDS IN MEMBRANES

FIG. 1. A schematic representation of an articial lipid membrane containing either the dihydroxy carotenoid zeaxanthin (left) or -carotene (right). These two carotenoids orientate themselves differently within the membrane so that the polar end groups of zeaxanthin anchor the molecule across the depth of the membrane, while -carotene occupies a position deep within the hydrophobic core (see 32, 36 for reviews).

In biological systems carotenoids rarely, if ever, occur as free (monomeric) molecules in solution. Rather they are predominantly found associated (often very tightly) with protein or lipoprotein structures. Perhaps the most well-known example of this is found in the carotenoprotein complexes found in many marine invertebrates in which the carotenoid molecule (typically astaxanthin) displays a profound bathochromic shift in its absorption spectrum on binding to the protein. Thus, the immediate environment of the carotenoid molecule may have a profound effect on its properties which, in turn, affects the way that these molecules function and governs, at least in part, their interaction with other compounds. Importantly, carotenoids also directly inuence their environment (see below) so that carotenoids generally bring about a degree of stabilization and are at the same time stabilized (10). The behavior of carotenoids in both model systems and in the membranes of algae and plants provides an insight into these interactions (32). Differences in the structure of two xanthophylls, namely, violaxanthin and

zeaxanthin, means that they interact differently with thylakoid membrane lipids, bringing about changes in membrane uidity and thermostability (33). The incorporation of zeaxanthin (in place of violaxanthin) into thylakoid membranes results in a lower susceptibility to lipid peroxidation (34, 35). The orientation of a carotenoid molecule in a membrane is dependent upon its structure and on the composition of the membrane itself (36). Both resonance Raman (37) and linear dichroism (38) have demonstrated that -carotene (and other carotenes such as lycopene) lies parallel with the membrane surface, deep within the hydrophobic core. In contrast, the dihydroxy carotenoid zeaxanthin entirely spans the membrane and therefore reactions with its conjugated CAC bond system are possible throughout the depth of the membrane (Fig. 1). Polar carotenoids such as zeaxanthin act like rivets, strengthening the membrane (36) but they also limit the penetration of oxygen into membranes (39). In contrast, the carotenes behave quite differently. The inuence of different carotenoids on the properties of membranes (including effects on signal transduction) is still not fully understood. For example, information is lacking on the behavior of cisisomers or mixtures of carotenoids. Not all xanthophylls behave the same and small differences in structure dictate their behavior. For example, the two dihydroxy carotenoids zeaxanthin and lutein orientate themselves quite differently in membranes. Lutein, with the ability to rotate the -end-group freely around the C(6, 7) single bond, adopts a position parallel with the membrane surface, effectively anchoring the molecule along one side of the membrane (36). Woodall and colleagues (27, 28) demonstrated that -cryptoxanthin and zeaxanthin had greater protective ability compared to -carotene or lycopene against

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AAPH-induced oxidation (by production of peroxyl radicals in the aqueous-phase) of liposomal membranes. However, both -carotene and zeaxanthin have the same conjugated CAC chain length (n 11) and behaved essentially the same when tested against peroxyl radicals in solution (28). The predicted collision rates of -carotene with aqueous peroxyl radicals within a membrane would, however, be predicted to be very low and, in this case, these molecules might therefore be expected to be poor antioxidants. Differences in their antioxidant behavior could be attributed in this case due to differences in their location and orientation within the lipid bilayer. It has been proposed that the prooxidant effects of carotenoids (or rather the lack of an antioxidant effect) may be due to their autoxidation (especially at high pO 2see below). A number of studies have tried to determine the nature of the products of carotenoid oxidation (20 22, 40, 41). The interaction of -carotene with cigarette smoke (in a model system) yields 1-nitro- -carotene and 4-nitro- -carotene (as both cis and all-trans forms) with the former as the major product (40). The products of Eqs. [2] and [3] may react with molecular oxygen to yield peroxyl radicals (31). These species can partake in lipid peroxidation reactions, and therefore any alteration in concentration of the carotenoid or pO 2 would inuence the formation of carotenoid peroxyl radicals or carotenoid autoxidation. Increased peroxide values in the plasma and the liver of rats fed -carotene have been recorded (42). However, there is still a lack of direct evidence that that the harmful effects of carotenoids in human diseases is related to their prooxidant effects.
CAROTENOID CONCENTRATION

One of the features of the recent intervention studies (ATBC and CARET; 4, 5) is the high dose of -carotene given as supplements. Mayne and colleagues (8) observed that this resulted in carotenoid levels in the blood (3.0 and 2.1 g/ml, respectively) well above that reported for the U.S. population (0.05 0.5 g/ml). What might the consequences of this be? Apparent prooxidant effects of carotenoids have been reported in vitro when high doses have been employed. However, the in vitro autooxidation of -carotene (see above) is dose-dependent only at 100% oxygen (43). At lower pO 2 (20% oxygen) the antioxidant effectiveness of -carotene was seen to decrease, but true prooxidative effects were not observed (31, 43). The procarcinogenic effects of the oxidative products (typically epoxides but also apo-carotenals produced via oxidative cleavage) of carotenoids has been proposed (see 8, 9). For example, the binding of benzo[a]pyrene metabolites to DNA is promoted in the presence of oxidative products of -carotene, but inhibited in the presence of

-carotene itself (44). At high concentrations of carotenoid, high levels of oxidative products may be expected to form in the presence of ROS. Elevated levels of such oxidative products have been observed in vitro in the lung tissues from smoke-exposed compared to control ferrets (9, 45). High levels of these products may also result in an acceleration of malignant transformation in lung tissues due to downregulation of the RAR -gene (45). Clearly, carotenoid structure inuences more than its antioxidant behavior per se. -Carotene has been shown to be effective against tert-butyl hydroperoxide-induced lipid peroxidation (46) and DNA damage (47). The mode of action of this articial peroxide is thought to be via degradation to alkoxyl and peroxyl radicals (1). However, in the presence of H 2O 2 or xanthine/xanthine oxidase (an exoge nous source of H 2O 2 and O 2 ) in HT29 adenocarcinoma cells, the ability of -carotene and lycopene to protect the cells against DNA damage (single strand breaks as measured by the Comet assay) was seen only at low doses ( 12 M; 48). This protective effect was lost as the dose of carotenoid was increased ( 4 M) so that at the highest doses tested (10 M) the carotenoid afforded no protection against DNA damage (Fig. 2a). Relatively high doses of -carotene also failed to protect against H 2O 2-induced DNA damage in HepG2 cells (47). The carotenoid concentration also affected the properties of the cell membrane so that at high doses, the membrane of the HT29 cells became increasingly permeabilized (48). A close correlation was observed between DNA damage and membrane permeabilization, indicating perhaps that the presence of carotenes may increase permeability to aqueous ROS. In contrast, the dihydroxy carotenoid, (3R, 3R)-zeaxanthin, exhibited dose-dependent protection of HT29 cells against xanthine/xanthine-oxidase-mediated DNA damage (Fig. 2b; 49). Prooxidative effects were not seen in these studies, rather a signicant decrease in antioxidant effectiveness. How can these observations be explained? Zeaxanthin is known to decrease membrane uidity, effectively lowering the rate of lipid peroxyl radical chain propogation and decreasing membrane permeabilisation (27). Alternatively, the different orientations adopted by zeaxanthin and the carotenes in these membranes may, in itself, allow more effective protection by the xanthophyll molecule against aqueous ROS (see above). While there are a number of criticisms that can be levelled at such in vitro studies, it is clear that different carotenoids can behave very differently against the same ROS. Such differences between the antioxidant behavior of -carotene and zeaxanthin have been seen before in model systems (e.g., 27) but not in human cells. While these carotenoids have an identical chomophore and very similar electron density proles

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FIG. 2. The effect of carotenoid concentration on the extent of single-strand DNA breaks (as measured by the Comet assay) in HT29 adenocarcinoma cells induced by ROS generated by xanthine/xanthine-oxidase. (A) -Carotene ({) and lycopene (F) (see 48). (B) Zeaxanthin (49).

(10, 27) so that their inherent antioxidant ability is effectively the same, they do behave quite differently once incorporated into a model or biological membrane. It is known that these carotenoids not only inuence the properties of membranes into which they are incorporated in a different manner (36; see above) but also that their effectiveness against ROS in the aqueous and lipid phases is quite different (27, 28). It is worth noting that both H 2O 2 and O 2 are components of cigarette smoke (50) and also produced in inammatory cells from asbestosis sufferers (51) and that neither -carotene nor lycopene exhibits in vitro antioxidant behavior against these particular ROS at high doses (48). At high concentrations, carotenoids also exhibit a tendency to aggregate or crystallize out of solution, with different compounds behaving differently, depending on their structure (36, 52). Cis-isomers have a lower tendency for aggregation compared with their all-trans counterparts (10). Such carotenoid aggregates have been directly observed in membranes, and their presence is thought to have a profound effect on the properties of the membrane itself by leading to an increase in membrane uidity and permeability, ultimately, perhaps, resulting in prooxidant-type effects (36). The biophysical and chemical properties of aggregates are quite different from that of the monomeric form of the carotenoid in solution (10, 36).
INTERACTION WITH OTHER DIETARY ANTIOXIDANTS

the cell. Many antioxidants are known to operate synergistically to provide an effective barrier against oxidation (1), including -carotene (and by inference other dietary carotenoids) and -tocopherol (53). Truscott (54) rst proposed a plausible integrated mechanism for the interaction of vitamins C and E with -carotene. In this model the carotenoid molecule repairs the vitamin E CAR CAR CAR TOH 3 TOH ASCH2 3 CAR ASCH 3 CAR CAR ASCH ASCH H H [4] [5] [6]

As there is no such thing as a universal antioxidant (1), different compounds act as different lines of defence against ROS. This may be based on their mode of action as an antioxidant and/or on their location within

radical (Eq. [4]) and the resulting carotenoid cation radical is, in turn, repaired by vitamin C (Eqs. [5] and [6]). Bohm et al. (55) demonstrated that the interaction of carotenoid with both vitamins E and C is possible in vitro. An additive response was seen for -carotene and vitamin E, but a synergistic response was only seen when vitamin C was added. The much reduced levels of vitamin C in the plasma of smokers compared with nonsmokers (56) is therefore clearly of signicance as (according to the above model) the -carotene radical cation would not be readily repaired. A carotenoid such as zeaxanthin whose conjugated system spans the membrane (see above) would, in theory, be able to interact with both lipid- and water-soluble antioxidants. In contrast, carotenes such as -carotene and lycopene would be expected to be much less effective because interaction with vitamin C would be much less likely as the resulting carotenoid radical cation would rst have to migrate from the hydrophobic core to the

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membrane surface. The relatively long lifetime and polarity of the carotenoid radical cation may, however, permit this, and there is some preliminary evidence that indicates that in a model membrane radicals formed from carotenes within the hydrophobic core may indeed react effectively with vitamin E (29). Vitamin C has been shown to protect both the carotenoid and the vitamin E pools in low-density lipoproteins from Cu 2 -mediated oxidative damage (57). One of the consequences of the administration of high doses of carotenoids in intervention studies is the resulting effect on the uptake and subsequent tissue distribution of coantioxidants (see 53). The synergistic protection afforded by carotenoids and other co-antioxidants is dependent upon a balance between all these components. An increase in the concentration of one of these might disturb this balance, reducing antioxidant effectiveness. An increase in carotenoid content may result in the formation of carotenoid cation radicals or adducts at a level beyond which the tocopherol/ascorbate pool can effectively repair, resulting in prooxidant effects. CAR1 CAR2 3 CAR1 CAR2 [7]

Carotenoids in human plasma lipoproteins and tissues do not exist on their own but rather as a heterogeneous mixture of xanthophylls and carotenes, yet few studies have considered the potential for interaction between different carotenoids. A synergistic response between different carotenoids has been reported (58), with a combination of lutein and lycopene proving to be most effective against AMVN-induced oxidation in multilamellar liposomes. Interestingly, of the carotenoids tested, lutein was least effective on its own. Electron transfer between different carotenoids (Eq. [7]) has been examined by Edge and Truscott (29, 59), highlighting the potential for carotenoid carotenoid interactions in biological systems, which typically have a heterogeneous carotenoid composition. Of the carotenoids studied, lycopene was the strongest reducing agent and astaxanthin the weakest, but, while lycopene was able to reduce the radical cations of lutein and zeaxanthin, -carotene could not.
PARTIAL PRESSURE OF OXYGEN

-carotene. At low pO 2, the -carotene radical cation was readily formed, while in air- and oxygen-saturated solutions a carotene radical adduct was also formed (53, 60). This second species decayed to the relatively unreactive carotene radical cation. The interaction of carotenoids with peroxyl radicals was described earlier (Eqs. [1][3]). At low pO 2 this process consumes peroxyl radicals and the carotenoid acts as a chain-breaking antioxidant. At high pO 2 the carotenoid radical could react with oxygen to produce a carotenoid peroxyl radical (autoxidation; Eq. [8]), which is capable of acting as a pro-oxidant. Peroxidation is promoted in the presence of unsaturated lipid (Eqs. [9] and [10]). The autoxidation of carotenoids is most pronounced at high concentrations (30). While these reactions have not yet been seen in vivo, it is appropriate to consider the possible physiological consequences of this as the different tissues and organs within the human body are very different in terms of distribution of pO 2: e.g., the lung alveoli 100 mm Hg, venous blood 40 mm Hg, and 515 mm Hg in tissues (1). Tumor tissues also have different pO 2 compared to normal, healthy, tissues (61). A number of in vitro and in vivo studies have been performed at very high pO 2 (760 mm Hg) but while this might promote effective prooxidation of carotenoids it CAR CAR OO L O2 3 CAR LH 3 CAR O2 3 L OO OOH L [8] [9] [10]

OO

Burton and Ingold (30) rst demonstrated that the antioxidant behavior of -carotene was, in part, dependent upon the partial pressure of oxygen ( pO 2). They showed that at low pO 2, -carotene acted as a chainbreaking antioxidant (consuming peroxy radicals; Eq. [1]), while at higher pO 2 the carotenoid lost its antioxidant ability and actually exhibited prooxidant behavior due to autoxidation. This was demonstrated in solution using the trichloromethylperoxyl radical and

is nonphysiological. It might therefore be expected that carotenoids may function differently in different parts of the body, so that they may, for example, be less effective antioxidants in the lung compared to other tissues. It does not necessarily mean that the carotenoids will act as prooxidants. The relative effectiveness of carotenoids compared to that of other antioxidants, especially -tocopherol, is also dependent on pO 2 (62). -Tocopherol is much more effective as an antioxidant at high pO 2, but -carotene is more effective at low pO 2. This serves to demonstrate how these co-antioxidants act together to provide an effective defence against oxidation in different tissues.
CONCLUSIONS

The function of dietary carotenoids and of their metabolites in the human body is clearly dependent upon a wide range of factors other than the basic chemical properties of these molecules per se. While in vitro studies provide an insight into these properties and into the interactions of carotenoids with ROS and co-

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MINIREVIEW 20. Handleman, G. J., van Kuijk, F. J., Chatterjee, A., and Krinsky, N. I. (1991) Free Radical Biol. Med. 10, 427 437. 21. Liebler, D. C., and Kennedy, T. A. (1992) Methods Enzymol. 213, 472 479. 22. King, T. J., Khachik, F., Bortkiewicz, H., Fukushima, L. H., Morioka, S., and Bertram, J. S. (1997) Pure Appl. Chem. 69, 21352140. 23. Di Mascio, P., Kaiser, S., and Sies, H. (1989) Arch. Biochem. Biophys. 274, 532538. 24. Conn, P. F., Schalch, W., and Truscott, T. G. (1991) J. Photochem. Photobiol. B. 11, 41 47. 25. Baltschun, D., Beutner, S., Briviba, K., Martin, H-D., Paust, J., Peters, M., Rover, S., Sies, H., Stahl, W., Steigel, A., and Sten horst, F. (1997) Liebigs Ann-Recueil 9, 18871893. 26. Wilkinson, F., and Ho, W. T. (1978) Spectrosc. Lett. 11, 425 436. 27. Woodall, A. A., Britton, G., and Jackson, M. J. (1997) Biochim. Biophys. Acta 1336, 575586. 28. Woodall, A. A., Lee, S. W., Weesie, R. J., Jackson, M. J., and Britton, G. (1997) Biochim. Biophys. Acta 1336, 33 42. 29. Edge, R., and Truscott, T. G. (1999) in The Photochemistry of Carotenoids (Frank, H. A., Young, A. J., Britton, G., and Cogdell, R. J., Eds.), pp. 223234, Kluwer Academic, Dordrecht, The Netherlands. 30. Mortensen, A., Skibsted, L. H., Sampson, J., Rice-Evans, C., and Everett, S. A. (1997) FEBS Lett. 418, 9194. 31. Burton, G. W., and Ingold, K. U. (1984) Science 224, 569 573. 32. Havaux, M. (1998) Trends Plant Sci. 3, 147151. 33. Havaux, M., Tardy, F., Ravenel, J., Chanu, D., and Parot, P. (1996) Plant Cell Environ. 19, 1359 1368. 34. Havaux, M., Gruszecki, W. I., Dupont, I., and Leblanc, R. M. (1991) J. Photochem. Photobiol. B 8, 361370. 35. Sarry, J. E., Montillet, J. L., Sauvaire, Y., and Havaux, M. (1994) FEBS Lett. 353, 147150. 36. Gruszecki, W. I. (1999) in The Photochemistry of Carotenoids (Frank, H. A., Young, A. J., Britton, G., and Cogdell, R. J., Eds.), pp. 363379, Kluwer Academic, Dordrecht, The Netherlands. 37. van de Ven, M., Kattenberg, M., van Ginkel, G., and Levine, Y. K. (1984) Biophys. J. 45, 12031209. 38. Johansson, L. B.-A., Lindblom, G., Wieslander, A., and Arvidson, G. (1981) FEBS Lett. 128, 9799. 39. Subczynski, W. K., Markowska, E., and Sielewiesiuk, J. (1991) Biochim. Biophys. Acta 1068, 68 72. 40. Bakeg, D. L., Krol, E. S., Jacobsen, N., and Liebler, D. C. (1999) Chem. Res. Toxicol. 12, 535543. 41. Kennedy, T. A., and Liebler, D. C. (1991) Chem. Res. Toxicol. 4, 290 295. 42. Alam, S. Q., and Alam, B. S. (1983) J. Nutr. 113, 2608 2614. 43. Palozza, P., Calviello, G., and Bartoli, G. M. (1995) Free Radical Biol. Med. 19, 887 892. 44. Salgo, M. G., Cueto, R., Winston, G. Q., and Pryor, W. A. (1999) Free Radical Biol. Med. 26, 162173. 45. Wang, X-D., Liu, C., Bronsen, R. T., Smith, D. E., Krinsky, N. I., and Russell, M. (1999) J. Natl. Cancer Inst. 91, 60 66. 46. Palozza, P., Luberto, C., Ricci, P., Sgarlata, E., Calviello, G., and Bartoli, G. M. (1996) Arch. Biochem. Biophys. 325, 145151. 47. Woods, J. A., Young, A. J., and Bilton, R. F. (1999) FEBS Lett. 449, 255258. 48. Lowe, G. M., Booth, L. A., Bilton, R. F., and Young, A. J. (1999) Free Radical Res. 30, 141151. 49. Lowe, G., Booth, L., and Young, A. J., unpublished data.

antioxidants, it is dangerous to extrapolate the results from such studies too far as the scenario within the body is highly complex. There is no evidence to support the hypothesis that carotenoids may act as prooxidants within a biological system (i.e., at physiological relevant pO 2s). What is more probable is that, on the basis of their performance in vitro, a number of factors may serve to reduce the antioxidant effectiveness of carotenoids in vivo, rendering them ineffective against certain ROS.
ACKNOWLEDGMENT
This work was partly supported by The Association for International Cancer Research.

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