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9 Antioxidant Vitamins and
Immune Function
DAVID A. HUGHES
Nutrition and Consumer Science Division, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK
Introduction
Oxidative stress, resulting from cumulative damage caused by reactive oxygen species (ROS), is present throughout life and is thought to be a major contributor to the ageing process. The immune system is particularly vulnerable to oxidative damage, since many immune cells produce these reactive compounds as part of the body’s defence mechanisms. Higher organisms have evolved a variety of antioxidant defence systems either to prevent the generation of ROS or to inter- cept any that are generated. These defence systems exist in both the aqueous and membrane compartments of cells and can be enzymic or non-enzymic in nature. The enzymes contain metal ions at their active sites – these must be obtained from the diet – while the diet is the source of many non-enzymic com- ponents of the body’s antioxidant defence system (e.g. antioxidant vitamins).
Reactive Oxygen Species and Antioxidant Defences
Reactive oxygen species
Free radicals are highly reactive molecules containing one or more unpaired elec- trons. Examples of free radicals are the superoxide anion (O 2 ·) and the hydroxyl radical (OH·). The term ‘reactive oxygen species’ is a collective one that includes not only oxygen-centred radicals but also some non-radical derivatives of oxygen, such as hydrogen peroxide (H 2 O 2 ), singlet oxygen and hypochlorous acid (HOCl). Hydrogen peroxide can very easily break down, particularly in the pres- ence of transition-metal ions (e.g. ferrous (Fe2+) iron), to produce the hydroxyl radical, the most reactive and damaging of the oxygen free radicals: H 2 O 2 + Fe 2+^ → OH· + OH^ + Fe 3+
© CAB International 2002. Nutrition and Immune Function (eds P.C. Calder, C.J. Field and H.S. Gill) 171
Exogenous sources of free radicals include ozone, UV radiation and ciga- rette smoke. Free radicals are also generated endogenously, mainly from two sources. The first is by leakage from the mitochondrial electron-transfer chain, as part of normal cellular metabolism. The second is as part of the respiratory- burst activity of leucocytes, which is involved in microbial killing. ROS can cause damage to all of the major classes of macromolecules. They cause strand breaks in DNA (Halliwell and Aruoma, 1991), which can potentially lead to subsequent misrepair, mutation and tumour-cell formation. An example of free-radical-mediated damage to proteins is the formation of cataracts, resulting from the damage to the crystallins in the lens of the eye. However, lipids are probably most susceptible to free radical attack, particularly long chain polyunsaturated fatty acids (PUFA) that contain several double bonds. The oxidative destruction of PUFA, known as lipid peroxidation, can be extremely damaging, since it proceeds as a self-perpetuating chain reaction. Generation of ROS in excess of the amounts that can be dealt with by the body’s antioxidant protective mechanisms is thought to be a major contributor to several degenerative disorders, such as cancer and cardiovascular diseases (Table 9.1), and to the ageing process. Strong associations between diets rich in antioxidant nutrients and a reduced incidence of cancer have been observed in several epidemiological studies (Block et al. , 1992; Giovannucci, 1999), and it has been suggested that a boost to the body’s immune system by antioxidants might, at least in part, account for this (Bendich and Olson, 1989). Indeed, it is probably crucial to attempt to balance the production of ROS and the antioxi- dant defence system, ideally by dietary means rather than by taking supple- ments, from as early an age as possible, in order to delay the onset of, if not prevent, many age-related disorders. The immune system appears to be particularly sensitive to oxidative stress. Immune cells rely heavily on cell–cell communication, particularly via mem- brane-bound receptors, to work effectively. Cell membranes are rich in PUFA, which, if peroxidized, can lead to a loss of membrane integrity, altered mem- brane fluidity (Baker and Meydani, 1994) and alterations in intracellular sig- nalling and cell function. It has been shown that exposure to ROS can lead to a reduction in cell-membrane-receptor expression (Gruner et al. , 1986). In addi- tion, the production of ROS by phagocytic immune cells can damage the cells themselves if they are not sufficiently protected by antioxidants.
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Table 9.1. Degenerative disorders associated with oxidative damage. Cancer Cardiovascular disease Stroke Cataract Degeneration of the macula region of the retina Immunosenescence Ageing
between 2.5 and 15.2 (median 6.7) mg day^1 -tocopherol equivalents for women (Department of Health, 1991). Another group of lipid-soluble com- pounds that can act as antioxidants are the carotenoids, such as -carotene, lycopene and lutein, found in highly pigmented fruits and vegetables (Mangels et al. , 1993). The major water-soluble free radical scavenger is ascorbic acid (vitamin C), which also plays a role in ‘sparing’ vitamin E, by regenerating -tocopherol from the oxidized tocopheroxyl radical (Bendich et al. , 1986). The estimated average requirement for vitamin C in adults in the UK is 25 mg day^1 (Department of Health, 1991). More recently, atten- tion has also focused on the antioxidant properties of plant polyphenols, found in tea and red wines (Rice-Evans, 1995), but considerably more infor- mation on the absorption, metabolism and excretion of these compounds in humans is required before their relative contribution to preventing oxidative damage can be assessed. A balanced diet, containing at least five or six varied portions of fruits and vegetables per day, should provide an adequate supply of antioxidants for healthy individuals. Concerns regarding the taking of supplements centre around the possibilities that certain compounds might have a toxic effect if taken in doses significantly higher than can be obtained from a healthy diet and that a reliance on supplements will lead to a reduced consumption of fresh fruit and vegetables, which probably contain a multitude of compounds whose health benefits we have yet to appreciate. However, in elderly individ- uals, whose diet might be restricted (e.g. by loss of appetite, dental con- ditions) and where absorption of nutrients is impaired, there might be a case for supplementation with certain nutrients. The case is probably strongest for vitamin E, because it is impossible to obtain high intakes of this nutrient with- out consuming a high-fat diet. Table 9.2 identifies dietary sources of anti- oxidant vitamins.
Antioxidant Vitamins and Immune Function
Vitamin C and immune function
Vitamin C is found in high concentrations in white blood cells and is rapidly uti- lized during infection; reduced plasma concentrations are often associated with reduced immune function (see Siegel, 1993). Animal and human studies have suggested that the dietary requirements for vitamin C are increased in cancer, sur- gical trauma and infectious diseases (see Siegel, 1993). The belief that high intakes of vitamin C will prevent the onset of the common cold has not been substantiated scientifically, although the associated symptoms following infection appear to be reduced by a moderate intake (Coulehan et al. , 1974). Pauling’s claims regarding the effects of vitamin C on the common cold (Pauling, 1970) inspired a great deal of research into its effect on immune function in the 1970s and early 1980s (reviewed by Thomas and Holt, 1978; Siegel, 1993). Vitamin C deficiency in the guinea pig impairs lymphocyte proliferation, the delayed-type hypersensitivity (DTH) response to tuberculin, the ability of neutrophils to kill bacteria and the
174 D.A. Hughes
activity of cytotoxic T-cells and delays the rejection of skin allografts, but has little effect on antibody responses (for references, see Siegel, 1993). Providing vitamin C to mice increased spleen lymphocyte proliferation in response to mitogens but did not affect natural killer (NK)-cell activity or the antibody response to sheep red blood cells or lipopolysaccharide (LPS) (see Siegel, 1993). Vitamin C decreased or slowed tumour development in some animal models, but not others (see Siegel, 1993). Vitamin C deficiency in humans did not impair lymphocyte proliferation or alter the number of CD4+ or CD8+ cells in the circulation (Kay et al. , 1982). However, Vitamin C (1–5 g daily for 3 days to several weeks) increased human T lymphocyte proliferation (Yonemoto et al. , 1976; Anderson et al. , 1980; Panush et al. , 1982) and neutrophil motility towards LPS-activated autologous serum (Anderson et al. , 1980). Some studies indicate that vitamin C increases circulating immunoglobulin (Ig) levels in humans (Prinz et al. , 1977; Vallance, 1977; Ziemlanski et al. , 1986), but other studies fail to show this (Anderson et al. , 1980; Panush et al. , 1982; Kennes et al. , 1983). Jacob et al. (1991) studied the effect of vitamin C at different levels in the diet on immune function in a group of young, healthy non-smokers. The subjects first consumed a vitamin C-deficient diet and then gradually increased their vitamin C intake (from 5 to 250 mg day^1 ). The vit- amin C-deficient diet decreased plasma and white-cell vitamin C concentrations by 50% and decreased the DTH response to seven recall antigens, but did not alter lymphocyte proliferation. Sixty or 250 mg vitamin C day^1 led to recovery of the DTH response, but did not affect lymphocyte proliferation. The authors sug- gest that the inconsistency regarding the influence of vitamin C on these two out- comes, both indicators of cell-mediated immunity, may result from the higher sensitivity of the DTH test, involvement of cells other than those isolated for in vitro cultures in the in vivo DTH response or other unknown factors. The lack of an effect on lymphocyte proliferation at an intake of 250 mg day^1 suggests that, at least in young individuals, only levels of vitamin C that approach pharmacologi- cal doses can produce a quantifiable effect on this parameter of immune function. It has been suggested that vitamin C intakes of 600 mg day^1 may be beneficial in reducing infections in individuals who undertake a large amount of physical activ- ity: for instance, studies of marathon runners have found a significantly lower incidence of post-race upper respiratory infections in runners taking a daily supple- ment of 600 mg vitamin C (Peters, 1997). One of the major problems in assessing the beneficial effects of dietary components on the immune system is the lack of a reliable marker of immune function that is known to be indicative of a long-term beneficial effect in terms of reducing the incidence of degenerative disorders in later life. Although not an immunological one, one recent study does provide an excellent example of the potential need to maintain adequate intakes of antioxidant nutrients in the middle years of life to prevent the accumulative damage caused by ROS being made manifest in later years. Jacques et al. (1997) examined the cross-sec- tional relationship between age-related lens opacities and vitamin C supple- ment use over a 10–12-year period in women without diagnosed cataract or diabetes. Use of vitamin C supplements for 10 years or more was associated with a 77% lower prevalence of early lens opacities and an 83% lower preva- lence of moderate lens opacities, compared with women who did not use sup-
Antioxidant Vitamins and Immune Function 175
(Baehner et al. , 1977); this latter effect is most probably due to a vitamin E- induced decrease in production of free radicals and related reactive species. In laboratory animals, vitamin E deficiency decreased spleen lymphocyte prolifer- ation in response to mitogens, NK-cell activity, specific antibody production fol- lowing vaccination and phagocytosis by neutrophils (for a review, see Meydani and Beharka, 1998). Vitamin E deficiency also increases susceptibility of ani- mals to infectious pathogens (for references, see Meydani and Beharka, 1998; Han and Meydani, 1999). Vitamin E supplementation of the diet of laboratory animals enhances antibody production, lymphocyte proliferation, NK-cell activ- ity, and macrophage phagocytosis (for references, see Meydani and Beharka, 1998). Dietary vitamin E promoted resistance to pathogens in chickens, turkeys, mice, pigs, sheep and cattle (for references, see Meydani and Beharka, 1998; Han and Meydani, 1999); some of these studies report improved immune-cell functions in the animals receiving additional vitamin E (see Han and Meydani, 1999). For example, vitamin E prevented the retrovirus-induced decrease in production of interleukin-2 (IL-2) and interferon- (IFN-) by spleen lymphocytes and in NK-cell activity in mice (Wang et al. , 1994). One application of the effects of vitamin E on immune function is in the elderly. This has been investigated in both murine models and human trials. Adding vitamin E to the diet of aged mice increased lymphocyte proliferation, IL-2 production and the DTH response (Meydani et al. , 1986). A high level of vitamin E in the diet (500 mg kg^1 food) also increased NK-cell activity of spleen cells from old (but not young) mice (Meydani et al. , 1988). In another study, young and old mice were fed diets containing adequate (30 mg kg^1 diet) or high (500 mg kg^1 diet) levels of vitamin E for 6 weeks and infected with influenza A virus: young mice and old mice fed the high level of vitamin E had lower lung titres of virus than old mice fed the adequate vitamin E diet (Hayek et al. , 1997). The high level of vitamin E caused increased production of IL- and IFN- by spleen lymphocytes from influenza-infected old mice (Han et al. , 1998; Han and Meydani, 2000). Supplementation of the diet of elderly human subjects with 800 mg vitamin E day^1 for 4 weeks increased lymphocyte prolif- eration stimulated by concanavalin A, IL-2 production and the DTH response, but did not affect IL-1 production, the number of CD4 cells or circulating Ig con- centrations (Meydani et al. , 1990). In a more recent study, 60, 200 and 800 mg vitamin E day^1 increased DTH response in elderly subjects, with 200 mg day^1 having the maximal effect (Meydani et al. , 1997). The two higher vitamin E doses improved antibody responses to hepatitis B, but only the 200 mg day^1 dose increased the antibody response to tetanus toxoid (Meydani et al. , 1997). The authors conclude that 200 mg vitamin E day^1 represents the optimal level for the immune response. In another study, young and elderly individuals were supplemented with 800 mg vitamin E day^1 for 48 days before being asked to run down an inclined treadmill for 45 min. Vitamin E supplementation was found to eliminate the age-associated difference in exercise-induced neutrope- nia, to prevent the exercise-induced increase in IL-1 production and to inhibit IL-6 production (Cannon et al. , 1991). Since these cytokines are involved in the inflammatory process and in exercise-induced muscle damage, their inhibition by vitamin E during exercise might have important implications. However, on a
Antioxidant Vitamins and Immune Function 177
cautious note, studies have reported that prolonged high intakes of vitamin E ( 1000 mg day^1 ) can lead to inhibition of neutrophil phagocytosis (Boxer, 1986). Further research is needed to assess the optimal intake of this nutrient required to provide benefit for different groups of individuals. Cigarette smoke contains millions of free radicals per puff, and other com- pounds present can stimulate the formation of other highly reactive molecules (Pryor and Stone, 1993). Serum levels of vitamin E (as well as of vitamin C and -carotene) and lung vitamin E concentrations are significantly lower in smokers compared with non-smokers and even supplementation with 2400 mg -tocopherol equivalents day^1 for 3 weeks failed to restore the lung vitamin E level to that found in non-smokers (Pacht et al. , 1986). Circulating phagocytes from smokers produce high levels of free radicals, which probably in part accounts for the depressed immune function observed in smokers (Johnson et al. , 1990), and there is some evidence that vitamin E supplementation can reduce the overproduction of ROS by phagocytic cells from smokers (Richards et al. , 1990). Reduced vitamin E status has also been reported in human immunodefi- ciency virus (HIV)-infected individuals. Passi et al. (1993) found that plasma vitamin E concentrations were significantly lower in a group of 200 HIV-posi- tive individuals compared with controls, but whether this is related to an inade- quate intake of this vitamin is unclear. Dietary diaries from a group of 100 HIV-infected asymptomatic men did not indicate an inadequate intake of vita- min E, but plasma levels were low or marginally low in 74% of the men (Beach et al. , 1992). In a study of patients who had developed acquired immune defi- ciency syndrome (AIDS), an inverse relationship was observed between serum vitamin E levels and severity of disease (Favier et al. , 1994). A recent study of 49 HIV-infected subjects provided with vitamin E and vitamin C observed a significant reduction in oxidative stress and a trend towards a reduction in viral load after 3 months (Allard et al. , 1998). These studies suggest that larger trials of these and other antioxidant nutrients in the treatment of HIV-infected per- sons should be encouraged, since there is a need to find alternative, cheaper, treatments than the combination therapies currently employed. In terms of mechanisms of action, in addition to its role as a protective antioxidant, vitamin E, at higher intakes, is associated with a reduced production of prostaglandin E 2 (PGE 2 ) (e.g. Meydani et al. , 1986, 1988, 1990). Since PGE 2 inhibits lymphocyte proliferation and NK-cell activity, it is possible that this may be one immunomodulatory mechanism of vitamin E action. It is also possible that vitamin E and, indeed, other antioxidant nutrients can influence a variety of inflammatory processes by inhibiting the activity of a transcription factor called nuclear factor kappa B (NFB). Transcription factors are intracellular regulators of gene expression. Once activated, the transcription factor binds to the promoter region of a specific gene within the DNA in the nucleus, resulting in that gene being ‘turned on’. NFB is required for maximal transcription of many proteins that are involved in inflammatory responses, including several cytokines, such as IL-1, IL-2 and tumour necrosis factor (TNF)-. NFB is a redox-sensitive tran- scription factor and it is thought that the generation of ROS is a vital link in medi- ating NFB activation by a variety of stimuli (Lavrovsky et al. , 2000).
178 D.A. Hughes
have reported increases in the numbers of CD4+ cells or in the ratio of CD4+ to CD8+ cells in the circulation, in the percentages of lymphocytes expressing the IL- and transferrin receptors, and in NK-cell activity (Alexander et al. , 1985; Watson et al. , 1991; Murata et al. , 1994), particularly in elderly subjects. The potential for increasing the numbers of CD4+ cells led to the suggestion that -carotene might be useful as an immunoenhancing agent in the treatment of HIV infection. Preliminary studies have shown a slight but insignificant increase in CD4+ numbers in response to -carotene (60 mg day^1 for 4 weeks) in patients with AIDS (Fryburg et al. , 1995), but long-term effectiveness has not been reported. Other investigators have been unable to confirm the increase in T-cell-medi- ated immunity in healthy individuals following -carotene supplementation. Santos et al. (1996, 1997) reported the results of two studies in the elderly: a short- term, high-dose study (90 mg day^1 for 21 days) in women and a longer-term, lower-dose trial (50 mg alternate day^1 for 10–12 years) in men. Both studies concluded that there was no significant difference in T-cell function as assessed by DTH response, lymphocyte proliferation, IL-2 and PGE 2 production and composi- tion of lymphocyte subsets (Santos et al. , 1997). However, these workers also examined the effect of -carotene supplementation on NK-cell activity in the longer-term trial with male volunteers and observed that supplementation of the diet of older males ( 65 years) with -carotene resulted in significantly greater NK-cell activity compared with subjects of a similar age given placebo treatment (Santos et al. , 1996). Since patients with Chediak–Higashi syndrome, a disorder associated with defective NK-cell function, show a higher susceptibility to tumour formation (Roder et al. , 1980), and homozygous mice genetically deficient in NK cell activity grow tumours and develop leukaemia more rapidly than do heterozy- gous littermates with normal NK-cell function (Lotzova, 1993), the enhancing effect of -carotene on NK-cell activity has been postulated to be a link between raised intakes of this nutrient and cancer prevention. As shown in Fig. 9.2, the study by Santos et al. (1996) highlighted both the reduction in NK-cell activity that is observed with age and the fact that the increase in NK-cell activity observed in older males (65–86 years) following -carotene supplementation restored it to the level seen in a group of younger males (51–64 years). The mechanism for this is unclear, but it was not due to an increase in the percentage of NK cells or to an increase in IL-2 production. The authors suggest that -carotene may be acting directly on one or more of the lytic stages of NK-cell cytotoxicity or on NK-cell activity-enhancing cytokines other than IL-2, such as IL-12. Individuals who are repeatedly exposed to UV light show suppression of immune function (Rivers et al. , 1989). Because carotenoids can provide photo- protection, several studies have assessed the ability of -carotene to protect the immune system from UV-induced free radical damage. In one study, a group of young males were placed on a low-carotenoid diet ( 1.0 mg day^1 total carotenoids) and given either placebo or 30 mg -carotene day^1 for 28 days prior to periodic exposure to UV light. DTH responses were significantly sup- pressed in the placebo group after UV treatments and the suppression was inversely proportional to plasma -carotene concentrations in this group (Fuller et al. , 1992). In contrast, no significant suppression of DTH responses was seen in the -carotene-treated group (Fuller et al. , 1992). The ability of -carotene
180 D.A. Hughes
Antioxidant Vitamins and Immune Function 181
to protect against the harmful effects of natural UV sunlight has also been demonstrated by exposing healthy female students to time- and intensity- controlled sunlight exposure: a Berlin-based study involved taking volunteers to the Red Sea and exposing areas of their skin to the sunlight by lifting discretely placed flaps in their specially designed swimsuits (Gollnick et al. , 1996)!
Specific lysis (%)
0
20
40
60
80
100
12.5:1 25.0:1 50.0:1 100.0:
51–64 year 65–86 year
E : T Placebo group
Specific lysis (%)
0
20
40
60
80
100
12.5:1 25.0:1 50.0:1 100.0:
51–64 year 65–86 year
E : T -Carotene group
Fig. 9.2. Natural killer cell activity in different age-groups of subjects consuming placebo or -carotene. Natural killer cell activity was determined at several effector-to-target cell ratios (E: T), using effector cells from subjects consuming placebo ( n = 17 for 51–64 years and n = 13 for 65–86 years) or -carotene ( n = 21 for 51–64 years, and n =8 for 65–86 years). Data are expressed as % target-cell lysis. Reprinted from Santos et al. (1996) with permission by the American Journal of Clinical Nutrition. © American Journal of Clinical Nutrition, American Society for Clinical Nutrition.
The effect of -carotene supplementation (15 mg day^1 for 26 days; equivalent to 150 g carrots day^1 ) on expression of MHC class II and adhesion molecules on the monocyte surface has been investigated. Following dietary supplementation, there were significant increases in plasma levels of -carotene and in the percentages of monocytes expressing the MHC class II molecule HLA-DR and the adhesion molecules ICAM-1 and LFA-3 (Hughes et al. , 1997). These results suggest that moderate increases in the dietary intake of - carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anti-carcinogenic prop- erties attributed to this compound. The increase in surface molecule expression may also, in part, account for the ability of -carotene to prevent the reduction in DTH response following exposure to UV radiation, since the latter can inhibit both HLA-DR and ICAM-1 expression. This finding could certainly be relevant to the preventive action of -carotene towards skin cancer (Mathews-Roth, 1989), since immunosuppressed individuals, such as renal-transplant patients, have an increased risk of skin cancer. As well as preventing oxidative damage, it has been suggested that - carotene, like vitamin E, can influence immune cell function by modulating the production of PGE 2. This eicosanoid is the major prostaglandin (PG) synthe- sized by monocytes and macrophages and is known to possess a number of immunosuppressive properties (see Calder and Field, Chapter 4, this volume). It has been suggested that -carotene might enhance immune responses by altering the activation of the arachidonic acid cascade (from which PGE 2 is derived), since it has been shown to be capable of suppressing the generation of arachidonic acid products in vitro from non-lymphoid tissues (Halevy and Sklan, 1987). This possibility requires further investigation. There have been very few studies examining the influence of carotenoids other than -carotene on human immune function, even though there is strong epidemiological evidence to suggest that lycopene (found in tomatoes) and lutein (found in peas, watercress and other vegetables) can protect against the development of prostate and lung cancer, respectively (Le Marchand et al. , 1993; Gann et al. , 1999). In addition, tomato intake has been found to be inversely associated with the risk of diarrhoeal and respiratory infections in young children in Sudan (Fawzi et al. , 2000). In terms of mechanisms of action, the effect of dietary supplementation with lycopene and lutein on the expression of monocyte surface molecules involved in antigen presentation has been inves- tigated. It was found that these carotenoids appear to be less influential than - carotene, when given at the same level in the diet (Hughes et al. , 2000). In addition, enriching the diet with lycopene (by drinking 330 ml of tomato juice daily) for 8 weeks did not modify cell-mediated immune responses in the elderly (Watzl et al. , 2000). In another study, performed in a group of older volunteers (over 65 years) living in Ireland, the effects of placebo, -carotene (8.2 mg day^1 ) and lycopene (13.3 mg day^1 ) for 12 weeks on various parameters of cell-mediated immunity were examined. There were no significant changes in circulating T-cell subsets, mitogen-stimulated lymphocyte proliferation or surface molecule expression following any of these interventions, in spite of significant increases in the plasma levels of the carotenoids (Corridan et al. , 2001). The
Antioxidant Vitamins and Immune Function 183
authors concluded that in well-nourished, free-living, healthy individuals, supple- mentation with relatively low levels of -carotene or lycopene is not associated with either beneficial or detrimental effects on cell-mediated immunity. Other investigators have shown an opposing effect of -carotene and lutein upon human lymphocyte proliferation (Watzl et al. , 1999), emphasizing further the fact that different carotenoids might affect immune function in differ- ent ways. Therefore, in fruits and vegetables, the influence of the combination of carotenoids they contain on immune function may represent the sum total of these different effects and, indeed, the potential for synergistic effects remains to be investigated. One possible factor to explain the different effects seen with different carotenoids might be the preferred location of these compounds within the cell and within the body. Carotenoids are lipid-soluble and thus it is thought that most will be concentrated in the lipid-rich membranes of the cell. However, their exact location may influence their effectiveness in modulating specific cel- lular events. Within the body, lycopene appears to be selectively taken up within the prostate, a finding that may help explain the association between higher intakes of lycopene and a reduced incidence of prostate cancer (Giovannucci, 1999). Thus, it is possible that tests on peripheral blood cells to determine immune function will not detect any localized effects, suggesting that there might be ‘hidden’ benefits associated with certain dietary components that we have yet to discover. The strongest epidemiological evidence supporting a beneficial effect of carotenoids in preventing cancer is the protective effect of -carotene intake in reducing the incidence of cancer of the lung. Carotenoid intake has been asso- ciated with a reduced lung-cancer risk in eight of eight prospective studies and in 18 of 20 retrospective studies (for a review, see Zeigler et al. , 1996). As a result, three major intervention trials were initiated, examining the efficacy of - carotene in the prevention of lung cancer (Alpha-tocopherol Beta-carotene Cancer Prevention Study Group, 1994; Hennekens et al. , 1996; Omenn et al. , 1996). The failure of these trials to show a protective effect, with two of the studies showing an increase in lung cancer in smokers receiving -carotene supplementation, has been widely publicized. The mechanism for the increased lung-cancer risk associated with the supplementation is unclear, but several suggestions have been made. Since the participants in these studies could be classified as ‘high-risk’ for developing lung cancer (long-term smokers or previ- ously exposed to asbestos), it is possible that many of them had undetected tumours prior to the commencement of supplementation. The stage (or stages) of carcinogenesis that -carotene might be effective against is unclear, but, if the effect is mediated via the immune system, it is likely to occur during the promotional stages preceding the formation of a malignant tumour. A recent analysis of the Cancer Prevention Study II (CPS-II), a prospective mortality study of more than 1 million US adults, investigated the effects of supplementa- tion with multivitamins and/or vitamins A, C and/or E on mortality during a 7- year follow-up period. The use of a multivitamin plus vitamins A, C and/or E significantly reduced the risk of cancer in former smokers and in never- smokers, but increased the risk of lung cancer in male smokers who had used a
184 D.A. Hughes
Acknowledgement
The author would like to thank the Biotechnology and Biological Sciences Research Council for financial support.
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