NUTRITION AND CANCER, 56(2), 232–240 Copyright © 2006, Lawrence Erlbaum Associates, Inc.

Redox Molecules and Cancer Prevention: The Importance of Understanding the Role of the Antioxidant Network
Mauro Serafini, Debora Villano, Giovanni Spera, and Nicoletta Pellegrini

Abstract: Cancer has a complex etiology with multiple risk factors that involve the interplay between genetic and environmental influences. There is compelling evidence that dietary plant foods appear to be protective against certain type of cancers. Among a number of mechanistic hypotheses, diet-derived antioxidants have been proposed to contribute to explain these findings. However, contrasting results from intervention trials have raised strong concerns about the influence of antioxidants on human health. A vulnerable point of the research on antioxidants is the lack of information on the effect of the whole array of dietary antioxidants in cancer prevention because so far mainly single molecules have been investigated. Total antioxidant capacity (TAC) considers the single antioxidant activity as well as the synergistic interactions of the redox molecules present in complex matrixes, giving an insight into the assessment of the non-enzymatic antioxidant network. This article will outline the state of art of the research on TAC and cancer, describing the plasma non-enzymatic antioxidant network and its association with diet. The feasibility of TAC assessment as an innovative tool for investigating the association between dietary antioxidants, oxidative stress, and cancer will be also discussed.

the basis of their ability to reduce DNA damage caused by RONS. In the absence of adequate and efficient cellular protection by the antioxidant network, oxidative stress can result in DNA damage and mutations. In monitoring the antioxidant defences, the body’s non-enzymatic antioxidant network can be assessed through the measurement of total antioxidant capacity (TAC), defined as the moles of radicals neutralized per 1 L of tested sample (3). TAC represents the result of many variables such as redox potentials of the compounds present in the matrix, their cumulative and synergistic interaction, the nature of the oxidizing substrate, and antioxidants localization (Fig. 1). Moreover, TAC considers the cumulative action of all the antioxidants present in the matrix (plasma, saliva, food extracts, tissues, etc.), providing an integrated parameter rather than the simple sum of measurable antioxidants and giving an insight into the assessment of the antioxidant network. This article will outline the state of art of the research on TAC and cancer, describing the plasma non-enzymatic antioxidant network and its association with diet. The feasibility of TAC assessment as an innovative tool for investigating the association between dietary antioxidants, oxidative stress, and cancer will be also discussed.

Introduction Human antioxidant defenses have evolved to protect biological systems against reactive oxygen and nitrogen species (RONS), and a sophisticated cooperative array of antioxidant defense mechanisms is found in biological systems (1). However, despite the high grade of complexity and efficiency of the endogenous antioxidant complex, there is a need to optimize the defense strategy against RONS with dietary antioxidants. There is compelling evidence that dietary plant foods appear to be protective against certain kinds of cancers (2), however it is still unclear which are the compounds involved in this effect and their mechanism of action. Among a number of mechanistic hypotheses, diet-derived antioxidants have been proposed to be protective against cancer mainly on Galenic Antioxidant Supplementations and Cancer Prevention On the basis of the postulated role of antioxidants in cancer prevention, randomized placebo-controlled intervention studies have been carried out supplementing subjects with different doses of galenic antioxidants, either in combination or alone. Results from many of these clinical trials have been contrasting, with some intervention studies showing a protective effect of antioxidants, whereas in others no benefits were reported or even an enhancement of cancer risk was observed (Table 1) (4–13). The Linxian trial conducted in China showed that an antioxidant cocktail (15 mg β-carotene + 30 mg vitamin E + 50 µg selenium) was able to reduce can-

M. Serafini and D. Villano are affiliated with Antioxidant Research Laboratory at the Unit of Human Nutrition, National Institute for Food and Nutrition Research, Rome, Italy. N. Pellegrini is affiliated with Internal Medicine, Department of Medical Pathophysiology, University of Rome La Sapienza, Rome, Italy. G. Spera is affiliated with the Human Nutrition Unit, Department of Public Health, University of Parma, Parma, Italy.

Figure 1. “The TAC concept”: Multiple factors affecting total antioxidant capacity. Adapted from Serafini and Del Rio (3).

cer mortality after 5 yr of supplementation (4). In the Supplémentation en Vitamines et en Minéraux Antioxydants (SUVIMAX) study, where a cocktail of antioxidants, minerals, and vitamins was used at nutritional dosages for 8 yr, supplementation was effective only in men where cancer incidence was reduced by about 30% in the supplemented group (11,12). A randomized controlled trial with a 4.5 yr supplementation of 200 µg of selenium per day showed a significant reduction in total cancer mortality and incidence in the selenium group as compared with the placebo group. Incidences of lung, colorectal, and prostate cancers also were significantly lower (8). Conversely, selenium treatment did not protect against the development of skin carcinomas. Contrasting findings were obtained from the Alpha-Tocopherol, Beta Carotene Cancer Prevention study (5) where supplementation with 20 mg/day of β-carotene was significantly associated with a higher incidence of lung cancer (18%) in male smokers. However, a decrease in the incidence of prostate cancer (32%) and mortality (41%) was observed in α-tocopherol supplemented group. Also the beta-Carotene and Retinol Efficacy Trial (CARET), conducted in the United States, produced similar disappointing results, with an increase in lung cancer being observed in asbestos-exposed workers after 4 yr of supplementation with β-carotene and retinol (6). Moreover, in the Physician’s Health Study 50 Vol. 56, No. 2

mg β-carotene for 12 yr were ineffective in reducing cancer incidence (7). The Womens’ Health Study did not find any effects on cancer risk and cardiovascular mortality with the supplementation of 50 mg β-carotene for 2 yr (9). An intervention study in patients with cardiovascular disease or diabetes showed that long-term vitamin E supplementation did not prevent cancer or major cardiovascular events and even increased the risk of heart failure (13). The overall picture becomes a little bit clearer once we analyze results from recent meta-analyses. Bjelakovic et al. (14) reviewed 14 randomized trials to establish whether antioxidant supplements might have a beneficial effect on the incidence of gastrointestinal cancers. The authors did not find any evidence that antioxidant supplementation can prevent gastric cancers; on the contrary, a negative effect on overall mortality was described. In seven trials, the fixed-effect model clearly showed that β-carotene and the association between β-carotene and vitamin A or vitamin E significantly increased mortality. A review of 8 intervention trials of antioxidant supplements on the progression of aged-related macular degeneration found a beneficial effect of supplementation only in one trial (15). The lack of protective effects of antioxidants was also reported by meta-analyses reviewing the role of antioxidant supplements for the prevention of lung cancer (16), all cause mortality (17), and cardio233

Table 1. Overview of Randomized Human Intervention Studies on Antioxidant Supplementation and Risk of Cancer: Characteristics and Main Resultsa
Study (Ref.) Blot et al., 1993 (4) China Country Subjects 29,584 subjects, 40-69 years Intervention 5-year supplementation, 15 mg/d β-carotene + 30 mg/d vitamin E + 50 µg Se 8-year supplementation, 50 mg/d α-tocopherol, 20 mg/d β-carotene, combination of both 4-year supplementation, 30 mg/d β-carotene + 25000 IU retinol 12-year supplementation, 50 mg/d β-carotene 4.5-year supplementation, 200 µg/d Se Outcome Decrease in total cancer rates (RR = 0.87); decrease in stomach cancer rates (RR = 0.79) β-carotene group: significant increase (18%) in lung cancer incidence Significant increase in lung cancer incidence (28%; RR = 1.36) No changes in lung cancer incidence and mortality No significant changes on incidence of skin cancer, no significant reduction in all-cause mortality, significant reduction in total cancer mortality (RR = 0.50)and incidence (RR = 0.63), significant reduction in prostate cancer (RR = 0.37), colorectal cancer (RR = 0.42) and lung cancer (RR = 0.54) No significant differences in cancer risk and cardiovascular death No changes in cancer incidence

Alpha-Tocopherol, Beta Carotene Cancer Prevention Study, 1994 (5) β-Carotene and Retinol Efficacy Trial, Omen et al., 1996 (6) Physician’s Health Study, Hennekens et al., 1996 (7) Clark et al., 1996 (8)


29,133 chronic heavy smokers (M) 50-69 years

United States

United States

18,314 subjects, smokers, former smokers, and asbestos-exposed workers, 45-74 years 11,000 healthy physicians (M), 40-84 years 1,312 patients with lesions on skin, mean age 63 years, 18-80 years

United States

Women’s Health Study, Lee et al., 1999 (9) MRC/BHF Heart Protection Study, 2002 (10)

United States

39,876 female health professionals ≥45 years 20,000 patients with coronary disease, cardiovascular events, diabetes, 40-80 years 12,741subjects (M,W), 45-60 years

2-year supplementation, 50 mg/d β-carotene 5-year supplementation, 600 mg/d vitamin E, 250 mg/d vitamin C, 20 mg/d β-carotene 7.5-year supplementation, 120 mg/d vitamin C, 30 mg/d vitamin E, 6 mg/d β-carotene, 100 µg/d Se, 20 mg/d zinc 6-year supplementation (9,541 subjects), 4-year ongoing follow-up (3,994 treated, 738 passive follow-up), 400 IU/d Vitamin E

United Kingdom

SUVIMAX Study, Hercberg et al., 2004 (11) Meyer et al., 2005 (12)


Heart Outcomes Prevention Evaluation, Lonn et al., 2005 (13)


Patients with cardiovascular disease or diabetes mellitus, ≥55 years

Men: significant decrease in total cancer incidence (RR = 0.69) and total cancer mortality (RR = 0.63) No differences in cancer incidence and mortality, higher risk of heart failure (RR = 1.21)

a: Abbreviations are as follows: RR, relative risk; M, men; W, women; Se, selenium.

vascular diseases (18). Taken as a whole, these findings raise strong concerns about the use of galenic antioxidants for cancer prevention and limit the contribution of antioxidants to the protective effects of fruit and vegetables. Vitamin C, vitamin E, and β-carotene, due to their undoubted antioxidant properties, have gained considerable importance during the last 20 yr. However, despite the well recognized role as antioxidant vitamins and provitamins, their role as anticancer agents remain ambiguous. If we consider their contribution to the antioxidant properties of fruit and vegetables, the “noble” vitamins are relatively small contributors of the total an234

tioxidant capacity of plant food where hundreds of different antioxidants are present. Moreover, antioxidant molecules do not act in isolation and synergistic interactions must be taken into account to properly assess the impact of antioxidants on health. The antioxidant network might represent the physiological combination of molecules endowed of different redox potentials synergistically networking against oxidative stress. On this basis, the process of understanding the role played by the antioxidant molecules in the anticancer effect of fruit and vegetables requires more information on the overall antioxidant properties of body fluids and plant foods. Nutrition and Cancer 2006

Non-Enzymatic Antioxidant Network Antioxidant defenses of the body are composed of molecular and enzymatic players; however, the composition of the network markedly differs in terms of concentration and components in different environments. Protection at the cellular level is mainlyguaranteed byenzymes (superoxide dismutase, catalase, and glutathione peroxidase) and glutathione, whereas in plasma, non-enzymatic antioxidants are playing the major role. Antioxidant molecules are located in both the hydrophilic and hydrophobic compartments of plasma and work in concert to decrease RONS concentration and to inactivate transition metal ions. Metal chelating antioxidants such as transferrin, albumin, and ceruloplasmin avoid radical production by inhibiting the Fenton reaction catalyzed by copper or iron. Chain breaking or free radical scavenging antioxidants include low-molecular weight compounds such as uric acid, bilirubin, thiols, vitamin E, ascorbic acid, carotenoids, and coenzyme Q10. It has been recently suggested that phenolic compounds, a class of secondary metabolites widely present in plant foods, might contribute to the plasma antioxidant network. However, the low concentration of phenolics in plasma and the lack of identification of their active metabolites do not allow for firm conclusions about their role as possible components of the in vivo antioxidant network to be drawn. In terms of the participation of individual components to the network, calculated on the basis of the single individual concentration and respective antioxidant stochiometric coefficient, the main contributor to TAC is uric acid (40–55%), followed by thiol groups (10–24%), ascorbic acid (8–15%), and vitamin E (less than 10%) (19–21). Contribution of single antioxidants to overall TAC leaves unexplained a percentage ranging from 20–40% that might be accounted for by unknown components, unmeasured molecules (Coenzyme Q10), and/or synergistic interactions. It has been shown that the tocopheroxyl radical, produced by the reaction in the outer layer of membranes between α-tocopherol and RONS, is reduced back to α-tocopherol by ascorbic acid located in the cytoplasm (22). The antioxidant network, due to its wide range of redox potential and localization, might allow a complete and efficient protection against radical attack generated in the different compartments. In this sense, when plasma is exposed to a continuous flux of aqueous peroxyl radicals from cigarette smoke, a chronological order of consumption is observed. The first antioxidant to be consumed is ascorbic acid, dropping its concentration from 45 µM to less than 1 µM after two cigarette puffs, followed by protein thiols, bilirubin, uric acid, and α-tocopherol (23). When oxidation is driven by lipid radical inducers in a lipid environment, α-tocopherol and carotenoids behave as the first line of defence followed by uric acid (24,25). The multifunctional properties and the versatility of the antioxidant network highlight the efficiency of the dynamic interactions between the component of the network in protecting plasma from radical attack driven by heterogeneous sources. Vol. 56, No. 2

TAC Measurement in Body Fluids: A Note In the last decade a large number of assays for measuring TAC in foods and biological matrixes have been developed (3). These assays are extremely diverse and choosing the appropriate analytical method and deciphering the results obtained can be extremely complex. Without going into detail about the technical meaning of TAC measurements, which is out of the aims of this article and can be found in more specific review (3), we would like to point out that the “perfect” method does not exist and every single assay likely furnishes specific and unique information. To best fit the generally accepted principle that antioxidant related processes are multifactorial, a battery of tests should be performed on biological samples. Keeping in mind the principal characteristics of each assay, information should be merged to have a correct picture of the phenomenon. We have decided to focus on the chain-breaking antioxidant potential, which utilizes a hydrogen atom transfer mechanism, as measured by the total radical-trapping antioxidant parameter (TRAP) assay (26) and on the reducing power, which utilizes an electron transfer reaction mechanism, as measured by the ferric reducing antioxidant potential (FRAP) assay (19).

Dietary Modulation of the Antioxidant Network Different studies have investigated the ability of diet to modulate plasma TAC following consumption of foods rich in antioxidants in human subjects. The first among these acute intervention studies, where TAC was monitored before and after food ingestion, were the works of Serafini et al. (27) and Maxwell et al. (28), which successfully demonstrated that both tea and red wine were able to boost plasma TAC. This preliminary evidence has been followed by studies showing the ability of different fruit juices, onions, lettuce, chocolate, honey, and so forth to modulate plasma TAC (3). The kinetics of these effects on TAC varies depending on the foods: liquids such as wine and tea show a peak immediately after consumption (30–60 min), solid foods such as lettuce increase TAC after 2–3 h, with levels returning to baseline in about 4–5 h (26). Although the acute approach is useful for obtaining information about the ability of food items to increase TAC in vivo, long-term dietary intervention trials are needed to fully understand the role of diet in modulating the plasma antioxidant network (Table 2) (29–38). For the first time in 1998, Cao et al. (29) observed an increase in plasma TAC after consumption of 10 servings of fruit and vegetables per day for 2 wk. Interestingly, the daily intake of TAC from fruit and vegetables during the previous 12 mo was significantly correlated with fasting plasma TAC suggesting a link between diet and the plasma antioxidant network. In agreement with this evidence, Lee et al. (33) observed an increase in plasma TAC after 7 days of consumption of tomato products with extra virgin olive oil. Leighton et al. (30) showed that plasma 235

Table 2. Overview of Chronic Human Intervention Studies on Diet and Plasma TACa
Results Study (Ref.) Cao et al., 1998 (29) Design Volunteers: 36 healthy subjects Duration: 2 wk Diet: 10 servings fruits and vegetables per day Volunteers: 21 healthy subjects Duration: 1 mo Diet: Mediterranean diet Volunteers: 5 healthy subjects Duration: 1 wk Diet: Fruit juice (up to 1500 mL) Volunteers: 23 healthy subjects Duration: 8 wk Diet: vegetable juice 330mL, spinach (10 g) Volunteers: 6 healthy subjects Duration: 5 wk Diet: tomato products (430 g) Volunteers: 25 healthy subjects Duration: 2 wk Diet: 5–7 servings of fruits and vegetables per day Volunteers: 22 healthy smokers Duration:3 wk Diet: vegetable burger, fruit drink Volunteers: 18 healthy smoking subjects Duration: 3 wk Diet: 5 servings of fruits and vegetables per day Volunteers: 43 healthy subjects Duration: 25 days Diet: 600 g fruits and vegetables per day Volunteers: 3,042 healthy subjects Duration: 1 yr Diet: Mediterranean diet TAC ↑ ↑

Method ORAC

Leighton et al., 1999 (30)


Young et al., 1999 (31)



Bub et al., 2000 (32)



Lee et al., 2000 (33)


Record et al., 2001 (34)



Van den Berg et al., 2001 (35)

↑ ↑


Roberts et al., 2003 (36)


Dragsted et al., 2004 (37)



Pitsavos et al., 2005 (38)

ImAnOx kit

a: Abbreviations are as follows: TAC, total antioxidant capacity; TEAC, Trolox equivalent antioxidant capacity; FRAP, ferric reducing antioxidant power; ORAC, oxygen radical antioxidant capacity; ImAnOx, Immunodiagnostik AG, Bensheim, Germany. b: Significant increments (↑) or no change (=) in plasma TAC.

TAC levels were increased after 1 mo in 21 healthy male volunteers following a plant-based Mediterranean diet. Roberts et al. (36) reported a notable increase in TAC after consumption of five portions of fruits and vegetables per day for 3 wk in 18 free-living healthy smoking volunteers. Similarly, in healthy smoking subjects consuming a daily vegetable burger and a fruit drink for 3 wk, plasma TAC showed a significant increase (35). Conversely, no change in plasma TAC was observed following consumption of a diet high in antioxidants, five to seven servings of fruit and vegetables per day for 2 wk in 25 healthy nonsmokers (34). The same conclusion was also reached by Young et al. (31) who were unable to observe any increase in plasma TAC after 1 wk of supplementation with 1500 ml of fruit juice per day. Dragsted et al. conducted an intervention trial on 43 young subjects randomly divided in 3 groups (37). The control group was asked to abstain from eating food with antioxidants for 25 days, where the other groups were supplemented with 600 g/day of fruits and vegetables and with pills containing the amount of vitamins and minerals contained in 600 g of fruit and vegetables. Results showed that

in all three groups plasma TAC and markers of oxidative damage and antioxidant enzymes were unaffected by the supplementation with plant foods, pills, or by the withdrawal of antioxidants from the diet (37). Serafini et al. (39) pointed out that, due to their ability to cope with light dietary stress, plasma antioxidant defences may need more than 25 days or specific and stronger dietary stresses, such as a high fat diet, to be challenged significantly in healthy young subjects. Moreover, the lack of changes in plasma TAC levels in the plant foods group could be due to physiological regulatory mechanisms that in the short-term buffers against a significant decline in plasma TAC. The only existing evidence based on a large number of subjects (3,042 subjects) is the ATTICA (Greek province) study where a significant association between plasma TAC and the Mediterranean diet score, an index of the adherence of the diet to a standard Mediterranean pattern, was observed (38). Plasma TAC was also positively correlated with the consumption of fruit, vegetables, and olive oil. Despite the lack of effect found in some studies, most of the evidence suggests a direct role of dietary antioxidants in


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modulating the plasma antioxidant network. Modality of ingestion, existence of homeostatic mechanisms of regulation, and physiological diversity in the absorption and disposal of antioxidants are variables that might affect the ability of diet to modulate plasma TAC in vivo.

that dietary associations can affect the in vivo antioxidant activity of phenolic rich food cannot be ignored and needs to be further investigated.

Plasma TAC and Cancer: In Vivo Evidence Does the Modality of Ingestion Affect the In Vivo Antioxidant Properties of Plant Food? The possibility that the in vivo efficiency of dietary antioxidants might be affected by the consumption of different foods in combination was introduced for the first time in 1996 when Serafini et al. (40) reported that the increase in plasma TAC following the consumption of both green and black tea was markedly reduced when the teas were consumed with milk. In agreement with this finding, a report by Langley-Evans showed a marked inhibition of milk addition on the antioxidant properties of black tea (41). These inhibitory effects of milk are in keeping with a report that the catechin content, antioxidant capacity, and antimutagenic activity of jejunal dialysates obtained after passage of teas through an in vitro gastrointestinal model were much reduced by the addition of milk to the teas (42). The antioxidant capacity of teas is related to the concentration of phenolic compounds (43), however, in vivo, the fate of dietary phenolics in the body show a low (1%) degree of absorption (44). The effect of the matrix in which phenolics are ingested might further affect their bioavailability through the strong interactions between phenolics and proteins leading to the formation of secondary bonds (45). These interactions might reduce the biological accessibility of the phenolics and therefore their potential antioxidant properties in vivo. However, results not supporting this hypothesis came from the Unilever Research Group in the Netherlands who reported that consumption of a single dose of either green or black tea resulted in an increase in plasma TAC that was not abolished by the addition of milk to the tea (46). However, it is important to underline the fact that the increases in plasma TAC reported by Leenen et al. (46) following tea consumption without milk (+3%) were much smaller than those observed by Serafini et al. (40) and Langley-Evans (+65% and +42%, respectively) (41). Moreover, lyophilized tea was used instead of tea leave infusions, which were used in the other trials. In a recent work the Serafini group showed that consumption of dark chocolate results in an increase in both the TAC and (-)-epicatechin content of plasma but that these effects are markedly reduced when either the dark chocolate was consumed with milk or milk chocolate is eaten (47). However, according to the reply to our study from Schroeter et al. (48), when a chocolate beverage drink rather than solid chocolate is given to subjects, the food matrix effect disappeared. However, the very low amount of lipids (3% as opposed to 30% in Serafini’s work) might represent the main reason for the lack of a matrix effect. Thus, the possibility Despite the likely importance of plasma TAC as a protective factor, its concerted impact on the incidence of cancer has not been adequately investigated and in vivo studies on TAC in people affected by cancer are very limited. Di Giacomo et al. (49) showed a significant decrease in TAC accompanied by a significant increase in oxidized proteins and lipid peroxides in colon cancer patients as well as in patients with precancerous lesions. An impairment of the antioxidant network also has been observed in a group of patients with cervical intraepithelial neoplasia (CIN), who had significantly lower plasma levels of TAC and vitamin C with respect to the control group. This imbalanced condition suggests degeneration in the antioxidant system and an overproduction of oxidants that was also confirmed by higher plasma malondialdehyde (a marker of lipid oxidation) concentrations in CIN patients than in controls (50). An altered redox balance seems to affect the TAC of body fluids as well as of organs. Liu et al. (51) assessed the TAC of plasma and of malignant pleural effusion (MPE) and DNA damage of tumour-associated lymphocytes in MPE from patients with carcinoma. Most of MPE are the results of malignant tumour metastasised to pleura, such as lung and breast cancer. They observed a decline in the TAC either in plasma or MPE, suggesting an impairment of the antioxidant network in cancer patients, and an ongoing oxidative insult present at the systemic level as well as at the tumour site. Moreover, a negative relationship was shown between the TAC of the MPE supernatant and DNA damage of lymphocytes suggesting that impairment of the antioxidant network might be related to a high degree of DNA damage. Erhola et al. (52) observed significantly lower TAC values as well as levels of all the TAC components (i.e., protein SH-groups, ascorbic acid and vitamin E) in lung cancer patients when compared to healthy controls. Mantovani et al. (53) showed that plasma TAC in 82 advanced-stage cancer patients was lower than in control subjects, whereas serum levels of pro-inflammatory cytokines (i.e., IL-6 and TNFα) and C-reactive protein were significantly higher in cancer patients suggesting an involvement of inflammatory cytokines in the oxidative conditions associated with cancer development. In a case control study, Ching et al. (54) showed that increased serum concentrations of β-carotene, retinol, bilirubin, and TAC were associated with significant reductions in breast cancer risk. There was a significant reduction in the odds ratio for breast cancer risk at the 2nd quartile of plasma levels of TAC (OR 0.52) while the risk did not change markedly in the 2nd and 3rd quartile (OR 0.47 and 0.41) suggesting the existence of threshold levels associated with the protective effect of

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plasma TAC. However, despite these evidences, more studies on a large number of subjects are needed to understand the role of plasma TAC during cancer development.

Specific Databases for Epidemiological Approach of TAC The use of TAC in epidemiological studies represents a new approach that has been proposed for the first time for investigating the relationship between the dietary antioxidant network and gastric cancer risk in a population-based case-control study (55). Results from this study showed that the intake of dietary TAC through plant food was inversely associated with the risk of gastric cancer with a significant dose-response trend. Moreover, a clear dose-response relationship for increasing levels of TAC was found in smokers, while never-smokers displayed a threshold effect with a similar lower risk (odds ratios of 0.48, 0.46, and 0.44 in the last 3 quartiles of intake, respectively, compared to the first quartile of 0.56). In the gastric cancer study, a TAC database of only 12 food items was utilized, however, to properly study this at an epidemiological level complete, homogenous, and versatile databases of TAC in foods are needed. Recently three databases in which the main food contributors to dietary TAC were analyzed have been published (56,57). The database published by Halvorsen et al. (58) included data on the FRAP, measuring the reducing power of 32 vegetables, 23 fruits, 19 berries,

11 tubers, 18 cereals, 10 pulses, 6 nuts, and 4 dried fruits from different countries all around the world. In the Wu et al. (59) database, 24 fruits, 22 vegetables, 10 nuts, 4 dried fruits, 16 spices, 19 breakfast cereals, breads, and snack foods from the United States were measured by the oxygen radical absorbance capacity (ORAC) assay. In the Italian database 34 vegetables, 30 fruits, 34 beverages, 6 vegetable oils, 11 spices, 5 dried fruits, 7 sweets, 18 cereal products, 5 pulses, and 6 nuts were evaluated with 3 different methods: TRAP, FRAP, and trolox equivalent antioxidant capacity assays for measuring, respectively, the chain-breaking antioxidant potential, the reducing power, and the quenching ability (57,60). One of the important points that has not been considered by the existing TAC database is represented by the effect of cooking procedure on TAC values in vegetables. Previous results have shown that the cooking procedure might change the content and the pattern of dietary antioxidants and, thus, should be considered to properly assess the “real” TAC intake. We have assessed the effect of cooking procedure (i.e., boiling, pan-frying, and deep-frying) on the TAC values of the main commonly consumed cooked vegetables in the Italian diet. In Table 3, the TAC values, expressed per kilogram of raw or cooked vegetable, are presented. The overall results showed a general increase of TAC after cooking, independently on the TAC assays used. Among the applied cooking methods, boiling resulted in the smallest increase in TAC, whereas the deep-frying resulted in the highest increase in TAC. The increase of TAC values due to the heating could be

Table 3. Ferric Reducing Antioxidant Potential (FRAP), Total Radical-Trapping Antioxidant Parameter (TRAP), and Trolox Equivalent Antioxidant Capacity (TEAC) of Raw (uncooked) and Cooked Vegetable Extractsa,b
Vegetable Artichoke Cooking Method raw boil pan-fry deep-fry raw boil pan-fry deep-fry raw boil pan-fry raw boil pan-fry raw boil pan-fry raw pan-fry deep-fry raw boil pan-fry FRAP (mmol Fe /kg) 17.06 60.10 28.45 86.01 12.06 12.34 16.79 39.49 1.47 1.08 2.38 8.89 7.17 10.86 7.58 10.55 31.09 4.29 5.77 6.58 25.50 14.37 61.68

TRAP (mmol Trolox/kg) 6.30 29.03 11.93 85.49 3.77 3.02 7.04 9.35 0.34 0.27 0.73 1.59 1.05 1.89 1.47 3.04 11.49 1.52 2.31 2.87 8.58 4.12 24.42

TEAC (mmol Trolox/kg) 3.04 9.52 4.51 30.36 2.35 2.90 3.37 9.55 0.59 0.47 0.99 1.57 2.02 2.52 2.07 2.38 6.25 0.93 1.27 1.57 5.59 3.62 15.55





Onion (yellow)

Turnip tops

a: Values represent the sum of the water- and lipo-soluble extracts. b: Values are referred to wet weight.


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explained by a major release of antioxidant compounds from the food matrix, more evident for fiber-rich vegetables such as artichokes and turnip tops where the TAC increase after cooking was remarkable (Table 3). This result clearly shows that TAC values are deeply affected by the cooking procedure, and this aspect cannot be ignored when the intake of dietary TAC is determined.




Conclusions The potential importance of the antioxidant network in cancer prevention is a relatively new topic of research. Although there are still aspects that need to be further investigated, evidence is mounting that TAC measurement might represent a promising tool to monitor the non-enzymatic antioxidant network in vivo and in epidemiological studies. Dietary and plasma TAC assessment in large clinical trials represents a crucial and unavoidable step to clarify the associations between diet, antioxidant status, and cancer prevention. In conclusion, after the contrasting results of clinical trials involving galenic antioxidants, the road to understanding the biological and chemopreventive actions of dietary antioxidants cannot be focused only on the search of a “magic bullet” but needs to consider all the redox players and their interactions.







Acknowledgments and Notes
Address correspondence to M. Serafini, Antioxidant Research Laboratory at the Unit of Human Nutrition, Istituto Nazionale di Ricerca Alimenti e Nutrizione, Via Ardeatina 546, 00178 Rome, Italy. Phone: +390651494557. FAX: +390651494550. E-mail: 18.



1. Halliwell B and Gutteridge JMC (eds): Free radicals in Biology and Medicine. Oxford, UK: Clarendon Press, 1989. 2. Krinsky NI and Johnson EJ: Carotenoid actions and their relation to health and disease. Molecular Aspects of Medicine 26, 459–516, 2005. 3. Serafini M and Del Rio D: Understanding the association between dietary antioxidants, red-ox status and disease: is the Total Antioxidant Capacity the right tool?. Redox report 9, 145–152, 2004. 4. Blot WJ, Li JY, Taylor RR, Guo W, Dawsey S, Wang GQ, et al.: Nutrition intervention trials in Linxian, China. Supplementation with specific vitamin and mineral combinations, cancer incidence and disease-specific mortality in the general population. J Natl Cancer Inst 85, 1483–1492, 1993. 5. The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. New Engl J Med 330, 1029–1035, 1994. 6. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass, A, et al.: Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. New Engl J Med 334, 1150–1155, 1996. 7. Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, et al.: Lack of effect of long-term supplementation with beta caro21.







tene on the incidence of malignant neoplasms and cardiovascular disease. New Engl J Med 334, 1145–1149, 1996. Clark LC, Combs GF, Turnbull BW, Slate EH, Chalker DK, Chow J, et al.: Effects of Selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized control trial. JAMA 276, 1957–1963, 1996. Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, et al.: Vitamin E in the primary prevention of cardiovascular disease and cancer: the Women’s Health Study: a randomized controlled trial. JAMA 694, 56–65, 2005. Heart Protection Study Collaborative Group: MRC/BHF heart protection study of antioxidant vitamin supplementation in 20536 high-risk individuals: a randomised placebo-controlled trial. Lancet 360, 23–33, 2002. Hercberg S, Galan P, Perziosi P, Bertrais S, Mennen L, Malvy D, et al.: The Su.VI.MAX. study – a randomised, placebo-controlled trial of the health effects of antioxidant vitamins and minerals. Arch Int Med 164, 2335–2342, 2004. Meyer F, Galan P, Douville P, Bairati I, Kegle P, Bertrais S, et al.: Antioxidant vitamin and mineral supplementation and prostate cancer prevention in the SU.VI.MAX trial. Int J Cancer 116, 182–186, 2005. Lonn E, Bosch J, Yusuf S, Sheridan P, Pogue J, Arnold JM, et al.: Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. JAMA 293, 1338–1347, 2005. Bjelakovic G, Nikolova D, Simonetti RG, and Gluud C: Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis. Lancet 364,1219–1228, 2004. Evans JR: Antioxidant vitamin and mineral supplements for slowing the progression of age-related macular degeneration. Cochrane Database Syst Rev 19, CD000254, 2006. Caraballoso M, Sacristan M, Serra C, and Bonfill X: Drugs for preventing lung cancer in healthy people. In: The Cochrane Library, Issue 2, 2004. Oxford: Wiley/Uodate Software. Miller ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel L, and Guallar E: Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Inter Med 142, 37–46, 2004. Vivekananthan DP, Penn MS, Sapp SK, Hsu A, and Topol EJ: Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomized trials. Lancet 361, 2017–2023, 2003. Benzie IFF and Strain JJ: The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal Biochem 239, 70–76, 1996 Valkonen M and Kuusi T: Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum. J Lipid Res 38, 823–833, 1997 Wayner DD, Burton GW, Ingold KU, Barclay LR, and Locke SJ: The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochem Biophys Acta 924, 408–419, 1987. Chaudiere J and Ferrari-Iliou R: Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 37, 949–962, 1999. Frei B, Forte TM, Ames BN, and Cross CE: Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma. Biochem J. 277, 133–138, 1991. Aldini G, Yeum KJ, Russell RM, and Krinsky NI: A method to measure the oxidizability of both the aqueous and lipid compartments of plasma. Free Rad Biol Med 31, 1043–1050, 2001. Yeum K-J, Aldini G, Chung H-Y, Krinsky NI, and Russell RM: The activities of antioxidant nutrients in human plasma depend on the localization of attacking radical species. J Nutr 133, 2688–2691, 2003. Serafini M, Bugianesi R, Salucci M, Maiani G, and Azzini, E: Effect of acute ingestion of fresh and stored lettuce on plasma total antioxidant capacity and antioxidant levels in humans. Brit. J. Nutr. 88, 615–623, 2002. Serafini M, Ghiselli A, and Ferro-Luzzi A: Red wine, tea and antioxidants. Lancet 344, 626, 1994.

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28. Maxwell S, Cruickshank A, and Thorpe G: Red wine and antioxidant activity in serum. Lancet 344, 193–194, 1994. 29. Cao G, Booth SL, Sadowski JA, and Prior RL: Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. Am J Clin Nutr 68, 1081–1087, 1998. 30. Leighton F, Cuevas A, Guasch V, Perez DD, Strobel P, San Martin A, et al.: Plasma polyphenols and antioxidants, oxidative DNA damage and endothelial function in a diet and wine intervention study in humans. Drugs Exp Clin Res 25, 133–41, 1999. 31. Young JF, Nielsen SE, Haraldsdottir J, Daneshvar B, Lauridsen ST, Knuthsen P, et al.: Effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status. Am J Clin Nutr 69, 87–94, 1999. 32. Bub A, Watzl B, Muller H, and Rechkemmer G: Moderate intervention with carotenoid-rich vegetable products reduces lipid peroxidation in men. J Nutr 130, 2200–2206, 2000. 33. Lee A, Thurnham DI, and Chopra M: Consumption of tomato products with olive oil but not sunflower oil increases the antioxidant activity of plasma. Free Radic Biol Med 29, 1051–1055, 2000. 34. Record IR, Dreosti IE, and McInerney JK: Changes in plasma antioxidant status following consumption of diets high or low in fruit and vegetables or following dietary supplementation with an antioxidant mixture. Br J Nutr 85, 459–464, 2001. 35. Van den Berg R, van Vliet T, Broekmans WM, Cnubben NH, Vaes WH, Roza L, et al.: A vegetable/fruit concentrate with high antioxidant capacity has no effect on biomarkers of antioxidant status in male smokers. J Nutr 131, 1714–1722, 2001. 36. Roberts WG, Gordon MH, and Walker AF: Effects of enhanced consumption of fruit and vegetables on plasma antioxidant status and oxidative resistance of LDL in smokers supplemented with fish oil. Eur J Clin Nutr 57, 1303–1310, 2003. 37. Dragsted LO, Pedersen A, Hermetter A, Basu S, Hansen M, Haren GR, et al.: The 6-a-day study: effects of fruit and vegetables on markers of oxidative stress and antioxidative defence in healthy nonsmokers. Am J Clin Nutr 79, 1060–1072, 2004. 38. Pitsavos C, Panagiotakos DB, Tzima N, Chrysohoou C, Economou M, Zampelas A, et al.: Adherence to the Mediterranean diet is associated with total antioxidant capacity in healthy adults: the ATTICA study. Am J Clin Nutr 82, 694–699, 2005. 39. Serafini M, Del Rio D, Crozier A, and Benzie IF: Effect of changes in fruit and vegetable intake on plasma antioxidant defenses in humans. Am J Clin Nutr 81, 531–532, 2005. 40. Serafini M, Ghiselli A, and Ferro-Luzzi A: In vivo antioxidant effect of green and black tea in man. Eur J Clin Nutr 50, 28–32, 1996. 41. Langley-Evans SC: Consumption of black tea elicits an increase in plasma antioxidant potential in humans. Int J Food Sci Nutr 51, 309–315, 2000. 42. Krul C, Schuite AL, Tenfelde A, van Ommen B, Verhagen H, and Havenaar R: Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model. Mut Res 474, 71–85, 2001. 43. Mukhtar H and Ahmad N: Tea polyphenols: prevention of cancer and optimizing health. Am J Clin Nutr 71, 1698S-1702S, 2000. 44. Manach C, Williamson G, Morand C, Scalbert A, and Remesy C: Bioavailability and bioefficacy of polyphenols in humans: I: Review of 97 bioavailability studies. Am J Clin Nutr 81, 230S–242S, 2005

45. Charlton AJ, Baxter NJ, Khan ML, Moir AJ, Haslam E, Davies AP, et al.: Polyphenol/peptide binding and precipitation. J Agric Food Chem 50, 1593–1601, 2002. 46. Leenen R, Rodenburg AJ, Tijburg LB, and Wiseman SA: A single dose of tea with or without milk increases plasma antioxidant activity in humans. Eur J Clin Nutr 54, 87–92, 2000. 47. Serafini M, Bugianesi R, Maiani M, Valtueòa S, De Santis S, and Crozier A: Plasma antioxidants from chocolate. Nature 424, 1013, 2003. 48. Schroeter H, Holt RR, Orozco TJ, Schimtz HH, and Keen CL: Nutrition: milk and absorption of dietary flavanols. Nature 426, 787–788. 49. Di Giacomo C, Acquaviva R, Lanteri R, Licata F, Licata A, and Vanella A: Nonproteic antioxidant status in plasma of subjects with colon cancer. Exp Biol Med 228, 525–528, 2003. 50. Lee GJ, Chung HW, Lee KH, and Ahn HS: Antioxidant vitamins and lipid peroxidation in patients with cervical intraepithelial neoplasia. J Korean Med Sci 20, 267–272, 2005. 51. Liu X, Zhao J, and Zheng R: DNA damage of tumor-associated lymphocytes and total antioxidant capacity in cancerous patients. Mutat Res 539, 1–8, 2003. 52. Erhola M, Nieminen MM, Kellokumpu-Lehtinen P, Metsa-Ketela T, Poussa T, and Alho H: Plasma peroxyl radical trapping capacity in lung cancer patients: a case-control study. Free Rad Res 26, 439–447, 1997. 53. Mantovani G, Maccio A, Madeddu C, Mura L, Gramignano G, Lusso MR, et al.: Quantitative evaluation of oxidative stress, chronic inflammatory indices and leptin in cancer patients: correlation with stage and performance status. Int J Cancer 98, 84–91, 2002. 54. Ching S, Ingram D, Hahnel R, Beilby J, and Rossi E: Serum levels of micronutrients, antioxidants and total antioxidant status predict risk of breast cancer in a case control study. J Nutr 132, 303–306, 2002. 55. Serafini M, Bellocco R, Wolk A, and Ekstrom AM: Total antioxidant potential of fruit and vegetables and risk of gastric cancer. Gastroenterology 123, 985–991, 2002 56. Dragland S, Senoo H, Wake K, Holte K, and Blomhoff R: Several culinary and medical herbs are important sources of dietary antioxidants. J Nutr 133, 1286–1290, 2003. 57. Pellegrini N, Serafini M, Salvatore S, Del Rio D, Bianchi M, and Brighenti F: Total antioxidant capacity of spices, dried fruits, nuts, pulses, cereals and sweets consumed in Italy assessed by three different in vitro assays. Mol Nutr Food Res 50, 1030–1038, 2006. 58. Halvorsen BL, Holte K, Myhrstad MCW, Barikmo I, Hvattum E, Fagertun Remberg S, et al.: A systematic screening of total antioxidants in dietary plants. J Nutr 132, 461–471, 2002. 59. Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, and Prior RL: Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J Agric Food Chem 52, 4026–4037, 2004. 60. Pellegrini N, Serafini M, Colombi B, Del Rio D, Salvatore S, Bianchi M, et al.: Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. J Nutr 133, 2812–2819, 2003.


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