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Effects of diet energy level and tomato powder consumption on antioxidant status in rats
ARTICLE IN PRESS Clinical Nutrition (2005) 24, 1038–1046
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Effects of diet energy level and tomato powder consumption on antioxidant status in rats Emilia A.M. Moreiraa,, Regina L.M. Fagundesa, Danilo Wilhelm Filhob, Daniela Nevesa, Fabı´ola Sellb, France Bellislec, Emil Kupekd Departamento de Nutric- a ˜o, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil b Departamento de Ecologia e Zoologia, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil c Centre National de la Recherche Scientifique, Inserm U 341, Hotel Dieu, Paris, France d Departamento de Sau ´de Pu ´blica, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil a
Received 18 January 2005; accepted 9 August 2005
KEYWORDS Energy intake; Tomato powder; Lycopene; Antioxidants; Oxidative stress
Summary Background and aims: Evaluate the influence of tomato powder in diets differing in energy level on antioxidant status in blood and liver of rats. Methods: Twenty-four adult male Wistar rats weighing 150–180 g were placed four groups (n ¼ 6). For 28 days, animals were fed a diet that was either hyper energetic or hypo energetic. Some diets were supplemented with tomato powder. Liver and blood were collected for analysis of antioxidant enzymes, non-enzymatic antioxidants, thiobarbituric acid reactive species, ubiquinol 9, a-tocopherol, lycopene and b-carotene. Data were analysed by two-way ANOVA. Results: Food intake and thiobarbituric acid-reactive substances contents in liver and plasma were significantly decreased by tomato powder at both energy levels. After tomato powder supplementation, the hepatic levels of ubiquinol 9, a-tocopherol, lycopene and b-carotene were significantly enhanced. In plasma, only the contents of lycopene and b-carotene were enhanced. The erythrocytic and hepatic activities of catalase were lower, while those of glutathione peroxidase were higher after the ingestion of tomato powder. Total and reduced glutathione contents in liver showed lower levels in cafeteria-fed rats compared to the hypo energetic diet.
Corresponding author. Tel.: +55 48 3319784; fax: +55 48 3319542.
E-mail address: [email protected] (E.A.M. Moreira). 0261-5614/$ - see front matter & 2005 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2005.08.005
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Conclusions: The data suggest that the lycopene and b-carotene component in the tomato power supplement might be beneficial for the prevention of oxidative damage in rats fed both types of energetic diets. & 2005 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction More than 700 carotenoids have been identified from plants and microorganisms.1 Among them, lycopene and b-carotene possess the most pronounced antioxidant properties.1–5 In fact, lycopene has a higher antioxidant potential than a-tocopherol and b-carotene.3,6,7 The mechanism ultimately responsible for the antioxidant property of carotenoids is still unknown.1,6 Since the in vitro demonstration by DiMascio et al.3 indicating that lycopene is the most efficient biological carotenoid quencher of singlet oxygen, research involving lycopene and tomato-derived products has increased strikingly.1,7 Lipophylic antioxidants such as tocopherols and carotenoids inhibit reactive oxygen species (ROS) propagation reactions through their chain-breaking ability.1,2,4,5 Lycopene also increases the antioxidant potential and lowers the generation of ROS such as hydroxyl radical and peroxides.1,7,8 The most abundant carotenoid present in tomato is lycopene, followed by b- and g-carotene.8,9 Compared to fresh tomatoes, processed tomato products show decreased antioxidant capacity in the hydrophilic fraction due mostly to vitamin C losses; however, they possess a higher antioxidant capacity in their lipophylic fraction, which contains carotenoids and tocopherols.10 Another advantage in the consumption of processed tomato products is that they are more bioavailable sources of lycopene compared to fresh tomatoes.7,11,12 The metabolism of oxygen in all aerobic organisms implies the continuous production of large amounts of ROS, which are deleterious to biomolecules such as DNA, lipids, sugars and proteins.13 ROS can elicit lipoperoxidation, a propagation reaction that damages cell membranes, provokes alterations in normal cell and tissue functions and contributes to many disease processes, including carcinogenesis and coronary diseases as well as the aging process.8,14,15 Oxygen toxicity is counteracted by endogenous enzymatic and non-enzymatic antioxidants and also by the presence of several nutritional antioxidants such as vitamins E and C, carotenoids and flavonoids.7,16–20 Oxidative stress5 is characterized by an imbalance between antioxidant capacity and ROS \generation. Because a nutritional deficiency of
antioxidants can be responsible for such a condition,21 the balance between ROS generation and antioxidant availability should be taken into consideration in diet studies. In most Western countries, excessive fat ingestion is common and is associated with the prevalence of obesity, cardiovascular diseases and cancer. Some studies have shown that hypo energetic diets can decrease the incidence of spontaneous or chemically induced degenerative diseases. On the other hand, hyper energetic diets seem to enhance lipoperoxidation and, therefore, elicit oxidative stress in various tissues.14 The main objective of the present study is to evaluate the influence of tomato powder (Lycopersicon esculentum) in diets differing in energy content on antioxidant status in the blood and liver of rats.
Material and methods Animals and treatment Adult male Wister rats weighing 150–180 g were used. Twenty-four animals were randomly assigned in four groups (n ¼ 6 in each group). During one week of acclimatization, they were kept in metabolic cages under controlled temperature and dark–light cycles (12:12, lights on at 06:00 h) with water and food ad libitum. Standard diets were manufactured and stored at 20 1C under vacuum to prevent oxidation. The contents of antioxidant vitamins in the basal diet were in accordance to AIN-93M22,23: vitamin E (a-tocopherol acetate) 75 IU kg1 and vitamin A, 4000 IU kg1. After the first week, the animals were treated for 4 weeks, which is sufficient time to obtain a dietary effect on lipid metabolism.24 The following diets were used: (a) group 1—hypo energetic diet; (b) group 2—‘‘cafeteria’’ diet (hyper energetic); (c) group 3—hypo energetic diet plus tomato powder; and (d) group 4—‘‘cafeteria’’ diet plus tomato powder. The hypo energetic diets were defined according to a consumption limit (30% lower in grams) using the standard animal diet based on the AIN-93M recommendations.22,23 In the ‘‘cafeteria’’ diet (hyper energetic/hyper lipidic), the control diet was available ad libitum together with a variety of
ARTICLE IN PRESS 1040 highly palatable foods using the model of ‘‘incomplete counterbalanced blocks’’25 in which three different foods such as cookies, chocolate and snacks were offered containing 5 g portions each, totaling 15 g per day, and 15 g of control diet ad libitum. The composition of the cafeteria diet can be found elsewhere.26,27 In the lycopene groups, 5 g of tomato powder (corresponding to approximately 20–30 mg lycopene kg1 day1) were given daily to rats mixed in the control diet in a proportion of 25% of tomato powder (5 g lycopene:15 g control diet), also containing the same amount of lycopene. Tomato powder has the highest lycopene concentration (1.1–1.3 mg g1 weight),28 but rats absorb carotenoids less efficiently than humans and rapidly convert carotenoids in the gut.5–7 Therefore, in the present study the amount of lycopene present in the tomato powder given to the experimental animals was used to reach values near to those found in plasma of human subjects. The experimental protocols were in accordance with the Helsinki Declaration.
Parameters analysed Fasted animals were killed by cervical dislocation. After weighing the animals, the livers were rapidly removed, kept on ice, and perfused with ice-cold saline (0.9% NaCl) solution for 5 min before being homogenised (1:9 w/v) in a buffer comprising 0.1% Triton X-100, 0.12 M NaCl, 30 mM Na2PO4, and pH 7.4. Homogenization was carried out at 4 1C, employing 20 strokes of a Potter–Elvehjen homogenizer. This was followed by centrifugation at 10,000g for 10 min at 5 1C. Supernatants were stored in liquid nitrogen until analysis by enzymatic assays. Blood was drawn from the caudal vein in heparinized syringes. After plasma removal, red cells were washed three times in saline solution. Hemolysates were obtained after addition of three volumes of 20 mM Tris–HCl, pH 8.0 and centrifuged at 3000g for 5 min at 5 1C. Hemolysate supernatants were used for enzymatic analysis.
Antioxidant enzymes Catalase activity (CAT): To measure CAT activity in blood, the hemolysates were further diluted 500 times.29 CAT was quantified by measuring the decay of a 10 mM hydrogen peroxide solution at 240 nm.29 Superoxide dismutase (SOD): SOD was measured after treating the hemolysates with a mixture of chloroform/ethanol (3:5 v:v), or using the supernatants from liver homogenates, and quantified at 550 nm by measuring the rate of cytochrome c
E.A.M. Moreira et al. reduction.30 Glutathione reductase (GR): this activitiy was assayed at 340 nm by measuring the rate of NADPH oxidation according to Carlberg and Mannervik.31 Glutathione S-transferase (GST): the activity was determined at 340 nm using CDNB (1-chloro-2,4-dinitrobenzene) as substrate and a 0.15 M GSH concentration.32 Glutathione peroxidase (GPx): GPx was measured at 340 nm through the glutathione/NADPH/GR system by the dismutation of tert-butylhydroperoxide.33
Non-antioxidant enzymes Reduced form of glutathione (GSH): Non-protein thiols, mostly present as the GSH, were measured at 412 nm according to Beutler34 using the Elmann’s reagent (DTNB: 2-dithionitrobenzoic acid). Immediately after liver excision tissue acid extracts were obtained by adding liver portions to 12% trichloroacetic acid (1:4 w/v), and then centrifuging the resulting solution at 5000g for 5 min at 5 1C. Supernatants from the acid extracts were added to a buffer containing 0.25 mM DTNB in 0.1 M Na2PO4, pH 8.0, and the formation of thiolate anion was immediately determined. Total glutathione (TG): was also measured at 412 nm in acid extracts according to the enzymatic method of Tietze.35 Oxidised glutathione (GSSG): was determined by calculating the difference (in equivalents of GSH) between TG and reduced glutathione contents.
Lipoperoxidation assay Thiobarbituric acid-reactive substances (TBARS): TBARS contents were determined to assess endogenous lipid oxidation in the liver according to Ohkawa36 and Bird and Draper.37 After excision, liver slices were immediately added to 12% trichloroacetic acid (1:4 v/v) and then centrifuged at 15,000g for 3 min at 5 1C. Supernatants were added to 50 mM Tris–HCl pH 7.0, vortexed for 20 s, added to 0.67% (w/v) 2-thiobarbituric acid, maintained in boiling water for 60 min, cooled at 5 1C for 30 min, and then analysed spectrophotometrically at 535 nm. Absorbance was expressed as equivalent to nmol TBARS g1 wet tissue (E535 ¼ 153 mM1 cm1). The biochemical parameters described above were measured in duplicate, except TBARS determinations, which were performed in triplicate.
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Antioxidant capacity: ubiquinol 9, a-tocopherol, lycopene and b-carotene Plasma antioxidant determinations were carried out by high performance liquid chromatography (HPLC) with in line electrochemical and UV detection. To evaluate plasma concentrations of atocopherol, lycopene and b-carotene, an aliquot of 200 ml of plasma or tissue homogenate was added to 500 ml of methanol and 4 ml of hexane. The mixture was vortexed for 1 min and then the tubes were centrifuged for 5 min at 1000g at 5 1C. A 3 ml aliquot from the hexane layer was dried under N2 flux, and the residue dissolved in 200 ml methanol:ethanol (1:1, v/v) and filtered through a 0.22 mmpore nylon membrane. To determine total ubiquinol 9 content, the extracts were reduced as follows38: extract aliquots (100 ml) were mixed with 0.5 ml of methanol, 0.5 ml of MilliQ water, and approximately 20 mg of NaBH4. The mixture was vortexed and incubated at room temperature in the dark for 30 min and then extracted into 4 ml of hexane following the steps described above. Measurements of antioxidants in the methanol:ethanol extract were made by reversed phase HPLC, using a Supelcosil LC-8 column (33 4.6 mm; 3 mm packing material), and 20 mM LiClO4 in methanol:H2O (97.7:2.5, v/v) as mobile phase. An in line electrochemical detector with a glassy-carbon working electrode (Bioanalytical System LC4C) at an applied oxidation potential of +0.6 V, and UV detector at 290 nm (Waters model 460, Milford, MA, USA) were used. Standard solutions of antioxidants were prepared according to Nierenberg et al.39 All the chemicals used in the present study were purchased from Sigma-Aldrich Co. (Ohio, USA).
Statistical methods Results are expressed as means7SEM. Differences between the four dietary groups were analysed by
Table 1
two-way ANOVA with energy level and supplementation as factors followed by Dunnett multiple range test for comparisons of means. Tests were performed using the STAT software, and P-values less than 5% (Po0:05) were considered to be statistically significant.
Results Energy intake and body weight gain Body weight gain over 4 weeks was affected by food intake and the energy content of the diets. The tomato powder supplementation decreased food intake, probably through an effect of palatability of the diet, and also energy intake. Table 1 illustrates the significant effects of energy level (high vs. low) on all three dependent variables: food intake (g day1), energy intake (kcal day1) and weight gain (g) (Po0:0001). Supplementation with tomato powder also had a significant effect on food intake (g) and energy intake (kcal) (Po0:0001). The main effect of tomato powder supplementation was not significant for body weight gain.
Liver biochemical parameters In liver (Tables 2 and 3), significant main effects of energy level were found for GSH and a-tocopherol contents and CAT, GPx, GST and SOD activities (Po0:01, Po0:05, Po0:01, Po0:05, Po0:0001, and Po0:0001, respectively). More specifically, in liver GSH (Table 2) and a-tocopherol (Table 3) contents, and also the activities of CAT, GPx, GST and SOD (Table 2) showed decreased values in cafeteria fed rats compared to rats fed the hypo energetic diet. Supplementation with tomato powder also had a significant effect on TBARS, UQ9, a-tocopherol,
Food intake, energy intake and weight gain of rats treated with different diets for 28 days.
Parameter
1
Food intake (g day ) Energy intake (kcal day1) Weight gain (g day1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 3
a
1370.1 5470.5a 2772.9a
Group 2 b
1770.4 7671.5b 9278.3b
c
1270.1 5270.5c 3273.0a
Significant effectsy
Group 4 1570.2d 6671.1d 8273.7b
E, L E, L E
E: main effect of energy level (Po0:05); L: main effect of supplementation with tomato powder (Po0:05). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). Group 1—hypo energetic diet, group 2—‘‘cafeteria’’ diet (hyper energetic), group 3—hypo energetic diet plus tomato powder, and group 4—‘‘cafeteria’’ diet plus tomato powder. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
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E.A.M. Moreira et al.
Table 2 GSH, GT, GSSG, TBARS contents and CAT, GR, GPx, GST, SOD activities in liver of rats treated with different diets for 28 days. Parameter
GSH (mmol g1) TG (mmol g1) GSSG (mmol g1) TBARS (nmol g1) CAT (mmol min1 g1) GR (mmol min1 g1) GPx (mmol min1 g1) GST (mmol min1 g1) SOD (U SOD g1)
Basal valuesy
No supplementationz
Tomato powder supplementationz
Group 1
Group 2
Group 3
Group 4
Significant effectsz
3.770.1a 4.470.1 0.770.1 341.2735.2a 1.570.1a
3.970.5a 4.670.2 0.770.4 336.6716.6a 1.670.2a
2.370.2b 3.170.2 0.870.3 359.5736.7a 1.370.1b
2.770.1b 3.870.2 1.170.2 265.1718.5b 0.770.1c
2.770.2b 4.470.2 1.770.4 272.1719.7b 0.270.1d
3.976.6
4.370.6
3.670.5
4.370.5
4.270.1
26.370.8b
42.170.5a
19.371.6b
55.578.7c
50.172.6d
E, L
109.379.8a
120.772.2a
66.4717.8b
164.8720.2a
52.572.3b
E
296.7739.0b
1289.67189.0a 319.3754.4b
962.77196.9a 405.7741.9b
E I NS L E, L NS
E
E: main effect of energy level (Po0:01); L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Basal values found in rats fed a normal chow. z Two-way ANOVA followed by Dunnett post hoc analysis.
Table 3 Ubiquinol 9, a-tocopherol, b-carotene and lycopene contents in liver of rats treated with different diets for 28 days. Parameter
Ubiquinol 9 (nmol g1) a-tocopherol (nmol g1) b-carotene (nmol g1) Lycopene (pmol g1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 2
Group 3
Group 4
1.6770.35a 1.1670.31a 0.2170.11a 3.2870.30a
0.8770.26a 0.7870.13b 0.1670.05a 3.8170.25a
5.3371.51b 3.5770.48c 1.3470.27b 10.8571.09b
4.9070.31c 2.0270.28d 0.8670.32c 6.0270.86b
Significant effectsy L E,L L L
E: main effect of energy level (Po0:01); L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
lycopene and b-carotene contents and on the activities of CAT and GPX in liver (Po0:01, Po0:001, Po0:001, Po0:01, Po0:001, Po0:0001, and Po0:001, respectively). Hepatic TBARS showed lower contents, CAT showed decreased and GPx increased activities (Table 2), whilst liver UQ9, a-tocopherol, and b-carotene showed elevated concentrations after supplementation with tomato powder, irrespective of the energy content of the diet
(Table 3). No significant differences were detected in the hepatic contents of total and oxidized glutathione among the fours groups (Table 2).
Blood biochemical parameters As shown in Tables 4 and 5, no main effect of energy level appeared in blood parameters. However,
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Table 4 GSH, GT, GSSG, TBARS contents and CAT, GR, GPx, GST activities in erythrocytes of rats treated with different diets for 28 days. Parameter
Basal valuesy No supplementationz Group 1
0.570.1a GSH (mmol ml1) TG (mmol ml1) 0.870.5 GSSG (mmol ml1) 0.370.4 TBARS (nmol ml1) 6.870.9 a 1 1 CAT (mmol min ml ) 1.170.1a GR (mmol min1 ml1) 1.670.3 GPx (mmol min1 ml1) 65.177.3a GST (mmol min1 ml1) 7.270.9a
Group 2
Tomato powder supplementationz Group 3
0.470.1a 0.670.1a 0.970.0b 1.170.5 0.870.1 1.270.1 0.770.4 0.270.1 0.370.1 6.271.2a 7.070.7a 3.570.4b a a 1.070.1 1.070.1 0.670.1b 1.970.2 1.470.1 1.870.1 72.074.1a 66.5710.0a 108.274.2b 6.270.6a 4.870.5a 6.670.3b
Significant effectsz
Group 4 0.970.1b 1.570.2 0.670.1 4.070.3b 0.670.1b 2.070.2 99.073.9b 6.770.3b
L NS I L L I L L
L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Basal values found in rats fed a normal chow. z Two-way ANOVA followed by Dunnett post hoc analysis.
Table 5 days.
Ubiquinol 9, lycopene and a-tocopherol contents in plasma of rats treated with different diets for 28
Parameter
Ubiquinol 9 (nmol ml1) a-tocopherol (nmol ml1) b-carotene (pmol ml1) Lycopene (pmol ml1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 2
Group 3
Group 4
0.1670.01 3.2770.92 (*) 11.0172.04a
0.1670.01 10.4270.79 (*) 10.8572.55a
0.0870.03 12.1970.57 (*) 34.4873.83b
0.1370.10 13.6171.29 (*) 21.5974.08b
Significant effectsy NS NS (*) L
L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. (*) Means under the detection limit. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
tomato powder supplementation significantly affected GSH, TBARS contents and CAT, GPx and GST activities (Po0:0001, Po0:001, Po0:0001, Po0:0001 and Po0:05, respectively). Contrary to response found in liver, erythrocytic GSH contents were higher after tomato powder supplementation (Table 4) in rats fed different energy level diets. Conversely, as found in the liver, TBARS contents and the activities of CAT were decreased, whilst the activities of GPx and GST were increased after tomato powder supplementation (Table 4). As in liver, no significant differences were observed for TG and GSSG contents and for GR activity (Table 4). Plasma lycopene contents showed relatively low values (nM range) when compared to hepatic
contents (mM range), and tomato powder fed rats showed higher lycopene contents compared to rats that received no supplementation (Table 5). bcarotene contents in plasma were under the detection limit (Table 5).
Discussion Recent evidence suggests that tomato-derived products have potential health benefits in relation to several human diseases and carotenoid intake is associated with a lower risk of ophthalmic degenerative diseases, cancer and cardiovascular
ARTICLE IN PRESS 1044 diseases.1,15 A relatively recent review has shown that 57 out of 72 studies indicated an association between tomato consumption or increased plasma lycopene and lower risk of various cancers.40 Estimates of daily intake of lycopene in humans are scanty in the literature41 but plasma levels for habitual consumers of tomato products fall within the range of 0.5–1.0 mM, while the range is 0.2–21.6 mM for other tissues.1,42 For b-carotene values for human plasma are in the range of 0.3–0.6 mM.1 Even though rats absorb carotenoids less efficiently than humans,4,5 relatively high lycopene contents of 20 mM and 40 nM were found in the liver and serum of rats, respectively. According to data available in the literature,43 the liver showed much higher concentrations compared to plasma. These lycopene contents were reached probably as a consequence of the relatively high concentration of lycopene present in the diet. Nevertheless, beyond a certain dietary concentration (50 mg kg1), which is slightly higher than that used in the present study, no further increases of serum or tissue lycopene concentration are observed.44 In the present study, tomato powder supplementation modified food intake over 4 weeks and altered several biochemical parameters. Tomato powder generally exerted a similar effect in the hypo energetic and the cafeteria diets. Few factor interactions occurred in blood and liver parameters. Tomato powder effects were seen on TBARS, suggesting that the lycopene present in tomato powder can attenuate the lipid peroxidation process. Beside lycopene, the hepatic levels of ubiquinol 9, a-tocopherol and b-carotene were enhanced after tomato powder ingestion, although significant differences in plasma were found only in the augmented b-carotene levels in rats supplemented with tomato powder. Apparently the liver revealed a better supplementation effect of tomato powder in relation to the non-enzymatic antioxidants than blood, which showed significant effects on the enzymatic antioxidants. The lycopene found in blood and liver of rats without tomato powder supplementation apparently came from the basal diet, which contains fibers, including remains from papaya and guava that probably account for the relatively small amounts of lycopene detected in these tissues. Hyper energetic diets can elicit oxidative stress probably via the enhancement of the lipoperoxidation process,14 while fat consumption influences oxidative stress21 by modifying the cellular levels of antioxidants and prooxidants, making membranes more susceptible to oxidation reactions. Lee et al.45 showed that the ingestion of olive oil
E.A.M. Moreira et al. together with tomato-derived products can also decrease the oxidative stress. The lower hepatic contents of reduced glutathione found in the cafeteria group compared to the hypo energetic diet, and also when compared this group after supplementation with tomato powder, suggest that GSH is mitigating the lipoperoxidation process in the liver. These results are in agreement with the diminished TBARS contents found in the liver and plasma from the groups supplemented with tomato powder. Similar results were found in related studies on tomato diets.7,46 Nevertheless, tomato powder supplementation apparently allowed the restoration of intraerythrocytic GSH levels in rats fed both diets, whilst in the liver this did not occur in rats fed the cafeteria diet. The lower GSH content found in the liver of cafeteria-fed rats may account for the lower GPx activity also found in the liver. GSH is a co-substrate for GPx function, and its depletion in the cafeteria group could be responsible for this decrease. This explanation also applies for red cells, where a good correlation between GSH contents and GPx activities after tomato powder supplementation was found, but not for liver after supplementation. The decreased concentrations of total and reduced glutathione found in rat hepatocytes from the cafeteria group without supplementation suggest that glutathione is depleted by the oxidative challenge associated with this diet. The reduced form of glutathione is usually found in high cellular concentrations in several animal tissues, and it is a very efficient and generalist ROS scavenger.47 Relatively small portions of ingested tomato puree promote an increase in plasma carotenoid levels and decrease induced oxidative stress in lymphocytes.48 In a study involving volunteers from several countries ingesting a high carotenoid diet, a persistent increase in serum carotenoids was detected in all subjects; although, in general, no increase or decrease in other enzymatic or non-enzymatic antioxidants was detected.49 The main finding from that study was that the natural balance of carotenoids achieved through normal diets is more effective than carotenoid supplementation.49 Lee and collaborators45 found an improved plasma antioxidant capacity in six subjects fed tomatoes (approximately 46 mg lycopene day1) together with olive oil, while other investigators analysing 11 female subjects given tomato puree (approximately 6.5 mg lycopene day1) found no increase in plasma antioxidant capacity.50 These authors explained this absence of antioxidant
ARTICLE IN PRESS Tomato powder and oxidative stress in rats improvement by a counterbalancing effect of exogenous antioxidants on the endogenous antioxidants, which is plausible. In the present study this only applies to the decreased catalase activities found in liver and red cells of rats fed both diets supplemented with tomato powder. Interestingly, hepatic and red cell catalase activities were diminished after tomato powder supplementation both in the hypo energetic group and also in the cafeteria group. Also, considering the same comparison, GPx activities were elevated in both tissues. The sustained elevation observed in liver and red cell GPx activities after tomato powder supplementation, is difficult to explain. However, the same response appeared in GST activities of red cells, and even taken into account that TBARS levels showed decreased contents in liver and red cells after supplementation, it might be still a consequence of the detoxification of hydroperoxides, which are targets for both enzymes.14 In accordance to the present study, induction of GPx was also found in female rats in a dose-dependent manner, after 2 weeks of lycopene consumption.51 Interestingly, in that work Breinholt and collaborators51 found that a dose of 5 mg of lycopene kg1 b.w. per day promoted enzymatic induction but higher doses (50 or 100 mg) did not.51 In the last decade several epidemiological and laboratory studies have yielded evidence for the protective antioxidant role of fruits, vegetables, and tea.1,15,52 One example of such nutritional habits is found in the Mediterranean region, which is characterized by a high consumption of olive oil and tomato-derived products.53 It is well accepted that this dietary tradition is responsible for a lower risk of coronary heart disease and several types of cancer, especially prostate cancer.54 The present study supports the notion that beside the contribution of b-carotene, food sources of highly bioavailable lycopene, such as cooked tomatoes or tomato-derived products,55,56 consumed in the context of both types of energetic diets might be beneficial in the prevention of oxidative damage related to ROS generation.
Acknowledgements DN and FS received Grants from PIBIC-CNPq, Brazil, and DWF is a recipient of a CNPq fellowship (521108/97-7). We thank Dr. Gareth Cuttle (Universidade Federal de Santa Catarina) and Marilyn Hammond (PBRC/LSU) for English revision.
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References 1. Aust O, Sies H, Stahl W, et al. Analysis of lipophilic antioxidants in human serum and tissues: tocopherols and carotenoids. J Chromatogr 2001;936:83–93. 2. Burton GW, Ingold KU. b-carotene an unusual type of lipid antioxidant. Science 1984;224:569–73. 3. DiMascio P, Kraiser S, Sies H. Lycopene as the most effective biological carotenoid singlet oxygen quencher. Arch Biochem Biophys 1989;274:532–8. 4. Krinsky NI. Carotenoids as antioxidants. Nutrition 2001;17: 815–7. 5. Sies H, Stahl W. Lycopene: antioxidant and biological effects and its bioavailability in humans. Proc Soc Exp Biol Med 1998;218:121–4. 6. Woodall AA, Lee SW, Weesie RJ, et al. Oxidation of carotenoids by free radicals: relationship between structure and reactivity. Biochem Biophys Acta 1997;1336:33–42. 7. Rao AV, Agarwal S. Role of lycopene as antioxidant carotenoid in the prevention of chronic disease: a review. Nutr Res 1999;19:305–23. 8. Clinton SK. Lycopene: chemistry, biology, and implications for human health and disease. Nutr Res 1998;56(2):35–51. 9. Bramley PM. Is lycopene beneficial to human health? Phytochemistry 2000;54:233–6. 10. Lavelli V, Peri C, Rizollo A. Antioxidant activity of tomato products as studied by model reactions using xanthine oxidase, myeloperoxidase, and copper-induced lipid peroxidation. J Agric Food Chem 2000;48(5):1442–8. 11. Ga ¨rtner C, Stahl W, Sies H. Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am J Clin Nutr 1997;66:116–22. 12. Rosati C, Aquilani R, Dharmapuri S, et al. Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J 2000;24:413–20. 13. Halliwell B, Gutteridge JMC. Oxygen is toxic gas—an introduction to oxygen toxicity and reatictive oxygen species. In: Free radicals in biology and medicine. 3rd ed. Oxford: Clarendon Press; 1999. p. 1–35. 14. Halliwell B, Gutteridge JMC. Free radicals, other reactive species and disease. In: Free radicals in biology and medicine. 3rd ed. Oxford: Clarendon Press; 1999. p. 617–783. 15. Kris-Etherton PM, Hecker KD, Bonanome A, et al. Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am J Med 2002;113(9B): 71S–88S. 16. Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 1991;91:14–22. 17. Salonen JT, Salonen R, Seppanen K, et al. Interactions of serum copper, selenium, and low density lipoprotein cholesterol in atherogenesis. Brit J Nutr 1991;302: 756–60. 18. Niki E, Yamamoto Y, Komuro E, et al. Membrane damage due to lipid oxidation. Am J Clin Nutr 1991;53:201–5. 19. Ascherio A, Stampfer MJ, Colditz GA, et al. Correlations of vitamin A and E intakes with the plasma concentrations of carotenoids and tocopherols among American men and women. J Nutr 1992;122:1792–801. 20. Menzel DB. Antioxidant vitamins and prevention of lung disease. Ann N Y Acad Sci 1992;669:141–55. 21. Chow CK. Vitamin E and oxidative stress. Free Radic Biol Med 1991;11:215–32. 22. Committee on Laboratory Animal Diets. Assembly of life sciences national research council. Nutr Abstr Rev 1979;40: 413–9.
ARTICLE IN PRESS 1046 23. Reeves PG, Nielsen FH, Fahey Jr GC. AIN-93, purified diets for laboratory rodents: final report of American Institute of Nutrition ad hoc Writing Committee on the reformulation of the AIN-76 rodent diet. J Nutr 1993;123:1939–51. 24. Basu S, Rise ´rus U, Turpeinen A, et al. Conjugated linoleic acid induces lipid peroxidation in men with abdominal obesity. Clin Sci 2000;99:511–6. 25. Bussab WO, Morettin PA. Estatı´stica Ba´sica, 4th ed. Sa ˜o Paulo: Editora Atual; 1991. 26. Rothwell NJ, Stock MJ. Effects of feeding a ‘‘cafeteria’’ diet on energy balance and diet-induced thermogenesis in four strains of rats. J Nutr 1982;112:1515–24. 27. Fagundes RLM, Kretzer IR. Tecido adiposo marrom e sua implicac- a ˜o no desenvolvimento da obesidade. Rev Ciencia Sau ´de 1993;12:59–68. 28. Rodriguez-Amaya DB. Latin American food sources of carotenoids. Arq Latinoam Nutr 1999;49(3):74S–84S. 29. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–5. 30. Flohe ´ L, Otting F. Superoxide dismutase assays. Methods Enzymol 1984;105:93–104. 31. Carlberg I, Mannervik B. Glutathione redutase. In: Meister A, editor. Methods in enzymology. New York: Academic Press; 1985. p. 484–90. 32. Habig WH, Pasbst MJ, Jakoby WB. Glutathione-S-transferases, the first enzymatic step in mercapturic acid formation. J Biol Chem 1976;249:7130–9. 33. Flohe ´ L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984;105:114–21. 34. Beutler E. Red cell metabolism: a manual of biochemical methods. New York: Grune & Straton; 1975. 35. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 1969;27:502–22. 36. Ohkawa H, Ohisi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reation. Anal Biochem 1979; 95:351–8. 37. Bird RP, Draper AH. Comparative studies on different methods of malondyaldehyde determination. Methods Enzymol 1984; 90:105–10. 38. Motchnik PA, Frei B, Ames BN. Measurements of antioxidants in human blood plasma. Methods Enzymol 1994;234:269–79. 39. Nierenberg DW, Nann SL. A method for determining concentrations of retinol, tocopherol, and five carotenoids in human plasma and tissue samples. Am J Clin 1992;56(2):417–26. 40. Giovannucci E. Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst 1999;91:317–31. 41. Rao AV, Waseem Z, Agarwal S. Lycopene content of tomatoes and tomato products and their contribution to dietary lycopene. Free Radical Res 1998;31:737–41. 42. Pateu I, Khachic F, Brown ED, et al. Chronic ingestion of lycopene-rich tomato juice or lycopene supplements significantly increases plasma concentrations of lycopene and
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related tomato carotenoids in humans. Am J Clin Nutr 1998;68:1187–95. Jain CK, Agarwal S, Rao AV. The effect of dietary lycopene on bioavailability, tissue distribution, in vivo antioxidant properties and colonic preneoplasia in rats. Nutr Res 1999; 19(9):1383–91. Boileau TWM, Clinton SK, Erdman Jr W. Tissue lycopene concentrations and isomer patterns are affected by androgen status and dietary lycopene concentration in male F344 rats. J Nutr 2000;130:1613–8. Lee A, Thurnham DI, Chopra M. Consumption of tomato products with olive oil but not sunflower oil increases the antioxidant activity of plasma. Free Radical Biol Med 2000; 29:1051–5. Velmurugan B, Bhuvaneswari V, Balasenthil S, et al. Lycopene, an antioxidant carotenoid, modulates glutathione-dependent hepatic biotransformation enzymes during experimental gastric carcinogenesis. Nutr Res 2001; 21:1117–24. Halliwell B, Gutteridge JMC. Antioxidant defences. In: Free radicals in biology and medicine. 3rd ed. Oxford: Clarendon Press; 1999. p. 105–245. Porrini M, Riso P. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased after a short period of tomato consumption. J Nutr 2000;130: 189–92. Southon S. Increased fruit and vegetable consumption within the EU: potential health benefits. Food Res Int 2000;33: 211–7. Pellegrini N, Riso P, Porrini M. Tomato consumption does not affect the total antioxidant capacity of plasma. Nutrition 2000;16(4):268–71. Breinholt V, Lauridsen ST, Daneshvar B, Jakobsen J. Doseresponse effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett 2000; 154(2):201–10. Rao AV, Agarwal S. Role of antioxidant lycopene in cancer and heart disease. J Am Coll Nutr 2000;19(5):563–9. Weisburger JH. Evaluation of the evidence on the role of tomato products in disease prevention. Exp Biol Med 1998; 218:140–3. Willis MS, Wians Jr FH. The role of nutrition in preventing prostate cancer: a review of the proposed mechanism of action of various dietary substances. Clin Chim Acta 2003; 330:5783. Shi J. Lycopene is tomato: chemical and physical properties affected by food processing. Crit Rev Biotechnol 2003; 20(4):293–334. Tyssandier V, Feillet-Coudray C, Caris-Veyrat C, et al. Effect of product consumption on the plasma status of antioxidant microconstituents and on the plasma total antioxidant capacity in healthy subjects. J Am Coll Nutr 2004;23(2): 148–56.
http://intl.elsevierhealth.com/journals/clnu
ORIGINAL ARTICLE
Effects of diet energy level and tomato powder consumption on antioxidant status in rats Emilia A.M. Moreiraa,, Regina L.M. Fagundesa, Danilo Wilhelm Filhob, Daniela Nevesa, Fabı´ola Sellb, France Bellislec, Emil Kupekd Departamento de Nutric- a ˜o, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil b Departamento de Ecologia e Zoologia, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil c Centre National de la Recherche Scientifique, Inserm U 341, Hotel Dieu, Paris, France d Departamento de Sau ´de Pu ´blica, Centro de Cie ˆncias da Sau ´de, Universidade Federal de Santa Catarina, Campus Universita´rio, Trindade, 88.040-970 Floriano ´polis, Brazil a
Received 18 January 2005; accepted 9 August 2005
KEYWORDS Energy intake; Tomato powder; Lycopene; Antioxidants; Oxidative stress
Summary Background and aims: Evaluate the influence of tomato powder in diets differing in energy level on antioxidant status in blood and liver of rats. Methods: Twenty-four adult male Wistar rats weighing 150–180 g were placed four groups (n ¼ 6). For 28 days, animals were fed a diet that was either hyper energetic or hypo energetic. Some diets were supplemented with tomato powder. Liver and blood were collected for analysis of antioxidant enzymes, non-enzymatic antioxidants, thiobarbituric acid reactive species, ubiquinol 9, a-tocopherol, lycopene and b-carotene. Data were analysed by two-way ANOVA. Results: Food intake and thiobarbituric acid-reactive substances contents in liver and plasma were significantly decreased by tomato powder at both energy levels. After tomato powder supplementation, the hepatic levels of ubiquinol 9, a-tocopherol, lycopene and b-carotene were significantly enhanced. In plasma, only the contents of lycopene and b-carotene were enhanced. The erythrocytic and hepatic activities of catalase were lower, while those of glutathione peroxidase were higher after the ingestion of tomato powder. Total and reduced glutathione contents in liver showed lower levels in cafeteria-fed rats compared to the hypo energetic diet.
Corresponding author. Tel.: +55 48 3319784; fax: +55 48 3319542.
E-mail address: [email protected] (E.A.M. Moreira). 0261-5614/$ - see front matter & 2005 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2005.08.005
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Conclusions: The data suggest that the lycopene and b-carotene component in the tomato power supplement might be beneficial for the prevention of oxidative damage in rats fed both types of energetic diets. & 2005 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Introduction More than 700 carotenoids have been identified from plants and microorganisms.1 Among them, lycopene and b-carotene possess the most pronounced antioxidant properties.1–5 In fact, lycopene has a higher antioxidant potential than a-tocopherol and b-carotene.3,6,7 The mechanism ultimately responsible for the antioxidant property of carotenoids is still unknown.1,6 Since the in vitro demonstration by DiMascio et al.3 indicating that lycopene is the most efficient biological carotenoid quencher of singlet oxygen, research involving lycopene and tomato-derived products has increased strikingly.1,7 Lipophylic antioxidants such as tocopherols and carotenoids inhibit reactive oxygen species (ROS) propagation reactions through their chain-breaking ability.1,2,4,5 Lycopene also increases the antioxidant potential and lowers the generation of ROS such as hydroxyl radical and peroxides.1,7,8 The most abundant carotenoid present in tomato is lycopene, followed by b- and g-carotene.8,9 Compared to fresh tomatoes, processed tomato products show decreased antioxidant capacity in the hydrophilic fraction due mostly to vitamin C losses; however, they possess a higher antioxidant capacity in their lipophylic fraction, which contains carotenoids and tocopherols.10 Another advantage in the consumption of processed tomato products is that they are more bioavailable sources of lycopene compared to fresh tomatoes.7,11,12 The metabolism of oxygen in all aerobic organisms implies the continuous production of large amounts of ROS, which are deleterious to biomolecules such as DNA, lipids, sugars and proteins.13 ROS can elicit lipoperoxidation, a propagation reaction that damages cell membranes, provokes alterations in normal cell and tissue functions and contributes to many disease processes, including carcinogenesis and coronary diseases as well as the aging process.8,14,15 Oxygen toxicity is counteracted by endogenous enzymatic and non-enzymatic antioxidants and also by the presence of several nutritional antioxidants such as vitamins E and C, carotenoids and flavonoids.7,16–20 Oxidative stress5 is characterized by an imbalance between antioxidant capacity and ROS \generation. Because a nutritional deficiency of
antioxidants can be responsible for such a condition,21 the balance between ROS generation and antioxidant availability should be taken into consideration in diet studies. In most Western countries, excessive fat ingestion is common and is associated with the prevalence of obesity, cardiovascular diseases and cancer. Some studies have shown that hypo energetic diets can decrease the incidence of spontaneous or chemically induced degenerative diseases. On the other hand, hyper energetic diets seem to enhance lipoperoxidation and, therefore, elicit oxidative stress in various tissues.14 The main objective of the present study is to evaluate the influence of tomato powder (Lycopersicon esculentum) in diets differing in energy content on antioxidant status in the blood and liver of rats.
Material and methods Animals and treatment Adult male Wister rats weighing 150–180 g were used. Twenty-four animals were randomly assigned in four groups (n ¼ 6 in each group). During one week of acclimatization, they were kept in metabolic cages under controlled temperature and dark–light cycles (12:12, lights on at 06:00 h) with water and food ad libitum. Standard diets were manufactured and stored at 20 1C under vacuum to prevent oxidation. The contents of antioxidant vitamins in the basal diet were in accordance to AIN-93M22,23: vitamin E (a-tocopherol acetate) 75 IU kg1 and vitamin A, 4000 IU kg1. After the first week, the animals were treated for 4 weeks, which is sufficient time to obtain a dietary effect on lipid metabolism.24 The following diets were used: (a) group 1—hypo energetic diet; (b) group 2—‘‘cafeteria’’ diet (hyper energetic); (c) group 3—hypo energetic diet plus tomato powder; and (d) group 4—‘‘cafeteria’’ diet plus tomato powder. The hypo energetic diets were defined according to a consumption limit (30% lower in grams) using the standard animal diet based on the AIN-93M recommendations.22,23 In the ‘‘cafeteria’’ diet (hyper energetic/hyper lipidic), the control diet was available ad libitum together with a variety of
ARTICLE IN PRESS 1040 highly palatable foods using the model of ‘‘incomplete counterbalanced blocks’’25 in which three different foods such as cookies, chocolate and snacks were offered containing 5 g portions each, totaling 15 g per day, and 15 g of control diet ad libitum. The composition of the cafeteria diet can be found elsewhere.26,27 In the lycopene groups, 5 g of tomato powder (corresponding to approximately 20–30 mg lycopene kg1 day1) were given daily to rats mixed in the control diet in a proportion of 25% of tomato powder (5 g lycopene:15 g control diet), also containing the same amount of lycopene. Tomato powder has the highest lycopene concentration (1.1–1.3 mg g1 weight),28 but rats absorb carotenoids less efficiently than humans and rapidly convert carotenoids in the gut.5–7 Therefore, in the present study the amount of lycopene present in the tomato powder given to the experimental animals was used to reach values near to those found in plasma of human subjects. The experimental protocols were in accordance with the Helsinki Declaration.
Parameters analysed Fasted animals were killed by cervical dislocation. After weighing the animals, the livers were rapidly removed, kept on ice, and perfused with ice-cold saline (0.9% NaCl) solution for 5 min before being homogenised (1:9 w/v) in a buffer comprising 0.1% Triton X-100, 0.12 M NaCl, 30 mM Na2PO4, and pH 7.4. Homogenization was carried out at 4 1C, employing 20 strokes of a Potter–Elvehjen homogenizer. This was followed by centrifugation at 10,000g for 10 min at 5 1C. Supernatants were stored in liquid nitrogen until analysis by enzymatic assays. Blood was drawn from the caudal vein in heparinized syringes. After plasma removal, red cells were washed three times in saline solution. Hemolysates were obtained after addition of three volumes of 20 mM Tris–HCl, pH 8.0 and centrifuged at 3000g for 5 min at 5 1C. Hemolysate supernatants were used for enzymatic analysis.
Antioxidant enzymes Catalase activity (CAT): To measure CAT activity in blood, the hemolysates were further diluted 500 times.29 CAT was quantified by measuring the decay of a 10 mM hydrogen peroxide solution at 240 nm.29 Superoxide dismutase (SOD): SOD was measured after treating the hemolysates with a mixture of chloroform/ethanol (3:5 v:v), or using the supernatants from liver homogenates, and quantified at 550 nm by measuring the rate of cytochrome c
E.A.M. Moreira et al. reduction.30 Glutathione reductase (GR): this activitiy was assayed at 340 nm by measuring the rate of NADPH oxidation according to Carlberg and Mannervik.31 Glutathione S-transferase (GST): the activity was determined at 340 nm using CDNB (1-chloro-2,4-dinitrobenzene) as substrate and a 0.15 M GSH concentration.32 Glutathione peroxidase (GPx): GPx was measured at 340 nm through the glutathione/NADPH/GR system by the dismutation of tert-butylhydroperoxide.33
Non-antioxidant enzymes Reduced form of glutathione (GSH): Non-protein thiols, mostly present as the GSH, were measured at 412 nm according to Beutler34 using the Elmann’s reagent (DTNB: 2-dithionitrobenzoic acid). Immediately after liver excision tissue acid extracts were obtained by adding liver portions to 12% trichloroacetic acid (1:4 w/v), and then centrifuging the resulting solution at 5000g for 5 min at 5 1C. Supernatants from the acid extracts were added to a buffer containing 0.25 mM DTNB in 0.1 M Na2PO4, pH 8.0, and the formation of thiolate anion was immediately determined. Total glutathione (TG): was also measured at 412 nm in acid extracts according to the enzymatic method of Tietze.35 Oxidised glutathione (GSSG): was determined by calculating the difference (in equivalents of GSH) between TG and reduced glutathione contents.
Lipoperoxidation assay Thiobarbituric acid-reactive substances (TBARS): TBARS contents were determined to assess endogenous lipid oxidation in the liver according to Ohkawa36 and Bird and Draper.37 After excision, liver slices were immediately added to 12% trichloroacetic acid (1:4 v/v) and then centrifuged at 15,000g for 3 min at 5 1C. Supernatants were added to 50 mM Tris–HCl pH 7.0, vortexed for 20 s, added to 0.67% (w/v) 2-thiobarbituric acid, maintained in boiling water for 60 min, cooled at 5 1C for 30 min, and then analysed spectrophotometrically at 535 nm. Absorbance was expressed as equivalent to nmol TBARS g1 wet tissue (E535 ¼ 153 mM1 cm1). The biochemical parameters described above were measured in duplicate, except TBARS determinations, which were performed in triplicate.
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Antioxidant capacity: ubiquinol 9, a-tocopherol, lycopene and b-carotene Plasma antioxidant determinations were carried out by high performance liquid chromatography (HPLC) with in line electrochemical and UV detection. To evaluate plasma concentrations of atocopherol, lycopene and b-carotene, an aliquot of 200 ml of plasma or tissue homogenate was added to 500 ml of methanol and 4 ml of hexane. The mixture was vortexed for 1 min and then the tubes were centrifuged for 5 min at 1000g at 5 1C. A 3 ml aliquot from the hexane layer was dried under N2 flux, and the residue dissolved in 200 ml methanol:ethanol (1:1, v/v) and filtered through a 0.22 mmpore nylon membrane. To determine total ubiquinol 9 content, the extracts were reduced as follows38: extract aliquots (100 ml) were mixed with 0.5 ml of methanol, 0.5 ml of MilliQ water, and approximately 20 mg of NaBH4. The mixture was vortexed and incubated at room temperature in the dark for 30 min and then extracted into 4 ml of hexane following the steps described above. Measurements of antioxidants in the methanol:ethanol extract were made by reversed phase HPLC, using a Supelcosil LC-8 column (33 4.6 mm; 3 mm packing material), and 20 mM LiClO4 in methanol:H2O (97.7:2.5, v/v) as mobile phase. An in line electrochemical detector with a glassy-carbon working electrode (Bioanalytical System LC4C) at an applied oxidation potential of +0.6 V, and UV detector at 290 nm (Waters model 460, Milford, MA, USA) were used. Standard solutions of antioxidants were prepared according to Nierenberg et al.39 All the chemicals used in the present study were purchased from Sigma-Aldrich Co. (Ohio, USA).
Statistical methods Results are expressed as means7SEM. Differences between the four dietary groups were analysed by
Table 1
two-way ANOVA with energy level and supplementation as factors followed by Dunnett multiple range test for comparisons of means. Tests were performed using the STAT software, and P-values less than 5% (Po0:05) were considered to be statistically significant.
Results Energy intake and body weight gain Body weight gain over 4 weeks was affected by food intake and the energy content of the diets. The tomato powder supplementation decreased food intake, probably through an effect of palatability of the diet, and also energy intake. Table 1 illustrates the significant effects of energy level (high vs. low) on all three dependent variables: food intake (g day1), energy intake (kcal day1) and weight gain (g) (Po0:0001). Supplementation with tomato powder also had a significant effect on food intake (g) and energy intake (kcal) (Po0:0001). The main effect of tomato powder supplementation was not significant for body weight gain.
Liver biochemical parameters In liver (Tables 2 and 3), significant main effects of energy level were found for GSH and a-tocopherol contents and CAT, GPx, GST and SOD activities (Po0:01, Po0:05, Po0:01, Po0:05, Po0:0001, and Po0:0001, respectively). More specifically, in liver GSH (Table 2) and a-tocopherol (Table 3) contents, and also the activities of CAT, GPx, GST and SOD (Table 2) showed decreased values in cafeteria fed rats compared to rats fed the hypo energetic diet. Supplementation with tomato powder also had a significant effect on TBARS, UQ9, a-tocopherol,
Food intake, energy intake and weight gain of rats treated with different diets for 28 days.
Parameter
1
Food intake (g day ) Energy intake (kcal day1) Weight gain (g day1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 3
a
1370.1 5470.5a 2772.9a
Group 2 b
1770.4 7671.5b 9278.3b
c
1270.1 5270.5c 3273.0a
Significant effectsy
Group 4 1570.2d 6671.1d 8273.7b
E, L E, L E
E: main effect of energy level (Po0:05); L: main effect of supplementation with tomato powder (Po0:05). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). Group 1—hypo energetic diet, group 2—‘‘cafeteria’’ diet (hyper energetic), group 3—hypo energetic diet plus tomato powder, and group 4—‘‘cafeteria’’ diet plus tomato powder. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
ARTICLE IN PRESS 1042
E.A.M. Moreira et al.
Table 2 GSH, GT, GSSG, TBARS contents and CAT, GR, GPx, GST, SOD activities in liver of rats treated with different diets for 28 days. Parameter
GSH (mmol g1) TG (mmol g1) GSSG (mmol g1) TBARS (nmol g1) CAT (mmol min1 g1) GR (mmol min1 g1) GPx (mmol min1 g1) GST (mmol min1 g1) SOD (U SOD g1)
Basal valuesy
No supplementationz
Tomato powder supplementationz
Group 1
Group 2
Group 3
Group 4
Significant effectsz
3.770.1a 4.470.1 0.770.1 341.2735.2a 1.570.1a
3.970.5a 4.670.2 0.770.4 336.6716.6a 1.670.2a
2.370.2b 3.170.2 0.870.3 359.5736.7a 1.370.1b
2.770.1b 3.870.2 1.170.2 265.1718.5b 0.770.1c
2.770.2b 4.470.2 1.770.4 272.1719.7b 0.270.1d
3.976.6
4.370.6
3.670.5
4.370.5
4.270.1
26.370.8b
42.170.5a
19.371.6b
55.578.7c
50.172.6d
E, L
109.379.8a
120.772.2a
66.4717.8b
164.8720.2a
52.572.3b
E
296.7739.0b
1289.67189.0a 319.3754.4b
962.77196.9a 405.7741.9b
E I NS L E, L NS
E
E: main effect of energy level (Po0:01); L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Basal values found in rats fed a normal chow. z Two-way ANOVA followed by Dunnett post hoc analysis.
Table 3 Ubiquinol 9, a-tocopherol, b-carotene and lycopene contents in liver of rats treated with different diets for 28 days. Parameter
Ubiquinol 9 (nmol g1) a-tocopherol (nmol g1) b-carotene (nmol g1) Lycopene (pmol g1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 2
Group 3
Group 4
1.6770.35a 1.1670.31a 0.2170.11a 3.2870.30a
0.8770.26a 0.7870.13b 0.1670.05a 3.8170.25a
5.3371.51b 3.5770.48c 1.3470.27b 10.8571.09b
4.9070.31c 2.0270.28d 0.8670.32c 6.0270.86b
Significant effectsy L E,L L L
E: main effect of energy level (Po0:01); L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
lycopene and b-carotene contents and on the activities of CAT and GPX in liver (Po0:01, Po0:001, Po0:001, Po0:01, Po0:001, Po0:0001, and Po0:001, respectively). Hepatic TBARS showed lower contents, CAT showed decreased and GPx increased activities (Table 2), whilst liver UQ9, a-tocopherol, and b-carotene showed elevated concentrations after supplementation with tomato powder, irrespective of the energy content of the diet
(Table 3). No significant differences were detected in the hepatic contents of total and oxidized glutathione among the fours groups (Table 2).
Blood biochemical parameters As shown in Tables 4 and 5, no main effect of energy level appeared in blood parameters. However,
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Table 4 GSH, GT, GSSG, TBARS contents and CAT, GR, GPx, GST activities in erythrocytes of rats treated with different diets for 28 days. Parameter
Basal valuesy No supplementationz Group 1
0.570.1a GSH (mmol ml1) TG (mmol ml1) 0.870.5 GSSG (mmol ml1) 0.370.4 TBARS (nmol ml1) 6.870.9 a 1 1 CAT (mmol min ml ) 1.170.1a GR (mmol min1 ml1) 1.670.3 GPx (mmol min1 ml1) 65.177.3a GST (mmol min1 ml1) 7.270.9a
Group 2
Tomato powder supplementationz Group 3
0.470.1a 0.670.1a 0.970.0b 1.170.5 0.870.1 1.270.1 0.770.4 0.270.1 0.370.1 6.271.2a 7.070.7a 3.570.4b a a 1.070.1 1.070.1 0.670.1b 1.970.2 1.470.1 1.870.1 72.074.1a 66.5710.0a 108.274.2b 6.270.6a 4.870.5a 6.670.3b
Significant effectsz
Group 4 0.970.1b 1.570.2 0.670.1 4.070.3b 0.670.1b 2.070.2 99.073.9b 6.770.3b
L NS I L L I L L
L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Basal values found in rats fed a normal chow. z Two-way ANOVA followed by Dunnett post hoc analysis.
Table 5 days.
Ubiquinol 9, lycopene and a-tocopherol contents in plasma of rats treated with different diets for 28
Parameter
Ubiquinol 9 (nmol ml1) a-tocopherol (nmol ml1) b-carotene (pmol ml1) Lycopene (pmol ml1)
No supplementationy
Tomato powder supplementationy
Group 1
Group 2
Group 3
Group 4
0.1670.01 3.2770.92 (*) 11.0172.04a
0.1670.01 10.4270.79 (*) 10.8572.55a
0.0870.03 12.1970.57 (*) 34.4873.83b
0.1370.10 13.6171.29 (*) 21.5974.08b
Significant effectsy NS NS (*) L
L: main effect of supplementation with tomato powder (Po0:01); I: interaction of energy level and tomato powder supplementation (Po0:01). Within a row, values with a superscript not sharing a letter are significantly different (Po0:05). NS ¼ not significant. (*) Means under the detection limit. Groups as in Table 1. Values are means7SEM, n ¼ 6 per group. y Two-way ANOVA followed by Dunnett post hoc analysis.
tomato powder supplementation significantly affected GSH, TBARS contents and CAT, GPx and GST activities (Po0:0001, Po0:001, Po0:0001, Po0:0001 and Po0:05, respectively). Contrary to response found in liver, erythrocytic GSH contents were higher after tomato powder supplementation (Table 4) in rats fed different energy level diets. Conversely, as found in the liver, TBARS contents and the activities of CAT were decreased, whilst the activities of GPx and GST were increased after tomato powder supplementation (Table 4). As in liver, no significant differences were observed for TG and GSSG contents and for GR activity (Table 4). Plasma lycopene contents showed relatively low values (nM range) when compared to hepatic
contents (mM range), and tomato powder fed rats showed higher lycopene contents compared to rats that received no supplementation (Table 5). bcarotene contents in plasma were under the detection limit (Table 5).
Discussion Recent evidence suggests that tomato-derived products have potential health benefits in relation to several human diseases and carotenoid intake is associated with a lower risk of ophthalmic degenerative diseases, cancer and cardiovascular
ARTICLE IN PRESS 1044 diseases.1,15 A relatively recent review has shown that 57 out of 72 studies indicated an association between tomato consumption or increased plasma lycopene and lower risk of various cancers.40 Estimates of daily intake of lycopene in humans are scanty in the literature41 but plasma levels for habitual consumers of tomato products fall within the range of 0.5–1.0 mM, while the range is 0.2–21.6 mM for other tissues.1,42 For b-carotene values for human plasma are in the range of 0.3–0.6 mM.1 Even though rats absorb carotenoids less efficiently than humans,4,5 relatively high lycopene contents of 20 mM and 40 nM were found in the liver and serum of rats, respectively. According to data available in the literature,43 the liver showed much higher concentrations compared to plasma. These lycopene contents were reached probably as a consequence of the relatively high concentration of lycopene present in the diet. Nevertheless, beyond a certain dietary concentration (50 mg kg1), which is slightly higher than that used in the present study, no further increases of serum or tissue lycopene concentration are observed.44 In the present study, tomato powder supplementation modified food intake over 4 weeks and altered several biochemical parameters. Tomato powder generally exerted a similar effect in the hypo energetic and the cafeteria diets. Few factor interactions occurred in blood and liver parameters. Tomato powder effects were seen on TBARS, suggesting that the lycopene present in tomato powder can attenuate the lipid peroxidation process. Beside lycopene, the hepatic levels of ubiquinol 9, a-tocopherol and b-carotene were enhanced after tomato powder ingestion, although significant differences in plasma were found only in the augmented b-carotene levels in rats supplemented with tomato powder. Apparently the liver revealed a better supplementation effect of tomato powder in relation to the non-enzymatic antioxidants than blood, which showed significant effects on the enzymatic antioxidants. The lycopene found in blood and liver of rats without tomato powder supplementation apparently came from the basal diet, which contains fibers, including remains from papaya and guava that probably account for the relatively small amounts of lycopene detected in these tissues. Hyper energetic diets can elicit oxidative stress probably via the enhancement of the lipoperoxidation process,14 while fat consumption influences oxidative stress21 by modifying the cellular levels of antioxidants and prooxidants, making membranes more susceptible to oxidation reactions. Lee et al.45 showed that the ingestion of olive oil
E.A.M. Moreira et al. together with tomato-derived products can also decrease the oxidative stress. The lower hepatic contents of reduced glutathione found in the cafeteria group compared to the hypo energetic diet, and also when compared this group after supplementation with tomato powder, suggest that GSH is mitigating the lipoperoxidation process in the liver. These results are in agreement with the diminished TBARS contents found in the liver and plasma from the groups supplemented with tomato powder. Similar results were found in related studies on tomato diets.7,46 Nevertheless, tomato powder supplementation apparently allowed the restoration of intraerythrocytic GSH levels in rats fed both diets, whilst in the liver this did not occur in rats fed the cafeteria diet. The lower GSH content found in the liver of cafeteria-fed rats may account for the lower GPx activity also found in the liver. GSH is a co-substrate for GPx function, and its depletion in the cafeteria group could be responsible for this decrease. This explanation also applies for red cells, where a good correlation between GSH contents and GPx activities after tomato powder supplementation was found, but not for liver after supplementation. The decreased concentrations of total and reduced glutathione found in rat hepatocytes from the cafeteria group without supplementation suggest that glutathione is depleted by the oxidative challenge associated with this diet. The reduced form of glutathione is usually found in high cellular concentrations in several animal tissues, and it is a very efficient and generalist ROS scavenger.47 Relatively small portions of ingested tomato puree promote an increase in plasma carotenoid levels and decrease induced oxidative stress in lymphocytes.48 In a study involving volunteers from several countries ingesting a high carotenoid diet, a persistent increase in serum carotenoids was detected in all subjects; although, in general, no increase or decrease in other enzymatic or non-enzymatic antioxidants was detected.49 The main finding from that study was that the natural balance of carotenoids achieved through normal diets is more effective than carotenoid supplementation.49 Lee and collaborators45 found an improved plasma antioxidant capacity in six subjects fed tomatoes (approximately 46 mg lycopene day1) together with olive oil, while other investigators analysing 11 female subjects given tomato puree (approximately 6.5 mg lycopene day1) found no increase in plasma antioxidant capacity.50 These authors explained this absence of antioxidant
ARTICLE IN PRESS Tomato powder and oxidative stress in rats improvement by a counterbalancing effect of exogenous antioxidants on the endogenous antioxidants, which is plausible. In the present study this only applies to the decreased catalase activities found in liver and red cells of rats fed both diets supplemented with tomato powder. Interestingly, hepatic and red cell catalase activities were diminished after tomato powder supplementation both in the hypo energetic group and also in the cafeteria group. Also, considering the same comparison, GPx activities were elevated in both tissues. The sustained elevation observed in liver and red cell GPx activities after tomato powder supplementation, is difficult to explain. However, the same response appeared in GST activities of red cells, and even taken into account that TBARS levels showed decreased contents in liver and red cells after supplementation, it might be still a consequence of the detoxification of hydroperoxides, which are targets for both enzymes.14 In accordance to the present study, induction of GPx was also found in female rats in a dose-dependent manner, after 2 weeks of lycopene consumption.51 Interestingly, in that work Breinholt and collaborators51 found that a dose of 5 mg of lycopene kg1 b.w. per day promoted enzymatic induction but higher doses (50 or 100 mg) did not.51 In the last decade several epidemiological and laboratory studies have yielded evidence for the protective antioxidant role of fruits, vegetables, and tea.1,15,52 One example of such nutritional habits is found in the Mediterranean region, which is characterized by a high consumption of olive oil and tomato-derived products.53 It is well accepted that this dietary tradition is responsible for a lower risk of coronary heart disease and several types of cancer, especially prostate cancer.54 The present study supports the notion that beside the contribution of b-carotene, food sources of highly bioavailable lycopene, such as cooked tomatoes or tomato-derived products,55,56 consumed in the context of both types of energetic diets might be beneficial in the prevention of oxidative damage related to ROS generation.
Acknowledgements DN and FS received Grants from PIBIC-CNPq, Brazil, and DWF is a recipient of a CNPq fellowship (521108/97-7). We thank Dr. Gareth Cuttle (Universidade Federal de Santa Catarina) and Marilyn Hammond (PBRC/LSU) for English revision.
1045
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