Effects of Commonly Consumed Fruit Juices and Carbohydrates on

NUTRITION AND CANCER, 45(1), 46–52
Copyright © 2003, Lawrence Erlbaum Associates, Inc.
Effects of Commonly Consumed Fruit Juices and Carbohydrates on
Redox Status and Anticancer Biomarkers in Female Rats
Vibeke M. Breinholt, Salka E. Nielsen, Pia Knuthsen, Søren T. Lauridsen,
Bahram Daneshvar, and Annemarie Sørensen
Abstract: Administration of apple juice, black currant juice,
or a 1:1 combination of the two juices significantly decreased
the level of the lipid peroxidation biomarker malondialdehyde
in plasma of female rats, whereas the protein oxidation
biomarker 2-amino-adipic semialdehyde, was significantly
increased following administration of orange juice, black currant juice, or the 1:1 combination of apple and black currant
juice. A significant increase in 2-amino-adipic semialdehyde
was also observed in control rats given sucrose, fructose, and
glucose in the drinking water at concentrations approximating the average carbohydrate levels in the employed fruit
juices. None of the fruit juices were found to affect the activities
of antioxidant enzymes in red blood cells or hepatic
glutathione S-transferase. Hepatic quinone reductase activity, on the other hand, was significantly increased by grapefruit juice, apple juice, and black currant juice. The total daily
intake of a selected subset of flavonoid aglycones ranged from
0.2 to 4.3 mg, and quercetin was found to be a minor constituent of all the juices investigated. In a parallel study, rats were
fed quercetin at doses ranging from 0.001 to 10 g/kg of diet.
However, no effects were observed on hepatic glutathione
S-transferase or quinone reductase activities, plasma redox
status, or the activity of red blood cell antioxidant enzymes.
Overall, the results of the present study suggest that commonly
consumed fruit juices can alter lipid and protein oxidation
biomarkers in the blood as well as hepatic quinone reductase
activity, and that quercetin may not be the major active principle. The observation that natural carbohydrates are capable
of mediating oxidative stress in vivo warrants further studies
due to the central role refined and unrefined carbohydrates
play in human nutrition.
Introduction
Many experimental animal studies, human intervention
studies, and epidemiological studies suggest that inclusion of
fruits and vegetables in a basal diet affords a significant pro-
tection against a wide range of common human cancer types
(1–7). However, despite extensive research in the area of cancer chemoprevention for several decades, it is yet not possible to pinpoint specific food items or single dietary components that are particularly efficient in protecting against
cancer. The observed protective effect of a high intake of
fruits and vegetables is thus only weakly correlated to single
dietary constituents or to distinct groups of phytochemicals.
The findings that several cancer intervention studies with single or simple combinations of promising anticarcinogens
have failed to show a cancer protective effect against several
cancer types (8–12) indicate that the protective action of
fruits and vegetables most likely arise from the combined exposure to several dietary components, rather than being the
result of the action of single anticarcinogenic components.
Overall, it thus seems more appropriate and less risky to obtain “an increased protected state” by increasing the consumption of fruits and vegetables rather than obtaining the
potentially protective factors from enriched fractions of plant
material or as synthetic or purified plant components.
Epidemiological investigations repeatedly show that regular consumption of dark green and cruciferous vegetables,
tomatoes, and citrus fruits in particular is related to a reduced
risk of several cancers (7,13–18). That fruit juices are readily
accessible throughout the world, palatable to most people
and a major source of flavonoids in several countries (19,20),
warrants further studies on the health promoting effects of
commercially available fruit juices.
The aim of the present study was to investigate the ability
of four different fruit juices, containing a wide range of potentially cancer protective flavonoids, to affect selected
biomarkers for redox status and phase 2 enzyme capacity in
female rats, to gain information on the potentially health promoting effects of fruit juices. These particular biomarkers
were chosen due to the central role oxidative stress play in the
cancer process and the presumed important role carcinogen
detoxification play in cancer protection. The potentially
modulatory role of the most commonly occurring carbohy-
V. M. Breinholt, S. E. Nielsen, S. T. Lauridsen, and B. Daneshvar are affiliated with the Institute of Food Safety and Nutrition, Division of Biochemical and
Molecular Toxicology, The Danish Veterinary and Food Administration, Mørkhøj Bygade 19, 2860 Søborg, Denmark. P. Knuthsen and A. Sørensen are affiliated
with Institute of Food Research and Nutrition, The Danish Veterinary and Food Administration, Mørkhøj Bygade 19, 2860 Søborg, Denmark.
drates present naturally in the employed fruit juices or added
to the juice during processing was also investigated. Furthermore, a parallel study was conducted to investigate the role of
the flavonoid quercetin, present in all juices investigated, to
impact on the selected biomarkers.
Materials and Methods
Materials
The chemicals used in this study were obtained from the
following: Sigma Chemical (St. Louis, MO): butylated
hydroxytoluene, 2-thiobarbituric acid, NADPH, glutathione,
FAD, purpald, potassium periodate, β-naphthoflavone
(BNF) (>97%), UDP-glucuronic acid, NADH, pyruvate
kinase, lactate dehydrogenase, phosphoenolpyruvate,
HEPES, Tween 20, glucose-6-phosphate, cytochrome c,
menadione, glucose-6-phosphate dehydrogenase, and dicoumarol; Aldrich Chemical (Steinheim, Germany):
malondialdehyde and quercetin (>98%). Ethoxyresorufin,
methoxyresorufin, pentoxyresorufin, benzyloxyresorufin,
and resorufin were obtained from Molecular Probes (Eugene, OR). 1-Chloro-2,4-dinitrobenzene (CDNB) was purchased from Riedel-de Häen (Seeize, Germany). The following commercial kits were used: superoxide dismutase (SOD)
(Randox, cat. no. SD 125), glutathione peroxidase (GPx)
(Randox, cat. no. RS 505), hemoglobin (Randox, cat. no. HG
980), fructose (BoehringerMannheim, kit. no. E0139106),
and protein (Roche, cat. no. 0736783). All other chemicals
were from Merck (Darmstadt, Germany).
Animals, Exposure Protocol, and
Preparation of Subcellular Fractions
Forty female Wistar rats, aged 6–7 wk (106–138 g) from
Møllegård Breeding Center Ltd. (Lille Skensved, Denmark),
were acclimatized for 7 days while maintained on a powdered semisynthetic diet based on casein (18%) and
carbohydrates (cornstarch, potato starch, dextrin, and sucrose) (68%) (21) and assigned to eight different treatment
groups of 5 animals each. Animals were kept in a 12-h light
and 12-h dark cycle at an average temperature and humidity
of 22°C and 55%, respectively. The animals in Group 8 were
administered BNF by gavage at a concentration of 0.1 g/kg of
body weight (kg b.w.) in a total volume of 2 ml of dimethyl
sulfoxide per kilogram of body weight during the last 4 days
of the study. In Groups 1 (control rats) and 8, the animals
were administered tap water, whereas the tap water in Groups
2–7 was substituted with fruit juices (Groups 2–6) or tap water containing different carbohydrates (Group 7) as shown in
Table 1. During the study the animals had free access to the
semisynthetic diet. After 7 days the animals were anesthetized and approximately 2–3 ml of blood was drawn from the
heart. The animals were subsequently killed by decapitation.
The liver was rinsed in ice-cold phosphate-buffered saline,
blotted dry on paper towel, weighed, and submerged in liquid
nitrogen until storage at –80°C. The microsomal and
cytosolic fractions from the liver were prepared as described
by others (22). Aliquots of 0.5 ml were stored at –80°C until
use. The protein concentration was determined by using the
BCA method (23) adapted to a Cobas Mira (Roche Diagnostic System, Basel, Schwizerland). The heparinized blood
samples were separated into plasma and red blood cells
(RBC) by centrifugation at 1,500 g for 10 min. The blood
cells were washed three times with 1 volume of cold 0.9%
NaCl followed by centrifugation at 1,500 g for 10 min. The
washed cells were lysed with 1 volume of deionized water.
Blood lysate and plasma were stored at –80°C until use.
In the quercetin dose-response study, 30 female Wistar
rats (116–138 g) from Møllegård Breeding Center Ltd. (Lille
Skensved, Denmark) were randomized according to weight
into six groups of 5 animals and acclimatized for 7 days
Table 1. Concentration of Total and Individual Flavonoids and Carbohydrates in Various Fruit Juicesa
Concentration of Flavonoids or Carbohydrates g/l Juice
[Mean Flavonoid (mg) or Mean Carbohydrate (g) Intake/Animal/Day]
Flavonoid/Carbohydrate
Quercetin
Naringenin
Hesperetin
Kaempferol
Isorhamnetin
Myricetin
Phloretin
Glucose
Fructose
Sucrose
Total flavonoids
Total carbohydrates
Group 2
(Grapefruit)
Group 3 (Apple)
0.003 (0.062)
0.196 (4.0)
0.007 (0.14)
0.003 (0.062)
0.003 (0.062)
n.d.
n.d.
22.8 (0.5)
23.4 (0.5)
20.0 (0.5)
0.212 (4.326)
66.2 (1.5)
0.002 (0.036)
n.d.
n.d.
n.d.
n.d.
n.d.
0.011 (0.2)
23.2 (0.3)
58.2 (0.5)
15.9 (1.1)
0.013 (0.236)
97.3 (1.9)
Group 4 (Black
Currant)
0.004 (0.078)
n.d.
n.d.
0.001 (0.019)
Trace
0.011 (0.21)
n.d.
51.4 (0.6)
51.6 (1.1)
27.6 (1.1)
0.016 (0.307)
130.4 (2.8)
Group 5
(Apple/Bl.
Currant)
0.003 (0.057)
n.d.
n.d.
0.001 (0.019)
Trace
0.006 (0.21)
0.006 (0.20)
37.3 (0.4)
54.9 (0.8)
21.8 (1.1)
0.016 (0.486)
114
(2.3)
Group 6 (Orange)
Group 7
(Carbohydrate
Control)
0.003 (0.062)
0.012 (0.25)
0.057 (1.17)
n.d.
0.007 (0.14)
n.d.
n.d.
23.4 (0.6)
25.1 (1.1)
41.3 (1.1)
0.079 (1.622)
89.8 (2.8)
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
21.7 (0.5)
54.9 (0.9)
37.3 (1.3)
n.d.
113.9 (2.7)
a: The content of anthocyanins and catechins that may have been present and thus contributed to the overall flavonoid concentration in the juices was not determined in the present study. n.d., not detected.
Vol. 45, No. 1
47
while maintained on a powdered semisynthetic diet with free
access to tap water. After acclimatization the animals received the semisynthetic diet containing 0, 0.001, 0.01, 0.1,
1, or 10 g of quercetin per kilogram of diet for 2 consecutive
weeks. The animals were killed at Day 15 and the liver and
blood processed as described above. Two samples from each
animal were analyzed and each individual analysis was conducted in duplicate.
In both studies the intake of water or juice as well as food
was recorded weekly. The body weights were likewise recorded once a week. Animal care and use complied with the
recommendations of the National Institutes of Health (24).
Determination of Drug-Metabolizing and
Antioxidant Enzyme Activities
The activities of cytosolic quinone reductase (QR) and
glutathione S-transferase (GST) were determined on a Cobas
Mira analyzer as described previously (25,26), according to
the methods by Ernster (27) and Habig et al. (28). QR and
GST activities were measured by using cytochrome c and
CDNB as substrates, respectively. Automated assays for the
activities of the antioxidant enzymes SOD, GPx, catalase
(CAT), and glutathione reductase (GR) in blood hemolysate
were performed on a Cobas Mira analyzer. SOD and GPx activities and hemoglobin were determined by using commercially available kits, whereas the activity of GR was determined by the method of Goldberg and Spooner (29). CAT
activity was determined according to a method described by
Wheeler et al. (30). The enzymatic activities in RBC were
calculated relative to the amount of hemoglobin, measured
by using Drabkins reagent (Randox Laboratories Ltd.,
Crumlin, UK).
Assessment of Lipid and Protein Oxidation
The biomarker of lipid peroxidation, malondialdehyde
(MDA), and the protein oxidation biomarker, 2-amino adipic
semialdehyde (AAS), were measured as described by
Lauridsen and Mortensen (31) and Daneshvar and colleagues
(32), respectively.
Quantification of Flavonoids and Individual
Carbohydrates in Fruit Juice
All fruit juices were purchased from three different grocery stores in Denmark. A grapefruit juice from Cadiso
(Frederikssund, Denmark) and a freshly squeezed orange
juice from Irma (Rødovre, Denmark) containing high concentrations of the flavanones naringenin and hesperetin were
selected for the study. Both juices contained additional
flavonoids as shown in Table 1. The apple juice used was produced by Nutana (Bjæverskov, Denmark) and the black currant juice by Cadiso (Frederikssund, Denmark). The concentration of a selected set of flavonoids and carbohydrates
present in the juices is given in Table 1.
48
Flavonoids were determined by applying fruit juice (5 ml)
to a Bond Elute column (500 mg, Varian, Harbor City, CA),
activated with 10 ml of methanol and 20 ml of water containing 1% formic acid. The column was rinsed with another 10
ml of water and finally the sample was eluted with 20 ml of
methanol, evaporated in vacuo, and the reconstituted sample
hydrolyzed in 1.2 M HCl. The hydrolysate was analyzed by
liquid chromatography/mass spectrometry (LC/MS) as described previously (19). Fructose was determined enzymatically by the ultraviolet light method of Boehringer
Mannheim (kit no. E0139106). Glucose and sucrose were
determined by high-pH anion-exchange chromatography
with pulsed amperometric detection using a CarboPac MA1
column from Dionex (Sunnyvale, CA) as described by
Andersen and Sørensen (33).
LC/MS Analysis of Plasma Flavonoids
Aliquots of 300 µl of plasma were added, 250 ng of morin
and daidzein as internal standards. The samples were enzymatically hydrolyzed essentially as described previously
(34) by the addition of 50 µl of β-glucuronidase/arylsulfatase
(0.55 U and 0.26 U, respectively) and incubation in a sealed
vial for 1 h at 37oC under continuous shaking. After hydrolysis, the samples were added 2.0 ml of m-phosphoric acid and
extracted with 2 ml of ethyl acetate. The samples were centrifuged for 10 min at 10,000 g at 4oC and the ethyl acetate
phase was transferred to a new vial. This procedure was repeated twice. After pooling of the two organic phases the
sample was evaporated to dryness under vacuum. The dry
residue was stored under Argon (g) at –20oC until analysis.
Prior to LC/MS the sample was reconstituted in 10% aqueous methanol containing 1% formic acid, giving a final volume of 250 µl, that was all injected onto the LC/MS. The
methodology used for the LC/MS analysis of the samples
with regard to quercetin and its metabolites isorhamnetin and
tamarixetin has been described elsewhere (35).
Statistical Analysis
Statistical analyses were conducted by using SAS version
6.08 (SAS Institute Inc., Cary, NC). Analysis of variance
with Dunnett’s test was employed for comparison between
the means of the treated groups and the controls. A P value of
less than 0.05 was used as the level of significance.
Results
The concentration of a selected subset of flavonoids and
carbohydrates present in the investigated fruit juices is given
in Table 1. The same brands of apple and black currant juice,
as used in a previous human intervention study conducted in
collaboration with our laboratory (36), were preselected for
the study to be able to compare data between the two studies.
The orange juice and the grapefruit used in the study were selected as they contained the highest concentration of total
Nutrition and Cancer 2003
flavonoids in comparison with other juices analyzed. Depending on the juice the total daily intake of flavonoids per
animal per day ranged between 0.2 and 4.3 mg.
In addition to the flavonoids, the concentrations of the
three carbohydrates, sucrose, glucose, and fructose, were determined in the juices. The average daily intake of total and
individual carbohydrates is presented in Table 1. From Table
1 it is evident that each fruit juice has a unique carbohydrate
profile, and that the intake of both total and individual carbohydrates varied between the experimental groups. Despite
the increased carbohydrate intake in groups 2–7 relative to
the control Group 1 and the BNF Group 8, final body weights
and the average weight gain did not differ among the various
groups (data not shown). The relative and total liver weights
in BNF-induced animals (Group 8), however, were significantly increased compared to Groups 2–7 (data not shown).
The antioxidant enzymes SOD, GR, GPx, and CAT measured in RBC were not found to be altered by any of the fruit
juices or by quercetin administered in the diet at doses ranging from 0.001 to 10 g/kg of diet (Fig. 1A and B).
Hepatic QR was significantly elevated above the control
level in rats administered grapefruit juice, apple juice, or
black currant juice (Fig. 2). Hepatic QR in the apple plus
black currant juice group also tended to be elevated compared to the control group; however, statistical significance
was not reached (P > 0.05). Hepatic GST activity for these
same juices was also above the control level but again statistical significance was not reached (P > 0.05). Across the employed dose range of quercetin, no effects were observed on
GST or QR in the liver. BNF was found to significantly induce QR and GST (Fig. 2), whereas BNF had no effects on
the remaining investigated biomarkers (data not shown).
Apple, black currant, and the 1:1 combination of these
two juices were found to significantly decrease the lipid
peroxidation biomarker MDA in plasma (Fig. 3A). Borderline significant (P = 0.07) reductions in plasma MDA in the
orange and grapefruit juice groups were also observed. In
contrast, increased levels of protein oxidation products,
measured as AAS, were found in plasma of animals exposed to orange, black currant, and black currant plus apple
juice. In the group of animals that received sucrose, glucose, and fructose in the drinking water (Group 7), the level
of AAS was also significantly increased compared to the
control. Neither the level of MDA, nor the level of AAS,
was altered by quercetin at doses ranging from 1 mg to 10
g/kg of diet (Fig. 3B).
The bioavailability of quercetin and its major metabolites,
isorhamnetin and tamarixetin, methoxylated at the 3 and 4
positions of quercetin, respectively, was analyzed for by
LC/MS in the quercetin dose-response study as described in
Materials and Methods. Quercetin and isorhamnetin could
be detected at all doses investigated, whereas tamarixetin was
only found at quercetin doses of 0.1 g/ kg of diet or higher
(Fig. 4). The concentrations of quercetin, isorhamnetin, and
tamarixetin in plasma were found to increase in a dose-dependent fashion. The maximal plasma concentrations of the
three flavonoids obtained at the 10 g/kg of diet dose were 2.3,
Vol. 45, No. 1
Figure 1. Activity of the antioxidant enzymes glutathione peroxidase
(GPx), superoxide dismutase (SOD), glutathione reductase (GR), and
catalase (CAT) in red blood cells from juice (A) and quercetin (B) fed female
rats. Gr.juice, grapefruit juice; Bl.cur., black currant juice; Carb., carbohydrate control.
8.4, and 2.4 µM for quercetin, isorhamnetin, and tamarixetin,
respectively. The average daily intake of quercetin (per kg
b.w.) based on recorded food consumption data was estimated to be 0, 0.13, 1.3, 12.9, 128, and 1,397 mg, corresponding to total daily quercetin intakes of 0, 0.016, 0.161,
1.654, 16.6, and 177.4 mg per animal. The plasma flavonoid
concentration in the juice study was not determined due to an
insufficient amount of sample. However, neither quercetin,
isorhamnetin, nor tamarixetin could be detected in urine
from animals exposed to the various juices (data not shown),
and it is thus questionable whether quercetin and its metabolites in plasma would have exceeded the detection limit of 2.5
ng/ml of plasma and thus allowed quantification of the three
compounds.
Discussion
Several fruit juices were analyzed for their specific
flavonoid content by LC/MS. From these analyses it was evident that the total flavonoid content varied markedly from
brand to brand. Similar observations on other juices are re49
Figure 2. Glutathione S-transferase (GST) (white bars) and quinone
reductase (QR) (black bars) activity in liver cytosol from juice (A) or
quercetin (B) fed female rats. The GST (µmol of glutathione-conjugate
formed/min/mg of protein) and QR (µmol of cytochrome c reduced/min/mg
of protein) activities in hepatic cytosol from BNF-induced rats were 4,032 ±
289 and 3,721 ± 412, respectively. The bars indicate the mean ± SD of duplicate samples from 5 animals.
ported in the literature (37,38). The content of individual
flavonoids was also found to vary within the same brand and
type of juice. The concentration of quercetin (2 and 4 mg/l) in
the currently employed batches of apple and black currant
juice, respectively, was thus somewhat lower than the concentrations previously determined (7 and 6 mg/l) in juices of
the same brand but from a different batch (36). Kaempferol
was likewise found at a concentration of 0.2 mg/l in the apple
juice used in the study by Young and colleagues (36),
whereas this flavonoid could not be detected in the present
study. The chalcone phloretin found in apple juice at a concentration of 11 mg/l in the present study, on the other hand,
was not detected in the particular batch of apple juice used in
the human intervention study. These results suggest that the
flavonoid content of fruit juices vary considerably between
different brands but also among different batches of juice.
The observed differences presumably reflect variations in the
quality of the fruits, the cultivar, the manufacturing practices
used, storage conditions, and duration as well as the presence
of other antioxidants in the juice that may function to stabilize the flavonoids. The flavonoid determinations in this and
50
Figure 3. Effect of fruit juices (A) or quercetin (B) on the lipid and protein
peroxidation biomarkers malondialdehyde (MDA) (black bars) and 2-amino
adipic semialdehyde (AAS) (white bars) in plasma from female rats. The
bars indicate the mean ± SD of duplicate samples from 5 animals.
in the previously published juice intervention study (36)
were conducted by the same laboratory and even by the same
personnel, so only little variation from this source is expected. It should be noted that the employed fruit juices also
contain anthocyanins, catechins, and other potentially active
phytochemicals, and that the measured set of flavonoids only
represents a fraction of the total flavonoid or phytochemical
content.
It is noteworthy that the effects observed on the
biomarkers of lipid and protein peroxidation, MDA and
AAS, follow the same trend in the human study (36) and the
present rat study, despite that both the total and the specific
flavonoid content of the measured subset of flavonoids varied between the two studies. In both studies MDA was thus
significantly decreased by the 1:1 combination of black currant and apple juice and plasma AAS was concordantly significantly increased. This observation strongly suggests that
specific components in apple and/or black currant juice trigger identical mechanisms or pathways in humans and in the
particular rat model used in the present study. The different
qualities of the fruit juices in the two studies, judged from the
content of specific flavonoids, furthermore suggest that other
components of the fruit juice rather than the measured set of
Nutrition and Cancer 2003
Figure 4. Concentration of quercetin and its two metabolites isorhamnetin
and tamarixetin in plasma from quercetin-fed female rats as determined by
liquid chromatography/mass spectrometry. The bars indicate the mean ± SD
of duplicate samples from 5 animals.
flavonoids mediate the observed effects on redox status. The
administration of either black currant or apple juice to rats
suggested that both juices alone could reduce the level of
lipid peroxidation, whereas only black currant juice administration increased the level of plasma AAS. Orange juice was
also found to have this effect. That AAS was affected in the
same direction in the human intervention study (36) and in
the present rat study, despite that the juices used were qualitatively very different with respect to flavonoid content, might
be a further indication that other juice components, such as
carbohydrates, vitamin C, or other potentially prooxidative
species, rather than the investigated flavonoids may be responsible for the observed effects on this particular protein
oxidation marker. It has previously been speculated that vitamin C or other prooxidant compounds coexisting with vitamin C in fruits may, at least in part, be responsible for the
prooxidant effect of fruit juices (36). The animals in Group 7,
however, which received the pure carbohydrates sucrose,
glucose, and fructose were also found to have an elevated
level of plasma AAS compared to the control. This observation strongly suggests that carbohydrates present in the respective fruit juices may also be fully or in part responsible
for the observed prooxidant effect on plasma proteins observed following fruit juice treatment. These results may be
related to the well-established observations that high glucose
levels result in nonenzymatic glycation of both extra- and
intracellular proteins, accumulation of sorbitol via the
aldose-reductase pathway, activation of protein kinase C
isoforms, as well as reduced bioavailability of nitric oxide
(39). The generation of reactive oxygen species may thus be
a common downstream mechanism by means of which the
multiple by-products of glucose exert their adverse effects on
Vol. 45, No. 1
plasma proteins. More recent data furthermore suggest that
sucrose enhances the number of preneoplastic lesions in
azoxymethane-initiated rats (40) and act as a mutagen in rat
colon (41). Further studies are thus strongly warranted to
more firmly establish the role of carbohydrates and carbohydrate-containing products in mediating protein oxidation in
humans, due to the central role refined and unrefined carbohydrates play in human nutrition today.
In several studies dealing with the health protective effects
of flavonoids, a major focus has been on the flavonol
quercetin, which is present at particularly high concentrations in onions, cruciferous vegetables, wine, and tea (42,43).
In the present study, quercetin was found in all fruit juices investigated, but in relatively small amounts. The quercetin
dose-response study conducted in the present investigation
spanned a wide range of quercetin doses, including those obtained in rats dosed with the various fruit juices (0.04–0.08
mg per day). The results revealed that quercetin alone was
not responsible for the observed alterations in redox status or
phase 2 enzyme activity, as no effect on the respective biochemical markers was observed following daily quercetin intakes between 0.016 and 177.4 mg. However, it cannot be
ruled out that quercetin together with other phytoprotectants
present in the juices may play a role in mediating the observed effects.
Investigation of the activities of the two phase 2 enzymes
QR and GST in liver and RBC antioxidative enzymes revealed that hepatic QR activity was significantly increased
by grapefruit, apple, and black currant juice, whereas none of
the remaining enzymes were affected by the juice administrations, by quercetin given in the diet as a pure compound, or
by selected carbohydrates. It is noteworthy that administration of moderate doses of fruit juices for only 1 wk is sufficient to increase hepatic QR by 40–60% above the control.
These data could be taken to suggest that fruit juices at reasonable dietary levels may favorably affect resistance to
chemical carcinogens and other dietary or environmental
toxicants detoxified by QR.
Overall, the present study provides evidence that commonly consumed fruit juices may alter important biochemical pathways involved in maintaining redox and phase 2 detoxification status in experimental animals. However,
whether alterations of these redox and detoxification enzyme
biomarkers do impact on the resistance of the animal toward
chemical carcinogens, what particular fruit juice components
are responsible for the observed effects, and whether these alterations impact on the cancer process in humans must still
be clarified.
Acknowledgments and Notes
The authors would like to thank Anita Nielsen and Katrin Christiansen
for excellent technical assistance. Address correspondence to V. M.
Breinholt, Mørkhøj Bygade 19, 2860 Søborg, Denmark. Phone: +45 33 95
66 05. FAX: +45 33 95 66 99. E-mail: [email protected]
Submitted 5 February 2002; accepted in final form 4 November 2002.
51
References
1. Ferguson LR: Prospects for cancer prevention. Mutat Res 428,
329–338, 1999.
2. Yip I, Heber D, and Aronson W: Nutrition and prostate cancer. Urol
Clin North Am 26, 403–411, 1999.
3. Young KJ and Lee PN: Intervention studies on cancer. Eur J Cancer
Prev 8, 91–103, 1999.
4. Faivre J and Bonithon-Kopp C: Chemoprevention of colorectal cancer.
Recent Results Cancer Res 151, 122–133, 1999.
5. La Vecchia C and Tavani A: Fruit and vegetables, and human cancer.
Eur J Cancer 7, 3–8, 1998.
6. Wargovich MJ: Experimental evidence for cancer preventive elements
in foods. Cancer Lett 114, 11–17, 1997.
7. Steinmetz KA and Potter JD: Vegetables, fruit, and cancer prevention:
a review. J Am Diet Assoc 96, 1027–1039, 1996.
8. Patrick L: Beta-carotene: the controversy continues. Altern Med Rev 5,
530–545, 2000.
9. Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, et al.:
Lack of effect of long-term supplementation with beta-carotene on the
incidence of malignant neoplasms and cardiovascular disease. N Engl
J Med 334, 1145–1149, 1996.
10. The Alpha-Tocopherol Beta-Carotene Study Group: The effects of vitamin E and beta-carotene on the incidence of lung cancer and other
cancers in male smokers. N Engl J Med 330, 1029–1035, 1994.
11. Greenberg ER: A clinical trial of antioxidant vitamins to prevent
colorectal cancer. N Engl J Med 331, 141–147, 1994.
12. McLennan R: Randomized trial of intake of fat, fiber, and beta-carotene to prevent colorectal adenomas. J Natl Cancer Inst 87,
1760–1766, 1995.
13. Bueno De Mesquita HB, Maisonneuve P, Runia S, and Moerman CJ:
Intake of foods and nutrients and cancer of the exocrine pancreas: a
population-based case-control study in the Netherlands. Int J Cancer
48, 540–549, 1991.
14. Cohen JH, Kristal AR, and Standord JL: Fruit and vegetable intakes
and prostate cancer risk. J Natl Cancer Inst 92, 61–68, 2000.
15. Gupta PC, Hebert JR, Bhonsle RB, Sinor PN, Mehta H, et al.: Dietary
factors in oral leukoplakia and submucous fibrosis in a population-based case control study in Gujarat, India. Oral Dis 4, 200–206,
1998.
16. Jain MG, Hislop GT, Howe GR, and Ghadirian P: Plant foods, antioxidants, and prostate cancer risk: findings from case-control studies in
Canada. Nutr Cancer 34, 173–184, 1999.
17. Levi F, Pasche C, LaVecchia C, Lucchini F, and Franceschi S: Food
groups and colorectal cancer risk. Br J Cancer 79, 1283–1287, 1999.
18. Michaud DS, Spiegelman D, Clinton SK, Rimm EB, Willett WC, et
al.: Fruit and vegetable intake and incidence of bladder cancer in a
male prospective cohort. J Natl Cancer Inst 91, 605–613, 1991.
19. Justesen U, Knuthsen P, and Leth T: Quantitative analysis of flavonols,
flavones, and flavanones in fruits, vegetables and beverages, by
high-performance liquid chromatography with diode array and mass
spectrometric detection. J Chromatogr A 799, 101–110, 1998.
20. Kumpulainen JT, Lehtonen M, and Mattila P: Trolox equivalent antioxidant capacity of average flavonoids intake in Finland. In Natural
Antioxidants and Anticarcinogens in Nutrition, Kumpulainen JT and
Salonen JT (eds). Cambridge, UK: The Royal Society of Chemistry,
1999, pp 141–150.
21. Meyer O, Blom L, and Søndergård J: The influence of minerals and
proteins on the nephrocalcinosis potential for rats of semisynthetic diets. Lab Anim 16, 271–273, 1982.
22. Lake BG: Preparation and characterization of microsomal fractions for
studies on xenobiotic metabolism. In Biochemical Toxicology: A Practical Approach, Snell K and Mullock B (eds). Oxford: IRL Press,
1987, pp 183–215.
52
23. Redinbaugh MG and Rurly RB: Adaption of the bichinchoninic acid
protein assay for use with microtiter plates and sucrose gradient fractions. Anal Biochem 153, 267–271, 1986.
24. National Research Council: Guide For the Care and Use of Laboratory
Animals. Bethesda, MD: National Institutes of Health, 1985 (NIH
Publ 85–23).
25. Breinholt V, Lauridsen ST, and Dragsted LO: Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat. Xenobiotica 29, 1227–1240, 2000.
26. Breinholt VM, Lauridsen ST, Daneshvar B, and Jakobsen J: Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat. Cancer Lett 154, 201–219, 2000.
27. Ernster L: DT Diaphorase. Methods Enzymol 10, 309–317, 1967.
28. Habig WH, Pabst MJ, and Jacoby WB: Glutathione S-transferases.
The first enzymatic step in mercapturic acid formation. J Biol Chem
249, 7130–7139, 1974
29. Goldberg DM and Spooner RJ: In Methods of Enzymatic Analysis,
Bergmeyer HU (ed). Deerfield Beach, Florida: Anonymous Verlag
Chemie, 1983, pp 258–265.
30. Wheeler CR, Salzman JA, Elsayed NM, Omaye ST, and Korte DW:
Automated assays for superoxide dismutase, catalase, glutathione,
peroxidase and glutathione reductase activity. Anal Biochem 184,
193–199, 1990.
31. Lauridsen ST and Mortensen A: Probucol selectively increases oxidation of atherogenic lipoproteins in cholesterol-fed mice and in
Watanabe heritable hyperlipidemic rabbits. Atherosclerosis 142,
169–178, 1999.
32. Daneshvar B, Frandsen H, and Dragsted LO: Gamma-glutamyl
semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins. Biomarkers 2, 117–123, 1997.
33. Andersen R and Sørensen A: Separation and determination of alditols
and sugars by high-pH anion-exchange chromatography with pulsed
amperometric detection. J Chromatogr A. 897, 195–204, 2000.
34. Nielsen SE and Dragsted LO: Column-switching high-performance
liquid chromatographic assay for the determination of quercetin in human urine with ultraviolet absorbance detection. J Chromatogr B 707,
81–89, 1998.
35. Nielsen SE, Freese R, Cornett C, and Dragsted LO: Identification and
quantification of flavonoids in human urine samples by column-switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry. Anal Chem 72,
1503–1509, 1999.
36. Young JF, Nielsen SE, Haraldsdottir J, Daneshvar B, Lauridsen ST, et
al.: Effect of fruit juice intake on urinary quercetin excretion and
biomarkers of antioxidative status. Am J Clin Nutr 69, 87–94, 1999.
37. Ho PC, Saville DJ, Covill PF, and Wanwimolruk S: Content of
CYP3A4 inhibitors naringin, naringenin and bergapten in grapefruit
and grapefruit juice products. Pharm Acta Helv 74, 379–385, 2000.
38. Ross SA, Ziska DS, Zhao K, and ElSohly MA: Variance of common
flavonoids by brand of grapefruit juice. Fitoterapia 71, 154–161, 2000.
39. Gugliucci A: Glycation as the glucose link to diabetic complications. J
Am Osteopath Assoc 100, 621–634, 2000.
40. Poulsen M, Mølck A-M, Thorup I, Breinholt V, and Meyer O: The influence of simple sugars and starch given during pre- or postinitiation
on aberrant crypt foci in rat colon. Cancer Lett 167, 135–143, 2001.
41. Dragsted LO, Daneshvar D, Vogel U, Autrup HN, Wallin H, et al.: A
sucrose-rich diet induces mutation in rat colon. Cancer Res 62,
4339–4345, 2002.
42. Hertog MGL, Hollman PCH, and Katan MB: Content of potentially
anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly
consumed in the Netherlands. J Agric Food Chem 40, 2379–2383,
1992.
43. Hertog MGL, Hollman PCH, and van de Putte B: Content of potentially anticarcinogenic flavonoids in tea infusions, wine and fruit
juices. J Agric Food Chem 41, 1242–1246, 1993.
Nutrition and Cancer 2003