Tomato juice intake suppressed serum concentration of 8-oxodG after extensive physical activity

Abstract

Background
DNA is constantly exposed to reactive oxygen species (ROS), spontaneously arising during the normal oxygen metabolism. ROS may result in temporary as well as permanent modifications in various cellular components such as lipids, proteins and DNA, which may have deleterious consequences. Demonstrating that a dietary supplementation of antioxidants can reduce oxidative DNA damage may provide evidence for the value of such supplementation in prevention of cancer and age related diseases.

Findings
The present study was conducted to address whether tomato juice protects against ROS induced by extensive physical exercise in untrained individuals. As a marker of oxidative stress, serum levels of 8-oxodG were monitored using a modified ELISA. An intervention was performed involving 15 untrained healthy subjects who performed a 20 min physical exercise at 80% of maximum pulse using an ergometer bicycle. Blood samples were taken before and one hour after the exercise. The procedure was repeated after 5 weeks with a daily intake of 150 ml tomato juice and followed by a 5 weeks wash-out period and another 5 weeks with a daily intake of tomato juice. The results indicated that a daily intake of tomato juice, equal to 15 mg lycopene per day, for 5 weeks significantly reduced the serum levels of 8-oxodG after an extensive physical exercise.

Conclusion
These data strongly suggest that tomato juice has a potential antioxidant effect and may reduce the elevated level of ROS induced by oxidative stress.

Full text

S H O R T R E P O R T Open Access
Tomato juice intake suppressed serum
concentration of 8-oxodG after extensive
physical activity
Mats Harms-Ringdahl, Dag Jenssen and Siamak Haghdoost
*
Abstract
Background: DNA is constantly exposed to reactive oxygen species (ROS), spontaneously arising during the normal
oxygen metabolism. ROS may result in temporary as well as permanent modifications in various cellular
components such as lipids, proteins and DNA, which may have deleterious consequences. Demonstrating that a
dietary supplementation of antioxidants can reduce oxidative DNA damage may provide evidence for the value of
such supplementation in prevention of cancer and age related diseases.
Findings: The present study was conducted to address whether tomato juice protects against ROS induced by
extensive physical exercise in untrained individuals. As a marker of oxidative stress, serum levels of 8-oxodG were
monitored using a modified ELISA. An intervention was performed involving 15 untrained healthy subjects who
performed a 20 min physical exercise at 80% of maximum pulse using an ergometer bicycle. Blood samples were
taken before and one hour after the exercise. The procedure was repeated after 5 weeks with a daily intake of
150 ml tomato juice and followed by a 5 weeks wash-out period and another 5 weeks with a daily intake of
tomato juice. The results indicated that a daily intake of tomato juice, equal to 15 mg lycopene per day, for
5 weeks significantly reduced the serum levels of 8-oxodG after an extensive physical exercise.
Conclusion: These data strongly suggest that tomato juice has a potential antioxidant effect and may reduce the
elevated level of ROS induced by oxidative stress.
Keywords: Reactive oxygen species, ROS, Free radicals, Exercise,Lycopene,Tomatojuice,Lifestyle,ELISA,
hMTH1, 8-oxo-dG
Findings
It has been suggested that dietary antioxidants reduce the
level of oxidative DNA damage induced by reactive oxy-
gen species. However, there are limited in vivo studies
which support this hypothesis as a number of epidemio-
logical studies showed no such effect following dietary
supplementation with carotenoids, vitamin C, or E [1,2].
Urinary concentration of 8-hydroxy-2′-deoxyguanosine
(8-oxodG, a base damage formed by reactive oxygen spe-
cies) has been used as a non-invasive biomarker of oxida-
tive DNA base damage in a number of studies [3,4].
Reactive oxygen species (ROS) have been suggested to
play an important role in mutagenesis, carcinogenesis,
and aging processes. ROS can react with different cellu-
lar components e.g., proteins, lipids and nucleic acids,
and give rise to chemical modifications. Under normal
conditions cellular antioxidant enzymes and other anti-
oxidants in the cell detoxify elevated levels of ROS and
minimize damage to intracellular components. However,
under extensive physiological activity ATP consumption
will increase followed by increased oxygen consumption
and, as a consequence, the production of ROS will
increase [5]. Healthy and/or well-trained persons and
vegetarians seem to have increased protection against
ROS-induced damages [6]. It suggests that regular phys-
ical exercise [7] and a diet rich in antioxidant [8] may have
a protective effect towards ROS-induced damage, in par-
ticular DNA base damage. One of the frequently studied
DNA base damages is 8-oxodG. Highly effective repair
mechanisms are operating both on DNA (e.g., hOGG1)
* Correspondence: Siamak.Haghdoost@gmt.su.se
Centre for Radiation Protection Research (CRPR), Department of Genetics,
Microbiology and Toxicology, Stockholm University, SE-106 91, Stockholm,
Sweden
© 2012 Harms-Ringdahl et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Harms-Ringdahl et al. Nutrition Journal 2012, 11:29
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and the nucleotide pool (hMTH1) to remove 8-oxodG/8-
oxodGTP from the cell [9,10]. Previously, we have shown
that extracellular 8-oxodG originates from the nucleotide
pool and may serve as a sensitive marker of oxidative
stress [11]. We have developed an ELISA method that
allows the determination of the very low concentrations of
8-oxodG present in human blood serum [11-13].
In the present study we aimed to investigate the pro-
tective effect of tomato juice intake towards ROS
induced by 20 min of extensive physical exercise. A
novel finding in the present intervention study was that
the level of 8-oxodG in human blood serum was
increased significantly after 20 min acute physical activ-
ity possibly caused by an increase of the intracellular
ROS level. No increase was observed when individuals
had been drinking 150 ml tomato juice per day during a
period of 5 weeks suggesting that the intracellular nu-
cleic acids and, in particular, the nucleotide pool were
unaffected and well protected from the deleterious effect
of ROS. The intervention study support the hypothesis
that antioxidants (e.g. lycopene) supplied from tomato
juice may protect against oxidative stress induced by ex-
tensive physical exercise.
Materials and methods
In this study 15 healthy and untrained donors (Table 1)
were asked to drink 150 ml tomato juice per day in 2 peri-
ods of 5 weeks each as follows: 5 weeks with tomato juice,
5 weeks without tomato juice and 5 weeks with tomato
juice. Blood samples were collected before and after start of
each period during the intervention study. At the day for
blood collection, the individuals were asked to do 20 min
physical exercise with 80% max pulse using an ergometer
bicycle. To calculate the individual maximum pulse, the fol-
lowing generally accepted formula was used: 220 - age =
maximum pulse. Blood samples were collected before and
after exercise. The serum level of 8-oxodG was analyzed as
a marker of oxidative stress. Of note, this intervention study
was designed according to recommendations which have
been published by Loft and co-workers [14]. The study was
performed in accordance with the ethical standards and
approved by the Swedish Ethical Committee at the
Karolinska University Hospital (Dnr 03–621).
Analysis of 8-oxodG in blood serum
Just before and one hour after each training session
(20 min cycling), blood samples were collected in tubes
Table 1 Main characteristics of the donors
Donors Age Sex Smoking Vegetarian Vitamins Background diseases
1 30 F No no no no
2 28 M No no no no
3 35 M No no yes no
4 28 F No no no no
5 29 F No no yes no
6 46 M No no no no
7 29 M No no no no
8 24 F No no yes no
9 27 M No no yes no
10 25 F No no no no
11 25 F 3-5 cig/day no no no
12 27 M No no no no
13 43 F No no no no
14 38 M No no no no
15 24 M No no no no
Table 2 Concentration of 8-oxodG before and after 20 min physical activity (mean ± SE) in blood serum after periods
with and without tomato juice intake
without TJ 5 weeks with TJ 5 weeks without TJ 5 weeks with TJ
8-oxodG ng/ml A1 A2 B1 B2 C1 C2 D1 D2
0.52±0.11 0.74±.0.14 0.56±0.13 0.46±0.06 0.45±0.08 0.83±0.16 0.31±0.06 0.39±0.06
A1, A2, B1 and B2; n=15. For C1 and C2; n=11 while for D1 and D2; n=9.
TJ: tomato juice.
Samples A1, B1, C1 and D1: the level of 8-oxodG before 20 min physical activity.
Samples: A2, B2, C2 and D2: the level of 8-oxodG 1h after 20 min physical activity.
Harms-Ringdahl et al. Nutrition Journal 2012, 11:29 Page 2 of 5
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without anticoagulant. After complete coagulation of the
blood samples, the blood serum was isolated. 8-OxodG
concentration of blood serum was measured using ELISA
as described previously [11,15]. The ELISA kit was pro-
vided by Health Biomarkers Sweden AB. Briefly, 1 ml
blood serum was purified using a C18 solid phase extrac-
tion column (Varian, CA) according to a previously
published method [16]. This step is necessary to remove
products other than 8-oxodG which could cross-react
with the monoclonal antibody used in the kit. A standard
curve for 8-oxodG (0.05 - 10 ng/ml) was established for
each plate covering the range of 8-oxodG in the samples.
One sample in each experiment was mixed with 1 ng
8-oxodG before purification which served as internal
standard. Validation of the modified ELISA method
was performed by HPLC-EC (r
2
:0.87,p<0.05) [15].
Comparisons between the ELISA and the HPLC-EC
methods showed a linear correlation at the concentration
range found in the human blood serum [15]. There was
no correlation between ELISA and HPLC-EC when unfil-
tered samples were used.
Determination of the lycopene content in tomato
juice
The lycopene content in tomato juice was determined
spectrophotometrically essentially as described in refer-
ence by Fish et al. [17]. The concentration of lycopene
was calculated using the molar extinction coefficient of
lycopene in hexane (17.2 × 10
4
M
-1
cm
-1
).
Statistical method
Student’s t-test was used for testing statistical signifi-
cance for serum levels of 8-oxodG. A p-value below 0.05
was deemed as significant.
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
1D-2D1C-2C1B-2B1A-2A
8-oxodG (ng/ml)
p<0.05
p<0.05
p=0.07
p=0.07
Figure 1 Mean values of the individual changes in 8-oxodG serum concentrations (values before exercise are subtracted from values
after exercise) after 20 minutes of exercise. White bars show the changes when exercise was performed without previous tomato juice
supplementation. Dotted bars show the values after five weeks of lycopene supplementation. For bars A2-A1 and B2-B1 n = 15 and for C2-C1
n = 11 while for D2-D1 n = 9.
)D+B()C+A(
8-oxodG (ng/ml)
Figure 2 Shows mean values of individual changes of 8-oxodG level after periods without(mean of A and C periods; n =26) and after
periods with tomato juice intake (mean of B and D values; n = 24) before and after exercise respectively. Bars show means ± SE.
Harms-Ringdahl et al. Nutrition Journal 2012, 11:29 Page 3 of 5
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Results
The participants were asked to have a daily intake of
150 ml tomato juice (containing 0.1 mg lycopene per ml
juice) for 35 days in 2 interventions with a wash-out
period between these periods. The main characteristics
of the donors are presented in Table 1. Blood samples
were collected day 0 (period A), day 35 (end of period
B), day 70 (end of period C; washout) and day 105 (end
of period D). Two blood samples were taken at each oc-
casion, one before and the second 60 minutes after the
20 min physical exercise. The serum level of each par-
ticipant was compared to his/her own control value.
Thus, each participant served as his/her own control.
Table 2 shows the concentrations of 8-oxodG in serum
at the different sampling times during the intervention
study. The background levels (before exercise) of 8-oxodG
did not deviate significantly during the interventions.
As shown in Table 2, 20 min extensive physical activity
increased the level of 8-oxodG in serum above the back-
ground level by an average of 42% (from 0.52 up to
0.74 ng/ml). In contrast, following 5 weeks (B) intake of
150 ml tomato juice (corresponding to a daily intake of
15 mg lycopene) the levels of 8-oxodG remained essen-
tially unchanged after compared to before the exercise.
After the 5-week washout period (C), the level of 8-
oxodG increased again (84%) after exercise in average
from 0.45 up to 0.83 ng/ml. After the second period of
tomato juice intake (D), the levels of 8-oxodG after ex-
ercise were almost the same as before the exercise (from
0.31 up to 0.39 ng/ml).The results are summarized and
presented in Table 2 and in Figures 1 and 2.
The data in Figure 2 are the average changes of 8-
oxodG induced by physical activity (the concentration of
8-oxodG before exercise is subtracted from the concen-
tration after activity exercise) without tomato juice in-
take (periods A and C) and with tomato juice intake
(periods B and D).
As shown in Table 1, four subjects took vitamin sup-
plementation. Also when these subjects were excluded
from the statistical analysis, the effects of tomato juice
intake remained significant.
Conclusion
Based on the results from this intervention study it is con-
cluded that 8-oxodG is significantly increased in blood
serum of 15 healthy donors after an acute physical exer-
cise, suggesting that there is a positive correlation between
8-oxodG in serum and the intracellular ROS production.
The study also demonstrates that 150 ml tomato juice in-
take (15 mg lycopene) per day significantly protects the
nucleotide pool from ROS produced in response to exten-
sive physical activity. The proposed explanation for the
observed results is that ROS induced by extensive physical
activity react with intracellular dNTP and give rise to
production of 8-oxodGTP. 8-OxodGTP is excreted from
the intra- to the extra-cellular matrix by the action of
hMTH1 protein [18] to inhibit its incorporation into the
DNA. Lower levels of 8-oxodG in serum during tomato
juice intake show that tomato juice intake protects dNTP
from ROS induced modification.
It is important to mention that beside lycopene toma-
toes also contain vitamin C, tocopherols and polyphe-
nols [19]. It has been shown that among all antioxidants
(in particular carotenoids) present in tomato juice, lyco-
pene is the most abundant and stable during industrial
food processing [19]. Vitamin C and tocopherols in fresh
tomato are destroyed by heating during food processing.
Not much is known about the polyphenols in tomato
juice [19]. Therefore, we believe that the antioxidant ac-
tivity of tomato juice is primarily due to its content of
lycopene.
It might be hypothesized that long term intake of tomato
juice may reduce oxidative stress levels in patients with
enhanced level of oxidative stress, for example, patients
with diabetes, cardiovascular diseases or inflammation. A
study is in progress to test the hypothesis on patients with
diabetes using the experimental model system described
with 8-oxodG as biomarker.
Competing interests
Authors have no competing interests.
Acknowledgements
This work was supported by the Commission of European Union (Grant
agreement 245030). We would like to thank associated professor Siv
Osterman Golkar for valuable comments and discussions. We also thank
Christofer Andersson for his contribution and all the volunteers who
participated in this project. Finally we thank Kiviks Musteri AB for providing
the tomato juice.
Authors’contributions
SH served as Principal Investigator and contributed to design of the
experiment, manuscript preparation and determination of 8-oxodG in
human blood. MH-R contributed to writing the manuscript, procurement of
external funding and study design. DJ contributed to study design,
manuscript preparation and data presentation. All authors read and
approved the manuscript.
Received: 2 November 2011 Accepted: 9 April 2012
Published: 2 May 2012
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doi:10.1186/1475-2891-11-29
Cite this article as: Harms-Ringdahl et al.:Tomato juice intake
suppressed serum concentration of 8-oxodG after extensive
physical activity. Nutrition Journal 2012 11:29.
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