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Antioxidants in Fruits and Vegetables: a Study of Cellular Availability and

Direct Effects on Human DNA

Article  in  Bioscience Biotechnology and Biochemistry · November 2006

DOI: 10.1271/bbb.60224 · Source: PubMed

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Antioxidants in Fruits and Vegetables: a Study of Cellular Availability
and Direct Effects on Human DNA
Yim Tong S ZETO,1;2; y Wing Kwan C HU,1 and Iris F. F. B ENZIE1
1
Department of Health Technology & Informatics, The Hong Kong Polytechnic University,
Hung Hom, Kowloon, Hong Kong
2
School of Health Sciences, Alameda Dr. Carlos D’ Assumpção, No. 335–341, Edif. Centro Hotline,
5
Andar, Macao Polytechnic Institute, Macau
The effects of several types of whole fruits and
vegetables on human lymphocytic DNA were investi-
gated by using two versions of the comet assay. The total
antioxidative capacity, as the FRAP value, and ascorbic
acid (AA) content were also measured to explore the
relationship between the effect and antioxidant content.
Key words: antioxidant; ascorbic acid; comet assay;
fruit; lysed cell
Fruits and vegetables contain antioxidative vitamins
and various phytochemicals with antioxidative activi-
ty.1–4) Fruits, e.g., lemon, orange and strawberry, have a
rich content of such flavonoids as eriocitrin, hesperidin
and anthocyanins,5–8) while quercetin, apigentin, myr-
icetin and luteolin are commonly found in vegetables.9)
Epidemiological studies have clearly shown that a high
intake of fruits and vegetables is associated with a lower
risk of cancer, and 30–40% of all cancers are estimated
to be preventable through dietary modification and an
increased intake of fruits and vegetables.10,11) The
aetiology of many types of cancer and chronic diseases
is thought to be mediated, at least partly, by oxidative
stress, and the protective effects of fruits and vegetables
ahs been suggested to be mediated in part by their
antioxidative micronutrient content.12,13) In vitro studies
have shown that antioxidative vitamins (vitamin C and
vitamin E) protected DNA against oxidative chal-
lenge.14,15) However, in vitro and supplementation
studies have not always demonstrated positive or
beneficial results.16–18) This may have been due to
the low cellular bioavailability of antioxidants, or to
protection only being seen under certain concentrations
or conditions. However, it may be that vitamin C is not
a key player, and that protection is conferred by other, as
yet undefined, components of healthy diets that are
found in association with vitamin C but that work in
y
To whom correspondence should be addressed. School of Health Sciences, Alameda Dr. Carlos D’ Assumpção, No. 335–341, Edif. Centro
Hotline, 5
Andar, Macao Polytechnic Institute, Macau; Tel: +852-92892854; Fax: +853-28753159; E-mail: ytszeto@ipm.edu.mo or
savio.yim.tong.szeto@inet.polyu.edu.hk
combination with or independently of vitamin C.19) The
aim of this study is to investigate potential DNA
protective effects of aqueous homogenates of whole
fruits and vegetables of known (measured) ascorbic acid
and total antioxidant content. To investigate the effects
on DNA, two versions of the comet assay were used: the
standard-whole cell comet assay,20,21) and a lysed cell
version4) in which ‘naked’ DNA is exposed to putative
genoprotectants or DNA-damaging agents. In this way,
the cellular bioavailability and mechanism of action
could be explored.
Agarose 3:1 (high-resolution blend) was purchased
from Amresco (Solon, OH, USA), and potassium
dihydrogen phosphate and potassium hydrogen phos-
phate were from Merck (New Jersey, USA). All other
reagents were purchased from Sigma (St. Louis, MO,
USA) and were of the highest grade available. Five
types of fruit, one type of commercial orange juice (with
no added preservative) and four types of vegetable were
tested. On the day purchased, a 100 g portion of the
edible part of each fruit or vegetable to be tested was
homogenized in 100 ml of cold phosphate-buffered
saline (PBS) at pH 7.4. Each homogenate was mixed
well and stored at 20
C until the day of testing, which
was within two months. Three ml of a homogenate was
mixed with 10 ml of a 0.4 M phosphate buffer (pH 7.4).
This mixture was centrifuged for 10 min at 2500 rpm and
4
C, the resulting supernatant was termed the ‘initial
extract’ of each fruit or vegetable. These were diluted
with PBS (for the standard comet assay) or with the
0.4 M phosphate buffer (for the lysed cell comet assay) in
10-fold stepwise dilutions to give a testing extract of
each fruit and vegetable concentration range of 102 to
105 relative to initial extract. The whole fruit orange
juice was also tested across a range of concentration.
Human lymphocytes from venous blood of consenting
adults were harvested according to our usual protocol.22)
Whole cells and matching cells that had been embedded
in agarose on a microscope slide and lysed with lysis
solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1%
Triton X-100, pH 10) were pre-treated for 30 min with
various dilutions of the fruit and vegetable extracts.4)
Whole cells were pre-incubated in Eppendorf tubes for
30 min at 37
C. For the lysed cells, 60 ml of an extract
was added to the gel containing the lysed cells. The gel
was covered with a coverslip, and the slide was then put
in a moist chamber at 37
C for 30 min. The whole cells
were then centrifuged and washed in PBS. The gel
samples containing the lysed cells were rinsed in a 0.4 M
phosphate buffer for 10 min. Some of the pre-treated
whole cells and some of the gel samples containing
‘naked’ DNA from the lysed cells were then subjected to
a standard oxidative challenge induced by exposure to
30 mM H2 O2 for 5 min (H2 O2 in PBS and on ice for the
standard comet assay; and in the phosphate buffer and at
RT for the lysed cell comet assay). The standard comet
assay was then performed on the whole cells, and the
lysed cell assay performed on the exposed nucleoids, as
described previously in detail.4,22) Three to five individ-
ual experiments were done for each tested extract of
each fruit and vegetable. The two versions of the comet
assay were performed in separate experiments. The
lysed cell comet assay employed here together with the
standard version provided insight into the possible effect
of the cell membrane and intracellular constituents in the
antioxidative action. The cell membrane was removed
before treatment with a fruit or vegetable extract so that
those antioxidatie molecules present in the extract were
able to directly access the nucleus. The total antioxidant
power (as the FRAP value, Ferric Reducing/Antioxidant
Power) and ascorbic acid (AA) content of each extract
were also measured.23,24)
The possibility of a catalase-mediated protective
effect in the fruit and vegetable extracts was investigated
by incubating an extract in a known amount of H2 O2 .
The initial extract of each fruit and vegetable was
diluted 10 fold with water, and then further diluted in
PBS containing H2 O2 to give a final concentration of
45 mM H2 O2 . The mixture was incubated at 37
C for
30 min, and the amount of H2 O2 remaining was then
measured by a modified version of the xylenol orange
assay.25,26)
Finally, exploratory stability studies were performed
with a lemon extract because it was found to be
protective in both versions of the comet assay. The
extract was put in a boiling water bath for 30 min to
inactivate the enzyme activity and destroy the heat-
liable antioxidants. Extracts with and without the heat
treatment were then run in parallel in both versions of
the comet assay. In addition, one part of the initial
extract was diluted 10 fold in an ascorbate oxidase
solution (final concentration of 4 IU/ml) and left at room
temperature for 15 min to degrade the ascorbic acid.
This was followed by further dilution with PBS or a
phosphate buffer for the standard or lysed cell comet
assay as appropriate, and the effects on DNA were
measured by using both versions of the assay. Pooled
lymphocytes from heparinised venous blood of appa-
rently healthy, consenting subjects were used in this
study. The lymphocytes were cryopreserved according
to the procedure of Collins et al.21) Cells were used
within 2 months of collection.
The results are presented in Table 1 and show that,
with the cell membrane removed (the lysed cell comet
assay), the extracts of lemon, persimmon, strawberry,
apple, broccoli and celery each conferred DNA protec-
tion at very low concentration. Protection in the standard
comet assay was seen with several extracts. However,
lemon (Fig. 1) and celery were the only fruit and
vegetable tested that showed protection in both versions
of the comet assay (Table 1). No statistically significant
protection or damage was seen with choy sum (a
Chinese green leafy vegetable), lettuce or orange in
either version of the comet assay at any of the
concentrations tested. The ascorbic acid content and
FRAP values were found to be negligible at the
concentration of the extracts tested. Of the 10 types of
extract tested, three protected DNA from oxidative
challenge as assessed by using the standard comet assay.
The results show that there was no significant decrease
in H2 O2 stability in the presence of any of these extracts,
indicating that the protective effect was not due to
intrinsic catalase activity.
Removing ascorbic acid by ascorbate oxidase from
the lemon extract did not remove the protective effect
seen in both versions of the comet assay. However, the
protective effect was not apparent in the heat-treated
lemon extract (Fig. 2).
The comet assay is a widely used biomonitoring tool
to assess the genotoxic and genoprotective effects of
various compounds.27) The use of both the standard and
lysed cell versions enabled further investigation of the
effects and possible mechanism of action of these
compounds: For example, as seen in this study, the
absence of a protective effect against oxidative chal-
lenge in the standard whole-cell comet assay after
exposure to a food, but with apparent protection in the
directly exposed ‘naked DNA’ lysed cell version implies
that the cellular bioavailability or response to antiox-
idative components of the food was low in whole cells.4)
Only the lemon extract and commercial orange juice
sample (no preservative added) showed significant
protective effects on intact cells. Interestingly, no
protective effect was seen from the orange extract. It
is worth investigating the commercial production of this
orange juice sample to identify the unexpected benefi-
cial effect. Furthermore, the FRAP and AA values for
the protective extracts were very low, and the protective
effect of lemon juice was not removed by enzymatic
destruction of its ascorbic acid content. This indicates
that the protection was not conferred by ascorbic acid.
Nor was the effect due to any intrinsic catalase-like
activity in the fruit. The disappearance of the protective
Table 1. Antioxidant Content and Effect of Pre-Treating with a Fruit or Vegetable Extract on the DNA Damage in Stressed Cells by the Whole Cell
and Lysed Cell Comet Assay; Results Are Expressed as the Percentage of DNA Damage Seen in Stressed Cells without Any Pre-Treatment

%DNA damage after H2 O2 treatment Calculated FRAP and AA content of

compared to control cells: tested extracts (based on measured values
mean (SD), n ¼ 3 to 5 of initial extracts)
Fruit Dilution Whole cell comet Lysed cell comet FRAP (mM) (by AA (mM) (by
of assay assay measurement of measurement of
initial initial extract and initial extract and
extract extrapolation) extrapolation)
Lemon 102 94(38) 76(21) 7 1.5
103 82(8) 59(18) 0.7 0.15
104 53(19) 66(16) 0.07 0.015
105 67(16) 71(17) 0.007 0.0015
Persimmon 102 89(20) 101(17) 7 2.8
103 67(12) 55(5) 0.7 0.28
104 89(42) 67(10) 0.07 0.028
105 73(27) 59(22) 0.007 0.0028
Strawberry 102 101(26) 76(21) 13 3.3
103 93(33) 59(18) 1.3 0.33
104 63(27) 66(16) 0.13 0.033
105 60(6) 71(17) 0.013 0.0033
Orange 102 69(30) 51(36) 7 2.8
103 71(13) 57(14) 0.7 0.28
104 86(21) 55(33) 0.07 0.028
105 86(37) 95(46) 0.007 0.0028
Commercial 102 78(23) 109(38) 12 5.4
orange 103 66(13) 82(41) 1.2 0.54
juice 104 60(7) 52(23) 0.12 0.054
105 71(6) 111(3) 00012 0.0054
Choy sum 102 96(18) 60(49) 6 0.3
103 69(19) 68(18) 0.6 0.03
104 91(32) 76(19) 0.06 0.003
105 80(19) 78(22) 0.006 0.0003
Broccoli 102 96(21) 97(27) 2 <0:2
103 78(25) 104(23) 0.2
104 77(39) 50(25) 0.02
105 88(7) 78(24) 0.002
Celery 102 65(13) 73(32) <2 <0:2
103 79(11) 74(18)
104 57(11) 61(15)
105 86(11) 82(16)
Lettuce 102 75(8) 72(32) <2 <0:2
103 85(9) 62(36)
104 77(25) 56(31)
105 88(28) 83(31)
Apple, red 102 75(21) 77(12) 3
103 70(40) 95(21) 0.3
104 68(8) 72(13) 0.03
105 89(9) 98(12) 0.003
Significantly different from the control (stressed, with no treatment);  p < 0:05; 
p < 0:01; Dunnett’s t-test.

effect after boiling the lemon extract indicates the
presence of heat-labile active ingredient(s). Further
work is needed in this area.
No protection was conferred by any fruit or vegetable
extract at the highest concentration tested, and those that
showed protection did so only with very dilute extracts.
It is possible that the pro-oxidant effect of antioxidants
at a high concentration would outweigh or counter-
balance the protective antioxidant effect under certain
circumstances. An in vitro pro-oxidant effect of ascorbic
acid has been shown in the comet assay,28 and our unpublished
observations)
and pro-oxidant activity of other antioxidants
has been shown in other tests.29–31) However, it should
be noted that we saw no evidence in this study of a pro-
oxidant effect in either version of the comet assay used
for any extract of the fruits and vegetables tested.
In summary, the new data presented here demonstrate
a DNA protective agent being present in at least some
fruits and vegetables that was heat sensitive and not
ascorbic acid. Further study is required to identify the
active ingredient(s) providing the genoprotective effects.
The results also show that the use of both versions
(whole cell and lysed cell) of the comet assay in parallel
may be a useful approach in elucidating the mechanism
for genoprotection.
 A A
125 100
DNA damage left (%)

DNA damage left (%)

100

75
* 50
50

25

0 0
control 10 -5 10 -4 10 -3 10 -2 control 10 -5 10 -4 10 -3
Dilution of initial extract Dilution of initial extract

B B
125
100
DNA damage left (%)

DNA damage left (%)

100
* **
75
**
* *
50
50

25

0
control 10 -5 10 -4 10 -3 10 -2 0
control 10 -5 10 -4 10 -3
Dilution of initial extract
Dilution of initial extract
Fig. 1. Effect of a Lemon Extract Treatment on DNA of Human
Fig. 2. Nature of the DNA Protectant in Lemon Assessed by the
Lymphocytes Assessed by the Comet Assay in (A) Standard ‘Whole
Lysed Cell Comet Assay.
Cell’ Version; (B) Lysed Cell Version.
Results were obtained in at least 3 separate experiments (mean þ
Results are expressed as the percentage of DNA damage in
1SD). The cells were pre-incubated in different dilutions of the
control cells and are mean þ 1SD of data obtained in at least 3
lemon extract and then challenged by H2 O2 (30 mM); (A) the lemon
separate experiments. The cells were pre-incubated in different
extract was boiled for 30 min before use; (B) the lemon extract was
dilutions of the lemon extract and then challenged by H2 O2 (30 mM);
 incubated with ascorbate oxidase (final concentration 4 U/l) for 15
p < 0:05 compared to control cells, Dunnett’s t-test.
min before use.  = p < 0:001, Dunnett’s t-test.

Acknowledgment Physiol., 136, 113–126 (2003).

The authors thank The Hong Kong Polytechnic
University and Macau Science & Technology Develop-
ment Fund for funding this work.
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