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Rowett Research Institute, Bucksburn, Aberdeen, Scotland, U.K. AB21 9SB
1Correspondence: Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen, Scotland, U.K. AB21 9SB. E-mail: aj{at}rri.sari.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol acetate/day; otherwise total fat,
carbohydrates, protein, and basal vitamin E contents remained
unchanged. DNA damage induced by 200 µM H2O2
in lymphocytes from volunteers as well as endogenous DNA damage in the
form of oxidized pyrimidines, measured by alkaline single-cell gel
electrophoresis (the comet assay), significantly decreased after
consumption of the 5% PUFA diet (P<0.001 and
P=0.01, respectively), but significantly increased after
consumption of the 15% PUFA diet when -tocopherol levels were in
the range of 5–7 mg/day (P=0.008 and
P=0.03, respectively). These changes were abolished by
an additional 80 mg d-tocopherol/day. This study indicates that
increasing dietary levels of PUFA to 15% may adversely affect some
indices of DNA stability. However, increasing the dietary intake of
vitamin E by 80 mg/day ameliorates the damaging effects of
PUFA.—Jenkinson, A. McE., Collins, A. R., Duthie, S. J.,
Wahle, K. W. J., Duthie, G. G. The effect of increased
intakes of polyunsaturated fatty acids and vitamin E on DNA damage in
human lymphocytes.
Key Words: PUFA • lipid peroxidation • comet assay • oxidized pyrimidines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Conversely, PUFAs are
highly susceptible to free radical-mediated lipid peroxidation, a
process thought to be involved in the initiation and progression of
certain diseases including atherosclerosis and some cancers
(3)
.
In animal studies, increased PUFA intake without concomitant
antioxidant supplementation exceeds the protective capacity of the
antioxidant defense systems, causing myopathies and neuropathies
(4)
. In addition, consumption of diets deficient in
antioxidants such as vitamin E and selenium increases DNA damage in
rats, particularly when the level of dietary unsaturated fatty acids is
raised (5
, 6)
.
In humans, levels of DNA adducts of malondialdehyde are increased after
consumption of a 13% PUFA diet while etheno-DNA adducts from lipid
peroxidation products, a proposed biomarker of DNA damage in
vivo, are also increased (7
, 8)
. This suggests that
increased lipid peroxidation in vivo or its products can
cause damage to DNA and therefore may be involved in the process of
carcinogenesis. Since dietary PUFA intake appears to enhance damage to
DNA, increased intake of nutritional antioxidants may have a protective
function. Vitamin E is the major lipid peroxidation chain-breaking
antioxidant in cell membranes and may provide protection against the
damaging effects of PUFAs, possibly indicating that intakes of both
dietary PUFA and antioxidants in combination may influence
carcinogenesis.
The aim of this study was to assess whether increased PUFA intake affects DNA damage in humans and whether any changes can be influenced by additional intake of antioxidant vitamins such as vitamin E.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods
Twenty-one healthy male nonsmokers aged 28.9 ± 1.3 years
were recruited. None were taking medication or vitamin/mineral
supplements. Fasting cholesterol concentrations were below 5.5 mmol/l.
The study was approved by the Joint Ethical Committee of the Grampian
Health Board and the University of Aberdeen. Volunteers also gave their
informed consent in writing.
Subjects participated in a free-living, split plot/change over trial in
which half consumed diets containing 5% PUFA as food energy for 4 wk
and, after a 10 wk washout period, subsequently consumed a 15% PUFA
diet for an additional 4 wk. The other subjects followed an identical
protocol except that they consumed the 15% PUFA diet first. Diets
consisted of normal foods available from local stores and were provided
either with or without an additional 80 mg of vitamin E/day in the form
of d-tocopherol acetate. Total fat, protein, and carbohydrate levels
were similar in both diets and reflected typical daily intakes in the
Scottish diet (Table 1
) (9)
. However, whereas monounsaturated fat levels were
constant, the amounts of polyunsaturated and saturated fats expressed
as a percentage of energy varied (Table 1)
. PUFA levels were designed
to be either below the normal intake of ~6% of energy as PUFA or
more than double that intake, up to 15% of energy (9)
.
Amounts of -tocopherol in both diets were calculated to approximate
to the U.S. RDA of 10 mg/day and vitamin C levels were kept within the
range 65–75 mg/day (Table 2
) (10)
.
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Blood collection and storage
Blood (up to 50 ml) was removed by venipuncture from the arm,
into evacuated tubes, with EDTA as anticoagulant. The blood samples
were obtained after an overnight fast at weeks 0, 4, 14, and 18, weeks
0–4 and 14–18 being the times that the volunteers consumed the
experimental diets.
Blood samples were stored on ice for a maximum of 1 h (minimizing loss of cellular antioxidants or PUFAs) before centrifugation (4°C, 2400 x g, 15 min). Plasma was aliquoted, frozen in liquid N2, and stored at -80°C. The ‘buffy coat’, enriched in white cells, was removed, diluted 1:1 with RPMI- 1640 medium, layered onto an equivalent volume of Histopaque, and centrifuged at 700 x g for 30 min at 20°C. The lymphocyte layer was removed and washed in 15 ml of RPMI- spun at 700 x g, 15 min, 20°C; the supernatant was poured off and the pellet was resuspended in 5 ml RPMI + 10% fetal calf serum (FCS). Cells were again centrifuged at 700 x g, 15 min, 20°C, the supernatant removed, and the pellet resuspended in 2 ml of freezing mix [9 ml FCS + 1 ml dimethyl sulfoxide; FCS from the same batch was used throughout the trial]. Lymphocytes were aliquoted immediately and frozen at approximately -1°C/min in polystyrene at -80°C before storage in liquid nitrogen.
Plasma vitamin E
Plasma vitamin E was measured by reverse-phase HPLC using a
method adapted from Hess et al. (11)
. Plasma (200 µl),
water (200 µl) and ethanol (400 µl) were mixed and added to 700
µl hexane and 100 µl echinenone (internal standard). Samples were
shaken for 10 min before centrifugation (9500 x g, 5
min). Six hundred microliters of the hexane layer was removed and dried
before dissolving in 200 µl DEA [20% (v/v) 1,4 dioxan, 20% (v/v)
ethanol, 60% (v/v) acetonitrile], then mixed and applied to the HPLC
column.
Single-cell gel electrophoresis
DNA damage was measured using the alkaline ‘comet assay’, or
single-cell gel electrophoresis. The lymphocytes in freezing mix were
quickly thawed at room temperature and centrifuged at 200 x
g for 3 min at 4°C. The samples were kept on ice to
minimize any potential interaction between lymphocytes and FCS; the
freezing mix was removed, 400 µl RPMI + 10% FCS was added, and the
pellet was resuspended. The cell suspension (50 µl) was added to 950
µl phosphate-buffered saline (PBS) in order to wash the cells and the
tubes were gently inverted before incubation for 5 min on ice and
centrifugation at 200 x g, 3 min, 4°C. For
H2O2 treatment, 50 µl of
cells were added to 750 µl PBS + 200 µl 1 mM
H2O2 (200 µM final
concentration) before incubation and centrifugation as described above.
After PBS was removed, the cells were suspended in 85 µl 1% (w/v)
LMP agarose in PBS, pH 7.4, at 37°C and immediately pipetted onto a
frosted glass microscope slide precoated with a layer of 1% (w/v) NMP
agarose in PBS. Slides were left at 4°C for 5 min to allow the
agarose to set and then incubated in lysis solution [2.5 M NaCl, 10 mM
Tris, 100 mM Na2EDTA, NaOH to pH 10, and
1% (v/v) Triton X-100] for 1 h at 4°C. This removes cytoplasm
and most nuclear proteins, leaving DNA as nucleoids. Slides were then
placed in a 260 mm wide horizontal electrophoresis tank with 0.3 M NaOH
and 1 mM Na2EDTA, pH 12.7, for 40 min at 4°C before
electrophoresis for 30 min at 25 V. The slides were then washed three
times for 5 min at 4°C with neutralizing buffer (0.4 M Tris-HCL, pH
7.5) before staining with 20 µl (5 µg/ml) DAPI.
We have modified this assay using the bacterial DNA repair enzyme,
endonuclease III, to detect specifically oxidized pyrimidines in DNA
(12)
. This gives a specific and sensitive measure of
oxidative DNA damage. For certain gels, the slides were washed three
times after lysis for 5 min at 4°C with buffer (40 mM HEPES-KOH, 0.1
M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8); after blotting dry with
tissue paper, the gels were incubated for 45 min at 37°C with either
50 µl buffer or endonuclease III in buffer (1 µg protein/ml).
Slides were incubated in a moist box to prevent slides from drying out
before electrophoresis as described above. All samples were analyzed in
duplicate.
DNA damage was quantified using a visual scoring method and validated
using computerized image analysis as described previously
(13)
. One hundred fluorescently stained nucleoids from
each gel were assessed and classified according to the relative
intensity of fluorescence in the tail into 1 of 5 classes, giving a
score of between 0 (all undamaged) and 400 (all maximally damaged).
Statistics
Paired t tests were carried out using Microsoft Excel
7.0 and the statistical package Genstat (IACR-Rothamsted, Herts, U.K.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol concentrations were unchanged after
consumption of the diets containing -tocopherol in the range of 5–7
mg/day. However, intake of an additional 80 mg -tocopherol/day
significantly increased plasma vitamin E concentrations after
consumption of both the 15% and 5% PUFA diets (P<0.001)
(Table 3
).
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Lymphocyte DNA damage induced in vitro by 200 µM
H2O2 was significantly
decreased after consumption of the 5% PUFA diet (P<0.001)
by the volunteers and significantly increased after consumption of the
15% PUFA diet (P=0.008), when vitamin E levels were 5–7
mg/day (Fig. 1
). These effects were much less marked after vitamin E supplementation,
and no significant differences were seen (Fig. 1)
. Levels of endogenous
oxidized pyrimidines followed a similar pattern. When dietary vitamin E
levels were in the range of 5–7 mg/day, oxidized base damage decreased
when the volunteers consumed the 5% PUFA diet (P=0.01) and
increased after consumption of the 15% PUFA diet (P=0.03)
(Fig. 2
). Vitamin E supplementation (additional 80 mg d-tocopherol/day)
eliminated these effects (Fig. 2)
. Curiously, endogenous DNA strand
breakage in lymphocytes showed a significant decrease after consumption
of the high PUFA diet (P=0.002) when dietary vitamin E
levels were 5–7 mg/day, and increased vitamin E consumption prevented
this effect (Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 2)
. Consumption of a high PUFA diet (15%)
significantly increased endogenous levels of oxidized pyrimidines in
lymphocytes and also enhanced the ability of
H2O2 in vitro to
induce DNA strand breakage when vitamin E intake was relatively low
(range of 5–7 mg/day). It was recently shown that levels of etheno-DNA
and malondialdehyde-DNA adducts, alternative markers of oxidative
damage, increase after consumption of an n-6 PUFA diet compared with a
monounsaturated fatty acid (MUFA) diet (7
, 8)
.
In the current study, the 5% PUFA diet with a relatively low vitamin E
intake (5–7 mg -tocopherol/day) caused a decrease in endogenous
levels of oxidized pyrimidines in lymphocytes and in the ability of
H2O2 in vitro to
induce strand breakage (Figs. 1
and 2)
. This suggests that a lowering
of PUFA intake modulates the detrimental effect of PUFA and may be
beneficial. Lower PUFA intake may reduce the amount of substrate
available in cell membranes for lipid peroxidation, lessening the
oxidative load and DNA damage.
As anticipated, plasma -tocopherol concentrations did not change
when intakes of vitamin E were low but increased significantly in the
groups provided with an additional 80 mg d-tocopherol/day.
Increasing dietary -tocopherol ameliorated the damaging effect of
the high PUFA diet. Vitamin E consumption decreases the DNA damage
detected after exhaustive exercise, suggesting that additional vitamin
E can prevent oxidative damage to DNA (14)
.
The level of lymphocyte DNA strand breakage did not change
significantly after consumption of the 5% PUFA diet either with or
without additional vitamin E. Similarly, there was no change after
consuming the 15% PUFA diet with added vitamin E. However, the 15%
PUFA/5–7 mg -tocopherol/day diet was associated with a significant
decrease in endogenous strand breaks. The biological significance of
this is unclear; strand breaks are a broad indicator of various kinds
of DNA damage, but also occur as transient intermediates in cellular
DNA repair (12)
. Increased levels of DNA damage after
consumption of a 15% PUFA diet suggests that the process of oxidation
of dietary PUFA may be linked to DNA damage in humans and that
increasing PUFA intake could increase damage. This, however, does not
necessarily imply that such damage increases the risk of
carcinogenesis, since DNA repair mechanisms may be able to compensate.
However, the influence of diet on DNA repair is complex to measure and
therefore is difficult to interpret. Moreover, to date there is little
information on the effects of vitamin E and PUFA on DNA repair.
In humans, lymphocytes are the only cell type available for estimating
oxidative DNA damage in the body as a whole. That it is reasonable to
regard them as a useful indicator is suggested by recent studies in
rats, in which consumption of cholesterol increased oxidative DNA
damage in both lymphocytes and endothelial cells of the aorta
(15)
.
In conclusion, using a novel application of a sensitive molecular
epidemiological technique to investigate the influence of nutrition on
DNA stability, we have shown that dietary PUFA, within a nutritionally
relevant range, may have potentially damaging effects and, by
implication, may increase the risk of developing certain cancers if
repair mechanisms are overwhelmed. Increasing intakes of lipid soluble
antioxidants in the form of -tocopherol protects against this
oxidative DNA damage and suggests that increasing PUFA intakes should
only be recommended when an adequate antioxidant intake is
ensured.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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). Data presented are mean ±
SE. ***Significance from baseline
P < 0.001. **Significance from baseline
P < 0.01.
