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Nucleotide supplements and DNA damage from high-fat diets
135
Animal Science 2005, 81: 135-140 1357-7298/05/42290135$20.00
© 2005 British Society of Animal Science
Effect of nucleotide supplementation on lymphocyte DNA damage induced
by dietary oxidative stress in pigs
J. Salobir †, V. Rezar, T. Pajk and A. Levart
Institute of Nutrition, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale,
Slovenia
†E-mail: janez.salobir@bfro.uni-lj.si
Abstract
The aim of the present study was to evaluate the effect of nucleotide supplementation on the oxidative stress induced
by a high proportion of dietary polyunsaturated fatty acids ( PUFAs) in pigs. Twenty-four male growing pigs were penned
individually and after an adaptation period divided into three groups. All groups received isocaloric daily rations composed
of a basal diet supplemented with either : starch (CONT), linseed oil (LIN) and LIN and nucleotides (LIN + NUC). The
experimental period lasted 21 days. Oxidative stress was evaluated by measuring the degree of lymphocyte nuclear DNA
damage, the urine malondialdehyde ( MDA) excretion rate, erythrocyte glutathione peroxidase concentration and the total
anti-oxidant status of plasma. Malondialdehyde concentrations in the blood and MDA urinary excretion rates were higher
( P < 0·01) in animals supplemented with LIN and LIN + NUC compared with CONT animals. The degree of DNA damage
in the LIN-supplemented animals was also higher ( P < 0·01). Compared with the LIN-supplemented animals, nucleotide
supplementation reduced ( P < 0·01) the degree of DNA damage in lymphocytes to the level of the CONT group. Erythrocyte
glutathione peroxidase concentration and plasma total anti-oxidant status were similar across treatments. The results of
this experiment indicate that nucleotide supplementation effectively eliminates the genotoxic effects of high PUFA intakes
on blood lymphocytes and demonstrates new evidence for the immunonutritive effect of nucleotides.
Keywords: damage, malondialdehyde, nucleotides, oxidative stress.
Diet has a great influence on the formation of free radicals
in animals and man. Free radicals can cause oxidative
stress, a disturbance in the pro-oxidant-anti-oxidant
balance potentially leading to tissue damage that can
play an important rôle in the etiology of many diseases.
Previous studies have shown that oxidative stress also has
detrimental effects on the immune system ( Vider et al., 2001).
Additionally, our own study in pigs has shown that oxidative
stress induced by a high intake of dietary polyunsaturated
fatty acids ( PUFAs) also increases damage to leukocyte
DNA (Rezar et al., 2003).
Nucleotides and nucleosides are not known to be anti-
oxidative substances per se, but they could nevertheless
play an important rôle in the prevention of DNA damage
induced by an elevated oxidative load. They could be
involved in the mechanisms of excision and repair of
fragmented DNA molecules. In some studies nucleotide
supplementation has been revealed to enhance T-cell
function including accelerated T-cell-dependent antibody
production, to increase in vitro proliferative response of
blood mononuclear cells and to promote the immune
response after vaccination (Jyonouchi, 1994; Yamamoto et
Introduction
Generally, it has been assumed that all living cells are capable
of meeting their requirements for nucleotides via de novo
synthesis. However, recent studies revealed that in many
tissues, except the liver, the requirements for nucleotides
are covered not only by de novo synthesis but also by the
salvage pathway (Godderis et al., 2002). There is increasing
evidence indicating that especially under various conditions
such as certain disease states, limited nutrient intake, rapid
growth etc. the endogenous supply is not sufficient for
normal cell function and that dietary sources are required
(Uauy et al., 1994; Lopez-Navarro et al., 1996; Carver,
1999). Numerous reports have demonstrated that, in these
circumstances, dietary supplementation with nucleotides,
nucleosides, or nucleic acids improves the efficacy of the
immune system, gastro-intestinal tract, liver, brain and
metabolism of lipids not only in humans but also in animals
( Martinez-Augustin et al., 1997; Yamauchi et al., 1998;
Cameron et al., 2001; Bastian and Weimann, 2002; Carver
et al., 2004). For instance Yu et al. (2002) demonstrated that
nucleotides in combination with glutamine improve food
intake, intestinal villous height and promote the immune
responses and foot and mouth disease neutralizing antibody
titres of weaned pigs.
Salobir, Rezar, Pajk and Levart
136
al., 1997; Cameron et al., 2001). Therefore nucleotides are
classified as immunomodulatory nutrients ( McCowen and
Bistrian, 2003).
Since oxidative stress is known to induce genotoxic effects on
immune cells and since nucleotides are known to play a rôle
as immunomodulatory nutrients, their rôle in the prevention of
the genotoxic effects of oxidative stress on immune cells could
be of interest. Dietary supplementation with nucleotides may
increase the repair mechanism and/or cellular proliferation
and thus optimize immune system function.
The aim of the present study was to evaluate the effect
of nucleotide supplementation on the oxidative stress
induced by a high proportion of dietary polyunsaturated
fat in the diet. In order to evaluate the oxidative stress, the
following parameters were measured in pigs : the degree
of the lymphocyte nuclear DNA damage, the plasma
malondialdehyde ( MDA) concentration, the 24-h urine
MDA excretion rate, erythrocyte glutathione peroxidase
concentration and the total anti-oxidant status of plasma.
Material and methods
Animals and diets
Twenty-four young growing castrated male crossbreed
pigs (Duroc × Landrace × Large White, live weight 11·9
±0·5 kg) were utilized in this experiment. The animals were
penned individually in balance cages that allowed separate
collection of urine and faeces. The room temperature was
maintained at 22°C. Animals were housed under 12 h
light/dark conditions. The experiment was divided into
adaptation and experimental periods that lasted for 8 and
21 days, respectively. The animals were given food at
2·5 times the maintenance requirement for growing pigs
(National Research Council, 1998). At the beginning of the
experimental period, the animals were randomly assigned to
three treatment groups. All groups received isocaloric daily
rations ( Table 1) composed of an equal amount of the basal
diet which was supplemented according to the different
dietary treatments with : starch (control; CONT), linseed
oil (LIN) and LIN and nucleotides (LIN + NUC). Nucleotides
were supplied with preparation of Ascogen (Chemoforma
Ltd, Switzerland) containing RNA extracted from yeast,
nucleotides, precursors of nucleotides, organic acids and
thermolysed yeast. The proportion of energy requirements
which were met by fat in the CONT group and in both linseed
oil supplemented groups was 0·05 and 0·30, respectively.
The composition and analysis of daily rations in different
groups are presented in Table 1. The food was given in the
form of a mix. All ingredients of the mixture, except linseed
oil, were mixed together weekly. The linseed oil was added
and mixed to the diet of individual animals before every
feeding.
During the adaptation phase all pigs received the CONT diet
and had ad libitum access to water by automatic waterer.
The animals were given food twice daily. At the beginning
and at the end of the experiment, the pigs were weighed.
Food analysis
The content of protein and fat was determined by standard
procedures published by Naumann and Bassler (1997).
The content of total dietary fibre was analysed by the
enzymatic-gravimetric method ( Prosky et al., 1992). The
fatty acid compositions of diets were analysed via gas
chromatographic method after transesterification of lipids
as described by Fidler et al. (2000). In brief, fatty acid methyl
esters (FAME) were prepared by the procedures described
by Park and Goins (1994). FAME were separated on an
Omegawax™ 320 Capillary column (Supelco, 30 m × 0·32
mm i.d.) by using an Agilent GC 6890 Series GC System
equipped with Agilent 7683 Series Injector and 7683 Series
Auto sampler. A 30 to 1 split ratio was used for injection of
1 µl of hexane containing FAME. The carrier gas was Ar-
CH4 (5%), and inlet pressure was maintained at 13·3 p.s.i.,
which corresponds to a nominal initial flow of 1·5 ml/min.
Table 1 Ingredient, food, energy and nutrient intakes of experimental pigs on the different diets (estimated for a 12-kg pig)
Diet
CONT LIN LIN+NUC
Ingredient intake
Linseed oil (g/day) 0·0 53·1 53·1
Wheat starch (g/day) 227·8 111·6 107·2
Maize (g/day) 59·8 59·8 59·8
Soya-bean meal (g/day) 117·6 117·6 117·6
Skimmed milk powder (g/day) 91·1 91·1 91·1
Mineral-vitamin-amino acid supplement (g/day)† 9·9 9·9 9·9
Nucleotide preparation (g/day)‡ 0·0 0·0 4·4
Total food intake (g/day) 506·2 443·1 443·1
Nutrient intake
Metabolizable energy (kJ/day)§ 7423 7423 7423
Proportion of energy from fat§ 0·050 0·300 0·300
Proportion of energy from PUFA§ 0·029 0·209 0·209
Protein (g/day) 86·5 88·7 87·9
Fat (g/day) 8·7 56·0 57·1
Total dietary fibre (g/day) 44·0 45·2 45·9
† Calculated to meet nutritional requirements according to NRC (1998). Mineral-vitamin-amino acid supplement provided daily: 2·0 g Ca, 3·4 g
P, 0·15 g Na, 1·65 mg retinol, 5·09 mg alpha-tocopherol, 0·03 g lysine and 0·6 g methionine.
‡ Ascogen; Chemoforma Ltd, Switzerland.
§ Proportions of energy from fat and from polyunsaturated fatty acids (PUFA) were estimated. The energy values of foodstuffs and diets were
according to Gesellschaft für Ernährungsphysiologie (1988).
Nucleotide supplements and DNA damage from high-fat diets
137
Injector temperature was maintained at 250ºC, and FID
detector temperature was maintained at 290ºC. During the
chromatographic run the oven temperature was raised from
170ºC to 215ºC at a rate of 1ºC/min.
Blood and urine samples
At the beginning (before division into experimental groups)
and at the end of the 21-day experimental period fasting
blood samples were taken from the jugular vein into
evacuated tubes and 48-h urine was collected.
Lymphocyte nuclear DNA damage – comet assay
Blood samples for single cell gel electrophoresis (comet
assay) were collected in evacuated tubes containing EDTAK3
anticoagulant (367654, Brand, Plymouth, UK). Lymphocytes
were isolated from the fresh blood samples according to a
modified procedure described by Singh (1997); 2·5 ml of
Histopaque-1077 (Sigma H-8889, Sigma-Aldrich, Steinheim,
Germany) was added to 14-ml Eppendorfer centrifuge tube
and overlaid with 8 ml of a mixture of blood and RPMI-
1640 (Sigma R-8758, Sigma-Aldrich, Steinheim, Germany)
medium, which had been gently mixed at a ratio of 1 to 1.
Centrifugation (300 × g, 35 min, room temperature) followed.
Lymphocytes, white pellet approximately 3 mm under red-
cell pellet, were transferred to another centrifuge tube and
washed twice in 5 ml sterile RPMI-1640 medium, followed
by centrifugation (300 × g, 5 min, room temperature). The
pellet of lymphocytes was finally mixed with 0·5 ml of RPMI-
1640 medium and used as lymphocyte isolate. A partially
modified procedure by Singh et al. (1988) was implemented
for the comet assay. Rough microscopic slides were used
for the microgel preparation. The isolated lymphocytes were
suspended briefly in low melting point agarose. The first
layer of agarose was left to dry at normal room temperature,
the others were left on ice for 10 min. The gels were then
immersed in a lysing solution (0·03 mol/l NaOH, 1·2 mol/l NaCl,
0·5% laurylsarcosine, 1% Triton X-100, 10% DMSO) at 4ºC
for 1 h. The gels were then transferred to an electrophoresis
buffer (0·03 mol/l NaOH, 2 mmol/l ethylene diamine tetra-
acetic acid, pH 13) for 40 min before electrophoresis at 25 V
for 20 min. Following electrophoresis, the gels were washed
three times for 5 min with 0·4 mol/l tris-HCl, pH 7·5 at 4ºC
before staining with ethidium bromide (2 µg/ml) for 20 min,
and then rinsed in 400 mmol/l tris-HCl at 4ºC. An Olympus
CH 50 epifluorescent microscope at 200× magnification
was used for the examination of lymphocyte nuclei in the
microgels (100 W Hg lamp, excitation filter of 480 to 550
nm and barrier filter of 590 nm). The images were captured
by a Hamamatsu Orca 1 CCD camera, analysed and the
nuclear DNA damage estimated by the Comet 5 dedicated
computer program (Single Cell Gel Electrophoresis; Kinetic
Imaging Ltd, UK).
Plasma and urine malondialdehyde ( MDA) concentration
Blood samples for MDA concentration analysis were
collected in 10-ml evacuated tubes containing EDTAK3
anticoagulant (368457, Brand, Plymouth, UK). The blood
was centrifuged for 10 min at 400 × g and 4ºC. Plasma
supernatants were transferred to microcentrifuge tubes and
stored at -70ºC. To increase the accuracy of determination
of the 24-h urine MDA excretion rate a 48-h urine collection
was applied instead of standard 24-h urine collection.
Urine was filtered into test tubes (10 to 15 ml) through filter
paper (520 A, Schleicher and Schuell, Dassel, Germany),
transferred to microcentrifuge tubes and stored at –70ºC.
The methodology of Wong et al. (1987) modified by Chirico
(1994) and Fukunaga et al. (1995) was used to measure
the concentrations of MDA in blood plasma and urine by
high-performance liquid chromatography using a Waters
Symmetry C18 chromatography column (5 µm, 4·6 × 150 mm)
and a Waters Symmetry C18
guard
column (5 µm, 3·9 × 20 mm).
A Waters Alliance 2690 apparatus equipped with a Waters
474 scanning fluorescence detector was applied ( Waters
Corporation, Milford, MA). The mobile phase consisted of
50 mmol/l KH2PO4 buffer (pH 6·8) and methanol in a gradient
mode. Flow rate of the mobile phase was 1 ml/min at an
ambient temperature and the resulting backpressure was
2000 p.s.i. The results of the analysis were evaluated by the
Millenium32 Chromatography Manager program.
Glutathione peroxidase (GPx) and total antioxidant status
( TAS)
Blood samples for GPx and TAS concentration analysis
were collected in evacuated tubes containing heparin
(367685, Brand, Plymouth, UK). Fifty µl of whole blood for
GPx analysis was transferred to microcentrifuge tubes and
stored at –70ºC. Blood for TAS was centrifuged for 10 min at
3000 × g and 4ºC. Plasma supernatants were transferred to
microcentrifuge tubes and stored at –70ºC. The methodology
of Paglia and Valentine (1967) was used for measurements
of GPx and the methodology of Miller and Rice-Evans (1996)
was used for measurements of TAS. Samples were assayed
with commercially available glutathione peroxidase and TAS
kits (Randox, Crumlin, UK), following the instructions of the
kits.
Statistical analysis
The data were analysed by the ANOVA procedure of Statistical
Analysis Systems Institute (2000). Monofactorial analysis
of variance (effect of dietary treatment) was used. When
ANOVA revealed a significant effect, the differences among
treatment groups were tested using Scheffe’s test. The level
of significance was set at P < 0·05.
Results
The animals adapted well to the experimental conditions.
During the experiment, the animals in all groups had no
health or other problems, consumed food continuously
and normal live-weight gain for this level of feeding was
observed (315±35 g/day). The live-weight gain was at the
same level ( P > 0·05) in all groups (CONT 306 g/day, LIN 310
g/day, LIN + NUC 329 g/day).
While at the beginning of the experimental period no
statistical differences among treatments in any of the
measured parameters could be observed, at the end of the
experiment some important differences were found.
Nuclear DNA damage to lymphocytes
The rate of DNA damage ( Table 2) is presented as the
proportion of DNA in the head of the comet and as the Olive
tail moment (Olive et al., 1992), defined as the product of
the amount of DNA in the tail and the mean distance of
migration in the tail (higher values for Olive tail moment
Salobir, Rezar, Pajk and Levart
138
represent higher rates of DNA damage). The results showed
that isocaloric replacement of starch in the diet with linseed
oil increased the rate of lymphocyte DNA damage in the LIN
group by reducing ( P < 0·01) the percentage of DNA in the
head and by increasing ( P < 0·01) the Olive tail moment. In
comparison with the LIN group, nucleotide supplementation
in the LIN + NUC group improved ( P < 0·01) both parameters
of the degree of lymphocyte DNA damage to the level of the
CONT group.
Plasma MDA concentration and MDA excretion rate in urine
At the end of the experimental period, the MDA concentration
in plasma and the MDA excretion rate in urine (per 24 h) in
both linseed oil supplemented groups were higher ( P < 0·01)
than in the CONT group ( Table 3). In comparison with the
LIN group, the nucleotide supplementation in the LIN + NUC
group did not influence either MDA concentration in plasma
or the MDA excretion rate in urine.
Erythrocyte glutathione peroxidase and total anti-oxidant
status
The concentration of erythrocyte glutathione peroxidase and
total anti-oxidant status at the end of the experiment were
similar across all three experimental groups ( Table 4).
Discussion
The effect of nucleotide supplementation on oxidative stress
induced by high polyunsaturated fat intake is currently
not known. In order to measure the effect of nucleotide
supplementation on oxidative stress in our experiment,
nutritive oxidative stress was firstly induced by increasing
the proportion of energy supply from fats from 0·05 to 0·30.
Since the recommendations for the upper limit of fat intake
in pigs is not known, the recommendations for humans were
used ( World Health Organization ( WHO), 2003) Secondly,
oxidative stress was additionally induced by selection of
linseed oil, which contains 0·73 g/g of polyunsaturated
fatty acids ( PUFAs) as indicated by analysis. The energy
supply from PUFAs was approximately 0·21of total energy,
which is significantly higher than the proportion 0·06 to 0·10
proposed by WHO (2003). Polyunsaturated fatty acids are
lipids that are highly susceptible to peroxidation. The latter
was demonstrated by Dhanakoti and Draper (1987) who
found significantly higher 24-h urinary MDA excretion levels
in rats given a PUFA-rich diet than in rats given a diet rich in
saturated fatty acids. Thirdly, linseed oil contains absolutely
and in relation to its PUFA content, a very low amount of α-
tocopherol. It is known that a high intake of PUFAs increases
the nutritive requirements for anti-oxidative vitamins (Duthie
et al., 1996). The oxidative stress in both groups given
Table 2 Rate of lymphocyte DNA damage measured by comet assay
Diet
CONT LIN LIN+NUC s.e. Significance
Proportion of DNA in head of comets
Day 0 0·958 0·958 0·957 0·0044
Day 21 0·964a 0·853b 0·954a 0·0060 ***
Olive tail moment†
Day 0 0·57 0·59 0·63 0·088
Day 21 0·71a 5·09b 0·86a 0·583 ***
a,b Means without the same superscripts in the same row differ significantly (P < 0·05).
† Defi‟
Table 3 Plasma malondialdehyde (MDA) concentration and 24-h MDA excretion in urine
Diet
CONT LIN LIN+NUC s.e. Significance
Plasma MDA (nmol/ml)
Day 0 0·354 0·347 0·434 0·034
Day 21 0·179a 0·855b 0·809b 0·051 ***
MDA excretion rate in urine (nmol per 24 h)
Day 0 1889 1815 1804 190
Day 21 1646a 6110b 6751b 522 ***
a,b Means without the same superscripts in the same row differ significantly (P < 0·05).
Table 4 The concentration of glutathione peroxidase (GPx) in erythrocytes and total antioxidant status ( TAS) in plasma
Diet
CONT LIN LIN+NUC s.e. Significance
Erythrocyte GPx (U/l)
Day 0 25027 26496 22939 1367
Day 21 24425 22295 23040 2425
Plasma TAS (mmol/l)
Day 0 0·633 0·592 0·609 0·029
Day 21 0·643 0·656 0·661 0·014
Nucleotide supplements and DNA damage from high-fat diets
139
linseed oil (LIN and LIN + NUC) was additionally increased
by the fact that the supply of supplemented anti-oxidative
vitamins was not increased, i.e. it remained at the level of the
CONT group ( Table 1).
As expected, 21-day linseed oil feeding in the LIN group
increased the oxidative stress by increasing ( P < 0·01) not
only the formation of lipid peroxidation products measured
as plasma MDA concentration and the urinary MDA excretion
rate, but also by inducing genotoxic changes of lymphocyte
on the basis of the studies of Dhanakoti and Draper (1987)
and our own study (Rezar et al., 2003).
By confirming the oxidative stress induced by of high fat
intake, the basis for studying the effect of nucleotides was
achieved. The positive effect of nucleotides on oxidative
status can be seen from the results of the comet assay. The
results show that the degree of lymphocyte DNA damage
in the nucleotide-supplemented group was lower ( P < 0·01)
than in the LIN group. Moreover, the degree of lymphocyte
DNA damage in this group was at the same level as in the
CONT group. It is evident that nucleotide supplementation
can prevent the genotoxic effect of high fat intake on DNA of
lymphocytes. Previously, protection of DNA integrity under
increased oxidative stress has been demonstrated for other
nutrients like vitamin E, vitamin C, β-carotene, polyphenols
(Duthie et al., 1996; Brennan et al., 2000; Pajk et al., 2002;
Salobir et al., 2002; Bub et al., 2003).
Nucleotide supplementation had no effect ( P > 0·05) on the
plasma MDA concentration, urinary MDA excretion rate,
erythrocyte glutathione peroxidase concentration and on
the total antioxidant status of the plasma. Since nucleotides
are not known to have anti-oxidative properties per se, a
sparing effect on GPx synthesis or increased plasma TAS
because of lower expenditure of anti-oxidative substances
already present could not be expected. A possible mode of
action of nucleotides to prevent lipid oxidation could be via
improved synthesis of RNA responsible for the synthesis
of enzymes required to cope with oxidative stress. But at
least in this experiment this was not the case. Possibly also
the absence of an effect of linseed oil supplementation
on erythrocyte GPx concentration and on the TAS of the
plasma in LIN group was a inappropriate basis from which
to measure such an effect.
The results indicate that nucleotide supplementation is not
able to prevent increased lipid oxidation in the body. Thus the
mode of action of supplemented nucleotides in prevention
of DNA damage induced by high oxidative load is most likely
the improved supply of nucleotides for the mechanisms of
excision and repair of damaged parts of DNA molecules
of immune and possibly other cells. The results indicate
also that in the case of oxidative stress the demand for
nucleotides increases over the endogenous supply and that
dietary sources are required. Consequently, the results show
that beside an increased cellular (Carver, 1994; Zomborszky-
Kovacs et al., 2000; Cameron et al., 2001; Yu et al., 2002)
and humoral immune response (Jyonouchi, 1994; Cordle et
al., 2002; Yu et al., 2002), the repair of damaged parts of
DNA molecules could most likely be an important step in the
optimization of immune system function as a consequence
of nucleotide supplementation.
Immune cells play a central rôle in the immune response.
They are susceptible to the DNA-damaging effects of a wide
variety of agents (Brennan et al., 2000). Alteration in T cells
may have implications for subsequent immune response,
when for example T memory cells are required to undergo
rapid proliferation in response to rechallenge with a specific
antigen. A decline in T-cell function is thought to play a
critical part in the age-related decline in immune function
( Pawelec et al., 1998) and genetic damage that accumulates
over time in vivo within lymphocytes is thought to contribute
to age-related decline in T-cell function (Barnett and Barnett,
1998). The question of whether nucleotide supplementation
is able to protect immune cells against genetic damage in
other conditions like certain disease states, food and other
toxins, limited nutrient intake, ageing as effectively as in the
case of oxidative stress remains to be established.
In conclusion, the results of this study demonstrated for
the first time that nucleotide supplementation effectively
eliminates the genotoxic effect of high PUFA intake on
blood lymphocytes. Nucleotide supplementation in these
experimental conditions may be an important component for
the repair mechanism of immune cells and thus underlines
the immunomodulatory rôle of nucleotides.
Acknowledgements
This work was supported by a grant from the Ministry of Agriculture,
Food and Forestry and the Ministry of Education, Science and
Sports of the Republic of Slovenia.
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(Received 29 December 2004 – Accepted 5 March 2005)
