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The effect of oxidative stress on nucleotide excision repair in colon tissue of
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newborn piglets.
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Sabine A.S. Langie1, Pawel Kowalczyk2,3, Barbara Tudek2,4, Romuald Zabielski5, Tomasz
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Dziaman6, Ryszard Oliński6, Frederik J. van Schooten1, Roger W.L. Godschalk1*.
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Risk Analysis and Toxicology, Maastricht University, The Netherlands,
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2
Institute of Biochemistry and Biophysics PAS, Warsaw, Poland
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3
Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw
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University, Poland
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University, of Life Sciences, Warsaw, Poland
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University, Bydgoszcz, Poland.
Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Health
Institute of Genetics and Biotechnology, Warsaw University, Poland
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus
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Key words: oxidative stress, antioxidants, DNA repair capacity, and 8-oxodeoxyguanine
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*Corresponding author:
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P.O. Box 616, 6200MD Maastricht
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Tel: +31(0)43-388 1104, Fax: +31(0)43-388 4146
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R.Godschalk@GRAT.unimaas.nl
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1
1
Abstract (300 words max.)
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Nucleotide excision repair (NER) is important for the maintenance of genomic integrity
3
and preventing the onset of carcinogenesis. Oxidative stress was previously found to
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inhibit NER in vitro, and dietary antioxidants could thus protect DNA not only by reducing
5
levels of oxidative DNA damage, but also by protecting NER against oxidative stress
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induced inhibition. To obtain further insight in this relation between oxidative stress and
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NER activity in vivo, oxidative stress was induced in newborn piglets by means of
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intramuscular injection of iron (200 mg) at day 3 after birth. Indeed, injection of iron
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significantly increased several markers of oxidative stress, such as 8-oxo-7,8-dihydro-2’-
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deoxyguanosine (8-oxodG) levels in colon DNA and urinary excretion of 8-oxo-7,8-
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dihydroguanine (8-oxoGua). In parallel, the influence of maternal supplementation with an
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antioxidant enriched diet was investigated in their offspring. Supplementation resulted in
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reduced iron concentrations in the colon (P=0.004) at day 7 and a 40% reduction of 8-
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oxodG in colon DNA (P=0.044) at day 14 after birth. NER capacity in animals that did not
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receive antioxidants was significantly reduced to 32% at day 7 as compared to the initial
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NER capacity on day 1 after birth. This reduction in NER capacity was less pronounced in
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antioxidant supplemented piglets (69%). Overall, these data indicate that NER can be
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reduced by oxidative stress in vivo, which can be compensated for by antioxidant
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supplementation.
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1
Introduction
2
The general population is constantly exposed to environmental carcinogens that may cause
3
damage to a variety of molecular targets, including DNA. If these DNA lesions are not
4
removed by DNA repair mechanisms in time, they can become self-perpetuating mutations
5
that contribute to ageing and human degenerative diseases such as cancer [1-3]. Therefore,
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DNA repair impairment is an important risk factor in the pathogenesis of certain diseases.
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One of the major DNA repair processes is nucleotide excision repair (NER), which is
8
involved in the removal of bulky-DNA adducts resulting from exposure to chemical
9
carcinogens like polycyclic aromatic hydrocarbons.
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An important modulator of DNA repair activity seems to be oxidative stress, which
11
results from an imbalance between formation of reactive oxygen species (ROS) and the
12
available antioxidant defences. ROS are produced as a consequence of normal cellular
13
metabolism, but may also arise from pathological processes and extra-cellular sources. It
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has been shown that these ROS and reactive nitrogen species (RNS) can affect
15
transcription of repair enzymes, but may also directly inactivate DNA repair enzymes by
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oxidation and nitrosation, respectively [4-7]. Furthermore, several factors that are released
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during oxidative stress, such as lipid peroxidation products (e.g. 4-hydroxynonenal,
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malondialdehyde), have been reported to inhibit NER [8-10]. Moreover, our previous in
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vitro studies have shown an inhibition of NER by oxidative stress inducers (e.g. hydrogen
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peroxide) [11,12]. A balance between oxidants and antioxidants is thus crucial to prevent
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oxidative stress and subsequent adverse effects on DNA repair processes.
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Therefore, we hypothesize that oxidative stress reduces nucleotide excision repair
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capacities in vivo and that an antioxidant rich diet can compensate for this effect. The in
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vivo model used in this study was newborn piglets, because of their similarities to humans
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regarding: i) morphology and function of organs, ii) DNA damage removal, and iii)
3
1
metabolic rates [13,14]. Newborns were chosen because their antioxidant systems, as in
2
humans and other mammals, are shown to be poorly developed and thus oxidative stress
3
can be modulated more easily [15,16]. Furthermore, in this particular study the colon was
4
selected as target organ, because large amounts of ROS are produced in inflammatory
5
diseases like ulcerative colitis that predispose patients to colorectal cancer [17-20].
6
Moreover, a few studies have suggested that abnormalities or deficiency in NER play a
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crucial role in the formation of sporadic colon carcinomas [21,22]. Our first aim of this
8
study was to evaluate the effect of increased oxidative stress on the nucleotide excision
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repair capacity (NERC) in colon tissue of newborn piglets. The second aim was to
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investigate whether these effects can be influenced by supplementation of mother sows
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with an antioxidant rich diet.
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1
Materials and Methods
2
Chemicals
3
Dulbecco’s Modified Eagles Medium (DMEM), Tris, KAc, alkaline phosphatase, (low
4
melting point = LMP) agarose, TritonX-100, glycerol and BSA were obtained from Sigma
5
(St. Louis, USA). Fetal Calf Serum (FCS), trypsin, TRIzol, Hanks Balanced Salt Solution
6
(HBSS; with and without Ca/Mg), Penicillin/streptomycin and ethidium bromide were
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purchased from Gibco Invitrogen (Scotland, UK). Benzo[a]pyrene-7,8-dihydrodiol-9,10-
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epoxide (BPDE) was obtained from NCI Chemical Carcinogen Reference Standard
9
Repository (Midwest Research Institute, Kansas City). Dimethyl sulfoxide (DMSO),
10
EDTA, DTT, SDS, H2O2, KOH, KCL, NaOH, NaCl, chloroform, iso-propanol, ethanol
11
and mercapto-ethanol were obtained from Merck (Germany). Proteinase K and RNase
12
were supplied by Roche (New York, USA).
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Animals and design of the study
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The experiments described here were conducted in compliance with the European Union
16
regulations concerning the protection of experimental animals. The study protocol was
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approved in advance by the Local Ethical Committee, Warsaw University of Life Sciences
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- SGGW (WULS), Warsaw, Poland. Care of the animals during the duration of the study
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was in accordance with the committee guidelines. A total of 12 pregnant sows (Sus scrofa
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domesticus, Landrace x Pietrain) were kept in standard farm conditions (state farm
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Dobrzyniewo, Poland) with approximate 70% humidity and a temperature of 22 ± 2°C in
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standard cages with straw bedding. At day 80 of pregnancy, the sows were randomly
23
divided into 2 groups, control (n=6) and supplemented with antioxidant rich food
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ingredients (n=6). The sows from the control group were fed with the standard diet for
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pregnant sows (dry matter (DM) 87.6%, mean energy (ME) 11.35 MJ/kg, crude protein
5
1
(CP) 13.1%) and lactating sows (DM 87.3%, ME 12.93 MJ/kg, CP 15.4%). The sows from
2
the supplemented group received the standard diet for pregnant and lactating sows
3
supplemented with a blend of substances (Table 1) that contribute to an increased
4
antioxidant status. This blend contained taurine (Otis, Poland), L-carnitine (Lonza,
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Poland), tocopherol acetate (Sigma-Aldrich, Poland), flaxseed and rapeseed providing -
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linolenic (C18:3n-3) and linoleic (C18:2n-6) fatty acids, and linden inflorescence (Kawon,
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Poland) as a source of flavonoids and other antioxidants, e.g. phenolic acids. The
8
supplementation diet was modified in such a way that energy and protein content was
9
similar to that in the control diet as published before [23,24]. Blood plasma, colostrum and
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milk analysis demonstrated substantial transfer of n-3 fatty acids, plant polyphenols and
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other antioxidants from the supplemented diet into sow blood and milk. Only plasma and
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milk concentrations of carnitine were found not to be influenced [23,24]. Sows were
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supplemented from day 80 of pregnancy (pregnancies in pigs have duration of approx. 115
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days) up to the 14th day of lactation. Fresh diet and water were provided each day ad
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libitum. After a short time of adaptation (4-5 days) in which the consumption of the
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supplemented diet was slightly decreased (P=0.095), we did not see any significant
17
difference between the supplemented and control group, both in pregnant and lactating
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sows for the rest of the study.
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Piglets were delivered at term and clinically healthy. The average number of delivered
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piglets was 11.3 and 10.5 per sow, respectively, in the control and supplemented group
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(P=0.88). At birth, the unsuckling piglets from the supplemented group showed a tendency
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toward smaller body weight as compared to control (P=0.07) [23]. However, this
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difference disappeared within 4 days after birth. To induce oxidative stress, newborn
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piglets were intra-muscularly injected with 200 mg iron dextran (FeDex) on day 3 post-
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partum, which is known to catalyze the generation of ROS [25]. Note that this is a standard
6
1
procedure found in every production farm worldwide to prevent anaemia, however, in our
2
study design it was considered as an oxidative stress inducer. One piglet from each litter
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was sacrificed for tissue sampling by CO2 inhalation on postnatal days 1, 2 (before FeDex
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injection), 4, 7, and 14. Unsuckling newborns sacrificed at day one after birth (did not
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receive FeDex injection) served as untreated controls. A schematic overview of the study
6
design is shown in Figure 1. Urine was collected by urinary bladder puncture and stored at
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-80°C until analyses. Colon tissue was isolated, snap frozen and stored at -80°C. Tissues
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were subsequently ground using mortar and tamper that were cooled in liquid nitrogen.
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Ground tissues were divided over eppendorf tubes containing 50-100 mg of tissue, snap
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frozen and stored at -80°C until analysis of the repair capacities or 8-oxodG levels.
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Measurement of iron levels in colon tissue
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Total amount of iron was measured in pig colon tissues by atomic absorption spectrometry.
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Ground tissues (n=3-9 per group per day) were hydrolyzed overnight in 1 ml of 7M HNO3
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at 60°C. After centrifugation, 20 µL of the supernatant was directly injected into a graphite
16
furnace atomic absorption spectrometer with Zeeman background correction (Varian,
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Bergen op Zoom, The Netherlands). Fe concentrations (ng/ml) were determined at 372 nm.
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Solutions with known concentrations of Fe were used for calibration. All the glassware
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was rinsed with 1% HNO3 to avoid contamination. Total Fe-concentration in ng Fe per mg
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colon tissue was calculated from the calibration curves and the weight of tissue.
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Determination of 8-oxodG in colon tissues
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To detect the base oxidation product 8-oxodG, HPLC with electrochemical detection
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(ECD) was performed. Ground frozen colon tissues (50-100 mg, n=3-4 per group per day)
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were thawed and genomic DNA was obtained using standard phenol extraction [26]. The
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DNA extraction procedure was optimized to minimize artificial induction of 8-oxodG, by
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using radical-free phenol, minimizing exposure to oxygen and by addition of 1 mM
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deferoxamine mesylate and 20 mM TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl),
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according to the European Standards Committee on Oxidative DNA Damage (ESCODD,
5
reference [27]). DNA concentrations were quantified by spectrophotometry and samples
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were frozen at -20°C until further use. HPLC-ECD of 8-oxodG was based on a method
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described earlier [28]. Briefly, 30 µg DNA was digested to deoxyribonucleosides by
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treatment with, 6 l 0.5M NaAc, 9 l 10mM ZnCl2 and 1.5 l nuclease P1 (stock: 1 U/µl),
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and incubation for 90’ at 37 oC. Subsequently, 30 l 0.5M Tris-HCl (pH 7.4) and 1.5 l
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alkali phosphatase (0.014 U/µl) was added followed by incubation at 37 oC for 45’. The
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digest was then analysed by HPLC-ECD, using a SupelcosilTM LC-18S column (250 x 4.6
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mm) (Supelco Park, Bellefonte, PA) and a DECADE electrochemical detector (Antec,
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Leiden, The Netherlands). The ECD-signal was first stabilized with mobile phase (94mM
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KH2PO4, 13mM K2HPO4, 26mM KCL and 0.5mM EDTA, 10% methanol) for
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approximately 3 hours at a flow rate of 1 ml/min. After stabilization, 8-oxodG was detected
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at a potential of 400 mV and dG was simultaneously monitored by UV absorption at 260
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nm.
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Urine analysis
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Processing of urine samples for estimation of 8-oxoGua levels was performed according to
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Dziaman et al. [29]. Briefly, 0.5 nmol of [15N3,
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(Sigma, HPLC grade, concentration 99%) were added as internal standards to 2 ml of
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urine. After centrifugation (2000 x g, 10 min), supernatant was filtered using a Millipore
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GV13 0.22 µm syringe filter and 500 µl of this solution was injected into the HPLC
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system. Purification of 8-oxoGua by HPLC was performed as described by Gackowski et
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C]-8-oxoGua and 10 µl of acetic acid
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al. [30]. GC/MS analysis was performed according to the method described by Dizdaroglu
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[31], adapted for [15N5]-8-oxoGua analyses (m/z 445 and 460 ions were monitored).
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Measurement of NER capacity in colon tissues
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To asses NER capacity in the piglets’ colon tissues, we applied a modified comet assay
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[32]. Basically, this assay measures the ability of NER-related enzymes that are present in
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cell/tissue extracts, to incise substrate DNA containing benzo[a]pyrene-diolepoxide
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(BPDE)-DNA adducts. The substrate nucleoids were prepared from untreated A549 cells
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(human epithelial lung carcinoma cells), which were purchased from the American Tissue
10
Culture Collection (ATCC) and were cultured in T75 flasks in DMEM supplemented with
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10% heat inactivated FCS and 1% penicillin/streptomycin. Cells were maintained at 37oC
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in a 5% CO2 atmosphere. A549 cells were tripsinized at approximately 80% confluency,
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embedded in LMP agarose on glass microscopy slides and subsequently lysed overnight in
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cold (4oC) lysis buffer (2.5 M NaCl, 0.1M EDTA, 0.01M Tris, 0.25M NaOH plus 1%
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Triton X-100 and 10% DMSO added just before use). The resulting nucleoids were then
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exposed to BPDE (1 µM in PBS) or vehicle control (DMSO, 0.5%) for 30 minutes at 4oC.
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To prepare protein/enzyme extracts, 50-100 mg of ground frozen colon tissues were
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thawed and resuspended in buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM
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dithiothreitol, 10% glycerol, adjusted to pH 7.8 using KOH, 100 µL per 50 mg tissue).
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Resulting aliquots were snap-frozen, thawed again and lysis was completed by adding 30
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µL of 1% Triton X-100 in buffer A per 100 µL of extract. The lysate was centrifuged at
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11,000 rpm for 5 minutes at 4C. Next, protein concentrations were determined by means
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of the BioRAD DC protein assay (Veenendaal, The Netherlands), using bovine serum
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albumin as a standard. Tissue extracts were diluted to a concentration of 0.3 mg/ml, and
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stored at -80C overnight. The next day, protein extracts were thawed and 4 volumes of
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1
reaction buffer B (45 mM HEPES, 0.25 mM EDTA, 2% glycerol, 0.3 mg/mL BSA,
2
adjusted to pH 7.8 with KOH) were added. From this mixture, 50 µl aliquots were added to
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the gel-embedded nucleoids containing high levels of BPDE-DNA adducts, and incubated
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for 10 minutes on a heating plate at 37°C. Alkaline treatment and electrophoresis, each 20
5
minutes, were conducted as in the standard comet assay. The increase in DNA breaks at 10
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minutes, leading to increased tail moments (TM), is indicative for the NERC of the cell
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extracts. After subtracting background levels from all data, the final repair capacity was
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calculated according to Langie et al. [32]. Samples of control and supplemented piglets,
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isolated at the same time points, were paired for analysis. Each sample was tested in two
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independent incubations within a single experiment. Nucleoids exposed to 1µM of BPDE
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were used as positive controls to correct for inter-assay variations (TMs of BPDE exposed
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cells ranged between experiments from 0.50 to 1.36, with a mean of 1.01±0.29).
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Statistical analysis
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Results are presented as mean values ± standard error. Differences in iron content, levels of
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oxidative DNA damage, repair capacities, and urinary excretion were analyzed by T-tests.
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The statistical analysis of the urine 8-oxoguanine levels was performed using ANOVA
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with post hoc multiple comparison LSD testing. Relationships between various parameters
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were assessed by linear regression (R), unless the relation was not linear. In the latter case,
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relationships were determined by means of spearman rank correlation (Rs). Statistical
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analysis was performed using SPSS v.15.0. A P-value ≤0.05 was considered statistically
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significant.
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Results
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Iron-induced oxidative stress in animal model
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The iron-induced oxidative stress was studied in the newborn piglets by assessment of; i)
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the iron content in colon tissue, ii) 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) levels
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in colon tissue and iii) urinary excretion of 8-oxo-7,8-dihydroguanine (8-oxoGua). Upon
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injection of 200 mg FeDex at day 3, the iron levels in the colon significantly increased at
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day 7 (P<0.001) to 5-times the initial level (Figure 2, white bars). At day 14 after birth, the
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iron concentrations in the colon had decreased again to the initial background levels. 8-
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oxodG levels in colon DNA were significantly ~3-fold increased at day 7 and 14 after birth
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and iron treatment (Figure 3, white bars). These elevated levels of 8-oxodG correlated with
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the amount of iron in colon tissue, using the data of control animals at all time points after
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birth (Rs=0.612, P=0.02). Urinary excretion of 8-oxoGua increased acutely upon FeDex
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exposure with 50% of the initial level, after which it stayed at constant levels up to day 14
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(Table 2).
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Antioxidant enriched dietary supplementation
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The intervention with antioxidant enriched diet was shown to be effective in reducing iron
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levels in the colon tissues of the supplemented piglets; 53% lower levels were observed at
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day 7 as compared to the controls (Figure 2, grey bars), which is expected to result in
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reduced levels of oxidative stress in the colon. Indeed, the level of 8-oxodG in colon of the
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supplemented group was significantly reduced to 60% at day 14 as compared to the
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unsupplemented group (Figure 3, grey bars). No significant differences in urinary
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excretion of 8-oxoGua was observed between the control and supplemented piglets.
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However, urinary excretion seemed to be slightly enhanced in the supplemented piglets
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compared to the controls (Table 2).
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Effects of oxidative stress, with and without antioxidant intervention, on DNA repair
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First of all, it should be noted that the NERC at day 2 (prior to FeDex injection) already
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non-significantly decreased to ~80% in both the supplemented and control group in
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comparison to the first day of life. However, the subsequent iron-induced oxidative stress
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in the piglets was paralleled by a further decline of NERC to 32% at day 7 as compared to
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day 1 after birth, which partly restored at day 14 to 54% (Table 3). A significant beneficial
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effect of the antioxidant supplementation on phenotypic NERC was observed at day 7 after
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birth (Table 3, P=0.050), which was significantly higher in the group of supplemented than
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unsupplemented animals (reduced to 69% in supplemented piglets versus 32% in non-
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supplemented). Moreover, using the data of control and supplemented animals at all time
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points after birth, significant inverse correlations of NERC with the levels of Fe (R=-0.734,
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P=0.016) and the levels of 8-oxodG in the colon were found (R=-0.701, P=0.024).
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1
Discussion
2
Ongoing research has focused on factors that can modulate the rate of DNA repair and one
3
of these factors is thought to be oxidative stress or its products. Already in the 1980’s, the
4
role of oxidants and antioxidants in DNA repair was evaluated in vitro and in vivo [33,34].
5
Our previous in vitro studies showed that increased oxidative stress can inhibit NER
6
[11,12], which was confirmed in vivo in the current study. The modulation of oxidative
7
stress by antioxidant rich diets could theoretically protect NER against reduction of its
8
activity and could thus improve the maintenance of genomic integrity. Indeed, in this
9
study, supplementation of mother sows with an antioxidant enriched diet seemed to partly
10
compensate the oxidative stress-induced reduction of the NERC in colon tissues of their
11
newborn offspring.
12
In this study, we have used iron (FeDex) to evaluate the effect of oxidative stress on
13
NERC. This transition metal is essential for normal cell function, however, it can also
14
catalyze the generation of reactive oxygen species via the Fenton reaction, leading to the
15
induction of lipid peroxidation and oxidative DNA damage [25,35,36]. Although the
16
oxidative stress observed in the newborn piglets may partly be a consequence of a sudden
17
increase in oxygenation after birth [29], the intramuscular injection of 200 mg FeDex
18
resulted in elevated amounts of iron in the colon and subsequent increased oxidative stress,
19
as measured by increased levels of 8-oxodG in colon DNA and increased urinary excretion
20
of 8-oxoGua. Similar observations upon injection with FeDex were previously reported in
21
rats [37], underscoring the suitability of our model.
22
In parallel, the influence of supplementation of mother sows with an antioxidant rich
23
diet, from day 80 of pregnancy until the end of the study at day 14 post-partum, was
24
investigated on oxidative stress parameters in their newborn offspring.
25
supplementation consisted of a blend of bioactive compounds (Table 1). Most of these
The
13
1
compounds posses antioxidant activity, mainly by acting as scavengers of ROS and lipid
2
peroxidation products [38-41]. Moreover, studies have shown that this antioxidant rich diet
3
improved the development of intestinal mucosa in comparison to the commercial diets
4
used in the pig industry [23] and some of the dietary compounds (e.g. fibres, phytic acid
5
and polyphenols) were found to bind pro-oxidant metals like iron resulting in decreased
6
absorption by intestinal epithelial cells [42-44]. Probably due to a combination of these
7
effects, the dietary supplementation in the current study succeeded to significantly lower
8
the oxidative stress in the colon of the newborn piglets. Indeed, lower levels of total iron in
9
colon tissue and a lower amount of 8-oxodG in the colon DNA from offspring of
10
supplemented sows were observed. Other studies have reported similar effects of relevant
11
dietary compounds on the levels of 8-oxodG in cellular DNA [45-47], but it should be
12
mentioned that this could not be reproduced by others [38,48,49].
13
The increase in oxidative stress in the newborn piglets was paralleled by a decline in
14
NER capacity to 32% of the initial levels (Table 3). Although, it is not yet clear which
15
factors contribute to this reduction of NERC in vivo, several products of oxidative stress
16
and inflammation can affect DNA repair. For instance, nitric oxide [50], lipid peroxidation
17
products (e.g. 4-hydroxynonenal, malondialdehyde) [8-10] and hypochlorous acid [12,51]
18
have the ability to inhibit NER, most likely by direct inactivation of NER proteins.
19
Moreover, particulate matter that is rich in metal and aldehyde content and able to induce
20
oxidative stress, was recently found to greatly inhibit NER in human lung cells [52].
21
We proposed that the inhibiting effects of oxidative stress on DNA repair could be
22
compensated for by antioxidant supplementation. As hypothesised, NERC was beneficially
23
affected by the supplementation with the blend of bioactive compounds. Although the
24
repair capacity still decreased in time after birth and iron administration, this reduction was
25
significantly smaller as compared to animals that were not supplemented. Until now, a
14
1
small number of studies has assessed the influence of diet and dietary compounds on DNA
2
repair processes [53,54]. One study in particular showed an increased BER capacity in
3
human lymphocytes upon supplementation with kiwifruits in vivo [55]. In the current
4
study, the restoration of the suppressed NERC can be explained by affecting signal
5
transduction pathways and gene transcription, leading to de-novo synthesis of NER related
6
proteins. Several studies have shown that dietary antioxidants like quercetine and vitamin
7
C were able to induce different DNA damage responsive signalling pathways (e.g. p53 and
8
AP-1/NFB) that can subsequently enhance the expression of NER-genes [56,57].
9
However, results from various in vivo intervention studies have been equivocal [58].
10
Therefore, further studies are needed to clarify possible correlations between antioxidant
11
intake and phenotypic DNA repair capacity.
12
On the basis of these observations, one could argue that the lower levels of 8-oxodG in
13
the colon tissues in the supplemented group are the direct result of improved DNA repair
14
capacities. Although it is widely believed that the main pathway to repair 8-oxodG is base
15
excision repair pathway, it has been suggested that the NER pathway is involved in the
16
repair of oxidized bases [59-61]. However, in the present study it is not possible to
17
distinguish cause and effect. Therefore, our data rather suggest that the formation of 8-
18
oxodG in the colon is merely an indicator of increased oxidative stress, which inhibits
19
DNA repair. Another observation supporting this, is the inverse correlation between the
20
levels of Fe, as an inducer of oxidative stress, and NERC (R=-0.734, p=0.016).
21
Since DNA repair is one of the first steps that can intervene between DNA damage and
22
mutagenesis, it might be a relevant biomarker in the assessment of individual cancer risks.
23
Therefore, studies on the modulation of DNA repair by exogenous or endogenous agents
24
are of great interest, especially DNA repair modulation by nutrition and dietary
25
compounds. In the process of evaluating and validating DNA repair as a biomarker, more
15
1
information needs to be gathered on intra- and inter-individual variations in DNA repair
2
capacity in a healthy human population. In the current study, variations in NERC between
3
piglets were in the range of ~4 to 10-fold, which is similar to variations reported previously
4
in human lymphocytes (~8 to 11-fold) [32,62,63]. These observations underscore the
5
similarities between pigs and humans and indicate that pigs/piglets would be a suitable in
6
vivo model to further substantiate the role of DNA repair in the development of cancer and
7
to provide answers to important questions on the modulation of DNA repair and human
8
health.
9
Collectively, our data demonstrate that oxidative stress inhibits nucleotide excision
10
repair in vivo. These deleterious effects can be compensated for by an antioxidant enriched
11
diet. Although, the underlying mechanisms remain unclear, a reduction in repair capacity
12
during diseases that involve oxidative stress (e.g. chronic inflammatory diseases) might
13
contribute to mutagenesis and carcinogenesis. Dietary interventions could guard against
14
these effects by protecting the DNA repair processes that maintain the integrity of the
15
DNA. Therefore, considering the prominent role of DNA damage and repair in health and
16
disease, further research on the modulation of DNA repair is urgently needed. Especially
17
studies involving oxidatively stressed subjects will clarify the link between oxidative stress
18
and defective DNA repair.
19
16
1
Acknowledgments
2
This work was financed by the Polish Ministry of Science and Higher Education grant
3
(PBZ-KBN-093/P06/2003). Part of the studies was supported by the European Network of
4
Excellence (NoE) “Environmental Cancer risk, Nutrition and Individual Susceptibility”
5
(ECNIS), operating within European Union sixth Framework Programme (FP6), Priority 5:
6
“Food Quality and Safety”, FOOD-CT-2005-513943.
7
8
17
1
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1
Legends to the Tables
2
Table 1. The composition of the standard diet and the bioactive substances used for
3
supplementation of pregnant and lactating sows, in g per 1 kg of feed.
4
5
6
7
Table 2. Urinary excretion rates of 8-oxoGua in newborn piglets. Data are presented as the
8
mean values (±standard error).
9
10
Asterisks indicate significant differences in excretion of 8-oxoGua versus day 1, within
11
each group separately (*P=0.042, **P<0.015, ***P<0.007).
12
25
1
Table 3. Mean repair capacities in colon tissues of newborn piglets as calculated from tail
2
moment values (± standard errors) and as percentage of day 1.
3
4
*
Significant difference in NER capacity between these groups (P=0.050).
5
6
26
1
2
Figure Legends
3
Fig. 1. Schematic overview of supplementation and sampling time points. Dietary
4
intervention of mother sows started at day 80 of the pregnancy and continued until day 14
5
of lactation. After birth, newborn piglets from the control as from the supplemented group
6
were injected with 200 mg of iron dextran. At days 1, 2, 4, 7 and 14 after birth newborn
7
piglets were sacrificed, and urine samples and tissues were collected.
8
9
Fig. 2. Iron concentrations in the colon of non-supplemented controls () and
10
supplemented piglets (). The level of iron in the colon tissues of the control group was
11
significantly increased at day 7 (*P=0.002). The supplementation of piglets resulted in a
12
lower iron concentration in the colon tissues at day 7 as compared to the controls
13
(**P=0.004). Data are presented as the mean values (n=3-9) and bars indicate standard
14
errors of the mean.
15
16
Fig. 3. Levels of 8-oxodG in the colon tissues; from piglets not supplemented with
17
antioxidant enriched diet (), and from supplemented piglets (). In both groups 8-oxodG
18
in the colon tissues was significantly increased at day 7 and 14 as compared to day 1 after
19
birth (**P=0.016, ***P=0.003 for the controls, and **P=0.012, *P=0.050 for the
20
supplemented piglets, respectively). Moreover, supplementation significantly reduced the
21
8-oxodG levels on day 14 (*P=0.044 supplemented vs. controls). Data are presented as the
22
mean values (±standard error).
23
24
27
1
2
3
4
5
6
Figure 1.
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Figure 2.
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1
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Figure 3.
6
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