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Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs
Mutation Research 657 (2008) 146–149
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs b ´ a , Teresa Bartłomiejczyk a , Jarosław Wolinski ´ Marcin Kruszewski a,∗ , Teresa Iwanenko , c d d c ´ ´ Rafał R. Starzynski , Mikołaj A. Gralak , Romuald Zabielski , Paweł Lipinski a
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warszawa, Poland Department of Gastrointestinal Physiology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrz˛ebiec, ul. Post˛epu 1, 05-552 Wólka Kosowska, Poland d Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, ul. Nowoursynowska 166, 02-787 Warszawa, Poland b c
a r t i c l e
i n f o
Article history: Received 20 March 2008 Received in revised form 25 July 2008 Accepted 27 August 2008 Available online 9 September 2008 Keywords: Hydrogen peroxide X-radiation Hypochlorous acid Tert-butyl hydroperoxide Comet assay
a b s t r a c t The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H2 O2 and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Living organisms use iron in many essential biological processes, including electron transfer, oxygen transport and DNA synthesis. Despite being an indispensable component of living cells, iron is dangerous when in excess. In the presence of ferrous ions, hydrogen peroxide undergoes Fenton reaction to produce an extremely reactive hydroxyl radical (• OH). Radical reactions initiated by • OH may result in damage to macromolecules, e.g., DNA, lipids and proteins. Toxicity attributable to excess iron can occur either acutely after a single large dose of iron or chronically because of excessive accumulation of iron in the body from either diet or blood transfusions, or both. Acute iron poisoning was linked with increased morbidity, coronary artery disease, inflammation, neurodegenerative disease, and stroke [1]. High stored iron levels also are related to increased risk for cancer and coronary heart disease [2,3]. Interestingly, it was also reported that body-iron status correlates with the urinary excretion of 8-hydroxy-2 -deoxyguanosine
∗ Corresponding author. Tel.: +48 225041118/064; fax: +48 225041341. E-mail address: [email protected] (M. Kruszewski). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.08.020
(8-OHdG) [4]. Since it was proposed that the urinary concentration of 8-OHdG is a reliable marker of oxidative stress [5], this suggests a correlation of the level of body-iron stores with the level of cellular DNA damage. Indeed, a correlation of the concentration of 8-OHdG in liver biopsies with the hepatic iron content was found in an animal model [6,7] and in patients with chronic hepatitis C [8]. Moreover, limited supplies of iron in the case of anemia result in a lower hepatic iron content but an increase in DNA damage in lymphocytes, likely due to the greater oxidative stress [9]. However, most data are from subjects who suffer from iron-overload or other pathologies and it is not clear whether DNA damage is correlated with disturbances in iron metabolism or due to other processes, e.g., inflammation. Here, we used newborn piglets as a natural animal model of infant anemia to evaluate the relation between hepatic iron content and lymphocyte susceptibility to oxidative stress. The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body-iron stores during the first few days of postnatal life. Since hepatic iron concentrations correlate well with iron levels in serum and with transferrin saturation [10], and since the ability of lymphocytes to acquire serum iron is well documented [11], it is reasonable to assume that iron concentrations in lymphocytes are also related to the hepatic iron content. Thus, hep-
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atic iron levels may reflect the susceptibility of lymphocytes to the oxidative stress that is unquestionably associated with iron-driven Fenton chemistry. As a result of our investigations, for the first time we show that lowered hepatic iron corresponds with a decreased susceptibility of blood cells to oxidative stress. 2. Materials and methods 2.1. Materials All chemicals were from Sigma Chemical, Co. (USA), unless otherwise indicated. FCS was from Gibco (UK). Polish Landrace male pig neonates were used in the experiment. The piglets were delivered healthy and without complications. All animals were kept in controlled conditions and allowed to nurse the sow ad libitum. The experimental protocol was approved by the Local Ethical Committee. 2.2. Isolation of mononuclear cells Animals were sacrificed by CO2 inhalation and blood was collected by heart puncture at day 1 (3–4 h after birth) and at day 7 after birth. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation on Histopaque 1007, resuspended in RPMI 1640 medium supplemented with 20% of FCS. Cells isolated from four 1-day-old and three 3-days-old animals were frozen in a medium containing 90% FCS and 10% DMSO. Before treatment with the DNA-damaging agents, cells were thawed, immediately resuspended in RPMI1640 medium supplemented with 20% of FCS, and left overnight. The cell viability was checked by use of the trypan blue exclusion method just prior to the treatment, and was 93 ± 3%. 2.3. Determination of total hepatic iron and serum iron content Liver and serum samples were collected from animals sacrificed by CO2 inhalation at day 1, 2, 4, 7 and 14 after birth, frozen in liquid nitrogen and stored at −80 ◦ C until analyses. Samples (0.5 g) were mineralized in a mixture of 5 ml of 65% HNO3 (Merck) and 1 ml of 30% H2 O2 (Merck) in hermetic high-pressure vessels by heating in a microwave oven (Ethos 900, Milestone). Iron content was estimated by flame (air–acetylene) atomic-absorption spectrophotometry (PerkinElmer 1100B) using hollow cathode lamps (248.3 nm) with deuterium background correction. The external standards were prepared using a 9972 Titrisol Iron Standard (Merck) and the sensitivity level was calculated to be 0.051 mg/l. 2.4. Irradiation with X-rays Approximately 2 × 104 MNCs were suspended in 100 l of 1% low melting-point agarose Type VII in PBS, cast on microscope slides pre-coated with 100 l of agarose (0.5% agarose Type I-A, in redistilled water) and allowed to set on ice. Once the agarose was solidified, slides were irradiated in an X-ray machine (dose 0–5 Gy, 200 kV, 5 mA, dose rate 1.2 Gy/min) on ice. Immediately after irradiation, slides were placed in the lysis solution and the extent of DNA damage was evaluated by the alkaline comet assay. 2.5. Treatment with H2 O2 , tert-butyl hydroperoxide or HOCl Cell suspensions in agarose gel were prepared as described above. Once the agarose was set, slides were placed in glass jars containing different concentrations of H2 O2 (0–250 M), tert-butyl hydroperoxide (TBH) (0–500 M) or HOCl (0–1000 M) in PBS pH 7.4 for 15 min at 4 ◦ C. The HOCl solution was prepared from a 25-mmol stock solution of NaOCl in PBS. At pH 7.4, this solution contains a mixture of HOCl and OCl− at approximately 1:1 ratio and is subsequently referred to as HOCl. The concentration of OCl− was determined spectrophotometrically using an absorption coefficient of 350 mol−1 cm−1 (292 nm) at pH 9.0. After treatment, slides were washed in ice-cold PBS and the extent of DNA damage was evaluated by the alkaline comet assay. 2.6. Comet assay DNA damage was assessed by the alkaline comet assay, which allowed to estimate DNA strand lesions, such as single-strand breaks, double-strand breaks and alkali-labile sites. The alkaline comet assay was performed according to the standard protocol described in detail in Ref. [12]. Briefly, the cells on the microscope slides were lysed in 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10 for 1 h at 4 ◦ C, in the dark. Slides were placed in an electrophoresis apparatus in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH >13) and left for 40 min at 10 ◦ C for DNA unwinding. Electrophoresis was then carried out for 30 min at 1.2 V/cm. All steps of slide preparation and electrophoresis were conducted in red light to avoid induction of additional DNA damage. After electrophoresis, the slides were washed three times in neutralizing buffer (0.4 M Tris, pH 7.5), dried from the excess buffer and stained with 1 M 4 ,6-
Fig. 1. Hepatic (closed symbols) and serum (open symbols) iron content in neonatal sucking piglets. Mean ± S.D., n = 5. a Denotes statistically significant difference from 1-day-old animals (Kruskal–Wallis test followed by Dunn’s multiple comparison test, p < 0.05). diamidino-2-phenylindole. Slides were left at 4 ◦ C in a humid chamber overnight and scored. Comets were scored with the computer-aided image-analysis system Comet v.3.0 (Kinetic Imaging Ltd., Liverpool, UK). The assay’s experimental design included an internal standard as a control for inter-experimental variability, which consisted of an additional slide with peripheral blood lymphocytes from a single donor irradiated with 3 Gy of X-radiation. The percentage DNA in the comet tail was taken as a measure of DNA damage. Fifty comets were measured per experimental point. 2.7. Statistical analysis The data are expressed as their means and standard deviation (S.D.). Kruskal–Wallis test followed by Dunn’s multiple comparison test was used to indicate the statistical differences in iron content in liver and serum (Graph Pad Software ver4, San Diego, CA, USA). Pair-wise comparison of the means was made by the Student’s t-test for dependent samples. In all statistical analysis P < 0.05 was taken as the level of significance.
3. Results and discussion The pig is born with limited supplies of iron and depends on iron from sows’ milk. Because this is not sufficient for fast-growing neonates, they also depend on supplementation to the diet from soil iron in the wild, or from extra given iron during indoor feeding. If not iron-supplemented, piglets show significantly higher pre-weaning morbidity and mortality and lower blood-hemoglobin concentration than the iron-supplemented ones [13]. In our experimental design, piglets were not supplemented with iron. Hence, as expected, the shortage of iron supplies combined with low iron diet resulted in a dramatic decline in the total iron content in the liver during the first 4 days post-partum (Fig. 1). At day 4 the decrease in hepatic iron content reached a minimum and no further decline was observed. At that time piglets had almost six times less iron in the liver than newborn ones. The difference was statistically significant (P = 0.004). This observation is in accordance with other published data. Since during the first 2–3 weeks of life pigs depend heavily on scarce iron from sows’ milk, their body stores are usually low [14]. In parallel to the dramatic decline of hepatic iron content, the level of serum iron also decreased, albeit with a 1-day delay. A statistically significant difference with newborn animals was observed for 7- and 14-days-old animals (Fig. 1). Apparently, hepatic iron was used to maintain the level of iron in serum. It was reported that hepatic iron stores correlated with the extent of DNA base damage [6–8] and with enhanced lipid peroxidation [15] in the hepatic tissue. To investigate whether hepatic iron content affects the susceptibility of lymphocytes to oxidative stress, we exposed the lymphocytes taken from newborn and 7-
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Fig. 2. DNA damage induced by H2 O2 in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-dayold and 7-days-old animals (Student’s t-test, p < 0.05).
days-old piglets to different physiological oxidative stressors and ionizing radiation. Interestingly, no differences in background DNA damage were found between untreated cells, in spite of the different hepatic iron content of the donor animals (Figs. 2–4). This indicates that hepatic iron content has no effect on blood lymphocytes in non-pathological conditions. All DNA-damaging agents used in this study induced DNA damage in a dose-dependent manner. As shown in Figs. 2 and 3, DNA damage induced by hydrogen peroxide and X-radiation, respectively, was significantly higher in lymphocytes taken from newborn piglets than in those taken from 7-days old animals for all tested doses. Interestingly, no difference in susceptibility was found in lymphocytes taken from newborn and 7-days old animals treated with NaOCl or TBH (Fig. 4). It is widely accepted that the toxicity of hydrogen peroxide is linked with cellular iron level [16], and that iron chelators diminish H2 O2 -induced toxicity [17,18]. The decrease in labile iron pool due to over-expression of the heavy ferritin subunit correlates with the decrease in cellular susceptibility to hydrogen peroxide [19]. The redox-active form of iron is responsible for H2 O2 -induced DNA damage [20]. Iron also seems to be involved in radiation-induced cellular injury and death. Iron chelators facilitate tissue-repair processes and recovery from radiation injury of lethally irradiated mice and rats [21]. Moreover, it was suggested that iron released
Fig. 3. DNA damage induced by X-radiation in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-day-old and 7-days-old-animals (Student’s t-test, p < 0.05).
Fig. 4. DNA damage induced by HOCl and tert-butyl hydroperoxide (TBH) in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-day- and 7-days-old animals (Student’s t-test, p < 0.05).
from ruptured lysosomes [22] or iron-containing proteins [23] is involved in radiation genotoxicity. In contrast, although HOCl is able to generate hydroxyl radicals in the presence of transition metal ions [24], only a small effect of iron ions on HOCl-induced DNA base damage was observed in vitro [25]. HOCl has been shown to react directly with different cellular components and induce DNA fragmentation likely due to the chlorination of DNA bases [25]. In our hands, HOCl-induced DNA damage to a similar extent in both iron-rich and iron-poor conditions, indicating that an iron-independent mechanism may be involved in the induction of DNA damage by HOCl. The available information about the role of iron in TBH-induced genotoxicity is also ambiguous. Although iron chelators diminish TBH-induced DNA genotoxicity [26,27], TBH-induced DNA-break formation is linked with enhanced lipid peroxidation and formation of DNA-adducts or a-basic sites [28–30] rather than with the direct action on DNA, as TBH does not cause strand scission in partially purified DNA [31]. Indeed, in our hands, the effect of hepatic iron content on cellular susceptibility to TBH was the smallest of all oxidative stressors studied. This points at an indirect role of iron ions and to other factors as potential modulators of TBHinduced genotoxicity, e.g., changes in antioxidant capacity during early neonatal development. Indeed, a postnatal increase in the extra-cellular antioxidant defense in blood of neonates accompanied by the decrease in the serum iron was observed in humans [32]. Hence, it cannot be excluded that changes in the antioxidant capacity during early neonatal life may also contribute to the observed changes in cellular susceptibility to oxidative stress. To avoid the confounding effects of serum antioxidant capacity, our experiments were conducted on isolated cells in a serum-free environment. While maximum care was taken to eliminate the effect of serum antioxidant capacity on the results, it cannot be excluded that changes in antioxidant capacity have taken place in the cells themselves. However, this is rather unlikely, as Crissinger et al. [33] reported no differences in the peroxidase activity per circulating granulocyte, when comparing 1-day old and 1-month old pigs. In summary, the data reported here show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to hydrogen peroxide and ionizing radiation. However, the absence of a difference in the background DNA damage between the control (untreated) cells indicates that hepatic iron content has no effect on blood lymphocytes under normal, nonpathological conditions.
M. Kruszewski et al. / Mutation Research 657 (2008) 146–149
Conflict of interest None. Acknowledgments The work was supported by the Ministry of Science and Higher 239 Education Grant No. PBZ-KBN-093/PZ06/2003. References [1] J. Emerit, C. Beaumont, F. Trivin, Iron metabolism, free radicals, and oxidative injury, Biomed. Pharmacother. 55 (2001) 333–339. [2] R.G. Stevens, D.Y. Jones, M.S. Micozzi, P.R. Taylor, Body iron stores and the risk of cancer, N. Engl. J. Med. 319 (1988) 1047–1052. [3] J.L. Sullivan, Interactions of stored iron with traditional and inflammatory cardiovascular risk factors, Atherosclerosis 167 (2003) 169. [4] M. Nakano, Y. Kawanishi, S. Kamohara, Y. Uchida, M. Shiota, Y. Inatomi, T. Komori, K. Miyazawa, K. Gondo, I. Yamasawa, Oxidative DNA damage (8hydroxydeoxyguanosine) and body iron status: a study on 2507 healthy people, Free Radic. Biol. Med. 35 (2003) 826–832. [5] R. Olinski, R. Rozalski, D. Gackowski, M. Foksinski, A. Siomek, M.S. Cooke, Urinary measurement of 8-OxodG, 8-OxoGua, and 5HMUra: a noninvasive assessment of oxidative damage to DNA, Antioxidant Redox Signal. 8 (2006) 1011–1019. [6] J.O. Kang, C. Jones, B. Brothwell, Toxicity associated with iron overload found in hemochromatosis: possible mechanism in a rat model, Clin. Lab. Sci. 11 (1998) 350–354. [7] M. Barollo, R. D’Inca, M. Scarpa, V. Medici, R. Cardin, M. Bortolami, C. Ruffolo, I. Angriman, G.C. Sturniolo, Effects of iron manipulation on trace elements level in a model of colitis in rats, World J. Gastroenterol. 11 (2005) 4396– 4399. [8] N. Fujita, S. Horiike, R. Sugimoto, H. Tanaka, M. Iwasa, Y. Kobayashi, K. Hasegawa, N. Ma, S. Kawanishi, Y. Adachi, M. Kaito, Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients, Free Radic. Biol. Med. 42 (2007) 353–362. [9] M. Aslan, M. Horoz, A. Kocyigit, S. Ozgonul, H. Celik, M. Celik, O. Erel, Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia, Mutation Res. 601 (2006) 144–149. [10] T.J. Lin, L.Y. Liao, S.Y. Lin, C.L. Lin, T.A. Chang, Influence of iron on the severity of hepatic fibrosis in patients with chronic hepatitis C, World J. Gastroenterol. 12 (2006) 4897–4901. [11] H. Artac, M. Coskun, I. Karadogan, O. Yegin, A. Yesilipek, Transferrin receptor in proliferation of T lymphocytes in infants with iron deficiency, Int. J. Lab. Hematol. 29 (2007) 310–315. [12] M. Wojewodzka, M. Kruszewski, T. Iwanenko, A.R. Collins, I. Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation. I. Strand breakage, Mutation Res. 416 (1998) 21–35. [13] P. Szabo, G. Bilkei, Iron deficiency in outdoor pig production, J. Vet. Med. A: Physiol. Pathol. Clin. Med. 49 (2002) 390–391. [14] J.E. Smith, K. Moore, D. Boyington, D.S. Pollmann, D. Schoneweis, Serum ferritin and total iron-binding capacity to estimate iron storage in pigs, Vet. Pathol. 21 (1984) 597–600.
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[15] J.G. Fischer, H.P. Glauert, T. Yin, M.L. Sweeney-Reeves, N. Larmonier, M.C. Black, Moderate iron overload enhances lipid peroxidation in livers of rats, but does not affect NF-kappaB activation induced by the peroxisome proliferator, Wy14,643, J. Nutr. 132 (2002) 2525–2531. [16] J.L. Pierre, M. Fontecave, Iron and activated oxygen species in biology: the basic chemistry, Biometals 12 (1999) 195–199. [17] A.C. Mello-Filho, M.E. Hoffmann, R. Meneghini, Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron, Biochem. J. 218 (1984) 273–275. [18] M. Kruszewski, M.H. Green, J.E. Lowe, I. Szumiel, Comparison of effects of iron and calcium chelators on the response of L5178Y sublines to X-rays and H2 O2 , Mutation Res. 326 (1995) 155–163. [19] A. Cozzi, B. Corsi, S. Levi, P. Santambrogio, A. Albertini, P. Arosio, Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity, J. Biol. Chem. 275 (2000) 25122–25129. [20] M. Kruszewski, Labile iron pool: the main determinant of cellular response to oxidative stress, Mutation Res. 531 (2003) 81–92. [21] J.R. Sorenson, Cu, Fe, Mn, and Zn chelates offer a medicinal chemistry approach to overcoming radiation injury, Curr. Med. Chem. 9 (2002) 639–662. [22] H.L. Persson, T. Kurz, J.W. Eaton, U.T. Brunk, Radiation-induced cell death: importance of lysosomal destabilization, Biochem. J. 389 (2005) 877–884. [23] D. Ghosal, M.V. Omelchenko, E.K. Gaidamakova, V.Y. Matrosova, A. Vasilenko, A. Venkateswaran, M. Zhai, H.M. Kostandarithes, H. Brim, K.S. Makarova, L.P. Wackett, J.K. Fredrickson, M.J. Daly, How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress, FEMS Microbiol. Rev. 29 (2005) 361–375. [24] L.P. Candeias, M.R. Stratford, P. Wardman, Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron(II) complex, Free Radic. Res. 20 (1994) 241–249. [25] M. Whiteman, A. Jenner, B. Halliwell, Hypochlorous acid-induced base modifications in isolated calf thymus DNA, Chem. Res. Toxicol. 10 (1997) 1240–1246. [26] P. Sestili, A. Guidarelli, M. Dacha, O. Cantoni, Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide: free radical scavenging versus iron chelating mechanism, Free Radic. Biol. Med. 25 (1998) 196–200. [27] S.A. Aherne, N.M. O’Brien, Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells, Free Radic. Biol. Med. 29 (2000) 507–514. [28] W. Breuer, E. Greenberg, Z.I. Cabantchik, Newly delivered transferrin iron and oxidative cell injury, FEBS Lett. 403 (1997) 213–219. [29] A. Kanazawa, T. Sawa, T. Akaik, H. Maeda, Formation of abasic sites in DNA by t-butyl peroxyl radicals: implication for potent genotoxicity of lipid peroxyl radicals, Cancer Lett. 156 (2000) 51–55. [30] S. Hix, O. Augusto, DNA methylation by tert-butyl hydroperoxide-iron (II): a role for the transition metal ion in the production of DNA base adducts, Chem. Biol. Interact. 118 (1999) 141–149. [31] A. Guidarelli, F. Cattabeni, O. Cantoni, Alternative mechanisms for hydroperoxide-induced DNA single strand breakage, Free Radic. Res. 26 (1997) 537–547. [32] M. Szabo, B. Vasarhelyi, G. Balla, T. Szabo, T. Machay, T. Tulassay, Acute postnatal increase of extracellular antioxidant defence of neonates: the role of iron metabolism, Acta Paediatr. 90 (2001) 1167–1170. [33] K.D. Crissinger, M.B. Grisham, D.N. Granger, Developmental biology of oxidantproducing enzymes and antioxidants in the piglet intestine, Pediatr. Res. 25 (1989) 612–616.
Contents lists available at ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs b ´ a , Teresa Bartłomiejczyk a , Jarosław Wolinski ´ Marcin Kruszewski a,∗ , Teresa Iwanenko , c d d c ´ ´ Rafał R. Starzynski , Mikołaj A. Gralak , Romuald Zabielski , Paweł Lipinski a
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warszawa, Poland Department of Gastrointestinal Physiology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrz˛ebiec, ul. Post˛epu 1, 05-552 Wólka Kosowska, Poland d Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, ul. Nowoursynowska 166, 02-787 Warszawa, Poland b c
a r t i c l e
i n f o
Article history: Received 20 March 2008 Received in revised form 25 July 2008 Accepted 27 August 2008 Available online 9 September 2008 Keywords: Hydrogen peroxide X-radiation Hypochlorous acid Tert-butyl hydroperoxide Comet assay
a b s t r a c t The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H2 O2 and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Living organisms use iron in many essential biological processes, including electron transfer, oxygen transport and DNA synthesis. Despite being an indispensable component of living cells, iron is dangerous when in excess. In the presence of ferrous ions, hydrogen peroxide undergoes Fenton reaction to produce an extremely reactive hydroxyl radical (• OH). Radical reactions initiated by • OH may result in damage to macromolecules, e.g., DNA, lipids and proteins. Toxicity attributable to excess iron can occur either acutely after a single large dose of iron or chronically because of excessive accumulation of iron in the body from either diet or blood transfusions, or both. Acute iron poisoning was linked with increased morbidity, coronary artery disease, inflammation, neurodegenerative disease, and stroke [1]. High stored iron levels also are related to increased risk for cancer and coronary heart disease [2,3]. Interestingly, it was also reported that body-iron status correlates with the urinary excretion of 8-hydroxy-2 -deoxyguanosine
∗ Corresponding author. Tel.: +48 225041118/064; fax: +48 225041341. E-mail address: [email protected] (M. Kruszewski). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.08.020
(8-OHdG) [4]. Since it was proposed that the urinary concentration of 8-OHdG is a reliable marker of oxidative stress [5], this suggests a correlation of the level of body-iron stores with the level of cellular DNA damage. Indeed, a correlation of the concentration of 8-OHdG in liver biopsies with the hepatic iron content was found in an animal model [6,7] and in patients with chronic hepatitis C [8]. Moreover, limited supplies of iron in the case of anemia result in a lower hepatic iron content but an increase in DNA damage in lymphocytes, likely due to the greater oxidative stress [9]. However, most data are from subjects who suffer from iron-overload or other pathologies and it is not clear whether DNA damage is correlated with disturbances in iron metabolism or due to other processes, e.g., inflammation. Here, we used newborn piglets as a natural animal model of infant anemia to evaluate the relation between hepatic iron content and lymphocyte susceptibility to oxidative stress. The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body-iron stores during the first few days of postnatal life. Since hepatic iron concentrations correlate well with iron levels in serum and with transferrin saturation [10], and since the ability of lymphocytes to acquire serum iron is well documented [11], it is reasonable to assume that iron concentrations in lymphocytes are also related to the hepatic iron content. Thus, hep-
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atic iron levels may reflect the susceptibility of lymphocytes to the oxidative stress that is unquestionably associated with iron-driven Fenton chemistry. As a result of our investigations, for the first time we show that lowered hepatic iron corresponds with a decreased susceptibility of blood cells to oxidative stress. 2. Materials and methods 2.1. Materials All chemicals were from Sigma Chemical, Co. (USA), unless otherwise indicated. FCS was from Gibco (UK). Polish Landrace male pig neonates were used in the experiment. The piglets were delivered healthy and without complications. All animals were kept in controlled conditions and allowed to nurse the sow ad libitum. The experimental protocol was approved by the Local Ethical Committee. 2.2. Isolation of mononuclear cells Animals were sacrificed by CO2 inhalation and blood was collected by heart puncture at day 1 (3–4 h after birth) and at day 7 after birth. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation on Histopaque 1007, resuspended in RPMI 1640 medium supplemented with 20% of FCS. Cells isolated from four 1-day-old and three 3-days-old animals were frozen in a medium containing 90% FCS and 10% DMSO. Before treatment with the DNA-damaging agents, cells were thawed, immediately resuspended in RPMI1640 medium supplemented with 20% of FCS, and left overnight. The cell viability was checked by use of the trypan blue exclusion method just prior to the treatment, and was 93 ± 3%. 2.3. Determination of total hepatic iron and serum iron content Liver and serum samples were collected from animals sacrificed by CO2 inhalation at day 1, 2, 4, 7 and 14 after birth, frozen in liquid nitrogen and stored at −80 ◦ C until analyses. Samples (0.5 g) were mineralized in a mixture of 5 ml of 65% HNO3 (Merck) and 1 ml of 30% H2 O2 (Merck) in hermetic high-pressure vessels by heating in a microwave oven (Ethos 900, Milestone). Iron content was estimated by flame (air–acetylene) atomic-absorption spectrophotometry (PerkinElmer 1100B) using hollow cathode lamps (248.3 nm) with deuterium background correction. The external standards were prepared using a 9972 Titrisol Iron Standard (Merck) and the sensitivity level was calculated to be 0.051 mg/l. 2.4. Irradiation with X-rays Approximately 2 × 104 MNCs were suspended in 100 l of 1% low melting-point agarose Type VII in PBS, cast on microscope slides pre-coated with 100 l of agarose (0.5% agarose Type I-A, in redistilled water) and allowed to set on ice. Once the agarose was solidified, slides were irradiated in an X-ray machine (dose 0–5 Gy, 200 kV, 5 mA, dose rate 1.2 Gy/min) on ice. Immediately after irradiation, slides were placed in the lysis solution and the extent of DNA damage was evaluated by the alkaline comet assay. 2.5. Treatment with H2 O2 , tert-butyl hydroperoxide or HOCl Cell suspensions in agarose gel were prepared as described above. Once the agarose was set, slides were placed in glass jars containing different concentrations of H2 O2 (0–250 M), tert-butyl hydroperoxide (TBH) (0–500 M) or HOCl (0–1000 M) in PBS pH 7.4 for 15 min at 4 ◦ C. The HOCl solution was prepared from a 25-mmol stock solution of NaOCl in PBS. At pH 7.4, this solution contains a mixture of HOCl and OCl− at approximately 1:1 ratio and is subsequently referred to as HOCl. The concentration of OCl− was determined spectrophotometrically using an absorption coefficient of 350 mol−1 cm−1 (292 nm) at pH 9.0. After treatment, slides were washed in ice-cold PBS and the extent of DNA damage was evaluated by the alkaline comet assay. 2.6. Comet assay DNA damage was assessed by the alkaline comet assay, which allowed to estimate DNA strand lesions, such as single-strand breaks, double-strand breaks and alkali-labile sites. The alkaline comet assay was performed according to the standard protocol described in detail in Ref. [12]. Briefly, the cells on the microscope slides were lysed in 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10 for 1 h at 4 ◦ C, in the dark. Slides were placed in an electrophoresis apparatus in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH >13) and left for 40 min at 10 ◦ C for DNA unwinding. Electrophoresis was then carried out for 30 min at 1.2 V/cm. All steps of slide preparation and electrophoresis were conducted in red light to avoid induction of additional DNA damage. After electrophoresis, the slides were washed three times in neutralizing buffer (0.4 M Tris, pH 7.5), dried from the excess buffer and stained with 1 M 4 ,6-
Fig. 1. Hepatic (closed symbols) and serum (open symbols) iron content in neonatal sucking piglets. Mean ± S.D., n = 5. a Denotes statistically significant difference from 1-day-old animals (Kruskal–Wallis test followed by Dunn’s multiple comparison test, p < 0.05). diamidino-2-phenylindole. Slides were left at 4 ◦ C in a humid chamber overnight and scored. Comets were scored with the computer-aided image-analysis system Comet v.3.0 (Kinetic Imaging Ltd., Liverpool, UK). The assay’s experimental design included an internal standard as a control for inter-experimental variability, which consisted of an additional slide with peripheral blood lymphocytes from a single donor irradiated with 3 Gy of X-radiation. The percentage DNA in the comet tail was taken as a measure of DNA damage. Fifty comets were measured per experimental point. 2.7. Statistical analysis The data are expressed as their means and standard deviation (S.D.). Kruskal–Wallis test followed by Dunn’s multiple comparison test was used to indicate the statistical differences in iron content in liver and serum (Graph Pad Software ver4, San Diego, CA, USA). Pair-wise comparison of the means was made by the Student’s t-test for dependent samples. In all statistical analysis P < 0.05 was taken as the level of significance.
3. Results and discussion The pig is born with limited supplies of iron and depends on iron from sows’ milk. Because this is not sufficient for fast-growing neonates, they also depend on supplementation to the diet from soil iron in the wild, or from extra given iron during indoor feeding. If not iron-supplemented, piglets show significantly higher pre-weaning morbidity and mortality and lower blood-hemoglobin concentration than the iron-supplemented ones [13]. In our experimental design, piglets were not supplemented with iron. Hence, as expected, the shortage of iron supplies combined with low iron diet resulted in a dramatic decline in the total iron content in the liver during the first 4 days post-partum (Fig. 1). At day 4 the decrease in hepatic iron content reached a minimum and no further decline was observed. At that time piglets had almost six times less iron in the liver than newborn ones. The difference was statistically significant (P = 0.004). This observation is in accordance with other published data. Since during the first 2–3 weeks of life pigs depend heavily on scarce iron from sows’ milk, their body stores are usually low [14]. In parallel to the dramatic decline of hepatic iron content, the level of serum iron also decreased, albeit with a 1-day delay. A statistically significant difference with newborn animals was observed for 7- and 14-days-old animals (Fig. 1). Apparently, hepatic iron was used to maintain the level of iron in serum. It was reported that hepatic iron stores correlated with the extent of DNA base damage [6–8] and with enhanced lipid peroxidation [15] in the hepatic tissue. To investigate whether hepatic iron content affects the susceptibility of lymphocytes to oxidative stress, we exposed the lymphocytes taken from newborn and 7-
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Fig. 2. DNA damage induced by H2 O2 in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-dayold and 7-days-old animals (Student’s t-test, p < 0.05).
days-old piglets to different physiological oxidative stressors and ionizing radiation. Interestingly, no differences in background DNA damage were found between untreated cells, in spite of the different hepatic iron content of the donor animals (Figs. 2–4). This indicates that hepatic iron content has no effect on blood lymphocytes in non-pathological conditions. All DNA-damaging agents used in this study induced DNA damage in a dose-dependent manner. As shown in Figs. 2 and 3, DNA damage induced by hydrogen peroxide and X-radiation, respectively, was significantly higher in lymphocytes taken from newborn piglets than in those taken from 7-days old animals for all tested doses. Interestingly, no difference in susceptibility was found in lymphocytes taken from newborn and 7-days old animals treated with NaOCl or TBH (Fig. 4). It is widely accepted that the toxicity of hydrogen peroxide is linked with cellular iron level [16], and that iron chelators diminish H2 O2 -induced toxicity [17,18]. The decrease in labile iron pool due to over-expression of the heavy ferritin subunit correlates with the decrease in cellular susceptibility to hydrogen peroxide [19]. The redox-active form of iron is responsible for H2 O2 -induced DNA damage [20]. Iron also seems to be involved in radiation-induced cellular injury and death. Iron chelators facilitate tissue-repair processes and recovery from radiation injury of lethally irradiated mice and rats [21]. Moreover, it was suggested that iron released
Fig. 3. DNA damage induced by X-radiation in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-day-old and 7-days-old-animals (Student’s t-test, p < 0.05).
Fig. 4. DNA damage induced by HOCl and tert-butyl hydroperoxide (TBH) in lymphocytes isolated from 1-day-old (n = 4) and 7-days-old (n = 3) piglets. Control values were subtracted from those for treated samples. Mean ± S.D. a Denotes a statistically significant difference between treated and control cells and b denotes a statistically significant difference between 1-day- and 7-days-old animals (Student’s t-test, p < 0.05).
from ruptured lysosomes [22] or iron-containing proteins [23] is involved in radiation genotoxicity. In contrast, although HOCl is able to generate hydroxyl radicals in the presence of transition metal ions [24], only a small effect of iron ions on HOCl-induced DNA base damage was observed in vitro [25]. HOCl has been shown to react directly with different cellular components and induce DNA fragmentation likely due to the chlorination of DNA bases [25]. In our hands, HOCl-induced DNA damage to a similar extent in both iron-rich and iron-poor conditions, indicating that an iron-independent mechanism may be involved in the induction of DNA damage by HOCl. The available information about the role of iron in TBH-induced genotoxicity is also ambiguous. Although iron chelators diminish TBH-induced DNA genotoxicity [26,27], TBH-induced DNA-break formation is linked with enhanced lipid peroxidation and formation of DNA-adducts or a-basic sites [28–30] rather than with the direct action on DNA, as TBH does not cause strand scission in partially purified DNA [31]. Indeed, in our hands, the effect of hepatic iron content on cellular susceptibility to TBH was the smallest of all oxidative stressors studied. This points at an indirect role of iron ions and to other factors as potential modulators of TBHinduced genotoxicity, e.g., changes in antioxidant capacity during early neonatal development. Indeed, a postnatal increase in the extra-cellular antioxidant defense in blood of neonates accompanied by the decrease in the serum iron was observed in humans [32]. Hence, it cannot be excluded that changes in the antioxidant capacity during early neonatal life may also contribute to the observed changes in cellular susceptibility to oxidative stress. To avoid the confounding effects of serum antioxidant capacity, our experiments were conducted on isolated cells in a serum-free environment. While maximum care was taken to eliminate the effect of serum antioxidant capacity on the results, it cannot be excluded that changes in antioxidant capacity have taken place in the cells themselves. However, this is rather unlikely, as Crissinger et al. [33] reported no differences in the peroxidase activity per circulating granulocyte, when comparing 1-day old and 1-month old pigs. In summary, the data reported here show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to hydrogen peroxide and ionizing radiation. However, the absence of a difference in the background DNA damage between the control (untreated) cells indicates that hepatic iron content has no effect on blood lymphocytes under normal, nonpathological conditions.
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