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Protective effects of GH and IGF-I against iron-induced lipid peroxidation in vivo
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 60 (2008) 453–458 www.elsevier.de/etp
Protective effects of GH and IGF-I against iron-induced lipid peroxidation in vivo Agnieszka Kokoszkoa,c, Jan Da˛ browskia,c, Andrzej Lewin´skia,c, Małgorzata Karbownik-Lewin´skab,c, a
Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, 281/289 Rzgowska Street, 93-338 Lodz, Poland b Department of Oncological Endocrinology, Medical University of Lodz, 7/9 Zeligowski Street, 90-752 Lodz, Poland c Polish Mother’s Memorial Hospital – Research Institute, 281/289 Rzgowska Street, 93-338 Lodz, Poland Received 27 February 2008; accepted 29 April 2008
Abstract Iron overload may enhance oxidative damage. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are involved in oxidative processes, lipid peroxidation (LPO) included. The aim of the study was to evaluate the in vivo effects of GH, IGF-I and/or iron on LPO in rat tissues. Male Wistar rats were administered iron (Fe2+; 3 mg/100 g b.w., i.p., on the 8th day) and/or GH (0.2 IU/100 g b.w.), and/or IGF-I (2 mg/100 g b.w.) once daily for 8 days. LPO products (malondialdehyde+4-hydroxyalkenals) were measured in rat brain, lung, small intestine, liver, kidney, testis, spleen and serum. Iron injection increased LPO only in the small intestine and that effect was completely prevented by either GH or IGF-I. In the brain, GH decreased, whereas IGF-I increased, the basal LPO. GH and IGF-I possess some ability to prevent iron-induced oxidative damage in iron sensitive tissues, but contribute to oxidative imbalance in other tissues. r 2008 Elsevier GmbH. All rights reserved. Keywords: Growth hormone (GH); Insulin-like growth factor-I (IGF-I); Iron; Oxidative damage; Lipid peroxidation (LPO)
Introduction Reactive oxygen species (ROS), free radicals included, are products of normal cellular metabolism. Overproduction of ROS results in enhanced oxidative stress and – in consequence – in increased damage to macromolecules, lipids included (Valko et al., 2007, Corresponding author at: Department of Oncological Endocrinology, Medical University of Lodz, 7/9 Zeligowski Street, 90-752 Lodz, Poland. Tel./fax: +48 42 271 13 43. E-mail address: [email protected] (M. Karbownik-Lewin´ska).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.04.012
2005). In turn, the products of oxidative damage to macromolecules may further enhance oxidative stress and damage all the components in the organism. Iron is a transition metal, which contributes to oxidative balance, although iron overload is associated with enhanced oxidative damage and cancer initiation. This occurs via different mechanisms. Among others, iron participates in the Fenton reaction (Fe2++ H2O2+H+-Fe3++dOH+H2O), the most basic reaction of oxidative stress, which is frequently used to in vitro induce oxidative damage to macromolecules, lipid peroxidation (LPO) included (Karbownik et al., 2001;
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A. Kokoszko et al. / Experimental and Toxicologic Pathology 60 (2008) 453–458
Gitto et al., 2001). When the whole organism is exposed to iron, not only Fenton reaction but also more complex mechanisms are involved in oxidative effects, due to the action of this transition metal. Growth hormone (GH) is a polypeptide, released in a pulsatile manner, by the anterior pituitary. The growthpromoting and metabolic actions of GH are mediated mainly by the insulin-like growth factor-I (IGF-I). There is some evidence that both GH and IGF-I are involved in oxidative processes. In patients with active acromegaly, an increased level of plasma lipid peroxide (the index of oxidative damage to low-density lipoprotein cholesterol) was found, the abnormality being directly related to increased GH concentration (Yarman et al., 2003). At the same time, increased LPO was also observed in children (Mohn et al., 2005) and adults (Ozbey et al., 2003; Scacchi et al., 2006; Kokoszko et al., 2006; Karbownik-Lewinska et al., 2008) with GH deficiency, the change being partially reversed, due to GH replacement in the latter group (KarbownikLewinska et al., 2008). Under in vitro conditions, GH and/or IGF-I (used in different concentrations) were found to contribute to oxidative balance in the rat liver and in the porcine thyroid and, in case of induced oxidative stress, they may cause even pro-oxidative effects (Kokoszko et al., personal communication). The aim of the study was to evaluate the in vivo effect of GH, IGF-I and/or iron on LPO in different rat tissues.
Materials and methods The procedures, used in the study, were approved by the Ethical Committee of the Polish Mother’s Memorial Hospital – Research Institute.
Chemicals GH, isolated from porcine pituitary glands, the human recombinant IGF-I expressed in Escherichia coli, and ferrous sulphate (FeSO4), were purchased from Sigma-Aldrich (St. Louis, MO). The LPO-586 kit for LPO was purchased from Calbiochem (La Jolla, CA). Other chemicals were of analytical grade and came from commercial sources.
In vivo experiment A total of 52 male Wistar rats (weighing about 150 g each) were used in the study. The animals were randomized into six groups and administered: freshly prepared 0.9% NaCl (Control; n ¼ 9), or FeSO4 in freshly prepared 0.9% NaCl (3 mg Fe2+/100 g b.w. (Hemalatha et al., 2004), on the 8th day of the
experiment; n ¼ 9), or GH in freshly prepared 50 mM Tris–HCl buffer (pH 7.4) (0.2 IU/100 g b.w. (Jung et al., 2003), once daily, for 8 days; n ¼ 9), or IGF-I in freshly prepared Millipore H2O (2 mg/100 g b.w. (CastillaCortazar et al., 1997), once daily, for 8 days; n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8). The control rats, which did not receive either FeSO4 or GH or IGF-I, were treated with 0.9% NaCl at the abovementioned time points. All the rats received drinking water ad libitum. All the substances were administered i.p. in a volume of 0.5 ml/injection. The rats were killed by decapitation 24 h after FeSO4 injection. Brains, lungs, small intestines, livers, kidneys, testes and spleens were collected and frozen on solid CO2. Peripheral blood was collected, and centrifuged at 3000g (10 min, 4 1C) to obtain serum. The collected tissues and serum were stored at 70 1C until assay.
Measurement of lipid peroxidation products The concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA), as an index of LPO, were measured in the brain, lungs, small intestine, liver, kidneys, testes and spleen homogenates, as well as in blood serum. The tissue from each animal (approximately 100 mg) was homogenized in ice-cold 50 mM Tris–HCl buffer (pH 7.4, 10%, w/v – in the final incubation volume). The homogenates were centrifuged at 3000g for 10 min at 4 1C. The supernatant or blood serum was mixed with 650 ml of a methanol:acetonitrile (1:3, v/v) solution, containing a chromogenic reagent, N-methyl-2-phenylindole, and vortexed. After addition of 150 ml of methanesulphonic acid (15.4 M), the incubation was carried out at 45 1C for 40 min. The reaction between MDA+4-HDA and N-methyl-2-phenylindole yields a chromophore, which is spectrophotometrically measured at the absorbance of 586 nm, using a solution of 4-hydroxynonenal (10 mM) as standard. The level of LPO in tissue homogenates was expressed as the amount of MDA+4-HDA (nmol) per mg protein, or – in case of serum – as the amount of MDA+4-HDA (nmol) per 1 ml of serum.
Measurement of protein concentration Protein concentration was measured using the Bradford’s method (Bradford, 1976), with bovine albumin as standard.
Statistical analysis The data were statistically analysed, using one-way analysis of variance (ANOVA) followed by Student– Newman–Keuls’ test. Statistical significance was
ARTICLE IN PRESS A. Kokoszko et al. / Experimental and Toxicologic Pathology 60 (2008) 453–458
determined at the level of po0.05. The results are presented as means7SEM.
Results Among several examined tissues, only the small intestine and the brain appeared to be sensitive to oxidative effects of GH, or IGF-I, or iron. The i.p. administration of FeSO4 resulted in a significant (p ¼ 0.009) increase in the basal LPO only in the rat small intestine, and that effect was completely prevented by either GH (p ¼ 0.001) or IGF-I (p ¼ 0.0003) (Fig. 1). In the rat brain, GH significantly (p ¼ 0.034) decreased the basal LPO, whereas IGF-I, either used alone or together with iron, significantly (p ¼ 0.046; p ¼ 0.002, respectively) increased LPO level (Fig. 2). No changes of LPO were found in any other tissues, due to GH, or IGF-I, or FeSO4 treatment (Table 1).
Discussion Our study has been the first attempt to evaluate the in vivo effect of GH, or IGF-I, administered alone or together with iron on LPO in rat tissues. The selection of FeSO4, as a causative factor of oxidative damage, for such a study was justified by some facts. It had been confirmed in several in vitro
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experiments that ferrous ion (Fe2+) had the capacity to induce LPO in different tissues (in the liver – Karbownik et al., 2000; in the testes – Karbownik et al., 2001; in the thyroid gland – Karbownik and Lewinski, 2003); however, such an effect has never been examined in the small intestine. The pro-oxidative effect of iron was also observed under in vivo condition. The injection of ferrous chloride (2.4 mg Fe2+/100 g b.w.) (Hu et al., 1990) or ferrous sulphate (3 mg Fe2+/100 g b.w.) (Hemalatha et al., 2004) significantly increased the content of LPO products (measured as the concentration of thiobarbituric acid-reactive substances) in the rat liver. LPO induction in the rat liver, found in those two cited studies (Hemalatha et al., 2004; Hu et al., 1990), and the lack of such an effect in rat liver in the present study could result from the two main differences: different strain (weanling WNIN rats (Hemalatha et al., 2004; Hu et al., 1990) vs. Wistar rats in our study) and different body mass (35 g (Hemalatha et al., 2004; Hu et al., 1990) vs. 150 g in our study). It is worth mentioning that the administration of ferric ions, i.e. Fe3+ (as ferric nitrate – 0.2 mg/100 g b.w.), also enhanced LPO in liver and induced acute hepatic injury in mice (Hsu et al., 2007). In our study, we found a statistically significant increase in LPO, due to FeSO4 treatment, but, unexpectedly, only in one tissue, namely in rat small intestine. In other examined tissues, we did not observe iron-induced changes in the basal level of LPO. Those findings may indicate that, under in vivo conditions, the sensitivity of particular tissues to iron-induced oxidative
♦ ♦
Fig. 1. Concentrations of MDA+4-HDA in the small intestine, collected from the rats, administered 0.9% NaCl (Control; n ¼ 9), or FeSO4 (n ¼ 9), or GH (n ¼ 9), or IGF-I (n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8). Data are expressed as nmol of MDA+4-HDA per mg of protein. Bars represent the means7SEM. *po0.05 vs. Control; ~po0.05 vs. Fe.
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Fig. 2. Concentrations of MDA+4-HDA in the rat brain. The experimental protocol corresponds to that described in the legend to Fig. 1. Data are expressed as nmol of MDA+4-HDA per mg of protein. Bars represent the means7SEM. *po0.05 vs. Control.
Table 1. Concentrations of MDA+4-HDA (means7SEM) in tissues collected from the rats, administered 0.9% NaCl (Control; n ¼ 9), or FeSO4 (n ¼ 9), or GH (n ¼ 9), or IGF-I (n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8) Tissues
Lung Spleen Liver Kidney Testis Serum
Groups Control
Fe
GH
IGF-I
Fe+GH
Fe+IGF-I
0.8970.03 2.1870.16 4.2470.52 2.2570.25 1.1470.27 22.0372.17
0.8770.06 2.2770.17 3.9970.42 2.1170.22 1.0070.15 22.7571.90
0.9470.06 2.2170.16 4.0170.23 1.8070.25 0.5770.08 17.1171.83
0.9470.04 1.6570.06 4.2470.65 2.3770.20 1.7070.77 17.8771.34
1.0470.11 2.0670.27 3.9970.33 1.9470.29 0.5970.07 20.1071.41
0.9870.07 2.4370.21 3.2070.32 2.2770.21 1.2470.36 24.4772.16
Data are expressed as nmol of MDA+4-HDA per mg of protein, or, in case of serum as nmol of MDA+4-HDA per 1 ml of serum (differences not significant).
damage demonstrates significant differences. It is well known that the small intestine is particularly vulnerable to several pro-oxidative agents, such as, for example, ionizing radiation (Guney et al., 2007; Sener et al., 2003) or streptozotocin-induced diabetes (Bhor et al., 2004; Shirpoor et al., 2007). Additionally, different sensitivity of particular tissues could possibly result from different availability of ‘‘free iron’’ under certain conditions. Although our experiment was definitely not associated with acute stress, 8-day-injections could have presumably redistributed – to a certain extent – blood flow from the intestine. It is well documented that the intestine is susceptible to ischaemia, reperfusion (Halliwell et al., 2000) and also surgical stress (Prabhu et al., 2000) – conditions resulting in enhanced oxidative stress. Thus, the increase in LPO after FeSO4 administration, observed in the intestine but not in other tissues, may
indicate that the intestine is more prone to iron-induced oxidative stress under the condition of ischaemia. A similar effect was also observed in rats during exercising, in which the effect of iron excess on oxidative damage to lipids in the intestine was enhanced by physical exercise (Zunquin et al., 2006). In our study, the observation of the ‘‘lack’’ of GH or IGF-I influence on the basal LPO in almost all (except brain) rat tissues was very desirable. This finding remains in agreement with the assumption that under physiological conditions any perfect antioxidant should not change the basal level of oxidative damage. Additionally, in rat small intestine, both GH and IGF-I were able to protect against iron-induced oxidative damage; thus, they did appear to behave as excellent antioxidants. Also, other authors observed protective effects of GH against LPO in rat intestine.
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In rats with septic shock, induced by E. coli intra-abdominal infection, GH administration (0.225 IU/100 g b.w./day) showed beneficial effects in maintaining the integrity of intestinal mucosa barrier, diminishing bacteria and endotoxin translocation and also decreasing endotoxin level in plasma (Huang et al., 2002). Similarly, GH treatment (0.2 IU/100 g b.w./day) decreased LPO, expressed as the level of MDA, and reduced bacterial translocation in septic rat intestine (Jung et al., 2003). Unfortunately, the effects of GH or IGF-I, found by us in another rat tissue, in the brain, are – not as those in the intestine – undesirable. We, surprisingly, observed that GH did decrease, whereas IGF-I increased the basal LPO in rat brain. Those effects exclude GH and IGF-I from the list of perfect antioxidants. LPO-enhancing effect due to IGF-I seen in the brain is worth discussing more broadly. IGF-I has been found to pass the blood–brain barrier (Yu et al., 2006), which means that this compound may directly influence oxidative processes in the brain, with pro-oxidative effects when used in pharmacological doses (which concerns the present study). The divergent results of external IGF-I observed in our study, namely pro-oxidative in the brain and protective in the small intestine, are not surprising. This apparent divergence may potentially result from the possible low physiological IGF-I concentration in the brain and comparatively high IGF-I concentration in the small intestine. (However, this should be experimentally proved.) In agreement with the above observation, the prooxidative effects of IGF-I have been confirmed in several in vivo studies, performed both in animals and in humans. GH excess (obviously followed by IGF-I excess) is associated with increased oxidative stress and decreased activity of antioxidative enzymes in transgenic mice with GH overexpression (Hauck and Bartke, 2001; Andersson et al., 2006). Also, patients with active acromegaly and – at the same time – high basal values of GH and IGF-I – demonstrate increased LPO level, expressed as plasma level of MDA (Yarman et al., 2003). A question arises if either GH or IGF-I could be used as an external protective factor in case of disorders, other than GH deficiency, associated with increased oxidative stress. Our in vivo results do not support such a use of either of the two substances. Also, other results do not support such a recommendation. For example, GH treatment, administered in patients with critical illnesses, associated with increased oxidative stress, appeared to double mortality and worsen morbidity (Takala et al., 1999). Concerning IGF-I, there is currently unambiguous evidence for an increased risk of cancer, a condition always associated with elevated oxidative stress, in patients with high-normal IGF-I
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concentrations (Hankinson et al., 1998; Kurek et al., 2000). In conclusion, not all rat tissues are equally sensitive to iron-induced LPO, with the small intestine clearly revealing such a property. GH and IGF-I possess some ability to prevent iron-induced oxidative damage in iron-sensitive tissues, but contribute by themselves to oxidative imbalance in other tissues.
Acknowledgements This research was supported by a grant from the Ministry of Education of Poland (Project no. 2 P05B 098 30) and by a grant from the Medical University of Lodz (503-1107-5).
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Hsu D-Z, Chien S-P, Chen K-T, Liu M-Y. The effect of sesamol on systemic oxidative stress and hepatic dysfunction in acutely iron-intoxicated mice. Shock 2007;28:596–601. Hu ML, Frankel EN, Tappel AL. Effect of dietary menhaden oil and vitamin E on in vivo lipid peroxidation induced by iron. Lipids 1990;25:194–8. Huang Y, Wang SR, Yi Ch, Ying MY, Lin Y, Zhi MH. Effects of recombinant human growth hormone on rat septic shock with intraabdominal infection by E coli. World J Gastroenterol 2002;8:1134–7. Jung SE, Youn YK, Lim YS, Song HG, Rhee JE, Suh GJ. Combined administration of glutamine and growth hormone synergistically reduces bacterial translocation in sepsis. J Korean Med Sci 2003;18:17–22. Karbownik M, Lewinski A. Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue. J Cell Biochem 2003;90:806–11. Karbownik M, Reiter RJ, Garcia JJ, Tan D-X. Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: evidence for the involvement of iron. Int J Biochem Cell Biol 2000;32:1045–54. Karbownik M, Gitto E, Lewin´ski A, Reiter RJ. Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes. J Cell Biochem 2001;81:693–9. Karbownik-Lewinska M, Kokoszko A, Lewandowski KC, Shalet SM, Lewinski A. Growth hormone replacement reduces increased lipid peroxidation in growth hormonedeficient adults. Clin Endocrinol 2008;68:957–64. Kokoszko A, Karbownik M, Lewin´ski A. Increased lipid peroxidation in growth hormone-deficient adult patients. Neuroendocrinol Lett 2006;27:225–30. Kurek R, Tunn UW, Eckart O, Aumu¨ller G, Wong J, Renneberg H. The significance of serum levels of insulinlike growth factor-1 in patients with prostate cancer. BJU Int 2000;85:125–9. Mohn A, Di Marzio D, Giannini C, Capanna R, Marcovecchio M, Chiarelli F. Alterations in the oxidant–antioxidant status in prepubertal children with growth hormone deficiency: effect of growth hormone replacement therapy. Clin Endocrinol 2005;63:537–42.
Ozbey N, Telci A, Cakatay U, Yurci A, Molvalilar S. Determination of oxidative protein and lipid damage in adult hypopituitary patients with GH deficiency. J Endocrinol Invest 2003;26:1001–7. Prabhu R, Anup R, Balasubramanian KA. Surgical stress induces phospholipid degradation in the intestinal brush order membrane. J Surg Res 2000;94: 178–84. Scacchi M, Valassi E, Pincelli AI, Fatti LM, Pecori Giraldi F, Ascoli P, et al. Increased lipid peroxidation in adult GHdeficient patients: effects of short-term GH administration. J Endocrinol Invest 2006;29:899–904. Sener G, Jahovic N, Tosun O, Atasoy BM, Yegen BC. Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats. Life Sci 2003;74:563–72. Shirpoor A, Khadem Ansari MH, Salami S, Ghaderi Pakdel F, Rasmi Y. Effect of vitamin E on oxidative stress status in small intestine of diabetic rat. World J Gastroenterol 2007; 13:4340–4. Takala J, Ruokonen E, Webster NR, Nielsen MS, Zandstra DF, Vundelinckx G, et al. Increased mortality associated with growth hormone treatment in critically ill adults. N Engl J Med 1999;341:785–92. Valko M, Morris H, Cronin MTD. Metals, toxicity and oxidative stress. Curr Med Chem 2005;12:1161–208. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44–84. Yarman S, O¨zden TA, Go¨kkus- u C. The evaluation of lipid peroxidation and acute effect of octreotide on lipid peroxidation in patients with active acromegaly. Clin Chim Acta 2003;336:45–8. Yu Y, Kastin AJ, Pan W. Reciprocal interactions of insulin and insulin-like growth factor I in receptor-mediated transport across the blood–brain barrier. Endocrinology 2006;147:2611–5. Zunquin G, Rouleau V, Bouhallab S, Bureau F, Theunynck D, Rousselot P, et al. Iron and exercise induced alterations in antioxidant status, protection by dietary milk proteins. Free Radic Res 2006;40:535–42.
Experimental and Toxicologic Pathology 60 (2008) 453–458 www.elsevier.de/etp
Protective effects of GH and IGF-I against iron-induced lipid peroxidation in vivo Agnieszka Kokoszkoa,c, Jan Da˛ browskia,c, Andrzej Lewin´skia,c, Małgorzata Karbownik-Lewin´skab,c, a
Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, 281/289 Rzgowska Street, 93-338 Lodz, Poland b Department of Oncological Endocrinology, Medical University of Lodz, 7/9 Zeligowski Street, 90-752 Lodz, Poland c Polish Mother’s Memorial Hospital – Research Institute, 281/289 Rzgowska Street, 93-338 Lodz, Poland Received 27 February 2008; accepted 29 April 2008
Abstract Iron overload may enhance oxidative damage. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are involved in oxidative processes, lipid peroxidation (LPO) included. The aim of the study was to evaluate the in vivo effects of GH, IGF-I and/or iron on LPO in rat tissues. Male Wistar rats were administered iron (Fe2+; 3 mg/100 g b.w., i.p., on the 8th day) and/or GH (0.2 IU/100 g b.w.), and/or IGF-I (2 mg/100 g b.w.) once daily for 8 days. LPO products (malondialdehyde+4-hydroxyalkenals) were measured in rat brain, lung, small intestine, liver, kidney, testis, spleen and serum. Iron injection increased LPO only in the small intestine and that effect was completely prevented by either GH or IGF-I. In the brain, GH decreased, whereas IGF-I increased, the basal LPO. GH and IGF-I possess some ability to prevent iron-induced oxidative damage in iron sensitive tissues, but contribute to oxidative imbalance in other tissues. r 2008 Elsevier GmbH. All rights reserved. Keywords: Growth hormone (GH); Insulin-like growth factor-I (IGF-I); Iron; Oxidative damage; Lipid peroxidation (LPO)
Introduction Reactive oxygen species (ROS), free radicals included, are products of normal cellular metabolism. Overproduction of ROS results in enhanced oxidative stress and – in consequence – in increased damage to macromolecules, lipids included (Valko et al., 2007, Corresponding author at: Department of Oncological Endocrinology, Medical University of Lodz, 7/9 Zeligowski Street, 90-752 Lodz, Poland. Tel./fax: +48 42 271 13 43. E-mail address: [email protected] (M. Karbownik-Lewin´ska).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.04.012
2005). In turn, the products of oxidative damage to macromolecules may further enhance oxidative stress and damage all the components in the organism. Iron is a transition metal, which contributes to oxidative balance, although iron overload is associated with enhanced oxidative damage and cancer initiation. This occurs via different mechanisms. Among others, iron participates in the Fenton reaction (Fe2++ H2O2+H+-Fe3++dOH+H2O), the most basic reaction of oxidative stress, which is frequently used to in vitro induce oxidative damage to macromolecules, lipid peroxidation (LPO) included (Karbownik et al., 2001;
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Gitto et al., 2001). When the whole organism is exposed to iron, not only Fenton reaction but also more complex mechanisms are involved in oxidative effects, due to the action of this transition metal. Growth hormone (GH) is a polypeptide, released in a pulsatile manner, by the anterior pituitary. The growthpromoting and metabolic actions of GH are mediated mainly by the insulin-like growth factor-I (IGF-I). There is some evidence that both GH and IGF-I are involved in oxidative processes. In patients with active acromegaly, an increased level of plasma lipid peroxide (the index of oxidative damage to low-density lipoprotein cholesterol) was found, the abnormality being directly related to increased GH concentration (Yarman et al., 2003). At the same time, increased LPO was also observed in children (Mohn et al., 2005) and adults (Ozbey et al., 2003; Scacchi et al., 2006; Kokoszko et al., 2006; Karbownik-Lewinska et al., 2008) with GH deficiency, the change being partially reversed, due to GH replacement in the latter group (KarbownikLewinska et al., 2008). Under in vitro conditions, GH and/or IGF-I (used in different concentrations) were found to contribute to oxidative balance in the rat liver and in the porcine thyroid and, in case of induced oxidative stress, they may cause even pro-oxidative effects (Kokoszko et al., personal communication). The aim of the study was to evaluate the in vivo effect of GH, IGF-I and/or iron on LPO in different rat tissues.
Materials and methods The procedures, used in the study, were approved by the Ethical Committee of the Polish Mother’s Memorial Hospital – Research Institute.
Chemicals GH, isolated from porcine pituitary glands, the human recombinant IGF-I expressed in Escherichia coli, and ferrous sulphate (FeSO4), were purchased from Sigma-Aldrich (St. Louis, MO). The LPO-586 kit for LPO was purchased from Calbiochem (La Jolla, CA). Other chemicals were of analytical grade and came from commercial sources.
In vivo experiment A total of 52 male Wistar rats (weighing about 150 g each) were used in the study. The animals were randomized into six groups and administered: freshly prepared 0.9% NaCl (Control; n ¼ 9), or FeSO4 in freshly prepared 0.9% NaCl (3 mg Fe2+/100 g b.w. (Hemalatha et al., 2004), on the 8th day of the
experiment; n ¼ 9), or GH in freshly prepared 50 mM Tris–HCl buffer (pH 7.4) (0.2 IU/100 g b.w. (Jung et al., 2003), once daily, for 8 days; n ¼ 9), or IGF-I in freshly prepared Millipore H2O (2 mg/100 g b.w. (CastillaCortazar et al., 1997), once daily, for 8 days; n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8). The control rats, which did not receive either FeSO4 or GH or IGF-I, were treated with 0.9% NaCl at the abovementioned time points. All the rats received drinking water ad libitum. All the substances were administered i.p. in a volume of 0.5 ml/injection. The rats were killed by decapitation 24 h after FeSO4 injection. Brains, lungs, small intestines, livers, kidneys, testes and spleens were collected and frozen on solid CO2. Peripheral blood was collected, and centrifuged at 3000g (10 min, 4 1C) to obtain serum. The collected tissues and serum were stored at 70 1C until assay.
Measurement of lipid peroxidation products The concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA), as an index of LPO, were measured in the brain, lungs, small intestine, liver, kidneys, testes and spleen homogenates, as well as in blood serum. The tissue from each animal (approximately 100 mg) was homogenized in ice-cold 50 mM Tris–HCl buffer (pH 7.4, 10%, w/v – in the final incubation volume). The homogenates were centrifuged at 3000g for 10 min at 4 1C. The supernatant or blood serum was mixed with 650 ml of a methanol:acetonitrile (1:3, v/v) solution, containing a chromogenic reagent, N-methyl-2-phenylindole, and vortexed. After addition of 150 ml of methanesulphonic acid (15.4 M), the incubation was carried out at 45 1C for 40 min. The reaction between MDA+4-HDA and N-methyl-2-phenylindole yields a chromophore, which is spectrophotometrically measured at the absorbance of 586 nm, using a solution of 4-hydroxynonenal (10 mM) as standard. The level of LPO in tissue homogenates was expressed as the amount of MDA+4-HDA (nmol) per mg protein, or – in case of serum – as the amount of MDA+4-HDA (nmol) per 1 ml of serum.
Measurement of protein concentration Protein concentration was measured using the Bradford’s method (Bradford, 1976), with bovine albumin as standard.
Statistical analysis The data were statistically analysed, using one-way analysis of variance (ANOVA) followed by Student– Newman–Keuls’ test. Statistical significance was
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determined at the level of po0.05. The results are presented as means7SEM.
Results Among several examined tissues, only the small intestine and the brain appeared to be sensitive to oxidative effects of GH, or IGF-I, or iron. The i.p. administration of FeSO4 resulted in a significant (p ¼ 0.009) increase in the basal LPO only in the rat small intestine, and that effect was completely prevented by either GH (p ¼ 0.001) or IGF-I (p ¼ 0.0003) (Fig. 1). In the rat brain, GH significantly (p ¼ 0.034) decreased the basal LPO, whereas IGF-I, either used alone or together with iron, significantly (p ¼ 0.046; p ¼ 0.002, respectively) increased LPO level (Fig. 2). No changes of LPO were found in any other tissues, due to GH, or IGF-I, or FeSO4 treatment (Table 1).
Discussion Our study has been the first attempt to evaluate the in vivo effect of GH, or IGF-I, administered alone or together with iron on LPO in rat tissues. The selection of FeSO4, as a causative factor of oxidative damage, for such a study was justified by some facts. It had been confirmed in several in vitro
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experiments that ferrous ion (Fe2+) had the capacity to induce LPO in different tissues (in the liver – Karbownik et al., 2000; in the testes – Karbownik et al., 2001; in the thyroid gland – Karbownik and Lewinski, 2003); however, such an effect has never been examined in the small intestine. The pro-oxidative effect of iron was also observed under in vivo condition. The injection of ferrous chloride (2.4 mg Fe2+/100 g b.w.) (Hu et al., 1990) or ferrous sulphate (3 mg Fe2+/100 g b.w.) (Hemalatha et al., 2004) significantly increased the content of LPO products (measured as the concentration of thiobarbituric acid-reactive substances) in the rat liver. LPO induction in the rat liver, found in those two cited studies (Hemalatha et al., 2004; Hu et al., 1990), and the lack of such an effect in rat liver in the present study could result from the two main differences: different strain (weanling WNIN rats (Hemalatha et al., 2004; Hu et al., 1990) vs. Wistar rats in our study) and different body mass (35 g (Hemalatha et al., 2004; Hu et al., 1990) vs. 150 g in our study). It is worth mentioning that the administration of ferric ions, i.e. Fe3+ (as ferric nitrate – 0.2 mg/100 g b.w.), also enhanced LPO in liver and induced acute hepatic injury in mice (Hsu et al., 2007). In our study, we found a statistically significant increase in LPO, due to FeSO4 treatment, but, unexpectedly, only in one tissue, namely in rat small intestine. In other examined tissues, we did not observe iron-induced changes in the basal level of LPO. Those findings may indicate that, under in vivo conditions, the sensitivity of particular tissues to iron-induced oxidative
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Fig. 1. Concentrations of MDA+4-HDA in the small intestine, collected from the rats, administered 0.9% NaCl (Control; n ¼ 9), or FeSO4 (n ¼ 9), or GH (n ¼ 9), or IGF-I (n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8). Data are expressed as nmol of MDA+4-HDA per mg of protein. Bars represent the means7SEM. *po0.05 vs. Control; ~po0.05 vs. Fe.
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Fig. 2. Concentrations of MDA+4-HDA in the rat brain. The experimental protocol corresponds to that described in the legend to Fig. 1. Data are expressed as nmol of MDA+4-HDA per mg of protein. Bars represent the means7SEM. *po0.05 vs. Control.
Table 1. Concentrations of MDA+4-HDA (means7SEM) in tissues collected from the rats, administered 0.9% NaCl (Control; n ¼ 9), or FeSO4 (n ¼ 9), or GH (n ¼ 9), or IGF-I (n ¼ 8), or FeSO4+GH (n ¼ 9), or FeSO4+IGF-I (n ¼ 8) Tissues
Lung Spleen Liver Kidney Testis Serum
Groups Control
Fe
GH
IGF-I
Fe+GH
Fe+IGF-I
0.8970.03 2.1870.16 4.2470.52 2.2570.25 1.1470.27 22.0372.17
0.8770.06 2.2770.17 3.9970.42 2.1170.22 1.0070.15 22.7571.90
0.9470.06 2.2170.16 4.0170.23 1.8070.25 0.5770.08 17.1171.83
0.9470.04 1.6570.06 4.2470.65 2.3770.20 1.7070.77 17.8771.34
1.0470.11 2.0670.27 3.9970.33 1.9470.29 0.5970.07 20.1071.41
0.9870.07 2.4370.21 3.2070.32 2.2770.21 1.2470.36 24.4772.16
Data are expressed as nmol of MDA+4-HDA per mg of protein, or, in case of serum as nmol of MDA+4-HDA per 1 ml of serum (differences not significant).
damage demonstrates significant differences. It is well known that the small intestine is particularly vulnerable to several pro-oxidative agents, such as, for example, ionizing radiation (Guney et al., 2007; Sener et al., 2003) or streptozotocin-induced diabetes (Bhor et al., 2004; Shirpoor et al., 2007). Additionally, different sensitivity of particular tissues could possibly result from different availability of ‘‘free iron’’ under certain conditions. Although our experiment was definitely not associated with acute stress, 8-day-injections could have presumably redistributed – to a certain extent – blood flow from the intestine. It is well documented that the intestine is susceptible to ischaemia, reperfusion (Halliwell et al., 2000) and also surgical stress (Prabhu et al., 2000) – conditions resulting in enhanced oxidative stress. Thus, the increase in LPO after FeSO4 administration, observed in the intestine but not in other tissues, may
indicate that the intestine is more prone to iron-induced oxidative stress under the condition of ischaemia. A similar effect was also observed in rats during exercising, in which the effect of iron excess on oxidative damage to lipids in the intestine was enhanced by physical exercise (Zunquin et al., 2006). In our study, the observation of the ‘‘lack’’ of GH or IGF-I influence on the basal LPO in almost all (except brain) rat tissues was very desirable. This finding remains in agreement with the assumption that under physiological conditions any perfect antioxidant should not change the basal level of oxidative damage. Additionally, in rat small intestine, both GH and IGF-I were able to protect against iron-induced oxidative damage; thus, they did appear to behave as excellent antioxidants. Also, other authors observed protective effects of GH against LPO in rat intestine.
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In rats with septic shock, induced by E. coli intra-abdominal infection, GH administration (0.225 IU/100 g b.w./day) showed beneficial effects in maintaining the integrity of intestinal mucosa barrier, diminishing bacteria and endotoxin translocation and also decreasing endotoxin level in plasma (Huang et al., 2002). Similarly, GH treatment (0.2 IU/100 g b.w./day) decreased LPO, expressed as the level of MDA, and reduced bacterial translocation in septic rat intestine (Jung et al., 2003). Unfortunately, the effects of GH or IGF-I, found by us in another rat tissue, in the brain, are – not as those in the intestine – undesirable. We, surprisingly, observed that GH did decrease, whereas IGF-I increased the basal LPO in rat brain. Those effects exclude GH and IGF-I from the list of perfect antioxidants. LPO-enhancing effect due to IGF-I seen in the brain is worth discussing more broadly. IGF-I has been found to pass the blood–brain barrier (Yu et al., 2006), which means that this compound may directly influence oxidative processes in the brain, with pro-oxidative effects when used in pharmacological doses (which concerns the present study). The divergent results of external IGF-I observed in our study, namely pro-oxidative in the brain and protective in the small intestine, are not surprising. This apparent divergence may potentially result from the possible low physiological IGF-I concentration in the brain and comparatively high IGF-I concentration in the small intestine. (However, this should be experimentally proved.) In agreement with the above observation, the prooxidative effects of IGF-I have been confirmed in several in vivo studies, performed both in animals and in humans. GH excess (obviously followed by IGF-I excess) is associated with increased oxidative stress and decreased activity of antioxidative enzymes in transgenic mice with GH overexpression (Hauck and Bartke, 2001; Andersson et al., 2006). Also, patients with active acromegaly and – at the same time – high basal values of GH and IGF-I – demonstrate increased LPO level, expressed as plasma level of MDA (Yarman et al., 2003). A question arises if either GH or IGF-I could be used as an external protective factor in case of disorders, other than GH deficiency, associated with increased oxidative stress. Our in vivo results do not support such a use of either of the two substances. Also, other results do not support such a recommendation. For example, GH treatment, administered in patients with critical illnesses, associated with increased oxidative stress, appeared to double mortality and worsen morbidity (Takala et al., 1999). Concerning IGF-I, there is currently unambiguous evidence for an increased risk of cancer, a condition always associated with elevated oxidative stress, in patients with high-normal IGF-I
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concentrations (Hankinson et al., 1998; Kurek et al., 2000). In conclusion, not all rat tissues are equally sensitive to iron-induced LPO, with the small intestine clearly revealing such a property. GH and IGF-I possess some ability to prevent iron-induced oxidative damage in iron-sensitive tissues, but contribute by themselves to oxidative imbalance in other tissues.
Acknowledgements This research was supported by a grant from the Ministry of Education of Poland (Project no. 2 P05B 098 30) and by a grant from the Medical University of Lodz (503-1107-5).
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