Biometals (2014) 27:349–361 DOI 10.1007/s10534-014-9717-8

Iron toxicity mediated by oxidative stress enhances tissue damage in an animal model of diabetes Ana Fla´via S. Sampaio • Maisa Silva • Waleska C. Dornas • Daniela C. Costa • Marcelo E. Silva • Rinaldo C. dos Santos Wanderson G. de Lima • Maria Lu´cia Pedrosa



Received: 31 January 2014 / Accepted: 10 February 2014 / Published online: 19 February 2014 Ó Springer Science+Business Media New York 2014

Abstract Although iron is a first-line pro-oxidant that modulates clinical manifestations of various systemic diseases, including diabetes, the individual tissue damage generated by active oxidant insults has not been demonstrated in current animal models of diabetes. We tested the hypothesis that oxidative stress is involved in the severity of the tissues injury when iron supplementation is administered in a model of type 1 diabetes. Streptozotocin (Stz)-induced diabetic and non-diabetic Fischer rats were maintained with or without a treatment consisting of iron dextran ip at 0.1 mL day-1 doses administered for 4 days at intervals of 5 days. After

A. F. S. Sampaio Program of Health and Nutrition, Federal University of Ouro Preto, Ouro Preto, Minas Gerais, Brazil Present Address: M. Silva  W. C. Dornas  D. C. Costa  M. E. Silva  W. G. de Lima  M. L. Pedrosa (&) Research Center in Biological Sciences (NUPEB), Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro s/n, Ouro Preto, Minas Gerais 35400-000, Brazil e-mail: [email protected] W. C. Dornas e-mail: [email protected] D. C. Costa  W. G. de Lima  M. L. Pedrosa Department of Biological Sciences, Federal University of Ouro Preto, Ouro Preto, Minas Gerais, Brazil M. E. Silva  R. C. dos Santos Department of Foods, Federal University of Ouro Preto, Ouro Preto, Minas Gerais, Brazil

3 weeks, an extensive increase (p \ 0.001) in the production of reactive oxygen species (ROS) in neutrophils of the diabetic animals on iron overload was observed. Histological analysis revealed that this treatment also resulted in higher (p \ 0.05) tissue iron deposits, a higher (p \ 0.001) number of inflammatory cells in the pancreas, and apparent cardiac fibrosis, as shown by an increase (p \ 0.05) in type III collagen levels, which result in dysfunctional myocardial. Carbonyl protein modification, a marker of oxidative stress, was consistently higher (p \ 0.01) in the tissues of the iron-treated rats with diabetes. Moreover, a significant positive correlation was found between ROS production and iron pancreas stores (r = 0.42, p \ 0.04), iron heart stores (r = 0.54, p \ 0.04), and change of the carbonyl protein content in pancreas (r = 0.49, p \ 0.009), and heart (r = 0.48, p \ 0.02). A negative correlation was still found between ROS production and total glutathione content in pancreas (r = -0.50, p \ 0.03) and heart (r = -0.45, p \ 0.04). In conclusion, our results suggest that amplified toxicity in pancreatic and cardiac tissues in rats with diabetes on iron overload might be attributed to increased oxidative stress. Keywords Diabetes  Heart  Iron  Pancreas  Reactive oxygen species Abbreviatons AGEs Advanced glycation end products ANOVA Analysis of variance AOAC Association of official analytical chemists

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BSA C CAT CI DM D DI DNPH DPI DTNB GSH-Px GSSG GSH H2O2 LIBC OHO 2PB PPAR-a RLU/min ROS Stz SOD TCA TIBC TNB ZC3b

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Bovine serum albumin Control group Catalase Control iron group Diabetes mellitus Diabetic group Diabetic iron group 2,4-Dinitrophenylhydrazine Diphenylene iodonium 5,5-Dithiobis(2-nitrobenzoic acid) Glutathione peroxidase Oxidized glutathione Reduced glutathione Hydrogen peroxide Latent iron-binding capacity Hydroxyl radicals Superoxide anions Pearl’s Prussian blue Peroxisome proliferator activated receptor-a Relative units of light/min Reactive oxygen species Streptozotocin Superoxide dismutase Trichloroacetic acid Total iron-binding capacity 5-Thio-2-nitrobenzoic acid Zymosan

Introduction Iron deficiency has been considered an important risk factor for illness (WHO 2002) and it is estimated to affect 2 billion people in the world (Stoltzfus and Dreyfuss 1998). Concerns have been raised about the effects of iron deficiency on health, which have led to recommendations for supplementation in populations with a high prevalence of anemia (Stoltzfus and Dreyfuss 1998). Diabetic patients who due to neglecting or ignorance, do not follow the appropriate dietary regimes, are at-risk of developing nutritional anaemia, especially on iron deficiency. Moreover, diabetics persons with poor glycaemic control are susceptible to recurrent attacks of ketoacidosis which may be accompanied by anorexia, severe returning with frequent hospitalization and excessive calorie losing (Dikow et al. 2002).

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Chronic hyperglycemia, a common feature of all forms of diabetes, involves the overproduction of reactive oxygen species (ROS) such as superoxide anions (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-), contributing to the auto-oxidation of glucose, shifts in redox balances, decreased tissue concentrations of reduced glutathione (GSH), and impaired activities of antioxidant enzymes (Ro¨sen et al. 2001). In particular, the balance between ROS production and the antioxidant defence systems determines the degree of oxidative stress once free radical reactions are essential for the host defence mechanisms of cells of the immune system; however, if free radicals are overproduced, they can cause tissue injury (Halliwell et al. 1992). It has been proposed that increased production of O2- may be the unifying mechanism by which hyperglycemia exerts its role in producing tissue damage, via its dismutation to produce H2O2, which in the presence of redox active transition metals, such as iron, produces OH(Thomas et al. 2009). Increasing scientific evidence suggests a link between glucose and iron metabolism and that this relationship is influenced by oxidative stress (Ferna´ndez-Real et al. 2002; Cooksey et al. 2004). Conversely, despite a considerable amount of data indicating that an increase of both O2- and iron content in tissue leads to increased OH- formation, the pro-oxidant effects of iron on the tissues of diabetic animal models have not been well documented. We have previously identified significantly higher oxidative stress in diabetic animals indicated by increasing carbonyl protein levels and decreasing antioxidant enzyme activity, and, unexpectedly iron supplementation altered the redox balance hepatic in favour of increased antioxidant levels (Silva et al. 2011). Additionally, peroxisome proliferator activated receptor-a (PPAR-a) was differentially expressed in diabetic and iron-supplemented hamsters and promoted different effects on the liver. Higher expression of PPAR-a and lower oxidative stress played an important role in hepatic integrity with histological data demonstrating that iron supplementation in the model under diabetic conditions caused less damage to the liver. However, extra-hepatic tissues have different kinetics of iron uptake and clearance compared to the liver (Noetzli et al. 2008) because they selectively, or near selectively, load circulating non-transferrinbound iron (Oudit et al. 2006).

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Iron overload is associated with progressive iron deposition in a variety of tissues, including the endocrine organs and heart. The toxic effects of iron overload, involving cellular apoptosis or necrosis in the heart, pancreas, and other organs are owing to the formation of highly reactive free radicals implicated in the pathogenesis of these lesions (Whittaker et al. 1996). Additionally, the pancreas appears to load iron earlier than the heart, providing an early marker of inadequate chelation regimens, which suggests that the pancreas leads the heart in iron loading (Noetzli et al. 2009). Myocardial cell death, hypertrophy, and fibrosis are the most frequently proposed mechanisms to explain cardiac changes, and increased ROS production is suspected to be involved in diabetic cardiomiopathy (Watanabe et al. 2010). In fact, the interaction between iron and diabetes can produce differential effects on different tissues under persistent oxidative stress due to the overproduction of ROS (Nishikawa and Araki 2007). It is possible that increased oxidative stress in diabetes involves elevated O2-, which is readily converted to more reactive species such as OHthrough the actions of superoxide dismutase (SOD) and transition metal-catalyzed Fenton reactions, when iron overload is present in diabetic rats. Thus, the central importance of iron in the pathophysiology of disease is derived from the ease with which iron is reversibly oxidized and reduced. This property, while essential for its metabolic functions, makes iron potentially hazardous because of its ability to participate in the generation of powerful oxidant species. Therefore, to explore the idea that iron excess might contribute to the production and intensify the extrahepatic tissue injury caused by free radicals in experimental diabetes, the present study investigated the effect of iron overload on hyperglycemia, which can play a role in the development and progression of diabetes with pancreatic and cardiac damage associated with enhanced generation of ROS.

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acid) (DTNB), and iron dextran were purchased from Sigma-Aldrich. Leukopaque and Monopaque gradients were obtained from Bion LTDA. Animals and treatments

Materials and methods

Male Fischer rats weighing on average 200 g (8 week old, obtained from the Experimental Nutrition Laboratory of the Federal University of Ouro Preto) were housed in plastic cages with controlled temperature and light-regulated space with 12-h light-and dark cycle. The rats were fed on free access to water and the formulated AIN-93 M diet and divided into four experimental groups (n = 7–8) as follows: the control group (C), the control group, which received iron dextran (CI), the diabetic group (D), and the diabetic group which received iron dextran (DI). Diabetes was induced with a single ip injection of streptozotocin (Stz), 35 mg kg-1 body weight in citrate buffer and the control animals were randomly assigned to a nondiabetic group that was injected with vehicle (sodium citrate buffer). Three days after the Stz injection, blood glucose level was measured by using a portable glucometer Accu-chek (Roche, Hvidovre, Denmark) in the blood collected from tail vein. Animals with glucose levels above 250 mg dL-1 were considered as diabetic. The injections ip of iron dextran, 100 g L-1, were given during week 3 at a total dose of 40 mg, divided into 0.1 mL day-1 doses for 4 days at intervals of 5 days. Control rats were administered 0.1 mL of sterile saline. Rats were killed 5 days later once part of the iron is transferred to transferrin and part is removed from circulation within 48 h, thus increasing tissue stores (Holbein 1980). At the end of all experiments, fasting rats were anesthetized using isoflurane and euthanized. Liver, pancreas and the heart were removed, weighed and stored either in liquid nitrogen or buffered formaldehyde for biochemical and histopathological analysis, respectively. All procedures were reviewed and approved by the Ethical Committee of Ouro Preto University, Ouro Preto, MG, Brazil, with protocol number 2010/24.

Chemicals

Metabolic variables

Streptozotocin (2-deoxy-2-{[methyl(nitroso)amino] carbonyl}amino-b-D-glycopyranose), luminol (5-amino-2, 3-dihydro-1,4-phthalazinedione), diphenylene iodonium (DPI), Zymosan (ZC3b), 5,5-dithiobis(2-nitrobenzoic

Blood samples were collected and centrifuged for determination of plasma and serum components. Glucose and fructosamine concentrations, serum iron, total iron-binding capacity (TIBC), and latent iron-

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binding capacity (LIBC) were determined by spectrophotometric analysis using commercial kits from Labtest Diagnostica SA kits (Lagoa Santa MG, Brazil). Serum concentrations of insulin were measured with the Ultra-Sensitive Rat Insulin Elisa Kit (Crystal Chem, Downers Grove, IL). Tissue samples: 100 mg, were digested in 2 mL of nitric acid at 100 °C. Excess acid was evaporated and iron levels were quantified by colorimetric analysis according to the Association of Official Analytical Chemists (AOAC), (1980) using the orthophenanthroline assay with an iron solution of 500 lg dL-1 as an external iron standard. Transferrin saturation was determined using the formula:  Serum iron lmol L 1 100%  1 TIBC ROS production ROS production was measured by luminol-amplified chemiluminescence, as described previously (Chaves et al. 2000). Briefly, 1 9 106 neutrophils were placed in Hank’s solution, pH 7.4, with 500 ll of luminol (10-4 M). Neutrophils were isolated using two different density gradients, Monopaque (d = 1.08) and Leucopaque (d = 1.12), in accordance with the procedures described by Bicalho et al. (1981). Cell viability of each sample was greater than 95 % as determined by the exclusion test with trypan blue. Photon emission was determined each minute for 30 min using a luminometer (Lumat, LB 9507, Berthold, Germany). Values were expressed as relative units of light/min (RLU/min).

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protein. The content of DNPH incorporated was calculated using the molar absorption coefficient of DNPH (22,000 M-1 cm-1) and expressed as nmol mg-1 of protein. The amount of tissue total proteins was determined by method described by Lowry et al. (1951) using bovine serum albumin (BSA) as a standard. Antioxidant defences Catalase (CAT) activity was measured by the Aebi (1984) method. H2O2 decomposition was calculated using the molar absorption coefficient 39.4 M-1 cm-1. Results were expressed as activity per milligram of protein. One unit of CAT is equivalent to the hydrolysis of 1 lmol of H2O2 per min. Initial reaction rate was measured from the decreasing decomposition of H2O2 observed spectrophotometrically at 240 nm for 5 min. Total glutathione content of homogenates was determined using a kit (CS0260) from Sigma (St. Louis, MO). This assay uses a kinetic method based on the reduction of DTNB (5,5’-dithiobis-(2-nitrobenzoic acid) to TNB (5-thio-2-nitrobenzoic acid), which can be determined spectrophotometrically at 412 nm. Sigma G4251 GSH was used as a standard. The activity of SOD was assayed by the spectrophotometric method of Marklund and Marklund (1974) using an improved pyrogallol autoxidation inhibition assay. SOD reacts with the O2- and this slows down the rate of formation of o-hydroxy-o-benzoquinone and other polymer products. One unit of SOD is defined as the amount of enzyme that reduces the rate of autoxidation of pyrogallol by 50 %.

Carbonyl protein determination Tissue preparation Determination of carbonyl content in oxidatively modified protein was performed according to the method described by Levine et al. (1994). Each sample was precipitated with 10 % (w/v) trichloroacetic acid (TCA). After centrifugation, the precipitate was treated with 10 mmol of 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl, incubated in the dark for 30 min at room temperate and then treated with 10 % TCA. After centrifuging, the precipitate was washed twice with ethanol/ethyl acetate (1:1) and dissolved in 1 ml of SDS 6 % followed by centrifugation. Absorbance in the supernatant was determined at 370 nm. Results were expressed in nmol of DNPH incorporated/mg of

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For histological light microscopy, pancreas and the heart of the rats were immediately excised and tissues were fixed in a solution of neutral buffered 10 % formalin. Sections of tissues were dehydrated through increasing concentrations of ethanol, and the fixed sections were embedded in paraffin. The paraffin blocks were sectioned (4 mm) and the sections were placed on albumin-coated slides, deparaffinized, hydrated through graded xylene and alcohol, processed, and stained with Perls’ Prussian blue method for iron pigments, Hematoxylin-Eosin, and Picrosirius solution (0.1 % solution of Sirius Red F3BA in

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saturated aqueous picric acid) to calculate the collagen content. Sections were then cleared and mounted and stained sections from each rat were used for morphometric analysis with Leica DM5000 optical microscope and Leica analysis software Qwin Plus. Samples on Sirius Red staining were also observed under polarized light to quantify as the collagen area. Statistical analysis Statistical evaluation of data was performed using the statistical program GraphPad Prism 5 for Windows (San Diego, CA, USA). After testing for normal distribution using the Kolmogorov–Smirnov test, descriptive statistics were carried out. Data were analyzed using univariate analysis of variance (one-way ANOVA) followed by Bonferroni post hoc test. Data in which the distributions were not considered normal were subjected to the non-parametric Kruskal–Wallis test followed by Dunns post hoc test. Differences between means to histological analysis of pancreas and the heart were evaluated using the unpaired t test. Correlation calculations were done using the Pearson correlation coefficient. A p value \ 0.05 was considered statistically significant.

Results Diabetes caused failure in body weight gain which led to a lower (p \ 0.001) final body weight in D and DI than in C and CI rats at the end of the 3-week experimental period. An increase (p \ 0.05) in the blood glucose level in DI rats was shown. This confirmed that the injection of Stz in normal rats significantly elevated blood glucose levels as compared with rats that were injected with citrate buffer alone, although only the DI group had significantly higher glucose levels than C and CI groups. In addition, insulin level decreased (p \ 0.001) and fructosamine concentrations were elevate (p \ 0.001) in diabetic rats as compared to control groups. No statistical differences were detected in serum iron, transferrin saturation and TIBC among groups, whilst tissues iron stores were higher (p \ 0.05) in groups CI and DI than in their respective controls. LIBC was significantly lower (p \ 0.01) in DI group as compared to C and D groups (Table 1).

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Histological sections were stained by Perl’s Prussian blue method for the observation of ‘‘hemosiderin granules’’. Pearl’s Prussian blue (PB) staining made Fe3? apparent. PB positive iron was present in the form of typical cytoplasmic granules in the cytoplasm of pancreatic cells. The pancreas of DI rats showed marked (p \ 0.01) iron deposits in relation to CI rats with granular iron found along the pancreatic acinis exocrine cells. Pancreatic macrophages were also stained, principally in connective tissues. In heart, heavy granular iron deposits were found in parenchymal cells with higher (p \ 0.05) area occupied by iron deposits in the heart of diabetic than control rats treated with iron dextran. Perl’s staining of cardiac sections was used to expand on these findings and demonstrated that iron was deposited both within and outside cardiomyocytes of the iron-overloaded heart (Fig. 1). Alterations in the pancreas of Stz-induced diabetic rats evidenced with Hematoxylin-Eosin staining apparent infiltration of lymphocytes or macrophages in the pancreas of control or diabetic iron dextran treated rats, suggesting an inflammatory response with observable higher (p \ 0.01) number of cells in the DI group. Diabetic cardiac muscle fibers were disordered and many of them were collapsed and focal coalescent areas of ischemic myocyte degeneration in the subendocardial, subepicardial region and papillary muscles of the myocardium were seen. Correspondingly, the two diabetic groups showed higher (p \ 0.05) number of inflammatory cells than control groups (Fig. 2). Histochemical analysis of fibrosis development was performed by Sirius-Red staining. Pancreatic fibrosis no occurs in diabetic animals or on iron treatment, but, revealed fibrosis in different heart regions, and was mainly replacement collagen. A typical appearance of the connective tissue staining demonstrated intense fibrosis development in the diabetic hearts and a quantitative analysis of the sections by imaging showed a statistically significant (p \ 0.05) higher fibrotic areas to D and DI group when compared to control rats (Fig. 3). Figure 4 shows the groups stained with Sirius-Red with polarization. The photomicrographs presented to observe collagen fibers in the heart demonstrated that type I collagen dominated in all animals for each of the study groups with no differences detected between any of the groups. Analysis of the type III collagen content demonstrated that the DI group had more (p \ 0.02) type III

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Table 1 Body weight, glucose, insulin, fructosamine and serum and tissue iron variables in experimental rats Control

Diabetic

C

CI

252.5 ± 5.9a

Body weight (g) -1

Glucose (mmol L )

6.2 ± 0.9

Insulin (pmol L-1)

251.2 ± 29.8a

b

b

7.3 ± 0.4

28.0 ± 10.1a -1

Fructosamine (mmol L ) -1

Serum iron (lg dL ) Hepatic iron (mg/100 mg tissue)

3.5 ± 0.4

b

220.0 ± 52.2 0.07 ± 0.02b b

Pancreas iron (mg/100 mg tissue)

0.029 ± 0.02

Heart iron (mg/100 mg tissue)

0.088 ± 0.006b

Transferrin saturation (%)

D

DI

169.2 ± 36.0b 10.3 ± 2.7

ab

169.5 ± 27.9b 13.9 ± 7.8a

20.8 ± 8.4a

8.5 ± 5.0b

6.2 ± 1.3b

b

3.9 ± 0.4

7.0 ± 0.5

a

6.8 ± 0.5a

229.8 ± 40.2

222.1 ± 56.6

262.6 ± 81.8

1.02 ± 0.21a ab

0.07 ± 0.04b

1.09 ± 0.15a

b

0.150 ± 0.08a

0.104 ± 0.09

0.010 ± 0.01

0.134 ± 0.026a

0.084 ± 0.036b

0.150 ± 0.02a

65.9 ± 7.2

72.9 ± 6.8

63.1 ± 7.8

75.2 ± 8.9

TIBC (lg dL-1)

323.7 ± 50.1

325.9 ± 51.6

348.1 ± 47.9

345.9 ± 83.6

LIBC (lg dL-1)

113.3 ± 12.3a

85.0 ± 11.3ab

123.5 ± 17.0a

83.3 ± 25.6b

Values are mean ± SD. Control group (C), control iron group (CI), diabetic group (D), and diabeticiron group (DI). Statistical differences are shown by different superscript letters. Total iron-binding capacity (TIBC); latent iron-binding capacity (LIBC)

Fig. 1 a Representative photomicrographs of pancreas (a, b) and heart (d, e) sections, and area occupied by iron deposits in pancreas (c) and the heart (f) of control and diabetic rats and control rats treated with iron dextran of experimental rats. Perl’s

staining. Control iron group (CI); diabetic iron group (DI). Note the presence of iron deposits in the pancreas and heart of control and diabetic rats treated with iron dextran (black arrows). Magnification 9440. * p \ 0.05

collagen than control group, which suggest changes in the collagen fiber organization. Iron overload in Stz-induced diabetes showed that neutrophils isolated from peripheral blood produce significantly (p \ 0.001) more ROS than the other groups. Carbonyl protein, which derives from the

glycation of proteins, especially under chronic hyperglycemia, and is implicated in tissue damage in vascular complications of diabetes, was demonstrated in pancreas and in the heart of experimental rats. Group DI showed a significant (p \ 0.01) increase of carbonyl protein content in tissues as compared to the

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Fig. 2 a Representative photomicrographs of pancreas (a, b, c, d) and heart (f, g, h, i) sections, and number of cells in the pancreas and heart (e, j, respectively) of experimental rats. H&E staining. Control group (C); Control iron group (CI); diabetic

group (D), and diabetic iron group (DI). Magnification 9440. * p \ 0.05 vs. C and # p \ 0.05 vs. D group. (dotted lines) limit of cell number of normal pancreatic tissue compared in C group

control group. It has not observed differences among glutathione levels, SOD, and CAT activities in tissues in the evaluating antioxidant system components, although CAT activity in pancreas tended to be lower in group DI than in control group, but it had just failed to reach statistical significance (Table 2). In the Fig. 5, linear regression analysis in the whole groups showed that ROS production was positively correlated with iron stores in pancreas and the heart (r = 0.42, p \ 0.04; r = 0.54, p \ 0.04, respectively), and carbonyl protein content in pancreas and the heart (r = 0.49, p \ 0.009; r = 0.48, p \ 0.02,

respectively). An association among ROS was still significant in relation to total glutatione measure in tissues. In particular, ROS correlated negatively with total glutathione pancreas and the heart (r = -0.50, p \ 0.03; r = -0.45, p \ 0.04, respectively).

Discussion In this study, we demonstrated that iron overloading caused the accumulation of granular iron deposits in pancreatic and cardiac tissues. In addition, we showed

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Fig. 3 Representative photomicrographs of pancreas (a, b, c, d) and heart (f, g, h, i), and area occupied by collagen in the pancreas and heart (f, j, respectively) of experimental rats. Control group (C); control iron group (CI); diabetic group (D); and diabetic iron group (DI). Note the presence of connective

tissue control and diabetic group rats treated with iron dextran. Magnification 9440. * p \ 0.05 vs. C group. (dotted lines) normal range between the filing of connective tissue than in group C, in the heart. * p \ 0.05 vs. C group

that high blood glucose level caused by defects in insulin production was associated with greater oxidative injury in diabetic rats on iron supplementation. Iron stores with higher ROS production was consistent with previous studies that show that the suggestion that redox-active iron may play a role in catalyzing OH- production from elevated O2-, which results in higher tissue damage in diabetic rats. We used an Stz-induced type 1 diabetic model and the pathophysiology of the Stz-induced rats was similar to that of animal models used in previous

studies (Junod et al. 1982; Porte and Schwartz 1996; Dong et al. 2002; Erejuwa et al. 2011). Stz-injected rats showed elevated plasma glucose and serum fructosamine levels, low serum insulin levels, and weight loss, which are indicative of diabetes mellitus (DM). The Stz-induced diabetic rats induces a hyperglycaemia with diminished insulin production, and large amounts of iron in the regulation of adequate serum iron levels lead to iron overload in liver diabetic. In DI rats, the LIBC was lower than that in controls, in accordance with a previous study which

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Fig. 4 Representative photomicrographs of quantitative collagen distribution in the heart (a, b, c, d), and box plot of the area occupied by type I collagen (e) and type II collagen (f) of experimental rats. Picrosirius-polarization method. Control

group (C); control iron group (CI); diabetic group (D); and diabetic iron group (DI). * Significantly different levels as calculated by post hoc Dunn’s analysis

Table 2 Reactive oxygen species, carbonyl protein, and antioxidant system components in experimental rats Control

ROS (RLU/30 min)

Diabetic

C

CI

D

DI

191.3 ± 41.1

363.3 ± 130.2

552.8 ± 103.3

1566.0 ± 386.9*#

14.2 ± 6.4

27.6 ± 10.5

30.9 ± 8.2

52.3 ± 12.3*

Pancreas Carbonyl protein (nmol mg-1ptn) -1

Glutathione total (nmol L )

0.09 ± 0.06

0.08 ± 0.02

0.06 ± 0.05

0.04 ± 0.03

SOD (% inhibition)

0.28 ± 0.04

0.29 ± 0.04

0.31 ± 0.04

0.28 ± 0.09

CAT (U mg-1 ptn)

3.6 ± 2.1

3.0 ± 1.1

2.0 ± 1.5

1.8 ± 0.6

16.6 ± 3.8

18.2 ± 2.7

19.0 ± 0.7

28.8 ± 7.6*

Glutathione total (nmol L-1)

56.8 ± 4.3

59.0 ± 2.9

62.7 ± 6.9

59.8 ± 5.0

SOD (% inhibition)

0.34 ± 0.05

0.35 ± 0.04

0.30 ± 0.03

0.30 ± 0.04

CAT (U mg-1 ptn)

12.2 ± 1.8

11.5 ± 2.7

9.4 ± 3.8

10.7 ± 4.0

Heart Carbonyl protein (nmol mg-1 ptn)

Values are mean ± SD. Control group (C), control iron group (CI), diabetic group (D), and diabetic iron group (DI). * p \ 0.05 vs. C. Catalase (CAT), reactive oxygen species (ROS), relative light unit (RLU), superoxide dismutase (SOD)

showed that iron dextran disappears from the circulation within 48 h and that it is distributed in various tissues because released iron can return to circulation bound to transferrin (Holbein 1980). Our results indicate that iron was released slowly into the circulatory system of DI rats. Lower LIBC leads to significant iron deposition in the liver without prior appearance in the systemic circulation. As proposed by Fawwaz et al. (1967), low plasma LIBC levels

hinder iron binding to transferrin or cause iron and transferrin to bind abnormally; iron is subsequently transported to the liver. Perl staining of tissue segments revealed Fe3? deposits in both control and Stz-diabetic rats, although the deposits were more prevalent in Stz rats than in controls. The staining pattern appeared to be lumenrelated and non-uniform, indicating that macrophageassociated hemosiderin may be involved. We

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Fig. 5 Correlations between reactive oxygen species and organ weight, iron stores, carbonyl protein, and total glutathione content of experimental animals. The Pearson r value was

calculated and a positive or negative correlation is statistically significant when p \ 0.05. Superoxide dismutase (SOD); catalase (CAT) and reactive oxygen species (ROS)

hypothesized that the elevated iron content in diabetes may contribute to tissue injury progression by increasing oxidative stress in tissue. Histological data of diabetic rat tissue with iron overload showed inflammation. Unlike acinar cells, which are only slightly affected, islets modifications indicate that the b-cell nuclei undergo karyolysis, the cytoplasm components disintegrate, the cell boundaries disappear, and a mass of debris containing nuclei fragments may appear during the necrotic process. These histological changes may be caused by cellular injury incurred by increased ROS production demonstrated in this study. Iron-administered diabetic animals showed changes in heart morphology and developed cardiac fibrosis, which was connected with chronic inflammatory processes. DI and D groups showed an increased number of inflammatory cells and a greater extent of fibrosis. Extracellular matrix (ECM) components, notably fibrillar collagens of types I and III, are essential for tissue architecture and cardiac function (Brower et al., 2006) and an increase in type III collagen level in myocardium was shown in our study. Under pathological conditions, distinct cells

designated myofibroblasts participate in collagen turnover. Both enhanced deposition of interstitial collagen and degradation of endomysial and permysial collagen contribute to cardiac dysfunction. Weber et al. (1988) reported an early but transient increase in type III collagen relative to type I in hypertensive primates, which corresponded to an increase in myocardial stiffness. In addition to alterations in the collagen types, the causes of tissue injury after iron overload are not fully understood. However, earlier studies (Whittaker et al. 1996; Oudit et al. 2004) have shown that iron overload is associated with increases in apoptosis and the development of fibrosis. Elevated ROS levels have been proposed to be an initiating event in diabetes development. The immune system is especially vulnerable to oxidative damage because several immune cells produce ROS (Marin et al. 2011). Rossoni-Ju´nior et al. (2012) showed that ROS are excessively produced by activated neutrophils in diabetes and they may damage essential cellular components. Our data showed that poorly controlled diabetes leads to hyperglycaemia, which induces an exacerbated inflammatory response and

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cardiac damage in animals when administered iron supplementation. Diabetes increases free radical production, causes organ failure, induces oxidation of glucose and causes non-enzymatic and progressive glycation of proteins that may lead to elevated levels of glucose-derived advanced glycation end products (AGEs) (Vlassara and Palace 2002). Our data indicate that serum albumin glycation (fructosamine) and protein oxidation in the pancreas and heart was significantly increased in DI rats in the tissues. Protein carbonyls are routinely used as a biomarker for protein damage caused by oxidized amino acid residues in stress conditions, and we have shown that there is an increase in glycoxidation in DM and other diabetic animals (Guerra et al. 2011 and Silva et al. 2011). In addition, a significant link between glycoxidation and ROS was found. Therefore, we speculate that increase in iron stores and tissue iron deposits may play a role that redox-active iron within the tissue catalyzing OH- production from elevated O2- and induce accelerated tissue damage in diabetics. In the presence of redox active transition metals, such as iron, occurs OH- production that acts locally and elicits tissue damage (Lipinski 2011). Furthermore, tissue OHradical production depends on the free iron availability and on H2O2 production, primarily via dismutation of O2-. The increase of both O2- and iron content in the organs may lead to increased OH- formation. SOD and CAT significantly decrease OH- production (Sies 1997). However, an oxidative environment is created in cells by impairment of endogenous antioxidant defences. During the oxidative stress that occurs under diabetic conditions, endogenous antioxidant defence systems not provided sufficient protection by minimizing cellular oxidation; as demonstrated by no change in the results for SOD and CAT activities for the different experimental groups. Under normal physiological conditions, antioxidant enzymes minimize cellular oxidations due to ROS. SOD converts O2- to H2O2 and enhanced SOD activity may lead to increased turnover of H2O2. Furthermore, CAT has been reported to be highly susceptible to increased O2(Kono and Fridovich 1982) and in normal cells, H2O2 is further metabolized to H2O and O2 by CAT. Nonetheless, in the event of increased H2O2 generation, CAT may be unable to sufficiently scavenge the high levels of H2O2. On the other hand, a decrease in the glutathione peroxidase (GSH-Px) activity may also result in

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increased production of OH- (Gutteridge 1994). As GSH is a cofactor of GSH-Px, it is important to note that total glutathione concentration was slightly lower in the pancreas of DI rats than that in the control group. Thus, the involvement of iron in diabetes pathophysiology can indicate that there may be higher oxidation of GSH to the reduction of H2O2. Glutathione, an important intracellular free radical scavenger and co-substrate for several enzymes, plays a prominent role in the degradation of H2O2 undergoing oxidation from its reduced form to an oxidized state (GSSG) (Johnson et al. 2012). Low GSH levels often occur in diabetic patients with aggravated complications (Thornalley et al. 1996), and in this study, ROS levels of rats showed a negative correlation with the total glutathione content of pancreas and the heart. Thus, in diabetic rats, tissues with heavy iron stores may have lower antioxidant defences due to an increase in ROS production. Consequently, a reduced antioxidant defences can be significant, given which have suggested that in the situation in which H2O2 is presented at a high concentration, antioxidant defence, offer an ineffective protection against the toxicity of extracellular. In conclusion, our data demonstrate that iron supplementation in diabetes involves greater oxidative stress resulting in severe pancreatic and heart damage. Furthermore, this study revealed that iron stores could produce morphological changes in a relatively short period of time and that the pancreas and heart tissues are particularly susceptible to an imbalance of oxidants/antioxidants in favor of oxidants contributing to the aggravation of diabetes. Acknowledgments This work received financial support from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais, Universidade Federal de Ouro Preto and research fellowships from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (Pedrosa, M. L., Silva, M. E.) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (Sampaio, A. F. S., Silva, M., Dornas, W.C.).

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Iron toxicity mediated by oxidative stress enhances tissue damage in an animal model of diabetes.

Although iron is a first-line pro-oxidant that modulates clinical manifestations of various systemic diseases, including diabetes, the individual tiss...
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