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Curcumin Protects Liver and Spleen from Oxidative Stress induced by

Excessive Dietary Iron Intake in a Rat Model of Nutritional Iron

Overload

Farid A. Badria1*, Ahmed S Ibrahim2 , and Adel F. Badria3

35516, and 3Medical Technology Center, Alexandria University, Egypt

*Corresponding authors: Farid A Badria, PhD, Pharmacognosy Department, Mansoura University,

Mansoura 35516 Egypt, email: faridbadria@yahoo.com;

Running Title: Curcumin As Anti-Oxidative Stress In Iron overload-Induced Toxicity.

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Pharmacognosy Department, 2Department of Biochemistry, Faculty of Pharmacy, Mansoura University

Keywords: Curcumin, Oxidative Stress, iron overload, liver toxicity, spleen toxicity.

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ABASTRACT

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Background: Iron overload (IO) is now being recognized as a health problem in industrialized countries

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where cultural practices encourage acquisition of excess iron from diets. Excess iron is highly toxic for

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vital organs especially liver and spleen. Curcumin, a superb nature's medicine with still unfolding health

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benefits, has been shown to possess iron chelation property that has not been studied in nutritional IO.

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Objective: A three-year field study was conducted by our team on the impact of environmental excess

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iron in drinking water and its health hazard on liver diseases. Here, we evaluate the efficacy of curcumin

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as nutritional supplement to alleviate the hepatospleno-disorders and their sequelae on IO-induced rats.

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Design/Results: Initial studies revealed that the treatment of iron-overloaded rats with curcumin

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resulted in marked decreases in iron accumulation within liver/spleen. To further assess the role of

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curcumin, we studied its effects on iron overload-induced oxidative stress, protein loss, and anti-oxidant

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depletion. Iron-overloaded rats had significantly more TBARS, NO and OH radicals in liver and spleen

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compared with control group. These effects of IO were all reduced by intervention treatment with

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curcumin. Following this further, the bulk protein content in liver and spleen as well as their endogenous

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anti-oxidants (SOD, CAT, GSH, and ASA) were highly significantly decreased in chronic iron

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intoxication. Inversely, the administration of curcumin improved the alterations in these biochemical

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parameters.

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Conclusions: These findings implicate the therapeutic potential of the multifunctional, nutritional iron

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chelator, curcumin, in dampening IO-induced hepatosplenic disorders. This beneficial effect of

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curcumin represents a new horizon in managing nutritional iron overload.

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INTRODUCTION

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Iron, one of the most abundant metals on earth, is an integral part of many proteins and enzymes that

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maintain good health (1). However, excess amounts of iron has a destructive nature if not properly

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diagnosed and treated (2). Undiagnosed iron overload can lead to hemochromatosis, in which the excess

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iron stored in body organs causing serious tissue damage that leads to life-threatening conditions.

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Nevertheless, the majority of public are unaware of the problem and unconcerned due to the mistaken

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idea that the majority of hemochromatosis cases are genetic in origin, which affects only about 1 million

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of the estimated 275 million Americans. In fact, the potential threat of iron overload is universal and

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comes with advancing age regardless of genetic factors (3). Badria et al. reported the impact of

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environmental excess iron on human health among Egyptians who are inhabitants an area with high iron

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content in drinking water (4).

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The content of body iron is regulated primarily by absorption since humans have no

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physiological mechanism by which excess iron is excreted. The quantity of dietary iron and the

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composition of the diet significantly influence iron absorption. Prolonged intake of high doses of iron

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leads to the accumulation of excess iron (5). This phenomenon has been well demonstrated in the

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episode of siderosis among the Bantu tribe in Africa who ingested excessive amounts of iron from their

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diet and beer. Their use of iron pots for cooking and brewing beer increased the iron content (6).

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Another important factor in iron absorption relates to the form of iron present in a diet. Herne iron and

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nonheme iron are the two major sources of iron. Heme iron, that is found in meat, fish, and poultry, is

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more effectively absorbed because this iron is associated with the porphyrin ring (7). A large population

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survey from Australia showed that normal volunteers have average iron storage is about twice as much

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as the optimal iron store for normal adults. Heavy ethanol intake and high meat consumption are

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believed to be the critical factors (8). Such practices have occurred in industrialized countries where

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alcohol intake as well as red meat consumption are high, and the use of iron fortification is widespread

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(9). Hepatotoxicity and spleen dysfunction are the most common findings in patients with iron

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overloading and in severe cases, iron is found within the Kupffer cells in the liver and the macrophage

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cells of the spleen (10). The etiology of these multiple organ dysfunctions has been attributed to the

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presence of excess non-protein bound free iron released to spark uncontrolled oxidation via generation

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of reactive oxygen species (ROS) through Fenton and Haber-Weiss reactions (11). ROS react directly

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with proteins, lipids, and nucleic acids and induce oxidative stress by depleting cellular stores of

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antioxidants (12). Consequently, it is of utmost importance to maintain iron homeostasis to ensure iron

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supply but to prevent accumulation of excess iron.

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In all iron-overload-induced diseases, iron removal by iron chelation therapy is an effective life-

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saving strategy. The currently available iron-chelating agents used clinically are deferoxamine, and

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deferasirox. However, such compounds show several side effects and limitations (13) that direct towards

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the finding of a more effective and safe drug which may rise the therapeutic benefits for patients. As

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there is still no safe oral iron chelator agent, the use of nutritional therapy in the area of drug discovery

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proved to be an efficient, safe, and economic tool in the area of health care. Curcumin, Figure 1A (14),

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has been reported to be among the most efficient and safe chemopreventive agents investigated in

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recent years and is currently in human trials to prevent cancer (15). The mechanism of action of

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curcumin is complex and likely multifactorial (16). Previously, Curcumin has shown antioxidant and

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iron-chelating properties in vitro (16, 17). However, the pre-clinical effects of curcumin on hepatic and

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splenic sequelae during nutritional iron overload toxicity have not been investigated in depth.

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Accordingly, the present study aimed to evaluate the ability of the iron chelator, curcumin, to attenuate

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excessive dietary iron intake-induced hepatic and splenic oxidative stress. Furthermore, this study

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pursues to gain insight into the mechanistic basis behind this effect.

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MATERIALS AND METHODS

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Materials

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Na2HPO4, KH2PO4, p-phenylenediamine.HCl, and NaNO2 were purchased from Merck Company

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(Darmstadt, Germany). Nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), NADH, 5,5\-

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dithiobis(2-nitrobenzoic acid) [DTNB], thiobarbituric acid (TBA) were from Sigma (St Louis, MO,

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USA).

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Animals

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All procedures with animals were performed in accordance with the National Institutes of Health Guide

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for the Care and Use of Laboratory Animals and Mansoura University guideline. Adult male rats with

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average body weight of 120 150 g were divided into the following groups (five animals each; n=5):

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1. Control rats (no iron was administered).

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2. Rats treated with iron (100 M FeSO4 in a drinking water for 90 days) and treated with or

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without 100 mg/kg curcumin in a drinking water for a further 90 days.

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The dose of curcumin (100 mg/kg, b.w.) was selected for hepatoprotective studies by referring the

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published report (18). In various animal studies, a dose range of 100 200 mg/kg body weight exhibited

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good antiinflammatory activity and seemed to have negligible adverse effect on human systems. Oral

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LD50 in mice was found to be more than 2.0 g/kg body weight (18).

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Samples Collection:

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All these rat groups were under experiment for (3 months), then were anesthetized and scarified. Liver

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and spleen were collected immediately, dissected, rinsed with isotonic saline and dried between two

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o
pieces of filter papers and then stored at 20 C in plastic vials containing 0.5 ml of ice cold sterile

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isotonic saline.

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Preparation of homogenates

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An accurately weighed piece of each organ (1g tissue in 10 ml homogenate buffer) was homogenized in

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ice cold homogenate buffer (0.3 M sucrose and 0.1 mM potassium dihydrogen phosphate; pH 7.0) using

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a Teflon pestle connected to a Braun Homogenizer Motor (25 strokes/min at 1000 rpm). The

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homogenate was centrifuged at 30,000x g for 30 min at 4oC to remove cell debris and nuclei. The

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resulting supernatant was used for biochemical analysis.

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Methods

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Enzymesassays

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The activity of superoxide dismutase (SOD) was assessed spectrophotometrically by the procedure of

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Nishikimi et al. (19). The results were expressed as percentage of inhibition of formazane formation per

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g wet tissue. The SOD-like activity of curcumin was assessed by the same method and its activity was

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expressed as percent of inhibition of formazan (19). Catalase (CAT) activity was measured according to

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the method described by Bergmeyer (20). The results were expressed as unit per g wet tissue. The CAT-

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like activity of curcumin was determined by using the same method and its activity was determined as

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unit/min. The activity of ceruloplasmin (Cp) was assayed as described by Henry et al. (21). The results

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were expressed as mg of oxidation products per g wet tissue.

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Biochemical analysis

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The concentration of reduced glutathione (GSH) was estimated spectrophotometrically at 412 nm by

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using DTNB as a color reagent (22). The results were expressed as mg GSH per g wet tissue. The level

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of total ascorbic acid (AsA) in tissue homogenates was determined spectrophotometrically according to

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the method of Zebrowski et al., (23). The results were expressed as mg ascorbic acid per g wet tissue.

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Lipid peroxidation (LPO) in liver and spleen homogenates was estimated by measuring the formation of

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thiobarbituric acid reactive substance (TBARS) (24). Nitric oxide (NO) concentration was determined

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spectrophotometrically according to the method of Hortelano et al., (25). The results were expressed as

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nmol nitrite level per g wet tissue. The level of hydroxyl radicals (.OH) was determined by Abou-Seif

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and Elgendy (26). The results were expressed as nmol of thiobarbituric acid reactive substance

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(TBARS) formed per g wet tissue.

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Quantitative determination of total proteins

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Protein content of homogenized samples was performed according to Lowry et al., method (27). The

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results were expressed as g protein per g wet tissue.

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Estimation of iron and copper levels

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Iron and copper concentrations in liver and spleen homogenates were determined using an Atomic

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Absorption Spectrophotometer ( Perkin-Elmer 2380, Norwalk, CT 06859-0012, USA) using an air

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acetylene flame.

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Statistical analysis

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All analyses were performed with the GraphPad Prism 3 package. Data were checked for skewness, and

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an unpaired t-test was performed if the distribution of the values was Gaussian. If the distribution was

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not normal, a Mann-Whitney test was used. P-values less than 0.05 were considered to be statistically

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significant.

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RESULTS

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Iron overload-induced oxidative stress in the liver as well as in the spleen and the protective effect

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of curcumin as an iron chelator

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Numerous pathological alterations in liver and spleen have been reported in cases with primary or

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secondary iron overload. Furthermore, it has been well established that iron-catalyzed free radical-

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mediated lipid peroxidation plays a significant role in these abnormal events. Therefore, the iron-

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overloaded rats were used in this study to investigate whether curcumin, a potential iron chelator, could

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ameliorate hepatic and splenic oxidative stress induced by excess dietary iron intake and the underlying

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mechanisms involved therein. To test this, we first sought to verify the potential of curcumin as an iron

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chelator in this preclinical study. This necessarily entails to get a more exact measure of iron overload.

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Measurement of hepatic or splenic iron concentration is the most quantitative, specific, and sensitive

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method for determining the body iron burden (28). Accordingly, these tissues were removed to

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determine how much iron is present. Employing this measurement, the organ iron concentrations for the

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liver and spleen are shown in Figure 1 B and C, where the chronic supplementation with iron led to

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about a 2.6- and 3.9-fold, respectively, elevation of total hepatic and splenic iron content over control

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pair-fed values. Treatment with curcumin resulted in a highly significant decrease in iron concentration

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in the liver as well as in the spleen (P < 0.001 for each).

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After

having shown the iron chelating properties of curcumin, we proposed its natural

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implementation in preventing metal-catalyzed prooxidative effects. Thereby we next aimed to assess the

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effect of curcumin on iron overloadinduced MDA, a terminal compound of lipid peroxidation that is

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commonly used as an index of oxidative stress status. As expected from excessive iron deposition in the

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liver, lipid peroxidation was induced locally as reflected by the significant increase in MDA in the

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untreated iron-overloaded group compared with the normal group, and that increase in tissue MDA level

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was reduced significantly by the curcumin treatment (Figure 2A and B).

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Furthermore, the generation of hydroxyl radical (HO), the highly reactive and cytotoxic free

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radical, is facilitated by iron, which catalyzes the interaction of superoxide (O2) and hydrogen peroxide

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(H2O2) by Fenton or Haber-Weiss reactions (11). Consequently, hepatic or splenic hydroxyl radical

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level was evaluated in iron-overloaded rats. The results showed that hepatic and splenic hydroxyl radical

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levels were (11.75-fold and 10.5-fold, respectively) higher in iron-overloaded rats compared to non-

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iron-overloaded. The curcumin treatment resulted in a significant decrease in hydroxyl radical level

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(approximately 62%) compared to untreated iron-overloaded rats (Figure 2C and D). Following this

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further, the hepatic and splenic iron overload was accompanied by a marked elevation of tissue nitric

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oxide (NO) concentrations up to 3.5- fold in both liver and spleen (Figure 2E and F). NO is an

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inorganic free-radical gaseous molecule that contributes to the oxidative burden and its concentrations

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were reduced significantly by the curcumin treatment (Figure 2E and F). These observations are

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consistent with the less severe lipid peroxidation phenotype observed in curcumin-treated rats, as free

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radicals play a critical role in the regulation of oxidative stress.

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Given the oxidative stress nature of iron overload-induced toxicity and the fact that free radicals

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signaling cascade plays a pivotal role in initiating protein loss (12), we finally examined the bulk

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degradation of proteins in liver and spleen under the chronic iron intake condition. As the continuation

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of the work, we hypothesized that curcumin may also be effective in attenuation of iron overload-

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induced hepatic and splenic oxidative damage and protein loss. To test this, the effect of oral ingested

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curcumin on hepatic and splenic protein levels were determined. Iron-overload significantly (P< 0.001)

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promoted protein loss in liver and spleen to be 137.174.1 g/wet liver tissue versus 164 8.28 g/wet

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liver tissue in control rats and 1504.1 g/wet spleen tissue versus 185.32 3.65 g/wet spleen tissue in

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control rats. Curcumin intervention resulted in a significant inhibition of iron overload-induced hepatic

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and splenic protein loss to be 1504.6 g/wet tissue and 171.56.76 g/wet tissue, respectively.

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In vitro antioxidant properties of Curcumin per se in scavenging harmful free radicals seen in

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iron overload

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In light of the aforementioned curcumins iron chelating property and anti-lipid peroxidation effects,

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interest in its mechanisms of action has been expanded to investigate whether curcumin per se has a free

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radical scavenging capability against the most potent reactive species, which has been implicated in the

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pathogenesis of liver and spleen dysfunction seen in iron overload. To address this point, the capacity of

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curcumin to scavenge hydroxyl, H2O2, and superoxide radicals was evaluated, respectively, by

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thiobarbituric acid reactive substance (TBARS), catalase-like, and the superoxide dismutase-like

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reaction in vitro assays. The efficiency curcumin in inhibition of hydroxyl radical generation was

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quantified as the amount of TBARS formed, and the results are depicted in Figure 3A. Furthermore,

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The ability of curcumin to scavenge H2O2 (catalase-like activity) and its ability to scavenge O2- (SOD-

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like activity) were measured. As shown in Figure 3B and C, respectively, the rate of H2O2 and

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superoxide degradation was significantly enhanced by curcumin in all doses tested. Collectively, these

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results implied that curcumin limits iron-overload induced oxidative stress both by chelating excess iron

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and by simultaneously functioning as an efficient radical scavenger .

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Curcumin boosts levels of endogenous antioxidants that excessive dietary iron-intake depletes

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The induction of oxidative stress in iron overloaded model can be caused by increased free-radical

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generation, antioxidant depletion, or both. Given the fact that cells are armed with a variety of

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antioxidants acting as a multilevel defenses against free radical damage, we next sought to determine

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whether curcumin alleviation of the oxidative stress associated with iron accumulation is also mediated

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by restoring endogenous antioxidant defenses. These defense systems include glutathione (GSH),

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ascorbic acid (ASA), and enzymes such as SOD, and catalase (29).

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As shown in Figure 4A and B, chronic iron intake was clearly associated with a significant

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depletion of hepatic and splenic GSH when compared to non-iron overloaded rats. The curcumin

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supplementation resulted in a great improvement of liver and spleen levels of GSH by approximately

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65% and 90%, respectively. Moreover, the storage of ASA in livers of rats fed with excessive iron

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intake for 12 weeks was 66% less, and spleen ascorbic acid was 70% less than that of the controls fed a

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diet with normal iron level (Figure 4C and D). The administration of curcumin to rats receiving

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excessive dietary iron intake has been shown to increase liver and spleen content of ascorbic acid as it

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did with glutathione by approximately 1.5-fold.

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Furthermore, excess iron in liver lowers the activity of catalase in homogenates of both liver and

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spleen of chronic iron intoxication group by 40 and 50%, respectively, compared to the corresponding

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values of normal control group, whereas the treatment with curcumin arrested the iron-induced depletion

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of CAT enzyme activity significantly (Figure 5A and B).

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Similarly, treatment with curcumin dramatically boosted activity of hepatic and splenic Cu-

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SOD activity by approximately 2.4- and 2.2-fold, respectively, in chronic iron overloaded rats, whereas

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a significant decrease in SOD activity has been observed in chronic iron overloaded rats (Figure 5C and

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D). In practice, the most common cause of poor SOD activity in rats with chronic dietary iron overload

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appears to be copper deficiency. At this stage, it is unclear whether the enhancement of liver and splenic

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SOD activity seen after curcumin treatment was also achieved through modulation of organ

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copper content and its antioxidant protein (ceruloplasmin). To delineate this preclinical association,

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Figure 6 demonstrates the average levels of copper and ceruloplasmin in both liver and spleen

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homogenates of iron intoxication rats either were treated with curcumin or served as non-treated controls

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group. Compared to the corresponding values of the control rat group, the administration of curcumin

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significantly restored the average of copper (Figure 6A and B) as well as ceruloplasmin levels (Figure

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6C and D) in both liver and spleen homogenates.

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DISCUSSION

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Iron overload is seen in increased absorption from diets, hereditary hemochromatosis, iron loading

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anemias, chronic liver disease, porphyria cutanea, congenital defects associated with iron overload,

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transfusions and therapeutic injections (30). In this study, we put forward a new pre-clinical evidence for

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introducing curcumin, an abundant and cheap micronutrient, as a multi-target therapeutic agent for

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treating hepatosplenic complications associated with excessive dietary iron intake. Curcumin has a wide

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therapeutic index with very low toxicity (31) and its efficacy has been demonstrated in different animal

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models of multiple diseases associated with oxidative injury (32, 33). The most interesting feature of

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curcumin is the lack of gastrointestinal side effects despite being an anti-inflammatory agent (18).

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Excess iron in liver is potentially detrimental to the cell because of its propensity to participate in

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oxidation-reduction reactions that promote tissue injury by catalyzing lipid peroxidation (34, 35). Iron

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initiates peroxidation by producing highly reactive hydroxyl radicals from hydrogen peroxide via Fenton

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type reactions or by complexing with oxygen directly to yield reactive perferryl and ferryl ions (12, 36).

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The role of iron in lipid peroxidation has been well studied both in vivo and in vitro (37, 38). In

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addition, it is also thought that a chronic increase in tissue iron level leads to depletion of antioxidants

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which, if not restored, can by itself lead to further ROS elevation and provide a mechanism for further

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dysfunction (12). Given the crucial participation of oxidative stress in liver and spleen failure seen in

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chronic iron overload, an iron chelator represents an important player in modulating these pathogeneses.

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This input has originated partly from histopathologic studies that show collagen fibrils were

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demonstrated immediately adjacent to the hepatocytes in the iron-overloaded animals but not the

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controls (39). These initial observations have been supported in postmortem human liver

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specimens

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mitochondrial and microsomal lipid peroxidation

(40) and reinforced by additional studies showing that oral iron loading induced
that was associated with selective decreases

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in microsomal hemoprotein concentrations and cytochrome P450-dependent enzymes (41, 42). The

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treatment with curcumin or antioxidants has demonstrated beneficial effects in the prevention of iron

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induced liver toxicity (43, 44). Moreover, the in vitro studies on cultured hepatocytes with high dose of

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iron have shown that iron induced free radical generation that kills liver cells (45).

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All of this seemingly overwhelming evidence has sculpted the concept of iron overload-mediated

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oxidative stress as a major contributing factor in the pathogenesis of its associated liver injury.

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Consistent with these previous studies, we observed similar findings regarding the oxidative stress and

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bulk protein degradation of liver and spleen under the chronic iron intake condition. However, this study

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is the first to show the preclinical utility and translational relevance of the specific curcumins iron-

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chelating potency during excessive iron intake. This notion was strengthen by the observation that the

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decrease in liver and spleen iron deposition induced by curcumin treatment was associated with

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remission of the aforementioned oxidative stress and protein loss.

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The specific metal chelator effect of curcumin against iron which was established previously in

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vitro (46) might be attributed to the presence of chemical groups such as -diketonate group. This

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assertion is supported by using spectrophotometry to quantify curcumin affinity for copper, zinc, and

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iron ions. Zn2+ showed little binding, but each Cu2+ or Fe2+ appeared to bind at least two curcumin

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molecules. The interaction of curcumin with copper exhibited positive cooperativity, with Kd

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approximately 10-60 M, while curcumin-iron interaction exhibited negative cooperativity, with Kd

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approximately 0.5-1.6 M (47). A recent study reported with the intracellular free copper tracer

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indicates that curcumin brings copper into the cells and releases it thereafter, resulting in an elevated

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intracellular free copper level. The dissociation of copper ions from curcumin in the cells supports the

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previous finding that the binding affinity of curcumin for copper is relatively moderate (48).

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Because excess iron is known to be responsible for the functional abnormalities associated with

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chronic iron overload, any beneficial effects of curcumin might be secondary to its action as a an iron

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chelator. Nevertheless, the demonstration that curcumin reduces the oxidative stress in other diseases

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(49, 50) has raised the possibility of multi-target properties beyond its role as an iron-chelating agent.

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An alternative mechanism of these effects is the possession of antioxidant properties. These properties

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were endorsed by the presence of chemical groups like hydroxyl, methoxy and 1,3-diketone conjugated

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diene system (51). In the context of chronic iron overload, these two effects of curcumin cannot be

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separated. Therefore, we studied the redox properties of curcumin against different reactive species

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generated in vitro. From these experimental results it can be derived that curcumin is also able to reduce

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oxidative stress during iron overload by scavenging, and/or neutralizing free radicals (such as hydroxyl

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radical, superoxide anions and H2O2 (52) known to participate in the pathogenesis of liver and spleen

291

dysfunctions during iron overload. Accordingly, the hepato- and splenic-protective potential of

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curcumin comes from its iron chelating property as well as its antioxidant effect.

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Further, besides curcumins ability to chelate iron and its antioxidant potential other reported

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effects might have contributed to our finding of curcumins salutary effects, such as restoring activities

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of endogenous antioxidants (53). In agreement, the oxidative stress in our experimental model due to

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chronic iron intake resulted in the depletion of putative non-enzymatic (GSH, ASA) and enzymatic

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(CAT and SOD) antioxidants in target tissues. This depletion of antioxidants was significantly restored

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after curcumin treatment. Thus, curcumin significantly helping revival from hepatic and splenic

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oxidative stress associated with excessive dietary iron intake both directly or indirectly via mending the

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levels of endogenous antioxidants.

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Taken together, because curcumin has an attractive safety profile and appreciable iron chelating

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property as well as anti-oxidant activity, it may represent a potential therapeutic agent for managing

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excessive dietary iron intake sequelae long before the occurrence of hepatic and splenic failure among

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those at risk for iron accumulation.

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Disclosures

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No conflicts of interest are declared by authors.

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Author contributions: All the authors contributed to the researched data as well as the discussion,

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reviewed and edited manuscript.

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424

FIGURE LEGENDS

425

Figure 1. The potential of curcumin as an iron chelator in damping iron accumulation in liver and

426

spleen. A) Structural characteristic of curcumin as an iron chelator. B, C) Level of iron (g/g wet tissue)

427

in liver and spleen, respectively, from iron-overloaded rats treated or untreated with curcumin. Data

428

shown are the meanSD, (n = 5).

429

Figure 2. Iron overload-induced oxidative stress in the liver as well as in the spleen and the

430

protective effect of curcumin. A, B) Level of MDA (N mol/g wet tissue) in liver and spleen,

431

respectively, in iron overloaded rats with or without curcumin treatment. Levels of MDA in these tissues

432

are significantly increased in iron overloaded rats than control rats. Curcumin treatment significantly

433

decreased MDA levels in iron overloaded rats. C, D) Level of hydroxyl radical (N mol/g wet tissue) in

434

liver and spleen, respectively, in iron overloaded rats with or without curcumin treatment. Levels of

435

hydroxyl radical in these tissues are significantly increased in iron overloaded rats than control rats.

436

Curcumin treatment significantly decreased hydroxyl radical levels in iron overloaded rats. E, F) Level

437

of NO (N mol/g wet tissue) in liver and spleen, respectively, in iron overloaded rats with or without

438

curcumin treatment. Levels of NO in these tissues are significantly increased in iron overloaded rats than

439

control rats. Curcumin treatment significantly NO levels in iron overloaded rats. Data shown are the

440

meanSD, (n = 5).

441

Figure 3. In vitro antioxidant properties of Curcumin per se in scavenging harmful free radicals

442

seen in iron overload. A) Hydroxyl radical (OH)-Scavenging activity of curcumin measured as % of

443

TBAR inhibition. B, C) Catalase-like and Superoxide dismutase-like activities of curcumin,

444

respectively. Data shown are the meanSD, (n = 5).

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445

Figure 4. Curcumin improves levels of endogenous non enzymatic antioxidants that excessive

446

dietary iron-intake depletes. A, B) Levels of glutathione (GSH; mg/g wet tissue) in liver and spleen,

447

respectively; C, D) Levels of ascorbic acid (ASA; mg/g wet tissue) in liver and spleen, respectively; in

448

iron overloaded rats with or without curcumin treatment. Levels of GSH and ASA in these tissues are

449

significantly decreased in iron overloaded rats than controls. Curcumin treatment significantly increased

450

GSH and ASA levels in iron overloaded rats. Data shown are the meanSD, (n = 5).

451

Figure 5. Curcumin boosts activities of endogenous enzymatic antioxidants that excessive dietary

452

iron-intake depletes. A, B) Activity of catalase (CAT; U/Min g wet tissue) in liver and spleen,

453

respectively; C, D) Activity of Superoxide dismutase (SOD; U/Min g wet tissue) in liver and spleen,

454

respectively; in iron overloaded rats with or without curcumin treatment. Activities of CAT and SOD in

455

these tissues are significantly decreased in iron overloaded rats than controls. Curcumin treatment

456

significantly boosted activities of both CAT and SOD in iron overloaded rats. Moreover, Activities of

457

CAT and SOD of curcumin-treated iron overloaded rats remained higher than those of control rats. Data

458

shown are the meanSD, (n = 5).

459

Figure 6. Curcumins modulation of organ copper content and its antioxidant protein

460

(ceruloplasmin). Levels of copper (A, B) and ceruloplasmin (C, D) in both liver and spleen

461

homogenates of iron intoxication rats either were treated with curcumin or served as non-treated controls

462

group. Compared to the corresponding values of the control rat group, the administration of curcumin in

463

iron overloaded rats significantly enhance the average of copper as well as ceruloplasmin levels in both

464

liver and spleen homogenates. Data shown are the meanSD, (n = 5).

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Figure 6
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