Abstract
The aim of this research was to test the chelation potency of deferasirox (DFS or ICL670)) and deferiprone (L1) in mobilization of arsenic in arsenic-exposed rats. Male Wistar rats were exposed to 60 mg/kg body weight sodium arsenite in drinking water for six weeks, followed by treatment with DFS (80 mg/kg body weight, oral, once daily), L1(100 mg/kg body weight, oral, once daily) alone or in combination, for six consecutive days. After chelation therapy, these rats were anesthetized by ether vapor, were immobilized by cervical dislocation and then their heart, liver, kidneys, intestine and blood were sampled for clinical hematological variables and determination of arsenic and iron concentration by Inductively Coupled Plasma/Optical Emission Spectrometry (ICP-OES). Comparison of single and combined therapy showed that the combined chelation therapy (DFS+L1) is more effective in depleting arsenic concentration from soft tissues. No effects of arsenic or any of the two treatments (L1 or DFS) on WBC, RBC and Hb were observed. Interestingly, platelet counts showed a decrease in arsenic exposure. The obtained Results of combined treatment should be confirmed by a different experimental model before extrapolation to other systems.
Introduction
Arsenic is a toxic metalloid widely present around the world particularly in soil, water or contaminated food. Although, humans can be exposed to arsenic via various routes, the major source for exposure to inorganic arsenic is through contaminated drinking water. Concentrations of arsenic in drinking water in several regions of the world exceed the standard WHO (World Health Organization) guidelines of 0.010 mg/L. Chronic exposure to arsenic-containing compounds has been associated with various disease manifestations, such as internal cancers [1], vascular diseases and diabetes [2]. One of the most vulnerable targets of chronic arsenicosis is the central nervous system [3]. The only remedial measure for chronic arsenicosis is chelation therapy. British Anti-Lewsite (BAL or 2, 3-dimercaprol), which was developed as an antidote against ‘Lewisite’, an arsenic based chemical warfare agent. British Anti-Lewsite has numerous serious effects including neurotoxicity and hence meso 2, 3-dimercaptosuccinic acid (DMSA), a safer derivative of British Anti-Lewsite has been tested in animals [4]. However, subsequent human trials in West Bengal (India) with dimercaptosuccinic acid failed to provide clinical recoveries in patients chronically exposed to arsenic [5]. Clinical investigations of some chelators in removing various toxic metals in rats have been previously published by [6, 7]. Deferasirox{4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-l-yl]-benzoic acid, ICL670 or DFS, Scheme 1} is a tridentate chelator with high selectivity for Fe3+. DFS possesses a pFe3+ value of 22.5 and can penetrate membranes easily and possesses good oral availability. Indeed, when orally administered to hyper transfused animals, deferasirox promotes the excretion of chelatable iron from hepatocellular iron stores four to five times more effectively than desferrioxamine [8, 9]. Another developed orally active chelating agent was deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one or L1), which is rapidly absorbed in the gastrointestinal tract and normally appears in serum a few minutes after oral administration. The main excretion route is the kidneys. Deferiprone is a bidentate iron chelator forming a 3 : 1 complex with iron and it is likely to act intracellularly [10]. With this background, for the first time, we investigated the single and combined therapeutic efficacy of deferasirox and deferiprone in reducing arsenic concentration in blood and soft tissues.
Experimental section
Reagents
All the chemicals used in this work were of either analytical grade or of extra pure grade of highest purity available locally. Sodium arsenite, deferiprone and other materials were purchased from Merck Chemicals Co. and deferasirox was purchased from Novartis Co. (Basel, Switzerland).
Maintenance of the animals
Male Wistar rats were obtained from animal house facility of Kerman Neuroscience Research Center (Kerman, Iran). The animals were kept under a controlled light: dark (12 : 12 h) schedule. The animals were divided randomly to control and were treated in groups and were housed in well-cleaned sterilized cages in an air-conditioned room with temperature maintained at 23 ± 2°C and 50% humidity. Animal ethical committee of Payame Noor University and Kerman Neuroscience Research Center approved the protocols for the experiments.
Experimental groups
In order to evaluate abilities of chelators deferasirox and deferiprone in removing of arsenic in arsenic-exposed rats, experiments were done on Wistar male rats (200 ± 10 g). Animals were classified as follows: The control group (n = 5) was given normal food and distilled water to drink. The concentrations of arsenic and iron in control group were compared with the groups that received arsenic and chelators. The toxic groups (n = 25) were given water containing 60 mg /kg body weight As3+ as sodium arsenite for 42 days. In order to compare the arsenic and iron concentrations in tissues, before and after chelation therapy, one group was selected as before chelation therapy (Vehicle As or Vehicle Fe) and was sacrificed before chelation therapy. Other arsenic-exposed animals were divided into 4 sub- groups of 5 rats each and given the following treatment for six consecutive days: Group control chelator (No treatment) Group deferiprone, L1 (100 mg/kg body weight, oral, once daily) Group deferasirox, DFS (80 mg/kg body weight, oral, once daily) Group deferasirox and deferiprone, DFS+L1 (40 and 50 mg/kg body weight, respectively, oral, once daily)
All animals of each group were sacrificed under light ether anesthesia, 48 h after the last dosing. Kidneys, heart, liver and intestine samples were weighed, dried and collected for determination of arsenic and iron concentration. The samples were put in an oven at 60°C for 3 days. Then, 1 g of each samples were digested by 1 ml of HNO3 (10 M). After digestion, the solutions were vaporized with the addition of 0.5 ml of H2O2 (30%) under the hood. Afterwards, the fragment was diluted with distilled water to 10 ml volume. Determination of arsenic and iron in samples were performed by using ICP-OES (Model: Varian VISTA-MPX). The values are expressed as mean values (at least three separate determinations) ± standard error of the mean (SEM). The data were subjected to statistical analysis by Student’s t-test; P < 0.05 was considered significant.
Clinical hematological variables
Blood was collected by cardiac puncture in heparinized tubes and level of hemoglobin (Hb), platelets (PLT), red blood cell (RBC) count and white blood cell (WBC) count were measured using a Sysmex hematology analyzer (model K4500).
Results and discussion
It was found that as the arsenic concentration increased in tissues, iron concentration decreased in kidneys and liver, which is probably due to an interference that could take place by arsenic through iron uptake mechanism. The maximum amount of arsenic accumulation was found in kidneys and liver, respectively. In order to investigate the spontaneous elimination of arsenic from body by the biological system, control chelator group was treated without chelation therapy and removal of arsenic by the biological system in this group was not noticeable. After the chelation therapy, the obtained results indicated that arsenic concentration in all tissues was significantly reduced. There is a statistical difference between deferasirox and deferiprone in reducing the amount of arsenic in various tissues. As single therapy efficiencies of chelators were compared in this study, it was found that deferasirox was more effective in decreasing arsenic level in all tissues, whereas, deferasirox was more effective in kidneys. Comparison of single and combined therapy showed that the combined chelation therapy (deferasirox + deferiprone) is more effective in reducing the arsenic concentration in all tissues. The effects of chelators deferasirox and deferiprone on arsenic concentration in the various tissues are shown in Table 1. The results of iron concentrations before and after chelation therapies are shown in Table 2. The iron concentration after the chelation therapy was significantly decreased. Thus, consumption of iron tablet is recommended for returning the iron level to its normal state. Combination of deferasirox and deferiprone shows more efficiency in decreasing iron level. The effects of exposure to arsenite and treatment with chelators either individually or in combination on some hematological variables are shown in Table 3.
Most of the arsenic content was accumulated in kidney and liver. Chelation therapy is one of the most effective ways to remove toxic metals from the biological system. It has been reported that the chelating agents who have higher stability constants with a metal in aqueous solution may also be successful in reducing the body burden of the metal. The combined therapy procedure can likely increase metal excretion, target specific metal tissues, minimize side effects and improve compliance. Many studies have now reported the high absorption/distribution, long-term efficacy and safety of deferasirox and deferiprone in removing some toxic metal ions and treating iron overload in patients with β-thalassemia major [11, 12]. After administration of chelators, the arsenic content was reduced. The result shows that both of chelators deferasirox and deferiprone increase the removal of arsenic in the tissues. Deferiprone is able to redistribute iron in mammals and by virtue of its bidentate nature and the ability for iron (III) at neutral pH values, the iron-deferiprone complex will donate iron to competing ligands [13, 14]. Deferasirox, by virtue of its small size and the ability to penetrate cells, has capability of efficiency scavenging excess toxic iron [15]. No effects of arsenite or any of the two treatments (deferasirox or deferiprone) on WBC, RBC and Hb were observed and platelet counts showed a decreased in arsenite exposure rats. There was no effect of arsenite on these variables in the present. This can be attributed to short duration of the arsenite exposure. Treatment with deferasirox and deferasirox + deferiprone provided significant recovery in platelet counts.
Conclusion
Comparison of the results indicates that the combined therapy (deferasirox + deferiprone) enhances the removal of arsenic from rat organs considerably. Each of chelators deferasirox and deferiprone has a different target tissue, therefore, combination of them can help to remove arsenic from various tissues effectively.
Therefore, their combination can help the removal arsenic from various tissues effectively. This study might be effective for preliminary testing of the ability of chelating agent in removing arsenic. Therefore, after essential preclinical experiments, the same study can be suggested for human administration.