BioMetals

, Volume 26, Issue 3, pp 465–471

Chelation of chromium(VI) by combining deferasirox and deferiprone in rats

Authors

    • Chemistry DepartmentShahid Bahonar University of Kerman
  • S. Jamil A. Fatemi
    • Chemistry DepartmentShahid Bahonar University of Kerman
  • Roza Ebrahimpour
    • Chemistry DepartmentShahid Bahonar University of Kerman
  • Faezeh Dahooee Balooch
    • Chemistry DepartmentShahid Bahonar University of Kerman
Article

DOI: 10.1007/s10534-013-9631-5

Cite this article as:
Iranmanesh, M., Fatemi, S.J.A., Ebrahimpour, R. et al. Biometals (2013) 26: 465. doi:10.1007/s10534-013-9631-5
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Abstract

The present research is aimed to characterize the potential efficiency of two chelators after chromium(VI) administration for 60 days following two doses of 15 and 30 mg/kg chromium(VI) per body weight daily to male rats. However, the hypothesis that the two chelators might be more efficient as combined therapy than as single therapy in removing chromium(VI) from rat organs was considered. In this way, two known chelators deferasirox and deferiprone were chosen and given orally as a single or combined therapy for a period of 1 week. Chromium(VI) and iron concentrations in tissues were determined by flame atomic absorption spectroscopy. The combined chelation therapy results show that deferasirox and deferiprone are able to remove chromium(VI) ions from various tissues while iron concentration returned to normal levels and symptoms also decreased.

Keywords

DeferasiroxDeferiproneChromium toxicityChelation therapyRats

Introduction

Chromium is an important metal, which is used in a variety of industrial applications e.g. textile dying, tanneries, metallurgy, metal electroplating, wood preserving and preparation of chromate compounds. It hence, large quantities of chromium have been discharged into the environment due to accidental releases or inadequate precautionary measures (Kimbrough et al. 1999). The chemical and toxicological behaviors of chromium depend on its oxidation state. The most important concern from the human health point of view is chromium(VI) for both acute and chronic exposures (ATSDR 1998). Chromium(VI) is known to enter cells readily via non-specific anion channels and it is thereafter reduced by intracellular reductants to the more stable Cr(III) with the concomitant formation of reactive intermediate species of Cr, Cr(V) and (IV), and reactive oxygen species (ROS) (Codd et al. 2001). These reactive species can cause DNA damage and lipid peroxidation (Bagchi et al. 2002; O’Brien et al. 2003). Thus, removal of chromium and especially chromium(VI), is an essential pollution abatement process that should be applied to all industrial effluents that contain this contaminant.

One way to remove toxic elements, such as chromium, from the body is chelation therapy. Chelation therapy involves the use of ligating drugs that bind metal for the treatment of potentially fatal conditions. These ligands promote the excretion and subsequent depletion of this transition metal in biological systems. Clinical evaluations of some chelators for the removal of toxic metal ions in rats have been previously reported by Amiri et al. (2007), Fatemi et al. (2007, 2009), Shokooh Saljooghi and Fatemi (2010b), Tubafard and Fatemi (2008). These chelating agents consist of a range of bidentate, tridentate and hexadentate ligands in which two, three or six atoms are able to coordinate, respectively (Clarke and Martell 1992; Gomez et al. 1988). In this procedure, chelator is added to the blood through a vein or administered orally in order to remove toxic element. Deferasirox (4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid, or ICL670; Fig. 1a was first reported in 1999 (Heinz et al. 1999). It is a tri-dentate chelator with high selectivity for Fe3+. It selectively binds Fe3+ over Fe2+ and shows little affinity for other divalent ions such as Zn2+ or Cu2+ (Steinhauser et al. 2004). In vivo, this selectivity is demonstrated by conserved plasma Zn and Cu levels in patients taking deferasirox and while its efficacy is rather low for inducing negative iron balance, it is effective and well tolerated (Nisbet-Brown et al. 2003). In 2005 deferasirox became the first FDA-approved oral alternative for treatment of iron overload and was subsequently approved in the EU in 2006 (Yang et al. 2007). Deferasirox possesses a pFe3+ value of 22.5 and can penetrate membranes easily and possesses good oral availability. Indeed, when orally administered to hypertransfused rats, deferasirox promotes the excretion of chelatable iron from hepatocellular iron stores four to five times more effectively than desferrioxamine (Hershko et al. 2001). The other chelator for iron overload is deferiprone (1,2-dimethy1-3-hydroxypyride-4-one) that has been shown in Fig. 1b. Deferiprone is water soluble and can be given orally (Hider et al. 1984; Kontoghiorghes et al. 1987; Gyparaki et al. 1987). It possesses a pFe3+ value of 20.5 and its important property is its ability to penetrate cells, coordinate iron, forming a neutral complex, which is also capable of permeating membranes (Glickstein et al. 2006). At present, combination therapy with deferiprone and desferoxamine, that is highly selective for iron(III) under biological conditions (pFe+3=26.6), is reported to be the most effective treatment for many patients (Galanello et al. 2010). The combined therapy procedure is likely to enhance iron excretion, target specific iron compartments, minimize side-effects (by virtue of the use of lower doses), facilitate individualization of therapy and improve compliance (Ma et al. 2012). Desferrioxamine with a higher pFe+3 value acting as a sink. Presumeably deferasirox, also possessing a higher pFe+3 value than deferiprone, behaves in a similar manner. Recently successful chelation therapy using both deferasirox and deferiprone has been reported (Voskaridou et al. 2011). This kind of therapy by combining two chelators is based on the assumption that various chelating agents mobilize toxic elements from different tissue compartments and therefore better results are expected (Flora et al. 1995). Results of this kind of combined chelation therapy have been confirmed by Amiri et al. (2007), Fatemi et al. (2007, 2009), Tubafard and Fatemi (2008). The aim of the present research was to test the chelation potency of deferasirox and deferiprone in combination, given to animals after chromium loading. Testing was performed by using a chronic poisoning model on rats with individual and combined chelators given shortly after chromium application.
https://static-content.springer.com/image/art%3A10.1007%2Fs10534-013-9631-5/MediaObjects/10534_2013_9631_Fig1_HTML.gif
Fig. 1

Chemical structures of deferasirox (a) and deferiprone (b)

Experimental section

Apparatus

A Varian atomic absorption spectrometer (FAAS) was used for measurement of chromium and iron concentrations in various organs. Also a Mettler analytical balance Model AE 160 was used in this research.

Maintenance of the animals

Male Wistar rats were obtained from Razi Institute (Karaj, Iran). They were bred in animal house at Kerman Neuroscience Research Center, Iran. The rats were maintained under a controlled light:dark (12:12 h) schedule at 23 ± 1 °C and the humidity of 55 %. The animals were assigned to control and treated groups and were kept in well cleaned sterilized cages. The rat food was purchased from Razi Institute. This study was approved by the ethics committee of Shahid Bahonar University of Kerman, Iran and Kerman Neuroscience Research Center, Iran.

Materials

K2Cr2O7, deferiprone and other materials were purchased from Merck Chemicals Co. and deferasirox was purchased from Novartis Co. (Basel, Switzerland).

Experimental design

In our model, we used two different doses of chromium followed by an early administration of chelating agents. Experiments were performed on 7-weeks-old Wistar male rats.

There were slight differences between the groups in the initial body weight of the rats (mean 200 g), but at the end of chromium administration experiment, those given chromium in their diet had significant weight loss (Table 1). Comparison of the weights in this experiment showed that dietary treatment affected the food intake, whereby animals that were given normal diet consumed more food than those given chromium. Also because of the slight (but significant) differences in body weight of rats at the start of the research, the results can be influenced by the initial classification and assignment of rats to treated groups. Therefore, the day 1 groups’ body weights are notable and they must be considered. Consequently after acclimatization of the animals, we assigned them randomly to control and treated groups.
Table 1

Body weights over 60 days for the rats in different groups (values are mean for the number of observation in parentheses)

Group

Control

Low dose drinking of chromium

High dose drinking of chromium

Initial body weighta (g)

205 ± 7(5) (day 1)

200 ± 4(5) (day 1)

195 ± 3(5) (day 1)

Final body weighta (g)

275 ± 6(5) (day 60)

255 ± 8(5) (day 67)

225 ± 7(4) (day 67)

aMean of five determination ± standard deviation

Two doses of 15 and 30 mg/kg chromium(VI) per body weight were given to treated groups for 60 days. Chelation therapy was carried out after chromium application.

In this part of the research, treated groups were divided into five groups: before chelation therapy, without chelation therapy, chelation therapy with deferasirox, chelation therapy with deferiprone and chelation therapy with deferasirox + deferiprone (Table 2). Chelators (deferasirox and deferiprone) were given orally after chromium application during 1 week. Doses of deferasirox and deferiprone were 140 and 300 mg/kg body weight, respectively. Observed chromium toxicity symptoms in rats were removed in short term (7 days) after drug administration. After chelation therapy, these rats were anesthetized with ether vapor and immobilized by cervical dislocation and their liver, kidneys, intestine, spleen and testicles samples were collected, weighed and dried for determination of their chromium contents. The samples were placed in an oven at 60 °C for 3 days. They were then digested by 1.5 ml of HNO3 per 1 g of dry weight tissues. After digestion, the solutions were evaporated with the addition of 1.0 ml of H2O2 under the hood. Then, the residue was diluted with water to 10 ml volume.
Table 2

Classification of animals

All rats

Control group

Treated groups

 Low level drinking group: 15 mg/kg Cr(VI) per body weight

  Before chelation therapy

  Without chelation therapy

  Chelation therapy with deferasirox (140 mg/kg body weight)

  Chelation therapy with deferiprone (300 mg/kg body weight)

  Chelation therapy with deferasirox (70 mg/kg body weight) + deferiprone (150 mg/kg body weight)

 High level drinking group: 30 mg/kg Cr(VI) per body weight

  Before chelation therapy

  Without chelation therapy

  Chelation therapy with deferasirox (140 mg/kg body weight)

  Chelation therapy with deferiprone (300 mg/kg body weight)

  Chelation therapy with deferasirox (70 mg/kg body weight) + deferiprone (150 mg/kg body weight)

Statistical analysis

Determination of chromium and iron in samples were carried out by FAAS. 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.

Result

Results of chromium raising and iron reduction in organs of two chromium doses groups were statistically different. The chromium accumulation in tissues at 30 mg/kg dose was more than the group at 15 mg/kg dose. A significant difference between control and treated groups was observed. The general symptoms of toxicity appeared after 60 days of chromium administration. Abnormal clinical signs in animals were appeared as follows: darkening of the eyes, yellowish discoloration of hair, flaccid, hypotonic muscles, irritability, weakness and loss of hair. Also the body weights of all animals in treated groups were significantly decreased. The highest amount of chromium was found in the intestine and then in the kidneys. After the chelation therapy, the obtained results showed that present chromium levels in all tissues were significantly reduced whereas, iron concentration returned to normal levels and the symptoms also reduced. Iron level is lowest in the groups having the highest chromium concentration, which is probably because of a significant interference that could take place by chromium through iron uptake mechanism. There is statistical difference between deferasirox and deferiprone in reducing the amount of chromium in various tissues. The t test was applied to the results assuming the certified values were the true values. At both lower and higher doses, deferasirox + deferiprone groups were more effective than deferiprone or deferasirox. When comparing efficiencies of mono chelators in this experiment, deferasirox was more efficient than deferiprone in decreasing chromium concentration in tissues. Comparison of mono and combining chelators in this experiment show more efficiency of deferasirox + deferiprone in reducing the chromium level in all tissues. The results of organ distribution of chromium before and after chelation therapies for chromium are shown in (Table 3). Furthermore, iron concentration after chromium administration was significantly decreased. The difference between iron values before and after chelation therapy is notable. Combination of deferasirox + deferiprone shows more efficiency in returning iron level to its normal state. The results of iron concentrations before and after chelation therapies are summarized in (Table 4). In order to investigate the effect of passing time in removing chromium from the body spontaneously, one group was treated as without chelation therapy. The results of chelation therapy group are shown in (Tables 3, 4). Comparison of the results obtained from both (before and without chelation therapy) groups indicate that the passing of time has no significant effect on the removal of chromium.
Table 3

The results of chromium levels before and after chelation therapies

Group

Before chelation therapy

Without chelation therapy

Chelation therapy with deferiprone

Chelation therapy with deferasirox

Combination

Liver (mg/kg)

 Control

0.112 ± 0.001

 Drinking (low dose)

0.311 ± 0.023

0.292 ± 0.019

0.224 ± 0.021

0.172 ± 0.018

0.146 ± 0.019

 Drinking (high dose)

0.943 ± 0.021

0.914 ± 0.023

0.643 ± 0.024

0.514 ± 0.017

0.192 ± 0.018

Kidney (mg/kg)

 Control

0.154 ± 0.019

 Drinking (low dose)

0.783 ± 0.027

0.713 ± 0.021

0.271 ± 0.011

0.251 ± 0.012

0.176 ± 0.012

 Drinking (high dose)

1.612 ± 0.029

1.544 ± 0.031

0.583 ± 0.017

0.472 ± 0.016

0.195 ± 0.022

Intestine (mg/kg)

 Control

0.104 ± 0.011

 Drinking (low dose)

1.531 ± 0.014

1.472 ± 0.013

0.204 ± 0.027

0.183 ± 0.023

0.124 ± 0.011

 Drinking (high dose)

2.163 ± 0.012

2.001 ± 0.014

0.254 ± 0.029

0.202 ± 0.021

0.154 ± 0.022

Spleen (mg/kg)

 Control

0.183 ± 0.019

 Drinking (low dose)

0.734 ± 0.012

0.692 ± 0.023

0.413 ± 0.011

0.274 ± 0.014

0.201 ± 0.031

 Drinking (high dose)

1.481 ± 0.024

1.411 ± 0.019

0.782 ± 0.029

0.621 ± 0.017

0.242 ± 0.024

Testicle (mg/kg)

 Control

0.154 ± 0.032

 Drinking (low dose)

0.272 ± 0.021

0.252 ± 0.011

0.213 ± 0.024

0.191 ± 0.027

0.172 ± 0.019

 Drinking (high dose)

0.451 ± 0.019

0.412 ± 0.022

0.231 ± 0.011

0.226 ± 0.013

0.221 ± 0.012

Five rats were placed in each group. Results are represented as arithmetic mean ± SEM and are significant at p < 0.05 when compared with control

Table 4

The results of iron levels before and after chelation therapies

Group

Before chelation therapy

Without chelation therapy

Chelation therapy with deferiprone

Chelation therapy with deferasirox

Combination

Liver (mg/kg)

 Control

5.45 ± 0.27

 Drinking (low dose)

5.1 ± 0.24

5.12 ± 0.24

5.26 ± 0.26

5.21 ± 0.31

5.31 ± 0.29

 Drinking (high dose)

4.56 ± 0.21

4.58 ± 0.19

5.31 ± 0.28

5.27 ± 0.27

5.41 ± 0.21

Kidney (mg/kg)

 Control

7.51 ± 0.25

 Drinking (low dose)

6.21 ± 0.21

6.27 ± 0.31

7.48 ± 0.27

7.06 ± 0.33

7.52 ± 0.24

 Drinking (high dose)

6.1 ± 0.23

6.15 ± 0.27

7.45 ± 0.24

7.31 ± 0.37

7.50 ± 0.26

Intestine (mg/kg)

 Control

1.25 ± 0.12

 Drinking (low dose)

1.14 ± 0.21

1.15 ± 0.21

1.19 ± 0.23

1.22 ± 0.14

1.24 ± 0.17

 Drinking (high dose)

1.11 ± 0.17

1.13 ± 0.19

1.17 ± 0.24

1.20 ± 0.19

1.22 ± 0.27

Spleen (mg/kg)

 Control

16.86 ± 0.22

 Drinking (low dose)

15.22 ± 0.37

15.30 ± 0.42

16.68 ± 0.39

16.72 ± 0.34

16.81 ± 0.32

 Drinking (high dose)

15.00 ± 0.38

15.90 ± 0.31

16.57 ± 0.32

16.61 ± 0.47

16.79 ± 0.39

Testicle (mg/kg)

 Control

4.10 ± 0.27

 Drinking (low dose)

2.51 ± 0.21

2.53 ± 0.17

3.73 ± 0.19

3.86 ± 0.24

3.99 ± 0.21

 Drinking (high dose)

2.47 ± 0.23

2.49 ± 0. 22

3.81 ± 0.24

3.90 ± 0.28

3.95 ± 0.22

Five rats were placed in each group. Results are represented as arithmetic mean ± SEM and are significant at p < 0.05 when compared with control

Discussion

The aim of the present research was to evaluate the ability of deferasirox + deferiprone in removing chromium from rat organs. 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-thalassaemia major (Cappellini 2008; Neufeld 2006). In this investigation, a short-term experimental model was used in order to speed up the preliminary testing procedure.

The effects of these chelators on chromium and iron levels were remarkable. It has been reported that the chelating agents having higher stability constants with a metal in aqueous solution may also prove successful in reducing the body burden of the metal (Kaur et al. 1984). Gastrointestinal absorption and uptake of chromium after oral exposure show the accumulation of chromium in various tissues as well as decrease of iron concentration in them. In order to understand the abilities of mentioned chelators, we have done the distribution of chromium and observed accumulation of direct toxic effect of chromium in rat organs. After the administration of chelating agents, the chromium content returned to nearly normal level of the control group, which indicates that deferasirox and deferiprone increase the elimination of chromium in rat organs and the symptoms are greatly disappeared. A comparison of the results obtained from with and without chelation therapies indicate that combined (deferasirox + deferiprone) therapy increases the elimination of chromium from rat organs effectively. The important finding that deferasirox + deferiprone leaves tissue iron levels close to normal is fundamental and would suggest that the proposed use of these two chelators will not be highly toxic. Our finding in Table 4 shows that there is virtually no difference in iron levels when the animals are treated with either deferiprone alone or deferasirox + deferiprone. This is particularly true in the kidney. The reason for this important observation is that deferiprone is able to redistribute iron in mammals (Evans et al. 2012). In comparison to the results obtained by Fatemi et al. (2007, 2009), Amiri et al. (2007), Tubafard and Fatemi (2008) it can be also concluded that the two chelators (deferasirox + deferiprone) are more efficient as combined therapy than single therapy in removing chromium from rat organs. Therefore combined therapy could eliminate chromium from rat organs and treat side-effects and the general symptoms of toxicity caused by chromium. Thus combination of deferasirox + deferiprone represent a promising drug of chromium-mobilizing agent. Also with considering that, their toxicities are relatively low, therefore they could be recommended for human administration.

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© Springer Science+Business Media New York 2013