chelation of chromium(vi) by combining deferasirox and deferiprone in rats

Chelation of chromium(VI) by combining deferasiroxand deferiprone in rats

Marzieh Iranmanesh • S. Jamil A. Fatemi •

Roza Ebrahimpour • Faezeh Dahooee Balooch

Received: 13 February 2013 / Accepted: 2 May 2013 / Published online: 14 May 2013

� Springer Science+Business Media New York 2013

Abstract The present research is aimed to charac-

terize 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 chro-

mium(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 Deferasirox � Deferiprone �Chromium toxicity � Chelation therapy � Rats

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 toxico-

logical 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). Chro-

mium(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 chro-

mium, 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

M. Iranmanesh (&) � S. J. A. Fatemi � R. Ebrahimpour �F. Dahooee Balooch

Chemistry Department, Shahid Bahonar University

of Kerman, 22 Bahman Blvd, PO BOX 76961,

Kerman, Iran

e-mail: [email protected]

S. J. A. Fatemi

e-mail: [email protected]

123

Biometals (2013) 26:465–471

DOI 10.1007/s10534-013-9631-5

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), Tubaf-

ard 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 pro-

motes the excretion of chelatable iron from hepatocel-

lular 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 mem-

branes (Glickstein et al. 2006). At present, combination

therapy with deferiprone and desferoxamine, that is

highly selective for iron(III) under biological condi-

tions (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. Presume-

ably 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 (Vosk-

aridou et al. 2011). This kind of therapy by combining

two chelators is based on the assumption that various

chelating agents mobilize toxic elements from differ-

ent 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 deferasi-

rox and deferiprone in combination, given to animals

after chromium loading. Testing was performed by

using a chronic poisoning model on rats with individ-

ual and combined chelators given shortly after chro-

mium application.

Experimental section

Apparatus

A Varian atomic absorption spectrometer (FAAS) was

used for measurement of chromium and iron concen-

trations 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

Fig. 1 Chemical structures of deferasirox (a) and deferiprone

(b)

466 Biometals (2013) 26:465–471

123

(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 pur-

chased 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 acclimatiza-

tion of the animals, we assigned them randomly to

control and treated groups.

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 administra-

tion. After chelation therapy, these rats were anesthe-

tized 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.

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 determina-

tions) ± 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 statisti-

cally 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

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)

a Mean of five determination ± standard deviation

Biometals (2013) 26:465–471 467

123

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 chro-

mium 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 con-

centration returned to normal levels and the symptoms

also reduced. Iron level is lowest in the groups having

the highest chromium concentration, which is proba-

bly 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 ? deferi-

prone 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 dif-

ference between iron values before and after chelation

therapy is notable. Combination of deferasirox ? def-

eriprone 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 spontane-

ously, 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.

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 b-thalassaemia major (Cap-

pellini 2008; Neufeld 2006). In this investigation, a

short-term experimental model was used in order to

speed up the preliminary testing procedure.

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)

468 Biometals (2013) 26:465–471

123

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 deferi-

prone 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

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 – – – –



Intestine (mg/kg)

Control 0.104 ± 0.011 – – – –



Spleen (mg/kg)

Control 0.183 ± 0.019 – – – –



Testicle (mg/kg)

Control 0.154 ± 0.032 – – – –



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

Biometals (2013) 26:465–471 469

123

that, their toxicities are relatively low, therefore they

could be recommended for human administration.

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chelation of chromium(vi) by combining deferasirox and deferiprone in rats

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