ORI GINAL RESEARCH

Removal of vanadium by combining desferrioxamineand deferiprone chelators in rats

Solmaz Tubafard Æ S. Jamilaldine Fatemi ÆAmir Shokooh Saljooghi Æ Masoud Torkzadeh

Received: 21 November 2008 / Accepted: 20 August 2009 / Published online: 17 September 2009

� Birkhauser Boston 2009

Abstract Investigations were conducted to evaluate the ability of two chelators,

desferrioxamine (DFO), and deferiprone (1,2-dimethy1-3-hydroxypyride-4-one, L1),

for the excretion of vanadium after a period of administration of vanadium salts in 6-

week-old male Wistar rats. Immediately after 60 days of vanadium administration, the

rats received chelators (L1, DFO or L1 ? DFO) for a period of 1 week. Chelators were

given orally (L1), intraperitoneally (DFO), or both to different groups of rats at two

different dosage levels. After chelation therapy, animals were sacrificed by exsan-

guination from abdominal aorta. Blood, kidney, liver, and heart samples were col-

lected and prepared for determination of vanadium and iron concentrations by graphite

furnace and flame atomic absorption spectroscopy (GF AAS, and F AAS) methods,

respectively. These chelators significantly enhanced the urinary and biliary excretion

of vanadium and restored the altered levels of iron. Furthermore, the hypothesis that

these two known chelators might be more effective in removing vanadium from the

body as a combined treatment than as monotherapy also was tested in this study.

Although there is no significant difference between these two chelators in reducing the

vanadium concentration, combination therapy (L1 ? DFO) may cause higher efficacy

and lower toxicity compared with monotherapies. Collectively, the results indicate

that the designed procedure might be useful for preliminary in vivo testing of the

efficiency of a chelating agent. However, our findings regarding the efficacy of

combination therapy should be confirmed in more experiments. This preliminary

study does not provide all answers to the magnitude of the efficiency of chelating

agents in vanadium toxicity, and thus, further research is warranted.

S. Tubafard � S. J. Fatemi (&) � A. S. Saljooghi

Chemistry Department, Shahid Bahonar University of Kerman, Kerman 76169, Iran

e-mail: fatemijam@yahoo.com

M. Torkzadeh

International Center of Science and High Technology and Environmental Sciences, Kerman, Iran

Med Chem Res (2010) 19:854–863

DOI 10.1007/s00044-009-9235-3

MEDICINALCHEMISTRYRESEARCH

Keywords Deferiprone � Desferrioxamine � Detoxification � Vanadium toxicity

Introduction

Vanadium ion is toxic to animals. Under environmental conditions, vanadium may

exist in oxidation states ?3, ?4, and ?5. Natural sources, in order of importance, are

continental dusts, volcanoes, sea salt spray, forest fires, and biogenic processes. The

air level of vanadium in industrial areas where high-vanadium fossil fuels are burnt

may be as much as 64 ng/m3. Water discharged from metallurgical plants may

contain hundreds of milligrams of vanadium per liter. In many industrial operations,

V2O5 is produced as a fume (condensation aerosol) consisting of small, respirable

particles with a potential for overexposure through inhalation. Inhaled vana-

dium compounds persist in, and are absorbed from, the lungs to different extents,

depending on their solubility; and absorbed vanadium can be distributed to all

organs. Vanadium compounds are acutely toxic by most routes of exposure, in most

species. Although dermal absorption of vanadium is not significant, toxicity has been

shown to be much higher by the oral route (Szakmary et al., 2002). Repeated

administration of vanadium compounds produces changes indicative of effects on

protein metabolism, such as a decrease in serum albumin concentrations, increase in

serum globulin, and changes in plasma amino acid concentrations. Various changes

in enzyme activities in blood and monoamine also have been described (Domingo,

1996). In human beings, long-term overexposure to vanadium causes wheezing.

Marked inflammation of the whole respiratory tract with pulmonary edema as well as

conjunctivitis, enteritis, and fatty infiltration of the liver has been reported. Mild

cases show sensory irritation, variable fever, conjunctivitis, and increased intestinal

motility. In moderate cases, there may be bronchospasm, cough, and vomiting and/or

diarrhea. Bronchitis or bronchopneumonia and signs of systemic toxicity, including

tremor and irreversible renal tubular damage, changes in the heart rhythm, right axis

deviation, and P-wave changes in the electrocardiogram have all been reported in

human beings, indicating the toxicity of vanadium compounds (Domingo, 1996).

Chelation therapy is a medical treatment in which a chelator is added to the blood

through a vein or administered orally to remove toxic elements that may be potentially

fatal. Chelation therapy involves the use of ligating drugs that bind to metals. These

ligands promote the excretion and subsequent depletion of the transition metal in

biological systems (Gomez et al., 1988a). 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 (Gomez et al., 1988a; Clarke and Martell, 1992).

Desferrioxamine (DFO) has been the most widely used chelator for the treatment

of iron overload. Its oral use is restricted because of its oral inactivity and therefore

subcutaneous or intravenous administration is usual. It has numerous side effects

and a high production cost. DFO also was found to be a maternal, embryo, and

terato-toxic agent in some animal species (Bosque et al., 1995; Kontoghiorghes,

1995). The simple synthesis of a new chelator (L1) for iron overload was described

previously by Kontoghiorghes and Sheppard in 1987 (Fig. 1). The human studies

showed that efficiency of L1 was comparable to that of DFO (Kontoghiorghes, 1995).

Med Chem Res (2010) 19:854–863 855

L1 is water soluble and can be given orally. These two chelators have different

abilities through the organism (Berdoukas et al., 1993). Hence, they may be used as a

combination. This kind of combination therapy is based on the assumption that

various chelating agents can mobilize toxic element from different tissue compart-

ments and therefore better overall results could be expected. Recent studies with

chelators that have different lipophilic properties given in combination have shown

favorable efficacy to mobilize lead (Flora et al., 1995), mercury (Kostial et al., 1996),

and cadmium (Kostial et al., 1997).

This study was designed to test the chelation potency of DFO and L1 while given

to animals solely or in combination after vanadium loading. Testing was performed

by using an acute experimental model on rats with mono or combined chelators

given shortly after vanadium application.

Materials and methods

Maintenance of the animals

Male Wistar rats were obtained from the Razi Institute of Karaj. They were bred in

the animal house at the Department of Biology in Shahid Bahonar University of

Kerman. The rats were kept in well-cleaned cages under a controlled light [dark

(12:12 h)] scheduled at 23 ± 1�C and humidity of 55%. Body weight of the rats

was measured in different stages of the study. Rat food was purchased from the

Karaj Institute, Tehran. This study was approved by the ethics committee of the

Shahid Bahonar University, Kerman, Iran.

Materials

Deferiprone (1,2-dimethyl-3-hydroxypyride-4-one, L1) was synthesized using a

previously described method (Kontoghiorghes and Sheppard, 1987). DFO and other

materials were purchased from Merk Chemicals Co.

Fig. 1 Simple synthesis of a new chelator (L1) for iron overload

856 Med Chem Res (2010) 19:854–863

Instrument

A microwave oven, model CEM MDS 200, was used to remove the water content

present in organs and to facilitate digestion. Varian model graphite furnace and

flame atomic absorption spectrometer (GFAAS and FAAS) were used to measure

the vanadium and iron concentrations in organs, respectively. A Mettler analytical

balance model AE 160 also was used in this study.

Methods

Study population consisted of 40 male Wistar rats that were all 6 weeks old. The rats

were individually caged in stainless steel and plastic cages with griddled bottoms.

Ten rats were recruited and named as the ‘‘control’’ group. These rats received

normal food and drink during the period of the study. Five of them were killed at the

end of the vanadium administration stage (day 60), whereas the remaining were

killed at the end of the study (day 67). The concentration of vanadium and iron in

blood and tissues were compared with that of the groups that received vanadium and

chelators. This was done to find out the normal amount of vanadium and iron in the

body of rats during the study period and to ensure that there were no unknown roots

of exposure to vanadium and iron during the full course of study. The remaining 30

rats were divided into two categories of 15 rats each and received vanadium salt at

two different dosage levels (low-dose category = 20 mg/kg, high-dose cate-

gory = 40 mg/kg) during a period of 60 days. After this stage, three rats from each

category were randomly selected and named as the ‘‘before chelation therapy’’ group

(n = 6). They were anesthetized with ether vapor, immobilized by cervical

dislocation, and sacrificed. Liver, kidneys, and heart were removed and dried.

Blood samples were taken by cardiac puncture and allowed to clot, and subsequently,

serum was removed by centrifugation. The vanadium and iron contents of the

mentioned tissues were determined, indicating the concentration of vanadium and

iron immediately after 60 days of vanadium administration at two different dosage

levels. Three rats from the two aforementioned categories also were recruited at this

stage and named as ‘‘without chelation therapy’’ group (n = 6). In this group, after

those 60 days of vanadium administration, the rats were given normal food and drink

until the end of the study without any chelating drugs. They were killed at the end of

the study to show the effect of passing time in concentration of vanadium and iron in

rat organs. The remaining rats in low-dose and high-dose categories (9 rats in each

category) were divided into three groups of three rats each. These rats were given L1

(orally), DFO (intraperitoneally), or both, respectively. Chelators were administered

during a period of 1 week immediately after vanadium administration, similar to that

explained by Gomez et al. (1988b). Doses of chelators were calculated based on the

rats’ weight (150 mg/kg body), and they were dissolved in deionized water or saline

solution.

After chelation therapy, animals were killed by exsanguinations from abdominal

aorta, and tissue and blood samples were collected for determination of vanadium and

iron contents to evaluate the efficacy of chelators in removing the toxic metal. The

blood samples were allowed to clot and the serum was removed by centrifugation. The

Med Chem Res (2010) 19:854–863 857

samples were placed in oven at 60�C for digestion for 3 days (1.5 ml HNO3 was added

per 1 g of dry weight). After digestion, the solutions were evaporated with the addition

of 1.0 ml H2O2 under the hood. The residue was then diluted with water to a volume of

100 ml. These samples were analyzed by graphite furnace atomic absorption

spectroscopy (GF AAS) on Shimadsu instrument (Ishida et al., 1989). The data were

statistically analyzed by Student’s t test (ref). P \ 0.05 was considered significant.

Determination of vanadium and iron

Determination of vanadium and iron in samples was performed by GFAAS and

FAAS spectrometry, respectively. Values are depicted as means and their standard

deviation were for at least three separate measurements.

Results

Mean initial body weight of the rats was 143 g, and after 60 days of vanadium

administration those given vanadium weighed significantly less (Table 1). Dietary

treatment also affected the food intake, whereby animals given normal diet

consumed more food than those given vanadium. Some of the vanadium toxicity

symptoms that appeared during the period of vanadium uptake included appearance

of black line on gums, loss of appetite and weight, loss of hair, skin reactions, and

reduction in food consumption and weight of organs. The highest amount of

vanadium was found in the liver followed by blood and kidney.

The increase in vanadium and the reduction in iron in blood and other organs

were statistically different in two dose groups. The accumulation of vanadium in

tissues at the 40-mg/kg dose was greater than that in the group that received 20 mg/

kg of vanadium. From the obtained data, it is clear that there is a significant increase

in vanadium concentration (p \ 0.05) in various tissues compared with the control

group.

The effects of administration of the chelators on vanadium and iron concentra-

tions in the various tissues are summarized in Tables 2 and 3. It is obvious that the

vanadium concentration after chelation therapy was significantly decreased

(p \ 0.05). Spontaneous elimination of vanadium by the biological system in the

groups without the chelation therapy is not noticeable (Table 2). The vanadium

concentration of the diet had a significant effect on iron status, as assessed by

deposited iron in various tissues (Table 3). Iron level was lowest in the group that

had the highest vanadium concentration, which is probably due to a significant

interference that could take place by vanadium through iron uptake mechanism.

Table 1 Body weight of the rats after 60 days of vanadium intake

Group Control Low-level vanadium High-level vanadium

Final body weight (g) 260 (n = 10) 215 (n = 15) 170 (n = 15)

Values are mean for number of observation

858 Med Chem Res (2010) 19:854–863

After chelation therapy, vanadium level present in tissues was significantly reduced,

and simultaneously, iron concentrations returned to the normal level and the

symptoms of toxicity also were reduced. Interactions between vanadium and iron

have not been previously reported. It is not clear whether vanadium interferes with

iron absorption and/or the subsequent metabolism, but it may be possible that

vanadium absorption takes place along with pathways for essential metals. If this is

the case, iron deficiency could result in increased absorption, as it is observed in

several organic elements, such as lead, cobalt, vanadium, and indium. Vanadium

levels in different groups and categories in different organs are depicted In Table 2.

The results of vanadium removal by chelators in the blood and liver of two doses

categories were statistically different. After chelation therapy, a significantly lower

value of vanadium was observed in both two doses groups. There is a statistically

significant difference between DFO and L1 in reducing the amount of vanadium in

liver. The results were found to be in agreement with the certified values at the 95%

confidence interval. The t test was applied to the results assuming the certified

values are the true values. The result of the experiment passed the t test at the 95%

confidence level and was significant for liver. At both lower and higher doses,

DFO ? L1 groups were more effective than DFO or L1. When comparing the

efficacy of monotherapies with each other, we found that DFO was more efficient in

decreasing vanadium concentration except in kidney. However, the efficiency of L1

Table 2 Results of vanadium contents in various tissues of rats before and after single and combined

chelation therapies

Group Before chelation

therapy

(day 60)

Without

chelation

therapy

(day 67)

Chelation

therapy

with DFO

(day 67)

Chelation

therapy

with L1

(day 67)

Combination

therapy

(DFO ? L1)

(day67)

Kidney (lg/kg)

Control 12.80 ± 1.5 12.55 ± 3.4

Low-dose group 43.01 ± 3.1 42.8 ± 2.2 23.1 ± 2.1 22.01 ± 1.7 12.99 ± 1.5

High-dose group 68.02 ± 5.4 67.5 ± 4.2 40.01 ± 3.5 39.01 ± 4.8 13.03 ± 2.7

Liver (lg/kg)

Control 33.0 ± 4.2 32.8 ± 5.6

Low-dose group 130.0 ± 3.5 128.0 ± 4.1 99.0 ± 1.8 115.0 ± 3.6 35.0 ± 4.2

High-dose group 184.0 ± 6.2 183.1 ± 6.8 115.01 ± 3.1 118.05 ± 5.2 36.0 ± 2.2

Heart (lg/kg)

Control 3.80 ± 0.36 3.71 ± 0.42

Low-dose group 11.01 ± 2.9 10.51 ± 3.3 7.02 ± 1.5 7.26 ± 3.3 4.21 ± 2.8

High-dose group 26.03 ± 2.5 25.14 ± 1.9 16.01 ± 2.7 18.02 ± 3.4 4.81 ± 2.4

Blood (lg/l)

Control 2.80 ± 0.29 2.68 ± 0.35

Low-dose group 12.10 ± 1.5 11.56 ± 2.4 7.42 ± 1.4 7.98 ± 3.6 3.61 ± 2.3

High-dose group 14.02 ± 2.3 13.22 ± 3.6 7.01 ± 1.6 8.05 ± 3.5 3.82 ± 2.4

Results are presented as arithmetic means ± SEM; p \ 0.05 compared with control

Med Chem Res (2010) 19:854–863 859

has been observed to reduce vanadium concentration in the kidney. Comparison of

mono and combining chelators in this experiment shows more efficiency of

DFO ? L1 in reducing the vanadium level in all tissues (Table 2).

After administration of vanadium, iron concentration was significantly decreased.

The difference between iron values before and after chelation therapy is notable.

Combination of DFO and L1 shows more efficiency in returning iron level to normal

state. The results of iron concentrations before and after chelation therapies

are summarized in Table 3. The decreases were statistically significant after the

coadministration.

To investigate the effect of passing time in removing vanadium from the body

spontaneously, one group was treated without chelation therapy. The results passed

the t test at 95% confidence level and were significant.

Discussion

Chelation therapy is one of the most effective methods to remove toxic elements

from a biological system. Despite the fact that the efficacy of DFO is well

documented, not all patients are able to cope with the rigorous requirements of the

long-term use of portable pumps. In addition, the high cost of this treatment is a

Table 3 Result of iron level in various tissues of rats before and after single and combined chelation

therapies

Group Before

chelation

therapy

(day 60)

Without

chelation

therapy

(day 67)

Chelation

therapy with

DFO (day 67)

Chelation

therapy

with L1

(day 67)

Combination

therapy

(DFO ? L1)

(day 67)

Kidney (mg/kg)

Control 4.326 ± 0.36 4.330 ± 0.41

Low-dose group 2.410 ± 0.24 2.418 ± 0.38 3.390 ± 0.18 3.495 ± 0.22 3.985 ± 0.17

High-dose group 2.400 ± 0.26 2.411 ± 0.34 2.638 ± 0.41 2.544 ± 0.29 4.995 ± 0.31

Liver (mg/kg)

Control 5.425 ± 0.15 5.439 ± 0.58

Low-dose group 3.193 ± 0.11 3.201 ± 0.44 3.693 ± 0.23 3.861 ± 0.16 4.885 ± 0.21

High-dose group 3.415 ± 0.25 3.421 ± 0.42 4.509 ± 0.31 4.267 ± 0.13 4.948 ± 0.22

Heart (mg/kg)

Control 6.745 ± 0.46 6.750 ± 0.52

Low-dose group 2.861 ± 0.29 2.865 ± 0.36 4.722 ± 0.33 4.400 ± 0.16 5.924 ± 0.24

High-dose group 3.945 ± 0.25 3.949 ± 0.39 4.257 ± 0.17 4.995 ± 0.31 5.965 ± 0.42

Blood (mg/l)

Control 22.341 ± 0.54 22.348 ± 0.61

Low-dose group 13.752 ± 0.39 13.755 ± 0.47 17.004 ± 0.42 16.064 ± 0.28 20.947 ± 0.25

High-dose group 11.133 ± 0.52 11.139 ± 0.68 15.905 ± 0.18 15.589 ± 0.35 21.509 ± 0.37

Results are presented as arithmetic means ± SEM; p \ 0.05 compared with control

860 Med Chem Res (2010) 19:854–863

serious obstacle to its more widespread use (Hershko, 2002). Therefore, there is a

great need for the development of alternative, orally effective chelating drugs.

Recently more than 1,000 candidate compounds were screened in animal models.

Of all the new chelating drugs available today, only deferiprone has been used as a

substitute for DFO in clinical trials involving many hundreds of patients (Barman

Balfour and Foster, 1999). However, the high cost and rigorous requirements of

DFO therapy and the significant toxicity of deferiprone underline the need for the

continued development of new methods, such as combined therapy. In view of these

considerations, the goal of the present study was to characterize the ability of two

chelators in removing this toxic element. Gastrointestinal absorption and uptake of

vanadium after oral exposure lead to the accumulation of vanadium in the kidney

and liver together with tissue damage and a reduction in iron concentration of blood.

These results show a direct toxic effect of vanadium and also indicate that both DFO

and L1 effectively increased the elimination of vanadium in rats.

In Table 2, a significant increase of vanadium in the liver among control and

drinking groups is shown. After chelation therapy, the vanadium level returned to

vanadium level of the control group, which indicates the efficacy of both chelators

in the elimination of vanadium from rats. The coadministration of two chelators

shows more ability to reduce vanadium element because of its relationship to their

stability constants for this element. The L1 appears to mobilize vanadium from the

tissues. L1 may have a better cardioprotective effect than DFO due to the ability of

deferiprone to penetrate cell membranes (Hershko et al., 2005).

In this investigation, a short-term experimental model was used to speed up the

preliminary testing procedure. The effect of chelators on iron level was remarkable.

There is no significant difference between DFO and L1 in increasing the iron level,

whereas DFO ? L1 are more efficient to enhance the iron level.

Our results indicate that this procedure might be useful for preliminary testing

of the efficiency of chelating agents in removing vanadium in vivo for several

reasons. Although a significant mobilization of vanadium occurred after both DFO

and L1 individual applications, the effect of their coadministration was observed to

be significantly potentate. Combined therapy by L1 and DFO provides synergistic

effects in vanadium burden reduction. The basis for this effect is that L1 easily

enters cells and is subsequently able to transfer the intracellular chelated vanadium

to the stronger chelator DFO in tissues. The combination of a weak chelator, which

has a better ability to penetrate cells, with a stronger chelator, which penetrate cells

poorly but has a more efficient urinary excretion, may result in a synergistic effect

through vanadium shuttling between the two compounds. The ability of deferi-

prone to penetrate cell membranes because of its low molecular weight and easy

use with oral administration and decreasing the side effect of DFO along with

mobilization property of DFO in tissues has proved the combination use of these

two chelators.

This study suggests an interaction between deferiprone and DFO and may have

important implications to the design of new strategies in toxic metals chelating

treatments. Therefore, chelating therapy could change the quality of life and life

expectancy of patients. However, development and the evolution of improved

Med Chem Res (2010) 19:854–863 861

strategies of chelating therapy require better understanding of the pathophysiology

of metal toxicity and the mechanism of action of vanadium chelating drugs.

We obtained the dose of decreasing vanadium concentration for the two known

chelating agents, L1 and DFO, as expected. We observed a higher efficiency of

DFO ? L1 in enhancing iron level as expected in relation to their stability constant

for iron. A comparison of the results obtained with and without chelation therapy

indicate that L1 and DFO effectively increase the elimination of vanadium,

therefore, it was not greatly time dependent. This testing procedure does not provide

all of the relevant answers for evaluating the efficiency of a chelating agent for

vanadium toxicity, such as kinetic data, vanadium dosing, etc. Despite these

shortcomings, the results indicate whether a new chelating agent or chelating agent

mixtures deserves further testing.

Conclusions

The results of this study demonstrate that the addition of L1 increases the effect of

DFO on vanadium elimination. This might provide additional information about

the potential usefulness of combined chelation treatment of vanadium toxicity.

However, the effects of the chelating agents, including Na2Ca-ethylen diam-

inetetraacetate (EDTA), Na3Ca-diethylen triaminepentaacetate (DTPA) (Tor et al.,1982), L-cysteine, 4, 5-dihydroxy-1, 3-benzene-disulfonic acid (Tiron) (Domingo

et al., 1986; Gomez et al., 1991), and the reducing agent ascorbic acid, on the

toxicity, excretion, and distribution intraperitoneally have been studied in male

Swiss mice. Chelating and reducing agents have been administered intraperitoneally

at doses equal to one-fourth of their respective LD50. Significant increases in

survival were noted with ascorbic acid, Tiron, and desferrioxamine (Gomez et al.,1988c). However, their clinical uses have encountered several limitations, including

low efficacy, toxicity, and side effects. Iron has been shown to be a potent inducer of

cell differentiation and apoptotic cell death in human promyelotic HL-60 leukemia

cell (Kim et al., 2006). ICL670 is the most advanced in development and appears to

reduce effectively the iron in the liver in some patients but is overall ineffective in

causing negative iron balance. It also is suspected that it is not effective for cardiac

iron removal. Combination therapies using L1 and DFO may cause higher efficacy

and lower toxicity compared with monotherapies (Kontoghiorghes, 2006).

To understand the abilities of the two mentioned chelators, we have done the

distribution of vanadium and observed accumulation of vanadium in the various

tissues and also early administration of chelating agents. Due to these consider-

ations, combination therapy using L1 and DFO causes higher efficacy and lower

toxicity compared with monotherapy. Our results support the usefulness of this

animal model for preliminary in vivo testing of vanadium chelators. The results of

combined chelator treatment confirmed the use of this method.

Acknowledgment The authors thank the head and director of International Center of Science, High

Technology and Environmental Science and Shahid Bahonar University of Kerman Faculty Research

Funds for their support of these investigations.

862 Med Chem Res (2010) 19:854–863

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