Mutagenesis vol. 18 no. 5 pp. 457-463,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Chromosomal aberration frequencies in patients with thalassaemia major undergoing therapy with deferiprone and deferoxamine in a comparative crossover study*
1Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK, 2Apotex Research Inc., 150 Signet Drive, Weston, Ontario, Canada M9L 1T9 and 3Istituto di Clinica e Biologia dell’Età Evolutiva/Ospedale Regionale Microcitemie ASL, Cagliari, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Measurements of chromosomal aberrations were made in 10 thalassaemia major patients treated long-term with deferiprone (at least 5 years) and compared with an equal number of patients matched for age, sex and iron overload, treated long-term with deferoxamine. Two blood samples were collected from each patient, 7 and 20 days after a transfusion episode, and the frequency of chromosomal aberrations (gaps, breaks and exchanges) in the patients’ circulating lymphocytes analysed in both samples using standard cytogenetic staining techniques. The frequency of reciprocal translocations was also analysed using fluorescence in situ hybridization. Relatively low frequencies of cells with stable and unstable aberrations were seen at both sampling times in all patients, with no statistically significant differences between sexes. Chromosomal aberrations were less frequent in patients treated long-term with deferiprone than in patients treated with deferoxamine, although the difference did not reach statistical significance. After the second blood sample had been collected, all patients had their iron chelation therapy switched to the other chelator. Patients treated long-term with deferiprone had their therapy switched to deferoxamine and patients treated long-term with deferoxamine had their therapy switched to deferiprone. After the switch, two further blood samples were collected 7 and 20 days after transfusion for each of the next two transfusion cycles in all patients. Analysis of the post-switch samples also revealed a slightly higher frequency of chromosomal aberrations during therapy with deferoxamine than with deferiprone at all time points. A small, but statistically significant, increase in cells with aberrations was observed at the first post-switch assessment in the group of patients whose therapy was switched from deferiprone to deferoxamine, whereas the switch from deferoxamine to deferiprone was associated with a decrease in the frequency of chromosomal aberrations. The results of the study demonstrate that, in a clinical setting, deferiprone has no greater clastogenic activity than that of deferoxamine.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
ß-Thalassaemia major is a genetic blood disease caused by mutations on chromosome 11 that cause a decrease in the production of ß-globin chains. The ensuing abnormal globin chain ratio results in ineffective erythropoiesis, haemolysis and severe anaemia. To survive, patients with ß-thalassaemia major require regular blood transfusions, which lead to a progressive tissue accumulation of iron. As there is no natural means for the body to eliminate the excess iron, the progressive increase in iron load results in damage to the heart, liver and endocrine organs. Ultimately death occurs, mainly due to cardiac haemosiderosis. In addition to the organ damage, excess iron can also lead to DNA damage by catalysing the production of reactive oxygen species within the cell, leading to the induction of chromosome aberrations (Whiting et al., 1981
; Hartwig et al., 1992; Hartwig and Schlepegrell, 1995). This was suggested to account for the observation of increased frequencies of micronuclei (Krishnaja and Sharma, 1994) and chromosomal aberrations (Côté and Papadakou-Lagoyanni, 1979) in cultured lymphocytes from thalassaemia patients. For these patients, the use of chelating agents, such as those described in this article, may protect not only against iron-induced organ damage but also against the excessive iron-catalysed, oxidative DNA damage.
Currently there are two iron chelating agents available for the management of iron overload in thalassaemia: deferoxamine, which needs to be administered parenterally, and deferiprone, which has the advantage that it is taken orally. Both agents have been shown to have antioxidant and cytoprotective effects (van der Kraaij et al., 1989; Balla et al., 1990; Morel et al., 1992, 1995; Stinson et al., 1992; Chenoufi et al., 1995; Shalev et al., 1995; Beall et al., 1996; Hagar et al., 1996; Matthews et al., 1997; Niihara et al., 1998; Anderson et al., 2000). On the other hand, some studies have indicated that these agents may have clastogenic effects (Juckett et al., 1998; Gille et al., 1992; Cragg et al., 1998). This clastogenic effect may reflect a non-genotoxic mechanism due to the chelation of iron from cellular systems that depend on iron for normal functioning. A likely candidate for this is ribonucleotide reductase, an important enzyme in DNA synthesis. Although genotoxicity via this mechanism would be unlikely to occur in conditions of iron overload, little information is available on the potential of deferoxamine in inducing chromosome aberrations in the clinical setting (Vig and Eyring, 1971) and, to the best of our knowledge, no information is available on the clastogenic effect of deferiprone in patients with ß-thalassaemia major. The present study was undertaken, therefore, to determine whether deferiprone could induce chromosome aberrations in a clinical setting and, if so, what was its effect compared with deferoxamine.
Although the induction of aberrations in a clinical setting would be best addressed by looking for evidence of clastogenicity before and during therapy, this approach is not practical because virtually all subjects are treated with chelation therapy from early in childhood. The approach used in this study, therefore, was to assess the existing frequency of chromosomal aberrations (using conventional cytogenetic techniques for gaps, breaks and exchanges) in patients receiving long-term therapy on either chelator and then determine if there was a significant change in the frequency of chromosomal aberrations in circulating lymphocytes in these same patients following a switch in therapy to the alternative chelator.
In order to determine the ability of the chelators to induce stable chromosomal rearrangements (i.e. reciprocal translocations, not easily detected by conventional cytogenetic techniques) following long-term therapy, the frequencies of cells with reciprocal translocations (between chromosomes 1, 2 and 4 and the remainder of the genome) were evaluated in each group of patients prior to the switch in therapy by fluorescence in situ hybridisation (FISH) (Swiger and Tucker, 1996).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Study medication
Deferiprone (Ferriprox, L1) was supplied by Apotex Research Inc. (Toronto, Canada) as 500 mg tablets. Deferoxamine (Desferal®) (DFO), prepared by Novartis (Basel, Switzerland) was obtained as vials of 500 mg sterile, lyophilized powder and dissolved in water for injection prior to use.
Study design
To minimize the influence of potential confounding factors, the subjects were stratified in such a way so as to ensure equal numbers of males and females and equal numbers of subjects with high body iron burdens (i.e. serum ferritin >2500 µg/l). All patients studied had been treated with their current chelator for a minimum of 5 years prior to enrolment in this study. By conducting a randomized, controlled, crossover study, the influence of various potential contributors to clastogenicity were minimized, or at least accounted for, providing a better opportunity to evaluate the role of either chelator treatment. Each subject acted as his/her own control. Subjects were sampled while taking their pre-study chelator and then again after switching to the other chelator. In order to determine the frequency of chromosomal aberrations before and after switching, blood was collected on days 7 and 20 of each of three transfusion cycles (i.e. during therapy with each chelator) (Figure 1). To avoid carryover of the chelating agents into the lymphocyte cultures, subjects were instructed to complete their deferoxamine infusion by 08:00 a.m. or were not prescribed their morning dose of deferiprone on the days of sampling. The two sampling times prior to switching chelation therapy served to establish steady-state aberration frequencies for the respective chelators, while the four post-switch samples provided information on the effect of a change in chelation therapy.
|
Group 1 Subjects in this group continued to receive deferiprone (their pre-study chelator) for the first full transfusion cycle. Deferiprone (25 mg/kg) was administered three times per day for a total daily dose of 75 mg/kg. The evening of transfusion number 2 (day 0, cycle 2), the subjects in this group began to take deferoxamine at 20–60 mg/kg body wt delivered by s.c. infusion, 4–7 days/week, until day 20 of the second transfusion cycle. The attending physician determined the dose of deferoxamine that the subject received according to the specific needs of the subject.
Group 2 Subjects in this group continued to receive deferoxamine (their pre-study chelator) for the first full transfusion cycle (a period of at least 20 days, starting from the day of transfusion 1, until the morning of transfusion 2). The evening of the second transfusion (day 0, cycle 2), this group began to take deferiprone daily, administered as 25 mg/kg, three times per day for a total daily dose of 75 mg/kg, and continued that therapy until day 20 of transfusion cycle 3.
To ensure that the clinical effects of the drugs, rather than any in vitro artefacts, were being evaluated, the time point for the collection of blood was selected such that there would be minimal amounts of the respective drugs in the blood. Both deferoxamine and deferiprone have relatively short elimination half-lives (t
0.6 h for deferoxamine and 2–3 h for deferiprone). Subjects were therefore sampled at midday (13:00 h) to avoid carryover of drug into the lymphocyte cultures. Subjects on deferiprone forwent their early morning dose on the day of sampling. Subjects on deferoxamine had their previous evening infusion completed by 08:00 h on the day of sampling.
Study population
The patients consenting to participate in this study were undergoing treatment at the Istituto di Clinica e Biologia dell’Età Evolutiva/Ospedale Regionale Microcitemie A.S.L. (Cagliari, Italy). The following broad inclusion and exclusion criteria were used: subjects had a diagnosis of thalassaemia major and were regularly transfused with blood filtered by a blood bank, for white blood cells; subjects were 
10 years of age; subjects were receiving ongoing chelation therapy with deferoxamine or deferiprone; subjects were non-smokers and had not been smokers (>20 cigarettes/day) for the 6 months prior to initiation of the study; subjects were not receiving or were not known to have received therapy known to have clastogenic effects.
The median age of the patients at the time of enrolment was 18.5 years (range 16–23 years) in Group 1 and 18.0 years (range 10–27 years) in Group 2. After completion of the first cycle of the study, chelation therapy of those patients treated with deferiprone (Group 1) were switched to deferoxamine, with a median daily dose of 29 mg/kg (range 18–43 mg/kg) for the subsequent two study cycles. Patients in Group 2 were being treated with deferoxamine (median dose 31 mg/kg/day, range 23–44 mg/kg) at their enrolment in the study and were switched to deferiprone (75 mg/kg/day) after completion of the first cycle of the study.
Blood cultures
Blood samples were collected into tubes containing sodium heparin and shipped, on ice, to Covance Laboratories (Harrogate, UK). Shipments were arranged such that, in general, cultures were established within 48 h of sampling.
Whole blood cultures were established in disposable centrifuge tubes by placing 0.4 ml of heparinized blood into 9.6 ml of HEPES-buffered RPMI medium containing 20% (v/v) foetal calf serum, 50 µg/ml gentamycin and 2% phytohaemagglutinin (PHA) (reagent grade). Blood cultures were then incubated at 37°C and rocked continuously. Up to 10 cultures were established per blood sample. All cultures were incubated for 72 h such that a mixture of cells in first and second division after culture initiation could be sampled. 5-Bromodeoxyuridine (BrdU) was added to some cultures 24 h prior to harvest such that the final concentration was 50 µM. The addition of BrdU allowed measurement of cell cycle times to ensure that, in each case, broadly similar populations of cells were being analysed.
Harvesting and slide preparation
Approximately 3 h prior to harvest, colchicine was added to give a final concentration of 1 µg/ml to arrest dividing cells in metaphase. Cultures were then harvested, slides prepared and, where applicable, stained in Giemsa using standard cytogenetic procedures for chromosome aberration analysis.
The slides from the BrdU cultures were stained in Hoescht 33258 (26.7 µg/ml) in McIlvaines buffer at pH 8 for 
25 min and rinsed twice in McIlvaines buffer. Slides were then irradiated with UV light under McIlvaines buffer for approximately 35 min at 50°C. Slides were rinsed in buffer, pH 6.8, and stained for 10 min in filtered 4% (v/v) Giemsa in buffer.
FISH
Rhodamine-labelled whole chromosome DNA probes for chromosomes 1, 2 and 4 (Appligene-Oncor Ltd, Harefield, UK) were used. In most cases, slides were pre-treated with RNase (100 µg/ml, prepared in 2x SSC) for 1 h at 37°C, and pepsin (0.005% in 10 mM HCl for 10 min at 37°C).
The slides were washed for 5 min at room temperature in 2x SSC and 0.05% (v/v) Tween 20. They were then dehydrated in an ethanol series (normally 70, 80 and 95%) for 2 min each at room temperature, then allowed to dry at room temperature. The cells were denatured in 70% formamide/2x SSC, pH 7.0 at 72°C for 2 min, then dehydrated in an ethanol series as described. The DNA probe was pre-warmed at 37°C then denatured at 72°C for 5 or 10 min. The probe (15–30 µl/slide) was aliquoted and a coverslip placed on top and sealed with rubber cement. Slides were placed in a moist chamber at 37°C overnight. The slides were washed in 0.5x SSC at 72°C for 5 min then transferred to 4x SSC with 0.5% Tween 20 for 1–2 min. Slides were counterstained with DAPI antifade.
Cytogenetic analysis
Slides were coded such that time of sampling and patient identity were not revealed to the analysts.
Chromosome aberrations Where possible, 300 metaphases from each blood sample were analysed for chromosome aberrations. In general, this was done by scoring 100 cells from each of three cultures. Analyses were divided such that 50 cells/culture (i.e. 150 cells/sample) were scored by each of two analysts.
Only cells with 44–46 chromosomes were considered acceptable for aberration analysis. Polyploid, hyperdiploid or endoreduplicated cells were noted and recorded separately. Structural aberrations were classified according to the ISCN scheme (Mitelman, 1995) into chromosome and chromatid gaps, breaks and exchanges.
Cell cycle kinetics Slides from cultures incubated in the presence of BrdU from pre-switch samples were examined for the proportion of cells in the first (M1), second (M2) and third or higher (M3) divisions, based on the pattern of differential staining of the chromosomes. One hundred metaphases were analysed and the average generation time (AGT) determined using the formula:
AGT = [time of BrdU incorporation (24 h)]/[M1 + (2 x M2) + (3 x M3)]
Slides were also examined for mitotic index (MI), i.e. the percentage of cells in mitosis, based on 1000 cells analysed per culture to further evaluate cell proliferation.
FISH It was intended that 500 cells should be analysed from each pre-switch sample for reciprocal translocations only (which cannot usually be distinguished using standard cytogenetic techniques and transmitted light microscopy). The limited number of cells on some of the slides or inadequate fluorescence signal strength meant that it was not always possible to analyse 500 cells from each pre-switch sample. In most cases, however, the combined pre-switch cell samples totalled 1000 for each patient because additional cells were analysed from the other pre-switch sample to make up cell numbers. Because the day 7 and day 20 samples may be considered identical, however, for the purposes of evaluation of translocation frequency, this would have had no impact on the interpretation of the study.
Statistical methods
At the end of the study, the proportions of cells with aberrations, excluding gaps, in each of the two pre-switch blood samples for each subject were combined. In this way, the normal variability of chromosomal aberration frequencies was taken into account. Comparisons were made between the frequency of aberrant cells in the combined pre-switch data and the frequency of aberrant cells in each of the post-switch samples for each individual and for each drug group using analysis of variance (ANOVA). To evaluate not only an increase but also a decrease in the frequency of aberrant metaphases, a two-sided test was used. Probability values of P < 0.05 were accepted as significant.
Prior to the initiation of the study, it was decided that a power analysis should be performed on the ‘pre-switch’ data (obtained by scoring slides ‘blind’) from the first 10 subjects (five male and five female) in each group (10 subjects treated long-term with deferiprone and 10 subjects treated long-term with deferoxamine) for a total number of 20 subjects. Recruitment of subjects was interrupted during the analysis to determine if this number would give adequate statistical power. Based on the frequencies of cells with aberrations, the analysis revealed that 10 patients per group gave 90% power of detecting a 2-fold increase and that a larger treatment group was not required.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell cycle kinetics and chromosomal aberration analysis
Although variability in mitotic index was observed between cultures at each of the six sampling times for all individuals, no individual had consistently low mitotic indices. The accepted normal range for average generation time in human lymphocyte cultures at Covance laboratories is 13 ± 1.5 h. With one exception, mean data for the ‘pre-switch’ samples fell within this range. Relatively low frequencies of cells with chromosome aberrations were seen at all sampling times in all patients and there were no consistent differences in cell cycle kinetics attributable to treatment/transfusion regime (Table I).
|
The number of cells with aberrations was consistently higher for deferoxamine therapy than deferiprone therapy at each time point (Figure 2 and Table I)
|
Statistical analysis of the data reveals a small but statistically significant (P < 0.01) increase in cells with aberrations (excluding gaps) in Group 1 (patients switched from deferiprone to deferoxamine) when the proportion of cells with aberrations at day 7, cycle 2 was compared with the pre-switch sample by Fisher’s exact test (Table II). This was attributable to an increase in aberrant cells in males. Group mean frequencies of cells with aberrations in males and females at subsequent sampling times were lower than in the pre-switch samples.
|
In patients whose chelation therapy was switched from deferoxamine to deferiprone (Group 2), a significant decrease in the proportion of cells with aberrations was observed on three occasions post-switch [day 20, cycle 2 (P < 0.05); day 7, cycle 3 (P < 0.01); day 20, cycle 3 (P < 0.01)] when compared with the pre-switch samples (Table II).
FISH
No clonal rearrangements were apparent and a low frequency of cells with translocations was observed in all individuals (Table III). Theoretically, exchanges between chromosomes 1, 2 and 4 and the remainder of the genome comprised 34.4% of all exchanges (Tucker et al., 1994). After correcting the data for the whole genome, the mean translocation frequencies for Group 1 (deferiprone) males and females were 0.12 and 0 per 100 cells, respectively, and 0.12 and 0.07 per 100 cells for Group 2 (deferoxamine) males and females.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Relatively low frequencies of cells with chromosome aberrations were seen at all sampling times in all patients. The means of the historical negative control data for cells with aberrations (excluding gaps) in human lymphocyte cultures at Covance Laboratories are 0.87% (range 0–4%) for males and 1.13% (range 0–5%) for females. These values are based on data from 206 or 104 cultures (males and females, respectively) from normal individuals. The frequency of cells with aberrations in the cultures prepared from all thalassaemia patients in this study fell within these ranges. Our data, therefore, are not in agreement with those of Côté and Papadakou-Lagoyanni (1979
), who found an increase in aberrations in thalassaemia patients, but do concur with the results of Krishnaja and Sharma (1994). In most cases, frequencies of cells with chromosomal aberrations in patients receiving deferoxamine or deferiprone were similar. Nevertheless, a statistically significant decrease in the frequency of chromosomal aberrations was observed following a switch in therapy from deferoxamine to deferiprone at three of the four post-switch sampling times. Furthermore, a statistically significant increase in chromosomal aberrations was seen in patients switched from deferiprone to deferoxamine in the first post-switch sample, but thereafter differences were non-significant.
The results of translocation analysis using FISH support the conclusion that low frequencies of heritable aberrations are associated with long-term treatment with deferiprone or deferoxamine. The methods used in this study are similar to those described by Tucker et al. (1994), who examined frequencies of translocations in a control population of 53 individuals by scoring translocations between chromosomes 1, 2 and 4 and the remaining genome. The authors concluded that there was a relationship between translocation frequency and age. This relationship would predict a translocation frequency of 0.16% at age 18, the median age for the thalassaemia patients in this study. The frequencies of translocations that we observed, 0 and 0.12% in males and females, respectively, receiving long-term deferiprone therapy and 0.07 and 0.12% for males and females receiving long-term deferoxamine, are consistent with what would be expected in normal individuals.
All the cytogenetic data, therefore, indicate that therapy with deferiprone is not associated with a greater frequency of chromosomal aberrations than that observed with deferoxamine therapy. The data also provide no evidence that treatment with either chelator results in frequencies of aberrations which are higher than would be expected in normal individuals.
Deferiprone is a new iron chelating agent and its genetic toxicology has been investigated in a number of standard in vitro and in vivo mutagenicity assays (internal studies on file). Deferiprone was negative in the bacterial reverse mutation assay with TA98, TA100, TA1538, TA1537 and Escherichia coli WP2 uvrA. It was positive, however, in both the absence and presence of metabolic activation in the mouse lymphoma mutation assay with L5178Y TK+/– cells. It was also positive in a chromosome aberration assay in vitro in CHO cells after a 4 h exposure to 1250 µg/ml both in the absence and presence of S9. This activity was also seen in a mouse micronucleus test, where significant increases in polychromatic erythrocytes with micronuclei were observed 24 h after a single i.p. administration at 140 or 280 mg/kg.
Care should be taken, however, when extrapolating results from non-iron loaded to iron loaded systems and the activity of deferiprone in standard mutagenicity tests may not be relevant in the clinical setting, where iron overload is prevalent. Although no direct measurements of DNA binding have been made, it is likely that the genetic toxicity of deferiprone is not the result of interaction with DNA.
Ribonucleotide reductase is a key enzyme in DNA precursor synthesis. It functions to supply balanced pools of precursors and is therefore critical during DNA replicative and repair synthesis. Deferiprone is known to inhibit this enzyme, which contains a labile iron prosthetic group. The iron in the molecule is in equilibrium with intracellular iron and ribonucleotide reductase inhibition by deferiprone is caused by chelation of the intracellular iron pool. Insufficient availability of one or more deoxyribonucleotides could lead to discontinuities in DNA synthesis and hence chromosomal breaks and rearrangements. Genotoxicity following ribonucleotide reductase inhibition has been described for two other inhibitors of ribonucleotide reductase, hydroxyurea and paracetamol (Bruce and Heddle, 1979; Hart and Hartley-Asp, 1983; Giri, 1993). Although in non-iron loaded systems deferiprone might be expected to show positive mutagenic results, in iron loaded systems deferiprone may act as an antimutagenic agent. It is now well established that iron, present in a bioavailable form, is a potent inducer of oxidative damage. Due to the redox chemistry of iron, it is able to catalyse the formation of reactive oxygen species. For example, the induction of oxidative damage by different ferric iron complexes in the presence of H2O2 has been shown in isolated DNA (Aruoma et al., 1989) and ferric iron complexes have been shown to induce DNA base damage, gene mutations and chromosomal aberrations in mammalian cells (Whiting et al., 1981; Hartwig and Schlepegrell, 1995; Dunkel et al., 1999).
Several studies have shown that deferoxamine can protect against DNA strand breaks and damage in renal tubular epithelial cells (Hagar et al., 1996), human colon carcinoma cells (Beall et al., 1996) and liver cells (Stinson et al., 1992), due to chemical hypoxic or metal ion-induced peroxidation. In general, comparative studies have shown that deferiprone and deferoxamine are equivalent in terms of their antioxidant and cytoprotective effects. Most particularly, it has been demonstrated that in some iron loaded situations where the chelation of intracellular iron is important, deferiprone can prevent the formation of reactive oxygen species and, hence, cytotoxicity (Balla et al., 1990; Shalev et al., 1995; Hagar et al., 1996; Niihara et al., 1998).
The current study is consistent with a recent paper by Whittaker et al. (2001), which showed that deferoxamine has a greater toxicity and mutagenicity than deferiprone in the mouse lymphoma assay.
It is concluded that therapy with deferiprone is not associated with a greater frequency of chromosomal aberrations than that observed with deferoxamine therapy. The results of clastogenicity studies with deferiprone in non-iron loaded situations where the chelation of iron may be involved may not be relevant to the clinical setting in patients with iron overload.
|
Acknowledgements |
|---|
The skilful assistance of Mrs Morna Murphy, Dr Caroline Trott and Mrs Yvonne Stoddart with the cytogenetic analysis is gratefully acknowledged. The contribution of Dr L.Ruvolleto to the conduct of the study is also gratefully acknowledged.
|
Notes |
|---|
4To whom correspondence should be addressed. Tel: +44 1423 848401; Fax: +44 1423 569595; Email: david.kirkland{at}covance.com
*The study was sponsored by Apotex. M.Spino is the Senior Vice President of Scientific Affairs and F.Tricta is the Medical Director of Apotex, the company that developed and manufactures deferiprone, the compound tested in this study
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Anderson,D., Yardley-Jones,A., Vives-Bauza,C., Chua-Anusom,W., Cole,C. and Webb,J. (2000) Effect of iron salts, haemosiderins and chelating agents on the lymphocytes of a thalassaemia patient without chelation therapy as measured in the comet assay. Teratog. Carcinog. Mutagen., 20, 251–264.[CrossRef][Web of Science][Medline]
Aruoma,O.I., Helliwell,B., Laughton,M.J., Quinlan,G.J. and Gutteridge,J.M. (1989) The mechanism of initiation of lipid peroxidation. Evidence against a requirement for an iron(II)–iron(III) complex. Biochem. J., 258, 617–620.[Web of Science][Medline]
Balla,G., Vercellotti,G.M., Eaton,J.W. and Jacob,H.S. (1990) Iron loading of endothelial cells augments oxidant damage. J. Lab. Clin. Med., 116, 546–554.[Web of Science][Medline]
Beall,H.D., Liu,Y., Siegel,D., Bolton,E.M., Gibson,N.W. and Ross,D. (1996) Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. Biochem. Pharmacol., 51, 645–652.[CrossRef][Web of Science][Medline]
Bruce,W.R. and Heddle,J.A.(1979) The mutagenic activity of 61 agents as determined by the micronucleus, salmonella and sperm abnormality assays. Can. J. Genet. Cytol., 21, 319–334.[Web of Science][Medline]
Chenoufi,N., Hubert,H., Loréal,O., Morel,I., Pasdeloup,N., Cillard,J., Brissot,P. and Lescoat,G. (1995) Inhibition of iron toxicity in rat and human hepatocyte cultures by the hydroxypyridin-4-ones CP20 and CP94. J. Hepatol., 23, 166–173.[CrossRef][Web of Science][Medline]
Côté,G.B. and Papadakou-Lagoyanni,S. (1979) ß-Thalassaemia: increased chromosomal anomalies in lymphocyte cultures. J. Med. Genet., 16, 52–55.
Cragg,L., Hebbel,R.P., Miller,W., Solovey,A., Selby,S. and Enright,H. (1998) The iron chelator L1 potentiates oxidative DNA damage in iron-overloaded liver cells. Blood, 92, 632–638.
Dunkel,V.C., San,R.H.C., Seifried,H.E. and Whitaker,P. (1999) Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells. Environ. Mol. Mutagen., 33, 28–41.[CrossRef][Web of Science][Medline]
Gille,J.J., van Berkel,C.G. and Joenje,H. (1992) Effect of iron chelators on the cytotoxic and genotoxic action of hyperoxia in Chinese hamster ovary cells. Mutat. Res., 275, 31–39.[CrossRef][Web of Science][Medline]
Giri,A.K. (1993) The genetic toxicology of paracetamol and aspirin: a review. Mutat. Res., 296, 199–210.[Web of Science][Medline]
Hagar,H., Ueda,N. and Shah,S.V. (1996) Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury of LLC-PK1 cells. Am. J. Physiol., 27, 209–215.
Hart,J.W. and Hartley-Asp,B. (1983) Induction of micronuclei in the mouse. Revised timing of the final stage of erythropoesis. Mutat. Res., 120, 127–132.[CrossRef][Web of Science][Medline]
Hartwig,A. and Schlepegrell,R. (1995) Induction of oxidative DNA damage by ferric iron in mammalian cells. Carcinogenesis, 16, 3009–3013.
Hartwig,A., Schlepegrell,R. and Beyersmann,D. (1992) Genotoxicity of Fe (III)–NTA in mammalian cells. Mutagenesis, 7, 159.
Juckett,M.B., Shadley,J.D., Zheng,Y. and Klein,J.P. (1998) Desferrioxamine enhances the effects of gamma radiation on clonogenic survival and the formation of chromosomal aberrations in endothelial cells. Radiat. Res., 149, 330–337.[Web of Science][Medline]
Krishnaja,A.P. and Sharma,N.K. (1994) Heterogeneity of chromosome damage in ß-thalassaemia traits. An evaluation of spontaneous and 
-ray induced micronuclei and chromosome aberrations in lymphocytes in vitro after G0 and G2 phase irradiation. Int. J. Radiat. Biol., 66, 29–39.[Web of Science][Medline]
Matthews,A.J., Vercellotti,G.M., Menchaca,H.J., Bloch,P.H.S., Michaleck,V.N., Marker,P.H., Murar,J. and Buchwald,H. (1997) Iron and atherosclerosis: inhibition by the iron chelator deferiprone (L1). J. Surg. Res., 73, 35–40.[CrossRef][Web of Science][Medline]
Mitelman,F. (ed.) (1995) An International System for Human Cytogenetic Nomenclature. Karger, Basel, Switzerland.
Morel,I., Cillard,J., Lescoat,G., Sergent,O., Pasdeloup,N., Ocaktan,A., Abdallah,M.A., Brissot,P. and Cillard,P. (1992) Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: comparison of their mechanism of protection with that of desferrioxamine. Free Radic. Biol. Med., 13, 499–508.[CrossRef][Web of Science][Medline]
Morel,I., Sergent,O., Cogrel,P., Lescoat,G., Pasdeloup,N., Brissot,P., Cillard,P. and Cillard,J. (1995) EPR study of antioxidant activity of the iron chelators pyoverdin and hydroxypyrid-4-one in iron-loaded hepatocyte culture: comparison with that of desferrioxamine. Free Radic. Biol. Med., 18, 303–310.[CrossRef][Web of Science][Medline]
Niihara,Y., Ge,J., Shalev,O., Hebbel,R.P. and Tanaka,K.R. (1998) Desferal (DFO) incubation of intact red blood cell (RBC) leads to decrease in NAD redox potential: evidence for Desferal as an inducer of oxidant stress in intact RBC. Blood, 92, 7b.
Shalev,O., Repka,T., Goldfarb,A., Grinberg,L., Abrahamov,A., Olivieri,N.F., Rachmilewitz,E.A. and Hebbel,R.P. (1995) Deferiprone (L1) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo. Blood, 86, 2008–2013.
Stinson,T.J., Jaw,S., Jeffery,H. and Plewa,M.J. (1992) The relationship between nickel chloride-induced peroxidation and DNA strand breakage in rat liver. Toxicol. Appl. Pharmacol., 117, 98–103.[CrossRef][Web of Science][Medline]
Swiger,R.R. and Tucker,J.D. (1996) Fluorescence in situ hybridisation: a brief review. Environ. Mol. Mutagen., 27, 245–254.[CrossRef][Web of Science][Medline]
Tucker,J.D., Lee,D.A., Ramsey,M.J., Briner,J., Olsen.L. and Moore,D.H. (1994) On the frequency of chromosome exchanges in a control population measured by chromosome painting. Mutat. Res., 313, 193–202.[CrossRef][Web of Science][Medline]
van der Kraaij,A.M.M., van Eijk,H.G. and Koster,J.F. (1989) Prevention of postischemic cardiac injury by the orally active iron chelator 1,2-cimethyl-3-hydroxy-4-pyridone (L1) and the antioxidant (+)-cyanidanol-3. Circulation, 80, 158–164.
Vig,B.K. and Eyring,E.J. (1971) Absence of chromosome aberrations in cultured human leukocytes treated by two metal chelators-deferoxamine and penicillamine. Mutat. Res., 12, 214–218.[Web of Science][Medline]
Whittaker,P., Seifried,H.E., San,R.H.C., Clarke,J.J. and Dunkel,V.C. (2001) Genotoxicity of iron chelators in L5178Y mouse lymphoma cells. Environ. Mol. Mutagen., 38, 347–356.[CrossRef][Web of Science][Medline]
Whiting,R.F., Wei,L. and Stich,H.F. (1981) Chromosome damaging activity of ferritin and its relation to chelation and reduction of iron. Cancer Res., 41, 1628–1636.
Received on March 25, 2003; revised on April 30, 2003; accepted on May 2, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K.M. Mitchell, A.L. Dotson, K.M. Cool, A. Chakrabarty, S.H. Benedict, and S.M. LeVine Deferiprone, an orally deliverable iron chelator, ameliorates experimental autoimmune encephalomyelitis Multiple Sclerosis, November 1, 2007; 13(9): 1118 - 1126. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||