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Inhibition of Fe2+- and Fe3+- induced hydroxyl radical production by the iron-chelating drug deferiprone
Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Original Contribution
Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the iron-chelating drug deferiprone V.A. Timoshnikov a,b, T. Kobzeva a, N.E. Polyakov a,n, G.J. Kontoghiorghes c a
Institute of Chemical Kinetics and Combustion, 630090 Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia c Postgraduate Research Institute of Science, Technology, Environment, and Medicine, Limassol, Cyprus b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 22 September 2014 Accepted 13 October 2014
Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe2 þ and Fe3 þ ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe2 þ have reported ferroxidase activity by L1 followed by the formation of Fe3 þ chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1–Fe3 þ complexes exhibit intense absorption bands up to 800 nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe2 þ and Fe3 þ ions and (2) does UV–Vis irradiation of the L1–Fe3 þ complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe3 þ aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO–OH, a(N)¼ 14.15 G, a(H) ¼ 16.25 G; PBN–OH, a(N) ¼16.0 G, a(H) ¼ 2.7 G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe2 þ ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage. & 2014 Elsevier Inc. All rights reserved.
Keywords: EPR Spin traps Deferiprone Iron chelation Fenton reaction Photo-Fenton reaction Hydroxyl radical Phototoxicity Free radicals
Introduction Deferiprone (L1) is an effective iron-chelating drug developed for the treatment of iron-overload toxicity in thalassemia and other ironrelated toxicity conditions. Deferiprone is orally active and is widely distributed in the body, thus targeting toxic iron in many tissues and organs [1,2]. Moreover, L1 is supposed to be an effective antioxidant that prevents oxidative stress and biomolecular, subcellular, cellular, and tissue damage caused mostly by iron- and copper-induced free radical formation in vivo [1] and references there in. Free radical formation is a natural process leading to the production of highly reactive species such as superoxide, nitric oxide, and hydroxyl radicals. Free radicals and other active oxygen species can also cause modification or damage of all known organic biomolecules, including
n
Corresponding author. Fax: þ 7 383 3307350. E-mail address: [email protected] (N.E. Polyakov).
DNA, sugars, proteins, and lipids [3–6]. Deferiprone has been shown to be effective and safe in the prevention of oxidative stress-related tissue damage under iron-loading and non-iron-loading conditions such as cardiomyopathy in thalassemia, acute kidney disease, and Friedreich ataxia see for review [1], for example. Similarly, the iron and other metal binding properties of L1 at various pH's, stability constants, inhibition of iron-catalyzed free radical toxicity, etc., have also been previously reported, but no information is available on the molecular mechanism of its antioxidant activity [1,2,7–13]. Also, there are no experimental data on the phototoxicity of the L1 chelate complexes, or the ability of L1 to prevent the phototoxic effects of other iron complexes. The absorption spectra of the L1– Fe(III) complexes exhibit intense absorption bands between 300 and 800 nm [7,8,11,14]. This light can penetrate through the skin and reach the capillaries. It may be possible that irradiation of the L1–Fe(III) complexes with visible light can result in formation of free radicals or toxic secondary products, which might cause toxicity such as photodermatitis, a toxic side effect observed with
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513 0891-5849/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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other drugs but not so far reported for L1 [15,16]. This information is particularly important for the thousands of patients treated with L1 and other chelating drugs, because iron-overload diseases such as thalassemia are mainly distributed in the Mediterranean, Middle East, and South East Asian countries, where extremely high sunlight irradiation levels are present. Usually the therapeutic dose of L1 ranges between 75 and 100 mg/kg/day divided into two or three doses per day; its peak blood concentration can range between 50 and 500 μΜ, and the drug is cleared from the blood within about 6 h [2,10,12]. Recently it was demonstrated using the CIDNP method that L1 undergoes fast decomposition via an electron transfer mechanism under direct and photosensitized UV irradiation ( o320 nm) [17]. But no changes in the absorption spectrum of the L1 iron complexes were detected under irradiation by visible light [17]. There are several examples of the photochemical activity of Fe (III) complexes [18–21]. For example, irradiation of Fe(III) complexes with various carboxylic organic acids by visible light results in electron transfer from the ligand to the Fe(III) ion and subsequently the formation of a redox-active Fe(II) ion and a set of free radicals [19]: [Fe–OOC–R]2 þ þhν-Fe2 þ þ R–COO, R–COO-R þ CO2. Irradiation of Fe(III) aqua complexes results in the formation of OH radicals with high quantum yield (0.1–0.3) depending on the irradiation wavelength and the pH of the solution [20,21]: [Fe(H2O)6]3 þ þhν-[Fe(H2O)5]2 þ þ OH þH þ , [Fe(OH)(H2O)5]2 þ þhν-[Fe(H2O)5]2 þ þ OH. Hydroxyl radical is known to be extremely reactive and is an intermediate in the oxidative damage of DNA and other biomolecules see for review [22] and references therein. In the present study we have applied an electron paramagnetic resonance (EPR) spin trapping technique to answer the question whether chelation of Fe ions by L1 results in the inhibition of dark and photochemically induced generation of active oxygen radicals. The method of spin trapping allows the transformation of an extremely short-lived radical to a long-lived one and is successfully used in the study of biologically relevant free radicals, especially active oxygen species: hydroxyl radical, peroxyl radical, and singlet oxygen [23–26]. Application of TMIO (2,2,4-trimethyl-2H-imidazole-1-oxide) and PBN (α-phenyl N-t-butyl nitrone) was shown to have certain advantages compared to the widely used DMPO (5,5-dimethylpyrroline-N-oxide) in the trapping of several types of radicals and in the interpretation of the resulting molecular mechanisms involved [23,24]. The second question we have tried to answer is whether L1 itself can produce reactive oxygen species under UV irradiation via electron or energy transfer.
Materials and methods Deferiprone was received from Lipomed, Inc., Switzerland. The spin traps used were PBN 98% from Sigma, TMIO 98% from Fluka, and TEMP (2,2,6,6-tetramethylpiperidine) 99% from Aldrich. The optical spectra of L1 were taken in distilled water in a 1-cm quartz cuvette using a Shimadzu UV-2401-PC spectrophotometer. EPR spectra of spin adducts were recorded on a Bruker EMX EPR spectrometer. The following EPR parameters were used: microwave bridge, frequency 10.112 GHz, power 20.65 mW; signal channel, receiver gain 2 105, modulation frequency 100 kHz, modulation amplitude 1.00 G.
The Fenton reaction was carried out in phosphate buffer (50 mM, pH 7.4), with 10 mM TMIO, 1 mM FeSO4, 1 mM H2O2, and 10 mM L1. The irradiation of samples was carried out directly in the probe of the EPR spectrometer by a 1000-W mercury lamp through water and optical filters Hg366 and Hg546. The concentration of the spin traps was 10 mM, the concentration of Fe(ClO4)3 was 1–3 mM, and the ratio L1:Fe(III) was 3:1. Irradiation time was 15 s for DMPO, TMIO, and PBN solutions and up to 20 min for the TEMP solution.
Results and discussion The formation of the L1 complex with Fe(III) could be readily detected from the appearance of a new absorption band between 300 and 700 nm (Fig. 1). This complex was well characterized earlier [2,8–13]. In particular, it was demonstrated that the absorption spectrum of L1 itself depends significantly on the solvent acidity due to the shift of equilibrium between protonated and deprotonated forms of L1: pKa1 ¼3.5 and pKa2 ¼10.2 (Scheme 1). The stoichiometry of the L1–Fe(III) complex is pH sensitive. In a neutral solution (pH 46) only the FeL13 complex exists. However, in acidic solution (2 opH o 6) a mixture of complexes can be detected (FeL13, FeL12, and FeL1) [2,8–13]. In the first experiment it was found that the widely used spin trap DMPO is photochemically unstable and produces spin adducts under irradiation by the mercury lamp without addition of L1 or Fe ions. Moreover, DMPO can react with Fe3 þ ions under dark conditions, which was evident from the change of the color of the Fe(ClO4)3 solution after addition of the spin trap. In earlier studies a Fe–DMPO complex was also detected by K. Makino et al. [27]. These reactions make it difficult to analyze the EPR spectra obtained in the presence of the iron ion. Therefore other spin traps, namely PBN and TMIO, were used for further experiments (Scheme 2). The aqua complex of the Fe(III) ion has an intense absorption band up to 400 nm [20,21]. Irradiation of aqueous Fe(ClO4)3 solution in the presence of spin traps in the pH range from 3.0 to 10.8 by the full light of a high-pressure mercury lamp or through an optical filter
Fig. 1. Optical absorption spectra of 0.6 mM L1 and 0.2 mM Fe(ClO4)3 aqueous solutions at pH 5.5. Inset: optical absorption spectrum of 1 mM aqueous L1 solution in the presence of 0.3 mM Fe(ClO4)3 at pH 5.5.
OH
O OH
+ N
K1 +H+
O OH
N
O
K2 -H+
N
Scheme 1. Equilibrium between protonated and deprotonated forms of deferiprone (L1).
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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3
H3C OH
HO
N
CH3
H
O
N O
CH3
TMIO
N
tBu
OH
O
OH N
tBu
H O
PBN Scheme 2. Structures of spin traps TMIO and PBN and their spin adducts with the OH radical.
Fig. 2. EPR spectra detected during irradiation (full light of mercury lamp through water filter) of 1 mM solution of Fe(ClO4)3 in distilled water at pH 3.5 with 10 mM TMIO in the absence and in the presence of 3 mM L1.
at 366 nm produces EPR signals of spin adducts. As an example, Fig. 2 shows the EPR spectrum of a spin adduct detected in the presence of TMIO. The determined HFC constants of the main spin adducts correspond to the published data for adducts with the OH radical (TMIO–OH, a(N)¼14.15 G, a(H)¼16.25 G; PBN–OH, a(N)¼ 16.0 G, a(H)¼2.7 G) [28–30]. These results are in accordance with the previously published data on the formation of OH radical under photolysis of iron aqua complexes [20,21]. Although there are many published examples of PBN–OH adduct formation in Fenton reaction or UV irradiation of H2O2, some studies indicate that the same adduct can be formed indirectly through interaction of the spin trap with metal ions and water molecules [28–31]. Formation of spin adducts of hydroxyl radicals with nitrones can be explained by: (1) oxidation of the trap with iron in its excited state followed by interaction of the radical cation of the trap with water or (2) addition of water molecules to the trap resulting in the corresponding hydroxylamine with its subsequent oxidation to the spin adduct of the hydroxyl radical [31]. To prove OH radical formation in the reaction under study a control experiment in the presence of dimethyl sulfoxide (DMSO) was done. It is known that in the presence of DMSO hydroxyl radicals are rapidly transformed to the methyl radical via the following reaction [28–30]:
OHþ DMSO-CH3 þCH3(OH)SO.
In air-saturated solution the OCH3 radical is usually detected [30]. The formation of CH3 and OCH3 radicals was proven in the systems
Fig. 3. EPR spectra detected during irradiation (mercury lamp through water filter and optical filters Hg366) of a 1 mM solution of Fe(ClO4)3 in 10% DMSO at pH 3.5 with 10 mM PBN in the absence and in the presence of 3 mM L1.
under study by the detection of corresponding spin adducts with HFC constants: TMIO–CH3, a(N)¼ 15.61 G, a(H)¼22.27 G; TMIO– OCH3, a(N)¼13.84 G, a(H)¼15.78 G; PBN–OCH3, a(N)¼15.10 G, a(H)¼3.29 G [28–30]. Fig. 3 depicts the EPR spectra detected during irradiation (mercury lamp through water filter and optical filters Hg366) of a 1 mM solution of Fe(ClO4)3 in 10% DMSO at pH 3.5 with 10 mM PBN in the absence and in the presence of 3 mM L1. The unidentified single signal at 3437 G in Fig. 3 is the signal from the quartz cuvette, and the minor signal (four lines with a (N)¼14.33 G, a(H) ¼14.11 G, Fig. 3) is t-Bu-aminoxyl formed by degradation of PBN [28]. Irradiation of the same solutions in the presence of L1 (the ratio L1:Fe(III) was 3:1) demonstrates a total absence of OH or OCH3 adducts (see Figs. 2 and 3 as examples). It is important to note that L1 inhibits OH radical formation in a wide range of pH from 3.0 to 10.8. Similarly, irradiation with visible light at 546 nm does not produce any spin adducts. It should be noted that under our experimental conditions all ligand is bound with Fe(III) owing to high binding constants: logK1 ¼15.1, logK2 ¼11.51, logK3 ¼9.27 [37]. So no free ligand is present in the solution, and our experimental observations cannot be explained by the reaction of free L1 with OH radicals. Concentrations of 1 mM Fe3 þ and 3 mM L1 were used in this study to get more intensive EPR spectra. Inhibition at lower concentrations of L1 was shown in vivo and in vitro in other published studies see, for example, [12,38]. Because the mechanism of inhibition of free radical production was determined as L1 iron complex formation, the lower concentration limit can be suggested to be in the micromolar range from the published binding constants of iron complexes with L1 [37]. Irradiation of L1 in the presence of TMIO without Fe ions does not show any spin adduct formation. It can be concluded that L1 itself does not produce oxygen radicals under irradiation via electron transfer or hydrogen transfer. It can also be suggested that the L1 radicals detected by the CIDNP technique under UV irradiation are not trapped by spin traps [17]. The absence of spin adduct signals in this reaction leads to the conclusion that there is no electron transfer from the L1 radical anion to the dissolved oxygen. It is known that another source of drug phototoxicity can be singlet oxygen formed usually by energy transfer from the triplet excited state of drug molecules [22,25]. Singlet oxygen might, as well as hydroxyl radical, participate in lipid and protein membrane oxidation or induce DNA damage see for review [21] and references therein. In an attempt to detect the formation of singlet oxygen under the photolysis of L1 the standard approach of the TEMP spin trap was used [25]. However, no spin adduct was detected in the EPR spectra during 20 min irradiation of L1 or the L1–Fe complex.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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in vivo models and in clinical studies involving various clinical conditions [1,3,12,39,41]. The Fe(II)-catalyzed free radical effects, which are inhibited by L1 through oxidation of Fe(II) to Fe(III) followed by Fe(III) complex formation, are also exhibited by other chelators such as deferoxamine and also by plasma transferrin and lactoferrin [2,41]. This is considered one of the major antioxidant mechanisms exhibited by these proteins under normal conditions and also under conditions of oxidative stress, inflammation, and infections [41].
Conclusion
Fig. 4. EPR spectra of TMIO–OH spin adduct detected after addition of 1 mM water solution of FeSO4 (in the absence and in the presence of 10 mM L1) to phosphate buffer solution (50 mM, pH 7.4) of H2O2 (1 mM) and TMIO (10 mM).
To date there is no published evidence of the formation of L1– Fe(II) complexes. One-electron reduction of the L1–Fe(II) complex by hydrated electrons results in a characteristic decay of absorption at 450 nm in a microsecond time scale [34]. However, it was not evident from this experiment if the absorption decay is due to complex dissociation or chemical transformation of L1. On the other hand, in the presence of excess L1 the formation of a red/ orange L1–Fe(III) complex was observed in the absorption spectrum due to the oxidation of Fe(II) to Fe(III) [17]. A number of EPR spin trapping studies see, for example, [35] and references therein have demonstrated the presence of reactive oxygen radical production during the oxidation reaction of Fe(II) to Fe(III) ions with hydrogen peroxide [36,37]: As an illustration, Fig. 4 shows TMIO–OH spin adduct formation during the reaction of FeSO4 with H2O2 in phosphate buffer at pH 7.4. The EPR parameters of the spin adduct are the same as in Fig. 2. The addition of excess L1 to Fe(II) solution completely inhibited hydroxyl radical formation. When the reaction is carried out in 10% DMSO solution the total yield of the spin adducts is reduced, which may be due lower spin trapping rates for CH3 and OCH3 radicals. Nevertheless, a significant decrease in the spin adduct yield was detected in the presence of L1 in these systems too. This result confirms the earlier suggestions that L1 oxidizes Fe(II) to Fe(III) and does not confirm the mechanism suggested by Merkofer et al. in which the L1–Fe(III) complex is formed in the presence of L1 owing to reaction of Fe(II) with hydrogen peroxide [17,34]. The EPR evidence presented in this study indicates that the FeL13 complex is redox inactive, as previously suggested by Devanur et al. [38]. Iron, and in particular Fe(II), is the major catalyst of free radical reactions in biological systems and of free radical damage in diseases related to oxidative stress. No free iron circulates in blood under normal conditions. However, excess “focal” iron has been detected in various organs in many diseases [39]. Non-transferrinbound iron is well documented in thalassemia and other iron-load diseases, in which transferrin is also found to be fully saturated with iron [40]. The presence of excess iron and of labile, nonprotein-bound iron is a source of continuous toxicity, which has been implicated in the pathogenesis of many diseases, including iron-overload diseases, cancer, atherosclerosis, and neurodegenerative and kidney diseases, as well as in aging. Within this context, L1 has been shown in this in vitro molecular model to be an effective inhibitor of OH radical production and to confirm earlier reports of its antioxidant capacity in various in vitro and
Deferiprone is widely used in iron-overload diseases such as thalassemia, which is mainly distributed in the Mediterranean, Middle East, and South East Asian countries [42,43]. In the same areas extremely high sun light irradiation levels are present. Taking into account that the usual therapeutic dose of L1 ranges between 75 and 100 mg/kg/day, and its peak blood concentration can range between 50 and 500 μΜ, it is important to control possible phototoxic effects of L1 and other iron chelators. This study shows that L1 can inhibit OH radical production via a photoFenton reaction. In addition, no reactive oxygen species were detected under direct UV irradiation of the L1 water solution or of L1–iron complexes during irradiation by UV and visible light. The most important finding of this study is the oxidation of Fe(II) to Fe(III) by deferiprone, with subsequent formation of an Fe(III)–L13 complex, which is redox inactive under dark physiological conditions. It is well known that iron and in particular Fe(II) is the major catalyst of free radical reactions in biological systems and of free radical damage in diseases related to oxidative stress. Overall, L1 works as an antioxidant by preventing iron-induced free radical formation (via Fenton reaction). These findings confirm previous findings of the antioxidant targeting potential of L1 in diseases related to oxidative damage [39].
Uncited references [32]; [33].
Acknowledgments This work was supported by the Council for Grants of the President of the Russian Federation for Support of Leading Scientific Schools (Project NSh 2272.2012.3). Special thanks to Dr. Igor Kirilyuk (Novosibirsk Institute of Organic Chemistry, Russia) and Dr. Irina Slepneva (Institute of Chemical Kinetics and Combustion, Russia), for the samples of spin traps, and Dr. Dmitri Stass (Institute of Chemical Kinetics and Combustion) for technical support and general discussion. References [1] Kontoghiorghes, G. J. Prospects for introducing deferiprone as potent pharmaceutical antioxidant. Front. Biosci. (Elite Ed.) 1:161–178; 2009. [2] Kontoghiorghes, G. J.; Eracleous, E.; Economides, C.; Kolnagou, A. Advances in iron overload therapies: prospects for effective use of deferiprone (L1), deferoxamine, the new experimental chelators ICL670, GT56-252, L1NA11 and their combination. Curr. Med. Chem. 12:2663–2681; 2005. [3] Oxidation and Antioxidants in Organic Chemistry and Biology. In: Denisov, E. T., Afanas’ev, I. B., editors. Boca Raton: CRC Press/Taylor & Francis; 2005. [4] Halliwell, B.; Gutteridge, J. M. C.; Cross, C. E. Free radicals, antioxidants and human disease: where are we now? J. Lab. Clin. Med. 119:598–620; 1992. [5] Kontoghiorghes, G. J. Iron chelation in biochemistry and medicine. In: RiceEvans, C., editor. Free Radicals, Oxidant Stress and Drug Action. London: Rechelieu Press; 1987. p. 277–303.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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Original Contribution
Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the iron-chelating drug deferiprone V.A. Timoshnikov a,b, T. Kobzeva a, N.E. Polyakov a,n, G.J. Kontoghiorghes c a
Institute of Chemical Kinetics and Combustion, 630090 Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia c Postgraduate Research Institute of Science, Technology, Environment, and Medicine, Limassol, Cyprus b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 22 September 2014 Accepted 13 October 2014
Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe2 þ and Fe3 þ ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe2 þ have reported ferroxidase activity by L1 followed by the formation of Fe3 þ chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1–Fe3 þ complexes exhibit intense absorption bands up to 800 nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe2 þ and Fe3 þ ions and (2) does UV–Vis irradiation of the L1–Fe3 þ complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe3 þ aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO–OH, a(N)¼ 14.15 G, a(H) ¼ 16.25 G; PBN–OH, a(N) ¼16.0 G, a(H) ¼ 2.7 G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe2 þ ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage. & 2014 Elsevier Inc. All rights reserved.
Keywords: EPR Spin traps Deferiprone Iron chelation Fenton reaction Photo-Fenton reaction Hydroxyl radical Phototoxicity Free radicals
Introduction Deferiprone (L1) is an effective iron-chelating drug developed for the treatment of iron-overload toxicity in thalassemia and other ironrelated toxicity conditions. Deferiprone is orally active and is widely distributed in the body, thus targeting toxic iron in many tissues and organs [1,2]. Moreover, L1 is supposed to be an effective antioxidant that prevents oxidative stress and biomolecular, subcellular, cellular, and tissue damage caused mostly by iron- and copper-induced free radical formation in vivo [1] and references there in. Free radical formation is a natural process leading to the production of highly reactive species such as superoxide, nitric oxide, and hydroxyl radicals. Free radicals and other active oxygen species can also cause modification or damage of all known organic biomolecules, including
n
Corresponding author. Fax: þ 7 383 3307350. E-mail address: [email protected] (N.E. Polyakov).
DNA, sugars, proteins, and lipids [3–6]. Deferiprone has been shown to be effective and safe in the prevention of oxidative stress-related tissue damage under iron-loading and non-iron-loading conditions such as cardiomyopathy in thalassemia, acute kidney disease, and Friedreich ataxia see for review [1], for example. Similarly, the iron and other metal binding properties of L1 at various pH's, stability constants, inhibition of iron-catalyzed free radical toxicity, etc., have also been previously reported, but no information is available on the molecular mechanism of its antioxidant activity [1,2,7–13]. Also, there are no experimental data on the phototoxicity of the L1 chelate complexes, or the ability of L1 to prevent the phototoxic effects of other iron complexes. The absorption spectra of the L1– Fe(III) complexes exhibit intense absorption bands between 300 and 800 nm [7,8,11,14]. This light can penetrate through the skin and reach the capillaries. It may be possible that irradiation of the L1–Fe(III) complexes with visible light can result in formation of free radicals or toxic secondary products, which might cause toxicity such as photodermatitis, a toxic side effect observed with
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513 0891-5849/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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other drugs but not so far reported for L1 [15,16]. This information is particularly important for the thousands of patients treated with L1 and other chelating drugs, because iron-overload diseases such as thalassemia are mainly distributed in the Mediterranean, Middle East, and South East Asian countries, where extremely high sunlight irradiation levels are present. Usually the therapeutic dose of L1 ranges between 75 and 100 mg/kg/day divided into two or three doses per day; its peak blood concentration can range between 50 and 500 μΜ, and the drug is cleared from the blood within about 6 h [2,10,12]. Recently it was demonstrated using the CIDNP method that L1 undergoes fast decomposition via an electron transfer mechanism under direct and photosensitized UV irradiation ( o320 nm) [17]. But no changes in the absorption spectrum of the L1 iron complexes were detected under irradiation by visible light [17]. There are several examples of the photochemical activity of Fe (III) complexes [18–21]. For example, irradiation of Fe(III) complexes with various carboxylic organic acids by visible light results in electron transfer from the ligand to the Fe(III) ion and subsequently the formation of a redox-active Fe(II) ion and a set of free radicals [19]: [Fe–OOC–R]2 þ þhν-Fe2 þ þ R–COO, R–COO-R þ CO2. Irradiation of Fe(III) aqua complexes results in the formation of OH radicals with high quantum yield (0.1–0.3) depending on the irradiation wavelength and the pH of the solution [20,21]: [Fe(H2O)6]3 þ þhν-[Fe(H2O)5]2 þ þ OH þH þ , [Fe(OH)(H2O)5]2 þ þhν-[Fe(H2O)5]2 þ þ OH. Hydroxyl radical is known to be extremely reactive and is an intermediate in the oxidative damage of DNA and other biomolecules see for review [22] and references therein. In the present study we have applied an electron paramagnetic resonance (EPR) spin trapping technique to answer the question whether chelation of Fe ions by L1 results in the inhibition of dark and photochemically induced generation of active oxygen radicals. The method of spin trapping allows the transformation of an extremely short-lived radical to a long-lived one and is successfully used in the study of biologically relevant free radicals, especially active oxygen species: hydroxyl radical, peroxyl radical, and singlet oxygen [23–26]. Application of TMIO (2,2,4-trimethyl-2H-imidazole-1-oxide) and PBN (α-phenyl N-t-butyl nitrone) was shown to have certain advantages compared to the widely used DMPO (5,5-dimethylpyrroline-N-oxide) in the trapping of several types of radicals and in the interpretation of the resulting molecular mechanisms involved [23,24]. The second question we have tried to answer is whether L1 itself can produce reactive oxygen species under UV irradiation via electron or energy transfer.
Materials and methods Deferiprone was received from Lipomed, Inc., Switzerland. The spin traps used were PBN 98% from Sigma, TMIO 98% from Fluka, and TEMP (2,2,6,6-tetramethylpiperidine) 99% from Aldrich. The optical spectra of L1 were taken in distilled water in a 1-cm quartz cuvette using a Shimadzu UV-2401-PC spectrophotometer. EPR spectra of spin adducts were recorded on a Bruker EMX EPR spectrometer. The following EPR parameters were used: microwave bridge, frequency 10.112 GHz, power 20.65 mW; signal channel, receiver gain 2 105, modulation frequency 100 kHz, modulation amplitude 1.00 G.
The Fenton reaction was carried out in phosphate buffer (50 mM, pH 7.4), with 10 mM TMIO, 1 mM FeSO4, 1 mM H2O2, and 10 mM L1. The irradiation of samples was carried out directly in the probe of the EPR spectrometer by a 1000-W mercury lamp through water and optical filters Hg366 and Hg546. The concentration of the spin traps was 10 mM, the concentration of Fe(ClO4)3 was 1–3 mM, and the ratio L1:Fe(III) was 3:1. Irradiation time was 15 s for DMPO, TMIO, and PBN solutions and up to 20 min for the TEMP solution.
Results and discussion The formation of the L1 complex with Fe(III) could be readily detected from the appearance of a new absorption band between 300 and 700 nm (Fig. 1). This complex was well characterized earlier [2,8–13]. In particular, it was demonstrated that the absorption spectrum of L1 itself depends significantly on the solvent acidity due to the shift of equilibrium between protonated and deprotonated forms of L1: pKa1 ¼3.5 and pKa2 ¼10.2 (Scheme 1). The stoichiometry of the L1–Fe(III) complex is pH sensitive. In a neutral solution (pH 46) only the FeL13 complex exists. However, in acidic solution (2 opH o 6) a mixture of complexes can be detected (FeL13, FeL12, and FeL1) [2,8–13]. In the first experiment it was found that the widely used spin trap DMPO is photochemically unstable and produces spin adducts under irradiation by the mercury lamp without addition of L1 or Fe ions. Moreover, DMPO can react with Fe3 þ ions under dark conditions, which was evident from the change of the color of the Fe(ClO4)3 solution after addition of the spin trap. In earlier studies a Fe–DMPO complex was also detected by K. Makino et al. [27]. These reactions make it difficult to analyze the EPR spectra obtained in the presence of the iron ion. Therefore other spin traps, namely PBN and TMIO, were used for further experiments (Scheme 2). The aqua complex of the Fe(III) ion has an intense absorption band up to 400 nm [20,21]. Irradiation of aqueous Fe(ClO4)3 solution in the presence of spin traps in the pH range from 3.0 to 10.8 by the full light of a high-pressure mercury lamp or through an optical filter
Fig. 1. Optical absorption spectra of 0.6 mM L1 and 0.2 mM Fe(ClO4)3 aqueous solutions at pH 5.5. Inset: optical absorption spectrum of 1 mM aqueous L1 solution in the presence of 0.3 mM Fe(ClO4)3 at pH 5.5.
OH
O OH
+ N
K1 +H+
O OH
N
O
K2 -H+
N
Scheme 1. Equilibrium between protonated and deprotonated forms of deferiprone (L1).
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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3
H3C OH
HO
N
CH3
H
O
N O
CH3
TMIO
N
tBu
OH
O
OH N
tBu
H O
PBN Scheme 2. Structures of spin traps TMIO and PBN and their spin adducts with the OH radical.
Fig. 2. EPR spectra detected during irradiation (full light of mercury lamp through water filter) of 1 mM solution of Fe(ClO4)3 in distilled water at pH 3.5 with 10 mM TMIO in the absence and in the presence of 3 mM L1.
at 366 nm produces EPR signals of spin adducts. As an example, Fig. 2 shows the EPR spectrum of a spin adduct detected in the presence of TMIO. The determined HFC constants of the main spin adducts correspond to the published data for adducts with the OH radical (TMIO–OH, a(N)¼14.15 G, a(H)¼16.25 G; PBN–OH, a(N)¼ 16.0 G, a(H)¼2.7 G) [28–30]. These results are in accordance with the previously published data on the formation of OH radical under photolysis of iron aqua complexes [20,21]. Although there are many published examples of PBN–OH adduct formation in Fenton reaction or UV irradiation of H2O2, some studies indicate that the same adduct can be formed indirectly through interaction of the spin trap with metal ions and water molecules [28–31]. Formation of spin adducts of hydroxyl radicals with nitrones can be explained by: (1) oxidation of the trap with iron in its excited state followed by interaction of the radical cation of the trap with water or (2) addition of water molecules to the trap resulting in the corresponding hydroxylamine with its subsequent oxidation to the spin adduct of the hydroxyl radical [31]. To prove OH radical formation in the reaction under study a control experiment in the presence of dimethyl sulfoxide (DMSO) was done. It is known that in the presence of DMSO hydroxyl radicals are rapidly transformed to the methyl radical via the following reaction [28–30]:
OHþ DMSO-CH3 þCH3(OH)SO.
In air-saturated solution the OCH3 radical is usually detected [30]. The formation of CH3 and OCH3 radicals was proven in the systems
Fig. 3. EPR spectra detected during irradiation (mercury lamp through water filter and optical filters Hg366) of a 1 mM solution of Fe(ClO4)3 in 10% DMSO at pH 3.5 with 10 mM PBN in the absence and in the presence of 3 mM L1.
under study by the detection of corresponding spin adducts with HFC constants: TMIO–CH3, a(N)¼ 15.61 G, a(H)¼22.27 G; TMIO– OCH3, a(N)¼13.84 G, a(H)¼15.78 G; PBN–OCH3, a(N)¼15.10 G, a(H)¼3.29 G [28–30]. Fig. 3 depicts the EPR spectra detected during irradiation (mercury lamp through water filter and optical filters Hg366) of a 1 mM solution of Fe(ClO4)3 in 10% DMSO at pH 3.5 with 10 mM PBN in the absence and in the presence of 3 mM L1. The unidentified single signal at 3437 G in Fig. 3 is the signal from the quartz cuvette, and the minor signal (four lines with a (N)¼14.33 G, a(H) ¼14.11 G, Fig. 3) is t-Bu-aminoxyl formed by degradation of PBN [28]. Irradiation of the same solutions in the presence of L1 (the ratio L1:Fe(III) was 3:1) demonstrates a total absence of OH or OCH3 adducts (see Figs. 2 and 3 as examples). It is important to note that L1 inhibits OH radical formation in a wide range of pH from 3.0 to 10.8. Similarly, irradiation with visible light at 546 nm does not produce any spin adducts. It should be noted that under our experimental conditions all ligand is bound with Fe(III) owing to high binding constants: logK1 ¼15.1, logK2 ¼11.51, logK3 ¼9.27 [37]. So no free ligand is present in the solution, and our experimental observations cannot be explained by the reaction of free L1 with OH radicals. Concentrations of 1 mM Fe3 þ and 3 mM L1 were used in this study to get more intensive EPR spectra. Inhibition at lower concentrations of L1 was shown in vivo and in vitro in other published studies see, for example, [12,38]. Because the mechanism of inhibition of free radical production was determined as L1 iron complex formation, the lower concentration limit can be suggested to be in the micromolar range from the published binding constants of iron complexes with L1 [37]. Irradiation of L1 in the presence of TMIO without Fe ions does not show any spin adduct formation. It can be concluded that L1 itself does not produce oxygen radicals under irradiation via electron transfer or hydrogen transfer. It can also be suggested that the L1 radicals detected by the CIDNP technique under UV irradiation are not trapped by spin traps [17]. The absence of spin adduct signals in this reaction leads to the conclusion that there is no electron transfer from the L1 radical anion to the dissolved oxygen. It is known that another source of drug phototoxicity can be singlet oxygen formed usually by energy transfer from the triplet excited state of drug molecules [22,25]. Singlet oxygen might, as well as hydroxyl radical, participate in lipid and protein membrane oxidation or induce DNA damage see for review [21] and references therein. In an attempt to detect the formation of singlet oxygen under the photolysis of L1 the standard approach of the TEMP spin trap was used [25]. However, no spin adduct was detected in the EPR spectra during 20 min irradiation of L1 or the L1–Fe complex.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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in vivo models and in clinical studies involving various clinical conditions [1,3,12,39,41]. The Fe(II)-catalyzed free radical effects, which are inhibited by L1 through oxidation of Fe(II) to Fe(III) followed by Fe(III) complex formation, are also exhibited by other chelators such as deferoxamine and also by plasma transferrin and lactoferrin [2,41]. This is considered one of the major antioxidant mechanisms exhibited by these proteins under normal conditions and also under conditions of oxidative stress, inflammation, and infections [41].
Conclusion
Fig. 4. EPR spectra of TMIO–OH spin adduct detected after addition of 1 mM water solution of FeSO4 (in the absence and in the presence of 10 mM L1) to phosphate buffer solution (50 mM, pH 7.4) of H2O2 (1 mM) and TMIO (10 mM).
To date there is no published evidence of the formation of L1– Fe(II) complexes. One-electron reduction of the L1–Fe(II) complex by hydrated electrons results in a characteristic decay of absorption at 450 nm in a microsecond time scale [34]. However, it was not evident from this experiment if the absorption decay is due to complex dissociation or chemical transformation of L1. On the other hand, in the presence of excess L1 the formation of a red/ orange L1–Fe(III) complex was observed in the absorption spectrum due to the oxidation of Fe(II) to Fe(III) [17]. A number of EPR spin trapping studies see, for example, [35] and references therein have demonstrated the presence of reactive oxygen radical production during the oxidation reaction of Fe(II) to Fe(III) ions with hydrogen peroxide [36,37]: As an illustration, Fig. 4 shows TMIO–OH spin adduct formation during the reaction of FeSO4 with H2O2 in phosphate buffer at pH 7.4. The EPR parameters of the spin adduct are the same as in Fig. 2. The addition of excess L1 to Fe(II) solution completely inhibited hydroxyl radical formation. When the reaction is carried out in 10% DMSO solution the total yield of the spin adducts is reduced, which may be due lower spin trapping rates for CH3 and OCH3 radicals. Nevertheless, a significant decrease in the spin adduct yield was detected in the presence of L1 in these systems too. This result confirms the earlier suggestions that L1 oxidizes Fe(II) to Fe(III) and does not confirm the mechanism suggested by Merkofer et al. in which the L1–Fe(III) complex is formed in the presence of L1 owing to reaction of Fe(II) with hydrogen peroxide [17,34]. The EPR evidence presented in this study indicates that the FeL13 complex is redox inactive, as previously suggested by Devanur et al. [38]. Iron, and in particular Fe(II), is the major catalyst of free radical reactions in biological systems and of free radical damage in diseases related to oxidative stress. No free iron circulates in blood under normal conditions. However, excess “focal” iron has been detected in various organs in many diseases [39]. Non-transferrinbound iron is well documented in thalassemia and other iron-load diseases, in which transferrin is also found to be fully saturated with iron [40]. The presence of excess iron and of labile, nonprotein-bound iron is a source of continuous toxicity, which has been implicated in the pathogenesis of many diseases, including iron-overload diseases, cancer, atherosclerosis, and neurodegenerative and kidney diseases, as well as in aging. Within this context, L1 has been shown in this in vitro molecular model to be an effective inhibitor of OH radical production and to confirm earlier reports of its antioxidant capacity in various in vitro and
Deferiprone is widely used in iron-overload diseases such as thalassemia, which is mainly distributed in the Mediterranean, Middle East, and South East Asian countries [42,43]. In the same areas extremely high sun light irradiation levels are present. Taking into account that the usual therapeutic dose of L1 ranges between 75 and 100 mg/kg/day, and its peak blood concentration can range between 50 and 500 μΜ, it is important to control possible phototoxic effects of L1 and other iron chelators. This study shows that L1 can inhibit OH radical production via a photoFenton reaction. In addition, no reactive oxygen species were detected under direct UV irradiation of the L1 water solution or of L1–iron complexes during irradiation by UV and visible light. The most important finding of this study is the oxidation of Fe(II) to Fe(III) by deferiprone, with subsequent formation of an Fe(III)–L13 complex, which is redox inactive under dark physiological conditions. It is well known that iron and in particular Fe(II) is the major catalyst of free radical reactions in biological systems and of free radical damage in diseases related to oxidative stress. Overall, L1 works as an antioxidant by preventing iron-induced free radical formation (via Fenton reaction). These findings confirm previous findings of the antioxidant targeting potential of L1 in diseases related to oxidative damage [39].
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Acknowledgments This work was supported by the Council for Grants of the President of the Russian Federation for Support of Leading Scientific Schools (Project NSh 2272.2012.3). Special thanks to Dr. Igor Kirilyuk (Novosibirsk Institute of Organic Chemistry, Russia) and Dr. Irina Slepneva (Institute of Chemical Kinetics and Combustion, Russia), for the samples of spin traps, and Dr. Dmitri Stass (Institute of Chemical Kinetics and Combustion) for technical support and general discussion. References [1] Kontoghiorghes, G. J. Prospects for introducing deferiprone as potent pharmaceutical antioxidant. Front. Biosci. (Elite Ed.) 1:161–178; 2009. [2] Kontoghiorghes, G. J.; Eracleous, E.; Economides, C.; Kolnagou, A. Advances in iron overload therapies: prospects for effective use of deferiprone (L1), deferoxamine, the new experimental chelators ICL670, GT56-252, L1NA11 and their combination. Curr. Med. Chem. 12:2663–2681; 2005. [3] Oxidation and Antioxidants in Organic Chemistry and Biology. In: Denisov, E. T., Afanas’ev, I. B., editors. Boca Raton: CRC Press/Taylor & Francis; 2005. [4] Halliwell, B.; Gutteridge, J. M. C.; Cross, C. E. Free radicals, antioxidants and human disease: where are we now? J. Lab. Clin. Med. 119:598–620; 1992. [5] Kontoghiorghes, G. J. Iron chelation in biochemistry and medicine. In: RiceEvans, C., editor. Free Radicals, Oxidant Stress and Drug Action. London: Rechelieu Press; 1987. p. 277–303.
Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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Please cite this article as: Timoshnikov, VA; et al. Inhibition of Fe2 þ - and Fe3 þ - induced hydroxyl radical production by the ironchelating drug deferiprone. Free Radic. Biol. Med. (2014), http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.513i
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