Chem. Res. Toxicol. 1999, 12, 137-143
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Articles Inhibition of Skeletal Sarcoplasmic Reticulum Activity by Deferoxamine Nitroxide Free Radical
Ca2+-ATPase
Masanori Kiyose, Chang-il Lee, and Eiichiro Okabe* Department of Pharmacology and ESR Laboratory, Kanagawa Dental College, Yokosuka, Kanagawa 238-0003, Japan Received September 9, 1998
Deferoxamine is an inhibitor of iron-dependent free radical reactions. Despite the antioxidant roles, prolonged clinical use of the chelator is far from benign, and paradoxically, deferoxamine has been shown to promote lipid peroxidation. The possible toxicity of the drug’s metabolites, such as deferoxamine nitroxide free radical, deserves attention. We, therefore, tested the hypothesis that deferoxamine nitroxide radicals produced as a result of enzymatic one-electron oxidation of deferoxamine by horseradish peroxidase in the presence of H2O2 are capable of inactivating Ca2+-ATPase of skeletal sarcoplasmic reticulum microsomes as a model system with which to explore the effect of the radical on a biological membrane. Ca2+-ATPase activity of sarcoplasmic reticulum was depressed by exposure to Fenton’s reagent (H2O2/FeSO4); the observed effect was significantly enhanced by deferoxamine. We found that the Fenton reaction produced hydroxyl radical, as determined by electron spin resonance spectroscopy. The formation of hydroxyl radical was completely inhibited by deferoxamine; instead, under the same experimental conditions (in the presence of sarcoplasmic reticulum vesicles with or without FeSO4 but without spin trap 5,5-dimethyl-1-pyrroline N-oxide), the spectral shape and hyperfine coupling constants of electron spin resonance signals confirmed to be long-lived deferoxamine radical were obtained. Furthermore, exposure of sarcoplasmic reticulum vesicles to deferoxamine radical formed by horseradish peroxidase via reaction with H2O2 caused an inhibition of the Ca2+-ATPase activity. The findings show that the sarcoplasmic reticulum vesicles can act as peroxidases and suggest that deferoxamine enhances the decreased Ca2+ATPase activity afforded by H2O2/FeSO4 due to formation of its metabolites, possibly deferoxamine nitroxide free radical.
Introduction The iron chelator deferoxamine (DFX1), a bacterial siderophore isolated from Streptomyces pilosus, is used in the treatment of pathological disorders where the effective removal of excess iron is desired (1). The excess of iron ions can promote membrane lipid peroxidation through the generation of cytotoxic reactive oxygen species (2-4) and, in particular, the highly reactive HO• radical (5). Since reactive oxygen species are suspected of playing a role in human diseases, the hypothesis that * Address all correspondence to Eiichiro Okabe, D.D.S., Ph.D., Professor and Chairman, Department of Pharmacology and ESR Laboratory, Kanagawa Dental College, 82 Inaoka-Cho, Yokosuka, Kanagawa 238-0003, Japan. Telephone: +81-468-22-8836. Fax: +81468-22-8868. E-mail:
[email protected]. 1 Abbreviations: DFX, deferoxamine; HO•, hydroxyl radical; HRP, horseradish peroxidase; ATPase, adenosine triphosphatase; SR, sarcoplasmic reticulum; EGTA, ethylene glycol bis(oxyethylenenitrilo)tetraacetic acid; ESR, electron spin resonance; DMPO, 5,5-dimethyl1-pyrroline N-oxide; MnO, manganese oxide marker; TEMPOL, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DMSO, dimethyl sulfoxide; DMTU, 1,3-dimethyl2-thiourea.
iron-chelating therapy could be an effective means of reducing tissue damage is currently under investigation (6, 7). Apart from the ability of DFX to bind iron in such a way as to inhibit its catalytic activity, this drug is thought to play an antioxidant role through its properties as a scavenger of oxygen free radicals. Although questionable (8), the possibility that DFX may play an antioxidant role independent of iron chelation is supported by studies showing the ability of DFX to scavenge peroxyl radicals (9), to act as a lipid chain-breaking antioxidant (10), and to donate electrons to purified oxidases (11). Despite these antioxidant roles, prolonged clinical use of the chelator is far from benign (12, 13), and paradoxically, DFX has been shown to promote lipid peroxidation (13, 14). The mechanisms involved in toxic reactions of metal-chelating agents are not completely understood and may depend on metal ion decompartmentalization (12, 15-17). In addition, the possible toxicity of the drug’s metabolites, such as the toxic DFX radical (17), deserves attention. It is unknown whether the DFX radical occurs
10.1021/tx980212y CCC: $18.00 © 1999 American Chemical Society Published on Web 12/30/1998
138 Chem. Res. Toxicol., Vol. 12, No. 2, 1999
in vivo, but it has been produced in vitro by various purified peroxidases (11) and via reaction with H2O2 (18). Peroxidase enzymes capable of oxidizing DFX to yield a mixture of DFX nitroxide radicals include HRP (18). In view of the widespread use of DFX in vivo and in vitro, we thought it was important to investigate, in detail, this proposed reaction of DFX with H2O2 and HRP and to determine the effects of DFX and reactive oxygen species interaction on Ca2+-ATPase of skeletal SR microsomes as a model system with which to explore the effect of DFX on a biological membrane. This study demonstrates that generation of HO• from Fenton’s reagent (H2O2/FeSO4) is inhibited by DFX; however, decreased Ca2+-ATPase activity of skeletal SR vesicles afforded by H2O2/FeSO4 reaction is potentiated by DFX, in part, due to formation of its metabolites, possibly DFX radical.
Experimental Procedures Reagents. The following drugs and chemicals were used: DFX (Desferal, Ciba-Geigy Pharmaceutic Division, Hyogo, Japan), HRP and catalase (Sigma, St. Louis, MO), DTNB and hydrogen peroxide (Wako Chemicals, Osaka, Japan; the H2O2 concentration was estimated spectrophotometrically by measuring its absorbance at 240 nm with the extinction coefficient of 81 L mol-1 cm-1, and then it was used for experiments), TEMPOL (Aldrich, Milwaukee, WI), and DMPO (Labotec, Tokyo, Japan; 99-100% pure, GC assay by Dojindo Laboratories, Kumamoto, Japan). All other reagents were analytical grade. SR Preparation. In accordance with our institutional Animal Care Committee guidelines, masseter muscles were taken from beagle dogs during anesthesia with sodium pentobarbital (25 mg/kg iv). The SR vesicles were prepared from minced masseter muscle as previously described by Okabe et al. (19). Briefly, the muscle was cleaned of fat and connective tissue, minced, and then homogenized twice [1 g of muscle/4 volumes of 10 mM imidazole buffer (pH 7.0)] for 1 min at 4 °C in an Excel autohomogenizer (DX-8, Nihon Seiki, Tokyo, Japan). The homogenate was centrifuged at 10000g for 20 min. The pellet was rehomogenized in 4 volumes of 10 mM imidazole and centrifuged at 10000g for 20 min. The supernatant fractions from this and the previous centrifugation were combined, poured through four layers of cheesecloth, and then centrifuged at 12000g for 15 min. The supernatant fraction was filtered through eight layers of cheesecloth and centrifuged at 31000g for 90 min. The pellets from this spin were rehomogenized, by use of a Potter-Elvehjem homogenizer with a Teflon pestle, in 1 M KCl and 10 mM imidazole buffer (pH 7.0), and then centrifuged at 145000g for 60 min. The SR pellet was rehomogenized in 30% sucrose and 20 mM Tris-HCl (pH 7.0) and stored at -80 °C. The protein concentration of this SR preparation was determined by the method of Lowry et al. (20). Ca2+-ATPase Activity. Ca2+-ATPase activity of SR was determined as the rate at which inorganic phosphate (Pi) was liberated during the incubation. The incubation bath (5 mL) was kept at 27 °C and contained 100 mM KCl, 20 mM imidazole buffer (pH 7.0), 10 mM NaN3, 10 mM potassium oxalate, 5 mM Na2ATP, 5 mM MgCl2, and 200 µM CaCl2 (21). The released phosphate in the filtrate was assayed by a colorimetric method (22). The Ca2+-ATPase activity was calculated as the difference in the ATPase rate in a bath containing 200 µM Ca2+ compared to one containing 0.02 M EGTA. ESR Analysis. ESR observations were made at room temperature (22 °C) with a JES-RE 3X, X-band spectrometer (JEOL, Tokyo, Japan) connected with the JEOL computer system Esprit at the following instrument settings: modulation amplitude, 0.063 mT; recording range, 5 mT; recording time, 2 min; time constant, 0.1 s for spin trapping studies with spin trap DMPO and 0.03 s for DFX radical detection; microwave power, 8 mW for spin trapping studies with DMPO and 21 mW for DFX
Kiyose et al. radical detection; and magnetic field, 334.2 ( 5 mT. After the ESR spectra were recorded, the signal intensity was normalized as a relative height against the standard signal intensity of the manganese oxide marker. Hyperfine coupling constants were calculated on the basis of the resonance frequency measured with a microwave frequency counter and the resonance field measured with the field measurement unit model ES-FC5 (JEOL). A quantitative analysis of the DFX radical spin concentration was performed as described previously (23); an absolute concentration of DFX radical was determined by a double integration of the ESR spectrum, in which a 1.0 µM TEMPOL solution was used for a primary standard of ESR absorption. Determination of Thiol Groups. The sulfhydryl group oxidation of SR by DFX radical was determined using DTNB or Ellman’s reagent. This reagent has been used extensively to determine the sulfhydryl concentration at pH 8.0 (24). DTNB provides a convenient optical assay for studying sulfhydryl group oxidation because it does not interact with disulfides and exhibits a large change in absorption at 412 nm on reacting with sulfhydryls. SR vesicles were incubated with DFX radical for 0.5 min at 27 °C. After exposure, the SR was added to the reaction mixture (2 mL) containing 0.1 M Tris-HCl buffer (pH 8.0), 1 mM EDTA, 1% sodium dodecyl sulfate, 1 mM DTNB, and 0.5 mg/mL SR. At the end of 10 min, the absorption was read at 412 nm and the concentration of thiol groups was calculated by using a molar extinction coefficient of 13.6 × 103 L mol-1 cm-1 for the product of the reaction (p-nitrothiophenol anion) (25). Data Presentation. In Ca2+-ATPase and thiol group studies, the SR vesicles (for control and experimental groups) prepared from the same pooled masseter muscles were studied in parallel. When data were analyzed, one-way analysis of variance was used and comparisons with control were made by Dunnett’s test. Statistical analyses were performed by SuperANOVA (Abacus Concepts, Inc., Barkeley, CA). A significance level p of
